Langmuir 1997, 13, 1677-1681
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Excess Free Energies of Interaction between 10,12-Pentacosadiynoic Acid (PDA) and Its Mannoside Derivative (MPDA). A Mixed-Monolayer Study Shaopeng Wang, Yajun Li, Lei Shao, Johnny Ramirez, Peng G. Wang, and Roger M. Leblanc* Department of Chemistry, University of Miami, Miami, Florida 33124 Received June 26, 1996. In Final Form: December 20, 1996X As potential materials for optic biosensors, the surface and optical properties of bio-functionalized polydiacetylene Langmuir-Blodgett films are interesting and important. p-10,12-Pentacosadiyne-1-Nphenylamide R-D-mannopyranoside (MPDA) was synthesized from 10,12-pentacosadiynoic acid (PDA). Pressure-area isotherms of pure and mixed MPDA/PDA monolayers were obtained. The analysis of the collapse pressures and the excess free energies of the isotherms shows that MPDA/PDA monolayers have good miscibility.
Introduction Real time detection of molecular recognition processes is a very important step in the investigation of biological phenomena, as well as for biosensor development. Diacetylene lipid monolayers can be polymerized by UV irradiation into a blue-colored film.1,2 Lipid chain disorder and tangling can decrease the effective conjugation length of the polydiacetylene backbone and cause the film color to change to red.3 This color change has been known to occur in response to a variety of environmental perturbations, such as change of pH, temperature, or mechanical stress.4-7 By adding a special receptor to the diacetylene lipid headgroup and mixing this receptor molecule into the diacetylene monolayer, the binding of a specific ligand to the receptor can disorder the film and change its color.8,9 Bacterial toxins are associated with many diseases in humans and animals.10 The binding of toxins to carbohydrate receptors on human cells represents the initial step in the invasion and infection of many bacterial toxins.11 By choosing a suitable oligosaccharide as the receptor, it is possible to develop an optical fiber biosensor to detect a virus, bacteria, and toxins, as well as real time monitoring of molecular recognition processes in biological systems. As the first step, p-10,12-pentacosadiyne-1-N-phenylamide R-D-mannopyranoside (MPDA) was synthesized from 10,12-pentacosadiynoic acid (PDA). Surface pressure-area isotherms of mixed MPDA/PDA monolayers at the air-water interface were studied. The excess free * Corresponding author. Fax: (305) 284-4571. Phone: (305) 2842282. E-mail:
[email protected]. X Abstract published in Advance ACS Abstracts, February 15, 1997. (1) Bloor, D.; Chance, R. R. J. Polym. Sci., Polym. Lett. Ed. 1978, 16, 205. (2) Tieke, B.; Lieser, G. J. Colloid Interface Sci. 1982, 88, 471. (3) Mino, N.; Tamura, H.; Ogawa, K. Langmuir 1991, 7, 2336. (4) Mino, N.; Tamura, H.; Ogawa, K. Langmuir 1992, 8, 594. (5) Chance, R. R.; Patel, G. N.; Witt, J. D. J. Chem. Phys. 1979, 71, 206. (6) Kaneko, F.; Shibata, M.; Kobayashi, S. Thin Solid Films 1992, 210, 548. (7) Shibata, M.; Kaneko, F.; Aketagawa, M.; Kobayashi, S. Thin Solid Films 1989, 179, 433. (8) Charych, D. H.; Nagy, J. O.; Spevak, W.; Bednarski, M. D. Science 1993, 261, 585. (9) Charych, D.; Cheng, Q.; Reichert, A.; Kuziemko, G.; Stroh, M.; Nagy, J. O.; Spevak, W.; Stevens, R. C. Chem. Biol. 1996, 3, 113. (10) Wilson, B. A.; Collier, R. J. Curr. Top. Microbiol. Immunol. 1992, 175. (11) Brenner, S.; Lerner, R. A. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 5381.
