enzymatic transformation
TRANSCRIPT
Enzymatic Transformation
Soundar Divakar
Enzymatic Transformation
Soundar Divakar Central Food Technological Research Institute Mysore, Karnataka , India
ISBN 978-81-322-0872-3 ISBN 978-81-322-0873-0 (eBook) DOI 10.1007/978-81-322-0873-0 Springer New Delhi Heidelberg New York Dordrecht London
Library of Congress Control Number: 2012951119
© Springer India 2013 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, speci fi cally the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on micro fi lms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. Exempted from this legal reservation are brief excerpts in connection with reviews or scholarly analysis or material supplied speci fi cally for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Duplication of this publication or parts thereof is permitted only under the provisions of the Copyright Law of the Publisher’s location, in its current version, and permission for use must always be obtained from Springer. Permissions for use may be obtained through RightsLink at the Copyright Clearance Center. Violations are liable to prosecution under the respective Copyright Law. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a speci fi c statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. While the advice and information in this book are believed to be true and accurate at the date of publication, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein.
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Springer is part of Springer Science+Business Media (www.springer.com)
Viky and Sasi
vii
This book is a summary of the research work carried out by me with my students and collaborators at the Central Food Technological Research Institute, Mysore, India. The work was carried out in a period spanning 18 years or so from 1995 onwards. The essence of the work from 70 or so publications is presented here in brief under 11 chapters.
The objective of this book is to explain the various techniques and strategies involved in enzymatic transformation reactions. Esteri fi cation of acids using lipases and glycosylation of alcohols and phenols using glycosidases are the two enzymatic reactions highlighted in this book. Both esteri fi cation and glycosylation reactions involve multifunctional substrates. Challenges using underivatised polar and nonpolar substrates in terms of product speci fi city/selectivity, yields and feasibility in such enzymatic reactions carried out in nonpolar media are explained with several examples of divergent multifunc-tional substrates. Hence, the information provided would be a source of useful information for not only established researchers but also for beginners who eye this as a potential and fruitful research area.
The author is sure that this book has brought out the trials and tribulations of carrying out enzymatic transformations clearly.
Preface
ix
Dr. Divakar is working as a Chief Scientist in the Central Food Technological Research Institute, Mysore, where he joined as a Scientist. Before this, he served as CSIR Pool Of fi cer at Molecular Biophysics Unit, Indian Institute of Science, Bangalore. After completing his post-graduation in 1976 from Pachaiyappa’s College, Chennai, he had a brief stint in a polymer industry, M/S Reichhold Chemicals, Ind. Ltd., Chennai. He completed his Ph.D. degree in Chemistry from the Australian National University, Canberra, Australia in 1982.
The author is actively involved in research for the past 36 years. While working as a Research and Development chemist at Ms. Reichhold Chemicals (Ind.) Ltd., he was actively involved in research in the fi elds of alkyd resins, unsaturated polyesters, phenol-formaldehyde resins (resoles and novolaks), urea-formaldehyde resins and epoxy resins.
After completing his Ph.D., he was involved in research work on three important areas, namely, 1. NMR investigations of some biological systems 2. Host-guest complexation chemistry with special reference to cyclodextrins 3. Enzyme catalyzed transformation reaction employing lipases and
glycosidases He has so far guided various Ph.D. and M.Sc. students and has published about 149 research articles including reviews. His work on Enzymatic Transformations has resulted in about 70 publications, which prompted him to write this book on Enzymatic Transformation.
About the Author
xi
Acknowledgements
The author gratefully acknowledges Central Food Technological Research Institute, Mysore, for providing the facilities and sanctioning the projects. Acknowledgement is also due to the Department of Biotechnology, India, and Department of Science and Technology, India, for providing the fi nancial assistance.
Author expresses his gratitude and appreciation to the following students and the other contributors: Dr. B. Manohar, Dr. P. Ravi, Dr. K.R. Kiran, Dr. C.V. Suresh Babu, Dr. H.H. Pattekhan, M.S. Pramila Rao, Dr. K. Lohith, Dr. G.R. Vjayakumar, Dr. B.R. Somashekar, Dr. R. Sivakumar, Mr. Mallikarjuna, Mr. Swaminathan and Mr. G. Vadivelan.
The author also acknowledges Springer for their prompt response and support in publishing this work.
xiii
1 Introduction ................................................................................... 1 1.1 Introduction .......................................................................... 1 1.2 Scope of the Book ................................................................ 2 References ..................................................................................... 2
2 Glycosidases ................................................................................... 5 2.1 Introduction .......................................................................... 5 2.2 Amylolytic Enzymes ............................................................ 5 2.3 Glucoamylase ....................................................................... 6 2.4 Sources of Glucoamylases ................................................... 6 2.5 Sources of Other Glycosidases ............................................ 7 2.6 Structural Features of Glucoamylase ................................... 7 2.7 Structural Features of β-Glucosidase ................................... 8 2.8 Glycosylation ....................................................................... 9 2.9 Mechanism of Glycosylation ............................................... 10 2.10 Glycosylation Reactions ...................................................... 11 2.11 Advantages of Enzymatic Glycosylation
over Chemical Methods ....................................................... 13 References ....................................................................................... 17
3 Lipases ............................................................................................ 23 3.1 Lipases ................................................................................. 23 3.1.1 Porcine Pancreas Lipase (PPL) ................................ 24 3.1.2 Rhizomucor miehei Lipase (RML) ........................... 25 3.1.3 Candida rugosa Lipase (CRL) ................................. 26 3.2 Lipase Specificity ................................................................. 27 3.3 Reactions Catalysed by Lipases ........................................... 28 3.3.1 Hydrolysis ................................................................ 28 3.3.2 Esterification ............................................................ 29 3.3.3 Transesterification .................................................... 29 3.4 Mechanism of Lipase-Catalysed Esterification
in Organic Solvents .............................................................. 29 3.5 Esterification Reactions ....................................................... 30 3.6 Advantages of Lipase Catalysis over
Chemical Catalysis ............................................................... 30 References ....................................................................................... 34
Contents
xiv Contents
4 Enzymatic Esterification of Compounds Possessing Multifunctional Hydroxyl and Carboxyl Groups ...................... 39
4.1 Introduction .......................................................................... 39 4.2 2-O-Acyl Esters of Lactic Acid ........................................... 39 4.2.1 Lactic Acid ............................................................... 40 4.2.2 2-O-Stearoyl Lactic Acid ......................................... 40 4.2.3 2-O-Palmitoyl Lactic Acid ....................................... 41 4.2.4 Optimisation of Reaction Parameters
Using Response Surface Methodology .................... 42 4.2.5 Effect of Acid Carbon Chain Length
on Esterification with Lactic Acid ........................... 45 4.2.6 Reusability of Porcine Pancreas Lipase ................... 45 4.2.7 Food Chemical Codex Specifications
for Enzymatically Synthesised 2-O-Acyl Esters of Lactic Acid ............................................... 46
4.3 Tolyl Esters .......................................................................... 47 4.3.1 Application of Central Composite Rotatable
Design to Lipase-Catalysed Synthesis of m-Cresyl Acetate ................................................. 49
4.4 Application of Plackett–Burman Design for Lipase-Catalysed Esterification of Anthranilic Acid .............................................................. 50
4.5 Kinetic Study of Porcine Pancreas Lipase Inhibition by p-Cresol (p-Cresyl acetate) and Lactic Acid (2-O-Stearoyl Lactate) ......................................................... 50
4.6 Thermostability of Porcine Pancreas Lipase ........................ 52 4.7 Scanning Electron Microscopy ............................................ 53 4.8 Hydrogen Ions in Micro-aqueous Phase During
Lipase-Catalysed Esterification in Nonaqueous Media ....... 55 4.9 Acetylation of Protocatechuic Aldehyde ............................. 58 4.10 4-t-Butylcyclohexyl Acetate ................................................ 58 4.11 Esterifi cation of b-Cyclodextrin........................................... 61 References ....................................................................................... 62
5 Enzymatic Polymerisation ............................................................ 65 5.1 Introduction .......................................................................... 65 5.2 Polylactic Acid ..................................................................... 66 5.2.1 Shake-Flask Level .................................................... 66 5.2.2 Bench-Scale Level ................................................... 66 5.2.3 Further Molecular Weight Build-Up ........................ 68 5.2.4 Nuclear Magnetic Resonance Spectroscopy ............ 70 5.2.5 Polylactic Acid Films .............................................. 71 5.3 Poly-e-caprolactone ............................................................. 72 5.3.1 Shake-Flask- and Bench-Scale-Level
Experiments ............................................................. 72 5.3.2 Nuclear Magnetic Resonance Spectroscopy ............ 74 5.3.3 Polycaprolactone Ester Films .................................. 74 5.4 Poly-p-hydroxybenzoate ...................................................... 74 5.5 Poly-p-benzamide ................................................................ 76 5.6 Polyadipates ......................................................................... 77 References ....................................................................................... 78
xvContents
6 Lipase-Catalysed Preparation of Aminoacyl Esters of Carbohydrates ............................................................... 81
6.1 Introduction .......................................................................... 81 6.2 l-Alanyl-d-Glucose ............................................................. 83 6.2.1 Optimisation Studies ................................................ 83 6.2.2 Reusability of Lipases .............................................. 83 6.2.3 Syntheses of l-Alanyl Esters of Carbohydrates ....... 85 6.3 Synthesis of l-Valyl-d-Glucose ........................................... 86 6.3.1 Syntheses of l-Valyl Esters of Carbohydrates ......... 90 6.4 l-Leucyl-d-Glucose ............................................................. 91 6.4.1 Optimum Conditions ................................................ 91 6.4.2 Selectivity................................................................. 91 6.4.3 Determination of Critical Micellar
Concentration (CMC) .............................................. 91 6.4.4 Syntheses of l-Leucyl Esters of Carbohydrates ...... 91 6.5 Syntheses of l-Isoleucyl Esters of Carbohydrates ............... 91 6.6 Synthesis of l-Phenylalanyl-d-Glucose ............................... 98 6.6.1 Optimum Conditions ................................................ 98 6.6.2 Reusability of Lipases .............................................. 100 6.6.3 Determination of Critical Micellar
Concentration (CMC) .............................................. 100 6.6.4 Optimisation of l-Phenylalanyl-d-Glucose
Synthesis Using Response Surface .......................... 100 6.6.5 l-Phenylalanyl Esters of Carbohydrates .................. 106 6.7 l-Prolyl Esters of Carbohydrates ......................................... 106 6.8 l-Tryptophanyl Esters of Carbohydrates .............................. 107 6.9 l-Histidyl Esters of Carbohydrates ....................................... 109 6.10 Spectral Characterisation of l-Alanyl, l-Valyl,
l-Leucyl, l-Isoleucyl, l-Prolyl, l-Phenylalanyl, l-Tryptophanyl and l-Histidyl Esters of Carbohydrates .................................................................. 109
6.11 Discussion ............................................................................ 112 References ....................................................................................... 120
7 Enzymatic Glycosylation of Alcohols .......................................... 123 7.1 Introduction .......................................................................... 123 7.2 n-Octyl-d-Glucoside ............................................................ 124 7.3 Synthesis of n-Octyl-d-Glucoside Using β-Glucosidase ..... 126 7.4 Determination of Critical Micellar Concentration (CMC) .. 127 7.5 Synthesis of n-Octyl Glycosides .......................................... 127 7.6 Spectral Characterisation ..................................................... 127 7.7 Synthesis of n-Alkyl Glucosides Using
Amyloglucosidase ................................................................ 129 7.7.1 Shake-Flask Method ................................................ 129 7.7.2 Re fl ux Method ......................................................... 129 7.8 Cetyl and Stearyl Glucosides ............................................... 130 7.9 Optimisation of n-Octyl-D-Glucoside Synthesis
Using Response Surface Methodology ................................ 130 References ....................................................................................... 134
xvi Contents
8 Glycosylation of Some Selected Phenols and Vitamins ............. 137 8.1 Phenols ................................................................................. 137 8.1.1 Guaiacyl Glycosides .............................................. 138 8.1.2 Eugenyl Glycosides ............................................... 141 8.1.3 Curcuminyl Glycosides.......................................... 141 8.1.4 Syntheses of N-Vanillyl-Nonanamide
Glycosides .............................................................. 149 8.1.5 Capsaicin Glycosides ............................................. 151 8.1.6 Syntheses of Vanillyl Glycosides ........................... 152 8.1.7 Syntheses of dl-Dopa Glycosides ......................... 165 8.1.8 l-Dopa Glycosides ................................................. 167 8.1.9 Syntheses of Dopamine Glycosides ....................... 171 8.1.10 Serotonyl Glycosides ............................................. 177 8.1.11 Epinephryl Glycosides ........................................... 182 8.2 Vitamins ............................................................................... 183 8.2.1 Glucosylation of Thiamin ...................................... 184 8.2.2 Syntheses of Ribo fl avinyl Glycosides ................... 187 8.2.3 Pyridoxine Glycosides ........................................... 191 8.2.4 Glycosylation of Retinol ........................................ 195 8.2.5 Syntheses of Ergocalciferyl Glycosides ................ 198 8.2.6 Cholecalciferol Glycosides .................................... 202 8.2.7 Syntheses of α-Tocopheryl Glycosides ................. 204 References ....................................................................................... 209
9 Glycosylation of Phenols and Vitamins: An Overview .............. 215 9.1 General ................................................................................. 215 9.2 n-Alkyl Glycosides .............................................................. 219 9.3 Curcuminyl-bis-Glycosides ................................................. 219 9.4 N-Vanillyl-Nonanamide Glycosides .................................... 220 9.5 Vanillyl Glycosides .............................................................. 220 9.6 dl-Dopa Glycosides ............................................................. 221 9.7 Dopamine Glycosides .......................................................... 221 9.8 Riboflavinyl Glycosides ....................................................... 221 9.9 Retinyl Glycosides ............................................................... 222 9.10 Ergocalciferyl Glycosides .................................................... 222 9.11 Cholecalciferol Glycosides .................................................. 223 9.12 α-Tocopheryl Glycosides ..................................................... 223 References ....................................................................................... 224
10 Kinetics of Some Selected Enzyme-Catalysed Reactions in Organic Solvents ....................................................................... 225
10.1 Introduction .......................................................................... 225 10.2 Methodology ........................................................................ 226 10.2.1 Esterification Procedure ......................................... 226 10.2.2 Glycosylation Procedure ........................................ 226 10.3 Esteri fi cation Kinetics .......................................................... 226 10.3.1 Esterification Kinetics of l-Alanine
and d-Glucose: Single Substrate Inhibition ........... 226 10.3.2 Esterification Kinetics of l-Phenylalanine
and d-Glucose: Double Substrate Inhibition ......... 232
xviiContents
10.3.3 Esterification Kinetics of l-Phenylalanine and d-Glucose: Single Substrate Inhibition ........... 236
10.4 Glycosylation Kinetics ......................................................... 239 10.4.1 Glycosylation Kinetics of Curcumin
and d-Glucose: Single Substrate Inhibition ........... 239 10.4.2 Glucosylation Kinetics of Vanillin
and d-Glucose: Single Substrate Inhibition ........... 244 10.5 Discussion ............................................................................ 246 10.5.1 Esterification Kinetics ............................................ 246 10.5.2 Glycosylation Kinetics ........................................... 248 References ....................................................................................... 249
11 ACE Inhibition and Antioxidant Activities of Enzymatically Synthesised Aminoacyl Esters and Glycosides ................................................................... 251
11.1 ACE Inhibition by Aminoacyl Esters of Carbohydrates .................................................................. 251
11.2 ACE Inhibition of Glycosides .............................................. 258 11.3 Antioxidant Activity of Glycosides ..................................... 268 References ....................................................................................... 277
Index ....................................................................................................... 279
xix
List of Abbreviations and Symbols
A Absorbance α Alpha ANOVA Analysis of variance ACE Angiotensin-converting enzyme Å Angstrom β Beta BSA Bovine serum albumin CRL Candida rugosa lipase 13 C Carbon-13 CCRD Central composite rotatable design J Coupling constant CMC Critical micellar concentration °C Degree centigrade d Delta DMSO-d
6 Deuterated dimethyl sulphoxide
CH 2 Cl
2 Dichloromethane
DMSO Dimethyl sulphoxide eV Electron volt EC Enzyme commission γ Gamma g Gram Hz Hertz HMQCT Heteronuclear multiple quantum coherence transfer HSQCT Heteronuclear single quantum coherence transfer HPLC High-performance liquid chromatography h Hour IR Infrared K
cat Catalytic rate constant
K i Inhibitor constant v Initial velocity KDa Kilodalton MS Mass spectroscopy V
max Maximum velocity
MHz Mega hertz K
M Michaelis–Menten constant
List of Abbreviations
xx List of Abbreviations
K mA
Michaelis–Menten constant for the lipase– l -alanine complex K
m L -alanine Michaelis–Menten constant for the lipase– l -alanine complex
K mB
Michaelis–Menten constant for the lipase– d -glucose complex K
m D -glucose Michaelis–Menten constant for the lipase– d -glucose complex
K i Dissociation constant for the lipase–inhibitor ( d -glucose) complex
K i D -glucose
Dissociation constant for the lipase–inhibitor ( d -glucose) complex mM Millimolar mol Mole [M] + Molecular ion nm Nanometre N Normality NMR Nuclear magnetic resonance [ a ] Optical rotation ppm Parts per million % Percentage p Pi PAGE Polyacrylamide gel electrophoresis PPL Porcine pancreas lipase KBr Potassium bromide 1 H Proton RSM Response surface methodology R
f Retention factor
RT Retention time RML Rhizomucor miehei lipase σ Sigma SDS Sodium dodecyl sulphate TMS Tetra-methyl silane TLC Thin layer chromatography 2D Two-dimensional UV Ultraviolet v/v Volume by volume a
w Water activity
cm −1 Wave per centimetre w/w Weight by weight
1S. Divakar, Enzymatic Transformation, DOI 10.1007/978-81-322-0873-0_1, © Springer India 2013
1.1 Introduction
The diverse biochemical reactions taking place in living systems are all mediated by series of enzymes (Michal 1999 ) . Almost all the bio-chemical reactions are catalysed by enzymes, from both within and outside the cells. Hence, the subject of enzymatic transformation is not new. However, the term enzymatic transforma-tion largely refers to those reactions conducted in vitro, using enzymes either in the form of microbial cells or isolated from intact cells or. Only about 2,800 enzymes have been charac-terised of the estimated 25,000 enzymes pres-ent in nature. Of which, 400 enzymes, mainly hydrolases, transferases and oxido-reductases have been identi fi ed as commercially potential ones. However, only 50 different kinds of enzymes fi nd application on an industrial scale (Winterhalter and Schreier 1993 ; Schreier and Winterhalter 1993 ; Berger 1995 ) . The enzymes of commer-cial importance in food industry are amylases,
proteases, pectinases, cellulases, hemicellulases, lipases and lactases.
Enzymes are protein speci fi c in their catalytic action, simple or conjugated capable of catalysing biochemical reactions. An enzyme in a reaction lowers the amount of activation energy required by the reaction. Enzymes are thermolabile and like proteins are inactivated at certain tempera-tures in a manner characteristic for protein dena-turation. Substances on which enzymes act speci fi cally are substrates, and their speci fi city plays a role in enzyme classi fi cation.
In the past two decades, the application of enzymes in organic synthesis has increased enor-mously as an extremely important synthetic strategy, with a wide variety of enzymes as tool in enzymatic and chemo-enzymatic synthesis (Ward and Singh 2000 ; Davis and Boyer 2001 ) . As biocatalysts save additional reaction steps compared to chemical synthesis, most of the organic chemists use biocatalysts. Highly chemo-, regio- and stereospeci fi c reactions under safer
1
Abstract
General concept on enzymes and enzymatic transformation along with the importance of carrying out enzymatic reactions in organic solvents is mentioned in this introductory chapter. A concluding Scope of the Book indicates that the book deals mainly with the transformations mediated by two important hydrolytic enzymes – glycosidases and lipases.
Introduction
2 1 Introduction
and eco-friendly conditions are possible with enzymes and can be used for the preparation of wide range of organic compounds, especially in food and pharmaceutical preparations. Oxido-reductases, hydralases (lipases, esterases, glycosidases, transglycosidases, peptidases, acy-lases, amidases, epoxide hydralases, nitrilases and hydantoinases), lyases and isomerases are used in organic synthesis (Drauz and Waldmann 2002 ; Faber 2004 ; Buchholz et al. 2005 ) .
Synthetic chemistry involving chemical routes requires drastic conditions, like use of acids or alkali, high temperature, hazardous chemicals, toxic metals and catalysts leading to high energy consumption, colouring of products, low selectivity and environmental pollution. It is also tedious to synthesise regio- and stereoselec-tive compounds, as they require continuous pro-tection and deprotection steps. Thus, chemical syntheses include multistep processes and result in a number of by-products and hence economi-cally inef fi cient. Some chemically synthesised products also require exhaustive and cost-inten-sive puri fi cation steps.
Enzymes give better solutions to all these problems. Enzymes can function under milder reaction conditions, without requiring high tem-peratures and use of hazardous chemicals, and they are totally ‘eco friendly’ in nature. Enzymatic activity in nonaqueous solvents offers a new methodology for the production of many useful compounds which are not feasible in aqueous media. Klibanov ( 1986 ) initiated this novel approach and an outstanding synthetic strategy which now fi nd use in a large number of applica-tions in organic synthesis. Enzymes are employed in organic solvents for the synthesis of esters (Santaniello et al. 1993 ) , chiral compounds (Orrenius et al. 1995 ) , surfactants (Plou et al. 1999 ; Sarney and Vulfson 1995 ; Sarney et al. 1996 ) , pharmaceutical intermediates (Duan et al. 1997 ) , biotransformations of oils and fats (Bosley and Clayton 1994 ) and sugar-based polymers (Patil et al. 1991 ) . Thus, the use of enzymes as biocatalysts in biotechnology has found potential applications in pharmaceutical, food, cosmetic, fl avour and fragrance and beverage industries.
1.2 Scope of the Book
In the last two decades, literature on biotrans-formations has been ever growing enriching our knowledge on the subject. A lot of information on the behaviour of enzymes under diverse reaction conditions is available. However, only very few general rules have emerged from these studies, indicating that the fi eld is wide open for creative minds to explore.
This book intends to throw some more light in this ever-growing area with a large number of examples on structurally diverse substrates and reaction conditions through investigations involv-ing different strategies dealing with random para-metric studies, optimization, response surface methodology and kinetics. Attempts have been made in this documentation to bring out the broad speci fi city of such enzyme-catalysed transforma-tions involving two commercially important class of enzymes – lipases and glycosidases.
Both glycosidase and lipases, among all the known enzymes, have come a long way in estab-lishing themselves as important synthetic tools to bio-organic reactions. The main scope of the present book is to discuss the use of glycosi-dases and lipases in reactions carried out in our laboratory for more than a decade or so, to pre-pare commercially important compounds and the underlying problems associated with the same. Thus, all efforts have been made to create interest in researchers about certain intricate details of the approaches in order to reap fruit-ful results as the problems are many and complex.
References
Berger RG (1995) Aroma biotechnology. Springer, New York/Berlin/Heidelberg
Bosley JA, Clayton JC (1994) Blueprint for a lipase support: use of hydrophobic controlled-pore glasses as model system. Biotechnol Bioeng 43:934–938
Buchholz K, Kasche V, Bornscheuer UT (2005) Biocatalysts and enzyme technology. Willey-VCH, Weinheim
Davis BG, Boyer V (2001) Biocatalysis and enzymes in organic synthesis. Nat Prod Rep 18:618–640
3References
Drauz K, Waldmann H (eds) (2002) Enzyme catalysis in organic synthesis, 2nd edn, vols 1–3. Willey-VCH, Weinheim
Duan G, Ching CB, Lim E, Ang CH (1997) Kinetic study of enantioselective esteri fi cation of ketoprofen with n -propanol catalysed by an lipase in an organic medium. Biotechnol Lett 19:1051–1055
Faber K (2004) Enzymes in organic solvents. In: Faber K (ed) Biotransform. Organic chemistry, 5th edn. Springer, Berlin
Klibanov AM (1986) Enzymes that work in organic solvents. Chem Technol 16:354–359
Michal G (1999) Biochemical pathways. Wiley, New York Orrenius C, Norin T, Hult K, Carrea G (1995) The Candida
antartica lipase B catalysed kinetic resolution of seudenol in non-aqueous media of controlled water activity. Tetrahedron Asym 12:3023–3030
Patil DR, Rethwisch DG, Dordick JS (1991) Enzymatic synthesis of sucrose containing liner polyester in nearly anhydrous organic media. Biotechnol Bioengg 37:639–646
Plou FJ, Cruces MA, Pastor E, Ferrer M, Bernabe M, Ballesterose A (1999) Acylation of sucrose with vinyl esters using immobilized hydrolysis demonstration
that chemical catalysis may interfere with enzymatic catalysis. Biotechnol Lett 21:635–639
Santaniello E, Ferraboschi P, Grisenti P (1993) Lipase-catalyzed transesteri fi cation in organic solvents. Applications to the preparation of enantiomerically pure compounds. Enzyme Microb Technol 15:367–382
Sarney DB, Vulfson EN (1995) Application of enzymes to the synthesis of surfactants. Trends Biotechnol 13:164–172
Sarney DB, Barnard MJ, MacManus DA, Vulfson EN (1996) Application of lipases to the regioselective synthesis of sucrose fatty acid monoesters. J Am Oil Chem Soc 73:1481–1487
Schreier P, Winterhalter P (1993) Progress in fl avor precursor studies. Allured, Carol Stream
Ward OP, Singh A (2000) Enzymatic asymmetric synthe-sis by decarboxylases. Curr Opin Biotechnol 11:520–526
Winterhalter P, Schreier P (1993) Biotechnology – challenge for the fl avor industry. In: Acree TE, Teranishi R (eds) Flavor science sensible principle and techniques. American Chemical Society, Washington, DC, pp 225–258
5S. Divakar, Enzymatic Transformation, DOI 10.1007/978-81-322-0873-0_2, © Springer India 2013
2.1 Introduction
In order to carry out an enzymatic transformation reaction, one requires a profound knowledge on enzymes themselves. Although several enzymes have been employed in such reactions, this book will deal mainly with two most well-known hydrolytic enzymes, glycosidases and lipases. A detailed description on glycosidases is outlined in this chapter.
Among the enzymes dealing with carbohy-drates, glycosidases and transglycosidases play an important role in the synthesis of glycosides. They belong to the group of carbohydrate-processing enzymes, widely employed in the regio- and stereoselective glycosylation reactions. Glycosidases are carbohydrases – enzymes that catalyse the hydrolysis of glycosidic bonds to liberate monosaccharides and oligosaccharides of lower molecular weight than the native simple as well complex carbohydrate substrates. These
enzymes are very widely distributed in nature and found in all organisms. These large and important groups of enzymes, now known as amylase, were investigated long back by Payen and Persoz ( 1833 ) , who were probably the fi rst to recognise this enzyme in 1833 as ‘diastase’ ( 1833 ) . Subsequently, a detailed study on glycosidases was carried out by many eminent chemists and biochemists including Fischer ( 1894 ) .
2.2 Amylolytic Enzymes
Starch-degrading enzymes have been broadly classi fi ed into two groups – endo-acting enzymes or endohydralases and exo-acting enzymes or exohydralases (Berfoldo and Anthranikian 2001 ) . a -Amylase ( a -1,4-glucan-4-glucanohydralse; EC 3.2.1.1) is an endo-acting enzyme which hydrolyses linkages in the starch polymer chain randomly, leading to the generation of linear and
2
Abstract
Glycosidases catalyse transformations leading to the attachment of carbo-hydrate molecules to aglycons. Hence, a detailed description of glycosi-dases is made in this chapter which includes their classi fi cation, nature, source, structural features, mechanism of glycosylation and advantages of such reactions. Also mentioned are examples of glycosylation reactions involving a wide variety of aglycons with different carbohydrate molecules in the form of a table.
Glycosidases
6 2 Glycosidases
branched oligosaccharides. Most starch-hydrolyzing enzymes belong to the a -amylase family contain-ing a characteristic catalytic ( b / a )
8 barrel domain.
Exo-acting starch hydrolases such as b -amylase, glucoamylase, a -glucosidase and isoamylase attack the substrate from the nonreducing end, producing oligosaccharides. b -Amylase (EC 3.2.1.2), also referred to as a -1,4- d -glucan malto-hydrolase or saccharogen amylase, hydrolyses a -1,4-glucosidic linkages of the starch chain to liberate successive maltose units from the nonre-ducing end, thereby producing b -maltose units by an inversion of con fi guration. a -Glucosidase (EC 3.2.1.20) attacks a -1,4 linkages of oligosac-charides and liberates glucose by retaining a -anomeric con fi guration.
2.3 Glucoamylase
Glucoamylase (E.C 3.2.1.3) is a fungal enzyme which goes under the names amyloglucosidase, 1,4- a - d -glucan hydrolase and g -amylase. Enzyme code assigned for this enzyme by the Enzyme Commission (IUBMB 1992 ) is EC 3.2.1.3 where number 3 denotes hydrolases, referring to cata-lytic hydrolytic cleavage of large molecules with the addition of water; number 2 indicates glyco-sidic bond-cleaving glucosidases, and number 1 refers to hydrolysis of O-glycosyl compounds. There are several enzymes under the group 3.2.1, of which glucoamylase is number 3 which forms the fourth number in the nomenclature. Glucoamylase refers to hydrolysis of terminal a -1,4-linked- d -glucose residues successively from nonreducing ends of the carbohydrate chains from starch and malto-oligosaccharides, releasing d -glucose with inversion of con fi guration to b - d -glucose (Fogarty 1983 ) .
When the next bond sequence is a -1,4, most forms of the enzyme can hydrolyse a -1,6- d -glu-cosidic bonds also. However, in vitro, this enzyme hydrolyses a -1,6- and a -1,3- d -glucosidic bonds also in other polysaccharides with high molecu-lar weights. Since this enzyme is capable of com-pletely hydrolyzing starch under long incubation periods, it is also called the saccharifying enzyme. Glucoamylases have the capacity to degrade large
oligosaccharides up to about 90% a -1,6 linkages depending on the size of the substrate and the position of the a -1,6 linkages. Reverse reactions involving synthesis of saccharides and glycosides from d -glucose occur with a very high glucoamy-lase concentration for prolonged incubation periods and high concentrations of substrates.
2.4 Sources of Glucoamylases
The main source of glucoamylases is fungi although they are derived from a wide variety of plants, animals and microorganisms. Commercial enzymes originate from strains of either Aspergillus niger or Rhizopus sp. where they are used for the conversion of malto-oligosaccharides into glucose (Fogarty 1983 ) . Since the discovery of two forms of glucoamylase from black koji mould in the 1950s, many reports have appeared on the multiplicity of glucoamylases, envisaged to be the result of several mechanisms, namely, mRNA modi fi cations, limited proteolysis, variation in carbohydrate content or presence of several structural genes (Pretorius et al . 1991 ) .
Fungal glucoamylases are usually one to fi ve forms of glycoproteins. Aspergillus niger is being used widely in the commercial production of an extracellular glucoamylase. Two forms of glu-coamylase – AG-I (glucoamylase I,99 kDa) and AG-II (glucoamylase II (112 kDa) – isolated from A. niger differed in their carbohydrate con-tent, pH, temperature stabilities and activity (Williamson et al . 1992 ; Stoffer et al . 1993 ) .
Gucoamylase from Aspergillus terreus strains was examined for the production of d -glucose and corn syrups (Ghosh et al . 1990 ; Ali and Hossain 1991 ) . A glucoamylase from Rhizopus sp. released glucose from starch with 100% ef fi ciency (Yu and Hang 1991 ) . Takahashi et al . ( 1985 ) isolated three forms of glucoamylase from Rhizopus sp., GA-I (74 kDa), GA-II (58.6 kDa) and GA-III (61.4 kDa). Glucoamylases from other mould strains are Humicola lanuginosa (Taylor et al . 1978 ) , Thermomyces lanuginosa (Haasum et al . 1991 ) , Myrothecium sp. M1 (Malek and Hossain 1994 ) and a phytopathogenic fungus Colletotrichum gloeosporiodes (Krause et al . 1991 ) .
72.6 Structural Features of Glucoamylase
There are several reports on the production of yeast glucoamylases (Saha and Zeikus 1989 ; Pretorius et al . 1991 ) . Glucoamylase has been identi fi ed in Saccharomyces cerevisiae (Pugh et al . 1989 ) , Saccharomyces cerevisiae var. dia-staticus (Kleinman et al . 1988 ; Pretorius et al . 1991 ) , Saccharomycopsis fi buligera (Itoh et al . 1989 ) , Schwanniomyces castellii (Sills et al . 1984 ) , Schwanniomyces occidentalis (Gellissen et al . 1991 ) , Pichia burtonii and Talaromyces sp.
Bacterial glucoamylases have also been identi fi ed from aerobic strains such as B. stearo-thermophilus (Srivastava 1984 ) , Flavobacterium sp. (Bender 1981 ) , Halobacterium sodamense (Chaga et al . 1993 ) and Arthrobacter globiformis I42 (Okada and Unno 1989 ) . Anaerobic strains include Clostridium thermohydrosulfuricum (Hyun and Zeikus 1985 ) , Clostridium sp. G0005 (Ohinishi et al . 1991 ) , Clostridium acetobutyli-cum (Chojecki and Blaschek 1986 ; Soni et al . 1992 ) , Clostridium thermosaccharolyticum (Specka et al . 1991 ) and the microaerophile, Lactobacillus amylovorus (James and Lee 1995 ) .
2.5 Sources of Other Glycosidases
Among the thermostable glycosidases used in the synthesis of glycosides, the most remarkable one is the b -glucosidase from the hyperthermophilic archeon Pyrococcus furiosus (Kengen et al . 1993 ) which is relatively easy to grow, and the enzyme is stable for 85 h at 100 °C. The enzyme has been cloned and over-expressed in Escherichia coli (Voorhorst et al . 1995 ) . b -Galactosidase from Aspergillus oryzae was ef fi cient towards alkyla-tion (Stevenson et al . 1993 ) . b -Galactosidase from Streptococcus thermophilus (Stevenson and Furneaux 1996 ) was employed for the synthesis of ethyl glycoside. b -Galactosidase from Bacillus circulans was also exploited by number of work-ers for synthetic purposes (Kojima et al . 1996 ) . Enzymatic synthesis of butylglycoside via a transglycosylation reaction of lactose was carried out using b -galactosidase from A. oryzae (Ismail et al . 1999a ) . With primary as well as secondary alcohols, b -xylosidase from A. niger is an ef fi cient glycosyl transfer catalyst that gave high (> 80%)
yields of alkyl xylosides (Shinoyama et al . 1988 ) from methanol up to butanol. Almond glucosi-dase has been widely employed for the synthesis of alkyl and phenolic glycosides (Ljunger et al . 1994 ; Vic and Crout 1995 ; Vic et al . 1995 ; Ducret et al . 2002 ) .
2.6 Structural Features of Glucoamylase
The structure of different glucoamylases showed a common subsite arrangement with seven in total and the catalytic site was located between subsite 1 and 2 (Hiromi et al . 1973 ; Ohinishi 1990 ; Fagerstrom 1991 ; Ermer et al . 1993 ) . Subsite 2 has the highest af fi nity for oligomeric substrates and glucose, followed by decreasing af fi nity towards subsites 3–7 (Fagerstrom 1991 ) . The glu-coamylase G1 of A. niger consists of three parts: (1) Ala-1-Thr-440, containing the catalytic site; (2) Ser-441-Thr-551, a highly O-glycosylated linker segment; and (3) Pro-512-Arg-616, a C-terminal domain responsible for substrate bind-ing (Stoffer et al . 1995 ; Svensson et al . 1983 ) . Functionally important carboxyl groups in glu-coamylase G2 from A. niger were identi fi ed to be Asp176, Glu179 and Glu180 in the catalytic site (Svensson et al . 1990 ) . Tryptophan residues have been proposed to be essential for enzymatic activ-ity (Rao et al . 1981 ) in A. niger glucoamylase and essentially tryptophan120 is reported to be respon-sible for binding of substrate and maintaining the structural integrity necessary for catalysis (Clarks and Svensson 1984 ) .
Aspergillus awamori GA-I has also three cata-lytic domains (Svensson et al . 1983 ) like A. niger , a catalytic domain (residues 1–440), an O-glycosylated domain (residues 441–512) and a starch-binding domain (residues 513–616). Aleshin et al . ( 1994 ) produced a structural model for the catalytic domain of glucoamylase from A. awamori from a 2.2-Å resolution crystal struc-ture of a proteolized form of GA-I A. awamori var X100, which contained the complete catalytic domain plus GA-II domain the N-terminal half of the O-glycosylated domain (residue 1–471). Amino acid sequence of three glucoamylases from
8 2 Glycosidases
Rhizopus, Aspergillus and Saccharomyces were compared (Tanaka et al . 1986 ) , of which the glucoamylases from Rhizopus and Aspergillus were highly homologous in both the nucleotide sequence and the amino acid sequence suggesting that these two glucoamylases were the most closely related among the three. The catalytic site in glu-coamylase is believed to consist of two carboxyl groups (Hiromi et al . 1966a, b ) , where one acts as a general acid, protonating the glucosidic oxygen, while the other in the ionised carboxylate form sta-bilises the substrate intermediary oxonium ion (Braun et al . 1977 ; Matsumura et al . 1984 ; Post and Karplus 1986 ; Rantwijk et al . 1999 ) .
Itoh et al . ( 1989 ) reported that in S. fi buligera glucoamylase, Ala-81, Asp-89, Trp-94, Arg-96, Arg-97 and Trp-166 were required for wild-type levels of activity, and Ala-81 and Asp-89 were not essential for catalytic activity which however played a role in thermal stability.
Complexes of glucoamylase from A. awamori with acarbose and d -gluco-dihydroacarbose indi-cate hydrogen bonds between sugar OH groups and Arg-54, Asp-55, Leu-177, Try-178, Glu-180 and Arg-305 of subsites 1 and 2 (Aleshin et al . 1994 ; Stoffer et al . 1995 ) . Glu-179 (Sierks et al. 1990 ) and Glu-400 are positioned geometrically for general acid and base catalysis, ideal for the glucoside bond cleavage and assistance in the nucleophilic attack of water at the anomeric cen-tre of the carbohydrate (Harris et al . 1993 ; Frandsen et al . 1994 ) . Both the active sites of A. niger and Rhizopus oryzae glucoamylases are very much identical (Stoffer et al . 1995 ) . In the active site of R. oryzae, the amino acid residues Arg-191, Asp-192, Leu-312, Trp-313, Glu-314, Glu-315 and Arg-443 are responsible for substrate binding through hydrogen bonds, whereas Glu-314 and Glu-544 are for glucosidic bond cleavage (Ashikari et al . 1986 ; Sierks et al. 1990 ) .
2.7 Structural Features of b -Glucosidase
Sweet almond b -glucosidase has been known to hydrolyse glycosides resulting in the net reten-tion of anomeric con fi guration (Eveleigh and
Perlin 1969 ) . It has followed the standard mechanism of such retaining glycosidases (McCarter and Withers 1994 ; Sinnot 1990 ) . Assignment of sweet almond b -glucosidase as a family 1 gly-cosidase and identi fi cation of its active site nucleophilic residues sequence Ile-Thr-Glu-Asn-Gly were done by He and Withers ( 1997 ) .
The primary structures of maize and sorghum b -glucosidases possess highly conserved peptide motifs TENEP and ITENG, which contain the two glutamic acids (Glu-191 and Glu-406) involved in the general acid/base catalysis and the respective family 1 b -glucosidases nucleo-philes (San-Aparicio et al . 1998 ) . A part slot-like active site (Davies and Henrissat 1995 ) was formed by these residues necessary for the sub-strate hydrolysis (Withers et al . 1990 ) .
In the glycosylation step, the nucleophile Glu-406 attacks the anomeric carbon (C-1) of the sub-strate and forms a covalent glycosyl–enzyme intermediate with concomitant release of the aglycon after protonation of the glucosidic oxy-gen by the acid catalyst Glu-191 (Withers et al . 1990 ) . In the next deglycosylation step, Glu-191 acts as a base, and a water molecule functions as the nucleophile and attacks the covalent glyco-syl–enzyme, releasing the glucose and regenerat-ing the nucleophilic Glu-406. In maize b -glucosidase isozyme Glu-1, these two catalytic glutamic acids are positioned within the active site at expected distances of ~5.5 Å for this mech-anism (Czjzek et al . 2001 ) . Verdoucq et al . ( 2003 ) from co-crystals of enzyme substrate and enzyme aglycon complexes of maize b -glucosidase isozyme Glu1 (ZmGlu1) have shown that fi ve amino acid residues – Phe-198, Phe-205, Try-378, Phe-466 and Ala-467 – are located in the aglycon-binding site of ZmGlu1 which form the basis of aglycon recognition and binding and hence the substrate speci fi city. Kaper et al . ( 2000 ) have studied the substrate speci fi city of a family 1 glycosyl hydrolase – the b -glucosidase (CelB) from the hyperthermophilic archean Pyrococcus furiosus, at a molecular level exhibiting a homo-tetramer con fi guration, with subunits having a typical ( b a )
8 -barrel fold. Comparison of the 3D
model of the Pyrococcus furiosus b -glucosidase and the 6-phospho- b -glycosidase (LacG) from
92.8 Glycosylation
the mesophillic bacterium Lactococcus lactis (Kaper et al . 2000 ) showed that the positions of the active site residues in LacG and CelB are very well conserved, and the conserved residues involved in substrate binding are Asn-17, Arg-77, His-150, Asn-206, Tyr-307 and Trp-410. The average distance between the oxygen atoms of these glutamate carboxylic acids is 4.3 Å (±1 Å) in CelB, which is very much in the range of the general observed distance in retaining glycosyl hydrolases (McCarter and Withers 1994 ) .
Investigation by Hays et al . ( 1998 ) of the cata-lytic mechanism, substrate speci fi city and trans-glycosylation acceptor speci fi city of guinea pig liver cytosolic b -glucosidase (CBG) indicated that CBG employed a two-step catalytic mecha-nism with the formation of a covalent enzyme–sugar intermediate and that CBG transferred sugar residues to primary hydroxyls and equato-rial but not axial C-4 hydroxyls of aldopyranosyl sugars (Hays et al . 1998 ) . Also the speci fi city of CBG for transglycosylation reactions was differ-ent from its speci fi city for hydrolytic reactions (Hays et al . 1998 ) and that CBG possessed a sin-gle active site nucleophile, speci fi cally the gluta-mate residue in the sequence TITENG.
2.8 Glycosylation
Hydrolysis is the natural reaction for glucosi-dases and glucoamylases, whereas glycosylation is a forced, reversed reaction. Glycosides are asymmetric mixed acetals formed by the reaction of the anomeric carbon atom of the intermolecu-lar hemiacetal or pyranose/furanoses form of the aldohexoses or aldoketoses with a hydroxyl group furnished by an alcohol (Lehinger 1975 ; Ernst et al . 2000 ) . The bond formed is called glycosidic bond, and the reaction is called glycosylation. Because of multiple hydroxyl groups of similar reactivity, controlled glycosylation remains a challenge to organic chemists. Classical chemical approaches inevitably require quite a number of protection, activation, coupling and deprotection steps (Igarashi 1977 ; Konstantinovic et al . 2001 ) . In contrast, enzymes (glycosidases and transgly-cosidases) offer one-step synthesis under mild
conditions in a regio- and stereoselective manner (Vic and Thomas 1992 ) . Enzyme-catalysed glycoside and oligosaccharide synthesis involves two types of reaction – a reverse hydrolytic glycosidase and a glycosyl-transferase-catalysed glycoside bond formation. A sugar donor and acceptor are incubated with the appropriate glycosidase or glycosyl-transferase that catalyses the ef fi cient and selective transfer of the glycosyl residue to the acceptor. Glycosyl-transferases are often dif fi cult to obtain (Auge et al . 1990 ) , while, in contrast, the glycosidase approach uses simpler glycosyl donors, the free monosaccharide itself. This method has the advantage of using relatively simple glycosyl donors and readily available commercial enzymes at the expense of the absence of region selectivity in some instances (Trincone et al . 2003 ) .
There are three types of reactions catalysed by glycosidases such as hydrolysis, reverse hydroly-sis and transglycosylation (Scheme 2.1 ). In aque-ous media, when there is large excess of water, glycoside or oligosaccharide or polysaccharide, hydrolysis is the dominant reaction (Scheme 2.1 A). Other two reactions, namely, reverse hydrolysis and transglycosylation, lead to synthesis of gly-cosides, and the difference depends on the nature of the glycosyl donor.
The reverse hydrolytic approach is an equilib-rium-controlled synthesis where the equilibrium is shifted towards synthesis (Panintrarux et al . 1995 ; Vic et al . 1997 ; Rantwijk et al . 1999 ) of a glycoside from a carbohydrate and an alcohol (Scheme 2.1 B). This can be achieved by reducing the water activity, increasing the substrate con-centrations and removing, if possible, the prod-ucts of reaction (Vic and Crout 1995 ) . This is a widely employed method for the enzymatic syn-thesis of alkyl glycosides and phenolic glycosides in an organic co-solvent (Vic and Crout 1995 ; Vic et al . 1997 ; Ducret et al . 2002 ) .
The transglycosylation method is a kinetically controlled synthesis where the enzyme catalyses the transfer of a glycosyl residue from a glycosyl donor to the glycosyl acceptor (Scheme 2.1 C). The reaction yield depends on the relative rate of product synthesis to that of hydrolysis. An ef fi cient acceptor used in a high concentration
10 2 Glycosidases
should favour the synthesis (Ismail et al . 1999b ; Rantwijk et al . 1999 ; Vulfson et al . 1990 ) although this may not be true with all the acceptors.
2.9 Mechanism of Glycosylation
In general, every hydrolysis of a glycosidic linkage by glycosidase is a reaction in which the product retains ( a → a or b → b ) or inverts ( a → b or b → a ) the anomeric con fi guration of the sub-strate (Chiba 1997 ) . In the normal hydrolytic reaction, the leaving group is an (oligo)saccharide and the nucleophile (glycosyl acceptor) is water (Scheme 2.1 A). However, an alcohol or a mono-saccharide can also act as a glycosyl acceptor (glycosylation). In the reversed hydrolysis, the condensation of a monosaccharide and an alcohol in which water is the leaving group (Scheme 2.1 B) was fi rst reported in 1913 (Rantwijk et al . 1999 ) . A recent review by Zechel and Withers ( 2001 ) focuses on the recent developments in the under-standing of nucleophilic and general acid–base catalysis in glycosidase-catalysed reactions. Various models have been proposed for the cata-lytic reaction mechanisms of carbohydrate hydro-lase in the transition state, but an unequivocal model remains to be established. Two signi fi cant models, such as nucleophilic displacement mech-anism (Scheme 2.2 ) and an oxo-carbenium ion intermediate mechanism (Scheme 2.3 ), were sug-gested for the hydrolytic reaction where glycosyl acceptor is water (Chiba 1997 ) .
The double displacement mechanism was found to be applicable to the enzymes, which
retain the anomeric con fi guration of the substrate. The two catalytic ionisable groups, a carboxyl, –COOH, and a carboxylate, -COO - , cleave the glucosidic linkage cooperatively by direct elec-trophilic and nucleophilic attacks against the gly-cosyl oxygen and anomeric carbon atoms, respectively, resulting in a covalent glucosyl–enzyme complex by a single displacement. Subsequently glucosyl–acetal bond is attacked with the hydroxyl group of the water (alcohol hydroxyl group in glycosylation) by retaining the anomeric con fi guration of the product by the double displacement. The double displacement mechanism is adequate for explaining the reac-tion, where the anomeric con fi guration of the substrates is retained (Chiba 1997 ) .
In the oxo-carbenium intermediate mecha-nism, the two catalytic groups of the carboxyl and carboxylate ion participate cooperatively in the departure of the leaving group by a proton transfer to the anomeric oxygen atom (Scheme 2.3 ). An enzyme-bound oxonium ion intermediate has been detected by NMR (Withers and Street 1988 ) . The second carboxylate, which is deprotonated in the resting state, stabilises the oxonium ion inter-mediate. In the next step, a nucleophile adds to the same face of the glycosyl–enzyme intermediate from which the leaving group was expelled, resulting in the net retention of the anomeric con fi guration at the anomeric centre. The addition of the nucleophile is assisted by the fi rst carboxy-late which in this step reverts to carboxylic acid. The oxo-carbenium intermediate mechanism has been applied to interpret the catalytic mechanism of many carbohydrate-degrading enzymes. This
Scheme 2.1 Reactions catalysed by glycosidases
112.10 Glycosylation Reactions
mechanism is applicable to both ‘retaining’ and ‘inverting enzymes’ (Chiba 1995 ) . Mutagenesis and X-ray structural studies have con fi rmed that the mechanism of retaining glycosidases is simi-lar (Sinnot 1990 ; Jacobson et al . 1994, 1995 ) .
2.10 Glycosylation Reactions
Biological activities of a naturally occurring gly-coside (Robyt 1998 ; Schmid et al . 2001 ; Akao et al . 2002 ) are primarily due to an aglycon moi-ety of that molecule. It is generally accepted that
glycosides are more water-soluble than most of the respective aglycons. Attaching a glycosidic moiety into the molecule increases its hydrophi-licity and thereby in fl uences physicochemical and pharmacokinetic properties of the respective compound like circulation, elimination and con-centrations in the body fl uids (Kren 2001 ) . Glycosides with unsaturated alkyl chains like ter-penes are claimed to possess antifungal and anti-microbial activity (Tapavicza et al . 2000 ; Zhou 2000 ) although it is unclear why the activity of these aglycons is improved by glycosylation. Chemical preparation of glycosides cannot meet
O
OOH
HO
O
CH2OH CH2OH
CH2OHCH2OH
R
-
C=O
OH H-O-R
O
OOH
OH
O
R1
C=O
O -
C
O
OO
OH
OH
O
C=O
O-
C
O
OO
HO
OH
O
OH
R1
C
O
O
- C
O
O
C=O
OH
Scheme 2.2 Nucleophilic double displacement mechanism (Chiba 1997 )
12 2 Glycosidases
EC food regulations, and therefore, chemical preparation of glycosides is not applicable in the food industry.
Many glycosides are used in broad range of applications as surfactants (Busch et al . 1994 ) , as food colourants and fl avouring agents (Sakata et al . 1998 ) , sweeteners (Shibata et al . 1991 ) , antioxidants, anti-in fl ammatory (Gomes et al . 2002 ) , antitumor (Kaljuzhin and Shkalev 2000 ) , antibiotics (Ikeda and Umezawa 1999 ) , antifun-gal (Tapavicza et al . 2000 ) , antimicrobial (Zhou 2000 ) and cardiac-related drugs (Ooi et al . 1985 ) . Glycosylation renders lipophilic compounds
more water-soluble and thereby increases bio-availability of biologically active compounds besides imparting stability to the aglycon (Kren and Martinkova 2001 ) . Alkyl glycosides are mainly used as nonionic surfactants in food, pharmaceuticals, chemical, cosmetic and deter-gent industries. These types of nonionic surfac-tants exhibit several interesting properties in detergency, foaming, wetting, emulsi fi cation and antimicrobial effect (Matsumura et al . 1990 ; Balzar 1991 ) . Alkyl glycosides are non-toxic, non-skin-irritating and biodegradable (Matsumura et al . 1990 ; Busch et al . 1994 ; Madsen et al .
O
OOH
HO
O
R
-
C=O
OH
H-O-R
O
OOH
HO
O
R1
C=O
O-
C
O
O
-C
O
O
C=O
OH
C
O
OCH2OH
O
OH
O
-+
C=O
O-
C
O
OO
OH
O
-+
OH R1
..CH2OH
HO
CH2OHCH2OH
OH
Scheme 2.3 Oxo-carbenium ion intermediate mechanism (Chiba 1997 )
132.11 Advantages of Enzymatic Glycosylation over Chemical Methods
1996 ) . Further alkyl glycosides are used as raw materials for sugar fatty acid ester synthesis (Mutua and Akoh 1993 ) .
2.11 Advantages of Enzymatic Glycosylation over Chemical Methods
There are many advantages of using glycosidases (Vijayakumar 2007 ; Sivakumar 2009 ) : 1. Exploitation of regio- and stereospeci fi city
and selectivity 2. Milder reaction conditions 3. Non-generation of by-products associated
with the use of several chemical procedures 4. Improved product yield and better product
quality 5. Use of nonpolar solvents which impart stabil-
ity to glycosidases, renders insolubility of the enzyme, solubility of alcohols and products in organic solvents and easy product workout procedures
6. No protection activation and deprotection required
7. Less environmental pollution The use of organic solvent in enzyme catalysis has attracted much attention due to several desir-
able factors such as solubilities of the organic compounds, shifting equilibrium towards the synthesis, increasing the enzyme stability and recovery of the enzyme (Rubio et al . 1991 ; Mohri et al . 2003 ) . Poor solubility of the carbohydrate substrate in the organic phase is a limiting factor especially when hydrophobic alcohol (glycosyl acceptor) itself is used as a substrate and in some cases as a solvent media (Laroute and Willemot 1992 ; Vic and Crout 1995 ; Crout and Vic 1998 ) . There are reports where glycosylations were car-ried out either in biphasic systems of a water-immiscible alcohol and water (that maintains sugar substrate and enzyme) or water and water-miscible monophasic system (Monsan et al . 1996 ) . The process of glycosylation can be effected under nonaqueous, solvent-free, high-substrate, high-temperature and moderate to high water activity conditions to achieve good yield of glycosides (Nilsson 1987 ; Roitsch and Lehle 1989 ; Gygax et al . 1991 ; Laroute and Willemot 1992 ; Vic and Thomas 1992 ; Shin et al . 2000 ) .
Table 2.1 lists some of the important surfac-tants, phenolic, fl avonoid, terpinyl, sweetener and medicinal glycosides, which have been pre-pared by the use of glycosidases, glucoamylases and glycosyl-transferases.
14 2 Glycosidases
Tab
le 2
.1
Gly
cosi
des
from
enz
ymat
ic g
lyco
syla
tions
Nam
e of
the
com
poun
d So
urce
of
enzy
me
App
licat
ions
R
efer
ence
s
A. S
urfa
ctan
t gly
cosi
des
(1)
b - D
-Gly
copy
rano
side
s of
n-h
epta
nol,
n-oc
tano
l, 2-
phen
yl h
exan
ol, 3
-phe
nyl
prop
anol
, 4-p
heny
l but
anol
, 5-p
heny
l pet
anol
, 6-
phen
yl h
exan
ol, 2
-pyr
idin
e m
etha
nol,
isob
utan
ol, i
sope
ntan
ol, p
-met
hoxy
cin
amyl
al
coho
l, is
opro
pano
l, cy
cloh
exan
ol, 1
-phe
nyl
etha
nol,
1,5-
pent
aned
iol,
1,6-
hexa
nedi
ol,
1,7-
hept
aned
iol,
1,8-
octa
nedi
ol, 1
,9-n
onan
e-di
ol, s
alic
yl a
lcoh
ol a
nd 4
-nitr
ophe
nol
b -G
luco
sida
se f
rom
alm
onds
A
s no
nion
ic s
urfa
ctan
ts, i
n de
terg
ents
and
cos
met
ics
Kat
usum
i et a
l. ( 2
004 )
(2)
b - D
-Glu
copy
rano
side
s of
pro
pano
l, he
xano
l and
oct
anol
R
aw a
lmon
d m
eal
In d
eter
gent
s an
d co
smet
ics
Cha
hid
et a
l . ( 1
992,
199
4 )
(3)
a / b
-Glu
copy
rano
side
s of
eth
anol
, 1-
prop
anol
, 2-p
ropa
nol,
2-m
ethy
l 2-p
ropa
nol,
1-bu
tano
l, 2-
buta
nol,
1-pe
ntan
ol, 1
-hex
anol
, 1,
3-bu
tane
diol
, 1,4
-but
aned
iol,
2,3-
buta
ne-
diol
, 1,2
-pen
tane
diol
, 1,5
-pen
tane
diol
Glu
coam
ylas
e an
d b -
gluc
osid
ase
In d
eter
gent
s an
d co
smet
ics
Lar
oute
and
Will
emot
( 19
92 )
(4)
Ally
l and
ben
zyl b
- D -g
luco
pyra
nosi
de,
ally
l- b -
D -g
alac
topy
rano
side
A
lmon
d b -
D -g
luco
sida
se
Use
d in
the
synt
hesi
s of
gly
copo
lym
ers,
as
tem
pora
ry a
nom
eric
-pro
tect
ed
deri
vativ
es in
car
bohy
drat
e ch
emis
try
Vic
and
Cro
ut (
1995
)
(5)
n-O
ctyl
glu
cosi
de, n
-oct
yl g
alac
tosi
de
b -G
alac
tosi
dase
fro
m A
. ory
zae ,
al
mon
d m
eal
In d
eter
gent
s an
d co
smet
ics
Cha
hid
et a
l . ( 1
994 )
(6)
n-O
ctyl
- b -
D -g
luco
side
, 2-h
ydro
xy
benz
yl g
luco
pyra
nosi
de.
Alm
ond
b -gl
ucos
idas
e In
det
erge
nts
and
cosm
etic
s V
ic e
t al .
( 199
7 )
(7)
n-O
ctyl
- b - D
-glu
cosi
de, n
-oct
yl- b
- D -
xylo
bios
ide,
n-o
ctyl
- b - D
-xyl
osid
e A
s bi
olog
ical
det
erge
nts
and
as
emul
sify
ing
agen
ts in
cos
met
ics
Nak
amur
a et
al .
( 200
0 )
B. P
heno
lic
glyc
osid
es
(1)
Eug
enol
- a -g
luco
side
a
-Glu
cosy
l tra
nsfe
r en
zym
e of
Xan
thom
onas
cam
pest
ris
WU
-970
1
As
a pr
odru
g of
a h
air
rest
orer
, as
a d
eriv
ativ
e of
spi
ces
Sato
et a
l . ( 2
003 )
2. E
ugen
ol- b
-glu
cosi
de
Bio
tran
sfor
mat
ion
by c
ultu
red
cells
of
Euc
alyp
tus
perr
inia
na
As
a pr
odru
g of
a h
air
rest
orer
O
riha
ra e
t al .
( 199
2 )
(3)
Van
illin
- b - D
-mon
oglu
copy
rano
side
B
y su
spen
sion
-cul
ture
d ce
lls
of C
offe
a ar
abic
a A
s a
food
add
itive
fl av
our
Kom
etan
i et a
l . ( 1
993a
)
152.11 Advantages of Enzymatic Glycosylation over Chemical Methods
(con
tinue
d)
Nam
e of
the
com
poun
d So
urce
of
enzy
me
App
licat
ions
R
efer
ence
s
(4)
Cap
saic
in- b
- D -g
luco
pyra
nosi
de
By
susp
ensi
on-c
ultu
red
cells
of
Cof
fea
arab
ica
Food
ingr
edie
nt a
nd p
harm
acol
ogic
al
appl
icat
ions
K
omet
ani e
t al .
( 199
3b )
By
cultu
red
cells
of
Phy
tola
cca
amer
ican
a H
amad
a et
al .
( 200
3 )
(5)
a -S
alic
in, a
-iso
salc
in, b
-sal
icin
B
acil
lus
mac
eran
s cy
clod
extr
in
gluc
anyl
tran
sfer
ase
and
Leuc
onos
toc
mes
ente
roid
es
B-7
42C
B d
extr
ansu
cras
e
Ant
i-in
fl am
mat
ory,
ana
lges
ic a
ntip
yret
ic
prod
rug
Yoo
n et
al .
( 200
4 )
(6)
Cur
cum
in g
lyco
side
s B
y ce
ll su
spen
sion
cul
ture
s of
C
atha
rant
hus
rose
us
Food
col
oura
nt, a
s an
tioxi
dant
K
amin
aga
et a
l . ( 2
003 )
C. F
lavo
noid
gly
cosi
des
(1)
Que
rcet
in-3
-O- b
- D -x
ylop
yran
osyl
(1
→ 2
)- b -
D -g
alac
topy
rano
side
Is
olat
ed f
rom
Tri
foli
um r
epen
s L
U
V-B
rad
iatio
n pr
otec
tion
Hof
man
n et
al.
( 200
0 )
(2)
Kae
mpf
erol
-3-O
- b - D
-xyl
opyr
anos
yl
(1 →
2)-
b - D
-gal
acto
pyra
nosi
de
D. S
wee
tene
r gl
ycos
ides
(1
) St
evio
side
, ste
viob
iosi
de, r
ebau
dios
ide
A,
reba
udio
side
B
Isol
ated
fro
m th
e le
aves
of
Stev
ia r
ebau
dian
a A
s a
natu
ral s
wee
tene
r, ut
ilise
d in
be
vera
ges
Koh
da e
t al .
( 197
6 )
(2)
Stev
iol-
13-O
-glu
copy
rano
side
, ste
viob
io-
side
, ste
vios
ide
and
reba
udio
side
E
nzym
e fr
actio
ns p
repa
red
from
th
e so
lubl
e ex
trac
ts o
f st
evia
A
s a
natu
ral f
ood
swee
tene
rs
Shib
ata
et a
l . ( 1
991 )
E. T
erpi
nyl g
lyco
side
s
(1)
Ger
enio
l b -g
luco
side
, ner
ol b
-glu
cosi
de,
citr
onio
l b -g
luco
side
b -
Glu
cosi
dase
fro
m A
. nig
er,
Tric
hode
rma
rees
ei, C
andi
da
mol
isch
iana
and
alm
ond
Goo
d bi
oava
ilabi
lity,
ant
ifun
gal a
nd
antim
icro
bial
act
ivity
G
unat
a et
al .
( 199
4 )
(2)
Ger
enio
l b -g
alac
tosi
de, n
erol
b -g
alct
o-si
de, c
itron
iol b
-gal
acto
side
b -
Gal
acto
sida
se f
rom
A. o
ryza
e G
ood
bioa
vaila
bilit
y, a
ntif
unga
l and
an
timic
robi
al a
ctiv
ity
Don
ho e
t al .
( 199
6 )
F. G
lyco
side
s in
med
icin
e (1
) E
nedi
yne
antib
iotic
s –
calic
heam
icin
Is
olat
ed f
rom
the
culti
vatio
n br
oth
of M
icro
mon
ospo
ra
echi
nosp
ora
Ant
itum
or a
gent
s L
ee e
t al .
( 198
7 ) ,
Gol
ik e
t al .
( 198
7 )
(2)
Vita
min
gly
cosi
des
5 ¢ -O
-( b -
D -g
alac
topy
rano
syl)
-thi
amin
A
. ory
zae
b -ga
lact
osid
ase
Exc
elle
nt n
utri
tiona
l ef fi
cien
cies
, mor
e st
able
aga
inst
UV
and
ligh
t. Su
zuki
and
Uch
ida
( 199
4 )
16 2 Glycosidases
Tab
le 2
.1
(con
tinue
d)
Nam
e of
the
com
poun
d So
urce
of
enzy
me
App
licat
ions
R
efer
ence
s
5 ¢ -O
-( b -
D -g
luco
pyra
nosy
l)-t
hiam
in
Cyc
lom
alto
dext
rin
gluc
anot
rans
-fe
rase
fro
m B
acil
lus
stea
roth
erm
ophi
lus
Plea
sant
tast
e an
d od
our,
good
bi
oava
ilabi
lity.
Mor
e st
able
tow
ards
ox
idat
ive
stre
ss a
nd U
V ir
radi
atio
n
Uch
ida
and
Suzu
ki (
1998
)
4- a
- D -g
luco
pyra
nosy
l rut
in
Cyc
lom
alto
dext
rin
gluc
anot
rans
-fe
rase
fro
m B
acil
lus
stea
roth
erm
ophi
lus
Suzu
ki a
nd S
uzuk
i ( 19
91 ) ,
A
ga e
t al.
( 199
0 )
2-O
- a -g
luco
pyra
nosy
l- l -
asco
rbic
aci
d
Alk
aloi
d gl
ycos
ides
– e
lym
ocla
vine
-O- b
- D -
fruc
tofu
rano
side
Is
olat
ed f
rom
a s
apro
phyt
ic
cultu
re o
f C
lavi
ceps
sp.
In
the
trea
tmen
t ort
host
atic
cir
cula
tory
di
stur
banc
es, h
yper
tens
ion,
hyp
erpr
o-la
ctin
aem
ia, a
ntib
acte
rial
and
cyt
osta
tic
effe
cts
and
hypo
lipae
mic
act
ivity
Kre
n an
d C
vak
( 199
9 )
Ster
oida
l gly
cosi
des
– gl
ycos
ides
of
dios
geni
n, s
olas
odin
e, s
olas
onin
e Is
olat
ed f
rom
Sol
anum
sp.
A
ntic
arci
noge
nic
activ
ity
Nak
amur
a et
al .
( 199
6 )
17References
References
Aga H, Yoneyama M, Sakai S, Yamamoto I (1990) Synthesis of 2-O- a -D-glucopyranosyl L-ascorbic acid by cyclomaltodextrin glucotransferase from Bacillus stearothermophilus . Agric Biol Chem 55:1751–1756
Akao T, Yoshino T, Kobashi K, Hatlori M (2002) Evaluation of salicin as an antipyretic prodrug that does not cause gastric injury. Planta Med 68:714–718
Aleshin AE, Firsov LM, Honzatko RB (1994) Re fi ned structure for the complex of acarbose with glucoamy-lases from Aspergillus awamori var. X100 to 2.4 Å resolution. J Biol Chem 269:15631–15639
Ali S, Hossain Z (1991) Characteristics of glucoamylase from Aspergillus terreus . J Appl Bacteriol 71:144–146
Ashikari T, Nakamura N, Tanaka Y, Kiuchi N, Shibano Y, Tanaka T, Amachi T, Yoshizumi H (1986) Rhizopus raw-starch-degrading glucoamylase Its cloning and expression in yeast. Agric Biol Chem 50:957–964
Auge C, Fernandez RF, Gautheron CM (1990) The use of immobilized glycosyltransferases in the synthesis of sialyl oligosaccharides. Carbohydr Res 200:257–268
Balzar D (1991) Alkylglucosides, their physico-chemical properties and their uses. Tenside Surf Det 28:419–427
Bender H (1981) A bacterial glucoamylase degrading cyclodextrins. Eur J Biochem 115:287–291
Berfoldo C, Anthranikian G (2001) Amylolytic enzymes from hyperthermophiles. Methods Enzymol 330:269–289
Braun H, Cogoli A, Semenza G (1977) Carboxyl groups at the two active centers of sucrose-isomaltoase from rabbit small intestine. Eur J Biochem 73:437–442
Busch P, Hensen H, Khare J, Tesmann H (1994) Alkylpolyglycosides-a new cosmetic concept for mil-derness. Agro-Food-Ind Hi-Tech 5:20–28
Chaga G, Porath J, Illeni T (1993) Isolation and puri fi cation of amyloglucosidase from Halobacterium sodome-nase . Biomed Chromatogr 7:256–261
Chahid Z, Montet D, Pina M, Graille J (1992) Effect of water activity on enzymatic synthesis of alkylglyco-sides. Biotechnol Lett 14(4):281–284
Chahid Z, Montet D, Pina M, Bonnot F, Graille J (1994) Biocatalyzed octylglycoside synthesis from a disac-charide. Biotechnol Lett 16:795–800
Chiba S (1995) In: The Amylase Research Society of Japan (ed) Enzyme chemistry and molecular biology of amylase and related enzymes. CRC Press, Boca Raton/Ann arbor/London/Tokyo, pp 68–82
Chiba S (1997) Molecular mechanism in a -glucosidase and glucoamylase. Biosci Biotech Biochem 61:1233–1239
Chojecki A, Blaschek HP (1986) Effect of carbohydrate source on alpha-amylase and glucoamylase formation by Clostridium acetobutylicum SA-1. Ind Microbiol 1:63–67
Clarks AJ, Svensson B (1984) Identi fi cation of an essen-tial tryptophanyl residue in the primary structure of glucoamylase G2 from Aspergillus niger . Carlesberg Res Commun 49:559–566
Crout DHG, Vic G (1998) Glycosidases and glycosyl transferases in glycoside and oligosaccharides synthe-sis. Biocatal Biotransform 2:98–111
Czjzek M, Cicek M, Zamboni V, Bevan DR, Henrissat B, Esen A (2001) The mechanism of substrate (aglcone) speci fi city in b -glucosidase –DIMBOA, – DIMBOA Glc and –dhurrin complexes. Proc Natl Acad Sci USA 97:13555–13560
Davies G, Henrissat B (1995) Structures and mechanisms of glycosyl hydrolases. Structure 3:853–859
Donho M, Kimura T, Hara H (1996) Methods of produc-ing geranyl b -D-galactopyranoside as fl avoring mate-rial by enzymatic galactosylation of citronellol. Jpn Kokai Tokkyo Konho JP 8188589–8188591 (CA 125 222344)
Ducret A, Carriere JF, Trani M, Lortie R (2002) Enzymatic synthesis of octyl glucoside catalysed by almond b -glu-cosidase in organic media. Can J Chem 80:653–656
Ermer J, Rose K, Huber G, Schhellenenberger A (1993) Subsite af fi nities of Aspergillus niger glucoamylase II determined with p -nitophenylmaltooligosaccharides. Biol Chem Hoppe Seyler 374:123–128
Ernst B, Hart GW, Sinay P (2000) Carbohydrates in chem-istry and biology, vol 1. Willey-VCH, Weinheim, pp 177–193
Eveleigh DE, Perlin AS (1969) A proton magnetic reso-nance study of the anomeric species produced by D-glucosidases. Carbohydr Res 10:87–95
Fagerstrom R (1991) Subsite mapping of Hormoconis resinae glucoamylase and their inhibition by glu-conolactone. J Gen Microbiol 137:1001–1008
Fischer E (1894) Ein fl uss der kon fi guration auf die wirkung der enzyme. Ber Chem Ges 27:2985–2993
Fogarty WM (ed) (1983) Microbial amylases. Microbial enzymes and biotechnology. Appl Science Publishers, London, pp 1–92
Frandsen TP, Dupont C, Lehmbeck J, Stoffer B, Sierks MR, Honzatko RB, Svensson B (1994) Site-directed mutagenesis of the catalytic base Glutamic acid 400 in glucoamylase from Aspergillus niger and of Tyrosine 48 and Glutamine 401, both hydrogen bonded to the gamma-carboxylate group of Glutamic acid 400. Biochemistry 33:13808–13816
Gellissen G, Janowicz ZA, Merckelbach A, Piontek M, Keup P, Weydemann U, Hollenberg CP, Srasser AWM (1991) Heterologus gene expression in Hansenula polymorpha : ef fi cient secretion of glucoamylase. Biotechnology 9:291–295
Ghosh A, Chatterjee BS, Das A (1990) Characterization of glucoamylase from Aspergillus terreus 4. FEMS Microbial Lett 66:345–349
Golik J, Clardy J, Dubay G, Groenewold G, Kawaguchi H, Konishi M, Krishnan B, Ohkuma H, Saitoh K, Dobye TW (1987) Esperamicins, a novel class of potent anti-tumor antibiotics. 3. Structures of esperamicins A1, A2 and A1b. J Am Chem Soc 109:3461–3464
Gomes DCF, Alegrio LV, Leon LL, de Lima MEF (2002) Total synthesis and anti-leishmanial activity of some curcumin analogues. Arzneim-Forsch 52:695–698
Gunata Z, Vallier MJ, Sapis JC, Baumes R, Bayonove C (1994) Enzymic synthesis of monoterpeny b -D-gluco-sides by various b -glucosidases. Enzyme Microb Technol 16:1055–1058
18 2 Glycosidases
Gygax D, Spies P, Winkler T, Pfaar U (1991) Enzymatic synthesis of b -D-glucuronides with in situ regeneration of uridine 5’-diphosphoglucuronic acid. Tetrahedron 47:5119–5122
Haasum I, Ericksen SH, Jensen B, Olsen J (1991) Growth and glucoamylase production by the thermophilic fun-gus Thermophilus lanuginose in a synthetic medium. Appl Microbiol Biotechnol 34:656–660
Hamada H, Nishida K, Furuya T, Ishihara K, Nakajima N (2003) Preparation of a new pepper: chemoenzymatic synthesis of capsaicin oligosaccharide and 8-nordihy-drocapsaicin. J Mol Catal B: Enzym 16:115–119
Harris EMS, Aleshin AE, Firsov LM, Honzatko RB (1993) Re fi ned structure of the complex of 1-deoxynojirimy-cin with glucoamylase from Aspergillus awamori var X100. Biochemistry 32:1618–1626
Hays WS, Vander Jagt DJ, Bose B, Serianni AS, Glew RH (1998) Catalytic mechanism and speci fi city for hydro-lysis and transglycosylation reactions of cytosolic b -glucosidase from guinea pig liver. J Biol Chem 273:34941–34948
He S, Withers SG (1997) Assignment of sweet almond b -glucosidase as a family 1 glycosidase and identi fi cation of its active site nucleophile. J Biol Chem 272:24864–24867
Hiromi K, Kawai M, Ono S (1966a) Kinetic studies on glucoamylase IV. Hydrolysis of isomaltose. J Biochem 59:476–480
Hiromi K, Takahashi K, Hamazu Z, Ono S (1966b) Kinetic studies on glucoamylase III. The in fl uence of pH on the rates of hydrolysis of maltose and panose. J Biochem 59:469–475
Hiromi K, Nitta Y, Numata C, Ono S (1973) Subsite af fi nities if glucoamylase examination of the validity of the subsite theory. Biochem Biophys Acta 302:362–375
Hofmann RW, Swinny EE, Bloor SJ, Markham KR, Ryan KG, Campbell BD, Jordan BR, Fountain DW (2000) Responses of nine Trifolium repens L. populations to ultraviolet-B radiation. Differential fl avonol glycoside accumulation and biomass production. Ann Bot 86:527–537
Hyun HH, Zeikus JG (1985) General biochemical charac-terization of thermostable pullulanase and glucoamy-lase from Clostridium thermohydrosulfuricum . Appl Environ Microbiol 49:1168–1173
Igarashi K (1977) The Koenigs-Knorr reaction. Adv Carbohydr Chem Biochem 34:243–283
Ikeda D, Umezawa S (1999) Aminoglycoside antibiotics. In: Ikan R (ed) Naturally occurring glycosides. Wiley, England, pp 1–42
Ismail A, Linder M, Ghoul M (1999a) Optimization of butyl-galactoside synthesis by b -galactosidase from Aspergillus oryzae . Enzyme Microb Technol 25:208–213
Ismail A, Soultani S, Ghoul M (1999b) Enzymatic-catalyzed synthesis of alkylglycosides in monophasic and biphasic systems. I. The transglycosylation reac-tion. J Biotechnol 69:135–143
Itoh T, Sakata Y, Akada R, Nimi O, Yamshita I (1989) Construction and characterization of mutant glucoam-ylases from the yeast Saccharomycopsis fi buligera . Agric Biol Chem 53:3159–3168
IUBMB (1992) Enzyme nomenclature. Academic Press, San Diego, California, ISBN 0-12-227164-5
Jacobson RH, Zhang X-J, DuBose RF, Matthews BW (1994) Three dimensional structure of b -galactosidase from E. Coli . Nature 369:761–766
Jacobson RH, Kuroki R, Weaver LH, Zhang X-J, Matthews BW (1995) In: Saddler JN, Penner MH (eds) Enzymatic degradation of insoluble carbohydrates, vol 618. ACS Symposium Series, Washington, DC, pp 38–50
James JA, Lee BH (1995) Cultural conditions for produc-tion of glucoamylase from Lactobacillus amylovorus ATCC 33621. J Appl Bacteriol 79:499–505
Kaljuzhin OV, Shkalev MV (2000) Immunomodulator and pharmaceutical compositions with antitumor properties, and a food additive. Patent EP1038532 (CA 129 335732)
Kaminaga Y, Nagatsu A, Akiyama T, Sugimoto N, Yamazaki T, Maitani T, Mizukami H (2003) Production of unnatural glucosides of curcumin with drastically enhanced water solubility by cell suspension cultures of Catharanthus roseus . FEBS Lett 555:311–316
Kaper T, Lebbink JHG, Pouwels J, Kopp J, Schulz GE, Oost JV, Vos WM (2000) Comparative structural anal-ysis and substrate speci fi city engineering of the hyper-thermostable b -glucosidase CelB from Pyrococcus furious . Biochemistry 39:4963–4970
Katusumi K, Mikio F, Yoshiteru I, Hiroyuki A (2004) Simple synthesis of b -D-glycopyranosides using b -glycosidase from almonds. Chem Pharm Bull 52:270–275
Kengen SWM, Luesink EJ, Stams AJM, Zehnder AJB (1993) Puri fi cation and characterization of an extremely thermostable b -glucosidase from the hyper-thermophilic archaeon Pyrococcucus furious . Eur J Biochem 213:305–312
Kleinman MJ, Wilkinson AE, Wright IP, Evans IH, Bevan EA (1988) Puri fi cation and properties of an extracel-lular glucoamylase from a diastatic strain of Saccharomyces cerevisiae . Biochem J 249:163–170
Kohda H, Kasai R, Yamasaki K, Tanaka O (1976) New sweet diterpene glucosides from Stevia rebaudiana . Phytochemistry 15:981–983
Kojima M, Maruo S, Ohgi T, Ezure Y (1996) Enzymatic synthesis of 4-O- b -D-glactopyranosylmoranoline and 3-O- b -D-galactopyranosylmoranoline by using b -galactosidase from Bacillus circullans . Biosci Biotech Biochem 60:694–696
Kometani T, Tanimoto H, Nishimura T, Kanbara I, Okada S (1993a) Glucosylation of capsaicin by cell suspen-sion cultures of Coffea arabica . Biosci Biotech Biochem 57:2192–2193
Kometani T, Tanimoto H, Nishimura T, Okada S (1993b) Glucosylation of vanillin by cultured plant cells. Biosci Biotech Biochem 57:1290–1293
Konstantinovic S, Predojevic J, Gojkovic S, Ratkovic Z, Mojsilovic B, Pavlovic V (2001) Synthesis of C7-C16 alkyl 2,3 dideoxy glucosides from glucose and fatty acids. Ind J Chem 40B:1242–1244
Krause DR, Wood CJ, MacLean DJ (1991) Glucoamylase (exo-1,4- a -D-gluanohydrolase, E.C. 3.2.1.3) is the
19References
major starch-degrading enzyme secreted by the phyto-pathogenic fungus Colletotrichm gloeosporiodes . J Gen Microbiol 137:2463–2468
Kren V (2001) Chemical biology and biomedicine of gly-cosylated natural compounds. In: Fraser-Reid B, Tatsuta K, Thiem J (eds) Glycoscience chemistry and chemical biology, vol 3. Springer, Berlin, pp 2471–2529
Kren V, Cvak L (1999) Ergot genus Claviceps , medicinal and aromatic plants-industrial pro fi les. Harwood Publ. Ltd., Amsterdam/London
Kren V, Martinkova L (2001) Glycosides in medicine: the role of glycosidic residue in biological activity. Curr Med Chem 8:1313–1338
Laroute V, Willemot RM (1992) Glucoside synthesis by glucoamylase or b -glucosidase in organic solvents. Biotechnol Lett 14:169–174
Lee MD, Dunne TS, Chang CC, Ellestad GA, Siegel MM, Morton GO, McGahren WJ, Borders DB (1987) Calichemicines, a novel family of antitumor antibiot-ics 2. Chemistry and structure of calichemicin, g I . J Am Chem Soc 109:3466–3468
Lehinger AL (1975) Sugars, storage polysaccharides and cell walls. In: Biochemistry. Worth Publishers Inc., New York, pp 249–276
Ljunger G, Adlercreutz P, Mattiasson B (1994) Enzymatic synthesis of octyl- b -glucoside in octanol at controlled water activity. Enzyme Microb Technol 16:751–755
Madsen T, Petersen G, Seiero C, Torslov J (1996) Biodegradability and aquatic toxicity of glycoside sur-factants and a nonionic alcohol etherate. J Am Oil Chem Soc 73:929–933
Malek SAS, Hossain Z (1994) Puri fi cation and character-ization of a thermostable glucoamylase from Myrothecium isolate. J Appl Bacteriol 76:210–215
Matsumura Y, Kasunoki M, Harada W, Kakudo M (1984) Structure and possible catalytic residues of taka amy-lase A. J Biochem 95:697–702
Matsumura S, Imai K, Yoshikawa S, Kawada K, Uchibori T (1990) Surface activities, biodegradability and anti-microbial properties of n-alkyl glucosides, manosides and galactosides. J Am Oil Chem Soc 67:996–1001
McCarter J, Withers SG (1994) Mechanisms of enzymatic glycoside hydrolysis. Curr Opin Struct Biol 4:885–892
Mohri K, Watanabe Y, Yoshida Y, Satoh M, Isobe K, Sugimoto N, Tsuda Y (2003) Synthesis of glycosyl-curcuminoids. Chem Pharm Bull 51:1268–1272
Monsan PF, Paul F, Pelenc P, Bouler E (1996) Enzymatic production of a -butyl glucoside and its fatty acid esters. Ann NY Acad Sci 799:633–641
Mutua LN, Akoh CC (1993) Synthesis of alkyl glucoside fatty acid esters in non aqueous media by Candida sp. lipase. J Am Oil Chem Soc 70:43–46
Nakamura T, Komori C, Lee Y-Y, Hashimoto F, Yohara S, Nohara T, Ejima A (1996) Cytotoxic activities of sola-num steroidal glycosides. Biol Pharm Bull 19:546–566
Nakamura T, Toshima K, Matsumura S (2000) One-step synthesis of n-octyl b -D-xylotrioside, xylobioside and xyloside from xylan and n-octanol using acetone pow-
der of Aureobasidium pullulans in supercritical fl uids. Biotechnol Lett 22:1183–1189
Nilsson KGI (1987) A simple strategy for changing the regio selectivity of glycosidase catalyzed formation of disaccharides. Carbohydr Res 167:95–103
Ohinishi M (1990) Subsite structure of Rhizopus niveus glucoamylase, estimated with the binding parameters for maltooligosaccharides. Starch/Starke 42:311–313
Ohinishi H, Sakai H, Ohta T (1991) Puri fi cation and some properties of a glucoamylasse from Clostridium sp. G0005. Agric Biol Chem 55:1901–1902
Okada G, Unno T (1989) A glucodextranase accompanied by glucoamylase activity from Arthrobacter globi-formis I 42. Agric Biol Chem 53:223–228
Ooi Y, Hashimoto T, Mitsuo N, Satoh T (1985) Enzymatic formation of b -galactosidase from Aspergillus oryzae and its application to the synthesis of chemically unstable cardiac glycosides. Chem Pharm Bull 33:1808–1814
Orihara Y, Furuya T, Hashimoto N, Deguchi Y, Tokoro K, Kanisawa T (1992) Biotransformation of isoeugenol and eugenol by cultured cells of Eucalyptus perrini-ana . Phytochemistry 31:827–831
Panintrarux C, Adachi S, Araki Y, Kimura Y, Matsuno R (1995) Equilibrium yield of n-alkyl- b -D-glucoside through condensation of glucose and n-octanol by b -galactosidase in a biphasic system. Enzyme Microb Technol 17:32–40
Payen A, Persoz JF (1833) Mémoire sur la diastase, les principaux produits de ses reactions et leur applica-tions aux arts industriels. Annales de chimie et de phy-sique 53:73–92
Post CB, Karplus M (1986) Does lusozyme follow the lusozyme pathway? An alternative based on dynamic structural and stereoelectronic considerations. J Am Chem Soc 108:1317–1319
Pretorius IS, Lambrechts MG, Marmur J (1991) The glu-coamylase multigene family in Saccharomyces cerevi-siae var. diastaticus an overview. CRC Crit Rev Biochem Mol Biol 26:53–76
Pugh TA, Shah JC, Magee PT, Clancy MJ (1989) Characterization and localization of the sporulation glucoamylase from Saccharomyces cerevisiae . Biochem Biophys Acta 994:200–209
Rantwijk FV, Oosterom MW, Sheldon RA (1999) Glycosidase-catalyzed synthesis of alkyl glycosides. J Mol Catal B: Enzym 6:511–532
Rao VB, Sastri NVS, Rao PVS (1981) Puri fi cation and characterization of a thermostable glucoamylase from the thermophilic fungus Thermomyces lanuginose . Biochem J 193:379–385
Robyt JF (1998) Essentials of carbohydrate chemistry. Springer, New York, pp 64–68
Roitsch T, Lehle L (1989) Structural requirements for pro-tein N-glycosylation. In fl uence of acceptor peptides on cotranslational glycosylation of yeast in fl uence and site-directed mutagenesis around a sequon sequence. Eur J Biochem 181:525–529
Rubio E, Fernandez MA, Klibanov AM (1991) Effect of the solvent on enzyme regio selectivity. J Am Chem Soc 113:695–696
20 2 Glycosidases
Saha BC, Zeikus JG (1989) Microbial glucoamylases biochemical and biotechnological features. Starch/Starke 41:57–64
Sakata I, Maruyama I, Kobayashi A, Yamamoto I (1998) Production of phenethyl alcohol glycoside. Jpn Kokai Tokkyo Konho, Japan Patent JP 10052297 (CA 128 229438)
San-Aparicio J, Hermoso JA, Martinz-Ripoll M, Laquerica JL, Polaina J (1998) Crystal structure of b -glucosidase A from Bacillus polymyxa insights into the catalytic activity in family1 glycosyl hydrolases. J Mol Biol 275:491–502
Sato T, Takeuchi H, Takahashi K, Kurosu J, Yoshida K, Tsugane T, Shimura S, Kino K, Kirimura K (2003) Selective a -glucosylation of eugenol by a -glucosyl transfer enzyme of Xanthomonas campestris WU-9701. J Biosci Bioeng 96:199–202
Schmid B, Kotter I, Heide L (2001) Pharmacokinetics of salicin after oral administration of a standard willow bark extract. Eur J Clin Pharmacol 57:387–391
Shibata H, Sonoke S, Ochiai H, Nishihashi H, Yamada M (1991) Glucosylation of steviol and steviol glucosides in extracts from Stevia rebaudiana Bertoni . Plant Physiol 95:152–156
Shin HK, Kong JY, Lee JD, Lee TH (2000) Synthesis of hydroxy benzyl- a -glucosides by amyloglucosidase-catal-ysed transglycosylation. Biotechnol Lett 22:321–325
Shinoyama H, Kamiyama Y, Yasui T (1988) Enzymatic synthesis of alkyl b -xylosides from xylobiose by application of the transxylosyl reaction of Aspergillus niger b -xylosidase. Agric Biol Chem 52:2197–2202
Sierks MR, Ford C, Reilly PJ, Svensson B (1990) Catalytic mechanism of fungal glucoamylases as de fi ned by mutagenesis of Asp 176, Glu179, and Glu180 in the enzyme from Aspergillus awamori . Protein Eng 3:193–198
Sills AM, Saunder ME, Stewart GG (1984) Isolation and characterization of the amylolytic system of Schwanniomyces castellii . J Inst Brew 90:311–316
Sinnot ML (1990) Catalytic mechanism of glycosyl trans-fer. Chem Rev 90:1171–1202
Sivakumar R (2009) Enzymatic synthesis of selected phe-nolic and vitamin glycosides. PhD thesis, University of Mysore
Soni BK, Kapp C, Goma G, Soucaille P (1992) Solvent production from starch effect of pH on a -amylase and glucoamylase localization and synthesis in synthetic medium. Appl Microbiol Biotechnol 37:539–543
Specka U, Mayer F, Antranikian G (1991) Puri fi cation and properties of thermoactive glucoamylase from Clostridium thermosaccharolyticum . Appl Environ Microbiol 57:2317–2323
Srivastava RAK (1984) Studies on extracellular and intra-cellular puri fi ed amylases from a thermophilic Bacillus stearothermophilus . Enzyme Microb Technol 6:422–426
Stevenson DE, Furneaux RH (1996) High yield synthesis of ethyl and 2- fl uroethyl b -D-galactopyranosides using Streptococcus thermophilus b -galactosidase. Enzyme Microb Technol 18:513–518
Stevenson DE, Stanley RA, Furneaux RH (1993) Optimization of alkyl b -D-galactopyronoside synthe-sis from lactose using commercially available b -galac-tosidase. Biotechnol Bioeng 42:657–666
Stoffer B, Frandsen T, Busk P, Schneider P, Svendsen I, Svensson B (1993) Production, puri fi cation and char-acterization of the catalytic domain of glucoamylase from Aspergillus niger . Biochem J 292:197–202
Stoffer B, Aleshin AE, Firsov LM, Svensson B, Honzatko RB (1995) Re fi ned structure for the complex of D-gluco-dihydroacarbose with glucoamylases from Aspergillus awamori var. X100 to 2.2 Å resolution dual conformation for extended inhibitors bound to the active site of glucoamylases. FEBS Lett 358:57–61
Suzuki Y, Suzuki K (1991) Enzymatic formation of 4 G- a -D-glucopyranosyl rutin. Agric Biol Chem 55:181–187
Suzuki Y, Uchida K (1994) Enzymatic formation of a new derivative of thiamin, b -galactosylthiamin. Biosci Biotech Biochem 58:1273–1276
Svensson B, Larsen K, Svendsen I, Boel E (1983) The complete amino acid sequence of the glycoprotein glucoamylase G1 from Aspergillus niger . Carlsberg Res Commun 48:529–544
Svensson B, Clarke AJ, Svendsen I, Moller H (1990) Identi fi cation of carboxylic acid residues in glucoamylase G2 from Aspergillus niger that participate in the catalysis and substrate binding. Eur J Biochem 18:29–38
Takahashi T, Kato K, Ikegami Y, Irie M (1985) Different behavior towards raw starch of three forms of glucoam-ylase from a Rhizopus sp. J Biochem 98:663–671
Tanaka Y, Ashikari T, Nakamura N, Kiuchi N, Shibano Y, Amachi T, Yoshizumi H (1986) Comparison of amino acid sequences of three glucoamylases and their struc-ture-function relationships. Agric Biol Chem 50:965–969
Tapavicza SV, Bell D, Kopp-Holtwiesche B (2000) Plant growth enhancement against phytopathogenic fungi and/or soil borne pests. Patent WO 0002451 (CA 132 60488)
Taylor PM, Napier EJ, Fleming ID (1978) Some proper-ties of a glucoamylase produced by the thermophilic fungus Humicola lanuginose . Carbohydr Res 16:301–308
Trincone A, Pagnotta E, Giordano A, Perugino G, Rossi M, Moracci M (2003) Enzymatic synthesis of 2-deox-yglycosides using the b -glycosidase of the archaeon Sulfolobus solfataricus . Biocatal Biotransform 21:17–24
Uchida K, Suzuki Y (1998) Enzymatic synthesis of a new derivative of thiamin, O- a -glucosylthiamin. Biosci Biotech Biochem 62(2):221–224
Verdoucq L, Czjzek M, Moriniere J, Beven DR, Esen A (2003) Mutational and structural analysis of aglycone speci fi city in maize and sorghum b -glucosidase. J Biol Chem 278:25055–25062
Vic G, Crout DHG (1995) Synthesis of allyl and benzyl b -D-glucopyranosides and allyl b -D-galactopyranoside from D-glucose or D-galactose and the corresponding
21References
alcohol using almond b -D-glucosidase. Carbohydr Res 279:315–319
Vic G, Thomas D (1992) Enzyme-catalyzed synthesis of alkyl- b -D-glucosides in organic media. Tetrahedron Lett 33:4567–4570
Vic G, Biton J, Beller DL, Michel JM, Thomas D (1995) Enzymatic glycosylation of hydrolytic alcohols in organic medium by the reverse hydrolysis reaction using almond b -D-glucosidase. Biotechnol Bioeng 46:109–116
Vic G, Thomas D, Crout DHG (1997) Solvent effect on enzyme-catalyzed synthesis of b -D-glucosides using the reverse hydrolysis method application to the pre-parative-scale synthesis of 2-hydroxybenzyl and octyl b -D-glucopyronosides. Enzyme Microb Technol 20:597–603
Vijayakumar GR (2007) Enzymatic synthesis of selected glycosides. PhD thesis, University of Mysore
Voorhorst WGB, Eggen RIK, Luesink EJ, De Vos WM (1995) Characterization of the Cel B gene coding for b -glucosidase from the hyperthermophilic archean Pyroccus furiosus and its expression and site directed mutation in Escherichia coli . J Bacteriol 177:7105–7111
Vulfson EN, Patel R, Beecher JE, Andrews AT, Law BA (1990) Glycosidases in organic solvents I. Alkyl- b -glucoside synthesis in a water-organic two-phase sys-tem. Enzyme Microb Technol 12:950–954
Williamson G, Belshaw NJ, Williamson MP (1992) O-Glycosylation in Aspergillus glucoamylase. Con fi rmation and role in binding. Biochem J 282:423–428
Withers SG, Street IP (1988) Identi fi cation of a covalent a -D-glucopyranosyl enzyme intermediate formed on a b -glucosidase. J Am Chem Soc 110:8551–8553
Withers GG, Warren RAJ, Street IP, Rupitz K, Kempton JB, Abersold R (1990) Unequivocal demonstration of the involvement in the mechanism of a retaining gly-cosidase. J Am Chem Soc 112:5887–5889
Yoon SH, Fulton DB, Robyt JF (2004) Enzymatic synthe-sis of two salicin analogues by reaction of salicyl alco-hol with Bacillus macerans cyclomaltodextrin glucanyltransferase and Leuconostoc mesenteroides B-742CB dextransucrase. Carbohydr Res 339:1517–1529
Yu RC, Hang YD (1991) Puri fi cation and characterization of a glucoamylase from Rhizopus oryzae . Food Chem 40:301–308
Zechel DL, Withers SG (2001) Dissection of nucleophilic and acid–base catalysis in glycosidases. Curr Opin Chem Biol 5:643–649
Zhou JH (2000) Herbal sweetening and preservative composition comprising licorice extract and mogro-sides obtained from plants belonging to cucurbita-ceae and/or momordica. Patent US 6103240 (CA 133 168393)
23S. Divakar, Enzymatic Transformation, DOI 10.1007/978-81-322-0873-0_3, © Springer India 2013
3.1 Lipases
Lipases (EC 3.1.1.3) catalyse hydrolysis of trig-lycerides at the oil/water interface (Lehninger 1977 ) . Under reverse hydrolytic conditions, lipases exhibit their ability to catalyse various other types of reactions like esteri fi cation, transesteri fi cation, polymerisation and lactonisa-tion. High selectivity and mild conditions associ-ated with lipase-mediated transformations have made them very attractive for the synthesis of a wide range of natural products, pharmaceuticals, fi ne chemicals, food ingredients and bio-lubri-cants (Schreier 1997 ; Dorm et al. 2004 ; Gill and Valivety 1997 ; Belarbi et al . 2000 ; Sharma et al. 2001 ) . The main reason for the use of lipases is the growing interest and demand for the produc-
tion of products through environmentally com-patible natural means. Lipases are regarded as enzymes with high commercial potential due to their versatility in application. Lipase-catalysed esteri fi cation in organic solvents offers synthetic challenges, which if dealt with successfully can result in the generation of several useful compounds.
Lipases are hydrolases which catalyse the hydrolysis of triacylglycerols to glycerol and free fatty acids. In eukaryotes, lipases are involved in various stages of lipid metabolism including fat digestion, absorption, reconstitution and lipopro-tein metabolism. In plants, lipases are found in energy reserve tissues. Lipases contain a hydro-phobic oligopeptide lid, covering the entrance of its active site which requires interfacial activation
3
Abstract
Among the hydrolytic enzymes, transformations mediated by lipases have been extensively studied. This chapter begins with a brief description on the three lipases – porcine pancreas lipase, Rhizomucor lipase and Candida rugosa lipase – employed extensively in the transformation work described in this book. This is followed by speci fi city of lipases and the reactions – both hydrolytic and esteri fi cation – catalysed by them with respect to their mechanism. After a brief mention about the advantages of using lipases in organic solvents over chemical reactions, this chapter concludes with a table furnished with literature data on mainly diverse esteri fi cation reac-tions carried out in the a past three decades using lipases in nonaqueous media.
Lipases
24 3 Lipases
at lipid–water interface (Martinelle et al. 1995 ) . This lid opens up in presence of hydrophobic interfaces, due to conformational change of lipases acquiring an open structure in which the active site residues become accessible to sub-strates. However, in the absence of interfaces, the lid covers the active site, making it inaccessible to substrates (Brzozowski et al. 1991 ) . The catal-ysis by lipase encompasses a linear substrate concentration gradient at the interface, amenable orientation of a scissile ester bond, reduction in the micro-aqueous phase around the substrate ester molecules and the conformational change of the enzyme (Derewenda and Sharp 1993 ) . Since lipases tolerate organic solvents in the reac-tion mixture, lipases fi nd promising position in organic chemical processing (Kiran and Divakar 2001 ; Kiran et al. 2001 ; Therisod and Klibanov 1986 ; Berglund and Hutt 2000 ; Harikrishna and Karanth 2001 ) , detergent formulations (Jaeger and Reetz 1998 ) , synthesis of biosurfactants (Plou et al. 1999 ; Sarney and Vulfson 1995 ; Sarney et al. 1996 ) , oleochemical industry (Bornscheuer 2000 ; Undurraga et al. 2001 ) , dairy industry (Vulfson 1994 ) , paper manufac-
ture (Jaeger and Reetz 1998 ) , nutrition (Pabai et al. 1995a, b ; Undurraga et al. 2001 ) and cos-metics and pharmaceutical processing (Berglund and Hutt 2000 ; Liese et al. 2000 ) . Other biotech-nological applications of lipases are shown in Table 3.1 .
3.1.1 Porcine Pancreas Lipase (PPL)
Pancreatic lipases exhibit high molecular activity with one molecule of lipase capable of cleaving nearly 7,000 ester bonds per second (Scharpe et al . 1997 ) under optimal conditions. Porcine pancreas has been the source of lipase for most of the work on pancreatic lipase. It is also the richest source for pancreatic lipases, and it is the fi rst puri fi ed lipase (Peschke 1991 ) .
Pancreatic lipase, a serine hydrolase, con-tains a single-chain glycoprotein of about 48-KDa molecular weight (Winkler and Gubernator 1994 ) . From pH titration and photo-oxidation studies, among the catalytic triad, Ser-152, His-263 and Asp-176, found to be responsible for the catalytic activity, histidine
Table 3.1 Biotechnological applications of lipases (Vulfson 1994 )
Industry Action Product or application
Detergents Hydrolysis of fats Removal of oil stains from fabrics Dairy Hydrolysis of milk fat, cheese ripening,
modi fi cation of butter fat Development of fl avouring agents in milk, cheese and butter
Bakery Flavour improvement and shelf life elongation Bakery products Beverages Improved aroma Beverages Food Quality improvement, transesteri fi cation Mayonnaise, dressings and whippings, health
foods Meat and fi sh Flavour development and removal of fats Meat and fi sh products Fats and oils Transesteri fi cation, hydrolysis Cocoa butter, margarine, fatty acids, glycerol,
mono- and diglycerides Chemicals Enantioselectivity, synthesis Chiral building blocks and chemicals Cosmetics Synthesis Emulsi fi ers, moisturisers Leather Hydrolysis Leather products Paper Hydrolysis Paper with improved quality by removing wax Cleaning Synthesis and hydrolysis Removal of cleaning agents like surfactants Food dressing Quality improvement Mayonnaise, dressing and whipping Pharmaceuticals Transesteri fi cation, hydrolysis Speciality lipids, digestive aids Health food Transesteri fi cation Health foods
253.1 Lipases
residue was the most important (Winkler et al . 1990 ) . Donner ( 1976 ) puri fi ed and measured some of the physical parameters: molecular weight 52 KDa, sedimentation coef fi cient (s degrees 20, w) 4.0 S, diffusion coef fi cient (D degrees 20, w) 6.7 × 10 −7 cm 2 s −1 , Stokes radius ( r ) 30.3 Å, partial speci fi c volume ( v ) 0.72 cm 3 g −1 , frictional ratio ( f / f
0 ) 1.23 and isoelectric point
(pI) 5.18. It consists of a central parallel b -sheet having a helical link (van Tilbeurgh et al. 1992 ; Hermoso et al. 1996 ) bound by prolipase at its edge with the plane of the prolipase roughly perpendicular to the C-terminal b -sheet domain of the lipase molecule (Fig. 3.1 ). Prolipase is a fl attened molecule with dimensions of about 33 Å × 24 Å × 16 Å consisting of mainly three fi nger-shaped regions constituted by residues 26–39, 47–64 and 67–87 held together by disul-phide bonds. Majority of hydrophobic amino acids are found in the region opposite of the lipase binding site. The catalytic active site also contains a surface helix amphipathic lid consist-
ing of residues 248–258 covering the active site (van Tilbeurgh et al. 1992 ) .
3.1.2 Rhizomucor miehei Lipase (RML)
Crystallographic study of Rhizomucor miehei lipase (RML) has shown a single polypeptide chain with 269 amino acid residues, and the molecular weight is 29.4 KDa (Brady et al. 1990 ; Brzozowski et al. 1991 ) . Brady et al. ( 1990 ) identi fi ed that RML is an a -/ b -type protein with three disulphide bonds responsible for stabilising the two terminal strands. The catalytic triad con-tains Ser-144, His-257 and Asp-203, buried under a single 17-residue lid (82–96 residues) that occludes the active site in the native structure (Derewenda et al. 1992 ) . Brzozowski et al. ( 1991 ) , studying an atomic model of inhibitor–RML complex, showed a direct covalent bond formation between nucleophilic O
g of Ser-144
and substrate. Carbonyl oxygen of the substrate may be stabilised by the interaction of amide nitrogen, and the hydroxyl of Ser-82 (Leu-145 may also be involved in amide interaction) through hydrogen bonding and thus Ser-82 exhib-iting a favourable conformation for the oxyanion interaction (Fig. 3.2a, b ).
The pentapeptide sequence – Gly 142 -His 143 -Ser 144 -Leu 145 -Gly 146 – corresponds to a tight turn between the fourth strand of the central b -sheet and a buried a -helix (Fig. 3.3 ). The catalytically active Ser-144 found in the middle of the turn is in the rare e -conformation ( j = 62; f = 121 o ). This structural motif consisting of a b -strand followed by a tight turn containing the active Ser and an a -helix was called as b - e Ser- a -motif (Fig. 3.3 ; Derewenda and Sharp 1993 ) .
The inhibition study of lipase by serine protease inhibitors like di-isopropyl phosphoro fl uoridate indicated the role of a Ser residue in the active site. It has been found that all known amino acid sequence of neutral lipases share a consensus pen-tapeptide GX
1 SX
2 G (where X represents any
amino acid, G represents glycine and S represents serine) which contain an essential Ser residue (Derewenda and Sharp 1993 ) .
Fig. 3.1 Schematic ribbon diagram of the porcine lipase–colipase structure. The glycan chain, connected to the lipase N-terminal domain (N domain), is drawn as a stick model. One tetraethylene glycol monooctyl ether inhibitor molecule, located in the open active site, is represented by balls and sticks . Colipase interacts with the lipase C-terminal domain (C domain) and with the fl ap (Adopted from Hermoso et al. 1996 )
26 3 Lipases
3.1.3 Candida rugosa Lipase (CRL)
Candida rugosa lipase is the fi rst example of a native interface-activatable lipase in ‘open’ form (Fig 3.3 ). CRL is a single-domain protein belong-ing to the family of a -/ b -hydrolase proteins con-sisting of a central hydrophobic eight-stranded b -sheet packed between two layers of amphiphilic a -helices (Fig. 3.3 ). CRL is made up of a single polypeptide chain with 534 amino acid residues with a molecular weight of 57 kDa. CRL appears in fi ve isoforms, which have been cloned and sequenced (Kawaguchi et al. 1989 ; Longhi et al. 1992 ) .
In CRL, the active site triad consists of Ser-209, Glu-341 and His-449 proximate to three surface loops (62–92, 122–129 and 294–305) very important for catalytic activity (Fig. 3.3 ). A characteristic super secondary structure
motif, found in all lipases, houses an embed-ded Ser-209. In the open conformation of CRL, the lid extends nearly perpendicular to the protein surface, forming a large depression that surround the active site. Uncharged polar residues constitute the hydrophilic area. The hydrophobic face of the fl ap facing the active site is mainly composed of aliphatic side chain amino acid residues, and the fl ap facing oppo-site the active site is hydrophilic in character. The geometry of loops 13 and 4 together with the active site of CRL suggests that the oxyan-ion hole O
g is formed by the backbone amide
of Gly-123, Gly-124 and Ala-210, involved in hydrogen bond formation with the substrates (Fig. 3.3 ). Presence of two acyl binding ‘pock-ets’ in the active site of CRL depicts the sub-strate speci fi city for carbon chain lengths: a small pocket for short-chain acids and a bigger
Fig. 3.2 ( a ) The hydrogen-bonding network in the active site of the Rhizomucor miehei lipase. The crystal structure (at 3-Å resolution) of a complex of RML lipase with n-hexylphosphonate ethyl ester in which the enzyme’s active site is exposed by the movement of the helical lid. The catalytic Ser-144 is immediately beneath the phos-phorus atom of the inhibitor. His-257 is clearly displaced towards the ethyl oxygen consistent with the proposed ori-entation of the substrate and the mechanism of hydrolysis. Hydrogen bond contacts between O
g atom of Ser-82, and
its amide NH are indicated (Adopted from Brzozowski et al. 1990 , 1991 ) ( b ) A schematic drawing showing the
packing within the b - e Ser- a -motif. The helix and strand pack against each other with four amino acids (tinted) forming the interface. The residues nearer the turn are in closer contact, and therefore, their side chains are restricted to those of smaller hydrophobic amino acids. The plane of the central peptide of the turn is perpendicular to the axis of the motif, which forces the catalytic Ser to adopt a strained e conformation. The two stars show the positions that b -carbons of amino acids other than Gly would occupy if the two invariant Gly residues of the GX
1 SX
2 G
pentapeptide were mutated (Adopted from Derewenda and Sharp 1993 )
273.2 Lipase Specificity
pocket for binding longer-chain acids (Parida and Dordick 1993 ) .
3.2 Lipase Speci fi city
Enzymes possesses extraordinary ability to exhibit regioselectivity and stereospeci fi city in reactions catalysed by them. Based on positional speci fi city, lipases can be divided into fi ve differ-ent classes (Camp et al. 1998 ) .
Lipases of fi rst group do not exhibit regiose-lectivity in that they catalyse hydrolysis of fatty acyl triglycerides independent of their type or position. Examples of this class are lipases from Candida cylindracea , Corynebacterium acnes and Staphylococcus aureus (Camp et al . 1998 ) .
Lipases from Aspergillus niger , Rhizopus delemar , Rhizomucor miehei , Candida rugosa and porcine pancreas catalyse reaction at sn -1 and sn -3 positions of triacylglycerides
Fig. 3.3 Overall structure of Candida rugosa lipase. ( a ) Ribbon representation with a-helices, b -strands and coils coloured in red, green and grey, respectively. The helical and coil segments forming the fl ap region are shown in dark blue and orange, respectively. The catalytic triad residues (Ser-209, Glu-341 and His-449), the disul-
phide bridges and the Asn-attached N-acetylglucosamine moieties are shown in ball-and-stick representation. ( b ) A representation of the lipase 2 topology with the sec-ondary structure elements identi fi ed ( b , strands; a , helix) (Adopted from Mancheno et al. 2003 )
28 3 Lipases
(Macrea 1985 ) , hence known as 1,3-speci fi c lipases.
