Amphiphilic dendritic peptides: Synthesis and behavior as an organogelator and liquid crystal

New amphiphilic dendritic peptides on dendritic polyaspartic acid were designed and synthesized. The organogel and liquid crystal properties of these amphiphilic dendritic peptides were fully studied by field-emission SEM, temperature dependent FT-IR, differential scanning calorimetry, polarization optical microscopy and X-ray diffraction experiments. Amphiphilic dendritic peptides G3 show good organogel properties with a minimum gelation concentration as low as 1 wt %. Furthermore, amphiphilic dendritic peptides G3 can form a hexagonal columnar liquid crystal assembly over a wide temperature range.


Introduction
Peptide self-assembly has drawn a significant attention due to potential applications, especially in the fields of biomedicine and bionanotechnology [1][2][3]. Programmed self-assembly of peptides into highly ordered nanostructures creates biomaterials that display a wide range of physical properties often exceeding those of synthetic polymers [4,5]. Peptideamphiphiles (PAs) represent an attractive class of bioactive molecules as they self-assemble into a variety of nanostructures, many of which have promising biological activity due to the exposed peptide regions on their outer surface [6,7]. The selfassembly of amphiphilic oligopeptide systems is thus emerging as a particularly powerful strategy to direct the self-assembly of relatively simple peptide building blocks toward sophisticated nanostructures [8]. Furthermore, the natural amino acid based dendrons or dendrimers are of great significance because of their similarity to proteins in composition and topology [9][10][11], as well as their architectural difference from currently prevalent linear model peptides. However, little attention has been paid to the self-assembly of natural amino acid based dendrimers, and especially to their gelation and liquid crystal properties [12,13]. Recently, a type of amphiphilic dendritic dipeptide was described by Percec et al. as self-assembling in helical pores  [14]. In addition, Takashi Kato and co-workers have recently reported that dendritic oligopeptides can act as useful building blocks for chiral supramolecular liquid crystals [15,16].
Herein, we present the synthesis of amphiphilic dendritic peptides (ADPs) composed of an aspartic acid core and an aliphatic periphery, and their self-assembly which leads not only to organogels but also to liquid crystals.

Synthesis and characterization
The amphiphilic dendritic peptides were synthesized convergently as depicted in Scheme 1 via standard EDCI coupling of N-carbobenzyloxy-L-aspartic acid and L-aspartic acid dodecyl ester (readily prepared by the esterification of aspartic acid with 1-dodecanol). The second (G2) and third generations (G3) were synthesized convergently in 75 and 60% yields, respectively, by repeating a two-reaction cycle, i.e., by removal of the carbobenzyloxy group of the lower-generation dendron by hydrogenation, and then coupling the resulting N-deprotected intermediate to the C-deprotected G1, prepared by hydrogenolysis of the benzylated peptide. 1 H NMR, MALDI-TOF mass spectrometry, and elemental analyses were used to verify the structure and purity of the amphiphilic dendritic peptides.

