COMMUNICATION DOI: 10.1002/chem.201303015
Photocontrolled Self-Assembly of a Bis-AzobenzeneContaining a-Amino Acid Miriam Mba,[a] Daniela Mazzier,[a] Simone Silvestrini,[a] Claudio Toniolo,[a, b] Paola Fats,[c] Ana I. Jimnez,*[c] Carlos Cativiela,[c] and Alessandro Moretto*[a, b]
Self-assembly has emerged as a powerful process for the production of well-ordered supramolecular structures at the nanometer scale. Among them, spherical (micellar and vesicle-like) architectures are attracting much attention, due to their promising applications in biomedicine and nanotechnology.[1] Peptides offer important advantages as building blocks for the construction of self-assembled nanostructures,[2] because of their biocompatibility and structural/ functional diversity. In the literature, there are many examples of supramolecular vesicles and micelles formed by block copolymers containing at least one high-molecularweight polymeric peptide domain[1–3] as well as by small- or medium-size peptides conjugated with molecules of nonpeptidic nature.[2, 4] In comparison, spherical nanostructures based exclusively on low-molecular-weight peptides are much less common.[2, 5, 6] Nanotubes and nanofibers predominate among the self-assemblies formed by the latter compounds.[2] The chemical diversity of peptides may be further expanded with the use of nonproteinogenic a-amino acid residues. We have already described[7] a symmetrically a, adisubstituted glycine, bis[p-(phenylazo)benzyl]glycine (pazoDbg), that contains two side-chain azobenzene moieties and undergoes reversible cis–trans isomerization upon exposure to light of the appropriate wavelength. In the present work, we explored the self-assembly properties of derivatives and short peptides based on this photoswitchable aamino acid. A surrogate of the N-protected dipeptide Fmoc-Phe-PheOH,[8] namely Fmoc-Phe-pazoDbg-OH (Fmoc: 9-fluorenylmethyloxycarbonyl) (1, Figure 1 A), was selected for this study. Intermolecular p–p stacking interactions involving ar-
[a] Dr. M. Mba, D. Mazzier, Dr. S. Silvestrini, Prof. C. Toniolo, Dr. A. Moretto Department of Chemistry, University of Padova via Marzolo 1, 35131 Padova (Italy) E-mail: [email protected] [b] Prof. C. Toniolo, Dr. A. Moretto Institute of Biomolecular Chemistry, Padova Unit, CNR via Marzolo 1, 35131 Padova (Italy) [c] Dr. P. Fats, Dr. A. I. Jimnez, Prof. C. Cativiela Instituto de Sntesis Qumica y Catlisis Homognea (ISQCH) CSIC-Universidad de Zaragoza, 50009 Zaragoza (Spain) E-mail: [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201303015.
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omatic side chains are a potent driving force for peptide self-assembly.[2, 6] Indeed, Phe-Phe has been recognized as the minimal self-assembling peptide sequence.[2, 9] The Fmoc group provides additional aromatic interactions that further contribute to the formation of supramolecular nanostructures, usually fibrillar in shape.[10, 11] Fmoc-Phe-pazoDbg-OH (1) was synthesized by solutionphase methods (see the Supporting Information). The reversible isomerization between the all trans (t) and all cis (c) forms [(t)1$(c)1] upon irradiation with UV (350 nm) or visible (450 nm) light was studied in MeOH solution by HPLC, CD, and UV/Vis absorption experiments (Figure 1 B; see also Figure S1 in the Supporting Information). The (t)1!(c)1 conversion was complete within 3 min. The HPLC profile obtained after 1-minute UV irradiation of (t)1 (Figure 1 B, left) shows the presence of intermediate species in which only one azobenzene group has isomerized (note that pazoDbg becomes chiral in this state[7]). The UV/Vis absorption and CD spectra of (t)1 and (c)1 exhibit significant differences in the region above 220 nm (Figure 1 B, center and right) that we assigned primarily to different relative dispositions of the aromatic ring in the chiral Phe residue with respect to those in the achiral pazoDbg for the (c)1 and (t)1 forms, which allow for a more effective chirality transfer in (t)1. However, it should be noted that the Fmoc chromophore is also known to contribute to the electronic absorption and CD above 250 nm.[12] The self-assembly properties of 1 were investigated in an aqueous environment. The lyophilized dipeptide was dissolved in DMSO at a concentration of 50 mg mL1. A 20 mL aliquot of this solution was dropped into 1980 mL of water and vortexed for a few seconds.[9] The resulting suspension was submitted to size-exclusion chromatography by gel filtration (Sephadex G75) and eluted with water. The yellowish fraction collected showed increased CD intensity when compared to (t)1 in solution (see Figure S2 A in the Supporting Information), which is a first indication of the selforganization of the system. Dynamic light scattering (DLS; see Figure S3 in the Supporting Information), transmission electron microscopy (TEM), environmental and field-emission scanning electron microscopy (E-SEM, FE-SEM), and atomic force microscopy (AFM) revealed that (t)1 forms spherical vesicle-like nanostructures with diameters of 50– 200 nm (Figure 2).
