Crown Ether Conjugate upon

monolayer (SAM) of ammonium-terminated alkanethiolate on a gold surface. ... Since the helix peptide without the crown ether unit was oriented in para...
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Langmuir 1998, 14, 2761-2767

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Self-Assembly of r-Helix Peptide/Crown Ether Conjugate upon Complexation with Ammonium-Terminated Alkanethiolate Yoshiko Miura and Shunsaku Kimura* Department of Material Chemistry, Graduate School of Engineering, Kyoto University, Yoshida Honmachi, Kyoto 606-01, Japan

Yukio Imanishi Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara 630-01, Japan

Junzo Umemura Institute for Chemical Research, Kyoto University, Gokanosho, Uji, Kyoto 611, Japan Received September 2, 1997. In Final Form: March 4, 1998 Hydrophobic helical crowned peptides were synthesized and investigated on formation of an oriented thin layer of the helix peptides. The helix peptides in ethanol were incubated with a self-assembled monolayer (SAM) of ammonium-terminated alkanethiolate on a gold surface. The thickness of the peptide thin layer formed on the gold surface was determined by the surface plasmon resonance method. Complexation of the crowned peptide with the ammonium-terminated alkanethiolate facilitated formation of a peptide monolayer or multilayers on gold depending on the peptide concentration. The orientation of the helix peptide on the gold surface was investigated by FT-IR reflection-absorption spectroscopy. The tilt angle of the helix axis from the normal of the gold surface was estimated to be 28°, when the crowned peptide complexed with the SAM of N-(-aminocaproyl)aminoethyl disulfide. This degree of orientation is more vertical to the surface than that of the complexed peptide with a SAM of 2-aminoethanethiol hydrochloride. Since the helix peptide without the crown ether unit was oriented in parallel to the surface, the crown ether/ammonium complexation should promote the vertical orientation of the helix peptides on the gold surface.

Introduction The R-helix is a major structural unit of proteins,1 and it frequently associates with other helices to form a helixbundle structure,2 suggesting self-assembling property of helices. Molecular packing of helices in a condensed state is also tight, and Boc-(Ala-Aib)8-OMe and Boc-(LeuAib)8-OBzl were shown to form two-dimensional crystals when they were spread at the air-water interface.3-5 It is, therefore, expected that a self-assembling membrane of R-helices will be stably formed on the surface of substrates and in water by suitable molecular designing. Several groups have so far reported on the self-assembly of R-helices on substrates. For example, Whitesell et al. have synthesized R-helices directly on a gold surface by using N-carboxyanhydride (NCA), whose polymerization was initiated by amino groups immobilized on the gold substrate.6,7 Worler et al. attached lipoic acid to the N terminal of poly(γ-L-benzyl glutamate), which was self* To whom correspondence should be addressed. Telephone: +8175-753-5628. Fax: +81-75-753-4911. E-mail: h54519@ sakura.kudpc.kyoto-u.ac.jp. (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) Fujita, K.; Kimura, S.; Imanishi, Y.; Okamura, E.; Umemura, J. Langmuir 1995, 11, 1675. (5) Higashi, N.; Sunada, M.; Niwa, M. Langmuir 1995, 11, 1864. (6) Whitesell, J. K.; Chang, H. K. Science 1993, 261, 73.

Figure 1. Schematic illustration of self-assembly of a helix peptide/crown ether conjugate on a self-assembled monolayer.

assembled on a gold substrate under an electric field.8 Although R-helices were successively attached on the surface by these methods, the molecular orientation and packing were not well-ordered. That is, poly(L-alanine) prepared by the NCA method showed an apparently random orientation of helices, which is deduced from the similarity of the absorption ratios of amide I and amide (7) Whitesell, J. K.; Chang, H. K.; Whitesell, C. S. Angew. Chem., Int. Ed. Engl. 1994, 33, 871. (8) Worley, C. G.; Linton, R. W.; Samulski, E. T. Langmuir 1995, 11, 3805.

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Figure 2. Molecular structure of helix peptides.

