Synthesis of broad-specificity activity-based probes for exo-β-mannosidases

Exo-beta-mannosidases are a broad class of stereochemically retaining hydrolases that are essential for the breakdown of complex carbohydrate substrates found in all kingdoms of life. Yet the detection of exo-beta-mannosidases in complex biological samples remains challenging, necessitating the development of new methodologies. Cyclophellitol and its analogues selectively label the catalytic nucleophiles of retaining glycoside hydrolases, making them valuable tool compounds. Furthermore, cyclophellitol can be readily redesigned to enable the incorporation of a detection tag, generating activity-based probes (ABPs) that can be used to detect and identify specific glycosidases in complex biological samples. Towards the development of ABPs for exo-beta-mannosidases, we present a concise synthesis of beta-manno-configured cyclophellitol, cyclophellitol aziridine, and N-alkyl cyclophellitol aziridines. We show that these probes covalently label exo-beta-mannosidases from GH families 2, 5, and 164. Structural studies of the resulting complexes support a canonical mechanism-based mode of action in which the active site nucleophile attacks the pseudoanomeric centre to form a stable ester linkage, mimicking the glycosyl enzyme intermediate. Furthermore, we demonstrate activity-based protein profiling using an N-alkyl aziridine derivative by specifically labelling MANBA in mouse kidney tissue. Together, these results show that synthetic manno-configured cyclophellitol analogues hold promise for detecting exo-beta-mannosidases in biological and biomedical research.