Thixotropic Hydrogelators Based on a Cyclo(dipeptide) Derivative

Oct 17, 2013 - ABSTRACT: Thixotropic hydrogelators have great potential in biomedical and biotechnological applications. In this study, we report new ...
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Thixotropic Hydrogelators Based on a Cyclo(dipeptide) Derivative Hiroko Hoshizawa,*,† Yuta Minemura,‡ Katsunori Yoshikawa,‡ Masahiro Suzuki,† and Kenji Hanabusa† †

Interdisciplinary Graduate School of Science and Technology and ‡Faculty of Textile Science and Technology, Shinshu University, 3-15-1 Tokida, Ueda-shi, Nagano 386-8567, Japan S Supporting Information *

ABSTRACT: Thixotropic hydrogelators have great potential in biomedical and biotechnological applications. In this study, we report new hydrogelators and their behavior during gel− sol−gel transitions. In particular, cyclo(L-O-hydroxyhexylaspartyl-L-phenylalanyl), which was synthesized with 1,6hexanediol, formed a thermally/isothermally reversible physical gel with several solvents, including pure water, saline, alcohols, as well as 1.0 M aqueous NaCl, KCl, CaCl2, and MgCl2 solutions. TEM observations showed self-assembled fibers with diameters of 10−100 nm. FT-IR results revealed that the gels were mainly formed by hydrogen bonding and van der Waals forces; thixotropic behavior resulted from the disruption of the van der Waals forces between the alkylene chains under shearing. These results were repeatedly and reproducibly observed at room temperature, even when measurements were repeated many times.

1. INTRODUCTION Gelation is caused by fibrous self-assembly of low-molecularweight gelators (LMWGs) driven by noncovalent intermolecular forces such as hydrogen bonding, van der Waals forces, π−π stacking, and electrostatic interactions. A gel results from solvent entrapment into the three-dimensional network formed by entangled fibers. Recently, numerous studies have reported extensively on low molecular-weight compounds that form physical gels. Such low molecular-weight starting materials are known as gelators and may be amino acids, saccharides, or steroids; they include asymmetric carbons in their structures.1−6 We have previously reported that cyclo(dipeptide) derivatives formed from acidic and neutral amino acids have strong gelation abilities. For example, cyclo(L-aspartyl-L-phenylalanyl), a branched alkyl ester of a cyclo(dipeptide) formed from the acidic amino acid L-aspartic acid and the neutral amino acid L-phenylalanine, has the ability to gelatinize organic solvents and oils at low concentrations. We demonstrated that the introduced alkyl chain increased the solubility of the cyclo(dipeptide) derivative in the solvent during the heating process, and that two of the amide groups in the six-membered 2,5-diketopiperazine ring prevent crystallization through rapid formation of intermolecular hydrogen bonding, thus causing gelation.7 However, the above-mentioned alkyl ester is highly hydrophobic and water-insoluble and could not form gels with aqueous solutions. As one approach for converting organogelators into hydrogelators, we proposed the introduction of a hydrophilic site on the terminus of the organogelator structure. In particular, it is known that when a hydrophilic cationic moiety is introduced onto an L-lysine-type organogelator, the resulting compound readily becomes water-soluble during heating and forms a gel upon cooling.8,9 Hydrogelators are © 2013 American Chemical Society

industrially used as rheological additives in food and paint as well as for medical materials, such as contact lenses; they are expected to be commercialized as novel biomaterials in biomineralization, drug delivery systems,10 and cell-growing scaffolds for tissue engineering.11,12 The unique viscoelastic behavior called thixotropy is widely used in industries. Thixotropy refers to an isothermal, reversible sol−gel phase transition, in which a gel-state substance converts to a sol under physical stimuli such as mechanical shearing (for example, shaking or stirring). On standing quiescent over time, the material returns to the gel state. This phenomenon was first described in 1923 by Schalek et al., who discovered that aqueous iron oxide gels could be turned into sols by mere shaking. The sols retransform into gels on standing still.13 Since then, gelling, thickening, and swelling abilities as well as thixotropic properties have been observed in paints and clay suspensions like bentonites and their main constituent montmorillonite. Thixotropic properties have also been observed in synthetic detergents, lubricants, foods, and cosmetics, attracting attention from both academia and industry. Today, thixotropic agents are commercially available as rheology control agents and are used in paints, inks, and cosmetics.14 Academically, thixotropy has been observed in gels prepared by LMWGs based on cholesterol derivatives,15,16 cyclodextrins,17 imidazole derivatives,18 porphyrin derivatives,19 naphthalenediimide,20 dianthracene,21 simple alkyl amides,22 and urea.23−25 However, thixotropy has only been observed with limited solvents because low-molecular-weight compounds Received: June 20, 2013 Revised: September 27, 2013 Published: October 17, 2013 14666

