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.

Portable capillary LC for in-line UV monitoring and MS detection: Comparable sensitivity and much lower solvent consumption.

Pharmaceutical development currently relies on quality separation methods from early discovery through to line-of-site manufacturing. There have been significant advancements made regarding the column particle packing, internal diameter, length connectivity, the understanding of the impact key parameters like void volume, flow rate, and temperature all that affects the resultant separation quality, that is, resolution, peak shape, peak width, run time, and signal-to-noise ratio. There is however a strong need to establish better alternatives to large bulky high-performance liquid chromatography racks either for process analytical reaction monitoring or mass spectrometry analysis in establishing product quality. Compact, portable high-pressure liquid chromatography can be a more efficient alternative to traditional ultra-high pressure liquid chromatography and traditional liquid chromatography. The compact versatile instrument evaluated here allows good separation control with either the on-board column with fixed ultra-violet wavelength cartridge or for use with a high-resolution mass spectrometry. Significant space reduction results in greener lab spaces with improved energy efficiency for smaller labs with lower energy demands. In addition, this compact liquid chromatography was used as a portable reaction monitoring solution to compare forced degradation kinetics and assess portable liquid chromatography-mass spectrometry capability for the analyses required for pharmaceutical drug product testing.

Electrochemical Recycling of Adenosine Triphosphate in Biocatalytic Reaction Cascades.

Adenosine triphosphate (ATP) provides the driving force necessary for critical biological functions in all living organisms. In synthetic biocatalytic reactions, this cofactor is recycled in situ using high-energy stoichiometric reagents, an approach that generates waste and poses challenges with enzyme stability. On the other hand, an electrochemical recycling system would use electrons as a convenient source of energy. We report a method that uses electricity to turn over enzymes for ATP generation in a simplified cellular respiration mimic. The method is simple, robust, and scalable, as well as broadly applicable to complex enzymatic processes including a four-enzyme biocatalytic cascade in the synthesis of the antiviral molnupiravir.