Design and Synthesis of Phosphatidylcholine Mimics and Their Mixing

exchangeable phospholipid dimers that mimic phosphatidylcholines significantly expands the scope of the nearest-neighbor recognition method and increa...
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Design and Synthesis of Phosphatidylcholine Mimics and Their Mixing Behavior with Phosphatidylglycerol Mimics in the Fluid Bilayer State Maki Uragami, Yasuhito Miyake, Nobuya Tokutake, Lan-hui Zhang, and Steven L. Regen* Department of Chemistry and Zettlemoyer Center for Surface Studies, Lehigh University, Bethlehem, Pennsylvania 18015 Received July 27, 2000 Two disulfide-based phospholipid dimers have been synthesized (PC14PC14 and PC16PC16), which have packing behavior, melting temperatures, and monomer unit structures that mimic those of 1,2-dimyristoylsn-glycero-3-phosphocholine (DMPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Examination of the mixing behavior of PC16 and an analogous phosphatidylglycerol (PG) mimic, by use of the nearestneighbor recognition method, has revealed that these lipids are ideally miscible in the physiologically relevant fluid phase. Addition of a hydrocarbon chain length mismatch of four methylenes per acyl chain and/or the addition of 29 mol % cholesterol do not alter such miscibility. In contrast, the inclusion of a basic peptide (pentalysine) affords a modest degree of nearest-neighbor recognition. The introduction of exchangeable phospholipid dimers that mimic phosphatidylcholines significantly expands the scope of the nearest-neighbor recognition method and increases its relevance to the study of animal cell membranes, whose two-dimensional structures remain to be elucidated.

Introduction Phospholipids that are present in naturally occurring cell membranes have compositions that are rich in diversity. Three general features that are common to all such lipids include: (i) a polar headgroup, (ii) two hydrocarbon chains, and (iii) a linkage that is composed of ester, amide, and/or ether bonds (Chart 1).1,2 Based on the compositional differences of naturally occurring phospholipids, one can imagine the possibility that cell membranes have two-dimensional organizations in which the lipids are segregated into discrete clusters or microdomains, according to their particular structure.1,3-5 One can also imagine the possibility that these clusters play a key role in many of the most important cellular processes: e.g., fusion, transport, surface recognition, and catalysis. Despite substantial interest in the notion of phospholipid segregation, obtaining evidence for such behavior, even for the simplest of model systems, has proven to be extremely difficult.3-5 In an effort to help clarify the relationship that exists between the structure of phospholipids and their mixing behavior, we have been investigating phospholipid mimics whose miscibility can be judged directly in the physiologically relevant fluid phase by chemical means. Specifically, we have devised a “nearest-neighbor recognition” (NNR) method that involves the equilibration of lipid bilayers made from disulfide-based dimers, using thiolate-disulfide interchange reactions (Chart 2).6,7 When dimer distributions are found to be statistical, such a (1) For a general discussion of each of the features that are noted in Chart 1, see: Gennis, R. B. Biomembranes: Molecular Structure and Function; Springer-Verlag: New York, 1989. (2) Phospholipids Handbook; Cevc, G., Ed.; Marcel Dekker: New York, 1993. (3) Welti, R.; Glaser, M. Chem. Phys. Lipids 1994, 73, 121. (4) Tocanne, J.-F.; Cezanne, L.; Lopez, A.; Piknova, B.; Schram, V.; Tournier, J.-F.; Welby, M. Chem. Phys. Lipids 1994, 73, 139. (5) For a brief review of a special type of lipid domains that has been proposed (i.e., “lipid rafts”), see: Simon, K.; Ikonen, E. Nature 1997, 387, 569. (6) Davidson, S. K. M.; Regen, S. L. Chem. Rev. 1997, 97, 1269.

Chart 1

Chart 2

finding indicates that the monomeric units are randomly distributed throughout the membrane. When homodimers are found to be in excess, this situation (by definition) reflects the presence of nearest-neighbor recognition. If nearest-neighbor interactions are independent of the dimer linkages that exist, then such NNR reflects the (7) For use of related PG-like thiolipid dimers for attachment to gold surfaces, see: Lang, H.; Duschl, C.; Vogel, H. Langmuir 1994, 10, 197.

10.1021/la001065i CCC: $19.00 © 2000 American Chemical Society Published on Web 09/09/2000

