Carbohydrate Structural Elucidation

Structural elucidation of carbohydrates
Strategy for polysaccharide structural analysis
• Monosaccharide composition: nature of the monosaccharide building blocks
• Sequences of monosaccharide residues and repeating units.
• Linkage patterns: linkage positions between the glycosidic linkages and branches
• Ring size: distinction of furanosidic and pyranosidic rings
• Anomeric configuration: α or β configuration of the glycosidic linkage
• Substitutions: position and nature of OH–modifications, such as O - phosphorylation,

acetylation, O - sulfation, etc.
Degradation of Polysaccharides
• Polysaccharides - hydrolysed into oligosaccharides by acid, and detailed characterization of

these oligosaccharides would help to elucidate the original polysaccharide structure.
Graded acid hydrolysis / Partial acid hydrolysis
• Hydrolysed by graded acid concentrations is applied to degrade the polysaccharide

progressively (0.05 M, 0.2 M, 0.5 M, and 2.0 M of trifluroacetic acid at 100 °C for 1 h
sequentially).
• The hydrolysates - ultra filtration.
• The detailed structural characterisation of resulting oligosaccharides /disaccharides/

monosaccharides would help to elucidate the original polysaccharide.
• Determines the branching pattern and the linkage between the sugar units.
Periodic acid oxidation - L. Malaprade 1928
• Periodic acid - is the highest oxoacid of iodine & iodine exists in oxidation state +7.
• Periodic acid will cleave the carbon-carbon bonds of vicinal diols / compounds containing two
or more OH or O groups attached to adjacent carbon atoms.
• By the formation of cyclic intermediate.
• Give rise to the formation of two carbonyl groups (aldehydes, ketones or acids).
Periodic acid does not cleave compounds in which the hydroxyl groups are separated by a -CH 2
group nor those in which a hydroxyl group is adjacent to an ether or acetal
• As the reaction is quantitative, valuable information can be deduced by estimating the moles
of HIO4 used and by identifying the product formed.
• Used to elucidate structural information of carbohydrates.

• Glucose consumes Five equivalents of HIO4 and yields five equivalents of formic acid and
one equivalent of formaldehyde.
• Open-chain form - complete oxidative cleavage takes place.

• The periodic acid reaction can be applied to glycosides for determining the ring size, i.e.,
whether they have pyranose or furanose structure.
• OH group attached to the anomeric carbon atom should be methylated (methanol +HCl).
• Under mild conditions (0.01 M to 0.05 M of HIO4).

• To determine the ring size of a monosaccharide, it is necessary to maintain the ring and prevent
it from equilibrating with other isomeric structures.
• Monosaccharide is treated with methanol and HCl to obtain the methyl glycoside.
• Glycosides with protected anomeric centers - do not undergo mutarotation, and they do not
react with most reagents under neutral or basic conditions.
• Chemical reactions can be carried out at other sites in the glycoside to determine the ring size
and configuration of the monosaccharides.
• Periodate oxidation can be used to estimate the degree of polymerization
• A quantitative determination of periodate consumed and the formic acid formed, combined with
the information on the sugar units surviving the oxidation reaction, will provide clues to the
nature of the glycosidic linkage and other structural features of the polysaccharides.
• Sugar units with different linkage patterns will vary significantly in the way they react with
Methylation (Etherification).
• The alcoholic OH groups of monosaccharides and disaccharides are converted to ether groups upon treatment with
methylating agents.
• This reaction has been extensively used to find out which OH groups in a sugar are free and available for reaction.
• Thus, α-methyl glucoside is methylated at carbon 2, 3, 4 and 6 but not at carbon 5 which is involved in hemiacetal
link.
• The cyclic forms of monosaccharides (hemiacetals) react with alcohols in acid solution to form
acetals.
• Glucose reacts with methyl alcohol in the presence of HCl to form methyl glucoside.
Acetylation (Esterification) - The glycosidic and alcoholic OH groups of monosaccharides and
disaccharides react with acetylating agents to form acetate derivatives called esters.
• The ability of sugars to form esters indicates the presence of alcohol groups in their molecule.
Determination of the Extent of Branching in Polysaccharides (Ex: Amylopectin)
• The amount of branching (number of (1→6) glycosidic bonds) in amylopectin can be

determined by the following procedure.
• A sample of amylopectin is exhaustively methylated—treated with a methylating agent (methyl

