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Helical Superstructures from a Poly(γ-benzyl-L-glutamate)-Poly(L-glutamic acid) Amphiphilic Diblock Copolymer: Monolayer Formation on Water and Its Specific Binding of Amino Acids Nobuyuki Higashi,* Tomoyuki Koga, and Masazo Niwa* Department of Molecular Science & Technology, Faculty of Engineering, Doshisha University, Kyo-tanabe, Kyoto 610-0321, Japan Received September 17, 1999. In Final Form: December 15, 1999 This study examines the monolayer properties of a novel amphiphilic diblock polymer of poly(γ-benzyl(PBLG)-block-poly(L-glutamic acid) (PLGA) (PBLG-b-PLGA) at the air-water interface and the binding of R-amino acids such as tryptophan, tyrosine, and phenylalanine to the monolayer surfaces. The block polymer, PBLG-b-PLGA, was synthesized via two steps: at first the PLGA block was prepared by polymerization of γ-benzyl-L-glutamate-N-carboxylic anhydride (BLG-NCA) with propylamine and then by removal of benzyl group, and subsequently the polymerization of BLG-NCA initiated with the amino group at the chain end of the PLGA was performed and gave a final product. PBLG-b-PLGA formed stable monolayers, in which the PLGA segment took an R-helix structure with above 95% helicity over the wide range of surface pressures and pHs. The helix orientaion was in almost perpendicular to the surface, being variable slightly by changing the surface pressure. These helix assemblies were found to have the ability to capture D-isomers of R-amino acids enantiomerically, and the enantioselectivity depended strongly on the hydrophobicity of the side groups of the amino acids. L-glutamate)
Introduction We report here the first synthesis of an amphiphilic diblock copolymer of poly(γ-benzyl-L-glutamate) (PBLG)block-poly(L-glutamic acid) (PLGA) (PBLG-b-PLGA), along with its monolayer properties including orientational features of the helix and the specific binding of R-amino acids. In the previous studies, we have established a wellorganized amphiphilic polymer assembly system at the air-water interface.1 Two-dimensionally aligned polyelectrolyte segments have provided peculiar properties different from those in bulk: for example, the monolayers of poly(methacrylic acid)-based amphiphiles have the ability to read out the chain length of the corresponding guest polymers such as poly(ethylene glycol).2 The basic structure of biopolymers such as protein also consists of polyelectrolytes. To fabricate such a structural model of protein, an amphiphilic PLGA, whose one terminus is modified with two long alkyl chains, has been aligned on water.3 The organized PLGA assemblies are expected to offer a field in which guest molecules can be specifically captured. In fact, it has been reported that surface monolayers carrying oligopeptide moieties as polar headgroups can bind guest peptides specifically in the aqueous phase.4 In addition, enantiomeric permeations of R-amino acids were reported to be accomplished through thick polymer films based on an R-helical PLGA having an oligo(oxyethylene) side chain5 and a bundle structure of helical (1) For a recent review, see: Higashi, N.; Niwa, M. Colloids Surf. A 1997, 123/124, 433. (2) (a) Higashi, N.; Shiba, H.; Niwa, M. Macromolecules 1989, 22, 4650. (b) Higashi, N.; Matsumoto, T.; Niwa, M. Langmuir 1994, 10, 4651. (3) Higashi, N.; Shimoguchi, M.; Niwa, M. Langmuir 1992, 8, 1509. (4) (a) Cha, X.; Ariga, K.; Onda, M.; Kunitake, T. J. Am. Chem. Soc. 1995, 117, 11833. (b) Cha, X.; Ariga, K.; Kunitake, T. J. Am. Chem. Soc. 1996, 118, 9545. (c) Ariga, K.; Kamino, A.; Cha, X.; Kunitake, T. Langmuir 1999, 15, 3875. (5) Maruyama, A.; Adachi, N.; Takatsuki, T.; Torii, M.; Sanui, K.; Ogata, N. Macromolecules 1990, 23, 2748.
