四氢噻吩-3-酮还原酶的筛选与表征
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摘要
通过基因挖掘技术获得一种来源于炭疽芽胞杆菌的短链脱氢酶BaCR1。它能够催化四氢噻吩-3-酮的不对称还原反应,进而合成碳青霉烯类抗生素硫培南的重要手性中间体(R)-四氢噻吩-3-醇。对BaCR1进行了催化性能表征,结果表明最适反应pH为7.0,最适反应温度为30℃,催化剂转化数(k_(cat))与米氏常数(K_M)之比(k_(cat)/K_M)为0.25 L/(mmol·s)。在100 mL的反应体系中,偶联葡萄糖脱氢酶用于辅酶再生,在底物上载量为50 mmol/L、重组大肠杆菌整细胞(干重)上载量为20 g/L的条件下,无需额外添加辅因子NADPH,反应24 h,转化率大于99%,还原产物的e.e.值为84%,实现了产物的克级制备,体现了酶法不对称还原制备(R)-四氢噻吩-3-醇的潜力。
(R)-Tetrahydrothiophene-3-ol, a key intermediate of sulopenem(a novel 2-sulfanyl pennan compound, used for therapy of livestock bacterial diseases), has been highly desired for pharmaceutical use over the last three decades. Apart from the complicated and environmentally unfriendly chemical synthesis, the most straightforward approach for this synthesis is the biological asymmetric reduction of tetrahydrothiophene-3-one. A high enantioselective ketoreductase is needed to accomplish this reaction. However, it is difficult to screen a high enantioselective ketoreductase towards the nearly spatially symmetrical substrate, tetrahydrothiophene-3-one. Some researches have been conducted to work on this issue, where mere enantiomeric excess(e.e.) or long reaction time was observed. In this study, a new enzyme(designated as BaCR1) from Bacillus anthracis str. Ames by library screening has been explored, which can be used for asymmetric synthesis of(R)-tetrahydrothiophene-3-ol. The characteristics of this enzyme were further investigated after protein purification by affinity adsorption separation with Ni-column. After performing reaction optimization, we found that the optimal pH value and temperature of BaCR1 for catalysis reaction was 7.0 and 30 ℃, respectively. k_(cat)/K_M was 0.25 L/(mmol·s), using a substrate concentration ranging from 0.125 mmol/L to 50 mmol/L. The half-life of purified BaCR1 was 9.63 h and 3.04 h at 30 ℃ and 40 ℃, respectively, suggesting good thermostability of BaCR1 in the catalysis reaction system. Finally,(R)-tetrahydrothiophene-3-ol was prepared in 100 mL scale. Without extra addition of NADPH, the reaction conversion could reach 99% with 84% of e.e. at the substrate loading of 50 mmol/L. Considering that this is a nature parent enzyme, the result is reasonably acceptable, which also reveals the potential of enzyme-catalyzed asymmetric synthesis of(R)-tetrahydrothiophen-3-ol.
(R)-Tetrahydrothiophene-3-ol, a key intermediate of sulopenem(a novel 2-sulfanyl pennan compound, used for therapy of livestock bacterial diseases), has been highly desired for pharmaceutical use over the last three decades. Apart from the complicated and environmentally unfriendly chemical synthesis, the most straightforward approach for this synthesis is the biological asymmetric reduction of tetrahydrothiophene-3-one. A high enantioselective ketoreductase is needed to accomplish this reaction. However, it is difficult to screen a high enantioselective ketoreductase towards the nearly spatially symmetrical substrate, tetrahydrothiophene-3-one. Some researches have been conducted to work on this issue, where mere enantiomeric excess(e.e.) or long reaction time was observed. In this study, a new enzyme(designated as BaCR1) from Bacillus anthracis str. Ames by library screening has been explored, which can be used for asymmetric synthesis of(R)-tetrahydrothiophene-3-ol. The characteristics of this enzyme were further investigated after protein purification by affinity adsorption separation with Ni-column. After performing reaction optimization, we found that the optimal pH value and temperature of BaCR1 for catalysis reaction was 7.0 and 30 ℃, respectively. k_(cat)/K_M was 0.25 L/(mmol·s), using a substrate concentration ranging from 0.125 mmol/L to 50 mmol/L. The half-life of purified BaCR1 was 9.63 h and 3.04 h at 30 ℃ and 40 ℃, respectively, suggesting good thermostability of BaCR1 in the catalysis reaction system. Finally,(R)-tetrahydrothiophene-3-ol was prepared in 100 mL scale. Without extra addition of NADPH, the reaction conversion could reach 99% with 84% of e.e. at the substrate loading of 50 mmol/L. Considering that this is a nature parent enzyme, the result is reasonably acceptable, which also reveals the potential of enzyme-catalyzed asymmetric synthesis of(R)-tetrahydrothiophen-3-ol.
引文
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[7] SUN Z, LONSDALE R, ILIE A, et al. Catalytic asymmetric reduction of difficult-to-reduce ketones: Triple-code saturation mutagenesis of an alcohol dehydrogenase [J]. ACS Catalysis. 2016, 6, 1598-1605.
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[9] MATSUO K, KAWABE S I, TOKUDA Y, et al. Asymmetric reduction of ketones with a germinated plant [J]. Tetrahedron: Asymmetry, 2008, 19(2): 157-159.
