pubs.acs.org/Langmuir © 2010 American Chemical Society
Supramolecular Structures Generated by a p-tert-Butylphenylamide Derivative of Deoxycholic Acid. From Planar Sheets to Tubular Structures through Helical Ribbons Francisco Meijide,† Alvaro Antelo,† Mercedes Alvarez Alcalde,† Aida Jover,† Luciano Galantini,‡ Nicolae Viorel Pavel,‡ and Jose Vazquez Tato*,† †
Departamento de Quı´mica Fı´sica, Facultad de Ciencias, Universidad de Santiago de Compostela, Avda. Alfonso X El Sabio s/n, 27002 Lugo, Spain, and ‡Dipartimento di Chimica, Research center SOFT-INFM-CNR, Universit a di Roma “La Sapienza”, P.le A. Moro 5, 00185 Roma, Italy Received December 2, 2009. Revised Manuscript Received February 19, 2010
The formation of supramolecular structures initiated by a p-tert-butylphenylamide derivative of deoxycholic acid (Na-t-butPhDC) is investigated. At 1.18 mM concentration of Na-t-butPhDC and 37 °C, initial flat ribbons are observed which self-transform into helical ribbons (with a mean pitch angle of 47 ( 6°) which finally originate molecular tubes with an external diameter of 241 ( 28 nm. Most of the molecular tubes show helical markings with a pitch angle value of 45 ( 4°, in full agreement with predictions of simple models based on chiral elastic properties of the membrane. A lateral association mechanism is proposed to account for the growth of the external diameter (from 225 ( 32 to 546 ( 59 nm) of tubes with time at 3.99 mM.
Introduction Low molecular lipid-based compounds and some block copolymer systems can self-assembly into tubular architectures, whose diameters lie in the range ∼10-1000 nm.1 Concerning to organic amphiphiles, molecular tubes are obtained by chiral molecular self-assembly (the importance of chirality in the formation of superstructures was recognized by Furhop et al.2), generally requiring highly ordered molecular packing and anisotropic intermolecular interactions. This is the case for fatty acid salts,3 phospholipids,4-8 diacetylenic lipids,9 glycolipids,10-13 peptidic *Corresponding author. (1) Shimizu, T.; Masuda, M.; Minamikawa, H. Chem. Rev. 2005, 105, 1401. (2) Fuhrhop, J. H.; Schnieder, P.; Rosenberg, J.; Boekema, E. J. Am. Chem. Soc. 1987, 109, 3387. (3) Douliez, J.-P.; Gaillard, C.; Navailles, L.; Nallet, F. Langmuir 2006, 22, 2942. (4) Thomas, B. N.; Lindemann, C. M.; Clark, N. A. Phys. Rev. E 1999, 59, 3040. (5) Singh, A.; Markowitz, M. A.; Tsao, L. I. Chem. Phys. Lipids 1992, 63, 191. (6) Patil, A. J.; Muthusamy, E.; Seddon, A. M.; Mann, S. Adv. Mater. 2003, 15, 1816. (7) Pakhomov, S.; Hammer, R. P.; Mishra, B. K.; Thomas, B. N. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 3040. (8) Lauf, U.; Fahr, A.; Westesent, K.; Ulrich, A. S. ChemPhysChem 2004, 5, 1246. (9) Schnur, J. M.; Ratna, B. R.; Selinger, J. V.; Singh, A.; Jyothi, G.; Easwaran, K. R. K. Science 1994, 264, 945. (10) Frusawa, H.; Fukagawa, A.; Ikeda, Y.; Araki, J.; Ito, K.; John, G.; Shimizu, T. Angew. Chem., Int. Ed. 2003, 42, 72. (11) Frankel, D. A.; O’Brien, D. F. J. Am. Chem. Soc. 1994, 116, 10057. (12) John, G.; Masuda, M.; Okada, Y.; Yase, K.; Shimizu, T. Adv. Mater. 2001, 13, 715. (13) Yui, H.; Minamikawa, H.; Danev, R.; Nagayama, K.; Kamiya, S.; Shimizu, T. Langmuir 2008, 24, 709. (14) Boettcher, C.; Schade, B.; Fuhrhop, J.-H. Langmuir 2001, 17, 873. (15) Vauthey, S.; Santoso, S.; Gong, H.; Watson, N.; Zhang, S. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5355. (16) Lu, K.; Jacob, J.; Thiyagarajan, P.; Conticello, V. P.; Lynn, D. G. J. Am. Chem. Soc. 2003, 125, 6391. (17) Neuzil, E.; Fourche, J.; Jensen, R.; Jensen, H.; Morin, G. Biochim. Biophys. Acta 1981, 641, 11. (18) Fourche, J.; Jensen, H.; Neuzil, E. Biochem. Soc. Trans. 1987, 15, 925. (19) Fourche, J.; Neuzil, E. Colloq. INSERM 1988, 166, 255. (20) Fourche, J.; Neuzil, E.; Jensen, H. Mol. Cryst. Liq. Cryst. 1988, 164, 1. (21) Terech, P.; Talmon, Y. Langmuir 2002, 18, 7240. (22) Terech, P.; De Geyer, A.; Struth, B.; Talmon, Y. Adv. Mater. 2002, 14, 495.
