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Langmuir 1998, 14, 5712-5718
Synthesis and Characterization of a New Generation of Cryptand-Based Triple-Tailed Amphiphiles: Spontaneous Formation of Vesicles and X-ray Crystallographic Studies Pradyut Ghosh, Sayam Sengupta, and Parimal K. Bharadwaj* Department of Chemistry, Indian Institute of Technology Kanpur, Kanpur 208016, India Received September 3, 1997. In Final Form: June 16, 1998 A cryptand incorporating three secondary amino groups has been derivatized by reacting with acid chlorides of different alkyl chain lengths (between C4 and C15) to get a new generation of six cryptand-based triple-tailed neutral amphiphiles. The cavity of the cryptand headgroup accommodates a Cu(II) ion giving another set of six amphiphiles. These amphiphiles can aggregate spontaneously as vesicles in 4% ethanolic water medium. They form vesicles by the extrusion method as well. In both methods, vesicles formed are mostly unilamellar. No distinguishable change can be seen in the shape of the vesicular structures between free and complexed amphiphiles. Spontaneously formed vesicles are less stable compared to those formed by the extrusion method. Stability of the vesicles decreases from weeks to hours with the lowering of the hydrophobic chain lengths. The amphiphile with three octanoyl groups attached (L4) crystallizes in the monoclinic space group P21/c with a ) 10.013(9) Å, b ) 30.923(7) Å, c ) 18.298(4) Å, β ) 92.67(4)°, Z ) 4, Rf ) 0.092, Rwf ) 0.120, and GOF ) 4.572. The solid-state structure of L4 shows the hydrophobic tails pack in the lattice by a mixed interdigitizing and noninterdigitizing manner, which is rare. We have also found in this study that spontaneous vesicular aggregates are possible with cryptandbased amphiphiles even when each hydrophobic tail has only four carbon atoms.
Introduction The process of vesicle formation via self-assembly of amphiphiles is an important event in all biosystems. Natural as well as synthetic amphiphiles can selforganize1-4 into vesicular structures where solubility properties and other physical factors such as alignment of hydrophobic tails, intermolecular packing of rigid segments, hydrogen bonding among neighboring amphiphiles, etc. play crucial roles. Synthetic vesicles are potentially important in many areas1,2 of biology and chemistry like photoinduced charge-separation, drugdelivery, peptidomimetic chemistry, materials research, etc. Although vesicles are formed spontaneously in vivo, most of the synthetic amphiphiles require either considerable mechanical energy like sonication1, extrusion,4 etc., or chemical treatments such as detergent dialysis,5 reverse-phase evaporation,6 etc., for vesicle formation. Of course, few systems7,8 are known that spontaneously form vesicles in solution. These are mostly mixed type surfactants to control bilayer curvature.9 While the physics of vesicular structure formation has been investigated quite thoroughly, the chemical problems involving synthesis and studies of such structures remain tardy.10 (1) Kunitake, T. Angew. Chem., Int. Ed. Engl. 1992, 31, 709. (2) Menger, F. M.; Gabrielson, K. D. Angew. Chem., Int. Ed. Engl. 1995, 34, 2091. (3) Wang, K.; Mun˜oz, S.; Zhang, L.; Castro, R.; Kaifer, A. E.; Gokel, G. W. J. Am. Chem. Soc. 1996, 118, 6707. (4) Ghosh, P.; Khan, T. K.; Bharadwaj, P. K. Chem. Commun. 1996, 189. (5) Matsumoto, S.; Khoda, M.; Murata, S. J. Colloid Interface Sci. 1977, 62, 149. (6) Szoka, F., Jr.; Papahadjopoulos, D. Proc. Natl. Acad. Sci. U.S.A. 1978, 75, 4194. (7) Chiruvolu, S.; Warriner, H. E.; Naranjo, E.; Idziak, S. H. J.; Ra¨dler, J. O.; Plano, R. J.; Zasadzinski, J. A.; Safinya, C. R. Science 1994, 266, 1222. (8) Herve, P.; Roux, D.; Bellocq, A.-M.; Nallet, F.; Gulik-Krzwicki, T. J. Phys. II 1993, 3, 1255. (9) Safran, S. A.; Pincus, P.; Andelman, D.; MacKintosh, F. C. Phys. Rev. A 1991, 43, 1071. (10) Maddox, J. Nature 1993, 363, 205.
