An Engineered Cytidine Deaminase for Biocatalytic Production of a Key Intermediate of the Covid-19 Antiviral Molnupiravir.

The Covid-19 pandemic highlights the urgent need for cost-effective processes to rapidly manufacture antiviral drugs at scale. Here we report a concise biocatalytic process for Molnupiravir, a nucleoside analogue recently approved as an orally available treatment for SARS-CoV-2. Key to the success of this process was the development of an efficient biocatalyst for the production of N-hydroxy-cytidine through evolutionary adaption of the hydrolytic enzyme cytidine deaminase. This engineered biocatalyst performs >85 000 turnovers in less than 3 h, operates at 180 g/L substrate loading, and benefits from in situ crystallization of the N-hydroxy-cytidine product (85% yield), which can be converted to Molnupiravir by a selective 5'-acylation using Novozym 435.

Engineering an enantioselective amine oxidase for the synthesis of pharmaceutical building blocks and alkaloid natural products.

The development of cost-effective and sustainable catalytic methods for the production of enantiomerically pure chiral amines is a key challenge facing the pharmaceutical and fine chemical industries. This challenge is highlighted by the estimate that 40-45% of drug candidates contain a chiral amine, fueling a demand for broadly applicable synthetic methods that deliver target structures in high yield and enantiomeric excess. Herein we describe the development and application of a "toolbox" of monoamine oxidase variants from Aspergillus niger (MAO-N) which display remarkable substrate scope and tolerance for sterically demanding motifs, including a new variant, which exhibits high activity and enantioselectivity toward substrates containing the aminodiphenylmethane (benzhydrylamine) template. By combining rational structure-guided engineering with high-throughput screening, it has been possible to expand the substrate scope of MAO-N to accommodate amine substrates containing bulky aryl substituents. These engineered MAO-N biocatalysts have been applied in deracemization reactions for the efficient asymmetric synthesis of the generic active pharmaceutical ingredients Solifenacin and Levocetirizine as well as the natural products (R)-coniine, (R)-eleagnine, and (R)-leptaflorine. We also report a novel MAO-N mediated asymmetric oxidative Pictet-Spengler approach to the synthesis of (R)-harmicine.