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Langmuir 1998, 14, 4978-4986
Dicarboxylic Oligopeptide Bolaamphiphiles: Proton-Triggered Self-Assembly of Microtubes with Loose Solid Surfaces Masaki Kogiso, Satomi Ohnishi, Kiyoshi Yase, Mitsutoshi Masuda, and Toshimi Shimizu* National Institute of Materials and Chemical Research, 1-1 Higashi, Tsukuba, Ibaraki 305, Japan Received February 27, 1998. In Final Form: April 20, 1998 A new family of oligopeptide-based bolaamphiphiles, glycylglycine- (1a-h), glycylglycylglycine- (2a-b), sarcosylsarcosine- (3), L-prolyl-L-proline- (4), glycylsarcosylsarcosine- (5), and glycyl-L-prolyl-L-proline (6)based bolaamphiphiles with a dicarboxylic headgroup at each end, has been synthesized. The oligopeptide fragments were linked via amide bond to a long-chain R,ω-dicarboxylic acid as a hydrocarbon spacer. Self-assembling properties of these bolaamphiphiles in water have been studied by light and cryogenic temperature transmission electron microscopy, infrared spectroscopy, and pH titration. Only sodium or potassium salts (acid soap) of the bolaamphiphiles 1a, 1c, 1e, 2a, and 2b produced well-defined microtubes of 1-3-µm diameter with closed ends. All the tubes encapsulated a number of vesicular assemblies inside the aqueous compartment. The tube formation strongly depends on the connecting alkylene chain length, the alkylene even-odd carbon numbers, and constituent amino acid residues. Vectorial formation of acid-anion dimers and loose interpeptide hydrogen-bond networks are responsible for the microtube self-assembly. The atomic force microscopic observation of the microtube made of 1e revealed a distorted hexagonal arrangement of the headgroups on the surface. A self-assembling model and the tube formation mechanism are also discussed from the viewpoint of proton-triggered self-assembly.
Introduction Aggregation behavior of long-chain fatty acids strongly depends on the ionization state of end carboxyl groups.1-3 In dilute aqueous solutions, common fatty acid soaps4 and amino acid amphiphiles5,6 can produce stable micellar fibers. They also cause a prominent viscoelastic effect.7,8 From pH titration and infrared spectroscopy, the moderate hydration and protonation proved to be responsible for the stable formation of such fibers. The surfaces of these fibers should be fluid.9 On the other hand, the formation of amide hydrogen-bond chains allows the headgroup arrangement of amphiphiles to be solidlike.9,10 As a consequence, well-defined helical structures in rods and ribbons can be constructed.11-13 * Corresponding author: e-mail,
[email protected]; fax, Int. code +81-298-54-4422. (1) Feinstein, M. E.; Rosano, H. L. J. Phys. Chem. 1969, 73, 601607. (2) Hargreaves, W. R.; Deamer, D. W. Biochemistry 1978, 17, 37593768. (3) Cistola, D. P.; Atkinson, D.; Hamilton, J. A.; Small, D. M. Biochemistry 1986, 25, 2804-2812. (4) Tra¨ger, O.; Sowade, S.; Bo¨ttcher, C.; Fuhrhop, J.-H. J. Am. Chem. Soc. 1997, 119, 9120-9124. (5) Imae, T.; Takahashi, Y.; Muramatsu, H. J. Am. Chem. Soc. 1992, 114, 3414-3419. (6) Imae, T.; Trend, B. Langmuir 1991, 7, 643-646. (7) Tsujii, K.; Saito, N.; Takeuchi, T. J. Colloid Interface Sci. 1984, 99, 553-560. (8) Clausen, T. M.; Vinson, P. K.; Minter, J. R.; Davis, H. T.; Talmon, Y.; Miller, W. G. J. Phys. Chem. 1992, 96, 474-484. (9) Fuhrhop, J.-H.; Helfrich, W. Chem. Rev. 1993, 93, 1565-1582. (10) Fuhrhop, J.-H.; Ko¨ning, J. Membranes and Molecular Assemblies: The Synkinetic Approach; The Royal Society of Chemistry: Cambridge, 1994; Vol. 5. (11) Nakashima, N.; Asakuma, S.; Kunitake, T. J. Am. Chem. Soc. 1985, 107, 509-510. (12) Fuhrhop, J.-H.; Schnieder, P.; Bo¨kema, E.; Helfrich, W. J. Am. Chem. Soc. 1988, 110, 2861-2867. (13) Yanagawa, H.; Ogawa, Y.; Furuta, H.; Tsuno, K. J. Am. Chem. Soc. 1989, 111, 4567-4570.
We have studied chiral fibers with solid surfaces and reported a stereochemical effect of the connecting links for 1-glucosamide bolaamphiphiles.14 The fibers rearrange their hydrogen-bond networks between the sugar headgroups to form a platelet crystal.15 These characteristics largely reflect the even and odd carbon numbers of the hydrocarbon spacers. In this paper, we detail protontriggered self-assembling properties of a new family of dicarboxylic oligopeptide bolaamphiphiles, 1a-h, 2a-b, 3, 4, 5, and 6 (Chart 1) in alkaline aqueous solutions. In particular, our attention was focused on the solid surfaces of the microtubes16,17 made of 1a, 1c, and 1e, that is, interpeptide hydrogen-bond networks and headgroup arrangements. A quaternary ammonium amphiphile with an oligoglycine headgroup only produced a crystalline precipitate in aqueous solution.18 Therefore, supramolecular tube structures are discussed with respect to the ionization state of terminal carboxyl groups and hydrogenbond formation in the headgroup. The even-odd effect of the carbon numbers in the connecting hydrocarbon links is also discussed. Results Light Microscopic Observation. Aqueous solutions (10 mM) of each bolaamphiphile and 2 equiv of alkaline or alkaline earth hydroxide (pH < 8) gave a clear solution upon sonication at 25 °C. Physical and self-assembling properties of a family of oligopeptide-based bolaam(14) Shimizu, T.; Masuda, M. J. Am. Chem. Soc. 1997, 119, 28122818. (15) Masuda, M.; Shimizu, T. Carbohydr. Res. 1997, 302, 139-147. (16) Shimizu, T.; Kogiso, M.; Masuda, M. Nature 1996, 383, 487488. (17) Shimizu, T.; Kogiso, M.; Masuda, M. J. Am. Chem. Soc. 1997, 119, 6209-6210. (18) Shimizu, T.; Hato, M. Biochim. Biophys. Acta 1993, 1147, 5058.
