pubs.acs.org/Langmuir © 2009 American Chemical Society
Fmoc-Diphenylalanine Self-Assembly Mechanism Induces Apparent pKa Shifts Claire Tang,†,‡ Andrew M. Smith,†,‡ Richard F. Collins,‡ 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, and §WestCHEM, University of Strathclyde, Thomas Graham Building, 295 Cathedral Street, Glasgow G1 1XL, United Kingdom Received February 23, 2009. Revised Manuscript Received May 20, 2009
We report the effect of pH on the self-assembly process of Fmoc-diphenylalanine (Fmoc-FF) into fibrils consisting of antiparallel β-sheets, and show that it results in two apparent pKa shifts of ∼6.4 and ∼2.2 pH units above the theoretical pKa (3.5). Using Fourier transform infrared (FTIR) spectroscopy, transmission electron microscopy (TEM), wide angle X-ray scattering (WAXS), and oscillatory rheology, these two transitions were shown to coincide with significant structural changes. An entangled network of flexible fibrils forming a weak hydrogel dominates at high pH, while nongelling flat rigid ribbons form at intermediate pH values. Overall, this study provides further understanding of the self-assembly mechanism of aromatic short peptide derivatives.
Introduction Over the past decade, significant efforts have been made to develop a new generation of biomaterials based on the selfassembly of peptides and their derivatives.1,2 By exploiting the spontaneous or induced molecular arrangement of peptides and their derivatives in aqueous solutions, nanostructured hydrogels can be formed under specific conditions.3-7 These highly hydrated scaffolds have potential applications in tissue engineering,5,8 3D cell culture,9-11 and templating.12,13 Such peptide based biomaterials exploit known biological architectures such as the β-sheet and are usually composed of peptide components exceeding 10 amino acids in length. A relatively new class of hydrogel *Corresponding author. E-mail:
[email protected]. (1) Lehn, J.-M. Supramolecular Chemistry: Concepts and Perspectives; VCH Press: New York, 1995. (2) Whitesides, G. M.; Grzybowski, B. Science 2002, 295, 2418–2421. (3) Zhang, S. Nat. Biotechnol. 2003, 21, 1171–1178. (4) Mart, R. J.; Osborne, R. D.; Stevens, M. M.; Ulijn, R. V. Soft Matter 2006, 2, 822–835. (5) Haines-Butterick, L.; Rajagopal, K.; Branco, M.; Salick, D.; Rughani, R.; Pilarz, M.; Lamm, M. S.; Pochan, D. J.; Schneider, J. P. Proc. Natl. Acad. Sci. U.S. A. 2007, 104, 7791–7796. (6) Tsonchev, S.; Niece, K. L.; Schatz, G. C.; Ratner, M. A.; Stupp, S. I. J. Phys. Chem. B 2008, 112, 441–447. (7) Saiani, A.; Mohammed, A.; Frielinghaus, H.; Collins, R.; Hodson, N.; Kielty, C. M.; Sherratt, M. J.; Miller, A. F. Soft Matter 2009, 5, 193–202. (8) Lee, K. Y.; Mooney, D. J. Chem. Rev. 2001, 101, 1869–1880. (9) Beniash, E.; Hartgerink, J. D.; Storrie, H.; Stendahl, J. C.; Stupp, S. I. Acta Biomater. 2005, 1, 387–397. (10) Jayawarna, V.; Richardson, S. M.; Hirst, A. R.; Hodson, N. W.; Saiani, A.; Gough, J. E.; Ulijn, R. V. Acta Biomater. 2009, 5, 934–943. (11) Zhou, M.; Smith, A. M.; Das, A. K.; Hodson, N. W.; Collins, R. F.; Ulijn, R. V.; Gough, J. E. Biomaterials 2009, 30, 2523–2530. (12) Hartgerink, J. D.; Beniash, E.; Stupp, S. I. Science 2001, 294, 1684–1688. (13) Reches, M.; Gazit, E. Science 2003, 300, 625–627. (14) Vegners, R.; Shestakova, I.; Kalvinsh, I.; Ezzell, R. M.; Janmey, P. A. J. Pept. Sci. 1995, 1, 371–378. (15) Jayawarna, V.; Ali, M.; Jowitt, T. A.; Miller, A. F.; Saiani, A.; Gough, J. E.; Ulijn, R. V. Adv. Mater. 2006, 18, 611–614. (16) Mahler, A.; Reches, M.; Rechter, M.; Cohen, S.; Gazit, E. Adv. Mater. 2006, 18, 1365–1370. (17) Schnepp, Z. A. C.; Gonzalez-McQuire, R.; Mann, S. Adv. Mater. 2006, 18, 1869–1872. (18) Toledano, S.; Williams, R. J.; Jayawarna, V.; Ulijn, R. V. J. Am. Chem. Soc. 2006, 128, 1070–1071.
