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Chun, Stephanie

DiRocco, Daniel

Galanie, Stephanie

Gangam, Rekha

Kolev, Joshua N.

Kosjek, Birgit

Ling Cheung-Lee, Wai

Maloney, Kevin

McIntosh, John

Moore, Jeffrey

Morris, William

Murphy, Grant

Salehi Marzijarani, Nastaran

Velásquez, Juan E.

Winston, Matthew

Zhong, Wendy

English

Engineering Conferences International

ENGINEERING HYDROXYLASE AND KETOREDUCTASE ACTIVITY, SELECTIVITY, AND STABILITY FOR A SCALABLE CONCISE SYNTHESIS OF BELZUTIFAN  Stephanie Galanie, Process Research and Development, MSD Stephanie.Galanie@merck.com Wai Ling Cheung-Lee, Process Research and Development, MSD Joshua N. Kolev, Process Research and Development, MSD Stephanie Chun, Process Research and Development, MSD Birgit Kosjek, Process Research and Development, MSD Jeffrey Moore, Process Research and Development, MSD John McIntosh, Process Research and Development, MSD Rekha Gangam, Analytical Research and Development, MSD Matthew Winston, Process Research and Development, MSD Juan E. Velásquez, Process Research and Development, MSD Nastaran Salehi Marzijarani, Process Research and Development, MSD Wendy Zhong, Analytical Research and Development, MSD Daniel DiRocco, Process Research and Development, MSD William Morris, Process Research and Development, MSD Grant Murphy, Process Research and Development, MSD Kevin Maloney, Process Research and Development, MSD   Key Words: Hydroxylation, Biocatalysis, Directed Evolution, α-ketoglutarate-dependent dioxygenase, ketoreductase  Exquisite stereo- and regioselectivity is a key advantage of biocatalytic reactions. In this talk, we present the identification and development of two enzymes, an α-ketoglutarate-dependent dioxygenase and a ketoreductase, that together enable the installation of three adjacent stereocenters in the oncology treatment belzutifan. Biocatalytic oxidations a powerful tool for selective C-H bond activation. However, industrial use of enzymes catalyzing aerobic hydroxylation is presently limited by their substrate scope and stability under practical conditions. We report the engineering and application of an Fe/αKG-dependent dioxygenase for the direct stereo- and regio-selective hydroxylation of a non-native fluoroindanone en route to belzutifan, replacing a five-step chemical synthesis with a direct enantioselective hydroxylation. Desired product formation was found to be limited by enzyme stability and product overoxidation, and these properties were subsequently improved by directed evolution, yielding a biocatalyst capable of >15,000 total turnovers. We further report the identification and directed evolution of a ketoreductase (KRED) to perform a dynamic kinetic resolution in tandem with chemical fluorination with 50% organic co-solvent. Highlighting the industrial utility of these biocatalysts, the high-yielding, green, and efficient oxidation and dynamic kinetic resolution were demonstrated at kilogram scale for the synthesis of belzutifan.     RSO OHOORSO OO RSO OHO FOHRSO OOORSO OOOFoPip4HO2 KREDiPrOHSelectfluorRSO OOOBrRSO OOOOHOR = OAr, FOSO OF FOHFCN

Engineering Hydroxylase and Ketoreductase Activity, Selectivity, and Stability for a Scalable Concise Synthesis of Belzutifan

https://dc.engconfintl.org/cgi/viewcontent.cgi?filename=0&article=1024&context=enzyme_xxvii&type=additional

Engineering Hydroxylase and Ketoreductase Activity, Selectivity, and Stability for a Scalable Concise Synthesis of Belzutifan

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Engineering Conferences International