Langmuir 2006, 22, 7793-7797
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Silk-Inspired Low-Molecular-Weight Organogelator Beatriu Escuder* and Juan F. Miravet* Departament de Quı´mica Inorga` nica i Orga` nica, UniVersitat Jaume I, 12071 Castello´ , Spain ReceiVed February 21, 2006. In Final Form: June 30, 2006 A minimal sequence from natural silks, the tetrapeptide GAGA, has been used as inspiration for the design of a new organogelator. Gels were obtained in several organic solvents, and their microscopic aspects were studied by transmission and cryo-scanning electron microscopies (TEM and cryo-SEM). FT-IR spectroscopy, circular dichroism, and wide-angle X-ray diffraction were used to study the self-assembly features of this molecule, and evidence of an antiparallel β-sheet organization was obtained. Remarkably, the precise secondary structure uniqueness found in Nature was successfully transferred into a small synthetic analogue that self-assembles driven only by noncovalent interactions.
Nature uses noncovalent interactions in many ways to obtain functional and structural materials. Thus, H-bonding, van der Waals, and hydrophobic interactions, among others, play a fundamental role in the hierarchical assembly of biomolecules. For instance, the storage of genetic information or the function of proteins would not be possible without the occurrence of such weak intermolecular forces. Scientists have been always inspired by Nature, whose efficiency and precise design are a chimerical goal at the forefront of science. Biomimetic systems have been designed not only to understand the natural world but also to find technological and biomedical applications.1 In this sense, one of the most interesting examples is protein structure. The particular secondary structure domains found in proteins have served as inspirations in the search for artificial materials with improved mechanical properties.2 The case of silks is a relevant example for the relationship between molecular design and material properties. In these structural proteins, β-sheet domains coexist with random-coil regions, modulating their mechanical features. For instance, the regular crystalline domains give strength, whereas the latter confer flexibility.3 It has been widely reported that many peptides and peptide derivatives form fibrous aggregates with different secondary structural motifs.4 The precisely oriented H-bond arrays found in R-helices and β-sheets and the hydrophobic interactions involving apolar residues lead to the formation of elongated aggregates that coil into fibers. These fibrilar networks are able to entrap solvent, leading to the formation of gels (hydrogels and organogels).5,6 Moreover, some examples of low-molecularweight peptidic gelators have also been reported, mainly including * Address correspondence to either author. E-mail:
[email protected] (B.E.),
[email protected] (J.F.M.). (1) (a) Breslow, R.; Dong, S. D. Chem. ReV. 1998, 98, 1997-2011. (b) Kool, E. T.; Morales, J. C.; Guckian, K. M. Angew. Chem., Int. Ed. 2000, 39, 990-1009. (c) Thordarson, P.; Bijsterveld, E. J. A.; Rowan, A. E.; Nolte, R. J. M. Nature 2003, 424, 915-918. (2) Self-Assembling Peptide Systems in Biology, Medicine and Engineering; Aggeli, A., Boden, N., Zhang, S., Eds.; Kluwer Academic Publishers: Dordrecht, The Netherlands, 2001. (3) van Hest, J. C. M.; Tirrell, D. A. Chem. Commun. 2001, 1897-1904 and references therein. (4) (a) Lashuel, H. A.; LaBrenz, S. R.; Woo, L.; Serpell, L. C.; Kelly, J. W. J. Am. Chem. Soc. 2000, 122, 5262-5277. (b) Aggeli, A.; Nyrkova, I. A.; Bell, M.; Harding, R.; Carrick, L.; McLeish, T. C. B.; Semenov, A. N.; Boden, N. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 11857-11862. (c) Lo´pez de la Paz, M.; Goldie, K.; Zurdo, J.; Lacroix, E.; Dobson, C. M.; Hoenger, A.; Serrano, L. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 16052-16057. (d) Behanna, H. A.; Donners, J. J. J. M.; Gordon, A. C.; Stupp, S. I. J. Am. Chem. Soc. 2005, 127, 1193-1200. (e) Deechongkit, S.; Powers, E. T.; You, S.-L.; Kelly, J. W. J. Am. Chem. Soc. 2005, 127, 8562-8570.
