Double-Chain Surfactants with Two Carboxylate Head Groups That

(a) Glasoe, P. K.; Eberson, L. J. Phys. Chem. 1964, 68, 1560. [ACS Full Text ACS Full Text ], [CAS] . Deuterium isotope effect in the ionization of su...
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Langmuir 1996, 12, 1976-1980

Double-Chain Surfactants with Two Carboxylate Head Groups That Form Vesicles David A. Jaeger* and Ethan L. G. Brown1 Department of Chemistry, University of Wyoming, Laramie, Wyoming 82071-3838 Received October 16, 1995. In Final Form: January 16, 1996X Double-chain, double-head group surfactants, 1, were prepared by dispersal of a 1:1 mixture of diastereomeric diacids 3a (r-2-(carboxymethyl)-2,t-5-dipentadecyl-c-4-(carboxymethyl)-1,3-dioxolane) and 3b (r-2-(carboxymethyl)-2,c-5-dipentadecyl-t-4-(carboxymethyl)-1,3-dioxolane) into pH 9.2 and 10.7 carbonate buffers. The resultant small unilamellar vesicles were characterized by 1H NMR spectroscopy, dynamic laser light scattering, differential scanning calorimetry, and gel filtration chromatography. The vesicles at pH 9.2 were larger and had a greater phase transition temperature than those at pH 10.7. These differences were attributed to a greater fraction of carboxyl groups among the carboxyl and carboxylate head groups at the lower pH.

Introduction Double-chain surfactants with two head groups comprise an active research area.2,3 Examples with dianionic,2 dicationic,2a,3 and dinonionic2a head groups have been reported. The dianionic surfactants contain carboxylate, phosphate, sulfate, or sulfonate groups, the dicationic surfactants contain quaternary ammonium groups, and the dinonionic surfactants contain carbohydrate and amino groups. These surfactants exhibit interesting solution behavior, and, in particular, some dicationic systems display bactericidal activity greater than that of single-chain quaternary ammonium surfactants.3f Most double-chain surfactants with two head groups reported to date form micelles. Only a few form vesicles/ bilayers.2a,b,e,3a The aggregate morphologies of doublechain, double-head group surfactants are of interest because the established relationship4 between surfactant structure and morphology cannot always be routinely applied to them. Herein we report the synthesis and characterization of the aggregates of diastereomeric surfactants 1a and 1b, which contain two carboxylate head groups and two alkyl chains connected by a ketal group.

* To whom correspondence may be addressed: e-mail, [email protected]. X Abstract published in Advance ACS Abstracts, March 15, 1996. (1) Present address: Hickson Danchem Corp., Danville, VA. (2) (a) Bosch, M. A.; Parra, J. L.; Sa´nchez-Baeza, F. Can J. Chem. 1993, 71, 2095. (b) de Groot, R. W.; Wagenaar, A.; Sein, A.; Engberts, J. B. F. N. Recl. Trav. Chim. Pays-Bas 1995, 114, 371. (c) Zhu, Y.-P.; Masuyama, A.; Kobata, Y.; Nakatsuji, Y.; Okahara, M.; Rosen, M. J. J. Colloid Interface. Sci. 1993, 158, 40. (d) Porter, N. A.; Ok, D.; Huff, J. B.; Adams, C. M.; McPhail, A. T.; Kim, K. J. Am. Chem. Soc. 1988, 110, 1896. (e) Ringsdorf, H.; Schlarb, B.; Venzmer, J. Angew. Chem., Int. Ed. Engl. 1988, 27, 113 and references therein. (3) (a) Danino, D.; Talmon, Y.; Zana, R. Langmuir 1995, 11, 1448. (b) Zana, R.; Talmon, Y. Nature 1993, 362, 228. (c) Menger, F. M.; Littau, C. A. J. Am. Chem. Soc. 1993, 115, 10083. (d) Devı´nsky, F.; Lacko, I.; Imam, T. J. Colloid Interface. Sci. 1991, 143, 336. (e) Abid, S. K.; Hamid, S. M.; Sherrington, D. C. J. Colloid Interface. Sci. 1987, 120, 245. (f) Imam, T.; Devı´nsky, F.; Lacko, I.; Mlynarcik, D.; Krasnec, L. Pharmazie 1983, 38, 308 and references therein. (4) (a) Israelachvili, J. N.; Mitchell, D. J.; Ninham, B. W. J. Chem. Soc., Faraday Trans. 2 1976, 72, 1525. (b) Israelachvili, J. N. Intermolecular and Surface Forces, 2nd ed.; Academic Press: New York, 1992; Chapter 17.

