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Vertically Aligned Multilayer Films of Monodispersed Helical Polypeptides with Micrometer Thickness via Simple Cast Masanobu Naito,*,‡,§ Kiyonobu Kishihara,†, and Yoshio Okahata*,† †
Department of Biomolecular Engineering, Tokyo Institute of Technology, Yokohama 226-8501, Japan, Graduate School of Materials Science, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan, and §PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama, Japan. Current affiliation: Orion Cosmetics Industry Co., LTD. )
‡
Received March 4, 2010. Revised Manuscript Received May 14, 2010 Utilizing the zwitterionic R-helix peptide bearing a cationic and anionic group at the N- and C-terminus, respectively, we first demonstrated that the vertically aligned multilayer film can be prepared by a simple cast and slow evaporation. The tilt angle of the peptide remained unchanged with ca. 30° in the range between submicrometer and several micrometers in thickness. The key designs allowing simple vertical alignment of the helical peptide multilayer films were (i) monodispersity of the peptide, (ii) electrostatic interaction between anionic substrate and the cationic group bearing at the N-terminus of the peptide, and (iii) interlayer electrostatic interaction among terminal groups of the peptide.
Introduction The R-helix is an essential secondary structural motif in proteins, which plays an important role in determining protein structure and function. The biological function of the R-helix is mainly attributed to its large macrodipole, originating from the alignment of individual dipole moments of peptide bonds.1 As well as their biological importance, R-helix peptides have been regarded as promising building blocks in fabrications of nanomaterials or nanodevices, taking advantage of their prominent self-assembling abilities.2 Among various applications, vertically aligned peptide monolayer films with distinct R-helical structures have been studied extensively using various preparation techniques and/or sophisticated molecular designs, including Langmuir-Blodgett (LB) technique,3 self-assembly,4 and surface-initiated polymerization.5 In these monolayers, each helical peptide has macromolecular dipole moments originating from the small dipoles of the amino acid repeating along its main chain axis. Therefore, when the R-helix peptide forms a vertically aligned monolayer, a large net dipole arises from the collection of noncancelable individual *To whom correspondence should be addressed. E-mail: mnaito@ ms.naist.jp (M.N.);
[email protected] (Y.O.). (1) Hol, W. G. J. Prog. Biophys. Mol. Biol. 1985, 45, 149. (2) (a) Yang, Y. L.; Khoe, U.; Wang, X. M.; Horii, A.; Yokoi, H.; Zhang, S. G. Nano Today 2009, 4, 193. (b) Ulijn, R. V.; Smith, A. M. Chem. Soc. Rev. 2008, 37, 664. (c) Floudas, G.; Spiess, H. W. Macromol. Rapid Commun. 2009, 30, 278. (3) (a) Kishihara, K.; Kinoshita, T.; Mori, T.; Okahata, Y. Chem. Lett. 1998, 27, 951. (b) Boncheva, M.; Vogel, H. Biophys. J. 1997, 73, 1056. (c) Toyotama, A.; Kugimiya, S.; Yonese, M.; Kinoshita, T.; Tsujita, Y. Chem. Lett. 1997, 443. (d) Hosokawa, H.; Kinoshita, T.; Tsujita, Y.; Yoshimizu, H. Chem. Lett. 1997, 745. (e) Yokoi, H.; Kinoshita, T. Chem. Lett. 2004, 33, 426. (f) Nguyen, L.-T. T.; Ardana, A.; ten Brinke, G.; Schouten, A. J. Langmuir 2009, 26, 6515. (4) (a) Geng, Y.; Discher, D. E.; Justynska, J.; Schlaad, H. Angew. Chem., Int. Ed. 2006, 45, 7578. (b) Miura, Y.; Kimura, S.; Imanishi, Y.; Umemura, J. Langmuir 1999, 15, 1155. (c) Sakamoto, M.; Ueno, A.; Mihara, H. Chem. Commun. 2000, 1741. (5) (a) Whitesell, J. K.; Chang, H. K. Science 1993, 261, 73. (b) Wang, Y. L.; Chang, Y. C. J. Am. Chem. Soc. 2003, 125, 6376. (c) Chang, Y.-C.; Frank, C. W. Langmuir 1998, 14, 326. (d) Wieringa, R. H.; Siesling, E. A.; Werkman, P. J.; Angerman, H. J.; Vorenkamp, E. J.; Schouten, A. J. Langmuir 2001, 17, 6485. (e) Luijten, J.; Groeneveld, D. Y.; Nijboer, G. W.; Vorenkamp, E. J.; Schouten, A. J. Langmuir 2007, 23, 8163. (f) Niwa, M.; Morikawa, M.; Higashi, N. Angew. Chem., Int. Ed. 2000, 39, 960. (g) Wain, A. J.; Do, H. N. L.; Mandal, H. S.; Kraatz, H.-B.; Zhou, F. J. Phys. Chem. C 2008, 112, 14513. (6) (a) Whitesell, J. K.; Chang, H. K.; Fox, M. A.; Galoppini, E.; Watkins, D. M.; Fox, H.; Hong, B. Pure Appl. Chem. 1996, 68, 1469. (b) Chang, Y. C.; Frank, C. W.; Forstmann, G. G.; Johannsmann, D. J. Chem. Phys. 1999, 111, 6136.
