| To investigate the optimal glycosyl ring conformation and other important features for inhibitor design of glycosidase enzymes, three classes of compounds were synthesised and tested as potential competitive reversible glycosidase inhibitors.;The kinetic and binding parameters were determined for various 2-deoxy- D-arabino-hexopyranosyl pyridinium salts. It was concluded that a hydrophobic aglycon could be utilised to enhance binding but had essentially no effect on catalysis. This led to the design of carbocyclic compounds possessing a hydrophobic aglycon. The target compounds (1 S,2S,3R,4S,5 S)-1-(hydroxymethyl)-5-(1'-indolyl)-1,2,3,4-cyclohexane-tetraol, (1S,2S,3R,4S,5 S)-1-(hydroxymethyl)-5-isoquinolinium-1,2,3,4-cyclohexanetetraol bromide and valienamine isoquinolinium bromide were synthesised and tested as glycosidase inhibitors. The results further illustrate the utility of a hydrophobic aglycon towards inhibitor binding.;A second class of compound based on the bicylo[2.2.2]octane framework was investigated. The compound 2,6-anhydro-1-deoxynojirimycin (2.5) locked in a 2,5B boat conformation, was chosen to investigate the optimum glycosyl ring conformation. Although the K i values for 2.5 were high, compounds of this nature are particularly interesting in view of the recently reported 2,5B boat conformations for covalently bound intermediates with xylanase enzymes. A third class of compounds was designed based on the bicyclo[4.1.0]heptane skeleton. The compounds (1R,2R,3 S,4S,5S,6S)-5-Amino-1-(hydroxymethyl)bicyclo[4.1.0]-2,3,4-heptanetriol (4.1) and (1S,2R,3 S,4S,5S,6R)-5-Acetamido-1-(hydroxymethyl)bicyclo[4.1.0]-2,3,4-heptanetriol (4.2) were synthesised. The fused cyclopropyl rings of the carbocyclic compounds force the 6-membered rings to adopt 2H3 half-chair conformations. Analysis of the inhibition kinetic data found 4.1 to be the most potent inhibitor of yeast alpha-glucosidase reported. The results from this study clearly illustrate the importance of a positive charge and the appropriate glycosyl ring conformation.;In conjunction with the above inhibition study a kinetic isotope effect study was also conducted on the spontaneous and yeast alpha-glucosidase-catalysed hydrolysis of various isotopomers of alpha-D-glucopyranosyl pyridinium salts. The results from this mechanistic study suggest that there is a nucleophilic component for the enzyme catalysed hydrolysis reaction, which is absent in the spontaneous hydrolysis reaction. It is also apparent from this study that mechanistic generalisations cannot be made when comparing substrates with different leaving groups in glucosidase-catalysed hydrolysis reactions. |
