The chemical synthesis of 2-deoxy-2-fluorodisaccharide probes of the hen egg white lysozyme mechanism.
2,4-Dinitrophenyl 2-acetamido-2-deoxy-beta-d-glucopyranosyl-(1-->4)-2-deoxy-2-fluoro-beta-d-glucopyranoside (GN2FG-DNP) and 2-acetamido-2-deoxy-beta-d-glucopyranosyl-(1-->4)-2-deoxy-2-fluoro-beta-d-glucopyranosyl fluoride (GN2FG-F) were prepared using a divergent synthetic approach involving 10 steps. The key steps involved the preparation of 1-O-acetyl-3,6-di-O-benzyl-2-deoxy-2-fluoro-alpha/beta-d-glucopyranose using Selectfluor(trade mark) in the presence of acetic acid and the subsequent glycosylation of this acceptor to generate the core 2-fluorodisaccharide. After further elaboration, the target molecules were obtained and tested as probes of the mechanism of hen egg white lysozyme (HEWL). Compound GN2FG-DNP is not a substrate for the enzyme while compound GN2FG-F is cleaved slowly with an apparent K(m) greater than 5mM and a second-order rate constant of k(cat)/K(m)=9.6s(-1)M(-1). Comparison of this value to that estimated for the hydrolysis of beta-chitobiosyl fluoride by HEWL (1200s(-1)M(-1)) [Ballardie, F. W.; Capon, B.; Cuthbert, M. W.; Dearie, W. M. Bioorg. Chem.1977, 6, 483-509] revealed a 126-fold rate decrease upon substitution of a fluorine group for the 2-acetamido group of beta-chitobiosyl fluoride. This decrease resulted in the steady-state accumulation of an intermediate as visualized by mass spectrometry and the ultimate crystallographic determination of its structure [Vocadlo, D. J.; Davies, G. J.; Laine, R.; Withers, S. G. Nature2001, 412, 835-838].
Vocadlo
David J
DJ
Department of Chemistry, University of British Columbia, Vancouver, BC, Canada V6T 1Z1.
Withers
Stephen G
SG
D019297:2,4-Dinitrophenol / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D000818:Animals; D002384:Catalysis; D002475:Cellobiose / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D004186:Disaccharidases / Q000138:chemical synthesis; D015335:Molecular Probes / Q000138:chemical synthesis*; D009113:Muramidase / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity
C503189:2,4-dinitrophenyl 2-acetamido-2-deoxyglucopyranosyl-(1-4)-2-deoxy-2-fluoroglucopyranoside; C503190:2-acetamido-2-deoxyglucopyranosyl-(1-4)-2-deoxy-2-fluoroglucopyranosyl fluoride; D015335:Molecular Probes; D002475:Cellobiose; D004186:Disaccharidases; C421864:hen egg lysozyme; D009113:Muramidase; D019297:2,4-Dinitrophenol