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ARTICLES Molecular Fibers Based on the Honeycomb-Like Self-Assembly of an r-Helical Polypeptide Gahee Kim,†,§ Jinhwan Yoon,†,§ Jong-Seong Kim,† Heesoo Kim,‡ and Moonhor Ree†,* Department of Chemistry, National Research Laboratory for Polymer Synthesis & Physics, Center for Integrated Molecular Systems, and BK School of Molecular Science, Pohang UniVersity of Science & Technology (Postech), Pohang 790-784, Republic of Korea, and Department of Microbiology, Dongguk UniVersity College of Medicine, Gyeongju 780-714, Republic of Korea ReceiVed: March 26, 2008; ReVised Manuscript ReceiVed: April 27, 2008
We have succeeded in fabricating well-grown molecular fibers of a polypeptide on substrates by using a conventional solution spin-coating and drying process. These molecular fibers were found to consist of a honeycomb-like molecular assembly formed via the hexagonal close packing of the polypeptide chains in the R-helix conformation. Peptides are of significant interest because of their assembly into stable, ordered molecular conformations.1–3 Much research effort has been applied to the production of ordered peptide thin films and the characterization of their structures.1–3 Further, well-ordered thin polymer films on surfaces have also attracted significant attention as a novel class of materials because of their potential applications in various fields.4 In the present study, we aimed to fabricate well-ordered molecular assemblies based on a polypeptide, poly(benzyloxycarbonyl-lysine), in which the peptide linkages are chemically integrated into the polymer, and then to determine their structures and the mechanism of their evolution. The polypeptide was synthesized with n ) 20 (n is the number-average degree of polymerization) by carrying out the ring-opening polymerization of benzyloxycarbonyl-lysine N-carboxyl anhydride,3 producing poly(benzyloxycarbonyllysine)20 (PBCL20) (for details of the synthesis, see the Supporting Information). PBCL20 solutions (solid content 2 wt %) were prepared in N,N-dimethylformamide (DMF) and tetrahydrofuran (THF), and then spin-coated onto precleaned silicon substrates, followed by drying under vacuum at room temperature for 1 day. The resulting polypeptide films with a thickness of 80-100 nm were quantitatively analyzed by using atomic force microscopy (AFM), Fourier transform infrared (FTIR) spectroscopy, and grazing incidence X-ray scattering (GIXS)5 using synchrotron X-ray sources and a two-dimensional (2D) detector. Figure 1 shows typical AFM surface images of films fabricated from solutions of PBCL20 in DMF and THF. Interestingly, fibrils were developed in the films, as can be seen in the figure. Furthermore, such fibrils were found to form through the whole films (see the optical microscopy (OM) images in the Supporting Information). In the case of the film fabricated from the polypeptide solution in DMF, the fibrils are well-developed with diameters near 1.0 µm and lengths of * To whom correspondence should be addressed. E-mail:
[email protected]. Tel: +82-54-279-2120. Fax: +82-54-279-3399. † Pohang University of Science & Technology. ‡ Dongguk Univeristy. § These authors contributed equally to this work.
Figure 1. AFM height images of PBCL20 thin films deposited on silicon substrates by spin-coating solutions of the polypeptide in (a,b) DMF and (c,d) THF.
several tens of micrometers (Figure 1a). Each fibril consists of rectangular domains with edges several hundreds of nanometers in length and long axes that are aligned along the fibril axis (Figure 1b). Similar fibrils are found in the films fabricated from the polypeptide solution in THF (Figure 1c,d). However, these fibrils are much shorter and thinner than those developed in the film prepared from the polypeptide solution in DMF. Moreover, the rectangular domains in these fibrils have dimensions in the range of 50 × 100 to 100 × 300 nm2, and so are at least 3 times smaller than those in the film obtained from the polypeptide solution in DMF. These differences might be due to the differences between the characteristics of DMF and THF. DMF has a higher boiling point (153 °C) than THF (65 °C) and a lower vapor pressure (2.7 mmHg at 20 °C) than THF (143 mmHg at 20 °C). Thus DMF molecules are present for longer in the film during the film formation process, and the polypeptide chains are more mobile during film formation in the film fabricated from the polymer solution in DMF. In general, a certain degree of
10.1021/jp802628p CCC: $40.75 2008 American Chemical Society Published on Web 07/09/2008
Molecular Fibers Formed from R-Helix Polypeptides
Figure 2. (a) 2D GIXS pattern measured at Ri ) 0.2° for a PBCL20 thin film deposited on a silicon substrate from a solution of the polypeptide in DMF.
