Molecular Recognition of Nucleolipid Monolayers of 1 - American

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Langmuir 2003, 19, 5389-5396

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Molecular Recognition of Nucleolipid Monolayers of 1-(2-Octadecyloxycarbonylethyl)cytosine to Guanosine at the Air-Water Interface and Langmuir-Blodgett Films Wangen Miao, Xuezhong Du, and Yingqiu Liang* Laboratory of Mesoscopic Materials and Science and State Key Laboratory of Coordination Chemistry, Nanjing University, Nanjing 210093, People’s Republic of China Received April 3, 2003 Molecular recognition of 1-(2-octadecyloxycarbonylethyl)cytosine monolayers to guanosine at the airwater interface and Langmuir-Blodgett films has been studied in detail using surface pressure-area isotherm, UV-vis, FTIR, and surface-enhanced Raman scattering spectroscopy techniques. The direct FTIR spectroscopic evidence for the molecular recognition of Watson-Crick complementary base pairs is observed. It is shown that the molecular recognition occurs between the cytosine and guanosine bases through triple hydrogen bonds at the expense of the intermolecular hydrogen-bonding network between the adjacent cytosine moieties in the monolayers. The cytosine rings undergo a change in orientation and aromatic stacking from the flat-on geometry without stacking interaction before molecular recognition to an end-on one in the J-aggregate fashion after molecular recognition. The triple hydrogen bonds between the cytosine and guanosine base pairs is stable below 60 °C and is completely dissociated above 120 °C, while the intermolecular hydrogen-bonding interaction between the adjacent cytosine moieties in the monolayers significantly strengthens the interaction between the corresponding hydrocarbon chains, so that the phase transition temperature of the LB films is increased to 145 °C. The process of molecular recognition is revealed in all directions using various molecular spectroscopic techniques.

Introduction Many biological molecules are found to have specific molecular recognition properties to present various biological functions. The most notable example of biological recognition based on hydrogen-bonding interaction is complementary base pairing in nucleic acids.1 The mutual recognition of Watson-Crick complementary bases in nucleic acids by means of multiple hydrogen bonds is the most efficient mechanism of accumulating, storing, reproducing, and evolving genetic information.2 Base-base interactions might be driven by π-π stacking and/or intermolecular hydrogen-bonding interactions, both necessary for nucleic acid stabilization, but it is worthwhile to recall that the hydrogen-bonding interaction does not provide significant driving force for molecular recognition in bulk water due to the strong competitive binding of water molecules.1,3 A great deal of experimental and theoretical work has been performed in the past decades to model and mimic biological complexity through supramolecular assemblies,2,4-6 such as, micelles,7-9 vesicles,10-12 and mono* To whom correspondence should be addressed. Fax: 86-253317761. E-mail: [email protected]. (1) Saenger, W. Principles of Nucleic Acid Structure; SpringerVerlag: New York, 1984. (2) Lehn, J. M. Supramolecular Chemistry; VCH: Weinheim, 1995. (3) Fersht, A. R. Trends Biochem. Sci. 1987, 12, 301. (4) Lehn, J. M. Angew. Chem., Int. Ed. Engl. 1990, 29, 1304. (5) Lawrence, D. S.; Jiang, T.; Levitt, M. Chem. Rev. 1995, 95, 2229. (6) Ariga, K.; Kunitake, T. Acc. Chem. Res. 1998, 31, 371. (7) Novick, J. S.; Chen, J. S.; Noronda, G. J. Am. Chem. Soc. 1993, 115, 7636. (8) Novick, J. S.; Cao, T.; Noronda, G. J. Am. Chem. Soc. 1994, 116, 3285. (9) Berti, D.; Barbaro, P.; Bucci, H.; Baglioni, P. J. Phys. Chem. B 1999, 103, 4916. (10) Onda, M.; Yoshihara, K.; Koyano, H.; Ariga, K.; Kunitake, T. J. Am. Chem. Soc. 1996, 118, 8524. (11) Berti, D.; Barbaro, P.; Bonaccio, S.; Barsacchi-Bo, G. Luisi, P. L. J. Phys. Chem. B 1998, 102, 303.