dipeptides and amphiphiles containing one amino acid, where H-bonding involving the amide groups and hydrophobic interactions are the driving forces for the assembly process in organic and aqueous solutions, respectively.7,8 In this work, considering all of the structural information cited above, our goal was to obtain low-molecular-weight gelators (LMWGs) by exploiting the one-dimensional H-bonding motif found in Bombix mori silk β-sheet fragments, namely, segments with the amino acid sequence Gly-Ala-Gly-Ala. Terminal groups were flanked with long tails to regulate their solubility and introduce additional van der Waals interactions. This simple sequence has been used for the design of polymeric silk-mimetic materials.9 However, to our knowledge, this is the first time that it has been employed for the construction of LMWGs. Synthesis of compound 4 was accomplished by conventional solution peptide synthesis as presented in Scheme 1 (also see Supporting Information). The self-assembly features of compound 4 were studied in different solvents, revealing a poor solubility in most of them. In fact, in all of the cases studied (see Table 1), either a gel or a viscous precipitate was formed (see Supporting Information for gelation details). Thus, almost transparent gels were obtained in tetrahydrofuran, chloroform, toluene, and cyclohexane, whereas incomplete gelation occurred in acetone and dioxane and some of the material remained as a precipitate. In acetonitrile, an opaque (5) (a) Aggeli, A.; Bell, M.; Boden, N.; Keen, J. N.; Knowles, P. F.; McLeish, T. C. B.; Pitkeathly, M.; Radford, S. E. Nature 1997, 386, 259-262. (b) Petka, W. A.; Harden, J. L.; McGrath, K. P.; Wirtz, D.; Tirrell, D. A. Science 1998, 281, 389-392. (c) Schneider, J. P.; Pochan, D. J.; Ozbas, B.; Rajagopal, K.; Pakstis, L.; Kretsinger, J. J. Am. Chem. Soc. 2002, 124, 15030-15037. (d) Pochan, D. J.; Schneider, J. P.; Kretsinger, J.; Ozbas, B.; Rajagopal, K.; Haines, L. J. Am. Chem. Soc. 2003, 125, 11802-11803. (e) Aggeli, A.; Bell, M.; Boden, N.; Carrick, L. M.; Strong, A. E. Angew. Chem., Int. Ed. 2003, 42, 5603-5606. (f) Yokoi, H.; Kinoshita, T.; Zhang, S. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 8414-8419. (6) Aggeli, A.; Bell, M.; Boden, N.; Keen, J. N.; McLeish, T. C. B.; Nyrkova, I.; Radford, S. E.; Semenov, A. J. Mater. Chem. 1997, 7, 1135-1145. (7) Molecular Gels. Materials with Self-Assembled Fibrillar Networks; Weiss, R. G., Terech, P., Eds.; Springer: Dordrecht, The Netherlands, 2006. (8) (a) Terech, P.; Weiss, R. G. Chem. ReV. 1997, 97, 3133-3159. (b) Estroff, L. A.; Hamilton, A. D. Chem. ReV. 2004, 104, 1201-1217. (9) (a) Panitch, A.; Matsuki, K.; Cantor, E. J.; Cooper, S. J.; Atkins, E. D. T.; Fournier, M. J.; Mason, T. L.; Tirrell, D. A. Macromolecules 1997, 30, 42-49. (b) Winningham, M. J.; Sogah, D. Y. Macromolecules 1997, 30, 862-876. (c) Rathore, O.; Sogah, D. Y. Macromolecules 2001, 34, 1477-1486. (d) Rathore, O.; Sogah, D. Y. J. Am. Chem. Soc. 2001, 123, 5231-5239. (e) Klok, H.-A.; Ro¨sler, A.; Go¨tz, G.; Mena-Osteritz, E.; Ba¨uerle, P. Org. Biomol. Chem. 2004, 2, 3541-3544. (f) Smeenk, J. M.; Otten, M. B. J.; Thies, J.; Tirrell, D. A.; Stunnenberg, H. G.; van Hest, J. C. M. Angew. Chem., Int. Ed. 2005, 44, 19681971.
10.1021/la060499w CCC: $33.50 © 2006 American Chemical Society Published on Web 08/01/2006
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Escuder and MiraVet Scheme 1
Table 1. Gelation Studies for Compound 4 (5 mg/mL)
a
solvent
resulta
methanol i PrOH acetone CH3CN THF DME CHCl3 CH2Cl2 dioxane toluene cyclohexane DMF
S Pc WG + Pc G opaque (10 mg/mL) G Pc G P WG + P G G P
Key: S, soluble; P, precipitate; Pc, colloid; G, gel; WG, weak gel.
