Enantiospecific synthesis of 1-azafagomine.

For the first time the two enantiomeric forms of the glycosidase inhibitor 1-azafagomine have been synthesised starting from D- and L-xylose. D-Xylose was converted to the 2,3,5-tribenzylfuranose, which upon reductive amination with tert-butyl carbazate gave the protected 1-hydrazino-1-deoxypentitol in high yield. N-acetylation, mesylation of the 4-OH, removal of the Boc group, cyclisation and deprotection gave (+)-1-azafagomine ((+)-1).

Direct NMR-spectroscopic determination of active-enzyme concentration by titration with a labeled inhibitor: determination of the k(cat) value of almond beta-glucosidase.

A new method for the determination of active-enzyme concentration of a glucosidase by using (13)C NMR spectroscopy is reported. The method consists of quantifying the binding between a (13)C-labelled, strong competitive inhibitor, [5-(13)C]-1-azafagomine (1), and the enzyme. The concentration of free inhibitor 1 is measured in a series of binding experiments from the intensity of its NMR signal relative to that of a reference.

Accurate determination of rate constants of very slow, tight-binding competitive inhibitors by numerical solution of differential equations, independently of precise knowledge of the enzyme concentration.

This paper is concerned with the determination of rate constants characterizing the binding and release of a slow binding inhibitor to and from an enzyme, here almond beta-glucosidase. We demonstrate the inability of the conventional method to yield reliable rate constants when one or more of these is less than 1 x 10(-4) per second. Instead one must use the much more accurate fitting of rate constants of the set of simultaneous differential equations characterizing the kinetic model.

Slow inhibition of almond beta-glucosidase by azasugars: determination of activation energies for slow binding.

The thermodynamic and activation energies of the slow inhibition of almond beta-glucosidase with a series of azasugars were determined. The inhibitors studied were isofagomine ((3R,4R,5R)-3,4-dihydroxy-5-hydroxymethylpiperidine, 1), isogalactofagomine ((3R,4S,5R)-3,4-dihydroxy-5-hydroxymethylpiperidine, 2), (-)-1-azafagomine ((3R,4R,5R)-4,5-dihydroxy-3-hydroxymethylhexahydropyridazine, 3), 3-amino-3-deoxy-1-azafagomine (4) and 1-deoxynojirimycin (5). It was found that the binding of 1 to the enzyme has an activation enthalpy of 56.

Investigation of the slow inhibition of almond beta-glucosidase and yeast isomaltase by 1-azasugar inhibitors: evidence for the 'direct binding' model.

(-)-1-Azafagomine [(3R,4R,5R)-4,5-dihydroxy-3-hydroxymethylhexahydropyridazine; inhibitor 1] is a potent glycosidase inhibitor designed to mimic the transition state of a substrate undergoing glycoside cleavage. The inhibition of glycosidases by inhbitor 1 and analogues has been found to be a relatively slow process. This 'slow inhibition' process was investigated in the inhibition of almond beta-glucosidase and yeast isomaltase by inhibitor 1 and analogues.