Langmuir 1998, 14, 6935-6940
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Formation of Oriented Helical Peptide Layers on a Gold Surface Due to the Self-Assembling Properties of Peptides Yoshiko Miura and Shunsaku Kimura* Department of Material Chemistry, Graduate School of Engineering, Kyoto University, Yoshida Honmachi Sakyo-ku, Kyoto 606-8501, Japan
Yukio Imanishi Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara 630-0101, Japan
Junzo Umemura Institute for Chemical Research, Kyoto University, Gokanosho, Uji, Kyoto 611-0011, Japan Received September 22, 1998. In Final Form: September 29, 1998 Several hydrophobic R-helical peptides containing a disulfide group were synthesized, and the formation of oriented self-assembled monolayers (SAMs) on gold surface was investigated. The orientation of helices in the SAMs was determined by Fourier transform infrared reflection-absorption spectroscopy measurements. The tilt angle of the helix axis from the surface normal was sensitively affected by the choice of solvent used in the preparation of SAMs, the nature of component amino acids, the molecular structure (either one-helix or two-helix peptide), and the length of peptide chain. The tilt angle was smaller when ethanol was used in the preparation of SAMs rather than N,N-dimethylformamide. Lipo-(Ala-Aib)8-OBzl (Lipo and OBzl represent a lipoic acid group and a benzyl ester group, respectively) showed a smaller tilt angle than Lipo-(Lys(Z)-Aib)8-OCH3. A one-helix peptide with a lipoic acid group at the N-terminal showed a smaller tilt angle than a two-helix peptide, in which the two helix peptides were connected by a disulfide linkage. The longest peptide, Lipo-(Ala-Aib)12-OBzl, showed the smallest tilt angle of 30° when the SAM was prepared in an ethanol solution. Taken together, helix-helix interaction should play a more important role in regulating the orientation of helical peptides in the SAM than the Au-disulfide interaction. Lipo(Ala-Aib)12-OBzl formed a nearly vertically oriented SAM with a parallel orientation of helices due to the highly self-assembling properties of the peptide.
Introduction The R-helix is a major structural unit of proteins and frequently associates with other helices in proteins to form a helix-bundle structure,2 reflecting the good self-assembling properties of helical peptides. Peptide helices have been shown to be neatly packed into monolayers. For example, Boc-(Ala-Aib)8-OBzl and its derivatives formed a two-dimensional crystal when they were spread at the air-water interface.3,4 Helical peptides can, therefore, be considered as possible materials for the construction of supramolecular systems.5 We have reported the formation of a thin layer helical peptide, where the helices are oriented nearly vertically to the surface of substrate (the tilt angle of helices from the surface normal was 28°).6 The helices were immobilized on the surface 1
* E-mail,
[email protected]; fax, +81-75-7534911, tel, +81-75-753-5628. (1) Branden, C.; Tooze, J. Introduction to Protein Structure; Garland Publishing: New York & London, 1991. (2) Otoda, K.; Kimura, S.; Imanishi, Y. J. Chem. Soc., Perkin Trans. 1 1993, 23, 3011. (3) (a) Fujita, K.; Kimura, S.; Imanishi, Y.; Rump, E.; Ringsdorf, H. Langmuir 1994, 10, 2731. (b) Fujita, K.; Kimura, S.; Imanishi, Y.; Rump, E.; Ringsdorf, H. Langmuir 1995, 11, 253. (4) (a) Fujita, K.; Kimura, S.; Imanishi, Y.; Okamura, E.; Umemura, J. Langmuir 1995, 11, 1675. (b) Imanishi, Y.; Fujita, K.; Miura, Y.; Kimura, S. Supramol. Sci. 1996, 3, 171. (5) (a) Imanishi, Y.; Kimura, S. Fundamental Investigations on the Creation of Biofunctional Materials; Kagaku-Dojin: Kyoto, 1991; pp 173-181. (b) Imanishi, Y.; Kimura, S. Proc. Jpn. Acad., Ser. B 1992, 68, 121.