The third group of lipases covers lipases with different rates of hydrolysis of monoacyl, diacyl and triacylglycerides. Some of these lipases are located in the tissues of rats and humans.
The fourth group of lipases catalyses the exchange of speci fi c type of fatty acids exempli fi ed by the extracellular lipases from the fungus Geotrichum candidum which preferentially releases unsaturated cis - n -9 fatty acid groups (Macrea 1985 ) .
The fi fth group of lipases shows stereospe-ci fi city, namely, a faster rate of hydrolysis of fatty acids placed at the sn -1 position than the sn -3 position or vice versa. Examples of this group are lipoprotein lipases from milk, adipose tissues and postheparin plasma which preferentially cleave the ester bond in sn -1 and human and rat lingual
lipases which react preferentially with the fatty acids at sn -3 position (Jensen et al. 1983 ) .
3.3 Reactions Catalysed by Lipases
The ranges of substrates with which lipases react and also the range of reactions they catalyse are probably far more than any other enzymes stud-ied till date.
Lipases catalyse three types of reactions (Scheme 3.1 ):
3.3.1 Hydrolysis
Ester hydrolysis is the dominant reaction in aqueous media, when there is large excess of water.
Scheme 3.1 Types of reactions catalysed by lipases
293.4 Mechanism of Lipase-Catalysed Esterification in Organic Solvents
3.3.2 Esteri fi cation
Esteri fi cation is achieved under low water conditions such as in nearly anhydrous sol-vents, and if the water content of the medium is controlled, relatively better product yields can be obtained.
3.3.3 Transesteri fi cation
The acid moiety of an ester is exchanged with another one. If the acyl donor is a free acid, the reaction is called acidolysis , and if the acyl donor is an ester, the reaction is called interesteri fi cation . In alcoholysis , the nucleo-phile alcohol acts as an acyl acceptor.
3.4 Mechanism of Lipase-Catalysed Esteri fi cation in Organic Solvents
Lipases show lipid splitting nature and the mechanism is same as that of serine proteases (Pleiss et al. 1998 ) . Catalytic triad in lipases contains Ser, His and Asp/Glu residues. The serine residue in active centre is activated by histidine and aspartic acid/glutamic acid resi-dues. The substrate acid forms a tetrahedral acyl–enzyme intermediate by reaction with the OH group of the catalytic serine residue. The resulting excess of negative charge that develops on the carbonyl oxygen atom is sta-bilised by the oxyanion hole (Brzozowski et al. 1991 ) . The tetrahedral intermediate I forms a serinate ester with elimination of water molecule. Subsequent nucleophilic attack of alcohol to the acyl–enzyme intermediate leads to tetrahedral intermediate II. Finally, the product ester is released, and enzyme is free for the next molecule to attack. Grochulski et al. ( 1994 ) , Cygler et al. ( 1994 ) and Schrag and Cygler ( 1997 ) proposed this mechanism for the ester formation in case of RML (Scheme 3.2 ).
Step I. Acylation Step Initially, serine hydroxyl group forms a tetra-
hedral intermediate complex I with acyl donor; the negative charge that is formed in the tetrahe-dral intermediate is stabilised by hydrogen bonding with the acid given which are respon-sible for the oxyanion hole formation.
Step II. Formation of Acyl–Enzyme Complex After the formation of the tetrahedral interme-
diate I, an acyl–enzyme complex is formed through covalent bond with Ser residue by losing one molecule of water.
Step III. Nucleophilic Attack by Alcohol (Carbohydrate)
Nucleophile alcohol attacks the carbonyl cen-tre of the tetrahedral intermediate forming a tetra-hedral complex II, forming an enzyme–acid–alcohol complex.
O
O H N HN
CH2
O
Ser-144His-257
Asp- 203_
R
O
HO
O
O H N HN
CH2
O
Ser-144His-257
Asp- 203R
OHO_
Tetrahedral complex I
O
O H N HN
CH2
O
RO
Ser-144His-257
Asp- 203
Acyl enzyme complex
30 3 Lipases
Step IV. Release of Ester (L-Amino Acyl Ester of Carbohydrate)
Finally, the ester is released, and the enzyme will be ready for the next molecule to attack.
3.5 Esteri fi cation Reactions
Table 3.2 lists some of the commercially impor-tant fl avour, fragrance, surfactant and sweetener esters prepared through lipase catalysis.
3.6 Advantages of Lipase Catalysis over Chemical Catalysis
There are many advantages of using lipases as biocatalysts (Lohith 2007 ; Somashekar 2009 ) :
1. Speci fi city of the reaction. 2. Milder reaction conditions under which the
lipolytic process can be operated. 3. Non-generation of by-products associated
with the use of several chemical procedures. 4. Improved product yield and better product
quality. 5. Exploitation of the stereo- and regiospeci fi cities
shown by lipases to produce high-value chiral synthons.
6. Success in immobilisation techniques that have enabled the reuse of lipases leading to economically viable processes.
7. Good conversion yields. 8. Lipases are highly thermostable, exhibiting
activity at 100°C. 9. Use of nonpolar solvents, which impart stability
to lipase rather than in water, renders insolubility
O
O H N HN
CH2
O-----
Ser-144His-257
Asp - 203_------
O
O
+ R C R'
Scheme 3.2 Lipase mediated esteri fi cation (Grochulski et al. 1993 ; Cygler et al. 1994 ; Schrag and Cygler 1997 )
O
N
CH2
O
RO
Ser-144His-257
Asp- 203
-H+
O
O H N HN
CH2
O
Ser-144His-257
Asp- 203
O H N HOH
RO
_
R
R O
Tetrahedral complex II
313.6 Advantages of Lipase Catalysis over Chemical Catalysis
Table 3.2 Commercially important esters synthesised by lipase mediated catalysis
Compound Use Lipase References
Flavour esters Isoamyl acetate Banana fl avour Pseudomonas
fl uorescence Takahashi et al. ( 1988 )
Candida antarctica Langrand et al. ( 1990 ) Rhizomucor miehei Rizzi et al. ( 1992 ) ,
Chulalaksananukul et al. ( 1993 ) Rhizomucor miehei Raza fi ndralambo et al. ( 1994 ) Candida cylindracea , PPL
Divakar et al. ( 1999 ), Harikrishna et al. ( 2001a, b )
Aspergillus niger Welsh and Williams ( 1990 ) , Welsh et al. ( 1990 )
Novozyme 435 Rhizomucor miehei, Candida antarctica
Gubicza et al. ( 2000 )
Pseudomonas pseudomallei
Guvenc et al. ( 2002 )
Porcine liver lipase Romero et al. ( 2005 ) Kanwar and Goswami ( 2002 ) , Ngrek ( 1974 ) , Kumar et al. ( 2005 )
Isoamyl butyrate Banana fl avour Candida antarctica Langrand et al. ( 1988, 1990) Rhizomucor miehei Mestri and Pai ( 1994a, b ) Candida cylindracea , PPL, Aspergillus niger
Welsh and Williams ( 1990 ) , Welsh et al. ( 1990 )
Candida antarctica, Gubicza et al. ( 2000 ) Geotrichum sp . and Rhizopus sp.
Macedo et al. ( 2004 )
Isoamyl propionate Banana fl avour Candida antarctica Langrand et al. ( 1988 ) Isoamyl isovalerate Apple fl avour Rhizomucor miehei Chowdary et al. ( 2002 ) Isobutyl isobutyrate Pineapple
fl avour Rhizomucor miehei Hamsaveni et al. ( 2001 )
Methyl propionate Fruity fl avour Rhizomucor miehei Perraud and Laboret ( 1989 ) Ethyl butyrate Pineapple
fl avour Candida cylindracea Gubicza et al. ( 2000 ) , Gillies
et al. ( 1987 ) Butyl isobutyrate Sweet fruity
odour Candida cylindracea , PPL and Aspergillus niger
Yadav and Lathi ( 2003 ) , Welsh and Williams ( 1990 ) , Welsh et al. ( 1990 )
Protocatechuic aldehyde Rhizomucor miehei , PPL Divakar ( 2003 ) Short-chain alcohol esters of C
2 –C
18 acids
Fruity odour Staphylococcus warneri Talon et al. ( 1996 )
Short-chain fatty acid esters Fruity odour Staphylococcus xylosus Mestri and Pai ( 1994a ) , Macedo et al. ( 2003 ) , Xu et al. ( 2002 )
Long-chain alcoholic esters of lactic acids
Flavour Candida antarctica From et al. ( 1997 ) , Torres and Otero ( 1999 ) , Parida and Dordick ( 1991 )
Novozyme 435 Rhizomucor miehei
Bousquet et al. ( 1999 )
Methyl benzoate Exotic fruit and berries fl avour
Candida rugosa Leszczak and Tran-Minh ( 1998 )
Tetrahydrofurfuryl butyrate Fruity favour Novozyme 435 Yadav and Devi ( 2004 )
(continued)
32 3 Lipases
Table 3.2 (continued)
Compound Use Lipase References
Cis-3-hexen-1-yl acetate Fruity odour Rhizomucor miehei Chang et al. ( 2003 ) Fragrance esters Tolyl esters Honey note Rhizomucor miehei , PPL Burdock ( 1994 ) , Suresh-Babu
et al. ( 2002 ) , Manohar and Divakar ( 2002 )
Anthranilic acid esters of C 2 –C
18
alcohols Flowery odour of jasmine
Candida cylindracea , PPL
Kittleson and Pantaleone ( 1994 ) , Suresh-Babu and Divakar ( 2001 ) , Manohar and Divakar ( 2004a )
4-t-Butylcyclohexyl acetate Woody and intense fl owery notes
PPL Manohar and Divakar ( 2004b )
Geranyl methacrylate Floral fruity odour
Rhizomucor miehei, PPL, Pseudomonas cepacia
Athawale et al. ( 2002 )
Citronellyl acetate Fruity rose odour
Candida antarctica SP435
Claon and Akoh ( 1994b ) Citronellyl propionate Citronellyl valerate Pseudomonas fragi Mishio et al. ( 1987 )
Marlot et al. ( 1985 ) Geranyl butyrate Fruity odour Candida rugosa Akoh et al. ( 1992 ) , Shieh et al.
( 1995 ) Geranyl propionate Geranyl valerate Farnesol butyrate Farnesol propionate Farnesol valerate Phytol butyrate Phytol propionate Phytol valerate Citronellyl laurate Fruity,
characteristic lavender and bergamot-like fragrance
Novozyme SP Yadav and Lathi ( 2004 )
a -Terpinyl acetate
a -Terpinyl propionate Rhizomucor miehei Rao and Divakar ( 2002 )
a -Terpinyl esters of fatty acids Rhizomucor miehei Rao and Divakar ( 2001 )
a -Terpinyl esters of short-chain acids Terpinyl esters of triglycerols Aspergillus niger,
Rhizopus delemar, Geotrichum candidum,
Iwai et al. ( 1980 )
Penicillium cyclopium Claon and Akoh ( 1994a ) Surfactant esters Oleic acid esters of short-chain alcohols
Surfactants Novozyme 435 Dorm et al. ( 2004 )
Butyl oleate Surfactants Rhizomucor miehei Knez et al. ( 1990 ) 2-O-Alkanoyl lactic acid esters of C
2 –C
18 alcohols
Surfactants Rhizomucor miehei , PPL Kiran and Divakar ( 2001 ) , Kiran et al. ( 1998 )
Surfactants and sweetener esters N-Acetyl- l -leucyl– d -glucose Surfactants Mucor javanicus ,
Pseudomonas cepacia , subtilisin
Maruyama et al. ( 2002 ) N-Acetyl- l -methionyl– d -glucose N-Acetyl- l -tyrosinyl– d -glucose N-Acetyl- l -tryptophanyl– d -glucose
(continued)
333.6 Advantages of Lipase Catalysis over Chemical Catalysis
Table 3.2 (continued)
Compound Use Lipase References
N-Acetyl- l -phenylalanyl– d -glucose
Surfactant Subtilisin Maruyama et al. ( 2002 ) , Riva et al. ( 1988 )
N-Acetyl- l -phenylalanyl– d -galactose N-Acetyl- l -phenylalanyl–fructose N-Acetyl- l -phenylalanyl–mannose N-Acetyl- l -phenylalanyl–lactose N-t-Boc- l -phenylalanyl–glucose
N-Acetyl- l -methionyl–methyl- b -galactopyranoside
Surfactants Optimase M-440, Proleather, APG 380
Park et al. ( 1996 )
N-t-Boc- l -phenylalanyl–galactose N-t-Boc- l -phenylalanyl–fructose N-t-Boc- l -phenylalanyl–methyl a - d -glucopyranoside N-t-Boc- l -phenylalanyl–sorbitol Surfactants Optimase M-440 Park et al. ( 1999 ) N-t-Boc- l -phenylalanyl–sucrose N-t-Boc- l -phenylalanyl–cellobiose N-t-Boc- l -phenylalanyl–raf fi nose N-t-Boc- l -phenylalanyl–trehalose N-t-Boc- l -phenylalanyl–maltose N-t-Boc- l -phenylalanyl–lactose N-t-Boc- l -leucyl–sucrose Surfactants Optimase M-440 Jeon et al. ( 2001 ) N-t-Boc- l -tyrosinyl–sucrose N-t-Boc- l -methionyl–sucrose N-t-Boc- l -aspartyl–sucrose Di-N-t-Boc- l -lysyl–sucrose N-t-Boc- l -phenylalanyl–xylitol N-t-Boc- l -phenylalanyl–arabitol N-t-Boc- l -phenylalanyl–mannitol N-t-Boc- l -phenylalanyl–N-acetyl– d -glucosamine l -Prolyl, l -phenylalanyl, l -tryptophanyl and l -histidyl esters of carbohydrates
Surfactants, sweeteners
Rhizomucor miehei , PPL, Candida rugosa
Vijayakumar et al. ( 2004 ) , Somashekar and Divakar ( 2007 ) , Lohith et al. ( 2003 ) , Lohith and Divakar ( 2005 ) 1- O -ester, 2- O -ester, 3- O -ester,
4- O -ester, 5- O -ester, 6- O -ester, 6 ¢ - O -ester 2,5-di- O - ester, 3,5-di- O -ester 2, 6-di- O -ester, 3, 6-di- O -ester
6,6 ¢ -di- O -ester
N-Acetyl- l -alanyl-methyl- b - d -galactopyranoside
Surfactants, sweeteners
Subtilisin Riva et al. ( 1988 ) Rhodotorula lactosa Suzuki et al . ( 1991 )
2- O -ester, 3- O -ester, 4- O -ester, 6- O -ester 6- O -Butyl glucose Surfactants PPL Therisod and Klibanov ( 1986 )
Kirk et al. ( 1992 ) Zaks and Dodds ( 1997 )
Subtilisin
(continued)
34 3 Lipases
Table 3.2 (continued)
Compound Use Lipase References
6- O -Acetyl glucose 6- O -Capryloyl glucose 6- O -Acetyl galactose Klibanov ( 1986 ) 6- O -Acetyl maltose Dordick ( 1989 ) 6- O -Acetyl fructose Schlotterbeck et al. ( 1993 ) 1- O -Acetyl fructose Boyer et al. ( 2001 ) Fructose oleate Surfactant Lipozyme, Rhizomucor
miehei Khaled et al . ( 1991 )
Fatty acid esters of glycosides Candida antarctica Adlerhorst et al. ( 1990 ) Candida rugosa Zaidi et al. ( 2002 ) Butyl oleate
Oleyl butyrate Oleyl oleate 6- O -Lauroyl sucrose Surfactants Humicola lanuginosa Ferrer et al. ( 1999 ) 6- O -Lauroyl glucose Candida antarctica B Ferrer et al. ( 2005 ) 6- O -Lauroyl maltose 6- O -Palmitoyl maltose 1,6-di- O -Lauroyl sucrose 6,6-di- O -Lauroyl sucrose
6 ¢ - O -Palmitoyl maltose
b -Methylglucoside methacrylate/acrylate
Surfactants Candida antarctica Kim et al. ( 2004 )
of the enzyme, solubility of substrates and products in organic solvents resulting in homogenous reaction conditions, easy prod-uct workout procedures and easy removal of water formed as a by-product.
References
Adlerhorst K, Björking F, Godtfredsen SE, Kirk O (1990) Enzyme catalyzed preparation of 6- O -acylglucopyranosides. Synthesis 1:112–115
Akoh CC, Copper C, Nwosu CV (1992) Lipase-G cata-lyzed synthesis of monoglycerides in organic solvent and analysis by HPLC. J Am Oil Chem Soc 69:257–260
Athawale V, Manjrekar N, Athawale M (2002) Lipase-catalyzed synthesis of geranyl methacrylate by transesteri fi cation study of reaction parameters. Tetrahedron Lett 43:4797–4800
Belarbi EH, Molina E, Chisti Y (2000) A process for high yield and scaleable recovery of high purity eicosapen-taenoic acid esters from micro algae and fi sh oil. Enzyme Microb Technol 26:516–529
Berglund P, Hutt K (2000) Biocatalytic synthesis of enantio-pure compounds using lipases. In: Patel RN (ed) Stereoselective biocatalysis. Marcel Dekker, New York
Bornscheuer UT (2000) Enzymes in lipid modi fi cation. Wiley-VCH, Weinheim/Berlin
Bousquet M, Willemot R, Monsan P, Boures E (1999) Enzymatic synthesis of unsaturated fatty acid gluco-side esters for dermo-cosmetic applications. Biotechnol Bioeng 63:730–736
Boyer V, Stanchev M, Fairbanks AJ, Davis BG (2001) Ready protease catalysed synthesis of carbohydrate-amino acid conjugates. Chem Commun 19:1908–1909
Brady L, Brzozowski AM, Derewenda U, Derewenda ZS, Dodson GG, Tolley S, Turkenburg JP, Christiansen L, Huge-Jensen B, Nashkov L, Thim L, Menge U (1990) A serine protease triad forms the catalytic center of triglycerol lipase. Nature 343:767–770
Brzozowski AM, Derewenda U, Derewenda ZS (1990) A model for interfacial activation in lipases from the structure of a fungal lipase-inhibitor complex. Nature 351:491–494
Brzozowski AM, Derewenda U, Derewenda ZS, Dodson GG, Lawson DM, Turkenburg JP, Bjorkling F, Huge-Jensen B, Patkar SA, Thim L (1991) A model for interfacial activation in lipases from the structure of a fungal lipase-inhibitor complex. Nature 351:491–494
Burdock GA (1994) In Fenaroli’s Handbook of fl avor ingredients, vol II, 3rd edn. CRC Press, Boca Raton
Camp JV, Huyghebaert A, Goeman P (1998) In: Christope AB (ed) Structural modi fi ed food fats synthesis bio-chemistry and use. AOCS Press, Champaign
35References
Chang SW, Shaw JF, Shieh CJ (2003) Optimization of enzymatically prepared hexyl butyrate. Food Technol Biotechnol 41:237–242
Chowdary GV, Divakar S, Prafulla SG (2002) Modeling on isoamyl isovalerate synthesis from Rhizomucor miehei lipase in organic media optimization studies. World J Microbiol Biotechnol 18:179–185
Chulalaksananukul W, Condoret JS, Combes D (1993) Geranyl acetate synthesis by lipase catalysed transesteri fi cation in supercritical carbon dioxide. Enzyme Microb Technol 15:691–698
Claon PA, Akoh CC (1994a) Lipase catalyzed synthesis of terpene esters by trans esteri fi cation in n -hexane. Biotechnol Lett 16:235–240
Claon PA, Akoh CC (1994b) Effect of reaction parameters on SP435 lipase-catalyzed synthesis of citronellyl acetate in organic solvent. Enzyme Microb Technol 16:835–838
Cygler M, Grochulski P, Kazlauskas RS, Schrag JD, Bouthillier F, Rubin B, Serreqi AN, Gupta AK (1994) A structural basis for the chiral preferences of lipases. J Am Chem Soc 116:3180–3186
Derewenda ZS, Sharp AM (1993) News from the inter-face the molecular structure of triacyl glyceride lipases. Trends Biochem Sci 18:20–25
Derewenda U, Brzozwski AM, Lawson DM, Derewenda ZS (1992) Catalysis at the interface The anatomy of a conformational change in a triglyceride lipase. Biochemistry 31:1532–1541
Divakar S, Kiran KR, Harikrishna S, Karanth NG (1999) An improved process for the preparation of esters of organic acids and alcohols. Indian Patent, 1243/DEL/99 No. 191078
Divakar S (2003) Lipase catalysed regioselective esteri fi cation of protocatechuic aldehyde. Indian J Chem Sect B 42B:1119–1122
Donner M (1976) Preparation of porcine pancreatic lipase free of co-lipase activity. Acta Chem Scand B 30:430–434
Dordick JS (1989) Enzymatic catalysis in monophasic organic solvents. Enzyme Microb Technol 11:194–211
Dorm N, Bela fi -Bak K, Bartha L, Ehrenstein U, Gubicza L (2004) Manufacture of an environmental-safe bio-lubricant from fusel oil by enzymatic esteri fi cation in solvent-free system. Biochem Eng J 21:229–234
Ferrer M, Cruces MA, Bernable M, Ballesteros A, Plou FJ (1999) Lipase catalysed regioselective acylation of sucrose in two solvent mixtures. Biotechnol Bioeng 65:10–16
Ferrer M, Soliveri J, Plou FJ, Cortes NL, Duarte DR, Christensenc M, Patinob JLC, Ballesterosa A (2005) Synthesis of sugar esters in solvent mixtures by lipases from Thermomyces lanuginosus and Candida antarc-tica B, and their antimicrobial properties. Enzyme Microb Tech 36:391–398
From M, Adlercreutz P, Mattiasson B (1997) Lipase cata-lyzed esteri fi cation of lactic acid. Biotechnol Lett 19:315–317
Gill I, Valivety R (1997) Polyunsaturated fatty acids Part 1. Occurrence, biological activities and applications. Trends Biotechnol 15:401–409
Gillies B, Yamazaki H, Armstrong DW (1987) Production of fl avor esters by immobilized lipase. Biotechnol Lett 9:709–714
Grochulski P, Li Y, Schrag JD, Bouthillier F, Smith P, Harrison D, Rubin B, Cygler M (1993) Insight into interfacial activation from an open structure of Candida rugosa lipase. J Biol Chem 268:12843–12847
Grochulski P, Bouthillier F, Kazlauskas RJ, Serreqi AN, Schrag JD, Ziomek E, Cygler M (1994) Analogs of reaction intermediates identify a unique substrate binding site in Candida rugosa lipase. Biochemistry 33:3494–3500
Gubicza L, Kabiri-Badr A, Keoves E, Bela fi -Bako K (2000) Large-scale enzymatic production of natural fl avour esters in organic solvent with continuous water removal. J Biotechnol 84:193–196
Guvenc A, Kapucu N, Mehmetoglu I (2002) The produc-tion of isoamyl acetate using immobilized lipases in a solvent-free system. Process Biochem 38:379–386
Hamsaveni DR, Prafulla SG, Divakar S (2001) Optimization of isobutyl butyrate synthesis using cen-tral composite rotatable design. Process Biochem 36:1103–1109
Harikrishna S, Karanth NG (2001) Lipase-catalyzed syn-thesis of isoamyl butyrate. A kinetic study. Biochim Biophys Acta 1547:262–267
Harikrishna S, Manohar B, Divakar S, Prapulla SG, Karanth NG (2001a) Optimization of isoamyl acetate production using immobilized lipase from Mucor mie-hei by response surface methodology. Enzyme Microb Technol 26:132–138
Harikrishna S, Divakar S, Karanth NG (2001b) Enzymatic synthesis of isoamyl acetate using immobilized lipase from Rhizomucor miehei . J Biotechnol 87:193–201
Hermoso J, Pignol D, Kerfelec B, Crenon I, Chapus C, Fontecilla-camps JC (1996) Lipase activation by non-ionic detergents. The crystal structure of the porcine lipase co-lipase-tetraethylene glycol monooctyl ether complex. J Biol Chem 270:18007–18016
Iwai M, Okumura S, Tsujisaka Y (1980) Synthesis of ter-pene alcohol esters by lipase. Agri Biol Chem 44:2731–2732
Jaeger KE, Reetz TM (1998) Microbial lipases from ver-satile tools for biotechnology. Trends Biotechnol 16:396–403
Jensen RG, de-Jong FA, Clark RM (1983) Determination of lipase speci fi city. Lipid 18:239–252
Jeon GJ, Park OJ, Hur BK, Yang JW (2001) Enzymatic synthesis of amino acid-sugar alcohol conjugates in organic media. Biotechnol Lett 23:929–934
Kanwar L, Goswami P (2002) Isolation of a Pseudomonas lipase produced in pure hydrocarbon substrate and its application in the synthesis of isoamyl acetate using membrane-immobilised lipase. Enzyme Microb Technol 31:727–735
36 3 Lipases
Kawaguchi V, Honda H, Toniguchi-Morimura J, Iwasaki S (1989) The codon CUG is read as serine in an asporogenic yeast Candida cylindracea . Nature 341:164–166
Khaled N, Montet D, Pina M, Graille J (1991) Fructose oleate synthesis in a fi xed catalyst bed reactor. Biotechnol Lett 13:167–172
Kim J, Haam S, Park DW, Ahn IS, Lee TG, Kim HS, Kim WS (2004) Biocatalytic esteri fi cation of methylgluco-side for synthesis of biocompatible sugar-containing vinyl esters. Chem Eng J 99:15–22
Kiran KR, Karanth NG, Divakar S (1998) An improved enzymatic process for the preparation of fatty acid hydroxyacid ester. Indian Patent, 1978/DEL/98 187313
Kiran KR, Divakar S (2001) Lipase catalysed esteri fi cation of organic acids with lactic acid. J Biotechnol 87:109–121
Kiran KR, Manohar B, Divakar S (2001) A central com-posite rotatable design analysis of lipase catalysed synthesis of lauroyl lactic acid at bench-scale level. Enzyme Microb Technol 29:122–128
Kirk O, Bjorkling F, Godfredsen SE, Larsen TS (1992) Fatty acid speci fi city in lipase catalysed synthesis of glucoside esters. Biocatalysis 6:127–134
Kittleson JR, Pantaleone (1994) Enzymic biphasic pro-cess for the synthesis of aromatic esters fl avoring agents from corresponding carboxylic acid and alco-hol by esteri fi cation mediated by a lipase from Candida cyclindracea . US. Patent, 5,437,991
Klibanov AM (1986) Enzymes that work in organic sol-vents. Chem Technol 16:354–359
Knez Z, Leitgeb M, Zavrsnik D, Lavrie B (1990) Synthesis of oleic acid esters with immobilized lipase. Fat Sci Technol 4:169–172
Kumar R, Modak J, Madras G (2005) Effect of the chain length of the acid on the enzymatic synthesis of fl avors in supercritical carbon dioxide. Biochem Eng J 23:199–202
Langrand G, Triantaphylides C, Baratti J (1988) Lipase catalysed formation of fl avor esters. Biotechnol Lett 10:549–554
Langrand G, Rondot N, Triantaphylides C, Baratti J (1990) Short-chain fl avor esters synthesis by micro-bial lipases. Biotechnol Lett 12:581–586
Lehninger LA (1977) Biochemistry, 3rd edn. Worth Publishers Inc., New York, pp 355–357
Leszczak JP, Tran-Minh C (1998) Optimized enzymatic synthesis of methyl benzoate in organic medium. Operating conditions and impact of different factors on kinetics. Biotechnol Bioeng 60:556–561
Liese A, Seelbach K, Wandrey C (2000) Industrial biotransformations. Wiley-VCH, Weinheim
Lohith K, Vijaya-kumar GR, Manohar B, Divakar S (2003) An improved enzymatic process for the prepa-ration of amino acyl esters of mono and disaccharides. Indian Patent, NF-492/03, PCT/03/00466
Lohith K, Divakar S (2005) Lipase catalysed synthesis of L-phenylalanine esters of D-glucose. J Biotechnol 117:49–56
Lohith K (2007) Enzymatic synthesis of selected amino acid esters of sugars. PhD thesis, University of Mysore
Longhi S, Fasetti F, Grandori R, Lotti M, Vanoni M, Alberghina L (1992) Cloning and nucleotide sequences of two lipase genes from Candida cylindracea . Biochim Biophys Acta 1131:227–232
Macedo GA, Lozano MMS, Pastore GM (2003) Enzymatic synthesis of short chain citronellyl esters by a new lipase from Rhizopus sp. Electron J Biotechnol 6(1):0717–3458
Macedo GA, Pastore GM, Rodrigues MI (2004) Optimising the synthesis of isoamyl butyrate using Rhizopus sp. lipase with a central composite rotatable design. Process Biochem 39:687–692
Macrea AR (1985) In: Tramper J, Vander-Plas HC, Linko P (eds) Biocatalyst in organic synthesis. Elsevier, Amsterdam, pp 195–208
Mancheno JM, Pernas MA, Martinez MJ, Ochoa B, Ruo ML, Hermosa JA (2003) Structural insights Into the lipase/esterase behavior in the Candida rugosa family crystal structure of the lipase 2 isoenzyme at 1.97 A resolution. J Mol Biol 332:1059–1069
Manohar B, Divakar S (2002) Application of central com-posite rotatable design to lipase catalyzed syntheses of m -cresyl acetate. World J Microbiol Biotechnol 18:745–751
Manohar B, Divakar S (2004a) Application of surface plots and statistical designs to selected lipase cataly-sed esteri fi cation reactions. Process Biochem 39:847–851
Manohar B, Divakar S (2004b) Porcine pancreas lipase acetylation of beta-cyclodextrin anchored 4-t-butylcy-clohexanol. Indian J Chem Sect B 43B:2661–2665
Marlot C, Langrand G, Triantaphylides C, Baratti J (1985) Ester synthesis in organic solvent catalyzed by lipase immobilized on hydrophilic supports. Biotechnol Lett 7:647–650
Martinelle M, Holmquist M, Hult K (1995) On the inter-facial activation of Candida antarctica lipase A and B as compared with Humicola lanuginosa lipase. Biochim Biophys Acta 1258:272–276
Maruyama T, Nagasawa SI, Goto M (2002) Enzymatic synthesis of sugar esters in organic solvents. J Biosci Bioeng 94:357–361
Mestri S, Pai JS (1994a) Effect of moisture on lipase cata-lyzed esteri fi cation of geraniol palmarosa oil in non-aqueous system. Biotechnol Lett 17:459–461
Mestri S, Pai JS (1994b) Synthesis of isoamyl butyrate by lipase by lipase of Mucor miehei . PAFAI J 2:24–26
Mishio T, Takahashi K, Yoshimoto T, Kodera Y, Saito Y, Inada Y (1987) Terpene alcohol ester synthesis by polyethylene glycol modi fi ed lipase in benzene. Biotechnol Lett 9:187–190
Ngrek S (1974) Synthesizing power of liver lipase. Acta Biol Expt 14:157–174
Pabai F, Kermasha S, Morin A (1995a) Interesteri fi cation of butter fat by partially puri fi ed extracellular lipases from Pseudomonas putida , Aspergillus niger and Rhizopus oryzae . World J Microbiol Biotechnol 11:669–677
37References
Pabai F, Kermasha S, Morin A (1995b) Lipase from Pseudomonas fragi CRDA 323 partial puri fi cation, characterization and interesteri fi cation of butter fat. Appl Microbiol Biotechnol 43:42–51
Parida S, Dordick JS (1991) Substrate structure and solvent hydrophobicity control lipase catalysis and enantioselectivity in organic media. J Am Chem Soc 113:2253–2259
Parida S, Dordick JS (1993) Tailoring lipase speci fi city by solvent substrate chemistries. J Org Chem 58:3238–3244
Park OJ, Park HG, Yang JW (1996) Enzymatic transesteri fi cation of monosaccharides and amino acid esters in organic solvents. Biotechnol Lett 18:473–478
Park OJ, Jeon GJ, Yang JW (1999) Protease catalysed synthesis of disaccharide amino acid esters in organic media. Enzyme Microb Technol 25:455–462
Perraud R, Laboret F (1989) Optimization of methyl pro-pionate production catalyzed by Mucor miehei lipase. Appl Microbiol Biotechnol 44:321–326
Peschke G (1991) Active components and galenic aspects of enzyme preparations. In: Lankisch PG (ed) Pancreatic enzymes in health and disease. Springer, Berlin, pp 55–64
Pleiss J, Fisher M, Schimid RD (1998) Anatomy of lipase binding site. Chem Phys Lipids 93:67–80
Plou FJ, Cruces MA, Pastor E, Ferrer M, Bernabe M, Ballesterose A (1999) Acylation of sucrose with vinyl esters using immobilized hydrolysis demonstration that chemical catalysis may interfere with enzymatic catalysis. Biotechnol Lett 21:635–639
Rao P, Divakar S (2001) Lipase catalysed esteri fi cation of a -terpineol with various organic acids application of the Plackett-Burman design. Process Biochem 36:1125–1128
Rao P, Divakar S (2002) Response surface methodologi-cal approach for the Rhizomucor miehei lipase-medi-ated esteri fi cation of a -terpineol with propionic acid and acetic anhydride. World J Microbiol Biotechnol 18:341–345
Raza fi ndralambo H, Blecker C, Lognoy G, Marlier M, Wathlet JP, Severin M (1994) Improvement of enzy-matic synthesis yields of fl avor acetates the example of isoamyl acetate. Biotechnol Lett 16:247–250
Riva S, Chopineau J, Kieboom APG, Klibanov AM (1988) Protease catalysed regioselective esteri fi cation of sug-ars and related compounds in anhydrous dimethylfor-mamide. J Am Chem Soc 110:584–589
Rizzi M, Stylos P, Riek A, Reuss M (1992) A kinetic study of immobilized lipase catalyzing the synthesis of isoamyl acetate by transesteri fi cation in n-hexane. Enzyme Microb Technol 14:709–714
Romero MD, Calvo L, Alba C, Habulin M, Primozic M, Knez Z (2005) Enzymatic synthesis of isoamyl acetate with immobilized Candida antarctica lipase in super-critical carbon dioxide. J Supercrit Fluids 33:77–84
Sarney DB, Vulfson EN (1995) Application of enzymes to the synthesis of surfactants. Trends Biotechnol 13:164–172
Sarney DB, Barnard MJ, MacManus DA, Vulfson EN (1996) Application of lipases to the regioselective
synthesis of sucrose fatty acid monoesters. J Am Oil Chem Soc 73:1481–1487
Scharpe S, Uyttenbroeck W, Samyn N (1997) Pancreatic enzyme replacement. In: Lauwers A, Scharpe S (eds) Pharmaceutical enzymes. Marcel Dekker, New York, pp 187–221
Schlotterbeck A, Lang S, Wray V, Wagner F (1993) Lipase catalyzed monoacylation of fructose. Biotechnol Lett 15:61–64
Schrag JD, Cygler M (1997) Lipase and a / b hydrolase fold. Methods Enzymol 284:85–107
Schreier P (1997) In: Berger RG (ed) Biotechnology of aroma compounds. Adv Biochem Eng Biotechnol 17:52
Sharma R, Chisti Y, Banerjee UC (2001) Production, puri fi cation, characterization, and applications of lipases. Biotechnol Adv 19:627–662
Shieh CJ, Akoh CC, Koehler PE (1995) Four-factor response surface optimization of the enzymatic modi fi cation of triolein to structured lipids. J Am Oil Chem Soc 72:619–623
Somashekar BR (2009) Lipase catalysed esteri fi cation of sugars with alkyl side chain containing amino acids. PhD thesis, University of Mysore
Somashekar BR, Divakar S (2007) Synthesis of L-valyl, L-leucyl and L-isoleucyl esters of carbohydrates using lipase as biocatalyst. Indian J Chem Sect B 46B:2026–2044
Suresh-Babu CV, Divakar S (2001) Selection of alcohols through Plackett-Burman design in lipase catalyzed synthesis of anthranilic acid. J Am Oil Chem Soc 78:49–52
Suresh-Babu CV, Karanth NG, Divakar S (2002) Lipase catalysed esteri fi cation of cresols. Ind J Chem Sect B 41B:1068–1071
Suzuki Y, Shimizu T, Takeda H, Kanda K (1991) Fermentative or enzymatic manufacture of sugar amino acid esters. Japan Patent, 03216194 A2
Takahashi K, Saito Y, Inada Y (1988) Lipases made active in hydrophobic media. J Am Oil Chem Soc 65:911–916
Talon R, Montel MC, Berdague JL (1996) Production of fl avor esters by lipases of Staphylococcus warneri and Staphylococcus xylosus . Enzyme Microb Technol 19:620–622
Therisod M, Klibanov AM (1986) Facile enzymatic prep-aration of mono acylated sugars in pyridine. J Am Chem Soc 108:5638–5640
Torres C, Otero C (1999) Part I Enzymatic synthesis of lactate and glycolate esters of fatty alcohols. Enzyme Microb Technol 25:745–752
Undurraga D, Markovits A, Erazo S (2001) Cocoa butter equivalent through enzymic interesteri fi cation of palm oil mid-fraction. Process Biochem 36:933–939
van Tilbeurgh H, Sarda L, Verger R, Cambillau C (1992) Structure of the pancreatic lipase-procolipase com-plex. Nature 359:159–162
Vijayakumar GR, Lohith K, Somashekar BR, Divakar S (2004) Lipase catalysed synthesis of L-alanyl, L-leucyl and L-phenylalanyl esters of D-glucose using unpro-tected amino acids. Biotechnol Lett 26:1323–1328
38 3 Lipases
Vulfson EN (1994) Industrial applications of lipases. In: Wolley P, Petersen SB (eds) Lipases their structure biochemistry and application. Cambridge University Press, New York
Welsh FW, Williams RE (1990) Lipase-mediated produc-tion of ethyl butyrate and butyl butyrate in nonaqueous systems. Enzyme Microb Technol 12:743–748
Welsh FW, Williams RE, Dawson KH (1990) Lipase-mediated synthesis of low molecular weight fl avor esters. J Food Sci 55:1679–1682
Winkler F, D’Arcy A, Hunziker W (1990) Structure of human pancreatic lipase. Nature 343:771–775
Winkler FK, Gubernator K (1994) Structure and mecha-nism of human pancreatic lipase. In: Wooley P, Peterson SB (eds) Lipase: their structure, biochemis-try and applications. Cambridge University Press, Cambridge, pp 139–157
Xu Y, Wang D, Qing-Mu X, Ao-Zhao G, Zhang KC (2002) Biosynthesis of ethyl esters of short chain fatty acids using whole-cell lipase from Rhizopus Chinensis CCTCCM 201021 in non aqueous phase. J Mol Catal B Enzyme 18:29–37
Yadav GD, Devi KM (2004) Immobilized lipase-catalysed esteri fi cation and transesteri fi cation reactions in non-aqueous media for the synthesis of tetrahydrofurfuryl butyrate comparison and kinetic modeling. Chem Eng Sci 59:373–383
Yadav GD, Lathi PS (2003) Kinetics and mechanism of synthesis of butyl isobutyrate over immobilised lipases. Biochem Eng J 16:245–252
Yadav GD, Lathi PS (2004) Synthesis of citronellol lau-rate in organic media catalyzed by immobilized lipases kinetic studies. J Mol Catal B Enzyme 27:113–119
Zaidi A, Gainer JL, Carta G, Mrani A, Kadiri T, Belarbi Y, Mir A (2002) Esteri fi cation of fatty acids using nylon-immobilized lipase in n -hexane kinetic param-eters and chain length effects. J Biotechnol 93:209–216
Zaks A, Dodds DR (1997) Applications of biocatalysts and biotransformations to the synthesis of pharmaceu-ticals. Drug Dev Today 2:513–531
39S. Divakar, Enzymatic Transformation, DOI 10.1007/978-81-322-0873-0_4, © Springer India 2013
4.1 Introduction
Besides catalysing the hydrolysis of triglycerides at the oil/water interface, lipases (triacyl glycerol hydrolases (E.C.3.1.1.3)) are also known to catalyse various other types of reactions like esteri fi cation, transesteri fi cation, polymerisation and lactonisation.
4.2 2-O-Acyl Esters of Lactic Acid
Multifunctional hydroxy compounds contain one or more functional groups along with a hydroxyl group. Esters of multifunctional hydroxy com-pounds like polyhydric alcohols and hydroxy acids play an important role in the area of food, since they comprise a wide range of compounds from biodegradable plastics to emulsi fi ers.
4
Abstract
This entire chapter is devoted to the esteri fi cation reactions involving compounds containing multifunctional groups like OH, COOH, CH
3 and
CHO. The description involves optimisation of reaction conditions for use of lipases in nonpolar solvents under low water activity by both conven-tional and response surface methodological conditions. It attempts to bring out the superiority of lipase catalysis over chemical synthesis in the few reactions discussed. Esteri fi cation of the OH group of lactic acid with the COOH group of few long-chain fatty acids to synthesise 2-O-alkanoyl acids is discussed in detail. This is followed by the work on the preparation of tolyl esters, protocatechuic aldehyde esters, 4-t-butylcyclohexyl acetate and acetylation of b -cyclodextrin. State of the lipases employed under nonaqueous solvents in the above-mentioned reactions is best brought by studies on thermostability and scanning electron microscopy. The role of water, constituting the micro-aqueous phase around the enzyme during its employment under nonpolar conditions, attempts to explain the integrity of the enzyme under such conditions, in terms of developing a theoretical model on the micro-aqueous pH and various equilibria occurring at and associated with the micro-aqueous phase.
Enzymatic Esteri fi cation of Compounds Possessing Multifunctional Hydroxyl and Carboxyl Groups
40 4 Enzymatic Esterifi cation of Compounds Possessing Multifunctional Hydroxyl and Carboxyl Groups
Fatty acid esters of lactic acid like 2-O-palmitoyl lactic acid and 2-O-stearoyl lactic acid are commercially important, biocompatible, ionic esters which fi nd wide applications in food, phar-maceutical and cosmetic industry as surfactants.
The reaction involves the hydroxyl group of lactic acid at the 2- position and the carboxyl group of the fatty acid. Till date only chemical routes for the synthesis of fatty acid esters of lactic acid are known. Enzymatic synthesis of these esters is not known till date. Fatty acid esters of lactic acid like 2-O-palmitoyl lactic acid, 2-O-stearoyl lactic acid, 2-O-lauroyl lactic acid and other 2-O-acyl esters of lactic acid have been synthesised using immobi-lised lipases from Rhizomucor miehei (RML) and porcine pancreas (PPL), Scheme 4.1 . For method-ologies, the readers are recommended to refer Kiran and Divakar ( 2001 ) .
4.2.1 Lactic Acid
Lactic acid invariably contained 12% water, which hindered the esteri fi cation reaction as it facilitated transesteri fi cation reactions involv-ing water of reaction present as well as that accumulated during the reaction. Crystalline
lactic acid, prepared by removing water by azeotropic re fl ux using benzene, gave better yields in enzymatic reactions.
Due to its highly hydrophilic nature, lactic acid was found to be insoluble in many nonpolar solvents. Polar solvents like dioxane and ethylm-ethyl ketone were found to dissolve both lactic acid and fatty acids. Ethylmethyl ketone was found to be a better solvent than polar solvents like dioxane which gave only 4% esteri fi cation only with RML as they strip the water off the enzyme rendering catalytically inactive.
4.2.2 2-O-Stearoyl Lactic Acid
The enzymatic reaction was controlled by several factors. Optimisation of the reaction by varying enzyme/substrate (E/S) ratios showed a maximum yield of 99% at an E/S ratio of 22.6 AU mmol −1 (Table 4.1 , Kiran and Divakar 2001 ) . For a lactic acid/fatty acid concentration of 0.06 M, an E/S ratio of 125.0 gL mole −1 gave maximum esteri fi cation in case of RML (50 and 40%, respec-tively, for 2-O-palmitoyl and 2-O-stearoyl lactic acids). In case of PPL, it was 40 and 30%, respec-tively, at an E/S ratio of 40 gL mole −1 .
Scheme 4.1 Lipase-catalysed esteri fi cation of organic acids with lactic/lactylic acids
414.2 2-O-Acyl Esters of Lactic Acid
4.2.3 2-O-Palmitoyl Lactic Acid
When hexane was used as the solvent, though lactic acid remained insoluble, 18% esteri fi cation was observed after 48 h (Kiran and Divakar 2001 ) . When chloroform was employed as the solvent, 18.2 and 76% ester formation were observed with RML and PPL, respectively (Table 4.2 ).
A maximum esteri fi cation of 85.1% was obtained with chloroform. A chloroform to hexane (30:70) mixture at (Table 4.2 ) an enzyme/
Table 4.1 Preparation of 2-O-stearoyl lactic acid (bench-scale) a
Solvent Enzyme/substrate ratio (E/S) b (PPL) Time (h) Ester yield (%) Initial rate (mmol h −1 )
CHCl 3 /hexane 11.3 64.5 43.1 0.32
CHCl 3 14.7 70 63.2 0.40
CHCl 3 /hexane 18.6 68 34.5 0.31
CHCl 3 20.5 89.5 95.8 0.36
CHCl 3 22.6 68 99.1 0.32
CHCl 3 23.6 86 90.4 0.40
CHCl 3 27.0 67 34.5 0.32
CHCl 3 29.5 63 40.2 0.33
a Lactic acid and stearic acid concentrations: 0.025 mole b Activity in hydrolytic units
Table 4.2 Preparation of 2-O-palmitoyl lactic acid by using RML and PPL (bench-scale)
Palmitic acid conc. (mole)
Lactic acid conc. (mole) Solvent
Enzyme/substrate ratio (E/S) a (AU mmol −1 )
% yield and time (h)
Initial rate (mmol h −1 )
0.11 0.11 Ethylmethyl ketone
RML (7.2) 0 (45) -
0.11 0.11 Hexane RML (7.2) 17.9 (44) 0.29 0.055 0.055 CHCl
3 PPL (10.3) 18.2 (39) 0.32
0.025 0.025 CHCl 3 PPL (22.6) 76 (40) 0.59
0.025 0.025 CHCl 3 PPL (22.6) 72 (112) 0.21
0.028 0.025 CHCl 3 /hexane PPL (10.1) 48.1 (65) 0.32
0.025 0.025 CHCl 3 PPL (14.8) 64.1 (70) 0.35
0.025 0.025 CHCl 3 PPL (18.1) 75.6 (94) 0.29
0.025 0.025 CHCl 3 /hexane PPL (18.2) 45.9 (68) 0.30
0.025 0.025 CHCl 3 PPL (21.3) 85.1 (92) 0.21
0.025 0.028 CHCl 3 PPL (27.1) 51.7 (68) 0.28
0.025 0.025 CHCl 3 PPL (29.4) 40.2 (36) 0.14
a Activity in hydrolytic units
Table 4.3 Comparison of ester yields as determined by titration, HPLC and 1 H NMR
Ester Ester yield (%)
Titration a HPLC b 1 H NMR c
2-O-stearoyl lactic acid
83.6 38.2 34.1
2-O-palmitoyl lactic acid
61.6 24.9 22.2
2-O-lauroyl lactic acid
43.7 21.7 24.3
Error in measurements will be: a ±5–10% b ±5% c ±5%
42 4 Enzymatic Esterifi cation of Compounds Possessing Multifunctional Hydroxyl and Carboxyl Groups
substrate ratio of 18–21 AU mmol −1 of PPL gave a yield of about 85% after 96 h.
The ester proportions determined by NMR and HPLC showed good correspondence (Table 4.3 ).
In 1 H NMR the signals in the region 4.0–5.3 ppm corresponding to –CH–O– from free and ester samples were quite informative (Table 4.4 ). In 13 C NMR the signals in the region 60–70 ppm and 170–190 ppm corresponding to –CH–O– and –CO– region, respectively, were informative. A detailed analysis of the signals in the product samples showed unreacted fatty acid, lactic acid and lactylic acids along with esteri fi ed fatty acid, lactic acid and lactylic acids. The yields of the esters were also determined. However, the yields were lesser than those detected by titrimetry.
4.2.4 Optimisation of Reaction Parameters Using Response Surface Methodology
Response surface methodological analyses of the esteri fi cation reaction between palmitic acid (Kiran et al. 2000 ) and stearic acid (Kiran et al. 1999 ) with lactic acid in the presence of immobi-lised lipases from Rhizomucor miehei and por-cine pancreas lipase were attempted.
4.2.4.1 2-O-Stearoyl Lactic Acid Design : Box–Behnken design, 27 experiments, 3 variables at 3 levels
Variables : Enzyme/substrate ratio, incubation period, lactic acid/stearic acid
Equation :
Table 4.4 Yield of organic acid esters of lactic acid as deduced from 1 H NMR a
Sample Ester yield (%) (potentiometry)
Free lactic acid (%)
Lactylate esters b (%)
Organic acid esters of lactic/lactylic acids (%)
Commercial lactic acid 64.1 35.9 Crystalline lactic acid 28.1 71.9 Stearoyl lactic acid 83.6 40.9 25.0 34.1 Stearoyl lactic acid–water layer 83.6 85.5 2.72 10.8 Stearoyl lactic acid–bicarbonate layer 83.6 – – – Stearoyl lactic acid–chloroform layer 83.6 8.2 21.3 70.5 Palmitoyl lactic acid 61.6 44.4 33.3 22.2 Lauroyl lactic acid 43.7 28.2 45.6 24.3 Palmitoyl methyl lactate 57.0 69.3 18.4 12.3 Palmitoyl butyl lactate 44.0 80.7 14.5 2.8
a Percentage of organic acid esters of lactic/lactylic acids was determined from increase in the area of –CH–O– signals at 4.9–5.20 ppm region in the spectra of samples with respect to that of crystalline lactic acid at 4.05 ppm; error in NMR measurements will be ±5% b Oligomers of lactic acid
e t e e e sRML : 23.95 3.96 2.9 8.286 5.19Y X X X X X X+− −= +
s t s s s tPPL : 10.43 1.65 5.42 3.14 4.26Y X X X X X X= + + − +
where Y = percentage of esteri fi cation X
e = enzyme/substrate ratio (E/S), g mole −1
X t = incubation period, h
X s = lactic acid (LA)/stearic acid (SA), mole L −1
The maximum yields predicted by the theo-retical equations for both the lipases matched well with the observed experimental values. In case of RML, 2-O-stearoyl lactic acid formation was found to increase with incubation period and
434.2 2-O-Acyl Esters of Lactic Acid
lactic acid/stearic acid concentrations with maxi-mum esteri fi cation at an E/S ratio of 125 gL mole −1 (Fig. 4.1 ). In case of PPL, esteri fi cation showed steady increase with increase in incubation period and lactic acid/stearic acid concentrations inde-
pendent of the enzyme/substrate ratios employed (Fig. 4.2 ).
The optimum conditions predicted for esteri fi -cation showed good correspondence with experi-mental values.
24
48
72
0
30
0.03
0.06
0.09
Incubation period,h (Xt)
% E
ster
ific
atio
n
LA(SA) Concentration, M (Xs)
Fig. 4.1 Response surface plot predicting esteri fi cation percentage of lactic acid for its reaction with stearic acid using Lipozyme IM20 at a fi xed E/S ratio of 166.7 g mol −1
1
e s tRML : At 102.1 gL mole ; 0.03 M; 72 h
Predicted Yield : 29.43 % Experimental Yield : 2 0 %.
/
4.1
X X X−= = =
1e s tPPL : At 40 gL mole ; 0.0882 M; 72 h
Predicted Yield :18.86% Experimental Yield :1 5 %.
/
7.8
X X X−= = =
4.2.4.2 2-O-Palmitoyl Lactic Acid Design : Box–Behnken design, 27 experiments, 3 variables at 3 levels
Variables : Enzyme/substrate ratio, incubation period, lactic acid/palmitic acid
Equation :
e s t s s s t
e s t e s
RML : 17.02 4.85 4.32 4.39 10.72 3.15
PPL : 4.90 2.27 1.37 2.99 3.57
= + + + − +
= + + + +
Y X X X X X X X
Y X X X X X
44 4 Enzymatic Esterifi cation of Compounds Possessing Multifunctional Hydroxyl and Carboxyl Groups
where Y = percentage of esteri fi cation X
e = enzyme/substrate ratio (E/S), AU mmole −1
X t = incubation period, h
X s = lactic acid (LA)/palmitic acid (SA), mM
In case of RML, palmitoyl lactic acid forma-tion increased with increase in LA(PA) concen-tration up to 60 mM above which the esteri fi cation decreased. Esteri fi cation increased with increase in E/S ratios and incubation periods. In case of PPL, the three-dimensional plots showed almost linear relationships. Esteri fi cation increased with increase in E/S ratios and incubation peri-ods. PPL was found to give lower yields of the ester when compared to RML. The correspon-dence between experimental and predicted con-ditions for optimum esteri fi cation was found to be good.
4.2.4.3 2-O-Lauroyl Lactic Acid Design : Central composite rotatable design (CCRD), 32 experiments, 5 variables at 5 levels (Kiran et al. 2001a )
Variables : Enzyme/substrate ratio (0.09–1.14 AU mmol −1 ), lactic acid concentration
(5–25 mmol), incubation time (6–54 h), buffer vol-ume (0–0.2 mL) and buffer pH values (4.0–8.0)
Equation :
t e a b
p t t e e a a
b b p p t e t
t b t p e a e b
e p a b a p b p
1.562 0.338 0.013 0.135 0.081
0.062 0.031 0.178 0.105
0.028 0.044 0.059 0.082
0.150 0.038 0.335 0.102
0.081 0.036 0.007 0.044
= + − + −+ − + +
+ − − +
+ + − −
− − − +
a
Y X X X X
X X X X X X X
X X X X X X X X
X X X X X X X X
X X X X X X X X
where
Y = ester yield (mmol) X
t = incubation period
X e = E/S ratio (AU mmol −1 )
X a = lactic acid concentration (mmol)
X b = buffer volume (mL)
X p = buffer pH
A typical three-dimensional plot is given in Fig. 4.3 which shows the effect of E/S ratios and lac-tic acid concentrations at a fi xed buffer volume of 0 mL and an incubation period of 54 h on ester yield. Highest ester yield of 4.8 mmol was observed at the lowest E/S ratio of 0.09 AU mmol −1 . Lower E/S ratios gave higher yields, and higher E/S ratios gave lesser yields. This behaviour clearly explained the competitive nature of binding between lauric and lactic acids for
Fig. 4.2 Response surface plot predicting esteri fi cation of lactic acid with stearic acid using PPL as a function of LA(SA) concentrations at different incubation periods at all E/S ratios
454.2 2-O-Acyl Esters of Lactic Acid
5
10
15
20
25
0.0
5.0
1.14
0.87
0.61
0.35
0.09
Ester yield (m
mol)
Fig. 4.3 Response surface plot showing variation in pre-dicted yield of lauroyl lactic acid as a function of E/S ratios and lactic acid concentrations at an incubation
period of 54 h. Since the buffer volume is 0 mL, terms connected with buffer volume and pH values were ignored
the same binding site on the enzyme. Addition of buffer in terms of both volume and pH did not have a profound effect on increase in ester yield. Predicted yields showed good validation with experimental yields when experiments corresponding to selected points on the contour plots were carried out.
4.2.5 Effect of Acid Carbon Chain Length on Esteri fi cation with Lactic Acid
It was observed that increase in carbon chain of the organic acid increased the ester yield (Fig. 4.4 , Table 4.5 ).
The initial rates measured also showed a similar pattern. Only exception to this rule was in the case of propionic acid which showed as esteri fi cation of 3.4%, whereas acetic acid showed
an esteri fi cation of 19.3%. Highest yield of 99% was observed with stearic acid. The low molecular weight stronger acids (lesser K
a than long-chain
fatty acids) undergo more dissociation resulting in increased hydrogen ion concentration at the micro-aqueous interphase. Lower carbon chain length organic acids also are more soluble at the inter-phase than the long-chain ones, thereby enhancing the dissociation, leading to lower observed yields (Kiran and Divakar 2001 ) .
4.2.6 Reusability of Porcine Pancreas Lipase
The ester yield decreased with each cycle from 75.1 to 14.6% after 5th recycle (Table 4.6 and Fig. 4.5 ). Due to strenuous conditions like heat and incubation in nonpolar solvents for longer periods,
46 4 Enzymatic Esterifi cation of Compounds Possessing Multifunctional Hydroxyl and Carboxyl Groups
catalytic activity of the enzyme was affected due to probable structural changes. The initial rates of the reactions also showed a gradual decrease which was in correspondence with decrease in esteri fi cation.
4.2.7 Food Chemical Codex Speci fi cations for Enzymatically Synthesised 2-O-Acyl Esters of Lactic Acid
Enzymatically synthesised 2-O-acyl esters of lactic acid in comparison with the commercially avail-able sodium-2-O-stearoyl lactylate (from Enzyme India Ltd. Chennai) showed good correspondence
Table 4.5 Effect of organic acid carbon chain length 2-O-acyl ester of lactic acid preparation using PPL a
Acid employed
Maximum esteri fi cation (%)
Initial rate (mmol h −1 )
Ester yield (mmol)
Acetic C 2 19.2 0.06 4.8
Propionic C 3 3.8 0.01 0.9
Butyric C 4 15.1 0.08 3.8
Isobutyric C 4 20.4 0.13 5.1
Valeric C 5 24.5 0.11 3.4
Isovaleric C 5 14.1 0.06 4.4
Octanoic C 8 29.6 0.08 7.7
Decanoic C 10
48.4 0.11 9.4 Lauric C
12 63.6 0.13 15.9
Palmitic C 16
74.5 0.21 18.7 Stearic C
18 90.4 0.32 24.8
AU mmol −1 – E/S ratio is with respect to hydrolytic activity units; solvent – chloroform; incubation period – 96 h a Reaction conditions: lactic acid and organic acids – 0.025 mole; E/S ratio – 22.6
Table 4.6 Reusability of PPL for the bench-scale synthesis of 2-O-stearoyl lactic acid a
Recycle NO b
Maximum esteri fi cation percentage
Initial rate (mmol h −1 )
1 75.1 0.28 2 40.5 0.17 3 25.6 0.09 4 28.4 0.05 5 14.6 0.05 6 14.9 0.04
a Lactic acid and stearic acid concentrations : 0.025 moles E/S ratio: 22.6 AU mmol −1 b Incubation period: 72 h, solvent: chloroform Activity in hydrolytic units
0
0.07
0.14
0.21
0.28
0.35
0
20
40
60
80
100
C2 C3 C4 C4' C5 C5' C8 C10 C12 C16 C18
Initial rate (mm
ol/h)%
Est
erific
atio
n
Carbon chain length of organic acids
% EsterificationInitial rate
Fig. 4.4 Effect of acid carbon chain length on extent of esteri fi cation of lactic acid and initial rate of the reactions (bench-scale). Reactions conditions: lactic and organic
acids: 0.025 mol; solvent: chloroform; E/S ratio: PPL, 22.6 AU mmol −1 (activity units were with respect to hydrolytic activity units); incubation period: 96 h
474.3 Tolyl Esters
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0
20
40
60
80
100
1 2 3 4 5 6
Initial rate (mm
ol/h)%
Est
erific
atio
n
Recycle Number
% Esterification
Initial rate
Fig. 4.5 Effect of enzyme reusability on extent of esteri fi cation of lactic acid with stearic acid (bench-scale). Reaction conditions: lactic and stearic acids: 0.025 mol;
solvent: chloroform; E/S ratio: 22.6 AU mmol −1 (activity units were with respect to hydrolytic activity units); incu-bation period: 72 h
to food chemical codex ( FCC speci fi cations ) in terms of acid value, ester value, sodium content and lactic acid contents (Table 4.7 ).
Enzymatically prepared stearoyl lactic acid shows an acid value of 69.4, whereas com-mercial preparation shows 110.4. Those of palmitoyl and lauroyl lactic acids showed acid values of 79.5 and 91.5, respectively. In case of ester values also, all the three enzymati-cally prepared esters showed better values than the commercial sample. Free lactic acid and sodium contents of enzymatically pre-pared ones were also within the speci fi ed range. Unlike chemically prepared sample, the enzymatic ones are neat without any side products. These characteristics showed that the enzymatically prepared ester samples are better than the commercial preparation and will be more suitable for applications in food formulations.
4.3 Tolyl Esters
Phenolic esters of organic acids especially those of cresols are good fl avour compounds as they possess a combination of sweet, fl oral and fruity odours which are very much desired in food and cosmetic industry (Burdock 1994 ) . Enzymatic esteri fi cation of fatty acids with primary alcoholic groups of functionalised phenols and interesteri fi cation reactions of peracetylated polyphenolic compounds have been reported (Habulin and Krmelj 1996 ) . However, before this report, direct synthesis of phenolic esters involving phenolic OH groups mediated by lipases was practically nil (Scheme 4.2 ).
Porcine pancreas lipase and Rhizomucor miehei lipase showed very little esteri fi cation in the presence of solvents under shake- fl ask conditions, for the preparation of esters of m- and
48 4 Enzymatic Esterifi cation of Compounds Possessing Multifunctional Hydroxyl and Carboxyl Groups
p-cresols using organic acids of carbon chain lengths C
2 –C
18 (Table 4.8 ). Benchscale condi-
tions employed a specially devised experimen-tal set-up (Kiran et al. 1998 ; Suresh-Babu et al. 2002 ) which maintained azeotropic conditions where the solvent system CHCl
3 to petroleum
ether (30:70) not only maintained a temperature at 56–60°C (when the reaction mixture was under refl ux) but also aided effective removal
of water of reaction from the reaction mixture, thereby facilitating further esterifi cation. The reaction gave better yields of ester when 0.05–0.2 mL of 0.1 M phosphate buffer of pH 7.0 was present in the reaction mixture. In the absence of the buffer, ester yields were very poor.
Effect of increasing amounts of RML addition showed enhanced ester yields from 51.6% for
Scheme 4.2 Lipase-catalysed esteri fi cation of cresols
Table 4.7 Comparative data on commercially available and enzymatically prepared 2-O-acyl esters of lactic acids
Sample Acid value Ester value
Sodium content
Lactic acid Content Arsenic
Heavy metals
(%w/w) (%w/w) (ppm) (ppm)
Food chemical codex speci fi cations 60–80 150–190 3.5–5.0 31–34 <3 <10 Commercial 2-O-stearoyl lactate 110.4 149.9 ND 24.4 ND ND Enzymatic samples
2-O-stearoyl lactic acid Sodium 2-O-stearoyl lactic acid 69.6 169.8 3.2 22.2 ND ND 2-O-stearoyl lactic acid 144.7 203.6 0 22.5 ND ND 2-O-stearoyl lactic acid(free lactic acid removed)
109.1 159.2 0 24.1 ND ND
2-O-palmitoyl lactic acid Sodium 2-O-palmitoyl lactic acid 79.5 172.9 3.3 21.8 ND ND 2-O-palmitoyl lactic acid 162.3 120.3 0 32.3 ND ND 2-O-stearoyl lactic acid(free lactic acid removed)
147.2 148.3 0 24.2 ND ND
2-O-lauroyl lactic acid Sodium 2-O-lauroyl lactic acid 91.3 153.3 3.5 24.8 ND ND 2-O-lauroyl lactic acid 169.8 183.7 0 28.2 ND ND 2-O-lauroyl lactic acid(free lactic acid removed)
152.6 141.2 0 23.9 ND ND
ND not determined
494.3 Tolyl Esters
450 mg to 74% for 1 g of RML. A maximum esteri fi cation of 52.9% was observed with 100 mg RML at pH 7.0 at an optimum buffer volume of 0.1 mL. Under identical reaction conditions, m- and p-cresol gave conversion yields above 30% for organic acids up to butyric acid. With increase in organic acid carbon chain length, the conver-sion yields decreased.
It was generally observed that larger amounts of enzymes are required for better conversions when p-cresol was employed. A detailed analysis of m- and p-cresyl ester synthesis using lipases has shown that cresols (m- and p-) generally act as inhibitors for these enzymes. This may be true for other phenols also.
4.3.1 Application of Central Composite Rotatable Design to Lipase-Catalysed Synthesis of m-Cresyl Acetate
Design : Central composite rotatable design (CCRD), 32 experiments, 5 variables at 5 levels (Manohar and Divakar 2002 )
Variables : m -Cresol concentrations (0.005–0.025 mole), enzyme/substrate ratios (0.18–1.22 AU mmole −1 ), incubation periods (6–54 h), pH (4–8) and buffer volumes (0–0.2 mL)
Equation : A second-order polynomial equa-tion was developed.
t e a
b p t t e e
a a b b p p
t e t a t b t p
e a e b e p a b
a p
1.5692 0.59067 0.0532 0.096
0.2917 0.2 0.142 0.202
0.289 0.2216 0.05583
0.115 0.169 0.026 0.0134
0.1092 0.246 0.283 0.036
0.0089 0.0
= + + −+ + + +
+ + +
+ − − +
− + − +
+ +
Y X X X
X X X X X X
X X X X X X
X X X X X X X X
X X X X X X X X
X X b p282X X
where
X a = m -cresol concentration (moles)
X e = enzyme/substrate ratio (AU mmoles −1 )
X t = incubation period (h)
X b = buffer volume (mL)
X p = buffer pH
The predictability of the model is at 74% con fi dence level. Three-dimensional plots and con-tour plots were obtained using the above equation.
Lipase-catalysed reaction between m -cresol and acetic acid did not proceed in the absence of the buffer. It did not also occur in the absence of the enzyme. The reaction required presence of buffer for better conversions, and a minimum amount of 0.1 mL buffer was found necessary for this reaction. Increase in buffer volumes at all the pH values studied (in the range pH 4.0–8.0) showed decrease in ester yield up to a buffer vol-ume of 0.1 mL. Above 0.1 mL, increase in buffer volume increased ester yield up to 0.2 mL (Fig. 4.6 ). Buffer pH values around 4.0 and below appear to favour better esteri fi cation than those at pH values in the range 4.0–8.0. The methodology projected conditions for higher yields up to 7.2 mmoles. Validation experiments carried out under these predicted conditions gave a highest
Table 4.8 Porcine pancreas lipase-catalysed esteri fi cation of m- and p-cresols
Shake- fl ask a Bench-scale b
Esters % Yield % Yield
m-cresyl acetate 18.6 (51) 44.1 (95) m-cresyl propionate 31.4 (70) 19.7 (43) m-cresyl butyrate 38.4 (44) 52.6 (72) m-cresyl isobutyrate 18.9 (69) 10.5 (64) m-cresyl valerate 20.8 (45) 1.3 (72) m-cresyl isovalerate 32.9 (45) 8.6 (72) m-cresyl octanoate 12.8 (30) 10.0 (72) m-cresyl decanoate 0.0 (72) 3.1 (72) m-cresyl laurate 10.1 (30) 64.6 (46) m-cresyl palmitate 0.0 (72) 7.4 (72) m-cresyl stearate 15.5 (44) 0.0 (72) p-cresyl acetate 64.9 (66) 67.9 (88) p-cresyl propionate 31.4 (69) 33.7 (74) p-cresyl butyrate 8.13 (21) 12.2 (46) p-cresyl isobutyrate 15.8 (48) 8.1 (44) p-cresyl valerate 11.3 (70) 4.5 (63) p-cresyl isovalerate 14.7 (21) 10.4 (23) p-cresyl octanoate 22.1 (53) 0.0 (89) p-cresyl decanoate 28.7 (28) 8.3 (44) p-cresyl laurate 17.2 (40) 12.5 (40) p-cresyl palmitate 23.8 (50) 8.6 (72) p-cresyl stearate 7.9 (20) 0.0 (70)
a Equimolar concentrations of cresols and organic acids (0.0023 mole) with 60 mg PPL b Equimolar concentrations of cresols and organic acids (0.01 mole) with 400 mg PPL and 0.1 mL 0.1 M phos-phate buffer pH 7.0 Figures in parentheses indicate period of incubation in hours to achieve maximum yield
50 4 Enzymatic Esterifi cation of Compounds Possessing Multifunctional Hydroxyl and Carboxyl Groups
00.050.1
0.150.2
4567
0
7
Est
er y
ield
(m
mo
le)
Buffer (ml) pH
Fig. 4.6 Three-dimensional plot showing the effect of pH and buffer volume on esteri fi cation for an E/S ratio of 0.7 AU mmol −1 , 0.25 mole m-cresol concentration and 72 h incubation period
experimental yield of 4.3 mmoles. The CCRD treatment clearly showed inhibitory nature of m -cresol in the esteri fi cation process.
4.4 Application of Plackett–Burman Design for Lipase-Catalysed Esteri fi cation of Anthranilic Acid
In order to determine the effectiveness of esteri fi cation of anthranilic acid using alcohols of carbon chain length C
1 –C
18 , Plackett–Burman
(PB) design allowed the selection of better alcohols for esteri fi cation of anthranilic acid (Scheme 4.3 ).
Of the alcohols employed, methanol, decanol, cetyl alcohol and stearoyl alcohols showed 99.9% signi fi cance. Esteri fi cation of anthranilic acid with methanol showed the highest yield of 45.6% (Suresh-Babu and Divakar 2001 ) .
The experimental and predicted yields with experimental design are shown in Table 4.9 . Generally it was observed that alcohols with 99.9% signi fi cance levels showed better yields, with highest yield from methanol followed by stearyl alcohol and decanol.
The reaction between anthranilic acid and meth-anol showed that an optimum enzyme amount of 250 mg of PPL was required for the reaction. Higher amounts of enzyme did not increase the yield. Experiments were conducted at different concentra-tions of methanol (0.01, 0.02, 0.05, 0.1 and 0.2 mole). At lower concentrations of methanol, esteri fi cation was slow. This may be due to evapora-tion of methanol. But at higher methanol concentra-tions (0.05 and 0.1 mole), conversion yields were good. A maximum yield of 45.6% was obtained when 0.05 mole of methanol was employed.
4.5 Kinetic Study of Porcine Pancreas Lipase Inhibition by p -Cresol ( p -Cresyl acetate) and Lactic Acid (2-O-Stearoyl Lactate)
The enzymatic reactions between p-cresol and ace-tic acid and lactic acid and stearic acid required larger concentrations of enzymes for lesser concen-trations of substrates indicating the inhibitory nature of p-cresol and lactic acid towards porcine pancreas lipase (Kiran and Divakar 2002 ) . The results from these kinetics investigations are described below.
514.5 Kinetic Study of Porcine Pancreas Lipase Inhibition by p-Cresol (p-Cresyl acetate) and Lactic Acid…
Initial velocities of the reaction measured and double reciprocal plots constructed by plotting 1/v versus 1/[acetic acid] or 1/[stearic acid] showed that the lines obtained at different p-cresol (lactic acid) concentrations intersected on the Y -axis, giving the maximum velocity ( V
max ). The various kinetic
parameters determined, namely, K M(p-cresol)
(K M(PC)
), K
M(acetic acid) (K
M(AA) ), K
M(lactic acid) (K
M(LA) ), K
M(Stearic acid)
(K M(SA)
), K i(p-cresol)
(K i(PC)
) and K i(lactic acid)
(K i(LA)
), from experimental data are shown in Table 4.10 .
Scheme 4.4 indicates the mechanism of inhibition of lipase activity by p-cresol and lactic acid, respec-tively, due to the formation of dead-end enzyme–p-cresol or enzyme–lactic acid complexes. In this process, the acyl–enzyme intermediate formed between acetic acid and lipase (stearic acid–lipase) resulted in the products by attacking the nucleophile (p-cresol or lactic acid as the case may be). During this process, the formation of two products, namely, release of water molecule due to acyl–enzyme com-plex formation and release of product due to lipase–ester dissociation, resulted from two substrates.
The rate equation for the mechanism described is given as
{ }max
M[Y] i M[X]K [X] 1 [X] / K K [Y] [X] [Y]=
× × + + × + ×V
v
where [X] = p-cresol or lactic acid concentration [Y] = acetic acid or stearic acid concentration K
M[X] = Michaelis-Menten constant of p-cresol
or lactic acid K
M[Y] = Michaelis-Menten constant of acetic
acid or stearic acid K
i = inhibition constant of p-cresol or lactic acid
By suitably modifying the equation, theo-retical values for kinetic parameters were developed.
Table 4.10 lists the theoretical values which were in close agreement to the experimental ones. This indicates that Ping-Pong Bi-Bi mechanism is applicable in both cases and clearly illustrates the inhibitory nature of p-cresol and lactic acid in the respective reactions.
Scheme 4.3 Lipase-catalysed esteri fi cation of anthranilic acid
Table 4.9 Experimental design with experimental and predicted yields
Number of alcohol carbon atoms
C 1 C
2 C
3 C
4 C
5 C
6 C
8 C
10 C
12 C
16 C
18
Experimental yield (mmole)
Predicted yield (mmole)
1 −1 1 −1 −1 −1 1 1 1 −1 1 4.8 4.8 1 1 −1 1 −1 −1 −1 1 1 1 −1 4.4 4.7
−1 1 1 −1 1 −1 −1 −1 1 1 1 3.7 3.9 1 −1 1 1 −1 1 −1 −1 −1 1 1 3.8 3.8 1 1 −1 1 1 −1 1 −1 −1 −1 1 2.4 2.7 1 1 1 −1 1 1 −1 1 −1 −1 −1 3.1 3.4
−1 1 1 1 −1 1 1 −1 1 −1 −1 0.0 0.2 −1 −1 1 1 1 −1 1 1 −1 1 −1 3.9 3.3 −1 −1 −1 1 1 1 −1 1 1 −1 1 2.8 1.2
1 −1 −1 −1 1 1 1 −1 1 1 −1 2.7 3.0 −1 1 −1 −1 −1 1 1 1 −1 1 1 4.8 4.5 −1 −1 −1 −1 −1 −1 −1 −1 −1 −1 −1 1.1 1.4
52 4 Enzymatic Esterifi cation of Compounds Possessing Multifunctional Hydroxyl and Carboxyl Groups
4.6 Thermostability of Porcine Pancreas Lipase
In order to understand the behaviour of PPL under different temperatures for longer periods of incu-bations, which is essential for employment of lipase in esteri fi cation reactions which are time consuming and requires higher temperatures and nonpolar solvents, thermostability of porcine pan-creas lipase was studied through response surface analysis (Kiran et al. 2001b ) . Native enzyme showed loss in activity at 60°C which could be due to a conformation with a greater unfolding at 60°C
than at 40°C and 80°C (Fig. 4.7 ). Longer periods of incubation of PPL at especially 80°C did not affect the active conformation of PPL even after incubation for a period up to 10 days. However, presence of small amounts of buffer stabilised the enzyme at 60°C contrary to what was observed with the native enzyme. In the presence of 0.2 M lactic acid, there was a general loss in active con-formation and hence activity, at all temperatures and periods studied.
The equation obtained to study the thermal stability in terms of activity measurements of native PPL is as follows:
Scheme 4.4 Schematic representation of Ping-Pong Bi-Bi mechanism of esteri fi cation of p-cresol with acetic acid and stearic acid with lactic acid L – lipase; PC – p-cresol; AA – acetic acid; L–AA – lipase–acetic acid complex;
PCA – p-cresyl acetate; LA – lactic acid; SA – stearic acid; L–PC and L–LA – dead-end enzyme–inhibitor complex; L–SA – lipase–stearic acid complex; SLA – stearoyl lactic acid; L*- acyl enzyme; W – water (Segel 1993)
Table 4.10 Data on kinetic parameters
p-cresyl acetate Kinetic parameters
Stearoyl lactic acid
Kinetic parameters Experimental Theoretical Experimental Theoretical
V max
(mole h −1 ) 0.000143 0.0002 V max
(M h −1 ) 0.00154 0.00132 K
M (AA) (mole) 0.00311 0.0226 K
M (SA) (M) 0.0548 0.0647
K M (PC)
(mole) 0.000035 0.007 K M (LA)
(M) 0.0098 0.0286 K
i(PC) (mole) 0.00966 0.0355 K
i(LA) (M) 0.0951 0.2364
2 2
t p t p t pPercentage activity 408.17 12.73 0.219 0.108 0.005 0.001X X X X X X= − + + − −
where X
t = incubation temperature
X p = incubation period
The results from activity measurements were supported by ultraviolet (UV) spectroscopic data of the same in every respect indicating that
534.7 Scanning Electron Microscopy
variation in conformational changes is responsi-ble for loss in activity (Fig. 4.8 ).
The following equation was obtained from UV measurements:
4050
6070
80
0
100
48 96 144 192 240Incubation temperature
( °C)
% A
ctiv
ity
Incubation period (h)
Fig. 4.7 Three-dimensional plot showing the effect of incubation period and temperature on percentage activity. Reaction conditions: 10 mg PPL was incubated in 2 mL
MIBK at 40°C, 60°C and 80°C for 48 h, 144 h and 240 h, respectively. Percentage activity is with respect to esteri fi cation activity of native PPL
2 2290 t p t p t p1.154 0.038 0.003 0.299 0.001 0.032A X X X X X X= − − − − −
where X
t = incubation temperature
X p = incubation period
Table 4.11 shows the results of regression analysis with experimental and predicted percent-age activity and absorbance values at 290 nm.
4.7 Scanning Electron Microscopy
The state of three lipase preparations, two from Rhizomucor miehei and one from porcine pan-creas, employed in the esteri fi cation reactions of p -cresol and lactic acid in nonpolar solvents
at 50–60°C investigated by scanning electron microscopy (SEM) showed the compact nature of adsorption and distribution of lipase on matrix in case of Rhizomucor miehei and car-rier in case of porcine pancreas (Suresh-Babu et al. 2001 ) . All the three lipase preparations subjected to high temperatures and nonpolar solvents for a prolonged period of incubation of 72–120 h showed decrease in the ‘compact-ness’ when compared with unused lipase. Presence of buffer preserved the activity and compactness, and the absence of the same, in some cases, reduced the amount of enzyme per unit area on the support. Rhizomucor miehei
54 4 Enzymatic Esterifi cation of Compounds Possessing Multifunctional Hydroxyl and Carboxyl Groups
Table 4.11 Predicted and experimental yields in case of incubation of PPL in MIBK
Exp no. X
t (°C) X
p (h) Percentage activity Absorbance at 290 nm
Coded Actual Coded Actual Experimental Predicted Experimental Predicted
1 −1 40 −1 48 72.8 79.3 0.953 0.862 2 −1 40 0 144 90.5 84.2 0.851 0.894 3 −1 40 1 240 85.4 83.2 0.876 0.923 4 0 60 −1 48 50.5 40.1 0.974 1.156 5 0 60 0 144 41.6 44.9 1.243 1.154 6 0 60 1 240 32.8 39.8 1.244 1.151 7 1 80 −1 48 83.7 87.6 0.942 0.851 8 1 80 0 144 89.3 90.3 0.771 0.816 9 1 80 1 240 87. 83.1 0.734 0.780
Fig. 4.8 Three-dimensional plot showing the effect of incubation period and temperature on absorbance at 490 nm. Reaction conditions: 10 mg PPL was incubated
in 2 mL MIBK at 40°C, 60°C and 80°C for 48 h, 144 h and 240 h, respectively
554.8 Hydrogen Ions in Micro-aqueous Phase During Lipase-Catalysed Esterification in Nonaqueous Media
lipase samples subjected to reaction in the presence of 0.1 mL and 0.1 M buffer at differ-ent pH values (4.0–9.0) showed decrease in ‘compactness’ of the enzyme on the surface which correlated to increase in esteri fi cation activity of the same. Increase in volume of buf-fer (0.05–0.75 mL) in the reaction mixture at pH 7.0 showed a decrease in compactness and also reduction in activity. The studies indicated that a compromise between extent of adsorp-tion, distribution and activity could be achieved by varying the pH and volume of buffer in the reactions, for maximum conversions.
4.8 Hydrogen Ions in Micro-aqueous Phase During Lipase-Catalysed Esteri fi cation in Nonaqueous Media
Water plays a crucial role in both hydrolysis and esteri fi cation in lipase catalysis (Hahn-Hagerdal 1986 ) . While a critical amount of water is essen-tial for maintaining the active conformation of the enzyme, excess water facilitates hydrolysis (Halling 1989 ; Zaks and Klibanov 1988 ) . Though the former aspect has been extensively studied as water activity, information on the latter is very scanty. While exhaustive stripping of water from the enzyme resulted in lowering of reaction rate, presence of a critical amount of water favoured esteri fi cation due to enhancement of enzyme activity (Valivety et al. 1992 ) .
In a nonpolar solvent, excess water adds to the already existing hydration shell of the enzyme constituting a micro-aqueous enzyme–water–solvent phase (Fig. 4.9 ). Micro-aqueous phase, although being not a well-de fi ned thermody-namic structure, holds the key for problems asso-ciated with many equilibrium processes of the esteri fi cation reaction that can be only comple-mentary to the associated enzymatic factors like solvent polarity and desiccants which affect the micro-aqueous phase thereby the extent of esteri fi cation as well. Micro-aqueous phase pro-vides the appropriate solvent characteristics like dielectric constant and polarity that maintain the active conformation in a nonpolar solvent.
The kinetic aspects of lipase-catalysed esteri fi cation reactions have been thoroughly investigated (Chulalaksanaukul et al. 1992 ; Lee et al. 1995 ; Janssen et al. 1999 ) . However, this is not the case for non-enzymatic thermodynamic equilibrium processes associated with the same. Molecular surface area of the enzyme, volume/thickness of the micro-aqueous phase, total [H + ] in the micro-aqueous phase, polarity of the sol-vent, diffusion behaviour of the acid/alcohol/ester and partitioning of water/[H + ]/alcohol/acid between the organic solvent and the micro-aque-ous phase all play a crucial role in determining the extent of esteri fi cation for a given amount of the enzyme. Among these, the major equilibrium processes are dissolution and dissociation of the organic acid in the micro-aqueous phase and par-titioning of water/[H + ]/acid/alcohol/ester between the micro-aqueous phase and the organic solvent (Fig. 4.9 ). Dissociation of water soluble organic acids and their dissociation in the micro-aqueous phase lead to the formation of the conjugate acid, H
3 O + . In case of weak organic acids, H
3 O + is
stronger than the organic acid itself and hence, facilitates hydrolysis, thereby limiting the extent of esteri fi cation. Many features like the micro-aqueous phase are dif fi cult to measure. Attempts have also been made by a few researchers to measure [H + ] at the micro-aqueous phase (Brown et al. 1997 ) .
A theoretical model was developed based on mass transfer equations for arriving at the con-centration of [H+] in the micro-aqueous phase and other factors which regulate esteri fi cation (Kiran et al. 2002 ) . The reaction chosen was the esteri fi cation equilibria of 2-O-stearoyl lactic acid synthesis using immobilised lipase from Rhizomucor miehei and porcine pancreas.
Table 4.12 lists the extent of esteri fi cation observed for different concentrations of substrate and enzyme for both RML and PPL in ethylm-ethyl ketone. The water of reaction formed is less than that required to cause aggregation of the enzyme, but just suf fi cient to form a thin fi lm on the enzyme to regulate its activity.
A quantity termed hydrogen ion number (HIN) in the micro-aqueous phase was determined using the relation
Table 4.12 Theoretical HIN, pH and water for the enzymatic preparation of 2-O-steroyl lactic acid
Substrate Enzyme Ester Yield Water of reaction pH HIN Log HIN
M g L −1 M % mL L −1 (×10 −7 ) Lipozyme
0.03 3.8 0.008 26.7 0.144 2.86 1.20 17.08 0.03 3.8 0.010 33.3 0.179 2.88 1.43 17.16 0.03 3.8 0.012 40.0 0.216 2.90 1.63 17.51 0.03 2.5 0.008 26.7 0.144 2.86 1.20 17.08 0.06 5.0 0.014 23.3 0.252 2.70 3.03 17.48 0.06 7.5 0.018 30.0 0.324 2.72 3.73 17.57 0.06 10.0 0.008 13.3 0.144 2.67 1.84 17.26 0.09 7.5 0.020 22.2 0.360 2.61 5.34 17.73 0.09 11.3 0.020 22.2 0.360 2.61 5.34 17.73 0.09 15.0 0.022 24.4 0.396 2.62 5.79 17.76
PPL 0.03 0.8 0.006 20.0 0.108 2.84 0.94 16.97 0.03 1.2 0.004 13.3 0.072 2.82 0.65 16.81 0.06 0.6 0.006 10.0 0.108 2.66 1.41 17.15 0.06 1.5 0.010 16.7 0.180 2.68 2.26 17.35 0.06 2.4 0.018 30.0 0.324 2.72 3.73 17.57 0.09 2.3 0.002 2.5 0.040 2.60 6.74 16.83 0.09 3.6 0.021 23.0 0.369 2.61 5.51 17.74 0.09 3.6 0.018 20.0 0.320 2.60 4.88 17.69
Microaqueous phase
Active site
E+RCOOH ERCOOH
E+ROH EROH
E+RCOOR ERCOOR
R-COOH+R OH+RCOOR+H2O
H2O + H+ H3O+
R-COOH RCOO- + H+
Solvent
Fig. 4.9 Schematic representation of micro-aqueous phase around lipase
574.8 Hydrogen Ions in Micro-aqueous Phase During Lipase-Catalysed Esterification in Nonaqueous Media
Fig. 4.10 Theoretical plot log HIN versus percentage of esteri fi cation
23
THIN H 1.008 4.023 10 Xml /1,000+⎡ ⎤= × × × ×⎣ ⎦
where 1.008 = atomic weight of H + .
The total [H + ] in the water of reaction consti-tuting the micro-aqueous phase was arrived at (Atkins 1987 ) using the relation:
( ) ( )0.5
LA LA SA SATH K EC K EC 0.5+⎡ ⎤ = × + ×⎣ ⎦
6 4 1KSA 1.76 10 and KLA 1.37 10 M− − −= × = ×
From the partitioning coef fi cient values deter-mined for stearic and lactic acids in ethylmethyl ketone (0.058 and 1.46, respectively), the effec-tive concentration of the acid (EC
LA and EC
SA ) at
the micro-aqueous phase constituted by the water of reaction was determined.
Theoretical plots of log HIN versus % esteri fi cation from 0 to 100% were constructed for acid concentrations 0.01–1.0 M. Parenthesis-shaped curves were obtained (Fig. 4.10 ). Irrespective of the acid concentration, during the course of reaction, log HIN varied with extent of esteri fi cation in an explicable manner.
At the initial stage up to 10% esteri fi cation, dissociation of the more unreacted acid in the
lesser water of reaction contributed to the micro-aqueous phase registering higher log HIN. Further increase in esteri fi cation up to 50% consumed more free acid and registered a very slight net increase in log HIN, due to decrease in free acid concentration although water of reaction was larger. Beyond 55% esteri fi cation, decrease in free acid in larger volume of water of reaction registered marginal decrease in log HIN which became progressive as the extent of conversion reached 99%. Experimentally derived data were also fi tted on to the theoretical plots, and they cover a very narrow range of esteri fi cation (Fig. 4.10 ). The values outside this region could be obtained with higher enzyme concentrations. Higher yields are not possible due to hydrolysis of the ester formed by [H + ] build-up.
At any stage of esteri fi cation, an equilibrium concentration of [H + ], i.e. a net proton build-up, is always maintained at the micro-aqueous phase. Such a build-up could affect esteri fi cation by effecting hydrolysis. The question arises as what happens to such a large number of [H+] in the micro-aqueous phase? In real-time systems, log HIN values will be low and that this model clearly brings out the role of enzymes and buffers
58 4 Enzymatic Esterifi cation of Compounds Possessing Multifunctional Hydroxyl and Carboxyl Groups
employed to neutralise this excess [H + ] at the micro-aqueous phase. The depletion could occur by (1) ionisable groups from the amino acid resi-dues on the surface of the enzyme molecule like Asp, Glu, Tyr, Lys and Arg at different levels of ionisation capable of ‘consuming’ these ions and (2) partitioning of [H + ], stearic and lactic acids, ester and water between the micro-aqueous phase and the surrounding bulk organic solvent that reduces the effective load of all these constituents in the micro-aqueous phase. Thus, this study explained the processes at the micro-aqueous phase dictating the extent of esteri fi cation.
4.9 Acetylation of Protocatechuic Aldehyde
Selectivity in enzymatic acetylation of the two phe-nolic OH groups at positions 3 and 4 of protocate-chuic aldehyde (PA) using lipases from Rhizomucor miehei (RML and Chirazyme) and porcine pan-creas lipase (Scheme 4.5 ) in methyl isobutyl ketone/benzene (15:85) was investigated (Divakar 2003 ) . Chemical esteri fi cation using acetic acid and acetic anhydride did not produce any product as no cata-lyst was employed. Similarly under identical con-ditions, acetic acid did not react even in the presence of the three lipases (Table 4.13 ).
However, acetic anhydride in the presence of all the three lipases showed esteri fi cation (Table 4.13 ). Product formation of mono-acetoxy and diacetoxy products was monitored by the H-5 signal of the reactant and the esters. Yields of the order of 41.0–73.5% were obtained with good selectivity (Table 4.14 ) with the highest selectiv-ity of 97% for the PPL-catalysed reaction.
In order to enhance the selectivity and yield, enzymatic acetylation was also carried out by anchoring PA inside b -cyclodextrin cavity. b -Cyclodextrin ( b CD) is a cyclic oligosaccharide consisting of 7 glucose units joined in the form of a ring by virtue of it, possessing an hydrophobic cavity of 6–8 A in size capable of accommodating a plethora of organic molecules that fi t into this size stabilised by H bonding, hydrophobic and other interactions. Earlier structural studies car-ried out by us have shown that protocatechuic aldehyde could be anchored inside b -cyclodextrin ( b CD) cavity in a 1:1 stoichiometric proportion with a binding constant value of 6,700 ± 770 M − 1 in water and 5,100 ± 580 M − 1 in NaHCO
3 buffer
(pH 10.5) (Maheswaran and Divakar 1997 ) . b -Cyclodextrin was peracetylated to prevent
acetic anhydride being consumed by the OH groups of b CD. Reaction between PA and acetic anhydride in the presence of peracetylated b CD was carried out using the three lipases (Table 4.13 ) successfully. All the three lipases showed more than 30% yield. However, the acetylation with RML showed >99% selectivity by yield 3-ace-toxy derivative exclusively.
4.10 4-t-Butylcyclohexyl Acetate
4-t-Butylcyclohexanol exists as cis and trans iso-mers with different fl avour notes. The trans esters possess a woody note, whereas the cis esters exhibit a strong, intense, fl owery note. The esters are used in soaps, detergents, creams, lotions and perfumes and also as a solubility-reducing agent for surfactants. At room temperature, the trans / cis ratio of the alcohol is 3:1 (Karger et al. 1968 ) .
Scheme 4.5 Lipase-catalysed regioselective esteri fi cation of protocatechuic aldehyde
594.10 4-t-Butylcyclohexyl Acetate
Chemical synthesis of the esters of 4-t-butyl-cyclohexanol was carried out with organic acids from acetic to heptanoic acids (Karger et al. 1968 ) . The trans / cis ratio of acetate, propionate, n-butyrate, valerate, caproate, heptylate, isobu-tyrate and pivalate remained at approximately 3.5 in all the cases. Acetylation of 4-t-butylcyclo-hexanol anchored inside b CD was found to give a trans / cis ratio of 5.5 (Pattekhan and Divakar 2001, 2002 ) .
Enzymatic esteri fi cation of 4-t-butylcyclo-hexanol was carried out by using porcine pan-creas lipase in order to obtain better yield and selectivity (Scheme 4.6 ). The yields were low in nonpolar solvents (Manohar and Divakar 2004 ) .
However, when it was anchored inside b -cyclo-dextrin ( b CD) cavity, the esteri fi cation yields were higher (Table 4.14 ).
The low-yield 4-t-butylcyclohexyl acetate in nonpolar solvents was attributed to the formation of aggregate of 4-t-butylcyclohexanol with the buried alcohol OH groups, thereby preventing esteri fi cation. Hence, b CD was employed for breaking down the aggregates, by anchoring 4-t-butylcyclohexanol inside b CD cavity. Peracetylated b CD was used and in the presence of the enzyme to promote interesteri fi cation between the acetate groups of b CD and 4-t-butylcyclohexanol even when acetic anhydride was employed. With increase in incuba-tion period, the esteri fi cation was found to increase
Table 4.13 Lipase-catalysed esteri fi cation data on protocatechuic aldehyde a
Reactions Ester yield
(Moles) Titration b NMR b 3-acetoxy 3,4-diacetoxy
Selectivity Derivative Derivative
PA + acetic anhydride No rean No rean – – – (0.009 + 0.0044) PA + acetic acid + PPL 63.1 No rean (0.01+0.01) PA + acetic acid +chirazyme 54.9 No rean (0.01+0.01) PA + acetic acid + RML 60.1 No rean (0.01+0.01) PA + acetic anh.+PPL 54.8 41.0 39.8 1.2 97.0 (0.01+0.005) PA + acetic anh.+ chirazyme 51.2 54.5 52.2 4.3 92.4 (0.01+0.005) PA + acetic anh + RML 52.9 73.5 64.2 7.3 90.0 (0.01+0.005)
PA + acetic anh+ b CDA+PPL 35.2 60.5 58.2 2.3 94.2
(0.01+0.005+0.001)
PA + acetic anh+ b CDA+Chirazyme 31.8 50.0 44.2 3.8 92.4
(0.01+0.005+0.001)
PA + acetic anh+ b CDA+_RML 35.3 48.1 44.4 3.7 92.3
(0.01 + 0.005 + 0.001)
PA + acetic anh + b CDA + PPL 54.4 61.5 57.7 3.8 93.8
(0.01+0.01+0.001) PA + acetic anh + b CDA + Chirazyme
51.7 79.4 64.7 14.7 81.5
(0.01 + 0.01 + 0.001)
PA + acetic anh + b CDA + RML 55.1 17.6 17.6 0 >99
(0.01 + 0.01 + 0.001)
a PA – protocatechuic aldehyde; + b CDA − b CD acetate b Monitoring by titration and 1 HNMR
60 4 Enzymatic Esterifi cation of Compounds Possessing Multifunctional Hydroxyl and Carboxyl Groups
steadily from 18.5% after 12 h to a maximum of 65.9% after 120 h. From the initial
slope value, the rate of acetylation was deter-mined to be 4.36 m mole h −1 . Further, an increase in b CD–acetate concentration from 0 to 0.005 mole (0.5 M equivalent to 4-t-butylcyclo-hexanol) resulted in increase in esteri fi cation up to a maximum of 55.9%, and it decreased there-after up to 0.001 mole (1 M equivalent of 4-t-butylcyclohexanol). Esteri fi cation also increased with increase in PPL concentration reaching a maximum of 55.7% at 450 mg and decreased thereafter up to 1 g (Table 4.14 ).
Gas chromatographic analyses of the reaction mixtures showed that trans / cis ratios vary from 2.1 to 4.4 which were higher than that of the alcohol 2.47. However, no general trend was observed between the reaction conditions employed and trans / cis ratios. Inclusion of 4-t-butylcyclohexanol inside the peracetylated b CD cavity preferentially facilitates the esteri fi cation of the trans isomer of the alcohol as the cis isomer is sterically hindered from reacting with the incoming acyl group due to the presence of bulky acetyl groups on per-acetylated b CD.
Table 4.14 Conversion yield and trans / cis ratios of 4-t-butylcyclohexyl acetate
Acetic anhyd. b CD acetate PPL Time Conversion yield Trans / cis
mole mole mg hr % ratios
0.0016 0.00025 275 48 18.8 2 0.0039 0.00025 275 48 43.8 2.95 0.0016 0.00075 275 48 29.0 2.6 0.0039 0.00075 275 48 34.2 2.4 0.0016 0.00025 625 48 18.1 4.34 0.0039 0.00025 625 48 39.4 2.1 0.0016 0.00075 625 48 7.6 2.83 0.0039 0.00075 625 48 66.6 2.29 0.0016 0.00025 275 96 47.1 2.37 0.0039 0.00025 275 96 56.3 2.07 0.0016 0.00075 275 96 44.9 2.55 0.0039 0.00075 275 96 55.8 2.35 0.0016 0.00025 625 96 39.5 2.8 0.0039 0.00025 625 96 73.4 2.71 0.0016 0.00075 625 96 21.2 2.7 0.0039 0.00075 625 96 57.1 2.9 0.0005 0.0005 450 72 10.4 4.01 0.005 0.0005 450 72 86.6 2.38 0.0028 0 450 72 12.1 2.1 0.0028 0.001 450 72 36.5 3.5 0.0028 0.0005 100 72 41.5 2.9 0.0028 0.0005 800 72 45.7 2.61 0.0028 0.0005 450 24 25.7 3.02 0.0028 0.0005 450 120 68.6 2.51 0.0028 0.0005 450 72 57.1 2.45 0.0028 0.0005 450 72 47.3 2.02 0.0028 0.0005 450 72 55.0 2.35 0.0028 0.0005 450 72 58.8 2.26 0.0028 0.0005 450 72 59.9 2.35 0.0028 0.0005 450 72 57.2 2.37
614.11 Esterification of b-Cyclodextrin
4.11 Esteri fi cation of b -Cyclodextrin
b -Cyclodextrin ( b CD) is soluble in water only to the extent of 1.8 g L −1 in water, and its solubility in organic solvents other than DMSO, DMF and pyridine is negligible or low. Poor solubility of cyclodextrin in many solvents can be reduced by derivatisation. Hence, the hydroxyl groups of b CD (7 hydroxyl groups from 2-OH, 7 hydroxyl from 3-OH and 7 hydroxyl groups from 6-CH
2 OH) were esteri fi ed (Pattekhan and
Divakar 2002 ) using lipases (Scheme 4.7 ). Among the organic acids employed – acetic,
propionic, butyric, isobutyric, valeric, isovaleric, lauric, octanoic, palmitic and stearic acids – only acetic, propionic and isobutyric acids were found to undergo esteri fi cation with 58.8, 25.8 and 17.4% yields, respectively (Table 4.15 ). Derivatised b CD like DM b CD (heptakis-2,6-di-O-methyl b CD) and HP b CD (hydroxypropyl
b CD) with acetic acid gave 32.8 and 55.8% esters, respectively (Table 4.15 ).
All these esters were soluble in methanol. b CD was able to react with 4.78 molecules, DM b CD with 2.45 molecules and HP b CD with 2 molecules of acetic acid. b CD reacted with 0.38 molecules of propionic acid and 2.43 molecules of isobutyric acid.
Out 7 primary hydroxyl groups and 14 sec-ondary hydroxyl groups of b CD from 2-OH and 3-OH, very few hydroxyl groups were converted to the esters. While primary hydroxyl groups can react with ease, the secondary hydroxyl groups at 2-OH reacted with dif fi culty, and 3-OH groups may not react at all being buried inside the cavity. Very low substitution of ace-tic, propionic and isobutyric groups is due to steric hindrance of the incoming acyl groups by the b CD cavity and the disposition of free hydroxyl groups within it.
Scheme 4.6 Lipase-catalysed esteri fi cation of 4-t-butylcyclohexanol
62 4 Enzymatic Esterifi cation of Compounds Possessing Multifunctional Hydroxyl and Carboxyl Groups
Table 4.15 Esteri fi cation of b CD and its derivatives catalysed by Rhizomucor miehei lipase a
System Ratio Percentage esteri fi cation by titration
Percentage esteri fi cation by 1 H NMR
Degree of substitution by 1 H NMR b
b CD–acetic acid 0.1:5 58.8 48.4 1:6.78
b CD–propionic acid 0.1:1 25.8 2.7 1:0.38
b CD–isobutyric acid 0.1:1 17.4 17.4 1:2.43
DM b CD–acetic acid 0.1:5 32.8 17.5 1:2.45
HP b CD–acetic acid 0.1:5 55.8 14.3 1:2
a Degree of substitution – ratio of peak area for CH 3 protons of acid to the number of H-1 protons of glucose units of
b CD and its derivatives b Percentage esteri fi cation by 1 HNMR – number of hydroxyl groups of b CD esteri fi ed out of 14 per molecule of b CD or its derivatives. Error in 1 HNMR measurements ±5%
Scheme 4.7 Three-dimensional structure of b -cyclodextrin (Roquett 1991 )
References
Atkins PW (1987) Physical chemistry, 2nd edn. Oxford ELBS, Oxford Press, Oxford, p 280
Brown JR, Guther MLS, Field RA, Ferguson MAJ (1997) Hydrophobic mannosides act as acceptors for trypanosome a -mannosyltransferases. Glycobiology 7:549–558
Burdock GA (1994) In Fenaroli’s handbook of fl avor ingredients, vol II, 3rd edn. CRC Press, Boca Raton
Chulalaksanaukul W, Condort JS, Combes D (1992) Kinetics of geranyl acetate synthesis by lipase cata-lyzed transesteri fi cation in n -hexane. Enzyme Microb Technol 14:293–298
Divakar S (2003) Lipase catalysed regioselective esteri fi cation of protocatechuic aldehyde. Indian J Chem Sect B 42B:1119–1122
Food Chemical Codex speci fi cations Habulin M, Krmelj V (1996) Synthesis of oleic acid esters
catalyzed by immobilized lipase. J Agric Food Chem 44(1):338–342
63References
Hahn-Hagerdal B (1986) Water activity a possible exter-nal regulator in biotechnical processes. Enzyme Microb Technol 8:322–327
Halling PJ (1989) Organic liquids and biocatalysts theory and practice. Trends Biotechnol 7:50–52
Janssen AEM, Sjursnes BJ, Vakurov AV, Halling PJ (1999) Kinetics of lipase catalyzed esteri fi cation in organic media correct model and solvent effects on parameters. Enzyme Microb Technol 24:463–470
Karger BL, Stern RL, Zannucci JF (1968) Anal Chem 40(4):727
Kiran KR, Divakar S (2001) Lipase catalysed esteri fi cation of organic acids with lactic acid. J Biotechnol 87:109–121
Kiran KR, Divakar S (2002) Enzyme inhibition by p -cresol and lactic acid in lipase mediated syntheses of p -cresyl acetate and stearoyl lactic acid A kinetic study. World J Microbiol Biotechnol 18:707–712
Kiran KR, Karanth NG, Divakar S (1998) An improved enzymatic process for the preparation of fatty acid hydroxyacid ester. Indian Patent, 1978/DEL/98 187313
Kiran KR, Karanth NG, Divakar S (1999) Preparation of steroyl lactic acid catalysed by immobilized lipases from Mucor miehei and porcine pancreas optimization using response surface methodology. Appl Microbiol Technol 52:579–584
Kiran KR, Manohar B, Karanth NG, Divakar S (2000) Response Surface Methodological study of esteri fi cation of lactic acid with palmitic acid cataly-sed by immobilised lipases from Mucor miehei and porcine pancreas. Z Lebm Unt Fors 211:130–135
Kiran KR, Manohar B, Divakar S (2001a) A central com-posite rotatable design analysis of lipase catalysed synthesis of lauroyl lactic acid at bench-scale level. Enzyme Microb Technol 29:122–128
Kiran KR, Suresh-Babu CV, Divakar S (2001b) Thermostability of porcine pancreas lipase in non-aqueous media. Process Biochem 36:885–892
Kiran KR, Karanth NG, Divakar S (2002) Hydrogen ion con-centration at the microaqueous phase in lipase catalysed
esteri fi cation in non-aqueous organic media – steroyllactic acid. Ind J Biochem Biophys 39:101–105
Lee Y, Howard LR, Villalon B (1995) Flavonoids and antioxidant activity of fresh pepper ( C . annum ) culti-vars. J Food Sci 60:473–476
Maheswaran MM, Divakar S (1997) Structural studies on inclusion compounds of b -cyclodextrin with some substituted phenols. J Incln Phenomenon 27:113–126
Manohar B, Divakar S (2002) Application of central composite rotatable design to lipase catalyzed synthe-ses of m -cresyl acetate. World J Microbiol Biotechnol 18:745–751
Manohar B, Divakar S (2004) Porcine pancreas lipase acetylation of beta-cyclodextrin anchored 4-t-butylcy-clohexanol. Indian J Chem Sect B 43B:2661–2665
Pattekhan HH, Divakar S (2001) Regioselectivity in the preparation of 2-hydroxy-4-ethoxybenzaldehyde from resorcinol in presence of b - cyclodextrin and its derivatives. J Mol Catal A Chem 169(2001):185–191
Pattekhan HH, Divakar S (2002) Regioselective acetyla-tion of 4-t-butylcyclohexanol in the presence of b -cyclodextrin and its derivatives. J Mol Catal A Chem 184:79–83
Roquett Catalog (1991) Kleptose- b -Cyclodextrin www.roquette-food.com
Suresh-Babu CV, Divakar S (2001) Selection of alcohols through Plackett-Burman design in lipase catalyzed syn-theses of anthranilic acid. J Am Oil Chem Soc 78:49–52
Suresh-Babu CV, Kiran KR, Divakar S (2001) Scanning electron microscopic studies of lipase catalysed esteri fi cation catalysis for the synthesis of stearoyl lac-tate and p-cresyl laurate. World J Microbiol Biotechnol 17:659–665
Suresh-Babu CV, Karanth NG, Divakar S (2002) Lipase catalysed esteri fi cation of cresols. Ind J Chem Sect B 41B:1068–1071
Valivety RH, Halling PJ, Macrae AR (1992) Rhizomucor miehei lipase remains highly active at water activity below 0.001. FEBS Lett 301:258–260
Zaks A, Klibanov AM (1988) Enzyme catalysis in mono-phasic organic solvents. J Biol Chem 263:3194–3201
65S. Divakar, Enzymatic Transformation, DOI 10.1007/978-81-322-0873-0_5, © Springer India 2013
5.1 Introduction
The total world annual demand for synthetic poly-mers amounts to 1.3 million t. Due to their envi-ronmental hazard, the emphasis is shifting towards the production of biodegradable polymers which are eco-friendly in nature. Polyhydroxyalkanoates are one such class of biodegradable polymers. Hydroxy acids like glycolic acid, beta-hydroxy-propionic acid, lactic acid, gamma-hydroxybu-tyric acid, delta-hydroxyvaleric acid and their lactones which are bifunctional in nature can be polymerised to give biodegradable polymers. There are many chemical methods available for the preparation of polylactic acid (Seiji and Masahiro 1992 ; Rika et al. 1996 ; Voss and Spielan 1958 ) . Enzymatic production (in vitro) also leads to production of these polymers through milder
reaction conditions, good yield, clean product and pollution-free process conditions (Gross et al. 2001 ; Kumar and Gross 2000 ) .
Among the various polyhydroxyalkanoates studied, both low and high molecular weight polycaprolactone esters possess several desirable physicochemical properties. Ring opening polymerisation of lactones and macrolides of various sizes using lipases has been reported. Pseudomonas fl uorescens lipase-catalysed polyme-risation of a -methyl- b -propiolactone resulted in a polymer with a number average molecular weight of 600–2,900 (Svirkin et al. 1996 ) . Copolymerisation of b -propiolactone and e -caprolactone using Pseudomonas fl uorescens lipase resulted in a random copolymer with a number average molecular weight of 520 (Namekawa et al. 1996 ) . In these cases, the reaction rates and molecular
5
Abstract
Potentiality of lipases to effect esteri fi cation has been explored to prepare polymers of monomer molecules which possess hydroxyl and carboxyl functions like lactic acid, e -caprolactone, p-hydroxybenzoic acid, p-amin-obenzoic acid, adipic acid and 1,6-hexanediol. Ring opening polymerisa-tion of e -caprolactone gave a polycaprolactone polymer of molecular weight 10,000, which was the best compared to the low molecular weight polymers of molecular weight 400–5,000 obtained with the other above-mentioned monomers. Relative advantages and disadvantages of the use of lipases in such polymerisation reactions along with the fi lm-forming properties of polylactic acid and polycaprolactone and its blends are pre-sented in this chapter.
