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Effect of Glycine Substitution on FmocDiphenylalanine Self-Assembly and Gelation Properties Claire Tang,†,‡ Rein V. Ulijn,§ and Alberto Saiani*,† †
School of Materials, The University of Manchester, Grosvenor Street, Manchester M1 7HS, United Kingdom Manchester Interdisciplinary Biocentre (MIB), The University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom § WestCHEM, Department of Pure & Applied Chemistry, University of Strathclyde, Thomas Graham Building, 295 Cathedral Street, Glasgow G1 1XL, United Kingdom ‡
bS Supporting Information ABSTRACT:
We have investigated the self-assembly behavior of fluorenyl-9-methoxycarbonyl (Fmoc)FG, FmocGG, and FmocGF and compared it to that of FmocFF using potentiometry, fluorescence and infrared spectroscopy, transmission electron microscopy, wide-angle X-ray scattering, and oscillatory rheometry. Titration experiments revealed a substantially shifted apparent pKa transition for FmocFG, FmocGG, and FmocGF. The apparent pKa values observed correlated with the hydrophobicity (log P) of the Fmocdipeptide molecules. FmocGG and FmocGF were found to self-assemble only in their protonated form (below their apparent pKa), while FmocFG formed self-assembled structures above and below its apparent pKa. FmocGG and FmocFG were found to form hydrogels below their apparent pKa transitions in agreement with the entangled fibers morphologies revealed by TEM. Unlike FmocFF and FmocGG, FmocFG showed unusual gelation behavior as gels were found to form upon heating. FmocGF formed precipitates instead of a hydrogel below its apparent pKa in agreement with the formation of micrometer scale sheetlike structures observed by TEM. The fact that all four Fmocdipeptides were found to self-assemble suggests that the main driving force behind the self-assembly process is a combination of the hydrophobic and ππ interactions of the fluorenyl moieties with a secondary role for hydrogen bonding of the peptidic components. The nature of the peptidic tail was found to have a pronounced effect on the type of self-assembled structure formed. This work indicates that the substitution of phenylalanine by glycine significantly impacts on the mode of assembly and illustrates the versatility of aromatic peptide amphiphiles in the formation of structurally diverse nanostructures.
’ INTRODUCTION In the last two decades, various peptide-based systems capable of self-assembly into structures with micro- and nanometer dimensions have been developed.15 These materials are of particular interest because of their potential use in a range of applications, including supramolecular electronics,6,7 photonics,8 and other areas of bionanotechnology.912 A number of these structures have also shown the ability to support cell culture, demonstrating their potential for tissue engineering applications.13,14 The structures formed are stabilized through a combination of hydrogen bonding between peptide backbones, hydrophilic and hydrophobic cooperative effects of amphiphile molecules, or π-stacking of aromatic moieties.15,16 A relatively r 2011 American Chemical Society
recent development has been the use of short peptides (generally between two and six amino acids) appended with aromatic ligands, which are thought to self-assemble via a combination of aromatic π-stacking interactions and H-bonding.17 Efforts are underway to develop an understanding of the molecular and supramolecular structures of these materials. It is clear that both the self-assembly pathway and the molecular structure have dramatic effects on the properties of the materials formed.18
Received: June 6, 2011 Revised: October 10, 2011 Published: October 13, 2011 14438
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Figure 1. Equilibrium between Fmocdipeptide neutral (acid) and ionized (conjugated base) forms and notation.
