Synthesis of tethered trisaccharides to probe inter-saccharide flexibility in carbohydrate-protein interactions

Two crystal structures of the trisaccharide epitope alpha-D-Galp(1-->2)[alpha-D-Abep(1-->3)alpha-D-Manp1-->OCH3 1 bound to antibody Fab and single-chain Fv fragments have been used to guide the design of an intramolecular tether that constrains the trisaccharide ligand to conformations close to those of the bound state. Preorganization of the ligand should overcome entropic penalties to binding and provide enhanced affinity. Three tethers [O(CH2)(n)O, n = 6, 7, and 8] were used to anchor the solvent-exposed C6 atoms of the mannose and galactose residues. Two synthetic schemes were employed. The first utilized an ethyl 1-thiogalactoside bearing the tether protected as a trityl ether 6-8. Glycosylation of the mannopyranoside 5 that contains a methanesulfonate at C6 by any of the donors 6-8 afforded disaccharides 37-39. Subsequent removal of the trityl group allowed an alkoxide to be generated on the omega-hydroxyl group of the tether for displacement of the sulfonate in 38% yield. By comparison when a omega-methanesulfonyloxy tether was incorporated as the mannoside 9, the disaccharide product 52 after reaction with the galactosyl donor 10 was converted to the macrocycle 4 in 61% yield. The 3,6-dideoxy-xylo-hexopyranose residue was introduced by thioglycoside chemistry to yield the protected, tethered trisaccharide target molecules 49-51, which were deprotected in a single hydrogenation step. Solid-phase enzyme immunoassays showed the tethered trisaccharides 2-4 to be weaker inhibitors than trisaccharide 1 (+0.2-0.3 kcal mol(-1)). This suggests that preorganization of oligosaccharides provides negligible gains in binding affinity.