Self-Assembly of Surfactant-like Peptides - Langmuir (ACS Publications)

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Langmuir 2007, 23, 12729-12736

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Self-Assembly of Surfactant-like Peptides Dave J. Adams,* Kathrin Holtzmann, Christian Schneider, and Michael F. Butler UnileVer Corporate Research, Colworth Science Park, Sharnbrook, Bedford, MK44 1LQ, U.K. ReceiVed April 17, 2007. In Final Form: August 14, 2007 Inspired by recent work describing surfactant-like peptides, we have carried out a systematic study on peptides with the underlying composition of V6D2, altering the absolute sequence to determine the importance of the surfactant-like structure. All of the peptides examined here formed self-assembled structures in water. However, in contrast to other reports, we have found a surprising diversity of structures including fibers, tapes, and twisted ribbons but an absence of the vesicles and nanotubes described previously. Further investigations demonstrated that peptide purity plays a significant role in the outcome of the self-assembly. Different batches behave very differently, which can be linked to the compositions of these batches. This work shows that there is a need for not only rational design but also ease of synthesis of the building blocks for self-assembled structures.

1. Introduction The self-assembly of small peptides has been used to prepare a number of distinct morphologies.1-14 These include vesicles, hollow tubes, fibers, tapes, and helical structures. A number of applications have been mooted for materials prepared by the self-assembly of peptides including scaffolding for tissue repair, hydrogels, drug delivery vehicles, and surface modification. The use of small peptides as building blocks potentially allows control over the macroscopic properties and functionality and can produce biocompatible materials. Short peptides have advantages in that they are relatively easy to design and synthesize. Although many short peptides have been prepared and used as building blocks, generally they self-assemble into tapes and fibers. Of particular interest is the work of Zhang et al., who recently reported on the self-assembly of surfactant-like peptides.12-14 These are short peptides that have one or two charged amino acids as the polar head and a tail consisting of several (often six) hydrophobic amino acids. Zhang et al. reported that these peptides selfassembled to give nanotubes and vesicles, very different morphologies than usually adopted by short peptides. Inspired by this work, we have carried out a systematic study on the self-assembly of surfactant-like peptides. We have examined peptides with the underlying composition of V6D2 (V ) valine, D ) aspartic acid), altering the absolute sequence to determine the importance of the surfactant-like structure, examining V6D2, * Corresponding author. E-mail: [email protected]. (1) Aggeli, A.; Boden, N.; Zhang, S. Self-Assembling Peptide Systems in Biology, Medicine and Engineering; Kluwer Academic Publishers: Dordrecht, The Netherlands, 2001; Chapter 1. (2) Chen, P. Colloids Surf., A 2005, 261, 3. (3) Tu, R. S.; Tirrell, M. AdV. Drug DeliVery ReV. 2004, 56, 1537. (4) Zhang, S. Nat. Biotechnol. 2003, 21, 1171. (5) Zhang, S.; Marini, D. M.; Hwang, W.; Santoso, S. Curr. Opin. Chem. Biol. 2002, 6, 865. (6) MacPhee, C. E.; Woolfson, D. N. Curr. Opin. Solid State Mater. Sci. 2004, 8, 141. (7) Hartgerink, J. D.; Clark, T. D.; Ghadiri, M. R. Chem.sEur. J. 1998, 4, 1367. (8) Rajagopal, K.; Schneider, J. P. Curr Opin. Struct. Biol. 2004, 14, 480. (9) Gilead, S.; Gazit, E. Supramol. Chem. 2005, 17, 87. (10) Caplan, M. R.; Moore, P. N.; Zhang, Kamm, S., R. D.; Lauffenberger, D. A. Biomacromolecules 2000, 1, 627. (11) Mihara, H.; Matsumura, S.; Takahashi, T. Bull. Chem. Soc. Jpn. 2005, 78, 572. (12) Vauthey, S.; Santoso, S.; Gong, H.; Watson, N.; Zhang, S. Proc. Natl. Acad. Sci. 2002, 99, 5355. (13) Santoso, S.; Hwang, W.; Hartman, H.; Zhang, S. Nano Lett. 2002, 2, 687. (14) von Maltzahn, G.; Vauthey, S.; Santoso, S.; Zhang, S. Langmuir 2003, 19, 4332.

