Nucleotide-Promoted Morphogenesis in Amphiphile Assemblies

Apr 20, 2009 - The kinetics of the nucleotide molecular organization and the formation of supramolecular helices was followed. We have shown that heli...
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Nucleotide-Promoted Morphogenesis in Amphiphile Assemblies: Kinetic Control of Micrometric Helix Formation† )

Carole Aime,‡ Rumi Tamoto,‡ Takao Satoh,‡ Axelle Grelard,‡ Erick J. Dufourc,‡ Thierry Buffeteau, Hirotaka Ihara,§ and Reiko Oda*,‡ ‡

)

Institut Europ een de Chimie et Biologie, UMR 5248 CBMN, CNRS-Universit e de Bordeaux - ENITAB, 2 rue Robert Escarpit, F-33607 Pessac, France, §Department of Applied Chemistry and Biochemistry, Kumamoto University, 2-39-1 Kurokami, Kumamoto 860-8555, Japan, and Institut des Sciences Mol eculaires, UMR 5255, CNRS-Universit e de Bordeaux, 351 Cours de la Lib eration, 33405 Talence, France Received December 31, 2008. Revised Manuscript Received March 6, 2009

Anionic nucleotides adenosine monophosphate or guanosine monophosphate interact with cationic vesicles, exchange with the counteranions of the amphiphiles in situ, and organize themselves at the membrane surfaces. Such organized nucleotides reciprocally transfer their chirality to membranes of nonchiral amphiphiles to induce the formation of right-handed micrometric helices on the time scale of hours. The kinetics of the nucleotide molecular organization and the formation of supramolecular helices was followed. We have shown that helix formation is a kineticdependent process that does not primarily result from ion exchange but from conformational reorganization and formation of weak interactions between confined nucleotides.

Introduction Self-assembly is the driving force for nanostructure formation from a broad diversity of building blocks. Therefore, this phenomenon is of particular interest in a wide range of fields such as colloids, amphiphiles, polymers, and biomolecular materials. Nowadays, many developments in nanotechnology attempt to exploit the self-assembling properties of soft materials.1 In particular, self-assembly through biological molecules such as amino acids and nucleic acids has attracted much attention.2 Indeed, the organization of biological systems is mostly governed by multiple weak interactions. DNA and RNA are good examples because their structure results from specific (and nonspecific) hydrogen bonds along with weak nonspecific interactions such as stacking interactions of the nucleobases. Among numerous bioinspired self-assemblies designed to develop systems that mimic nature,3 nucleoamphiphiles have been investigated in particular to take advantage of the recognition properties4 and self-assembling properties5 of these biopolymers. † Part of the Molecular and Polymer Gels; Materials with Self-Assembled Fibrillar Networks special issue. *Corresponding author. E-mail: [email protected].

(1) Hamley, W. Angew. Chem., Int. Ed. 2003, 42, 1692. (2) (a) Lowe, C. R. Curr. Opin. Struct. Biol. 2000, 10, 428. (b) Niemeyer, C. M. Angew. Chem., Int. Ed. 2001, 40, 4128. (3) (a) Seeman, N. C. Nature (London) 2003, 421, 427. (b) Ding, B.; Sha, R.; Seeman, N. C. J. Am. Chem. Soc. 2004, 126, 10230. (c) Liao, S.; Seeman, N. C. Science 2004, 306, 2072. (d) Matsuura, K.; Yamashita, T.; Igami, Y.; Kimizuka, N. Chem. Commun. 2003, 376. (e) Aime, C.; Nishiyabu, R.; Gondo, R.; Kaneko, K.; Kimizuka, N. Chem. Commun. 2008, 6534. (4) (a) Onda, M.; Yoshihara, K.; Koyano, H.; Ariga, K.; Kunitake, T. J. Am. Chem. Soc. 1996, 118, 8524. (b) Berti, D.; Baglioni, P.; Bonaccio, S.; Barsacchi-Bo, G.; Luisi, P. L. J. Phys. Chem. B 1998, 102, 303. (c) Berti, D.; Luisi, P. L.; Baglioni, P. Colloids Surf., A 2000, 167, 95. (d) Baglioni, P.; Berti, D. Curr. Opin. Colloid Interface Sci. 2003, 8, 55. (5) (a) Haruta, O.; Nishida, J.; Ijiro, K. Colloids Surf., A 2006, 326, 284. (b) Moreau, L.; Barthelemy, P.; El Maataoui, M.; Grinstaff, M. W. J. Am. Chem. Soc. 2004, 126, 7533. (c) Moreau, L.; Ziarelli, F.; Grinstaff, M. W.; Barthelemy, P. Chem. Commun. 2006, 1661. (d) Gissot, A.; Camplo, M.; Grinstaff, M. W.; Barthelemy, P. Org. Biomol. Chem. 2008, 6, 1324.

