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

N-Acetylglucosaminyltransferase-V (GlcNAcT-V) transfers a beta-linked GlcNAc residue from UDP-GlcNAc to the 6-OH group of the alpha Man residue in oligosaccharides terminating in the sequence beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->6)-beta-D-Glcp-OR (5, R = (CH2)(7)CH3). 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-alpha-D-glucose (10) and 1,2-anhydro-3,4,6-tri-O-benzyl-beta-D-manno (13) as donors for the glycosylations. The thio analogs (7, 8) were kinetically evaluated as substrates for GlcNAcT-V and found to be accepters with two- to three-fold increase in V-max but higher K-m values (7, K-m = 376 mu M; 8, K-m = 300 mu M) than their parent compound 6 (K-m = 111 mu M), 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.