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Jul 13, 2017 - ABSTRACT: Peptide-π-peptide triblock molecules can self- assemble into 1-D nanostructures with extensive hydrogen-bonding networks ...
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Self-Assembly and Associated Photophysics of Dendron-Appended Peptide-π-Peptide Triblock Macromolecules Tejaswini S. Kale,† Jeannette E. Marine,∥ and John D. Tovar*,†,‡,§ †

Department of Chemistry, Krieger School of Arts and Sciences, ‡Institute for NanoBioTechnology, and §Department of Materials Science and Engineering, Whiting School of Engineering, Johns Hopkins University, 3400 N. Charles St., Baltimore, Maryland 21218, United States ∥ Department of Chemistry, Stony Brook University, Stony Brook, New York 11794-3400, United States S Supporting Information *

ABSTRACT: Peptide-π-peptide triblock molecules can selfassemble into 1-D nanostructures with extensive hydrogen-bonding networks under appropriate pH conditions. These materials are of interest due to the embedded π-electron units that can facilitate energy and charge transport within biocompatible peptide matrices. Interactions among amino acid residues presented along these hydrogen-bonded structures lead to hierarchical bundling into larger fibrillar assemblies. This complicates the analysis of individual fibrils, an understanding of which is important for tailoring the functionality of the resulting nanomaterials. Appending large bulky groups onto the peptides should frustrate these bundling interactions and significantly alter the self-assembly behavior by restricting the formation of higher order assemblies. Here we evaluate the self-assembly behavior of peptide-π-peptide molecules appended with poly(alkyl ether) dendrons containing tetraethylene glycol functionalities. These dendrons render the peptide− dendron hybrid (PDH) molecules incapable of assembly under conditions that typically promote assembly of the parent peptides. However, 1,1,1,3,3,3-hexafluoro-2-propanol, a fluorinated alcohol known to denature proteins, triggers the aqueous assembly of the PDH molecules, thus yielding platelike nanostructures with narrow size distributions. This presents a new methodology for generating self-assembled nanostructures from peptide-π-peptide materials and provides an opportunity for orthogonal functionalization using motifs with varying degrees of hydrophilicity and functions. The effects of peptide sequence and dendron position along the peptide backbone on the assembly behavior were investigated using UV−vis, photoluminescence, and circular dichroism spectroscopies, and the morphologies of the resulting self-assembled nanostructures were investigated using transmission electron microscopy.

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INTRODUCTION A priori control over the self-assembly of peptide-based functional nanomaterials is an as-yet unrealized challenge. Understanding macromolecular self-assembly behavior is essential for generating functional nanomaterials with controlled morphologies. Peptides are notorious for their propensity to engage in higher-order interactions which lead to a variety of morphologies including tapes, fibrils, and biologically relevant insoluble amyloid-like plaques.1−8 In peptide β-sheets, this is driven in part by the supramolecular, intersheet interaction of amino acid residues that are presented above and below the plane of the sheet. One strategy to control the long-range supramolecular packing of peptide nanostructures involves the introduction of opposing attractive and repulsive intermolecular interactions to attenuate the otherwise uncontrollable aggregation of β-sheet nanostructures. Peptide−dendron hybrids (PDHs) have drawn attention due to the high degree of structural control possible in these materials.9−18 Both peptides and dendrons are built with precise placement of functionalities and with well-defined © XXXX American Chemical Society

molecular weights, leading to highly tailored, monodisperse macromolecules in which the dendritic groups adopt local conformations that greatly affect the assembly behavior of the peptide backbone. Although the dendrons themselves do not necessarily drive the assembly processes of the PDH molecules, they do lead to stabilization of the assembly when installed on α-helix forming peptides owing to intermolecular dendritic packing.9 With appropriate interdendritic distance along the peptide backbone, these molecules undergo a transformation to form β-sheets when the dendrons were cofacially packed. The assembly behavior of PDH materials is solvent sensitive, and dynamic interconversion between nanotubes and fibrils has been observed by changing solution pH or ionic strength.19 A transition from α-helix to β-sheet rich forms was observed upon changing the solvation media from 2,2,2-trifluoroethanol (TFE) to water.10 PDH molecules containing tetraethylene glycolReceived: April 20, 2017 Revised: June 10, 2017

