Communication pubs.acs.org/Biomac
Differential Self-Assembly Behaviors of Cyclic and Linear Peptides Sung-ju Choi,† Woo-jin Jeong,† Seong-Kyun Kang,‡ Myongsoo Lee,‡ Eunhye Kim,§ Du Yeol Ryu,§ and Yong-beom Lim*,† †
Translational Research Center for Protein Function Control and Department of Materials Science & Engineering and §Department of Chemical and Biomolecular Engineering, Yonsei University, Seoul 120-749, Korea ‡ Center for Bio-Responsive Assembly and Department of Chemistry, Seoul National University, Seoul 151-747, Korea S Supporting Information *
ABSTRACT: Here we ask the fundamental questions about the effect of peptide topology on self-assembly. The study revealed that the self-assembling behaviors of cyclic and linear peptides are significantly different in several respects, in addition to sharing several similarities. Their clear differences included the morphological dissimilarities of the self-assembled nanostructures and their thermal stability. The similarities include their analogous critical aggregation concentration values and cytotoxicity profiles, which are in fact closely related. We believe that understanding topology-dependent self-assembly behavior of peptides is important for developing tailor-made self-assembled peptide nanostructures.
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INTRODUCTION Macromolecules that have cyclic structures (macrocycles) are interesting molecules not just because they do not have chain ends but because their topological features can significantly affect their chemical, physical, and biological properties. Macrocycles can more effectively maintain rigid and conformationally constrained structures than their linear counterparts.1−6 Many examples exist in nature in which such conformational constraints help to stabilize folded or self-assembled structures. One of the strategies typically used by nature to establish reversible covalent macrocyclic constraints in a linear polypeptide chain is by making disulfide bonds, which can assist in protein folding by reducing the conformational entropy of the unfolded state.7,8 Peptides have the same chemical structure as proteins but are shorter in length. Peptides have several advantages over proteins, including the fact that they can be synthesized and mass-produced more easily, and they have broader chemical diversity.9 However, peptides and peptide epitopes are often unstructured because of their short chain length and thermodynamic instability, limiting their usefulness when precise and stable conformations are required, as in specific molecular recognition events. Therefore, many attempts have been made to stabilize the peptide conformation using cyclization strategies.10,11 Several proteins, including cyclotides, topologically unique head-to-tail cyclized proteins in which the cyclic structure offers remarkable thermal stability and protease resistance, use a similar strategy.12,13 Self-assembled peptide nanostructures, constructed by the controlled self-assembly of peptide molecules, have become promising biomaterials.14−27 Self-assembled peptide nanostructures range in size from tens of nanometers to several © 2012 American Chemical Society
micrometers, which are comparable to the sizes of natural proteins or protein assemblies. Therefore, self-assembled peptide nanostructures might be regarded as nanosized artificial proteins. Research, even though still highly primitive when compared with natural proteins, are in progress to devise selfassembled peptide nanostructures that can mimic or displace the diverse biological functions of natural proteins, possibly with enhanced properties or with functions unprecedented in nature. One of the most simple, widespread, and effective ways of constructing self-assembled nanostructures is to make use of amphiphilic building blocks, in which one of the blocks is designed to be hydrophilic and the other is designed to be hydrophobic. Probably one of the most famous examples of amphiphilic building blocks for self-assembly is diblock copolymers. Likewise, many self-assembled peptide nanostructures are also based on the peptide molecules having amphiphilic character.28−30 Various types of self-assembling amphiphilic peptides have been developed to date and have reported to be useful in many bioapplications. However, despite the unique and advantageous properties of cyclic molecules, all of the self-assembling amphiphilic peptides developed to date make use of linear peptide molecules as building blocks, and the self-assembly behavior of cyclic peptides with two dissimilar and amphiphilic blocks has not been explored so far, to the best of our knowledge. It should be noted that self-assembling cyclic peptides with alternating D- and L-amino acids cannot be considered as amphiphiles and usually form nanostructures Received: April 17, 2012 Revised: May 25, 2012 Published: June 8, 2012 1991
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soluble only under hydrophobic lipid bilayer environment.6 Because linear peptides often have unstable conformations (vide ante), conformational stability of surface-exposed peptides within nanostructures self-assembled from linear peptides can be limited. In this Communication, we ask the fundamental questions about differential self-assembly behaviors of amphiphilic peptides with cyclic or linear structures.