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A majority of biomolecules are chiral, and their chirality is often expressed in the morphology of the molecular assemblies on a supramolecular scale of nanometers to micrometers.6 Among those, a number of examples can be found in the literature where nucleobase-derived amphiphiles self-assemble to form chiral ribbons.7 We are particularly interested in the chiral morphology control of self-assemblies of ionic amphiphilic molecules via counterion specificity. We have previously reported that nonchiral dicationic gemini surfactants self-assemble into chiral fibers in the presence of chiral tartrate counterions8 or oligoalanine peptides.9 Through these studies, it became clear that the strength of interaction between the amphiphiles and their counterions is a key factor in determining the assembling behavior in terms of kinetics and morphology. The same dicationic gemini surfactants in the presence of nucleotides formed a hydrogel with uridine 50 -monophosphate (UMP) without apparent chirality expression on the supramolecular level.10 More recently, we have reported micrometric helix formation with monocationic amphiphiles, didodecyle (noted hereafter as C12) or ditetradecyle (C14) dimethylammonium complexed with nucleotides, adenosine 50 -monophosphate (AMP), or guanosine (6) For a review on chiral fibrous structures, see aIhara H.; Takafuji, M.; Sakurai , T. In Encyclopedia of Nanoscience and Nanotechnology; Nalwa, H. S., Ed.; American Scientific Publishers: Stevenson Ranch, CA, 2004; Vol. 9, pp 473495.(b) Brizard, A.; Oda, R.; Huc, I. Top. Curr. Chem. 2005, 256, 167. (7) (a) Iwaura, R.; Yoshida, K.; Masuda, M.; Oshini-Kameyama, M.; Yoshida, M.; Shimizu, T. Angew. Chem., Int. Ed. 2003, 42, 1009. (b) Shimizu, T.; Iwaura, R.; Masuda, M.; Hanada, T.; Yase, K. J. Am. Chem. Soc. 2001, 123, 5947. (c) Itojima, Y.; Ogawa, Y.; Tsuno, K.; Handa, N.; Yanagawa, H. Biochemistry 1992, 31, 4757. (d) Yanagawa, H.; Ogawa, Y.; Furuta, H.; Tsuno, K. J. Am. Chem. Soc. 1989, 111, 4567. (8) (a) Oda, R.; Huc, I.; Candau, S. J. Angew. Chem., Int. Ed. 1998, 37, 2689. (b) Oda, R.; Huc, I.; Schmutz, M.; Candau, S. J.; MacKintosh, F. C. Nature (London) 1999, 399, 566. (c) Berthier, D.; Buffeteau, T.; Leger, J. -M.; Oda, R.; Huc, I. J. Am. Chem. Soc. 2002, 124, 13486. (d) Brizard, A.; Aime, C.; Labrot, T.; Huc, I.; Berthier, D.; Artzner, F.; Desbat, B.; Oda, R. J. Am. Chem. Soc. 2007, 129, 3754. (e) Oda, R.; Laguerre, M.; Huc, I.; Artzner, F. J. Am. Chem. Soc. 2008, 130, 14705. (9) Brizard, A.; Kiagus Ahmad, R.; Oda, R. Chem. Commun. 2007, 2275. (10) Wang, Y.; Desbat, B.; Manet, S.; Aime, C.; Labrot, T.; Oda, R. J. Colloid Interface Sci. 2005, 283, 555.

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50 -monophosphate (GMP).11 In these systems, the chirality of the sugar moiety of the nucleotide is expressed on the supramolecular level. In the present study, we have investigated the kinetic aspect of the assembling behavior and structural transition from vesicles to helices in situ in aqueous solution of the nucleoamphiphilic systems. Nonchiral C12 or C14 cationic amphiphiles in the presence of nonchiral acetate counterions (noted as C12Ac and C14Ac (Scheme 1A)) form vesicles in water. Upon addition of nucleotide AMP or GMP, ion exchange occurs in situ: nucleotides replace acetate counterions because their pKa (phosphate group) in water is lower than that of acetate (2 vs 4.76). This ion exchange leads to the formation of the resulting nucleoamphiphile (Scheme 1B) and volatile acetic acid. Thus, by simply adding nucleotides to nonchiral vesicle solutions, chiral helix formation was observed through ion exchange. The kinetics of chirality transfer is investigated both on the supramolecular scale (optical microscopy (OM) study) and on the molecular scale (NMR and infrared (IR) studies). These investigations clearly show that the interaction between the amphiphiles and their counterions is a key factor controlling the self-assembling kinetics and morphology, and interestingly, the ion exchange and the molecular organization kinetics operate on different time scales.