0743-7463/96/2412-1976$12.00/0

Results Synthesis. The synthesis of surfactants 1 is outlined in Scheme 1. The reaction of threo diol ester 4 with β-keto ester 5 gave diastereomeric diester ketals 2. In the 1H NMR spectrum of 2 (see Experimental Section), four singlets of equal intensity were observed for the CO2CH3 groups, corresponding to a 1:1 mixture of 2a and 2b. In the 13C NMR spectrum of 2, four equal signals for CO2CH3, and two equal signals for C-2 of the dioxolane rings also indicated a 1:1 mixture of 2a and 2b.5 This mixture was converted into diacid ketals 3. In the 1H NMR spectrum of 3 (see Experimental Section), two singlets of equal intensity were observed for CH2CO2H, corresponding to a 1:1 mixture of 3a and 3b. In the 13C NMR spectrum of 3, two equal signals for C-2 of the dioxolane rings also indicated a 1:1 mixture of 3a and 3b. A 1:1 mixture of surfactants 1a and 1b was generated in situ by the dispersal of 3 into a pH 9.2 or 10.7 KHCO3-K2CO3 buffer.6 Carbonate buffers with K+ rather than Na+ as counterion were used because the potassium surfactants were much more soluble than the sodium surfactants. Surfactant Characterization. Surfactant 1 was characterized by Krafft temperature (Tk) and critical aggregation concentration (cac) measurements. Aggregated 1 was characterized by 1H NMR spectroscopy, dynamic laser light scattering (DLLS), differential scanning calorimetry (DSC), and gel filtration chromatography. Unless noted otherwise, aggregated 1 was prepared by sonicating a mixture of 3 and a pH 9.2 or 10.7 buffer at 25 °C, heating it to 70 °C, and allowing it to cool to 25 °C, to give clear but translucent solutions. Tk and cac Measurements. Surfactant 1 did not precipitate at 25 °C from solutions prepared with either the pH 9.2 or 10.7 buffer. Therefore, the Tk values of 1 in the two buffers are likely e25 °C. However, the possibility that the Tk values are >25 °C, with 1 giving kinetically stable supersaturated solutions at 25 °C, cannot be discounted. The cac values of 1 were determined in the pH 9.2 and 10.7 buffers by a fluorescence probe method7 with 1,6diphenyl-1,3,5-hexatriene (DPH) as probe. The initial break in a plot of the relative fluorescence of DPH vs the (5) In comparisons of 13C NMR signals of diastereomers, it is reasonably assumed that the T1s and NOEs of the carbons involved are comparable, thus allowing quantitation based on their intensities. (6) On dissolution of 3 into the carbonate buffers, deprotonation of the two carboxyl groups is likely incomplete at pH 9.2 and may or may not be complete at pH 10.7 (see Discussion). In any event, the resulting system is designated as 1. (7) (a) Chattopadhyay, A.; London, E. Anal. Biochem. 1984, 139, 408. (b) Goodling, K.; Johnson, K.; Lefkowitz, L.; Williams, B. R. J. Chem. Ed. 1994, 71, A8.

© 1996 American Chemical Society

Double-Chain Surfactants

Langmuir, Vol. 12, No. 8, 1996 1977

Scheme Ia

a Key: (a) p-MeC H SO H, C H (Dean-Stark); (b) KOH, 6 4 3 6 6 MeOH; (c) HO2CCO2H, H2O; (d) KHCO3-K2CO3 buffer.

Figure 2. DLLS histograms of hydrodynamic diameter vs relative volume for 1 in the (a) pH 9.2 and (b) 10.7 buffers.

Figure 1. Plots of relative fluorescence vs the log of the concentration of 1 in the (a) pH 9.2 and (b) 10.7 buffers.

log of the concentration of 1 corresponds to the cac. Representative plots are given in Figure 1. This method is based on the partitioning of DPH from the bulk aqueous solution, below the cac, into surfactant aggregates, at and above the cac. A substantial increase in the fluorescence quantum yield of DPH accompanies this change in microenvironment. In each fluorescence measurement the buffer contained 5.0 × 10-7 M DPH and 0.010 vol % THF, which was present to facilitate complete solubilization of the probe below the cac. The cac values were (2.2 ( 0.6) × 10-6 and (2.0 ( 0.6) × 10-6 M in the pH 9.2 and 10.7 buffers, respectively. cac values comparable to these were obtained in the two buffers containing 1.5 × 10-5 M DPH and 0.10 vol % THF. Thus it is apparent that the probe is not seeding8 1’s aggregation. 1H NMR Spectroscopy. In the synthesis of 3, the saponification of 2 with KOH in MeOH (Scheme 1) was (8) Wang, G.-J.; Engberts, J. B. F. N. Langmuir 1994, 10, 2583.

