1,1-Difluoroalkyl glucosides: a new class of enzyme-activated irreversible inhibitors of .alpha.-glucosidases

Abstract

There is presently an upsurge of interest in glycosidase inhibitors. Such compounds have been shown to be important tools in mechanistic studies on glycohydrolases,1 as well as having promising therapeutic applications.2 However, only a few examples of enzyme-activated irreversible inhibitors of glycosidases have been described. They include conduritol epoxides,3 glycosylmethyltriazenes,4 and the aziridine triol derivatives of piperidine.5 These compounds are activated in the first protonation step of the enzyme-assisted glycoside hydrolysis. Due to their high intrinsic chemical reactivity, insufficient specificity could limit their use as drugs. Inhibitors activated only after or during enzymic cleavage of the glycosidic bond should be more selective.The first examples of this new class are 2-deoxy-2-fluoroglycosides.6 These inhibitors are believed to be cleaved before inactivating the enzyme by the formation of a covalent glycosyl-enzyme complex. We now wish to report a novel enzymeactivated irreversible inhibitor of yeast -glucosidase based on the activation, following hydrolytic cleavage, of the aglycon moiety of the molecule.-Glucosidases catalyze the hydrolysis reaction shown in Scheme I where R can be either a glycosidic residue or an aglycon leaving group. Our approach toward irreversible inhibition of -glucosidase was to design R in such a way that the glucosidase 1 would be a stable substrate, while the alcohol 2 would be spontaneously and rapidly converted into a reactive alkylating agent.1,1-Difluoroalkyl glucosides 3 (Scheme II) were chosen to illustrate this concept. The , -difluoro alcohols 4 (products of the -glucosidase-catalyzed hydrolysis of 3) are known7 to rapidly lose HF and to be transformed into acid fluorides 5. These acylating agents are then expected to form a covalent adduct 6 with a nucleophilic residue of the enzyme-active site.8

A Historical Perspective for the Catalytic Reaction Mechanism of Glycosidase; So As to Bring about Breakthrough in Confusing Situation

“…Available inhibitors, 41,42) 2-deoxy-2-fluoro--D-glucopyranosyl fluoride, 43) 1,1-difluoroalkyl-glucoside, 44) and 5-fluoro--D-glucopyranosyl fluoride 45) regarded as substrate analogs, and conduritol B epoxide 46) as a different type of inhibitor, 47,48) have been synthesized as a novel class of enzyme-activated irreversible inhibitors, which are all mechanism-based inhibitors (suicide substrate). In mechanism-based inhibitors, 49) usually glycone analog specifically binds to a carboxylate group at the active site directly involved in the activity, and then the glycosidase is irreversibly inactivated.…”

“…In mechanism-based inhibitors, 49) usually glycone analog specifically binds to a carboxylate group at the active site directly involved in the activity, and then the glycosidase is irreversibly inactivated. The mechanism-based inactivation reaction for many -and -glucosidases [43][44][45][46][47][48][50][51][52] and -glucan glucohydrolases [53][54][55] have been examined using the inhibitors named above. Most of the substrate analogs directly modify a catalytic carboxylate of enzyme, but 1,1-difluoroalkyl-glucoside 44) is distinct from the modification reaction of the other analogs.…”

Catalytic Reaction Mechanism Based on α-Secondary Deuterium Isotope Effects in Hydrolysis of Trehalose by European Honeybee Trehalase

“…Available inhibitors, 51,52) conduritol B epoxide, 53) 2-deoxy-2-fluoro-glucoside, 54) 1,1-difluoroalkyl-glucosides, 55) and 5-fluoro--D-glucopyranosyl fluoride, 56) have been synthesized as a novel class of enzyme-activated irrever-sible inhibitors, which are mechanism-base inhibitors (suicide substrates). 2-Deoxy-2-fluoro-glucosides, 1,1-difluoroalkyl-glucosides, and 5-fluoro--glucosyl fluoride are regarded as substrate analogs, and conduritol B epoxide, as an analog in the transition state.…”

“…57,58) In mechanism-base inhibition, 59) usually glycone analog binds to a carboxylate group in active site directly involved in the activity, and then the glycosidase is irreversibly inactivated. Mechanism-base inactivation reactions for many -and -glucosidases [54][55][56][57][58][60][61][62] and -glucan glucohydrolases [63][64][65] have been examined using the inhibitors mentioned above. The time-dependence for inactivation showed curves of definite pseudo first-order reaction.…”

Approaches to labeling and identification of active site residues in glycosidases

“…7A), which undergoes enzyme-catalyzed cleavage, releasing a fluorohydrin. This rapidly eliminates hydrogen fluoride, yielding an acyl fluoride that can react with the enzyme active site (Halazy et al, 1989). The second example is a difluorotolyl glycoside (Fig.…”