The self-assembly pathways exploited for aromatic peptide amphiphiles include enzymatic reactions1921 or changes in environmental conditions such as change in temperature,22 dilution from organic solvents into water,11,2327 or sequential pH change.28,29 Solubilization of the gelator molecules constitutes a key step to ensure formation of homogeneous gels and may be challenging due to the amphiphilic character of the peptide derivatives. The most common approach used to control the selfassembly of aromatic peptide amphiphiles involves solubilization at high pH (ensuring deprotonation of the C-terminal carboxylic acid), followed by a decrease in pH until gelation is observed (which is accommodated by the protonation of this acid). To achieve this, pH is adjusted either using sugar precursors which generate acids through in situ hydrolysis29,30 or by addition of hydrochloric acid (HCl).28,31 The latter method can lead to formation of acidic pockets composed of fibrillar networks constrained in domains within the gel samples.32 Complete dispersion of these domains is difficult by simply vortexing the gels but can be achieved by heating to dissolve the kinetically trapped aggregates. Thus, we previously reported on an optimized sample preparation method, involving stepwise addition of acid followed by a heat/cool cycle that led to reproducible and more homogeneous hydrogels that could subsequently be characterized. In addition to the self-assembly pathway, the nature of the nanostructures formed by self-assembly of aromatic peptide amphiphiles is controlled by molecular structure and peptide sequence. The most widely studied examples are the fluorenyl-9methoxycarbonyl (Fmoc)-functionalized peptides covering a range of hydrophobicities. Under specific conditions and depending on the peptide sequence, nanostructured materials with different morphological, physical, and mechanical properties could be formed. For example, FmocFF gave rise to ∼3 nm fibrils that laterally self-assembled into tapelike structures, FmocL3 formed ∼20 nm tubular structures,7 and FmocL5 formed microscopic sheetlike structures.33 Not all aromatic peptide amphiphiles lead to hydrogel formation. For instance, FmocFG formed hydrogels, whereas FmocGF formed precipitates under the test conditions.28 Gel formation was observed for both FmocVLK(Boc) and FmocK(Boc)LV—in which the lysine side chain was protected by tert-butyloxycarbonyl (Boc), a nonaromatic protecting group, to prevent any interferences arising from charges and additional aromatic moieties. These peptide derivatives exhibited distinct self-assembling behaviors and conformational differences.34 Modifying the order of the amino acid sequence therefore clearly alters the structural properties of the systems, which had a substantial impact on self-assembly.35
To gain further insights into the role of molecular structure and amino acid sequence in molecular self-assembly, we herein focus on Fmocdipeptides based on combinations of phenylalanine and glycine. The pH dependence of FmocFG, Fmoc GG, and FmocGF (Figure 1) self-assembly processes will be investigated and compared to that of the FmocFF system.32 We show that in all cases self-assembly of the studied Fmoc dipeptides results in a suppressed ionization leading to pKa shifts with respect to the theoretical pKath, suggesting that the terminal carboxylic acid is positioned in a hydrophobic environment upon self-assembly. The pKa of each system was found to correspond to significant structural transitions with distinct microscopic and molecular arrangements depending on the amino acid sequence. The transitions were studied using potentiometry, fluorescence and infrared spectroscopy, transmission electron microscopy, X-ray scattering, and shear rheometry. Overall, this paper provides further understanding of the self-assembly mechanism of aromatic peptide amphiphiles.
’ MATERIALS AND METHODS Materials. All Fmocdipeptides were purchased from Bachem (Bubendorf, Switzerland) and used without further purification. The purity of the compounds was verified by HPLC (>98%) and mass spectrometry. HPLC grade water was purchased from Merck and deuterated water (99.9 atom % D) from Sigma-Aldrich. Potentiometry. pH measurements were performed using a Hanna Instruments pH210 pH-meter equipped with a Hamilton Spintrode pH-probe (reference system, Ag/AgCl; electrolyte, 3 M KCl; diaphragm, ceramic; sensitivity, 58 mV/pH unit at 25 °C). The pH-meter was calibrated before each experiment to check the response of the electrode with two buffer solutions purchased from Fisher Scientific: phthalate pH 4.01 and phosphate pH 7.01 buffer solutions. “Titration” Experiments. For 5, 10, and 20 mmol L1 samples, the required amount of Fmocdipeptide was directly suspended into 2 mL of HPLC grade water. Sodium hydroxide (0.5 M) was added to the aqueous suspensions of Fmocdipeptide until pH 10.5 was reached (e.g., ∼55 μL for 10 mmol L1 sample). The samples were vortexed and sonicated (VWR ultrasonicator bath, 30 W) for 1 min until fully dissolved. For 1 mmol L1 samples, 5 mmol L1 samples at pH 10.5 were used as stock solutions and diluted to the desired concentration in a final volume of 2 mL. The pH of the final solutions was then adjusted to pH 10.5. The titration experiments were performed by stepwise addition of small volumes of diluted HCl (0.085 M; 260 μL depending on pH and system), up to a total added 14439
