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

Wai Ling Cheung-LeeJoshua N KolevJohn A McIntoshAgnieszka A GilWeilan PanLi XiaoJuan E VelásquezRekha GangamMatthew S WinstonShasha LiKotoe AbeEmbarek AlwediZachary E X DanceHaiyang FanKaori HiragaJungchul KimBirgit KosjekDiane N LeNastaran Salehi MarzijaraniKeith MatternJonathan P McMullenKarthik NarsimhanAjit VikramWei WangJia-Xuan YanRong-Sheng YangVictoria X ZhangWendy ZhongDaniel A DiRoccoWilliam J MorrisGrant S MurphyKevin M Maloney
Published in: Angewandte Chemie (International ed. in English) (2024)
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.
Keyphrases
  • heavy metals
  • wastewater treatment
  • hydrogen peroxide
  • structural basis
  • electron transfer