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Elemental analysis: calcd (%) for C19H26N2O5 (Mw 362.42): C, 62.97; H, 7.23; N, 7.73. Found: C, 62.95; H, 7.31; N, 7.91. Cyclo(L-O-hydroxydodecylasparaginyl-L-phenylalanyl) (3). A mixture of 22.55 g (0.191 mol) of 1,12-dodecanediol, 5.00 g (0.019 mol) of cyclo(L-aspartyl-L-phenylalanyl), 4.65 g (0.029 mol) of DiPC, and 2.55 g (0.021 mol) of DMAP was refluxed in 300 mL of CH2Cl2 for 10 h. After removing CH2Cl2 by evaporation, the waxy solid was dried using an oil pressure pump. To remove 1,12-dodecanediol, the obtained white solid was recrystallized from a mixture of ligroin and toluene (ratio 9:1). After the washing process was repeated 10 times, the crude product was dried. The obtained light yellow product was dissolved in 300 mL of MeOH and filtered to remove insoluble parts. After evaporating, the white product was dissolved in 100 mL of 1propanol. The objective substance was precipitated in excess diisopropyl ether and dried under vacuum. Yield: 1.53 g (18%). FT-IR (KBr): 1741 cm−1 (ν CO, ester). 1H NMR (400 MHz, DMSO-d6): δ = 8.18−7.98 (s, −NH−CO−), 7.30− 7.16 (m, aromatic), 4.31 (t, −OH), 4.21 (q, Phe−CH2−CH−), 4.03 (t, NH−CH−CH2−COO), 3.12−2.88 (q, Phe−CH2), 1.98−1.53 (q, CH2−COO−), 1.52−1.37 (m, −(CH2)2−CH2−CH2−(CH2)2−). Elemental analysis: calcd (%) for C25H38N2O5 (Mw 446.58): C, 67.24; H, 8.58; N, 6.27. Found: C, 68.46; H, 8.77; N, 6.53. 2.2. Characterization of Gelators. 1H NMR spectra were obtained using a Bruker AVANCE at 400 MHz for CDCl3 or DMSO-d6. The FT-IR spectra were obtained by a Jasco FT-IR FS-420 spectrometer using KBr pellets. Elemental analyses were performed with a Perkin-Elmer 240B analyzer. The thermal properties of the compounds were determined with a SHIMADZU DSC-60 differential scanning calorimeter (DSC) calibrated using an indium standard. Wide-angle X-ray diffraction (WAXD) profiles were obtained with a Rigaku Rotaflex RU-200B X-ray generator equipped with a Rigaku PMG-GA goniometer. The X-ray source was Ni-filtered Cu Kα radiation (0.15418 nm) generated at 40 kV and 150 mA. Compounds 1−3 were synthesized by coupling cyclo(L-aspartyl-Lphenylalanyl) with DiPC and DMAP as starting materials for the various diols (see Scheme 1). The compounds were identified by FT-

exhibit crystallization rather than gelation. In addition, these gelators mainly exhibit thixotropy and gelation with lipophilic solvents; they are weakly soluble or do not form gels in polar solvents such as pure water and alcohols. While thixotropy is often observed in natural aqueous compounds, it is extremely difficult to produce thixotropy in newly synthesized hydrogelators. The thixotropic hydrogels that have been previously reported are high molecular-weight compounds such as PEGylated indomethacin mixed with α-cyclodextrin,25 1,4bis[4-(chloromethyl)benzamide]benzene copolymer,26 poly(ethylene oxide)-b-poly(ε-captrolactone) diblock copolymer,27 guar gum,28,29 alginate derivative,30,31 and hyaluronic acid.32 In contrast, there are only a few low molecular-weight hydrogels; they include aluminum hydroxide,33 L-cysteine and silver nitrate mixtures,34 and metal alkoxide solutions.35 In addition, thixotropic behavior and its mechanism are not sufficiently understood in supramolecular systems. In this study, we investigated the gelation ability of hydrophilic cyclo(L-aspartyl-L-phenylalanyl) derivatives, a lowmolecular-weight cyclo(dipeptide) derivative, in pure water and in aqueous solution by introducing a terminal alkyl chain that contains an OH group. The resulting gel demonstrated both thermal and isothermal (thixotropy) phase transitions. We describe this novel hydrogelator and its properties using the results obtained from gelation tests, FT-IR, electron microscopy, as well as viscoelastic and thixotropic observations.

