Engineered Styrene Monooxygenase by the RRIPE Strategy for Efficient Enantioselective Synthesis of Efinaconazole Intermediate

Styrene monooxygenase (SMO) has been extensively employed in the epoxidation of aromatic alkenes for synthesizing pharmaceutical intermediates. We explored SMO for the biocatalytic conversion of aromatic alkenes with significant steric hindrances. For this purpose, we selected an efinaconazole intermediate as the target product of the epoxidation reaction via SMO catalysis. Herein, we describe a strategy for synthesizing the key chiral intermediate 1b of efinaconazole. A styrene monooxygenase (SMO) from Streptomyces exfoliatus (SeStyA) was employed as a candidate for the biocatalytic asymmetric epoxidation of substrate 1a to 1b. By employing the RRIPE strategy (residue–residue interactions-guided protein engineering strategy), we tailored the enzyme activity for synthesizing efinaconazole intermediate starting from a nonreactive SeStyA toward the substrate 1a. RRIPE involves saturation mutagenesis of residues in the substrate binding pocket and residues within 5 Å of the identified positive residue after each round of mutagenesis. After 13 rounds of enzyme evolution through the RRIPE strategy, we obtained a variant, M13, that demonstrated approximately a 1680-fold increase in conversion compared to the M1 variant, which initially displayed an extremely low activity. On a gram-scale synthesis, M13 achieved both conversion and diastereomeric excess (de) exceeding 99% after 48 h. This study unveils an efficient approach to providing high-quality intermediates for pharmaceutical synthesis, including efinaconazole and other important antifungal agents. Furthermore, the general approach for constructing suitable enzymes through the RRIPE strategy contributes to the advancement of enzyme engineering in the pharmaceutical industry.