Self‐assembly of heterochiral, aliphatic dipeptides with Leu

This work describes the self‐assembly behavior of heterochiral, aliphatic dipeptides, l‐Leu‐d‐Xaa (Xaa = Ala, Val, Ile, Leu), in green solvents such as acetonitrile (MeCN) and buffered water at neutral pH. Interestingly, water plays a structuring role because at 1% v/v, it enables dipeptide self‐assembly in MeCN to yield organogels, which then undergo transition towards crystals. Other organic solvents and oils were tested for gelation, and metastable gels were formed in tetrahydrofuran, although at high peptide concentration (80 mM). Single‐crystal X‐ray diffraction revealed the dipeptides' supramolecular packing modes in amphipathic layers, as opposed to water channels reported for the homochiral Leu–Leu, or hydrophobic columns reported for homochiral Leu–Val and Leu–Ile.


| INTRODUCTION
Over the last 20 years, dipeptides have become very popular cheap building blocks for nanostructured hydrogels or organogels for a variety of applications. 1They were reported to self-assemble into various types of nanostructures, of which the nanotubes and microtubes formed by Phe-Phe are the most popular. 2 Dipeptide gels typically consist of a matrix of elongated nanostructures, such as nanotubes and nanofibrils, 1 although dipeptides can form also other types of nanomorphologies, such as nanoparticles 3 or beaded strings. 4Among the many types of gels that can be formed, organogels and hydrogels differ for the nature of the liquid-phase used, which is organic in the former and aqueous in the latter.Although hydrogels are certainly attractive for their sustainability, organogels can also offer a number of advantages, such as the higher variety of potential solvents (including green options, such as acetonitrile) as well as various properties (e.g., conductivity, stability, absorption and release of molecules, responsiveness) towards many advanced applications, spanning from batteries to sensors and actuators, anti-fouling or ice-phobic coatings, and so on. 5merous examples of peptide gelators are based on aromatic units to favor self-assembly in polar solvents, 6 although exceptions do exist. 7It is widely known that aromatic N-caps are convenient appendages to favor self-organization in water through π-π stacking, 8 and Phe stands out among amino acids for its strong propensity towards self-assembly into nanostructures. 9[12] During the last decade, we have investigated the introduction of Damino acids especially in uncapped tripeptides to control the positioning of the side chains of hydrophobic amino acids, so as to attain amphiphilic conformers that can self-organize into gelling superstructures in water. 13,14In recent years, we extended the concept to dipeptides containing Phe, and realized that the sequence space of heterochiral, aliphatic dipeptides' self-assembly is still unexplored. 12Given that heterochiral dipeptides based on Leu and Phe revealed to be gelators regardless of the relative position of each amino acid in the sequence, 15 we chose to explore non-aromatic dipeptides, L-Leu-D-Xaa (Xaa = Ala, Val, Ile, Leu), for their ability to gel green solvents, such as buffered water or acetonitrile (MeCN).The choice fell on aliphatic hydrophobic amino acids for two reasons: first, they are more likely to yield dipeptides with sufficient hydrophobicity to self-assemble and gel; second, they would enable to fill an existing gap on the sequence-mapping of aliphatic, hydrophobic dipeptides for their ability to selfassemble into gels and/or form water-filled nanotubes. 12

| Materials and general methods
All solvents and reagents were obtained from Merck (Milan, Italy) and were used as provided.A 2-chlorotrytil chloride resin was bought from GL Biochem (Shanghai, China).All buffers and aqueous solutions were prepared by using pure water that was dispensed from a Millipore MilliQ-system RiOs/Origin (St. Louis, MS, USA) with a resistivity > 18.2 MΩÁcm at 25 C. 1 H-and 13 C-nuclear magnetic resonance (NMR) spectra were recorded on a Varian Innova spectrometer at 400 and 100 MHz, respectively, while LC-MS data were obtained with an Agilent 6120 Infinity. 14Optical microscopy images were acquired on a ZEN Primovert optical microscope with an Axiocam and a polarized filter, using Zen 3.3 (blue edition) software.

