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 2H3 conformations. We also report the synthesis and reactivity of a fluorinated analogue and the structure of its covalently linked intermediate (flattened 2H3 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.

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

Selective methods for introducing protein post-translational modifications (PTMs) within living cells have proven valuable for interrogating their biological function. In contrast to enzymatic methods, abiotic catalysis should offer access to diverse and new-to-nature PTMs. Herein, we report the boronate-assisted hydroxamic acid (BAHA) catalyst system, which comprises a protein ligand, a hydroxamic acid Lewis base, and a diol moiety. In concert with a boronic acid-bearing acyl donor, our catalyst leverages a local molarity effect to promote acyl transfer to a target lysine residue. Our catalyst system employs micromolar reagent concentrations and affords minimal off-target protein reactivity. Critically, BAHA is resistant to glutathione, a metabolite which has hampered many efforts toward abiotic chemistry within living cells. To showcase this methodology, we installed a variety of acyl groups in E. coli dihydrofolate reductase expressed within human cells. Our results further establish the well-known boronic acid− diol complexation as a bona f ide bio-orthogonal reaction with applications in chemical biology and in-cell catalysis.

Glycomimetics are structural mimics of naturally occurring carbohydrates and represent important therapeutic leads in several disease treatments. However, the structural and stereochemical complexity inherent to glycomimetics often challenges medicinal chemistry efforts and is incompatible with diversity-oriented synthesis approaches. Here, we describe a one-pot proline-catalyzed aldehyde α-functionalization/aldol reaction that produces an array of stereochemically well-defined glycomimetic building blocks containing fluoro, chloro, bromo, trifluoromethylthio and azodicarboxylate functional groups. Using density functional theory calculations, we demonstrate both steric and electrostatic interactions play key diastereodiscriminating roles in the dynamic kinetic resolution. The utility of this simple process for generating large and diverse libraries of glycomimetics is demonstrated in the rapid production of iminosugars, nucleoside analogues, carbasugars and carbohydrates from common intermediates.

Life emerges from networks of multiple chemical reactions mediated by enzymes. If abiotic chemical catalysis is implanted into the reaction network of life, such an integration would produce organisms generating...