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Self-Assembly, Hydrogelation and Nanotube Formation by Cation-Modified Phenylalanine Derivatives Annada Rajbhandary, Danielle M. Raymond, and Bradley L. Nilsson Langmuir, Just Accepted Manuscript • Publication Date (Web): 17 May 2017 Downloaded from http://pubs.acs.org on May 18, 2017

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Self-Assembly, Hydrogelation and Nanotube Formation by Cation-Modified Phenylalanine Derivatives

Annada Rajbhandary, Danielle M. Raymond, and Bradley L. Nilsson* Department of Chemistry, University of Rochester, Rochester, NY, 14627-0216, USA.

E-mail: [email protected] Fax: +1 585 276-0205; Tel. +1 585 276-3053

1 ACS Paragon Plus Environment

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Abstract Fluorenylmethoxycarbonyl-protected phenylalanine (Fmoc-Phe) derivatives are a privileged class of molecule that spontaneously self-assemble into hydrogel fibril networks. Fmoc-Phe derived hydrogels are typically formed by dilution of the hydrogelator from an organic cosolvent into water, by dissolution of the hydrogelator under basic aqueous conditions followed by adjustment of the pH with acid, or by other external triggering forces, including sonication and heating. These conditions complicate biological applications of these hydrogels. Herein, we report C-terminal cation-modified Fmoc-Phe derivatives that are positively charged across a broad range of pH values and that can self-assemble and form hydrogel networks spontaneously without the need to adjust pH or to use an organic cosolvent. In addition, these cationic FmocPhe derivatives are found to self-assemble into novel sheet-based nanotube structures at higher concentrations. These nanotube structures are unique to C-terminal cationic Fmoc-Phe derivatives; the parent Fmoc-Phe carboxylic acids form only fibril or worm-like micelle structures. Nanotube formation by the cationic Fmoc-Phe molecules is dependent on positive charge at the C-terminus, since at basic pH where the positive charge is reduced only fibrils/worm-like micelles are formed and nanotube formation is suppressed. These studies provide an important example of Fmoc-Phe derivatives that can elicit hydrogelation without organic cosolvent or pH modification and also provide insight into how subtle modification of structure can perturb the self-assembly pathways of Fmoc-Phe derivatives.

Introduction Self-assembled nanostructures derived from peptide and amino acid derivatives have attracted considerable attention due to their natural ability to form supramolecular networks that


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can be utilized for drug delivery, regenerative medicine, tissue engineering, and other biological applications.1-5 Research focused on investigating the mechanisms of formation of these nanomaterials is of increasing importance to enable intelligent design of next-generation biomaterials.6-9 Hydrogels derived from self-assembling low-molecular weight (LMW) amino acid-derivatives are attractive alternatives to more expensive peptide-derived systems. Fluorenylmethoxycarbonyl-protected phenylalanine (Fmoc-Phe) derivatives are a privileged example of these types of LMW hydrogelators.10-18 Fmoc-Phe derived gels that possess all the biochemical and material properties required for advanced biological applications are rare.19-22 For example, hydrogels for cell and tissuebased applications are most useful if they are formed directly in complex aqueous cell culture media. Fmoc-Phe derived hydrogels, which are inherently anionic at high pH, are typically formed by dilution of the hydrogelator from an organic cosolvent into water (the “solvent switch” method) or by dissolution of the hydrogelator under basic aqueous conditions followed by adjustment of the pH with acid to an acidity where self-assembly and hydrogelation occur (the “pH switch” method).23 Gelation of Fmoc-Phe derivatives has also been triggered by other methods, including sonication and heating.10, 13 Both the solvent switch and pH switch methods for triggering hydrogelation of Fmoc-Phe derivatives complicate use of these hydrogels in cellbased applications. Solvent switch methods rely on the use of toxic organic cosolvents (for example, DMSO) to solubilize the self-assembling amino acid derivatives.24 The high pH required to deprotonate the anionic C-terminal carboxylic acid of Fmoc-Phe derivatives for solubilization in water without organic cosolvents is problematic for cells and the most efficient hydrogelation of Fmoc-Phe derivatives is observed at pH values of 3–5, well below the optimal neutral pH preferred by most cells.13,

23, 25-26

Gel formation of anionic Fmoc-Phe derivatives


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promoted by changing the pH from basic to neutral/acidic, requires long incubation periods for gelation.17, 27-30 In addition, some studies have indicated that some degree of Fmoc removal can occur at basic pH where the Fmoc-Phe derivatives are anionic, soluble, and unassembled into fibril networks.26, 31 Fmoc-Phe derivatives that spontaneously form hydrogels without the need for organic cosolvents or pH changes would enable this family of gelators to be more effectively utilized for biological applications. Previous attempts to increase solubility of anionic Fmoc-Phe hydrogelators by appending polyethylene glycol (PEG) at the C-terminus resulted in only marginally improved water solubility and did not obviate the need for organic cosolvents to facilitate gelation.32 While anionic Fmoc-Phe derivatives have been limited in terms of required gelation conditions, we reasoned that cationic modification of Fmoc-Phe derivatives would impart improved water solubility of these molecules across a broader range of physiologically pH values compared to the parent carboxylic acids that are soluble in aqueous solutions only in their anionic state at basic pH.17,

27, 29, 33

Soluble cationic Fmoc-Phe derivatives may not

efficiently self-assemble in the charged state due to charge repulsion, but it has been demonstrated in cationic self-assembling peptides that self-assembly can occur efficiently in media with sufficient ionic strength (as a function of salt concentration) wherein charge screening reduces repulsive Coulombic effects between the constituent peptides of the selfassembled network.34-41 We expected that ionic strength could also be used as a trigger to initiate gelation of cationic Fmoc-Phe derivatives.


