Electrostatic and Aromatic Interaction-Directed Supramolecular Self

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Electrostatic and Aromatic Interaction-Directed Supramolecular SelfAssembly of a Designed Fmoc-Tripeptide into Helical Nanoribbons Yanyan Xie,† Xiangchao Wang,† Renliang Huang,*,‡ Wei Qi,*,†,§ Yuefei Wang,† Rongxin Su,†,§ and Zhimin He† †

State Key Laboratory of Chemical Engineering, School of Chemical Engineering and Technology, ‡School of Environmental Science and Engineering, and §Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin Key Laboratory of Membrane Science and Desalination Technology, Tianjin University, Tianjin 300072, PR China S Supporting Information *

ABSTRACT: Supramolecular self-assembly offers an efficient pathway for creating macroscopically chiral structures in biology and materials science. Here, a new peptide consisting of an N-(9-fluorenylmethoxycarbonyl) headgroup connected to an aromatic phenylalaninetryptophan dipeptide and terminated with zwitterionic lysine (FmocFWK) and its cationic form (Fmoc-FWK-NH2) were designed for selfassembly into chiral structures. It was found that the Fmoc-FWK peptide self-assembled into left-handed helical nanoribbons at pH 11.2−11.8, whereas it formed nanofibers at pH 5 and 12 and large flat ribbons composed of many nanofibers in the pH range of 6−11. However, only nanofibers were observed in the cases of Fmoc-FWKNH2 at different values. A series of structural characterizations based on CD, FTIR, UV−vis and fluorescence spectroscopy reveal that the electrostatic and aromatic interactions and the associated hydrogen bonding direct the self-assembly into various structures. The enhanced π−π stacking and hydrogen bonding were found in the helical nanoribbons. This difference in intermolecular interactions should be derived from the ionization of carboxyl and amino groups from lysine residues at different pH values. Furthermore, we performed molecular dynamics simulations to gain insight into the assembly mechanisms. The results imply that a relatively rigid molecular conformation and the strong intramolecular aromatic interaction between Trp and Fmoc groups favor chiral self-assembly. This study is the first attempt to design a Fmoctripeptide for the fabrication of helical structures with macroscopic chirality, which provides a successful example and allows us to create new peptide-based chiral assembly systems.

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INTRODUCTION Helical nanostructures abound in nature and exist in forms such as an α helix in proteins, a double helix in deoxyribonucleic acid (DNA), and a triple helix in collagen. They play a critical role in the construction of three-dimensional architectures and thus contribute to unique functions in biology. Inspired by these helices in natural biomolecules, many studies have been performed to design and fabricate artificial helical structures from polymers, π-conjugated molecules, and amphiphilic molecules over the past few years.1−4 One promising direction this research has taken is toward supramolecular self-assembly, which provides an efficient pathway to create such highly ordered nanostructures. Some helical structures, such as helical fibers, helical ribbons, twisted sheets, and helical tubes, were successfully obtained in the laboratory by the regulation of noncovalent interactions from chiral or achiral molecules.5−9 Additionally, the handedness and structural metric of helices could be rationally controlled in some cases by changing the enantiomer chirality and self-assembly conditions (e.g., solvent, temperature, and light).7,10−14 These sophisticated nanostructures are extremely attractive as potential building blocks in © 2015 American Chemical Society

various applications, for instance, templates for nanofabrication, enantioselective recognition, asymmetric catalysis, and chiroptical switches.15−17 Peptide materials created by supramolecular self-assembly have become a research hotspot because of their structural diversity, facile functionalization, and excellent biocompatibility.18−23 In recent years, many peptide-based molecules were reported as building blocks to generate helical nanostructures, as summarized in Table S1. A classic example is the peptide amphiphile containing a palmitoyl tail, the 2-nitrobenzyl group, and an oligopeptide segment.24 In this case, quadruple helices with a uniform width of ∼33 nm and a pitch of 92 nm were formed, which could further dissociate into single nonhelical fibrils in response to light. More recently, Liu and coworkers reported some peptide amphiphiles that consisted of amino acids (e.g., alanine, glutamine) and long alkyl chains.6,12,13,25,26 For example, a peptide bolaamphiphile of N,N-hexadecaneReceived: December 8, 2014 Revised: February 3, 2015 Published: February 18, 2015 2885

