Absorption and fluorescence properties and assignments of firefly bioluminescence substrate analog: <scp>seMpai</scp>

Electronic absorption and fluorescence properties of a red-emission firefly luciferin analog, seMpai, which is water-soluble and shows a neutral pH, were revealed by quantitative spectroscopic measurements in aqueous solutions of pH 2-10. They were analyzed by density functional theory (DFT) calculations, time-dependent DFT calculations, and vibrational analyses using the neutral form of seMpai and its conjugate acids and bases. In a pH 8 solution, which is commonly used for firefly bioluminescence, seMpai showed an absorption maximum at 380 nm. From the theoretical absorption spectra and pH dependence of the normalized concentrations of the neutral form of seMpai and its conjugate acids and bases, it was found that the most abundant chemical species at pH 8 is the carboxylate anion, and the 380-nm absorption band was assigned as the <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:semantics><mml:mrow><mml:mi>π</mml:mi></mml:mrow> </mml:semantics> </mml:math> - <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:semantics><mml:mrow><mml:mi>π</mml:mi></mml:mrow> </mml:semantics> </mml:math> * transition of this anion. The fluorescence spectrum of seMpai in the pH 8 aqueous solution showed an emission maximum at 534 nm for 380-nm excitation. According to the theoretical pH dependence of the normalized concentrations and theoretical absorption and fluorescence energies of the chemical forms of seMpai, the most reasonable fluorescence pathway at pH 8 is the emission from the first excited singlet state S₁ of the carboxylate anion through its excitation.