© Copyright 1997 by the American Chemical Society
VOLUME 101, NUMBER 15, APRIL 10, 1997
LETTERS Structure and Biomembrane Mimetic Behavior of the Water-Octanol Interface D. T. Cramb and S. C. Wallace* Department of Chemistry, 80 St. George Street, UniVersity of Toronto, Toronto, Ontario M5S 3H6, Canada ReceiVed: October 10, 1996; In Final Form: February 11, 1997X
A widely used screen to test the propensity of molecules to absorb into living tissue is the water-octanol emulsification test. The determination of the contribution of interfacial adsorption to bioavailability has been elusive using this standard assay. In this report, a novel technique of measuring molecular bioavailability is introduced. This technique employs laser light to probe molecular adsorption to the biomembrane mimetic water-octanol interface. It is shown that adsorption to the membrane plays a critical role in the process delivering molecules, such as pharmaceuticals, to tissue cells. Additionally, it has been possible to determine for the first time experimentally the structure of the water-octanol interface.
1. Introduction The use of octanol-water partitioning as an initial indicator of the bioavailability of molecules is almost universal in the development of pharmaceuticals1-3 and environmental pollutants.4-6 It has been believed for some time that emulsified solutions of water-saturated octanol and octanol-saturated water contain domains that mimic biomembranes and that the partitioning of molecules between these phases is indicative of the propensity of a molecule to absorb into living tissue. This would result from the amphiphilicity of octanol. To date, however, there has been no direct experimental measurement of the structure of the water-octanol interface. Furthermore, because of the desire for solubility in water, paired with the ability to cross the cell membrane, most drugs are designed to be amphiphilic, whereas the amphiphilicity of many pollutants is serendipitous. In addition to partitioning itself into the aqueous and organic phases, an amphiphilic molecule will also adsorb to the interface between these phases. Therefore, in order to assess bioavailability, one must not only consider partitioning between bulk phases but also consider the partitioning of biomolecules, pharmaceuticals, and pollutants between the bulk X
Abstract published in AdVance ACS Abstracts, April 1, 1997.
S1089-5647(96)03108-2 CCC: $14.00
and the water-octanol interface. This information is not contained in the results of a standard water-octanol emulsification test. In this report, we present an alternative method for assessing the bioavailability of molecules, which preserves information about a molecule’s propensity for membrane adsorption. Only very recently has it become possible to measure the adsorption of molecules at buried interfaces. The method employed is interface-specific second-harmonic generation of laser light.7-9 This is a technique that allows the determination of molecular orientation at liquid-liquid interfaces with submonolayer sensitivity. More recently, the technique of sum-frequency generation has been used to determine the orientation of a hexadecanol monolayer at the air-water interface.10 In our work, we have determined for the first time experimentally the structure of the neat water-octanol interface. Moreover, we have measured the structure and energetics of adsorption of the amphiphilic biomolecule L-tryptophan from the aqueous bulk medium to the interface. We find that the partitioning between bulk and interface is indeed different than that between the two bulk phases. 2. Experimental Procedures The experimental arrangement has been described previously.7,11 Only the relevant modifications will be presented here. © 1997 American Chemical Society
2742 J. Phys. Chem. B, Vol. 101, No. 15, 1997
Letters
Because octanol and water are virtually immiscible, a very stable liquid-liquid interface can be maintained between the two bulk media. In our experiment, a cylindrical quartz vessel contains the two liquids. Since water is denser, it is the lower phase. High-purity distilled and deionized water was used. Chromatographic grade octanol (Aldrich) and L-tryptophan (Aldrich) were used without further purification. The laser beam first passes through a polarization rotator and focusing lens (focal length ) 7.5 cm) before reflecting off the liquid-liquid interface. The reflected beam is then optically and spatially filtered, selected for polarization, and detected with a photomultiplier tube. For two-photon excitation, the laser wavelength was 660 nm. All experiments were performed at a controlled ambient room temperature of 18 °C. 3. Results and Discussion A. Structure of the Water-Octanol Interface. The structure of the water-octanol interface was determined using second-harmonic generation, SHG, of laser light. The theory behind SHG is well documented.12,13 Light impinges on an interface between two centrosymmetric media, and the interface can act as a nonlinear medium and allow two photons of the fundamental light frequency to combine to create one photon of double the fundamental frequency, i.e., the second harmonic of the laser light. Since this process only takes place at the interface, any phenomenon that changes the efficiency of this process will reveal changes occurring only at the interface. The incident laser beam interacts with the macroscopic interface, but the process is formulated in terms of the individual molecules at the interface. The intensity of the second-harmonic light can be modeled as being proportional to the square of the sum over the orientational average of the hyperpolarizabilities of all the molecules in the laser-interface interaction region. Formally, this may be written as14
