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UNIVERSITY OF MARIBOR
FACULTY OF CHEMISTRY AND CHEMICAL ENGINEERING
Doctoral thesis
Preparation of enantiomers using high-pressure technologies
Author: Paul Thorey
Mentor: prof. dr. Maja Habulin
Co-mentor: prof. dr. Béla Simándi
Maribor, 2010
Preparation of enantiomers using high-pressure technologies
Abstract
The study of two different methods of obtaining chiral alcohols is proposed herein.
The requirement of the relatively new paradigm of green chemistry associated with clean
technologies such as biocatalysis or non-conventional solvents, dense gases, was
focused at. Indeed, the two methods of production of chiral alcohols were:
the conversion of acetophenone into (R)-1-phenylethanol in dense gases
catalysed by Lactobacillus brevis alcohol dehydrogenase and its
coenzyme, NADP/H;
the resolution of (±)-trans-1,2-cyclohexanediol by cocrystal formation with
tartaric acid followed by supercritical extraction.
In both cases high enantiopurities were achieved (ee>99%).
Key words:
High-pressure technologies, enantiomers, green chemistry, R-1-phenylethanol,
Lactobacillus brevis, alcohol dehydrogenase, NADP, liquid propane, enzyme deactivation,
resolution, trans-1,2-cyclohexanediol, tartaric acid, cocrystal, supercritical carbon dioxide,
extraction, X-ray diffraction, differential scanning calorimetry.
UDK: 66 – 987 : 544 . 122 . 3 (043 . 3)
2
CONTENT FIGURES .............................................................................................................. 8 TABLES .............................................................................................................. 12 ABBREVATIONS................................................................................................ 14 ABBREVATIONS................................................................................................ 14 SYMBOLS .......................................................................................................... 15 THANKS ............................................................................................................. 17 DEDICATION ...................................................................................................... 18 1. INTRODUCTION: in search of asymmetry................................................... 19 2. Bibliographical review.................................................................................. 24
2.1. Production of enantiomers ................................................................... 24 2.1.1. Enantiomers and stereoselective synthesis-importance of the
catalyst. 24 2.1.2. Biocatalysis ..................................................................................... 27
2.1.2.1. Generalities on enzymes.......................................................... 27 2.1.2.2. Biocatalysis in industry ............................................................. 29 2.1.2.3. Membranes .............................................................................. 30 2.1.2.4. Biphasic systems...................................................................... 31 2.1.2.5. Improving the stability of enzymes: immobilisation techniques . 32 2.1.2.6. Immobilisation of ADHs. ........................................................... 34 2.1.2.7. Enzyme-catalysed reactions in non aqueous solvents.............. 35
2.1.3. Resolution of racemic mixture. ........................................................ 37 2.1.3.1. Chiral chromatographic separation........................................... 37 2.1.3.2. Resolution by selective crystallisation: conglomerates and
racemates. 39 2.1.3.3. Resolution by formation of diastereoisomers............................ 40 2.1.3.4. Resolution of enantiomer by formation of diastereoisomeric salt.
42 2.1.3.5. Other methods of resolution of alcohols ................................... 48
3
2.1.3.6. Resolution of enantiomers by formation of a diastereoisomeric
cocrystal with tartaric acid instead of a salt.......................................................... 49 2.2. Conversion of acetophenone to R-1-phenylethanol using alcohol
dehydrogenase from Lactobacillus brevis (LBADH) .................................................... 52 2.2.1. Alcohol dehydrogenases require a coenzyme NADH and NADPH that
must be regenerated. .............................................................................................. 52 2.2.1.1. Generalities about the coenzymes ........................................... 52 2.2.1.2. Regeneration by a second enzyme .......................................... 55 2.2.1.3. Regeneration by the same enzyme: sacrificial substrate method.
56 2.2.2. Different studies with alcohol dehydrogenase from Lactobacillus
brevis 57 2.2.3. Alcohol dehydrogenase in non aqueous solvent.............................. 59 2.2.4. Goal of this work concerning LBADH in dense gases...................... 61
2.3. Resolution via the formation of diastereomeric complexes with (+)-
tartaric acid followed by extraction with supercritical carbon dioxide applied (±)-trans-
1,2-cyclohexanediol .................................................................................................... 61 2.3.1. Different methods of resolution of (±)-trans-1,2-cyclohexanediol
based on the formation of a covalent bond.............................................................. 62 2.3.2. Resolution of (±)-trans-1,2-cyclohexanediol by selective formation of
a cocrystal 62 2.3.3. Physical properties of CHD. ............................................................ 64 2.3.4. Method of resolution of CHD by formation of a cocrystal CHD-TA and
optimisation of the parameters of extraction: temperature and pressure.................. 66 2.3.5. Issues concerning the resolution of CHD by cocrystallisation and SFE
to be addressed in the present work........................................................................ 68 3. Conversion of acetophenone to R-1-phenylethanol ..................................... 70
3.1. Materials and methods......................................................................... 70 3.1.1. Materials ......................................................................................... 70
3.1.1.1. Reagent ................................................................................... 70 3.1.1.2. Biocatalyst................................................................................ 70
3.1.2. Methods .......................................................................................... 70
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3.1.2.1. Preparation of the biocatalyst ................................................... 70 3.1.2.2. High-pressure view cell ............................................................ 71 3.1.2.3. Reaction with LBADH............................................................... 72 3.1.2.4. ADH activity test....................................................................... 73 3.1.2.5. Autoclave for incubation of biocatalyst...................................... 74
3.1.3. Analytical methods.......................................................................... 74 3.2. Results and discussion ........................................................................ 75
3.2.1. Reaction in water ............................................................................ 75 3.2.2. Preliminary test in heptane.............................................................. 75 3.2.3. Reaction at high-pressure ............................................................... 76
3.2.3.1. Reaction in propane with co-immobilised catalyst..................... 77 3.2.3.2. Reaction in biphasic system propane-water ............................. 78
3.2.4. Deactivation of LBADH.................................................................... 80 3.2.4.1. Deactivation of “untreated” LBADH .......................................... 80 3.2.4.2. Deactivation of LBADH in propane ........................................... 81 3.2.4.3. Reaction in biphasic systems propane-water............................ 81
3.3. Conclusion and future work.................................................................. 82 4. Resolution of (±)-trans-1,2-cyclohexanediol via the formation of
diastereomeric complexes with (+)-tartaric acid followed by extraction with supercritical
carbon dioxide. ............................................................................................................... 83 4.1. Materials and methods......................................................................... 83
4.1.1. Materials. ........................................................................................ 83 4.1.2. Determination of the structure of the co-crystal. .............................. 83 4.1.3. Supercritical fluid extractor. ............................................................. 83 4.1.4. Resolution of CHD with TA and SFE............................................... 85
4.1.4.1. Sample preparation. ................................................................. 85 4.1.4.2. Supercritical fluid extraction...................................................... 85 4.1.4.3. Raffination by alkaline treatment .............................................. 86
4.1.5. Analytical methods.......................................................................... 86 4.2. Results and discussion ........................................................................ 87
4.2.1. Characterisation of the cocrystal ..................................................... 87 4.2.1.1. The structure of the co-crystal. ................................................. 87
5
4.2.1.2. Characterisation of the co-crystal TA-RRCHD.......................... 91 4.2.2. Decomposition of the CoC in situ .................................................... 93 4.2.3. Description of the extraction............................................................ 96
4.2.3.1. Monitoring the evolution of the content of the extractor by XRD
and fractionning 96 4.2.3.2. Improving the enantiomeric excesses by leaving off an
intermediate fraction...........................................................................................102 4.2.4. Sample preparation........................................................................102
4.2.4.1. Investigation of the binaries RRCHD-TA and SSCHD-TA.
Coroboration by XRD. ........................................................................................102 4.2.4.2. Investigation of the cocrystallisation by XRD, ternary phase
diagram. 106 4.2.4.3. Two issues raised by the XRD studies: sodium hydrogen tartrate
(NaTA) and metastable compound.....................................................................109 4.2.4.4. Conditions of crystallisation/sample preparation......................110
4.2.5. Toward enantiopure products, molar ratio, double extraction .........113 4.2.5.1. Extraction with molar ratios varying .........................................113 4.2.5.2. Resolution repeated twice .......................................................115
4.3. Conclusion on the resolution of CHD by cocrystallisation and SFE and
further plan 118 5. CONCLUSIONS .........................................................................................120 6. LITTERATURE...........................................................................................121 7. APPENDIX .................................................................................................132
7.1. Challenge of green chemistry..............................................................132 7.1.1. Context of the development of green chemistry..............................132 7.1.2. 12 principle of green chemistry. Derived conceps. .........................134 7.1.3. Alternative solvent: supercritical fluids and SCCO2.........................136
7.2. Reaction run with coimmobilised NADP ad LBADH in non-aqueous
solvent, propane and heptane....................................................................................139 7.3. Miscibility of ACP, ISP, 1-phenylethanol, acetone and propane ..........140 7.4. Method of determination of the structure of the cocrystal CoC ............140 7.5. Theoretical ternary diagram with a liquid solution................................141
6
7.5.1. If no liquid solution between the CHD enantiomers exists. .............142 7.5.2. A liquid solution exists between the CHD enantiomers...................143
7.5.2.1. Gibbs free enthalpy of a binary mixture RacCHD and RRCHD or
SSCHD presenting a partial miscibility ...............................................................143 7.5.2.2. Ternary diagram......................................................................143
7.6. Results of the experiment for the determination of the phase diagram.
146 7.7. Diffractogram of samples prepared according to different methods.....152
7
"Perhaps looking-glass milk isn't good to drink" Said Alice to the cat.
(Lewis Caroll, Though the looking-glass)
8
FIGURES
Figure 1: Structure of RPE, SSCHD, and RRCHD, the asymmetric compound
targeted in this work........................................................................................................ 21 Figure 2: The first homogeneous asymmetric catalysis by a chiral metal complex.
....................................................................................................................................... 24 Figure 3: Enantioselective synthesis of (-)-mentol................................................ 25 Figure 4: Bifunctional Ru-BINAP(diamine) complexes for enantioselective
hydrogenation of simple ketones (acetophenone into R-1-phenylethanol). ..................... 26 Figure 5: Illustration of how fast the enzyme drives a reaction............................. 28 Figure 6: Emil Fischer’s substrates...................................................................... 29 Figure 7: Classification of membrane filtration processes .................................... 30 Figure 8: Example of a continous conversion with a membrane .......................... 31 Figure 9 : A two-phased system involving a coenzyme-dependent enzyme ........ 32 Figure 10: L-amino acid production catalyzed by aminoacylase. ......................... 33 Figure 11: Three examples of carrier-coupling using the amino group of an enzyme
(Enz in this figure)........................................................................................................... 34 Figure 12: Binary mixture melting point diagram for a conglomerate-forming pair of
enantiomers (a) and a racemate-forming (b)................................................................... 39 Figure 13: Chemical and physical equilibria in the racemization for N-(2-
methylbenzylidene)phenylglycine amide......................................................................... 40 Figure 14: The resolution of rac-CHD by Chatterjee. ........................................... 41 Figure 15: Separation of enantiomers (+)-A and (-)-A combined with racemisation.
....................................................................................................................................... 42 Figure 16: Common resolution of a base B by the acid (L)-HA. ........................... 44 Figure 17 : a) Binary melting point phase diagram of salt p and salp n and b) their
solubility diagram. ........................................................................................................... 45 Figure 18 : Resolution with a molar ratio of 0.5.................................................... 46 Figure 19: Resolution of amphetamine by distillation ........................................... 47 Figure 20: Formation of phtalate derivatives, useful intermediates for resolution of
alcohol by diastereomeric salt formation ......................................................................... 48
9
Figure 21: Resolution of chiral alcohol by enzyme catalysed acylatation ............. 49 Figure 22: General activity of an alcohol dehydrogenase (ADH).......................... 53 Figure 23 : NADP+: Nicotinamide adenine dinucleotide ....................................... 53 Figure 24 : NAD is a hydrid acceptor and NADH a hydrid donnor........................ 53 Figure 25 : Coenzyme regeneration in the case of a reduction with NAD(P)H. .... 55 Figure 26 : Reactions catalysed by LBADH, conversion of acetophenone and
regeneration of NADPH by addition of isopropanol. ........................................................ 57 Figure 27: Comparison of 2 solid-liquid melting phase diagrams a) a usual for a
couple of enantiomers and b) CHD’s case with a solid solution....................................... 65 Figure 28: Principle of the resolution of (±)-CHD by co-crystal formation followed
by an extraction in SCCO2 (mr=0.5)................................................................................ 66 Figure 29: Decomposition of the residuum........................................................... 67 Figure 30 : Scheme and picture of the high-pressure reactor. ............................. 72 Figure 31: Example of a test of enzyme activity................................................... 74 Figure 32: Autoclave for the measurement of ADH deactivation in propane ........ 74 Figure 33: Conversion of ACP to RPE in water.................................................... 75 Figure 34: Reaction in heptane with co-immobilised catalyst (reaction G) ........... 76 Figure 35: Bioconversion in propane with immobilised catalyst............................ 78 Figure 36: Bioconversion in the biphasic system water/dense propane at 100 bar
....................................................................................................................................... 78 Figure 37: Bioconversion in the biphasic system water/dense propane at 30 bar 79 Figure 38: Bioconversion in the biphasic system water/dense propane at 200 bar
....................................................................................................................................... 79 Figure 39: Deactivation of an aqueous solution of LBADH at atmospheric pressure
and 36°C......................................................................................................................... 80 Figure 40: Deactivation of LBADH in powder form at atmospheric conditions...... 80 Figure 41: Deactivation of the preparations of LBADH in propane at 30 bar. ....... 81 Figure 42: Deactivation of an aqueous solution of LBADH in a biphasic system with
dense propane at 30 bar................................................................................................. 82 Figure 43: Supercritical fluid extractor.................................................................. 84 Figure 44: ORTEP diagram (Spek 2003) of the CHD-TA co-crystal (1)................ 87
10
Figure 45: The two dimensional infinite hydrogen bonded plane of the CHD-TA co-
crystal (1)........................................................................................................................ 90 Figure 46 : The inner TA layer of the sheet (Macrae et al. 2006) presenting its
hydrogen bonding system of co-crystal 1. ....................................................................... 91 Figure 47: Theoretical (in black) and experimental (in red) diffraction pattern of
CoC ................................................................................................................................ 91 Figure 48: FTIR: spectrum of the CHD-TA co-crystal Coc. .................................. 92 Figure 49: DSC melting peak of the pure CHD-TA co-crystal CoC in sealed Al-pan
at 10°C/min (mass 2.59 mg). .......................................................................................... 92 Figure 50: Simultaneous TG/DTA curves of the pure CHD-TA co-crystal 1 ......... 93 Figure 51: Extraction curves at different temperature and pressure, study of the
decomposition of CoC in situ. ......................................................................................... 94 Figure 52: Experimental powder pattern of the compound involved in the resolution
system. ........................................................................................................................... 97 Figure 53 : Loss of weight of the extractor according to the weight of CO2 and
sampling. The spline line is only indicative...................................................................... 97 Figure 54: Theoretical loss of weight of the extractor if no sample had been taken.
This figure does not show more information than the previous but has the advantage to
show which aspect the extraction curve would have if no sample had been taken.......... 98 Figure 55: Diffractograms of the different samples from the material inside the
extractor over the extraction............................................................................................ 99 Figure 56: Different fractions during extraction, their enantiomeric excesses......101 Figure 57: DSC curve of binary mixture corresponding to 1:1 molar ratio of a)
(R,R)-CHD and co-crystal (1) and b) (S,S)-CHD and (R,R)-TA, both exhibiting eutectic
melting behavior. ...........................................................................................................104 Figure 58: Melting binary phase diagram SSCHD TA. ........................................104 Figure 59: The problematic melting point phase diagram of RRCHD-TA. ...........105 Figure 60: Two ternary phase diagrams TA-SSCHD-RRCHD ............................107 Figure 61: DSC analysis of a sample of composition RRCHD:TA 16:84 featuring
the metastable compound “X” ........................................................................................110 Figure 62: Diffractograms of sample of mr=0.5 evaporated at different temperature
......................................................................................................................................112
11
Figure 63: Yield and F parameter with varying molar ratio..................................114 Figure 64: Enantiomeic excess with varying molar ratio......................................114 Figure 65: Second resolution of mixture 1 presenting an ee of SSCHD..............116 Figure 66: Second resolution of mixture 2 presenting an enantiomeric excess of
RRCHD..........................................................................................................................116 Figure 67: Sustainable development as a confluence of three domains: social,
economy, and environment............................................................................................132 Figure 68: Two determinant catalytic steps in the “green” synthesis of ibuprofen.
......................................................................................................................................136 Figure 69: phase diagramm (P,T) of a fluid.........................................................137 Figure 70: “No solid solution CoC>racem” phase diagram of RRCHD, SSCHD and
TA supposing that a racemic compound is formed and not a solid solution....................142 Figure 71 : “No solid solution CoC<racem” alternative phase diagram with RRCHD,
SSCHD, CoC, RacCHD, and TA....................................................................................142 Figure 72 : Gibbs molar enthalpy of a binary RRCHD-SSCHD ...........................143 Figure 73: “Solid solution and CoC>solCHD” ternary phase diagram .................144 Figure 74 : Variation of free energy of the system CHD + dn TA when CoC forms.
......................................................................................................................................145 Figure 75 : “Solid solution and CoC=solCHDlim” ternary phase diagram with
solCHD 2 .......................................................................................................................145 Figure 76: Example of ternary phase diagram where CoC can intake a small
fraction of SSCHD..........................................................................................................146
12
TABLES Table 1: The opposite enantiomers have different biological activities. ................ 22 Table 2: Examples of pharmaceuticals resolved by diastereomeric crystallisation in
the process ..................................................................................................................... 43 Table 3 : Coenzyme and their associated group.................................................. 52 Table 4 : Price of some nicotine adenine dinucleotides (from Jülich Chiral Solution
GmbH’s product portfolio (february 2007)) ...................................................................... 54 Table 5 : Some properties of Formate Dehydrogenase (FDH) (Product Portfolio
2007) .............................................................................................................................. 56 Table 6: Melting point and structural data for crystalline phases of trans-1,2-
cyclohexanediol and references...................................................................................... 64 Table 7: Comparasion of different results obtained for the resolution of CHD...... 68 Table 8: Result of the three conversions run in biphasic systems. ....................... 80 Table 9: Summary of crystallographic data, data collections, structure
determination and refinement for CHD-TA co-crystal (1)................................................. 88 Table 10: Intermolecular interactions in the crystal structure of CHD-TA co-crystal
(1). .................................................................................................................................. 89 Table 11: Melting point and enthalpy of fusion of the applied chemicals .............103 Table 12: DSC and XRD data of binary mixtures in the ternary system ..............103 Table 13: Result of the different experiments of further enantioenrichment of
mixture 1 and mixture 2 .................................................................................................117 Table 14: Waste of the different segment of chemical industry ...........................135 Table 15: Critical points of fluid presenting an industrial interest.........................137 Table 16: Result of the bioconversion of ACP into RPE in heptane and propane.
......................................................................................................................................139 Table 17: Miscibility in propane...........................................................................140 Table 18: The different sample prepared for investigation of the ternary system,
their composition and the phase observed by XRD........................................................146 Table 19: Composition of the different sample if the ternary does not present solid
solution. .........................................................................................................................147
13
Table 20: Which deviation do we observe from the model "no solid solution
CoC>racem"? ................................................................................................................147 Table 21: Composition of the different sample if the ternary presents a solid
solution and the deviations observed. ............................................................................147 Table 22: Sample for sample preparation prepared in different condition ...........152
14
ABBREVATIONS ACP Acetophenone
ADH Alcohol dehydrogenase
CHD Trans-1,2-cyclohexanediol
CoC cocrystal CHD-TA 1:1
DBTA O,O′-dibenzoyl-(2R,3R)-tartaric acid
DSC differential scanning calorimetry
ee enantiomeric excess
FDA Food and drug administration
FDH Formate Dehydrogenase
GC Gas chromatography
HPLC High performance (or pressure) liquid chromatography
HTA Sodium hydrogen tartrate
ISP isopropanol
LBADH Alcohol dehydrogenase from Lactobacillus brevis
NAD Nicotine adenine dinucleotide
NADH Nicotine adenine dinucleotide hydrogene
NADP Nicotine adenine dinucleotide phosphate
NADPH Nicotine adenine dinucleotide phosphate hydrogene
NaTA Hydrogen tartrato sodium
racCHD Racemic (±)-trans-1,2-cyclohexanediol
RPE R-1-phenylethanol
RRCHD (R,R)-trans-1,2-cyclohexanediol
RT Room temperature
SCCO2 Supercritical carbon dioxide
SF Supercritical fluid
SFE Supercritical fluid extraction
SolCHD Solid solution of racCHD with SSCHD or RRCHD
SSCHD (S,S)-trans-1,2-cyclohexanediol
SSTA (S,S)-tartaric acid
TA (R,R)-tartaric acid
15
SYMBOLS a activity no unit
c concentration (mol/L)
CO2rel relative weight of CO2 (mCO2/mCHDini) (g/g)
ee enantiomeric excess (%)
eeext or ee1 enantiomeric excess of the extract (%)
(first extraction)
eeext2 or ee2 enantiomeric excess of the extract (%)
(second extraction)
eeraf enantiomeric excess of the raffinate (%)
Ea activation energy (kJ/mol)
F F parameter no unit
g Gibbs free molar enthalpy (kJ/mol)
G Gibbs free enthalpy (kJ)
H enthalpy (kJ/mol)
Keq equilibrium constant no unit
µ chemical potential (kJ/mol)
µ0 chemical potential of a pure compound (kJ/mol)
P pressure (MPa, bar)
Pc critical pressure (MPa)
t time (h, min)
T temperature (°C, K)
Teu Eutectic temperature (°C, K)
Tc critical temperature (°C, K)
v reaction rate (mmol/min,
mmol/(min.U),…)
vi initial reaction rate (mmol/min,
mmol/(min.U),…)
R ideal gas constant (kJ/(mol K))
S entropy (kJ/(mol K))
V volume (L)
16
x fraction (%)
X fraction in a binary (%)
Y yield (%)
Yext or Y1 yield of the first extraction (%)
Y2 yield of the second extraction (%)
Yraf yield of raffination (%)
17
THANKS
I would like to warmly thank the people who made this work possible.
Friends and family. Colleagues and administrations.
As a complement:
“Hvala lepa”,
“Köszönöm szepa”,
Merci bcp,
“danke schön”,
“muchas gracias”!
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DEDICATION
This work is dedicated to the memory of RENE HUBERT (1923-2010)
Introduction
19
1. INTRODUCTION: in search of asymmetry
Enantiomer comes from the Greek ἐνάντιος, opposite, and μέρος, part. Hands, feet are
enantiomeric pairs, they are mirror image to each other. A hand, a foot, snails are enantiomers for
an enantiomer is a molecule that does not superimpose on its mirror-image. Examples of synthetic
enantiomer and their biological activities are given in Table 1, where each line’s compounds are
mirror images. Whereas industrial production of enantiomerically pure products is a thorny
problem which has to be (partially) addressed herein, nature is remarkably able to perfectly
perform such a task with the helps of enzymes, the catalysts of life. The origin of the asymmetric
chirality in life is much discussed but we can more surely state that the need and the origin of
synthetic asymmetric molecules is to find in biological processes!
Biological organisms are built out of asymmetric compounds and the physiological
phenomena arise from highly precise molecular interactions in which chiral host molecules
recognize two enantiomeric guest molecules in different ways. Consequently many compounds
which are “active” are also chiral, and its stereoisomer often present dramatically different
activities toward a living organism as illustrated in Table 1 which includes the examples of the
present introduction. Industry found the source of chirality in natural products and still intensively
uses the building blocks produced in vivo. So many natural substances had applications in the fields
of chirotechnologies (Sheldon 1993): amino acid and sugar, which are eminently asymmetric, also
the biocatalyst and the secondary metabolites (alkaloids, essential oils…). Semisynthesis was
applied to create novel drug from traditional cures. A good illustration is the drug family derived
from opium poppy’s alkaloids. The catalytic potency of enzymes is taken advantage of by the
industry in processes based on biocatalysts or biotransformation as for antibiotics. Natural products
are the most prominent separation agents (for instance sugars for chromatographic columns) or
resolving agents.
The sectors of chemistry which needs asymmetric molecule are: pharmaceuticals, animals
heath products, agrochemical, electronic chemical, pheromones, flavours and fragrances. The scale
of this production spans from chiral synthons or highly active material produced at kilogram-scale
to amino acid which production reaches 105 tonnes per year. Agrochemical and pharmaceutical
commonly reaches tens of thousand of tonnes a year (Collins et al. 1992). The demand for
enantiomerically pure drugs has increased, so did the demand for stereospecific processes. Between
1983 and 1985, 95% of the drugs with an asymmetric carbon were sold under the racemic form,
while in 1992, only 25% of the drugs were sold as racemate, a equimolar mixture of both
Introduction
20
enantiomers (Collins et al. 1992) and the sale of enantiomer passed over the 120 billion marks in
the year 2000 (Kennedy et al. 2002). In 1998, 48% of the small molecule drugs approved by the
FDA were single enantiomers, and in less than half of those cases the chiral motif was made by
synthetic chemical methods. By 2007, the proportion of single enantiomers in small molecule drug
approvals had risen to 71%, and of these products, 70% had the chirality introduced by synthetic
chemical methodology. (Thayer 2008; Lennon et al. 2009)
Pairs of enantiomer generally have pharmaceutical activities (or efficacy) and only one
might be needed as for paclobutrazol (see Table 1: The opposite enantiomers have different
biological activities.) A similar issue is raised by essential oils as carvone or menthol or the
sweetener which taste or smell is rather different from one enantiomer to another: their
enantiopurification is compulsory. In some cases one of the enantiomers is not active and presents
no side effect and consequently the drug is often marked as racemic. This results in a poor yield:
half of the product is lost, as for ibuprofen, and consequently, when the production of a racemic is
shifted to a pure enantiomer the capacity of the process doubles, and the cost efficiency is
improved. Moreover the pharmacological study and validation of a drug is less complex for an
enantiomerically pure and the probability to have complex interaction between the active molecules
is reduced. Using the single active enantiomer can lower side effect of a drug, the same way it can
limit the environmental impact of agrochemicals.
Another reason for the development of chirotechnologies concerns the ownership of the
drugs. The development of a new drugs is extremely expensive : it costs about US$ 400 million
(2000 dollars) (Dimasi et al. 2003). When the patent of a drug sold as a racemate expires it is
possible to patent the sole active enantiomer to prolong the ownership on a molecule. The cost of
such an operation is obviously much lower than the discovery of a new molecule (Kennedy et al.
2002). For instance, the recently patented Xyzal (levocetirizine) is the active R-enantiomer of
cetirizine (Zyrtec, the racemate). Fluoxetine (prozac)) was subject of a dispute between two
companies about the commercialisation of fluoxetine as racemic or pure enantiomer (Kennedy et
al. 2002).
Thalidomide was a treatment for morning sickness. It possesses two enantiomers one
causes the desired sedative effect while the other was teratogenic and caused many foetal
disformation1 (Kennedy et al. 2002). Since this scandal the FDA and European Committee for
Proprietary Medicinal Products imposes that the activity of both enantiomers are known separately.
This decision increased a lot the cost of the development of a drug, therefore, pharmaceutical
1 Thalidomide racemises in physiological conditions. However they are used against leprosy and
might be again used against Aids disorders and tuberculosis.
Introduction
21
companies might decide to develop (and patent) an enantiomerically pure product. Hence, the other
enantiomer got the status of an undesired impurity.
On one hand, the authorities and the industrials’ interest for chirotechnologies has
motivated academic research in chemistry to study this relatively new topic. On the other hand, the
improvement of the legislation and products on the market took place because analytical
procedures allowed the determination of configurations or enantiomeric excesses, natural product
chemistry and biochemistry improved and stereoselective preparative methods were developed.
In this context much favourable to the development of chirotechnologies, the study of two
different methods of obtaining chiral alcohols is proposed herein. The requirement of the relatively
new paradigm of green chemistry associated with clean technologies such as biocatalysis or dense
gases as non-conventional solvents will be focused at. Indeed, the two proposed method of
production of chiral alcohol (see their structure on Figure 1) is the conversion of acetophenone into
(R)-1-phenylethanol in dense gases catalysed by Lactobacillus brevis alcohol dehydrogenase and
the resolution of (±)-trans-1,2-cyclohexanediol by cocrystal formation followed by supercritical
extraction.
OH
RPE
(R)-1-phenylethanol
OH
OH
SSCHD (S,S)- trans-1,2-cyclohexanediol
OH
OH RRCHD (R,R)- trans-1,2-cyclohexanediol
Figure 1: Structure of RPE, SSCHD, and RRCHD, the asymmetric compound targeted in
this work.
Introduction
22
Table 1: The opposite enantiomers have different biological activities.
H2NCOCH2
NH2 H
COOH
(S)-asparagine has a bitter taste.
H2NCOCH2
NH2 H
COOH
(R)-asparagine has a sweet taste.
O
(S)-carvone: caraway flavour
O
(R)-carvone: spearmint flavour
OH
O2N
OH
NHCOCHCl2
(R,R)-chloramphenicol is antibacterial.
OH
O2N
OH
NHCOCHCl2
(S,S)-chloramphenicol is inactive.
N NO
OH
O
Cl (R)-Levocetirizine is an antihistaminic.
N NO
OH
O
Cl S-Levocetirizine is not active.
ONH H
CF3
(R)-fluoxetine is antidepressant with
cardiac side-effect.
ONH H
CF3
(S)-fluoxetine is antidepressant with lower
side-effect.
NHO
ON
O
O (S)-thalidomide is teratogenic.
NHO
ON
O
O (R)-thalidomide has a sedative activity.
