| A monoclonal antibody, SYA/J6, that was produced against the lipopolysaccaride layer of variant Y Shigella flexneri was used as a model to investigate carbohydrate-protein interactions. Crystal structures of this protein and bound ligands, that showed the position of the trisaccharide Rha-Rha-GlcNAc, enabled the use of two different strategies to design tighter binding, low molecular weight ligands.; The first strategy involved the addition of novel contacts, such as putative hydrogen bond and hydrophobic residues, to the core disaccharide epitope via a two-carbon linker arm. The synthesis of seven test ligands was carried out and it was found using a solid phase competitive assay that three of the ligands had higher affinities to the protein than the native disaccharide 49. The best inhibitor, 4, had an association constant that was 50 times better than 49, but an order of magnitude less than the native trisaccharide 3.*; The second strategy involved the addition of intramolecular constraints to the native trisaccharide structure to decrease its flexibility in solution. The synthesis of three tethered trisaccharides was accomplished. Results from a solid phase assay indicated that one of the ligands, 51, was a better inhibitor than 3 by a factor of three. Microcalorimetry measurements provided a more accurate factor of twenty for this ligand. It was found that both the enthalpy and entropy of binding were improved with this design strategy, relative to 3.*; To account for the improvement in binding seen in biological assays, minimum energy calculations using the AMBER_PLUS forcefield were performed. The results indicated that 51 adopted a conformation that was similar to 3 in solution. Both 3 and 51 showed good overlap with the bound ligand in the crystal structures. Molecular dynamics simulations revealed that this ligand was more rigid in solution than 3. Distances obtained from nOe enhancements substantiated the results of molecular modeling.; *Please refer to dissertation for diagrams. |