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at room temperature for 1 day without disturbance to ensure complete self-assembly. For Fmoc-FWK-NH2 peptides, the peptide assemblies were obtained following the same procedure as described before. To investigate the effect of pH on self-assembly, aqueous solutions with different pH values ranging from 5 to 12 were used. Compositional Analysis. The chemical compositions of the hydrogels formed at pH 7, 9, 10 and 11.5, respectively, were measured by high-performance liquid chromatography (HPLC) and liquid chromatography−mass spectrometry (LC−MS). Specifically, the formed hydrogels were directly freeze dried and then dissolved in acetonitrile solvent. The concentrations of Fmoc-FWK and its hydrolysate (FWK) were determined using an Agilent HPLC 1200 system (Agilent Technologies, USA) equipped with a UV−vis detector operated at 220 nm on a Venusil MP-C18 column (5 μm, 4.6 mm × 250 mm, Tianjin Bonna-Agela Technologies Ltd., China). The mobile phase was composed of acetonitrile and water with increasing acetonitrile content from 20 to 70% over 25 min at a total flow rate of 1 mL min−1. The pure Fmoc-FWK and FWK were used as standards to produce the calibration curve for the quantitative calculation. Both chemicals were further confirmed by LC−MS analysis, which was performed on a Agilent 1200 HPLC system coupled to an Agilent 6310 ion-trap mass spectrometer (Agilent Technologies, USA) with an ESI source, operated in positive-ion mode. Rheological Measurement. Rheological measurements were carried out using a Stress Tech rheological instrument (Rheological Instruments AB CO., Sweden). The stock peptide solution (35 mg mL−1) was poured onto the plate of the instrument, which was held at 25 °C by using an integrated temperature controller. Then, an aqueous solution at pH 5, 7, or 11.5 was added immediately. The storage modulus G′ and loss modulus G″ were measured as a function of time at a constant frequency of 1 rad s−1. After 30 min, a frequency sweep (0.1−50 Hz) test was conducted to measure the frequency-dependent storage and loss shear moduli. Morphological Characterization. The hydrogels were frozen at −45 °C and subsequently dried under vacuum. All samples were sputter-coated with platinum using an E1045 Pt-coater (Hitachi Hightechnologies CO., Japan) and then imaged with an S-4800 field emission scanning electron microscope (SEM, Hitachi Hightechnologies CO., Japan) at an acceleration voltage of 3 keV. The morphology of the peptide assemblies was further assessed using a JEOL 100CX-II transmission electron microscope (TEM, JEOL Ltd., Japan) operated at 80 keV. To prepare samples, the hydrogel was dispersed in ddH2O, and a 10 μL aliquot of aqueous dispersion was placed onto a 300 mesh carbon-coated copper grid, air dried, and then negatively stained with 1 wt % phosphotungstic acid solution for 2 min before imaging. The samples for atomic force microscopy (AFM) measurement were prepared as follows: the hydrogel was dispersed in ddH2O, and a 100 μL aliquot of the aqueous dispersion was spread on a mica surface and air dried before observation. AFM images were recorded using an Agilent 5500 atomic force microscope (Agilent, USA) equipped with N9797 AU-1FP Pico software in tapping mode. Structural Characterization. Circular dichroism (CD) analysis was performed on a Jasco J-810 CD spectropolarimeter (Jasco Inc., Japan) over a wavelength range of 185−350 nm with a resolution of 1 nm. Spectra were recorded three times using a scanning speed of 100 nm min−1 with a bandwidth of 1 nm. Fourier transform infrared spectroscopy (FTIR) spectra of xerogels were recorded on a Nicolet560 FTIR spectrometer (Nicolet Co., USA) with a KBr pellet method across the range of 400−4000 cm−1. A total of 16 scans were accumulated with a resolution of 4 cm−1 for each spectrum. UV−vis spectra were monitored on a TU-1810 UV−vis spectrophotometer (Persee Instruments Ltd., China) with a wavelength range of 200−700 nm. Fluorescence spectra were recorded on a Cary Eclipse spectrofluorometer (Agilent CO., USA) with excitation at 280 nm. All fluorescence spectra were recorded at 25 °C. The excitation and emission slit widths were fixed at 5 and 5 nm, respectively. Zeta Potential Measurement. Zeta potential measurements of Fmoc-FWK peptide solutions and peptide assemblies were performed using a Zetasizer Nano-ZS (Malvern Instruments Ltd., U.K.). To

