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Langmuir 1999, 15, 3424-3429
Photoregulated Potassium Ion Permeation through Dihexadecyl Phosphate Bilayers Containing Azobenzene and Stilbene Surfactants Yabin Lei† and James K. Hurst* Department of Chemistry, Washington State University, Pullman, Washington 99164-4630 Received September 9, 1998. In Final Form: February 25, 1999 Photoresponsive asymmetrically organized systems based upon small unilamellar dihexadecyl phosphate (DHP) vesicles were constructed by entrapping high concentrations of potassium ion within the vesicular aqueous core and incorporating either an amphiphilic trans-azobenzene-containing phosphate monoester or an amphiphilic trans-stilbene-containing carboxylic acid into its membrane structure. Spectroscopic measurements indicated that the azobenzene derivative was molecularly dispersed in the hydrocarbon phase of the vesicle and that the extent of aggregation of the membrane-localized stilbene derivative was minor. Thermal K+ leak rates from the doped vesicles were very low, with calculated permeability coefficients (P) of ∼4 × 10-12 cm/s at 40 °C for DHP vesicles containing 5.5 mol % of the trans-azobenzene derivative and ∼1.5 × 10-11 cm/s at 38 °C for vesicles containing 5.5 mol % of the trans-stilbene derivative; for comparison, P = 2 × 10-12 cm/s for undoped vesicles at 40 °C. Photoexcitation of the azobenzene-doped vesicles at 360 nm caused >90% trans f cis photoisomerization over the measured temperature range (25-40 °C), with complete reversion to the trans isomer upon photoexcitation at 450 nm. Photoexcitation of deoxygenated suspensions of the stilbene-doped vesicles at 315 nm gave ∼80% conversion to the cis isomer in the photostationary state, which was not reversible. At 25 °C, K+ leak rates for the isomeric azobenzene-doped vesicles were nearly identical; at 40 °C, K+ leakage for the DHP vesicles containing the cis-azobenzene isomer corresponded to P = 2 × 10-11 cm/s, 5-fold greater than that of the trans isomer. In trans f cis f trans photocycling experiments, K+ leak rates alternately increased and decreased, indicating that the vesicles remained intact. At 40 °C, K+ leakage from the vesicles containing predominantly cis-stilbene was ∼2-fold greater than that from vesicles with the corresponding trans isomer. In electrochemical experiments, viologen-mediated reduction of the DHP-bound trans-azobenzene occurred at E < -0.44 V (NHE), with hydrazobenzene reoxidation at E = -0.16 V; addition of viologen radicals to aqueous suspensions of the trans-azobenzene-doped DHP vesicles caused immediate decolorization of the dye.
Introduction Recent research in our laboratory has been directed at developing photoregulated membrane-organized oxidation-reduction systems in which the active elements are coupled through membrane polarization in a manner similar to chemiosmotic energy transduction and signaling in living cells.1,2 The basic concept is illustrated in Figure 1, which describes an ion-impermeable closed bilayer membrane containing two functional elements, a vectorially organized electrogenic transmembrane oxidationreduction system and a reversible photoisomerizable dye capable of acting as an ion gate. Transmembrane redox leads to membrane polarization; in the absence of chargecompensating ion translocation, this developing potential impedes further reaction.3 Ion permeabilities are modulated by the dye, which in one conformation more extensively disrupts normal side-chain packing of the membrane-forming surfactant amphiphile than the other, thereby increasing diffusion rates. Thus, in the more disruptive conformation the membrane potential collapses at a greater rate; in the limit where the membrane is * To whom correspondence should be addressed. † Current address: Witco Corp., 5777 Franz Road, Dublin, Ohio 43017. (1) Harold, F. M. The Vital Force: A Study in Bioenergetics; W. H. Freeman: New York, 1986. (2) Nicholls, D. G.; Ferguson, S. J. Bioenergetics 2; Academic Press: San Diego, 1992. (3) Hurst, J. K. In Kinetics and Catalysis in Microheterogeneous Systems; Surfactant Science Series, Vol. 38; Gratzel, M., Kalyanasundaram, K., Eds.; Marcel Dekker: New York, 1991; Chapter 7.
