Irreversible inhibitors and activity-based probes as research tools in chemical glycobiology

In this review, we will discuss the enzymes that are involved in the synthesis and degradation of glycoconjugates and we will give an overview of the inhibitors and activity-based probes (ABPs) that have been used to study these. Following discussion of some general aspects of the biosynthesis and degradation of N-linked glycoproteins, attention is focused on the enzymes that hydrolyze the protein–carbohydrate linkage, peptide N-glycanase and glycosylasparaginase and their mechanism. We then focus on the biosynthesis of O-linked glycoproteins and glycolipids and in particular on the enzymes that hydrolyze the interglycosidic linkages in these, the glycosidases. Some important mechanism-based glycosidase inhibitors that form a covalent bond with the targeted enzyme(s), their corresponding ABPs and their application to study this class of enzymes are highlighted. Finally, alternative pathways for degradation of glycoconjugates and an ABP-based strategy to study these will be discussed

On the reactivity and selectivity of donor glycosides in glycochemistry and glycobiology:trapped covalent intermediates

The reactivity of sugar donors and the stability of covalent intermediates formed in both chemical and biological systems is an active subject of study in both glycochemistry and glycobiology. Knowledge of the structure of these intermediates is vital for understanding reactivity and stereoselectivity in glycosidic bond formation, and in glycosidic bond destruction in the case of enzymatic hydrolysis. For chemical reactions, tuning of the electron-withdrawing power of the carbohydrate side chains allows for stabilization of covalent anomeric triflates thereby enabling chemo-, regio- and stereoselective glycosylations. Retaining glycosidase-mediated hydrolysis reactions in turn often involve a covalent intermediate. The existence of such covalent intermediates was convincingly demonstrated at the beginning of this century by making use of modified glycosyl substrates tuned such that stable adducts are formed efficiently but the ensuing hydrolysis is slowed down. Recently this concept has also been used in the design of glycosidase activity-based probes. This review describes recent investigations on different carbohydrate decoration patterns to influence both chemical and biological reactivity and selectivity

1,6-Cyclophellitol Cyclosulfates : A New Class of Irreversible Glycosidase Inhibitor

The essential biological roles played by glycosidases, coupled to the diverse therapeutic benefits of pharmacologically targeting these enzymes, provide considerable motivation for the development of new inhibitor classes. Cyclophellitol epoxides and aziridines are recently established covalent glycosidase inactivators. Inspired by the application of cyclic sulfates as electrophilic equivalents of epoxides in organic synthesis, we sought to test whether cyclophellitol cyclosulfates would similarly act as irreversible glycosidase inhibitors. Here we present the synthesis, conformational analysis, and application of novel 1,6-cyclophellitol cyclosulfates. We show that 1,6-epi-cyclophellitol cyclosulfate (α-cyclosulfate) is a rapidly reacting α-glucosidase inhibitor whose 4C1 chair conformation matches that adopted by α-glucosidase Michaelis complexes. The 1,6-cyclophellitol cyclosulfate (β-cyclosulfate) reacts more slowly, likely reflecting its conformational restrictions. Selective glycosidase inhibitors are invaluable as mechanistic probes and therapeutic agents, and we propose cyclophellitol cyclosulfates as a valuable new class of carbohydrate mimetics for application in these directions

Functionalized cyclophellitols are selective glucocerebrosidase inhibitors and induce a bona fide neuropathic Gaucher model in zebrafish

Gaucher disease is caused by inherited deficiency in glucocerebrosidase (GBA, a retaining β-glucosidase), and deficiency in GBA constitutes the largest known genetic risk factor for Parkinson's disease. In the past, animal models of Gaucher disease have been generated by treatment with the mechanism-based GBA inhibitors, conduritol B epoxide (CBE), and cyclophellitol. Both compounds, however, also target other retaining glycosidases, rendering generation and interpretation of such chemical knockout models complicated. Here we demonstrate that cyclophellitol derivatives carrying a bulky hydrophobic substituent at C8 are potent and selective GBA inhibitors and that an unambiguous Gaucher animal model can be readily generated by treatment of zebrafish with these