IXYZ(2ω) )
32π3ω2 sec2(θ2ω) jωejω|2 I2 (1) |ej2ω‚χc(2) s (2ω):e c3(ω) e1/2(2ω)
where ejω is the electric field vector of the incident photon and χc(2) s (2ω) is the second-order surface nonlinear susceptibility tensor. It can be approximated as the sum of the number of molecules at specific surface orientations times their orientationally averaged second-order nonlinear polarizabilities, Nn〈R(2) ijk 〉n. By choosing appropriate incident light polarizations and by detecting selectively a single polarization of the secondharmonic light, one can measure a particular orientational component of the principal hyperpolarizability moment. From this, one can deduce the average orientation of the molecule with respect to the interface normal by taking the ratio of two different polarization arrangements. The ratio of the intensity of p-polarized input fundamental frequency radiation and p-polarized output second-harmonic radiation over s-polarized input and p-polarized output was experimentally determined to be 3.9 ( 0.8. From the equation7,15
[
]
Ipp 3〈cos3 θ〉 1 )R) 2 Isp 4 2〈sin θ cos θ〉
2
(2)
the angle of the principal moment of hyperpolarizability, R, to the surface normal is determined to be 39 ( 10°. From a semiempirical molecular orbital program (MOPAC 93) calculation, we find that the principal nonresonant moment of R lies along the long axis of octanol. The principal hyperpolarizability
Figure 1. Orientation of interfacial water and octanol. The water molecules have their electric dipole moments parallel to the interface. There is tetrahedral coordination around the alcohol oxygen where two H-bonds are formed with water. The coordinate system is the laboratory frame. We have included the simulated and measured octanol O-H bond angle with the interface normal.
of H2O is about one-tenth of that of octanol and can therefore be neglected. We conclude that the backbone of octanol is inclined at an average angle of 39 ( 10° to the surface normal. We can compare this result with a recent molecular dynamics simulation by Michael and Benjamin.16 In that study they found that the structure of the interface was dominated by octanols positioned with their polar head groups pointing toward the water phase in such a way that both the H-O and O-C bonds lie at angles of (30° to the surface normal. On average, the H2O molecules were oriented with their electric dipole moments parallel to the interface and their molecular planes perpendicular to the interface. We depict a slice of the calculated liquidliquid interface in Figure 1. For the orientation of H2O, we assume that the calculated structure16 is correct. Figure 1 shows that the agreement between experiment and theory is remarkable. It is of further interest to compare our result for octanol with those of Wolfrum and Laubereau10 for hexadecanol at the airwater interface. They found that the nonpolar backbone is within 8° of being perpendicular to surface. The difference in their system is that with the longer hydrophobic chain, one would expect the hydrophobic interchain interactions to dominate the backbone structure. In our system, interaction with bulk octanol must hinder the interfacial octanol from assuming a more erect orientation. B. Adsorption to the Water-Octanol Interface. In order to assess the biomembrane mimetic properties of the octanolwater interface and its contribution to molecular partitioning, we introduce a probe molecule to the system and observe its behavior. We have chosen L-tryptophan for its amphiphilicity and because of its biological relevance. At the neutral pH of this experiment we expect, tryptophan to be in its zwitterionic form. In this configuration, the ammonium and carboxylate moieties are highly hydrophilic and the indole side chain is highly hydrophobic.17 Thus, the molecule should show an energetic preference to being adsorbed at the octanol-water interface over being solvated in either of the bulk media. In standard partitioning assays using emulsification of the immiscible liquids, there is no way to extract any interface-specific information. The interfacial partitioning of L-tryptophan and the free energy