Figure 1. Stylized depiction (in cross section) of a bifunctional vesicle exhibiting light-regulated transmembrane oxidationreduction. In state a, reduction of internal viologen (V2+) is blocked by membrane polarization; in state b, photoisomerization of the membrane-localized dye increases leak rates of the electrolyte cation (M+) which, by collapsing the membrane potential, relieves inhibition of the transmembrane redox reaction.
ion-impermeable in one conformation but permeable in the other, the photoisomerizable dye acts as an on-off switch, toggling the membrane between conducting and nonconducting states. The likelihood of constructing a functioning device based upon these principles is supported by the recent demonstration of chemiosmotic ATP photosynthesis employing a synthetic vesicle containing only a quinone pool, the F0-F1 ATP synthase, and a vectorially oriented artificial electron transport chain.4 In this study, dihexadecyl phosphate (DHP) vesicles were used as the organizing matrix. A major advantage
10.1021/la981223u CCC: $18.00 © 1999 American Chemical Society Published on Web 04/02/1999
Photoregulated Transmembrane K+ Diffusion
of these membranes is that electrogenic transmembrane oxidation-reduction is tightly coupled to charge-compensating translocation of lipophilic ions,5-7 indicating that intrinsic permeabilities of electrolyte ions are very low. The low permeabilities of aqueous ions probably arise from relatively compact surfactant side-chain packing in the hydrocarbon bilayer, which causes the membrane to be in its gel phase at ambient temperatures.8 The relatively rigid DHP hydrocarbon matrix might be problematic for proper functioning of the ion gates, however, because thermodynamic barriers to bilayer-disrupting photoisomerizations could be prohibitively large9 or the photochromic dye could be excluded entirely from the hydrocarbon region of the bilayer.10,11 A wide variety of photoisomerizable compounds, including those that undergo reversible cis h trans isomerizations and ring opening-closing reactions,12,13 are potentially suitable as ion gates; many have been used to modulate the properties of materials,14 including altering ion permeabilities in phospholipid membranes. Preliminary studies using DHP vesicles have indicated that simple lipophilic spiropyrans and spirooxazines, compounds that undergo reversible ring opening to merocyanine forms, either are not incorporated or undergo relatively inefficient photoisomerization when incorporated into the vesicle (K. Giertz and R. F. Khairutdinov, unpublished observations). In contrast, as reported herein, cis h trans photoisomerization of an amphiphilic azobenzene can effectively modulate K+ permeabilities across DHP vesicle membranes. Expermental Section Preparation of Vesicles. The amphiphilic azobenzene derivative, 4-dodecyl-4′-(3-phosphate-trimethyleneoxy)azobenzene was synthesized with the assistance of Dr. J. M. Kim following general procedures developed elsewhere for similar compounds.15 Briefly, the synthetic route involved the five-step sequence given in Scheme 1.Intermediary products from each step were purified by chromatography on silica gel. For the final product, 90 MHz 1H NMR (CDCl3) gave δ 0.88 (t, 3), 1.26-1.40 (m, 18), 1.70 (m, 2), 2.05 (m, 2), 3.80 (m, 2), 4.02 (t, 2), 4.21 (t, 2), 6.86 (dd, 4), 7.90 (dd, 4). A more detailed description of the synthesis is provided as Supporting Information. The compound 4-hexylstilbene-4′-butyric acid
was a gift from David G. Whitten (Los Alamos National (4) Steinberg-Yfrach, G. S.; Rigaud, J.-L.; Durantini, E. N.; Moore, A. L.; Gust, D.; Moore, T. A. Nature 1998, 392, 479. (5) Patterson, B. C.; Thompson, D. H.; Hurst, J. K. J. Am. Chem. Soc. 1988, 110, 3656. (6) Lymar, S. V.; Hurst, J. K. J. Am. Chem. Soc. 1992, 114, 9498. (7) Lymar, S. V.; Hurst, J. K. J. Phys. Chem. 1994, 98, 989. (8) Humphry-Baker, R.; Thompson, D. H.; Lei, Y.; Hope, M. J.; Hurst, J. K. Langmuir 1991, 7, 2592. (9) Whitten, D. G. Acc. Chem. Res. 1993, 26, 502. (10) Armitage, B.; O’Brien, D. F. J. Am. Chem. Soc. 1992, 114, 7396. (11) Khairutdinov, R. F.; Giertz, K.; Hurst, J. K.; Voloshina, E. N.; Voloshin, N. A.; Minkin, V. I. J. Am. Chem. Soc. 1998, 120, 12707. (12) Durr, H.; Bouas-Lauren, T. H. Photochromism: Molecules and Systems; Elsevier: Amsterdam, The Netherlands, 1990. (13) Proceedings of the 1st International Symposium on Organic Photochromism. Mol. Cryst. Liq. Cryst. A 1994, 246, 1. (14) See, for example: Willner, I. Acc. Chem. Res. 1997, 30, 347. Tsivgoulis, G. M.; Lehn, J.-M. Chem. Eur. J. 1996, 2, 1399. Shinkai, S. In Cation Binding by Macrocycles: Complexation of Cationic Species by Crown Ethers; Inoue, Y., Gokel, G. W., Eds.; Marcel Dekker: New York, 1990; Chapter 9. Xie, S.; Natansohn, A.; Rochon, P. Chem. Mater. 1993, 5, 403. Barzykin, A. V.; Fox, M. A.; Ushakov, E. N.; Stanislavsky, O. B.; Gromov, S. P.; Fedorova, O. A.; Alfimov, M. V. J. Am. Chem. Soc. 1992, 114, 6381. Salbeck, J.; Kommisarov, V. N.; Minkin, V. I.; Daub, J. Angew. Chem., Int. Ed. Engl. 1992, 31, 1498. Liu, Z. F.; Hashimoto, K.; Fujishima, A. Nature 1990, 347, 658. Okahata, Y. Acc. Chem. Res. 1986, 19, 57 and references therein.