Materials and Methods Synthesis of C12 and C14 Acetate. Cationic surfactants and dialkyldimethylammonium bromide (n = 12, 14) were purchased from Fluka and used without any further purification. In a common procedure, the surfactant is mixed with silver acetate (1 equiv) in methanol. The mixture is stirred for 30 min at 50 °C until the formation of a black precipitate of silver bromide, whereas the acetate surfactant is soluble in methanol. Silver bromide is filtered on Celite to give a colorless solution. After evaporation, the product is dissolved in a mixture of chloroform/ methanol (9/1 v/v), precipitated with acetone, filtered, and dried under vacuum. Preparation of the Samples. Guanosine 50 -monophosphate and adenosine 50 -monophosphate were purchased from Fluka and Acros Organics, respectively, and used without any further purification. A vesicular solution of C14Ac is added to a solution of GMP and AMP with the final concentration of each reagent being 15 mM. The sample is neither heated nor mixed. Deionized water (Purelab Prima Elga, 18.2 MΩ 3 cm) was used to perform analysis by OM and TEM, and D2O (Eurisotop CEA Saclay, France) was used for NMR and IR studies. OM was used to ensure that no morphological variations occurred by exchanging H2O for D2O. The following notation was used for the 1H NMR splitting patterns: singlet (s), doublet (d), triplet (t), multiplet (m), and doublet of doublets (dd). C12GMP. 1H NMR (400 MHz, CD3OD, 25 °C, δ ppm): 8.08 (1H, s); 5.85 (1H, d, 3J = 6.36 Hz); 4.76 (1H, dd, 3J = 5.14 Hz, 3J = 6.36 Hz); 4.38 (1H, dd, 3J = 2.45 Hz, 3J = 5.14 Hz); 4.18 (1H, m); 4.14 (1H, m); 4.07 (1H, m); 3.28 (4H, m); 3.05 (6H, s); 1.74 (4H, m); 1.38 (4H, m); 1.29 (32H, m); 0.90 (6H, t, 3J = 6.85 Hz). (11) Aime, C.; Manet, S.; Satoh, T.; Park, K. Y.; Ihara, H.; Godde, F.; Oda, R. Langmuir 2007, 23, 12875. (12) From these images alone, it may be suggested that the ring-like shapes observed in Figures 1a and 2a are from toroid-shaped aggregates. However, such an ambiguity can be lifted as we follow the moving motions of these objects under the microscope, because the toroid objects should move to a side-view perspective as they move around with Brownian motion, whereas the projection of vesicles should remain spherical at all times, as observed with our samples. Also, C12Br, which is known as DDAB, was reported in the 1980s by Kunitake as the first non-natural molecule to form vesicles. Moreover, the freeze-fracture of these vesicles unambiguously shows the spherical curvature of the object (Figure S1)

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Aim e et al. Scheme 1. Molecular structure of the studied nucleoamphiphiles: (A) monocationic amphiphile molecule with an acetate counteranion (C12Ac and C14Ac surfactants) with added adenosine 50 -monophosphate (AMP) or guanosine 50 -monophosphate (GMP) and (B) monocationic amphiphile molecules complexed with GMP or AMP (C12GMP or C14AMP surfactant, respectively).

C NMR (400 MHz, CD3OD, 25 °C, δ ppm): 138.45; 89.25; 75.05; 72.36; 65.85; 65.22; 51.20; 49.64; 49.42; 49.21; 48.99; 48.79; 48.57; 48.36; 48.27; 48.11; 48.06; 47.98; 47.92; 47.88; 33.08; 30.75; 30.63; 30.52; 30.48; 30.20; 27.37; 23.74; 23.49; 14.43. C14AMP. 1H NMR (Bruker Avance 400, CD3OD, 25 °C, δ ppm): 8.56 (1H, s); 8.21 (1H, s); 6.09 (1H, d, 3J = 6.11 Hz); 4.67 (1H, m); 4.41 (1H, dd, 3J = 3.11 Hz, 3J = 4.76 Hz); 4.24 (1H, m); 4.12 (2H, m); 3.28 (4H, m); 3.05 (6H, s); 1.73 (4H, m); 1.38 (4H, m); 1.29 (40H, m); 0.89 (6H, t, 3J = 13.36 Hz). 13C NMR (400 MHz, CD3OD, 25 °C, δ): 152,76; 141,46; 141,43; 123,10; 123,08; 121,08; 107,86; 88,89; 86,14; 86,10; 86,06; 76,35; 72,44; 65,96; 65,91; 65,86; 65,20; 51,21; 49,64; 49,42; 49,32; 49,21; 48,99; 48,79; 48,57; 48,36; 48,15; 48,08; 48,00; 47,95; 47,90; 47,84; 47,80; 47,76; 47,70; 47,64; 47,59; 33,09; 30,77; 30,63; 30,49; 30,18; 27,35; 25,62; 23,74; 23,48; 14,44. 12

