Enzymatic synthesis of N-linked oligosaccharides terminating in multiple sialyl-Lewis x and GalNAc-Lewis x determinants: clustered glycosides for studying selectin interactions

Abstract

Galactosyltransferase, sialyltransferase, and fucosyltransferase were used to create a panel of complex oligosaccharides that possess multiple terminal sialyl-Le x (NeuAc α2–3Gal[Fuc α1–3] β1–4GlcNAc) and GalNAc-Le x (GalNAc[Fuc α1–3] β1–4GlcNAc). The enzymatic synthesis of tyrosinamide biantennary, triantennary, and tetraantennary N-linked oligosaccharides bearing multiple terminal sialyl-Le x was accomplished on the 0.5 μmol scale and the purified products were characterized by electrospray MS and 1H NMR. Likewise, biantennary and triantennary tyrosinamide oligosaccharides bearing multiple terminal GalNAc-Le x determinants were synthesized and similarly characterized. The transfer kinetics of human milk α3/4-fucosyltransferase were compared for biantennary oligosaccharide acceptor substrates possessing Gal β1–4GlcNAc, GalNAc β1–4GlcNAc, and NeuAc α2–3Gal β1–4GlcNAc which established NeuAc α2–3Gal β1–4GlcNAc as the most efficient acceptor substrate. The resulting complex oligosaccharides were chemically tethered through the tyrosinamide aglycone to the surface of liposomes containing phosphatidylthioethanol, resulting in the generation of glycoliposomes probe which will be useful to study relationships between binding affinity and the micro- and macro-clustering of selectin ligand.