Asymmetric Synthesis of (S)-N,N-Dimethyl-3-hydroxy-3-(2-thienyl)-1-propanamine by Cell-free System of Carbonyl Reductase CR2†

doi:10.7503/cjcu20170164

Abstract

Abstract:

(S)-N,N-Dimethyl-3-hydroxy-3-(2-thienyl)-1-propanamine[(S)-DHTP] is an important intermediate for the production of (S)-duloxetine, which is a new and effective antidepressant drug. Since the efficiency of biosynthesis of (S)-DHTP is yet limited, development of suitable biocatalytic system is necessary for (S)-DHTP production from asymmetric reduction of N,N-dimethyl-3-keto-3-(2-thienyl)-1-propanamine(DKTP). In this study, the cell-free biocatalytic system involving recombinant carbonyl reductase CR2 was constructed and the corresponding reaction conditions were optimized. (S)-DHTP with high optical purity(e.e. value>99.9%) and the yield of 61.70% was obtained from DKTP(10 g/L) at pH=8.0[0.1 mol/L triethanolamine(TEA) buffer] and 30 ℃ after reaction for 6 h, by employing glucose(120 g/L) as the co-substrate and coenzyme NADP⁺(0.1 mmol/L) to drive the cofactor regeneration system. During the reaction process, the pH value decreased significantly due to the formation of gluconic acid from glucose for cofactor recycling. Then the yield of (S)-DHTP was increased to 67.73% by controlling the reaction pH. Substrate inhibition was observed when investigating the conversion of DKTP under different concentrations, and the space time yield of 1.33 g·L^-1·h^-1 was achieved for DKTP(10 g/L).

Key words: Biocatalysis, Enzyme, Crude enzyme system, Asymmetric reduction, (S)-N, N-Dimethyl-3-hydroxy-3-(2-thienyl)-1-propanamin

CLC Number:

O621.25+4.2

TrendMD:

SUN Taiqiang, LI Bin, NIE Yao, WANG Dong, XU Yan. Asymmetric Synthesis of (S)-N,N-Dimethyl-3-hydroxy-3-(2-thienyl)-1-propanamine by Cell-free System of Carbonyl Reductase CR2^†[J]. Chem. J. Chinese Universities, 2017, 38(10): 1772.

Figures/Tables 7

Fig.1 Effect of reaction temperature on asymmetric synthesis of (S)-DHTP by CR2 cell-free systemAll reactions were carried out in 10 mL TEA buffer(0.1 mol/L) comprising DKTP(10 g/L) with addition of glucose(100 g/L) and NADP+(0.2 mmol/L). All the results were the average of three parallel replicates.

Fig.2 Effect of initial pH on asymmetric synthesis of (S)-DHTP by CR2 cell-free systemAll reactions were carried out in 10 mL TEA buffer(0.1 mol/L) comprising DKTP(10 g/L) with addition of glucose(100 g/L) and NADP+(0.2 mmol/L). All the results were the average of three parallel replicates.

Table 1 Effect of cofactor concentration on reduction of DKTP by CR2 cell-free system*

NADP⁺/(mmol·L^-1)	e.e.(%)	Yield(%)	TTN	NADP⁺/(mmol·L^-1)	e.e.(%)	Yield(%)	TTN
0.200	>99.9	45	103	0.010	>99.9	38	1512
0.100	>99.9	53	241	0.005	>99.9	33	3436
0.050	>99.9	50	325	0
0.020	>99.9	49	1129

Fig.3 Effect of co-substrates on reduction of DKTP by CR2 cell-free systemAll reactions were carried out in 10 mL TEA buffer(0.1 mol/L, pH=8.0) comprising DKTP(10 g/L) with addition of cosubstrate(10 g/L) and NADP+(0.1 mmolL) at 30 ℃. All the results were the average of three parallel replicates. a. Glycerol; b. ethanol; c. 2-propanol; d. sorbitol; e. mannitol; f. sucrose; g. fructose; h. lactose; i. maltose; j. glucose; k. xylose.

Fig.4 Effect of glucose concentration on reduction of DKTP by CR2 cell-free systemAll reactions were carried out in 10 mL TEA buffer(0.1 mol/L, pH=8.0) comprising DKTP(10 g/L) and NADP+(0.1 mmol/L) at 30 ℃. All the results were the average of three parallel replicates.

Fig.5 Curves of variation of pH value in the asymmetric reaction processAll reactions were carried out in 10 mL TEA buffer(0.1 mol/L, pH=8.0) comprising DKTP(10 g/L) with addition of glucose(120 g/L) and NADP+(0.1 mmol/L) at 30 ℃. All the results were the average of three parallel replicates.

Fig.6 Reaction process of CR2 cell-free system catalyzing asymmetric reduction of DKTP under different concentrationsAll reactions were carried out in 10 mL TEA buffer(0.1 mol/L, pH=8.0) comprising DKTP with addition of glucose(100 g/L) and NADP+(0.2 mmol/L) at 30 ℃. All the results were the average of three parallel replicates.

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