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Design, synthesis and evaluation of high affinity oligosaccharide ligands

by Gagné, Rodney A.

Abstract (Summary)
A monodonal antibody, SYA/J6, that was produced against the lipopolysaccharide layer of variant Y Shigella jemmi was used as a mode1 to investigate carbohydrate -protein interactions. Crystal structures of this protein and bound Iigands, 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 hydrophobie residues, to the core disaccharide epitope oia a two-carbon Mer m. The synthesis of seven test ligands was cded out and it was found using a solid ,hase competitive assay that three of the ligands had higher affinities to the protein than the native disaccharide 49. The best inhibitor, 4, had an assoaation constant that was 50 times better than 49, but an order of magnitude less than the native trisaccharide 3. 4- OnPr NHAc dao \NHAc HO HO OH OH 4 49 The second strategy involved the addition of intrarnolecular constraints to the native trisacdiaride structure to decrease its flexibility in solution. The synthesis of three tethered trisaccharides was accompkhed. 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 wexe improved with this design strategy, relative to 3. NHAc HO OH 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 dynarnics simulations revealed that this ligand was more rigid in solution than 3. Distances obtained from nOe enhancements substantiated the results of molecular modeling.
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Source Type:Master's Thesis

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Date of Publication:01/01/2000

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