Tuning of Morphology by Chirality in Self‐Assembled Structures of Bis(Urea) Amphiphiles in Water

Abstract We present the synthesis and self‐assembly of a chiral bis(urea) amphiphile and show that chirality offers a remarkable level of control towards different morphologies. Upon self‐assembly in water, the molecular‐scale chiral information is translated to the mesoscopic level. Both enantiomers of the amphiphile self‐assemble into chiral twisted ribbons with opposite handedness, as supported by Cryo‐TEM and circular dichroism (CD) measurements. The system presents thermo‐responsive aggregation behavior and combined transmittance measurements, temperature‐dependent UV, CD, TEM, and micro‐differential scanning calorimetry (DSC) show that a ribbon‐to‐vesicles transition occurs upon heating. Remarkably, chirality allows easy control of morphology as the self‐assembly into distinct aggregates can be tuned by varying the enantiomeric excess of the amphiphile, giving access to flat sheets, helical ribbons, and twisted ribbons.

S3 Cryo-TEM. The samples were prepared by depositing a few µL of amphiphile solution on holey carbon coated grids (Quantifoil 3.5/1, Quantifoil Micro Tools, Jena, Germany). After blotting the excess liquid, the grids were vitrified in liquid ethane (Vitrobot, FEI, Eindhoven, The Netherlands) and transferred to a FEI Tecnai T20 cryo-electron microscope equipped with a Gatan model 626 cryo-stage operating at 200 keV. Micrographs were recorded under low-dose conditions with a slow-scan CCD camera. The bilayer thickness was measured on slightly defocused cryo-electron microscopy images to obtain maximal phase contrast.
Sample Preparation: 1 mL of a 2 mM solution of the compound ((R,R)-U1 or (S,S)-U1) in technical grade CH2Cl2 or CHCl3 was placed in a 4 mL vial. The solvent was slowly evaporated using a nitrogen flow and a thin film of the amphiphilic compound was formed, which was subsequently hydrated with doubly distilled water and submitted to three consecutive cycles of freeze-sonication-heat-sonication. In order to exclude possible interferences given by electrolytes or solutes, we performed the first selfassembly studies in doubly distilled water which had an Electric Conductivity (EC) of units of nS prior to use. Enantiomerically enriched mixtures of U1 were prepared by mixing stock solutions of the separate enantiomers.

Synthesis of bis(urea) amphiphiles
Compound (R,R)-1 (95 mg, 0.15 mmol) was dissolved in CH2Cl2 (10 mL) under a nitrogen atmosphere. Dodecyl isocyanate (65 mg, 74 µL, 0.31 mmol) was added and the reaction mixture was left stirring under a nitrogen atmosphere for 16 h, after which it was concentrated in vacuo. Purification by precipitation in 1:1:1 (v/v) CH2Cl2/Et2O/Pentane mixture (6 mL) followed by column chromatography Compound (S,S)-U1 (139 mg, y = 89%) was obtained using the same procedure as for (R,R)-U1 and the analytical data was identical, with the exception of the NH peaks (highlighted in the spectra with a *), of which the chemical shift is concentration dependent in CDCl3; [ ] 20 = -25.68 (c 2.10*10 -1 , CH3CN). Figure S1: 1 H NMR of of (R,R)-U1, full spectrum. NH peaks are highlighted with *.        Upon heating, the shape of the CD signal of (S,S)-U1 changed to a signal that is similar to that of the monomeric amphiphile in solution (which is shown in Figure S7a), in correspondence with the thermal transitions observed by micro-DSC (above 30 °C). The same phenomenon is observed for amphiphile (R,R)-U1 and the results are comparable in the spectral region between 275 and 285 nm. The same trend is observed for both amphiphiles although the intensity of the bisignate CD signal (between 200 and 245 nm) seems to be higher in the case of (R,R)-U1 ( Figure S8b), which we cannot yet explain.

Temperature Dependent CD Spectroscopy
The decrease in intensity of the CD signal witnessed upon heating from 40 °C to 70 °C in both cases can be explained by a degree of precipitation occurring inside the cuvette (due to the LCST of the sample). Molecules are either in solution or in their self-assembled state below the Lower Critical Solution Temperature (LCST). However, above the LCST precipitation occurs due to weakened interactions with the aqueous medium, which is due to the ethylene glycol units expelling water molecules with increasing temperature in an entropically favourable process. [4,5] This process results in a collapse of the amphiphile, causing a change in the aggregation behaviour [6][7][8][9][10] and eventually precipitation.
Hence, we performed transmittance measurements to gain insight into the thermo-responsive behaviour. We measured the transmittance at 450 nm between 20 °C and 70 °C, at concentrations ranging from 0.5 to 5 mM ( Figure S11). As expected, the LCST shows a linear dependence on the sample concentration. Furthermore, transmittance values were quasi-reversible over three consecutive heating and cooling cycles between 20 °C and 70 °C (insert Figure S11). Since the data collected in the micro-DSC experiment, CD and Cryo-TEM measurements clearly pointed towards a specific ribbon-tovesicle transition temperature (34 °C for 2 mM sample), we hypothesize that a LCST transition possibly takes place at higher temperature.    Alongside with the formation of vesicles at the thermal transition observed with micro-DSC some precipitation occurs, also visible by Cryo-TEM ( Figure S14), in agreement with the LCST data ( Figure  S11).