Reaction of a Vesicular Functionalized Surfactant with 2-Chloroethyl

Elzbieta Kubicz-Loring. Department of Chemistry, University of Wyoming, Laramie, Wyoming 82071-3838. Received April 23, 1999. In Final Form: June 29, ...
0 downloads 0 Views 313KB Size
Download PDF
7180

Langmuir 1999, 15, 7180-7185

Reaction of a Vesicular Functionalized Surfactant with 2-Chloroethyl Phenyl Sulfide, a Mustard Simulant David A. Jaeger,* Curtis L. Schilling III, Alexander K. Zelenin, Bei Li, and Elzbieta Kubicz-Loring Department of Chemistry, University of Wyoming, Laramie, Wyoming 82071-3838 Received April 23, 1999. In Final Form: June 29, 1999 Functionalized double-chain surfactant 1 (potassium O,O′-didodecylphosphorodithioate) was synthesized. Its small unilamellar vesicles were characterized by dynamic laser light scattering and differential scanning calorimetry and its giant vesicles by phase-contrast optical microscopy. Also, 1’s giant vesicles containing fluorescent dye 4a (5-carboxyfluorescein) or 4b [5-(dodecanamido)fluorescein] were characterized by epifluorescence microscopy. In a pH 9.0 borate buffer at 25 °C, vesicular 1 reacted with 2 (2-chloroethyl phenyl sulfide), a simulant for the chemical warfare agent mustard [bis(2-chloroethyl) sulfide], to give 5 [S-[(2-phenylthio)ethyl] O,O′-didodecylphosphorodithioate], involving capture of reactive intermediate cation 9 (1-phenylthiocyclopropane) by the anion of 1. This reaction was accompanied by the precipitation of 5, which resulted in wounding/destruction of the vesicles and the release of dye 4a (from giant vesicles). The combination of the conversion of 2 into 5 and dye release suggests the potential of vesicular systems for simultaneous decontamination and signaling of chemical agents. 2 hydrolyzed to give only 6 [2-(phenylthio)ethanol] in the pH 9.0 buffer at 25 °C.

Introduction The destruction (decontamination) of chemical warfare agents such as mustard and VX remains a major practical concern.1 It would be desirable to have systems that can simultaneously decontaminate and signal the presence of chemical agents. Such a system based on bilayer vesicles would function as follows in this dual-purpose application. Vesicles containing a water-soluble signaling compound, entrapped within their aqueous compartments, are prepared from a functionalized surfactant. The vesicular surfactant reacts with a chemical agent, resulting in decontamination of the agent and wounding/destruction of the vesicles, thereby releasing the signaling compound to indicate the presence of the chemical agent. We have previously reported a study of the application of a vesicular system to the simultaneous decontamination and signaling of a VX simulant.2

Herein we report the results of an investigation that applies the above dual-purpose strategy to another chemical agent. In particular, we give an account of the synthesis and characterization of functionalized doublechain surfactant 1 and a study of the reaction of its vesicles with 2, a mustard simulant.

Results Synthesis. Surfactant 1 was prepared in two straightforward steps as illustrated. The reaction of 1-dodecanol with phosphorus pentasulfide gave acid 3, which was converted into 1 by reaction with K2CO3.

Surfactant Characterization. Surfactant 1 was characterized by measurement of its critical aggregation concentration (cac). Attempts to detect a Krafft temperature (Tk) for 1 in both H2O and a pH 9.0 borate buffer between ca. 12 and 95 °C were unsuccessful. Aggregated surfactant 1 was characterized by 1H NMR spectroscopy, dynamic laser light scattering (DLLS), differential scanning calorimetry (DSC), phase-contrast optical microscopy, and epifluorescence microscopy. Except for microscopy, aggregated 1 was prepared by sonicating 1 in H2O (D2O) or a pH 9.0 borate buffer. For microscopy, aggregated 1 was prepared by hydration of solid 1 in the same buffer. cac Measurement. In H2O at 25 °C, 1’s cac value, determined by surface tensiometry (du Nou¨y ring), is (6 ( 1) × 10-5 M. The surface tension of a solution of 1 in H2O above its cac is ca. 35 mN/m at 25 °C. The cac of 1 in the pH 9.0 buffer used in its reactions with 2 should be less than the cac in H2O.3 1H NMR Spectroscopy. The 1H NMR spectrum of 1 (1.9 × 10-2 M) sonicated 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.4 DLLS. Aggregates of 1 (4.8 × 10-4 M), prepared by sonication in H2O, were analyzed by DLLS (90° scattering angle) at 25 °C. Gaussian analysis established the presence of one population with a hydrodynamic diameter of 67 ( (1) (a) Yang, Y.-C.; Baker, J. A.; Ward, J. R. Chem. Rev. 1992, 92, 1729. (b) Yang, Y.-C. Chem. Ind. (London) 1995, 334. (c) Yang, Y.-C. Acc. Chem. Res. 1999, 32, 109. (2) Jaeger, D. A.; Li, B. Langmuir, in press. (3) The cac’s of ionic surfactants are typically less in aqueous electrolyte solutions than in pure H2O (Mukerjee, P.; Mysels, K. J. Natl. Stand. Ref. Data Ser. (U.S. Natl. Bur. Stand.) 1971, NSRDS-NBS 36). (4) Browning, J. L. In Liposomes: From Physical Structure to Therapeutic Applications; Knight, C. G., Ed.; Elsevier/North-Holland: New York, 1981; Chapter 7.

