Theoretical Study of Firefly Luciferin pKa Values—Relative Absorption Intensity in Aqueous Solutions

Ground‐state vibrational analyses of firefly luciferin and its conjugate acids and bases are performed. The Gibbs free energies obtained from these analyses are used to estimate pK_a values for phenolic hydroxy and carboxy groups and the N–H⁺ bond in the N‐protonated thiazoline or benzothiazole ring of firefly luciferin. The theoretical pK_a values are corrected using the experimental values. The concentrations of these chemical species in solutions with different pH values are estimated from their corrected pK_a values, and the pH dependence of their relative absorption intensities is elucidated. With the results obtained we assign the experimental spectra unequivocally. Especially, the small peak near 400 nm at pH 1–2 in experimental absorption spectra is clarified to be due to the excitation of carboxylate anion with N‐protonated thiazoline ring of firefly luciferin. Our results show that the pK_a values of chemical species, which are contained in the aqueous solutions, are effective to assign experimental absorption spectra.