S0743-7463(98)00241-8 CCC: $15.00 © 1998 American Chemical Society Published on Web 07/31/1998
Dicarboxylic Oligopeptide Bolaamphiphiles Chart 1. Molecular Structure of Oligopeptide Bolaamphiphiles with Dicarboxylic Headgroup at Each End
phiphiles are summarized in Table 1. When an aqueous solution is allowed to stand at room temperature for 2-3 weeks, the sodium salts of 1a, 1c, 1e, 2a, and 2b or the potassium salt of 1e produced cotton-like, fibrous assemblies. Light microscopic observations all revealed that these fibers are composed of microtubes of several hundreds micrometer length with closed ends. They are of uniform length, and the diameter is in the range of 1-3 µm. In particular, phase-contrast,16 dark-field,16 and confocal laser scanning light microscopy clearly showed the presence of a number of encapsulated spherical or ellipsoidal assemblies in the tubes (Figure 1). The enclosure distribution and size are random. Most of them did not move freely within the aqueous compartment. Only small spherical assemblies take a Brownian motion. Therefore, their surfaces seem to directly contact with the inner surface of the tubes. No spherical assemblies can be found outside the tubes. Low phase-contrast images of the spheroids suggest that these assemblies are not oil droplet or air bubble but vesicular assemblies composed of single lamellar or multilamellar. The tubes have a solid surface, which is quite different from the fluid bilayer structure exemplified by a myelin figure.9 The surface stiffness and thermal stability of the tubes can be confirmed by sonication and heating of the solution up to 90 °C. These two drastic treatments caused no morphological change and no phase transition in the microtubes, which are common for usual bilayer membranes.19 Dehydration of the tubes and subsequent drying (19) Kunitake, T.; Okahata, Y.; Shimomura, M.; Yasunami, S.; Takarabe, K. J. Am. Chem. Soc. 1981, 103, 5401-5413.
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in vacuo had no remarkable influence on the tube morphologies (Figure 1b). Only vesicle structures appeared as a collapsed flat disk on the inner surface of the tubes. Replacement of the glycylglycine or glycylglycylglycine fragment in the headgroup with sarcosylsarcosine, Lprolyl-L-proline, glycyl-L-prolyl-L-proline, or glycylsarcosylsarcosine produced no tube structures. Their alkaline aqueous solutions remained clear for several months (Table 1). In comparison with 2b, the bolaamphiphile 1e where one glycine residue is reduced at each end produced apparently thinner tubes of 1-2-µm diameter. In addition, we observed fine needle-shaped microcrystals of a few micrometers in length for 1e. The crystals are sitting on the outside of the tubes and projecting outward.17 The tube formation strongly depends on both the chain length (n) and even-odd carbon numbers of the hydrocarbon link in the bolaamphiphiles (Table 1). Glycylglycine bolaamphiphiles 1b, 1d, 1f, 1g, and 1h with odd-numbered (n ) 7 and 9) or longer alkylene chains (n > 11) produced no tubes but clear solutions or fine precipitates. Among countercations investigated, only monovalent Na+ and K+ were efficient for the tube formation. Li+, Ca2+, and (C2H5)4N+ were ineffective. Moreover, we found no remarkable effect of the ionic strength of Na+ (10-40 mM) on the tube formation. A further increase in the Na+ ionic strength (g50 mM) was also ineffective for the tube formation. FT-IR Spectroscopy. The CdO and N-H stretching vibration bands of the terminal carboxyl and amide groups directly reflect the ionization state and the formation of hydrogen bonds.4,20 In addition, the deformation vibration bands of methylene groups display the packing mode of a oligomethylene chain.21 The FT-IR spectrum of the dehydrated microtubes made of 1a was compared with those of its sodium salt and its carboxylic acid crystal. The obtained IR absorption bands and their assignments are summarized in Table 2, along with those of polyglycine I and II.22,23 Besides the carboxylate band at 1600 cm-1, we observed the new appearance of the carboxyl band at 1744 cm-1 for the microtube, which is absent in the sodium salt. The occurrence of amide I and II bands at 1640 and 1551 cm-1, respectively, supports the hydrogen bond formation between the peptide headgroups in the microtubes. However, the occurrence of both hydrogen-bonded (3325 cm-1) and non-hydrogen-bonded NH stretching bands (3419 and 3497 cm-1) shows the presence of an incomplete, somewhat loose network of hydrogen bonds. The microtube also showed a sharp peak at 1408 cm-1 attributable to the symmetric stretching vibration band of the COO- group. The methylene scissoring band was observed as a shoulder at 1434 cm-1. In contrast to a broad band of the acid crystal around 1430 cm-1 attributable to orthorhombic packing of the oligomethylene chain,24 its sharp appearance indicates a chain packing (20) Doan, V.; Ko¨ppe, R.; Kasai, P. H. J. Am. Chem. Soc. 1997, 119, 9810-9815. (21) Snyder, R. G.; Strauss, H. L. J. Phys. Chem. 1982, 86, 51455150. (22) Blout, E. R.; Linsley, S. G. J. Am. Chem. Soc. 1952, 74, 19461951. (23) Elliott, A.; Malcolm, B. R. Trans. Faraday Soc. 1955, 52, 528536. (24) Garti, N.; Sato, K. Crystallization and Polymorphism of Fats and Fatty Acids; Marcel Dekker: New York, 1988. (25) Yamada, N.; Okuyama, K.; Serizawa, T.; Kawasaki, M.; Oshima, S. J. Chem. Soc., Perkin Trans. 2 1996, 2707-2714. (26) Parikh, A. N.; Schivley, M. A.; Koo, E.; K.Seshadri; Aurentz, D.; Mueller, K.; Allara, D. L. J. Am. Chem. Soc. 1997, 119, 3135-3143. (27) Badger, R. M.; Pullin, A. D. E. J. Chem. Phys. 1954, 22, 11421142.