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dipoles,5a,6 which can be attributed to a variety of fields, especially in photonics,5a,7 molecular electronics,8 and catalysis.9 Although much attention has been paid to vertically aligned helical peptide monolayer films, multilayer films, with one exception,10 have not been reported due to the difficulty in assembly by conventional multilayer fabrication techniques, such as the LB or layer-by-layer method. However, multilayered peptide films sometimes involve more hierarchical complicated structures.2a,b,11 Therefore, if the multilayered films of vertically aligned R-helix peptides with flatness could be prepared easily and precisely, such a technique would contribute tremendously to a variety of applications, especially second-order nonlinear materials which require unidirectional alignment of molecular polarizability axes over wavelengths up to the micrometer range.12 In this Letter, we first demonstrate the simple fabrication of vertically aligned multilayer films by cast and slow evaporation on an anionic surface using a zwitterionic R-helix peptide bearing a cationic and anionic group at the N- and C-terminus, respectively. In the multilayer film of the helical peptide obtained by the simple cast and slow evaporation, the tilt angle remained unchanged with ca. 30° in the range of several tens of nanometers to several micrometers in thickness. For comparison, a conventional LB method was applied to prepare multilayered films of the cationic R-helix peptide with layers up to several tens of nanometers. Although monodispersed R-helix peptides nearly vertically aligned in the several layers, the orientation of the peptides became disordered when the number of layers exceeded 30 (ca. 100 nm). Thus, the key designs allowing vertical alignment of the helical peptide multilayer film were (i) monodispersity of the peptide, (ii) electrostatic interaction between the anionic substrate (7) Yasutomi, S.; Morita, T.; Kimura, S. J. Am. Chem. Soc. 2005, 127, 14564. (8) Strong, A. E.; Moore, B. D. J. Mater. Chem. 1999, 9, 1097. (9) (a) Licini, G.; Prins, L. J.; Scrimin, P. Eur. J. Org. Chem. 2005, 969. (b) Davie, E. A. C.; Mennen, S. M.; Xu, Y.; Miller, S. J. Chem. Rev. 2007, 107, 5759. (10) Miura, Y.; Xu, G. C.; Kimura, S.; Kobayashi, S.; Iwamoto, M.; Imanishi, Y.; Umemura, J. Thin Solid Films 2001, 393, 59. (11) (a) Wang, W. P.; Chau, Y. Soft Matter 2009, 5, 4893. (b) Sheparovych, R.; Roiter, Y.; Yang, J. Y.; Kopecek, J.; Minko, S. Biomacromolecules 2009, 10, 1955. (c) Giri, K.; Bhattacharyya, N. P.; Basak, S. Biophys. J. 2007, 92, 293. (d) Lomander, A.; Hwang, W. M.; Zhang, S. G. Nano Lett. 2005, 5, 1255. (12) Eaton, D. F. Science 1991, 253, 281.