| Gelation and oscillatory rheology
The dipeptides were dissolved (20 mM or 30 mM) in MeCN (or other organic solvents), and subsequent addition of 1% v/v of milliQ water with NaOH (equimolar to the peptide) led to gelation.The viscoelastic properties of the gels were probed using an oscillatory rheometer (Kinexus Ultra Plus), using a parallel-steel geometry (20-mm flat plates).In a typical experiment, the dipeptides were dissolved in the desired concentration (30 mM) of MeCN (400 μl).Before the analysis, 6 μl of 2 M NaOH were added to the solution, mixed with a pipette two times, and then the sample was transferred onto the rheometer parallel-plate (20 mm).Frequency sweeps were performed at 1 Pa while stress sweeps were recorded a frequency of 1 Hz, at 25 C using the Peltier temperature-controller accessory of the rheometer.

| Circular dichroism
Circular dichroism (CD) spectra were recorded on freshly prepared samples (1 mM) in quartz cells (1 mm) in milliQ water with the pH adjusted to 7.0 with 0.1 M NaOH.The temperature was set at 25 C on a Jasco J-815 with a scanning rate of 50 nm/min and a set resolution of 1 nm.

| Crystallization
The dipeptides were dissolved (20 mM) in MeCN, and subsequent addition of 1% v/v of milliQ water with NaOH (equimolar to the peptide) led to the formation of crystals of the zwitterionic peptide form that were collected after 24 h.The peptides were crystallized also in dimethyl sulfoxide (DMSO) and the structures were identical.Details of single-crystal X-ray diffraction (XRD) performed at the XRD1 beamline at Elettra synchrotron (Italy) can be found in the Supporting information S1.

| Dipeptides preparation and purification
The four dipeptides with the general sequence L-Leu-D-Xaa (Xaa = Ala, Val, Ile, Leu) shown in Scheme 1 were obtained in solid phase with a standard procedure using Fmoc as N-protecting group and 2-chlorotrytil chloride resin.They were purified by HPLC in reverse phase, and their purity and identity were confirmed by ESI-MS, 1 H-and 13 C-NMR spectroscopy (see Supporting information S1).Their HPLC retention times can be used as an experimental measure of their hydrophobicity, 16 and they are shown in Table 1 together with their logP.Interestingly, the HPLC retention times revealed that L-Leu-D-Ala and L-Leu-D-Val had analogous hydrophobicity, which was significantly lower than L-Leu-D-Leu and L-Leu-D-Ile.Hydrophobicity is indeed one important parameter to determine self-aggregation propensity in polar solvents. 11

| Dipeptides characterization and selfassembly
The CD spectra of the four dipeptides were recorded in milliQ water at neutral pH to gain insights into their conformations (Figure 1). 17ey were all very similar.In particular, the CD spectra of L-Leu-D-Ala, L-Leu-D-Val, and L-Leu-D-Ile presented a positive maximum centered at 197-198 nm, followed by a negative minimum at 228 nm.Analogously, the CD spectrum of L-Leu-D-Leu displayed a maximum centered at 195 nm, and a minimum at 225 nm, with the intensity of the CD signal being the lowest of the series.
The CD spectrum of L-Ala, L-Val, L-Ile, and L-Leu amino acids are characterized by a positive maximum at approximately 200 nm, which has been ascribed to the higher-energy π !π* transition of the carbonyl group chromophore. 18Their mirror-image enantiomers, that is, D-Ala, D-Val, D-Ile, and D-Leu, thus display a mirrored spectrum that is reflected on the y axis, thus with a negative minimum in the same region. 19However, in the case of aliphatic dipeptides, such as L-Ala-L-Ala, the situation is inverted. 20Furthermore, the presence of a lowerenergy n !π* transition of the amide chromophore of the peptidic bond can lead to a signal in the 210-230 nm range. 21This signal was reported to be positive for aliphatic L-dipeptides and negative for heterochiral dipeptides L-Ala-D-Xaa (Xaa = Arg, Asp, Glu, Lys, Met). 22We can conclude that the presence of D-amino acids at the C-terminal position of aliphatic dipeptides (see also Figure 1) results in spectral features typical of aliphatic D-dipeptides, and in our case the spectra correspond to those reported for random coils.The observation of the C-terminal amino acid dictating the CD spectra chirality was in agreement with the fact that D-Phe-L-Phe displayed analogous spectral features as L-Phe-L-Phe. 23However, care should be taken before generalizing this finding to other peptidic sequences, for it was found that in the case of dipeptide regio-and stereo-isomers containing Phe and an aliphatic amino acid (i.e., Val, 24 Leu, 25 or Ile 26 ), it was the stereoconfiguration of Phe that dictated the chirality (i.e., the sign) of the CD spectral features, regardless of its position along the sequence (i.e., at the N-or C-terminus).

