CD from E. coli was expressed in BL21(DE3) cells and purified to homogeneity via nickel affinity chromatography. To investigate whether this enzyme could serve as a biocatalyst for the preparation of N-hydroxy-cytidine 4, we initially established direct spectrophotometric assays to monitor the interconversion of cytidine 2, uridine 3 and N-hydroxy-cytidine 4, based on diagnostic differences between the UV-Vis spectra of these species. Such assays are valuable to allow high-throughput and real-time analysis of biotransformations. In particular, the N-hydroxy-cytidine 4 spectrum shows a diagnostic feature at >310 nm that is not present in either cytidine 2 or uridine 3, thus allowing Nhydroxy-cytidine 4 formation/decay to be easily monitored.