SYNTHESIS OF TETHERED TRISACCHARIDES TO PROBE INTER-SACCHARIDE FLEXIBILITY IN CARBOHYDRATE-PROTEIN INTERACTIONS

Two crystal structures of the trisaccharide epitope alp(1-->2)[alpha-D -Abep(1-->3)alpha-D-Manp1-->OCH3 1 bound to antibody Fab and single-ch ain Fv fragments have been used to guide the design of an intramolecul ar tether that constrains the trisaccharide ligand to conformations cl ose 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 so lvent-exposed C6 atoms of the mannose and galactose residues. Two synt hetic schemes were employed. The first utilized an ethyl 1-thiogalacto side bearing the tether protected as a trityl ether 6-8. Glycosylation of the mannopyranoside 5 that contains a methanesulfonate at C6 by an y of the donors 6-8 afforded disaccharides 37-39. Subsequent removal o f the trityl group allowed an alkoxide to be generated on the omega-hy droxyl group of the tether for displacement of the sulfonate in 38% yi eld. By comparison when a omega-methanesulfonyloxy tether was incorpor ated as the mannoside 9, the disaccharide product 52 after reaction wi th the galactosyl donor 10 was converted to the macrocycle 4 in 61% yi eld. The 3,6-dideoxy-xylo-hexopyranose residue was introduced by thiog lycoside chemistry to yield the protected, tethered trisaccharide targ et molecules 49-51, which were deprotected in a single hydrogenation s tep. Solid-phase enzyme immunoassays showed the tethered trisaccharide s 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.