Complete enzymic synthesis of the mucin-type sialyl Lewis x epitope, involved in the interaction between PSGL-1 and P-selectin

Sialyl Lewis x (sLe(x)) is an established selectin ligand occurring on N- and O-linked glycans. Using a completely enzymic approach starting from p-nitrophenyl N-acetyl-alpha-D-galactosaminide (GalNAc(alpha 1-pNp as core substrate, the sLe(x)-oligosaccharide Neu5Ac(alpha 2-3)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-6)[Gal(beta 1-3)]GalNAc(alpha 1-pNp, representing the O-linked form, was synthesized in an overall yield of 32%. In a first step, Gal(beta 1-3)GalNAc(alpha 1-pNp was prepared in a yield of 52% using UDP-Gal and an enriched preparation of beta 3-galactosyltransferase (EC 2.4.1.122) from rat liver. UDP-GlcNAc and a recombinant affinity-purified preparation of core 2 beta 6-N-acetylglucosaminyltransferase (EC 2.4.1.102) fused to Protein A were used to branch the core 1 structure, affording GlcNAc(beta 1-6)[Gal(beta 1-3)]GalNAc(alpha 1-pNp in a yield of > 85%. The core 2 structure was galactosylated using UDP-Gal and purified human milk beta 4-galactosyltransferase 1 (EC 2.4.1.38) (yield of > 85%), then sialylated using CMP-Neu5Ac and purified recombinant alpha 3-sialyltransferase 3 (EC 2.4.99.X) (yield of 87%), and finally fucosylated using GDP-Fuc and recombinant human alpha 3-fucosyltransferase 6 (EC 2.4.1.152) produced in Pichia pastoris (yield of 100%). Overall 1.5 mu mol of product was prepared. MALDI TOF mass spectra, and 1D and 2D TOCSY and ROESY H-1 NMR analysis confirmed the obtained structure.