layers13-22 at the air-water interface. Analysis and mimicry of these processes not only will facilitate better understanding of the corresponding biological functions and processes but also can be significant in the development of new medicines and chemical sensors.23,24 A pioneering work on the base pair mimic at the air-water interface was described by Kitano et al.;25 thereafter, much work about molecular recognition at the air-water interface between nucleolipids and their complementary bases dissolved in the subphase was reported.13-18,20-22 The Langmuir monolayers at the air-water interface provide unique environments for molecular interactions and consequently for molecular recognition.6,23,25-29 Bases in DNA interact mainly via hydrogen bonds with their Watson-Crick partners on the complementary strand, and each nucleic acid strand is greatly stabilized by stacking interactions between neighboring bases.1,2 The (12) Kimizuka, N.; Kawasaki, T.; Hirata, K.; Kunitake, T. J. Am. Chem. Soc. 1998, 120, 4094. (13) Alhers, M.; Ringsdorf, H.; Rosemeyer, H.; Seela, F. Colloid Polym. Sci. 1990, 268, 132. (14) Kurihara, K.; Ohto, K.; Honda, Y.; Kunitake, T. J. Am. Chem. Soc. 1991, 113, 5077. (15) Berndt, P.; Kurihara, K.; Kunitake, T. Langmuir 1995, 11, 3083. (16) Berti, D.; Franchi, L.; Baglioni, P. Langmuir 1997, 13, 3438. (17) Shimomura, H.; Nakamura, F.; Ijiro, K.; Taketsuna, H.; Tanaka, M.; Nakamura, H.; Hasbe, K. J. Am. Chem. Soc. 1997, 119, 2341. (18) Ra¨ler, U.; Heiz, C.; Luisi, P. L. Tampe´, R. Langmuir 1998, 14, 6620. (19) Week, M.; Fink, R.; Ringsdorf, H. Langmuir 1997, 13, 3515. (20) Huang, J.; Li, C.; Liang, Y. Langmuir 2000, 16, 3937. (21) Li, C.; Huang, J.; Liang, Y. Langmuir 2000, 16, 7701. (22) Li, C.; Huang, J.; Liang, Y. Langmuir 2001, 17, 2228. (23) Ringsdorf, H.; Schlarb, B.; Venzmer, J. Angew. Chem., Int. Ed. Engl. 1988, 27, 113. (24) Kimizuka, N.; Kawasaki, T.; Hirata, K.; Kunitake, T. J. Am. Chem. Soc. 1995, 17, 6360. (25) Kitano, H.; Ringsdorf, H. Bull. Chem. Soc. Jpn. 1985, 58, 2826. (26) Moy, V.; Florn, E.; Gaub, H. Science 1994, 266, 257. (27) Shimomura, M. Prog. Polym. Sci. 1993, 18, 295. (28) Kunitake, T. Supramol. Sci. 1996, 3, 45. (29) Bohanon, T.; Denzinger, S.; Fink, R. Angew. Chem., Int. Ed. Engl. 1993, 32, 1033.

10.1021/la0345690 CCC: $25.00 © 2003 American Chemical Society Published on Web 05/17/2003

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organized nucleolipid monolayers at the air-water interface might act as single nucleic acid strands, due to the hydrophobic interaction between the corresponding alkyl chains, to recognize complementary bases in the subphase, which not only provide more information on molecular recognition of nucleic acid bases in nature but also is suitable to being studied with molecular spectroscopy (UV-vis, FTIR, and Raman) using the LangmuirBlodgett (LB) technique. 1H NMR is also one of the techniques most frequently used to investigate molecular recognition between biologically relevant molecules, particularly in elucidating base-base interactions by this technique and discriminating between stacking and hydrogen-bonding interaction (stacking causes an upfield shift of the resonance of the base protons, while a hydrogen bond causes a downfield shift),30 and the 2D 1H NMR technique will provide direct information on interaction and distance between the protons of the bases.31 However, the 1H NMR technique is mostly limited to the systems of micelles and liposomes. Most work on molecular recognition of the base pairs is focused upon the studies of adenine and uracil/thymine moieties in nucleolipids;6,13-16,18,20-22,25 however, few studies on molecular recognition of guanine and cytosine moieties17,18,23 are only limited to the comparison of surface pressure (π)-area (A) isotherms. In this paper, nucleolipids with cytosine moieties were synthesized and used to form monolayers at the air-water interface to recognize complementary bases guanosine in the subphase. π-A isotherms were used to investigate monolayer behaviors prior to and after molecular recognition. A combination of UV-vis, FTIR, and surface-enhanced Raman scattering (SERS) spectroscopy techniques were employed to study the molecular recognition of the base pairs in the corresponding LB films, which provided abundant information on the molecular recognition in all directions. The direct spectroscopic evidence for the molecular recognition of Watson-Crick base pairs has been observed; moreover the formation of the complementary base paring was further demonstrated through variable-temperature FTIR spectroscopy. Experimental Section Synthesis of 1-(2-Octadecyloxycarbonylethyl)cytosine. 1-(2-Carboxyethyl)cytosine (183 mg, 1 mmol), which was prepared from N4-acetylcytosine (Sigma) with the method reported in previous literature,32 and KOH powder (56 mg, 1 mmol) in dry DMSO (10 mL) were stirred at 60 °C for 4 h. Octadecyl methanesufonate (348 mg, 1 mmol) was then added to the mixture, which was further stirred at the same temperature for another 10 h. After being cooled to room temperature, the mixture was poured into 200 mL of water. White precipitate was collected, washed with water, and dried in a vacuum over P2O5. The white solid was purified via silica gel column chromatography and eluted with methanol/chloroform (v/v, 1:8) solvents to give 270 mg of 1-(2-octadecoxycarboxylethyl)cytosine in 62% yield.1H NMR, 500 MHz (CDCl3): 7.45 (d, 1H, H-6); 5.91 (d, 1H, H-5); 3.99 (t, 2H, H-1′); 3.95 (t, 2H, H-2′); 2.75 (t, 2H, CH2(2)); 1.70 (m, 2H, CH2(3)); 1.22 (m, 28H, CH2(4-17)); 0.82 (t, 3H, CH3(18)). Anal. Calcd for C25H45N3O3: C, 68.93; H, 10.41; N, 9.65. Found: C, 69.37; H, 10.62; N, 9.61. Monolayer Spreading. The experiments for monolayer spreading were performed on a Nima Langmuir-Blodgett trough (Nima Technology, Coventry, England) equipped with computer controls. A paper wilhelmy plate was used as the surface pressure sensor and situated in the middle of the trough. Two barriers (30) Berti, D.; Barbaro, P.; Bucci, I.; Baglioni, P. J. Phys. Chem. B 1999, 103, 4916. (31) Bonaccio, S.; Capitani, D.; Segre, A. L.; Walde, P.; Luisi P. L. Langmuir 1997, 13, 1952. (32) Wada, T.; Inaki, Y.; Takemoto, K. Polym. J. 1989, 21, 11.