Investigation of gelation
All amphiphilic dendritic peptides were subjected to gelation tests in a variety of organic solvents. Each compound was combined with solvent in a screw-capped sample tube and heated until the solid completely dissolved. The solution was then cooled to room temperature, and gelation monitored by inverting the sample tube to see whether the solution flows or not. The observed gelation behavior of each solution is summarized in Table 1. Compound G1 showed good solubility in a range from non-polar (n-hexane) to polar solvents (ethanol), and showed no gelation in any solvent tested. Compound G2 can gel only in the nonpolar n-hexane. Compound G3 gelled in n-hexane, toluene, 1,4-dioxane, and ethanol but was soluble in dichloromethane.
The microscopic structure of the gel was investigated by fieldemission SEM (FE-SEM). Figure 1 shows representative FE-SEM images of the xerogel of G3 formed in n-hexane. These images clearly show that organogels assembled from G3 formed thin fibres that underwent further aggregation to form fibre bundles. These fibre bundles constitute a highly developed entangled network.  Figure 2 shows the XRD scan of organogelator G3 and liquid crystal G3. Being different from the liquid crystal, the XRD of organogelator shows no reflection peaks in the small-angle region which indicates that molecular arrangement in the organogelator is disordered. Figure 3 shows the FT-IR of G3 as an organogelator and as a liquid crystal. The absorption at 3081 cm −1 is ascribed to the N-H stretching frequency of the hydrogen bonds. The absorption intensity at 3081 cm −1 decreases in the organogelator which indicates the loss of hydrogen bonds. Hydrogen bonds as physical cross-linking points in these compounds are an essential factor for gelling. With more physical cross-linking points, compound G3 gels more easily. Compound G2 can gel only in n-hexane. Compared with the other solvents, n-hexane is a poor solvent, in which hydrogen bonds form more easily. It is therefore reasonable that the minimum gelation concentration of G3 (1 wt %) was lower than G2 (3 wt %) in the same (n-hexane) solvent.

Investigation of the liquid crystalline behavior
Thermotropic behavior of ADPs was investigated by a combination of differential scanning calorimetry (DSC), polarization optical microscopy (POM) and X-ray diffraction (XRD) experiments. Figure 4 shows DSC curves of amphiphilic dendritic peptides. In the DSC scan from −60 °C to 200 °C, G1 showed three phase transitions at 15 °C, 25 °C and 40 °C, which are attributed to phase transitions of recrystallization and crystal to isotropic melt. G2 only showed a phase transitions at 71 °C, which is attributed to the phase transition of crystal to isotropic melt. DSC analysis of G3 showed phase behavior, where a LC mesophase, on a second cooling, appeared at 40 °C and then disappeared at 145°C to form an isotropic melt ( Figure 4). The high temperature for the isotropic melt of G3 is the result of hydrogen bond interactions of the amide groups which reinforce the columnar organization. Cooling from 160 °C to 145 °C, G3 begins to form the conic fan-shaped textures ( Figure 6A), which are characteristic of hexagonal columnar liquid crystals. The conic fan-shaped textures of G3 grow ( Figure 6B), and no longer change at 50 °C ( Figure 6C). Figure 7 shows the XRD scan for the birefringent phase of G3 at 50 °C. The birefringent phases of G3 were further confirmed as a hexagonal columnar liquid crystal phase through assignment of the reflections. In the wide angle region, the diffused halo at around 4.5 Å is due to the disorder of the   terminal alkyl chains. In the small-angle region, the XRD profile of G3 shows two reflection peaks corresponding to d spacing of 24.8 and 12.5 corresponding to (100) and (200) reflections. The XRD results show the compound G3 selfassembles into columnar structures.

Conclusion
In summary, we designed and synthesized amphiphilic peptides on dendritic polyaspartic acid. These new amphiphilic dendritic peptides are capable of gelling a variety of organic solvents to form stable organogels via cooperative non-covalent forces, with a minimum gelation concentration as low as 1 wt %. It is interesting that amphiphilic dendritic peptides G3 can form a hexagonal columnar liquid crystals over a wide temperature range.
Experimental L-Aspartic acid and N-carbobenzyloxy-L-aspartic acid were purchased from Yangzhou Baosheng Bio-Chemical Co. Ltd. G3: 0.133g N-carbobenzyloxy-L-aspartic acid (0.5 mmol) was dissolved in 30 mL of DCM, 0.288 g EDCI (1.5 mmol) was added, and the mixture stirred at 0 °C for 30 min. 1.553 g of the second generation L-aspartic acid dodecyl ester (1.5 mmol) and 0.228 g HOBt (1.5 mmol) were added and the mixture stirred at room temperature for 24 h. Solvent was removed by rotary evaporation. The crude product was purified by column chromatography on silica gel with methylene chloride:ethanol