dioyl-di-L-glutamic acid (HDGA) could self-assemble into helical nanotubes in water.25 They also obtained twisted ribbons from a racemic mixture of AlaC17 in hexane through a heating−cooling process. These twisted ribbons were used to recognize the chirality of amino acid derivatives.26 Such alkylchain-containing molecules were also designed by other groups to create helical nanostructures.8,27−30 For instance, Song et al.30 fabricated left- and right-handed double helices via the supramolecular self-assembly of C11H23CO-AYSSGAPPMPPF. These helices were further used as templates to construct goldnanoparticle-based double-helical superstructures with controllable structural metrics and handedness.30 Another successful example is the ILQINS hexapeptide, which is derived from lysozymes in hen egg whites. This peptide could self-assemble into a right-handed helical ribbon in an aqueous solution with pH 2 at room temperature.5 Additionally, some unique peptidebased derivatives (e.g., dilysine peptides-NDI (1,4,5,8-naphthalenetetracarboxylic acid diimide),31 Table S1) were also designed to create helical structures. In view of the structural diversity of peptides, this study aims to design a new peptide molecule for the fabrication of helical nanostructures. DNA is a well-known double helix and serves as a carrier for biological information storage. This double helix is stabilized primarily by two forces: hydrogen bonds between nucleotides and base-stacking interactions among aromatic nucleobases.32 It exists in many possible conformations that include right-handed A-DNA, B-DNA, and left-handed Z-DNA forms.33,34 In particular, the DNA double helix presents a smart pH responsiveness due to its phosphate and basic groups. The double helix is stable over the pH range of 5−9 and starts to uncoil at pH 12. This unique structure and performance greatly inspire us to design a pH-responsive peptide-based helix via the regulation of hydrogen bonds (or electrostatic interactions) and aromatic interactions. Fmoc-based short peptides are a widely studied class of supramolecular hydrogelators. The Fmoc moiety provides strong aromatic interactions to drive Fmoc-peptide selfassembly into nanofibers or nanotubes,18,35−44 in which no macroscopic chirality was presented (Table S2). Herein, we designed two new peptide-based molecules containing Nfluorenyl-9-methoxycarbonyl (Fmoc) groups and phenylalanine-tryptophan-lysine (FWK) fragments: one is an FmocFWK-COOH (Fmoc-FWK) peptide, and the other is a cationic Fmoc-FWK-NH2 peptide. The self-assembly behaviors of these peptides at different pH values were investigated experimentally via morphological and structural characterization using several microscopy and spectroscopy techniques. Furthermore, molecular dynamics simulations were performed to investigate the molecular conformation of Fmoc-tripeptides at different pH values and thus to understand the self-assembly mechanism.

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EXPERIMENTAL SECTION

Materials. N-(9-Fluorenylmethoxycarbonyl)-phenylalanine-tryptophan-lysine (Fmoc-Phe-Trp-Lys-OH, Fmoc-FWK, 98%), N-(9-fluorenyl methoxycarbonyl)-phenylalanine-tryptophan-lysine-NH2 (FmocFWK-NH2, 98%), and phenylalanine-tryptophan-lysine (FWK, 98%) were synthesized by GL Biochem Ltd. (China). All other chemicals were of analytical grade and were obtained from commercial sources. Preparation of Fmoc-FWK Helical Nanoribbons. In a typical experiment, the lyophilized Fmoc-FWK peptide was dissolved in a mixed solvent of acetonitrile−H2O (v/v = 1) at a concentration of 35 mg mL−1 at 60 °C. The resulting peptide solution was then diluted to a final concentration of 7 mg mL−1 in an aqueous solution at pH 11.5. Within several seconds, a peptide hydrogel was formed and then aged 2886