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Figure 1. Chemical structures of (a) nucleolipid 1-(2-octadecyloxycarbonylethyl)cytosine and (b) complementary base guanosine. compressed or expanded symmetrically at the same speed from two sides of the trough. Monolayers were obtained by spreading 80 µL of chloroform solution of cytosine amphiphile with a concentration of 1 mM onto pure water or aqueous guanosine solution (1.0 mM). After the monolayer was spread, 15 min was allowed for solvent evaporation, and then the monolayer was compressed at a rate of 10 mm/min unless otherwise indicated. The subphase temperature was kept at 20 ( 1 °C. Each π-A curve was reproducible. Preparation of SERS-Active Silver Substrates. Fresh 5% NaOH solution (2.5 mL) was added to 50 mL of AgNO3 solution whereupon a dark brown precipitate was obtained. Concentrated NH3‚H2O was then added to the mixture dropwise until the precipitate was redissolved. D-Glucose (10%, 15 mL) was added to the prepared Tollen’s reagent with careful agitation to ensure mixing. Then the solution was immediately poured into a culture dish in which the cleaned glass substrates were placed horizontally. After 5 min, the solution was poured out. The silvercoated substrates were rinsed with distilled water and sonicated in distilled water for 1 min. The substrates were then stored in distilled water before LB film fabrication. Langmuir-Blodgett Film Transfer. The monolayers were compressed up to the surface pressure 25 mN/m, and 30 min was spent to establish equilibrium of the stable monolayers. The monolayers were then transferred onto quartz, CaF2, and silvercoated substrates by the vertical dipping method at a dipping rate of 2.0 mm/min. The substrate surface was set to be perpendicular to the direction of the moving barrier. Prior to transfer, the quartz and CaF2 substrates were cleaned by successive sonification in chloroform, acetone, ethanol, and pure water for 5 min each. The transfer ratio of Z-type LB films was nearly unity (1.0 ( 0.1). Spectrum Measurements. UV spectra of the LB films onto quartz plates were recorded on a Shimadzu UV-3100 spectrometer. FTIR transmission spectra of the LB films onto CaF2 substrates were collected through an IFS66V spectrometer (Bruker) equipped with a DTGS detector. All spectra were collected for 500 interferograms with the resolution 4 cm-1. FTRaman spectra of the LB films onto silver-coated substrates were detected on a Bruker RFS100 spectrometer with a Nd:YAG laser emitting at 1064 nm and an InGaAs detector working at the liquid nitrogen temperature. Five hundred scans were performed for LB films, and 50 scans were taken for solid powder with a laser power of 150 mW at 4-cm-1 resolution. For the measurements of infrared spectra at elevated temperatures, a CaF2 substrate with the transferred LB film was mounted into a heating cell, and temperature control was achieved with a P/N 21.500 automatic temperature controller (Graseby Specac Inc.) through a copper-constantan thermocouple with an accuracy of (1 °C. After the temperature was raised to the preset value, 15 min was allowed for thermal equilibrium.

