Firefly luciferin precursor 2-cyano-6-hydroxybenzothiazole: Fluorescence à la carte controlled by solvent and acidity

Abstract

2-Cyano-6-hydroxybenzothiazole (CBTOH) is a key intermediate in the biosynthesis of D-luciferin, also used for its chemical synthesis and as luciferin bioluminescence precursor for chemical analysis. We characterized CBTOH and its O-methylated derivative by UV–vis absorption, by steady-state and time-resolved fluorescence spectroscopy, and by density functional theory (DFT) quantum mechanical calculations. Both the acidity of the hydroxyl group and the basicity of the thiazole nitrogen strongly increase upon electronic excitation, triggering solvent- and acidity-dependent deprotonation and protonation processes, with concomitant changes in the fluorescence properties. DFT and time-dependent DFT calculations revealed the existence of a charge displacement that takes place in the excited state from the phenol moiety to the cyanothiazole group for the neutral and the cationic forms of CBTOH. This study reveals CBTOH as the first member of a new class of small dyes with strong photoacid and photobase character, its rich excited-state behavior allowing modulation of its fluorescence properties through solvent and acidity changes. These features make CBTOH a potentially useful fluorescent probe