by complexation between a surface ammonium group and a crown ether group at the peptide terminal. In the present study, the thin layer formation of helical peptides by spontaneous chemisorption of the peptide-thiolate conjugates to a gold surface was attempted.7 In the selfassembled monolayers (SAMs), a thin layer of helical peptides is immobilized by chemical bonding to the surface of the substrate, and the peptide molecules orient themselves in the same direction when they adopt a vertical orientation on the surface (Figure 1). Several research groups have reported the formation of thin membranes of R-helical peptides on suitable substrates.8-13 A helical peptide thin layer with nearly (6) Miura, Y.; Kimura, S.; Imanishi, Y.; Umemura, J. Langmuir 1998, 14, 2761. (7) Ulman, A. An Introduction to Ultrathin Organic Films: From Langmuir-Blodgett to Self-Assembly; Academic Press: Boston, MA, 1991. (8) Jaworek, T.; Neher, D.; Wegner, G.; Wieringa, R. H.; Schouten, A. J. Science 1998, 279, 57. (9) (a) Enriquez, E. P.; Gray, K. H.; Guarisco, V. F.; Linton, R. W.; Mar, K. D.; Samulski, E. T. J. Vac. Sci. Technol., A 1992, 10, 2775. (b) Enriquea, E. P.; Samulski, E. T. Mater. Res. Soc. Symp. Proc. 1992, 225, 423. (c) Worley, C. G.; Linton, R. W.; Samulski, E. T. Langmuir 1995, 11, 3805. (10) (a) Whitesell, J. K.; Chang, H. K. Science 1993, 261, 73. (b) Whitesell, J. K.; Chang, H. K.; Whitesell, C. S. Angew. Chem., Int. Ed. Engl. 1994, 33, 871. (11) (a) Wieinga, R. H.; Schouten, A. J. Macromolecules 1996, 29, 3032. (b) Heise, A.; Menzel, H.; Yim, H.; Foster, M. D.; Wieringa, R. H.; Schouten, A. J.; Erb, V.; Stamm, M. Langmuir 1997, 13, 723. (12) Chang, Y.; Frank, C. W. Langmuir 1996, 12, 5824. (13) Boncheva, M.; Vogel, H. Biophys. J. 1997, 73, 1056.
10.1021/la981296d CCC: $15.00 © 1998 American Chemical Society Published on Web 10/30/1998
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Figure 1. Schematic illustration of SAM of helical peptide.
Figure 2. Molecular structure of peptide derivatives.
vertical orientation has been reported with a heneicosapeptide.13 However, in most cases, the helices were laid down on a gold surface or were shown to adopt a nearly random orientation as indicated by the amide I/amide II absorbance ratio in the Fourier transform infrared reflection-absorption spectroscopy (FTIR-RAS) being less than 2. The molecular structure of the peptide thin membrane will reflect sensitively the self-assembly properties of the peptide. In other words, the helix alignment in the peptide thin membrane should be strongly dependent on the preparative conditions as well as the molecular structure of the peptide. Therefore, in the present study, peptide thin membranes were prepared by considering the nature of solvent used for the preparation of the peptide membranes, the nature of component amino acids, the molecular structure of the helical peptides (either one-helix or two-helix), and the length of the peptide chain. Our aim is to establish the molecular design and preparative conditions for a helix peptide membrane that is oriented vertically to the surface of substrate. Experimental Section Materials. Peptides. All peptides (Figure 2) were synthesized by the conventional liquid-phase method. Dicyclohexylcarbodiimide and 1-hydroxybenzotriazole were used for coupling reactions. Where the coupling reaction was difficult to initiate, the condensation reaction was carried out using O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate. The tert-butyloxycarbonyl (Boc) and benzyl ester (OBzl) groups for protection of peptide terminals were removed by trifluoroacetic acid or hydrogen chloride in dioxane and hydrogenation with palladium/carbon. Identification of the peptides was made by 1H NMR and mass spectroscopy as described below together with analytical TLC data which were obtained by using Merk silica gel 60 F254 aluminum plates. The solvent systems used for TLC were (I) CHCl3/methanol/ammonia water ) 65/25/5 v/v/v and (II) CHCl3/methanol ) 4/1 v/v.