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scaffolds formed from the self-assembly of much shorter peptides modified with aromatic ligands has recently been developed.14-20 The aromatic moieties play a key role in the self-assembly process through π-π interactions, while the peptide components are stabilized via hydrogen bonding. The main focus of our work to date has been to characterize the self-assembling behavior of dipeptides possessing an N-terminal fluorenyl-9-methoxycarbonyl (Fmoc) group. The overall aim is to establish design rules allowing the preparation of biomaterials, in particular hydrogels, with tailored properties. We have recently shown that Fmoc-diphenylalanine (Fmoc-FF) can form hydrogels at physiological pH on which cells have been shown to grow and proliferate.10,11,15 We subsequently proposed a molecular model for the self-assembled structure formed by this system.19 Fmoc-FF peptides were shown to form antiparallel β-sheets that self-assemble laterally through π-π stacking of the fluorenyl groups and phenyl rings (phenylalanine side chain) present on the edge of the β-sheets. Due to the natural twist of the β-sheets, four sheets come together to form a cylindrical fibril with an external diameter of ∼3.0 nm. These fibrils were then shown to further self-assemble laterally, forming large flat ribbons under specific pH conditions. For a detailed discussion of the model, we refer the reader to ref 19. The R-carboxylic acid groups of amino acids are in their anionic form at neutral pH. N-protected nonpolar peptides have pKa values of about 3.5.21 (SPARC web calculator to be found at http://ibmlc2.chem.uga.edu/sparc.) In dilute solutions, one would therefore expect terminal carboxylic acid groups to be ionized at pH values higher than 3.5 and the molecules to be negatively charged. Self-assembly would presumably be unfavored at pH higher than 3.5 due to electrostatic repulsion between the negatively charged molecules. Indeed, a (19) Smith, A. M.; Williams, R. J.; Tang, C.; Coppo, P.; Collins, R. F.; Turner, M. L.; Saiani, A.; Ulijn, R. V. Adv. Mater. 2008, 20, 37–41. (20) Zhang, Y.; Gu, H.; Yang, Z.; Xu, B. J. Am. Chem. Soc. 2003, 125, 13680– 13681. (21) Ulijn, R. V.; Moore, B. D.; Janssen, A. E. M.; Halling, P. J. J. Chem. Soc., Perkin Trans. 2 2002, 1024–1028.
Published on Web 06/19/2009
DOI: 10.1021/la900653q
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Figure 1. Equilibrium between the dipeptide neutral acidic form (Fmoc-FF) and its basic ionized form (Fmoc-FF-).
number of Fmoc-peptides only showed self-assembly at low pH.15 However, Fmoc-FF (Figure 1), a highly hydrophobic peptide derivative, was found to self-assemble at neutral pH.19 It is known that pKa can shift dramatically in protein and peptide self-assembly, especially in hydrophobic environments.22 For example, a shift of 6.1 has been observed for aspartic acid side chain carboxylic acid in a family of protein based polymers composed of the repeat of polypentapeptides.23 pKa shifts have also been encountered in fatty acids. For instance, palmitic acid (C16) revealed a pKa shift of up to 3.9 which is related to the formation of foam by these molecules.24 Here we show that Fmoc-FF self-assembly results in a suppressed ionization leading to dramatic pKa shifts related to significant structural transitions.