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Figure 1. A) Chemical structures of the pazoDbg-containing building block 1; the all trans (t), cis/trans, trans/ cis, and all cis (c) forms are shown. B) Left: HPLC profile after 1 min irradiation of (t)1 at 350 nm. Center: UV/Vis spectra of (t)1 and (c)1 (0.1 mm solution in MeOH). Right: CD spectra of (t)1 and (c)1 (1 mm solution in MeOH).
Figure 2. Left: TEM (A, unstained) and SEM (B, field emission; C, environmental) images of the nanostructures formed by (t)1 in water. Right (top): AFM image of the (t)1 nanostructures; the brightest spots correspond to the highest parts of the surface. Bottom: Height profiles of selected nanospheres.
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The close similarity of the microscopy images obtained after the sample was left standing at room temperature for several weeks indicates a high stability for the system. Irradiation at 350 nm for 5 min to generate (c)1 resulted in deformation of the nanospheres to yield elongated shapes (see Figure S4 in the Supporting Information). This morphological change proved to be reversible as the spherical assemblies were recovered after illumination with visible light [(c)1!(t)1]. The reversibility of the isomerization process was also confirmed by UV/Vis absorption spectroscopy along two cycles (see Figure S2 B in the Supporting Information). Longer UV-irradiation times resulted in complete disruption of the vesicles (see Figure S4 in the Supporting Information). These latter results suggest that the (t)1!(c)1 isomerization is somewhat hindered in the assembled structure (note that a 3 minute exposure to UV light allowed for complete isomerization in solution; Figure 1 B). Accordingly, the trans-to-cis isomerization of only a fraction of azobenzene moieties induces a slight rearrangement of the assembly, but the intermolecular attractive forces are still capable of essentially preserving the three-dimensional self-assembled structure. However, when the ratio of azobenzene moieties in the nonplanar cis form reaches a certain level, the intermolecular packing mode is so strongly perturbed (due to shape changes and less effective p–p stacking) that the ordered supramolecular architecture is not maintained and the spherical assemblies are transformed into amorphous aggregates. A potential application of this photosensitive process to induce the controlled release of substances entrapped in the
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nanoassemblies was evaluated. A 20 mL aliquot of the above mentioned solution of (t)1 in DMSO was mixed with 1980 mL of a 10 mg mL1 aqueous solution of gold nanoparticles (GNps, 2 nm diameter) coated with a helical undecapeptide.[13] After a short vortex agitation, the suspension obtained was submitted to gel filtration and thus separated from the nonencapsulated peptide-coated GNps. Microscopy studies revealed the presence of nanostructures similar in morphology to those formed by (t)1 in the absence of GNps. UV irradiation of the gel-filtered sample (10 min) resulted in partial disruption of the vesicles with concomitant release of the encapsulated peptide-coated GNps, as detected by TEM (Figure 3).
Figure 3. TEM images (unstained) showing: A) nanospheres formed by (t)1 and peptide-coated GNps, scale bar = 200 nm; B) the nanospheres disassembly induced by UV irradiation, scale bar = 500 nm; C) details of released GNps (black dots), scale bar = 10 nm.
To the best of our knowledge, this is the first example of a supramolecular vesicle-like system formed exclusively by peptides that exhibits photoresponsive properties.[2, 14] Indeed, the reported number of spherical nanoassemblies of any composition that are sensitive to light is rather limited in comparison to other external stimuli.[1–3, 15] Among peptide-based nanospheres, several structures have been shown to undergo morphological transitions induced by changes in pH[2, 4c,e, 14] or concentration[2, 5c, 6b,c, 14] or by the presence of metal ions.[2, 5a,b,e, 14] Quite recently, the first examples of light-sensitive block-copolymer micelles with polypeptide domains have been described.[16] However, there is no previous report on any photoresponsive vesicular or micellar nanostructure formed entirely by peptides or even by hybrid
