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Synthesis and Properties of a Poly-bolyte Fredric M. Menger* and Subramanian Marappan Department of Chemistry, Emory University, 1515 Pierce Drive, Atlanta, Georgia 30322 Received April 19, 2000. In Final Form: June 6, 2000 A “poly-bolyte” containing 11 C22 chains interconnected by 10 catechol units possessing water-solublizing sulfonate groups was synthesized and studied for its solubility, surface activity, dye adsorption, NMR, dynamic light scattering, electron microscopy, calorimetry, and effect on phospholipid bilayers. The amphiphile was not surface active, formed polydisperse vesicles at low concentrations, and did not disrupt phospholipid vesicles, in contrast to a conventional monomeric surfactant of similar structure. The polybolyte is one of a growing number of examples whereby synthetic chemistry is expanding the scope of colloid chemistry.
A “bolaamphiphile” or (more simply) a “bolyte” is defined as a long hydrocarbon chain bearing water-solublizing groups at both termini.1 Bolytes are well-known to assemble into aggregates such as micelles and vesicles.2 The task at hand here was to prepare and examine a polymeric version of a bolyte, that is, a poly-bolyte. Such a poly-bolyte (compound 5) was synthesized according to Scheme 1. It has 11 hydrocarbon chains with 22 carbons each plus multiple sulfonates to impart hydrophilicity. The synthesis was straightforward except for the fact that, to achieve molecular weights exceeding 3000, it was necessary to carry out the polymerization in two steps: (a) 2 was purified and characterized after chromatographically separating it from 1 (99:1 hexanes-EtOAc on silica); (b) 2 was converted into 3 (220 mg of catechol plus 1 g of fused K2CO3 in 20 mL of dry acetone to which was added 1.8 g of 2 in 30 mL of dry acetone with reflux for 48 h). Solvent removal and successive washes of the resulting solid with hexane, benzene, ethyl acetate, and methanol gave 3 as a white solid (1.2 g, 64%). Use of other bases (KOH or NaOH) or other solvents (acetonitrile, methanol, THF, and DMF at either room temperature or reflux) failed to produce sufficiently high molecular weights. A MALDI-TOF mass spectrum of 3 showed a peak at m/z ) 4633.84 (the sole peak above m/z ) 1000), corresponding to the structure of 3 given in Scheme 1. The content of the multiple washes used in the workup of 3 was not investigated. Sulfonation of 3 with SO3-DMF in chloroform at reflux produced 4 and 5. Elemental analysis and NMR indicated 70% of the aromatic rings had been monosulfonated with no disulfonation.3,4 The effect on self-assembly of interconnecting multiple bolyte units in 4 and 5 was difficult to predict (and hence intriguing). As seen in Scheme 2, the molecules could, in principle, fold into lamellar-like and starlike conformations and, thereby, aggregate into vesicles and micelles, respectively. Of course, the union between polymeric and colloidal systems is not new. Ringsdorf et al.5 have reviewed the substantial work on polymeric oriented systems made from polymerizable surfactants. More recent references in the area can be found in a paper dealing with a bolyte-like polymerizable triblock copoly(1) Fuhrhop, J.-H.; David, H.-H.; Mathieu, J.; Liman, U.; Winter, H.-J.; Boekma, E. J. Am. Chem. Soc. 1986, 108, 1785-1791. (2) Fuhrhop, J.-H.; Mathieu, J. J. Chem. Soc., Chem. Commun. 1983, 144-145. (3) Cerfontain, H.; Coenjaarts, N. J.; Koeberg-Telder, A. Recl. Trav. Chim. Pay-Bas 1989, 108, 7-13. (4) The sequence of sulfonates portrayed in Scheme 1 for 4 and 5 is arbitrary. (5) Ringsdorf, H.; Schlarb, B.; Venzmer, J. Angew. Chem., Int. Ed. Engl. 1988, 27, 113-158.
mer.6 But, with the possible exception of cationic ionene polymers,7,8 the amphiphilic framework of 4 and 5 is original. Although poly-bolytes 4 and 5 are insoluble in the common organic solvents (ranging in polarity from hexane to methanol), 5 mg will dissolve in 1 mL of warm water and remain there without precipitating upon cooling. Surface tension studies show that the poly-bolytes are not surface active. Thus, 1 g/L fails to lower the surface tension of water, whereas an equal concentration of a simple analogue, drawn below and henceforth called “ surfactant”, reduces the surface tension from 72 to 30
dyn/cm. Lack of surface activity of 4 and 5, which persists for many hours in undisturbed aqueous solutions, likely reflects a sluggish rate of molecular reorganization at the air/water interface. Pinacyanol chloride, a cationic dye, imparts a pale pink color to solutions of anionic surfactants below their critical micelle concentrations.9 Above the critical micelle con-
centration, 0.01 mM dye has a bright blue color with an absorbance near 600 nm. This remarkable transition is not fully understood, but it is no doubt related to microenvironmental effects and/or dye aggregation.10-12 When pinacyanol chloride was added to various concentrations of our anionic surfactant (Figure 1), the solutions changed from pink to blue as micelles formed above 0.4 mg/mL surfactant. A similar change was observed for 5 (6) Nardin, C.; Hirt, T.; Leukel, J.; Meier, W. Langmuir 2000, 16, 1035-1041. (7) Rembaum, A.; Noguchi, H. Macromolecules 1972, 5, 261-269. (8) Kunitake, T.; Nakashima, N.; Takarabe, K.; Nagai, M.; Tsuge, A.; Yanagi, H. J. Am. Chem. Soc. 1981, 103, 5945-5947. (9) Corrin, M. L.; Klevens, H. B.; Harkins, W. D. J. Chem. Phys. 1946, 14, 480-486. (10) Nemcova, I.; Duskova, M. Collect. Czech. Chem. Commun. 1992, 57, 296-302. (11) Sarkar, M.; Poddar, S. J. Colloid Interface Sci. 2000, 221, 181185. (12) Menger, F. M.; Keiper, J. S. Angew. Chem., Int. Ed. Engl. 2000, 39, 1906-1920.