followed by rotary evaporation of MeOH to give a solid residue that contained 1 and excess KOH. The 1H NMR spectrum of this material in D2O contained no discernible signals for 1. The lack of signals, due to line width broadening, is consistent with the absence of micelles and with the presence of vesicles, or other large aggregates.9 DLLS. Aggregates of surfactant 1 prepared in the two buffers were analyzed by DLLS (90˚ scattering angle) at 25 °C. In each buffer, distribution analysis of the autocorrelation function established the presence of two populations with the following hydrodynamic diameters and relative volumes: at pH 9.2, 36 ( 4 nm (97 ( 1 vol %) and 170 ( 8 nm (3 ( 1 vol %); at pH 10.7, 22 ( 1 nm (98 ( 2 vol %) and 158 ( 43 nm (2 ( 2 vol %). Representative histograms of hydrodynamic diameter vs relative volume (mass) for 1 in the pH 9.2 and 10.7 buffers are given in Figure 2. The procedure for preparation of solutions in the two buffers includes a step in which the samples are heated to 70 °C (see above). If this step is deleted, the resultant solutions by DLLS analysis contain unstable multimodal distributions of aggregates. However, after 3 days at 25 °C the solutions give results comparable to those of Figure 2. DSC. Thermograms were obtained for aggregates of 1 in the pH 9.2 and 10.7 buffers. The phase transition temperatures (Tc) and calorimetric enthalpies (∆Hcal) were as follows: at pH 9.2, Tc ) 48.6 ( 0.3 °C and ∆Hcal ) 20.6 (9) Browning, J. L. In Liposomes: From Physical Structure to Therapeutic Applications; Knight, C. G., Ed.; Elsevier/North Holland: New York, 1981; Chapter 7.

1978 Langmuir, Vol. 12, No. 8, 1996

Figure 3. DSC thermograms for 1 in the pH 9.2 and pH 10.7 buffers.

( 1.8 kJ/mol; at pH 10.7, Tc ) 39.8 ( 0.4 °C and ∆Hcal ) 23.7 ( 0.8 kJ/mol. At its Tc, a bilayer undergoes a transition from the gel to the less-ordered liquid crystalline state, corresponding to conformational changes of the alkyl groups.10 Micelles would not be expected to display phase transitions. Representative thermograms are given in Figure 3. Gel Filtration Chromatography. Aggregated 1 was prepared in the pH 10.7 buffer containing 0.87 mM 5(6)carboxyfluorescein (CF),11 a water-soluble dye. The resultant solution was subjected to gel filtration chromatography12 on Sephadex G-25 with the pH 10.2 buffer as eluant. A portion of CF eluted at the void volume of the column. Discussion In the DLLS results the major population in each buffer is assigned to small unilamellar vesicles (SUVs), which generally display a size range of 20-50 nm.13 Spherical micelles, which typically have a hydrodynamic diameter of ca. 3 nm,14 are not a reasonable possibility for the major population. No physical significance can be attributed to the minor population in each buffer. It probably corresponds to a few large particles that were grouped together as part of the bimodal distribution by the mathematical analysis of the light scattering data. The phase transitions observed in the DSC thermograms of Figure 3 for the dilute solutions of 1 are consistent with bilayer membranes and not with micelles.15 The elution of a portion of CF at the void volume in the gel filtration chromatography of sonicated solutions containing both 1 and CF is consistent with, but does not require, the presence of closed bilayer vesicles.16 Taken as a whole, the results from 1H NMR (10) Wilkinson, D. A.; Nagle, J. F. In ref 9; Chapter 9. (11) Weinstein, J. N.; Ralston, E.; Leserman, L. D.; Klausner, R. D.; Dragsten, P.; Henkart, P.; Blumenthal, R. In Liposome Technology; Gregoriadis, G., Ed.; CRC Press: Boca Raton, FL, 1984; Vol. 3, p 183. (12) Weinstein, J. N.; Yoshikami, S.; Henkart, P.; Blumenthal, R.; Hagins, W. A. Science (Washington, D.C.) 1977, 195, 489. (13) Fendler, J. H. Membrane Mimetic Chemistry; Wiley-Interscience: New York, 1982; Chapter 6. (14) Briggs, J.; Dorshow, R. B.; Bunton, C. A.; Nicoli, D. F. J. Chem. Phys. 1982, 76, 775. (15) However, endotherms due to clusters of micelles have been observed in DSC thermograms of moderately concentrated solutions of single-chain surfactants: Blandamer, M. J.; Briggs, B.; Burgess, J.; Butt, M. D.; Brown, H. R.; Cullis, P. M.; Engberts, J. B. F. N. J. Colloid Interface Sci. 1992, 150, 285. (16) CF could be adsorbed to the surface of open bilayers. For an example in a related system, see: Carmona-Ribeiro, A. M.; Chaimovich, H. Biochim. Biophys. Acta 1983, 733, 172.