Optical Microscopy with Differential Interferential Contrast (DIC). Samples sealed between a slide glass and cover glass were observed with a Nikon Eclipse PhysioStation E600FN with adequate condensers and a prism for DIC observations. Kinetic NMR Measurements on Aggregates. 1H NMR experiments were carried out on a Bruker Avance 500 spectrometer operating at 500.13 MHz and equipped with solid-state NMR facilities. Samples were enclosed in 4-mm-diameter Zirconia rotors (50 μL internal volume). Magic-angle sample spinning (MAS) was performed at 8 kHz. A H2O/D2O (80/20 v/v) mixture was used for sample preparation. A D2O lock was used, and the temperature was regulated to 290 K (heating due to air friction when spinning is taken into account). Acquisition was performed using a classical one-pulse sequence with water suppression (presaturation of 50 dB) on a spectral width of 5 kHz. Typically, 32 scans were recorded with a repetition time of 3 s. This leads to an acquisition time per experiment of 96 s. Spectra were obtained by the application of a Lorentzian filtering function of 1 Hz and Fourier transformation of time-dependent signals. IR Measurements. IR spectra were recorded with a ThermoNicolet Nexus 670 FTIR spectrometer, at a resolution of 4 cm-1, by coadding 50 scans. Samples were held in a demountable cell with CaF2 windows and a 50 μm Teflon spacer. Baseline corrections for absorption spectra were made by subtracting the absorption of D2O solvent.

Results and Discussion When CnAc (n = 12, 14) is dispersed in water, it spontaneously forms vesicles, as can be seen in Figures 1a and 2a.11 To this vesicle solution, a solution of nucleotides (AMP or GMP) is added. Different behaviors are observed depending on the nucleotide, the (13) Thomas, B. N.; Safinya, C. R.; Plano, R. J.; Clark, N. A. Science 1995, 267, 1635.

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system. OM images of vesicles of C14Ac (15 mM) (a) and after mixing C14Ac + AMP (15 mM) (b) just after mixing and after (c) 10 min, (d) 1 h, (e) 2 h, and (f) 3 days. The scale bars are 10 μm.

Figure 2. Vesicle to helix formation observed with C12Ac + GMP system. OM images of vesicles of C12Ac (a) and of C12Ac + GMP (15 mM) (b) just after mixing and after (c) 30 min, (d) 5 h, (e) 1 day, and (f) 7 days. The scale bar (10 μm) is common for all images.

hydrophobic chain length of the surfactant, and their concentrations. Hereafter, unless explicitly noted, 1 equiv of nucleotides was added to CnAc molecules. Kinetics of Helix Formation. C14Ac + AMP. When the solution of C14Ac was mixed with AMP solution, the formation of precipitates was observed with concentration-dependent kinetics. At a final concentration of 15 mM for each reagent, precipitation took place after only a few hours, whereas at 10 mM it took 4 weeks (Supporting Information Figure S2). We then followed the structural transformation of the aggregates using OM. C14Ac in aqueous solution formed vesicles (Figure 1a). Immediately after C14Ac and AMP were mixed, vesicles ruptured to form denser structures (Figure 1b,c). The sample became slightly opaque, and then fibers started growing from these dense cores and chiral right-handed helices formed along with needles (Figure 1d,e). The proportion of helices relative to needles increased with time (Figure 1f) for about a week. Images of the step-by-step transformation of aggregates at a fixed object are shown in Supporting Information (Figure S3). Both needles and microhelices were also observed with C14AMP (separately presynthesized, as previously reported12). Electron microscopy revealed that the needles are formed from rolled up multilayered cigarlike structures. However, interestingly, the comparison of C14Ac + AMP and C14AMP systems indicates a different needle to microhelix ratio, depending on the AMP complexation

method. Indeed, with precomplexed C14AMP, the needle to microhelix ratio was much higher than with the present system (Supporting Information Figure S4). This emphasizes the importance of kinetics of ion exchange and AMP reorganization in the morphologies of the aggregates. Interestingly, the formation of micrometric helices was limited in the surfactant concentration range of 10-15 mM. At 5 mM, no needles or helices were observed, and at 20 mM, mainly needles that do not show apparent chiral structure were observed under OM. C14Ac + GMP. Addition of GMP to C14Ac vesicles rapidly induced the transition from translucent to clouded suspension. After a few minutes, the suspension turned into a gel. The kinetics of gel formation was much quicker than what was observed with AMP at the same concentrations. Because the kinetics with C14Ac + GMP was too fast to capture the transition from vesicles to fibers, we studied the transition with C12Ac + GMP systems. C12Ac + GMP. Although the kinetics of the gel formation for this system was slightly slower than that with C14Ac, it was still much faster than the kinetics of precipitation observed with C14Ac + AMP. Right after mixing C12Ac and GMP, the viscosity of the solution increased, and vesicles transformed to continuous thin membranes (Figure 2b). Part of these membranes became fibers and aggregated into denser structures. After about half an

