Characterization of the Photophysical Behavior of DFHBI Derivatives: Fluorogenic Molecules that Illuminate the...

( Z)-5-(3,5-Difluoro-4-hydroxybenzylidene)-2,3-dimethyl-3,5-dihydro-4 H-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-4 H-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 absorption band near 420-460 nm and the nature of emission near 430 and 500 nm through various degrees of stabilization and the transformation between the neutral and the anionic species at both ground and excited states. These observations are confirmed by using a methoxy-substituted molecule (( Z)-5-(4-methoxybenzylidene)-2,3-dimethyl-3,5-dihydro-4 H-imidazol-4-one), where the 420-460 nm band is absent in the presence of methanol and the spectra are similar to those of PFP-DFHBI in noninteracting solvents, such as acetonitrile and dichloromethane. Thus, in addition to the major role of photoisomerization as a nonradiative process of deactivation of the excited state, the fluorescence of DFHBI-type molecules is very sensitively dependent upon the pH of the medium as well as upon solvent-specific interactions, such as hydrogen-bonding ability and polarity.