Miura et al. Lipo-A12B. 1H NMR (CDCl3, 270 MHz): δ 1.39 (m, 2H, CH2), 1.40-1.47 (m, 18H, Ala CβH3),1.60 (m, 36H, Aib CH3), 1.63 (m, 2H, CH2), 1.90 (m, 2H, CH2), 2.40 (t, 2H, CH2), 2.44 (m, 1H, CH), 3.10 (t, 2H, CH2), 3.50 (m, 2H, CH2), 3.93 (m, 6H, Ala CRH), 5.10 (s, 2H, benzyl CH2), 7.32 (AA′BB′, 5H, benzene), 7.5-7.8 (br, 12H, NH). FAB-MS [M + Na]+ 1256, TLC: Rf (I) ) 0.84, Rf (II) ) 0.81. Lipo-A16B. 1H NMR (CDCl3, 270 MHz): d 1.39 (m, 2H, CH2), 1.40-1.47 (m, 24H, Ala CβH3), 1.60 (m, 48H, Aib CH3), 1.63 (m, 2H, CH2), 1.90 (m, 2H, CH2), 2.40 (t, 2H, CH2), 2.44 (m, 1H, CH), 3.10 (t, 2H, CH2), 3.50 (m, 2H, CH2), 3.93 (m, 8H, Ala CRH), 5.10 (s, 2H, benzyl CH2), 7.32 (AA′BB′, 5H, benzene), 7.5-7.8 (br, 16H, NH). FAB-MS [M + H]+ 1546, TLC: Rf (I) ) 0.89, Rf (II) ) 0.87. Lipo-A24B. 1H NMR (CDCl3, 270 MHz): δ 1.39 (m, 2H, CH2), 1.40-1.47 (m, 36H, Ala CβH3), 1.60 (m,72H, Aib CH3), 1.63 (m, 2H, CH2), 1.90 (m, 2H, CH2), 2.40 (t, 2H, CH2), 2.44 (m, 1H, CH), 3.10 (t, 2H, CH2), 3.50 (m, 2H, CH2), 3.93 (m, 12H, Ala CRH), 5.10 (s, 2H, benzyl CH2), 7.32 (AA′BB′, 5H, benzene), 7.8-8.0 (br, 24H, NH). FAB-MS [M + Na]+ 2192. TLC: Rf (I) ) 0.98, Rf (II) ) 0.74. LipoKZ16M. 1H NMR (CDCl3, 270 MHz): δ 1.39 (m, 2H, CH2), 1.47 (m, 16H, Lys CγH2), 1.56 (m, 48H, Aib CH3), 1.7-1.9 (m, 32H, Lys CβH2 CδH2), 1.90 (m, 2H, CH2), 2.38 (t, 2H, CH2), 2.44 (m, 1H, CH), 3.08 (m, 16H, Lys CH2), 3.10 (t, 2H, CH2), 3.50 (m, 2H, CH2), 3.60 (s, 3H, methoxy CH3), 3.78 (m, 8H, Lys CRH), 5.01 (m, 16H, benzyl(Z) CH2), 7.28 (AA′BB′, 40H, phenyl), 7.58.1 (br, 24H, NH). FAB-MS [M + Na]+ 3021. TLC: Rf (I) ) 0.59, Rf (II) ) 0.86. SUA16B. 1H NMR (CDCl3, 270 MHz): δ 1.07-1.32 (m, 40H, CH2), 1.40-1.47 (m, 48H, Ala CβH3), 1.60 (m, 96H, Aib CH3), 3.88 (m, 16H,Ala CRH), 5.10 (s, 4H, benzyl CH2), 7.38 (AA′BB′, 10H, benzene), 7.5-7.8 (br, 32H, NH). FAB-MS [M + Na]+ 3135. TLC: Rf(I) ) 0.79, Rf(II) ) 0.95. BA16S2. 1H NMR (CDCl3, 270 MHz): δ 1.40-1.47 (m, 48H, Ala CβH3), 1.50 (s, 18H, Boc), 1.60 (m, 96H, Aib CH3), 2.50 (t, 4H, CH2), 3.22 (t, 4H, CH2), 3.93 (m, 16H, Ala CRH), 7.5-7.8 (br, 32H, NH). FAB-MS [M + Na]+ 2873. TLC: Rf (I) ) 0.83, Rf (II) ) 0.87. Deposition of Gold on Glass Plate. The substrate coated with gold was prepared by the vapor deposition of chromium and then gold (99.99%) onto a clean slide glass. Thicknesses of the chromium and gold layers, monitored by a quartz oscillator, were about 10 and 1000 Å, respectively. The slide glass was treated in advance with acetone and methanol followed by H2SO4 (97%):H2O2 (30%) ) 7:3 for 1 h. The plate was washed thoroughly with distilled water and ethanol. The gold substrate was used for self-assembling experiments of peptides within a few days after preparation. Preparation of Self-Assembled Monolayers. The gold substrate was incubated in an ethanol or N,N-dimethylformamide (DMF) solution of peptide (0.1 mM) for 24 h. After incubation, the substrate was rinsed rigorously with ethanol