Langmuir, Vol. 15, No. 10, 1999 3425 Scheme 1
Laboratory). The trans isomers of the dyes were incorporated into DHP vesicles as follows: 24 mg of solid DHP was dispersed using the microtip probe of a Heat Systems Ultrasonics model W-225 sonicator in 10 mL of 20 mM Tris (Cl), pH 8, by sonication for two 10 min periods with an intervening 5 min cooling period. This preformed DHP vesicle suspension was then added to a beaker containing 1-2 mg of the dye, which had been dispersed as a film on the glass surface by evaporation from warm tetrahydrofuran (azobenzene) or chloroform (stilbene) solutions using a stream of dry argon. Following additional sonication for 10 min, the suspension was filtered through 0.45 µm cellulose acetate membranes and then centrifuged for 90 min at 100 000g,8 and the top translucent layer was taken for subsequent experiments. Prepared in this fashion, the final solution contained a nearly monodisperse ∼1 µM population of small unilamellar DHP vesicles with hydrodynamic radii of 11-12 nm.8 Vesicles containing entrapped potassium ion were prepared by adding 20 mM KCl to the original sonication buffer. External KCl was removed by cation-exchange chromatography using a 10 × 1 cm column containing Chelex-100 resin (Biorad) that had been preequilibrated with Tris buffer. Physical Measurements. Photocontrolled release experiments were conducted by passing light from a medium-pressure Hg arc lamp through water and appropriate band-pass filters and then through the sample. Illumination at 360 nm was used to effect trans f cis photoisomerization of the azobenzene amphiphile, and 450 nm illumination was used for its cis f trans photoisomerization. The trans-stilbene-containing vesicles were illuminated at 315 nm; these suspensions were first deoxygenated by bubbling argon through them for ∼15 min to minimize photooxidation of the isomeric cis-stilbene to phenanthrenes.16 The samples were continuously stirred during illumination using a magnetic bar; the temperature was controlled by placing the reaction cuvette in a thermostated cell holder. Following illumination, the samples were rapidly cooled to room temperature, and the external K+ concentration was determined with a K+ specific-ion electrode connected to an Orion model 701A/digital IONALYZER. The total amount of K+ in the system was then determined by resonicating the vesicle suspension to release entrapped ions and repeating the measurement. The electrode was calibrated using 0.01-1.0 mM solutions of KCl in Tris buffer and suspensions of DHP, t-azo/DHP, or t-stil/DHP17 that had been passed through the cation-exchange column as reference blanks. Optical spectra were recorded using a Hewlett-Packard 8452A diode array spectrophotometer. The reference cuvette contained undoped vesicles at approximately the same concentration as those in the sample cuvette to correct for any scattering by the particles; in general, however, scattering by small unilamellar DHP vesicles prepared by our methods8 is negligible at wavelengths longer than 260 nm. DSC thermograms were obtained using a Perkin-Elmer model DSC 7 differential scanning calorimeter as previously described.8 A Princeton Applied Research model 273 potentiostat/galvanostat was used to provide (15) Kim, J. M. Ph.D. Dissertation, Kyushu University, Kyushu, Japan, 1990. (16) Mallory, F. B.; Wood, C. S.; Gordon, J. T. J. Am. Chem. Soc. 1964, 86, 3094. (17) The notations t-azo/DHP, c-azo/DHP, and t-stil/DHP are used to indicate DHP vesicles doped with the trans-azobenzene, cisazobenzene, and trans-stilbene compounds, respectively.