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Circular Dichroism. Circular dichroism (CD) spectra were measured using a Chirascan CD spectrometer equipped with peltier temperature controller (Applied Photophysics). Spectra were recorded from 260 to 190 nm using a 2 mm path-length cuvette. Scans were repeated five times and averaged. Molar ellipticity was calculated per amino acid residue. Fluorescence Spectroscopy. The steady-state fluorescence spectra were recorded using a Hitachi F-4500 fluorescence spectrophotometer in 1 cm path length quartz cuvettes. To measure fluorescence from tryptophan residues, we excited samples at 280 nm. Excitation and emission slits with a nominal bandpass of 5 nm were used for the measurements. Cytotoxicity Assay. HeLa cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS). For the cytotoxicity assay, cells were seeded in a 96-well plate at a density of 104 cells/well in 100 μL of culture medium and grown at 37 °C, 5% CO2 to reach 60−70% confluence. The culture medium was removed, and DMEM (90 μL) was added. Then, the varying concentrations of peptides (10 μL) were added to each well. Following 4 h of incubation at 37 °C, 5% CO2, 10 μL of WST-1 solution was added. The cells were further incubated for 4 h, and the absorbance of each sample was measured at a wavelength of 450 nm using a microplate reader.
EXPERIMENTAL SECTION
General. Fmoc-amino acids and coupling reagents were purchased from Novabiochem (Germany) and Anaspec (USA). General chemicals were obtained from Sigma-Aldrich (USA) and Merck (Germany). HPLC solvents were purchased from Fisher Scientific (USA). Tissue culture reagents were obtained from Invitrogen (USA). Although we did not perform detailed kinetic experiment, we found that spectroscopic signals and nanostructural morphologies are quite stable and reproducible soon after the dissolution of the peptides. To make it quite sure that the peptide assemblies were in thermodynamic equilibrium state, we incubated all samples for several days before taking measurements. Repeated experiments revealed that steady state has been reached after the incubation period. Cyclic Peptide Synthesis. The 2-Chlorotrityl resin was first preloaded with Fmoc-Gly-OH. Further couplings of amino acids were performed on a Tribute peptide synthesizer on 0.1 mmol scale (Protein Technologies). Standard amino acid protecting groups were employed for the synthesis. For the head-to-tail cyclization reaction, N-terminal Fmoc-group was deprotected following the completion of the final amino acid coupling. The protected peptide fragment (20 μmol) was liberated from the resin by AcOH/2,2,2-trifluoroethanol (TFE)/methylene chloride (MC) (2:2:6) treatment. After an appropriate time (∼1 to 2 h), the resin was removed by filtration, and the filtrate was recovered (4 mL × 2). Finally, the resin was washed three times with the cleavage mixture. Hexane was added to the filtrate to remove acetic acid as an azeotrope with hexane. The protected peptide fragment was obtained as a white powder following repeated evaporation cycles (the dissolution of the peptide fragment in MC, hexane addition, and evaporation). For cyclization, typically 5 μmol of the protected peptide fragment and 12 μmol DIPEA were dissolved in DMF (10 mL) and transferred into a syringe. To achieve pseudo-high dilution condition, this solution was added to a stirred solution of 5 μmol PyBOP and 5 μmol HOBt in DMF (10 mL) at a rate of 0.05 mL/min by using a syringe pump. Once the addition was completed, the reaction mixture was further stirred for ∼5 h. Following DMF evaporation, the residue was dissolved in MC, and then tert-butyl methyl ether/hexane was added to triturate the cyclized and protected peptide fragment (three times). The peptide fragment was treated with cleavage cocktail (TFA/TIS/water; 95:2.5 2.5) for 3 h and was triturated with tert-butyl methyl ether. The peptides were purified by reverse-phase HPLC (water−acetonitrile with 0.1% TFA). The molecular weight was confirmed by MALDI-TOF mass spectrometry. The purity of the peptides was >95%, as determined by analytical HPLC. Concentration was determined spectrophotometrically in water/acetonitrile (1:1) using a molar extinction coefficient of tryptophan (5502 M−1cm−1) at 280 nm. Yield: cyPA-1 (3.5%), liPA-1 (6.7%), cyPA-2 (2.6%), liPA-2 (4.5%). Transmission Electron Microscopy (TEM). Two μL of sample (typically, 5−50 μM) was placed onto a carbon-coated copper grid and dried completely. Then, 2 μL of water was added for 1 min to dissolve and remove unbound peptide and was wicked off by filter paper. The sample was stained with 1% uranyl acetate for negative staining. The specimen was observed with a JEOL-JEM 2010 instrument operating at 120 kV. The data were analyzed with DigitalMicrograph software. Atomic Force Microscopy. For atomic force microscopy (AFM), 1 μL of the sample in water was deposited onto a freshly cleaved mica surface for 1 min and dried in air. The images were obtained in tapping mode with a Nanoscope IV instrument (Digital Instruments). AFM scans were taken at set point of 0.8 to 1 V, and scanning speed was 1 to 2 Hz.