S0743-7463(96)00641-5 CCC: $14.00
energies and collapse pressures of the isotherms were obtained and analyzed; the results show some interactions between the molecules. The two constituents in the monolayer have a good miscibility in all of the ranges of the molar ratios of MPDA and surface pressures. Materials and Methods Synthesis of Monosaccharide-Lipid Conjugates. Material. 10,12-Pentacosadiynoic acid (PDA) was purchased from Farchan Laboratories (Gainsville, FL). The compound was purified by recrystallization with petroleum ether (36-60 °C). All chemicals for the syntheses were purchased from Fisher Scientific Co. (Pittsburgh, PA) as reagent grade. Thin-layer chromatography (TLC) was conducted on Baker silica gel 60 F254 TLC plates with fluorescent indicator. Column chromatography was conducted with Baker silica gel flash chromatography packing, 40 µm. The catalyst used for the hydrogenations was 5% Pd/C on a dry weight basis (Degussa-type E101 NO/W; Aldrich). Methods. A PDA derivative with a mannoside headgroup p-10,12-pentacosadiyne-1-N-phenylamide R-D-mannopyranoside (MPDA) was synthesized. The synthesis of MPDA (Figure 1) was finished in two steps: the first step involved the transformation of the aromatic nitro group in p-nitrophenylmannopyranoside, 1, to an amino group by using a catalytic hydrogenation. In the second step, this amino functionality will be used as the anchor to connect the carboxylic terminus of the lipid (PDA, 3). The coupling made via an amide bond would generate the desired final product 4. The yields for these two steps were 67% and 21.1%, respectively, and the final product was a fine white-pink powder. After the lipid was coupled to the sugar derivative, sodium methoxide was used in tandem to eliminate any byproducts resulting from the ester bond formation between the sugar and the lipid moieties. Experimental Details. A fraction of 2 (0.3 g, 1.1 mmol) was dissolved in a mixture of 8 mL of methanol and 13 mL of dichloromethane, to which the functionalized lipid 10,12-pentacosadiynoic acid (3, 0.50 g, 1.83 mmol) was added, followed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 0.26 g, 1.33 mmol) to act as an activator. The mixture was stirred at room temperature for 20 h, when TLC revealed the formation of 4. To destroy the ester bonds that may have formed between the hydroxyl groups and the PDA-25, after evaporating the CH2Cl2, the residue was dissolved in MeOH and a catalytic amount of NaOMe was added, which was just enough to make the solution basic (pH ∼9-10). The system was stirred at room temperature for ∼1-2 h, filtered, acidified with Dowex50W resin, stirred for several minutes to displace the sodium salts present, and then filtered again. The solvent was evaporated to give 0.15 g (0.23 mmol) of 4 after column chromatography for a yield of 21.1%. Physical Chemistry Properties. The purity of MPDA was characterized with 1H NMR, mass, and UV-vis spectroscopies.
© 1997 American Chemical Society
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Wang et al.
Figure 1. Synthetic path of the formation p-10,12-pentacosadiyne-1-N-phenylamide R-D-mannopyranoside (MPDA) from 10,12pentacosadiynoic acid (PDA). 1H NMR spectra of MPDA were obtained from a 400 MHz Varian
VXR400 spectrometer. Mass spectra of MPDA were recorded on a GC/MS Hewlett-Packard 5988A in the FAB mode. UV-vis spectra of PDA and MPDA were obtained with the UV-2101PC scanning spectrophotometer (Shimadzu Scientific Instruments, Inc.), with 1 cm optical path length quartz cells. Isotherms of Pure and Mixed PDA/MPDA Monolayer. All organic solvents used in this study are HPLC grade (Fisher Scientific Co.). Chloroform was purified (distillation, the component between 61.1-61.3 °C was collected) before use. Pure and mixed PDA/MPDA monolayers were dissolved in a mixture of chloroform and methanol (5:1 in molar ratio, the purpose of methanol is to increase the solubility of MPDA) to a concentration of 1.0 mM. The water used for the monolayer study was purified by a Modulab 2020 water purification system (Continental Water Systems Corp., San Antonio, TX). The water has a resistance of 18 MΩ cm and a surface tension of 72.6 mN/m at 20 °C. The surface tension was measured by a digital-tensiometer, model K10 (KRU ¨ SS GmbH Wissenschaftliche Labogera¨te, Hamburg, Germany). Cadmium chloride was certified ACS grade (Fisher Scientific Co.). All surface pressure-area isotherms were measured on a KSV minitrough system at room temperature (23.0 ( 0.5 °C), with a humidity of 40 ( 5%. The trough has an area of 225 cm2. The pressure sensor has a resolution of 0.02 mN/m. For each isotherm experiment, 40 µL of a 1.0 mM sample was spread on the water surface, waiting 10 min for solvent evaporation before compression. The barrier was compressed at a speed of 2.5 Å2 molecule-1 min-1. Isotherms of different PDA/MPDA monolayers on pure water (pH ) 5.8) and 10-4 M CdCl2 (pH ) 6.6) were obtained.
