A Biocatalytic Approach to a Key Intermediate for the Synthesis of the COVID-19 Experimental Drug Molnupiravir

Herein we report the conversion of cytidine 2 to N-hydroxycytidine 7 catalysed by cytidine deaminase (CD). The wild-type enzyme operates efficiently at high sustrate loadings and hydroxylamine concentrations to favor N-hydroxy-cytidine formation over uridine. Although the wild-type enzyme demonstrated good activity, we were able to further enhance the ratio of N-hydroxycytidine to uridine produced through directed evolution of CD. In particular, a T123G mutation close to the active site dramatically reduces cytidine hydrolysis activity whilst preserving desired amination activty. The approach reported provides a new route to a key intermediate for the COVID-19 experimental drug Molnupiravir 1.