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RESULTS AND DISCUSSION As a simplified model system, we designed several amphiphilic peptide building blocks, in which oligo-arginines and oligotryptophans represented hydrophilic and hydrophobic blocks, respectively (Figure 1). Linear peptides were synthesized using
Figure 1. Chemical structures and amino acid sequences of cyclic and linear peptides. Arginines (hydrophilic and charged amino acids) and tryptophan (hydrophobic and aromatic amino acids) are shown in blue and red, respectively.
standard Fmoc protocols on 2-chlorotrityl chloride resin. Cyclization of a protected peptide fragment was performed under pseudo-high-dilution conditions to prevent intermolecular multimerization reactions (Supporting Information).31 The peptides were purified by HPLC (>95%). All amphiphilic peptides were soluble in water. As an initial study, the peptide building blocks were dissolved in water (5−50 μM), and their nanostructural morphologies were investigated by TEM. As shown in Figure 2a, TEM revealed irregularly shaped nanoaggregates of the linear peptide liPA-1. By contrast, regularly shaped spherical objects of ∼10 nm in diameter were observed for the cyclic peptide cyPA-1 (Figure 2b). To address the question of whether the chain length affects the self-assembly behavior, we prepared peptide building blocks with longer hydrophilic blocks (liPA-2 and cyPA-2). A similar trend was observed during the self-assembly 1992
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Figure 2. Nanostructural morphologies of the self-assembling peptide supramolecular aggregates. TEM images of (a) liPA-1, (b) cyPA-1, (c) liPA-2, and (d) cyPA-2. (e) AFM image of cyPA-2. (f) DLS result for cyPA-2 (average RH = 5.89 nm).
Figure 3. Characterization of the self-assembly behavior of linear versus cyclic peptides. Temperature-dependent CD spectra of (a) liPA-2, 10 μM and (b) cyPA-2, 10 μM (4−84 °C, 10 °C interval). Insets: normalized mean residue ellipticity at 224 and 223 nm for liPA2 and cyPA-2, respectively. Concentration-dependent fluorescence emission spectra of (c) liPA-2 and (d) cyPA-2. Insets: plots of fluorescence intensity as a function of peptide concentrations (log scale).
of liPA-2 and cyPA-2 (Figure 2c,d). The aggregates of liPA-2 exhibited irregular morphologies, whereas those of cyPA-2 contained a fairly homogeneous population of spherical objects (diameter; ca. 11 nm). Investigation with AFM further corroborated the spherical morphologies and homogeneity (Figure 2e). Dynamic light scattering (DLS) examinations revealed that the average hydrodynamic radius (RH) of cyPA-2 supramolecular aggregates is 5.89 nm (so the diameter is 11.78 nm), which correlates well with the TEM result (Figure 2f). It should be noted that the nanostructure formation was instantaneous and prolonged storage of up to several months did not change the size and shape of the nanostructures. Therefore, all of these results clearly indicate that the selfassembly behaviors of linear and cyclic peptides are significantly different. In these specific examples of peptides, the linear peptides tended to aggregate into irregular objects, whereas cyclic peptides preferred to form regular spheres. Intrigued by these results, we further scrutinized the differential self-assembly behaviors of linear and cyclic peptides using liPA-2 and cyPA-2. First, the conformation of the peptides was probed with CD spectroscopy (Figure 3). The CD spectra of both liPA-2 and cyPA-2 displayed a distinct minimum of ellipticity around 200−203 nm, indicating that the peptides exist mostly in random coil states and do not have clearly defined secondary structures. In addition, both peptides showed the strong negative bands around 223 to 224 nm. These bands are the behaviors of tryptophan expected from exciton coupling produced by the aromatic chromophores of indoles stacking against one another.32−34 Therefore, these results indicate that the primary driving force for the selfassembly of both linear and cyclic peptides is the pure hydrophobic and π−π stacking interactions between tryptophan residues and that secondary structure formation, such as β-sheets, is not involved in the self-assembly process.
