Cyclic Transmembrane Charge Transport Mediated by Low-Potential

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Cyclic Transmembrane Charge Transport Mediated by Low-Potential Pyrylium Ions Song Liang, Linyong Zhu,† and James K. Hurst* Department of Chemistry, Washington State University, Pullman, Washington 99164-4630, United States S Supporting Information *

ABSTRACT: We have investigated the capacity of a series of Ndialkylaminophenyl-substituted pyrylium and thiopyrylium ions to act as photosensitizers and redox mediators between reactants separated by bilayer membranes. These studies were prompted by earlier results indicating that simple trimethy- and triphenyl-substituted analogues could promote efficient photosensitized transmembrane redox between vectorially organized reactants by an electroneutral e−/OH− antiport mechanism. Unlike the dyes used in the earlier studies, the ions investigated herein absorb strongly throughout the visible absorption region and are therefore potentially useful in solar photoconversion processes. We demonstrate that these ions can carry out cyclic electron transport between phase-separated electron donors and occluded Co(bpy)33+ in several transversely organized vesicles. The quantum yields obtained were relatively low, but were independent of the membrane microviscosity, suggesting that transmembrane diffusion was not rate-limiting. Triphenylpyrylium and triphenylthiopyrylium ions were shown to be capable of acting as combined photosensitizers/redox relays, apparently by direct oxidation of either solvent (water) or buffer (acetate) ions from their triplet-excited state. These reactions did not require addition of separate photosensitizers and electron donors; as such, they represent a minimal photochemical scheme for effecting transmembrane charge separation. The low-potential visible-absorbing pyrylium ions were unable to function in this dual capacity, consistent with thermodynamic limitations. However, redox titrations established that the pyranyl radicals of these dyes should be capable of reducing H+ to H2 in weakly acidic solutions. Consistent with their strongly reducing nature, these dyes were shown to be capable of forming methyl viologen radical in photoinitiated transmembrane redox reactions.

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INTRODUCTION Closed vesicles formed by self-assembly of surfactant molecules and ions can be effective mesoscopic constructs for controlling complex sequences of reactions.1,2 For example, microphase partitioning can minimize dissipative recombination reactions of immediate products formed in chemical or photochemical redox reactions,3,4 thereby allowing their efficient coupling to following reactions that are isolated in phase-separated compartments. As such, organized systems based upon vesicles have found interest among researchers concerned with direct solar photoconversion processes. Many of these assemblies duplicate fundamental aspects of chemiosmotic coupling found in biological energy-transducing organelles, which store photonic and chemical energy as transmembrane electrochemical gradients that are then used to drive secondary processes using other membrane-associated protein complexes. Both electrogenic, that is, charge separating, and electroneutral transmembrane reactions are involved in these processes, and artificial biomimetic systems have been constructed that utilized either or both of them to carry out similar transmembrane redox reactions.5,6 We have been interested in developing vesicle-based assemblies for catalytic solar photolysis of water, that is, 2H2O → 2H2 + O2, which employ diffusible redox carriers capable of cycling across bilayer membranes as uncharged © 2012 American Chemical Society

molecules in both of their oxidation states. This type of assembly seems inherently more suited to high-flux applications such as generation of fuels than designs based upon oriented electron transport chains because, unlike vectorial electron transport, their redox cycling between membrane-separated aqueous phases does not generate a rate-retarding transmembrane potential that must be dissipated by chargecompensating transmembrane ion diffusion. Moreover, the inherent need to dissipate energy to ensure directionality at each of the discrete electron-hopping steps comprising electron transport severely constrains the distance over which charge can be efficiently transported. Correspondingly, the photosynthetic electron transport chain appears to have evolved in a manner that allows optimal capture of solar energy via charge separation across a fluid bilayer membrane whose barrier width is ∼40 Å. This dimensional constraint may be part of what has been termed “legacy biochemistry”;7 that is, the intrinsic width of biological membranes was presumably established before the evolution of transmembrane electron transport protein complexes within them. In any event, the same fundamental bioenergetic constraints apply to covalently linked redox Received: April 24, 2012 Revised: July 17, 2012 Published: July 20, 2012 12171

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overall reaction cycle (Scheme 1). Moreover, the implied capacity to oxidize water without added catalysts is novel and potentially transforming, given the intensity of current efforts to find suitable catalysts for this reaction. The pyrylium ions used in our initial studies absorb appreciably only in the near-ultraviolet region, which renders them unsuitable for application to solar photoconversion processes. The phenyl-substituted pyranyl analogues are also only weakly reducing, and therefore could not drive production of solar fuels. In the present study, we have investigated the properties of a series of low-potential triphenylpyrylium analogues that absorb strongly in the visible region with the intent of assessing their suitability as combined photosensitizers/redox shuttles in membrane assemblies that could potentially be adapted to solar-driven water photolysis.

centers that form artificial electron transport chains such that transfer of charge becomes increasingly inefficient energetically with increasing distance of separation. In principle, this dimensional limitation does not exist for diffusion-based carrier-mediated redox systems, which might offer advantage in allowing consideration of a wider range of materials for membrane barriers. Our recent efforts have focused on the use of pyrylium ions as transmembrane redox carriers in photoinitiated reactions that lead to charge separation across membrane bilayers.8 As illustrated in Scheme 1, these ions are capable of functioning as Scheme 1. Reaction Schemes for Pyrylium-Mediated Photosensitized Transmembrane Redox by an Electroneutral e−/OH− Antiport Mechanisma

