Asymmetric Hydrolysis Of Styrene Oxide Catalyzed By Mung Bean Epoxide Hydrolases

Enantiopure chiral epoxides and ortho-diols are very important chiral synthons for finechemicals, playing a crucial role in the synthesis of medicines, pesticides and many others.Enantiopure ortho-diols can be prepared via the asymmetric hydrolysis of racemic epoxidescatalyzed by epoxide hydrolases (EHs). This process has attracted much attention due to itshigh enantioselectivity, mild reaction conditions and low environmental pollution. Owing toits cheapness and availability, Mung bean EHs showed great potential for the biosynthesis ofortho-diols. Up to now, however, only enantioconvergent hydrolysis of racemic p-nitrostyreneoxide using Mung bean EHs has been conducted in the aqueous monophasic system. Theasymmetric hydrolysis of epoxide carried out in aqueous monophasic system afforded verylow yield and product e.e., due to poor solubility of epoxide and its pronouncednon-enzymatic hydrolysis. To date there has been no report on the Mung bean EHs-mediatedasymmetric hydrolysis of epoxides in non-aqueous reaction media (organicsolvent-containing systems and ionic liquid-containing systems). On the other hand, ionicliquids (ILs) have recently gained increasing attention as green reaction media for biocatalysisand biotransformation. According to the reports available, many enzymes show higher activity,selectivity and stability in IL-containing systems. As a result, biocatalytic reactions inIL-containing systems exhibit tremendous application potential. In this dissertation, acomparative study was made of the biocatalytic asymmetric hydrolysis of styrene oxide (SO)to (R)-1-phenyl-1,2-ethanediol (PED) with Mung bean EHs in various reaction media, and theeffects of diverse reaction conditions on the biocatalytic hydrolysis of SO were exploredsystematically. The catalytic performances exhibited by Mung bean EHs in IL-containingsystems were also characterized. Additionally, the novel biocatalytic reaction system used forhighly efficient and enantioselective synthesis of enantiopure (R)-PED was established.The poor solubility of SO and the pronounced non-enzymatic hydrolysis of SO wereobserved in aqueous phase. The optimal temperature, buffer pH, shaking rate and substrateconcentration for the reaction performed in aqueous phase were35℃,6.5,220r/min and5mmol/L, respectively, under which the initial reaction rate, the yield and the product e.e.reached16.2mmol/(L·h),47.6%and92.6%. To further improve the reaction efficiency, phase transfer method was used in this work.The second phase was expected to effectively extract substrate and lower the concentration ofsubstrate in aqueous phase, thus relieving the substrate inhibition and the non-enzymatichydrolysis of SO. The effects of various organic solvents (ethyl acetate, trichloromethane,cyclohexane, n-hexane, n-octane and n-decane) on asymmetric hydrolysis of SO with Mungbean EHs were studied here. Owing to the excellent solvent property of n-hexane for SO, thesubstrate concentration was markedly increased in the n-hexane/buffer biphasic system.Additionally, the n-hexane/buffer biphasic system can inhibit effectively the non-enzymatichydrolysis of SO, thus significantly enhancing the product e.e.. Among all the tested organicsolvents, n-hexane was found to be the most suitable one for the reaction. For the reactionperformed in the n-hexane/buffer biphasic system, the optimal volume ratio of n-hexane tobuffer, reaction temperature, buffer pH, shaking rate and substrate concentration for thereaction were1:1,35℃,6.5,220r/min and20mmol/L, respectively, under which the initialreaction rate, the yield, and the product e.e. were5.4mmol/(L·h),49.2%, and94.3%,respectively.The addition of water-miscible ILs into the n-hexane/buffer biphasic system was thoughtto be able to improve the enzyme activity and eliminate the non-enzymatic hydrolysis of SO,thus improving the rate of the reaction. It was found that the effects of water-miscible ILs onthe asymmetric hydrolysis of SO varied widely. The ILs with cation containing a hydroxylgroup, namely1-(2’-hydroxyl)ethyl-3-methylimidazolium tetrafluoroborate (C2OHMIM·BF4)was found most suitable as the co-solvent for the reaction, owing to their low toxicity to theenzyme, leading to the highest initial rates, yield and product e.e.. In the n-hexane/bufferbiphasic system, addition of a small amount of the IL C2OHMIM·BF4partly eliminated thenon-enzymatic hydrolysis of SO. Also, the C2OHMIM·BF4-containing biphasic system canimprove the initial reaction rate and the concentration of substrate, thus significantlyincreasing reaction efficiency. The optimal ILs content, reaction temperature, buffer pH,shaking rate and substrate concentration for the reaction conducted in theC2OHMIM·BF4-containing biphasic system were found to be4%(v/v),35℃,6.5,220r/minand32mmol/L, respectively, under which the initial reaction rate, the yield, and the producte.e. were9.7mmol/(L·h),49.4%and95.3%, respectively. It was proved experimentally that the organic solvent-containing reaction system showedan obvious toxicity to EHs, leading to a substantial drop in catalytic activity of EHs withprolonged reaction time. To solve the aforementioned problems, the biocompatiblewater-immiscible ILs, instead of traditional organic solvents, were employed as the secondphase of the biphasic system for the EHs-mediated asymmetric hydrolysis of SO. Of theexamined nine hydrophobic ILs, the IL C4MIM·PF6showed the best biocompatibility withEHs and the excellent solubility for the substrate SO, and consequently inceased greatlysubstrate concentration in the reaction system. Also, the biphasic system containingC4MIM·PF6could effectively inhibit the non-enzymatic hydrolysis of SO and thus afforded amarkedly increased product e.e.. Therefore, C4MIM·PF6was regarded as the most suitablewater-immiscible IL for the reaction. In the C4MIM·PF6/buffer biphasic system, the optimalvolume ratio of IL to buffer, reaction temperature, buffer pH, shaking rate and substrateconcentration for the reaction were found to be1:6,35℃,6.5,220r/min and100mmol/L,respectively, under which the initial reaction rate, the yield and the product e.e. were18.4mmol/(L·h),49.4%and97.0%.This study provides not only a deeper understanding of enzyme catalysis in ILs, but also anovel and efficient route to enantiomerically pure ortho-diols.