Photophysics of Iodine-Substituted Oxyluciferins: Observation of Simultaneous High Fluorescence Quantum Yields and Singlet Oxygen Sensitization

The bioluminescence of firefly oxyluciferin is a subject of broad interest, but while its photophysics have been extensively studied within the singlet manifold, little is known about its ability to produce triplet states and whether this process can be enhanced by heavy atom substitution. Here, a combined experiment-theory effort clarifies the role of intersystem crossing (ISC) and competing nonradiative processes in dimethyloxyluciferin and its 7-iodo, 5-iodo, and 5,7-diiodo-luciferin derivatives. Fluorescence quantum yield (FLQY) and transient absorption measurements show that in the phenol form, all derivatives undergo picosecond relaxation to a dark state, which we assign to a triplet excitation. Deprotonation increases the lifetime of the lowest singlet state by a factor of 100 and, surprisingly, iodination increases both the FLQY and the singlet O₂ quantum yield (SOQY). Specifically, in the phenolate form, diiodo-oxyluciferin has FLQY = 0.42 and SOQY = 0.41, as compared to FLQY = 0.33 and SOQY <0.02 for unsubstituted oxyluciferin. Electronic structure calculations reveal that deprotonation reorders the low-lying singlet manifold, leading to a marked difference in the effective singlet-state charge localization for phenol and phenolate forms. Consequently, in the phenol form, a large difference in total orbital angular momentum between singlet and triplet states enhances ISC. Iodination predominantly affects the triplet state energetics and suppresses a competing nonradiative decay channel that likely involves internal conversion. Our results show how new functionalities can be engineered into the luciferin chromophore, unlocking the possibility of using this compound to sensitize reactive oxygen species for biomedical applications.