Figure 1. Vesicle to helix formation observed with C14Ac + AMP

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hour (for the solution of 15 mM), these fibers turned into chiral right-handed helices and continued to grow (Figure 2c-e) for about a week (Figure 2f). The formation of micrometric helices was limited to 7.5-15 mM surfactant solutions.These helices were also very similar to those previously observed with presynthesized C12GMP.11 Freeze-fracture images of these micrometric helices revealed their substructure consisting of bundles of nanometerscale fibers. For both nucleotides, the formation of chiral helical structures results from the addition of nucleotides to nonchiral vesicles. We then observed in detail how these helices grew. As described above, in the case of C14Ac + AMP, the vesicles first ruptured to form dense structures, and the helices grew out of them whereas in the case of C12Ac + GMP, continuous thin films were first formed before helices growth (Figure 3). In both cases, the stepby-step helix growth followed by OM (Figure 3c,d) showed that the helix growth seems to occur at their extremities with a coiling motion. However, from these images alone, we cannot unambiguously distinguish if helices indeed grow from the tip or from the base if the helices have a consistent pitch along the growth axis. We then followed the kinetics of molecular organization using NMR and IR spectroscopy. Kinetics of Molecular Reorganization during Helix Formation. NMR. C14Ac + AMP (15 mM). We followed the time evolution of the molecular environment using 500 MHz 1 H NMR. To have well-resolved resonances, we used the highresolution magic angle spinning technique (HR-MAS). To investigate the role of added AMP and its effect on acetate complexation, we focused on three key NMR signals corresponding to two nonexchangeable protons of the adenine base (H8 (0) and H2 (b), Figure 4) and to the three equivalent protons of the methyl group of the acetate ion (2 Figure 4). Right after sample preparation, the NMR spectrum showed that the signal from the acetate ion disappeared. This signal was replaced by the peak characteristic of acetic acid (1.952 ppm vs 1.816 for acetate) (Figure 4d,g). This indicates that ion exchange had occurred rapidly after mixing AMP and C14Ac. AMP has replaced the acetate counteranion at the cationic membrane, leading to the formation of acetic acid (Scheme 1). At this stage (about 10 min after mixing), OM studies show that no helix is formed. We have then followed the chemical shifts evolution of the two peaks corresponding to the protons of adenine nucleobase. Whereas the acetate ion peak quickly reached the final value after about 40 min (half-life of about 3 min) (Figure 4d) indicating rapid total ion exchange, the protons of the base showed much slower downfield chemical shift evolution (0.02 ppm for H2 and 0.035 ppm for H8) during a few thousand minutes with a half-life of 150-200 min (Figure 4b,c). This reflects the fact that the environment of confined nucleobases still evolves after a few hours. The chemical shielding reaches a plateau after about 3 days (Figure 4e,f). This corresponds to the kinetics of helix formation as observed using OM. C12Ac + GMP (7.5 mM). For this system, because the kinetics was too fast at a surfactant concentration of 15 mM we investigated the chemical shift variation with time at 7.5 mM. As observed with AMP, ion exchange occurred very quickly after mixing in less than 10 min (Figure 5d) (half-life ∼2 min). The protons of the guanine base also showed downfield chemical shift variation upon complexation with the surfactant (0.04 ppm for H2(b) and 0.08 ppm for H8(0)) (Figure 5b,c). In spite of shorter alkyl chains and the lower concentration, the transition was much faster than with C14Ac + AMP and was completed about 20 min after mixing C12Ac and GMP (half-life ∼5 min) (Figure 5e,f). 8492 DOI: 10.1021/la8043297

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Figure 3. Step-by-step helix formation of C14Ac + AMP (a, c) and C12Ac + GMP (b, d) followed by OM. (a, b) Global growth from vesicles and thin films, respectively. (c, d) Detailed views showing that the helices grow with rotating motion. (e) Schematic representation of the helices’ growth.

Meanwhile, the peak positions kept shifting very slightly until about 2 days (data not shown). The correlation of the morphological changes of the aggregates and the changes in molecular environment was further investigated using IR spectroscopy. IR Spectroscopy. C14Ac + AMP (15 mM). The frequencies for the antisymmetric and symmetric CH2 stretching vibrations (at 2924 and 2854 cm-1, respectively) of the hydrophobic Langmuir 2009, 25(15), 8489–8496

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Figure 4. Kinetics of chemical shifts variation by 500 MHz 1H NMR in C14Ac + AMP. (a) Spectra of C14Ac + AMP in H2O/ D2O (80/20 v/v). Parts of the 500 MHz 1H NMR spectra of C14Ac + AMP in H2O/D2O (80/20 v/v): (b) H2 and (c) H8 of the adenine base and (d) equivalent protons of the methyl group of acetate (for 1H NMR chemical shifts assignment, see Materials and Methods). Kinetics of chemical shifts variation for (e) H2 (b, halflife ∼190 min) and (f) H8 (0, half-life ∼150 min) protons of the nucleobases and (g) for the equivalent protons of the acetate counteranion (2, half-life ∼3 min).