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Figure 1. (a) Molecular structure of the Fmoc-FWK peptide. (b) SEM, (c, d) AFM, and (e, f) TEM images of the Fmoc-FWK helical nanoribbons formed at pH 11.5. prepare the peptide solutions, 1 mg of Fmoc-FWK peptide was dissolved in 1 mL of ddH2O. The pH of the resulting solution was adjusted with 0.5 M NaOH or 0.1 M HCl. A pH meter was used to measure the pH values of all of the solutions. For peptide assemblies, the peptide hydrogel was synthesized as mentioned before and then dispersed in ddH2O to a final concentration of 0.07 mg mL−1. pH Titration. Fmoc-FWK-OH or Fmoc-FWK-NH2 peptides were dissolved in ddH2O at a concentration of 10 mmol L−1 at pH 2.5 by adding 0.1 M HCl. The titration curves were recorded by monitoring the pH values during the stepwise addition of small volumes of 0.5 M NaOH. The samples were stirred to ensure complete mixing during the titration process. No significant difference (0.3 pH unit) was observed between the two pH measurements. The pH measurements were performed using a TitroLine Alpha Plus AutomaticTitrator (Scott CO., Germany). Molecular Dynamics Simulations. Hydrogen atoms of the Fmoc-tripeptide residues were added using Discovery Studio (version 2.5.5, Accelrys). The system was centered in a cubic box of TIP3P water molecules, and a sufficient number of Na+ ions and HPO42−/ H2PO4− were added to neutralize the negative charges. A CHARMm force field was used to assign molecules, and classical molecular dynamics (MD) simulations were performed using Discovery Studio (version 2.5.5, Accelrys). The system energy was first minimized by performing 5000 steps using the steepest descent algorithm, followed by an additional 2000 steps of minimization using the conjugate

gradient method. Then, the system was heated to the target temperature of 300 K for a period of 2 ps under constant-pressure periodic boundary conditions (NPT). Subsequently, the system was allowed to equilibrate for 1 ps at constant pressure and temperature (NPT) with a time step of 1 fs, followed by 1 ns of production simulation performed under the same conditions. A cutoff of 14 Å was used for nonbonded interactions, and long-range electrostatic interactions were treated by means of the particle mesh Ewald (PME) method. The MD simulation results were further analyzed using Discovery Studio software (version 2.5.5, Accelrys).

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RESULTS AND DISCUSSION Preparation and Morphological Characterization of Fmoc-FWK Helical Nanoribbons. In this study, our molecular design strategy focuses on an aromatic dipeptide (FW) protected at the N-terminus with an Fmoc group and modified at the C-terminus with a lysine residue (Figure 1a). We expect that the π−π association of the Fmoc-FW fragment and hydrogen bonding along the peptide backbone will strongly drive the supramolecular self-assembly in an aqueous solution, leading to the formation of well-ordered nanostructures. Meanwhile, the lysine residue at the C-terminus will contribute to the zwitterionic properties of the Fmoc-FWK peptide, which 2887

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Figure 2. (a) Photograph of the Fmoc-FWK hydrogels formed at different pH values. (b) Rheology analysis of the Fmoc-FWK hydrogels. The peptide concentration was held constant at 7 mg mL−1.

Figure 3. SEM images of Fmoc-FWK assemblies formed at different pH values: (a) 5, (b) 7, (c) 11, (d) 11.2, (e) 11.8, and (f) 12. The scale bar is 500 nm.

provide the possibility to control its self-assembly behavior structure by pH. To demonstrate the self-assembly ability of the designed peptide conjugates, the Fmoc-FWK peptides were initially dissolved in a mixed solvent of acetonitrile/H2O (v/v = 1:1) at 60 °C and then diluted in an aqueous solution of NaOH (pH 11.5), allowing them to self-assemble. Upon mixing the two solutions, a supramolecular hydrogel was formed within seconds (Figure 1a, right sample). Figure 1b and Figure S1 show the SEM micrographs of a freeze-dried gel. Highly uniform helical nanoribbons over a large area are observed. These helices have a width of ∼100 nm and a thread pitch of ∼200 nm. To further observe their morphology, atomic force