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amphiphiles,14-16 sterols,17-20 bile salts,21-28 and cholesterol (which evidence a prominent position).17-20,29,30 In fact, metastable helical and tubular intermediates have been observed in the pathway for cholesterol crystallization in native and model biles.31 Bile salts (natural biosurfactants highly widespread in the animal kingdom)32 play an important role in several physiological processes.33 Their amphipathic nature is due to the existence of a hydrophilic side and a hydrophobic side and, consequently, these steroidal compounds form aggregates of different size and structure.34,35 The presence in bile acids of the hydroxyl groups (commonly at positions 3, 7, and/or 12) and the carboxylate group at the side chain has made them very attractive in the design of chiral templates,36 soft materials,37 molecular baskets,38 etc., (23) Jean, B.; Oss-Ronen, L.; Terech, P.; Talmon, Y. Adv. Mater. 2005, 17, 728. (24) Terech, P.; Friol, S.; Sangeetha, N.; Talmon, Y.; Maitra, U. Rheol. Acta 2006, 45, 435. (25) Terech, P.; Sangeetha, N. M.; Bhat, S.; Allegraud, J.-J.; Buhler, E. Soft Matter 2006, 2, 517. (26) Terech, P.; Jean, B.; Ne, F. Adv. Mater. 2006, 18, 1571. (27) Soto Tellini, V. H.; Jover, A.; Meijide, F.; Vazquez Tato, J.; Galantini, L.; Pavel, N. V. Adv. Mater. 2007, 19, 1752. (28) Galantini, L.; Leggio, C.; Jover, A.; Meijide, F.; Pavel, N. V.; Soto, V. H.; Vazquez Tato, J.; Di Leonardo, R.; Ruocco, G. Soft Matter 2009, 5, 3018. (29) Jung, J. H.; Lee, S.-h.; Yoo, J. S.; Yoshida, K.; Shimizu, T.; Shinkai, S. Chem.;Eur. J. 2003, 9, 5307. (30) Jung, J. H.; Ono, Y.; Shinkai, S. Langmuir 2000, 16, 1643. (31) Chung, D. S.; Benedek, G. B.; Konikoff, F. M.; Donovan, J. M. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 11341. (32) Moschetta, A.; Xu, F.; Hagey, L. R.; van Berge-Henegouwen, G. P.; van Erpecum, K. J.; Brouwers, J. F.; Cohen, J. C.; Bierman, M.; Hobbs, H. H.; Steinbach, J. H.; Hofmann, A. F. J. Lipid Res. 2005, 46, 2221. (33) Monte, M. J.; Garcia Marin, J. J.; Antelo, A.; Vazquez Tato, J. World J. Gastroenterol. 2009, 15, 804. (34) Small, D. M. In The Bile Acids, Chemistry, Physiology, and Metabolism; Nair, P. P., Kritchevski, D., Eds.; Plenum Press: New York. 1971, Chapter 8, p 249. (35) Carey, M. C. In Sterols and bile Acids; Danielsson, H., Sj€ovall, J., Eds.; Elsevier Sci. Publ.: Amsterdam, 1985, 5. (36) Bandyopadhyaya, A. K.; Sangeetha, N. M.; Maitra, U. J. Org. Chem. 2000, 65, 8239. (37) Soto Tellini, V. H.; Jover, A.; Galantini, L.; Pavel, N. V.; Meijide, F.; Vazquez Tato, J. J. Phys. Chem. B 2006, 110, 13679. (38) Zhao, Y. Curr. Opin. Colloid Interface Sci. 2007, 12, 92.