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COMMUNICATION compounds containing a nonpolymeric peptide segment.[17] Recently, novel, stimulus-responsive supramolecular structures in the form of fibers, gels, and spheres, derived from an azobenzene-containing benzenetricarboxamide derivative, were described by Kim and co-workers.[18] Therefore, we next decided to conjugate H-(pazoDbg)-OEt to 1,3,5-trichlorotriazine with the aim at exploring the supramolecular stimuli response of a novel C3-symmetrical tris-azobenezene systems, namely compound 2 (Figure 4 A). H-(pazoDbg)OEt, 1,3,5-trichlorotriazine, and triethylamine were mixed in a DMF solution for 48 h, affording 2 in 69 % yield. All six azobenzene units were able to switch reversibly in a MeOH solution from the (t)$(c)states (Figure 4 A) under appropriate irradiation. The intermediates were detected by HPLC. Spherical microstructures were generated (in form of milklike suspension) from a 15 mm solution of 2 in DMF/ THF (1:7) dialyzed (membrane cutoff, 1 kDa, 48 h) against ultrapure water (Figure 4 B, left). Under UV irradiation (20 min), a morphological transition from the suspension to gel was observed. We recorded TEM images after different irradiation times. The analysis revealed a conversion from spheres to elongated spheres (5 min), and eventually to a mixture of spherical/elongated fibers (20 min; Figure 4 B, center and right, respectively). Irradiation of the gel with visible light resulted in gel disruption, followed by precipitation of the obtained amorphous aggregates, which do not undergo further light conversion. Vesicle and hydrogel microstructure formation were also confirmed by AFM analysis (Figure S5, in the Supporting Information) Finally, we expanded the use of our pazoDbg amino acid to the synthesis of the “octopus” system 3 (Figure 5 A) to generate photoresponsive C4-symmetric, dendrimeric-like, multiazobenzene systems. Tfa-(pazoDbg)-OH (Tfa, trifluoroacetyl), reacted with pentaerythritol in presence of N,N’-diisopropylcarbodiimide and N,N-dimethylaminopyridine afforded 3 in a remarkable 90 % yield. The resulting system is characterized by as many as eight azobenzene moieties and can be viewed as a central core surrounded by a shell of azobenzene groups at the periphery. Up to eleven (out of the possible fifteen, Figure 5 B) discrete states produced by trans-to-cis isomerizations of the individual azobenzene units were observed by reverse-phase HPLC depending on the time of exposure of 3 (in a MeOH solution) to the UV light (Figure 5 C). This process is fully reversible (cis-to-trans) under visible light irradiation for several cycles. Moreover, this system shows a high propensity to self-assemble in a variety of organic (DMF, DMSO, THF, MeOH, CH3CN)/aqueous solutions (even at low concentrations, below 1 mm) to generate supramolecular vesicles as revealed by TEM experiments (Figure 6 A). These vesicles were able to rearrange, depending on the exposure time to UV light, to: 1) a spongelike microstructure (10 min, Figure 6 B and inset) and 2) to fibers (40 min; Figure 6 C). These microstructure rearrangement was reversibly controlled under exposure to visible light, which produced spongelike microstructures (20 min; Figure 6 D), followed by a mixture of fiber, spongelike and spherical microstructures
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Figure 4. A) Chemical structure and isomerization process of the system (2) formed by our pazoDbg-containing building block tris-conjugated to the 1,3,5-triazine template. B) TEM images of the light-driven, reversible, microstructure morphological transition of this system, generated from a DMSO/ H2O suspension. Scale bars = 1 mm, 100 nm, and 100 nm, for the left, center, and right panels, respectively.
Figure 5. A) and B) Chemical structure and isomerization process of the system (3) formed by our pazoDbg-containing building block tetraconjugated to the pentaerythritol template. C) Reverse-phase HPLC of a 3 minute UV-irradiated solution of 3.
and fibers are reported, respectively, in Figure 7 A and B. The spongelike microstructures (Figure 7 C) were characterized in a mixture with fiber, under the same conditions as those reported for Figure 6 B. The height profiles of selected sponges reveal the irregular 3D morphology present on their surface. In summary, the ability of the compounds described in this paper to promote well-ordered supramolecular nanostructures highlights the importance of pazoDbg in this field, in particular: 1) the two side-chain azobenzene moieties give rise to aromatic stacking interactions strong enough to drive the self-assembly process; 2) these interactions can be disrupted by light, thus rendering the supramolecular structure photoresponsive. Such a combination of properties is, as far as we know, unique and makes the noncoded pazoDbg a-amino acid of remarkable interest in the fabrication of light-sensitive self-assembled nanostructures.