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Figure 1. Chemical structures of C-terminal cationmodified Fmoc-Phe derivatives. Based on this reasoning, Fmoc-Phe derivatives with C-terminal cationic groups (Figure 1, compounds 1-3) were prepared in order to assess whether these molecules would have improved self-assembly and hydrogelation properties relative to the parent anionic carboxylic acids. The parent Fmoc-Phe carboxylic acids of compounds 1-3 (Fmoc-Phe, Fmoc-3F-Phe, and Fmoc-F5Phe) have previously been shown to effectively self-assemble into hydrogel networks, with side chain halogen groups enhancing self-assembly by perturbing intermolecular aromatic interactions.16,

25, 32, 38, 42-46

However, the parent carboxylic acids require the use of organic

cosolvents or pH switching in order trigger efficient self-assembly and hydrogelation.17, 27, 29, 47-48 In the work reported herein, it was observed that Fmoc-Phe cationic amines 1-3 efficiently selfassemble into supramolecular fibrils without the need for organic cosolvents or pH switching. At high ionic strength, assemblies of 1-3 formed hydrogel networks. Significantly, it was also observed that at higher concentrations compounds 1-3 form novel sheet-like assemblies that evolve into nanotube structures. These nanotubes are not observed with the parent Fmoc-Phe 5 ACS Paragon Plus Environment

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carboxylic acids, which form only one-dimensional fibrils under most aqueous conditions. Herein, we detail these findings, which provide not only examples of Fmoc-Phe derivatives that can elicit hydrogelation without organic cosolvent or pH modification, but also offer insight into how subtle modification of structure can perturb the self-assembly pathways and supramolecular structures formed by Fmoc-Phe derivatives.

Materials and Methods Fmoc-amino acids and organic solvents were purchased commercially and used without further purification. Synthetic details and characterization data for all new compounds are reported in the accompanying Supporting Information. Water used for hydrogelation was purified using a nanopure filtration system (Barnstead NANOpure, 0.2 µm filter, 18 Ω).

Self-assembly and Hydrogelation Conditions Self-assembly and hydrogelation of compounds 1-3 was initiated by dissolution of each compounds at 2.5 mM, 5 mM, or 10 mM concentrations in unbuffered water. The solutions were then heated to 80 ºC and sonicated for 20–30 seconds to ensure complete dissolution of the compounds. Compounds 1 and 2 completely dissolved to form transparent solutions whereas compound 3 was only partially soluble. After the dissolution of the compounds, NaCl was added to each solution to a final [NaCl] of 114 mM. The solution was vigorously agitated and gelation occurred in 10–180 s. When no salt was added after dissolution, no gelation was observed but self-assembly was clearly visible as noted by TEM and CD experiments and described in detail in the Results and Discussion section.

Circular Dichroism (CD) Spectroscopy 6 ACS Paragon Plus Environment

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CD spectra were recorded on a Prodata Chirascan 202 circular dichroism spectrometer using a 0.1 mm path length quartz cuvette. Self-assembled and hydrogel samples were prepared as described above. Spectra of the transparent gels and solutions were collected at 25 °C from 350 to 190 nm with a 1.0 nm step, 1.0 nm bandwidth, and 3 s averaging time per step.

NMR Spectroscopy NMR spectra were recorded using Brüker Avance-400 MHz and 500 MHz spectrometers. For characterization of synthetic compounds, 1H,

13

C NMR and

19

F chemical shifts are reported as

δ with reference to TMS at 0 ppm for 1H, solvent for 13C NMR, and trifluoroacetic acid at -76.55 ppm for

19

F NMR. See the attached Supporting Information for 1H,

13

C and

19

F spectra and

tabulated data. To comparatively quantify the extent of self-assembly in solution and hydrogel states, compounds 1-3 were prepared in D2O solutions as described previously. Reference solutions of compounds 1-3 (5 mM) in DMSO-d6 were prepared as standards for unassembled states. NMR tubes were fitted with an internal capillary containing 24 mM DMF in DMSO-d6 as an external standard to enable quantification of monomeric, unassembled material in each solution. As compounds are incorporated into fibril, signal is reduced due to line broadening effects of the fibrils and remaining signal is attributed to remaining soluble monomer. The degree of assembly was assessed by comparative integration of signal peaks of the D2O and DMSO-d6 samples for each compound. Each signal was integrated relative to the external DMF standard.

