31P- and 13C-n.m.r.-spectral and chemical characterization of the end-group and repeating-unit components of oligosaccharides derived by acid hydrolysis of Haemophilus influenzae type b capsular polysaccharide.

Haemophilus influenzae type b capsular polysaccharide [repeating unit, leads to 3)-beta-D-Ribf-(1 leads to 1)-D-Ribol-5-(PO2H leads to] was partially hydrolyzed with HCl to give oligosaccharides that were isolated by size-exclusion chromatography, and then characterized by 31P- and 13C-n.m.r.-spectral and chemical methods, in order to determine the end-group composition and, hence, the number-average chain-length (L). The ratio (approximately 17:8) of monophosphate end-groups to D-ribofuranose end-groups revealed the relative rates of hydrolysis of the phosphoric diester linkage and the glycosidic linkage in the repeating-unit structure. Cleavage of the phosphoric diester linkage was approximately 92% regioselective, as indicated by the approximately 12:1 ratio of D-ribofuranose monophosphate end-groups to D-ribitol monophosphate end-groups. The n.m.r. spectra of the oligosaccharide repeating-unit provided evidence for partial stereomutation (approximately 3-8%) that involved rearrangement of the D-ribofuranose phosphoric diester linkage and anomerization at C-1 of D-ribofuranose. Variously sized oligosaccharides (L = 4, 7, and 12), that had D-ribofuranose end-groups reacted with bovine serum albumin that had an average of approximately 9 adipyl hydrazide functionalities, to give, within experimental error, quantitative yields of the corresponding hydrazone-linked, oligosaccharide-protein conjugates.