Dale A. Cumming

Separation of branched sialylated oligosaccharides using high-pH anion-exchange chromatography with pulsed amperometric detection

Ten characterized sialylated oligosaccharides from bovine fetuin (B. Bendiak, M. Harris-Brandts, S. W. Michnick, J. P. Carver, and D. A. Cumming, Biochemistry, in press; and D. A. Cumming, C. G. Hellerqvist, M. Harris-Brandts, S. W. Michnick, J. P. Carver, and B. Bendiak, Biochemistry, in press) were chromatographed using high-performance anion-exchange chromatography with pulsed amperometric detection. At near neutral pH values, oligosaccharides were separated according to their number of formal negative charges from sialic acid; however, at alkaline pH, the neutral portion of the oligosaccharides enhanced resolution due to oxyanion formation. Specifically, trisialylated triantennary oligosaccharides containing a Gal-beta(1,3)GlcNAc sequence were more retained and could be completely separated from those having only Gal-beta(1,4)GlcNAc units. Oligosaccharides containing the same number of sialic acids were separated according to the combination of alpha(2,6)- and alpha(2,3)-linked sialic acids (alpha(2,6)-linked sialic acid reduced retention time). The relative molar electrochemical responses for di-, tri-, tetra-, and pentasialylated oligosaccharides were found to be similar (4.8 +/- 14% relative to glucose). Coelution studies were performed with each of the characterized oligosaccharides and the mixture of oligosaccharides which were released from fetuin with N-glycanase. The relative proportion of the major classes of sialylated oligosaccharides (bi-, tri-, tetra-, and penta-) varied significantly in bovine fetuin from different sources.

Hydrazinolysis-N-reacetylation of glycopeptides and glycoproteins. Model studies using 2-acetamido-1-N-(l-aspart-4-oyl)-2-deoxy-β-d-glucopyranosylamine

2-Acetamido-1-N-(L-aspart-4-oyl)-2-deoxy-beta-D-glucopyranosyla mine (1) was used as a model glycopeptide to study the hydrazinolysis-N-reacetylation procedure. The major, initial product was the beta-acetohydrazide derivative of 2-acetamido-2-deoxy-D-glucose (2) which gave 2-acetamido-2-deoxy-D-glucose (5) after exposure to acidic conditions. Very mild conditions of hydrolysis of 2 gave a 75-80% overall yield of 5 from 1 after the hydrazinolysis-N-reacetylation procedure. Several other minor compounds were detected which were not converted into 5 upon mild acid hydrolysis, indicating that 20-25% of product cannot be recovered as 5 at the reducing end of oligosaccharides.

Purification of oligosaccharides having a free reducing-end from glycopeptide sources

A hydrazinolysis-N-reacetylation procedure, modified by the inclusion of a mild acid-hydrolysis step after N-acetylation, was used to prepare, in overall yields of 60-70%, pure oligosaccharides containing a reducing D-GlcNAc residue from glycopeptide sources. Three types of asparagine-linked glycopeptides were treated: a high-mannose type, a complex-type not containing sialic acid, and a complex-type containing sialic acid, linked both alpha-(2----3) and alpha-(2----6) to beta-D-Galp residues. After the hydrazinolysis-N-reacetylation procedure, there was often contamination of the reducing oligosaccharides with glycopeptide that remained intact through the procedure, as well as minor oligosaccharide products, altered in the nature of the residue at the reducing end. Oligosaccharides having a reducing D-GlcNAc residue were purified by standard liquid chromatography and high-pressure liquid chromatography (l.c.) 360-MHz 1H-n.m.r. was valuable in establishing common structural reporter signals which enabled major products to be identified at stages during the production of free reducing oligosaccharides, and their purity to be assessed.

On the utility of 13C-n.m.r. spectroscopy in the identification of the primary structures of manno-oligosaccharides and glycopeptides

The utility of 13C-n.m.r. spectroscopy in the identification of the primary structures of mannose-containing glycans is investigated. Unlike 1H resonances where the chemical shifts reflect multiple short- and long-range effects, the chemical shifts of 13C resonances are dependent largely upon short-range effects classified as glycosylation (linkage) and substitution effects. These effects are parametized for glycans composed of mannose and encoded in a FORTRAN algorithm. Applications of this program to unknown sets of experimental chemical shifts for the resonances of anomeric carbons gave the following conclusions. (1) This program can be used to produce a sub-set of possible structures inclusive of the known structure. (2) For other than simple oligosaccharides, it is unlikely that a single structure is consistent with the data for anomeric carbons alone, even when the linkage composition of the glycan has been assessed from other spectral data. (3) When used in conjunction with other chemical techniques, this program can provide a powerful tool for primary analysis of the structure of mannose-containing glycans.