Asymmetric Enzymatic Reduction Of Prochiral β-ketoesters By Enzyme-coupled System

Asymmetric reduction of prochiral ketones catalyzed by oxidoreductases is still one ofmost important methods for the preparation of chiral alcohols. Compared with whole-cellssystem, isolated enzyme systems are always superior due to fast reaction rate, high opticalpurity with absence of side reactions and mass transfer resistance. In this thesis, a carbonylreductase was selected from the tool-box containing13recombinant oxidoreductases forhighly stereoselective reduction of the target reaction from4-chloro-3-oxobutanoate ethylester (COBE) to (S)-4-chloro-3-hydroxybutanoate ethyl ester [(S)-CHBE], which is a keyintermediate in synthesis of pharmaceuticals. Enzymatic properties of the enzyme and aglucose dehydrogenase (GDH) used for coenzyme regeneration were investigated. By kineticsstudies of each enzyme, a reaction velocity model was developed for the optimization ofenzyme activity ratio in this enzyme-coupled system according to kcatwhich indicated thevelocity and efficiency of the reactions catalyzed by redox enzymes. An aqueous/organicbiphasic system was constructed for reduction of COBE in high concentration. Besides,different enzyme-coupled systems were applied to catalyze several kinds of β-ketoesters, andthe reaction principles including the influence of electronic effect and steric effect ofsubstituting groups were studied.(1) The NADPH dependent carbonyl reductase CR2from Candida macedoniensis AKU4588was selected from oxidoreductase tool-box to produce optically pure (>99.9%e.e.)(S)-4-chloro-3-hydroxybutanoate ethyl ester [(S)-CHBE] using GDH for coenzymeregeneration. By enzymatic properity studies of the two enzymes, diversities were found: theoptimal reaction pH and temperature of CR2were pH4.5and40℃, while that of GDH waspH7.0and42℃respectively. Enzyme activity of CR2kept stable under acid or neutral pHcondition while GDH was stable under neutral pH condition. Kinetics studies indicated thatboth of the enzymatic reactions catalyzed by each enzyme belonged to Ordered Bi Bimechanism or Theorell-Chance mechanism. The kcatvalue of CR2and GDH were8.45S-1and351.86S-1reapectively. Due to the influence of electronic effect and steric effect of thesubstituting groups, there were remarkable differences between the substrate selectivities ofCR2when catalyzed various β-ketoesters while COBE was the optimal substrate among them.This study had widened the applicability of CR2for reduction of β-ketoesers.(2) The enzyme-coupled system was constructed for coenzyme regeneration by CR2andGDH in aqueous phase with optimal reaction pH (7.0), temperature (37℃) and molar ratioof co-substrate/substrate (1.8/1). Then, a reaction velocity model was developed according tokinetics parameter kcatto optimize enzyme activity ratio of CR2/GDH. Under optimal reactionconditions catalyzed by the optimized system,50mmol/L COBE was completely convertedinto (S)-CHBE (>99.9%e.e.) with space-time yield of191.6mmol/L·h and specificspace-time yield of47.9mmol/L·h·U. Compared with the reported reaction systems, theimproved enzyme-coupled system had shown advantages of higher space-time yield andbetter economical efficiency of enzyme molecule.(3) Due to low solubility of COBE in water together with the toxic action and inhibitory action to each enzyme activity brought by COBE or (S)-CHBE, the organic solvent dibutylphthalate with aqueous/organic phase ratio of1:1was selected as an appropriate solvent for insitu extraction. With the combination of single aqueous reaction system and biphasic reactionsystem, COBE concentration was increased to300mmol/L by substrate feeding method.Product yield reached97.4%with optical purity of more than99.9%e.e. within6h, and the(S)-CHBE concentration was cumulated to475mmol/L with the specific space-time yield of16.2mmol/L·h·U which was much higher than the reported reaction systems. The productswere (S)-alcohols when the hydrogen atom at β-position was substituted by element ofhalogen family, and the e.e. value dropped regularly along with the decrease of substrateselectivity of CR2. The configurations were overturned and all products were (R)-alcoholswhen aromatic groups substituted groups at β-position. To different β-ketoesters when alkylgroup connected to the ester bond with methyl at β-position, the products once been detectedwere (R)-alcohols. On the other hand, the product yields would increase effectively whencatalyzed by biphasic reaction systems. The reaction medium of ionic liquid could efficientlyimprove the stereoselectivity of CR2when catalyzed most of the β-ketoesters with opticalpurity of more than95%e.e.. Application of ionic liquid into enzymatic reactions wouldprovide a new and available method for preparation of highly optically pure chiral alcohols.