Enhancement of asymmetric bioreduction of N,N-dimethyl-3-keto-3-(2-thienyl)-1-propanamine to corresponding (S)-enantiomer by fusion of carbonyl reductase and glucose dehydrogenase

Abstract

Background

(S)-(−)-N,N-Dimethyl-3-hydroxy-3-(2-thienyl)-1-propanamine (DHTP) is a key intermediate for the preparation of (S)-duloxetine, an important antidepressant drug. However, so far, the catalytic efficiency of (S)-DHTP synthesis by asymmetric bioreduction is yet limited. The present study aims to develop an efficient system for synthesis of (S)-DHTP by bioreduction.

Results

Various recombinant carbonyl reductases were evaluated for asymmetric reduction of N,N-dimethyl-3-keto-3-(2-thienyl)-1-propanamine (DKTP) to produce (S)-DHTP. The NADPH-dependent carbonyl reductase CR2 was identified as the suitable candidate, giving (S)-DHTP in absolute configuration. Then the fusion protein involving CR2 and glucose dehydrogenase (CR2-L-GDH) was constructed to further improve cofactor regeneration and resulted catalytic efficiency of the enzymatic reduction. By studying the effects of reaction conditions involving cofactor regeneration, suitable catalytic system was achieved for CR2-L-GDH catalyzing (S)-DHTP synthesis. Consequently, (S)-DHTP (>99.9% e.e.) with yield of 97.66% was obtained from 20 g L⁻¹ DKTP within 8-h reaction, employing 40 g L⁻¹ glucose and 0.1 mmol L⁻¹ NADP⁺ to drive the cofactor regeneration, resulting in the space–time yield of 2.44 g L⁻¹ h⁻¹.

Conclusion

Optically pure (S)-DHTP with improved yield was obtained by fusion enzyme CR2-L-GDH. Fusion enzyme-mediated biocatalytic system would be promising to enhance reaction efficiency of enzyme-coupled system for preparation of optically active alcohols.

Keywords

Asymmetric reduction / Carbonyl reductase / Glucose dehydrogenase / Fusion enzyme / Cofactor regeneration

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Funding

National High Technology Research and Development Program of China(2015AA021004)

National Natural Science Foundation of China(21376107)

Natural Science Foundation of Jiangsu Province(BK20151124)

High-end Foreign Experts Recruitment Program(GDT20153200044)

Program for Advanced Talents within Six Industries of Jiangsu Province(2015-NY-007)

Fundamental Research Funds for the Central Universities(JUSRP51504)