PLGA templated with phosphazene.6 The authors noted the importance of an assembled structure of PLGA helices within the polymer films for bringing about the enantioselectivity. We have discovered that the monolayer of the amphiphilic PLGA can capture R-amino acids enantiomerically in aqueous solution while the laterally attenuated monolayer with a PLGA segment-free amphiphile leads to less selectivity.7 This fact again points out the importance of such an assembled structures of helices for enhancement of enantioselectivity. The main purpose of this paper is therefore to fabricate a stable peptide assembly, in which the component block copolypeptide (PBLG-b-PLGA) is well-organized and retains a helical structure. The resultant copolypeptide assemblies are expected to possess an enhanced enantiomeric binding ability toward R-amino acids, which will be applicable to biosensors on the basis of molecular recognition. Experimental Section Synthesis of Amphiphilic Diblock Polymer (PBLG-bPLGA). Amphiphilic PLGA diblock polymer was synthesized by polymerization of γ-benzyl-L-glutamate-N-carboxylic anhydride (BLG-NCA). BLG-NCA was synthesized by reacting γ-benzylL-glutamate with triphosgen in a THF solution.8 First, a chloroform solution (20 mL) of BLG-NCA (2.7 g, 10.2 mmol) was mixed with a chloroform solution (10 mL) of n-propylamine (1.6 × 10-2 g, 0.27 mmol) at room temperature, and the mixture was stirred for 3 h. Then the reaction mixture was poured into a large excess of diethyl ether, and the precipitate (Pr-PBLG) was separated by centrifuge and then washed with diethyl ether repeatedly and dried: yield 2.2 g (97%). See Scheme 1. The number-average degree of polymerization of PBLG was estimated to be 38 by means of 1H NMR spectroscopy (400 MHz; JEOL JNM GX-400 spectrometer) on the basis of the area ratio of the (6) Inoue, K.; Miyahara, A.; Itaya, T. J. Am. Chem. Soc. 1997, 119, 6191. (7) Higashi, N.; Saitou, M.; Mihara, T.; Niwa, M. J. Chem. Soc., Chem. Commun. 1995, 2119. (8) Daly, W. H.; Poche, D. Tetrahedron Lett. 1988, 29, 5859.
10.1021/la991246o CCC: $19.00 © 2000 American Chemical Society Published on Web 02/18/2000
Monolayer Properties of PBLG-b-PLGA
Langmuir, Vol. 16, No. 7, 2000 3483
Scheme 1
Figure 1. Surface pressure-area isotherms of PBLG-b-PLGA on water at various pHs, 20 °C.
signal of ω-CH3 of propylamino group to that of benzyl CH2 in PBLG segment. Subsequently, the Pr-PBLG (2.2 g) thus obtained was dissolved in 20 mL of N,N-dimethylformamide (DMF), and the removal of benzyl groups was carried out by reduction with Pd(black)/H2. After removal of Pd(black) by filtration, the solution was poured into a large excess of diethyl ether. As a result, PrPLGA was obtained as a white powder: yield 1.2 g (92%). The structure of Pr-PLGA was confirmed by 1H NMR spectroscopy. Finally, polymerization of BLG-NCA (0.36 g, 1.4 mmol) initiated with the amino group at the chain end of the Pr-PLGA (0.40 g, 8.1 × 10-2 mmol) was carried out in 10 mL of DMF at room temperature for 24 h. The diblock polymer (PBLG-b-PLGA) was precipitated by pouring the solution into diethyl ether and then washed with chloroform and diethyl ether several times and dried: yield 0.66 g (94%). The average degree of polymerization of the PBLG block segment was determined to be 17 by 1H NMR spectroscopy in the same manner as mentioned above. Surface Pressure-Area Isotherms and LangmuirBlodgett (LB) Films. The monolayers are obtained by spreading a benzene/DMF (7/3 in vol.) solution (about 1 mg mL-1) of PBLGb-PLGA on water that was purified (r > 18 MΩ cm) using a Milli-Q system (Millipore Ltd.). Twenty minutes after spreading, the monolayer was compressed continuously with a rate of 1.20 cm2 s-1. Wilhelmy’s plate method and a Teflon-coated trough with a microprocessor-controlled film balance, FSD-220 (USI system Ltd., Japan) with a precision of 0.01 mN m-1, were used for surface pressure measurements. pH in the subphase was adjusted with aqueous HCl and NaOH (0.01 M). The subphase temperature was kept at 20 ( 0.1 °C. LB films were transferred in the vertical mode at various surface pressures and a transfer rate of 10 and 100 mm min-1 at up- and downstroke motions, respectively, onto a hydrophobically treated quartz plate or a CaF2 plate. The deposition of PBLG-b-PLGA onto a gold substrate was carried out in the horizontal-lifting method. Fourier Transfer Infrared Spectroscopy (FTIR) Measurements. The transmission FTIR spectra of the LB films transferred onto CaF2 plates were measured by a Nicolet System 800 using a Mercury-Cadmium-Tellurium (MCT) detector (resolution, 4 cm-1; number of scans, 1024). Reflection absorption spectra of the LB film were obtained on a Au-deposited glass plate using a MCT detector (resolution, 4 cm-1; number of scans, 1024). The p-polarized light was introduced onto the sample at 80° to the surface normal. The sample and the detector chamber were purged with dried air in both cases.
