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Electron Transfer through a Self-Assembled Monolayer of a Double-Helix Peptide with Linking the Terminals by Ferrocene Shinpei Okamoto, Tomoyuki Morita, and Shunsaku Kimura* Department of Material Chemistry, Graduate School of Engineering, Kyoto UniVersity, Kyoto-Daigaku-Katsura, Nishikyo-ku, Kyoto 615-8510, Japan ReceiVed October 20, 2008. ReVised Manuscript ReceiVed December 29, 2008 A unique molecular structure, a double-helix peptide, was self-assembled on gold, and the electron transfer through the monolayer was studied. The double-helix peptide consists of two 9mer 310-helical peptide chains having a disulfide group at each N terminal and being linked by a ferrocene dicarboxylic acid between the C terminals. Each helical peptide chain has three naphthyl groups in a linear arrangement along the helix. The monolayer properties and the electron transfer from the ferrocene unit to gold were studied with reference peptides with a similar double helix but without naphthyl groups, a single helix with a dicarboxylic ferrocene unit, and a single helix with a monocarboxylic ferrocene unit. It was demonstrated that the naphthyl groups on the side chains had no effect on electron transfer, and the electron-transfer rate in the double-helix monolayer was not promoted, despite the two electron pathways in the molecule. We propose that in the double-helix monolayer, molecular motions are suppressed, possibly by its rigid structure tethered by the two linkers on gold to cancel out acceleration effects of the 2-fold electron pathways and the ferrocene substitution number. The factors that affect the electron-transfer reaction across the helical peptide SAMs are discussed in depth.

Introduction Helical peptides have attracted much attention as components of well-defined molecular assemblies. Their importance is evident from their regular and rigid structure and excellent ability to form regular self-assemblies, as is well demonstrated in natural protein structures. In particular, it has been shown that helical structures are essential for locating chromophores in their proper positions and for mediating and controlling electron transfer in electron-transport proteins.1-3 To understand electron transport in biological systems on the molecular level, electron-transfer reactions through helical peptides have been extensively studied spectroscopically in solution4-9 and electrochemically10-17 and with scanning probe microscopy18-20 on the surface. These efforts have revealed significant features of helical peptides such * Corresponding author. Tel: +81-75-383-2400. Fax: +81-75-383-2401. E-mail: [email protected]. (1) Hol, W. G. J. Prog. Biophys. Mol. Biol. 1985, 45, 149–195. (2) Beratan, D. N.; Onuchic, J. N.; Winkler, J. R.; Gray, H. B. Science 1992, 258, 1740–1741. (3) Isied, S. S.; Ogawa, M. Y.; Wishart, J. F. Chem. ReV. 1992, 92, 381–394. (4) Vassilian, A.; Wishart, J. F.; Vanhemelryck, B.; Schwarz, H.; Isied, S. S. J. Am. Chem. Soc. 1990, 112, 7278–7286. (5) Ogawa, M. Y.; Wishart, J. F.; Young, Z. Y.; Miller, J. R.; Isied, S. S. J. Phys. Chem. 1993, 97, 11456–11463. (6) Sisido, M.; Hoshino, S.; Kusano, H.; Kuragaki, M.; Makino, M.; Sasaki, H.; Smith, T. A.; Ghiggino, K. P. J. Phys. Chem. B 2001, 105, 10407–10415. (7) Fox, M. A.; Galoppini, E. J. Am. Chem. Soc. 1997, 119, 5277–5285. (8) Galoppini, E.; Fox, M. A. J. Am. Chem. Soc. 1996, 118, 2299–2300. (9) Shin, Y. G. K.; Newton, M. D.; Isied, S. S. J. Am. Chem. Soc. 2003, 125, 3722–3732. (10) Morita, T.; Kimura, S. J. Am. Chem. Soc. 2003, 125, 8732–8733. (11) Watanabe, J.; Morita, T.; Kimura, S. J. Phys. Chem. B 2005, 109, 14416– 14425. (12) Galka, M. M.; Kraatz, H. B. ChemPhysChem 2002, 3, 356–359. (13) Kraatz, H. B.; Bediako-Amoa, I.; Gyepi-Garbrah, S. H.; Sutherland, T. C. J. Phys. Chem. B 2004, 108, 20164–20172. (14) Sek, S.; Tolak, A.; Misicka, A.; Palys, B.; Bilewicz, R. J. Phys. Chem. B 2005, 109, 18433–18438. (15) Mandal, H. S.; Kraatz, H. B. Chem. Phys. 2006, 326, 246–251. (16) Kai, M.; Takeda, K.; Morita, T.; Kimura, S. J. Pept. Sci. 2008, 14, 192– 202. (17) Long, Y. T.; Abu-Rhayem, E.; Kraatz, H. B. Chem.sEur. J. 2005, 11, 5186–5194. (18) Xiao, X. Y.; Xu, B. Q.; Tao, N. J. J. Am. Chem. Soc. 2004, 126, 5370– 5371.