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Langmuir volume of 750 μL. After each addition the samples were heated to 7580 °C for 1 min, vortexed, sonicated, and subsequently cooled back to room temperature using a water bath. pH values were recorded before and after heating of the samples. The samples were deemed fully mixed when no significant difference ((0.3 pH unit) was observed between two pH measurements. Due to the strong and constant agitation applied, samples were liquid at all times during the titration experiments. As a control, water was also titrated using the same methodology described above; that is, NaOH was added to the water in order to bring the pH of the solution to 10.5, and then HCl was added stepwise. Sample Preparation. Depending on the desired concentration, the required amount of Fmocdipeptide was suspended into 2 mL of HPLC grade water. Sodium hydroxide (0.5 M) was gradually added to the aqueous suspensions of Fmocdipeptide until pH 10.5 was reached (∼55 μL for 10 mmol L1 sample). The samples were vortexed and sonicated for 1 min to fully dissolve the short peptide amphiphiles. Depending on the concentration and on the target pH, a required volume of dilute hydrochloric acid (0.085 M) was then added dropwise while the solution was vortexed and sonicated until the target pH was obtained. Next, the samples were heated to 7580 °C until fully dissolved (2.5, 1.0, and 2.0 min for FmocFG, FmocGG, and FmocGF, respectively) and homogenized. The samples were subsequently cooled and maintained at 4 °C for ∼12 h (overnight) to promote or maintain gelation. To allow comparison between the studied samples, all were investigated after this same aging time. Reported pH values were those measured after storage. They were found to be identical to the pH values measured before heating within (0.3 units. Fluorescence Spectroscopy. Fluorimetry experiments were undertaken using a Perkin-Elmer LS55 luminescence spectrometer equipped with a Julabo F25 temperature control device. After their preparation, the samples were transferred into PMMA disposable cuvettes of 1 cm thickness (Fisher Scientific) and kept at 4 °C overnight. Emission and excitation slit widths were set at 3 and 10 nm, respectively. Emission spectra (excitation at 265 nm) were acquired at 25 °C in the 300 600 nm range with a scan speed of 300 nm min1 using FL WinLab software. Changes in the pH conditions and therefore in the self-assembled structures formed are likely to modify the refractive index of the samples and the quenching effect resulting from self-assembly. These effects would have a direct effect on the intensity of the transmitted light and hence on the emission peaks’ intensity. For this reason, fluorescence spectra were all normalized as a function of their respective maximum emission peak. The unnormalized spectra are presented in the Supporting Information. Fourier Transform Infrared Spectroscopy (FTIR). Multiple bounce attenuated total reflectance (ATR) FTIR experiments were undertaken using samples prepared in deuterated water. Spectra were recorded on a Thermo Nicolet 5700 spectrometer equipped with a trough plate comprising of a zinc selenide crystal, which permitted 12 reflections with a 45° angle of incidence. The samples were spread directly on the surface of the trough plate. Spectra were acquired in the 4000400 cm1 range with a resolution of 4 cm1 over 128 scans. The deuterated water spectrum was used as background and subtracted from all spectra (software used: Omnic version 7.2, Thermo Electron Corporation). Mechanical Properties. Mechanical properties were assessed using a stress-controlled rheometer (Bohlin C-VOR) equipped with a Peltier device (Bohlin Instruments) to control temperature
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using a parallel-plate geometry (40 mm diameter). To ensure the measurements were made in the linear regime, amplitude sweeps were performed and showed no variation in G0 and G00 up to a strain of 1%. The dynamic moduli of the hydrogel were measured as a function of frequency in the range 0.01100 rad s1 with a strain of 1%. To keep the sample hydrated, a solvent trap was used and the atmosphere within the sample chamber was saturated with water. Unless otherwise stated the experiments were performed at 25 °C and repeated at least three times (each time on new samples) to ensure reproducibility. The standard deviation of multiple experiments is represented by error bars in the dynamic frequency sweeps. Transmission Electron Microscopy (TEM) and Image Analysis. A carbon coated copper grid (400 mesh, Agar Scientific) was glow discharged for 30 s and then placed on 10 μL of sample for 15 s. After blotting on Whatman 50 filter paper, the loaded grid was washed in double distilled water for 30 s and blotted. The sample was then stained with 10 μL of 2% (w/v) uranyl acetate (centrifugated for 5 min beforehand) for 1 min and blotted for 10 s. Data were collected at high vacuum on a JEOL 1220 transmission electron microscope connected to a high resolution (up to 3 Å/pixel resolution) Gatan Orius CCD camera. Wide Angle X-ray Scattering (WAXS). Wide angle X-ray scattering experiments were conducted using 10 mmol L1 samples. Wet samples were spread onto glass slides as thin films and allowed to air-dry for 48 h prior to data collection. Experiments were performed on a Philips X’Pert diffractometer equipped with a copper source (wavelength of 1.54 Å), applying scans from Bragg angles of 1°33°. The glass slide spectrum was used as background and subtracted from all spectra.
