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Structure and Molecular Recognition Properties of a Poly(allylamine) Monolayer Containing Poly(L-alanine) Graft Chains Masahiro Higuchi,*,† Jonathan P. Wright,† Kazuhiro Taguchi,† and Takatoshi Kinoshita‡ Department of Organic Materials, National Institute of Materials and Chemical Research and CREST, JST (Japan Science and Technology), 1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan, and Department of Materials Science & Engineering, Nagoya Institute of Technology and CREST, JST (Japan Science and Technology), Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan Received February 29, 2000. In Final Form: May 22, 2000 An amphiphilic polymer, poly(allylamine) (Mw ) 10 000) containing 37 mol % poly(L-alanine) hydrophobic graft chains (PAAgPAla), was prepared. The average degree of polymerization of the poly(L-alanine) graft chains was determined to be 13. The amphiphilic polymer was observed to form a monolayer at the airwater interface. We investigated the structure and molecular recognition properties of the monolayer using π-A isotherm measurement, reflection-absorption Fourier transform infrared spectroscopy, fluorescence anisotropy, atomic force microscopy, and surface plasmon resonance. The structure of the monolayer was found to be strongly pH dependent in an aqueous solution. In acidic conditions, the protonated allylamine moieties of PAAgPAla dissolved readily into the aqueous phase, while the hydrophobic poly(L-alanine) graft chains acted as anchors to keep the monolayer at the interface, resulting in the formation of a collapsed monolayer. In alkaline conditions, the PAAgPAla chains were located entirely at the airwater interface, forming stable and closely packed uniform monolayers because of dehydration of the allylamine moieties. In the monolayer, the poly(L-alanine) graft chains self-assembled, because of the loss of electrostatic repulsions between the neighboring allylamine moieties, yielding β-sheet structure domains at the interface. These active domains interact specifically with aqueous L-alanine rather than with its corresponding D isomer. The binding of L-alanine to the PAAgPAla monolayer exhibited Langmuir-type saturation behavior, with its binding constant (K ) 1.0 × 105 M-1) being 20 times larger than that observed for the binding of D-alanine to the monolayer (K ) 4.9 × 103 M-1). The poly(L-alanine) domain in the PAAgPAla monolayer acted as a binding site for L-alanine molecules.
Introduction Molecular recognition between signaling molecules and receptors at the target cell surface is an initial event for signal transduction in living systems.1,2 It has been recognized that the specific high-order structure of the peptide chains yields the molecular recognition domain in the receptor and is closely related to the induced signal transfer on and through the biological membrane.3 Studies on synthetic molecular recognition systems not only may be important to the understanding of a simple and/or essential mechanism for signal reception and transduction through biological interfaces but also may provide the basis of a molecular device capable of transferring and receiving information. It has been reported4-9 that artificial systems composed of an ordered assembly of * To whom correspondence should be addressed. † National Institute of Materials and Chemical Research and CREST. ‡ Nagoya Institute of Technology and CREST. (1) Stern, L. J.; Brown, J. H.; Jardetzky, T. S.; Gorga, J. C.; Urban, R. G.; Strominger, J. L.; Wiley, D. C. Nature 1994, 368, 215. (2) Tama, J. R. H.; Murshudov, G. N.; Dodson, E. J.; Neil, T. K.; Dodson, G. G.; Higgins, C. F.; Wilkinson, A. J. Science 1994, 264, 1578. (3) Unwin, N. Nature 1995, 373, 37. (4) Kinoshita, T.; Doi, T.; Kato, A.; Tsujita, Y.; Yoshimizu, H. Chaos 1999, 9, 276. (5) Higashi, N.; Saitou, M.; Mihara, T.; Niwa, N. J. Chem. Soc., Chem. Commun. 1995, 2119. (6) Fijita, K.; Kimura, S.; Imanishi, Y. J. Am. Chem. Soc. 1994, 116, 2185. (7) Cha, X.; Ariga, K.; Kunitake, T. J. Am. Chem. Soc. 1996, 118, 9545. (8) Taguchi, K.; Ariga, K.; Kunitake, T. Chem. Lett. 1995, 701.
