Study On P450 Monooxygenase Catalyzed Resolution Of 2-Substituted-1,2,3,4-Tetrahydroquinoline

Objective: To kinetic resolution 2-substituted-1,2,3,4-tetrahydroquinoline using P450 monooxygenase as a biocatalyst by P450-catalyzed C-N bond oxidation,and develope a new biocatalytic synthesis route for the preparation of chiral 2-substituted-1,2,3,4-tetrahydroquinoline compounds.Methods: 1.Screening of enzymes: The 2-methyl-1,2,3,4-tetrahydroquinoline（2-MTHQ）was used as a template substrate to screen the laboratory-constructed self-supporting P450 monooxygenase and non-self-supporting P450 monooxygenase engineered bacteria and their mutants.Finally,a biological oxidative enzyme can be obtained which can generate 2-MTHQ with excellent stereoselectivity.It was found that P450PL2-SM3 was oxidized with excellent（S）-stereoselectivity to obtain（R）-2-MTHQ with an ee value of 88%.It was also found that P450_pyr-L-13 obtained（S）-2-MTHQ by（R）-stereoselective oxidative splitting of 2-MTHQ with an ee value of 41%.2.Optimization of reaction conditions: Using excellent P450 monooxygenase whole cells as biocatalysts,the enzyme culture system and reaction system for 2-MTHQ oxidation resolution were systematically investigated.The most suitable P450 monooxygenase optimal culture conditions and the most suitable reaction system for 2-MTHQ stereoselective oxidative resolution were established 3.Investigation of substrate scope: Using the most suitable reaction system,the multiple types of 2-substituted-1,2,3,4-tetrahydroquinoline substrates were investigated.Results: 1 Laboratory conserved P450 monooxygenases,including P450_PL2 series（5 strains）,P450_PL7 series（5 strains）,P450_pyr-L series（13 strains）,P450_PL2-2 mutants（9 strains）,P450DA and its mutants（13 strains）,were screened with（rac）-2-MTHQ as substrate and P450 whole cells as biocatalyst.2 An optimal culture system and optimal reaction system for（S）-configuration-selective P450PL2-SM3 to 2-MTHQ chiral oxidative resolution was constructed.The optimal culture system was as follows: inducer concentration 0.1 mM,induction temperature 25 ℃,induction time 8h;optimal reaction system: reaction temperature 15 ℃,reaction buffer PBS pH 8.0,cell concentration 10 g cdw/L,substrate concentration 2 mM,reaction time 1.5h,ee value 93%,conversion 80%.3 An optimal culture system and optimal reaction system for（R）-configuration-selective P450_pyr-L-13 to 2-MTHQ chiral oxidative resolution was constructed.The optimal culture system was as follows: inducer 0.2 mM,induction temperature 25 ℃,induction time 12h;optimal reaction system: 35 ℃,Gly-Na OH pH 8.0,10 g cdw/L,2 mM substrate,5 mL system reaction 24h,93% ee value,80% conversion.4.The substrate scope was expanded through the optimal reaction system for 2-substituted-1,2,3,4-tetrahydroquinoline compounds.It was found that P450PL2-SM3 has led tohigh enantioselectivity for benzene ring-substituted substrates（1f,1g,1j,1k）,and the ee value of（R）-configuration substrate can reach 60-92% after oxidative resolution.2-position substituents（cyclopropyl 1b,n-butyl 1c,isobutyl 1d and tert-butyl 1e）showed lower oxidation resolution activity.It appears that the P450_pyr-L-13 substrate was poorly tolerated in this stereoselectivity,except that 2-cyclopropyl substituted（2b）and 6-F substituted（1g）substrates furnished products with 48% ee and 37% ee,respectively.5.Conclusion: The（S）-stereo-selective biocatalyst P450PL2-SM3 and（R）-stereo-selective biocatalyst P450_pyr-L-13 were obtained by screening laboratory-conserved P450 monooxygenases with（rac）-2-MTHQ as substrates,establishing a stereo-selective complementary biocatalytic synthesis route for chiral 2-substituted-1,2,3,4-tetrahydroquinoline oxidative splitting.In the selective C-N oxidation of 2-MTHQ by P450PL2-SM3,benzyl hydroxylation of 2-MTHQ was performed at the same time due to the hybrid nature of its catalytic function.2-methyl-1,2,3,4-tetrahydroquinolin-4-ol was generated.However,P450_pyr-L-13 showed no hydroxylation activity against 2-MTHQ during its catalysis.In the present work,we discovered for the first time that P450 monooxygenase can achieve kinetic splitting of 2-substituted-1,2,3,4-tetrahydroquinoline by stereoselective oxidation of C-N bond,expanding the new application of P450 monooxygenase in biocatalytic reaction.