Transmission Electron Microscopy (TEM)


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TEM images were obtained using an Hitachi 7650 transmission electron microscope with an accelerating voltage of 80 kV. Samples of gel (10 µL) were applied directly onto 100 mesh carbon coated copper grids and allowed to stand for 1 min. Excess gel was carefully removed by capillary action using a filter paper and the grids were stained with uranyl acetate (10 µL) for 2 min. Excess stain was removed by capillary action, and the grids were allowed to air-dry for 15 min. Measurements for the dimensions of fibrils/aggregates were conducted using ImageJ64 software (http://rsbweb.nih.gov/ij/) and are reported as the average of at least 100 measurements of unique aggregates for each species with error reported as the standard deviation about the mean. Rheology Rheological measurements were conducted using a TA Instruments AR-G2 rheometer. A 20 mm parallel plate geometry equipped with a solvent trap filled with water was used for the experiments. Gels were formed directly on the Peltier plate. The experiments were performed using 500 µL of sample with a 1.4 mm gap size operating in oscillatory mode. Strain sweep experiments were performed to determine the linear viscoelastic at 25 °C for 0.1–100 % strain at a frequency of 6.283 rad s-1. Dynamic time sweep experiments were performed at 25 °C for 20 min with an angular frequency of 6.283 rad s-1 and 0.2% strain, which falls within the linear viscoelastic region for each gel. See Supporting Information for time and strain sweep data. Frequency sweep experiments were then performed from 0.1–100 rad s-1 with 0.2% strain at 25 °C. All measurements were performed in the linear viscoelastic region for each gel as determined by prior strain sweep experiments. Reported values for storage and loss moduli (G' and G" respectively) are the average of at least three distinct measurements on separate gels with error reported as the standard deviation about the mean.


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Results and Discussion The central hypothesis for the studies reported herein was that cationic Fmoc-Phe derivatives would be more amenable to the formation of hydrogels without the use of organic cosolvents and without the need for pH adjustment to trigger self-assembly and gelation than corresponding Fmoc-Phe amino acids. Accordingly, we synthesized compounds 1-3 (Figure 1) by coupling NBoc-1,3-propanediamine with the parent Fmoc-Phe derivatives. Detailed protocols and characterization data can be found in the Supporting Information (Scheme S1 and Figures S1– S11). The aqueous solubility and self-assembly properties of compounds 1-3 were then assessed as described in the following sections.

Self-assembly in salt-free conditions. Compounds 1-3 were readily dissolved in water without the use of organic cosolvents upon suspension of the solids in water followed by mild heating and sonication. These aqueous solutions of 1 and 2 were transparent, and slightly viscous at 5 mM concentrations. Aqueous solutions of 3 were slightly turbid due to the lower solubility of 3 compared to 1 and 2 based on the higher hydrophobicity of F5-Phe compared to Phe.49 The pH of these solutions was acidic (4.0–4.6) due to residual TFA from deprotection of the cation in the final synthetic step (Scheme S1, Supporting Information). It was anticipated that these cationic Fmoc-Phe derivatives would require higher ionic strength than is found in water to screen repulsive effects between the cations and trigger self-assembly. However, the increased viscosity of simple aqueous solutions of 1-3 suggested that self-assembly had occurred even in the absence of salts to increase solution ionic strength.


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Evidence for self-assembly of 1-3 in these solutions was obtained by transmission electron microscopy (TEM) analysis. TEM images (Figure 2) clearly show that all three cationic FmocPhe analogs formed fibrils or worm-like micelles upon dissolution in water. TEM images were obtained one day after dissolution in water at room temperature. Solutions of compounds 1 and 2 were found to contain one-dimensional aggregates that were 7.4 ± 1.5 nm in diameter (Figure 2A) and 4.5 ± 0.6 nm in diameter (Figure 2C) respectively. These are similar to the fibrils/wormlike micelles previously observed for the parent carboxylic acids of these Fmoc-Phe derivatives.16, 46 Solutions of compound 2 contained slighter wider aggregates that were 37.5 ± 3.4 nm in diameter as well as a smaller amount of two-dimensional sheet strutures that were as large as 199 ± 21 nm wide (Figure 2B). Interestingly, enforcement of cationic states in other selfassembling systems has also been found to promote formation of sheet-like assemblies compared to one-dimensional aggregates for the corresponding non-cationic forms.50 While cationic modification of these Fmoc-Phe derivatives increases water solubility of these compounds, the balance between charge and hydrophobicity is not sufficiently altered to completely preclude self-assembly.

Figure 2. TEM images of fibrils formed by each compound at 5 mM in water under salt-free conditions. A) Compound 1, B) compound 2, and C) compound 3. 10 ACS Paragon Plus Environment

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Circular dichroism (CD) spectroscopy of solutions of compounds 1-3 indicates that the assembly mode for these structures is similar to that of the parent Fmoc-Phe amino acids.16, 25, 32, 46, 51-52

CD spectra were obtained for all compounds at 2.5 mM, 5 mM, and 10 mM

concentrations in unbuffered water (Figure S12, Supporting Information). The CD data for 1-3 show absorbances from 200–220 nm characteristic of π to π* transitions that indicate probable intermolecular Phe-Phe interactions and at 270–310 nm characteristic of n to π* transitions that indicate probable Fmoc-Fmoc interactions (Figure S12).16, 18 This is consistent with CD spectra for our previously reported self-assembling Fmoc-Phe derivatives.

16, 25, 32, 46, 51-52

It has been

proposed, based on these previously reported CD spectra, that the fibril packing unit adopts an apparent parallel packing orientation stabilized by Fmoc-Fmoc and Phe-Phe interactions thus showing the strong absorbances at 200–220 and 270–370 nm.16,

43-44

Crystals of Fmoc-Phe

derivatives that are obtained from initial fibril states are consistent with the proposed parallel arrangement.42, 44 Thus, by analogy, we suspect that the fibrils of 1-3 also adopt these packing modes, although higher resolution techniques must be used to confirm this proposed assembly structure.