[10] ALEXANDRE F R, PANTALEONE D P, TAYLOR P P, et al. Amine-boranes: Effective reducing agents for the deracemisation of dl-amino acids using l-amino acid oxidase from Proteus myxofaciens [J]. Tetrahedron Letters, 2002, 43(4): 707-710.
[11] FARINA V, REEVES J T, SENANAYAKE C H, et al. Asymmetric synthesis of active pharmaceutical ingredients [J]. Chemical Reviews, 2006, 106(7): 2734-2793.
[12] JONES J B, SCHWARTZ H M. Enzymes in organic syntheses: 19. Evaluation of the stereoselectivities of horse liver alcohol dehydrogenase; catalyzed oxidoreductions of hydroxy-and ketothiolanes,-thianes, and-thiepanes [J]. Canadian Journal of Chemistry, 1981, 59(11): 1574-1579
[13] NAGAI H, KONUKI K, ITO M, et al. Method for manufacturing optically active tetrahydrothiophene derivative and method for crystallization of optically active tetrahydrothiophene-3-ol: US20080050787 [P]. 2008.
[14] LIANG J, LALONDE J, BORUP B, et al. Development of a biocatalytic process as an alternative to the(-)-DIP-Cl-mediated asymmetric reduction of a key intermediate of montelukast [J]. Organic Process Research & Development, 2010, 14(1): 193-198.
[15] LIANG J, MUNDORFF E, RAMA V, et al. Highly enantioselective reduction of a small heterocyclic ketone: Biocatalytic reducion of tetrahydrothiophene-3-one to the corresponding (R)-alcohol [J]. Organic Process Research & Development, 2010, 14(1): 188-192.
[2] BREUER M, DITRICH K, HABICHER T, et al. Industrial methods for the production of optically active intermediates [J]. Angewandte Chemie International Edition, 2010, 43(7): 788-824.
[3] 周伟佳, 郑高伟, 陈焕辉, 等. 利用脂肪酶Novozym 435催化转酯反应对映选择性合成(R)-硫辛酸的手性前体[J]. 华东理工大学学报(自然科学版), 2014, 40(1): 53-57.
[4] GHANEM A. Trends in lipase-catalyzed asymmetric access to enantiomerically pure/enriched compounds [J]. Tetrahedron, 2007, 63(8): 1721-1754.
[5] NAKAMURA K, YAMANAKA R, MATSUDA T, et al. Recent developments in asymmetric reduction of ketones with biocatalysts [J]. Tetrahedron: Asymmetry, 2003, 14(18): 2659-2681.
[6] XU G, NI Y. Bioreductive preparation of ACE inhibitors precursor(R)-2-hydroxy-4-phenylbutanoate esters: Recent advances and future perspectives [J]. Bioresources and Bioprocessing, 2015, 2: 15-25.
[7] SUN Z, LONSDALE R, ILIE A, et al. Catalytic asymmetric reduction of difficult-to-reduce ketones: Triple-code saturation mutagenesis of an alcohol dehydrogenase [J]. ACS Catalysis. 2016, 6, 1598-1605.
[8] REETZ M T, WU S. Greatly reduced amino acid alphabets in directed evolution: Making the right choice for saturation mutagenesis at homologous enzyme positions [J]. Chemical Communications, 2008, 43(43): 5499-5501.
[9] MATSUO K, KAWABE S I, TOKUDA Y, et al. Asymmetric reduction of ketones with a germinated plant [J]. Tetrahedron: Asymmetry, 2008, 19(2): 157-159.
[10] ALEXANDRE F R, PANTALEONE D P, TAYLOR P P, et al. Amine-boranes: Effective reducing agents for the deracemisation of dl-amino acids using l-amino acid oxidase from Proteus myxofaciens [J]. Tetrahedron Letters, 2002, 43(4): 707-710.
[11] FARINA V, REEVES J T, SENANAYAKE C H, et al. Asymmetric synthesis of active pharmaceutical ingredients [J]. Chemical Reviews, 2006, 106(7): 2734-2793.
[12] JONES J B, SCHWARTZ H M. Enzymes in organic syntheses: 19. Evaluation of the stereoselectivities of horse liver alcohol dehydrogenase; catalyzed oxidoreductions of hydroxy-and ketothiolanes,-thianes, and-thiepanes [J]. Canadian Journal of Chemistry, 1981, 59(11): 1574-1579
[13] NAGAI H, KONUKI K, ITO M, et al. Method for manufacturing optically active tetrahydrothiophene derivative and method for crystallization of optically active tetrahydrothiophene-3-ol: US20080050787 [P]. 2008.
[14] LIANG J, LALONDE J, BORUP B, et al. Development of a biocatalytic process as an alternative to the(-)-DIP-Cl-mediated asymmetric reduction of a key intermediate of montelukast [J]. Organic Process Research & Development, 2010, 14(1): 193-198.
[15] LIANG J, MUNDORFF E, RAMA V, et al. Highly enantioselective reduction of a small heterocyclic ketone: Biocatalytic reducion of tetrahydrothiophene-3-one to the corresponding (R)-alcohol [J]. Organic Process Research & Development, 2010, 14(1): 188-192.