Membrane Activity of Isophthalic Acid Derivatives - American

Department of Chemistry, University of Victoria, Victoria, ... and with mean specific conductance ranging from 9.2 pS for NaCl and 15.4 pS for KCl to ...
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Membrane Activity of Isophthalic Acid Derivatives: Ion Channel Formation by a Low Molecular Weight Compound T. M. Fyles,* R. Knoy, K. Mu¨llen, and M. Sieffert Department of Chemistry, University of Victoria, Victoria, British Columbia, Canada V8W 3P6, and Max-Planck-Institut fu¨ r Polymerforschung, Akermannweg 10, D-55128 Mainz, Germany Received April 23, 2001. In Final Form: July 26, 2001 A suite of 17 alkoxy-substituted isophthalic acid derivatives has been assessed for the ability to open well-defined ion channels in planar and vesicle bilayer membranes. One compound, 5-(12-tricosanoxy)isophthalic acid, forms nonrectified cation selective channels with open lifetimes of the order of seconds and with mean specific conductance ranging from 9.2 pS for NaCl and 15.4 pS for KCl to 31.0 pS for CsCl. In addition to this dominant channel behavior, occasional variations in conductance levels suggest that related oligomeric channel structures can form. Channel formation is under close structural control as a compound bearing only two more methylene groups is significantly less active. The active compound has the lowest molecular weight of any known ion channel forming compound.

Introduction Membrane transport processes play key roles in biological systems including the specific ion channels that are the functional components of the nervous system.1,2 The lure of artificial devices capable of regulating information flow across membranes has inspired a range of mimics of natural channels,3-6 which serve both technological and diagnostic roles. The molecular basis of ion transport in natural systems is only slowly emerging,7-9 so model systems and structure-activity studies can provide insights. Concurrently, simple systems are more likely to lead to ionic devices such as sensors,10 since the biochemical infrastructure of natural transport proteins is proving to be difficult to transpose to artificial systems.11 Synthetic ion conductors active in lipid bilayer membranes show a range of transport functions ranging from defined ion channels with specific activities akin to natural channels12,13 to disrupting agents functionally related to membrane-disrupting peptides.14-16 One of the continuing challenges is to design synthetic ion conducting systems * Corresponding author. T. M. Fyles, Department of Chemistry, University of Victoria, Box 3065, Victoria BC, Canada, V8W 3P6. Tel: (250) 721 7184. Fax: (250) 721 7147. E-mail: [email protected]. (1) Stein, W. Carriers, Channels, and Pumps: An Introduction to Membrane Transport; Academic Press: San Diego, 1990. (2) Nicholls, D. G. Proteins, Synapses, and Transmitters; Blackwell Science: Oxford, 1994. (3) Fyles, T. M. Curr. Opin. Chem. Biol. 1997, 1, 497-505. (4) Kobuke, Y. Advances in Supramolecular Chemistry; Gokel, G. W., Ed.; JAI Press: Greenwich, CT, 1997; Vol. 4, pp 163-210. (5) Voyer, N. Top. Curr. Chem. 1996, 184, 1-37. (6) Gokel, G. W.; Ferdani, R.; Liu, J.; Pajewski, R.; Shabany, H.; Uetrecht, P. Chem.sEur. J. 2001, 7, 33-39. (7) Chang, G.; Spencer, R. H.; Lee, A. T.; Barclay, M. T.; Rees, D. C. Science 1998, 282, 2220-2226. (8) Doyle, D. A.; Cabral, J. M.; Pfuetzner, R. A.; Kuo, A.; Gulbis, J. M.; Cohen, S. L.; Chait, B. T.; MacKinnon, R. Science 1998, 282, 69-77. (9) Miyazawa, A.; Fujiyoshi, Y.; Stowell, M.; Unwin, N. J. Mol. Biol. 1999, 288, 765-786. (10) Cornell, B. A.; Braach-Maksvytis, V. L. B.; King, L. G.; Osman, P. D. J.; Raguse, B.; Wieczorek, L.; Pace, R. J. Nature 1997, 387, 580584. Gu, L.-Q.; Braha, O.; Conlan, S.; Cheley, S.; Bayley, H. Nature 1999, 398, 686-690. (11) Sackmann, E. Science 1996, 271, 43-48. Cheng, Y.; Bushby, R. J.; Evans, S. D.; Knowles, P. F.; Miles, R. E.; Ogier, S. D. Langmuir 2001, 17, 1240-1242. (12) Baumeister, B.; Sakai, N.; Matile, S. Angew. Chem., Int. Ed. Engl. 2000, 39, 1955-1958. (13) Gokel, G. W. Chem. Commun. 2000, 1-9.