’ RESULTS AND DISCUSSION Titrations. FmocFF was previously shown to exhibit two dramatic pKa shifts that were found to be related to significant structural transitions, formation of fibrils, and their side-by-side alignment to form tapelike structures that eventually precipitate.32 Titration experiments were carried out in the same way on the FmocFG, FmocGG, and FmocGF systems at concentrations of 1, 5, 10, and 20 mmol L1 (Figure 2AC). The theoretical pKa values of the Fmocdipeptides were calculated using SPARC web calculator (http://archemcalc.com/sparc). All experiments started at pH 10.5 following addition of NaOH. The samples’ pH variations were then recorded as a function of added HCl. The samples were heated after each addition of acid. Due to the vigorous and constant agitation applied, samples were liquid at all times during the titration experiments, ensuring the absence of pH measurement artifacts related to gel formation and clogging of the pH meter membrane. Water was also titrated in the same way as a control. At pH 10.5 most of the FmocFG molecules were expected to be ionized. At 10 and 20 mmol L1 samples were slightly cloudy under these pH conditions and became clear upon heating. For all concentrations investigated, as HCl was added to the mixtures, the pH gradually decreased (Figure 2 A) while the samples became slightly cloudy. A first transition was observed at pH of ∼7.3 for the 10 and 20 mmol L1 samples, with transitions occurring at a pH of about 6.3 and 5.5 for 5 and 1 mmol L1 samples, respectively. Once the transition was completed, the pH dropped again and the samples turned more turbid with addition of HCl. From pH 3.8 onward a white precipitate started to appear 14440
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Figure 2. Titration curves (pH vs moles of added HCl) of water and (A) FmocFG, (B) FmocGG, and (C) FmocGF samples at 1, 5, 10, and 20 mmol L1. nHCl (moles of HCl added corrected for moles of HCl needed to titrate water) vs nFmocdipeptideCOOH (moles of Fmocdipeptide present in the sample) for the 1, 5, 10, and 20 mmol L1 samples of (D) FmocFG at pH values of 8.5 and 5.0, (E) FmocGG at pH values of 6.5 and 3.8, and (F) FmocGF at pH values of 8.5 and 5.0. Inset: Degree of ionization α derived from the slope of the fitted linear curves.
at 1 mmol L1, whereas at 5 mmol L1 and above the samples became more viscous and started to aggregate. As the pH was decreased further, precipitation was observed for all the samples, and all the titration curves were found to merge with the water titration curve. At all concentrations tested FmocGG amphiphiles were fully dissolved at pH 10.5. At 1 mmol L1, the sample remained clear over the range of pH studied and no transition was observed. At 5 mmol L1 and above, the samples were all clear at high pH and became slightly cloudy upon addition of HCl as the solutions’ pH was gradually lowered. pH transitions occurred at values of 4.8,
4.5, and 4.0 for 20, 10, and 5 mmol L1 samples, respectively (Figure 2 B). Toward the end of the transition, samples became more viscous and turbid. At low pH (
