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, →3)-β- d-Ribf-(1 → 1)- d-Ribol-5-(PO ₂H→] 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 がL. The ratio (～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 ～92% regioselective, as indicated by the ～ 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 (～3–8%) that involved rearrangement of the d-ribofuranose phosphoric diester linkage and anomerization at C-1 of d-ribofuranose. Variously sized oligosaccharides (がL = 4, 7, and 12) that had d-ribofuranose end-groups reacted with bovine serum albumin that had an average of ～9 adipyl hydrazide functionalities, to give, within experimental error, quantitative yields of the corresponding, hydrazone-linked, oligosaccharide-protein conjugates.