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but their ability of forming molecular tubes in solution has not been recognized until recently.21-28 We have recently proved that modifications of the hydrophilic/ hydrophobic balance of bile salts induce strong changes on the physicochemical properties of the native bile salt, as well as on the kind of supramolecular structures which can be obtained.37 This modification can be done by enlarging the hydrophobic region of the bile acid as a result of the attachment of a hydrophobic group to the steroid nucleus. Such an enlargement also facilitates the formation of tubular structures. This is the case of sodium cholate (NaC) modified at its 3-position with the hydrophobic p-tertbutylphenyl group (compound named Na-t-butPhC).27 Nanotubes from sodium lithocholate (which is the only unmodified bile salt which can form these supramolecular architectures) are single walled (with a wall thicknes of 1.5 nm which is close to the monomolecular length of the steroid) but the molecular tubes formed by Na-t-butPhC (at least at the most concentrated samples) seems to be multiwalled tubes spaced by 13 ( 1 nm.28 Mixed bilayers forming secondary multilamellar arrangements of concentric tubes are commonly formed by sterols in aqueous solutions of various water-soluble amphiphiles (sodium oleate and fatty amines).18-20 On the other hand, we have to remind that the physicochemical properties of the natural bile salts are highly dependent on the number, location and stereochemistry of the hydroxyl groups.34,39-41 In short, these effects may be referred to simply as “hydroxyl effect”. For instance, the only structural difference between NaC and sodium deoxycholate (NaDC) is the existence or not of a hydroxyl group at C7. Its absence enlarges the hydrophobicity of the molecule, allowing for stronger hydrophobic interactions.42 This leads to several physicochemical differences in their aggregation behavior: (i) the average aggregation number for NaC does not grow with the addition of inert salts,43 as NaDC does;44 (ii) only NaDC forms gels at pH values close to neutrality;45-47 and (iii) deoxycholate salts form fibers.48,49 Previous observations for natural bile salts suggest that the “hydroxyl effect” can also have strong influences on the associative behavior of bile salts modified with hydrophobic residues. Thus, it can be expected that the absence of the hydroxyl group at C-7 in Na-t-butPhDC (Figure 1), the monohydroxy homologous of Na-t-butPhC, can have a strong influence on the mechanism of tube formation. Even different structures could emerge as bile salt aggregation is a difficult task to predict. With such a confidence in mind, we have investigated the associative behavior of this derivative of deoxycholic acid. The obtained results are presented in this paper, focusing our interest in conditions where tubular structures are formed. Particular attention has been paid to the kinetics of the process. (39) Alvarez, M.; Jover, A.; Carrazana, J.; Meijide, F.; Soto, V. H.; Vazquez Tato, J. Steroids 2007, 72, 535. (40) Jover, A.; Meijide, F.; Soto, V. H.; Vazquez Tato, J.; Rodrı´ guez Nun~ez, E. R.; Ton-Nu, H.-T.; Hofmann, A. F. Steroids 2004, 69, 379. (41) Miyata, M.; Tohnai, N.; Hisaki, I. Acc. Chem. Res. 2007, 40, 694. (42) Ramos Cabrer, P.; Alvarez-Parrilla, E.; Al-Soufi, W.; Meijide, F.; Rodrı´ guez Nun~ez, E.; Vazquez Tato, J. Supramol. Chem. 2003, 15, 33. (43) Coello, A.; Meijide, F.; Rodrı´ guez Nun~ez, E.; Vazquez Tato, J. J. Phys. Chem. 1993, 97, 10186. (44) Esposito, G.; Giglio, E.; Pavel, N. V.; Zanobi, A. J. Phys. Chem. 1987, 91, 356. (45) Blow, D. M.; Rich, A. J. Am. Chem. Soc. 1960, 82, 3566. (46) Jover, A.; Meijide, F.; Rodrı´ guez Nun~ez, E.; Vazquez Tato, J.; Mosquera, M.; Rodrı´ guez Prieto, F. Langmuir 1996, 12, 1789. (47) Jover, A.; Meijide, F.; Rodrı´ guez Nunez, E.; Vazquez Tato, J. Langmuir 2002, 18, 987. (48) D’Archivio, A. A.; Galantini, L.; Giglio, E.; Jover, A. Langmuir 1998, 14, 4776. (49) Bonincontro, A.; D’Archivio, A. A.; Galantini, L.; Giglio, E.; Punzo, F. J. Phys. Chem. B 1999, 103, 4986.