II between the thin membrane by FTIR-RAS and the KBr pellet by FTIR transmission spectroscopy. Furthermore, other reports on the preparation of a thin membrane of poly(γ-L-benzyl glutamate) or poly(γ-L-methyl glutamate) by the NCA method showed helices lying on the surface rather than standing, which was suggested by strong amide I absorption in FTIR transmission spectroscopy.9 Therefore, helices should be nearly lying on the surface when the peptide membrane is prepared by the NCA method. A similar result was also obtained by Chang et al., who determined the tilt angle of helices toward the normal of the surface to be 64°, when poly(γ-L-benzyl glutamate) was covalently bonded to a silicon oxide surface.10 To the best of our knowledge helix thin layers with a vertical orientation against the surface have not been previously described. We considered when the chemical bonds are formed between peptides and substrate randomly, some peptides are forced to align in a parallel way, but most peptides will not take such an arrangement because of repulsive force between macrodipole moments. Thus, attachment of helix peptides to the surface through covalent bonds will cause severe disorder in the self-assembly of R-helices. We used a different method to fix helical peptides on substrates from that of previous reports, that is to use complex formation for anchoring peptides to surface (Figure 1). Complex formation may not be so strong as to destroy self-assembly of helices if it occurs at all in solution and on a substrate upon an anchoring process. 18-Crown-6-ether and an ammonium group were chosen for the pair complexation, and the former was connected to the C terminal of helix peptides (Figure 2) and the latter was fixed on a gold substrate. Two kinds of ammonium compounds were used; one is 2-amino(9) (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. (10) Chang, Y.; Frank, C. W. Langmuir 1996, 12, 5824.

Figure 3. Molecular structure of 2-aminoethanethiol hydrochloride and N-(-aminocaproyl)-2-aminoethyl disulfide hydrochloride (C8S2).

ethanethiol hydrochloride and the other is N-(-aminocaproyl)-2-aminoethyl disulfide hydrochloride (Figure 3). Both compounds formed a self-assembled monolayer (SAM) on a gold surface, but the coverage rates of the surface were different. The thicknesses of the SAM and the peptides were determined by surface plasmon resonance (SPR), and the molecular orientation on the gold substrate was determined by FTIR reflection-absorption spectroscopy (FTIR-RAS). Experimental Section Materials. Peptides. All peptides (Figure 2) were synthesized by the conventional liquid-phase method. Dicyclohexylcarbodiimide (DCC) and N-hydroxybenzotriazole (HOBt) were used for coupling reactions. When the coupling reaction was difficult, the condensation reaction was carried out by using O-(7azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU). The Boc group and the benzyl ester group for terminal protection were removed by trfiluoroacetic acid and hydrogenation using palladium/carbon, respectively. 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 a Merk silica gel 60 F254 aluminum plate. The solvent systems for TLC were (I) CHCl3/methanol/ammonia water ) 65/25/5 v/v/v and (II) CHCl3/methanol ) 8/2 v/v. BA16B. 1H NMR (CDCl3, 270 MHz) δ: 1.40-1.47 (m, 24H, Ala CβH3), 1.50 (s, 9H, Boc), 1.60 (m, 48H, Aib CH3), 3.93 (m, 8H, Ala CRH), 5.10 (s, 2H, benzyl CH2), 7.32 (AA′BB′, 5H, benzene),