Letters for the process were determined in the following way. If chromophores are aligned at an interface, it is possible to extract interface-specific fluorescence emission using a suitable choice of excitation polarization followed by selection of a single polarization component of the emitted fluorescence. Furthermore, by employing two-photon excitation, one can reduce the volume of interaction to those chromophores near the interfacial region. This is because a two-photon process depends quadratically on the intensity of the incident laser field and inversely on the laser beam cross section. The intensity of two-photon absorption will be greatest at the beam’s focus. Thus, a tightly focused laser beam can reduce the excitation dimensions easily to a spot size of 50 µm parallel to the surface maintained 1-3 µm perpendicular to the surface.11 Because the two-photon fluorescence signal was much larger than the second-harmonic signal, it was not possible to do SHG studies on this system. A laser beam of λ ) 660 nm was incident on the wateroctanol interface at an angle of 60° to the surface normal. The undispersed fluorescence, observed in reflection off the surface, was spatially filtered and passed through an optical filter to remove the fundamental wavelength. The polarization behavior of the emitted fluorescence was monitored with a Kodak polarizing film. The same experimental setup was used to assay the polarization characteristics of both light absorption and emission of bulk media tryptophan. The signal from the photomultiplier tube was found to vary as a function of the square of the input beam intensity, thus confirming a two-photon absorptive process. With all the polarization selection optics in place, the signal from the interface was the same order of magnitude as that from the bulk. Two-photon absorptions to both the La and Lb excited states of tryptophan are allowed quantum mechanically.18 It has been observed that the higher dipole moment La state is stabilized in polar solvents such as H2O and therefore lower in energy.19 This will be the case with bulk aqueous tryptophan and may also be the case with interfacial tryptophan. The wavelength of our incident radiation suggests that the state to which we are exciting is 30 300 cm-1 higher in energy than the ground electronic state. The onset of the S0 f La transition in H2O at room temperature has been measured20 to be as low as 31 750 cm-1. At 330 nm the excitation must be from vibrationally excited levels in the far red tail of the S0 f La transition. In the bulk, tryptophan displays no measurable variation of the fluorescence signal with input laser polarization. There is also no evidence of polarization of the emitted fluorescence. This would be expected from a sample of randomly oriented molecules. However, at the interface, there is a dependence of the fluorescence signal on the input laser beam polarization. When exciting with strictly s-polarized input radiation, the ratio of s-polarized to p-polarized fluorescence is 2:1. This is highly suggestive of preferential orientation of the indolyl chromophore at the water-octanol interface. In Figure 2, we present a depiction of the position of L-tryptophan at the water-octanol interface. In order to determine the bulk-interface partitioning and the free energy for the interface adsorption, an adsorption isotherm must be measured. Figure 3 shows the dependence of the octanol-water surface coverage, Θ, on the concentration of bulk aqueous tryptophan. Θ was determined by assuming that the two-photon fluorescence intensity is linearly dependent on concentration, in accordance with the Beer-Lambert approximation for the two-photon absorption process.21 The error bars on the data points represent 1 standard deviation from the mean. The statistics are generated by analysing 10 data sets for each concentration. The photons were counted for a period
J. Phys. Chem. B, Vol. 101, No. 15, 1997 2743
Figure 2. Orientation of L-tryptophan at the water-octanol interface. The hydrophobic indole side chain positions itself on the octanol side of the interface in the hydrophobic region. From fluorescence polarization results (see text), one expects the plane of the indole side chain to be oriented more parallel to the interface rather than perpendicular to it.
Figure 3. Adsorption isotherm for aqueous tryptophan at the wateroctanol interface. The error bars are 1 standard deviation determined from the average of 10 data sets of 30 s collection time each. The solid line is a fit using a Langmuir isotherm.
of 30 s. There was nearly zero background signal from the neat water-octanol interface. The solid line represents a fit to the data points of a Langmuir adsorption isotherm,22 which assumes that there is no dependence of the free energy of adsorption on the surface coverage. For the desorption process a
Trp(interface) S Trp(aq) the form of the Langmuir adsorption isotherm is
Θ)
CTrp CTrp +
a 55.6 M
(3)
Here, CTrp is the bulk aqueous molar concentration of tryptophan, a is the partitioning constant for desorption of tryptophan from the interface, and a/55.6 can be thought of as an equilibrium constant K for desorption. By extrapolation to infinite dilution, the solution can be assumed to behave ideally. Thus, the equilibrium constant can be used to determine the free energy of adsorption, ∆Gad, using the following equation22
∆Gad ) -RT ln(55.6/a)
(4)
where R is the ideal gas constant and T is temperature in kelvin. From our model we find a value for a of (4.5 ( 0.6) × 10-3, which leads to a value for the free energy of ∆Gad ) -23 ( 2 kJ/mol. This value can be compared with that derived for the partitioning of tryptophan between bulk H2O and bulk octanol.
