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The chemical synthesis of 2-deoxy-2-fluorodisaccharide probes of the hen egg white lysozyme mechanism

TitleThe chemical synthesis of 2-deoxy-2-fluorodisaccharide probes of the hen egg white lysozyme mechanism
Publication TypeJournal Article
Year of Publication2005
AuthorsVocadlo, DJ, Withers, SG
JournalCARBOHYDRATE RESEARCH
Volume340
Pagination379-388
Date PublishedFEB 28
ISSN0008-6215
Abstract

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-p-D-g lucopyranosyl 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-glueopyranose using Selectfluor(TM) 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., greater than 5 mM and a second-order rate constant of k(cat)/ K-m = 9.6 s(-1) M-1. Comparison of this value to that estimated for the hydrolysis of beta-chitobiosyl fluoride by HEWL (1200 s(-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. Nature 2001, 412, 835-838]. (C) 2005 Elsevier Ltd. All rights reserved.

DOI10.1016/j.carres.2004.12.015