Abstract：
Chiral epichlorohydrin (ECH) is an attractive intermediate for chiral pharmaceuticals and chemicals preparation. The asymmetric synthesis of chiral ECH using 1,3-dicholoro-2-propanol (1,3-DCP) catalyzed by a haloalcohol dehalogenase (HHDH) was considered as a feasible approach. However, the reverse ring opening reaction caused low optical purity of chiral ECH, thus severely restricts the industrial application of HHDHs. In the present study, a novel selective conformation adjustment strategy was developed with an engineered HheCPS to regulate the kinetic parameters of the forward and reverse reactions, based on site saturation mutation and molecular simulation analysis. The HheCPS mutant E85P was constructed with a markable change in the conformation of (S)-ECH in the substrate pocket and a slight impact on the interaction between 1,3-DCP and the enzyme, which resulted in the kinetic deceleration of the reverse reactions. Compared with HheCPS, the catalytic efficiency (kcat(S)-ECH/Km(S)-ECH) of the reversed reaction dropped to 0.23-fold (from 0.13 to 0.03 mM-1 s-1), while the catalytic efficiency (kcat(1,3-DCP)/Km(1,3-DCP)) of the forward reaction only reduced from 0.83 to 0.71 mM-1 s-1. With 40 mM 1,3-DCP as substrate, HheCPS E85P catalyzed the synthesis of (S)-ECH with the yield up to 55.35% and the e.e. increased from 92.54 to >99%. Our work provided an effective approach for understanding the stereoselective catalytic mechanism as well as the green manufacturing of chiral epoxides.
摘要：
手性表氯醇（ECH）是一种有吸引力的中间体，用于制备手性药物和化学品。在卤代醇脱卤酶（HHDH）催化下，使用1,3-二氯-2-丙醇（1,3-DCP）不对称合成手性ECH被认为是一种可行的方法。然而,反向开环反应导致手性ECH的光学纯度低，严重制约了HHDHs的工业应用。在本研究中,一种新的选择性构象调整策略与工程HheCPS开发调节动力学参数的正向和反向反应，基于位点饱和突变和分子模拟分析。HheCPS突变体E85P的构建具有底物口袋中（S）-ECH构象的可标记变化，并且对1,3-DCP与酶之间的相互作用有轻微影响，这导致了逆反应的动力学减速。与HheCPS相比,逆反应的催化效率（kcat（S）-ECH/Km（S）-ECH）降至0.23倍（从0.13降至0.03mM-1s-1），而正向反应的催化效率（kcat（1,3-DCP）/Km（1,3-DCP））仅从0.83降至0.71mM-1s-1。以40mM1,3-DCP为底物,HheCPSE85P催化合成（S）-ECH，收率高达55.35％，e.e。从92.54增加到>99％。我们的工作为理解立体选择性催化机理以及手性环氧化物的绿色制造提供了有效的方法。