However, recent years have seen an upsurge in the synthesis of surfactants with different chemical structures and composition.11-16 Earlier, we had reported one cryptand-based amphiphile which readily formed vesicles4 in alcoholic water via extrusion. It also formed a monolayer17 at the air-water interface in a Langmuir trough. Herein, we describe the syntheses and characterization of six homologous cryptand-based triple-tailed, triple-head amphiphiles and their copper(II) inclusion complexes. We show that these amphiphiles can form vesicles spontaneously in 4% ethanolic water and by the commonly used extrusion method as well. The cryptand18 headgroup is chosen for a number of reasons. It has (i) a preformed hydrophilic cavity whose topology can be tailored to recognize ion/molecule of interest, (ii) three phenyl rings as rigid segments that favor ordered structures, and (iii) a pseudo-3-fold symmetry passing through the two bridgehead nitrogens with three easily functionalizable secondary nitrogen atoms that will favor a specific arrangement of the hydrophobic chains in the molecule. Our interest in these amphiphiles stems from the observation that they can be potentially useful in a number of areas such as charge separation, drug-delivery, bursting cell walls of bacteria,19 etc. Here, we also present the first X-ray structure of one of the amphiphiles, which was (11) Kunitake, T.; Okahata, Y.; Shimomura, M.; Yasunami, S.; Takarabe, K. J. Am. Chem. Soc. 1981, 103, 5401. (12) Menger, F. M.; Yamasaki, Y. J. Am. Chem. Soc. 1993, 115, 3840. (13) Mun˜oz, S.; Malle´n, J.; Nakano, A.; Chen, Z.; Gay, I.; Echegoyen, L.; Gokel, G. W. J. Am. Chem. Soc. 1993, 115, 1705. (14) Schenning, A. P. H. J.; de Bruin, B.; Feiters, M. C.; Nolte, R. J. M. Angew. Chem., Int. Ed. Engl. 1994, 33, 1662. (15) Bhattacharya, S.; De, S. J. Chem. Soc., Chem. Commun. 1995, 651. (16) Jaeger, D. A.; Li, B.; Clark, T., Jr. Langmuir 1996, 12, 4312. (17) Das, G.; Ghosh, P.; Bharadwaj, P. K.; Singh, U.; Singh, R. A. Langmuir 1997, 13, 3582. (18) Ghosh, P.; Bharadwaj, P. K. Current Sci. 1997, 72, 797. (19) Behm, C. A.; Creaser, I. I.; Daszkiewick, B. K.; Geue, R. J.; Sargeson, A. M.; Walker, G. W. Chem. Commun. 1993, 1844.
S0743-7463(97)00991-8 CCC: $15.00 © 1998 American Chemical Society Published on Web 09/09/1998
Cryptand-Based Triple-Tailed Amphiphiles Scheme 1. Synthetic Scheme for the Amphiphiles L1-L6
undertaken to find out its crystal-packing characteristics to understand the solid-state packing. Experimental Section Materials. All chemicals were of reagent grade and were used without further purification unless otherwise noted. Triethanolamine, tris(2-aminoethyl)amine, salicylaldehyde, sodium borohydride, acid chlorides and the metal salts were obtained from Aldrich (U.S.) Sodium hydroxide, anhydrous sodium sulfate, and thionyl chloride were received from S.D.Fine Chemicals (India). Chloroform, methanol, propan-2-ol, acetonitrile, tetrahydrofuran, and dichloromethane, were received from Merck (India). The solvents and thionyl chloride were purified prior to use following standard methods. Synthesis of the Cryptand Headgroup. Synthesis of the cryptand was achieved as described earlier4 (Scheme 1). The final product was purified by crystallization from acetonitrile prior to its use. General Synthesis of the Triacylcryptands, L1-L6. The acyl derivatives of the cryptand were synthesized by reacting the cryptand with acid chlorides at low temperature in a 1:3 molar ratio as illustrated in Scheme 1. In a typical experiment, the