Published on Web 05/20/2010
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Figure 1. (A) Chemical structure and schematic representation of helical peptides. Helix1, Nþ-(Leu)20-OEt; Helix2, Nþ-(Leu)20-O-; and Helix3, Nþ-(Leu)n-NPr (n: 12-32). Color gradation from red to blue along the peptide main chain represents the direction of the dipole. (B) Schematic illustration of the helical peptides prepared on self-assembled monolayer (SAM)-pretreated gold surfaces. SAMs were prepared with 2-mercaptoethane sulfonic acid (SAM1) and dodecane-1-thiol (SAM2). The tilt angle (θ) was evaluated with FT-IR RAS.
and the cationic group bearing at the N-terminus of the peptide, and (iii) interlayer electrostatic interaction among terminal groups of the peptide.
Experimental Section Preparation of r-Helix Peptides. As the monodispersed helical peptides, L-leucine 20mer derivatives were synthesized using a conventional 9-fluorenylmethoxycarbonyl (Fmoc) solidphase peptide synthesis (SPPS).13 As monocationic and zwitterionic peptides, Helix1 with trimethylammonium (TMA) at the N-terminus and Helix2 with TMA at the N-terminus and carboxylate at the C-terminus were prepared, respectively (Figure 1A).14 In comparison, a cationic polydispersed helical peptide with TMA at the N-terminus was synthesized by N-carboxy amino acid anhydride (NCA) method (Helix3). From MALDI-TOF-MS measurements, the average number of polymerization (n) was estimated to be 12-32. Here, it is widely accepted that the SPPS15 and NCA16 methods are known to reduce the risk of racemization at a negligible level. Therefore, the peptides used in this study should form an optically pure R-helix structure without configrational defects. To confirm this issue, all samples were confirmed to have the R-helix structure in the solution using circular dichroism (CD) measurements.14 Characterization of Peptide Orientation. The orientation of helical peptides on substrates can be characterized by FT-IR RAS (reflection absorption spectroscopy) because the transition dipole moments of amide I (1663 cm-1) and amide II (1546 cm-1) are anisotropic with the helical backbone. Therefore, the dichroic ratio (Dobs) of amide I over amide II absorption area can be utilized to estimate the average tilt angle (θ) of the R-helical peptide from the surface normal (Figure 1B).17 FT-IR RAS (13) Fields, G. B.; Noble, R. L. Int. J. Pept. Protein Res. 1990, 35, 161. (14) See the Supporting Information. (15) Di Fenza, A.; Tancredi, M.; Galoppini, C.; Rovero, P. Tetrahedron Lett. 1998, 39, 8529. (16) (a) Deming, T. J. J. Am. Chem. Soc. 1998, 120, 4240. (b) Berger, A.; Katchalski, E. J. Am. Chem. Soc. 1951, 73, 4084. (c) Hanby, W. H.; Waley, S. G.; Watson, J. Nature 1948, 161, 132. (17) Enriquez, E. P.; Samulski, E. T. Mater. Res. Soc. Symp. Proc. 1992, 255, 423.
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Figure 2. FT-IR RAS spectra of six-layered helical peptide films of Helix1 prepared by the LB method. (I) X-type and (II) Y-type LB films. The tilt angles (θ) were evaluated by the dichroic ratio (Dobs) of amide I over amide II absorption area. spectra were measured by using a Bio-Rad RTS spectrometer equipped with a HgCdTe detector and P-polarized light. Preparation of Multilayered LB Films. Among various methods of LB technique, the horizontal lifting method, in which the substrate is horizontally placed against the surface, was applied following the method described in an aforementioned paper.3a Briefly, the helical peptide was spread on the hexanewater interface and then compressed to be 35 mN/m of surface pressure. While monitoring the π-A curve, the helical peptide monolayer film was transferred onto the gold substrate, in the manner of X-type (head-to-tail) or Y-type (head-to-head/tail-totail) multilayer film. The film thickness was estimated by frequency changes of a 9 MHz quartz crystal microbalance, and the transfer ratio was confirmed to be 1.0 ( 0.1.18 We also carried out the conventional vertical dipping method to prepare the multilayer film. However, the monolayer film was not successfully transferred, probably due to the stiff and brittle nature of the peptide film. Preparation of Multilayered Cast Films. In general, the helical peptides were dissolved in chloroform (0.2 mg/mL). Helix3 was dissolved in chloroform with 3.0 v/v % 1,1,1,3,3,3-hexafluoroisopropanol, due to the relatively low solubility in chloroform alone. To control the thickness of the peptide films, an O-ring was placed on the SAM-modified gold surfaces, followed by pouring the helical peptide/chloroform solutions. For simple cast/slow evaporation, these samples were kept for ca. 12 h under a chloroform-saturated atmosphere to slowly evaporate the solvent. For quick evaporation, peptide solution was quickly dried within ca. 1 h in open air. Here 2-mercaptoethane sulfonic acid (SAM1) and dodecane-1-thiol (SAM2) were used for the anionic and nonionic monolayers, respectively. The thickness of the helical peptide multilayers was estimated by frequency changes of a 9 MHz quartz crystal microbalance, in a way similar to that of the LB method.
