4480
Langmuir 1997, 13, 4480-4482
Notes Supramolecular Transformations of Vesicles from Amino Acid Based Double Chain Amphiphiles Claudio Cescato, Peter Walde, and Pier Luigi Luisi* Institut fu¨ r Polymere, ETH-Zentrum, Universita¨ tstrasse 6, CH-8092 Zu¨ rich, Switzerland Received February 21, 1997. In Final Form: May 5, 1997
Vesicles formed by surfactants containing amino acidic groups offer the interest of combining the spherical membrane lipidic structure with the binding and catalytic functions of proteins. Following this line of reasoning, several attempts were undertaken in the literature, and in fact, amino acid based amphiphiles containing two hydrophobic chains were the first synthetic, chiral bilayer forming amphiphiles described in the literature.1 These amphiphiles attracted much attention, especially after it was recognized that they exhibit morphological transformations between spherical, closed bilayers (vesicles), and helical superstructures if cooled below their phase transition temperatures.2 Similar observations were made with a variety of other chiral amphiphiles, including naturally occurring phospholipids,3 galactosylceramides,4 diacetylenephosphatidylcholines,5 phosphatidylnucleosides,6 and N-alkylaldonamides.7 The prerequisites for the formation and growth of helical superstructures in aqueous dispersion are believed to be (i) the amphiphilic character of the molecule, (ii) its chirality, and (iii) relatively strong attractive interactions between the amphiphiles (e.g., hydrogen bonds between amides).8 The chemical structure of most of the double chain amino acid amphiphiles studied thus far can be schematically * To whom to address correspondence. (1) Kunitake, T.; Nakashima, N.; Hayashida, S.; Yonemori, K. Chem. Lett. 1979, 1413-1416. (2) (a) Nakashima, N.; Asakuma, S.; Kim, J.-M.; Kunitake, T. Chem. Lett. 1984, 1709-1712. (b) Nakashima, N.; Asakuma, S.; Kunitake, T. J. Am. Chem. Soc. 1985, 107, 509-510. (c) Ishikawa, Y.; Nishimi, T.; Kunitake, T. Chem. Lett. 1990, 25-28. (d) Yamada, K.; Ihara, H.; Ide, T.; Fukumoto, T.; Hirayama, C. Chem. Lett. 1984, 1713-1716. (e) Ihara, H.; Fukumoto, T.; Hirayama, C.; Yamada, K. Polym. Commun. 1986, 27, 282-285. (f) Ihara, H.; Takafuji, M.; Hirayama, C.; O’Brien, D. F. Langmuir 1992, 8, 1548-1553. (3) (a) Binary mixtures of negatively charged cardiolipin and phosphatidylcholines in the presence of Ca2+: Lin, K.-C.; Weis, R. M.; McConnell, H. M. Nature 1982, 296, 164-165. (b) Negatively charged dimyristoylphosphatidylglycerol in the presence of 1 M NaCl: Kodama, M.; Miyata, T.; Yokoyama, T. Biochim. Biophys. Acta 1993, 1168, 243248. (4) Kulkarni, V. S.; Anderson, W. H.; Brown, R. E. Biophys. J. 1995, 69, 1976-1986. (5) (a) Georger, J. H.; Singh, A.; Price, R. R.; Schnur, J. M.; Yager, P.; Schoen, P. E. J. Am. Chem. Soc. 1987, 109, 6169-6175. (b) Rudolph, A. S.; Ratna, B. R.; Kahn, B. Nature 1991, 352, 52-55. (6) (a) Yanagawa, H.; Ogawa, Y.; Furuta, H.; Tsuno, K. J. Am. Chem. Soc. 1989, 111, 4567-4570. (b) Itojima, Y.; Ogawa, Y.; Tsuno, K.; Handa, N.; Yanagawa, H. Biochemistry 1992, 31, 4757-4765. (c) Bonaccio, S.; Wessicken, M.; Berti, D.; Walde, P.; Luisi, P. L. Langmuir 1996, 12, 4976-4978. (7) (a) Pfannemu¨ller, B.; Welte, W. Chem. Phys. Lipids 1985, 37, 227-240. (b) Fuhrhop, J.-H.; Schnieder, P.; Boekma, E.; Helfrich, W. J. Am. Chem. Soc. 1988, 110, 2861-2867. (c) Fuhrhop, J.-H.; Demoulin, C.; Rosenberg, J.; Boettcher, C. J. Am. Chem. Soc. 1990, 112, 28272829. (8) (a) Fuhrhop, J.-H.; Ko¨ning, J. Membranes and Molecular Assemblies: The Synkinetic Approach; The Royal Society of Chemistry: Cambridge, 1994. (b) Recently, a theoretical study on the possible molecular origin of the helix formation of amphiphiles has been