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DOI: 10.1021/acs.macromol.7b00821 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules based dendrons appended to α-helix forming peptides form αhelical bundles, with the peptide assembly driving the presentation of the dendrons on the solvent exposed, exterior residues.11,12 This provides the capability to design materials in which dendrons with different functionalities and compatibilities can be held in specific supramolecular arrangements, encapsulating folded protein-like cores. While dendrons have been appended on α-helix forming peptides, PDH materials based on parent peptides with high propensities toward 1-D fibrillar nanostructure formation are not well established. Here, we describe the self-assembly behavior of dendronappended peptide-π-peptide molecules. We previously reported peptide-based semiconducting materials which form β-sheet rich 1-D nanostructures under acidic conditions.20,21 These peptide-π-peptide molecules have the ability to transport energy and charge through the peptidic nanostructures, making them interesting scaffolds for bioelectronics applications.22−26 The embedded π-electron unit shows variations in photophysical properties based on differences in its local environment arising from different solvation states that arise from molecularly dissolved or assembled states, including subtleties of different amino acid compositions.27−29 Controlling the nanostructure morphologies of these materials has been a challenge as polydisperse higher-order fibrils are observed (e.g., nanostructures with widths significantly larger than the molecular length),26,27 which complicate the analysis of individual supramolecular polymers. We previously demonstrated that incorporating the π-electron unit oblique to the peptide backbone can limit these higher-order interactions and yield materials that form nanostructures with widths comparable to their molecular lengths.29 We hypothesized that this was the result of steric crowding by the π-electron units that frustrated the association of neighboring peptide sheets. We propose an alternate strategy to restrict these higher-order interactions by establishing steric bulk in the peptide domain of the peptide-π-peptide molecules. These bulky pendent groups should also hinder the higher-order sheet interactions and provide another means to minimize fibrillar bundling. Therefore, we appended dendrons to these peptide-π-peptide molecules and evaluated the spectroscopic (UV−vis absorption, photoluminescence, and circular dichroism, common measurements used to verify the electronic delocalization among the embedded π-units necessary for electronic function) behavior of the π-electron unit embedded within the dendron-appended peptide ensembles and their morphological characteristics (transmission electron microscopy). We find that the selfassembly properties of the PDH materials vary significantly from their parent peptides (being sensitive to the solvation environment) and provide nanostructures with narrow size distributions.