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Figure 1. Structure of the [3β,5β,12R]-3-(4-tert-butylbenzoilamine)12-hydroxycholan-24-oic acid. Sodium salt: Na-t-butPhDC.
Experimental Section The new derivative was obtained by reacting the p-tert-butylbenzoyl chloride with the 3β-amino derivative of deoxycholic acid.50 Na-t-butPhDC was obtained from stoichiometric neutralization of the corresponding acid. Other chemicals were used without further purification. Solutions of Na-t-butPhDC were prepared in D2O (99.8%, Panreac, Spain) to avoid bacterial growth. TEM images were obtained at room temperature in a JEOL JEM-1011, operated at 80 kV, equipped with a MegaView III camera. For the measurements, the samples were prepared by deposition of a drop of the thermostated mother solution onto carbon-coated copper grids. CD spectra were recorded on a JASCO model 715 and reported in molar ellipticity [Θ]. The samples were thermostated in cells of 0.1 cm path-length within 0.5 °C by a circulating water bath.
Results and Discussion Surface tension measurements of aqueous solutions of Na-tbutPhDC (see ESI) reveal that its critical aggregation concentration in aqueous solution (≈45 μM, 25 °C) is 130 times lower than the critical micelle concentration of NaDC.51 This result is in agreement with observations for the couple Na-t-butPhC/NaC.27 A 1.18 mM solution of Na-t-butPhDC was prepared and kept at constant temperature (37 °C). TEM images show that flat ribbons (Figure 2), and helical ribbons (Figure 3) are observed after 30 min and 2 h, respectively. The folding presented by many sheets (Figure 3a) indicates that these structures are precursors of the helical ribbons. At this stage, helical ribbons are predominant in the system but some of them are in an advanced state toward the formation of molecular tubes (Figure 3b, arrow). The helixes are often more loosely coiled near their ends than in their central parts, a result also observed by Georger et al.52 for polymerizable phosphatidylcholines. For that system, the authors have pointed out that the open helixes are flexible and bend easily under the influence of solvent shear, resulting in an appreciable polydispersity in the ribbon width at these stages. This could also be the case for Na-t-butPhDC since a high polydispersity is observed (average value for ribbon width at this time being 164 ( 43 nm) although the width is almost constant for a given ribbon. The average pitch angle measured is 47 ( 6o. Polydispersities in pitch and width have been reported by Lee et al.53 in the growth of helical ribbons of alanine-based amphiphiles and by Frankel and O’Brien11 for N-dodeca-5,7-diyne galactonamide assemblies. Bending is not uniform (see arrow in Figure 3a) and the longitudinal axis is not defined yet. A similar behavior has been reported for gem-oligoalanine surfactants.54 (50) Vazquez Tato, J.; Soto Tellini, V. H.; Trillo Novo, J. V.; Alvarez Alcalde, M.; Antelo Queijo, A.; Carrazana Garcı´ a, J.; Jover Ramos, A.; Meijide del Rı´ o, F. Spain, ES2296463A1, 2005. (51) Coello, A.; Meijide, F.; Rodrı´ guez Nun~ez, E.; Vazquez Tato, J. J. Pharm. Sci. 1996, 85, 9. (52) Georger, J. H.; Singh, A.; Price, R. R.; Schnur, J. M.; Yager, P.; Schoen, P. E. J. Am. Chem. Soc. 1987, 109, 6169. (53) Lee, S. J.; Kim, E.; Seo, M. L.; Do, Y.; Lee, Y.-A.; Lee, S. S.; Jung, J. H.; Kogiso, M.; Shimizu, T. Tetrahedron 2008, 64, 1301. (54) Brizard, A.; Ahmad, R. K.; Oda, R. Chem. Commun. 2007, 2275.