Self-assembly and hydrogelation at high ionic strength. Contrary to cationic self-assembling peptides, which require charge-screening salts to promote assembly in water,36,

38, 53

cationic

Fmoc-Phe hydrogelators 1-3 are able to self-assemble in aqueous solutions without added salts. While direct dissolution of 1-3 in water resulted in self-assembly, the extent of aggregate formation was not sufficient to establish hydrogel networks. This is primarily because the equilibrium between monomer and supramolecular assemblies is not sufficiently skewed to assembled states (NMR experiments to confirm this are discussed below) and also because the


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resulting cationic assemblies do not efficiently entangle (possibly due to charge repulsion between fibrils) to elicit emergent hydrogelation. In order to explore whether compounds 1-3 can form hydrogel networks, the self-assembly of these derivatives was next assessed in aqueous NaCl solutions. Experiments were performed by addition of NaCl salt (114 mM NaCl to approximate salt concentrations in common cell culture media) to solutions of compounds 1-3 (5 mM) in unbuffered water. Addition of salt resulted in rapid formation of hydrogels that were self-supporting as indicated by stability to vial inversion within 3 minutes (Figure 3A). Hydrogel formation was assessed at 2.5 mM, 5 mM, and 10 mM solutions of compounds 1-3 in 114 mM aqueous NaCl. These solutions were not externally buffered and had pH values from 3.9–4.5. Compounds 1 and 2 readily formed self-supporting hydrogels at all three concentrations. In contrast, compound 3 was found to be only partially soluble at concentrations greater than 2.5 mM. Compound 3 was only partially soluble at 5 and 10 mM concentrations, resulting in optically opaque hydrogels (Figure 3A). CD spectra of these hydrogels (Figure S13, Supporting Information) showed similar electronic structure to the assemblies observed in salt-free conditions, indicating that the packing structure is most likely not significantly altered by the addition of NaCl. Rheological experiments were performed on these hydrogels in order to characterize the emergent viscoelastic character of these materials. Strain sweep experiments (Figure S14, Supporting Information) were initially conducted on all gels in order to define the linear viscoelastic region for each hydrogel. Based on this data, time sweep experiments and frequency sweep experiments within the linear viscoelastic region were conducted in order to compare the rigidity of each gel. Time sweep experiments (Figure S15, Supporting Information) show that gelation occurs within 3 minutes upon addition of NaCl to the solutions of these cationic Fmoc-


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Phe derivatives. Frequency sweep experiments (Figure 3B-D, Table 1) reveal hydrogel rigidity increases as gelator concentration increases. In addition, hydrogels of the monofluorinated compound 2 tend to have marginally higher storage and loss modulus values (G' and G'' respectively) than hydrogels of compounds 1 or 3 at the same concentrations (Table 1). We have previously observed that halogen-substituted side chains in the phenyl group side chain of FmocPhe derivatives exhibit more rigid emergent viscoelasticity, presumably due to the increased hydrophobicity of these derivatives and perturbation in the electronic π-π interactions involving the phenyl ring in the packed structures.46,

51

Gels formed by compound 1 at 10 mM and

compound 2 at 5 and 10 mM have G' values an order of magnitude greater than G", which is commonly viewed as a necessary emergent property for biological cell based applications.54 Compound 3 forms much weaker gels. The low solubility of 3 precluded rheological analysis at concentrations greater than 5 mM. The lower solubility of compound 3 most likely accounts for the reduced rigidity of its hydrogels since less compound is in solution and able to contribute to formation of the self-assembled hydrogel network.


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Figure 3. A) Digital image of self-supporting hydrogels (as indicated by vial inversion) formed by compounds 1-3 (5 mM compound in 114 mM aqueous NaCl). B) Rheological frequency sweep data for hydrogels formed by compound 1 at concentrations ranging from 2.5–10 mM (114 mM aqueous NaCl), C) compound 2 (2.5–10 mM, 114 mM NaCl), D) compound 3 (2.5–10 mM, 114 mM NaCl).


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Table 1. Tabulated G' and G" values (Pa) for hydrogels of compounds 1, 2 and 3 at varying concentrations. Error is expressed as the standard deviation about the mean of at least 3 replicate experiments. Compound

G', G"

2.5 mMa

G' 215 ± 17 G" 66 ± 12 G' 234 ± 51 2 G" 61 ± 20 G' 383 ± 63 3 G' 56 ± 8 a G' and G'' are expressed in Pa units. 1

5 mMa 384 ± 66 178 ± 65 1118 ± 143 86 ± 7 517 ± 66 218 ± 65

10 mMa 1080 ± 142 86 ± 6 4094 ± 322 363 ± 30

TEM imaging of the various hydrogels revealed fibrils or worm-like micelles of similar dimensions comprising the respective hydrogel networks (Figure 4). TEM images were obtained 15 minutes after hydrogel formation. Aggregates in hydrogels of compound 1 were 12–28 nm in diameter Figure 4A-C) at all concentrations and fibrils/worm-like micelles of compound 3 were 4.5 ± 0.6 nm in diameter at 2.5 mM and 5 mM concentrations (Figure 4G and 4H). As was observed in salt free conditions, hydrogels of compound 2 were found to contain assemblies with several distinct morphologies. At 2.5 mM concentrations, worm-like micelles that were 9.5 ± 1.7 nm in diameter were observed (Figure 4D). At 5 mM and 10 mM concentrations, larger twodimensional sheets as large as 200 nm ± 12 nm in width were observed in addition to worm-like micelles ~9 nm in diameter (see Figures 4E and 4F). Interestingly, at even higher concentrations (20 mM), hydrogels of both compounds 1 and 2 were seen to be dominated by sheet-derived nanotubes ~200 nm in diameter. These structures are described more fully in the following section.