2. EXPERIMENTAL SECTION 2.1. Materials. Cyclo(L-aspartyl-L-phenylalanyl). The cyclo(dipeptide) derivative cyclo(L-aspartyl-L-phenylalanyl) was synthesized by cyclization of N-(L-α-aspartyl)-L-phenylalanine 1-methyl ester (Aspartame), a commercially available artificial sweetener.7 Cyclo(L-O-hydroxypropylaspartyl-L-phenylalanyl) (1). A mixture of 11.57 g (0.152 mol) of 1,3-propanediol, 4.00 g (0.0152 mol) of cyclo(L-aspartyl-L-phenylalanyl), 2.11 g (0.0167 mol) of DiPC, and 2.04 g (0.0167 mol) of DMAP was refluxed in 300 mL of CHCl3 for 10 h. After removing CHCl3 by evaporation, the waxy solid was dried under vacuum. To remove 1,3-propanediol, the obtained white solid was recrystallized from a mixture of ligroin and toluene (ratio 9:1). After the washing process was repeated 10 times, the crude product was dried. The obtained light yellow product was dissolved in 50 mL of MeOH. The objective substance was precipitated in excess diisopropyl ether and dried under vacuum. Yield: 2.73 g (56%). FT-IR (KBr): 1741 cm−1 (ν CO, ester). 1H NMR (400 MHz, DMSO-d6): δ = 8.19−7.98 (s, −CO−NH−), 7.31− 7.16 (m, aromatic), 4.48 (t, −OH), 3.12−2.88 (q, Phe−CH2), 1.99− 1.51 (q, CH2−COO−), 1.67 (quint, −CH2−CH2−CH2−). Elemental analysis: calcd (%) for C16H20N2O5 (Mw 320.14): C, 59.99; H, 6.29; N, 8.74. Found: C, 59.35; H, 6.51; N, 8.45. Cyclo(L-O-hydroxyhexylasparaginyl-L-phenylalanyl) (2). A mixture of 22.55 g (0.191 mol) of 1,6-hexanediol, 5.00 g (0.019 mol) of cyclo(L-aspartyl-L-phenylalanyl), 4.65 g (0.029 mol) of DiPC, and 2.55 g (0.021 mol) of DMAP was refluxed in 300 mL of CH2Cl2 for 10 h. After removing CH2Cl2 by evaporation, the waxy solid was dried using an oil pressure pump. To remove 1,6-hexanediol, the obtained white solid was recrystallized from a mixture of ligroin and toluene (ratio 9:1). After the washing process was repeated 10 times, the crude product was dried. The obtained light yellow product was dissolved in 300 mL of MeOH and filtered to remove insoluble parts. After evaporating, the white product was dissolved in 100 mL of 1-propanol. The objective substance was precipitated in excess diisopropyl ether and dried under vacuum. Yield: 5.61 g (81%). FT-IR (KBr): 1741 cm−1 (ν CO, ester). 1H NMR (400 MHz, DMSO-d6): δ = 8.17−7.98 (s, −CO−NH−), 7.60− 7.16 (m, aromatic), 4.33 (t, −OH), 4.21 (q, Phe−CH2−CH), 4.03(t, NH−CH−CH2−COO), 3.12−2.88 (q, Phe−CH2), 1.99−1.54 (q, CH2−COO−), 1.53−1.38 (m, −(CH2)2−CH2−CH2−(CH2)2−).

Scheme 1. Preparation of Hydrogelators 1−3

IR, NMR, and elemental analyses. The FT-IR results showed absorption at 1741 cm−1 assigned to the ester (ν CO) and absorption at 3200 cm−1 assigned to the O−H stretching vibrations. An NMR spectrum confirmed the OH proton peak of 4.33 ppm. The elemental analysis also confirmed the target compounds. 2.3. Gelation Tests. The gelation properties for 40 different solvents and pure water were tested. The typical procedure for gelation testing is as follows: a weighed sample is mixed with a solvent in a test tube with a screw cap (14 mm inner diameter) and heated until the solid is dissolved. The resulting organic solvent or aqueous solution is cooled to room temperature (25 °C) for 2 or 4 h, respectively. Gelation is examined visually; when no fluid runs down the walls of the tube upon inversion, the material is considered to be a “gel”. 2.4. Transmission Electron Microscopy (TEM) Observations. The TEM samples were prepared as follows: a toluene solution of the gelator and osmic acid was added dropwise onto a carbon-coated 400mesh copper grid. The grid was then dried under vacuum for 24 h. The dried sample was stored overnight in a sealed bottle. 2.5. Rheological Measurements. The rheological measurements were performed on a Rheologia A300 coaxial-cylinder-rotating rheometer (Elquest Co. Ltd., Japan) with double cylinder geometry. A water bath was equipped for temperature control. The dimensions 14667

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of the rheometer were 18.3 mm, 21.0 mm, and 26.7 mm for the inner diameter (static cylinder), outer diameter (rotational cup), and sheared height, respectively. 2.6. Observations of Thixotropy. The rheological measurements of thixotropic behavior were performed as noted above. The FT-IR spectra of gels were obtained on a Jasco FT-IR FS-420 spectrometer using a spectroscopic cell with a CaF2 window and 0.025 mm spacers operating at a 2 cm−1 resolution with 32 scans. The sol sample was magnetically stirred for 5 min and then immediately inserted into the CaF2 sample holder. After 12 h, the “reformed gel” was used for measurements. Morphological identification was performed by polarized-light optical microscopy (POM) using an Olympus BX51 optical microscope equipped with a thermostat. All POM images were taken at 25 °C. 2.7. FT-IR Spectra. The FT-IR spectra of gels were obtained on the same spectrometer and CaF2 cell, as described in section 2.6. The sol samples were magnetically stirred for 5 min and then immediately inserted into the CaF2 sample holder. After 12 h, the “reformed gel” was used for measurement.

GT = transparent gel, GTL = translucent gel, GO = opaque gel, P = precipitate, I = almost insoluble. bSaline contains 8.6 g L−1 NaCl, 0.3 g L−1 KCl, and 0.33 g L−1 CaCl2. cPBS contains 8.0 g L−1 NaCl, 0.2 g L−1 KCl, 2.9 g L−1 Na2HPO412H2O, and 0.2 g L−1 KH2PO4. The values denote MGC at 25 °C; the unit is g L−1 (gelator/liquid).