Synthesis of Phosphatidylcholine Mimics

presence of lateral heterogeneity. In other words, the phospholipids are nonrandomly arranged within the bilayer. To date, nearly all NNR studies that have been carried out have been based on phospholipids that bear anionic headgroups that mimic phosphatidylglycerols (PGs); the one exception being analogous phospholipids that mimic phosphatidylethanolamines (PEs).8 In all instances, the requisite dimers have been synthesized from the corresponding phosphatidylethanolamines using commercially available coupling agents. From these studies, we have been able to quantify the effects of hydrocarbon chain length mismatch, the presence of unsaturation, chirality, and the inclusion of other membrane components (e.g., nonexchangeable phospholipids of varying length, hydrophobic peptides, and cholesterol) on the mixing behavior of these phospholipid mimics. Although PGs and PEs play an important role in most biological membranes, it is the phosphatidylcholines (PCs) that have been used, most extensively, in model membrane studies. One reason for their popularity is the fact that PCs are major components in animal cell membranes. A second reason relates to their structural simplicity; i.e., mechanistic interpretations can be simplified. In contrast to PGs and PEs, for example, PCs are devoid of functionality in the headgroup that can form hydrogen bonds with other membrane components. Associative interactions, however, are possible between a negatively charged phospholipid and a neighboring PC by electrostatic association with the choline amino cation. For these same reasons, we have been keenly interested in creating a completely new class of exchangeable phospholipids that mimic phosphatidylcholines. In this paper, we report the design and synthesis of two such mimics. We also report their mixing behavior with analogous PG mimics in the absence and in the presence of cholesterol. Motivated by current interest in peripheral proteins, as agents that may affect the lateral organization of phospholipid bilayers, we have also carried out preliminary studies of the mixing behavior of PC and PG mimics in the presence of pentalysinesa hydrophilic peptide, whose strong association with negatively charged phospholipids is presumed to derive from electrostatic interactions.3,9,10 Results Phospholipid Design. Our design of a PC mimic was based on the hypothesis that replacement of a single methyl moiety in the headgroup of a conventional PC with a thioethyl unit would lead to minimal perturbation (Chart 3). The thioethyl unit would then provide a means for dimer interchange reactions, which is essential for NNR experiments. Specific phospholipid dimers that were chosen as synthetic targets are shown in Chart 4. Phospholipids 1 (PC14PC14) and 2 (PC16PC16), bearing (8) (a) Dewa, T.; Vigmond, S. J.; Regen, S. L. J. Am. Chem. Soc. 1996, 118, 3435. (b) Krisovitch, S. M.; Regen, S. L. J. Am. Chem. Soc. 1991, 113, 8175. (c) Inagaki, M.; Shibakami, M.; Regen, S. L. J. Am. Chem. Soc. 1997, 119, 7161. (d) Shibakami, M.; Inagaki, M.; Regen, S. L. J. Am. Chem. Soc. 1997, 119, 12354. (e) Shibakami, M.; Inagaki, M.; Regen, S. L. J. Am. Chem. Soc. 1998, 120, 3758. (f) Uragami, M.; Miyake, Y.; Regen, S. L. Langmuir 2000, 16, 3491. (g) Krisovitch, S. M., Regen, S. L. J. Am. Chem. Soc. 1992, 114, 9828. (h) Uragami, M.; Dewa, T.; Inagaki, M.; Hendel, R. A.; Regen, S. L. J. Am. Chem. Soc. 1997, 119, 3797. (9) Ben-Tal, N.; Honig, B.; Peltzsch, R. M.; Denisov, G.; McLaughlin, S. Biophys. J. 1996, 71, 561. (10) For a review of fluorescence microscopic methods that have been used to visualize lipid domains, including phase separation induced by the interaction of Ca2+ with negatively charged phospholipids, see: Glaser, M. In Comments Molec. Cell. Biophys. 1992, 8 (1, 2), 37.

Langmuir, Vol. 16, No. 21, 2000 8011 Chart 3

Chart 4

myristoyl and palmitoyl groups, respectively, were expected to mimic the packing and melting behavior of 1,2dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Phospholipids 5 (PG16PG16) and 6 (PG18PG18), containing the 3-thiopropionamide moiety in place of a secondary hydroxyl group, were chosen as mimics of phosphatidylglycerols (Charts 3 and 4).8f Phospholipids 3 (PC16PG16) and 4 (PC14PG18) represent the corresponding heterodimers that are necessary in order to produce equilibrium mixtures of dimers starting from the heterodimer side of the equilibrium. In principle, a nearest-neighbor recognition investigation of PC16/PG16-based membranes should provide insight into the effects of headgroup charge on lipid mixing. An examination of closely related PC14/ PG18-based bilayers should then yield information on the influence of a combination of headgroup charge plus a chain length mismatch of four methylene units on lipid mixing. (11) Harbison, G.; Griffin, R. G. J. Lipid Res. 1984, 25, 1140. (12) Findlay, E. J.; Barton, P. G. Biochemistry 1978, 12, 2401. (13) Vigmond, S. J.; Dewa, T.; Regen, S. L. J. Am. Chem. Soc. 1995, 117, 7838. (14) Vist, M. R.; Davis, J. K. Biochemistry 1990, 29, 451. (15) Mosior, M.; McLaughlin, S. Biochim. Biophys. Acta 1992, 1105, 185. (16) Raudino, A.; Zuccarello, F.; Buemi, G. J. Phys. Chem. 1987, 91, 6252. (17) Hartmann, W.; Galla, H.-J.; Sackmann, E. FEBS Lett. 1977, 78, 169. (18) Garidel, P.; Blume, A. Langmuir 2000, 16, 1662.

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Uragami et al. Table 1. Nearest-Neighbor Recognitiona entry 1 2 3 4 5 6 7 8

equilibrating monomers PC16/PG16 PC16/PG16 PC16/PG16 PC14/PG18 PC14/PG18 PC14/PG18 PG14/PG18 PG14/PG18

additive 29% cholesterol 5% pentalysine 29% cholesterol 5% pentalysine 29% cholesterol

heterodimerb homodimer 1.94 ( 0.07 1.98 ( 0.06 1.80 ( 0.07 1.96 ( 0.09 2.08 ( 0.04 1.80 ( 0.09 1.86 ( 0.05 1.59 ( 0.04

a

Chemical equilibrium was generally reached in ca. 6 h at 60 °C. b Molar ratio of heterodimer to the PG homodimer ( one standard deviation from the mean.