10.1021/la990488l CCC: $18.00 © 1999 American Chemical Society Published on Web 08/12/1999

Reaction of Surfactant with a Mustard Simulant

Langmuir, Vol. 15, No. 21, 1999 7181

Figure 2. Simultaneous phase-contrast and epifluorescence photomicrographs of GVs of 1 containing (a) hydrophilic dye 4a and (b) hydrophobic dye 4b.

Figure 1. Phase-contrast photomicrograph of a GV of surfactant 1 (bar here and in other figures ) 50 µm).

6 nm, which corresponds to small unilamellar vesicles (SUVs).5 The SUVs were stable for g2 days, as evidenced by the obtainment of comparable DLLS results for a given sample over this period. DSC. Thermograms were obtained for 1 (1.0 × 10-2 M) vortexed in H2O and the pH 9.0 buffer. The phase transition temperatures (Tc) and calorimetric enthalpies (∆Hcal) are as follows: Tc ) 16.3 ( 0.1 and 12.4 ( 0.1 °C for H2O and buffer, respectively; ∆Hcal ) 10.2 ( 0.3 and 9.6 ( 0.2 kJ/mol for H2O and buffer, respectively. At its Tc, a vesicle bilayer undergoes a transition from the gel to the less-ordered liquid crystalline state, corresponding to conformational changes of the alkyl groups.6 Micelles would not be expected to display phase transitions. Phase-Contrast Optical and Epifluorescence Microscopy. Phase-contrast optical microscopy revealed the presence of giant vesicles (GVs)7 after a thin smear of solid 1 was hydrated in the pH 9.0 buffer at 25 °C. A representative GV is illustrated in Figure 1; GVs were stable for over a week at 25 °C. GVs were also prepared in the pH 9.0 buffer in the presence of hydrophilic dye 4a and hydrophobic dye 4b, which are in deprotonated, anionic forms in the buffer. In each case a mixed thin film of 1 and dye 4 (molar ratios: 1/4a ) 900:1; 1/4b ) 400:1) was hydrated in the pH 9.0 buffer to give GVs that were observed by simultaneous phase-contrast optical and epifluorescence microscopy. Representative GVs containing 4a and 4b are illustrated in panels a and b of Figure 2, respectively. With both 4a and 4b about 33% of the GVs had fluorescence intensities greater than those of their buffer backgrounds. Within a GV water-soluble deprotonated 4a should be electrostatically repelled from the negatively charged bilayer membranes, resulting in its confinement within the aqueous compartments, consistent with the appearance of the GV in Figure 2a.8 Within a GV deprotonated hydrophobic 4b should be incorporated in the bilayer membranes, with its long-chain alkyl group directed into the hydrocarbon interior and its ionic headgroup at the bilayer-water (5) Fendler, J. H. Membrane Mimetic Chemistry; Wiley-Interscience: New York, 1982; Chapter 6. (6) McElhaney, R. N. Chem. Phys. Lipids 1982, 30, 229. (7) For reviews, see (a) Menger, F. M.; Gabrielson, K. D. Angew Chem., Int. Ed. Engl. 1995, 34, 2091. (b) Menger, F. M.; Angelova, M. I. Acc. Chem. Res. 1998, 31, 789. (c) Menger, F. M.; Keiper, J. S. Curr. Opin. Chem. Biol. 1998, 2, 726. (8) For another example of the appearance of a GV containing a watersoluble fluorescent dye, see: Menger, F. M.; Lee, S. J. Langmuir 1995, 11, 3685.

interface. The widespread fluorescence of the GV in Figure 2b suggests that it is multilamellar.9 The fluorescence intensity of a GV containing 4a, monitored by epifluorescence microscopy intermittently (to minimize dye photobleaching,10 if any), generally decreased by ca. 50% over 2-4 h. The fluorescence intensity of a GV containing 4b was not monitored with time.