because it either dissolved completely in solvents or it formed precipitates upon cooling to room temperature. The chain length of the hydroxypropyl segment of compound 1 is short and low in hydrophobicity (highly hydrophilic), making the compound readily soluble in polar solvents. However, the relatively short hydroxypropyl segment was insufficient to prevent intermolecular hydrogen bonding in the cyclo(dipeptide) segment, resulting in crystallization and precipitation upon cooling. Cyclo(dipeptide) derivatives with bulky segments such as branched alkyls, polypeptides, polycaprolactone, polypropylene glycol, polyethylene glycol, and polydimethylsiloxane chains have been previously reported to form gels that are stable over long periods of time.7,38,39 The introduction of long alkyl chains prevents crystallization caused by the hydrogen-bonded amide groups of the cyclo(dipeptide) derivatives by introducing appropriate intermolecular separations, but it is necessary to consider their solubility in polar solvents (e.g., water). From these results, compound 2 was determined to have the most suitable alkyl chain length and hydrophobicity for hydrogel formation. Table S1 in the Supporting Information displays the results of gelation tests with alcohol/water mixtures. Interestingly, when water was added to 1 or 3 ethanol solution, immediate gelation behavior was observed. Therefore, the gelation test was conducted using aqueous mixtures of methanol or ethanol at various ratios. Compounds 1 and 3 did not form gels in most aqueous solutions, but they demonstrated gelation ability in alcohol/water mixtures. Note that compound 3 has an extremely low MGC value of 5 g L−1 and yet was still able to form a gel. It appears that there is a good balance between hydrophobic and hydrophilic sites in the cyclo(dipeptide) derivative hydrogelator. The gelation test results with organic solvents are shown in Table S2. Compounds 1−3 formed gels in organic solvents such as ethyl acetate, chlorobenzene, nitrobenzene, and isopropyl myristate. 3.2. Electron Microscopic Observations. LMWGs are known to form a three-dimensional network via self-assembly of the molecules. We have previously reported that cyclo(Laspartyl-L-phenylalanyl) derivatives form self-assembled nanofibers with three-dimensional network structures.40 Therefore, to observe the microstructures in the hydrogel, we prepared dried samples for transmission electron microscopy (TEM). Figure 1 shows a TEM image of dried xerogel prepared from compound 2 and water. Compound 2 has a low molecular weight of 362, but the TEM image shows a fibrous structure very similar to that of polymer aggregates. The fiber diameter is

the table denote the minimum gel concentration (MGC) required to gelatinize the solvent at room temperature. Compound 2 demonstrated good gelation ability not only in methanol, ethanol, 1-propanol, water, and phosphate buffered saline used for cell culture but also in NaCl, CaCl2, and 1.0 M aqueous MgCl2. In contrast, compound 1 did not form gels in water, alcohols, or aqueous solutions. Compound 3 demonstrated gelation in some alcohols and aqueous acetic acid solution; this can probably be attributed to the alkyl chain length. Previous reports have described that introducing longer alkyl chains onto amino acids increases the lipophilicity of the compounds, thereby improving their ability to form gels that are stable for long periods of time.37,38 In the above-mentioned case, the hydroxydodecyl segment of compound 3 acts as a hydrophobic site in water, making the compound insoluble in aqueous solutions. In contrast, compound 1 did not form gels

Figure 1. TEM image of hydrogel prepared from compound 2 at 25 g L−1.

3. RESULTS AND DISCUSSION 3.1. Gelation Testing. The gelation abilities of the compounds were evaluated by test tube inversion,36 and the results for compounds 1−3 are shown in Table 1. The values in Table 1. Results of Gelation Tests and Minimum Gel Concentrations (g L−1) at 25 °C liquids

1

2

3

methanol ethanol 1-propanol ethyl acetate water HCl (1.0 M) H3PO4 (1.0 M) CH3COOH (1.0 M) salineb PBSc NaCl (1.0 M) KCl (1.0 M) MgCl2 (1.0 M) CaCl2 (1.0 M)

S S S S P P P P P P P P P P

GT(100) GT(50) GT(50) GO(25) GO(25) GO(20) GTL(10) GO(20) GTL(10) GO(10) GTL(10) GTL(10) GTL(10) GTL(10)

GT(100) GT(50) GT(50) GO(50) I I I GT(10) I I I I I I

a

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approximately 10−100 nm, and some sections display fiber assemblies that are similar to a wool ball. Figure 5 shows the images of the TEM samples obtained from 2 in toluene (see the Supporting Information). Similar fibrous structures and fringe micelle aggregation were clearly observed. This gelation is considered to be a result of supramolecular fiber formation by intermolecular bonding of gelators, which entraps solvent molecules within its three-dimensional network. 3.3. Dynamic Viscoelastic Measurements. Oscillatory testing is an important method for determining the rheology of viscoelastic materials, gels, low-viscous liquids, pastes, elastomeric polymers, and rigid solids. The strain amplitude sweep test, a type of oscillatory testing, can be used to establish the plateau region and is useful in determining properties such as colloidal dispersion. Figure 2 shows the results from an

unique property for its potential application in the biomedical field, such as thin-film formation and injectable gels.43,44 However, thixotropic behavior and its mechanism are not sufficiently understood in hydrogel systems. When determining their gelation ability, we found that all of the gels prepared from 1−3 are thixotropic. Figure 3 shows the isothermal phase

Figure 3. Images of an ethanol/water mixture (20% ethanol/80% water) containing compound 2 (25 g L−1). (A) Gel formed from hot solution upon cooling. (B) Low-viscosity fluid formed by vigorous hand shaking. (C) Gel reformed after standing for 1 h.

