These results highlight biotransformations with cytidine deaminase as a promising strategy for the production of N-hydroxy-cytidine 4. Unfortunately, under thermodynamic control significant quantities of uridine by-product are formed, even at elevated NH2OH concentrations. Furthermore, the use of such high NH2OH concentrations is undesirable for large scale applications and leads to the formation of impurities over extended reaction times. To address these shortcomings, we elected to engineer CD via directed evolution to optimize pathway A whilst minimizing pathway B. Similar strategies have been employed to control the partitioning of transglycosylation/hydrolysis by glycoside hydrolases . Accelerating the direct amination of cytidine in this way should allow accumulation of 4 under kinetic control at reduced NH2OH concentrations.