Figure 1. Surface pressure-area isotherms for (A) DMPC (left) and PC14PC14 (right) and (B) DPPC (left) and PC16PC16 (right) over a pure water subphase at 25 °C. Scheme 1

Scheme 2

Phospholipid Synthesis. Schemes 1 and 2 outline the synthetic approach that was used to prepare 1 and 2.

In brief, reaction of 2-(dimethylamino)ethanethiol with 2,2′-dithiodipyridine afforded 2-dimethylaminoethylpyridyldisulfide (7). Subsequent quaternization with 2-bromoethanol, anionic replacement with tetraphenylborate (to give 8), and condensation with 1,2-dimyristoyl-snglycero-3-phosphatidic acid or 1,2-dipalmitoyl-sn-glycero3-phosphatidic acid then furnished the activated forms of the desired thiol monomer (9).11 Finally, liberation of the thiol monomer (10), via reduction with dithiothreitol (DTT), and reaction with its precursors gave the corresponding homodimers 1 and 2, respectively.8a The synthesis of 6 has previously been described; the “shorter” homologue, 5, was prepared by similar means.8f Each of the heterodimers, 3 and 4, was synthesized by reacting the appropriate thiol monomer of the PG mimic (i.e., PGSH) with the activated form of the PC thiol monomer (9). PC14PC14 and PC16PC16 as Exchangeable Mimics of DMPC and DPPC. In Figure 1A, we show the monolayer behavior of DMPC and PC14PC14 over a pure water subphase at 25 °C. As is readily apparent, both isotherms exhibit very similar compressibility; the only difference being that the exchangeable dimer occupies approximately twice the area at all surface pressures. The compressibility for PC16PC16 is also very similar to that of DPPC (Figure 1B). In this case, PC16PC16 exhibits a liquid-to-solid transition region that is somewhat extended and less sharply defined as compared with DPPC (Figure 1B). The gel to liquid-crystalline phase-transition temperatures (Tm), which were measured by high sensitivity differential scanning calorimetry (hs-DSC), for DMPC, PC14PC,14 DPPC, and PC16PC16 were 24.0, 21.2, 41.5 and 40.9 °C. On the basis of their similar structures, packing behavior, and melting temperatures, we conclude that PC14PC14 and PC16PC16 are excellent exchangeable mimics of DMPC and DPPC, respectively. Mixing Behavior. Using experimental procedures similar to those previously reported, we measured the equilibrium heterodimer/homodimer ratio for membranes containing PC16/PG16 monomer units (Table 1, entries 1-3).8f Thus, large unilamellar vesicles (0.2-µm average diameter, extrusion method), which were initially composed of either a 1/1 molar ratio of PC16PC16/PG16PG16 or pure PC16PG,16 plus 20 mol % of the corresponding thiol monomers, were allowed to equilibrate at pH 7.0. The Tm values that were measured for PC14PG18, PC16PG16, PG16PG16, and PG18PG18 were 33.6, 39.8, 41.5, and 55.1 °C, respectively. In all cases, equilibration reactions were carried out at 60 °C in order to maintain the physiologically relevant fluid phase. Subsequent quenching (adjusting the pH to 5.0) and analysis of the dimer distributions by HPLC gave equilibrium values that converged at 1.94 ( 0.07. Inclusion of 29% cholesterol did not have a significant influence on the dimer distribution. When 5 mol % of

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pentalysine was included in the dispersion, however, nearest-neighbor recognition (i.e., enhanced homodimer formation) could be detected. In separate studies, binding measurements indicated that more than 95% of the pentalysine was associated with such vesicles under the conditions used (see Experimental Section). Nearestneighbor recognition experiments that were attempted using higher concentrations of pentalysine resulted in precipitation of the lipids from solution. Results that were obtained using membranes composed of PC14/PG18 monomer units were essentially the same as those found with the PC16/PG16 system (entries 4-6). Finally, fluid bilayers derived from PG14/PG,18 exhibited a modest degree of nearest-neighbor recognition. Similar to what has been found with related PG mimics, the inclusion of cholesterol led to an enhancement in nearest-neighbor recognition (entries 7 and 8).8a Discussion The appearance of a statistical equilibrium mixture of dimers derived from PC16/PG16 monomer units provides strong evidence that these exchangeable phospholipids are ideally mixed in the fluid phase. Similar conclusions have been reached for conventional (nonexchangeable) phosphatidylcholines and phosphatidylglycerols in the gelfluid coexistence region, based on DSC analyses, where 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phosphatidylglycerol were found to have identical gel to liquid-crystalline phase transition temperatures.12 Moreover, the width that was observed for the thermal transition for the phase change of an equimolar mixture of the two lipids was not appreciably greater than that observed with either of the two components alone. The fact that statistical equilibrium mixtures of dimers are also produced from PC14/PG18 further indicates that the combination of a chain length mismatch of four methylene units and a difference in headgroup charge are insufficient to produce nonideal mixing. The observation that cholesterol does not induce nearest-neighbor recognition in the PC14/PG18 system is noteworthy. In earlier studies, we have found that cholesterol promotes NNR in “first-generation” PG mimics, which are similar to PG14 and PG18 but which lacked the hydroxymethyl moiety in the headgroup.13 The present finding that cholesterol enhances NNR in PG14/PG18 bilayers is, therefore, an expected result (entries 7 and 8). Our working hypothesis has been that by moving the bilayer from a liquid-crystalline phase toward a condensed β-phase upon addition of cholesterol, tighter packing provides an environment in which the lipids are better able to recognize the chain-length mismatch.13,14 We presume, therefore, that bilayers made from PG14/PG18 behave similarly. The fact that cholesterol does not induce NNR in bilayers made from PC14/PG18 is intriguing. The simplest explanation for this result, we believe, is that in the more compact, cholesterol-rich membrane, a chain length mismatch is balanced by charge repulsion among the PG headgroups. Specifically, whereas the mismatch of the acyl chains favors homodimer formation, the repulsive forces between neighboring, negatively charged PGs favor heterodimer formation, the net result being the ideal mixing that is observed. The nearest-neighbor recognition that has been detected in bilayers made from PC16/PG16 (and also from PC14PG18) in the presence of pentalysine is a likely consequence of electrostatic association with the negatively charged lipids at the membrane-water interface.9,15 Such association