functional groups show a specific binding behavior for some substrates. For example, Higashi et al. reported5 an enantioselective binding of R-amino acid isomers to Langmuir-Blodgett (LB) films of poly(L-glutamic acid)based amphiphiles on quartz plates. They concluded that the highly assembled structure of the R-helical poly(L-glutamic acid) segments in the LB film plays an important role in causing the specific binding of the D and L isomers of R-amino acid. Kunitake et al. reported7 the specific binding of aqueous dipeptides onto peptidefunctionalized monolayers. They suggested that the recognition site is self-assembled on the surface of the monolayer via the interaction with guest dipeptides. In this study, we report the structure of a monolayer prepared from the amphiphilic polymer poly(allylamine) bound by poly(L-alanine) graft chains (PAAgPAla). We also report the molecular recognition capability of the monolayer toward certain aqueous amino acids. The structure of the monolayer was found to be strongly pH dependent in an aqueous solution. In alkaline conditions, the poly(L-alanine) graft chains self-assemble in the PAAgPAla monolayer because of interactions between the poly(L-alanine) graft chains which were in β-sheet structure form. These graft chains yielded sites which bind specifically with aqueous L-alanine rather than its D isomer. The specific binding of L-alanine onto the monolayer is entirely dependent on the structure of the monolayer, which could be regulated by pH adjustment. (9) Oishi, Y.; Torii, Y.; Kuramori, M.; Suehiro, K.; Ariga, K.; Taguchi, K.; Kamino, A.; Kunitake, T. Chem. Lett. 1996, 411.
10.1021/la000290x CCC: $19.00 © 2000 American Chemical Society Published on Web 07/20/2000
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Langmuir, Vol. 16, No. 17, 2000 Chart 1. Chemical Structure of PAAgPAla
Preliminary results of the morphology and the molecular recognition properties of the PAAgPAla monolayer at the air-water interface have been reported previously.10 Experimental Section Materials. An amphiphilic polymer containing poly(L-alanine) graft chains (PAAgPAla; Chart 1) was obtained by polymerization of N-carboxy-L-alanine anhydride with poly(allylamine) (Mw ) 10 000) as an initiator.11,12 Poly(allylamine) (0.1 g) was dissolved in water (100 mL) and the pH adjusted to 12.0. Ethyl acetate (100 mL) containing N-carboxy-L-alanine anhydride (0.1 g) was added gradually to the vigorously stirred poly(allylamine) aqueous solution. After 24 h, the solution was filtered and dialyzed overnight using a Spectra/Pore molecular porous membrane tube (Spectrum Medical Industries, Inc., MWCO 3500). After dialysis, the solution was lyophilized to obtain PAAgPAla. The graft chain, poly(L-alanine), content of PAAgPAla was determined to be 37 mol % by potentiometric counting of the unreacted allylamine groups. The average degree of polymerization of poly(L-alanine) was determined to be 13 by elemental analysis. The pKa of the PAAgPAla membrane, which was obtained by casting a trifluoroethanol solution of PAAgPAla, in a 0.1 M NaCl aqueous solution was 7.1. This value is less than the pKa of poly(allylamine), 8.0, in the same solution. This implies that the allylamine moieties of PAAgPAla are in a hydrophobic environment compared with those of poly(allylamine); i.e., the poly(L-alanine) graft chains are dispersed along the main chain rather than grouped together. Methods Surface Pressure-Area Isotherm Measurements. Surface pressure-area (π-A) isotherms of the PAAgPAla monolayers were measured using a Langmuir film balance (Nippon Laser & Electronics Lab., NL-LB-240-MWC). A Teflon trough was filled with Milli-Q-treated and doubly distilled water containing a 0.1 M NaCl solution. The pH of the subphase solution was adjusted by the addition of either HCl or NaOH. A small measured amount of a trifluoroethanol solution of PAAgPAla was spread onto the water surface with a Termo microsyringe. π-A isotherms were taken at a compression rate of 5 mm/min and a subphase temperature of 21 °C under a nitrogen atmosphere. Spectroscopic Measurements. The conformation of the poly(L-alanine) graft chains in the PAAgPAla monolayers, affixed to gold-deposited glass plates, was estimated from reflection-absorption Fourier transform infrared spectroscopy (FTIR-RAS; Perkin-Elmer, Spectrum 2000). The monolayer transfer was carried out using the vertical dipping method with the monolayers attaching to the plate on the upstroke, at a surface pressure of 10 mN/m. (10) Higuchi, M.; Taguchi, K.; Kinoshita, T. Chem. Lett. 1999, 1117. (11) Daly, W. H.; Poche, D. Tetrahedron Lett. 1988, 29, 5859. (12) Banfod, C. H.; Elliot, A.; Hangy, W. E. Synthetic Polypeptides; Academic: New York, 1956.