3426 Langmuir, Vol. 15, No. 10, 1999
Figure 2. Absorption spectra of t-azo/DHP vesicle suspensions at different dopant levels. Conditions: [vesicles] = 1 µM in 20 mM Tris (Cl), pH 8.0, containing 1.75 (a), 3.5 (b), or 5.5 mol % trans-azobenzene (c). electrochemical potentials for the cyclic voltammetric and spectroelectrochemical measurements; cell construction and experimental designs were as previously described.18 Luminescence spectra of samples in 4 mm rectangular fluorescence cells thermostated at 25 °C were recorded using a Perkin-Elmer MPF66 fluorescence spectrophotometer; excitation was at 310 nm with both entrance and exit slits at 0.5 nm.
Results and Discussion Morphology of the Azobenzene within DHP Vesicles. Azobenzene-containing surfactants that selfassemble to form supramolecular structures with two- or three-dimensional order often exhibit spectroscopic properties which indicate extensive aggregation of the chromophore. Both head-to-head (H-type) and head-totail (J-type) aggregates19,20 have been observed;20-23 however, when present in multicomponent bilayers, the azobenzene units usually form H aggregates or are molecularly dispersed.21,23 As described in the following paragraph, the state of aggregation of the DHP-bound azo amphiphile in both its cis and trans isomeric states was examined using primarily spectroscopic techniques. All of the derivatized azobenzene in an azo/DHP vesicle preparation coeluted with vesicles on Sephadex G-50 gel filtration columns, indicating that the dye was completely bound. In contrast, analogous preparations utilizing unsubstituted azobenzene in place of the surfactant dye gave no incorporation by the vesicles. As illustrated in Figure 2, the optical spectra of the t-azo/DHP vesicles showed two prominent absorption bands whose shapes, wavelength maxima, and relative intensities were characteristic of the monomeric trans-azobenzene chromophore in nonpolar solvents.9 Had the dye formed H aggregates, the major π-π transition at 358 nm would have been shifted to higher energies by as much as 50 nm; conversely, J aggregation would have caused the bands to red shift.20 The spectra were independent of dopant levels over azobenzene/DHP molar ratios ranging from 0.017 to 0.055, for which the relative band intensities were constant, with a 358/248 nm absorbance ratio of 1.75 ( 0.05. The spectra were also independent of temperature over the range 2560 °C. Photoexcitation at 360 nm caused immediate and (18) Lei, Y.; Hurst, J. K. J. Phys. Chem. 1991, 95, 7918. (19) Kasha, M.; Rawls, H. R.; El-Bayoumi, M. A. Pure Appl. Chem. 1965, 11, 271. (20) Shimomura, M.; Ando, R.; Kunitake, T. Ber. Bunsen-Ges. Phys. Chem. 1983, 87, 1134. (21) Shimomura, M.; Kunitake, T. J. Am. Chem. Soc. 1987, 109, 5175. (22) Kawai, T.; Umemura, J.; Takenaka, T. Langmuir 1989, 5, 1378. (23) Song, X.; Perlstein, J.; Whitten, D. G. J. Am. Chem. Soc. 1997, 119, 9144.
Lei and Hurst
Figure 3. Spectral changes following illumination of 1 µM suspensions of 5.5 mol % t-azo/DHP vesicles at 360 nm for 5 (a), 10 (b), 15 (c), and 30 s (d). Arrows indicate loss of the major band associated with the trans isomer and appearance of bands attributable to the cis isomer.
