The peptide was designed with two aliphatic D-aa at the N-terminus and the L-Phe-Phe self-assembling motif at the C-terminus, synthesized in solid phase, purified by HPLC and characterized by NMR and ESI-MS (Fig. and ESI Section S2). In particular, the design featured the elongation of the reported gelator DLeu-Phe-Phe[5b] with D-Nle, which displays a linear sidechain favoring intermolecular packing. While design rules for self-assembling D,L-tripeptides are emerging, to date there is limited understanding of the supramolecular behavior of unprotected D,L-tetrapeptides, and filling the gap answers an interesting and still open research question. It should be noted that even subtle chemical modifications of these systems can dramatically affect their supramolecular behavior. Indeed, Nacetylation of DLeu-Phe-Phe completely hindered self-assembly, and analogous results were obtained for the L-homochiral analog Nle-Leu-Phe-Phe (see ESI Section S9). These data confirmed the importance of both termini in establishing intermolecular salt bridges, as evident from the single-crystal XRD structure of a similar D,L-tripeptide, as well as heterochirality in hydrophobic short peptides to enable hydrogelation of amphipathic superstructures. MD simulations of DNle-DLeu-Phe-Phe in explicit water revealed that the most representative conformation of the zwitterion is a turn with all hydrophobic chains on the same side of the backbone (Fig. ). Such isotactic spatial arrangement was recently reported to be key for the self-assembly of hydrophobic D,L-tripeptides into stable hydrogels. In both cases, the backbone is kinked. Peptide molecules stack thanks to hydrogen bonding between amides, with a pattern similar to that characterizing β-sheets and the aromatic rings running up the stack in a helical arrangement (Fig. ). Dihedral angles of the peptide backbone were calculated for the 2nd (D-Leu) and the 3rd residue (L-Phe) and are shown in the Ramachandran plot (Fig. ). ). The presence of intramolecular H-bonds that could hold together the turn of the heterochiral tetrapeptide was verified by variable-temperature 1 H-NMR spectroscopy, firstly in DMSO as a non-aggregating solvent (Fig. ), and then in the presence of water (Fig. ). 1 H-NMR shifts of amide signals were visible and displayed a linear correlation with temperature from 298 K to 333 K, indicating no conformational loss. In particular, the NH chemical shifts of Leu and C-terminal Phe (Fig. ) displayed a temperature gradient Δδ/ΔT > -4.6 ppb/K, which is a strong indication of involvement in H-bonding. Remarkably, the presence of water did not lead to loss of amide signals (Fig. ), indicating they were not exchanging with the solvent, although only the NH of Leu maintained a temperature coefficient within the expected range for H-bonds (Fig. ). Conversely, the same experiment carried out on the homochiral analog did not lead to any indication of H-bonds (see ESI section S6).