Enzymatic Polymerisation
66 5 Enzymatic Polymerisation
weight build-up were found to be slow. In order to enhance the reaction rates and reduce the incuba-tion period, initiators like methanol and butanol were used. Use of methanol as initiator resulted in polycaprolactone with a degree of polymerisation of 35 at 40°C within 4 days (Knani et al. 1993 ) . In the presence of butanol as an initiator, PPL-catalysed polymerisation of e -caprolactone at 65°C in n-heptane resulted in an esteri fi cation of 33–100% with a number average molecular weight in the range 313–1,600. So far, the highest molecular weight achieved was 7,600 in case of polycaprolactone by PPL when butanol was used as initiator. Zhang et al. ( 1996 ) have used bacterial protease from Bacillus subtilis for the ring opening polymerisation of e -caprolactone which resulted in a polymer with a number average molecular weight of 810. Many reports are also available on lipase-catalysed polymerisation of macrolides, namely, octanolide (Kobayashi et al. 1998 ) , unde-canolide dodecanolide, pentadecanolide (Uyama and Kobayashi 1996 ; Bisht et al. 1998 ) and hexa-decanolide (Namekawa et al. 1996 ) . Generally, it was observed that lipases from Candida and Pseudomonas species showed higher activity and better molecular weight build-up. The highest molecular weight of 25,000 was achieved with undecanolide (Uyama and Kobayashi 1996 ) .
5.2 Polylactic Acid
Polylactic acid, a biodegradable polyhydroxyal-kanoate (PHA), is one of promising replacements to synthetic polymers. The advantages of poly-lactic acid are its high strength, thermoplasticity, fabricability, biodegradability and bioenviron-mental compatibility. The demand for polylactic acid amounts to 200 million pounds per year (Lipinsky and Sinclair 1986 ) .
Both low and high molecular weight polylactic acids are known. High molecular weight polylactic acid possesses very good mechanical properties. Low molecular weight polylactic acids, when blended with other polymers, also exhibit char-acteristics of high molecular weight polymers. A detailed study on its preparation enzymatically was carried out (Scheme 5.1 , Kiran and Divakar 2003 ) .
5.2.1 Shake-Flask Level
Reactions carried out at an incubation temperature of 60°C by employing crystalline lactic acid dissolved in EMK in the presence of Lipozyme IM20, Chirazyme or PPL showed lesser yields, and the molecular weights were in the range 217–384 irrespective of divergent conditions employed. Transesteri fi cation reaction involving water of reaction was responsible for the reduced yields.
5.2.2 Bench-Scale Level
At bench-scale level, most of these drawbacks were overcome in terms of both extent of esteri fi cation and the molecular weight build-up (Table 5.1 ).
Lipozyme IM20 : In the presence of Lipozyme IM20, higher E/S ratios of 4.39 and 9.21AU mmol −1 resulted in 45.4 and 48.6% esteri fi cation with molecular weights of 411 and 459, respectively.
Chirazyme : The highest esteri fi cation of 66.9% at an E/S ratio of 0.79 AU mmol −1 with a molecular weight of 783 was obtained.
Porcine Pancreas Lipase : Increase in E/S ratio from 0.43 to 1.07 AU mmol −1 resulted in improve-ment of molecular weight from 809 to 1,128. Further increase in E/S ratio to 1.28 AU mmol −1 resulted in lower yield of 35.1% and lesser molecular weight (901).
A typical reaction pro fi le of lactic acid polymerisation using PPL is shown in Fig. 5.1 . The molecular weight build-up was found to fol-low the relation
24.2 log 2.74E M= -
where E = the percentage of esteri fi cation and M = the molecular weight. Figure 5.1 shows the theoretical plot for this equation and experimen-tal points from Chirazyme and PPL-catalysed polymerisation reactions. These experimental points corresponded well with the theoretical line showing the validity of the prediction. During the initial stages of the reaction, decrease in acid content corresponded only to a small increase in
675.2 Polylactic Acid
Scheme 5.1 Reactions depicting the strategy employed for the preparation of polylactic acid and its polymers with linker molecules
CH3
CH3 CH3 CH3
CH3 CH3 CH3
CH3 CH3 CH3
CH3 CH3CH3
CH3 CH3 CH3
Lipase
Lactic acid n = 15 –20 Polylactic acid
Polylactic acid Dicarboxylic acid
Lipase
n = 15 - 20 and m = 0 – 6 Polylactic acid – dicarboxylic acid polymer
Polylactic acid
Lipase
HO–CH –COOH HO-CH-CO-[O-CH -CO]n-O-CH-COOH + H2O
a
b
c
HO-CH-CO-[O-CH -CO]n-O-CH-COOH +HOOC-[CH2]m –COOH
HOOC-CH-O-[OC-CH-O]n-OC-CH-O-OC-[CH2]m–CO-O-CH-CO-[O-CH -CO]n-O-CH-COOH + H2O
HO-CH-CO-[O-CH -CO]n-O-CH-COOH + Phthalic anhydride
Polylactic acid –phthalic anhydride polymer
68 5 Enzymatic Polymerisation
molecular weight due to predominant formation of dimers and trimers. However, above 85% esteri fi cation, a small increase in extent of esteri fi cation signi fi cantly improved the molecu-lar weight since it corresponded to formation of an ester linkage between already existing oligo-meric chains.
5.2.3 Further Molecular Weight Build-Up
A molecular weight of 1,423 probably might indicate the presence of 18 lactic acid monomers formed on an average from two polylactic acid chains of nine residues each. Lipases may not be able to bind longer chains, and hence, acyl trans-fer may not be possible beyond a nine-residue
chain length leading to decrease in extent of esteri fi cation. In order to further enhance the molecular weight, polylactic acid chains were linked by other molecules. Several molecules containing hydroxyl and carboxyl functionalities could serve as linker molecules as polylactic acid chains contained a free hydroxyl and a carboxyl group at their terminal positions. Of them, diols could not be used, as they might inhibit the enzyme (Chaudhary and Qadri 1990 ) . Dicarboxylic acids and anhydrides such as oxalic acid, succinic acid, succinic anhydride, adipic acid, malonic acid and phthalic anhydride at different molar equivalents in the range 0.1–1.0 (with respect to the polylactic acid concentration) were used along with enzymatically or chemically prepared polylactic acid of molecular weights 1,400–1,600 for the reactions.
e d g b a
CH3 CH3 CH3 O
n=15-20Polylacticacid e -Caprolactone
Lipase
CH3 CH CH3
n=5-20
d
HO-CH-CO-[O-CH -CO]n-O-CH-COOH +
-O-CH-CO-[O-CH -CO]n-O-CH2-(CH2)3-CH2-CO-[O-CH -CO]n-O-CH2-(CH2)3-CH2-CO-
Polylactic acid- e-caprolactone copolymer
CH2-CH2-CH2-CH2-CH2-CO
3
Scheme 5.1 (continued)
695.2 Polylactic Acid
Table 5.1 Data on polymerisation of lactic acid monomer by lipases at bench-scale level a
Lactic acid (mol)
E/S ratio b (AU mmol −1 )
Max. esteri fi cation c (%) and incubation period (h)
Final esteri fi cation c (%) and incubation period (h)
Molecular weight d
Lipozyme IM20 0.056 1.76 35.4 (148) 35.4 (148) 417 0.056 4.39 45.4 (188) 45.4 (166) 411 0.056 9.21 48.6 (117) 48.6 (188) 459
Chirazyme 0.056 0.75 52.2 (145) 31.4 (240) 430 0.056 1.51 39.5 (115) 39.5 (115) 486 0.056 2.27 34.5 (125) 34.5 (125) 480 0.056 e 0.82 46.5 (180) 46.5 (180) 447 0.055 f 0.79 50.0 (165) 50.0 (165) 531 0.055 g 0.79 66.9 (181) 62.5 (205) 783 0.055 g 1.59 45.9 (162) 42.8 (186) 532 0.056 g 2.25 59.3 (234) 22.2 (329) 833 0.056 h 1.52 36.8 (138) 15.8 (207) 683
PPL 0.056 0.43 39.7 (141) 39.7 (165) 468 0.056 i 0.43 15.6 (84) 15.6 (116) 705 0.056 0.9 46.2 (273) 41.9 (298) 799 0.056 g 0.43 51.4 (211) 49.6 (234) 809 0.056 g 0.86 74.4 (426) 65.8 (545) 1,040 0.056 g 1.07 73.8 (402) 73.8 (470) 1,128 0.056 g 1.28 35.1 (273) 35.1 (401) 901 0.056 j 1.28 81.1 (387) 81.1 (423) 1,295 0.056 j 0.43 79.9 (498) 79.9 (550) 1,300 0.556 j 0.43 80.2 (507) 80.2 (545) 1,423
a Procedure described in Kiran and Divakar ( 2003 ) b Enzyme/Substrate ratio c Esterifi cation at the end of reaction d Esterifi cation after product work out e Methanol was added as initiator at 0.1 M equivalent f Butanol was added as initiator at 0.1 M equivalent g 0.1-mL, 0.1-M pH 7 sodium phosphate buffer was added h 0.2-mL, 0.1-M pH 7 sodium phosphate buffer was added i Reactions were carried out in benzene at 78°C j Reactions were conducted in hexane–MIBK (7:1) mixture
5.2.3.1 Malonic Acid The highest esteri fi cation of 36.2% and the high-est molecular weight of 3,795 were observed with 1.0 M equivalent of malonic acid.
5.2.3.2 Succinic Acid In case of succinic acid, maximum esteri fi cation observed was 66.7% at 0.1 M equivalent with a molecular weight of 2,685. Higher succinic acid equivalents gave less than 41% esteri fi cation molecular weights less than 1,830.
5.2.3.3 Adipic Acid Use of 1.0 M equivalent of adipic acid resulted in the highest esteri fi cation of 19.9% with a molec-ular weight of 1,734.
5.2.3.4 Phthalic Anhydride Polylactic acid of molecular weight 1,600 gave the maximum esteri fi cation of 62.3% with 0.1 M equivalent, the molecular weight being 2,685. Maximum molecular weight of 1,980 was observed with 20% esteri fi cation with 0.2 equivalent.
70 5 Enzymatic Polymerisation
5.2.3.5 Succinic Anhydride Enzymatically prepared polylactic acid of molecular weight 1,300 used along with 0.1 M equivalent of succinic anhydride resulted in a maximum esteri fi cation of 79.8% after 545 h with a molecular weight of 3,102. A maximum yield of 80.1% was observed with 0.1 M equivalent of succinic anhydride with chemically prepared polylactic acid (1,600), giving a molecular weight of 3,300 (Table 5.2 ).
5.2.4 Nuclear Magnetic Resonance Spectroscopy
Polylactic acids prepared through lipase catalysis were characterised by 1 H and 13 C NMR.
Molecular weight of the polylactic acid was determined through 1 H NMR. The number of repeat units was determined from the ratios of areas of the signals of –CH-O- corresponding to the polymer
Table 5.2 Lipase-catalysed synthesis of polymers of lactic acid using linker succinic anhydride at bench-scale level a
Polylactic acid concentration
Max. esteri fi cation b (%) and incubation period (h)
Succinic anhydride (molar equivalents)
Molecular weight c
0.055 d 79.8 (545) 0.1 3,102 0.055 e 80.1 (544) 0.1 3,300 0.057 e 42.7 (500) 0.2 3,086 0.055 e 38.4 (552) 0.5 2,439 0.055 f 2.2 (374) 0.1 -
a PPL was employed at an E/S ratio of 0.43 AU mmol −1 ; activity units were with respect to esteri fi cation activity units; solvent: hexane, MIBK, chloroform mixture b Error in extent of esteri fi cation was ±5–10% c Molecular weight was determined by end-group analysis (Gowariker et al. 1992 ) d Enzymatically prepared 1,300-molecular weight polylactic acid was employed e Chemically prepared 1,600-molecular weight polylactic acid was employed f Chemically prepared 4,200 polylactic acid was employed
0
10
20
30
40
50
60
70
80
90
100
0 3000 6000 9000 12000 15000
% E
ster
ific
atio
n
Molecular weight
Theoretical
PPL
Chirazyme
Fig. 5.1 Theoretical plot for prediction of molecular weight from extent of esteri fi cation along with experimental points. Percentage of esteri fi cation = 24.2 log M − 2.74
715.2 Polylactic Acid
ester chain (5.08–5.15 ppm) and those correspond-ing to the –CH-O- from the hydroxyl terminal of the polymer chain (4.35 ppm). The enzymatically pre-pared polylactic acid with molecular weight of 1,300 gave a molecular weight of 288 by 1 H NMR. Molecular weights determined by viscometry and 1 H NMR were found to show good agreement. Both viscometry and 1 H NMR gave weight average molecular weight, M
w , whereas end-group analysis
gave number average molecular weight, M n .
5.2.4.1 13 C NMR Three signals observed at 15.1, 20.4 and 20.5 ppm, respectively, corresponded to –CH
3 signals from
polylactic acid chain, hydroxyl terminal of poly-lactic acid and from free lactic acid, respectively. Two down fi eld signals at 65.0 and 65.2 ppm cor-responded to those of free lactic acid and hydroxyl terminal of the polylactic acid, respectively. Those at 69.6 and 70.3 ppm corresponded to ester –CH-O- of polylactic acid and carboxyl terminal of polylactic acid chain, respectively. The ester –CH-O- region at 69.6 ppm was a huge signal compared to the others, indicating larger extent of polymerisation. In the carbonyl carbon region, while 178.8- and 175.3-ppm signals corresponded to free lactic acid and hydroxyl terminal -CO- of polylactic acid respectively, those at 174.1, 173.8, 170.4 and 170.2 ppm corresponded to carbonyl carbon signals from various polymeric species.
5.2.5 Polylactic Acid Films
It was found that polylactic acid of molecular weights 400–3,300 could be cast into fi lms only
when blended with polystyrene of molecular weight 2 × 10 5 . Various proportions of polysty-rene were mixed with polylactic acid in chloro-form and cast into fi lms. For polystyrene content less than 50%, mixing with plasticisers up to 10% was required to cast a fi lm with polylactic acid (1,600). Polylactic acids with molecular weight of 3,300 and above formed fi lms with polysty-rene without the addition of the plasticiser. However, addition of plasticiser slightly improved the mechanical properties of the fi lm.
Polylactic acid fi lms prepared were character-ised in terms of their tensile strength, percentage elongation and optical properties like transmit-tance and haze (Table 5.3 ). Tensile strength of 1:1 blends of enzymatically prepared polylactic acids (1,400 and 3,300) with polystyrene was better (186 and 160 psi) than those of chemically pre-pared polylactic acids with molecular weights of 1,600 and 4,200 (157 and 128 psi, respectively). Compared to 100% polystyrene fi lm, all the other blends of polylactic acid with polystyrene showed better elongation of around 1.5–2.0%. Higher elongation of 8.4% was observed with a blend of enzymatically prepared polylactic acid–succinic anhydride polymer (3,300). Due to non-transpar-ent nature, all the blended fi lms showed low transmittance (<5.0%) and high haze (>90%). Films were also tested for heat sealability. All the blended fi lms were found to be heat sealable.
Of all the fi lms tested, a 1:1 blend of enzy-matically prepared polylactic acid–succinic anhydride polymer (3,300) and polystyrene (2 × 10 5 ) showed the highest elongation of 8.4%, higher transmittance of 4.9%, comparatively lesser haze of 91.5% and good tensile strength
Table 5.3 Data on properties of polylactic acid fi lms
Films Tensile strength a (psi) (%) Elongation
Optical properties Heat sealability (%) Transmittance (%) Haze
E-1400 b 50% + PS c 50% 186 1.3 4.4 94.7 Yes E-3300 d 50% + PS c 50% 160 8.4 4.9 91.5 Yes PS c 100% 341 0.8 84.0 5.7 Yes
a Tensile strength was measured at 20°C at an R H of 55%
b Enzymatically prepared polylactic acid of molecular weight 1,400 c Polystyrene of molecular weight 2 × 10 5 d Enzymatically prepared polylactic acid–succinic anhydride polymer of molecular weight 3,300
72 5 Enzymatic Polymerisation
(160 psi) than the other enzymatically prepared polymer blends. However, for a good fi lm, these characteristics had to be improved further.
Use of linker molecules led to various possi-bilities in the formation of the polymer chain. The acyl transfer reaction by the enzyme might aid in the monomer dicarboxylic acid being attached to one end of the polylactic acid chain. This resulted in a free carboxylic acid terminal on one polylactic acid chain, which facilitated reac-tion with the hydroxyl terminal of another poly-lactic acid chain. This reaction, although dif fi cult, had occurred in case of malonic acid and succinic anhydride resulting in polymers of molecular weights of 3,795 and 3,300, respectively.
It is generally recognised that polymerisation of free hydroxy acids by enzymatic means is dif fi cult. In case of enzymatic polymerisation of dicarboxylic acids like adipic acid or vinyl adi-pate with diols like 1,4-butanediol, molecular weights achieved were less than 2,000 (Seymour and Carrea 1984 ) . In case of small- and medium-sized lactones and macrolides, polymerisation has been shown to be easier due to higher ring strain (Kobayashi et al. 1998 ) . Molecular weights of the order of 25,000 were, in fact, achieved with higher macrolides (Chaudhary and Qadri 1990 ) . Inoue ( 1996 ) has studied the chemical ring open-ing polymerisation of a large number of ring sys-tems with and without initiators. They have generally observed that while six, seven or even higher-membered lactone rings can be easily polymerised, the chemical ring opening polymeri-
sation of fi ve-membered lactones has never been successful even with initiators.
5.3 Poly- e -caprolactone
A typical polymerisation with e -caprolactone is depicted in Scheme 5.2 (Divakar 2004 ) .
5.3.1 Shake-Flask- and Bench-Scale-Level Experiments
At shake- fl ask level, the yields were lesser, and the molecular weight build-up was also low which were overcome at bench-scale level. The experimental set-up employed has been described before (Divakar et al. 1999 ) . Table 5.4 shows the results.
A typical reaction pro fi le of e -caprolactone polymerisation using PPL is shown in Fig. 5.2 . Polycaprolactone esters prepared showed a maxi-mum molecular weight of 11,004 by end-group analysis. This was much higher than what was observed with several monomers attempted so far. However, gel permeation chromatography showed a molecular weight of 3,448 for the same polymer.
A molecular weight of 10,000 probably might indicate the presence of 84 e -caprolactone mono-mers formed on an average from two polycapro-lactone ester chains of 42 residues each. This indicates limitation exhibited by lipase on the
O PPL
ε-Caprolactone
Polycaprolactone ester n = 84
Mol.wt.=9900
CH2-CH2-CH2-CH2-CH2- C O
Scheme 5.2 Preparation of polycaprolactone ester
735.3 Poly-e-caprolactone
Table 5.4 Data on polymerisation of e -caprolactone by porcine pancreas lipase a
System (mole)
PPL g
Yield %
Period of incubation Molecular weight b
e - Caprolactone 3 75.9 285 11,004 (3448) f
(0.26)
e - Caprolactone + Succinic anh. 0.5 59 620 7,696
( 0.03) (0.006)
e - Caprolactone + Malonic acid 0.5 59.2 370 8,700
(0.33) (0.003)
e - Caprolactone + Lactic acid d 0.5 31.3 518 6,197
(0.03) (0.03)
e - Caprolactone + Polylactic acid e 0.5 53.6 258 8,333
(0.03) (0.06)
e - Caprolactone + caprolactam 0.5 33.6 445 2,847
(0.03) (0.03) Polycaprolactone + Adipic acid 0.5 22.2 291 9,769 Ester (0.04) (0.004) Polycaprolactone + Erythritol 0.5 63.5 744 7,162 Ester (0.04) (0.004) Polycaprolactone + Erythritol 0.5 76.2 216 8,222 Ester (0.04) (0.008)
a Solvent 150-ml benzene with 10-ml pyridine in some cases. Error in molecular weights will be ±5–10% b Molecular weight determined by end-group analysis c Polycaprolactone ester of molecular weight 11,004 d Crystalline lactic acid containing 4% water e Polylactic acid of molecular weight 1,400 f Molecular weight determined by gel permeation chromatography
100
90
80
70
60
50
40
30
20
Per
cen
tag
e o
f E
ster
ific
atio
n
Period of Incubation in h
10
00 19 43 67 99 123140164188212236268292316331355379403427451475489
%..
Fig. 5.2 Reaction pro fi le of e -caprolactone polymerisation by PPL. Reaction conditions: e -caprolactone (0.26 mole); PPL: 3 g; solvent: benzene 150 mL
74 5 Enzymatic Polymerisation
length of acyl groups transferred. In order to fur-ther enhance the molecular weight, polycaprolac-tone ester chains could be linked to other molecules like succinic anhydride, adipic acid, malonic acid, glycolic acid, lactic acid, caprolac-tam and erythritol and polylactides.
These molecules did not result increase in molecular weight. However, few monomers like caprolactam, glycolic acid polylactide and higher equivalents of erythritol showed decrease in molecular weights. Since most of the monomers employed were capable of ef fi cient acyl transfer, they did not add to the terminal groups of the polymer chain to enhance further molecular weight build-up probably due to steric factors arising out of binding of long polymer chains as acyl acceptors in the active sites. Instead, the smaller molecules can effect transestri fi cation reactions (acidolysis) leading to reduction in molecular weights.
5.3.2 Nuclear Magnetic Resonance Spectroscopy
Polycaprolactone esters prepared through lipase catalysis were characterised by 1 H and 13 C NMR. In the 1 H NMR spectrum, e -caprolactone showed signals for 2-CH
2 at 2.58 ppm, 3,5-CH
2 at 1.86 ppm,
4-CH 2 at 1.60 ppm and 6-CH
2 at 4.32 ppm. On
polymerisation, no monomer signals could be observed. Similarly, in 13 C NMR, monomer signals on polymerisation showed up fi eld shifts. C-1 sig-nal showed shift due to conversion from lactone to ester. Similarly, other signals including C-6 showed up fi eld shifts due to polymerisation.
5.3.3 Polycaprolactone Ester Films
It was found that polycaprolactone esters of molecular weights in the range 4,000–11,000 could be cast into fi lms only when blended with polystyrene of molecular weight 2 × 10 5 and cel-lulose acetate of molecular weight 39,000. On their own, they could not be cast into fi lms. Various proportions of polystyrene and cellulose acetate were mixed with polycaprolactone ester
in chloroform and cast into fi lms and tested (Table 5.5 ).
Polycaprolactone ester copolymerised with polylactic acid (molecular weight 1,400) when blended with cellulose acetate (1:1) showed the highest tensile strength of 1,383 psi. This blend showed also better percentage elongation (2.59), percentage haze (5.1), percentage transmittance (83.9) and burst strength (0.527 kg cm −2 ). Most of polycaprolactone ester blends were heat seal-able and showed good heat seal strength
These studies clearly show that lipase-catalysed polymerisation of e -caprolactone possesses good potential to be developed as a biodegrad-able polymer.
5.4 Poly- p -hydroxybenzoate
Liquid crystal polymers (LCP) are basically aro-matic polyesters. They have crystalline structure both in molten and in the solid state. They fi nd wide applications in the fi eld of electronics. The most frequently used basic monomers in conven-tional LCP are hydroxybenzoic acid, aminoben-zoic acid, hydroxynaphthoic acid, terephthalic acid, isophthalic acid and bisphenol A. Poly- p -hydroxybenzoate and its copolymers are used for printed circuits, wire coating, in dyes and plas-tics, as hot-melt adhesive to bond PVC sheets, to improve the adhesion to rubber, in transparent bottles, as an interior part in automobile or air-plane and in fi lms. Poly- p -benzamides and its copolymers are used as fi bres, electrical insula-tion, in cosmetics and in fi lms (Huang et al. 1992 ; Taesler et al. 1996 ; Kumar et al. 1996 ; Preston et al. 1992 ) .
Porcine pancreas lipase-catalysed polymerisa-tion of p -hydroxybenzoic (PHBA) acid monomer and its copolymers with hydroxy acids and lac-tones (Scheme 5.3 ) showed a highest yield of 5.2% with a number average molecular weight ( M
n ) of 184 was observed for the homo-oligomer
(Divakar 2003 ) . This showed that the reaction mixture contained largely unreacted monomer and its dimer, the former being the major compo-nent. With the increase in PPL amount, there was no molecular weight build-up.
Tab
le 5
.5
Prop
ertie
s of
fi lm
s fo
rmed
with
pol
ycap
rola
cton
e es
ter
blen
ds
Sam
ples
a A
vg.,
max
and
min
th
ickn
ess
(mic
rons
) Te
nsile
st
reng
th (
psi)
Pe
rcen
tage
el
onga
tion
Perc
enta
ge h
aze
Perc
enta
ge
tran
smitt
ance
B
urst
str
engt
h (k
g cm
−2 )
H
eat
seal
abili
ty
Hea
t sea
l st
reng
th (
psi)
Poly
styr
ene b (
5%)
– 34
1 0.
836
5.7
84.0
–
Yes
–
Poly
( e
-CL
–pol
y L
A 1
400)
, mol
. wt.
8300
+ p
olys
tyre
ne b (
1:1,
10%
) 45
.0, 8
0, 3
0 34
1 0.
855
100
1.0
0.
070
Yes
23
5.2
Poly
( e -
CL
–cry
stal
line
LA
), m
ol. w
t. 62
00 +
pol
ysty
rene
b (1:
1, 1
0%)
52.0
, 100
, 25
190
1.03
8 10
0 0
.7
1.05
4 Y
es
88.
4
Poly
( e -
CL
), m
ol. w
t. 99
00
+ p
olys
tyre
ne b (
1:1,
10%
) 42
.7, 6
0, 2
5 21
0 2.
200
100
1.1
0.
070
Yes
20
9.7
Cel
lulo
se
acet
ate c (
5%)
32.5
, 95,
15
38
1.37
2 14
.9
70.1
0.
158
Yes
–
Poly
( e -
CL
–pol
y L
A 1
400)
, mol
. wt.
8300
+ c
ellu
lose
ace
tate
c (1:
1, 2
0%)
61.0
, 90,
30
1,38
3 2.
591
5.1
83.9
0.
527
Yes
14
5.1
Poly
( e -
CL
–cry
stal
line
LA
), m
ol. w
t. 62
00 +
cel
lulo
se a
ceta
te c (
1:1,
10%
) 12
6.0,
330
, 30
80
1.93
1 3.
1 85
.5
0.14
1 Y
es
45.
6
LA L
actic
aci
d, e
- CL
e -c
apro
lact
one;
all
mol
ecul
ar w
eigh
t val
ues
dete
rmin
ed b
y en
d-gr
oup
anal
ysis
a A
ll sa
mpl
es w
ere
prep
ared
enz
ymat
ical
ly. F
ilms
wer
e w
et c
ast a
fter
dis
solv
ing
in c
hlor
ofor
m a
nd w
ere
cond
ition
ed a
t 27°
C a
nd 6
5% R
H
b Mol
ecul
ar w
eigh
t 2 ×
10 5
c Mol
ecul
ar w
eigh
t 39,
000
(GPC
), 3
3% a
cety
late
d
76 5 Enzymatic Polymerisation
In order to improve the molecular weight, PHBA was reacted with different monomers like lactic acid, glycolic acid and e -caprolactone in presence of PPL. Lactic acid exhibited a conver-sion of 65.1% with M
n 195 indicating again a mix-
ture of unreacted monomers and probably their esters. Copolymerisation with glycolic acid was studied in different solvents at different ratios of the monomers. A maximum esteri fi cation of 60% with M
n 497 was obtained in 1:4 MIBK–hexane
(v/v) system which was found to be the best sol-vent system for this reaction. However, the high-est M
n of 525 was observed when 1:2 PHBA and
glycolic acid were employed. Such low M n values
indicate only mixture of oligomers. e -Caprolactone, as a comonomer with PHBA
gave higher M n than the other monomers. Thus,
PHBA and e -caprolactone showed 60.4% esteri fi cation with M
n 1,486. Also, a 1:2 M equiv-
alent of PHBA and e -caprolactone showed 65.2% yield with M
n 1,398. Increase in e -caprolactone
content did not aid in further molecular weight build-up. Thus, e -caprolactone along with PHBA showed greater propensity for copolymerisation.
5.5 Poly- p -benzamide
A homo-oligomer of p -aminobenzoic acid was prepared (Scheme 5.4 ) showing a conversion of 26.4% with M
n of 778 (Divakar 2003 ) . With
increase in incubation period, percentage conver-sion increased with molecular weight. In order to increase the molecular weight further,
HO COOH + HO COOH
HO COOH
Lipasep-Hydroxybenzoic acid
Mol Wt = 1500
CO-[O-R-CO]n-O
Polybenzoate n = 0 - 10 R = (CH3)-CH-, -(CH2)5-, phenyl
Scheme 5.3 Lipase-catalysed polymerisation of p-hydroxybenzoic acid
COOH + COOH
COOH
Lipase
Mol Wt = 1100
NH2 NH2
NH2 CO-[NH-R-CO]n-NH
Polybenzamide n = 0 - 12 R = (CH3)-CH-, -(CH2)5-, Phenyl
Scheme 5.4 Lipase-catalysed polymerisation of p-aminobenzoic acid
775.6 Polyadipates
p -aminobenzoic acid was polymerised with lac-tic acid and e -caprolactone. p -Aminobenzoic acid–lactic acid oligomer showed M
n 1,049 after
590 h at 52.6% conversion, and p -aminobenzoic acid– e -caprolactone oligomer showed M
n 706 after
494 h at 36.1% conversion. Copolymer reaction with lactic acid showed the highest conversion yield and molecular weight probably due to smaller molecular size of lactic acid acting as both acyl acceptor and donor. The copolymerisation potentiality of p -aminobenzoic acid was found to be slightly better than that of PHBA although the observed low M
n values indicated only oligomer
formation. It was found that the oligomers of PHBA with
e -caprolactone ( M n ) prepared enzymatically could
not be cast into fi lms as such even when a 10% solution in CHCl
3 was employed. But when blended
with polystyrene (molecular weight – 2 × 10 5 ), soya protein (molecular weight – 30,000) and cellulose acetate (molecular weight – 39,000), fi lms were obtained. Low molecular weight polymers of p -aminobenzoic acid (700–1,500) could not be cast into fi lms even after blending in varying propor-tions with polystyrene and cellulose acetate. This may be probably because the hydrophobic interac-tions between the benzene ring of PHBA copoly-mer and polystyrene or cellulose acetate may be stronger than that of p -aminobenzoic acid.
Enzymatic methods generally have shown low molecular weight polymers even with mono-mers like lactones (Gross et al. 2001 : Kumar and Gross 2000 ; Svirkin et al. 1996 : Namekawa et al. 1996 ) . The reason for obtaining low molecular weight copolymers by enzymatic methods in case of PHBA and e -caprolactone and p -amin-obenzoic acid and lactic acid (as in case of lac-tones also) is probably due to strong binding of especially the aromatic monomers to the active site of porcine lipase preventing facile acyl trans-fer to the hydroxyl or amino groups of the other monomers. Large amounts of enzymes than those employed in this work may probably be required for preparing higher molecular weight polymers. A better approach would be to deriva-tise the monomers to suitably activate the func-tional groups to attain higher molecular weight polymers.
5.6 Polyadipates
Adipic acid polymers (polyadipates) have recently been found to possess several desirable characteristics necessary for a wide variety of applications (Inoue 1996 ; Binns et al. 1994 ; Sandez-Adsuar and Martin-Martiz 2000 ; Bartz and Roehe 2003 ; Muhlfeld and Wagener 2000 ) . Reports have shown that even low molecular weight polymers (molecular weight around 2,000) of polyadipates can be used along with polyurethane and other such polymers to make automobile interior parts, hot-melt adhesives for textiles, moulding compositions, packaging materials and medical goods to name a few (Bohl et al. 2000 ) . A reaction between adipic acid and 1,6-hexanediol (Scheme 5.5 ) showed minimum yield of 33.7% with a number average molecular weight of 1,265. The highest number average molecular weight of 2,396 was obtained with 1,6-hexanediol. The reaction with 1,4-butane-diol showed 55.5% conversion after 568 h, with a number average molecular weight of 1,645. Reaction between adipic acid and ethylene gly-col showed 36.5% conversion with a number average molecular weight of 2,173 after 568 h. Although this reaction showed lower conversion, higher molecular weight polymers were obtained, when compared to reactions with the other diols (Table 5.6 ).
Other aromatic dicarboxylic acids like terephthalic acid and isophthalic acids were also reacted with ethylene glycol. In order to dissolve terephthalic acid and isophthalic acid, reactions were conducted with 10-mL pyridine and toluene–hexane (30:60) solvent mixture. Reactions were incubated for a period of 20–27 days. Reaction between ethylene glycol and terephthalic acid showed 36.6% conversion with a number average molecular weight of 927 after 666 h whereas reaction with isophthalic acid showed a conversion of 61.3% and a number average molecular weight of 1,488 after 735 h.
This study has shown that low molecular weight polyadipates of molecular weights 2,400 can be produced through lipase catalysis in
78 5 Enzymatic Polymerisation
organic media. Even with puri fi ed monomers, higher molecular weights could not be obtained. Further increase in molecular weight may not be possible because of limitations in acyl transfer by the enzyme due to increase in chain length. However, activation of -COOH and OH groups can yield higher molecular weight polyadipate copolymers.
References
Bartz T, Roehe P (2003) Ger Offen DE 19854404. A1, 31 May 2000, p 6
Binns F, Roberts SM, Taylor A, Williams CF (1994) Studies leading to the large scale synthesis of polyesters using enzymes. J Chem Soc Perkin Trans 1:899–904
Bisht KS, Deng F, Gross RA, Kaplan DL, Swift G (1998) Ethyl glucoside as a multifunctional initiator for
Lipase
Adipic acid 1,4-Hexane diol
Polyadipate n = 0 -15 Mol Wt = 2400
HOOC-(CH2)6-COOH + HO-(CH2)6-OH
HOOC-(CH2)4-CO-[O-(CH2)6-O]n-CO-(CH2)4-COOH
Scheme 5.5 Lipase-catalysed polymerisation of adipic acid and 1,4-hexanediol
Table 5.6 Data on enzymatically prepared polyadipates and glycolates
Monomers (mole) PPL (mg) Solvent (ml) (%) Yield (time – h)
Molecular weight
Adipic acid + 1,6-hexanediol 500 Benzene to pyridine 59 (568) 1,645 (0.01) (0.01) (9:1) Adipic acid + 1,6-hexanediol 500 EMK – 25 33.7 (473) 1,267 (0.01) (0.01) toluene to hexane (25:50) Adipic acid + 1,6-hexanediol 500 Benzene to pyridine 92 (500) 2,396 (0.025) (0.025) (9:1) Adipic acid + 1,6-hexanediol 500 Benzene to pyridine 65 (387) 570 (0.035) (0.035) (9:1) Adipic acid + 1,6-hexanediol 500 Benzene to pyridine 63 (360) 567 (0.05) (0.05) (9:1) Adipic acid + 1,4-butanediol 500 Benzene to pyridine 65 (744) 547 (0.025) (0.025) (9:1) Adipic acid + 1,4-butanediol 500 EMK – 25 55.5 (568) 1,645 (0.01) (0.01) toluene to hexane (25:50) Adipic acid + ethylene glycol 500 Benzene to pyridine 82 (620) 467 (0.025) (0.025) (9:1) Adipic acid + ethylene glycol 500 Benzene to pyridine 64 (616) 754 (0.025) (0.025) (9:1) Adipic acid + ethylene glycol 500 EMK – 25 36.5 (568) 2,173 (0.01) (0.01) toluene to hexane (25:50) Ethylene glycol + terephthalic acid 500 Pyridine – 10 36.6 (666) 927 (0.01) (0.01) toluene to hexane (30:60) Ethylene glycol + isophthalic acid 500 Pyridine – 10 61.3 (735) 1,488 (0.01) (0.01) toluene to hexane (30:60)
EMK Ethyl methyl ketone. Error in molecular weights will be ±10%
79References
enzyme-catalyzed regio-selective lactone ring-opening polymerization. J Am Chem Soc 120:1363–1367
Bohl W, Partusch G, Fleishmann S (2000) Ger Offen DE 19928608 A1, 28 Dec
Chaudhary AN, Qadri RB (1990) Higher fatty acid esters of lactic acid. Pak J Ind Res 33:334–336
Divakar S (2003) Porcine pancreas lipase catalysed prepara-tion of oligomers of p-hydroxybenzoic acid and p-amin-obenzoic acid. Ind J Chem Sect B 42B:1467–1470
Divakar S (2004) Porcine pancreas lipase catalysed ring-opening polymerization of e -caprolactone. J Macromol Sci Part A Pure Appl Chem A41(5):537–546
Divakar S, Kiran KR, Harikrishna S and Karanth NG (1999) An improved process for the preparation of esters of organic acids and alcohols. Indian Patent, 1243/DEL/99 No. 191078
Gowariker VR, Viswanathan NV, Sreedhar J (1992) Polymer science. Wiley Eastern Ltd., New Delhi
Gross RA, Kumar A, Kalra B (2001) Polymer synthesis by in vitro enzyme catalysis. Chem Rev 101(7):2097–2124
Huang K, Lin YG, Winter HH (1992) p-Hydroxy benzo-ate/ethylene terephthalate copolyester: structure of high-melting crystals formed during partially molten state annealing. Polymer 33:4533–4537
Inoue A (1996) JPN Kokai Tokkyo Koho Jp 08059847, A25, Mar 1996, Heirei 5, Japan (CA)
Kiran KR, Divakar S (2003) Lipase catalyzed polymeriza-tion of lactic acid and its fi lm forming properties. World J Microbiol Biotechnol 19:859–865
Knani D, Gutman AL, Kohn DH (1993) Enzymatic polyesteri fi cation in organic media enzyme – cata-lyzed synthesis of linear polyesters. 1. Condensation polymerization of linear hydroxyesters. J Polym Sci Part A Polym Chem 31:1221–1232
Kobayashi S, Uyama H, Namekawa S, Hayakawa H (1998) Enzymatic ring-opening polymerization and copolymerization of 8-octanolide by lipase catalyst. Macromolecules 31:5655–5659
Kumar A, Gross RA (2000) Candida antarctica lipase B-catalyzed transesteri fi cation: new synthetic routes to copolyesters. J Am Chem Soc 122:11767–11770
Kumar A, Kumar N, Parmar V (1996) Preparative and mechanistic aspects of interesteri fi cation reactions on diols and peracetylated polyphenolic com-pounds catalysed by lipases. Pure Appl Chem 68:749–752
Lipinsky ES, Sinclair RG (1986) Is lactic acid a commodity chemical? Chem Eng Progr 82:26–32
Muhlfeld H, Wagener S (2000) Eur Pat Appl EP 1043349, A2, 11 Oct 2000, 5 pp
Namekawa S, Uyama H, Kobayashi S (1996) Lipase-catalyzed ring-opening polymerization and copoly-merization of b -propiolactone. Polym J 28:730–731
Preston J, Kotek R, Krigbaum WR (1992) Macromol Synth 11:27
Rika M, Tadaki S, Kayoko Y (1996) Jpn Kokkai Tokkyo Koho JP 08:73,573
Sandez-Adsuar MS, Martin-Martiz JM (2000) J Adhes Sci Technol 14:8
Seiji O, Masahiro O (1992) Jpn Kokai Tokkyo Koho JP 06:172502
Seymour RB, Carrea CE Jr (1984) Structure relationship in polymers. Plannum Press, New York, pp 55–69
Svirkin YY, Xu J, Gross RA, Kaplan DL, Swift G (1996) Enzyme catalyzed stereo-selective ring- opening polymerization of a -methyl- b -propiolac-tone. Macromolecules 29:4591–4597
Taesler C, Wittich H, Jourgen C, Schulte K, Kricheldorf HR (1996) J Appl Polym Sci 61:783–792
Uyama H, Kobayashi S (1996) Enzymatic ring-opening polymerization of macrolides to polyesters. Front Biomed Biotechnol 3:5–15
Voss W, Spielan P (1958) German (East) Patent 14, 548 Zhang H, Deng X, Huang Z (1996) Ring-opening polym-
erization of e -caprolactone by bacterial protease. Biotechnol Lett 18:1051–1054
81S. Divakar, Enzymatic Transformation, DOI 10.1007/978-81-322-0873-0_6, © Springer India 2013
6.1 Introduction
Carbohydrates are information-rich molecules, which are suited well for modi fi cation to new types of compounds with expected biological activity (Hurtley et al. 2001 ) . Compared to fatty acid esters of carbohydrates, aminoacyl esters of carbohydrates can give added additional functionality to the side chain. Presence of hydroxyl as well as amine groups in the molecule helps in the polycondensation reac-tions (Park et al. 1999 ) . Recently Shiraki et al. ( 2004 ) reported that aminoacyl esters prevent ther-mal inactivation and aggregation of lysozyme.
Aminoacyl esters of carbohydrates are used as sweetening agents, surfactants, microcap-sules in pharmaceutical preparations, active nucleoside amino acid esters, antibiotics and in the delivery of biological active agents (Dordick 1989 ; Tamura et al. 1985 ; Kirk et al. 1992 ; Zaks and Dodds 1997 ) . Chemical acylation of carbohydrates regioselectively is complex due to the presence of multiple hydroxyl groups, which require protection and deprotection (Tamura et al. 1985 ; Haines 1981 ) . Use of lipases in the synthesis of sugar esters is indus-trially important due to regio- and stereoselectivity
6
Abstract
The esteri fi cation potentialities of lipases from Rhizomucor miehei , Candida rugosa and porcine pancreas (PPL) are discussed in detail with respect to the syntheses of l -alanyl, l -valyl, l -leucyl, l -isoleucyl, l -prolyl, l -phenylalanyl, l -tryptophanyl and l -histidyl esters of representative carbohydrates like hexo-pyranoses ( d -glucose, d -galactose, d -mannose), ketose ( d -fructose), pento-furanoses ( d -arabinose, d -ribose), disaccharides (lactose, maltose, sucrose) and sugar alcohols ( d -sorbitol, d -mannitol), using unprotected and unactivated amino acids and carbohydrates. Lipase-catalysed esteri fi cation reactions of l -alanyl- d -glucose, l -valyl- d -glucose, l -leucyl- d -glucose, l -phenylalanyl- d -glucose and l -phenylalanyl-lactose were optimised in terms of incubation period, solvents, lipase concentrations, substrate concen-trations, buffer pH and its concentrations and lipase reusability. Spectroscopic investigations discuss the regioselectivity of the product esters formed. The relative merits of the three lipases under the reaction conditions employed, in terms of incubation period, substrate and enzyme concentrations, solvent, pH and buffer concentrations, reusability leading to the observed yield and nature and types of products formed, are discussed in detail.
Lipase-Catalysed Preparation of Aminoacyl Esters of Carbohydrates
82 6 Lipase-Catalysed Preparation of Aminoacyl Esters of Carbohydrates
imparted by them (Ferrer et al. 1999 ) . Enzymatic methods do not require such elabo-rate and expensive procedures. Enzymatic synthesis of bond making reactions like esteri fi cation is usually carried out in organic media. When enzymes are used in organic media, they exhibit speci fi city (Wescott and Klibanov 1994 ) , thermostability (Ayala et al. 1986 ; Wheeler and Croteau 1986 ) , molecular memory (Stahl et al. 1991 ; Dabulis and Klibanov 1993 ) and capacity to catalyse reverse reactions (Kuhl et al. 1990 ; West et al. 1990 ) .
Hitherto, very few references are available on the lipase-catalysed esteri fi cation of aminoacyl esters of sugars. Most of the earlier workers used proteases and N-protected and carboxyl group activated amino acids for synthesising aminoacyl esters of carbohydrates (Riva et al. 1988 ; Park et al. 1996, 1999 ; Jeon et al. 2001 ) . Therisod and Klibanov ( 1986 ) used subtilisin to acylate carbo-hydrates with activated carboxylic acids in anhy-drous organic solvents. Riva et al. ( 1988 ) carried out subtilisin-catalysed synthesis of N-acetyl- l -alanyl-methyl- b - d -galactopyranoside in anhy-drous DMF, with a yield of 70% (4- O - 16% and 6- O - 84%), and N-acetyl- d -alanyl-methyl- b - d -galactopyranoside with a yield of 35% (2- O - 10%, 3- O - 10%, 4- O - 12% and 6- O - 68%). Suzuki et al. ( 1991 ) synthesised l -alanyl- d -glucose by using d -glucose, methyl- l -alaninate hydrochloride and intact cells of Rhodotorula lactosa . There are no reports on the synthesis of l -valyl- d -glucose esters enzymatically. There are some reports on the chemical synthe-sis of methyl 2- O -[N- t -boc]- l -valyl- d -glucose, methyl-2,3-di- O - l -leucyl- a - d -glucose and methyl-3- O -[N- t -boc]- l -valyl-glucose and dies-ters such as ethyl-2,3-di- O -[N- t -boc]- l -valyl- d -glucose, methyl-2,3-di- O - l -valyl- d -glucose, methyl-4,6-di- O - l -valyl- d -glucose and methyl-2,3-di- O - l -isoleucyl- a - d -glucose (Tamura et al. 1985 ) whose synthesis involved pro-tection and deprotection. Park et al. ( 1999 ) has carried out transesteri fi cation with Optimase M-440 to synthesise t -boc-leucyl-sucrose by using tert-butoxy-carbonyl- l -leucyl-cyanomethyl ester/tert-butoxy-carbonyl- l -leucyl-tri fl uoro
ethyl ester and sucrose. Maruyama et al. ( 2002 ) have investigated the synthesis of N-acetyl- l -leucyl- d -glucose in t -butanol containing 10% dimethyl sulfoxide by transesteri fi cation between N-acetyl- l -leucyl-cyanomethyl ester and d -glu-cose. Park et al. ( 1999 ) reported that lipase from porcine pancreas and Lipozyme IM20 gave very low yields (<2%), compared to proteases which gave conversion yields ranging from 15 to 98% when N-protected and carboxyl group activated amino acid was used for the acylation of d -glu-cose in pyridine. All these reactions were con-ducted in shake fl asks using lesser quantity of substrates and larger quantity of enzymes.
Hydrophobic amino acids, like l -phenylala-nine, l -tyrosine and l -tryptophan with carbohy-drate moiety may improve the water solubility (Maruyama et al. 2002 ) , which is very much essential for bioavailability of amino acids. Lipases from Candida rugosa , Mucor javani-cus , Pseudomonas cepacia and Pseudomonas fl uorescens ; proteases from Aspergillus melleus (Maruyama et al. 2002 ) ; and other proteases like subtilisin (Riva et al. 1988 ; Boyer et al. 2001 ) and Optimase M-440 (Park et al. 1996, 1999 ) catalysed the transesteri fi cation of N-blocked l -phenylalanine ester and d -glucose regiose-lectively and that too preferably at the primary hydroxyl groups.
Herein is described a detailed report on the lipase-catalysed synthesis of l -alanyl, l -valyl, l -leucyl, l -isoleucyl, l -phenylalanyl, l -trypto-phanyl, l -tyrosyl and l -histidyl esters of hexoses ( d -glucose, d -galactose, d -mannose), a ketose ( d -fructose), pentoses ( d -arabinose, d -ribose), disaccharides (maltose, sucrose, lac-tose) and carbohydrate alcohols ( d -mannitol and d -sorbitol). Attempts to synthesise the same using N-acetyl derivatives resulted in very little conversion with Rhizomucor miehei (RML), Candida rugosa (CRL) and porcine pancreas (PPL) as biocatalysts. Hence, unprotected and unactivated l -amino acids and carbohydrates were employed (Lohith et al. 2003, 2006a, b ; Lohith and Divakar 2005, 2007 ; Vijayakumar et al. 2004 ; Lohith and Divakar 2005 ; Somashekar and Divakar 2007 ) .
836.2 L-Alanyl-D-Glucose
6.2 L -Alanyl- D -Glucose
6.2.1 Optimisation Studies
Esteri fi cation was carried out in the presence of lipase by incubating d -glucose and l -alanine in an organic media (Scheme 6.1 ). Lipases from Rhizomucor miehei (RML) and porcine pancreas (PPL) were employed for the reaction (Somashekar and Divakar 2007 ) . The extent of esteri fi cation was monitored by HPLC (Fig. 6.1 ).
The esteri fi cation reaction between unpro-tected and unactivated l -alanine and d -glucose was studied in detail using RML and PPL in terms of incubation period, lipase concentrations, substrate concentrations, buffer (pH and concen-tration) and enzyme reusability. The esteri fi cation reactions described in present work did not occur without the use of enzymes. Optimum conditions determined were:
RML: 26% at 72-h incubation period with an initial rate of 0.004 mmol h −1 (Fig. 6.2 ); 50% (w/w d -glucose) RML; 0.1-mM (0.1 mL of 0.1 M) acetate buffer, pH 4.0; l -alanine 4 mmol and d -glucose 4 mmol (Fig. 6.3 , Tables 6.1 and 6.2 )
PPL: 18% at 40% (w/w d -glucose PPL); l -alanine, 2 mmol; buffer, 0.5-mM (0.5 mL of 0.1 M) acetate buffer pH 5.0 (Tables 6.2 and 6.3 )
Earlier workers have not studied the effect of buffer salts on this esteri fi cation reaction. Carrying
out this esteri fi cation reaction in the presence of buffers of certain pH not only imparted ‘pH mem-ory’ or ‘pH tuning’ to the enzyme but also pro-vided the optimum water activity necessary for better performance of the enzyme. Besides, addi-tion of buffer salts of certain concentration also affected the ionic activities of especially the micro-aqueous layer around the enzyme, where the buf-fer salts are concentrated during the course of the reaction. All these have been found to be operative in these esteri fi cation reactions.
6.2.2 Reusability of Lipases
After each cycle, in case of RML, there was a steady loss of 15–22% of enzyme concentration both in the presence as well as absence of 0.2-mM (0.2 mL of 0.1 M) acetate buffer (pH 4.0). In the absence of buffer salts, the esteri fi cation activity decreased from 24% (1st cycle: total enzyme activity, 99.4 m mol min −1 ) to 8% (4th cycle: total enzyme activity, 23.5 m mol min −1 ). The yields in 2nd and 3rd cycles were 17% (total enzyme activity – 66.4 m mol min −1 ) and 10% (total enzyme activity – 43.1 m mol min −1 ), respec-tively. In the presence of buffer salts (pH 4.0), the esteri fi cation activity decreased from 17% (1st cycle: total enzyme activity, 99.4 m mol min −1 ) to 5% (4th cycle: total enzyme activity of
Scheme 6.1 Lipase-catalysed regioselective synthesis of l -alanyl esters of carbohydrates
84 6 Lipase-Catalysed Preparation of Aminoacyl Esters of Carbohydrates
23 m mol min −1 ). The yields in 2nd and 3rd cycles were 11% (total enzyme activity – 66.9 m mol min −1 ) and 8% (total enzyme activity – 42.2 m mol min −1 ), respectively.
However, in case of PPL, there was a drastic loss of enzyme concentration from 20 to 60% after each cycle, as PPL was partially soluble in water and the reaction was stopped after the
2nd cycle due to reduction in enzyme. In the absence of buffer salts, esteri fi cation activity of the PPL decreased from 19% (1st cycle: total enzyme activity, 15 m mol min −1 ) to 3% (4th cycle: total enzyme activity, 0.7 m mol min −1 ). The 2nd and 3rd cycle yields were 12% (total enzyme activity – 5.6 m mol min −1 ) and 10% (total enzyme activity – 3.3 m mol min −1 ),
Fig. 6.1 HPLC chromatograph for the reaction mixture of l -alanine and d -glucose. ( a ) Column, aminopropyl; mobile phase, acetonitrile to water (80:20 v/v); fl ow rate, 1 mL min −1 ;
detector, refractive index. ( b ) Column, C-18; mobile phase, acetonitrile to water (v/v 20:80); fl ow rate, 1 mL min −1 ; detector, UV at 210 nm. Errors in yields are within ±10%
856.2 L-Alanyl-D-Glucose
respectively. However, in the presence of buffer salts (pH 5.0), the esteri fi cation activity decreased slightly from 11% (1st cycle: total enzyme activity, 15 m mol min −1 ) to 6% (2nd cycle: total enzyme activity, 4.1 m mol min −1 ).
6.2.3 Syntheses of L -Alanyl Esters of Carbohydrates
Syntheses of l -amino acid esters of different car-bohydrates (Somashekar and Divakar 2007 ) were carried out between different amino acids ( l -ala-
Fig. 6.2 Reaction pro fi le for l -alanyl- d -glucose synthesis. Reaction conditions, CH
2 Cl
2 ; DMF (v/v 90:10); RML,
54 mg (30% w/w D-glucose); L-alanine, 2 mmol; D-glucose,
1.0 mmol; buffer, 0.1-mM (0.1 mL of 0.1 M) acetate buffer (pH 4.0)
Fig. 6.3 Effect of substrate concentration on synthesis of l -alanyl- d -glucose. RML, 30% (w/w d -glucose); solvent, CH
2 Cl
2 :DMF (v/v 90:10), at
40°C; buffer, 0.1-mM (0.1 mL of 0.1 M) acetate buffer (pH 4.0). l -alanine (■), 1–5 mmol at 1 mmol d -glucose; d -glucose (▲), 1–5 mmol at 1 mmol l -alanine and a constant enzyme concentra-tion of 54 mg
86 6 Lipase-Catalysed Preparation of Aminoacyl Esters of Carbohydrates
Table 6.1 Effect of lipase concentration on the synthesis of l -alanyl- d -glucose a
Lipase concentration (% w/w d -glucose)
Rhizomucor miehei lipase (RML) b Yield % (mmol)
Porcine pancreas lipase (PPL) c Yield % (mmol)
Rhizomucor miehei lipase (RML) d yield % (mmol)
10 2 (0.04) 14 (0.27) 7 (0.14) 20 1 (0.02) 15 (0.29) 10 (0.21) 30 3 (0.05) 17 (0.35) 26 (0.53) 40 9 (0.17) 18 (0.36) 30 (0.60) 50 18 (0.37) 15 (0.29) 22 (0.44)
a d -Glucose, 1 mmol, and l -alanine, 2 mmol. Conversion yields from HPLC determined with respect to l -alanine. Error in yield measurements will be ±10–15%. This applies to all the yields given in the subsequent tables also b Solvent – CHCl
3 :hexane:DMF (v/v/v 45:45:10) – at 60°C
c Solvent – CH 2 Cl
2 :DMF (v/v 90:10) – at 40°C
d Carried out in the presence of buffer with 100 mL of solvent system b containing 0.1-mM (0.1 mL of 0.1 M) acetate buffer (pH 4.0)
Table 6.2 Effect of buffer salts (pH and buffer concentration) on the synthesis of l -alanyl- d -glucose a
RML PPL
pH b Yield % (mmol)
pH 4.0 c concn mM
Yield % (mmol) pH d
Yield % (mmol)
pH 5.0 e concn mM
Yield % (mmol)
4.0 26 (0.53) 0.05 25 (0.49) 4.0 9 (0.18) 0.05 13 (0.25) 5.0 7 (0.13) 0.1 26 (0.53) 5.0 17 (0.33) 0.1 17 (0.33) 6.0 20 (0.39) 0.2 8 (0.16) 6.0 15 (0.31) 0.2 11 (0.23) 7.0 8 (0.16) 0.3 7 (0.15) 7.0 11 (0.23) 0.3 10 (0.21) 8.0 10 (0.19) 0.4 17 (0.34) 8.0 No yield 0.4 12 (0.24) – – 0.5 18 (0.35) – – 0.5 17 (0.34)
a d -Glucose, 1 mmol, and l -alanine, 2 mmol; incubation period, 72 h; RML, 30% (w/w d -glucose). 100 mL of the sol-vent containing speci fi ed volumes, concentration and pH of the buffer b Solvent, 100 mL CHCl
3 :hexane:DMF (v/v/v 45:45:10) at 60°C; buffer, 0.1-mM (0.1 mL of 0.1 M) appropriate pH buffer
c Solvent, 100 mL CH 2 Cl
2 :DMF (v/v 90:10) at 40°C; buffer, 0.05–0.5 mL of 0.1 M acetate ( pH 4.0)
d Same solvent system as c. Buffer – 0.1 mM (0.1 mL of 0.1 M) appropriate pH e Solvent – same solvent system as c. 0.05 mL to 0.5 mL of 0.1 M acetate (pH 5.0)
nine, l -valine, l -leucine, l -isoleucine, l -prolyl, l -phenylalanyl, l -tryptophanyl and l -histidyl) and carbohydrates ( d -glucose, d -galactose, d -mannose, d -fructose, d -arabinose, d -ribose, lactose,
maltose, sucrose, d -mannitol, d -sorbitol). l -Alanine is a polar and a nonessential amino acid contain-ing methyl group as a side chain. Its esters with above-mentioned carbohydrates were synthesised. Table 6.4 shows the ester yields form HPLC, types of esters formed and percentage proportions of the individual esters by RML, and Table 6.5 shows the ester yields from HPLC by both CRL and PPL.
6.3 Synthesis of L -Valyl- D -Glucose
Lipases form Rhizomucor miehei (RML) and porcine pancreas (PPL) were employed for the reaction (Scheme 6.2 , Somashekar and Divakar
Table 6.3 Effect of partially puri fi ed PPL concentration on the synthesis of l -alanyl- d -glucose a
Partially puri fi ed PPL concentration (% w/w of d -glucose) Yield % (mmol)
55.6 41 (0.41) 111.1 78 (0.78) 166.7 64 (0.68) 222.2 50 (0.50) 277.8 39 (0.39)
a d -Glucose, 1 mmol, and l -alanine, 1 mmol; solvent CH
2 Cl
2 :DMF (v/v 90:10) at 40°C. Carried out in the pres-
ence of buffer with 100 mL of solvent system b containing 0.2-mM (0.2 mL of 0.1 M) acetate buffer (pH 5.0)
876.3 Synthesis of L-Valyl-D-Glucose Ta
ble
6.4
Sy
nthe
ses
of l
-ala
nyl e
ster
s of
car
bohy
drat
es u
sing
RM
L a
l -A
lany
l est
ers
of c
arbo
hydr
ates
(%
pro
port
ions
b )
Yie
ld (
%)
HO
OH
HO
OH
H
H
HH
H
O
O
O
H3C
H2N
O
HO
HHH
H
H
OH
OH
OH
O OH
3C
H2N
O
HO
HHH
H
H
OH
O
NH
2
CH
3
O
HO
HO
30 (
mon
oest
ers-
24,d
iest
ers-
6)
2- O
- l -a
lany
l- b -
d -gl
ucos
e (2
0)
3- O
- l -a
lany
l- b -
d -gl
ucos
e (1
2)
6- O
- l -a
lany
l- b -
d -gl
ucos
e (4
7)
O
O
HO
OH
OH
H
H
HH
H
O
O
CH
3
NH
2
O
H3C
H2N
O O
O
CH
3
NH
2
O
OH
OH
H
H
HH
OH
H
OH
2N H3C
2,6-
di- O
- l -a
lany
l- b -
d -gl
ucos
e (1
5)
3,6-
di- O
- l -a
lany
l- b -
d -gl
ucos
e (6
)
O
H3C
O
HO
OH
H
H
OH
HH
O
OH
H2N
H3C
O
OHH
H
OH
H
HO O
OH
OH
H2N
O
OHH
H
OH
HO
HH
O
NH
2
CH
3
O
OH
21 (
only
mon
oest
ers)
2- O
- l -a
lany
l- d -
gala
ctos
e (3
3)
3- O
- l -a
lany
l- d -
gala
ctos
e (3
2)
6- O
- l -a
lany
l- d -
gala
ctos
e (3
5)
O H
OH
H
H
H
HO
O
O
OH
OH
H2N H
3C
O
OH
H
H
H
O
OH
HO
HO
H
O
H2NH3C
O H
OH
H
H
HH
O
OH
O
NH
2
CH
3
O
OH
49 (
mon
oest
ers-
39, d
iest
ers-
10)
3- O
- l -a
lany
l- d -
man
nose
(25
) 4-
O - l
-ala
nyl-
d -m
anno
se (
25)
6- O
- l -a
lany
l- d -
man
nose
(30
)
O
O
O
CH
3
NH
2
O
OH
H
H
HO
H HO
OH
H3C
H2N
OC
H3
H3C
H2N
O
OH
HO
H
O
O
H
H
HO
HO
NH
2
3,6-
di- O
- l -a
lany
l- d -
man
nose
(9)
4,
6-di
- O - l
-ala
nyl-
d -m
anno
se (
11)
OH
O
OHH
O
OH
O
O
NH
2
CH
3 H
3C H2N
O
OO
OHH
O
OH
OH
H
3C H2N
O
O
OO
OH
CH
3
NH
2
O
HO
OH
52 (
mon
o es
ters
-35,
die
ster
-17)
1- O
- l -a
lany
l- d -
fruc
tose
(34
) 6-
O - l
-ala
nyl-
d -fr
ucto
se (
34)
1,6-
di- O
- l -a
lany
l- d -
fruc
tose
(32
)
(con
tinue
d)
88 6 Lipase-Catalysed Preparation of Aminoacyl Esters of Carbohydrates
O
OH
O
O
NH
2
CH
3
OH
HO
OO
OH
O
NH
2
H3C
OH
HO
O
O
OH
O
OH
3C
O
NH
2
CH
3
OH
H2N
9 c (
mon
oest
ers-
6, d
iest
er-3
)
2- O
- l -a
lany
l- d -
arab
inos
e (3
3)
5- O
- l -a
lany
l- d -
arab
inos
e(34
) 2,
5-di
- O - l
-ala
nyl-
d -ar
abin
ose
(33)
H3C
O
OH
OH
O
OH
O
H2N
O
H2N
H3C
OH
OH
OO
OH
CH
3
NH
2
O
OH
OH
OO
O
O
NH
2CH
3
48 (
mon
oest
ers-
23, d
iest
er-2
5)
3- O
- l -a
lany
l- d -
ribo
se (
16)
5- O
- l -a
lany
l- d -
ribo
se
3,5-
di- O
- l -a
lany
l- d -
ribo
se (
52)
OH
OH
OH
OH
HH
OH
OH
H
OO
OHH
H
H
H
O
NH
2
CH
3
OH
HO
CH
3
NH
2
O
HO
H
H
HH
OH
OO
H
H
HO
OHH
H
OH
HO
HO
O
OH
O
O
H
OH
HH
OH
OH
H
OO
OHH
H
H
H
O
NH
2
CH
3O
NH
2CH
3
OH
HO
HO
20
(m
onoe
ster
s-14
, die
ster
-6)
6- O
- l -a
lany
l-la
ctos
e (3
4)
6 ¢ - O
- l -a
lany
l-la
ctos
e (3
4)
6,6 ¢
-di-
O - l
-ala
nyl-
lact
ose
(32)
OH
HO
OH
H
HH
OH
OH
O
H
O
OHH
H
H
HH
OO
H
CH
3
NH
2
O
O
OH
H
H
HH
OH
OO
H
H
OH
O
OHH
H
H
HO
OH
OH
O
O
NH
2
CH
3
OH
OH
H
HH
OH
OO
H
H
O
OHH
H
H
HO
HO
H
CH
3
NH
2
O
OC
H3
NH
2
O O
56 (
mon
oest
ers-
38, d
iest
er-1
8)
6- O
- l -a
lany
l-m
alto
se (
34)
6 ¢ - O
- l -a
lany
l-m
alto
se (
34)
6,6 ¢
-di-
O - l
-ala
nyl-
mal
tose
(32
)
OO
OH
HOO
O
OH
HO
H
H
HH
OH
O
OH
HO
H2N
H3C
8 (o
nly
mon
oest
er)
6- O
- l -a
lany
l-su
cros
e
a l -A
lani
ne,
2 m
mol
; ca
rboh
ydra
tes,
1 m
mol
; R
ML
, 40
% (
w/w
car
bohy
drat
e);
buff
er,
0.1-
mM
(0.
1 m
L o
f 0.
1 M
) ac
etat
e bu
ffer
(pH
4.0
); C
H 2 C
l 2 :D
MF
(v/v
90:
10)
at 4
0°C
; in
cuba
tion
peri
od, 7
2 h.
Con
vers
ion
yiel
ds w
ere
from
HPL
C w
ith r
espe
ct to
l -a
lani
ne c
once
ntra
tion
b Per
cent
age
prop
ortio
ns o
f in
divi
dual
est
ers
wer
e de
term
ined
fro
m th
e pe
ak a
reas
or
from
thei
r cr
oss
peak
s of
the
carb
on-1
3 C
6 an
d C
5 (i
n ca
se o
f pe
ntos
es)
sign
als
in th
e 2-
D
HSQ
CT
spe
ctru
m
c Sev
eral
cro
ss p
eaks
, due
to o
peni
ng a
nd/o
r de
grad
atio
n of
the
fi ve-
mem
bere
d ri
ng d
urin
g es
teri
fi cat
ion
Tab
le 6
.4
(con
tinue
d)
896.3 Synthesis of L-Valyl-D-Glucose
Table 6.5 Preparation of l -alanyl ester of carbohy-drate using lipases from Candida rugosa and porcine pancreas
l -Alanyl ester of carbohydrate CRL a % yield (mmol)
Crude PPL b % yield (mmol)
l -Alanyl- d -glucose 25 (0.50) 78 (0.78) l -Alanyl- d -galactose 22 (0.44) 72 (0.72) l -Alanyl- d -mannose 33 (0.66) 10 (0.10) l -Alanyl- d -fructose 12 (0.24) 67 (0.67) l -Alanyl- d -arabinose 3 (0.06) 27 (0.27) l -Alanyl- d -ribose 12 (0.24) 38 (0.38) l -Alanyl-lactose 27 (0.54) 58 (0.58) l -Alanyl-maltose 17 (0.34) 28 (0.28) l -Alanyl-sucrose 8 (0.16) 8 (0.83)
a l -Alanine, 2 mmol; carbohydrates, 1 mmol; CRL, 40% (w/w carbohydrate); buffer, 0.1-mM (0.1 mL of 0.1 M) phosphate buffer pH 7.0; CH
2 Cl
2 :DMF (v/v 90:10) at 40°C;
incubation period, 72 h. Conversion yields were from HPLC with respect to l -alanine concentration b l -Alanine, 1 mmol; carbohydrates, 1 mmol; crude PPL, 111% (w/w carbohydrate); buf-fer, 0.2-mM (0.2 mL of 0.1 M) acetate buffer pH 5.0; CH
2 Cl
2 : DMF (v/v 90:10) at 40°C;
incubation period, 72 h
Scheme 6.2 Lipase-catalysed synthesis of l -valyl esters of carbohydrates
2007 ) and the esteri fi cation reaction was studied in detail using RML and PPL in terms of incu-bation period, lipase concentrations, buffer (pH and concentration) and substrate concentrations. Optimum conditions determined were:
A CRL concentration of 40% w/w d -glucose exhibited 68% conversion at 72 h at an initial rate
of esteri fi cation of 0.0425 mmol h −1 ; buffer – 0.1-mM (0.1 mL of 0.1 M) phosphate buffer (pH 7.0) – showed the highest yield of 68% (0.68 mmol); CRL steadily increased esteri fi -cation up to 30% enzyme concentration (84%, 0.84 mmol) (Table 6.4 ); both 10% (w/w d -glu-cose) RML and PPL in the presence of 0.1-mM
90 6 Lipase-Catalysed Preparation of Aminoacyl Esters of Carbohydrates
(0.1 mL of 0.1 M) acetate buffer (pH 5.0) showed a maximum yield of 59% (0.59 mmol) and 62% (0.62 mmol), respectively, which thereafter decreased up to 50% of lipase concentration (Table 6.6 ); 3–5 mmol of l -valine gave (Fig. 6.4 ) 0.49 mmol of the ester.
Similarly, 3 mmol d -glucose gave a yield of 0.83 mmol. With both l -valine and d -glucose variation, the esteri fi cation increased up to 3
equivalents, indicating that l -valine and d -glucose are not inhibitors to CRL.
6.3.1 Syntheses of L -Valyl Esters of Carbohydrates
l -Valine ( l -2-amino-3-methylbutanoic acid) is a polar and an essential dietary amino acid
Table 6.6 Effect of lipase concentration on the synthesis of l -valyl- d -glucose a
Lipase concentration (% w/w d -glucose)
Candida rugosa lipase (CRL) yield % (mmol)
Candida rugosa lipase (CRL) b yield % (mmol)
Rhizomucor miehei lipase (RML) c yield % (mmol)
Porcine pancreas lipase (PPL) c yield % (mmol)
10 2 (0.02) 73 (0.73) 59 (0.59) 62 (0.62) 20 6 (0.06) 77 (0.77) 41 (0.41) 45 (0.45) 30 14 (0.14) 84 (0.84) 31(0.31) 33 (0.33) 40 25 (0.25) 68 (0.68) 24 (0.24) 27 (0.27) 50 10 (0.10) 65 (0.65) 25 (0.25) 36 (0.36)
a d -Glucose, 1 mmol, and l -valine, 1 mmol; solvent, CH 2 Cl
2 :DMF (v/v 90:10) at 40°C
b Carried out in the presence of buffer with 100 mL of solvent containing 0.1-mM (0.1 mL of 0.1 M) phosphate buffer (pH 7.0) c Carried out in the presence of buffer with 100 mL of solvent containing 0.1-mM (0.1 mL of 0.1 M) acetate buffer (pH 5.0)
0.31
0.49 0.47
0.48
0.25
0.6
0.83
0.760.72
0
0.2
0.4
0.6
0.8
1
1 2 3 4 5
Est
erific
atio
n yi
eld
(mm
ol)
Substrate concentration (mmol)
L-valine
D-glucose
Fig. 6.4 Effect of substrate concentration on synthesis of l -valyl- d -glucose. CRL, 40% (w/w d -glucose); solvent, CH
2 Cl
2 :DMF (v/v 90:10), at 40°C. l -Valine (■), 1–5 mmol
at 1 mmol d -glucose; d -glucose (●), 1–5 mmol at 1 mmol l -valine and a constant enzyme concentration of 72 mg
916.5 Syntheses of L-Isoleucyl Esters of Carbohydrates
containing isopropyl group as a side chain. Esteri fi cation of l -valine with carbohydrates was carried out using CRL and crude PPL under opti-mal conditions. Table 6.7 shows the HPLC ester yields, types of esters formed and percentage proportions of the individual esters from CRL-catalysed reaction (Somashekar and Divakar 2007 ) .
6.4 L -Leucyl- D -Glucose
6.4.1 Optimum Conditions
In a Rhizomucor miehei (RML)- and porcine pancreas (PPL)-catalysed esterifi cation (Scheme 6.3 , Somashekar and Divakar 2007 ) optimisa-tion of reaction conditions showed the highest yield of 10.8% (0.22 mmol) at 20% enzyme concentration. Better conversions (RML- 36%, PPL-24%) could be achieved with both the enzymes at 5 equivalents of l -leucine. Effect of buffer salts in the range 4.0–8.0 with 40% (w/w d -glucose) RML at 1:2 molar equivalent of d -glucose and l -leucine (Table 6.8 ) showed that 0.2-mM (0.1 mL of 0.1 M) acetate buffer (pH 5.0) gave the highest yield of 63% (0.63 mmol). The effect of various buffer vol-umes in the range 0.05 mM (0.05 mL) to 0.6 mM (0.6 mL) of phosphate buffer (pH 7.0) showed that the esteri fi cation yields increased with increase in the buffer concentration from 0.05-mM (0.05 mL) to 0.6-mM (0.6 mL) phosphate buffer (pH 7.0) with the highest yield of 85% (1.7 mmol).
6.4.2 Selectivity
An attempt to improve the selectivity of ester formation made by reducing the incubation period in case of l -alanyl- d -glucose synthesis using RML gave a conversion yield of 15%. From two-dimensional HSQCT NMR, the formation of three monoesters (6- O - 42%, 3- O - 33% and 2 -O- 25%) with only b -anomer of d -glucose was found, the d -glucose employed being a 40:60 mixture of a - and b -anomers, respectively.
6.4.3 Determination of Critical Micellar Concentration (CMC)
Surfactant property of the aminoacyl esters of carbohydrate was evaluated by determining the critical micellar concentration (CMC) value for l -alanyl- b - d -glucose spectroscopically at 470 nm (Rosenthal and Loussale 1983 ) . The CMC of l -alanyl- b - d -glucose was found to be 2.25 mM (0.056%).
6.4.4 Syntheses of L -Leucyl Esters of Carbohydrates
l -Leucine ( l -2-amino-4-methylpentanoic acid) is a polar and an essential dietary amino acid containing 2-methylpropyl group as a side chain. Compared to l -alanine (solubility 127.3 g L −1 at 25°C) and l -valine (solubility 88.5 g L −1 at 25°C), the solubility of l -leucine in water is low (24.26 g L −1 at 25°C). However, the carbohydrate esters can be expected to exhibit higher solubility in water. Using opti-mum conditions, l -leucyl esters of different carbohydrates were prepared using CRL and crude PPL (Vijayakumar et al. 2004 ) . Table 6.9 shows the HPLC ester yields, types of esters formed and percentage proportions of the indi-vidual esters from CRL catalysis (Somashekar and Divakar 2007 ) .
6.5 Syntheses of L -Isoleucyl Esters of Carbohydrates
l -Isoleucine ( l -2-amino-3-methylpentanoic acid) is a polar and an essential dietary amino acid con-taining 3-methylpropyl group as a side chain. Here also, the solubility of l -isoleucine is low (41.2 g L −1 at 25°C) like of that of l -leucine. Using optimum conditions, l -isoleucyl esters of different carbohydrates were prepared (Somashekar and Divakar 2007 ) by employing CRL and crude PPL (Scheme 6.4 ). Table 6.10 shows the HPLC ester yields, types of esters formed and percentage proportions of the indi-vidual esters from CRL-catalysed reaction.
92 6 Lipase-Catalysed Preparation of Aminoacyl Esters of Carbohydrates
Table 6.7 Syntheses of l -valyl esters of carbohydrates a
l -Valyl esters of carbohydrates (% proportions b ) Yield (%)
HO
OH
HO
H
H
H H O
O
O
CH3
OH
H3C
H2N
O
OH
HH
H
H
HO
OH
OH2N
O
CH3
H3C
OH
O
OH
HH
H
HHO
O
NH2O
CH3
CH3
OH
HO
68 (monoesters-36, diesters-32)
2- O - l -valyl- d -glucose (10) 3- O - l -valyl- d -glucose (12) 6- O - l -valyl- d -glucose (31)
OH2N
O
HOHO
H
H
HHO
O NH2
O
CH3H3C
H3CCH3
OH
OH2N
O
O NH2
O
H
H
H H
OH
O
CH3
H3C
H3CCH3
OH
HO
2,6-di- O - l -valyl- d -glucose (23) 3,6-di- O - l -valyl- d -glucose (24)
O
O
H
H
OH
HHO
OH
CH3H3C
HO
N2H
O
OH
HH
OH
H
HONH2
O
OH
OH
CH3
CH3
CH3CH3
OH
O NH2
O
HOH
H
OH
H H
OH
O
30 (only monoesters)
2- O - l -valyl – d -galactose (48) 3- O - l -valyl- d -galactose (26) 6- O - l -valyl- d -galactose (26)
O
H
OHH
H
H
O
NH2O
OH
CH3
CH3
HOHO
51 (only monoester)
6- O - l -valyl- d -mannose
OOH
OH
HO
OH
O
O
NH2
CH3
CH3
NH2
O
O O
OH
OH
OH
CH3
CH3
HO
NH2
O
O O
OH
OH
O
H3C
CH3
CH3
CH3
NH2
O
HO
34 (monoesters-21, diester-13)
1- O - l -valyl- d -fructose (29) 6- O - l -valyl- d -fructose (34) 1,6-di- O - l -valyl- d -fructose (37)
O
OH
O
O
NH2
OH
CH3
CH3
HO
OO
OH
OH
O
NH2 OH
CH3
CH3
OO
OH
O
O
NH2
O
NH2
OH
CH3
CH3
CH3
CH3
25 c (monoesters-14, diester-11)
2- O - l -valyl- d -arabinose (32) 5- O - l -valyl- d -arabinose (25) 2,5-di -O - l -valyl- d -arabinose (43)
NH2
O
OH
OHO
OH O
CH3
CH3
O
NH2
OHOH
O OOH
CH3
CH3
NH2
O
O
O O
OH
OH
CH3
CH3
CH3
CH3
O
NH2
33 c (monoesters-17, diester-16)
3- O - l -valyl- d -ribose (26) 5- O - l -valyl- d -ribose (26) 3,5-di- O - l -valyl- d -ribose (48)
(continued)
936.5 Syntheses of L-Isoleucyl Esters of Carbohydrates
l -Valyl esters of carbohydrates (% proportions b ) Yield (%)
OH
OH OH
H
H H
OH
OOH
H
OO
OH
HH
H
HOH
O
NH2
OH
CH3
CH3
NH2
O
OHH
H
H H
OH
OOH
H
OHO
OH
HH
H
HOOH
O
OH
CH3
CH3
47 (only monoesters)
6- O - l -valyl-maltose (49) 6 ¢ -O - l -valyl-maltose (51)
O
NH2
O
OH
HO
OO
OH
OHOH
H
H
HH
OH
OOH
CH3
CH3
60 (only monoester)
6- O - l -valyl-sucrose
O
H
H
HH
OH
OH
OHOH
OHCH2
CH2
O NH2
CH3
CH3
52 (only monoester)
1- O - l -valyl- d -mannitol
a l -Valine, 2 mmol; carbohydrates, 1 mmol; CRL, 40% (w/w carbohydrate ); buffer, 0.1-mM (0.1 mL of 0.1 M) phos-phate buffer pH 7.0; CH
2 Cl
2 :DMF (v/v, 90:10) at 40°C; incubation period, 72 h. Conversion yields were from HPLC
with respect to l -valine concentration b Percentage proportions of individual esters were determined from the peak areas or from their cross peaks of the car-bon-13 C6 and C5 (in case of pentoses) signals in the 2-D HSQCT spectrum c Several cross peaks, due to opening and/or degradation of the fi ve-membered ring during esteri fi cation
Table 6.7 (continued)
Scheme 6.3 Lipase-catalysed synthesis of l -leucyl- d -glucose esters in anhydrous organic media
94 6 Lipase-Catalysed Preparation of Aminoacyl Esters of Carbohydrates
Table 6.8 Effect of buffer salts (pH and buffer concentration) on the synthesis of l -leucyl- d -glucose a
pH b Yield % (mmol) pH 7.0 c concn mM Yield % (mmol)
4.0 16 (0.31) 0.05 5 (0.1) 5.0 63 (1.26) 0.1 21 (0.42) 6.0 16 (0.33) 0.2 14 (0.28) 7.0 14 (0.27) 0.4 32 (0.64) 8.0 7 (0.14) 0.6 85 (1.70)
a d -Glucose, 1 mmol, and l -leucine, 2 mmol; incubation period, 72 h; RML, 40% (w/w d -glucose) 100 mL of the solvent containing speci fi ed volumes, concentration and pH of the buffer b Solvent, 100 mL C
6 H
6 :CHCl
3 :DMF (v/v/v 45:45:10) at 60°C. Buffer, 0.2-mM (0.2 mL
of 0.1 M) appropriate pH buffer c Solvent, 100 mL. Buffer, 0.05 mL to 0.6 mL of 0.1 M phosphate (pH 7.0)
Table 6.9 Syntheses of l -leucyl esters of carbohydrates a
l -Leucyl esters of carbohydrates (% proportions b ) Yield (%)
OH
OHOH
H
H
H H O
ONH2
O
CH3
CH3
OH
O
OH
HH
H
H
OHOH
ONH2
O
CH3
CH3
OH
O
OH
HH
H
HOH
OH
O
NH2O
CH3
CH3
OH
43 (monoesters-34, diesters-9)
2- O - l -leucyl- d -glucose (17) 3- O - l -leucyl- d -glucose (20) 6- O - l -leucyl- d -glucose 42)
ONH2
O
HOHO
H
H
H HO
O NH2
OCH3
CH3
CH3
CH3
OH
ONH2
O
O NH2
O
OH
H
H
H H
OH
O
CH3
CH3
CH3
CH3
OH
2,6-di- O - l -leucyl- d -glucose (10) 3,6-di- O - l -leucyl- d -glucose (11)
ONH2
O
OH
OH
H
H
OH
HHO
OH
CH3
CH3
OH
O NH2
O
H
OHH
OH
H H
OH
O
CH3
CH3
21 (only mono esters)
2- O - l -leucyl- d -galactose (48) 6- O - l -leucyl- d -galactose (52)
O
H
OHH
H
H
OHO
NH2
O
OH
OH
CH3
CH3
O
CH3CH3
O
NH2
OHOHH
H
HOHO
OH
H
O
H
OHH
H
HOH
OH
O
NH2O
OH
CH3
CH3
31 (only mono esters)
3- O - l -leucyl- d -mannose (28) 4- O - l -leucyl- d -mannose (30) 6- O - l -leucyl- d -mannose (42)
NH2
O
O O
OH
OH
OH
OH
CH3
CH3
48 (only monoester)
6- O - l -leucyl- d -fructose
(continued)
956.5 Syntheses of L-Isoleucyl Esters of Carbohydrates
Table 6.9 (continued)
l -Leucyl esters of carbohydrates (% proportions b ) Yield (%)
OOH
OH
O
O
NH2
OH
CH3
CH3
OO
OH
OH
O
NH2 OH
CH3
CH3
OO
OH
O
O
NH2
O
NH2
OHCH3
CH3
CH3
CH3
42 (monoesters-24, diester-18)
2- O - l -leucyl- d -arabinose (24) 5- O - l -leucyl- d -arabinose (33) 2,5-di- O - l -leucyl- d -arabinose (43)
NH2
O
OH
OHO
OH O
CH3
CH3
O
NH2
OHOH
O OOHCH3
CH3
NH2
O
O
O O
OH
OH
O
NH2CH3
CH3
CH3
CH3
38 (mono esters-18, diester-20)
3- O - l -leucyl- d -ribose (16) 5- O - l -leucyl- d -ribose (32) 3,5-di -O - l -leucyl- d -ribose (52)
OH
OH OH
H
HH
OH
OOH
H
OO
OH
HH
H
HOH
O
NH2
OH
CH3
CH3
44 (only monoester)
6- O - l -leucyl-maltose
O
NH2
O
OH
HO
OO
OH
OH
OH
H
H
HH
OH
OOH
CH3
CH3
38 (only monoester)
6- O - l -leucyl-sucrose
O
H
H
HH
OH
OH
OHOH
OHCH2
CH2
O NH2 CH3
CH3
ONH2
CH3
CH3
CH3
CH3NH2
O
CH2
CH2O
OHOH
OH
OH
HH
H
H
O
45 (monoester-25, diester-20)
1- O - l -leucyl- d -mannitol (56) 1,6-di- O - l -leucyl- d -mannitol (44)
O
OH
OH
OH
OHH
H
H
OHOH
CH2
CH2
O NH2 CH3
CH3
25 (only monoester)
1- O - l -leucyl- d -sorbitol
a l -Leucine, 2 mmol; carbohydrates, 1 mmol; CRL, 40% (w/w carbohydrate ); buffer, 0.1-mM (0.1 mL of 0.1 M) phos-phate buffer (pH 7.0); CH2Cl2:DMF (v/v 90:10) at 40°C; incubation period, 72 h. Conversion yields were from HPLC with respect to l -leucine concentration b Percentage proportions of individual esters were determined from the peak areas or from their cross peaks of the car-bon-13 C6 and C5 (in case of pentoses) signals in the 2-D HSQCT spectrum c Several cross peaks, due to opening and/or degradation of the fi ve-membered ring during esteri fi cation
96 6 Lipase-Catalysed Preparation of Aminoacyl Esters of Carbohydrates
Scheme 6.4 Lipase-catalysed synthesis of l -isoleucyl esters of carbohydrates
Table 6.10 Syntheses of l -isoleucyl esters of carbohydrates a
l -Isoleucyl esters of carbohydrates (% proportions b ) Yield (%)
O
OH
HH
H
H
OH
OH
ONH2
OCH3 CH3
OH
O
OH
HH
H
HOH
OH
O
NH2O
CH3CH3
OH
47 (only monoesters)
3- O - l -isoleucyl- d -glucose (42) 6- O - l -isoleucyl- d -glucose (58)
ONH2
O
OH
OH
H
H
OH
H HO
OH
CH3
CH3
NH2
O
CH3
OH
OHH
OH
H
H
OH
OOH
CH3
OH
O NH2
O
HOH
H
OH
H H
OH
O
CH3CH3
46 (only monoesters)
2- O - l -isoleucyl- d -galactose(78) 3- O - l -isoleucyl- d -galactose(10) 6- O - l -isoleucyl- d -galactose(12)
O
H
OHH
H
H
OHO
NH2
O
OH
CH3
CH3
O
CH3
O
NH2
OHOHH
H
HOHO
OH
H
CH3
O
H
OHH
H
HOH
OH
O
NH2O
OH
CH3CH3
55 (monoesters-25, diesters-30)
3- O - l -isoleucyl- d -mannose (19) 4- O - l -isoleucyl- d -mannose (13) 6- O - l -isoleucyl- d -mannose (13)
O
NH2
CH3
CH3
OH
O
O
NH2
OOH
H
H
H OH
H
O
CH3
CH3
O
NH2
CH3
H
OO
OHH
H
HOH OH
NH2
O
CH3
O
CH3
CH3
3,6-di- O - l -isoleucyl- d -mannose (27)
4,6-di- O - l -isoleucyl- d -mannose (28)
(continued)
976.5 Syntheses of L-Isoleucyl Esters of Carbohydrates
Table 6.10 (continued)
l -Isoleucyl esters of carbohydrates (% proportions b ) Yield (%)
OH O
OH
OH
OH
O
CH3CH3
O
NH2
O O
OH
OH
OH
OH
NH2
O
CH3CH3
NH2
O
CH3CH3
CH3CH3
O
NH2O
OH
OH
OH
OO
43 (monoesters-28, diester-15)
1- O - l -isoleucyl- d -fructose (36) 6- O - l -isoleucyl- d -fructose (30) 1,6-di- O - l -isoleucyl- d -fructose (34)
OOH
OH
OOH
CH3 CH3
NH2
O
O
NH2
CH3 CH3
OHOH
OH
O O
O
NH2
CH3 CH3
OHO
OH
O O
CH3 CH3
NH2
O
55 c (monoesters-31, diester-24)
2- O - l -isoleucyl- d -arabinose (24) 5- O - l -isoleucyl- d -arabinose (33) 2,5-di- O - l -isoleucyl- d -arabinose (43)
OH
OHO
OH O
CH3CH3
NH2
O
O
NH2
CH3 CH3
OHOO
OH OH
O
NH2
CH3 CH3
O
O O
OH
OH
NH2
53 c (monoesters-38, diester-15)
3- O - l -isoleucyl- d -ribose (52) 5- O - l -isoleucyl- d -ribose (20) 3,5-di -O - l -isoleucyl- d -ribose (28)
OH
OH OH
OH
HH
OH
OH
H
OHO
O
HH
H
HOH OH
ONH2
CH3
CH3
CH3 CH3
NH2
O
OHOHH
H
HH
OH
OO
H
H O
OH
HH
OH
HOOH
OH
CH3 CH3
NH2
O
OHOHH
H
HH
OH
OOH
H
HO
OH
HH
OH
HOOH
O
45 (only monoesters)
2- O - l -isoleucyl-lactose (39) 6- O - l -isoleucyl-lactose (40) 6 ¢ - O - l -isoleucyl-lactose (21)
OH
OH OH
H
HH
OH
OOH
H
OHO
O
HH
H
HOH OH
CH3 CH3
NH2
O
O
NH2
CH3CH3
OHOHH
H
HH
OH
OO
H
OHO
OH
HH
H
HOOH
OH
O
NH2
CH3CH3
OH
O
OH OH
H
HH
OH
OOH
H
OHO
OH
HH
H
HOH
54 (only monoesters)
2- O - l -isoleucyl-maltose (38) 6- O - l -isoleucyl-maltose (40) 6 ¢ - O - l -isoleucyl-maltose (22)
O
NH2
CH3CH3
OH
O
OH
HH
H
HOH
OH
OH
O O
HO
OH
O
22 (only monoester)
6- O - l -isoleucyl – sucrose
(continued)
98 6 Lipase-Catalysed Preparation of Aminoacyl Esters of Carbohydrates
l -Isoleucyl esters of carbohydrates (% proportions b ) Yield (%)
O
H
H
HH
OH
OH
OHOH
OHCH2
CH2
CH3
CH3NH2
O
O
NH2CH3
CH3
O
NH2CH3
CH3CH2
CH2O
OHOH
OH
OH
HH
H
H
O
52 (monoesters-32, diester-20)
1- O - l -isoleucyl- d -mannitol (62) 1,6-di- O - l -isoleucyl- d -mannitol (38)
a l -Isoleucine, 2 mmol; carbohydrates, 1 mmol; CRL, 40% (w/w carbohydrate); buffer, 0.1-mM (0.1 mL of 0.1 M) phosphate buffer (pH 7.0); CH2Cl2:DMF (v/v 90:10) at 40°C; incubation period, 72 h. Conversion yields were from HPLC with respect to l -isoleucine concentration b Percentage proportions of individual esters were determined from the peak areas or from their cross peaks of the car-bon-13 C6 and C5 (in case of pentoses) signals in the 2-D HSQCT spectrum c Several cross peaks, due to opening and/or degradation of the fi ve-membered ring during esteri fi cation
Table 6.10 (continued)
6.6 Synthesis of L -Phenylalanyl- D -Glucose
6.6.1 Optimum Conditions
Lipases from Rhizomucor miehei (RML) and porcine pancreas (PPL) were employed for the reaction (Scheme 6.5 , Lohith et al. 2003 ; Lohith
and Divakar 2005 ) . The extent of esteri fi cation was monitored by HPLC (Fig. 6.5 ).
Both d -glucose and l -phenylalanine are insoluble in many organic solvents. They require solvents like pyridine or DMSO for complete solu-bility. Since these solvents more health hazardous and are also dif fi cult to handle during work up, they were not employed. An attempt was made
Scheme 6.5 Lipase-catalysed synthesis of l -phenylalanyl esters of carbohydrates
996.6 Synthesis of L-Phenylalanyl-D-Glucose
Fig. 6.5 HPLC chromatograph for the reaction mixture of l -phenylalanine and d -glucose. ( a ) Column, aminopro-pyl; mobile phase, acetonitrile to water (80:20 v/v); fl ow rate, 1 mL min −1 ; detector, refractive index. ( b ) Column,
C-18; mobile phase, acetonitrile to water (20:80 v/v); fl ow rate, 1 mL min −1 ; detector, UV at 254 nm. Errors in yields are ±10–15%
to choose the right solvent, which should dissolve enough substrate to carry out the reaction without affecting the enzyme activity or stability. Presence of small amount of DMF in dichloromethane improved the solubility of substrates employed (Table 6.11 ). Presence of dichloromethane and
DMF (90:10 v/v) showed maximum yield of 98% (0.98 mmol). Very low esteri fi cation (<5%) was observed with heptane to DMF (90:10 v/v) and benzene to DMF (90:10 v/v).
Esteri fi cation increased with increasing DMF volume from 5 to 10 mL; thereafter, it decreased
100 6 Lipase-Catalysed Preparation of Aminoacyl Esters of Carbohydrates
Table 6.11 Effect of solvent mixtures on the synthesis of l -phenylala-nyl- d -glucose a
Solvent (90: 10 v/v) % yield RML
% yield PPL
Acetone: DMF 42 46 Benzene: DMF <5% <5% Chloroform: hexane: DMF b 57 32 Dichloromethane: DMF 98 68 Hexane: DMF 49 10 Heptane: DMF <5% <5%
a Conversion yields were from HPLC with respect to l -phenylalanine. Error in yield measurements will be ±10–15%. This applies to all the yields given in the subsequent tables also. Reaction conditions: l -phenylalanine, 1 mmol; d -glucose, 1 mmol; RML/PPL, 90 mg; incubation period, 72 h; solvent, 90 mL solvent with 10 mL of DMF b CH
2 Cl
2 : hexane: DMF (45:45:10 v/v/v)
gradually. At higher volume of DMF (increasing polarity of the solvent mixture), enzyme may undergo denaturation due to stripping of water molecule from the enzyme surface, which is very much essential for maintaining the three-dimensional structural stability. This could lead to lesser yields at higher volume of DMF.
Optimum conditions determined were RML: Incubation period, 72 h at an initial rate of 0.012 mmol h −1 ; enzyme, 40% (w/w enzyme); l -phe, 1 equivalent; buffer, 0.2-mM (0.2 mL of 0.1 M) buffer, pH 7.0 (Tables 6.12 and 6.13 , Fig. 6.6 )
PPL: Enzyme, 50% (w/w enzyme); buffer, 0.1-mM (0.1 mL of 0.1 M) buffer, pH 6.0 (Tables 6.12 and 6.13 )
6.6.2 Reusability of Lipases
Reusability of RML and PPL was studied at an equimolar (2.5 mmol) d -glucose and l -phenylala-nine concentration with PPL (50%, w/w based on d -glucose) and RML (40%, w/w based on d -glu-cose). After completion of each reaction, the enzyme separated from the reaction mixture was air dried and reused for the next reaction. After each cycle, total esteri fi cation activity ( m mol min −1 ) of the enzyme was determined. In case of RML, the enzyme loss after each cycle was about 20–30%, whereas in case of PPL, it was 50–70% as PPL dissolved in water more readily than RML. Reusability was examined up to fourth cycle for RML, but in case of PPL, it was only up to two cycles (Fig. 6.7 ). RML activity decreased gradually
from 91% (1st cycle: total enzyme activity, 99.0 m mol min −1 ) to 10% (4th cycle: total enzyme activity, 9.6 m mol min −1 ), esteri fi cation yields at second and third cycles are 80% (total enzyme activity – 86.4 m mol min −1 ) and 23% (total enzyme activity – 24.1 m mol min −1 ), respectively. However, PPL showed better activity in the second cycle also, 46% (total enzyme activity – 15 m mol min −1 ) in the fi rst cycle and 45% (total enzyme activity – 10 m mol min −1 ) in the second cycle.
6.6.3 Determination of Critical Micellar Concentration (CMC)
In order to evaluate the surfactant property of the aminoacyl esters of carbohydrates, critical micellar concentration (CMC) values were determined by spectroscopic method (Beyaz et al. 2004 ) for l -phe-nylalanyl- d -glucose and l -phenylalanyl-lactose.
Both l -phenylalanyl- d -glucose and l -pheny-lalanyl-lactose showed CMC of 3.25 mM (0.11%) and 9.5 mM (0.6%), respectively.
6.6.4 Optimisation of L -Phenylalanyl- D -Glucose Synthesis Using Response Surface
6.6.4.1 Methodology (RSM) It is widely employed in the lipase-catalysed syn-thesis of esters (Liao et al . 2003 ; Guvenc et al . 2002 ; Montogomery 1991 ) . Chang et al . ( 2003 ) used RSM to optimise the synthesis of hexyl
1016.6 Synthesis of L-Phenylalanyl-D-Glucose
Table 6.12 Effect of lipase concentration on the synthesis of l -phenylala-nyl- d -glucose
Enzyme concentration (w/w based on d -glucose)
Esteri fi cation a % (mmol) RML
Esteri fi cation b % (mmol) PPL
10% 64 (0.64) 62 (1.56) 20% 68 (0.68) 46 (1.15) 30% 67 (0.67) 42 (1.04) 40% 98 (0.98) 68 (1.70) 50% 57 (0.57) 76 (1.89)
a d -Glucose, 1 mmol; l -phenylalanine, 1 mmol; solvent, CH 2 Cl
2 : DMF (90:10 v/v) at
40°C b d -glucose, 2.5 mmol; l -phenylalanine, 2.5 mmol; incubation period, 72 h
Table 6.13 Effect of buffer salts (pH and concentration) on the synthesis of l -phenylalanyl- d -glucose a
pH RML PPL Buffer conc. (mM)
RML PPL
Esteri fi cation b % (mmol)
Esteri fi cation c % (mmol)
Esteri fi cation d % (mmol)
Esteri fi cation e % (mmol)
4.0 79 (0.79) 31 (0.31) 0.05 54 (0.54) 23 (0.23) 5.0 69 (0.69) 47 (0.47) 0.1 67 (0.67) 65 (0.65) 6.0 69 (0.69) 63 (0.63) 0.2 79 (0.79) 63 (0.63) 7.0 67 (0.67) 34 (0.34) 0.3 78 (0.78) 49 (0.49) 8.0 45 (0.45) 44 (0.44) 0.4 69 (0.69) 47 (0.47) – – – 0.5 70 (0.70) 06 (0.06)
a d -Glucose, 1 mmol; l -phenylalanine, 1 mmol; incubation period, 72 h; enzyme, 30% w/w based on d -glucose; solvent, CH
2 Cl
2 : DMF (90:10 v/v) at 40°C
b buffer concentration, 0.2 mM (0.2 mL of 0.1 M); solvent, CHCl 3 : hexane : DMF (45 : 45 : 10 v/v) at 60°C
c buffer concentration, 0.2 mM (0.2 mL of 0.1 M) d buffer, 0.1 M of pH 4.0 acetate buffer; solvent, CHCl
3 : hexane: DMF (45 : 45 : 10 v/v) 60°C
e buffer, 0.1-M pH 6.0 phosphate buffer
butyrate using Lipozyme IM-77. Synthesis of kojic acid monolaurate using lipase from Pseudomonas cepacia was optimised using RSM (Chen et al . 2002 ) . Hence, RSM was employed for the synthesis of l -phenylalanyl- d -glucose also (Lohith et al . 2006b ) .
Design : Central Composite Rotatable Design (CCRD), 32 experiments, 5 variables at 5 levels
Variables : l -phenylalanine concentration in mmol, RML amount in mg, pH, incubation period in h and buffer concentration in mM. Table 6.14 shows the coded and actual levels of the variables employed in the design matrix.
Equation : A second-order polynomial equa-tion was developed to study the effects of the variables on the esteri fi cation yields in terms of linear, quadratic and cross product terms.
Y = -3.13292 + 0.175788*X 1 + 0.004961*X
2 + 0.6
26492*X 3 + 0.004151*X
4 + 6.141212 *X
5 -
0.02203*X 1 *X
1 – 2.2E-05*X
2 *X
2 -0.05053
*X 3 *X
3 - 4.3E-05*X
4 *X
4 + 0.434091*
X 5 *X
5 + 0.001315*X
1 *X
2 - 0.013* X
1 *X
3 +
0.000115*X 1 *X
4 + 0.0825*X
1 * X
5 -0.00052
*X 2 *X
3 + 4.75E-05*X
2 *X
4 - 0.01787*X
2 *X
5 + 0.002052*X
3 *X
4 - 0.3475*X
3 *X
5 - 0.044
17* X 4 * X
5
Y = conversion yield in mmol, X 1 = l -phenylala-
nine concentration (mmol), X 2 = RML concentration
(mg), X 3 = buffer pH, X
4 = incubation period (h) and
X 5 = buffer concentration (mM). Coef fi cients : Microsoft Excel software,
Version 5.0 Analysis of variance (ANOVA) : Microsoft
Excel software
102 6 Lipase-Catalysed Preparation of Aminoacyl Esters of Carbohydrates
0
0.2
0.4
0.6
0.8
0 1 2 3 4 5 6
Yie
ld, m
mol
Substrate concentration, mmol
D-glucose
L-phenylalanine
Fig. 6.6 Effect of substrate concentration on synthesis of l -phenylalanyl- d -glucose. (□) l -phenylalanine , 1–5 mmol; d -glucose, 1 mmol; RML, 30% w/w based on d -glucose. (♦) d -glucose , 1–5 mmol; l -phenylalanine, 1 mmol; RML,
40% w/w based on d -glucose. Reaction conditions: tem-perature, 40°C; incubation period, 72 h; solvent, DMF and CH
2 Cl
2 (10:90 v/v)
91
80
23
10
46 45
0
25
50
75
100
1 2 3 4
Yie
ld, %
No. of cycles
RML PPL
Fig. 6.7 Enzyme reusability in the synthesis of l -pheny-lalanyl- d -glucose. d -glucose and l -phenylalanine, 2.5 mmol each; temperature, 40°C; incubation period, 72 h. RML amount: 1st cycle, 180 mg (total enzyme activ-ity – 99.0 m mol min −1 ); 2nd cycle, 173 mg (total enzyme
activity – 86.4 m mol min −1 ); 3rd cycle, 91 mg (total enzyme activity –24.1 m mol min −1 ); 4th cycle, 77 mg (total enzyme activity – 9.6 m mol min −1 ). PPL amount: 1st cycle, 225 mg (total enzyme activity – 15 m mol min −1 ); 2nd cycle, 66 mg (total enzyme activity 10 m mol min −1 )
Optimisation : Microsoft Excel Solver function The experimental data fi tted the second-order
polynomial equation well as indicated by an R 2 value of 0.7 (Table 6.15 ).
Effect of l -phenylalanine concentration and RML concentration on the extent of esteri fi cation is shown in Fig. 6.8 . At lower l -phenylalanine concentrations, the extent of esteri fi cation
1036.6 Synthesis of L-Phenylalanyl-D-Glucose
Table 6.14 Coded values of the variables and their corresponding actual values used in the design of experiments
Variables −2 −1 0 1 2
l -Phenylalanine in mmol 1 2 3 4 5 RML in mg 27 54 81 108 135 pH 4.0 5.0 6.0 7.0 8.0 Incubation period in h 24 48 72 96 120 Buffer concentration in mM 0.1 0.2 0.3 0.4 0.5
Table 6.15 Experimental design with experimental and predicted yields of l -phenylalanyl- d -glucose based on the response surface equation
Expt. No l -Phenylalanine RML pH Incubation period Buffer conc. Yield expt. mmol Yield pred. mmol
1 −1 −1 −1 −1 1 0.66 0.62 2 −1 −1 −1 1 −1 0.34 0.26 3 −1 −1 1 −1 −1 0.23 0.15 4 −1 −1 1 1 1 0.34 0.35 5 −1 1 −1 −1 −1 0.28 0.16 6 −1 1 −1 1 1 0.27 0.24 7 −1 1 1 −1 1 0.26 0.23 8 −1 1 1 1 −1 0.60 0.52 9 1 −1 −1 −1 −1 0.35 0.30 10 1 −1 −1 1 1 0.45 0.49 11 1 −1 1 −1 1 0.56 0.60
12 1 −1 1 1 −1 0.56 0.55 13 1 1 −1 −1 1 0.76 0.76 14 1 1 −1 1 −1 0.73 0.68 15 1 1 1 −1 −1 0.38 0.33 16 1 1 1 1 1 0.47 0.51 17 0 0 0 −2 0 0.27 0.37 18 0 0 0 2 0 0.45 0.48 19 0 0 −2 0 0 0.25 0.35 20 0 0 2 0 0 0.26 0.28 21 0 −2 0 0 0 0.41 0.44 22 0 2 0 0 0 0.38 0.47 23 −2 0 0 0 0 0.06 0.22 24 2 0 0 0 0 0.69 0.64 25 0 0 0 0 −2 0.27 0.47 26 0 0 0 0 2 0.76 0.69 27 0 0 0 0 0 0.42 0.52 28 0 0 0 0 0 0.63 0.52 29 0 0 0 0 0 0.67 0.52 30 0 0 0 0 0 0.32 0.52 31 0 0 0 0 0 0.81 0.52 32 0 0 0 0 0 0.40 0.52
decreased with increase in RML concentrations. However, beyond 2 mmol of l -phenylalanine (two equivalents to d -glucose employed), the extent of esteri fi cation increased with increase in
RML amounts from 27 mg to 135 mg. At less than 2 mmol of l -phenylalanine, increase in RML concentrations could result in total binding of d -glucose (present to the extent of 1 mmol) pre-
104 6 Lipase-Catalysed Preparation of Aminoacyl Esters of Carbohydrates
Fig. 6.8 Three-dimensional surface plot showing the effect of l -phenylalanine and RML concentration on the extent of esteri fi cation at 0.3-mM (0.3 mL of 0.1 M) pH 6.0 buffer and 72-h incubation period
dominantly to the active site of RML compared to l -phenylalanine, due to competition in binding between l -phenylalanine and d -glucose, thus reducing the possibility of facile transfer of l -phenylalanyl group to d -glucose.
Figure 6.9 shows the effect of RML concen-tration and incubation period on the extent of esteri fi cation. The extent of esteri fi cation decreased with increase in RML concentration at incuba-tion periods below 60 h. However, at incubation
Fig. 6.9 Three-dimensional surface plot showing the effect of RML concentration and incubation period on the extent of esteri fi cation at 3 mmol l -phenylalanine and 0.3-mM (0.3 mL of 0.1 M) pH 6.0 buffer
1056.6 Synthesis of L-Phenylalanyl-D-Glucose
periods above 60 h, esteri fi cation increased at all RML concentrations in the range of 27 mg to 135 mg. Effect of pH and incubation period also showed a similar behaviour.
Effect of l -phenylalanine concentration and pH on the extent of esteri fi cation is shown in Fig. 6.10 in the form of a contour plot. The high-est yield of 0.6 mmol was observed for a very narrow pH range of 4.5–6.5. At lower l -phenyla-lanine concentrations, lower iso-esteri fi cation (<0.6 mmol) regions were observed for a very broad pH range of 4.0–8.0. Increase in conver-sion yields thereafter resulted from a very narrow pH range around 6.0.
RSM study clearly showed that an optimum buffer volume (concentration) in the form of a
micro-aqueous layer sheathing the enzyme and pH essential for facilitating the ionisation states of the active site and charged amino acid residues on the surface (to maintain the active con fi rmation of the enzyme) are highly crucial for this esteri fi cation reaction. An optimum yield of 1.01 mmol was predicted from the optimised conditions of 3 mmol l -phenylalanine, 100 mg of RML, pH 4.8 acetate buffer, 24-h incubation period and 0.5-mM buffer concentration. The experimental yield under such conditions was found to be 0.97 mmol. Validation experiments were also carried out at various random condi-tions predicted by the response plots. Table 6.16 shows results from validation experiments to be in good agreement with predicted yields.
Fig. 6.10 Contour plot showing iso-esteri fi cation regions obtained due to the effect of l -phenylalanine concentra-tion and buffer pH on the extent of esteri fi cation at 84 mg
RML, 72-h incubation period and 0.3-mM (0.3 mL of 0.1 M) buffer concentration
Table 6.16 Validation of experimental data
Expt. No.
L − Phe mmol
RML (mg) pH
Incubation period (h)
Buffer conc. (mM)
Yield predicted mmol
Yield a expt. mmol
1 3.0 50 6.0 72 0.45 0.69 0.64 2 3.0 110 6.0 72 0.30 0.58 0.65 3 3.0 81 5.0 72 0.45 0.65 0.74 4 4.5 81 6.0 72 0.50 0.82 1.07 5 3.0 80 6.0 60 0.30 0.50 0.55 6 4.0 81 6.0 70 0.30 0.60 0.66 7 3.0 100 4.8 24 0.5 1.01 0.97
a Conversion yields were from HPLC. Experimental yields are an average from two experiments
106 6 Lipase-Catalysed Preparation of Aminoacyl Esters of Carbohydrates
6.6.5 L -Phenylalanyl Esters of Carbohydrates
l -Phenylalanine is one of the important aro-matic amino acid containing phenyl group as a side chain. The solubility in water is very less. Using optimum conditions, an attempt was made to prepare l -phenylalanyl esters of differ-ent carbohydrates. The carbohydrates employed were aldohexoses, ketohexose, pentose ( d -arab-inose and d -ribose), disaccharides and carbohy-drate alcohol (Lohith et al. 2007 ) .