as better mediation of electron tunneling than hydrocarbon chains,6,18,20-22 a dipole moment effect on electron-transfer direction,7,8 and feasible long-range electron transfer due to the mechanism change from simple electron tunneling to electron hopping.10,11,16,23-27 A molecular dynamics-associated process also affects the electron-transfer efficiency.13,15,17,28,29 With the aim of developing applications of helical peptides for electronically functional molecular materials, we have prepared selfassembled monolayers (SAMs) of helical peptides carrying photosensitizers and have demonstrated that the helical peptide acts as a molecular photodiode.30-35 Furthermore, for unique molecular architectures in solution and on the surface, we have prepared triangle structures composed of three helical peptide chains with covalent linkage36 and through dipole-dipole (19) Sek, S.; Swiatek, K.; Misicka, A. J. Phys. Chem. B 2005, 109, 23121– 23124. (20) Sek, S.; Misicka, A.; Swiatek, K.; Maicka, E. J. Phys. Chem. B 2006, 110, 19671–19677. (21) Antonello, S.; Formaggio, F.; Moretto, A.; Toniolo, C.; Maran, F. J. Am. Chem. Soc. 2003, 125, 2874–2875. (22) Polo, F.; Antonello, S.; Formaggio, F.; Toniolo, C.; Maran, F. J. Am. Chem. Soc. 2005, 127, 492–493. (23) Malak, R. A.; Gao, Z. N.; Wishart, J. F.; Isied, S. S. J. Am. Chem. Soc. 2004, 126, 13888–13889. (24) Petrov, E. G.; May, V. J. Phys. Chem. A 2001, 105, 10176–10186. (25) Petrov, E. G.; Shevchenko, Y. V.; Teslenko, V. I.; May, V. J. Chem. Phys. 2001, 115, 7107–7122. (26) Petrov, E. G.; Hanggi, P. Phys. ReV. Lett. 2001, 86, 2862–2865. (27) Petrov, E. G.; May, V.; Hanggi, P. Chem. Phys. 2002, 281, 211–224. (28) Dey, S. K.; Long, Y. T.; Chowdhury, S.; Sutherland, T. C.; Mandal, H. S.; Kraatz, H. B. Langmuir 2007, 23, 6475–6477. (29) Orlowski, G. A.; Chowdhury, S.; Kraatz, H. B. Langmuir 2007, 23, 12765– 12770. (30) Morita, T.; Kimura, S.; Kobayashi, S.; Imanishi, Y. J. Am. Chem. Soc. 2000, 122, 2850–2859. (31) Morita, T.; Kimura, S.; Kobayashi, S.; Imanishi, Y. Chem. Lett. 2000, 676–677. (32) Yasutomi, S.; Morita, T.; Imanishi, Y.; Kimura, S. Science 2004, 304, 1944–1947. (33) Yasutomi, S.; Morita, T.; Kimura, S. J. Am. Chem. Soc. 2005, 127, 14564– 14565. (34) Morita, T.; Yanagisawa, K.; Kimura, S. Polym. J. 2008, 40, 700–709. (35) Yanagisawa, K.; Morita, T.; Kimura, S. J. Am. Chem. Soc. 2004, 126, 12780–12781.

10.1021/la8034962 CCC: $40.75  2009 American Chemical Society Published on Web 01/30/2009

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Figure 1. Chemical structures of the peptide derivatives and schematic presentation of their self-assembled monolayers on gold.

interaction.37 Here we present a novel double-helix structure as a unique electron-transfer system. The double helix (Figure 1, DHN) is composed of two helical peptide chains connected by a dicarboxylic ferrocene unit at their C terminals. Ferrocene has been most widely used to study electron-transfer reactions through SAMs.38-43 Each peptide chain is a 9mer consisting of three repeats of a sequence of 2-naphthylalanine-R-aminoisobutyric acid-R-aminoisobutyric acid (NapAla-Aib-Aib). Because of the high content of Aib residues, the peptide chain forms a 310-helical structure that makes one turn with three residues and thus arranges the three naphthyl groups in a linear manner along the helix. Both helical peptide chains have a disulfide group at their N terminals for im(36) Yoshida, K.; Kawamura, S.; Morita, T.; Kimura, S. J. Am. Chem. Soc. 2006, 128, 8034–8041. (37) Ishikawa, T.; Morita, T.; Kimura, S. Bull. Chem. Soc. Jpn. 2007, 80, 1483–1491. (38) Chidsey, C. E. D. Science 1991, 251, 919–922. (39) Sikes, H. D.; Smalley, J. F.; Dudek, S. P.; Cook, A. R.; Newton, M. D.; Chidsey, C. E. D.; Feldberg, S. W. Science 2001, 291, 1519–1523. (40) Smalley, J. F.; Finklea, H. O.; Chidsey, C. E. D.; Linford, M. R.; Creager, S. E.; Ferraris, J. P.; Chalfant, K.; Zawodzinsk, T.; Feldberg, S. W.; Newton, M. D. J. Am. Chem. Soc. 2003, 125, 2004–2013. (41) Smalley, J. F.; Sachs, S. B.; Chidsey, C. E. D.; Dudek, S. P.; Sikes, H. D.; Creager, S. E.; Yu, C. J.; Feldberg, S. W.; Newton, M. D. J. Am. Chem. Soc. 2004, 126, 14620–14630. (42) Creager, S.; Yu, C. J.; Bamdad, C.; O’Connor, S.; MacLean, T.; Lam, E.; Chong, Y.; Olsen, G. T.; Luo, J. Y.; Gozin, M.; Kayyem, J. F. J. Am. Chem. Soc. 1999, 121, 1059–1064. (43) Liu, B.; Bard, A. J.; Mirkin, M. V.; Creager, S. E. J. Am. Chem. Soc. 2004, 126, 1485–1492. (44) Gray, H. B.; Winkler, J. R. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 3534–3539.

mobilization onto a gold surface. When the two peptide chains are tethered on gold by gold-sulfur linkages to form a SAM, they will stand parallel to each other by exposing the ferrocene unit on the monolayer surface (Figure 1). Immobilization with the two sites on gold will restrict the molecular motion on the surface compared to the case of tethering to one site. Because of a relatively short electron-transfer distance between the ferrocene unit and gold, electron tunneling may prevail in this monolayer. There are several interesting questions pertaining to this electron-transfer system: (i) Can the side-chain chromophores accelerate the electron transfer by strengthening the electronic coupling? (ii) Do the two electron pathways double the electrontransfer rate constant? (iii) How do restricted molecular motions affect the electron transfer? (iv) Is the substitution number of the ferrocene unit influential? To address these questions, three control peptides were also prepared (Figure 1): a similar double-helix having Ala residues for NapAla residues (DHA), a single helix with a disubstituted ferrocene unit (SHN), and single helix with a monosubstituted ferrocene unit (SHNm). Effects of intervening chromophores11,44-46 and molecular dynamics13,15,17,28,29,47-52 on electron transfer have been of great interest in the field of biological electron transfer. (45) Shih, C.; Museth, A. K.; Abrahamsson, M.; Blanco-Rodriguez, A. M.; Di Bilio, A. J.; Sudhamsu, J.; Crane, B. R.; Ronayne, K. L.; Towrie, M.; Vlcek, A.; Richards, J. H.; Winkler, J. R.; Gray, H. B. Science 2008, 320, 1760–1762. (46) Stubbe, J.; van der Donk, W. A. Chem. ReV. 1998, 98, 705–762. (47) Schlag, E. W.; Sheu, S. Y.; Yang, D. Y.; Selzle, H. L.; Lin, S. H. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 1068–1072.

