Hierarchical Self-Assembly of Di-, Tri-and Tetraphenylalanine Peptides Capped with Two Fluorenyl Functionalities: From Polymorphs to Dendrites

Homopeptides with 2, 3 and 4 phenylalanine (Phe) residues and capped with fluorenylmethoxycarbonyl and fluorenylmethyl ester at the N-and C-terminus, respectively, have been synthesized to examine their self-assembly capabilities. Depending on the conditions, the di-and triphenylalanine derivatives self-organize into a wide variety of stable polymorphic structures, which have been characterized: stacked braids, doughnuts-like, bundled arrays of nanotubes, corkscrew-like and spherulitic microstructures. These highly aromatic Phe-based peptides also form incipient branched dendritic microstructures, even though they are highly unstable, making their manipulation very difficult. In opposition,


INTRODUCTION
Fluorenylmethoxycarbonyl (Fmoc)-based short peptides are widely studied because of their particular supramolecular assembly capabilities. Thus, the Fmoc moiety provides strong aromatic interactions that drive the peptide self-assembly into nanofibers or nanotubes. [1][2][3][4][5][6][7][8][9][10][11] In peptide sequences containing aromatic residues, which intrinsically form -stacking interactions, the role of such type of interactions become predominant. A very illustrative example corresponds to the self-assembly of diphenylalanine (FF), as a minimal sequence to form peptide nanostructures, which organizes forming peptide nanotubes stabilized by a combination of hydrogen bonding and repeated phenyl stacking interactions. [12][13][14] In contrast, Fmoc-FF forms peptide fibrils 15 and very stable hydrogels 9,16,17 that were thought to arise from the stacking between Fmoc groups and between phenyl groups. The remarkable importance of stacking interactions induced by the Fmoc group at the N-terminus was also illustrated using a series of dipeptides and amino acids, 18 such aromatic moiety acting as a consistent facilitator of gelation in comparison to other simple hydrophobic groups, such as tert-butoxycarbonyl.
More recently, research on the self-assembly of triphenylalanine (FFF) and Fmoc-FFF also evidenced some important differences. 19,20 More specifically, FFF and Fmoc-FFF were found to self-assemble into solid fibrillary plate-like nanostructures 19 (also named "nanoplates") and hydrogels, 20 respectively. In all cases, - stacking interactions between aromatic rings were found to play a decisive role in the formation of such supramolecular aggregates. In a very recent study we examined the selfassembly of tetraphenylalanine (FFFF) and Fmoc-FFFF, which had never been reported before. 21 FFFF molecules were found to assemble into tubes, exhibiting structural imperfections in comparison to FF. Theoretical calculations suggested that these 4 structural defects in FFFF tubes are due to the fact that the increment in the conformational flexibility is accompanied by a reduction in the number of restrictions associated with hydrogen bonds. In opposition, Fmoc-FFFF organizes into a variety of polymorphs depending on the experimental conditions, which included ultra-thin nanoplates, fibrils and star-like submicrometric aggregates. 21 The applications of self-assembled peptides in the biomedical (e.g. as cargo to target delivery of drugs and genes, scaffolds in tissue engineering and regenerative biomedicine, and biosensors) and nanotechnological (e.g. fabrication of composite materials by controlled nucleation, electronic and magnetic nanonowires) fields have been extensively reviewed in the last years. [22][23][24][25] However, many of these applications are focused on a well-defined morphology. The control exerted by the environmental conditions in the morphology of a given system, regulating the apparition of different polymorphs, can enhance such applications, even leading to multifunctional systems. In this work we synthesize and study the self-assembly of phenylalanine-based peptides capped with Fmoc and 9-fluorenylmethyl ester (OFm) as N-and C-terminal aromatic components, respectively. More specifically, the results for a peptide series formed by FF, FFF and FFFF have been systematically compared. Furthermore, intermolecular interactions formed by these peptides, named Fmoc-FF-OFm, Fmoc-FFF-OFm and Fmoc-FFFF-OFm (Scheme 1), have been also investigated using theoretical calculations. It should be remarked that the interest of these systems lies not only in the high concentration of aromatic groups but also in the complete elimination of the normally free basic (N-terminus) and acidic (C-terminus) ends that are often important for gelation. 2,26 Accordingly, no hydrogel is a priori expected for these systems while the formation of multiple supramolecular self-assembled organizations may be reached through stacking interactions. It is worth noting that in a very recent study it was 5 reported a double Fmoc-functionalized low molecular weight peptide, which behaved very differently from the corresponding single Fmoc-functionalized analogue. 27 More specifically, Fmoc-Lys-Fmoc was found to form pH-controlled gels whereas single Fmoc-Lys failed under similar experimental conditions.