| Dipeptides self-assembly
Self-assembly of the four dipeptides was firstly probed using an established protocol. 14 sodium phosphate buffer typically triggers self-assembly of the zwitterions that can engage in electrostatic interactions between the positively charged N-termini and the anionic carboxylates at the C-termini.In all cases, rare instances of microfibrils were observed by optical microscopy (Figure 2), although they were very short and thin in the case of the least hydrophobic L-Leu-D-Ala, and progressively more visible for L-Leu-D-Val and L-Leu-D-Ile and especially longer for L-Leu-D-Leu.
In acetonitrile (MeCN), all the four dipeptides were completely soluble even at the high concentration of 20 mM.However, it was sufficient to add just 1% v/v of milliQ water with an amount of NaOH that was equimolar to the dipeptides (to neutralize the presence of TFA from the HPLC purification and obtain the zwitterions) to induce organogelation of the three dipeptides (Figure 3) L-Leu-D-Val (minimum gelling concentration, mgc, 30 mM), L-Leu-D-Ile (mgc 20 mM), and L-Leu-D-Leu (mgc 20 mM).For comparison, ( p-aminobenzoyl)-L-Phe-D-Ala-L-Phe-NH 2 also displayed an mgc of 30 mM in MeCN. 26Other solvents were also tested following the same procedure, but the dipeptides were highly soluble in alcohols (methanol or ethanol), insoluble in soybean or silicone oil.In DMSO, they all crystallized.L-Leu-D-Leu and L-Leu-D-Ile gelled tetrahydrofuran, but at the high concentration of 80 mM.
After 24 h, in all cases the metastable organogels in MeCN underwent a transition towards crystals.Only in the case of L-Leu-D-Leu, curved fibrils were firstly seen by optical microscopy (Figure 4A), before transitioning towards needle crystals (Figure 4B).
Conversely, in all the other cases, crystals rapidly formed with different morphologies (Figure 4C).The evolution from gels to crystals is a relatively common phenomenon in peptide selfassembly, whose kinetics and thermodynamics have been widely studied, 27 although exceptions do exist with gels representing the thermodynamic product. 28

| Dipeptide organogel characterization
Oscillatory rheometry is the most appropriate technique to assess the viscoelastic properties of soft matter and confirm the gel nature.
However, in the case of all peptides, but L-Leu-D-Leu, the transition towards crystals inevitably started during the rheological analyses, leading to extensive noise and impeding correct measurements.
Therefore, only the rheological analyses of the L-Leu-D-Leu organogel in MeCN are reported in Figure 5.In particular, it was not possible to monitor the gelation kinetics, which occurred immediately after the addition of water as described in the previous section.Subsequent frequency sweeps confirmed a soft gel nature with the storage (G 0 ) and loss (G 00 ) moduli corresponding to 1.6 ± 0.5 kPa and 0.3 ± 0.1 kPa, respectively, which were not dependent upon the applied frequency in the range of 0.1-10 Hz (Figure 5A).Furthermore, stress sweeps showed a linear viscoelastic interval until 1 Pa, and a subsequent solgel transition at approximately 20 Pa (Figure 5B).
T A B L E 2 Intermolecular distances (Å) and angles ( ) for the selected hydrogen bonds found in the crystal structures of the four dipeptides.

| XRD analyses
The single crystals obtained from the hydrogels as described above in Sections 2.5 and 3.2 were analyzed by XRD using synchrotron radiation (see Supporting information S1).The intermolecular distances and angles for the hydrogen bonds found in the crystal structures of the four dipeptides are listed in Table 2.
L-Leu-D-Ala (CCDC deposition number 2300851) crystallizes as a hydrate crystal form with a host: guest ratio of 1:2 (Figure 6).In contrast to the homochiral counterpart crystal structure, which is characterized by water channels, 29