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Synthesis of Helical Peptides. All of the peptide derivatives were synthesized by the liquid-phase method from DL-R-lipoic acid, (NapAla-Aib-Aib)3 derivatives,35 and monosubstituted11 and disubstituted10 ferrocene units prepared by literature procedures using O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) and N,N-diisopropylethylamine (DIEA) as coupling reagents. All of the intermediates were identified by 1H NMR spectroscopy, and the final products were further confirmed by FAB mass spectrometry. The purity of the compounds was checked by thin-layer chromatography (TLC). The identification data of the final products are shown below. DHN TLC (chloroform/methanol ) 5/1 v/v) Rf ) 0.58. 1H NMR (CDCl3, 400 MHz): δ 1.25-1.51 (84H, AibCH3, SSCH2CH2CH(CH2)3CH2CO), 1.60, 2.28 (4H, SSCH2CH2CH(CH2)3CH2CO), 2.47 (4H, SSCH2CH2CH(CH2)3CH2CO), 3.13 (4H, SSCH2CH2CH(CH2)3CH2CO), 3.18 (8H, NHCH2CH2NH), 3.49 (12H, NapAlaCβH2), 3.57 (2H, SSCH2CH2CH(CH2)3CH2CO), 4.00-4.11 (6H, NapAlaCRH), 4.41-5.00 (8H, ferrocene-H), 7.28-8.61 (64H, naphthyl-H, amideH). MS (FAB, matrix; nitrobenzylalcohol): m/z 2939.33 (calcd for C158H196FeN22O22S4 [(M + H)+] m/z 2938.32). DHA TLC (chloroform/methanol ) 5/1 v/v): Rf ) 0.80. 1H NMR (DMF-d7, 400 MHz): δ 1.37 (18H, AlaCH3), 1.45-1.52 (84H, AibCH3, SSCH2CH2CH(CH2)3CH2CO), 1.60, 1.70, 1.85, 2.47 (4H, SSCH2CH2CH(CH2)3CH2CO), 2.28 (4H, SSCH2CH2CH(CH2)3CH2CO), 3.13 (4H, SSCH2CH2CH(CH2)3CH2CO), 3.19 (8H, NHCH2CH2NH), 3.52 (2H,SSCH2CH2CH(CH2)3CH2CO),4.01-4.20(6H,AlaCRH),4.30-5.00 (8H, ferrocene-H), 7.29-8.10 (22H, amide-H). MS (FAB, matrix; nitrobenzylalcohol): m/z 2182.98 (calcd for C98H160FeN22O22S4 [(M + H)+] m/z 2182.03). SHN TLC (chloroform/methanol ) 10/1 v/v): Rf ) 0.33. 1H NMR (CDCl3, 400 MHz): δ 1.18-1.61 (42H, AibCH3, SSCH2CH2CH(CH2)3CH2CO), 1.82, 2.43 (2H, SSCH2CH2CH(CH2)3CH2CO), 1.99 (3H, CH3CO), 2.40 (2H, SSCH2CH2CH(CH2)3CH2CO), 3.00-3.65 (17H, SSCH2CH2CH(CH2)3CH2CO, NapAlaCβH2, NHCH2CH2NH), 3.68-4.10 (3H, NapAlaCHR), 4.39-4.54 (9H, ferrocene-H), 6.10-8.15 (34H, naphthyl-H, amide-H). MS (FAB, matrix; nitrobenzylalcohol): m/z 1690.75 (calcd for C89H112FeN13O13S2 [(M + H)+] m/z 1691.73). SHNm TLC (chloroform/methanol ) 5/1 v/v): Rf ) 0.67. 1H NMR (CDCl3, 400 MHz): δ 1.15-1.72 (42H, AibCH3, SSCH2CH2CH(CH2)3CH2CO), 1.82, 2.38 (2H, SSCH2CH2CH(CH2)3CH2CO), 2.29 (2H, SSCH2CH2CH(CH2)3CH2CO), 3.02-3.62 (17H, SSCH2CH2CH(CH2)3CH2CO, NapAlaCβH2, NHCH2CH2NH), 3.68-4.10 (3H, NapAlaCHR), 4.21-4.56 (9H, ferrocene-H), 6.22-8.20 (32H, naphthylH, amide-H). MS (FAB, matrix; nitrobenzylalcohol): m/z 1562.75 (calcd for C84H104FeN11O11S2 [(M + H)+] m/z 1562.67). Spectroscopy in Solution. The absorption and fluorescence spectra of the peptides carrying naphthyl groups (DHN, SHN, and SHNm) in ethanol were recorded on a Shimadzu UV-2450PC spectrometer and a Jasco FP-6600 fluorometer, respectively, at a naphthyl