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Terthiophene-5,5″]dicarboxylic acid (OT3 diacid) was synthesized following a previously reported procedure.30 Second-generation azidefunctionalized poly(alkyl ether) dendron, 2,2-bis(2,2-di[2-(2-[2-(2methoxyethoxy)ethoxy]ethoxy)ethoxymethyl]propyloxymethyl)ethyl azide was synthesized as previously reported.11 All other reagents and starting materials were used as received. General Solid Phase Peptide Synthesis (SPPS) Procedure. Peptides were synthesized via standard SPPS using Fmoc-protected amino acids, starting from Wang resin preloaded with the first amino acid (Wang-Val = 0.7 mmol/g; Wang-Glu(OtBu) = 0.5 mmol/g). Fmoc deprotection was carried out by treating the resin with a 20% piperidine solution in DMF for 2 min followed by filtering, adding fresh piperidine solution, and mixing for 10 min. The resin was washed thrice each with NMP, methanol, and dichloromethane (DCM). Amino acid couplings were performed by external activation of 3 equiv of the Fmoc-protected amino acid, relative to resin loading, with 2.9 equiv of HBTU and 10 equiv of diisopropylethylamine in NMP, which was then added to the peptide chamber and agitated for 60−90 min under ambient conditions. After coupling, the resin was again washed with NMP, methanol, and DCM. All couplings were monitored using the Kaiser test on a few dry resin beads. The procedure was repeated until the desired oligopeptide sequence was obtained. General N-Acylation Procedure of Peptides.30 Following completion of the oligopeptide sequence and deprotection of the last amino acid residue, the resin was treated with [2,2′:5′,2″terthiophene-5,5″]dicarboxylic acid (OT3 diacid, 1 equiv) which was activated by HBTU (0.97 equiv) and diisopropylethylamine (10 equiv) for 2 h. The solution was then filtered, and a blank coupling was carried out using HBTU (0.97 equiv) and DIPEA (10 equiv) for 2 h. After coupling, the resin was filtered and washed thrice with NMP, methanol, and DCM. General Workup and Cleavage Procedure of Peptides. The resin with completed sequence was treated with 9.5 mL of trifluoroacetic acid and 500 μL of water for 3 h. The solution was filtered from the resin beads, washed with DCM, and concentrated under reduced pressure. The crude π-electron conjugate was then precipitated from solution using cold diethyl ether and isolated through centrifugation. The resulting pellet was triturated twice with diethyl ether to yield crude product, which was dissolved in approximately 2 mL of water and 30 μL of ammonium hydroxide and lyophilized. Cu-Catalyzed 1,3-Dipolar Cycloaddition Reaction.11 To a solution of the azide-functionalized second-generation poly(alkyl ether) dendron (2.5 equiv), CuSO4·5H2O (0.4 equiv) and sodium L-ascorbate (1.75 equiv) in DMSO was added a 36−38 mmol/mL solution of pure peptide in Milli-Q water. The mixture was gently agitated at ambient conditions in a sealed container for 22−24 h, after which it was diluted using water, filtered, and purified using RP-HPLC. Reverse-Phase HPLC. Samples were prepared by dissolving lyophilized peptide solids in Millipore water containing a small amount of ammonium hydroxide (pH 8). Purification and analysis were performed using an Agilent SD1 PrepStar System with a Phenomenex C8 column (Luna 5 μm, 250 × 21.20 mm and 250 × 4.60 mm). The mobile phase consisted of ammonium formate aqueous buffer (1% (v/v), pH 8) and acetonitrile. Analytical traces were recorded at 390 nm using linear gradient of 10%−60% acetonitrile/buffer over 25 min at 1 mL/min. NMR Spectroscopy. 1H NMR spectra were obtained using a Bruker Avance 400 MHz FT NMR spectrometer and processed with Bruker Topspin 1.3. Peptide 1H NMR spectra were acquired using a presaturation pulse to suppress signal due to water. Chemical shifts are reported in parts per million relative to the residual protio solvent [D2O δ: 4.79 (1H)]. Electrospray Ionization Mass Spectrometry (ESI-MS). ESI-MS data were collected using a Thermo Finnigan LCQ Deca ion trap mass spectrometer in the negative ion mode. Samples were prepared using a 1:1 MeOH:water solution with 1% ammonium hydroxide. Matrix-Assisted Laser Desorption Ionization−Time-of-Flight (MALDI-ToF). MALDI-ToF spectra were acquired on a Bruker

EXPERIMENTAL DETAILS

Materials. N-Methylpyrrolidone (NMP), Wang resin loaded with Glu (OtBu) or Val, and 9-fluorenylmethoxycarbonyl (Fmoc) protected amino acids were obtained from Advanced ChemTech. Fmoc-L-propargylglycine (leading to the Gp) was purchased from ChemImpex Intl. O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU) was obtained from Oakwood Chemicals. N,N-Dimethylformamide (DMF), diisopropylethylamine, CuSO4·5H2O, sodium L-ascorbate, dimethyl sulfoxide (DMSO), methanol, and piperidine were purchased from Sigma-Aldrich. Solvents were degassed by sparging with nitrogen for 30 to 90 min and dried over 4 Å molecular sieves before use. Tetrakis(triphenylphosphine)palladium was obtained from Strem Chemicals. [2,2′:5′,2″B

DOI: 10.1021/acs.macromol.7b00821 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules Chart 1. Structure of Peptide−Dendron Hybrid (PDH) Molecules 1−4

