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 anti-viral 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 cyt-idine deaminase. This engineered biocatalyst performs >85,000 turnovers in less than 3 hours, 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.