cryptand 1 (0.56 g, 1 mmol) was dissolved with constant stirring at room temperature in 50 mL of dry tetrahydrofuran (THF) containing triethylamine (0.30 g, 3 mmol) to neutralize the acid formed. The solution was allowed to cool to 5 °C. A solution of an acid chloride (3 mmol) taken in 50 mL of dry THF was added dropwise to the cold cryptand solution over a period of 6 h under dinitrogen atmosphere with constant stirring and maintaining the reaction temperature at 5 °C. After the addition was complete, the reaction mixture was stirred for 3 h at room temperature and then refluxed for 1 h. Upon complete removal of the solvent a reddish-brown oily liquid was left which was shaken with 100 mL of water, and the desired compound was extracted with chloroform (4 × 50 mL). The organic layer after drying over anhydrous sodium sulfate was completely evaporated to obtain the amphiphile as a pale yellow/white product. Further purification could be achieved by recrystallization from dichloromethane/petroleum ether (1:5 v/v). Palmitoyl Derivative of the Cryptand, L1. Yield 81%. Pale yellow solid, mp 60 °C. 1H NMR (400 MHz, CDCl3, 25 °C, TMS) δ: 1.2 (m, 87H), 1.95 (t, 6H), 2.6 (s br, 6H), 2.9 (s br, 6H), 3.5 (s br, 6H), 4.2 (s, 6H), 4.9 (s br, 6H), 7.1 (m, 12H). FAB-MS m/z: 1275 [L1]+ (100%). IR (KBr pellet) 1645 cm-1 (s br). Anal. Calcd for C81H135N5O6: C, 76.30; H, 10.67; N, 5.49. Found: C, 76.04; H, 10.79; N, 5.32. Dodecanoyl Derivative of the Cryptand, L2. Yield 76%. White solid, mp 72 °C. 1H NMR (300 MHz, CDCl3, 25 °C, TMS) δ: 1.2 (m, 63H), 2.0 (t, 6H), 2.6 (s br, 6H), 2.9 (s br, 6H), 3.5 (s br, 6H), 4.2 (s, 6H), 4.9 (s br, 6H), 7.0 (m, 12H). FAB-MS m/z: 1106 [L2]+ (100%). IR (KBr pellet) 1645 cm-1 (s br). Anal. Calcd
Langmuir, Vol. 14, No. 20, 1998 5713 for C69H111N5O6: C, 74.89; H, 10.11; N, 6.33. Found: C, 75.16; H, 10.30; N, 6.52. Decanoyl Derivative of the Cryptand, L3. Yield 74%. White solid, mp 74 °C. 1H NMR (300 MHz, CDCl3, 25 °C, TMS) δ: 1.2 (m, 51H), 2.0 (t, 6H), 2.6 (s br, 6H), 2.9 (s br, 6H), 3.5 (s br, 6H), 4.2 (s, 6H), 4.9 (s br, 6H), 7.1 (m, 12H). FAB-MS m/z: 1022 [L3]+ (100%). IR (KBr pellet) 1645 cm-1 (s br). Anal. Calcd for C63H99N5O6: C, 74.00; H, 9.76; N, 6.85. Found: C, 74.17; H, 10.09; N, 6.80. Octanoyl Derivative of the Cryptand, L4. Yield 71%. White solid, mp 77 °C. 1H NMR (300 MHz, CDCl3, 25 °C, TMS) δ: 1.2 (m, 39H), 2.0 (t, 6H), 2.6 (s br, 6H), 2.95 (s br, 6H), 3.5 (s br, 6H), 4.2 (s, 6H), 4.95 (s br, 6H), 7.0 (m, 12H). FAB-MS m/z: 939 [L4]+ (100%). IR (KBr pellet) 1645 cm-1 (s br). Anal. Calcd for C57H87N5O6: C, 72.96; H, 9.34; N, 7.46. Found: C, 73.12; H, 9.46; N, 7.67. Heptanoyl Derivative of the Cryptand, L5. Yield 73%. White solid, mp 78 °C. 1H NMR (300 MHz, CDCl3, 25 °C, TMS) δ: 1.2 (m, 33H), 2.0 (t, 6H), 2.6 (s br, 6H), 2.95 (s br, 6H), 3.5 (s br, 6H), 4.2 (s, 6H), 4.95 (s br, 6H), 7.0 (m, 12H). FAB-MS m/z: 896 [L5]+ (100%). IR (KBr pellet) 1645 cm-1 (s br). Anal. Calcd for C54H81N5O6: C, 72.35; H, 9.11; N, 7.81. Found: C, 72.12; H, 8.96; N, 8.09. Pentanoyl Derivative of the Cryptand, L6. Yield 71%. White solid, mp 80 °C. 1H NMR (300 MHz, CDCl3, 25 °C, TMS) δ: 1.0 (m, 21H), 2.0 (t, 6H), 2.6 (s br, 6H), 2.95 (s br, 6H), 3.5 (s br, 6H), 4.2 (s, 6H), 4.95 (s br, 6H), 7.0 (m, 12H). FAB-MS m/z: 812 [L6]+ (100%). IR (KBr pellet) 1645 cm-1 (s br). Anal. Calcd for C48H69N5O6: C, 70.99; H, 8.56; N, 8.62. Found: C, 71.10; H, 8.87; N, 9.03 Preparation of the Cu(II) Perchlorate Salts of the Amphiphiles. An amphiphile was dissolved in methanol (0.1 mmol in 20 mL) and to it solid copper perchlorate hexahydrate (0.1 mmol) was added with constant stirring at room temperature for 30 min. Color of the solution changed to green. Evaporation of methanol afforded a green solid, which was washed with distilled water and filtered. Yields of the air-dried complexes of the amphiphiles were in the range 75-85%. The complexes were characterized by different spectral techniques (vide infra). General Procedures. 