Acknowledgements (1 h; Figure 6 E) and finally by almost exclusively spherical microstructures (2 h; Figure 6 F). The vesicle-to-sponge-to-fiber microstructure transition occurring for 3 was additionally studied by AFM. Vesicles
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Financial support from the Ministerio de Ciencia e Innovacin-FEDER (grant CTQ2010-17436, FPU fellowship to P.F.), Gobierno de AragnFSE (research group E40), and the University of Padova (PRAT A. M. C91 J11003560001 and M. M. CPDA119117) are gratefully acknowl-
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COMMUNICATION Keywords: amino acid · azobenzene · microstructure transition · photoswitch · selfassembly
[1] Recent reviews: a) G. Fuks, R. M. Talom, F. Gauffre, Chem. Soc. Rev. 2011, 40, 2475 – 2493; b) S. F. M. van Dongen, H.-P. M. DeHoog, R. J. R. W. Peters, M. Nallani, R. J. M. Nolte, J. C. M. van Hest, Chem. Rev. 2009, 109, 6212 – 6274; c) E. Soussan, S. Cassel, M. Blanzat, I. RicoLattes, Angew. Chem. 2009, 121, 280 – 295; Angew. Chem. Int. Ed. 2009, 48, 274 – 288. [2] Recent reviews: a) Z. Luo, S. Zhang, Chem. Soc. Rev. 2012, 41, 4736 – 4754; b) C. Valry, F. Artzner, M. Paternostre, Soft Matter 2011, 7, 9583 – 9594; c) X. Yan, P. Zhua, J. Li, Chem. Soc. Rev. 2010, 39, 1877 – 1890; d) I. W. Hamley, Angew. Chem. 2007, 119, 8274 – Figure 6. TEM images of the light-driven, reversible, vesicle-to-sponge-to-fiber microstructure transitions un8295; Angew. Chem. Int. Ed. dergone by 3 (see text). 2007, 46, 8128 – 8147. [3] Recent reviews: a) B. Tian, X. Tao, T. Ren, Y. Weng, X. Lin, Y. Zhang, X. Tang, J. Mater. Chem. 2012, 22, 17404 – 17414; b) C. Cai, L. Wang, J. Lin, Chem. Commun. 2011, 47, 11189 – 11203; c) A. Carlsen, S. Lecommandoux, Curr. Opin. Colloid Interface Sci. 2009, 14, 329 – 339. [4] a) N. Gour, D. Kedracki, I. Safir, K. X. Ngo, C. Vebert-Nardin, Chem. Commun. 2012, 48, 5440 – 5442; b) H. Wang, C. Yang, L. Wang, D. Kong, Y. Zhang, Z. Yang, Chem. Commun. 2011, 47, 4439 – 4441; c) F. Versluis, I. Tomatsu, S. Kehr, C. Fregonese, A. W. J. W. Tepper, M. C. A. Stuart, B. J. Ravoo, R. I. Koning, A. Kros, J. Am. Chem. Soc. 2009, 131, 13186 – 13187; d) Y. Zhou, M. Kogiso, T. Shimizu, J. Am. Chem. Soc. 2009, 131, 2456 – 2457; e) S. Ghosh, M. Reches, E. Gazit, S. Verma, Angew. Chem. 2007, 119, 2048 – 2050; Angew. Chem. Int. Ed. 2007, 46, 2002 – 2004; f) K. Matsuura, K. Murasato, N. Kimizuka, J. Am. Chem. Soc. 2005, 127, 10148 – 10149. [5] a) P. Koley, A. Pramanik, Soft Matter 2012, 8, 5364 – 5374; b) J. Naskar, S. Roy, A. Joardar, S. Das, A. Banerjee, Org. Biomol. Chem. 2011, 9, 6610 – 6615; c) S. Maity, P. Jana, S. K. Maity, D. Haldar, Langmuir 2011, 27, 3835 – 3841; d) A. Mishra, J. J. Panda, A. Basu, V. S. Chauhan, Langmuir 2008, 24, 4571 – 4576; e) S. Ghosh, S. K. Singh, S. Verma, Chem. Commun. 2007, 2296 – 2298; f) S. Vauthey, S. Santoso, H. Gong, N. Watson, S. Zhang, Proc. Natl. Acad. Sci. USA 2002, 99, 5355 – 5360. [6] a) R. Orbach, L. Adler-Abramovich, S. Zigerson, I. Mironi-Harpaz, D. Seliktar, E. Gazit, Biomacromolecules 2009, 10, 2646 – 2651; b) X. Yan, Y. Cui, Q. He, K. Wang, J. Li, W. Mu, B. Wang, Z. Ouyang, Chem. Eur. J. 2008, 14, 5974 – 5980; c) X. Yan, Q. He, K. Wang, L. Duan, Y. Cui, J. Li, Angew. Chem. 2007, 119, 2483 – 2486; Angew. Figure 7. AFM images of A) vesicles and B) fibers from 3. C) AFM Chem. Int. Ed. 2007, 46, 2431 – 2434; d) M. Reches, E. Gazit, Nano image of fibers from 3. C) AFM image of a mixture of sponges and Lett. 2004, 4, 581 – 585. fibers from 3, and height profiles of selected nanosponges (numbers in [7] P. Fats, E. Longo, F. Rastrelli, M. Crisma, C. Toniolo, A. I. Jimnez, the height profile correspond to the numbered structures in the AFM C. Cativiela, A. Moretto, Chem. Eur. J. 2011, 17, 12606 – 12611. image). [8] a) A. Mahler, M. Reches, M. Rechter, S. Cohen, E. Gazit, Adv. Mater. 2006, 18, 1365 – 1370; b) V. Jayawarna, M. Ali, T. A. Jowitt, A. F. Miller, A. Saiani, J. E. Gough, R. V. Ulijn, Adv. Mater. 2006, edged. A.M. thanks Dr. F. Caicci for assistance with TEM data record18, 611 – 614. [9] M. Reches, E. Gazit, Science 2003, 300, 625 – 627. ing.
