Application Of Enzymatic Asymmetric Reduction In The Preraration Of Atorvastatin's Pharmacophore Side Chain Tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate

Statins is a group of 3-hydroxy-3-methylglutaryl coenzyme A(HMG-CoA)reductase inhibitor,firstly launched in 1980s,which is extensively used in the therapy of osteoporosis and hypercholesterolemia.Recently,statins have dominated the market of cholesterol-lowering drugs.Atorvastatin(ATS),the third generation of statins,has been the best-selling pharmaceutical agent since 2006 with over $ 10 billion in annual sales.As the key chiral building block for atorvastatin,synthesis of t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate is of greatly economic and social benefits.Before 2000,t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate was exclusively synthesized by chemical synthesis,however,the developed chemical processes suffered from the use of costly,environment-unfriendly catalysts and extremely reaction conditions.With the developments of biotechnology and enzyme engineering,biocatalysis with excellent stereoselectivity has been the research focus of pharmaceutical industry and the powerful tool for production the chiral compound currently.To improve the ketoreductase production of strain ZJB-08109,maintained in our lab,its cultivation conditions were optimized in detail.Results showed that the optimal cultivation conditions are as follows:seed age 10 h,inoculum size 7.0%,and initial medium pH 5.5,culture temperature 32?,fermentation time 14 h.Under the optimal cultivation conditions,the biomass of strain ZJB-08109 reached 4.13 g/L,with specific activity of 54.48 U/g.The catalytic properties of strain ZJB-08109 were studied using t-butyl(5R)-6-cyano-5-hydroxyhexanoate as substrate.It was found that the enzyme activity of strain ZJB-08109 peaked at round pH 6.0,which was found to be beneficial to the ketoreductase pH-stability.The thermostablity of the strain ZJB-08109 was found to be weak at its optimal catalytic temperature of 30?.The KDecat of the ketoreductase at 25?,30?and 35? were 0.01934,0.03925 and 0.10537 h-1 with corresponding half-life times(t1/2)of 35.84,17.66 and 6.58 h,respectively.Outcomes demonstrated that ketoreductase activity of strain ZJB-08109 was inhibited by Fe2+,Fe3+,Zn2+,Mg2+ and Ni2+,especially Cu+ and Cu2+,however,other metal irons tested had insignificant impact on the ketoreductase activity.The ketoreductase activity of strain ZJB-08109 was enhanced by the presence of Tween-20 and Sorbitol,but inhibited by SDS.Adding 3.0%(w/v)Tween-20 improved the reductase activity by 1.46 times.Based on the results of above studies,a detailed study on the preparation of t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate via the bioreduction of t-butyl(5R)-6-cyano-5-hydroxyhexanoate using resting cells of strain ZJB-08109 was conducted in this paper.And the optimal reaction conditions were confirmed as follows:temperature 31?,buffer K2HPO4-KH2PO4(50.0 mM),pH 6.2,0.7%glucose acting as co-substrate,and concentration of substrate 31.5 mmol L-1,wet cells 30 g/L.Under the optimal reaction conditions,the accumulated t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate concentration reached 20.4 mM in 6 h,in a yield of 64.6%and d.e.>99.0%.