V5DVD, and V4D2V2. For V5DVD and V4D2V2, we do not have such a well-defined hydrophobic tail and hydrophilic head group. We have also compared both N-acetylated and non-acetylated peptides. 2. Materials and Methods 2.1. Materials. All peptides were purchased from two suppliers, Pepceuticals, Nottingham UK, and Suzhou Tianma, China. All batches were purchased at a nominal purity level of >95%. As in other reports,12-14 no further purification of peptide treatment was performed. Three different batches were purchased from Pepceuticals, designated N1, N2, and N3 in the text. One batch was purchased from Suzhou Tianama, designated ST in the text. Most peptide samples were white, fluffy powders. There was a distinct difference in morphology for the N3 batch for V6D2 and Ac-V6D2, which were crystalline solids. Single batches of both V5DVD and V4D2V2 were purchased from Pepceuticals and used as received. All other chemicals were purchased from Aldrich and used as received. Deionized water was used throughout (resistivity ) 18.2 MΩ). 2.2. Sample Preparation. The peptide was weighed with a Mettler MT5 balance into a DSC pan and suspended in filtered (Sartorius Minisart 200 nm) water in a 7 mL volume glass sample vial to a nominal concentration of 5 mM assuming the peptides are pure (which corresponds to 4.2 mg mL-1), and the pH was adjusted with filtered (Sartorius Minisart 200 nm) NaOH or HCl (0.1 M or 1 M solutions were used); then it was subjected to ultrasound (Bandelin Sonorex Ultrasonic bath) for 5 min. For all pH measurements, a Jenway 3020 pH meter was used after regular calibration. Experiments indicated that extending the period of ultrasound to 10 min had no effect on the self-assembly. Extended ultrasound for more than 20 min resulted in an increase in the temperature of the solution and was therefore avoided. 2.3. Analysis. TEM. Samples for TEM were prepared as described above in section 2.2. TEM was carried out by applying a drop of the sample solution to a plasma-treated (20 s) carbon-coated copper grid and negatively stained with methylamine tungstate. Samples were viewed using a JEOL 1200EX TEM at 100 kV. Images were taken on a bioscan 1000 CCD camera. NMR. 1H NMR spectra were recorded using a Bruker AMX 400 MHz spectrometer. The samples were prepared as above in section 2.2 in D2O and the pH was adjusted using 0.1 N NaOD solution (prepared by dilution of a 30 wt % commercially available stock solution). Sixteen scans were recorded. Circular Dichroism. Circular dichroism was carried out on an OLIS DSM CD with a 1 mm path length glass sample cell in the range from 190 to 270 nm. The subtracted background spectrum (water) consisted of an average of five scans. Samples were prepared

10.1021/la7011183 CCC: $37.00 © 2007 American Chemical Society Published on Web 11/08/2007

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Figure 1. 1H NMR spectrum of Ac-V6D2 (batch N1) at pD 11 in D2O. Inset is an expansion of the region between 2.8 and 3.3 ppm. as above in section 2.2. Because of excess scattering from samples at the original concentrations, the solutions were diluted by a factor of 10 to allow spectra to be collected. IR Spectroscopy. Samples for IR were prepared as described above in section 2.2. IR spectra were collected on an ATR FTIR Bio-Rad FTS 6000 spectrometer (Cambridge, MA). Spectra were measured on a diamond ATR with a resolution of 4 cm-1, using 256 scans and an open aperture. Spectra were recorded in transmission mode then transformed into absorbance. Using the GRAMS/AI software package, background and solvent spectra were subtracted and the spectra were Fourier self-convoluted and peak fitted. HPLC-MS. Samples were prepared as described above in section 2.2. HPLC-MS was carried out with HPLC-positive electrospray MS using a Waters/Alliance LC-MS system with a detector at 214 nm. The individual components were separated on a Vydac Protein C4 reversed-phase column with a gradient using 0.025% TFA in water and 0.025% TFA in acetonitrile with a flow rate of 1 mL/min, and the column was maintained at 30 °C. The eluent from the HPLC column was split at a ratio of 1:4, introducing 200 µL/min into the electrospray ion source of the mass spectrometer. Ionization was achieved using the following parameters: capillary voltage, 3.0 kV; cone, 30 V; extractor, 4 V; source temperature, 100 °C; desolvation temperature, 300 °C; desolvation gas flow, 500 L/h; and cone gas flow, 100 L/h. Full scan mass spectra were recorded across the mass range of m/z 50-2000 with a scan time of 2 s.