The Binding Experiment of r-Amino Acids. D- or LTryptophan (Trp), D- or L-tyrosine (Tyr), and D- or L-phenylalanine (Phe) were purchased from Wako Pure Chemical Industries, Ltd., and used without further purification. The binding experiment of R-amino acid was carried out as follows. One-layer LB film, in which the PLGA segments of PBLG-b-PLGA are exposed to water phase, was first deposited on each side of the hydrophobically treated quartz plate. Subsequently, these LB films were separately immersed in aqueous solution of D- or L-amino acids (1 mM, pH 4.0), and the binding processes were followed as an absorbance difference (∆A) in UV spectra before and after binding of R-amino acids. UV spectra of the LB films were measured with a Shimazu UV-2100. Fluorescence spectra of the LB films were measured in a quartz cell (path length 10 mm) filled with pure water (pH 4.0) by using a Jasco FP-770 spectrofluorophotometer.
Results and Discussion Monolayer Characteristics of PBLG-b-PLGA. We have reported in the previous paper that a PLGA amphiphile, having two long alkyl chains (C18H37) at one terminus as a hydrophobic part, shows the secondary structural transition from R-helix to β-sheet structure upon compression of its monolayer because of strengthening of the interhelix interaction.3 For the purpose of the present study, it must be required to avoid forming β-sheet structure and at the same time to assemble R-helices, two-dimensionally. Therefore, we have devised a diblocktype copolymer in which a rigid, rodlike PBLG helix segment is in place of two long alkyl chains incorporated into the chain end of the PLGA segment. The PLGA segment is readily expected to prefer to take an R-helix monolayer phase since the cross-sectional area of PBLG helix is surely larger than that of PLGA helix. Figure 1 shows surface pressure-area isotherms of PBLG-b-PLGA at different pHs, 20 °C. At every pH, the PBLG-b-PLGA forms a stable monolayer. It has been reasonably considered that hydrophobic polypeptide helices prefer to lie down at the air-water interface and eventually give a lower collapse pressure such as 20 mN m-1.9 In our case, surface pressure-area isotherms showed a steep increase part after phase transition from LE to LC between 20 and 30 mN m-1 and then the monolayer collapsed at around 50 mN m-1, suggesting that helices (9) (a) Loeb, G. I. J. Colloid Interface Sci. 1968, 26, 236. (b) Loeb, G. I.; Baier, R. E. J. Colloid Interface Sci. 1968, 27, 38.
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Figure 2. Transmission FTIR spectrum of the four-layer LB film of PBLG-b-PLGA on a CaF2 plate deposited at 20 mN m-1, pH 4.0. The results from computer generated peak resolution and curve fit using Gaussian peak shapes are also displayed; there appear four peaks in CdO stretching band region at 1735, 1711, 1658, and 1626 cm-1 of ester, carboxylic acid, amide I (R-helix), and amide I (β-sheet), respectively, and three peaks in N-H being band region (amide II) at 1551, 1535, and 1518 cm-1 of R-helix, random coil, and β-sheet, respectively.
