7.5 Working with Carbohydrates Lecture
SUMMARY 7.5 Working with Carbohydrates
■ Establishing the complete structure of oligosaccharides and polysaccharides requires
determination of the linear sequence, branching positions, the configuration of each
monosaccharide unit, and the positions of the glycosidic linkages—a more complex problem
than protein and nucleic acid analysis.
■ The structures of oligosaccharides and polysaccharides are usually determined by a
combination of methods: specific enzymatic hydrolysis to determine stereochemistry at the
glycosidic bond and to produce smaller fragments for further analysis; methylation to locate
glycosidic bonds; and stepwise degradation to determine sequence and configuration of
anomeric carbons.
■ Mass spectrometry and high-resolution NMR spectroscopy, applicable to small samples of
carbohydrate, yield essential information about sequence, configuration at anomeric and
other carbons, and positions of glycosidic bonds.
■ Solid-phase synthetic methods yield defined oligosaccharides that are of great value in
exploring lectin-oligosaccharide interactions and may prove clinically useful.
■ Microarrays of pure oligosaccharides are useful in determining the specificity and affinity
of lectin binding to specific oligosaccharides.
A growing appreciation of the importance of oligosaccharide structure in biological signaling
and recognition has been the driving force behind the development of methods for analyzing
the structure and stereochemistry of complex oligosaccharides. Oligosaccharide analysis is
complicated by the fact that, unlike nucleic acids and proteins, oligosaccharides can be
branched and are joined by a variety of linkages. The high charge density of many
oligosaccharides and polysaccharides, and the relative lability of the sulfate esters in
glycosaminoglycans, present further difficulties.
For simple, linear polymers such as amylose, the positions of the glycosidic bonds are
determined by the classical method of exhaustive methylation: treating the intact
polysaccharide with methyl iodide in a strongly basic medium to convert all free hydroxyls to
acid-stable methyl ethers, then hydrolyzing the methylated polysaccharide in acid.
The only free hydroxyls in the monosaccharide derivatives so produced are those that were
involved in glycosidic bonds. To determine the sequence of monosaccharide residues,
including any branches that are present, exoglycosidases of known specificity are used to
remove residues one at a time from the nonreducing end(s). The known specificity of these
exoglycosidases often allows deduction of the position and stereochemistry of the linkages.
FIGURE 7-38 Methods of carbohydrate analysis. A carbohydrate purified in the first
stage of the analysis often requires all four analytical routes for its complete
characterization.
For analysis of the oligosaccharide moieties of glycoproteins and glycolipids, the
oligosaccharides are released by purified enzymes— glycosidases that specifically cleave O-
SUMMARY 7.5 Working with Carbohydrates
■ Establishing the complete structure of oligosaccharides and polysaccharides requires
determination of the linear sequence, branching positions, the configuration of each
monosaccharide unit, and the positions of the glycosidic linkages—a more complex problem
than protein and nucleic acid analysis.
■ The structures of oligosaccharides and polysaccharides are usually determined by a
combination of methods: specific enzymatic hydrolysis to determine stereochemistry at the
glycosidic bond and to produce smaller fragments for further analysis; methylation to locate
glycosidic bonds; and stepwise degradation to determine sequence and configuration of
anomeric carbons.
■ Mass spectrometry and high-resolution NMR spectroscopy, applicable to small samples of
carbohydrate, yield essential information about sequence, configuration at anomeric and
other carbons, and positions of glycosidic bonds.
■ Solid-phase synthetic methods yield defined oligosaccharides that are of great value in
exploring lectin-oligosaccharide interactions and may prove clinically useful.
■ Microarrays of pure oligosaccharides are useful in determining the specificity and affinity
of lectin binding to specific oligosaccharides.
A growing appreciation of the importance of oligosaccharide structure in biological signaling
and recognition has been the driving force behind the development of methods for analyzing
the structure and stereochemistry of complex oligosaccharides. Oligosaccharide analysis is
complicated by the fact that, unlike nucleic acids and proteins, oligosaccharides can be
branched and are joined by a variety of linkages. The high charge density of many
oligosaccharides and polysaccharides, and the relative lability of the sulfate esters in
glycosaminoglycans, present further difficulties.
For simple, linear polymers such as amylose, the positions of the glycosidic bonds are
determined by the classical method of exhaustive methylation: treating the intact
polysaccharide with methyl iodide in a strongly basic medium to convert all free hydroxyls to
acid-stable methyl ethers, then hydrolyzing the methylated polysaccharide in acid.
The only free hydroxyls in the monosaccharide derivatives so produced are those that were
involved in glycosidic bonds. To determine the sequence of monosaccharide residues,
including any branches that are present, exoglycosidases of known specificity are used to
remove residues one at a time from the nonreducing end(s). The known specificity of these
exoglycosidases often allows deduction of the position and stereochemistry of the linkages.
FIGURE 7-38 Methods of carbohydrate analysis. A carbohydrate purified in the first
stage of the analysis often requires all four analytical routes for its complete
characterization.
For analysis of the oligosaccharide moieties of glycoproteins and glycolipids, the
oligosaccharides are released by purified enzymes— glycosidases that specifically cleave O-