Self-Assembly of R-Helix Peptide/Crown Ether Conjugate 7.5-7.8 (br, 16H, NH). FAB-MS: [M + Na]+ 1480. TLC: Rf(I) ) 0.96, Rf(II) ) 0.94. BA24B. 1H NMR (CDCl3, 270 MHz) δ: 1.40-1.47 (m, 36H, Ala CβH3), 1.50 (s, 9H, Boc), 1.60 (m, 72H, Aib CH3), 3.93 (m, 12H, Ala CRH), 5.10 (s, 2H, benzyl CH2), 7.32 (AA′BB′, 5H, benzene), 7.5-8.1 (br, 12H, NH). FAB-MS: [M + Na]+ 2104. TLC: Rf(I) ) 0.94, Rf(II) ) 0.83. BA16OH. 1H NMR (CDCl3, 270 MHz) δ: 1.40-1.47 (m, 24H, Ala CβH3), 1.50 (s, 9H, Boc), 1.60 (m, 48H, Aib CH3), 3.93 ( m, 8H, Ala CRH), 7.5-7.8 (m, 16H, NH). FAB-MS: [M + Na]+ 1390. TLC: Rf(I) ) 0.48, Rf(II) ) 0.80. BA16Cr. 1H NMR (CDCl3, 270 MHz) δ: 1.40-1.47 (m, 27H, Ala CδH3), 1.50 (s, 9H, Boc), 1.60 (m, 72H, Aib CH3), 3.65 (s, 4H, crown ether CH2), 3.72 (s, 4H, crown ether CH2), 3.80 (s, 4H, crown ether CH2), 3.82 (s, 4H, crown ether CH2), 3.93 (m, 9H, Ala CRH), 4.07 (br, 8H, crown ether CH2), 6.78 (AA′BB′, 1H, benzene),7.40 (AA′BB′, 1H, benzene), 7.5-8.0 (br, 17H, NH), 9.10 (AA′BB′, 1H, benzene). FAB-MS: [M + Na]+ 1770. TLC: Rf(I) ) 0.82, Rf(II) ) 0.57. BA24Cr. 1H NMR (CDCl3, 270 MHz) δ: 1.40-1.47 (m, 39H, Ala CβH3), 1.50 (s, 9H, Boc), 1.60 (m, 72H, Aib CH3), 3.65 (s, 4H, crown ether CH2), 3.72 (s, 4H, crown ether CH2), 3.80 (s, 4H, crown ether CH2), 3.82 (s, 4H, crown ether CH2), 3.93 (m, 13H, Ala CRH), 4.07 (br, 8H, crown ether CH2), 6.78 (AA′BB′, 1H, benzene), 7.40 (AA′BB′, 1H, benzene), 7.5-8.0 (br, 25H, NH), 9.10 (AA′BB′, 1H, benzene). FAB-MS: [M + Na]+ 2395. TLC: Rf(I) ) 0.84, Rf(II) ) 0.76. BA16E2Cr. 1H NMR (CDCl3, 270 MHz) δ: 1.4-1.7 (m, 83H, Ala CβH3, Boc, Aib CH3, Glu CβH2), 2.40 (m, 2H, Glu CγH2), 3.65(s, 8H, crown ether CH2), 3.70 (m, 16H, crown ether CH2), 3.82 (s, 8H, crown ether CH2), 3.93 (m, 9H, Ala CRH), 4.07 (br, 8H, crown ether CH2), 6.78 (AA′BB′, 2H, benzene), 7.28 (AA′BB′, benzene 1H), 7.60 (AA′BB′, 1H, benzene), 7.8-8.2 (br, 27H, NH), 9.10 (AA′BB′, 2H, benzene). FAB-MS: [M + H]+ 2115. TLC: Rf(I) ) 0.83, Rf(II) ) 0.49. BA24E2Cr. 1H NMR (CDCl3, 270 MHz) δ: 1.4-1.7 (m, 83H, Ala CβH3, 24H, Boc, Aib CH3, Glu CβH2), 2.4-2.7 (m, 2H, Glu CγH2), 3.65 (s, 8H, crown ether CH2), 3.70 (m, 16H, crown ether CH2), 3.82 (s, 8H, crown ether CH2), 3.93 (m, 13H, Ala CRH), 4.07 (br, 8H, crown ether CH2), 6.78 (AA′BB′, 2H, benzene),7.28 (AA′BB′, 1H, benzene), 7.60 (AA′BB′, 1H, benzene), 7.8-8.2 (br, 27H, NH), 9.15 (AA′BB′, 2H, benzene). FAB-MS: [M + H]+ 2740. TLC: Rf(I) ) 0.92. N-(-Aminocaproyl)-2-aminoethyl Disulfide Hydrochloride (C8S2). C8S2 (Figure 3) was synthesized from 2-aminoethanethiol hydrochloride and N-(tert-butyloxycarbonyl)--aminocaproic acid using DCC and N-hydroxysuccinimide (HOSu) as coupling reagents. The Boc group was removed with 4 N HCl/ dioxane. 1H NMR (d6-DMSO, 270 MHz) δ: 1.40 (m, 4H), 1.60 (m, 8H), 2.18 (t, 4H), 2.78 (t, 4H), 2.87 (t, 4H), 3.32 (t, 4H), 7.78.1 (br, 8H). FAB-MS: [M + H]+ 378. TLC: Rf(I) ) 0.25, Rf(II) ) 0.0. Preparation of Substrates. Gold substrates on clean glass microscope slides were prepared by thermal evaporation of gold with a 500-Å thickness. Prior to the deposition of gold, the slides were washed with acetone, soap water, doubly distilled water, and ethanol. Gold substrates were stored in ethanol and dried in a stream of nitrogen gas immediately before formation of SAMs. SAMs were formed by immersing the gold substrates in a 1 mM ethanol solution of 2-aminoethanethiol hydrochloride for 1 h or C8S2 for 3 h, and the substrates were rinsed with ethanol. Peptide Layer Formation. The gold substrate with a 2-aminoethanethiol hydrochloride SAM was incubated with an ethanol solution of peptide (1 mM) for 1 h. Then, the substrates were rinsed with ethanol and dried in a stream of nitrogen gas. On the other hand, the gold substrate with a C8S2 SAM was incubated with the ethanol solution of peptide (1 mM or 0.1 mM) for 3 h, and the excessive solution was removed by suction using filtration paper. The substrates were dried in a stream of nitrogen gas. FTIR-RAS Measurement. The FTIR spectra were recorded on a Nicolet Magna 850 Fourier transform infrared spectrophotometer. For RAS measurements, a Harrick Model RMA-1DG/ VRA reflection attachment was used, and the p-polarized beam was obtained through a Hitachi Au/AuBr wire-grid polarizer.

