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Development of probes for active GH29 α-l-fucosidases., GH29 α-l-fucosidases catalyze the hydrolysis of α-l-fucosidic linkages. Deficiency in human lysosomal α-l-fucosidase (FUCA1) leads to the recessively inherited disorder, fucosidosis. Herein we describe the development of fucopyranose-configured cyclophellitol aziridines as activity-based probes (ABPs) for selective in vitro and in vivo labeling of GH29 α-l-fucosidases from bacteria, mice and man. Crystallographic analysis on bacterial α-l-fucosidase confirms that the ABPs act by covalent modification of the active site nucleophile. Competitive activity-based protein profiling identified l-fuconojirimycin as the single GH29 α-l-fucosidase inhibitor from eight configurational isomers.

Insulin is a key hormone of human metabolism with major therapeutic importance for both types of diabetes. New insulin analogues with more physiological profiles and better glycemic control are needed, especially analogues that preferentially bind to the metabolic B-isoform of insulin receptor (IR-B). Here, we aimed to stabilize and modulate the receptor-compatible conformation of insulin by covalent intra-chain crosslinking within its B22-B30 segment, using the Cu I -catalyzed Huisgen 1,3-dipolar cycloaddition reaction of azides and alkynes. This approach resulted in 14 new, systematically crosslinked insulin analogues whose structures and functions were extensively characterized and correlated. One of the analogues, containing a B26-B29 triazole bridge, was highly active in binding to both IR isoforms, with a significant preference for IR-B. Our results demonstrate the potential of chemistry-driven modulation of insulin function, also shedding new light on the functional importance of hormones B-chain C-terminus for its IR-B specificity.