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would then be expected to favor separation of the anionic PG component from the zwitterionic PC analogues.16-18 Conclusions Two exchangeable phosphocholine mimics (PC14PC14 and PC16PC16) have been synthesized for nearest-neighbor recognition studies. On the basis of their overall structure, monolayer properties, and melting temperatures, we conclude that these lipids serve as excellent models for DMPC and DPPC, respectively.19 Examination of the mixing behavior of PC14PC14 and PC16PC16 with analogous PG mimics, by use of NNR methods, has provided strong evidence for ideal mixing when the hydrocarbon chain structures are identical and also when a chain mismatch of four methylene groups exists. Ideal mixing is also apparent when these membranes contain 29 mol % cholesterol. In the presence of a basic and hydrophilic peptide (pentalysine), however, nearest-neighbor recognition could be detected in bilayers made from PC14/PG18 and from PC16/PG16. The introduction of exchangeable phosphatidylcholine mimics, of the type reported herein, significantly expands the scope of the nearest-neighbor recognition method. It has allowed, for the first time, a direct assessment of nearest-neighbor interactions between zwitterionic PClike phospholipids and anionic PG-like phospholipids in the physiologically relevant fluid phase. Of special importance is that these PC mimics increase the relevance of the NNR method to the study of animal cell membranes, whose two-dimensional structures remain to be elucidated. Our efforts in this area are continuing. Experimental Section General Methods. Unless stated otherwise, all reagents were obtained from commercial sources and used without further purification. 1,2-Diacyl-sn-glycero-3-phosphatidic acids (monosodium salt) were obtained from Avanti Polar Lipids (Alabaster, AL). 2,4,6-Triisopropylbenzenesulfonyl chloride (TPSCl) was obtained from Sigma, purified by recrystallization from 1% thionyl chloride in hexane, and dried under vacuum. All 1H NMR spectra were recorded on a Bruker 360-MHz instrument; chemical shifts are reported in ppm and are referenced to residual solvent. Boc(R)-2-amino-3-phenylmethoxy-1-propanol, which was used to prepare the PG mimics, was obtained from Advanced ChemTech Co. (Louisville, KY). Pentalysine was obtained from Sigma Chemicals and used directly. 3-(N-Morpholino)propanesulfonic acid (MOPS) was used as a buffering agent for all nearestneighbor experiments. Specific procedures for the synthesis of 6, PG14PG14, and PG14PG18, as well as the corresponding thiol monomer, have previously been described.8f All nearest-neighbor recognition experiments, DSC analyses, and monolayer measurements were performed at least in duplicate. 2-N,N-Dimethylaminoethyl-2-pyridyl Disulfide (7). To a solution of 2.88 g (13 mmol) of 2,2′-dithiodipyridine in 3.5 mL of CH3OH was added a solution made from 1.85 g (13 mmol) of 2-(dimethylamino)ethanethiol hydrochloride plus 3.5 mL of CH3OH. After stirring the mixture for 15.5 h at room temperature, under an argon atmosphere, the solvent was removed under reduced pressure. The crude yellow product was then dissolved in 100 mL of CHCl3, washed with 30 mL of 10% aqueous NaOH and 30 mL of saturated aqueous NaCl, dried over Na2SO4, filtered, and concentrated under reduced pressure. The product was then purified by column chromatography (EM Science silica, CHCl3 followed by CHCl3/CH3OH, 20/1, v/v) to give 2.19 g (79%) of 7 as a pale yellow oil having Rf 0.34 (silica, CHCl3/CH3OH, 10/1, v/v). 1H NMR (360 MHz, CDCl ) δ: 2.21 (s, 6 H), 2.59 (t, J ) 7.2 Hz, 3 (19) The monolayer behavior of DMPC and DPPC reported herein are fully consistent with previously reported data for these lipids: (a) Longo, M. L.; Bisagno, A. M.; Zasadzinski, J. A. N.; Bruni, R.; Waring, A. J. Science 1993, 261, 453. (b) Gaines, G. L., Jr. Insoluble Monlayers at Liquid-Gas Interfaces, Interscience Publishers: New York, 1966; p 257.

