Molecular Recognition in Monolayers. Complementary Base Pairing in

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Langmuir 1997, 13, 3438-3444

Molecular Recognition in Monolayers. Complementary Base Pairing in Dioleoylphosphatidyl Derivatives of Adenosine, Uridine, and Cytidine Debora Berti, Lucia Franchi, and Piero Baglioni* Department of Chemistry, University of Florence, I-50121 Florence, Italy

Pier Luigi Luisi ETH-Zentrum, Institut fu¨ r Polymere, CH-8092 Zu¨ rich, Switzerland Received March 31, 1997X Chemical recognition by base complementarity in DNA and RNA is strictly related to their stereochemical order. The way in which this high stereoregular order has been achieved in a prebiotic world is not fully understood yet. More primitive systems that display complementary base recognition as a prerequisite to information and, eventually, self-replication might represent a possible route. This study investigates phosphatidylnucleosides bearing complementary bases, adenine and uridine, that can mutually recognize each other, giving mixed structures with features characteristic of complementary base pairing. Dioleoylphosphatidyl derivatives of adenosine (DOP-adenosine), uridine (DOP-uridine), and cytidine (DOP-Cytidine) have been studied at the water-air interface as a function of pH and subphase composition. When monovalent cations (Li+, Na+, and K+) are dissolved in the subphase, the phosphatidyl derivative monolayers show expansion or compression depending on the cation nature. In particular DOP-adenosine shows a preferential interaction with Li+. The properties of mixtures of the DOP-adenosine/DOPuridine complementary bases were investigated and compared to those of the non-complementary bases (DOP-adenosine/DOP-cytidine). The results indicate a preferential interaction in a hydrophilic environment only for complementary nucleophospholipids at physiological pH, suggesting that the specific interfacial orientation of the phospholiponucleoside imposed by the interface promotes the molecular recognition between the two complementary bases in a way that resembles the Watson-Crick pairing in natural nucleic acids. Moreover, mixed monolayers of adenosine-uridine derivatives show a minimum of the free energy of mixing for DOP-uridine rich mixtures (around the DOP-adenosine/DOP-uridine ) 0.2-0.3 mole fraction) close to the stoichiometry of the trimeric adduct (uridine)2‚adenosine that forms in highly concentrated solutions of uridine and adenosine, where adenosine displays simultaneously the Watson-Crick and the Hoogsten hydrogen bond patterns.

Introduction Molecular recognition is a fundamental characteristic of living systems. In order to achieve selective molecular recognition and binding in artificial systems, much effort has been devoted to design supramolecular devices that mimic the molecular organization and compartmentalization typical of biological systems.1-5 Nucleic acids store the information necessary for selfmaintenance and self-replication of a cell. At a molecular level the information is expressed via typical hydrogen bond patterns and stacking interactions that allow selective recognition between complementary nucleosides.6,7 Experimental and theoretical studies on complementary base recognition have been performed in noncompetitive solvents, i.e. organic solvents,8-13 while in an aqueous * Corresponding author. Phone: (voice) +39-55-2757567. Fax: +39-55-240865. E-mail: [email protected]. X Abstract published in Advance ACS Abstracts, May 15, 1997. (1) Lehn, J. M. Angew. Chem., Int. Ed. Engl. 1988, 27, 90. (2) Ringsdorf, H.; Schlarb, B.; Venzmer, J. Angew. Chem. 1988, 100, 117. (3) Fendler, J. H. Membrane Mimetic Chemistry; Wiley: New York, 1982. (4) Lehn, J. M. Supramolecular Chemistry, Concepts and Perspectives; VCH: New York, 1995. (5) Fuhrhop, J.-H.; Ko¨ning, J. Membranes and Molecular Assemblies: The synkinetic approach; The Royal Society of Chemistry: London, 1994. (6) Saenger, W. Principles of Nucleic Acid Structure; SpringerVerlag: New York, 1984. (7) Cantor, C. R.; Shimmel, P. R. Biophysical Chemistry; W. H. Freeman: San Francisco, 1980. (8) Katz, L.; Penman, S. J. Mol. Biol. 1966, 15, 220.

