Convenient synthesis of deazaflavin cofactor FO and its activity in F420-dependent NADP reductaseElectronic supplementary information (ESI) available. See DOI: 10.1039/c5ob00365b

F 420 and FO are phenolic 5-deazaflavin cofactors that complement nicotinamide and flavin redox coenzymes in biochemical oxidoreductases and photocatalytic systems. Specifically, these 5-deazaflavins lack the single electron reactivity with O 2 of riboflavin-derived coenzymes (FMN and FAD), and, in general, have a more negative redox potential than NAD(P) + . For example, F 420 -dependent NADP + oxidoreductase (Fno) is critical to the conversion of CO 2 to CH 4 by methanogenic archaea, while FO functions as a light-harvesting agent in DNA repair. The preparation of these cofactors is an obstacle to their use in biochemical studies and biotechnology. Here, a convenient synthesis of FO was achieved by improving the redox stability of synthetic intermediates containing a polar, electron-rich aminophenol fragment. Improved yields and simplified purification techniques for FO are described. Additionally, Fno activity was restored with FO in the absence of F 420 . Investigating the FO-dependent NADP + /NADPH redox process by stopped-flow spectrophotometry, steady state kinetics were defined as having a K m of 4.00 ± 0.39 μM and a k cat of 5.27 ± 0.14 s −1 . The preparation of FO should enable future biochemical studies and novel uses of F 420 mimics. Revised synthesis of FO, a 5-deazaflavin cofactor, and its activity as a surrogate for the F 420 cofactor in Fno.