Between Peptides and Bile Acids: Self-Assembly of Phenylalanine

Article pubs.acs.org/JPCB

Between Peptides and Bile Acids: Self-Assembly of Phenylalanine Substituted Cholic Acids Leana Travaglini,† Andrea D’Annibale,† Maria Chiara di Gregorio,† Karin Schillén,‡ Ulf Olsson,‡ Simona Sennato,§ Nicolae V. Pavel,† and Luciano Galantini*,† †

Department of Chemistry, “Sapienza” University of Rome, P.le Aldo Moro 5, 00185 Rome, Italy Division of Physical Chemistry, Department of Chemistry, Center for Chemistry and Chemical Engineering, Lund University, SE-221 00 Lund, Sweden § Department of Physics and CNISM, “Sapienza” University of Rome, P.le Aldo Moro 5, 00185 Rome, Italy ‡

S Supporting Information *

ABSTRACT: Biocompatible molecules that undergo self-assembly are of high importance in biological and medical applications of nanoscience. Peptides and bile acids are among the most investigated due to their ability to self-organize into many different, often stimuli-sensitive, supramolecular structures. With the aim of preparing molecules mixing the aggregation properties of bile acid and amino acid-based molecules, we report on the synthesis and self-association behavior of two diastereomers obtained by substituting a hydroxyl group of cholic acid with a L-phenylalanine residue. The obtained molecules are amphoteric, and we demonstrate that they show a pHdependent self-assembly. Both molecules aggregate in globular micelles at high pH, whereas they form tubular superstructures under acid conditions. Unusual narrow nanotubes with outer and inner cross-section diameters of about 6 and 3 nm are formed by the derivatives. The diasteroisomer with α orientation of the substituent forms in addition a wider tubule (17 nm cross-section diameter). The ability to pack in supramolecular tubules is explained in terms of a wedge-shaped bola-form structure of the derivatives. Parallel or antiparallel faceto-face dimers are hypothesized as fundamental building blocks for the formation of the narrow and wide nanotubes, respectively.

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INTRODUCTION The ability of some biocompatible molecules to self-assemble allows them to be used as fabrication tools in the production of many nanomaterials in a bottom-up approach.1 Among them, peptides are particularly studied, as they can be designed to provide many different self-organization behaviors by properly selecting the amino acid sequence and their chirality.2−4 Studying the aggregation of peptides allows moreover the rationalization of interactions between proteins, which are particularly important in the investigation of pathogenesis of some protein conformational diseases.5−7 Dipeptides such as phenylalanine−phenylalanine constitute the simplest peptide building block. They often self-assemble in nanotubes and have shown applicative interest in preparation of nanowires.4,8,9 More complex molecules are constituted by Lego-peptides, presenting two distinct surfaces being either hydrophobic or hydrophilic.10 The self-assembling process in water is determined by interactions between the hydrophobic sides and defined patterns of ionic bonds involving charged amino acid residues regularly arranged on the hydrophilic sides. The peptides can aggregate into nanofibers, which develop into hydrogels that are useful as a scaffold for tissue engineering. Another example is represented by surfactant-like peptides having a hydrophilic head with charged amino acids and a hydrophobic tail with hydrophobic amino acids.11 The peptide © 2013 American Chemical Society

monomers undergo self-assembly in water to form nanotubes and vesicles, which can be used as targeting delivery systems. Cyclic peptides with an even number of alternating D and L amino acids may also be prepared. They have been reported to form the narrowest peptide nanotube (inner diameters of 1.0− 1.5 nm) by stacking.12,13 The stacking occurs through intermolecular hydrogen bonding, and the end product is cylindrical structures with the amino acid side chains of the peptide defining the properties of the outer surface of the tube and the peptide backbone determining the properties of the inner surface. They are often observed in crystals, but some of them also occur in lipid bilayers, providing transmembrane channels.13 Another class of self-associating biological molecules is represented by bile acids, such as cholic acid (HC) (Figure 1). These molecules are steroid amphiphiles having a convex hydrophobic face and a concave more hydrophilic one, containing hydroxyl groups in specific positions together with a side-chain that ends with a carboxylic head. Because of their rigid structure and their unusual distribution of hydrophobic and hydrophilic regions, these molecules are able to form Received: May 30, 2013 Revised: July 10, 2013 Published: July 11, 2013 9248

dx.doi.org/10.1021/jp405342v | J. Phys. Chem. B 2013, 117, 9248−9257

The Journal of Physical Chemistry B

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to aggregate under the effect of forces of limited directionality, e.g., hydrophobic interactions, and the more geometrically constrained ones, such as π−π stacking and hydrogen bonds, involving both the bile acid and the amino acid moieties. Their self-assembly is therefore complicated and not explainable on the basis of the packing geometric rules of common surfactants or more conventional peptide-based amphiphiles, which are obtained by linking a nonpolar chain to a hydrophilic peptide.43 Due to their amphoteric nature, the self-association process of the two derivatives is sensitively influenced by pH. A previous study was reported on the self-assembly of β-L-PheC under acidic conditions.42 Here, the study on this compound was completed by extending the analysis to high pH values. In parallel, self-association of α-L-PheC has been characterized, both at high and low pH, in order to highlight the effect of C3 configuration (different orientation of the substituting residue). The presented study was performed by means of circular dichroism (CD), dynamic light scattering (DLS), small-angle X-ray scattering (SAXS), UV absorption, and surface tension measurements, in combination with atomic force and cryogenic transmission electron microscopy (AFM and cryo-TEM).