molecular mobility is critical for the formation of molecular assemblies and their growth. In conclusion, the larger domains and fibrils in the film fabricated with the DMF solution of the polypeptide are due to the additional mobility of the polypeptide chains provided by the DMF solvent during the film formation process. To examine the structures of the fibrils and their domains in the polypeptide films, GIXS analysis was performed. Figure 2 shows a representative 2D GIXS pattern, which was measured at a grazing incidence angle of Ri ) 0.2° for a 100 nm thick PBCL20 film fabricated from a polypeptide solution in DMF. Similar 2D GIXS patterns were observed for the films prepared from THF solutions of the polypeptide (data not shown). As can be seen in the figure, several scattering spots are present with regular spacing over the scattering angle region 5-15°, but there are no Debye-Scherrer rings. The scattering spots satisfy the Bragg conditions for a hexagonal reciprocal lattice of cylinders lying in the film plane (see the GIXS data analysis in the Supporting Information). The GIXS pattern also contains two specular reflection spots at the in-plane scattering angle 2θf ) 0° along the direction of the out-of-plane scattering angle Rf (Figure 2). The reflection spot in the low angle region was found to have a d-spacing of 1.50 nm, which can be assigned to the interdistance of the cylinders. The other reflection spot was determined to have a d-spacing of 0.75 nm, indicating that this spot is the second-order peak of the first spot in the lowangle region. Neither of the reflection spots have Debye-Scherrer rings. These GIXS results indicate that the rectangular domains in the polypeptide film consist of a well-ordered hexagonal packing structure of cylinders lying in the film plane and further that the hexagonally packed cylinders are well stacked in the out-of-plane direction without any rotational disorder along the cylinder axis. In addition to the above scattering spots, the GIXS pattern contains a weak, broad isotropic scattering ring near 20° (Figure 2); the d-spacing of this broad ring was estimated to be 0.47 nm. This scattering ring is discussed later in the paper. We also conducted an FTIR spectroscopy analysis of the films and molecular simulations in order to determine the molecular conformation and dimensions of a single chain of the polypeptide. The FTIR spectra of the films contain a vibration peak at 1652 cm-1 (Figure 3). This peak is characteristic of the carbonyl stretching vibrations of amide linkages (amide-I) in the R-helix conformation.6 In contrast, the films do not reveal vibration peaks in other amide-I regions, such as at 1620 cm-1 due to a β-sheet conformation. Thus the FTIR analysis confirms that the polypeptide chains in the films are all present in the R-helix conformation. We attempted to determine the molecular dimensions of these polypeptide chains in an R-helical conformation by carrying out a molecular simulation analysis with the Cerius2
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Figure 3. FT-IR spectrum of a PBCL20 thin film. The arrow denotes the amide bands indicative for the secondary structure of the peptide.
Figure 4. (a) Molecular model of a PBCL20 chain in the R-helix conformation: left, top view; right, side view. The atoms are colored as follows: C, gray; O, red; N, blue; H, white. The red cylinder denotes the main chain in the R-helix conformation. (b) Schematic representation of the 2D HCP structure formed in PBCL20 thin films with PBCL chains in the R-helix conformation: left, front view; right, side view. (c) Molecular packing model for PBCL20 molecules in the single domain shown in Figure 1: left, front view; right, top view.
software package (Accelrys, San Diego, CA). As shown in Figure 4a, the molecular dimensions of an R-helical single polypeptide chain are estimated to be a diameter of 1.53 nm, a length of 3.04 nm, and an R-helical pitch period of 0.50 nm. The R-helical pitches are composed of 4-(benzyloxycarbonylamino)butyl bristles positioned with a certain periodic rotational angle along the polypeptide backbone (Figure 1 and Figure 4a). Overall, the single polypeptide chain has a columnar shape (i.e., a cylinder) with an R-helical conformation (Figure 4a). Among the molecular parameters of the R-helical polypeptide chain, the diameter is comparable with the interdistance of the hexagonally packed cylinders that was determined with the
8870 J. Phys. Chem. B, Vol. 112, No. 30, 2008 GIXS analysis discussed above (Figure 2 Figure 4b). Further, the R-helical pitch period is comparable with the d-spacing of the broad isotropic ring in the GIXS pattern (Figure 2). These results suggest that the weak, broad isotropic scattering ring in the GIXS pattern is due to the periodic R-helical pitches of the bristles along the polypeptide backbone. The above GIXS, FTIR, and molecular simulation results confirm that the rectangular domains in the polypeptide film have a well-ordered hexagonal packing structure consisting of cylinders lying in the film plane and further that the hexagonally packed cylinders are well stacked in the outof-plane direction without any rotational disorder along the cylinder axis (Figure 4b). We propose the molecular packing model for the PBCL20 thin films shown in Figure 4c. In the single domains shown in Figure 1, R-helical PBCL20 molecules are well stacked along the thickness direction in a honeycomb-like structure; this model is strongly supported by the presence of several diffraction spots and an isotropic diffraction ring in the GIXS image and the unit cell lattice parameters, as well as by the calculated molecular dimensions of the R-helical PBCL20 chain. In conclusion, we have succeeded in this study in fabricating well-grown molecular fibers in films of PBCL20 with a conventional solution spin-coating process. These molecular fibers consist of a honeycomb-like molecular assembly resulting from hexagonal close packing (HCP) of PBCL20 molecules in the R-helix conformation. Acknowledgment. This study was supported by the Korea Science & Engineering Foundation (National Research Laboratory Program and Center for Integrated Molecular Systems) and by the Ministry of Education (BK21 program).