Results and Discussion Physical Chemistry Properties of PDA and MPDA. H NMR of MPDA (CD3OD): δ.0.90 (t, 3H, J ) 7.2 Hz, CH3), 1.29 (s, 20H, CH2), 1.36 (s, 12H, CH2), 1.50 (m, 4H, CH2), 1.69 (t, 2H, J ) 7.6 Hz, CH2), 2.23 (t, 4H, J ) 6.8 Hz, -CtC-CH2-), 2.34 (t, 2H, J ) 7.2 Hz, -C(O)-CH2), 3.57-3.62 (m, 1H), 3.68-3.78 (m, 3H), 3.88 (dd, 1H, Ja ) 3.2 Hz, Jb ) 8.0 Hz), 3.98 (s, 1H), 5.42 (s, 1H, anomeric H), 7.07 (d, 2H, J ) 9.2 Hz, Ar), 7.45 (d, 2H, J ) 9.2 Hz, Ar). The calculated molecular weight for C37H57O7N is 627.3; the molecular weight found from the mass spectra is 628.4. Figure 2A shows the UV-vis spectra of PDA in methanol and hexane. Because MPDA does not dissolve in hexane, only the UV-vis spectrum of MPDA in methanol was 1
Figure 2. (A) UV spectra of PDA in methanol and hexane; (B) UV spectrum of MPDA in methanol.
obtained (Figure 2B). The extinction coefficients in the figures were calculated based on four different concentrations of the sample.
A Mixed-Monolayer Study
Figure 3. Surface pressure-area isotherms of PDA/MPDA mixtures with different molar fractions of MPDA (χMPDA): (A) on pure water, pH 5.8; (B) on 10-4 M CdCl2, pH 6.6.
Figure 2A shows that in both polar (methanol) and nonpolar solvents (hexane), PDA has four major absorption peaks at 215, 226, 240 and 254 nm. This result is comparable with that from Tieke et al.12 The extinction coefficients in methanol are slightly higher than those in hexane, while all of the peak positions remain the same. This means that as an amphiphile molecule, PDA has almost the same behavior in both polar and nonpolar environments. MPDA has one large peak at 247 nm, with an extinction coefficient of 17 400 cm-1 M-1. Both molecules show no absorption in the visible range. Surface Pressure-Area Isotherms of Pure and Mixed PDA/MPDA Monolayers. Surface pressurearea (π-A) isotherms of pure and mixed PDA/MPDA monolayers on pure water (Figure 3A) and on 10-4 M CdCl2 (Figure 3B) were obtained. For pure PDA and MPDA, each curve shown in the figure represents the average of six different measurements done with two separate samples. For each PDA/MPDA mixture, each curve represents the average of three different measurements. The area variation of the individual measurement is within (0.6 Å2 molecule-1. (12) Tieke, B.; Lieser, G.; Wegner, G. J. Polym. Sci.: Polymer Chem. Edition, 1979, 17, 1631.
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Pressure-Area Isotherms of Pure PDA and MPDA Monolayers. Figure 3A curve a shows the π-A isotherms of pure PDA on pure water (pH 5.8, 23 °C). From the curve we can get the limiting area per molecule of PDA at π ) 0 mN/m, about 26 Å2 molecule-1, with a collapse pressure at 15 mN/m. This result is comparable to the work of Day and Ringsdorf.13 They measured an area of 24 Å2 molecule-1, with a collapse pressure at 20 mN/m on pure water at 20 °C. Also, Bicgajski and Cadenhead14 obtained a limiting area of 28 Å2 molecule-1 at 25.2 °C. These values show a linear increase of the limiting area with temperature and show that our result is consistent with the existing literature value. Curve e in Figure 3A shows the π-A isotherm of pure MPDA on pure water. The limiting area is about 32 Å2 molecule-1 at π ) 0 mN/ m, with a collapse pressure at about 55 mN/m. The large hydrophilic group of MPDA stabilized the monolayer and also increased the area of the molecule. Both the areas of PDA and MPDA were confirmed by the corresponding Corey-Pauling-Koltun model. Figure 3B curve a shows the π-A isotherm of pure PDA on 10-4 M CdCl2 (pH 6.6, 23 °C). From the curve we can get the limiting area of PDA to be about 22 Å2 molecule-1, with a collapse pressure at 45 mN/m. This result is comparable to the work of Mino et al.,14 which shows an area of 23 Å2 molecule-1 with a collapse pressure at 45 mN/m on 3.6 × 10-6 Cd2+ at pH 6.8. This isotherm is completely different compared to that on pure water, due to the divalent cation effect. Each divalent cation, Cd2+, can interact with two PD-, the hydrophilic headgroup of the dissociated PDA molecule; thus, the area per molecule of the monolayer was reduced and the metastability of the monolayer was enhanced. For MPDA on 10-4 M CdCl2 (Figure 3B curve e), the isotherm remains at the same limiting area per molecule and the same collapse pressure as on pure water. Since there is no charge on the MPDAs headgroup, pH and divalent cations have little effect on the behavior of the MPDA monolayers. Pressure-Area Isotherms of Mixed PDA/MPDA Monolayers. In