gel was found at higher concentration, and in methanol, the compound remained soluble after being cooled. The microscopic aspects of the gels formed by compound 4 were investigated by TEM, in all cases revealing the presence of an entangled network of fibers (Figure 1). Fibrils of less than 20 nm could be observed as the smaller nanoobjects that further assembled into bundles with a width of ca. 100 nm and a length of several micrometers. Cryo-scanning electron microscopy (cryo-SEM) is a useful technique for obtaining in situ images of the three-dimensional structure of gels and also determining whether drying of the solvent in TEM sample preparation had some effect on the observed structures. In this technique, the gel sample is frozen and cut; then, after a few minutes of solvent sublimation and sputtering, an image of the gel network is revealed (Figure 2; also see Supporting Information for details). As can be seen, the general aspect is that of a microporous material with fibrillar domains surrounding micrometer-sized pools filled with solvent. In Figure 2A, the observed section plane shows orthogonally oriented domains of fibers, revealing either transversal or longitudinal cross sections of the interfibrilar pores. At first, a preferred general orientation appears to occur within the different domains. A closer examination reveals the presence of tiny fibrils less than 100 nm in diameter assembled into the spongelike
fibrillar regions (Figure 2C). Comparing the results found with the two microscopic techniques, similar dimensionalities could be observed for both the swollen and the collapsed gel networks. FT-IR spectroscopy has been shown to be an effective analytical tool for establishing the protein secondary structure, and in the case of biology-inspired systems, it can easily be used for comparisons with the fingerprints of naturally occurring analogues. In this sense, the amide I carbonyl stretching vibration can be used not only to determine the presence of β-sheets and other secondary structures but also to clearly distinguish between antiparallel and parallel β-sheets. In general, the amide I band appears at 1656 cm-1 for random coil conformations; 1650 cm-1 for R-helices; and ca. 1630 cm-1 for β-sheets, with the difference between the two possible orientations of the latter reflected in an accompanying weak band at about 1690 cm-1 for antiparallel β-sheets and 1645 cm-1 for parallel β-sheets.8b To further investigate the secondary structure present in the gels formed by silk-mimetic compound 4, FT-IR spectra of the xerogels were recorded (see Supporting Information). It has been reported that the sequence GAGA shows a high tendency to form antiparallel β-sheets in the solid state.8 It could be observed that, in all cases in this work, the amide I vibrations appeared as a broad intense band centered at ca. 1625 cm-1, supporting the preference for β-sheet conformations. Furthermore, all of the xerogels clearly showed a weak accompanying band at 1695 cm-1 ascribable to an antiparallel orientation of the peptide chains. The breadth of the amide I band could be due to the presence of minor fractions with a random coil or parallel orientation. Thus, the FT-IR spectra suggests antiparallel β-sheet conformations as the main secondary structural motif in these gels, with a slight influence of the solvent in the degree of order achieved.10 Circular dichroism spectroscopy has been widely used to investigate the conformational preferences of proteins and peptides, and the study of model polypeptides has allowed the establishment of characteristic spectra for the different secondary (10) Gel-phase FT-IR spectra showed that there were no significant changes in the absorption bands studied with respect to the xerogels, suggesting that drying of the solvent did not have any significant effect on the antiparallel molecular arrangement (see Supporting Information).
Silk-Inspired Low-Molecular-Weight Organogelator
Figure 1. Transmission electron micrographs of gels formed by compound 4 in (A) chloroform, (B) tetrahydrofuran, and (C) toluene. Pt-shadowing.
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Figure 2. Cryo-electron scanning microscopy (cryoSEM) images of a gel formed by compound 4 in toluene.
structure arrangements.11 In the present case, CD was used to support the evidence for β-sheet formation in the organogels. Unfortunately, amide chromophores absorb in the UV region, below the cutoff wavelength of some of the solvents studied. Moreover, in other cases, scattering problems appeared at the critical gel concentration (cgc) because of the formation of a turbid gel. CD spectra of cyclohexane solutions could be recorded (not shown) at a concentration of ca. 0.4 mM, below the cgc, showing a negative band centered near 220 nm that can be assigned to the peptide n-π* transition characteristic of a β-sheet. However, a positive band centered at 195 nm, also typical of a β-sheet, could not be clearly observed. At this point, assuming that the drying process did not have a significant influence on
Figure 3. CD spectra of xerogels of compound 4 (KBr disks).
(11) Sreerama, N.; Woody, R. Circular Dichroism of Peptides and Proteins. In Circular Dichroism: Principles and Applications; Berova, N., Nakanishi, K., Woody, R. W., Eds.; Wiley: New York, 2000; pp 337-382.
the resulting aggregates, we considered the possibility of performing solid-state CD of the xerogels in KBr disks. The application of this particular technique has been limited to some
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Escuder and MiraVet
Figure 4. X-ray powder diffraction patterns of xerogels of compound 4 (left, low-angle region; right, wide-angle region).