concentration of ca. 1 × 10-5 M. The excitation wavelength for fluorescence spectra was 280 nm, and the slit widths were 3 nm for excitation and 6 nm for emission. The CD spectra of all of the peptides were measured in ethanol on a Jasco J-820 CD spectropolarimeter with an optical cell having a 0.1 cm optical path length. The residue concentration was set at ca. 1.0-3.0 × 10-4 M for the naphthyl-carrying peptides, and the residue concentration was set at ca. 1.0 × 10-3 M for DHA. All measurements were carried out at room temperature. Preparation of SAMs. A glass slide was immersed in sulfuric acid overnight, rinsed thoroughly with water and methanol, and dried in vacuum for 15 min. Chromium and then gold (99.99%) were deposited on the glass slide to make a gold substrate via an Osaka Vacuum N-KS350 vacuum deposition system. The thicknesses of the chromium and gold layers, monitored by a quartz oscillator, were approximately 300 and 2000 Å, respectively. The surface roughness of a similar gold substrate has been determined by atomic force microscopy to be less than 1.05. The prepared gold substrate was immediately used for self-assembly. The gold substrate was immersed in an ethanol solution of the peptide (ca. 0.1 mM) for 24 h, rinsed thoroughly with ethanol, chloroform, and chloroform/methanol (1/1 v/v) in this order, and dried in a steam of dry nitrogen gas and in vacuum for 15 min. To fill in monolayer defects, the peptidemodified substrate was placed in a test tube, the test tube was placed in a round-bottomed flask with dodecanethiol (ca. 3 mL) at the bottom, and the system was kept at 80 °C for 2 h in an oil bath.53 After the treatment, the substrate was rinsed thoroughly with ethanol and toluene and dried in a steam of dry nitrogen gas and in vacuum for 15 min. Monolayer Characterizations. IRRAS spectra were recorded on a Thermo Fisher Scientific Nicolet 6700 Fourier transform infrared spectrometer at room temperature with a Harrick RMA-1DG/VRA reflection attachment. The incident angle was set at 80-85° from the surface normal. The number of interferogram accumulations was 200. The tilt angles of the helix axis from the surface normal were determined from the amide I/II absorbance ratio by using an equation in the literature.10,30,32,54-56 Ellipsometry was carried out with a Mizojiri Optical DHA-OLX/S autoellipsometer at room temperature to determine the thicknesses of the monolayers. A helium-neon laser at a wavelength of 632.8 nm was used as the incident light, and its incident angle was set at 65°. The complex optical constant of the monolayer was assumed to be 1.50 + 0.00i. The thickness of the monolayer was calculated automatically by an equipped program. The thicknesses were measured on more than five different spots on the surface, and the data were averaged. The typical standard deviation was ca. 1 Å. Electrochemical Measurements. Electrochemical experiments were performed with a BAS model 604 voltammetric analyzer at room temperature on a three-electrode system with the monolayermodified gold substrate as the working electrode, Ag/AgCl/3 M NaCl(aq) as the reference electrode, and a platinum wire as the auxiliary electrode. Milli-Q water was used to prepare the solutions. The sealed electrochemical cell with the solution was flushed with nitrogen gas for 15 min prior to the experiments. All of the applied potentials on the working electrode reported here are with respect to the reference electrode. The area of the working electrode exposed to the electrolyte solution was 0.9-1.1 cm2. The uncompensated resistance of the cell, estimated by impedance spectroscopy, was ca. 3 to 4 Ω. The blocking experiment by CV to assess the monolayer defects was carried out in a 1 mM K4[Fe(CN)6] + 1 M KCl aqueous solution at a 0.1 V/s scan rate. CV and CA were used to study the electron transfer that was carried out in a 1 M HClO4 aqueous solution. CV was performed at a 0.1 V/s scan rate. The monolayer capacitances were determined by dividing the current at a certain potential away

(48) Schlag, E. W.; Yang, D. Y.; Sheu, S. Y.; Selzle, H. L.; Lin, S. H.; Rentzepis, P. M. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 9849–9854. (49) Meggers, E.; Michel-Beyerle, M. E.; Giese, B. J. Am. Chem. Soc. 1998, 120, 12950–12955. (50) Wan, C. Z.; Fiebig, T.; Kelley, S. O.; Treadway, C. R.; Barton, J. K.; Zewail, A. H. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 6014–6019. (51) O’Neill, M. A.; Barton, J. K. J. Am. Chem. Soc. 2004, 126, 13234–13235. (52) O’Neill, M. A.; Barton, J. K. J. Am. Chem. Soc. 2004, 126, 11471–11483.

(53) Donhauser, Z. J.; Mantooth, B. A.; Kelly, K. F.; Bumm, L. A.; Monnell, J. D.; Stapleton, J. J.; Price, D. W.; Rawlett, A. M.; Allara, D. L.; Tour, J. M.; Weiss, P. S. Science 2001, 292, 2303–2307. (54) Tsuboi, M. J. Polym. Sci. 1962, 59, 139. (55) Gremlich, H. U.; Fringeli, U. P.; Schwyzer, R. Biochemistry 1983, 22, 4257–4264. (56) Kennedy, D. F.; Crisma, M.; Toniolo, C.; Chapman, D. Biochemistry 1991, 30, 6541–6548.

The peptide derivatives were synthesized by the liquid-phase method. The electronic interactions and arrangement of the naphthyl groups and the peptide conformation were studied by absorption, fluorescence, and circular dichroism (CD) spectroscopy in solution. The peptides were immobilized on gold to form a SAM, and monolayer defects were filled in with dodecanethiol from the gas phase. The formed monolayers were characterized by infrared reflection-absorption spectroscopy (IRRAS), ellipsometry, and cyclic voltammetry (CV), and the electron transfer from the ferrocene unit to gold was studied by CV and chronoamperometry (CA).