Results and Discussion Behavior of Monolayers at the Air-Water Interface. Figure 1 shows chemical structures of nucleolipid with cytosine moiety and its complementary base guanosine. It is well-known that the shape, collapse pressure, and limiting area of a π-A isotherm depend on subphase, temperature, and molecular structure of amphiphiles investigated. For an amphiphile, when experimental

Molecular Recognition of Nucleolipid Monolayers

Figure 2. π-A isotherms of the nucleolipid monolayers at the air-water interface on (a) pure water and (b) aqueous 1 mM guanosine solution: temperature, 20 °C; compression rate, 10 mm/min.

conditions such as temperature are fixed, the shape of the isotherm is strongly influenced by the mutual interaction between the film-forming molecules and soluble molecules in the subphase, and this feature has been widely used in the studies on molecular recognition at the air-water interface. Figure 2 compares π-A isotherms of nucleolipid monolayers on pure water and guanosine-containing solution (1 mM). On pure water, the nucleolipids form a stable monolayer with a collapse pressure of 57 mN/m and a limiting area of 36 Å2/molecule, which is estimated from the π-A curve by extrapolating its condensed region to the zero surface pressure. Combining the limiting area and cytosine size, it is probable that the planes of the cytosine rings adopt a parallel orientation more or less to the monolayer surface. The shape of the isotherm suggests that the hydrocarbon chains in the monolayer are tilted from the normal of the monolayer at a certain angle. On aqueous guanosine solution, the monolayer is a little expanded in the region of lower molecular area, together with a collapse pressure of 68 mN/m and a limiting area of 38 Å2/molecule. The shape of the isotherm implies that the hydrocarbon chains in the monolayer seem to be vertical in comparison with those on pure water. An increase in collapse pressure suggests the occurrence of molecular recognition of base pairs at the air-water interface. In general, when complementary bases are dissolved in the subphase, the corresponding isotherm is shifted to a little larger molecular area, which indicates a decrease of packing density caused by the interaction of the nucleobases in the monolayer. In our case, the large hydrophilic headgroups of the hydrogen-bonded base pairs cannot give rise to an obvious expansion of the isotherm, instead, comparable limiting area to that on pure water. This suggests that the aromatic rings of the hydrogenbonded base pairs and the C-C-C planes of the corresponding hydrocarbon chains are preferentially oriented perpendicular to the monolayer surface in comparison with those in the absence of guanosine. Otherwise, if the aromatic rings remain the identical orientation prior to and after molecular recognition, the limiting area in the presence of guanosine should be much larger than that in the case of pure water due to the large hydrophilic headgroups of the hydrogen-bonded base pairs, and the corresponding hydrocarbon chains would be further tilted. The evidence for the aromatic ring reorientation will be

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Figure 3. UV-vis spectra of nine-monolayer nucleolipid LB films deposited from (a) pure water and (b) aqueous 1 mM guanosine solution, respectively, together with the spectra of (c) nucleolipid in chloroform and (d) guanosine in water.

further put forward in the following section of molecular spectroscopy. UV-vis Spectra of LB Films. Spectra a and b of Figure 3 show UV-vis spectra of nine-monolayer nucleolipid LB films deposited from pure water and aqueous guanosine solution, respectively. In the case of pure water, a broad band with the absorption maximum near 276 nm appears. The maximum is very close to that in chloroform solution (278 nm, Figure 3c), suggesting that no significant π-π interaction from aromatic stacking of cytosinecytosine moieties is formed. It is likely that the planes of the cytosine rings are preferentially oriented parallel to the film substrate. In our case, the possibility that the π-π stacking of the cytosine-cytosine moieties between neighboring layers is also ruled out, considering the fact that the multilayers in the LB films are transferred at the surface pressure 25 mN/m in the Z-type fashion. In the presence of guanosine in the subphase, the absorption maximum in the UV-vis spectrum of the corresponding LB film appears at 290 nm (Figure 3b), which is different neither from the spectrum of aqueous guanosine solution (Figure 3d) with a maximal absorption at 252 nm and a shoulder near 275 nm nor from the spectrum of the LB film in the case of pure water (276 nm). The red shifts of absorption maximum indicate the formation of π-π aromatic stacking in the J-aggregate fashion not only between the guanine rings but also between the cytosine rings. This suggests that the cytosine planes undergo a reorientation from the flat-on mode to an end-on one in order to promote aromatic stacking of cytosine-guanine rings. It may be inferred the occurrence of molecular recognition between the cytosine and guanosine base pairs at the air-water interface through intermolecular hydrogen-bonding interaction, which is supported by the similar studies of Kunitake et al.33 They reported that a complementary nucleobase cooperatively bonds to 1′,3′bis(octadecyloxy)isopropyl orotate monolayer, with the analogous headgroup, through both hydrogen bonding and aromatic stacking.33 The above results are consistent with those of the corresponding π-A isotherms. The molecular recognition of the cytosine-guanosine base pairing is accompanied with the formation of intermolecular hydrogen-bonding interaction and π-π interaction of aromatic stacking. (33) Kawahara, T.; Kurihara, K.; Kunitake, T. Chem. Lett. 1992, 1839.

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Figure 4. Comparison of FTIR transmission spectra of ninemonolayer nucleolipid LB films transferred from (a) pure water and (b) aqueous 1 mM guanosine solution, respectively.

