Influence of pH on spectral and photophysical properties of 9-methyl-5-deazaalloxazine and 10-ethyl-5-deaza-isoalloxazine

Absorption, emission, excitation and synchronous fluorescence spectra were used to describe protonation/deprotonation equilibria for a derivative of deazaalloxazine, 9-methyl-5-deazaalloxazine (9Me-5-DAll), and its flavin derivative 10-ethyl-5-deaza-isoalloxazine (10Et-5-DIAll). In addition, spectral and photophysical properties of 9Me-5-DAll and of 10Et-5-DIAll were compared to those of methyl derivatives of alloxazine, such as lumichrome or lumiflavin (10-methyl-isoalloxazine). ecies only is present in acidic and neutral solutions of 9Me-5-DAll, which absorbs and emits light, in contrast to basic solutions where three such species are present, identified as the two monoanions and one dianion. The analysis of steady-state and time-resolved spectra predict that the N(3) monoanion of 9Me-5-DAll has “alloxazinic” structure while its N(1) monoanion and N(1,3) dianion have predominantly isoalloxazinic structures. The lifetimes of these forms were determined at selected pH values. Similarly, for 10Et-5-DIAll there is one protonated species at low pH values. At higher pH, this species undergoes deprotonation forming the neutral molecule, which is present in aqueous solutions up to pH 12. At pH > 12 the monoanionic, isoalloxazinic form of 10Et-5-DIAll is predominant. Based on the analysis of the time-resolved spectra of the latter species, we conclude that its protonated form has no detectable fluorescence. Additionally, time-resolved spectra indicate that at pH > 11 there are two emitting species in solution. The first is identified as the isoalloxazinic monoanion of 10Et-5-DIAll, and the second as the tautomeric form of this monoanion, which is formed in the excited state. on the measurements, we also estimated pKa values of the two compounds for the equilibria between the individual species both in the ground and excited states.