In this contribution, the relative hyperfine couplings are determined for the 1H nuclei of the flavin mononucleotide (FMN) radical in an aqueous environment. In addition, three structural analogs with different methylation patterns are characterized and the influence of the substituents at the isoalloxazine moiety on the electronic structure of the radicals is explored. By exploiting nuclear hyperpolarization generated via the photo-CIDNP (chemically induced dynamic nuclear polarization) effect, it is possible to study the short-lived radical species generated by in situ light excitation. Experimental data are extracted by least-squares fitting and supported by quantum chemical calculations and published values from electron paramagnetic resonance and electron-nuclear double resonance. Furthermore, mechanistic details of the photoreaction of the investigated flavin analogs with l-tryptophan are derived from the photo-CIDNP spectra recorded at different pH values. Thereby, the neutral and anionic radicals of FMN and three structural analogs are, for the first time, characterized in terms of their electronic structure in an aqueous environment.

Deazaflavins are important analogues of the naturally occurring flavins: riboflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). The use of 5‐deazaflavin as replacement coenzyme in a number of flavoproteins has proven particularly valuable in unraveling and manipulating their reaction mechanisms. It was frequently reported that one‐electron‐transfer reactions in flavoproteins are impeded with 5‐deazaflavin as cofactor. Based on these findings, it was concluded that the 5‐deazaflavin radical is significantly less stable compared to the respective flavin semiquinone, and quickly re‐oxidizes or undergoes disproportionation. The long‐standing paradigm of 5‐deazaflavin being solely a two‐electron / hydride acceptor / donor – “a nicotinamide in flavin clothing” – needs now to be reevaluated with the indirect observation of a one‐electron reduced (paramagnetic) species using photo‐chemically induced dynamic nuclear polarization (photo‐CIDNP) 1H nuclear magnetic resonance (NMR) under biologically relevant conditions.