Materials and Methods Materials. Fmoc-FF peptides were purchased from Bachem (Bubendorf, Switzerland) and used without any further purification. The purity of the compound was checked 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 (Ag/AgCl reference system, 3 M KCl electrolyte, ceramic diaphragm, 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. Depending on the desired concentration, the required amount of Fmoc-FF was suspended into 2 mL of HPLC grade water. Sodium hydroxide (0.5 M, e.g., ∼55 μL for 10 mmol L-1 sample) was added to the aqueous suspensions of Fmoc-FF until pH 10.5 was reached. The samples were vortexed and sonicated (VWR ultrasonicator bath, 30 W) for 1 min to fully dissolve the modified peptide. To ensure that these conditions did not lead to significant hydrolysis of the carbamate and loss of the fluorenyl moiety, HPLC was used to estimate the cleavage percentage. After 10 min at pH 10.5, less than 1% cleavage was observed. The “titration” experiments were performed by stepwise addition of small volumes of diluted HCl (0.085 M). After each addition the samples were heated to 75-80 °C, vortexed, sonicated, and subsequently cooled back to room temperature using a water bath. pH values were recorded before and after heating the samples. The samples were deemed fully mixed when no significant difference ((0.3 pH unit) was observed between the 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 (22) Stryer, L. Biochemistry, 4th ed.; Freeman: New York, 1995. (23) Urry, D. W.; Peng, S. Q.; Parker, T. M.; Gowda, D. C.; Harris, R. D. Angew. Chem., Int. Ed. Engl. 1993, 32, 1440–1442. (24) Kanicky, J. R.; Poniatowski, A. F.; Mehta, N. R.; Shah, D. O. Langmuir 2000, 16, 172–177.
9448 DOI: 10.1021/la900653q
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 Fmoc-FF was suspended into 2 mL of HPLC grade water. Sodium hydroxide (0.5 M) was added to the aqueous suspensions of Fmoc-FF until pH 10.5 was reached (∼55 μL for 10 mmol L-1 sample). The samples were vortexed and sonicated for 1 min to fully dissolve the modified peptide. 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 75-80 °C for 1 min and homogenized. The samples were subsequently cooled and maintained at 4 °C for ∼12 h (overnight) to promote gelation. 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. Fourier Transform Infrared (FTIR) Spectroscopy. Multiple bounce attenuated total reflectance FTIR experiments were undertaken using samples prepared in deuterated water. Spectra were recorded on a Thermo Nicolet 5700 spectrometer equipped with a smart ark trough plate comprising a zinc selenide crystal. The samples were spread directly on the surface of the trough plate. Spectra were acquired in the 4000-400 cm-1 range with a resolution of 4 cm-1 over 128 scans. The deuterated water spectrum was used as background and subtracted from all spectra.
Transmission Electron Microscopy (TEM) and Image Analysis. A glow discharged carbon coated copper grid (400 mesh,
Agar Scientific) was placed on 10 μL of sample for 30 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 on a Tecnai 10 transmission electron microscope operating at 100 keV onto Kodak SO-163 films at a calibrated magnification of 43 200. Micrographs were scanned at 1600 dpi using a UMAX 2000 transmission scanner, giving a specimen level increment of 3.66 A˚ pixel-1. Data were converted to LINUX format and were then analyzed using EMAN.25 Using BOXER, a total of 550 straight and nonoverlapping fiber areas were interactively selected and a projection average was created using reference free alignment with a maximum shift value of (4 pixels. Wide Angle X-ray Scattering (WAXS). Wide angle X-ray scattering experiments were conducted using 10 mmol L-1 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 A˚). Mechanical Properties. Mechanical properties were assessed using a strain-controlled rheometer (Bohlin C-CVO) equipped with a Peltier device to control temperature. A parallel-plate geometry was used with a diameter of 40 mm. 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 (25) Ludtke, S. J.; Baldwin, P. R.; Chiu, W. J. Struct. Biol. 1999, 128, 82–97.