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Figure 4. FTIR transmission spectrum of BA16Cr in a KBr pellet (top) and FTIR-RAS spectrum of BA16Cr on a SAM of 2-aminoethanethiol hydrochloride (bottom). The incident angle was set at 85°. The number of interferogram accumulations was 500. Molecular orientation on a gold substrate was assessed using FTIR-RAS.11,12 The transition moments of amide I and amide II of BA16Cr and C8S2 were determined by FTIR transmission spectroscopy of KBr pellets of varying concentrations. The thickness of the peptide or C8S2 layer was determined by SPR measurement, details being explained in the following section. These values were used for theoretical calculation of infrared reflection absorbances according to Hansen’s optical theory on thin isotropic multilayers. By comparing the theoretical values with the experimental vales of FTIR-RAS absorbances, the orientation angles of the transition moments of the major infrared bands were evaluated.13,14 SPR Measurement. The surface plasmon resonance setup is based on the Kretschmann configuration, which is essentially the attenuated total reflection (ATR) technique. The goniometer is accurate up to 0.1°. The light signals were then reflected to a phase-sensitive photodiode array detector. The signal is stored and graphed in a computer after AD conversion to produce a reflectivity angle of incidence scan. Adsorption of compounds on a gold substrate changes the reflection minimum angle. The thickness of the adsorbed layer was calculated from the extent of the change of the angle and the refractive index of the layer, n ) 1.50 for peptides and n ) 1.49 for alkanethiolates. The total layer thickness was also confirmed by FTIR-RAS measurement, especially in the cases of the layers less than 10 Å. CD Measurement. CD spectra of the peptides were measured in an ethanol solution at room temperature on a JASCO J-600 CD spectrometer using an optical cell of 0.1-cm path length.

Results and Discussion Peptide Self-Assembly on a SAM of Aminoethanethiol. An ethanol solution of peptide was incubated in the presence of a SAM of 2-aminoethanethiol hydrochloride on a gold surface, and the thickness of the peptide layer was determined by SPR (Table 1). The thickness increased in the order BA16B < BA16OH < BA16Cr ∼ BA16E2Cr with hexadecapeptides and in the order BA24B (11) Hasegawa, T.; Takeda, S.; Kawaguchi, A.; Umemura, J. Langmuir 1995, 11, 1236. (12) Umemura, J.; Kamata, T.; Kawai, T.; Takenaka, T. J. Phys. Chem. 1990, 94, 62. (13) Hansen, W. N. Symp. Faraday Soc. 1970, 4, 27. (14) Drude, P. Ann. Phys. U. Chem. N. F. 1889, 32, 584.