S0743-7463(97)00190-X CCC: $14.00
represented as structure 1 shown in Figure 1. Either glutamic acid or aspartic acid is the central unit of the molecule, representing the building block between the head group and the hydrophobic part of the molecule. The two alkyl chains are linked with the two carboxylate groups of Glu and Asp via either ester or amide bonds. The hydrophilic head group finally is connected to the amino group of the amino acid. Notice that in this way the functional groups of the amino acids are protected, and therefore the original goal of having amino acid functions for binding and catalysis is not fulfilled. Here, we present alternative ways for the preparation of amino acid based amphiphiles that permit us to circumvent this situation, so as to have vesicles with free amino acid side chains.9 In the following, the synthesis of the two compounds 2 and 3 (Figure 1) will first be presented together with the physical characterization of the corresponding vesicles. In contrast to 1, the two amphiphiles 2 and 3 are characterized by a free amino acid side chain, either of Glu (2) or of Arg (3), which represent the polar head groups of the molecules. The two alkyl chains are connected through an amide bond with the R-carboxylate and with the R-amino group of the amino acid. In this way, the functional side chain groups remain free. 2 and 3 were synthesized by first acylating the R-amino group of the side chain protected amino acid (glutamic acid 5-benzyl ester and ω-N-nitroarginine) with the N-hydroxysuccinimide ester of lauric acid, followed by the coupling of laurylamine to the R-carboxyl group of the amino acid. In the final step, the corresponding protecting groups were removed by standard procedures (H2 with Pd/C). The purity of the compounds was ascertained by 1H-NMR spectroscopy, mass spectrometry, and elemental analysis. Aqueous dispersions of 2 or 3 were prepared by adding an appropriate amount of the amphiphile to a buffer solution and heating the mixture above the phase transition temperature Tc.10 Compound 2 was first deprotonated by dispersing it in the presence of 1.1 equiv of NaOH, followed by the addition of a 50 mM glycine buffer to reach a final pH of 10. Amphiphile 3 was directly dispersed in 50 mM Tris buffer (pH 8.0). The concentration of 2 or 3 in all experiments was in the range 0.1-4.3 mM. Aqueous dispersions of both amphiphiles 2 or 3 once heated to and kept above Tc, showed the presence of spherically closed bilayers (vesicles), as evidenced by electron micrographs. If a sample of 3 was repetitively passed through polycarbonate membranes with 100 nm pores, as routinely done, for example, with vesicles from presented: Nandi, N.; Bagchi, B. J. Am. Chem. Soc. 1996, 118, 1120811216. (c) The predicted sense of helicity seems to agree with known experimental results: Nandi, N.; Bagchi, B. J. Phys. Chem. A 1997, 101, 1343-1351. (9) (a) Formation of vesicles and helical fibers is also known for singlechain N-acyl amino acids (e.g., n-dodecyl-L-Asp) in which amino acid side chains and the carboxylate connected to the R-carbon atom are free: Imae, T.; Takahashi, Y.; Muramatsu, H. J. Am. Chem. Soc. 1992, 114, 3414-3419. (b) Vesicles from double-chain amphiphiles with oligoL-Glu or oligo-L-Asp head groups, in which all the side chains are free, have also been prepared, and it has been shown that these vesicles transform into fibrous aggregates.2d,e (10) Tc of aqueous dispersions of 2 and 3 (3%, wt/v) was determined by differential scanning calorimetry (DSC) using a TC 11 instrument from Mettler (Switzerland) at a heating rate of 2 °C/min. While 2 showed a single transition at 48 °C, the DSC trace of 3 was complex, showing multiple transitions above 25 °C, the highest being at 42 °C.
