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 currently in phase 3 clinical trials as an orally available treatment for SARS-CoV-2. Key to the success of this process was the development of a cytidine aminotransferase for the production of N-hydroxy-cytidine through evolutionary adaption of the hydrolytic enzyme cytidine deaminase. This engineered biocatalyst performs >100,000 turnovers in less than 30 minutes, operates at 180 g/L substrate loading and benefits from in situ crystallization of the N-hydroxy-cytidine product (>90% yield), which can be converted to Molnupiravir by a selective 5’-acylation using Novozym® 435.
The new version contains the full experimental details of the work and most importantly we include the DNA sequence of the most active cytidine deaminase (CAT3) which is used for the conversion of cytidine to N-hydroxycytidine en route to molnupiravir.