microscopy (AFM) and transmission electron microscopy (TEM) were employed to characterize the samples. As shown in Figure 1c,d, left-handed helices with a thread pitch of ∼200 nm are also observed clearly. In particular, the TEM images reveal that the helical ribbons exist either in individual form or are further intertwined with each other to form a superhelical structure (Figure 1e,f). This superhelix has some structural similarities to the triple helix that exists in collagen fibrils. Previously, Muraoka et al.24 reported a quadruple helix that was composed of two smaller helices, each of which was composed of two individual fibrils. pH Responsiveness of the Supramolecular SelfAssembly of Fmoc-FWK. We further investigated the pH 2888

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Figure 4. (a) CD spectra of the Fmoc-FWK solution and assemblies formed at different pH values ranging from 5 to 12. (b) Change in the intensity of CD peaks at 192, 222, and 275 nm with increasing pH values. (c−f) Spectroscopic characterization of the Fmoc-FWK solution and assemblies formed at different pH values: (c, d) FTIR, (e) UV−vis, and (f) fluorescence. The inset in panel f shows the intensity change in the fluorescence emission peaks at 330 nm. The peptide solution was prepared by dissolving the Fmoc-FWK peptide in a mixed solvent of acetonitrile/H2O (v/v = 1:1) to a final concentration of 7 mg mL−1. The same concentration (7 mg mL−1) was used to obtain peptide assemblies in all experiments.

shown in Figure 2b, show that the value of the storage modulus G′ is approximately 1 order of magnitude higher than the loss modulus G″, indicative of the elastic and solidlike hydrogels. Specifically, in the case of pH 5.0, the value of G′ reached 31.77 kPa, which increased to 48.51 and 82.46 kPa at pH 11.5 and 7.0, respectively. In comparison to other Fmocpeptide hydrogels (e.g., Fmoc-FF,38 the value of G′ is ∼10 kPa at ∼10 mg mL−1), Fmoc-FWK has a larger G′ value, which is characteristic of higher mechanical strength and is possibly attributed to the stronger intermolecular interactions (e.g., π−π, hydrogen bonding). In addition, it is worth noting that the G′ of the Fmoc-FWK hydrogel formed at pH 7 is much higher than for the cases where the pH was changed to 5 and 11.5. This difference should be derived from the different assemblies’ structures.

responsiveness of the supramolecular self-assembly of FmocFWK peptides. To demonstrate this issue, the peptide stock solution was diluted in an aqueous solution with different pH values ranging from 5 to 12 (the pH values were adjusted by HCl or NaOH). As shown in Figure 2a and Figure S2, the selfsupporting hydrogels were formed within seconds over a wide pH range from 5 to 11.8. When the pH value increased to 12, however, no gelation was observed, even after 24 h. The chemical compositions of hydrogels were measured by HPLC and LC−MS analysis. As shown in Figure S3, only 0.2 and 1.5% of Fmoc-FWK was hydrolyzed to FWK at pH 10 and 11.5, respectively, while no FWK was determined in the cases of pH 7 and 9, indicating a high stability of Fmoc-FWK at 60 °C and basic conditions. The viscoelastic properties of these peptide hydrogels were evaluated by small-deformation oscillatory measurements. The results of frequency sweep tests, as 2889

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Figure 5. (a) Molecular structure of the Fmoc-FWK-NH2 peptide. (b) Fluorescence emission spectra of the Fmoc-FWK-NH2 solution and assemblies formed at different pH values. (c, d) SEM images of peptide nanofibers formed at (c) pH 5 and (d) pH 11.5. The scale bar is 500 nm. The peptide solution was prepared by dissolving the Fmoc-FWK-NH2 peptide in a mixed solvent of acetonitrile/H2O (v/v = 1:1) to a final concentration of 7 mg mL−1. The same concentration (7 mg mL−1) was used to obtain self-assemblies.

