Department of Chemistry, Indiana University, Bloomington, Indiana 47405, United States.
J Am Chem Soc. 2022 Sep 14;144(36):16676-16682. doi: 10.1021/jacs.2c07422. Epub 2022 Aug 31.
In this study, we engineer a variant of the flavin-dependent halogenase RebH that catalyzes site- and atroposelective halogenation of 3-aryl-4(3)-quinazolinones via kinetic or dynamic kinetic resolution. The required directed evolution uses a combination of random and site-saturation mutagenesis, substrate walking using two probe substrates, and a two-tiered screening approach involving the analysis of variant conversion and then enantioselectivity of improved variants. The resulting variant, 3-T, provides >99:1 e.r. for the ()-atropisomer of the major brominated product, 25-fold improved conversion, and 91-fold improved site selectivity relative to the parent enzyme on the probe substrate used in the final rounds of evolution. This high activity and selectivity translate well to several additional substrates with varied steric and electronic properties. Computational modeling and docking simulations are used to rationalize the effects of key mutations on substrate binding. Given the range of substrates that have been used for atroposelective synthesis via electrophilic halogenation in the literature, these results suggest that flavin-dependent halogenases (FDHs) could find many additional applications for atroposelective catalysis. More broadly, this study highlights how RebH can be engineered to accept structurally diverse substrates that enable its use for enantioselective catalysis.
在这项研究中，我们对依赖黄素的卤化酶 RebH 进行了改造，使其能够通过动力学或动态动力学拆分，对 3-芳基-4(3)-喹唑啉酮进行位置和对映选择性卤化。所需的定向进化使用了随机和定点饱和突变、使用两种探针底物的底物游走以及涉及变体转化率分析然后提高变体对映选择性的两级筛选方法的组合。所得变体 3-T 对主要溴化产物 ()-对映异构体的对映体过量 (>99:1 e.r.)、转化率提高 25 倍、探针底物的位点选择性提高 91 倍，该探针底物用于进化的最后几轮。这种高活性和选择性很好地转化为具有不同空间和电子性质的几种其他底物。计算建模和对接模拟用于合理说明关键突变对底物结合的影响。鉴于文献中已经使用了多种通过亲电卤化进行对映选择性合成的底物，这些结果表明依赖黄素的卤化酶 (FDHs) 可能会在对映选择性催化方面找到许多其他应用。更广泛地说，这项研究强调了如何对 RebH 进行工程改造，以接受结构多样的底物，从而使其能够用于对映选择性催化。