| CONCLUSIONS
In conclusion, this work reports the preparation, characterization, and study of the self-assembly behavior of the four heterochiral dipeptides L-Leu-D-Xaa (Xaa = Ala, Val, Ile, Leu) in buffered water and MeCN.Water played a structuring role, for its addition to a MeCN solution of either one of the four dipeptides triggered self-assembly into metastable organogels, which then converted into crystals.The supramolecular packing of all the four compounds revealed the presence of amphipathic layers, as opposed to water-filled channels observed for other self-assembling hydrophobic dipeptides with Leu. 15 However, it should be noted that the crystals of L-Leu-D-Leu and L-Leu-D-Ile were obtained in DMSO, which is also present in the crystal structure, and it is possible that in water a different packing can be found.This work adds another piece to the puzzle of heterochiral dipeptide self-assembly 12 to complete the mapping of the nanostructures obtained by these simple building blocks towards functional and green materials.Potential applications could include molecular and solvent separation and environmental remediation, 32 on their own or together with other components in smart materials. 33 t R (min) HPLC %B c Solubility (M) (ESOL) b Solubility (M) (Ali) b Solubility (M) (SILICOS-IT) b Optical microscopy photographs using a polarized filter of the dipeptide fibrils in phosphate buffer at pH 7.3.(A) L-Leu- D-Ala; (B) L-Leu-D-Val; (C) L-Leu-D-Ile; (D) L-Leu-D-Leu.Scalebar = 200 microns.F I G U R E 1 Circular dichroism (CD) spectra of the four dipeptides at 1 mM in milliQ water at pH 7.0.S C H E M E 1 Chemical structures of the four heterochiral dipeptides studied in this work.
Briefly, the aliphatic sequences were first dissolved in their anionic form in an alkaline solution of sodium phosphate at pH 11.8, whereby the dipeptides' negative charge at the C-terminus ensures molecular repulsion.Next, lowering the pH to neutral through the addition of an equal volume of a mildly acidic F I G U R E 4 Optical microscopy photographs of (A) L-Leu-D-Leu fibrils, which then converted into (B) needle crystals, while the other dipeptides' crystals displayed different morphology, such as (C) L-Leu- D-Val crystals.F I G U R E 3 Photographs of dipeptide organogels at 20 mM in MeCN with 1% v/v water.F I G U R E 5 Oscillatory rheology of L-Leu-D-Leu organogel.(A) Frequency sweep, (B) stress sweep.
in the crystal structure of L-Leu-D-Ala water molecules are localized in layers parallel to the ab plane.Regarding the intermolecular interactions, L-Leu-D-Ala shows two hydrogen atoms of the -NH 3 + groups interacting with the À OOC-whereas the third hydrogen atom interacts with water molecules O1W localized inside the channel.Additionally, the dipeptides align one on top of the other along the b axis via hydrogen bonds involving the carbonyl oxygen atom O1, the hydrogen atom of the peptide bond and the hydrogen atom attached to the α-carbon.These two motifs promote the formation of amphipathic layers, which are separated into hydrophobic and hydrophilic regions defined by the side chains and backbone atoms, respectively.The latter contains the water molecules that interact with the dipeptides.Analogously, L-Leu-D-Val (CCDC deposition number 2300852) crystallizes as a hydrate crystal form with a host:guest ratio of 1:2.5, but in this case, water molecules occupy non-interconnected cavities (Figure 7).Crystals were obtained also in DMSO, and the structure F I G U R E 7 Crystal packing of L-Leu-D-Val (view along the b axis).Hydrophilic regions in light blue background, hydrophobic regions in light orange background.Atom types: C gray, H white, O red, N blue.F I G U R E 6 Crystal packing of L-Leu-D-Ala (view along the b axis).Hydrophilic regions in light blue background, hydrophobic regions in light orange background.Atom types: C gray, H white, O red, N blue.Only the atoms with the highest occupancy factors are shown.packing analysis performed on the two crystal structures reveals a very similar solid-state arrangement with the dipeptides forming a layered structure with channels surrounded by the hydrophobic side chains.L-Leu-D-Ile, in contrast to L-Leu-D-Leu, shows two independent dipeptide molecules in the asymmetric unit and two interconnected channels where the DMSO guest molecules are localized.In both crystal structures, two of three hydrogen atoms of the -NH 3 + interact head-to-tail with the À OOC-group defining the sheet structure while the third one directly interacts with the DMSO molecule localized inside the channel.Regarding the homochiral counterparts, Mitra and Subramanian 31 have reported the DMSO solvate crystal structure of L-Leu-L-Leu whereas, to the best of our knowledge, the DMSO solvate crystal structure of the dipeptide L-Leu-L-Ile has never been reported in literature.