FTIR Spectra of LB Films. Figure 4a shows FTIR transmission spectra of a nine-monolayer nucleolipid LB film transferred from pure water. Two strong bands at 2918 and 2850 cm-1 are attributed to the antisymmetric and symmetric CH2 stretching vibrations (υa(CH2) and υs(CH2)) of long hydrocarbon chains, respectively. It is known that the υa(CH2) and υs(CH2) frequencies are sensitive to the conformational order of alkyl chains.34,35 Lower wavenumbers are characteristic of highly ordered conformers in preferential all-trans chains, while the number of gauche conformers increases with frequencies and width of the bands. The low frequencies at 2918 and 2850 cm-1 are characteristic of highly ordered alkyl chains with almost all-trans conformation. The doublet peak around 1475 and 1462 cm-1 is assigned to the CH2 scissoring vibration (δ(CH2)) of the alkyl chains. The δ(CH2) vibrational mode is well-known to be extremely sensitive to the interaction between alkyl chains.36,37 The appearance of the δ(CH2) splitting band is indicative of an orthorhombic subcell packing where the C-C-C planes of adjacent hydrocarbon chains are oriented at about 90° to each other. The single band at 1732 cm-1 is assigned to the CdO stretching vibration (υ(CdO)) in the ester groups, while the peak at 1660 cm-1 is ascribed to the C(2)dO stretching mode in the cytosine moieties.38-40 The absorption band at 1621 cm-1 is due to the coupled vibration of C(5)dC(6) and C(4)dN(3) bonds in the cytosine rings,38-40 and the 1526- and 1490-cm-1 peaks are attributed to the coupled ring vibration.39,40 In the highfrequency region, the band around 3359 cm-1 is due to the NH2 stretching vibration (υ(NH2)) in the cytosine bases.39,40 Both position and bandwidth suggest that the cytosine moieties in the monolayers are intermolecularly hydrogen bonded, which will drive the planes of the cytosine rings to orient preferentially parallel to the film substrate, so that no π-π interaction of aromatic stacking occurs as mentioned in the above section. (34) Snyder, R. G.; Hsu, S. L.; Krimm, S. Spectrochim. Acta 1978, 34A, 395. (35) Snyder, R. G.; Strauss, H. L.; Elliger, C. A. J. Phys. Chem. 1982, 86, 5145. (36) Snyder, R. G. J. Mol. Spectrosc. 1961, 7, 116. (37) Tasumi, M.; Shimanouchi, T. J. Chem. Phys. 1965, 43, 124. (38) Mathlouthi, M.; Seuvre, A. M. Carbohydr. Res. 1986, 146, 1. (39) Floria´n, J.; Baumruk, V.; Leszczyn´ski, J. J. Phys. Chem. 1996, 100, 5578. (40) Lord, R. C.; Thomas, G. J., Jr. Spectrochim. Acta 1967, 23A, 2551.

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For a nine-monolayer LB film transferred from aqueous guanosine solution (Figure 4b), changes in characteristic vibrational bands are obviously observed. The υ(NH2) band undergoes a shift in frequency from 3359 to 3349 cm-1 and an increase in bandwidth, which provides direct spectroscopic evidence for the molecular recognition of cytosine-guanosine base pairing (Watson-Crick complementary bases) through intermolecular hydrogen-bonding interaction. It is shown that the hydrogen-bonding interaction between cytosine-guanosine base pairs is stronger, due to the formation of triple hydrogen bonds, than that between the cytosine-cytosine moieties in the monolayers, which is schematically illustrated in Figure 5. If the triple hydrogen bonds between the cytosine and guanosine base pairs are not formed, a relatively sharp peak at 3359 cm-1 should overlap with the broad band around 3349 cm-1 in Figure 4b. The broad band around 3349 cm-1, in fact, appears at the expense of the relatively sharp band at 3359 cm-1, which means that the hydrogenbonding interaction between the cytosine and guanosine base pairs at the air-water interface is formed and the hydrogen-bonding interaction between the cytosine moieties in the monolayers is destroyed at the same time. The spectral change reveals that the specific recognition does exist between the cytosine and guanosine base pairs. In the low-frequency region (Figure 4b), two new bands appear at about 1691 and 1597 cm-1, which are attributed to the C(6)dO stretching vibration40,41 and coupled mode of C(2)dN(3) and C(8)dN(7) vibrations40,41 in the guanine rings, respectively. The original absorption peaks related to the cytosine rings at 1660, 1621, and 1526 cm-1 undergo a frequency shift to 1659, 1630, and 1533 cm-1, respectively. These spectral changes also reflect the occurrence of molecular recognition between the cytosine and guanosine base pairs. It is worthwhile to notice that these bands at 1660, 1621, and 1490 cm-1 due to the skeletal vibrations of the cytosine rings are significantly reduced in intensity except the weak band at 1526 cm-1 in comparison with those prior to molecular recognition, which indicates that the cytosine planes undergo a change from a preferential flat-on orientation before molecular recognition to a favorable end-on one after molecular recognition. This result is in good agreement with that obtained from the UV-vis spectra of the corresponding LB films. In addition, the δ(CH2) band is accordingly changed from the doublet peak around 1475 and 1462 cm-1 to a singlet peak at 1468 cm-1. This indicates that the subcell packing of the alkyl chains is transformed from the orthorhombic structure before molecular recognition to a hexagonal one,36,37 where the hydrocarbon chains can freely rotate around their long axes, after molecular recognition, although the hydrocarbon chains still maintain almost ordered all-trans conformations. SERS Spectra of LB Films. An important aspect of SERS is its potential for probing the interaction between various adsorbates and metal surface, which can elucidate the surface orientation of the adsorbed molecules. A successful basis for analysis of SERS spectra with regard to orientation of adsorbates is worked out in the form of “surface selection rules”.42-48 Normal modes of the surface (41) Szczepaniak, K.; Szczepaniak, M. J. Mol. Struct. 1987, 156, 29. (42) Moskovits, M.; Suh, J. S. J. Phys. Chem. 1988, 92, 6327. (43) Suh, J. S.; Moskovits, M. J. Am. Chem. Soc. 1986, 108, 4711. (44) Moskovits, M.; Dilella, D. P.; Maynard, K. J. Langmuir 1988, 4, 67. (45) Moskovits, M. J. Chem. Phys. 1982, 77, 4408. (46) Greenler, R. G.; Snider, D. P.; Witt, D.; Sorbello, R. S. Surf. Sci. 1982, 118, 415. (47) Allen, C. S.; Van Duyne, R. P. Chem. Phys. Lett. 1979, 63, 455. (48) Creighton, J. A. Surf. Sci. 1983, 124, 209.

