State Key Laboratory of Chemical Resources Engineering, Beijing University of Chemical Technology, Beijing 100029, China; Bontac Bio-Engineering (Shenzhen) Co., Ltd., Shenzhen, Guangdong 518107, China.
State Key Laboratory of Chemical Resources Engineering, Beijing University of Chemical Technology, Beijing 100029, China.
J Biosci Bioeng. 2024 Aug;138(2):111-117. doi: 10.1016/j.jbiosc.2024.05.002. Epub 2024 May 31.
The synthesis of carbocyclic-ddA, a potent antiviral agent against hepatitis B, relies significantly on (1R,3R)-3-hydroxycyclopentanemethanol as a key intermediate. To effectively produce this intermediate, our study employed a chemoenzymatic approach. The selection of appropriate biocatalysts was based on substrate similarity, leading us to adopt the CrS enoate reductase derived from Thermus scotoductus SA-01. Additionally, we developed an enzymatic system for NADH regeneration, utilising formate dehydrogenase from Candida boidinii. This system facilitated the efficient catalysis of (S)-4-(hydroxymethyl)cyclopent-2-enone, resulting in the formation of (3R)-3-(hydroxymethyl) cyclopentanone. Furthermore, we successfully cloned, expressed, purified, and characterized the CrS enzyme in Escherichia coli. Optimal reaction conditions were determined, revealing that the highest activity occurred at 45 °C and pH 8.0. By employing 5 mM (S)-4-(hydroxymethyl)cyclopent-2-enone, 0.05 mM FMN, 0.2 mM NADH, 10 μM CrS, 40 μM formic acid dehydrogenase, and 40 mM sodium formate, complete conversion was achieved within 45 min at 35 °C and pH 7.0. Subsequently, (1R,3R)-3-hydroxycyclopentanemethanol was obtained through a simple three-step chemical conversion process. This study not only presents an effective method for synthesizing the crucial intermediate but also highlights the importance of biocatalysts and enzymatic systems in chemoenzymatic synthesis approaches.
作为一种有效的抗病毒药物，针对乙型肝炎的carbocyclic-ddA 的合成，主要依赖于（1R,3R）-3-羟基环戊烷甲醇作为关键中间体。为了有效地生产这种中间体，我们采用了化学酶法途径。根据底物相似性选择合适的生物催化剂，我们采用了来源于 Thermus scotoductus SA-01 的 CrS 烯醇还原酶。此外，我们还开发了一种利用 Candida boidinii 甲酸脱氢酶的 NADH 再生酶系统。该系统促进了（S）-4-（羟甲基）环戊-2-烯酮的高效催化，形成（3R）-3-（羟甲基）环戊酮。此外，我们成功地在大肠杆菌中克隆、表达、纯化和表征了 CrS 酶。确定了最佳反应条件，结果表明，在 45°C 和 pH 8.0 时，酶活性最高。采用 5mM（S）-4-（羟甲基）环戊-2-烯酮、0.05mM FMN、0.2mM NADH、10μM CrS、40μM 甲酸脱氢酶和 40mM 甲酸钠，在 35°C 和 pH 7.0 下，45min 内可实现完全转化。随后，通过简单的三步化学转化过程得到（1R,3R）-3-羟基环戊烷甲醇。本研究不仅提供了一种有效的合成关键中间体的方法，还强调了生物催化剂和酶系统在化学酶法合成方法中的重要性。
