Chemoenzymatic synthesis of (1R,3R)-3-hydroxycyclopentanemethanol: An intermediate of carbocyclic-ddA

Abstract

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.