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2 H), 2.89 (t, J ) 7.2 Hz, 2 H), 7.04 (q, J ) 4.3, 7.4 Hz, 1 H), 7.60 (t, J ) 7.2 Hz, 1 H), 7.73 (d, J ) 7.2 Hz, 1 H), 8.42 (d, J ) 5 Hz, 1 H). IR νmax (NaCl) 2943.0, 2818.3, 2773.1, 1568.8, 1453.6, 1419.5, 1288.6, 1120.9, 1050.2, 764.8 cm-1. 13C NMR (90.5 MHz, CDCl3) δ: 160.0, 149.1, 136.6, 120.1, 119.1, 57.7, 44.9, 36.5. 2-[2-(2-Pyridyldithioethyl)-N,N-dimethylamino]ethanol Tetraphenylborate (8). 2-Dimethylaminoethyl pyridyl disulfide (2.0 g, 9.33 mmol) and 2-bromoethanol (1.17 g, 9.33 mmol) were dissolved in 3.0 mL of toluene. The reaction mixture was then stirred for 38 h at room temperature under an argon atmosphere. Subsequent removal of solvent under reduced pressure and column chromatography (silica, CHCl3/CH3OH, 20/1 v/v to pure CH3OH) afforded 1.60 g of a pale yellow product having Rf 0.36 (CHCl3/CH3OH/H2O, 13/6/1 v/v/v). To an aqueous solution of this product (5.0 mL) was added a solution made from of NaBPh4 (1.60 g, 4.68 mmol) and 5.0 mL of water. Subsequent stirring for 30 min at room temperature afforded the desired product, 8, as a colorless powder (2.0 g, 74%), having mp 150151 °C (dec). 1H NMR (360 MHz, DMSO-d6) δ: 3.05 (s, 6 H), 3.24 (m, 2 H), 3.37 (m, 2 H), 3.68 (m, 2 H), 3.79 (m, 2 H), 5.24 (t, J ) 5.0 Hz, 1 H), 6.78 (t, J ) 7.2 Hz, 4 H), 6.91 (t, J ) 7.2 Hz, 8 H), 7.17 (m, 8 H), 7.27 (t, J ) 7.0 Hz, 1 H), 7.73 (d, J ) 7.2 Hz, 1 H), 7.84 (t, J ) 7.5 Hz, 1 H), 8.49 (d, J ) 5.7 Hz, 1 H). IR νmax (KBr) 3530.0, 3049.3, 2998.7, 1573.1, 1474.9, 1453.3, 1419.8, 740.9, 708.3 cm-1. 13C NMR (90.5 MHz, DMSO-d6) δ: 163.0, 149.8, 125.2, 121.5, 119.9, 64.9, 63.1, 54.9, 50.9, 30.5. HRMS for (C11H19O1N2S2)+ calcd: 259.0939. Found: 259.0945. 2-Pyridyl-2-dithioethyl PC14 (9a). 1,2-Dimyristoyl-snglycero-3-phosphate (100.0 mg, 0.16 mmol) was dissolved in 5.0 mL of pyridine and warmed to 35 °C. To this solution was added TPSCl (150.0 mg, 0.48 mmol), followed by addition of 8 (188.6 mg, 0.32 mmol). After stirring the reaction mixture for 4.5 h at 35 °C, 1.0 mL of water was added. The solvent was then removed under reduced pressure and the crude product was purified by column chromatography (EM Science silica, 30 g, CHCl3/CH3OH, 10/1 to CHCl3/CH3OH/H2O, 13/6/1 v/v/v) to give 89.2 mg (67%) of a colorless waxy oil having Rf 0.58 (CHCl3/CH3OH/H2O, 13/6/1 v/v/v). 1H NMR (360 MHz, CDCl3) δ: 0.85 (t, J ) 6.7 Hz, 6 H), 1.22 (s, 40 H), 1.54 (m, 4 H), 2.25 (q, J ) 7.2 Hz, 4 H), 3.29 (m, 2 H), 3.34 (s, 6 H), 3.82∼3.97 (m, 6 H), 4.10 (dd, J ) 7.1, 12.0 Hz, 1 H), 4.31 (m, 2 H), 4.37 (dd, J ) 2.9, 12.0 Hz, 1 H), 5.19 (m, 1 H), 7.16 (t, J ) 6.1 Hz, 1 H), 7.58 (d, J ) 8.0 Hz, 1 H), 7.65 (t, J ) 7.6 Hz, 1 H), 8.47 (d, J ) 4.6 Hz, 1 H). IR νmax (KBr) 2919.8, 2850.4, 1734.1, 1568.4, 1460.9, 1251.6, 1065.6, 670.3 cm-1. 13C NMR (90.5 MHz, CDCl3) δ: 173.1, 158.2, 149.9, 137.3, 121.4, 119.8, 70.4, 64.7, 63.1, 58.8, 51.9, 34.1, 31.8, 31.0, 29.6, 29.2, 24.8, 22.6, 14.0. HRMS for (C42H78O8N2P1S2)+ calcd: 833.4937. Found: 833.4926. 2-Pyridyl-2-dithioethyl PC16 (9b). Using procedures similar that described for the preparation of 9 (m ) 3), the desired product was obtained in a 55% isolated yield, having Rf 0.58 (CHCl3/ CH3OH/H2O, 13/6/1 v/v/v). 1H NMR (360 MHz, CDCl3) δ: 0.85 (t, J ) 6.7 Hz, 6 H), 1.22 (s, 48 H), 1.53 (m, 4 H), 2.24 (q, J ) 7.2 Hz, 4 H), 3.29 (m, 2 H), 3.33 (s, 6 H), 3.82 (m, 2 H), 3.88 (m, 2 H), 3.95 (m, 2 H), 4.09 (dd, J ) 7.1, 12.0 Hz, 1 H), 4.30 (m, 2 H), 4.36 (dd, J ) 2.6, 12.0 Hz, 1 H), 5.18 (m, 1 H), 7.14 (t, J ) 6.0 Hz, 1 H), 7.58 (d, J ) 7.9 Hz, 1 H), 7.64 (t, J ) 7.5 Hz, 1 H), 8.47 (d, J ) 4.6 Hz, 1 H). IR νmax (KBr) 2919.9, 2853.7, 1740.5, 1579.8, 1465.8, 1243.7, 1088.8 cm-1. 13C NMR (90.5 MHz, CDCl3) δ: 173.5, 158.2, 150.0, 137.3, 121.6, 70.3, 65.0, 62.8, 59.3, 52.1, 34.2, 31.9, 31.1, 29.7, 29.1, 24.9, 22.6, 14.1. HRMS for (C46H86O8N2P1S2)+ calcd: 889.5563. Found: 889.5587. PC14PC14 (1). Dithiothreitol (160.2 mg, 1.04 mmol) was dissolved in 5.0 mL of CHCl3, and the resulting solution was cooled to 0 °C. A solution made from of activated monomer (9, m ) 3) (30.0 mg, 0.035 mmol) plus 1.5 mL of CHCl3 was added dropwise to this solution, followed by addition of 1 mL of a CHCl3 rinse. The reaction mixture was stirred for 30 min at 0 °C and then for 1 h at room temperature. Subsequent removal of solvent under reduced pressure afforded a crude oil containing thiol monomer (10), which was purified by column chromatography (silica, 5 g, CHCl3/CH3OH, 10/1 v/v, and CHCl3/CH3OH/H2O, 13/6/1 v/v/v). To a solution of this monomer (1.0 mL of CHCl3) was added a solution made from the activated monomer (9a) (35.0 mg, 0.042 mmol) plus 3.0 mL of CHCl3 at room temperature. The mixture was stirred at room temperature for 30 min and the