Figure 1. Molecular structures of β- and α-L-PheC and its precursor HC. The substitution site (carbon C3) is shown on the HC molecule.

aggregates with specific supramolecular arrangement and to carry out biological functions.14 Depending on the conditions and type of bile acid, systems of globular15−17 or rod-like18,19 aggregates as well as gel fibrils and tubules20−23 can be obtained. Moreover, it has been recently shown that, by relatively small modifications of the molecular structure, new derivatives can be prepared that bear additional and sometimes uncommon self-assembly properties. For example, cationic hydrogelators can be obtained by changing the carboxylic group into an ammonium group.24−27 Furthermore, derivatives that self-assemble into tubular structures with different sizes and that sometimes show thermo-responsive aggregation28−31 can be prepared by specific substitutions of one hydroxyl group on the rigid four-ring system with a hydrophobic aromatic residue. The mixtures of anionic and cationic forms of some of these aromatic-substituted derivatives self-assemble into tubules whose charge can be tuned by controlling the mixture stoichiometry.32 Lamellar structures are formed when adamantyl residues are used as substituents,33 whereas vesicles are provided by self-assembly of some Gemini derivatives.34 Surfactant features as well as ion recognition are exploited in several derivatives for biomedical applications as antimicrobial agents,35,36 protein or drug nanoparticle stabilizers,37,38 drug carriers,39 and anion receptors.40,41 With this knowledge, this work was aimed at preparing new molecules that combine bile acid and amino acid blocks and merge bile acids and peptide aggregation features. To this purpose, two new diastereomeric derivatives were synthesized by substituting the OH group on C3 of the cholic acid (HC) with a L-Phe residue, thus providing 3β-(2′-(S)-amino-3′phenylpropanamido)-7α,12α-dihydroxy-5β-cholan-24-oic acid (β-L-PheC)42 and 3α-(2′-(S)-amino-3′-phenylpropanamido)7α,12α-dihydroxy-5β-cholan-24-oic acid (α-L-PheC) in Figure 1. The amino acid L-Phe was chosen, since it is well established that it is crucial in the self-assembly of peptides due to its hydrophobicity and/or ability to form π−π staking.8,9 Despite being attached to a large steroidic skeleton, L-Phe is therefore expected to sensitively bias the aggregation of the two derivatives. Unlike the amino acid-conjugated bile acids available in nature, α- and β-L-PheC present a free amino group, while keeping their carboxylic group unmodified, and they are therefore amphoteric. Indeed, the derivatives present a complex distribution of polar and apolar moieties and they are expected

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MATERIALS AND METHODS Synthesis of the Derivatives. The synthesis of β-L-PheC was previously reported.42 The novel derivative, denoted α-LPheC (1), was prepared by using a similar synthetic route (Scheme 1).42 Scheme 1. Synthesis of α-L-PheC

The 3α-aminoderivative (2) of cholic acid used as starting material was obtained by the procedures previously described in the literature.44,45 All the reagents were purchased from Sigma-Aldrich and used without further purification. Dry solvents, namely, dichloromethane, tetrahydrofuran (THF), and methanol, were distilled following standard procedures before use. Reactions and chromatographic separations were monitored by thin layer chromatography (TLC) on a 0.25 mm silica gel plate (Merck Kieselgel 60 F254). Phosphomolybdic acid 12% solutions in EtOH, I2 vapor, and UV light (254 nm) were used as revealing agents. Column chromatography was carried out on silica-gel (Merck Kieselgel 60, 70−230 mesh, 0.063−0.20 mm). 1H and 13 C NMR spectra were recorded on a Varian XL 300 Mercury (300 MHz) spectrometer using 5 mm tubes and chloroform-d (CDCl3), methanol-d4 (CD3OD), and dimethyl sulfoxide-d6 ((CD3)2SO) as internal standards. IR spectra of products (CHCl3 solution, 10 mg/mL) were recorded with a Shimadzu IR 470 spectrophotometer. High resolution ESI mass spectra were carried out on a Q-TOF Micro spectrometer operating in a positive ion mode. Melting points were determined using a 9249