nearly complete trans f cis conversion (Figure 3), which was completely reversible upon excitation at 460 nm. This trans h cis isomeric cyclization was repeated up to 10 times without any evidence of photodegradation of the dye or other spectral alterations or of development of hysteretic response to the photostimulating light.24 As with the t-azo/DHP vesicles, the absorption spectra of the c-azo/DHP vesicles were independent of dopant mole fraction and temperature over the entire range investigated. The t-azo/DHP vesicles gave no detectable fluorescence emission. DSC thermograms closely resembled those previously obtained for pure small unilamellar DHP vesicles in Tris (Cl), which comprised a broad endothermic band spanning 45-65° with peaks at ∼46 and ∼64 °C;8 no additional peaks that might be attributed to domain formation were detected. All of these featuress concentration and temperature-independent band shapes and energies, rapid reversible photoisomerization, absence of fluorescence emission, and unaltered phase transition enthalpiessare consistent with the azobenzene being molecularly dispersed within the DHP bilayer membrane.21 Potassium Ion Release Studies. Sonication of K+loaded vesicles caused release of the ion to the bulk aqueous medium; maximal concentration levels attained in the external medium of suspensions containing 1 µM vesicles were 190 ( 10 µM. Because the occluded volume at these vesicle concentrations was ∼0.1% of the total volume,8 the effective internal concentration before release, and therefore the initial transmembrane concentration gradient, was ∼190 mM. Diffusion of entrapped K+ from DHP vesicles was extremely slow. Typical thermal leak rates from DHP were ∼1% and ∼3% of the total occluded K+ after 60 min at 25 and 40 °C, respectively. Neither broad band nor 360 nm illumination had any effect upon the K+ release rate. These leak rates correspond to fluxes (J) across the DHP bilayer of ∼1 × 10-16 mol/cm2‚s at 25 °C and 4 × 10-16 mol/cm2‚s at 40 °C, which were calculated assuming a total inner membrane surface of ∼4 × 103 cm2/mL for a 1 µM suspension of vesicles.25 Corresponding values for the permeability coefficient (P), defined as P ) J/∆c, were calculated using 190 mM as the (24) In tetrahydrofuran with DHP absent, the azobenzene underwent rapid photodegradation. Reversible trans-cis photoisomerization was observed; however, after several cycles, photobleaching of the chromophore was evident and the solution became turbid.
Photoregulated Transmembrane K+ Diffusion
Figure 4. Cumulative release of K+ from vesicles accompanying reversible trans f cis photocycling. Conditions: 1 µM suspensions of 5.5 mol % t-azo/DHP vesicles illuminated alternately for 10 min intervals at 360 nm (solid line) and 450 nm (dashed line). Solid circles: freshly prepared sample at 41 °C. Open circles: sample incubated at 23 °C for 6 h prior to photolysis at 38 °C. The difference in initial K+ concentrations reflects thermal leakage during the incubation period.
K+ concentration gradient (∆c); values obtained were P ) ∼0.7 × 10-12 cm/s and ∼2 × 10-12 cm/s at the two temperatures. The membrane permeability first decreased and then increased as increasing amounts of the transazobenzene surfactant were added to the vesicle; P values obtained at 25 °C for 0.0175, 0.0375, and 0.055 mole fraction azobenzene surfactants were ∼0.2 × 10-12, ∼0.7 × 10-12, and ∼2 × 10-12 cm/s, respectively. Above ∼45 °C (the onset of the major transition), the vesicles containing entrapped K+ ion were unstable. Upon heating, the turbidity of the suspensions rapidly increased, ultimately giving rise to extensive flocculation of the DHP. In contrast, vesicles containing the trans-azobenzene surfactant, but without entrapped K+, retained their structural integrity in their membrane liquid-crystalline phase for at least several hours. The instability of the K+-loaded vesicles precluded their use in photochemical experiments at temperatures above ∼45 °C. Photoregulated Potassium Ion Release. At room temperature, the transmembrane K+ diffusion rate was only slightly enhanced by photoconversion of the dye to its cis form. However, at temperatures approaching the phase transition, K+ release was accelerated severalfold. This effect is depicted in Figure 4, where reversible switching between t-azo/DHP and c-azo/DHP conformers leads to alternately increasing and decreasing K+ diffusion rates. In Figure 4, the lower trace shows the response of vesicles immediately after preparation, and the upper trace is for vesicles that had been held for 6 h at 25 °C; the leak rates appear to be unaffected by this aging process.26 Permeability coefficients were estimated for vesicles containing the cis and trans forms of the dye from the accumulated amount of K+ released during the periods of illumination at 360 and 450 nm, respectively (Figure 4). For t-azo/DHP, P ∼ 4 × 10-12 cm/s, and for c-azo/DHP, (25) The vesicles are approximately spherical with a radius for the inner aqueous core of 7-8 nm.8 This gives an inner surface area of (6-8) × 10-12 cm2/vesicle or a total inner surface area of (3.7-4.8) × 103 cm2/mL for 6 × 1014 vesicles/mL. (26) In both aqueous solution and the DHP membranes, the cis isomer underwent slow thermal isomerization to the trans isomer (t1/2 ) 2-3 min at 25 °C). Consequently, in these experiments, the vesicles were illuminated continuously during the 10 min illumination cycles to maintain photostationary states in the vesicles that comprised nearly pure cis or trans isomers.