3. Results The self-assembly of a small family of surfactant-like peptides has been examined on the basis of V6D2 and Ac-V6D2. These two peptides differ only at the N terminus. For V6D2, the N terminus is a free NH2. For Ac-V6D2, the N terminus is acetylated. As a result, the peptides will differ with regard to their charge at a given pH. V6D2 will carry an extra positive charge on the N terminus below the pKa of this free NH2 (ca. 7.5). Hence, the net charge on the two peptides will differ below pH 7.5. To test the hypothesis that the surfactant-like nature of the peptides is important, we have also investigated V5DVD and V4D2V2. Here, the same absolute amino acids are used, but the sequences are such that there are not such well-defined hydrophobic tails and hydrophilic head groups. 3.1. Peptide Characterization. All peptides were purchased from peptide synthesis companies and prepared by wellestablished solid-phase synthetic approaches. As is standard, the only characterization provided by these companies was limited mass spectroscopic analysis demonstrating the presence of the required molecular weight in the sample. Hence, the peptides were characterized more thoroughly in-house. 1H NMR of the peptides demonstrated the presence of impurities. An example spectrum of batch N1 of the acetylated peptide is shown in Figure 1.

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Figure 2. 1H NMR spectrum of V6D2 (batch ST) at pD 11 in D2O.

All of the expected resonances can be seen (valine CH3 at 0.85 ppm, valine CH at 2 ppm with CH3 from the acetyl group at 1.95 ppm, CH2 from aspartic acid at 2.4-2.7 ppm, and backbone CH between 3.9 and 4.4 ppm; the NH protons do not appear to H-D exchange with the deuterated solvent). However, traces of impurities are also clearly detectable, for example, the peaks between 2.8 and 3.3 ppm. The identity of these species is unclear. Impurities were also detected in the samples of V6D2. The NMR of batch ST is shown in Figure 2. Interestingly, for this batch, the NMR spectrum is very weak compared to that expected considering the amount of material used in the initial sample preparation. It is possible that this is due to aggregation of the peptide, resulting in broadening of the line widths and hence an apparent decrease in signal to noise. However, when combined with the HPLC and TEM data (see below), it seems most likely that this batch is significantly less pure than the others. Further investigation of the purity of the peptides was carried out by HPLC-MS. Again, impurities could be easily detected. Example traces from the samples of V6D2 are shown in Figure 3. From Figure 3, it can be seen that the required peptide is only one component in the system. Indeed, for the N1 batch, V6D2 is a minority component. The mass spectra of the different components confirm the identity of the broad peak as V6D2 (M+ ) 843) highlighted on each spectrum in Figure 3. The peak running a minute earlier corresponds to V5D2 (M+ ) 744), whereas that at 2 min earlier corresponds to V4D2 (M+ ) 645). Data from the ST batch was poor in quality because of the low signal strength despite the samples being prepared at the same concentration, indicative of impurities that are not detected by HPLC. These impurities are likely to be inorganic in nature (as evidenced by crystalline material in the TEM image). The impurities in the other samples at longer times remain unidentified but appear oligomeric in nature (as suggested by the multiple repeating fragments in the mass spectra). It is difficult to determine quantitatively the amount of V6D2 present in each sample. Complications arise from the overlapping nature of the peaks and the fact that, at least for the ST batch, a significant amount of material is not HPLC-detectable. However, we can estimate from the integration of the peaks that batch N1 contains approximately 7% V6D2, N2 contains 38%, N3 contains 9%, and ST contains 42%. This of course assumes that all material is detectable by HPLC. Clearly, in all cases, the peptides are impure and contain V6D2 as minority components. Similar impurities were observed for the acetylated V6D2 as well as V5DVD and V4D2V2 (Supporting Information). 3.2. Self-Assembly in Aqueous Media. The self-assembly of these peptides was examined in water. All solutions were prepared at a concentration of 4.2 mg mL-1 (nominally 5 mM if the peptides

