Biocatalytic Aerobic Oxidation for Large-Scale Production of trans-3-Hydroxy-l-Proline

Abstract

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.