Reaction of 2 with SUVs of 1 in the pH 9.0 Buffer. Reactions of simulant 2 with SUVs of 1 (1:1 molar ratio) in the pH 9.0 borate buffer were performed at 25 °C under conditions in which 2 initially was not completely soluble (0.0099 M 2 if completely dissolved). 31P NMR analysis of the reaction mixtures indicated that 1 was converted into only 5 (eq 1) and that the reaction was ca. 70% complete after 19 h. 1H NMR analysis of the reaction mixtures indicated the presence of a trace of alcohol 6 after 48 h. In some instances reaction mixtures were lyophilized after 136 h. By 31P NMR analysis of the residue with added (C6H5)3P as an internal standard, the yield of 5 was 88 ( 10%. In the absence of 1, simulant 2 hydrolyzed in the pH 9.0 borate buffer to give alcohol 6 as the only product (eq 2).

When deuterium-labeled 2a11 was substituted for 2 in the above reaction with vesicular 1, a 50:50 mixture of 5a and 5b was obtained. Reaction of 2 with GVs of 1 in the pH 9.0 Buffer. Reactions of 2 with GVs of 1 (1:1 molar ratio) were followed by phase-contrast optical microscopy. Simulant 2 was added as an EtOH solution to the pH 9.0 borate buffer containing 1’s GVs; the resultant concentrations of 2 (if (9) Menger, F. M.; Lee, S. J.; Keiper, J. S. Langmuir 1996, 12, 4479. (10) (a) Song, L.; Varma, C. A. G. O.; Verhoeven, J. W.; Tanke, H. J. Biophys. J. 1996, 70, 2959. (b) Song, L.; Hennink, E. J.; Young, I. T.; Tanke, H. J. Biophys. J. 1995, 68, 2588. (11) (a) Taber, D. F.; Wang, Y. J. Org. Chem. 1993, 58, 6470. (b) McManus, S. P.; Karaman, R. M.; Sedaghat-Herati, R.; Hovanes, B. A.; Ding, X.-T.; Harris, J. M. J. Org. Chem. 1993, 58, 6466.

7182

Langmuir, Vol. 15, No. 21, 1999

completely dissolved) and EtOH were 8 × 10-4 M and 0.5 vol %, respectively. On addition of its EtOH solution, simulant 2 forms 1-5µm opaque droplets that disperse throughout the buffer and accumulate on the GVs. Figure 3a shows a GV with sorbed droplets of 2, which in real time are moving around the surface of the GV. Most of the GVs were destroyed during the period from 10 to 17 h after the addition of 2 as the result of degradation of their bilayer membranes. The destruction of an individual GV could be abrupt, involving a peeling process and collapse, as illustrated in Figure 3 for the GV of Figure 3a. In Figure 3b, obtained 56 s after Figure 3a, the GV is peeling back on itself along a front moving from right to left, as indicated by the arrow. The peeling is almost complete in Figure 3c, obtained 1 s after Figure 3b. The entire peeling process, from the onset to the end of the moving front, took less than 2 s. The GV has collapsed in Figure 3d, obtained 30 s after Figure 3c. The extent of the reaction of 2 with 1 at the time of peeling is unknown. Reactions of 2 with GVs of 1 containing dye 4a (900: 900:1 molar ratio, respectively) were followed by simultaneous phase-contrast optical and epifluorescence microscopy. Simulant 2 was added as an EtOH solution to the pH 9.0 borate buffer containing 1’s GVs with entrapped 4a; the concentrations of 2 and EtOH were the same as above. As in the reaction of 2 with GVs of 1 without 4a, the GVs with 4a were destroyed as the result of degradation of their bilayer membranes. However, for unknown reasons, GV destruction was slower with 4a, generally occurring more than 24 h after the addition of 2. The loss of 4a was often detected before GV destruction was complete. Panels a and b in Figure 4, obtained 72 s apart, show a GV undergoing reaction with 2. Over this 72-s period the fluorescence intensity of the GV decreased markedly, due to the loss of 4a from the GV as a consequence of the reaction of 2 with 1. In controls, oils 7 and 8, which do not react with surfactant 1, effected neither GV destruction nor the loss of dye 4a. Thus these processes observed with 1’s GVs upon the addition of 2 do not result simply from the physical interaction of 2 with the GVs.

Discussion A priori there are at least two possible mechanisms for the formation of 5 from the reaction of surfactant 1 and 2 (eq 1). The first involves SN2 attack of 1’s nucleophilic anionic headgroup directly on 2. The second possibility involves initial ionization of 2 with neighboring group participation by sulfur to give episulfonium ion 9, which is then captured by 1’s headgroup to give 5 (eq 3). The obtainment of a 50:50 mixture of 5a and 5b from the reaction of vesicular 1 with deuterium-labeled 2a indicates that the pathway to 5 proceeds through 9. Indeed, reactions

Jaeger et al.

Figure 3. Phase-contrast photomicrographs of a GV of 1 (a) undergoing reaction with simulant 2, (b) and (c) during a peeling process, and (d) after the peeling process; see the text for descriptions.