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EXPERIMENTAL SECTION

Materials. The pyrylium compounds, 4-(4′-diethylaminophenyl)2,6-diphenylpyrylium (DEAPφ2Py+) fluoroborate,9 4-(4′-diethylaminophenyl)-2,6-diphenylthiopyrylium (DEAPφ2PyS+) fluoroborate,9 2,6-di-tert-butyl-4-(4′-dimethylamino)-phenyl)pyrylium (DMAP(tBu) 2 Py + ) perchlorate, 10 2,6-di-tert-butyl-4-(4′-dimethylamino)phenyl)thiopyrylium (DMAP(t-Bu)2PyS+) perchlorate,10 and 2,6-bis(4′-dimethylamino)-phenyl)-4-phenylpyrylium ((DMAP)2φPy+) perchlorate,11 and the doubly o-xylyl-bridged cofacial diviologen, tetrahydro-6,9:10,13:18,21,22,1-tetraethenodibenzo-c,o-1,6,13,18-tetraazacyclotetracosinetetraium (DV4+) hexafluorophosphate,12 were synthesized following procedures described in the literature. All synthesized compounds gave 1 H NMR spectra and optical spectroscopic parameters that were in agreement with published values.9−13 Natural phosphatidylcholine (EPC) was extracted from fresh hen’s egg yolk and purified by column chromatography following published procedures.14 Other phospholipid surfactants and cholesterol were obtained from Avanti Polar Lipids. N,N′-Dimethylacetate4,4′-bipyridinium (DMAV2+) dibromide and N,N′-diacetonitrile-4,4′bipyridinium (DANV2+) dibromide were kindly donated by Dr. Brian Gregg (National Renewal Energy Laboratory). All other chemicals, including methyl viologen (N,N′-dimethyl-4,4′-bipyridinium (MV2+)) and benzyl viologen (N,N′-dibenzyl-4,4′-bipyridinium (BV2+)) dichlorides, were best available grade from commercial suppliers and used as received. In-house deionized water was further purified using a Milli-Q ion exchange/reverse osmosis system. Vesicle Preparation. To form asymmetrically organized liposomal systems, phosphatidyl surfactants, with or without cholesterol, were first dissolved in CHCl3 and rotoevaporated in a round-bottom flask to form a surfactant surface film. The film was detached and hydrated by introduction of aqueous buffer (40 mM Tris/Cl, pH 8.0, or 40 mM sodium acetate, pH 5.0) followed by repeated freeze−thaw recycling. Unilamellar vesicles with average diameters of ∼100 nm were then prepared from the mixture by high-pressure extrusion through tracketched micropore filters.15 Electron acceptors were introduced into the aqueous core by dissolving either Co(bpy)3(ClO4)3 (1 mM) or viologen dichlorides (0.1 mM) in the aqueous buffers prior to vesicle formation, after which the external cations were removed by chromatography of the suspensions on a Bio-Rad Chelex-100 column. Pyrylium and thiopyrylium salts, photosensitizers, and electron donors were then added to the external medium to complete the asymmetric assembly. Small unilamellar dihexadecylphosphate (DHP) vesicles incorporating occluded Co(bpy)33+ were prepared by sonication/ chromatography as described in detail elsewhere.6,16 To facilitate their formation, these vesicles were prepared in Tris buffers at pH 8; where more acidic reaction environments were desired, the final pH was adjusted by adding acetic acid (HOAc) to the suspending medium. Because HOAc is membrane-permeable, rapid pH equilibration of the aqueous core is expected to occur. The extent of binding of the pyrylium and thiopyrylium ions to preformed PC liposomes and DHP vesicles was determined by membrane ultrafiltration using 30 000 MW cutoff (Centricon-30) centrifugal microconcentrators.17 Vesicle

a

(Left) Zinc porphyrin-photosensitized transmembrane reduction of an occluded electron acceptor (A) by an external electron donor (D).8 The reaction cycle involves: (1) sensitizer photoexcitation (ZnTPPS4− = [5,10,15,20-(4-sulfonatophenyl)porphinato]zinc(II)); (2) oxidative quenching of the triplet-excited sensitizer, generating a porphyrin πcation and neutral pyranyl radical; (3) transmembrane diffusion of the radical and one-electron reduction of the electron acceptor (A); (4) addition of OH− to generate an uncharged pseudobase (shown here as the 1,5-diketone tautomer); (5) outward diffusion of the pseudobase; and (6) OH− release to the medium, re-forming the pyrylium cation. The oxidative half-cycle is closed by reaction of the zinc porphyrin πcation with the donor (D). (Right) Oxidative quenching of the triplet photoexcited pyrylium ion by solvent or acetate ion8 (S) leads to formation of the pyranyl radical, which carries out the same transmembrane redox cycle as described on the left (shown here in greater detail). In this case, neither an independent photosensitizer nor a sacrificial electron donor is required for the reaction to proceed. The driving force for transmembrane redox derives from the inherent asymmetry of the assembly, where the electron acceptor is confined to the inner aqueous compartment and membrane-impermeable electron donors and sensitizers are located in the external medium.

