Cysteine Nucleophiles in Glycosidase Catalysis: Application of a Covalent ?? <scp>l?</scp> Arabinofuranosidase Inhibitor

The recent discovery of zinc-dependent retaining glycoside hydrolases (GHs), with active sites built around a Zn(Cys)3(Glu) coordination complex, has presented unresolved mechanistic questions. In particular, the proposed mechanism, depending on Zn-coordinated cysteine nucleophile and passing through thioglycosyl enzyme intermediate, remains controversial. This is primarily due to expected stability intermediate C?S bond. To facilitate study this atypical we report synthesis cyclophellitol-derived ?-l-arabinofuranosidase inhibitor, hypothesised react catalytic form non-hydrolysable adduct analogous covalent intermediate. inhibitor reacts exclusively thiol nucleophiles representatives GH families 127 146. X-ray crystal structures determined for resulting adducts enable MD QM/MM simulations, which provide insight into mechanism breakdown. Leveraging unique chemistry cyclophellitol derivatives, simulations here support assignment zinc-coordinated as illuminate finely tuned energetics remarkable metalloenzyme clan.