transition (thixotropy) image for the gel prepared from hydrogelator 2. The gel (image A) transforms to sol (image B) at room temperature under vigorous shaking. When the sol is left standing at room temperature, it returns to the gel state (image C). This behavior can be observed repeatedly at 25 °C. Thixotropy is a reversible time-dependent property often observed in mixed systems such as colloidal suspensions; in these systems, the particulate network that was destroyed by shearing reforms after standing for some time. To evaluate thixotropy, Figure 4 shows the relationship between G′ and the recovery time of samples prepared from compound 2. Unfortunately, pure water gel is very brittle, and it was partially broken in the rotational cup. Therefore, we took the data of ethanol/water mixture gel rather than those of pure water gel. The plot in panel (A) shows the storage modulus of the gel prepared from the heating−cooling process in the manner of a gelation test; its value was approximately 8000 Pa. Following the G′ measurement in panel (A), a sol state was achieved by rotating the sample for 10 min and then G′ was immediately measured (panel (B)). The gel-to-sol transition resulted in a significant decrease in the G′ value to below 3000 Pa. However, after 1 h of standing, G′ increased to 5000 Pa (Figure 4C). The reformation of the gel after standing still is implied by the higher value of G′ in panel (C) than in panel (B). The value of G′ measured 3 h after the transition to sol is shown in Section D. The G′ value was restored to 8000 Pa, approximately equal to that in panel (A), indicating that the gel has reformed. In addition, if the gel of panel (D) is broken by reshaking, it reforms to the same (D) state. Furthermore, G′ is observed to increase with continuous sweep in each section, possibly owing to a unique phenomenon called “reopexy,” in which vibration induces the reorientation of molecules. Flocculation of molecules in gels is generally a very slow process. However, the vibration of sweep appears to induce rapid reorientation in this measurement. Similar behaviors were observed with most gels prepared from compounds 1−3. In addition, these results were repeatedly and reproducibly observed within the rest time, even when the measurements were repeated many times at 25 °C. 3.5. FT-IR Spectra. Hydrogen bonding and van der Waals interactions are known to act as major driving forces in the selfassembly of gelator molecules. To evaluate thixotropic behavior

Figure 2. Strain dependence of dynamic moduli in an ethanol/water mixture (25% ethanol/75% water) gel at 25 °C.

amplitude sweep test with the angular frequency fixed at 0.1 or 1 Hz. The figure shows the storage modulus (G′) and loss modulus (G″) of the hydrogels prepared from compounds 1−3 and an ethanol/water mixture (25% ethanol/75% water) for the strain amplitude range 0.01−2.5. All samples were prepared in MGC. The values of G′ were greater than those of G″ for all samples under minimum strain at γ = 0.01. However, when the strain was greater than 0.1, the G′ values for compounds 1 and 2 were smaller than the G″ values. This type of linearity at a relatively small strain range and the inversion of G′ and G″ values due to large strain are often observed in colloidal dispersion systems.41,42 As has been reported in detail in the past, because linear viscoelasticity of supramolecular gels is roughly similar to that of colloidal dispersion systems, they display linearity up to a certain strain range but show nonlinearity over larger strain ranges. The intersection of G′ and G″ at γ = 0.5 for compound 3 signifies the transition from gel to sol due to large shear strains. The above results suggest that in the supramolecular gel, the three-dimensional structure is disrupted by small amplitude strains. 3.4. Thixotropy. Many LMWGs are known to exhibit temperature-dependent sol−gel phase transitions.5 However, there are gelators that exhibit both temperature-dependent and isothermal sol−gel phase transitions. Such phase transition behavior due to the destruction of particulate network structure is also observed in consumer products such as cosmetics, paint, ink, coatings, detergents, food, and pharmaceutical products.13 In recent years, there has been a growing interest in this type of 14669

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Figure 4. Time-dependence of the storage modulus. The sample was prepared from gelator 2 in ethanol/water mixture (20% ethanol/80% water) at 20 g L−1. The plot shows the storage modulus of gel prepared by a heating and cooling process (A), sol state prepared by inner-cylinder rotation (B), and reformed gel after standing at rest for 1 h (C) and 2 h (D).

Figure 5. FT-IR spectra of compound 2 in D2O (5 g L−1) and CHCl3 (10 g L−1) at 25 °C.

The FT-IR measurements can also provide information on interalkyl chain packing. The absorption band of the CH2 stretching vibration of compound 2 was observed at 2982 cm−1 in the D2O hydrogel (Figure 5B). However, this band was not observed in the D2O sample liquefied by mechanical shear or in the CHCl3 solution. Interestingly, after standing still for 12 h, when the reformed gel was measured, the absorption band at 2982 cm−1 was once again observed. The CH2 bending (scissoring) vibration near 1470 cm−1 is known to be a useful measure of intermolecular interactions; it is used as a key band in investigating alkyl chain packing in crystalline and gel phases.45,46 Figure 5C shows the IR spectra of the D2O sample from compound 2 and CHCl3 near 1470 cm−1. No prominent peaks were observed in this region for CHCl3. However, the D2O hydrogel and sol shown in green

using infrared spectroscopy, the FT-IR spectra were obtained for D2O hydrogel prepared from compound 2 and a CHCl3 solution (Figure 5). The D2O hydrogel was prepared by the same method as the gelation test (green lines in Figure 5), the sol was prepared by applying mechanical shearing to the gel (red lines in the figure), and the gel was reformed by allowing the sol sample to stand still for 12 h (black lines in the figure). For the CHCl3 solution, the absorption band assigned to the nonhydrogen bond CO stretching vibration was observed at 1681 cm−1. In contrast, the absorption band assigned to hydrogen bond CO stretching at 1646 cm−1 was observed for all samples prepared from D2O (Figure 5A). These results suggest that compound 2 forms hydrogen bonds with solvents (such as water) to form gels in both liquid and gel phases. 14670

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Figure 6. POM images of the gel, sol, and reformed gel prepared from an ethanol/water mixture (20% ethanol/80% water) and compound 1 at a concentration of 50 g L−1.

