Asymmetric Synthesis Of Atorvastatin Intermediate By Pichia Pastoris X-33

Atorvastatin can inhibit the HMG-CoA reductase.The HMG-CoA reductase is the rate-limiting enzyme in the biosynthesis of cholesterol in liver. Atorvastatin can decrease the cholesterol level in human blood through inhibiting the combine of HMG-CoA reductase and its substrate HMG-CoA, therefore atorvastatin is able to reduce high lipid density. Ethyl (R)-3-hydroxy-5-(1,3-dioxoisoindolin-2-yl)-pentanoate is a important potential intermediate for the synthesis of atorvastatin. This makes the synthesis of ethyl (R)-3-hydroxy-5-(1, 3-dioxoisoindolin-2-yl)-pentanoate have important application value.In this study, HPLC methods for analysing ethyl 3-hydroxy-5-(1,3-dioxoisoindolin-2-yl)-pentanoate and ethyl (R)-3-hydroxy-5-(1,3-dioxoisoindolin-2-yl)-pentanoate were established. The structure of ethyl (R)-3-hydroxy-5-(1,3-dioxoisoindolin-2-yl)-pentanoate was proved by HPLC-MS,1H NMR and special rotation.In order to synthesize ethyl (R)-3-hydroxy-5-(1,3-dioxoisoindolin-2-yl)-pentanoate by bioreduction, the yeast strains in our lab were screened. Interestingly, Ethyl (R)-3-hydroxy-5-(1,3-dioxoisoindolin-2-yl)-pentanoate was found to be produced efficiently from ethyl 5-(1,3-dioxoisoindolin-2-yl)-3-oxopentanoate by Pichia pastoris X-33. This strain had well enantioselectivity and substrate tolerance. The fermentation conditions of the cell growth of Pichia pastoris X-33 were optimized for the carbonyl reduction. The optimum cultivation conditions were as follows:fermentation time of 30 h, temperature of 30℃and initial pH of 8.0.We studied the reaction conditions of the asymmetric reduction of ethyl (R)-3-hydroxy-5-(1,3-dioxoisoindolin-2-yl)-pentanoate. The effects of initial substrate concentration, reaction time, co-substrate, amount of yeast cells, initial pH, reaction temperature, as well as cosolvent on the yield and enantiomeric excesses (e.e. value) of product were examined in mono-phase system. The optimal reaction conditions are as fallows:substrate concentration 10 g/L, cell concentration 120 g/L, co-substrate glucose concentration 120 g/L, initial pH 6.5, reaction temperature 35℃, reaction time 12 h, cosolvent tween-80 40ml/L, and the yield 90.44% with the high e.e. value of 96.67%. The yield can be stable after three times reutilization.