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Table 1. Physical and Self-Assembling Properties of Oligopeptide-Based Bolaamphiphiles oligopeptide-based bolaamphiphile ZzzYyyXxx-NHCO(CH2)nCONH-XxxYyyZzz 1a 2a 1b 1c 1d 1e
3 4 2b 5 6 1f 1g 1h
n
XxxYyyZzza
6 6 7 8 9 10
GlyGly GlyGlyGly GlyGly GlyGly GlyGly GlyGly GlyGly GlyGly GlyGly GlyGly SarSar ProPro GlyGlyGly GlySarSar GlyProPro GlyGly GlyGly GlyGly
10 10 10 10 10 11 12 14
self-assembly mp (°C)
206 (dec) 234 (dec) 222 (dec) 222 (dec) 228 (dec) 224 (dec)
oil oil 234 (dec) oil oil 225 (dec) 224 (dec) 216 (dec)
hydroxideb
morphologyc
tube yield (%)
NaOH NaOH NaOH NaOH NaOH NaOH LiOH KOH Ca(OH)2 (C2H5)4NOH NaOH NaOH NaOH NaOH NaOH NaOH NaOH NaOH
tube tube (solution) tube precipitate tube (solution) tube (solution) (solution) (solution) (solution) tube (solution) (solution) precipitate precipitate precipitate
5-10 1-3 1-3 1-3 1-3
1-3
a Xxx, Yyy, and Zzz indicate one of glycine, sarcosine, or L-proline residue. b 2 equiv of each hydroxide was added to the corresponding aqueous dispersion (10 mM) of the bolaamphiphile. c Observed using light microscopy.
Figure 1. (a) Vesicle-encapsulated microtubes made of 1e in water, observed using confocal laser scanning microscopy. (b) Dried microtubes made of 1e, observed using phase-contrast light microscopy. Collapsed vesicles are denoted by arrows.
in a triclinic or hexagonal structure.25-27 Furthermore, the CH2 antisymmetric and symmetric stretching bands at 2927 and 2854 cm-1, respectively, suggested the presence of high trans conformational populations15,21 of the hydrocarbon link in the microtube.
To investigate an even-odd effect of the carbon numbers in the hydrocarbon link on the supramolecular tube assemblies, we compared FT-IR spectra of the dicarboxylic bolaamphiphiles containing the C6-C12 hydrocarbon link (see Supporting Information). The peak frequencies of the N-H stretching (the amide A and B bands27,28), the CH2 stretching, and the amide I and II bands clearly show an even-odd effect for all chain lengths (6 e n e 12) measured (Figure 2). This behavior is quite different from that of 1-glucosamide bolaamphiphiles,14 in which a definite even-odd effect can be observed only for the longer hydrocarbon link (n g 10). Difference in conformational rigidity of the connecting n-alkylene chains15,29 will be responsible for the chain-length dependence of the evenodd effect. Actually, X-ray single-crystal analyses of 1a,30 1c,30 and 1e17,31 revealed no difference in the alkylene chain conformation. The absorption frequencies of the CdO stretching band (amide I) should decrease with the hydrogen-bond formation, whereas those of the N-H deformation band (amide II) should increase. Therefore, the odd-numbered glycylglycine bolaamphiphiles 1b, 1d, and 1f form relatively stronger hydrogen bonds between the amide groups than the even-numbered derivatives 1a, 1c, 1e, 1g, and 1h. Interestingly, this even-odd behavior is similar to that of 1-glucosamide bolaamphiphiles.14 Hydrogen-bond networks in the headgroup play an important role in characterizing the hydrocarbon link conformation. Atomic Force Microscopic Observation. To characterize the membrane thickness and surface details with molecular resolution, we observed the microtubes made of 1e at 25 °C in air using atomic force microscopy (AFM). Figure 3 shows an AFM image (10 µm × 10 µm) of airdried microtubes and the cross-sectional profile of vesiclelacked and vesicle-encapsulated portions of a tube. The tube heights were evaluated to be 232 ( 31 and 444 ( 37 nm out of 8-10 samples, respectively. The flat shape of the cross section for the vesicle-lacked portion indicates the absence of an aqueous compartment. This means that the membrane thickness of the tube wall is at longest 116 (28) Miyazawa, T. Poly-R-amino acids; Marcel Dekker: New York, 1967. (29) Shimizu, T.; Masuda, M.; Shibakami, M. Chem. Lett. 1997, 267268. (30) Unpublished results. (31) Kogiso, M.; Masuda, M.; Shimizu, T. Supramol. Chem., in press.
Dicarboxylic Oligopeptide Bolaamphiphiles
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Table 2. FT-IR Absorption Bands and Their Assignments for the Three Different Ionization States of Glycylglycine Bolaamphiphile 1a and Two Polyglycine Structures glycylglycine bolaamphiphile 1a assignment
sodium salt (soap)
NH str. (amide A)
3325 3419 3495 3075 2927 2851
3325 3419 3497 3078 2927 2854 1744
amide I
1639
1640
COO- antisym str. amide II CH2 scissoring def. COO- sym str. amide IIId
1601 1550 1438 1407 1277 1251 1035
1600 1551 1434 (shc) 1408 1278 1250 1037
NH str. (amide B) CH2 antisym str. CH2 sym str. COOH
GlyGly skeletal vibration a
From ref 23. b From ref 22. c Shoulder.
d
microtube
polyglycine poly[Gly]n acid crystal
polyglycine I
polyglycine II
3300a
3290a
1685b 1630b
1641b
1553 1430 (shc)
1524b
1558b
1269 1248 1030
1297b
1309b
1015a
1026a
3312 3083 2924 2854 1706 1728 1643
Including OH def. + C-O str. (COOH).