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Figure 3. HPLC traces for different batches of V6D2 in water: N1 (top left), N2 (top right), N3 (bottom left), and ST (bottom right). The peak from V6D2 present in the mixture is highlighted in each case.

are pure) in deionized water and then adjusted to the required pH as described by Zhang et al.12 The non-acetylated peptides became soluble only at a pH of 7 or above. The acetylated peptides were less soluble, and clear solutions were obtained only at a pH of 9 or above. The self-assembled structures in solution at a pH of 7 or above were then imaged by TEM. Here, we visualized the self-assembled structures using a staining technique. This was shown not to affect the structures by comparison to samples shadowed with platinum rather than stained (data not shown). This technique differs from that used by Zhang et al. to image the structures formed from their samples of surfactant-like peptides. The technique used by Zhang et al. requires freezing of the samples, which could cause complicating artifacts. Representative TEM data is shown in Figure 4 for the V6D2 batches. Batch ST was found to give no apparent self-assembled structures. Instead, crystalline material was deposited on the grid. Coupled with the NMR and HPLC data, it was assumed that the peptides from this supplier were very impure. As a result, we concentrated on the self-assembly of the peptides supplied by Pepceuticals. The N1 batch was found to give a mixture of broad tapes and highly twisted structures. Batches N2 and N3 were found to give only nontwisted rods of uniform diameter. The highly twisted structures in batch N1 have regular helicity, as can be seen in Figure 5. The broad tapes can also be seen to twist regularly and, in some cases, break at this twist on the grid. Similar images were recorded for the Ac-V6D2 peptides (Figure 6). Here, the self-assembled structures were more similar, although batch N2 showed some crystalline material. Similar images were also recorded for V5DVD and V4D2V2 at a pH of 9 or above (Figure 7). The measured widths and, where appropriate, pitches, of the self-assembled structures are tabulated in Table 1. The measured widths and pitches are approximate because of difficulties in determining exact edges on the stained TEM images. From the data in Table 1 and Figures 4-7, it can

be seen that, whereas some variation in size is apparent, broadly similar structures are imaged for all peptides apart from V6D2 (N1), where helical structures are observed and batch ST shows where crystalline impurities dominate the images. The secondary structure adopted by the peptides in solution can be determined by both circular dichroism (CD)15 and infrared spectroscopy.16 Here, we find that IR indicated that all batches of V6D2 and Ac-V6D2 adopt β-sheet structures as shown by the peak at 1628 cm-1 (Figure 8). However, circular dichroism (carried out at a lower concentration because of scattering artifacts at the original concentration) indicates that a significant proportion of the peptides exist as random coils in solution with a limited amount of β-sheet structure present. The solutions were then dialyzed for 5 days (changing the water at least 4 times a day) through a 12 000 MWCO membrane, and the solutions were reanalyzed. Here, once again, IR showed the presence of β-sheet structure. However, after dialysis, CD also showed only the presence of β-sheets (Figure 9), although the solution strengths were very weak. Interestingly, neither CD nor IR showed any change in the spectra over several days, implying that the structures are not evolving.

4. Discussion The self-assembly of short peptides is an exciting area that offers significant promise with regard to the preparation of a wide range of different types of materials. Examples include hydrogel scaffolds for tissue repair and structurants.17 Peptides offer advantages over other building blocks in that they are potentially biocompatible and enzyme degradable. Also, with (15) See, for example, Holzwarth, G.; Doty, P. J. Am. Chem. Soc. 1965, 87, 218. (16) Bandekar, J. Biochim. Biophys. Acta 1992, 1120, 123. (17) Shin, H.; Jo, S.; Mikos, A. G. Biomaterials 2003, 24, 4353.