or DMF and dried in a stream of dry nitrogen gas. FTIR-RAS. The FTIR spectra were recorded on a Nicolet Magna 850 Fourier transform infrared spectrometer. For RAS measurements, a Harrick model RMA-1DG/VRA reflection attachment was used, and a p-polarized beam was obtained through a Hitachi Au/AuBr wire-grid polarizer. Incident angle was set at 85°. The number of interferogram accumulations was 500. Molecular orientation of the peptide layer on a gold surface was determined on the basis of the amide I/amide II absorbance ratio of FTIR-RAS according to eq 1 under the assumption of uniform crystal of the peptide-thin layer.13-18 (14) (a) Enriquez, E. P.; Gray, K. H.; Guarisco, V. F.; Linton, R. W.; Mar, K. D.; Samulski, E. T. J. Vac. Sci. Technol., A 1992, 10, 2775. (b) Enriquea, E. P.; Samulski, E. T. Mater. Res. Soc. Symp. Proc. 1992, 225, 423. (c) Worley, C. G.; Linton, R. W.; Samulski, E. T. Langmuir 1995, 11, 3805. (15) (a) Gremlich, H. U.; Fringeli, U. P.; Schwyzer, R. Biochemistry 1983, 22, 4257. (b) Fringeli, U. P.; Gunthard, H. H. Membrane Spectroscopy; Springer-Verlag: Belin and Heidelberg, Germany, 1981. (16) Greenler, R. G. J. Chem. Phys. 1966, 44, 310. (17) Okamura, E.; Umemura, J.; Takenaka, T. Can. J. Chem. 1991, 69, 1691.
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Figure 3. CD spectra of peptides in ethanol at a concentration of 1.5 × 10-5 mol/L at room temperature: (a) LipoA12B, LipoA16B, LipoA24B, and LipoKZ16M; (b) BA16S2 and SUA16B.
1 I1 C1 3(2SHSt1 + 1) Dobs ) ) ) I2 1 C2 (2St2 + 1) 3 1 2 (3 cos2 γ - 1) 2 C 1 2 (3 cos2 γ - 1) 2
[ [
][21(3 cos θ - 1)] + 1 (1) ][21(3 cos θ - 1)] + 1 2
1
2
2
Ii, Ci, SH, and Sti (i ) 1 or 2 corresponding to amide I or amide II) represent the observed absorbance, proportionality constant, order parameter of the helix axis, and order parameter of the transition moments, respectively. Dobs, γ, θi, and C represent the observed amide I/amide II absorbance ratio, the tilt angle of the helix axis from the surface normal, the angle between the transition moment (amide I or amide II) and the helix axis, and the scaling constant, respectively. For the angles of the transition moments from the helix axis, we have used averaged angles of transition moments along the helix axis, in which interactions between transition moments19 were neglected, in the previous paper.6 To be more exact, we used literature values of the angles of the transition moments from the helix axis as 39° of θ1 and 75° of θ2, respectively.20 The scaling factor, C, was determined from the amide I/amide II absobance ratio in a KBr pellet taken as a random orientation (in the present case, the ratios are in the range of 1.5 ( 0.2, which agree with the literature value, 1.5 ( 0.214b). CD Measurement. CD spectra of the peptides were measured in an ethanol solution at room temperature on a JASCO J-600 CD spectrometer using optical cell of 0.1 or 1 cm path length.