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Figure 5. Schematic illustrations of the hydrogen-bonding patterns, aromatic ring orientation, and the corresponding phase transition processes prior to and after molecular recognition.

molecule involving changes in molecular polarizability with a component normal to the surface are subject to the greatest enhancement. The normal modes of out-of-plane vibration involve a changing dipole moment that is perpendicular to the plane of the molecule. In contrast, the change of dipole moment accompanying an in-plane vibration is parallel to the plane of the molecule. Spectra a and b of Figure 6 show a normal Raman spectrum of nucleolipid powder and a SERS spectrum of a one-monolayer nucleolipid LB film on a silver-coated substrate transferred from pure water, respectively (no scattering peak is detected in the normal Raman spectrum of the one-monolayer LB film on a smooth silver-coated plate). The SERS spectrum of the LB monolayer is completely different from the normal spectrum of the powder. Only the scattering peaks near 1410 (a combination of C(5)-H and C(6)-H out-of-plane bending vibra-

tions),39,49 890 (C(2)dO out-of-plane bending mode),39 and 685 cm-1 (a combination of NH2 torsional and out-of-plane bending vibrations)39 related to the cytosine moieties in the monolayer are observed. While some of the stretching bands in Figure 6a due to the cytosine rings, such as, at 1673 (a combination of C(2)dO and C(5)dC(6) stretching vibrations),39,50 1521 (a combination of C(4)sC(5) and N(3)dC(4) stretching modes),39 and 1276 cm-1 (C(2)sN(3) stretching vibration),39 as well as the band at 785 cm-1 (ring-breathing vibration)39,50 are not detected in the SERS spectrum of the LB monolayer in comparison with the normal spectrum of the powder. On the basis of the surface selection rules, these spectral differences indicate that the cytosine rings are lying flat on the surface. In solution, (49) Nishimura, Y.; Tsuboi, M. J. Mol. Struct. 1986, 146, 123. (50) Otto, C.; van den Tweel, T. J. J.; de Mul, F. F. M.; Greve, J. J. Raman Spectrosc. 1986, 17, 289.

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Figure 6. Normal Raman spectrum of (a) nucleolipid powder and SERS spectra of one-monolayer LB films transferred from (b) pure water and (c) aqueous 1 mM guanosine solution.