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The fluorescence anisotropy, γ ) (I| - I⊥)/(I| + 2I⊥), of 1,6-diphenyl-1,3,5-hexatriene (DPH) in PAAgPAla monolayers was measured at the air-water interface using a photonic multichannel analyzer (Hamamatsu Photonics K.K., PMA-11) to elucidate the fluidity of the PAAgPAla monolayers. The fluorescence intensities are represented by I| and I⊥, polarized parallel and perpendicular to the direction of the polarized excitation beam, respectively. The excitation light source used was a 150 W deuterium lamp (Otuka Electronics Co. Ltd., MC-962A) equipped with a Toshiba UV-D33S glass filter. The polarized excitation light used was equipped with a fiber bundle array connected polarizer, with the incident angle fixed at 45° with respect to the monolayer surface. The I| and I⊥ emissions were collected by a second fiber bundle array connected analyzer, which was set at 90° to the excitation beam. Microscopic Measurements. The morphology of the PAAgPAla monolayers was observed using an atomic force microscope (Digital Instruments, Inc., Nanoscope IIIa) in the contact mode. The PAAgPAla monolayers were transferred onto freshly cleaved mica by the horizontal lifting method. The surface pressure of the monolayers was kept at 10 mN/m during the transfer. Atomic force microscopy (AFM) images were obtained in the “height” mode using V-shaped, oxide-sharpened silicon nitride cantilevers (120 µm in length), with a spring constant of 0.58 N/m. Imaging was performed using a 10 µm × 10 µm scanner. Surface Plasmon Resonance (SPR) Spectroscopic Measurements. The binding behavior of substrates L- and D-alanine toward PAAgPAla monolayers on golddeposited glass plates was measured using SPR spectroscopy (Nippon Laser & Electronics Lab., SPR670B). The PAAgPAla monolayers were transferred onto the gold-deposited glass plates using the following method. First, a self-assembled monolayer of butyric acid was formed on the gold surface by immersing the golddeposited glass plate in a 10 µM ethanol solution of 4,4-dithiodibutyric acid for 30 min and then rinsing with ethanol. The carboxyl groups were then converted to the corresponding succinimide esters by the coupling reaction between butyric acid and N-hydroxysuccinimide in the presence of [1-ethyl-3,3-bis(methylamino)propyl]carbodiimide. The PAAgPAla monolayer was then attached covalently to the gold-deposited glass plate and then rinsed with pure water. The PAAgPAla monolayers were transferred onto the modified gold-deposited glass plate by the horizontal lifting method from a 0.1 M NaCl aqueous subphase at both pH 3.0 and 10.0, respectively. The surface pressure of the monolayers was maintained at 10 mN/m during the transfer. Changes in the refractive index of the medium near the gold surface were measured using SPR.13 A plot of reflected intensity versus the angle of incidence shows a minimum, θm, corresponding to the excitation of surface plasmons at the gold-solution interface. The value θm shifts with changes in the refractive index of the interfacial region near the gold surface.14,15 This change in shift, ∆θm, is proportional to the amount of binding substrate at the surface. During the measurement, a 0.1 M NaCl aqueous solution at pH 3.0 and 10.0 was passed over the sample surface at 10 µL/min, respectively. This flow was periodically replaced with a 0.1 M NaCl aqueous solution containing either L- or (13) Otto, A. Z. Phys. 1968, 216, 398. (14) Eagen, C. F.; Weber, W. H. Phys. Rev. B 1979, 19, 5068. (15) Nylander, C.; Liedberg, B.; Lind, T. Sens. Actuators 1982/83, 3, 79.
