**1. Introduction**

Biological membranes play an essential role in almost every cellular process and are characterized by a variety of lipid species, which differ in their physicochemical properties. The immiscibility of these components causes heterogeneity of the membrane and the formation of so-called "lipid rafts". Rafts represent liquid-ordered domains (Lo). They consist mainly of saturated hydrocarbons (sphingolipids and phospholipids) and are free to move in the liquid disordered bilayer (Ld) of the membrane constituted by unsaturated fatty acids [1]. In recent years, there has been a growing interest in the study of membrane heterogeneity. Fluorescence techniques are one of the most widely used methods of characterization for membrane analysis, providing excellent tools for this purpose. These methods require the use of fluorescent probes, able to penetrate the membranes allowing a

precise and immediate analysis [2,3]. The number of probes developed for the study of raft membranes is continuously increasing; those so far reported have restrictions related to the type of membrane and the fluorescence technique used [4–6].

The most recently identified class of probes corresponds to the class of environment-sensitive probes, which can directly distinguish the Lo and Ld phases due to differences in their intrinsic properties. These probes can change their fluorescence properties, fluorescence intensity, and even the emission color in response to changes in the environment [5,7–9]. This class includes solvatochromic fluorescent probes that show variations in their fluorescence properties depending on the polarity and hydration of the environment. Very promising in this context are the probes of the family of 3-hydroxyflavones (3-HF), in which an electron-donating moiety is present in position 4′ . They show an excited state intramolecular proton transfer (ESIPT) reaction between the normal excited state N\* and the tautomeric proton transfer product T\*. The ESIPT process takes place by intramolecular proton transfer from the 3-hydroxy group to the 4-carbonyl (Scheme 1), thus resulting in two intense and well-separated emission bands [10,11].

**Scheme 1.** Diagram of ESIPT Reaction in 3HF.

The intensity and the position of the two emission bands depend on the solvent polarity. The states N\* and T\* have different charge distributions and interact differently with their environment. The N\* form shows a high charge separation and, therefore, a higher dipole moment than the T\* state. Proton transfer in the ESIPT process from the 3-hydroxy to the 4-carbonyl group causes a shift of the negative charge, reducing the polar character of the T\* species. On the other hand, the presence of the electron-donating N,N-diethylamino group, contributes to the increase of the dipole moment of the species N\* (this is not observed for the T\* state) [12]. This difference in the distribution of charge results in a higher dependence of the N\* band, located at shorter wavelengths, on the solvent polarity. An important parameter is the intensity ratio of emission maxima of the two bands IN\*/I T\*, which is directly correlated to the energy of the two states and provides an indication of the dependence on the solvent-polarity [13]. In aprotic solvents, the ESIPT process is speedy, because it is barrier-free, and this results in a decrease in intensity of the N\* state band. In protic solvents, the solvation and formation of H-intermolecular bonds impose a high activation barrier that leads to a redshift of the N\* band with an increase in intensity. In contrast, the fluorescence of the T\* state is almost indifferent to the solvent polarity and remains practically unchanged [14,15]. This common effect in protic solvents, such as methanol and ethanol, sometimes results in the presence of a single band in the emission spectra, because of the overlap of the N\* band, shifted at longer wavelengths, and T\* band poorly resolved [16,17].

It is well known that the insertion of a probe into a bilayer can result in a, sometimes drastic, perturbation of the membrane physical state. [18–20]. This perturbation can impact the experimental

results, so it is of primary importance to verify that the probe is capable to fit into the double layer without significantly changing its characteristics.

In this work, we report the design, synthesis, characterization, and the study in lipid vesicles and model membranes of a new solvatochromic probe for membrane analysis. The new probe, called 3HF18 and bearing the 3-hydroxyflavone moiety, has been provided with a saturated chain of 18 carbon atoms and a zwitterionic head so to facilitate the anchoring to the polar heads of the lipid bilayer and avoid the complete internalization, compared to similar existing probes [21]. The presence of the electron-donating N,N-diethylamino group on the 3-hydroxyflavone moiety, contributes to the increase of the dipole moment of the N\* form, and ensures a high solvatochromic effect. Also, absorption and fluorescence analyses in organic solvents with different polarities were carried out to investigate the solvatochromic properties. Finally, the behavior of the probe 3HF18 in the model membrane was studied through DFT analysis and by fluorescence microscopy on GUVs.

#### **2. Results and Discussion**
