ANALYSIS OF CARBOHYDRATE-PROTEIN INTERACTIONS WITH SYNTHETIC N-LINKEDNEOGLYCOCONJUGATE PROBES

Recently we have described a simple efficient chemical method of gener ating an asparagine side-chain linker with beta-stereochemistry at the anomeric position of neutral oligosaccharides. We now report the 1-N- glycyl beta-derivatization of sialylated saccharides. Several neoglyco conjugates formed using these N-linked intermediates were investigated for their usefulness in probing carbohydrate-protein interactions. Fi rst, biotinyl derivatives of two xylose/fucose class plant-type oligos accharides purified from horseradish peroxidase were effective in demo nstrating the carbohydrate specificity of polyclonal anti-(horseradish peroxidase) antibodies. Secondly, a fluorescein-labelled asialo- and digalactosylated biantennary complex sugar was synthesized and shown t o bind to a Ricinus communis agglutinin column. This galactose-specifi c recognition was abolished by treating this fluorescein-labelled olig osaccharide with jack bean beta-galactosidase. Finally, two 1-N-glycyl beta-saccharide derivatives were modified with thiophosgene to form t heir corresponding isothiocyanate derivatives. Coupling of these isoth iocyanate derivatives of sugars to BSA, amino-derivatized polystyrene plates and glass-fibre discs resulted in multiple sugar presentation. The binding of an anti-N-acetylglucosamine monoclonal antibody to N,N' -diacetylchitobiose residues presented on BSA and solid supports was s hown by e.l.i.s.a. Similarly the binding of concanavalin A to asialo-, agalactosylated biantennary complex oligosaccharide residues attached to BSA was demonstrated by a competitive e.l.i.s.a. Our results demon strate that N-linked neoglycoconjugates could be made readily availabl e and they are valuable tools for the detailed analyses of carbohydrat es and carbohydrate-binding proteins.