1H NMR spectra were recorded either on a Bruker WP-300 FT (300 MHz) or a Bruker WM-400 FT (400 MHz) instrument. FAB mass (positive ion) data were recorded on a JEOL SX 102/DA-6000 mass spectrometer using argon as the FAB gas at 6 kV and 10 mA. The accelerating voltage was 10 kV, and the spectra were recorded at 298 K. Melting points were obtained using an electrical melting point apparatus by PERFIT, India and were uncorrected. UV-vis absorption spectra of the Cu(II)-complexed amphiphiles were recorded on a Jasco V-570 spectrophotometer at 298 K. The electron paramagnetic resonance (EPR) spectra of the complexes were recorded on a Varian E-109 spectrometer operating at the X-band using DPPH as the external standard. Elemental analyses were obtained from the Central Drug Research Institute, Lucknow, India. X-ray Crystallography. Single crystals of L4 suitable for X-ray crystallographic work were grown by slow evaporation from a solution of the amphiphile at room temperature in dichloromethane: petroleum ether (1:5). A suitable crystal of the compound was mounted at the end of a glass fiber with epoxy cement. The lattice parameters, data collection method, structure solution, and refinement details are collected in Table 1. Cell parameters and reflection intensities were measured at 298 K on an Enraf-Nonius CAD4 Mach diffractometer with graphitemonochromated Mo KR radiation (λ ) 0.71073 Å). The cell parameters were determined by least-squares fitting of 25 centered reflections in the range, 18 e 2θ e 22. The structure was solved by the direct methods and completed by successive difference Fourier syntheses. Some of the H atoms could be located in the difference map while others were added at calculated positions. Temperature factors for the H atoms were assigned as follows: those associated with the cryptand headgroup were given a fixed Ueq of 0.06 Å2; those associated with the hydrophobic tails were given Ueq of 0.1 Å2. All the three hydrophobic tails were found to be heavily disordered as expected at 298 K. For each disorder atom, two or three satellite peaks appeared with different occupancy. However, we got the best result by not considering these satellite peaks except for two
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Table 1. Crystallographic and Refinement Data for L4 formula molecular weight crystal system space group a/Å b/Å c/Å β/deg V/Å3 Z Dc/g cm3 Dm/g cm-3 µ/mm-1 crystal size/mm corrections applied radiation, graphite monochromated/λ, Å transmission (max,min) scan type scan range 2θ range/deg structure solution package structure solution method no. of unique reflections no. of reflections used [I > 2σ(I)] no. of variables R R′ goodness of fit largest and mean shift/esd minimum, maximum peak in diff Fourier map/e Å-3
C57H87N5O6 938.352 monoclinic P21/c 10.013(9) 30.923(7) 18.298(4) 92.67(4) 5659(3) 4 1.10 1.19 0.07 0.4 × 0.3 × 0.2 Lp, decay Mo KR/0.71073 0.9790, 0.9584 θ-2θ 0.80 + 0.35 tan θ 2-50 XTAL 3.2 direct methods 9934 2935 580 0.092 0.120 4.572 0.080, 0.009 -0.69, +0.81
a R ) ∑||F | - |F ||/∑|F |. R′ ) [|∑ω(F | - |F |)2/∑ω(F )2]1/2, ω ) o c o o c o 1/σ(F).
