We initially employed this assay to investigate the conversion of N-hydroxy-cytidine 4 to uridine 3. Consistent with previous reports, cytidine deaminase promotes N-hydroxy-cytidine 4 hydrolysis under ambient conditions, with complete conversion of 100 μM substrate achieved in 60 minutes (0.1% enzyme loading, Fig. ). Encouraged by these results, we next turned our attention to the more challenging synthesis of N-hydroxy-cytidine 4 using either uridine 3 or cytidine 2 as a starting material. Pleasingly, biotransformations carried out in the presence of 1% NH2OH in water led to the accumulation of a product with spectral features consistent with N-hydroxy-cytidine 4. Similar final concentrations of 4 are formed using either 2 or 3 as a starting material, or in reactions starting from 4, suggesting that the enzyme establishes an equilibrium distribution of products (Fig. ). As anticipated, the ratio of 4:3 formed is dependent on NH2OH concentration and 1:6 and 3:1 mixtures were achieved using 10% and 50% NH2OH solutions, respectively (Fig. ). Significantly, the concentration of product 4 does not accumulate beyond the equilibrium distribution irrespective of the NH2OH concentration used (Fig. ), suggesting that cytidine hydrolysis followed by uridine amination (pathway B) outcompetes direct amine transfer (pathway A) with the wild type enzyme.