in the monolayers stand up on the water surface by incorporation of the PLGA moiety as one block segment. Extrapolation of the steep part of the isotherm to zero surface pressure gave a value of area per PBLG-b-PLGA molecule of 3.5 nm2. The diameter of PBLG helical rod including side chain region was reported to vary within the range of 1.6-2.6 nm, depending upon the conformation of the side chain.10,11 On the basis of these diameters, we can calculate the cross-sectional area of PBLG helices to range from 2.3 to 5.8 nm2 per molecule when they orient perpendicularly to the water surface. The value obtained from the extrapolated area (3.5 nm2) is in the range of the above theoretical cross-sectional areas, thus suggesting that the helices may “stand up” on the water surface. In the isotherms there is no remarkable difference in the pH at high surface pressures but below 20 mN m-1 a slight difference appears; i.e., the monolayer has a tendency to expand with lowering pH. This variation in isotherms must be ascribed to the extent of ionization and/or conformational change of the PLGA segment. Secondary Structure and Orientation of PBLGb-PLGA at the Air-Water Interface. To elucidate the secondary structure of the peptide segments of PBLGb-PLGA monolayers, the monolayers were transferred in the vertical-dipping method at various surface pressures (20-40 mN m-1), pH 4.0, onto CaF2 plates. The transfer ratio ((10%) was unity both in the downstroke and in the upstroke mode. Figure 2 shows a typical transmission FTIR spectrum of the four-layer Langmuir-Blodgett (LB) film deposited at a surface pressure of 20 mN m-1. In amide I and amide II regions, characteristic absorptions of R-helix appear at 1653 and 1550 cm-1, respectively,12,13 although a minor existence of β-sheet and random coil (10) Helfrich, J.; Hentschke, R. Macromolecules 1995, 28, 3831. (11) Chang, Y.; Frank, C. W. Langmuir 1996, 12, 5824. (12) Miyazawa, T.; Blout, E. R. J. Am. Chem. Soc. 1961, 83, 712. (13) Itoh, K.; Foxman, B. M.; Fasman, G. D. Biopolymers 1976, 15, 419.
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Figure 3. Fraction of secondary structures (b, R-helix; 4, β-sheet, 9; random coil) evaluated by peak resolution of the corresponding transmission FTIR spectra as a function of the surface pressure at which the LB films were deposited.
structures may not be excluded. The fraction of secondary structures (R-helix, β-sheet, and random coil), evaluated by peak resolution of the spectra obtained, is plotted in Figure 3 as a function of the surface pressure at which the LB films were prepared. It can been seen from the figure that at every surface pressure PBLG-b-PLGA takes R-helix with more than 95% content. Previously we have demonstrated that the conformation of PLGA segment, whose one terminus is connected with two alkyl chains in place of the PBLG segment as a hydrophobic part, is readily changed from R-helix to β-sheet structures by compressing the monolayer because of compulsory contact of helices.3 In the case of PBLG-b-PLGA monolayers, however, the high helix content was maintained even after compression of the monolayer to higher surface pressures. Such a stabilization of R-helix structure must be due to the fact that the PLGA helices as the hydrophilic part cannot interact over each other to cause conformational change even in the condensed phase (e.g., at a surface pressure of 40 mN m-1) because the cross-sectional area of helical PBLG segment is significantly larger than that of helical PLGA segment (ca. 1.3 nm2).14 Subsequently, the orientation of these PBLG-b-PLGA helices was examined by means of reflection-absorption (RA)-FTIR spectroscopy. Enriquez et al.15 demonstrated that the nature of the order and average orientation of the polypeptide R-helix in the self-assembled monolayers on gold substrates can be inferred from RA-FTIR data. Accordingly, the one-layer films of PBLG-b-PLGA were deposited onto gold substrates by using a horizontal-lifting method at constant surface pressures of 20, 30, and 40 mN m-1, pH 4.0, and their RA-FTIR spectra were then measured and displayed in Figure 4. By using the ratio of the amide I and amide II integrated intensities, D ) AI/AII, the average tilt of the helical axis from the surface normal, 〈θ〉15,16 (Scheme 2) was evaluated for the mono(14) Reda, T.; Hermel, H.; Holtje, H. D. Langmuir 1996, 12, 6452. (15) Enriquenz, E. P.; Samulski, E. T. Mater. Res. Soc. Symp. Proc. 1992, 255, 423. (16) Tsuboi, M. J. Polym. Sci. 1962, 59, 139.