The HPLC retention times for l -phenylalanine and the corresponding carbohydrate esters are shown in Table 6.17 . Ester formation was also monitored by TLC, and spots were detected by spraying ninhydrin and 1-naphthol (for reducing sugar detection), and the R
f values are shown in
Table 6.17 . Two-dimensional HSQCT NMR spectroscopy
of the l -phenylalanyl esters of carbohydrates pre-pared by using CRL gave good information on the nature and proportion of esters formed. Table 6.18 shows the ester yields from HPLC, percentage proportions of individual esters determined from the peak areas of the 13 C C6 or C5 (in case of d -arabinose) signals or cross peaks from 2-D NMR. It was also con fi rmed from NMR that out of 11 carbohydrates employed for the reaction, 8 were converted into esters with l -phenylalanine ( d -glucose, d -galactose , d -mannose , d -fructose,
d -arabinose, lactose , maltose and d -mannitol) and very less esteri fi cation (>5%) was observed with d -ribose, sucrose and d -sorbitol.
6.7 L -Prolyl Esters of Carbohydrates
l -Proline is the only amino acid where the amine group is part of a fi ve-membered ring. l -Proline is highly hygroscopic in nature. l -Proline is one of the important active constituents in many of the peptide drugs. Proline derivates like proline esters and its salts act as pharmaceutically active ingredients in tumour treatments (Zoser 2005 ) .
Esteri fi cation was carried out between l -proline and carbohydrates (Scheme 6.6 , d -glucose, d -galac-tose, d -mannose, d -fructose, d -arabinose, d -ribose, lactose, maltose, sucrose, d -mannitol, d -sorbitol) using Candida rugosa lipase (CRL) in the pres-ence of organic solvent (Lohith and Divakar 2007 ) . A 0.2-mM (0.2 mL) pH 4.0 acetate buffer was employed to the reaction mixture to impart the ‘pH memory’ to CRL. The reaction mixture containing the l -amino acid (1 mmol), carbohydrate (1 mmol), CRL (50% w/w based on respective carbohydrate) and the solvent were re fl uxed for a period of 72 h.
Table 6.19 shows the HPLC retention times and R
f values, and Table 6.20 shows ester yields
form HPLC, types of esters formed and percent-age proportions of the individual esters.
Table 6.17 Retention times and R f values of l -phenylalanyl esters of carbohydrates
Compound Retention time (min) a R f values b
l -Phenylalanine 2.2 0.58 l -Phenylalanyl- d -glucose 2.9 & 3.1 c 0.42 l -Phenylalanyl- d -galactose 3.3 & 3.8 c 0.44 l -Phenylalanyl- d -mannose 3.0 & 3.5 c 0.39 l -Phenylalanyl- d -fructose 3.4 & 4.1 c 0.45 l -Phenylalanyl- d -arabinose 4.6 0.47 l -Phenylalanyl-lactose 3.1 0.31 l -Phenylalanyl-maltose 4.5 0.36 l -Phenylalanyl- d -mannitol 2.9 0.52
a Conditions: column, C18; mobile phase, acetonitrile to water (20:80 v/v); fl ow rate, 1 mL min −1 ; detector, UV at 254 nm b A 20 × 20 cm silica plate (mesh size 60–120); mobile phase, butanol to acetic acid to water (70:20:10 v/v/v); peak identi fi cation, ninhydrin (amine); 1-naphthol (sugar) c In case of few esters two ester peaks were detected
1076.8 L-Tryptophanyl Esters of Carbohydrates
Table 6.18 l -Phenylalanyl Esters of Carbohydrates
l -Phenylalanyl esters of carbohydrates Esteri fi cation yield (%) a Esters (% proportion) b
l -Phenylalanyl- d -glucose 79 2-O- l -Phenylalanyl- d -glucose (19) Monoesters – 53 3-O- l -Phenylalanyl- d -glucose (23) Diesters -26 6-O- l -Phenylalanyl- d -glucose (25)
2,6-di-O- l -Phenylalanyl- d -Glucose (17) 3,6-di-O- l -Phenylalanyl- d -glucose (16)
l -Phenylalanyl- d -galactose 45 2-O- l -Phenylalanyl d -galactose (19) Monoesters – 33 3-O- l -Phenylalanyl- d -galactose (20) Diesters – 12 6-O- l -Phenylalanyl- d -galactose (32)
2,6-di-O- l -Phenylalanyl- d -galactose (16) 3,6-di-O- l -Phenylalanyl- d -galactose (13)
l -Phenylalanyl- d -mannose 62 3-O- l -Phenylalanyl d -mannose (19) Monoesters – 47 4-O- l -Phenylalanyl- d -mannose (19) Diesters – 15 6-O- l -Phenylalanyl- d -mannose(38)
3,6-di-O- l -Phenylalanyl- d -mannose ( 12 ) 4,6-di-O- l -Phenylalanyl- d -mannose (12)
l -Phenylalanyl- d -fructose 50 1-O- l -Phenylalanyl d -fructose (72) Monoesters only 6-O- l -Phenylalanyl- d -mannose(28)
l -Phenylalanyl- d -arabinose 64 2-O- l -Phenylalanyl d -arabinose (35) Monoesters – 51 5-O- l -Phenylalanyl- d -arabinose (44) Diesters –13 2,5-di-O- l -Phenylalanyl- d -arabinose (21)
l -Phenylalanyl-lactose 61 6-O- l -Phenylalanyl lactose (42) Monoesters – 46 6 ¢ -O- l -Phenylalanyl lactose (34) Diesters –15 6,6 ¢ di--O- l -Phenylalanyl lactose(24)
l -Phenylalanyl-maltose 60 6-O- l -Phenylalanyl maltose (59) Monoesters only 6 ¢ -O- l -Phenylalanyl maltose (41)
l -Phenylalanyl- d -mannitol 43 1-O- l -Phenylalanyl- d -mannitol(62) Monoesters – 29 1,6 ¢ -di-O- l -Phenylalanyl- d -mannitol (38) Diesters –14
a L-Phenylalanine, 1 mmol; carbohydrates, 1 mmol; CRL, 50% (w/w based on carbohydrate ); buffer, 0.2 mM (0.2 mL of 0.1 M) pH 4.0 acetate buffer; CH
2 Cl
2 : DMF (v/v 90: 10) at 40°C; incubation period, 72 h; conversion yields were
from HPLC with respect to l -phenylalanine concentration b Percentage proportions of individual esters determined from the peak areas of the 13 C C6, C5 (in case of pentoses) signals or from cross peaks of the 2-D HSQCT spectrum
6.8 L -Tryptophanyl Esters of Carbohydrates
l -Tryptophan is one of the important aromatic amino acid with benzopyrrole (indole) side chain. The solubility of l -tryptophan in water is 11.4 g L −1 at 25°C (Chapman Hall 1982 ) . The esteri fi cation reaction was carried out (Lohith and Divakar 2007 ) between l -tryptophan and carbohydrates using CRL under optimum reaction conditions (Scheme 6.7 ). The reaction mixture consisting of equimolar (1 mmol)
concentration of l -tryptophan, carbohydrates and 50% CRL (w/w of respective carbohydrate) in the presence of 0.2-mM (0.2 mL) pH 4.0 acetate buffer in CH
2 Cl
2 to DMF (90:10 v/v) was
re fl uxed for a period of 72 h. The HPLC retention times for l -tryptophan
and its corresponding carbohydrate esters are shown in Table 6.21 . Ester formation was also monitored by TLC, and spots were detected by spraying ninhydrin and 1-naphthol (for reducing sugar detection), and the R
f values are shown in
Table 6.21 .
108 6 Lipase-Catalysed Preparation of Aminoacyl Esters of Carbohydrates
Scheme 6.6 Candida rugosa lipase-catalysed syntheses of l -prolyl esters of carbohydrates
Table 6.19 Retention times and R f values of l -prolyl esters of carbohydrates
Compound Retention time (min) a R f values b
l -Proline 3.0 0.72 l -Prolyl- d -glucose 3.8 0.62 l -Prolyl- d -galactose 3.9 & 4.4 c 0.66 l -Prolyl- d -mannose 3.2 & 4.0 c 0.59 l -Prolyl- d -fructose 3.9 0.61 l -Prolyl- d -ribose 3.2 & 3.8 c 0.57 l -Prolyl-lactose 3.8 0.42 l -Prolyl-maltose 3.8 0.44 l -Prolyl- d -sorbitol 3.1 & 4.1 c 0.57
a Conditions: column, C18; mobile phase, acetonitrile to water (20:80 v/v); fl ow rate, 1 mL min −1 ; detector, UV at 210 nm b TLC, A 20 × 20 cm silica plate (mesh size 60–120); mobile phase, butanol to acetic acid to water (70:20:10 v/v/v); peak identi fi cation, ninhydrin (amino acid); 1-naphthol (sugar) c In case of few esters, two ester peaks were detected
Two-dimensional HSQCT NMR spectros-copy of the l -tryptophanyl esters of carbohy-drates prepared by using CRL gave good information on the nature and proportion of the esters formed. Multiplicities in the a CH of l -tryptophan indicated the esteri fi cation occurred
at more than one hydroxyl group. Table 6.22 shows the ester yields form HPLC, percentage proportions of individual esters determined from the peak areas of the C5 (in case of pentoses) or C6 13 C signals or from cross correlation peaks from 2-D NMR.
1096.10 Spectral Characterisation of L-Alanyl, L-Valyl, L-Leucyl, L-Isoleucyl, L-Prolyl…
Table 6.20 l -Prolyl esters of carbohydrates
l -Prolyl esters of carbohydrates Esteri fi cation yield (%) a Esters (% proportion) b
l -Prolyl- d -glucose 60 2-O- l -Prolyl- d -glucose (26) Monoesters only 3-O- l -Prolyl- d -glucose (26)
6-O- l -Phenylalanyl- d -glucose (38) l -Prolyl- d -galactose 52 2-O- l -Prolyl- d -galactose (20)
Monoesters only 3-O- l -Prolyl- d -galactose (12) 6-O- l -Prolyl- d -galactose (68)
l -Prolyl- d -mannose 62 3-O- l -Prolyl- d -mannose (21) Monoesters only 4-O- l -Prolyl- d -mannose (20)
6-O- l -Prolyl- d -mannose(24) 3,6-di-O- l -Prolyl- d -mannose (19) 4,6-di-O- l -Prolyl- d -mannose (16)
l -Prolyl- d -fructose 61 1-O- l -Prolyl- d -fructose (31) Monoesters – 45 6-O- l -Prolyl- d -mannose(42) Diesters – 16 1,6-di-O- l -Prolyl- d -fructose
l -Prolyl- d -ribose 41 3-O- l -Prolyl- d -ribose (35) Monoesters only 5-O- l -Prolyl- d -ribose (65)
l -Prolyl-lactose 68 6-O- l -Prolyl lactose (58) Monoesters only 6 ¢ -O- l -Prolyl lactose (42)
l -Prolyl maltose 66 2-O- l -Prolyl maltose (29) Monoesters only 6-O- l -Prolyl- d -ribose (38)
6 ¢ -O- l -Prolyl- d -ribose (33) l -Prolyl- d -sorbitol 20 1-O- l -Prolyl- d -sorbitol (73)
Monoesters only 6-O- l -Prolyl- d -sorbitol (27)
a l -Proline, 1 mmol; carbohydrates, 1 mmol; CRL, 50% (w/w based on respective carbohydrate); buffer, 0.2-mM (0.2 mL) pH 4.0 acetate buffer; CH
2 Cl
2 : DMF (90: 10 v/v) at 40°C; incubation period, 72 h; conversion yields were from
HPLC with respect to l -proline concentration b The ester proportions were calculated from the area of respective 13 C signals
6.9 L -Histidyl Esters of Carbohydrates
l -Histidine is a basic amino acid and the solubility in water is 41.9 g L −1 at 25°C (Chahid et al . 1982 ) . Esteri fi cation of l -histidine with carbohydrates was carried out using CRL under optimal reaction conditions of 1 mmol each of l -histidine and car-bohydrates with 50% (w/w based on respective carbohydrate) CRL and 0.2-mM (0.2 mL) pH 4.0 acetate buffer and incubated for 72 h (Scheme 6.8 , Lohith and Divakar 2007 ) . The HPLC retention time and R
f values are shown in Table 6.23 .
The percentage proportions of the individual esters formed were determined by taking the peak areas of the C6 or C5 (in case of pentoses) of 13 C
signals or cross peaks from 2-D NMR. Table 6.24 shows the ester yields form HPLC, types of esters formed and percentage portions of the individual esters.
6.10 Spectral Characterisation of L -Alanyl, L -Valyl, L -Leucyl, L -Isoleucyl, L -Prolyl, L -Phenylalanyl, L -Tryptophanyl and L -Histidyl Esters of Carbohydrates
Two-dimensional HSQCT NMR spectroscopy of all the eight amino acyl esters of carbohydrates gave good information on the nature and propor-
110 6 Lipase-Catalysed Preparation of Aminoacyl Esters of Carbohydrates
Sch
eme
6.7
C
RL
-cat
alys
ed s
ynth
eses
of
l -tr
ypto
phan
yl e
ster
s of
car
bohy
drat
es
1116.10 Spectral Characterisation of L-Alanyl, L-Valyl, L-Leucyl, L-Isoleucyl, L-Prolyl…
Table 6.21 Retention times and R f values of l -tryptophanyl esters of carbohydrates
Compound Retention time (min) a R f values b
l -Tryptophan 4.9 0.62 l -Tryptophanyl- d -glucose 6.9 0.51 l -Tryptophanyl- d -galactose 6.2 0.46 l -Tryptophanyl- d -mannose 6.2 0.49 l -Tryptophanyl- d -fructose 7.1 0.42 l -Tryptophanyl-lactose 5.3 0.39 l -Tryptophanyl-maltose 5.2 0.42 l -Tryptophanyl-sucrose 5.8 0.41 l -Tryptophanyl- d -sorbitol 5.8 0.58
a Conditions: column, C18; mobile phase, acetonitrile to water (20:80 v/v); fl ow rate, 1 mL min −1 ; detector, UV at 254 nm b TLC, 20 × 20 cm silica plate (mesh size 60–120); mobile phase, butanol to acetic acid to water (70:20:10 v/v/v); peak identi fi cation, ninhydrin (amino acid) and 1-naphthol (sugar)
Table 6.22 l -Tryptophanyl esters of carbohydrates
l -Tryptophanyl esters of carbohydrates Esteri fi cation yield (%) a Esters (% proportion) b
l -Tryptophanyl- d -glucose 42 2-O- l -Tryptophanyl- d -glucose (22) Monoesters – 34 3-O- l -Tryptophanyl- d -glucose (21) Diesters – 8 6-O- l -Tryptophanyl- d -glucose (38)
2,6-di-O- l -Tryptophanyl- d -Glucose (10) 3,6-di-O- l -Tryptophanyl- d -glucose (9)
l -Tryptophanyl- d -galactose 27 6-O- l -Tryptophanyl- d -galactose Monoesters only
l -Tryptophanyl- d -mannose 34 6-O- l -Tryptophanyl- d -mannose Monoesters only
l -Tryptophanyl- d -fructose 18 1-O- l -Tryptophanyl- d -fructose (55) Monoesters only 6-O- l -Tryptophanyl- d -mannose(45)
l -Tryptophanyl lactose 42 6-O- l -Tryptophanyl lactose (64) Monoesters only 6 ¢ -O- l -Tryptophanyl lactose (36)
l -Tryptophanyl maltose 70 2-O- l -Tryptophanyl maltose (13) Monoesters – 56 6-O- l -Tryptophanyl maltose (38) Diesters – 14 6 ¢ -O- l -Tryptophanyl maltose (29)
6,6 ¢ -di-O- l -Tryptophanyl maltose (20) l -Tryptophanyl sucrose 7 6-O- l -Tryptophanyl sucrose
Monoesters only l -Tryptophanyl sorbitol 8 1-O- l -Tryptophanyl- d -sorbitol (79)
Monoesters only 6-O- l -Tryptophanyl- d -sorbitol (21)
a l -Tryptophan, 1 mmol; carbohydrates, 1 mmol; CRL, 50% (w/w based on respective carbohydrate ); buffer, 0.2-mM (0.2 mL) pH 4.0 acetate buffer; CH
2 Cl
2 : DMF (90: 10 v/v) at 40°C; incubation period, 72 h; conversion yields were from
HPLC with respect to l -tryptophan concentration b The ester proportions were calculated from the area of respective 13 C signals
tion of the esters formed (Tables 6.25 and 6.26 ). Two-dimensional HSQCT NMR data showed that up fi eld chemical shift values for a -CH from l -alanyl and b -CH
2 from l -valyl, l -leucyl, l -iso-
leucyl, l -prolyl, l -phenylalanyl, l -tryptophanyl and l -histidyl units indicated that the respective amino acids reacted with the carbohydrates and multiple cross peaks indicated that the reaction
112 6 Lipase-Catalysed Preparation of Aminoacyl Esters of Carbohydrates
Scheme 6.8 CRL-catalysed syntheses of l -histidyl esters of carbohydrates
Table 6.23 Retention times and R f values of l -histidyl esters of carbohydrates
Compound Retention time (min) a R f values b
l -Histidine 4.2 0.56 l -Histidyl- d -glucose 4.7 0.43 l -Histidyl- d -mannose 4.7 0.41 l -Histidyl- d -fructose 4.8 0.38 l -Histidyl-maltose 4.8 0.32 l -Histidyl- d -mannitol 4.8 0.43
a Conditions: column, C18; mobile phase, acetonitrile to water (20:80 v/v); fl ow rate, 1 mL min −1 ; detector, UV at 254 nm b TLC, 20 × 20 cm silica plate (mesh size 60–120); mobile phase, butanol to acetic acid to water (70:20:10 v/v/v); peak identi fi cation, ninhydrin (amino acid) and 1-naphthol (sugar)
occurred at more than one hydroxyl group of the carbohydrate molecules employed. In the carbo-hydrates, the respective 1 H and 13 C chemical shifts of atoms corresponding to the reactive positions like C2, C3, C4 and C6 showed up fi eld/down fi eld shifts indicative of mono-/diester formation.
UV spectra of aminoacyl esters of carbohy-drates showed shifts in the s → s * band in the 194–228-nm range and IR carbonyl stretching frequencies in the 1,601–1,690-cm −1 range indi-cating that l -amino acid carboxylic group had
been converted into their corresponding carbo-hydrate esters.
6.11 Discussion
The esteri fi cation potentialities of lipases from Rhizomucor miehei (RML), Candida rugosa (CRL) and porcine pancreas (PPL) were explored in detail for the syntheses of l -alanyl, l -valyl, l -leucyl, l -isoleucyl, l -prolyl, l -phenylalanyl, l -tryptophanyl and l -histidyl esters of carbohy-
1136.11 Discussion
Table 6.24 l -Histidyl esters of carbohydrates
l -Histidyl esters of carbohydrates Esteri fi cation yield (%) a Esters (% proportion) b
l -Histidyl- d -glucose 32 2-O- l -Histidyl- d -glucose (25) Monoesters – 25 3-O- l -Histidyl- d -glucose (24) Diesters – 7 6-O- l -Histidyl- d -glucose (28)
2,6-di-O- l -Histidyl- d -glucose (12) 3,6-di-O- l -Histidyl- d -glucose (11)
l -Histidyl- d -mannose 72 3-O- l -Histidyl- d -mannose (28) Monoesters only 6-O- l -Histidyl- d -mannose (72)
l -Histidyl- d -fructose 58 6-O- l -Histidyl- d -fructose Monoesters only
l -Histidyl maltose 58 2-O- l -Histidyl maltose (38) Monoesters – 42 6 ¢ -O- l -Histidyl maltose (34) Diesters – 16 6,6 ¢ -di-O- l -Histidyl maltose (28)
l -Histidyl mannitol 62 1-O- l -Histidyl- d -mannitol Monoesters only
a l -Histidine, 1 mmol; carbohydrates, 1 mmol; CRL, 50% (w/w based on respective carbohydrate ); buffer, 0.2-mM (0.2 mL) pH 4.0 acetate buffer; dichloromethane: DMF (90: 10 v/v) at 40°C; incubation period, 72 h; conversion yields were from HPLC with respect to l -histidine concentration b The ester proportions were calculated from the area of respective 13 C signals
drates ( d -glucose, d -galactose, d -mannose, d -fruc-tose, d -arabinose, d -ribose, lactose, maltose, sucrose, d -sorbitol, d -mannitol) using unprotected and unactivated amino acids and carbohydrates.
An experimental set-up for these esteri fi cation reactions has been developed which maintains a very low water activity ( a
w = 0.0054). The set-up
involves re fl uxing an appropriate amount of l -amino acid and carbohydrate in the presence of buffer salts and lipases in the speci fi ed low boiling solvent mixture. The condensed vapours of the solvent were passed through a desiccant before being returned into the reaction mixture, which facilitates complete removal of water of reaction. This set-up uses larger concentrations of substrates and lesser amounts of the enzymes and results in higher conversions. The experi-mental set-up maintained a low water activity ( a
w = 0.0054) due to azeotropic distillation and
recycling the solvent back into the reaction sys-tem after passing through a bed of desiccant. Even the water of reaction formed could also be used to constitute the micro-aqueous layer around the enzyme and the excess water could be removed by azeotropic distillation. The same could occur even with the addition of added
enzyme (with little water content) and buffer volume. The added carbohydrate molecule could also reduce the water content of the reaction mixture. Adachi and Kobayashi ( 2005 ) have reported that the hexose which is more hydrated decreased the water activity in the system and shifts the equilibrium towards synthesis. All these factors lead to maintenance of an equilib-rium concentration of water around the enzyme all the time. Hence, thermodynamic binding equilibria interplayed by inactivation and inhi-bition along with maintenance of an optimum water activity could be governing this reaction as re fl ected by the extent of conversion under different reaction conditions of added buffer, enzyme and substrate concentrations.
Lipase-catalysed esteri fi cation reactions of l -alanyl- d -glucose, l -valyl- d -glucose, l -leucyl- d -glucose, l -phenylalanyl- d -glucose and l -phe-nylalanyl-lactose were optimised in terms of incubation period, solvents, lipase concentrations, substrate concentrations, buffer pH and its concentrations and lipase reusability. The optimum conditions determined for these esteri fi cation reactions by studying the effect of variables like incubation period, enzyme and
114 6 Lipase-Catalysed Preparation of Aminoacyl Esters of Carbohydrates
Tab
le 6
.25
Perc
enta
ge y
ield
s an
d pr
opor
tions
of l
-ala
nyl e
ster
s of
car
bohy
drat
es fr
om R
ML
and
l -v
alyl
, l -l
eucy
l and
l -i
sole
ucyl
est
ers
of c
arbo
hydr
ates
CR
L c
atal
ysed
reac
tions
a
Car
bohy
drat
e l -
Ala
nyl
l -V
alyl
l -
Leu
cyl
l -Is
oleu
cyl
Yie
ldb
% P
ropo
rtio
ns
Yie
ld b
% P
ropo
rtio
ns
Yie
ld b
% P
ropo
rtio
ns
Yie
ld b
% P
ropo
rtio
ns
d -gl
ucos
e 30
2-
O -E
ster
(20
) 68
2-
O -E
ster
(10
) 43
2-
O -E
ster
(17
) 47
3 -
O -E
ster
(42
)
3- O
-Est
er (
12)
3- O
-Est
er (
12)
3- O
-Est
er (
20)
6- O
-Est
er (
58)
6- O
-Est
er (
47)
6- O
-Est
er (
31)
6- O
-Est
er (
42)
2,6-
di- O
-Est
er (
15)
2,6-
di- O
-Est
er (
23)
2,6-
di- O
-Est
er (
10)
3,6-
di-O
-Est
er (
6)
3,6-
di- O
-Est
er (
24)
di- O
-Est
er (
12)
d -ga
lact
ose
21
2- O
-Est
er (
33)
30
2- O
-Est
er (
48)
21
2- O
-Est
er (
48)
46
2- O
-Est
er (
78)
3- O
-Est
er (
32)
3- O
-Est
er (
26)
6- O
-Est
er (
52)
3- O
-Est
er (
10)
6- O
-Est
er (
35)
6- O
-Est
er (
26)
6- O
-Est
er (
12)
d -m
anno
se
49
3- O
-Est
er (
25)
51
6- O
-Est
er
31
3- O
-Est
er (
28)
55
3- O
-Est
er (
19)
4- O
-Est
er (
25)
4- O
-Est
er (
30)
4- O
-Est
er (
13)
6- O
-Est
er (
30)
6- O
-Est
er (
42)
6- O
-Est
er (
13)
3,6-
di- O
-Est
er (
9)
3,6-
di- O
-Est
er (
27)
4,6-
di- O
-Est
er (
11)
4,6-
di- O
-Est
er (
28)
d -fr
ucto
se
52
1-O
-Est
er (
34)
34
1- O
-Est
er (
29)
48
6- O
-Est
er
43
1- O
-Est
er (
36)
6-O
-Est
er (
34)
6- O
-Est
er (
34)
6- O
-Est
er (
30)
1,6-
di-O
-Est
er (
32)
1,6-
di- O
-Est
er (
37)
1,6-
di- O
-Est
er (
34)
d -ar
abin
ose
9 2-
O -E
ster
(33
) 25
2-
O -E
ster
(32
) 42
2-
O -E
ster
(24
) 55
2-
O -E
ster
(24
)
5- O
-Est
er (
34)
5- O
-Est
er (
25)
5- O
-Est
er (
33)
5- O
-Est
er (
33)
2,5-
di- O
-Est
er (
33)
2,5-
di- O
-Est
er (
43)
2,5-
di- O
-Est
er (
43)
2,5-
di- O
-Est
er (
43)
d -ri
bose
48
3-
O -E
ster
(16
) 33
3-
O -E
ster
(26
) 38
3-
O -E
ster
(16
) 53
3-
O -E
ster
(52
)
5- O
-Est
er (
32)
5- O
-Est
er (
26)
5- O
-Est
er (
32)
5- O
-Est
er (
20)
3,5-
di- O
-Est
er (
52)
3,5-
di- O
-Est
er (
48)
3,5-
di- O
-Est
er (
52)
3,5-
di- O
-Est
er (
28)
lact
ose
20
6- O
-Est
er (
34)
45
2- O
-Est
er (
39)
6 ¢ - O
-Est
er (
34)
– –
– –
6- O
-Est
er (
40)
6,6 ¢
-di -
O -E
ster
(32
) 6 ¢
-O -E
ster
(21
)
mal
tose
56
6-
O -E
ster
(34
) 47
6-
O -E
ster
(49
) 44
6-
O-E
ster
54
2-
O -E
ster
(38
)
6 ¢ - O
-Est
er (
34)
6 ¢ - O
-Est
er (
51)
6- O
-Est
er (
40)
6,6 ¢
-di-
O -E
ster
(32
) 6 ¢
-O -E
ster
(22
)
sucr
ose
8 6-
O -E
ster
60
6-
O -E
ster
38
6-
O -E
ster
22
6-
O -E
ster
d -m
anni
tol
– 52
1-
O -E
ster
45
1-
O -E
ster
(56
) 52
1-
O -E
ster
(62
)
1,6-
di- O
-Est
er (
44)
1,6-
di- O
-Est
er (
38)
d -so
rbito
l –
– –
– 25
1-
O -E
ster
–
–
a Con
fi rm
atio
n of
est
ers
and
thei
r pe
rcen
tage
pro
port
ions
wer
e de
term
ined
by
2-D
HSQ
CT
NM
R. C
onve
rsio
n yi
elds
are
an
aver
age
from
two
expe
rim
ents
b Y
ield
s fr
om H
PLC
. Err
ors
in y
ield
mea
sure
men
ts w
ill b
e w
ithin
±10
%
1156.11 Discussion
Tab
le 6
.26
Pe
rcen
tage
yie
lds
and
prop
ortio
ns o
f l -
prol
yl, l
-phe
nyla
lany
l, l -
tryp
toph
anyl
and
l -h
istid
yl e
ster
s of
car
bohy
drat
es f
rom
CR
L c
atal
ysed
rea
ctio
ns a
Car
bohy
drat
e l -
Prol
yl
l -Ph
enyl
alan
yl
l -T
rypt
opha
nyl
l -H
istid
yl
Yie
ld b
% P
ropo
rtio
ns
Yie
ld b
% P
ropo
rtio
ns
Yie
ld b
% P
ropo
rtio
ns
Yie
ld b
% P
ropo
rtio
ns
d -gl
ucos
e 62
2-
O -E
ster
(26
) 79
2-
O -E
ster
(19
) 42
2-
O -E
ster
(22
) 32
2-
O -E
ster
(25
) 3-
O -E
ster
(26
) 3-
O -E
ster
(23
) 3-
O -E
ster
(21
) 3-
O -E
ster
(24
) 6-
O -E
ster
(48
) 6-
O -E
ster
(25
) 6-
O -E
ster
(38
) 6-
O -E
ster
(28
) 2,
6-di
- O -E
ster
(17
) 2,
6-di
- O -E
ster
(10
) 2,
6-di
- O -E
ster
(12
) 3,
6-di
- O -E
ster
(16
) 3,
6-di
- O -E
ster
(9)
3,
6-di
- O -E
ster
(11
) d -
gala
ctos
e 52
2-
O -E
ster
(20
) 45
2-
O -E
ster
(19
) 27
6-
O -E
ster
44
–
3- O
-Est
er (
12)
3- O
-Est
er (
20)
6- O
-Est
er (
68)
6- O
-Est
er (
32)
2,6-
di- O
-Est
er (
16)
3,6-
di- O
-Est
er (
13)
d -m
anno
se
40
3- O
-Est
er (
21)
62
3- O
-Est
er (
18)
34
6- O
-Est
er
72
3- O
-Est
er (
28)
4- O
-Est
er (
20)
4- O
-Est
er (
18)
6- O
-Est
er (
72)
6- O
-Est
er (
24)
6- O
-Est
er (
39)
3,6-
di- O
-Est
er (
19)
3,6-
di- O
-Est
er (
13)
4,6-
di- O
-Est
er (
16)
4,6-
di- O
-Est
er (
12)
d -fr
ucto
se
61
1- O
-Est
er (
31)
50
1- O
-Est
er (
28)
18
1- O
-Est
er (
45)
58
6- O
-Est
er
6- O
-Est
er (
42)
6- O
-Est
er (
72)
6- O
-Est
er (
55)
1,6-
di- O
-Est
er (
27)
d -ar
abin
ose
– –
64
2- O
-Est
er (
35)
– –
– –
5- O
-Est
er (
44)
2,5-
di- O
-Est
er (
21)
d -ri
bose
41
3-
O -E
ster
(35
) –
– –
– –
– 5-
O -E
ster
(65
) la
ctos
e 68
2-
O -E
ster
(27
) 61
6-
O -E
ster
(42
) 42
6-
O -E
ster
(64
) –
– 6-
O -E
ster
(38
) 6 ¢
- O -E
ster
(31
) 6 ¢
- O -E
ster
(36
)
6 ¢ - O
-Est
er (
35)
6,6 ¢
-di-
O -E
ster
(27
)
(con
tinue
d)
116 6 Lipase-Catalysed Preparation of Aminoacyl Esters of Carbohydrates
Car
bohy
drat
e l -
Prol
yl
l -Ph
enyl
alan
yl
l -T
rypt
opha
nyl
l -H
istid
yl
Yie
ld b
% P
ropo
rtio
ns
Yie
ld b
% P
ropo
rtio
ns
Yie
ld b
% P
ropo
rtio
ns
Yie
ld b
% P
ropo
rtio
ns
mal
tose
66
2-
O -E
ster
(29
) 60
6-
O -E
ster
(59
) 70
2-
O -E
ster
(13
) 58
6-
O -E
ster
(40
) 6-
O -E
ster
(38
) 6 ¢
- O -E
ster
(41
) 6-
O -E
ster
(38
) 6 ¢
- O -E
ster
(32
)
6 ¢ - O
-Est
er (
33)
6 ¢ - O
-Est
er (
29)
6,6 ¢
-di-
O E
ster
(28
)
6,6 ¢
-di-
O -E
ster
(20
) su
cros
e –
– –
– 7
6- O
-Est
er
– –
d -so
rbito
l 20
1-
O -E
ster
(73
) –
– 8
1- O
-Est
er (
79)
8 –
6- O
-Est
er (
27)
6- O
-Est
er (
21)
d -m
anni
tol
– –
43
1- O
-Est
er (
62)
– –
62
1- O
-Est
er
1,6-
di- O
-Est
er (
38)
a Con
fi rm
atio
n of
est
ers
and
thei
r pe
rcen
tage
pro
port
ions
wer
e de
term
ined
by
2-D
HSQ
CT
NM
R. C
onve
rsio
n yi
elds
are
an
aver
age
from
two
expe
rim
ents
b Y
ield
s fr
om H
PLC
. Err
ors
in y
ield
mea
sure
men
ts w
ill b
e ±
10%
Tab
le 6
.26
(c
ontin
ued)
1176.11 Discussion
substrate concentrations, pH and buffer concen-tration clearly explain the behaviour of the lipases.
Most of the parameters show that esteri fi cation increases up to a certain point, and thereafter they remain as such or decrease a little. This complex esteri fi cation reaction is not controlled by kinetic factors or thermodynamic factors or water activ-ity alone. Use of lower enzyme concentrations did not result in thermodynamic yields. The ther-modynamic binding equilibria regulate the con-centrations of the unbound substrates at different enzyme and substrate concentrations and thereby conversion as the reaction proceeds with time. At lesser enzyme concentrations, for a given amount of substrates (enzyme/substrate ratio low), rapid exchange between bound and unbound forms of both the substrates with the enzyme (on a weighted average based on binding constant val-ues of both the substrates) leaves substantial number of unbound substrate molecules at the start of the reaction which decrease progressively as conversion takes place (Romero et al . 2005 ; Marty et al . 1992 ) . This becomes more so, if one of them binds more fi rmly to the enzyme than the other (higher binding constant value) as the respective enzyme/substrate ratios change (dur-ing the course of the reaction) unevenly till the conversion stops due to total predominant bind-ing (inhibition). At intermediatory enzyme con-centrations, such a competitive binding results in a favourable proportion of bound and unbound substrates to effect quite a good conversion. At higher enzyme concentrations, most of the sub-strates would be in the bound form leading to inhibition and lesser conversion (higher enzyme/substrate ratios). Also, the esteri fi cation reaction requires larger amount of enzyme compared to hydrolysis. While this leads to lesser selectivity, they also give rise to varying bound and unbound substrate concentrations till the conversion ends. For a given amount of enzyme and substrates, there is no increase in conversion beyond 72 h to 120 h. Longer incubation periods of especially lesser enzyme concentrations could also result in partial enzyme inactivation. However, not all the enzymes are inactivated before the end of the reaction.
The present study investigated the effect of buf-fer salts on this esteri fi cation reaction which ren-dered ‘pH tuning’ of the enzyme, besides providing optimum water activity necessary for the better per-formance of the enzyme. All the three lipases, RML, CRL and PPL, showed higher conversions when a small amount of buffer salt was employed. By imparting ‘pH memory’ or ‘pH tuning’, the cata-lytic activity of the lyophilised subtilisin Carlsberg in the pH range 5.0–11.0, in organic solvents like acetonitrile and 3-pentanone was reported to be enhanced (Xu and Klibanov 1996 ) . The enzymatic activity of subtilisin cross-linked crystals in anhy-drous 3-pentanone was accelerated by the addition of organic soluble (a mixture of a suitable acid and sodium salt) buffers (Xu and Klibanov 1996 ) . The enantioselectivity of Candida antarctica lipase B in organic media was increased by ‘pH tuning’ of the enzyme by the addition of certain buffer salts which altered the protonation state of the enzyme and selectively tuned enantioselectivity and catalytic activity (Quiros et al . 2002 ) . Similarly, the present work also showed enhanced activity of RML and CRL in the presence of buffer salts. However, buffer salts did not enhance esteri fi cation with PPL.
Besides imparting ‘pH memory’ or ‘pH tun-ing’, added water is essential for the integrity of the three-dimensional structure of the enzyme molecule and therefore its activity (Dordick 1989 ) . Zaks and Klibanov ( 1986 , 1988 ) reported that at low water activities, the lower the solvent polarity, the higher the enzyme activity. Beyond the critical water concentration, esteri fi cation decreases because the size of the water layer formed around the enzyme retards the transfer of acyl donor to the active site of the enzyme (Humeau et al . 1998 ; Camacho-Paez et al . 2003a, b ) and also the water layer surrounding the enzymes makes enzyme to be more fl exible by forming multiple hydrogen bonds and interacting with organic solvent causing denaturation (Valiveti et al . 1991 ) . Increase in buffer volume affected this esteri fi cation reaction signi fi cantly. It could increase the water activity of the system in the initial stages by increasing the thickness of the micro-aqueous layer around the enzyme. Higher volumes of the buffer in the micro-aqueous layer could also cause slight inactivation of the enzyme
118 6 Lipase-Catalysed Preparation of Aminoacyl Esters of Carbohydrates
due to increase in salt concentration beyond a critical point. Partridge et al. ( 2001 ) reported that when an enzyme is suspended in a low-water organic solvent, the counter ions are in closer contact with the opposite charges on the enzyme because of the lower dielectric constant of the medium. Thus, protonation of the ionisable groups on the enzyme could be controlled by the type and availability of these ions as well as hydrogen ions resulting in a ‘pH memory’ or ‘pH tuning’. The third factor is the increase in ionic strength which could play a favourable role in esteri fi cation. Optimum pH were found to be: pH 4.0 for RML and pH 5.0 for PPL in case of l -alanyl- d -glucose, pH 7.0 for CRL in case of l -valyl- d -glucose and pH 5.0 for RML in case of l -leucyl- d -glucose reactions. This clearly indicate a slight unfavourable conformational change in the enzyme at about pH 4.0–6.0 lead-ing to lesser conversion beyond pH 4.0 and 5.0 for RML, 5.0 for PPL and 7.0 for CRL.
In case of alkyl side chain containing amino acids, l -alanine, l -valine, l -leucine and l -isoleu-cine, aldohexoses ( d -glucose, d -mannose and d -galactose), ketohexose ( d -fructose), pentose ( d -ribose) and the disaccharides (maltose) showed better conversions with all the four amino acids employed. Least conversions were observed for carbohydrate alcohols and sucrose esters. l -Valyl esters (25–78%) as well as l -leucyl esters (21–65%) showed better conversion than l -alanyl esters (3–78%) and l -isoleucyl esters (9–55%). Among the lipases employed, Candida rugosa lipase and porcine pancreas lipase have shown better conversions than Rhizomucor miehei lipase. l -Alanine, l -valine and l -leucine with d -glucose and l -alanine and l -isoleucine with d -mannose gave fi ve diastereomeric esters. Both d -arabinose and d -ribose have shown three diastereomeric esters with all the amino acids employed. Lactose did not react with l -valine and l -leucine and d -sorbitol with l -alanine, l -valine and l -isoleucine. l -Alanyl-sucrose, l -valyl- d -mannose, l -valyl-sucrose, l -leucyl-maltose, l -leucyl-sucrose and l -isoleucyl-sucrose formed only 6- O -ester. Carbohydrates like lactose, d -mannitol and d -sorbitol reacted selectively depending on the amino acid indicating that they
may not be good nucleophiles, probably could be due to more hydrogen bonding propensity for d -mannitol and d -sorbitol and more steric hindrance in case of lactose. Loss of speci fi city could be due to use of larger amount of enzymes (about 40% w/w carbohydrate), which gave a large number of esters.
Among the three aromatic amino acids and l -proline investigated, l -tryptophan showed lesser conversion (7–70%) to esters compared to the other three amino acids (20–79%). l -Trypto-phan could not form stable acyl enzyme complex due to its bulkiness and its transfer to the carbo-hydrate moiety could also be sterically dif fi cult. l -Prolyl as well as l -phenylalanyl esters showed better conversion (20–79%) followed by l -histi-dyl esters (32–72%, Table 6.24 ). l -Histidine reacted only with d -glucose, d -mannose, d -fruc-tose, maltose and d -mannitol. l -Histidine could be a less ef fi cient acyl donor compared to the other amino acids. This could be due to the extra hydrogen bonding potentialities of the imidazo-lium hydrogens of histidine compared to the other amino acids studied.
d -Arabinose, d -mannitol and sucrose in case of l -proline, d -ribose, sucrose and d -sorbitol in case of l -phenylalanine, d -arabinose, d -ribose and d -mannitol in case of l -tryptophan did not undergo any reaction. l -Tryptophanyl- d -galactose, l -trypto-phanyl- d -mannose, l -tryptophanyl-sucrose, l -his-tidyl- d -fructose and l -histidyl- d -mannitol formed only 6- O -ester. d -Ribose reacted only with l -pro-line and d -arabinose only with l -phenylalanine.
Certain carbohydrates containing axial hydroxyl groups, like C2 position in d -mannose and d -ribose and C4 position in d -galactose, have not reacted indicating that esteri fi cation with axial secondary hydroxyl group is dif fi cult to achieve, especially with bulky acyl donor amino acids. Carbohydrates like d -arabinose, d -ribose, sucrose, d -sorbitol and d -mannitol reacted selectively indicating that they are not very good acyl acceptors. This could be due to smaller size of the carbohydrate molecule in case of d -arabinose and d -ribose resulting in fi rm binding, more hydrogen bonding propensity in case of the linear carbohydrate alcohols, d -sorbitol and d -mannitol and steric hindrance in case of sucrose. In case of aldopentoses ( d -arabinose and
1196.11 Discussion
d -ribose), NMR spectrum clearly indicated degra-dation and/or ring opening during the reaction. In case of l -alanyl- d -ribose and l -valyl- d -ribose, opening of the fi ve-membered ring during esteri fi cation was noticed by observation of a large number of signals in the 3.0–5.0-ppm ( 1 H) and 63–75-ppm ( 13 C) region which could be due to excess strain on the ring due to introduction of bulky amino acid groups to d -ribose OH groups.
Two-dimensional HSQCT NMR con fi rmed that only monoesters (1-O-, 2-O-, 3-O-, 4-O-, 5-O-, 6-O- and 6 ¢ -O-) and in most of the cases few diesters (1,6-di-O-, 2,5-di-O-, 2,6-di-O-, 3,6-di-O-, 4,6 ¢ -di-O- and 6,6 ¢ -di-O-) were found to be formed in this esteri fi cation reaction. Nature of the products clearly indicated that primary hydroxyl groups of the carbohydrates (1- O -, 5- O -, 6- O - and 6 ¢ - O -) esteri fi ed predominantly over the secondary hydroxyl groups (2- O -, 3- O - and 4- O -). Among the secondary hydroxyl groups, 4- O -ester was formed only in case of d -mannose. Out of secondary hydroxyl groups, 2-O, 3-O and 4-O were found to be esteri fi ed to different extents depending on the l -amino acids, lipase and the carbohydrate employed. NMR spectroscopy also indicated that both a - and b -anomers reacted in case of d -glucose, d -galac-tose, d -mannose, d -arabinose, d -ribose, lactose and maltose. The anomeric hydroxyl groups did not react. Carbohydrates containing axial hydroxyl groups in axial position like C2 in d -mannose and d -ribose and C4 in d -galactose have not reacted, indicating that esteri fi cation with axial secondary hydroxyl groups are dif fi cult, especially with alkyl amino acyl donors. Lesser incubation periods gave rise to only monoesters. The anomeric hydroxyl groups of carbohydrate molecules did not react because of rapid glycosidic ring opening and closing process.
About 128 l -aminoacyl esters of carbohy-drates have been synthesised in the present work, and about 116 esters are reported for the fi rst time. The new esters reported are l -alanyl- d -glu-cose, l -alanyl- d -galactose, l -alanyl- d -mannose, l -alanyl- d -fructose, l -alanyl- d -arabinose, l -ala-nyl- d -ribose, l -alanyl-lactose, l -alanyl-maltose, l -alanyl-sucrose, l -valyl- d -glucose, l -valyl- d -
galactose, l -valyl- d -mannose, l -valyl- d -fruc-tose, l -valyl- d -arabinose, l -valyl- d -ribose, l -valyl-maltose , l -valyl-sucrose, l -valyl- d -man-nitol, l -leucyl- d -glucose, l -leucyl- d -galactose, l -leucyl- d -mannose, l -leucyl- d -fructose, l -leucyl- d -arabinose, l -leucyl- d -ribose, l -leucyl-maltose, l -leucyl-sucrose, l -leucyl- d -mannitol, l -leucyl- d -sorbitol, l -isoleucyl- d -glucose , l -iso-leucyl- d -galactose , l -isoleucyl- d -mannose, l -isoleucyl- d -fructose, l -isoleucyl- d -arabinose, l -isoleucyl- d -ribose, l -isoleucyl-lactose, l -iso-leucyl-maltose, l -isoleucyl-sucrose and l -isole-ucyl- d -mannitol, l -prolyl- d -glucose, l -prolyl- d -galactose, l -prolyl- d -mannose, l -prolyl- d -ribose, l -prolyl- d -fructose, l -prolyl-lactose, l -prolyl-maltose, l -prolyl- d -sorbitol, l -phenylalanyl- d -arabinose, l -tryptophanyl- d -mannose, l -tryptophanyl- d -galactose, l -tryptophanyl- d -fructose, l -tryptophanyl-lactose, l -tryptophanyl-malt-ose, l -histidyl- d -glucose, l -histidyl- d -mannose, l -histidyl- d -fructose, l -histidyl-maltose and l -histidyl- d -mannitol.
In case of l -alanyl- d -glucose, only b -anomer of d -glucose reacted, the d -glucose employed being a 40:60 mixture of a - and b -anomers, respectively. The anomeric composition of d -glucose employed for the reaction was 40:60 ( a : b ), and the equal peak areas of anomeric H-1 chemical shift values observed at 4.24 and 4.0 ppm indicated that either both the anomers have reacted to equal extent (1:1) or d -glucose had undergone mutarotation in case of l -valyl and l -leucyl esters of d -glucose. Only monoesters were formed with only b -anomer of d -glucose, when an attempt was made to improve the selectivity of ester formation by decreasing the incubation period in case of l -alanyl- d -glucose synthesis using RML.
Commercial crude PPL preparations contain variety of estero-/lipolytic enzymes with low PPL concentrations (Segura et al . 2006 ; Birner-Grunberger et al . 2004 ) which could also perform facile esteri fi cation. Hence, a small amount of esters formed from esterases along with those of lipases in the present reaction cannot be ruled out. Since the reactions were carried out at a low temperature of 40–60°C, the formation of pep-tide was less than 3%, even though unprotected l -amino acid was used for the reaction. NMR
120 6 Lipase-Catalysed Preparation of Aminoacyl Esters of Carbohydrates
data clearly indicated that no Maillard reaction occurred. Under these reaction conditions, for-mation of Maillard reaction products is quite likely. For instance, Maillard and Pictet–Spengler phenolic condensation products were reported in the reaction between phenolic amino acids and d -glucose in phosphate buffer at different pH from 5.0 to 9.0 at 90°C (Manini et al . 2005 ) . Similarly Maillard products from the reaction between d -glucose and N- t -boc- l -lysine incu-bated with aminoguanidine in phosphate buffer (pH 7.4) at 70°C was also reported (Reihl et al . 2004 ) . No such Maillard reaction type products were detected by mass as well as NMR in the present investigation. RML, CRL and PPL showed signi fi cant esteri fi cation (up to 84%) when unprotected l -amino acid was used. When N-acetyl- l -alanine was used in the present work, both RML and PPL gave <5% yield. Riva et al . ( 1988 ) have reported two monoesters (4- O - and 6 -O- ester) and no diester for l -alanine, using subtilisin, a protease. Our present study has shown that comparable esteri fi cation yields to others could be achieved by employing PPL, CRL and RML instead of protease. Lipases from Candida rugosa , Rhizomucor miehei and porcine pancreas showed broad substrate speci fi city towards amino acids as well as carbohydrates.
Response surface methodological studies carried out to optimise the RML-catalysed esteri fi cation reaction of l -phenylalanyl- d -glucose also brought out the salient features of this esteri fi cation reaction.
Thus this study has shown that unprotected and unactivated amino acids containing hydro-phobic alkyl side chain can serve as good acyl donors in the esteri fi cation reaction catalysed by lipases from Candida rugosa , Rhizomucor mie-hei and porcine pancreas.
References
Adachi S, Kobayashi T (2005) Synthesis of esters by immobilized lipase catalyzed condensation reaction of sugars and fatty acids in water-miscible organic sol-vent. J Biosci Bioeng 99(2):87–89
Ayala G, Gomez-Puyou T, Gomez-Puyou A, Darzon A (1986) Thermostability of membrane enzymes in organic solvents. FEBS Lett 20:41–43
Beyaz A, Oh WS, Reddy P (2004) Synthesis and CMC studies of 1-methyl-3-(penta fl uorophenyl)imidazo-lium quaternary salts. Colloids Surf B Biointerfaces 15:71–74
Birner-Grunberger R, Scholze H, Faber K, Hermetter A (2004) Identi fi cation of various lipolytic enzymes in crude porcine pancreatic lipase preparations using covalent fl uorescent inhibitors. Biotechnol Bioeng 85:147–154
Boyer V, Stanchev M, Fairbanks AJ, Davis BG (2001) Ready protease catalysed synthesis of carbohydrate-amino acid conjugates. Chem Commun 19:1908–1909
Camacho-Paez B, Robles Medina A, Camacho Rubio F, Gonzalez Moreno P, Molina Grim E (2003a) Modeling the effect of free water on enzyme activity in immobi-lized lipase-catalyzed reactions in organic solvents. Enzyme Microb Technol 33:845–853
Camacho-Paez B, Robles MA, Camacho RF, Esteban-Cerdán L, Molina GE (2003b) Kinetics of lipase-catal-ysed interesteri fi cation of triolein and caprylic acid to produce structured lipids. J Chem Technol Biotechnol 78(4):461–470
Chahid Z, Montet D, Pina M, Graille J (1982) Dictionary of organic compounds, vol 5, 5th edn. Chapman Hall, New York, p 5645
Chang SW, Shaw JF, Shieh CJ (2003) Optimization of enzymatically prepared hexyl butyrate. Food Technol Biotechnol 41:237–242
Chapman Hall (1982) Dictionary of organic compounds, vol 5, 5th edn. Chapman Hall, New York, p 5645
Chen QH, He GQ, Ali MAM (2002) Optimization of medium composition for the production of elastase by Bacillus sp.EL31410 with response surface methodol-ogy. Enzyme Microb Technol 30:667–672
Dabulis K, Klibanov AM (1993) Dramatic enhancement of enzymatic activity in organic solvents. Biotechnol Bioeng 41:566–571
Dordick JS (1989) Enzymatic catalysis in monophasic organic solvents. Enzyme Microb Technol 11:194–211
Ferrer M, Cruces MA, Bernable M, Ballesteros A, Plou FJ (1999) Lipase catalysed regio selective acylation of sucrose in two solvent mixtures. Biotechnol Bioeng 65:10–16
Guvenc A, Kapucu N, Mehmetoglu I (2002) The produc-tion of isoamyl acetate using immobilized lipases in a solvent-free system. Process Biochem 38:379–386
Haines AH (1981) Selective removal of protecting groups in carbohydrate chemistry. Adv Carbohydr Chem Biochem 39:13–70
Humeau M, Girardin B, Rovel AM (1998) Effect of the thermodynamic water activity and the reaction medium hydrophobicity on the enzymatic synthesis of ascorbyl palmitate. J Biotechnol 63:1–8
Hurtley S, Service R, Szuromi P (2001) Cinderella’s coach is ready. Science 291:2337
Jeon GJ, Park OJ, Hur BK, Yang JW (2001) Enzymatic synthesis of amino acid-sugar alcohol conjugates in organic media. Biotechnol Lett 23:929–934
121References
Kirk O, Bjorkling F, Godfredsen SE, Larsen TS (1992) Fatty acid speci fi city in lipase catalysed synthesis of glucoside esters. Biocatalysis 6:127–134
Kuhl P, Halling PJ, Jakubke H-D (1990) Chymotrypsin suspended in organic solvents with salt hydrates is a good catalyst for peptide synthesis from mainly undis-solved reactants. Tetrahedron Lett 31:5213–5216
Liao HF, Tsai WC, Chang SW, Shieh CJ (2003) Application of solvent engineering to optimize lipase-catalyzed 1,3-diglyacylcerols by mixture response surface meth-odology. Biotechnol Lett 25(21):1857–1861
Lohith K, Divakar S (2005) Lipase catalysed synthesis of L-phenylalanine esters of D-glucose. J Biotechnol 117:49–56
Lohith K, Divakar S (2007) Candida rugosa catalyzed preparation of L-prolyl, L-phenylalanyl, L-tryptophanyl and L-histidyl esters of carbohydrates. Biochem Eng J 34:28–43
Lohith K, Vijaya-kumar GR, Manohar B, Divakar S (2003) An improved enzymatic process for the prepa-ration of amino acyl esters of mono and disaccharides. Indian Patent, NF-492/03, PCT/03/00466
Lohith K, Somashekar BR, Manohar B, Divakar S (2006a) An improved enzymatic process for the preparation of amino acyl esters of disaccharides. Indian patent, 285/NF/2006
Lohith K, Manohar B, Divakar S (2006b) In fl uence of buffer and L-phenyl alanine concentration in the RML catalysed synthesis of L-phenyl alanyl-D-glucose ester investigated through response surface methodology. Eur Food Res Technol 224(2):219–224
Lohith K, Manohar B, Divakar S (2007) Competitive inhi-bition by substrates in Rhizomucor miehei and Candida rugosa lipases catalysed esteri fi cation reaction between L-phenylalanine and D-glucose. World J Microbiol Biotechnol 23:955–964
Manini P, Napolitano A, d’Ischia M (2005) Reaction of D-glucose with phenolic amino acids: further insights into competition between Maillard and Pictet-Spengler condensation pathways. Carbohydr Res 340:2719–2727
Marty A, Chulalaksananukul W, Condoret JS, Willemont RM, Durand G (1992) Comparison of lipase-catalyzed esteri fi cation in supercritical carbon dioxide and n-hexane. Biotechnol Lett 12(1):11–16
Maruyama T, Nagasawa SI, Goto M (2002) Enzymatic synthesis of sugar esters in organic solvents. J Biosci Bioeng 94:357–361
Montogomery DC (1991) Design and analysis of experi-ments. Wiley, New York, pp 542–547
Park OJ, Park HG, Yang JW (1996) Enzymatic transesteri fi cation of monosaccharides and amino acid esters in organic solvents. Biotechnol Lett 18:473–478
Park OJ, Jeon GJ, Yang JW (1999) Protease catalysed synthesis of disaccharide amino acid esters in organic media. Enzyme Microb Technol 25:455–462
Partridge J, Harper N, Moore B, Halling PJ (2001) Enzymes in nonaqueous solvents methods and protocols, Methods in Biotechnology. Humana Press, Totowa, pp 227–234
Quiros M, Parker MC, Turner NJ (2002) Tuning lipase enantioselectivity in organic media using solid-state buffers. J Org Chem 66:5074–5079
Reihl O, Bieme KM, Lederer MO, Schwach W (2004) Pyridinium-carbaldehyde: active maillard reaction product from the reaction of hexose with lysine resi-dues. Carbohydr Res 339:705–714
Riva S, Chopineau J, Kieboom APG, Klibanov AM (1988) Protease catalysed regioselective esteri fi cation of sug-ars and related compounds in anhydrous dimethylfor-mamide. J Am Chem Soc 110:584–589
Romero MD, Calvo L, Alba C, Habulin M, Primozic M, Knez Z (2005) Enzymatic synthesis of isoamyl acetate with immobilized Candida antarctica lipase in super-critical carbon dioxide. J Supercrit Fluids 33:77–84
Rosenthal K, Loussale F (1983) Critical micelle concentra-tion determination of non-ionic detergent with Coomassie Brilliant Blue – G 250. Anal Chem 55:1115–1117
Segura RL, Betancor L, Palomo JM, Hidalgo F-LG, Terreni Mateo C, Cortes A, Fernandez-Lafuente R, Guisan JM (2006) Puri fi cation and identi fi cation of dif-ferent lipases contained in PPL commercial extracts a minor contaminant is the main responsible of most esterase activity. Enzyme Microb Technol 39:817–823
Shiraki K, Kudou M, Nishikori S, Kitagawa H, Imanaka T, Takagi M (2004) Arginine ethyl esters prevents thermal aggregation and inactivation of lysozyme. Eur J Biochem 271:3242–3247
Somashekar BR, Divakar S (2007) Lipase catalyzed syn-thesis of L-alanyl esters of carbohydrates. Enzyme Microb Technol 40:299–309
Stahl M, Jeppsson-Wistrand U, Mansson MO (1991) Induced stereoselectivity and substrate selectivity of bioimprinted a -chymotrypsin in anhydrous organic solvents. J Am Chem Soc 113:9366–9368
Suzuki Y, Shimizu T, Takeda H, Kanda K (1991) Fermentative or enzymatic manufacture of sugar amino acid esters. Japan Patent, 03216194 A2
Tamura M, Shoji M, Nakatsuka T, Kinomura K, Okai H, Fukui S (1985) Methyl 2,3-di-(L- g - amimobutyryl)- a -D-glucopyranoside, a sweet substance and tastes of related compounds of neutral amino acids and D-glucose derivatives. Agric Biol Chem 49:2579–2586
Therisod M, Klibanov AM (1986) Facile enzymatic prep-aration of mono acylated sugars in pyridine. J Am Chem Soc 108:5638–5640
Valiveti RH, Johnston GA, Suckling CJ, Halling PJ (1991) Solvent effect on biocatalysis in organic systems equi-librium position and rates of lipase catalysed esteri fi cation. Biotechnol Bioeng 38:1137–1143
Vijayakumar GR, Lohith K, Somashekar BR, Divakar S (2004) Lipase catalysed synthesis of L-alanyl, L-leucyl and L-phenylalanyl esters of D-glucose using unprotected amino acids. Biotechnol Lett 26:1323–1328
Wescott CR, Klibanov AM (1994) The solvent depen-dence of enzyme speci fi city. Biochim Biophys Acta 1206:1–9
122 6 Lipase-Catalysed Preparation of Aminoacyl Esters of Carbohydrates
West JB, Hennen WJ, Lalonde JL, Bibbs J, Zhong Z, Meyer EF, Wong CH (1990) Enzymes as synthetic catalyst mechanistic and active-site considerations of natural and modi fi ed chymotrypsin. J Am Chem Soc 112:5313–5320
Wheeler CJ, Croteau R (1986) Terpene cyclase catalysis in organic solvent/minimal water media demonstra-tion and optimization of (+)- a -pinene cyclase activity. Arch Biochem Biophys 248:429–434
Xu K, Klibanov AM (1996) pH control of the catalytic activity of cross-linked enzyme 9crystals in organic solvents. J Am Chem Soc 118:9815–9819
Zaks A, Dodds DR (1997) Applications of biocatalysts and biotransformations to the synthesis of pharmaceu-ticals. Drug Dev Today 2:513–531
Zaks A, Klibanov AM (1986) Substrate speci fi city of enzymes in organic solvents vs. water is reversed. J Am Chem Soc 108:2767–2768
Zaks A, Klibanov AM (1988) Enzyme catalysis in monophasic organic solvents. J Biol Chem 263:3194–3201
Zoser (2005) Anti-cancer composition comprising proline or its derivatives as anti-tumor antibody. WO2005 120495. European patent A61K39/395C3
123S. Divakar, Enzymatic Transformation, DOI 10.1007/978-81-322-0873-0_7, © Springer India 2013
7.1 Introduction
Like acylation of carbohydrates, regioselective glycosylation involving carbohydrates is a quite challenging synthetic objective because of several hydroxyl groups in these molecules (Haines 1976 ) . Chemical methods of glycosylation involve protection and deprotection (Konstantinovic et al. 2001 ) . The use of enzymatic method for glyco-side synthesis in principle avoids selective protec-tion – deprotection and control of con fi guration (Chahid et al. 1992 ; Thiem 1995 ; Vic et al. 1997 ; Kosary et al. 1998 ) . Glycosyl transfer reactions for the synthesis of glycosides can be carried out under thermodynamically or kinetically controlled conditions (Ichikawa et al. 1992 ; Rantwijk et al. 1999 ) . Reverse hydrolytic method is a thermody-namically controlled method employed for the synthesis of alkyl glycosides (Chahid et al. 1992 ; Ismail and Ghoul 1996 ; Vic et al. 1996 ) . The
reaction comprises a monosaccharide with a nucleophile such as an alcohol to give the corre-sponding glycoside and water. Transglycosylation is kinetically controlled reaction wherein glyco-side (e.g. disaccharide) is used as a glycosyl donor (Stevenson et al. 1993 ; Ismail et al. 1998, 1999 ) .
The use of organic solvent in enzyme cataly-sis has been stimulated by several factors such as solubilities of the organic compounds, shift-ing equilibrium towards the synthesis, increas-ing the enzyme stability and recovery of the enzyme (Rubio et al. 1991 ) . One critical limita-tion in these systems is the poor solubility of the carbohydrate substrate in the organic phase especially when hydrophobic alcohol (glycosyl acceptor) itself is used as a substrate and in some cases as a solvent media to obtain surfac-tant with a long hydrocarbon chain (Laroute and Willemot 1992 ; Vic and Crout 1995 ; Crout and Vic 1998 ) . There are reports where glycosylations
7
Abstract
Chapters 7 , 8 and 9 deal with enzymatic glycosylation. Glycosylation of alcohols (Chap. 7 ), water-soluble and water-insoluble phenols (Chap. 8 ) and water-soluble and water-insoluble vitamins (Chap. 8 ) involve glycosi-dases like amyloglucosidases from a Rhizopus mould, b -glucosidase from sweet almond and b -glucosidases immobilised onto calcium alginate beads. The results from comparative studies of the preparation in shake fl asks under nonsolvent conditions – employing substrate itself as the sol-vent or in presence of solvents and by re fl ux method employing di-isopro-pyl ether – are discussed.
Enzymatic Glycosylation of Alcohols
124 7 Enzymatic Glycosylation of Alcohols
were carried out either in biphasic systems of a water-immiscible alcohol and water (that main-tains sugar substrate and enzyme) or water and water-miscible monophasic system (Monsan et al. 1996 ) . The process of glycosylation can be effected under nonaqueous, solvent-free condi-tions, high substrate, high temperature and mod-erate to high water activity to yield glycosides (Nilsson 1987 ; Roitsch and Lehle 1989 ; Gygax et al. 1991 ; Laroute and Willemot 1992 ; Vic and Thomas 1992 ; Shin et al. 2000 ) .
Recently, synthesis of glycosides has gener-ated much interest because of their broad range of applications in various fi elds. This class of compounds is mainly used as nonionic surfac-tants in food and pharmaceuticals. These types of nonionic surfactants exhibit several interesting properties in detergency, foaming, wetting, emulsi fi cation and antimicrobial effect (Matsumura et al. 1990 ; Balzar 1991 ) . Alkyl gly-cosides are non-toxic, non-skin-irritating and biodegradable (Matsumura et al. 1990 ; Busch et al. 1994 ; Madsen et al. 1996 ) . Further, alkyl glycosides are used as raw materials for sugar fatty acid ester synthesis (Mutua and Akoh 1993 ) . Because of these properties, alkyl glycosides fi nd great potential application in many diversi fi ed areas such as pharmaceutical, chemical, cosmetic and detergent industries.
Glycosylation, besides being an important method for the structural modi fi cation of com-pounds with useful biological activities, also allows the conversion of a water-insoluble com-ponent into water-soluble one, thereby improv-ing its pharmacological applications (Suzuki et al. 1996 ; Vijayakumar and Divakar 2005 ; Sivakumar and Divakar 2006 ) . The present
investigation has been undertaken to achieve this objective.
Enzymatic glycosylation described here was carried out using amyloglucosidase from Rhizopus sp., a commercially available enzyme known to cleave a (1 → 4) glycosidic linkage of starch to give glucose. Comparative studies of the prepara-tion in shake fl asks under nonsolvent conditions (employed alcohol itself acts as solvent) or in pres-ence of solvents and by re fl ux method involving re fl uxing and stirring the reaction mixture using di-isopropyl ether as solvent were carried out (Vijayakumar et al. 2005, 2007 ) . Glucosylation occurred with amyloglucosidase only in the presence of water, which is added in the form of buffer of certain pH, volume of water and salt concentra-tion. A general scheme for the glycosylation reac-tion involving synthesis of n-alkyl glucosides is shown in Scheme 7.1 .
7.2 n-Octyl- D -Glucoside
Synthesis of n-octyl- d -glucoside was studied in detail (Vijayakumar et al. 2007 ) . Effects of incubation period, pH, buffer concentration and enzyme concentration were studied by both shake- fl ask and re fl ux methods. The retention time for free d -glucose was found to be 5.2 and 7.2 min for n-octyl- d -glucoside (Fig. 7.1 ). Glycosylation pro fi le is shown in Fig. 7.2 .
Optimal conditions for the reaction are the following:
At shake- fl ask level, the optimum conditions were found to be 30% (w/w d -glucose) amyloglucosidase (Tables 7.1 and 7.2 , Fig. 7.3 ) concentration and 0.8 mM (0.4 mL), pH 6.0 phosphate buffer at an
Scheme 7.1 Synthesis of n-alkyl glucosides
1257.2 n-Octyl-D-Glucoside
Fig. 7.1 Typical HPLC chromatogram for the reaction mixture of d -glucose and n-octyl- d -glucoside. HPLC con-ditions: aminopropyl column (10- m m particle size, 3.9 × 300 mm length); solvent – CH
3 CN: H
2 O (80:20 v/v);
fl ow rate – 1 mL min −1 ; and RI detector. Retention times: solvent peak – 3.6 min; d -glucose – 5.2 min; and n-octyl- d -glucoside – 7.2 min
Table 7.1 Effect of amyloglucosidase concentration on the synthesis of n-octyl- d -glucoside a
Enzyme % (w/w d -glucose)
Shake- fl ask method Enzyme % (w/w d -glucose)
Re fl ux method
Yield – % ( m mol) Yield – % ( m mol)
10 26 (143) 10 11 (58) 20 28 (153) 20 8 (42) 30 28 (155) 30 46 (255) 40 28 (154) 40 26 (142) 50 17 (96) 50 40 (223) 80 19 (106) 75 27 (147) 100 10 (55) 100 20 (113)
a Conversion yields were from HPLC with respect to 0.555 mmol of d -glucose. d -Glucose – 0.555 mmol; n-octanol – 50 eq (0.027 mol); temperature – 60°C for shake- fl ask method and 68°C for re fl ux method; pH – 6.0; 0.01 M; and buffer volume – 0.4 mL (0.04 mM for re fl ux method and 0.8 mM for shake- fl ask method). Error in yield measurements will be ±5–10%. This applies to all the yields given in the subsequent tables also
Table 7.2 Effect of buffer concentration on the synthesis of n-octyl- d -glucoside a
Buffer concentration (mM)
Shake- fl ask method Buffer concentration (mM)
Re fl ux method
Yield – % ( m mol) Yield – % ( m mol)
0.2 15 (86) 0.01 5 (29) 0.4 17 (94) 0.02 8 (44) 0.8 20 (109) 0.04 40 (223) 1.2 4 (24) 0.06 31 (173) 1.6 4 (24) 0.08 No yield – – 0.1 No yield
a Conversion yields were from HPLC with respect to 0.555 mmol of d -glucose. d -Glucose – 0.555 mmol; n-octanol – 50 eq (0.027 mol); temperature – 60°C for shake- fl ask method and 68°C for re fl ux method; enzyme – 50% w/w d -glu-cose; and pH – 4.0 (0.2–1.6 mM) for shake- fl ask method and 6.0 (0.01–0.1 mM) for re fl ux method
126 7 Enzymatic Glycosylation of Alcohols
0
10
20
30
40
50
0 20 40 60 80 100 120 140
Con
vers
ion
yiel
d (%
)
Incubation time (hr)
Fig. 7.2 A typical reaction pro fi le for n-octyl- d -gluco-side synthesis by the re fl ux method. Conversion yields were from HPLC with respect to 0.555 mmol of d -glu-cose. Reaction conditions: d -glucose –0.555 mmol;
n-octanol – 0.027 mol; amyloglucosidase –50% (w/w d -glucose); 0.04 mM (0.4 mL of 10 mM buffer); pH 5.0 acetate buffer; solvent – di-isopropyl ether; and tempera-ture –68°C
0
10
20
30
40
50
4 5 6 7 8
Con
vers
ion
yiel
d (%
)
pH
shake flask
reflux
Fig. 7.3 Effect of pH on n-octyl- d -glucoside synthesis. Conversion yields were from HPLC with respect to 0.555 mmol of d -glucose. Reaction conditions: d -glucose –0.555 mmol: n-octanol –0.027 mol; buffer concentration –0.04 mM (0.4 mL of 10 mM buffer) for re fl ux method
and 0.8 mM (0.4 mL of 10 mM buffer) for shake- fl ask method; and incubation –72 h. Re fl ux method: solvent – di-isopropyl ether and temperature –68°C. Shake- fl ask method: temperature –60°C
incubation period of 72 h. Similarly, the optimum conditions for the re fl ux method were found to be 30% (w/w d -glucose) amyloglucosidase (Tables 7.1 and 7.2 , Fig 7.3 ) concentration and 0.04 mM (0.4 mL), pH 6.0 phosphate buffer at an incubation period of 72 h.
7.3 Synthesis of n-Octyl- D -Glucoside Using b -Glucosidase
n-Octyl- d -glucoside was synthesised by the re fl ux method using b -glucosidase isolated from sweet almond. The reaction mixture analysed by
1277.6 Spectral Characterisation
HPLC showed 23% (230 m mol) conversion with respect to the d -glucose employed. Two-dimensional NMR (HSQCT) con fi rmed forma-tion of n-octyl- b - d -glucoside.
7.4 Determination of Critical Micellar Concentration (CMC)
Since glycosides of long-chain alcohols serve as nonionic surfactants, critical micellar concentra-tion (CMC) of n-octyl- d -glucoside determined (Rosenthal and Loussale 1983 ) was found to be 16.1 mM (0.47%).
7.5 Synthesis of n-Octyl Glycosides
The optimum conditions worked out for the syn-thesis of n-octyl- d -glucoside by the re fl ux method were employed for the synthesis of n-octanol, glycosides of various carbohydrates. The various carbohydrates employed were d -glucose, d -galac-tose, d -mannose, d -fructose, d -arabinose, d -ribose, maltose, sucrose, lactose, d -mannitol and d -sorbitol. n-Octyl maltoside (conversion yield 15%, 150 m mol) and n-octyl sucrose (con-version yield 13%, 130 m mol) were obtained by this procedure (Scheme 7.2 ).
The retention times for free carbohydrate and n-octyl glycosides in HPLC were d -glucose –
5.2 min; n-octyl- d -glucoside – 7.2 min; maltose – 7.4 min; n-octyl maltoside – 10.5 min; sucrose – 6.4 min; and n-octyl sucrose – 7.1 min. The structures of the glycosides formed, HPLC yield and product proportions are presented in Table 7.3 . Other carbohydrates such as d -fruc-tose, d -arabinose, d -ribose, lactose and d -sorbitol did not form any glycoside with n-octanol. However, HPLC indicated glycosylation of d -galactose, d -mannose and d -mannitol with conversion yields less than 5%.
7.6 Spectral Characterisation
The glycosides were characterised by UV, IR, 2-D NMR (HSQCT) and optical rotation. Two-dimensional HSQCT NMR gave information on the nature and proportions of the products formed (Table 7.3 ). Since n-octyl glycosides are surfac-tant molecules, the proton signals are broad due to aggregation at concentration above CMC employed, and hence, coupling constant values could not be determined satisfactorily for these compounds.
UV spectrum of n-octyl- a and b - d -glucosides, maltoside and sucroside synthesised by b -glu-cosidase showed s → s * band between 190 and 205 nm and n → p * band between 275 and 278.5 nm. The IR spectral band in the region 1,027–1,053 cm −1 corresponded to the glycosidic
Scheme 7.2 Synthesis of n-octyl glycosides
128 7 Enzymatic Glycosylation of Alcohols
C–O–C symmetrical stretching and 1,225–1,259 cm −1 for the glycosidic C–O–C asymmetri-cal stretching frequencies.
In case of a -glucoside, the 1 H NMR area of the C1 anomeric cross peaks, con fi rmed that a : b ano-meric composition obtained was 63:25. Apart from the free C1 a and C1 b signals, the cross peaks in the anomeric region with 13 C chemical shift values at 98.5 and 103.2 ppm with the cor-responding 1 H values at 4.62 and 4.17 ppm clearly indicated C1 a of the glucoside and C1 b glucoside formation. The cross peak in the C6 region ( 13 C at 67.2 ppm and 1 H at 3.64 ppm) indicated C6-O-alkylated product. Mass value 294 [M + 2] obtained from mass spectrum further con fi rmed the product formation
Two-dimensional HSQCT showed the down fi eld chemical shift for C1 b at 103.2 ppm
and the corresponding 1 H value at 4.18 ppm, indi-cating the formation of only C1 b glucoside. No other product formation (C1 a glucoside or C6-O-alkylated) was detected unlike the amyloglucosi-dase-catalysed reaction.
For the maltoside, the down fi eld chemical shift for C1 a at 98.8 ppm and the corresponding 1 H value at 4.63 ppm indicated formation of only C1 a maltoside and no C-6-O-alkylated products. A mass value 455 was obtained for [M + 1] peak.
In case of the sucroside, two-dimensional HSQCT con fi rmed the formation of C1-O- and C6-O- of alkylated products. The chemical shift values for C1 at 62.8 ppm ( 1 H at 3.76 ppm) and C6 at 63.0 ppm ( 1 H at 3.25 ppm) indicated the formation C1-alkylated and C6-alkylated products, respectively. Also NMR data clearly showed that hydrolysis of sucrose has taken
Table 7.3 n -Octyl glycosides with conversion yields and product proportions a
Glycosides and product proportions b Glycosylation yield (%) c
O
OHOH
OH
OH
O CH3
O
OHOH
OH
OH
O CH3
46
n-Octyl- a - d -glucoside (63) n-Octyl- b - d -glucoside (25)
O
OHOH
OH
OH
O CH3
C6-O-octyl- d -glucose (12)
O
OHOH
OH
OH
O CH3
23
n-Octyl- b - d -glucoside d
OH
OH
OH H
H
H
HH
OHO
O
OH
H
H
HH
OH
OOH
O CH3
15
n-Octyl maltoside
OH
O
OHOH
OH
O
O
HO
OH
O
CH3
OH
OH
O
OHOH
OH
OH
O
HO
OH
OOCH3
13
C1-O-octyl sucrose (44) C6-O-octyl sucrose (56)
a Carbohydrate and n-octanol – 1:50 equivalents; amyloglucosidase – 30% (w/w carbohydrate); solvent – di-isopropyl ether; temperature – 68°C; and incubation period – 72 h b The product proportions determined from 2D-HSQCT NMR C1/C6 cross peak areas are shown in brackets c Conversion yields were from HPLC with respect to free carbohydrate d The compound was synthesised by using b -glucosidase from sweet almonds
1297.7 Synthesis of n-Alkyl Glucosides Using Amyloglucosidase
place and the hydrolyzed d -glucose is glyco-sylated at C1 a ( 13 C at 98.7 ppm and 1 H at 4.64 ppm) and alkylated at the C6-O- position ( 13 C at 67.5 ppm and 1 H at 3.54 ppm). The mass value 455 for [M + 1] peak con fi rmed the product formation.
7.7 Synthesis of n-Alkyl Glucosides Using Amyloglucosidase
n-Alkyl glucosides using alcohols of carbon chain length C1–C18 were synthesised with the following speci fi c alcohols, namely, methyl alcohol, ethyl alcohol, n-propyl alcohol, n-butyl alcohol, n-amyl alcohol, n-hexyl alco-hol, n-heptyl alcohol, n-octyl alcohol, n-nonyl alcohol, n-decyl alcohol, lauryl alcohol, cetyl alcohol and stearyl alcohol. The reaction mix-tures were analysed by HPLC. Retention times are d -glucose – 5.2 min; n-methyl- d -glucoside – 7.0 min; n-ethyl- d -glucoside – 7.1 min; n-propyl- d -glucoside – 7.1 min; n-butyl- d -glucoside – 7.1 min; n-amyl- d -glucoside – 7.1 min; n-hexyl- d -glucoside – 7.2 min; n-heptyl- d -glucoside – 7.2 min; n-nonyl- d -glucoside – 7.5 min; n-decyl- d -glucoside – 7.6 min; lauryl- d -glucoside – 7.6 min; cetyl- d -glucoside – 7.7 min; and stearyl- d -glucoside – 7.7 min.
7.7.1 Shake-Flask Method
Alcohols of carbon chain lengths C2–C18 were employed for the synthesis of glucosides. The yields obtained (with respect to d -glucose) were found to be in the range 3–28% (16–156 m mol). The results showed that the yields are higher for ethanol (10%, 57 m mol), n-propyl alcohol (13%, 73 m mol) and n-butanol (9%, 52 m mol). For other medium-chain-length alcohols like n-amyl alco-hol (3%, 16 m mol), n-hexyl alcohol (9%, 47 m mol) and n-heptyl alcohol (5%, 26 m mol), the yields were much lower. However, from n-octyl alcohol to n-decyl alcohol, the yields were the highest (20%, 109 m mol to 23%, 127 m mol) with n-nonyl alcohol giving the highest yield (28%, 155 m mol). The yields decreased slightly with further increase in alcohol chain lengths up to stearyl alcohol. The shake- fl ask method gave lesser yields at pH 4.0 in general for the carbon chain lengths up to C7. From n-octyl alcohol onwards, the yields increased with increase in chain lengths up to C10.
7.7.2 Re fl ux Method
Alkyl glucosides were also synthesised with vari-ous alcohols by the re fl ux method (Vijayakumar et al. 2007 ) in presence of 0.04 mM (0.4 mL), pH 4.0 and 5.0 acetate buffer (Figs. 7.4 and 7.5 ). At
0
50
100
150
200
250
300
C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C12 C16 C18
Yie
ld (
mic
ro m
ol)
Carbon chain length
Fig. 7.4 Synthesis of n-alkyl glucosides by re fl ux method at pH 4.0. d -glucose (0.000555 mol) and alcohol in 1:50 M proportions along with 50% w/w of amyloglucosi-
dase and 0.4 mL 0.01 M acetate buffer at pH 4.0 re fl uxed with 50 mL di-isopropyl ether (boiling point –68°C) for 72 h. Conversion yields by HPLC
130 7 Enzymatic Glycosylation of Alcohols
pH 4.0 the yields were lower for methyl (13%, 71 m mol), ethyl (5%, 27 m mol) and n-propyl alco-hols (7%, 39 m mol). However, the yields were higher for the remaining alcohols. The highest yield was observed for n-amyl alcohol (44%, 245 m mol). In general, except lauryl alcohol (10%, 55 m mol), the yields were higher for n-octyl alcohol (24%, 134 m mol) onwards towards higher-chain-length alcohols.
At pH 5.0, the yields obtained were found to be in the range of 12% (65 m mol)–44% (242 m mol). Higher yields were observed for ethyl alcohol (44%, 242 m mol) and lauryl alcohol (36%, 200 m mol).
7.8 Cetyl and Stearyl Glucosides
The effect of increasing enzyme concentration on the synthesis of cetyl and stearyl glucosides was investigated (Vijayakumar et al. 2007 ) . The yields obtained at 40% enzyme concentration was higher in case of both cetyl (6%, 34 m mol) and stearyl glucosides (19%, 105 m mol) compared to the other enzyme concentrations. The yields gen-erally were higher at all the enzyme concentra-tions for stearyl alcohol compared to cetyl alcohol. This could be because the longer-chain-
length alcohol functioned as a better nucleophile for accepting a d -glucose molecule than the shorter-chain-length alcohol.
7.9 Optimisation of n-Octyl- D -Glucoside Synthesis Using Response Surface Methodology
Ismail et al. ( 1998 ) have reported the synthesis of butyl glucoside by RSM. Using b -galactosidase, Chahid et al. ( 1994 ) have reported the synthesis of a mixture of octylglucoside and octylgalacto-side through a transglycosylation reaction involv-ing lactose and n-octanol. The present work deals with a detailed RSM analysis of the synthesis of n-octyl- d -glucoside using amyloglucosidase from Rhizopus sp. by the shake- fl ask method (Vijayakumar et al. 2005 ) .
Design : Central composite rotatable design (CCRD), 32 experiments, 5 variables at 5 levels
Variables : n-Octanol concentration, enzyme con-centration, pH, buffer concentration (buffer volume) and temperature. Coded and actual values are shown in Table 7.4 . Actual set of experiments undertaken as per the CCRD with coded values and the glucosyla-tion yields obtained is given in Table 7.5 .
0
50
100
150
200
250
300
C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C12 C16 C18
yiel
d (
mic
ro m
ol)
Carbon chain lengh
Fig. 7.5 Synthesis of n-alkyl glucosides by re fl ux method at pH 5.0. d -glucose (0.000555 mol) and alcohol in 1:50 M proportions along with 50% w/w of amyloglucosi-
dase and 0.4 mL 0.01 M acetate buffer at pH 5.0 re fl uxed with 50 mL di-isopropyl ether (boiling point – 68°C) for 72 h. Conversion yields by HPLC
1317.9 Optimisation of n-Octyl-D-Glucoside Synthesis Using Response Surface Methodology
Table 7.4 Coded values of the variables and their corresponding actual values used in the design of experiments
Variables −2 −1 0 1 2
n-Octanol (eq) 15 30 45 60 75 Amyloglucosidase (mg) 20 40 60 80 100 pH 4.0 5.0 6.0 7.0 8.0 Buffer volume (mL) 0.2 0.4 0.6 0.8 1.0 Temperature (°C) 30 40 50 60 70
Table 7.5 Experimental design with experimental and predicted yields of n-octyl- d -glucoside
Expt no n-Octanol Amylo glucosidase pH
Buffer volume Temperature
Yield a e xperimental
Yield predicted
1 −1 −1 −1 −1 1 20.0 22.4 2 −1 −1 −1 1 −1 10.7 12.3 3 −1 −1 1 −1 −1 27.2 30.3 4 −1 −1 1 1 1 0.9 5.8 5 −1 1 −1 −1 −1 37.5 35.6 6 −1 1 −1 1 1 1.8 1.8 7 −1 1 1 −1 1 14.4 13.8 8 −1 1 1 1 −1 13.1 11.6 9 1 −1 −1 −1 −1 31.6 30.8 10 1 −1 −1 1 1 1.2 4.2 11 1 −1 1 −1 1 26.6 27.1 12 1 −1 1 1 −1 17.8 17.4 13 1 1 −1 −1 1 18.1 15.6 14 1 1 −1 1 −1 17.9 14.5 15 1 1 1 −1 −1 37.2 33.3 16 1 1 1 1 1 2.5 2.4 17 0 0 0 −2 0 40.9 43.8 18 0 0 0 2 0 12.1 7.1 19 0 0 −2 0 0 14.1 15.9 20 0 0 2 0 0 20.0 18.1 21 0 −2 0 0 0 27.6 21.5 22 0 2 0 0 0 7.1 15.1 23 −2 0 0 0 0 26.3 24.3 24 2 0 0 0 0 26.2 28.1 25 0 0 0 0 −2 13.8 18.4 26 0 0 0 0 2 0 0 27 0 0 0 0 0 27.6 24.1 28 0 0 0 0 0 21.4 24.1 29 0 0 0 0 0 21.1 24.1 30 0 0 0 0 0 30.1 24.1 31 0 0 0 0 0 23.4 24.1 32 0 0 0 0 0 17.1 24.1
a Conversion yields were from HPLC with respect to 0.555 mmol of d -glucose. The experimental yields were an average from two experiments
132 7 Enzymatic Glycosylation of Alcohols
Equation : A second-order polynomial equation was developed to study the effects of the variables on the esteri fi cation yields in terms of linear, qua-dratic and cross product terms.
Y=-98.557-0.780X1 + 1.280X
2 + 17.752X
3 -
82.346X4 + 4.005X
5 + 0.002X
1X
1 - 0.004 X
2
X2 - 1.783 X
3X
3 + 14.526X
4X
4 - 0.043X
5X
5 -
0.002X1X
2 + 0.124X
1X
3 + 0.051X
1X
4 + 0.0003
X1X
5 - 0.066X
2X
3 + 0.049 X
2X
4 - 0.009 X
2
X5 + 1.341 X
3X
4 + 0.035 X
3X
5 + 0.166 X
4X
5
where X 1 – n-octanol concentration; X
2 – amylo-
glucosidase concentration; X 3 – pH; X
4 – buffer
volume; and X 5 – temperature.
Coef fi cients : Microsoft Excel software, Version 5.0
Analysis of Variance ( ANOVA ): Microsoft Excel software ANOVA showed the model is signi fi cant at P < 0.01.
Optimisation : Microsoft Excel Solver function
The experimental data fi tted the second-order polynomial equation well as indicated by a R 2 value of 0.895 (Table 7.5 ).
The maximum yield predicted based on the response model 53.5% is obtained at an n-octanol concentration of 75 eq 20 mg amyloglucosidase concentration, 0.30 mM (0.2 mL of 10 mM buf-fer) pH 7.8 buffer and 50°C. The experiments conducted at the above optimum conditions resulted in 53.8% yield. Apart from the above experiment, validation of the response model was carried out at selected random process conditions.
These validation experiments closely agreed with the predicted yields (Table 7.6 ).
The effect of n-octanol and enzyme concen-tration on the glucosylation of n-octanol to n-octyl- d -glucoside at 0.6 mL of 10 mM, pH 6.0 at 50°C is shown in Fig. 7.6 . The extent of con-version increased with the increase in n-octanol equivalents at lower enzyme concentrations. With increase in enzyme concentration from 20 to 60 mg (20–60% with respect to w/w d -glucose), the extent of glucosylation reached a maximum of 27.0% at 60 mg (60% w/w d -glucose) enzyme concentration. With further increase in enzyme concentration to 100 mg (100% w/w d -glucose), the yield decreased. This was the behaviour at all equivalents of n-octanol. Around an enzyme con-centration of 60 mg (60% w/w d -glucose), increase in n-octanol equivalents exhibited a trough with a slight dip around an n-octanol con-centration of 45 eq. The surface plot illustrates that at lower enzyme concentrations, suf fi cient amount of free n-octanol is available for transfer of d -glucose to n-octanol resulting in increase in glucosylation. At higher enzyme concentrations, a complete binding of n-octanol to the enzyme would effectively reduce the concentration of free n-octanol, thereby causing a reduction in the transfer of d -glucose molecule to n-octanol lead-ing to reduced glucosylation.
The catalytic ef fi ciency of the enzyme was found to be the highest at 50°C, and it decreased to very low values at 70°C at all enzyme con-centrations (Fig. 7.7 ). Other conditions kept constant were n-octanol – 45 eq, pH – 6.0 and
Table 7.6 Validation of experimental data
n-Octanol (eq) Amylo glucosidase (mg) pH
Buffer volume (mL) Temperature (°C) Yield predicted
Yield a experimental
50 50 4.5 0.3 55 27.3 23.8 40 30 6.5 0.5 65 13.6 6.09 65 50 7.5 0.3 65 25.2 25.3 55 45 4.5 0.3 45 34.2 30.1 55 45 4.5 0.3 35 31.0 20.9 10 65 5.5 0.7 65 2.8 4.2 50 55 4.5 0.9 65 0 5.0 75 20 7.8 0.2 50 53.5 53.8
a Conversion yields were from HPLC with respect to 0.555 mmol of d -glucose. The experimental yields were an average from two experiments
1337.9 Optimisation of n-Octyl-D-Glucoside Synthesis Using Response Surface Methodology
buffer concentration – 1.3 mM (0.6 mL of 10 mM buffer). At 30°C, glucosylation occurred at all enzyme concentrations. The highest con-version was with 60 mg (60% w/w d -glucose) of enzyme. At lower enzyme concentrations, the
conversions were lower. However, they increased with increase in temperature up to 50°C indicat-ing that the optimum temperature required for glucosylation is 50°C. At a higher temperature of 70°C, the enzyme most probably underwent
Fig. 7.6 Three-dimensional surface plot showing the effect of n-octanol concentration and enzyme concentration on the extent of glucosylation (pH –6.0, buffer volume –0.6 mL, temperature –50°C)
Fig. 7.7 Three-dimensional surface plot showing the effect of enzyme concentration and temperature on the extent of glucosylation (n-octanol – 45 eq, pH – 6.0, 0.6 mL of 10 mM buffer)
134 7 Enzymatic Glycosylation of Alcohols
denaturation leading to a loss in catalytic activity.
The effect of buffer volume and pH on the extent of glucosylation at an n-octanol concentra-tion of 45 eq, 60 mg enzyme and 50°C effectively indicated the role of concentration of buffer salts and their effect on importing ‘pH memory’ to amyloglucosidase. With increase in pH, glucosy-lation decreased at all buffer volumes. An opti-mum buffer volume of 0.6 mL buffer was found to be the best for this reaction.
Thus, this study showed that this model is very good in predicting the glucosylation of n-octanol by the amyloglucosidase enzyme.
References
Balzar D (1991) Alkylglucosides, their physico-chemical properties and their uses. Tenside Surfactant Deterg 28:419–427
Busch P, Hensen H, Khare J, Tesmann H (1994) Alkylpolyglycosides-a new cosmetic concept for mil-derness. Agro-Food-Ind Hi-Tech 5:20–28
Chahid Z, Montet D, Pina M, Graille J (1992) Effect of water activity on enzymatic synthesis of alkylglyco-sides. Biotechnol Lett 14(4):281–284
Chahid Z, Montet D, Pina M, Bonnot F, Graille J (1994) Biocatalyzed octylglycoside synthesis from a disac-charide. Biotechnol Lett 16:795–800
Crout DHG, Vic G (1998) Glycosidases and glycosyl transferases in glycoside and oligosaccharides synthe-sis. Biocatal Biotransform 2:98–111
Gygax D, Spies P, Winkler T, Pfaar U (1991) Enzymatic synthesis of b -D-glucuronides with in situ regeneration of uridine 5 ¢ -diphosphoglucuronic acid. Tetrahedron 47:5119–5122
Haines AH (1976) Relative reactivities of hydroxyl groups in carbohydrates. Adv Carbohydr Chem Biochem 33:11–109
Ichikawa Y, Look GC, Wong CH (1992) Enzyme-catalysed oligosaccharide synthesis. Anal Biochem 202:215–238
Ismail A, Ghoul M (1996) Enzymatic synthesis of butyl glycosides by glycosidases. Biotechnol Lett 18:1199–1204
Ismail A, Soultani S, Ghoul M (1998) Optimization of the enzymatic synthesis of butyl glucoside using response surface methodology. Biotechnol Prog 14:874–878
Ismail A, Linder M, Ghoul M (1999) Optimization of butylgalactoside synthesis by b -galactosidase from Aspergillus oryzae . Enzyme Microb Technol 25:208–213
Konstantinovic S, Predojevic J, Gojkovic S, Ratkovic Z, Mojsilovic B, Pavlovic V (2001) Synthesis of C7-C16 alkyl 2,3 dideoxy glucosides from glucose and fatty acids. Indian J Chem 40B:1242–1244
Kosary J, Stefanovits-Banyai E, Boross L (1998) Reverse hydrolytic process for O-alkylation of glucose cata-lyzed by immobilized a - and b -glucosidases. J Biotechnol 66:83–86
Laroute V, Willemot RM (1992) Glucoside synthesis by glucoamylase or b -glucosidase in organic solvents. Biotechnol Lett 14:169–174
Madsen T, Petersen G, Seiero C, Torslov J (1996) Biodegradability and aquatic toxicity of glycoside sur-factants and a nonionic alcohol etherate. J Am Oil Chem Soc 73:929–933
Matsumura S, Imai K, Yoshikawa S, Kawada K, Uchibori T (1990) Surface activities, biodegradability and antimicrobial properties of n-alkyl glucosides, man-nosides and galactosides. J Am Oil Chem Soc 67:996–1001
Monsan PF, Paul F, Pelenc P, Bouler E (1996) Enzymatic production of a -butyl glucoside and its fatty acid esters. Ann N Y Acad Sci 799:633–641
Mutua LN, Akoh CC (1993) Synthesis of alkyl glucoside fatty acid esters in non aqueous media by Candida sp. lipase. J Am Oil Chem Soc 70:43–46
Nilsson KGI (1987) A simple strategy for changing the regioselectivity of glycosidase catalyzed formation of disaccharides. Carbohydr Res 167:95–103
Rantwijk FV, Oosterom MW, Sheldon RA (1999) Glycosidase-catalyzed synthesis of alkyl glycosides. J Mol Catal B: Enzym 6:511–532
Roitsch T, Lehle L (1989) Structural requirements for pro-tein N-glycosylation. In fl uence of acceptor peptides on cotranslational glycosylation of yeast in fl uence and site-directed mutagenesis around a sequon sequence. Eur J Biochem 181:525–529
Rosenthal KS, Loussale F (1983) Critical micelle concen-tration determination of non-ionic detergent with comassive brilliant blue-G250. Anal Chem 55: 1115–1117
Rubio E, Fernandez MA, Klibanov AM (1991) Effect of the solvent on enzyme regio-selectivity. J Am Chem Soc 113:695–696
Shin HK, Kong JY, Lee JD, Lee TH (2000) Synthesis of hydroxy benzyl- a -glucosides by amyloglucosidase-catalysed transglycosylation. Biotechnol Lett 22:321–325
Sivakumar R, Divakar S (2006) Glycosylation of vanillin by amyloglucosidase in organic media. Tetrahedron Lett 47:695–699
Stevenson DE, Stanley RA, Furneaux RH (1993) Optimization of alkyl b -D-galactopyranoside synthe-sis from lactose using commercially available b -galac-tosidase. Biotechnol Bioeng 42:657–666
Suzuki Y, Ki YH, Uchida K, Takami M (1996) Enzymatic synthesis of glucosylated and phosphatidylated bio-logically active compounds. J Appl Glycosci 43:273–282
Thiem J (1995) Applications of enzymes in synthetic carbo-hydrate chemistry. FEMS Microbiol Rev 16:193–211
Vic G, Crout DHG (1995) Synthesis of allyl and benzyl b -D-glucopyranosides and allyl b -D-galactopyrano-side from D-glucose or D-galactose and the corre-
135References
sponding alcohol using almond b -D-glucosidase. Carbohydr Res 279:315–319
Vic G, Thomas D (1992) Enzyme-catalyzed synthesis of alkyl- b -D-glucosides in organic media. Tetrahedron Lett 33:4567–4570
Vic G, Hastings JJ, Crout DHG (1996) Glycosidase cata-lyzed synthesis of glycosides by an improved proce-dure for reverse hydrolysis. Application to the chemoenzymatic synthesis of galactopyrano-syl-(1 → 4)-O- a -galactopyranoside derivatives. Tetrahedron Asymmetry 7:1973–1984
Vic G, Thomas D, Crout DHG (1997) Solvent effect on enzyme-catalyzed synthesis of b -D-glucosides using the reverse hydrolysis method application to the
preparative-scale synthesis of 2-hydroxybenzyl and octyl b -D-glucopyranosides. Enzyme Microb Technol 20:597–603
Vijayakumar GR, Divakar S (2005) Synthesis of guaiacol- a -D-glucoside and curcumin- bis- a -D-glucoside by an amyloglucosidase from Rhizopus . Biotechnol Lett 27:1411–1415
Vijayakumar GR, Manohar B, Divakar S (2005) Amyloglucosidase catalyzed synthesis of n-octyl-D-glucoside-analysis using response surface methodol-ogy. Eur Food Res Technol 220:272–277
Vijayakumar GR, George C, Divakar S (2007) Synthesis of n-alkyl glucosides by amyloglucosidase. Indian J Chem B 46B:314–319
137S. Divakar, Enzymatic Transformation, DOI 10.1007/978-81-322-0873-0_8, © Springer India 2013
8.1 Phenols
Biological activities of a naturally occurring gly-coside (Robyt 1998 ; Schmid et al. 2001 ; Akao et al. 2002 ) are primarily due to an aglycon moiety of that molecule. It is generally accepted that gly-cosides are more water-soluble than most of the respective aglycons. Attaching a glycosidic moi-ety into the molecule increases its hydrophilicity and thereby in fl uences physicochemical and phar-macokinetic properties of the respective compound like circulation, elimination and concentrations in the body fl uids (Kren 2001 ) . Glycosides with unsaturated alkyl chains like terpenes are claimed to possess antifungal and antimicrobial activity (Tapavicza et al. 2000 ; Zhou 2000 ) although it is
unclear why the activity of these aglycons is improved by glycosylation. Glycosides of pep-tides and steroids are used in antitumor formula-tions (Kaljuzhin and Shkalev 2000 ) and in cardiac-related drugs (Ooi et al. 1985 ) , respec-tively. Curcumin [1E,6E-1,7-di(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione], a yellow pigment of turmeric (the dried rhizome of Curcuma longa belonging to Zingiberaceae), is used primarily as a food colourant, but it is also a pharmacologically active principle of turmeric with potent antioxidative, anti-in fl ammatory and anti-leishmanial (Gomes et al. 2002 ) activities. Eugenol, main part of the clove oil (more than 90%), is used as a precursor of vanillin. It was found that eugenol possesses pharmacological
8
Abstract
Section 8.1 describes the synthesis of glycosides of water-insoluble phenols such as guaiacol, eugenol, curcumin, N-vanillyl-nonanamide and capsaicin and water-soluble phenols like vanillin, dl -dopa, l -dopa, dopamine, serotonin and epinephrine with carbohydrates: d -glucose, d -galactose, d -mannose, d -fructose, d -arabinose, d -ribose, maltose, sucrose, lactose, d -mannitol and d -sorbitol.
Section 8.2 deals with enzymatic syntheses of more water-soluble thiamin (vitamin B1), ribo fl avin (vitamin B2) and pyridoxine (vitamin B6) and fat-soluble retinol (vitamin A), ergocalciferol (vitamin D2), cholecalciferol (Vitamin D3) and a -tocopherol (vitamin E). The reac-tions were investigated and discussed (Chap. 9 ) in terms of incubation period, pH, buffer, enzyme and substrate concentrations and regio- and stereoselectivity.
Glycosylation of Some Selected Phenols and Vitamins
138 8 Glycosylation of Some Selected Phenols and Vitamins
activity and modi fi cation is needed in its application. Guaiacol, eugenol, curcumin and a -tocopherol are lipophilic and insoluble in water, which limits their further pharmacological exploitation and practical applications. Currently, very few reports are available on the synthesis of curcuminyl glyco-sides (Kaminaga et al. 2003 ; Mohri et al. 2003 ) and eugenol glycosides (Orihara et al. 1992 ; Sato et al. 2003 ) and that too by cell suspension and chemical methods.
Chemical preparation of glycosides cannot meet EC food regulations, and therefore chemi-cal preparation of glycosides is not applicable in the food industry. Further, regio- and stereoselec-tivity are two main criteria which are dif fi cult to achieve by chemical methods due to multiple hydroxyl groups in sugar molecule which require protection and deprotection (Mohri et al. 2003 ) . Hence, enzyme-catalysed reactions appear to be good alternatives.
This chapter describes synthesis of glycosides of phenols such as guaiacol, eugenol, curcumin, N-vanillylnonanamide, capsaicin, vanillin, dl -dopa, l -dopa, dopamine, serotonin and epinephrine by re fl ux method using amyloglucosidase from Rhizopus mould, b -glucosidase and immobilised b -glucosidase with carbohydrates: d -glucose, d -galactose, d -mannose, d -fructose, d -arabinose, d -ribose, maltose, sucrose, lactose, d -mannitol and d -sorbitol (Vijayakumar et al. 2006 ; Vijayakumar and Divakar 2005, 2007, 2011a, b ; Sivakumar et al. 2006a, b, 2009 ; Sivakumar and Divakar 2007, 2009a, b ). The reactions were inves-tigated in terms of incubation period, pH, buffer, enzyme and substrate concentrations, regio- and stereoselectivity.
8.1.1 Guaiacyl Glycosides
8.1.1.1 Guaiacyl- D -Glucoside Synthesis of guaiacyl- a - d -glucoside was stud-ied in detail to understand phenolic glycosyla-tion of such molecules (Vijayakumar and Divakar 2005 ) . Guaiacyl- a - d -glucoside synthe-sis in di-isopropyl ether using amyloglucosidase was optimised in terms of incubation period, pH, buffer concentration, enzyme concentration
and d -glucose concentration (Scheme 8.1 ). Optimum conditions determined were 1:50 equivalents of d -glucose and guaiacol, 50% (w/w d -glucose) of amyloglucosidase, 0.06-mM (0.6 mL) pH 7.0 phosphate buffer and 72-h incubation period (Table 8.1 , Fig. 8.1 ).
8.1.1.2 Synthesis of Guaiacyl Glucoside Using b -Glucosidase
Guaiacyl- d -glucoside synthesised by the re fl ux method using b -glucosidase isolated from sweet almonds involved considering d -glucose and guaiacol in 1:50 M ratio along with 50% (w/w d -glucose) enzyme and 0.06-mM (0.6-mL) pH 7.0 buffer in di-isopropyl ether solvent and re fl uxed for 72 h. The reaction mixture analysed by HPLC showed 22% conversion with respect to the d -glucose concentration employed.
8.1.1.3 Synthesis of Guaiacyl Glycosides Aldohexoses ( d -galactose and d -mannose), keto-hexose ( d -fructose), aldopentoses ( d -arabinose and d -ribose), disaccharides (maltose, sucrose and lactose) and carbohydrate alcohols ( d -mannitol and d -sorbitol) were employed for the synthesis of guaiacyl glycosides under the optimum conditions obtained for guaiacyl- a - d -glucoside synthesis (Scheme 8.1 ). d -Galactose reacted with guaiacol and gave the conversion yield of 17%. The reten-tion times for d -galactose and guaiacyl- d -galacto-side were 5.2 and 8.1 min, respectively. Other carbohydrates did not undergo any glycosylation with guaiacol (Table 8.2 ).
8.1.1.4 Spectral Characterisation UV spectrum of guaiacyl- a - d -glucoside showed s → s * band in the region 201–210 nm and n → p * band between 270 and 282 nm (free gua-iacol 279.5 nm). The hypsochromic shift observed for the glycoside indicated that guaiacol had undergone glycosylation. IR spectral band in the region 1,030–1,041 cm −1 corresponded to glyco-sidic aryl–alkyl C–O–C symmetrical stretching and 1,236–1,250 cm −1 to glycosidic aryl–alkyl C–O–C asymmetrical stretching frequencies. NMR spectral data con fi rmed that guaiacol formed C1 a -glucoside, C1 b - d -glucoside and C6-O-arylated product with d -glucose. Mass
1398.1 Phenols
Scheme 8.1 Synthesis of guaiacol, eugenol and curcumin glycosides
Table 8.1 Effect of buffer pH and buffer concentration on guaiacyl- a - d -glucoside synthesis a
pH b Yield, % Buffer concentration c (mM) at pH 7.0 Yield, %
4.0 30 0.01 3 5.0 43 0.02 11 6.0 31 0.04 51 7.0 51 0.06 52 8.0 32 0.08 39
0.1 11
a Conversion yields from HPLC with respect to 0.55 mmol of d -glucose. Error in yield mea-surements ± 5–10%. Guaiacol, 0.027 mol; enzyme, 50% (w/w d -glucose); incubation, 72 h; temperature, 68 °C b 0.04 mM (0.4 mL of 10-mM buffers added in 100-mL reaction mixture) c 0.1–1.0 mL of pH 7.0 phosphate buffer
spectrum for guaiacyl- d -glucoside showed m/z peak at 286 [M] + supporting the NMR data for the monoglycoside formation.
Down fi eld chemical shift of C1 a signal at 95.2 ppm ( 1 H at 4.97 ppm) indicated that a C1
glycosylated product was formed with galactose and C6-O- signal at 67.0 ppm ( 1 H at 3.70 ppm) indicated C6-O-arylated product. Mass spectrum showed m/z peak at 286 [M] + indicating monoga-lactoside formation.
140 8 Glycosylation of Some Selected Phenols and Vitamins
0
10
20
30
40
50
60
10 20 30 40 50 75
Con
vers
ion
yiel
d (%
)
Enzyme % (w/w D-glucose)
Fig. 8.1 Effect of amyloglucosidase concentration on guaiacyl- a - d -glucoside synthesis. Conversion yields were from HPLC with respect to 0.555 mmol of d -glucose. Reaction conditions: d -glucose, 0.555 mmol; guaiacol,
0.027 mol; 0.05-mM (0.5 mL of 10-mM buffer in 100-mL reaction mixture) pH 7.0 phosphate buffer; solvent, di-isopropyl ether; incubation, 72 h; temperature, 68°C
Table 8.2 Guaiacyl glycosides with conversion yields and product proportions
Glycosides and product proportions (%) a Glycosylation yield b (%)
O
OH
HH
H
H
OH
OH
OH
O
CH3O
O
OH
O
OH
OH
H
H
HH
OH
O
H3C
52
Guaiacyl- a - d -glucoside (52) C6-O-guaiacyl- d -glucose (48)
O
OH
HH
H
H
OH
OH
OH
OO
CH3
22
Guaiacyl- b - d -glucoside c
O
OH
HH
H
OH
OH OH
OO
HCH3
O
OH
OOH
OH
H
HH
OH
OH
H3C
17
Guaiacyl- a - d -galactoside (95) C6-O-guaiacyl- d -galactose (5)
a Product proportions shown in brackets were determined from 2-D HSQCT NMR C1/C6 cross peak areas b Conversion yields were from HPLC with errors in yield measurements ±5–10% c The compound was synthesised by using b -glucosidase from sweet almonds
1418.1 Phenols
8.1.2 Eugenyl Glycosides
Eugenyl glycosides were synthesised using amyloglucosidase (Vijayakumar and Divakar 2007 ) .
8.1.2.1 Eugenyl Maltoside Amyloglucosidase-catalysed synthesis of eugenyl maltoside was optimised in terms of incubation period, pH, buffer concentration and enzyme con-centration as a prototype reaction for detailed inves-tigation. The optimum conditions determined were as follows: glycosylation yield increased with the increase in incubation period from 3 h (7% yield) to 72 h (39% yield) and was the highest at 72 h at an initial rate of 19.2 m mol/h; conversion yield increased from 14% for 0.04 mM (0.4 mL) to 39% for 0.1 mM (1.0 mL); maximum glycosylation (39% yield) occurred at pH 5.0 acetate buffer; between 10 and 80% (w/w maltose) of enzyme concentration, 40% enzyme was found to be the best (conversion yield of 39%); and all other enzyme concentrations gave very less conversion yields (less than 19%).
8.1.2.2 Synthesis of Eugenyl Glucoside Using b -Glucosidase
b -Glucosidase isolated from sweet almond employed for the synthesis of eugenol glucoside in di-isopropyl ether solvent gave 19% conver-sion (from HPLC).
8.1.2.3 Synthesis of Eugenyl Glycosides Eugenyl glycosides of carbohydrates were syn-thesised (Scheme 8.1 ). Retention times for the carbohydrates and glycosides are d -glucose, 5.20 min; eugenyl- d -glucoside, 7.47 min; d -man-nose, 4.9 min; eugenyl- d -mannoside, 5.87 min; sucrose, 6.4 min; eugenyl sucrose, 8.02 min; d -man-nitol, 5.3 min; and eugenyl- d -mannitol, 7.12 min. From HPLC results, it was con fi rmed that d -galactose, d -fructose, d -arabinose, d -ribose, lactose and d -sorbitol did not undergo glycosyla-tion with eugenol (Table 8.3 ).
8.1.2.4 Spectral Characterisation UV spectra of the eugenyl glycosides showed shifts in the wavelength for p → p * (extended con-jugation) band in the range 265–279 nm (275.5 nm for free eugenol) con fi rming product formation.