order to study the miscibility of the mixed PDA/MPDA monolayers, isotherms of different molar fractions of MPDA in mixed monolayers on pure water and on 10-4 M CdCl2 were recorded (Figure 3). The characteristics of the mixing can be analyzed from the change in the collapse pressures and molecular areas of the isotherms. Figure 4 shows the collapse pressures of the monolayers as a function of the mole fraction of MPDA. For the pure water subphase, the collapse pressures of the isotherms show a nearly linear relationship with the molar ratio of MPDA (Figure 4A). According to the method developed by Crisp and presented by Gaines,16 this indicates either complete miscibility and nearly ideal behavior or complete immiscibility of the two components in the mixed monolayers. The collapse pressures of the isotherms increase when the molar ratios of MPDA in the mixture increase; this means that MPDA molecules increase the stability of the mixed monolayer. The most likely reason is that the relatively large hydrophilic headgroup of MPDA causes the molecule to more easily stand at the air/water interface. For mixed PDA/MPDA monolayers on 10-4 M CdCl2 (Figure 4B), the difference of the collapse pressures (13) Day, D.; Ringsdorf, H. J. Polymer Sci., Polym. Lett. Ed. 1978, 16, 205. (14) Frontiers of Polymers and Advanced Materials. International Conference on Frontiers of Polymers and Advanced Materials, Jakarta, Indonesia, 1993; Plenum: New York, 1994; p 699. (15) See ref 4. (16) Gaines, G. L., Jr. Insoluble Monolayers at Liquid-Gas Interfaces; Wiley-Interscience: New York, 1966.
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Figure 4. Collapse pressure of PDA/MPDA mixed monolayers as a function of the molar fraction of MPDA: (A) on pure water, pH 5.8; (B) on 10-4 M CdCl2, pH 6.6.
between the different mixtures is relatively small because the collapse pressure of pure PDA is only slightly lower than that of MPDA. We can still see the nearly linear relationship between the collapse pressure and the molar fraction of MPDA. Although the cadmium substrate may only affect the charged molecules and could enhance the segregation of the molecules, the segregation is not observed, since only about 15% of the PDA molecules are ionized at pH 6.6, according to Mino et al.15 Comparing parts A and B of Figure 4, it is also found that at a lower χMPDA, the collapse pressures for pure water are lower than those for 10-4 M CdCl2. Again, this is due to the divalent cation, Cd2+, which has a strong interaction with PDA molecules, stabilizing the monolayer. An understanding of the interaction between PDA and MPDA is also provided by comparing the molecular areas of the mixing, which is calculated through the additivity rule with the experimental molecular areas. Figure 5 shows the experimental molecular areas plotted as a function of the mole fraction of MPDA at different surface pressures. For an immiscible monolayer, the molecular area of the mixed monolayer should follow the additivity rule16
A12 ) χ1A1 + χ2A2
Wang et al.
Figure 5. Molecular area of the mixed PDA/MPDA monolayers as a function of the molar fraction of MPDA at different surface pressures: (A) on pure water, pH 5.8; (B) on 10-4 M CdCl2, pH 6.6.
where A12 is the mean molecular area expected at a given surface pressure in the two-component film, χ1 and χ2 are the mole fractions of the components in the mixed monolayer, and A1 and A2 are the molecular areas of the pure components at the same surface pressure. Thus, a linear correlation between the molecular area and the molar fraction of one component could mean either immiscibility or miscibility with nearly ideal behavior. Figure 5A shows the molecular area versus the molar fraction of MPDA of the mixed monolayers on pure water at three different surface pressures, 5, 10, and 15 mM/m. Positive deviations of the molecular areas from the additivity rule are observed for all molar fractions at all surface pressures studied. The maximum value of the deviation is located around χMPDA ) 0.75. The deviations are larger at lower pressure. The positive deviations indicate that there are some kinds of repulsions or disorders between the PDA and MPDA molecules. It means that the two types of molecules that are mixed with each other and the monolayer are miscible. The positive deviations also indicate that the interaction between PDA/MPDA is weaker than those of PDA/PDA and MPDA/MPDA, and there is the possibility of partial miscibility at low temperatures.