Jaeger and Brown

spectroscopy, DLLS, DSC, and gel filtration chromatography are consistent with the formation of only vesicles from 1 in the pH 9.2 and 10.7 buffers. On going from pH 10.7 to 9.2, the fraction of carboxyl groups among the carboxyl and carboxylate head groups almost certainly increases (see below), resulting in lesser electrostatic repulsion among them. As a consequence of lesser head group repulsion, there is a decrease in aggregate curvature and thus a larger aggregate size4 at the lower pH. An increase in the efficiency of alkyl chain packing also accompanies these changes, reflected by an increase in Tc on going from pH 10.7 to 9.2. Engberts and co-workers2b have also detected an increase in the size of SUVs of a double-chain surfactant with two carboxylate head groups on going from higher to lower pHs. Vesicles have been prepared from C8 to C18 singlechain saturated and unsaturated carboxylic acids above their Tc values in aqueous solutions at pHs where the carboxyl groups are not completely deprotonated.17 Thus both carboxyl and carboxylate head groups are present within the vesicles. The importance of hydrogen-bonding among these head groups as a structural feature of the vesicles has been disputed.17ab,18 The pH range over which vesicles are formed is a function of temperature and the hydrocarbon chain length and concentration of the carboxylic acid.17 We have made vesicles from diacids 3 above pH 10 and below Tc, in contrast to the conditions required for vesicle formation from single-chain carboxylic acids.17 At temperatures below Tc, vesicles could not be prepared from any of the single-chain carboxylic acids studied, and at pH >10, they formed micelles and not vesicles.17 Each of the diacids 3a and 3b in vesicular form will display two pKas, corresponding to ionization of its two carboxyl groups. Since these four pKa values are unknown, the extents of deprotonation of 3a and 3b at pH 9.2 and 10.7 are unknown. The apparent pKas of dodecanoic and oleic acids in bilayer form are 8.0 and 8.0-8.5,17a respectively, compared to a typical pKa value of 4.8 for a monomeric long-chain carboxylic acid in water.19 If the first and second pKas of vesicular 3a and 3b are e8.5, deprotonation is incomplete at pH 9.2 and essentially complete at pH 10.7. However, if a second pKa is substantially greater than 8.5, deprotonation of 3 is incomplete even at pH 10.7. Only with incomplete deprotonation could there be hydrogen bonding among carboxyl and carboxylate head groups. If deprotonation is in fact complete at pH 10.7, the formation of vesicles from 3 contrasts with the formation of only micelles from fully deprotonated single-chain carboxylic acids,17 as noted above. In this case, the covalent attachment of two hydrocarbon chains within 3, to give, in effect, a dimerlike structure, must be largely responsible for the formation of vesicles with only carboxylate head groups. It is possible that the second pKa of 3a and/or 3b is >8.5. The second pKa of a dicarboxylic acid can indeed be quite large.20,21 For example, the second pKa of d,l(17) For examples, see: (a) Cistola, D. P.; Hamilton, J. A.; Jackson, D.; Small, D. M. Biochemistry 1988, 27, 1881. (b) Edwards, K.; Silvander, M.; Karlsson, G. Langmuir 1995, 11, 2429. (c) Hargreaves, W. R.; Deamer, D. W. Biochemistry 1978, 17, 3759. (d) Wick, R.; Walde, P.; Luisi, P. L. J. Am. Chem. Soc. 1995, 117, 1435 and references therein. (18) Haines, T. H. Proc. Natl. Acad. Sci. U.S.A. 1983, 80, 160. (19) Cistola, D. P.; Small, D. M.; Hamilton, J. A. J. Biol. Chem. 1987, 262, 10971. (20) (a) Glasoe, P. K.; Eberson, L. J. Phys. Chem. 1964, 68, 1560. (b) Glasoe, P. K.; Hutchison, J. R. J. Phys. Chem. 1964, 68, 1562. (c) Eyring, E. M.; Haslam, J. L. J. Phys. Chem. 1966, 70, 293. (d) Haslam, J. L.; Eyring, E. M.; Epstein, W. W.; Jensen, R. P.; Jaget, C. W. J. Am. Chem. Soc. 1965, 87, 4247. (21) Rebek, J., Jr.; Duff, R. J.; Gordon, W. E.; Parris, K. J. Am. Chem. Soc. 1986, 108, 6088.