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[10] Incorporation of Fmoc at the N-terminus of dipeptides often results in the formation of nanofibers (hydrogels): Y. Zhang, H. Gu, Z. Yang, B. Xu, J. Am. Chem. Soc. 2003, 125, 13680 – 13681. [11] Also Na-Fmoc protection of aromatic amino acids (Phe, Tyr) induces formation of fibrillar hydrogels: a) S. Sutton, N. L. Campbell, A. I. Cooper, M. Kirkland, W. J. Frith, D. J. Adams, Langmuir 2009, 25, 10285 – 10291; b) Z. Yang, H. Gu, D. Fu, P. Gao, J. K. Lam, B. Xu, Adv. Mater. 2004, 16, 1440 – 1444. [12] a) C.-D. Chang, M. Waki, M. Ahmad, J. Meienhofer, E. O. Lundell, J. D. Haug, Int. J. Pept. Protein Res. 2009, 15, 59 – 66; b) H. Cao, X. Zhu, M. Liu, Angew. Chem. 2013, 125, 4216 – 4220; Angew. Chem. Int. Ed. 2013, 52, 4122 – 4126. [13] I. M. Rio-Echevarria, R. Tavano, V. Causin, E. Papini, F. Mancin, A. Moretto, J. Am. Chem. Soc. 2011, 133, 8 – 11. [14] Reviews on stimuli-responsive peptide-based materials: a) D. W. P. M. Lçwik, E. H. P. Leunissen, M. van den Heuvel, M. B. Hansen, J. C. M. van Hest, Chem. Soc. Rev. 2010, 39, 3394 – 3412; b) R. J. Mart, R. D. Osborne, M. M. Stevens, R. V. Ulijn, Soft Matter 2006, 2, 822 – 835.
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[15] Reviews on stimuli-responsive polymer micelles/vesicles: a) Y. Zhao, Macromolecules 2012, 45, 3647 – 3657; b) J. Du, R. K. OReilly, Soft Matter 2009, 5, 3544 – 3561; c) M.-H. Li, P. Keller, Soft Matter 2009, 5, 927 – 937; d) F. Meng, Z. Zhong, J. Feijen, Biomacromolecules 2009, 10, 197 – 209. [16] a) G. Liu, C.-M. Dong, Biomacromolecules 2012, 13, 1573 – 1583; b) S. Kumar, J.-F. Allard, D. Morris, Y. L. Dory, M. Lepage, Y. Zhao, J. Mater. Chem. 2012, 22, 7252 – 7257; c) V. K. Kotharangannagari, A. Sanchez-Ferrer, J. Ruokolainen, R. Mezzenga, Macromolecules 2011, 44, 4569 – 4573. [17] A few examples of light-responsive peptide self-assemblies exhibiting non-spherical (mainly fibrillar) morphologies have been published. See ref. [14]. [18] S. Lee, S. Oh, J. Lee, Y. Malpani, Y.-S. Jung, B. Kang, J. Y. Lee, K. Ozasa, T. Isoshima, S. Y. Lee, M. Hara, D. Hashizume, J.-M. Kim, Langmuir 2013, 29, 5869 – 5877.
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Received: July 31, 2013 Published online: October 21, 2013
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Photocontrolled Self-Assembly of a Bis-AzobenzeneContaining a-Amino Acid Miriam Mba,[a] Daniela Mazzier,[a] Simone Silvestrini,[a] Claudio Toniolo,[a, b] Paola Fats,[c] Ana I. Jimnez,*[c] Carlos Cativiela,[c] and Alessandro Moretto*[a, b]
Self-assembly has emerged as a powerful process for the production of well-ordered supramolecular structures at the nanometer scale. Among them, spherical (micellar and vesicle-like) architectures are attracting much attention, due to their promising applications in biomedicine and nanotechnology.[1] Peptides offer important advantages as building blocks for the construction of self-assembled nanostructures,[2] because of their biocompatibility and structural/ functional diversity. In the literature, there are many examples of supramolecular vesicles and micelles formed by block copolymers containing at least one high-molecularweight polymeric peptide domain[1–3] as well as by small- or medium-size peptides conjugated with molecules of nonpeptidic nature.[2, 4] In comparison, spherical nanostructures based exclusively on low-molecular-weight peptides are much less common.[2, 5, 6] Nanotubes and nanofibers predominate among the self-assemblies formed by the latter compounds.[2] The chemical diversity of peptides may be further expanded with the use of nonproteinogenic a-amino acid residues. We have already described[7] a symmetrically a, adisubstituted glycine, bis[p-(phenylazo)benzyl]glycine (pazoDbg), that contains two side-chain azobenzene moieties and undergoes reversible cis–trans isomerization upon exposure to light of the appropriate wavelength. In the present work, we explored the self-assembly properties of derivatives and short peptides based on this photoswitchable aamino acid. A surrogate of the N-protected dipeptide Fmoc-Phe-PheOH,[8] namely Fmoc-Phe-pazoDbg-OH (Fmoc: 9-fluorenylmethyloxycarbonyl) (1, Figure 1 A), was selected for this study. Intermolecular p–p stacking interactions involving ar-
[a] Dr. M. Mba, D. Mazzier, Dr. S. Silvestrini, Prof. C. Toniolo, Dr. A. Moretto Department of Chemistry, University of Padova via Marzolo 1, 35131 Padova (Italy) E-mail: [email protected] [b] Prof. C. Toniolo, Dr. A. Moretto Institute of Biomolecular Chemistry, Padova Unit, CNR via Marzolo 1, 35131 Padova (Italy) [c] Dr. P. Fats, Dr. A. I. Jimnez, Prof. C. Cativiela Instituto de Sntesis Qumica y Catlisis Homognea (ISQCH) CSIC-Universidad de Zaragoza, 50009 Zaragoza (Spain) E-mail: [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201303015.