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Figure 5B shows the molecular area versus the molar fraction of MPDA of the mixed monolayers on 10-4 CdCl2 at five different surface pressures, 5, 10, 20, 30 and 40 mN/m. Again, the positive deviations of the molecular areas from the additivity rule are observed for all molar fractions at all surface pressures studied. The maximum value of the deviations is located around χMPDA ) 0.75 for the pressure below 20 mN/m and χMPDA ) 0.9 for the pressure above 20 mN/m. For χMPDA < 0.2, the deviations are very small; thus, the mixed monolayers in this range may have less disorder and are, therefore, better organized. Excess Free Energies of PDA/MPDA Mixed Monolayers. The interaction between PDA and MPDA molecules in the mixed monolayer can be analyzed more quantitatively if we evaluate the excess free energy of mixing, ∆GXSπ, at a given surface pressure. Our experimental setup did not allow us to monitor the vapor-liquid transition. As previously noted by Gershfeld,17 the Helmholtz free energy of film compression is underestimated if one does not monitor the surface pressure at a sufficiently large molecular area. So it is possible that some error was introduced in ∆GXSπ by assuming that the components in the mixed monolayer behave ideally in the limit of zero surface pressure. With that in mind,
∆GXSπ )
∫0π(A12 - χ1A1 - χ2A2)dπ
where A1, A2, and A12 are the molecular areas at surface pressure π for the pure components 1 and 2 and for the mixture, respectively. χ1 and χ2 are the molar fractions of the components in the monolayer. From this equation, we deduce that if the two components are immiscible and follow the additivity rule, the excess free energy will be zero at any pressure and molar fraction. The excess free energies of the mixed PDA/MPDA monolayers are presented in Figure 6 as a function of the molar fraction of MPDA at different surface pressures. Figure 6A shows the excess free energies of mixing for a pure water subphase at three pressures, 5, 10, and 15 mN/m. The excess free energies are positive at all different molar fractions of MPDA and pressures of the monolayer. The maximum value is about 440 J/mol at χMPDA ≈ 0.6 and 15 mN/m. Figure 6B shows the excess free energies of a mixed PDA/MPDA monolayer on a 10-4 M CdCl2 subphase at four different surface pressures, 5, 10, 20, and 40 mN/m. The excess free energies are positive at all different molar fractions of MPDA and pressures of the monolayer. The maximum value is about 700 J/mol at χMPDA ≈ 0.7 and 40 mN/m. The optimum value of the excess free energies for both pure water and 10-4 M CdCl2 decreased steadily with the surface pressure. This is due to the fact that the excess molecular areas (A12 - χ1A1 - χ2A2) are positive over all ranges of pressures. From all of the above discussions, we conclude that PDA/MPDA monolayers have good miscibilities over all of the ranges of pressures and molar fractions for both pure water and 10-4 M CdCl2 at 23 °C. The expansion of the molecular areas from the additivity rule of the mixed monolayer, especially at higher χMPDA, indicates some kind of disorder or repulsion between PDA and MPDA molecules. This is possibly due to the (17) Gershfeld, N. L. In Techniques of Surface and Colloid Chemistry and Physics; Good, R. J., Stromberg, R. R., Patrick, R. L., Eds.; Marcel Dekker: New York, 1972; Vol. 1, p 1.
Figure 6. Excess free energies of mixing as a function of the molar fraction of MPDA for mixed PDA/MPDA monolayers at different surface pressures: (A) on pure water, pH 5.8; (B) on 10-4 M CdCl2, pH 6.6.
headgroup repulsion between the two different molecules. Although the expansion is very small at low χMPDA, which indicates a weak repulsion between PDA and MPDA, the change of the collapse pressures is still strong evidence supporting the miscibility of the monolayer. The weak molecular area expansion at low χMPDA may also mean a well-ordered monolayer. Conclusion We have shown that the PDA/MPDA mixed monolayers have good miscibilities at all molar ratios of MPDA and surface pressures for both pure water and 10-4 CdCl2 subphases. There is some sort of interaction between PDA and MPDA molecules, especially at high molar fractions of MPDA. Subphase divalent cations Cd2+ increased the stability of the PDA monolayer. Thus, with 10-4 M CdCl2 in the subphase and at a low MPDA molar ratio, we obtained a better organized and miscible PDA/MPDA monolayer for further biosensor studies. LA9606411