Figure 4. Simultaneous phase-contrast and epifluorescence photomicrographs of a GV of 1 containing dye 4a undergoing reaction with 2; photomicrograph (b) was obtained 72 s after (a).

of nucleophiles with 2, other mustard simulants, and mustard itself generally proceed through episulfonium ions.1,11b,12

Since the reaction of 2 in vesicular 1 gave 5 accompanied by only a trace of alcohol 6, 1’s headgroup must be a much better nucleophile than H2O in capturing episulfonium ion 9. This conclusion is consistent with the competition factor of 2600 M-1 reported13 for nonsurfactant anion 10 relative to H2O at 25 °C in the capture of 11, the episulfonium ion derived from mustard. Yang and coworkers14 found that thiosulfate (S2O32-) captured 12, the episulfonium ion derived from 2-chloroethyl ethyl sulfide, to the exclusion of capture by H2O in 50 vol % aqueous Me2CO at 20 °C. Thiosulfate has a reported13 competition factor of 27 000 M-1 relative to H2O in the capture of 11. Anion 13, the presumed product15 of the capture of 12 by thiosulfate, was stable for 3 months in 50 vol % aqueous Me2CO. (12) (a) McManus, S. P.; Karaman, R. M.; Sedaghat-Herati, R.; Harris, J. M. J. Org. Chem. 1995, 60, 4764. (b) Sedaghat-Herati, M. R.; McManus, S. P.; Harris, J. M. J. Org. Chem. 1988, 53, 2539. (13) Ogston, A. G.; Holiday, E. R.; Philpot, J. St L.; Stocken, L. A. Trans. Faraday Soc. 1948, 44, 45. (14) Yang, Y.-C.; Szafraniec, L. L.; Beaudry, W. T.; Ward, J. R. J. Org. Chem. 1988, 53, 3293. (15) Distler, H. Angew Chem., Int. Ed. Engl. 1967, 6, 544.

Reaction of Surfactant with a Mustard Simulant

Langmuir, Vol. 15, No. 21, 1999 7183

2a. The combination of the conversion of 2 into 5 and the release of dye 4a suggests the potential of vesicular systems for simultaneous signaling and decontamination of chemical agents. Experimental Section Reported strategies for the decontamination of mustard and its simulants include hydrolysis,1,11b,16 other nucleophilic substitutions,1,13 oxidation,1,17 and elimination.1 In the present study, the decontamination of simulant 2 corresponds to its conversion into 5 by nucleophilic substitution (eq 1). However, since the anion of surfactant 1 is a potential leaving group, 5 is an alkylating agent, like 2. Thus the conversion of 2 into 5 does not represent complete decontamination. But 14, an analogue of 5 that should be both more water-soluble and more reactive than 5, was stable for >21 days in the pH 9.0 borate buffer at 25 °C,18 in contrast to the near complete hydrolysis of 2 to 6 under the same conditions after 3 days. The signaling component of the vesicular system corresponds to the release of dye 4a as the result of the wounding of the vesicles effected by the reaction of 2 with surfactant 1.

The peeling and collapse of a GV, as observed in Figures 3, could be responses to the conversion of a portion of surfactant 1 in the outer leaflet of the (presumed multilamellar) GV’s outermost bilayer membrane into neutral 5. The resulting asymmetrical bilayer membrane undergoes a peeling process that brings the inner leaflet, with its hydrophilic headgroups intact, outside and the less hydrophilic outer leaflet containing 1 and 5, inside, followed by collapse. Wounding of GVs effected by other means has been observed previously.8 The formation of GVs by the hydration of surfactants is well-known.19 But the origin of the preferential incorporation of dyes 4 into ca. 33% of the GVs formed upon hydration of mixed thin films of surfactant 1 and 4 was not anticipated. Summary Double-chain surfactant 1 was synthesized. Its SUVs were characterized by DLLS, to give vesicle sizes, and by DSC, to give Tc and ∆Hcal values, and its GVs were characterized by phase-contrast optical microscopy and by epifluorescence microscopy with the use of dyes 4a and 4b. In a pH 9.0 borate buffer at 25 °C, vesicular 1 reacted with mustard simulant 2 to give neutral 5, with resultant wounding/destruction of the SUVs and GVs and the release of entrapped dye 4a from the latter. The formation of 5 involved the capture of episulfonium ion 9 by the anion of 1, as demonstrated with the use of deuterium-labeled (16) (a) Yang, Y.-C.; Ward, J. R.; Luteran, T. J. Org. Chem. 1986, 51, 2756. (b) McManus, S. P.; Neamati-Mazaeh, N.; Hovanes, B. A.; Paley, M. S.; Harris, J. M. J. Am. Chem. Soc. 1985, 107, 3393. (c) Bartlett, P. D.; Swain, C. G. J. Am. Chem. Soc. 1949, 71, 1406. (17) (a) Menger, F. M.; Rourk, M. J. Langmuir 1999, 115, 309. (b) Menger, F. M.; Elrington, A. R. J. Am. Chem. Soc. 1991, 113, 9621. (c) Yang, Y.-C.; Szafraniec, L. L.; Beaudry, W. T.; Davis, F. A. J. Org. Chem. 1990, 55, 3664. (d) Bacaloglu, R.; Blasko, A.; Bunton, C. A.; Foroudian, H. J. J. Phys. Org. Chem. 1992, 5, 171. (e) Wagner, G. W.; MacIver, B. K.; Yang, Y.-C. Langmuir 1995, 11, 1439. (f) Gall, R. D.; Hill, C. L.; Walker, J. E. J. Catal. 1996, 159, 473. (18) Jaeger, D. A.; Zelenin, A. K. To be published. (19) (a) Menger, F. M.; Gabrielson, K. J. Am. Chem. Soc. 1994, 116, 1567. (b) Reeves, J. P.; Dowben, R. M. J. Cell Physiol. 1969, 73, 49. (c) Hub, H. H.; Zimmerman, U.; Ringsdorf, H. FEBS Lett. 1982, 140, 254.