atoms. H-atoms were not added for the disordered atoms. All non-hydrogen atoms except those with high temperature factors were anisotropically refined; the rest were isotropically refined. The positional or thermal parameters of the H atoms were not refined but they were included in the structure factor calculation. The refinements were made on F by full-matrix least-squares calculations. All computations were made with the XTAL 3.2 program package20 on a 486-DX personal computer (IBMcompatible, PCL, India) operating under MS-DOS version 5 at 66 MHz. General Methods of Preparation of Vesicles. To form vesicles, 1 mL of ethanolic solution of an amphiphile (5 mM) was slowly added at room temperature to 24 mL of deionized water. The dispersion was not subjected to any type of mechanical agitation other than gentle mixing for ∼5 min at room temperature to obtain a turbid dispersion. In another method, 1 mL solution of an amphiphile in ethanol (5 mM) was injected into 24 mL of a buffer solution at room temperature whereby a vesicular suspension was obtained. The buffer solution was made as follows: To 50 mM Tris-buffer solution dilute HCl was added to make the pH 7.4. Then the required amounts of histidine and NaCl were added to the buffer solution to maintain their concentrations at 5 and 50 mM, respectively. The NaCl was added to keep the ionic strength at the desired level. This suspension was extruded through a polycarbonate membrane of porosity 0.1 µm at a constant temperature of 303 K. After five cycles of extrusion, unilamellar vesicles of almost equal size were obtained. The particular composition of the buffer solution was made with the future goal(s) of using such vesicular suspensions in biomimetic studies. Transmission Electron Microscopy. Vesicular structures were characterized by negatively stained (0.5% uranyl acetate) electron micrographs. The respective samples were prepared by placing a drop of the vesicular suspension on a Formvarcoated copper grid that was then allowed to be air-dried. A drop of 0.5% uranyl acetate solution in water was placed on the sample grid and dried again at room temperature. The sample was examined by means of a JEOL TEM-2000F instrument operating at 100 kV. (20) Hall, S. R.; Stuart, J. M.; Flack, H. B. The XTAL 3.2 Reference Manual; Universities of Western Australia, and Maryland, 1993.
Static Turbidimetry. A 2.50 mL aliquot of the stock analyte containing the suspension of extruded or spontaneously formed vesicles was transferred into a quartz cuvette, and the turbidimetric profile at 25 °C was recorded in the range between 300 and 600 nm using a JASCO V-570 spectrophotometer. Ethanolic water (4%) was used as the reference in a cuvette in case of vesicular suspension in 4% ethanolic water; a buffered solution containing 4% ethanol was used when the vesicular suspension was in the alcoholic buffered mixture. In all cases, triple-distilled water was used. Note: Because absorption spectrophotometers are not ideal turbidimeters, the corresponding turbidity measurements will vary somewhat according to the instrument employed. For the present work, a JASCO model V-570 UV-vis-NIR spectrophotometer with single grating monochromator, Czerny-Turner mount, and double beam type was used.
