Biocatalytic Synthesis Of Ethyl (R)-2-Hydroxy-4-Phenylbutyrate

The biocatalysis and chiral synthesis are attracting more and more attentions nowadays because of the escalating concerns on different pharmacological effects of different optical isomers and the limitations on the registration and application of racemic drugs. Biocatalytic synthesis of optically pure pharmaceuticals and their intermediates is one of the most investigated and exploited methods due to its remarkable advantages such as mild reaction conditions, high enantioselectivity and environmental benign. In this thesis, the whole-cell asymmetric bioreduction of ethyl 2-oxo-4-phenylbutyrate (OPBE) to prepare ethyl (R)-2-hydroxy-4-phenylbutyrate ((R)-HPBE) was studied.The GC analytic methods for ethyl 2-oxo-4-phenylbutyrate and ethyl 2-hydroxy-4-phenylbutyrate were established. Among 63 microorganisms tested in aqueous medium, Candida krusei SW2026 was isolated as a highly effective biocatalyst in the reduction process due to its excellent enantioselectivity and substrate tolerance. The fermentation conditions for the carbonyl reductase production and cell growth of Candida krusei SW2026 were optimized. The optimum fermentation medium comprised: 4.5% glucose, 3% peptone, 1.5% beef extract, and 0.05% Mn2+; and the optimum cultivation conditions were: initial pH of 6.0, temperature of 28°C, rotation speed of 180 r/min, and fermentation time of 48 h.The optimum reaction conditions of the asymmetric reduction of OPBE were as follows: temperature 30 oC, buffer pH 6.6. The effect of the co-substrate on the reaction was investigated and glucose was selected to be the most suitable co-substrate. The optimum cell concentration is 1.5 g/ 10 mL. The substrate inhibitory effect was identified in the study of effect of substrate concentration. Substrate fed-batch strategy was adopted to mitigate substrate toxicity and deactivation effect on the enzyme. The yield of the product was improved when substrate concentration was raised to 20 g/L. Under the optimal conditions, (R)-HPBE was obtained with ee of 97.55% and yield of 81.24% after 14 h of reaction. The product (R)-HPBE was purified by silica gel column chromatography and characterized by 1H and 13C NMR spectra.In order to achieve higher product concentration with desired enantiopurity and yield, nonaqueous medium biocatalysis was carried out. In the aqueous/organic biphasic system, dibutyl phthalate was the optimum organic solvent. It was shown that at 20 g/L of OPBE, ee, yield, and product concentration reached to 97.4%, 82.0% and 16.6 g/L respectively. In the aqueous/ionic liquid biphasic system, high product yield was achieved when [Bmim]PF6 was chosen to be the ionic liquid phase with isopropyl ether as the extractant. In 1 L reaction system, the product was obtained with 96.5% ee and 71.5% yield. After the purification, the purity and overall yield of the product was 99% and 86.7%, respectively.