© 1997 American Chemical Society
Notes
Langmuir, Vol. 13, No. 16, 1997 4481
Figure 2. Video-enhanced light micrographs of aqueous dispersions of 2 (pH 9.2). The samples were analyzed by an Axioplan microscope (from Zeiss) after storage at room temperature for 13 days (A) and 30 days (B), respectively. The length of the bar corresponds to 10 µm.
Figure 1. Chemical structures of some of the amino acid based amphiphiles studied before2 1 and of the compounds 2-5 prepared and investigated in the present work.
phosphatidylcholines,11,12 the size of the vesicles was reduced to approximately 100 nm, remaining stable for several days as long as the temperature was kept above about 45 °C. Another strategy for the preparation of amphiphiles containing free functional groups of the amino acid side chains is exemplified by 4 and 5. In this case, the reactive imidazole group present in His is attached to a phosphatidyl moiety through a methylene spacer. These two new imidazole-containing phospholipids 4 (1,2-dimyristoyl-sn-glycerol-3-phosphomethylimidazole, DMPMI) and 5 (1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphomethylimidazole, POPHMI), were synthesized from the corresponding phosphatidylcholines (DMPC and POPC) and 4-(hydroxymethyl)imidazole.13 From 4 and 5 stable unilamellar (11) Mayer, L. D.; Hope, M. J.; Cullis, P. R. Biochim. Biophys. Acta 1986, 858, 161-168. (12) Dorovska-Taran, V.; Wick, R.; Walde, P. Anal. Biochem. 1996, 240, 37-47. (13) 4 and 5 were synthesized with phospholipase D from Streptomyces sp. AA586 (from Genzyme) using phosphatidylcholines (from Avanti Polar Lipids) and 4-(hydroxymethyl)imidazole (from Fluka) in 1:1 (v/v) CHCl3/0.1 M sodium acetate buffer, pH 4.5, containing 0.1 M Ca2+ at 45 °C (2 h), following the procedure described by Shuto et al. for 5′-phosphatidylnucleosides: Shuto, S.; Ueda, S.; Imamura, S.; Fukukawa, K.; Matsuda, A.; Ueda, T. Tetrahedron Lett. 1987, 28, 199202.
liposomes could be formed by the extrusion technique10 with a Tc of the liposomes of 4 being at ∼30 °C, as determined by DSC and fluorescence depolarization measurements using 1,6-diphenyl-1,3,5-hexatriene (DPH) as fluorescence probe. Mixed liposomes containing DMPC/ DMPHMI or POPC/POPHMI (1:1, molar ratio) showed a weak catalytic activity toward the hydrolysis of pnitrophenyl octanoate in comparison to the values obtained in the presence of imidazole in the buffer.14 It appears that under appropriate conditions both classes of compounds 2 and 3, and 4 and 5, are able to form stable vesicles that contain free functional groups of amino acid side chains. Concerning the stability of these (chiral) vesicles, a general important question is whether time dependent structural transformations occur to form other forms of supramolecular aggregates, in particular if the samples are stored below Tc. Indeed, if samples of 2 aged at room temperature (T < Tc), the number of submicrometer-sized vesicles decreased and more and more elongated structures appeared (Figure 2). These helical and tubular assemblies had diameters on the order of dozens of micrometers. A light micrograph of an aqueous dispersion of 2 kept for 30 days at room temperature is shown in Figure 2B. Similarly, vesicles of 3 transformed into nonvesicular, elongated aggregates that, however, were considerably smaller than in the case (14) The pseudo-first-order rate constants at 25 °C were (5.4 ( 0.3) × 10-4 s-1 (for 4) and (8.1 ( 0.1) × 10-4 s-1 (for 5). The corresponding value in buffer alone (65 mM sodium phosphate, pH 8) was (1.0 ( 0.2) × 10-4 s-1 and in the presence of 60 µM imidazole (0.9 ( 0.2) × 10-4 s-1. The initial concentration of p-nitrophenyl octanoate was 6 µM.
