Synthesis and evaluation of thio-trisaccharides as acceptors for N-acetylglucos-aminyltransferase-V

N-Acetylglucosaminyltransferase-V (GlcNAcT-V) transfers a β-linked GlcNAc residue from UDP-GlcNAc to the 6-OH group of the αMan residue in oligosaccharides terminating in the sequence β- D -GlcpNAc-(1 → 2)-α- D -Manp-(1 → 6)-β- D -Glcp-OR ( 5 , R = (CH 2 ) 7 CH 3 ). The terminal GlcNAc moiety may be replaced by a Glc residue to produce trisaccharide 6 . Two thio analogs ( 7 , 8 ) of trisaccharide 6 , where the oxygen atoms in the glycosidic linkages between sugar residues were replaced by sulfur, were prepared by multistep chemical synthesis that made use of the key intermediates 1,2-anhydro-3,4,6-tri-O-benzyl-α- D -glucose ( 10 ) and 1,2-anhydro-3,4,6-tri-O-benzyl-β- D -mannose ( 13 ) as donors for the glycosylations. The thio analogs ( 7 , 8 ) were kinetically evaluated as substrates for GlcNAcT-V and found to be acceptors with two- to three-fold increase in V max but higher K m values ( 7 , K m = 376 μM; 8 , K m = 300 μM) than their parent compound 6 (K m = 111 μM), which has the natural oxygen linkage. The thio analogs 7 and 8 could be quantitatively converted into the expected product tetrasaccharides ( 27 , 28 ) by incubation with GlcNAcT-V and UDP-GlcNAc. The enzymatic results indicate that GlcNAcT-V tolerates the substitution of the natural oxygen linkage of the acceptor by a sulfur linkage. Keywords: N-acetylglucosaminyltransferase-V, enzyme acceptors, trisaccharide analogs, thioglycosides.