Cascade Biocatalysis Asymmetric Aminohydroxylation Of Alkenes To Chiral β-Amino Alcohols

Chiralβ-amino alcohols,which have been extensively applied in medicine,fine chemicals,chiral auxiliaries and optical materials,are important components of many pharmaceutically active molecules and natural products.Aminohydroxylation of olefins is one of the most powerful strategies to access chiralβ-amino alcohols.In the past few years,traditional chemical methods have emerged as selective catalysts for aminohydroxylation of olefins,but these methods have some problems,such as low selectivity,expensive and toxic catalysts,and unfriendly environment.Biocatalysis has attracted much attention in aminohydroxylation of olefins as its significant advantages include mild reaction conditions,high selectivity and environment friendly.In this study,one-pot of four enzymes(styrene monooxygenase,epoxide hydrolase,alcohol dehydrogenase andω-transaminase)cascade catalytic reaction system was constructed,and asymmetric aminohydroxylation of styrene compounds was successfully catalyzed to synthesize enantiomerically pureβ-amino alcohols.This study develops a new high selective,economic and efficient catalytic synthesis approach for the green synthesis of chiralβ-amino alcohols,and provides theoretical guidance and practical reference for the establishment of low-cost and large-scale biological cascade catalytic synthesis of chiral compounds.Firstly,a(S)-ω-transaminase(BMTA)(from Bacillus megaterium SC6394)was cloned and overexpressed in E.coli,and the BMTA was purified and characterized.(S)-ω-transaminase BMTA was screened out from seven kinds of transaminases,and the BMTA was purified and characterized.The results showed that the maximum activity of BMTA was detected at pH 7.5and 55 ~oC,had good stability at pHs from 6.5 to 8.0 and temperatures from 4 ~oC to 30 ~oC.The BMTA had high activity toward(R)-β-amino alcohols with the specific activity of 1.107 U/mg,but no activity toward(S)-β-amino alcohols.Kinetic resolution of a set of racemicβ-amino alcohols by BMTA resulted in 50%conversion of substrates and leaved the(S)-β-amino alcohols in 50-99%ee.Asymmetric reductive amination of severalα-hydroxy ketones by BMTA resulted in 58-96%conversions and>99%ee of(R)-β-amino alcohols.Secondly,four kinds of element enzymes of the cascade catalytic system were screened out.This system includes the styrene monooxygenase(SMO)needed for the first step reaction,which could catalyze the synthesis of(S)-epoxides from olefins.The epoxide hydrolase(Sp EH)catalyzed hydrolysis of(S)-epoxide to(S)-1,2-diol in the second step reaction.The alcohol dehydrogenase(Go SCR)catalyzed the(S)-1,2-diol toα-hydroxy ketone in the third step reaction.The last step of the reaction was catalyzed by theω-transaminase MVTA or BMTA.MVTA is a(R)-ω-transaminase that catalyzed the synthesis of(S)-β-amino alcohols fromα-hydroxy ketones,whereas the BMTA is a(S)-ω-transaminase that catalyzed the synthesis of(R)-β-amino alcohols fromα-hydroxy ketones.NADH/NAD~+cycle would be accomplished under the catalysis of SMO and Go SCR.The reaction conditions of the multi-enzyme cascade catalysis system(SMO/SPEH/GOSCR/MVTA)were optimized by the catalytic styrene(20m M).The optimal reaction conditions were at pH 7.5 and 25 ~oC,and the enzyme ratio was SMO:Sp EH:Go SCR:MVTA=4:1:3:2.When the concentration of NADH was 0.02 m M,(S)-phenylglycoalcohol could be obtained in>99%ee and 93.5%conversion from styrene.The constructed multi-enzyme cascade systems(SMO/Sp EH/Go SCR/MVTA and SMO/Sp EH/Go SCR/BMTA)were successfully used to catalyze the synthesis of ten different(S)-or(R)-β-amino alcohols from styryl olefins(10-20 m M)with conversion of 13.9-93.5%and ee value of 86-99%.Finally,E.coli(CGS-DEM)and E.coli(CGS-DEB)co-expressing SMO,Sp EH,Go SCR and MVTA or BMTA were constructed for whole-cell catalytic reaction in the phosphate buffer(100 m M,pH 7.5)-hexadecane(1:1)reaction system,which could reduce the toxicity of the substrate olefins to the enzymes,and further reduce reaction costs without adding NADH.The co-expressing cells of E.coli(CGS-DEM)and E.coli(CGS-DEB)were successfully used to synthesize ten different(S)-or(R)-β-amino alcohols from styryl olefins(10 m M)with conversion of 16.5-99.7%and ee value of 86-99%.In a 100 m L reaction scale,E.coli(CGS-DEM)and E.coli(CGS-DEB)were used to catalyze the preparation of(S)-phenylglycinol(63.1mg),(R)-phenylglycinol(64.3 mg),(S)-2-amino-2-(4-fluorophenyl)ethanol(50.9 mg),(R)-2-amino-2-(4-fluorophenyl)ethanol(61.9 mg)and(R)-2-amino-2-(3-fluorophenyl)ethanol(55.2mg)from the substrate styrene(105 mg),4-fluorophenyl(122 mg)and 3-fluorophenyl(123 mg)in>99%ee.