General Procedures and Materials. 1H (270 and 400 MHz) and 13C NMR (67.9 MHz) spectra recorded in CDCl3 employed Me4Si and CDCl3 (center line at δ 77.00 relative to Me4Si) as internal standards, respectively. 1H NMR spectra recorded in D2O and the pH 9.0 borate buffer in D2O employed HOD (δ 4.65 relative to Me4Si) as internal standard. J values are in hertz. 31P NMR (109.4 MHz) spectra were recorded in CDCl3 and the pH 9.0 borate buffer with 85% H3PO4 as external standard. Silica gel (J. T. Baker 3405) was used for column chromatography. The pH 9.0 borate buffer was prepared from 0.025 g (0.12 mmol) of anhydrous Na2B4O7 [or 0.046 g (0.12 mmol) of Na2B4O7‚10H2O], 5.0 mL of H2O, and 0.20 mL of 0.10 M HCl in H2O. The same buffer in D2O was prepared with anhydrous Na2B4O7 and 0.10 M DCl in D2O. Sonication was performed with a Branson 2200 (125 W) ultrasonic cleaner and vortexing with a Thermolyne M16715 mixer. Extracts were dried over MgSO4. The cac of 1 was determined from plots of surface tension (du Nou¨y ring) vs log[surfactant] using a Fisher model 20 tensiometer. The attempts to determine Tk values for 1 in H2O and the pH 9.0 borate buffer followed a literature procedure.20 All melting points were measured in open capillary tubes and are uncorrected. Elemental analyses were performed by Atlantic Microlab, Atlanta, GA. Potassium O,O′-Didodecylphosphorodithioate (1). Modified literature procedures were used.21 To a stirred solution of 16.0 g (85.9 mmol) of 1-dodecanol in 100 mL of C6H5Me at 25 °C, 4.77 g (10.7 mmol) of phosphorus pentasulfide was added in portions over 30 min. The resultant opaque mixture was refluxed under N2 until it became clear (ca. 48 h), and then it was rotary evaporated to afford O,O′-didodecylphosphorodithioic acid (3), which was used without purification. To a stirred mixture of the acid in 100 mL of Et2O, 6.67 g (48.3 mmol) of K2CO3 was added in several portions. After 12 h at 25 °C the mixture was filtered, and the resultant solids were recrystallized two times from Me2CHOH (5 °C) to yield 12.3 g (57%) of 1 as a white solid: mp 169-170 °C; 1H NMR (270 MHz, CDCl3) δ 3.97 (m, 4 H, 2 CH2O), 1.68 (m, 4 H, 2 CH2CH2O), 1.26 (m, 36 H, 2 (CH2)9), 0.88 (t, 6 H, 2 CH3); 13C NMR δ 67.12, 67.01, 31.95, 30.60, 30.49, 29.77, 29.55, 29.40, 25.95, 22.69, 14.09; 31P NMR (CDCl3) δ 110.9 (s). Anal. Calcd for C24H50O2PS2K: C, 57.10; H, 9.98. Found: C, 57.10; H, 9.73. DLLS. Measurements were made at 25 °C on the Nicomp particle sizer described previously22 with samples prepared as follows. A solution of 6.3 mg (0.012 mmol) of 1 in 5.0 mL of CHCl3 (stored over Na2CO3) was rotary evaporated to give a thin film of 1 that was dried for 12 h (25 °C, 0.1 mmHg). Then 25.0 mL of HPLC-grade H2O was added, and the mixture was sonicated for 2 h at 60 °C. The resultant sample was filtered through a 0.45-µm filter (Durapore) into a 6-mm × 50-mm culture tube (Kimble 73500-650). Immediately thereafter, the tube was capped (5-mm NMR tube cap) and inserted into the particle sizer, and the run was begun with a photopulse rate of 300-350 kHz. Data were analyzed by Nicomp software (version 12.3); the hydrodynamic diameter is an average of five runs with different samples. DSC. Calorimetry was performed on a Calorimetry Sciences 4100 multicell differential scanning calorimeter. Measurements were made with 0.5000-g portions of samples prepared in H2O or the pH 9.0 borate buffer as follows. A solution of 11.8 mg (0.023 mmol) of 1 in 5.0 mL of CHCl3 (stored over Na2CO3) was (20) De´marcq, M.; Dervichian, D. Bull. Soc. Chim. Fr. 1945, 12, 939. (21) (a) Bolotova, G. I.; Kotova, G. G.; Zimina, K. I.; Isagulyants, V. I. Zh. Prikl. Khim. 1965, 38, 1580 (Chem. Abstr. 1965, 63, 13028a). (b) Komkov, I. P.; Levitskaya, V. M. Izv. Vysshikh Uchebn. Zavedenii, Khim. Khim. Tekhnol. 1966, 9, 424 (Chem. Abstr. 1967, 66, 18488n). (c) Komkov, I. P.; Levitskaya, V. M. Izv. Vysshikh Uchebn. Zavedenii, Khim. Khim. Tekhnol. 1966, 9, 254 (Chem. Abstr. 1966, 65, 13526b). (d) Levin, I. S.; Sergeeva, V. V.; Tarasova, V. A.; Varentsova, V. I.; Rodina, T. F.; Vorsina, I.A.; Kozlova, N. E.; Kogan, B. I. Russ. J. Inorg. Chem. 1973, 18, 867. (22) Jaeger, D. A.; Sayed, Y. M. J. Org. Chem. 1993, 58, 2619.