2744 J. Phys. Chem. B, Vol. 101, No. 15, 1997 This partitioning was determined by measuring spectrophotometrically the equilibrium bulk concentrations. A value of Kow ) (1.0 ( 0.02) × 10-2 was obtained suggesting ∆Gow ) -5.8 ( 0.1 kJ/mol. The higher accuracy of this number reflects the higher accuracy of the spectrophotometric measurement of concentration. Kow is the ratio of the concentration of tryptophan in octanol to that in water. ∆Gow is calculated by converting Kow in terms of concentrations to Kow′ in terms of mole fractions. Thus, Kow′ ) 0.11Kow. By comparing ∆Gad to ∆Gow, one finds a strong indication of preference for tryptophan to position itself at the amphiphilic interface compared with being dissolved in either bulk medium. Since we were exciting a vibrationally hot chromophore, it was not possible to do any temperaturedependent experiments with this system. The strong preference for interface adsorption found here has significant implications for typical octanol-water partitioning studies. A standard octanol-water partitioning assay involves an emulsification step followed by centrifugation to separate the phases. The concentration of the molecule of interest is then measured for each phase. In this process, a large variation in droplet size is to be expected ranging from small micelles to multilamellar vesicles. A recent molecular dynamics simulation has indicated that this is indeed the case, by showing that octanol micelles are formed in water solutions and reverse octanol micelles are formed in octanol solution.23 During emulsification, amphiphilic molecules will minimize their free energy by adsorbing to the interface. At suitably high concentrations of solute, solute monolayers may exist on many of the interfaces. In the subsequent separation of phases, the molecules must decide which bulk phase is more favorable. From our results, the test molecule L-tryptophan positions itself preferentially at the octanol-water interface in comparison with either bulk phase. If the interfacial surface area were decreased, the tryptophan would be forced into both bulk phases according to the surface area to volume ratios and the adsorption isotherms of each phase. If this is extended to an infinitely small interfacial area, the concentrations in each bulk phase would represent the partitioning between the phases, and all information about the interface itself would be lost. This is what occurs during the centrifugation step in standard wateroctanol partitioning assays. This is unfortunate because the interfacial partitioning parameters have direct relevance to the binding of molecules such as pharmaceuticals to cell membranes. 4. Conclusions In conclusion, we have determined for the first time experimentally the structure of the octanol-water interface. We find that octanol molecules are oriented at an angle of 39 ( 10° to the surface normal. The octanol-water interface displays
Letters sufficient organization to make it a valuable model for a biomembrane. Additionally, we have introduced a two-photon excitation/fluorescence polarization technique in order to measure selectively liquid interface-specific adsorption phenomena. An isotherm for the adsorption of L-tryptophan from the bulk aqueous phase to the H2O-C8H17OH interface was determined. Associated with this process is a change in free energy of ∆Gad ) -23 ( 2 kJ/mol, which is significantly larger in magnitude than the free energy of transfer between the bulk media, ∆Gow ) -5.8 ( 0.1 kJ/mol. Thus, in order to study membrane adsorptive effects, one must use an interface-specific experimental technique such as that described in this Letter. Acknowledgment. Financial support from the Natural Sciences and Engineering Research Council of Canada and from the Ontario Laser and Lightwave Research Centre is gratefully acknowledged. The authors thank Steven Martin for his help with the molecular orbital calculations. References and Notes (1) Hansch, C.; Dunn, W. J. J. Pharm. Sci. 1972, 61, 1-19. (2) Gaspari, F.; Bonati, M. J. Pharm. Pharmacol. 1987, 39, 252-260. (3) Smith, R. N.; Hansch, C.; Ames, M. M. J. Pharm. Sci. 1975, 64, 599-606. (4) Leyder, F.; Boulanger, P. Bull. EnVironm. Contam. Toxicol. 1983, 30, 152-157. (5) Doucette, W. J.; Andren, A. W. EnViron. Sci. Technol. 1987, 21, 821-824. (6) Brinck, T.; Murray, J. S.; Politzer, P. J. Org. Chem. 1993, 58, 7070-7073. (7) Cramb, D. T.; Martin, S. C.; Wallace, S. C. J. Phys. Chem. 1996, 100, 446-448. (8) Eisenthal, K. B. Acc. Chem. Res. 1993, 26, 636-653. (9) Kott, K. L.; Higgins, D. A.; McMahon, R. J.; Corn, R. M. J. Am. Chem. Soc. 1993, 115, 5342-5343. (10) Wolfrum, K.; Laubereau, A. Chem. Phys. Lett. 1994, 228, 83-88. (11) Cramb, D. T.; Wallace, S. C. Submitted for publication in J. Am. Chem. Soc. (12) Raising, Th.; Shen, Y. R.; Fim, M. W.; Grubb, S. Phys. ReV. Lett. 1985, 55, 2903-2910. (13) Heinz, T. F.; Chen, C. K.; Richard, D.; Shen, Y. R. Phys. ReV. Lett. 1982, 48, 478-481. (14) Shen, Y. R. The Principles of Nonlinear Optics; Wiley: New York, 1984. (15) Mazely, T. L.; Heatherington, W. M. J. Chem. Phys. 1987, 86, 3640-3655. (16) Michael, D.; Benjamin, I. J. Phys. Chem. 1995, 99, 16810-16813. (17) Tanford, C. The Hydrophobic Effect; Wiley: New York, 1980. (18) Meech, S. R.; Phillips, D. R.; Lee, A. G. Chem. Phys. 1983, 80, 317-328. (19) Creed, D. Photochem. Photobiol. 1984, 39, 537-562. (20) Rehms, A. A.; Callis, P. R. Chem. Phys. Lett. 1993, 208, 276282. (21) Birge, R. R.; Zhang, C.-F. J. Chem. Phys. 1990, 92, 7178-7195. (22) Adamson, A. W. Physical Chemistry of Surfaces; Wiley: New York, 1990. (23) DeBolt, S. E.; Kollman, P. A. J. Am. Chem. Soc. 1995, 117, 53165340.