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solid was collected by vacuum filtration and then dried in a desiccator for 24 h. This procedure gave the pure product 1 (360 mg, y = 65%). m.p. 180−183°; [α]D 4.03 deg cm3 g−1 dm−1 (c = 1.46 in MeOH). 1H NMR (300 MHz, CD3OD, δ): 7.26 (m, 5H, aromatic); 3.99 (bs, 1H, CH-12); 3.80 (bs, 2H, CH-7, Phe CH); 3.47 (m, 1H, CH-3); 3.05 (m, 2H, Phe CH2); 1.00−2.30 (m, 24 H, CH2 and CH of steroid skeleton and side chain); 0.91 (d, 3H, J = 6.6 Hz, CH3-21); 0.82 (s, 3H, CH319); 0.61 (s, 3H, CH3-18). 13C NMR (300 MHz, CD3OD, δ) 180.9, 170.7, 136.8, 130.6, 129.8, 128.5, 73.9, 68.9, 56.5, 51.4, 47.5, 43.6, 43.0, 41.0, 40.1, 37.2, 37.1, 37.0, 35.9, 35.7, 34.3, 33.2, 29.6, 28.8, 28.1, 27.9, 24.2, 23.3, 17.7, 13.0. HRMS (ESITOF): calcd for [C33H50N2O5 + H]+ 555.3792; found 555.3797. Sample Preparation. Solubility tests showed that the new derivatives α-L-PheC and β-L-PheC are not soluble in water (pH 5.9). The aggregation was studied in basic (pH 10.0) and acidic (pH 1.1) conditions, where the molecules are negatively and positively charged, respectively. Titrated solutions of HCl and a 6.0 × 10−2 M carbonate/bicarbonate buffer, prepared by mixing equimolar amounts of sodium carbonate (3.0 × 10−2 M) and bicarbonate (3.0 × 10−2 M), were used as solvents in acidic and basic conditions, respectively. All the compounds were from Carlo Erba Reagents. Surface Tension Mesurements. The surface tension γ as a function of concentration was measured by the ring detachment method on solutions of α- and β-L-PheC and HC at high pH. Measurements were performed with a computerized Lauda instrument maintaining the temperature at 25.0 ± 0.1 °C by a thermostatic apparatus. The cac values were inferred from the break points in the γ vs logarithm(surfactant concentration) curves. Cryo-TEM Measurements. The vitrification of the cryoTEM sample was carried out using a controlled environment vitrification system,46 keeping the relative humidity close to saturation at around 26 °C. A thin film of the gel on a lacey carbon-coated copper grid was rapidly vitrified by plunging into liquid ethane (−180 °C) and stored in liquid nitrogen before the examination. The micrographs were recorded using a Philips CM120 Bio TWIN electron microscope equipped with a Gatan MSC791 cooled-CCD camera detection system, operating at 120 kV, under low electron dose conditions. AFM Measurements. AFM experiments were carried out with a DIMENSION ICON controlled by a Nanoscope V Controller (Bruker AXS, Germany) equipped with a closed loop scanner. The images were obtained in AFM intermittent contact (tapping) mode in air. High resolution rotated-tapping mode etched silicon probes (RTESPs) with a nominal tip radius of 8 nm and resonant frequency around 300 kHz were used. The sample was prepared by smearing the gel through a gentle rotation by spin coating at around 600 rpm for several minutes, until the surface appeared dried. No rinsing was employed in order to avoid modification of network structure. SAXS Measurements. SAXS measurements were performed at the MAX II SAXS beamline I911-4 at MAXIV Laboratory in Lund, Sweden.47,48 Calibration measurements were carried out using a LaB6 sample. The solutions were injected into thermostatted quartz capillary sample holders and equilibrated for at least 20 min before measurement. The scattered intensity was recorded at wavelength λ = 0.91 Å, on a 165 mm diameter MarCCD detector. The two-dimensional (2D) SAXS patterns were processed using the Fit2D software.49 For the micellar solutions at high pH, SAXS

