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

Abstract

2,4-Dinitrophenyl 2-acetamido-2-deoxy-β- d-glucopyranosyl-(1→4)-2-deoxy-2-fluoro-β- d-glucopyranoside (GN2FG-DNP) and 2-acetamido-2-deoxy-β- d-glucopyranosyl-(1→4)-2-deoxy-2-fluoro-β- 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-α/β- d-glucopyranose using Selectfluor ™ 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 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 β-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 β-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].