Results and Discussion Conformation of Peptide. The CD spectra in ethanol of all the peptide derivatives are shown in Figure 3. All the peptide derivatives showed a double minimum profile, which is characteristic of an R-helical structure.21,22 The (18) (a) Debe, M. K. J. Appl. Phys. 1984, 55, 3354. (b) Debe, M. K. Appl. Surf. Sci. 1982-1983, 14, 1. (19) Nevskay, N. A.; Chirgadze, Y. N. Biopolymers 1976, 15, 637. (20) Tsuboi, M. J. Polym. Sci. 1965, 59, 139. (21) Holzwarth, G.; Doty, P. J. Am. Chem. Soc. 1965, 87, 218. (22) (a) Greenfield, N.; Fasman, G. D. Biochemistry 1969, 8, 4108. (b) Chen, Y. H.; Yang, J. T.; Chau, K. H. Biochemistry 1974, 13, 3350.
helix content of LipoA12B was less than that of other peptide derivatives, showing that the chain length of LipoA12B is not long enough to stabilize R-helical conformation. The helical conformation of the peptides was also confirmed by FTIR spectroscopy. The absorptions appeared about 1660 and 1540 cm-1 and were assigned to amide I and amide II bands of R-helical conformation, respectively.23 Aib residues in the sequence contribute to stabilization of the R-helical conformation.24 Kinetics of the Formation of Peptide SAM. The time frame of chemisorption of LipoA16B to a gold surface was studied by following the change of amide I absorbance in the FTIR-RAS spectra. The reaction occurred so fast that nearly 70% of the peptide reacted in the initial 30 min (Figure 4). The absorbance reached saturation after 12 h of incubation. However, the reaction profile cannot be explained by the Langmuir isothermal absorption equation of SAM.7 It is composed of the initial fast part and the later slow part. The peptides initially adsorbed on to a gold surface might obstruct the binding of the remaining peptides. The incubation was continued for 24 h to complete chemisorption. It should be noted that the SAM was washed rigorously with solvent to remove physically adsorbed peptides from the SAM. It was confirmed that the RAS spectra of the SAM did not change with repeated washings, indicating that only chemically bound peptides existed on a gold surface. Effect of Solvent. Despite the tendency for the present helical peptides to associate together, association in solution is sensitively influenced by the nature of the solvent. We prepared the SAM of LipoA16B from ethanol or DMF solution (Figure 5). When the SAM was prepared from an ethanol solution of LipoA16B, the amide I/amide II absorbance ratio in the FTIR-RAS spectrum was larger (23) (a) Krimm, S.; Bandeker, J. Adv. Protein Chem. 1986, 38, 181. (b) Kennedy, D. F.; Chrisma, M.; Chapman, T. D. Biochemistry 1991, 30, 6541. (24) (a) Burgess, A. W.; Leach, S. J. Biopolymers 1973, 12, 2599. (b) Karle, I. L.; Balaram, P. Biochemistry 1990, 29, 6747.
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Miura et al. Table 1. The Absorbance of Amide I Band, Amide I/Amide II Absorbance Ratio, Order Parameter of Helices in SAM, and the Tilt Angle (degree) of the Various Peptide SAMs
peptide
absorbance of amide I (10-3)a
amide I/ amide IIa
LipoA12B LipoA16Bb LipoA16Bc LipoA24B LipoKZ16M BA16S2 SUA16B
6.54 ( 0.04 15.7 ( 0.9 4.13 ( 0.09 21.4 ( 2.5 6.70 ( 0.77 4.60 ( 0.81 7.54 ( 0.76
1.87 ( 0.06 3.44 ( 0.17 1.12 ( 0.01 4.41 ( 0.49 1.33 ( 0.13 1.13 ( 0.15 1.85 ( 0.17
order parameter tilt angle of helix (deg) 0.17 0.49 -0.18 0.63 -0.009 -0.024 0.16
48 36d 63 30d 55 66 49
a The values are an average of more than three different samples. The peptide SAM was prepared from an ethanol solution. c The peptide SAM was prepared from a DMF solution. d The tilt angles of 30° and 36° in this table are calculated to be 24° and 33°, respectively, by using the angles of transition moments described in the previous paper.6
b
Figure 4. Amounts of LipoA16B immobilized on a gold surface with different incubation durations as determined by the amide I absorbance in FTIR-RAS: (b) experimental data; (s) calculation according to the Langmuir isothermal adsorption.
