Synthesis and enzymatic evaluation of alpha-galactosidase inhibitors

The compounds containing a structural motif of a cyclopropyl ring fused onto a carba-galactopyranose skeleton were synthesized and tested as potential reversible and irreversible £\-galactosidase inhibitors. (1R,2S,3S,4S,5S,6S)-5-amino-1-(hydroxymethyl)bicyclo[4.1.0]heptan-2,3,4-triol and (1S,2S,3S,4S,5S,6R)-5-amino-1-(hydroxymethyl)bicyclo[4.1.0]heptan-2,3,4-triol were synthesized from methyl £\-D-galactopyranoside. A kinetic study showed the later compound is not a inhibitor. However the former is a potent reversible inhibitor of coffee bean £\-galactosidase with a Ki value of 541 nM. (1R,2S,3S,4R,5S,6S)-5-(3,5-difluorophenoxy)-1-(hydroxymethyl) bicyclo[4.1.0]heptan-2,3,4-triol and (1S,2S,3S,4R,5S,6R)-5-(3,5-difluorophenoxy)-1-(hydroxymethyl)bicyclo[4.1.0]heptan-2,3,4-triol were synthesized as irreversible inhibitors of £\-galactosidases. Analysis of inhibitory kinetic data illustrated that the former compound is an irreversible inhibitor for coffee bean £\-gal! actosidase with inactivation rate constant (kinact) of 5.6 „e 10-4 s-1 and has a dissociation constant (Ki) of 4.1 mM. Furthermore, using the competitive inhibitor, (1R,2S,3S,4S,5S,6S)-5-amino-1-(hydroxymethyl)bicyclo[4.1.0]heptan-2,3,4-triol, to protect the enzyme from inactivation confirmed that inactivation of the £\-galactosidase enzyme by (1R,2S,3S,4R,5S,6S)-5-(3,5-difluorophenoxy)-1-(hydroxymethyl)bicyclo[4.1.0]heptan-2,3,4-triol occurred in the active site.