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Figure 2. TEM images of flat ribbons observed after 30 min. [Na-t-butPhDC] = 1.18 mM, 37 °C.
Figure 3. TEM images of helical ribbons observed after 2 h. [Na-t-butPhDC] = 1.18 mM, 37 °C. The folding presented by many sheets (arrow in Figure 3a) indicates that these structures are precursors of the helical ribbons. Although helical ribbons are predominant, some of them are in an advanced state toward the formation of molecular tubes (arrow in Figure 3b).
Figure 4. TEM images of helical ribbons observed after (a) 6 h and (b) 19 days. [Na-t-butPhDC] = 1.18 mM, 37 °C.
After 6 h, TEM images (Figure 4a) show that molecular tubes are becoming predominant. Although helical ribbons are still distinguished, their presence decreases with time. The coexistence of helical ribbons and tubular structures has been reported for different systems.52,55 After 19 days, TEM images show molecular tubes highly stacked (Figure 4b). Along this interval of time, the outer mean diameter of the molecular tubes does not vary significantly, the final average value being 241 ( 28 nm (see Supporting Information). Most of the molecular tubes show helical markings, allowing for the measurement of the pitch angle, the average value being 45 ( 4°. This fact confirms that helical ribbons are the precursors of tubes. Thus, the mechanism of the formation of tubes follows the sequence flat ribbon f helical ribbon f tube. This sequence has
been also pointed out by Song et al.56 for a bolaamphiphilic conjugated polymer. In other cases, helical ribbons and tubules structures have been proposed as metastable intermediates but the step from helical ribbons to tubes was difficult to observe because of its extremely fast growing rate.29 TEM images show that the formation of molecular tubes takes place during the period 3-6 h. These conclusions are fully compatible with measurements of the evolution of the circular dichroism spectrum of a solution of Na-t-butPhDC in very similar experimental conditions. The absorption UV spectrum of Na-tbutPhDC exhibits two main bands around 200 and 240 nm, both related to π-π* transitions involving essentially the phenyl amide group. For a 1.0 mM solution of Na-t-butPhDC, the circular dichroism spectra recorded at the earlier times of the process show
(55) Blanzat, M.; Massip, S.; Speziale, V.; Perez, E.; Rico-Lattes, I. Langmuir 2001, 17, 3512.
(56) Song, J.; Cheng, Q.; Kopta, S.; Stevens, R. C. J. Am. Chem. Soc. 2001, 123, 3205.
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Figure 5. CD spectra corresponding to the data at times 30, 1815, and 4610 min.
Figure 6. Temporal evolution of the molar ellipticities of the 208 and 244 nm bands of the circular dichroism spectra of Na-tbutPhDC (1.0 mM) at 37 °C.
two positive bands centered at 201.5 and 244 nm and other two negative at 195 and 208 nm, which are the result of the superposition of the Cotton effect for the absorption bands (Figure 5). The temporal evolution of the circular dichroism spectra show an initial fast increase of the molar ellipticities of the bands (in Figure 6 are plotted those associated with the bands of 208 and 244 nm), which are related to the development of the helical ribbons in the system observed by TEM. The increase is observed for about 500 min. From this time, some changes in the circular dichroism spectra are observed: (i) a little red-shift (≈2.5 nm in magnitude) of the positive band at 244 nm, (ii) a broadening of the positive band at 201.5 nm, and (iii) a decrease in the molar ellipticities of the bands. The two first effects are evident in Figure 5 and the third one in the second part of curves in Figure 6. These facts clearly indicate that a further reorganization of the helical arrangement is now taking place, and probably related to the relaxation of the surfactant packing accompanying the reduction of the pitch angle in the transformation helical ribbon f molecular tube observed by TEM. The formation of tubular structures from helical ribbons can be related to two simple mechanisms: (i) formation of the tube by changing of the pitch angle of the helical ribbon (with constant ribbon width); or (ii) formation of the tube by the growth in width of the helical ribbon (with constant pitch length). The first one seems to be involved in the formation of nanotubular structures by long-chain sugar-based amphiphiles57 and the second one has been claimed in the formation of tubes by crown-appended cholesterol.29 (57) Jung, J. H.; Do, Y.; Lee, Y.-A.; Shimizu, T. Chem.;Eur. J. 2005, 11, 5538.
