Spectral−Structural Effects of the Keto−Enol−Enolate and Phenol−Phenolate Equilibria of Oxyluciferin

The effects of environmental polarity on the enolization of the keto form and the deprotonation of the enol, and the role of the neutral and ionized 6′-OH group in the fluorescence of the firefly emitter, oxyluciferin, were assessed through a detailed study of the structure and absorption and fluorescence spectra of its 6′-dehydroxylated analogue. It was found that the deprotonated 6′-O− group is a necessary, albeit insufficient, factor in accounting for the observed yellow-green and red emissions of oxyluciferin. Its negative charge is essential for effective excited-state charge transfer, which lowers the emission energy and broadens the emission spectrum. Deprotonation of the 6′-OH group changes its effect on the emission energy from blue- to red-shifting. Furthermore, the combination of these opposite effects and resonance stabilization of the phenolate−keto form causes switching of the order of maximum emission wavelengths of the three species involved in the keto−enol−enolate equilibrium from enol ≪ keto < enolate in absence of 6′-OH to keto < enol ≪ enolate with 6′-OH, to enol < enolate < keto with 6′-O−. If only the keto−enol−enolate equilibrium is considered, solvents of medium polarity are the most effective in decreasing the excited-state energy. Polar or very polar environments also stimulate shift of the ground-state equilibrium toward the enol form. Under such circumstances, the enol group can be partly or completely deprotonated in the ground state or from the excited state: a polar environment facilitates the ionization, while a less polar environment requires the presence of a stronger base. In the absence of bases, the ground-state keto form exists only in solvents of very weak to medium polarity, but with stronger bases, it can also exist in a nonpolar or very weakly polar environment, usually together with the enolate anion. The phenol−enolate form of oxyluciferin, a species that could not be experimentally detected prior to this study, was identified as a yellow-emitting species.