IR C–O–C symmetrical stretching frequencies in the range 1,020–1,066 cm −1 and asymmetrical stretching frequencies in the range 1,262–1,268 cm −1 indicated that eugenol had undergone glycosyla-tion. Allylic C = C stretching frequency bands for the glycosides were also detected in the range 1,631–1,676 cm −1 . 2-D HSQCT spectral data showed formation of C1 glycoside and C6-O-arylated products. The mass m/z peaks (experi-mental section) further con fi rmed the formation of above-mentioned monoglycosides.
In the presence of b -glucosidase, d -glucose reacted with eugenol, and the only product, C1 b -glucoside, was found to be formed. In the ano-meric region, the only cross peak ( 13 C at 103.3 ppm and 1 H at 4.19 ppm) apart from the free C1 a and C1 b cross peaks indicated the for-mation of C1 b -glucoside.
8.1.3 Curcuminyl Glycosides
8.1.3.1 Synthesis of 1,7- O -(bis- b - D -Glucopyranosyl)Curcumin Using b -Glucosidase
Synthesis of 1,7- O -(bis- b - d -glucopyranosyl)cur-cumin using b -glucosidase from sweet almond was optimised in terms of incubation period, pH, buffer, enzyme and curcumin concentration in di-isopropyl ether solvent (Vijayakumar and Divakar 2005, 2007 ) . In summary the essential fi ndings are as follows: incubation period showed increase in conversion yields – 4% (12 h), 8% (24 h), 12% (48 h) and 25% (72 h) with increase in incubation period (Fig. 8.2a ) at an initial rate of 3.24 m mol/h; variation in buffer pH from pH 4.0 to 8.0 at 0.04 mM (0.4 mL) showed that the highest con-version yield of 29% was obtained at pH 4.0; effect of buffer concentration at pH 4.0 showed that conversion yield increased with increase in buffer concentration from 0.01 to 0.1 mM (0.1–1.0 mL) with the highest yield of 48% at 0.1 mM (1.0 mL); increasing amyloglucosidase concen-tration showed that (Fig. 8.2b ) 50% (w/w d -glu-cose) enzyme was required to achieve a maximum conversion of 48%; higher curcumin concentra-tions could be inhibitory to the enzyme as the conversion yield decreased from 37% for 0.2 and
142 8 Glycosylation of Some Selected Phenols and Vitamins
Table 8.3 Eugenyl glycosides with conversion yields and product proportions
Glycosides and product proportions (%) a Glycosylation yield b (%)
O
OH
HH
H
H
OH
OH
OH
O
CH3O
CH2
O
OH
O
OH
OHH
H
HH
OH
O
CH3
CH2
32
Eugenyl- a - d -glucoside (53) C6-O-eugenyl- d -glucose (47)
O
OH
HH
H
H
OH
OH
OH
O
CH2
OCH3
19
Eugenyl- b - d -glucoside c
O
H
OHH
H
H
OH
OH
OH
O
CH3O
CH2
8
Eugenyl- a - d -mannoside
OH
OHOH H
H
H
HH
OHO
O
O
OH
H
H
HH
OH
OOH
HCH2
CH3
O
O CH3
CH2
H
OO
OH
HH
H
H
OH
OH
O
OOH
HH
H
H
HOH
OH
OH
O
OH
OH H
H
H
HH
OHO
O
OH
OH
H
H
HH
OH
OOH
H
CH2
CH3O
17
Eugenyl maltoside (52) C6-O-eugenyl maltose (28) C6″-O-eugenyl maltose (20)
OHO
OHOH
OH
O
OH O
HO
OH
OCH2
CH3
O
OHO
OHOH
OH
OH
O
HO
OH
O
CH2
CH3
O
O
OCH3
CH2
O
OH
HO
OOH
OH
OHOH
OH
O
O
C1-O-eugenyl sucrose (35) C6-O-eugenyl sucrose (45) C6″-O-eugenyl sucrose (20) 7
OH
OH HOH HH OHH OH
H
OH
CH2
CH3O
7
C1-O-eugenyl mannitol
a Product proportions determined from 2-D HSQCT NMR C1/C6 cross peak areas are shown in brackets b Conversion yields were from HPLC with errors in yield measurements ±5–10% c The compound was synthesised by using b -glucosidase from sweet almonds
1438.1 Phenols
0.4 mmol curcumin to 34% (0.8 mmol), 29% (1.0 mmol), 22% (1.2 mmol) and 9% at 1.5 mmol curcumin; and maximum glycoside yield of 42% was obtained between 1.5 and 2.0 mmol of d -glu-cose concentrations.
The optimum conditions for this reaction were found to be curcumin and d -glucose in the 1:2 M ratio, 50% (w/w d -glucose) amyloglucosidase,
0.1-mM (1.0-mL), pH 4.0 acetate buffer and 72-h incubation period.
8.1.3.2 Antioxidant Activity Antioxidant activity of curcumin and curcuminyl-bis- a - d -glucoside was evaluated by DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging method (Moon and Terao 1998 ). An antioxidant
4
8
12
25
0
5
10
15
20
25
30
0 20 40 60 80
Con
vers
ion
yiel
d (%
)
Incubation period (h)
19.020.0
28.029.0
48.0
29.0
0
10
20
30
40
50
60
0 10 20 30 40 50 60 70 80
Con
vers
ion
yiel
d (%
)
Enzyme %(w/w D-glucose)
a
b
Fig. 8.2 Curcuminyl-bis- a - d -glucoside synthesis. Conversion yields were from HPLC with respect to 1-mmol d -glucose. ( a ) Effect of incubation period. Reaction conditions: d -glucose, 1 mmol; curcumin, 0.5 mmol; 0.06-mM (0.6-
mL), pH 6.0 phosphate buffer. ( b ) Effect of enzyme con-centration. Reaction conditions: d -glucose, 1 mmol; curcumin, 0.5 mmol; incubation period, 72 h; 0.1 mM (1.0 mL) of pH 4.0 acetate buffer
144 8 Glycosylation of Some Selected Phenols and Vitamins
activity of 80% for curcuminyl-bis- a - d -glucoside was obtained. The antioxidant activity of the glyco-side was found to be comparable to that of free curcumin (79%). Standard BHA activity was found to be 82%.
8.1.3.3 Solubility Test Determination of the water solubility of curcum-inyl-bis- a - d -glucoside showed that it is soluble to the extent of 14 g L −1 . Hence, curcuminyl-bis- a - d -glucoside was found to exhibit higher solubility than curcumin in water which exhib-ited negligible solubility.
8.1.3.4 Total Colour Test Curcuminyl-bis- a - d -glucoside exhibited a total colour of 8.8 in DMSO with curcumin as the stan-dard. The colour test was carried out according to the AOAC Of fi cial Methods of Analysis ( 1995 ) .
8.1.3.5 Curcuminyl Glycosides of Other Carbohydrates
Amyloglucosidase-catalysed synthesis of curcuminyl glycosides was synthesised (Scheme 8.1 ). The extent of glycosylation was analysed by HPLC. The retention times are d -glucose, 5.20 min; curcuminyl-bis- d -gluco-side, 7.52 min; d -mannose, 4.9 min; curcuminyl-bis- d -mannoside, 6.78 min; maltose, 7.4 min; curcuminyl-bis-maltoside, 8.3 min; sucrose, 6.4 min; curcuminyl-bis-sucrose, 8.44 min; d -mannitol, 5.3 min; and curcuminyl-bis- d -man-nitol, 8.35 min. From HPLC results, it was con fi rmed that d -galactose, d -fructose, d -arabi-nose, d -ribose, lactose and d -sorbitol did not undergo glycosylation with curcumin (Table 8.4 ). The carbohydrates, which underwent glycosyla-tion, are d -glucose, d -mannose, maltose, sucrose and d -mannitol.
Table 8.4 Curcuminyl glycosides with conversion yields and product proportions
Glycosides and product proportions (%) a Glycosylation yield b (%)
O
OOHOH
OH
CH2OH
CH3CH3OO
OOCH2OH
OHOH
OHO
O
48
Curcuminyl-bis- a - d -glucoside (62)
OH
OHOHOH
O O
OHOHOH
OH
O
OO
OOCH3 CH3
O
C6-O-curcuminyl-bis- d -glucose (38)
O
OHOH
OH
CH2OH
CH3CH3OO
OO
OO
O
OHOH
OH
CH2OH
11
Curcuminyl-bis- b - d -glucoside c
O
O
OH
OHOH
CH2OH
CH3CH3OO
OOCH2OH
OHOH
OH
O
O
9
Curcuminyl-bis- a - d -mannoside
(continued)
1458.1 Phenols
Table 8.4 (continued)
Glycosides and product proportions (%) a Glycosylation yield b (%)
OH
OHOH
OH O
O
O
OHOH
OOH
OH
OHOH
OHO
O
O
OHOH
OOHOO
OOCH3 CH3
19
Curcuminyl-bis-maltoside (37)
OH
OHOH
OH O
O
OHOH
O
O
OH
OO
OOCH3 CH3
OH
O
O
OHOH
O
O OHOH
OH
OH
C6-O-curcuminyl-bis-maltose (36)
OOH
OHOHO
O
OHOH
OOH
OH
OOH
OHOH O
O
OHOH
OOH
OH
OO
OOCH3 CH3
C6″-O-curcuminyl-bis-maltose (27)
OHO
OHOH
OH
O
OH O
HO
OH
O
OHO
OHOH
OH
O-
OHO
HO
OH
OOO
OOCH3 CH3
OO
OHOH
OH
OH
OH O
HO
OH
O
OO
OHOH
OH
OH
OHO
HO
OH
O
OO
OOCH3 CH3
19
C1-O-curcuminyl-bis-sucrose (12) C6″-O-curcuminyl-bis-sucrose (18)
OHO
OHOH
OH
O
OH
HO
O O
OHCH3CH3
OO
OO
OH
O O
HO
OH
O
OHOH
OH
OOH
Curcuminyl-bis-sucrose (70)
O
CH3CH3OO
OO
O
OH
OHHOHHHOHHOH
H
HH
H
OHHOHHHOHHOH
OH
14
C1-O-curcuminyl-bis- d -mannitol
a Product proportions determined from 13 C 2-D HSQCT NMR C1/C6 peak areas or their cross peaks are shown in brackets b Conversion yields were from HPLC with errors in yield measurements ±5–10%
146 8 Glycosylation of Some Selected Phenols and Vitamins
8.1.3.6 Spectral Characterisation The structures of the glycosides formed, HPLC yield and product proportions are presented in Table 8.4 . Curcumin, a higher phenyl propanoid homologue of eugenol, also reacted with the same carbohydrate molecules d -glucose, d -mannose, maltose, sucrose and d -mannitol as eugenol.
From the UV spectra, shift in the wavelength for the p → p * extended conjugation band in the 410–430 nm (433 nm for free curcumin) con fi rmed glycoside formation. IR C–O–C sym-metrical stretching frequencies in the range 1,020–1,071 cm −1 and asymmetrical stretching frequencies in the range 1,254–1,262 cm −1 also indicated that curcumin had undergone glycosy-lation. The symmetrical chemical shift values in the curcumin region of the product glycosides in the 2-D HSQCT spectra indicated that bis-gly-cosylated and bis-C6-O-arylated products were formed. Curcumin glycosides did not change colour on treatment with dilute alkali indicating that both the phenolic OH groups are glycosy-lated. Two-dimensional HSQCT NMR spectrum for the glucoside showed down fi eld chemical shift values for C8 and C8 ¢ at 150 ppm (149.4 ppm for free curcumin) and C1 a at 99.0 ppm ( 1 H at 4.66 ppm), respectively, con fi rming that curcumin was glucosylated symmetrically at the C8 and C8 ¢ position with the C1 carbon of the a - d -glucose anomer. Chemical shift values of the C6 cross peak ( 13 C at 66.5 ppm and 1 H at 3.52 and 3.70 ppm) con fi rmed C6-O-arylated product also being formed. Mass spectrum showed m/z value 691.5 [M-1] + for the bis-glycosylated products.
d -Mannose formed only a bis-C1 a product ( 13 C at 101 ppm and 1 H at 4.99 ppm). Mass spec-trum showed m/z value at 692.4 [M] + for the bis- d -mannoside. Maltose formed C1 a -maltoside, C6-O-arylated and C6″-O-arylated products. In the mass spectrum, the mass m/z peak at 1,016 [M] + corresponded to the bis product. A nonre-ducing sugar, sucrose, reacted with curcumin, which resulted in the formation of three arylated products (Table 8.4 ) as evidenced from the shift in the peaks for C1-O- ( 13 C at 65.1 ppm and 1 H at 3.54 ppm), C6-O- ( 13 C at 63.2 ppm and 1 H at
3.25 ppm) and C6″-O- ( 13 C at 65.0 ppm and 1 H at 3.41 ppm). Mass spectrum showed the m/z peak at 1,039 [M + Na] + con fi rming the bis-ary-lated product. In d -mannitol the C1 cross peak at 65.0 ppm ( 1 H at 3.45 ppm) indicated the for-mation of bis-C1-O-arylated product, which was also con fi rmed by the m/z peak at 719 [M + Na] + . Curcuminyl-bis- b - d -glucoside syn-thesised by using b -glucosidase from sweet almonds was con fi rmed from the C1 13 C value at 103.2 ppm and the corresponding 1 H value at 4.16 ppm.
8.1.3.7 Optimisation of Curcuminyl-bis- a - D -Glucoside Synthesis Using Response Surface Methodology
Design : Central Composite Rotatable Design (CCRD), 32 experiments, 5 variables at 5 levels (Vijayakumar et al. 2006 )
Variables : A myloglucosidase amount (% w/w of d -glucose), curcumin concentration (mmol), incu-bation period (h), buffer concentration (mM) and pH coded and actual values are shown in Table 8.5 . Actual set of experiments undertaken as per the CCRD with coded values and the glucosylation yields obtained is given in Table 8.6 .
Equation : A second-order polynomial equation was developed to study the effects of the vari-ables on the esteri fi cation yields in terms of lin-ear, quadratic and cross product terms.
1 2
3 4 5
1 1 2 2 3 3
4 4 5 5 1 2
1 3 1 4 1 5
2 3 2 4 2 5
3 4 3 5
61.7385 2.064 28.920
0.264 195.663 4.439
0.014 22.954 0.0006
168.79 0.330 0.446
0.003 8.333 0.039
0.213 381.2 5.062
4.661 0.022 22.
Y X X
X X X
X X X X X X
X X X X X X
X X X X X X
X X X X X X
X X X X
= - + +
+ + +
- - -
+ - +
- - -
- - +
+ - + 4 55X X
where X 1 , amyloglucosidase concentration; X
2 ,
curcumin concentration; X 3 , incubation period;
X 4 , buffer concentration; and X
5 , pH.
Coef fi cients : Microsoft Excel software, Version 5.0
1478.1 Phenols
Table 8.5 Coded values of the variables and their corresponding actual values used in the design of experiments
Variables −2 −1 0 1 2
Amyloglucosidase (%) 10 25 40 55 70 Curcumin (mmol) 0.2 0.4 0.6 0.8 1.0 Incubation period (h) 24 48 72 96 120 Buffer concentration (mM)
0.02 0.04 0.06 0.08 0.1
pH 4.0 5.0 6.0 7.0 8.0
Table 8.6 Experimental design with experimental and predicted yields of glucosylation based on the response surface equation
Expt. no. Enzyme %
Curcumin (mmol)
Incubation (h)
Buffer concn (mM) pH
Yield a experimental (%)
Yield predicted (%)
1 25 0.4 48 0.08 5.0 28.6 29.9 2 25 0.4 48 0.04 7.0 17.4 19.9 3 25 0.4 96 0.04 5.0 20.9 20.7 4 25 0.4 96 0.08 7.0 42.9 44.5 5 25 0.8 48 0.04 5.0 22.1 22.2 6 25 0.8 48 0.08 7.0 35.3 37.1 7 25 0.8 96 0.08 5.0 37.8 37.0 8 25 0.8 96 0.04 7.0 25.6 26.0 9 55 0.4 48 0.04 5.0 28.1 27.2 10 55 0.4 48 0.08 7.0 28.6 29.4 11 55 0.4 96 0.08 5.0 36.8 34.9 12 55 0.4 96 0.04 7.0 24.5 23.9 13 55 0.8 48 0.08 5.0 33.1 31.5 14 55 0.8 48 0.04 7.0 42.1 41.8 15 55 0.8 96 0.04 5.0 35.0 32.0 16 55 0.8 96 0.08 7.0 40.2 38.9 17 10 0.6 72 0.06 6.0 23.6 19.6 18 70 0.6 72 0.06 6.0 21.3 25.2 19 40 0.2 72 0.06 6.0 28.9 27.0 20 40 1.0 72 0.06 6.0 34.4 36.1 21 40 0.6 24 0.06 6.0 33.8 31.4 22 40 0.6 120 0.06 6.0 33.8 36.1 23 40 0.6 72 0.06 4.0 27.7 30.7 24 40 0.6 72 0.06 8.0 40.3 37.2 25 40 0.6 72 0.02 6.0 28.7 29.2 26 40 0.6 72 0.10 6.0 47.1 46.5 27 40 0.6 72 0.06 6.0 38.2 35.3 28 40 0.6 72 0.06 6.0 40.6 35.3 29 40 0.6 72 0.06 6.0 33.3 35.3 30 40 0.6 72 0.06 6.0 35.0 35.3 31 40 0.6 72 0.06 6.0 30.3 35.3 32 40 0.6 72 0.06 6.0 34.9 35.3
a Conversion yields were from HPLC with respect to 1 mmol of d -glucose. The experimental yields are an average from two experiments. Error in yield measurements ±5–10%
148 8 Glycosylation of Some Selected Phenols and Vitamins
Analysis of Variance ( ANOVA ): Microsoft Excel software. ANOVA shows the model is signi fi cant at P < 0.01.
Optimisation : Microsoft Excel Solver function The experimental data fi tted the second-order
polynomial equation well as indicated by an R 2 value of 0.9.
Average absolute deviation between predicted and experimental yields was found to be 6.0. Amyloglucosidase requires some amount of water to be present for its optimum activity, and this is achieved by adding buffer of certain volume, salt concentration and pH (Vic et al. 1997 ; Chahid et al. 1992 ) . Salient features of this amyloglucosi-dase-catalysed reaction are described in the three-dimensional surface and contour plots.
Effect of various amyloglucosidase and cur-cumin concentrations on the extent of glucosyla-tion of curcumin shows a maximum conversion of 35% for curcumin concentrations above 0.55 mmol at 35–60% (w/w d -glucose) amylog-lucosidase concentrations (Fig. 8.3 ). Iso-glucosylation regions of 15–25% yield could be predicted for amyloglucosidase concentrations below 30% and above 60% at all curcumin con-
centrations in the range 0.2–1.0 mmol. While lower amyloglucosidase converted less, higher amyloglucosidase could be inhibitory to cur-cumin at a constant d -glucose concentration.
Effect of amyloglucosidase concentration and pH on the extent of glycosylation also exhibited a similar pattern. A narrow range of amyloglucosi-dase concentration of 35–55% showed maximum glucosylation of 35% at above pH 5.5. Above and below this enzyme concentration range, the con-version yield was lesser in the pH range 4.0–8.0. For the hydrolytic activity, the pH optimum for amyloglucosidase is 5.0, and the isoelectric point for the enzyme is 4.2. Above pH 5.5 and towards higher pH 8.0, the ionisable groups especially from acidic amino acid residues (Glu314 and Glu544) in the active site could exist in anionic forms, enabling abstraction of the anomeric hydroxyl proton of glucose to aid in the facile transfer of glucose molecule to curcumin (Frandsen et al. 1994 ; Sierks et al. 1990 ) .
Figure 8.4 shows the effect of amyloglucosi-dase concentration and buffer concentration on the conversion yield of curcuminyl-bis- a - d -glucoside. Buffer concentration included both the effects of the concentrations of the buffer salts and volume
Fig. 8.3 Three-dimensional surface and contour plot showing the effect of amyloglucosidase concentration and curcumin concentration on the extent of glucosylation at incubation period, 72 h; buffer concentration, 0.06 mM (0.6 mL of 0.01-M buffer), pH 6.0
Fig. 8.4 Three-dimensional surface and contour plot showing the effect of amyloglucosidase concentration and buffer concentration on the extent of glucosylation at cur-cumin, 0.6 mmol; incubation period, 72 h; and pH, 6.0
1498.1 Phenols
of the buffer. While buffer volume determined the effect of water activity on amyloglucosidase, pH controlled the extent of ionisation of the charged amino acid residues of the amyloglucosidase in the active site and on the surface, the latter being also affected by the buffer concentration. Together, both these quantities explain the role of the active conformation of the enzyme, on catalysis at the re fl uxing temperature of the solvent (at 68°C). After 120 h of incubation period, amyloglucosi-dase was found to lose only 20% of its activity. Between an amyloglucosidase concentration range of 15–45%, maximum conversion of 45% could be observed in the buffer concentration range 0.095–0.1 mM (0.95–1.0 mL of 0.01-M pH 6.0 buffer). The same amyloglucosidase concentration range could not give good conversion yields at buffer concentrations less than 0.08 mM (0.8 mL of 0.01-M pH 6.0 buffer). Besides, amyloglucosi-dase concentrations above 60% also showed lesser yields in the buffer concentration range 0.03–0.1 mM (0.3–1.0 mL of 0.01-M pH 6.0). This feature clearly shows that the extent of gluco-sylation could be dictated by a critical buffer (0.95–1.0 mL of 0.01-M pH 6.0) to enzyme ratio (15–45% w/w d -glucose).
A maximum yield of 65.6% was predicted based on the response model for an amyloglu-cosidase concentration of 16.9% (w/w d -glucose), 0.33-mmol curcumin concentration, 120-h incu-bation period and 0.1-mM (1.0 mL of 0.01-M pH 6.0 buffer) buffer concentration at pH 7.5. Experiment conducted at the above optimum conditions resulted in a yield of 56.3%. Validation of the response model was also tested by carry-ing out experiments at selected random condi-tions. The yields obtained from validation experiments also agreed with the predicted yields with an average absolute deviation of 8.5% (Table 8.7 ).
Thus, this study showed that this model is very good in predicting the glucosylation of curcumin by the amyloglucosidase enzyme.
8.1.4 Syntheses of N-Vanillyl-Nonanamide Glycosides
Capsaicin {( E )-N-[(4-hydroxy-3-methoxyphenyl)methyl]-8-methyl-6-nonemide} (Ohnuki et al. 2001 ) , a fat-soluble phenolic compound present as the major pungent principle in fruits of Capsicum species, can also be used as a hypoglycemic
Table 8.7 Validation of experimental data
Expt. no. Enzyme (%)
Curcumin (mmol)
Incubation period (h)
Buffer conc. (mM) pH
% yield predicted
% yield a experimental
1 40 0.6 72 0.06 6.0 35.4 39.2 2 40 0.3 72 0.06 5.0 29.5 31.0 3 25 0.6 72 0.07 6.0 34.6 37.1 4 40 0.9 110 0.06 6.0 35.2 28.3 5 40 0.5 20 0.06 6.0 28.5 31.4 6 40 0.6 70 0.06 6.0 35.3 35.7 7 60 0.9 72 0.06 6.0 35.5 36.7 8 20 0.5 72 0.06 6.0 27.3 31.3 9 35 0.6 72 0.06 6.5 35.4 28.0 10 20 0.6 72 0.06 5.5 26.5 32.5 11 40 0.6 65 0.06 6.0 35.0 41.5 12 15 0.6 50 0.06 6.0 20.9 27.5 13 40 0.6 72 0.09 7.0 45.3 46.5 14 40 0.6 72 0.04 5.0 30.1 34.2 15 16.9 0.33 120 0.1 7.5 65.6 56.3
a Conversion yields were from HPLC with respect to 1 mmol of d -glucose. The experimental yields are an average from two experiments. Error in yield measurements ±5–10%
150 8 Glycosylation of Some Selected Phenols and Vitamins
(Lan et al. 2004 ) , mutagenic and carcinogenic agent (Gannett et al. 1988 ) ; as a topical analgesic in phar-maceutical preparations (Rashid et al. 2003 ) ; and as antioxidant (Lee et al. 1995 ) , anti-in fl ammatory (Heyes et al. 2004 ) and antifungal agent (Bhabadesh et al. 1996 ) . It also can increase cat-echolamine secretion and suppress body fat accu-mulation (Watanabae et al. 1994 ) , inhibit NADH oxidase in plasma membranes (Morre et al. 1995 ) and reduce the peri-renal adipose tissue weight and serum triacylglycerol by enhancing energy metabolism through a b -adrenergic action (Kawada et al. 1986 ) . N-Vanillyl-nonanamide {N-[(4-hydroxy-3-methoxyphenyl)methyl]nonanamide}, a synthetic substitute of capsaicin, exhibits hypotensive and anti-nociceptive effects as that of natural capsaicin (Chen et al. 1992 ). Capsaicin (Iorizzi et al. 2001 ) has also been con-verted into its corresponding glucoside by cell suspension cultures (Kometani et al. 1993a ; Hamada et al. 2003 ) and chemical methods (Hamada et al. 2001 ) . Capsaicin and usage of its derivatives have been limited due to its low water solubility and high pungency.
Hence, a study on the synthesis of 4- O -( d -glucopyranosyl)N-vanillyl-nonanamide was under-taken (Scheme 8.2 , Sivakumar and Divakar 2007 ) .
HPLC analysis (Fig. 8.5 ) showed the following retention times: d -glucose, 6.9 min; 4- O -( d -
glucopyranosyl)N-vanillyl-nonanamide, 8.2 min; d -galactose, 7.1 min; 4- O -( d -galactopyranosyl)N-vanillyl-nonanamide, 8.3 min; d -mannose, 6.7 min; 4- O -( b - d -mannopyranosyl)N-vanillyl-nonanamide, 8.9 min; d -ribose, 7.4 min; 4- O -( d -ribofuranosyl)N-vanillyl-nonanamide, 8.9 min; maltose, 8.5 min; 4- O -( a - d -glucopyranosyl-(1 ¢ → 4) d -glucopyranosyl)N-vanillyl-nonanamide, 15.2 min; lactose, 9.3 min; and 4- O -( b - d -galactopyranosyl-(1 ¢ → 4) b - d -glucopy-ranosyl)N-vanillyl-nonanamide, 17.9 min.
8.1.4.1 Synthesis of 4- O -( D -Glucopyranosyl)N-Vanillyl-Nonanamide Using Amyloglucosidase
Glucosylation of N-vanillyl-nonanamide with d -glucose using amyloglucosidase (Table 8.8 ) showed the following optimum conditions: incu-bation period, 72 h, and buffer, 0.2-mM, pH 7 phosphate buffer (2 mL of 0.01-M buffer in 100-mL di-isopropyl ether solvent); at pH 7, increase in buffer concentration from 0.03 to 0.25 mM (0.3–2.5 mL) showed increase in conversion with increase in buffer concentration to a maximum of 56% at 0.2-mM (2-mL) buffer; at 10% (w/w d -glu-cose) amyloglucosidase concentration conversion yield was the lowest of 16% which then increased to a maximum yield of 56% at 40% (w/w d -glu-cose) enzyme concentration (Table 8.8 ); increase
Scheme 8.2 Syntheses of N-vanillyl-nonanamide glycosides
1518.1 Phenols
Fig. 8.5 HPLC chromatogram for the reaction mixture of d -glucose and 4- O -( d -glucopyranosyl)N-vanillyl-nonanamide. HPLC conditions: aminopropyl column (10 m m, 250 mm × 4.6 mm); solvent, CH
3 CN:H
2 O
(70:30 v/v); fl ow rate, 1 mL min −1 ; RI detector. Retention times: d -glucose, 7 min, and 4- O -( d -glucopyranosyl)N-vanillyl-nonanamide, 8.2 min
in N-vanillyl-nonanamide concentration (Table 8.8 ) exhibited increase in glucosylation from 0.2 mmol (12% yield) to 0.6 mmol (23%) and thereafter remained constant up to 1 mmol (23%).
8.1.4.2 Solubility of 4- O -( D -Glucopyranosyl)N-Vanillyl-Nonanamide
4- O -( d -Glucopyranosyl)N-vanillyl-nonanamide was found to be soluble in water to the extent of 7.7 g L −1 . Thus the water-insoluble and predomi-nantly fat-soluble N-vanillyl-nonanamide has been rendered water-soluble through this glyco-sylation reaction.
8.1.4.3 Syntheses of N-Vanillyl-Nonanamide Glycosides of Other Carbohydrates Using Amyloglucosidase and b -Glucosidase and Their Spectral Characterisation
Other N-vanillyl-nonanamide glycosides were pre-pared using amyloglucosidase and b -glucosidase.
UV spectra of N - vanillyl-nonanamide glycosides showed shifts for s → s * band in the 192.5–203.5-nm (201.5 nm for free N - vanillyl-nonanamide)
region, s → p * band in the 225–227-nm (225 nm for free N - vanillyl-nonanamide) region and p → p * band in the 263–283.5-nm (279 nm for free N - vanillyl-nonanamide) region and IR C–O–C asymmetrical stretching frequencies in the 1,254–1,279-cm −1 region and symmetrical stretching fre-quencies in the 1,027–1,038-cm −1 region indicating that N - vanillyl-nonanamide had undergone glycosy-lation. From the 2-D HSQCT spectra of the N - vanillyl-nonanamide glycosides, C1 a - and C1 b -glycosides besides C6- O -arylated products were detected. Mass spectra also con fi rmed the formation of the above-mentioned glycosides (Table 8.9 ).
8.1.5 Capsaicin Glycosides
Capsaicin glycosides were synthesised enzymati-cally using b -glucosidase from sweet almond to obtain capsaicin derivatives whose water solubil-ity is greater than that of capsaicin (Scheme 8.3 ). Optimum reaction conditions used for the syn-thesis of capsaicin glycosides are capsaicin 0.25 mmol; carbohydrate 0.25 mmol; b -glucosi-dase 40% (w/w carbohydrate); 1-ml (0.01-M) pH
152 8 Glycosylation of Some Selected Phenols and Vitamins
7.0 phosphate buffer; 50-ml di-isopropyl ether; and 72-h incubation period. Glycosylation of capsaicin was carried out with d -glucose, d -galactose and lactose using b -glucosidase. Capsaicin glyco-sides yields were in the 15–29% range. Excellent regioselectivity was observed with d -glucose and lactose (Table 8.10 ).
8.1.6 Syntheses of Vanillyl Glycosides
Vanillin(4-hydroxy-3-methoxybenzaldehyde) is used as an additive in food and beverages (60%), considerable amounts as fl avour and fragrances (20–25%) and 5–10% as an intermediate for pharmaceuticals. It possesses a wide range of
Table 8.8 Optimisation of reaction conditions for the synthesis of 4- O -( d -glucopyranosyl)N-vanillyl-nonanamide
Reaction conditions Variable parameter b Yield (%) c
Incubation period (h) N-Vanillyl-nonanamide – 0.5 mmol 3 25 d -Glucose – 0.5 mmol 6 29 pH – 7 12 31 Buffer concentration – 0.2 mM (2 mL) 24 33 Enzyme – 40% w/w d -glucose 48 45
72 56 96 34
120 28 pH (0.01 M)
N-Vanillyl-nonanamide – 0.5 mmol a 4 18 d -Glucose – 0.5 mmol 5 18 Enzyme – 40% w/w d -glucose 6 25 Buffer – 0.1 mM (1 mL) 7 39 Incubation period – 72 h 8 19
Buffer concentration (mM) N-Vanillyl-nonanamide – 0.5 mmol 0.03 26 d -Glucose – 0.5 mmol 0.06 37 Enzyme – 40% w/w d -glucose 0.1 42 pH – 7 0.15 49 Incubation period – 72 h 0.2 56
0.25 50 Enzyme concentration (% w/w d -glucose)
N-Vanillyl-nonanamide – 0.5 mmol 10 16 d -Glucose – 0.5 mmol 20 24 pH – 7 30 39 Buffer concentration – 0.2 mM (2 mL) 40 56 Incubation period – 72 h 50 21
75 10 N-Vanillyl-nonanamide (mmol)
pH – 7 0.2 12 Buffer concentration – 0.2 mM (2 mL) 0.4 14 d -Glucose – 0.2 mmol 0.6 23 Enzyme – 40% w/w d -glucose 0.8 24 Incubation period – 72 h 1 23
a Initial reaction conditions b Other variables are the same as under reaction conditions, except the speci fi ed ones c HPLC yields expressed with respect to 0.5-mmol d -glucose employed except for the last experiment where N-vanillyl-nonanamide concentrations were varied with respect to only 0.2-mmol d -glucose
1538.1 Phenols
Tab
le 8
.9
Synt
hese
s of
N-v
anill
yl-n
onan
amid
e gl
ycos
ides
a
Gly
cosi
des
Am
ylog
luco
sida
se c
atal
ysis
b -
Glu
cosi
dase
cat
alys
is
Prod
uct (
% p
ropo
rtio
n) b
Yie
lds
(%) c
Prod
uct
(% p
ropo
rtio
n) b
Yie
lds
(%) c
OCH
3
O
H
OH
OH
H
H
HH
OH
O
NH
CH
3O
OH
OO
CH
3
HO
HO
HH
H
HH
OH
O
NH
CH
3O
OH
O
OHH
OH
OH
H
H
HH
OH
O
OCH
3
NH
CH
3O
C1 a
-glu
cosi
de (
70),
C
6- O
-ary
late
d (3
0)
56
C1 b
-glu
cosi
de
35
4- O
-( a
- d -G
luco
pyra
nosy
l)N
-van
illyl
-non
anam
ide
4- O
-(6-
d -G
luco
pyra
nosy
l)N
-van
illyl
-non
anam
ide
4- O
-( b -
d -G
luco
pyra
nosy
l)N
-van
illyl
-non
anam
ide
O
OCH
3
HH
OH
H
OH
OH
HH
OH
O
OCH
3NH
OCH
3
H
OH
O
H
OH
HH
OH
H
O
NH
CH
3O
OH
C1 a
-gal
acto
side
(42
),
C1 b
-gal
acto
side
s (5
8)
14
C1 b
-gal
acto
side
26
4- O
-( a
- d -G
alac
topy
rano
syl)
N-v
anill
yl-n
onan
amid
e
4- O
-( b -
d -G
alac
topy
rano
syl)
N-v
anill
yl-n
onan
amid
e
OCH
3
HO
H2C
OH
OH
O
H
H
HO
HHH
O
NH
CH
3O
– –
C1 b
-man
nosi
de
24
4- O
-( b -
d -M
anno
pyra
nosy
l)N
-van
illyl
-non
anam
ide
(con
tinue
d)
154 8 Glycosylation of Some Selected Phenols and VitaminsTa
ble
8.9
(c
ontin
ued)
Gly
cosi
des
Am
ylog
luco
sida
se c
atal
ysis
b -
Glu
cosi
dase
cat
alys
is
Prod
uct (
% p
ropo
rtio
n) b
Yie
lds
(%) c
Prod
uct
(% p
ropo
rtio
n) b
Yie
lds
(%) c
OC
H3
CH
2OH
H
OH
OH
HH
HO
O
NH
CH
3O
OC
H3
CH
2OH
O
OH
OH
HH
HH
O
NH
CH
3O
C1 a
rib
osid
e (3
3),
C1 b
rib
osid
e (6
7)
9 C
1 a r
ibos
ide
(30)
, C
1 b r
ibos
ide
(70)
10
4- O
-( a
- d -R
ibof
uran
osyl
)N-v
anill
yl-n
onan
amid
e
4- O
-( b -
d -R
ibof
uran
osyl
)N-v
anill
yl-n
onan
amid
e
OC
H3
OO
OH
HH
H
H
OH
HO
H2C
OH
OH
H
H
H
HH O
HO
O
H
OH
NH
CH
3O
OC
H3
O
HO
OH
HH
H
H
OH
HO
H2C
HO
H2C
OH
OH
H
H
H
HH
OH
O
O
NH
CH
3O
OC
H3
OHH
O
OO
HHH
H
HH
OH
OH
OH
H
H
HH
OH
OO
H
O
NH
CH
3O
OC
H3
O
OO
H
HH
H
H
HO
HOH
HO
H2C
HO
H2C
OH
H
H
HH O
H
OO
HNH
CH
3O
C1 a
-mal
tosi
de (
11),
C6-
O -
aryl
ated
(40
), C
6 ¢ - O
-ary
late
d (4
9)
15
C1 b
-mal
tosi
de
9
4- O
-( a
- d -G
luco
pyra
nosy
l-(1
¢ →4)
a - d
-glu
copy
rano
syl)
N-v
anill
yl-n
onan
amid
e
4- O
-( a
- d -G
luco
pyra
nosy
l-(1
¢ →4)
6- d -
gluc
opyr
anos
yl)N
-van
illyl
-non
anam
ide
4- O
-( a
- d -G
luco
pyra
nosy
l-(1
¢ →4)
6 ¢ - d
-glu
copy
rano
syl)
N-v
anill
yl-n
onan
amid
e
4- O
-( a
- d -G
luco
pyra
nosy
l-(1
¢ →4)
b - d -
gluc
opyr
anos
yl)N
-van
illyl
-non
anam
ide
OC
H3
NH
CH
3O
OH
OH
O
H
OH
HH O
H
OH
H
O
OH
HH
H
H
OH
HO
H2C
O
H
– –
C1 b
-lac
tosi
de
28
4- O
-( b -
d -G
alac
topy
rano
syl-
(1 ¢ →
4) b -
d -gl
ucop
yran
osyl
)N-v
anill
yl-n
onan
amid
e
a N-V
anill
yl-n
onan
amid
e an
d ca
rboh
ydra
te, 0
.5 m
mol
eac
h; e
nzym
e co
ncen
trat
ion,
40%
w/w
of c
arbo
hydr
ate;
sol
vent
, di-
isop
ropy
l eth
er; b
uffe
r, 0.
2-m
M (2
-mL
) pH
7 p
hosp
hate
bu
ffer
; inc
ubat
ion
peri
od, 7
2 h
b The
pro
duct
pro
port
ions
wer
e de
term
ined
fro
m th
e ar
ea o
f re
spec
tive
1 H/ 13
C s
igna
ls
c Con
vers
ion
yiel
ds w
ere
from
HPL
C w
ith r
espe
ct to
fre
e ca
rboh
ydra
te. E
rror
in y
ield
mea
sure
men
ts is
±10
%
1558.1 Phenols
pharmacological activities such as antimicrobial (Lopez-Malo et al. 1998 ) , anticarcinogenic (Stephan and Peter 2003 ) , antioxidant (Burri et al. 1999 ) , antifungal (Fitzgerald et al. 2005 ) and antimutagenic (Kometani et al. 1993b ) . The
solubility of vanillin in water varies from 3 g L −1 at 4.4°C to 62.5 g L −1 at 80°C (The Merck Index 2006 ) . Thus the solubility and bioavailability of vanillin limit its pharmacological applications. Glycosylation is a useful tool to improve the
Scheme 8.3 Syntheses of capsaicin glycosides
Table 8.10 b -Glucosidase-catalysed syntheses of capsaicin glycosides a
Glycosides Product (% proportion) b Yields (%) c
NH
O
OO
CH3
CH3
CH3H
CH2OHOH
OHH
H
HH
OH
O
C1 b 15
3- O -( b - d -Glucopyranosyl)capsaicin
NH
O
OO
CH3
CH3
CH3
H
CH2OH
H
OH
H
OH
HH
OH
O
C1 a (42) and C1 b (58) 13
3- O -( a - d -Galactopyranosyl)capsaicin
NH
O
OO
CH3
CH3
CH3H
CH2OHH
OHH
OH
HH
OH
O
3- O -( b - d -Galactopyranosyl)capsaicin
NH
O
OO
CH3
CH3
CH3H
OHH
H
HH
OH
OOH
H
HO
OH
HH
OH
H
OOH
OH
C1 b 29
3- O -( b - d -Galactopyranosyl-(1 ¢ →4) b - d -glucopyranosyl)capsaicin
Capsaicin, 0.25 mmol; carbohydrate, 0.25 mmol; solvent, di-isopropyl ether a b -glucosidase concentration, 40% w/w carbohydrates; 0.10-mM (1.0-ml) pH 7.0 phosphate buffer; incubation period, 72 h b product proportions were calculated from the area of respective 1 H/ 13 C signals. Conversion yields were from HPLC with respect to the carbohydrate. Error in yield measurements is ±10%
156 8 Glycosylation of Some Selected Phenols and Vitamins
Scheme 8.4 Synthesis of vanillyl glycosides
water solubility and bioavailability (Kometani et al. 1993a ; Tietze et al. 2003 ) of vanillin.
Preparation of vanillyl glycosides has been reported by cell suspension cultures (Tietze et al. 2003 ) , plant cell tissue cultures, organ cultures (Sommer et al. 1997 ) and chemical methods (Reichel and Sckickle 1943 ) . However, prepara-tion by enzymatic methods has not been previ-ously reported. This section deals with the enzymatic method using amyloglucosidase from a Rhizopus mould and b -glucosidase from sweet almond for the preparation of vanillyl glycosides in a nonpolar solvent (Scheme 8.4 , Sivakumar and Divakar 2006 ) .
HPLC analysis of the glycosides (Fig. 8.6 ) showed the following retention times: d -glucose, 5.4 min; 4- O -( d -glucopyranosyl)vanillin, 7.8 min; d -galactose, 5.3 min; 4- O -( d -galactopyranosyl) vanillin, 7.5 min; d -mannose, 4.9 min; 4- O -( d -mannopyranosyl)vanillin, 7.8 min; maltose, 8.5 min; 4- O -( a - d -glucopyranosyl-(1 ¢ → 4) d -glucopyranosyl)vanillin, 17.1 min; sucrose, 9.8 min; 4- O -( d -
fructofuranosyl-(2 → 1 ¢ ) a - d -glucopyranosyl)vanillin, 14.3 min; lactose, 9.9 min; 4- O -( b - d -galactopyranosyl-(1 ¢ → 4) b - d -glucopyranosyl) vanillin, 8.2 min; d -sorbitol, 6.7 min; and 4- O -( d -sorbitol)vanillin, 10.1 min.
8.1.6.1 Synthesis of 4- O -( D -Glucopyranosyl)Vanillin Using Amyloglucosidase
Optimum conditions determined are vanillin (1 mmol) and d -glucose (1 mmol); 72-h incuba-tion period (Fig. 8.7a , Table 8.11 ); re fl ux tem-perature of di-isopropyl ether at 68°C; pH 4.0; buffer concentration, 0.1 mM (1 mL); and 53% at 40% (w/w d -glucose) enzyme concentration (Fig. 8.7b , Table 8.11 ).
8.1.6.2 Solubility of 4- O -( D -Glucopyranosyl)Vanillin
Determination of water solubility of 4- O -( d -glucopyranosyl)vanillin showed that it is solu-ble to the extent of 35.2 g L −1 . Hence,
1578.1 Phenols
4- O -( d -glucopyranosyl)vanillin was found to be more soluble than vanillin (2 g L −1 ) at 25°C in water.
8.1.6.3 Syntheses of Vanillyl Glycosides of Other Carbohydrates Using Amyloglucosidase and b -Glucosidase and Their Spectral Characterisation
Vanillyl glycosides of other carbohydrates (Scheme 8.4 ) were synthesised using amyloglu-cosidase and b -glucosidase (Table 8.12 ).
UV spectra of vanillyl glycosides showed shifts in s → s * band in the 193.5–198.5-nm (195 nm for free vanillin) range, s → p * band in the 222–224.5-nm (228.5 nm for free vanillin) range, p → p * band in the 273–283.5-nm (272 nm for free vanillin) range, IR C–O–C asymmetrical stretching frequen-cies in the 1,249–1,265-cm −1 range and symmetri-cal stretching frequencies in the 1,024–1,038-cm −1 range indicating that vanillin had undergone glycosylation. In 2-D Heteronuclear Single Quantum Coherence Transfer (HSQCT) spectra, the respective chemical shift values of carbon
atoms C1 a and b , C6 a and b showed glycoside formation and arylation (Table 8.12 ).
8.1.6.4 Synthesis of 4- O -( a - D -Glucopyranosyl-(1 ¢ → 4) D -Glucopyranosyl)Vanillin Using Glucosidases by Response Surface Methodology
Design : Central Composite Rotatable Design (CCRD), 32 experiments, 5 variables at 5 levels (Sivakumar et al. 2006a )
Variables : G lucosidase concentration, vanillin concentration, incubation period, buffer volume and pH in case of both the glucosidases. Table 8.13 shows the coded and actual levels of the variables employed in the design matrix. Actual set of experiments undertaken as per CCRD with coded values and the maltosylation yields obtained are given in Table 8.14 .
Equation : A second-order polynomial equation was developed to study the effects of the vari-ables on the esteri fi cation yields in terms of lin-ear, quadratic and cross product terms.
Fig. 8.6 HPLC chromatogram for the reaction mixture of d -glucose and 4- O -( d -glucopyranosyl)vanillin. HPLC conditions: aminopropyl column (10 m m, 300 mm × 3.9 mm);
solvent, CH 3 CN:H
2 O (80:20 v/v); fl ow rate, 1 mL min −1 ; RI
detector. Retention times: d -glucose, 5.4 min, and 4- O -( d -glucopyranosyl)vanillin, 7.8 min
158 8 Glycosylation of Some Selected Phenols and Vitamins
Coef fi cients : Microsoft Excel software, Version 5.0
Analysis of Variance ( ANOVA ): Microsoft Excel software ANOVA shows the model is signi fi cant at P < 0.01.
Optimisation : Microsoft Regression software A second-order polynomial equation was devel-oped to study the effect of the variables on the maltoside yields.
8.1.6.4.1 Amyloglucosidase-Catalysed Synthesis of 4- O -( a - D -Glucopyranosyl-(1 ¢ → 4) D -Glucopyranosyl)Vanillin
The data obtained using amyloglucosidase were fi tted to a second-order polynomial equation, and the predictive equation obtained with coef fi cients exhibited an R 2 value of 0.81.
0
10
20
30
40
50
60
70
0
Con
vers
ion
yiel
d (%
)
20 40 60 80 100Incubation period (h)
0
10
20
30
40
50
60
10 20 30 40 50 75
Con
vers
ion
yiel
d (%
)
Amyloglucosidase concn (% w/w D-glucose)
a
b
Fig. 8.7 ( a ) Reaction pro fi le for 4- O -( d -glucopyranosyl)vanillin synthesis by the re fl ux method. Conversion yields were from HPLC with respect to 1 mmol of d -glucose. Reaction conditions: d -glucose, 1 mmol; vanillin, 1 mmol; amyloglucosidase, 40% (w/w d -glucose); 0.1-mM pH 4 acetate buffer; solvent, di-isopropyl ether; temperature,
68°C; and ( b ) effect of amyloglucosidase concentration for 4- O -( d -glucopyranosyl) vanillin synthesis. Reaction conditions: d -glucose, 1 mmol; vanillin, 1 mmol; 0.1-mM pH 4 acetate buffer; solvent, di-isopropyl ether; tempera-ture, 68°C; and incubation period, 72 h
1598.1 Phenols
Equation :
1 5
3 3 5 5
1 4 1 5
2 3 3 4
4 5
3.7258 25.3461
0.0051 - -3.7238
0.6985 0.4896
0.0281 0.7730
5.7750
Y X X
X X X X
X X X X
X X X X
X X
t
= - +
+
+ +
- -
+
where X 1 , enzyme concentration; X
2 , vanillin
concentration; X 3 , incubation period; X
4 , buffer
volume; X 5 , pH; and Y , yield. Table 8.14 shows
the predicted yields obtained by using the reduced equation.
The effect of amyloglucosidase and buffer con-centration on the extent of maltosylation at 1.5-mmol vanillin, pH 6 and 72-h incubation period
Table 8.11 Optimisation of reaction conditions for the synthesis of 4- O -( d -glucopyranosyl)vanillin
Reaction conditions a Variable parameter b Yield (%) c
Incubation period (h) Vanillin – 1 mmol 3 28 d -Glucose – 1 mmol 6 26 pH – 4 12 29 Buffer concentration – 0.1 mM (1 mL) 24 31 Enzyme – 40% w/w d -glucose 48 38
72 53 96 46 pH (0.01 M)
Vanillin – 1 mmol a 4 51 d -Glucose – 1 mmol 5 28 Enzyme – 40% w/w d -glucose 6 22 Buffer concentration – 0.1 mM (1 mL) 7 18 Incubation period – 72 h 8 17
Buffer concentration (mM) Vanillin – 1 mmol 0.02 13 d -Glucose – 1 mmol 0.04 46 Enzyme – 40% w/w d -glucose 0.06 48 pH – 4 0.08 50 Incubation period – 72 h 0.1 51
0.12 42 Enzyme concentration (% w/w d -glucose)
Vanillin – 1 mmol 10 17 d -Glucose – 1 mmol 20 32 pH – 4 30 35 Buffer concentration – 0.1 mM (1 mL) 40 53 Incubation period – 72 h 50 30
75 31 Vanillin (mmol)
pH – 4 0.5 49 Buffer concentration – 0.1 mM (1 mL) 1 53 d -Glucose – 1 mmol 1.5 47 Enzyme – 40% w/w d -glucose 2 48 Incubation period – 72 h 2.5 46
a Initial reaction conditions b Other variables are the same as under reaction conditions, except the speci fi ed ones c HPLC yields expressed with respect to 1-mmol d -glucose employed
160 8 Glycosylation of Some Selected Phenols and Vitamins
Table 8.12 Syntheses of vanillyl glycosides using amyloglucosidase and b -glucosidase
Glycosides
Amyloglucosidase catalysis a b -Glucosidase catalysis b
Product (% proportion) c
Yields (%) d
Product (% proportion) c
Yields (%) d
O
OH
HH
H
HOH
OHH
O
CH3OO
OH
O
CH3OO
OH
HH
H
HOH
OH
H
O
OH
O
CH3 OO
OH
HOH
OHH
H
HH
OH
O
C1 a -glucoside (52), C1 b -glucoside (17), C6- O- arylated (31)
53 C1 b -glucoside 10
4- O -( a - d -Glucopyranosyl)vanillin
4- O -( b - d -Glucopyranosyl)vanillin 4- O -(6- d -Glucopyranosyl)vanillin
O
OH
HH
OH
HOH
HHOH2C
H
O
OCH3
O
O
CH3OO
OH
HH
OH
HOH
HCH2OH
H
O
C1 a -galactoside 18 C1 a -galactoside (23), C1 b -galactoside (77)
6
4- O -( a - d -Galactopyranosyl)vanillin
4- O -( b - d -Galactopyranosyl)vanillin
O
OCH3
O
H
HOH2COH
OH
H
H
HOH
H
O
O
CH3OO
H
OHH
H
H
OHOH
CH2OH
H
O
C1 a -mannoside 13 C1 a -mannoside (44), C1 b -mannoside (56)
13
4- O -( a - d -Mannopyranosyl)vanillin
4- O -( b - d -Mannopyranosyl)vanillin
O
O OH
H H
H
HH OH
OHHOH2C
HOH2C
OHH
H
HH
OH
OH
OO
CH3
O
OCH3
O
H
OO
OH
H H
H
HOH
HOH2C
HOH2COH
OHHH
H
HH
OHO
O
C1 a -maltoside (42), C6- O- arylated (30), C6 ¢ - O- arylated (28)
29 C1 b -maltoside 8
4- O -( a - d -Glucopyranosyl-(1 ¢ →4) a - d -glucopyranosyl)vanillin
4- O -( a - d -Glucopyranosyl-(1 ¢ →4) b - d -glucopyranosyl)vanillin
O
CH3O
OH
H
O
OOH
HH
H
HHOH
OHCH2OH
OHH
H
HH
OH
OO
O
CH3OO
OHO
OH
HH
H
HOH
OHOH H
H
H
HH
OH O
O
H
OH
4- O -( a - d -Glucopyranosyl-(1 ¢ →4)6- d -glucopyranosyl)vanillin
4- O -( a - d -Glucopyranosyl-(1 ¢ →4)6 ¢ - d -glucopyranosyl)vanillin
O
CH3O
O
OH
HO
OOH
OH
OHOH
H
H
HH
OH
OO
O
CH3O
OHO
OH
HH
H
HOH
OH
O
OH OHO
OH
O
C1- O- arylated (39), C6 ¢ - O- arylated (61)
23 – -
4- O -(1- d -Fructofuranosyl-(2→1 ¢ ) a - d -glucopyranosyl)vanillin
4- O -(6 ¢ - d -Fructofuranosyl-(2→1 ¢ ) a - d -glucopyranosyl)vanillin
O
CH3 OO
OH
HH
HO
HOH
H
HOH2C
H
HOH2C
OOHO
H
H
HH
OHH
O
4- O -( b - d -Galactopyranosyl-
(1 ¢ →4) b - d -glucopyranosyl)vanillin
– – C1 b -lactoside 25
(continued)
1618.1 Phenols
Glycosides
Amyloglucosidase catalysis a b -Glucosidase catalysis b
Product (% proportion) c
Yields (%) d
Product (% proportion) c
Yields (%) d
O
CH3O
CH2
CH2OH
H OHOH HH OHH OH
O
O
CH3O
OHHOHHHOHOHH
CH2
CH2OH
O
O
CH3O
O
CH2
CH2
H OHOH HH OHH OH
O
OCH3
O
C1- O- arylated (13), C6- O- arylated (25), C1,6 di- O- arylated (62)
13 – –
4- O -(1- d -Sorbitol)vanillin 4- O -(6- d -Sorbitol)vanillin 1,6- O -(Bis-4- O -vanillin) d -sorbitol
a Vanillin and carbohydrate, 1 mmol each; amyloglucosidase concentration, 40% w/w of carbohydrate; solvent, di-iso-propyl ether; buffer, 0.1-mM (1-mL) pH 4 acetate buffer; incubation period, 72 h b Vanillin, 1 mmol; carbohydrate, 0.5 mmol; b -glucosidase concentration, 50% w/w of carbohydrate; solvent, di-isopro-pyl ether; buffer, 0.17-mM (1.7-mL) pH 4.2 acetate buffer; incubation period, 24 h c Conversion yields were from HPLC with respect to free carbohydrate. Error in yield measurements is ±10% d The product proportions were determined from the area of respective 1 H/ 13 C signals
Table 8.12 (continued)
Table 8.13 Coded values of the variables and their corresponding actual values used in the design of experiments
Variables −2 −1 0 1 2
Amyloglucosidase/ b -glucosidase (% w/w maltose) 10 20 30 40 50
Vanillin (mmol) 0.5 1 1.5 2 2.5 Incubation period (h) 24 48 72 96 120 Buffer volume (mL) 0.4 0.8 1.2 1.6 2 pH (0.01 M) 4 5 6 7 8
showed that (Fig. 8.8a ) at a buffer concentration of 0.05 mM (0.5 mL), increase in amyloglucosidase concentration from 10 to 50% (w/w maltose) decreased the maltosylation yield. However, at higher buffer concentrations beyond 0.125 mM (1.25 mL), the maltosylation yield increased with increase in amyloglucosidase concentration. A critical enzyme to buffer concentration could be dictating the extent of maltosylation, as there is a crossover point clearly depicting the reversal of the maltosylation behaviour at 30% (w/w maltose) amyloglucosidase concentration and a buffer vol-ume of 0.125 mM (1.25 mL).
Effect of amyloglucosidase concentration and incubation period at 1.5-mmol vanillin, pH 6 and 0.125-mM (1.25 mL) buffer concentration is shown in Fig 8.8B . With increase in incubation
period from 24 to 120 h, maltosylation yield decreases. At all the other incubation periods, increase in amyloglucosidase concentration from 10 to 50% (w/w maltose) showed a very slight increase in the maltosylation yields.
Effect of vanillin and incubation period on the maltosylation yield at 30% (w/w maltose) amylo-glucosidase, pH 6 and 0.125-mM (1.25 mL) buffer concentration is shown in Fig 8.9a . With increase in incubation period, the maltosylation yield decreases at all the vanillin concentrations in the range 0.5–2.5 mmol. This decrease in yield is quite steep up to 84 h and thereafter gradual. However, at all the incubation periods in the range 24–120 h, increase in vanillin concentration causes a mar-ginal decrease in yields indicating that vanillin could be mildly inhibitory to amyloglucosidase.
162 8 Glycosylation of Some Selected Phenols and Vitamins
Effect of amyloglucosidase concentration and pH on the extent of maltosylation at 0.125-mM (1.25 mL) buffer, 1.5-mmol vanillin and 72-h incubation period is shown in Fig. 8.9b . An optimum pH of 6 observed at 10% (w/w malt-ose) amyloglucosidase concentration is main-tained throughout up to 50% (w/w maltose) amyloglucosidase concentration with an increas-ing extent of conversion from 10 to 50% (w/w
maltose) enzyme concentration. However, a crossover point exhibiting reversal in the malto-sylation yields is observed around pH 6 and 30% (w/w maltose) amyloglucosidase concen-tration. Above pH 6 and above 30% (w/w malt-ose) amyloglucosidase, the yield increases with enzyme concentration and pH. The extent of conversion is lesser below 30% (w/w maltose) enzyme and pH 6.
Table 8.14 Experimental design with experimental and predictive yields of maltosylation based on response surface methodology a
Expt. no.
Glucosidase (% w/w maltose)
Vanillin(mmol)
Incubation period (h)
Buffer volume (mL)
pH (0.01 M)
Amyloglucosidase b -Glucosidase
Experimental Predicted Experimental Predicted Yield (%) Yield (%) Yield (%) Yield (%)
1 20 1 48 0.8 7 13.6 13.2 24.3 23.8 2 20 1 48 1.6 5 16.7 27.6 15.6 13.5 3 20 1 96 0.8 5 21.8 27.2 20.1 8.8 4 20 1 96 1.6 7 7.92 1.5 8.4 8.6 5 20 2 48 0.8 5 19.1 21.7 37.7 30.1 6 20 2 48 1.6 7 17.4 25.7 21.8 22.9 7 20 2 96 0.8 7 17.3 14.6 30.6 29.2 8 20 2 96 1.6 5 8.2 0 13.1 8.9 9 40 1 48 0.8 5 8.9 8.6 21.9 17.5 10 40 1 48 1.6 7 59.6 43.4 7.6 14.5 11 40 1 96 0.8 7 15.8 22.5 15.2 13.8 12 40 1 96 1.6 5 9.2 0 24.4 24.1 13 40 2 48 0.8 7 19.2 17 19.3 15.2 14 40 2 48 1.6 5 8.8 23.1 37.7 32.5 15 40 2 96 0.8 5 13.7 10.1 29.5 24.9 16 40 2 96 1.6 7 23.9 15.2 32.4 28.7 17 10 1.5 72 1.2 6 27.5 16.7 16.7 19.5 18 50 1.5 72 1.2 6 14.6 18.6 20.9 23.2 19 30 0.5 72 1.2 6 16.8 19.7 22.8 16.1 20 30 2.5 72 1.2 6 16.4 15.6 25.7 31.8 21 30 1.5 24 1.2 6 68.1 40.8 23.6 22.8 22 30 1.5 120 1.2 6 8.8 18.1 17.4 19.8 23 30 1.5 72 0.4 6 14.8 17.7 10.6 22.2 24 30 1.5 72 2 6 9.7 17.6 19.5 20.5 25 30 1.5 72 1.2 4 10.1 0 15.7 16.3 26 30 1.5 72 1.2 8 10.7 7.3 28.1 14.7 27 30 1.5 72 1.2 6 13.4 17.7 17.6 21.3 28 30 1.5 72 1.2 6 8.9 17.7 15.8 21.3 29 30 1.5 72 1.2 6 8.5 17.7 18.1 21.3 30 30 1.5 72 1.2 6 13.3 17.7 14.2 21.3 31 30 1.5 72 1.2 6 8.8 17.7 17.9 21.3 32 30 1.5 72 1.2 6 8.5 17.7 16.2 21.3
a Conversion yields obtained from HPLC with respect to 0.5 mmol of maltose. Error in yield measurement will be ±10%. This applies to all the yields given in the subsequent tables also
1638.1 Phenols
Fig. 8.8 Three-dimensional surface plots showing the effect of variables in the amyloglucosidase-catalysed reaction: ( a ) amyloglucosidase and buffer concentrations on the extent of maltosylation yield (pH 6; vanillin,
1.5 mmol; incubation period, 72 h) and ( b ) amyloglucosi-dase and incubation period on the extent of maltosylation yield (pH 6; buffer concentration, 0.125 mM/1.25 mL; vanillin concentration, 1.5 mmol)
Fig. 8.9 Three-dimensional surface plots showing the effect of variables in the amyloglucosidase-catalysed reaction: ( a ) vanillin and incubation period on the extent of maltosylation yield (pH 6; buffer concentration, 0.125 mM/1.25 mL; amyloglucosidase, 30% w/w malt-
ose) and ( b ) amyloglucosidase concentrations and pH on the extent of maltosylation yield (vanillin concentration, 1.5 mmol; buffer concentration, 0.125 mM/1.25 mL; incubation period, 72 h)
Maximum yield predicted based on the response model is 43.9% for the amyloglucosi-dase-catalysed reaction under the following conditions: amyloglucosidase, 30% (w/w of maltose); vanillin, 1 mmol; buffer concentra-
tion, 0.125 mM (1.25 mL) pH 6; and incubation period, 24 h. The experiments conducted at the above optimum conditions resulted in 49.4% yield. Further validation of the response model carried out at certain selected random process
164 8 Glycosylation of Some Selected Phenols and Vitamins
conditions showed that (Table 8.15 ) there appears to be a good correspondence between predicted and experimental yields at yields less than 40% and the correspondence appears to deviate a little at higher predictive yields.
8.1.6.4.2 b -Glucosidase-Catalysed Synthesis of 4- O -( a - D -Glucopyranosyl-(1 ¢ → 4) b - D -Glucopyranosyl)Vanillin
Equation :
3 4 5
2 2 5 5 1 3
1 4 1 5 3 5
0.7408 30.6961 20.7320
2.6099 - -1.4606 0.0092
0.9891 0.2937 0.0720
Y X X X
X X X X X X
X X X X X X
= - - +
+ +
+ - +
where X 1 , enzyme concentration; X
2 , vanillin
concentration; X 3 , incubation period; X
4 , buffer
volume; X 5 , pH; and Y , yield. Table 8.14 shows
the predicted yields obtained by using the reduced equation. Average absolute deviation between the experimental and predicted yields using this model is 22.3%.
Correlation Coef fi cient : R 2 − 0.69 Figure 8.10a shows the effect of b -glucosidase
and vanillin concentrations on the maltoside yield at pH 6, 0.125-mM (1.25-mL) buffer concentra-tion and 72-h incubation period. At all the b -glu-cosidase (10–50% w/w maltose) concentrations, the maltosylation yield increases with increase in vanillin concentration. However, increase in b -glucosidase concentration exhibited very little
enhancement in yield. Maximum yield is depicted at 50% (w/w maltose) b -glucosidase concentra-tion and 2–2.5 mmol of vanillin.
Effect of b -glucosidase and buffer concentration at pH 6, 1.5-mmol vanillin and 72-h incubation period on the maltosylation yield is shown in Fig. 8.10b . In the saddle-shaped surface obtained, a crossover point indicating reversal in the maltosyla-tion behaviour is observed at 30% (w/w maltose) b -glucosidase concentration and 0.125-mM (1.25-mL) buffer concentration. Up to 30% (from 10% w/w maltose) b -glucosidase and 0.125-mM to 0.04-mM (1.25-mL to 0.4-mL) buffer concentration, the maltosylation yield decreases with increase in enzyme concentration. Above this crossover values in both the variables, the yield increases. Here also, a critical b -glucosidase to buffer concentration appears to in fl uence the extent of maltosylation.
The maximum yield predicted based on the response model is 33.6% for the b -glucosidase-catalysed reaction under the following conditions: b -glucosidase, 50% (w/w of maltose); vanillin, 2.5 mmol; buffer concentration, 0.125 mM (1.25 mL) pH 6; and 72-h incubation period. The experiment conducted at the above optimum con-ditions gave 27.1% yield. Validation experiments performed at certain random selected conditions also showed good correspondence between exper-imental and predicted yields (Table 8.16 ).
Hence, the present RSM study has clearly showed the usefulness of CCRD technique in clearly bringing out the most salient features of this maltosylation reaction.
Table 8.15 Validation data for the amyloglucosidase-catalysed reactions at selected random conditions a
Expt. no. Amyloglucosidase (% w/w maltose)
Vanillin (mmol)
Incubation period (h)
Buffer volume (mL) pH
Predicted yield (%)
Experimental yield (%)
1 30 1.5 72 1.2 6 17.6 10.7 2 15 2 72 1.2 6 15.9 22.1 3 30 1 24 1.2 6 41.1 49.6 4 30 0.5 72 2 6 19.6 29.7 5 30 1.5 72 0.8 6 17.7 8.6 6 45 1.5 48 1.2 6 27 30.2 7 30 2 72 1.2 6 16.6 13.9 8 30 1.5 48 1.2 7 24.8 22.1 9 30 1.5 36 1.6 6 43.9 38.7 10 20 1.5 24 1.2 6 40.3 42.3
a Conversion yields obtained from HPLC with respect to 0.5 mmol of maltose
1658.1 Phenols
8.1.7 Syntheses of DL -Dopa Glycosides
dl -Dopa ( dl -3,4-dihydroxy phenylalanine), an aromatic amino acid precursor of dopamine, is the most effective drug for Parkinson’s disease (Yar 1993 ) . Parkinson’s disease is characterised by a severe and progressive degeneration of nigrostriatal dopamine (DA) neurons (Angerlacenci 2007 ) associated with the de fi ciency of catecholamine and dopamine (Shetty et al. 2002 ) . It is generally accepted that after administration, l -dopa in Parkinson’s dis-ease is converted into dopamine by aromatic
l -amino acid decarboxylase (AADC) within the serotonergic (5-HT) fi bres in the striatum and substantia nigra pars reticulate (Yamada et al. 2007 ) . Glucose is the brain source of energy, and this and other hexoses are also transferred across the blood–brain barrier (BBB) by the glucose carrier GLUT1. Such a transport will be facili-tated if l -dopa is converted into the glycoside as the l -dopa converted product glucosyl dopamine is able to interact with the glucose transporter (GLUT1) and absorbed into the central nervous system (CNS) from the blood stream (Dalpiaz et al. 2007 ) . During chronic treatment with
Fig. 8.10 Three-dimensional surface plots showing the effect of variables in the b -glucosidase-catalysed reaction: ( a ) vanillin and b -glucosidase concentrations on the extent of maltosylation yield (pH 6; buffer concentration,
0.125 mM/1.25 mL; incubation period, 72 h) and ( b ) b -glucosidase concentration and buffer concentration on the extent of maltosylation yield (pH 6; incubation period, 72 h; vanillin concentration, 1.5 mmol)
Table 8.16 Validation data for the b -glucosidase-catalysed reactions at selected random conditions a
Expt. no. b -Glucosidase (% w/w maltose)
Vanillin (mmol)
Incubation period (h)
Buffer volume (mL) pH
Predicted yield (%)
Experimental yield (%)
1 50 2.5 72 1.2 6 33.6 27.1 2 10 0.5 72 1.2 6 14.3 22.5 3 30 1.5 72 1.2 6 21.3 18.7 4 40 1.5 72 1.5 6 22.3 20.6 5 25 1.5 80 1.2 6 20.2 17.5 6 45 1.5 100 1.2 6 25.7 21.3 7 35 1.5 72 1.2 6 21.8 15.7 8 30 1.5 48 0.6 6 22.7 27.6 9 30 1.5 108 1.2 7 20.9 15.5 10 30 1.5 72 0.5 6 22.1 24.7
a Conversion yields obtained from HPLC with respect to 0.5 mmol of maltose
166 8 Glycosylation of Some Selected Phenols and Vitamins
l -dopa, a variety of transport problems like involuntary movements occur which can be over-come by the employment of glucosyl derivatives (Pardridge 2002 ; Madrid et al. 1991 ) . Side effects of the drugs can be reduced, and drug stability can be increased by modi fi cation of the aglycon molecule. For example, l -dopa and new dopamin-ergics can be modi fi ed to improve bioactivity properties (Pras et al. 1995 ; Giri et al. 2001 ) .
l -Dopa was fi rst isolated from Vicia faba (Guggenheim 1913 ; Vered et al. 1994 ) as a b -anomer (Nagasawa et al. 1961 ; Andrews and Pridham 1965 ) . Vicia faba has been incorporated into dietary strategies to manage Parkinsonian motor oscillations (Kempster et al. 1993 ) . A crude extract from the petals of Mirabilis jalapa was mixed with cyclo -Dopa and UDP-glucose to give cyclo -Dopa-5- O -glucoside by the action of glucosyl transferases (Sasaki et al. 2004 ; Wyler et al. 2004 ) . However, no chemical or enzymatic methods have been reported yet for the synthesis
of dl -dopa glycosides. Enzymatic method could be the alternative one which does not require protection and deprotection process (Fernandez et al. 2003 ; Roode et al. 2003 ) and provide milder reaction conditions, easy workup, less pollution, higher yields and selectivity. It can also give rise to stable dopa derivatives with enhanced stability and pharmacological activity (Suzuki et al. 1996 ; Vijayakumar and Divakar 2007 ) . This section describes the synthesised dl -dopa glycosides using amyloglucosidase from Rhizopus mould and b -glucosidase from sweet almond in organic media (Scheme 8.5 , Sivakumar et al. 2009 ) .
HPLC analysis of the glycosides prepared exhibited the following retention times for the substrates and products: dl -dopa, 8.5 min; d -glucose, 6.2 min; dl -dopa- d -glucoside, 13 min; d -galactose, 7.1 min; dl -dopa- d -galactoside, 8.9 min; d -mannose, 6.7 min; dl -3-hydroxy-4- O -( b - d -mannopyranosyl) phenylalanine, 8.7 min; lactose, 9.3 min; dl -3-hydroxy-4- O -( b - d -galac-
Scheme 8.5 Syntheses of dl -dopa glycosides
1678.1 Phenols
topyranosyl-(1 ¢ → 4) b - d -glucopyranosyl)pheny-lalanine, 1.9 min; d -sorbitol, 6.7 min; dl -3-hydroxy-4- O -(6- d -sorbitol)phenylalanine, 7.9 min; d -mannitol, 6.8 min; and dl -dopa- d -mannitol, 7.8 min.
8.1.7.1 Synthesis of DL -Dopa- D -Glucoside Using Amyloglucosidase
Glucosylation reaction between dl -dopa and d -glucose catalysed by amyloglucosidase from Rhizopus mould showed optimum conditions (Table 8.17 ) of incubation period, 72; pH 6.0, 0.06–0.14-mM (0.6–1.4-mL) buffer and yield in the range 62–59%. Up to 40% (w/w d -glucose) enzyme, the conversion yield more or less remained the same (yields 59%, 65%, 62% and 59% for 10%, 20%, 30% and 40% w/w d -glucose/enzyme, respectively). About 62% yield was obtained at 0.5-mmol dl -dopa.
8.1.7.2 Syntheses of DL -Dopa Glycosides of Other Carbohydrates Using Amyloglucosidase and b -Glucosidase and Their Spectral Characterisation
dl -Dopa glycosides with other eleven carbohy-drates using amyloglucosidase and b -glucosidase (Scheme 8.5 ) gave bis products of glycosides and C6-O-arylated products.
The glycosides were characterised by UV, IR, mass, melting point, optical rotation and 2-D HSQCT, which provided good information on the nature and proportions of the products formed (Table 8.18 ).
UV spectra of dl -dopa glycosides showed s → s * band ranging from 191.5 to 198.5 nm (199.5 nm for dl -dopa), s → p * band ranging from 221.5 to 226 nm (221 nm for dl -dopa) and n → p * band from 295 nm to 297.5 nm (280 nm for dl -dopa). IR spectra showed 1,019–1,040-cm −1 band for the glycosidic C–O–C aryl–alkyl symmetrical stretching and 1,307-1,320-cm −1 band for the asymmetrical stretching frequen-cies. In 2-D Heteronuclear Single Quantum Coherence Transfer (HSQCT) spectra, the respective chemical shift values of carbon atoms C1 a and b , C6 a and b showed glycoside forma-tion and arylation.
8.1.8 L -Dopa Glycosides
Syntheses of l -dopa glycosides were carried out under the optimum conditions of 0.5 mmol, car-bohydrates - 1 mmol, amyloglucosidase/immobi-lised b -glucosidase - 10% w/w of carbohydrate, 0.1 mM (1 mL) of 0.01-M pH 6 phosphate buffer for an incubation period of 72 h in 100-mL di-isopropyl ether (Scheme 8.6 , Vadivelan and Divakar 2011a ) . Diverse carbohydrate molecules, aldohexoses ( d -glucose, d -galactose and d -mannose), ketohexose ( d -fructose), pentoses ( d -ribose and d -arabinose), disaccharides (lactose, maltose, sucrose) and carbohydrate alcohol ( d -mannitol), were employed in the presence of amyloglucosi-dase and immobilised b -glucosidase.
UV spectra of l -dopa glycosides (Tables 8.19 and 8.20 ) showed shift in the s → s * band in the 198.5–205-nm (28.5 nm for l -dopa) range, shift in the p → p * band in the 267.5–295-nm (281 nm for l -dopa) range and shift in the n → p * band around 388 nm (390 nm for l -dopa). IR spectra showed shifts in the 1,015–1,047-cm −1 band for the glycosidic C–O–C aryl–alkyl symmetrical stretching and 1,206–1,312-cm −1 band for the asymmetrical stretching frequencies. Mass spec-tral data also con fi rmed product formation.
In 2-D Heteronuclear Single Quantum Coherence Transfer (HSQCT) spectra, the respec-tive chemical shift values of carbon atoms C1 a and b , C6 a and b showed glycoside formation and arylation.
However, there are distinct differences in this enzymatic reaction by both amyloglucosidase and b -glucosidase between the two substrates l -dopa and dl -dopa as described below. The nature and proportion of the l -dopa glycosides are shown in Tables 8.19 and 8.20 . Amyloglucosidase catalysed l -dopa reaction with all the eleven carbohydrate molecules employed. However, b -glucosidase catalysed the reaction with only d -glucose, d -galactose, d -ribose and lactose. Amyloglucosidase showed selectivity in the case of d -mannose, d -fructose, d -ribose, maltose, sucrose, d -mannitol and d -sor-bitol. About 20 individual glycosides were syn-thesised enzymatically using the glucosidases, of which 16 are being reported for the fi rst time. The
168 8 Glycosylation of Some Selected Phenols and Vitamins
new glycosides reported are l -dopa- d -glucoside, l -dopa- d -galactoside, l -dopa-mannoside, l -dopa-fructoside, l -dopa-riboside, l -dopa-arabinoside, l -dopa-lactoside, l -dopa-maltoside, l -dopa-sucroside and d -mannitolide.
Only monoglycosylated/arylated products were formed. No bis products with both the OH
groups at 3rd and 4th positions glycosylated/arylated could be detected. This indicated that steric effects are responsible for the formation of only monoglycosylated products. Under the reaction conditions employed, l -dopa gave a mixture of 3- O - and 4- O -glycosylated products with many of the carbohydrates employed. C6-O-arylated
Table 8.17 Optimisation of reaction conditions for the synthesis of dl -dopa- d -glucoside using amyloglucosidase
Reaction conditions a Variable parameter b Conversion yields (%) c
Incubation period (h) dl -Dopa – 0.5 mmol 3 42 d -Glucose – 1 mmol 6 48 pH – 6 12 58 Buffer concentration – 0.1 mM (1 mL) 24 60 Amyloglucosidase – 10% w/w d -glucose 48 63
72 62 96 57
120 49 pH (0.01 M)
dl -Dopa – 0.5 mmol a 4 16 d -Glucose – 1 mmol 5 38 Amyloglucosidase – 30% w/w d -glucose 6 62 Buffer concentration – 0.1 mM (1 mL) 7 48 Incubation period – 72 h 8 22
Buffer concentration (mM) dl -Dopa – 0.5 mmol 0.03 51 d -Glucose – 1 mmol 0.06 62 Amyloglucosidase – 30% w/w d -glucose 0.1 63 pH – 6 0.14 59 Incubation period – 72 h 0.18 52
0.22 47 Amyloglucosidase (% w/w d -glucose)
dl -Dopa – 0.5 mmol 10 59 d -Glucose – 1 mmol 20 65 pH – 6 30 62 Buffer concentration – 0.1 mM (1 mL) 40 59 Incubation period – 72 h 50 47
75 45 dl -Dopa (mmol)
pH – 6 0.2 22 Buffer concentration – 0.1 mM (1 mL) 0.4 41 d -Glucose – 1 mmol 0.5 62 Amyloglucosidase – 10% w/w d -glucose 0.8 53 Incubation period – 72 h 1.2 56
1.6 55 2 54
a Initial reaction conditions b Other variables are the same as under reaction conditions, except the speci fi ed ones c HPLC yields expressed with respect to 1-mmol d -glucose employed
1698.1 Phenols
(con
tinue
d)
Tab
le 8
.18
Sy
nthe
ses
of d
l -3,
4-di
hydr
oxyp
heny
lala
nine
( dl
-dop
a) g
lyco
side
s us
ing
amyl
oglu
cosi
dase
and
b -g
luco
sida
se a
Gly
cosi
des
Am
ylog
luco
sida
se c
atal
ysis
b -
Glu
cosi
dase
cat
alys
is
Prod
uct (
% p
ropo
rtio
n) b
Yie
lds
(%) c
Prod
uct (
% p
ropo
rtio
n) b
Yie
lds
(%) c
O OH
H HH
H
H
OH
OH
OH
OOH
NH
2
CO
OH
OOH
NH
2O
H
OH
OH
H
HH
H
OH
O
H
CO
OH
OH
OH
H
HHH
OH
O
OH
NH
2
OH
OH
CO
OH
4- O
-C1 a
(24
), 4
- O -C
1 b
(48)
, 4- O
-C6-
O -a
ryla
ted
(6),
3- O
-C1 b
(22
)
62
4- O
-C1 b
(28
),
4- O
-C6-
O -a
ryla
ted
(72)
33
dl -3
-Hyd
roxy
-4- O
-( a
- d -g
luco
pyra
nosy
l)ph
enyl
alan
ine
dl -3
-Hyd
roxy
-4- O
-( b -
d -gl
ucop
yran
osyl
)phe
nyla
lani
ne
dl -3
-Hyd
roxy
-4- O
-(6-
d -gl
ucop
yran
osyl
)phe
nyla
lani
ne
O
H
O
NH
2
CO
OH
H
O OH
HH
H
HO
HO
H
OH
dl -4
-Hyd
roxy
-3- O
-( b -
d -gl
ucop
yran
osyl
)phe
nyla
lani
ne
O OH
HH
OH
H
H
OH
H
OH
OOH
NH
2CO
OH
OOH
NH
2O
H
HO
HH
OH
HH
OH
O
H
CO
OH
HO
HH
OH
HH
OH
O
OH
NH
2
OH
OH
CO
OH
4- O
-C1 a
(27
), 4
- O -C
1 b
(25)
, 4- O
-C6-
O -a
ryla
ted
(29)
, 3- O
-C1 a
(10
),
3- O
-C1 b
(9)
46
4- O
-C1 a
(17
),
4- O
-C1 b
(35
),
4- O
-C6-
O -a
ryla
ted
(23)
, 3- O
-C1 a
(21
),
3- O
-C1 b
(4)
31
dl -3
-Hyd
roxy
-4- O
-( a
- d -g
alac
topy
rano
syl)
phen
ylal
anin
e
dl -3
-Hyd
roxy
-4- O
-( b -
d -ga
lact
opyr
anos
yl)p
heny
lala
nine
dl
-3-H
ydro
xy-4
- O -(
6- d -
gala
ctop
yran
osyl
)phe
nyla
lani
ne
OH
HO
HH
OH
HH
OH
O
H
CO
OH
NH
2
O OH
O OH
HH
OH
H
H
OH
H
OH
CO
OH
NH
2
O OH
dl -4
-Hyd
roxy
-3- O
-( a
- d -g
alac
topy
rano
syl)
phen
ylal
anin
e
dl -4
-Hyd
roxy
-3- O
-( b -
d -ga
lact
opyr
anos
yl)p
heny
lala
nine
OOH
NH
2O
H
OH
OH
H
HH
OH
HO
H
CO
OH
4- O
-C1 b
61
4-
O -C
1 b
32
dl -3
-Hyd
roxy
-4- O
-( b -
d -m
anno
pyra
nosy
l)ph
enyl
alan
ine
170 8 Glycosylation of Some Selected Phenols and Vitamins
Tab
le 8
.18
(c
ontin
ued)
Gly
cosi
des
Am
ylog
luco
sida
se c
atal
ysis
b -
Glu
cosi
dase
cat
alys
is
Prod
uct (
% p
ropo
rtio
n) b
Yie
lds
(%) c
Prod
uct (
% p
ropo
rtio
n) b
Yie
lds
(%) c
CO
OH
NH
2
OH
O
H
OH
H
H
HH
OH
OO
H
H
HO
OHH
H
OH H
OO
H
OH
– –
4- O
-C1 b
17
dl -3
-Hyd
roxy
-4- O
-( b -
d -ga
lact
opyr
anos
yl-(
1 ¢ →
4) b -
d - g
luco
pyra
nosy
l)ph
enyl
alan
ine
OH
NH
2
CO
OH
O
OH
HO
HH
HO
HO
HH
CH
2OH
CH
2
dl
-3-H
ydro
xy-4
- O -(
6- d -
sorb
itol)
phen
ylal
anin
e
4- O
-C6-
O -a
ryla
ted
12
– –
OH
NH
2
CO
OH
O
HO
HH
OH
OH
HO
HH
CH
2OH
CH
2
OH
NH
2
CO
OH
O
HO
HH
OH
OH
HO
HH
CH
2
CH
2
O
CO
OH
NH
2
OH
4- O
-C1-
O -a
ryla
ted
(70)
, B
is 4
- O -C
1,6-
di- O
-ary
late
d (3
0)
20
– –
dl -3
-Hyd
roxy
-4- O
-(1-
d -m
anni
tol)
phen
ylal
anin
e 1,
6- O
-(B
is d
l -3-
hydr
oxy-
4- O
-phe
nyla
lani
ne) d
-man
nito
l
a dl -
3,4-
Dih
ydro
xyph
enyl
alan
ine,
0.5
mm
ol; c
arbo
hydr
ate,
1 m
mol
; enz
yme
conc
entr
atio
n, 1
0% w
/w o
f ca
rboh
ydra
te; s
olve
nt, d
i-is
opro
pyl e
ther
; buf
fer,
0.1-
mM
(1-
mL
) pH
6
phos
phat
e bu
ffer
; inc
ubat
ion
peri
od, 7
2 h
b The
pro
duct
pro
port
ions
wer
e de
term
ined
fro
m th
e ar
ea o
f re
spec
tive
1 H/ 13
C s
igna
ls
c Con
vers
ion
yiel
ds w
ere
from
HPL
C w
ith r
espe
ct to
fre
e ca
rboh
ydra
te. E
rror
in y
ield
mea
sure
men
ts is
±10
%
1718.1 Phenols
compounds are observed with d -glucose and d -galactose. While d -fructose gave 2-O, sucro-side and d -mannitol gave 1-O aryl derivatives.
d -Glucose exhibited 33.3% a and 66.7% b compared to the 60:40 a : b -anomeric composi-tion of free d -glucose employed. d -Galactose showed 42.3% a and 57.7% b compared to the 92:8 a : b -anomeric composition of free d -galac-tose employed. Similarly d -arabinose showed 25.9% a and 74.1% b compared to the 95:5 a : b -anomeric composition of the d -arabinose employed.
8.1.9 Syntheses of Dopamine Glycosides
Parkinson’s disease is a neurodegenerative dis-ease characterised by bradykinesia, tremors, rigidity and dif fi culty in walking (Geng et al. 2004, 2007 ) . The usual treatment for this disor-der is the use of l -dopa, which enters the central nervous system (CNS) through active transport
and is enzymatically cleaved in the brain to release dopamine. To overcome the drawbacks of l -dopa treatment in transport problems across blood–brain barrier (BBB), new derivatives of dopamine able to penetrate the BBB, by making use of speci fi c transport systems (Pardridge 2002 ; Audus et al. 1992 ; Madrid et al. 1991 ) , are employed. Glucose is the brain’s source of energy and other hexoses are transported across the BBB by the glucose carrier GLUT1. Glycosyl dop-amine derivatives bearing the sugar moiety linked to either the amino group or the catechol ring of dopamine through amide, ester or glycosidic bonds were synthesised chemically with potent anti-Parkinsonian properties (Fernandez et al. 2003 ) . The glucosyl dopamine derivative is able to interact with the glucose transporter (GLUT1) and absorbed into the CNS from the blood stream (Dalpiaz et al. 2007 ) . Glycosylated form of dop-amine is stable in the periphery and, after reach-ing the brain through GLUT1 carrier, gets hydrolyzed by the action of brain enzymes to release dopamine (Fernandez et al. 2003 ) .
Scheme 8.6 Syntheses of l -dopa glycosides
172 8 Glycosylation of Some Selected Phenols and Vitamins
Table 8.19 Synthesis of l -dopa glycosides using amyloglucosidase and immobilised b -glucosidase
S. no. Glycosides
Amyloglucosidase catalysis
% of product (proportion) % of product (conversion)
1
OO
OH
H
H
HH
H
OH
OH
OH
O
OH
NH2
OH
12 54
l -3-Hydroxy-4-O-( a - d -glucopyranosyl) phenylalanine
O
H
OHH
H
H
HOH
OH
CH2OH
NH2
OH
O
OH
O
42
l -3-Hydroxy-4-O-( b - d -glucopyranosyl)phenylalanine
O
OHOH
H
HH
H
H
OH
OHO O OH
NH2OH
14
l -3-Hydroxy-4-O-(6-O- d -glucopyranosyl)phenylalanine
O
HOH
H
HH
H
O
OH
OH CH2OH
OH O
NH2
OH
22
l -4-Hydroxy-3-O-( b - d -glucopyranosyl)phenylalanine 2
O
O
OH
H
HOH
H
H
OHH
CH 2OH
NH2
O
OH
OH
l -3-Hydroxy-4-O-( a - d -galactopyranosyl) phenylalanine
22 44
O
NH2
O
OH
OH
H
CH2OHH
OH
H
OHH
HOH
O
l -3-Hydroxy-4-O-( b - d -galactopyranosyl)phenylalanine
30
O
OHOH
H
HOH
H
H
OH
HO O OH
NH2
28
l -3-Hydroxy-4-O-(6-O- d -galatopyranosyl)phenylalanine
O
OH
NH2
OH
OCH2OH
H
OH
H
HOH
H
H
OH
O
20
l -4-Hydroxy-3-O-( a - d -galactopyranosyl) phenylalanine
(continued)
1738.1 Phenols
S. no. Glycosides
Amyloglucosidase catalysis
% of product (proportion) % of product (conversion)
3
O
H
OH
H
H H
H
OHOH
CH2OH
O
OH
O
OH
NH2
15 46.3
l -3-Hydroxy-4-O-( a - d -mannopyranosyl)phenylalanine 4
OH
O
OH
NH2
OH
O
OH
OH
OH
O
16 8.7
l -3-Hydroxy-4-O-(2-O- d -fructofuranosyl) phenylalanine 5
O
O
OH
OH
OH
NH2
OH
O
OH
17 62.7
l -3-Hydroxy-4-O-( a - d -ribofuranosyl)phenylalanine 6
O
O
OH
OH
OH
NH2
OH
O
OH
25.9 63.8
l -3-Hydroxy-4-O-( a - d -arabinofuranosyl) phenylalanine
O
OH
OHOH
O
OH
O
OH
NH2
74.1
l -3-Hydroxy-4-O-( b - d -arabinofuranosyl) phenylalanine 7
NH2
OH
O
OH
O
OOH H
HH
H
HOH
CH2OHCH2OH
H OH
O
HOH
H
H
OH
H
O
41.6 29
l -3-Hydroxy-4-O-( a -lactosyl) phenylalanine
NH2
OH
O
OH
OO
H
OH
H
H
H
HOH
CH2OH
CH2OH
H
OH
O
H
OH
H
HOH
HO
58.4
l -3-Hydroxy-4-O-( b -lactosyl) phenylalanine 8
O
O
OH
H
H
H
H
H
OHOH
CH2OH
CH2OH
OH
O
HH
H
H
OH
H
O
OH
O OH
OH
l -3-Hydroxy-4-O-( b -maltosyl) phenylalanine
35.7
Table 8.19 (continued)
(continued)
174 8 Glycosylation of Some Selected Phenols and Vitamins
S. no. Glycosides
Amyloglucosidase catalysis
% of product (proportion) % of product (conversion)
9
O
O
OHH
H
H
H
H
OH
OH
CH2OH
O
OH
OH
OH
O
OHO
OH
NH2
8.5
l -3-Hydroxy-4-O- (1-O-sucrose) phenylalanine 10
OH
H
OH
H
H
OH
H
OH
H
OH
O
NH2
OH
O
OH
5
l -3-Hydroxy-4-O-(1-O- d -mannitolide) phenylalanine
Table 8.19 (continued)
Table 8.20 Synthesis of l -dopa glycosides using immobilised b -glucosidase
S. no. Glycosides % of product (proportion) % of conversion
1
OO
OH
H
H
HH
H
OH
OH
OH
O
OH
NH2
OH
24 57.6
l -3-Hydroxy-4-O-( a - d -glucopyranosyl) phenylalanine
O
H
OHH
H
H
HOH
OH
CH2OH
NH2
OH
O
OH
O
48
l -3-Hydroxy-4-O-( b - d -glucopyranosyl)phenylalanine
O
OHOH
H
HH
H
H
OH
OHO O OH
NH2OH
6
l -3-Hydroxy-4-O-(6-O- d -glucopyranosyl)phenylalanine
(continued)
1758.1 Phenols
S. no. Glycosides % of product (proportion) % of conversion
O
HOH
H
HH
H
O
OH
OH CH2OH
OH O
NH2
OH
22
l -4-Hydroxy-3-O-( b - d -glucopyranosyl)phenylalanine 2
O
O
OH
H
HOH
H
H
OHH
CH 2OH
NH2
O
OH
OH
43.9 55
l -3,-Hydroxy-4-O-( a - d -galactopyranosyl)phenylalanine
O
NH2
O
OH
OH
H
CH2OHH
OH
H
OHH
HOH
O
56.1
l -3-Hydroxy-4-O-( b - d -galactopyranosyl)phenylalanine 3
O
O
OH
OH
OH
NH2
OH
O
OH
46.2 44
l -3-Hydroxy-4-O-( a - d -ribofuranosyl)phenylalanine
OH
O
OH
NH2
OH
OH
OH
O
O
53.8
l -3-Hydroxy-4-O-( b - d -ribofuranosyl)phenylalanine 4
NH2
OH
O
OH
O
OOH H
HH
H
HOH
CH2OHCH2OH
H OH
O
HOH
H
H
OH
H
O
8.5 25
l -3-Hydroxy-4-O-( a - d -lactosyl) phenylalanine
NH2
OH
O
OH
OO
H
OH
H
H
H
HOH
CH2OH
CH2OH
H
OH
O
H
OH
H
HOH
HO
87.5
l -3-Hydroxy-4-O-( b - d -lactosyl) phenylalanine
Table 8.20 (continued)
Employing chemical methods for the synthesis of dopamine glycosides require protection and deprotection steps (Fernandez et al. 2003 ) . Enzymatic method is the alternative one which provides milder reaction conditions, easy workup,
less pollution, higher yields and selectivity (Suzuki et al. 1996 ; Vic and Thomas 1992 ; Vijayakumar et al. 2007 ) .
It is in this context that dopamine glycosides were prepared using amyloglucosidase from
176 8 Glycosylation of Some Selected Phenols and Vitamins
Rhizopus mould and immobilised b -glucosidase in organic media (Scheme 8.7 , Sivakumar and Divakar 2009a ) .
8.1.9.1 Amyloglucosidase-Catalysed Glucosylation of Dopamine
Enzymatic glucosylation between dopamine and d -glucose was optimised in terms of incubation period, pH, buffer, enzyme and dopamine con-centrations using both amyloglucosidase and immobilised b -glucosidase. Optimum conditions determined were as follows: 58% at 24-h incuba-tion (Table 8.21 ) at pH 6.0 and 40% (w/w d -glu-cose) amyloglucosidase showed the highest yield of 45% at 0.5 mmol of dopamine.
8.1.9.2 Immobilised b -Glucosidase-Catalysed Glucosylation of Dopamine
Optimum conditions for the glucosylation of dopamine using immobilised b -glucosidase were worked out. Optimum conditions are 58% yield at 72 h in 0.04-mM (0.4-mL) pH 5.0 buffer and a yield of 65% with 50% (w/w d -glucose) immobi-lised b -glucosidase (Table 8.22 ) with 0.5-mmol dopamine.
8.1.9.3 Syntheses of Dopamine Glycosides of Various Carbohydrates Using Amyloglucosidase and Immobilised b -Glucosidase and Their Spectral Characterisation
Syntheses of the other dopamine glycosides were carried out under optimised conditions mentioned above, with dopamine and carbohy-drates: d -glucose, d -galactose and d -mannose (Scheme 8.7 ) using amyloglucosidase and immo-bilised b -glucosidase.
Ultraviolet-visible spectra of dopamine glyco-sides showed s → s * band ranging from 191.5 to 201 nm (199 nm for dopamine), s → p * band ranging from 217 to 225.5 nm (218.5 nm for dop-amine) and p → p * and n → p * band from 276 to 286 nm (280 nm for dopamine). IR spectra showed 1,028–1,079-cm −1 band for the glyco-sidic C–O–C aryl–alkyl symmetrical stretching and 1,196–1,204-cm −1 band for the asymmetrical stretching frequencies. In 2-D Heteronuclear Single Quantum Coherence Transfer (HSQCT) spectra, the respective chemical shift values of carbon atoms C1 a and b , C6 a and b showed gly-coside formation and arylation (Table 8.23 ).
Scheme 8.7 Syntheses of dopamine glycosides
1778.1 Phenols
Table 8.21 Optimisation of reaction conditions for the synthesis of dopamine- d -glucoside using amyloglucosidase
Reaction conditions Variable parameter b Conversion yields (%) c
Incubation period (h) Dopamine – 0.5 mmol 3 44 d -Glucose – 1 mmol 6 45 pH – 6 12 47 Buffer concentration – 0.06 mM (0.6 mL) 24 58 Amyloglucosidase – 40% w/w d -glucose 48 48
72 52 96 48
120 37 pH (0.01 M)
Dopamine – 0.5 mmol a 4 No yield d -Glucose – 1 mmol 5 16 Amyloglucosidase – 30% w/w d -glucose 6 35 Buffer concentration – 1 mL (0.1 mM) 7 29 Incubation period – 72 h 8 23
Buffer concentration (mM) Dopamine – 0.5 mmol 0.03 21 d -Glucose – 1 mmol 0.06 39 Amyloglucosidase – 30% w/w d -glucose 0.1 27 pH – 6 0.15 22 Incubation period – 72 h 0.2 9
0.25 9 Amyloglucosidase (% w/w d -glucose)
Dopamine – 0.5 mmol 10 13 d -Glucose – 1 mmol 20 26 pH – 6 30 34 Buffer concentration – 0.06 mM (0.6 mL) 40 45 Incubation period – 72 h 50 19
75 21 Dopamine (mmol)
pH – 6 0.25 33 Buffer concentration – 0.06 mM (0.6 mL) 0.5 50 d -Glucose – 1 mmol 0.75 48 Amyloglucosidase – 40% w/w d -glucose 1 31 Incubation period – 72 h 1.5 25
2 16
a Initial reaction conditions b Other variables are the same as under reaction conditions, except the speci fi ed ones c HPLC yields expressed with respect to 1-mmol d -glucose employed
8.1.10 Serotonyl Glycosides
Serotonin is primarily found in the gastrointestinal tract and central nervous system of humans as well as animals. In addition to animals, it is also found in insects, fungi and plants (Kang et al. 2009 ) . Serotonin is an important neurotransmitter in the
central and peripheral nervous systems. It has been reported to modulate the regulation of a variety of major physiological functions includ-ing affective behaviour, memory and thermoreg-ulation via the interaction at serotonin receptor subtypes (Hoyer and Martin 1997 ) . Basically 80% of the human body’s total serotonin is
178 8 Glycosylation of Some Selected Phenols and Vitamins
located in the enterochromaf fi n cells in the gut, where it is used to regulate intestinal movements (Berger et al. 2009 ) and eventually fi nds its way out of tissues into the blood. Serotonin present in the blood then stimulates cellular growth to repair liver damage (Matondo et al. 2009 ) . In blood, serotonin is collected from plasma, by platelets which store it and activate it whenever platelets
bind in damaged tissue to stop bleeding and to aid in healing (Marieb 2009 ) . Serotonin, which is also chemically unstable, has an important role to play in the mechanistic action of antipsychotic agents, a topic of intense research, which prom-ises better treatments for schizophrenia in the forthcoming years (Jones and Blackburn 2002 ; Katrien et al. 1997 ) .
Table 8.22 Optimisation of reaction conditions for the synthesis of 3-hydroxy-4- O -( b - d -glucopyranosyl)phenyleth-ylamine using immobilised b -glucosidase
Reaction conditions Variable parameter b Conversion yields (%) c
Incubation period (h) Dopamine – 0.5 mmol 3 9 d -Glucose – 1 mmol 6 12 pH – 5 12 12 Buffer concentration – 0.04 mM (0.4 mL) 24 19
Immobilised b -glucosidase – 25% w/w d -glucose 48 26
72 58 96 35 120 14 pH (0.01 M)
Dopamine – 0.5 mmol a 4 62 d -Glucose – 1 mmol 5 64
Immobilised b -glucosidase – 50% w/w d -glucose 6 60
Buffer concentration – 0.1 mM (1 mL) 7 48 Incubation period – 72 h 8 42
Buffer concentration (mM) Dopamine – 0.5 mmol 0.04 65 d -Glucose – 1 mmol 0.08 57
Immobilised b -glucosidase – 50% w/w d -glucose 0.12 56
pH – 5 0.18 35 Incubation period – 72 h 0.25 27
Immobilised b -glucosidase concentration (% w/w d -glucose)
Dopamine – 0.5 mmol 10 56 d -Glucose – 1 mmol 25 58 pH – 5 40 59 Buffer concentration – 0.04 mM (0.4 mL) 50 65 Incubation period – 72 h 75 21
Dopamine (mmol) pH – 5 0.25 26 Buffer concentration – 0.04 mM (0.4 mL) 0.5 58 d -Glucose – 1 mmol 1 15
Immobilised b -glucosidase – 25% w/w d -glucose 1.5 16
Incubation period – 72 h 2 14
a Initial reaction conditions b Other variables are the same as under reaction conditions, except the speci fi ed ones c HPLC yields expressed with respect to 1-mmol d -glucose employed
1798.1 Phenols Ta
ble
8.2
3
Synt
hese
s of
dop
amin
e gl
ycos
ides
usi
ng a
myl
oglu
cosi
dase
and
imm
obili
sed
b -gl
ucos
idas
e
Gly
cosi
des
Am
ylog
luco
sida
se c
atal
ysis
a Im
mob
ilise
d b -
gluc
osid
ase
cata
lysi
s b
Prod
uct (
% p
ropo
rtio
ns) c
Yie
lds
(%) d
Prod
uct (
% p
ropo
rtio
ns) c
Yie
lds
(%) d
O OH
HH
H
H
H
HO
HO
OH
O
HO
NH
2
O
HO
NH
2O
H
HO H
OH
H
HH
OHO
H
N
H2
O
HO
OH
HO
HO
H
H
HH
H
OHO
4- O
-C1 a
(57
), 4
- O -C
1 b
(29)
, 3- O
-C1 b
(14
) 58
4-
O -C
1 b
65
3-H
ydro
xy-4
- O -(
a - d
-glu
copy
rano
syl)
phen
ylet
hyla
min
e
3-H
ydro
xy-4
- O -(
b - d -
gluc
opyr
anos
yl)p
heny
leth
ylam
ine
4-H
ydro
xy-3
- O -(
b - d -
gluc
opyr
anos
yl)p
heny
leth
ylam
ine
O OH
HH
HO
H
H
HO
H
OH
OHO
NH
2
OHO
NH
2O
H
HH
OH
HO
HH
OHO
H
HH
OH
HO
HH
OHO
OH
NH
2
HO O
H
4- O
-C1 a
(21
), 4
- O -C
1 b
(32)
, 4- O
-C6-
O -a
ryla
ted
(36)
, 3- O
-C1 a
(11
)
32
4- O
-C1 a
(31
), 4
- O -C
1 b
(26)
, 4- O
-C6-
O -a
ryla
ted
(33)
, 3- O
-C1 a
(10
)
29
3-H
ydro
xy-4
- O -(
a - d
-gal
acto
pyra
nosy
l)ph
enyl
ethy
lam
ine
3-H
ydro
xy-4
- O -(
b - d -
gala
ctop
yran
osyl
)phe
nyle
thyl
amin
e 3-
Hyd
roxy
-4- O
-(6-
d -ga
lact
opyr
anos
yl)p
heny
leth
ylam
ine
O OH
HH
HO
H
H
HO
H
OH
NH
2
O
HO
4-H
ydro
xy-3
- O -(
a - d
-gal
acto
pyra
nosy
l)ph
enyl
ethy
lam
ine
O H
HO
H
H
H
H
HO
HO
OH
OHO
NH
2
OHO
NH
2O
H
HO
HO
H
H
H
HO HO
H
O H
HO
H
H
H
H
HO
HO
OH
NH
2
O
HO
4- O
-C1 a
(29
), 4
- O -C
1 b
(57)
, 3- O
-C1 a
(14
) 40
4-
O -C
1 a (
29),
4- O
-C1 b
(5
5), 3
- O -C
1 a (
16)
28
3-H
ydro
xy-4
- O -(
a - d
-man
nopy
rano
syl)
phen
ylet
hyla
min
e
3-H
ydro
xy-4
- O -(
b - d -
man
nopy
rano
syl)
phen
ylet
hyla
min
e
4-H
ydro
xy-3
- O -(
a - d
-man
nopy
rano
syl)
phen
ylet
hyla
min
e
a 3,4
-Dih
ydro
xyph
enyl
ethy
lam
ine,
0.5
mm
ol;
carb
ohyd
rate
, 1 m
mol
; am
ylog
luco
sida
se c
once
ntra
tion,
40%
w/w
of
carb
ohyd
rate
; so
lven
t, di
-iso
prop
yl e
ther
; bu
ffer
, 0.0
6-m
M
(0.6
-mL
) pH
6 p
hosp
hate
buf
fer;
incu
batio
n pe
riod
, 24
h b 3
,4-D
ihyd
roxy
phen
ylet
hyla
min
e, 0
.5 m
mol
; ca
rboh
ydra
te,
1 m
mol
eac
h; i
mm
obil
ised
b -g
luco
sida
se c
once
ntra
tion
, 25
% w
/w o
f ca
rboh
ydra
te;
solv
ent,
di-i
sopr
opyl
eth
er;
buff
er, 0
.04-
mM
(0.
4-m
L)
pH 5
ace
tate
buf
fer;
incu
bati
on p
erio
d, 7
2 h
c The
pro
duct
pro
port
ions
wer
e de
term
ined
fro
m th
e ar
ea o
f re
spec
tive
1 H/ 13
C s
igna
ls
d Con
vers
ion
yiel
ds w
ere
from
HPL
C w
ith r
espe
ct to
fre
e ca
rboh
ydra
te. E
rror
in y
ield
mea
sure
men
ts is
±10
%
180 8 Glycosylation of Some Selected Phenols and Vitamins
Scheme 8.8 Glycosylation of serotonin and epinephrine
Serotonin can be made more water-soluble (solubility of serotonin in water 2 g/100 mL at 27°C) and stable by derivatising. Serotonin sup-plements especially derivatised ones could be better substituents for external administration in diet which resulted in the preparation of seroto-nyl glycosides (Scheme 8.8 , Vadivelan and Divakar 2011b ) .
The reaction did not take place without the use of the enzyme amyloglucosidase from Rhizopus mould under the reaction conditions employed. Serotonin (Scheme 8.8 ) contains phenolic –OH group at position 5. The carbohydrates employed were d -glucose, d -galactose, d -mannose, d -ribose, d -arabinose, d -fructose, d -maltose, lactose, sucrose, d -sorbitol and d -mannitol. The reaction did not occur at the primary and second-ary amino groups, respectively. In case of
serotonin, reaction took place with d -glucose, d -galactose, d -mannose and d -ribose only. The other carbohydrate molecules did not react. This could be due to competitive binding of serotonin and carbohydrates to the enzyme restricting reac-tions with certain carbohydrates as facile transfer of tightly bound serotonin to the lesser nucleo-philic carbohydrates rendered impossible. Glycosylation yields were generally in the range 13–29% (Table 8.24 ).
UV spectra of serotonin glycosides showed UV
max between 205 and 215 nm ( s - s * transition),
274 and 296 nm (n- p * transition) and around 367 nm for extended n- p * transition (270 nm for free serotonin). IR C–O–C glycosidic aryl–alkyl symmetrical stretching frequencies in the 1,016–1,088-cm −1 range and asymmetrical stretching frequencies in the 1,200–1,208-cm −1 range indicated
1818.1 Phenols
Table 8.24 Amyloglucosidase-catalysed syntheses of serotonin and epinephrine glycosides
Glycoside Product (% proportion) a
Yield (%) b
NH
H2N
O
OOH
HH
H
H
HHO
HO
OH
NH
O
HOH
HH
H
H
HO
HO
OH
O
NH2
C1 a (26), C1 b (39), 6- O - a (17) and 6- O - b (19) monoglycosides
15
7a : Serotoninyl-5- O (− a - d -glucopyranoside)
7b : Serotoninyl-5- O (− b - d -glucopyranoside)
O
HOOH
HH
H
H
HHO
O
NH
H2N
HO
O
OH
HH
H
H
OHHOHO
O
NH
H2N
H
7c : Serotoninyl-5- O -(6- a - d -glucopyranoside)
7d Serotoninyl-5- O -(6- b - d -glucopyranoside)
NH
O
NH2
O
HOH
HH
HO
H
HO
H
OH
O
NH
H2N
O
OHOH
HH
HO
H
HHO
H
O
NH
H2N
O
HOH
HH
HO
H
OHHOH
C1 a (49) and 6- O - a (25), 6- O - b (26) monogalactoside
13
8a : Serotoninyl-5- O (− b - d -galactopyranoside)
8b : Serotoninyl-5- O -(6- a - d -galactopyranoside)
8c : Serotoninyl-5- O -(6- b - d -galactopyranoside)
NH
O
NH2
O
HH
OHH
H
H
HO
HO
OH
O
NH
H2N
O
OHH
OHH
H
H
HHO
OH
O
NH
H2N
O
HH
OHH
H
H
OHHO
HO
C1 b (48) and 6- O - a (22) and 6- O - b (20) mono-mannoside
18
9a Serotoninyl-5- O (− b - d -mannopyranoside)
9b : Serotoninyl-5- O -(6- a - d -mannopyranoside)
9c : Serotoninyl-5- O- (6- b - d -mannopyranoside)
NH
H2N
O
O
H
OH
H
OH
H
HOH
NH
H2N
O
HH
OH
H
OH
H
HO
O
C1 a (72) and C1 b (28) monoriboside
29
10a : Serotoninyl-5- O (− a - d -ribofuranoside)
10b : Serotoninyl-5- O (− b - d -ribofuranoside)
(continued)
that serotonin has undergone glycosylation. Mass spectra also con fi rmed glycosylation through detection of the parent M + ion for the glycosides. In 2-D Heteronuclear Single Quantum Coherence
Transfer (HSQCT) spectra, the respective chemi-cal shift values of carbon atoms C1 a and b , C6 a and b showed glycoside formation and arylation.