Figure 7. Schematic of a model for thixotropic behavior in gel−sol−gel transitions.

and red, respectively, contain slight absorption peaks at 1465 cm−1, indicating that there are relatively weak intermolecular interactions among the alkyl chains of compound 2. However, the gel reformed from the D2O sol after 12 h of standing still showed a significant increase in the 1465 cm−1 peak compared to the green and red lines. These results suggest that thixotropic behavior is caused by intermolecular interactions among alkyl chains. 3.6. Polarized-Light Optical Microscopy (POM). To investigate the structural formations and intermolecular interactions, phase transitions in samples prepared from compound 2 and water were observed by TEM. Although differences between gel and reformed gel are not clearly confirmed, fibers in the sheared sol are clearly shorter than those in other samples. Therefore, we acquired the phase transitions of the samples prepared from compound 1 and ethanol/water mixture, and they were monitored using a polarized optical microscope. Figure 6 shows the POM images of the gel, sol, and reformed gel prepared from an ethanol/ water solution and compound 1 at a concentration of 50 g L−1. Small spherulite structures were observed in the gel, as shown in Figure 6a. To demonstrate the aging effect in the spherulite structure, the gel (Figure 6a) was retained in the POM stage at 25 °C before stirring. The small spherulite structures were stable after standing for 24 h. However, the spherulites disappeared when the sol was formed by stirring the gel (Figure 6b); presumably, this occurred because the spherulite

structures were disrupted by mechanical shearing, so the Maltese cross was no longer observed. After 2 h of standing, nonspherulite structures were observed in the void areas that were originally in the sol state (Figure 6c). Because the birefringence that emerged in the process of gel reformation from the sol was not a Maltese cross, it appears that the destroyed spherules formed a new lamella-like, higher-order structure. Similar phenomena were observed by POM for samples prepared with compounds 2 and 3; unfortunately, useful images could not be obtained because of poor image contrast. Figure 7 illustrates a model for thixotropic behavior. When the gelator is heated/cooled in solvents such as pure water, fibrous supramolecules self-assemble via hydrogen bonding and the solvent is trapped in the three-dimensional spherulite, forming a gel (Figure 7(i)). By applying a shear such as by shaking or stirring, the fiber structure unravels and loses viscoelasticity, forming a sol (Figure 7(ii)). However, when the sol was allowed to stand, a three-dimensional lamella-like structure forms by van der Waals forces, resulting in gelation (Figure 7(iii)). Heated hydrogel molecules with high kinetic energy can easily form highly ordered conformation such as spherulite. In contrast, the molecules of sheared sol, which lack the kinetic energy to form spherules, form flocculate lamellalike structures by van der Waals interactions. 14671