Materials and Methods

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Figure 2. (a) Absorption and (b) fluorescence spectra of the peptides carrying naphthyl groups (DHN, SHN, and SHNm) at ca. 1 × 10-5 M of the naphthyl group. The excitation wavelength for fluorescence spectroscopy was 280 nm.

from the ferrocene oxidation, where the charging current is dominant, by the scan rate in the cyclic voltammograms. Assuming the monolayer to be a plate condenser, the relative dielectric constant (εm) was determined by εm ) Cmd/(εvS) in which Cm is the monolayer capacitance, d is the ellipsometry thickness, εv is the vacuum permittivity, and S is the electrode area. In CA, the time constant in the current follower of the potentiostat was set at 10-4 s. At time zero, the potential was stepped from formal oxidation potential overpotential (V) to formal oxidation potential + overpotential (V). The SHNm monolayer with a monosubstituted ferrocene unit was examined at +0.04, +0.08, +0.12, and +0.16 V overpotentials, and the obtained current-time curves were fitted by a double-exponential function in the region of 0.001-0.01 s to afford two decay components. The slow decay components serving as the electrontransfer rate constants (ket) were plotted on overpotentials (Tafel plot) to determine the standard rate constant (ket0). Unfortunately, because the disubstituted ferrocene unit of the other peptides (DHN, DHA, and SHN) was unstable under a positive potential, the experiments at multiple overpotentials failed. During the experiments, the ferrocene unit rapidly decomposed; accordingly, the current response from electron transfer was attenuated. Therefore, the ket values at a +0.08 V overpotential obtained with fresh monolayer samples were determined and compared. At least three separate experiments were carried out on different monolayer samples for each molecule, and the results were averaged. To validate the rate constant determined by CA, electrochemical impedance spectroscopy (EIS) was performed on the SHNm SAM by the method reported in our previous work.57 Briefly, the Bode plot obtained at the formal oxidation potential was fitted by an equivalent circuit consisting of the solution resistance (Rs), monolayer constant phase element,58,59 and electron-transfer resistance (Ret) and capacitance (Cet),60-62 and ket0 was calculated by ket0 ) 1/(2RetCet). Similar measurements on the other SAMs failed because of the instability of the disubstituted ferrocene unit when it was oxidized. Molecular Modeling. Molecular modeling was performed on Fujitsu CAChe WorkSystem 6.1.1 software. Initial geometry was generated for each molecule. The dihedral angles of the peptide backbone were set to be ω ) 180°, φ ) -60°, and ψ ) -30°, respectively, to produce a 310-helical structure. A parallel arrangement of helices was assumed for the double-helix peptides (DHN and (57) Takeda, K.; Morita, T.; Kimura, S. J. Phys. Chem. B 2008, 112, 12840– 12850. (58) Wu, X. Z.; Zhang, W. Z.; Hou, Y. J. Electroanal. Chem. 1995, 398, 1–4. (59) Protsailo, L. V.; Fawcett, W. R. Electrochim. Acta 2000, 45, 3497–3505. (60) Steinem, C.; Janshoff, A.; Ulrich, W. P.; Sieber, M.; Galla, H. J. Biochim. Biophys. Acta 1996, 1279, 169–180. (61) Yamada, T.; Nango, M.; Ohtsuka, T. J. Electroanal. Chem. 2002, 528, 93–102. (62) Creager, S. E.; Wooster, T. T. Anal. Chem. 1998, 70, 4257–4263.

DHA). The initial geometry was then optimized by the Molecular Mechanics program 2 (MM2) method and the semiempirical Austin model 1 (AM1) method in the MOPAC 2002 package on the same software. The length along the molecular long axis (helix axis) was estimated to be ca. 33 Å for each molecule, and thus the theoretical monolayer thickness was estimated by 33[cos(IRRAS tilt angle)] (Å). On the other hand, the theoretical molecular surface densities were calculated from the tilt angles and the cross-sectional molecular areas with hexagonal packing of the molecules. The cross-sectional molecular areas were estimated from the optimized geometries and their inverses correspond to the limiting surface densities with a 0° tilt angle, which are 5.4 × 10-11 mol/cm2 for DHN, 11.5 × 10-11 mol/cm2 for DHA, and 11.0 × 10-11 mol/cm2 for SHN and SHNm, respectively. The limiting surface density × cos(IRRAS tilt angle) gave the theoretical surface density of the molecules.

Results and Discussions Synthesis and Spectroscopy. The peptide derivatives (DHN, DHA, SHN, and SHNm; Figure 1) were synthesized by the liquidphase method. The electronic interactions between the naphthyl groups in the peptides carrying naphthyl groups (DHN, SHN, and SHNm) were studied in ethanol. Their absorption spectra at the same naphthyl concentrations are shown in Figure 2a. Characteristic absorption peaks for a naphthalene monomer are observed at 268, 276, and 288 nm (shoulder).63 The fluorescence spectra at the same naphthyl concentrations are shown in Figure 2b. Only monomer emission of naphthalene is observed at 329, 337, and 354 nm (shoulder),63 and the spectra are free from excimer emission at longer wavelengths. These results indicate that there is no strong interaction between the neighboring naphthyl groups either in the ground state or in the excited state and suggest that the naphthyl groups are regularly spaced along the helical peptide. However, the fluorescence intensities are different. DHN shows weaker emission than SHN. This may suggest self-quenching between the naphthyl groups on different peptide chains in DHN. The emission from SHNm is also weaker than that from SHN. The reason is unclear, but one possibility is that the monosubstituted ferrocene unit may partially quench the nearby naphthyl group. The arrangement of the naphthyl groups and the peptide conformation were examined by CD spectroscopy. The results for the naphthyl-carrying peptides are shown in Figure 3a. A pair (63) Du, H.; Fuh, R. C. A.; Li, J. Z.; Corkan, L. A.; Lindsey, J. S. Photochem. Photobiol. 1998, 68, 141–142.