cyclical carriers via ring-opening pseudobase equilibration in their oxidized forms. In prior research, we demonstrated that 2,4,6-trimethylpyrylium and 2,4,6-triphenylpyrylium ions and their thiopyrylium analogs are effective oxidative quenchers of triplet-photoexcited Zn porphyrins, generating in the process the corresponding neutral pyranyl radicals, which then reduce occluded electron acceptors on time scales associated with transmembrane diffusion. It was also noted in these studies that triplet-excited states of the pyrylium ions were quenched by water or acetate ion, forming long-lived transients with absorption bands characteristic of pyranyl radicals. Decay of these species was accelerated in the presence of dihexadecylphosphate (DHP) vesicles that contained occluded Co(bpy)33+ (bpy = 2,2′-bipyridine).8 Collectively, these results imply that the photoexcited pyrylium ions might be capable of direct photosensitized oxidation of water to give the membranetraversing pyranyl radicals. If so, this dual capability as combined photosensitizer/transmembrane redox shuttle is very appealing because it obviates the need for additional photosensitizers, eliminating two of the redox steps in the 12172

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viologen2+/+ potentials were corrected to reference against NHE by adding 0.51 V to the experimentally determined values. Relative one-electron reduction potentials for the pyrylium ions and viologens in vesicle-containing media were determined by anaerobically mixing preformed solutions of the viologen radical cations with pyrylium-doped vesicle suspensions and determining by optical spectrophotometry the composition of the product solutions. These studies made use of a double-chambered 1 cm path length optical cuvette whose outer chamber was purged of O2 prior to introducing the vesicle suspensions; control studies using undoped vesicles established that adventitious O2 introduced during syringe transfer of the vesicles was negligible. The viologen radical cations were formed by stoichiometric reduction of the corresponding dications with sodium dithionite and were stable over the course of the titration experiments. In practice, only MV•+ was found to react with the pyrylium ions; the equilibrium constants for its reactions were determined by decreases in the MV•+ absorption band at 398 nm6 following addition of the vesicle suspensions assuming 1:1 reaction stoichiometry, that is, Py+ + MV•+ → Py• + MV2+. The pyrylium ion potential was then calculated from the measured equilibrium constant and the known values for the methyl viologen reduction potentials in the vesicle-containing media.20

integrity was verified by dynamic light scattering measurements made with a Coulter N4 Plus Submicrometer Particle Sizer; SDP analysis of the autocorrelation decay curves indicated formation of narrowly distributed unimodal populations of vesicles whose average diameters for PC liposomes were ∼125 nm; diameters of DHP vesicles formed by ultrasonication are typically ∼25 nm.16 The extent of lipid peroxidation in EPC vesicles at various stages of preparation was examined by using standard thiobarbituric acid reactive substances (TBARS) analysis for malondialdehyde. Photochemical Analyses. Continuous photolyses were performed by using filtered light from 1.5 kW or 200 W xenon lamps essentially as previously described.17 For experiments utilizing ZnTPPS4− as photosensitizer, a 420 nm interference filter was placed in the optical path to limit illumination to the Soret band; absolute quantum yields were measured at a light intensity of ∼2.5 × 10−9 einstein/s, determined using a calibrated bolometer. For experiments utilizing pyrylium ions as photosensitizers, aqueous CuSO4 and Schott GG 455 filters were used to provide 450 nm < λ < 650 nm broadband illumination at (4−5.5) × 10−8 einstein/s. In these instances, chemical actinometry using ferrioxalate was used to estimate the light intensity. The extent of reduction of Co(bpy)33+ was determined spectrophotometrically at 320 nm using Δε320 = 2.85 × 104 M−1 cm−1 for the Co(bpy)33+−Co(bpy)32+ difference extinction coefficient.6 The amounts of accumulated DV3+ and MV•+ radical cations were similarly determined at 632 nm using ε632 = 1.48 × 104 M−1 cm−1 and at 398 nm using ε398 = 4.33 × 104 M−1 cm−1.6,13 According to Scheme 1, the vesicle interior is expected to acidify as OH− is transported outwardly during redox cycling of the mediator. The buffering capacity of the medium is sufficient to prevent buildup of a large pH gradient in experiments utilizing PC liposomes, where the extent of Co(bpy)33+ loading is relatively low and the occluded volume is relatively large. However, under the prevailing reaction conditions, the internal volume of the DHP vesicle suspensions is ∼0.1% of the total volume,16 so that the internal concentration of occluded Co(bpy)33+ is ∼103-fold greater than its apparent analytical concentration (maximally, ∼40 μM). Because these reactions were routinely run in ∼40 mM acetate ion (e.g., Figures S2,S3), the buffering capacity of the medium may have been exceeded in the final reaction stages. Earlier studies have indicated that passive diffusion of protons across DHP bilayers is relatively slow and probably limited by charge-compensating movement of other ions, that is, dependent upon the medium composition.8 Nonetheless, in the present studies, reduction of occluded Co(bpy)33+ under continuous photolysis went to completion, and the photoreaction profiles gave no evidence of ratemodulating effects arising from developing transmembrane electrochemical gradients (Figure S3). Equilibrium Constants for Pseudobase Formation. Pyrylium ring-opening−closing reactions (Scheme 1) were monitored spectrophotometrically as previously described;8 equilibrium constants, expressed as acid dissociation constants for the reactions Py+ + H2O → PyOH + H+, were estimated from the pH-dependent loss of absorption in the characteristic visible band of the cation assuming a simple two-species equilibrium. Note, however, that the pseudobases can exist in several tautomeric forms, including ring-closed OH adducts and ring-open ketohydroxy and diketo structures. As such, the reported equilibrium constants are operationally defined and are not necessarily true ionization constants.18 Electrochemical Analyses. Cyclic voltammograms were obtained in dry acetonitrile containing 0.1 M tetrabutylammonium tetrafluoroborate by using a PAR model 273 potentiostat/galvanostat. Measurements were made in a standard two-compartment, threeelectrode cell consisting of a glassy carbon working electrode, a coiled platinum wire auxiliary electrode, and an Ag/0.1 M AgNO3 reference electrode in DMSO, which was isolated from the reaction chamber by a Vycor frit. Half-cell potentials were measured at a scan rate of 100 mV/s. The reference electrode was calibrated vs NHE by including ferrocene (Fc) as an internal standard in several of the analyte solutions. The Fc+/0 half-cell potential appeared at 0.18 V in these solutions. Because the accepted value for this couple in CH3CN is 0.69 V vs NHE,19 the half-cell potentials for the pyrylium+/0 and