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Figure 2. “Titration” curves (pH versus moles of added HCl) of water and Fmoc-FF samples at 0.01, 0.1, 1, 5, and 10 mmol L-1. as a function of frequency in the range 10-2-102 rad s-1 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. The experiments were performed at 25 °C and repeated at least three times (each time on new samples) to ensure reproducibility.
Results and Discussion Hydrogels of aromatic short peptide derivatives were previously formed by sequential pH change,15 dilution from fluorinated solvents,16 as well as enzymatic hydrolysis of corresponding esters.26 Here we focus on the pH dependence of the Fmoc-FF self-assembly process using a modified sample preparation method, described above, in order to accurately control the pH of the samples. This new method enabled us to improve the homogeneity of the samples and to reduce the formation of kinetically trapped aggregates. To investigate the ionization behavior of Fmoc-FF, we performed “titration” experiments by measuring the variation of the samples’ pH as a function of added HCl. The experiments were carried out on samples at 0.01, 0.1, 1, 5, and 10 mmol L-1 concentrations. Water was also titrated in the same way as a control (Figure 2). At 0.01 mmol L-1, Fmoc-FF was found to be soluble at all pH values. The titration curve obtained at this concentration was similar to that of water. At 0.1 mmol L-1, Fmoc-FF was found to be soluble at pH values above 7, while at lower pH values the peptide was found to precipitate. The overall pH variation as a function of added HCl was in this case too similar to that of water. For samples prepared at 1 mmol L-1 and above, Fmoc-FF was found to be fully soluble at high pH (g10.5) only. At all concentrations tested, the peptide derivative was fully dissolved at pH 10.5. At this pH, most of the Fmoc-FF molecules are expected to be ionized. When HCl was added to the solutions, the pH gradually dropped for all the samples (Figure 2). Once a pH of ∼9.2 was reached, the pH of the 5 and 10 mmol L-1 samples was found to increase slightly to 9.5-10.2 and in the case of the 10 mmol L-1 sample to become constant. For the 1 mmol L-1 sample, no increase in pH was observed and a transition was observed at a slightly lower pH of ∼8.6. As the samples went through this first transition, they became slightly cloudy. Once the first transition was complete, the pH of all samples was found to decrease again with addition of HCl. The samples became more (26) Das, A. K.; Collins, R. F.; Ulijn, R. V. Small 2008, 4, 279–287.
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Figure 3. nHCl (moles of HCl added corrected for moles of HCl needed to titrate water) versus nFmoc-FF (moles of peptide present in the sample) for the 1, 5, and 10 mmol L-1 samples at pH values of 8.9, 7.0, and 4.3. Inset: Degree of ionization, R, derived from the slope of the fitted linear curves (for more details, see the text).
Figure 4. FTIR spectra of Fmoc-FF samples at 10 mmol L-1 prepared in D2O at pH 10.5 starting point of the “titration” experiment, pH 9.1 below apparent pKa 1, and pH 6.8 and 4.2 above and below apparent pKa 2, respectively.
turbid as the pH decreased. At a pH of 6.2-5.2, a second transition was observed and the pH of the 5 and 10 mmol L-1 samples was found to become constant. As the samples went through this second transition, a white precipitate appeared. For samples with pH values below 5, phase separation occurred with the emergence of a clear liquid phase at the top and a white precipitate at the bottom of the test tube when left at room temperature. As the pH was decreased further, the precipitation and phase separation were found to become more pronounced and rapid. At low pH (