that offer both a well-defined activity and a simple synthesis. There has been a marked trend from the complex designs reported a decade ago17,18 to the much simpler systems reported in recent years.19,20 Even so, most active structures remain large, and structural variations are limited. At the same time, the activity of many synthetic ion-conducting compounds is increasingly understood as a result of stabilized aqueous defects that span the bilayer membrane.21,22 Although structurally less elaborate than natural channels, such defects can show significant functional regularity including ion selectivity, high specific activity, and voltage rectification.23 Even some conventional detergents, such as Triton, enhance membrane permeability at concentrations lower than those required for detergent dissolution of lipids and show channel-like membrane conductance.24 The general conclusion is that a sophisticated transport function does not require a correspondingly complex structure. One approach to the design of new ion conductors focuses on the potential of very simple amphiphilic compounds to induce transmembrane transport. From an evolutionary perspective, the earliest cellular organisms would have required transporters that must have been vastly simpler than modern transport proteins.25 The simplest functionally competent structures might offer insight into the (14) Jayasuriya, N.; Bosak, S.; Regen, S. L. J. Am. Chem. Soc. 1990, 112, 5844-5850. (15) Nagawa, Y.; Regen, S. L. J. Am. Chem. Soc. 1991, 113, 72377240. (16) Fyles, T. M.; Zeng, B. Chem. Commun. 1996, 2295-2296. (17) Pregel, M. J.; Jullien, L.; Canceill, J.; Lacombe, L.; Lehn, J.-M. J. Chem. Soc., Perkin Trans. 2 1995, 417-426. (18) Fyles, T. M.; James, T. D.; Kaye, K. C. J. Am. Chem. Soc. 1993, 115, 12315-12321. (19) Yoshino, N.; Satake, A.; Kobuke, Y. Angew. Chem., Int. Ed. Engl. 2001, 40, 457-459. (20) Bandyopadhyay, P.; Janout, V.; Zhang, L.; Sawko, J. A.; Regen, S. L. J. Am. Chem. Soc. 2000, 122, 12888-12889. (21) Fyles, T. M.; Loock, D.; van Straaten-Nijenhuis, W. F.; Zhou, X. J. Org. Chem. 1996, 61, 8866-8874. (22) Abel, E.; Maguire, G. E. M.; Meadows, E. S.; Murillo, O.; Jin, T.; Gokel, G. W. J. Am. Chem. Soc. 1997, 119, 9061-9062. (23) Fyles, T. M.; Loock, D.; Zhou, X. J. Am. Chem. Soc. 1998, 120, 2997-3003. (24) Rostovtseva, T. K.; Bashford, C. L.; Lev, A. A.; Pasternak, C. A. J. Membr. Biol. 1994, 141, 83-90. (25) Das, S.; Lengweiler, U. D.; Seebach, D.; Reusch, R. N. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 9075-9079.