7184

Langmuir, Vol. 15, No. 21, 1999

rotary evaporated to give a thin film of 1 that was dried for 12 h (25 °C, 0.1 mmHg). Then 2.36 mL of H2O or the pH 9.0 buffer was added, and the mixture was vortexed for 6 min at 60 °C and held at 25 °C for 30 min in a vacuum oven (1 mmHg) to effect degassing. Scans were made from 0 to 30 °C and from 30 to 0 °C at 30 °C/h. 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.6,23 The Tc and ∆Hcal values are averages of three runs with different samples. Formation of GVs of 1 in the pH 9.0 Borate Buffer. Two methods were used as described below, which involved the hydration of solid 17a,19a and of mixed films of 1 and dye 4.19b,c A rubber garden hose washer [25 mm (o.d.) × 15 mm (i.d.) × 2.5 mm] was cemented (Dow Corning 100% silicone rubber aquarium sealant) onto a glass microscope slide (75 mm × 25 mm × 1 mm). To one side of the glass bottom of the well formed by the washer, ca. 0.2 mg (0.0004 mmol) of 1 was smeared over an area of ca. 8 mm × 2 mm. Then 0.50 mL of the pH 9.0 borate buffer was added, and the well was covered by a glass cover slip and allowed to stand undisturbed at 25 °C for at least 60 min. Thereafter, GVs were observed in the mixture for over a week by phase-contrast optical microscopy. Figure 1 was obtained 2 h after adding the buffer to the smear of 1. GVs were also prepared in the pH 9.0 borate buffer in the presence of dye 4a (Molecular Probes C-1359) as follows. In the well of the microscope slide, ca. 0.2 mg (0.0004 mmol) of 1 was dissolved in 20 µL of Me2CHOH, which was allowed to evaporate at 25 °C to form a thin film of 1 on the glass surface. After the thin film was further dried for 2 h (25 °C, 0.1 mmHg), 6.4 µL of 0.069 mM 4a (0.44 × 10-6 mmol) in EtOH was added to the well, and the EtOH was allowed to evaporate to give a thin film of 1 and 4a. The thin film was further dried for 2 h (25 °C, 0.1 mmHg), followed by the careful addition of 0.50 mL of the pH 9.0 buffer. The well was covered by a cover slip and allowed to stand undisturbed at 25 °C for 60 min. Then the mixture was observed by phase-contrast optical microscopy and simultaneous phasecontrast optical and epifluorescence microscopy. The latter (Figure 2a) demonstrated that 4a was concentrated within the aqueous compartments of ca. 33% of the GVs. GVs were also prepared in the pH 9.0 borate buffer in the presence of dye 4b (Molecular Probes D-109) as follows. In the well of the microscope slide, 0.7 mg (0.001 mmol) of 1 was dissolved in 20 µL of Me2CHOH, which was allowed to evaporate at 25 °C to form a thin film of 1 on the glass surface. After the thin film was further dried for 2 h (25 °C, 0.1 mmHg), 27 µL of 0.088 mM 4b (2.4 × 10-6 mmol) in EtOH was added to the well, and the EtOH was allowed to evaporate to give a thin film of 1 and 4b. Thereafter, the procedure was the same as for the GVs containing 4a. The resultant mixture was observed by phase-contrast optical microscopy and simultaneous phase-contrast optical and epifluorescence microscopy. The latter (Figure 2b) demonstrated that 4b was concentrated within ca. 33% of the GVs. Reaction of 2 with SUVs of 1 in the pH 9.0 Borate Buffer. A mixture of 5.0 mg (0.0099 mmol) of 1 and 1.00 mL of the pH 9.0 borate buffer in H2O (D2O) was sonicated for 15 min at 55 °C in a 5-mm NMR tube to give a translucent solution. The 31P NMR spectrum of this solution contained one singlet at δ 111.2 for 1. Then 1.7 mg (0.0099 mmol) of 2 (Aldrich) was added, and the tube was shaken vigorously to give a cloudy mixture, containing small droplets of undissolved 2, that was held at 25 °C. At various times the mixture was shaken and analyzed by 31P NMR to monitor the formation of 5 (δ 94.7) from the reaction of 1 with 2, and in some instances by 1H NMR, which was less informative. After 136 h some reactions were lyophilized. Then 0.75 mL of CDCl3 was added to the residue, followed by 4.2 mg (0.016 mmol) of (C6H5)3P as an internal standard. The mixture was analyzed by 31P NMR to give the relative intensities of the singlets for 5 and (C6H5)3P (δ -4.8) and, in turn, the yield of 5. Isolation of 5 from the Reaction of SUVs of 1 with 2 in the pH 9.0 Borate Buffer. A mixture of 40 mg (0.079 mmol) of 1 and 4.0 mL of the pH 9.0 borate buffer in D2O was sonicated for 2 h at 55 °C to give a translucent solution. Then 14 mg (0.081 mmol) of 2 was added, followed by vigorous shaking to give a cloudy mixture containing small droplets of undissolved 2. At (23) Takahashi, K.; Sturtevant, J. M. Biochemistry 1981, 20, 6185.