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omatic side chains are a potent driving force for peptide self-assembly.[2, 6] Indeed, Phe-Phe has been recognized as the minimal self-assembling peptide sequence.[2, 9] The Fmoc group provides additional aromatic interactions that further contribute to the formation of supramolecular nanostructures, usually fibrillar in shape.[10, 11] Fmoc-Phe-pazoDbg-OH (1) was synthesized by solutionphase methods (see the Supporting Information). The reversible isomerization between the all trans (t) and all cis (c) forms [(t)1$(c)1] upon irradiation with UV (350 nm) or visible (450 nm) light was studied in MeOH solution by HPLC, CD, and UV/Vis absorption experiments (Figure 1 B; see also Figure S1 in the Supporting Information). The (t)1!(c)1 conversion was complete within 3 min. The HPLC profile obtained after 1-minute UV irradiation of (t)1 (Figure 1 B, left) shows the presence of intermediate species in which only one azobenzene group has isomerized (note that pazoDbg becomes chiral in this state[7]). The UV/Vis absorption and CD spectra of (t)1 and (c)1 exhibit significant differences in the region above 220 nm (Figure 1 B, center and right) that we assigned primarily to different relative dispositions of the aromatic ring in the chiral Phe residue with respect to those in the achiral pazoDbg for the (c)1 and (t)1 forms, which allow for a more effective chirality transfer in (t)1. However, it should be noted that the Fmoc chromophore is also known to contribute to the electronic absorption and CD above 250 nm.[12] The self-assembly properties of 1 were investigated in an aqueous environment. The lyophilized dipeptide was dissolved in DMSO at a concentration of 50 mg mL1. A 20 mL aliquot of this solution was dropped into 1980 mL of water and vortexed for a few seconds.[9] The resulting suspension was submitted to size-exclusion chromatography by gel filtration (Sephadex G75) and eluted with water. The yellowish fraction collected showed increased CD intensity when compared to (t)1 in solution (see Figure S2 A in the Supporting Information), which is a first indication of the selforganization of the system. Dynamic light scattering (DLS; see Figure S3 in the Supporting Information), transmission electron microscopy (TEM), environmental and field-emission scanning electron microscopy (E-SEM, FE-SEM), and atomic force microscopy (AFM) revealed that (t)1 forms spherical vesicle-like nanostructures with diameters of 50– 200 nm (Figure 2).
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Figure 1. A) Chemical structures of the pazoDbg-containing building block 1; the all trans (t), cis/trans, trans/ cis, and all cis (c) forms are shown. B) Left: HPLC profile after 1 min irradiation of (t)1 at 350 nm. Center: UV/Vis spectra of (t)1 and (c)1 (0.1 mm solution in MeOH). Right: CD spectra of (t)1 and (c)1 (1 mm solution in MeOH).
Figure 2. Left: TEM (A, unstained) and SEM (B, field emission; C, environmental) images of the nanostructures formed by (t)1 in water. Right (top): AFM image of the (t)1 nanostructures; the brightest spots correspond to the highest parts of the surface. Bottom: Height profiles of selected nanospheres.
