Chemoenzymatic synthesis of pentalenolactones via stereoselective Riley oxidation by engineered P450_BM3

Riley oxidation is a powerful method for introducing oxygen functionality but typically relies on toxic reagents and generally lacks enantiocontrol. This limitation has historically restricted the utility of this transformation for the direct asymmetric construction of complex scaffolds. Here, we show that fusion library screening and directed evolution enable the highly enantio- and diastereoselective Riley-type oxidation of a cis-diquinane substrate catalyzed by engineered P450_BM3. This enzymatic desymmetrization strategy provides streamlined access to key chiral cis-diquinane building block on a gram scale, facilitating rapid downstream diversification. Furthermore, by merging this enzymatic desymmetrization with chemical ring formation and late-stage biosynthetic oxidation, we achieve asymmetric total synthesis of pentalenolactone D and neo-pentalenolactone D. This work highlights the power of integrating protein engineering with synthetic chemistry to synthesis of complex natural products.