The hydrolysis of epoxides by epoxide hydrolases (EHs) is a sustainable approach to synthesize chiral vicinal diols. However, few EHs hitherto reported can catalyse the enantioconvergent synthesis of enantiopure alkane- and alkene-1,2-diols, due to the strict desire for regiocomplementarity. To actualize the enantioconvergent hydrolysis of rac-1,2-epoxyoctane (1a), the regioselectivity (α_S, 55.3%) of SlEH1 for (S)-1a was manipulated via site-directed mutagenesis. Ten specific residues lining the substrate-binding pocket of SlEH1 were identified based on the computer-aided design, each of which was replaced by seven residues, respectively. Among 66 single-site mutants, SlEH1^W106L, SlEH1^W106T, SlEH1^F109I, SlEH1^M180V, SlEH1^F189I and SlEH1^F189L were selected, by which rac-1a was hydrolysed into (R)-octane-1,2-diol (1b) with an ee_p range of 62.1–82.4%. After combinatorial mutagenesis and screening, one double-site mutant, SlEH1^W106T/F189L, was obtained having the highest α_S of 96.7%. The gram-scale enantioconvergent hydrolysis of 400 mM (51.3 g L⁻¹) rac-1a was carried out using 200 mg mL⁻¹ wet cells of E. coli/sleh1^W106T/F189L at 20 °C for 24 h, producing (R)-1b with 94.7% ee_p and 95.6% yield. The substrate spectrum assay of SlEH1^W106T/F189L towards 20 mM rac-1a–6a was conducted, producing (R)-1b–6b with 81.1–97.4% ee_p values. Furthermore, the molecular dynamics simulation analysis indicated that the regiopreference of SlEH1^W106T/F189L attacking on the C_α of (S)-1a was greater than that of SlEH1, which was consistent with their α_S values measured experimentally. This work engineered a superior double-site mutant, SlEH1^W106T/F189L, for the enantioconvergent synthesis of a variety of chiral alkane- and alkene-1,2-diols, especially (R)-1b and 6b, with high ee_p values.

中文翻译：

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操纵茄茄环氧化物水解酶的区域选择性，以对映体合成对映纯的烷烃-和烯烃-1,2-二醇

通过环氧化物水解酶（EH）水解环氧化物是合成手性邻位二醇的可持续方法。然而，由于对区域互补性的严格要求，迄今报道的很少有EHs可以催化对映纯的烷烃-和烯烃-1,2-二醇的对映收敛合成。为了具体化的水解对映外消旋-1,2-环氧辛烷（1A），区域选择性（α_小号，55.3％）的S1中EH1为（小号） - 1A被操纵通过位点定向诱变。Sl的底物结合口袋内衬的十个特定残基EH1是根据计算机辅助设计确定的，每个分别被七个残基取代。在66个单点突变体中，选择了Sl EH1 ^W106L，Sl EH1 ^W106T，Sl EH1 ^F109I，Sl EH1 ^M180V，Sl EH1 ^F189I和Sl EH1 ^F189L，将rac - 1a水解为（R）-辛烷-1,2 -二醇（1b）的ee _p范围为62.1–82.4％。经过组合诱变和筛选后，有一个双位点突变体，SL EH1 ^{W106T / F189L}，得到具有最高α_小号的96.7％。使用200 mg mL ^-1的E. coli / sleh1 ^{W106T / F189L}湿细胞在20°C下进行400 mM（51.3 g L ^-1）rac - 1a的克级对映体水解24 h，产生（R）-1b，ee _p为94.7％，产率为95.6％。进行了针对20 mM rac - 1a-6a的Sl EH1 ^{W106T / F189L}的底物光谱测定，产生（R）-1b–6b的ee _p值为81.1–97.4％。此外，分子动力学模拟分析表明的regiopreference S1中EH1 ^{W106T / F189L}在C攻击_α（的小号） - 1A比的更大的S1中EH1，这是与它们一致的α_小号实验测量值。这项工作设计了一个优异的双位点突变体Sl EH1 ^{W106T / F189L}，用于对映体合成各种具有高ee值的手性链烷烃和链烯基1,2-二醇，尤其是（R）-1b和6b_p值。