Enzyme Screening and Engineering for N- and O-Demethylation: Key Steps in the Synthesis of Buprenorphine

Benzylisoquinoline alkaloids are valuable active ingredients in medicines that are typically extracted from plants and subsequently derivatized. Buprenorphine is a member of this family of compounds and is an effective analgesic and is also used for the treatment of opioid use disorder. The commercial route of synthesis for buprenorphine starts from thebaine and uses toxic reagents and harsh reaction conditions for the N- and O-demethylation steps.

Here we 1propose an alternative approach for buprenorphine synthesis via enzymatic N- and Odemethylation reactions. Utilizing rational enzyme design and directed evolution, two oxygenase enzymes were identified and engineered. For the N-demethylation reaction, the best variant achieved a cumulative improvement in conversion of 567-fold, while for the Odemethylation the best variant achieved 22-fold cumulative improvement in conversion. A separate variant was able to efficiently catalyse both the N-demethylation and the Odemethylation reactions.