3-Hydroxy-4′-[di-(2-hydroxyethyl)amino]flavone as a new step in search of an ideal membrane ratiometric fluorescent probe

We report on the synthesis and fluorescence properties, in solvents and lipid vesicles, of a new 3-hydroxyflavone derivative: 2-[4-di(2-hydroxyethyl)aminophenyl]-3-hydroxy-4H-4-chromenone (FHE). Like others 3-hydroxyflavones, this probe is characterized by an excited-state intramolecular proton transfer reaction displaying two tautomeric excited states, which results in two emission bands well separated on the wavelength scale. Their relative intensities are highly environment-sensitive, allowing a two-band ratiometric analysis. The novel and original structural characteristics of FHE consist in the presence of two hydroxyethyl groups able to form H-bonds with phospholipid ester groups, thus conferring it anchoring properties at the membrane interface without any electrostatic interactions. FHE was tested in a series of organic solvents and demonstrates a good response to solvent polarity, with a peculiar sensitivity to solvents with strong H-bonding ability. In lipid vesicles, FHE displays a good sensitivity to membrane dipole potential, as shown by the electrochromism of both the excitation and the tautomer T* emission spectra. FHE is also sensitive to changes in membrane fluidity and to the binding of amphiphilic polyanions of biological importance as adenosine triphosphate (ATP). Finally, a tentative comparison of spectral parameters was made between the data obtained in solvents and in vesicles by using as “criterion coordinates” the logarithm of the two bands intensity ratio versus the difference in wavenumbers of their maxima.