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Figure 1. Surface pressure-area isotherms of PAAgPAla monolayers at various pH in 0.1 M NaCl, at 21 °C. Figure 3. FTIR-RAS spectra of the PAAgPAla monolayer transferred onto a gold-deposited glass plate at pH 3.0, 7.0, and 10.0.
Figure 2. pH dependence of the limiting area per monomer unit of the PAAgPAla monolayer. D-alanine
at increasing concentration, while the temperature was kept constant at 28 °C. Results and Discussion Structure of PAAgPAla Monolayer. Figure 1 shows the surface pressure-area (π-A) isotherms for the PAAgPAla monolayer at various pH values, conducted at a constant temperature of 21 °C. The abscissa indicates the area per monomer unit of PAAgPAla molecule. The limiting area per monomer unit of PAAgPAla, given by extrapolation of the linear portion of the π-A isotherm, is strongly dependent on the pH of the subphase (Figure 2). The decrease in the limiting area of the PAAgPAla monolayer in acid pH can be explained in terms of the dissolution of a part of the chains into the aqueous phase because of the ionization and/or hydration of their allylamine moieties. The midpoint for the pH-induced changes in the limiting area is found to be located at approximately pH 8.0, which is a little higher than the pKa, 7.1, of the allylamine moiety of PAAgPAla. This means that the degree of ionization of the allylamine groups at around their pKa is sufficient for a part of the PAAgPAla chains to stay in the aqueous phase; however, a slight decrease in their ionization degree from that at their pKa resulted in the cooperative accumulation of PAAgPAla chains at the interface. This may be reflected as the steep increase in the limiting area at around pH 8.0. The conformation of the poly(L-alanine) graft chains in the monolayer was estimated from FTIR-RAS after the PAAgPAla monolayer was transferred onto the gold-
Figure 4. Surface pressure dependence of fluorescence anisotropy (γ) of DPH in the PAAgPAla monolayer at pH 3.0 and 10.0 in 0.1 M NaCl, at 21 °C.
deposited glass plate. The transfer was carried out using the vertical dipping method, with monolayers formed on the upstroke motion at a surface pressure of 10 mN/m, drawn from a 0.1 M NaCl aqueous subphase at pH 3.0, 7.0, and 10.0, respectively. The conformation of the poly(L-alanine) graft chains in the PAAgPAla monolayer was confirmed by the position of the amide I band (R-helix, 1652 cm-1; β-sheet, 1636 cm-1; random coil, 1658 cm-1)16 in the FTIR-RAS spectra of the PAAgPAla monolayer on the gold-deposited glass plate (Figure 3). It was found that the poly(L-alanine) graft chains in the monolayer adopt an R-helix conformation with considerable amounts of random coil at pH 3.0. On the other hand, the formation of β-sheet structures for the graft chains was confirmed in the FTIR-RAS spectra at both pH 7.0 and 10.0. The fluidity of the PAAgPAla monolayer at the airwater interface was estimated with the fluorescence anisotropy using DPH as a probe. It is common knowledge that the fluorescence anisotropic value, γ, for DPH reflects the environmental fluidity around the fluorescence probe.17 The surface pressure dependences of γ for DPH in the PAAgPAla monolayer at pH 3.0 and 10.0 are shown in Figure 4. The γ values for DPH at pH 10.0 are larger than those at pH 3.0 in the experimental surface pressure (16) Miyazawa, T.; Blout, E. R. J. Am. Chem. Soc. 1961, 83, 712. (17) Shinitzky, M.; Barenholz, Y. J. Biol. Chem. 1974, 249, 2652.
