*2.7. The Binding Energy Between Probe and Membrane*

In order to check whether either tautomeric form has a preference for the Lo or Ld phase of the membranes, the binding energy with the membrane/water system has been calculated. The binding energies for the last 5 ns of the simulation are shown in Table 4.

**Table 4.** Binding energy values for the 3HF18 (form N\* and T\*) in POPC and DPPC-Chol membrane.


<sup>1</sup> The binding energy of the 3HF18 probe in POPC membrane N\* form; <sup>2</sup> Binding energy of the 3HF18 probe in POPC membrane T\* form; <sup>3</sup> Binding energy of the 3HF18 probe in DPPC-Chol membrane N\* form; <sup>4</sup> Binding energy of the 3HF18 probe in DPPC-Chol membrane T\* form.

As we can see from Table 4 and Figure 9, each tautomeric form has a preference for a membrane: N\* has better interaction with the POPC membrane while T\* interacts better with DPPC-Chol. To validate the energy binding results, a t-test statistical analysis was performed for the different POPC and DPPC systems with the two forms of the 3HF18 probe, and the results are reported in the Supplementary Materials section. The analysis shows that the energies are significantly different, with a probability of statistical significance of over 98%.

**Figure 9.** Box plot of binding energies on four systems: the 3HF18 probe in the N\* form in DPPC-Chol membrane (blue); the 3HF18 probe in the N\* form in POPC membrane (orange); the 3HF18 probe in the T\* form in DPPC-Chol membrane (grey); the 3HF18 probe in the T\* form in POPC membrane (yellow). The binding energy expressed in kcal/mol is shown on the *y*-axis.

#### **3. Materials and Methods**

All the reagents and solvents were purchased from Sigma Aldrich (Milan, Italy) and used without further purification. Optical observations of the probe in GUVs were performed by using a TCS SP8 Confocal laser (Leica, Wetzlar, Germany), UV laser λ = 405 nm, emission range for the N\* transition λ = 477–527 nm, emission range for the T\* transition λ = 550–600 nm). The images were acquired with a sequential scan between lines and an average of three frames. UV–vis absorption spectra of the samples were recorded at 25 ◦C in acetonitrile solution, on a Lambda 800 spectrophotometer (Perkin Elmer, Rodgau, Germany). The spectral region 650–240 nm was investigated by using a cell path length of 1.0 cm. Probe concentration of about 3.0 × 10−<sup>5</sup> mol L−<sup>1</sup> was used. Fluorescence spectra were performed at 25 ◦C in acetonitrile solution, on a Jasco FP-750 Spectrofluorometer (JASCO Europe, Cremella (LC) Italy), at a concentration of about 3.0 × 10−<sup>7</sup> mol L−<sup>1</sup> . <sup>1</sup>H-NMR spectra were recorded with a DRX/400 spectrometer (Bruker, Billerica, MA, USA). Chemical shifts are reported relative to the residual solvent peak (chloroform-d: H = 7.26 ppm). The following abbreviations are used to express spin multiplicities in <sup>1</sup>H NMR spectra: s = singlet; d = doublet; dd = double doublet; t = triplet; m = multiplet. High-resolution mass spectra were acquired on an LTQ-Orbitrap instrument (Thermo-Fisher, Waltham, MA, USA) operating in positive ion mode. The probe was dissolved in methanol at a concentration of 0.1 mg/mL and injected into the MS ion source. Spectra were acquired in the 150–800 *m*/*z* range.