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Figure 4. TEM images of different batches of V6D2: N1 (top left), N2 (top right), N3 (bottom left), and ST (bottom right) at pH 9 (scale bars are 200 nm). All samples were prepared at a concentration of 4.2 mg mL-1.

all of the available amino acids as building blocks for the peptides themselves, a huge number of permutations of peptides can be prepared. Hence, extremely tunable systems are possible. Here, we discuss our results on the self-assembly of so-called surfactantlike peptides. These short peptides have a hydrophobic tail consisting of six hydrophobic valine residues and a head group consisting of two negatively charged aspartic acid residues. We have investigated both N-acetylated and non-acetylated peptides. Zhang et al. have reported that these specific peptides selfassemble in dilute aqueous solution to give tubes and vesicles.12-14 One disadvantage of such peptides is their synthesis. The peptides used here with several valine residues (four batches purchased from two different suppliers) are very hydrophobic. Conventionally, it is recommended by peptide synthesis companies that such numbers of sequential hydrophobic amino acids such as valine are kept to a minimum. This is because β-sheet structures can be formed during synthesis, leading to incomplete

coupling and hence impure products. In addition, the high hydrophobicity also means that solubilizing and purifying the peptide postsynthesis is extremely difficult. During this study, it became clear that batch reproducibility is a key concern. An analysis of the peptides by NMR showed that whereas the majority of the material consisted of valine and aspartic acid residues as expected, unidentified impurities were also present. Analysis by HPLC coupled with mass spectroscopy demonstrated that all batches were very impure. Indeed, in the case of batch N1, the required V6D2 was only a minor component of the system. Significant amounts of V5D2 were identified in all systems, and in batch N1, V4D2. Presumably, the lower number of valine residues in the system indicates a very inefficient coupling reaction during synthesis, in agreement with the generally accepted notion of keeping the number of hydrophobic residues in one sequence to a minimum. Quantifying the absolute amount of the required peptide in each batch was difficult because

Self-Assembly of Surfactant-like Peptides

Figure 5. TEM image of batch N1 of V6D2 at a pH of 9 showing the regular helical pitch of the twisted structures. The scale bar represents 200 nm. The sample was prepared at a concentration of 4.2 mg mL-1.

of some overlap in the HPLC trace and, most importantly, because it was clear that at least one batch (batch ST) contained a significant amount of material that was not detectable by either NMR or HPLC. Although absolute quantification was not possible, we estimate from simple integration of the HPLC trace that the amount of V6D2 in the batches is perhaps as low as 9%. It is clear that there are significant differences in both the composition and purity of all batches. It would obviously have been desirable to compare these systems to an example of the pure peptides. However, despite repeated synthesis attempts, no pure samples were produced. Purification postsynthesis also proved ineffective, with the peptides being so hydrophobic that all attempts to separate the components failed. Direct comparisons to the peptides used in the studies by Zhang et al. are also impossible because of the lack of any information provided regarding the purity of their peptides. Despite the amount of impurity in the systems, the selfassembly of the peptides did lead to structures similar to those reported for many peptide self-assembled systems. We examined the self-assembly at a pH of 7 or above (non-acetylated) or pH 9 or above (acetylated) where the peptides give visually clear solutions at concentrations of 4.2 mg mL-1. Below these pH values, the peptides are clearly insoluble. However, above these pH values, rods, tapes, and helical structures were imaged by TEM. All of these structures have been observed previously via peptide self-assembly. It is unclear if the surfactant-like peptides can pack in a similar way to that proposed for more conventional short peptides.18 However, with the levels of impurities in the current systems, modeling was deemed inappropriate. The different batches of peptides gave rise to different structures. Batch N1 of V6D2 gave rise to helical structures, whereas most other peptides gave rods of relatively uniform width and variable length. The structures formed in these cases are rods. Were they tubes, stain would be expected to ingress into the hydrophilic interior as has been shown by Reches and Gazit.19 This is not (18) Fishwick, C. W. G.; Beevers, A. J.; Carrick, L. M.; Whitehouse, C. D.; Aggeli, A.; Boden, N. Nano Lett. 2003, 3, 1475. (19) Reches, M.; Gazit, E. Science 2003, 300, 625.