Mettler FP 80 apparatus, interfaced with a Microstar IV microscope. Synthesis of Compound 3. N,N′-diisopropylcarbodiimide (DIC) (0.11 mL, 0.7 mmol) was added to an ice-bath cooled solution of 2 (421 mg, 1.0 mmol), N-tert-butoxycarbonyl-(S)phenylalanine (318 mg, 1.2 mmol), and 1-hydroxybenzotriazole (HOBt) (121 mg, 0.9 mmol) in 10 mL of dry THF under an inert atmosphere. The reaction mixture was kept under stirring at 0 °C for 30 min and then at r.t. for 48 h. After reaction completion, the white suspension was filtered, and the filtrate concentrated in vacuo. The resulting oily residue was dissolved in ethyl acetate (20 mL), and the organic layer washed with 2N citric acid (2 × 20 mL), with saturated NaHCO3 (2 × 20 mL) and with brine (2 × 20 mL). After drying over Na2SO4, the solvent was removed under reduced pressure, affording a crude residue that was purified by silica-gel column (EtOAcpetroleum ether 3:7), giving methyl 3α-(2′-(S)-(tertbutoxycarbonyl)amino-3′-phenylpropanamido)-7α,12α-dihydroxy-5β-cholan-24-oate (3) (354 mg, y = 53%). mp 89−91 °C [a]D 31.92 deg cm3 g−1 dm−1 (c = 0.99 in MeOH). 1H NMR (300 MHz, CDCl3, δ): 7.19 (m, 6H, aromatic and −NHCO−), 6.10 (bs, 1H, −NHCOO−), 5.12 (bs, 1H, Phe CH), 4.23 (m, 1H, CH-3), 3.94 (m, 1H, CH-12), 3.81 (m, 1H, CH-7), 3.64 (s, 3H, OCH3), 2.98 (m, 2H, Phe CH2), 1.35 (s, 9H, t-Bu); 1.00− 2.50 (m, 26H, CH2 and CH of steroid skeleton and side chain, 2 OH), 0.95 (d, J = 5.0 Hz, 3H, CH3-21), 0.86 (s, 3H, CH3-19), 0.65 (s, 3H, CH3-18). 13C NMR (300 MHz, CDCl3, δ): 174.7, 170.0, 155.3, 136.7, 129.3, 128.5, 126.7, 80.0, 72.7, 68.0, 55.7, 51.4, 49.5, 46.9, 46.4, 41.9, 41.7, 39.4, 39.0, 36.4, 35.7, 35.0, 34.6, 34.5, 30.9, 30.8, 28.2, 27.5, 27.3, 26.7, 23.4, 23.1, 22.7, 17.3, 12.4. IR (CHCl3): 3440, 3020, 1713, 1669, 1530, 1190 cm−1. HRMS (ESI, m/z): calcd for [C39H60N2O7 + Na]+ 691.4293; found 691.4299. Synthesis of Compound 4. Trifluoroacetic acid (1 mL) was added dropwise at 0 °C to a stirred solution of compound 3 (668 mg, 1.0 mmol) in dry CH2Cl2 (19 mL). The mixture was stirred at r.t. for 4 h and then washed with saturated NaHCO3 (3 × 25 mL) water and brine (2 × 20 mL). The organic layer was dried over Na2SO4 and concentrated in vacuo. Purification by silica-gel column chromatography (eluting first with ethyl acetate and then with MeOH−Et3N 99.5:0.5) afforded the methyl 3α-(2′-(S)-amino-3′-phenylpropanamido)7α,12α-dihydroxy-5β-cholan-24-oate (4) (506 mg, y = 89%) as white solid. mp 120−123 °C; [α]D 32.30 deg cm3 g−1 dm−1 (c = 0.43 in MeOH). 1H NMR (300 MHz, CDCl3, δ): 7.26 (m, 6H, aromatic, −NHCO−), 3.97 (m, 1H, CH-12) overlapped with 4.03 (m, 1H, Phe CH), 3.84 (bs, 1H, CH-7), 3.64 (s, 3H, OCH3), 3.54 (m, 1H, CH-3), 3.19 (m, 2H, Phe CH2), 1.10− 2.70 (m, 28H, CH2 and CH of steroid skeleton and side chain, 2 OH, NH2), 0.96 (d, 3H, J = 6.0 Hz, CH3-21), 0.88 (s, 3H, CH3-19), 0.67 (s, 3H, CH3-18). 13C NMR (300 MHz, CDCl3, δ): 174.7, 172.7, 137.7, 129.3, 128.6, 126.7, 72.8, 68.2, 56.3, 51.4, 49.1, 47.1, 46.4, 41.9, 41.6, 40.9, 39.4, 36.4, 35.7, 35.1, 34.6, 34.4, 30.9, 30.8, 28.2, 27.5, 27.4, 26.6, 23.1, 22.6, 17.3, 12.5. IR (CHCl3): 3445, 3015, 1729, 1655, 1040 cm−1. HRMS (ESI, m/z): calcd for [C34H52N2O5 + H]+ 569.3949; found 569.3944. Synthesis of Compound 1. Compound 4 (568 mg, 1 mmol) was dissolved in MeOH (5 mL), and 2N LiOH solution (5 mL) was added. The mixture was stirred for 24 h at r.t. The mixture was concentrated to half volume under reduced pressure, and 1N HCl was added dropwise until the precipitation of product was complete (pH 6.7). The white 9250



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μm were used. The sample temperature was kept constant within 0.5 °C by a circulating water bath. The time-dependent light scattering correlation function was analyzed only at the 90° scattering angle through cumulant expansion. The apparent hydrodynamic diameters were calculated by the Stokes− Einstein relationship. Data did not show any relevant dependence on the exchanged wave vector in the range 30− 150° under our experimental conditions.

