Asymmetric Reducing Of 6-cyano-(5R)-hydroxy-3-oxohexanoic Acid Tert-butyl Ester With Carbonyl Reductase

Atorvastatin calcium is the active ingredient of the best-selling cholesterol lowering drug Lipitor(?),having a pharmacophore side chain(3 R,5R)-dihydroxyhexanoate.t-butyl 6-cyano-(3R,5R)-dihydroxy-hexanoa te was important building block for the pharmacophore side chain.The stereoselective synthesis of the building block t-butyl 6-cyano-(3R,5R)-dihydroxy-hexanoate has been a very valuable and competitive research area due to a high cost by the current synthetic route.Its traditional chemical synthesis relies on expensive borane stoichiometric reagents,or in the presence of catalytic of metal and organics,and is conducted at low temperature below-60 ?,concomitant with considerable amounts of energy comsuption and waste formation.So it's very important to developed a successful biocatalytic process for t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate.An isolate X25 displaying moderate diastereoselective ketoreductase activity towards t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate was screened using the enrichment culture technique.It was identified belonging to Pichia guilliermondii based on phenotypic,biochemical characteristics and 18S rDNA sequence comparison.Using one-factor-at-a-time experimental design,culture conditions and media composition were optimized.Under the optimized conditions:glucose 40.0 g/L,yeast extract 30.0 g/L,NaCk 1.0g/L,Na2PO4 3.0 g/L,CuCl2 50.0 mg/L,pH7.0,t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate 4.0 g/L,loading size 30.0 ml,inoculum size 4.0%,temperature 28 ?,culture time 40 h,P.guilliermondii X25 ketoreductase activity was 61.3 U/L,improved 2 times,de 92.9%,And a biocatalytic process for t-butyl 6-cyano-(3R,5R)-dihydroxyhe-xanoate was successfully developed with Pichia guilliermondii X25.The best condition for biocatalytic process was 35 ?,pH7.0 phosphate buffer(50 mM),t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate 10.0 g/L,glucose 50.0 g/L acting as NAD(P)H regeneration cosubstrate,resting cells 50.0 g/L,after 25 h the yield of t-butyl 6-cyano-(5R)-hydroxy-3-oxo hexanoate was 95.7%,de 93.2%.As the best condition was similar to initial condition,the yield just improved 7.2%.