Synthesis of a di-O-methylated pentasaccharide for use in the assay of N-acetylglucosaminyltransferase III activity.

The biantennary oligosaccharide analogue beta-D-Glc pNAc-(1-->2)-alpha-D-Man p-(1-->3)-[beta-D-Glc pNAc-(1-->2)-alpha- D-Man p-(1-->6)]-beta-D-man p-O(CH2)8COOMe (3) is a potential substrate for N-acetylglucosaminyltransferases (GlcNAcTs) III-V which are present in mammalian cells. The di-O-methylated analogue of 3, beta-D-Glc pNAc-(1-->2)-[4-O-methyl-alpha-D-Man p]-(1-->3)-[beta-D-Glc pNAc-(1-->2)-[6-O-methyl-alpha-D-Man p]-(1-->6)]-beta-D-Man p-O-(CH2)8COOMe (5), was prepared by a block synthesis approach involving sequential addition of two O-methylated disaccharide donors to a protected central beta-D-Man residue. The OH groups acted on by GlcNAcT-IV and -V are protected from glycosylation in 5 since they are present as their methyl ethers. Pentasaccharide 5 was found to be an excellent substrate for GlcNAcT-III (EC 2.4.1.144) from rat kidney with Km = 0.15 mM. The product formed by incubation of 5 with a rat kidney extract, in the presence of UDP-GlcNAc, was isolated, structurally characterized by NMR spectroscopy and confirmed to be the expected di-O-methyl hexasaccharide where a beta-D-Glc pNAc residue had been added to OH-4 of the central beta-D-Man p unit.