Enzymatic C(sp³)‑H Amination: P450-Catalyzed Conversion of Carbonazidates into Oxazolidinones

Cytochrome P450 enzymes can effectively promote the activation and cyclization of carbonazidate substrates to yield oxazolidinones via an intramolecular nitrene C–H insertion reaction. Investigation of the substrate scope shows that while benzylic/allylic C–H bonds are most readily aminated by these biocatalysts, stronger, secondary C–H bonds are also accessible to functionalization. Leveraging this “non-native” reactivity and assisted by fingerprint-based predictions, improved active-site variants of the bacterial P450 CYP102A1 could be identified to mediate the aminofunctionalization of two terpene natural products with high regio- and stereoselectivity. Mechanistic studies and KIE experiments show that the C–H activation step in these reactions is rate-limiting and proceeds in a stepwise manner, namely, via hydrogen atom abstraction followed by radical recombination. This study expands the reactivity scope of P450-based catalysts in the context of nitrene transfer transformations and provides first-time insights into the mechanism of P450-catalyzed C–H amination reactions

Evolution of a Green and Sustainable Manufacturing Process for Belzutifan: Part 1Process History and Development Strategy

An improved synthesis has been developed for belzutifan, a novel HIF-2α inhibitor for the treatment of Von Hippel–Lindau (VHL) disease-associated renal cell carcinoma (RCC). The efficiency of previous supply and commercial routes was encumbered by a lengthy 5-step sequence, needed to install a chiral benzylic alcohol by traditional methods. Identification and directed evolution of FoPip4H, an iron/α-ketoglutarate dependent hydroxylase, enabled a direct enantioselective C–H hydroxylation of a simple indanone starting material. While this enabling transformation set the stage for a greatly improved synthesis, several other key innovations were made including the development of a base-metal-catalyzed sulfonylation, a KRED-catalyzed dynamic kinetic resolution, and a facile SNAr reaction in water. Together, these improvements resulted in a significantly shorter synthesis (9 steps) versus the supply route (16 steps) and a 75% reduction in process mass intensity (PMI), while also removing the reliance on third-row transition metals and toxic solvents

Evolution of a Green and Sustainable Manufacturing Process for Belzutifan: Part 4Applications of Process Analytical Technology in Heterogeneous Biocatalytic Hydroxylation

Belzutifan has been approved recently by the U.S. Food and Drug Administration (FDA) for treating patients with certain types of Von Hippel-Lindau (VHL) disease-associated tumors. Although a commercial synthetic process has been established to make belzutifan, a further optimized process with fewer steps, improved cost-effectiveness, and a smaller environmental footprint is always in demand. In the new commercial synthetic route, a single-step biocatalytic hydroxylation reaction was used to replace the five chemical steps required in the previous route. In developing this new biocatalytic reaction, multiple process analytical technologies (PATs), such as Fourier transform infrared spectroscopy (FTIR), in situ imaging, dissolved oxygen monitoring, etc., were used to track important reaction parameters under complex reaction conditions (e.g., multiphases and dense slurry). With quantitative modeling, the product concentration and yield can be tracked in real time based on FTIR. This is particularly important for dense slurry reactions, for which offline sampling becomes challenging due to the sample inhomogeneity. In-depth mechanistic insights were also obtained using PATs revealing reaction kinetics controlled by different mass transfer limited processes, as well as the unique role of 1-octanol. These PAT-enabled capabilities for reaction tracking and understanding facilitated process development from the laboratory to the pilot scale and ensured a robust process for the hydroxylation reaction