Autoflex III Smartbeam instrument using α-cyano-4-hydroxycinnamic acid matrix. UV−Vis and Photoluminescence Spectroscopy. UV−vis spectra were obtained using a Varian Cary 50 Bio UV−vis spectrophotometer. Photoluminescence spectra were obtained using a PTi Photon S3 Technology International fluorometer with an Ushio xenon short arc lamp. Spectroscopic samples were prepared by diluting peptide stock solutions to the appropriate concentration (ca. 3 μM) in Millipore water to achieve an optical density near 0.1. The pH was then adjusted by adding 10 μL of 1 M KOH (basic, pH 10) followed by 20 μL of 1 M HCl (acidic, pH 1). The excitation wavelengths correspond to the λmax observed in the absorption spectra of the peptides. Quantum yields are reported relative to quinine sulfate in 1 N sulfuric acid (0.55). Circular Dichroism (CD). CD spectra were obtained using an AVIV circular dichroism spectrometer (Model 420). Spectroscopic samples were prepared by diluting the peptide stock solutions to the appropriate concentration (7−18 μM) in Millipore water. The pH was then adjusted by adding 10 μL of 1 M KOH (basic, pH 10) followed by 20 μL of 1 M HCl (acidic, pH 1). Transmission Electron Microscopy (TEM). Imaging was performed on a Philips EM 420 transmission electron microscope equipped with an SIS Megaview III CCD digital camera at an accelerating voltage of 100 kV. The samples were prepared by floating a 200 mesh copper grid coated with Formvar film (Electron Microscopy Sciences) onto a drop of 1 mg/mL solution of assembled peptide in water for 5 min at room temperature. The grid was washed with DI water, and the excess solution was wicked off by touching the side of the grid to filter paper. The sample was then stained using 2% uranyl acetate solution, washed with DI water and excess moisture was wicked off. The grid was allowed to dry in air before imaging. Dynamic Light Scattering (DLS). Samples were prepared by dissolving lyophilized peptide solids in Milli-Q water (ca. 3 μM). The pH of the samples was adjusted using 1 M HCl or 1 M KOH (pH 1 for acidic samples and pH 10 for basic samples). All measurements were recorded using a Malvern Instruments Zetasizer Nano-ZS90 at 25 °C.

sequence with hydrophilic or hydrophobic amino acid residues relative to the dendron and (ii) varying the position of the dendron along the backbone. Thus, hydrophilic glutamic acid residues (Glu, E) were used to provide solubility under alkaline pH and undergo protonation under acidic conditions to trigger the self-assembly process. Valine (Val, V) and alanine (Ala, A) residues were used for their known propensity toward forming β-sheets,31,32 while the L-propargylglycine (Gp) unit was incorporated as an alkyne handle to attach azide-functionalized tetraethylene glycol-based dendrons via Huisgen-type 1,3dipolar cycloadditions. All peptides were synthesized using solid-phase peptide synthesis followed by “on-resin” dimerization via amidation with [2,2′:5′,2″-terthiophene-5,5″]dicarboxylic acid, followed by reverse phase high-performance liquid chromatography (RPHPLC) purification, according to our previous report (see Supporting Information).30 As has been shown previously, neighboring residues on a peptide that forms a β-sheet-like structure will be presented on opposite faces of the resulting tape.33,34 Therefore, varying the peptide sequence from VEVAGp (1p) to EVEAGp (2p), as shown in Chart S1, would alter the placement of the reactive Gp residue (and thus the placement of the dendron) to be on a hydrophobic valinecontaining face or on a hydrophilic glutamic acid-containing face, respectively. To change the position of the dendron with respect to the embedded π-electron unit, VGpEVA (3p) and EGpVEA (4p) peptide sequences were utilized. The target PDH molecules 1−4 (Chart 1) were obtained by reacting the appropriate purified Gp-containing peptide with a secondgeneration poly(alkyl ether) dendron containing an azide functionality at the focal point using the copper-catalyzed 1,3dipolar cycloaddition reaction.11 The PDH molecules were purified via RP-HPLC to obtain analytically pure materials. All Gp-containing precursors and the resulting dendron-installed peptides were characterized by 1H NMR and ESI or MALDIToF mass spectrometry (Supporting Information). pH Driven Assembly Attempts. The peptides used in this study were designed to undergo pH-triggered self-assembly under acidic conditions arising from protonation of the glutamic acid residues. The process of self-assembly was monitored by recording signature changes in the spectroscopic behavior of the embedded π-electron unit under molecularly

