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Synthesis and testing of nitrogen-containing sugar analogues as inhibitors of thermoanaerobacterium saccharolyticum b-xylosidase Hiebert, Timothy J.

Abstract

l-Deoxyxylonojirimycin, several N-substituted 1-deoxyxylonojirimycin derivatives, D-xylonolactam, and D-xylonojirimycin tetrazole were synthesised and tested as inhibitors of Thermoanaerobacteriwn saccharolyticum β-xylosidase to investigate the influence of substitution at the endocyclic nitrogen, the importance of basicity at the ring nitrogen to inhibitory potency, and the influence of sp² hybridisation at the anomeric centre on inhibitor binding in this model system. l-Deoxyxylonojirimycin is a competitive inhibitor of T. saccharolyticum β-xylosidase with Ki values ranging from 1.5 mM at pH 4.5 to 13.5 μM at pH 6.5. This pH dependence is consistent with the inhibitor binding in its cationic form to the enzyme acid/base catalyst in its deprotonated form. N-Acetyl and JV-formyl- 1-deoxyxylonojirimycin bind poorly to T. saccharolyticum β-xylosidase, as would be expected if the endocyclic nitrogen must be protonated for good inhibition of this enzyme by 1-deoxynojirimycin-type inhibitors. However, N-benzyloxycarbonyl- 1-deoxyxylonojirimycin also binds relatively tightly to the enzyme (K[sub i] = 110 μM, pH 5.5) while Af-benzyl- 1-deoxyxylonojirimycin is a poor inhibitor (K[sub i] = 7.2 mM, pH 5.5). The inhibitory potency of Af-benzyloxycarbonyl-ldeoxyxylonojirimycin is therefore surprising, but may be the result of fortuitous noncovalent interactions between the pendant aromatic group and a hydrophobic site on the enzyme. Apparently these interactions are not accessible to the N-benzyl group. D-Xylonolactam is a weak inhibitor of T. saccharolyticum β-xylosidase (K[sub i] = 3 mM, pH 5.5), while Dxylonojirimycin tetrazole binds relatively tightly (K[sub i] = 100 μM, pH 5.5) consistent with the notion that proton transfer by T. saccharolyticum β-xylosidase may take place in the plane of the pyranose ring. Surprisingly, several of these inhibitors bind well to the Glul60Ala mutant of T. saccharolyticum β-xylosidase in which the putative acid/base catalyst has been replaced, suggesting that a third carboxyl group in the active site may assume a role in binding and possibly also catalysis in the mutant enzyme. N-Benzyloxycarbonyl- 1-deoxyxylonojirimycin was used as a glycosyl acceptor in transglycosylation reactions catalysed by the Glu358Ala mutant of Agrobacterium sp. Β- glucosidase. Both β-1,3-linked and β-1,4-linked products were formed in a total yield of 50.4%. While the enzyme initially formed β-l,4-linkages to the inhibitor only slowly, the 'disaccharide' product was a superior glycosyl acceptor and was quickly converted to tri- and tetrasaccharide products. Hexa-O-acetyl xylobiose was isolated on a gram scale by treatment of birchwood xylan with T. viride xylanase, followed by acetylation and column chromatography. This intermediate provided a convenient route to the xylanase substrates 2,5-dinitrophenyl (3-Dxylobioside and 2-nitrophenyl P-D-xylobioside in 41 and 48% overall yields respectively.

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