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Figure 4. TEM images of hydrogels formed by compounds 1-3 at various concentrations obtained 15 min after hydrogelation. A) 1 at 2.5 mM, B) 1 at 5 mM, C) 1 at 10 mM, D) 2 at 2.5 mM, E) 2 at 5 mM, F) 2 at 10 mM, G) 3 at 2.5 mM, H) 3 at 5 mM.


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We next conducted experiments to compare the emergent viscoelastic properties of solutions of compounds 1-3 without added salt (non-hydrogel) and those with salt (hydrogels). Since it was observed that fibril self-assembly occurred in salt-free conditions, we wished to compare the degree of self-assembly at low and at high ionic strength in order to understand the basis for gelation in the presence of salt. Is emergent hydrogelation due to higher monomer incorporation into fibrils/worm-like micelles in the presence of salt (and thus higher aggregate network density), or due to fibril entanglement and noncovalent cross-linking being favored at high ionic strength? NMR spectroscopy was used to explore these possibilities. Solution state NMR has been used for comparative quantification of the extent of monomer incorporation into selfassembled networks.25,

55-56

These experiments take advantage of slow tumbling rates of fibril

structures compared to monomer on NMR timescales. Monomer is readily observed in solution state NMR experiments, whereas aggregate signal is lost due to line broadening effects.25 Examination of comparative signal integrations (against an external standard) of solutions in which monomer states are maintained compared to solutions that are hydrogels and nonhydrogel fibrils/worm-like micelles can enable an analysis of the amount of monomer that is incorporated into aggregate in each state. It should be noted that these experiments do not independently confirm the formation of a specific type of aggregate, but only that aggregates are generally formed. We conducted solution state NMR experiments comparing samples of compounds 1-3 (5 mM) in DMSO-d6 (unassembled monomer) with samples in deuterated water with NaCl (114 mM) (hydrogel aggregates) and without (non-hydrogel aggregates) NaCl (Figure S16, Supporting Information). DMF in DMSO-d6 was used as an external standard (inserted into the NMR samples in a sealed capillary, see Materials and Methods for details). As shown in Figure


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S16, spectra A are hydrogels of each compound (D2O, 114 mM NaCl, 5 mM Fmoc-Phe derivative), spectra B are non-hydrogel solutions of each compound (DMSO-d6, 5 mM FmocPhe derivative), and spectra C are unassembled. As can be observed, the aqueous no-salt solution signals (spectra B) are considerably broader than the unassembled monomeric forms (spectra C) implying that the solution forms of the compounds have assembled morphologies that precludes display of distinct proton peaks. Also, it can be observed that there is a significant upfield chemical shift from monomeric to assembled form. This difference in chemical shift is most likely due to the different DMSO and D2O solvents, although the degree of shifting may be indicative of equilibrium between assembled and unassembled states. Comparative integration of the indicated peaks indicates that the amount of monomer present in the aqueous solution is 3% lower than in the DMSO solution for compound 1 and 31% lower for compound 2, suggesting that 0.14 mM of compound 1 and 1.95 mM of compound 2 monomer is in an aggregated state in these solutions. This degree of aggregation is relatively low however, this data is in agreement with our past results which suggest that halogen-substitued side chains in the phenyl group of Fmoc-Phe derivative self-assemble better than the parent Fmoc-Phe carboxylic acid.46 Only compound 3 shows significant loss of peak intensity in the salt-free solution, although this is most likely due to the lower solubility of this compound than to a higher degree of assembly (although both scenarios could account for the data). In comparison NMR spectra of each compound in hydrogel states (spectra A) show extreme line-broading and almost complete disappearance of signal, indicating that almost all monomer (5 mM based on comparative integrations) is incorporated into the aggregate network. Thus, while self-assembly of these cationic Fmoc-Phe derivatives in salt free conditions is evident based on TEM images and CD spectroscopy, the equilibrium between monomer and assembled stated is tilted heavily towards


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monomer. Upon increasing the solution ion strength by addition of NaCl, the equilibrium is strongly shifted towards the assembled state, accounting for efficient gelation under these conditions.

Nanosheet and nanotube formation by cationic Fmoc-Phe derivatives. The observation of sheet-like structures mixed with fibrils/worm-like micelles in many of these samples prompted additional study of these assemblies. Two-dimensional sheets have not been observed in the selfassembly of the parent amino acids for these cationic Fmoc-Phe derivatives. In addition, the sheet morphology formed by cationic compounds 1-3 seemed to become more prominent at higher concentrations and longer incubation times. Based on these observations, we studied the self-assembly of compounds 1 and 2 at 20 mM concentrations (114 mM NaCl) to further understand the formation of these sheets. Interestingly, it was observed that compound 1 formed unique nanotube structures at 20 mM concentrations (114 mM NaCl). After a 15 minute incubation period, TEM images of 1 showed a mixture of fibrils (Figure 5A, 6.9 ± 0.9 nm wide) and sheets (Figure 5B, 194.9 ± 13.3 nm wide, hundreds of nanometers to micrometers in length). These sheets appear to be folding and twisting into nascent thin-walled nanotube structures. After 1 hour, more mature nanotube structures emerge in which the sheet edges in the twisted structures observed at earlier time points (Figure 5B) have fused. After longer incubation periods (4 hours and 24 hours, Figures 5C,D and 5E,F respectively), these nanotube structures dominate and fibrils are only sparsely observed, suggesting that the sheets and subsequent nanotubes arise from the initially formed fibrils. The nanotubes of compound 1 are 196.7 ± 17.7 nm in diameter. These nanotube solutions formed opaque hydrogels at these high concentrations of the Fmoc-Phe derivative. Detailed rheological analysis was not conducted on these gels since they would not be of utility for most 19 ACS Paragon Plus Environment

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biological cell-based applications due to their opacity. These nanotube studies thus primarily focused on understanding the transition from fibril assemblies to the nanotube structures.

