Enzyme screening and engineering for N- and O-demethylation: key steps in the synthesis of buprenorphine

Alexandra T. P. Carvalho, Daniel F. A. R. Dourado, Jenny Spratt, Jill M. Caswell, Timofey Skvortsov, Derek J. Quinn, John S. Carey*, Thomas S. Moody*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

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Abstract

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 propose an alternative approach for buprenorphine synthesis via enzymatic N- and O-demethylation reactions. Utilizing rational enzyme design and directed evolution, we identified and engineered two oxygenase enzymes. For the N-demethylation reaction, the best variant achieved a cumulative improvement in conversion of 567-fold, while for the O-demethylation, the best variant achieved 22-fold cumulative improvement in conversion. A separate variant was able to efficiently catalyze both the N-demethylation and the O-demethylation reactions.
Original languageEnglish
JournalOrganic Process Research & Development
Early online date16 Feb 2024
DOIs
Publication statusEarly online date - 16 Feb 2024

Bibliographical note

doi: 10.1021/acs.oprd.3c00417

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