Figure 6. CD spectra of LipoA16B in ethanol with varying concentrations.
Figure 5. FTIR-RAS spectra of SAMs of LipoA16B prepared from (a) DMF solution and (b) ethanol solution.
(3.44) than that from a DMF solution (1.12). Order parameters of helix axis in SAMs were calculated by using eq 1, and are summarized in Table 1 together with amide I/amide II absorbance ratios. Although helices are arranged in SAMs with a distribution, the tilt angles of helix axis from the surface normal were calculated on the basis of the order parameters with an assumption of a uniform orientation of the helices with a fixed tilt angle (Table 1). Notably, the tilt angle of the helix axis from the surface normal is 36°, indicating a nearly vertical orientation of the helix axis to a gold surface. It has been reported that the tilt angles of a poly(γ-benzyl-L-glutamate) SAM and a heicosapeptide SAM were 64°12 and 30°,13 respectively. The amide I absorbance of peptide SAMs is proportion-
ally related to the amideI/amide II absorbance ratio (Table 1). This relationship is explained by two factors: (i) When the helices take a more vertical orientation, the amount of peptide attached to a gold surface becomes larger. (ii) The amide I absorbance of helical peptides gets strong with a vertical orientation due to a strong transition moment of the longitudinal optical amide I mode along the main helix axis being excited in FTIR-RAS spectroscopy.13,16,25 Therefore, the strong amide I absorbance of LipoA16B also supports the formation of a SAM with nearly vertical orientation. The negative intensity of the Cotton effect of LipoA16B in ethanol increased with increasing peptide concentration (Figure 6). LipoA16B should, therefore, form aggregates in ethanol by adopting a more regular helical conformation. On the other hand, aggregation of peptides was not evident in DMF, which is a good solvent for peptides. It is concluded that in ethanol solution LipoA16B easily associates together due to the self-assembling properties, resulting in the formation of SAM with a nearly vertical orientation. Effect of Component Amino Acid. Helix-helix interaction by van der Waals force will be favored by (25) Yamamoto, K.; Ishida, H. Vib. Spectrosc. 1994, 8, 1.
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Figure 7. FTIR-RAS spectra of LipoKZ16M SAM. The peak at 1720 cm-1 is assigned to a urethane group.
increasing the contact area between the helices. However, it is still difficult to choose the best sequence of amino acids to maximize this contact area. On the other hand, the monolayer of alkylated polysilane has been reported to be highly stable because the alkyl chains fill up defects in the monolayer of rodlike molecules.26 LipoKZ16M, which has the repeating sequence of Lys(Z) and Aib with a lipoic acid group at the N-terminal, was synthesized. The side chain of Lys(Z) is considered to behave as a solvent to stabilize the membrane of the helical peptide. The RAS spectrum of LipoKZ16M SAM showed a low amide I/amide II absorbance ratio (1.33), which indicates random orientation of helices on a gold surface (Figure 7). Therefore, the side chain of Lys(Z) was shown not to be effective in promoting self-assembly of helices with a vertical orientation, even though the helix membrane is stable. Boc-(Ala-Aib)8-OCH3 and Boc-(Lys(Z)-Aib)8-OCH3 monolayers showed different types of π-A isotherms.4 The former showed a steeper increase of surface pressure upon compression to form a two-dimensional crystal, while the latter stayed in a liquid condensed state. Therefore, the side chain of Lys(Z) should prevent transformation of the peptide membrane into a two-dimensional crystal. Samulski et al. have reported that the SAM of poly(γbenzyl-L-glutamate) having lipoic acid group at the N-terminal was laid down on a gold surface due to hindrance of molecular packing by the bulky side chains of Glu(OBzl).9 Taking these points into consideration, flexible and bulky side chains cause the helices in SAMs to be lying down on a gold surface. The LipoA16B SAM, displaying a vertical orientation, is therefore the result of tight packing of the helices due to the self-assembling properties. Effect of One-Helix or Two-Helix Structure. Two helix chains embeded in a molecule was considered to promote self-assembly of helices due to less entropy change accompanied with side-by-side arrangement of two helices. Two-helix peptides, BA16S2 and SUA16B, were synthesized, in which two Ala-Aib sequences are connected by a disulfide linkage. SUA16B has a long alkyl chain at the N-terminal, which might contribute to intramolecular selfassembly of two sequences due to the favorable packing of alkyl chains. The RAS spectra of BA16S2 and SUA16B showed low amide I/amide II absorbance ratios (1.13 and 1.85), indicating the helices nearly lying down on gold surface or adopting a random orientation (Figure 8). The smaller (26) Wegner, G. Thin Solid Films 1992, 216, 105.