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RESULTS AND DISCUSSION Pyrylium-Sensitized Transmembrane Reduction Reactions. The capacity of simple pyrylium ions to act as combined photosensitizer/redox relays (Scheme 1) was demonstrated in continuous illumination studies using a variety of asymmetrically organized vesicle systems. A typical result is illustrated in Figure 1, where 2,4,6-triphenylthiopyrylium ion (ϕ3PyS+) is used as sensitizer/relay in mixed cholesterol/ dipalmitoylphosphatidylcholine (DPPC) vesicles that contain Co(bpy)33+ as an internal electron acceptor. Under the reaction conditions, complete reduction to Co(bpy)32+ rapidly occurred upon illumination into the pyrylium absorption band, as

Figure 1. Spectral changes upon 300−500 nm broadband illumination of 70/30 mol fraction DPPC/cholesterol vesicles (1.2 mg/mL DPPC and 0.2 mg/mL cholesterol) containing occluded Co(bpy)33+ ion in 40 mM Tris/Cl, pH 8.0, with added 5 μM ϕ3PyS+ and 1.0 mM EDTA. Light from a 200 W Xe source was passed through a CuSO4 solution and blue bandpass filter (Corning 1-64). Spectra were recorded every 30 s during illumination. As shown in the inset, the reaction abruptly ceased upon blocking the light and resumed unabated upon reillumination 20 min later. The bold arrow identifies the spectrum of the dark solution. Spectra of the illumination window and pyrylium absorption bands are shown in Figure S1. The overall initial concentration of Co(bpy)33+, calculated from the total absorption change at 320 nm, is 19 μM. 12173

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shown is the structure of the cofacial diviologen (DV4+) used as an electron acceptor:

indicated by the diagnostic shift in the near-UV optical absorption bands from 320 to 285 nm.6,8 No reaction was observed in the absence of illumination or if the ϕ3PyS+ ion was omitted from the vesicle suspension. Very similar reactions were observed when other vesicles (EPC, dimyristoylphosphatidylcholine (DMPC), dihexadecyl phosphate (DHP)) or the analogous 2,4,6-triphenypyrylium ion (ϕ3Py+) were substituted for their respective components. The reaction could be run in the absence of added electron donors, but was more efficient when EDTA or dithiothreitol were included in the external solution. Leakage of Co(bpy)33+ from DPPC or DHP vesicles was negligible during the several hours time interval associated with sample preparation and illumination, as determined by column chromatography; slow leakage of the acceptor was observed for DMPC vesicles at room temperature, however, which compromised quantitation of the transmembrane redox process for these assemblies. This leakage could be minimized by cooling the vesicle suspensions below the gel−liquid crystalline phase transition (23 °C). Considerably larger amounts of Co(bpy)33+ can be entrapped within DHP vesicles than the PC liposomes because the transition metal cations adsorb strongly to the anionic DHP interface, but not the zwitterionic PC interfaces. Using heavily loaded DHP vesicles, we were able to demonstrate complete reduction of up to ∼40 μM occluded Co(bpy)33+ (expressed as a total solution concentration) with as low as 2 μM ϕ3PyS+, corresponding to an average of ∼20 transmembrane cycles per sensitizer ion. These reactions exhibited slow relaxation phenomena that were not observed in the photochemical reactions involving PC liposomes; that is, slow continued reduction of Co(bpy)33+ was observed following blocking the light path. Illumination was accompanied by partial bleaching of the pyrylium ion, which recovered during the dark period, and then again bleached upon resumption of illumination, which was accompanied by accelerated reduction of the internal electron acceptor. This behavior is illustrated in Figures S2,S3. A plausible interpretation is that the pyranyl radical and/or pseudobasic forms of the pyrylium ion accumulate to relatively high steady-state levels during illumination (Scheme 1), which then continue to react following interruption of the illumination source. The immediate oxidized product formed in the photochemical reduction of the pyrylium ions by water8 has not been identified. Hydrogen peroxide has been shown to accumulate upon irradiation of ϕ3Py+ solubilized in anionic sodium dodecyl sulfate micelles or encapsulated in zeolite cages,21,22 and formation of ring-hydroxylated aromatic products has been demonstrated by both transient spectrophotometry21 and chemical identification of reaction products.23 On the basis of these observations, hydroxyl radical has been suggested to be the reactive intermediate. As we have previously shown for DHP-bound pyrylium ions,8 the photoexcited triplet state was identified as the reactive species in the micellar suspensions, suggesting analogous chemistry.21 Despite considerable effort, we have not yet found conditions for which the low-potential pyrylium and thiopyrylium ions described in the following sections will function effectively as photosensitizers in these assemblies. As discussed below, reactions of these ions in their photoexcited states with the medium components are thermodynamically unfavorable. Physical Properties of the Low-Potential Pyrylium Ions. The pyrylium ions under investigation are listed below together with our abbreviated notations for their names. Also