and in the solid, a ring-breathing vibration usually corresponds to a strong band. In the SERS spectrum, on the other hand, a high intensity is only expected for this band when the ring is standing up or is tilted with respect to the surface.43 On the basis of the fact that the strong band at 785 cm-1 due to the cytosine ring-breathing vibration is absent in the SERS spectrum, the cytosine moieties are deduced to be lying flat on the substrate surface, and the molecular motion is restricted due to the interaction between the ring and silver surface. In addition, a new broad peak appears around 234 cm-1 corresponding to Ag-N bonds, indicating the formation of nucleobase-metal complex. In the SERS spectrum of a one-monolayer LB film transferred from aqueous guanosine solution (Figure 6c), a scattering peak around 668 cm-1 overlapped with the 685-cm-1 band is clearly observed due to the guanine ringbreathing vibration,50,51 indicating that the guanine rings are standing up or are tilted with regard to the surface. At the same time, new enhanced bands attributed to the guanine rings appears at 1577 (a combination of N(3)s C(4) and C(4)dC(5) stretching),50 1508 (C(5)dC(4)sN(9) stretching),50 1482(C(6)sN(1)-C(2) stretching),50 1430 (N(3)dC(2)sNH2 stretching),50 1335 (N(7)dC(8)sN(9) stretching),50 546, and 506 cm-1 (a combination of C(2)s N(1)sC(6) and C(5)dC(4)sN(3) bending).50 The contribution from the cytosine ring stretching vibration could not be ruled out in the region 1600-1300 cm-1. The coexistence of these bands of ring stretching and out-of-plane bending vibrations suggests that the planes of the hydrogen-bonded base pairs are tilted with respect to the surface after molecular recognition. The enhanced band at 1355 cm-1 originating from the N(7)dC(8)sN(9) stretching mode of the guanine moieties indicates that the imidazole rings in the guanine moieties would be in direct contact with the silver surface. Indeed, it has been confirmed that, for purine nucleotides, the nitrogen at N(7) is preferable to other atoms in the heterocyclic moieties to interact with metal.52 A strong sharp band owing to the formation of Ag-N bonds appears at 240 cm-1, which implies the occurrence of the strong interaction between the guanine rings (at N(7)) and the silver surface in comparison with the weak broad peak around 234 cm-1 in Figure 6b. The (51) Mathlouthi, M.; Seuvre, A. M. Carbohydr. Res. 1986, 146, 15. (52) Watanabe, T.; Kawanami, O.; Katoh, H.; Honda, K.; Nishimura, Y.; Tsuboi, M. Surf. Sci. 1985, 158, 341.

Figure 7. Temperature-dependent FTIR transmission spectra of a nine-monolayer nucleolipid LB film transferred from pure water in the regions (a) 3500-1200 cm-1 and (b) 3000-2800 cm-1.

flat-on geometry of the cytosine rings in Figure 6b could be due to the π electron bonded to the surface with a preference of nitrogen at N(3) interaction with the surface, and the end-on geometry of the guanine rings in Figure 6c could be owing to a nitrogen lone pair (N(7)) bonded to the surface. Certainly, the orientation of the base moieties is influenced more or less by the interaction between the headgroups and the substrate surface; however, the Raman results are consistent with those obtained from the corresponding UV-vis and FTIR spectra. Phase Transition Behaviors of LB Films. The phase behaviors of LB films are closely correlated with the interchain interaction and molecular structure of the filmforming molecules. It is well-known that the υa(CH2) frequency is very sensitive to the conformation of hydrocarbon chains.34,35 FTIR spectroscopy is a powerful tool to monitor precisely subtle changes in conformation and orientation of the film-forming molecules. Figure 7a shows temperature-dependent FTIR spectra of a nine-monolayer nucleolipid LB film deposited from pure water (prior to molecular recognition), and Figure 7b displays the corresponding variable-temperature spectra in the region 3000-2800 cm-1. At room temperature, the υa(CH2) and υs(CH2) frequencies are located at 2918 and 2850 cm-1, respectively, indicative of almost ordered all-trans conformation in the alkyl chains. The two frequencies basically remain unchanged with temperature before the film is heated to 140 °C and then increase

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Figure 8. Temperature dependence of antisymmetric CH2 stretching frequencies of nine-monolayer nucleolipid LB films transferred from (9) pure water and (0) aqueous 1 mM guanosine solution.

abruptly to 2924 and 2854 cm-1, respectively, in the temperature range 140-150 °C, in final, keep constant above 150 °C. The υa(CH2) frequency as a function of temperature is plotted in Figure 8. It is clear that the order-disorder phase transition of the LB film prior to molecular recognition occurs at the temperature about 145 °C. The corresponding υ(NH2) band (around 3359 cm-1 at room temperature) gradually undergoes an increase in intensity and a decrease in bandwidth with temperature prior to the transition temperature (145 °C), which indicates that the hydrogen-bonding interaction between the adjacent cytosine headgroups is progressively reduced. When the temperature is elevated above 150 °C, the sharp band is suddenly changed to a broad one, which indicates that the intermolecular hydrogen-bonding interaction is completely destroyed and that the film is in the melted state. The high transition temperature (145 °C) of the LB film is regarded to result from the intermolecular hydrogen-bonding interaction between the adjacent cytosine headgroups. It is found that the intermolecular hydrogenbonding interaction between hydrophilic headgroups can significantly strengthen the interaction between the corresponding hydrophobic chains, so that the transition temperature of the corresponding LB film is obviously increased.53,54 On the other hand, the high transition temperature of the nucleolipid LB film demonstrates the formation of hydrogen-bonded network between the cytosine headgroups (schematically represented in Figure 5), which is the driving force for the cytosine rings to orient favorably parallel to the film substrate without π-π aromatic stacking. The bands at 1659, 1621, and 1490 cm-1 related to the υ(CdO) and skeletal vibrations in the cytosine bases increase in intensity with temperature, so that their intensities are higher than that of the υ(CdO) band (in the ester groups) at elevated temperatures, particularly above the transition temperature (145 °C). The changes in the transmission spectra are closely related to the alternation of ring orientation. It is inferred that the cytosine planes gradually lie flat on the surface with temperature, which is schematically illustrated in Figure 5. (53) Du, X.; Shi, B.; Liang, Y. Langmuir 1998, 14, 3631. (54) Du, X.; Liang, Y. J. Phys. Chem. B 2000, 104, 10047.