Used against leprosy
Introduction
23
O
OH
(S)-ibuprofen is an anti-inflammatory drug.
O
OH
(R)-ibuprofen is inactive.
NH
NH
OH
Et
H
OH
H Et (S,S)-ethambutol is tuberculostatic.
NH
NH
OH
Et
H
OH
H Et (R,R)-ethambutol causes blindness.
NN
N
tBu
Cl
OH
(R,R)-paclobutrazol is a fungicide.
NN
N
tBu
Cl
OH
(S,S)-paclobutrazol is a plant growth
regulator.
NO OF3C
COOBu
MeH
(S)-fluazifop butyl is inactive.
NO OF3C
COOBu
MeH
(S)-fluazifop butyl is an herbicide.
Bibliographical review
24
2. Bibliographical review
2.1. Production of enantiomers
2.1.1. Enantiomers and stereoselective synthesis-importance
of the catalyst.
Enantiomeric excess (ee) is a common way to describe the composition in enantiomers of a
mixture. Its formula is given in Equation 1. A common industrial standard for ee is above 70-80 %
(Collins et al. 1992). But higher grade can be demanded for certain purposes.
SRSR
ee
Equation 1 : Enantiomeric excess
The development of stereoselective chiral catalyst was the object of much effort and recent
development of not only biocatalysts (Sheldon 1993) but also abiological catalysts that present the
advantage to avoid the substrate limitation imposed by enzymes2. The catalytic asymmetric
synthesis is mainly based on chiral transition metal complexes or chiral acid and base. An example
is given in the Figure 2. The catalyst was improved by screening of Schiff base, ligand for the
metallo complex, leading to enantioselectivity up to 94%.
Figure 2: The first
homogeneous
asymmetric catalysis
by a chiral metal
complex.
Reproduced from Chirotechnology: the Industrial Synthesis of Optically Active
Compounds (Sheldon 1993)
An industrially-relevant synthesis based on stereoselective catalyst is the synthesis of (-)-
mentol that was developed by Noyori who was then rewarded with Nobel Prize in chemistry. The
2 For the importance of the catalyst in green chemistry and an overall introduction to this topic, see
annex 7.1.
Bibliographical review
25
key step was the asymmetric isomerisation of geranyldiethylamine catalyzed by an (S)-BINAP–Rh
complex in THF forming (R)-citronellal enamine, which upon hydrolysis gives (R)-citronellal in
96–99% ee (see Figure 3). The asymmetric reaction is performed on a nine-ton scale (Noyori
2001).
Figure 3: Enantioselective synthesis of (-)-mentol
Reproduced from (Noyori 2001)
Concerning the production of chiral alcohols green chemistry has presented different tools.
There are several techniques based on the stereoselective reduction, hydrogenation of ketones.
Several homogenous catalysts can be used for this purpose. The most effective homogeneous
hydrogenation catalysts are complexes consisting of a central metal ion, one or more (chiral)
ligands and anions which are able to activate molecular hydrogen and to add the two H atoms to an
acceptor substrate (Sheldon et al. 2007). Ru, Rh and Ir complexes stabilized by tertiary (chiral)
phosphorus ligands are the most active and the most versatile catalysts. The halogen-containing
BINAP–Ru(II) complexes is a precious catalyst for this reaction and allow ee superior to 90%
(Noyori 2001). Noyori rendered possible the enantioselective hydrogenation of aromatic ketones by
introducing a new class of Ru-BINAP (diamine) complexes (Noyori et al. 2001). In this case
hydrogen transfer is facilitated by ligand assistance. The company Takasago used this catalyst for
Bibliographical review
26
the production of (R)-1-phenylethanol in 99% ee using only 4 bar of hydrogen (see Figure 4), a
very moderate pressure compared to the 100 bar commonly used. There is consensus that the
transfer of the two H atoms occurs in a concerted manner as depicted in Figure 4 (Sheldon et al.
2007). This hypothesis explains the need for an N–H moiety in the ligand.
Figure 4: Bifunctional
Ru-BINAP(diamine)
complexes for
enantioselective
hydrogenation of
simple ketones
(acetophenone into
R-1-phenylethanol).
Reproduced from
Green Chemistry and
Catalysis (Sheldon et
al. 2007)
More rarely, heterogenous catalysts can catalyse the stereoselective reduction of ketone.
They are metal with chiral natural modifier such as Raney nickel system for β-functionalized
ketones (with tartaric acid (TA) as a modifier) or Pt catalysts modified with cinchona alkaloids for
α-functionalized ketones. In this last case, acetic acid or toluene as solvent, close to ambient
temperature and medium to high-pressure (10–70 bar) are sufficient to ensure high
enantioselectivities of 95 to 97.5% (Sheldon et al. 2007).
Some chiral catalysts were given as examples. Before concentration our attention to
biocatalyst and more specifically alcohol dehydrogenase it is necessary to give some definitions
necessary for the evaluation of the catalytic capacity. The activity of a catalyst is its ability to
perform a typical reaction in known condition: the activity is defined according to a certain
protocol that is widely accepted, shared by the researchers keen on comparing their results. The
activity is the initial rate of this reaction expressed in and µmol.min-1 and µmol.min-1/mgCatalyst is
often abbreviated as U and U/mgCatalyst, respectively. The activity is a very important parameter that
accounts for the deactivation (or hyper activation) of a catalyst. The half-life of catalyst is the time
after which half of the initial activity remains. The turnover is the number of catalytic cycles the
Bibliographical review
27
catalyst undertaken by unit of time. The total turnover is the number of catalytic cycle for a
conversion, this to say the number of synthesized molecules per number of catalyst molecules used.
2.1.2. Biocatalysis
2.1.2.1.Generalities on enzymes
The enzymes ARE the catalysts of life. These proteins are long chains from 100 to several
hundreds α-amino acids whose sequences are encoded in the DNA. This polypeptide chain forms
the primary structure of enzymes. Intramolecular bounds maintain the three-dimensional
structure of the protein. Secondary structure is the term given to local regions (10–20 amino
acids) of stable, ordered three-dimensional structures held together by hydrogen bonding, that is
non-covalent bonding between acidic hydrogens (O-H, N-H) and lone pairs. The three-dimensional
structure of protein sub-units, known as the tertiary structure, arises from packing together
elements of secondary structure to form a stable global conformation, which in the case of enzymes
is catalytically active. The packing of secondary structural units usually involves burying
hydrophobic amino acid side chains on the inside of the protein and positioning hydrophilic amino
acid side chains on the surface. The quaternary structure of an enzyme is the final structure of the
enzyme that can involve several proteins and cofactor or coenzymes (see 2.2.1.1). For more detail
please refer to Introduction to Enzyme and Coenzyme Chemistry (Bugg 2004).
Because of each enzyme’s millions year evolution, they are extremely good catalysts when
compared with man-made catalysts. They present three qualities that are speed, selectivity and
specificity. The speed of the enzyme in catalysing biochemical reaction can be illustrated by the
example found in Introduction to Enzyme and Coenzyme Chemistry (Bugg 2004): The rate of acid-
catalysed glycoside catalysis is accelerated 103-fold by intramolecular acid catalysis, but enzyme-
catalysed glycoside hydrolysis is 104-fold faster still – some 107 faster than the uncatalysed
reaction carried out at pH 1 (see Figure 5).
Bibliographical review
28
Figure 5: Illustration of how fast the enzyme drives a reaction
Reproduced from Introduction to Enzyme and Coenzyme Chemistry (Bugg 2004)
The selectivity and specificity of the enzyme is one of their best-known properties – and
also the most looked for. Actually, most enzymes have a limited range of accepted substrates and
the reaction they catalyse leads often to a unique product. Maybe the most striking of the extreme
stereoselectivity of the enzyme is the fact that in living organisms only one orientation is given to
the amino acid, the nucleic acid and so on. The discovery of this property of the enzyme was
underlined by Emil Fischer in 1894. He observed that the enzyme known as emulsin catalyzes the
hydrolysis of β-methyl-D-glucoside, while the enzyme known as maltase is active towards the α-
methyl-D-glucoside substrate (see Figure 6). This led Fischer to suggest his famous “lock-and-key”
theory of enzyme specificity, which he described in his own words as follows: “To use a picture, I
would say that enzyme and the glucoside must fit into each other like a lock and key, in order to
effect a chemical reaction on each other” (Vasic-Racki 2006)
Bibliographical review
29
Figure 6: Emil Fischer’s substrates
Reproduced from (Vasic-Racki 2006)
2.1.2.2.Biocatalysis in industry
They are important catalyst for industrial purposes, especially food industry. The
advantages of biocatalysis are the following: Enzymes (or micro-organisms) are renewable,
biodegradable and non toxic catalysts; they present good kinetic (high turnover) and high
selectivity (important enantiomeric excess and conversion and no protection step needed).
Moreover, they require only mild reaction conditions (low temperature, moderate pH and so on).
The History of Biostranformation – Dreams and Reality is told by Durda Vasic-Racki in a
thrilling way in Industrial Biotransformations (Liese et al. 2006) and we cannot report all
inventions and developments, and rather use some historical hallmarks as illustration and invite the
reader to this excellent historical introduction (Vasic-Racki 2006). The origin of biotransformation
can not be dated because the first microbial biotransformations, the production of alcoholic
brewage, cheese, vinegar were known before writing. As we need the restrain the scope of this
presentation we will refer only to enzyme-catalysed reaction, this is to say we exclude microbial
biotransformation and all those based on whole cell.
5 classes of enzyme are used for bioconversion: oxidoreductases (EC 1), transferases (EC
2), hydrolases (EC 3), Lyases (EC 4), isomerases (EC 5). Many oxidoreductases include the
NAD(P) dependent alcohol dehydrogenase, which will be considered latter. The enzyme class of
oxidoreductases possesses also enzyme which are not NAD(P)-dependent and whose use is
consequently easier - especially in non-aqueous solvent. Peroxidases and Polyphenol oxidase
consume hydrogen peroxide (H2O2) and O2, respectively. Desaturase are able to unsaturate an alkyl
chain (releasing H2) (Klibanov 2003; Liese et al. 2006). An example of liase that is industrially
relevant is the Penicillin amidase for manufacture of semi-synthetic β-lactam antibiotics. The
Bibliographical review
30
worldwide capacity is more than 20,000 t.a–1. Lipases are a relevant example of this class and will
be treated later for the reason of their prominence in non aqueous media. We would like to mention
some points on the different bioreactor setups which are relevant to our studies
2.1.2.3.Membranes
Filtration is the operation of separating solid from liquid. Membranes are filters which pore
size is so small that is can retain a fraction of the constituent of a mixture. They main application in
biocatalysis is to maintain an enzyme in solution but it exists also membranes that can keep the
coenzyme as well, as shown on Figure 7. It can also be functionalised to present a selectivitity
more specific than size as charge, polarity and so on.
Figure 7: Classification of membrane filtration processes
Reproduced from Industrial Biotransformations (Liese et al. 2006)
An example of continuously operated stirred tank reactor that uses enzyme membrane
reactor for the separation of the product from the enzyme 2-oxo-4-phenyl-butyric acid 2-hydroxy-
4-phenyl-butyric acid with D-lactate dehydrogenase and regeneration of coenzyme performed with
formate dehydrogenase is showed on Figure 8.
Bibliographical review
31
Figure 8: Example of a continous conversion with a membrane
Reproduced from Industrial Biotransformations (Liese et al. 2006)
2.1.2.4.Biphasic systems
If low solubility of substrate imposes a large reaction volume or if a substrate or product is
instable in water so that it is important to reduce the time it spends in the aqueous phase, a biphasic
system can be chosen instead of a single aqueous phase. Such two phase systems are depicted in
Figure 9. An example of a system that requires a coenzyme was chosen and includes apolar
products and substrates. The two phases are:
Aqueous phase. It contains the catalysts (the enzyme and the coenzyme if needed) and
hydrophilic substrates and products. The reaction takes place in this medium.
Organic water-immiscible phase. It “stores” the apolar/hydrophobic substrates and
products and it exchanges them with the other phase. Ideally, it provides the substrates to
the aqueous phase for the reaction to occur and extract the products.
Enzyme should be carefully chosen for this kind of system because the interface
water/organic solvent can deactivate them (Groger et al. 2003). A way to prevent the deactivation
of enzymes at this interface is to prevent them from reaching it with a membrane. If a membrane
had to be added on the reaction setup depicted in Figure 9 it would be in the aqueous phase close to
the organic phase so that the enzyme remains below the interface between the phases. Important
disadvantages of this technique are that it slows down the mass transfer and it increases pressure
drop.
Bibliographical review
32
Figure 9 : A two-phased system involving a coenzyme-dependent enzyme
The stereoselective hydrogenations of a ketone into an alcohol are often performed in such
media because the solubility of the product and substrate in water is usually low. (This is the case
of ACP and RPE.) It is also a simple way to separate the product from the catalysts. However
running reaction batch-wise and extract the product at the end is also common (Liese et al. 2006).
2.1.2.5.Improving the stability of enzymes: immobilisation
techniques
An important inconvenient of enzyme and biocatalyst in general is their instability that
represent an important cost. The catalyst is expensive and must be renewed often. Immobilisation is
a technique that improve the half-life of an enzyme. Another advantages is that the catalyst is easily
separable from the products. For more detail on immobilisation of enzyme please refer to Carrier-
bound Immobilized Enzymes (Cao 2005) where much information on this topic is found.
Many methods of enzyme immobilisation have been developed. The first is the simplest:
the enzyme is linked to the carrier by physical bounds. The advantages of this method are the
simplicity, the reversibility that allows the recyclig of the carrier and the fact that the enzyme does
not undergo much modification may lead to an unaltered activity. The main disadvantages come
from the weakness of the bound that often results in an important release of the enzymes into the
reaction media especially in presence of high desorption forces such as high ionic strength, pH and
so on. In organic solvent, where the enzyme is no soluble, physical absorption is a convenient
Bibliographical review
33
technique. The quantity of adsorbed enzyme is critical and a minimum monolayer coverage is often
required. Indeed protein molecules tends to maximize contact with the carrier surface by deforming
or unfolding, thus resulting in loss of activity, because of conformation changes, when coverage of
the carrier surface by the protein is below the monolayer. Additive as an inactive protein can fill the
surface on the carrier unoccupied by a too little quantity of enzyme. The absorbent should be
chosen with care because the interaction between the enzyme and the carrier will determine the
properties of the enzyme. The hydrophobicity/aquaphilicity balance of the support is decisive.
Hence, hydrophobic carriers is suitable for lipase immobilisation while some enzymes were more
favourably immobilised on more polar surface carrier able to form H-bond with enzyme or ionic
interaction. The first full scale industrial use of an immobilized enzyme was based on aminoacylase
immobilized on DEAE–Sephadex (weak ion exchanger) in a packed bed reactor. In 1969, they
started the industrial production of L-methionine as shown on Figure 10. This reaction is also an
example of resolution coupled with a racemisation. More examples will be given in 2.1.3
Figure 10: L-amino acid production catalyzed by aminoacylase.
Reproduced from History of Industrial Biotransformations (Vasic-Racki 2006)
Another technique of immobilisation is known as “entrapment”: a matrix is formed around
the enzyme with pores so fine that the enzyme can not leave. The matrix establishes multiple
physical bounds around the enzyme. Alginate gel formation is the more common technique of
entrapment. The water soluble alginate is mixed with the biocatalyst solution and dropped into a
calcium chloride solution in which water-insoluble alginate beads are formed. Carrageenan and
polyacrylamide gels are also widely used. Enzyme entrapment in sol-gel was applied to lipases
from Candida rugosa and porcine pancreas for reaction in SCCO2 and near critical propane. The
stability of the enzyme was improve and the reaction rate was much enhanced compared to reaction
with non-immobilised enzyme (Novak et al. 2003). Yeast ADH was immobilised by entrapment in
poly(AAmco-HEMA) gel (Soni et al. 2001)
The last method of immobilisation is the formation of a chemical bound between the carrier
and the enzyme. Several methods are possible and three are given as example in Figure 11. The
two first methods correspond to the coupling of the amino group of the enzyme, whereas in the last
Bibliographical review
34
case a carboxylic acid of the enzymes is activated with a diimide to be bounded to the carrier. It
should be emphasised that many other techniques are possible and many use a spacer, a longer
chain molecule that links the enzyme to the carrier. The enzyme is bounded to the carrier by
multiple bounds to the carrier and physical interaction takes place as for adsorption. The
advantages are numerous: the leakage of enzyme is slight, the conformation of the enzyme is
“frozen” on the carrier and this technique alters or improves the enzymes properties the more
radically.
OO
OH
NH2
NH2 Enz
NH2 Enz
EnzHOOC N NR
R
Enz O NR
O NHR
NH
Enz
O RHN NHR
O
O NH
OH
Enz
O NH
NH
EnzSupports SupportsCNBr
+
+
Figure 11: Three examples of carrier-coupling using the amino group of an enzyme (Enz
in this figure).
Another way to form a biocatalyst insoluble in water is the crosslinking technique: covalent
bound are formed between enzyme. The most common reagent is glutardialdehyde . This technique
was combined with precipitation: CLEA, cross-linked enzyme aggregates, are formed (Sheldon
2008).
2.1.2.6.Immobilisation of ADHs.
ADHs were seldom immobilised (Soni et al. 2001; Bolivar et al. 2006) for many processes
using them are run in a single phase (that is followed by an extraction), in biphasic systems or rely
on membranes (see 2.1.2). LBADH was immobilised by adsorption on glass bead for gas phase
reaction as it will be detailed in the next chapter. This method gave very good results for gas phase
reaction, allowing to lengthen its half-life considerably. LBADH (see 2.2 for more detail about this
enzyme.) was also immobilised for plug-flow reactor (Hildebrand et al. 2006) where the
regeneration is performed by isopropanol. This example is good example of how the different
technique can be combined: when a covalent bound is formed the enzyme establishes physical
Bibliographical review
35
bound to the carrier and the stability of the enzyme can be futher improved by crosslinking the
different enzyme’s residue left unreacted after immobilisation. In this example LBADH was
immobilised on amino-epoxy carrier supports prior to covalent binding to the epoxy groups of the
support, the protein physically adsorbed to the surface. Through additional amino groups on the
support, this adsorption process is facilitated and proceeds at low buffer concentrations. After the
adsorption process, bonds are formed between the epoxy groups and the nucleophilic groups of the
enzyme, resulting in a covalent multi-point attachment in which the enzyme conformation is more
rigid and therefore more stable against inactivation. At the end of this process of immobilization
the half-life of the enzyme was still the same as free in buffer, about 20 hours at 30°C. The half-life
of the preparation was increased over 500 h while 20 % of the initial enzyme activity remains by
proper immobilisation technique which combines the blocking of the remaining epoxy group of the
support with mercatoethanol and the cross-linking of enzyme with glutardialdehyde. A process run
with this catalyst achieved a conversion of 60% for enantiomerically pure RPE with TON of
2,500,000 and the catalyst was used over 10 weeks (Hildebrand et al. 2006).
2.1.2.7.Enzyme-catalysed reactions in non aqueous
solvents
The “natural” solvent for biocatalysis is water. However, it was discovered in the mid-
1980’s that enzymes are surprisingly active in organic solvents (Zaks et al. 1984; Zaks et al. 1985)
for only a small quantity of water is necessary to the maintaining of the ternary structure (as low as
0.02%, few water molecules for an enzyme molecule). Hydratation gives flexibility to the enzyme.
This flexibility is not only responsible for the enzyme to catalyse a reaction, the shape of the
enzyme adapting to the substrate, but also at the origin of the deactivation of the enzyme.
Deactivation of an enzyme corresponds to the unfolding of its chain. The deactivation can be of
two kinds: denaturation which is reversible and inactivation that is reversible (Fágáin 1995). The
water content of the non-aqueous media, or water activity, has a dramatic effect on the enzyme
stability and activity, water being exchanged between the surface of the solvent and the solvent.,
and must be optimised: too low water activity lead to too rigid enzymes laking activity and high
water activity can lead to fast deactivation. Too polar solvents are not suitable for enzyme-
catalysed reaction in non-aqueous solvents as they strip away water molecules from the surface of
the enzyme.
The advantages for the use of organic solvents instead of water in biocatalysis can be listed
(Vulfson et al. 2001; Klibanov 2003): the selectivity or activity can be modified and so can be more
beneficial to a desired reaction (Zaks et al. 1986), the slight solubility in water of certain
compounds, and/or stability in water limit the performance of the reaction in aqueous solvent.
Bibliographical review
36
Moreover, an extraction of the medium by an organic solvent, possibly contaminant and toxic
solvents like hexane, is necessary for recovering the products. The solubility of reactants is
increased, the non-enzymatic (spontaneous) and side reactions (products not stable in water)
eliminated. Enzyme stability can be enhanced, as well as the stability of coenzymes. But the
following problems may arise: many enzymes are quickly deactivated in organic solvents, the
reaction rate is low sometimes, solvents strongly inhibit certain enzymes, and aggregation of the
enzyme is a limit to mass transfer. Immobilisation generally considered as necessary in organic
solvent unless enzyme aggregate. A proper immobilisation improves the stability and/or activity of
biocatalysts.
Most reactions developed in non-aqueous solvents involve ester formation,
transesterification, hydrolysis. There are several reasons for this development. The enzyme
involved in those processes, as lipases, are found in vivo at the interface between phase, oil or fat,
and an aqueous phase, which explain their good stability and activity in non-aqueous solvents.
Indeed they are considered as “hard” enzymes: their structure is less flexible and, nonetheless,
active. This fact also explains why their immobilisation is particularly easy and efficient (Cao
2005). Lipases are well-known enzymes and have found many industrial applications such as large
scale used in detergent. Synthesis with lipase often involved apolar molecule which are not soluble
in water and so in this solvent the mass transfer of would slow down the reaction too much. In
absence of water the hydrolysis does not take place: in water many reactions are simply impossible
thermodynamically for the ester is hydrolysed. The case of ADH-catalysed reaction will be treated
in more details in 2.2.3.
Despite all those efforts to develop stereoselective catalysts, production of enantiomerically
pure molecule is often not feasible. Indeed, the result of most synthesis remains a racemic
compound of which only half is required. The separation of one enantiomer from the other is called
resolution. The main methods of resolution of enantiomers are presented in the next part. To the
point of view of the atom economy developed in a resolution3 is inferior to a neat stereoselective
synthesis for twice too many molecules are consumed and the atom efficiency is automatically
below 0.5 and decreases exponentially with the increase in the number of the asymmetric centres in
a molecule.
3 or, better, a resolution where one of the enantiomer is not desired or where the unwanted
enantiomer is recycled by racemisation. The case of the racemisation is important to the green chemisty’s
paradigm and would be consider in the coming part.
Bibliographical review
37
2.1.3. Resolution of racemic mixture.
This part will focus at the methods that enable the resolution of enantiomeric mixtures:
chromatographic method 2.1.3.1, separation of conglomerate 2.1.3.2, the resolution by formation of
diastereomers 2.1.3.3 (especially salts 2.1.3.4 and co-crystal 2.1.3.6). An example of a method
based on a biocatalyst is given in 2.1.3.5.
2.1.3.1.Chiral chromatographic separation
Chromatography is defined as a physical method of separation in which the components to
separate are distributed between two phases, one of which is stationary (stationary phase) while the
other (the mobile phase) moves in a definite direction. Separation of solutes injected into the
system arises from differential retention of the solutes by the stationary phase. The term liquid
chromatography is used when the stationary phase is a solid and the mobile phase a liquid. In the
case of gas chromatography the mobile phase is a gas while the stationary solid. Chromatographic
techniques, HPLC and GC, are routinely used as quantitative analytical method and now a wide
range of chiral columns allows the separation of the enantiomers. After a column with a high
loading capacity is successfully developped an analytical method can scaled-up.
Liquid chromatography and especially HPLC is the most important preparative method
used for the separation of enantiomers. The classical open-column liquid chromatography was
improved by the use of very small particles for the solid adsorbent stationary phase. Because of this
bed of packing material had much lower permeability, it became necessary to use a pump to
generate sufficient pressure to produce a flow rate high enough (Fekete 2008). The pressures used
for an analysis by HPLC are generally about 200 bar while the column can stand about 400 bar. In
the case of Ultra High Performance Liquid Chromatography the pressure are generally about 800
bar and the maximum pressure is about 1000 bar. Those techniques are usually gentle and
appropriate for unstable molecules. Although they have the reputation to be expensive and
ineffective for large scale separation, their quality has improved and chromatography is sometime a
first choice for resolution (Subramaniam 2001) and, according to this author, the separation are
rather easy to develop and appropriate for the small scale. The first HPLC column used for chiral
resolutions are based on polysaccharide, cellulose and starch, and the next developments were
based on substituted polysaccharides. Then, column came based on emulsion polymerisation of
acrylamides from amino acids. Fundamentally, three kinds of chiral stationary phases are
distinguished: chiral polymers, achiral matrices (mainly silica gel) modified with chiral moieties
(amino acid derivatives, crown ethers, cinchona alkaloids, carbohydrates, amines, tartaric acid
derivatives, cyclodextrins and binaphthol), and imprinted materials (chiral cavities) (Francotte
2005).
Bibliographical review
38
A first problem for enantiomeric resolution based on HPLC is the fact that separation is
performed batch-wise. Some methods allow a more intensive use of the equipment and the saving
of solvent: multiple close injection (the injection are performed repeatedly so that the support is
always involved in separation), recycling and peak shaving (the faction of solvent that contained
the overlapping of the two enantiomers is reinjected i.e. recycled). Another improvement is the
techniques of simulated moving beds which allows a continuous separation rather than proceeding
by batches (Rodrigues et al. 2001). The method of true moving bed is intuitive but hard to set:
liquid (equivalent to the mobile phase in HPLC) and a solid (absorbent equivalent to the stationary
phase) flow in opposite direction. The more retained molecule exits the separator with the solid
while the less goes out with the liquid. The process based on simulated moving beds are based on
this concept but there is no movement of the solid phase but the position of the inlet and outlet
steams move periodically simulating the movement of the solid absorbent (Rodrigues et al. 2001).
This technique was used by the pharmaceutical industry for the resolution of chiral drugs at large
scale production (>100g) including chiral buiding blocks as 1-phenylethanol (Negawa et al. 1992).
Despite its versatility, this technique represents a large investment prohibitive for small companies.
The inconvenient of chromatographic method is the high cost, notably in solvent that came
out very diluted out of the column and might contained additive such as a buffer, the matter related
to the scale-up, as the quality of the resolution might degrade at a larger scale. (Chiral) preparative
GC is rare (Subramaniam 2001). The column used was γ-cyclodextrine, cyclodextrine being the
most common type of (analytical) column for chiral GC. Chiral GC coupled with simulated moving
beds was applied to the resolution of (±)-enflurane, a volatile anesthetic. The use of membrane for
chiral resolution is at the moment limited, as well.
Chromatographic methods using supercritical fluids
Supercritical fluids got more and more important in the domain of chromatography
(Villeneuve et al. 2005) despite the investment that represents the equipment and the complexity
(or the lack of formation) regarding those techniques. The unique properties of supercritical fluids4
improves resolutions previously performed with HPLC (Phinney 2001): the higher diffusivity for
solute and the lower solubility compared to liquids leads to a higher efficiency and a shorter
analysis time. The pressure drop along the column which is a limit to scaling up liquid
chromatography processes is much lower using SF. The recovery of the product is easier and the
solvent consumption lowered. Considering all those advantages, the operation cost for preparative
SFC will be half of that of preparative HPLC (Villeneuve et al. 2005).It is to notice that the used of
4 The annexes contains a more general presentation of supercritical fluids (7.1.3).
Bibliographical review
39
SCCO2 is limited to apolar product, even if this difficulty might be overcome by using a modifier
such as methanol. SF chromatography was also applied to the preparation of drug (Fuchs et al.
1992; Perrut 1994). The columns used for SF chromatography are the same as for HPLC.
The readers who would like to know more about this field are advised to consult the book
Preparative Enantioselective Chromatography (Cox 2005) and Techniques in Preparative Chiral
Separation (Subramaniam 2001). Both have a section on SF chiral chromatography.
2.1.3.2.Resolution by selective crystallisation:
conglomerates and racemates.
a) b)
Figure 12: Binary mixture melting point diagram for a conglomerate-forming pair of
enantiomers (a) and a racemate-forming (b).
When a racemic mixture is liquid it forms a homogenous phase that is not optically active.
Solid enantiomers present two main behaviours: conglomerate or racemate. If they form
conglomerate they crystallise separately with different symmetry and this was exploited by Pasteur
for the resolution of tartaric acid (Pasteur 1922-1939). However, most enantiomers don’t form a
conglomerate but rather a racemate (Jacques et al. 1981): The racemate is a compound of the two
enantiomers in an equimolar ratio5 whereas in the case of conglomerate no such compound is stable
and the enantiomers crystallise separately as shown on Figure 12a). Most enantiomers are
racemate.
A conglomerate can be separated by differential crystallisation, the crystallisation of the
desired enantiomer is promoted by seeding (Jacques et al. 1981) as used for the preparation of α-
methyl-DOPA by Merk (Collins et al. 1992). A very interesting enantioseparation method
combines with racemisation gave enantiomerically pure product. It is a new concept and was
applied N-(2-Methylbenzylidene)phenylglycine Amide (Noorduin et al. 2008a; Noorduin et al.
5 except for the rare case of liquid solutions.
Bibliographical review
40
2008b) A saturated solution of this amine containing a fraction of solid crystal is ground until a
only one crystal is present Figure 13. This is among the convincing explanations for the origin of
asymmetry in life but, to the point of view of the chemical engineer, a process based on such
method has an impressive efficiency: virtually no waste is generated.
Figure 13: Chemical and
physical equilibria in the
racemization for N-(2-
methylbenzylidene)phenyl
glycine amide
1: N-(2-methylbenzylidene)phenylglycine amide, for this figure only. The enantiomers
have different colors, blue and red.
