碳环-ddA的合成,一种有效的抗乙型肝炎的抗病毒药物,显著依赖于(1R,3R)-3-羟基环戊烯乙醇作为关键中间体。为了有效地生产这种中间体,我们的研究采用了化学酶的方法。选择合适的生物催化剂是基于底物相似性,导致我们采用源自ThermusscotogutusSA-01的CrS烯酸还原酶。此外,我们开发了一种用于NADH再生的酶系统,利用博伊氏念珠菌的甲酸脱氢酶。该系统促进了(S)-4-(羟甲基)环戊-2-烯酮的有效催化,导致形成(3R)-3-(羟甲基)环戊酮。此外,我们成功克隆,表达,纯化,并对大肠杆菌中的CrS酶进行了表征。确定了最佳反应条件,显示最高活性发生在45°C和pH8.0。通过使用5mM(S)-4-(羟甲基)环戊-2-烯酮,0.05mMFMN,0.2mMNADH,10μMCrS,40μM甲酸脱氢酶,和40mM甲酸钠,在35°C和pH7.0下在45分钟内实现完全转化。随后,(1R,通过简单的三步化学转化过程获得3R)-3-羟基环戊烯乙醇。这项研究不仅提供了合成关键中间体的有效方法,而且还强调了生物催化剂和酶系统在化学酶合成方法中的重要性。
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.