Bioinformatic-Guided Discovery of O -Dealkylation Activity in Cytochrome P450 Monooxygenases

Abstract The O-dealkylation of alkyl ethers—or the removal of small alkyl protecting groups (PGs)—remains a significant challenge in organic synthesis. While traditional chemical methods require harsh reaction conditions and lack sufficient selectivity, biocatalytic approaches represent valuable alternatives. However, enzymes facilitating the desired O-dealkylation reactions are scarce. In this work, we applied a sequence similarity network (SSN)-guided approach to identify cytochrome P450 monooxygenases (CYPs) with O-dealkylation activity within the growing sequence diversity found in public databases. We investigated six CYPs, three of which exhibited significant activity toward structurally diverse ethers bearing methyl, ethyl, propyl, or allyl groups. While P450ThcB and P450Terp enabled the deprotection of aromatic and aliphatic primary and secondary ethers, P450Sca2-mut complemented the substrate scope by effectively deprotecting sterically demanding tertiary ethers. In particular, P450ThcB and P450Terp exhibited high regioselectivity, preferentially cleaving phenolic over benzylic ethers. Furthermore, P450ThcB enabled the regioselective deprotection of veratryl alcohol. Preparative scale reactions of three substrates yielded up to 74% of the isolated and (regioselectively) cleaved alcohol product. Together, these findings substantially expand the enzymatic toolbox for the selective removal of alkyl PGs and highlight the suitability of SSN-guided database mining approaches for the identification of biocatalysts with the desired function.