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

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.

Benzylic Photobromination for the Synthesis of Belzutifan: Elucidation of Reaction Mechanisms Using In Situ LED-NMR.

A detailed mechanistic understanding of a benzylic photobromination en route to belzutifan (MK-6482, a small molecule for the treatment of renal cell carcinoma associated with von Hippel-Lindau syndrome) has been achieved using in situ LED-NMR spectroscopy in conjunction with kinetic analysis. Two distinct mechanisms of overbromination, namely, the ionic and radical pathways, have been revealed by this study. The behavior of the major reaction species, including reactants, intermediates, products, and side products, has been elucidated. Comprehensive understanding of both pathways informed and enabled mitigation of a major process risk: a sudden product decomposition. Detailed knowledge of the processes occurring during the reaction and their potential liabilities enabled the development of a robust photochemical continuous flow process implemented for commercial manufacturing.

Continuous Partial Oxidation of Methane to Methanol Catalyzed by Diffusion-Paired Copper Dimers in Copper-Exchanged Zeolites.

Copper-exchanged zeolites can continuously and selectively catalyze the partial oxidation of methane to methanol using only oxygen and water at low temperatures, but the genesis and nature of the active sites are currently unknown. Herein, we demonstrate that this reaction is catalyzed by a [Cu-O-Cu]2+ motif that forms via a hypothesized proton-aided diffusion of hydrated Cu ions within the cages of SSZ-13 zeolites. While various Cu configurations may be present and active for methane oxidation, a dimeric Cu motif is the primary active site for selective partial methane oxidation. Mechanistically, CH4 activation proceeds via rate-determining C-H scission to form a surface-bound C1 intermediate that can either be desorbed as methanol in the presence of H2O/H+ or completely oxidized to CO2 by gas-phase O2. High partial oxidation selectivity can be obtained with (i) high methane and water partial pressures and (ii) maximizing Cu dimer formation by using zeolites with high Al content and low Cu loadings.

Catalytic Oxidation of Methane into Methanol over Copper-Exchanged Zeolites with Oxygen at Low Temperature.

The direct catalytic conversion of methane to liquid oxygenated compounds, such as methanol or dimethyl ether, at low temperature using molecular oxygen is a grand challenge in C-H activation that has never been met with synthetic, heterogeneous catalysts. We report the first demonstration of direct, catalytic oxidation of methane into methanol with molecular oxygen over copper-exchanged zeolites at low reaction temperatures (483-498 K). Reaction kinetics studies show sustained catalytic activity and high selectivity for a variety of commercially available zeolite topologies under mild conditions (e.g., 483 K and atmospheric pressure). Transient and steady state measurements with isotopically labeled molecules confirm catalytic turnover. The catalytic rates and apparent activation energies are affected by the zeolite topology, with caged-based zeolites (e.g., Cu-SSZ-13) showing the highest rates. Although the reaction rates are low, the discovery of catalytic sites in copper-exchanged zeolites will accelerate the development of strategies to directly oxidize methane into methanol under mild conditions.

Methane to acetic acid over Cu-exchanged zeolites: mechanistic insights from a site-specific carbonylation reaction.

The selective low temperature oxidation of methane is an attractive yet challenging pathway to convert abundant natural gas into value added chemicals. Copper-exchanged ZSM-5 and mordenite (MOR) zeolites have received attention due to their ability to oxidize methane into methanol using molecular oxygen. In this work, the conversion of methane into acetic acid is demonstrated using Cu-MOR by coupling oxidation with carbonylation reactions. The carbonylation reaction, known to occur predominantly in the 8-membered ring (8MR) pockets of MOR, is used as a site-specific probe to gain insight into important mechanistic differences existing between Cu-MOR and Cu-ZSM-5 during methane oxidation. For the tandem reaction sequence, Cu-MOR generated drastically higher amounts of acetic acid when compared to Cu-ZSM-5 (22 vs 4 µmol/g). Preferential titration with sodium showed a direct correlation between the number of acid sites in the 8MR pockets in MOR and acetic acid yield, indicating that methoxy species present in the MOR side pockets undergo carbonylation. Coupled spectroscopic and reactivity measurements were used to identify the genesis of the oxidation sites and to validate the migration of methoxy species from the oxidation site to the carbonylation site. Our results indicate that the Cu(II)-O-Cu(II) sites previously associated with methane oxidation in both Cu-MOR and Cu-ZSM-5 are oxidation active but carbonylation inactive. In turn, combined UV-vis and EPR spectroscopic studies showed that a novel Cu(2+) site is formed at Cu/Al <0.2 in MOR. These sites oxidize methane and promote the migration of the product to a Brønsted acid site in the 8MR to undergo carbonylation.