Vocadlo David J, Withers Stephen G
Department of Chemistry, University of British Columbia, Vancouver, BC, Canada V6T 1Z1.
Carbohydr Res. 2005 Feb 28;340(3):379-88. doi: 10.1016/j.carres.2004.12.015.
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].
2,4-二硝基苯基 2-乙酰氨基-2-脱氧-β-D-吡喃葡萄糖基-(1→4)-2-脱氧-2-氟-β-D-吡喃葡萄糖苷（GN2FG-DNP）和 2-乙酰氨基-2-脱氧-β-D-吡喃葡萄糖基-(1→4)-2-脱氧-2-氟-β-D-吡喃葡萄糖基氟化物（GN2FG-F）采用一种涉及 10 步的发散合成方法制备。关键步骤包括在乙酸存在下使用 Selectfluor（商标）制备 1-O-乙酰基-3,6-二-O-苄基-2-脱氧-2-氟-α/β-D-吡喃葡萄糖，以及随后将该受体进行糖基化以生成核心 2-氟二糖。经过进一步修饰，获得了目标分子，并作为鸡蛋清溶菌酶（HEWL）作用机制的探针进行了测试。化合物 GN2FG-DNP 不是该酶的底物，而化合物 GN2FG-F 则被缓慢裂解，其表观 K(m)大于 5mM，二级速率常数 k(cat)/K(m)=9.6s(-1)M(-1)。将该值与通过 HEWL 对β-壳二糖基氟化物水解估计的值（1200s(-1)M(-1)）[巴拉迪，F.W.；卡彭，B.；卡斯伯特，M.W.；迪里，W.M.生物有机化学 1977，6，483 - 509]进行比较，结果表明，用氟原子取代β-壳二糖基氟化物的 2-乙酰氨基后，反应速率降低了 126 倍。这种降低导致通过质谱可视化的中间体稳态积累，并最终通过晶体学确定了其结构[沃卡德洛，D.J.；戴维斯，G.J.；莱恩，R.；威瑟斯，S.G.自然 2001，412，835 - 838]。