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RESULTS AND DISCUSSION Peptide Design and Synthesis. Properties of peptide-πpeptide molecules are sensitive to the peptide composition as well as the structure of the π-electron unit. Here, terthiophene was used as a representative π-electron unit with established photophysical properties as an isolated chromophore and within exciton-coupled aggregates. The peptide backbones were designed to evaluate the effect of (i) varying the peptide C

DOI: 10.1021/acs.macromol.7b00821 Macromolecules XXXX, XXX, XXX−XXX

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indicate the presence of amorphous structures (Figure 2b and Figure S4). This is in agreement with the lack of self-assembled

dissolved (pH 10) or self-assembled (pH 1) conditions. We previously reported similar peptide-π-peptide materials that exhibit “H-like” aggregation of the π-electron unit upon selfassembly, characterized by a distinct blue-shift in the absorption spectrum and quenching and red-shifting in the PL spectrum upon acidification.26 Such behavior was also observed in the alkyne-containing parent peptides 1p−4p (Figure S1). Figure 1

Figure 2. Representative TEM images obtained from two separate batches of nanostructures of (a) peptide 3p and (b) PDH 3 generated at pH 1.

nanostructures being generated under these conditions with the dendron functionality presenting well-solvated, hydrophilic moieties hindering their formation. Under similar conditions, the parent peptides formed large aspect ratio 1-D nanostructures (Figure 2a and Figure S4). HFIP Driven Assembly. The relatively small changes observed upon acidification in the absorption, PL, and CD spectra of the π-electron unit in these PDH molecules as well as the similarity in DLS profiles are not indicative of the selfassembly process being triggered. Therefore, we infer that molecules 1−4 remain essentially molecularly dissolved, even under the acidic conditions which were sufficient to trigger the self-assembly of the parent peptides. A combination of hydrophilicity of the peptide backbone and the hydrophilic nature of the nonionic, oligo ethylene glycol dendrons (albeit less hydrophilic than the peptide itself) does not yield the appropriate hydrophilic−lipophilic balance required to form assemblies in aqueous media, at least at the concentrations used in this study. If this were the case, disruption of solvation of the dendron should increase the hydrophobicity of the PDH molecules and trigger the formation of self-assembled nanostructures at some critical point in the transition from good to poor solvation. This trigger point can be achieved by changing the pH of solution or by adding a nonsolvent to a molecularly dissolved solution. Addition of a solvent that preferentially interacts with only a part of the molecule, therefore disrupting its overall solvation in the good solvent that it was previously dissolved in can also trigger assembly. Indeed, dendron-triggered self-assembly of PDH molecules has been documented before, where transitions among α-helical and β-sheet-type assemblies were observed upon changing the solvation media from phosphate buffered saline (PBS) to a mixture of buffer and TFE.10 Fluorinated alcohols such as TFE and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) are known to be strong hydrogen-bond donors and have been employed to manipulate peptide−peptide interactions.9,10,35 The dendron functionality, being relatively more hydrophobic as compared to the peptide, likely interacts with the HFIP molecules forming water excluded clusters in solution and allows for peptide assembly to be triggered under appropriate pH conditions.9,36 In the case of PDH molecules 1−4, addition of HFIP led to a drastic change in the photophysical properties (Figure 3) and the aggregation state (DLS, Figure S3). When ca. 3% (v/v) HFIP was added to an acidified solution of the PDH molecules 1 and 2, quenching in both absorption and PL intensities was observed, but with no accompanying spectral shifts. In the case

Figure 1. UV−vis (solid lines) and PL (dashed lines) spectra (a, c) and CD spectra (b, d) recorded in aqueous media at 25 °C.