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Figure 5. AFM images of the PAAgPAla monolayer transferred onto mica at (a) pH 3.0 and (b) pH 10.0.
region. This implies that PAAgPAla forms closely packed monolayers at pH 10.0, although the surface area of the PAAgPAla molecule at pH 10.0 is larger than that at pH 3.0 at the same surface pressure (Figure 1). The morphology of the PAAgPAla monolayers was directly observed using AFM. The monolayers were transferred onto mica from a 0.1 M NaCl aqueous subphase at pH 3.0 and 10.0, respectively, using the horizontal lifting method at a surface pressure of 10 mN/m. Figure 5a shows the AFM image for the PAAgPAla monolayer at pH 3.0, where small domains with heights of several nanometers are clearly visible in the image. At pH 10.0, however, no domains are observed and a uniform monolayer structure is confirmed (Figure 5b). Similar AFM images were also observed in LB films affixed to gold-deposited mica (Picosubstrate, Molecular Imaging Co.). These results suggest that, in acidic conditions, protonation of the allylamine moieties occurs, allowing a part of the PAAgPAla chains to dissolve into the aqueous phase, resulting in a decrease in the monolayer area. In other words, the hydrophobic poly(L-alanine) graft chains orientate the monolayer toward the interface by anchoring itself in the air phase. The hydrated sections of the monolayer beneath the interface may be observed as domains in the AFM image in Figure 5a. In alkaline conditions, dehydration of the allylamine moieties occurs, and as a result, the PAAgPAla chains locate themselves entirely at the air-water interface. This is reflected in the AFM image (Figure 5b) as a uniform monolayer structure. In the monolayer, the poly(L-alanine) graft chains self-assemble, because of the loss of the electrostatic repulsion between the neighboring allylamine moieties, to yield β-sheet structure domains at the interface, resulting in the formation of stable and closely packed monolayers (Figure 4). Molecular Recognition Properties of the PAAgPAla Monolayers. We investigated the selective binding of both D- and L-alanine molecules to the PAAgPAla monolayers by π-A isotherm measurements. Figure 6 shows the π-A isotherms for the PAAgPAla monolayer in 0.1 mM aqueous solutions of D- and L-alanine (containing 0.1 M NaCl), taken at pH 3.0 and 10.0, respectively. The π-A isotherms for the monolayer at pH 3.0 exhibit expansions with both 0.1 mM D-alanine (Figure 6a(ii)) and 0.1 mM L-alanine solutions (Figure 6a(iii)). This result implies that the aqueous alanine molecules are nonselectively incorporated into the PAAgPAla monolayer.
Higuchi et al.
Figure 6. (a) Surface pressure-area isotherms of the PAAgPAla monolayer at pH 3.0 (at 21 °C): (i) in 0.1 M NaCl; (ii) in 0.1 mM D-alanine containing 0.1 M NaCl; (iii) in 0.1 mM L-alanine containing 0.1 M NaCl. (b) Surface pressure-area isotherms of the PAAgPAla monolayer at pH 10.0 (at 21 °C): (i) in 0.1 M NaCl; (ii) in 0.1 mM D-alanine containing 0.1 M NaCl; (iii) in 0.1 mM L-alanine containing 0.1 M NaCl.
Figure 7. Binding kinetics of L- and D-alanine molecules to the PAAgPAla monolayer at (a) pH 3.0 and (b) pH 10.0. The concentration of alanine molecules introduced to the PAAgPAla monolayer surface was 80 µM.