Materials.
Boc and Fmoc-aminoacids were supplied by PolyPeptide group, N- [3-(dimethylamino)-propyl]-N'-ethylcarbodiimide hydrochloride was a product from Bachem and all other reagents for peptide synthesis were purchased from Sigma-Aldrich.

Peptide synthesis and characterization.
The preparation of Fmoc-FF-OFm, Fmoc-FFF-OFm and Fmoc-FFFF-OFm peptides was carried out following standard procedures of peptide synthesis in solution starting from the corresponding F-derivative and using the Boc or Fmoc group as protection for the amino moieties. A general procedure for the coupling reactions is given in Figure 1.
Melting points were determined on a Gallenkamp apparatus and are uncorrected. IR spectra were registered on a Thermo Nicolet Avatar 360 FTIR spectrophotometer;  max is given for the main absorption bands. 1 H and 13 C NMR spectra were recorded on a Bruker AV-400 or ARX-300 instrument at room temperature unless otherwise indicated and using the residual solvent signal as the internal standard; chemical shifts (δ) are expressed in ppm and coupling constants (J) in Hertz. Optical rotations were measured on a JASCO P-1020 polarimeter. High-resolution mass spectra were obtained on a Bruker Microtof-Q spectrometer.

Preparation of initial solutions of peptides. Organic solvents were purchased from
Sigma-Aldrich, Fisher Scientific and Scharlab. The peptide concentration in the 6 prepared solutions ranged from 0.05 to 5 mg/mL. Solutions or dispersions (25 or 100 μL) of the peptides were prepared from 4-5 mg/mL stocks. The solvents used to dissolve the synthesized peptides were hexafluoroisopropanol (HFIP) and dimethylformamide (DMF). Milli-Q water, methanol (MeOH), ethanol (EtOH), isopropanol ( i PrOH) or acetone was added as co-solvents to reduce the peptide concentration and alters the polarity of the environment. More specifically, as usual for Phe-based aromatic peptides, the three investigates compounds were soluble in HFIP and DMF, while they were only partially soluble or insoluble in alcohols, water and acetone. Accordingly, self-assembly studies were conducted using as solvents pure HFIP and mixtures HFIP:alcohol (alcohol= MeOH, EtOH and i PrOH), H 2 O being added to complete the series of mixtures. As the main objective of these co-solvents was to increase the hydrophilicity of the environment and, for this purpose, the ratio solvent:co-solvent was systematically varied from 4:1 to 1:99 (i.e. 4:1, 2:3, 1:4, 1:9, 1:19; 1:24, 1:49 and 1:99). In addition, as peptides were also soluble in DMF and partially soluble in acetone, some trials were performed using pure DMF, DMF:acetone and HFIP:acetone mixtures, even though in this case the number of explored ratios was lower because the amount of observed microstructures was relatively infrequent with respect to HFIP and HFIP:alcohol. Finally, 10 or 20 μL aliquots were placed on microscope coverslips or glass slides (glass sample holders) and kept at room temperature (25 ºC) or inside a cold chamber (4 ºC) until dryness. The humidity was kept constant in both laboratories at 50%.

Optical microscopy. Morphological observations were performed using a Zeiss
Axioskop 40 microscope. Micrographs were taken with a Zeiss AxiosCam MRC5 digital camera.

Scanning electron microscopy (SEM). SEM studies were performed in a Focussed
Ion Beam Zeiss Neon 40 scanning electron microscope operating at 5 kV and equipped with an EDX spectroscopy system. Samples were mounted on a double-side adhesive carbon disc and sputter-coated with a thin layer of carbon to prevent sample charging problems.