Jaeger et al. various times the mixture was shaken and analyzed by 1H and31P NMR to follow the conversion of 1 (δ 111.2) into 5 (δ 94.7). The reaction was ca. 70% complete after 19 h. The white cloudy reaction mixture was lyophilized, and the residue was extracted with CHCl3. After rotary evaporation the residue was chromatographed on a 20-cm × 1-cm column of silica gel packed in hexane and eluted with 6:4 (v/v) CHCl3-hexane to give 30 mg (63%) of 5 as an oil: 1H NMR (270 MHz, CDCl3) δ 7.17-7.44 (m, 5 H, Ar H), 4.05 (m, 4 H, 2 CH2O), 2.96-3.22 (m, 4 H, SCH2CH2), 1.67 (m, 4 H, 2 CH2CH2O), 1.26 (s, 36 H, 2 (CH2)9), 0.88 (t, 6 H, 2 CH3); 13C NMR δ 134.60, 130.08, 129.06, 126.67, 68.14, 68.06, 34.44, 32.79, 31.90, 30.02, 29.89, 29.63, 29.56, 29.48, 29.34, 29.14, 25.57, 22.67, 14.11; 31P NMR (CDCl3) δ 94.87 (s). Anal. Calcd for C32H59O2S3P: C, 63.74; H, 9.86. Found: C, 63.52; H, 9.83. Reaction of 2a with SUVs of 1 in the pH 9.0 Borate Buffer. Deuterium-labeled 2a was prepared by the literature procedure.11a The reaction of 85 mg (0.49 mmol) of 2a in a solution of 242 mg (0.48 mmol) of vesicular 1 in 24.0 mL of the pH 9.0 buffer was performed as above for the isolation of 5 to give 173 mg (60%) of product, which by 1H NMR analysis was a 50:50 mixture of 5a and 5a: 1H NMR (400 MHz, CDCl3) δ 7.19-7.40 (m, 10 H, Ar H), 4.06 (m, 8 H, CH2O), 3.17 (s, 2 H, CD2CH2SC6H5), 3.04 (d, J ) 18, 2 H, CH2CD2SC6H5), 1.68 (m, 8 H, CH2CH2O), 1.27 (s, 72 H, (CH2)9), 0.86 (t, 12 H, CH3). The relative intensities of the signals at δ 3.04 and 3.17 were 50:50. Reaction of 2 with GVs of 1 in the pH 9.0 Buffer. To 0.50 mL of a GV sample, containing ca. 0.2 mg (0.0004 mmol) of 1, prepared by solid hydration as above in the microscope slide well, was added 2.6 µL of 0.15 M 2 (0.0004 mmol) in EtOH. To avoid evaporation from the reaction mixture, the entire microscope slide, covered by a cover slip, was placed in a chamber containing an aliquot of the pH 9.0 borate buffer. From time to time the reaction mixture was observed by phase-contrast optical microscopy; see the text and Figure 3. Reactions of 2 in the pH 9.0 buffer were also performed with GVs of 1 containing dye 4a. To 0.50 mL of a GV sample, containing ca. 0.2 mg (0.0004 mmol) of 1 and 0.44 × 10-6 mmol of 4a, prepared by mixed thin film hydration as above in the microscope slide well, was added 2.6 µL of 0.15 M 2 (0.0004 mmol) in EtOH. Evaporation from the reaction mixture was avoided as above. From time to time the reaction mixture was observed by phasecontrast optical microscopy and simultaneous phase-contrast optical and epifluorescence microscopy; see the text and Figure 4. Controls on the Stability of GVs of 1. In the well of the microscope slide, ca. 0.2 mg (0.0004 mmol) of 1 was dissolved in 20 µL of Me2CHOH, which was allowed to evaporate at 25 °C to form a thin film of 1 on the glass surface. The thin film was dried for 2 h (25 °C, 0.1 mmHg), followed by the careful addition of 0.50 mL of the pH 9.0 borate buffer. The well was covered by a cover slip and allowed to stand undisturbed at 25 °C for 60 min. After the mixture was observed by phase-contrast optical microscopy to confirm the presence of GVs, 0.7 µL of 0.60 M 1,2-dimethoxy4-(1-propenyl)benzene (0.0004 mmol) (7) (Aldrich, mixture of cis and trans isomers) in EtOH was added. The resultant mixture, which contained a 1:1 molar ratio of 7 and 1, was monitored by phase-contrast optical microscopy. There was no difference in the nature and stability of the GVs relative to GVs of 1 in the absence of 7. Controls with the same results were also performed with 3.5 and 7.0 µL additions of 0.60 M 7 in EtOH, which gave mixtures with 5:1 and 10:1 molar ratios of 7 to 1. Analogous controls were also performed with the substitution of 3-(3,4-dimethoxyphenyl)-1-propanol (8) (Aldrich) for 7. With 1:1, 5:1, and 10:1 molar ratios of 8 to 1, there was no difference in the nature and stability of the GVs relative to GVs of 1 in the absence of 8. Others controls demonstrated that 7 does not effect the loss of dye 4a from GVs of 1 containing 4a prepared as above (7:1:4a molar ratio ) 900:900:1). Reaction of 2 in the pH 9.0 Borate Buffer. A mixture of 0.59 mg (0.0034 mmol) of 2 and 1.0 mL of the pH 9.0 borate buffer in D2O at 25 °C contained fine oil drops of undissolved 2 even after vigorous shaking. Within 72 h, ca. 95% of 2 was consumed to give 6 as the only product, as determined by 1H NMR analysis of the reaction mixture. The identity of 6 was confirmed by comparison with the 1H NMR spectrum of an authentic sample (Aldrich) in the pH 9.0 borate buffer in D2O.