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At the beginning of the process, in ribbons where flat and helical regions are simultaneously observed (see Supporting Information) the width is smaller in the helical region that in the flat one. The helical pitch is also larger in the extremes of the ribbon than in helical regions. Thus, the decrease in the pitch even has to compensate the decrease in width, suggesting that reduction of the pitch angle of the helical ribbon is crucial at the first steps of the formation of tubes. A similar situation has been reported by Lee et al.53 when studying the helical ribbon growth process of alanine-based amphiphiles. Different theoretical approaches for the conversion chiral membrane f ribbon f tubule have been proposed. The subject has been partially reviewed by Shimizu et al.1 The Helfrich-Prost model58 introduces the concept of an intrinsic chiral bending force which arises from the fact that long chiral molecules (bile salts are chiral compounds59 which allow the resolution of enantiomers60) pack at a nonzero twist angle with respect to their neighbors. If the molecules lie in bilayers and are tilted with respect to the local layer normal, the favored twist from neighbor to neighbor leads the whole membrane to twist in a cylinder. The optimum shape of the helical ribbon is controlled by the balance between the bending of the ribbon and the torsion of its edges. The model predicts that the elastic energy is at a minimum at a pitch angle of 45° for an elastically isotropic ribbon. This model has been extended by other authors (see ref 1 for a review) in particular to account for high, low and intermediate pitch angles.31,58,61,62 It is noteworthy that the average value for the pitch angle measured from the helical markings (at the end of the process) in Na-tbutPhDC molecular tubes (see above) is in full agreement with previous prediction. This agreement suggests that ribbons are elastically isotropic. When the Na-t-butPhDC concentration is increased to 3.99 mM, at the same temperature, TEM images collected after 24 h (Figure 7a) reveal the presence of molecular tubes without helical markings. Their edges are best defined with aging. A first conclusion is that the tube formation process takes place faster when the self-assembly steroid concentration is increased. Examples of how concentration can affect the mechanism of the self-assembly of an amphiphile leading to molecular tubes are rarely found in the literature. To our knowledge only two papers can be mentioned.28,63 In a recent communication,28 we have reported that a sharp increase in the steroid concentration of Na-t-butPhC leads to multilamellar tubes. (58) Helfrich, W.; Prost, J. Phys. Rev. A 1988, 38, 3065. (59) Miyata, M.; Tohnai, N.; Hisaki, I. Molecules 2007, 12, 1973. (60) Bortolini, O.; Fantin, G.; Fogagnolo, M. Chirality 2005, 17, 121. (61) Komura, S.; Zhong-can, O.-Y. Phys. Rev. Lett. 1998, 81, 473. (62) Zastavker, Y. V.; Asherie, N.; Lomakin, A.; Pande, J.; Donovan, J. M.; Schnur, J. M.; Benedek, G. B. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 7883. (63) Spector, M. S.; Easwaran, K. R.; Jyothi, G.; Selinger, J. V.; Singh, A.; Schnur, J. M. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 12943.
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Figure 7. TEM images of tubes observed after 1 (a), 8 (b), 14 (c), and 17 (d) days. [Na-t-butPhDC] = 3.99 mM, 37 °C.
Figure 8. Histograms showing the distribution of the diameter of the tubes analyzed at 1 (a), 8 (b), and 14 (c) days. [Na-t-butPhDC] = 3.99 mM, 37 °C.
Figure 9. Lateral association observed after 2 days. [Na-t-butPhDC] = 3.99 mM, 37 °C. Arrows indicate two examples where the fusion is taking place.
