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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.
Item Metadata
Title |
Synthesis and testing of nitrogen-containing sugar analogues as inhibitors of thermoanaerobacterium saccharolyticum b-xylosidase
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Creator | |
Publisher |
University of British Columbia
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Date Issued |
1999
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Description |
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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Extent |
4974486 bytes
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Genre | |
Type | |
File Format |
application/pdf
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Language |
eng
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Date Available |
2009-06-16
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Provider |
Vancouver : University of British Columbia Library
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Rights |
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.
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DOI |
10.14288/1.0061512
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URI | |
Degree | |
Program | |
Affiliation | |
Degree Grantor |
University of British Columbia
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Graduation Date |
1999-05
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Campus | |
Scholarly Level |
Graduate
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Aggregated Source Repository |
DSpace
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Item Media
Item Citations and Data
Rights
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.