Given the results described above, intensity of the band at 223 to 224 nm can be used as a measure of peptide aggregation.34 As shown in the inset of Figure 3a, temperature-dependent CD spectra of liPA-2 showed a rapid and linear decrease in mean residue ellipticity at 224 nm. At a high temperature (84 °C), the normalized degree of aggregation was decreased to 62% compared with that at 4 °C. In contrast, the ellipticity at 223 nm for cyPA-2 stopped decreasing above 54 °C, and the degree of aggregation was maintained at around 82%, even at a high temperature of 84 °C (an inset in Figure 3b). Taking all of these results into account, the cyclic peptide forms more robust and thermally stable nanostructures than the linear peptide, even though they make use of the same aggregation mechanism (i.e., pure hydrophobic and π−π stacking interactions, vide ante). It is well known that the indole chromophore in tryptophan is highly sensitive to the nature of its local environment, which has led to the wide use of tryptophan’s intrinsic fluorescence in probing protein conformational changes and interactions with other molecules.35,36 As shown in Figures 3c,d, a clear concentration-dependent increase in tryptophan fluorescence was observed at concentrations ranging from 0.1 to 20 μM for both liPA-2 and cyPA-2. Plots of the fluorescence intensity at 350 nm as a function of peptide concentrations are shown in the insets of Figure 3c,d. Above certain concentrations, there were sudden increases in fluorescence intensity for both peptides. These discontinuous changes in intensity most likely reflect the onset of aggregation and critical aggregation concentration (CAC). The point of intersection of the 1993
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assay. As shown in Figure 5, both liPA-2 and cyPA-2 are not toxic within the concentration range tested, and they exhibited
extrapolated linear regression lines was used to calculate the CACs of the peptides. The calculation shows that CACs for liPA-2 and cyPA-2 are 5.7 and 3.3 μM, respectively. Although the CAC value was slightly lower for the cyclic peptide than for the linear counterpart, the difference was rather small. Taken together, these results allow us to propose a model for the spherical nanostructures (Figure 4). The spherical nano-
Figure 5. Cytotoxicity profiles of the self-assembling peptides in HeLa cells (WST-1 cell proliferation assay). liPA-2 (filled circle) and cyPA-2 (open circle). Mean ± SD (n = 4).
Figure 4. Models of self-assembled cyPA-2 supramolecular aggregates. (a) Left, a micelle model; right, a vesicle model. (b) Detailed view of the vesicle model. Modeling was performed using Materials Studio (Accelrys).
similar levels of cytotoxicity. It has been shown that stable molecular assembly prevents free diffusion of the individual amphiphilic components to the cell surface and lipid bilayer interior, and the cytotoxicity of amphiphilic peptides is largely proportional to their CAC values.29,40 Therefore, the similar cytotoxicity profiles of the linear and cyclic peptides are most likely due to their analogous CAC values.
