The design of covalent inhibitors in glycoscience research is important for the development of chemical biology probes. Here we report the synthesis of a new carbocyclic mechanism-based covalent inhibitor of an α-glucosidase. The enzyme efficiently catalyzes its alkylation via either an allylic cation or a cationic transition state. We show this allylic covalent inhibitor has different catalytic proficiencies for pseudoglycosylation and deglycosylation. Such inhibitors have the potential to be useful chemical biology tools.

Mechanism-based glycoside hydrolase inhibitors are carbohydrate analogs that mimic the natural substrate’s structure. Their covalent bond formation with the glycoside hydrolase makes these compounds excellent tools for chemical biology and potential drug candidates. Here we report the synthesis of cyclohexene-based α-galactopyranoside mimics and the kinetic and structural characterization of their inhibitory activity toward an α-galactosidase from Thermotoga maritima (TmGalA). By solving the structures of several enzyme-bound species during mechanism-based covalent inhibition of TmGalA, we show that the Michaelis complexes for intact inhibitor and product have half-chair (2H3) conformations for the cyclohexene fragment, while the covalently linked intermediate adopts a flattened half-chair (2H3) conformation. Hybrid QM/MM calculations confirm the structural and electronic properties of the enzyme-bound species and provide insight into key interactions in the enzyme-active site. These insights should stimulate the design of mechanism-based glycoside hydrolase inhibitors with tailored chemical properties.

Retaining glycoside hydrolases cleave their substrates through stereochemical retention at the anomeric position. Typically, this involves two-step mechanisms using either an enzymatic nucleophile via a covalent glycosyl enzyme intermediate or neighboring-group participation by a substrate-borne 2-acetamido neighboring group via an oxazoline intermediate; no enzymatic mechanism with participation of the sugar 2-hydroxyl has been reported. Here, we detail structural, computational, and kinetic evidence for neighboring-group participation by a mannose 2hydroxyl in glycoside hydrolase family 99 endo-α-1,2-mannanases. We present a series of crystallographic snapshots of key species along the reaction coordinate: a Michaelis complex with a tetrasaccharide substrate; complexes with intermediate mimics, a sugar-shaped cyclitol β-1,2-aziridine and β-1,2-epoxide; and a product complex. The 1,2-epoxide intermediate mimic displayed hydrolytic and transfer reactivity analogous to that expected for the 1,2-anhydro sugar intermediate supporting its catalytic equivalence. Quantum mechanics/molecular mechanics modeling of the reaction coordinate predicted a reaction pathway through a 1,2-anhydro sugar via a transition state in an unusual flattened, envelope (E 3 ) conformation. Kinetic isotope effects (k cat /K M ) for anomeric-2 H and anomeric-13 C support an oxocarbenium ion-like transition state, and that for C2-18 O (1.052 ± 0.006) directly implicates nucleophilic participation by the C2-hydroxyl. Collectively, these data substantiate this unprecedented and long-imagined enzymatic mechanism.

Glycoside hydrolases (GHs) catalyze hydrolyses of glycoconjugates in which the enzyme choreographs a series of conformational changes during the catalytic cycle. As a result, some GH families, including α-amylases (GH13), have their chemical steps concealed kinetically. To address this issue for a GH13 enzyme, we prepared seven cyclohexenyl-based carbasugars of α-d-glucopyranoside that we show are good covalent inhibitors of a GH13 yeast α-glucosidase. The linear free energy relationships between rate constants and pK a of the leaving group are curved upward, which is indicative of a change in mechanism, with the better leaving groups reacting by an SN1 mechanism, while reaction rates for the worse leaving groups are limited by a conformational change of the Michaelis complex prior to a rapid SN2 reaction with the enzymatic nucleophile. Five bicyclo[4.1.0]­heptyl-based carbaglucoses were tested with this enzyme, and our results are consistent with pseudoglycosidic bond cleavage that occurs via SN1 transition states that include nonproductive binding of the leaving group to the enzyme. In total, we show that the conformationally orthogonal reactions of these two carbasugars reveal mechanistic details hidden by conformational changes that the Michaelis complex of the enzyme and natural substrate undergoes which align the nucleophile for efficient catalysis.

Glycoside hydrolases (GHs) have attracted considerable attention as targets for therapeutic agents, and thus mechanism-based inhibitors are of great interest. We report the first structural analysis of a carbocyclic mechanism-based GH inactivator, the results of which show that the two Michaelis complexes are in 2 H 3 conformations. We also report the synthesis and reactivity of a fluorinated analogue and the structure of its covalently linked intermediate (flattened 2 H 3 half-chair). We conclude that these inactivator reactions mainly involve motion of the pseudo-anomeric carbon atom, knowledge that should stimulate the design of new transition-state analogues for use as chemical biology tools. Lifeissupportedbyamyriadofenzyme-catalyzedreactions;one such life-sustaining activity is the transfer of carbohydrate groups from one biomolecule to another.[1] Understanding how these fundamentally important transfer reactions occur in nature guides researchers in the design of compounds (inhibitors/activators) that modulate the activity of these biological catalysts. Glycoside hydrolases (GHs or glycosidases) are a type of carbohydrate-processing enzyme used in the reshaping of biomolecules.[2] Most GHs catalyze glycoside hydrolysis through one of two distinct processes that are reliant on a pair of active-site aspartic and/or glutamic acid residues. Hydrolysis by such retaining glycosidases involves two sequential inversions of configuration at the anomeric center, the first of which results in the formation of a covalent glycosyl-enzyme intermediate (Figure 1 a). In contrast, inverting glycosidases operate via a single inversion of configuration at the anomeric center. In both cases, pyranosylium ion like transition states (TSs), which can have half-chair ( 4 H 3 / 3 H 4 ), boat ( 2,5 B/B 2,5 ), or envelope ( 4 E and 3 E) conformations (Figure 1 b), [2,3] are implicated. By exploiting this knowledge, we recently reported that the cyclopropyl-containing carbasugar 1 is a mechanism-based inactivator of an a-d-galactosidase from Thermotoga maritima (TmGalA; Figure 1 c). [4] Within the enzymatic active site, 1 likely forms a transient bicyclobutenium ion (1 + ), and enzyme inactivation occurs through alkylation of the catalytic nucleophile Asp 327.Given the current desire for small-molecule transitionstate analogues (TSAs) as leads for therapeutic development, [5] it is important to understand how GHs stabilize cationic TSs.[4] Of note, GHs are among the most catalytically proficient enzymes, since they accelerate hydrolysis of glycosidic bonds by up to 10 17 -fold.[6] Therefore, an understanding of the distinct ring conformations of the substrate