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Figure 3. (a) CD spectra of the naphthyl-carrying peptides (DHN, SHN, and SHNm) at a residue concentration of ca. (1.0-3.0) × 10-4 M, (b) optimized geometry of the peptide chain carrying naphthyl groups where the terminal groups and hydrogen atoms are omitted for clarity, and (c) CD spectrum of DHA at a residue concentration of ca. 1.0 × 10-3 M.

of strong splitting peaks with a positive sign at a shorter wavelength (ca. 219 nm) and a negative sign at a longer wavelength (ca. 229 nm) are observed. The center wavelength is around 225 nm, which is the 1Bb transition of a naphthyl group along its long axis.64,65 On the basis of the exciton chirality theory,64-69 this type of CD pattern indicates that the chromophores are closely arranged in a counterclockwise manner from the view along the helix axis. The plausible peptide conformation was estimated by computational molecular modeling. The initial geometry was generated using typical dihedral angles for a 310-helix and was optimized by the MM2 and AM1 methods. The calculation gave a 310-helical structure with counterclockwise arrangement of the naphthyl groups as an energy-minimized geometry (Figure 3b), which agrees with the CD observation. On the other hand, DHA shows a typical CD pattern of a 310-helix with a negative peak at 205 nm and a shoulder at 225 nm (Figure 3c).70 The molar ellipticity of the peptide backbone is on the order of 103 deg cm2 dmol-1 residue whereas that of the naphthyl groups is on the order of 105 deg cm2 dmol-1 residue at the peaks. Thus, the CD signal from the naphthyl groups is dominant over that from the peptide backbone in the spectra of DHN, SHN, and SHNm. We conclude that these peptides take the 310-helical conformation as designed. Monolayer Characterization. The peptide SAMs were prepared on gold by the immersion of a gold substrate in an ethanol solution of the respective peptides and filling in the monolayer defects with dodecanethiol from the gas phase. Molecular orientation was studied by IRRAS spectroscopy. The spectra are shown in Figure 4. Amides I and II are observed at around 1670 and 1540 cm-1 and are characteristic wave numbers for the helical conformation.56 The tilt angles of the helices from the surface normal were determined on the basis of the amide I and II absorbance ratio.10,30,32,54-56 The tilt angles are 42° for DHN, 45° for DHA, 45° for SHN, and 49° for SHNm SAMs, (64) Sisido, M.; Egusa, S.; Imanishi, Y. J. Am. Chem. Soc. 1983, 105, 4077– 4082. (65) Sisido, M.; Egusa, S.; Imanishi, Y. J. Am. Chem. Soc. 1983, 105, 1041– 1049. (66) Woody, R. W. J. Chem. Phys. 1968, 49, 4797–4806. (67) Sisido, M.; Imanishi, Y. Macromolecules 1985, 18, 890–894. (68) Inai, Y.; Hirabayashi, T. Biopolymers 2001, 59, 356–369. (69) Inai, Y.; Oshikawa, T.; Yamashita, M.; Hirabayashi, T.; Hirako, T. Biopolymers 2001, 58, 9–19. (70) Toniolo, C.; Polese, A.; Formaggio, F.; Crisma, M.; Kamphuis, J. J. Am. Chem. Soc. 1996, 118, 2744–2745.

Figure 4. IRRAS spectra of the peptide SAMs.

respectively (Table 1). This result shows that the helical peptide chains have a comparably upright orientation in all of the monolayers. The theoretical thicknesses of the monolayers estimated from the molecular length (33 Å) and the tilt angles agree well with the experimental thicknesses determined by ellipsometry (Table 1). This good agreement indicates that the peptides form a well-defined monolayer with uniform molecular orientation. Monolayer packing was examined by CV in an aqueous solution with redox ferrocyanide ions. The cyclic voltammograms of the control bare gold and the SAMs are shown in Figure 5. Clear redox peaks of the ferricyanide/ferrocyanide couple are observed for the bare gold, whereas they are completely suppressed in the presence of the peptide SAMs on gold. This shows that the monolayers do not have defects that allow the ferrocyanide ions to diffuse and make contact with the gold surface. A monotonic increase in current is observed in the SHN and SHNm monolayers at above 0.3-0.35 V. This current may be attributed to the tunneling current between the redox species in the aqueous phase and gold through the monolayer,71 which is allowed by the smaller thicknesses of the single-helix monolayers. (71) Finklea, H. O. In Electroanalytical Chemistry; Bard, A. J., Rubinstein, I., Eds.; Marcel Dekker: New York, 1993; Vol. 19, pp 177-220.

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Okamoto et al. Table 1. Data Summary

tilt angle (deg) theoretical monolayer thickness (Å) experimental thickness (Å) formal oxidation potential (V) peak separation (mV) peak full width at half-maximum (mV) theoretical surface density (× 10-11 mol/cm2) experimental surface density (× 10-11 mol/cm2) monolayer capacitance per unit area (µF/cm2) dielectric constant of the monolayer electron-transfer rate constant at +0.08 V overpotential (s-1)

