Mechanistic Investigation of UPO-Catalyzed O-Dealkylation via Kinetic and ¹³C-Labeling Studies

Alkyl ethers are attractive protecting groups for alcohols due to their small size, low cost, and stability under a wide range of reaction conditions. However, their use in synthetic chemistry is limited because deprotection typically requires harsh, non-selective conditions. To address this challenge, we aim to establish enzymatic O-dealkylation as a mild and selective strategy to make alkyl ethers more viable as protecting groups. Unspecific peroxygenases (UPOs) are promising candidates for this task, catalyzing diverse oxidative transformations using H₂O₂ as the sole oxidant. In initial screening experiments, we evaluated a library of UPOs against a structurally diverse panel of alkyl ether substrates. One enzyme displayed broad activity, effectively cleaving methyl, ethyl and propyl ethers from aliphatic and aromatic substrates. Product analysis from these reactions revealed substrate-dependent overoxidation levels that suggested the presence of different underlying reaction pathways. This observation prompted detailed mechanistic studies using kinetic profiling and 13C-labeling experiments. Our findings provide new mechanistic insight into UPO-catalyzed O-dealkylation and demonstrate the enzyme's potential for enabling selective, mild deprotection of alkyl ethers—unlocking their broader use as protecting groups in synthetic applications.