S0743-7463(97)00334-X CCC: $14.00

environment stacked structures are more stable than hydrogen-bonded ones. However, a base pairing in water induced by an intercalating agent has been reported.14 Some authors have reported studies of complementary binding in micelles,15 where the competition of the protic solvent is overcome by the compartmentalization of the partners into a micellar hydrophobic core. A challenging approach to combine the lipid compartmentalization with the nucleotide structures has been developed, and phosphatidylnucleosides, which are able to form stable liposomes, have been prepared.16 In liposomes, the nucleotides are spread over spherical surfaces. Another possible approach is offered by lipid monolayers and bilayers, whose supramolecular architecture has been widely exploited by Kunitake and coworkers17-19 and Ringsdorf and co-workers.20 Recently, (9) Hamlin, R. M., Jr.; Lord, R. C.; Rich, A. Science 1966, 148, 1734. (10) Pitha, J.; Jones, R. N.; Pithova, P. Can. J. Chem. 1966, 44, 1044. (11) L. Williams, G. W.; Williams, L. D.; Shaw, B. R. J. Am. Chem. Soc. 1989, 111, 7205. (12) Pranata, J.; Wierschke, S. G.; Jorgensen, W. L. J. Am. Chem. Soc. 1991, 113, 2810. (13) Pistolis, G.; Paleos, C. M.; Malliaris, A. J. Phys. Chem. 1995, 99, 8896. (14) Constant, J. F.; Fahy, J.; Lhomme, J. Tetrahedron Lett. 1987, 28, 1777. (15) Novick, J. S.; Chen, J. S.; Noronha, G. J. Am. Chem. Soc. 1993, 115, 7636. (16) Bonaccio, S.; Walde, P.; Luisi, P. L. L. J. Phys. Chem. 1994, 98, 6661-6663. (17) Honda, Y.; Kurihara, K.; Kunitake, T. Chem. Lett. 1991, 681. (18) Kawahara, T.; Kurihara, K.; Kunitake, T. Chem. Lett. 1992, 1839. (19) Berndt, P.; Kurihara, K.; Kunitake, T. Langmuir 1995, 11, 3083.

© 1997 American Chemical Society

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Langmuir, Vol. 13, No. 13, 1997 3439

Figure 1. Schematic drawing of the chemical structures of the DOP-nucleoside derivatives studied at the air-water interface.

Kunitake et al. have presented a new investigation of the recognition between bases and the guanidinium group.19 In this paper, which is a part of a comprehensive study of the behavior of the above mentioned phosphatidylnucleosides, we report the surface characterization of a novel group of functionalized synthetic phospholipid amphiphiles bearing a nucleoside moiety on the polar head group, phospholiponucleosides. Dioleoylphosphatidyl derivatives of adenosine, uridine, and cytidine were synthesized according to the method proposed by Shuto and co-workers21-23 by following the modifications proposed by Bonaccio et al.16 In this class of compounds the phospholipid group, the major component of cell membranes, is connected to a nucleobase, capable of displaying selective recognition. The molecule bears a negative charge on the phosphate group, thus reproducing in the polar head the charge of the repeating unit in nucleic acids. The occurrence of such a negative phosphate group plays a fundamental role on the DNA structure and of course on its interactions with cellular cations, whose changes in concentration can in turn alter the structure. Surface pressure and surface potential vs area isotherm measurements were performed in order to elucidate the effect of pH and monovalent cations in the subphase on surface parameters of dioleoylphosphatidyl derivatives of adenosine (DOP-adenosine), uridine (DOP-uridine), and cytidine (DOP-cytidine). The miscibility properties of mixtures of the complementary bases DOP-adenosine/ DOP-uridine were also investigated and compared to those of the noncomplementary bases DOP-adenosine/ DOP-cytidine. The results show a preferential interaction for complementary nucleophospholipids at physiological pH, whereas at lower pHs no specific interaction is detectable. Moreover, weaker interactions for DOPadenosine/DOP-cytidine mixtures are detectable. Experimental Section KCl, NaCl, and LiCl pure products for analysis were supplied by Fluka Chemie; Tris was purchased by Sigma Aldrich Chemie. Bidistilled water was purified with a MilliQ water system supplied by Millipore. Spreading solutions were prepared by (20) Ahlers, M.; Ringsdorf, H.; Rosemeyer, H.; Seela, F. Colloid Polym. Sci. 1990, 268, 132. (21) Shuto, S.; Ueda, S.; Imamura, S.; Fukukawa, K.; Matsuda, A.; Ueda, T. Tetrahedron. Lett. 1987, 28, 199. (22) Shuto, S.; Ito, H.; Ueda, S.; Imamura, S.; Fukukawa, K.; Tsujino, M.; Matsuda, A.; Ueda, T. Chem. Pharm. Bull. 1988, 36, 209. (23) Shuto, S.; Imamura, S.; Fukukawa, K.; Ueda, T. Chem. Pharm. Bull. 1988, 36, 5020.