object should be either a spherical micelle or a vesicle.37,38 In the case of cyPA-2, the length of the molecule determined by Corey−Pauling−Koltun space-filling modeling (CPK modeling) was ca. 2.5 to 3.0 nm, depending on the peptide conformation. Fitting such a molecular length into a spherical micelle model would result in micelles of roughly 6 nm in diameter (Figure 4a), which is far smaller than the diameter of the spherical nano-object observed (∼11 nm). Instead, the vesicle model agrees nicely with the peptide’s molecular length, wherein the cyclic peptides form an extended conformation along the axis, connecting the hydrophilic and hydrophobic blocks and indole rings of tryptophans, which are stacked against each other in a bilayered structure (Figure 4b). The stacking of the indole residues is in good agreement with the CD data (vide ante). Likewise, the diameter of the selfassembled spherical nano-object of cyPA-1 also fits well with the vesicle model. Therefore, the results suggest that the cyclic peptides are likely to self-assemble into bilayered vesicular nanostructures, which are unusually small considering their molecular length. The unique self-assembly behaviors of the cyclic peptides, their high thermal stability, and their formation of remarkably small vesicular structure likely result from their constrained structures and the entropic advantage of their cyclic topology during the self-assembly process.39 It would be interesting to more systematically investigate the intrinsic driving force underlying unique self-assembly behavior of cyclic peptides, possibly using self-assembling peptides with different amino acid composition and molecular weight; this will be the subject of future study. After establishing the self-assembly behavior of linear and cyclic peptides, it was of interest to investigate the manner in which these amphiphilic peptides interact with mammalian cells and to obtain their differential cytotoxicity profiles for potential cellular applications. To this end, a comparative toxicity assay was performed in HeLa cells using the WST-1 cell proliferation
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CONCLUSIONS In this work, we have shown that the properties of selfassembling cyclic and linear peptides are significantly different in several respects, in addition to sharing several similarities. Their clear differences included the morphological dissimilarities of the self-assembled nanostructures and their thermal stability. Moreover, the study has revealed that cyclic peptides can make unique and unusual assemblies because of their constrained structure. The similarities include their analogous CAC values and cytotoxicity profiles, which are likely to be closely related. Although it is an open question whether these trends can be applied to self-assembling peptides with different structures and molecular weights, it is clear that the topology of amphiphilic peptides profoundly affects their self-assembly behaviors. Understanding this principle is important for developing tailor-made self-assembled peptide nanostructures.
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ASSOCIATED CONTENT
* Supporting Information S
Spectral data. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest. 1994
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(29) Lim, Y. B.; Lee, E.; Lee, M. Angew. Chem., Int. Ed. 2007, 46, 9011−9014. (30) Tovar, J. D.; Claussen, R. C.; Stupp, S. I. J. Am. Chem. Soc. 2005, 127, 7337−7345. (31) Malesevic, M.; Strijowski, U.; Bachle, D.; Sewald, N. J. Biotechnol. 2004, 112, 73−77. (32) Dempsey, C. E.; Mason, P. E.; Jungwirth, P. J. Am. Chem. Soc. 2011, 133, 7300−3. (33) Grishina, I. B.; Woody, R. W. Faraday Discuss. 1994, 245−262. (34) Ladokhin, A. S.; Selsted, M. E.; White, S. H. Biochemistry 1999, 38, 12313−12319. (35) Chen, Y.; Barkley, M. D. Biochemistry 1998, 37, 9976−9982. (36) Robbins, R. J.; Fleming, G. R.; Beddard, G. S.; Robinson, G. W.; Thistlethwaite, P. J.; Woolfe, G. J. J. Am. Chem. Soc. 1980, 102, 6271− 6279. (37) Lim, Y. B.; Moon, K. S.; Lee, M. J. Mater. Chem. 2008, 18, 2909−2918. (38) Shimizu, T.; Masuda, M.; Minamikawa, H. Chem. Rev. 2005, 105, 1401−1443. (39) Honda, S.; Yamamoto, T.; Tezuka, Y. J. Am. Chem. Soc. 2010, 132, 10251−10253. (40) Pakstis, L. M.; Ozbas, B.; Hales, K. D.; Nowak, A. P.; Deming, T. J.; Pochan, D. Biomacromolecules 2004, 5, 312−318.
ACKNOWLEDGMENTS We thank National Research Foundation (NRF) of Korea for funding through the Future-based Technology Development Program (Nano Fields; 2011-0019125), the Basic Science Research Program (2011-0003540, 2011-0003065, and 2012R1A1A2006453), the Translational Research Center for Protein Function Control, Yonsei University (2012-0000888), and Seoul R&BD program (ST110029M0212351).
