The design and synthesis of a new diazotransfer reagent, imidazole-1-sulfonyl azide hydrochloride, are reported. This reagent has proven to equal triflyl azide in its ability to act as a "diazo donor" in the conversion of both primary amines into azides and activated methylene substrates into diazo compounds. Crucially, this reagent can be prepared in a one-pot reaction on a large scale from inexpensive materials, is shelf-stable, and is conveniently crystalline.

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The design and synthesis of transition-state mimics reflects the growing need both to understand enzymatic catalysis and to influence strategies for therapeutic intervention. Iminosugars are among the most potent inhibitors of glycosidases. Here, the binding of 1-deoxynojirimycin and (+)-isofagomine to the "family GH-1" beta-glucosidase of Thermotoga maritima is investigated by kinetic analysis, isothermal titration calorimetry, and X-ray crystallography. The binding of both of these iminosugar inhibitors is driven by a large and favorable enthalpy. The greater inhibitory power of isofagomine, relative to 1-deoxynojirimycin, however, resides in its significantly more favorable entropy; indeed the differing thermodynamic signatures of these inhibitors are further highlighted by the markedly different heat capacity values for binding. The pH dependence of catalysis and of inhibition suggests that the inhibitory species are protonated inhibitors bound to enzymes whose acid/base and nucleophile are ionized, while calorimetry indicates that one proton is released from the enzyme upon binding at the pH optimum of catalysis (pH 5.8). Given that these results contradict earlier proposals that the binding of racemic isofagomine to sweet almond beta-glucosidase was entropically driven (Bülow, A. et al. J. Am. Chem. Soc. 2000, 122, 8567-8568), we reinvestigated the binding of 1-deoxynojirimycin and isofagomine to the sweet almond enzyme. Calorimetry confirms that the binding of isofagomine to sweet almond beta-glucosidases is, as observed for the T. maritima enzyme, driven by a large favorable enthalpy. The crystallographic structures of the native T. maritima beta-glucosidase, and its complexes with isofagomine and 1-deoxynojirimycin, all at approximately 2.1 A resolution, reveal that additional ordering of bound solvent may present an entropic penalty to 1-deoxynojirimycin binding that does not penalize isofagomine.

Enzyme inhibition through mimicry of the transition state is a major area for the design of new therapeutic agents. Emerging evidence suggests that many retaining glycosidases that are active on alpha- or beta-mannosides harness unusual B2,5 (boat) transition states. Here we present the analysis of 25 putative beta-mannosidase inhibitors, whose Ki values range from nanomolar to millimolar, on the Bacteroides thetaiotaomicron beta-mannosidase BtMan2A. B2,5 or closely related conformations were observed for all tightly binding compounds. Subsequent linear free energy relationships that correlate log Ki with log Km/kcat for a series of active center variants highlight aryl-substituted mannoimidazoles as powerful transition state mimics in which the binding energy of the aryl group enhances both binding and the degree of transition state mimicry. Support for a B2,5 transition state during enzymatic beta-mannosidase hydrolysis should also facilitate the design and exploitation of transition state mimics for the inhibition of retaining alpha-mannosidases--an area that is emerging for anticancer therapeutics.