A common feature of the pyrylium ions is the presence of one or two dialkylaminophenyl substituents which, when deprotonated, strongly donate electron density to the pyrylium ring. As a consequence, the characteristic low-energy optical absorption bands of the ring-closed forms are shifted from the near-UV into the visible region,24 and the one-electron potentials for reduction to the neutral pyrylium radicals are shifted to values significantly lower than that of the 2,4,6triphenylpyrylium ion.25,26 Additionally, pyrylium ions bearing these substituents have very low fluorescence quantum yields (φ < 0.01), a feature that is advantageous for photoredox reactions utilizing them as sensitizers. The presence of bulky substituents in the 4-position of the heterocyclic ring also tends to block radical coupling to form less-reactive bipyrans, a reaction that is prominent among low-potential pyranyl radicals.26 Optical spectra taken in 1:1 CH3CN/H2O (v/v) are shown in Figure 2, and visible absorption maxima measured for these

Figure 2. Optical absorption spectra of pyrylium and thiopyrylium ions in 1:1 (v/v) CH3CN/H2O. Concentrations were adjusted to give approximately equal maximal intensities in the visible bands.

compounds in this mixed solvent and in EPC liposomes and DHP vesicles are listed in Table 1. The absorption band for ϕ3Py+ is weakly solvatochromic, undergoing a small bathochromic shift upon decreasing the solvent dielectric; a ∼10 nm shift is also observed for ϕ3Py+ bound to DHP vesicles, which can be attributed to its localization within the hydrocarbon core of the vesicle.8 Shifts of similar magnitude are observed for the derivatized pyrylium ions upon exposure to DHP vesicle-containing solutions (Table 1), suggesting that they also are incorporated within the membrane interior. 12174

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biphasic character. Optical changes could be completely or partially reversed upon prompt acidification with HCl, depending upon the ion, but the extent of recovery of the pyrylium ion absorption generally diminished at longer times, indicating subsequent degradation of the initially formed pseudobase had occurred. The apparent pKa values listed in Table 1 were estimated by assuming that the relatively rapid initial changes approximated equilibration of the cation and pseudobase forms and that the subsequent changes were associated with spectral shifts caused by product degradation. These changes could be described by the equilibrium: Py+ + H2O = PyOH + H+. The pKa values calculated in this fashion were insensitive to the time in kinetic traces that was selected to represent the equilibration point. For example, for DMAP(tBu)2Py+, the pKa determined from spectra taken 3 h after increasing the pH was ∼8.6, whereas at 12 h, it was ∼8.1. For the thiopyrylium ions, bleaching of the cation optical band occurred only in strongly alkaline solution and proceeded with apparent rates that were an order of magnitude slower than observed for the pyrylium ions under comparable conditions. Moreover, the optical changes were not reversible, indicating that bleaching was accompanied by irreversible degradation of the compounds. In these cases, the pKa values were estimated from the pH where bleaching was first detected. The dynamical behavior of these ions was unchanged when solutions were purged of O2 and rigorously protected from light exposure, suggesting that degradation was hydrolytic in character. The pKa values for these low-potential prylium and thiopyrylium ions are higher by several units than those typically measured for higher potential analogues (cf., ϕ3Py+, ϕ3PyS+ in Table 1). As noted for the relative rates, these trends undoubtedly reflect stabilization of the cation by the electrondonating ring substituents; that is, the ring is progressively deactivated toward nucleophilic addition of OH− by addition of increasing numbers of electron-donating substituents and by substitution of the less electronegative S atom for O in the heterocyclic ring. These effects can account, at least qualitatively, for the relative ordering in Ka values given in Table 1.18,24 Transmembrane Reduction of Co(bpy) 3 3+ Ion. 3 ZnTPPS4− photosensitized transmembrane reduction of occluded Co(bpy)33+ by external donors was observed in all cases when any of the pyrylium or thiopyrylium ions were introduced to suspensions of reductant-loaded PC or DHP vesicles; no net transmembrane redox occurred under these illumination conditions (excitation into the ZnTPPS4− Soret band) when either the pyrylium ions or the electron donors were omitted, indicating that the dyes functioned as combined oxidative quenchers of 3ZnTPPS4− and transmembrane redox mediators (Scheme 1). The efficiencies of ascorbic acid, triethanolamine (TEOA), EDTA, and dithiothreitol (D(SH)2) as electron donors were surveyed in preliminary studies. Systems containing EDTA gave greater photosensitivity than either TEOA (60%) or ascorbate (∼4−20-fold) under comparable conditions and reactivity nearly identical to that of D(SH)2. With reactions organized on PC liposomes, D(SH)2 suffered the disadvantage of slowly diffusing across the bilayer, where it thermally reduced the Co(bpy)33+ ion. This background thermal reaction is not an issue with EDTA as donor because it is membrane-impermeable on the time scales of interest and its reduction of Co(bpy)33+ is highly endergonic.27 Consequently, EDTA was used as the sacrificial donor in instances where quantitative comparisons among different