Figure 8. FTIR-RAS spectra of SAMs of (a) BA16S2 and (b) SUA16B.
Figure 9. FTIR-RAS spectra of SAMs of (a) LipoA12B and (b) LipoA24B.
tilt angle of SUA16B SAM in comparison with BA16S2 SAM is presumably due to the long alkyl chain of SUA16B which should promote association of the helices. However, the tilt angle of SUA16B (49°) was significantly larger than that of LipoA16B (36°) (Table 1). The cystamine group of BA16S2 and the ω-carboxyldecyl disulfide group of SUA16B do not provide a good linker to promote association of the two helices. Cystamine is considered to be too short to accommodate two helices in a bent form, whereas the ω-carboxyldecyl disulfide group is rigid and
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the distance between two alkyl chains is short compared with that between two helices side-by-side. Chain-Length Effect of Peptide Carrying Terminal Lipoic Acid Group. The effect of the chain length of the peptides on the helix orientation in the SAMs was investigated using dodeca-, hexadeca-, and tetradocosapeptides containing alternating Ala and Aib sequences with N-terminal lipoic acid groups. RAS spectra of the SAMs of these peptides revealed that the amide I/amide II absorbance ratio increased with increasing chain length, LipoA12B < LipoA16B < LipoA24B (Figure 9). Since Boc(Ala-Aib)12-OBzl formed a monolayer with vertical orientation due to the high self-assembling properties,4b the large absorbance ratio of LipoA24B (4.41) should be due to the high self-assembling properties of the tetracosapeptide. The tilt angle of the LipoA24B SAM was 30°, which is the smallest among the peptides examined here. Conclusion The tilt angles of the helix axis from the surface normal were determined for various peptide SAMs. The tilt angle moved toward zero in the following cases: (i) use of ethanol as solvent for the SAM preparation, (ii) Ala as a component amino acid, (iii) one helix peptide with terminal lipoic acid group, and (iv) elongation of peptide chain. These points are well explained in terms of conditions for
Miura et al.
obtaining good self-assembly of the peptides. For example, LipoA16B was shown to form helix aggregates in an ethanol solution. Boc-(Ala-Aib)8-OCH3 formed a monolayer of two-dimensional crystal. Boc-(Ala-Aib)12-OBzl formed a particularly regular monolayer. In summary, peptide SAMs are considered to take a vertical orientation on a gold surface when they form a helix-bundle structure in the preparative solution. Terminal lipoic acid groups of peptide chains cluster on one face of the helix bundle. The cluster reacts with a gold surface, resulting in fixation of the peptides with a vertical orientation. The reaction of the peptide cluster with a gold surface proceeded rapidly in the initial stage followed by a slow process. The reaction in its final stages should be hindered by steric factors, as access of peptide clusters to a surface nearly completely covered with the peptides becomes statistically more unlikely. Acknowledgment. This work is partly supported by a Grant-in-Aid for Scientific Research on Priority Area from the Ministry of Education, Science and Culture, Japan, and Yazaki Memorial Foundation for Science and Technology, Japan. The authors thank Dr. Katsuhiko Fujita, Kyushu University, for his comments. LA981296D
