Separation of partially desialylated branched oligosaccharide isomers containing alpha (2-->3)- and alpha (2-->6)-linked Neu5Ac.

The following two tri-sialylated triantennary oligosaccharides, which differ only in the linkage of the Neu5Ac to the uppermost branch were, individually, partially desialylated to produce all possible di- and mono-sialylated isomers. [formula: see text] A tetra-sialylated triantennary isomer, which contained an alpha (2-->6)-linked Neu5Ac to the GlcNAc on branch III, was also converted to all possible trisialylated isomers by mild acid hydrolysis as previously described (Roher et al., Anal. Biochem., 212, 7-16, 1993). The resulting branch isomers were separated using high-pH anion-exchange chromatography (HPAEC). Structures were assigned to peak fractions on the basis of the previously described effect of alpha (2-->6)- and alpha (2-->3)-linked Neu5Ac on the elution order of branched lactosamine-type oligosaccharides (Townsend et al., Anal. Biochem., 182, 1-8, 1989). No differences in the acid lability of the Neu5Ac linkage to either Gal (alpha (2-->3) or alpha (2-->6)) or GlcNAc (alpha (2-->6)) were observed. Our studies show that chemical desialylation and HPAEC is a useful approach to prepare and identify all possible sialylated branch isomers and should prove useful for defining the branch specificity of sialyltransferases and sialidases.