Reaction of Surfactant with a Mustard Simulant Phase-Contrast Optical and Epifluorescence Microscopy. The following instrumentation was used. A Nikon DiaphotTMD inverted microscope, equipped with Nikon TMD-EF epifluorescence attachments and Hg lamp, was connected, in order, to a Cohu 4915-2110 CCD solid-state camera, Panasonic WJ-180 time-date generator, Sanyo GVR-S5955 SVHS videocassette recorder, Sony PVM-1343MD color video monitor, and Gateway P-5-133 PC fitted with a FlashPoint PCI 2MB (3030) video capture board. Digital images of vesicles observed at 200× magnification were formatted and printed with Abobe Photoshop 5.0 and Adobe PageMaker 6.5 software. A Nikon BV-2B filter cube was used for epifluorescence microscopy (excitation, 400-

Langmuir, Vol. 15, No. 21, 1999 7185 440 nm; dichroic mirror, 455 nm; barrier filter, 480 nm). Samples were thermostated on a Physitemp TS-4ER thermal microscope stage, controlled with Physitemp TPC-WIN software installed on the PC. The images of Figures 1-4 were taken of GV samples thermostated at 15 °C. Other than when a sample was observed by microscopy, it was held at 25 °C.

Acknowledgment. Acknowledgment is made to the U.S. Army Research Office for the support of this research. We thank Dr. Theotis Clark, Jr., for performing the controls with 7 and 1’s GVs containing dye 4a. LA990488L