10.1021/la0105937 CCC: $20.00 © 2001 American Chemical Society Published on Web 09/18/2001

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minimum requirements of ion channels, both for reflecting on early biology and for creating future technology. One appealing class of amphiphiles is the isophthalic acid ether derivatives explored by a number of groups. In the solid state, 5-alkoxy isophthalic acids form extended hydrogenbonded arrays consisting of linear “tapes” with interdigitated alkyl chains or macrocyclic hexamers.26 The latter structures apparently persist in solution in nonpolar solvents.27,28 The hexamers open a cavity about 1.4 nm in diameter that is suggestive of one segment of a tubular channel.26 The same class of compounds form well-ordered two-dimensional bilayer structures on graphite surfaces in which the alkyl chains interpenetrate.29 Some partly neutralized 5-alkoxy isophthalates form self-supporting fibers.30 The available structural data support an arrangement of monomers in these aggregates involving alkyl group interpenetration in a bilayerlike structure.31 Thus, alkoxy isophthalates show several appealing structural features as potential ion conductors: (1) The dianionic headgroup will establish a strong amphiphilic character that will orient the compound in the bilayer membrane. (2) The compounds have demonstrated an ability to form aggregates that may lead to oligomeric structures sufficiently large to span the membrane. (3) The aromatic headgroup at the membrane-solution interface may assist cation entry into a channel.32 (4) They are easily prepared, and a large suite of compounds is available for examination.26,29-36 Some of the available structures are illustrated (compounds 1-6). 5-n-Alkoxy substituted isophthalic acids (1, n ) 10-18) and partly fluorinated derivatives such as 2 offer a range of lipophilicities. As amphiphiles, these structures are structurally similar to the corresponding N-alkylpyridinium salts and would thus be expected to form micellar dispersions in water. The 4,6-dialkoxy (3) and 5-branched alkoxy derivatives (4) have a larger alkyl chain volume for the same headgroup area and thus would be expected to form lamellar structures on dispersion in water. A partially neutralized 4 (n ) m ) 11) is reported to form vesicles.30 Finally, a number of derivatives such as 5 and 6, with aromatic or macrocyclic units within the tail of the amphiphile, have been prepared. One example, the diaza 18-crown-6 derivative 5, is akin to Gokel’s trismacrocycle channel system,13 lacking the “portal” macrocycles. The goal of this study is to survey a suite of compounds, 1-6, for ion-conducting activity in bilayer membranes. The focus is on well-behaved ion channel behavior rather (26) Valiyaveettil, S.; Mu¨llen, K. New J. Chem. 1998, 89-95. (27) Yang, J.; Marendez, J.-L.; Geib, S.; Hamilton, A. D. Tetrahedron Lett. 1994, 35, 3665-3668. (28) Zimmerman, S. C.; Zeng, F.; Reichert, D. E. C.; Kolotuchin, S. V. Science 1996, 271, 1095-1098. (29) De Feyter, S.; Gesquie`re, A.; Abdel-Mottaleb, M. M.; Grim, P. C. M.; De Schryver, F. C.; Meiners, C.; Sieffert, M.; Valiyaveettil, S.; Mu¨llen, K. Acc. Chem. Res. 2000, 33, 520-531. (30) Menger, F. M.; Lee, S. J. J. Am. Chem. Soc. 1994, 116, 59875988. (31) Meiners, C.; De Feyter, S.; Lieser, G.; van Stam, J.; Solterman, A.; Berghmans, H.; De Schryver, F. C.; Mu¨llen, K. Langmuir 1999, 15, 3374-3380. (32) Maguire, G. E. M.; Meadows, E. S.; Murray, C. L.; Gokel, G. W. Tetrahedron Lett. 1997, 38, 6339-6342. (33) De Feyter, S.; Gesquie`re, A.; Greim, P. C. M.; De Schryver, F. C.; Valiyaveettil, S.; Meinres, C.; Seiffert, M.; Mu¨llen, K. Langmuir 1999, 15, 2817-2822. (34) Gesquie`re, A.; Abdel-Mottaleb, M. M.; De Feyter, S.; De Schryver, F. C.; Sieffert, M.; Mu¨llen, K.; Calderone, A.; Lazzaroni, R.; Bre´das, J.-L. Chem.sEur. J. 2000, 6, 3739-3746. (35) Valiyaveettil, S.; Gans, C.; Klapper, M.; Gereke, R.; Mu¨llen, K. Polym. Bull. 1994, 34, 13-15. (36) Valiyaveettil, S.; Enkelmann, V.; Mu¨llen, K. Chem. Commun. 1994, 2097-2098.

