Determination of protonation states of iminosugar-enzyme complexes using photoinduced electron transfer.

A series of -alkylated analogues of 1-deoxynojirimycin containing a fluorescent 10-chloro-9-anthracene group in the -alkyl substituent were prepared. The anthracene group acted as a reporting group for protonation at the nitrogen in the iminosugar because an unprotonated amine was found to quench fluorescence by photoinduced electron transfer. The new compounds were found to inhibit β-glucosidase from and α-glucosidase from , with values in the low micro- to nanomolar range. Fluorescence and inhibition pH studies of the β-glucosidase-iminosugar complexes revealed that the amino group in the inhibitor is unprotonated when bound, while one of the active site carboxylates is protonated.