Figure 5. TEM images of hydrogels formed by compound 1 (20 mM, 114 mM NaCl) at various time intervals. A) 15 min, B) 15 min, C) 4 h, D) 4 h, E) 24 h, F) 24 h.

Similar results were observed with compound 2 at 20 mM concentrations (114 mM NaCl). After 15 minutes, long sheets 283.6 ± 36.3 nm wide have formed and appear to be twisting and folding onto themselves into nascent tube-like morphologies (Figure 6A and 6B). After 1 hour, a mixture of the “immature” nanotubes (Figure 6C) and more mature nanotubes 205.6 ± 19.4 nm wide (Figure 6D) were observed. After 4 hours and 24 hours (Figure 6E–H), only mature 20 ACS Paragon Plus Environment

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nanotube structures were observed. Interestingly, while 1 initially showed thinner fibrils mixed with sheets, solutions of 2 showed only sheet morphologies at 20 mM concentrations. This is consistent with previous studies of the parent Fmoc-3F-Phe amino acid, which has been shown to self-assemble more efficiently than Fmoc-Phe.46

Figure 6. TEM images of hydrogels formed by compound 2 at 20 mM (114 mM NaCl) at various time intervals. A) 15 min, B) 15 min, C) 1 h, D) 1 h, E) 4 h, F) 4 h, G) 24 h, H) 24 h.

Similar to Hamley’s description of nanotube formation in surfactant-like peptides (SLPs),57 TEM images of assemblies of compounds 1 and 2 at 20 mM concentrations showed similar sheets folding into tubes transtions, presumbably due to the surfactant-like nature of the molecules. It was evident that nanotubes of 1 and 2 are highly uniform (200 nm in diameter). It can also be noted from the TEM images (Figures 5 and 6) that multiple ultrathin sheets can wrap


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around to form the tubes, indicating that these nanotubes are most likely multi-walled. It was also observed that TEM images show the accumulation of the negative stain (uranyl acetate) around the nanotubes (for example, Figures 6E and 6G) which suggests that the lamination of fibrils to form sheets and nanotubes probably places the cationic hydrophilic segment of the molecules exposed to the water allowing it to efficiently crystallize the negative stain on the surface. Additional studies were performed to explore the temperature dependent assembly of sheetderived nanotubes assembled from Fmoc-Phe cation-modified derivatives. Compounds 1 and 2 (20 mM, 114 mM NaCl) were dissolved and allowed to assemble at 40 ºC, 25 ºC, and 4 ºC (Figure 7 for compound 2, Figure S17 in Supporting Information for compound 1). TEM images were obtained of assemblies formed at each of these temperatures various time intervals. At 40 ºC, some mature nanotubes along with less mature “folding” nanotubes were evident after 15 minutes (Figure 7A). After 4 hours and 24 hours, mature nanotubes were the only assembly observed (Figure 7B and 7C). Similar results were observed in the samples that were dissolved at 25 ºC (Figure 7D-F). After 15 minutes at 25 ºC, tubes in advanced folding states were observed, but these were less mature than those observed at 40 ºC (Figure 7D). After 4 hours and 24 hours at 25 ºC, only mature nanotubes were observed (Figure 7E and 7F). Finally, at 15 minutes at 4 ºC, folding events were in the early stages, with sheets clearly evident that were just beginning to curve into laminated tube structures (Figure 7G). At 4 ºC, immature tubes were the primary products even 4 hours and 24 hours after dissolution. Similar results were observed in parallel studies with compound 1 (Supporting Information, Figure S17). These studies give significant insight into the mechanism of nanotube assembly by these compounds, showing clear evidence


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of initial formation of sheets that then fold into immature nanotubes in which the sheet edges finally fuse to provide the mature nanotube structures.


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Figure 7. TEM images of nanotubes formed by compound 2 at 20 mM (114 mM NaCl) at varying temperatures and time intervals. A) 40 °C , 15 min; B) 40 °C, 4 h; C) 40 °C, 24 h; D) 25 ºC, 15 min; E) 25 ºC, 4h; F) 25 ºC, 24 h; G) 4° C, 15 min; H) 4 °C, 4 h; I) 4 °C, 24 h.