dissolving about 6 mg of product in 10 mL of CHCl3 and stored at -19 °C. The solution was refreshed after 1 week in order to prevent product degradation. Spreading isotherms, obtained by discontinuous compression of about 4 Å2/molecule per step, were recorded using a computer-controlled MGW-Lauda Filmwaage balance. The barrier speed was 4 cm/min. For each area value, three readings of surface pressure were carried out every 100 s to ensure that thermodynamic equilibrium had been attained. The accuracy of the measurements was (0.03 mN/m for surface pressures and (1 Å2/molecule for molecular areas. All isotherms were recorded at 20 °C and the temperature was kept constant within (0.2 deg. Surface potential measurements were carried out using two 241Am electrodes (1.04 cm2, 200 µC) by following the procedure described elsewhere.24 Surface potentials and surface pressures were recorded simultaneously as a function of molecular area. The accuracy was (25 mV. The results are presented in terms of ∆V/n, where n is the surface concentration of the surfactant expressed in number of film molecules per square centimeter of the film. Collapse pressures were calculated as the surface pressure corresponding to the plateau in the Π/A isotherms. The compressibility modulus has been obtained from the relationship, Cs-1 ) -A(δΠ/δA). 1H and 31P{1H} NMR spectra were recorded at 500.132 and 202.46 MHz, respectively, on a Bruker Avance DRX-500 spectrometer equipped with a variable temperature control unit accurate to (0.1 deg. Chemical shifts are relative to tetramethylsilane and to 85% H3PO4, respectively, as external references. Downfield values are reported as positive. Doublequantum filtered 1H COSY experiments25 were recorded using degassed nonspinning samples with 1024 increments of size 2 K covering the full range (ca. 5000 Hz) in both dimensions. Synthesis. 5′-(3-sn-Phosphatidyl)nucleosides were synthesized according to the procedure described by Shuto et al.21-23 A chloroform solution of DOPC was added to an acetate buffer solution containing the nucleoside and PLD-P ex Streptomyces sp (E.C.3.1.4.4.). The mixture was stirred vigorously at 45 °C for at least 3 h. HCl (2 N), MeOH, and CHCl3 were added; then the organic layer was washed with water and dried. The product was purified by stepwise elution flash chromatography, and the acidic form of the phosphatidylnucleoside was converted into the ammonium salt, in order to avoid product degradation. The chemical structures of the synthesized molecules are shown in Figure 1. NMR Data. DOP-Uridine. 1H-NMR (DMSO): δ ) 0.90 (t, 6H, CH3), 1.30 (m, 40H, aliphatic CH2), 1.60 (m, 4H, CH2CH2COO), 2.05 (m, 8H, CH2CHdCH), 2.35 (m, 4H, CH2COO), 3.90 (m, 4H, H5′/5′′, sn-3-CH2), 4.01 (m, 1H, H4′), 4.10 (m, 1H, H3′), (24) Jaycock, M. J.; Parfitt, G. D. Chemistry of Interfaces; Ellis Horwood Series in Physical Chemistry; Ellis Horwood: Chichester, U.K., 1981. (25) Shaka, A. J.; Freeman, R. J. Magn. Reson. 1983, 51, 169.

3440 Langmuir, Vol. 13, No. 13, 1997

Berti et al. Table 1. Surface Parameters for DOP-Uridine Monolayers at the Air/Water Interface subphase limiting area 1012∆VN compressibility pH (Å2/molecule) [(mV cm2)/molecule] modulus (mN/m) 2.0 5.5 7.5

100 110 125

1.5

52 52 60

3.0

Figure 2. Surface pressure-area isotherms (Π/A) of DOPuridine at 20 °C on water and on different pH subphases. There is a stepwise increase of the limiting area passing from an acidic to a slightly basic pH. 4.20 (m, 2H, H2′, sn-1-CH2), 4.40 (dd, 1H, sn-1-CH2), 5.18 (m, 1H, sn-2-CH), 5.35 (m, 1H, 3′ OH), 5.40 (m, 4H, CHdCH), 5.50 (m, 1H, 2′OH), 5.70 (d, 1H, H5), 5.90 (d, 1H, H1′), 7.1 (NH4+), 7.90 (d, 1H, H6), 11.40 (m, 1H, H3). 31P-NMR (DMSO): δ ) -0.33. DOP-Adenosine. 1H-NMR (DMSO): δ ) 0.90 (t, 6H, CH3), 1.30 (m, 40H, aliphatic CH2), 1.60 (m, 4H, CH2CH2COO), 2.05 (m, 8H, CH2CHdCH), 2.35 (m, 4H, CH2COO), 3.90 (m, 4H, H5′/ 5′′, sn-3-CH2), 4.10 (m, 1H, H3′), 4.20 (m, 2H, sn-1-CH2, H2′), 4.30 (m, 1H, H4′) 4.4 (dd, 1H, sn-1-CH2), 4.60 (m, 1H, 3′OH), 5.18 (m, 1H, sn-2-CH), 5.40 (m, 4H, CHdCH) 5.55 (m, 1H, 2′ OH), 6.00 (d, 1H, H1′), 7.00-7.50 (NH4+, NH2′) 8.15 (s, 1H, H2), 8.59 (s, 1H, H8). 31P-NMR (DMSO): δ ) -0.69. DOP-Cytidine. 1H-NMR (DMSO): δ ) 0.90 (t, 6H, CH3), 1.30 (m, 40H, aliphatic CH2), 1.60 (m, 4H, CH2CH2COO), 2.05 (m, 8H, CH2CHdCH), 2.40 (m, 4H, CH2COO), 3.90 (m, 4H, H5′/5′′; sn-3-CH2), 4.00 (m, 1H, H4′), 4.05 (m, 2H, H2′, H3′), 4.20 (m, 1H, sn-1-CH2), 4.40 (dd, 1H, sn-1-CH2), 5.18 (m, 1H, sn-2-CH), 5.35 (m, 1H, 3′OH), 5.40 (m, 5H, 2′OH, CHdCH), 5.70 (d, 1H, H5), 5.90 (d, 1H, H1′), 7.00-7.50 (NH4+, NH2), 8.00 (d, 1H, H6).

