characterization of carbohydrates

Carbohydrates – Characterization Overview

Methods for Structure and Property Deduction Chemical methods for functional group identification Chemical methods for ring size identification Methylation analysis X-ray crystallography Analytical methods for characterizationy f

UV-Vis spectroscopy Circular dichroism spectroscopy NMR spectroscopy NMR spectroscopy Mass spectrometry

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Monosaccharides

M i Mutarotation

OHHCHO

CH OHOHHOHHHOHOHH

CH2OHD-glucose

-D-glucopyranose(-D-glucose)

-D-glucopyranose(-D-glucose)

Optical activity[] = +112 2O [] = +18 7O[]D +112.2 []D +18.7

At equilibrium …. []D = +52.7O (Not an average!But weighted average; 63.6% of the -anomer)

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Simple Tests for Reducing / Non-reducing Sugars

I t f R d i S Case of hemoglobin (Hb)

Hb is glycated ….. HbA1c levels should be < 6.5%; Reaction of high glucose ith NH t th N t i (V l) f Hb N ti R fl t

Importance of Reducing Sugars

with NH2 group at the N-terminus (Val) of Hb; Non-enzymatic; Reflects Glc levels for the past 3 months (typical life of erythrocytes)

Can serve as sugar modifying handleLabel with chromogenic groups or fluorophores

Tollen’s Reagent

g g p pPreparation of sugar-labeled solid matrix

COOH

OHH

CHO

OHH

HHO

H OH

HHO

H OH Mild Oxidizing Agente.g., Tollen’s reagent

+ Metallic Silver

CH2OH

H OH

CH2OH

H OH

D Gl cose D Gl conic Acid

(Ag+ / NH3 – H2O

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D-Glucose D-Gluconic Acid

Simple Tests for Reducing / Non-reducing Sugars

Benedict’s reagent

RCHO 2C 2+ 5HO RCOO C O 3H O

• Benedict's reagent is a solution of the citrate complex of CuSO4 in

RCHO + 2Cu2+ + 5HO‐ RCOO‐ + Cu2O + 3H2O

g p 4water. Cu2+ is a weak oxidizing agent.

• A positive test is the formation of a red precipitate of Cu2O.

Sugars that exhibit positive Tollen’s / Benedict’s tests• Aldoses• Ketoses, if they exhibit good keto-enol tautomerism• Glycosides do not show positive test because they cannot exhibit

aldehyde equlibriumO

HOHO

HOCH2aldehyde equlibrium

4OCH3

OH

UV-Vis Spectroscopy of Carbohydrates

Glycans are UV-Vis transparent; groups include –OH, –CHO, –C=O, –OCH3, –COOH, etc.

Some glycans have a conjugated double bond; e.g., heparin g y j g ; g , pdisaccharide. Such groups absorb at 232 nm, which allows identification and quantitation of oligosaccharides

Have to be typically labeled for visualization Use reducing end labels such as 2-aminopyridine (2-AP) 2- Use reducing end labels such as 2-aminopyridine (2-AP), 2-

aminoacridone (AMAC), 7-amino-1,3-naphthalenedisulphonic acid (AGA), p-aminobenzoic acid (PABA), 2-aminobenzamide (2ABA), 4-aminobenzonitrile 8 aminonaphthalene 1 3 6 trisulfonic acid (ANTS)aminobenzonitrile, 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS), 1-phenyl-3-methyl-5-pyrazolone (PMP), 1-(4-methoxy)phenyl-3-methyl-5-pyrazolne (PMPMP), 6-aminoquinoline (6-AQ), 1-

lt h l 1 5 di i hth l 8 i 1 3 65

maltohepaosyl-1,5-diaminonaphthalene, 8-aminopyrene-1,3,6-trisulfonate (APTS), etc.

UV-Vis Spectroscopy of Carbohydrates

Labeling reaction ... typically Schiff base formation followed by reduction to a stable covalent bond

O

CH2OH

OH OHOH

CH2OH

OH HN

OH

RO

OH

OH

OH

RO

NAcOH,

2-AP

CH2OH

NaCNBH3

OHOH

RO

OH

OH

CH2OH

OH NH

N

OH

RO

OH

OH

RO

NH

d d S hiff b6

reduced Schiff base

NMR Spectroscopy of Carbohydrates

Every polysaccharide has a unique 1D NMR spectrum, which contains all of the information about the structure

Additional NMR experiments are performed to assign the resonances Additional NMR experiments are performed to assign the resonances, e.g., 1H,1H-COSY and 1H,1H-TOCSY for 1H resonances or 1H,13C-HETCOR or HSQC for 13C resonances

Sequence determination is performed through experiments such as NOESY (1H,1H through space correlations) or HMBC (3JCOCH)

M th d f i t t ti f 1H NMR t h ti Methods for interpretation of 1H NMR spectrum can save much time and effort

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Basic Approach for NMR Structure Determination

1D 1H-NMR+ 1D 13C NMR+ 1D 13C-NMR

2D 1H 1H-COSY2D H, H COSY

8Taken from Desai et al. Carbohydr. Res. 1993, 241, 249-259.