measurements were carried out in a thermostatically controlled (25.0 ± 0.1 °C) quartz capillary of 1 mm by using a Kratky Compact camera (Anton Paar), containing a slit collimation system, equipped with a NaI scintillation counter. Ni-filtered Cu Kα radiation (λ = 1.54 Å) was used. In all cases, scattering curves were recorded within the range 0.1 nm−1 < k < 4.0 nm−1 (k = 4π sin(θ)/λ, where 2θ is the scattering), and were corrected for solvent and capillary contributions. The indirect Fourier transform method developed in the ITP program was used for interpreting the spectra of the micellar solutions. The scattered intensity I(k) can be related to the pair distribution function p(r) of the single scattering particle. The p(r) function is strongly dependent on the shape and size of the scattering particles and vanishes at the maximum particle size. Furthermore, it permits the determination of the electronic radius of gyration Rg. Moreover, in the case of worm-like micelles, the pair distribution function pc(r) and the gyration radius Rc of the cross section can be estimated.50 The SAXS spectra of the gels were analyzed using the expression of the intensity of a hollow cylinder, obtained starting from the general equation of the form factor for a cylinder with shells as51,8−10

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RESULTS AND DISCUSSION Basic Conditions. The results of the surface tension measurements on the derivatives and the precursor at pH 10.0 are reported in Figure 2, as a graph of the surface tension as a function of concentration c.

2 ⎡ J (kDo /2) J (kDi /2) ⎤ ⎥ I(k) ∝ ⎢Do 2 1 − Di 2 1 kDo /2 kDi /2 ⎦ ⎣

where Do and Di are the outer and inner diameters of the tube, respectively, and Jn(x) is the n order Bessel function of the first kind. A theoretical equation for the intensity of a collection of N tubules can be estimated by extending the expression proposed by Oster and Riley for bundles of cylinders,52,53 in analogy with the Debye formula, as N

Figure 2. Surface tension γ of α- (full circles) and β-L-PheC (open triangle) and HC (open squares) in sodium carbonate/bicarbonate buffer, as a function of the logarithm of the concentration c (in M) at 25 °C.

N

1 IN (k) ∝ 2 P(k) ∑ ∑ Jo (kdij) N i=1 j=1

where dij is the distance between the centers of the ith and jth tubules. The fits of the experimental curves of Figure 3 were performed assuming

The curves do not show any minima, which indicates the absence of surface-active impurities in the samples, and point out cac values of 5.0, 8.0, and 10.0 mM for α-L-PheC, β-L-PheC, and HC, respectively. DLS measurements performed on β- and α-L-PheC samples at c = 50 mM indicate that micelles are formed with hydrodynamic diameters of 2.7 ± 0.4 and 2.8 ± 0.4 nm, respectively. These values are slightly larger than the one obtained for the HC micelles (2.2 ± 0.4 nm). In agreement with these results, globular shapes and gyration radii values of 1.09 ± 0.03, 1.51 ± 0.02, and 1.00 ± 0.02 nm were inferred from the SAXS pair correlation functions for β-L-PheC, α-LPheC, and HC, respectively (Figure 3). Due to their peculiar molecular structure, the aggregate morphologies of bile salts and derivatives cannot be explained on the basis of the conventional geometric rules of surfactant packing and, even in the simple case of natural bile salts, the definition of aggregate models is still an open question. The self-assembly of the reported derivatives is probably based on hydrophobic forces involving mainly the nonpolar moieties (phenyl group and steroid hydrophobic face) and hydrogen bonds involving amine, amide, and hydroxyl groups. The reported DLS and SAXS results suggest that the presence of LPhe does not change remarkably the molecular packing in the micelles. Slightly larger micelles are in general expected for the derivatives because of their larger molecular sizes as inferred by

K

I (k ) ∝

∑ wN N ⟨IN(k)⟩ N =1

where wN is the weight fraction of the bundle of N tubules. The average in the last equation is considered with respect to a distribution of diameters. In our calculations, a Gaussian distribution with a 10% standard deviation was considered. UV and CD Measurements. CD spectra were recorded on a JASCO model 715 and reported in molar ellipticity [θ]. The UV spectra were reported in molar extinction coefficient ε, after correction for the solvent contribution. The spectra were recorded in the wavelength (λ) range 200−300 nm, by using quartz cuvettes with path lengths ranging from 0.1 to 1 mm, depending on the sample concentration. The spectral resolution was 1 nm. The reported spectra are the results of four scans. DLS Measurements. A Brookhaven instrument constituted by a BI-2030AT digital correlator with 136 channels and a BI200SM goniometer was used. The light source was a Uniphase solid-state laser system model 4601 operating at 532 nm. Dust was eliminated by means of a Brookhaven ultra filtration unit (BIUU1) for flow-through cells, the volume of the flow cell being about 1.0 cm3. Nuclepore filters with a pore size of 0.1 9251



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Figure 4. (a) CD and (b) UV spectra of α- (dashed line) and β-LPheC (full line) at a concentration of 2.0 mM, pH 10.0 and T = 20 °C.