Results and Discussion Synthesis. The cryptand headgroup can be synthesized by allowing the two tripodal units to react at 5 °C under moderate dilution conditions without using any templating metal ion. The solvent system for the condensation reaction is quite crucial, and in the present case, MeOH:THF (40:1 v/v) is found empirically to be the solvent of choice. The Schiff base is reduced in situ with sodium borohydride. Isolation of the pure product in high yields (65%) from the reaction workup suggests that the cryptand formed is the thermodynamically stable product under the reaction conditions. The cryptand is designed to have three secondary amino groups in their framework which can be functionalized. To synthesize an amphiphile, the cryptand is treated with an acid chloride in 1:3 molar ratio in dry THF medium under a dinitrogen blanket at 5 °C. The reaction goes cleanly affording the desired amphiphiles in very high yields. The 1H NMR spectra of the amphiphiles are consistent with their structures and the fast atom bombardment (FAB) mass spectra exhibit the molecular ion peak (100%) in all cases. Hence, the amphiphiles behave as robust molecules and can be handled with ease without decomposition. The copper(II) salts of the amphiphiles can be isolated when copper(II) perchlorate hexahydrate is added to a solution of an amphiphile in methanol. The green solid obtained with each amphiphile has ligand field band positions at ∼785 (max, 80 M-1 cm-1) and ∼950 nm (max, 50 M-1 cm-1). These complexes also display broad EPR signals in the solid state which becomes axial in MeOH (ca. 1 × 10-3 M) at 298 K. Both the EPR and electronic spectral characteristics match closely with those obtained on the well-characterized [ Cu(1)](ClO4)2 (1 is the unsubstituted cryptand) in which copper ion enters the cavity forming a distorted tetrahedral geometry. Molecular and Crystal Structure of L4. The molecular structure of L4 was carried out to probe the disposition of the hydrophobic tails with respect to the cryptand headgroup and its packing characteristics in the solid state. A perspective view of the molecule showing the atom numbering scheme is shown in Figure 1. The bridgehead nitrogen atoms of the cryptand headgroup exist in an endo-endo conformation with a distance of 6.570 Å between them, which is slightly longer compared to that found in the unsubstituted cryptand (6.249 Å). The distances between any two amido nitrogens are similar; the same is true in case of the ethereal oxygens (Table 2). In the unsubstituted cryptand, however, these distances are quite dissimilar. Thus, upon substitution with the acyl groups, the cryptand assumes a better 3-fold symmetry about the axis passing through the bridgehead nitrogen atoms. The bond distances and angles in the cryptand headgroup are within normal statistical errors.
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Langmuir, Vol. 14, No. 20, 1998 5715
Figure 1. An ORTEP21 drawing of L4 viewing along the two bridgehead nitrogen atoms. Some of the atoms are not labeled for clarity. H-atoms are also omitted for clarity. Table 2. Selected Non-Bonded Distances (Å) for Unsubstituted Cryptand and L4
N(1)‚‚‚N(3) N(2)‚‚‚N(4) N(2)‚‚‚N(5) N(4)‚‚‚N(5) O(1)‚‚‚O(2) O(1)‚‚‚O(3) O(2)‚‚‚O(3)
unsubstituted cryptand
L4
6.249 4.036 4.336 3.536 4.862 5.365 3.992
6.570 4.549 4.644 4.601 4.301 4.271 4.168
The distances and angles associated with the disordered atoms of the hydrophobic tails deviate to within (10% from the normal values. The crystal packing of the amphiphile indicates that the hydrophobic chains are aligned in an intermolecular digitizing and noninterdigitizing manner (Figure 2) in the crystal lattice. The crystal structures22-24 of amphiphiles hitherto determined have head-to-head bilayers with mostly interdigitizing hydrophobic chains. In the present case, the 3-fold symmetry of the amphiphiles prevents them from aligning only in intermolecular digitizing fashion. The chains are curved in one direction and the carbonyl linkages do not allow the hydrophobic tails to spread laterally out of the cryptand. The ability to form vesicular structures spontaneously by the amphiphiles L1-L6 and their copper(II) complexes may be due to special topology of the cryptand architecture and the presence of carbonyl (21) Johnson, C. K. ORTEP, Report ORNL-3794, Oak Ridge National Laboratory, Oak Ridge, TN, 1971. (22) Okuyama, K.; Soboi, Y.; Iijima, N.; Hirabayashi, K.; Kunitake, T.; Kajiyama, T. Bull. Chem. Soc. Jpn. 1988, 61, 1485. (23) Fahrnow, A. M.; Saenger, W.; Fritsch, D.; Schnieder, P.; Fuhrhop, J.-H. Carbohydr. Res. 1993, 242, 11. (24) Abe, Y.; Harata, K.; Fujiwara, M.; Ohbu, K. Langmuir 1996, 12, 636.