Atomic Force Microscopy (AFM).
Topographic AFM images were obtained using either a Dimension 3100 Nanoman AFM or a Multimode, both from Veeco (NanoScope IV controller) under ambient conditions in tapping mode. AFM measurements were performed on various parts of the morphologies, which produced reproducible images similar to those displayed in this work. Scan window sizes ranged from 55 m 2 to 8080 m 2 .  The interaction energy, E int , for each complex formed by three peptide molecules was computed as the difference between the energy of the complex and the sum of the energies calculated for each of the three peptide molecule: The cooperative energy, E coop , for the -sheets formed by three strands was estimated as the difference between E int and the expected interaction energies (Eq. 2).
The expected interaction energy, E int (E), was supplied as the sum of the DFT interaction energies of all dimers contained in the complex (Eqs. [3][4][5][6]. Accordingly, E coop provides an evaluation of the many-body (non-additive) effects.
Interaction and cooperative energies were corrected with the basis set superposition error (BSSE) by mean of the standard counterpoise method.
FTIR spectroscopy. Infrared transmittance spectra were recorded with a Jasco FTIR 4100 Fourier Transform spectrometer in a 4000-650 cm -1 interval. An MKII Golden Gate attenuated total reflection (ATR) accesory from Specac was used. The measurements were taken using 4 cm-1 resolution and 1000 scans averaging.

Peptide synthesis
To a solution of the appropriately N  -protected -amino acid (4.00 mmol) in CH 2 Cl 2 , Description of all intermediates is provided in the Electronic Supporting Information (ESI). Table 1 summarizes the different conditions required for the formation of the morphologies identified in this work. Accordingly, assemblies with different morphologies were obtained from solutions of Fmoc-FF-OFm in HFIP alone or mixed with a co-solvent. Morphology drastically depends not only on the co-solvent but also on the peptide concentration and temperature. This is evidenced in Figure S1, which displays optical micrographs of the most representative morphologies. This enormous morphological variability is in contrast with observations on Fmoc-FF, which tends to self-assemble into a hydrogel based on - interlocked -sheets. Thus, the formation of external filled region seems to precede the appearance of the hole ( Figure S2c). It should be noted that the presence of gelatinous rounded aggregates is supported by the role played by aromatic end groups as facilitators of gelation. 9,[16][17][18] Accordingly, the majority of the peptide molecules located at the central region of such gelatinous structures could migrate through diffusion towards the external region, allowing the development of the hollow microstructure after solubilization or precipitation of the remaining peptide molecules.

Self-assembly of Fmoc-FF-OFm
This proposed mechanism agrees with the recent observations of Ulijn and co- tubes present a hexagonal-like symmetry that resembles that found for well-ordered microtubes formed by self-assembled FF. 36,37 The formation of robust hexagonal microtubes was attributed to the confined organization of nanoscale tubular structures at long range during slow crystallization or aggregation (i.e. kinetic control of nucleation and growth). 37,38 In general, FF-based nanotubes prepared at room temperatures by rapid dispersion of molecules exhibit circular shape that is thermodynamically more stable. 12,21,39 Hexagonal-like peptide microtubes were also obtained in 4 Nevertheless, the stability of these dendritic microstructures, which formed very rapidly only after perturbing the equilibrium conditions (i.e. removal of the upper thin glass cover of the glass slip), was very poor, thus disappearing after only 15 min. In spite of such instability, these results suggest that Phe-based peptides could be used to tune the morphology of macromolecules and inorganic materials. In a recent study Tendler and 14 co-workers 40 described FF unstable dendritic structures obtained by spin-casting a HFIP peptide solution (0.5 or 1 mg/mL) onto mica. However, such morphologies, which transformed into needle-like crystals upon exposure to humid air, corresponded to starlike dendritic assemblies rather than tree-like structures like those displayed in Figure   S3. Highly ordered dendritic assembly of FF was also reported by Kim and coworkers, 41 who used a buffer peptide solution with pH= 1 and a silicon wafer substrate.
In this case, the morphology of the self-assembled dendrites, which resembled ice crystal structures in snowflakes, 42 was also very different from the tree-like arrangements achieved for Fmoc-FF-OFm. The instability of the dendritic structures formed by Fmoc-FF-OFm has been attributed to the effects induced by the environmental humidity and the surface charge, which experienced drastic changes when the cover glass slide was removed.

