9-Fluorenylmethoxycarbonyl (Fmoc)-glycine coupling of saccharide beta-glycosylamines for the fractionation of oligosaccharides and the formation of neoglycoconjugates.

We have devised a new saccharide derivatization scheme to provide not only the temporary attachment of a chromophore for detecting and facilitating the chromatographic separation of carbohydrates, but also the intermediates for further derivatization to produce neoglycoconjugates. Several neutral unprotected saccharide beta-glycosylamines were formed by the direct condensation of the reducing saccharides with aqueous ammonium bicarbonate. The beta-glycosylamine derivatives of N-acetylglucosamine, di-N-acetylchitobiose, and asialo-, digalactosylated biantennary complex oligosaccharide were N-acylated separately with excess 9-fluorenylmethoxycarbonyl (Fmoc)-glycine. The Fmoc-glycinamido beta-derivatives of these unprotected saccharides were well separated by normal-phase high-performance liquid chromatography and detected by ultraviolet absorption. Similar derivatization and fractionation of a partial acid hydrolyzate of chitin were equally successful resulting in the separation of Fmoc-glycinamido derivatives of di-N-acetylchitobiose to hepta-N-acetylchitoheptaose in the hydrolyzate. The reversibility of the Fmoc derivatization was demonstrated by treating the Fmoc-glycinamido derivative of N-acetylglucosamine with piperidine to generate its 1-N-glycyl-beta-saccharide derivative. The structure and stereochemistry of this product was confirmed by proton nuclear magnetic resonance spectroscopy. The 1-N-glycyl-beta-saccharide derivatives are stable intermediates for the formation of asparagine-linked neoglycoconjugates.