chains (Supporting Information Figure S5) indicate that the nucleoamphiphiles are in the fluid phase with a high rate of gauche conformations. No variation in hydrophobic chain organization was observed even 7 days after mixing AMP and C14Ac. This result is particularly interesting when compared to previously reported systems in which the vesicle-to-helix transition was induced upon a temperature decrease with the diacetylenic lipids.13 In this case, the transition was associated with the gaucheto-trans conformation change of the hydrophobic chains. We have also recently reported that the hydrophobic chains of gemini-tartrate or gemini-aligo-alanine forming chiral ribbons are in the trans conformation.8d, 9 This loose molecular organization is possibly the origin of much larger-scale assemblies observed with these molecules compared to the two systems mentioned above (a few micrometers compared to tens to hundreds of nanometers). In contrast with the gemini-tartrate system, we could not observe finely resolved peaks with SAXS/WAXS that would have allow us to access information on the molecular organization. However, important variations are observed concerning the IR bands characteristic of the complexed counterions. First, the black and white arrows (at 1570 and 1711 cm-1 in Figure 6a) represent the bands characteristic of the acetate ion and acetic acid formed during ion exchange, respectively. Figure 6a shows that the band characteristic of acetic acid is not observed with C14Ac without any added AMP. This confirms that its presence in C14Ac + AMP results from the replacement of the acetate counterion with AMP and consecutive acetic acid Langmuir 2009, 25(15), 8489–8496

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Figure 5. Kinetics of chemical shifts variation by 500 MHz 1H NMR in C12Ac + GMP (7.5 mM). (a) Spectra of C12Ac + GMP in H2O/D2O (80/20 v/v). Parts of 500 MHz 1H NMR spectra: (b) H8 and (c) H2 of the guanine base and (d) equivalent protons of the methyl group of acetate. Kinetics of variation of chemical shifts are shown for (e) H8 (0, half-life ∼ 5 min) and (f) H2 (b, half-life ∼ 6 min) protons of the nucleobases.

formation. In addition, Figure 6a shows the absence of the band at 1570 cm-1 (acetate counterion) upon addition of AMP to C14Ac. The rapid kinetics of ion exchange as observed in Figure 6b by the replacement of the band at 1570 cm-1 (acetate) with the band at 1711 cm-1 (acetic acid) supports the NMR data discussed above: ion exchange occurs less than 10 min after mixing AMP and C14Ac . AMP is complexed with the surfactant, and acetic acid is formed. The black and white stars (at 1623 and 1666 cm-1, Figure 6b) are characteristic of the aromatic nucleobase. The origin of the band at 1623 cm-1 can be associated with CdN (C(2)dN(3), C(6)dN(1), and C(8)dN(7)) stretching vibrations with contributions from N-D bending vibrations. The intensity of this band decreases with time along with the increase in the intensity of the band at 1666 cm-1. This band at 1666 cm-1 may be attributed to the formation of hydrogen bonds between neighboring bases. (This increase could be associated with the protonation of the nucleobase. However, in our case, the sample exhibits a pH of about 4. At this pH, the nucleotide bears a single anionic charge.) Indeed, this band has previously been reported as a marker of double helix formation of polyAMP through hydrogen bonds.14 The increase in intensity stabilizes after 3 days, again exhibiting kinetics similar to that for helix formation as observed by OM. In summary, ion exchange occurs a few minutes after mixing AMP and C14Ac, allowing confinement of the nucleotides at the cationic membrane surface. However, the reorganization of the nucleotides as a result of hydrogen bonding between neighboring nucleobases at the membrane surface takes much longer as revealed by the behavior of the band at 1666 cm-1, and on the macroscopic scale, helices are formed after only 3 days. (14) Petrovic, A. G.; Polavarapu, P. L. J. Phys. Chem. B 2005, 109, 23698.

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Figure 6. (a) Comparison of IR spectra obtained with C14Ac (15 mM) and C14Ac + AMP (15 mM) samples. (b) Kinetics study by IR spectroscopy. IR spectra after mixing C14Ac and AMP (15 mM) in the range of the (a) bands characteristic of the counterions acetate and AMP. (b) Comparison of IR spectra obtained with C14Ac (15 mM) and C14Ac + AMP (15 mM) samples