Figure 2. Dependence of (a) the N-H stretching (amide A), (b) the N-H stretching (amide B), (c) the CH2 antisymmetric stretching, (d) CH2 symmetric stretching, (e) amide I, and (f) amide II IR absorption bands on the chain length (n) of 1a-g.
Figure 3. (a) AFM image (10 µm × 10 µm) of vesicle-lacked and vesicle-encapsulated portions of a microtube made of 1e, (b) their cross-section profile, and (c) a schematic representation of the cross sections. In this AFM image, the vertical distances are 226.6 and 406.9 nm for the vesicle-lacked and vesicleencapsulated portions, respectively.
nm. Similarly, the thickness of the vesicle wall is less than 106 nm. Figure 4 shows a high-resolution AFM image (5 nm × 5 nm) of the tube surface. The surface layers were not damaged by the AFM tip even after repeated scanning. The brighter portions, which correspond to individual terminal headgroups of 1e, appear to be arranged in a two-dimensional (2D) oblique array. Nearest- and next-neighboring spacings can be roughly estimated to be 0.44 (a-axis) and 0.44 nm (b-axis),
respectively, and interaxis angle γ is 103°. Therefore, the lattice dimension of the headgroup arrangement is analogous to a pseudohexagonal packing of 1e in the crystal.17,31 However, the molecular array in the AFM image is distorted. Cryogenic Temperature Transmission Electron Microscopic Observation. Aqueous solutions of the anionic bolaamphiphiles have been characterized using
4982 Langmuir, Vol. 14, No. 18, 1998
Figure 4. (a) AFM image (5 nm × 5 nm) of a microtube surface made of 1e and (b) a processed AFM image of the headgroups with Grain size analysis.
Figure 5. Cryo-TEM micrograph of a swarm of rodlike micelles in the supernatant of aqueous solution containing the sodium salt of 1e.
low-dose cryogenic temperature transmission electron microscopy (cryo-TEM) at high magnification. Cryo-TEM for the clear solution (10 mM) of 1e obviously indicated that the charged species of the bolaamphiphile exist as a swarm of rodlike micelles of ca. 10-nm width (Figure 5). This cryo-TEM image is different from the micellar fibers obtained so far.4,5 Such an aggregation state of 1e can be
Kogiso et al.
Figure 6. (a) pH dependence of the ionization degree (R) when aqueous dispersions of 1a-g (10 mM) were titrated with 1 N NaOH aqueous solution at 23 °C: (3) 1a; (1) 1b; (O) 1c; (b) 1d; (4) 1e; (2) 1f; (]) 1g. (b) Detailed titration profile (R > 0.6) for 1a and 1g. The pH and R range, at which we observed microtubes, are denoted by arrows. Appearance of the aqueous solution: (- - -) clean; (s) opalescent.
also confirmed by the occurrence of the IR amide I (CdO str.) and II (N-D def.) bands at 1638 and 1460 cm-1, respectively, in alkaline deuterium oxide solution (D2O + 2 equiv of NaOD), which is ascribed to hydrogen bond formation. The cmc (critical micelle concentration) value was estimated to be >50 mM for the pure sodium salt of 1e in water at 20 °C. Therefore, immediate protonation to the end carboxylate groups will promote the molecules to associate together via hydrogen bonds and to form such rodlike micelles. Ionization Degree (r) as a Function of pH. To investigate the ionization profile and its chain-length dependence, we evaluated the ionization degree (R) of a family of dicarboxylic bolaamphiphiles as a function of pH from titration method. Figure 6a shows a plot of the ionization degree (R) against pH for the aqueous solutions of 1a-h. Each curve obtained has a sigmoidal shape and shifts to acidic pH with decreasing the chain length (n) of the connecting hydrocarbon links. A similar tendency of the titration curves was already reported for dicarboxylic amino acid amphiphiles.5,32 We can see that the longer the hydrocarbon link is, the more easily the bolaamphiphile can be protonated to form a carboxylic acid precipitate. Effective hydrophobic interaction between the alkylene chain is the reason for the promoted aggregation of the longer bolaamphiphiles. During the titration experiment, 1a gave an aqueous dispersion below R ) 0.81, whereas 1g yielded fine precipitates below R ) 0.99 (Figure 6b). The low yield of the microtubes formed
Dicarboxylic Oligopeptide Bolaamphiphiles
Figure 7. Schematic representation of (a) dicarboxylate species, (b) intralayer acid-anion dimers, and (c) interlayer acid-acid dimers that respectively result in the formation of rodlike micelles, microtubes, and needle-shaped microcrystals.
with 1c and 1e (Table 1) can be attributed to the prompt formation of acid species and their low solubility in water. In Figure 6b, we denoted the pH and R range on the titration curve, at which we actually observed the microtubes of 1a on the light microscopic scale. We notice that the tube formation takes place in the R range just before precipitation. Discussion Self-Assembling Model. In a similar way to dicarboxylic acid bolaamphiphiles reported previously,33,34 the present oligoglycine bolaamphiphiles should form monolayered multilamellar membranes. The 116-nm membrane thickness of the microtubes evaluated from AFM analysis corresponds to 30-40 layers of a fully extended molecule. The FT-IR study suggests that the microtube membrane includes intralayer acid-anion dimers35 (Figure 7b). Smith and Tanford have shown that hydrogen bonds between R-COO- and R-COOH are stabilized (32) Imae, T.; Suzuki, S.; Abe, A.; Ikeda, S.; Fukui, Y.; Senoh, M.; Tsuji, K. Colloids Surf. 1988, 33, 75-83. (33) Fuhrhop, J.-H.; David, H. H.; Mathieu, J.; Liman, U.; Winter, H. J.; Bo¨kema, E. J. Am. Chem. Soc. 1986, 108, 1785-1791. (34) Fuhrhop, J.-H.; Demoulin, C.; Rosenberg, J.; Bo¨ttcher, C. J. Am. Chem. Soc. 1990, 112, 2827-2829. (35) Haines, T. H. Proc. Natl. Acad. Sci. U.S.A. 1983, 80, 160-164.