Results and Discussion It is well-known that phospholipids form stable monomolecular films at the water-air interface.26 The phospholipid phase at the water-air interface is controlled by several factors including the chain length of the phospholipid, the polar head group nature, and in particular the electrostatic surface potential of the monomolecular film, which mainly depends on the distribution of protons, cations, and anions with respect to that of the bulk aqueous phase. Both phosphate and base groups of the compounds studied in this work (DOP-adenosine, DOP-uridine, and DOP-cytidine) can be protonated or deprotonated according to the pH values at the water-lipid interface. A general feature of the surface behavior of these compounds is the temperature independence and the excellent reproducibility of the spreading isotherms. Monolayer of Pure DOP-Adenosine, DOP-Uridine, and DOP-Cytidine at the Water-Air Interface. Figure 2 shows surface pressure vs area isotherms (Π/A) of DOP-uridine at the air-water interface at different pHs. The increase of the pH of the subphase produces shifts of the spreading isotherms toward larger areas. The values of the limiting areas, A0 (see Table 1), gradually increase from 100 Å2/molecule at pH ) 2 to 125 Å2/molecule at pH ) 7.5. The increase of the limiting (26) Mo¨hwald, H. In Phospholipids Handbook; Cevc, G., Ed.; Marcel Dekker: New York, 1993; pp 579-602 (see also references therein).

Figure 3. Surface pressure-area isotherms (Π/A) of DOPadenosine at 20 °C on water and on different pH subphases. Table 2. Surface Parameters for DOP-Adenosine Monolayers at the Air/Water Interface collapse subphase limiting area 10-12∆VN Ks max pressure pH (Å2/molecule) [(mV cm2)/molecule] (mN/m) (mN/m) 2.0 5.5 7.5

107 104 139

5.0 1.9

52 52 50

35.8 35.9 40.5

area A0 of the phospholiponucleoside, in agreement with the phospholipid behavior,27 is consistent with the presence of a net negative charge on the polar head groups and, therefore, with repulsive electrostatic interaction, as expected from the pK relative to the deprotonation of the of the uridine-N3 (pK < 2) and of the phosphate group (pK ) 6.4). This is confirmed by the trend of the surface potential, that increases from 1.53 × 10-12 (mV cm2)/ molecule for pH ) 2 to 3.03 × 10-12 (mV cm2)/molecule for pH ) 7.5. The compressibility moduli indicate a liquidexpanded phase for the phospholiponucleoside. In Figure 3 spreading isotherms of the complementary phospholiponucleoside, DOP-adenosine, are reported. DOP-adenosine, in addition to the phosphate group, possesses an amino group that is protonated at acidic pH (pKa ) 3.9 in adenosine 5′-phosphate6). The limiting areas do not show noteworthy modifications in passing from the subphase at pH ) 2 to pH ) 5.5, while there is a significant increase at pH ) 7.5 ascribable similarly to DOP-uridine to the full deprotonation of the phosphate group and to the deprotonation of adenosine-N1. This is also supported by the value of the surface potential, which is higher at acidic pH than at physiological values (see Table 2). The smaller area value found for acidic pH can be attributed to the favorable interactions, electrostatic as well as hydrogen bonding, which could be established between the protonated ring and the partially deprotonated phosphate groups. At physiological pH, where the only charged group is the negative phosphate, repulsive interactions seem to be overwhelming. It is worthwhile to recall that the molecular arrangement in the monolayer (27) Papahadjopoulos, D. Biochim. Biophys. Acta 1968, 163, 240.