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(5) Richard G. Weiss, P. T. Molecular Gels; Dordrecht: Springer, 2006. (6) Dastidar, P. Supramolecular Gelling Agents: Can They Be Designed? Chem. Soc. Rev. 2008, 37, 2699−2715. (7) Hanabusa, K.; Matsumoto, M.; Kimura, M.; Kakehi, A.; Shirai, H. Low Molecular Weight Gelators for Organic Fluids: Gelation Using a Family of Cyclo(dipeptide)s. J. Colloid. Interface. Sci. 2000, 224, 231− 244. (8) Suzuki, M.; Owa, S.; Shirai, H.; Hanabusa, K. Supramolecular Hydrogel Formed by Glucoheptonamide of L -lysine: Simple Preparation and Excellent Hydrogelation Ability. Tetrahedron 2007, 63, 7302−7308. (9) Hanabusa, K.; Nakayama, H.; Kimura, M.; Shirai, H. Easy Preparation and Prominent Gelation of New Gelator Based on LLysine. Chem. Lett. 2000, 29, 1070−1071. (10) Li, J.; Li, X.; Ni, X.; Wang, X.; Li, H.; Leong, K. W. Selfassembled Supramolecular Hydrogels Formed by Biodegradable PEOPHB-PEO Triblock Copolymers and Alpha-cyclodextrin for Controlled Drug Delivery. Biomaterials 2006, 27, 4132−4140. (11) Temenoff, J. S.; Mikos, A. G. Injectable Biodegradable Materials for Orthopedic Tissue Engineering. Biomaterials 2000, 21, 2405−2412. (12) Tan, H.; Chu, C. R.; Payne, K. A.; Marra, K. G. Injectable in Situ Forming Biodegradable Chitosan-hyaluronic Acid Based Hydrogels for Cartilage Tissue Engineering. Biomaterials 2009, 30, 2499−2506. (13) Mewis, J. Thixotropy - A General Review. J. Non-Newtonian Fluid Mech. 1979, 6, 1−20. (14) Barnes, A. Thixotropy-A Review. J. Non-Newtonian Fluid Mech. 1997, 70, 1−33. (15) Xue, M.; Gao, D.; Liu, K.; Peng, J.; Fang, Y. Cholesteryl Derivatives as Phase-selective Gelators at Room Temperature. Tetrahedron 2009, 65, 3369−3377. (16) Huang, X.; Raghavan, S. R.; Terech, P.; Weiss, R. G. Distinct Kinetic Pathways Generate Organogel Networks with Contrasting Fractality and Thixotropic Properties. J. Am. Chem. Soc. 2006, 128, 15341−15352. (17) Araki, J.; Ito, K. Strongly Thixotropic Viscosity Behavior of Dimethylsulfoxide Solution of Polyrotaxane Comprising A-Cyclodextrin and Low Molecular Weight Poly(ethylene glycol). Polymer 2007, 48, 7139−7144. (18) Weng, W.; Jamieson, A. M.; Rowan, S. J. Structural Origin of the Thixotropic Behavior of a Class of Metallosupramolecular Gels. Tetrahedron 2007, 63, 7419−7431. (19) Shirakawa, M.; Fujita, N.; Shinkai, S. A Stable Single Piece of Unimolecularly Pi-stacked Porphyrin Aggregate in a Thixotropic Low Molecular Weight Gel: a One-dimensional Molecular Template for Polydiacetylene Wiring up to Several Tens of Micrometers in Length. J. Am. Chem. Soc. 2005, 127, 4164−4165. (20) Dawn, A.; Shiraki, T.; Ichikawa, H.; Takada, A.; Takahashi, Y.; Tsuchiya, Y.; Lien, L. T. N.; Shinkai, S. Stereochemistry-Dependent, Mechanoresponsive Supramolecular Host Assemblies for Fullerenes: a Guest-Induced Enhancement of Thixotropy. J. Am. Chem. Soc. 2012, 134, 2161−71. (21) Mukhopadhyay, P.; Fujita, N.; Takada, A.; Kishida, T.; Shirakawa, M.; Shinkai, S. Regulation of a Real-Time Self-Healing Process in Organogel Tissues by Molecular Adhesives. Angew. Chem., Int. Ed. Engl. 2010, 49, 6338−6342. (22) Lescanne, M.; Grondin, P.; D’Aléo, A.; Fages, F.; Pozzo, J.-L.; Monval, O. M.; Reinheimer, P.; Colin, A. Thixotropic Organogels Based on a Simple N-hydroxyalkyl Amide: Rheological and Aging Properties. Langmuir 2004, 20, 3032−3041. (23) Brinksma, J.; Feringa, B. L.; Kellogg, R. M.; Vreeker, R.; Esch, J. van Rheology and Thermotropic Properties of Bis-Urea-Based Organogels in Various Primary Alcohols. Langmuir 2000, 16, 9249− 9255. (24) van Esch, J. H.; Schoonbeek, F.; de Loos, M.; Kooijman, H.; Spek, A. L.; Kellogg, R. M.; Feringa, B. L. Cyclic Bis-Urea Compounds as Gelators for Organic Solvents. Chem.Eur. J. 1999, 5, 937−950.

4. CONCLUSIONS We have developed novel hydrogelators by reacting the cyclo(dipeptide) derivative cyclo(L-aspartyl-L-phenylalanyl) with diols. From the gelation tests, compound 2 was determined to have the most suitable alkyl chain length and hydrophobicity for hydrogel formation. In particular, cyclo(L-Ohydroxyhexylaspartyl-L-phenylalanyl), which was synthesized with 1,6-hexanediol, formed a thermally/isothermally reversible physical gel with several solvents, including pure water, saline, alcohols, as well as 1.0 M aqueous NaCl, KCl, CaCl2, and MgCl2 solutions. The TEM observations of samples prepared from compounds 1−3 showed self-assembled fibers with diameters of 10−100 nm. The FT-IR results revealed that the gels were mainly formed by hydrogen bonding and van der Waals forces; thixotropic behavior was caused by the disruption of the van der Waals forces between the alkylene chains under shearing. These results were repeatedly and reproducibly observed at room temperature, even when measurements were repeated many times. The POM results showed birefringence image of three patterns: spherulite, broken spherical structures, and lamella-like structures, respectively. On a macroscopic scale, thixotropy refers to an isothermal, reversible gel−sol−gel phase transition due to reforming of the particulate network. However, in this study, we found that the microscopic structures are different before and after shearing. We expect that our study will help understand the viscoelastic behavior of supramolecular systems.

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ASSOCIATED CONTENT

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AUTHOR INFORMATION

S Supporting Information *

Additional gelation tests, TEM, field emission scanning electron microscopy (FE-SEM), thixotropic/hysteresis loops, differential scanning calorimetry (DSC), and powder X-ray diffraction (PXRD). This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a research fellowship of the Japan Society for the Promotion of science for Young Scientists (to H.H.) and Grant-in-Aid for Global COE Program by the Ministry of Education, Culture, Sports, Science, and Technology. The authors wish to thank Prof. Masato Takahashi (Shinshu University) and Mr. Kousaku Ohno (Elquest Co. Ltd., Japan) for their generous support of the rheological measurements.