Langmuir, Vol. 15, No. 10, 1999 3427
P ∼ 2 × 10-11 cm/s, indicating that trans f cis photoisomerization caused a 5-fold increase in transmembrane diffusion of K+ at ∼40 °C. Redox Properties of the Dye. In protic solvents, simple azobenzenes undergo two-electron reduction to the corresponding hydrazobenzenes; because two protons are taken up in the process, the reduction potentials are pHdependent.27 In aprotic media, one-electron reduction to the corresponding radical is observed.28 Cyclic voltammetry of trans-azobenzene in 9/1 (v/v) ethanol/aqueous 20 mM Tris (pH 8) gave a single reduction wave at -0.54 V with reoxidation at -0.24 V (all potentials vs NHE), similar to previously reported values in pure ethanol.27 The ∼300 mV peak separation between the reduction and oxidation waves reflects a complex reaction sequence involving stepwise electrochemical and chemical processes. The corresponding reduction wave for the azobenzene surfactant was -0.34 V in the aqueous/ethanol solution and -0.45 V in 20 mM Tris (pH 8) when deposited as a Langmuir-Blodgett monolayer film on an In-doped SnO2 working electrode.29 As expected, aqueous suspensions of t-azo/DHP vesicles gave no voltammetric waves, presumably because interfacial electron transfer to the vesicles was slow. Reduction potentials for the vesiclebound azobenzene were therefore obtained by spectroelectrochemical measurements using optically transparent thin-layer electrodes.18 In the absence of redox mediators,30 no reduction of the azobenzene was observed at potentials as low as -0.66 V; however, when either ∼10 µM methyl viologen (E°(MV2+/+) ) -0.44 V) or benzyl viologen (E°(BV2+/+) ) -0.36 V) was present in solution, azobenzene reduction was observed at E < -0.44 V and proceeded to completion within 1 h. In these experiments, 1 µM suspensions of 0.055 dopant mole fraction t-azo/DHP vesicles were used, for which [azobenzene] ∼ 240 µM; consequently, the added viologens were clearly functioning as cyclic redox mediators between the electrode and vesicles. The reduced dye was slowly reoxidized in ∼80% yield to the azobenzene at -0.16 V but was not oxidized at -0.26 V, indicating that reduction to hydrazobenzenes in the vesicles was chemically reversible but proceeded by electrochemically irreversible mechanisms. Viologens are potential candidates for use in constructing artificial ion-gated electron transport systems (e.g., Figure 1) because electrogenic transmembrane redox pathways can be selected by appropriate structural modifications31 and/or manipulation of the overall redox poise of the system.6,7 One problem apparent from the spectroelectrochemical studies is that the viologen radical cations are sufficiently reducing to cross-react with the azobenzene chromophore. Correspondingly, addition of viologen radicals formed by reduction of methyl viologen or the N-hexyl-N′-methyl-4,4′-bipyridinium (E°(C6MV2+/+) ) -0.45 V) or N-hexadecyl-N′-methyl-4,4′-bipyridinium (E°(C16MV2+/+) ) -0.51 V) analogues gave immediate decolorization of the azobenzene surfactant-doped vesicles. (Potentials cited are for one-electron reduction of DHPbound monoalkyl viologens.)18 Efforts to circumvent this problem are currently directed at developing electrogenic transmembrane pathways using less reducing electron carriers and strongly oxidizing electron acceptors within the inner aqueous core. Photoregulated Release by an Amphiphilic Stilbene Fatty Acid. Rates of K+ release from DHP vesicles (27) Lavrion, E.; Mugnier, Y. J. Electroanal. Chem. 1980, 111, 337. (28) Lavrion, E.; Mugnier, Y. J. Electroanal. Chem. 1978, 93, 69. (29) Liu, Z.-F.; Hashimoto, K.; Fujishima, A. Chem. Lett. 1990, 2177. (30) Dutton, P. L. Methods Enzymol. 1978, 54, 411. (31) Patterson, B. C.; Hurst, J. K. J. Phys. Chem. 1993, 97, 454.
