Characterization of the Photophysical Behavior of DFHBI Derivatives: Fluorogenic Molecules that Illuminate the Spinach RNA Aptamer

(Z)-5-(3,5-Difluoro-4-hydroxybenzylidene)-2,3-dimethyl-3,5-dihydro-4H-imidazol-4-one (DFHBI) and its analogues are fluorogenic molecules that bind the Spinach aptamer (a small RNA molecule), which was selected for imaging RNA. They are extremely weakly fluorescent in liquid solvents. It had been hypothesized that photoisomerization is a very efficient nonradiative process of deactivation. We show, consistent with the results of other studies, that if the isomerization is impeded, the fluorescence signal is enhanced significantly. In addition, we provide a thorough characterization of the photophysical behavior of DFHBI and its derivatives, notably that of (Z)-5-(3,5-difluoro-4-hydroxybenzylidene)-2-methyl-3-((perfluorophenyl)methyl)-3,5-dihydro-4H-imidazol-4-one (PFP-DFHBI) in various solvent environments. Solvent-dependent studies were performed with various mixtures of solvents. The results suggest that hydrogen bonding or strong interactions of the solvents with the phenolic-OH group change the absorpt...