C12Ac + GMP (10 mM). Figure 7a shows the infrared spectra of C12Ac, GMP, and C12Ac + GMP at 10 mM. Again, we chose a concentration lower than 15 mM used for C14Ac + AMP because the reaction at 15 mM was too fast in the presence of GMP. The white and black arrows (at 1570 and 1711 cm-1), which represent the characteristic bands of the acetate ion and acetic acid, respectively, again confirm the rapid exchange of counterions. The bands at 1670, 1580, and 1568 cm-1 are characteristic of the guanine nucleobase (Figure 7a, –1–). The band at 1670 cm-1 is associated with CdO stretching vibration whereas the bands at 1580 and 1568 cm-1 are associated with CdC and CdN (C(2)dN(3) and C(8)dN(7)) stretching vibrations with contributions from N-D bending vibrations. When C12Ac and GMP are mixed, the bands at 1580 and 1568 cm-1 disappeared rapidly, and new bands at 1587 and 1556 cm-1 were observed. These important shifts may be associated with the ordering of guanine residues in the supramolecular structures as previously reported.15 Meanwhile, in this case, the nature of the interaction (hydrogen bonds or π-π stacking) between the guanine residues is not straightforward to understand. In addition, this transition was again too fast for a kinetics investigation, confirming the NMR measurements. After this transition, the intensities of these bands show very little variation with time. Their intensity initially decreases rapidly, but after 2 h, it starts to increase again (Figure 7c), reaching a stationary value after 2 days. This initially rapid (10 min) transition followed by much slower (2 days) and slight modifications also confirms the NMR observations as well as the helix formation kinetics observed with OM. In the systems investigated above, helix formation is simply induced by the addition of nucleotides AMP or GMP. This process involves different transition steps at the macroscopic level (fluid solution to gel or precipitate), the mesoscopic level (15) (a) Setnicka, V.; Urbanova, M.; Volka, K.; Nampally, S.; Lehn, J. M. Chem. Eur. J. 2006, 12, 8737. (b) Howard, F. B.; Frazier, J.; Miles, T. Biopolymers 1977, 16, 791.

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Figure 7. (a) Comparison of IR spectra obtained with C12Ac (10 mM), GMP solution (10 mM) at pH 4, and C12Ac (10 mM) + GMP (10 mM) (pH 4). (b) Kinetics study by IR spectroscopy after mixing C12Ac and GMP in the range of bands characteristic of the counterions. (c) Intensity evolution of the band at 1550 cm-1.

(vesicle to helix transition), and the molecular level as summarized in Table 1. The molecular organization kinetics is quite different and much faster for the C12Ac + GMP system than for the C14Ac + AMP system. Starting from similar (or even identical) amphiphiles, this underlines the importance and specificity of the counterion nature in ionic assembly properties. In particularly, while adenine and guanine have similar stacking propensities, the guanine nucleobase exhibits edges having self-complementary hydrogen-bond donors and acceptors, responsible for the higher degree of intermolecular interactions between GMP than between AMP.16 These factors may account for the very fast kinetics observed with GMP-amphiphiles in spite of their shorter hydrophobic chains. Despite these differences, in both systems helix formation occurs through a similar molecular organization mechanism as evidenced by NMR and IR. First, ion exchange occurs in a few minutes, leading to the confinement of nucleotides, followed by their reorganization through cooperative internucleotide interactions at the membrane surface. Helix formation and growth as observed by OM followed much slower kinetics after ion exchange, indicating that it is a cooperative process due to molecular reorganization. Effect of Stoichiometry on the Morphology of the Aggregates. We have then varied the AMP or GMP to CnAc ratio from 0.25 to 1.5. The concentration of added AMP or GMP is varied with a constant amphiphile (C14 or C12) final concentration set at 15 mM. Figure 8 show the macroscopic aspect of the samples as well as their OM images. C14Ac + AMP. Depending on the AMP to C14Ac ratio, the macroscopic aspect of the samples varies from solution to precipitate (Figure 8A). Macroscopically, the higher the ratios, the (16) Davis, J. T.; Spada, G. P. Chem. Soc. Rev. 2007, 36, 296.

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Table 1. Summary of the Kinetics of CnAc + Nucleotides at Different Levels in the Bulk (Macroscopic), in Supramolecular Structures (Mesoscopic), and at the Molecular Level macroscopic aspect

C12Ac + GMP 15 mM C14Ac + AMP 15 mM

mesoscopic aspect

molecular kinetics (IR, RMN)

right after the nucleotides addition

gel formation/ precipitate

helix: formation (OM) beginning

helix: end of the growth

acetate to nucleotide ion exchange

nucleotide organization

viscosity increase

1 day (gel)

30 min

1 week

∼2 min (half-life)

∼10 min

no immediate change

1 h (precipitate)

30 min (needles) 1 h (helices)

1 week

∼4 min (half-life)

∼2.5 h

Figure 9. IR spectra of C14Ac (40 mM) + AMP (10 mM) (vesicles) vs C14Ac (15 mM) + AMP (15 mM) (helices). When the proportion of AMP is much inferior to that of C14Ac, the peak at 1666 cm-1 disappears, which relates the absence of interbase interactions with the morphology of the aggregates (vesicles).