iodide) that replaces the hydrogen of every sugar hydroxyl with a methyl group, converting OH
to OCH3.
• All the glycosidic bonds in the treated sample are then hydrolyzed in aqueous acid, and the
amount of 2, 3-di-methylglucose so formed is determined.
Structural elucidation of carbohydrate by GC MS
• GAS chromatography, coupled with mass spectrometry (GC-MS)
• Compositional analysis using GC-MS requires the polymer to be hydrolysed into its constituent
sugars which can then be analysed.
• A fundamental requirement for the separation and analysis of carbohydrates by GC is that they
must be thermally stable and volatile.
• Carbohydrates must therefore be converted into stable, volatile compounds prior to

chromatography.
• Mixture is vaporized in a heated chamber – gas chromtograph
column
• Components gets separated after interaction with the column
• Fractions come out at different times -leads to ionization chamber of
the mass analyzer (quadrapole mass analyzer)
• provides mass spectra corresponding to the effluents
1. Methylation adds a methyl group to all free hydroxyl groups on the sugar. (methyl iodide in
dry dimethyl sulfoxide/silver oxide solution)
2. Methanolysis will hydrolyse the sugar leaving a free hydroxyl group on the sugar which can
then be silylated. (an acidic solution of methanol with the sample being heated at 80°C for
16–20 hours—a method known as methanolysis)
3. Silylation - Thermally stable and highly volatile derivatives can be easily obtained by the
silylation reaction (trimethylsilyl [−Si(CH3)3] - trimethylsilyl chloride or bis(trimethylsilyl)
acetamide)
4. The derivatised sugars are separated by GC-MS and characterised.
(Silyl derivatives - most widely used derivatives for GC applications)

• Methylation adds a methyl group to all free hydroxyl groups on the sugar.
• Methanolysis will hydrolyse the sugar leaving a free hydroxyl group on the sugar which can
then be silylated.
• An acidic solution of methanol with the sample being heated at 80°C for 16–20 hours - a
method known as methanolysis.
Methanolysis – O-CH3 at C1 position

(Reagent – Trimethylsilyl chloride)
TMSi- Trimethylsilyloxy ether
The position of the TMSi

group will indicate the linkages on
the particular sugar
• Characterisation is based on the retention time, height of the peaks and molecular weight
• Anomers have to be identified based on the difference in retention time on the GC column.
• Analysis of Glycans; Polysaccharide Functional Properties: J.P. Kamerling, G.J. Gerwig,
in Comprehensive Glycoscience, 2007
• The combination of periodate oxidation, reduction, and mild acid hydrolysis is known as the
Smith Degradation.
• Periodate oxidation
• Reduction – sodium borohydride (NaBH4)
to reduce the aldehyde group to give the
corresponding alcohols.
• The end product (acetal) is sensitive to acid.
• Mild acid hydrolysis - 3-O-α-D-
glucopyranosyl - D-glycerol.
• Determination of linkage pattern

Carbohydrate Structural Elucidation

Uploaded by

Copyright:

Available Formats

Carbohydrate Structural Elucidation

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Carbohydrate Structural Elucidation

Uploaded by

Copyright:

Available Formats

Structural elucidation of carbohydrates

Strategy for polysaccharide structural analysis

• Monosaccharide composition: nature of the monosaccharide building blocks

• Sequences of monosaccharide residues and repeating units.

• Ring size: distinction of furanosidic and pyranosidic rings

• Anomeric configuration: α or β configuration of the glycosidic linkage

• Substitutions: position and nature of OH–modifications, such as O - phosphorylation,

• Polysaccharides - hydrolysed into oligosaccharides by acid, and detailed characterization of

Graded acid hydrolysis / Partial acid hydrolysis

• Hydrolysed by graded acid concentrations is applied to degrade the polysaccharide

• The hydrolysates - ultra filtration.

• The detailed structural characterisation of resulting oligosaccharides /disaccharides/

• Used to elucidate structural information of carbohydrates.

• Open-chain form - complete oxidative cleavage takes place.

• Under mild conditions (0.01 M to 0.05 M of HIO4).

• The amount of branching (number of (1→6) glycosidic bonds) in amylopectin can be

• A sample of amylopectin is exhaustively methylated—treated with a methylating agent (methyl

• Carbohydrates must therefore be converted into stable, volatile compounds prior to

dry dimethyl sulfoxide/silver oxide solution)

16–20 hours—a method known as methanolysis)

silylation reaction (trimethylsilyl [−Si(CH3)3] - trimethylsilyl chloride or bis(trimethylsilyl)

4. The derivatised sugars are separated by GC-MS and characterised.

(Silyl derivatives - most widely used derivatives for GC applications)

Methanolysis – O-CH3 at C1 position

TMSi- Trimethylsilyloxy ether

The position of the TMSi

You might also like