Reproduced from (Noorduin et al. 2008a)
Unlike congomerate-forming chiral molecule, racemate-forming enantiomers yields a
racemate only when crystallised and a non-equimolar mixture of both (ee≠0) can be purified only if
the enantiomeric excess is over the ee of the eutectic mixture: in this case the enantiomer
precipitates before the racemate (Jacques et al. 1981; Lorenz et al. 2006). This is why a resolution
of a racemate should yield, prior to recrystallisation, a mixture of racemate and enantiomer which
ee is higher than the eutectic’s (Wilen et al. 1977). However, the purification by recrystallisation is
a supplementary step that provokes the use of supplementary solvent, its evaporation if it is wanted
to recycle the molecule that did not crystallise. Consequently, a resolution should yield the
enantiomer with an ee as high as possible. If the ee is high enough no further refinement is needed.
If it is too low a recrystallisation is required and the higher the ee, the less material to recycle is
produced.
2.1.3.3.Resolution by formation of diastereoisomers
A common strategy for the separation of a racemate is the use of a resolving agent that is
asymmetric. A resolving agent is a compound that is added to the targeted product for its
resolution. It is generally removed from the product at the end of the separation and recycled for
another use and, to this point view, comparable to a protecting agent in organic chemistry. The
resolving agent forms a diastereoisomer with each enantiomer. The two diastereoisomers possess
different physical properties and consequently can be separated. The different interaction between
the enantiomers and the resolving agent that are used for the resolution are covalent (the present
Bibliographical review
41
part and 2.1.3.5) or ionic (see 2.1.3.4) bonds or weaker as intermolecular bonds (see 2.1.3.6). The
chiral molecule often belongs to the so-called chiral pool: this is a set of molecule mostly from
natural origin (but not only) which are readily available and cheap. They are often used as the
building block that introduce the asymmetry in a synthesis or as resolving agent. The chiral pool
includes aminoacids, hydroxyl acid as tartaric acid (TA), carbohydrates, alkaloids and so on
(Collins et al. 1992). Examples of resolution applied to the resolution of our targeted enantiomer,
R-1-phenylethanol and trans-1,2-cyclohexanediols, will be given in 2.1.3.5 and 2.3.1.
At the moment we would like to give and example of resolution of Rac-CHD by formation
of covalent bound with a chiral resolving agent. The method was presented in a publication
(Chatterjee et al. 2007) and summarised in Figure 14, the resolving agent is (S)-O-Acetylmandelic
Acid which forms an ester with CHD. The two diastereoisomeric esters are separated by
chromatography (or preferential crystallisation).
AcOH
Ph
COOH
OH
OH
OH
OH
OR
OH
OH
RO
OR
OH
OH
RO
OH
OH
OH
OH
(R,R)-(-)-CHD
(S,S)-(+)-CHD
2 + esterification
+
hydrolysis
hydrolysis
(R,R)-(-)-CHDee=96%Y=32%
(S,S)-(+)-CHDee=97%Y=32%
Silica gel
chromatography
Figure 14: The resolution of rac-CHD by Chatterjee.
A practical way to measure the quality of a resolution is to use the selectivity of Fogassy
parameter that combines the yield and the ee in one value. Equation 2 is used when only one of
the enantiomers is required and Equation 3 when both are. When there is no resolution, S and F
are nil. At best, S and F can be 0.5 and 1, respectively. They can be calculated with the data from
Chatterjee’s method of resolution S= 0.31 for SSCHD and F=0.62.
eeYS * Equation 2
eeYeeYF 2211 ** Equation 3
Racemisation
An elegant method of exceeding the maximum theoretical yield of 50 % is to treat the
unwanted enantiomer with a catalytic amount of a substance which leads to its racemisation as
illustrated in Figure 15. In this figure a general scheme of resolution combined with racemisation is
Bibliographical review
42
presented while other examples will be given the text. This method allows to overcome the
limitation of resolution to a yield of 50 % and the yield can be theoretically as high as 100 %. The
method of separation of the enantiomers of Figure 10 or Figure 13 were two good first examples of
resolution combined with racemisation. Another will be given in 2.1.3.5 for phenylethanol.
Figure 15: Separation of enantiomers (+)-A and (-)-
A combined with racemisation.
Pure (+)-A is obtained for all (-)-A is transformed
into (+)-A. S can be superior to 0.5.
2.1.3.4.Resolution of enantiomer by formation of
diastereoisomeric salt.
The importance of this method was given in the introduction and examples are given in the
Table 2. Many reviews and books exist on this topic that remains the main technique of production
of enantiomers despite the importance that asymmetric synthesis and biocatalysis took and the fact
it can be applied only to molecules that present acidobasic properties. The following works can be
mentioned: Enantiomers, racemates, and resolution (Jacques et al. 1981), Strategies in
optical resolutions (Wilen et al. 1977); Optical resolution via diastereoisomeric salt
formation (Kozma 2002), Strategies in optical resolution: a practical guide (Faigl et al.
2008), Chirality in industry (Collins et al. 1992), Stereochemistry of organic compounds
(Eliel et al. 1994), and Optical resolution procedures of chiral compounds (Newman 1978-
1984). Those text books helped me for the general presentation given below.
(+/-)-Anon stereoselective
separation
reaction
racemisation
(-)-A
(+)-A
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43
Table 2: Examples of pharmaceuticals resolved by diastereomeric crystallisation in the
process
(reproduced from (Collins et al. 1992))
Pharmaceutical Resolving agent
Ampicillin D-camphorsulphonic acid Ethambutol L-tartaric acid (natural form) TA Chloramphenicol D-camphorsulphonic acid Dextropropoxyphene D-camphorsulphonic acid Dexbrompheniramine D-phenylsuccinic acid Fosfomycin R-(+)-phenethylamine Thiamphenicol L-tartaric acid (natural form) Naproxen Cinchonidine Diltiazem R-(+)-phenethylamine
In Figure 16, the most common resolution procedure is shown. It presents the resolution of
a base (DL)-B, whose enantiomer (L)-B is desired. The resolving agent is a chiral acid (L)-HA,
whose pKa is lower than BH+ , i.e. HA is an acid which is stronger than (DL)-BH+: (DL)-B and HA
react together and form salts that precipitate. In the most general case the separation of the salts is
performed by the selective crystallisation of the less soluble salt, n-salt. Other method of separation
of the diastereoisomeric salt exists as chromatography. The salts are then decomposed to free the
enantiomers. The decomposition is often realised with a base (or acid) that are strong enough to
take the place of an ion in the diastereoisomeric salt (In the example of Figure 16 NaOH reacts with
the n-salt to give (L)-B). To the perspective of the atom economy, any kind of waste should be
minimised but to the point of view of the profitability of the process, the separation of the free
enantiomer and the recovery of the resolving agent are essential. Indeed the loss of enantiomer or
resolving agent can be an important cost and the waste should be treated. It is difficult to give a
general method to do it (This is to say to recover (L)-HA and (L)-B in the figure below.). However,
an example of such a process for the case of a resolution with TA, a very common polar acidic
resolving agent, is given in 2.3.4 Figure 29. The workout of aqueous solution is generally done by
liquid-liquid extraction and energy intensive differential crystallisations. These steps of separation
or purification are based on simple acidobasic chemical reactions and equilibria and is the source of
much waste and energy consumption along the chemical process. TA is so cheap a resolving agent
that it is sometimes disposed of. There is also the possibility to use ion exchange resins to this
purpose or chromatographic method.
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44
(D)-B + (L)-B + 2 (L)-HA (D)-BH+,(L)-A- + (L)-BH+,(L)-A- racemate resolving
agentn-salt p-salt
n-salt p-salt
separation
decomposition of the salt by NaOH
(L)-B
Na+,(L)-A-
Figure 16: Common resolution of a base B by the acid (L)-HA.
Formation of 2 diastereoisomeric salts, their separation and the decomposition of a
diastereoisomeric salt by a strong base, NaOH in this case.
Partial crystallisation has several advantages: it is relatively simple and flexible as
appropriate to intermittent batch production and only standard equipment is required. However it
can use many tanks for the storage of mother liquor and presents disposal problem because much
waste can finally be produced (Collins et al. 1992) It is possible for bases and acids only and when
the pair of salts forms a conglomerate6 but no compound nor liquid solution (or if a liquid solution
occurs it should be of a limited extend). The ee of the eutectic point must be high (Es on Figure 17
b)). A first estimation of the selectivity (Equation 4) can be derived from the eutectectic
composition xeu of the binary melting point phase diagram (Figure 17 a)), that is generally closed to
eutectic composition. The investigation of the position of the eutectic point by DSC allows an easy
and fast screening of the resolving agent (Kozma et al. 1992). The procedure is simple: a DSC scan
of an equimolar mixture of the salt p and salt n is run. So it is not necessary at this moment to have
the enantiomerically pure compounds. This scan will provide 4 values that are the temperature of
the eutectic and its enthalpy of melting and the temperature of the liquidus and its melting enthalpy:
these are the two points marked on Figure 17 for xn=0.5. With a calculation based on the Schröder-
van Laar equation, cf Equation 5 (Jacques et al. 1981), the selectivity can be evaluated (Madarász
et al. 1994) by extrapolating the eutectic composition xeu.
eu
eu
xxF
1
21 Equation 4
6 The same way that two enantiomers can form a racemic compound as shown in 2.1.3.2.
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45
)11()1ln(1
12 TTR
Hx
)11(ln2
22 TTR
Hx
Equation 5: Schröder-van Laar equations
0.0 0.5 1.0
Xn
T
Salt pXeu Salt n
a)
b)
Figure 17 : a) Binary melting point phase diagram of salt p and salp n and b) their
solubility diagram.
Figure 17 b) is the solubility diagram of the salt (B-HA+) in the solvent used for
crystallisation. The line PEN is the limit of solubility of the salt, there is only a liquid phase above
it. E is the eutectic composition (Es on b) is the same xeu on b)), at which the liquid is in
equilibrium with the two salts (in the domain salt p-salt-E) where A is found. In the two last
triangular domains of the ternary P-E-salt p and N-E-salt n (where C is), the mother liquor, the
liquid that contains the unprecipitated diastereoisomeric salt, is in equilibrium with one salt. The
amount of solvent is very important during the crystallisation of the diastereomeric salts M. when
the amount of solvent is increasing from A to C the salt p disappears, the quantity of precipitated
salt n then decreases. The optimum of a resolution is found at the point B (eu
eu
xxF
1
21) where
the maximum amount of salt n has precipitated without containing any salt p. The mother liquor at
this point at the composition E, so some salt n is lost with the mother liquor and contaminated the
salt p. Consequently, the more on the left E is the better the resolution is. The lower E is the less
solvent should be used.
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46
Molar ratio
The molar ration (Equation 6) is defined as the ratio between resolving and agent and
enantiomer.
nn
enantiomer
agentresolvingmr _
Equation 6: general definition of the molar ratio, mr
In the case of the most classic resolution, ie by formation of two diastereoisomeric salts, p
and n, 1 mol of resolving agent was used for 1 mol of racemic compound (molar ratio=1). Both
Enantiomers are thoroughly transformed into salt. After the mixing of the two molecules, no
species remains as a molecule (without mentioning the case where the molecule are able to
exchange more than an electron, as for TA) it was developed a technique where a non
stoechiometric amount of resolving agent is added to the racemate.
(D)-B + (L)-B + (L)-HA (D)-BH+,(L)-A- + (L)-Bracemate resolving
agentn-salt free enantiomer
Figure 18 : Resolution with a molar ratio of 0.5
The goal is to allow a better separation of the enantiomers not based on the difference of
solubility between the salts but between a salt, the more stable, and the enantiomer that did not
react. In the most typical case mr=0.5, half quantity of resolving agent is added to the enantiomers,
the method is named Pope and Peachey in reference of the two researcher who were the first to use
it. This supposed, and this is most favourable case, that all the resolving agent reacts with only one
of the enantiomers. For instance and in the most common (and favourable) case, the formed salt
precipitates completely, and the unreacted enantiomers remains in solution as shown on Figure 18.
Actually several equilibriums take place: acido basic reaction and precipitation (Ács et al. 1985).
The system of equation was solved and the optimal molar ratio is found slightly above 0.5: it
corresponds to the quantity of resolving agent for which all the enantiomer has reacted, this is to
say half the quantity of enantiomers plus a generally neglectable portion that corresponds to the
resolving agent consumed by the other equilibrium (Kozma 2002; Faigl et al. 2008).
A resolution can also run with half an equivalent of resolving agent (molar ratio of 0.5) but
also half an equivalent of a stronger base or acid, so that both enantiomers form a salt but with very
dissimilar physical properties, for instance solubility in different solvents which the further
separation can be based on. The case of the resolution of 1-(4-Fluorophenyl)-2-Methylamino)-
propane with tartaric acid (TA) is detailed in Optical resolution via diastereoisomeric salt
formation (Kozma 2002).
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47
mr=0.5 and evaporation of the solvent
Several problems can arise while trying to separate the n-salt from the free enantiomer. The
mother liquor that is obtained after filtering out the precipitate may contain a variable amount of
impurities, which are unprecipitated salts, unreacted resolving agent and unwanted enantiomer.
This is why an alternative technique was developed where the solvent is evaporated. After half an
equivalent of resolving agent is added to the system and the salt n is formed the solvent is
evaporated until a mixture of solid is obtained: mostly salt and free enantiomer.
At this point two strategies are possible. The first is to pursue the distillation, and the free
enantiomer is the next compound to get out of the column. In some case a further increase in
temperature can lead to the decomposition of the salt immediately followed by it. This method
allows a very simple separation of the enantiomer as well as the regeneration of the resolving
agent. However it implies that the enantiomer is distillable, the salt releases the enantiomer before
degrading. Thus, amphetamine was resolved with its hemiamide with phtalic acid (Figure 19).
After distillation of the first enantiomer the temperature is raised and diastereoisomeric salt
decomposes: the hemiphtalate is transformed into a phthalimide (loss of CO2 and cyclisation) and
release the second enantiomer (Kozma 2002). The resolving agent can be recovered by careful
hydrolysis.
Figure 19: Resolution of amphetamine by distillation
Reproduced from Optical resolution via diastereoisomeric salt formation (Kozma 2002).
The second strategy is the following: the free enantiomer is extracted from the solid by a
solvent that presents a better selectivity for this system than the solvent used for the acidobasic
reaction with resolving agent. At the end of the extraction only diastereoisomeric salt is left and it
can be treated by an acid or a base for recovering the opposite enantiomer and the resolving agent
as previously described. For all the reasons given in 7.1.3 SCCO2 was particularly suitable solvent
for the extraction of the free enantiomer (Fogassy et al. 1994). This technique that combines
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48
diastereomeric salt formation, evaporation of the solvent and SFE was used for the extraction of
(±)-cis- and (±)-trans-permetric acids when resolved by R-α-phenylethylamine and S-2-
kenzylaminobutan-1-ol (Simándi et al. 1998), of tetramisole with DBTA (Keszei et al. 1999;
Székely et al. 2002), of ibuprofene with R-phenylethylamine (Molnar et al. 2006), N-
methylamphetamine with DBTA and O,O’-di-p-toluyltartaric acid (Kmecz et al. 2007). This
technique afforded better results than using the more classical method of partial crystallisation,
notably due to the adjustable selectivity of the solvent.
2.1.3.5.Other methods of resolution of alcohols
Resolution by formation of diastereoisomeric salts is impossible for product which cannot
give or accept a proton. This is the case of alcohols. Nonetheless, they are often derivatised for this
purpose. The salt forming derivative used to this purpose are phtalates, oxalates, glycolic acid
esters, hemisulphate (Jacques et al. 1981). Phthalate derivatisation is presented on Figure 20. The
phthalate is often resolved with brucine. The alcohol is released by saponification.
O
O
OCOOH
O
OR(+/-)ROH (+/-)+
Figure 20: Formation of phtalate derivatives, useful intermediates for resolution of alcohol
by diastereomeric salt formation
A second strategy based on esterification with a carboxylic acid as a resolving agent was
presented in 2.1.3.3 on Figure 14. A third is to form a covalent bond between one of enantiomers
and an achiral molecule selectively, the asymmetry is introduced via a chiral catalyst such a
biocatalyst (see 2.1.2). An interesting instance of this method is the stereoselective acylation of
alcohol via a lipase. Only one of the enantiomer is acylated and then separated from the other. This
reaction is run in non-aqueous solvent because water would lead to the hydrolysis of the ester, an
unfavourable equilibrium. Vinyl acetate is often used for reacting with an alcohol because no water
is formed but formaldehyde that renders the reaction irreversible (as in Figure 21). However the use
of salt hydrate allows to control the water activity and to absorb the water formed during the
esterification (Halling 1992; Zacharis et al. 1997) and other techniques exists (Rosell et al. 1996).
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49
OH
OH
O
OOH
O CH3
O
O
R-phenylethanol
S-phenylethanol
+
S-phenylethanol
R-phenylethanyl acetate
+
Figure 21: Resolution of chiral alcohol by enzyme catalysed acylatation
Rac-1-phenylethanol was resolved using this technique over Candida antarctica lipase
B in ionic liquids (Figure 21). The result was good as the enantiomeric was very high 99% and the
yield was 50%. A similar process with Pseudomonas cepacia lipase was developed in SCCO2 and
combined with the racemisation of the unreacted R-1-phenylethanol with the metal catalyst or the
acid catalyst Nafion SAC 13 (Benaissi et al. 2009), fairly good enantiomeric excesses and yield
were achieved, up to 95% and 85%, respectively. (R)-1-phenylethanyl acetate is sold as a fragrance
and has a floral, fresh, green note (Sheldon et al. 2007).
There is a parallel between this method of resolution and the resolution via partial
diastereomer formation, in both cases the concept of molar ratio can be applied as vinyl acetate
associated with a lipase is equivalent to a chiral resolving agent.
2.1.3.6.Resolution of enantiomers by formation of a
diastereoisomeric cocrystal with tartaric acid instead of a
salt.
TA and the related compounds have been widely used as an acidic resolving agent, their
price and availability made them the preferential source of chirality not only for resolution but also
as a catalyst for asymmetric catalysis (Collins et al. 1992). A comprehensive review about the uses
of TA and its main derivatives, diacyls (dibenzoyl- or O,O’-di-p-toluyltartaric acid) and anhydrids
for resolution exists (Synoradzki et al. 2008). Another advantage of TA is that (-)-tartaric acid, the
unnatural form, is available but more expensive.
The use of tartaric acid is not restricted to the resolution of bases. Indeed, in some cases
where even the existence of a salt was expected, IR spectroscopy did not show an exchange of
proton, and the formed compound was a complex or co-crystal rather than a salt (Nemák et al.
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50
1996). No ionic but a strong system of hydrogen bonds were responsible of the co-crystallisation,
(+)-tartaric acid (TA) being a good hydrogen bond acceptor and donor. This opened the way for the
resolution of compounds without basic properties with (+)-tartaric acid derivatives and the
screening of some alcohols with O,O′-dibenzoyl-(2R,3R)-tartaric acid was realised (Kassai et al.
2000). DBTA forms also cocrystal with ethers (Szczepańska et al. 1995) and a trans-
bicyclodiamine (Hatano et al. 1994). Among TA derivatives DBTA is by far the most used for
forming diastereomeric cocrystals. And, to my best knowledge, no such compound was found with
TA.
In most cases the resolution is performed by precipitation of the diastereoisomeric
compounds, which the free enantiomer is then separated from. To my best knowledge, the
resolution of enantiomer with TA derivative that is based on a soluble complex rather than a crystal
is rare, although and example exist mentioned in (Kozma 2002) who referred to enantioselective
extraction and the partition of salts of chiral bases between water and solvents containing a
lipophilic tartaric acid ester (Prelog et al. 1983). The application is partition chromatography.
TA derivatives are obviously not the only compounds which form cocrystal susceptible to
help resolving enantiomeric pairs. The presentation of supramolecular compound was done in the
treatise Crystalline Molecular Complexes and Compounds (Herbstein 2005) and especially the
chapter 12 Hydrogen bonded molecular complexes and compounds (Vol 2) is of our interest. H
bond are undoubtedly the most widespread of the specific interactions linking molecules with
suitable functional groups together in the solid state and in the gas and liquid phases. The
enzymatic molecular complexes are examples. The cocrystal that involved TA derivative are made
of weak bonding, especially H-bond and Van der Waals interaction instead of the ionic bonds that
was responsible for the formation of the diastereiosomeric salt. The term cocrystal was chosen as
very general for this kind of compound. Herbstein proposes a more precise nomenclature based on
the crystallographic structure. So a difference is made between the rare case of molecular
complexes AB where A is mostly bounded to other A molecule by H bonds and forms only one
with B and the common case of molecular compound where the framework structures, with the two
components in alternating array The arrays may extend in zero, one, two or three dimensions
(Herbstein 2005).
In relation to the production of enantiomerically pure (R)-1-phenylethanol, an very
interesting method of resolution based on inclusion complex was described: 1-phenylethanol was
subject of a screening for resolution with a cocrystal of (1S,2R)-2-amino-1,2-diphenylethanol and
benzoic acid that possesses three-dimensionally dissymmetric cavities (Kobayashi et al. 2004). An
interesting ee of 87% was obtained for RPE, as well as a yield of 91% (to be divided by two if
referred to (±)-1-phenylethanol instead of RPE). Another example with the resolving agent (R,R)-
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51
1,2-cyclohexanediamine (for CHD) will be given in 2.3.4.
The resolution by diastereomeric cocrystal formation seems to be theoretically usable to
more compounds than only acid or base if compared with salts. In practice this is not true because
it is difficult to find a resolving agent which forms a diastereomeric cocrystal and whose
cocrystallisation with the targeted enantiomer is stereoselective enough.
The separation of the cocrystal from the uncocrystallised enantiomer by extraction or
selective crystallisation is more difficult when the crystal structure is stabilised by H bonds which
are less strong than ionic bondings. That’s why the extraction with SCCO2 is so valuable in this
case, it allows to adjust finely the extraction fluid to the optimised selectivity by playing on
pressure and temperature.
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52
2.2. Conversion of acetophenone to R-1-phenylethanol using alcohol
dehydrogenase from Lactobacillus brevis (LBADH)
2.2.1. Alcohol dehydrogenases require a coenzyme NADH
and NADPH that must be regenerated.
2.2.1.1.Generalities about the coenzymes
Table 3 : Coenzyme and their associated group
Coenzyme Abbreviation Group transferred
nicotine adenine dinucelotide
NAD - partly composed of niacin Hydride (H-)
nicotine adenine dinucelotide phosphate
NADP -Partly composed of niacin Hydride (H-)
flavine adenine dinucelotide
FAD Partly composed of riboflavin (vit. B2)
electron (hydrogen atom)
coenzyme A CoA Acyl groups
Coenzyme Q CoQ electrons (hydrogen atom)
thiamine pyrophosphate thiamine (vit. B1) Aldehydes
pyridoxal phosphate pyridoxine (vit B6) amino groups
3-phosphoadenosine-5’-phosphosulfate PAPS Sulphate
Biotin Biotin carbon dioxide
carbamide coenzymes vit. B12 alkyl groups
Contrary to the enzymes which require only their amino acid residues, some needs a co-
factor or a coenzyme to be active. The co-factors are inorganic ions such as Fe2+, Mg2+, Mn2+, Zn2+
and so on. For instance, Mg2+ is necessary for glucose 6-phosphatase or Cu2+ for cytochrome
oxidase. A coenzyme is a complex organic or metalloorganic molecule and brings a group that is
exchanged between the substrate and the product of the reaction (see Table 3). For instance ATP
and ADP are important coenzyme related to phosphate transfers and, thus, energy exchanges
(Nelson et al. 2004).
From now on, only the case of nicotine adenine dinucelotides NAD(P)(H) will be
envisaged. Alcohol dehydrogenases (ADH) catalyse the reaction shown on Figure 22 and requires
the coenzyme: NAD(P)H. NAD(P)H can be used for the production of other products such as
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53
aminoacids, ketones from α,β-unsaturated carbonyl compounds, via D-amino acid dehydrogenase,
ene reductases respectively. The structure NADP is shown on Figure 23.
R
O
R' R R'
HOH+ NAD(P)H + H+ + NAD(P)+
ADH
* Figure 22: General activity of an alcohol dehydrogenase (ADH)
Figure 23 : NADP+: Nicotinamide adenine dinucleotide
The group that is given by NAD(P)H to the ketone to form an alcohol is an hydride (H-) (cf
Figure 24) and this reaction is consequently a reduction. To an industrial point view, the main
interest is that the alcohol is asymmetric.
Figure 24 : NAD is a hydrid acceptor and NADH a hydrid donnor.
“H-“ is used symbolically (it is not stable).
NAD is a coenzyme and its presence is necessary for the reaction to occur: no other
hydride exchanger is accepted by the enzyme. But to a certain point of view, regarding to the
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54
kinetic of the process or the structural information concerning the enzyme-coenzyme bond, NAD
can be considered as a substrate for the reaction. This bond is not so strong and nicotinamidic
coenzymes are found in solution free from the enzyme. The KM(NAD)=0,01-0,1mmol/L for
commercially available formate dehydrogenase (Cordes et al. 1994). For acetophenone reduction
by NADPH by Lactobacillus brevis (LBADH): KM(acetophenone)=0.85 mmol/L and
KM(NADPH)=0.16 mmol/L (Hummel 1997). It is not the case for every coenzyme. As an example, the
heme in hemoproteins is strongly bound to the apoenzyme through a single coordination bond
between the heme iron and an amino acid side-chain. Actually the bound between the NAD(H)(P)
and the apoenzyme, the enzyme without the coenzyme, is established by weak bonds, specific (H-
bonds) and non-specific bonds (Niefind et al. 2003; Schlieben et al. 2005) for LBADH. Most
NAD(P)-dependent enzymes follow compulsory-order ternary-complex mechanism, which means
that the coenzyme-enzyme complex is formed at first and then it binds the substrate (Cornish-
Bowden 1995).
The coenzymes are expensive chemicals. Most of them are available on the market but
their price is high. As a matter of fact, their structure is complicated and their synthesis is
consequently expensive. (They are synthesised using biocatalytic steps or by purification of cell
extracts.) Table 4 gives some example of the prices of the nicotine adenine dinucleotide
coenzymes.
Table 4 : Price of some nicotine adenine dinucleotides (from Jülich Chiral Solution
GmbH’s product portfolio (february 2007))
Coenzyme Formula and molecular weight (g/mol)
Price Price by mol of exchangeable Hydride
NAD C21H27N7O14P2 × 3 H2O 717.47
850 €/250g 2,500 €/mol
NADH C21H29N7O14P2Na2
709.4 1150 €/100g 8,200 €/mol
NADP C21H26N7O17P3Na2 787.4
1330 €/100g 10,500 €/mol
NADPH C21H26N7O17P3Na4
833.4 1450 €/10g 120,000 €/mol
At those prices no process is viable if the coenzymes are used stoichiometrically. That’s
why the coenzymes need to be regenerated. This means that the form of the coenzyme which is
used for the synthesis, NAD(P)H, is given back by a second chemical reaction from NAD(P), see
Figure 25. The number of cycles undertaken by the coenzyme, or the number of time the coenzyme
has been regenerated is called “coenzyme total turnover number”. This concept is symmetrical to
Bibliographical review
55
any turnover used for the description of a catalyst, an enzyme for instance: actually in this case the
coenzyme is also a catalyst, it does not appear in the chemical equation of the overall reaction.
Figure 25 : Coenzyme regeneration in the
case of a reduction with NAD(P)H.
There are three main ways to
regenerate coenzymes this is to say how to
perform the “second reaction” of Figure 25:
Regeneration by a second enzyme that depends on the same coenzyme: a second enzyme
and its associated substrate(s) is added to the reaction medium.
Regeneration by the same enzyme as for the synthesis of the product: a sacrificial reactant
is added to the reaction medium.
Chemical method and electrochemical method.
At the moment the last case is rare, the coenzyme stability being still an issue but the two
first techniques are used.
2.2.1.2.Regeneration by a second enzyme
The system that is the most used for the regeneration of NAD(P)H is based on formate
dehydrogenase:
O
OH+ NAD(P)H + NAD(P)+CO2
Equation 7: Formate dehydrogenase
Formate Dehydrogenase (FDH) is the most used system for NADH regeneration in
bienzymatic system (Eckstein et al. 2004a). Indeed, the equilibrium constant of the reaction
presented in Equation 7, which gives NADH back, has an equilibrium constant of 15000 (Liese et
al. 2006), so that the reaction is almost irreversible because the by-product of the reaction, CO2, is
easily separated as a gas. This is not the case of the by-product of every regeneration method
(compare with Glucose 6-phosphate). Formate is an available and very cheap product. Its
consumption over the reaction leads to a modification of the pH, which should be controlled
therefore. Properties and price of a NAD-specific FDH and a NADP-specific is given in Table 5.
NAD(P)H NAD(P)+
PRODUCTSUBSTRATEfirst reaction
second reaction
Bibliographical review
56
Table 5 : Some properties of Formate Dehydrogenase (FDH) (Product Portfolio 2007)
Coenzyme NAD-specific FDH
From Candida boidinii (E. coli recombinant)
NADP-specific FDH from Pseudomonas spec. 101 (E. coli
mutant recombinant)
KM(formate) 13 mmol/L 12 mmol/L
KM(NAD(P)) 0.09 mmol/L 0.29 mmol/L
References (Schütte et al. 1976; Cordes et al. 1994) (Tishkov et al. 1993)
Price 38€/kU 500€/kU
As it can be seen in the table, NADPH regeneration is not as good as for NADH. First the
enzyme is more expensive, and the KM(NAD(P)) is higher.
Other way of regenerating NAD(P)H are given in the Equation 8 (Gu et al. 1990), Equation
9 and Equation 10 (Van Der Donk et al. 2003).
+ NAD(P)H + H+ + NAD(P)+gluconolactone glucose Equation 8: regeneration with glucose dehydrogenase.