Figure 2. Packing of L4 projected onto the ab-plane.
unit as a spacer between the headgroup and the alkyl chains which allows compact chain packing without conformational constraint on the headgroup. Transmission Electron Micrographs. Negatively stained electron micrographs show that the shape of the vesicles formed spontaneously (Figure 3a,b) either from the free or the Cu(II)-complexed amphiphiles are spherical in shape. Similar shapes can be observed for the vesicles prepared by the extrusion method (Figure 3c,d). The average diameter of the vesicles approximately lies in the range 225-350 nm. The copper(II) inclusion complexes
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Figure 3. Representative negative-stain transmission electron micrograph of the spontaneously made vesicles of (a, top left) L4 (×10000), (b, top right) its Cu(II) inclusion complex (×12000) along with micrographs of extruded vesicles of (c, bottom left) L4 (×20000) and (d, bottom right) its Cu(II) complex (×20000).
of the amphiphiles do not lead to any significant structural changes since the cryptand cavity is preorganized,17 and hence they behave like free amphiphiles. Stability of the Bilayer Structures and Colloidal Stability. In liposome research, the term stability is not well-defined.25 In a broad sense, it refers to the stability of the particles and its constituents including the entrapped molecules. It also refers to the stability of the size and shape of the vesicles. In the present work, the stability of the vesicular dispersions refers to the stability of the size of the vesicles. With time, the vesicles fuse, they form clusters, and finally coagulate. We have probed the stability of the vesicles mainly by measuring the turbidity of the dispersions at 400 nm. A stable vesicular dispersion would not show any perceptible variation of turbidity. On the other hand, for a metastable dispersion the turbidity first increases due to formation of vesicular aggregates and then slowly decreases to almost zero as the aggregates form clusters and settle at the bottom. (25) Lasic, D. D. Angew. Chem., Int. Ed. Engl. 1994, 33, 1685.
In the present case, stability of the vesicular suspension is found to be dependent on the method adopted for their formation. For the vesicular dispersions made by the extrusion method, turbidity increases only by 5-10% after a month in the case of L1 and L2; Dispersions made from L3 and L4 show less stability and the turbidity increases by the same amount within 15 days at 298 K (Table 3). In each case, however, the turbidity decreases to almost zero after about 3 months. For L5 sedimentation is almost complete at room temperature within 2-3 days, while for L6, which has the shortest hydrophobic chains (Scheme 1), the sample crashes out as clumps when extruded through the polycarbonate membrane. The same trend is observed in the case of Cu(II)-complexed amphiphiles. The spontaneously formed vesicular dispersions in 4% ethanolic water are found to be less stable compared to the corresponding samples prepared by extrusion. This could be due to the fact that extruded dispersions give smaller vesicles, which is also seen in the TEM photographs (Figure 3). Interestingly, among all the spontane-
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Langmuir, Vol. 14, No. 20, 1998 5717
Figure 4. Representative negative-stain transmission electron micrograph of the spontaneously made vesicles of (a) L4 (×10000) after 1 week. Table 3. Turbidity Measurements of the Vesicular Suspension Made by Extrusion Method of the Free Amphiphiles L1-L4 (Concentrated 2 × 10-4 M, Temperature 298 K) in 4% Ethanolic Medium at 400 nm with Time optical density amphiphile
fresh
15 days
30 days
3 months
L1 L2 L3 L4
0.65 0.60 0.70 0.73
0.67 0.63 0.75 0.78
0.70 0.67 0.37 0.30
0.008 0.006 0.004 0.005
Table 4. Turbidity Measurements of the Spontaneously Made Vesicular Suspension of the Free Amphiphiles L1-L5 (Concentration 2 × 10-4 M, Temperature 298 K) in 4% Ethanolic Medium at 400 nm with Time optical density amphiphile
fresh
4h
1 day
2 days
4 days
7 days
L1 L2 L3 L4 L5
0.70 0.65 0.72 0.77 0.82
0.90 0.84 0.92 0.84 1.10
0.85 0.75 0.60 0.97 0.60
0.75 0.55 0.45 1.00 0.20
0.65 0.40 0.25 1.20 0.05
0.10 0.08 0.08 1.40 0.05
Table 5. Influence of Ionic Strength (50 mM NaCl) on the Spontaneously Made Vesicles of Free L1-L5 (Concentration 2 × 10-4 M, Temperature 298 K) in 4% Ethanolic Medium optical density amphiphile
fresh
1/ 4
h
5h
10 h
72 h
L1 L2 L3 L4 L5
0.72 0.68 0.75 0.75 0.83
0.65 0.58 0.66 0.68 0.72
0.58 0.50 0.40 0.35 0.15
0.35 0.20 0.10 0.07 0.01
0.012 0.008 0.008 0.002 0.002
ously made vesicular dispersions, those of L4 and its Cu(II) complex show higher stability. For others, turbidity values show a general pattern (Table 4). Vesicular suspensions of L6 and its Cu(II) derivative crash out within an hour due to formation of large clumps and the dispersions become almost clear at 298 K. However, unlike other cases, the clumps do not settle at the bottom. In the
Figure 5. Turbidity vs 1/λ4 measured at 25 °C for vesicular suspension from L4 stored at 25 °C for the lengths of time indicated.
