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.

In recent years, there have been numerous incidents of serious accidents related to impurities of optical isomers, particularly in pharmaceutical treatment, environmental problems, and pesticide application. Among these impurities, chiral epichlorohydrin (ECH) is an important C3 synthon and a potentially genotoxic impurity. The enantiopure forms of S-ECH and R-ECH are key raw materials for synthesizing many drugs, which make it important to accurately quantify the specific conformation of chiral epichlorohydrin in pharmaceuticals. In this paper, we achieved the separation of chiral ECH by gas chromatography (GC) and based on the combination of vibrational circular dichroism (VCD) experiments and theoretical calculations, the qualitative method of chiral ECH was achieved without relying on a single enantiomeric standard.