+ NAD(P)H + H+ Glucose-6-phosphate + NAD(P)+6-phospho-D-gluconate
Equation 9: regeneration with Glucose-6-P dehydrogenase.
+ NAD(P)H + H+ Lactate + NAD(P)+pyruvate
Equation 10 : regeneration with Lactose dehydrogenase.
2.2.1.3.Regeneration by the same enzyme: sacrificial
substrate method.
This is the technique that was used in the experimental work. Only one enzyme is used
during the process. A second substrate which is called “sacrificial substrate” and possesses a group
belonging to the same family as the product is added to the system. The second substrate is not
transformed into a valuable product: it is “sacrificed”. In our case, R-phenylethanol is produced
from acetophenone and NADPH according to the first equation of Figure 26. NADPH is
regenerated using isopropanol as sacrificial substrate. It undergoes the reaction opposite to the
substrate’s (second equation of Figure 26), which gives NADPH back. In the last line of the Figure
26, the final yield is given where both NADP and LBADH are catalysts.
Bibliographical review
57
Figure 26 : Reactions catalysed by LBADH, conversion of acetophenone and regeneration
of NADPH by addition of isopropanol.
Isopropanol is in a large excess so that the equilibrium of the reaction is favourable to the
production of R-1-phenylethanol: The final equilibrium of such a reaction is determined by
Equation 11. Enzyme catalysed reactions were used for the determination of thermodynamic
equilibrium in different solvents including SCCO2 (Tewari et al. 2005).
aaaa
lisopropanoACP
acetoneRPEKeq
Equation 11: Thermodynamic equilibrium of the conversion of ACP into RPE.
To increase the final concentration of the product, different possibilities exist:
Increasing the sacrificial substrate initial concentration (but it can lead to enzyme inhibition
or deactivation),
Removing the sacrificial product during the reaction. As an example, when isopropanol is
used as sacrificial substrate in the case of ketone reduction, acetone, the sacrificial product,
can be eliminated by evaporation (acetone is more volatile than isopropanol) or by
pervaporation (for instance, the reaction media is in contact with a membrane which has a
reduced pressure on its other side (Stillger et al. 2002)).
2.2.2. Different studies with alcohol dehydrogenase from
Lactobacillus brevis
LBADH was chosen because of its availability, low price, the rare orientation it gives to its
products, and promising application in gas phase reactor. Moreover, no trial of synthesis in organic
media has ever been published.
Bibliographical review
58
The discovery of LBADH went back to the preliminary work on LBADH (Hoshino 1960).
The enzyme which will be used for this study was latter patented by Hummel (Hummel et al. 1997)
and also presented in a publication (Hummel 1997). The gene coding for Alcohol dehydrogenase
(ADH) from Lactobacillus brevis (LBADH) was expressed in E. coli. LBADH is commercialised
by Jülich Chiral Solution GmbH, Codexis, Jülich, Germany (Codexis 2008), as a lyophilised
powder (crude enzyme preparation).
The structure of LBADH was resolved and the crystallographic structure of LBADH was
presented in three works (Niefind et al. 2000; Niefind et al. 2003; Schlieben et al. 2005): it is a
homotetramere bound by Mg2+, its molecular weight is 4*26,6 kDa=106.4 kDa. It catalyses the
conversion of acetophenone (ACP) to R-1-phenylethanol (RPE) using the coenzyme
NADP/NADPH (first equation in Figure 26). LBADH presents a high enantiomeric excess for the
synthesis of R-1-phenylethanol (RPE) which is generally enantiopure. More generally, LBADH has
a broad substrate range and the synthesis could be therefore applied or adapted to another product,
notably substrates having a relatively high molecular weight. LBADH accept other carbonyls as
substrate, such as acetophenone derivatives, propiophenones, aliphatic open chain ketones, 2- and
3-ketoesters, cyclic ketones and so on (Hummel 1997). LBADH give the anti-Prelog orientation to
their products, as well as LKADH (Lactobacillus kefir). This is an advantage because most ADHs
lead to Prelog orientation, for instance HLADH (from horse liver), TBADH or ADH T (from
Thermoanaerobacter brockii), and YADH (Bakers’ yeast from Saccharomyces cerevisiae).
The specific activity of LBADH is 490 U/mg (Hummel 1997). No complete kinetic study
of LBADH is available. Nonetheless, pieces of kinetic data are available: Hummel measured the
following kinetic parameters for LBADH: Km(acetophenone)=0.85 mM and Km(NADPH)=0.16 mM
(Hummel 1997). In a publication (Schlieben et al. 2005) two LBADH are investigated: the wild-
type and a mutant. Only the values concerning the wild-type are given here: for the NADP
reduction Km(NADP)=0.015 mM, kcat=4.4 s-1 (substrate saturation), and for the NADPH oxidation
Km(NADPH)=0.04 mM, kcat=38.1 s-1 (substrate saturation). For the acetophenone reduction by
NADPH, Km(acetophenone)=2.8 mM, kcat=44.5 s-1 (cosubstrate saturation), and for the oxidation of
phenylethanol by NADP Km(phenylethanol)=2.9 mM, kcat=5.4 s-1. A simple kinetic model is proposed
(initial rate only) for the study of substrate inhibition (butan-2-one as the substrate) (Schumacher et
al. 2006). The issue was to find out whether a cosolvent could limit the inhibition. An important
deactivation due to the cosolvent was noticed.
An unusal purification of LBADH based on extraction in an ionic liquid was patented
(Dreyer et al. 2008b; Dreyer et al. 2008a). This enzyme was recently further improved by
engineering (Ching et al. 2008). Lactobacillus brevis was also engineered for the production of
ethanol out of biomass (Liu et al. 2007).
Bibliographical review
59
The stability of LBADH was presented in most publications presenting a process involving
it. A more specific research were done about its stability with a cosolvent as acetonitrile or 1,4-
dioxane in water (Schumacher et al. 2006). The deactivation of LBADH in biphasic system
water/organic solvent for storage was investigated (Villela Filho et al. 2003).
LBADH was used in many processes. In most cases it was used dissolved in water, as well
as the coenzyme. Some processes were based on whole cell with regeneration by sacrificial
isopropanol and removal of acetone in continuous flow (Schroer et al. 2007b), with formate
dehydrogenase (Ernst et al. 2005). The different methods of regeneration are compared in (Schroer
et al. 2007a) and the method with isopropanol gave the best results. In a case the enzyme was
coimmobilised on an amino-epoxy support plus treatment with glycine, mercaptoethanol
glutardialdehyde and applied to a plug-flow reactor (Hildebrand et al. 2006) and the stability of the
catalyst was much improved (This case of immobilisation is detailed in 2.1.2.5). Crosslinked
enzyme particles were made using a polyaldehyde prepared from a polysaccharide as the water-
soluble crosslinking agent (Mateo et al. 2004). Stabilisation with ionic liquid were also investigated
(Braeutigam et al. 2007; Dreyer et al. 2008a). A biphasic system of ionic liquid and water can
present an advantageous coefficient of partition that improve the yield of the conversion (Eckstein
et al. 2004b).
Most patented processes involve an aqueous phase which is extracted at the end of the
reaction and, sometimes continuously. Never a dense gas was used for this purpose. Isopropanol
regenerates NADPH, for instance: (Mueller et al. 2003; Peschko et al. 2005; Groeger et al. 2006;
Meudt et al. 2006; Peschko et al. 2006; Pfaller et al. 2007; Yasohara et al. 2007; Groeger et al.
2008).
No process was run in the absence of water except for gas phase reaction run at
atmospheric pressure (Ferloni et al. 2004; Trivedi et al. 2005; Trivedi et al. 2006) presented in next
paragraph.
2.2.3. Alcohol dehydrogenase in non aqueous solvent.
The general case of enzyme-catalysed reaction in organic solvent was treated in 2.1.2.7. In
the case of coenzyme-dependent reactions, permanent complexes between enzyme and coenzyme
are formed in organic solvent; hence no dissociation takes place, even at low affinity as in the case
of LBADH and NADP. LBADH was successfully immobilised on glass beads using sugar as a
stabiliser (Ferloni et al. 2004; Trivedi et al. 2005; Trivedi et al. 2006) and a residual activity
superior to 300 % was observed probably because of “structural changes in the enzyme molecule
during the drying process” (Trivedi et al. 2005). The method for co-immobilising the enzyme
Bibliographical review
60
and the coenzyme on glass beads that will be used herein was adapted from this work
about gas-phase continuous reaction.
Several studies about ADH in non aqueous media were carried out. Except from Deetz’s
preliminary study (Deetz et al. 1988), no successful way for coenzyme regeneration with a second
enzyme was proposed : the regeneration is undertaken by the same enzyme with a sacrificial
substrate. The conversion was catalysed by HLADH (Horse Liver alcohol dehydrogenase, an NAD
dependent ADH) in isopropyl ether, butyl acetate, chloroform (Grunwald et al. 1986). The reaction
was catalysed by YADH (Yeast ADH) co-lyophilised with NAD in heptane (Yang et al. 1993). The
stability of YADH and TBADH (Thermoanaerobacter brockii ADH) in different organic solvents
(n-dodecane, n-octane, toluene, and pyridine) was measured (Miroliaei et al. 2002). Fluorinated
NAD was used with HLADH in SCCO2 (Panza et al. 2002) . The conversion and the total turnover
was investigated with YADH and HLADH (Snijder-Lambers et al. 1991) on different carrier and in
different solvents. The influence of the water activity was tested (Adlercreutz 1991; Jonsson et al.
1999; Jönsson et al. 1999a) in hexane with TBADH and HLADH and kinetic constants evaluated.
The equilibrium between 2 pairs of related alcohol and ketone (for instance isopropanol and
acetone or 2-pentanol and 2-pentanone) was studied using a commercially available preparation of
ADH and coenzyme (Tewari et al. 2005). Polymere (ethyl cellulose)-aided solubilisation of
HLADH and NAD is tested in different solvents at different water activities with different enzyme-
coenzyme ratios (Virto et al. 1995). Generally YADH presents the advantage of its price and
availability while it deactivates rapidly. HLADH and TBADH are more stable and TON as high as
a million were obtained with them. Many studies showed that the water activity the most
favourable to the initial is the highest (Deetz et al. 1988; Yang et al. 1993; Virto et al. 1995;
Jönsson et al. 1999b). Indeed the maximum of activity of HLADH is observed when a monolayer
of water surrounds it (about thousand water molecule) (Adlercreutz 1996). However, only few
studies investigated the variation of stability according to the water activity (Yang et al. 1993). In
this study a high water activity led to a faster deactivation, but the low reaction TON is
counterbalanced by the increase in the reaction rate.
The hydrogenation of acetophenone by coimmobilised LBADH and NADP in gas phase
(Ferloni et al. 2004) gave very good results (high turnover, enantiomeric excess and space-time-
yield.) NADPH was regenerated by isopropanol, while the enzyme and the coenzyme were
coimmobilised on glass beads. The conditions of co-immobilisation were optimised: the choice of
the support, the temperature and pressure of drying, the amount of NAPD and the addition of
stabiliser (sucrose) (Trivedi et al. 2005). The influence of the water activity was investigated and
the results fit the previous well: when the water activity is higher, the initial rate is higher and the
half life is smaller. The effect of the temperature was also investigated and an increase in
Bibliographical review
61
temperature increased the reaction rate but decreased the half-life of LBADH. They eventually
chose to perform the reaction at 30°C, water activity at 0.55: excellent TON as high as 4 million
were thus afforded (Trivedi et al. 2006).
Dense gases have been used as solvents for enzyme-catalysed reactions, with lipases in
most cases (Habulin et al. 2007; Knez 2009). Whereas ADH were relatively often tested in non
aqueous solvent (Klibanov 2003), processes with ADH in non-aqueous solvents are rather rare due
to their instability in them (Lavandera et al. 2008) and they were seldom tested in dense gases
(Matsuda et al. 2000; Panza 2001; Tewari et al. 2005). Matsuda set up a process with immobilised
resting cell of Geotrichum candidum in SCCO2 but the biocatalyst was too prone to deactivation
(Matsuda et al. 2003).
2.2.4. Goal of this work concerning LBADH in dense gases
As showed previously, very few processes based on ADH were run in dense gases and little
is known about their stability in those fluids. Our laboratory has an expertise in enzyme-catalysed
reaction in non-aqueous solvent and especially dense gases. However, processes using ADHs were
not treated at the moment. So an important part of the practical work is the development of
protocols in relation to this family of enzyme, as techniques of immobilisation, reaction setup in
gas phases. The goal was to address those issues: whether a conversion is possible, which
enantiomeric excess presents the product, what stability the enzyme has in this medium. LBADH,
as a first target, was tested.
2.3. Resolution via the formation of diastereomeric complexes with (+)-
tartaric acid followed by extraction with supercritical carbon
dioxide applied (±)-trans-1,2-cyclohexanediol
(R,R)-trans-1,2-cyclohexanediol (RRCHD) and (S,S)-trans-1,2-cyclohexanediol
(SSCHD) are important building blocks or chiral auxiliaries (Groaning et al. 1998; Tanaka et al.
2001; Tiecco et al. 2003; Wojaczynska et al. 2008). This bibliographical review was limited to
CHD and resolution. Rac-CHD is a cheap product that is almost free of cis-1,2-cyclohexanediol.
Indeed the organists possess several anti addition to produce CHD from cyclohexene (Clayden et
al. 2001). It is interesting to mention that enantioselective synthesis of CHD were described, as
example the asymmetric hydrogenation of cyclohexane-1,2-dione over cinchonidine-modified
platinum (Sonderegger et al. 2003). Biocatalytic methods exists for the synthesis of an enantiomer
of CHD, the hydrolysis of the corresponding epoxyde by an epoxide hydrolase (Chiappe et al.
2007). Many chromatographical methods were described but in an analytical purpose in most
cases.
Bibliographical review
62
2.3.1. Different methods of resolution of (±)-trans-1,2-
cyclohexanediol based on the formation of a covalent bond.
Numerous methods of resolution of (R,R)-CHD and (S,S)-CHD have been developed. The
majority of them involved the formation or the hydrolysis of an ester. Those resolution techniques
found a source of asymmetry in a biocatalyst, a chiral reagent or a chiral non biological catalyst.
The biocatalytic resolution techniques based on an ester bound are :
Selective hydrolysis: hydrolysis of their racemic acetates and chloroacetates in the
presence of a highly selective ester hydrolase from Pseudomonas sp. (Laumen et al. 1989),
by fermentation with Rhizopus nigricans (Kawai et al. 1981), monoacetates were also
preparated. from the racemic diacetates by lipase-catalyzed hydrolysis (Bodai et al. 2003)
and the 2 step hydrolysis of diacetate CHD by porcine liver esterase (PLE) and then lipase
from Pseudomonas cepacia (Caron et al. 1991).
By transesterification with Pseudomonas cepacia lipase (Kaga et al. 1998) or by fungal
lipases (Bodai et al. 2003).
By a selective esterification: acylation by lipase YS (from Pseudomonas fluorescens)
(Naemurz et al. 1995), lipase from Pseudomonas cepacia, and lipase from Candida rugosa
(Kazlauskas et al. 1991).
The resolution was also realized by esterification with chiral carboxylic acid such as o-
acetyl mandelic acid (Chatterjee et al. 2007) or Ma NP acid (Kasai et al. 2004). Chiral catalysts
were used in those cases: acylation catalysed by Cu(II)(borabox) (Mazet et al. 2006) or by amine-
phosphinite bifunctional organocatalysis derived from quinidine (Mizuta et al. 2006) and
monobenzoylation catalyzed by organotin compounds (Iwasaki et al. 2000).
Dispiroketals were used for the resolution of CHD, a chiral diketone was used as a
protecting group for CHD by formation of 1,2-diacetal.
Other resolution techniques were developed: Dispiroketals selectively reacted with CHD
(Ley et al. 1996a; Ley et al. 1996b). A chiral diketone was used as a protecting group for CHD by
formation of 1,2-diacetal (Lenz et al. 1998). The hydroboration-oxidation with
diisopinocampheylborane of benzyl or diphenylmethyl vinyl ethers, followed by cleavage
(Peterson et al. 1988), the kinetic resolution of CHD by Bacillus stearothermophilus diacetyl
reductase (Bortolini et al. 1998).
2.3.2. Resolution of (±)-trans-1,2-cyclohexanediol by selective
formation of a cocrystal
CHD was resolved (Kawashima et al. 1991) by reacting with an other resolving agent
(R,R)-1,2-cyclohexanediamine that formed a co-crystal with SSCHD preferentially. This method
Bibliographical review
63
gave SSCHD in a yield of 73% based on the enantiomer present in the racemic compound. Its
optical purity was 67%.
The structure of the co-crystal between SSCHD and (R,R)-1,2-cyclohexanediamine was
established and discussed in publication by Hanessian (Hanessian et al. 1994; Hanessian et al.
1995; Hanessian et al. 1999). Other co-crystals were found between RRCHD and (R,R)-2,3-
diaminobutane (Hanessian et al. 1999) or N-methylmorphine-N-oxide (Chanzy et al. 1982). These
structures present an inner core based on hydrogen bonds between the diamine and diol moieties,
which are responsible for the stereoselectivity of the crystallisation and the geometry of the crystal,
and an outer region formed of the hydrophobic groups.
Inconvenient separation of the product from the resolving agent.
A matter with the method of resolution with (R,R)-1,2-cyclohexanediamine
(Kawashima et al. 1991) is that the diol and the diamine have comparable solubility in most
solvents and the decomposition of the cocrystal and the separation of (R,R)-1,2-
cyclohexanediamine from CHD required a silica-gel short column. The problem was
encountered in the case of the resolution of ibuprofene with the resolving agent R-(+)-
phenylethylamine, where the separation of the salt and the free enantiomer was done by SFE
(SCCO2) (Molnar et al. 2006): in both case the resolving agent and the racemic compound
present similar polarities and, thus, solubility. The SFE of the free enantiomer lead to an
extract that is polluted with R-(+)-phenylethylamine that was removed by liquid-liquid
extraction: the acidic aqueous solution that allows to have R-(+)-phenylethylamine as a
hydrophilic cation and ibuprofen as a neutral acid that is extracted in an apolar organic
solvent. In both cases (Kawashima et al. 1991; Molnar et al. 2006), the extracts presented
good enantiomeric excesses but the extra step of purification to remove the resolving agent
induces disadvantages: the resolving agent is lost (in one case absorbed on silica gel and in
the second dissolved into liquid solution) and its recovery is possible but expensive, possibly
energy-intensive. Tartaric acid (TA) comparatively presents an important advantage: its
solubility is extremely low in SCCO2.
Our group already studied the resolution of 1,2-disubstituted cyclohexane using TA
derivative (Székely et al. 2004) based on the screening of the resolution of secondary alcohols
with DBTA (Illés et al. 2002). In the case of the resolution of trans-2-chloro-cyclohexanediol
with DBTA it was possible to decompose the diastereomeric cocrystal in the extractor at
moderate temperature and to extract the released (S,S)-enantiomer.
Bibliographical review
64
2.3.3. Physical properties of CHD.
Table 6: Melting point and structural data for crystalline phases of trans-1,2-
cyclohexanediol and references.
Tfus (K)
Reference(s) for Tfus
Space group, cell parameters: a b c (Å) α β γ (°)
Refcode in CSD (Allen 2002),
PDF file No. from ICDD T(K)
RacCHD Stable
polymorph
376–377
Verkade et al. (1928) quoted in (Lloyd et
al. 2007)
Pbca 7.885 19.301 8.498
90 90 90
ZZZKPE01, 02-064-1664
ca 295 K (Sillanpaa et al. 1984)
377 Lettré & Lerch
(1952) quoted in (Lloyd et al. 2007)
Pbca 7.888 19.333 8.501
90 90 90
ZZZKPE02, 02-073-8658
ca 295 K (Jones et al. 1989)
376.4 (Leitao et al. 2002) Pbca
7.885 19.301 8.498 90 90 90
ZZZKPE04 02-093-6744
299 K (Lloyd et al. 2007)
377 White (1931) quoted in (Lloyd et al. 2007)
RacCHD Metastable polymorph
C12/c1 18.578 10.007 7.272
90 96.32 90 subliming at RT
ZZZKPE06 299 K (Lloyd et al. 2007)
RRCHD or SSCHD Stable
polymorph
382.5 (Leitao et al. 2002) P3121
10.191 10.191 10.821 90 90 120
PIWXIK, 215 K (Hanessian et al. 1994)
P3221
10.229 10.229 10.909 90 90 120
RIHMAX01 02-093-3042
299 K (Lloyd et al. 2007) RRCHD or
SSCHD Metatable polymorph
352.8 (Leitao et al. 2001)
CHD was the subject of several publications because of its industrial importance and also
different features very interesting to a theoretic point of view (see
Table 6). The pure enantiomers crystallised according to two different forms that melts at
382.5 K and 352.8 K and the crystal are generally a mixture of the two forms whose difference in
melting can be observed (Leitao et al. 2001). Only the stable polymorph’s structure was determined
(Hanessian et al. 1994; Lloyd et al. 2007). A racemic compound (racCHD) of (R,R)- and (S,S)-1,2-
cyclohexanediol is present under two polymorphic forms, as well. The stable polymorph’s structure
was determined by three research groups (Sillanpaa et al. 1984; Jones et al. 1989; Lloyd et al.
2007) and the metastable’s by one (Lloyd et al. 2007).
Bibliographical review
65
The solid-liquid melting phase diagram for mixtures of RRCHD and SSCHD was
investigated by DSC methods (Leitao et al. 2002). This binary system although resembled a type of
melting phase diagram including a racemic compound (Jacques et al. 1981), but had an unusual
feature. Usually, a racemic compound, as a co-crystal of both enantiomers, can form a eutectic
mixture, i.e. crystal conglomerate with one of the enantiomers as presented Figure 27 a).
a) usual case
b) the case of RR and SSCHD
Figure 27: Comparison of 2 solid-liquid melting phase diagrams a) a usual for a couple of
enantiomers and b) CHD’s case with a solid solution.
Actually, in this case, eutectic compositions were found at about molar fractions of XSSCHD
= 0.2 and 0.8. Furthermore the corresponding eutectic temperature of Teu=371 K was observed in
the composition ranges 0 < XSS-CHD < 0.2 and 0.8 < XSS-CHD < 1, but at intermediate composition,
0.2 < XSS-CHD < 0.8, the endothermic heat effect of eutectic melting does not occur (Leitao et al.
2002), see Figure 27b. This phenomenon was explained by Leitao et al. as the proof of the
formation of a solid solution for compositions within the latter wide range around the 1:1 molar
ratio. A metastable phase was also found below eutectic temperature (xSS-CHD < 0.2 and xSS-CHD >
0.8), as the continuation of the liquidus curve of solid solution (Leitao et al. 2002). In this figure the
liquidus and solidus of the solid solution are not distinguished. Indeed DSC measurements showed
only a broad peak probably because these two effects occurred at very closed temperature by DSC.
The interval of composition where the liquid solution is found rather than two compounds
according to the temperature is not precisely known. However, according to their results to Leitao,
the solid solution should occupy the interval 0.2-0.8 for all the studied temperatures because no
transition was observed below.
Bibliographical review
66
2.3.4. Method of resolution of CHD by formation of a cocrystal
CHD-TA and optimisation of the parameters of extraction:
temperature and pressure.
This part was subject of the paper I co-authored with Peter Molnar (Molnar et al. 2008) and
is included to his PhD thesis (Molnár 2009).
Resolving agents of TA’s family were screened and TA was eventually found to be the
best. The method of resolution of CHD with TA and extraction with SCCO2 is schematically
represented on Figure 28.The source of asymmetry in this method of resolution comes from TA.
This method consists in three steps:
1. The selective cocrystallisation.
2. The extraction by SCCO2.
3. The raffination of the residuum, decomposition of the co-crystal.
OHOH
O
OH OH
H OH
OH
OH
OH
OHOH
OH
OH
OHOH
OHO
OH OH
H OH
(R,R)-(+)-tartaric acid
(S,S)-(+)-CHD
(S,S)-(+)-CHD(R,R)-(-)-CHD
+ +
,
evaporation of the solvent (ethanol)
Co-crystal to be raffinated
Extracted inSCCO2
Figure 28: Principle of the resolution of (±)-CHD by co-crystal formation followed by an
extraction in SCCO2 (mr=0.5)
The selective cocrystallisation consists in the dissolution of racCHD and TA in two
separate ethanolic solutions that are then mixed. The solution gets cloudy, which presumably
shows that the cocrystal has precipitated – this interpretation was the most natural at this moment
of the research but will reveal wrong as shown in the experimental part (and more precisely in
4.2.4.3). An inert filtering agent, perfil, is added to it. Ethanol is then evaporated. A cocrystal is
preferentially formed between RRCHD and TA: it “captures” RRCHD but no or little SSCHD
because only no or only little cocrystal between SSCHD and TA is formed. The SS enantiomer
remains “free”, this is to say not “cocrystallised” or “uncocrystallised” and is due to be extracted
with SCCO2. The quantity of TA in relation to the quantity of CHD is an important factor during
the extraction and the molar ration (mr) is defined as :
Bibliographical review
67
nn
CHD
TAmr
Equation 12: definition of the molar ratio, mr
At the moment only the more usual mr of 0.5 was studied. This molar ratio gives the best
result in the ideal case. This is to stay when the perfect cocrystallisation occurs between 1 mol of
CHD (0.5 mol SSCHD + 0.5 mol RRCHD) and 0.5 mol of TA, 0.5 mol of CoC is formed and 0.5
mol of SSCHD are free (ideal case).
The free CHD, mostly SSCHD, is extracted with SCCO2. What is left over in the extractor
mostly consists in the cocrystal presumably. Those methods, that will be used herein again, are
detailed in 4.1.4 and 4.1.4.2.
The residuum is treated in order to recover RRCHD. The decomposition of the cocrystal or
raffination is based on the acidic properties of tartaric acid: the tartaric acid treated with a basic
aqueous solution. Water was then rotoevaporated and CHD was extracted by chloroform. The
detailed protocol (precisely given in 4.1.4.3) is presented in Figure 29. This method presents
several disadvantages: it includes many steps, is time-consuming, uses much solvent, generates
wastes.
Figure 29: Decomposition of the residuum.
A perfect extraction selectivity, in this case, means that a) the partial cocrystallisation is
perfect so that CoC contains all and only RRCHD consequently all free CHD is SSCHD b) all and
Bibliographical review
68
only SSCHD is extracted. This extracts presents no RRCHD released by CoC so the cocrystal
RRCHD-TA does not decompose at all over the supercritical extraction. The yield of extraction is
50%. c) No CoC is lost and the decomposition of CoC is total. So the yield of the decomposition is
50%. The temperature and the pressure play an important role in the extraction. Indeed, the
solubility of CHD in SCCO27, the speed of decomposition of the cocrystal varies with temperature
and pressure. The density, viscosity and diffusivity changes with the temperature as well. The study
of the influence of the pressure and temperature of extraction on the F-parameters (Equation 3) was
the subject of an experimental design (Molnar et al. 2008) and the best resolutions within the range
of the experimental design gave F about 0.6 and were achieved under the following conditions: P =
10 MPa, T = 63 °C; or P =20 MPa, T = 33 °C (see Table 7).
Table 7: Comparasion of different results obtained for the resolution of CHD
According to the result published in (Molnár 2009). The “middle point” (150 bar, 48°C) was
repeated 4 times, the standard deviation is given.
Yext (%) eeext (%) Yraf (%) eeraf (%) F 0≤F≤1
Ideal resolution with mr=0.5 50 100 50 100 1
Middle point mr=0.5,
Pext=150 bar Text=48°C
54.0
±1 55.5
±3
31.5
±3
76.4
±4
0.54
±0.025
mr=0.5, Pext=100 bar Text=63°C
47.8 61.4 36.9 91.9 0.61 Experimental
resolution
mr=0.5, Pext=200 bar Text=33°C
50.6 62.1 33.7 81.9 0.59
2.3.5. Issues concerning the resolution of CHD by
cocrystallisation and SFE to be addressed in the present
work.
The resolution of CHD by cocrystallisation and SFE is performed in three steps as shown
previously (in 2.3.4). The second steps, the extraction, was optimised and is not the concern of the
present work. The last step (Figure 29) presented several disadvantages: it is time consuming and
7 It should not be forgotten that the solubility of CHD depend on its enantiomeric excess.
Bibliographical review
69
has very bad contribution to the E-factor, while all the previous steps are essentially green. It is
necessary to develop an alternative method for the decomposition of the cocrystal.
The eeext seemed to be limited at about 60% (cf Table 7). It should be investigated if this is
due to a thermodynamic limitation during the sample preparation or another matter arising during
the sample preparation and/or the SFE. And generally, it is aimed at a better knowledge of what
occurred during the extraction. Thus other issues arise: what can be learnt by analytical techniques
and what is the structure of the cocrystal RRCHD - TA responsible for the extraction the
stereoselectivity and what is the structure of its counterpart cocrystal of SSCD – TA. One of the
goal is to observe which kind of data can be afforded by DSC and to check if it corroborates with
other techniques such as XRD.
Conversion of acetophenone to R-1-phenylethanol
70
3. Conversion of acetophenone to R-1-phenylethanol
3.1. Materials and methods
3.1.1. Materials
3.1.1.1.Reagent
NADPH and NADP were provided by Jülich chiral solution GmbH, Codexis, Jülich,
Germany (Codexis 2008). Heptane, acetone, isopropanol, phosphate buffer solution (pH = 7), were
provided by Merck, and MgCl2 was added so that [Mg2+]=1 mM. MgCl2.6H2O, orange silica gel
were provided by Riedel-deHaën. Acetophenone, acetophenone standard, and Na2CO3.10H2O were
provided by Fluka. Decane was provided by Aldrich. Sucrose, glass beads (425-600 µm), R- and S-
1-phenylethanol standards were provided by Sigma.
