DOI: 10.1002/anie.202316133 ISSN: 1433-7851

Engineering Hydroxylase Activity, Selectivity, and Stability for a Scalable Concise Synthesis of a Key Intermediate to Belzutifan

Wai Ling Cheung-Lee, Joshua N. Kolev, John A. McIntosh, Agnieszka A. Gil, Weilan Pan, Li Xiao, Juan E. Velásquez, Rekha Gangam, Matthew S. Winston, Shasha Li, Kotoe Abe, Embarek Alwedi, Zachary E. X. Dance, Haiyang Fan, Kaori Hiraga, Jungchul Kim, Birgit Kosjek, Diane N. Le, Nastaran Salehi Marzijarani, Keith Mattern, Jonathan P. McMullen, Karthik Narsimhan, Ajit Vikram, Wei Wang, Jia-Xuan Yan, Rong-Sheng Yang, Victoria Zhang, Wendy Zhong, Daniel A. DiRocco, William J. Morris, Grant S. Murphy, Kevin M. Maloney
  • General Chemistry
  • Catalysis

Biocatalytic oxidations are an emerging technology for selective C‐H bond activation. While promising for a range of selective oxidations, practical use of enzymes catalyzing aerobic hydroxylation is presently limited by their substrate scope and stability under industrially relevant conditions. Here, we report the engineering and practical application of a non‐heme iron and α‐ketoglutarate‐dependent dioxygenase for the direct stereo‐ and regio‐selective hydroxylation of a non‐native fluoroindanone en route to the oncology treatment belzutifan, replacing a five‐step chemical synthesis with a direct enantioselective hydroxylation. Mechanistic studies indicated that formation of the desired product was limited by enzyme stability and product overoxidation, with these properties subsequently improved by directed evolution, yielding a biocatalyst capable of >15,000 total turnovers. Highlighting the industrial utility of this biocatalyst, the high‐yielding, green, and efficient oxidation was demonstrated at kilogram scale for the synthesis of belzutifan.

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