10.1021/la0005816 CCC: $19.00 © 2000 American Chemical Society Published on Web 07/28/2000
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Letters Scheme 1
Scheme 2
(Figure 1) except that it occurred at much lower concentrations of 5, indicating a greater propensity of the polybolyte to aggregate. Multimethod data overwhelmingly support the presence of vesicular 5 as opposed to micellar 5. Thus, NMR spectra of aggregated 5 (600 MHz, 1 mg/mL in D2O) gave only a highly broadened methylene signal and an almost indiscernible aromatic signal. Micelles of the surfactant analogue, on the other hand, have sharp NMR peaks. Dynamic light scattering (preheated 1 mg/mL of 5 passed through a 200 nm pore-size filter and examined with a Coulter N4 instrument) showed polydisperse paricles 10130 nm in diameter. Electron microscopy (cryo-HRSEM
on an extruded sample with a Cr coating) confirmed the presence of spherical particles in this size range (Figure 2). While such results are traditionally interpreted as signifying “vesicles”, we must point out that there are no hard data proving bilayer-type organization within the particles. Interaction of the poly-bolytes with bona fide phospholipid bilayers was an issue worth investigating. Dispersions of pure 5 have no phase transition from 20 to 90 °C that is detectable by differential scanning calorimetry (DSC), in contrast to dipalmitoylphosphatidylcholine (DPPC), which has a gel-to-liquid crystal transition of 41 °C (Figure 3). Addition of 5 to DPPC multilamellar
Letters
Figure 1. Plot of the absorbance of 1 × 10-5 M pinacyanol chloride at 610 nm versus concentration of surfactant (9) and poly-bolyte 5 (b). Data were recorded within 15 s of dye addition to the sample. Longer times caused an absorbance decrease with the surfactant but not with poly-bolyte. Intense investigation (impurities, aggregation changes, etc.) led to no satisfactory explanation of this decrease.
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Figure 3. (A) DSC heating scan of a multilamellar suspension of DPPC. (B) DSC heating scan of a mixed suspension of polybolyte 5/DPPC (10:90, wt %). The concentration of the aqueous dispersion was 2 mg/mL. Scans were run at 30 °C/h.
Figure 4. Leakage of carboxyfluorescein entrapped in POPC vesicles upon addition of surfactant (9) and poly-bolyte 5 (b). As the dye leaks out, its self-quenching is reduced and fluorescence is enhanced. POPC vesicles were made 2 mg of lipid/mL in 20 mM phosphate buffer (pH 7.5) and 1 mM EDTA. The initial concentration of entrapped dye was 20 mM. Arrows indicate the addition of 0.1 mg/mL surfactant or poly-bolyte. Figure 2. Cryo-HRSEM images of vesicles prepared from the poly-bolyte 5 in water (1 mg/mL).
dispersions (10:90 wt %) caused the sharp endothermic peak at 41 °C to deteriorate into a broad signal at 38 °C. This result suggests an intimate coexistence between 5 and DPPC as opposed to discrete domains of each component within the bilayer that would have allowed the 41 °C peak to remain in tact. Given the results of the previous paragraph, the question arises as to how addition of 5 to a vesicular phospholipid affects the permeability of the bilayer. To address this question, 100 nm vesicles of 1-palmitoyl-2oleoylphosphatidylcholine (POPC) were prepared by extrusion in the presence of 20 mM carboxyfluorescein. Fluorescence dye not encapsulated by the vesicles was removed by elution through a Sephadex G-50 column. When 0.2 mg surfactant was added to 2 mL of this vesicle preparation, the weakened vesicles released dye into bulk water, where dilution destroyed the self-quenching of the dye. As a consequence, the fluorescence intensity was
increased (Figure 4). Yet, as seen, addition of the same weight amount of 5 failed to disrupt the vesicles, once again demonstrating the apparent compatibility of the poly-bolyte with the phospholipid membrane. In summary, a polymeric surfactant was examined whose behavior differs from that of a conventional surfactant with regard to solution and membrane behavior. This poly-bolyte is one of a growing number of structurally novel amphiphiles that are appearing on the scene and expanding the scope of colloid chemistry.12 Acknowledgment. This work was supported by the National Institutes of Health. We appreciate the technical assistance of Dr. Robert Apkarian and Mr. Kevin Caran with the electron microscopy. Supporting Information Available: Details on the synthetic procedures, physical studies, vesicle preparation, and entrapment method. This material is available free of charge via the Internet at http://pubs.acs.org.
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