Monolayer Properties of PBLG-b-PLGA
Langmuir, Vol. 16, No. 7, 2000 3485 Table 2. Enantioselective Interaction between PBLG-b-PLGA LB Film and r-Amino Acids R-amino acids
enantiomer
(∆Aeq/) × 107
Phe
D
12.0 1.2 3.8 0.6 7.1 2.4
L
Trp
D
Tyr
D
L L
Ra
hydropathyb (kJ mol-1)
10.0
2.5
6.5
1.5
3.0
0.08
a R is the ratio of the amount of surface-bound D-isomer and that of L-isomer. b Free energy changes for the transfer of amino acid side chains from a hydrophobic to a hydrophilic phase.19
Figure 4. Reflection-absorption (RA)-FTIR spectra for the onelayer LB films of PBLG-b-PLGA deposited onto gold substrates by using a horizontal-lifting method at prescribed surface pressures, pH 4.0. Scheme 2
Table 1. Amide I to Amide II Integrated Intensity Ratio (D ) AI/AII) and the Average Tilt Angle of the Helix Axes from the Surface Normal 〈θ〉 at Various Surface Pressures surface pressure (mN m-1)
D ) AI/AII
〈θ〉a deg
20 30 40
2.4 2.8 3.8
45 40 33
a The θ values were estimated from the D values obtained from RA-FTIR, with amide I ) 1658 cm-1, amide II ) 1551 cm-1.
layers deposited at the prescribed surface pressures and summarized in Table 1. It is apparent from this table that with increasing surface pressure the orientation of helix rods is considerably improved and becomes much closer
to the perpendicular alignment with a tilt angle of 33°. A similar situation has been observed for the monolayers of a diblock polymer amphiphile of poly(γ-methyl-Lglutamate)-b-poly(ethylene glycol),17 in which at a lower surface pressure the polypeptide helix lies down on the water surface and then by compressing the monolayer has a tendency to stand up perpendicularly. It should be noted that in our monolayer the helices tilted slightly from the surface normal even in the highly condensed phase at 40 mN m-1. The helices are assembled in the monolayer with a parallel packing. The helical polypeptides are known to have a macrodipole moment along the helix axis.18,19 In particular, the macrodipole moment of PBLGb-PLGA is expected to be much larger than that of poly(γ-methyl-L-glutamate)-b-poly(ethylene glycol), since PLGA block as a hydrophilic part would take an R-helical structure under these conditions and also behave as a rigid rod similar to the PBLG block segment. The parallel packing of helices must be, therefore, energetically unfavorable, and consequently led to the slight tilt of helices. Enantiomeric Binding of r-Amino Acids to PBLGb-PLGA Monolayers. The PBLG-b-PLGA monolayercovered surfaces were subsequently subjected to binding experiments of R-amino acids such as tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe), all of which have an aromatic ring in the side chain. One-layer LB films, in which the PLGA segments of PBLG-b-PLGA are exposed to water phase, were deposited on each side of the hydrophobically treated quartz plates. A set of the LB films was separately immersed in aqueous solution of Lor D-amino acid (1 mM, pH 4.0) for a prescribed period, and the binding process was followed as an absorbance difference (∆A) at λmax of each R-amino acid in the UV spectrum before and after binding of R-amino acid. The values of ∆A for all R-amino acids used in this study were observed to increase gradually upon immersion owing to binding of R-amino acids at the monolayer surfaces, and then leveled off within about 1 h. The amount of monolayerbound R-amino acid at equilibration (∆Aeq), which is normalized with the molar extinction coefficient () for each R-amino acid, is listed in Table 2. For D-isomers, the value of ∆Aeq/ is found to decrease in the order of Phe, Tyr, and Trp. The molecular size of the side group of R-amino acids evaluated by a CPK molecular model decreases in the order of Trp, Tyr, and Phe. Therefore, the observed order in the amount of monolayer-bound R-amino acid demonstrates that the smaller R-amino acid could adsorb more easily onto the monolayer surface owing to depression of steric hindrance. The amounts of monolayer(17) Toyama, A.; Kugumiya, S.; Yonese, M.; Kinoshita, T.; Tsujita, Y. Chem. Lett. 1997, 443. (18) Wada, A. Adv. Biophys. 1976, 9, 1. (19) Hol, W. G. J.; van Duijnen, P. T.; Berendsen, H. J. C. Nature 1978, 273, 443.
