|dc.description.abstract||Herein we describe a novel methodology for the synthesis of tri-antennary complex N-glycans. This methodology entails the use of a chemoenzymatic approach whereby a core pentasaccharide functionalized with the four orthogonal protecting groups - levulinoyl (Lev), fluorenylmethyloxycarbonate (Fmoc), allyloxycarbonate (Alloc), and 2-naphthylmethyl (Nap), at key branching positions, was synthesized chemically. Upon selective deprotection of those orthogonal protecting groups, three unique saccharide structures were attached by chemical glycosylations to form two decasaccharides with different branching patterns. The decasaccharides were used as precursors for enzymatic extension using glycosyltransferases, which yielded a library of various symmetrical and asymmetrical complex N-glycans.
The novelty of this approach enatils the judicious manipulation of the substrate specificities of various glycosyltransferases, which donot recognize N-acetyllactosmine masked with acetyl protecting groups. Furthermore, certain glycosyltransferases do not recognize terminal N-acetylglucosamine residues as a substrate and hence renders it inactive towards glycosylation. However upon removal of the acetyl esters, and conversion of the N- acetylglucosamine residue to an N-acetyllactosmine, it would serve as a substrate for
enzymatic modification for further elaboration of the glycan. Using this novel chemoenzymatic approach, a library of glycans were synthesized, which were printed as microarrays and screened for binding to lectins and influenza-virus hemagglutinins, which showed that recognition is modulated by presentation of minimal epitopes in the context of complex N-glycans. We employed the use of our novel chemoenzymatic methodology for the synthesis of triantennary glycans found on human egg cell zona pellucidas (ZPs), to study the binding affinities of the ZP glycans to the sperm. The preliminary results indicated that the presence of Sialyl Lewisx (SLex) on an extended branch as a SLexLex moiety enhanced the inhibition.
We also, report the chemical synthesis of the core trisaccharide-glycopeptide found as a posttranslational modification on a specific hydroxyproline (HyPro) residue of the protein Skp1, in an organism called Dictyostelium. The trisaccharide-HyPro and trisaccharide- glycopeptide were used to study the substrate specificity for the enzyme AgtA in order to ascertain its substrate specificity. The synthetic trisaccharide-glycopeptide was conjugated to a carrier protein (KLH), which will be used for generating monoclonal antibodies to study glycosylation changes during development in Dictyostelium.||