aggregate (Figure S1, Supporting Information) and are not involved in π−π stacking. Acidic Conditions. Under acidic conditions, the carboxylic groups are undissociated and the protonation of the amine groups occurs. With respect to the basic samples, the derivatives present therefore a complete different location of the charge which determines significant differences in the selfassembly properties of the derivatives. Hydrogels are formed by both compounds at the analyzed concentrations of 18.0 and 36.0 mM in the presence of added electrolyte (0.15 M NaCl). The cryo-TEM experiments demonstrated that the network of β-L-PheC gel is constituted by elongated structures that have an inhomogeneous contrast with dark edges, which is typical of tubular morphologies. The tubules have an outer diameter of about 6 nm and are sometimes intertwined (Figure 5a). The images confirm the results of the previous study.42 The α-LPheC gel is also formed by nanotubes with a similar cross section (6 nm) (Figure 5b). SAXS was used as complementary technique to characterize the gel structure. The spectra show a similar pattern for both derivatives (Figure 6). After a smooth decreasing part at low k, oscillations characterized by an intensity maximum around 1.2 nm−1 may be observed. The oscillating profile is typical of dispersions of elongated aggregates with a monodisperse cross section, such as those observed by microscopy techniques.20−22,48−51 The SAXS data were analyzed using the form factor of a hollow cylinder. As previously reported, in the case of β-L-PheC, the theoretical scattering curve for a single tube is able to reproduce the data at high k values, but it slightly deviates from the experimental profile in low k region (Figure 6a). This inconsistency can be solved by assuming that the tubes are partly associated in small bundles, sometimes intertwined, in agreement with the cryoTEM experiments; see Figure 5a. A satisfactory fit was achieved for tubular structures with outer and inner diameters of 6.2 ± 0.1 and 2.8 ± 0.1 nm and by assuming the presence of bundles formed by couples or groups of three tubes triangularly packed, with a spacing of 7.2 nm in a weight fraction of 0.10 and 0.08, respectively (Figure 6a). The value of the outer diameter is in good agreement with the one inferred by cryo-TEM. At high k

Figure 3. Experimental (dots) and smeared calculated (full line) scattering intensities (top) and corresponding pair correlation functions p(r) (bottom) for samples of (a) α-L-PheC (pink), (b) HC (green), and (c) β-L-PheC (red) 50 mM in buffer solution NaHCO3/Na2CO3 60 mM (pH 10.0), at 25.0 °C. The intensity curves are reported in arbitrary units. For clarity, the patterns b and c are shifted by an arbitrary constant. The residuals are reported in the insets.

both techniques. Some small distortions of the packing induced by the different orientations of the substituting Phe group could be invoked to justify the slightly bigger size of the aggregates of α-L-PheC with respect to those of β-L-PheC in agreement with the SAXS data. UV absorption spectra for solutions below the cac of the derivatives are very similar (Figure 4). The main contribution is given by the L-Phe group for which a weak band around 255 nm and a main peak in the range 200−210 nm, related to the Lb and La transitions of the aromatic group, are expected.54 Some differences are detected, instead, in the corresponding CD profiles, presenting asymmetric bands with maxima around 215 and 200 nm for β- and α-L-PheC, respectively. In general, positive CD bands with a maximum in the range 213−220 nm are reported in the literature for model compounds containing the phenylalanine side chain. This signal is ascribed mainly to the La transition, but it contains also a significant contribution of the peptide n−π transition, and it is very sensitive to the molecule conformation. The differences in the profiles of the two derivatives show that they present a different conformation of the L-Phe group in solution. At concentrations higher than the cac, no differences are observed for both the UV and CD curves, thus showing that the L-Phe residues preserve their conformations in the 9252



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type of nanotubes in both samples, as shown by cryo-TEM images. As a matter of fact, a satisfactory fit of the α-L-PheC experimental data is achieved with a theoretical curve of single nanotubes with outer and inner cross-section diameters of 6.4 ± 0.1 and 3.0 ± 0.1 nm, respectively (Figure 6b). However, no deviations between theoretical and experimental curves indicating the formation of bundles are visible in this case. In some cryo-TEM samples, the coexistence of tubules with a larger cross-section diameter (16−19 nm) with the thinner tubules was also observed (Figure 7).

Figure 5. Cryo-TEM images of (a) β-L-PheC gel (36 mM, pH 1.1, 0.15 M NaCl) and (b) α-L-PheC (36 mM, pH 1.1, 0.15 M NaCl). The bar corresponds to 100 nm.

Figure 7. Cryo-TEM images of α-L-PheC gel (36 mM, pH 1.1, NaCl 0.15 M) showing the presence of tubules with a bigger cross section (16−19 nm). The bar corresponds to 100 nm.