Figure 5. Energy-minimized models for the self-assembly of compound 4 in the gels (MACROMODEL 7.0, AMBER*, CHCl3).15
metal complexes and a few organic crystalline compounds.12 In the field of peptide science, it has been used to determine the helical screw orientation in derivatized peptides.13 In our case, we believed that we could obtain direct information on the aggregation in the xerogels. For that purpose, the organogels were dried under a vacuum, mixed with dry KBr in an agate mortar (ca. 0.5-1 wt %), and pressed at 10 ton in a standard press for 10 min. Transparent disks of less than 0.5-mm width were obtained, and their CD spectra were recorded in at least six different positions on the disk and averaged. Figure 3 shows the (12) Minguet, M.; Amabilino, D. B.; Wurst, K.; Veciana, J. J. Chem. Soc., Perkin Trans. 2 2001, 670-676. (13) Formaggio, F.; Crisma, M.; Toniolo, C.; Kamphuis, J. Biopolymers 1996, 38, 301-304.
averaged spectra for cyclohexane, tetrahydrofuran, chloroform, and toluene xerogels. In all cases, bands typical for a β-sheet were observed, centered at ca. 220 nm (negative lobe) and 195 nm (positive lobe). The distinction between parallel and antiparallel β-sheets is more difficult to establish, and although some authors report differences in the zero-crossover wavelengths, the difference seems to be only very slight and could be very sensitive to solvent or small structural changes. Furthermore, in our case, spectra could not be recorded below 190 nm as a result of technical limitations. X-ray powder diffraction patterns of xerogels of compound 4 in chloroform, tetrahydrofuran, and toluene were recorded, revealing the crystalline nature of these materials (Figure 4). In
Silk-Inspired Low-Molecular-Weight Organogelator
all cases, low-angle reflections at ca. 46 Å were observed, assignable to the interlamellar spacing proposed in Figure 5. Signals in the wide-angle region at 7.1 and 4.4 Å can be assigned to the packing of an apolar antiparallel β-sheet.14 Small differences at long distance were observed for the different solvents, suggesting tighter packing in chloroform than in the other solvents. In fact, the broad backgrounds observed for toluene and tetrahydrofuran xerogels could be due to a higher mobility of the alkyl tails. An energy-minimized model for the molecular self-assembly using a solvent simulation of CHCl3 was constructed as shown in Figure 5. In the model, one can see a one-dimensional array of H-bonds in which each molecule is involved in 10 H-bonds, thus forming an antiparallel β-sheet, in agreement with the results obtained by FT-IR spectroscopy. Moreover, the calculated antiparallel periodicity in that direction corresponds to the observed diffraction peak at 7.1 Å. In this model of aggregation, long alkyl tails would reinforce one-dimensional assembly by van der Waals interactions. a hierarchical process would follow, with β-sheets assembling into apolar antiparallel layers, as described for silks and other silk mimetics, and finally, into flat nanoribbons that merge into thicker fibers capable of entrapping solvent in their voids. The high preference shown by this system for antiparallel assembly is remarkable. In natural examples as well as in reported (14) Atkins, E. D. T. Ribbonlike Lamellar Structures from Chain-Folded Polypeptides. In Self-Assembling Peptide Systems in Biology, Medicine and Engineering; Aggeli, A., Boden, N., Zhang, S., Eds.; Kluwer Academic Publishers: Dordrecht, The Netherlands, 2001; pp 19-33. (15) Mohamadi, F.; Richards, N. G. J.; Hendrickson, T.; Still, W. C. J. Comput. Chem. 1990, 11, 440-467.
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polymeric analogues, the presence of a turn in the sequence plays an important role in the control of the β-sheet orientation. In our case, the intramolecular folding effect is not present, but the enthalpy gained in the self-assembly process seems to be sufficient to overcome the unfavorable entropic factor. In conclusion, we have used a well-established natural structural motif for the design of new LMWGs. We have demonstrated that gels can be obtained in organic solvents of different polarity through the formation of β-sheetlike aggregates and that a clear preference for their antiparallel organization, as in natural counterparts, is observed in most cases. Currently, work is in progress to introduce different functional groups into the side chains in order to obtain materials with additional properties (responsive and catalytic materials), taking advantage of the precise packing of functionalities that can be achieved through the antiparallel molecular organization. Acknowledgment. The authors thank Prof. R. J. M. Nolte and Dr. M. C. Feiters at Radboud University Nijmegen (The Netherlands) for providing access to electron microscopy facilities and G. -J. Janssen for technical assistance. The technical assistance of the SCIC (Universitat Jaume I) is also acknowledged. Funding was provided by Generalitat Valenciana (Project GV05/ 089). Supporting Information Available: Synthetic procedures, characterization of new compounds, additional FT-IR spectra, and technical details. This material is available free of charge via the Internet at http://pubs.acs.org. LA060499W