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The close similarity of the microscopy images obtained after the sample was left standing at room temperature for several weeks indicates a high stability for the system. Irradiation at 350 nm for 5 min to generate (c)1 resulted in deformation of the nanospheres to yield elongated shapes (see Figure S4 in the Supporting Information). This morphological change proved to be reversible as the spherical assemblies were recovered after illumination with visible light [(c)1!(t)1]. The reversibility of the isomerization process was also confirmed by UV/Vis absorption spectroscopy along two cycles (see Figure S2 B in the Supporting Information). Longer UV-irradiation times resulted in complete disruption of the vesicles (see Figure S4 in the Supporting Information). These latter results suggest that the (t)1!(c)1 isomerization is somewhat hindered in the assembled structure (note that a 3 minute exposure to UV light allowed for complete isomerization in solution; Figure 1 B). Accordingly, the trans-to-cis isomerization of only a fraction of azobenzene moieties induces a slight rearrangement of the assembly, but the intermolecular attractive forces are still capable of essentially preserving the three-dimensional self-assembled structure. However, when the ratio of azobenzene moieties in the nonplanar cis form reaches a certain level, the intermolecular packing mode is so strongly perturbed (due to shape changes and less effective p–p stacking) that the ordered supramolecular architecture is not maintained and the spherical assemblies are transformed into amorphous aggregates. A potential application of this photosensitive process to induce the controlled release of substances entrapped in the
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Photocontrolled Self-Assembly of a Bis-Azobenzene
nanoassemblies was evaluated. A 20 mL aliquot of the above mentioned solution of (t)1 in DMSO was mixed with 1980 mL of a 10 mg mL1 aqueous solution of gold nanoparticles (GNps, 2 nm diameter) coated with a helical undecapeptide.[13] After a short vortex agitation, the suspension obtained was submitted to gel filtration and thus separated from the nonencapsulated peptide-coated GNps. Microscopy studies revealed the presence of nanostructures similar in morphology to those formed by (t)1 in the absence of GNps. UV irradiation of the gel-filtered sample (10 min) resulted in partial disruption of the vesicles with concomitant release of the encapsulated peptide-coated GNps, as detected by TEM (Figure 3).
Figure 3. TEM images (unstained) showing: A) nanospheres formed by (t)1 and peptide-coated GNps, scale bar = 200 nm; B) the nanospheres disassembly induced by UV irradiation, scale bar = 500 nm; C) details of released GNps (black dots), scale bar = 10 nm.
To the best of our knowledge, this is the first example of a supramolecular vesicle-like system formed exclusively by peptides that exhibits photoresponsive properties.[2, 14] Indeed, the reported number of spherical nanoassemblies of any composition that are sensitive to light is rather limited in comparison to other external stimuli.[1–3, 15] Among peptide-based nanospheres, several structures have been shown to undergo morphological transitions induced by changes in pH[2, 4c,e, 14] or concentration[2, 5c, 6b,c, 14] or by the presence of metal ions.[2, 5a,b,e, 14] Quite recently, the first examples of light-sensitive block-copolymer micelles with polypeptide domains have been described.[16] However, there is no previous report on any photoresponsive vesicular or micellar nanostructure formed entirely by peptides or even by hybrid
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COMMUNICATION compounds containing a nonpolymeric peptide segment.[17] Recently, novel, stimulus-responsive supramolecular structures in the form of fibers, gels, and spheres, derived from an azobenzene-containing benzenetricarboxamide derivative, were described by Kim and co-workers.[18] Therefore, we next decided to conjugate H-(pazoDbg)-OEt to 1,3,5-trichlorotriazine with the aim at exploring the supramolecular stimuli response of a novel C3-symmetrical tris-azobenezene systems, namely compound 2 (Figure 4 A). H-(pazoDbg)OEt, 1,3,5-trichlorotriazine, and triethylamine were mixed in a DMF solution for 48 h, affording 2 in 69 % yield. All six azobenzene units were able to switch reversibly in a MeOH solution from the (t)$(c)states (Figure 4 A) under appropriate irradiation. The intermediates were detected by HPLC. Spherical microstructures were generated (in form of milklike suspension) from a 15 mm solution of 2 in DMF/ THF (1:7) dialyzed (membrane cutoff, 1 kDa, 48 h) against ultrapure water (Figure 4 B, left). Under UV irradiation (20 min), a morphological transition from the suspension to gel was observed. We recorded TEM images after different irradiation times. The analysis revealed a conversion from spheres to elongated spheres (5 min), and eventually to a mixture of spherical/elongated fibers (20 min; Figure 4 B, center and right, respectively). Irradiation of the gel with visible light resulted in gel disruption, followed by precipitation of the obtained amorphous aggregates, which do not undergo further light conversion. Vesicle and hydrogel microstructure formation were also confirmed by AFM analysis (Figure S5, in the Supporting Information) Finally, we expanded the use of our pazoDbg amino acid to the synthesis of the “octopus” system 3 (Figure 5 A) to generate photoresponsive C4-symmetric, dendrimeric-like, multiazobenzene systems. Tfa-(pazoDbg)-OH (Tfa, trifluoroacetyl), reacted with pentaerythritol in presence of N,N’-diisopropylcarbodiimide and N,N-dimethylaminopyridine afforded 3 in a remarkable 90 % yield. The resulting system is characterized by as many as eight azobenzene moieties and can be viewed as a central core surrounded by a shell of azobenzene groups at the periphery. Up to eleven (out of the possible fifteen, Figure 5 B) discrete states produced by trans-to-cis isomerizations of the individual azobenzene units were observed by reverse-phase HPLC depending on the time of exposure of 3 (in a MeOH solution) to the UV light (Figure 5 C). This process is fully reversible (cis-to-trans) under visible light irradiation for several cycles. Moreover, this system shows a high propensity to self-assemble in a variety of organic (DMF, DMSO, THF, MeOH, CH3CN)/aqueous solutions (even at low concentrations, below 1 mm) to generate supramolecular vesicles as revealed by TEM experiments (Figure 6 A). These vesicles were able to rearrange, depending on the exposure time to UV light, to: 1) a spongelike microstructure (10 min, Figure 6 B and inset) and 2) to fibers (40 min; Figure 6 C). These microstructure rearrangement was reversibly controlled under exposure to visible light, which produced spongelike microstructures (20 min; Figure 6 D), followed by a mixture of fiber, spongelike and spherical microstructures
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Figure 4. A) Chemical structure and isomerization process of the system (2) formed by our pazoDbg-containing building block tris-conjugated to the 1,3,5-triazine template. B) TEM images of the light-driven, reversible, microstructure morphological transition of this system, generated from a DMSO/ H2O suspension. Scale bars = 1 mm, 100 nm, and 100 nm, for the left, center, and right panels, respectively.