Self-assembly of Fmoc-FFF-OFm
Although previous experiments on Fmoc-FFF proved that such peptide selfassembles hydrogels, 42 a variety of morphologies have been obtained for Fmoc-FFF-OFm solutions using HFIP alone or mixed with a co-solvent (Table 1). Again, the morphology changes not only with the co-solvent but also with the peptide concentration and temperature. Optical micrographs of the most representative morphologies are provided in Figure S4. A result that deserves special attention is the apparition of dendritic-like microstructures in HFIP:EtOH mixtures ( Figure S4e

Self-assembly of Fmoc-FFFF-OFm
Optical micrographs of the most representative assemblies identified for Fmoc-FFFF-OFm are displayed in Figure S6. Although in this case the variety of polymorphic structures decreases with respect to Fmoc-FFF-OFm and, especially, Fmoc-FF-OFm (Table 1) Thus, in absence of growth front nucleation processes, crystallization is known to yield highly symmetric dendrites, whereas intricately structured and locally disordered polycrystalline spherulite patterns often form. 47  were identified in thin films of poly(styrene)-block-poly(methyl methacrylate) diblock copolymer after annealing in block selective solvent vapor. 48 Besides, Fmoc-FFFF-OFm molecule self-assemble forming stacked braids morphologies, similar to those reached for the smaller peptides, in 1:99 to 1:9 HFIP:EtOH at 4 ºC and peptide concentrations ranging from 0.05 to 0.5 mg/mL ( Figures S6b and 6b). The length of the braids, which ranged from 9 to 16 m, was practically independent of the peptide concentration (Figure 6b and S7). Interestingly, higher concentrations provoked the assembly of peptide molecules in soft and metastable dendritic-like architectures ( Figure S6c). Although the formation of such assemblies was highly repetitive, they frequently disappeared in the conditions required for AFM and, especially, SEM characterization.
The most spectacular assembly was obtained for peptide 1:4 HFIP:EtOH solutions at room temperature. In this case, well-defined multidimensional dendritic microarchitectures were obtained, as it is shown in Figure 7 for the 0.5, 1 and 2 mg/mL Fmoc-FFFF-OFm solutions. These structures appeared very fast and spontaneously by rubbing a dried mass of the material obtained from the drop-cast of the peptide in the alcohol mixture with a glass slip. Although these branched structures present some irregularities, inspection of the AFM images suggest that in the early stage of growth primary frameworks were nucleated from the center. Thus, these framework structures exhibit a 4-fold pseudo-symmetry with a typical branching angle of 90º. As growth continued, the dendrites formed highly branched structures with a specific branching angle of 45º. Branching angles are schematically depicted in Figure 7 for the dendritic structure derived from the 1 mg/mL peptide solution, which exhibits the highest regularity. Finally, it is worth noting that the length of branches decreases with increasing distance from the primary framework, evidencing that the self-similarity of these hierarchical microarchitectures is not very high.
It should be mentioned that polymorphism was also reported for Fmoc-FFFF. 21 In HFIP:water this N-Fmoc-protected peptide was found to assemble into ultra-thin nanoplates that aggregate in microclusters. Replacement of water by EtOH as cosolvent resulted in the formation of peptide fibrils at peptide concentrations < 0.5 mg/mL, while irregular star-like structures of submicrometric dimensions appeared at higher peptide concentrations. Finally, poorly defined nanospherical aggregates were obtained at Fmoc-FFFF concentrations of 1 mg/mL in HFIP:water.