Results and Discussion Multilayered LB Films. In a previous paper,3a we reported that, using the LB technique, cationic Helix1 forms a vertically aligned monolayer film with 34° of the tilt angle at a hexane-water interface and the monolayer film is successfully transferred onto the gold substrate, whereas at an air-water interface cationic Helix1 is disordered with an average tilt angle of 80°. Therefore, we first attempted the LB technique at the hexane-water interface in order to prepare the multilayer film of cationic Helix1. Figure 2 shows FT-IR RAS spectra of six layers of Helix1 with (I) X-type and (II) Y-type multilayer films. From the values of (18) Ariga, K.; Okahata, Y. Langmuir 1994, 10, 3255.
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Figure 4. Effect of thickness on tilt angle (θ). Blue and red solid lines represent the tilt angles of the Helix1 (blue line) and Helix2 (red line) on the SAM1, respectively. Thickness of the peptide films was estimated by QCM technique.
Figure 3. FT-IR RAS spectra of multilayered cast films of (I) cationic Helix1 on anionic SAM1, (II) zwitterionic Helix2 on cationic SAM1, (III) cationic Helix1 on nonionic SAM2, (IV) polydispersed cationic Helix3 on anionic SAM1, and (V) cationic Helix1 on gold substrate. The thickness of the peptide multilayers was adjusted to be ca. 30 nm which was estimated by frequency changes of a 9 MHz quartz crystal microbalance.
Dobs of X-type (3.5) and Y-type (3.3), the tilt angles θ were estimated to be 38° and 39°, respectively. These values are nearly identical to those of the monolayers.3a However, the orientation of the helical peptide became disordered when the number of the layers exceeded 30 (ca.100 nm). This may result from subtle structural defects being enhanced through multistep LB fabrication. Here, it is noteworthy that, up to several tens of layers, the orientation of the helical peptide followed the orientation of the first layer on the surface. Here, it was revealed that ordering behavior was significantly affected by the dispersity of the R-helix peptide. It is known that the dispersity of the molecular length significantly affected the ordering of the rodlike molecules.19 In particular, in the case of phase behavior of liquid crystalline rodlike molecules, the monodispersity stimulates entropy-driven closed-packing of the rodlike molecules, regardless of the synthetic polymer20 or biopolymer.21 For example, although poly(R,L-glutamic acid) (PLLG) derivatives usually form a nematic liquid crystalline phase, monodispersed PLLG derivatives synthesized by genetic engineering can form smectic liquid crystalline phases, in which helical peptides are organized into layers perpendicular to the molecular orientation.21 Similarly, in the present case, polydispersed cationic Helix3 could not form the monolayer film even through the same LB preparation as that of the monodispersed cationic Helix1 (data not shown). Multilayered Cast Films. Figure 3 shows the FT-IR RAS spectra of the multilayered cast films of helical peptides on various substrates. Here the thickness of the multilayer was adjusted to be approximately 30 nm, using a 9 MHz quartz crystal microbalance (QCM). Unfortunately, cationic Helix1 was fairly disordered on the nonionic SAM2 and gold substrates, with tilt angles of 58° and (19) Bates, M. A.; Frenkel, D. J. Chem. Phys. 1998, 109, 6193. (20) Okoshi, K.; Kamee, H.; Suzaki, G.; Tokita, M.; Fujiki, M.; Watanabe, J. Macromolecules 2002, 35, 4556. (21) Yu, S. J. M.; Conticello, V. P.; Zhang, G. H.; Kayser, C.; Fournier, M. J.; Mason, T. L.; Tirrell, D. A. Nature 1997, 389, 167.