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than on nonspecific detergent or membrane-disrupting activity. Previous surveys of transport activity have relied on vesicle bilayer membranes.18,23 The main weakness of vesicle techniques is the lack of mechanistic information they provide: discrete ion channels, ion carriers, membrane-disrupting agents, or simple detergents all give rise to apparently the same observed enhancement of membrane permeability. Only through extensive experimentation can mechanistic hints emerge.18,20 Survey of the activity of a suite of compounds using planar bilayers is not commonly reported as the throughput is limited, the concentration range is restricted, and the day-to-day repeatability is difficult to establish.37 Despite these drawbacks, we elected to survey the available compounds using the voltage-clamp technique, as this is the sole method to unequivocally establish the presence of channellike activity, in contrast to the detergent effects that might be expected for amphiphilic compounds such as 1-6. Results and Discussion The survey relies on the reliable formation of a goodquality lipid bilayer membrane and a predictable response from known channel-forming compounds. Bilayers were formed across an aperture (0.25 mm) in a polystyrene cuvette that defined one-half of the cell. The cell was initially filled with 1 M electrolyte solutions (alkali metal chlorides; 2.8 mL in the cuvette, 5.0 mL in the holder). Lipid (8:1:1 phosphatidyl choline/phosphatidic acid/ cholesterol) in decane solution was applied to the aperture and thinned using a fine brush to produce bilayers with a typical capacitance of 200-220 pF. From the aperture dimensions, this capacitance indicates only a very small torus of solvent surrounding the actual bilayer. The lipid mixture was chosen to be compatible with vesicle experiments using the same mixture. Once lipid had been introduced to the cell, good-quality bilayers could also be formed by a dipping technique in which the cuvette was slowly raised to a point where the aperture was above the lipid-saturated surface of the remaining electrolyte in the cell. Lowering the cuvette created a bilayer across the aperture. Usually, a slight brushing produced highcapacitance bilayers. Once formed, all bilayers were (37) Sackmann, B.; Neher, E. Single-Channel Recording; Plenum Press: New York, 1983.

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Figure 1. Transmembrane current as a function of time at +100 mV applied potential and 1 M KCl electrolyte showing different types of membrane activity: (A) baseline following addition of 50 µL DMSO, (B) example of “irregular” activity, (C) example of a step conductance change ignored as too small to be significant, and (D) regular step conductance change indicating channel formation.

monitored for 40-60 min at an applied transmembrane potential of (100 mV (alternate periods of 10 min at each polarity). Typically, 2-5 spikes of 0.1-0.3 pA magnitude would be observed during the monitoring period. Bilayers that showed baseline drift or irregular noise, had more frequent (>5) small current spikes, or had spikes in excess of 0.4 pA were rejected. The compound to be tested was introduced in a few microliters of a millimolar solution in methanol or DMSO, as required to dissolve the compound. Compounds were added to the cis side of the cell; the trans side was the ground for applied potentials. Electrical contact was established via agar salt bridges leading to Ag/AgCl electrodes in concentrated electrolyte. Although the reliability of any one bilayer could not be directly probed, the overall reliability of the process was assessed from bimonthly replication with the channel-forming peptide gramicidin. Every experiment with gramicidin produced the expected single-channel openings after a single injection, with the same specific conductance. The day-to-day reliability of the bilayer formation procedure is therefore suitable for the survey of activity. In addition to reliable membrane formation, there is a need to define which types of transmembrane electrical conductivity will indicate that the tested compound is “active”. In addition to the usual step conductance changes observed with natural channels, there are a variety of unusual conductance changes seen with synthetic ion conductors. We took a conservative view: only step conductance changes were considered as indicating activity. Some clarifying examples are shown in Figure 1. The baseline (Figure 1A) shows a noise band after hardware filtering at 1 kHz. Some compounds and experiments produced an irregular and noisy baseline as shown in Figure 1B. This type of activity was ignored. It might indicate the onset of detergent behavior, but this is not the focus of this report. Figure 1C shows a single steplike change in conductance. This type of event was ignored as well, as the two levels are too closely similar compared to the width of the noise band. Only step changes as illustrated in Figure 1D, that are significantly larger than the noise band, would be considered as an indication of activity under the conditions of the survey. This restrictive criterion will mean that some compounds will be ignored that under other conditions of lipid, concentration, and experimental protocol might well form regular ion channels. It also means that a practical survey can be completed over a period of a few months.