illustrates the typical photophysical spectra obtained when the parent peptides and PDH molecules (3p and 3 shown, respectively) were subjected to different pH conditions in aqueous media. Surprisingly, PDH molecules 1−4 did not show any meaningful difference in the photophysical behavior of the embedded terthiophene unit under basic or acidic conditions (Figure 1c). The absorption spectra (λmax at ca. 395 nm) and the PL spectra (λmax at ca. 450 and 470 nm) remained unchanged, and PL lifetimes were ca. 0.3 ns for all PDH molecules, under both basic and acidic conditions (Table S1). Furthermore, no significant changes were observed in the CD spectra under both basic and acidic conditions, with a notable absence of excitonic coupling under acidic conditions in the low energy (π−π*) spectral region (Figure 1d). The parent propargyl precursor peptides, 1p−4p, exhibit a strong bisignate feature in this spectral window, clearly indicating the persistence of chiral local environment in the nanostructures (Figure 1b and Figure S2). The absence of this feature in the PDH molecules may be indicative of either the molecules remaining in the dissolved state or the presence of disorganized aggregates lacking or with limited intermolecular electronic interactions, in agreement with the steady state and timeresolved spectroscopic measurements. DLS was used to probe the state of assembly under the different pH conditions. These data (Figure S3) indicate no apparent difference in sizes of the structures present under alkaline and acidic conditions, suggesting no appreciable changes in aggregation under acidic conditions. The bulky dendron groups likely hinder the formation of well-organized aggregates of the PDH molecules by disrupting the hydrophilic−lipophilic balance, owing to the hydrophilicity of the oligo ethylene glycol units. Accordingly, TEM images of the acidified solutions containing 1 mg/mL of PDH molecules 1−4 D

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DLS data indicate a difference in aggregation state of this peptide upon addition of HFIP to the acidic solution, while peptides 1p, 3p, and 4p do not show any significant variation (Figure S6). Addition of 2-propanol to the acidic solutions of 1−4 did not trigger assembly (Figure S7). This indicates that the assembly behavior is due to solvation by HFIP and not simply due to the addition of an alcoholic solvent. The PL decay profile of molecule 3 at pH 1 in the presence of HFIP fitted best to a biexponential decay profile with components of 3.63 ns (15%) and 0.53 ns (85%) (Table 1 and Table 1. Photophysical Properties of PDH Molecules 1−4 in the Presence of 3.2% (v/v) HFIPa absorption (λmax/nm)

Figure 3. UV−vis (solid lines) and PL (dashed lines) spectra of PDH molecules (a) 1, (b) 2, (c) 3, and (d) 4 generated at 25 °C.

of molecules 3 and 4, a blue-shift and quenching in absorption spectra and quenching accompanied by red-shifting of the PL spectra were observed. This suggests that the relative placement of dendrons and π-electron unit is critical to the type of aggregation observed upon assembly. The close proximity of the dendron to the π-electron core unit in the case of molecules 1 and 2 may cause steric hindrance to the electronic communication among neighboring units, hence not allowing for the classical “H-like” aggregation outcomes to be observed. Placing these dendritic units farther away from the core of the molecule, as in 3 and 4, relieves this strain and allows for such electronic communication among the terthiophene units, thus leading to the observed “H-like” aggregation behavior. In the case of PDH molecule 3 (Figure 1c), wherein the dendron is placed closer to the periphery of the molecule, the absorption spectra shifted from 398 nm at pH 10 and 400 nm at pH 1 to 384 nm (pH 1, 3.2% HFIP). The PL spectra that showed two peaks under basic as well as acidic conditions at 471 and 450 nm changed to a broad, featureless peak with λmax at 503 nm (pH 1, 3.2% HFIP). In the case of molecule 4, wherein the dendron position was same as in 3 but the peptide sequence was different, on the other hand, the absorption spectra shifted from 394 nm at pH 10 and 393 nm at pH 1 to 369 nm (pH 1, 3.2% HFIP). The PL spectra that had a peak at 469 nm with a broad shoulder at 451 nm under basic as well as acidic conditions changed to a broad, featureless peak with λmax at 519 nm (pH 1, 3.2% HFIP). When HFIP was added under basic conditions, the PL intensity was reduced, but the structure and position of the peak remained unchanged (Figure S5). Since the characteristic “H-like” aggregation behavior was not observed in this case, a combination of pH-triggered peptide aggregation and alteration of the dendron solvation sphere are both required to achieve the close electronic packing. As control experiments, we evaluated the effects of HFIP on the alkyne-containing peptides along with effects of 2-propanol on the formation of the PDH nanostructures. While peptides 3p and 4p maintained their aggregation state previously achieved under acidic conditions even in the presence of HFIP, peptides 1p and 2p underwent a change in spectral properties as seen from blue-shift in the absorption spectra and corresponding shift in the CD spectra as well as further quenching of PL intensity (Figures S1 and S2). Peptide 2p, in particular, showed large variations in the self-assembly behavior.