4482 Langmuir, Vol. 13, No. 16, 1997
Notes
Figure 3. CD spectrum (A) and negative staining electron micrographs (B, C) of an aqueous dispersion of 3 (50 mM Tris/HCl, pH 8). (A) Temperature dependence of the CD spectrum after extrusion through polycarbonate membranes with 100 nm pore diameters. Curve 1: immediately after extrusion, measured at 53 °C. The same sample, stored at room temperature for 53 h and measured at 25 °C (curve 2), and then reheated to 53 °C and measured again (curve 3). Curve 4: 3 dissolved in methanol. [3] ) 0.5 mM, path length ) 0.5 cm. Molar ellipticities are plotted against the wavelength, measured on a JASCO J-600 spectropolarimeter. An electron micrograph of a freshly extruded sample, [3] ) 0.1 mM, is shown in (B), and that of a sample that had been stored at room temperature for 5 days, [3] ) 1 mM, is given in (C). The length of the bar corresponds to 0.5 µm.
of 2 (see the negative staining electron micrograph in Figure 3). The initially spherical vesicles transformed within hours at room temperature into helical fragments and finally into helically twisted or tubelike structures. These assemblies seemed to be rather flexible since many curved segments were observed (Figure 3B). The general behavior of aqueous dispersions of 2 and 3 was rather similar to what has been observed before for other chiral amphiphiles.2-7 Changes in the molecular conformation on cooling dispersions of 3 from 53 to 25 °C were also studied by circular dichroism (CD) (see Figure 3A). The intensity of the CD signal at ∼208 nm (n f π* transitions of the amide bonds) of 3 dissolved in methanol (no aggregates) was comparatively small, suggesting a large number of conformations in equilibrium with each other. Above Tc, the CD signal of 3 dispersed in aqueous solution was also rather small (molar ellipticity [Θ] at 206 nm ∼ -16 × 103 deg cm2 dmol-1), which may indicate a considerably high conformational flexibility of the molecules within the vesicles. When the sample was cooled below Tc, the CD signal became more negative ([Θ]206 nm ) -11 × 104 deg cm2 dmol-1), which suggests a shift of the conformational equilibrium toward highly organized helical structures. As seen from Figure 3, the morphological vesicle f helical structure transformation was completely reversible (curve 3 of Figure 3, obtained after reheating the sample). In summary, for both amino acid based amphiphiles 2 and 3, vesicle f helical structure transformations were observed by changing the temperature. While above Tc vesicles are predominant, at a temperature below Tc helical strands and tubes are formed upon storage. This trans-
formation process was faster in the case of 3 (a matter of hours) compared with 2 (a matter of days). Our investigation indicated that this morphological transformation process, initially observed by Kunitake et al.2a-c and Ihara et al.,2d-f seems to be a rather general phenomenon for aqueous dispersions of chiral amphiphiles with complex head groups. In other words, no stable vesicles of 2 or 3 could be obtained below Tc. One may even wonder why liposomes of saturated phosphatidylcholines, such as 1,2dipalmitoyl-sn-glycero-3-phosphocholine or 1,2-distearoylsn-glycero-3-phosphocholine, under appropriate conditions do not undergo similar transformations. Furthermore, no helix formation could be observed with 4 or 5. In conclusion, we have shown here that it is possible to prepare vesicles containing free amino acid side chains on the lipidic surface. Preliminary data show that the enhancement of catalytic activity brought about by the imidazole-containing phospholipids on the vesicle surface is rather small.15 It is possible that better results could be obtained by the synergistic actions of different amino acid side chains in proximity to each other, a possibility that appears technically feasible by preparing vesicles containing two or more cosurfactant molecules bearing different amino acid side chain residues, e.g., those present in the active site of enzymes. Studies in this direction are in progress. Acknowledgment. The authors would like to thank Michaela Wessicken for the electron microscope analysis. LA9701900 (15) Cescato C. Untersuchungen an Aggregaten aus Aminosa¨ ureamphiphilen, Dissertation ETH-Zu¨rich Nr. 11709, 1996.