Figure 2. Single-channel activity of 7 at -120 mV applied potential (1 M KCl): (A) current-time record showing three levels and (B) all-points histogram of the same data.

Initially, a total of 15 compounds represented by 1-6 were tested at least in triplicate in planar bilayers and subsequently in vesicles using a pH-stat method.18 Only three compounds showed any sign of membrane activity, and only one was consistently active as defined above. The aza-crown compound 5 increases the permeability of vesicle bilayer membranes but produces only irregular current-time traces of the type shown in Figure 1B. It is probable that it acts in a nonspecific manner to disrupt bilayers, possibly by a detergent action (dissolution). Compound 2 also shows a low level of membrane activity in vesicles and does show regular step conductance changes. However, the observed openings were very infrequent. Only 26 events with a total duration of about 20 s were observed in three experiments lasting over an hour each. This level of activity is too low to characterize. In contrast, compound 4 (m ) n ) 11, redrawn as 7) produced numerous step changes in conductance every time it was examined. Ironically, compound 7 showed no hint of activity in vesicle bilayer membranes and so would have been missed in a conventional vesicle-based survey. Figure 2A shows a portion of a current-time record for 7 recorded in KCl electrolyte at an applied potential of -120 mV. Three conductance levels more negative than the baseline are apparent. There also appears to be some variation in the magnitude of the three levels dependent upon which section of the data is examined. However, a histogram constructed from these data (Figure 2B) clearly shows that the minor differences are insignificant within the envelope of the fitted Gaussian distributions. This same conclusion may be drawn from a plot of mean current per level as a function of level number (event plot) that shows that all conductance changes fall on a single line within the error of the Gaussian widths (r2 ) 0.997). Thus,

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Figure 3. Single-channel activity of 7 at +120 mV applied potential (1 M KCl) recorded 3 min later than Figure 2: (A) current-time record showing three levels and (B) all-points histogram of the same data.

the data of Figure 2 indicate that three copies of the same type of channel have formed in this time window.

At positive applied potential, the same experiment shows additional complexity (KCl electrolyte, +120 mV applied potential). Figure 3A was recorded 3 min later than Figure 2A. Here, the differences between the levels within the window presented are large, and it appears that the transition marked “1” is distinctly smaller than that marked “2”. The all-points histogram (Figure 3B) bears out this qualitative observation: there is no regular progression of maxima as in Figure 2B. The arrows indicate that the observed transitions occur from only two of the three lower levels. An event plot of mean current as function of level number is distinctly nonlinear (r2 ) 0.97). Figure 3A thus arises from two different types of channels: a channel giving transitions 2 and 3 and a “smaller” channel giving transition 1. The same type of behavior was evident for other electrolytes (NaCl, CsCl) in every set of experiments done. Some periods of observation would show a regular progression of levels, as typified by Figure 2. Another period of observation would show two or more distinct classes of transition as typified by Figure 3. The regular behavior was reproducible between days; the variable behavior was reproducible at different times within a single experiment but differed on subsequent days. Note that the slope of a regular event plot (derived from Figure 2B for example) gives an average value for the magnitude of the channel conductance whereas only discrete values