The absence of a typical oscillating profile for tubules with these sizes in the SAXS experimental pattern suggests that the wide tubules are not always formed in the samples with SAXS detectable fractions. By performing a fit where the contribution of the wide tubes is also considered, we observed that the shape of the curve remain not sensitively changed and a satisfactory fit is obtained, up to a weight fraction percentage of wide tubules of 4.0% (Figure S3, Supporting Information). Atomic force microscopy performed on the dried samples (Figure 8) indicates the presence of elongated structures in agreement with cryo-TEM, and also demonstrates that the tubes are quite resistant to loss of water. The comparison of the images of the two derivatives shows immediately that the cross-section diameters of the structures are significantly more polydisperse in the case of α-L-PheC. The height distribution of the β-L-PheC nanotubes calculated from the image in Figure 8a shows a peak around 6.6 nm (Figure S2a, Supporting Information) in accordance to what was previously reported and in agreement with the diameters inferred from cryo-TEM, whereas the α-L-PheC distribution shows a wider main peak around 7−9 nm, which is consistent with the presence of the wider tubes (Figure S2b, Supporting Information, and Figure 7). SAXS has also been recorded on low pH samples at high temperature (55 °C) where the gel is broken and low viscosity solutions are formed (Figure 9). The results show that in this case micelles are present, with different shapes for the two derivatives. Here, the SAXS data collected on the β-L-PheC sample is consistent with globular micelles with gyration radius and maximum dimension of 1.0 ± 0.1 and 2.7 ± 0.1 nm, respectively.42 Conversely, for α-L-PheC, the extracted p(r) shows the typical asymmetric profile of cylindrical scattering particles, suggesting that the surfactant is organized in wormlike micelles (Figure 9). From the calculated pair correlation function pc(r) of the micelles, we extract that the micellar cross section has probably an elliptical shape and/or presents an inhomogeneous electron

Figure 6. Experimental SAXS spectra of (a) 36 mM β-L-PheC gel (white circles) together with best fits for single tubules (green full line) and mixtures of single tubules and small bundles (red dashed line), (b) 36 mM α-L-PheC gel (white circles) and the best fit, by assuming the presence of single tubes (blue line).

values, the SAXS experimental curves of α-L-PheC and β-LPheC gels are very similar, suggesting the presence of the same 9253



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determined from the ITP analysis. A very similar Rc value (1.05 ± 0.03 nm) was inferred by the best linear fit slope of ln I(k) vs k2, according to the Guiner plot for rodlike scatterers ln(Ik) ∝ −(Rc2k2/2) (Figure S4, Supporting Information). Broad CD signals are provided by the two derivatives at the wavelength of the main absorption band, presenting maxima around 218 and 200 nm for β- (Figure 10) and α-L-PheC

Figure 8. AFM images of (a) β-L-PheC and (b) α-L-PheC gels. The bars are 500 nm.

Figure 10. CD (upper panel) and UV (lower panel) spectra of β-LPheC at a concentration of 18.0 mM and pH 1.1 recorded at different temperature values. The arrows indicate the evolution of the profiles by increasing the temperature.

(Figure 11), respectively. By increasing the temperature, the breaking of the gels is determined and an evolution of the CD curves is observed. For α-L-PheC, a decrease of the signal occurs around 200 nm; conversely, an increase around 218 nm takes place for β-L-PheC. For the latter, an increase of the UV absorption main band is observed, too. These observations suggest that a chiral arrangement of Phe residues, different for

Figure 9. Experimental (dots) and calculated (full line) SAXS intensity (upper panel) and pair correlation function P(r) (lower panel) of the worm-like micelles of the α-L-PheC sample at a concentration of 36.0 mM, pH 1.1 and T = 55 °C. The residuals and the pair correlation function pc(r) of the micelle cross section are reported in the insets.

Figure 11. CD spectra of α-L-PheC at a concentration of 36.0 mM and pH 1.1 recorded at different temperatures. The arrow indicates the evolution of the profiles by increasing the temperature. In the lower panel, the UV curves at the two limit temperatures of 20 and 50 °C are reported.

density distribution, and has a maximum distance of 3.6 nm. A cross-section gyration radius of Rc of 1.11 ± 0.02 nm was 9254


The Journal of Physical Chemistry B the two derivatives, is present in the nanotubes and changes when the nanotubes break into micelles. For both the derivatives, the UV intensity of the gels is remarkably lower than for the basic micellar solution, meaning that π−π stacking stabilizes the gel nanotubes. In the case of β-L-PheC, an increase of the UV absorption indicates that the π−π stacking breaks as the system changes from tube to micelle (Figure 10). On the contrary, in the case of α-L-PheC, no sensitive variation of the UV intensity is observed. This indicates that π−π stacking is preserved in the worm-like micelles (Figure 11). In our previous study of the self-assembly of the β-L-PheC derivate, it has been hypothesized that the formation of the narrow nanotubes is due to the peculiar bola-form structure of these molecules.42 This structure is related to the presence of two main polar domains (ammonium ion and carboxylic groups) positioned in opposite extremes of the molecule. The bola-form surfactant is strongly asymmetric, since the ammonium ion bears a charge and is situated close to a bulky phenyl group, whereas the carboxylic group is neutral and linked to an alkyl chain. As a consequence, the whole molecule has a wedge shape morphology which allows it to pack in tubular monolayers.13 By applying a more detailed model of the supramolecular organization in the narrow nanotubes, we suggest that the tubules may be constituted by wedge-shaped parallel face-toface dimers held together by back-to-back interactions (Figure 12a).