Uragami et al. solvent then removed under reduced pressure. Purification by column chromatography (silica, 3 g, CHCl3/CH3OH/H2O, 13/6/1 v/v/v) afforded 38.6 mg (76%) of 1 as a colorless amorphous solid having Rf 0.10 (CHCl3/CH3OH/H2O, 13/6/1 v/v/v). 1H NMR (360 MHz, CDCl3) δ: 0.83 (t, J ) 6.6 Hz, 12 H), 1.22 (s, 80 H), 1.54 (m, 8 H), 2.25 (q, J ) 8.0 Hz, 8 H), 3.34 (m, 16 H), 3.7∼4.0 (m, 12 H), 4.09 (dd, J ) 7.4, 11.6 Hz, 2 H), 4.28 (m, 4 H), 4.37 (d, J ) 10.6 Hz, 2 H), 5.17 (m, 2 H). IR νmax (KBr) 2919.3, 2850.4, 1736.9, 1466.9, 1254.5, 1172.6, 1099.7, 1072.4 cm-1. 13C NMR (90.5 MHz, CDCl3) δ: 173.3, 70.5, 64.3, 63.2, 59.2, 51.7, 34.2, 31.9, 30.6, 29.7, 29.3, 24.8, 22.6, 14.0. HRMS for (C74H147O16N2P2S2)+ calcd: 1445.9667. Found: 1445.9710. PC16PC16 (2). Using procedures similar to that used for the preparation of 1, the analogous homodimer, 2, was obtained in 76% yield, having Rf 0.10 (CHCl3/CH3OH/H2O, 13/6/1 v/v/v). 1H NMR (360 MHz, CDCl3) δ: 0.84 (t, J ) 6.7 Hz, 12 H), 1.22 (s, 96 H), 1.54 (m, 8 H), 2.25 (q, J ) 8.0 Hz, 8 H), 3.35 (m, 16 H), 3.86∼3.91 (m, 12 H), 4.08 (dd, J ) 7.9, 11.0 Hz, 2 H), 4.27 (m, 4 H), 4.37 (d, J ) 10.6 Hz, 2 H), 5.16 (m, 2 H). IR νmax (KBr) 2920.7, 2853.3, 1737.1, 1593.5, 1469.1, 1253.6, 1173.5, 1098.9, 1068.5 cm-1. 13C NMR (90.5 MHz, CDCl3) δ: 173.5, 70.5, 64.5, 63.2, 59.1, 51.9, 34.2, 31.9, 30.7, 29.7, 29.3, 24.9, 22.6, 14.1. HRMS for (C84H163O16N2P2S2)+ calcd: 1558.0919. Found: 1558.0859. PG16PG16 (5). Using procedures similar to those previously described, an activated thiol monomer of PG16SH was prepared, containing the 2-pyridyldithio moiety in 33% yield, starting from the corresponding phosphatidic acid.8f The activated monomer exhibited an Rf 0.60 (CHCl3/CH3OH/H2O, 13/6/1 v/v/v). 1H NMR (360 MHz, CDCl3): δ 0.85 (t, J ) 6.6 Hz, 6 H), 1.22 (m, 48 H), 1.53 (m, 4 H), 2.25 (m, 4 H), 2.62 (m, 2 H), 3.01 (m, 2 H), 3.61 (m, 1 H), 3.73 (m, 1 H), 3.90 (m, 4 H), 4.13 (m, 1 H), 4.36 (d, J ) 11.1 Hz, 1 H), 5.18 (m, 1 H), 7.06 (m, 1 H), 7.62 (m, 2 H), 8.42 (m, 1 H). IR νmax (KBr) 3274.8, 2918.5, 2849.0, 1738.5, 1652.3, 1568.9, 1465.4, 1419.4, 1221.1, 1068.8 cm-1. 13C NMR (90.5 MHz, CDCl3) δ: 173.6, 171.2, 150.9, 149.5, 137.1, 120.8, 119.8, 70.4, 63.5, 62.7, 60.5, 50.7, 35.3, 34.2, 31.9, 29.7, 29.3, 24.8, 22.6, 14.1. HRMS for (C46H82O10N2P1S2)+ calcd: 917.5149. Found: 917.5133. Using procedures similar to those described for the synthesis of 1, the activated thiol monomer was reduced to the corresponding thiol (PG16SH) and then reacted with additional quantity of its precursor (the activated thiol monomer) to yield PG16PG16 in 64%, having Rf 0.50 (CHCl3/CH3OH/H2O, 13/6/1 v/v/v). 1H NMR (360 MHz, CDCl3): δ 0.85 (t, J ) 6.6 Hz, 12 H), 1.23 (m, 96 H), 1.55 (m, 8 H), 2.28 (m, 8 H), 2.61 (m, 4 H), 2.90 (m, 4 H), 3.6 (m, 4 H), 3.92 (m, 6 H), 4.11 (m, 4 H), 4.36 (s, 2 H), 5.19 (m, 2 H). IR νmax (KBr) 3300.0, 2918.3, 2849.8, 1737.8, 1647.8, 1555.1, 1465.1, 1231.8, 1099.7, 1066.0, 846.8 cm-1. 13C NMR (90.5 MHz, CDCl3) δ: 173.7, 171.5, 70.5, 63.6, 62.7, 60.4, 50.9, 35.7, 34.2, 29.7, 29.3, 24.9, 22.6, 14.0. HRMS for (C82H157O20N2P2S2)+ calcd: 1616.0246. Found: 1616.0167. PC16PG16(3). This heterodimer was prepared by reacting PG16SH with 9 (m ) 5), using procedures similar to those described above. The desired product exhibited an Rf 0.27 (CHCl3/ CH3OH/H2O, 13/6/1 v/v/v). 1H NMR (360 MHz, CDCl3): δ 0.85 (t, J ) 6.6 Hz, 12 H), 1.23 (m, 96 H), 1.55 (m, 8 H), 2.27 (m, 8 H), 2.70 (m, 2 H), 3.08 (m, 2 H), 3.29 (m, 8 H), 3.67 (m, 4 H), 3.82 (m, 2 H), 3.90 (m, 6 H), 4.09 (m, 4 H), 4.37 (m, 4 H), 5.18 (m, 2 H). IR νmax (KBr) 3219.4, 2919.0, 2848.8, 1737.0, 1696.3, 1652.7, 1545.2, 1504.5, 1463.8, 1228.4, 1190.6, 1103.4 cm-1. 13C NMR (90.5 MHz, CDCl3) δ: 173.4, 171.1, 70.3, 63.7, 62.8, 60.9, 59.1, 51.9, 35.4, 34.2, 31.9, 29.7, 29.3, 29.2, 24.8, 22.6, 14.0. HRMS for (C82H159O18N2P2S2)+ calcd: 1586.0505. Found: 1586.0581. PC14PG18 (4). This heterodimer was prepared by reacting PG18SH with 9 (m ) 3), using procedures similar to those described above. The desired product exhibited an Rf 0.26 (CHCl3/ CH3OH/H2O, 13/6/1 v/v/v). 1H NMR (360 MHz, CDCl3): δ 0.85 (t, J ) 6.6 Hz, 6 H), 1.23 (m, 92 H), 1.55 (m, 8 H), 2.26 (m, 8 H), 2.69 (m, 2 H), 3.07 (m, 2 H), 3.30 (m, 8 H), 3.67 (m, 4 H), 3.82 (m, 2 H), 3.92 (m, 6 H), 4.08 (m, 4 H), 4.36 (m, 4 H), 5.18 (m, 2 H). IR νmax (KBr) 3453.4, 2921.3, 2853.4, 1737.8, 1647.8, 1622.5, 1465.1, 1243.1, 1172.8, 1105.4, 1068.8 cm-1. 13C NMR (90.5 MHz, CDCl3) δ: 173.5, 170.7, 70.4, 63.7, 62.9, 59.1, 52.0, 35.4, 34.2, 31.9, 29.7, 29.3, 29.2, 24.9, 22.6, 14.0. HRMS for (C82H159O18N2P2S2)+ calcd: 1586.0504. Found: 1586.0476. Nearest-Neighbor Recognition Analysis. In a typical experiment, 0.24 µmol of PC16PC16, 0.24 µmol of PG16PG16, 0.12

