Acceptor hydroxyl group mapping for calf thymus alpha-(1-->3)-galactosyltransferase and enzymatic synthesis of alpha-D-Galp-(1-->3)-beta-D-Galp-(1-->4)-beta D-GlcpNAc analogs.

The epitope of the acceptor substrate for alpha-(1-->3)-galactosyltransferase from calf thymus has been mapped by using a series of mono-deoxygenated and mono-O-alkylated Type II (beta-D-Ga1p-(1-->4)-beta-D-G1cpNAc) disaccharides. The 4-OH group of the beta-D-galactopyranosyl residue is a key polar group essential for glycosyl transfer, tolerating neither deoxygenation nor O-alkylation. Substitution at positions 6 and 6' by a variety of polar alkyl substituents was readily tolerated, allowing the preparative enzymatic synthesis of a series of trisaccharide derivatives carrying polar substituents on each of these hydroxyl groups. These new analogs are potential inhibitors of Clostridium difficile toxin A and of a human anti-alpha-Gal antibody.