ABSTRACT
After N-sulfation, GlcA may be epimerized at C5 into IdoA. An important consequence of this reaction is that IdoA, unlike GlcA or the adjacent glucosamine residue, exists in equilibrium between different conformations. Epimerization thus gives the sugar chain new structural properties. The IdoA may be 2-O-sulfated at the C2 position, and this may also occur to a lesser extent on GlcA. The 2-O-sulfated hexuronic acids are almost always found in contiguous NS domains of the polysaccharide. The 2-O-sulfotransferase has been shown to form molecular complexes with the C5-epimerase. Three HS glucosamine 6-O-sulfotransferases have been identified, with sulfate at C6 on glucosamine residues adjacent to NS glucosamine [15]. Sulfation at C3 of glucosamine by one of the five 3-O-sulfotransferases, though rare, is critical for forming structures with anticoagulant activity and more recently has been found to be important for at least some other activities too [15]. 16.3 Oligosaccharide PreparationThe determination of the detailed structures of heteropolysaccha-rides such as heparin and HS, including the positions and anomeric configurations of the glycosidic linkages, can only be accomplished on small fragments, obtained by depolymerization and purification. By virtue of the presence of the unique N-sulfate groups in heparin and HS, the polysaccharide may be degraded by chemical methods, which specifically targets these groups. Thus, deaminative cleavage by nitrous acid cleaves at NS glucosamines or, under more gentle conditions, at N-unsubstituted glucosamine [16]. In addition, enzy-matic depolymerization by bacterial lyases allows further sequencespecific cleavage of the sugar chains. Together, these methods have allowed heparin researchers to degrade the polysaccharide to frag-ments of various sizes, which are amenable to structural and func-tional analysis. 16.3.1 Nitrous Acid CleavageHeparin and HS can be depolymerized with nitrous acid at pH 1.5. The modification of unsubstituted glucosamine or GlcNS residues by nitrous acid forms pair-wise oligosaccharides with 2,5-anhydro-D-mannose derivatives at the reducing end [17]. Treatment of nitrous acid cleaves 2-amino-2-deoxy-D-glucosidic bonds, initiated
by nitrosation of the amino group of the sugar. In this reaction, the GlcNSO3 residues are converted to 2,5-anhydro-D-mannose residues. The N-sulfates and the amino groups of GlcNSO3 residues are released as SO2-4 and N2 [16, 18]. Nitrous acid is prepared by addition of an inorganic nitrite salt to a solution of acid. Shively and Conrad stated that different lower concentrations preparations of nitrous acid will cleave D-glucosamine glycosides only when the amino group of the D-glucosamine is unsubstituted, while more concentrated solutions will cleave also the glycosides of NS D-glucosamine. However, all cleavages of amino sugar glycosides convert the D-glucosamine residue to an anhydro-D-mannose residue, which becomes the new reducing terminal of the oligosaccharide formed in the deamination reaction [16]. The advantage of using nitrous acid scission is that it is useful in increasing the reactivity of the oligosaccharide products toward nucleophiles at the reducing end of anhydromannose residues. However, the problem is that it is difficult to detect the oligosaccharides because these oligosaccharides lack strong chromophores [18].