Synthesis of Phosphatidylcholine Mimics µmol of PG16SH, and 0.12 µmol of PC16SH in dichloromethane were transferred to a test tube. The dichloromethane was then evaporated under a stream of argon, to give a thin film of the lipids. The test tube was then placed under reduced pressure (0.4 mmHg) for at least 12 h in order to remove residual dichloromethane. After addition of 2.0 mL of a buffer [1 mM 3-(N-morpholino)propanesulfonic acid (MOPS), 10 mM KCl, and 30 µM pentalysine, pH 5.0], the test tube was immersed in a water bath for 30 min at 60 °C. The lipid was dispersed in a buffer via vortex mixing. To prepare a more homogeneous liposomal dispersion, the solution was frozen (77 K), thawed in a water bath (60 °C), and vortex mixed. This procedure was repeated 4 times. The resulting liposomal dispersion was then extruded, sequentially, through 0.4- and 0.2-µm polycarbonate filters. The thiolate-disulfide interchange reaction was initiated after the sample had thermally equilibrated at the desired temperature, by increasing the pH to 7.0 with the addition of 10 µL of 0.15 M KOH. All dispersions were maintained under an argon atmosphere throughout the course of the interchange reaction. Aliquots (0.30 mL) were removed at desired time intervals and quenched with 25 µL of 0.01 M HCl (final pH 5.0). After removal of water under reduced pressure, the resulting white solid was triturated with 2 mL of chloroform and centrifuged, and the chloroform then removed under reduced pressure to yield a clear film. Samples were dissolved in 5 µL of chloroform plus 95 µL of mobile phase (HPLC) prior to injection. Analysis of Dimer Distributions by High-Performance Liquid Chromatography. Mixtures of lipid dimers were analyzed by HPLC using a Beckman Ultrasphere C18 reverse phase column (4.6 × 250 mm, 5-µm particle size). In general, the premixed mobile phase contained 80% 10 mM tetrabutylammonium acetate (TBA) in denatured ethanol, 12% water, and 8% hexane (v/v/v). For the PC14/PG18 system, the premixed mobile phase contained 80% 3 mM tetrabutylammonium acetate (TBA) in denatured ethanol, 12% water, and 8% hexane (v/v/v) was used. The flow rate was 0.9 mL/min, and the column was maintained at 31.2 °C. Peaks were monitored at 205 nm using a Waters 996 photodiode array detector. Data were collected and processed by using a Millennium workstation (Waters Corp.). Retention times for PG16PG16, PC16PG16, PC14PG18, and PG18PG18 were 11.3, 26.5, 10.3, and 14.0 min, respectively (Scheme 2). Binding of Pentalysine. Using procedures similar to those described above, large unilamellar vesicles (200 nm) were prepared from PC16PC16 (0.237 µmol), PG16PG16 (0.237 µmol),