Table 1. Relevant Physical Properties of Substituted Pyrylium Ions ion +

DMAP(t-Bu)2Py DMAP(t-Bu)2PyS+ DEAPϕ2Py+ DEAPϕ2PyS+ (DMAP)2ϕPy+ ϕ3Py+ ϕ3PyS+

λmax (nm)a

E1/2(P+/0) (V)b

pKac

492 (490, 502) 530 (530, 544) 545 (550, 558) 588 (592, 602) 612 (618, 630) 355 (---, 366)d 372 (---, 386)d

−0.54 −0.44 −0.36 −0.22 −0.46 −0.10 0

8.6 ∼12 8.6 ∼10 9.3 4.6d 6.3d

a

In 1:1 (v/v) CH3CN/H2O (in EPC, in DHP). bBy cyclic voltammetry in dry CH3CN vs NHE, as determined using ferrocene/ferricinium ion as internal standard. cPseudoacid dissociation constant, Ka = [H+][PyOH]/[Py+] = Kw/Kb, with Kb defined as the pseudobase dissociation constant, measured spectrophotometrically at 23 °C in aqueous buffers. dIn 40 mM sodium acetate;8 dashed lines indicate that spectra in EPC were not measured.

Smaller shifts were observed for PC suspensions, suggesting either that binding was localized to the membrane−aqueous interface or that the ions were partitioning between the aqueous and hydrocarbon phases. Strong association of these ions to the DHP vesicles was confirmed by membrane ultrafiltration measurements where, in each instance, association was essentially complete (>90%) in weakly acidic to neutral 40 mM acetate or Tris buffers (pH 4−8). However, a considerable fraction of each pyrylium ion diffused through the semipermeable membranes in analogous studies using PC liposomes, indicating that binding in these instances was only partial (Figures S4,S5). Substitution of a sulfur atom for oxygen also caused a ∼20−40 nm bathochromic shift in the lowestenergy absorption band, consistent with the general reported behavior for this class of compounds.24 Cyclic voltammograms taken in CH3CN displayed quasireversible one-electron reduction waves whose midpoint potentials decreased with increasing electron-donating character of the substituent groups (Table 1). Thus, replacing an electron-withdrawing phenyl substituent by either tert-butyl or dialkylamino electron-donating groups decreased the potential by 100−260 mV; substituting S for O in the heterocyclic ring also increased the potential by 100−140 mV, consistent with the general reported behavior for these compounds.25 For these pyrylium ions, the amplitudes of the reoxidation waves in the cathodic sweeps were nearly identical to the anodic waves, indicating that radical dimerization to bipyrans or decomposition reactions were inconsequential on the seconds time scales under the conditions of the electrochemical experiments. Apparent pseudoacid dissociation constants (Ka) in aqueous solution were estimated by monitoring loss of the characteristic visible absorption of the cations upon addition to buffers of varying alkalinity. Unlike the higher potential ions previously studied,8 these ions underwent only slow spectral changes following application of a pH jump. The reaction rates increased with increasing pH, but were highly dependent upon the identities of the individual ions; under identical reaction conditions, the order of the initial rate of change was: DMAP(t-Bu)2Py+ ≈ DEAPϕ2Py+ > (DMAP)2ϕPy+ > DMAP(tBu)2PyS+ ≈ DEAPϕ2PyS+. These rates parallel the effects expected for addition of electron-donating substituents and substitution of S for O in the heterocyclic ring upon nucleophilic attack of the chromophore ring.18,24 For the pyrylium ions, first-order plots of the spectral losses exhibited 12175

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vesicles were desired. The relative capacities of the pyrylium ions to mediate transmembrane redox across EPC bilayer membranes under a common set of conditions are illustrated in Figure 3. Upon prolonged excitation at 420 nm, ∼20 μM

Table 2. Quantum Yields for Transmembrane Reduction of Co(bpy)33+a mediator DMAP(t-Bu)2Py+ DMAP(t-Bu)2PyS+ DEAPϕ2Py+ DEAPϕ2PyS+ (DMAP)2ϕPy+ ϕ3Py+ ϕ3PyS+

EPC 0.026 0.032 0.016 0.026 0.014

(0.017) (0.013) (0.015) (0.012) (0.013)

DMPC

DPPC

DHP

0.026 0.032 0.016 0.027 0.014

0.025 0.031 0.016 0.026 0.013

0.0049 0.014 0.0088 0.011 0.0040 (0.10)b (0.10)b

Conditions: 5 mg/mL surfactant vesicles ([PC] ≈ 0.08 μM, [DHP] ≈ 2.3 μM) in 40 mM Tris/Cl, pH 8.0, or 40 mM sodium acetate, pH 5.0 (values in parentheses), plus ∼1 mM occluded Co(bpy)33+ and 5 mM EDTA in the external medium; when present, [ZnTPPS4−] = 2 μM; [Py+] = 2 μM. All reactions were at ambient temperature. Individual data are averages of at least three individual determinations, whose ranges were ±30%. bIn 30 mM sodium acetate, pH 5.0.8 a