182 8 Glycosylation of Some Selected Phenols and Vitamins
Except for the serotoninyl glucosides, all the other glycosides and 6-O-derivatives are being reported for the fi rst time. Since the phenolic group at position 5 in serotonin is not sterically hindered, all the four carbohydrates d -glucose, d -galactose, d -mannose and d -ribose underwent facile reaction with serotonin.
8.1.11 Epinephryl Glycosides
Epinephrine is a hormone and also a neurotrans-mitter, brie fl y called as adrenaline. As a hor-mone, it acts on nearly all body tissues. Epinephrine’s various functions result from its binding to a variety of adrenergic receptors trig-gering a number of metabolic changes. Binding to a -adrenergic receptors inhibits insulin secre-tion, stimulates glycogenolysis in the liver and muscle and stimulates glycolysis in muscle
(Sircar 2007 ) . b -Adrenergic receptor binding triggers glucagon secretion in the pancreas, increased adrenocorticotropic hormone secre-tion by the pituitary gland and increased lipoly-sis by adipose tissue. All these effects lead to increased blood glucose and fatty acids, provid-ing substrates for energy production within cells throughout the system (Sircar 2007 ) . Due to its vasoconstrictive effects, epinephrine is the drug of choice for treating anaphylaxis and also use-ful in treating sepsis. It is also used as a broncho-dilator for asthma as its speci fi c b -agonists are unavailable or ineffective.
Epinephrine can be made more water-soluble (solubility of epinephrine 0.01 g/100 mL at 18°C) and stable by derivatising. Epinephrine supple-ments like glycosides could be better supple-ments, and hence epinephryl glycosides were prepared enzymatically (Scheme 8.8 , Vadivelan and Divakar 2011b ) .
Glycoside Product (% proportion) a
Yield (%) b
O
HOH
HH
H
H
HO
HO
OH
OH
N
H3C H
O
HO
OH
N
H3C H
O
HOH
HH
H
H
HO
HO
OH
O
HO
3- O -C1 b (63) and 4- O -C1 b (37) monoglucoside
18
11a: Epinephrinyl-3- O -(− b - d -glucopyranoside)
11b : Epinephrinyl-4- O -(− b - d -glucopyranoside)
O
HH
OHH
H
H
HO
HO
OH
OH
NH3C H
O
HO
OH
N
CH3
H
HOO
OOH
HH
H
HHO
HO
OH
H
OH
N
H3C H
O
HOH
HH
H
H
HO
HO
OH
O
HO
C1 a (72) and C1 b (28) monoriboside
29
12a: Epinephrinyl-3- O (− b - d -mannopyranoside)
12b: Epinephrinyl-4- O (− a - d -mannopyranoside)
12c: Epinephrinyl-4- O (− b - d -mannopyranoside)
a Product proportions were determined by 13 C 2-D HSQCT NMR C1/C6 peak areas or their cross peaks b Conversion yields were from HPLC with errors in yield measurements of ±5–10%
Table 8.24 (continued)
1838.2 Vitamins
In case of epinephrine, out of the 11 carbohydrates employed, only d -glucose and d -mannose reacted effectively. The other carbohydrate molecules did not react. Structural comparison between serotonin and epinephrine clearly shows that epinephrine is more polar compared to serotonin and hence could bind much more strongly to the enzyme compared to sero-tonin, inhibiting the enzyme towards reaction with other carbohydrate molecules. Compared to the other carbohydrate molecules besides d -glucose and d -mannose, epinephrine could bind more strongly than those other carbohydrate molecules thereby inhibiting reaction.
While the reaction with glucose exhibited a conversion yield of 18%, d -mannose gave only 29% yield. Both 3-O- and 4-O-products were formed. Only phenolic –OH groups of epineph-rine at position 3 and 4 reacted in a facile manner. Epinephrine contains two phenolic OH molecules and alcoholic OH group, all three capable of undergoing glycosylation. No product formation arising out of reaction at the secondary hydroxyl group at position 7 was detected.
UV spectra showed l max
at 208 nm and 231 nm ( s - s * transition) for the reaction products of d -glucose and d -mannose, respectively, and another l
max at 266 nm for d -glucoside and
285 nm (n- p * transition) for d -mannoside (279 nm for epinephrine itself). Similarly IR, glycosidic aryl–alkyl C–O–C stretching frequen-cies at 1,048 cm −1 for d -glucoside and 1,021 cm −1 for d -mannoside and asymmetrical stretching frequencies at 1,212 cm −1 and 1,223 cm −1 for the respective glucoside and mannosides indicated glycosylation. 2-D HSQCT spectra conformed the product formation further from the respective chemical shift values (Table 8.24 ). The C3 and C4 carbons of epinephrine showed shifts at around 144–145 ppm in their respective deriva-tives indicating reaction involving these phenolic hydroxyl groups.
Presence of hydrophobic/hydrophilic phenolic –OH group epinephrine bestowed excellent nucleophilicity to these molecules promoting reaction with few diverse carbohydrate mole-cules. Only monoglycosylated/arylated products were detected. However, loss of regiospeci fi city
in case of epinephrine could be due to employ-ment of large concentration of the enzyme. Reaction with only few carbohydrate molecules indicated that both serotonin and epinephrine possess the propensity to inhibit the enzyme strongly. The glycosides prepared are more water-soluble and stable than their respective aglycons, hence portending excellent pharmaceutical prop-erties for the prepared glycosides.
8.2 Vitamins
Vitamins are organic compounds that are required in small amounts for the normal functioning of the body and maintenance of metabolic integrity. As with many other biologically active com-pounds, glycoside derivatives of vitamins have been identi fi ed and their properties investigated. The formation of glycosylated derivatives of vitamins, whether natural in plants, animals or microorganisms or by intentional chemical modi fi cations, represents a process that may cause dramatic changes in chemical, nutritional and metabolic properties of vitamins.
Hence, water-soluble vitamins were glycosy-lated to improve their water solubility, and fat-soluble vitamins were glycosylated also to improve their water solubility. Both classes of vitamins on glycosylation could possess better nutritional and pharmacological properties that are very much desired.
Enzymatic syntheses of more water-soluble thiamin (vitamin B1), ribo fl avin (vitamin B2) and pyridoxine (vitamin B6) and fat-soluble retinol (vitamin A), ergocalciferol (vitamin D2), cholecalciferol (vitamin D3) and a -tocopherol (vitamin E) with carbohydrates ( d -glucose, d -galactose, d -mannose, d -fructose, d -ribose, d -arabinose, maltose, sucrose, lactose, d -sorbitol and d -mannitol) were carried out using amylog-lucosidase from Rhizopus mould and b -glucosi-dase isolated from sweet almond. The usual 11 carbohydrate molecules were employed (Sivakumar and Divakar 2009b ; Ponrasu et al. 2008, 2009 ; Einstein Charles et al. 2009a, b ; Manohar and Divakar 2010 ) .
184 8 Glycosylation of Some Selected Phenols and Vitamins
8.2.1 Glucosylation of Thiamin
Thiamin (3-[(4 ¢ amino-2 ¢ -methyl-pyrimidine-5 ¢ -yl-methyl)-5-(2-hydroxy-ethyl)]-4-methylthiaz-olium chloride hydrochloride, vitamin B
1 ), a
water-soluble vitamin belonging to the B com-plex group, is an important cofactor of decarbox-ylase, transketolase and carboxylase (Kren et al. 1998 ) . The characteristic odour and a strong tongue-pricking taste of thiamin could be reduced by preparing derivatives of this vitamin (Suzuki and Uchida 1994 ) .
Enzymatic synthesis of O- a -glucosylthiamin has been reported by a consecutive action of cyclomaltodextrin glucanotransferase from Rhizopus stearothermophilus and glucoamylase from Rhizopus sp . (Uchida and Suzuki 1968 ) . Glucosylation of thiamin (vitamin B1) is dis-cussed here (Scheme 8.9 , Ponrasu et al. 2009 ) .
8.2.1.1 Response Surface Methodology Design : Central Composite Rotatable Design (CCRD), 32 experiments, 5 variables at 5 levels (Ponrasu et al. 2009 )
Variables : Five variables were employed. Actual set of experiments undertaken and the glucosyla-tion yields obtained are shown in Table 8.25 .
Equation : A second-order polynomial equation was developed to study the effects of the variables
on the esteri fi cation yields in terms of linear, quadratic and cross product terms.
279.21 1.93* Xe 33.99* Xc 2.37*
Xt 58.46* Xv 37.13* Xp 0.007*
Xe* Xe 57.88* Xc* Xc 0.007* Xt *
Xt 6.15* Xv* Xv 0.74* Xp*
Xp 0.05* Xe* Xc 0.0005* Xe*
Xt 0.02* Xe* Xv 0.20* Xe*
Xp 0.11* Xc* Xt 33.50* Xc*
Xv 15.42* Xc* Xp 0.12* Xt
Y = - + -- - +
+ ++ +- -- ++ -- + *
Xv 0.21* Xt * Xp 11.92* Xv* Xp+ +
where Xe is the enzyme concentration, Xc is the thiamin concentration, Xt is the incubation period, Xv is the buffer concentration, Xp is the pH and Y is the yield.
Coef fi cients : Kyplot Software Version 2.0 Beta
Analysis of Variance ( ANOVA ): Kyplot, Software. R 2 value of 0.74. ANOVA shows the model is signi fi cant at P < 0.01.
Optimisation : Microsoft Regression software Contour plots (Fig. 8.11a–e ) clearly brought
out the glucosylation behaviour of the enzyme under the reaction conditions employed.
Glucosylation reaction gave a mixture of a - and b -glucosides. b -Glucosidase is a retaining
Scheme 8.9 Synthesis of thiamin glucoside
1858.2 Vitamins
enzyme which gives rise to b -glucosides exclu-sively. Formation of a mixture of a - and b -glu-cosides indicated that the b -glucosidase prepared by the tannin precipitation method could contain contaminant a -glucosidase responsible for such products. This was con fi rmed by detecting an a -glucosidase activ-ity of 305.3 AU ( m mol/min·mg of enzyme prep-aration) for the native enzyme and 101.3 AU
( m mol/min·mg of enzyme preparation) for the immobilised enzyme.
NMR data clearly con fi rmed the formation of two glucosides: 2- O -( a - d -glucopyranosyl)thia-min and 2- O -( b - d -glucopyranosyl)thiamin (Scheme 8.9 ). A doublet at 4.86 ppm with an equatorial–equatorial coupling constant value of 2.6 Hz corresponded to the a -glucoside, and another at 4.37 ppm with an axial–equatorial
Table 8.25 Experimental design with experimental and predictive yields of glucosylation based on response surface methodology a
Expt. no.
Immobilised b -glucosidase (% w/w d -glucose) Thiamin(mmol)
Incubation period (h)
Buffer concentration (mM)
pH (0.01 M)
Experimental yield (%)
Predicted yield (%)
1 40 0.5 48 0.08 7.0 11.9 13.2 2 40 0.5 48 0.16 5.0 31.3 32.6 3 40 0.5 96 0.08 5.0 20.6 20.6 4 40 0.5 96 0.16 7.0 57.9 52.3 5 40 1.0 48 0.08 5.0 41.0 39.3 6 40 1.0 48 0.16 7.0 24.3 17.1 7 40 1.0 96 0.08 7.0 31.0 22.3 8 40 1.0 96 0.16 5.0 37.0 28.4 9 80 0.5 48 0.08 5.0 17.2 24.5 10 80 0.5 48 0.16 7.0 45.0 46.7 11 80 0.5 96 0.08 7.0 35.2 35.4 12 80 0.5 96 0.16 5.0 22.0 22.3 13 80 1.0 48 0.08 7.0 20.0 18.6 14 80 1.0 48 0.16 5.0 24.2 22.9 15 80 1.0 96 0.08 5.0 29.3 26.6 16 80 1.0 96 0.16 7.0 53.4 45.2 17 20 0.75 72 0.12 6.0 16.2 26.5 18 100 0.75 72 0.12 6.0 32.8 30.6 19 60 0.25 72 0.12 6.0 42.4 34.9 20 60 1.25 72 0.12 6.0 12.5 28.1 21 60 0.75 24 0.12 6.0 32.5 28.3 22 60 0.75 120 0.12 6.0 25.6 37.9 23 60 0.75 72 0.04 6.0 14.0 12.6 24 60 0.75 72 0.20 6.0 19.8 29.3 25 60 0.75 72 0.12 4.0 17.4 15.8 26 60 0.75 72 0.12 8.0 14.5 24.2 27 60 0.75 72 0.12 6.0 18.4 17.0 28 60 0.75 72 0.12 6.0 17.9 17.0 29 60 0.75 72 0.12 6.0 18.0 17.0 30 60 0.75 72 0.12 6.0 19.7 17.0 31 60 0.75 72 0.12 6.0 17.7 17.0 32 60 0.75 72 0.12 6.0 18.8 17.0
a Conversion yields obtained from HPLC with respect to 1.0 mmol of glucose. Error in yield measurement will be ±5%. This applies to all the yields given in the subsequent tables also. Data are an average from two measurements
186 8 Glycosylation of Some Selected Phenols and Vitamins
coupling constant value of 6.4 Hz corresponded to the b -anomer. Carbon-13 chemical shift values also con fi rmed the chemical shift value of the a -anomer at 98 ppm and b -anomer at 103 ppm.
Effect of immobilised b -glucosidase and thia-min concentration on the extent of glucosylation (Fig. 8.11a ), enzyme concentration and pH on the
extent of glucosylation (Fig. 8.11b ) and buffer concentration and pH on the extent of glucosyla-tion (Fig. 8.11d ) showed reversal in glucosylation at 60% (w/w d -glucose) enzyme concentration, 0.12-mM buffer concentration and pH 6. Below this crossover point, the yields gradually decreased which later increased beyond this
20 40 60 80 100 20 40 60 80 100
20 40 60 80 100
0.4
0.6
0.8
1
1.2
Thi
amin
(m
mol
)
20
25
3035
4045
20
25
30
35
4
5
6
7
8
p H
45
15
25
30
20
3540
15
20
25
30
35
0.2 0.5 0.75 1 1.254
5
6
7
8
pH
15
25
35
45
55
20
30
40
50
2025
3035
40
0.04 0.08 0.12 0.16 0.24
5
6
7
8
pH10
20
30
2010
15
15
25
5
0.04
0.08
0.12
0.16
0.2
Buf
fer
conc
entr
atio
n (m
M)
15
20
30
40
25
35
20
25
30
35
40
Enzyme (%w/w D-glucose) Enzyme (%w/w D-glucose)
Buffer concntration (mM)Thiamin (mmol)
Enzyme (% w/w D-glucose)
a b
c d
e
Fig. 8.11 Contour plots showing the effect of ( a ) con-centration of thiamin and enzyme, ( b ) pH and enzyme concentration, ( c ) pH and thiamin concentration, ( d ) pH
and buffer concentration and ( e ) buffer and enzyme con-centration on the glycoside yields
1878.2 Vitamins
point. Such a crossover point indicated that under the experimental conditions corresponding to this point, the enzyme behaviour enhanced the yields thermodynamically and kinetically. An enzyme concentration of 60% (w/w d -glucose) and 0.75 mmol of thiamin corresponded to a com-plete binding of thiamin to the enzyme. Similarly a buffer concentration of 0.12 mM at pH 6 also could favourably dispose the enzyme for enhanced conversion through a favourable conformational change.
Figure 8.11c illustrating the effect of thiamin concentration and pH and Fig. 8.11d that of buf-fer concentration and pH bring out the effect of thiamin ionisation on the extent of glucosylation. Thiamin possesses two ionisable moieties – a thi-azolium group with a pK of 16.9–18.9 and an amino group with a pK of 7.0–8.5. At a buffer concentration of 0.12 mM, lower concentrations of thiamin could result in complete neutralisation of amine hydrochloride as the pH and buffer con-centrations are increased, leading to higher con-versions (Fig. 8.11c, d ). Below pH 6, positively charged thiamin could bind strongly to the enzyme, thus acting as an ef fi cient inhibitor, whereas above pH 6, it could act as an ef fi cient acceptor molecule for d -glucose due to loss of most of its charge on the free amino group. However, the thiazolium moieties with a pK of 16.9–18.9 will be least affected at pH 6. Figure 8.11e illustrates the effect of enzyme and buffer concentration on the extent of glucosyla-
tion at 0.75-mmol thiamin concentration, 72-h incubation period and pH 6. Higher buffer con-centrations favoured enhanced glucosylated yields at all the enzyme concentrations.
A maximum conversion yield of 52% predicted by the response model for the glucosylation of thiamin at 40% (w/w d -glucose) immobilised b -glucosidase, 0.5-mmol thiamin, 96-h incuba-tion period, 0.16-mM buffer concentration and pH 7 was found to yield 58% under the above experimental conditions. Validation experiments performed at certain selected random conditions gave moderate to good correspondence between experimental and predicted yields (Table 8.26 ). Thus the present response surface methodology study has brought the enzymatic behaviour of b -glucosidase clearly in this glucosylation reaction.
8.2.2 Syntheses of Ribo fl avinyl Glycosides
Ribo fl avin [1-deoxy-1-(3,4-dihydro-7,8-dime-thyl-2,4-dioxobenzo{g}pteridine-10(2 H)-yl)- d -ribitol], vitamin B
2 , is a constituent member
of the vitamin B complex (LeBlane et al. 2005 ) . Ribo fl avin is a necessary growth factor impor-tant for erythrocyte production (Bates 1993 ) . As a prosthetic group of oxido-reductive fl avoenzymes, it functions as a fl avin nucleotide in the process of electron transport, leading to
Table 8.26 Validation data for the immobilised b -glucosidase-catalysed reactions at selected random conditions a
Expt. no.
Immobilised b -glucosidase (% w/w d -glucose)
Thiamin (mmol)
Incubation period (h)
Buffer concentration (mM) pH
Predicted yield (%)
Experimental yield (%)
1 50 0.75 72 0.12 6.0 17.0 27.0 2 20 0.75 72 0.12 4.0 41.0 31.0 3 20 0.25 72 0.12 6.0 43.0 49.4 4 30 1.0 72 0.10 5.0 35.0 33.0 5 40 0.40 72 0.10 5.0 23.6 21.5 6 30 0.25 60 0.11 7.0 37.6 34.0 7 40 0.75 60 0.08 5.0 26.0 21.0 8 60 0.50 60 0.14 5.0 21.3 22.6 9 100 0.75 72 0.12 6.0 31.0 32.3 10 60 0.50 72 0.12 6.0 23.0 24.0
a Conversion yields obtained from HPLC with respect to 1.0 mmol of d -glucose. Data are an average from two measurements
188 8 Glycosylation of Some Selected Phenols and Vitamins
oxidative degradation of pyruvate, fatty acids and amino acids (Werner et al. 2005 ) . Ribo fl avin is a potent antioxidant, protective against many diseases including cancer (Sengodan et al. 2003 ) . Also it degrades nicotine into a pharmacologi-cally inactive substance (Dickerson et al. 2004 ) . Recent researches focus on the role of plasma ribo fl avin in determining the homocysteine concentration, which is a risk factor in cardiovas-cular diseases (Powers 2003 ) . Because of its limited gastrointestinal absorption, large doses of ribo fl avin remain unabsorbed.
Ribo fl avin is synthesised in plants and few microorganisms (Bacher et al. 2000 ) . It is also obtained from animal source (Whitby 1971 ) . As early as 1952, Whitby reported 5 ¢ - d -ribo fl avinyl- d -glucopyranoside from ribo fl avin by using homogenates of rat liver. Enzymes from different microorganisms have also been employed (Suzuki and Uchida 1969 ) for the preparation of 5 ¢ - d -ribo fl avinyl- a - d -glucopyranoside. Uchida and Suzuki ( 1968 ) and Suzuki and Uchida ( 1983 )
have reported preparation of ribo fl avin glucoside in growing cultures of Ashbya gossypii and Eremothecium ashbyii and in germinating barley seeds. Tachibana ( 1955 ) has reported preparation of ribo fl avinyl- b - d -galactoside in cell suspension cultures of Aspergillus oryzae . In 1971 , Tachibana again showed ribo fl avin glucoside and other oli-gosaccharides from Escherichia coli, Clostridium acetobutyricum, Leuconostoc mesenteroides and cotyledons of pumpkin Cucurbita pepo and of sugar beet Beta vulgaris . However, on the follow-ing pages, an exclusive enzymatic synthesis of ribo fl avinyl glycosides is discussed (Scheme 8.10 , Sivakumar and Divakar 2009b ) .
HPLC retention times for the substrates and products (Fig. 8.12 ) are as follows: ribo fl avin, 5.5 min; d -glucose, 6.5 min; 5- O -( d -glucopyranosyl)ribo fl avin, 8.2 min; d -galactose, 7.1 min; 5- O -( d -galactopyranosyl)ribo fl avin, 12.4 min; d -man-nose, 6.7 min; 5- O -( d -mannopyranosyl)ribo fl avin, 13.4 min; d -ribose, 7.4 min; 5- O -( d -ribofuranosyl)ribo fl avin, 9.1 min; maltose, 11.5 min; 5- O -( a - d -
Scheme 8.10 Syntheses of ribo fl avinyl glycosides
1898.2 Vitamins
glucopyranosyl-(1 ¢ → 4) d -glucopyranosyl)ribo fl avin, 17.2 min; sucrose, 9.6 min; 5- O -(1- d -fructofuranosyl-(2 → 1 ¢ ) a - d -glucopyranosyl)ribo fl avin, 16.1 min; lactose, 9.3 min; and 5- O -( b - d -galactopyranosyl-(1 ¢ → 4) b - d -glucopyranosyl)ribo fl avin, 15.1 min.
8.2.2.1 Synthesis of 5- O -( D -Glucopyranosyl)Ribo fl avin Using Amyloglucosidase
Optimum conditions for the synthesis of 5- O -( d -glucopyranosyl)ribo fl avin (Table 8.27 ) are: maxi-mum yield - 31% between 12 and 72 h (Table 8.27 , Fig. 8.13 ), pH-7, Amyloglucosidase - 50% (w/w d -glucose), ribofl avin - 1 mmol and Buffer- 0.1-mM (1-mL).
8.2.2.2 Synthesis of 5- O -( b - D -Glucopyranosyl)Ribo fl avin Using b -Glucosidase
5- O -( b - d -Glucopyranosyl)ribo fl avin using b -glucosidase isolated from sweet almond was also synthesised (Scheme 8.10 and Table 8.28 and Fig. 8.14 ).
Determination of water solubility of 5- O -( d -glucopyranosyl)ribo fl avin showed that it is soluble to the extent of 8.2 g L −1 at room temperature (25°C). Hence, 5- O -( d -glucopyranosyl)ribo fl avin was found to be more soluble than ribo fl avin itself (0.2 g L −1 at 25°C) in water under identical conditions.
8.2.2.3 Syntheses of Ribo fl avinyl Glycosides of Other Carbohydrates Using Amyloglucosidase and b -Glucosidase and Their Spectral Characterisation
Syntheses of ribo fl avinyl glycosides with other carbohydrates ( d -glucose, d -galactose, d -mannose, d -ribose, maltose and sucrose) were attempted under (Scheme 8.10 ) optimum conditions using amyloglucosidase and b -glucosidase (Table 8.29 ).
Spectral characterisation showed the follow-ing features. Ultraviolet-visible spectra of ribo fl avinyl glycosides showed shifts in s → s * band in the 193–196.5-nm (191 nm for free ribo fl avin) range, s → p * band in the 223–230.5-nm (224 nm for free ribo fl avin) range and p → p *
Fig. 8.12 HPLC chromatogram for the reaction mixture of 5- O -( d -glucopyranosyl)ribo fl avin. HPLC conditions: aminopropyl column (10 m m, 300 mm × 3.9 mm); solvent, CH
3 CN: H
2 O
(70:30 v/v); fl ow rate, 1 mL min −1 ; RI detector. Peak retention times: solvent peak, 4.4 min; ribo fl avin, 5.5 min; d -glucose, 6.5 min; and 5- O -( d -glucopyranosyl)ribo fl avin, 8.2 min
190 8 Glycosylation of Some Selected Phenols and Vitamins
band in the 253.5–283.5-nm (266 nm for free ribo fl avin) range and IR glycosidic C–O–C symmetrical stretching frequencies in the 1,031–1,071-cm −1 range and glycosidic C–O–C asymmetrical stretching frequencies in the 1,148–1,298-cm −1 range indicating that ribo fl avin had undergone glycosylation. From the 2-D HSQCT spectra of the ribo fl avinyl glycosides, the glycoside
formation was con fi rmed from their respective 1 H and 13 C chemical shift values of carbon atoms C1 a , C1 b and C6- O -arylated com-pounds. Mass spectra also con fi rmed the forma-tion of the glycosides. Two-dimensional HSQCT data clearly indicated that the glycosy-lation has occurred at the 5-CH
2 OH of ribitol
moiety of ribo fl avin.
Table 8.27 Optimisation of reaction conditions for the synthesis of 5- O -( d -glucopyranosyl)ribo fl avin using amyloglucosidase
Reaction conditions Variable parameter b Yield (%) c
Incubation period (h) Ribo fl avin – 0.5 mmol 3 18 d -Glucose – 1 mmol 6 17 pH – 7 12 25 Buffer concentration – 0.1 mM (1 mL) 24 25 Amyloglucosidase – 50% w/w d -glucose 48 23
72 25 96 8 pH (0.01 M)
Ribo fl avin – 0.5 mmol a 4 11 d -Glucose – 1 mmol 5 14 Amyloglucosidase – 40% w/w d -glucose 6 15 Buffer concentration – 0.1 mM (1 mL) 7 17 Incubation period – 72 h 8 14
Buffer concentration (mM) Ribo fl avin – 0.5 mmol 0.05 17 d -Glucose – 1 mmol 0.1 19 Amyloglucosidase – 40% w/w d -glucose 0.15 6 pH – 7 0.2 5 Incubation period – 72 h 0.25 2
Amyloglucosidase concentration (% w/w d -glucose)
Ribo fl avin – 0.5 mmol 10 18 d -Glucose – 1 mmol 20 19 pH – 7 30 23 Buffer concentration – 0.1 mM (1 mL) 40 24 Incubation period – 72 h 50 25
75 21 Ribo fl avin (mmol)
pH – 7 0.25 19 Buffer concentration – 0.1 mM (1 mL) 0.5 25 d -Glucose – 1 mmol 1 31 Amyloglucosidase – 50% w/w d -glucose 1.5 25 Incubation period – 72 h 2 25
a Initial reaction conditions b Other variables are the same as under reaction conditions, except the speci fi ed ones c HPLC yields expressed with respect to 1-mmol d -glucose employed
1918.2 Vitamins
8.2.3 Pyridoxine Glycosides
Vitamin B 6 is a collective term for a group of
three related compounds, pyridoxine, pyridoxal, pyridoxamine and their phosphorylated deriva-tives. Pyridoxal 5 ¢ -phosphate is an important cofactor in a wide range of biochemical processes including amino acid metabolism and antibiotic biosynthesis (Studart et al. 2005 ) and as regula-tory molecules in signal transduction.
Pyridoxine is a light and heat sensitive water-soluble vitamin. However, pyridoxine glycosides are less heat and light sensitive than pyridoxine (Kawai et al. 1971a, b ) . Synthesis of pyridoxine-7- O - a - d -glucoside by transglycosylation using Coriolus and Verticillium cultured cells (Asano and Wada 2003 ) and enzyme from the marine mol-lusc Aplysia fasciata (Andreotti et al. 2006 ) was reported. In this section a systematic study was
undertaken to prepare pyridoxine glycosides using b -glucosidase isolated from sweet almond in di-isopropyl ether nonpolar medium (Scheme 8.11 , Einstein Charles and Divakar 2009 ) .
Under optimum conditions of 40% (w/w d -glucose), 0.18-mM (1.8-ml) pH 5 acetate buf-fer for 72-h incubation period b -glucosidase gave a maximum yield of 36 for pyridoxine- d -gluco-side (Sivakumar et al. 2006a, b ; Ponrasu et al. 2008 ) . Table 8.30 describes the optimum yields obtained.
Syntheses of the other pyridoxine glycosides were carried out at the optimised conditions, with d -glucose, d -galactose, d -mannose, d -fructose, d -ribose, d -arabinose, maltose, lactose, sucrose, d -sorbitol and d -mannitol, and the reaction occurred with only the three aldohexoses employed (Table 8.31 ) with yields in the range 23–40%. Non-reactivity of the other carbohydrate
Fig. 8.13 ( a ) Reaction pro fi le for 5- O -( d -glucopyranosyl)ribo fl avin synthesis by the re fl ux method. Conversion yields were from HPLC with respect to 1 mmol of d -glucose. Reaction conditions: d -glucose, 1 mmol; ribo fl avin, 0.5 mmol; amyloglucosi-dase, 50% (w/w d -glu-cose); 0.1-mM (1-mL) pH 7 phosphate buffer; solvent, di-isopropyl ether; and temperature, 68°C. ( b ) Effect of buffer concentration for 5- O -( d -glucopyranosyl)ribo fl avin synthesis. Reaction conditions: d -glucose, 1 mmol; ribo fl avin, 0.5 mmol; amyloglucosidase, 40% (w/w d -glucose); pH 7 phosphate buffer; solvent, di-isopropyl ether; temperature, 68°C; and incubation period, 72 h
192 8 Glycosylation of Some Selected Phenols and Vitamins
molecules could be due to stronger binding of pyridoxine to the active site of b -glucosidase than the above-mentioned carbohydrate molecules, thereby preventing their facile transfer to the nucleophilic primary OH of pyridoxine.
UV spectra of pyridoxine glycosides showed shifts in the s → s * band ranging from 191 to 197 nm (194 nm for pyridoxine), s → p * band between 220 and 226.5 nm, p → p * at 278–297 (291 nm for pyridoxine) nm and n → p * band
Table 8.28 Optimisation of reaction conditions for the synthesis of 5- O -( b - d -glucopyranosyl)ribo fl avin using b -glucosidase
Reaction conditions Variable parameter b Yield (%) c
Incubation period (h) Ribo fl avin – 0.5 mmol a 3 11 d -Glucose – 1 mmol 6 13 pH – 6 12 14 Buffer concentration – 0.1 mM (1 mL) 24 16
b -Glucosidase – 30% w/w d -glucose 48 18
72 22 96 18 pH (0.01 M)
Ribo fl avin – 0.5 mmol 4 6 d -Glucose – 1 mmol 5 7
b -Glucosidase – 50% w/w d -glucose 6 8
Buffer concentration – 0.1 mM (1 mL) 7 8 Incubation period – 72 h 8 7
Buffer concentration (mM) Ribo fl avin – 0.5 mmol 0.03 8 d -Glucose – 1 mmol 0.06 11
b -Glucosidase – 50% w/w d -glucose 0.1 18
pH – 6 0.14 8 Incubation period – 72 h 0.2 No yield
0.25 No yield
b -Glucosidase concentration (% w/w d -glucose)
Ribo fl avin – 0.5 mmol 10 5 d -Glucose – 1 mmol 20 12 pH – 6 30 23 Buffer concentration – 0.1 mM (1 mL) 40 20 Incubation period – 72 h 50 17
75 10 Ribo fl avin (mmol)
pH – 6 0.25 21 Buffer concentration – 0.1 mM (1 mL) 0.5 24 d -Glucose – 1 mmol 0.75 17
b -Glucosidase – 30% w/w d -glucose 1 16
Incubation period – 72 h 1.5 13 2 13
a Initial reaction conditions b Other variables are the same as under reaction conditions, except the speci fi ed ones c HPLC yields expressed with respect to 1-mmol d -glucose employed
0
5
10
15
20
25
0 20 40 60 80 100Incubation period (h)
Con
vers
ion
yiel
d (%
)
0
5
10
15
20
25
30
0.25 0.5 0.75 1 1.5 2Riboflavin concn (mmol)
Con
vers
ion
yiel
d (%
)
a
b
Fig. 8.14 ( a ) Reaction pro fi le for 5- O -( b - d -glucopyranosyl)ribo fl avin synthesis by the re fl ux method. Conversion yields were from HPLC with respect to 1 mmol of d -glucose. Reaction conditions: d -glucose, 1 mmol; ribo fl avin, 0.5 mmol; b -glucosidase, 30% (w/w d -glucose); 0.1-mM (1-mL) pH 6 phosphate buffer; solvent, di-isopropyl ether; and temperature, 68°C. ( b ) Effect of ribo fl avin concentration for 5- O -( b - d -glucopyranosyl)ribo fl avin synthesis. Reaction conditions: d -glucose, 1 mmol; b -glucosidase, 30% (w/w d -glucose); 0.1-mM (1-mL) pH 6 phosphate buffer; solvent, di-isopro-pyl ether; temperature, 68°C; and incubation period, 72 h
Table 8.29 Syntheses of ribo fl avinyl glycosides using amyloglucosidase and b -glucosidase
Glycosides
Amyloglucosidase catalysis a b -Glucosidase catalysis b
Product (% proportion) c Yields (%) d
Product (% proportion) c
Yields (%) d
OH
OHOH
H
H
HH
HO
O
O
H
H
CH2
OH
CH2
OH
OH
H
CH3
CH3
O
O
NH
N
N
N
OH
OHOH
H
H
HH
HO
O
N
N
N
NH
O
O
CH3
CH3
H
OH
OH
CH2
OH
CH2
H
H
O
H
N
N
N
NH
O
O
CH3
CH3
H
OH
OH
CH2
OH
CH2
H
H
O
H
OH
O
HO
HH
H
HOH
OH
C1 a -glucosides (43), C1 b -glucosides (22), C6- O -arylated (35)
25 C1 b -glucoside 24
5- O -( a - d -Glucopyranosyl)ribo fl avin
5- O -( b - d -Glucopyranosyl)ribo fl avin 5- O -(6- d -Glucopyranosyl)ribo fl avin
OH
HOH
H
OH
HH
HO
O
O
H
H
CH2
OH
CH2
OH
OH
H
CH3
CH3
O
O
NH
N
N
N
OH
HOH
H
OH
HH
HO
O
N
N
N
NH
O
O
CH3
CH3
H
OH
OH
CH2
OH
CH2
H
H
O
H
C1 a -galactosides (52), C1 b -galactosides (48)
14 C1 a -galactosides (47), C1 b -galactosides (53)
9
5- O -( a - d -Galactopyranosyl)ribo fl avin
5- O -( b - d -Galactopyranosyl)ribo fl avin
OH
OHOH
H
H
HOH
H
O
OH
H
CH2
OH
CH2
OH
OH
H
CH3
CH3
O
O
NH
N
N
N
OH
OHOH
H
H
HOH
H
O
N
N
N
NH
O
O
CH3
CH3
H
OH
OH
CH2
OH
CH2
H
H
O
H
C1 a -mannoside 11 C1 a -mannoside (59) C1 b -mannoside (41)
7
5- O -( a - d -Mannopyranosyl)ribo fl avin
5- O -( b - d -Mannopyranosyl)ribo fl avin
(continued)
194 8 Glycosylation of Some Selected Phenols and Vitamins
between 321 and 329 nm (325 nm for pyridoxine). IR spectra showed shifts in the 1,033–1,088-cm −1 range for the glycosidic C–O–C aryl–alkyl sym-metrical stretching and 1,279–1,280-cm −1 range
for the asymmetrical stretching frequencies. 2-D HSQCT spectra con fi rmed the glycoside forma-tion from C1 a , C1 b and C6-O-arylations. Shifts in the 6th and 7th primary OH carbon chemical
Table 8.29 (continued)
Glycosides
Amyloglucosidase catalysis a b -Glucosidase catalysis b
Product (% proportion) c Yields (%) d
Product (% proportion) c
Yields (%) d
N
N
N
NH
O
O
CH3
CH3
O
H
OHOH
HHH
O
H
OH
OH
CH2
OH
H
H
OH
N
N
N
NH
O
O
CH3
CH3
H
OH
OH
CH2
OH
H
H
O
OH
OHOH
HHH
H
O
C1 a riboside (23), C1 b riboside (77)
40 – –
5- O -( a - d -Ribofuranosyl)ribo fl avin
5- O -( b - d -Ribofuranosyl)ribo fl avin
OHO
OH
HH
H
HOH
O
O OH
HH
H
HH OH
OH
OH
O
H
H
CH2
OH
CH2
OH
OH
H
CH3
CH3
O
O
NH
N
N
N
HH
CH2
OH
CH2
OHOH
H
CH3
CH3
O
O
NH
N
N
N
H
OH
OHOH
OHHH
H
HH
OHO
O
OHH
H
HH
OH
OO
H
H
CH2
OH
CH2
OH
OH
H
CH3
CH3
O
O
NH
N
N
N
OHO
OH
HH
H
HOH
O
O OH
HH
H
HH OH
OHO
OH
H
C1 a -maltoside (35), C6- O -arylated (48), C6 ¢ - O -arylated (17)
5 – –
5- O -( a - d -Glucopyranosyl-(1 ¢ →4) a - d -glucopyranosyl)ribo fl avin
5- O -( a - d -Glucopyranosyl-(1 ¢ →4)6- d -glucopyranosyl)ribo fl avin
5- O -( a - d -Glucopyranosyl-(1 ¢ →4)6 ¢ - d -glucopyranosyl)ribo fl avin
N
N
N
NH
O
O
CH3
CH3
H
OH
OH
CH2
OH
CH2
H
H O
OH
OHO OH
O
OHOH
H
H
HHHO
OOH
C1- O -arylated 12 – –
5- O -(1- d -Fructofuranosyl-(2→1 ¢ ) a - d -glucopyranosyl)ribo fl avin
OH
H
CH2
OH
CH2
OH
OH
H
CH3
CH3
O
O
NH
N
N
N
OHH
H
HH
OH
OOH
H
HO
OH
HH
OH
HO OH
OH
H
– – C1 b -lactoside 9
5- O -( b - d -Galactopyranosyl-(1 ¢ →4) b - d -glucopyranosyl)ribo fl avin
a Ribo fl avin, 0.5 mmol; carbohydrate, 1 mmol; amyloglucosidase concentration, 50% w/w of carbohydrate; solvent, di-isopropyl ether; buffer, 0.1-mM (1-mL) pH 7 phosphate buffer; incubation period, 72 h b Ribo fl avin, 0.5 mmol; carbohydrate, 1 mmol; b -glucosidase concentration, 30% w/w of carbohydrate; solvent, di-isopropyl ether; buffer, 0.1-mM (1-mL) pH 6 phosphate buffer; incubation period, 72 h c Conversion yields were from HPLC with respect to free carbohydrate. Error in yield measurements is ±5–10% d The product proportions were determined from the area of respective 1 H/ 13 C signals
1958.2 Vitamins
Scheme 8.11 Syntheses of pyridoxine glycosides
shift values to 60.6 and 62.8 ppm, respectively, from 56.4 and 58.6 ppm for free pyridoxine, indi-cated that glucosylation occurred at the primary OH groups of the pyridoxine. Mass spectral data also con fi rmed product formation.
No bis-glycosylated products where both the OH groups at 6th and 7th positions of pyridoxine reacted were detected. Phenolic OH of pyridox-ine has not also reacted. Besides, only C1 glyco-sylated products were detected. In d -galactose, a / b proportions were 74:26 ( a : b ) with respect to 60:40, for free d -galactose, thus showing a slight excess of a - d -galactoside formation. However, d -mannose showed a marginally high b -manno-side formation as detected from an a / b propor-tion of 58:42 compared to 77:23 for the free d -mannose.
8.2.4 Glycosylation of Retinol
Components of vitamin A, retinol and retinoic acid, are fat-soluble micronutrients essential for growth, vision, cell differentiation and integrity of the immune system (Nsoukpoe-Kossi et al. 2007 ; Kren and Martinkova 2001 ) . Its derivatives show therapeutic utility in several types of cancer and skin disorders (Ong et al. 1994 ) . Different metabolic forms of vitamin A show different activities – 11-cis-retinol is the major ligand for the opsins in vision. 9-cis-Retinoic acid is active in cell differentiation and embryogenesis, and retinyl esters serve as transport and storage forms of vitamin A (Ong et al. 1994 ) . Need to synthesise
and evaluate newer and more ef fi cient glycosides of vitamins including vitamin A is strong due to several factors including unmet therapeutic needs, remarkable diversity of both chemical structures and biological activities of naturally occurring secondary metabolites, the utility of bioactive natural products as biochemical and molecular probes and advances in solving the demand for supply of complex natural products (Clark 1996 ) . One such glycosidic derivative of retinol, N-(4-hydoxyphenyl)retinamide(4-HPR), has been shown to be effective in numerous types of tumour models and has been included in phase III clinical trials also (Walker et al. 2002 ) .
Our enzymatic reactions with vitamin A in shake fl asks resulted in degradation of vitamin A (Einstein Charles et al. 2009a ) . Hence, still more milder conditions are required for compounds like vitamin A (Scheme 8.12 ). Supercritical fl uid carbon dioxide media (SCCO
2 ) were employed.
Besides maintaining the integrity of the com-pounds during reaction, it would also be very effective to carry out such reactions in SCCO
2
media. Performing reactions in supercritical fl uids (SCF) with carbon dioxide also have other advantages like energy reduction, ease of product recovery, lesser cost of downstream processing and reduction in side reactions (Tai et al. 2001 ; Marty et al. 1990 ; Russell and Beckman 1991 ) .
Syntheses of retinol glycosides were carried out under SCCO
2 pressure of 120 bar at 50°C.
A reaction vessel of 100-ml capacity controlled thermostatically fi tted with a magnetic stirrer and a recirculating fl uid pressure differential loop
196 8 Glycosylation of Some Selected Phenols and Vitamins
with a Rheodyne valve capable of delivering 0.5-ml sample was employed. Retinol (0.5 mmol), carbohydrate (1 mmol), 40% (w/w of carbohy-drate) b -glucosidase, 0.12-mM (1.2-ml) pH 6 phosphate buffer and 24-h incubation period in 10 mL of DMF under 120-bar SCCO
2 at 50°C
were employed for the reactions. A concentration of 40% (w/w carbohydrate) of b -glucosidase cor-responded to 224.6 AU of the enzyme. After the reaction, carbon dioxide was released slowly and the enzyme denatured at 100°C by holding in boiling water bath for 5–10 min. The reaction
Table 8.30 Optimisation of reaction conditions for the synthesis of pyridoxine- d -glucoside using b -glucosidase
Reaction conditions a Variable parameter b Conversion yields (%) c
pH (0.01 M) Pyridoxine – 0.5 mmol 4 20 d -Glucose – 0.5 mmol 5 25
b -Glucosidase – 40% w/w d -glucose 6 14
Buffer volume – 1 mL (0.1 mM) 7 7 Incubation period – 72 h 8 17
Buffer concentration (mM) d Pyridoxine – 0.5 mmol 0.4 7 d -Glucose – 0.5 mmol 0.8 19
b -Glucosidase – 40% w/w d -glucose 1.2 23
Incubation period – 72 h 1.8 36 pH – 5.0 2.5 27
b -Glucosidase concentration (% w/w d -glucose)
Pyridoxine – 0.5 mmol 10 23 d -Glucose – 0.5 mmol 25 27 Buffer volume – 0.18 mM (1.8 mL) 40 36 Incubation period – 72 h 50 30 pH – 5.0 75 31
Pyridoxine concentration (mmol) d -Glucose – 0.5 mmol 0.25 19 Buffer volume – 0.18 mM (1.8 mL) 0.5 36
b -Glucosidase – 40% w/w d -glucose 1 25
Incubation period – 72 h 1.5 17 pH – 5.0 2 15
Incubation period (h) Pyridoxine – 0.5 mmol 3 0 d -Glucose – 0.5 mmol 6 0 Buffer volume – 0.18 mM (1.8 mL) 12 0
b -Glucosidase – 40% w/w d -glucose 24 7
pH – 5.0 48 8 72 36 96 37
a Initial reaction conditions b Other variables are the same as the initial reaction conditions, except for the speci fi ed ones c HPLC yields expressed with respect to 1-mmol d -glucose employed, HPLC on a 250 mm × 4.6 mm aminopropyl column with acetonitrile:water (70:30 v/v) as mobile phase and detecting using an RI detector, values an average from two independent reactions d Buffer: 0.01-M acetate buffer for pH 4 and 5, phosphate buffer for pH 6 and 7 and borate buffer for pH 8
1978.2 Vitamins
Table 8.31 b -Glucosidase-catalysed syntheses of pyridoxine glycosides a
Glycosides b -Glucosidase catalysis b
Product c (% proportion) Conversion d yields (%)
7- O -( a - d -Glucopyranosyl)pyridoxine 7- O -C1 a (39) 36
7- O -( b - d -Glucopyranosyl)pyridoxine 7- O -C1 b (41)
6- O -( a - d -Glucopyranosyl)pyridoxine 6- O -C1 a (20)
7- O -( a - d -Galactopyranosyl)pyridoxine 7- O -C1 a (27) 40
7- O -( b - d -Galactopyranosyl)pyridoxine 7- O -C1 b (26)
6- O -( a - d -Galactopyranosyl)pyridoxine 6- O -C1 a (47)
7- O -( a - d -Mannopyranosyl)pyridoxine 7- O -C1 a (34) 23
7- O -( b - d -Mannopyranosyl)pyridoxine 7- O -C1 b (42)
6- O -( a - d -Mannopyranosyl)pyridoxine 6- O -C1 a (24)
a Pyridoxine, 0.5 mmol; carbohydrate, 1.0 mmol; enzyme concentration 40% w/w carbohydrates, solvent, di-isopropyl ether; DMF, 5.0 mL; 0.18-mM (1.8-ml) pH 5.0 acetate buffer; incubation period, 72 b The product proportions were determined from the area of respective 1 H/ 13 C signals c Unreacted carbohydrate and unreacted pyridoxine were separated from the product glycosides by passing through Sephadex G15 column (100 cm × 1 cm); eluting with water at 1 mL h −1 , individual glycosides could not be separated satisfactorily due to similar molecular weights of the glycosides formed d Conversion yields were from HPLC peak areas of the glycosides and carbohydrates and expressed as percentage gly-coside formed with respect to concentration of the carbohydrate. Error in yield measurements is ±10%. Retention times are: pyridoxine, 8.4 min; d -glucose, 8.1 min; d -galactose, 8.2 min; d -mannose, 8.3 min; pyridoxine- d -glucoside, 10.4 min; pyridoxine- d -galactoside, 10.3 min; and pyridoxine- d -mannoside, 10.1 min
Scheme 8.12 Syntheses of retinol glycosides using b -glucosidase
mixture was extracted with hexane to remove unreacted retinol. The aqueous reaction mixture was evaporated in the dark to get a mixture of the glycoside and unreacted carbohydrate. Retinol being light and air sensitive, workup and isola-tion were also carried out in the dark.
HPLC retention times for retinol and its gly-cosides are as follows: retinol, 3.5 min; d -glu-cose, 7.5 min (Fig. 8.15 ); 18- O -( d -glucopyranosyl)retinol, 8.9 min; d -galactose, 7.0 min; 18- O -( d -galactopyranosyl) retinol, 7.6 min; d -mannose, 6.7 min; 18- O -( b - d -mannopyranosyl)retinol,
7.7 min; d -fructose, 6.8 min; 18- O -( d -fructofuranosyl)retinol, 7.9 min; d -sorbitol, 6.7 min; and 18- O -(1- d -sorbitol)retinol, 7.7 min.
2-D HSQCT spectra and mass spectra con fi rmed the formation of monoglycosides of retinol.
8.2.4.1 Retinol Glycosides Glycosylation did not take place without the enzyme in SCCO
2 , under the reaction conditions of
temperature and low solvent content employed. Conducting the reaction in shake fl asks, under such reaction conditions, gave degraded products of
198 8 Glycosylation of Some Selected Phenols and Vitamins
vitamin A. Hence, carrying out reactions in SCCO 2
was found to be extremely advantageous as the conditions employed were milder and the yield and selectivity were extremely good (Table 8.32 ).
Glycosylation reaction between retinol and carbohydrates – aldohexoses ( d -glucose, d -galac-tose and d -mannose), ketohexose ( d -fructose) and carbohydrate alcohol ( d -sorbitol) (Scheme 8.12 ) – was a facile reaction, and the yields obtained were in the 9–34% range. Highest yield of 34% was obtained with d -fructose.
No oxidative or polymerised products of retinol were detected from the NMR spectra and mass spec-tra. Ultraviolet-visible spectra of retinol glycosides showed shifts in s → s * band in the 196–205-nm (192 nm for free retinol) range, s → p * band in the 214–237.5-nm (236 nm for free retinol) range, p → p * band in the 260–261.5-nm (266.5 nm for free retinol) range and n → p * band in the 280–286.5-nm (286.5 nm for free retinol) range. IR glycosidic C–O–C symmetrical stretching frequencies were observed in the 1,026–1,080-cm −1 range and glyco-sidic C–O–C asymmetrical stretching frequencies in the 1,220–1,298-cm −1 range indicating that retinol had undergone glycosylation. 2-D HSQCT spectra showed formation of C1 a , C1 b and C1-O and C6-O products with the corresponding carbohydrate mol-ecules. Mass spectra also con fi rmed formation of the above-mentioned glycosides. NMR data clearly indicated that the glycosylation occurred at the 18-CH
2 OH of the ole fi nic moiety of retinol and C1
position of d -glucose, d -galactose, d -mannose and the CH
2 OH groups of d -fructose and d -sorbitol.
Glycosylation gave rise to 38% of a - d -gluco-side and 62% of b - d -glucoside compared to the 40:60 a : b -anomeric composition of d -glucose and 42% a - d -galactoside along with 58% b - d -galactoside compared to the 92:8 a : b -anomeric composition of d -galactose. In the oxocarbenium ion mechanism for the glycosylation (Chiba 1997 ) , a planar carbenium ion centre formed with d -glucose and d -galactose is available for attack by the nucleophilic retinol ole fi nic OH from both above and below the plane giving rise to a mixture of a / b -anomeric products. Unlike d -glucose, d -galactose yielded more of the b product. b -Glucosidase catalysis gave exclu-sively C1 b -mannoside and C1- O -derivatised d -sorbitol, indicating its capability to exhibit excellent regioselectivity (Table 8.32 ). However, d -fructose gave reaction products from both the CH
2 OH groups. Presence of a -glucosidase in the
enzyme could be responsible for formation of the a -glycosides in case of d -glucose and d -galactose.
8.2.5 Syntheses of Ergocalciferyl Glycosides
Vitamin D, belonging to the class of seco-steroids, comprises ten substances, each with different level of activity (Holick 2004 ; Maurizio et al. 2007 ) .
Fig. 8.15 HPLC chromatogram for the reaction mixture of d -glucose, retinol and 18- O -( d -Glucopyranosyl)retinol. HPLC conditions: aminopropyl column (10 m m, 300 mm × 3.9 mm), solvent, CH
3 CN: H
2 O (70:30 v/v);
fl ow rate, 1 mL min −1 ; RI detector. Retention times: retinol, 3.15 min; d -glucose, 6.69 min; and 18- O -( d -Glucopyranosyl)retinol, 8.89 min
1998.2 Vitamins
Table 8.32 b -Glucosidase-catalysed syntheses of retinol glycosides a
Glycosides Product (% proportion) a
Yields (%) b
O
CH3
CH3CH3CH3 CH3
HOH2C
OHOHH
H
H
HH
OH
O
O
CH3
CH3CH3CH3 CH3 HOH2C
OHOH
H
H
HH
OHH
O
C1 a (38) and C1 b (62) glucoside
21
18- O -( a - d -Glucopyranosyl)retinol
18- O -( b - d -Glucopyranosyl)retinol
O
CH3
CH3CH3CH3 CH3
HOH2CH
OHHH
OH
HH
OH
O
O
CH3
CH3CH3CH3 CH3 HOH2C
HOH
H
OH
HH
OHH
O
C1 a (26) and C1 b (74) galactoside
28
18- O -( a - d -Galactopyranosyl)retinol
18- O -( b - d -Galactopyranosyl)retinol
O
CH3
CH3CH3CH3 CH3 HOH2C
OH
OHH
H
HOH
HH
O
C1 b -mannoside 18
18- O -( b - d -Mannopyranosyl)retinol
O
CH3
CH3CH3CH3 CH3
OH
HO
HOH
HHHO
O
O
CH3
CH3CH3CH3 CH3
OH
HO
HOH
HHHO
O
C1- O - (35) and C6- O - (65) derivatised
34
18- O -(1- d -Fructofuranosyl)retinol 18- O -(6- d -Fructofuranosyl)retinol
O
CH3
CH3CH3CH3 CH3
HH
H
H
CH2
CH2OH
OH
OH
OHOH
C
C
CC
C1- O -derivatised 9
18- O -(1- d -Sorbitol)retinol
a Product proportions were calculated from the area of respective proton signals b Conversion yields were from HPLC with respect to free carbohydrate 2 – 6 . Error in yield measurements is ±5%
Seco-steroids are those in which one of the ring is broken and in vitamin D by ultraviolet B light (UV-B, sunlight). Fat-soluble vitamin D occurs mainly in two active forms: ergocalciferol or acti-vated ergosterol (vitamin D
2 ) found in plants and
irradiated yeast and cholecalciferol or activated 7-dehydrocholesterol (vitamin D
3 ) formed in human
skin after exposure to UV-B rays from the sun. Ergocalciferol (vitamin D2) is a plant sterol,
derived from ergosterol which is the most common dietary source (Mello 2003 ) . Vitamin D is not only a nutrient but also a precursor of a steroid hormone
with a wide range of activities that include an impor-tant role in calcium metabolism and cell differentia-tion. Vitamin D derivatives can be useful in treatment of several forms of cancer, and their mode of action is currently under scrutiny (Smith et al. 1999 ; James et al. 1999 ; Peehl et al. 2003 ; Wieder et al. 2003 ; Chen et al. 2003 ) . Ergocalciferol prevents infantile rickets, capable of healing adult osteomalacia and osteonecrosis during renal transplantation (Houghton and Vieth 2006 ; Scholz et al. 1983 ) . Active metabolite of vitamin D3 is 1 a , 25-dihy-droxyvitamin D3 which regulates a wide variety of
200 8 Glycosylation of Some Selected Phenols and Vitamins
Scheme 8.13 Synthesis of 20- O -( d -glucopyranosyl)ergocalciferol
biological activities like intestinal calcium absorp-tion, bone resorption and mineralisation (Feldman et al. 2000 ) . 1,25-Dihdroxyvitamin D3-glycoside has been identi fi ed in the plant. For example, Solanum malacoxylon possess a vitamin d -like cal-cinogenic principle, which is water-soluble (Hausslera et al. 1976 ) . Hence, ergocalciferol gly-cosides were prepared enzymatically using amylo-glucosidase from Rhizopus mould (Scheme 8.13 ).
Since ergocalciferol is sensitive to light, the reaction and the workout were carried out under the dark. The glycoside was also stored in the dark. The dried residue was subjected to HPLC, and the conversion yields were determined from HPLC peak areas (Fig. 8.16 ). HPLC retention times for the substrates and products are as follows: ergocalciferol, 4.5 min; d -glucose, 6.5 min; and 20- O -( d -glucopyranosyl)ergocal-ciferol, 9.3 min. An attempt was made to synthe-sise the ergocalciferyl glycosides using various other earlier mentioned carbohydrates also, but
no other carbohydrate except d -glucose under-went glycosylation with ergocalciferol.
8.2.5.1 Synthesis of 20- O -( D -Glucopyranosyl)Ergocalciferol Using Amyloglucosidase
Optimisation conditions for the synthesis of 20- O -( d -glucopyranosyl)ergocalciferol using amyloglucosidase are as follows: maximum glu-cosylation of 32% yield was obtained at pH 6 (Table 8.33 and Fig. 8.17A ), conversion yield increased from 30% for 0.04-mM (0.4-mL) buf-fer to 43% for 0.12-mM (1.2-mL) buffer and 40% enzyme was found to give the best conversion yield of 42% (Table 8.33 , Fig. 8.17b ).
8.2.5.2 Solubility of 20- O -( D -Glucopyranosyl)Ergocalciferol
Determination of water solubility of 20- O -( d -glucopyranosyl)ergocalciferol showed that it is soluble to the extent of 6.4 g L −1 . Hence,
Fig. 8.16 HPLC chromatogram for the reaction mixture of 20- O -( d -glucopyranosyl)ergocalciferol. HPLC conditions: aminopropyl column (10 m m, 300 mm × 3.9 mm), solvent, CH
3 CN: H
2 O
(70:30 v/v); fl ow rate, 1 mL min −1 ; RI detector. Peak retention times: solvent peak, 3.2 min; d -glucose, 6.5 min; and 20- O -( d -glucopyranosyl)ergocalciferol, 9.3 min
Table 8.33 Optimisation of reaction conditions for the synthesis of 20- O -( d -glucopyranosyl)ergocalciferol using amyloglucosidase
Reaction conditions Variable parameter b Yield (%) c
pH (0.01 M) Ergocalciferol – 0.5 mmol a 4 23 d -Glucose – 1 mmol 5 27 Amyloglucosidase – 40% w/w d -glucose 6 32 Buffer concentration – 0.1 mM (1 mL) 7 27 Incubation period – 48 h 8 21
Buffer concentration (mM) Ergocalciferol – 0.5 mmol 0.04 30 d -Glucose – 1 mmol 0.08 37 Amyloglucosidase – 40% w/w d -glucose 0.12 43 pH – 6 0.16 30 Incubation period – 48 h 0.2 8
Amyloglucosidase concentration (% w/w d -glucose)
Ergocalciferol – 0.5 mmol 10 10 d -Glucose – 1 mmol 20 27 pH – 6 30 30 Buffer concentration – 0.12 mM (1.2 mL) 40 42 Incubation period – 48 h 50 29
75 11
a Initial reaction conditions b Other variables are the same as under reaction conditions, except the speci fi ed ones c HPLC yields expressed with respect to 1-mmol d -glucose employed
202 8 Glycosylation of Some Selected Phenols and Vitamins
0
10
20
30
40
3.0 4.0 5.0 6.0 7.0 8.0 9.0
pH (0.01M)
Con
vers
ion
yiel
d (%
)
0
10
20
30
40
50
10 20 30 40 50 75
Amyloglucosidase concn (% w/w D-glucose)
Con
vers
ion
yiel
d (%
)a
b
Fig. 8.17 ( a ) Effect of pH for 20- O -( d -glucopyranosyl)ergocalciferol synthesis by the re fl ux method. Conversion yields were from HPLC with respect to 1 mmol of d -glu-cose. Reaction conditions: d -glucose, 1 mmol; ergocalcif-erol, 0.5 mmol; amyloglucosidase, 40% (w/w d -glucose); 0.1 mM (1 mL) solvent, di-isopropyl ether; temperature, 68°C; and incubation period, 48 h. ( b ) Effect of enzyme concentration for 20- O -( d -glucopyranosyl) ergocalciferol synthesis. Reaction conditions: d -glucose, 1 mmol; ergo-calciferol, 0.5 mmol; 0.12-mM (1.2-mL) pH 6 phosphate buffer; solvent, di-isopropyl ether; temperature, 68°C; and incubation period, 48 h
20- O -( d -glucopyranosyl)ergocalciferol was found to be soluble than the water-insoluble ergocalciferol.
8.2.5.3 Spectral Characterisation Ultraviolet-visible spectra of 20- O -( d -glucopyranosyl)ergocalciferol (Table 8.34 ) showed shifts in s → s * band at 193 nm (194 nm for free ergocalciferol), s → p * band at 216 nm (216.5 nm for free ergocalciferol) and p → p * band in the 265 nm (265.5 nm for free ribo fl avin), IR gly-cosidic C–O–C symmetrical stretching frequency at 1,033 cm −1 and glycosidic C–O–C asymmetrical stretching frequency at 1,258 cm −1 indicating that
ergocalciferol had undergone glycosylation. From 2-D HSQCT spectra of 20- O -( d -glucopyranosyl)ergocalciferol, C1 a -glucoside, C1 b -glucoside and C6- O -arylated were con fi rmed by the 1 H and 13 C chemical shift values of the respective atoms. Mass spectra also con fi rmed the formation of the above-mentioned glucosides. Two-dimensional HSQCT data clearly indicated that glucosylation has occurred at the acyclic OH of 20th position of ergocalciferol.
8.2.6 Cholecalciferol Glycosides
Fat-soluble, light sensitive vitamin D 3 (Lehninger
1977 ) supplementation is very dif fi cult, and it can be solved by synthesising glycosyl deriva-tives of vitamin D
3 with enhanced solubility and
stability. Glycosylation reaction can be effected by many methods – chemical (Du et al. 2004 ; Sophie et al. 2004 ) and cell culture (Hamada et al. 2003 ) . Among these, enzymatic synthesis involves milder reaction conditions, simple workup procedure, easy recovery, less pollution and a cost-effective process with good selectivity and yield (Vijayakumar and Divakar 2007 ) . Enzymatic glycosylation can be effected by glucosidases (Sivakumar et al. 2006a, b ; Vijayakumar et al. 2006 ) . Glucosidases are hydrolytic in nature, but unusual conditions involving organic solvents with little quantity of water direct the enzymes towards glycosy-lation (Ponrasu et al. 2008 ) .
This section (Scheme 8.14 ) discusses the preparation of water-soluble glycosides of chole-calciferol (Manohar and Divakar 2010 ) .
8.2.6.1 17- O -( D -Glucopyranosyl)Cholecalciferol
This enzymatic reaction did not take place with-out the presence of enzymes under the reaction conditions employed. Under optimum condi-tions, 60% (w/w d -glucose) b -glucosidase gave a maximum yield of 14% at 0.12-mM (1.2-ml) pH 6 phosphate buffer in 30-h incubation period (Scheme 8.14 , Fig. 8.18 , Tables 8.35 and 8.36 ). Under the above-mentioned optimum conditions, synthesis of cholecalciferol glycosides was
2038.2 Vitamins
Scheme 8.14 Synthesis of cholecalciferol glycosides
Table 8.34 Synthesis of 20- O -( d -glucopyranosyl)ergocalciferol using amyloglucosidase
Glycosides Amyloglucosidase catalysis a
Product (% proportion) b Yields (%) c
O
OH
HH
H
HOH
CH2OHOH CH2
CH3
O
CH3
CH3
CH3
CH3
HH
H
H
O
OH
HH
H
HOH
CH2OHOH
HH
CH3
CH3
CH3
CH3
O
CH3
CH2
C1 a -glucoside (22), C1 b -glucoside (52), C6- O -arylated (26)
42
20- O -( a - d -Glucopyranosyl)ergocalciferol
20- O -( b - d -Glucopyranosyl)ergocalciferol
OH
O
OH
HH
H
HOH
OHH
O
CH2
CH3
CH3
CH3
CH3
CH3
HH
20- O -(6- d -Glucopyranosyl)ergocalciferol
a Ergocalciferol, 0.5 mmol; d -glucose, 1 mmol; amyloglucosidase, 40% (w/w d -glucose); solvent, di-isopropyl ether; buffer, 0.12-mM (1.2-mL) pH 6 phosphate buffer; incubation period, 48 h b The product proportions were determined from the area of respective 1 H/ 13 C signals c Conversion yields were from HPLC with respect to free d -glucose. Error in yield measurements is ±5–10%
204 8 Glycosylation of Some Selected Phenols and Vitamins
Fig. 8.18 HPLC chromatogram for the reaction mixture of 6- O -( b - d -glucopyranosyl) a -tocopherol. HPLC conditions: aminopropyl column (10 m m, 300 mm × 3.9 mm), solvent, CH
3 CN: H
2 O
(70:30 v/v); fl ow rate, 1 mL min −1 ; RI detector. Peak retention times: a -tocopherol, 4.3 min; solvent peak, 5.2 min; d -glucose, 7.5 min; and 6- O -( b - d -glucopyranosyl) a -tocoph-erol, 9.3 min
attempted with other carbohydrate molecules also to evaluate the propensity of glycosylation by b -glucosidase with the usual diverse carbohy-drate molecules (Table 8.35 ).
8.2.6.2 Spectral Characterisation UV spectra of cholecalciferol glycosides (Table 8.36 ) showed shifts in the s → s * band in the 190.5–192.0-nm (191.0 nm for cholecal-ciferol) range, s → p * band in the 222.5–226.5-nm range, p → p * in the 268.5–272.0-nm (291 nm for cholecalciferol) range and n → p * band in the 282.5–327.5-nm (323 nm for chole-calciferol) range. IR spectra showed shifts in the 1,060–1,080-cm −1 range for the glycosidic C–O–C aryl–alkyl symmetrical stretching and in the 1,355–1,391-cm −1 range for the asym-metrical stretching frequencies. Once again 2-D HSQCT spectra con fi rmed the formation of C1 a , C1 b and C6-O- derivatives of d -glu-cose, d -galactose, d -mannose, d -ribose and
d -fructose. The carbon chemical shift value at 60 ppm (59.7 ppm for free cholecalciferol) indicated that glycosylation occurred at the ali-cyclic OH group at position 17 of cholecalcif-erol. Mass spectral data also con fi rmed product formation.
8.2.7 Syntheses of a -Tocopheryl Glycosides
a -Tocopherol [2,5,7,8-tetramethyl-2-(4 ¢ ,8 ¢ ,12 ¢ -trimethyltridecyl)-6-chromanol, vitamin E], an oil-soluble vitamin, is a very important compo-nent of biological membranes, stabilising them by acting as a potent antioxidant and free radi-cal scavenger (Burton et al. 1983 ) . Vitamin E has been frequently used since 1970 to treat various diseases (Satoh et al. 2001 ) , which includes treatment against gynaecological internal secretion, control against sterility, heart
2058.2 Vitamins
Table 8.35 Optimisation of reaction conditions for the synthesis of 17- O -( d -glucopyranosyl)cholecalciferol using b -glucosidase
Reaction conditions a Variable parameter b Conversion yields (%) c
pH (0.01 M) Cholecalciferol – 0.5 mmol 4 10 d -Glucose – 1 mmol 5 13
b -Glucosidase (60% w/w d -glucose) 6 14
Buffer concentration – 0.12 mL (1.2 mL) 7 12 Incubation period – 30 h 8 8
Buffer concentration (mM) Cholecalciferol – 0.5 mmol 0.04 11 d -Glucose – 1 mmol 0.08 12
b -Glucosidase (60% w/w d -glucose) 0.12 14
pH – 6 0.16 13 Incubation period – 30 h 0.2 13
b -Glucosidase concentration (w/w d -glucose)
Cholecalciferol – 0.5 mmol 20 3 d -Glucose – 1 mmol 40 10 Buffer concentration – 0.12 mM (1.2 mL) 60 14 pH – 6 80 12 Incubation period – 30 h 100 4
Incubation (h) Cholecalciferol – 0.5 mmol 10 11 d -Glucose – 1 mmol 20 13
b -Glucosidase (60% w/w d -glucose) 30 14
pH – 6 40 13 Buffer concentration – 0.12 mM (1.2 mL) 50 12
a Initial reaction conditions b Other variables are the same as the initial reaction conditions, except for the speci fi ed ones c HPLC yields expressed with respect to 0.5-mmol d -glucose employed
Table 8.36 b -Glucosidase-catalysed syntheses of cholecalciferol glycosides
b -Glucosidase catalysis Product proportion a (%) Conversion yields b (%)
17- O- ( a - d -Glucopyranosyl)cholecalciferol 17- O - a (34) 14
17- O- ( b - d -Glucopyranosyl)cholecalciferol 17- O - b (48) 17- O- (6- d -Glucopyranosyl)cholecalciferol 17- O -6 (18)
17- O- ( a - d -Galactopyranosyl)cholecalciferol 17- O - a (29) 30
17- O- ( b - d -Galactopyranosyl)cholecalciferol 17- O - b (56) 17- O- (6- d -Galactopyranosyl)cholecalciferol 17- O -6 (15)
17- O- ( a - d -Mannopyranosyl)cholecalciferol 17- O - a (52) 26
17- O- ( b - d -Mannopyranosyl)cholecalciferol 17- O - b (48) 17- O -(1- d -Fructofuranosyl)cholecalciferol 17- O - a (42) 13
17- O -( b - d -Fructofuranosyl)cholecalciferol 17- O - b (58)
a Product proportions were determined from the area of respective 1H/13C signals b Conversion yields were from HPLC with respect to the carbohydrate. Error in yield measurements is ±5%
206 8 Glycosylation of Some Selected Phenols and Vitamins
circulation, liver diseases, peripheral blood cir-culation, thrombosis, drug poisoning, radiation damage, aging and carcinogenesis. Vitamin E is also used as a therapeutic agent against acute lung and aspirin-induced gastric mucosal inju-ries (Ochiai et al. 2002 ; Isozaki et al. 2005 ; Ichikawa et al. 2003 ) .
Vitamin E has been reported to exhibit poor water solubility, stability and absorbtivity. Glycosylation improves the pharmacological property by increasing the water solubility of vitamin E. Attachment of b -glucosyl, b -malto-syl and b -oligomaltosyl units via 6-OH group of a -tocopherol (Trolox) was achieved by Lahman and Thiem ( 1997 ) . One-step enzymatic glycosylation is useful for the preparation of glycosides rather than chemical glycosylation, which requires a large number of protection–deprotection steps. Water-soluble a -tocopherol derivatives 2,5,7,8-tetramethyl-2-(4-methyl-pentyl)chroman-6-yl- b - d -glucopyranoside and 2,5,7,8-tetramethyl-2-(4methyl pentyl)chro-man-6-yl-6- O - b - d -glucopyranosyl- b - d -glu-copyranoside using cultured plant cells of Phytolacca americana and Catharanthus roseus (Hamada et al. 2002 ; Kondo et al. 2006 ; Shimoda et al. 2006 ) were prepared. Enzymatic glycosylation of vitamin E using an a -glucosi-
dase from Saccharomyces sp. (Murase et al. 1997 ) showed that the glycosylated product is water-soluble (>1 g mL −1 ) and its free radical scavenging activity is similar to that of a -tocopherol.
Herein presented is the preparation of the much desired water-soluble a -tocopherol glyco-sides (Scheme 8.15 , Ponrasu et al. 2008 ) .
Syntheses of a -tocopherol glycosides involved carrying out reactions under a nitrogen atmosphere. The reaction residues were subjected to HPLC analysis (Fig. 8.18 ): a -tocopherol, 4.3 min; d -glu-cose, 7.5 min; d -galactose, 7.1 min; d -mannose, 6.7 min; 6- O -( d -glucopyranosyl) a -tocopherol, 9.3-min; 6- O -( d -galactopyranosyl) a -tocopherol, 10.1-min; and 6- O -( d -mannopyranosyl) a -tocopherol, 9.1-min.
8.2.7.1 Synthesis of 6- O -( b - D -Glucopyranosyl) a -Tocopherol Using b -Glucosidase
Optimal conditions for the reaction are as follows: conversion yields increased with increasing incubation periods from 3 to 72 h and decreased at 96 h of incubation period (Table 8.37 , Fig. 8.19a ); conversion yield was the highest at pH 6, being 24%, maximum yield of 23% at 0.1-mM (1-mL) buffer
Scheme 8.15 Syntheses of a -tocopheryl glycosides
2078.2 Vitamins
Table 8.37 Optimisation of reaction conditions for the synthesis of 6- O -( b - d -glucopyranosyl) a -tocopherol using b -glucosidase
Reaction conditions Variable parameter b Conversion yields (%) c
Incubation period (h)
a -Tocopherol – 0.5 mmol a 3 17
d -Glucose – 0.5 mmol 6 17 pH – 6 12 16 Buffer concentration – 0.1 mM (1 mL) 24 16
b -Glucosidase – 40% w/w d -glucose 48 18
72 22 96 20
120 17 pH (0.01 M)
a -Tocopherol – 0.5 mmol 4 18
d -Glucose – 0.5 mmol 5 20
b -Glucosidase – 40% w/w d -glucose 6 24
Buffer concentration – 0.1 mM (1 mL) 7 20 Incubation period – 72 h 8 17
Buffer concentration (mM)
a -Tocopherol – 0.5 mmol 0.05 21
d -Glucose – 0.5 mmol 0.1 23
b -Glucosidase – 40% w/w d -glucose 0.15 19
pH – 6 0.2 13 Incubation period – 72 h 0.25 8
b -Glucosidase concentration (% w/w d -glucose)
a -Tocopherol – 0.5 mmol 10 10
d -Glucose – 0.5 mmol 20 18 pH – 6 30 17 Buffer concentration – 0.1 mM (1 mL) 40 23 Incubation period – 72 h 50 8
75 7
a -Tocopherol (mmol) pH – 6 0.25 18 Buffer concentration – 0.1 mM (1 mL) 0.5 21 d -Glucose – 0.5 mmol 0.75 17
b -Glucosidase – 40% w/w d -glucose 1 15
Incubation period – 72 h 1.5 15 2 15 2.5 16
a Initial reaction conditions b Other variables are the same as under reaction conditions, except the speci fi ed ones c HPLC yields expressed with respect to 0.5-mmol d -glucose employed
concentration (Table 8.37 , Fig. 8.19b ); 40% (w/w d -glucose) b -glucosidase at 0.1-mM (1-mL) pH 6 buffer gave the maximum con-version yield of 23%; and b -glucosidase gave the highest conversion yield of 21% at 0.5-mmol a -tocopherol.
8.2.7.2 Solubility of 6- O -( D -Glucopyranosyl) a -Tocopherol
Determination of water solubility of 6- O -( d -glucopyranosyl) a -tocopherol showed that it is soluble to the extent of 25.9 g L −1 . Hence, a water-soluble 6- O -( d -glucopyranosyl) a -tocopherol
208 8 Glycosylation of Some Selected Phenols and Vitamins
was prepared in the present work from water-insoluble a -tocopherol.
8.2.7.3 Syntheses of a -Tocopheryl Glycosides of Other Carbohydrates Using b -Glucosidase and Their Spectral Characterisation
Syntheses of the other a -tocopheryl glycosides were carried out at the above-determined opti-mised conditions, with a -tocopherol and carbo-hydrates: d -glucose, d -galactose and d -mannose (Table 8.38 ).
Ultraviolet-visible spectra of a -tocopheryl glycosides showed s → s * band in the 193–
198.5-nm (199 nm for a -tocopherol) range, s → p * band in the 223–224.5-nm (228 nm for a -tocopherol) range and p → p * band in the 270.5–273.5-nm (292 nm for a -tocopherol) range; IR spectra showed 1,028–1,084-cm −1 range band for the glycosidic C–O–C aryl–alkyl symmetrical stretching and 1,259–1,260-cm −1 range band for the asymmetrical stretching fre-quencies indicating that a -tocopherol had under-gone glycosylation. NMR and Mass data con fi rmed the product formation. The phenolic carbon chemical shift value at 150.5 ppm (145.4 ppm for free a -tocopherol) indicated that glucosylation occurred at the phenolic OH group of a -tocopherol.
0
5
10
15
20
25
0 30 60 90 120
Incubation period (h)
Con
vers
ion
yiel
d (%
)
0
5
10
15
20
25
30
0.05 0.1 0.15 0.2 0.25
Buffer concn (mM)
Con
vers
ion
yiel
d (%
)
a
b
Fig. 8.19 ( a ) Reaction pro fi le for 6- O -( b - d -glucopyranosyl) a -tocoph-erol synthesis by the re fl ux method. Conversion yields were from HPLC with respect to 0.5 mmol of d -glucose. Reaction conditions: d -glucose, 0.5 mmol; a -tocopherol, 0.5 mmol; b -glucosidase, 40% (w/w d -glucose); 0.1-mM (1-mL) pH 6 phosphate buffer; solvent, di-isopropyl ether; and temperature, 68°C. ( b ) Effect of buffer concentra-tion for 6- O -( b - d -glucopyranosyl) a -tocoph-erol synthesis. Reaction conditions: d -glucose, 0.5 mmol; a -tocopherol, 0.5 mmol; b -glucosidase, 40% (w/w d -glucose); pH 6 phosphate buffer; solvent, di-isopropyl ether; temperature, 68°C; and incubation period, 72 h
209References
References
Akao T, Yoshino T, Kobashi K, Hatlori M (2002) Evaluation of salicin as an antipyretic prodrug that does not cause gastric injury. Planta Med 68:714–718
Andreotti G, Giordano A, Tramice A, Mollo E, Trincone A (2006) Hydrolyses and transglycosylations per-formed by puri fi ed a -D-glucosidase of the marine mollusk Aplysia fasciata. J Biotechnol 122:274–284
Andrews RS, Pridham JB (1965) Structure of dopa gluco-side from Vicia faba . Nature 205:1213–1214
Angerlacenci M (2007) Dopamine dysregulation of movement control in L-dopa induced dyskinesia. Trends Neurosci 30(5):236–243
AOAC Of fi cial Methods of Analysis (1995) Chapter 46. 16th edn, vol II, p 12
Asano Y, Wada K (2003) Regioselective glucosylation of pyridoxine by microorganisms. Biosci Biotechnol Biochem 67:499–507
Audus KL, Chikhale PJ, Miller WD, Thompson SE, Borchardt RT (1992) Brain uptake of drugs. The in fl uence of chemicals and biological factors. Adv Drug Res 23:1–64
Table 8.38 Syntheses of a -tocopheryl glycosides using b -glucosidase
Glycosides b -Glucosidase catalysis a
Product (% proportion) b Yields (%) c
O
OH
HH
OH
H
OH
HCH2OH
O
CH3CH3CH3
CH3
CH3
CH3
CH3 CH3
O
C1 b -glucoside 23
6- O -( b - d -Glucopyranosyl) a -tocopherol
O
OH
HH
OH
H
OH
HCH2OH
O
CH3CH3CH3
CH3
CH3
CH3
CH3 CH3
O
C1 a -galactosides (41), C1 b -galactosides (59)
11
O
H
OHH
H
H
OH
OHCH2OH
O
CH3CH3CH3
CH3
CH3
CH3
CH3 CH3
O
6- O -( a - d -Galactopyranosyl) a -tocopherol
6- O -( b - d -Galactopyranosyl) a -tocopherol
O
H
OHH
H
H
OH
OHCH2OH
O
CH3CH3CH3
CH3
CH3
CH3
CH3 CH3
O
C1 a -mannoside (46), C1 b -mannoside (54)
18
O
H
OHH
H
H
HO
HOCH2OH
O
CH3 CH3 CH3
CH3
CH3
CH3
H3C CH3
O
6- O -( a - d -Mannopyranosyl) a -tocopherol
6- O -( b - d -Mannopyranosyl) a -tocopherol
a a -Tocopherol, 0.5 mmol; carbohydrate, 0.5 mmol; b -glucosidase concentration 40% (w/w d -glucose); solvent, di-isopropyl ether; buffer, 0.1-mM (1-mL) pH 6 phosphate buffer; incubation period, 72 h b The product proportions were determined from the area of respective 1 H/ 13 C signals c Conversion yields were from HPLC with respect to free carbohydrate. Error in yield measurements is ±5–10%
210 8 Glycosylation of Some Selected Phenols and Vitamins
Bacher A, Eberhardt S, Fischer M, Kis K, Richter G (2000) Biosynthesis of vitamin B 2 (ribo fl avin). Annu Rev Nutr 20:153–167
Bates C (1993) Ribo fl avin. Int J Vitam Nutr Res 63:274–277
Berger M, Gray JA, Roth C (2009) The expanded biology of serotonin. Ann Rev Med 60:355–366
Bhabadesh C, Somnath M, Dehdas B, De Amit K (1996) Capsaicin a unique antioxidant, antifungal, anti-in fl ammatory analgesic compound with antifungal activity against dermatophytes. Med Sci Res 24:669–670
Burri J, Graf M, Lambelet P, Loliger J (1999) Vanillin more than a fl avoring agent – a potential antioxidant. J Sci Food Agric 48:49–56
Burton GW, Joyce A, Ingold KU (1983) Is vitamin E the only lipid soluble, chain-breaking antioxidant in human blood plasma and erythrocyte membrane. Arch Biochem Biophys 221:281–290
Chahid Z, Montet D, Pina M, Graille J (1992) Effect of water activity on enzymatic synthesis of alkylglyco-sides. Biotechnol Lett 14(4):281–284
Chen CW, Ou-Yang C-C, Yeh C-W (2003) Synthesis of galactooligosaccharides and transgalactosylation modeling in reverse micelles. Enzyme Microb Technol 33:497–507
Chen IJ, Yang JM, Yeh JL, Wu BN, Lo YC, Chen SJ (1992) Hypotensive and antinociceptive effects of ether linked and relatively non pungent analogs of N-nonanoyl-vanillylamide. Eur J Med Chem 27:187–192
Chiba S (1997) Molecular mechanism in a -glucosidase and glucoamylase. Biosci Biotech Biochem 61:1233–1239
Clark AM (1996) Natural products as a resource for new drugs. Pharm Res 13:1133–1141
Dalpiaz A, Filosa R, de Caprariis P, Conte G, Bortolotti F, Biondi C, Scatturin A, Prasad PD, Pavanc B (2007) Molecular mechanism involved in the transport of a prodrug dopamine glycosyl conjugate. Int J Pharm 336:133–139
Dickerson TJ, Yamamoto N, Janda Kim D (2004) Antibody-catalysed oxidative degradation of nicotine using ribo fl avin. Bio Org Med Chem 12:4981–4987
Du Y, Wei G, Linhardt RJ (2004) Total synthesis of quer-cetin 3-sophorotrioside. J Org Chem 69:2206–2209
Einstein Charles R, Divakar S (2009) b -Glucosidase cata-lyzed syntheses of pyridoxine glycosides. Biosci Biotech Biochem 73:233–236
Einstein Charles R, Ponrasu T, Udaya Sankar K, Divakar S (2009a) Syntheses of retinol glycosides using b -glu-cosidase in SCCO2 media. Appl Biochem Biotech 159:199–207
Einstein Charles R, Ponrasu T, Sivakumar R, Divakar S (2009b) Angiotensin converting enzyme inhibitory and antioxidant activities of enzymatically synthesized phenolic and vitamin glycosides. Biotech Appl Biochem 52:177–184
Feldman D, Xiao-Yan Z, Krishnan AV (2000) Vitamin D and prostate cancer. Endocrinology 141:5–9
Fernandez C, Nieto O, Fontenla JA, Rivas E, de Ceballos ML, Fernandez-Mayoralas A (2003) Synthesis of glycosyl
derivatives as dopamine prodrugs Interaction with glucose carrier GLUT-1. Org Biomol Chem 1:767–771
Fitzgerald DJ, Stratford M, Gasson MJ, Narbad A (2005) Structure function analysis of the vanillin molecule and its antifungal properties. J Agric Food Chem 53:1769–1775
Frandsen TP, Dupont C, Lehmbeck J, Stoffer B, Sierks MR, Honzatko RB, Svensson B (1994) Site-directed mutagenesis of the catalytic base Glutamic acid 400 in glucoamylase from Aspergillus niger and of Tyrosine 48 and Glutamine 401, both hydrogen bonded to the gamma-carboxylate group of Glutamic acid 400. Biochemistry 33:13808–13816
Gannett PM, Nagel DL, Reilly PJ, Lawson T, Sharpe J, Toth B (1988) The capsaicinoids their separation, syn-thesis and mutagenicity. J Org Chem 53:1064–1071
Geng X, Song L, Pu X, Tu P (2004) Neuroprotective effects of phenylethanoid glycosides from Cistanches salsa against 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic toxicity in C57 Mice. Biol Pharm Bull 27(6):797–801
Geng X, Tian X, Tu P, Pu X (2007) Neuroprotective effects of echinacoside in the mouse MPTP model of Parkinson’s disease. Eur J Pharmacol 564:66–74
Giri A, Dhingra V, Giri CC, Singh A, Ward OP, Narasu ML (2001) Biotransformations using plant cells, organ cultures and enzyme systems current trends and future prospects. Biotechnol Adv 19:175–199
Gomes DCF, Alegrio LV, Leon LL, de Lima MEF (2002) Total synthesis and anti-leishmanial activity of some curcumin analogues. Arzneim-Forsch 52:695–698
Guggenheim M (1913) Dioxyphenylalanine neue Aminosaureaus Vicia faba . Hoppe-Seylers’s Zeitschr Physiol Chem 88:276–284
Hamada H, Tomi R, Asada Y, Furuya T (2001) Phytoremediation of bisphenol A by cultured suspen-sion cells of Eucalyptus perriniana regioselective hydroxylation and glycosylation. Tetrahedron Lett 43:4087–4089
Hamada H, Nishida K, Furuya T, Ishihara K, Nakajima N (2002) Preparation of a new pepper chemoenzymatic synthesis of capsaicin oligosaccharide and 8-nordihy-drocapsaicin. J Mol Catal B: Enzym 16:115–119
Hamada H, Ohiwa S, Nishida T, Katssuragi H, Takeda T, Hamada H, Nakajima N, Ishihara K (2003) One step glucosylation of capsaicinoids by cultured cells of Phytolacca americana . Plant Biotechnol 20:253–255
Hausslera MR, Wasserman RH, McCaina TA, Peterlik M, Bursaca KM, Hughesa MR (1976) 1,25-Dihydroxyvitamin D3-glycosides Identi fi cation of a calcinogenic principle of Solanum malacoxylon . Life Sci 18(10):1049–1056
Heyes Z, Szabo A, Nemeth J, Jakab B, Pinter E, Banvolgyl A, Kereskai L, Keri G, Szolcsanyl J (2004) Antii fl ammatory and analgesic effects of somatostatin released from chronic arthritis model in the rat. Arthritis Rheum 50:1677–1685
Holick MF (2004) Sunlight and vitamin D for bone health and prevention of autoimmune diseases, cancers, and cardiovascular disease. Am J Clin Nutr 6:1678S–1688S
211References
Houghton LA, Vieth R (2006) The care against ergocalcif-erol (vitamin D2) as a vitamin supplement. Am J Clin Nutr 84:694–697
Hoyer D, Martin G (1997) 5-HT receptor classi fi cation and nomenclature towards a harmonization with the human genome. Neuropharmacology 36(4–5):419–428
Ichikawa H, Yoshida N, Takano H, Ishikawa T, Handa O, Takagi T, Nito Y, Murase H, Yoshikawa T (2003) A novel water-soluble vitamin E derivative (TMG) pro-tects against gastric mucosal damage induced by isch-emic and reperfusion in rats. Dig Sci 48(1):54–58
Iorizzi M, Lanzotti V, De Marino S, Zollo F, Blanco-Molina M, Macho A, Munoz E (2001) New glycosides from Capsicum annum L. Var. accuminatum . Isolation, structure determination and biological activity. J Agric Food Chem 49:2022–2029
Isozaki Y, Yoshida N, Ichikawa H, Kuroda M, Kokura S, Naito Y, Okanoue T, Yoshikawa T (2005) A novel water-soluble vitamin E derivative protect against aspirin-induced gastric mucosal injury in rats. Int J Mol Med 16(6):1035–1040
James SY, Williams MA, Newland AC, Colston KW (1999) Leukemia cell differentiation cellular and molecular interactions of retinoids and vitamin D. Gen Pharmacol 32:143–154
Jones BJ, Blackburn TP (2002) The medical bene fi t of 5-HT research. Pharmcol Biochem Behav 71(4):555–681
Kaljuzhin OV, Shkalev MV (2000) Immunomodulator and pharmaceutical compositions with antitumor properties, and a food additive. Patent EP1038532 (CA 129 335732)
Kaminaga Y, Nagatsu A, Akiyama T, Sugimoto N, Yamazaki T, Maitani T, Mizukami H (2003) Production of unnatural glucosides of curcumin with drastically enhanced water solubility by cell suspension cultures of Catharanthus roseus . FEBS Lett 555:311–316
Kang K, Park S, Kim YS, Lee S, Back K (2009) Biosynthesis and biotechnological production of serotonin deriva-tives. Appl Micorb Biotechnol 83(1):27–34
Katrien T, Miloslav P, Sophie S, Guy E, Yvette M (1997) New antioxidant mixture for long term stability of serotonin, dopamine and their metabolites in auto-mated microbore liquid chromatography with dual electrochemical detection. J Chromatograph B Biomed Sci Appl 694(2):297–303
Kawada T, Watanabae T, Takahashi T, Tanaka T, Iwai K (1986) Effects of capsaicin on lipid metabolism in rats fed a high fat diet. J Nutr 116:1272–1278
Kawai F, Yamada H, Ogata K (1971a) Properties of pyri-doxine glucoside. J Vitaminol 17:121–124
Kawai F, Yamada H, Ogata K (1971b) Studies on trans-glycosidation to vitamin B 6 by microorganisms Part IV. Puri fi cation of a bacterial enzyme catalyzing pyridoxine glucoside synthesis. Agric Biol Chem 35:184–190
Kempster PA, Bogetic Z, Secombei JW, Martin HD, Balazs NDH, Wahlquist MC (1993) Motor effect ofbroad beans Vicia faba in Parkinson’s disease Single dose studies. Asia Paci fi c J Clin Nutr 2:85–89
Kometani T, Tanimoto H, Nishimura T, Kanbara I, Okada S (1993a) Glucosylation of capsaicin by cell suspen-sion cultures of Coffea arabica . Biosci Biotech Biochem 57:2192–2193
Kometani T, Tanimoto H, Nishimura T, Okada S (1993b) Glucosylation of vanillin by cultured plant cells. Biosci Biotech Biochem 57:1290–1293
Kondo Y, Shimoda K, Takimura J, Hamada H, Hamada H (2006) Glycosylation of vitamin E homologue by cul-tured plant cells. Chem Lett 35:324–326
Kren V (2001) Chemical biology and biomedicine of gly-cosylated natural compounds. In: Fraser-Reid B, Tatsuta K, Thiem J (eds) Glycoscience chemistry and chemical biology. Springer, Berlin, 3: 2471-2529
Kren V, Martinkova L (2001) Glycosides in medicine: the role of glycosidic residue in biological activity. Curr Med Chem 8:1303–1328
Kren V, Hunkova Z, Halada P, Suzuki Y (1998) Transglycosylation of thiamine by fungal b -N-Acetyl-hexosaminidases. Biosci Biotechnol Biochem 62(12):2415–2417
Lahmann M, Thiem J (1997) Synthesis of a -tocopheryl oligosaccharides. Carbohydr Res 299:23–31
Lan T, Dalip R, Errol MY (2004) Isolation and puri fi cation of the hypoglycemic principle present in Cap. frute-scens. Phytotherap Res 18:95–96
LeBlane JG, Burgees C, Sesma F, de Giori GS, Van Sinderen D (2005) Lactococcus lactis is capable of improving the ribo fl avin status in de fi cient rats. British J Nutr 94:262–267
Lee Y, Howard LR, Villalon B (1995) Flavonoids and anti-oxidant activity of fresh pepper ( C. annum ) cultivars. J Food Sci 60:473–476
Lehninger LA (1977) Biochemistry, 3rd edn. Worth Publishers Inc., New York, pp 355–357
Lopez-Malo A, Alzamora SM, Argaiz A (1998) Vanillin and pH synergistic effects on mold growth. J Food Sci 63:143–146
Madrid Y, Langer LF, Brem H, Langer R (1991) New directions in the delivery of drugs and other substances to the central nervous system. Adv Pharmacol 22:299–324
Manohar B, Divakar S (2010) Enzymatic synthesis of cholecalciferol glycosides. J Food Sci Technol 46(5):469–475
Marieb EN (2009) Essentials of human anatomy and physiology, 8th edn. Pearson/Benjamin Cummings, San Fransisco, p 336. ISBN 321-51342-8
Marty A, Chulalaksananukul W, Condoret JS, Willemont RM, Durand G (1990) Comparison of lipase-catalyzed esteri fi cation in supercritical carbon dioxide and n-hexane. Biotechnol Lett 12(1):11–16
Matondo RB, Punt C, Homberg J, Toussaint MJ, Kisjes R, Korporaal SJ, Akkerman JW, Cuppen E, de Bruin A (2009) Deletion of the serotonin transporter in rats dis-turbs serotonin homeostasis without impairing liver regeneration. Am J Physiol Gastrointest Liver Physiol 296(4):G963–G968
Maurizio C, Kati O, Maria O, Sabrina P, Bruno S (2007) Vitamin D in rheumatoid arthritis. Autoimmun Rev 7:59–64
212 8 Glycosylation of Some Selected Phenols and Vitamins
Mello JRB (2003) Calcinosis-calcinogenic plants. Toxicon 41:1–12
Mohri K, Watanabe Y, Yoshida Y, Satoh M, Isobe K, Sugimoto N, Tsuda Y (2003) Synthesis of glycosyl-curcuminoids. Chem Pharm Bull 51:1268–1272
Moon JH, Terao J (1998) Antioxidant activity of caffeic acid and dihydrocaffeic acid in Lard and human low-density lipoprotein. J Agri Food Chem 46: 5062–5065
Morre DJ, Chueh PJ, Morre DM (1995) Capsaicin inhibits preferentially the NADH oxidase and growth of trans-formed cells in culture. Proc Natl Acad Sci USA 92:1831–1835
Murase H, Yamauchi R, Kato K, Kunieda T, Terao J (1997) Synthesis of a novel vitamin E derivative, 2-( a -D-glucopyranosyl) methyl-2,5,7,8-tetramethyl chroma- 6-ol transglycosylation. Lipids 32:73–78
Nagasawa T, Takagi H, Kawakami K, Suzuki T, Shahaghi Y (1961) The browning compounds of bean III isola-tion of dopa-O- b -D-glucoside and the enzymic mech-anism for the color change of broad bean. Agric Biol Chem 25:441–447
Nsoukpoe-Kossi CN, Sedaghat-Herati R, Ragi C, Hotchandani S, Tajmir-Riahi HA (2007) Retinol and retinoic acid bind human serum albumin: stability and structural features. Int J Biol Macromol 40:484–490
Ochiai J, Takano H, Ichikawa H, Naito Y, Yoshida N, Yanagisawa R, Yoshino S, Murase H, Yoshikawa T (2002) A novel water-soluble vitamin E derivative, 2-( a -D-glucopyranosyl)methyl-2,5,7,8-tetramethyl-chroman-6-ol, protects against acute lung injury and mortality in endotoxemic rats. Int J Mol Med 18(6):580–584
Ohnuki K, Haramizu S, Oki K, Watanabe T, Yazawa S, Fushiki T (2001) Administration of capsiate, a non-pungent capsaicin analog, promotes energy metabo-lism and suppresses body fat accumulation in mice. Biosci Biotechnol Biochem 65:2735–2740
Ong DE, Newcomer ME, Chytil F (1994) Biology chem-istry and medicine. In: Sporn MB, Roberts AB, Goodman DS (eds) The retinoids. Raven Press Ltd., New York, pp 283–318
Ooi Y, Hashimoto T, Mitsuo N, Satoh T (1985) Enzymatic formation of b -galactosidase from Aspergillus oryzae and its application to the synthesis of chemically unstable cardiac glycosides. Chem Pharm Bull 33:1808–1814
Orihara Y, Furuya T, Hashimoto N, Deguchi Y, Tokoro K, Kanisawa T (1992) Biotransformation of isoeugenol and eugenol by cultured cells of Eucalyptus perrini-ana . Phytochemistry 31:827–831
Pardridge WM (2002) Targeting neurotherapeutic agents through the blood–brain barrier. Arch Neurol 59:35–40
Peehl DM, Krishnan AV, Feldman D (2003) Pathways mediating the growth-inhibitory actions of vitamin D in prostate cancer. J Nutr 133:2461–2469
Ponrasu T, Charles RE, Sivakumar R, Divakar S (2008) Syntheses of a -tocopheryl glycosides by glucosidases. Biotechnol Lett 30:1431–1439
Ponrasu T, Manohar B, Divakar S (2009) A response surface methodological study on prediction of gluco-sylation yields of thiamin using immobilized b - glu-cosidase. Process Biochem 44:251–255
Powers HJ (2003) Ribo fl avin and health. Am J Clin Nutr 77:1352–1360
Pras N, Woerdenbag J, Van Uden W (1995) Bioconversion potential of plant enzymes for the production of phar-maceuticals. Plant Cell Tissue Org Cult 43:117–121
Rashid MH, Inoue M, Kondo S, Kawashima T, Bakoshi S, Ueda H (2003) Novel expression of vanilloid receptor I on capsaicin-insensitive fi bres accounts for the anal-gesic effect of capsaicin in neuropathic pain. J Pharmacol Exp Ther 304:940–948
Reichel L, Sckickle R (1943) Synthesen neuer methoxyli-erter Chalkon-Flavanon-glucoside unter physiologis-chen Bedingungen. Chemie und Biochemie der P fl anzenstoffe, XII. Mitteilung Plant Chem 76B:1134–1137
Robyt JF (1998) Essentials of carbohydrate chemistry. Springer, New York, pp 64–68
Roode BM, Franssen MCR, van der Padt A, Boom RM (2003) Perspective for the industrial enzymatic produc-tion of glycosides. Biotechnol Prog 19:1391–1402
Russell AJ, Beckman EJ (1991) Should the high diffusiv-ity of a supercritical fl uid increase the rate of an enzyme-catalyzed reaction? Enzyme Microbiol Technol 13(12):1007–1007
Sasaki N, Adachi T, Koda T, Ozeki Y (2004) Detection of UDP-glucose Cyclo-dopa 5-O-glucosyltransferase activity in four O’ clocks ( Mirabilis jalapa L .). FEBS Lett 568:159–162
Sato T, Takeuchi H, Takahashi K, Kurosu J, Yoshida K, Tsugane T, Shimura S, Kino K, Kirimura K (2003) Selective a -glucosylation of eugenol by a -glucosyl transfer enzyme of Xanthomonas campestris WU-9701. J Biosci Bioeng 96:199–202
Satoh T, Miyataka H, Yamamoto K, Hirano K (2001) Synthesis and physiological activity of novel tocoph-eryl glycosides. Chem Pharm Bull 49(8):948–953
Schmid B, Kotter I, Heide L (2001) Pharmacokinetics of salicin after oral administration of a standard willow bark extract. Eur J Clinic Pharmacol 57:387–391
Scholz D, Mebel M, Topelmann J, Grossmann I, Scholze J, Mrochen H (1983) Prevention of osteonecrosis follow-ing renal transplantation by using vitamin D2 (ergocal-ciferol). Proc Eur Dial Transplant Assoc 20:331–337
Sengodan S, Palanivel S, Panchanadham S (2003) Curative effect of ribo fl avin, niacin and ascorbic acid on tamox-ifen mediated endometrial carcinoma bearing Sprage-Dawley rats with reference to lipid peroxidation and antioxidant status. J Clin Biochem Nutr 33:39–45
Shetty P, Atallah MT, Shetty K (2002) Effects of UV treat-ment on the proline-linked pentose phosphate pathway for phenolics and L-DOPA synthesis in dark germi-nated Vicia faba . Process Biochem 37:1285–1295
Shimoda K, Konda Y, Abe K, Hamada H, Hamada H (2006) Formation of water-soluble vitamin derivatives from lipophilic vitamins by cultured plant cells. Tetrahedron Lett 47:2695–2698
213References
Sierks MR, Ford C, Reilly PJ, Svensson B (1990) Catalytic mechanism of fungal glucoamylases as de fi ned by mutagenesis of Asp 176, Glu179, and Glu180 in the enzyme from Aspergillus awamori . Protein Eng 3:193–198
Sircar S (2007) Medical physiology. Thieme Publishing Group, Stuttgart, p 536. ISBN 3-13-144061-9
Sivakumar R, Divakar S (2006) Glycosylation of vanillin by amyloglucosidase in organic media. Tetrahedron Lett 47:695–699
Sivakumar R, Divakar S (2007) Syntheses of N-vanillyl-nonanamide glycosides using amyloglucosidase from Rhizopus mold and b -glucosidase from sweet almond. Biotechnol Lett 29:1537–1548
Sivakumar R, Divakar S (2009a) Enzymatic syntheses of dopamine glycosides. Enzyme Microb Technol 44:33–39
Sivakumar R, Divakar S (2009b) Amyloglucosidase from Rhizopus mold and b - glucosidase from sweet almond catalysed syntheses of ribo fl avinyl glycosides. Indian J Biotechnol 8:400–407
Sivakumar R, Manohar B, Divakar S (2006a) Synthesis of vanillyl-maltoside using glucosidases by response sur-face methodology. Eur Food Res Technol 226:255–263
Sivakumar R, Vijayakumar GR, Manohar B, Divakar S (2006b) Competitive substrate inhibition of amyloglu-cosidase from Rhizopus mold by vanillin and curcumin in respective glucosylation reactions. Biocatal Biotrans 24:299–305
Sivakumar R, Ponrasu T, Divakar S (2009) Syntheses of DL-dopa glycosides using glucosidases. Glycoconj J 26:199–209
Smith DC, Johnson CS, Freeman CC, Muindi J, Wilson JW, Trump DL (1999) A phase I trial of calcitriol (1,25-dihydroxycholecalciferol) in patients with advanced malignancy. Clin Cancer Res 5:1339–1345
Sommer J, Schroeder C, Stockigt J (1997) In vivo forma-tion of vanillin glucoside. Plant Cell Tiss Org Cult 50:119–123
Sophie L, Robert G, Pierre K (2004) A new method of solvent free O – and N – glycosilation using activated carbon fi bers (ACF) as a promoter. Applications to the synthesis of saponin and nucleoside analogues. Chem Commun 5:586–587
Stephan D, Peter K (2003) Vanillins – a novel family of DNA-PK inhibitors. Nucleic Acid Res 31:5501–5512
Studart MT, Raschle T, Forster G, Amrhein N, Fitzpatrick TB (2005) Vitmin B6 biosynthesis in higher plants. Proc Natl Acad Sci USA 102:13687–13692
Suzuki Y, Uchida K (1969) Biosynthesis of ribo fl avin- a -glucoside by plant grains. Arch Biochem Biophys 130(1):683–684
Suzuki Y, Uchida K (1983) Formation of glucosyl ribo fl avin in germinating barley seeds. Nogaku Kenkyu 9:285–292
Suzuki Y, Uchida K (1994) Enzymatic formation of a new derivative of thiamin, b -galactosylthiamin. Biosci Biotech Biochem 58:1273–1276
Suzuki Y, Ki YH, Uchida K, Takami M (1996) Enzymatic synthesis of glucosylated and phosphatidylated bio-logically active compounds. J Appl Glycosci 43:273–282
Tachibana S (1955) Sugar compounds of ribo fl avin II Biosynthesis of ribo fl avinyl galactoside by Aspergillus oryzae . Vitamins 9:119–124
Tachibana S (1971) Isolation of ribo fl avin glycosides. In: Vitamin and coenzymes. McCormick CDB, Wright LD (eds) Methods in enzymology. Academic Press, New York, 18B: 413–416
Tai CY, Huang SC, Huang MS, Liu HS (2001) Hydrolysis of amylopectin by isoamylase under supercritical car-bon dioxide. J Chin Inst Chem Eng 32(3):269–275
Tapavicza SV, Bell D, Kopp-Holtwiesche B (2000) Plant growth enhancement against phytopathogenic fungi and/or soil borne pests. Patent WO 0002451 (CA 132)
The Merck Index (2006) In: Neil MJO (ed) An encyclope-dia of chemicals, drugs and biologicals, 14th edn. Merck and Co., Whitehouse Station
Tietze LF, Griesbach U, Schuberth I, Bothe U, Marra A, Dondoni A (2003) Novel carboranyl C-glycosides for the treatment of cancer by boron neutron capture ther-apy. Chem Eur J 9:1296–1302
Uchida K, Suzuki Y (1968) Production of ribo fl avin glu-coside in growing cultures of Ashbya gossypii and Eremothecium ashbyii . Denpon Nogei Kagaku Kaishi 42(4):233–237
Vadivelan G, Divakar S (2011a) Enzymatic synthesis of L-Dopa glycosides. Indian J Chem Sect B 50B:1779–1785
Vadivelan G, Divakar S (2011b) Enzymatic Synthesis of serotonyl and epinephryl glycosides. Int J Fund Appl Life Sci 1(2):15–21
Vered Y, Rabey JM, Paleveitch D, Grosskopf I, Harsat A, Yanowski A, Shabtai H, Graff E (1994) Bioavailability of levodopa after consumption of Vicia faba seedlings by Parkinsonian patients and control subjects. Clin Neuropharmacol 17:138–146
Vic G, Thomas D (1992) Enzyme-catalyzed synthesis of alkyl- b -D-glucosides in organic media. Tetrahedron Lett 33:4567–4570
Vic G, Thomas D, Crout DHG (1997) Solvent effect on enzyme-catalyzed synthesis of b -D-glucosides using the reverse hydrolysis method application to the pre-parative-scale synthesis of 2-hydroxybenzyl and octyl b -D-glucopyronosides. Enzyme Microb Technol 20:597–603
Vijayakumar GR, Divakar S (2005) Synthesis of guaiacol- a -D-glucoside and curcumin- bis- a -D-glucoside by an amyloglucosidase from Rhizopus . Biotechnol Lett 27:1411–1415
Vijayakumar GR, Divakar S (2007) Amyloglucosidase catalyzed synthesis of eugenyl and curcuminyl glyco-sides. Biotechnol Lett 29:575–584
Vijayakumar GR, Manohar B, Divakar S (2006) Amyloglucosidase catalyzed synthesis of curcumin-bis- a -D-glucoside-A response surface methodological study. Eur Food Res Technol 223:725–730
Vijayakumar GR, George C, Divakar S (2007) Synthesis of n-alkyl glucosides by amyloglucosidase. Ind J Chem B 46B:314–319
Walker J, Alsha fi e G, Abou-Issa H, Curley R Jr (2002) An improved synthesis of the C-linked glucuronide of
214 8 Glycosylation of Some Selected Phenols and Vitamins
N-(4-hydroxyphenyl) retinamide. Bioorg Med Chem Lett 12:2447–2450
Watanabae T, Kawada T, Kato T, Harada T, Iwai K (1994) Effects of capsaicin analogs on adrenalin cate-cholamine secretion in rats. Life Sci 54:369–374
Werner R, Manthey KC, Grif fi n JB, Zempleni J (2005) HepG2 cells develop signs of ribo fl avin de fi ciency within 4 days of culture in ribo fl avin-de fi cient medium. J Nutr Biochem 16:617–624
Whitby LG (1971)Glycosides or ribo fl avin. In: Vitamin and coenzymes. McCormick CDB, Wright LD (eds) Methods in enzymology. Academic Press, New York, 18B:404–413
Wieder R, Novick SC, Hollis BW, Bryan M, Chanel SM, Owusu K, Camastra D, Saunders T, Pliner L, Harrison J, Bonate P, Williams T, Soignet S (2003) Pharmacokinetics and safety of ILX23-7553, a non-calcemic-vitamin D 3 analogue, in a phase I study of
patients with advanced malignancies. Invest New Drugs 21:445–452
Wyler H, Meuer U, Bauer J, Stravs-Mombelli L (2004) Cyclodopa glucoside ((2S)-5-( b -D-glucopyranosyloxy)-6-hydroxyindoline-2-carboxylicacid) and its occurrence in red beet ( beta Vulgaris Varnn rubra L .). Helvetica Chim Acta 67(5)):1348–1355
Yamada H, Aimi Y, Nagatsu I, Taki K, Kudo M, Arai R (2007) Immunohistochemical detection of L-dopa derived dopamine within serotonergic fi bres in the striatum and the substantia nigra pars reticulata in Parkinsonian model rats. Neurosci Res 59:1–7
Yar MD (1993) Parkinson’s disease, the L-dopa era. Adv Neurobiol 60:11–17
Zhou JH (2000) Herbal sweetening and preservative com-position comprising licorice extract and mogrosides obtained from plants belonging to cucurbitaceae and/or momordica. Patent US 6103240 (CA 133 168393)
215S. Divakar, Enzymatic Transformation, DOI 10.1007/978-81-322-0873-0_9, © Springer India 2013
9.1 General
Glycosylation potentialities of amyloglucosi-dase from Rhizopus sp., b -glucosidase from sweet almond and calcium alginate bead-entrapped b -glucosidase were explored in detail in the synthesis of a wide variety of carbohydrate acceptor molecules such as alcohols (alcohols of carbon chain length C1–C18), phenols (guaiacol, eugenol, curcumin, vanillin, N-vanillyl-nonanamide, capsaicin, l -dopa, dl -dopa, dop-amine, serotonin and epinephrine) and vitamins (retinol (vitamin A), thiamin (vitamin B1), ribo fl avin (vitamin B2), pyridoxine (vitamin B6), ergocalciferol (vitamin D2), cholecalciferol (vitamin D3) and a -tocopherol (vitamin E)). The donor carbohydrates include aldohexoses ( d -glucose, d -galactose and d -mannose), keto-hexose ( d -fructose), pentoses ( d -arabinose and d -ribose), disaccharides (maltose, sucrose and lactose) and carbohydrate alcohols ( d -mannitol and d -sorbitol).