Figure 4. Current-voltage relationships for 7 in three electrolytes: (A) 1 M NaCl, (B) 1 M KCl, and (C) 1 M CsCl. Filled circles are slopes of event plots of a sequence of three or more conductance levels. Open circles are discrete observations.

for transitions can be derived from histograms such as Figure 3B. These two measures of channel conductance can be combined to derive a current-voltage relationship for compound 7. Figure 4 shows the results for three electrolytes (A ) NaCl, B ) KCl, C ) CsCl). The average values are plotted as solid circles, while the open circles show the more scattered discrete values obtained over a number of experiments. The fitted lines given in Figure 4 are derived from the averaged data (solid points) only. The inherent ion selectivity of the channels formed by 7 is seen in Figure 4 as the order Cs+ > K+ > Na+. The specific conductance is as follows: Na+, 9.2 ( 0.1 pS; K+, 15.4 ( 0.2 pS; Cs+, 31.0 ( 0.1 pS. Both the selectivity and the magnitudes of the specific conductance are closely similar to those observed with anionic bis-macrocyclic bolaamphiphile channels.21 These latter act via small membrane-spanning aggregates that can bear no more than a single anionic charge per headgroup. More highly negatively charged monomers should give rise to more marked Na+/K+ selectivity as selectivity is related to the electrostatic interactions with the headgroups.21 This suggests that the isophthalate headgroups are not fully protonated in the active channel structure. A second point that emerges from Figure 4 is that the occurrence of “discrete” transitions is somewhat more

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common at positive applied potentials than at negative and somewhat more common as the absolute magnitude of the applied potential increases. A positive applied potential has the electric field gradient aligned in parallel to a monomeric amphiphile inserted from the cis side of the cell. This orientation would be less stable than the antiparallel one achieved for the same monomer at negative applied potential. Collections of oriented monomers would be even more sensitive to the sign of the applied potential.23 There is no evidence of nonlinear currentvoltage behavior, nor less of a current rectification. Nonetheless, the scatter to higher positive potentials could be a reflection that the channels are less stable under this polarity. The original suite of compounds containing 7 offered only one other similar structure (4, m ) 4, n ) 6) with substantially shorter alkyl chains. Many of the inactive compounds of types 1, 3, 5, and 6 were substantially longer than 7. Against this backdrop of inactivity, we chose to prepare two additional compounds of type 4 that might provide additional structure-activity information. Accordingly, we prepared the shorter derivative 8 and the longer derivative 9 and surveyed their transport activity. We were both surprised and disappointed that neither compound showed any evidence of activity according to the procedures outlined above. Eventually, we were able to observe very infrequent openings by 9, but this required addition to both sides of the bilayer. Openings were observed only at negative potentials. The few openings were all step conductance changes, of somewhat larger magnitude than previously observed for 7. Regen has recently reported a system that is extremely sensitive to the overall length of the transporter with respect to the bilayer lipid composition.20 An analogous experiment based on variation of lipid thickness in planar bilayers is certainly possible, but exploration of this issue will require a more conventional approach to activity optimization that lies outside the scope of this survey study.

Conclusions It is unlikely that a single molecule of 7 would be capable of the regular conductance changes observed. The conducting species is more likely to be a small aggregate consisting of a few molecules (2-10). A stack of cyclic hexamers, derived from the reported solid and solution state structures,26,27 can be ruled out as the specific conductance observed is far too small for such a large pore. A structure related to the tapes reported previously26 might effectively line a transmembrane pore, but this would project the alkyl chains of 7 roughly perpendicular to the lipid chains. From the packing perspective, it is more likely that the alkyl chains of 7 are parallel to the lipid chains. Certainly, a monomer of 7 dissolved in a bilayer would be expected to be oriented with the long axis parallel to the lipid tails. However, the length of the alkyl groups in 7 (C12 from the secondary carbon) is significantly shorter than the lipid alkyl chains (average C16). Thus, a likely location for the aromatic groups would be at the level of the fatty acid carbonyls (the “midpolar region”6) rather than within the phosphocholine “headgroup” layer of the bilayer. Side-to-side dimers or trimers would then produce a bundle that could stabilize water molecules to a depth below the fatty acid carboxyl groups.

