*2.3. General Synthetic Procedure*

All compounds have been synthesized as follows: 0.2 mmol 4-aminobenzo-15-crown-5 are mixed with the corresponding 1.1 equiv. isocyanate in 10 mL of chloroform. The solution was stirred under reflux overnight. Solution was concentrated under rotary evaporator. Hexane was introduced to precipitate the product, after filtration, the filter cake was dried in the air and yields as final product, yields are around 65%.

*1-(Benzo-15-crown-5-15-yl)-3-octylurea* (**1**), 1H NMR (DMSO-*d*6, 300 MHz, 298 K) δ 8.19 (s, 1H), 7.13 (d, *J* = 2.1 Hz, 1H), 6.77 (dt, *J* = 8.6, 5.4 Hz, 2H), 6.00 (t, *J* = 5.6 Hz, 1H), 3.98 (dd, *J* = 8.6, 4.1 Hz, 4H), 3.74 (dd, *J* = 10.7, 6.0 Hz, 4H), 3.60 (s, 8H), 3.04 (dd, *J* = 12.6, 6.6 Hz, 2H), 1.40 (s, 2H), 1.26 (s, 10H), 0.86 (t, *J* = 6.6 Hz, 3H). 13C NMR (75 MHz, CDCl3, 298 K) δ 156.89, 149.56, 145.71, 132.33, 114.99, 114.49, 108.70, 70.89, 70.79, 70.42, 70.26, 69.61, 69.38, 68.55, 40.43, 31.81, 30.12, 29.31, 29.25, 26.93, 22.64, 14.09. ESI-MS Calcd. for C23H39N2O6 (M + H<sup>+</sup>) 439.28, Found 439.38; Calcd. for C23H38N2O6Na (M + Na<sup>+</sup>) 461.55, Found 461.35. IR (cm−1): 3297, 2924, 2855, 1627, 1559, 1516, 1457, 1417, 1269, 1233, 1135, 985, 937, 847, 628. HR-MS (ESI+) calcd. for C23H39N2O6 439.2808 found *m*/*z* 439.2814.

*(R)-(Benzo-15-crown-5-15-yl)-3-(octan-2-yl)urea* (**r2**), 1H NMR (300 MHz, 298 K, DMSO-*d*6) δ 8.08 (s, 1H), 7.14 (d, *J* = 2.3 Hz, 1H), 6.81 (d, *J* = 8.7 Hz, 1H), 6.73 (dd, *J* = 8.6, 2.3 Hz, 1H), 5.83 (d, *J* = 8.1 Hz, 1H), 3.98 (dd, *J* = 8.9, 4.5 Hz, 4H), 3.75 (dd, *J* = 10.8, 6.1 Hz, 4H), 3.60 (s, 9H), 1.31 (d, *J* = 29.3 Hz, 10H), 1.05 (d, *J* = 6.5 Hz, 3H), 0.86 (t, *J* = 6.6 Hz, 3H). 13C NMR (75 MHz, 298 K, CDCl3) δ 156.19, 149.77, 145.87, 132.69, 115.25, 114.69, 108.97, 71.09, 71.00, 70.62, 70.45, 69.79, 69.54, 68.77, 46.29, 37.46, 31.94, 29.36, 26.22, 22.74, 21.60, 14.22. ESI-MS Calcd. for C23H39N2O6 (M + H<sup>+</sup>) 439.3, Found 439.3; Calcd. for C23H38N2O6Na (M + Na<sup>+</sup>) 461.3, Found 461.3; Calcd for C23H38N2O6K (M + K<sup>+</sup>) 477.2, Found 477.3. HR-MS (ESI+) calcd. for C23H39N2O6 439.2808 found *m*/*z* 439.2813.