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observed in any case here. Batch ST gave few rodlike structures although a large amount of crystalline material was imaged. This agrees with the HPLC and NMR traces that suggest that a significant amount of this batch is not peptidic (the significant amount of crystalline material imaged by TEM indicates that there is probably significant inorganic material present in this sample). It is tempting to link the observation that batch N1 of V6D2 contained a significant amount of V4D2 to this being the only peptide sample to give helical structures, suggesting that it may be possible to induce such structures by mixing peptides with unequal numbers of residues. Clearly, in no case did we observe vesicles or nanotubes as reported for Ac-V6D2 by Zhang et al.12-14 One explanation for this discrepancy is the different TEM techniques used here and in the work of Zhang et al. However, vesicles can easily be imaged by staining techniques,20 implying that, were vesicles to exist in the solutions, it would be possible to image them using the technique used in this work. Hence, the lack of observed vesicles and nanotubes is presumably due to differing batch composition to the peptides used in this work and that of Zhang et al. The importance of the surfactantlike nature of the peptides was probed by comparing V6D2 with V5DVD and V4D2V2. Here, the same amino acids are used in the peptides, but the sequence is such that these peptides no longer have such defined hydrophilic head groups and hydrophobic tails. In these cases, we find that broadly the same structures are formed as for V6D2 and Ac-V6D2. As for these peptides, purity is an issue for V5DVD and V4D2V2. The greatest difference between the peptides seems to be due to purity rather than the self-assembled structures. This implies that the surfactant-like nature of the peptides is less important than might be expected. Certainly, for such hydrophobic peptides where achieving high purity is difficult, one must first consider the levels of purity that can be achieved and then, after this, consider the importance of the absolute sequence of the amino acids. IR demonstrates that all of the peptides have a predominantly β-sheet structure. CD showed a significant amount of random coil in solution with only a small amount of β-sheet structure. This signal did not change with time. Because the peptides are expected to self-assemble by the formation of β-sheets, it would be expected that the random coil signal would decrease with time to be replaced with an increasing β-sheet signal. Further work showed that the structures giving rise to the random coil could be removed by dialysis through a 12K MWCO membrane. This shows that, in these systems, there are two populations of peptides: those existing in small aggregates or free/monomeric states capable of diffusing through the dialysis membranes, which give rise to a random coil signal, and those that are too large to diffuse through the membranes, which give rise to a β-sheet signal. However, it is possible that these effects arise from the lower concentrations used for CD as compared to the TEM and IR data. If the peptides are close to their critical aggregation concentration (CAC), then they will not be in the form of selfassembled structures. Yang and Zhang have reported that the CAC of their samples of V6D2 is approximately 1.1 mM.21 Hence, the samples used here at a nominal concentration of 5 mM will most likely be below this concentration in the solutions used for CD. The presence of β-sheet structures that are not removed by dialysis strongly suggests that there are self-assembled structures present and hence the peptides are above their CAC. However, because the peptides used are actually a mixture of different peptides, it is possible that some of the constituents of the mixture are now below their CAC and others are above their CAC. This (20) Adams, D. J.; Butler, M. F.; Weaver, A. C. Langmuir 2006, 22, 4534. (21) Yang, S. J.; Zhang, S. Supramol. Chem. 2006, 18, 389.

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Figure 6. TEM images of different batches of Ac-V6D2: N1 (top left), N2 (top right), and N3 (bottom left) at pH 9 (scale bars are 500 nm). Crystalline material is highlighted in the N2 batch. All samples were prepared at a concentration of 4.2 mg mL-1.

would explain the mixture of random coil and β-sheet structure seen in the CD. Alternatively, this discrepancy between IR and CD could be due to the sampling method. IR was carried out using an ATR crystal and hence measures what is on the surface of this crystal. CD is carried out in transmittance and so measures the bulk solution. This observation could therefore indicate that the β-sheet structures either preferentially adsorb to the surface or are formed at the surface. TEM is obviously also a technique where adsorbed materials are analyzed and hence the TEM images may not be representative of the totality of the solution. All of this data shows that batch reproducibility is a key issue with such hydrophobic peptides. The variation in composition of each batch makes comparison extremely difficult. Although the precise composition of the impurities in the mixtures is unknown, the impurities do consist of surfactant-like peptides (e.g., V4D2) and hydrophobic valine sequences. It is likely,

