Elucidation of oligosaccharide function by use of chemical synthesis of glycoprotein

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We developed convenient synthetic method of asparagine-linked sialylglycopeptides. The typical feature of our strategy for the synthesis of a sialylglycopeptide is to employ undecadisialyloligosaccharyl Fmoc-asparagine (Fmoc-Asn(CHO)-OH) without protecting groups on its hydroxyl groups except for the benzyl ester of NeuAc residues. Our synthetic methodology solved the problem of esterification toward sugar hydroxyl groups by activated amino acid during the elongation of a peptide chain. When employing high concentrations of Fmoc-amino acid, esterification markedly occurred, but the esterification scarcely occurred when employing low concentrations of reactants. Taking advantage of these findings, we examined the synthesis of a high molecular sialylglycopeptide, CTLA-4 fragment (113-150) having two complex-type sialyloligosaccharides, by use of native chemical ligation (NCL). As a result, we succeeded in the synthesis of a sialylglycopeptide having a cysteine residue at the N-terminus (CTLA-4:129-150 fragment) and a sialylglycopeptide-thioester (CTLA-4:113-128 fragment). Finally, these synthesized sialylglycopeptides were applied to NCL reaction and the reaction successfully afforded a desired product, CTLA-4 (113-150), having mature and pure complex-type sialyloligosaccharides in excellent purity.