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As noted above, the cation selectivity data suggest that the isophthalates are partially protonated, as would be expected in an aligned oligomer where the isophthalate carboxylates would be in close proximity. The limited activity of 9 required addition to both sides of the bilayer. Although 7 was active from one side only, the conductance changes observed following addition to both sides of the bilayer were indistinguishable from those produced from single-side addition. If the transport activity requires only a single-leaflet perturbation, there should be some indication of nonlinear current-voltage response and some dependence on the location of the addition, as in other systems.24 Neither was observed; thus, 7 can apparently penetrate the bilayer and would lead to aggregates in both bilayer leaflets. This opens the possibility that the active structure is a membrane-spanning tail-to-tail dimer of small aggregates. Similar tail-to-tail structures have been recently proposed for cholate-derived synthetic channels.19,20 On average, a tail-to-tail structure would have to form with equal numbers of monomers in both bilayer leaflets in order to give the overall linear current-voltage response. However, differing numbers of monomers in the two leaflets of the bilayer might produce the discrete behavior discussed above. Compound 7 is the lowest molecular weight compound to form demonstrated ion channels in bilayer membranes. The significant differences between 7 and any of the other compounds investigated in the survey indicate the formation of a specific active structure with defined structural constraints. Given the close similarity of 7 and 9, compounds that differ in chain length by a single methylene in each branch of the alkyl chain, the active structure is under remarkably tight structural control. This level of control is unexpected and provides a fertile starting point for more focused examination of mechanism. Experimental Section Materials. Egg phosphatidylcholine, diphytanoyl phosphatidylcholine, and cholesterol were purchased from Avanti Polar Lipids. Other salts and reagents were purchased from SigmaAldrich at the highest purity available. Electrolyte solutions were made up with deionized, doubly distilled water. Synthesis of Alkoxy Isophthalic Acids. Alkoxy isophthalic acids were prepared by alkylation of dimethyl 5-hydroxyisophthalate with suitable alkyl halides under basic conditions, followed by ester hydrolysis with LiOH in methanol. The following 15 derivatives were available from previous work:29-36 n-decanoxy (1, n ) 10); n-tetradecanoxy (1, n ) 14); n-hexadecanoxy (1, n ) 16); n-octadecanoxy (1, n ) 18); 2; bis-(n-octadecanoxy) (3, m ) n ) 18); bis(n-docosanoxy) (3, m ) n ) 22); n-docosanoxy-npentyloxy (3, m ) 5, n ) 22); 5-undecanoxy (4, m ) 4, n ) 6); 12-tricosanoxy (4, m ) n ) 11; 7); 5; 6 (2,6-naphthyl, R ) S-2methylbutyl); 6 (2,6-naphthyl, R ) n-C22H45); 6 (1,5-naphthyl, R ) n-C20H41); 6 (2,6-naphthyl, R ) (CH2)11(CF2)5CF3). 5-(9-Heptadecanoxy) Isophthalic Acid. 5-(9-Heptadecanoxy) isophthalic acid (4, n ) m ) 8) was prepared by reduction of 9-heptadecanone (LiAlH4, THF; 90%), conversion of the alcohol to the bromide (PPh3-Br2; 58%), alkylation of dimethyl 5-hydroxyisophthalate (NaH, DMF followed by bromide plus catalytic NaI; 53%), and ester hydrolysis (KOH, ethanol; 41%) as a white powder; mp, 141-144 °C. 1H NMR (CDCl3): 8.4 (1H, s), 7.7 (2H, s), 4.35 (1H, br. quin.), 1.75-1.3 (28H, br. m.), 0.85 (6H, br. t.). 13C NMR (CDCl ): 171.5, 158.7, 130.9, 124.4, 122.0, 78.6, 33.7, 3 31.9, 29.8, 29.6, 29.3, 25.3, 22.7, 14.1. MS (+LSIMS, mNBA): 421.2. Exact mass (+LSIMS, mNBA): 421.2955. Calculated for C25H41O5: 421.2943. 5-(13-Pentacosanoxy) Isophthalic Acid. 5-(13-Pentacosanoxy) isophthalic acid (4, n ) m ) 12) was similarly prepared from 13-pentacosanone as a white powder; mp, 77-78.5 °C. 1H NMR (CD3OD): 8.1 (1H, s), 7.6 (2H, s), 4.8 (1H, s), 4.3 (1H, br. quin.), 1.65-1.1 (44H, br. m.), 0.8 (6H, br. t.). 13C NMR (CD3OD): 168.8, 160.3, 133.8, 124.0, 121.9, 79.6, 34.8, 33.1, 30.8, 30.7, 30.6, 30.5,