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bound L-isomers are found to be much less than those for the corresponding D-isomers. Our preliminary results20 concerning the binding experiment have demonstrated that chirality of the component glutamic acid of the polymer segment is not the primary crucial factor that causes such an enantioselectivity since the PLGA monolayers, being mainly in β-sheet structure, show no selectivity between D- and L-isomers. Preferably a highly helical conformation and its assembled structure have been suggested to be more important in causing such an enantiomeric binding. We have no conclusive mechanism for explaining the predominant binding of D-isomer so far, but helical sense of the polymer segment is considered to play one of the key roles, which will require further exploration. The selectivity between D- and L-isomers estimated as a ratio of ∆Aeq(D-isomer)/∆Aeq(L-isomer) ()R) is also listed in Table 2. It is clear that the organized PLGA surface of the PBLG-b-PLGA monolayer has the ability to discriminate R-amino acids enatiomerically although the value of R varies depending on the kind of R-amino acids. This effect on R value is considered to be probably due to differences in hydrophobicity as well as steric factors of the side chain of R-amino acids. Table 2 includes the value of hydrophathy, which indicates a hydrophobicity scale corresponding to the free energy of transfer of the side chain of the R-amino acid from water to an apolar environment.21 The larger value of hydrophathy means the side chain is more hydrophobic. The hydrophathy of these three amino acids is in the order Phe > Trp > Tyr. The observed enantioselectivity (R) is found to be in the same order as that of hydrophathy, suggesting that hydrophobic interaction plays an important role in enhancing such a selectivity. To elucidate the microenvironment of the monolayerbound R-amino acids, fluorescence spectroscopy was employed. Trp is a suitable candidate for this purpose since Trp is fluorescent. Figure 5 shows the fluorescence emission spectra of Trp, excited at 270 nm, in water of pH 4.0 and in an LB film of PBLG-b-PLGA immersed in water of pH 4.0. In water, Trp gave an emission maximum (λem) at 354 nm. On the other hand, in the LB film the λem showed a remarkable blue-shift to 335 nm. The fluorescence spectrum of Trp is considerably influenced by its surrounding polarity.22 In fact, when spectra of Trp were (20) Koga, T.; Higashi, N.; Niwa, M. Proc. 2nd Int. Symp. Polyelectrolytes 1999, 423. (21) Eisenberg, D.; Weiss, R. M.; Terwillinger, T. C.; Wilcox, W. Faraday Symp. Chem. Soc. 1982, 17, 109. (22) Maste, M. C. L.; Norde, W.; Visser, A. J. W. D. J. Colloid Interface Sci. 1997, 196, 224.
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Figure 5. Fluorescence spectra of D-Trp, excited at 270 nm, in water of pH 4.0 (dotted line) and in an LB film of PBLGb-PLGA immersed in water of pH 4.0 (solid line).
measured in water/dioxane mixtures with various compositions, λem moved to a shorter wavelength with decreasing solvent polarity; i.e., the observed blue-shift of λem suggests that Trp prefers to exist in a more hydrophobic environment with the LB film. These spectral features also strongly support the importance of hydrophobicity of R-amino acids to enhance the enantioselectivity. In summary, the present study demonstrates that a novel amphiphilic diblock polymer PBLG-b-PLGA is successfully prepared and forms a stable monolayer, in which the PLGA segment takes R-helix structure over the wide range of surface pressures but its orientation is variable by changing the surface pressure. In addition, these helix assemblies can capture D-isomers of R-amino acids predominantly, and such a selectivity is found to depend strongly on hydrophobicity of the R-amino acids. This diblock copolypeptide would be applicable to biosensors on the basis of such an enantiomeric binding of R-amino acids. LA991246O