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Table 1. Thickness (Å) of Peptide Layer Formed on Gold Treated with 2-Aminoethanethiol Hydrochloride by SPR Measurement peptide

thickness (Å)

BA16Cr BA16E2Cr BA16OH BA16B BA24Cr BA24E2Cr BA24B

11 12 5 0 16 15 0

< BA24Cr ∼ BA24E2Cr with tetradocosapeptides. The same result was obtained from the absorption intensities of amides by FTIR-RAS measurements. Since BA16B and BA24B having the C terminal blocked by a benzyl ester did not form any peptide layer on the surface, the formation of peptide layers with BA16Cr, BA16E2Cr, BA24Cr, and BA24E2Cr should be due to interaction of the crown ether unit of the peptides with an ammonium group of a SAM. The peptide having a carboxyl group at the C terminal, BA16OH, showed slight formation of a peptide layer. Interaction of an ammonium group with a carboxylate group might be weaker than that with a crown ether under the present conditions. The conformation and orientation of the peptides held on the surface were investigated by FTIR-RAS (Figure 4).15 The absorptions appeared at 1660 and 1540 cm-1, which were assigned to amide I and amide II absorptions of an R-helical conformation, respectively.16 The R-helical conformation should be stabilized by Ala and Aib residues. The molecular orientation of the helix peptide can be estimated by FTIR-RAS, in which an absorption band with the transition moment oriented vertically to the surface will be enhanced. In helix peptides, the transition moment of amide I absorption lies nearly parallel to the helix axis and that of amide II adsorption lies perpendicular to the helix axis.16 The observed ratio of the amide I and II absorbances, AobsI/AobsII (Dobs), of the BA16Cr peptide layer on a gold surface with a SAM of 2-aminoethanethiol hydrochloride was similar to that for a KBr dispersion (Figure 4). Therefore, the orientation of the peptide on a gold substrate is obviously random. It has been reported that cystamine dihydrochloride, which forms the same SAM with 2-aminoethanethiol hydrochloride, formed a loosely packed SAM on a gold substrate and that the maximum surface coverage of gold is about 35%.17 This behavior is due to the absence of Van der Waals interaction, which is necessary to produce a dense SAM. We have synthesized C8S2 to increase the surface coverage and have investigated peptide complexation with a SAM of C8S2. SAM of C8S2. The thickness of the SAM of C8S2 was 5 Å by SPR when the gold substrate was incubated with an ethanol solution of C8S2 for 3 h. The thickness increased with elongation of incubation time and reached the maximum value after 12 h of incubation. The gold substrate with half the coverage of the saturated one was used for further investigation. The molecular orientation of C8S2 on the gold surface was investigated by FTIR-RAS (Figure 5). When a KBr pellet was used, the amide I absorption was stronger than the amide II absorption, indicating the molecular extinction coefficient of amide I is larger than that of amide II. (15) Greenler, R. G. J. Chem. Phys. 1996, 44, 310. (16) (a) Krimm, S.; Bandeker, J. Adv. Protein Chem. 1986, 38, 181. (b) Kennedy, D. F.; Chrisma, M.; Chapman, T. D. Biochemistry 1991, 30, 6541. (17) Arias, F.; Godeinez, L. A.; Wilson, S. R.; Kaifer, A. E.; Echegoyen, L. J. Am. Chem. Soc. 1996, 118, 6086.

Figure 5. FTIR transmission spectrum of C8S2 in a KBr pellet (top) and FTIR-RAS spectrum of C8S2 SAM (bottom).

Figure 6. Relation of the (a) amide I and (b) amide II absorbances of C8S2 with the orientation angle of the transition moment toward the surface normal of the three-layer specimen calculated by Hansen’s optical theory.