Double-Chain Surfactants

2,3-di-tert-butylsuccinic acid in H2O at 25 °C is 10.25.20a The surprisingly low acidity of the monoanion of this acid derives from intramolecular hydrogen bonding between its carboxyl and carboxylate groups, which is sterically enforced by the tert-butyl groups. The apparent second pKa of d,l-2,3-di-tert-butylsuccinic acid in the microenvironment presented by the interface of an anionic vesicle would be even higher. Intramolecular hydrogen bonding is possible between the cis carboxyl and carboxylate groups of the monoanion of 3a, but not between the trans groups of the monoanion of 3b. It is interesting to speculate about the dispositions of 1 within a bilayer membrane. There are two limiting orientations of the 1,3-dioxolane ring with respect to an aggregate-water interface: parallel and perpendicular. With the former arrangement, both carboxyl/carboxylate head groups and the two pentadecyl chains of 1a can readily extend into their preferred microenvironments, water and bilayer, respectively. For 1b, only one head group and one alkyl chain can extend directly into their preferred microenvironments. With a perpendicular orientation of the 1,3-dioxolane ring, both carboxyl/ carboxylate groups and the two pentadecyl chains of both 1a and 1b can readily extend into their preferred microenvironments. Overall, for 1 in the parallel orientation, a bilayer should more readily accommodate 1a than 1b, whereas for 1 in a perpendicular orientation, a bilayer should readily accommodate both 1a and 1b. Surfactant 1 in principle represents a cleavable surfactant22 since it contains a ketal group, which is stable under neutral and basic conditions but labile under acidic conditions. The cleavable nature of 1 was not demonstrated because upon acidification of an aqueous solution of 1, water-insoluble diacid 3 would precipitate before hydrolysis of the ketal group.

Langmuir, Vol. 12, No. 8, 1996 1979

General Procedures and Materials. 1H (270 and 400 MHz) and 13C (67.9 and 100.6 MHz) NMR spectra, unless noted otherwise, were recorded in CDCl3 with Me4Si and CDCl3 (center line at 77.00 ppm relative to Me4Si) as internal standards, respectively. 1H NMR spectra recorded in DMSO-d6 and D2O employed CD3SOCD2H (δ 2.49) and Me3Si(CH2)2CO2Na as internal standards, respectively. J values are in hertz. Infrared spectra were recorded with Nujol mull samples between NaCl plates. The high-resolution FAB mass spectrum was obtained at the Washington University Resource for Biomedical and Bioorganic Mass Spectrometry. Sonication was performed with a Branson 2200 (125 W) ultrasonic cleaner. Flash and open column chromatography on silica gel were performed as previously described.23 The buffers were prepared as follows with HPLCgrade H2O: pH 9.2 (I ) 0.030), a mixture 50.0 mL of 0.050 M KHCO3 and 2.6 mL of 0.10 M KOH was diluted to 100 mL with HPLC-grade H2O; pH 10.7 (I ) 0.035), a mixture of 50.0 mL of 0.050 M KHCO3 and 22.7 mL of 0.10 M KOH was diluted to 200 mL. Solutions were dried over Na2SO4. Melting points are uncorrected. Elemental analyses were performed by Atlantic Mircolab, Atlanta, GA. In general, yields were not maximized.