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26.2, 23.8, 14.5. MS (+LSIMS, mNBA): 533.3. Exact mass (+LSIMS, mNBA): 533.4223. Calculated for C33H57O5: 533.4191. Bilayer-Clamp Experiments. The apparatus and general procedures for single-channel recording have been previously reported.21,23 Agar salt bridges were used to stabilize junction potentials and were employed between the electrolyte in each well of the cell and the Ag/AgCl electrode. New bridges were prepared for each compound tested. Bilayers were formed by brushing as described previously and by dipping. After lipid in decane had been introduced by brushing, a lipid/decane film formed on the surface of the electrolyte, and bilayers could then be formed by the dipping method. The dipping method is usually used for forming bilayers in an experimental setup where an aperture in a thin sheet partition (PTFE) is clamped between pools of electrolyte, and the bilayer is formed by lowering and then raising the air-water interface.38 In this apparatus, the entire cuvette was pulled vertically to expose one face of the aperture to the air-water interface held in the cell holder to oppose monolayers. Bilayers were also formed by initially dipping to form a bilayer that was brushed with downward strokes to sufficiently high capacitance. Once a bilayer of sufficient quality was formed, aliquots of the isophthalates (typically 1.5 mM in methanol, trifluoroethanol, or DMSO) were injected with a microliter syringe as closely as possible to the bilayer in the free well of the cuvette holder (cis side). The measured voltage was applied with respect to the trans (cuvette) side of the bilayer, making the trans side the relative ground. Every 10-15 min, successive aliquots of isophthalate (38) Benz, R.; Fro¨hlich, O.; Lau¨ger, P.; Montal, M. Biochim. Biophys. Acta 1975, 394, 323-334.

Fyles et al. were injected and the polarity was switched. For a typical experiment, 10 µL of test solution was injected and a potential of +120 mV was applied for 15 min. At that point, another 10 µL aliquot was injected and a potential of -120 mV was applied for another 15 min. If no activity had been observed by this point, another 10 µL of solution was injected and the polarity was switched for the third time. As soon as activity was observed, data files of current as a function of time were acquired using the Fetchex program of the pClamp 6 suite. The files were imported into Origin 6.0, and low pass filtering was performed at 100 Hz using a fast Fourier transform. Vesicle Experiments. The general methods for vesicle preparation and pH-stat titration have been reported previously.18,21 Vesicles were sized using a LiposoFast membrane apparatus (Avestin, Ontario, Canada) to give a bimodal population of vesicles (80-85% 230 nm diameter, 20-15% 80 nm diameter). A stock solution of the alkoxy isophthalic acid in methanol or DMSO was neutralized with the theoretical amount of choline hydroxide in water to eliminate the initial pH jump following injection to the vesicle solution. Proton efflux rates were determined as previously18,21 in K2SO4 solution (k0 × 109 mol H+ s-1; total transporter concentration, µM): 2 (5.9; 16); 5 (9.3; 16); gramicidin (3.3; 0.013).

Acknowledgment. This work was supported by the Natural Sciences and Engineering Research Council (Canada) and the NATO Collaborative Research Grants Program. LA0105937