Molecular Recognition in Monolayers

is known to affect the acid-base equilibrium,28 and therefore the phospholiponucleoside pK at the interface can be slightly different from those in the bulk. Compressibility moduli are very similar to those obtained for DOP-uridine and are slightly affected by pH (Table 2), whereas collapse pressures and surface potential show significant variations as the subphase approaches pH ) 7.5, indicating a higher stability of the film in the latter case. The different limiting areas of the two derivatives (DOP-uridine and DOP-adenosine) at physiological pH (125 and 139 Å2/molecule for DOP-uridine and DOPadenosine, respectively) can be attributed only to the different nucleosidic moiety. This is also confirmed by the analysis of the surface potential. Uridine has a dipole moment slightly higher than adenosine, 3.9 and 3.0 D, respectively.6 Since the surface potential, normalized by the area occupied at the interface by the phospholiponucleoside molecule, is higher for DOP-uridine, we conclude that both phosphate group and base contribute to the surface potential. In addition, the above results seem to support that the presence of the nucleobase induces a change on the orientation of the polar head group with respect to the interface. Cytidine is also a pyrimidine base, but in natural nucleic acids it is not the Watson-Crick partner of adenosine. DOP-cytidine has a surface limiting area at physiological pH of 121 Å2/molecule, which means a surface sterical hindrance very similar to DOP-uridine, as expected from their similar chemical structures. Interactions of Monovalent Cations with Nucleoside Derivatives. The interactions of monovalent cations with nucleic acids largely affect equilibrium processes, like conformational transitions or binding processes. The observed effects, which have been extensively investigated in aqueous solutions, are very different from those observed for the interactions of ions with mononucleotides or other biopolymers, since DNA and RNA are polyelectrolytes bearing a high density of negative charge.29 The investigation of ion interactions with phospholiponucleosides arranged in a supramolecular array appears to be of some relevance due to the presence of the interface that consistently affects the ion distribution around the phospholiponucleosides. In fact, in living systems, ion concentrations may change in response to cellular conditions, and even slight changes can have consistent effects on the charge distribution of a cylindrical polyelectrolyte, altering its structure and the extent of other equilibrium processes such as binding to other charged molecules targeting DNA, i.e. drugs or proteins. Monovalent cations are known to shift to higher temperature the transition from the helix form to a random coiled structure.6 NMR relaxation rates of K+ and Na+ have highlighted a Coulombic interaction of the cation with double-stranded nucleic acids, occurring without the displacement of the ion hydration shell water molecules.31 Other ions, as Mg2+, exhibit more specific interactions.31 Phospholiponucleoside surface behavior is affected by monovalent cations dissolved in the subphase. Figure 4 shows the Π/A isotherm of DOP-uridine on 10-3 M saline subphases. In the case of KCl, a significant expansion (130 Å2/molecule), compared to pure water (110 Å2/ molecule), is present. The limiting areas of the DOP(28) Tocanne, J-F.; Teissie´, J. Biochim. Biophys. Acta 1990, 1031, 111. (29) Anderson, C. F.; Record, M. T., Jr. Annu. Rev. Phys. Chem. 1995, 46, 657. (30) Braunlin, W. H. Adv. Biophys. Chem. 1995, 15, 89. (31) Holbrook, S. R.; Sussman, J. L.; Warrant, R. W.; Church, G. M.; Kim, S. H. Nucl. Acids Res. 1977, 4, 2811.

Langmuir, Vol. 13, No. 13, 1997 3441

Figure 4. Surface pressure-area isotherms (Π/A) of DOPuridine at 20 °C on pure water and on a 10-3 M XCl solutions (X ) Li+, Na+, K+).

Figure 5. Surface pressure-area isotherms (Π/A) of DOPadenosine at 20 °C on pure water and on 10-3 M XCl solutions (X ) Li+, Na+, K+).

uridine monolayer in the presence of 10-3 M LiCl and 10-3 M NaCl, show a weaker increase to 116 and 121 Å2/molecule, respectively. This supports that monovalent ions interact with the DOP-uridine monolayer in the order Li+ e Na+ , K+, which correlates with the dimensions of the hydrated ions in solution. Therefore, a possible interpretation can be related to the different dimensions of the hydration shells of the ions. Hydrated potassium ions have the smallest radius, which allows us to interact fully with the polar head groups of the phospholiponucleoside in the monolayer. The overall effect is an increase of the area occupied by the phospholiponucleoside molecule. It is interesting to note that the spreading isotherms coincide at higher pressures, close to the collapse region, suggesting that the cation interacts weakly with the nucleoside, and it is expelled from the monolayer at high lateral pressures (high packing). DOP-adenosine, due to the different polar head group, is affected by the presence of monovalent cations in a totally different way (Figure 5). As expected for a pure Coulombic interaction with the phosphate anionic group of the phospholiponucleoside, KCl and NaCl cause a consistent compression of the surface areas; LiCl, likewise KCl for DOP-uridine, produces a consistent expansion of the monolayer and a decrease of the surface potential with respect to the film on pure water. It is interesting to note that Li+ produces a strong increase of the collapse pressure of the DOP-adenosine monolayer (about 4 mN/m),

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Berti et al.