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These results are interesting in light of other cationic self-assembling systems that have been observed to form thin-walled nanotube assemblies. For example, Zhang and coworkers have reported surfactant-like peptides (SLP), including A6K and V6K that form nanotubes with helical twists.58-59 Hamley and coworkers have studied the self-assembly transition of such SLPs and have reported that these materials form ultrathin sheets that curve onto themselves to form helical ribbons and eventually form nanotubes.57 The amyloid-β 16–22 fragment (Aβ(16-22)) forms fibrils under neutral conditions, but under acidic conditions in which the peptide is cationic, it forms nanotubes.50 The amyloid-β-derived Phe-Phe dipeptide also has been reported to form nanotubes under conditions in which the peptide is cationic.60-64 Amyloid peptides share many characteristics with surfactant-like gelators, as do compounds 1-3 and the parent carboxylic acids from which 1-3 are derived. The similarity of these compounds to simple surfactants should not be discounted in attempts to explain the assembly and gelation of 1-3, since recent literature is replete with examples of similar molecules that exhibit surfactant-like behavior.65-68 Thus, the compounds described herein may be behaving as dynamic surfactants which share the properties of amyloid assemblies, including the formation of fibril or fibril derived architectures, formation of these fibrils after initial hydrophobic collapse into surfactantlike micelles,25, 69 and the exhibition of amyloid-like properties, including binding to amyloidspecific dyes like thioflavin T.42,

70

These similarities also illustrate that amyloid materials

behave as surfactants as well in many respects. Influence of pH on self-assembly of cationic Fmoc-Phe derivatives. The assembly data for cationic Fmoc-Phe derivatives, taken together with literature precedent for nanotube formation with peptide systems, suggests that the appended ammonium cation in these compounds drives the formation of distinctive sheet-like structures that eventually mature into nanotubes. In order


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to test this, we conducted several additional studies. Fmoc-Phe derivatives, including the parent Fmoc-3F-Phe and Fmoc-F5-Phe, have been previously observed to assemble only into fibril-like structures, with no prior evidence of nanotube formation.16, 46 Previous studies, however, have typically been at lower concentrations (~5 mM). In order to examine whether nanotube formation by compounds 1, 2 and 3 is only a concentration effect, the parent Fmoc-Phe, Fmoc3F-Phe and Fmoc-F5-Phe carboxylic acids were assembled by dilution from DMSO stocks into water (2% DMSO/water, v/v) at 20 mM final amino acid concentration. The amino acids were not fully soluble under these conditions, but TEM images obtained after 3 days showed only formation of fibrils/worm-like micelles (Figure S18, Supporting Information) and no evidence of nanotube formation. Next, we examined the assembly of these materials at basic pH values at which the positive charge would be reduced by deprotonation of the ammonium cation. For these experiments, compounds 1 and 2 were dissolved in unbuffered water at 20 mM (114 mM NaCl) and 0.5 M NaOH solution was immediately added to adjust the pH to 10.5. The pKa of propylamine is ~10.5, so at this pH we expect the compounds to be in only a partially protonated state. TEM images of these solutions were obtained after 1 day and after 5 days to assess nanotube assembly (Figure 8). Even after 5 days, only fibrils/worm-like micelles ~11.5 nm in diameter were observed, with no evidence of nanotube formation. These studies confirm that cationic character is essential to promote the formation of two-dimensional sheets that fold into nanotubes from these Fmoc-Phe derivatives.


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Figure 8. TEM images of compounds 1 and 2 in basic conditions (pH 10.5) at 20 mM (114 mM NaCl) at various time intervals. A) Compound 1, day 1; B) compound 1, day 5; C) compound 2, day 1; B) compound 2, day 5. Finally, we explored the self-assembly of Fmoc-Phe cationic derivatives at pH 7. At pH 7, the charge state of the C-terminal amino group should still be primarily posivitely charged, although the equilibrium between charged and uncharged will be very modestly shifted relative to acidic pH, where the equilibrium is expected to almost entirely favor the protonated form. These studies were performed by dissolving compound 1 in water (5 mM) and adjusting the pH to 7 by addition of NaOH. After the pH was adjusted to 7, 114 mM NaCl was added to induce self-assembly and hydrogelation of 1. Interestingly, even at neutral pH, no mature nanotube structures were observed after 24 hours or even after 5 days. At 24 hours, fibrils (Figure S19) were observed similar to those described above for self-assembly at pH 10.5. The fibrils/worm27 ACS Paragon Plus Environment

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like micelles observed at pH 7 after 24 hours were of two morphogies: fibrils ~11 nm in diameter and slightly broader fibrils ~20 nm in diameter (Figure S19A and B). After 5 days, wider, flat tape-like structures were observed, some as large as 60 nm in diameter (Figure S19C). These wider tapes are consistent with nanotube precursors observed at acidic pH; it may be expected that given longer incubation times, nanotubes may appear at pH 7 as well. The slower evolution of tapes and nanotubes at this pH is a function of concentration (5 mM); at higher concentrations (20 mM) nanotube formation happens at faster rates. Self-assembly of 1 at neutral pH occurs in a similar fashion to self-assembly at pH ~4.5. This is logical, since the cationic charge state should be similar at these pH values. Influence of counterion on self-assembly and hydrogelation of cationic Fmoc-Phe. The studies reported above all feature trifluoroacetate (TFA) counterions for the cationic amine group of the Fmoc-Phe derivatives. The TFA counterions are a function of the use of trifluoroacetic acid to deprotect the Boc group from the amino groups (see Schemes S1–S3 in Supporting Information). In order to determine whether the TFA counterion influences the self-assembly properties, we conducted the Boc deprotection reaction to form compound 1 using HCl in dioxane, provide compound 1 as the hydrochloride salt. This material was dissolved in water (5 mM) after which NaCl was added (114 mM final concentration). No pH adjustment was performed and the pH of the solutions was less acidic (pH 6.6) than that observed with the TFA salts (~4.5). This is due to the relative ease with which residual HCl is removed under vacuum relative to residual TFA. The solution formed a self-supporting hydrogel within minutes that was indistinguishable from the TFA salt hydrogels described earlier (Figure 3). TEM images of these hydrogels (Figure S20, Supporting Information) showed fibrils/worm-like micelles 4–8 nm in diameter after 24 h (Figure S20A) and nanotape/nanoribbon structures dominating after 60 h 28 ACS Paragon Plus Environment

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(Figure S20B–C). These nanoribbons are beginning to twist into nanotubes under these conditions. These results are virtually identical to those observed for the TFA salts of compound 1, indicating that self-assembly and hydrogelation are insensitive to the ammonium counterion.