A novel experimental set-up was developed for even large-scale synthesis of glycosides using lesser enzymes and larger concentrations of sub-strates to give higher yields. This set-up involved re fl uxing the reaction mixture containing the
alcohol/phenol/vitamin and carbohydrate with appropriate concentrations of amyloglucosidase and b -glucosidase (native/immobilised) in the presence of speci fi ed pH and buffer concentra-tions in 100 mL of di-isopropyl ether solvent at 68°C for a speci fi ed incubation period. This set-up gave higher yields and selectivity than the conventional shake- fl ask experiments.
Optimisation of reaction parameters for the synthesis of above-mentioned phenolic and vita-min glucosides was carried out in terms of incu-bation period, pH, buffer, enzyme and phenol/vitamin concentrations.
The glycosylation yields were better with phe-nols compared to those with the vitamins employed. Vitamins are not better nucleophiles compared to the phenols employed. Amyloglucosidase from Rhizopus mould gave higher yield with lesser selec-tivity, and b -glucosidase from sweet almond gave lesser yield and greater selectivity. In general, con-version yields ranging from 5 to 62% for amyloglu-cosidase catalyses and 7 to 65% for b -glucosidase (native/immobilised) catalyses were obtained for various phenols/vitamins. Invariably dl -dopa and dopamine gave high yields with less regioselectivity with both the glucosidases employed. Loss of regi-oselectivity in many of the glycosylation/arylation
9
Abstract
The reactions were discussed in this chapter in terms of incubation period, pH, buffer, enzyme and substrate concentrations, regio- and stereoselectivity.
Glycosylation of Phenols and Vitamins: An Overview
216 9 Glycosylation of Phenols and Vitamins: An Overview
reactions could be due to the employment of large amount of the enzymes. This is inevitable, as the reversible reaction requires such large concentra-tions of enzymes for conversions.
Phenols employed for the glycosylation pos-sesses 3,4-dihydroxy phenyl derivatives, where a substituent para to the hydroxyl group at position 4 of the phenyl ring is substituted by -CHO or -CH═CH- or -CH
2 and the 3rd position substituted
with -OCH 3 and -OH. All the phenols employed in
this study possess one or two phenolic groups – one invariably at the 4th position and another as such as in dl -dopa and dopamine or modi fi ed to a OCH
3 group (vanillin, N-vanillyl-nonanamide and
curcumin). However, both the OH groups have reacted giving monoglycosides and arylated prod-ucts, but no bis derivatives in case of dl -dopa, l -dopa, dopamine and epinephrine were observed. In curcumin both the phenolic OH groups on either side reacted to give a bis products. Hence, the pres-ence of bulky groups para to 4-OH did not hinder formation of glycoside. A reasonable extent of selectivity exhibited by these enzymes has been utilised for the preparation of the glycosides, thereby eliminating the need for elaborate protec-tive and deprotective strategies.
Effect of incubation period showed that the yield increased up to a time period of 72 h and then showed a remarkable drop at still higher incubation periods, which could be due to partial hydrolysis of the glycosides formed after 72 h. Since the enzymes could not be recovered, they were not reused again. Hence, the activity of the enzyme after the reaction could not be deter-mined. Re fl uxing di-isopropyl ether for a speci fi ed incubation period did not produce any peroxides in these glycosylation reactions.
Glycosylation described in the present work did not occur without the use of enzyme. Glycosidase reactions occur only in the presence of certain amount of water (Vic and Crout 1995 ; Vijayakumar et al. 2007 ) , whose presence in the reaction mixture could be regulated carefully to get a good glycosylation yield. Besides imparting ‘pH memory’, added water is essential for main-taining the integrity of the three-dimensional structure of the enzyme molecule and therefore its activity (Dordick 1989 ) in a nonpolar solvent
like di-isopropyl ether (Vijayakumar and Divakar 2005 ) . Water has been added in the form of 10 mM buffer. When buffer concentration (buffer volume) was varied, the lower and higher buffer concentrations (buffer volume) resulted in lesser conversion yields. A lower buffer concentration may not be suf fi cient to keep the active confor-mation of the enzyme, and a higher buffer con-centration could result in hydrolysis of the product.
Beyond a critical water concentration, glyco-sylation decreased due to the size of the water layer formed around the enzyme retarding the transfer of the glycosyl donor to the active site of the enzyme (Humeau et al. 1998 ; Camacho-Paez et al. 2003a, b ) . Besides it renders the water layer surrounding the enzymes more fl exible by form-ing multiple H bonds and interacting with organic solvent causing denaturation (Valiveti et al. 1991 ) . The protonation of the ionisable groups during the addition of buffer on the enzyme could be controlled by the type and availability of the counter ions as well as hydrogen ions resulting in ‘pH memory’, and the increase in ionic strength could play a favourable role in glycosylation (Pardridge 2002 ) . The carbohydrate molecule could also reduce the water content of the reaction mixture. Adachi and Kobayashi ( 2005 ) have reported that the hexose, which is more hydrated, decreased the water activity in the sys-tem and shifts the equilibrium towards synthesis. The effect of pH showed that pH 4 for vanillin (Table 8.11 ) and curcumin (Table 8.6 ), pH 7 for N-vanillyl-nonanamide (Table 8.8 ), pH 6 for dl -dopa (Table 8.17 ) and pH 5 for dopamine (Table 8.21 ) are the best for obtaining maximum con-version. The three-dimensional structure of the enzyme up to pH 7 may still retain a highly active conformation. However, pH 8 was not the optimum one for any of the phenols employed.
At lesser enzyme concentrations, for a given amount of substrates (enzyme/substrate ratio low), rapid exchange between bound and unbound forms of both the substrates (carbohydrates and the aglycon) with the enzyme (on a weighted average based on binding constant values of both the sub-strates) leaves substantial number of unbound sub-strate molecules at the start of the reaction which
2179.1 General
decrease progressively as conversion takes place (Romero et al. 2003 ; Marty et al. 1992 ) . This becomes more so, if one of them binds more fi rmly to the enzyme than the other (higher binding con-stant value) as the respective enzyme/substrate ratios keep changing (during the course of the reaction) unevenly till the conversion stops due to complete binding (inhibition) of the predominant substrate. At intermediatory enzyme concentra-tions, such a competitive binding results in favour-able proportions of bound and unbound substrates to effect quite a good conversion. At higher enzyme concentrations, most of the substrates would be in the bound form leading to inhibition and lesser conversion (higher enzyme/substrate ratios). Also, the glycosylation reaction requires larger amount of enzyme compared to hydrolysis. Effect of enzyme concentration at a fi xed d -glucose concen-tration showed a maximum conversion at around 40% (w/w d -glucose) in many cases. At higher concentrations of both glucosidases, conversion yields decreased probably due to inhibition of the enzyme by the phenols employed. While this leads to lesser selectivity, they also give rise to varying bound and unbound substrate concentrations till the conversion ends. For a given amount of enzyme and substrates, there is no increase in conversion beyond 72–120 h. Longer incubation periods of especially lesser enzyme concentrations could also result in partial enzyme inactivation. However, not all the enzyme is inactivated before the end of the reaction.
Amyloglucosidase-catalysed synthesis of n-octyl- a - d -glucoside, guaiacyl- a - d -glucoside, eugenyl maltoside, curcuminyl-bis- a - d -gluco-side, dl -dopa glucoside, dopamine glucoside, ribo fl avinyl glucoside and tocopheryl- a - d -glucoside was optimised in terms of incubation period, buffer pH, buffer concentration, enzyme concentration and substrate concentrations. n-Octanol reacted with d -glucose, maltose and sucrose; guaiacol reacted with d -glucose and d -galactose; eugenol and curcumin reacted with d -glucose, d -mannose, maltose, sucrose and d -mannitol, and a -tocoph-erol reacted with only d -glucose. Phenols/vitamins underwent glycosylation/arylation with the following respective carbohydrate molecules. A few examples are vanillin ( d -glucose, d -galactose,
d -mannose, maltose, sucrose, lactose and d -sorbitol), N-vanillyl-nonanamide ( d -glucose, d -galactose, d -mannose, d -ribose, maltose and lactose), curcumin ( d -glucose, d -galactose, d -mannose and lactose), dl -dopa ( d -glucose, d -galactose, d -mannose, d -sorbitol and d -mannitol), dopamine ( d -glucose, d -galactose and d -mannose), ribo fl avin ( d -glucose, d -galactose, d -mannose, d -ribose , maltose, sucrose and lactose) and ergocalciferol only with d -glucose and a -tocopherol ( d -glucose, d -galactose and d -mannose). All the phenols employed invariably reacted with aldohexoses ( d -glucose, d -galactose and d -mannose) employed to yield respective glycosides except in the reaction between N - vanillyl-nonanamide and d -mannose. Dopamine showed exclusively gly-cosylation only with aldohexoses.
Thus water-insoluble N-vanillyl-nonanamide, curcumin, capsaicin, retinol, ergocalciferol, chole-calciferol and a -tocopherol and less water-soluble vanillin and ribo fl avin were converted to more water-soluble glycosides thereby improving their potential bioavailability and pharmacological properties. Water solubility of the curcuminyl-bis- a - d -glucoside prepared was found to be 14 g L −1 , whereas curcumin itself is practically insoluble in water. Curcuminyl-bis- a - d -glucoside exhibited a total colour of 10.8 in DMSO.
Lack of stereo- and regiospeci fi city could be due to employment of larger amounts of the enzymes. This is inevitable, as this reversible reaction required such large concentrations of the enzymes to effect glycosylation.
Amyloglucosidase, besides effecting glycosy-lation, also facilitated the hydrolysis of maltose, lactose and sucrose. Lactose did not hydrolyze at all. Only in case of sucrose, the resultant glucose formed underwent trans-glucosylation with van-illin in the presence of amyloglucosidase to yield C1 b glucosylated and C6- O -arylated product.
Both amyloglucosidase and b -glucosidase (native/immobilised) did not catalyse the reac-tion with d -fructose and d -arabinose with any of the phenols employed. Also, while b -glucosidase (native/immobilised) catalysed reaction with lactose, amyloglucosidase did not. Hence, d -fructose and d -arabinose could not result in not-so-facile formation of the
218 9 Glycosylation of Phenols and Vitamins: An Overview
required oxocarbenium ion intermediate (Chiba 1997 ) during the catalytic action of the enzyme, which is an essential requirement for glycosy-lation. In both the glucosidase-catalysed reac-tions, other than d -fructose and d -arabinose, the remaining above-mentioned carbohydrates underwent glycosylation/arylation with different phenols/vitamins to different extents. Such selec-tive reactions could be due to stronger binding of the phenols/vitamins to the active site of enzymes than the respective unreacted carbo-hydrate molecules, thereby preventing facile transfer of the carbohydrate molecules to the nucleophilic phenolic or vitamin OH groups. During hydrolysis of amylase, the a -1,4 linked glucose units get hydrolysed which give rise to b - d -glucose by amyloglucosidase. This clearly showed that amyloglucosidase possesses ‘inverting’ potentiality. The same behaviour was observed in the glycosylation reaction also. In general amyloglucosidase catalyses gave C1 a - and b -glycosides along with C6- O -aryl derivatives. However, b -glucosidase (native/immobilised) catalyses gave exclusively C1 b -glycosides in several cases, indicating its capa-bility to exhibit excellent regioselectivity in this glycosylation with the carbohydrate molecules.
In most cases, C1 glycosylated products were detected. Only few carbohydrate molecules showed C1- O -/C6- O -arylation. d -Sorbitol and d -mannitol gave arylated products by reacting only to the primary OH groups. No reaction occurred at the secondary hydroxyl groups of the carbohydrate molecules. Only monoglycosylated or monoarylated products were detected. No car-bohydrate molecule gave bis products. Among the phenols employed, only curcumin showed bis-glycosylated products by reacting at both the hydroxyl moieties of the feruloyl units. Even dl -dopa, l -dopa, dopamine and epinephrine did not give bis products. b -Glucosidase showed gener-ally b -glycosides and in very few cases C1- O -/C6- O -arylated products. However, amyloglucosi-dase on the other hand showed both C1 a - and C1 b -glycosylated and/or C6-arylated products. Phenolic OH at the 4th position readily reacted with the carbohydrate molecules employed and
wherever possible dl -dopa. l -dopa, dopamine and epinephrine underwent reaction at the 3rd phenolic OH also. Totally hydrophobic acceptor molecules like curcumin, capsaicin N-vanillyl-nonanamide, retinol, ergocalciferol, cholecalcif-erol and a -tocopherol reacted with only very few carbohydrate molecules like d -glucose, d -galactose, d -mannose and lactose where as the remaining hydrophilic phenols and vitamins employed vanillin, l -dopa, dl -dopa, dopamine and ribo fl avin reacted with large number of carbohydrate molecules.
Out of 99 individual phenolic and vitamin gly-cosides synthesised enzymatically all the synthe-sised C1-glycosides and C6-O-alkylated/arylated of the carbohydrates, about 89 new compounds are reported in the present work: n-octyl-sucrose, guaiacyl- a - d -glucoside, guaiacyl- b - d -glucoside, guaiacyl- a - d -galactoside, eugenyl- a - d -manno-side, eugenyl maltoside, eugenyl sucrose, euge-nyl- d -mannitol, 1,7- O -(bis- b - d -glucopyranosyl) curcumin, 1,7- O -(bis- a - d -galactopyranosyl)cur-cumin, 1,7- O -(bis- b - d -galactopyranosyl)cur-cumin, 1,7- O -(bis- a - d -mannopyranosyl)curcumin, 1,7- O -(bis- b - d -mannopyranosyl)cur-cumin, 1,7- O -(bis- b - d -galactopyranosyl-(1 ¢ → 4 ) a - d -g lucopyranosy l )curcumin , 1,7- O -(bis- b - d -galactopyranosyl-(1 ¢ → 4) b - d -glucopyranosyl) curcumin, curcuminyl-bis- a - d -mannoside, curcuminyl-bis-maltoside, curcuminyl-bis-sucrose and curcuminyl-bis- d -mannitol, 4- O -( d -galactopyranosyl)vanillin, 4- O -( d -mannopyranosyl)vanillin, 4- O -( a - d -glucopyranosyl-(1 ¢ → 4) d -glucopyranosyl)vanillin, 4- O -( d -fructofuranosyl-(2 → 1 ¢ ) a - d -glucopyranosyl)vanillin , 4- O -( b - d -galacto-pyranosyl - (1 ¢ → 4 ) b - d -g lucopyranosyl )vanillin, 4- O -( d -sorbitol) vanillin, 4- O -( d -galactopyranosyl)N-vanillyl-nonanamide, 4 - O - ( b - d -mannopyranosy l )N-van i l l y l -nonanamide, 4- O -( d -ribofuranosyl)N-vanillyl-nonanamide, 4- O - ( a - d -glucopyranosyl- (1 ¢ → 4 ) d -g lucopyranosy l )N-van i l ly l -nonanamide, 4- O -( b - d -galactopyranosyl- (1 ¢ → 4) b - d -glucopyranosyl)N-vanil lyl-nonanamide, 1,7- O -(bis- b - d -galacto-pyranosyl)curcumin, 1,7- O -(bis- b - d -mannopyranosyl)curcumin, 1,7- O -(bis- b - d -galactopyranosyl-
2199.3 Curcuminyl-bis-Glycosides
(1 ¢ → 4) a - d -glucopyranosyl)curcumin, dl -Dopa- d -galactoside, dl -3-hyDroxy-4- O -( b - d -mannopyranosyl)phenylalanine, dl -3-hydroxy-4- O -( b - d -galactopyranosyl-(1 ¢ → 4) b - d -glu-copyranosyl)phenylalanine, dl -3-hyDroxy-4- O -(6- d -sorbitol) phenylalanine, dl -Dopa- d -mannitol, dopamine- d -galactoside, dopamine- d -mannoside, serotoninyl-5-O- a and b d -glucopyranoside, serotoninyl-5-O-6-O- a and b d -glucopyranoside, serotoninyl-5-O- b - d -galac-topyranoside, serotoninyl-5-O-6-O- a and b d -galactopyranoside, serotoninyl-5-O- b d -man-nopyranoside, serotoninyl-5-O-6-O- a and b d -mannopyranoside, serotoninyl-5-O- a and b d -ribopfuranoside, epinephryl 3-O and 4-O b - d -glucopyranoside, epinephryl 3-O- b - d -manno-pyranoside, 4-O- a and b - d -mannopyranoside, 18- O -( a - d -glucopyranosyl)retinol, 18- O -( b - d -glucopyranosyl)retinol, 18- O -( a - d -galactopyranosyl)retinol, 18- O -( b - d -galactopyranosyl)retinol, 18- O -( b - d -mannopyranosyl)retinol, 18- O -(1- d -fructofuranosyl)retinol, 18- O -(6- d -fructofuranosyl)retinol, 18- O -(1- d -sorbitol)retinol, 7- O -( a - d -glucopyranosyl)pyridoxine, 7- O -( b - d -glucopyranosyl)pyridoxine, 6- O -( a - d -glucopyranosyl)pyridoxine, 7- O -( a - d -galactopyranosyl)pyridoxine, 7- O -( b - d -galactopyranosyl)pyridoxine, 7- O -( a - d -galactopyranosyl)pyridox-ine, 7- O -( a - d -mannopyranosyl)pyridoxine, 7- O -( b - d -mannopyranosyl)pyridoxine , 6- O -( a - d -mannopyranosyl)pyridoxine 5- O -( a - d -galactopyranosyl)ribo fl avin, 5- O -( b - d -galactopyranosyl)ribo fl avin, 5- O -( a - d -mannopyranosyl)ribo fl avin, 5- O -( b - d -mannopyranosyl)ribo fl avin, 5- O -( a - d -ribofuranosyl)ribo fl avin , 5- O -( b - d -ribofuranosyl)ribo fl avin , 5- O -( a - d -glucopyranosyl-(1 ¢ → 4) a - d -glucopyranosyl)ribo fl avin, 5- O -( a - d -gluco-pyranosyl - (1 ¢ → 4 )6- d -g lucopyranosyl ) ribo fl avin, 5- O -( a - d -glucopyranosyl-(1 ¢ → 4)6 ¢ - d -glucopyranosyl)ribo fl avin, 5- O -(1- d -fruc to-furanosyl - (2 → 1 ¢ ) a - d -g lucopyranosyl ) ribo fl avin, 5- O -( b - d -galactopyranosyl-( 1 ¢ → 4 ) b - g l u c o p y r a n o s y l ) r i b o fl av i n , 6- O - ( a - d -galactopyranosyl) a -tocopherol, 6- O -( b - d -galactopyranosyl) a -tocopherol, 6- O -( a - d -mannopyranosyl) a -tocopherol,6- O -( b - d -mannopyranosyl) a -tocopherol, 17- O -( a - d -galactopyranosyl)cholecalciferol, 17- O -( b - d -
galactopyranosyl)cholecalciferol, 17- O -(6- d -g a l a c t o p y r a n o s y l ) c h o l e c a l c i f e r o l , 17- O -( a - d -mannopyranosyl)cholecalciferol, 17- O -( b - d -mannopyranosyl)cholecalciferol, 17- O -(1- d -fructofuranosyl)cholecalciferol and 1 7 - O - ( b - d - f r u c t o f u r a n o s y l ) c h o l e c a l -ciferol.
Response surface methodological studies, use-ful for scaling up, were carried out for the opti-misation of n-octyl- a - d -glucoside, curcuminyl glucoside, vanillyl maltoside and thiamin gluco-side synthesis by shake- fl ask method and curcum-inyl- a - d -glucoside synthesis by re fl ux method
9.2 n-Alkyl Glycosides
The present work (Chap. 7 ), especially the re fl ux method with amyloglucosidase, showed that the glucoside yields were much higher (methanol 25%, ethyl alcohol 44%, n-butanol 28%, n-hexanol 36% and n-octanol 46% with respect to d -glucose concentration employed) than those reported compared to those reported by Vic and Thomas ( 1992 ) – 13.1% for methanol, 9.8% for ethyl alcohol, 6.6% for n-butanol, 4.9% for n-hexanol and 3.6% for n-octanol in the reactions carried out with almond b -glucosidase. Among the nucleophilic straight-chain alcohols employed, while shake fl ask favoured medium-chain-length alcohols, the re fl ux method at pH 4.0 favoured shorter-chain-length alcohols and pH 5.0 more or less favoured almost all the chain length. This clearly shows that since pH affected ionisation states of the surface amino acid residues in amy-loglucosidase between pH 4.0 and pH 5.0, the enzyme should be favouring a more open active site at pH 5.0, accommodating alcohols of differ-ent chain length favourably for glycosylation.
9.3 Curcuminyl-bis-Glycosides
Although b -glucosidase catalysis (Sect. 8.1.3 ) does not exhibit an inversion, it had signi fi cantly altered the a , b composition: d -galactose, 39% a - d -galactoside and 61% b - d -galactoside (compared to 92:8 a : b for free d -galactose), and d -mannose,
220 9 Glycosylation of Phenols and Vitamins: An Overview
35% a - d -mannoside and 65% b - d -mannoside (compared to 27:73 a : b for free d -mannose) . Also, curcumin showed bis-glycosylated prod-ucts with the carbohydrates with which it reacted.
d -Galactose and d -mannose gave very low conversions ( £ 12%). Hence, they could possess very low binding potentiality (low binding constant value) to the enzyme. Presence of hydrophobic propanoid group para to the phe-nolic OH bestows good nucleophilicity in these molecules promoting reaction with carbohydrate molecules.
9.4 N-Vanillyl-Nonanamide Glycosides
All these glycosides were soluble in water to dif-ferent degrees and also less pungent. They could hence be used in pharmaceutical applications. Both amyloglucosidase and b -glucosidase did not catalyse the reaction with d -fructose, d -arab-inose, sucrose, d -sorbitol and d -mannitol (Sect. 8.1.4 ). Also, while b -glucosidase catalysed reac-tion with lactose, amyloglucosidase did not. N-Vanillyl-nonanamide could bind to these enzymes more strongly than the above carbohy-drate molecules to the nucleophilic phenolic OH of N-vanillyl-nonanamide.
Amyloglucosidase clearly exhibited its ‘invert-ing’ potentiality in the glycosylation giving rise to the a - d -glucoside (70% of the a -glucoside without the b component compared to the 40:60 a : b anomeric composition of d -glucose employed), a - d -galactoside (42% a and 58% b compared to the 92:8 a : b anomeric composition of d -galactose employed) and a - d -riboside (33% a and 67% b compared to the 34:66 a : b ano-meric composition of d -ribose employed) . Both the glucosidases gave low conversions with d -ribose and maltose ( £ 10%). However, b -glu-cosidase catalysis gave exclusively C1 b -glycosides with the exception of d -ribose, indicating its capability to exhibit excellent regi-oselectivity in this glycosylation with the carbo-hydrate molecules d -glucose, d -galactose, d -mannose, maltose and lactose. Even the
presence of hydrophobic bulky alkyl side chain in N-vanillyl-nonanamide did not pose much of a steric hindrance when the carbohydrate mole-cules were transferred to its phenolic OH group.
9.5 Vanillyl Glycosides
d -Glucose employed was an a , b anomeric mix-ture (40:60); the glycosides formed showed pre-dominant proportions of the a -anomer (>80%), indicating the potential for ‘inverting’ amyloglu-cosidase (from Rhizopus mould) to convert the majority of b - d -glucose into its respective a - d -glucoside (Sect. 8.1.6 ). In hydrolysis, amyloglu-cosidase hydrolyses a -1,4 linked glucose units in amylose to give b -glucose.
A maximum yield of 53% was obtained for a mixture of three mono-glucosides of 4- O -( d -glucopyranosyl)vanillin (Table 8.12 ). Also, amy-loglucosidase catalysis gave C1 a - and b -glycosides along with arylated derivatives in many cases except d -galactose and d -mannose. However, b -glucosidase catalysis gave exclu-sively C1 b -glycosides with the exception of d -galactose and d -mannose, indicating its capa-bility to exhibit excellent regioselectivity in this glycosylation with the carbohydrate mole-cules d -glucose , maltose and lactose. In case of b -glucosidase, it signi fi cantly altered the anomeric composition of d -galactose (23% a - d -galactoside and 77% b - d -galactoside com-pared to the 92:8 a : b anomeric composition of d -galactose employed) and d -mannose (44% a - d -mannoside and 56% b - d -mannoside com-pared to the 27:73 a : b anomeric composition of d -mannose employed) . Among the carbohydrates employed, sucrose and d -sorbitol gave C1- O - and C6- O -arylated products with vanillin.
In case of sucrose, the C6- O -arylated product proportion was more compared to the C1- O -arylated product, which could be due to the steric hindrance offered by the C2 position of the fructose moiety when it is transferred to such phenolic nucleophiles. Even the presence of a hydrophobic aldehydic group in vanillin did not pose much of a steric hindrance when the carbohydrate molecules were transferred to its
2219.8 Ribofl avinyl Glycosides
phenolic OH group. Both amyloglucosidase and b -glucosidase did not catalyse d -fructose, d -arabinose, d -ribose and d -mannitol. Vanillin could be a better inhibitor to these enzymes com-pared to these carbohydrate molecules, binding strongly to the enzyme, thereby blocking the facile transfer of the carbohydrate molecule to the nucleophilic phenolic OH of vanillin.
9.6 DL -Dopa Glycosides
Under the reaction conditions employed, dl -dopa gave a mixture of 3- O - and 4- O -glycosylated/arylated products with many of the carbohydrates employed (Sect. 8.1.7 ). Amyloglucosidase catal-ysed the reaction with d -glucose, d -galactose, d -mannose, d -sorbitol and d -mannitol. b -Glucosidase catalysed the reaction with d -glucose, d -galactose, d -mannose and lactose.
Amyloglucosidase gave selectivity with d -mannose to give 4- O -C1 b and d -sorbitol to give 4- O -C6- O -arylated product. b -Glucosidase gave selectivity with d -mannose to give 4- O -C1 b and lactose to give 4- O -C1 b product. Glycosylated/arylated products at 3- O - and 4- O - positions were detected from d -glucose and d -galactose in the catalysis with both the enzymes. No other reacted carbohydrate molecule has formed products at the 3rd OH position.
Glucosidases employed did not catalyse the reaction with d -fructose, d -arabinose, d -ribose, maltose and sucrose. Amyloglucosidase exhibited ‘inverting’ potentiality in reacting with d -galactose. d -Galactose showed 66% a and 34% b compared to the 92:8 a : b anomeric composition of free d -galactose employed.
9.7 Dopamine Glycosides
Only monoglycosides were detected (Sect. 8.1.9 ). Bis glycosides involving both the 4-OH and 3-OH phenolic groups simultaneously were not detected. Under the reaction conditions employed, immobilised b -glucosidase showed selectivity with d -glucose to give rise to the 4- O -C1 b product. Dopamine gave a mixture of 3- O - and
4- O -glycosylated products with d -galactose and d -mannose with both the enzymes and 4- O -C6- O -arylated product detected only with d -galactose.
Amyloglucosidase exhibited its ‘inverting’ potentiality in the glycosylation of d -galactose giving rise to 64% 4- O -products and 36% exclu-sively 3- O -C1 b product compared to the 92:8 a : b anomeric composition of the d -galactose employed. Both amyloglucosidase and immobil-ised b -glucosidase did not catalyse the reaction with d -fructose, d -arabinose, d -ribose, maltose, sucrose, lactose, d -sorbitol and d -mannitol under the conditions employed. Dopamine could be a better inhibitor to these enzymes compared to the carbohydrate molecules, thereby blocking the transfer of the carbohydrate molecules to the phe-nolic OH group of dopamine.
9.8 Ribo fl avinyl Glycosides
Ribo fl avinyl glycosides have been formed from many of the carbohydrates employed (Sect. 8.2.2 ). The yields are shown in Table 8.29 . In case of amyloglucosidase catalysis, 5- O -( d -ribofuranosyl) ribo fl avin was found to give the highest yield (40%) and 5- O -( a - d -glucopyranosyl-(1 ¢ → 4) d -glucopyranosyl)ribo fl avin gave the lowest yield of 5% (Table 8.29 ). This clearly showed that d -ribose being a smaller carbohydrate mole-cule acts as a better acceptor than the bulkier maltose. b -Glucosidase gave the highest yield of 24% for 5- O -( b - d -glucopyranosyl)ribo fl avin and the lowest yield of 7% for 5- O -( d -mannopyranosyl)ribo fl avin. Also, while b -glucosidase favoured lactoside formation, amyloglucosidase did not.
In spite of possessing a ribitol group, ribo fl avin is soluble to the extent of only 0.2 g L −1 (Whitby 1971 ) , which is due to the strong nonpolar nature of the aglycon molecule. However, attachment of a monosaccharide unit to ribitol improved the water solubility of ribo fl avin to 8.2 g L −1 . This shows that glycosylation is capable of counter-acting the nonpolar aglycon characteristics to a great extent. Ribo fl avin showed glycosylation with many of the carbohydrates compared to the other vitamins employed (ergocalciferol and
222 9 Glycosylation of Phenols and Vitamins: An Overview
a -tocopherol) in spite of the bulky acceptor molecule. This could be due to the presence of primary OH present at the ribitol moiety in ribo fl avin, nucleophilic enough to serve as an ef fi cient acceptor towards certain carbohydrate molecules which get glycosylated.
Amyloglucosidase gave the following a : b proportions: d -glucose (66% of a - d -glucoside and 44% b - d -glucoside compared to the 40:60 a : b anomeric composition of free d -glucose), d -galactose (52% a - d -galactoside and 48% b - d -galactoside compared to the 92:8 a : b of free d -galactose) and d -ribose (23% a - d -riboside and 77% b - d -riboside compared to the 34:66 a : b of free d -ribose) . Although b -glucosidase in general does not exhibit inversion, it had signi fi cantly altered the a - b composition: d -galactose (47% a - d -galactoside and 53% b - d -galactoside) and d -mannose (59% a - d -mannoside and 41% b - d -mannoside comparable to 27:73 a : b of free d -mannose).
Amyloglucosidase catalysis showed selectivity in case of d -mannose by yielding the C1 a - d -mannoside and with sucrose yielding C1- O -arylated product. It gave C1 a - and b -glycosides in case of d -glucose, d -galactose and d -ribose. With d -glucose and maltose, C6- O -arylated prod-ucts were also formed. b -Glucosidase showed selectivity with d -glucose yielding C1 b - d -glucoside and with lactose yielding C1 b -lactoside. However, it gave a mixture of C1 a - and b -glycosides in case of d -galactose and d -mannose.
9.9 Retinyl Glycosides
b -Glucosidase did not catalyse the reaction with d -ribose, d -arabinose, maltose, sucrose, lactose and d -mannitol (Sect. 8.2.4 ). This could be stron-ger due to binding of retinol to the active site of b -glucosidase than the above-mentioned carbo-hydrate molecules, thereby preventing the facile transfer of these carbohydrate molecules to the nucleophilic ole fi nic OH of retinol. Our work on the preparation of several glycosides with different aglycons showed that diverse reactivity of the carbohydrate molecules was with respect to the aglycons employed (Sivakumar 2009 ;
Vijayakumar 2007 ) . With different aglycons, different reactivities were observed even under conventional reaction conditions. In most of the reactions carried out in our laboratory, glycosyla-tion did not take place with d -ribose, d -arabinose, lactose and sucrose, however, with very few exceptions. This type of selectivity is not due to the SCCO
2 conditions employed.
A one-step synthesis involved milder reaction conditions, eliminating the need of protective and deprotective strategies to convert light and air sensi-tive, fat-soluble retinol into more stable and water-soluble glycosyl derivative, to be useful as a pharmacologically and therapeutically active water-soluble component of vitamin A. Such derivatisa-tion of vitamin A was possible due to the potentiality of b -glucosidase to act in SCCO
2 media.
9.10 Ergocalciferyl Glycosides
Ergocalciferyl- d -glucosides (20- O -( a - d -glucopyranosyl)ergocalciferol, 20- O -( b - d -glucopyranosyl)ergocalciferol and 20- O -(6- d -glucopyranosyl)ergocalciferol) were detected in the reaction when amyloglucosidase was employed (Sect. 8.2.5 , Table 8.34 ). However, amyloglucosidase did not show much regioselectivity in this reac-tion. Amyloglucosidase did not catalyse the reac-tion with the other carbohydrate molecules. This could be due to either steric hindrance caused by the bulkier hydrophobic vitamin D2 molecule when the carbohydrate molecules were trans-ferred to its acyclic OH group or due to the stron-ger binding of vitamin D2 compared to the other carbohydrate molecules employed. The maxi-mum conversion was found to 42% for 20- O -( d -glucopyranosyl)ergocalciferol. Being ‘inverting’ in nature, amyloglucosidase gave the following a : b proportions – 48% of a - d -glucoside and 52% b -glucoside compared to the 40:60 a : b anomeric composition of the d -glucose employed.
At a boiling temperature of 68 °C, the enzymes were stable, losing only about 20% of their activity during an incubation period of 50 h. Re fl uxing di-isopropyl ether during the incubation periods up to 50 h did not also produce peroxides in these glycosylation reactions.
2239.12 α-Tocopheryl Glycosides
b -Glucosidase isolated from sweet almond was entrapped into calcium alginate beads and used in the glycosylation reaction to facilitate recovery and reuse. In the reaction mixture, the enzymes dissolved in small volume of the buffer employed. They did not get precipitated by the di-isopropyl ether solvent. During workup the solvent in the reaction mixture was distilled off leaving a residue consisting of the enzyme, prod-uct and the unreacted substrates. Hence, the amount of the enzyme leached out into the buffer or the di-isopropyl ether solvent could not be determined. Reuse of the calcium alginate bead-entrapped b -glucosidase showed 30% reduction in activity, which could be due to loss of the enzyme.
9.11 Cholecalciferol Glycosides
Synthesis of other cholecalciferol glycosides (Table 8.36 ) showed that apart from the three aldo-hexoses and aldopentose employed, d -ribose, d -arabinose, maltose, sucrose, lactose, d -sorbitol and d -mannitol did not undergo glycosylation under the conditions employed. Although the reac-tions were conducted with disaccharides maltose, lactose and sucrose, transglycosylation did not occur under the conditions employed (Sect. 8.2.6 ). However, the disaccharides were hydrolyzed by the enzymes, the reason for which is not known.
In case of d -glucose and d -galactose, C-6 derivatives were also detected. Anomeric propor-tions were found to be greatly altered. In d -glucose the proportion of 52:48 is different from that of free d -glucose (60:40). However, in d -galactose b -anomer has been formed (45:56) compared to 92:8 of free d -galactose. In d -mannose, more of a -glycoside is formed (46:54) compared to (27:73) free d -mannose. The formation of a and b anomeric mixture is due to the a -glucosidase present in sweet almond isolate, which exhibited an activity of 359.7 AU ( m mol (min·mg) −1 enzyme preparation). Thus through glycosylation, a water-insoluble cholecalciferol (vitamin D
3 ) was
transformed into a water-soluble cholecalciferol glycoside in this study.
9.12 a -Tocopheryl Glycosides
a -Tocopheryl glycosides have been synthesised using b -glucosidase (Sect. 8.2.7 ). The yields and product proportions are shown in Table 8.37 . b -Glucosidase 40% (w/w d -glucose) gave a max-imum conversion yield of 23% for 6- O -( b - d -glucopyranosyl)- a -tocopherol at 0.1 mM (1 mL) of pH 6 phosphate buffer, indicated excellent regioselectivity.
The a and b forms of glycosides were deter-mined based on the 1 H and 13 C chemical shift values of the anomeric proton and carbon signals, respectively. Besides, the coupling constant val-ues of the anomeric 1 H protons were also useful in arriving at the anomeric con fi guration. In an a con fi guration, the anomeric carbon exhibited chemical shift values between 92 and 94 ppm. In a b con fi guration, such values are between 96 and 104 ppm. In an a con fi guration, the coupling between the anomeric and C2 proton is equato-rial–axial corresponding to coupling constant values of 2–4 Hz. In case of a b con fi guration, the coupling between the anomeric and C2 proton is axial–axial corresponding to a value of 6–8 Hz. However, in d -mannose, since the OH at C2 posi-tion is axial, the coupling for the C2 and the ano-meric protons is equatorial–equatorial (1–2 Hz) for the a con fi guration and axial–equatorial (2–4 Hz) for the b con fi guration.
Under the reaction conditions, 11% 6- O -( a - d -galactopyranosyl)- a -tocopherol, 6- O -( b - d -galactopyranosyl) a -tocopherol, 18% 6- O -( a - d -mannopyranosyl) a -tocopherol and 6- O -( b - d -mannopyranosyl) a -tocopherol were also obtained with b -glucosidase (Table 8.38 ). In case of d -galactose, the a / b proportions were found to be 41:59 ( a : b ) with respect to a / b proportions of 92:8, for the free d -galactose, thus favouring a slight excess of b - d -galactoside formation. However, b -glucosidase only marginally favoured a - d -mannoside forma-tion as evidenced from the a / b proportions of 46:54 ( a : b ) for the glycosides compared to an a / b proportion of 27:73 for free d -mannose.
224 9 Glycosylation of Phenols and Vitamins: An Overview
Syntheses of the other a -tocopheryl glycosides with carbohydrate molecules showed that except for the three aldohexoses employed, d -fructose, d -arabinose, d -ribose, maltose, sucrose, lactose, d -sorbitol and d -mannitol did not undergo glycosylation under the conditions employed. b -Glucosidase is not an inverting enzyme. In the present work, b -glucosidase gave b -glucoside and a / b anomeric mixture of glycosides with d -galactose and d -mannose. In the oxocarbenium ion mechanism (Chiba 1997 ) , a planar carbenium ion centre formed with d -galactose and d -mannose could be available for attack by the nucleophilic a -tocopherol phenol from both above and below the plane giving rise to a mixture of a / b anomeric products. With d -glucose, b -glucosi-dase gave its natural b -glucoside. Since d -galactose and d -mannose are not natural products of b -glucosidase, the same selectivity could not be achieved by this enzyme.
There are not many reports on the glyco-sylating potential of amyloglucosidase in the literature. The results from this investigation have shown conclusively that amyloglucosidase and b -glucosidase (native/immobilised) from sweet almond are excellent enzymes for carrying out effective glucosylation of such diverse alcohols, phenols and vitamins. Thus the present work has brought out the multifaceted characteristics of these enzymes in the glycosylation of selected phenols and vitamins with structurally diverse carbohydrate molecules employed. Also such glycosylation produces more water-soluble and stable vitamin derivatives with diverse carbohy-drate molecules.
References
Adachi S, Kobayashi T (2005) Synthesis of esters by immobilized lipase catalyzed condensation reaction of sugars and fatty acids in water-miscible organic sol-vent. J Biosci Bioeng 99(2):87–89
Camacho-Paez B, Robles Medina A, Camacho Rubio F, Gonzalez Moreno P, Molina Grim E (2003a) Modeling the effect of free water on enzyme activity in immobilized
lipase- catalyzed reactions in organic solvents. Enzyme Microb Technol 33:845–853
Camacho-Paez B, Robles MA, Camacho RF, Esteban-Cerdán L, Molina GE (2003b) Kinetics of lipase-catal-ysed interesteri fi cation of triolein and caprylic acid to produce structured lipids. J Chem Technol Biotechnol 78(4):461–470
Chiba S (1997) Molecular mechanism in a -glucosidase and glucoamylase. Biosci Biotechnol Biochem 61:1233–1239
Dordick JS (1989) Enzymatic catalysis in monophasic organic solvents. Enzyme Microb Technol 11:194–211
Humeau M, Girardin B, Rovel AM (1998) Effect of the thermodynamic water activity and the reaction medium hydrophobicity on the enzymatic synthesis of ascorbyl palmitate. J Biotechnol 63:1–8
Marty A, Chulalaksananukul W, Condoret JS, Willemont RM, Durand G (1992) Comparison of lipase-catalyzed esteri fi cation in supercritical carbon dioxide and n-hexane. Biotechnol Lett 12(1):11–16
Pardridge WM (2002) Targeting neurotherapeutic agents through the blood–brain barrier. Arch Neurol 59:35–40
Romero MD, Calvo L, Alba C, Daneshfar A, Ghaziaskar HS (2003) Enzymatic synthesis of isoamyl acetate with immobilized Candida antarctica lipase in n -hexane. Enzyme Microb Technol 37:42–48
Sivakumar R (2009) Enzymatic synthesis of selected phe-nolic and vitamin glycosides. PhD thesis, University of Mysore
Valiveti RH, Johnston GA, Suckling CJ, Halling PJ (1991) Solvent effect on biocatalysis in organic systems equi-librium position and rates of lipase catalysed esteri fi cation. Biotechnol Bioeng 38:1137–1143
Vic G, Crout DHG (1995) Synthesis of allyl and benzyl b - D -glucopyranosides and allyl b - D -galactopyrano-side from D -glucose or D -galactose and the corre-sponding alcohol using almond b -D-glucosidase. Carbohydr Res 279:315–319
Vic G, Thomas D (1992) Enzyme-catalyzed synthesis of alkyl- b - D -glucosides in organic media. Tetrahedron Lett 33:4567–4570
Vijayakumar GR (2007) Enzymatic synthesis of selected glycosides. PhD thesis, University of Mysore
Vijayakumar GR, Divakar S (2005) Synthesis of guaiacol- a -D-glucoside and curcumin-bis- a - D-glucoside by an amyloglucosidase from Rhizopus . Biotechnol Lett 27:1411–1415
Vijayakumar GR, George C, Divakar S (2007) Synthesis of n-alkyl glucosides by amyloglucosidase. Indian J Chem B 46B:314–319
Whitby LG (1971) Glycosides or ribo fl avin. In: McCormick CDB, Wright LD (eds) Vitamin and coen-zymes. Methods in enzymology, 18B. Academic, New York, pp 404–413
225S. Divakar, Enzymatic Transformation, DOI 10.1007/978-81-322-0873-0_10, © Springer India 2013
10.1 Introduction
In recent years, lipases (EC 3.1.1.3) have been suc-cessfully used in organic solvents for the synthesis of esters. Many reports available on lipase-catalysed
reactions in organic solvents deal with reaction kinetics of esteri fi cation (Yadav and Trivedi 2003 ; Kiran and Divakar 2002 ; Janssen et al. 1999 ) , racemisation (Duan et al. 1997 ) and hydrolysis (Van-Tol et al. 1992 ) . In some esteri fi cation
10
Abstract
Kinetics of few selective enzymatic esteri fi cation and glycosylation reac-tions in organic solvents is discussed. In all the kinetics, initial rates were determined, and from the pattern of the double reciprocal plots of 1/[S] versus 1/ v , appropriate kinetic models were identi fi ed and the equations worked out. Iterative procedures adopted to carry out curve- fi tting of the experimental plots resulted in determination of the four kinetic parameters K
i , K
mA , K
mB and k
cal corresponding to the best fi t. All the enzymes studied
showed Ping-Pong Bi-Bi mechanism. In the enzymatic esteri fi cation reac-tion between L -alanine and D -glucose in dichloromethane, d -glucose was found to be inhibitory to both Rhizomucor miehei lipase and Candida rugosa lipase. However, both l -phenylalanine and d -glucose in dichlo-romethane solvent were found to exhibit competitive double substrate inhibition of Rhizomucor miehei lipase, leading to dead-end inhibition by RML– d -glucose complex and RML– l -phenylalanyl complexes. On the other hand, esteri fi cation of l -phenylalanine with d -glucose using Candida rugosa lipase in dichloromethane showed that only d -glucose functions as a competitive inhibitor forming dead-end CRL– d -glucose complex.
Similarly, kinetics of glucosylation was investigated in detail for the glu-cosylation of curcumin and vanillin using amyloglucosidase in di-isopropyl ether solvent. Both kinetics could be best described by the Ping-Pong Bi-Bi model with a single competitive substrate inhibition by respective curcumin and vanillin of amyloglucosidase leading to dead-end inhibition. Observed kinetic picture was explained in terms of the active site geometry and the geometry of binding of the substrate/s to the enzyme.
Kinetics of Some Selected Enzyme-Catalysed Reactions in Organic Solvents
226 10 Kinetics of Some Selected Enzyme-Catalysed Reactions in Organic Solvents
reactions, lipases follow Ping-Pong Bi-Bi mechanism (Zhang et al. 2005 ; Zaidi et al. 2002 ; Yadav and Lathi 2004 ) . Chulalaksanaukul et al. ( 1990 ) fi rst proposed that kinetics of Rhizomucor miehei lipase-catalysed esteri fi cation of ethyl oleate fol-lowed Ping-Pong Bi-Bi mechanism. This mecha-nism involves binding of acid and alcohol in successive steps releasing water and the product ester again in succession. The kinetic investigation of RML-catalysed synthesis of isoamyl acetate followed Ping-Pong Bi-Bi mechanism with com-petitive inhibition by the substrates and product forming dead-end inhibitor complexes (Rizzi et al. 1992 ) . Bousquet-Dubouch et al. ( 2001 ) reported a competitive inhibition in CRL-catalysed alcoholy-sis of methylpropionate, in which water was also found to be a competitive inhibitor with a higher inhibition constant than n -propanol. Kinetic behav-iour of CRL in the esteri fi cation of long-chain fatty acids and alcohols (Zaidi et al. 2002 ) and tetrahy-drofurfuryl alcohol with butyric acid (Yadav and Devi 2004 ) showed that both reactions followed Ping-Pong Bi-Bi mechanism with competitive inhibition by both substrates. In citronellyl laurate synthesis, RML follows the ordered Bi-Bi mecha-nism wherein b -citronellol binds to the enzyme to yield the b -citronellol–enzyme complex, which again binds to lauric acid to form the ternary enzyme– b -citronellol–lauric acid complex. Finally, it decomposes to give b -citronellyl laurate and water as products in this process (Yadav and Lathi 2004 ) . A series of dead-end RML–lauric acid complexes were also reported in this process.
Kinetic studies on few enzymatic hydrolytic reactions involving glucosidases and amyloglucosi-dase are known (Hiromi et al. 1983 ; Tanaka et al. 1983 ; Ohnishi and Hiromi 1989 ; Goto et al. 1994 ) . However, report on glycosylation kinetics involving a glycosylating enzyme is practically nil.
In this chapter, kinetics of esteri fi cation between l -alanine and d -glucose and l -phenyla-lanine and d -glucose to form l -alanyl– d -glucose and l -phenylalanyl– d -glucose, respectively, with RML and CRL was carried out (Lohith et al. 2007 ; Somashekar et al. 2007 ) . Similarly, a detailed kinetic investigation on the glucosylation reaction between curcumin and d -glucose and vanillin and d -glucose involving an amyloglucosidase from Rhizopus mould leading to the synthesis of
1,7- O -bis- d -glucopyranosyl–curcumin and vanil-lin– d -glucose, respectively, was also carried out, and the results from these investigations are described below (Sivakumar et al. 2006 ) .
10.2 Methodology
10.2.1 Esteri fi cation Procedure
Kinetic experiments were carried out by re fl uxing 5 mM to 0.1 M of l -alanine/ l -phenylalanine and 5 mM to 0.1 M of d -glucose along with 100 mL CH
2 Cl
2 :DMF (90:10 v/v, 40°C) in the presence
of 0.75–0.18 g (50% w/w carbohydrate employed) of Rhizomucor miehei lipase (RML) in the pres-ence of 0.1 mM (0.1 mL of 0.1 M) of acetate buf-fer (pH 4.0) and Candida rugosa lipase (CRL) in the presence of 0.1 mM (0.1 mL of 0.1 M) of phosphate buffer (pH 7.0) l for the speci fi ed peri-ods of time between 3 and 48 h. An experimental set-up which rendered the reaction mixture dry was employed (Lohith and Divakar 2005 ; Lohith et al. 2007 ) . The product formation was analysed by HPLC and NMR. NMR data clearly indicated the presence of mono- and di - O- esters.
10.2.2 Glycosylation Procedure
Kinetic experiments were carried out by re fl uxing 5 mM to 0.1 M of vanillin/curcumin and 5 mM to 0.1 M of d -glucose in 100 mL di-isopropyl ether at 68°C in the presence of amyloglucosidase (10–75% w/w of carbohydrate) and 0.5–2.5 mL of 0.01 M pH 4.0–8.0 buffer for an incubation period of 72 h. Here also, the product formation was analysed by HPLC and NMR.
10.3 Esteri fi cation Kinetics
10.3.1 Esteri fi cation Kinetics of L -Alanine and D -Glucose: Single Substrate Inhibition
Enzymatic esteri fi cation reaction between l -alanine and d -glucose was studied in dichlo-romethane using Rhizomucor miehei and Candida
22710.3 Esterification Kinetics
rugosa lipases (Somashekar et al . 2007 ) . For the concentrations of d -glucose and l -alanine, indi-vidual experiments in duplicate (30 × 2 lipases) were performed for incubation periods of 3, 6, 12, 24 and 36 h. Initial rate (speci fi c reaction rate, v ) was determined from the initial slope of the plot of the amount of esters formed (M) versus incubation period (h) and expressed as M h −1 (mg protein) −1 (Fig. 10.1 ). R 2 obtained from least-square analysis for the initial rate in each case was found to be within 0.88–0.95. Each plot shown in this work was constructed from all experimentally determined values; a few initial rates were obtained by curve- fi tting.
The initial rates ( v ) for RML were found to be in the range of 15–176 × 10 −6 M h −1 (mg pro-tein) −1 . CRL experiments showed the initial rates to be in the range of 20–460 x 10 −6 M h −1 (mg protein) −1 . The effects of external mass transfer phenomena – internal and external diffusions (Yadav and Devi 2004 ; Marty et al. 1990 ) – if any, on the RML and CRL enzymes employed were not tested in this work.
Using initial rates, double reciprocal plots were constructed to graphically evaluate the apparent values of k
cat , K
m l -alanine
, K m d -glucose
and K i :
RML, Fig. 10.2 (1/ v versus 1/[ d -glucose]) and Fig. 10.3 (1/ v versus 1/[ l -alanine]), and CRL, Fig. 10.4 (1/ v versus 1/[ d -glucose]) and Fig. 10.5 (1/ v versus 1/[ l -alanine]). Figure 10.6 shows a replot of the slopes from Fig. 10.3 (RML), and Fig. 10.7 shows the replot of the slopes from Fig. 10.5 (CRL). Figure 10.2 from RML reac-tions and Fig. 10.4 from CRL reactions show a series of curves obtained for different fi xed con-centrations of l -alanine for varying d -glucose concentration, in which slight increase in initial rates is observed at lower d -glucose concentra-tions. At higher d -glucose concentrations, the rates markedly decrease. Also, increasing l -alanine concentration increases the initial rates at all d -glucose concentrations. Figure 10.3 from RML reactions and Fig. 10.5 from CRL reactions show a series of parallel lines for different fi xed low d -glucose concentrations at varying l -alanine concentration. These change to lines with differ-ent slopes at higher d -glucose concentrations.
The plots in Figs. 10.2 , 10.3 , 10.4 , 10.5 , and 10.6 show that the kinetics could be best described by Ping-Pong Bi-Bi model in which l -alanine and d -glucose bind in subsequent steps releasing water and l -alanyl– d -glucose also in subsequent
Fig. 10.1 Time courses of esteri fi cation reactions – concen-trations of l -alanyl– d -glucose versus incubation periods; RML (90 mg) or CRL (90 mg) was allowed to react with 0.005 M l -alanine and 0.020 M d -glucose in 100 mL of
mixture of dichloromethane/dimethylformamide (v/v 90:10) mixed with 0.1 mM (0.1 mL of 0.1 M) sodium acetate buffer (pH 4.0) for RML or 0.1 mM (0.1 mL of 0.1 M) sodium phosphate buffer (pH 7.0) for CRL
228 10 Kinetics of Some Selected Enzyme-Catalysed Reactions in Organic Solvents
Fig. 10.2 Double reciprocal plots for RML-catalysed l -alanyl– d -glucose reaction: 1/ v versus 1/[ d -glucose] plots; a series of curves show the effect of varying d -glucose concentration at different fi xed l -alanine concentrations
in the range of 0.005–0.05 M. The inset shows plots obtained by the computer simulation for 0.0003 and 0.0006 M l -alanine concentrations
Fig. 10.3 Double reciprocal plots for RML-catalysed l -alanyl– d -glucose reaction: 1/ v versus 1/[ l -alanine] plots; a series of plots shows the effects of varying l -alanine concentration at different fi xed d -glucose concentrations
in the range of 0.005–0.025 M; plots shown for 0.0003 and 0.0006 M d -glucose concentrations are from computer simulation
22910.3 Esterification Kinetics
Fig. 10.4 Double reciprocal plots for CRL-catalysed l -alanyl– d -glucose reaction: 1/ v versus 1/[ l -alanine] plots 1/ v versus 1/[ d -glucose] plots; a series of plots show the effect of varying d -glucose concentration at different fi xed
l -alanine concentrations in the range of 0.005–0.1 M; the plots shown for 0.0003 and 0.0006 M l -alanine concentra-tions are from computer simulation
Fig. 10.5 Double reciprocal plots for CRL-catalysed l -alanyl– d -glucose reaction: 1/ v versus 1/[ l -alanine]; a series of plots show the effect of varying l -alanine con-centrations at different fi xed d -glucose concentrations in
the range of 0.005–0.05 M. The plots shown for 0.0003 and 0.0006 M d -glucose concentrations are from the computer simulation
230 10 Kinetics of Some Selected Enzyme-Catalysed Reactions in Organic Solvents
steps, (Scheme 10.1 ) with competitive substrate inhibition that lead to dead-end inhibition (Segel 1993 ) . Both RML and CRL were found to be inhibited by d -glucose.
This model could be described by the follow-ing rate equation:
(10.1)
where v is initial rate, V max
is maximum velocity, [ A ] is l -alanine concentration, [ B ] is d -glucose concentration, K
mA is Michaelis–Menten con-
stant for the lipase– l -alanine complex, K i is dis-
sociation constant for the lipase–inhibitor ( d -glucose) complex and K
mB is Michaelis–
Menten constant for the lipase– d -glucose com-plex. Because the initial rates are in M h −1 (mg protein) −1 , V
max is expressed as k
cat = V
max /enzyme
concentration.
maxmA mB
i
[ ][ ]
[ ][ ] 1 [ ] [ ][ ]
=⎛ ⎞
+ + +⎜ ⎟⎝ ⎠
v A B
V BK B K A A B
K
Fig. 10.6 Replot of slopes obtained from Fig. 10.3 versus [ d -glucose] (RML)
Fig. 10.7 Replot of slopes obtained from Fig. 10.5 versus [ d -glucose] (CRL)
23110.3 Esterification Kinetics
The apparent values of the four important kinetic parameters K
i d -glucose , K
m l -alanine , K
m d -glucose
and k cat
were graphically evaluated. The intercepts of the positive slopes of the curves in Figs. 10.2 and 10.4 on the Y -axis, particularly at the highest l -alanine concentration (0.05 M/0.1 M) employed, gave 1/ k
cat for RML and CRL (Table 10.1 ).
Figure 10.5 (RML) and Fig. 10.6 (CRL) show a replot of the slopes from Figs. 10.3 and 10.5 ver-sus [ d -glucose], respectively, for which slope = K
m l -alanine /( k
cat K
i ), Y-intercept = K
m l -alanine / k
cat and
X-intercept = - K i , where K
i represents the dissoci-
ation constant for the lipase– d -glucose complex. K
m d -glucose was obtained using Eq. 10.2 derived by
rearranging Eq. 10.1 :
(10.2)
where K mB
= Michaelis–Menten constant for the lipase– d -glucose complex.
To con fi rm that the kinetics of the RML- and CRL-catalysed syntheses of l -alanyl– d -glucose follows the above-mentioned model, the apparent values of the four important kinetic parameters k
cat , K
i , K
mA and K
mB were also estimated through
curve- fi tting using Eq. 10.1 . The range of values tested for these parame-
ters and the constraints employed for the iteration
procedure are as follows: k cat
< 1 M h −1 mg −1 , K
i d -glucose > K
m d -glucose , K
m d -glucose < K
m l -alanine and
K m l -alanine
< 10 M. The iteration procedure for the curve- fi tting
method involved non-linear optimisation through minimising the sum of squares of deviations between v
exptl and v
pred , such that values for the
four kinetic parameters mentioned above corre-spond to the best fi t achieved.
Table 10.1 lists graphical as well as the curve- fi tted values for comparison. Tables 10.2 and 10.3 show the comparison between experimental and predictive initial rate obtained under different reac-tion conditions. Although computer-simulated v
pred
values showed R 2 values of 0.84 for RML and 0.86 for CRL, the discrepancy between v
exptl and v
pred
appeared to be signi fi cant at several substrate con-centrations. This could be due to (1) the constraints employed in the iteration procedure (curve- fi tting method), which limits the fl exibility required to examine the real system in solution; (2) the error in the experimental graphical methods based on HPLC measurements, which itself could involve errors of the order of ±10–16%; and (3) the hetero-geneous experimental conditions employed involv-ing undissolved carbohydrate and enzyme, on one hand, and dissolved amino acid, on the other, in dichloromethane containing dimethylformamide in a 50:1 proportion.
2cat mA mA
mBi
[ ] [ ] [ ][ ]
[ ] [ ]
k B K B K BK B
v A A K= − − −
A B
EF
P Q
E
EB BKi
(FB EQ)(EA FP)
Scheme 10.1 Ping-Pong Bi-Bi mechanism of RML and CRL-catalysed synthesis of l -alanyl d -glucose showing inhibition by d -glucose (Segel 1993 )
Table 10.1 Apparent values of kinetic parameters for RML- and CRL-catalysed synthesis of l -alanyl– d -glucose
Name of the lipase k cat
× 10 3 (M h −1 mg −1 ) K mA
× 10 3 (M) K mB
× 10 3 (M) K i × 10 3 (M)
RML a 0.29 ± 0.028 4.9 ± 0.51 0.21 ± 0.018 1.76 ± 0.19 b 0.4 ± 0.038 11.2 ± 1.23 0 ± 0.96 5.5 ± 0.59
CRL a 0.75 ± 0.08 56.2 ± 5.7 16.2 ± 1.8 21.0 ± 1.9 b 1.0 ± 0.11 56.2 ± 5.4 16.1 ± 1.5 21.0 ± 2.3
A l -alanine, B d -glucose, a graphical method, b curve- fi tted values
232 10 Kinetics of Some Selected Enzyme-Catalysed Reactions in Organic Solvents
Table 10.2 Experimental and predicted initial rate values for the synthesis of l -alanyl– d -glucose by RML
l -Alanine (M) d -Glucose (M) v
experimental × 10 6
(M h −1 mg −1 ) a v
predictive × 10 6
(M h −1 mg −1 ) b
0.005 0.005 30 51 0.005 0.01 37 45 0.005 0.015 28 37 0.005 0.02 25 31 0.005 0.025 15 27 0.01 0.005 38 73 0.01 0.01 51 72 0.01 0.015 55 64 0.01 0.02 49 56 0.01 0.025 55 49 0.015 0.005 48 84 0.015 0.01 46 91 0.015 0.015 72 84 0.015 0.02 91 75 0.015 0.025 88 67 0.02 0.005 65 92 0.02 0.01 97 104 0.02 0.015 41 99 0.02 0.02 76 91 0.02 0.025 127 83 0.025 0.005 71 97 0.025 0.01 146 114 0.025 0.015 116 112 0.025 0.02 132 104 0.025 0.025 111 96 0.05 0.005 79 109 0.05 0.01 176 142 0.05 0.015 158 149 0.05 0.02 176 147 0.05 0.025 176 141
a Graphical method b Curve- fi tted values
10.3.2 Esteri fi cation Kinetics of L -Phenylalanine and D -Glucose: Double Substrate Inhibition
Comparative kinetic investigations were carried out involving lipases from Rhizomucor miehei and Candida rugosa on the esteri fi cation reaction between l -phenylalanine and d -glucose to form l -phenylalanyl– d -glucose (Lohith et al. 2007 ) . The results from the investigations using RML are described below.
R 2 values obtained from least-square analysis for the initial velocities in both cases were found to be 0.85–0.95. In case of RML-catalysed reac-tions, initial velocities ( v ) were found to be in the range 35 × 10 −6 to 245 × 10 −6 M h −1 (mg protein) −1 , and CRL-catalysed experiments showed initial velocities in the range 40 × 10 −6 to 325 × 10 −6 M h −1 (mg protein) −1 .
Double reciprocal plots were constructed, and the reciprocal plot patterns for the two substrates in RML-catalysed reactions are almost symmet-
23310.3 Esterification Kinetics
rical (Figs. 10.8 and 10.9 ). The double reciprocal plots shown in the present work were constructed from all the experimentally determined initial rate values and few initial rate values determined from the curve- fi tting procedure. Figure 10.8 shows 1/ v versus 1/[ d -glucose] plots, exhibiting series of curves obtained for different fi xed level concentrations of l -phenylalanine at varying d -glucose concentrations. Figure 10.9 shows 1/ v versus 1/[ l -phenylalanine] plots, exhibiting series of curves obtained for different fi xed level
concentrations of d -glucose at varying l -pheny-lalanine concentrations. Figures 10.10 and 10.11 show the replots of slopes from Figs. 10.8 and 10.9 , respectively.
The plots in Figs. 10.8 and 10.9 showed that the kinetics could be best described by (Segel 1993 ) Ping-Pong Bi-Bi model with competitive double substrate inhibition leading to dead-end inhibition by RML– d -glucose complex and l -phenylalanyl–RML– l -phenylalanine complex (Scheme 10.1 ).
Table 10.3 Experimental and predicted initial rate values for the synthesis of l -alanyl– d -glucose by CRL
l -Alanine (M) d -Glucose (M) v
experimental × 10 6
(M h −1 mg −1 ) a v
predictive × 10 6
(M h −1 mg −1 ) b
0.005 0.005 35 55 0.005 0.01 38 52 0.005 0.02 37 42 0.005 0.035 26 32 0.005 0.05 20 25 0.01 0.005 52 89 0.01 0.01 66 92 0.01 0.02 76 78 0.01 0.035 53 61 0.01 0.05 44 49 0.02 0.005 99 130 0.02 0.01 157 148 0.02 0.02 158 137 0.02 0.035 162 112 0.02 0.05 90 92 0.035 0.005 164 161 0.035 0.01 275 201 0.035 0.02 267 202 0.035 0.035 168 174 0.035 0.05 228 148 0.05 0.005 269 178 0.05 0.01 314 234 0.05 0.02 347 250 0.05 0.035 370 224 0.05 0.05 404 195 0.1 0.005 269 203 0.1 0.01 320 291 0.1 0.02 362 345 0.1 0.035 449 338 0.1 0.05 460 310
a Graphical method b Curve- fi tted values
234 10 Kinetics of Some Selected Enzyme-Catalysed Reactions in Organic Solvents
Fig. 10.8 Double reciprocal plots for RML-catalysed l -phenylalanyl– d -glucose reaction: 1/ v versus 1/[ d -glu-cose], series of curves showing the effect of varying d -glucose concentrations at different fi xed concentrations
of l -phenylalanine in the range 0.005–0.05 M. Inset shows plots obtained from the computer simulation pro-cedure for l -phenylalanine at 0.0003 and 0.0006 M concentrations
Fig. 10.9 Double reciprocal plots for RML-catalysed l -phenylalanyl– d -glucose reaction: 1/ v versus 1/[ l -phenylalanine], series of curves showing effect of varying l -phenylalanine concentrations at different
fi xed concentrations of d -glucose in the 0.005–0.05 M range. Inset shown for 0.0003 M and 0.0006 M concen-trations of d -glucose is from the computer simulation procedure
23510.3 Esterification Kinetics
Fig. 10.10 Replot of slopes of 1/[ l -phenylalanine] versus [ d -glucose] from Fig. 10.8 from RML-catalysed reaction
Fig. 10.11 Replot of slopes of 1/[ d -glucose] versus [ l -phenylalanine] from Fig. 10.9 from RML-catalysed reaction
This model could be described by the following rate equation 10.3 :
(10.3)
where v = initial rate, V max
= maximum velocity, A = l -phenylalanine concentration, B = d -glucose concentration, K
mA = Michaelis–Menten constant for
l -phenylalanine, K mB
= Michaelis–Menten constant for d -glucose, K
iB = dissociation constant of the
RML– d -glucose complex and K iA
= dissociation con-stant of the RML– l -phenylalanine complex. Since the initial rates are in M h −1 mg −1 of the protein, V
max
is expressed as k cat
= V max
/enzyme concentration. The fi ve important kinetic parameters K
i d -glucose ,
K i l -phenylalanine
, K m l -phenylalanine
, K m d -glucose
and k cat
were evaluated graphically. Intercept of the positive slopes of Fig. 10.10 on the Y -axis, especially at the higher concentrations of l -phenylalanine employed,
maxmA mB
iB iA
[ ][ ]
[ ] [ ][ ] 1 [ ] 1 [ ][ ]
=⎛ ⎞ ⎛ ⎞
+ + + +⎜ ⎟ ⎜ ⎟⎝ ⎠ ⎝ ⎠
v A B
V B AK B K A A B
K K
236 10 Kinetics of Some Selected Enzyme-Catalysed Reactions in Organic Solvents
gave 1/ k cat
(Table 10.4 ). From Fig. 10.10 , slope = K
m l -phenylalanine /( k
cat K
iB ), Y -intercept = K
m l -phenylalanine / k
cat
and X -intercept = − K iB.
From Fig. 10.11 , slope = K
m d -glucose /( k
cat K
iA ), Y -intercept = K
m d -glucose / k
cat and
X -intercept = − K iA
, where K iA
and K iB
represent dis-sociation constant for RML– l -phenylalanine and RML– d -glucose complexes, respectively.
As a con fi rmatory approach, the values of the fi ve important kinetic parameters K
mA , K
mB , k
cat ,
K iA
and K iB
were also estimated through a curve- fi tting procedure by using Eq. 10.3 . The range of values tested for these parameters and the con-straints employed for the iteration procedure are k
cat d -glucose < 10 mM h −1 mg −1 , K
i d -glucose > K
m d -glucose ,
K m d -glucose
< K m l -phenylalanine
and K m l -phenylalanine
< 90 mM. The iteration procedure involved determination of initial velocities ( v
pred ) by incrementing the above-
mentioned fi ve kinetic parameters in Eq. 10.3 from their lowest approximations (bound by the above-mentioned constraints) and subjecting the v
pred
(obtained for all the concentrations of d -glucose and l -phenylalanine) to non-linear optimisation, by minimising the sum of squares of deviations between v
pred and v
exptl. The set of fi ve kinetic
parameters which resulted in minimum sum of squares of deviation between v
pred and v
exptl were
considered the best set (Table 10.4 ). Table 10.5 shows the comparison between experimental and predictive initial rate values obtained under differ-ent reaction conditions. Computer-simulated v
pred
values showed an R 2 value of 0.65.
10.3.3 Esteri fi cation Kinetics of L -Phenylalanine and D -Glucose: Single Substrate Inhibition
The results from the above kinetic studies using Candida rugosa lipase are described below.
Double reciprocal plots constructed for the CRL-catalysed reaction showed that Figs. 10.12 and 10.13 are not similar unlike those of the RML-catalysed reaction. Figure 10.12 repre-sents 1/ v versus 1/[ d -glucose], and Fig. 10.13 represents 1/ v versus 1/[ l -phenylalanine]. Figure 10.14 shows the replot of slopes from Fig. 10.12 . Figure 10.12 shows series of curves obtained for different fi xed levels concentra-tions of l -phenylalanine at varying d -glucose concentrations where slight increase in rates is observed at lower d -glucose concentrations and, at higher concentrations of d -glucose, the rates reduce drastically. Figure 10.13 shows series of parallel lines for different fi xed lower d -glucose concentrations at various l -phenyla-lanine concentrations, and this changes to lines with different slopes at higher d -glucose concentrations.
Here also, the plots in Figs. 10.12 and 10.13 showed that the kinetics could be best described by (Segel 1993 ) Ping-Pong Bi-Bi model (Scheme 10.1 ), however, with a difference that only one substrate ( d -glucose) functions as a competitive inhibitor forming dead-end CRL– d -glucose complex.
This model could be described by the follow-ing rate equation 10.1 .
The four important kinetic parameters K i d -glu-
cose, K
m l -phenylalanine , K
m d -glucose and k
cat were evaluated
graphically. Intercept of the positive slopes of Fig. 10.12 on the Y -axis, especially at the higher concentration of l -phenylalanine, gave 1/ k
cat
(Table 10.6 ). From Fig. 10.14 , slope = K m l -phenylalanine
/( k
cat K
i ), Y -intercept = K
m l -phenylalanine / k
cat and
X -intercept = − K i where K
i represents dissociation
constant for the CRL– d -glucose complex. K
m d -glucose was obtained from Eq. 10.2 obtained by
rearranging Eq. 10.1 .
Table 10.4 Apparent values of kinetic parameters for RML-catalysed synthesis of l -phenylalanyl– d -glucose
Name of the compound k
cat (mM h −1
(mg protein) −1 ) K mA
(mM) K mB
(mM) K iA
(mM) K iB
(mM)
l -Phenylalanyl– d -Glucose a 0.13 ± 0.002 3.13 ± 0.33 2.13 ± 0.2 6.0 ± 0.58 9.0 ± 0.86 b 2.24 ± 0.23 95.6 ± 9.7 80.0 ± 8.5 90.0 ± 9.2 13.6 ± 1.42
A l -phenylalanine, B d -glucose, a graphical method, b computer-simulated values
23710.3 Esterification Kinetics
Here also, a curve- fi tting procedure was car-ried out using Eq. 10.1 to estimate k
cat, K
i, K
mA and
K mB,
to con fi rm that the kinetics of CRL-catalysed l -phenylalanyl– d -glucose reactions followed the above-mentioned model. The range of values tested and the constraints employed are k
cat d -glucose <
1 mM h −1 mg −1 , K i d -glucose
> K m d -glucose
, K m d -glucose
< K m l -phenylalanine
and K m l -phenylalanine
< 10 mM. Here also, the iteration procedure was the same as that employed for the RML-catalysed reaction. Table 10.6 lists graphical as well as the computer-simulated values for comparison. Table 10.7 shows the comparison between experimental and predictive initial rate values obtained under
Table 10.5 Experimental and predicted initial rate values for the synthesis of l -phenylalanyl– d -glucose by RML
l -Phenylalanine conc. (M)
d -Glucose conc. (M)
K cat-experimental
a (10 −6 M h −1 (mg protein) −1 )
K cat-predictive
b (10 −6 M h −1 (mg protein) −1 )
0.005 0.005 46 51 0.005 0.01 79 53 0.005 0.015 54 48 0.005 0.02 35 43 0.005 0.025 50 38 0.005 0.050 40 24 0.01 0.005 75 70 0.01 0.01 75 84 0.01 0.015 74 83 0.01 0.02 135 77 0.01 0.025 123 71 0.01 0.050 46 47 0.015 0.005 109 79 0.015 0.01 100 705 0.015 0.015 116 709 0.015 0.02 117 105 0.015 0.025 113 98 0.015 0.050 67 69 0.02 0.005 83 83 0.02 0.01 107 117 0.02 0.015 89 127 0.02 0.02 96 126 0.02 0.025 105 121 0.02 0.050 84 89 0.025 0.005 51 84 0.025 0.01 74 125 0.025 0.015 153 141 0.025 0.02 123 144 0.025 0.025 116 140 0.025 0.050 86 107 0.05 0.01 92 133 0.05 0.015 245 168 0.05 0.02 224 187 0.05 0.025 186 196
a Graphical method b Curve- fi tted values Average absolute deviation in initial velocities – 21.7%
238 10 Kinetics of Some Selected Enzyme-Catalysed Reactions in Organic Solvents
Fig. 10.12 Double reciprocal plots for CRL-catalysed l -phenylalanyl– d -glucose reaction: 1/ v versus 1/[ d -glu-cose], series of curves showing the effect of varying d -glucose concentrations at different fi xed concentrations
of l -phenylalanine in the 0.005–0.05 M concentration range. Inset shown for 0.0003 and 0.0006 M concentra-tions of l -phenylalanine is from the computer simulation procedure
Fig. 10.13 Double reciprocal plots for CRL-catalysed l -phenylalanyl– d -glucose reaction: 1/ v versus 1/[ l -phenylalanine], series of plots showing effect of varying l -phenylalanine concentrations at different fi xed concen-
trations of d -glucose in the 0.005–0.025 M concentration range, and the plots for 0.0003 and 0.0006 M concentra-tions of d -glucose are from the computer simulation procedure
23910.4 Glycosylation Kinetics
different reaction conditions. Computer-simulated v
pred values showed an R 2 of value 0.63.
In case of CRL, at increasing fi xed concen-trations of l -phenylalanine (Fig. 10.12 ), the rate increases at lower concentrations of d -glucose. At higher concentrations of d -glu-cose corresponding to minimum 1/ v , the rate decreases; the plots tend to become closer to 1/ v axis. Figure 10.13 (CRL) also re fl ects the same behaviour, where at lower concentra-tions of d -glucose, the plots appear parallel probably so for as K
i > K
mB . However, at higher
fi xed concentrations of d -glucose, the slopes vary drastically. Thus the kinetic data clearly shows the inhibitory nature of d -glucose in this reaction.
10.4 Glycosylation Kinetics
10.4.1 Glycosylation Kinetics of Curcumin and D -Glucose: Single Substrate Inhibition
Typical rate plot for the curcumin glucosidic reaction is shown in Fig. 10.15 (Sivakumar et al. 2006 ) . Initial velocities ( v ) were found to be in the range 0.08 to 5.2 × 10 −5 M h −1 (mg protein) −1 .
Double reciprocal plots were constructed by plotting 1/ v versus 1/[curcumin] and 1/ v versus 1/[ d -glucose]. The plots are shown in Figs. 10.16 and 10.17 . The plots in Figs. 10.16 and 10.17 showed that the kinetics could be best described
Fig. 10.14 Replot of slopes of 1/[ l -phenylalanine] versus [ d -glucose] from Fig. 10.12 from CRL-catalysed reaction
Table 10.6 Apparent values of kinetic parameters for CRL-catalysed synthesis of l -phenylalanyl– d -glucose
Name of the compound k cat
(mM h −1 (mg protein) −1 ) K
mA (mM) K
mB (mM) K
iB (mM)
l -Phenylalanyl– d -glucose a 0.40 ± 0.005 5.6 ± 0.58 6.48 ± 0.69 7.0 ± 0.73 b 0.51 ± 0.06 0 ± 0.98 6.0 ± 0.64 8.5 ± 0.81
A l -phenylalanine, B d -glucose, a graphical method, b computer-simulated values
240 10 Kinetics of Some Selected Enzyme-Catalysed Reactions in Organic Solvents
by (Segel 1993 ) Ping-Pong Bi-Bi model (Scheme 10.1 ) with competitive substrate inhibi-tion leading to dead-end inhibition.
The rate equation describing the model is shown in Eq. 10.1 .
The four important kinetic parameters K curcumin
, K
m d -glucose , K
m curcumin and k
cat curcumin were evaluated
graphically. Intercept of the positive slopes of Fig. 10.16 on the Y -axis, especially at the highest concentration of d -glucose (0.1 M) employed,
gave 1/k cat
for curcumin (Table 10.8 ). Figure 10.18 shows the replot of slopes (Fig. 10.17 ) of 1/[ d -glucose] versus [curcumin], from which slope = K
m d -glucose /( k
cat K
i ), Y -intercept = K
m d -glucose /
k cat
and X -intercept = − K i , where K
i represents dis-
sociation constant for the amyloglucosidase–cur-cumin complex. K
m curcumin was obtained by
Eq. 10.2 generated by rearranging Eq. 10.1 . The values of the four important kinetic
parameters, k cat,
K i , K
mA and K
mB , were also
Table 10.7 Experimental and predicted initial rate values for the synthesis of l -phenylalanyl– d -glucose by CRL
l -Phenylalanine conc. (M)
d -Glucose conc. (M)
K cat-experimental
(10 −6 M h −1 (mg protein) −1 ) a
K cat-predictive
(10 −6 M h −1 (mg protein) −1 ) b
0.005 0.005 40 96 0.005 0.01 67 86 0.005 0.015 90 74 0.005 0.02 89 64 0.005 0.025 44 56 0.01 0.005 56 136 0.01 0.01 92 136 0.01 0.015 123 123 0.01 0.02 133 110 0.01 0.025 62 99 0.015 0.005 85 158 0.015 0.01 187 168 0.015 0.015 173 158 0.015 0.02 155 145 0.015 0.025 128 133 0.02 0.005 93 171 0.02 0.01 224 191 0.02 0.015 317 185 0.02 0.02 100 173 0.02 0.025 213 160 0.025 0.005 155 181 0.025 0.01 243 208 0.025 0.015 155 205 0.025 0.02 100 195 0.025 0.025 191 182 0.05 0.005 256 204 0.05 0.01 325 252 0.05 0.015 258 263 0.05 0.02 269 261 0.05 0.025 291 253
a Graphical method b Curve- fi tted values Average absolute deviation in initial velocities – 26.3%
24110.4 Glycosylation Kinetics
0.000
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0 5 10 15 20 25 30
Con
vers
ion
yiel
d (M
)
Time (h)
Fig. 10.15 Initial rate ( v ) plot – conversion yields versus incubation period – curcuminyl-bis- a - d -glucoside: d -glucose 20 mM, curcumin 5 mM, amyloglucosidase 90 mg and 0.6 mL (0.06 mM) of 0.01 M, pH 6.0 phosphate buffer
Fig. 10.16 Double reciprocal plot: 1/ v versus 1/[cur-cumin], series of plots from experimentally measured initial rate values showing the effect of varying cur-cumin concentrations at different fi xed concentra-
tions of d -glucose in the range 5 mM to 0.1 M. Insets show plots obtained from the computer simulation procedure for 0.3 and 0.6 mM concentrations of d -glucose
242 10 Kinetics of Some Selected Enzyme-Catalysed Reactions in Organic Solvents
Fig. 10.17 Double reciprocal plot: 1/ v versus 1/[ d -glucose], series of plots from experimentally mea-sured initial rate values showing the effect of varying d -glucose concentrations at different fi xed concentra-
tions of curcumin in the range 5 mM to 0.025 M. The plots shown for 0.3 and 0.6 mM concentrations of curcumin are from the computer simulation procedure
Table 10.8 Kinetic parameters for the synthesis of curcuminyl-bis- a - d -glucoside
Name of the compound k cat
(10 −5 M h −1 mg −1 ) K mA
(mM) K mB
(mM) K i (mM)
Curcuminyl-bis- a - d -glucoside
a 5.0 ± 0.48 88.9 ± 8.7 25.1 ± 2.3 7.0 ± 0.75 b 6.07 ± 0.58 0 ± 0.9 4.6 ± 0.5 3.0 ± 0.28
A = d -glucose, B = curcumin, a graphical method, b computer-simulated values
0
2000
4000
6000
8000
-0.01 -0.005 0 0.005 0.01 0.015 0.02 0.025 0.03
Slo
pe 1
/[D-g
luco
se]
[Curcumin], M
Fig. 10.18 Replot of slope (from Fig. 10.17 ) – slope 1/[ d -glucose] versus [curcumin]
24310.4 Glycosylation Kinetics
estimated mathematically through computer simulation as described earlier. The range of values tested for these parameters and the constraints employed are k
cat curcumin < 0.01 M h −1 mg −1 , K
i cur-
cumin < K
m curcumin, K
m curcumin < K
m d -glucose and K
m d -glu-
cose < 0.1 M. The set of four kinetic parameters
which resulted from minimum sum of squares of deviation between v
pred and v
exptl were considered
to be the best set, and they are shown in Table 10.8 which lists graphical as well as the computer-simulated values for comparison. Table 10.9 shows the comparison between experimental and predictive initial rate values obtained under
different reaction conditions. Computer simulation showed v
predicted values with R 2 value of 0.83
emphasising that this model is reasonably good in explaining the kinetics of this reaction.
Here also, with increasing concentrations of d -glucose (Fig. 10.16 ), the rate increases at lower concentrations of curcumin. At higher concentra-tions of curcumin corresponding to minimum 1/ v , the rate decreases; the plots tend to become closer to 1/ v axis. Figure 10.17 also re fl ects the same behaviour, where at lower concentrations of cur-cumin, the lines appear parallel probably so for as K
i > K
mB . However, at higher fi xed concentrations
Table 10.9 Experimental and predicted initial rate values for the synthesis of curcuminyl-bis- a - d -glucoside
d -Glucose (M) Curcumin (M) v
experimental
(10 −5 M h −1 mg −1 ) v
predictive
(10 −5 M h −1 mg −1 )
0.005 0.005 0.153 0.837 0.005 0.01 0.105 0.600 0.005 0.015 0.084 0.456 0.005 0.02 0.076 0.367 0.005 0.025 0.250 0.306 0.01 0.005 0.238 1.324 0.01 0.01 0.336 1.048 0.01 0.015 0.225 0.831 0.01 0.02 0.153 0.682 0.01 0.025 0.153 0.577 0.015 0.005 0.352 1.643 0.015 0.01 0.543 1.396 0.015 0.015 0.411 1.144 0.015 0.02 0.611 0.957 0.015 0.025 1.204 0.820 0.02 0.005 0.723 1.867 0.02 0.01 1.168 1.674 0.02 0.015 1.835 1.410 0.02 0.02 0.795 1.199 0.02 0.025 1.223 1.038 0.025 0.005 1.456 2.034 0.025 0.01 2.202 1.902 0.025 0.015 1.758 1.638 0.025 0.02 0.568 1.413 0.025 0.025 0.847 1.235 0.1 0.005 3.000 2.779 0.1 0.01 4.580 3.208 0.1 0.015 5.230 3.185 0.1 0.02 2.820 3.041 0.1 0.025 4.580 2.868
244 10 Kinetics of Some Selected Enzyme-Catalysed Reactions in Organic Solvents
of curcumin, the slopes vary drastically where K
i < K
mB . Thus the kinetic data clearly shows the
inhibitory nature of curcumin in this reaction. Competition between d -glucose and curcumin for the active site (binding site) of amyloglucosidase could result in predominant curcumin binding at higher concentrations, displacing d -glucose, lead-ing to the formation of the dead-end amyloglu-cosidase–curcumin complex.
10.4.2 Glucosylation Kinetics of Vanillin and D -Glucose: Single Substrate Inhibition
Here also amyloglucosidase was employed (Sivakumar et al. 2006 ) . To graphically evaluate the apparent values of the kinetic parameters V
max,
K i , K
m vanillin and K
m d -glucose , initial rates (speci fi c
reaction rate) were evaluated by measuring of 4- O -( d -glucopyranosyl)vanillin formation at dif-ferent incubation periods. For each concentration of vanillin (5 mM–0.1 M) and d -glucose (5 mM–0.1 M), individual experiments were performed for incubation periods of 3, 6, 12 and 24 h (30 × 4 for each system). R 2 values obtained from least-square analysis for the initial veloci-ties in both cases were found to be around 0.95. The plots shown in this work were constructed from all the experimentally determined and few computer-generated initial rate values. A typical rate plot for vanillin glucosidic reactions is shown in Fig. 10.19 , and the initial velocities ( v ) were found to be in the range 0.17 to 5 × 10 −5 M h −1 (mg protein) −1 . The enzyme lost only 10% of its activ-ity after incubation for 24 h.
Double reciprocal plot was constructed by plotting 1/ v versus 1/[vanillin]. The plot is shown in Fig. 10.20 , which shows a series of curves obtained for different fi xed concentrations of d -glucose at varying vanillin concentrations, where slight increase in initial rates at lower van-illin concentrations is observed and at higher concentrations of vanillin the rates reduce drasti-cally. Figure 10.21 shows a series of lines obtained for different fi xed concentrations of vanillin at varying d -glucose concentrations where at fi xed lower vanillin concentrations, the lines were par-allel and at fi xed higher vanillin concentrations, lines with different slopes were observed. The plots in Figs. 10.20 and 10.21 showed that the kinetics could be best described by (Segel 1993 ) Ping-Pong Bi-Bi model (Scheme 10.1 ) with com-petitive substrate inhibition leading to dead-end inhibition (Eq. 10.1 ).
The four important kinetic parameters K i vanillin
, K
m d -glucose , K
m vanillin and k
cat vanillin were evaluated
graphically. Intercept of the positive slope of Fig. 10.20 on the Y -axis, especially at the highest concentration of d -glucose (0.1 M) employed, gave 1/ k
cat for vanillin (Table 10.10 ).
Figure 10.22 shows the replot of slope of Fig. 10.21 (1/[ d -glucose] versus [vanillin] plot) from which slope = K
m d -glucose /( k
cat K
i ),
Y-intercept = K m d -glucose
/ k cat
and X-intercept = - K i ,
where K i represents dissociation constant for the
amyloglucosidase–vanillin complex. K m vanillin
was obtained from Eq. 10.2 generated by rear-ranging Eq. 10.1 .
The values of the four important kinetic parameters, k
cat, K
i , K
mA and K
mB , were also esti-
mated mathematically through computer simula-
0.000
0.001
0.002
0.003
0.004
0 10 20 30
Con
vers
ion
yiel
d (M
)
Time (h)
Fig. 10.19 Initial rate ( v ) plot: d -glucose 10 mM, vanillin 5 mM, amyloglucosi-dase 90 mg and 0.1 mM (1 mL) of 0.01 M, pH 4 acetate buffer
24510.4 Glycosylation Kinetics
Fig. 10.20 Double reciprocal plot: 1/ v versus 1/[vanil-lin]. Series of plots from experimentally measured ini-tial rate values showing the effect of varying vanillin concentrations at different fi xed concentrations of
d -glucose in the 5 mM to 0.1 M range. Insets show plots obtained from the computer simulation pro-cedure for 0.3 and 0.6 mM concentrations of d -glucose
Fig. 10.21 Double reciprocal plot: 1/ v versus 1/[ d -glucose]. Series of plots from experimentally measured initial rate values showing the effect of varying d -glucose concentrations at different fi xed
concentrations of vanillin in the 5 mM to 0.05 M range. The plots shown for 0.3 and 0.6 mM concen-trations of vanillin are from the computer simulation procedure
Table 10.10 Kinetic parameters for the synthesis of 4- O -( d -glucopyranosyl)vanillin
k cat
10 −5 M h −1 mg −1 K m d -glucose
(mM) K m vanillin
(mM) K i (mM)
Graphical method 0 ± 1 65.0 ± 6.7 45.6 ± 4.4 12.5 ± 1.3 Computer-simulated values 35.0 ± 3.2 60.0 ± 6.2 50.0 ± 4.8 5 ± 1.1
246 10 Kinetics of Some Selected Enzyme-Catalysed Reactions in Organic Solvents
tion. The range of values tested for these parameters and the constraints employed for the iteration procedure are as follows: k
cat vanil-
lin < 0.01 M h −1 mg −1 , K
i vanillin < K
m vanillin, K
m vanillin < K
m
d -glucose and K
m d -glucose < 0.1 M. The set of four
kinetic parameters which resulted from minimum sum of squares of deviation between v
pred and v
exptl
were considered to be the best set, and they are shown in Table 10.10 which lists graphical as well as the computer-simulated values for com-parison. Table 10.11 shows the comparison between experimental and predictive initial rate values obtained under different reaction condi-tions. Computer simulation showed v
pred values
with R 2 values of 0.85 for vanillin reaction empha-sising that this model is reasonably good in explaining the kinetics of this reaction.
10.5 Discussion
10.5.1 Esteri fi cation Kinetics
In case of l -alanine and d -glucose reaction, the kinetic data clearly shows the inhibitory nature of d -glucose towards both RML and CRL. With increasing l -alanine concentration (Fig. 10.2 for RML and Fig. 10.4 for CRL), the initial rate increases with decreasing d -glucose concentra-tion. With increasing d -glucose concentration up to the minimum 1/ v , the initial rate decreases, and
the plots tend to become closer to the 1/ v axis ( Y -axis).
Figure 10.3 (RML) and Fig. 10.5 (CRL) also show the same behaviour, in which at low d -glucose concentrations, the plots appear paral-lel probably as long as K
i > K
mB is concerned.
However, at high fi xed d -glucose concentrations, the slopes of the plots drastically vary. Thus, in these reactions, the kinetic data clearly shows the inhibitory nature of d -glucose. The competition between l -alanine and d -glucose for the active site (binding site) of lipases (RML/CRL) could result in a predominant binding of d -glucose at high concentrations, displacing l -alanine and thus leading to the formation of the dead-end lipase– d -glucose complex.
For the RML reaction, K mA
(4.9 ± 0.51 × 10 −3 M) is always higher than K
mB (0.21 ± 0.018 × 10 −3 M,
Table 10.1 ), which shows that l -alanine is bound to RML less fi rmly than d -glucose ( K
mA / K
mB = 23.3). A similar behaviour is also
observed with CRL (Table 10.1 ) K mA
(56.2 ± 5.7 × 10 −3 M), K
mB (16.2 ± 1.8 × 10 −3 M)
and K mA
/ K mB
= 3.5. However, the respective val-ues are very much higher for CRL than for RML, indicating that CRL can yield better con-versions than RML. Between RML and CRL, the K
i for d -glucose is lower for RML
(5.5 ± 0.59 × 10 −3 M) than for CRL (21.0 ± 2.3 × 10 −3 M), indicating that the RML is inhibited by d -glucose far more ef fi ciently than
0
1000
2000
3000
4000
-0.02 -0.01 0.00 0.01 0.02 0.03 0.04 0.05 0.06
[Vanillin], M
Slo
pe 1
/[D-g
luco
se]
Fig. 10.22 Replot of slope (from Fig. 10.21 ): 1/[ d -glucose] versus [vanillin]
24710.5 Discussion
CRL. This could also explain the better conversion observed with CRL than with RML.
The kinetic data of l -phenylalanine and d -glu-cose reaction clearly shows the inhibitory nature of both d -glucose and l -phenylalanine towards RML. Competition between l -phenylalanine and d -glu-cose for the active site (binding site) of RML could result in predominant binding of either substrate at their higher concentrations leading to the formation of the dead-end complexes (RML– d -glucose/ l -phenylalanine). K
mA (95.6 ± 9.7 mM) is slightly
higher than K mB
(80.0 ± 8.5 mM, Table 10.4 ), which shows that both substrates possess almost equal
propensity for the reaction ( K mA
/ K mB
= 1.19). This could also inferred from the dissociation constant values, K
i l -phenylalanine (90.0 ± 9.2 mM) > K
i d -glucose
(13.6 ± 1.42 mM). In case of CRL, competition between l -phe-
nylalanine and d -glucose for the active site of CRL could result in predominant binding of d -glucose at higher concentrations, displacing l -phenylalanine, leading to the formation of the dead-end lipase– d -glucose complex. Here also, K
mA (0 ± 0.98 mM) is slightly higher (Table 10.6 )
than K mB
(6.0 ± 0.64 mM, K mA
/ K mB
= 1.67). The respective kinetic parameter values ( K
mA and K
mB )
Table 10.11 Experimental and predicted initial rate values for the synthesis of 4- O d -glucopyranosyl)vanillin
d -Glucose (M) Vanillin (M) v
experimental
(10 −5 M h −1 mg −1 ) v
predictive
(10 −5 M h −1 mg −1 )
0.005 0.005 0.218 1.219 0.005 0.01 0.300 1.189 0.005 0.02 0.280 0.913 0.005 0.035 0.214 0.643 0.005 0.05 0.168 0.492 0.01 0.005 0.506 1.763 0.01 0.01 0.774 1.976 0.01 0.02 0.702 1.672 0.01 0.035 0.422 1.231 0.01 0.05 0.366 0.957 0.02 0.005 1.003 2.269 0.02 0.01 1.746 2.952 0.02 0.02 1.666 2.866 0.02 0.035 1.033 2.269 0.02 0.05 0.917 1.815 0.035 0.005 2.069 2.587 0.035 0.01 4.128 3.745 0.035 0.02 2.757 4.128 0.035 0.035 1.987 3.551 0.035 0.05 1.774 2.947 0.05 0.005 3.475 2.741 0.05 0.01 1.618 4.195 0.05 0.02 4.968 5.010 0.05 0.035 3.027 4.588 0.05 0.05 2.401 3.926 0.1 0.005 6.110 2.945 0.1 0.01 7.340 4.881 0.1 0.02 7.510 6.676 0.1 0.035 9.170 6.961 0.1 0.05 480 6.414
248 10 Kinetics of Some Selected Enzyme-Catalysed Reactions in Organic Solvents
are higher for RML compared to CRL indicating that RML can give better conversions than CRL.
Lipase is a single-domain molecule belonging to the family of a / b -hydrolase proteins (Derewenda et al. 1992 ; Grochulski et al. 1994 ) . Most of the lipases reported contain Ser-His-Asp/Glu catalytic triads in their active site (Grochulski et al. 1994 ) with exceptions like esterases from Streptomyces scabies which contain only Ser 14 and His 283 (Wei et al. 1995 ) .
Both RML and CRL contain amino acids in their active sites capable of forming hydrogen bonds with suitable donor molecules. The cata-lytic triad in RML consists of Ser-144, His-257 and Asp-203 (Brady et al. 1990 ) . CRL contains Ser-209, Glu-341 and His-449 (Grochulski et al. 1994 ) . Brzozowski et al. ( 1991 ) showed in an atomic model of the inhibitor n -hexylchlorophos-phonate ethyl ester–RML complex that in the oxyanion hole, which is directly responsible for the substrate binding, a direct covalent bond for-mation between the nucleophilic O
g of Ser-144
and the phosphorous atom of n -hexylchlorophos-phonate ethyl ester is possible. In CRL, the oxya-nion hole O
g (Ser-209) is formed by the amide
backbones of Gly-123, Gly-124 and Ala-210 through the hydrogen bonding between the amide –CO–NH– and the hydroxyl of Ser-209, which is stabilised by the helix dipole (Grochulski et al. 1994 ) . d -Glucose possesses fi ve hydroxyl groups, and l -alanine possesses carboxyl and amino groups capable of forming hydrogen bonds with polar side chains of amino acids. Ser-144 hydroxyl and Asp-203 carboxyl groups of RML and Ser-209 and Glu-341 of CRL (Grochulski et al. 1993 ) residues are very good candidate molecules for exhibiting hydrogen-bonding interactions.
Between d -glucose and l -alanine/ l -phenylala-nine, the former possesses more hydrogen-bond-ing functional groups than the amino or carboxyl groups of l -alanine/ l -phenylalanine. Ser-144 in RML and Ser-209 in CRL can form hydrogen bonds with the amino N atom of l -alanine as well as the O atom of d -glucose. Because the K
m l -alanine
value is higher than the K m d -glucose
values for both enzymes, d -glucose could strongly bind to these enzymes than l -alanine/ l -phenylalanine.
Zaidi et al. ( 2002 ) reported that the interaction between nylon-immobilised CRL and alcohol through hydrogen bonding could block the nucleophilic site of the enzyme engaged in acyla-tion, leading to inhibition. A similar behaviour can also be envisaged between d -glucose hydroxyl groups and the above-mentioned oxy-gen of serine and the carboxylate groups of glu-tamic acids. Hence, this kinetic study could clearly explain the inhibition of both RML and CRL by d -glucose. Also, for the fi rst time, it has been found that d -glucose could be inhibitor to both lipases at higher concentrations.
10.5.2 Glycosylation Kinetics
In the kinetic reaction between d -glucose and curcumin, K
m d -glucose (0 ± 0.9 mM, Table 10.8 ) is
always higher than K mB
(4.6 ± 0.5 mM) which shows that while glucose binding could lead to product formation, curcumin binding to the active site could result in inhibition of the amyloglu-cosidase activity.
The inhibitory nature of vanillin towards amy-loglucosidase from Rhizopus mould can be deduced clearly from the kinetic data of d -glu-cose and vanillin reaction. With increasing con-centrations of d -glucose (Fig. 10.20 ), the rate increases at lower concentrations of vanillin. At higher concentrations of vanillin corresponding to minimum 1/ v , the rate decreases; the plots tend to become closer to 1/ v axis. Figure 10.21 also re fl ects the same behaviour, where at lower con-centrations of vanillin, the lines appear parallel probably so for as K
i > K
mB . However, at higher
fi xed concentrations of vanillin, the slopes vary drastically where K
i < K
mB . Thus the kinetic data
clearly shows the inhibitory nature of vanillin in this reaction. Competition between d -glucose and vanillin for the active site (binding site) of amyloglucosidase could result in predominant vanillin binding at higher concentrations, displac-ing d -glucose, leading to the formation of the dead-end amyloglucosidase–vanillin complex. In this reaction, K
m d -glucose (60.0 ± 6.2 mM, Table 10.4 )
is always higher than K mB
(50.0 ± 4.8 mM) which shows that while glucose binding could lead to
249References
product formation, vanillin binding to the active site could result in inhibition of the amyloglu-cosidase activity.
Glucoamylases possess ( a / a ) 6 -barrel fold
structure which is different from the ( b / a ) 8 -barrel
fold structure of a -amylase, b -amylase and a -glucosidase (Svensson et al. 1990 ; Aleshin et al. 1992, 1994 ; Chiba 1997 ) . In the catalytic domain, two glutamic acids Glu314 and Glu544 in Rhizopus oryzae (Ashikari et al. 1986 ; Aleshin et al. 1992 ) are reported to be the catalytic amino acid residues directly involved as acid–base cata-lysts in the hydrolytic reaction (Sierks et al. 1990 ; Chiba 1997 ) . It has also been shown that oxocar-benium ion mechanism is the most suitable for the hydrolytic reaction in both ‘retaining’ and ‘inverting’ enzymes (Chiba 1995 ) . Although no decisive mechanism has been proposed so far, for the glycosylation reactions, it is generally believed that the oxocarbenium ion mechanism could be the most probable one.
Catalysis occurs mainly between subsites 1 and 2 of glucoamylase, and the active site of Aspergillus niger glucoamylase is identical to that of Rhizopus oryzae (Stoffer et al. 1995 ) . Sugar OH groups are held fi rmly in the active site subsites 1 and 2 of Rhizopus oryzae through hydrogen bonds with Arg191, Asp192, Leu312, Trp313, Glu314, Glu315 and Arg443 (Ashikari et al. 1986 ; Aleshin et al. 1994 ) . The above-men-tioned residues can also stabilise planar cur-cumin/vanillin bound to the active site through hydrogen bonds. Curcumin/vanillin could form effective hydrogen bonds between the enolic, phenolic OH and the carbonyl group of curcumin/vanillin and the Arg191, Asp192, Trp313, Glu315 and Arg443 residues. Hence, higher concentra-tions of curcumin/vanillin are capable of displac-ing the glucose–oxocarbenium ion from the active site and occupy its position instead, lead-ing thereby to dead-end inhibition. This may not happen at lower concentrations of curcumin/vanillin.
Several lipase-catalysed esteri fi cation reac-tions have been described to follow Ping-Pong Bi-Bi mechanism, which deals with two sub-strates (acid and alcohol) and two products (water and ester). So far, enzyme-mediated glycosylation,
especially the one involving a carbohydrate molecule and an aglycon molecule, has not been reported to follow Ping-Pong Bi-Bi model. This could be the fi rst report of its kind.
References
Aleshin AE, Golubev A, Firsov LM, Honzatko RB (1992) Crystal structure of glucoamylase from Aspergillus awamori var X100 to 2.2-Å resolution. J Biol Chem 267:19291–19298
Aleshin AE, Firsov LM, Honzatko RB (1994) Re fi ned structure for the complex of acarbose with glucoamy-lases from Aspergillus awamori var. X100 to 2.4 A° resolution. J Biol Chem 269:15631–15639
Ashikari T, Nakamura N, Tanaka Y, Kiuchi N, Shibano Y, Tanaka T, Amachi T, Yoshizumi H (1986) Rhizopus raw-starch-degrading glucoamylase: ts cloning and expression in yeast. Agric Biol Chem 50:957–964
Bousquet-Dubouch MP, Graber M, Sousa N, Lamare S, Legoy MD (2001) Alcoholysis catalysed by Candida rugosa lipase B in a gas/solid system obeys a Ping-Pong Bi-Bi mechanism with competitive inhibition by the alcohol substrate and water. Biochem Biophys Acta 1550(1):90–99
Brady L, Brzozowski AM, Derewenda U, Derewenda ZS, Dodson GG, Tolley S, Turkenburg JP, Christiansen L, Huge-Jensen B, Nashkov L, Thim L, Menge U (1990) A serine protease triad forms the catalytic center of triglycerol lipase. Nature 343:767–770
Brzozowski AM, Derewenda U, Derewenda ZS, Dodson GG, Lawson DM, Turkenburg JP, Bjorkling F, Huge-Jensen B, Patkar SA, Thim L (1991) A model for interfacial activation in lipases from the structure of a fungal lipase-inhibitor complex. Nature 351:491–494
Chiba S (1995) Enzyme chemistry and molecular biology of amylase and related enzymes. The amylase research society of Japan (ed). CRC Press, Boca raton/Ann arbor/London/Tokyo, pp 68–82
Chiba S (1997) Molecular mechanism in a -glucosidase and glucoamylase. Biosci Biotech Biochem 61:1233–1239
Chulalaksanaukul W, Condort JS, Delorme P, Willemot RM (1990) Kinetic study of esteri fi cation by immobi-lized lipase in n -hexane. FEBS Lett 276:181–184
Derewenda U, Brzozwski AM, Lawson DM, Derewenda ZS (1992) Catalysis at the interface the anatomy of a conformational change in a triglyceride lipase. Biochemistry 31:1532–1541
Duan G, Ching CB, Lim E, Ang CH (1997) Kinetic study of enantioselective esteri fi cation of ketoprofen with n-propanol catalysed by an lipase in an organic medium. Biotechnol Lett 19:1051–1055
Goto M, Tanigava K, Kanlayakrit W, Hayashida S (1994) The molecular mechanism of binding of glucoamy-lases I from A spergillus awamori var. kawachi to cyclodextrin and raw starch. Biosci Biotech Biochem 58:49–54
250 10 Kinetics of Some Selected Enzyme-Catalysed Reactions in Organic Solvents
Grochulski P, Li Y, Schrag JD, Bouthillier F, Smith P, Harrison D, Rubin B, Cygler M (1993) Insight into interfacial activation from an open structure of Candida rugosa lipase. J Biol Chem 268:12843–12847
Grochulski P, Bouthillier F, Kazlauskas RJ, Serreqi AN, Schrag JD, Ziomek E, Cygler M (1994) Analogs of reaction intermediates identify a unique substrate binding site in Candida rugosa lipase. Biochemistry 33:3494–3500
Hiromi K, Ohnishi M, Tanaka A (1983) Subsite structure and ligand binding mechanism of glucoamylase. Mol Cell Biochem 51:79–95
Janssen AEM, Sjursnes BJ, Vakurov AV, Halling PJ (1999) Kinetics of lipase catalyzed esteri fi cation in organic media correct model and solvent effects on parameters. Enzyme Microb Technol 24:463–470
Kiran KR, Divakar S (2002) Enzyme inhibition by p -cresol and lactic acid in lipase mediated syntheses of p -cresyl acetate and stearoyl lactic acid: a kinetic study. World J Microbiol Biotechnol 18:707–712
Lohith K, Divakar S (2005) Lipase catalysed synthesis of l -phenylalanine esters of d -glucose. J Biotechnol 117:49–56
Lohith K, Manohar B, Divakar S (2007) Competitive inhi-bition by substrates in Rhizomucor miehei and Candida rugosa lipases catalysed esteri fi cation reaction between l -phenylalanine and d -glucose. World J Microbiol Biotechnol 23:955–964
Marty A, Chulalaksananukul W, Condoret JS, Willemont RM, Durand G (1990) Comparison of lipase-catalyzed esteri fi cation in supercritical carbon dioxide and n-hexane. Biotechnol Lett 12(1):11–16
Ohnishi M, Hiromi K (1989) Binding of maltose to Rhizopus niveus glucoamylases in the pH range where the catalytic carboxyl groups are ionized. Carbohyd Res 195:138–144
Rizzi M, Stylos P, Riek A, Reuss M (1992) A kinetic study of immobilized lipase catalyzing the synthesis of isoamyl acetate by transesteri fi cation in n-hexane. Enzyme Microb Technol 14:709–714
Segel IH (1993) Enzyme kinetics, 2nd edn. Wiley, New York, pp 826–882
Sierks MR, Ford C, Reilly PJ, Svensson B (1990) Catalytic mechanism of fungal glucoamylases as de fi ned by muta-genesis of Asp 176, Glu179, and Glu180 in the enzyme from Aspergillus awamori . Protein Eng 3:193–198
Sivakumar R, Vijayakumar GR, Manohar B, Divakar S (2006) Competitive substrate inhibition of amyloglu-cosidase from Rhizopus mold by vanillin and curcumin in respective glucosylation reactions. Biocatal Biotrans 24:299–305
Somashekar BR, Lohith K, Manohar B, Divakar S (2007) Inhibition of Rhizomucor miehei and C andida rugosa lipases by d -glucose in the esteri fi cation reaction between l -alanine and d -glucose. J Biosci Bioeng 103(2):122–128
Stoffer B, Aleshin AE, Firsov LM, Svensson B, Honzatko RB (1995) Re fi ned structure for the complex of d -gluco-dihydroacarbose with glucoamylases from Aspergillus awamori var. X100 to 2.2 Å resolution dual conformation for extended inhibitors bound to the active site of glucoamylases. FEBS Lett 358:57–61
Svensson B, Clarke AJ, Svendsen I, Moller H (1990) Identi fi cation of carboxylic acid residues in glucoamy-lase G2 from Aspergillus niger that participate in the catalysis and substrate binding. Eur J Biochem 18:29–38
Tanaka A, Yamashita T, Ohnishi M, Hiromi K (1983) Steady-state and transient kinetic studies on the bind-ing of maltooligosaccharides to glucoamylases. J Biochem 93:1037–1043
Van-Tol JBA, Odenthal JB, Jongejan JA, Duine JA (1992) Relation of enzyme reaction rate and hydrophobicity of the solvent. In: Tramper J, Vermue MH, Beetink HH, Von-Stocker U (eds) Biocatalysis in non-conven-tional media. Elsevier, Amsterdam, pp 229–235
Wei Y, Schottel JL, Derewenda U, Swenson L, Patkar S, Derewenda ZS (1995) A novel variant of the catalytic triad in the Streptomyces scabies esterase. Nat Struct Biol 2:218–223
Yadav GD, Lathi PS (2004) Synthesis of citronellol lau-rate in organic media catalyzed by immobilized lipases kinetic studies. J Mol Cat B Enzyme 27:113–119
Yadav GD, Devi KM (2004) Immobilized lipase-cataly-sed esteri fi cation and transesteri fi cation reactions in non-aqueous media for the synthesis of tetrahydro-furfuryl butyrate comparison and kinetic modeling. Chem Eng Sci 59:373–383
Yadav GD, Trivedi AH (2003) Kinetic modeling of immo-bilized-lipase catalysed transesteri fi cation of n -octanol with vinyl acetate in non-aqueous media. Enzyme Microb Technol 32:783–789
Zaidi A, Gainer JL, Carta G, Mrani A, Kadiri T, Belarbi Y, Mir A (2002) Esteri fi cation of fatty acids using nylon-immobilized lipase in n -hexane kinetic parameters and chain length effects. J Biotechnol 93:209–216
Zhang T, Yang L, Zhu Z (2005) Determination of internal diffusion limitation and its macroscopic kinetics of the transesteri fi cation of CPB alcohol catalyzed by immo-bilized lipase in organic media. Enzyme Microb Technol 36:203–209
251S. Divakar, Enzymatic Transformation, DOI 10.1007/978-81-322-0873-0_11, © Springer India 2013
11.1 ACE Inhibition by Aminoacyl Esters of Carbohydrates
Angiotensin-converting enzyme (dipeptidyl car-boxypeptidase, EC 3.4.15.1) is a zinc-containing nonspeci fi c dipeptidyl carboxypeptidase widely distributed in mammalian tissues (Li et al. 2004 ) . Angiotensin-converting enzyme (ACE) regulates the blood pressure by modulating renin–angio-tensin system as shown in Scheme 11.1 (Vermeirssen et al. 2002 ) . This enzyme increases the blood pressure by converting the decapeptide angiotensin I into the potent vasoconstricting octapeptide, angiotensin II. Angiotensin II brings about several central effects, all leading to a fur-ther increase in blood pressure. ACE is a multi-functional enzyme that also catalyses the degradation of bradykinin (blood pressure-lower-
ing nanopeptide), and therefore inhibition of ACE results in an overall anti-hypertensive effect (Li et al. 2004 ; Johnston 1992 ) .
