Modulation of antibody affinity by synthetic modifications of the most exposed pyranose residue of a trisaccharide epitope.

When the Salmonella trisaccharide epitope, methyl 3-O-(3,6-dideoxy-alpha-D-xylo-hexopyranosyl)-2-O-(alpha-D-galactopyra nos yl)- alpha-D-mannopyranoside 12 is bound by a monoclonal antibody Se155.4, the 3,6-dideoxy-alpha-D-hexose is completely buried, while the galactopyranosyl mannopyranosyl units lay across the protein surface. Crystallography of an antibody complexed with 12 also shows that the galactose residue is the most exposed saccharide. A simplified strategy to synthesize 12 and analogues modified at the galactose residue is described. Monosaccharide building blocks containing benzyl ether and ester protecting groups were used for efficient assembly of trisaccharides that can be deprotected by a single hydrogenolysis step, or occasionally preceded by a transesterification stage. Glycosylation of methyl 2-O-benzoyl-4,6-di-O-benzyl-alpha-D- mannopyranoside 4 by 2,4-di-O-benzyl-3,6-dideoxy-D-xylo-hexopyranosyl chloride 8 affords after transesterification the disaccharide acceptor 10. This disaccharide serves as a universal acceptor for glycosylation by glycosyl donors that lead, following facile deprotection, to the alpha- and beta-D-galacto, alpha- and beta-D-gluco, and 2-amino-2-deoxy-alpha-D-galacto trisaccharides 12, 14, 17, 18 and 21. Only a small change in binding energy delta (delta G) occurs when the alpha-D-galactopyranosyl residue of 12 is replaced by either an alpha-D-glucopyranosyl 17 or a 2-amino-2-deoxy alpha-D-galactopyranosyl unit 21. Whereas binding of the beta-D-glucopyranose congener 18 was tolerated by the antibody, the beta-D-galactopyranose analogue 14, showed a 250 fold loss of affinity.(ABSTRACT TRUNCATED AT 250 WORDS)