Table 3. Surface Behavior of DOP-Adenosine on Different Saline Subphases limiting area 10-12∆VN compressibility subphase (Å2/molecule) [(mV cm2)/molecule] modulus (mN/m) LiCl NaCl KCl

119 78 80

1.6 1.5 1.7

56 52 51

indicating that a strong interaction between the ion and the nucleoside moiety is present. Limiting areas, surface potential, and compressibility moduli are reported in Table 3. These results support that specific interactions are present between phospholipid nucleosides and monovalent ions. These interactions seem to be related to the size of the interacting ion and to the nucleoside moiety. The larger DOP-adenosine group can better accommodate larger hydrated ions with respect to DOP-uridine. Probably, a different conformation of the nucleoside phosphate group is induced by the bulky hydrated Li+. Bidimensional Mixtures between Complementary and Noncomplementary Nucleobases. Ringsdorf et al.,20 describing the surface behavior of some synthetic uncharged nucleolipids and their interactions with polynucleotides, showed that when a complementary base is dissolved in the subphase, an expansion of the monolayer is detectable. This has been attributed to specific phospholipobase-base interactions. In our case, since both partners are negatively charged, the presence of repulsive and nonspecific interactions between the base and the phospholiponucleoside monolayer is likely to prevent any binding. As expected, the surface Π/area isotherms of DOP-nucleosides (uridine, adenosine, and cytidine) on a 10-2 M Tris subphase (pH ) 7.5) are actually not affected by either the complementary base (10-2 M) or the complementary polynucleotide, present in the subphase. This behavior is similar to that of mononucleosides in water, where molecular recognition is hampered by the hydrogen-bonding competition displayed by the solvent, while in polynucleotides, despite the very high charge density, the bases, thanks to the covalent backbone they are attached to, succeed in cooperative base pairing with a complementary polynucleotide, showing the so called zipper effect.6 This suggests that specific interactions can take place when a supramolecular support is available. According to this point of view, we have investigated the surface behavior of different mixtures of the DOPadenosine/DOP-uridine complementary bases and of the DOP-adenosine/DOP-cytidine noncomplementary bases. In Figure 6 the spreading isotherm for an equimolar mixture is reported and compared to the curves of the pure components. The two nucleolipids are miscible in the whole range of mole ratios, as demonstrated by the trend of the collapse pressure. The mixing behavior has been calculated according to the analysis of Goodrich.32 The Gibbs free energy of mixing for an ideal monolayer is given by

∑i xi ln xi

∆Gideal ) RT m

(1)

and for a nonideal mixing monolayer

∆GΠ m )

∫0Π(A12 - x1A1 - x2A2) dΠ + kBT(x1 ln x1 + x2 ln x2) (2)

(32) Goodrich, F. C. Proceedings of the 2nd International Congress on Surface Activity; Butterworth: London, 1957.

Figure 6. Surface pressure-area isotherms (Π/A) isotherm of DOP-adenosine, DOP-uridine and the 1:1 mixture at 20 °C on a pH ) 7.5 subphase.

Figure 7. Excess ∆G of mixing (kJ mol-1) plotted as a function of DOP-adenosine mole fraction. Each plot corresponds to a different surface pressure. The continuous lines correspond to the ideal behavior.

The excess of the Gibbs free energy of mixing is given by Π ideal ∆GΠ ) xs ) ∆Gm - ∆Gm

∫0Π(A12 - x1A1 - x2A2) dΠ

(3)

where Ai is the area per molecule of the component i and xi is its mole fraction. In this way, the ideal free energy of mixing to the stability of the mixed film can be separated from the contribution arising from specific interactions between the components. The monolayers of the DOP-adenosine/DOP-uridine mixtures are miscible in the whole range of composition and pH studied. Interestingly, monolayers on DOPadenosine/DOP-uridine show ideal behavior at pH ) 2 and 5.5 (pure water), while a deviation from ideality is detectable pH ) 7.5, revealing the presence of attractive interactions between polar head groups only at physiological pH. This is highlighted in Figure 7 where ∆GΠ xs values are plotted as a function of the mole ratio at different surface pressures. A sharp minimum is not clearly recognizable, though the strongest attractive interactions can be found for DOP-uridine rich mixtures, around the DOP-adenosine/DOP-uridine ) 0.2-0.3 mole fraction region, while DOP-adenosine rich mixtures have a behavior close to ideality. This peculiar behavior found with DOPuridine rich mixtures can be related to the hexagonal packing of surfactant molecules at the interface as