presence of 50 mM dm-3 of NaCl, the stability of the vesicles decreases (Table 5) due to higher rate of flocculation. On the other hand, by lowering the temperature to 278 K, corresponding vesicular suspensions are found to be more stable due to slowing down of the process of fusion of the vesicles. We have probed the spontaneously formed dispersions by transmission electron microscopy after aging. A typical micrograph is shown in Figure 4 for L4 taken after 1 week. The photograph clearly shows fusion of vesicles to form larger vesicles. Turbidity measurements in the range 300-600 nm were carried out at 298 K according to the method of Barrow and Lentz26 on the spontaneously made vesicles. According to this method, a plot of turbidity between 300 and 650 nm against the reciprocal fourth power of the scattering wavelength is empirically found to be linear with zero intercepts (extrapolated to infinite wavelength). In the presence of minute quantities of large multilamellar vesicles, the plot remains linear with intercepts quantitatively proportional to the amount of large vesicles. Although this technique is not a popular one, it is quite convenient27 and we have used it to supplement our electron micrograph data on the aging vesicular suspension. These studies indicate that vesicles in 4% ethanolic water medium are mostly unilamellar. When studied as a variation of time, L4 show that large multilamellar aggregates form at 303 K after 1 week (Figure 5). Other systems (except L6 ) form multilamellar aggregates within days; L6 forms multilamellar structures and precipitates out in an hour. Cu(II) salts of the amphiphiles behave like the corresponding free amphiphiles. Conclusion In conclusion, we have shown that cryptand-based triple-tailed neutral and Cu(II)-complexed amphiphiles form vesicles spontaneously. This may be due to special topology of the cryptand architecture and the presence of a carbonyl unit as a spacer between the cryptand and the (26) Barrow, D. A.; Lentz, B. R. Biochim. Biophys. Acta 1980, 597, 92. (27) Fendler, J. H. Membrane Mimetic Chemistry; Wiley: New York, 1982; pp 117-120.
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hydrophobic chains which allows compact chain packing without conformational constraint on the headgroup. The disposition of the hydrophobic chains in these amphiphiles favor bilayer curvature leading to the formation of spontaneous vesicles. The solid-state structure of L4 provides the example where the hydrophobic tails pack in the lattice by a mixed interdigitizing and noninterdigitizing manner, which is quite uncommon. We have also shown that spontaneous vesicular aggregates are possible with cryptand-based amphiphiles even when each tail is only four carbon atoms long.
Ghosh et al.
Acknowledgment. This work was supported by the Department of Science and Technology, New Delhi, India (Grant No. SP/S1/F-13/91 to P.K.B.). Supporting Information Available: 1H NMR, FABMS, X-ray data, and packing diagrams (27 pages). This material is contained in many libraries on microfiche, immediately follows this article in the microfilm version of the journal, can be ordered from the ACS, and can be downloaded from the Internet. See any current masthead page for ordering information and Internet access instructions. LA9709913