PL (λmax/nm)

PL lifetime (τ/ns)

molecule

pH 1

pH 1 + HFiP

pH 1

pH 1 + HFiP

1 2 3

398 397 400

404 394 384

450/470 450/471 451/473

453/470 453/476 503

0.29 0.30 0.29

4

393

369

451/469

519

0.28

0.24 0.41 3.63 (15%) + 0.53 (85%) 4.41 (33%) + 1.02 (67%)

pH 1

pH 1 + HFiP

a PL lifetimes were measured using λex = 375 nm and λem = 470 nm (for 1 and 2); 505 nm (for 3 and 4).

Figure S8), similar to that observed in the case of selfassembled nanostructures of peptide 3p (Table S1 and Figure S9). Similarly, in the case of molecule 4, a biexponential decay profile was observed with components at 4.41 ns (33%) and 1.02 ns (67%). Molecules 1 and 2, on the other hand, had PL lifetimes of 0.24 and 0.41 ns, respectively, similar to those observed under basic and acidic conditions in the absence of HFIP. A combination of these steady-state and lifetime measurements indicates electronic delocalization within the terthiophene domains in peptide−dendron hybrid nanostructures is achieved in the presence of HFIP in PDH molecules 3 and 4, but not in molecules 1 and 2. The CD spectra of molecules 1−4 are consistent with observations made from the steady state photophysical studies. As shown in Figure 4, molecules 1 and 2 did not show the characteristic bisignate features in the terthiophene absorption region (290−430 nm) under acidic conditions. Upon addition of ca. 3% (v/v) HFIP, a weak spectral feature emerged in the spectrum of 1, with multiple crossover points at 400, 332, and 255 nm. In the higher-energy spectral region, two minima at 244 and 220 nm were observed. This indicates that the local environment of the π-electron unit changed upon addition of the HFIP, and a net chirality was persistent in the nanostructures. PDH molecule 2 did not show the bisignate feature under similar conditions, suggesting a lack of electronic coupling even in the presence of HFIP. On the other hand, PDH molecule 3, which also did not show the characteristic bisignate feature at pH 1 (Figure 2c), showed a bisignate feature with negative Cotton effect (consistent with peptide 3p, Figure S2c) with a crossover point at 380 nm (close to its absorption λmax of 384 nm) upon addition of HFIP. The higher energy peak around 200 nm observed in the case of acidified solution disappeared, but the broad feature at ca. 225 nm was retained in position and magnitude. PDH molecule 4 also shows a bisignate feature with crossover point at 370 nm (UV− vis λmax = 369 nm), but the sign was reversed as compared to E

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large dispersion of nanostructure lengths. The average widths of nanostructures from 25 measurements were 8.55 ± 0.6 nm (peptide 1p), 6.02 ± 0.4 nm (peptide 2p), 10.39 ± 0.8 nm (peptide 3p), and 5.97 ± 0.3 nm (peptide 4p), and their average lengths varied between 300 and 900 nm. These widths are significantly larger than their corresponding molecular lengths, estimated using low level energy minimization calculations to be ca. 4 nm, and reflect the presence of hierarchical bundling of individual fibrils to form larger nanostructures. On the contrary, the nanostructures in all PDH molecules appeared to be short platelike nanostructures with rounded ends. The average nanostructure widths calculated from 25 measurements were 7.1 ± 0.8 nm (PDH 1), 6.9 ± 0.4 nm (PDH 2), 7.2 ± 0.9 nm (PDH 3), and 7.3 ± 0.9 nm (PDH 4), and the average lengths were 50−80 nm. The molecular length of these molecules was estimated to be ca. 5 nm. The difference in the nanostructure morphology caused by the addition of HFIP again indicates dendron-driven assembly variation in these molecules, consistent with spectroscopic measurements. The nature of these assemblies and the differences therein have to be investigated in further computational and experimental detail to understand the mechanism of the transitions observed here. However, the remarkable similarity in the dimensions and shapes of these structures, independent of the peptide sequence, suggests that the dendron functionality dominates the assembly formation and yields nanostructures with much narrower size distributions.

