Biocatalytic Aerobic Oxidation for Large-Scale Production of <i>trans</i>-3-Hydroxy-<scp>l</scp>-Proline
Direct, stereoselective hydroxylation of unactivated C–H bonds has the potential to dramatically streamline organic synthesis, and enzymes are particularly well-suited for facilitating these transformations, enabling clean and efficient processes that are scalable for industrial applications. Here, we report the development of a chemoenzymatic process for producing trans-3-hydroxy-l-proline (1), a key intermediate in synthesizing the proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor enlicitide decanoate (MK-0616). This process proceeds by direct biocatalytic aerobic C(sp3)–H hydroxylation of l-proline, mediated by an engineered Fe(II)- and α-ketoglutarate (α-KG)-dependent oxygenase. Through integrated enzyme discovery, protein engineering, and process development, we achieved a robust large-scale biocatalytic oxidation using minimal enzyme loading at high substrate concentrations. In combination with a high-yielding in situ protection and an efficient isolation procedure, this methodology has been used to produce high-purity N-Cbz-trans-3-hydroxy-l-proline 5 at >400 kg scale.
Authors
- Gregory A. Cope
- Keith Mattern (ORCID: https://orcid.org/0000-0002-4693-0698)
- Jeffrey T. Kuethe (ORCID: https://orcid.org/0000-0001-8955-6010)
- Mengbin Chen (ORCID: https://orcid.org/0000-0001-6135-8661)
- Sotheby Parke Bernet and Co.
- Kai‐Jiong Xiao (ORCID: https://orcid.org/0000-0001-5613-2035)
- Xue Zheng (ORCID: https://orcid.org/0000-0003-2990-6714)
- Wen-Qiu Xi
- E. B. Sirota (ORCID: https://orcid.org/0000-0002-6205-0295)
- Summer A. Baker Dockrey
- Yangzhong Qin (ORCID: https://orcid.org/0000-0002-2450-521X)
- Srikanth Saripalli (ORCID: https://orcid.org/0000-0002-3906-7574)
- Yingju Xu (ORCID: https://orcid.org/0000-0002-1984-9501)
- Jonathan Vroom
- Duan Jian-jun
- Peng Teng (ORCID: https://orcid.org/0000-0002-7412-9646)
- Anna Fryszkowska (ORCID: https://orcid.org/0000-0003-0872-1497)
- Erica L. Schwalm (ORCID: https://orcid.org/0000-0002-1719-6121)
- Alexandra C. Sun (ORCID: https://orcid.org/0000-0002-5583-8068)
- Nikki Kruse (ORCID: https://orcid.org/0000-0003-4989-3789)
- Christopher K. Prier (ORCID: https://orcid.org/0000-0003-0902-1636)
- David A. Thaisrivongs (ORCID: https://orcid.org/0000-0002-0387-8885)
- Yonggang Chen (ORCID: https://orcid.org/0000-0002-0970-6363)
- Lushi Tan
- Mary Stanik
- Jack Liang
Institutions
- Merck & Co., Inc., Rahway, NJ, USA (United States) (US)
- Codexis (United States) (US)
- Analytical Services (US)
- Analytical Engineering (United States) (US)
- Delta Electronics (China) (CN)
- Molecules for Health (United States) (US)
Publication Details
- Journal
- Organic Process Research & Development
- Published
- 2025-07-14
- DOI
- https://doi.org/10.1021/acs.oprd.5c00136
- Citations
- 2
- Primary Topic
- Enzyme Catalysis and Immobilization
- Type
- article
- Field-Weighted Citation Impact
- 1.61