employed to characterize the molecular-scale structural information on Fmoc-FWK assemblies. As observed from Figure 4a and Figure S6, the nanofibers (pH 5) and flat ribbons (pH 6−11) have similar CD spectra, showing a positive peak at 190 nm and two negative peaks at 205 and 222 nm, indicative of α-helical π → π* and superhelical n → π* transitions.37 In the cases of helical nanoribbons (pH 11.2−11.8), the negative Cotton effect at 216, 275, 285, and 293, and 304 nm indicates the superhelical arrangements formed by the Lys, Phe, and Try residues and the fluorenyl groups, respectively.8,46 Similar superhelical arrangement was also found previously in nonhelical (e.g., Fmoc-AA36) or helical (Fmoc-K-C16H338) nanofibers. Figure 4b shows the change in intensity of the CD peaks at 190, 216, and 275 nm. The significant increase in peak intensity is clearly observed in the pH range of 11.2−11.8, indicating the presence of strong chirality. It is worth noting that the CD spectra of nanofibers show a weak positive Cotton effect (275−304 nm), whereas the helices show a strong negative Cotton effect. This transition implies that aromatic residues and fluorenyl groups exist in different helical orientations between these structures. In addition, the CD spectra of helical nanoribbons provide further evidence in support of the left-handed conformation, which is consistent with the morphological analysis described above (Figure 1). Figure 4c,d shows the FTIR spectra of Fmoc-FWK assemblies formed at pH 5, 7, and 11.5. As observed from Figure 4c, one dominant peak is located at a wavelength of 1654 cm−1, suggesting that the main secondary structures are all α-helix arrangements, confirming the results obtained by CD.5,7,12 Although the nanofibers, flat ribbons, and helical nanoribbons exhibit different morphologies and chirality, they adopt a similar helical arrangement on the molecular scale. We expect that the hierarchical self-assembly of these small, helixarranged molecular clusters further produces larger nanorib-

To directly observe the assembly morphology, we employed scanning electron microscopy (SEM) to characterize the freezedried samples. As observed from Figure 3a, at a pH of 5, a number of nanofibers with a highly uniform diameter of 10−20 nm are observed. It is worth noting that no macroscopic chirality was presented in these nanofibers. With increasing pH, large, flat ribbons resulting from the lateral association of many fibers were formed over a pH range of 6−11 (Figure 3b,c, Figure S4). A similar pH-induced transition from fiber to flat ribbon was also reported previously in the case of Fmoc-FF.45 In these assemblies, as the pH increased from 11 to 11.2 or 11.8, a unique and uniform nanohelix was observed (Figure 2d,e), similar to those observed at pH 11.5, as mentioned before. However, a further increase in the pH value to 12 led to an uncoiled nanofiber. At this high pH, no helical nanoribbon was found (Figure 3f). More importantly, we found that the helical nanoribbons formed at a given pH were very stable at different pH values ranging from 5 to 12, indicating the irreversibility of such a self-assembly pathway. To gain insight into this self-assembly phenomenon, the zeta potentials of the Fmoc-FWK solutions and assemblies were measured (Figure S5). At a low or high pH (e.g., 5 or 12), a fraction of FmocFWK molecules are expected to be ionized (e.g., zeta potentials are 40 mV at pH 5 and −52 mV at pH 12); therefore, the selfassembled nanofibers exist in individual form due to the electrostatic repulsion between peptides. As the pH values shifted to 6−11, the degree of ionization of Fmoc-FWK and the fiber surface charge decreased (e.g., 10 mV at pH 6), allowing them to grow laterally and form large, flat ribbons. At pH 11.2− 11.8, the formation of Fmoc-FWK helices may be driven synergistically by many factors such as electrostatic interactions, hydrogen bonding, and aromatic stacking. Structural Characterization. Circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR) were 2890

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Figure 6. Molecular dynamics simulations of (a, b) Fmoc-FWK and (c, d) Fmoc-FWK-NH2. (a, c) The final molecular conformations at pH 5 (yellow) and pH 11.5 (gray). (b, d) Time evolutions of C root-mean-square deviations (rmsd’s) with respect to the final structures.