Molecular Recognition in Monolayers

Langmuir, Vol. 13, No. 13, 1997 3443

described by Shah.35 According to this author, when molecules with the smallest molecular area (DOP-uridine, in our case) represent the main component in the 3/1 mole ratio mixture, they occupy the corners of hexagons, while the largest surfactants (DOP-adenosine) are in the centers of the hexagonal structure. This particular and regular arrangement of the two components at the interface provides sufficient intermolecular interactions for the ordered structure.35 Very interestingly, the monolayer composition at which a minimum of the free energy of mixing is detected is close to the stoichiometry of the trimeric adduct (uridine)2‚adenosine that forms in highly concentrated solutions of uridine and adenosine, where adenosine displays simultaneously the WatsonCrick and the Hoogsten hydrogen bond patterns.6 As a complement to the Goodrich analysis, the attractive interactions leading to molecular recognition between the conjugated nucleobases in the mixed films can be quantified according to Joos’ theory, which calculates the surface pressure of collapse for the mixed monolayers.33 According to this treatment, when the two amphiphiles are completely miscible, the collapse pressure of a mixed monolayer, πc,m, satisfies the following formula:

(

x1γ1 exp

)

πc,m - πc,1 RTΓ∞1

(

+ x2γ2 exp

)

πc,m - πc,2 RTΓ∞2

)1

(4)

with

Γ∞1 )

1016 ; NAA1,lim

Γ∞2 )

1016 NAA2,lim

where x1, x2, πc,1, πc,2, Alim,1, and Alim,2 are the mole fractions, the collapse pressures, and the limiting area values at the collapse point of the two components; R, T, and NA are the universal constant of gases, the absolute temperature, and the Avogadro number, respectively; and γ1 and γ2 indicate the surface activity coefficients of the two components at the collapse point. If the two components show an ideal miscibility, γ1 ) γ2 ) 1, then the experimental values of πc,m should be identical to the theoretical predictions (i.e. πc,m ) x1πc,1 + x2πc,2). On the other hand, for a real mixture, the difference between the experimental and the theoretical values is directly related to the interactions between the two components. When interactions occur, the γ coefficients cannot be equal to unity, and a regular behavior can be assumed as a first approximation. Denoting by Rβ the potential energy of interaction between a molecule “R” and a molecule “β”, the energy of mixing (W) can be calculated as33,34

(

W ) nz Rβ -

)

RR + ββ x1x2 ) nz∆x1x2 2

(5)

where n is the number of moles and z is the number of the nearest molecules. The surface activity coefficients can be expressed as γ1 ) exp(ξx22) and γ2 ) exp(ξx12), where

∆ )

RTξ 6

(6)

(assuming z ) 6, in the closely packed arrangement of the monolayer). Substituting these values for γ1 and γ2 in eq 4, we obtain (33) Joos, P.; Demel, R. A. Biochim. Biophys. Acta 1969, 183, 447. (34) Joos, P. Bull. Soc. Chim. Belg. 1969, 78, 207. (35) Shah, D. O. J. Colloid Interface Sci. 1971, 37, 745.

[

x1 exp

]

Alim,1(πc,m - πc,1) exp(ξx22) + kBT Alim,2(πc,m - πc,2) exp(ξx12) ) 1 (7) x2 exp kBT

[

]

The interaction parameter ξ can be adjusted to calculate πc,m from eq 7, knowing all the other experimental parameters. The collapse pressures for the mixtures are higher than the pressures calculated according to the ideal behavior between the two components, 40.5 and 36.7 mN/m for DOP-adenosine and DOP-uridine, respectively. The application of the Joos theory highlights two distinct regions, depending on the monolayer composition. In fact, the Joos parameter has a negative value (ζ ) -2.88) for uridine rich mixtures (DOP-adenosine mole fractions 0 e x < 0.5 and collapse pressures ranging from 36.7 to 40.9 mN/m), while for adenosine rich mixtures the behavior is still attractive but approaches the ideal behavior (ζ ) -0.36). We can thus conclude that attractive interactions are present throughout the whole range of monolayer compositions and that they are enhanced in the DOPuridine rich region, as already deduced by the Goodrich analysis. To distinguish between a specific stacking interactions and molecular recognition, we studied DOP-adenosine/ DOP-cytidine mixtures. DOP-cytidine, as already reported, has a surface limiting area at physiological pH of 121 Å2/molecule. The collapse pressure for the DOP-cytidine/DOP-adenosine mixture is 40.1 mN/m, lower than that of the pure components (40.5 for DOP-adenosine and 40.3 for DOPcytidine). The application of Joos’ theory to this mixture shows the presence of repulsive interactions (ξ ) +0.24) between the two bases.36 This repulsive interaction would prevent the specific recognition between the bases, reported for the DOP-adenosine/DOP-uridine complementary pair. However, a negative deviation from area additivity is also present. This effect can be due to steric hindrance of the polar groups and/or to specific interactions between the phospholiponucleosides at the interface.37,38 The ∆GΠ xs values corresponding to various surface pressures are compared in Figure 8 with ∆GΠ xs of the complementary base pair at pH 5.5 and pH 7.5. As shown, ∆GΠ xs values for the complementary base pair (DOP-adenosine/DOPuridine) are higher than for DOP-adenosine/DOPcytidine, showing that specific interactions are present in the complementary bases. Stacking may play a role, but it is not the only driving force responsible for nonideal mixing. In fact, we should expect that, as occurs in polynucleotide strands or nucleic acids,6 the stacking between Π electrons of the nucleobases also contributes to the “homolipids” monolayer stabilization. The Watson-Crick molecular recognition is not the only possible pattern of H-bonding pairing between complementary bases, but thanks to its symmetry properties it is by far the most favorite one in a helical structure. Of course, we do not expect that this holds for a bidimensional organization, where other hydrogen bond network patterns can be displayed, and the nonequimolar stoichi(36) Baglioni, P.; Dei, L.; Puggelli, M. Colloid Polym. Sci. 1985, 263, 266. (37) Dei, L.; Baglioni, P.; Carla´, M.; Martini, E. In Surfactants in Solution; Mittal K. L., Ed.; Plenum: New York, 1989; Vol. 8, pp 435444. (38) Baglioni, P.; Carla´, M.; Dei, L.; Martini, E. J. Phys. Chem. 1987, 91, 1460.