Kim et al. Supporting Information Available: Experimental Section, GIXS data analysis, and OM analysis. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) (a) Weisenforn, A. L.; Romer, D. U.; Lorenzi, G. P. Langmuir 1992, 8, 3145–3149. (b) Auduc, N.; Ringenbach, A.; Stevenson, I.; Jugnet, Y.; Duc, T. M. Langmuir 1993, 9, 3567–3573. (c) Powers, E. T.; Kelly, J. W. J. Am. Chem. Soc. 2001, 123, 775–776. (d) Powers, E. T.; Yang, S. I.; Lieber, C. M.; Kelly, J. W. Angew. Chem., Int. Ed. 2002, 41, 127–130. (2) Yoon, J.; Ree, M.; Hwang, Y.-T.; Lee, S. W.; Lee, B.; Kim, J.-S.; Kim, H.; Magonov, S. N. Langmuir 2004, 20, 544–549. (3) Jiang, H.; Stupp, S. I. Langmuir 2005, 21, 5242–5246. (4) (a) Knobler, C. M.; Schwartz, D. K. Curr. Opin. Colloid Interface Sci. 1999, 4, 46–51. (b) Rapaport, H.; Kjaer, K.; Jensen, T. R.; Leiserowitz, L.; Tirrel, D. A. J. Am. Chem. Soc. 2000, 122, 12523–12529. (c) Gamboa, A. L. S.; Filipe, E. J. M.; Brogueira, P. Nano Lett. 2002, 2, 1083–1086. (5) (a) Lee, B.; Park, Y.-H.; Hwang, Y.-T.; Oh, W.; Yoon, J.; Ree, M. Nat. Mater. 2005, 4, 147–150. (b) Lee, B.; Oh, W.; Hwang, Y.; Park, Y.H.; Yoon, J.; Jin, K. S.; Heo, K.; Kim, J.; Kim, K.-W.; Ree, M. AdV. Mater. 2005, 17, 696–701. (c) Lee, B.; Yoon, J.; Oh, W.; Hwang, Y.; Heo, K.; Jin, K. S.; Kim, J.; Kim, K.-W.; Ree, M. Macromolecules 2005, 38, 3395– 3405. (d) Park, I.; Lee, B.; Ryu, J.; Im, K.; Yoon, J.; Ree, M.; Chang, T. Macromolecules 2005, 38, 10532–10536. (e) Lee, B.; Oh, W.; Yoon, J.; Hwang, Y.; Kim, J.; Landes, B. G.; Quintana, J. P.; Ree, M. Macromolecules 2005, 38, 8991–8995. (f) Lee, B.; Park, I.; Yoon, J.; Park, S.; Kim, J.; Kim, K.-W.; Chang, T.; Ree, M. Macromolecules 2005, 38, 4311–4323. (g) Heo, K.; Jin, K. S.; Yoon, J.; Jin, S.; Oh, W.; Ree, M. J. Phys. Chem. B 2006, 110, 15887–15895. (h) Heo, K.; Park, S.-G.; Yoon, J.; Jin, K. S.; Jin, S.; Rhee, S.-W.; Ree, M. J. Phys. Chem. C 2007, 111, 10848–10854. (i) Jin, S.; Yoon, J.; Heo, K.; Park, H.-W.; Shin, T. J.; Chang, T.; Ree, M. J. Appl. Crystallogr. 2007, 40, 950–958. (j) Yoon, J.; Jin, K. S.; Kim, H. C.; Kim, G.; Heo, K.; Jin, S.; Kim, J.; Kim, K.-W.; Ree, M. J. Appl. Crystallogr. 2007, 40, 476–488. (k) Yoon, J.; Yang, S. Y.; Heo, K.; Lee, B.; Joo, W.; Kim, J. K.; Ree, M. J. Appl. Crystallogr. 2007, 40, 305–312. (6) (a) Kricheldorf, H. R. R-Amino Acid N-Carboxyanhydride and Related Materials; Springer: New York, 1987. (b) Jackson, M.; Mantsch, H. H. Crit. ReV. Biochem. Mol. Biol. 1995, 30, 95–120.
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