Highly Stereoselective Reduction of Bulky Carbonyl Compounds by Carbonyl Reductase Variant†

doi:10.7503/cjcu20180214

Abstract

Abstract:

RCR-F285A/W286A, the variant of the (R)-specific carbonyl reductase(RCR), was used as the biocatalyst for asymmetric reduction of carbonyl compounds with bulky groups. In this study, ketoesters and heterocyclic ketone, including ethyl 2-oxo-4-phenylbutyrate(OPBE), ethyl benzoylacetate(EBA) and 1-benzyl-3-pyrrolidone, were employed. Catalytic property(including enzyme activity and kinetic parameters) and enantioselectivity of RCR-F285A/W286A towards tested bulky substrates were measured. RCR-F285A/W286A exhibited enzymatic activity towards all of the tested substrates. The corresponding chiral alcohols of the tested substrates acting as important pharmaceutical intermediates were prepared with high optical purity(e.e.>99%). Moreover, the effects of reaction conditions, including pH, temperature, substrate concentration, cosolvents and ratio of the optimum cosolvent, on the asymmetric reduction of OPBE with the highest yield were investigated. The results showed that the yield of 80.3% was obtained at pH=7.0, 30 ℃, 1 g/L substrate and isopropanol with volume fraction of 3.5%. Furthermore, fed-batch strategy was adopted to weaken substrate inhibition with the space-time yield 0.062 g·L^-1·h^-1 at 10 g/L OPBE. This result would provide the experimental basis for the industrial application of the enzyme-mediated asymmetric synthesis of ethyl (R)-2-hydroxy-4-phenylbutanoate.

Key words: Carbonyl reductase, Ethyl 2-oxo-4-phenylbutyrate, Ethyl benzoylacetate, 1-Benzyl-3-pyrrolidone, Asymmetric transformation

TrendMD:

LIANG Chen, NIE Yao, XU Yan. Highly Stereoselective Reduction of Bulky Carbonyl Compounds by Carbonyl Reductase Variant^†[J]. Chem. J. Chinese Universities, 2018, 39(11): 2438.

Figures/Tables 7

Table 1 Catalytic property of RCR-F285A/W286A towards bulky substrates

Specific activity/ (U·mg^-1)	K_m/ (mmol·L^-1)	k_cat/s^-1	k_cat/K_m/ (L·mmol^-1·s^-1)
1.33±0.1	3.38	0.43	0.13
1.2±0.09	3.89	1.04	0.27
0.36±0.06	8.76	4.35	0.50

Fig.1 Effect of pH on asymmetric reduction of OPBEThe reactions were carried out in 2 mL PBS buffer(0.1 mmol/L, pH=6.0—8.0) or Tris-HCl buffer(0.1 mmol/L, pH=8.5) with addition of 5 mmol/L NADH, 1 g/L OPBE, and 0.5%(volume fraction) isopropanol at 30 ℃, 12 h.

Fig.2 Effect of temperature on asymmetric reduction of OPBEReaction conditions: 5 mmol/L NADH, 1 g/L OPBE, and 0.5%(volume fraction) isopropanol in 2 mL PBS buffer(0.1 mmol/L, pH=7.0), 15—40 ℃, 12 h.

Fig.3 Effect of substrate concentration on asymmetric reduction of OPBEReaction conditions: 5 mmol/L NADH, OPBE(0.5—3 g/L) and 0.5%(volume fraction) isopropanol in 2 mL PBS buffer(0.1 mmol/L, pH=7.0), 30 ℃, 12 h.

Table 2 Effect of cosolvents on asymmetric reduction of OPBE*

Cosolvent	Ethanol	Isopropanol	N-Hexane	Ethyl acetate	DMSO	THF	Toluene	Blank
lgP	-0.24	0.38	3.50	0.68	-1.30	0.49	2.50	—
Yield(%)	15.7	64.79	0.22	3.94	3.54	0.48	0.41	0.21

Fig.4 Effect of isopropanol ratio on asymmetric reduction of OPBEReaction condition: 5 mmol/L NADH, 1 g/L OPBE and isopropanol(0.5%—5.5%, volume fraction) in 2 mL PBS buffer(0.1 mmol/L, pH=7.0), 30 ℃, 12 h.

Fig.5 Time course of asymmetric reduction of OPBE in fed-batch by crude enzymeReaction conditions: 5 mmol/L NADH, 1 g/L OPBE and isopropanol(0.35%, volume fraction) in 100 mL PBS buffer(0.1 mmol/L, pH=7.0), 30 ℃. 1 g/L OPBE dissolved in 350 μL isopropanol was added every 12 h and freeze-dried powder of crude enzyme was added every 30 h.

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