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that had been doped with 4-hexylstilbene-4′-butyric acid were also briefly examined. Whitten and co-workers have deduced from photophysical studies that this compound intercalates within the bilayer structure of vesicles (including DHP), which would place the stilbene unit within the hydrocarbon phase aligned collinearly with the n-alkane chains of the vesicle-forming surfactant;32 this general conclusion is supported by bromination kinetics studies, although the specific measurements with DHP were done using vesicles whose membranes had apparently been breached.33 In organized films prepared from stilbene-containing amphiphiles, the stilbene unit tends to form H aggregates;34-38 however, when highly diluted in most vesicles, the dopant appears to be monomerically dispersed.32 For our studies, spectra of the stilbene derivative in DHP vesicles were very similar to spectra of the monomer obtained in nonpolar solvents,34 exhibiting two vibronically structured absorption bands with maxima at 317 and 236 nm and strong fluorescence emission at 364-370 nm. The absorption at the maximum followed the Lambert-Beer law over the experimental range of dopant concentrations used (stilbene/DHP molar ratio ) 0.012-0.060). These spectral features are quite distinct from stilbene H aggregates, which typically exhibit 40-50 nm blue-shifted absorption spectra and substantially red-shifted fluorescence bands.34,35,37,38 Taken alone, these data would suggest that the stilbene compound is monomerically dispersed in the DHP vesicles. However, the fluorescence intensity decreased with increasing dopant mole fractions and was accompanied by a progressive red shift in the position of maximum emission and changing relative intensities of the individual vibronic bands; at the highest loadings, small changes were also observed in the shape of the 317 nm absorption band. This behavior might be attributable to a form of aggregation other than H aggregation or, more likely, to heterogeneity in the location of intravesicular binding sites, e.g., surface-exposed vs interior sites.32,33 Thermal leakage of K+ from DHP vesicles containing 0.055 mole fraction of the trans-stilbene compound and 350 mM internal K+ ion was also slow, with a calculated permeability coefficient at 38 °C of P ∼ 1.5 × 10-11 cm/s. Photoirradiation of deoxygenated suspensions at 315 nm resulted in ∼80% conversion to the cis isomer in the photostationary state, accompanied by a 2-fold increase in K+ permeability, i.e., P ∼ 2.5 × 10-11 cm/s. As with t-azo/DHP, vesicle integrity was maintained over the course of the experiments, as indicated by the absence of increased turbidity in the suspensions. General Observations. The primary objective of the present studies was to evaluate the capacity for modulation of transmembrane electrolyte leakage across DHP bilayers by reversible cis f trans photoisomerization. The amphiphilic compounds used were chosen because prior physical studies in other self-assembling films suggested that the chromophores would be located within the hydrocarbon phase, hence, optimally positioned to disrupt the local bilayer structure. Indeed, the physical properties (32) Suddaby, B. R.; Brown, P. E.; Russell, J. C.; Whitten, D. G. J. Am. Chem. Soc. 1985, 107, 5609. (33) Mizutani, T.; Whitten, D. G. J. Am. Chem. Soc. 1985, 107, 3621. (34) Mooney, W. F.; Brown, P. E.; Russell, J. C.; Costa, S. B.; Pedersen, L. G.; Whitten, D. G. J. Am. Chem. Soc. 1984, 106, 5659. (35) Furman, I.; Geiger, H. C.; Whitten, D. G.; Penner, T. L.; Ulman, A. Langmuir 1994, 10, 837. (36) Spooner, S. P.; Whitten, D. G. J. Am. Chem. Soc. 1994, 116, 1240. (37) Song, X.; Geiger, C.; Furman, I.; Whitten, D. G. J. Am. Chem. Soc. 1994, 116, 4103. (38) Song, X.; Geiger, C.; Leinhos, U.; Perlstein, J.; Whitten, D. G. J. Am. Chem. Soc. 1994, 116, 10340.