second factor that could play a role relates to the different sizes of the two types of vesicles used in these studies. Specifically, the DHP vesicles are much smaller (∼25 nm average diameters,16 as compared to ∼100 nm for the PC liposomes). This leads to significant differences in radii of curvature, with corresponding greater perturbation of the normal bilayer structure within DHP membranes. It is uncertain whether this condition leads to a greater intrinsic barrier to transverse diffusion, for example, by steric crowding of alkane chains in the inner bilayer leaflet, that might retard overall transmembrane redox rates. The permeability coefficients measured for diffusion of neutral ϕ3Pyo, ϕ3PySo, MVo, and N-alkyl-4cyanopyridine radicals across DHP vesicles all range between 0.16 and 6.0 × 10−4 cm/s,6,8,17 roughly 10−100-fold lower than for similar neutral molecules across PC bilayers.29 A more extensive study of the influence of various reaction parameters upon the initial rates of Co(bpy)32+ formation was carried out in DHP assemblies using ZnTPPS4‑-sensitized DMAP(t-Bu)2PyS+ reduction by D(SH)2. Initial overall quantum yields calculated from these rates increased linearly with pH from φCo(II) = 0.003 at pH 3.1 to a high of φCo(II) = 0.025 at pH 8, and then declined slightly at higher pH values. The initial rate also exhibited an influence of the buffer composition; at pH 9.0, φCo(II) = 0.013, 0.015, and 0.021 in 100 mM borate, TAPS (3-tris(hydroxymethyl)methylamino-1-propanesulfonic acid), and Tris-Cl, respectively. These buffers were chosen because they differentially perturb the DHP membrane ultrastructure, as indicated by the major phase transition temperatures (Tc = 67, 67, and 45 °C); this difference may reflect selective adsorption of the cationic buffer (Tris) to the anionic membrane surface.16 Quantum yields were linearly dependent upon the thiopyrylium ion concentration to ∼10 μM, but independent of the quencher concentration at higher concentrations. Minor dependencies were also noted for the concentrations of vesicles (φCo(II) = 0.025−0.018 over the range 2.2−6.5 mg/mL) and sensitizer ion (φCo(II) = 0.025−0.015, pH 8.3, and 0.008−0.012, pH 5.0, over the range [ZnTPPS4−] = 2−33 μM). Identical quantum yields were measured for illumination into the porphyrin Soret and Q-bands. Data on which these summaries are based are given in Figures S7−S8 and Tables S1−S2. The general trend is for slightly greater φCo(II) values with increasing membrane disorder. The relatively strong influence of base, for which φCo(II) increases 10-fold upon increasing the pH from 3 to 8 (Figure S8), again suggests

Figure 3. 3ZnTPPS4−-photosensitized transmembrane reduction of Co(bpy)33+ by EDTA mediated by low-potential pyrylium and thiopyrylium ions. Conditions: 5 mg/mL EPC liposomes (∼8 × 10−8 M); initial internal [Co(bpy)33+] ≈ 1 × 10−3 M; external [ZnTPPS4−] = 2 × 10−6 M; external [EDTA] = 5 × 10−3 M; [Py+] = 2 × 10−6 M; in 40 mM Tris/Cl, pH 8.0, at ambient temperature; λex = 420 nm.

Co(bpy)33+ was reduced to Co(bpy)32+, which is the total amount of occluded acceptor ion, based upon changes in the optical spectra (Figure S6) and calculations based upon the vesicle dimensions28 and amount of complex ion added to the vesicle-forming buffer. Because the quantity of the occluded Co(bpy)33+ ion was ∼10-fold greater than the amount of added pyrylium ions, it follows that, on average, each mediating ion must have undergone ∼10 transmembrane redox cycles over this period. As is also evident from the data, quantum yields for the overall reaction (φCo(II)) calculated from the initial slopes of the plots were insensitive to the identity of the ion. Furthermore, their relative values bore no apparent relationship to either their reduction potentials or their intrinsic pseudoacid dissociation (pKa) constants (Table 1). The same reactivity characteristics were observed at different pH values and using different surfactant materials to form the vesicles; these data are summarized in Table 2. The overall quantum yields were ∼(2−5)-fold higher for corresponding reactions across EPC liposomes than DHP vesicles. This relative order parallels the microviscosities of the two membranes; specifically, the EPC membrane is in its relatively disordered, or liquid crystalline, phase at room temperature, whereas the DHP membrane is in its ordered, or gel, phase.16,29 However, no differences in φCo(II) were observed when the PC liposomes were prepared from dimyrystoylphosphatidylcholine (DMPC) and dipalmitoyl-phosphatidylcholine (DPPC) surfactants (Table 2), which are in their liquid crystalline (Tc = 23 °C) and gel (Tc = 41 °C) phases, respectively, at room temperature.29 Thus, at least for the phosphatidylcholinebased membranes, net transmembrane redox does not appear to involve rate-limiting transmembrane diffusion of the electron carrier because, if that were the case, one might expect lower quantum yields for the more viscous microenvironments. A 12176

