Synthesis of the acceptor analog alphaFuc(1-->2)alphaGal-O(CH2)7 CH3: a probe for the kinetic mechanism of recombinant human blood group B glycosyltransferase.

We report the chemical synthesis of alphaFuc(1-->2)alphaGal-O(CH2)7CH3 (1) an analog of the natural blood group (O)H disaccharide alphaFuc(1-->2)betaGal-OR. Compound 1 was a good substrate for recombinant blood group B glycosyltransferase (GTB) and was used as a precursor for the enzymatic synthesis of the blood group B analog (alphaGal(-->3)alphaFuc(1-->2)]alphaGal-O(CH2)7CH3+ ++ (2). To probe the mechanism of the GTB reaction, kinetic evaluations were carried out employing compound 1 or the natural acceptor disaccharide alphaFuc(1-->2)betaGal-O(CH2)7CH3 (3) with UDP-Gal and UDP-GalNAc donors. Comparisons of the kinetic constants for alternative donor and acceptor pairs suggest that the GTB mechanism is Theorell-Chance where donor binding precedes acceptor binding. GTB operates with retention of configuration at the anomeric center of the donor. Retaining reactions are thought to occur via a double-displacement mechanism with formation of a glycosyl-enzyme intermediate consistent with the proposed Theorell-Chance mechanism.