Enzymatic Asymmetric Synthesis Of Tert Butyl(3R,5R)-6-Cyano-3,5-Dihydroxhexanoate

(3R,5R)-tert-butyl-6-cyano-3,5-dihydroxyhexanoate(ATS-7)is a valuable chiral synthon for atorvastatin calcium,which is used for the synthesis of the cholesterol-lowering drugs.The method of enzyme catalysis for the preparation of chiral drugs is now recognized as an environmental friendly and efficient mean.This thesis designs a new potential synthetic route of ATS-7,which is via two-step asymmetric reduction process using tert-butyl 6-cyano-3,5-dioxohexanoate(CDOH)as the starting material.The main contents of this work were as follows:1.Asymmetric reduction of CDOH to(5R)-tert-butyl-6-cyano-5-hydroxyl-3-oxohexanoate(ATS-6)as the model reaction,adopting the strategy of gene mining,build the enzyme library which contains nine carbonyl reductases.But no catalytic activity is detected for CDOH.2.Asymmetric reduction of ATS-6 to ATS-7 as the model reaction,adopting the strategy of gene mining,build the enzyme library which contains six aldo-keto reductases.These aldo-keto reductases were named SCAKR,PKAKR,CGAKR,KMAKR,NCAKR,ZBAKR.Whole cell lysate these aldo-keto reductases are soluble in expression.And aldo-keto reductase SCAKR can convert ATS-6 to ATS-7 with the enzyme activity of 672U/L.3.The molecular weight of the aldo-keto reductase SCAKR is approximately 38kDa.It is active and stable at 25? and pH 7.0.The Michaelis-Menten constant(Km),maximal reaction rate(vmax)and catalytic number(kcat)for ATS-6 were 32.04mM,1.99U/mg and 0.259s-1.Then the medium and induction conditions were optimized and the enzyme activity of 1.281 U/mL were obtained.4.Asymmetric reduction of ATS-6 to ATS-7 need to add NADPH as co-enzyme.In order to solve co-enzyme NADPH regeneration during reaction,glucose dehydrogenase was over expressed and used to regenerate NADPH.After the comparison of several strategies of enzymatic regeneration of co-enzyme,recombinant strain E.coli BL21(DE3)/pET28a-scakr1-gdh-2 was the best regeneration of co-enzyme.And E.coli BL21(DE3)/pET28a-scakr1-gdh-2 reduce 14.75g/L substrate with conversion rate of 100%after 2.5 hours,the space-time yield was 9.16g·L-1 h-1.