3.1.1.2.Biocatalyst
LBADH expressed in E. coli, was provided by Jülich Chiral Solution GmbH, Codexis,
Jülich, Germany as a lyophilised powder (crude enzyme preparation).
2 different batches of lyophilised powder were used. The first was used for the conversion
of ACP into RPE. The provider indicated that the activity before shipping was of 9.3 U/mg while
we measured it at 8 U/mg at the reception. From the value of the specific activity we deduce that
the content of active enzyme of this enzyme preparation was about 1.6% and that 1 mg of this
preparation contains 0.15 µmol. This value was used for calculation of TONE.
The second batch was used for the study of deactivations. Its activity was 69 U/mg before
shipping and 21 after. The meaning of U and the way to measure it is given in 3.1.2.4.
3.1.2. Methods
3.1.2.1.Preparation of the biocatalyst
The preparation is indicated for the catalyst used for the reactions in propane. The catalyst
for reactions in heptane was prepared according to the same method but with different quantity of
material. The details are given in Table 16 in annex 7.2. The co-immobilisation of LBADH and
NAPD on glass beads was done the following way: enzyme (25 mg) and coenzyme (11 mg) were
diluted in the buffer in a beaker. The solution was stirred for 10 minutes at 4°C. The support, 5 g of
glass beads, was then added. This mixture was stirred for 1 hour at 4°C. The beaker was placed into
Conversion of acetophenone to R-1-phenylethanol
71
a desiccator at the pressure of about 50 kPa for 2 hours and then at the pressure of about 10 kPa
until it dried. This protocol was adapted from the work done in gas phase (Trivedi et al. 2005).
3.1.2.2.High-pressure view cell
The reactions at high-pressure were performed in a high-pressure reactor with sapphire
windows which is presented in Figure 30. This apparatus provided by NWA GmbH (Lörrach,
Germany) (NWA (http://www.nwa-highpressure.de)) consists of a large piston, equipped with a
mechanical stirrer, heater and thermostat. It is closed by a sapphire window at each extremity, and
can be seen through. The back sapphire window is mobile and the volume of the cell thus variable
(from 60 to 30 cm3). The pressure inside the cell is controlled by the operator. The back sapphire
window is pushed by hydraulic oil whose pressure is set and balances the pressure inside the cell.
Pressurized propane was supplied by a high-pressure membrane pump (PM-101, NWA
GmbH, Lörrach, Germany). When the catalyst was immobilised on glass beads it was introduced
into the opened cell. The cell was assembled and the liquid reactants, isopropanol and
acetophenone with decane, the internal standard (as well as the aqueous phase in the case of the
biphasic system) were injected through an opening at the top of the cell. The cell, which had been
sealed, was filled with dense gas until the gas phase was present only in a small amount. When the
desired temperature was reached the pressure was adjusted by changing the position of the back
sapphire window. Sampling without depressurisation was made possible with an autosampler of the
type of HPLC injector. The volume of the cell was reduced by the volume of the sample as the
movable sapphire windows moved towards the other extremity. The samples were sprayed into a
test tube which was then rinsed with hexane. This solution was analysed by Gas chromatograph.
Conversion of acetophenone to R-1-phenylethanol
72
Figure 30 : Scheme and picture of the high-pressure reactor.
1 liquid propane, 2 high-pressure pump, 3 one-way valve, 4 needle valve at the inlet of the
view cell, 5 needle valve (sampling of the lower phase) 6 static sapphire window (the view
cell can be opened at this level), 7 mobile sapphire (It slides inside the cell), 8 mechanical
stirrer, 9 temperature indicator and temperature regulation, 10 heaters, 11 pressure
indicator, 12 autosampler, 13 safety rupture disk, 14 air, 15 compressor, 16 air pressure
regulator, 17 hydraulic oil system, 18 hydraulic oil, 19 pressure regulator, 20 safety pane.
3.1.2.3.Reaction with LBADH
The reaction was performed using the regeneration method described in 2.2.1.3. It relies on
the “sacrificial substrate” isopropanol. This reaction was run in different media: water, heptane,
propane with coimmobilised catalysts and in the biphasic system water-propane.
The reaction run in water at 30°C was catalysed by 8 mg NADP and 15 mg LBADH.
50mL of buffer contained 10 mmol of acetophenone 1.5 mol of isopropanol. The reaction started
by adding the 2 solid catalysts that dissolved immediately. The evolution of the concentration of
ACP and RPE was measured by GC. The samples taken from the reaction mixture regularly were
extracted with 5 times with their volume of ethyl acetate The organic phase is then dried with
molecular sieve (4 Å) prior to GC analysis.
Conversion of acetophenone to R-1-phenylethanol
73
Reactions were performed in heptane. The conditions varied and are given in Table 16 in
annex 7.2. The reaction started when the prepared biocatalyst (see 3.1.2.1) was added to the
reaction mixture (heptane, ACP and ISP) followed by water.
The reaction of acetophenone with isopropanol catalysed by LBADH and NADP was
investigated in two media that involved high-pressure: in propane, the enzyme and coenzyme being
co-immobilised and in a biphasic system water/propane (enzyme and coenzyme dissolved in the
aqueous phase). Reactions catalysed by immobilised biocatalyst were run in dense propane.
1.5 mmol of acetophenone and 25 mmol of isopropanol reacted at 3 MPa and 30 °C in propane
inside the “high-pressure view cell”. It was checked before the reaction that it forms a
homogeneous mixture. The water activity was set with Na2SO4 10/0 (Zacharis et al. 1997). The
hydrate Na2SO4.10H2O can release water while the anhydrous Na2SO4 can absorb some.
Equilibrium took place so that the activity coefficient of water was buffered at 0.8.
In the case of the reactions in the biphasic system water/dense propane the reactions were
catalysed by 25 mg LBADH, 10 mg NADP dissolved in water, to which 7 g isopropanol and 0.18 g
acetophenone was added and, then, liquid propane. The temperature was set at 30 °C and the
pressures at 30 bar. The concentration of ACP and RPE was measured in the propane phase.
3.1.2.4.ADH activity test
The activity of ADH solution was measured according to the protocol given by the
provider. The method is based on chemical reaction presented at the first line on Figure 26. The
disappearance of NADPH in the presence of acetophenone and ADH was quantified by UV-Vis
spectrometry. The molar extinction coefficient of NADPH is 6220 M-1cm-1 at 340 nm while NADP
does not absorb at this wavelength. 20 µL of NADPH (9.5 mM) was added to 970 µL of a solution
of acetophenone (11 mM) in phosphate buffer (pH = 7) containing 1 mM of Mg2+ at 30°C. The
reaction started when 10 µL of a solution containing the enzyme was added to it. The absorbance
was regularly measured over the first minute. The activity of ADHs is given in the unit U. 1 U
corresponds to the disappearance of 1 µmol/min of NADPH.
The measurement of the stability of an enzyme preparation consists of measuring its
remaining activity after incubation in certain conditions. Hence, samples from the enzyme
preparation are taken regularly and the rest of their activity is measured. When the deactivation of
solid preparation was investigated the samples were dissolved in the appropriate quantity of buffer
before activity test.
Conversion of acetophenone to R-1-phenylethanol
74
Figure 31: Example of a test of enzyme
activity
3.1.2.5.Autoclave for incubation of biocatalyst
A set-up was developed for the measurement of the deactivation of LBADH in two media
involving high-pressure, propane and propane/aqueous phase. LBADH was treated in a
thermostated autoclave presented in Figure 32. When the enzyme was dissolved in aqueous
solution, the liquid sample was introduced in the autoclave at the desired temperature. The seal was
then screwed and the propane pumped into the autoclave. After the sample was taken through the
capillary, a small amount of propane was added to maintain the pressure to balance the decrease in
pressure. For the activity measurement of the lyophilised powder, the autoclave was disassembled
in order to take samples.
Figure 32: Autoclave for the measurement of
ADH deactivation in propane
1 liquid propane, 2 aqueous solution of ADH, 3
liquid propane from high-pressure pump, 4 blow
disk, 5 manometer, 6 thermocouple, 7
sampling, 8 seal, 9 magnetic stirrer. It is represented the case of a biphasic system.
In case of biocatalyst under the form of a solid
no stirrer was used.
3.1.3. Analytical methods
The solution were analysed by Gas chromatograph HP 5890 equipped with the integrator
HP 3392A and the column BetadexTM 120 30 m × 0.25 mm, 0.25 µm film (Supelco). The
quantification and the calibration were done using decane as an internal standard.
Conversion of acetophenone to R-1-phenylethanol
75
3.2. Results and discussion
3.2.1. Reaction in water
The conditions for this reaction (i.e. concentration of acetophenone, isopropanol and
NADP) were taken from an article dedicated to the optimisation of the conversion of acetophenone
to S-1-phenylethanol by ADH T (Findrik et al. 2005). In this case the concentration of ISP was
very high so that it acted not only as a sacrificial substrate but also as a cosolvent that allowed the
dilution of ACP, a compound rather insoluble in water. The reaction catalysed by LBADH (Figure
33) exhibited a very good yield (98.5 %) and enantioselectivity (RPE was enantiopure). The initial
rate is high (0.25 mmol/(L.min), 12.5 µmol/min, or 0.68 µmol/(min.mgE)). The total turnover
related to the enzyme was TONE = 150 and related to the coenzyme TONCoE = 50. We cannot
conclude from this experiment that the TON were limited to those values neither they could be
higher.
Figure 33: Conversion of ACP to RPE in water
3.2.2. Preliminary test in heptane
We decided to use heptane as a solvent because it was shown that this solvent is suitable
for bioconversion with coimmobilised ADH and coenzyme (Snijder-Lambers et al. 1991; Yang et
al. 1993). Reactions at high-pressure require expensive equipment and special safety conditions so
the early development of the co-immobilisation with usual organic solvent is preferable. The
reactions run in heptane are presented in Table 16 of annex 7.2. As an example the bioconversion is
shown below in Figure 34.
Conversion of acetophenone to R-1-phenylethanol
76
Figure 34: Reaction in heptane with co-immobilised catalyst (reaction G)
In each case RPE was enantiopure, but the conversion was limited (small yield) as well as
the total turnovers (<100). The constant K was calculated for each reaction, according to the
equation:
][*][][*][
ISPACPacetoneRPEK
Equation 13
K did not reach a limit, Keq (see 2.2.1.3) and as the co-immobilised catalyst could not be
reused for a second conversion it was deduced that the TONs were probably limited because of the
deactivation of the enzyme.
The biocatalyst required to be co-immobilised to show some activity. Mixed powders of
LBADH and NAPD without any further preparation led to no conversion. Such an experiment was
also performed in dense propane and gave the same result. This confirms the need for a delicate
preparation of the catalyst for reaction in medium where they are not soluble.
3.2.3. Reaction at high-pressure
The reaction of acetophenone with isopropanol catalysed by LBADH and NADP was
investigated in two media : in propane, the enzyme and coenzyme being co-immobilised and in a
biphasic system water/propane (enzyme and coenzyme dissolved in the aqueous phase).
Conversion of acetophenone to R-1-phenylethanol
77
3.2.3.1.Reaction in propane with co-immobilised catalyst
When the reation is run with catalysts immobilised on a solid carrier it is important that the
reaction medium presents only a single liquid phase. In case there is no good solubility of the
reactants and products in the solvent, a second liquid phase could provoke the deactivation of the
enzyme by a more polar phase. Indeed, it could strip off the essential water or inhibit the enzyme
by a too high concentration of the substrate. Mixtures with the substrates and products at different
concentrations with propane at 30°C and 30 bar were observed in the view cell (described in
3.1.2.2) in order to check how many phases were present (see results in annexe 7.3 Table 17). In
every case the miscibility was perfect and no second phase was seen except for a gaseous at low
pressure.
The water activity was buffered –set at a certain activity- using salt hydrates. The water
activity can be set at 0.8 using Na2SO4.10H2O and Na2SO4 ((Zacharis et al. 1997) and more
generally (Halling 1992)). This high water activity could influence the miscibility between the
different substances: a water-rich phase could appear. However such a second phase was not
observed.
The catalyst and the salt pair were placed separately in folded filter paper into the view
cell. The initial rate in propane when enzyme and co-enzyme were co-immobilised was about
0.05 µmol/min/mgenzyme . As an example for comparison, it was found with TBADH (ADH from
Thermoanaerobium brockii) initial rate up to 0.15 µmol/min/mgenzyme at 25°C for the reduction of
2-pentanone in hexane after optimising the water activity (Jönsson et al. 1998). The comparison is
interesting because the activity of the lyophilised ADH powder used for preparation of the catalyst
were comparable, 7.3 U/mg in their case, 8 U/mg in our case. So the recovery of LBADH activity
in propane after immobilisation (by comparison with the activity test presented in 3.1.2.4, so in
water) was less than 1 %. Low recoveries in organic media of the same order of magnitude were
found for YADH (ADH from yeast) but about 100% with Horse liver ADH (HLADH) (Snijder-
Lambers et al. 1991). The TON of our reaction related to the enzyme was 180 and related to the
coenzyme 50 and the yield was 45 %. To compare, other studies showed that after an optimised
immobilisation LBADH gave TON above 106 in aqueous solvent in a continuous reactor. If certain
studies in organic solvents presented such low order of magnitude for TON, other presented values
as high as 106 (Grunwald et al. 1986; Snijder-Lambers et al. 1991) (both with HLADH). A study of
hydrogenation done in dense gases with a biocatalyst based on ADH have shown limited yield
(Matsuda et al. 2003). Contrary to our results, the immobilisation of LBADH with a similar method
gave good results with gas phase continuous flow reactor: the TONs were above 106 (Ferloni et al.
2004; Trivedi et al. 2006). As the stability of the enzyme was already an important issue in heptane,
the investigation of the stability of LBADH was undertaken and the results are presented in 3.2.4.2.
Conversion of acetophenone to R-1-phenylethanol
78
Figure 35: Bioconversion in propane with immobilised catalyst
3.2.3.2.Reaction in biphasic system propane-water
The bioconversion in propane was run at three different pressures, 3, 10 and 20 MPa. The
evolution of the concentration of ACP and RPE are given in Figure 36, Figure 37 and Figure 38
and these results are summarised in Table 8.
Figure 36: Bioconversion in the biphasic system water/dense propane at 100 bar
Conversion of acetophenone to R-1-phenylethanol
79
Figure 37: Bioconversion in the biphasic system water/dense propane at 30 bar
Figure 38: Bioconversion in the biphasic system water/dense propane at 200 bar
The syntheses in the biphasic system gave high yield of about 90 %. The initial rate , 0.1-
0.2 µmol/min/mgenzyme., were higher than in the previous case but lower than in water. The yield
was about 90 %. The fact that the initial rate is lower than in water can be explain easily:
the mass transfer in a stirred single phase is higher than in the biphasic system. The total
turnover was about 80 related to coenzyme and 300 related to enzyme. The yield was satisfactory
but the catalyst could not be reused. LBADH seemed to have a limited stability in the biphasic
system. That’s why the study of its deactivation in this medium was needed.
Conversion of acetophenone to R-1-phenylethanol
80
Table 8: Result of the three conversions run in biphasic systems.
Pressure (bar) 30 100 200
Temperature (°C) 30 30 30
Yield (%) 93 Not determined 85
Enantiomeric excess (%) >99 >99 >99
µmol/min 5 3.6 3.3 Initial rate
µmol/(min/mgE) 0.2 0.15 0.13
Total turnover/ tetramere 320 Not determined 285
Total turnover/ active sites 80 Not determined 80
Total turnover/ coenzyme 100 Not determined 93
3.2.4. Deactivation of LBADH
The deactivation of LBADH was measured at 4 different conditions, in a water solution, as
a native powder at atmospheric pressure and in dense propane, finally in the biphasic system
water/propane.
3.2.4.1.Deactivation of “untreated” LBADH
The deactivation of LBADH was measured in an aqueous phase and at atmospheric
conditions. The plot of the deactivation of the enzyme, the remaining activity according to the
incubation time is presented in Figure 39 and Figure 40 , respectively.
Figure 39: Deactivation of an aqueous
solution of LBADH at atmospheric
pressure and 36°C.
Figure 40: Deactivation of LBADH in
powder form at atmospheric conditions.
Conversion of acetophenone to R-1-phenylethanol
81
The half-life of the enzyme in a phosphate buffer (pH = 7) was about 1.5 hours at 36 °C.
When lyophilised LBADH powder was incubated at atmospheric conditions a fast deactivation
occurred at first but its activity remained almost constant at 6 U/mgenzyme after 20 hours at 30 °C.
The activity decreased fast at 40 °C and, after 7 hours, more slowly. The deactivation is faster
when the enzyme is dissolved into water than when treated as a powder. Solvation in water gives
enzymes more plasticity and LBADH can unfold and deactivate more rapidly. Lack of water
renders its unfolding harder (Fágáin 1995), the good stability LBADH possesses as a solid is
exploited for storage and gas phase processes.
3.2.4.2.Deactivation of LBADH in propane
Figure 41: Deactivation of the preparations
of LBADH in propane at 30 bar.
The deactivation of LBAHD in
propane is shown in Figure 41. The trend of
this deactivation was similar to the
deactivation at atmospheric condition. A fast
decrease took place at first and was followed by a quasi steady remaining activity. This special
phenomenon was observed when lyophilised LBADH preparation was incubated at atmospheric
pressure (Figure 40) or in dense propane (Figure 41). After a fast deactivation, a portion of activity
remained. Such a residual activity is usually explained by the presence of different enzyme
populations with different stability. The work by Rees and Halling seems to provide a good
explanation for the presence of the residual activity (Rees et al. 2001). They elegantly demonstrated
that “the lyophilized powders contain different populations of protein molecules. Some are
relatively exposed, whereas others are protected by contact with other protein molecules and/or
other components of the biocatalyst powder”. Enzymes inside the particles are less sensitive to
deactivation than those on surface. Finally, the most relevant to the description of stability of non-
immobilised LBADH is the slope at the beginning of deactivation test and not the residual activity.
We noticed that LBADH (at the surface of the lyophilised powder) is prone to deactivate whether it
is in dense propane or at atmospheric conditions.
3.2.4.3.Reaction in biphasic systems propane-water
The deactivation of the enzyme was measured in the biphasic system water/dense propane
(Figure 42). The half-life of an aqueous solution of LBADH in phosphate buffer in contact with
dense propane was 6.5 hours at 29°C, and 0.2 hours at 35°C. At 40°C, the second sample of the
Conversion of acetophenone to R-1-phenylethanol
82
enzyme solution taken after 25 minutes presented no activity. When comparing with the result
obtained in aqueous phase we conclude: the stability of the enzyme in an aqueous buffer was
lowered when propane was added. The enzyme was not only deactivated in an aqueous solvent but
also at the interface with propane (Halling 1994). The deactivation of LBADH in biphasic systems
at 4°C was the subject of another study and half-life from 1.5 hours with dicloromethane up to
2000 hours in tert-buthyl methyl ether. Indeed, the stability was enhanced by this solvent.
Cyclohexane and nonane gave half-life of 4 and 9 hours, respectively (Villela Filho et al. 2003).
This is comparable with the fast deactivation in water/propane medium. The deactivation of ADH
at the interface between aqueous solution and organic solvent is usually fast and a method for
overcoming it is the use of membranes that prevent the contact between enzyme and organic
solvent (Kruse et al. 1996).
Figure 42: Deactivation of an
aqueous solution of LBADH in a
biphasic system with dense
propane at 30 bar.
3.3. Conclusion and future work
Protocols for the testing of the potential of ADHs in dense gases were successfully
developed and applied to LBADH and the conversion of acetophenone to R-1-phenylethanol.
Synthesis and deactivation determination were done in propane, enzyme and coenzyme being co-
immobilised, and in biphasic system water/propane where enzyme and coenzyme are solubilised in
an aqueous phase. LBADH presented a high enantioselectivity but also a fast deactivation in those
media.
Due to this fast deactivation LBADH is probably not a good candidate for performing this
bioconversion industrially. However protocols are ready and other ADH could be tested such as
ADH T or HLADH which stability was shown to be higher in organic solvent.
Resolution of (±)-trans-1,2-cyclohexanediol
83
4. Resolution of (±)-trans-1,2-cyclohexanediol via the formation
of diastereomeric complexes with (+)-tartaric acid followed by
extraction with supercritical carbon dioxide.
4.1. Materials and methods
4.1.1. Materials.
(+)-Tartaric acid (>99.5%, ref. 251380), RacCHD (>96%) was provided by Sigma-Aldrich
Corp. RacCHD (>96%, ref. 29005) and SSCHD (>99% (sum of enantiomers), ref. 29003) were
provided by Fluka. RRCHD (>99%, 421790) was provided by Aldrich. Pure ethanol was provided
by Reanal Ltd, (Budapest, Hungary). Hungary. CO2 (99.5 w/w% pure) was supplied by Linde Ltd.
Perfil 250™ (expanded and milled perlite for use as a filtering aid with specific surface area of 2.9
m2/g) was kindly given by Baumit Co.
4.1.2. Determination of the structure of the co-crystal.
The transparent monocrystals of the co-crystal (1) of TA and (R,R)-CHD were grown by a
slow evaporation at room temperature of an equimolar solution of TA and (R,R)-CHD in water and
ethanol (1:1). The measurement and the calculation of the sructure that lead to the resolution of the
structure were done by Petra Bombics and are presented in annex 7.4.
4.1.3. Supercritical fluid extractor.
The supercritical fluid extractor is presented in Figure 43. A more detailed presentation of
the plant was given in (Simándi et al. 1998). The extractions were run at a semi-preparatory scale
(gram-scale) with extraction pressure from 10 to 20 MPa (25 MPa being the pressure limit of the
equipment). The separator pressure was set 4 MPa and at the temperature of 40°C. Both extractor
and separator had a volume of 25 mL and were thermostated via their heating jackets where
thermostated water circulated. The liquid carbon dioxide at -10°C was supplied to the constant flow
diaphragm Lewa® pump via the by-pass vessel. There were two working modes for the extractor:
in the stand-by mode CO2 remained in the closed by-pass circuit that included the by-pass vessel,
the cooler and the pump (the fluid circulates according to the blue arrows on Figure 43). When the
extraction was due to start, the by-pass valve was closed and the extractor valve opened: this is the
extraction mode. The direction of the fluids in the extraction mode is indicated by the orange
arrows. After the extractor valve the liquid CO2 was heated up to the desired extraction temperature
and, consequently became supercritical if the temperature was higher than Tc. The CO2 expanded.
The pressures inside the extractor and inside the first separator were manually controlled by
Resolution of (±)-trans-1,2-cyclohexanediol
84
playing on the control valves 1 and 2. The pressure dropped dramatically at the outlet of the control
valve 1: the separator pressure was set below Pc and CO2 is gaseous. Most of the extracts is
insoluble in gaseous CO2 and precipitated in the separator, mostly, and in the pipe between the
control valve 1 and the separator. The expansion of CO2 imposed the thermostating of the valve
and the heating of the pipe between the control valve 1 and the extractor with a heat gun. Two
supplementary separators kept at atmospheric pressure and room temperature were added after the
main for insuring that no extract powder were released in the atmosphere (only one is represented
on Figure 43). A flow meter measured the flow of CO2 at atmospheric condition before the exhaust
pipe.
Figure 43: Supercritical fluid extractor
On this figure typical conditions of extraction are indicated. The blue arrow indicates the
recirculation of the fluid in the bypass when the extractor is in the stand-by mode, whereas
the orange indicates the direction of the flow of CO2 during an extraction.
The plant involving high-pressure, risk assessment was a permanent concern while
conceiving and using the plant. The temperature of the extractor and the separator cannot exceed a
temperature set on the water thermostat. The equipment was tested by the providers to support a
pressure higher than 25 MPa. The pressure is maintained below 25 MPa by safety blow disk that
burst above this pressure. Two are placed on the plant. The first is in the bypass and in case the
pressure after the pump exceed 250 bar it bursts and the liquid CO2 is released into the bypass
vessel that buffer the pressure. The second is placed before the extractor. The SCCO2 would be
released directly to the exhaust pipe in case the pressure exceeds 25 MPa. This might happen in
case the extractor or the control valve or the pipe between the extractor and the separator get
Resolution of (±)-trans-1,2-cyclohexanediol
85
cogged with precipitated extract, for instance or if the operator is too slow to open the control valve
1 in case of increasing pressure.
4.1.4. Resolution of CHD with TA and SFE
The resolution of CHD by formation of a cocrystal with TA followed by supercritical
extraction consists in three steps: the sample preparation, the supercritical fluid extraction and the
raffination by an alkaline treatment. The general principle of an extraction was described in 2.3.4.
4.1.4.1.Sample preparation.
For a typical sample due to be extracted by SFE, i.e the sample in this work (unless the
contrary is stated) and also the samples used in previous works (Molnar et al. 2008; Molnár 2009),
were prepared according to this protocol: 1g of CHD was dissolved in 15 mL ethanol and 0.646 g
TA, as well. These two solutions were mixed and then 1.5 g Perfil 250tm might be added to it.
Ethanol was evaporated at 40-50°C in a rotoevaporator at a reduced pressure of about 160 Torr and
the solid was scratched out from the round-bottomed flask with a spatula and let to dry out
overnight at room temperature and atmospheric pressure in an open Petri dish.
The samples of the binary mixtures of TA and enantiomeric CHDs were prepared by
evaporation of the solvent from the ethanolic solutions of CHD and TA at reduced pressure (about
160 Torr) and 40-45°C when no other condition is specified.
4.1.4.2.Supercritical fluid extraction.
The extractions performed on a sample as described below were carried out at pressure
from 100 to 200 bar and temperature from 33 to 63°C with an average flow of CO2 of about 20
g/min and the total weight of CO2 was 470 g. It was checked by a second extraction in the same
condition but with 180 g of CO2 that the “extractable” part of the CHD which mostly corresponds
to the uncomplexed (S,S)-CHD had been extracted. Indeed only a small amount, inferior to 20 mg,
was extracted in this second step. When other conditions are applied to the extraction they are
mentioned.
The yield of extraction of Yext was calculated as the loss of weigh of extractor during the
considered extraction divided by the weight of CHD initially put into the extractor. In order to
allowg a comparison between the different extractions, the amount of CO2 is given as the weight of
CO2 divided by the weight of CHD contained initially in the extractor. This value is CO2rel defined
as:
Resolution of (±)-trans-1,2-cyclohexanediol
86
Equation 14: definition of the relative weight of CO2 during an extraction
ww
initial
CHD
CrelCO 022
4.1.4.3.Raffination by alkaline treatment
A schematic presentation of the alkaline treatment of the residuum, the remainder of the
sample after extraction, is shown on Figure 29. The raffinate was removed from the extractor and
40 ml of methanol was added to it, in order to dissolve the remaining diastereomeric complexes.
After 1 h of stirring, the inert support was filtered and the methanol was evaporated at 40-50 °C in
30 mbar vacuum. The complex was decomposed by a saturated aqueous solution of Na2CO3 (6 ml)
during the stirring for 15 min. The water then was evaporated from the residue at 70–80°C in
30 mbar vacuum. The solid particles were crushed and 20 ml of trichloromethane was added to it.
After 30 min of stirring, the sodium-tartrate salt was filtered out and the organic phase was
evaporated. The product obtained contains the RRCHD in excess.
The yield of raffination Yraf was calculated as the weight of CHD isolated after the
rotoevaporation of chloroform divided by weight of CHD initially put into the extractor.
4.1.5. Analytical methods
The GC analysis were done with an Agilent 4890D chromatograph using Hydrodex-β-6-
TBDPM column (25 m × 0.25 mm × 0.25 µm film with permethylated β-cyclodextrin, Macherey &
Nagel, No.: 21519/11). The analysis was performed at isotherm conditions (130 °C), the carrier gas
was helium, the split ratio 1:50, detector: FID at 250 °C, injector temperature at 250 °C.
FTIR spectra of cocrystal (1) was measured by Excalibur Series FTS 3000 (Biorad) FTIR
spectrophotometer in KBr between 700 and 4000 cm-1.
Powder X-ray diffraction patterns were recorded with a PANalytical X’pert Pro MDP X-
ray diffractometer using CuKα and Ni filter.
Differential scanning calorimetry (DSC) measurements were performed using a Modulated
DSC 2920 device (TA Instruments). The samples (1-2 mg) were measured in sealed Al-pans at a
heating rate of 10 K/min. Simultaneous thermogravimetry and differencial thermal analysis
(TG/DTA) tests were conducted using an STD 2960 Simultaneous TG/DTA equipment (TA
Instruments), a heating rate of 10 K/min, open Pt crucibles and an air purge of 130 ml/min.
Resolution of (±)-trans-1,2-cyclohexanediol
87
4.2. Results and discussion
4.2.1. Characterisation of the cocrystal
4.2.1.1.The structure of the co-crystal.
The crystal system of co-crystal 1 is orthorhombic, the space group is P212121 (No 19),
having one TA and one CHD molecules in the asymmetric unit (Z=4, Z’=1) (Figure 44). Detailed
crystallographic data, beyond the parameters of data collection, structure determination and
refinement are presented in Table 9. The orientation of the molecule is given by RRCHD and
(2R,3R)-(+)-tartaric acid which were the starting material. Both alcoholic oxygen atoms in the
CHD are in equatorial positions. The hydrogen atomic positions of the alcoholic and acidic OH-s
were determined by difference Fourier calculations (for measurement and calculation see 7.4)
Figure 44: ORTEP diagram (Spek 2003) of the CHD-TA co-crystal (1)
Represented at 50 % probability level, heteroatoms are shaded. The chiral centres are C2
R, C3 R, C21 R and C26 R, respectively.
Resolution of (±)-trans-1,2-cyclohexanediol
88
Table 9: Summary of crystallographic data, data collections, structure determination and
refinement for CHD-TA co-crystal (1).
