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.

Abstract 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 1 H and 13 C 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.