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when carboxyl groups are linked to long alkyl chains.36 The protonated bolaamphiphiles form stable acid-anion pairs with deprotonated species through unusually strong hydrogen bonds. Vectorial formation of such dimers is considered to be a driving force for the tube formation. The bolaamphiphile 1e is in the micellar form above pH ≈8 and it forms crystals below pH ≈6.5. In the pH range of 6.5-8.0, the molecules self-assemble to form the microtubes, based on the presence of both protonated and deprotonated species. From FT-IR results and AFM analysis, the three-dimensional (3D) hydrogen bond networks between amide groups in the headgroups are not so regularly constructed as those in the acid crystal structure.17,31 Furthermore, the L-prolyl-L-proline or sarcosylsarcocine fragments having no hydrogen-bond ability do not contribute to the tube formation. Thus, the glycylglycine fragment is a necessary condition for the tube formation. The reason would be based on the diglycyl character that can form versatile hydrogen bonds running in three different directions.37 For the dehydrated tube of 1a, we found a broad band at 1037 cm-1 attributable to skeletal vibration of glycylglycine residue (Table 2), which can be similarly observed for polyglycine II.22,23 The peak frequencies of the amide I and II bands are also in good agreement with those of polyglycine II. Actually, the direct observation of tube surfaces by AFM supports the presence of distorted hexagonal molecular array. The surface is not analogous to that of usual 3D crystals, but solidlike. However, further protonation to the headgroups rearranges the hydrogen bond scheme to form interlayer acid-acid dimers (Figure 7c).17 Even-Odd Effect. Besides an even-odd effect of the connecting links on the FT-IR absorption bands (Figure 2), a similar effect was observed when each pH value at R ) 0.5 in the titration curve (Figure 6a) is plotted against the corresponding chain length (n). The odd-numbered bolaamphiphiles are slightly difficult to be protonated when compared with the even-numbered derivatives. Furthermore, the former derivatives form relatively stronger hydrogen bonds between amide groups. These findings suggest that the molecular arrangements in the odd-numbered derivatives are unsuitable for the formation of intralayer acid-anion dimers. Their hydrogen-bonded networks may allow the headgroups to be suitably arranged for the acid-acid dimers. Consequently, no microtubes were produced from the odd-numbered bolaamphiphiles irrespective of the chain length (n). A Possible Mechanism for the Tube Formation. From light and cryo-electron microscopy, FT-IR spectroscopy, and pH titration, we schematically illustrated a possible microtube formation mechanism in Figure 8. Just after the beginning of incubation, anionic bolaamphiphiles are being dissolved as rodlike micelles (Figure 8a). Immediately, protonation takes place at the fluid surfaces of the micelles and causes efficient aggregation between charged and uncharged species (Figure 8b). Thus, the vectorial formation of intralayer acid-anion dimers effectively produces a stable lamellar membrane with solid surfaces (Figure 8c). The membrane curvature may be caused by an asymmetric protonation environment at the outer and inner surfaces of the tube. The passage mentioned above slowly proceeds in solution because this protonation process requires several days to weeks, perhaps by the dissolution of carbon dioxide in air. Perturbation or vigorous mixing of the sample solution (36) Smith, R.; Tanford, C. Proc. Natl. Acad. Sci. U.S.A. 1973, 70, 289-293. (37) Bella, J.; Puiggali, J.; Subirana, J. A. Polymer 1994, 35, 12911297.
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Figure 8. A possible mechanism for the proton-triggered self-assembly of a vesicle-encapsulated microtube and a needle-shaped microcrystal. For clarity, multilayers of the microtubes and vesicles are tentatively represented by double or monolayers.
destroys the preorganization of the tubes. We found that the pH range where the microtube formation occurred corresponds to R ) 0.81-0.84 and R ) 0.98-0.99 for 1a and 1e, respectively. Protons added from outside are localized at the headgroup surfaces. Namely, the pH at the surfaces is lower than the bulk pH. A similar decrease in pH at highly charged surfaces was also observed for the vesicle formation with some fatty acids.2,38 The proton concentration in the headgroup region may vary widely, and the region of negatively charged headgroups functions as a proton buffer. Finally, the self-assembled microtubes, which are less soluble in aqueous media, precipitate at a certain pH (Figure 8d). The rearrangement of a hydrogen bond network into interlayer acid-acid dimers results in the growth of needlelike crystals17 on the tube surfaces. Usual fatty acid vesicles can be prepared by titrating alkaline micellar solutions with a dilute acid. However, no microtubes can be prepared for the present bolaamphiphiles by this titration method. Only precipitate can be obtained. The long-chain bolaamphiphiles (n g 11) also gave no tubes. Higher surface charge density created by condensed packing will allow them to form acid-acid dimer precipitates. On the other hand, one may envisage few possible mechanisms for the vesicle formation. One mechanism is based on a defect in the inner layers of the tube walls. For example, amputated multilayers may lead to the formation of energetically favorable spherical assemblies within the aqueous compartment (parts b and c of Figure 8). Actually, we observed a number of constricted portions on the outer surfaces of microtubes over a period of 1-2 years (Figure 9a). Consequently, the tube fission slowly took place to yield several ellipsoidal assemblies (Figure 9b). This morphological change indicates that the most energetically stable shape is spheroidal for the assemblies of 1a, 1c, and 1e. In summary, the proton-triggered self-assembly of the dicarboxylic oligoglycine bolaamphiphiles produced organic microtubes. Their solid surfaces are characterized by both incomplete 3D hydrogen-bond networks between peptide headgroups and a distorted hexagonal lattice. Rearrangement of the hydrogen-bond network converted the surface from a solidlike one to a crystalline one. Experimental Section Synthesis of Oligopeptide-Based Bolaamphiphiles. Amino acid derivatives were purchased from Kokusan Chemicals. Other chemicals were commercially available. Melting points (38) Gebicki, J. M.; Hicks, M. Chem. Phys. Lipids 1976, 16, 142160.