DHN SAM

DHA SAM

SHN SAM

SHNm SAM

42 25 26 0.65 63 125 4.0 4.4 6.1 18 69 ( 29

45 23 25 0.66 21 105 8.1 8.0 10.8 31 71 ( 4

45 23 18 0.64 13 105 7.8 6.2 26.3 53 209 ( 46

49 22 22 0.45 26 95 7.2 6.3 7.9 19 73 ( 6

Electrochemical Measurements. To study the electron transfer between the ferrocene unit and gold, cyclic voltammetry was performed in an HClO4 solution. Figure 6 shows the cyclic voltammograms of the SAMs. Reversible redox peaks of the ferrocene oxidation are observed at the formal potential of 0.64-0.66 V for the disubstituted ferrocene unit (DHN, DHA, and SHN) and 0.45 V for the monosubstituted ferrocene unit (SHNm, Table 1). The peak full widths at half-maximum are 95-125 mV (Table 1), some of which are slightly larger than the ideal value (90 mV), suggesting that there is a slight interaction among the ferrocene units.28,72,73 This is reasonable because the ferrocene units are arranged at a relatively high density on the surface. The surface densities of the ferrocene unit were calculated by integration of the anodic peaks. The obtained experimental molecular densities agree well with the calculated densities estimated from the IRRAS tilt angles (Table 1), supporting the regularity and homogeneity of the monolayers. The monolayer capacitances were determined from the charging currents in the cyclic voltammograms, and the dielectric constants of the monolayers were estimated (Table 1). The high capacitance and accordingly high dielectric constant of the SHN SAM indicate that this monolayer has defects that allow the penetration of water molecules and ionic species from the aqueous phase. The dangling chain on the ferrocene unit might have disturbed molecular packing. The capacitances of the other SAMs are much smaller than that of the SHN SAM but are still larger than those of well-packed long-chain alkanethiol SAMs (typically 1-5 µF/ cm2).71,74 However, taking into consideration the fact that the dielectric constants of peptides are higher than those of alkyl chains, the penetration of solvent molecules, and the hydrated layer at the monolayer surface, the dielectric constants of these SAMs (18-31) look reasonable for a well-packed helical peptide monolayer. The slightly larger dielectric constant of the DHA SAM may be due to the absence of hydrophobic side-chain naphthyl groups. It should be noted that the ferrocene surface densities did not change upon dodecanethiol treatment and thus the peptides were not replaced by dodecanethiol. To discuss the electron-transfer rate constants, CA was performed in an HClO4 solution. Time courses of the currents of the SHNm SAM with a monosubstituted ferrocene unit at various overpotentials are shown in Figures 7a. The 1-10 ms regions where the charging current is negligible are successfully fitted by a double-exponential function to give two decay constants. The logarithmic natural values of the decay constants are plotted over the overpotentials in Figure 7b (Tafel plot). The time constant of the charging current must be less than 0.1 ms under the conditions of the uncompensated resistance of 3 to 4 (72) Brown, A. P.; Anson, F. C. Anal. Chem. 1977, 49, 1589–1595. (73) Sek, S.; Palys, B.; Bilewicz, R. J. Phys. Chem. B 2002, 106, 5907–5914. (74) Sek, S.; Sepiol, A.; Tolak, A.; Misicka, A.; Bilewicz, R. J. Phys. Chem. B 2004, 108, 8102–8105.

Ω, the monolayer capacitance being less than 10 µF/cm2, and the time constant of the current follower of 0.1 ms. Therefore, the fast decay components with time constants of a few milliseconds cannot be attributed to the charging current or to any artifact by the current follower. As a significant difference, the fast decay components are independent of the overpotentials, and the slow decay component exponentially increases as the overpotential increases. The latter overpotential dependence is typical for electron tunneling, and thus the slow decay components should be assigned to the electron-transfer rate constants due to ferrocene oxidation (ket).73,75-78 Extrapolation to the zero overpotential gave a standard electron-transfer rate constant (ket0) of ca. 40 s-1. Furthermore, the EIS measurement was also carried out for the SHNm SAM (data not shown) to give ket0 of ca. 30 s-1, which coincides well with the value from the CA analysis. This agreement confirms that the slow decay components originate from the ferrocene oxidation, although the fast decay components are yet to be identified. The slope of the Tafel plot (6.5/V) is substantially smaller than the typical slope reported for ferrocene-terminated alkanethiol SAMs76 (ca. 18/V with a reorganization energy of ca. 0.9 eV at low overpotentials). One possibility is that not only electron tunneling but also electron hopping are operative. Our previous study suggested that the transition from electron tunneling to hopping occurs somewhere in the 12-24 Å monolayer thickness as the helical peptide chain length increases.16 It is thus considered that the contributions of the electron tunneling and hopping mechanisms to the rate constants may become comparable in the SHNm SAM because the monolayer thickness is 22 Å. The rate constants of electron hopping are independent of overpotentials, and thus the mixing of electron tunneling and hopping decreases the apparent slope of the Tafel plot. The ferrocene surface densities were determined from the preexponential factors of the slow decay components to be 4.7 × 10-11, 8.7 × 10-11, 11.1 × 10-11, and 12.3 × 10-11 mol/cm2 at 0.04, 0.08, 0.12, and 0.16 V overpotentials, respectively.71 The determined ferrocene density at a 0.04 V overpotential is less than the ferrocene density determined by CV (6.3 × 10-11 mol/cm2), but this is acceptable because not all of the ferrocene units are oxidized at a low overpotential. However, it is a problem that the ferrocene densities at the other overpotentials are larger than that determined by CV. One possible explanation is that the charging current may increase with the penetration of water and ions into the monolayer. Such an effect becomes more significant when the potential gets more positive, as shown by the widened (75) Finklea, H. O.; Ravenscroft, M. S.; Snider, D. A. Langmuir 1993, 9, 223–227. (76) Sek, S.; Misicka, A.; Bilewicz, R. J. Phys. Chem. B 2000, 104, 5399– 5402. (77) Finklea, H. O.; Yoon, K.; Chamberlain, E.; Allen, J.; Haddox, R. J. Phys. Chem. B 2001, 105, 3088–3092. (78) Finklea, H. O.; Hanshew, D. D. J. Am. Chem. Soc. 1992, 114, 3173–3181.

Electron Transfer through a Double-Helix Peptide

Figure 5. Cyclic voltammograms of a bare gold substrate and the SAMmodified substrates in a 1 mM K4[Fe(CN)6] + 1 M KCl aqueous solution at a scan rate of 0.1 V/s.

Figure 6. Cyclic voltammograms of the SAMs in a 1 M HClO4 aqueous solution at a scan rate of 0.1 V/s.