Basic Approach for NMR Structure Determination

2D 1H,1H-NOESY 2D 1H,13C-HETCOR

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Basic Principle

= Through-bond correlation

= Through-space correlation

= Anomeric proton= Anomeric proton

3J couplings may needed for conformational assignment Requires knowledge of saccharide residues Requires knowledge of saccharide residues Additional experiments needed for larger structures

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NMR Tools on the Internet(Taken from a lecture on ‘Internet tools for the Interpretation of NMR spectra’

b

Polysaccharide structure:

byDr. Roland Stenutz ([email protected]), University of Stockholm, Sweden)

yComponents ”type” Hex or HexNAc

relative configuration Glc or Manabsolute configuration D- or L-absolute configuration D or Lring size -p or –f

Linkages position 4) or 6)stereochemistry - or -stereochemistry or

Sequence 4)Glc(4)Gal(or

4)Gal(4)Glc(4)Gal(4)Glc(

NMR can be used to perform each of these steps except for theNMR can be used to perform each of these steps, except for the determination of the absolute configurationMost saccharides are typically present in one form in nature, which serves to identify the configurationidentify the configuration

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Internet-based Approaches to NMR Analysis

comparison with a database (SugaBase)simple and accurate but limited to known structures or sub-structures.

comparison with simulated NMR spectra (CASPER)requires information about the components and linkages to limit the q f p gnumber of possible structures.

Just a few examples of tools available in polysaccharide analysisp p y y

Taken from ‘Internet tools for the Interpretation of NMR spectra’ by Dr. Roland Stenutz([email protected]), University of Stockholm, Sweden)

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SugaBasehttp://www.boc.chem.uu.nl/sugabase/databases.html)p g )


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Sweet-DB

http://glycosciences.de hosts several tools and databases for the carbohydrate chemist

Sweet-DB is an interface to CarbBank/SugaBase


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Searching With NMR Data on Sugabase


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Search Results


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Best Search Result


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CASPER(http://www.casper.organ.su.se)( p p g )


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CASPER


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CASPER

Calculated shifts

Assignment

Experimental shifts


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CASPER

CASPER also allows incorporation of glycosylation and substituent shifts, positional shifts

Sequences can also be derived using CASPERq g


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Mass Spectrometry in the Structure Determination of Glycans

The premier structure determination tool today Highly sensitivity (g quantities) Highly information rich full sequence with detailed stereochemical Highly information rich ... full sequence with detailed stereochemical

structure possible Widely applicable ... N- & O-glycans, sialylated glycans, GAGs, etc. May be used in tandem with chemical or enzymatic pre-treatment Various MS technologies are available for ionization

MALDI (Matrix assisted laser desorption ionization)( p ) ESI or nESI (Electrospray or nanoElectrospray ionization) FAB (Fast atom bombardment)

Various approaches are available for structure elucidation Various approaches are available for structure elucidation HR TOF (high resolution time of flight) MS/MS or MS/MSn (tandem MS) Ion Mobility Ion Mobility

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A Strategy for MS Sequencing of N-Glycans

Taken from Azadiand Heiss. Methods Mol. Biol. 2009, 534, 37 5137-51.

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Exoglycosidases in Sequencing of Glycans

24A good ref: Kannicht et al. Methods Mol. Biol. 2008, 446, 255-266.

Permethylation/Peracetylation and Linkage Analysis

Permethylation advantages include easier determination of linkages and branching

Allows simultaneous analysis of sialic acid and neutral glycans Allows simultaneous analysis of sialic acid and neutral glycans Better ionization in MS chamber Makes analysis possible with pM quantities of sample

CH3I, dry DMSOCH2Cl2 2M TFA, >100 OC

1M NH4OH, NaBD4

Ac2O, base, heat

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The Mass Spectrometer

l l b i d h ( ) Molecules can be imparted charge (+ve or –ve) Charged molecules can be made to move (accelerate) A moving object (e.g., a molecule) can be made to deflect Deflection is related to mass

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Deflection is related to mass

The Tandem Mass Spectrometer

27Image from Access Science from McGraw Hill

Nomenclature of Product Ions

Fragment ions containing the non-reducing terminus are labeled A, B, C Fragment ions containing the reducing terminus are labeled X, Y and Z Subscripts 1, 2, 3, …. refer to the number of residue from either the reducing

or the non-reducing terminus Subscript 0 refers to cleavage of the bond with the aglycon Fragment labels A and X are reserved for intra-ring cleavage Superscripts refer to intra-ring bonds cleaved (clockwise numbering)

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Superscripts refer to intra ring bonds cleaved (clockwise numbering) Products ions can be [M+nH]n+, [M-nH]n-, [M+nNa+mH](n+m)+,

Zaia, J., Mass Spectrom. Rev. 2004, 23, 161-227.

Formation of Product Ions

29Zaia, J., Mass Spectrom. Rev. 2004, 23, 161-227.


30Zaia, J., Mass Spectrom. Rev. 2004, 23, 161-227.


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Glycan Characterization Using SimGlycanTM

32Taken from Apte and Meitei, Methods Mol. Biol. 2010, 600, 269-281.

characterization of carbohydrates

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