Studies of the hydrolysis mechanisms of sialosides and synthesis and evaluation of a bicyclo[4.1.0]heptyl analogue of glucose -1 -phosphate.

摘要

Sialic acids, which are nine-carbon acidic keto sugar residues, are very often terminally linked to complex oligosaccharide chains in living organisms. Their highly exposed positions allow them to be actively involved in cell-cell and cell-protein recognition processes. Sialidases are enzymes that catalyze the hydrolysis of natural a linked sialosides of these glycoconjugates with retention of configuration going via a tyrosinyl beta-sialoside intermediate.;A panel of seven aryl beta-D-sialosides, were synthesized and characterized in order to study their hydrolysis mechanisms in aqueous medium. 4-nitrophenyl beta-D-N-sialoside was chosen for a comparative mechanistic study with its corresponding alpha-anomer. It was found that the beta anomer was more than hundred times more stable than the alpha-anomer. Detailed kinetic and product studies pointed towards a dissociative mechanism (SN1) for both anomers with solvation of the carboxylate moiety being the driving force in the hydrolysis reaction. Our result disproves the alpha lactone intermediate proposed for the alpha anomer by Ashwell et al. The biological significance of these results will be discussed. The aryl beta-D-sialosides were screened against the Y370G mutant of M. viridifaciens and it was found that 3-chlorophenyl beta-D-sialoside was the best inhibitor with a Ki 1.7 muM.;Finally, the synthesis of a bicyclo[4.1.0]heptyl analogue of glucose-1-phosphate, (1R, 2R, 3S, 4 S, 5S, 6S)-5-phosphate-2,3,4-trihydroxy-1-hydroxymethyl)-bicyclo[4.1.0]heptan-2-yl dihydrogen phosphate (4.5) in eleven steps is reported. Methyl alpha- D-glucopyranoside was used as starting material. Compound 4.5 was tested as a potential substrate of UTP: alpha-D-glucose-1-phosphate uridylyltransferase, the enzyme that converts alpha-D-glucose 1-phosphate into UDP-glucose. However, compound 4.5 was found to be a weakly binding inhibitor (12% inhibition at a concentration of 0.1 mM).;A series of aryl alpha-D-sialosides were synthesized in order to characterize the wild type M. viridifaciens enzyme and its mutants Y370G and D92G. The tyrosine residue, the nucleophile, formed the beta-linked enzyme-substrate intermediate in the active site while the aspartate residue acted as the general acid base catalyst.