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CONCLUSIONS

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ASSOCIATED CONTENT

Article

We have investigated the pH-dependent self-assembly behavior of the amino acid-substituted bile acids α- and β-L-PheC in aqueous solutions. At high pH, both derivatives form globular micellar aggregates similar to those of the precursor cholic acid, whereas, under acidic conditions, they self-assemble into tubular structures that form hydrogels at higher concentrations. This suggests that an introduction of a L-Phe residue in place of the OH group in C3 of cholic acid provides diastereisomers that display a rich spectrum of pH-dependent self-assembled structures. Both derivatives form very narrow nanotubes with outer and inner cross-section diameters of about 6 and 3 nm, respectively, as observed by cryo-TEM and AFM and confirmed by SAXS measurements. The diasteroisomer with α orientation of the substituent also forms wider tubules (17 nm cross-section diameter) that coexist with the narrow tubules, although to a lesser extent. The wedge-shaped bolaform structure of the derivatives and their ability to pack in parallel or antiparallel face-to-face dimers can be invoked to explain the formation of the narrow and wide nanotubes, respectively. The supramolecular organic tubules formed by amphiphiles have generally diameters between 10 and 1000 nm.55 The narrow tubules formed by α- and β-L-PheC studied in this work represent an exception among the surfactant tubules and show that amino acid-substituted bile acids can reach very low values of cross-section diameters. In general, tubular nanoaggregates have a wide applicative interest in nanotechnologies.56−61 Long and narrow nanotubes such as those formed by the reported derivatives can therefore open up for new application areas based on these structures. As a matter of fact, narrow nanotubes are already particularly suitable for the encapsulation of arrays of compounds, such as fullerenes62,63 or the preparation of nanosized wires, which are used in tissue engineering64 or optoelectronics.65 The biological nature of the phenyalaninesubstituted cholic acids, as those studied here, makes these compounds interesting for the preparation of biomaterials. Our results also point out an additional and useful advantage, namely, that, by a simple peptide substitution on a bile acid molecule, we are able to combine the versatile self-assembly properties which are already provided by systems of pure peptides or bile acids, separately.

Figure 12. Wedge-shaped structure of the molecule and aggregation model for the narrow (a) and wide (b) tubes of α-L-PheC. The different colors in the molecule represent H (white), C (cyan), N (blue), and O (red).

S Supporting Information *

One figure reporting the distribution of the heights of AFM images and one figure reporting a Guiner plot for the SAXS of worm-like micelles. This material is available free of charge via the Internet at http://pubs.acs.org.

The charged ammonium and carboxylic groups are expected to lay on the outer and inner surfaces of the tube wall, respectively. The wall thickness, determined by SAXS (1.7 nm), is similar to the head-to-head molecule length (∼2.0 nm), supporting a monolayer packing in the tube wall. In the case of the α-L-PheC wider tubes, an antiparallel face-to-face dimer with a slightly truncate wedge shape could be the building block (Figure 12b). In both cases, π−π stacking could stabilize the dimers or take place between dimers along the tubules, justifying the CD and UV behaviors of Figures 10 and 11. The absence of wider tubules in the β-L-PheC gel suggests that this derivative is unable to form stable antiparallel face-to-face dimers. Reasonably, the axial orientation of the substituent on C3 does not allow the aromatic moiety to be close enough to form π−π stacking in the dimer.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest. 9255



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ACKNOWLEDGMENTS We are grateful to Gunnel Karlsson for the cryo-TEM experiments. Beamline manager Tomás Plivelic is acknowledged for assistance at the MAX II SAXS beamline I911-4 at MAXIV Laboratory. We also thank Università Sapienza di Roma (project C26A098K4X) and the Swedish Research Council for financial support.

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ABBREVIATIONS HC, cholic acid; β-L-PheC, 3β-(2′-(S)-amino-3′-phenylpropanamido)-7α,12α-dihydroxy-5β-cholan-24-oic; α-L-PheC, 3α(2′-(S)-amino-3′-phenylpropanamido)-7α,12α-dihydroxy-5βcholan-24-oic acid; CD, circular dichroism; SAXS, small-angle X-ray scattering; Cryo-TEM, cryogenic transmission electron microscopy; AFM, atomic force microscopy; DLS, dynamic light scattering

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