Tk Measurements. A mixture of 1.0 mg of diacids 3 and 1.0 mL of the pH 9.2 or 10.7 buffer was sonicated at 25 °C for 15 or 5 min, respectively, to disperse the solid diacids, and then it was heated to 70 °C to effect the deprotonation to yield 1. The resulting solution was then allowed to cool to 25 °C. Since with each buffer the solution remained clear indefinitely at 25 °C, the Tk values in the two buffers are likely e25 °C, although it is possible that the solutions at 25 °C are supersaturated but kinetically stable. cac Measurements. Modified literature procedures7 were followed. A given solution of DPH (Aldrich) (5.5 × 10-3 M) in spectral-grade THF was stored at 5 °C and used for no longer than 3 days. A 10.0 µL portion of the DPH solution was diluted to 100 mL with buffer to give a stock solution used in a cac measurement. The initial surfactant solution (4.0 × 10-4 M) for a cac measurement was prepared as follows. A 2.4 mg (4.0 mmol) portion of 3 was added to 10.0 mL of buffer, and the mixture was sonicated at 25 °C until the large particles dissolved (ca. 8 min), and then it was held at 70 °C for 5 min and allowed to cool to 25 °C. Then 1.0 µL the probe stock solution was added to the resultant solution to give the initial solution. Solutions with lower [1] were prepared by sequential dilution with the stock solution. Prior to a fluorescence measurement, a labeled surfactant solution was allowed to stand in the dark at 25 °C for 1 h to ensure that the probe was completely solubilized. Fluorescence measurements were made with a Perkin-Elmer LS-5 fluorescence spectrophotometer. The excitation wavelength was 358 nm and the emission wavelength 250 nm. The excitation and emission slit widths were 3 and 5 nm, respectively. Measurements were made at room temperature using a waterjacketed, 1-cm path length quartz cell (NSG Precision Cells T-54FL). The cell, filled with a probe-containing surfactant, was kept in the dark in the spectrophotometer for 15 min before the excitation shutter was opened and the fluorescence intensity measured. The cac values reported in the Results are averages of two runs with different samples. DLLS. Measurements were made at 25 °C on the instrumentation described previously.23 The surfactant solutions for DLLS analysis were prepared as follows. A 0.96 mg (1.6 mmol) portion of 3 was added to 4.0 mL of buffer, and the mixture was sonicated at 25 °C until the large particles dissolved (ca. 8 min), and then it was held at 70 °C for 5 min and allowed to cool to 25 °C. After standing at 25 °C for 1 h, the resultant solution was filtered through a 5-µm filter (Millipore SVLP01300) and then through a 0.45-µm filter (Millipore SJHV004NS) into a 6-mm × 50-mm culture tube (Kimble 73500-650). The tube was capped (5-mm NMR tube cap) and centrifuged for 10 min at 12 000 rpm. Immediately thereafter, the capped tube was inserted into the particle sizer and the run begun with a photopulse rate of 300400 kHz. Data were analyzed by Nicomp distribution analysis software (version 12.3), which gave histograms of relative volume vs diameter. Runs with and without centrifugation gave the same results. The hydrodynamic diameters and vol % values reported in the Results are averages of three runs with different samples. DSC. Calorimetry was performed on a Hart Scientific Model 7708 differential scanning calorimeter. Scans were made from 23 to 73 °C and from 73 to 23 °C at 1 °C/min. Each transition was reversible as evidenced by the same Tc value for successive up-scans. The values of ∆Hcal were determined by integration of the thermograms.24 Measurements were made with 0.65-g portions of surfactant samples prepared as follows. To 2.0 mg (3.4 mmol) of 3 was added 2.00 or 1.00 mL of the pH 9.2 or pH 10.7 buffer, respectively. The mixture was then sonicated at 25 °C until the large particles dissolved (ca. 5-8 min). The Tc and ∆Hcal values reported in the Results are averages of three runs with different samples. Gel Filtration Chromatography. Sephadex G-25 (medium, 50-150 µm, Pharmacia) was allowed to swell in the pH 10.7 buffer for 3 h and was then degassed for 30 min at 20 mmHg. A 1.0-cm × 8.0-cm column of the resultant slurry was washed with the pH 10.7 buffer. The column outflow was attached to an ISCO UA-5 absorbance detector (254 nm). The void volume of the column (5.0 mL) was determined by chromatography of

(22) Jaeger, D. A. Supramol.Chem. 1995, 5, 27. (23) Jaeger, D. A.; Sayed, Y. M. J. Org. Chem. 1993, 58, 2619.

(24) (a) McElhaney, R. N. Chem. Phys. Lipids 1982, 30, 229. (b) Takahashi, K.; Sturtevant, J. M. Biochemistry 1981, 20, 6185.

Summary A 1:1 mixture of surfactants 1a and 1b in pH 9.2 and 10.7 carbonate buffers forms SUVs, which were characterized by 1H NMR spectroscopy, DLLS, DSC, and gel filtration chromatography. The SUVs at pH 9.2 were larger and had a greater Tc than those at pH 10.7. These differences were ascribed to a greater fraction of carboxyl groups among the carboxyl and carboxylate head groups at pH 9.2 than at the higher pH. Experimental Section