Lei and Hurst
of the DHP-doped membranes described above are consistent with both surfactant probes being intercalated within the bilayer structure of the membrane. The ∼5fold difference in K+ leak rates for c-azo/DMP and t-azo/ DMP achieved in these studies (Figure 4) is comparable to maximal differences for solute and ion difffusion for cis- and trans-isomeric forms of azobenzene-derivatized materials reported for other systems where catastrophic destruction of the membrane was avoided.39-42 Although maximum achievable cis/trans diffusion rate ratios are remarkably similar in various membrane assemblies, the actual conditions under which these are attained vary widely. For example, it was found using ternary composite membranes containing an azobenzenedoped dialkyldimethylammonium bilayer that K+ leak rates for cis and trans isomers were indistinguishable when the membrane was in its solid (gel) phase but that substantive differences were seen when the membrane was in its liquid (liquid-crystalline) phase;41 in contrast, cis-trans discrimination for solute diffusion was greater in the gel phase than in liquid phase for liposomes constructed from very similar membranes.39 In our case, discrimination was observed in the gel phase, but only at temperatures very near the DHP gel f liquid-crystalline phase transition. Solute and ion diffusion rates through phosphatidylcholine liposomes are often maximal at the phase transition or in the prephase transition region,43-45 which has been associated with maximal fluxional disorder;45 whether this behavior describes K+ release from DHP vesicles could not be ascertained, because the K+loaded vesicles were unstable in their liquid-crystalline phase. Multicomponent phosphatidylcholine (PC) liposomes containing a molecularly dispersed analogue possessing azobenzene units within their hydrocarbon chains exhibited negligible release of encapsulated carboxyfluorescein (CF) with either cis- or trans-isomeric forms when the membrane was in its gel phase but slow preferential release of CF in liquid-crystalline membranes when the dopant was in its cis conformation.23 Thus, the photoresponse of individual systems is highly variable and must depend on subtle molecular interactions within the supramolecular assemblies. Much greater discrimination in diffusion rates accompanying trans f cis photoisomerization have been reported for PC liposomes containing photosensitive units in their hydrocarbon chains.23,46,47 The most thoroughly characterized of these has involved the azobenzenemodified phosphatidylcholine analogue mentioned above. When the analogue was aggregated and the host PC was in its gel phase, illumination led to immediate release of all of the occluded CF.23 This release, however, was accompanied by destruction of the vesicles. Similarly, partial rapid release of calcein from a multicomponent liposome containing a structurally similar azobenzene analogue was observed upon trans f cis photoisomer(39) Kano, K.; Tanaka, Y.; Ogawa, T.; Shimomura, M.; Kunitake, T. Photochem. Photobiol. 1981, 34, 323. (40) Okahata, Y.; Lim, H.-J.; Hachiya, S. J. Chem. Soc., Perkin Trans. 2 1984, 989. (41) Kumano, A.; Niwa, O.; Kajiyama, T.; Takayanagi, M.; Kunitake, T.; Kano, K. Polym. J. 1984, 16, 461. (42) Aoyama, M.; Watanabe, J.; Inoue, S. J. Am. Chem. Soc. 1990, 112, 5542. (43) Blok, M. C.; van Deenen, L. L. M.; De Gier, J. Biochim. Biophys. Acta 1976, 433, 1. (44) Braganza, L. F.; Blott, B. H.; Coe, T. J.; Melville, D. Biochim. Biophys. Acta 1983, 731, 137. (45) Georgallas, A.; MacArthur, J. D.; Ma, X.-P.; Nguyen, C. V.; Palmer, G. R.; Singer, M. A.; Tse, M. Y. J. Chem. Phys. 1987, 86, 7218. (46) Pidgeon, C.; Hunt, C. A. Photochem. Photobiol. 1983, 37, 491. (47) Morgan, C. G.; Thomas, E. W.; Sandhu, S. S.; Yianni, Y. P.; Mitchell, A. C. Biochim. Biophys. Acta 1987, 903, 504.
Photoregulated Transmembrane K+ Diffusion
Langmuir, Vol. 15, No. 10, 1999 3429
Acknowledgment. The authors thank David G. Whitten, Los Alamos National Laboratory, for his generous gift of the amphiphilic stilbene compound used in these studies and Dr. J. M. Kim for his assistance in the synthesis of the amphiphilic azobenzene compound. Funding for this research was provided by the Division of Chemical Sciences, Office of Basic Energy Sciences, U.S. Department of Energy, under Grant DE-FG06-95ER14851.
ization, but this appeared to be a consequence of vesicle fusion.47 In the present studies, care was given to maintaining the photoisomerizable dopants in their molecularly dispersed forms to maintain vesicle integrity, which is essential to maintaining reversibility. Because it appears that substantive increases in cis/trans discrimination in electrolyte leak rates will not be easily achieved in these systems, we have turned our attentions to surfactants containing photochromic units which undergo reversible ring opening-closing reactions and for which we anticipate larger volume changes upon isomerization and,48 hence, larger perturbation of the membrane structure.
Supporting Information Available: A description of the synthesis of 4-dodecyl-4′(3-phosphate-trimethylene-oxy)azobenzene. See any current masthead page for ordering information and WEB access instructions.
(48) Holden, D. A.; Ringsdorf, H.; Deblauwe, V.; Smets, G. J. Phys. Chem. 1984, 88, 716.
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