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The treatment of benzyl 2,3-O-isopropylidene-β-L-xylopyranoside with N-hydroxyphthalimide under Mitsunobu conditions, followed by protecting-group interchange, gave benzyl 4-O-[(tert-butoxycarbonyl)amino]-2,3- O-isopropylidene-α-D-arabinoside. Mild acid hydrolysis and catalytic hydrogenolysis afforded 4-O-[(tert-butoxycar bonyl)amino]-D-arabinose that, upon heating in water, gave the dihydrooxazine [(4R,5S,6R)-5,6-dihydro-4,5-dihydroxy-6-hydroxymethyl-4H-1,2-oxazine] as a crystalline solid. A single-crystal structure determination of this solid showed it to exist in the [sup 5] H[sub 6] conformation. Reduction of the dihydrooxazine gave the tetrahydrooxazine [(4R,5S,6R)-4,5-dihy droxy-6-hydroxymethyl-3,4,5,6-tetrahydro-2H-1,2-oxazine]. The dihydrooxazine was an effective inhibitor of two β-glucosidases (K[sub i] = 27 and 35 µM). Benzyl 2,3-O-isopropylidene-β-L-xylopyranoside, via the derived imidazylate, was converted into a nitrile that, upon reduction and protecting-group manipulations, gave benzyl 4-C-aminomethyl-4-deoxy-α-D-arabinoside. Treatment of this amine with hydrogen and palladium-on-carbon gave isofagomine.Key words: dihydrooxazine, tetrahydrooxazine, isofagomine, iminosugars, glycosidase inhibitors.Le traitement du 2,3-O-isopropylidène-β-L-xylopyranoside de benzyle par du N-hydroxyphtalimide dans des conditions de Mitsunobu, suivi d'un échange des groupes protecteurs, conduit à la formation du 4-O-[(tert-butoxycarbonyl)amino]-2,3-O-isopropylidène-α-D-arabinoside de benzyle. Soumis à une hydrolyse acide légère et une hydrogénolyse catalytique, celui-ci fournit alors le 4-O-[(tert-butoxycarbonyl)amino]-D-arabinose qui, par chauffage dans l'eau, livre la dihydrooxazine [(4R,5S,6R)-5,6-dihydro-4,5-dihydroxy-6-hydroxyméthyl-4H-1,2-oxazine] sous la forme de solide cristallin. Une détermination de structure sur un cristal unique montre qu'elle existe dans la conformation [sup 5] H[sub 6] . La réduction de la dihydrooxazine fournit la tétrahydrooxazine [(4R,5S,6R)-4,5-dihydroxy-6-hydroxyméthyl-3,4,5,6-tétrahydro-2H-1,2-oxazine]. La dihydrooxazine est un inhibiteur efficace de deux β-gluco sidases (K[sub i] = 27 et 35 µM). Le 2,3-O-isopropylidène-β-L-xylopyranoside de benzyle, par le biais de l'imidazylate qui en dérive, a été transformé en un nitrile qui, après réduction et des manipulations des groupes protecteurs, fournit le 4-C-aminométhyl-4-désoxy-α-D-arabinoside de benzyle. Le traitement de cette amine avec de l'hydrogène et du palladium sur du charbon conduit alors à l'isofagomine.Mots clés : dihydrooxazine, tétrahydrooxazine, isofagomine, iminosucres, inhibiteurs de glycosidases.[Traduit par la Rédaction] [ABSTRACT FROM AUTHOR]

The inhibition of glycoside hydrolases, through transition-state mimicry, is important both as a probe of enzyme mechanism and in the continuing quest for new drugs, notably in the treatment of cancer, HIV, influenza, and diabetes. The high affinity with which these enzymes are known to bind the transition state provides a framework upon which to design potent inhibitors. Recent work [for example, Below, A. et al. J. Am. Chem. Soc. 2000, 122, 8567-8568; Zechel, D. L. et al. J. Am. Chem. Soc. 2003, 125, 14313-14323] has revealed quite confusing and counter-intuitive patterns of inhibition for a number of glycosidase inhibitors. Here we describe a synergistic approach for analysis of inhibitors with a single enzyme ‘model system’, the Thermotoga maritima family 1 β-glucosidase, TmGH1. The pH dependence of enzyme activity and inhibition has been determined, structures of inhibitor complexes have been solved by X-ray crystallography, with data up to 1.65 Å resolution, and isothermal titration calorimetry was used to establish the thermodynamic signature. This has allowed the characterization of 18 compounds, all putative transition-state mimics, in order to build an ‘inhibition profile’ that provides an insight into what governs binding. In contrast to our preconceptions, there is little correlation of inhibitor chemistry with the calorimetric dissection of thermodynamics. The ensemble of inhibitors shows strong enthalpy-entropy compensation, and the random distribution of similar inhibitors across the plot of ΔH°a vs TΔS°a likely reflects the enormous contribution of solvation and desolvation effects on ligand binding. [ABSTRACT FROM AUTHOR]

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" β-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 β-glucosidase was entropically driven (B&uamul;low, 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 β-glucosidases is, as observed for the T. maritima enzyme, driven by a large favorable enthalpy. The crystallographic structures of the native T. maritima β-glucosidase, and its complexes with isofagomine and 1-deoxynojirimycin, all at ∼2.1 Å resolution, reveal that additional ordering of bound solvent may present an entropie penalty to 1-deoxynojirimycin binding that does not penalize isofagomine. [ABSTRACT FROM AUTHOR]

Analyzes the inhibition of the endocellulase Cel5A from bacillus agaradhaerens by a series of oligosaccharide glycosidase inhibitors. Background on the Cel5a endocellulase; Examination of the three-dimensional structure of Cel5A; Atomic resolution structure.