Discovery and Engineering <i>R</i> uCHMO for Enantioselective Construction of Chiral Quaternary and Tertiary Centers via Baeyer–Villiger Oxidation: Chemoenzymatic Synthesis of (+)-Kopsiyunnanine K

The stereoselective Baeyer–Villiger (BV) oxidation of 4,4-disubstituted cyclohexanones to chiral ε-lactones bearing quaternary stereocenters remains an unsolved challenge. Existing chiral metal complex catalysts, organocatalysts, and Baeyer–Villiger monooxygenases (BVMOs) are largely ineffective against these sterically hindered substrates despite working well with 4-monosubstituted analogues. Herein, we report the discovery and engineering of a cyclohexanone monooxygenase from Ruegeria sp. R14 (RuCHMO). It catalyzes the asymmetric BV oxidation of both 4,4-disubstituted and 4-monosubstituted cyclohexanones, providing efficient access to chiral S-configured ε-lactones bearing either quaternary or tertiary stereocenters. Through semirational engineering, the optimized variant Mu2 (RuCHMO-F490M/L201I) exhibits enhanced catalytic efficiency (up to 97% isolated yield and >99% ee) and broad substrate scope. The synthetic utility of this methodology is demonstrated by a concise chemoenzymatic total synthesis of the complex alkaloid (+)-kopsiyunnanine K, highlighting its potential in the synthesis of complex natural products.