Figure 4. CD spectra of PDH molecules (a) 1, (b) 2, (c) 3, and (d) 4 generated at 25 °C.

the parent peptide 4p. This indicates that in both these cases an organized nanostructure with net chirality is obtained upon addition of HFIP, which allows for electronic communication among the terthiophene units within the nanostructure, supported by steady-state photophysical studies. The disappearance of the peak at ca. 200 nm and retention of that at 225 nm likely indicate a transition to predominantly β-sheet rich structures in molecule 3, whereas in the case of molecule 4, the peak at 219 nm is retained, suggesting the presence of βsheet-rich structures. While a typical β-sheet signal has a maximum in the 195−210 nm spectral window and a minimum at 216 nm, red-shifts in these peak positions have been associated with twisting of the β-sheet.3,37 TEM images of nanostructures generated by adding HFIP (3.2% (v/v)) to acidified solutions of PDH molecules 1−4 are as shown in Figure 5. These 1-D nanostructures were structurally significantly different from those observed from their respective peptide precursors (Figure S4). The parent peptides formed nanostructures with varying aspect ratios and

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CONCLUSIONS Appending dendrons to peptide-π-peptide molecules leads to the formation of PDH molecules with drastically different selfassembly properties and yields nanostructures with narrow size dispersion. Self-assembly of PDH molecules was only triggered by addition of HFIP to acidic aqueous solutions of these molecules, while the parent peptides self-assembled in aqueous solutions under acidic conditions. The difference in solvation properties of the dendrons and the peptide backbones altered the overall hydrophilic−lipophilic balance in the PDH molecules, only allowing self-assembly when the oligo ethylene glycol containing poly(alkyl ether) dendrons were solvated by HFIP. This presents a new methodology for generating selfassembled nanostructures from peptide-π-peptide materials and provides an opportunity for orthogonal functionalization using motifs with varying degrees of hydrophilicity. We found that subtle variations in the peptide sequence led to changes in the local environment of the π-electron unit. Location of the dendron on the peptide backbone was also critical for electronic communication among the core units within the self-assembled nanostructures. While “H-like” aggregation was not observed when the dendron was adjacent to the π-electron unit, it was observed when the dendron was near the exterior of the molecule. In all cases, the nanostructure widths were ca. 7 nm, indicating a significant degree of control in this size dimension. While the lengths varied between 50 and 80 nm, this variance is significantly lower than that observed in the case of the parent peptides. The PDH molecules described here constitute a class of peptide-based materials with controlled nanostructure morphologies. This design principle for controlling the dimensions of nanostructures can be used as a broader methodology for generating size controlled functional biomaterials.

Figure 5. Representative TEM images obtained from two separate batches of nanostructures of PDH molecules (a) 1, (b) 2, (c) 3, and (d) 4 generated at pH 1 in the presence of 3.2% (v/v) HFiP. F

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.7b00821. Synthetic procedures, characterization details including 1 H NMR, ESI-MS, MALDI, and HPLC, photophysical data for peptides, DLS data for peptides and PDH molecules, and Table S1 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (J.D.T.). ORCID

John D. Tovar: 0000-0002-9650-2210 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Johns Hopkins University and the National Science Foundation (DMR-1407493) for generous support and Professor Jonathan G. Rudick (Stony Brook University) for supplying the poly(alkyl ether) dendron and for helpful discussion. We also thank the Center for Molecular Biophysics (JHU) for the use of the circular dichroism spectrometer and Prof. Hai-Quan Mao for the use of Zetasizer for dynamic light scattering measurements.

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REFERENCES

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DOI: 10.1021/acs.macromol.7b00821 Macromolecules XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.macromol.7b00821 Macromolecules XXXX, XXX, XXX−XXX