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70°, respectively (Figure 3III and V). Considering the results of the multilayered LB films, we hypothesize that if the first layer of the monodispersed helical peptide is vertically aligned, the upper layers spontaneously form vertical alignment even through a simple cast. To achieve the vertically aligned first layer, we chose the sulfonic acid bearing SAM1 as an anionic surface. This was expected to stimulate vertical alignment via electrostatic interaction between the anionic surface and cationic terminus of the helical peptides.4b Consequently, Helix1 was vertically aligned normal to the anionic SAM1 surface with a tilt angle of 16°, which suggests that the cast film has a much higher order than that from the LB technique (ca. 40°) (Figure 3I). However, the orientation of the peptide was gradually disordered with increased thickness (Figure 4, blue line). Conversely, polydispersed cationic Helix3 exhibited an omnidirectional multilayer film with a tilt angle of 75° on SAM1 (Figure 3IV). This result was consistent with the abovementioned results on the multilayered LB film. To overcome the thickness-dependent disordering, we redesigned zwitterionic Helix2, based on the following expectation: when cationic TMA interacts with the anionic SAM1, the carboxylate group at the opposite C terminus would appear at the surface, and could act as another anionic surface for the incoming Helix2. Consequently, the tilt angle of Helix2 remained unchanged with ca. 30° up to ca. 1.3 μm in thickness even through the simple cast and slow evaporation (Figure 4, red line). Here, it is noteworthy that the tilt angle for Helix1 (θ = 16°) implies that its monolayer on SAM1 is more organized than that of Helix2 on the same substrate (θ = 31°). This may be caused by differences in geometrical directions of electrostatic interaction and dipoledipole interaction. Thus, the tilt angle of Helix2 may be originated from the geometrical direction between cationic TMA and anionic carboxylate group between Helix1 in the multilayered film. On the other hand, the tilt angle of Helix1 on the SAM1 was probably determined by the bonding geometry between the sulfonic acid of SAM1 and the TMA group of Helix1 on the substrate surface only, which was enhanced through the dipoledipole interaction up to several tens of nanometers. In addition, the morphologies of the multilayered films were observed with atomic force microscopy, which showed relatively smooth surfaces with less than 1 nm in vertical intervals.14 The helical peptides may spontaneously rearrange to become the most stable closed-packing structure during slow evaporation, similar to the rodlike helical conjugated polymers on the substrates.22 On the other hand, the orientation of the peptides became greatly disordered when the solvent was quickly evaporated within ca. (22) Sakurai, S. I.; Okoshi, K.; Kumaki, J.; Yashima, E. Angew. Chem., Int. Ed. 2006, 45, 1245.
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1 h in open air. Furthermore, we also attempted to prepare the zwitterionic polydispersed peptide samples to further clarify the effect of polydispersity. However, the use of these samples was limited due to low solubility, even in chloroform with 3.0 v/v % 1,1,1,3,3,3-hexafluoroisopropanol. This is probably because aggregation among disordered peptides occurred during sample preparation.
helical peptide multilayer films may be utilized in designing wellordered multilayer films of helical peptides with various functionalities. Furthermore, we believe that knowledge gained from this study will serve as a guide toward designing vertically aligned multilayers for electrically and optically active surfaces.
Conclusion
Acknowledgment. The authors dedicate this article to the memory of Mr. Taiji Kishihara. He benevolently supported our research and education.
Using the monodispersed and zwitterionic R-helix peptide, we first demonstrated that a vertically aligned multilayer film several micrometers in thickness was prepared using a very simple cast and slow evaporation. This approach to obtaining highly ordered
Supporting Information Available: Experimental details, CD spectra, and AFM observations. This material is available free of charge via the Internet at http://pubs.acs.org.
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