However, the amide II absorption became stronger than the amide I absorption in the C8S2 SAM spectrum (Figure 5). Thus, C8S2 is oriented nearly perpendicularly to the gold substrate.18 The precise molecular orientation of C8S2 was further investigated as follows.11,12 With varying C8S2 concentrations in KBr pellets, the extinction coefficients of C8S2, kC8S2, were determined by FTIR measurement to be 0.164 (amide I) and 0.104 (amide II). In FTIR-RAS measurement, the specimen is composed of three layers: air, C8S2, and gold. The optical constants of each layer were taken as nair ) 1.00, kair ) 0.0, nC8S2 ) 1.49, kC8S2 ) 0.164 (amide I), ngold ) 5.11, and kgold ) 36.5. The thickness of the C8S2 layer was 5.0 Å by SPR. According to the theoretical equation for reflection absorbance of a three-layer system (air, C8S2, and gold), the relation between reflection absorption and the tilt angle of the transition moment of amide I from the surface normal was obtained (Figure 6). The experimental value of reflection absorbance indicates the orientation angle of the amide transition moment of amide I to be 60° from the calculated relation curve (Figure 6). Similarly, taking the optical constants of nair ) 1.00, kair ) 0.0, nC8S2 ) 1.49, kC8S2 ) 0.104 (amide II), ngold ) 5.79, and kgold ) 38.6, the orientation angle of the amide (18) Clegg, R. S.; Hutchison, J. E. Langmuir 1996, 12, 5239.

Self-Assembly of R-Helix Peptide/Crown Ether Conjugate

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Figure 7. Schematic illustration of self-assembled monolayer of C8S2.

II transition moment was found to be 23°. It is, thus, concluded that the molecular tilt angle of C8S2 from the substrate normal is 16°, assuming the all-trans conformation of C8S2 (Figure 7). The amide I and amide II absorptions of C8S2 on the gold substrate appeared at 1651 and 1551 cm-1, respectively. The amide I absorption appeared at higher wavenumber than that for a KBr pellet (1632 cm-1), whereas the amide II absorption appeared at lower wavenumber. These shifts are opposite to those predicted for hydrogen-bonded amides.18 Therefore, C8S2 in a SAM should not form hydrogen bonds between neighboring amides, suggesting the absence of clear phase separation into a C8S2 cluster and an uncovered gold surface under the condition of half coverage. Peptide Self-Assembly on SAM of C8S2. An ethanol solution of peptide was incubated in the presence of a C8S2 SAM, and the gold substrate was taken out of the solution followed by suction of solution adsorbed on the plate. The thickness of the peptide thin layer was determined by SPR. When a concentrated peptide solution of BA16Cr (1 mM) was used for incubation with the gold substrate, the thickness of the peptide layer was 115 Å, suggesting the formation of the peptide multilayer. This is probably due to the self-assembling property of the helix peptides. In order to prepare a peptide monolayer by specific complexation of the crown ether unit with an ammonium group on gold, the peptide concentration in ethanol solution was reduced to 0.1 mM. The thickness of the BA16Cr layer became 29 Å, which was three times that of the BA16B layer (10 Å) prepared under the same experimental conditions. On the other hand, the BA16Cr peptide layer became thin (13 Å) by rinsing the plate with ethanol, suggesting reversible dissociation of the peptide from the plate. However, the thickness was still significant compared with that of the BA16B peptide layer after rinsing with ethanol, which was 0 Å. Thus, complexation should occur between BA16Cr and an ammonium group of the plate. The binding constant of BA16Cr with an ammonium compound was determined by CD measurements in ethanol. The molar ellipticity at 222 nm of BA16Cr increased the negative intensity with the addition of n-heptylamine hydrochloride (Figure 8), indicating an increase of helix content. On the other hand, BA16B did not show any change in CD measurements with the addition of the ammonium compound. These observations are well explained by complexation of n-heptylamine

Figure 8. CD spectra of BA16Cr in ethanol (1.5 × 10-4 M) with varying concentrations of n-heptylamine hydrochloride at room temperature. Table 2. Absorbance of Amide I of Peptide Layer by FTIR-RAS Measurementa BA16Cr on C8S2 SAM on bare gold a

BA16B 10-2

(1.42 ( 0.02) × (3.25 ( 0.49) × 10-3

(3.27 ( 0.09) × 10-3 (4.65 ( 0.19) × 10-3

The values are the average of at least three different samples.