exceed 11; in these cases, the helical structure was formed. This red shift together with an increased intensity should be attributed to the enhanced aromatic interactions (or π−π stacking) between fluorenyl groups and also suggests the presence of J-type π−π aggregation.50,51 Therefore, we expect that strong π−π stacking is an important force in directing the supramolecular self-assembly of Fmoc-FWK from nanofibers to large, flat ribbons to such helical nanoribbons. As mentioned before, the dissociation equilibrium of FmocFWK molecules at different pH values leads to a significant difference in electrostatic interactions and π−π stacking as well as hydrogen bonding in the supramolecular self-assembly process, thus forming nanofibers, large, flat ribbons, or helical nanoribbons. The results indicate that the terminal groups (COOH, NH2) play important roles in directing the formation of various self-assemblies. Supramolecular Self-Assembly of Fmoc-FWK-NH2. To further demonstrate this point, we designed another cationic Fmoc-FWK-NH2 peptide (Figure 5a), which differs only in that the terminal group of the lysine residues is either COOH or NH2 compared to the Fmoc-FWK peptide as described before. As expected, these two peptides gave rise to a quite different self-assembly behavior. As shown in Figure 5c,d and Figure S8, Fmoc-FWK-NH2 self-assembled into hydrogels over a pH range of 5 to 12, but all of these gels were composed of nanofibers. No large, flat ribbons or helical nanoribbons were formed. On the one hand, as observed from Figure S9, FmocFWK has pKa1 at 2.9 and pKa2 at 11.5, while Fmoc-FWK-NH2 has pKa1 at 9.7 and pKa2 at 11.8. As mentioned before, the Fmoc-FWK helical nanoribbons were formed when the pH range (11.2−11.8) was near pKa2. In these cases, the carboxyl group ionized to its negative form (COO−), whereas the amino group existed in both forms of NH2 and NH3+, leading to extensive electronic interactions. However, Fmoc-FWK-NH2 is positive charged in the pH range of 5−12 due to the

bons with macroscopic chirality. Such hierarchical self-assembly was also demonstrated recently in a chiral supramolecular gel.47 Additionally, a new peak at 1604 cm−1 starts to appear when the pH values increase to 7 and 11.5, which should be due to the side-chain COO− groups of lysine.12,48,49 In the N−H stretching band (Figure 4d), two sharp peaks at 3415 and 3418 cm−1 are observed for the nonhelical assemblies. However, for helical nanoribbons, only a broad band centered at 3408 cm−1 dominated, which indicates that an enhanced hydrogenbonding interaction exists in the helices.8 The results provide strong evidence in support of the fact that the hydrogen bond is a main driving force for the formation of helical nanoribbons. UV−vis and fluorescence spectra were further recorded to monitor the change in aromatic interactions between peptide molecules at different pH values. As shown in Figure 4e, the UV−vis absorption spectrum of the Fmoc-FWK peptide solution exhibits a peak centered at 266 nm with two shoulders at 289 and 299 nm. The peak and shoulders shift to 273, 294, and 304 nm, respectively, along with the formation of supramolecular structures at pH 5−12. This red shift reveals the presence of face-to-face stacking of aromatic groups within the architecture.26 Figure 4f shows the fluorescence emission spectra of the Fmoc-FWK solution and assemblies formed at different pH values. Two characteristic emission peaks at 306 and 314 nm with a broad shoulder at 351 nm are observed in the case of the peptide solution, indicative of Fmoc groups and Trp residues, respectively. In the cases of peptide nanofibers and large ribbons formed at pH 5−11, the emission peaks shift to 330 and 355 nm, but their intensity decreases significantly compared to that of peptide molecules (Figure S7). Interestingly, the emission spectra of Fmoc-FWK helices exhibit a strong peak centered at 330 nm with a shoulder at 355 nm. The inset in Figure 4f shows the intensity change of the peak at 330 nm with increasing pH values. A significant increase in peak intensity is observed when the pH values 2891

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spectra of Fmoc-FWK hydrogels. SEM images of the FmocFWK flat ribbons that were formed at different pH values. Zeta potential of Fmoc-FWK solution and the assemblies formed. CD and fluorescence spectra of the Fmoc-FWK assemblies obtained at different pH values. SEM images of the FmocFWK-NH2 nanofibers that were formed at different values. Titration curves of Fmoc-FWK. UV−vis spectra of Fmoc-FWKNH2 solution and hydrogels that were formed at different pH values. Final molecular conformations of Fmoc-FWK and Fmoc-FWK-NH2. MDS time evolutions of distances with respect to the final structure. Summary of the peptide-based molecules and the corresponding helical assemblies and the Fmoc-based peptides and the corresponding assemblies morphology. Mean values of rmsd and distances calculated between 850 and 1000 ns. This material is available free of charge via the Internet at http://pubs.acs.org.