Formula C6 H12 O2, C4 H6 O6
Formula Weight 266.24 Crystal System Orthorhombic Space group P212121 (No. 19)
a, b, c [Angstrom] 6.7033(13) 7.2643(16) 24.863(5) V [Ang**3] 1210.7(4)
Z 4 D(calc) [g/cm**3] 1.461 Mu(MoKa) [ /mm ] 0.128
F(000) 568 Crystal Size [mm] 0.20 x 0.20 x 0.20 Temperature (K) 295
Radiation [Angstrom] MoKa 0.71073 Theta Min-Max [°] 3.2, 26.4
Dataset -8: 8 -9: 9 -31: 31 Tot., Uniq. Data, R(int) 25226, 2468, 0.121
Observed data [I > 2.0 sigma(I)] 2216 Nref, Npar 2468, 170 R, wR2, S 0.0640, 0.1369, 1.05
Max. and Av. Shift/Error 0.00, 0.00 Flack x -0.5(18)
Min. and Max. Resd. Dens. [e/Ang^3] -0.26, 0.28 * w = 1/[\s^2^(Fo^2^)+(0.0222P)^2^+1.1376P] where P=(Fo^2^+2Fc^2^)/3
The two moieties of the co-crystal contain six donors and eight acceptors of hydrogen
bond. Thus a rather complex hydrogen bonding pattern (Table 10) is constructed in the crystal
structure (Figure 45). A two dimensional sheet is formed in the ab crystallographic plane with the
width of c/2. The 2D hydrogen bonded sheet is like a “double sandwich”. The inner part is
constructed from TA-s (Figure 46). It is “covered” on both upper and bottom side by CHD-s
(Figure 45a and b). Thus the inner part is hydrophilic, the outer coat is hydrophobic.
Resolution of (±)-trans-1,2-cyclohexanediol
89
Table 10: Intermolecular interactions in the crystal structure of CHD-TA co-crystal (1).
D-H...A D-H (Å) H...A (Å) D...A (Å) D-H...A (°) symmetry operation
O2 -H2O ...O12 0.8200 2.3000 2.615(3) 104.00 Intra O3 -H3O ...O41 0.8200 2.3800 2.707(3) 105.00 Intra O12-H12O...O21 0.8200 1.7700 2.593(3) 176.00 Within asym unit O26-H26O...O11 0.8200 2.1300 2.923(3) 162.00 Within asym unit O2 -H2O ...O26 0.8200 2.1000 2.873(3) 157.00 x,-1+y,z O3 -H3O ...O41 0.8200 2.0200 2.740(4) 146.00 1/2+x,-1/2-y,-z O21-H21O...O3 0.8200 1.9700 2.786(3) 171.00 1/2+x,1/2-y,-z O26-H26O...O41 0.8200 2.5000 2.882(3) 109.00 1/2+x,1/2-y,-z O42-H42O...O26 0.8200 1.9900 2.773(3) 160.00 -1+x,-1+y,z C2 -H2 ...O21 0.9800 2.3900 3.280(4) 150.00 -1+x,y,z
There are two intramolecular hydrogen bonded loops in TA stabilizing the conformation of
the molecule: …O12-C1-C2-O2-H2O… and …O41-C4-C3-O3-H3O… both are S(5) by the graph set
analysis (Grell et al. 2000). Within the asymmetric unit the two constituents are hydrogen bonded
forming a R22(9) homodromic loop: …O11=C1-O12-H12O…O21-C21-C26-O26-H26…. A further
hydrogen bonded loop exists between CHD and TA: …H21O-O21-C21-C26-O26-H26O…O41=C4-
C3-O3… which is heterodromic R22(10). In the inner part of the “sandwich” the TA molecules are
connected by strong intermolecular interaction to each other (Figure 46) and to CHD molecules.
There is no hydrogen bond between CHD molecules. One TA molecule within the sheet is
connected to four other TA-s, to three TA-s directly, to one TA via a CHD. One TA molecule is
connected to four CHD molecules with six hydrogen bonds. The hydrogen bond loops are: …H3O-
O3-C3-C2-O2-H2O…O26-H26…O41… R23(9) and …O41-C4-C3-O3-H3O…O41=C4-O42-
H42…O26-H26… R33(11). Finally, there is a weak C-H…O type interaction also, C2-H2…O21 within
the sheet.
Resolution of (±)-trans-1,2-cyclohexanediol
90
A
B
C
Figure 45: The two dimensional infinite hydrogen bonded plane of the CHD-TA co-crystal
(1).
View from the a, b crystallographic axis, respectively, are side views, while view from the
c crystallographic axis is a perpendicular view to the sheet. TA is coloured red, while CHD
is blue. Hydrogen atoms are omitted for clarity(Macrae et al. 2006).
Resolution of (±)-trans-1,2-cyclohexanediol
91
Figure 46 : The inner TA layer of the sheet
(Macrae et al. 2006) presenting its
hydrogen bonding system of co-crystal 1.
View from the c crystallographic axis.
Hydrogen atoms are omitted for clarity.
The experimental powder X-ray diffraction pattern of sample with 1:1 molar ratio of (R,R)-
CHD and (R,R)-TA has been checked with comparison with the simulated powder diffraction
pattern of co-crystal CHD-TA (CoC) generated from the single crystal data presented above: the
agreement was good as shown on Figure 47.
Figure 47: Theoretical (in black) and
experimental (in red) diffraction pattern of
CoC
4.2.1.2.Characterisation of the co-crystal TA-RRCHD
The FT-IR spectrum of the co-crystal (1) has been measured and presented an interesting
feature (Figure 48). A splitting of the single carboxyl carbonyl C=O stretching vibration (at 1740
cm-1) of pure TA, occurred in to two C=O bands at 1738 and 1698 cm-1 in the spectrum of co-
crystal 1. The former band (1736 cm-1) belongs certainly to the C4O41O42H42 carboxylic group
despite its carbonyl oxygen O41 is involved in three of relatively strong hydrogen bonds as
acceptor, nevertheless its H12 proton is kept also relatively strongly, based on donor – acceptor
Resolution of (±)-trans-1,2-cyclohexanediol
92
distances (Table 2). The latter band (at 1698 cm-1), meanwhile, should belong to C1O11O12H12
carboxylic group, whose O11 oxygen is involved only in a weak hydrogen bond, and the H12
proton is loosely kept and very intensely shared with O21 oxygen of O21H21 hydroxyl group of
CHD. The quasi-anionic feature (which stabilized with strong intramolecular hydrogen bond of
O12 to O2 of O2H2 hydroxyl group) resulted in large decrease of carbonyl frequency.
Figure 48: FTIR: spectrum of the CHD-TA co-crystal Coc.
The DSC analysis of CHD-TA co-crystal (1) in sealed Al-pan showed a melting point at
133.2°C and its enthalpy of fusion was 56.7 kJ/mol.
Figure 49: DSC melting peak of the pure
CHD-TA co-crystal CoC in sealed Al-pan at
10°C/min (mass 2.59 mg).
Resolution of (±)-trans-1,2-cyclohexanediol
93
Figure 50: Simultaneous TG/DTA curves of the
pure CHD-TA co-crystal 1
(open Pt crucible, air flow of 130 ml/min, heating
rate 10°C/min, mass 8.68 mg).
In an open Pt crucible of the simultaneous
TG/DTA apparatus, the co-crystal 1 shows the same
melting point (133.2 °C). Mass loss of 3 % in the TG
curve shows some sublimation of CHD from 100°C to
the melting point. A further evaporation of CHD seems
to be overlapping with the decomposition process of
tartaric acid above 170°C.
Remark: Growing crystal from mixture of SSCHD and TA gave monocrystal large enough to be
submitted to single crystal X-ray diffraction measurement. Only SSCHD enantiomer and TA
crystals were found.
4.2.2. Decomposition of the CoC in situ
It was shown previously many disadvantages of the technique of decomposition of the
residuum of the extraction that consists mostly in CoC and perfil (this will be demonstrated in
4.2.3.1). It was intended to decompose the cocrystal “in situ” in a two step extraction. The first step
is exactly the same as in 2.3.4 and follow by the second step that is run at higher pressure and
temperature. The idea was that at a temperature and pressure that are high enough CoC will
decompose by releasing RRCHD that is extracted by CO2, TA being insoluble in SCCO2. A
cocrystal involves less strong interaction between the two molecules than a salt. That’s why such a
method of decomposition seemed possible. Also an experiment in a view cell (cf Figure 30)
allowed observing the behaviour of a monocrystal of CoC in SCCO2. At 93°C and 20 MPa the
shape of the crystal was changing. This indicates that the RRCHD is extracted from it and that TA
forms from CoC(this was demonstrated by XRD).
22
SCCOSolidSCCO RRCHDTACoC
Equation 15: decomposition of CoC in SCCO2.
Experiment in the view cell was a visual proof for the reaction written above and it was
decided to test this by an extraction. This was proven possible by a preliminary work at an
Resolution of (±)-trans-1,2-cyclohexanediol
94
extraction in two steps (curve 1 in Figure 51) : the sample was prepared in the condition of 4.1.4
but with 50% more CHD, TA, perfil and ethanol (solvent), ie in the molar ratio 0.5 with 1.5 g of
CHD. The first extraction step was run at 33°C and 100 bar. Y1 was equal to 43% with ee=58%.
After taking the sample and rinsing the pipes the extraction (second extraction step) is continued at
a higher pressure and temperature, 20 MPa and 93°C. The yield of the second extraction (Y2) is
calculated as Y1 is. Y2 was found equal to 52% and ee2=54%. This result was promising in the
sense that the overall yield of extraction is high (95% whereas it is about 85% with the raffination
step) even if the ee2 is lower than result found with the raffination method described before.
Figure 51: Extraction curves at different temperature and pressure, study of the
decomposition of CoC in situ.
This experiment was reproduced but with an extraction performed at 15 MPa and 48°C cf
Figure 51 curve 2). Y1 was equal to 52%. The second extraction step was performed at temperature
and pressure of 95°C and 200 bar, both the maximum values possible within the safety range of the
equipment. The result was very good: Y2=43% and eeext2=91%. This method of decomposition is a
clear improvement. The Y2 is higher than Yraf and eeext2 higher than eeraf. Consequently, in this
case a higher F parameter is obtained F=0.68 (while in the middle point F=0.54±0.025). The fact
Yext2 is higher than Yraff. The first reason is that the raffination of the residuum had probably
quite a low yield, ie much CHD is lost over the process. The second is that in the case of the
Resolution of (±)-trans-1,2-cyclohexanediol
95
raffination by alkaline treatment Yraf is calculated on the base of the weight of CHD isolated
contrary to the Yext2 which is calculated the same way as Yext, based on the decrease in weight of
the extractor. This is a “maximised” yield because it does not include all the lost of extract inherent
to any extraction process and so does not represent the quantity of CHD isolated. ee2 is higher than
eeraf probably because the sublimation of CHD might be stereoselective and induce a variation of
ee. Indeed, the raffination includes an evaporation of water at reduced pressure and temperature as
high 80°C and CHD is known to sublimate easily. It was discovered that many substances’
enantiomeric excess varies during sublimation(Fletcher et al. 2007). The sublimate usually presents
a higher enantiomeric excess and, consequently, the residue’s ee decreases. The reason might be
that the excess of enantiomer crystallises in a form that retains the molecule less by comparison to
the racemic. Another explanation is that the molecule in the gas phase forms clusters which are
more or less stable if the molecule presents the same orientations or not (Borho et al. 2001; Perry et
al. 2007). 1,2-diols structure is favourable to the formation of dimers which stability is gas phase is
different if they are formed of twice the same enantiomers (RR and RR or SS and SS) or of one
another (RR and SS). (Interestingly, this might be another explanation for the origin of asymmetry
in life: amino acid brought by meteorite would present an enantiomeric enrichment by sublimation
in the atmosphere.)
The second step of the extraction that corresponds to the decomposition of CoC was tested
at 20 MPa and two other temperatures, 73 and 83°C, see extraction curves 3 and 4 in Figure 51.
The extraction took place at a very similar rate in those two last cases whereas at 93 °C the
extraction was completed with less solvent. Both extractions were achieved in high yields, 96 %
and >99 %.
The advantages of SCCO2 were applied here for the decomposition of CoC in situ. The
insolubility of TA in CO2 is an advantage here again: because other solvent, as chloroform or
water, are able to decompose CoC but they also dissolve a part of TA. High yield were obtained.
The ee of the second extract are rather high but the eeext are still limited. Can analytical method
explain the limitation on ee1 and ee2? Which phases are encountered during the extraction process?
Remark: Even if SCCO2 is a privileged solvent for its “greenness” (see in the Annex 7.1.3) other
were tested. The solubility of CHD is so low in hexane that too much would be necessary. Diethyl
ether and chloroform posed two problems, they presented little selectivity and they dissolved a
fraction of TA.
Resolution of (±)-trans-1,2-cyclohexanediol
96
4.2.3. Description of the extraction
4.2.3.1.Monitoring the evolution of the content of the
extractor by XRD and fractionning
The different constituents of the material present in the extractor are distinguishable by
XRD but their precise quantification is not feasible and amorphous phases are invisible. The
experimental diffractograms of the different pure substances are given in Figure 52.
We wanted to take advantage of XRD for understanding what occurred during an
extraction better. This experience consisted in taking sample of the material partially extracted to
see which phase got dissolved and taken away from the extractor with CO2. The extraction curve is
plotted in the Figure 53 at each points of the extraction where a sample is indicated on this figure
the extractor was dismantled, the material left into the extractor was mixed and consequently
correspond to the average content of the extractor and 2 samples were taken in order to limit the
problem due to sampling. They were similar in every case this is why only one diffractogram was
presented in Figure 55 for each sampling. As the samples taken for XRD were a significant part of
the material to be extracted, some CHD is not extracted, lost with the sample for XRD and the
aspect of the extraction curve is modified, actually flattened at the end. That’s why a corrected
curve was plotted (Figure 54) that shows the course of the extraction as if no sample had been
taken.
Resolution of (±)-trans-1,2-cyclohexanediol
97
Figure 52: Experimental powder pattern of the compound involved in the resolution
system.
ANA 12 is defined in Table 18 of the annex 7.6. It presents the peak of the “X” compound
whose case is treated in 4.2.4.3.
Figure 53 : Loss of
weight of the extractor
according to the weight
of CO2 and sampling.
The spline line is only
indicative.
Resolution of (±)-trans-1,2-cyclohexanediol
98
Figure 54: Theoretical
loss of weight of the
extractor if no sample
had been taken. This
figure does not show
more information than
the previous but has
the advantage to show
which aspect the
extraction curve would
have if no sample had
been taken.
The sample to be extracted is done as in 4.1.4, mr=0.5 but with 1,5g CHD. One initial
sample was taken: Sample 0 from the material to be extracted. In a first step, the sample was
extracted with CO2 at 200 bar and 33°C. This corresponded to the best extraction condition as
demonstrated in the previous work (Molnar et al. 2008). 2 samples were taken, the first (Sample 1’)
at the bottom of the extractor where CO2 had entered the extractor first, as it went up in it and the
other after mixing the content of the extractor (Sample 1). After reassembling the extractor and
pursuing the extraction another sample, Sample 2, was taken the same way as Sample 1. A last
sample was taken for the first step of the extraction when the decrease in weight of the extractor,
that is equivalent to the quantity of extracted CHD, was smaller than 20 mg CHD for 200g of CO2:
it corresponded to the end of the first step of the extraction.
The complex was decomposed by a second extraction as in 4.2.2 (95°C and 200 bar). Two
samples were taken in the course of this extraction, the first in its middle of the second extraction
and the other at the end when 360 g of CO2 extracted less than 10mg of CHD.
Resolution of (±)-trans-1,2-cyclohexanediol
99
Figure 55: Diffractograms of the different samples from the material inside the extractor
over the extraction.
The origin of the most important peaks is indicated. When two peaks cannot be
distinguished both origins are given.
The analysis of the sample results allowed a good understanding of the phenomenon taking
place over the extraction. The different diffractograms are shown in the Figure 55, where the
substances responsible for the different peaks are indicated.
The initial material (sample 0 before extraction) contained enantiomeric CHD, (S,S)-CHD,
the cocrystal plus racemic CHD and traces of other material. The presence of racemate indicated
that the cocrystallisation of TA with CHD was partial only because a total cocrystallisation would
lead to only CoC and SSCHD (enantiomer). Consequently, the preparation of the sample is an
important factor that lowered the ee of the first extract and will be treated in more details later.
Over the first extraction step the enantiomer, SSCHD, and the racemate were dissolved and
withdrawn by CO2 as the progressive decrease in the intensity of their peaks indicated. In the last
sample, sample 3, the peaks referring to the enantiomer and the racemate had completely
disappeared. SSCHD could not be distinguished from racemate by extraction as shown by the
sample 1’ at the bottom of the extractor whose diffraction pattern was similar to Sample 3’s (except
Resolution of (±)-trans-1,2-cyclohexanediol
100
there is less TA) that was fully extracted at the beginning of the extraction already. The difference
in solubility between the liquid solution of racCHD and of the enantiomer was not important
enough to allow a separation of those two phases by extraction.
Very little TA can be seen in the sample 0 and the quantity of TA increased over the
extraction. At the end of the first extraction, after the enantiomer and the racemate are fully
extracted, the cocrystal decomposed slowly, leaving more TA which cannot be extracted due to its
insolubility in CO2. The decomposition of the cocrystal continued in the second step of the
extraction faster because of the higher temperature, as it was shown in Figure 51. Indeed, the
amount of TA increased from sample 3 to sample 5 while the cocrystal disappeared until
completion for sample 5 that is actually only composed of TA and perfil. So the rest of a resolution
of CHD by cocrystallisation and SFE can be recycled for a new resolution: the quantity of waste is
considerably low and the resolving agent does not require any expensive regeneration.
Thus the decomposition of CoC started from the first extraction steps on and this can be
compared to the first experiment of decomposition in two steps in 4.2.2 (see Figure 51). During the
first extraction step fractions, whose ee was measured, were taken. The ee are given on Figure 56,
curve 2 and 5. The trend followed what we found by XRD: at the beginning mostly free CHD was
extracted. The ee’s of the fractions taken during the first extraction (excess of SSCHD) decreased
when most free CHD had been extracted because the contribution in RRCHD by the decomposition
of CoC was more important. The extraction of free CHD as racemate or pure enantiomer seemed
total according to the diffractogram as we cannot see their peaks any longer in Sample 3. However,
an incomplete extraction of free CHD in the first extraction cannot be excluded based on XRD.
Indeed the sensibility of this method is limited and only the compounds at the surface of the
powder diffract X-rays are seen; so crystals of free CHD covered up by CoC or TA or trapped into
Perfil pores are likely to be unseen and to be last portion of free CHD to be extracted.
To summarise, the ee of the first extracts was relatively low due to two phenomena. The
first is the only partial formation of cocrystal, some TA remained not cocrystallised, as well as
RRCHD that forms racemate with SSCHD. The second is the slow decomposition of the cocrystal
that was the most visible at the end of the first extraction but probably occurred all over in different
proportions.
Remark: there is a difference between the evolution of the ee of curve 2 and 5. In the first case the
ee did not simply decrease but increased and then decreased. The explanation for such an evolution
might be a fine difference if the solubilities of the two free CHD species: SolCHD and SSCHD.
Resolution of (±)-trans-1,2-cyclohexanediol
101
Figure 56: Different fractions during extraction, their enantiomeric excesses
Curve 2 and 3 were fully presented in Figure 51. Only the fraction of the curve where ee
were measured are given. In the case of the extraction 3 the first step of extraction lead to
an enantiomeric excess of 63 %. The ee indicated for certain segment of the extraction
curve corresponds to the ee of this fraction.
The second extraction step has shown good results because the whole cocrystal was
extracted and only TA was left at the end of it (Sample 5). The limitation of the ee of the second
extraction step at about 90% can have several reasons.
A first explanation is that a small amount of free CHD unseen with XRD had not been
completely extracted at the first extraction or, from another point of view, the impurity of SSCHD
found in the second extraction are due the incompleteness of the first extraction. The ee increase
over the second extraction step because the rest of free CHD (mostly RRCHD) is mostly extracted
at the beginning of the second step.
A second extraction is that some other cocrystal is formed between SSCHD and TA or the
co-crystal could also intake a small amount of SSCHD in addition to RRCHD i.e. CoC forms a
solid solution, possibly a lattice compound where a little proportion of RRCHD can be replaced by
SSCHD. This happens very often in the case of diastereomeric salt and a common limitation to this
resolution method (Jacques et al. 1981; Kozma 2002). The reason for the variation of ee over the
Resolution of (±)-trans-1,2-cyclohexanediol
102
second step could be the following: The rest of unextracted free enantiomer is extracted before the
complete decomposition/extraction of CoC than at lower temperature and pressure or the fact that a
CoC containing a portion of (or only) SSCHD would be less stable than CoC with pure RRCHD.
4.2.3.2.Improving the enantiomeric excesses by leaving
off an intermediate fraction
The evolution of the ee over the extraction indicates that it is lower at the end of the first
extraction and at the beginning of the second extraction. That’s why it was intended to perform an
extraction similar to the previous except that the intermediate fraction between the first and the
second extract is left off. The first extraction gave a fraction rich in SSCHD whose ee1=75 % and
the second rich in RRCHD ee2=91 %. The intermediate fraction presents a sligh ee of 14 % and
accound for 15-20% of the sample. Removing this small fraction allow an improvement of the ee
but reduces Y1 and Y2 (they could not be calculated precisely.). However it should be noted that
this step does not lead to the extra waste because in an industrial process this intermediate fraction
would be recycled in another resolution as will be shown later in 4.2.5.2. However, the
improvement of ee1 is limited.
4.2.4. Sample preparation
4.2.4.1.Investigation of the binaries RRCHD-TA and
SSCHD-TA. Coroboration by XRD.
The stereoselective formation of a cocrystal is the base of this resolution. The difference in
stability between the two cocrystals of SSCHD-TA and RRCHD-TA will partially determine the
quality of a resolution: if one is much more stable only a small amount of the other is formed and
the resolution is good while if their stability is similar no resolution occurs. At this point we know
that the cocrystal RRCHD-TA is more stable than SSCHD-TA (otherwise the resolution would not
take place). The formation of a cocrystal SSCHD-TA is questionable as we could not grow a
monocrystal of it and, concerning this issue, we have investigated both binary systems between
RRTA and RRCHD or SSCHD with DSC and powder X-ray diffraction. This work served as a
background for thermal investigation of the ternary RRCHD-SSCHD-TA. This work was presented
in an article (Thorey et al.). The melting point and enthalpy of fusion of the initial chemicals
applied in preparation of binary mixtures are listed in Table 11, while the temperatures of the
observed thermal heat effect(s) and the initial crystalline phase composition of various mixtures at
room temperature are given in Table 12 for some examples.
Resolution of (±)-trans-1,2-cyclohexanediol
103
Table 11: Melting point and enthalpy of fusion of the applied chemicals
Initial chemicals
Tfus observed by DSC K, (°C)
Hfus measured by DSC (kJ/mol)
Reference Tfus (K) Ref.
(±)-CHD
372 (99) (small pre-melting
endothermic peak at 87°C, as well)
34.4 376.4 (Leitao et al. 2002)
(R,R)-CHD 381 (108) 18.0 382.5 (Leitao et al.
2002) (S,S)-CHD 377 (104) 15.0 Ibid ibid
(R,R)-tartaric acid 444 (171) (decomposes) 22.4 (estimated)
(Martin Britto Dhasa et al. 2007; Stanton et al. 2008;
Takata et al. 2008)
Co-crystal (1) 406 (133) 56.7 This work
Table 12: DSC and XRD data of binary mixtures in the ternary system
Binary mixtures Molar ratio
Eutectic temperature Teu K,
(°C)
Liquidus temperature Teu K (°C)
Crystalline phases present
(XRD)*
1 (R,R)-CHD: (R,R)-TA 3:1 373.3 (100.2) Ca. 396 (123) (R,R)-CHD and
Co-crystal (1)
2 (RR)-CHD: (R,R)-TA 1:3 404.7 (131.6) Ca. 433 (160) Co-crystal (1)
and (R,R)-TA
3 (S,S)-CHD: (R,R)-TA 3:1 359.7 (86.6) - (S,S)-CHD and
(R,R)-TA
4 (S,S)-CHD: (R,R)-TA 1:1 358.6 (85.5) Ca. 410 (137) (S,S)-CHD and
(R,R)-TA
5 (S,S)-CHD: (R,R)-TA 1:3 253.6 (80.5) Ca. 433 (160) (R,R)-TA and (S,S)-
CHD
The XRD profile of the samples formed from the 1:3 and 3:1 binary mixtures of (R,R)-
CHD and (R,R)-TA corresponded to the 1:1 mixtures of co-crystal (1) and (R,R)-CHD or (R,R)-TA,
respectively. These samples showed eutectic melting behavior, eutectic temperatures were found
by DSC lower than the melting point of co-crystal (1): one between (R,R)-CHD and co-crystal (1)
at 100°C (Fig. 8a); and the second between co-crystal (1) and (R,R)-TA at 131.6°C. DSC showed,
in all the cases of the binary (S,S)-CHD – (R,R)-TA system, a constant eutectic temperature of 85-
86°C. It is lower than the melting point of (S,S)-CHD and the eutectic temperature of (1) and TA or
(1) and (R,R)-CHD. This indicates that no co-crystal is formed between (S,S)-CHD and (R,R)-TA.
This result is corroborated by the XRD profile of the sample for the binary mixtures of (R,R)-TA
Resolution of (±)-trans-1,2-cyclohexanediol
104
and (S,S)-CHD that presented 1:3, 1:1, and 3:1 only patterns of (R,R)-tartaric acid (PDF No. 00-
033-1883; 00-020-1901; 00-031-1911) and (S,S)-CHD (PDF No. 02-093-3042), and no other
reflections occurred.
After the eutectic melting, an elongated dissolution of the excess phase has been continued,
as it is shown for both the 1:1 molar ratio of (R,R)-CHD and co-crystal (1) or (S,S)-CHD and (R,R)-
TA in Fig. 8a and 8b, respectively. In case of 3:1 molar ratio of (S,S)-CHD and (R,R)-TA, only the
single eutectic melting effect occurred, i.e. it represents almost a eutectic composition between
(S,S)-CHD and (R,R)-TA.
Figure 57: DSC curve of binary mixture
corresponding to 1:1 molar ratio of a) (R,R)-
CHD and co-crystal (1) and b) (S,S)-CHD and
(R,R)-TA, both exhibiting eutectic melting
behavior.
For this kind of study, DSC is very
efficient. Indeed only few scans were sufficient to
demonstrate that no cocrystal and, moreover, are
corroborated by XRD.
It is possible to represent the binary phase
diagram TA-SSCHD: see Figure 58 with the
calculated liquidus and eutectic temperature (for
detail see the related article (Thorey et al.)). The
eutectic temperature is well defined and rather constant but the liquidus is a bit low compared to
the calculated value. It can be argued that the rather high experimental value of the liquidus is due
to the fact that SSCHD sublimate easily so that a part of it is lost for dissolving TA so that the point
should be actually shifted to the left as if there was less SSCHD in the sample.
Figure 58: Melting binary phase
diagram SSCHD TA.
Resolution of (±)-trans-1,2-cyclohexanediol
105
a)
b)
Figure 59: The problematic melting point phase diagram of RRCHD-TA.
a) Binary RRCHD and TA with formation of CoC. First set of data with the interpretation
with two eutectic points.
b) More data with the interpretation with a eutectic point (RRCHD-CoC) and a peritectic
point (CoC-TA). The open circle corresponds to the end of the melting of the unique peak
of the DSC scan of CoC.
An example of DSC analysis will be given for XTA = 84% in Figure 61: DSC analysis of a
sample of composition RRCHD:TA 16:84.
A binary diagram RRCHD-TA with two eutectics, RRCHD-CoC and CoC-TA,
corresponds to the interpretation given above and is presented in the Figure 59 (a) with the
calculated liquidus and eutectic point. The value of the eutectic melting temperature of the binary
CoC-TA is very close to the melting point of pure CoC and the fitting is generally bad. An
explanation can be similar to the one given for the binary SSCHD-TA: the sublimation of CHD
causes its non interaction with TA and so suppresses the eutectic melting between TA and CoC. So
we added some points to the binary to confirm the trend. The points are given in the Figure 59 (b).
Another interpretation of the binary RRCHD-TA can be based on the presence of a peritectic point
between CoC and TA or incongruent melting of CoC rather than a eutectic. This could explain why
the first melting in the CoC-TA region equals the melting of CoC: this melting would correspond to
the decomposition of CoC. However this explanation has its flaws, especially regarding the melting
of CoC that is neat and so does not seem to take place parallely to the crystallisation of a fraction
TA followed by its dissolution into the melt. It is also possible that transitions are missing by the
kinetic of the transition that ends with an amorphous phase unable to crystallise. For instance, if the
interpretation with a peritectic point is exact it is possible that the crystallisation of TA from molten
Resolution of (±)-trans-1,2-cyclohexanediol
106
CoC is difficult and so is of a limited extend. The presence of amorphous and metastable phase will
be raised in the next part.
The thermoanalytical study allowed us to determine that no cocrystal was formed between
TA and SSCHD, while a complete knowledge of the binary RRCHD-TA revealed complicated and
subject of different interpretations. Complex issues such as amorphicity and sublimation arise.
4.2.4.2.Investigation of the cocrystallisation by XRD,
ternary phase diagram.
The goal of this study is to give some tool for the fast determination of suitability of a
sample for resolution of CHD and also to know more about the limitation on eeext. We have shown
that the limitation was of two types: the first is the slow decomposition of CoC, the second the not
total formation of CoC. The question we desire to answer here is: is the partial cocrystallisation is
due to thermodynamical reasons or rather kinetic? In case the ee is thermodynamically limited the
low ee cannot be overcome whereas if the reason is kinetic more appropriate conditions of
crystallisation can lead to improvements and effort are worse spending on this issue. The other
reason why the understanding of the phase diagram is important is to know whether a non-racemic
CHD can be resolved a second time for enantiomeric enrichment.