3444 Langmuir, Vol. 13, No. 13, 1997

Figure 8. Excess ∆G of mixing (kJ mol-1) of the DOPadenosine/DOP-uridine 1:1 mixture at pH ) 7.5 and 5.5 (b and 0, respectively) compared to the 1:1 DOP-adenosine/DOPcytidine ∆G curve (pH ) 7.5, 2) at various surface pressures.

ometry corresponding to the more stable mixed film should not to be regarded as surprising. Our results point out that stacking is not the only interaction occurring in mixed monolayers of phospholiponucleosides, and we can conclude that mixed films of DOP-adenosine/DOP-uridine are stabilized by stacking and probably by hydrogen bond interactions occurring between polar head groups. These results are also in agreement with a forthcoming study of phospholiponucleosides in liposomal structures, in which it is demonstrated that only complementary bases are recognized in these supramolecular structures. Conclusions The surface behavior of dioleoylphosphatidyl derivatives of adenosine, uridine, and cytidine has been investigated. Surface parameters showed variations depending on the nucleosidic moiety covalently attached to the phospholipid backbone and on the subphase pH in a way suggesting that the protonation-deprotonation equilibria of the base and the phosphate group are the main factors controlling bidimensional parameters. Monovalent cations (Li+, Na+, and K+) dissolved in the subphase affect the nucleolipid film. Monolayers of DOPuridine interact with monovalent cations in the order Li+ e Na+ , K+, which correlates with the dimensions of the

Berti et al.

hydrated ions in solution. DOP-adenosine monolayers have a different behavior. Na+ and K+ produce a compression of the DOP-adenosine monolayer due to Coulombic interactions, while Li+ produces an expansion in the monolayer, a decrease of the surface potential, and a strong increase of the collapse pressure of the DOPadenosine monolayer (about 4 mN/m), indicating that a specific strong interaction between the ion and the nucleoside moiety is present. Miscibility between two complementary bases has been investigated. The nonideal behavior shown by the complementary nucleobases at physiological pH only has been investigated with Goodrich and Joos’ analyses. Both point out the presence of attractive interactions, not fully ascribable to the stacking of the aromatic moieties. Repulsive interactions are present in DOP-adenosine/ DOP-cytosine mixtures. These interactions would prevent recognition between the noncomplementary bases. We attributed the stabilization of the mixed films of DOPadenosine/DOP-uridine to both stacking interactions and hydrogen bonds, which can be established between polar heads with a specificity that resembles the Watson-Crick pairing in natural nucleic acids. Moreover, mixed monolayers of adenosine-uridine derivatives show a minimum of the free energy of mixing for DOP-uridine rich mixtures (around DOP-adenosine/DOP-uridine ) 0.2-0.3 mole fraction) close to the stoichiometry of the trimeric adduct (uridine)2‚adenosine that forms in highly concentrated solutions of uridine and adenosine, where adenosine displays simultaneously the Watson-Crick and the Hoogsten hydrogen bond patterns.6 These results are also in agreement with a forthcoming study of the liposome of a phospholiponucleobase where specific recognition between the nucleobase is found.39 Moreover, the above results indicated that the interfacial orientation of nucleolipids provides a suitable environment for base-base pairing. Acknowledgment. This work has been supported with EU grants ERBCHRX920007 and CIPDCT940005, the Consortium for the Study of Large Interface Systems (CSGI), MURST, and the National Council of Research (CNR). LA970334A (39) Berti, D.; Baglioni, P.; Bonaccio, S.; Barsacchi, G.; Luisi, P. L. Submitted for publication.