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Jaeger and Brown

0.10 mL of an aqueous 1% (w/w) solution of blue dextran (Sigma, average MW 2 000 000) with the pH 10.7 buffer as eluant. Samples for chromatography were prepared as follows. A mixture of 0.99 mg (0.0026 mmol) of 5(6)-carboxyfluorescein, 10.0 mg (0.0724 mmol) of K2CO3, and 3.0 mL of the pH 10.7 buffer was shaken until all of the solid dissolved. Then 12.5 mg (0.021 mmol) of 3 was added, and the mixture was sonicated for 10 min at 25 °C. After the resultant solution sat at 25 °C for 15 min, a 1.0 mL aliquot was subjected to chromatography with the pH 10.7 buffer as eluant. trans-2-Octadecenoic Acid. By the literature procedure,25 octadecanoic acid (Aldrich) was converted (26%) into the title compound: mp 54-55 °C (lit.25 mp 55 °C); 1H NMR (270 MHz) δ 7.08 (d of t, J ) 15.8, 6.6, 6.6, 1 H, CH2CHdCH), 5.82 (d, J ) 15.8, 1 H, CH2CHdCH), 2.22 (q, J ) 6.6, 2 H, CH2CHdCH), 1.46 (m, 2 H, CH2CH2CH), 1.26 (s, 24 H, (CH2)12), 0.88 (t, 3 H, CH3). threo-2,3-Dihydroxyoctadecanoic Acid. By a modified literature procedure,25 the above acid was converted into the title compound. A mixture of 5.60 g (19.8 mmol) of trans-2octadecenoic acid, 7.40 g (43.6 mmol) of dry AgNO3, 5.00 g (19.7 mmol) of I2, and 130 mL of glacial MeCO2H was stirred at 25 °C for 2 h, followed by the addition of 0.400 mL (22.2 mmol) of H2O. The reaction mixture was refluxed for 2 h and then cooled to 25 °C. The precipitate was removed by filtration, MeCO2H was removed by rotary evaporation, and the solid residue was dissolved in Et2O. The Et2O solution was washed with 6 N HCl and filtered, and then it was washed with H2O and saturated aqueous NaCl, followed by rotary evaporation. A mixture of the solid residue, 8.4 g (0.15 mol) of KOH, and 50 mL of 1:1 (v/v) EtOH-H2O was refluxed for 1 h and then added to a solution of 14 mL of concentrated HCl in 250 mL of H2O. This mixture was held at 0 °C for 30 min and then filtered. The resultant solid was washed with Et2O, collected by filtration, and recrystallized (0 °C) from EtOAc to yield 0.738 g (12%) of the title compound: mp 123-125 °C (lit.25 mp 125 °C); 1H NMR (270 MHz, DMSO-d6) δ 4.71 (br s, 1 H, OH), 4.41 (br s, 1 H, OH), 3.81 (d, J ) 2.6, 1 H, CH(OH)CO2H), 3.64 (m, 1 H, CH2CH(OH)), 1.38 (m, 2 H, CH2CH(OH)), 1.23 (br s, 26 H, (CH2)13), 0.84 (t, 3 H, CH3). Methyl threo-2,3-Dihydroxyoctadecanoate (4). A mixture of 0.387 g (1.22 mmol) of the above diol acid, 5 drops of concentrated H2SO4, and 70 mL of MeOH was refluxed for 4 h and cooled to 25 °C. Then the H2SO4 was neutralized with solid NaHCO3, and the mixture was filtered and rotary evaporated. An Et2O solution of the residue was washed with 10% NaHCO3 and dried. The ether was rotary evaporated to give 0.298 g (74%) of product that was recrystallized (25 °C) from hexane to give 4: mp 94-95 °C; 1H NMR (400 MHz) δ 4.11 (d, J ) 2.9 Hz, 1 H, CH(OH)CO2CH3), 3.89 (m, 1 H, CH2CH(OH)), 3.84 (s, 3 H, CH3O), 3.04 (d, J ) 4.9, 1 H, OH), 1.89 (d, J ) 8.8, 1 H, OH), 1.60 (m, 2 H, CH2CH(OH)), 1.26 (s, 26 H, (CH2)13), 0.88 (t, 3 H, CH3). Anal. Calcd for C19H38O4: C, 69.05; H, 11.59. Found: C, 68.91; H, 11.49. Methyl 3-Oxooctadecanoate (5).26 By a literature procedure,27 MeCOCH2CO2Me and C14H29Br (Aldrich) were converted