dx.doi.org/10.1021/la301675t | Langmuir 2012, 28, 12171−12181

Langmuir

Article

BV2+/+ = −0.10 V; DV4+/3+ = 0.08 V, where the value for the cofacial diviologen is the average of two nearly independent, very closely spaced potentials for one-electron reduction of each viologen ring, that is, E1/2(DV4+/3+) ≈ E1/2(DV3+/2+).12,13 The corresponding aqueous viologen potentials are −0.48 V (MV2+/+),31 −0.37 V (BV2+/+),32 −0.24 V (DMAV2+/+), −0.21 V (DANV2+/+), and ∼0 V (DV4+/3+)13 vs NHE. These latter values are displayed in Figure 5. Comparison with the values in Table 1 indicates that electron transfer from all of the lowpotential pyranyl radicals to these viologens is thermodynamically favorable in CH3CN and for reduction of aqueous DV4+, DMAV2+, and DANV2+; in contrast, reaction with aqueous BV2+ and MV2+ could be limited to the more strongly reducing pyranyl radicals. For a variety of reasons, these comparisons based upon homogeneous potentials may not accurately reflect the actual potentials in the microheterogeneous medium. The viologen potentials are strongly dependent upon the medium polarity. The ∼300 mV shift to lower values measured for the MV2+/+ and BV2+/+ potentials in H2O relative to CH3CN reflects the strong solvation of the dication in aqueous media. For DV4+/3+ and DV3+/2+ this shift is much smaller, perhaps reflecting more extensive charge delocalization, and hence more lipophilic character, in this dimeric ion. The higher reduction potentials for this ion can be attributed, at least in part, to “pimer” formation,12,33 a configuration that is enforced by the o,o-xylyl bridges that constrain the two viologen segments to an overlapping cofacial arrangement. Similar aggregated structures can form between monomeric viologens within membrane bilayers,34 which could also raise their effective viologen reduction potentials. One notes that reduction potentials for the lipophilic8 ϕ3Py+ and ϕ3PyS+ ions are also insensitive to solvent (E1/2 = −0.10 and 0 V in CH3CN and −0.13 and 0.03 V in H2O,25,26 respectively), again probably reflecting the relatively small charge/radius ratio of the cations. The interfacial charge can also have a large effect upon potentials of bound viologens, with shifts of ∼(160−180) mV for both first and second reduction potentials being measured for binding of monoalkylviologens to anionic (DHP) versus cationic (didodecyldimethylammonium) bilayer membranes.20 The magnitudes of these potentials are also sensitive to the extent of binding of the viologen dications to the vesicles, which can depend upon the medium conditions. Finally, we note that the effective medium polarity exhibits a sharp gradient in passing from the bulk aqueous phase (ε = 78) to the membrane interior (ε ≈ 4),35 so that relocation of neutral pyranyl radicals toward the nonpolar hydrocarbon phase in the bilayer membranes might dramatically increase their reduction potentials to levels where electron transfer to viologens located in polar microenvironments is no longer possible. Given the uncertainties in redox potentials for both pyrylium ions and viologens introduced by the microheterogeneous media, we directly determined their relative potentials in vesicle-containing solutions by spectrophotometric titration, as described in the Experimental Section. A result typical of those obtained upon adding the vesicle-bound low potential pyrylium ions to solutions of MV•+ is shown in Figure 4. From the magnitude of the change in MV•+ absorption, one calculates apparent equilibrium constants for the reaction, Py+ + MV•+ → Py• + MV2+, ranging from K ≈ (0.005−0.4) for different combinations of DMAP(t-Bu)2Py+ or DMAP(t-Bu)2PyS+ bound to DHP vesicles or DMPC liposomes. Using potentials determined by thin-layer spectroelectrochemistry for vesicle-

that the ring-opening pseudobase step (Scheme 1) is at least partially rate-limiting.8 The quantum yields in DHP vesicles are (7−25)-fold lower for these mediators than were reported for ϕ3Py+ and ϕ3PyS+ under similar conditions.8 In the earlier study, as well as studies using N-alkyl-4-cyanopyridinium ions as transmembrane redox mediators,17 the apparent rate constant for oxidative quenching of 3ZnTPPS4− was shown to be proportional to the fraction of unbound quencher; this fraction, in turn, was the determining factor in the overall quantum yields for transmembrane redox. The low-potential pyrylium ions strongly adsorbed to DHP vesicles, precluding accurate determination of the unbound fraction (Figures S4,S5). Thus, the very low values of φCo(II) measured in these studies probably reflect the strong partitioning of the redox mediators onto the DHP bilayer membranes. A major contributing factor to the lower quenching rates of the bound pyrylium ions is likely to be the strong electrostatic repulsion between the photoexcited 3 ZnTPPS4− ion and the highly charged membrane interface, which has previously been shown to markedly impede electron transfer from 3ZnTPPS4− to DHP-bound viologens.30 Similar observations of the dramatic influence of electrostatics upon photoredox dynamics have been reported for quenching reactions of ϕ 3 Py + incorporated into anionic sodium dodecylsulfate micelles. Accordingly, the micellarized 3ϕ3Py+ was rapidly quenched by aqueous cationic reductants, whereas no quenching was observed when anionic reductants were used.21 This explanation could also account in part for the ∼(2−5)fold higher overall quantum yields measured for PC liposomes under comparable conditions (Table 2). As demonstrated by membrane ultrafiltration studies, pyrylium ion binding to the liposomes was considerably less extensive, presumably because electrostatic attraction of the cationic dye to the formally neutral, zwitterionic PC interface is markedly attenuated relative to the anionic DHP interface, leaving a substantial fraction of the ions in solution where they can effectively quench the 3ZnTPPS4− ion. Thus, for example, binding of DMAP(t-Bu)2Py+ and DMAP(t-Bu)2PyS+ to EPC and 0.7/0.3 mol fraction DPPC/cholesterol was