*(S)-(Benzo-15-crown-5-15-yl)-3-(octan-2-yl)urea* (**s2**), 1H NMR (300 MHz, 298 K, DMSO-*d*6) δ 8.09 (s, 1H), 7.14 (s, 1H), 6.81 (d, *J* = 8.7 Hz, 1H), 6.73 (d, *J* = 8.5 Hz, 1H), 5.84 (d, *J* = 8.1 Hz, 1H), 3.97 (s, 4H), 3.75 (s, 4H), 3.60 (s, 9H), 1.30 (d, *J* = 29.2 Hz, 10H), 1.05 (d, *J* = 6.5 Hz, 3H), 0.86 (t, *J* = 6.3 Hz, 3H). 13C NMR (75 MHz, 298 K, CDCl3) δ 156.09, 149.73, 145.63, 133.07, 115.34, 114.42, 108.74, 71.04, 70.94, 70.59, 70.41, 69.86, 69.78, 69.52, 68.74, 46.19, 37.49, 31.95, 29.38, 26.23, 22.74, 21.62, 14.22. ESI-MS Calcd. for C23H39N2O6 (M + H<sup>+</sup>) 439.3, Found 439.4; Calcd. for C23H38N2O6K (M + K<sup>+</sup>) 477.2, Found 477.3. HR-MS (ESI+) calcd. for C23H39N2O6 439.2808 found *m*/*z* 439.2810.

*1-(2-Ethylhexyl)-3-(benzo-15-crown-5-15-yl)urea* (**3**), 1H NMR (300 MHz, 298 K, DMSO-*d*6) δ 8.18 (s, 1H), 7.13 (d, *J* = 2.1 Hz, 1H), 6.80 (d, *J* = 8.6 Hz, 1H), 6.74 (dd, *J* = 8.6, 2.1 Hz, 1H), 5.98 (t, *J* = 5.5 Hz, 1H), 4.07 – 3.93 (m, 4H), 3.74 (dd, *J* = 10.6, 5.9 Hz, 4H), 3.60 (s, 8H), 3.01 (d, *J* = 3.2 Hz, 2H), 1.40 – 1.13 (m, 9H), 0.86 (q, *J* = 7.0 Hz, 6H). 13C NMR (75 MHz, 298 K, CDCl3) δ 156.81, 149.72, 145.73, 132.87, 115.18, 114.47, 108.78, 71.06, 70.95, 70.61, 70.43, 69.78, 69.53, 68.72, 43.28, 39.77, 31.07, 29.00, 24.28, 23.18, 14.21, 10.99. ESI-MS Calcd. for C23H39N2O6 (M + H<sup>+</sup>) 439.3, Found 439.3; Calcd for C23H38N2O6Na (M + Na<sup>+</sup>) 461.3, Found 461.3; Calcd. for C23H38N2O6K (M + K<sup>+</sup>) 477.2, Found 477.2. HR-MS (ESI+) calcd. for C23H39N2O6 439.2808 found *m*/*z* 439.2809.

### *2.4. Lipid Bilayer Transport Experiments*

### 2.4.1. LUV Preparation for HPTS Experiments

The large unilamellar vesicles (LUVs) were formed using egg yolk <sup>l</sup>-<sup>α</sup>-phosphatidylcholine (EYPC chloroform solution, 800 μL, 20 mg). To this solution was added 800 μL of methanol MeOH and the solvent was slowly removed by evaporation under vacuum at room temperature and dried overnight under high vacuum. The resulting thin film was hydrated with 400 μL of bu ffer (10 mM sodium phosphate, pH 6.4, 100 mM NaCl) containing 10 μM HPTS. During hydration, the suspension was submitted to seven freeze-thaw cycles (liquid nitrogen, water at room temperature). The obtained white suspension was extruded 21 times through a 0.1 μm polycarbonate membrane in order to transform the large multilamellar liposome suspension (LMVs) into LUVs with an average diameter of 100 nm. The LUVs suspension was separated from extra-vesicular HPTS dye by using size exclusion chromatography (SEC, stationary phase Sephadex G-50, mobile phase: phosphate bu ffer with 100 mM NaCl) and diluted with mobile phase to give 2.8 mL of 11mM lipid stock solution (considering all the lipids have been incorporated) [15].