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REFERENCES

(1) Estroff, L. A.; Hamilton, A. D. Water Gelation by Small Organic Molecules. Chem. Rev. 2004, 104, 1201−1218. (2) Sangeetha, N. M.; Maitra, U. Supramolecular Gels: Functions and Uses. Chem. Soc. Rev. 2005, 34, 821−836. (3) George, M.; Weiss, R. G. Molecular Organogels. Soft Matter Comprised of Low-molecular-mass Organic Gelators and Organic Liquids. Acc. Chem. Res. 2006, 39, 489−497. (4) Fages, F. Low Molecular Mass Gelators: Design, Self-Assembly, Function; Topics in Current Chemistry, Vol. 256; New York: Springer, 2005. 14672

dx.doi.org/10.1021/la402333h | Langmuir 2013, 29, 14666−14673

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Article

(25) Ma, D.; Zhang, L.-M. Supramolecular Gelation of a Polymeric Prodrug for Its Encapsulation and Sustained Release. Biomacromolecules 2011, 12, 3124−3130. (26) Misawa, Y.; Koumura, N.; Matsumoto, H.; Tamaoki, N.; Yoshida, M. Hydrogels Based on Surfactant-Free Ionene Polymers with N,N′-(p-Phenylene)dibenzamide Linkages. Macromolecules 2008, 41, 8841−8846. (27) Li, X.; Li, J. Supramolecular Hydrogels Based on Inclusion Complexation Between Poly(ethylene oxide)-b-poly(ε-caprolactone) Diblock Copolymer and α-cyclodextrin and Their Controlled Release Property. J. Biomed. Mater. Res. A 2008, 86, 1055−1061. (28) Barbucci, R.; Pasqui, D.; Favaloro, R.; Panariello, G. A Thixotropic Hydrogel from Chemically Cross-linked Guar Gum: Synthesis, Characterization and Rheological Behaviour. Carbohydr. Res. 2008, 343, 3058−3065. (29) Panariello, G.; Favaloro, R.; Forbicioni, M.; Caputo, E.; Barbucci, R. Synthesis of a New Hydrogel, Based on Guar Gum, for Controlled Drug Release. Macromol. Symp. 2008, 266, 68−73. (30) Leone, G.; Torricelli, P. Amidic Alginate Hydrogel for Nucleus Pulposus Replacement. J. Biomed. Mater. Res. A. 2007, 84A, 391−401. (31) Chhatbar, M. U.; Prasad, K.; Chejara, D. R.; Siddhanta, A. K. Synthesis of Sodium Alginate Based Sprayable New Soft Gel System. Soft Matter 2012, 8, 1837−1844. (32) Picard, J.; Giraudier, S.; Larreta-Garde, V. Controlled Remodeling of a Protein-polysaccharide Mixed Gel: Examples of Gelatin-Hyaluronic Acid Mixtures. Soft Matter 2009, 5, 4198−4205. (33) Freunlich, H. Thixotropie; Hermann: Paris, 1935. (34) Pakhomov, P.; Khizhnyak, S.; Ovchinnikov, M.; Komarov, P. Supramolecular Hydrogels Based on Silver Mercaptide. Self-Organization and Practical Application. Macromol. Symp. 2012, 316, 97−107. (35) Sakka, S.; Kozuka, H. Rheology of sols and fiber drawing. J. Non.-Cryst. Solids 1988, 100, 142−153. (36) Menger, F. M.; Caran, K. L. Anatomy of a Gel. Amino Acid Derivatives That Rigidify Water at Submillimolar Concentrations. J. Am. Chem. Soc. 2000, 122, 11679−11691. (37) Suzuki, M.; Setoguchi, C.; Shirai, H.; Hanabusa, K. Organogelation by Polymer Organogelators with a L-Lysine Derivative: Formation of a Three-Dimensional Network Consisting of Supramolecular and Conventional Polymers. Chem.Eur. J. 2007, 13, 8193−8200. (38) Suzuki, M.; Hanabusa, K. Polymer Organogelators That Make Supramolecular Organogels Through Physical Cross-linking and SelfAssembly. Chem. Soc. Rev. 2010, 39, 455−463. (39) Gibson, M. I.; Cameron, N. R. Organogelation of Sheet-Helix Diblock Copolypeptides. Angew. Chem., Int. Ed. Engl. 2008, 47, 5160− 5162. (40) Yang, Y.; Suzuki, M.; Kimura, M.; Shirai, H.; Hanabusa, K. Preparation of Cotton-like Silica. Chem. Commun. (Cambridge, U.K.) 2004, 1332−1333. (41) Ackerson, B. Shear Induced Order and Shear Processing of Model Hard Sphere Suspensions. J. Rheol. 1990, 34, 553−590. (42) Watanabe, H.; Yao, M.-L.; Yamagishi, A.; Osaki, K.; Shitata, T.; Niwa, H.; Morishima, Y. Nonlinear Rheological Behavior of a Concentrated Spherical Silica Suspension. Rheol. Acta 1996, 35, 433−445. (43) Truyen, D.; Courty, M.; Alphonse, P.; Ansart, F. Catalytic Coatings on Stainless Steel Prepared by Sol−gel Route. Thin Solid Films 2006, 495, 257−261. (44) Barbucci, R.; Giardino, R.; De Cagna, M.; Golini, L.; Pasqui, D. Interpenetrating Hydrogels (IPHs) as a New Class of Injectable Polysaccharide Hydrogels with Thixotropic Nature and Interesting Mechanical and Biological Properties. Soft Matter 2010, 6, 3524−3532. (45) Snyder, R. G. Vibrational Spectra of Crystalline n-Paraffin Part I. Methylene Rocking and Wagging Modes. J. Mol. Spectrosc. 1960, 434, 411−434. (46) Cameron, D. G.; Umemura, J.; Patrick, T. T. W.; Mantsch, H. H. A Fourier Transform Infrared Study of the Coagel Micelle Transitions of Sodium Laurate and Sodium Oleate. Colloids Surf. 1982, 4, 131−145. 14673

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