Synthesis of pentasaccharide analogues of the N-glycan substrates of N-acetylglucosaminyltransferases III, IV and V using tetrasaccharide precursors and recombinant β-(1 → 2)-N-acetylglucosaminyltransferase II

Recombinant human UDP-GlcNAc:α-Man-(1 → 6)R β-(1 → 2)-N-acetylglucosaminyltransferase II (EC 2.4.1.143, GlcNAc-T II) was produced in the Sf9 insect cell/baculovirus expression system as a fusion protein with a (His)6 tag and partially purified by affinity chromatography on a metal chelating column. The partially purified enzyme was used to catalyze the transfer of GlcNAc from UDP-GlcNAc to R-α-Man(1 → 6)(β-GlcNAc(1 → 2)α-Man(1 → 3))β-Man-O-octyl to form β-GlcNAc(1 → 2)R-α-Man(1 → 6)(β-GlcNAc(1 → 2)α-Man(1 → 3))β-Man-O-octyl where there is either no modification of the α-Man(1 → 6) residue (7), or where R is 3-deoxy (8), 4-deoxy (9) or 6-deoxy (10). The yields ranged from 64–80%. Products were characterized by 1H and 13C nuclear magnetic resonance spectroscopy and fast atom bombardment mass spectrometry. Compounds 7–10 are pentasaccharide analogues of the biantennary N-glycan substrates of N-acetylglucosaminyltransferases III, IV and V.