TEM images were also collected after 8 (Figure 7b), 14 (Figure 7c), and 17 days (Figure 5d). The images show that the diameter of the tubular structures increases with time (Figure 8). At the beginning and at the end of the process (≈14 days) monomodal distributions are observed (the average diameter values being 225 ( 32 and 546 ( 59 nm, respectively), and a bimodal one at intermediate stages. It is noteworthy that the average diameter values of the two populations in the bimodal distribution agree with the values of the monomodal distributions, probing the remarkable diameter growth with time. 7772 DOI: 10.1021/la904548k
The mean diameter of the tubes initially formed is compatible with that obtained at lower Na-t-butPhDC concentration (see above), while the tubes with larger diameter are very similar to those reported for Na-t-butPhC.27 Although helical markings are not observed, the agreement in the diameter size suggests that the formation mechanism is the same at both steroid concentrations. Several examples can be found in the literature where clear tubes are accompanied by a much less proportion of tubes with helical striations.11,14,21,63 As we have shown above, these helical markings are reminiscent from the helical ribbons precursors of tubes Langmuir 2010, 26(11), 7768–7773
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and suggest a chiral order in the organization of the tubular structures. So it is evident that the initial small tubes self-transform into larger ones, a process which is concluded in 2 weeks. Diameters have grown by an average factor of 2.4. Two mechanisms may be invoked to explain this transformation. First, large tubes could be formed by lateral association of the small ones, as proposed for the Na-t-butPhC system.27 Second, a self-reorganization from multilamellar tubes to single-wall tubes could occur gradually with time. Simple geometrical analysis indicates that the number of small tubes or the number of lamellae involved in the transformation would be equal to the measured average factor of growth. The first mechanism seems to be more plausible since (i) small tubes are observed at both concentrations studied here, (ii) the larger tubes are observed at high steroid concentrations, while the smaller ones disappear with time, and (iii) the probability of parallel rearrangements (see Figure 7) increases with the rising of the concentration. Following this hypothesis, we have carefully checked the TEM images in order to observe any evidence in favor of this mechanism. Since tubes are fully superimposed, this observation is not easy. Even so, some images, taken after 2 days, suggest a lateral association (see Figure 9) supporting the first mechanism.
Conclusions As we have shown, Na-t-butPhDC has a remarkable ability of forming tubular structures, but the mechanism leading to the final tubes clearly differs from that of Na-t-butPhC. This fact can be related to the key role that location, orientation and number of hydroxy groups34,39-41 play in crystal patterns of cholanic acids and salts. On the other hand, SAXS studies on Na-t-butPhC (64) Yoswathananont, N.; Sada, K.; Nakano, K.; Aburaya, K.; Shigesato, M.; Hishikawa, Y.; Tani, K.; Tohnai, N.; Miyata, M. Eur. J. Org. Chem. 2005, 5330. (65) Aburaya, K.; Hisaki, I.; Tohnai, N.; Miyata, M. Chem. Commun. 2007, 4257. (66) Miragaya, J.; Jover, A.; Fraga, F.; Meijide, F.; Vazquez Tato, J. Crystal Growth Des. 2010, 10.1021/cg9009064.
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suggest that multiwalled tubes with well spaced layers are formed gradually with time.28 However, the structure of each single wall for the two bile salt derivatives is still unknown, although a wellorganized structure, resembling the layer structures frequently found for the crystal structure of bile salts,64-66 would not be unexpected. So, the wall structure can probably be inferred from the knowledge of the crystal structure of the compound (as it was done for an adamantyl derivative of cholic acid).37 Unfortunately these compounds do not crystallized easily in water and crystals with quality enough to be properly resolved have not been yet obtained. Present and previous results27 also suggest that derivatives of the p-tert-butylphenyl residue with other bile salts (having hydroxyl groups with different location or orientation, such as ursodeoxycholic and other epimers) must be undertaken in order to understand the role of the hydrogen bond and hydrophobic interactions in directing the formation of tubes. In this context, we have to remind that the number and location of the hydrogenbonding sites are crucial features to the tessellation, the stability and selectivity of many supramolecular assemblies.67 This is a general fact for all bile salt aggregates. Acknowledgment. We thank Alba Roman from the Electronic Microscopy service of USC (Campus of Lugo). The authors from USC thank the Ministerio de Ciencia y Tecnologı´ a, Spain, (Project MAT2006-61721) for financial support. The authors from Sapienza Universita di Roma thank the MIUR financial supports (PRIN Project 2006 039789-001). Supporting Information Available: A figure showing surface tension measurements, a table of outer average diameter of molecular tubes at different times at high concentration and a figure showing width and helical pitch of helical ribbons at different times. This material is available free of charge via the Internet at http://pubs.acs.org. (67) Lehn, J.-M. Supramol. Chem.; VCH: Weinheim, Germany, 1995.
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