therefore, that mixed bilayers form (although whether the bilayer exists as phase-separated regions of different peptide moieties or whether all components are intimately mixed is not known). In general, headgroup repulsion favors the bending of bilayer sheets whereas chain repulsion and the presence of charge opposes bending.22,23 If the present system were pure, then the nature of the peptides, namely, the likelihood of attractive hydrogen bonding interactions and the rigid, interacting valine tail groups would tend to favor flat sheets (as observed in some cases). However, it is known that bilayers formed in mixed systems with different and asymmetically arranged chain lengths can adopt a spontaneous curvature.24,25 The presence of impurities in the current case (22) Israelachvili, J. Intermolecular and Surface Forces, 2nd ed.; Academic Press: London, 1992; pp 382-385. (23) Pincus, P.; Joanny, J. F.; Andelmann, D. Europhys. Lett. 1990, 11, 763. (24) May, S.; Ben-Shaul, A. J. Chem. Phys. 1995, 103, 3839. (25) Porte, G.; Ligoure, C. J. Chem. Phys. 1995, 102, 4290.

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Figure 8. Example IR spectra of V6D2 and Ac-V6D2. The peak at approximately 1628 cm-1 indicates the presence of β sheets. All samples were prepared at a concentration of 4.2 mg mL-1.

Figure 9. CD of peptides before (top b, Ac-V6D2 (batch N1); O, V6D2 (batch N2); 1, V6D2 (batch N3); 4, V6D2 (batch ST)) and after dialysis (bottom b, Ac-V6D2 (batch N1); O, V6D2 (batch N2); 1, V6D2 (batch N3)). β-Sheet structures are indicated by the single minimum at approximately 220 nm. Samples were prepared at a concentration of 4.2 mg mL-1 and then diluted by a factor of 10. The dialyzed samples were dialyzed for 5 days. Table 1. Width and Pitch of Self-Assembled Structures from the Different Peptides Prepared at a Concentration of 4.2 mg mL-1a peptide

Figure 7. TEM images of V5DVD (left) and V4D2V2 (right) at pH 9 (scale bars are 200 nm). Both samples were prepared at a concentration of 4.2 mg mL-1.

may therefore result in the bending and twisting of the bilayers. Without prior knowledge of the interchain interactions and distribution, however, the prediction of these effects is not possible.

5. Conclusions Our work on the self-assembly of surfactant-like peptides has demonstrated that peptide purity is of the utmost importance. Different batches behave very differently, which is undoubtedly linked to the different compositions of these batches. The peptides used in this study are very hydrophobic and hence are very difficult to prepare as pure products by solid-phase synthesis. This work uses similar peptides to those used in Zhang et al., and ideally we would compare our results to the data presented by these authors. Unfortunately, Zhang et al. give no indication of the

V6D2 (N1) V6D2 (N2) V6D2 (N3) V6D2 (ST) Ac-V6D2 (N1) Ac-V6D2 (N2) Ac-V6D2 (N3) Ac-V6D2 (ST) V5DVD V4D2V2 a

width of fibers of selfassembled structure/nm

pitch of selfassembled structure/nm

broad tapes, 18-36 fibers 9-22 9-15 14-18

150-400 30-35

18-20 13-16 9-12 6-11 6-11

All data comes from solutions prepared at pH 9.

purity of the peptides nor do they provide analytical data for the materials. As a result, it is impossible to directly compare to the peptides reported previously. In this work, we describe the use of multiple batches, each of which contained different impurities, and show that in each case the structures formed differ greatly from those described by Zhang et al. This may well be due to the differences in purity between any of our batches and the literature examples. Nonetheless, this work has shown that a

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number of interesting structures can be prepared by self-assembly using such peptides as building blocks but highlights the need not only for rational design but also ease of synthesis of homogeneous samples when considering such an approach. Acknowledgment. We thank Paul Pudney and Craig Gregor for the IR spectra, Paul Sanderson for the NMR spectra, Chris Tier for the HPLC spectra, and Steve Furzeland and Tony Weaver for the TEM images. We thank the Leonardo da Vinci scheme

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for funding K.H. while at Colworth. We thank Dek Woolfson (University of Bristol) and Mark Kirkland (Unilever R&D) for helpful discussions. Supporting Information Available: Example HPLC traces for Ac-V6D2 and V5DVD; example IR data showing the effect of underand oversubtracting water from the raw spectrum. This material is available free of charge via the Internet at http://pubs.acs.org. LA7011183