protonation of amino groups, resulting in the electronic repulsion between peptide molecules. On the other hand, the characteristic peaks in the UV−vis and fluorescence spectra, similar to those of Fmoc-FWK, were also observed in the case of Fmoc-FWK-NH2 (Figure S10 and Figure 5b); however, they displayed a weak fluorescence intensity. These results imply that Fmoc-FWK-NH2 nanofibers undergo different electrostatic interactions and π−π stacking compared to Fmoc-FWK helical nanoribbons. It provides further evidence that the carboxyl group at the terminal ends plays a key role in the pH-responsive self-assembly and formation of helical nanoribbons. Molecular Dynamics Simulations. Finally, molecular dynamics simulations (MDS) were performed to understand the self-assembly behaviors at pH 5 and 11.5 with a concentration of 7 mg mL−1. The final molecular conformations, which were stable under their surrounding conditions, are shown in Figure 6a,c and Figure S11. It can be observed that the four conformations were very different compared to each other. Specifically, the four characteristic distances between groups Trp and Fmoc (d1), Phe and Trp (d2), NH2 and Phe (d3), and NH2 and Trp (d4) were collected, as summarized in Table S3. It is worth noting that Fmoc-FWK has a shorter distance between the Trp and Fmoc groups (d1) at pH 11.5, suggesting a stronger intramolecular aromatic interaction consistent with the results from fluorescence analysis. Additionally, the time evolutions of C root-mean-square deviations (rmsd’s) with respect to the final structure were also collected. As observed from Figure 6b,d, Fmoc-FWK has the smallest rmsd value of 10.4 for the final conformation at pH 11.5, indicating its rigid conformation. According to the TEM results, we infer that the nanoribbons must twist into helices to stabilize the structure, whereas this process is not favored for the peptide molecules with a flexible configuration. On the basis of the simulation results together with the experimental findings, we expect that the formation of helical nanoribbons requires a rigid molecular conformation and a strong intramolecular aromatic interaction between the Trp and Fmoc groups.

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Corresponding Authors

*(R.H.) E-mail: [email protected]. Tel: +86 22 27407799. Fax: +86 22 27407599. *(W.Q.) E-mail: [email protected]. Tel: +86 22 27407799. Fax: +86 22 27407599. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Natural Science Foundation of China (nos. 21476165, 51173128, and 21306134), the 863 Program of China (nos. 2012AA06A303 and 2013AA102204), the Ministry of Science and Technology of China (no. 2012YQ090194), the Ministry of Education (no. 20130032120029), Beiyang Young Scholar of Tianjin University (2012), and the Program of Introducing Talents of Discipline to Universities of China (no. B06006).

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CONCLUSIONS We have successfully demonstrated the rational design and supramolecular self-assembly of a simple Fmoc-FWK peptide, producing a unique left-handed helical nanoribbon. The selfassembly is responsive to changes in pH and results in different assemblies, including nanofibers, large, flat ribbons, and helical nanoribbons. Structural characterization reveals that the electrostatic interactions, π−π stacking, and hydrogen bonding induced by the ionization of carboxyl and amino groups from lysine residues play important roles in directing the assembly process and thus forming various self-assemblies. Moreover, the MDS results imply the rigid molecular conformation of FmocFWK at pH 11.5, and the strong intramolecular π−π stacking between Trp and Fmoc groups also favors the hierarchically self-assembly into helical nanoribbons with macroscopic chirality. The results demonstrated in this study have given us a better understanding of the molecular design and chiral assembly of Fmoc-peptides and have laid the foundation for their further application as chiral nanomaterials.

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AUTHOR INFORMATION

REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

SEM image of the Fmoc-FWK helical nanoribbons that were formed at pH 11.5. Photographs of Fmoc-FWK assemblies formed at different pH values. HPLC profiles and LC−MS 2892

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