into 5: mp 48-49 °C (lit.26 46.2-48.0 °C); 1H NMR (400 MHz) δ 3.74 (s, 3 H, CH3O), 3.46 (s, 2 H, CH2CO2CH3), 2.53 (t, J ) 7.6, 2 H, CH2CO), 1.59 (m, 2 H, CH2CH2CO), 1.25 (s, 26 H, (CH2)13), 0.89 (t, 3 H); 13C NMR (100.6 MHz) δ 202.75, 167.67, 52.26, 49.01, 43.08, 31.92, 29.65, 29.43, 29.35, 29.01, 23.49, 22.68, 14.08. r-2-(Methoxycarbonylmethyl)-2,t-5-dipentadecyl-c-4(methoxycarbonyl)-1,3-dioxolane (2a) and r-2-(Methoxycarbonylmethyl)-2,c-5-dipentadecyl-t-4-(methoxycarbonyl)1,3-dioxolane (2b). A mixture of 0.229 g (0.693 mmol) of 4, 0.14 g (0.448 mmol) of 5, 50 mg of p-MeC6H4SO3H‚H2O, and 6 mL of C6H6 was refluxed under a Dean-Stark trap and N2 for 25 h. The reaction mixture was then diluted with 25 mL of C6H6, washed with aqueous 10% NaHCO3, and dried. Rotary evaporated left an oil that was flash chromatographed on a 3-cm × 25-cm column of silica gel packed in, and eluted with, 2:1 (v/v) hexane-Et2O to yield 0.213 g (76%) of a 1:1 mixture of 2a and 2b as an oil: 1H NMR (400 MHz) δ 4.04-4.24 (m, 4 H, 2 CH(O)CH(O)), 3.78 (s, 3 H, CH3O), 3.77 (s, 3 H, CH3O), 3.68 (s, 3 H, CH3O), 3.67 (s, 3 H, CH3O), 2.79 (AB, J ) 14.1, 2 H, CH2CO2CH3), 2.73 (s, 2 H, CH2CO2CH3), 1.62-1.90 (m, 8 H, 2 CH2CH(O)CH(O) and 2 CH2C(O)(O)), 1.26 (s, 104 H, 4 (CH2)13), 0.88 (t, 12 H, 4 CH3); 13C NMR (67.8 MHz) δ 170.88 (CO2CH3), 170.53 (CO2CH3), 169.67 (CO2CH3), 169.47 (CO2CH3), 111.46 (C(O)(O)), 111.22 (C(O)(O)), 79.98, 79.56, 79.30, 52.25, 51.54, 42.99, 42.68, 38.30, 37.87, 33.00, 32.87, 31.87, 29.64, 29.52, 29.42, 29.30, 25.52, 25.41, 23.69, 22.87, 22.62, 14.03. Anal. Calcd for C38H72O6: C, 73.03; H, 11.61. Found: C, 72.80; H, 11.62. r-2-(Carboxymethyl)-2,t-5-dipentadecyl-c-4-(carboxymethyl)-1,3-dioxolane (3a) and r-2-(Carboxymethyl)-2,c-5dipentadecyl-t-4-(carboxymethyl)-1,3-dioxolane (3b). A mixture of 0.486 g (0.778 mmol) of 2, 1.3 g (23 mmol) of KOH, and 25 mL of MeOH was stirred 25 °C under N2 for 16 h. The MeOH was rotary evaporated to leave a solid that was dissolved in 80 mL of H2O. This solution was then acidified to pH 3-4 with aqueous 5% oxalic acid and extracted twice with Et2O. The extracts were dried and rotary evaporated to leave 0.377 g of solid that was recrystallized (0 °C) three times from hexane to yield 0.147 g (32%) of a 1:1 mixture of 3a and 3b: mp 75-77 °C; 1H NMR (400 MHz) δ 4.10-4.30 (m, 4 H, 2 CH(O)CH(O)), 2.81 (s, 2 H, CH2CO2H), 2.77 (s, 2 H, CH2CO2H), 1.62-1.89 (m, 8 H, 2 CH2CH(O)CH(O) and 2 CH2C(O)(O)), 1.26 (s, 104 H, 4 (CH2)13), 0.88 (t, 12 H, 4 CH3); 13C NMR (100.6 MHz) δ 174.00 (CO2H), 172.85 (CO2H), 111.68 (C(O)(O)), 111.39 (C(O)(O)), 80.24, 80.07, 79.42, 78.78, 42.57, 42.32, 38.45, 38.03, 33.54, 33.07, 31.93, 29.70, 29.46, 29.36, 25.63, 25.48, 23.93, 22.98, 22.69, 14.08. Anal. Calcd for C36H68O6: C, 72.44; H, 11.48. Found: C, 72.30; H, 11.56. FAB HRMS (3-nitrobenzyl alcohol-Na2CO3 matrix) calcd for C36H68O6Na (M + Na+) 619.4913, found 619.4883. In another preparation following the above procedure, a 1H NMR spectrum (400 MHz, D2O) of the solid residue left after rotary evaporation of MeOH, containing 1 and excess KOH(KOD), contained only low intensity, broadened signals (for (CH2)13 and CH3), consistent with the formation of vesicles.9

(25) Palameta, B.; Prostenik, M. Tetrahedron 1963, 19, 1463. (26) Utaka, M.; Watabu, H.; Higashi, H.; Sakai, T.; Tsuboi, S.; Torii, S. J. Org. Chem. 1990, 55, 3917. (27) Huckin, S. N.; Weiler, L. J. Am. Chem. Soc. 1974, 96, 1082.

Acknowledgment is made to the U.S. Army Research Office for the support of this research. LA950872S