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REFERENCES
(1) Lim, Y. B.; Moon, K. S.; Lee, M. Angew. Chem., Int. Ed. 2009, 48, 1601−1605. (2) Choi, S. J.; Jeong, W. J.; Kim, T. H.; Lim, Y. B. Soft Matter 2011, 7, 1675−1677. (3) Hackenberger, C. P. R. Angew. Chem., Int. Ed. 2008, 47, 5984− 5988. (4) Mylne, J. S.; Colgrave, M. L.; Daly, N. L.; Chanson, A. H.; Elliott, A. G.; McCallum, E. J.; Jones, A.; Craik, D. J. Nat. Chem. Biol. 2011, 7, 257−259. (5) Zhang, K.; Lackey, M. A.; Wu, Y.; Tew, G. N. J. Am. Chem. Soc. 2011, 133, 6906−6909. (6) Hartgerink, J. D.; Granja, J. R.; Milligan, R. A.; Ghadiri, M. R. J. Am. Chem. Soc. 1996, 118, 43−50. (7) Betz, S. F. Protein Sci. 1993, 2, 1551−1558. (8) Zavodszky, M.; Chen, C. W.; Huang, J. K.; Zolkiewski, M.; Wen, L.; Krishnamoorthi, R. Protein Sci. 2001, 10, 149−160. (9) Marx, V. Chem. Eng. News 2005, 83, 17−24. (10) Hili, R.; Rai, V.; Yudin, A. K. J. Am. Chem. Soc. 2010, 132, 2889−2891. (11) Kawakami, T.; Ohta, A.; Ohuchi, M.; Ashigai, H.; Murakami, H.; Suga, H. Nat. Chem. Biol. 2009, 5, 888−890. (12) Gustafson, K. R.; Sowder, R. C., II; Henderson, L. E.; Parsons, I. C.; Kashman, Y.; Cardellina, J. H.; Mcmahon, J. B.; Buckheit, R. W., Jr; Pannell, L. K.; Boyd, M. R. J. Am. Chem. Soc. 1994, 116, 9337−9338. (13) Poth, A. G.; Colgrave, M. L.; Lyons, R. E.; Daly, N. L.; Craik, D. J. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 10127−10132. (14) Lim, Y. B.; Moon, K. S.; Lee, M. Chem. Soc. Rev. 2009, 38, 925− 934. (15) Banwell, E. F.; Abelardo, E. S.; Adams, D. J.; Birchall, M. A.; Corrigan, A.; Donald, A. M.; Kirkland, M.; Serpell, L. C.; Butler, M. F.; Woolfson, D. N. Nat. Mater. 2009, 8, 596−600. (16) Gazit, E. Chem. Soc. Rev. 2007, 36, 1263−1269. (17) Jeong, W. J.; Lim, Y. B. Macromol. Biosci. 2012, 12, 49−54. (18) König, H. M.; Kilbinger, A. F. Angew. Chem., Int. Ed. 2007, 46, 8334−8340. (19) Lim, Y. B.; Lee, E.; Lee, M. Angew. Chem., Int. Ed. 2007, 46, 3475−3478. (20) Lim, Y. B.; Lee, E.; Yoon, Y. R.; Lee, M. S.; Lee, M. Angew. Chem., Int. Ed. 2008, 47, 4525−4528. (21) Kwon, S.; Shin, H. S.; Gong, J.; Eom, J.-H.; Jeon, A.; Yoo, S. H.; Chung, I. S.; Cho, S. J.; Lee, H.-S. J. Am. Chem. Soc. 2011, 133, 17618− 17621. (22) Mart, R. J.; Osborne, R. D.; Stevens, M. M.; Ulijn, R. V. Soft Matter 2006, 2, 822−835. (23) Percec, V.; Dulcey, A. E.; Balagurusamy, V. S. K.; Miura, Y.; Smidrkal, J.; Peterca, M.; Nummelin, S.; Edlund, U.; Hudson, S. D.; Heiney, P. A.; Hu, D. A.; Magonov, S. N.; Vinogradov, S. A. Nature 2004, 430, 764−768. (24) Nagy, K. J.; Giano, M. C.; Jin, A.; Pochan, D. J.; Schneider, J. P. J. Am. Chem. Soc. 2011, 133, 14975−14977. (25) Shao, H.; Parquette, J. R. Angew. Chem., Int. Ed. 2009, 48, 2525− 2528. (26) Bowerman, C. J.; Nilsson, B. L. J. Am. Chem. Soc. 2010, 132, 9526−9527. (27) Matsuura, K.; Murasato, K.; Kimizuka, N. J. Am. Chem. Soc. 2005, 127, 10148−10149. (28) Hartgerink, J. D.; Beniash, E.; Stupp, S. I. Science 2001, 294, 1684−1688. 1995
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