Figure 9. (a) Microtubes of 1e with several constricted portions on the outer surfaces and (b) isolated ellipsoidal or spherical vesicle assemblies by the slow tube fission, observed using phase-contrast light microscopy. were recorded on a Yanaco micro melting point apparatus, MP500D, and are uncorrected. 1H NMR spectra were recorded with a JEOL NX-270 spectrometer by using trimethylsilane as an internal standard for the organic solutions. FT-IR spectra were recorded on a Perkin-Elmer Paragon 1000 FT-IR spectrometer (resolution ) 4 cm-1). Glycylglycine-, glycylglycylglycine-, L-prolyl-L-proline-, sarcosylsarcosine-, glycyl-L-prolyl-L-proline-, and glycylsarcosylsarcosine benzyl ester hydrochlorides were all prepared by conventional peptide synthesis. The oligopeptide-based bolaamphiphiles were synthesized by the coupling of corresponding diand tripeptide benzyl ester hydrochloride with a long-chain R,ω-
Dicarboxylic Oligopeptide Bolaamphiphiles dicarboxylic acid at -5 to -10 °C in dimethylformamide (DMF). Water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1-hydroxylbenzotriazole were effective as a condensation reagent and an additive, respectively. The terminal benzyl groups of 1a-h, 2a, and 2b were removed by alkaline hydrolysis with 0.1 N NaOH, and those of other bolaamphiphiles containing sarcosine or proline residue were deprotected by catalytic hydrogenation under 5% Pd-carbon. Glycylglycine 1a-h and glycylglycylglycine bolaamphiphiles 2a-b were obtained as a white powder after workup by washing with water and acetone. The other bolaamphiphiles 3, 4, 5, and 6 were purified by silica gel column chromatography (eluent, CHCl3/MeOH/H2O ) 5/4/1) to yield oily products. The typical synthetic procedure and related analytical data are as follows. Bis(N-r-amido-glycylglycylglycine) 1,10-Decane Dicarboxylate (2b). To a solution of 1,10-decanedicarboxylic acid (0.50 g, 2.17 mmol) and 1-hydroxylbenzotriazole (0.65 g, 4.77 mmol) in DMF was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.92 g, 4.77 mmol) dissolved in CHCl3 (10 mL) with stirring at -5 °C. After 1 h, a solution of glycylglycylglycine benzyl ester hydrochloride (1.51 g, 4.77 mmol) in MeOH (10 mL) and triethylamine (0.67 mL, 4.77 mmol) was added to the reaction mixture. It was stirred for 24 h at 0 °C and allowed to gradually warm to room temperature. Evaporation of the solvent gave a white powder, which was washed with 10% citric acid, water, 4% sodium hydrogencarbonate, and water. Recrystallization from DMF gave the benzyl ester of 2b as a white powder (1.17 g). To a solution of the powder (1.17 g, 1.55 mmol) in DMF (300 mL) was added 0.1 N NaOH (35 mL, 3.5 mmol) at 85 °C. After being stirred for 4 h, the solution was acidified with 1 N HCl (4 mL, 4 mmol). The solvent was evaporated under reduced pressure to leave a white solid residue. The product was thoroughly washed with water and acetone to give a white powder of 2b (0.89 g, 71%). 1H NMR (DMSO-d6): δ 1.36 (m, 12H, CH2(CH2)6CH2); 1.59 (m, 4H, CH2(CH2)6CH2); 2.24 (t, J ) 7.3 Hz, 4H, COCH2(CH2)8CH2CO); 3.83 (d × 3, J ) 5.4 Hz, 12H, NHCH2CO × 3); 8.18 (t × 3, J ) 5.4 Hz, 6H, NH). Anal. Calcd for C24H40O10N6: C, 50.34; H, 7.04; N, 14.68. Found: C, 50.11; H, 7.07; N, 14.14. In the same way, other peptide-based bolaamphiphiles, 2a, 1a-h, 3, 4, 5, and 6, were also synthesized. 1H NMR spectra and elemental analyses for these bolaamphiphiles gave satisfactory data. Bis(N-r-amido-glycylglycylglycine) 1,6-Hexane Dicarboxylate (2a). Yield 86%. 1H NMR (DMSO-d6): δ 1.36 (m, 4H, CH2(CH2)2CH2); 1.60 (m, 4H, CH2(CH2)2CH2); 2.24 (t, J ) 7.3 Hz, 4H, COCH2(CH2)4CH2CO); 3.83, 3.85, and 3.87 (d × 3, J ) 5.4 Hz, 12H, NHCH2CO × 3); 8.18, 8.24, and 8.27 (t × 3, J ) 5.4 Hz, 6H, NH). Anal. Calcd for C20H32O10N6: C, 50.34; H, 7.04; N, 14.68. Found: C, 50.11; H, 7.07; N, 14.14. Bis(N-r-amido-glycylglycine) 1,6-Hexane Dicarboxylate (1a). Yield 81%. 