Langmuir, Vol. 16, No. 21, 2000 8015 and PC16PG16 (0.426 µmol) plus 3.0 mL of buffer (1 mM MOPS and 10 mM KCl, pH 7.0). After addition of 15.7 µL of pentalysine (0.045 µmol, 2.859 mM), the vesicle dispersion was incubated for 2 h at 50 °C. A portion of this dispersion (1.5 mL) was then dialyzed against 1.5 mL of the same buffer, using a Spectra/Por Membrane (MWCO 50,000). Aliquots (0.1 mL) were withdrawn as a function of time and mixed with 3.0 mL of a the same buffer containing 20 µL (164 µmol) of fluorescamine. The resulting solution was then stirred for 2 h at room temperature, and its fluorescence intensity measured at λmax 480 nm, using a spectral bandwidth of 15 nm and an excitation wavelength of λex 390 nm. Calibration curves were made by using varying concentrations of pentalysine. Differential Scanning Calorimetry. All calorimetry measurements were performed on a Microcal MC-2 calorimeter with DA-2 data acquisition and analysis software. Multilamellar vesicles were prepared by dispersing a thin lipid film (0.9 µmol) in 1.8 mL of 10 mM borate buffer (140 mM NaCl and 2.0 mM NaN3, pH 7.4), and their melting behavior was measured after four freeze-thaw (-196/+60 °C) cycles, using the same buffer solution as a reference. Heating scans were recorded between 10 and 60 °C at a scan rate of 30 deg/h. A borate buffer baseline was also collected and subtracted from each thermogram. Surface Pressure-Area Isotherms. Surface pressure-area isotherms were recorded by use of an MGW Lauda film balance, which was equipped with a computerized data acquisition station. All isotherms were measured at 25 °C. Water (ca. 1 L), which was used as a subphase, was purified via a Milli-Q filtration system and purged with nitrogen for 15 min. Before addition to the film balance, the surface of this degassed water was aspirated in order to remove surface-active contaminants. All surfactant solutions (1 mg/mL in CHCl3) were spread onto the aqueous subphase having a surface area of 600 cm2, using a gastight, 50-µL Hamilton syringe. Actual concentrations were determined by direct weighing of aliquots after evaporation of solvent using a Cahn 27 electrobalance. In all cases, spreading solvents were allowed to evaporate for at least 30 min prior to compression (2 × 10-3 nm2/molecule‚s) under a flow of nitrogen.

Acknowledgment. We are grateful to the National Institutes of Health (PHS Grant GM56149) for support of this research. LA001065I