Study On Enzymatic Asymmetric Synthesis Of 2-Fluorocyclohexanone

Chirality is a widespread phenomenon and plays many important roles in nature.Chiral compounds have been used widely in a variety of fields involved pesticides,pharmaceuticals,fine chemicals and so on.Due to the different physiological activities of different enantiomers,compounds with high chiral purity are necessary achieved via separation and synthesis.Among the known methods,enzymetical methods have been attracted attention for the advantages of high catalytic efficiency,high specificity（stereoselectivity and regioselectivity）,mild reaction conditions and environmental friendly.Baeyer-Villiger monooxygenases（BVMO）belong to a type of flavin-dependent monooxygenase family,which catalyze a variety of ketones to convert into esters or lactones in the presence of O₂.P450-BM3is a type of cytochrome P450（P450-BM3）that is also a monooxygenase with multifunction.These two types of enzymes are widely distributed in nature,swith broad substrate-scope and strong catalytic function,they are potential industrial catalysts in consideration.Based on properties of BVMO and P450-BM3,this study focuses on directed evolution of these two monooxygenases for chiral resolution and asymmetric synthesis of 2-fluorocyclohexanone.It contains following parts.1.BVMO-Tm CHMO（cyclohexanone monooxygenase from Thermocrispum municipale DSM 44069）screening system was constructed by optimizing expression conditions of the enzyme,testing the reaction activity of wild-type Tm CHMO,and screening the NADPH（nicotinamide adenine dinucleotide phosphate）cycling systems.2.In view of the poor stereoselectivity of Tm CHMO wild-type enzyme,molecular docking simulation of Tm CHMO and racemic fluorocyclohexanone were performed,and the mutation site was determined based on the amino acids around the substrate-binding pocket.Three rounds of directed evolution were carried out on Tm CHMO by combining site-specific saturation mutation and iterative saturation mutation.Finally,two mutants with configuration reversal and configuration retention were obtained,with the ee values>85%.3.To solve the low rate of P450-BM3 converting fluorocyclohexane to 2-fluorocyclohexanone asymmetrically,we optimized the reaction conditions and attempted to use the hydrogenase Ts ADH for constructing a tandem reaction system,which increased the conversion rate to>40%.