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.

中文翻译：

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通过羰基还原酶和葡萄糖脱氢酶的融合增强N，N-二甲基-3-酮-3-（2-噻吩基）-1-丙胺的不对称生物还原成相应的（S）-对映体

背景

（S）-（-）-N，N-二甲基-3-羟基-3-（2-噻吩基）-1-丙胺（DHTP）是制备（S）-度洛西汀（一种重要的抗抑郁药）的关键中间体。然而，到目前为止，通过不对称生物还原合成（S）-DHTP的催化效率仍然受到限制。本研究旨在开发一种通过生物还原合成（S）-DHTP的有效系统。

结果

评价了各种重组羰基还原酶的不对称还原N，N-二甲基-3-酮-3-（2-噻吩基）-1-丙胺（DKTP）以产生（S）-DHTP。鉴定出NADPH依赖性羰基还原酶CR2是合适的候选物，以绝对构型给出（S）-DHTP。然后构建包含CR2和葡萄糖脱氢酶的融合蛋白（CR2-L-GDH），以进一步改善辅因子的再生并产生酶促还原的催化效率。通过研究涉及辅因子再生的反应条件的影响，获得了适合CR2-L-GDH催化（S）-DHTP合成的催化体系。结果，从20g L ^-1获得产率为97.66％的（S）-DHTP（＞ 99.9％ee）。在8小时反应中的DKTP，使用40 g L ^-1葡萄糖和0.1 mmol L ^-1 NADP ⁺驱动辅因子再生，导致时空产率为2.44 g L ^-1  h ^-1。

结论

通过融合酶CR2-L-GDH获得具有提高的收率的光学纯的（S）-DHTP。融合酶介导的生物催化系统有望提高制备光学活性醇的酶偶联系统的反应效率。