Several synthetic drugs and biomolecules are available for ACE inhibition. Captopril is a suc-cessful synthetic anti-hypertensive drug, and similar such molecules like enalapril, perindopril, ceranopril, ramipril, quinapril and fosinopril also show ACE inhibitory activities (Hyuncheol et al. 2003 ; Dae-Gill et al. 2003 ) . The mechanism of ACE inhibition by captopril is shown in Scheme 11.2 (De Lima 1999 ) . The hypothetical representation of inhibitors (hydrolysed products of peptides) binding to the ACE is shown in Scheme 11.3 and also reported that glycine, valine and leucine at the carboxyl terminus of the peptide inhibitor are the potent inhibitors (De
11
Abstract
Since most of the ACE inhibitory drugs are peptides, this chapter deals with exploration of ACE inhibitory activities for some enzymatically syn-thesised l -alanyl, l -valyl, l -leucyl, l -isoleucyl, l -proline, l -phenylala-nine, l -tryptophan and l -histidine esters of carbohydrates and glycosides of n -octanol, phenolic glycosides of guaiacol, eugenol, curcumin, vanillin, N-vanillyl-nonanamide, dl -dopa, dopamine and vitamin glycosides of retinol, thiamin, ribo fl avin, pyridoxine, ergocalciferol, cholecalciferol and a -tocopherol. A few glycosides were also tested for antioxidant activities. These results exhibiting both the phenolic and vitamin glycosides holding promising potential as antioxidants and ACE inhibitors are discussed in terms of structure–function relationship between these compounds and those of the commercially known inhibitors.
ACE Inhibition and Antioxidant Activities of Enzymatically Synthesised Aminoacyl Esters and Glycosides
252 11 ACE Inhibition and Antioxidant Activities of Enzymatically Synthesised…
Rennin-angiotensin systemAngiotensinogen
Kallikrein-Kinin SystemKininogen
Rennin Kallikrein
Angiotensin I
Angiotensin II
BradykininIncreased
prostaglandin synthesis
ACE
Inactive fragments Vasodilatation
Decreased peripheral vascular resistance
Decreased blood pressure
Vasoconstriction Aldosterone secretion
Increased peripheral vascular resistance Increased Na+and
water retention
Increased blood pressure
Scheme 11.1 Role of angiotensin-converting enzyme (ACE) in regulating blood pressure (Li et al . 2004 )
Scheme 11.2 Hypothetical representation of ACE inhibition by captopril binding to the active sites (De Lima 1999 )
Scheme 11.3 Hypothetical representation of ACE active sites and binding of inhibitors (De Lima 1999 )
25311.1 ACE Inhibition by Aminoacyl Esters of Carbohydrates
Lima 1999 ; Wu and Liu 2002 ; Kim et al. 2001, 2003 ) .
Some naturally occurring ‘biologically active peptides’ also act as ACE inhibitors. Deloffre et al. ( 2004 ) reported that a neuropeptide from leach brain showed ACE inhibition with an IC
50
value of 19.8 m M. The N-terminal dipeptide (Tyr-Leu) of b -lactorphin was found to be the most potent inhibitor (Mullally et al. 1996 ) . Many pep-tide inhibitors are derived from different food proteins like Asp-Leu-Pro and Asp-Gly from soy protein hydrolysis (Wu and Liu 2002 ) and Gly-Pro-Leu and Gly-Pro-Val from bovine skin gela-tin hydrolysis (Kim et al. 2001 ) . Cooke et al. ( 2003 ) prepared 4-substituted phenylalanyl esters of alkyl or benzyl derivatives, which exhibited ACE inhibitory activity.
Aminoacyl esters of carbohydrates fi nd wide variety of applications in food and pharmaceuti-cal industries. Aminoacyl esters have not been shown so far to exhibit ACE inhibition activity. Since most of the ACE inhibitory drugs are pep-tides, it was envisaged that the aminoacyl esters of carbohydrates also could possess ACE inhibi-tion activities as they contain aminoacyl groups as part of their structure. Hence, this chapter deals with exploration of ACE inhibition activities for some enzymatically synthesised l -alanyl, l -valyl, l -leucyl, l -isoleucyl, l -proline, l -phenylalanine, l -tryptophan and l -histidine esters of carbohy-drates using lipases in organic media. ACE inhi-bition activity of the above-mentioned aminoacyl
esters of carbohydrates was carried out by the Cushman and Cheung method ( 1969, 1971 ) . Since hippuryl- l -histidyl- l -leucine (HHL) mim-ics the carboxyl dipeptide of angiotensin I, it has been routinely used as the substrate for screening ACE inhibitors.
Underivatised l -amino acids and carbohy-drates were also tested for ACE inhibition as con-trols, and they did not show any ACE inhibitory activities (Vasudeva Kamath et al. 2006 ; Lohith et al. 2006 ) . Only esters showed activities. Isolated ACE inhibitor tested for lipase and pro-tease activity (Table 11.1 ) showed a small extent of protease activity (13.3%) compared to ACE activity but no lipase activity. In presence of aminoacyl esters prepared, the isolated ACE showed 8.9% protease activity (Table 11.1 ) com-pared to the ACE activity. This con fi rmed that the ACE inhibition observed in the presence of amin-oacyl esters prepared is more due to ACE inhibi-tion rather than protease inhibition.
Figure 11.1 shows a typical ACE inhibition plot for captopril which showed an IC
50 value of
0.060 ± 0.006 mM. ACE inhibition plots for all the tested esters, such as carbohydrate esters of l -alanine (Fig. 11.2 ), l -valine (Fig. 11.3 ), l -leu-cine (Fig. 11.4 ) and l -isoleucine (Fig. 11.5 ), l -prolyl esters (Fig. 11.6 ), l -phenylalanyl esters (Fig. 11.7 ), l -tryptophanyl esters (Fig. 11.8 ) and l -histidyl esters (Fig. 11.9 ), are shown. Tables 11.2 and 11.3 show the compounds tested, their conversion yields from the respective enzy-
Table 11.1 Protease inhibition assay for d -glucose ester a
System Protease activity (min −1 mg −1 enzyme protein b )
Percentage of protease activity with respect to ACE activity c
Control ACE – 0.5 mL + 0.5 mL of 0.6% haemoglobin + 0.5 mL Buffer
0.0436 13.3
l -Isoleucyl- d -glucose – 0.5 mL ester + ACE – 0.5 mL + 0.5 mL of 0.6% haemoglobin
0.0267 8.9
a Conditions: ACE – 0.5 mL (0.5 mg). All the solutions were prepared in 0.1 M tris-HCl (pH 7.5): incubation period, 30 min; temperature, 37°C; 0.5 mL of 10% trichloroacetic acid added to arrest the reaction; Blank performed without enzyme and ester; absorbance measured at 440 nm; ester – 0.5 mL of 0.8 mM b Average absorbance values from three individual experiments c Percentage protease activity with respect to an ACE activity of 0.327 m mol (min mg protein) −1
254 11 ACE Inhibition and Antioxidant Activities of Enzymatically Synthesised…
matic reactions, proportions and nature of the esters formed and ACE inhibitory activities for these compounds.
The compounds were characterised by two-dimensional Heteronuclear Single Quantum Coherence Transfer (2D-HSQCT) NMR spectra recorded for the samples. From NMR, it was con fi rmed that mono- and diesters in different proportions were detected (Tables 11.2 and 11.3 ). In some cases like l -valyl-maltose, l -valyl- d -mannitol, l -leucyl- d -fructose, l -leucyl- d -manni-tol and l -isoleucyl- d -glucose, l -prolyl- d -glucose, l -prolyl- d -ribose, l -prolyl-lactose, l -phenylala-nyl- d -fructose, l -tryptophanyl- d -fructose, l -his-tidyl- d -fructose and l -histidyl- d -mannitol, only monoesters were found to be formed. A 1- O -monoester was formed in case of l -valyl- d -fructose, l -valyl- d -mannitol, l -leucyl- d -sorbitol, l -isoleucyl- d -fructose, l -prolyl- d -fructose, l -phenylalanyl- d -fructose, l -phenylalanyl- d -mannitol, l -tryptophanyl- d -fructose and l -histi-dyl- d -mannitol. A 2- O -monoester was found to be formed in case of l -alanyl- b - d -glucose, l -valyl- d -glucose, l -leucyl- d -glucose and l -iso-leucyl-maltose, l -prolyl- d -glucose, l -prolyl- d -
ribose, l -phenylalanyl- d -glucose, l -phenylalanyl- d -galactose, l -tryptophanyl- d -glucose and l -histidyl- d -glucose. A 3- O -monoester was found to be formed in case of l -alanyl- b - d -glucose, l -valyl- d -glucose, l -leucyl- d -glucose and l -isoleucyl- d -glucose. A 6 ¢ - O -monoester was found to be formed in case of l -alanyl-lactose, l -valyl-maltose and l -isole-ucyl-maltose. All the esters invariable showed formation of 6- O -monoester except l -alanyl- d -ribose, l -leucyl- d -ribose, l -isoleucyl- d -ribose and l -prolyl- d -ribose where the primary C-5 hydroxyl group reacted to form 5- O -ester. Diesters such as 1,6-di- O -, 2,6-di- O -, 3,6-di- O -, 3,5-di- O - and 6,6 ¢ -di- O - were found to be formed in case of l -alanyl- b - d -glucose, l -alanyl- d -ri-bose, l -alanyl-lactose, l -valyl- d -glucose, l -valyl- d -fructose, l -leucyl- d -glucose, l -leucyl- d -ribose, l -isoleucyl- d -fructose and l -isoleucyl- d -ribose. Similarly 1,6-di-O-, 2,6-di-O-, 3,5-di-O- and 6,6 ¢ -di-O- were found to be formed in case of l -prolyl- d -fructose, l -phenylalanyl- d -glucose, l -phenylalanyl- d -galactose, l -phenylalanyl-lac-tose, l -phenylalanyl- d -mannitol, l -tryptophanyl- d -glucose and l -histidyl- d -glucose. It was not
0
20
40
60
80
0 10 20 30 40 50 60 70 80
% I
nhib
itio
n
Captopril (μM)
Fig. 11.1 A typical ACE inhibition plot for captopril. Concentration range, 6.7–66.7 m M; substrate, 0.1 mL hip-puryl-histidyl-leucine (5 mM); buffer, 100 mM phosphate
buffer (pH 8.3) containing 300 mM NaCl; incubation period, 30 min; temperature, 37°C. IC
50 value – 0.060 ± 0.006 mM
25511.1 ACE Inhibition by Aminoacyl Esters of Carbohydrates
possible to separate the individual esters from their reaction mixtures even through chromatog-raphy on Sephadex G-10 or Bio Gel P2. Thus, the activities described are for the mixtures of these mono- and diesters. From NMR, it was con fi rmed that mono- and diesters in different proportions were detected (Table 11.3 ).
Among the esters, l -isoleucyl- d -glucose (0.7 ± 0.07 mM) was found to exhibit the best inhibitory activity. With increase in alkyl side chain branching, d -glucose esters of l -alanine (3.1 ± 0.30 mM), l -valine (6.0 ± 0.59 mM), l -leu-cine (2.8 ± 0.27 mM) and l -isoleucine (0.7 ± 0.07 mM) showed better inhibition (lesser
IC 50
values) than the other esters, which could be directly correlated to increase in hydrophobicity (Table 11.2 ). IC
50 values £ 1.0 mM were detected
for l -valyl- d -mannitol (1.0 ± 0.09 mM), l -isole-ucyl- d -glucose (0.7 ± 0.07 mM), l -isoleucyl- d -fructose (0.9 ± 0.09 mM), l -isoleucyl-maltose (0.9 ± 0.09 mM) and l -leucyl- d -fructose (0.9 ± 0.08 mM). Similarly, the best IC
50 values
£ 1.0 mM were obtained for l -phenylalanyl- d -glucose (1.0 ± 0.09 mM), l -tryptophanyl- d -fruc-tose (0.9 ± 0.09 mM) and l -histidyl- d -fructose (0.9 ± 0.09 mM). Among aminoacyl esters tested for ACE inhibition activity, l -phenylalanyl- d -glucose (1.0 ± 0.09 mM), l -tryptophanyl- d -fruc-
0
10
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30
40
50
0.0 0.5 1.0 1.5 2.0 2.5
% I
nhib
itio
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0 0.5 1 1.5 2 2.5
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L-Alanyl-D-glucose (mM) L-Alanyl-D-ribose (mM)
L-Alanyl-lactose (mM)
a b
c
Fig. 11.2 ACE inhibition plots for l -alanyl esters of car-bohydrates. ( a ) l -alanyl- d -glucose – concentration range, 0.25–2.0 mM; substrate, 0.1 mL hippuryl-histidyl-leucine (5 mM); buffer, 100 mM phosphate buffer (pH 8.3) con-
taining 300 mM NaCl; incubation period, 30 min; tem-perature, 37°C. ( b ) l -alanyl- d -ribose – concentration range, 0.25–2.0 mM. ( c ) l -alanyl- d -lactose – concentra-tion range, 0.25–2.5 mM
256 11 ACE Inhibition and Antioxidant Activities of Enzymatically Synthesised…
tose (0.9 ± 0.09 mM) and l -histidyl- d -fructose (0.9 ± 0.09 mM) were found to exhibit the best inhibitory activity. Among the carbohydrates employed, d -fructose and d -mannitol esters showed better ACE inhibition (Table 11.3 ) than the other carbohydrate esters. l -Prolyl esters con-taining prolyl unit, an active unit of captopril, showed IC
50 values in the 1.4–4.4-mM concen-
trations range (Table 11.3 ). Although, aminoacyl esters were separated from the reaction mixture by column chromatography, it was dif fi cult to separate the individual esters. Hence, the actual potency of the individual esters could not be unequivocally established in the present work.
The present work for the fi rst time has shown the ACE inhibitory potency of the above-men-tioned aminoacyl esters prepared enzymatically. Since milder reaction conditions were employed, the products formation did not suffer due to side reactions. Captopril is N-[(S)-3-mercapto-2-methylpropionyl]- l -proline containing prolyl unit as essential for ACE inhibition (De Lima 1999 ) . Some naturally occurring ‘biologically active peptides’ such as N-terminal dipeptide (Tyr-Leu) of b -lactorphin, Asp-Leu-Pro and Asp-Gly from soy protein and Gly-Pro-Leu and Gly-Pro-Val from bovine skin gelatin hydrolysis also act as ACE inhibitors (Deloffre et al . 2004 ;
0
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itio
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itio
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-1.33E-15 0.3 0.6 0.9 1.2
% I
nhib
itio
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L-Valyl-D-glucose (mM) L-Valyl-D-fructose (mM)
L-Valyl-D-mannitol (mM)L-Valyl-maltose (mM)
a b
c d
Fig. 11.3 ACE inhibition plots for l -valyl esters of carbohydrates. ( a ) l -valyl- d -glucose – concentra-tion range, 0.5–2.5 mM; substrate, 0.1 mL hippuryl-histidyl-leucine (5 mM); buffer, 100 mM phosphate buffer (pH 8.3) containing 300 mM NaCl; incuba-
tion period, 30 min; temperature, 37°C. ( b ) l -valyl- d -fructose – concentration range, 0.5–2.5 mM. ( c ) l -valyl-maltose – concentration range, 0.5–2.5 mM. ( d ) l -valyl- d -mannitol concentration range, 0.2–1.0 mM
25711.1 ACE Inhibition by Aminoacyl Esters of Carbohydrates
Mullally et al . 1996 ; Wu and Liu 2002 ; Kim et al . 2001 ) . Although the aliphatic aminoacyl esters of d -glucose, d -fructose, d -ribose and lactose were prepared and tested, mere presence of a alkyl unit does not give rise to a high level of ACE inhibi-tion. Overall, it was clear that alkyl side chains can be accommodated in the hydrophobic S
1 and
S 2 subsites of angiotensin I-converting enzyme
(Michaud et al . 1997 ; De Lima 1999 ) . The free amino group in the amino acid esters can also serve as good ligands for Zn 2+ in the ACE active site. Carbohydrates in esters could also bind to the hydrophobic and/or hydrophilic subsites of angiotensin I-converting enzyme, as they possess
both hydrophobic and hydrophilic groups in their structure.
Although the prolyl esters of d -glucose, d -fructose, d -ribose, lactose and d -mannitol were prepared and tested, mere presence of a prolyl unit does not give rise to a high level of ACE inhibition. However, the esters tested in the pres-ent work clearly possess groups like pyrrolidine ring and aromatic groups, which can be accom-modated in the hydrophobic S
1 and S
2 subsites of
angiotensin I-converting enzyme (Michaud et al . 1997 ; De Lima 1999 ) . The results indicate that the esters hold promise as the potential inhibitors for ACE.
0
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L-Leucyl-D-ribose (mM) L-Leucyl-D-sorbitol (mM)
L-Leucyl-D-glucose (mM) L-Leucyl-D-fructose (mM)
a b
c d
Fig. 11.4 ACE inhibition plots for l -leucyl esters of carbohydrates. ( a ) l -leucyl- d -glucose – concentra-tion range, 0.25–2.0 mM; substrate, 0.1 mL hippuryl-histidyl-leucine (5 mM); buffer, 100 mM phosphate buffer (pH 8.3) containing 300 mM NaCl; incubation
period, 30 min; temperature, 37°C. ( b ) l -leucyl- d -fructose – concentration range, 0.25–2.0 mM; ( c ) l -leucyl- d -ribose – concentration range, 0.25–2.0 mM. ( d ) l -leucyl- d -sorbitol – concentration range, 0.25–2.0 mM
258 11 ACE Inhibition and Antioxidant Activities of Enzymatically Synthesised…
11.2 ACE Inhibition of Glycosides
Glycosides from the leaves of Abeliophyllum dis-tichum like acteoside, isoacteoside, rutin and hir-sutin moderately inhibited the angiotensin I-converting enzyme activity (Hyuncheol et al . 2003 ) . Glycosides like 3 -O- methyl crenatoside from Microtoena prainiana also showed more than 30% ACE inhibitory activity. Phenylpropanoid glycosides from Clerodendron trichotomum such as acteoside, leucosceptoside A, martynoside, acteoside isomer and isomar-
tynoside also showed ACE inhibitory effect (Dae-Gill et al . 2003 ) .
Phenolic glycosides found in a variety of fruits, vegetables and other food materials have been studied extensively for their antioxidant properties (Moon and Terao 1998 ; Moon et al. 2007 ) . Antioxidative action is reported to protect living organisms from oxidative damages, result-ing in the prevention of various diseases such as cancer, cardiovascular diseases, diabetes and aging (Azuma et al. 1999 ) . Flavonols and their glycosides protect red blood cells against free
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60
70
0 0.25 0.5 0.75 1 1.25
L-Isoleucyl-maltose (mM)
L-Isoleucyl-D-fructose (mM)
a b
c d
Fig. 11.5 ACE inhibition plots for l -isoleucyl esters of carbohydrates. ( a ) l -isoleucyl- d -glucose – con-centration range, 0.2–0.8 mM; substrate, 0.1 mL HHL (5 mM); buffer, 100 mM phosphate buffer (pH 8.3) containing 300 mM NaCl; incubation period,
30 min; temperature, 37°C. ( b ) l -isoleucyl- d -fructose – concentration range, 0.2–0.8 mM; ( c ) l -isoleucyl- d -ribose – concentration range, 0.25–2.0 mM. ( d ) l -isoleucyl-maltose – concentration range, 0.2–1.0 mM
25911.2 ACE Inhibition of Glycosides
radical-induced oxidative haemolysis (Dai et al. 2006 ) . The key role of phenols as antioxidants stems from the presence of hydroxyl groups attached to their aromatic rings, which enable them to scavenge free radicals (Kefalas et al. 2003 ; Villano et al. 2007 ) . The present work describes the ACE inhibition and antioxidant activities of the synthesised glycosides.
Several glycosides were tested for the ACE inhibitory activities. ACE was isolated from pig lung. The enzymatic reactions were carried out under optimised conditions worked out for these reactions. The enzymatic procedure employed unprotected and unactivated alcohols, phenols and carbohydrates. ACE inhibition activity of the above-mentioned glycosides of carbohydrates was
determined by the Cushman and Cheung method ( 1971 ) . Since hippuryl- l -histidyl- l -leucine (HHL) mimics the carboxyl dipeptide of angiotensin I, it has been used as the substrate for screening ACE inhibitors. Here also, eugenyl- d -glycoside showed inhibition of protease in ACE (Table 11.4 ).
Glycosides of n -octanol, phenolic glycosides of guaiacol, eugenol, curcumin, vanillin, N-vanillyl-nonanamide, DL-dopa, dopamine and vitamin glycosides of retinol, thiamin, ribo fl avin, pyridoxine, ergocalciferol, cholecalciferol and a -tocopherol were tested for ACE inhibition (Tables 11.5 and 11.6 and 11.7 , Vasudeva, Kamath et al. 2006 ; Lohith et al. 2006 ; Einstein Charles et al. 2009 ) . A few glycosides were also tested for antioxidant activities. Antioxidant
0
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L-Prolyl-D-ribose (mM)L-Prolyl-lactose (mM)
L-Prolyl-D-glucose (mM) L-Prolyl-D-frucose (mM)
a b
c d
Fig. 11.6 ACE inhibition plots for l -prolyl esters of car-bohydrates. ( a ) l -prolyl- d -glucose – concentration range, 0.2–1.6 mM; substrate, 0.1 mL HHL (5 mM); buffer, 100 mM phosphate buffer pH 8.3 containing 300 mM
NaCl; incubation period, 30 min; temperature, 37°C. ( b ) l -prolyl- d -fructose – concentration range, 0.33–2.64 mM. ( c ) l -prolyl-lactose – concentration range, 0.2–1.6 mM. ( d ) l -prolyl- d -ribose – concentration range, 0.2–1.8 mM
260 11 ACE Inhibition and Antioxidant Activities of Enzymatically Synthesised…
activities of few glycosides prepared are also shown in Table 11.8 .
Typical ACE inhibition plot for captopril, which showed an IC
50 value of 0.060 ± 0.006 mM,
is shown in Fig. 11.10 . Typical ACE inhibition plots for all the tested glycosides such as n -octyl glycosides (Fig. 11.11 ), guaiacyl glycosides (Fig. 11.12 ), eugenyl glycosides (Fig. 11.13 ), curcuminyl and a -tocopheryl glycosides (Fig. 11.14 ) are shown 4- O -( d -galactopyranosyl)vanillin (Fig. 11.15 ), 4- O -( d -sorbitol)vanillin
(Fig. 11.15 ), 4- O -( a - d -ribofuranosyl)N-vanillyl-nonanamide (Fig. 11.15 ), 4- O -( a - d -gluco py ranosy l - (1 ¢ →4) b - d - g lucopyranosy l )N-vanillyl-nonanamide (Fig. 11.15 ), 1,7- O -(bis- d -galactopyranosyl)curcumin (Fig. 11.15 ), 1,7- O -( b i s - b - d - g a l a c t o p y r a n o s y l - ( 1 ¢ → 4 ) d -glucopyranosyl) curcumin (Fig. 11.15 ), DL-3-hydroxy-4- O -( d -gluco pyranosyl)phenylalanine (Fig. 11.16 ), DL-3-hydroxy-4- O -(6- d -sorbitol) phenyl alanine (Fig. 11.16 ), 3-hydroxy-4- O -( d -glucopyranosyl)phenylethylamine (Fig. 11.16 ),
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tion
L-Phenylalanyl-D-fructose L-Phenylalanyl-lactose (mM)
L-Phenylalanyl-D-galactose (mM)L-Phenylalanyl-D-glucose (mM)
a b
c d
Fig. 11.7 ACE inhibition plots for l -phenylalanyl esters of carbohydrates ( a ) l -phenylalanyl- d -glucose – concen-tration range, 0.17–1.36 mM; substrate, 0.1 mL hippuryl-histidyl-leucine (5 mM); buffer, 100 mM phosphate buffer pH 8.3 containing 300 mM NaCl; incubation period,
30 min; temperature, 37°C. ( b ) l -phenylalanyl- d -galac-tose – concentration range, 0.14–1.02 mM. ( c ) l -phenyla-lanyl- d -fructose – concentration range, 0.13–1.36 mM. ( d ) l -phenylalanyl-lactose – concentration range, 0.13–1.06 mM
0
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L-Tryptophanyl-D-fructose (mM)L-Tryptophanyl-D-glucose (mM)
a b
Fig. 11.8 ACE inhibition plots for l -tryptophanyl esters of carbohydrates ( a ) l -tryptophanyl- d -glucose – concen-tration range, 0.13–1.06 mM; substrate, 0.1 mL hippuryl-histidyl-leucine (5 mM); buffer, 100 mM phosphate buffer
pH 8.3 containing 300 mM NaCl; incubation period, 30 min; temperature, 37°C. ( b ) l -tryptophanyl- d -fructose – concentration range, 0.13–1.06 mM, concentration range – 0.2–1.2 mM
0
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L-Histidyl-D-mannitol (mM)
L-Histidyl-D-fructose (mM)L-Histidyl-D-glucose (mM)
a b
c
Fig. 11.9 ACE inhibition plots for l -histidyl esters of carbohydrates ( a ) l -histidyl- d -glucose – concentration range, 0.2–1.6 mM; substrate, 0.1 mL hippuryl-histidyl-leucine (5 mM); buffer, 100 mM phosphate buffer pH 8.3
containing 300 mM NaCl; incubation period, 30 min; temperature, 37°C. ( b ) l -histidyl- d -fructose – concentra-tion range, 0.2–1.6 mM. ( c ) l -histidyl- d -mannitol, con-centration range – 0.2–1.6 mM
262 11 ACE Inhibition and Antioxidant Activities of Enzymatically Synthesised…
Table 11.2 IC 50
values for ACE inhibition by alkyl side chain containing aminoacyl esters of carbohydrates a
Aminoacyl ester of carbohydrates
Conversion yield (%) b
Products (% proportion) c IC 50
value (mM) d
l -Alanyl- b - d -glucose 30 2- O- l -Alanyl- b - d -glucose (47) 3.1 ± 0.30
3- O- l -Alanyl- b - d -glucose (12)
6- O- l -Alanyl- b - d -glucose (20)
2,6-di- O- l -Alanyl- b - d -glucose (15)
3,6-di- O- l -Alanyl- b - d -glucose (6) l -Alanyl- d -ribose 48 3- O- l -Alanyl- d -ribose (16) 2.7 ± 0.25
5- O- l -Alanyl- d -ribose (32) 3,5-di- O- l -Alanyl- d -ribose (52)
l -Alanyl-lactose 20 6- O- l -Alanyl-lactose (34) 2.0 ± 0.20
6 ¢ - O- l -Alanyl-lactose (34)
6,6 ¢ -di- O- l -Alanyl-lactose (32) l -Valyl- d -glucose 68 2- O- l -Valyl- d -glucose (10) 6.0 ± 0.59
3- O- l -Valyl- d -glucose (12) 6- O- l -Valyl- d -glucose (31) 2,6-di- O- l -Valyl- d -glucose (23) 3,6-di- O- l -Valyl- d -glucose (24)
l -Valyl- d -fructose 34 1- O- l -Valyl- d -fructose (29) 2.8 ± 0.28 6- O- l -Valyl- d -fructose (34) 1,6-di- O - l -Valyl- d -fructose (37)
l -Valyl-maltose 42 6- O- l -Valyl-maltose (49) 3.1 ± 0.30
6 ¢ - O- l -Valyl-maltose (51) l -Valyl- d -mannitol 56 1- O- l -Valyl- d -mannitol 1.0 ± 0.09 l -Leucyl- d -glucose 43 2- O- l -Leucyl- d -glucose (17) 2.8 ± 0.27
3- O- l -Leucyl- d -glucose (20) 6- O- l -Leucyl- d -glucose (48) 2,6-di- O- l -Leucyl- d -glucose (8) 3,6-di- O- l -Leucyl- d -glucose (7)
l -Leucyl- d -fructose 48 6- O - l -Leucyl- d -fructose 0.9 ± 0.08 l -Leucyl- d -ribose 38 3- O - l -Leucyl- d -ribose (16) 1.5 ± 0.14
5- O - l -Leucyl- d -ribose (32) 3,5-di -O - l -Leucyl- d -ribose (52)
l -Leucyl- d -sorbitol 60 1- O - l -Leucyl- d -sorbitol 2.7 ± 0.25 l -Isoleucyl- d -glucose 47 3- O - l -Isoleucyl- d -glucose (42) 0.7 ± 0.07
6- O - l -Isoleucyl- d -glucose (58) l -Isoleucyl- d -fructose 42 1- O - l -Isoleucyl- d -fructose (36) 0.9 ± 0.09
6- O - l -Isoleucyl- d -fructose (30) 1,6-di- O - l -Isoleucyl- d -fructose (34)
l -Isoleucyl- d -ribose 53 3- O - l -Isoleucyl- d -ribose (52) 3.8 ± 0.37 5- O - l -Isoleucyl- d -ribose (20) 3,5-di -O - l -Isoleucyl- d -ribose (28)
l -Isoleucyl-maltose 54 2- O - l -Isoleucyl-maltose (38) 0.9 ± 0.09 6- O - l -Isoleucyl-maltose (40)
6 ¢ - O - l -Isoleucyl-maltose (22)
a Respective amino acids and carbohydrates as controls showed no ACE inhibition activity b Conversion yields were from HPLC within ±10% errors in HPLC yield measurements c Product proportions determined from 13 C, 2D HSQCT NMR C6 peak areas (C5 cross peaks in case of ribose) or their cross peaks d IC
50 values compared to that of captopril 0.060 ± 0.006 mM determined by Cushman and Cheung method
26311.2 ACE Inhibition of Glycosides
Table 11.3 IC 50
values for ACE inhibition by aminoacyl esters of carbohydrates a
Aminoacyl ester of carbohydrates
Conversion yield (%) b Products (% proportion) c IC
50 value (mM) d
l -Prolyl- d -glucose 62 2-O- l -Prolyl- d -glucose (26) 1.7 ± 0.17 3-O- l -Prolyl- d -glucose (26) 6-O- l -Prolyl- d -glucose (48)
l -Prolyl- d -fructose 61 1-O- l -Prolyl- d -fructose (31) 4.4 ± 0.43 6-O- l -Prolyl- d -fructose (42) 1,6-di-O- l -Prolyl- d -fructose (27)
l -Prolyl- d -ribose 41 3-O- l -Prolyl- d -ribose (35) 2.0 ± 0.19 5-O- l -Prolyl- d -ribose (65)
l -Prolyl-lactose 68 6- O - l -Prolyl-lactose (58) 1.6 ± 0.15
6 ¢ - O - l -Prolyl-lactose (42) l -Phenylalanyl- d -glucose 79 2-O- l -Phenylalanyl- d -glucose (19) 1.0 ± 0.09
3-O- l -Phenylalanyl- d -glucose (23) 6-O- l -Phenylalanyl- d -glucose (25) 2,6- di- O - l -Phenylalanyl- d -glucose (17) 3,6-di-O- l -Phenylalanyl- d -glucose (16)
l -Phenylalanyl- d -galactose 45 2-O- l -Phenylalanyl- d -galactose (32)
4.6 ± 0.45
3-O- l -Phenylalanyl- d -galactose (20) 6-O- l -Phenylalanyl- d -galactose (19) 2,6-di-O- l -Phenylalanyl- d -galactose (16) 3,6-di-O- l -Phenylalanyl- d -galactose (13)
l -Phenylalanyl- d -fructose 50 1-O- l -Phenylalanyl- d -fructose (72) 13.6 ± 1.35 6-O- l -Phenylalanyl- d -fructose (28)
l -Phenylalanyl-lactose 61 6-O- l -Phenylalanyl-lactose (42) 7.8 ± 0.77 6 ¢ -O- l -Phenylalanyl-lactose (31) 6,6 ¢ -di-O- l -Phenylalanyl-lactose (27)
l -Phenylalanyl- d -mannitol 43 1-O- l -Phenylalanyl- d -mannitol (62) 2.6 ± 0.25 1,6-di-O- l -Phenylalanyl- d -mannitol (38)
l -Tryptophanyl- d -glucose 42 2-O- l -Tryptophanyl- d -glucose (22) 7.4 ± 0.73 3-O- l -Tryptophanyl- d -glucose (21) 6-O - l -Tryptophanyl- d -glucose (38) 2,6-di-O- l -Tryptophanyl- d -glucose (10) 3,6-di-O- l -Tryptophanyl- d -glucose (9)
l -Tryptophanyl- d -fructose 18 1-O- l -Tryptophanyl- d -fructose (45) 0.9 ± 0.09 6-O- l -Tryptophanyl- d -fructose (55)
(continued)
264 11 ACE Inhibition and Antioxidant Activities of Enzymatically Synthesised…
Aminoacyl ester of carbohydrates
Conversion yield (%) b Products (% proportion) c IC
50 value (mM) d
l -Histidyl- d -glucose 42 2-O- l -Histidyl- d -glucose (25) 3.5 ± 0.34 3-O- l -Histidyl- d -glucose (24) 6-O- l -Histidyl- d -glucose (28) 2,6-di-O- l -Histidyl- d -glucose (12) 3,6-di-O- l -Histidyl- d -glucose (11)
l -Histidyl- d -fructose 58 6-O- l -Histidyl- d -fructose 0.9 ± 0.09 l -Histidyl- d -mannitol 8 1-O- l -Histidyl- d -mannitol 1.7 ± 0.16
a Respective amino acids and carbohydrates as controls showed no ACE inhibition activity b Conversion yields were from HPLC with ±10–15% errors in HPLC yield measurements c Product proportions determined from 13 C, 2D HSQCT NMR C6 peak areas (C5 cross peaks in case of ribose) or their cross peaks d IC
50 values compared to that of captopril 0.060 ± 0.006 mM determined by Cushman and Cheung ( 1969 ) method
Table 11.3 (continued)
Table 11.4 Inhibition of protease in ACE by eugenyl- a - d -glucoside a
System Protease activity (min −1 mg −1 enzyme protein) b
Percentage of protease activity with respect to ACE activity c
Control: ACE – 0.5 mL + 0.5 mL of 0.6% haemoglobin + 0.5 mL buffer
0.0436 13.3
Eugenyl- a - d -glucoside: 0.5 mL glycoside + ACE – 0.5 mL + 0.5 mL of 0.6% haemoglobin
0.0292 8.2
a Conditions: ACE – 0.5 mL (0.5 mg). All the solutions were prepared in 0.1 M pH 7.5 tris-HCl: incubation period, 30 min; temperature, 37°C; 0.5 mL of 10% trichloroacetic acid added to arrest the reaction; Blank performed without enzyme and glycoside; absorbance measured at 440 nm; eugenyl- a - d -glucoside – 0.5 mL of 0.8 mM b Average absorbance values from three individual experiments c Percentage protease activity with respect to an ACE activity of 0.327 m mol (min mg protein) −1
Table 11.5 IC 50
values for ACE inhibition by glycosides a
Glycoside Yield b (%) Products formed (% proportions) c IC 50
value (mM) d
n -Octyl- d -glucoside 46 C1 a -glucoside (63), C1 b -glucoside (25), C6-alkylated (12)
1.0 ± 0.09
n -Octyl maltoside 22 C1 a -maltoside 1.5 ± 0.13
n -Octyl sucrose, 13 C1- O -alkylated (44), C6- O -alkylated (56)
1.7 ± 0.15
Guaiacyl- a - d -glucoside 52 C1 a glucoside (52), C6- O -arylated (48)
3.7 ± 0.36
Guaiacyl- a - d -galactoside 17 C1 a -galactoside (95), C6- O -arylated (5)
2.3 ± 0.22
Eugenyl- a - d -glucoside 32 C1 a glucoside (53), C6- O -arylated (47)
0.5 ± 0.04
Eugenyl- a - d -mannoside 8 C1 a -mannoside 5.3 ± 0.51
Eugenyl maltoside 17 C1 a -maltoside (52), 6- O -arylated (28), C6″- O -arylated (20)
0.7 ± 0.06
Eugenyl sucrose 7 C1- O -arylated (45), C6- O -arylated (35), C6″- O -arylated (20)
1.7 ± 0.15
(continued)
26511.2 ACE Inhibition of Glycosides
Table 11.6 Antioxidant and angiotensin-converting enzyme inhibitory activities of various phenolic and vitamin glycosides a
Compounds Antioxidant activity IC
50 value (mM) b
ACE inhibition IC
50 value (mM) c
Butylated hydroxy anisole (BHA) 0.046 ± 0.002 – Enalapril – 0.071 ± 0.004 Vanillin 1 1.65 ± 0.08 1.87 ± 0.09 4- O -( d -Glucopyranosyl)vanillin 2.66 ± 0.13 1.11 ± 0.06
4- O -( b - d -Glucopyranosyl)vanillin 0.9 ± 0.45 0.61 ± 0.03
4- O -( a - d -Galactopyranosyl)vanillin 1.62 ± 0.08 1.12 ± 0.06
4- O -( d -Galactopyranosyl)vanillin 1.18 ± 0.06 0.61 ± 0.03
4- O -( a - d -Mannopyranosyl)vanillin 1.55 ± 0.08 1.02 ± 0.05
4- O -( d -Mannopyranosyl)vanillin 1.08 ± 0.05 2.3 ± 0.1
4- O -( a - d -Glucopyranosyl-(1 ¢ →4) d -glucopyranosyl)vanillin
1.17 ± 0.06 1.63 ± 0.08
4- O -( a - d -Glucopyranosyl-(1 ¢ →4) b - d -glucopyranosyl)vanillin
2.64 ± 0.13 1.89 ± 0.09
4- O -( d -Fructofuranosyl-(2→1 ¢ ) a - d -glucopyranosyl)vanillin
1.23 ± 0.06 15.7 ± 0.79
4- O -( b - d -Galactopyranosyl-(1 ¢ →4) b - d -glucopyranosyl)vanillin
0.8 ± 0.04 0.92 ± 0.05
4- O -( d -Sorbitol)vanillin 1.24 ± 0.06 0.81 ± 0.04 N-Vanillyl-nonanamide 0.054 ± 0.003 1.53 ± 0.08 4- O -( d -Glucopyranosyl)N-vanillyl-nonanamide 1.18 ± 0.06 1.33 ± 0.07
4- O -( b - d -Glucopyranosyl)N-vanillyl-nonanamide 1.4 ± 0.07 3.33 ± 0.17
4- O -( d -Galactopyranosyl)N-vanillyl-nonanamide 2.9 ± 0.15 2.05 ± 0.1
4- O -( b - d -Galactopyranosyl)N-vanillyl-nonanamide 0.94 ± 0.05 2 ± 0.1
4- O -( b - d -Mannopyranosyl)N-vanillyl-nonanamide 1.14 ± 0.06 2.57 ± 0.13
4- O -( a - d -Ribofuranosyl)N-vanillyl-nonanamide 0.98 ± 0.05 1 ± 0.05
4- O -( a - d -Glucopyranosyl-(1 ¢ →4) d -glucopyranosyl)N-vanillyl-nonanamide
0.8 ± 0.04 2.41 ± 0.12
(continued)
Glycoside Yield b (%) Products formed (% proportions) c IC 50
value (mM) d
Eugenyl- d -mannitol 7 C1- O -arylated 2.1 ± 0.21
Curcuminyl-bis- a - d -glucoside 48 C1 a -glucoside (62), C6- O -arylated (38) 1.5 ± 0.13
Curcuminyl-bis- a - d -mannoside 9 C1 a -mannoside 1.0 ± 0.09
Curcuminyl-bis-maltoside 19 C1 a -maltoside (37), C6- O -arylated (36), C6″- O -arylated (27)
1.2 ± 0.11
Curcuminyl-bis-sucrose 19 C1- O -arylated (12), C6- O -arylated (70), C6″- O -arylated (18)
1.8 ± 0.17
Curcuminyl-bis- d -mannitol 14 C1- O -arylated 1.8 ± 0.17
a -Tocopheryl- a - d -glucoside 52 C1 a -glucoside 1.2 ± 0.11
a Respective alcohols, phenols and carbohydrates as controls did not show any ACE inhibition activities; nonreducing sugar unit carbons of disaccharide are double primed b Conversion yields were from HPLC c Product proportions determined from 2D-HSQCT NMR C1/C6 cross-peak areas d IC
50 values compared to that of captopril 0.060 ± 0.005 mM determined by Cushman and Cheung method
Table 11.5 (continued)
266 11 ACE Inhibition and Antioxidant Activities of Enzymatically Synthesised…
Table 11.6 (continued)
Compounds Antioxidant activity IC
50 value (mM) b
ACE inhibition IC
50 value (mM) c
4- O -( a - d -Glucopyranosyl-(1 ¢ →4) b - d -glucopyranosyl)N-vanillyl-nonanamide
0.75 ± 0.04 0.8 ± 0.04
4- O -( b - d -Galactopyranosyl-(1 ¢ →4) b - d -glucopyranosyl)N-vanillyl-nonanamide
1.04 ± 0.05 1.82 ± 0.09
Curcumin 0.053 ± 0.003 0.83 ± 0.04
1,7- O -(Bis- b - d -glucopyranosyl)curcumin 0.8 ± 0.04 1.09 ± 0.05
1,7- O -(Bis- d -galactopyranosyl)curcumin 0.92 ± 0.05 0.88 ± 0.04 1,7- O -(Bis- d -mannopyranosyl)curcumin 0.75 ± 0.04 1.9 ± 0.1
1,7- O -(Bis- b - d -galactopyranosyl-(1 ¢ →4) d -glucopyranosyl)curcumin
0.95 ± 0.05 0.67 ± 0.03
DL-Dopa 0.045 ± 0.002 0.6 ± 0.03 DL-3-Hydroxy-4- O -( d -glucopyranosyl)phenylalanine
1.11 ± 0.06 1.2 ± 0.06
DL-3-Hydroxy-4- O -( d -glucopyranosyl)phenylalanine
0.98 ± 0.05 1.26 ± 0.06
DL-3-Hydroxy-4- O -( d -galactopyranosyl)phenylalanine
2.26 ± 0.11 1.71 ± 0.08
DL-3-Hydroxy-4- O -( b - d -mannopyranosyl)phenylalanine
1.13 ± 0.06 1.87 ± 0.09
DL-3-Hydroxy-4- O -( b - d -galactopyranosyl-(1 ¢ →4) b - d -glucopyranosyl)phenylalanine
0.9 ± 0.05 3.33 ± 0.17
DL-3-Hydroxy-4- O -(6- d -sorbitol)phenylalanine 1.86 ± 0.09 0.56 ± 0.03 DL-Dopa- d -mannitol 1.9 ± 0.09 1.58 ± 0.08 Dopamine 0.04 ± 0.002 1.93 ± 0.1 3-Hydroxy-4- O -( d -glucopyranosyl)phenylethylamine 1.45 ± 0.07 1.27 ± 0.06
3-Hydroxy-4- O -( b - d -glucopyranosyl)phenylethylamine 0.98 ± 0.05 2.38 ± 0.12
3-Hydroxy-4- O -( d -galactopyranosyl)phenylethylamine 0.93 ± 0.05 2.38 ± 0.12 3-Hydroxy-4- O -( d -mannopyranosyl)phenylethylamine 1.8 ± 0.09 1.93 ± 0.1 Ribo fl avin – 1.08 ± 0.05 5- O -( d -Glucopyranosyl)ribo fl avin – 1.27 ± 0.06
5- O -( b - d -Glucopyranosyl)ribo fl avin – 1.75 ± 0.09
5- O -( d -Galactopyranosyl)ribo fl avin – 0.83 ± 0.04
5- O -( a - d -Mannopyranosyl)ribo fl avin – 2.08 ± 0.1
5- O -( d -Mannopyranosyl)ribo fl avin – 1.92 ± 0.1 5- O -( d -Ribofuranosyl)ribo fl avin – 1.11 ± 0.06
5- O -( a - d -Glucopyranosyl-(1 ¢ →4) d -glucopyranosyl)ribo fl avin – 0.8 ± 0.04
5- O -(1- d -Fructofuranosyl-(2→1 ¢ ) a - d -glucopyranosyl)ribo fl avin – 1.03 ± 0.05
5- O -( b - d -Galactopyranosyl-(1 ¢ →4) b - d -glucopyranosyl)ribo fl avin – 1.09 ± 0.05
Ergocalciferol – 1.2 ± 0.06 20- O -( d -Glucopyranosyl)ergocalciferol 0.9 ± 0.05 1.17 ± 0.06
a -Tocopherol 0.054 ± 0.003 1.07 ± 0.05
6- O -( b - d -Glucopyranosyl) a -tocopherol 1.2 ± 0.06 1.33 ± 0.07
6- O -( d -Galactopyranosyl) a -tocopherol 0.72 ± 0.04 2.59 ± 0.13
6- O -( d -Mannopyranosyl) a -tocopherol 0.5 ± 0.03 1.8 ± 0.09
a Glucosidases catalysed synthesis of phenolic glycosides of vanillin (Table 8.12), N-vanillyl-nonanamide (Table 8.9), curcumin (Table 8.4), DL-dopa (Table 8.18), dopamine (Table 8.23) and vitamin glycosides of ribo fl avin (Table 8.29 ), ergocalciferol (Table 8.34 ) and a -tocopherol (Table 8.38 ) where the conversion yields and product proportions are shown. Carbohydrates did not show any antioxidant and ACE inhibition activity. Error in measurements is ±5% b Antioxidant activity values determined by DPPH radical scavenging method (Moon and Terao 1998 ) c ACE activity determined by Cushman and Cheung method ( 1971 )
26711.2 ACE Inhibition of Glycosides
Table 11.7 IC 50
values for ACE inhibition by glycosides a
Compounds Product (% proportion)
Amyloglucosidase catalysis (% yield)
b -glucosidase catalysis (% yield)
IC 50
values (mM) b
Enalapril – – – 0.071 ± 0.004 Vanillin – – – 1.87 ± 0.09 Vanillyl- d -glucoside 4- O -( b - d -Glucopyranosyl)vanillin
– 10 0.61 ± 0.03
Vanillyl- d -galactoside 4- O -( a - d -Galactopyranosyl)vanillin (23) 4- O -( b - d -Galactopyranosyl)vanillin (77)
– 6 0.61 ± 0.03
DL-Dopa – – – 0.6 ± 0.03 DL-Dopa- d -sorbitol 3-Hydroxy-4- O -(6- d -sorbitol)
phenylalanine
12 – 0.56 ± 0.03
Curcumin – – – 0.83 ± 0.04 Curcuminyl-bis- d -glucoside 1,7- O -(Bis- b - d -glucopyranosyl)
curcumin
– 44 1.09 ± 0.05
Pyridoxine – – 1.05 ± 0.06 Pyridoxine- d -glucoside 7- O -( a - d -Glucopyranosyl)
pyridoxine (39) 7- O -( b - d -Glucopyranosyl)
pyridoxine (41) 6- O -( a - d -Glucopyranosyl)
pyridoxine (20)
– 35 0.84 ± 0.04
Thiamin – – – 3.33 ± 0.17 Thiaminyl- d -fructoside 11- O -(1- d -Fructofuranosyl)
thiamin (54) 11- O -(6- d -Fructofuranosyl)
thiamin (46)
– 54 0.52 ± 0.03
Ribo fl avin – – – 1.08 ± 0.05
Ribo fl avinyl-maltose 5- O -( a - d -Glucopyranosyl-(1 ¢ →4) a - d -
glucopyranosyl)ribo fl avin (35) 5- O -( a - d -Glucopyranosyl-(1 ¢ →4)6- d -
glucopyranosyl)ribo fl avin (48) 5- O -( a - d -Glucopyranosyl-(1 ¢ →4)6 ¢ - d -
glucopyranosyl)ribo fl avin (17)
25 – 0.8 ± 0.04
a No activity for carbohydrates b Values are an average from two measurements
3-hydroxy-4- O -( d -mannopyranosyl)phenyl-ethylamine (Fig. 11.16 ), 5- O -( d -galacto-pyranosyl)ribo fl avin (Fig. 11.16 ), 5- O -( a - d -glucopyranosyl-(1 ¢ 4) d -gluco pyranosyl)ribo fl avin (Fig. 11.16 ), 20- O -( d -glucopyranosyl) ergocal-ciferol (Fig. 11.17 ), 6- O -( b - d -glucopyranosyl) a -tocopherol (Fig. 11.17 ) and 6- O -( d -manno-pyranosyl) a -tocopherol (Fig. 11.17 ) are shown.
The compounds were characterised by two-dimensional Heteronuclear Single Quantum Coherence Transfer (2D-HSQCT) NMR spectra. In case of glycoside syntheses, the major product was the glycosylated product, and relatively lesser amounts of C6- O -alkylated or C6- O -arylated products were also detected. Thus, the activities described are for the mixtures of these compounds.
268 11 ACE Inhibition and Antioxidant Activities of Enzymatically Synthesised…
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Captopril (μM)
Fig. 11.10 A typical ACE inhibition plot for capto-pril: concentration range, 6.7–33.3 m M; substrate, 0.1 mL hippuryl-histidyl-leucine (5 mM); buffer, 100 mM phosphate buffer pH 8.3 containing 300 mM NaCl; incubation period, 30 min; temperature, 37°C. IC
50 value – 0.060 ± 0.006 mM
Table 11.8 IC 50
values for antioxidant activities of glycosides a
Compound IC 50
value (mM) b
Butylated hydroxyanisole 0.046 ± 0.002 Vanillin 1.65 ± 0.08
4- O -( b - d -Glucopyranosyl)vanillin 0.9 ± 0.05
N-vanillyl-nonanamide 0.054 ± 0.003
4- O -( a - d -glucopyranosyl)N-vanillyl-nonanamide 1.18 ± 0.06
DL-Dopa 0.045 ± 0.002 DL-Dopa- d -glucoside 0.98 ± 0.05
3-Hydroxy-4- O -( b - d -galactopyranosyl-(1 ¢ →4) b - d -glucopyranosyl)phenylalanine
0.9 ± 0.05
Dopamine 0.045 ± 0.002
3-Hydroxy-4- O -( b - d -glucopyranosyl)phenylethylamine 0.98 ± 0.05
Dopamine- d -galactoside 0.93 ± 0.05 Curcumin 0.79 ± 0.03
1,7- O -(Bis- b - d -glucopyranosyl)curcumin 0.8 ± 0.04
a -Tocopherol 0.054 ± 0.003
6- O -( a - d -Glucopyranosyl) a -tocopherol 1.04 ± 0.05
20- O -( d -Glucopyranosyl)ergocalciferol 0.9 ± 0.05
a No activity for carbohydrates b Values are an average from two measurements
11.3 Antioxidant Activity of Glycosides
DPPH (2,2-diphenyl-1-picrylhydrazyl) is a highly coloured commercially available radical source, widely used for rough estimation of the ability of antioxidants to trap potentially damaging one-electron oxidants, i.e. the number of DPPH mol-ecules reduced by one molecule of an antioxidant
(Potier et al. 1999 ) . Many methods to evaluate the antioxidative activity of speci fi c compounds have been described, but the most widely documented one deals with DPPH radical (Portes et al. 2007 ; Roche et al. 2005 ) . The radical scavenging ef fi ciency of phenolic and vitamin glycosides tested in this investigation is listed in Table 11.8 .
Butylated hydroxy anisole (BHA) was used as a control. The plot obtained is shown in Fig. 11.18 . Plots for the antioxidant activity of a few selected glycosides, 4- O -( b - d -glucopyranosyl)vanillin (Fig. 11.19 ), 4- O -( b - d -galactopyranosyl-(1 ¢ →4) b - d -gluco pyranosyl)vanillin (Fig. 11.19 ), 4- O -( b - d -galactopyranosyl)N-vanillyl-nonanamide (Fig. 11.19 ), 4- O -( a - d -glucopyranosyl-(1 ¢ →4) b - d -glucopyranosyl)N-vanillyl-nonanamide (Fig. 11.19 ), 1,7- O -(bis- b - d -glucopyranosyl)curcumin (Fig. 11.19 ), 1,7- O -(bis- d -manno-pyranosyl) curcumin (Fig. 11.19 ), DL-3-hydroxy-4- O -( d -gluco pyra-nosyl)phenylalanine (Fig. 11.20 ), DL-3-hydroxy-4- O - ( b - d -ga lac topyranosyl- (1 ¢ →4) b - d -glucopyranosyl)phenylalanine (Fig. 11.20 ), 3-hydroxy-4- O -( b - d -gluco pyranosyl) phenyl-ethylamine (Fig. 11.20 ), 3-hydroxy-4- O -( d -galactopyranosyl) phenylethylamine (Fig. 11.20 ),
26911.3 Antioxidant Activity of Glycosides
05
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nn-octyl-D-glucoside (mM) n-Octyl maltoside (mM)
n-Octyl sucrose (mM)
a b
c
Fig. 11.11 ACE inhibition plots for n -octyl glycosides ( a ) n -octyl- d -glucoside, ( b ) n -octyl maltoside, ( c ) n -octyl sucrose. Concentration range, 0.13–1.06 mM; substrate,
0.1 mL hippuryl-histidyl-leucine (5 mM); buffer, 100 mM phosphate buffer pH 8.3 containing 300 mM NaCl; incu-bation period, 30 min; temperature, 37°C
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Guaiacyl-α-D-glucoside (mM) Guaiacyl-α-D-galactoside (mM)
a b
Fig. 11.12 ACE inhibition plots for guaiacyl glycosides ( a ) guaiacyl- a - d -glucoside, ( b ) guaiacyl- a - d -galactoside. Concentration range, 0.13–1.06 mM; substrate, 0.1 mL
hippuryl-histidyl-leucine (5 mM); buffer, 100 mM phos-phate buffer pH 8.3 containing 300 mM NaCl; incubation period, 30 min; temperature, 37°C
20- O -( d -glucopyranosyl) ergocalciferol (Fig. 11.20 ) and 6- O -( d -mannopyranosyl) a -tocoph-erol (Fig. 11.20 ), are shown.
Totally, 99 glycosides were tested for antioxi-dant activity and 49 glycosides tested for angio-tensin-converting enzyme (ACE) inhibitory
activities. Enzymatic glycosylation produced only monoglycosides, and no diglycosides were detected except curcumin, which showed bis gly-cosylation. In spite of possessing OH groups at third and fourth positions, DL-dopa and dopamine gave a mixture of 4-OH, 3-OH and 4- O- C6-
270 11 ACE Inhibition and Antioxidant Activities of Enzymatically Synthesised…
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Eugenyl maltoside (mM)
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Eugenyl sucrose (mM)
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Eugenyl-α-D-glucoside (mM) Eugenyl-α-D-mannoside (mM)
Eugenyl-D-mannitol (mM)
a b
c d
e
Fig. 11.13 ACE inhibition plots for eugenyl glycosides ( a ) eugenyl- a - d -glucoside, ( b ) eugenyl- a - d -mannoside, ( c ) eugenyl maltoside, ( d ) eugenyl sucrose, ( e ) eugenyl- d -mannitol. Concentration range, 0.13–1.06 mM; substrate,
0.1 mL hippuryl-histidyl-leucine (5 mM); buffer, 100 mM phosphate buffer pH 8.3 containing 300 mM NaCl; incu-bation period, 30 min; temperature, 37°C
arylated compounds (Tables 11.6 and 11.7 ) but no bis glycosides. With many phenols and vitamins, C1 a - and/or C1 b -glycosides were formed, and in some case, C6- O -arylated products were also formed. Among the phenols employed, vanillin, N-vanillyl-nonanamide, curcumin, DL-dopa and dopamine possess structural similarity by having hydroxyl group at the fourth position and hydroxyl
or methoxy group at third position besides having a CH= or CH
2 carbon para to the fourth OH posi-
tion. Such a structural similarity is responsible for better antioxidant activities of the free phenols compared to their glycosides which also have not lost much of their activities even after glycosyla-tion. However, these glycosides did not show high ACE inhibition activities. Some like vanillin and
27111.3 Antioxidant Activity of Glycosides
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Curcuminyl-bis-D-mannitol (mM)
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a-Tocopheryl-a-D-glucoside (mM)
Curcuminyl-bis-a-D-glucoside (mM) Curcuminyl-bis-a-D-mannoside (mM)
Curcuminyl-bis-maltoside (mM) Curcuminyl-bis-sucrose (mM)
a b
c d
e f
Fig. 11.14 ACE inhibition plots for curcuminyl and a -tocopheryl glycosides. ( a ) Curcuminyl-bis- a - d -glucoside, ( b ) curcuminyl-bis- a - d -mannoside, ( c ) cur-cuminyl-bis-maltoside, ( d ) curcuminyl-bis-sucrose, ( e ) curcuminyl-bis- d -mannitol, ( f ) a -tocopheryl- a - d -
glucoside 36 . Concentration range, 0.13–1.06 mM; sub-strate, 0.1 mL hippuryl-histidyl-leucine (5 mM); buffer, 100 mM phosphate buffer pH 8.3 containing 300 mM NaCl; incubation period, 30 min; temperature, 37°C
DL-dopa glycosides showed reasonable extent of ACE inhibition activities. Phenolic OH was found to be very essential for antioxidant activity. Although introduction of a carbohydrate molecule at the phenolic OH decreases the antioxidant activity, some of the glycosides still possess sub-stantial amount of antioxidant activities. Presence of free OH group in case of DL-dopa and dop-
amine did not show good antioxidant activity of the glycosides where one of the OH group is modi fi ed leaving the other free. Since ribo fl avin did not contain a phenolic OH group, its antioxi-dant activities were not measured. However, introduction of the carbohydrate molecules at the phenolic OH did not alter the ACE inhibition activities much.
272 11 ACE Inhibition and Antioxidant Activities of Enzymatically Synthesised…
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4-O-(D-Galactopyranosyl)vanillin (mM)
1,7-O-(Bis-β-D-galactopyranosyl-(1´→4)D-glucopyranosyl)curcumin (mM)
1,7-O-(Bis-D-galactopyranosyl)curcumin(mM)
4-O-(α-D-Ribofuranosyl)N-vanillyl-nonanamide (mM)
4-O-(α-D-Glucopyranosyl-(1´→4)β-D-glucopyranosyl)N-vanillyl-nonanamide (mM)
4-O-(D-Sorbitol)vanillin (mM)
a b
c d
e f
Fig. 11.15 ACE inhibition plots for phenolic glycosides: ACE, 0.1 mL (10 mg in 25-mL stock solution); glycoside concentration range, 0.2–1.8 mM; substrate, 0.1 mL hippuryl- l -histidyl- l -leucine (5 mM); buffer, 100 mM phosphate buffer pH 8.3 containing 0.3 M sodium chlo-ride; incubation period, 30 min; and temperature, 37°C. ( a ) 4- O -( d -Galactopyranosyl)vanillin. ( b ) 4- O -( d -Sorbitol)
vanillin. ( c ) 4- O -( a - d -Ribofuranosyl)N-vanillyl-nonanamide. ( d ) 4- O -( a - d -Glucopyranosyl-(1 ¢ →4) b - d -glucopyranosyl)N-vanillyl-nonanamide. ( e ) 1,7- O -(bis- d -Galactopyranosyl)curcumin; and ( f ) 1,7- O -(bis- b - d -Galactopyranosyl-(1 ¢ →4) d -gluco pyranosyl)curcumin
27311.3 Antioxidant Activity of Glycosides
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5-O-(D-Galactopyranosyl)riboflavin (mM) 5-O-(α-D-Glucopyranosyl-(1´→4)D-glucopyranosyl)riboflavin (mM)
3-Hydroxy-4-O-(D-glucopyranosyl)phenylethylamine (mM)
3-Hydroxy-4-O-(D-mannopyranosyl)phenylethylamine (mM)
DL-3-Hydroxy-4-O-(D-glucopyranosyl)phenylalanine (mM)
DL-3-Hydroxy-4-O-(6-D-sorbitol)phenylalanine (mM)
a b
c d
e f
Fig. 11.16 ACE inhibition plots for phenolic and vita-min glycosides: ACE, 0.1 mL (10 mg in 25-mL stock solution); glycoside concentration range, 0.2–1.8 mM; substrate, 0.1 mL hippuryl- l -histidyl- l -leucine (5 mM); buffer, 100 mM phosphate buffer pH 8.3 containing 0.3 M sodium chloride; incubation period, 30 min; and tempera-ture, 37°C. ( a ) DL-3-hydroxy-4- O -( d -Glucopyranosyl)
phenylalanine. ( b ) DL-3-Hydroxy-4- O -(6- d -sorbitol)phe-nylalanine. ( c ) 3-Hydroxy-4- O -( d -glucopyranosyl)phenyl ethylamine. ( d ) 3-Hydroxy-4- O -( d -mannopyranosyl)phe-nylethylamine. ( e ) 5- O -( d -Galactopyranosyl)ribo fl avin. ( f ) 5- O -( a - d -Glucopyranosyl-(1 ¢ →4) d -glucopyranosyl)ribo fl avin
0
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6-O-(β-D-Glucopyranosyl)α-tocopherol(mM)
20-O-(D-Glucopyranosyl)ergocalciferol(mM)
6-O-(D-Mannopyranosyl)α-tocopherol(mM)
a b
c
Fig. 11.17 ACE inhibition plots for phenolic and vita-min glycosides: ACE, 0.1 mL (10 mg in 25-mL stock solution); glycoside concentration range, 0.2–1.8 mM; substrate, 0.1 mL hippuryl- l -histidyl- l -leucine (5 mM); buffer, 100 mM phosphate buffer pH 8.3 containing 0.3 M
sodium chloride; incubation period, 30 min; and tempera-ture, 37°C. ( a ) 20- O -( d -Glucopyranosyl)ergocalciferol. ( b ) 6- O -( b - d -Glucopyranosyl) a -tocopherol. ( c ) 6- O -( d -Mannopyranosyl) a -tocopherol
0
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Butylated Hydroxy Anisole (μM)
Fig. 11.18 Antioxidant inhibition plot for buty-lated hydroxyanisole (BHA). Concentration range, 0–120 m M; DPPH, 1 mL (3.6 mM); buffer, 0.1-M
tris-HCl (pH 7.4); incubation period, 20 min; and temperature, 37°C. IC
50 value – 0.046 ± 0.002 mM
27511.3 Antioxidant Activity of Glycosides
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4-O-(β-D-Glucopyranosyl)vanillin (mM)4-O-(β-D-Galactopyranosyl-(1´→4)β-D-
glucopyranosyl)vanillin (mM)
4-O-(b-D-Galactopyranosyl)N-vanillyl-nonanamide (mM)
4-O-(a-D-Glucopyranosyl-(1´→4)b-D-glucopyranosyl)N-vanillyl-nonanamide (mM)
1,7-O-(Bis-β-D-glucopyranosyl)curcumin(mM)
1,7-O-(Bis-D-mannopyranosyl)curcumin(mM)
a b
c d
e f
Fig. 11.19 Antioxidant activity plot for phenolic glyco-sides. DPPH, 1 mL (3.6 mM); glycoside concentration range, 5–10 mM; buffer, 0.1-M tris-HCl (pH 7.4); incuba-tion period, 20 min; and temperature, 37°C. IC
50 value for
the antioxidant activity was obtained as the concentration of the glycoside corresponding to 50% decrease in DPPH absorbance from these plots. ( a ) 4- O -( b - d -Glucopyranosyl)
vanillin. ( b ) 4- O -( b - d -Galactopyranosyl-(1 ¢ →4) b - d -glucopyranosyl)vanillin. ( c ) 4- O -( b - d -Galactopyranosyl)N-vanillyl-nonanamide. ( d ) 4- O -( a - d -Glucopyranosyl-(1 ¢ 4) b - d -glucopyranosyl)N-vanillyl-nonanamide. ( e ) 1,7- O -(bis- b - d -Glucopyranosyl)curcumin. ( f ) 1,7- O -(bis- d -Mannopyranosyl)curcumin
276 11 ACE Inhibition and Antioxidant Activities of Enzymatically Synthesised…
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20-O-(D-Glucopyranosyl)ergocalciferol (mM)
6-O-(D-Mannopyranosyl)α-tocopherol (mM)
3-Hydroxy-4-O-(D-galactopyranosyl) phenylethylamine (mM)
DL-3-Hydroxy-4-O-(β-D-galactopyranosyl-(1´Æ4)β-D-glucopyranosyl)phenylalanine (mM)
DL-3-Hydroxy-4-O-(D-glucopyranosyl) phenylalanine (mM)
3-Hydroxy-4-O-(β-D-
a b
c d
e f
Fig. 11.20 Antioxidant activity plot for phenolic and vitamin glycosides. DPPH, 1 mL (3.6 mM); glycoside concentration range, 5–10 mM; buffer, 0.1 M tris-HCl (pH 7.4); incubation period, 20 min; and temperature, 37°C. IC
50 value for the antioxidant activity was obtained
as the concentration of the glycoside corresponding to 50% decrease in DPPH absorbance from these plots.
( a ) DL-3-Hydroxy-4- O -( d -glucopyranosyl)phenylalanine. ( b ) DL-3-Hydroxy-4- O -( b - d -galactopyranosyl-(1 ¢ →4) b - d -glucopyranosyl)phenylalanine. ( c ) 3-Hydroxy-4- O -( b - d - g l u c o p y r a n o s y l ) p h e n y l e t h y l a m i n e . ( d ) 3-Hydroxy-4- O -( d -galactopyranosyl) phenylethylam-ine. ( e ) 20- O -( d -Glucopyranosyl)ergocalciferol. ( f ) 6- O -( d -Mannopyranosyl) a -tocopherol
277References
The aglycon phenols and free vitamins were also subjected to measurement of antioxidant activity and ACE inhibition as controls. Antioxidant activities were determined (Table 11.8 ) for phenols and also vitamins pos-sessing phenolic OH group and alicyclic OH group like ergocalciferol (vitamin D2). Antioxidant activities of the phenolic and vitamin glycosides were in the 0.5 ± 0.03 mM to 2.66 ± 0.13 mM range when compared to the free aglycons whose values were quite high (0.053 ± 0.003 mM to 1.65 ± 0.08 mM). Butylated hydroxyanisole (BHA) showed the lowest IC
50
value at 0.046 ± 0.002 mM, and no other glyco-sides could come closer to this value. Many of the glycosides showed lesser IC
50 values of
<1 mM (Table 11.6 ). The best IC 50
values ( £ 0.75 mM) observed are 4- O -( a - d -glucopyranosyl-(1 ¢ →4) b - d -glucopyranosyl)N-vanillyl-nonanamide, 0.75 ± 0.04 mM; 1,7- O -(bis- d -mannopyranosyl)curcumin, 0.75 ± 0.04 mM; 6- O -( d -galactopyranosyl) a -tocopherol, 0.72 ± 0.04 mM; and 6- O -( d -mannopyranosyl) a -tocopherol, 0.5 ± 0.03 mM.
IC 50
values for ACE inhibition of glycosides range from 0.56 ± 0.03 mM to 3.33 ± 0.17 mM (Tables 11.6 and 11.7 ). Enalapril, a known syn-thetic drug, showed an IC
50 value of
0.071 ± 0.004 mM, but no other phenolic or vitamin glycosides could exhibit such a low value. The present work for the fi rst time has shown the ACE inhibitory potency of the above-mentioned glycosides prepared enzymatically. Among the glycosides tested, eugenyl- a - d -glucoside (0.5 ± 0.04 mM), eugenyl maltoside (0.7 ± 0.06 mM), octyl- d -glucoside (1.0 ± 0.09 mM) and curcuminyl-bis- a - d -manno-side (1.0 ± 0.09 mM) exhibited the best ACE inhibitory activities (IC
50 £ 1.0 mM; Table 11.4 ).
Among the carbohydrates employed, both gluco-sides and maltosides showed the best ACE inhib-itory activities. Alkyl glycosides showed better inhibitory activities than the phenolic glycosides. However, the best ACE inhibitory activities for the glycosides (<0.75mM) detected were 4- O -( b - d -glucopyranosyl) vanillin, 0.61 ± 0.03 mM; 4- O -( d -galacto pyranosyl)vanillin, 0.61 ± 0.03 mM; 1,7- O -(bis- b - d -galactopyranosyl-
(1 ¢ →4) d -glucopyranosyl) curcumin, 0.67 ± 0.03 mM; and DL-3-hydroxy-4- O -(6- d -sorbitol) phenylala-nine, 0.56 ± 0.03 mM. Among the glycosides tested, phenolic glycosides showed better ACE activities than the vitamin glycosides. Both phe-nolic and vitamin glycosides showed comparable IC
50 values to aglycon units. Derivatisation by the
introduction of carbohydrate molecule to the phenolic/alcoholic OH had very little effect on the IC
50 values compared to the control. Hence, it
can be concluded that the presence of free pheno-lic or derivated OH groups is not essential for ACE inhibition. Many commercial inhibitors like enalapril are peptides containing the essential prolyl units. Among the carbohydrates employed, aglycons modi fi ed with d -glucose, d -galactose and d -sorbitol showed less IC
50 values (from
0.56 ± 0.03 mM to 0.75 ± 0.04 mM) than those modi fi ed with the other carbohydrate molecules.
These results suggest that both the phenolic and vitamin glycosides hold promising potential as antioxidants and ACE inhibitors although the IC
50 values are slightly on the higher side, a draw-
back which could be corrected through suitable modi fi cations.
References
Azuma K, Nakayama M, Koshioka M, Ippoushi K, Yamaguchi Y, Kohata K, Yamauchi Y, Ito H, Higashio HJ (1999) Phenolic antioxidants from the leaves of Corchorus olitorius L. Agric Food Chem 47:3963–3966
Cooke JWB, Hayes D, Henson RA, Hermitage SA, Ward RA, Whitehead AJ (2003) Process for the preparation of 4-substituted phenylalanyl ester derivatives. PCT Int Appl WO 2003068725:A2 (CA 138 180345)
Cushman DW, Cheung HS (1969) A simple substrate for assay for dog lung angiotensin converting enzyme. Fed Proc 28:3019
Cushman DW, Cheung HS (1971) Spectrophotometric assay and properties of the angiotensin-converting enzyme of rabbit lung. Chem Pharmacol 20:1637–1638
Dae-Gill K, Yong-Sup L, Hyoung-Ja K, Yun-Mi L, Ho-Sub L (2003) Angiotensin converting enzyme inhibitory phenylpropanoid glycosides from Clerodendron trichotomum . J Ethnopharmacol 89:151–154
Dai F, Miao Q, Zhou B, Yang L, Liu ZL (2006) Protective effects of fl avanols and their glycosides against free
278 11 ACE Inhibition and Antioxidant Activities of Enzymatically Synthesised…
radical induced oxidative hemolysis of red blood cells. Life Sci 78:2488–2493
De Lima DP (1999) Synthesis of angiotensin converting enzyme (ACE) inhibitors an important class of anti-hypertensive drugs. Quim Nova 22:375–381
Deloffre L, Sautiere PE, Huybrechts R, Hens K, Vieau D, Salzet M (2004) Angiotensin converting enzyme inhi-bition studies by natural leech inhibitors by capillary electrophoresis and competition assay. Eur J Biochem 271:2101–2106
Einstein Charles R, Ponrasu T, Sivakumar R, Divakar S (2009) Angiotensin converting enzyme inhibitory and antioxidant activities of enzymatically synthesized phenolic and vitamin glycosides. Biotech Appl Biochem 52:177–184
Hyuncheol O, Dae-Gill KC, Hun-Taeg L, Ho-Sub L (2003) Four glycosides from the leaves of Abeliophyllum distichum with inhibitory effects on angiotensin converting enzyme. Phytother Res 17:811–813
Johnston CI (1992) Renin-angiotensin system: a dual tis-sue and hormonal system for cardiovascular control. J Hypertens 10:13–26
Kamath V, Rajini PS, Lohith K, Somashekar BR, Divakar S (2006) Angiotensin Converting enzyme inhibitory activity of amino acid esters of carbohydrates. Int J Biol Macromol 38:89–93
Kefalas P, Kallithraka S, Parejo I, Makris DP (2003) A comparative study on the in vitro antiradical activity and hydroxyl free radical scavenging activity in aged red wines. Food Sci Technol Int 9:383–387
Kim SK, Byun HG, Park PJ, Shahidi F (2001) Angiotensin I converting enzyme inhibitory peptides puri fi ed from bovine skin gelatin hydrolysate. J Agric Food Chem 49:2992–2997
Kim WD, Kaneko S, Park GG, Tanaka H, Kusakabe I, Kobayashi H (2003) Puri fi cation and characterization of a -galactosidase from sun fl ower seeds. Biotechnol Lett 25:353–358
Li GH, Le GW, Yong-Hui S, Shrestha S (2004) Angiotensin I–converting enzyme inhibitory peptides derived from food proteins and their physiological and pharmaco-logical effects. Nutr Res 24:469–486
Lohith K, Vijayakumar GR, Somashekar BR, Sivakumar R, Divakar S (2006) Glycosides and amino acyl esters
of carbohydrates as potent inhibitors of Angiotensin Converting Enzyme. Eur J Med Chem 41:1059–1072
Michaud A, Williams TA, Chauvet MT, Corvol P (1997) Substrate dependence of angiotensin I-converting enzyme inhibition captopril displays a partial selectiv-ity for inhibition of N-acetyl-seryl-aspartyl-lysyl-proline hydrolysis compared with that of angiotensin I. Mol Pharmacol 51:1070–1076
Moon JH, Terao J (1998) Antioxidant activity of caffeic acid and dihydrocaffeic acid in Lard and human low-density lipoprotein. J Agr Food Chem 46:5062–5065
Moon YH, Lee JH, Jhon DY, Jun WJ, Kang SS, Sim J, Choi H, Moon JH, Kim D (2007) Synthesis and char-acterization of novel quercetin- b - d -glucopyranosides using glucansucrase from Leuconostoc mesenteriodes . Enzyme Microb Technol 40:1124–1129
Mullally MM, Meisel H, FitzGerald RJ (1996) Synthetic peptides corresponding to a lactalbumin and b -lacto-globulin sequences with angiotensin-I-converting enzyme inhibitory activity. J Biol Chem 377:259–260
Portes E, Gardrat C, Castellan A (2007) A comparative study on the antioxidant properties of tetrahydrocur-cuminoids and curcuminoids. Tetrahedron 63:9092–9099
Potier P, Maccario V, Giudicelli MB, Queneau Y, Dangles O (1999) Gallic esters of sucrose as a new class of antioxidants. Tetrahedron Lett 40:3387–3390
Roche M, Dufour C, Mora N, Dangles O (2005) Antioxidant activity of olive phenols Mechanistic investigation and characterization of oxidation prod-ucts by mass spectrometry. Org Biomol Chem 3:423–429
Vermeirssen V, Van-Camp J, Verstraete W (2002) Optimization and validation of an angiotensin-con-verting enzyme inhibition assay for the screening of bioactive peptides. J Biochem Biophys Methods 51:75–87
Villano D, Fernandez-Pachon MS, Moy ML, Troncoso AM, Garca-Parrilla MC (2007) Radical scavenging ability of polyphenolic compounds towards DPPH free radical. Talanta 71:230–235
Wu JY, Liu SW (2002) In fl uence of alcohol concentration on lipase-catalyzed enantioselective esteri fi cation of racemic naproxen in isooctane under controlled water activity. Enzyme Microb Technol 26:124–130
279
Index
A ACE inhibition plots , 253, 255–261, 274 ACE inhibitory activity , 253, 258 Acidolysis , 28, 74 Active site , 8, 23–26, 77, 91, 104, 105, 117, 148, 149,
192, 216, 218, 219, 222, 244, 246–249, 257 Acylation , 29, 81, 82, 123, 248 Adipic acid , 68, 69, 72–74, 77, 78 Aglycons , 11, 137, 183, 222, 277 Alcohols , 7, 13, 39, 50, 82, 118, 123–135, 138, 215, 219,
224, 226, 259, 265 Alcoholysis , 29, 226 Amino acids containing aliphatic side chains , 26 Amino acylation , 82 Amino acyl esters
1- O -amino acyl esters , 33 1,6-di- O -amino acyl esters , 114–116, 119, 254 2,5-di- O -amino acyl esters , 33, 114, 115, 119 2,6-di- O -amino acyl esters , 33, 114, 115, 119, 254 3- O -amino acyl esters , 33 3,6-di- O -amino acyl esters , 33, 114, 115, 119, 254 4,6-di- O -amino acyl esters , 114, 115, 119 5- O -amino acyl esters , 33, 254 6- O -amino acyl esters , 33 6’- O -amino acyl esters , 33, 114 6,6’-di- O -amino acyl esters , 33, 114–116, 119, 254
Analysis of variance (ANOVA) , 101, 132, 148, 158, 184
Angiotensin converting enzyme (ACE) , 251–277 Anomeric carbon , 8–10, 223 Anomers , 91, 119 Anthranilic acid , 32, 50, 51 Antioxidant activity , 143–144, 251–277 Antioxidants , 12, 150, 155, 188, 204, 251–277 Arylated , 146, 168, 183, 216, 218, 220, 221 Aspergillus sp. , 8 Azeotropic , 40, 48, 113
B Bench-scale conditions , 48 Bioavailability , 12, 15, 16, 82, 155, 156, 217 Bis glycosides , 221, 270 Box–Behnken design , 42, 43 Buffer , 44, 69, 83, 124, 138, 215, 226, 253, Butylated hydroxyanisole , 268, 274, 277
4-t-Butylcyclohexanol , 58–61 4-t-Butylcyclohexyl acetate , 58–61
C Calcium alginate beads , 223 Candida rugosa lipase (CRL) , 25–27, 90, 106, 108, 118,
226, 236 Capsaicin , 138, 149–155, 215, 217, 218 Capsaicin glycosides
3- O -( d -galactopyranosyl)capsaicin , 155 3- O -( d - d -galactopyranosyl-(1 ¢ →4) b - d -
glucopyranosyl)capsaicin , 155 3- O -(D-glucopyranosyl)capsaicin , 155
Captopril , 251–254, 256, 260, 262, 264, 265, 268 Carbohydrates , 5, 33, 81–120, 123, 127, 138, 141,
144–145, 151, 155, 157, 167, 168, 176–177, 180, 182, 183, 189–190, 197, 198, 200, 208–209, 215, 216, 218, 220, 221, 251–268, 277
Carboxyl groups , 7, 8, 39–62, 68, 82, 248 Catalysis , 7, 8, 10, 13, 24, 30–34, 55, 70, 74, 77, 91,
123, 149, 153, 154, 160, 161, 169, 170, 172–174, 179, 189, 193, 194, 197, 198, 203, 205, 209, 219–222, 249, 267
Central composite rotatable design (CCRD) , 44, 49–50, 101, 130, 146, 157, 164, 184
Cetyl alcohol , 50, 129, 130 Cetyl glucoside , 130 Chemo enzymatic method , 1 Chiral compounds , 2 Chirazyme , 58, 59, 66, 69 Chloroform , 41, 42, 46, 47, 70, 71, 74, 75, 99 Cholecalciferol glycosides
17- O -(1- d -fructofuranosyl)cholecalciferol , 205, 219 17- O -( a - d -galactopyranosyl)cholecalciferol , 205,
219 17- O -(6- d -galactopyranosyl)cholecalciferol , 205, 219 17- O -(6- d -glucopyranosyl)cholecalciferol , 205 17- O -( d -glucopyranosyl)cholecalciferol , 202–205 17- O -( d -mannopyranosyl)cholecalciferol , 205, 219
13 C NMR , 42, 70, 71 C-O-C asymmetrical stretching , 128, 138, 141, 146, 151,
157, 190, 198, 202 C-O-C symmetrical stretching , 128, 138, 146, 190,
198, 202 Color test c, 144,
S. Divakar, Enzymatic Transformation, DOI 10.1007/978-81-322-0873-0, © Springer India 2013
280
Competitive inhibition , 226 Computer simulation , 228, 229, 234, 238, 241–243,
245, 246 Contour plots , 45, 49, 148, 184, 186 Conversion yields , 30, 49, 50, 82, 86, 88, 89, 93, 95, 98,
100, 105, 107, 109, 111, 113, 114, 116, 125–132, 139–145, 147, 149, 154, 155, 158, 161, 162, 164, 165, 168, 170, 177–179, 182, 185, 187, 191, 193, 194, 196, 197, 199, 200, 202, 203, 205–209, 215–217, 241, 253, 262, 264–266
Co-polymerization , 65–66, 76, 77 Cresols , 47–49 Critical micellar concentration (CMC) , 91, 100, 127 Critical water concentration , 117, 216 Curcumin , 137, 138, 141–144, 146–149, 215–220,
226, 239–244, 248, 249, 259, 260, 266–270, 272, 275, 277
Curcuminyl glycosides Curcuminyl-bis- d -glucoside , 144, 265 Curcuminyl-bis- d -mannoside , 144, 265 C1- O -curcuminyl-bis-maltoside , 145 C1- O -curcuminyl-bis-mannitol , 145 C1- O -curcuminyl-bis-sucrose , 145 C6- O -curcuminyl-bis-maltose , 145 C6’’- O -curcuminyl-bis-maltose , 145 C6’’- O -curcuminyl-bis-sucrose , 145 C6- O -curcuminyl-bis- d -glucose , 144
Curve fi tting , 227, 231, 233, 236, 237 b -Cyclodextrin , 58, 59, 61–62 b -Cyclodextrin acetate , 58
D d -Arabinose , 82, 86, 106, 113–115, 118, 119, 127,
138, 141, 144, 167, Dead-end amyloglucosidase-vanillin complex , 244, 248 Dead-end complexes , 247 Dead-end lipase- d -glucose complex , 246, 247 Deglycosylation, 8 d -Fructose , 86, 106, 113–115, 118, 127, 138, 141, 144,
167, 171, 180, 183, 191, 197, 198, 204, 215, 217, 218, 220–222, 224, 256, 257
d -Galactose , 82, 86, 106, 113, 118, 119, 127, 138, 141, 144, 150, 152, 156, 166, 167, 171, 176, 180, 182, 183, 188, 189, 191, 195, 197, 198, 204, 206, 208, 215, 217–224, 277
d -Glucose , 6, 32, 82, 124, 138, 215, 226, 253, Diastereomers , 118 Di esters , 82, 119, 254, 255 Di-isopropyl ether , 124, 126, 128–130, 138, 140, 142,
150, 154–156, 158, 161, 167, 170, 179, 191, 193, 194, 196, 197, 202, 203, 208, 209, 215, 216, 222, 223, 226
2,2-Diphenyl-1-picrylhydrazyl (DPPH) , 143, 266, 268, 274–276
Dissociation constant , 230, 231, 235, 236, 240, 244, 247
2D HSQCT spectra , 146, 151, 183, 190, 194, 197, 198, 202, 204
171 , 180, 183, 191, 215, 217, 218, 220–224 dl -Dopa glycosides
1,6- O -(bis dl -3-hydroxy-4- O -phenylalanine) d -mannitol , 170
dl -3-hydroxy-4- O -( b -D-galactopyranosyl-(1 ¢ →4) b - d -glucopyranosyl)phenylalanine , 166–167, 170, 219, 266, 268
dl -3-hydroxy-4- O -( d -galactopyranosyl)phenylalanine , 169
dl -3-hydroxy-4- O -(6- d -galactopyranosyl)phenylalanine , 169
dl -4-hydroxy-3- O -( d -galactopyranosyl)phenylalanine , 169
dl -3-hydroxy-4- O -( d -glucopyranosyl)phenylalanine , 169, 273, 276
dl -3-hydroxy-4- O -(6- d -glucopyranosyl)phenylalanine , 169
dl -4-hydroxy-3- O -( d -glucopyranosyl)phenylalanine , 169
dl -3-hydroxy-4- O -(1- d -mannitol)phenylalanine , 170 dl -3-hydroxy-4- O -( d -mannopyranosyl)
phenylalanine , 169 dl -3-hydroxy-4- O -(6- d -sorbitol)phenylalanine , 167,
170, 266, 273 d -Mannitol , 82, 86 d -Mannose , 81, 82, 106, 114, 115, 118, 119, 127, 138,
141, 144, 146, 150, 156, 166, 167, 176, 180, 182, 183, 188, 189, 191, 195, 197, 198, 204, 206, 208, 215,–219–223
DMF , 61, 82, 85, 86, 88–90, 93–95, 98–102, 107, 109, 111, 113, 196, 197, 226
DMSO , 61, 98, 144, 217 Dopamine , 138, 165, 171, 176–178, 215–218, 221, 259,
266, 268–271 Dopamine glycosides
3-hydroxy-4- O -( d -galactopyranosyl)phenylethylamine , 266
3-hydroxy-4- O -(6- d -galactopyranosyl)phenylethylamine , 179
4-hydroxy-3- O -( d -galactopyranosyl)phenylethylamine , 266
3-hydroxy-4- O -( d -glucopyranosyl)phenylethylamine , 178, 179, 260, 266
4-hydroxy-3- O -( b - d -glucopyranosyl)phenylethylamine , 179
3-hydroxy-4- O -( d -mannopyranosyl)phenylethylamine , 179, 266, 267
4-hydroxy-3- O -( a - d -mannopyranosyl)phenylethylamine , 179
Double displacement mechanism , 10, 11 Double reciprocal plots , 51, 227–229, 232–234, 236,
238, 239 Double substrate inhibition , 233–236 d -Ribose , 82, 86, 106, 113–114, 118, 119, 127, 138, 141,
144, 150, 167, 180, 182, 183, 188, 189, 191, 204, 215, 217, 220–224, 257
d -Sorbitol , 82, 86, 106, 113, 114, 116, 118, 127, 138, 141, 144, 156, 161, 167, 180, 183, 191, 197, 198, 215, 217, 218, 220, 221, 223, 224, 277
Index
281
E Enzyme
catalysis , 13, 123 reusability , 47, 83, 102
Enzyme/substrate ratio , 41–44, 49, 117, 216, 217 Epinephrine , 137, 138, 180–183, 215, 216, 218 Epinephrinyl glycosides
epinephrinyl-3- O -( d -glucopyronoside) , 182 epinephrinyl-4- O -( d -glucopyronoside) , 182 epinephrinyl-3- O- ( d -mannopyronoside) , 182 epinephrinyl-4- O -( d -mannopyronoside) , 182
Ergocalciferyl glycosides 20- O -(6- d -glucopyranosyl)ergocalciferol ,
203, 222 20- O -( d -glucopyranosyl)ergocalciferol , 200–203,
222, 266 Ester , 2, 13, 24, 39, 65, 81, 124, 171, 225, 251, Ester hydrolysis , 28 Esteri fi cation
activity , 53, 55, 70, 83–85, 100 kinetics , 226–239
Ethanol , 14, 129 Ethylmethyl ketone , 40, 41, 55, 57 Eugenol , 137–139, 141, 146, 215, 217, 259 Eugenyl glycosides
C1- O -eugenyl-mannitol , 142 C1- O -eugenyl-sucrose , 142 C6- O -eugenyl- d -glucose , 142 C6- O -eugenyl- d -maltose , 142 C6’’- O -eugenyl- d -maltose , 142 C6- O -eugenyl-sucrose , 142 C6’’- O -eugenyl-sucrose , 142 eugenyl- d -glucoside , 141, 142 eugenyl- d -maltoside , 141, 142 eugenyl- d -mannoside , 141, 142
Experimental yields , 45, 105, 131, 132, 147–149, 164
F Fat soluble , 149, 151, 183, 195, 199, 202, 222 Food , 1, 2, 12, 14, 15, 23, 24, 39, 40, 46–48, 124, 137,
138, 152, 253, 258 Food additives , 14 Fragrance esters , 31
G Gauaicyl glycosides
guaiacyl- d -galactoside , 138 guaiacyl- d -glucoside , 138, 139
Glucosylation , 124, 130, 132–134, 146–151, 167, 176, 184–187, 195, 200, 202, 208, 217, 224, 226, 244–246
Glycosidases , 2, 5–16 Glycoside , 5, 34, 123, 137, 215, 251, Glycosyl acceptor , 9, 10, 13, 123 Glycosylation , 5, 123, 137, 215, 226, 269, Glycosylation kinetics , 226, 239–246, 248–249 Glycosyl donor , 9, 123, 216
Glycosyl transferase , 9, 13 Guaiacol , 138–140, 215, 217, 259
H Hexane 1,6-diol , 41 Hippuryl-l-histidyl-l-leucine (HHL) , 253, 258, 259 1 H NMR , 41, 42, 62, 70, 71, 74, 128 HPLC , 41, 42, 83, 84, 86, 88, 89, 91, 93, 95,
98–100, 105–109, 111, 113, 114, 116, 125–132, 138–147, 149–152, 154–159, 161, 162, 164–166, 168, 170, 177–179, 182, 185, 187–194, 196–208, 226, 231, 262, 264, 265
Hydrogen ions , 55–58, 118, 216 Hydrolase , 1, 6, 8–10, 23, 24 Hydrolytic activity , 46, 47, 148 Hydrophobic amino acids , 25, 26, 81 Hydroxyl groups , 9, 61, 62, 81, 82, 118, 119, 123, 138,
183, 218, 248, 259
I Immobilised b -glucosidase , 138, 167, 172–176, 178,
179, 185–187, 221 Immobilised enzyme , 185 Inclusion complexes , 60 Incubation period , 6, 42, 66, 83, 124, 138, 215,
226, 253, Inhibitor , 25, 26, 49, 90, 187, 221, 226, 236, 248,
251–253, 257, 259, 277 Inhibitor constant , 51, 226 Initial rates , 41, 46–47, 83, 89, 100, 141, 227, 230–233,
235–237, 240–247 Iso-butanol , 14 Iso-propanol , 14 Iteration procedure , 231, 236, 237, 246
K Kinetically controlled , 9, 123 Kinetics
data , 239, 244, 246–248 models , 225 parameters , 51, 52, 231, 235, 236, 239, 240,
242–246
L Lactic acid
2- O -acyl esters of lactic acid , 39–47 2- O -lauroyl lactic acid , 44–45 2- O -palmitoyl lactic acid , 41–42 2- O -stearoyl lactic acid , 40–41
Lactose , 7, 82, 106, 113–115, 118, 119, 127, 130, 138, 141, 144, 150, 152, 156, 166, 167, 180, 183, 189, 191, 215, 217, 218, 220–224, 257
Lactylates , 42, 46 Lactylic acid , 40, 42
Index
282
l -Alanine l -alanyl- d -arabinose , 89, 119 l -alanyl- d -fructose , 89, 119 l -alanyl- d -galactose , 89, 119 l -alanyl- d -glucose , 89, 119 l -alanyl-lactose , 89, 119 l -alanyl-maltose , 89, 119 l -alanyl- d -mannose , 89, 119 l -alanyl- d -ribose , 89, 119 l -alanyl-sucrose , 89, 119
Lauryl alcohol , 129, 130 l -dihydroxyphenylalanine , 169 l -dopa , 138, 165, 166, 168, 171, 215, 216, 218 l -Dopa glycosides
l -3-hydroxy-4- O -( d -arabinofuranosyl)phenylalanine , 173
l -3-hydroxy-4- O -(2- O - d -fructofuranosyl)phenylalanine , 173
l -3-hydroxy-4- O -( d -galactopyranosyl)phenylalanine , 172
l -4-hydroxy-3- O -( b - d -galactopyranosyl)phenylalanine , 172
l -3-hydroxy-4- O -(6- O - d -galactopyanosyl)phenylalanine , 172
l -3-hydroxy-4- O -( d -glucopyranosyl)phenylalanine , 172, 174
l -3-hydroxy-4- O -(6- O - d -glucopyranosyl)phenylalanine , 174
l -4-hydroxy-3- O -( b - d -glucopyranosyl)phenylalanine , 172
l -3-hydroxy-4- O -(lactosyl)phenylalanine , 173 l -3-hydroxy-4- O -( b -maltosyl)phenylalanine , 173 l -3-hydroxy-4- O -(1- O - d -mannitolide)
phenylalanine , 174 l -3-hydroxy-4- O -( d -mannopyranosyl)
phenylalanine , 173 l -3-hydroxy-4- O -( d -ribofuranosyl)
phenylalanine , 173, 175 l -3-hydroxy-4- O -(1- O -sucrose)
phenylalanine , 174 l -Histidine
l -histidyl- d -fructose , 112, 113, 119, 264, l -histidyl- d -glucose , 112, 113, 119, 264 l -histidyl-maltose , 112, 113, 119 l -histidyl- d -mannitol , 112, 113, 119 l -histidyl- d -mannose , 112, 113, 119
Linker molecules , 67, 68, 72 Lipase , 1, 5, 23, 39, 65, 81, 253, Lipozyme , 34, 43, 56, 66, 69, 82, 101 l -Isoleucine
l -isoleucyl- d -arabinose , 119 l -isoleucyl- d -fructose , 119 l -isoleucyl- d -galactose , 119 l -isoleucyl- d -glucose , 119 l -isoleucyl-lactose , 119 l -isoleucyl-maltose , 119 l -isoleucyl- d -mannitol , 119 l -isoleucyl- d -mannose , 119 l -isoleucyl- d -ribose , 119 l -isoleucyl-sucrose , 119
l -Leucine l -leucyl- d -arabinose , 119 l -leucyl- d -fructose , 119 l -leucyl- d -galactose , 119 l -leucyl- d -glucose , 119 l -leucyl-lactose , 119 l -leucyl-maltose , 118, 119 l -leucyl- d -mannitol , 119 l -leucyl- d -mannose , 119 l -leucyl- d -ribose , 119 l -leucyl- d -sorbitol , 119 l -leucyl-sucrose , 118, 119
Low molecular weight polymers , 77 Low water solubility , 150 l -Phenylalanine
l -phenylalanyl- d -arabinose , 106, 107 l -phenylalanyl- d -fructose , 106, 107 l -phenylalanyl- d -galactose , 106, 107 l -phenylalanyl- d -glucose , 106, 107 l -phenylalanyl-lactose , 106, 107 l -phenylalanyl-maltose , 106, 107 l -phenylalanyl- d -mannitol , 106, 107 l -phenylalanyl- d -mannose , 106, 107
l -Proline l -prolyl- d -fructose , 108, 109, 119 l -prolyl- d -galactose , 108, 109, 119 l -prolyl- d -glucose , 108, 109, 119 l -prolyl-lactose , 108, 109, 119 l -prolyl-maltose , 108, 109, 119 l -prolyl- d -mannose , 108, 109, 119 l -prolyl- d -ribose , 108, 109, 119 l -prolyl- d -sorbitol , 108, 109, 119
l -Tryptophan l -tryptophanyl- d -fructose , 111, 119 l -tryptophanyl- d -galactose , 111, 119 l -tryptophanyl- d -glucose , 111, 119 l -tryptophanyl-maltose , 111, 119 l -tryptophanyl- d -mannose , 111, 119 l -tryptophanyl- d -sorbitol , 111, 119 l -tryptophanyl-sucrose , 111, 119
l -Valine l -valyl- d -arabinose , 119 l -valyl- d -fructose , 119 l -valyl- d -galactose , 119 l -valyl- d -glucose , 119 l -valyl-maltose , 119 l -valyl- d -mannitol , 119 l -valyl- d -mannose , 119 l -valyl- d -ribose , 119 l -valyl-sucrose , 119
M Malonic acid , 68, 69, 72–74 Maltose , 6, 86, 106, 113, 114, 117–119, 127, 138,
144, 146, 150, 156, 183, 188, 189, 191, 215, 217, 220–224
Mass spectrometry , 128, 139, 146, 151, 167, 181, 190, 195, 197, 198, 202, 204
Methanol , 7, 14, 50, 61, 66, 69, 219
Index
283
Methylisobutyl ketone , 58 Michaelis–Menten constant , 51, 230, 231, 235 Microaqueous phase , 24, 55–59 Milder reaction conditions , 2, 13, 30, 65, 166, 175,
202, 222, 256 Mono esters , 87, 88, 91–98, 107, 109, 111, 113, 119,
120, 154 Mono glycosylation , 10, Multi-functional compounds , 251
N n-alkyl glucosides , 124, 129–130 n-butanol , 129, 219 n-heptane , 66 n-hexanol , 219 n-Octanol , 14, 125–128, 130–134, 146, 217, 219, 259 n-Octyl- d -glucoside , 124–127, 130–134, 264, 269 n-Octyl glycosides
C1- O -Octyl sucrose , 128 C6- O -Octyl sucrose , 128
n-Octyl maltoside , 127, 128, 264, 269 Non-aqueous conditions , 2 Non-ionic surfactants , 12, 14, 124, 127 Non-polar medium , 191 Non-polar solvents , 13, 30, 40, 45, 52, 53, 55, 59,
156, 216 Nuclear magnetic resonance (NMR) , 10, 42, 70–71, 74,
106, 119, 120, 127, 128, 138, 139, 185, 198, 208, 226, 254, 255
N-Vanillyl nonamide , 138, 150–152, 215, 218–220, 259, 265–266, 268, 270
N-Vanillyl nonamide glycosides 4- O -( d -galactopyranosyl)N-vanillyl-nonanamide ,
150, 265 4- O -( a - d -galactopyranosyl-(1 ¢ →4) a - d -
glucopyranosyl)N-vanillyl-nonanamide , 150, 154 4- O -( d -glucopyranosyl)N-vanillyl-nonanamide ,
150–152 4- O -( a - d -glucopyranosyl-(1 ¢ →4) a - d -
glucopyranosyl)N-vanillyl-nonanamide , 154 4- O -( d -mannopyranosyl)N-vanillyl-nonanamide ,
150, 153 4- O -( d -ribofuranosyl)N-vanillyl-nonanamide ,
150, 218
O Optical rotation , 127, 167 Optimisation studies , 83 Oxo-carbenium ion , 10, 12
P p-cresol , 48–53 Petroleum ether , 48 pH , 6, 44, 69, 83, 124, 138, 215, 226, 253, Pharmaceutical , 2, 12, 23, 24, 40, 81, 124, 150, 152, 183,
220, 253 Pharmacological applications , 15, 124, 155
Phenolic glycosides , 7, 9, 14, 258, 259, 266, 272, 275, 277
Phenols vitamins , 215, 217, 218 pH memory , 83, 106, 117, 118, 134, 216 Phthalic anhydride , 67–70 Ping-Pong Bi-Bi mechanism , 51, 226,
231, 249 Plackett-Burman design , 50 Polyadipates , 77–78 Poly- e -caprolactone , 72–74 Polyhydroxy alkanoates , 65, 66 Poly lactic acid , 65–74 Polymer , 5, 65–67, 70–72, 74 Polymer fi lms , 71 Poly-p-benzamide , 74, 76–77 Poly-p-hydroxybenzoate , 74–76 Porcine pancreas lipase (PPL) , 24–25, 27, 40, 42,
45–47, 49–53, 55, 58, 59, 66, 73, 74, 82, 83, 86, 88–90, 98, 112, 118, 120
Predicted yields , 43, 45, 50, 51, 103, 105, 131, 132, 147, 149, 159, 162–165, 185, 187
Propanol , 14 Protocatechuic aldehyde , 31, 58, 59 Pungency , 150 Pyridoxine
6- O -( d -galactopyranosyl)pyridoxine , 197 7- O -( d -galactopyranosyl)pyridoxine , 197, 219 6- O -( d -glucopyranosyl)pyridoxine , 197, 219 7- O -( d -glucopyranosyl)pyridoxine , 197, 219 6- O -( d -mannopyranosyl)pyridoxine , 197, 219 7- O -( d -mannopyranosyl)pyridoxine , 197, 219
R Re fl ux method , 124–127, 129–130, 138, 158, 191,
193, 202, 208, 219 Regioselectivity , 26, 27, 152, 198, 215, 218, 220,
222, 223 Response surface methodology , 2, 42–45, 130–134,
146–149, 157–165, 184–187 Retention time , 106–109, 112, 124, 125, 127, 129, 138,
141, 144, 150, 151, 156, 157, 166, 188, 189, 197, 198, 200, 201, 204
Retinyl glycosides , 222 Reverse hydrolysis , 9 Rhizomucor miehei lipase , 25, 26, 47, 62, 86, 90,
118, 226 Rhizopus sp. , 6, 31, 124, 130, 184, 215 Ribo fl avinyl glycosides
5- O -(1- d -fructofuranosyl-(2→1 ¢ ) a - d -glucopyranosyl)ribo fl avin , 188–190, 194, 266
5- O -( d -galactopyranosyl)ribo fl avin , 188, 266, 267 5- O -( b - d -galactopyranosyl-(1 ¢ →4) b - d -
glucopyranosyl)ribo fl avin , 189, 266 5- O -(6- d -glucopyranosyl)ribo fl avin , 193, 266 5- O -( d -glucopyranosyl)ribo fl avin , 189–193, 266 5- O -( a - d -glucopyranosyl-(1 ¢ →4) a - d -glucopyranosyl)
ribo fl avin , 188, 194, 219, 266, 267 5- O -( a - d -glucopyranosyl-(1 ¢ →4)6 ¢ - d -
glucopyranosyl)ribo fl avin , 194, 219, 266, 267
Index
284
Riboflavinyl glycosides (cont.) 5- O -( a - d -glucopyranosyl-(1 ¢ →4)6- d -
glucopyranosyl)ribo fl avin , 194, 267 5- O -( d -mannopyranosyl)ribo fl avin , 188, 221, 266 5- O -( d -ribofuranosyl)ribo fl avin , 188, 266
Ring-opening polymerization , 65, 66, 72
S Scanning electron microscopy , 53–55 Serotonin , 138, 177, 178, 180–183, 215 Serotoninyl glycosides
serotoninyl-5- O -( d -galactopyranoside)181 , 219 serotoninyl-5- O -(6- d -galactopyranoside) , 181, 219 serotoninyl-5- O -( d -glucopyranoside) , 181, 219 serotoninyl-5- O -(6- a - d -glucopyranoside) , 181, 219 serotoninyl-5- O -( d -mannopyranoside) , 181, 219 serotoninyl-5- O -(6- d -mannopyranoside) , 181, 219 serotoninyl-5- O -( d -ribofuranoside) , 181, 219
Shake- fl ask method , 125, 126, 129, 130, 219 Single substrate inhibition , 226–232, 236–246 Solubility test , 144 Spectral characterization , 109–112, 127–129, 138–141,
144, 151, 157, 167, 176–177, 189–191, 202, 204, 208–209
Stearyl alcohol , 50, 129, 130 Stearyl glucoside , 130 Stereoselectivity , 81–82, 138 Stereospeci fi city , 13, 26, 28 Substrate , 1, 2, 5–10, 13, 24–26, 28, 29, 34, 40–42, 50, 51,
55, 56, 82, 83, 85, 90, 99, 102, 113, 117, 120, 123, 124, 138, 166, 167, 182, 188, 200, 215–217, 223, 226–236, 239–244, 247, 249, 253–261, 268–274
Subtilisin , 32, 33, 82, 117, 120 Succinic anhydride , 68, 70–72, 74 Sucrose , 82, 86, 106, 113, 114, 116, 118, 127, 128,
138, 141, 144, 146, 156, 167, 180, 183, 188, 189, 191, 215, 217, 220–224
Sugar donor , 9 Sugar molecule , 138 Surfactants , 2, 12–14, 24, 32–34, 40, 58, 81, 124, 127 Sweet almond b -glucosidase , 8
T Tensile strength , 71, 74, 75 Thermodynamically controlled , 123 Thermostability , 52–53, 82 Three-dimensional surface plots , 163, 165 a -Tocopherol
6- O -( d -galactopyranosyl) a -tocopherol , 206, 209, 219, 223, 266
6- O -( d -glucopyranosyl) a -tocopherol , 204, 206–209, 223, 266–269
6- O -( d -mannopyranosyl) a -tocopherol , 206, 209, 219, 223, 266, 267, 269, 276
a -Tocopheryl glycosides , 204–209, 223–224, 260, 271
Tolyl esters , 32, 47–50 Transesteri fi cation , 23, 24, 28–29, 39, 40, 66, 82 Transformation , 1, 5 Trans glycosylation , 9
U Ultraviolet-visible spectroscopy , 176, 189, 198,
202, 208 Underivatized and unprotected amino acids , 253 Underivatized and unprotected carbohydrates , 253
V Validation , 45, 49, 105, 132, 149, 163–165, 187 Vanillin , 137, 138, 155–159, 161–165, 215–218, 220,
221, 226, 244–249, 259, 260, 265–268, 270 Vanillyl glycosides
1,6- O -(bis-4- O -vanillin) d -sorbitol , 161 4- O -( d -glucopyranosyl-(1 ¢ →4) a - d -glucopyranosyl)
vanillin , 160 4- O -( b - d -galactopyranosyl-(1 ¢ →4) b - d -
glucopyranosyl)vanillin , 160 4- O -( d -glucopyranosyl-(1 ¢ →4)6 ¢ - d -glucopyranosyl)
vanillin , 160 4- O -( d -glucopyranosyl-(1 ¢ →4)6- d -glucopyranosyl)
vanillin , 160 4- O -(1- d -fructofuranosyl-(2→1 ¢ ) a - d -
glucopyranosyl)vanillin , 160 4- O -(1- d -sorbitol)vanillin , 161 4- O -(6 ¢ - d -fructofuranosyl-(2→1 ¢ ) a - d -
glucopyranosyl)vanillin , 160 4- O -(6- d -glucopyranosyl)vanillin , 160 4- O -(6- d -sorbitol)vanillin , 161 4- O -( d -galactopyranosyl)vanillin , 218, 260, 265 4- O -( d -glucopyranosyl)vanillin , 156–159, 220 4- O -( d -mannopyranosyl)vanillin , 156, 218, 265
Vitamin glycosides , 15, 218, 259, 265, 266, 268, 273, 274, 276, 277
Vitamins Vitamin A-retinol , 195–198 Vitamin B1-thiamin , 184–187 Vitamin B2-ribo fl avin , 187–191 Vitamin B6-pyridoxine , 183, 191–195 Vitamin D2-ergocalciferol , 198–202 Vitamin D3-cholecalciferol , 202–204 Vitamin E- a -tocopherol , 204–209
W Water insoluble phenols , 138 Water insoluble vitamins , 123 Water soluble phenols , 137, 151 Water soluble vitamins , 183
Index