1H NMR (DMSO-d6): δ 1.35 (m, 4H, CH2(CH2)2CH2); 1.59 (m, 4H, CH2(CH2)2CH2); 2.23 (t, J ) 7.3 Hz, 4H, COCH2(CH2)8CH2CO); 3.82 (d, J ) 5.4 Hz, 4H, CONHCH2CO × 2); 3.87 (d, J ) 5.4 Hz, 4H, CH2COO × 2); 8.19 (t × 2, J ) 5.4 Hz, 4H, NH). Anal. Calcd for C16H26O8N4: C, 52.38; H, 7.47; N, 12.22. Found: C, 52.40; H, 7.47; N, 12.11. Bis(N-r-amido-glycylglycine) 1,7-Heptane Dicarboxylate (1b). Yield 67%. 1H NMR (DMSO-d6): δ 1.35 (m, 6H, CH2(CH2)3CH2); 1.60 (m, 4H, CH2(CH2)3CH2); 2.24 (t, J ) 7.3 Hz, 4H, COCH2(CH2)5CH2CO); 3.82 (d, J ) 5.4 Hz, 4H, CONHCH2CO × 2); 3.87 (d, J ) 5.4 Hz, 4H, CH2COO × 2); 8.20 (t × 2, J ) 5.4 Hz, 4H, NH). Anal. Calcd for C17H28O8N4: C, 49.03; H, 6.78; N, 13.46. Found: C, 49.15; H, 6.78; N, 13.34. Bis(N-r-amido-glycylglycine) 1,8-Octane Dicarboxylate (1c). Yield 83%. 1H NMR (DMSO-d6): δ 1.35 (m, 8H, CH2(CH2)4CH2); 1.60 (m, 4H, CH2(CH2)4CH2); 2.23 (t, J ) 7.3 Hz, 4H, COCH2(CH2)6CH2CO); 3.82 (d, J ) 5.4 Hz, 4H, CONHCH2CO × 2); 3.87 (d, J ) 5.4 Hz, 4H, CH2COO × 2); 8.18 (t × 2, J ) 5.4 Hz, 4H, NH). Anal. Calcd for C18H30O8N4 0.5H2O: C, 49.19; H, 7.11; N, 12.75. Found: C, 49.64; H, 6.97; N, 12.68. Bis(N-r-amido-glycylglycine) 1,9-Nonane Dicarboxylate (1d). Yield 75%. 1H NMR (DMSO-d6): δ 1.36 (m, 10H, CH2(CH2)5CH2); 1.61 (m, 4H, CH2(CH2)5CH2); 2.24 (t, J ) 7.3 Hz, 4H, COCH2(CH2)7CH2CO); 3.83 (d, J ) 5.4 Hz, 4H, CONHCH2CO × 2); 3.88 (d, J ) 5.4 Hz, 4H, CH2COO × 2); 8.18
Langmuir, Vol. 14, No. 18, 1998 4985 (t × 2, J ) 5.4 Hz, 4H, NH). Anal. Calcd for C19H32O8N4: C, 51.34; H, 7.26; N, 12.61. Found: C, 51.43; H, 7.21; N, 12.49. Bis(N-r-amido-glycylglycine) 1,10-Decane Dicarboxylate (1e). Yield 83%. 1H NMR (DMSO-d6): δ 1.25 (m, 12H, CH2(CH2)6CH2); 1.49 (m, 4H, CH2(CH2)6CH2); 2.09 (t, J ) 7.3 Hz, 4H, COCH2(CH2)8CH2CO); 3.70 (d, J ) 5.4 Hz, 4H, CONHCH2CO × 2); 3.75 (d, J ) 5.4 Hz, 4H, CH2COO × 2); 7.96 (t × 2, J ) 5.4 Hz, 4H, NH). Anal. Calcd for C20H34O8N4: C, 52.38; H, 7.47; N, 12.22. Found: C, 52.40; H, 7.47; N, 12.11. Bis(N-r-amido-glycylglycine) 1,11-Undecane Dicarboxylate (1f). Yield 55%. 1H NMR (DMSO-d6): δ 1.36 (m, 10H, CH2(CH2)7CH2); 1.60 (m, 4H, CH2(CH2)7CH2); 2.24 (t, J ) 7.3 Hz, 4H, COCH2(CH2)9CH2CO); 3.83 (d, J ) 5.4 Hz, 4H, CONHCH2CO × 2); 3.88 (d, J ) 5.4 Hz, 4H, CH2COO × 2); 8.18 (t × 2, J ) 5.4 Hz, 4H, NH). Anal. Calcd for C21H36O8N4: C, 53.37; H, 7.68; N, 11.86. Found: C, 53.25; H, 7.75; N, 11.73. Bis(N-r-amido-glycylglycine) 1,12-Dodecane Dicarboxylate (1g). Yield 83%. 1H NMR (DMSO-d6): δ 1.35 (m, 10H, CH2(CH2)8CH2); 1.59 (m, 4H, CH2(CH2)8CH2); 2.23 (t, J ) 7.3 Hz, 4H, COCH2(CH2)10CH2CO); 3.82 (d, J ) 5.4 Hz, 4H, CONHCH2CO × 2); 3.87 (d, J ) 5.4 Hz, 4H, CH2COO × 2); 8.18 (t × 2, J ) 5.4 Hz, 4H, NH). Anal. Calcd for C22H38O8N4: C, 54.30; H, 7.87; N, 11.52. Found: C, 54.24; H, 7.94; N, 11.35. Bis(N-r-amido-glycylglycine) 1,14-Tetradecane Dicarboxylate (1h). Yield 83%. 1H NMR (DMSO-d6): δ 1.36 (m, 10H, CH2(CH2)10CH2); 1.60 (m, 4H, CH2(CH2)10CH2); 2.24 (t, J ) 7.3 Hz, 4H, COCH2(CH2)12CH2CO); 3.82 (d, J ) 5.4 Hz, 4H, CONHCH2CO × 2); 3.87 (d, J ) 5.4 Hz, 4H, CH2COO × 2); 8.17 (t × 2, J ) 5.4 Hz, 4H, NH). Anal. Calcd for C24H42O8N4 0.5H2O: C, 55.05; H, 8.28; N, 10.70. Found: C, 55.34; H, 8.34; N, 10.56. Bis(N-r-amido-sarcosylsarcosine) 1,10-Decane Dicarboxylate (3). A pale yellow oil. Yield 23%. 1H NMR (CDCl3): δ 1.22 (m, 4H, CH2(CH2)2CH2); 1.52 (m, 4H, CH2(CH2)2CH2); 2.23 (t, J ) 7.3 Hz, 4H, COCH2(CH2)8CH2CO); 2.98 (s, 12H, NCH3 × 4); 4.02-4.23 (m, 8H, CH2COO × 4). Bis(N-r-amido-L-prolyl-L-proline) 1,10-Decance Dicarboxylate (4). A colorless oil (0.24 g, 0.39 mmol). Yield 20%. 1H NMR (CDCl3): δ 1.27 (m, 12H, CH2(CH2)6CH2); 1.60 (m, 4H, CH2(CH2)6CH2); 2.09 (t, J ) 7.3 Hz, 4H, COCH2(CH2)8CH2CO); 1.95-2.37 (m, 16H, β- and γ-CH2 × 4); 3.46-3.84 (m, 8H, δ-CH2 × 4); 4.55-4.72 (m, 4H, R-CH × 4). Bis(N-r-amido-glycylsarcosylsarcosine) 1,10-Decane Dicarboxylate (5). A pale yellow oil. Yield 36%. 1H NMR (CDCl3): δ 1.25 (m, 4H, CH2(CH2)2CH2); 1.60 (m, 4H, CH2(CH2)2CH2); 2.21 (t, J ) 7.3 Hz, 4H, COCH2(CH2)8CH2CO); 2.97, 3.02, and 3.07 (s, 12H, NCH3 × 4); 4.11-4.26 (m, 12H, CH2COO × 6); 7.98 (t, J ) 5.4 Hz, 2H, NH). Bis(N-r-amido-glycyl-L-prolyl-L-proline) 1,10-Decane Dicarboxylate (6). A colorless oil. Yield 60%. 