Examples on the analysis of diffractograms were presented in 4.2.3.1 and 4.2.4.1. The
spectrum of CoC fits with the simulated powder pattern simulated (Table 6) well. It should be
noted that the racemate has a structure that fits with the metastable racemic CHD (ZZZKPE06) and
not with the more stable structure. The racemate CHD corresponds to the liquid solution that is
formed between the enantiomers for ee<60 % according to Leitao’s work (Leitao et al. 2002) (this
will be again verified on sample ANA4 and ANA5 or ANA19 in Annex 7.6). It is possible that the
less stable polymorph of racCHD is the only which can accept an ee, this is to say that can form a
solid solution. In Sample 0 (Figure 55) the solid solution is in equilibrium with the SSCHD, so the
solid solution is saturated with SSCHD and if Leitao’s values for the limit of the solid solution is
good the solid solution contains mostly SSCHD (ee=60%) and consequently the free enantiomer
(solid solution + pure enantiomer) has an enantiomeric excess superior to 60 %. A more detailed
presentation of the solid solution that forms RacCHD is given in annex 7.5.2.1 because of it
implication for the ternary phase diagram. The phase diagram presented in Figure 60 a) is the more
favourable to the resolution of CHD by TA because CoC is formed in every part of the diagram and
the sample for mr=0.5 is composed only of CoC and SSCHD. We call this type of ternary phase
diagram “solid solution and CoC>solCHD” because it includes the formation of a solid solution
and CoC is always more stable than SolCHD. We thought that the partial crystallisation can be
Resolution of (±)-trans-1,2-cyclohexanediol
107
explained by a ternary phase diagram between TA-SSCHD-RRCHD (Figure 60 b), solid solution
and CoC=solCHDlim) where an area is found where the CoC formation is less favourable than the
formation of SolCHD . The demonstration of the theoretical existence of those phase diagrams is
presented in appendix 7.5.
a)
b)
Figure 60: Two ternary phase diagrams TA-SSCHD-RRCHD
a) “solid solution and CoC>solCHD” ternary phase diagram.
b) “solid solution and CoC=solCHDlim” ternary phase diagram.
To this purpose samples were prepared and analysed by XRD. Their composition and the
phases observed by XRD is given in Annex 7.6. ANA 8 correspond to the cocrystal grown for
powder XRD, DSC, TGDTA and IR in 4.2.1. The sample ANA20-21-22 and the sample ANA7
and 8 were used for the binaries of 4.2.4.1 (ANNA20-21-22 are similar to ANA1-2-3 but the
quality of the analytical SSCHD used in the 20-21-22 was higher and the DSC scan gave better
results.). In those case the interpretation is straightforward: TA forms CoC with RRCHD (and the
excess of TA or RRCHD crystallises by its own) but not with SSCHD as shown in 4.2.4.1. We
should remark that the proportion between the peaks of TA, enantiomer and CoC follows
proportion of the different compound reasonably well.
The interpretation of the diffractogram of the next samples was more complicated. The
formation of CoC for mixture of TA and CHD with an excess of SSCHD was difficult (ANA23-24-
25) which sees to fit better to the case of “solid solution CoC=solCHDlim” ternary phase diagram.
Mixture of racemCHD with different amount of TA shows results that might confirm this
interpretation as little enantiomer is shown (ANA 13 to 17). The samples done with CHD
presenting an excess of RRCHD and different amount of TA (ANA 10-11-12, right side of he
phase diagram) gave at first surprising results as new compound was formed, named “X”, while in
Resolution of (±)-trans-1,2-cyclohexanediol
108
the repetition of this experiment (ANA26-27-28) no X was found but CoC did not form in every
case (“X” is a metastable compound and will be treated below in 4.2.4.3). In fact, several
experiments showed that no CoC was formed in area where it was expected to form in every type
of phase diagram as ANA13-25. Actually many samples presented too low quantities of CoC
compared to what is expected as ANA14. Also many experiments present SSCHD in the region
where, in the second type of diagram, CoC is in equilibrium with SolCHD, as for sample 0 of
Figure 55 for instance. The crystallisation of the enantiomerically pure fraction of SSCHD is
actually a problem as is shown on Table 11 where SSCHD melting enthalpy, low compared to the
value found for RRCHD or in the literature, is due to a low crystallinity. Consequently, if this
interpretation is right, the samples on the line TA-racemCHD of the ternary (ANA 13 to17) present
no or little SSCHD not because they belong to the second type of ternary but rather because the
SSCHD is present as an amorphous phase. Actually the fact the cocrystallisation is mediated by a
metastable phase is a common phenomenon reported for the preparation of pharmaceuticals
(Jayasankar et al. 2006). The presence of an liquid, amorpheous phase is obvious for many samples
which are sticky and difficult to spread out for on the sample holder for XRD. Moreover at the end
rotoevaporation a vitreous phase is present from which the crystals germinates and grow slowly.
The cocrystallisation might be impeded by the high viscosity of this amorphous phase. For all those
reasons we think the formation of CoC is prevented because the kinetic of its growth is smaller
than TA or SolCHD’s and the liquid phase remains amorphous, as will be shown below. Other
proofs of the hypothesis stating that the limited formation of CoC is due to kinetic problems and
not thermodynamical problems will come from the result of the last part of this chapter. During the
preparation of a sample containing racCHD and TA in mr=0.5 we could isolate a fraction of
amorphous phase and analyse it by GC: it presented an ee of SSCHD close to 50%. This finding
corroborates the hypothesis of the amorphous phase which contains the excess of SSCHD well.
The composition of this amorphous phase is close to the ternary eutectic composition that contains
also a small amount of TA: the composition of a ternary eutectic between CoC, SSCHD and
RacCHD was found equal to XCoC = 0.051 xSSCHD = 0.542 xracCHD = 0.405 (calculated by Janos
Madarasz see (Thorey et al.))
The investigation of the ternary phase diagram indicates us that the solid solution is formed
but cannot give a clear answer about the thermodynamic equilibrium as it presents difficulties to be
reached and is of the type of “solid solution CoC>solCHD” ternary phase diagram Figure 60 a)
while some “trend” of “solid solution and CoC=solCHDlim” ternary phase diagram (Figure 60 b)) is
seen because of the low difference of stability of the different species (see annex 7.6). The research
of condition of crystallisation that are the most favourable to the formation of CoC was
investigated and the results are presented below in 4.2.4.4.
Resolution of (±)-trans-1,2-cyclohexanediol
109
The study of molar ratio is generally important for the optimisation of a resolution
technique. In our case this study is particularly important for two reasons: SSCHD-TA cocrystal
does not exist, so an excess of TA should not decrease the eeext and an excess of TA might allow a
better formation of CoC. The study of the variation of the molar ratio will be done in 4.2.5.1. at the
moment 2 problems left untreated have to be considered:
4.2.4.3.Two issues raised by the XRD studies: sodium
hydrogen tartrate (NaTA) and metastable compound.
Sodium hydrogen tartrate (NaTA)
When the ethanolic solution of TA and the solution of racCHD were mixed together
precipitation occurred immediately. The first interpretation proposed was that this precipitate is
CoC because the concentration of RRCHD and TA would have been above the molar solubility of
CoC. However if TA was the compound that precipitates it might explain why TA is found in the
solid sample whereas it is not expected : TA would be not stable but precipitates faster than CoC
from the ethanolic solution and a fraction of TA would not be available to transform into CoC.
To test this hypothesis a large sample in the molar ratio 0.5 was prepared. As usual a
precipitate formed after mixing the CHD and TA solutions; it was filtrated out. The analysis by
XRD showed that, although its pattern looked liked TA it was the anhydrous salt catena-(hydrogen-
(+)-tartrato)-sodium. This was demonstrated by dissolving the precipate into distillated water, and
analysing the salt obtained after evaporation: it was hydrated hydrogen tartrato sodium.
Interestingly the solid formed after filtering out the salt and evaporating ethanol gave no trace of
TA or NaTA. We first thought that sodium impurity came from TA but using a higher grade TA
gave the same result. Eventually the sodium impurities came from the technical CHD and reacted
with TA during the formation of Coc. The equation of the reaction that took place in case if the
impurities are sodium hydroxide (only used as an example-it could be another base) is given in
Equation 16.
NaOHNaOH 6542664 OHCOHC
Equation 16
That’s why the next experiments with racCHD were conducted with racCHD extracted
with SCCO2 because SCCO2 does not dissolve salts generally. Solution of this purified CHD did
not lead to any precipitation when mixed with solution of TA. Also a resolution run with this
material instead of technical CHD gave better results: The yield (Y1+Y2) are higher simply
Resolution of (±)-trans-1,2-cyclohexanediol
110
because initially there were more CHD compared with a technical preparation. eeext are higher this
is probably explained by the fact there is more TA for forming Coc i.e. the molar ratio was superior
to the previous experiments (see 4.2.5.1). An example is given latter in Table 13.
“X” compound
A compound called “X” was found in some cases see annex 7.6. This compound is
identified without ambiguity by XRD as it possesses peak distinguishable from the other at about
2θ = 8.85, 12.2, and 16.8. The diffractogram of ANA 12 presented in Figure 55 possesses the peak
of X. The structure of this compound is not known as it could not be isolated. The metastability
was demonstrated by different methods. Sample presenting X’s peaks lost them after been heated at
temperature above 100°C. The XRD pattern of the sample CHDG (see annex 7.6) were measured
at different temperatures and X disappeared at about 90°C. Eventually, a DSC scan of a sample of
the binary RRCHD-TA (xTA=0.84) see Figure 61. X melted at about 98°C and recrystallised
afterwards. This behaviour is typical for a metastable compound (Jacques et al. 1981; Leitao et al.
2001; Leitao et al. 2002).
Figure 61: DSC analysis of a sample of
composition RRCHD:TA 16:84 featuring
the metastable compound “X”
4.2.4.4.Conditions of crystallisation/sample preparation
We showed in 4.2.4.1 that the problems linked to the preparation of the samples are of a
kinetic nature rather than thermodynamic. In this paragraph we would like to show experiments
that, first of all, confirm the fact that the limitation is of a kinetic nature and, secondly, which
conditions play important roles.
As indicated in the bibliographic review, some diastereoisomeric salts for resolution were
prepared by precipitation into the melt without adding a solvent (the enantiomeric pair and the
resolving agent were heated together until they melted). In some cases higher F parameters were
afforded and it also saves solvent. We intended it for the system of resolution TA CHD. It was
Resolution of (±)-trans-1,2-cyclohexanediol
111
impossible to prepare the CoC from the melt because of the sublimation of CHD: CHD tends to
precipitated at the top of the flask where melting was intended. Also such an operation requires
high temperature (>120°C) that could be deleterious effect on the compounds. Other solvents were
tested, water was not satisfactory because it was difficult to evaporate and lead to low yield. Less
polar solvents than ethanol could not dissolve TA. We eventually focused on ethanol because it
gave the best results, it allows a fairly good dissolution of those rather polar compounds, its
evaporation is easy and finally ethanol is also a green solvent.
The preparation of the sample was done at different temperatures, different molar ratios
and with and without perfil. Examples of diffractograms are presented on Figure 62 where the
peaks that can be focused at for a quick evaluation of the diagram are indicated. CoC being the
species responsible for the resolution its easily noticeable peak is the most important. The data are
collected in the annex 7.7 in Table 22.
It shows that a complete formation CoC where no RRCHD is left uncocrystallised (this is
to say no SolCHD) is possible as in the case of “40°C with perfil”. Another point is shown by this
study the moment of the sample preparation when the solvent is rotoevaporated is not the only
determinant step of the crystallisation: the latter evaporation of ethanol remaining after
rotoevaporation in an open Petri dish gave good results whereas sample let to evaporate in a round-
bottomed flask gave only little formation of CoC. Indeed the cocrystallisation did not occur in
some cases (55°C) and only SolCHD and TA were detected. A too slow or partial evaporation of
ethanol might be the reason. In two cases the crystallinity was really poor and surprisingly in one
case solid was mostly made of CoC and SolCHD (30°C rbf) while in the other it was SolCHD and
TA (40°C rbf). The conditions that may hinder CoC formation are not well understood, and perfil
did not seem to have a very determinant influence. The problem is a matter of difference in the
crystal growth kinetics of the different species.
Resolution of (±)-trans-1,2-cyclohexanediol
112
Figure 62: Diffractograms of sample of mr=0.5 evaporated at different temperature
Only a characteristic peak useful to the determination and evaluation of the quantity of a
compound is marked. The preparation of the sample at 40°C was done with and without
perfil.
The study of crystallinity is often done by DSC that could give quantitative results. Indeed
DSC is the technique the most used for determination of crystallinity especially in the case of
polymers. The study of the melting of samples at mr=0.5 gives different peaks that difference in
size can be explained by the fact that the crystallinity of certain phase is more or less important.
However the presence of many phases (CoC, enantiomer (SSCHD), solCHD and TA) renders this
type of analysis delicate. Sampling is difficult for this kind of sample and the present of an achiral
support as perfil will lead to smaller sample. The interpretation of the scans is difficult because
many parameters influence the size and the temperature of the peaks: quantity of uncrystalised
RRCHD, quantity of precipitated TA, sublimation of CHD, crystal size. It is also important to be
able to run the study with technical CHD for the optimisation of an industrial process and DSC is
very sensitive to the purity of the material. A DSC scan at 10°C/min (usual scan speed used over
this work) was eventually run showed that the eutectic melting was not separated well from the
Resolution of (±)-trans-1,2-cyclohexanediol
113
subsequent dissolving of CoC into it. For all those reason the study of crystallinity by DSC looks
tedious and uncertain even if we cannot assume that valuable results would not arise from such a
study.
XRD analysis has the advantage of rapidity and easiness of analysis and interpretation
without destroying the samples. This method allowed us, first of all, to validate our hypothesis of
4.2.4.2. The second indication given by XRD is semi-quantitative: the sample is the best when the
peak of CoC is the highest compared to solCHD and TA. The best results were obtained by
rotoevaporation of ethanol at 40°C followed by evaporation of ethanol overnight from a Petri dish.
It is important to remember that XRD does not show amorpheous phases and their presence is an
important source of limitation of the resolution process.
Concerning the preparation of the sample due to be extracted for the resolution of CHD
with TA, we identified the limitations on the ee1 and found empirical condition the most favourable
to a good resolution. However, the obtained extracts have ees too low for commercialisation and
should be improved.
4.2.5. Toward enantiopure products, molar ratio, double
extraction
The aim of this last part concerning the resolution is the practical use of the more
theoretical research presented above. They are based on two principles, the first is the variation of
the molar ratio that can be realised due to the fact that no cocrystal is formed between RRCHD and
TA, the second is to perform the resolution twice on the same material with (+)- or (-)-TA.
4.2.5.1.Extraction with molar ratios varying
The concept of molar ratio, nn
CHD
TAmr , was introduced in 2.1.3.4 and in the case of the
resolution of CHD by TA in 2.3.4.
The first extraction step was performed at 20 MPa and 33°C. The second was based on the
acidobasic treatment shown in 4.1.4.3. The result of these series of resolution is presented in Figure
63 and Figure 64.
Resolution of (±)-trans-1,2-cyclohexanediol
114
Figure 63: Yield and F parameter with varying molar ratio
Figure 64: Enantiomeic excess with varying molar ratio
Resolution of (±)-trans-1,2-cyclohexanediol
115
If the phase diagram of Figure 60 a) is correct, the CHD that is extracted corresponds to all
the CHD that is uncocrystalised consequently:
if mr<0.5 : mrYext 1 and mr
mreeext
1
if mr>0.5: Yext=0.5 and 1extee .
Yext-1Yraff and 1Rafee . All those equation are plotted on the graphs to allow
comparison. The theoretical Yext fits with the points well, while Yraf is a bit below the theoretical
curve. This is probably due to the loss of material over the raffination of the residuum, as stated in
4.2.2. The theoretical eeext is not so bad for small mr values but for mr>0.4 it goes wrong.
Explanations were proposed for this in 4.2.3.1. The last value of eeraf stay close to 0.8 and shows a
decrease for the small value of mr (this could fit with a phase diagram where CoC intakes some
SSCHD see 7.5.2.2) and then slightly increase maybe because of a better crystallisation (this can
also explain the increase in eeext). The point for mr=0.1 is very low and this might be due to an
uncomplete first extraction step or a matter of crystallisation.
F is increased by increasing mr. At best, it reached 0.8, which represents a good
improvement. However the ee are still too limited and a double resolution seemed necessary. The
interpretation of the resolution with varying molar ratio is enabled by the work done in the previous
part. The corroboration is good.
4.2.5.2.Resolution repeated twice
We did not manage to produce an extract with satisfying ee by a single resolution. In this
context it was intended to increase the enantiopurity by performing a second resolution performed
on 2 typical extracts obtained according to the resolution with TA: mixture 1 that presents an ee of
75% of SSCHD and mixture 2 with an ee of 85% of RRCHD.
ee2 is higher than ee1 so, the same way that TA was used to produce RRCHD of good
enantiopurity, SSTA was used to produce SSCHD: this is to say we expected to obtain SSCHD or
RRCHD of high ee from the second extract only. The further enantiopurification of SSCHD
(mixture 1) and RRCHD (mixture 2) is shown on Figure 65 and Figure 66, respectively. Different
molar ratios were applied and the results are collected in Table 13. The molar ratio is again defined
as nn
CHD
TAmr ( nnn chdrrchdsschd because nnn chdrrchdsschd 2 ) The next figures are
intended to give a visual idea of the quantity of resolving agent compared to the targeted
Resolution of (±)-trans-1,2-cyclohexanediol
116
compound. The height of the rectangles is proportion to the quantity of RRCHD and SSCHD or TA
(or SSTA).
Figure 65: Second resolution of mixture 1 presenting an ee of SSCHD
Figure 66: Second resolution of mixture 2 presenting an enantiomeric excess of RRCHD
Resolution of (±)-trans-1,2-cyclohexanediol
117
Table 13: Result of the different experiments of further enantioenrichment of mixture 1 and
mixture 2
First extraction Second extraction mr
Y1 ee1 Y2 ee2
RacCHD
ee=0 0.5 50% 87% 50% 79%
0.5 48% 52.4% 52% 96.6% Mixture 1
SSCHD ee=74.6% 0.58 42.6% 39.0% 55.3% 99.6%
0.85 14.1% 4.2% 83.8% 99.3% Mixture 2
RRCHD ee=85.0% 1 7.1% 50% 92.9% 99.2%
In every case, the extraction was complete (Y1 + Y2 close to 100%) and the targeted final
extract presented high ee, superior to 99% in three cases. The crystallisation occurred well and
when TA (or SSTA) was not in excess (last line of Table 13) the formation of CoC was nearly
complete as Y2 was close to mr. This is what was expected as Y2 roughly corresponds to the
quantity of CHD cocrystallised which is egal to the quantity of TA, as long as TA is not in excess.
If we compare those last four extractions to the extraction made with racCHD we noticed
that Xrr2, thecomposition in RRCHD in the second extract, (when TA was used) increased when
Xrr of the sample (and Xrr1 of the first extract) increased. This is the principle which the double
resolution is based on: the successive improvement of the enantiomeric excess. 2 explanations for
the increase of ee2 are given below.
Referring to the limitation indicated on the ee2 before, the two explanation invoked
precedently are both satisfying in this latter case. The first was that some free enantiomer remains
unextracted at the end of the first extraction and is recovered at the second. In the case of a
resolution where the starting material is not a racemic the remaining of the first extraction
recovered in the second would have a higher Xrr so that the obtained second extract would be
contaminated with residue less harming to the enantiopurity of the second extract: ee2 is
consequently higher. The second explanation is that CoC intakes a small amount of SSCHD. This
explanation fits well with the result. Actually equilibrium would take place between the two liquid
solutions of CHD and CoC. The equilibrium would be of that kind: when Xrr of one phase
increases Xrr of the other also does, as proposed in the annex 7.5.2.2 with the example of Figure
76. This explanation is convincing but should be handled with care as no demonstration of the
existence of CoC containing SSCHD was intended at the moment. Generally the exhibition of a
Resolution of (±)-trans-1,2-cyclohexanediol
118
solid solution is treated by DSC. This is difficult and should be handled with care because many
wrong interpretations might arise from DSC scan and precise measurement are required for
showing small variations in the melting temperature. Generally it is tedious work and coupling with
microscopic observations might be helpful. The presence of amorphous phase, metastable
compound “X” but also polymorphic forms of racCHD or enantiomeric CHD, the sublimation of
CHD render this task difficult.
A resolution repeated twice using TA and SSTA afforded ee above 99% for the preparation
of RRCHD and SSCHD, respectively, while a simple variation of mr could not. The highly
enantiopure CHD consists in the second extract. These are suitable for industrial use. But if higher
enantiopurity is required further enantioenrichment is feasible by simple fractional recrystallisation
as the ee of the obtained fraction is much higher than the eutectic’s (Jacques et al. 1981). It should
be also added that due to the feasibility of repeated resolution no CHD fraction is lost for any
fraction can be recycled in the next resolution. And the mother liquor used for recrystallisation can
be reused for preparing a new sample with TA or SSTA. The resolution of CHD is thus flexible
and the E-factor of a process of preparation of enantiopure CHD would be very low.
4.3. Conclusion on the resolution of CHD by cocrystallisation and SFE and further plan
The cocrystal responsible for the resolution was characterised and its structure resolved. A
good understanding of the resolution was afforded by analytical method. The fact that SSCHD and
TA do not cocrystallise leads to the development of resolution based on an excess of TA.
Eventually a resolution based on resolutions repeated twice brought products with high
enantiomeric excess above 99%.
Further work
The probable existence of a liquid solution of CoC that allows CoC to intake a small
amount of SSCHD was not demonstrated at the moment. The conditions of preparation of the
initial sample are determinant and seem an interesting matter but the study seemed also very
difficult. However, we have shown that a second resolution overcomes the limitation of a one-step
process.
Beyond those two theoretical issues, it should be emphasised that the resolution of CHD by
TA using SFE afforded almost enantiomerically pure CHD in a very competitive way: no
expensive reagent is needed, the resolution agent is completely recovered, CHD is never wasted as
Resolution of (±)-trans-1,2-cyclohexanediol
119
it is recycled in a new resolution. No hazardous solvent is used. Hence a perfectly green process is
ready to scale-up for the commercial production of SSCHD and RRCHD.
Conclusion 120
5. CONCLUSIONS
Two different ways of producing enantiomers were presented in this work. They
correspond to the 2 most important chirotechnologies: the use of a chiral catalyst and resolution by
precipitation of enantiomeric compounds. In both cases high enantiopurity was achieved (ee>99%).
The last important chirotechnlogy is the chromatography and it was used for analysis in this work.
The first method was based on an enzyme-catalysed reaction and involved the development
of novel protocols for testing alcohol dehydrogenases in dense gases. Those protocols were applied
to the alcohol dehydrogenases from Lactobacillus brevis and the production of enantiopure R-1-
phenylethanol. The second consisted in the development of the method of resolution of trans-1,2-
cyclohexanediol by crystallisation with tartaric acid followed by extraction with supercritical
carbon dioxide. The work included an analytical monitoring of the process that afforded a better
understanding and, eventually, the improvement of results with ee superior to 99%. Throughout
this work a special stress was given to green techniques. Indeed alternative solvent as dense gases
were used and the matter of waste minimisation was considered.
The result of the study of production of R-1-phenylethanol is very positive if the
enantiopurity of the obtained product is only considered but the fast deactivation of LBADH
renders an industrial process using this enzyme in dense gases unviable. A set of methods and
protocols were successfully developed. They are available for the screening of other alcohol
dehydrogenases for stereoselective hydrogenation in dense gases.
The resolution of trans-1,2-cyclohexanediol by crystallisation with tartaric acid followed
by extraction with supercritical carbon dioxide is fully developed and perfectly green. Both
enantiomers are separated and their enantiopurity is high. The resolving agent, tartaric acid, is
recovered after extraction. So the next step would be the scaling up of this process.
Litterature 121
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Litterature 131
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Appendix 132
7. APPENDIX
7.1. Challenge of green chemistry
A generally well accepted definition of green chemistry is : “The design, development,
and implementation of chemical processes and products to reduce or eliminate substances
hazardous to human health and the environment.” (Anastas et al. 1998; Poliakoff et al. 2002)
Interestingly, EPA, Environmental Protection Agency (USA), has adopted this definition.
Another definition is: “Green chemistry efficiently utilizes (preferably renewable) raw
materials, eliminates waste and avoids the use of toxic and/or hazardous reagents and
solvents in the manufacture and application of chemical products.(Sheldon et al. 2007 )” This
definition has the advantage of introducing the issues of limiting the waste and saving the raw
material.
It should be noticed that the notion of “endangering” should be considered in a broad
acceptation: danger can be physical (explosition, flammability, …), toxic (or toxicological :
carcinogenic, mutagenic…) or global (ozone depletion, global warming…). The notion of danger
should be enlarged according to the new research and knowledge in safety, toxicity and ecology.
The global warming has only been known for a short period of time and research in toxicology is
improving and reveals hazard where the responsible substances were not even detected few
decades ago (Khetan et al. 2007).
7.1.1. Context of the development of green chemistry
Green chemistry is to connect to the paradigm of the “sustainable development” which was
adopted by the united nation in 1987 as “a development which fits to the needs of the present
day without endangering next generation’s capacity to fulfil their needs.” Report of the World
Commission on Environment and Development: Our Common Future (Brundtland 1987) Figure
67 shows the theoretical set of sustainable development as the merging of considerations which are
social, ecological and economical.
Figure 67: Sustainable development as a confluence
of three domains: social, economy, and environment
Accordingly, sustainable chemistry is defined as:
“Within the broad framework of Sustainable
Development, we should strive to maximise resource
efficiency through activities such as energy and non-renewable resource conservation, risk
Appendix 133
minimisation, pollution prevention, minimisation of waste at all stages of a product life-cycle,
and the development of products that are durable and can be re-used and recycled.
Sustainable Chemistry strives to accomplish these ends through the design, manufacture and
use of efficient and effective, more environmentally benign chemical products and processes.
(Curzons et al. 2001)”
Public opinion about chemical industry is generally negative (Fahrenkamp-Uppenbrink
2002). The strong distrust of chemical industry can stand as a limit to its activity or growth. The
European Chemical Industry Council (CEFIC) survey in 1994 showed that 60% of the general
public had an unfavourable view of the chemical industry and in the USA, a survey carried out for
the Chemical Manufacturers Association (CMA) in 1993 showed that only 26% were favourably
disposed towards the industry (Clark 1999). Many industrial accident got an important media
coverage such as Flixborough disaster (1974, England), Seveso disaster (1976, Italy), Love Canal
disaster, hazardous waste disposal close to a school (from 1978 on, USA), Bhopal Disaster (1984,
India), accident at the Chernobyl nuclear power plant (1986, Soviet Union, Ukraine), Toulouse
AZF, explosion of chemical factory (2001, France). To this, it should be added the arising
consciousness that air (from housing to cities), water (from the ground water to oceans) and soils
are contaminated with chemical wastes is not restricted to the specialists (Ramade 2005). The first
examples of public concern are probably acid rains, which were followed by oil slick or adverse
effects of DDT, and currently global warming. Facing a strong opposition, different actors of
chemical industry have to adopt a “counter-propaganda” strategy. Green chemistry has got this first
aspect: a reassuring concept that targets the non specialist. Green, the colour of chlorophyll, is
opposition to the black colour of the petrol : Total has changed its logo, it is green now.
Legislation has been effective in improving environmental conditions. The waste and their
disposal are better controlled. The maximal level of emission of contaminant in air, rivers, ground
water and so on, has been progressively lowered, as an example we can mentioned the dust or SOx
in the air, the heavy metals or dioxines in the rivers. But toxic materials are still discharged in
considerable amounts. For 2007, the latest year for which data are available, disposal or
other releases of TRI chemicals totalled almost 1.9 million tons from about 22,000 U.S.
facilities submitting approximately 84,900 chemical forms (Epa 2007). Preventing waste is
(sometimes) wrongly seen as a cost without profit. For instance, the USA annually spends $115
billions in 1992 treating this enormous quantity of waste (Clark 1999). The cost provoked by waste
are from different nature as retreatment, recycling or disposal and continuously increases because
of new legislation. To this, should be added the cost of raw material that is not transformed into
valuable products (this factor gets more important with petrol rising price), cost in energy and so
Appendix 134
on. In 1990, USA passed a law called Pollution Prevention Act. It represents a shift, as it promotes
the idea that instead of treating the waste, it is better to avoid them.
7.1.2. 12 principle of green chemistry. Derived conceps.
Those 12 principles were found at first in the book Green Chemistry: Theory and Practice
(Anastas et al. 1998) . There are found in many books and articles thereafter.
1. It is better to prevent waste than to treat or clean up waste after it is formed.
2. Synthetic methods should be designed to maximise the incorporation of all
materials used in the process into the final product.
3. Wherever practicable, synthetic methodologies should be designed to use and
generate substances that possess little or no toxicity to human health and the
environment.
4. Chemical products should be designed to preserve efficiency of function while
reducing toxicity.
5. The use of auxiliary substances (e.g. solvents, separation agents, etc) should be
made unnecessary wherever possible and, innocuous when used.
6. Energy requirements should be recognised for their environmental and economic
impacts and should be minimised. Synthetic methods should be conducted at
ambient temperature and pressure.
7. A raw material or feedstock should be renewable rather depleting wherever
technically and economically practicable.
8. Unnecessary derivatisation (blocking group, protection/deprotection, temporary
modification of physical/chemical processes) should be avoided whenever
possible.
9. Catalytic reagents (as selective as possible) are superior to stoichiometric reagents.
10. Chemical products should be designed so that at the end of their function they do
not persist in the environment and break down into innocuous degradation
products.
11. Analytical methodologies need to be further developed to allow for real-time, in-
process monitoring and control prior to the formation of hazardous substances.