hydrochloride with the crown ether moiety of BA16Cr, which stabilizes the helix conformation by favorable interaction of the positive charge of n-heptylamine hydrochloride with the negative end of the macrodipole of the helical peptide BA16Cr.19 The binding constant of n-heptylamine hydrochloride and BA16Cr was determined to be (1.14 ( 0.34) × 103 (L/mol) from the change of the molar ellipticity at 222 nm of BA16Cr under the assumption of a 1:1 complex. The moderate binding strength is agreeable with the previous observation that the peptide layer anchored at the C8S2 SAM was partly washed out by rinsing with ethanol. However, the complexation is strong enough to keep the peptide monolayer on the surface. Furthermore, the following observations support the consideration that complexation is a key factor for preparation of the peptide layer. The absorbances of amide I of the various peptide layers on the gold substrate with a C8S2 SAM are summarized in Table 2. The absorbance of amide I of the BA16Cr layer on a C8S2 SAM [(1.42 ( 0.02) × 10-2] was significantly larger than those of the BA16Cr layer on bare gold [(3.25 ( 0.09) × 10-3], the BA16B layer on a C8S2 SAM [(3.27 ( 0.09) × 10-3], and the BA16B layer on bare gold [(4.65 ( 0.19) × 10-3]. Namely, both crown ether and ammonium groups are necessary for the formation of a thick BA16Cr peptide layer. The helix orientation of the peptide layers was evaluated by the ratio of the amide I and II absorbances, Dobs, in FTIR-RAS spectra (Table 3). The Dobs value of BA16Cr on a C8S2 SAM was 2.19 ( 0.06. On the other hand, the Dobs values of other peptide layers were in the range 0.8(19) Otoda, K.; Kimura, S.; Imanishi, S. J. Chem. Soc., Perkin Trans. 1 1993, 3011.

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Table 3. Amide I to Amide II Intensity Ratios (Dobs) of the Peptide Layers on a Gold Substrate by FTIR-RAS Measurementa on C8S2 SAM on bare gold a

BA16Cr

BA16B

2.19 ( 0.06 1.07 ( 0.20

1.14 ( 0.15 0.862 ( 0.098

The values are the averages of at least three different samples.

Figure 11. Relation of the (a) amide I and (b) amide II absorbances of BA16Cr with the orientation angle of the transition moment toward the surface normal of the four-layer specimen calculated by Hansen’s optical theory. Figure 9. FTIR-RAS spectrum of the BA16B layer formed on a SAM of C8S2.

Figure 10. FTIR-RAS spectrum of the BA16Cr layer formed on a SAM of C8S2.

1.2, which was even lower than that for a random orientation of helix peptides (around 1.6). Only a BA16Cr layer on a C8S2 SAM oriented rather vertically to the substrate, and in other cases the peptide nearly lay down on the surface. For example, in the case of a BA16B peptide layer on a C8S2 SAM, amide I absorption was slightly weaker than amide II absorption (Figure 9), indicating a parallel orientation of the helix axis to the surface of the gold substrate. The thickness of the BA16B peptide layer was determined by SPR to be 10 Å, which also supports the orientation parallel to the surface. This difference in the helix peptide orientation is, therefore, brought about by the result of complexation of the crown ether unit of BA16Cr with the ammonium group of the C8S2 SAM, which forms a contrast to the nonspecific interaction in other cases. The tilt angle of the helix axis from the surface normal was calculated from the results of FTIR-RAS measurements. The amide I absorption of BA16Cr on a C8S2 SAM was more than twice as large as the amide II absorption (Figure 10). Using the theoretical equation for the reflection absorption of a four-layer specimen (air, BA16Cr, C8S2, and gold) and the following optical constants (nair ) 1.00, kair ) 0.0, nBA16Cr ) 1.50, kBA16Cr ) 0.375 (amide I), nC8S2 ) 1.49, kC8S2 ) 0.0963, ngold ) 5.11,

Figure 12. Schematic illustration of the orientation of the helix peptide BA16Cr on gold. The z-axis and H represent the normal of the gold substrate and the helix axis, respectively. The helix molecule with its amide I transition moments ti at angles θ rotates around the helix axis H, which, in turn, rotates around the z-axis with a narrow distribution of γ.