Figure 5. A) and B) Chemical structure and isomerization process of the system (3) formed by our pazoDbg-containing building block tetraconjugated to the pentaerythritol template. C) Reverse-phase HPLC of a 3 minute UV-irradiated solution of 3.
and fibers are reported, respectively, in Figure 7 A and B. The spongelike microstructures (Figure 7 C) were characterized in a mixture with fiber, under the same conditions as those reported for Figure 6 B. The height profiles of selected sponges reveal the irregular 3D morphology present on their surface. In summary, the ability of the compounds described in this paper to promote well-ordered supramolecular nanostructures highlights the importance of pazoDbg in this field, in particular: 1) the two side-chain azobenzene moieties give rise to aromatic stacking interactions strong enough to drive the self-assembly process; 2) these interactions can be disrupted by light, thus rendering the supramolecular structure photoresponsive. Such a combination of properties is, as far as we know, unique and makes the noncoded pazoDbg a-amino acid of remarkable interest in the fabrication of light-sensitive self-assembled nanostructures.
Acknowledgements (1 h; Figure 6 E) and finally by almost exclusively spherical microstructures (2 h; Figure 6 F). The vesicle-to-sponge-to-fiber microstructure transition occurring for 3 was additionally studied by AFM. Vesicles
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Financial support from the Ministerio de Ciencia e Innovacin-FEDER (grant CTQ2010-17436, FPU fellowship to P.F.), Gobierno de AragnFSE (research group E40), and the University of Padova (PRAT A. M. C91 J11003560001 and M. M. CPDA119117) are gratefully acknowl-
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COMMUNICATION Keywords: amino acid · azobenzene · microstructure transition · photoswitch · selfassembly
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[10] Incorporation of Fmoc at the N-terminus of dipeptides often results in the formation of nanofibers (hydrogels): Y. Zhang, H. Gu, Z. Yang, B. Xu, J. Am. Chem. Soc. 2003, 125, 13680 – 13681. [11] Also Na-Fmoc protection of aromatic amino acids (Phe, Tyr) induces formation of fibrillar hydrogels: a) S. Sutton, N. L. Campbell, A. I. Cooper, M. Kirkland, W. J. Frith, D. J. Adams, Langmuir 2009, 25, 10285 – 10291; b) Z. Yang, H. Gu, D. Fu, P. Gao, J. K. Lam, B. Xu, Adv. Mater. 2004, 16, 1440 – 1444. [12] a) C.-D. Chang, M. Waki, M. Ahmad, J. Meienhofer, E. O. Lundell, J. D. Haug, Int. J. Pept. Protein Res. 2009, 15, 59 – 66; b) H. Cao, X. Zhu, M. Liu, Angew. Chem. 2013, 125, 4216 – 4220; Angew. Chem. Int. Ed. 2013, 52, 4122 – 4126. [13] I. M. Rio-Echevarria, R. Tavano, V. Causin, E. Papini, F. Mancin, A. Moretto, J. Am. Chem. Soc. 2011, 133, 8 – 11. [14] Reviews on stimuli-responsive peptide-based materials: a) D. W. P. M. Lçwik, E. H. P. Leunissen, M. van den Heuvel, M. B. Hansen, J. C. M. van Hest, Chem. Soc. Rev. 2010, 39, 3394 – 3412; b) R. J. Mart, R. D. Osborne, M. M. Stevens, R. V. Ulijn, Soft Matter 2006, 2, 822 – 835.
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Received: July 31, 2013 Published online: October 21, 2013
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