12. Substances and the form of a substance used in a chemical process should be
chosen so as to minimise the potential for chemical accidents, including releases,
explosions and fires.
The chemical syntheses should be designed to prevent waste, leaving no waste to treat or
clean up. In many cases, the improvement must not only be done on a step of the synthesis but the
Appendix 135
whole synthesis should design from start. From this principle, can be deduce other indicators for
the evaluation of a process than the yield: the E-factor and the atom efficiency.
The E factor is the weight of waste divided by the weight of desired product (Equation 17).
It must be minimised. Average E factors according to industry segment are presented in the table
below.
WW
productdesired
wastefactorE_
_ Equation 17: E-factor
Table 14: Waste of the different segment of chemical industry
Adapted from (Poliakoff et al. 2002)
Industry segment Product tonnage Kg waste/kg
product Oil refining 106-108 <0.1
Bulk chemicals 104-106 <1-5
Fine chemical 102-104 5 >50
Pharmaceuticals 10-103 25 >100
The concept of atom utilisation (Sheldon 2000) atom efficiency, or atom economy concept
(Trost 1991) is an extremely useful tool for rapid evaluation of the amount of waste generated by
alternative routes to a specific product. It is calculated by dividing the molecular weight of the
desired product by the sum total of the molecular weights of all substances produced in the
stoichiometric equation for the reaction(s) involved.
iii
productdesired
MnMefficiencyAtom __
Equation 18: atom efficiency
where ni is the stoechiometric coeffient of the molecule i when ndesired product =1.
The comparison is made on a theoretical basis (i.e., 100% chemical yield). The theoretical
E factor is readily derived from the atom efficiency, for example, an atom efficiency of 40%
corresponds to an E factor of 1.5. In practice, the E factor is much higher as the yield is not 100%,
as an excess of reagent(s) is often used, and solvent losses and salt generation in subsequent
neutralisation steps have to be taken into account. More developed tool can also be used as the Life
Cycle Assessment (LCA) or adding indicator as the energy consumed or the quantity of CO2
released by the process.
Appendix 136
We cannot develop every aspects of green chemistry because virtually every different
fields of chemistry are involved. The case of the synthesis of ibuprofene is interesting. The “green”
synthesis is based on two catalysts step (see Figure 68). The influence of the catalyst will find other
examples in the case of biocatalysis (see 2.1.2).
Figure 68: Two
determinant catalytic steps
in the “green” synthesis of
ibuprofen.
Reproduced from (Sheldon
2000)
Other subject of attention is the use of biomass as a feedstock (Corma et al. 2007) which
possess many large scale applications such the conversion of vegetable oil into biofuel. It should be
noticed that in this case as for others nothing is perfect the matter is more the greening of the
chemical industry rather than to obtain a industry labelled “green”: the production of biofuel
generate much glycerol that has not find an use yet and the conversion of biomass into fuel is the
competition with the traditional use of such crop: food supply.
7.1.3. Alternative solvent: supercritical fluids and SCCO2
Solvent is a burning issue due to the large quantity used and the difficult they present to be
contained and recycled. Almost the 15 billion kilograms of organic and halogenated solvent that
are produced worldwide inevitably end up leaching in the environment (Desimone 2002). A
process can be simply improved by the replacement of a solvent by another belonging to the same
family with more favourable properties such as higher boiling point - hence they generate less
waste as vapour - or a lower toxicity. A simple ways of limiting solvent consumption consist in the
recycling or the development of processes using less or even no solvent at all. A process can also
be redesigned in order to implement the use of an alternative solvent. Among them, it can be
mentioned the ionic liquids, the fluorinated phases, or the supercritical fluids.
Supercritical fluids are fluids at temperature and pressure above their critical points (Tc,
Pc) and at a pressure below the solidification (see Figure 69 and Table 15). Properties of these
fluids are unique. Density, viscosity, diffusivity and so on are intermediate between a gas and a
solid. They allow better mass transfer properties than using conventional solvents. The physical
properties of SCF can be tuned by playing on pressure and temperature (Kerton 2009). Some well
known reaction have been running under supercritical conditions for several decades: the Born–
Haber process for ammonia synthesis operates under supercritical conditions as do the
Appendix 137
polymerisation of low density polyethylene (LDPE) (Adams et al. 2004 ) and the synthesis of
methanol (Perrin et al. 2002).
Figure 69: phase diagramm (P,T)
of a fluid
Reproduced from (Kerton 2009)
Table 15: Critical points of fluid presenting an industrial interest.
Compound Tc (◦C) Pc (bar) ρc (kg/m3)
Carbon dioxide 31.3 72.9 468
Nitrogen oxide 36.5 71.4 457
Xenon 16.6 58.8 1155
SF6 45.5 37 734
Ethane 32.4 48 203
Propane 97 42 217
Butane 152 38 225
Pentane 197 33.3 237
Diethyl ether 193.6 41.7 265
Methanol 240.5 78.9 272
Ethanol 243.4 63 276
Ammonia 132.3 111.3 235
Water 374.4 226.8 323
Supercritical carbon dioxide (SCCO2) presents several supplementary advantages. While
its Pc is not so high (72.8 bar) Its Tc is low (31°C) and is compatible with heat sensitive
Appendix 138
compounds. CO2 is an “ideal solvent”, being non toxic, non inflammable, readily accessible, and
less expensive than organic solvents. That’s why it is classified as “Generally recognized as safe”
(GRAS) by the FDA. This solvent is easily separated from the product. When the pressure is
released SCCO2 become a gas where the solubility of most products is very low. Running a
reaction in a dense gas instead of a conventional solvent allows to save energy and to produce
compounds free of solvent. Moreover SCCO2 is a renewable substance readily available in large
amount (product of fermentation).
However it should be emphasised that the high-pressure involved with those fluids requires
a special care about safety and also the equipment is more expensive. Running batch reactions in
SCCO2 is often said impossible because a large volume reactor requires very large walls and,
consequently, an important investment.
Providers of equipment for high-pressure technologies exist and plants using this reaction
medium exist. The most important application SCCO2 concerns the extraction of product meant to
human consumption. One of the first extraction is the decaffeination of coffee (Dean et al. 2000).
Many extraction concern extraction of essential oils (Mchugh et al. 1994). It is also used for
separation techniques, such as resolution of enantiomer (Simándi et al. 1998) and chromatography
(Phinney 2001). Differences in solubilities, dissociation constant, and stability may be more
pronounced than in ordinary solvents and can be adjusted by fine setting of temperature and
pressure.
SCCO2 is also used for the formation of fine particles in a process as expansion from gas
saturated solutions (PGSS). A solute dissolved in SCCO2 is sprayed through a nozzle where the
pressure drops and the CO2 becomes a gas in which the solute is not soluble anymore and,
consequently, precipitates or crystallises (Kerc et al. 1999).
SCCO2 is also used for chemical reaction (Jessop et al. 1999). Thomas Swan & Co (UK)
performs the continuous hydrogenation in this solvent; they take advantage of its ability to dissolve
H2. Its capacity is up to 1000 tonnes per year (Adams et al. 2004 ). This fluid afforded better
selectivity than with conventional solvent because it was possible to control the selectivity
kinetically instead of thermodynamically. The homogeneity of the reaction medium plays an
important role as well as the good mass transport properties which allow good kinetics. Enzyme-
catalysed reaction in this SCCO2 are also the object of much research (Aaltonen 1999; Habulin et
al. 2007; Knez 2009) and industrial process are currently running.
A negative point encountered with SCCO2 is its extremely low polarity and also it weak
solvent power which limits the range of possible solute to apolar compound of relatively low
molecular weight (Kerton 2009). Those difficulties can be overcome by adding a modifier, a low
Appendix 139
molecular weight solvent that is soluble in SCCO2 improves it solvent properties. Ethanol,
methanol are commonly used.
7.2. Reaction run with coimmobilised NADP ad LBADH in non-aqueous solvent, propane and heptane
Table 16: Result of the bioconversion of ACP into RPE in heptane and propane.
WEI
GH
T of
LB
ADH
mg
Wei
ght o
f NAD
P m
g
Con
cent
ratio
n of
AC
P m
M
ratio
isop
ropa
nol-
acet
ophe
none
YIEL
D %
TON
(EN
ZYM
E)
TON
(CO
-EN
ZYM
E)
INIT
IAL
RAT
E µm
ol/min/mgE
cons
tant
K*1
03
A 2.2 13 40 1 3.7 120 3 - 1.6
B 4 5.2 40 0.9 0.8 18 2 - 0.082
C 3.3 4.2 30 1.2 0.0 0 0 0 0
D 2.9 6.2 46 1.1 4.2 100 7 0.2 1.8
E *** *** 40 1 0.0 0 0 0 0
F 2.4 3.2 40 2 8.4 116 13 0.2 4.1
Hep
tane
G 2.4 3.2 40 2 8.8 120 14 0.2 4.5
H 25 11.10 28 16 45.3 180 49 0.056 24 Propane
I 25 11.10 28 16 0 0 0 0 0
The activity of the preparation of LBADH used the experiences reported in Table 16 was
8 U/mg and contained 0.15µmol of active LBADH for 1 mg.
Reaction condition: in 25ml heptane (except for C 38 mL) with 50µL (A, B, D, E) or 30 µL
(F, G) water. In C’s case, no water was added the enzyme and coenzyme were not coimmobilised,
the two powder were introduce in the reactor without any further preparation. For A, B, D, E, F and
G, the catalyst were prepared according to 6.3, 500 mg silica (A, B) coarse glass beads (D, F, G
(7g)) were used with sugar (A (2 mg), F (12 mg)), G (12 mg)) or not (B, D, ). The catalyst for E
was the same as for D reused after a two-day reaction. Reaction H was run in the view cell (about
60 mL) with glass beads (5 g) the water activity was set at 0,8 with salt hydrate Na2SO4, 10/0.
Enzyme was incubated in propane with the salts for 2 hours. Reaction I was run in the same
Appendix 140
conditions as H, except from the catalysts which was made of the two powder with no further
preparation
7.3. Miscibility of ACP, ISP, 1-phenylethanol, acetone and propane
GL : equilibrium gas-liquid
L : liquid phase
Table 17: Miscibility in propane
% in weight of the different compounds
Propane Aceto-
phenone
Phenyl-
ethanol
Iso-
propanol Acetone T °C P (bar) Phase
76 % 24 % 0 % 0 % 0 % 37 12 GL
76 % 24 % 0 % 0 % 0 % 30 30 L
76 % 24 % 0 % 0 % 0 % 30 50 L
61 % 18 % 0 % 21 % 0 % 25 9 GL
61 % 18 % 0 % 21 % 0 % 30 9 L
61 % 18 % 0 % 21 % 0 % 30 52 L
61 % 18 % 0 % 21 % 0 % 30 30 L
79 % 0 % 21 % 0 % 0 % 29 11 GL
79 % 0 % 21 % 0 % 0 % 30 50 L
79 % 0 % 21 % 0 % 0 % 30 30 L
82.5 % 5.5 % 5.5 % 4.5 % 1.5 % 30 10 GL
82.5 % 5.5 % 5.5 % 4.5 % 1.5 % 30 30 L
82.5 % 5.5 % 5.5 % 4.5 % 1.5 % 30 50 L
7.4. Method of determination of the structure of the cocrystal CoC
The selected transparent crystal of 1 for single crystal X-ray diffraction measurement had
the size of 0.55 x 0.55 x 0.34 mm. 1 was mounted on a loop with parathon oil. Cell parameters
were determined by least-squares of all reflections in the whole measured range. Intensity data
Appendix 141
were collected on a RIGAKU RAXIS-RAPID diffractometer (graphite monochromator; Mo-K
radiation, = 0.71073Å). Empirical absorption correction was applied to the data. The structure
was solved by direct methods (Sheldrick 1997b). Anisotropic full-matrix least-squares refinements
(Sheldrick 1997a; Barbour 2001) on F2 for all non-hydrogen atoms were performed. Neutral atomic
scattering factors were taken from the International Tables for X-ray Crystallography (Wilson
1992). Crystallographic data, parameters of data collection, structure solution and refinement can
be found in Table 1. Since there are no strong anomalous scattering centres in the constituents and
the diffraction measurement was performed using Mo-K radiation, the Flack x parameter (Flack
1983) is not reliable. The O-H hydrogen atomic positions could be located in the difference Fourier
maps. Hydrogen atoms were included in structure factor calculations but they were not refined. The
isotropic displacement parameters of the hydrogen atoms were approximated from the U(eq) value
of the atom, to which they were bonded.
Crystallographic data (excluding structure factors) for the cocrystal structure of CoC have
been deposited with the Cambridge Crystallographic Data Centre as supplementary publication
number CCDC 728882.
7.5. Theoretical ternary diagram with a liquid solution
The study of the phase diagram is not only interesting to a theoretical point of view but can
allow to determine in which proportion the sample to be extracted by SFE will give the best yield
and ee. The phase diagram are investigated are room temperature and atmospheric. The hypothesis
is done that over the extraction process the equilibrium between the solid phase is not drastically
modified. This hypothesis should be rather correct as only condensed phase are present and
pressure has little effect on them and the applied temperature is closed to room temperature.
The aim of this part is to show what type can be expected. The different phase diagram are
proposed will the hypothesis on the chemical potential made. The cocrystal exist and forms from an
equimolar mixture of RRCHD and TA. We can conclude that:
TARRCHDCoc Equation 19
No cocrystal is formed between SSCHD and TA (see 4.2.4.1). As RacCHD is formed when
there are RRCHD and SSCHD we can conclude the same way that:
2 RRCHDRacCHD Equation 20
Appendix 142
At first we will not consider the fact that a solid solution is formed between the SSCHD
and RRCHD but a more usual racemic compound.
7.5.1. If no liquid solution between the CHD enantiomers
exists.
The Equation 19 and the Equation 20 are not sufficient for determining the phase diagram:
we do not know if CoC or RacCHD will formed in an equimolar mixture of TA, RRCHD, SSCHD.
We need another hypothesis that is given by the very fact the resolution works and consequently
we know that in the precious case RRCHD is more stabilised by forming a compound with TA
compared to with SSCHD, this is to say:
TARacCHDCoC SSCHD Equation 21
Now the phase diagram may be plotted.
Figure 70: “No solid solution
CoC>racem” phase diagram of
RRCHD, SSCHD and TA
supposing that a racemic
compound is formed and not a
solid solution.
The central point corresponds
to the composition of the sample used
for the first experiment (mr=0.5) (Molnar et al. 2008). At this point this phase diagram is respected
it would lead to CoC and SSCHD only.
Remark: if TARacCHDCoC SSCHD we obtain:
Figure 71 : “No solid solution CoC<racem”
alternative phase diagram with RRCHD,
SSCHD, CoC, RacCHD, and TA.
Appendix 143
The central point corresponds to the composition of the sample used in (Molnar et al.
2008): if this phase diagram is respected it would lead to mixture of TA and RacCHD.
7.5.2. A liquid solution exists between the CHD enantiomers.
7.5.2.1.Gibbs free enthalpy of a binary mixture RacCHD
and RRCHD or SSCHD presenting a partial miscibility
We know from (Leitao et al. 2002) that a solid solution is formed between RacCHD and
one of its enantiomers, RRCHD or SSCHD (see 2.3.3). We can plot the molar entropy of a mixture
of RRCHD and SSCHD according to the composition.
Figure 72 : Gibbs
molar enthalpy of a
binary RRCHD-
SSCHD
In the Figure
72, the fact that a
second polymorph of
RacCHD exists
(Lloyd et al. 2007) is
not presented. It
does not affect much the Gibbs enthalpy of CHD and add a phase for molar fraction close to 0.5.
7.5.2.2.Ternary diagram
The ternary phase diagram is more complicated when applied to solid solution and
different cases can be envisaged. The first is that CoC is formed when TA added to CHD that
contain any fraction of RRCHD or that the reaction of Equation 22 takes place for every X (X
being XSSCHD).
dnCoCRRdnXXSSCHDdndnTARRXXSSCHD ))1(,()1())1(,(
Equation 22
Appendix 144
Figure 73: “Solid solution and
CoC>solCHD” ternary phase
diagram
Figure 73 represents a
theoretical phase diagram of RRCHD,
SSCHD and TA supposing that a solid
solution is formed between RacCHD
and SSCHD or RRCHD. The central
point correspond to the composition of
the sample mr=0.5 (Molnar et al. 2008): if this phase diagram is respected it would lead to CoC and
SSCHD only.
The hypothesis taken in the previous diagram can be written using the chemical potential
and the molar enthalpy plotted in Figure 72.
If in the ternary we have 1 mole of CHD in a mixture of X mole SSCHD and 1-X mole
SSCHD (so CHD contribution to the total free energy of the system is gCHD(X) and add an infinity
decimal quantity of TA dn, dn Coc forms so the quantity of CHD has decreased to 1-dn and its
molar fraction of SSCHD is dn
X1
which is egal to X(1+dn) at the first order (Equation 22). The
variation of free enthalpy dG associated to this transformation is inferior to zero and is egal to (at
the first order):
Xgg SSCHD
CHDCHDTACoC d
dXdndndG )(
00
And we introduce the chemical potential of SSCHD (equal to RRCHD’s) we obtain:
Xg
Xg SSCHDCHDSSCHD
CHDSSCHDTACoC d
dSSCHDdn
dG ))()0( ( 000
Equation 23
Then the hypothesis of the last ternary was that dndG
would be always inferior to zero, this
is to say that the formation of CoC is always favourable for it leads to a decrease in the free energy.
Appendix 145
dndG
is a increasing function of XSSCHD that present a plateau for X=0.8 to 1. Theoretically,
there is a possibility that the sign of dndG
changes between 0.5 and 0.8.
Figure 74 : Variation of free energy of the
system CHD + dn TA when CoC forms.
If we accept that the sign of dndG
changes this means that for a certain
composition of SolCHD, that is named Xlim
(SolCHDlim correspond to its Xlim where
0dndG
) the formation of CoC is as favourable as the formation of SolCHD and free TA. Above
Xlim, CoC does not form and, below Xlim, the CoC forms. So another ternary phase diagram can be
plotted taking into account this new possibility.
Figure 75 : “Solid solution and
CoC=solCHDlim” ternary phase
diagram with solCHD 2
The central point corresponds to
the composition of the sample mr=0.5
(Molnar et al. 2008): if this phase
diagram is respected it would lead to
SolCHDlim, CoC and TA. Let’s see what
happen at this point of the ternary for 2
extreme values: if Xlim=0.5 there are only SolCHDlim (equivalent to RacCHD is this very case) and
if Xlim=0.8 there would be TA, CoC and SolCHDlim . In the last case if 1 mol of RacCHD (this is to
say 1 mol of RRCHD and 1 mol of SSCHD) and 1 mole of TA are mixed we would obtain a
SolCHD that contains 1 mol of SSCHD and 0.25 mol of RRCHD, 0.25 mol TA and 0.75 mol Coc
(0.75 mol TA + 0.75 mol RRCHD).
Appendix 146
Remark: the function dndG
indicates the difficulty of formation of CoC: if 0dndG
CoC
cannot form and, then, the lower the easier the formation of CoC. Deviation to ideality (or
thermodynamical equilibrium) is found for values of XSS close to the saturation of the liquid
solution with SSCHD. This is to say that if the system is out equilibrium, the phase diagram Figure
75 might represent a metastable phase diagram. If amorphous phase is included it have much
chance to have a composition close to the ternary eutectic.
Remark 2: A ternary phase diagram where CoC can accept a fraction of SSCHD can also
be constructed, an example is given in Figure 76. This phase diagram is purely indicative and given
just to illustrate the discussion where incorporation of SSCHD in CoC is envisaged.
Figure 76: Example of ternary phase
diagram where CoC can intake a small
fraction of SSCHD
The dotted line on the CoC+SolCHD
indicates the equilibrium between a
solCHD of a certain composition and the
CoC that contain the corresponding
amount of SSCHD. This scheme is only an
example.
7.6. Results of the experiment for the determination of the phase
diagram.
In this annex the different results of analysis for the determination of the ternary phase
diagram is presented as well as different hypothesis that allow interpretation. The explanation
retained is that the ternary phase diagram is of the type of Figure 73: “Solid solution and
CoC>solCHD” ternary phase diagram that presents the formation of a metastable compound called
“X”, and difficulties of formation of CoC. Amorphicity is also to expect.
Table 18: The different sample prepared for investigation of the ternary system, their
composition and the phase observed by XRD
Appendix 147
Table 19: Composition of the different sample if the ternary does not present solid
solution.
It would obey to the phase diagram Figure 70: “No solid solution CoC>racem” phase
diagram of RRCHD, SSCHD and TA supposing that a racemic compound is formed and
not a solid solution.
Table 20: Which deviation do we observe from the model "no solid solution CoC>racem"?
How can we interpret it? Two explanation are tested : phase diagram of the type of Figure
75 : “Solid solution and CoC=solCHDlim” ternary phase diagram with solCHD 2 or the fact
that the system is out of equilibrium and the formation of CoC does not take in place in the
full extent.
Table 21: Composition of the different sample if the ternary presents a solid solution and
the deviations observed.
It would obey to the phase diagram Figure 73: “Solid solution and CoC>solCHD” ternary
phase diagram.
*CHDG was prepared with SSTA and SSCHD. So this is the enantiomer of CoC. We fit
this result in the table as if it was done of TA and RRCHD.
Appendix 148
analytical composition phases observed by XRD name quality? sschd rrchd ta enantiomer racem TA CoC X ANA 1 no 25 0 75 yes yes ANA 2 no 50 0 50 yes yes ANA 3 no 75 0 25 yes yes ANA 4 no 66 34 0 little yes ANA 5 no 35 65 0 very little yes ANA 6 yes 16 84 0 yes yes ANA 7 yes 0 25 75 yes yes ANA 8 yes 0 50 50 trace trace mostly ANA 9 yes 0 75 25 yes yes ANA 10 yes 4 21 75 little little yes yes ANA 11 yes 8 42 50 little Very little little yes yes ANA 12 yes 12 63 25 little Very little little yes much ANA 13 no 3 3 95 yes yes no Very little ANA 14 no 8 8 85 yes yes little ANA 15 no 25 25 50 little yes yes yes ANA 16 no 43 43 15 little yes little yes ANA 17 no 48 48 5 yes trace ANA 19 yes 73 27 0 little yes ANA 20 yes 25 0 75 yes yes ANA 21 yes 50 0 50 yes yes ANA 22 yes 75 0 25 yes yes ANA 23 yes 18 7 75 little yes much ANA 24 yes 37 13 50 not much yes yes ANA 25 yes 55 20 25 little yes little ANA 26 yes 10 45 45 yes not much a lot ANA 27 yes 50 25 25 yes yes a lot yes yes ANA 28 yes 80 10 10 yes a lot yes little chdg* yes 50 50 yes yes mr=0.5 low qual. 33 33 33 yes yes little yes
Appendix 149
the simpler model: CoC is formed then TA then racCHD then the free enantiomer left for 100 mol of RRCHD + SSCHD + TA in % in %w name CoC TA racCHD RRCHD SSCHD CoC TA racCHD RRCHD SSCHD CoC TA racCHD RRCHD SSCHD ANA 1 0 75 0 0 25 0 75 0 0 25 0 79 0 0 21 ANA 2 0 50 0 0 50 0 50 0 0 50 0 56 0 0 44 ANA 3 0 25 0 0 75 0 25 0 0 75 0 30 0 0 70 ANA 4 0 0 34 0 31 0 0 52 0 48 0 0 69 0 31 ANA 5 0 0 35 30 0 0 0 54 46 0 0 0 70 30 0 ANA 6 0 0 16 67 0 0 0 19 81 0 0 0 33 67 0 ANA 7 25 50 0 0 0 33 67 0 0 0 47 53 0 0 0 ANA 8 50 0 0 0 0 100 0 0 0 0 100 0 0 0 0 ANA 9 25 0 0 50 0 33 0 0 67 0 53 0 0 47 0 ANA 10 21 54 0 0 4 26 68 0 0 5 39 57 0 0 3 ANA 11 42 8 0 0 8 72 14 0 0 14 84 9 0 0 7 ANA 12 25 0 12 26 0 40 0 19 41 0 53 0 23 24 0 ANA 13 3 92 0 0 3 3 95 0 0 3 4 94 0 0 2 ANA 14 8 77 0 0 8 8 84 0 0 8 14 80 0 0 6 ANA 15 25 25 0 0 25 33 33 0 0 33 50 28 0 0 22 ANA 16 15 0 28 0 15 26 0 48 0 26 33 0 53 0 14 ANA 17 5 0 43 0 5 10 0 81 0 10 11 0 84 0 5 ANA 19 0 0 27 0 46 0 0 37 0 63 0 0 54 0 46 ANA 20 0 75 0 0 25 0 75 0 0 25 0 79 0 0 21 ANA 21 0 50 0 0 50 0 50 0 0 50 0 56 0 0 44 ANA 22 0 25 0 0 75 0 25 0 0 75 0 30 0 0 70 ANA 23 7 68 0 0 18 7 73 0 0 20 13 72 0 0 15 ANA 24 13 37 0 0 37 16 42 0 0 42 27 41 0 0 32 ANA 25 20 5 0 0 55 25 6 0 0 69 43 6 0 0 51 ANA 26 45 0 0 0 10 82 0 0 0 18 91 0 0 0 9 ANA 27 25 0 0 0 50 33 0 0 0 67 53 0 0 0 47 ANA 28 10 0 0 0 80 11 0 0 0 89 22 0 0 0 78 chdg* 50 0 0 0 0 100 0 0 0 0 100 0 0 0 0 mr=0.5 33 0 0 0 33 50 0 0 0 50 70 0 0 0 30
Appendix 150
How it can be explained? name ok? comments pb formation coc solid solution ANA 1 yes ANA 2 yes ANA 3 yes ANA 4 no not enough enantiomer yes ANA 5 no not enough enantiomer yes ANA 6 yes ANA 7 yes ANA 8 yes ANA 9 yes ANA 10 no X instead TA no no
ANA 11 yes but with X no no
ANA 12 no X instead racem and RR no ANA 13 no no CoC but racem yes no
ANA 14 no not enough CoC too much racem yes no
ANA 15 no racem but no SSCHD partially partially ANA 16 yes ANA 17 no no CoC but racem yes no ANA 19 no enan missing no yes ANA 20 yes ANA 21 yes ANA 22 yes ANA 23 no no coc but racem yes partially ANA 24 no not enough enantiomer/coX yes yes ANA 25 no no coc but racem yes partially ANA 26 yes ANA 27 no too much racem yes partially ANA 28 no no coc but racem yes partially chdg* no X! no no mr=0.5 no racem and little TA unexpected yes partially
Appendix 151
with solid solution limit 0.2 < Xss < 0.8) in %w name CoC TA racsolsolCHD RRCHD SSCHD ok? explanation? ANA 1 0 79 0 0 21 yes ANA 2 0 56 0 0 44 yes ANA 3 0 30 0 0 70 yes ANA 4 0 0 100 0 0 no but better trace of enantiomer ANA 5 0 0 100 0 0 no but better trace of enantiomer ANA 6 0 0 82 19 0 yes ANA 7 47 53 0 0 0 yes ANA 8 100 0 0 0 0 yes ANA 9 53 0 0 47 0 yes ANA 10 39 57 0 0 3 no X ANA 11 84 9 0 0 7 yes ANA 12 53 0 47 0 0 no X ANA 13 4 94 0 0 2 no no CoC formation ANA 14 14 80 0 0 6 no bad CoC formation ANA 15 50 28 0 0 22 no bad CoC formation ANA 16 33 0 67 0 0 yes ANA 17 11 0 89 0 0 no no CoC formation ANA 19 0 0 100 0 0 no but better trace of enantiomer ANA 20 0 79 0 0 21 yes ANA 21 0 56 0 0 44 yes ANA 22 0 30 0 0 70 yes ANA 23 13 72 0 0 15 no no CoC formation ANA 24 27 41 0 0 32 no no CoC formation ANA 25 43 6 0 0 51 no no CoC formation ANA 26 91 0 0 0 9 yes ANA 27 53 0 0 0 47 no bad CoC formation ANA 28 22 0 0 0 78 no no CoC formation chdg* 100 0 0 0 0 no X mr=0.5 70 0 0 0 30 no bad CoC formation
Bibliography 152
7.7. Diffractogram of samples prepared according to different methods
Table 22: Sample for sample preparation prepared in different condition
Rbf: the evaporation of the ethanol in the second step in done over night in the same
round-bottomed flask (rbf) as for the rotoevaporation. pd: the evaporation of the ethanol in
the second step in done over night in a petri dish (pd) or beaker (bk).
Sam
ple
nam
e
Con
ditio
n of
ev
apor
atio
n of
eth
anol
Tem
pera
ture
of
roto
evap
orat
ion
(°C
)
Furth
er e
vapo
ratio
n
perfi
l
mr
Muc
h m
orph
ous
phas
e by
vis
ual o
bser
vatio
n?
Obs
erva
tion
by X
RD
75°C Rotoevap. 75 rbf no 0.5 no Much CoC and enantiom Little racem
55°C Rotoevap. 55 rbf no 0.5 no TA and SolCHD Traces of CoC
40°C Rotoevap. 40 rbf no 0.5 no Only TA and SolCHD
40°C Rotoevap. 40 rbf yes 0.5 no Only TA and SolCHD
30°C Rotoevap. 30 rbf no 0.5 yes Much CoC and SolCHD little enantiom. no TA
40°C Rotoevap. 40 rbf no 1 no Only TA and SolCHD
40°C Rotoevap. 40 rbf yes 1 no TA and SolCHD little CoC
40°C Rotoevap. 40 pd no 0.5 no Much CoC Little Enantiom
40°C Rotoevap. 40 pd yes 0.5 no CoC and Enantiom No SolCHD
Pat 6 none bk no 0.5 yes Much racem, TA traces of Coc no enantiom
Pat 7 none rbf no 0.5 yes Much CoC, racem, trace of enantiom, no TA