kgold ) 36.5), the relation between the reflection absorbance and the tilt angle of the transition moment of amide I from the surface normal was obtained (Figure 11). The experimental value of the reflection absorbance indicates the orientation angle of the transition moment of amide I from the normal to be 45°. On the other hand, taking the following optical constants (nair ) 1.00, kair ) 0.0, nBA16Cr ) 1.50, kBA16Cr ) 0.221 (amide II), nC8S2 ) 1.49, kC8S2 ) 0.100, ngold ) 5.91, kgold ) 38.8), the orientation angle of the amide II transition moment was found to be 54°. It is, thus, concluded that the tilt angle of the helix axis toward the normal of the gold substrate (γ) is 28°. In this calculation, the angles between the direction of the helix axis and the amide I and amide II transition moment vectors were taken to be 33° and 67°, respectively (Figure 12).20,21 The determined tilt angle is consistent with the thickness of the peptide layer determined by SPR, in the case that the peptides taking a fully R-helical structure (20) Schwyzer, R.; Minakakis, P. M.; Kimura, S.; Gremilich, H. J. Pept. Sci. 1997, 3, 65. (21) Momany, F.; McGuire, R. F.; Burgess, A. W. J. Phys. Chem. 1975, 79, 2361.

Self-Assembly of R-Helix Peptide/Crown Ether Conjugate

form a dense self-assembled monolayer. The tilt angle here, however, was obtained under the assumption of a uniform crystal of the peptide layer. Since the orientations of the helix peptides are considered to be distributed in the peptide layer, the results of FTIR-RAS measurement would be better analyzed by using a distribution function, as Enriquez et al. have proposed.22 This point remains for future work. The order parameter S of BA16Cr was determined for the following model: (i) BA16Cr takes an R-helical conformation, and the helix axis is uniaxially oriented with an average angle of γ (28°) with respect to the surface normal; (ii) the amide I transition moment is uniformly distributed around the helix axis with the angle θ (33°) (Figure 11). Then, S is determined by eq 1.23

S)

3 cos2 γ - 1 3 cos2 θ - 1 2 2

(1)

to be 0.37. This value means that a relatively well-oriented helix-peptide layer was prepared in the present investigation. It is notable that the orientation of a helix peptide on a gold substrate is nearly perpendicular to the surface. The major factor needed to realize a vertical orientation should be complex formation of the crown ether unit with the ammonium group of the C8S2 SAM. In addition, the self-assembling tendency of BA16Cr is considered to be an important factor for the formation of an oriented peptide layer. One of the driving forces of the self-assembly of the peptide may be the macrodipole moment of the helix. In the peptide assembly, the helices should be arranged in an antiparallel orientation to tolerate dipole repulsion. Therefore, it is speculated that half of the BA16Cr molecules adsorbed on the gold surface are involved in the crown-ammonium complex formation and that the other half of the peptide directs the crown ether unit to the air. This speculation is supported by the observation that the peptide layer prepared from an ethanol solution containing BA16Cr and BA16B (1/1, 0.1 mM in total) (22) (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. (23) Okamura, E.; Umemura, J.; Takenaka, T. Biochim. Biophys. Acta 1986, 856, 68.

Langmuir, Vol. 14, No. 10, 1998 2767

Figure 13. FTIR-RAS spectrum of a BA16Cr/BA16B equimolar mixture layer formed on a SAM of C8S2.

showed nearly the same amide I/amide II ratio as that of BA16Cr (Figure 13), indicating that the replacement of half of the BA16Cr helices with BA16B helices did not influence the peptide layer formation on a C8S2 SAM. Conclusion Boc-(Ala-Aib)8-Ala-Cr formed a self-assembled peptide layer of R-helices with a nearly vertical orientation toward the gold substrate. Complexation of the terminal crown ether unit of the peptide with the ammonium group immobilized on the gold substrate played an important role in the formation of the peptide monolayer. The peptides are considered to take an antiparallel orientation in the monolayer. The use of crown/ammonium complexation for anchoring a self-assembled monolayer to a substrate will also be effective for other self-assembling compounds. Furthermore, fabrication of a self-assembled monolayer such as patterning on a gold substrate may be possible by immobilization of one component in a prescribed pattern and molecular recognition at complexation of the other component. 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. LA970989B