Figure 8. Photographs showing the macroscopic aspect of (A) C14Ac (15 mM) + AMP samples with different AMP/C14Ac ratios, from 0.25 to 1.50 (numbers below the samples) and OM images of the samples: (B) ratios from 0.25 to 0.50, (C) ratios from 0.75 to 1.25, and (D) ratios above 1.50. The image in the insert shows the TEM image of the fiber showing nanometer-scale chiral structure. (E) C12Ac (15 mM) + GMP samples with different GMP/C12Ac ratios from 0.25 to 1.50 . OM images of the samples: (F) ratio of 0.25 and ratios (G) from 0.50 to 0.75 and (H) from 1.00 to 1.50.

more opaque the solutions. The proportion of vesicles, helices, and needles as observed by OM depends on the AMP/C14Ac ratio and can roughly be separated into three phases (Figure 8A). For AMP/C14Ac ratios lower than 0.50, C14Ac + AMP solutions remain almost clear. Only vesicles are observed (Figure 8B). The presence of a large population of acetate counterions that remain complexed to the amphiphiles may prevent the formation of interbase interaction between neighboring nucleobases. IR spectra comparing vesicle solutions (AMP/ C14Ac = 0.25) and helix suspensions (AMP/C14Ac = 1) show the absence of the absorption band at 1666 cm-1 for the vesicle solution (Figure 9), which confirms that this band can be considered to be a marker for supramolecular helical structure formation through hydrogen bonds. Langmuir 2009, 25(15), 8489–8496

In the 0.75 -1.25 range, precipitation and opacity increase with the ratio. Within this range, micrometric helices are observed along with needles (Figure C) and the proportion of needles relative to helices increases with the ratio. Finally, with an excess amount of AMP, for an AMP/C14Ac ratio higher than 1.50, only needles are observed by OM (Figure 8D). TEM images show nanometric chiral cigar-like structure on the nanometer scale (Figure 8D insert). These microneedles could then be viewed as nanohelices. Depending on the stoichiometry, the formation of either microhelices or nanohelices is favored. Precipitation occurs more rapidly in samples having the highest AMP/C14Ac ratios. As described before, when AMP interacts with vesicles, the fibers/ needles form first, which then transform to microhelices. The presence of excess AMP resulting in a higher density of needles could account for the faster precipitation. C12Ac + GMP. For a GMP/C12Ac ratio up to 0.25, C12Ac + GMP remained a clear solution, and thin films and fibers were observed under OM (Figure 8F). In contrast to the AMP system, no vesicles were observed at such a low ratio. For a ratio between 0.5 and 0.75, the solutions are still fluid, and micrometric helices appear along with fibers (Figure 8G). For a ratio between 1.00 and 1.50, the solutions become gels and do not flow when the bottle is inverted (Figure 8E). Within this range, micrometric helices grow in diameter (Figure 8H), and helix density increases with the ratio. Moreover, the formation of helices is much more rapid for a higher ratio of GMP to surfactant (about 3 min for a ratio of 1.5 whereas about 30 min is required for a ratio of 1).

Conclusions The weak interactions between nucleic acids AMP or GMP confined at the surfaces of amphiphile assemblies induce chiral supramolecular structures. This cooperative interaction between nucleotides and amphiphiles is much stronger than the ionic interaction between acetates and amphiphiles. This allowed us to follow the formation of micrometric chiral structures directly when a nucleotide solution is added to the vesicles solution of cationic DOI: 10.1021/la8043297

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nonchiral amphiphiles CnAc. We have observed in situ that vesicles first rupture to form large aggregates (AMP) or continuous films (GMP), from which micrometric helices grow. A detailed microscopy observation showed the growth of these helices . Spectroscopic measurements showed that micrometric helical structures of nucleoamphiphiles result from cooperative effects in the molecular organization of nucleobases. Ion exchange is the important step, which induces the confinement of nucleotides at cationic membrane surfaces. This process occurs within minutes after mixing the two molecules CnAc and nucleotides. Subsequently, the nucleotides confined at membrane surfaces reorganize themselves through weak interactions such as H bonds and π-π stacking. This reorganization and the formation of directional intermolecular interaction is the driving force for helix formation, and the CnAc to nucleotide ratio is an important factor that determines the helix dimensions (i.e., microhelices to nano-

8496 DOI: 10.1021/la8043297

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helices). The main difference between AMP and GMP systems is the kinetics of helix formation and the helix dimensions as well as the solution properties (precipitate vs gel). Once confined at the cationic membrane surfaces, GMP organizes much more rapidly (on the order of minutes) than AMP (∼100 min). This could be attributed to the nature of intermolecular interactions between GMP or AMP. The formation of supramolecular chiral structures clearly requires fine matching between surfactant structures and their counterions. The control of the nature and kinetics of the interactions between counterions gives access to the morphological tuning of the self-assembly of these new helical bioarchitectures. Supporting Information Available: Additional experimental information as described in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

Langmuir 2009, 25(15), 8489–8496