1H NMR (CDCl3): δ 1.23 (m, 12H, CH2(CH2)6CH2); 1.59 (m, 4H, CH2(CH2)6CH2); 2.09 (t, J ) 7.3 Hz, 4H, COCH2(CH2)8CH2CO); 1.95-2.21 (m, 16H, β- and γ-CH2 × 4); 3.35-3.77 (m, 8H, δ-CH2 × 4); 3.95, 3.96, 4.11, and 4.13 (m, 4H, COCH2NH); 4.55-4.72 (m, 4H, R-CH × 4); 7.96 (t, J ) 5.4 Hz, 2H, NH). Preparation of Tubes and Light Microscopy. The 10 mM aqueous solutions (10 mL) were obtained by sonicating the corresponding bolaamphiphiles and 2.1 equiv of alkaline and alkaline earth hydroxide in water at 25 °C (Branson ultrasonicator model 1210, 47 kHz, 80 W). Each solution was allowed to stand at room temperature for 2-3 weeks. Perturbation of the solutions by stirring or sampling affected the yield of the tubes. Light microscopic measurement was carried out in a similar way to those described in detail elsewhere.14 The samples were also examined by confocal laser-scanning light microscopy (Carl Zeiss LSM-410, differential interference mode) at room temperature. Atomic Force Microscopy. A drop of aqueous suspension containing microtubes was placed on a clean slide glass and dried overnight at atmospheric pressure in an electric dry desiccator. The shape, morphology, and surface details of the tubes were observed using a commercial atomic force microscope (Digital Instruments, Inc., Nanoscope IIIa) with a silicon nitride cantilever (spring constant, 0.12 N/m) using contact mode. AFM images (10 µm × 10 µm) of the microtubes were produced in the height mode without any image processing except flattening. A highresolution AFM image (5 nm × 5 nm) was shown in the height mode without any image processing except lowpass filtering.
4986 Langmuir, Vol. 14, No. 18, 1998 Then, the image was processed with Grain size analysis (height threshold ) 0.218 nm) to draw the outline of the bright portions, from which we evaluated spacings and internal angle in the molecular array. Cryo-Transmission Electron Microscopy. Thin films of sample were formed by placing a 3-µL drop of the supernatant of the aqueous solution on a holey polymer support film that had been coated with silver and carbon and mounted on the surface of a standard TEM grid. The drop was blotted off with filter paper. The assembly was then vitrified by jet-freezing in cooled liquid ethane. Vitrified specimens were examined at 120 kV in the electron spectroscopic imaging by using an analytical electron microscope (Carl Zeiss EM-912 Omega) with a cryo transfer holder (Gatan, Inc., model 626). The cryo transfer holder temperature was maintained below -165 °C. Images were recorded on an imaging plate at 20000-50000× and digitally enlarged. pH Titration. Aqueous dispersions (50 mL) were prepared by adding degassed distilled water to a weighed powder (10 mM) of 1a-h and succeeding sonication at 25 °C. The aqueous dispersions were then titrated with 1 N NaOH. The alkaline aqueous solutions of 1a-h were also prepared by adding an adequate amount of standardized solution of NaOH to the aqueous dispersions and titrating with 1 N HCl. However, the occurrence of precipitates in the course of titration gave poor reproducibility of the titration curve in the latter manner. Therefore, we employed the former, reproducible titration method with the alkaline solution. Titration was accomplished using
Kogiso et al. Mitsubishi Chemical Industries, Ltd., automatic titrator GT-05 at 23 °C. The degree of ionization (R) was calculated by the following equation in the same manner as that reported previously
R ) 1 - (CH+,total - CH+,free)/(2C) where CH+,total and CH+,free are the total and free molar amount of protons, respectively, and C is the molar concentration of bolaamphiphile
Acknowledgment. We thank Professor Martin Mueller of the Institute of Cell Biology, Eidgenoessische Technische Hochshule (ETH, Switzerland), and Mr. Erhard Zellmann of LEO Elektronenmikroskopie GmbH for their excellent help with cryo-transmission electron microscopy and Mr. Hirokazu Aizawa of Carl Zeiss, Ltd., for his excellent help with confocal laser-scanning microscopy. Supporting Information Available: FT-IR spectra of the dicarboxylic bolaamphiphiles 1a-g mentioned in the text (7 pages). Ordering information is given on any current masthead page. LA9802419