Figure 9. Temperature-dependent FTIR transmission spectra of a nine-monolayer nucleolipid LB film transferred from aqueous 1 mM guanosine solution in the regions (a) 35001200 cm-1 and (b) 3000-2800 cm-1.

The δ(CH2) band is gradually changed from the doublet peak around 1475 and 1462 cm-1 to a singlet peak near 1468 cm-1 with temperature, which indicates that the subcell packing of the alkyl chains is transformed from orthorhombic structure at room temperature to a hexagonal one at the elevated temperatures.36,37 Figure 9a shows temperature-dependent FTIR spectra of a nine-monolayer nucleolipid LB film deposited from aqueous guanosine solution (after molecular recognition), and Figure 7b displays the corresponding variabletemperature spectra in the region 3000-2800 cm-1. The corresponding υa(CH2) frequency as a function of temperature is also plotted in Figure 8. To one’s surprise, the υa(CH2) frequency increases from 2918 cm-1 at 60 °C to 2922.5 cm-1 at 100 °C and then decreases rapidly to 2918 cm-1 again when the film is heated to 120 °C, and the following changes are the same as those prior to molecular recognition above 120 °C, where an abrupt increase in the frequency occurs at 140-150 °C. Above 120 °C, the features of the spectral changes in Figure 9a are the same as those in Figure 7a, which exhibits the phase transition behavior of the LB film transferred from pure water. The spectral features in Figure 9a in the temperature range 30-100 °C are indicative of molecular recognition of cytosineguanosine base pairing as shown in Figure 4b. When the LB film after molecular recognition is heated to 60 °C, the hydrocarbon chains almost maintain the ordered all-trans conformation, and the corresponding spectra basically

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remain unchanged. The above results indicate that the intermolecular hydrogen-bonding interaction between the cytosine and guanosine base pairs exists below 60 °C, meaning that the base paring is stable at the temperature of the human body (around 37 °C). Although the intermolecular hydrogen-bonding interaction between the cytosine and guanosine base pairs is stronger than that between the cytosine moieties in the monolayers, the hydrogen-bonding interaction between the base pairs after molecular recognition cannot strengthen the interaction between the hydrophilic headgroups or the interaction between the corresponding hydrophobic chains. When the temperature is elevated above 60 °C, the gauche conformers in the alkyl chains increase, and the triple hydrogenbonding interaction starts weakening; at the same time, the hydrogen-bonding interaction between the adjacent cytosine moieties in the monolayers forms gradually. The FTIR spectral features at 110 °C in Figure 9a already exhibit the information on transformation process of the intermolecular hydrogen-bonding interaction as mentioned above. The gradual increase of the υa(CH2) frequency in the wide temperature range 60-100 °C in comparison with the abrupt change at 140-150 °C is considered to result from two probable reasons: one is due to the influence of the transformation of hydrogenbonding interaction, and the other arises from the influence of the π-π interaction of aromatic stacking between the hydrophilic headgroups. When the temperature is increased to 120 °C, the hydrogen-bonding interaction between the cytosine and guanosine base pairs is completely dissociated, and the hydrogen-bonded network between the adjacent cytosine moieties in the monolayers is formed and the almost all-trans conformation in the alkyl chains is recovered, accompanied with the change of the cytosine ring orientation from the end-

Miao et al.

on fashion to the flat-on one. The above phase transition behavior not only provides powerful evidence for molecular recognition of the cytosine and guanosine base pairs at the air-water interface through strong hydrogen-bonding interaction but also reveals the process of the molecular recognition in all directions. Conclusions The behavior of 1-(2-octadecyloxycarbonylethyl)cytosine monolayer at the air-water interface in the presence of guanosine in the subphase indicates that the cooperative interaction between the cytosine moieties and guanosine bases takes place. The direct evidence for molecular recognition of cytosine-guanosine base pairs is observed through FTIR spectroscopy. Various molecular spectroscopic studies (UV-vis, FTIR, and SERS) indicate that the molecular recognition occurs between the cytosine and guanosine bases through triple hydrogen bonds at the expense of the intermolecular hydrogen-bonding network between the adjacent cytosine moieties in the monolayersand that the cytosine rings undergo a change in orientation and aromatic stacking from the flat-on geometry without stacking interaction before molecular recognition to an end-on one in the J-aggregate fashion after molecular recognition. The temperature-dependent FTIR spectral results further demonstrate the formation of the WatsonCrick complementary base pairs, together with the transformation of hydrogen-bonding patterns from cytosine-cytosine moieties to cytosine-guanosine base pairs after molecular recognition. Acknowledgment. This work was financially supported by the National Natural Science Foundation of China (NSFC, No. 20273029). LA0345690