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charging current traces after the ferrocene oxidation in the cyclic voltammogram (Figure 6). If the aqueous species into the monolayer would penetrate slowly into the monolayer because of limited diffusion, then this process might occur in the same time region of ferrocene oxidation, which should increase the apparent electron-transfer current, resulting in an overestimation of the surface coverage. Unfortunately, similar CA measurements at multiple overpotentials and EIS measurements for the other monolayers were unsuccessful because of the instability of the disubstituted ferrocene unit when it was oxidized. The disubstituted ferrocene unit rapidly decomposed under a positive potential. Therefore, single CA experiments on fresh monolayer samples were carried out at a +0.08 V overpotential (Figure 7c), and the ket values determined from the slow decay components were compared. More than three separate experiments were carried out using different monolayer samples for each molecule, and the obtained ket values were averaged (Table 1). The ket values are 69 ( 29 s-1 for DHN, 71 ( 4 s-1 for DHA, 209 ( 46 s-1 for SHN, and 73 ( 6 s-1 for SHNm SAMs, respectively. Discussion of Electron-Transfer Rate Constants. The electron-transfer rate constant of the DHN SAM with the sidechain naphthyl groups is statistically the same as that of the DHA SAM without the chromophores. This result indicates that there is no enhancement of electronic coupling between the ferrocene unit and gold through the chromophores. The energy of the tunneling electron is at +0.73 V with the application of a 0.08 V overpotential versus the Ag/AgCl reference, whereas the energy levels of the frontier molecular orbitals, HOMO and LUMO, of the naphthyl group are roughly approximated to be ca. +1.6 and -2.3 V79 versus the Ag/AgCl reference, respectively, which are the oxidation and reduction potentials of naphthalene in solution. Therefore, no effect of the naphthyl groups on electron transfer in these peptide SAMs is attributed to poor coupling of the tunneling electron with the molecular orbitals of the naphthyl group. The electron transfer in the single-helix SHN SAM is 3-fold faster than that of the double-helix DHN SAM. The capacitance determined by the CV measurement has shown that the SHN SAM has substantial defects. The faster electron transfer may be attributed to the difference in molecular dynamics: molecular motions occur more freely in the loosely packed SHN SAM to

Figure 7. CA results: (a) time courses of the currents in the SHNm SAM at various positive overpotentials, (b) Tafel plots of the two decay constants obtained for the SHNm SAM (open circles are the fast components and closed circles are the slow components), and (c) time courses of the currents in all of the SAMs at a 0.08 V overpotential. The circles are the experimental data, and the black solid lines are the fitting results with a doubleexponential function.

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accelerate the electron transfer.13,15 However, it is also possible that the terminal ferrocene unit has more chances to be located closer to gold in the loosely packed monolayer, leading to the acceleration of electron transfer by simple shortening of the electron-transfer distance. At the moment, the difference in the rate constants between the DHN and SHN SAMs cannot be discussed only from the point of view of the different number of electron-transfer pathways. For the same reason, the result of the SHN SAM cannot be compared with that of the SHNm SAM only from the point of view of the substitution number of the ferrocene unit. However, we can have an in-depth discussion of the electrontransfer reaction as follows. The rate constant of the DHN SAM is comparable to that of the SHNm SAM despite multiple differences between the two SAMs: the substitution number of the ferrocene unit, the number of electron-transfer pathways, and the degrees of freedom in molecular motion. It was reported that electron transfer with a disubstituted ferrocene unit is slightly faster than that with a monosubstituted ferrocene unit because of its smaller reorganization energy.29 The activation energy of electron tunneling between a ferrocene unit and gold in a SAM is influenced by solvent reorganization around the ferrocene unit, and a disubstituted ferrocene unit has less contact area with surrounding ions and solvent molecules than does a monosubstituted one.29,76,80,81 It was estimated at most a 2-fold difference in the electron-transfer rate constants between the disubstituted and monosubstituted ferrocenes.29 The double helix has another acceleration factor for the 2-fold electron-transfer pathways against the single helix. It is thus expected that the electrontransfer rate in the DHN SAM would show 4-fold acceleration of the SHNm SAM. However, the experimental result of the rate constant of the DHN SAM was comparable to that of the SHNm SAM. Some deceleration effect is therefore operative in the DHN SAM to cancel out the above two acceleration effects. To explain this situation, we propose that molecular motions are restricted by the double-helix structure tethered by two linkers on gold, which should have a deceleration effect on electron transfer in the DHN SAM.

Okamoto et al.

chromophores are connected by a ferrocene unit at their C terminals. The remaining terminals were tethered to the gold surface by gold-sulfur linkages to form a self-assembled monolayer. The monolayer defects were filled in with dodecanethiol. Three other reference molecules were also prepared and similarly self-assembled on gold. They formed a homogeneous monolayer with upright molecular orientation clarified by infrared spectroscopy, ellipsometry, and cyclic voltammetry. Electron transfer from the ferrocene unit to gold was studied by electrochemical methods. Comparison between the double-helix monolayers with and without side-chain naphthyl groups indicates that there is no effect of the side-chain chromophores on the enhancement of the electronic coupling to accelerate electron tunneling. Interestingly, the double-helix monolayer with a disubstituted ferrocene unit showed an electron-transfer rate constant comparable to that of the single-helix monolayer with a monosubstituted ferrocene unit. The restricted molecular motions in the double-helix monolayer should decelerate the electron transfer to cancel out acceleration effects of the small reorganization energy of the disubstituted ferrocene unit and the 2-fold electron-transfer pathways in the double helix. These findings will enable us to control the electron transfer in a helical peptide monolayer in many ways. A system to accelerate the electron transfer is of course important, but a multicomponent system with multiple electron-transfer rates may also be interesting. The preparation of such systems is in progress. Acknowledgment. This work was partially supported by Grants-in-Aid for Young Scientists B (16750098), for Young Scientists A (20685009), for Exploratory Research (17655098), and for Scientific Research B (15350068) and by the Global COE Program, International Center for Integrated Research and Advanced Education in Materials Science, from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. LA8034962

Conclusions In this study, we synthesized a unique molecular structure, a double helix, in which two helical peptides carrying side-chain

(79) Kavarnos, G. J.; Turro, N. J. Chem. ReV. 1986, 86, 401–449. (80) Weber, K. S.; Creager, S. E. J. Electroanal. Chem. 1998, 458, 17–22. (81) Orlowski, G. A.; Kraatz, H. B. Electrochim. Acta 2006, 51, 2934–2937.