Illarionova Victoria, Kaiser Johannes, Ostrozhenkova Elena, Bacher Adelbert, Fischer Markus, Eisenreich Wolfgang, Rohdich Felix
Lehrstuhl für Organische Chemie und Biochemie, Department Chemie, Technische Universität München, Lichtenbergstrasse 4, D-85747 Garching, Germany.
J Org Chem. 2006 Nov 10;71(23):8824-34. doi: 10.1021/jo061466o.
The nonmevalonate isoprenoid pathway is an established target for antiinfective drug development. This paper describes high-throughput methods for the screening of 2C-methyl-D-erythritol synthase (IspC protein), 4-diphosphocytidyl-2C-methyl-D-erythritol synthase (IspD protein), 4-diphosphocytidyl-2C-methyl-D-erythritol kinase (IspE protein), and 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF protein) against large compound libraries. The assays use up to three auxiliary enzymes. They are all monitored photometrically at 340 nm and are robust as documented by Z-factors of >or=0.86. 13C NMR assays designed for hit verification via direct detection of the primary reaction product are also described. Enzyme-assisted methods for the preparation, on a multigram scale, of isoprenoid biosynthesis intermediates required as substrates for these assays are reported. Notably, these methods enable the introduction of single or multiple 13C labels as required for NMR-monitored assays. The preparation of 4-diphosphosphocytidyl-2C-methyl-D-erythritol 2-phosphate in multigram quantities is described for the first time.
非甲羟戊酸类异戊二烯途径是抗感染药物开发的一个既定靶点。本文描述了针对大型化合物库筛选2C-甲基-D-赤藓糖醇合酶（IspC蛋白）、4-二磷酸胞苷-2C-甲基-D-赤藓糖醇合酶（IspD蛋白）、4-二磷酸胞苷-2C-甲基-D-赤藓糖醇激酶（IspE蛋白）和2C-甲基-D-赤藓糖醇2,4-环二磷酸合酶（IspF蛋白）的高通量方法。这些测定使用多达三种辅助酶。它们均在340nm处通过光度法进行监测，并且如Z因子≥0.86所证明的那样具有稳健性。还描述了通过直接检测主要反应产物来进行命中验证的13C NMR测定法。报告了以多克规模制备这些测定所需的类异戊二烯生物合成中间体作为底物的酶辅助方法。值得注意的是，这些方法能够根据NMR监测测定的需要引入单个或多个13C标记。首次描述了多克量的4-二磷酸胞苷-2C-甲基-D-赤藓糖醇2-磷酸的制备方法。