Conclusion Herein, we have described simple Fmoc-Phe derivatives that have been modified by appending cationic amines at the C-terminus. The rationale for creating these derivatives was to identify Fmoc-Phe based molecules that can self-assemble into hydrogel networks without the need for organic cosolvents or in situ pH modification. It was observed that compounds 1-3 were indeed competent to form self-supporting hydrogels in simple aqueous solutions with sufficient ionic strength. Hydrogels were formed at salt concentrations that mimic those found in commonly used cell culture media, giving rise to the possibility that these materials may be appropriate for cell-based biological applications. It was also observed that these cationic Fmoc-Phe derivatives assemble into distinctive sheetbased nanotube structures. At lower concentrations (10 mM), sheets were observed along with fibrils, but at higher concentrations (20 mM), sheets that folded into nanotubes were favored. These nanotube structures are unique to C-terminal cationic Fmoc-Phe derivatives for this family of compounds. The parent Fmoc-Phe carboxylic acids form only fibril/worm-like micelles structures. Nanotube formation by the cationic Fmoc-Phe molecules is dependent on positive charge at the C-terminus, since at basic pH where the positive charge is reduced only fibrils are formed and nanotube formation is suppressed. These studies provide empirical insight into how subtle modification of structure can perturb the self-assembly pathways of Fmoc-Phe derivatives and other self-assembling molecules. Future studies will focus on defining the packing structure


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of these derivatives in order to more fully understand the mechanistic basis for these interesting empirical observations.

Supporting Information Supporting information includes synthetic protocols and characterization data for all compounds, spectroscopic data for all assemblies, NMR spectra for assembly characterization, and additional TEM images.

Acknowledgments This work was supported by the National Science Foundation (DMR-1148836). We gratefully acknowledge Karen Bentley (URMC Electron Microscope Research Core) for her assistance in the TEM and SEM imaging experiments.

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Synopsis: We report C-terminal cation-modified Fmoc-Phe derivatives that have the ability to form hydrogel networks without the need to adjust pH or to use an organic cosolvent for inducing self-assembly. These cationic Fmoc-Phe derivatives also form sheet-based nanotube structures under appropriate conditions. 35 ACS Paragon Plus Environment

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Figure 1. Chemical structures of C-terminal cation-modified Fmoc-Phe derivatives.

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Figure 2. TEM images of fibrils formed by each compound at 5 mM in water under salt-free conditions. A) Compound 1, B) compound 2, and C) compound 3.


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Figure 3. A) Digital image of self-supporting hydrogels (as indicated by vial inversion) formed by compounds 1-3 (5 mM compound in 114 mM aqueous NaCl). B) Rheological frequency sweep data for hydrogels formed by compound 1 at concentrations ranging from 2.5–10 mM (114 mM aqueous NaCl), C) compound 2 (2.5– 10 mM, 114 mM NaCl), D) compound 3 (2.5–10 mM, 114 mM NaCl). 167x176mm (300 x 300 DPI)


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Figure 4. TEM images of hydrogels formed by compounds 1-3 at various concentrations obtained 15 min after hydrogelation. A) 1 at 2.5 mM, B) 1 at 5 mM, C) 1 at 10 mM, D) 2 at 2.5 mM, E) 2 at 5 mM, F) 2 at 10 mM, G) 3 at 2.5 mM, H) 3 at 5 mM.


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Figure 5. TEM images of hydrogels formed by compound 1 (20 mM, 114 mM NaCl) at various time intervals. A) 15 min, B) 15 min, C) 4 h, D) 4 h, E) 24 h, F) 24 h.


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Figure 6. TEM images of hydrogels formed by compound 2 at 20 mM (114 mM NaCl) at various time intervals. A) 15 min, B) 15 min, C) 1 h, D) 1 h, E) 4 h, F) 4 h, G) 24 h, H) 24 h.


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Figure 7. TEM images of nanotubes formed by compound 2 at 20 mM (114 mM NaCl) at varying temperatures and time intervals. A) 40 °C , 15 min; B) 40 °C, 4 h; C) 40 °C, 24 h; D) 25 ºC, 15 min; E) 25 ºC, 4h; F) 25 ºC, 24 h; G) 4 °C, 15 min; H) 4 °C, 4 h; I) 4 °C, 24 h.


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Figure 8. TEM images of compounds 1 and 2 in basic conditions (pH 10.5) at 20 mM (114 mM NaCl) at various time intervals. A) Compound 1, day 1; B) compound 1, day 5; C) compound 2, day 1; B) compound 2, day 5.


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Self-Assembly, Hydrogelation, and Nanotube ... - ACS Publications

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