Fractal analysis of Fmoc-FFFF-OFm dendritic microarchitectures
In this section we introduce a fractal analysis of the geometrical structures of the dendritic microarchitectures obtained for Fmoc-FFFF-OFm. Fractal objects are selfsimilar structures for which increasing magnifications reveal similar features on different length scales. 49 The fractal dimension (FD), which indicates how a fractal pattern changes with the scale at which it is measured, was determined by the boxcounting method 9 analyzing binary images ( Figure S8) derived from the AFM images displayed in Figure 7. Fmoc-FFFF-OFm is 2, 3, and 4, respectively, per pair of interacting molecules either in parallel or antiparallel. However, in all cases hydrogen bonding parameters, especially the H···O distance, are more favourable for the antiparallel than for the parallel assembly. This feature agrees with the fact that the antiparallel sheet is energetically favoured with respect to the parallel one for the three investigated peptides. Moreover, the instability of the parallel assembly increases from 1.8 to 9.8 kcal/mol when the number of Phe residues in the peptide increases from 2 to 4. This behavior is fully consistent with the interaction energies (E int ) listed in Table 2, which only take into account the non-covalent interactions between the three peptides of each system. Thus, the E int values calculated for the antiparallel / parallel assemblies decrease from -52.8 / -51.0 kcal/mol to -88.6 / -78.7 kcal/mol when the number of Phe per molecule increases from 2 to 4.
The cooperative energy (E coop ) values for all complexes, which were calculated as it is described in the ESI, are included in Table 2 an antiparallel arrangement of the -sheets, respectively. 53 However, although the positions of these two peaks are typical to an antiparallel arrangement of the β-sheets, their relative intensity is not. Thus, for ideally defined antiparallel β-sheet arrangements, usually find in longer peptide sequences and proteins, the band at 1695 cm -1 is expected to be much weaker. 54 This unusual feature was also detected for the Fmoc-FF 26 and, by analogy, has been attributed to the short peptide sequence. Thus, the antiparallel β-sheet obtained for these two amino acids peptide (Fmoc-FF-Fmoc and Fmoc-FF) is not ideal. 26 Interestingly, the spectra recorded for fibrous Fmoc-FFF-Fmoc and Fmoc-FFFF-Fmoc samples also display the peak at 1695 cm -1 , even though in these cases a very intense peak appears at 1640-1645 cm -1 ( Figure S13). The latter absorption, which has been also identified in fibrous amyloids organized in antiparallel -sheets, 55 is related with the twist angle of -sheets formed by a large number of -strands. These observations are fully consistent with previously discussed theoretical calculations.
Significant findings are also detected from the comparison of the antiparallel preferences found in this work for Fmoc-FF-OFm, Fmoc-FFF-OFm and Fmoc-FFFF-OFm with those of their unblocked homologues or with a single Fmoc group. Thus, Xray crystallography proved a parallel -sheet assembly for FF, 56 while which was confirmed by FTIR studies. 57 In contrast, intermolecular hydrogen bonds and antiparallel sheets were found for Fmoc-FF. 6 This change was attributed to the interlocking the Fmoc groups from alternate -sheets to create -stacked pairs. On the other hand, Tamamis et al. 42,58 and Guo et al. 59 used atomistic and coarse-grained molecular dynamics (MD) simulations, respectively, to study the assembly mechanism and the molecular basis for the structural features of FFF-based peptides nanostructures.
Authors found that FFF-based peptides spontaneously assembled into solid nanometer-sized nanospheres and nanorods with substantial content of anti-parallel -sheet.
Furthermore, theoretical calculations on FFFF and Fmoc-FFFF using the methodology that in the present study revealed a parallel -sheets for FFFF and Fmoc-FFFF. 21 The work reflects not only self-similarity but also that the dendritic assembly occurs through a diffusion limited aggregation mechanism onto a plane (i.e. the surface substrate).
Theoretical calculations considering a model -sheet with three interacting strands indicate that the studied peptides adopt an antiparallel arrangement, which is more stable than the parallel one, with intermolecular hydrogen bonds and - interactions.
Moreover, such stability increases with the length of the Phe-segment. Unfortunately, extension of these theoretical studies to 2D and/or 3D nano-architectures represents a very complex task that, in addition, is severely limited by the large influence of the 26 environmental conditions (e.g. solvent, peptide concentration and temperature) in the assembly.
In summary, our results suggest that Phe-homopeptides capped with two fluorenyl functionalities are a novel class of material that can be used to achieve a wide variety of desirable structures at the very small length-scale by simply controlling the assembly conditions. In particular, the well-defined and stable dendritic structures formed by Fmoc-FFFF-OFm indicate that highly aromatic Phe-homopeptides with four, or even more, residues should be considered as powerful building blocks for the fabrication of complex and relatively infrequent structures. Potential applications of peptide assemblies in nanotechnology and nanobiology should be explored along next decades.          Table 2.  Table 2. Summary of the results derived from M06L/6-31G(d) calculations on antiparallel and parallel -sheets of Fmoc-FF-OFm, Fmoc-FFF-OFm and Fmoc-FFFF-OFm: relative energy (E), number of hydrogen bonds in the model with three strands (#N hbonds ), hydrogen bonding distance (d H···O ) and angle (N-H···O), interaction energy (E int ), and cooperative energy associated with three-body non-additive effects (E coop ). Although the number of starting geometries for each peptide was around 20-30, only results for the most stable antiparallel and parallel arrangements are displayed (Figures 9, S9 and S10).