The π-A isotherm for the monolayer with 0.1 mM Dalanine at pH 10.0, however, shows little effect (Figure 6b(ii)), a result consistent with that of amino acid free water (Figure 6b(i)). In contrast, the π-A isotherm for the monolayer with the corresponding L-alanine solution (pH 10.0) displayed a significant expansion (Figure 6b(iii)), which suggests that the self-assembled poly(L-alanine) domain in the PAAgPAla monolayer can specifically bind aqueous L-alanine molecules. The binding behavior of alanine molecules to PAAgPAla monolayers was examined more closely by SPR. Figure 7 shows the kinetics for the binding of L- or D-alanine to the PAAgPAla monolayer (affixed to gold-deposited glass plates) when the flow passed over the monolayer was replaced with a 0.1 M NaCl aqueous solution containing 80 µM alanine, at pH 3.0 and 10.0, respectively. The introduction of L- and D-alanine into the PAAgPAla
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Figure 9. Proposed binding site structure for aqueous amino acid formed by the self-assembly of poly(L-alanine) graft chains in the PAAgPAla monolayer. Table 1. Binding Constants for L- and D-Alanine Molecules to the PAAgPAla Monolayer at pH 3.0 and 10.0 pH of the subphase 3.0
substrate
binding constant/M-1
L-Ala
8.5 × 103 7.9 × 103 1.0 × 105 4.9 × 103
D-Ala
10.0
L-Ala D-Ala
are represented by eq 3, where ∆θm,max represents the saturated value of ∆θm, obtained from Figure 8, when the Figure 8. Binding isotherms of L- and D-alanine to the PAAgPAla monolayer at (a) pH 3.0 and (b) pH 10.0.
monolayer at pH 3.0 induced relatively small shifts in the resonance angle of the surface plasmon, ∆θm (Figure 7a). At pH 10.0, however, the introduction of L-alanine into the PAAgPAla monolayer leads to a large shift in ∆θm compared to that induced by its corresponding D isomer (Figure 7b). Figure 8 shows the binding isotherms of L- and D-alanine to the PAAgPAla monolayers at pH 3.0 and 10.0, respectively. The binding isotherm for L-alanine with the PAAgPAla monolayer at pH 10.0 exhibits a typical Langmuir-type saturation behavior. This indicates that the PAAgPAla monolayer provides a specific binding site for L-alanine molecules. The equilibrium between the binding and dissociation of alanine molecules to the PAAgPAla monolayer can be represented by eq 1, where kbin and kdiss represent the
[PAAgPAla monolayer] + kbin
} [PAAgPAla-alanine] (1) [alanine] {\ k diss
rate of binding and dissociation of the alanine molecules to the PAAgPAla monolayer, respectively. The binding constant, K, is denoted by eq 2. Shift changes in the SPR
K ) kbin/kdiss
(2)
angle (∆θm) for the PAAgPAla monolayer, based on the introduction of alanine molecules to the monolayer surface,
d(∆θm)/dt ) kbin[alanine]∆θm,max (kbin[alanine] + kdiss)∆θm (3) binding site on the PAAgPAla monolayer is completely occupied by alanine molecules. Table 1 gives the binding constants, K (calculated using eq 3), for the binding of L- and D-alanine to the PAAgPAla monolayer at pH 3.0 and 10.0, respectively. The binding constants, K, for the binding of L- and D-alanine to the PAAgPAla monolayer at pH 3.0 are similar and small in value. In contrast, the binding constant for the binding of Lalanine to the PAAgPAla monolayer at pH 10.0 was very large, ca. 20 times larger than that of its D isomer. These results suggest that, in alkaline conditions, the selfassembled poly(L-alanine) domain in the PAAgPAla monolayer can form specific binding sites for aqueous L-alanine rather than its D isomer; i.e., aqueous L-alanine molecules can specifically incorporate into the selfassembled domain of the poly(L-alanine) graft chains of the PAAgPAla monolayer. In conclusion, we demonstrated a possibility for the molecular recognition of certain aqueous amino acids using a monolayer membrane system constructed from poly(allylamine), bound by poly(L-alanine) graft chains (PAAgPAla). Figure 9 shows the proposed binding site structure for aqueous amino acid, formed by the self-assembly of poly(L-alanine) graft chains in the PAAgPAla monolayer. In alkaline conditions, PAAgPAla forms stable and closely packed uniform monolayers in which the poly(L-alanine) graft chains self-assembled at the interface. The domain formed by the poly(L-alanine) graft chains in the monolayer acts as a specific binding site for L-amino acid. LA000290X
