A series of cyclopeptoid-based iminosugar clusters has been evaluated to finely probe the ligand content-dependent increase in α-mannosidase inhibition. This study led to the largest binding enhancement ever reported for an enzyme inhibitor (up to 4700-fold on a valency-corrected basis), which represents a substantial advance over the multivalent glycosidase inhibitors previously reported. Electron microscopy imaging and analytical data support, for the best multivalent effects, the formation of a strong chelate complex in which two mannosidase molecules are cross-linked by one inhibitor.

Multivalent design of glycosidase inhibitors is a promising strategy for the treatment of diseases involving enzymatic hydrolysis of glycosidic bonds in carbohydrates. An essential prerequisite for successful applications is the atomic-level understanding of how outstanding binding enhancement occurs with multivalent inhibitors. Herein we report the first high-resolution crystal structures of the Jack bean α-mannosidase (JBα-man) in apo and inhibited states. The three-dimensional structure of JBα-man in complex with the multimeric cyclopeptoid-based inhibitor displaying the largest binding enhancements reported so far provides decisive insight into the molecular mechanisms underlying multivalent effects in glycosidase inhibition.