Low-Molecular-Weight Organo- and Hydrogelators Based on Cyclo(l

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Low-Molecular-Weight Organo- and Hydrogelators Based on Cyclo(L‑Lys‑L‑Glu) Huimin Geng,† Lin Ye,†,‡ Ai-ying Zhang,†,‡ Jingbo Li,†,‡ and Zeng-guo Feng*,†,‡ †

School of Materials Science and Engineering and ‡Beijing Key Laboratory of Construction Tailorable Advanced Functional Materials and Green Applications, Beijing Institute of Technology, No. 5 South Street Zhongguancun, Beijing 100081, China S Supporting Information *

ABSTRACT: Four cyclo(L-Lys-L-Glu) derivatives (3−6) were synthesized from the coupling reaction of protecting L-lysine with L-glutamic acid followed by the cyclization, deprotection, and protection reactions. They can efficiently gelate a wide variety of organic solvents or water. Interestingly, a spontaneous chemical reaction proceeded in the organogel obtained from 3 in acetone exhibiting not only visual color alteration but also increasing mechanical strength with the progress of time due to the formation of Schiff base. Moreover, 6 bearing a carboxylic acid and Fmoc group displayed a robust hydrogelation capability in PBS solution. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) revealed the characteristic gelation morphologies of 3D fibrous network structures in the resulting organo- and hydrogels. FT-IR and fluorescence analyses indicated that the hydrogen bonding and π−π stacking play as major driving forces for the self-assembly of these cyclic dipeptides as lowmolecular-weight gelators. X-ray diffraction (XRD) measurements and computer modeling provided information on the molecular packing model in the hydrogelation state of 6. A spontaneous chemical reaction proceeded in the organogel obtained from 3 in acetone exhibiting visual color alteration and increasing mechanical strength. 6 bearing an optimized balance of hydrophilicity to lipophilicity gave rise to a hydrogel in PBS with MGC at 1 mg/mL.

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INTRODUCTION Supramolecular gels, which are held together by noncovalent bonds exhibiting stimuli-responsiveness and tunable characteristics,1,2 have shown the great potential applications in the area such as nanofabrication, catalysts, sensing, tissue engineering, and sewage treatments.1,3,4 The low-molecular-weight gelators (LMWGs) possessing in general a molecular weight of less than 2000 Da are capable of absorbing a vast amount of organic solvents or water to generate physical organo- and hydrogels in which the molecules are self-assembled into a fixed 3D network of fibers.5 Among those LMWGs, peptide-based hydrogels have been widely exploited as additives in the food, cosmetic, and pharmaceutical industries, carriers for the controlled drug delivery, and scaffolds for the tissue engineering and regenerative medicine on account of their high biocompatibility and low toxicity.1,6,7 For example, Law et al. recently described a series of nontoxic peptide-based self-assembled matrices that can degrade following enzyme reaction,8 in which the core peptide sequence consisted of a protease cleavable region is flanked by two self-assembly motifs. In addition, enzymecatalyzed hydrogels based on Fmoc-dipeptides were reported by Xu et al. showing the promising application in the controlled drug release.6,9 Ulijn et al. demonstrated that chemically modified Fmoc-diphenylalanines (Fmoc-FF) have good biocompatibility toward different cell types, and the formed © XXXX American Chemical Society

hydrogels possess tunable chemical and mechanical properties suitable for in vitro cell culture.10,11 At the same time, the diphenylalanine (FF) can self-assemble into organogel to incorporate quantum dots showing photoluminescent,12 and its cationic form (FF-NH2·HCl) can form biocompatible waterdispersible 3D colloidal spheres under ultrasound.13 However, this unmodified smallest dipeptide cannot directly gelate water to give access to hydrogel.11 Meanwhile, cyclic dipeptides or 2,5-diketopiperazines (2,5DKPs) which frequently occur as natural peptide derivatives and are often prepared from the condensation of two corresponding α-amino acids have been attracting considerable attention for their physiological and pharmacological activities.5,14,15 For instance, as a typical cyclic dipeptide, cyclo(LTyr-L-Lys) was reported to hold antinociceptive activity.16 Feng et al. serendipitously found that this cyclic dipeptide can not only give rise to organogels but also yield strong hydrogel under ultrasound.14 After N-acylated with D-(+)-gluconic acid, a shear-triggered hydrogel with thixotropic nature was generated by applying shear force on its metastable supersaturated solution.17 Additionally, fumaryl-modified cyclo(LReceived: March 18, 2016 Revised: April 19, 2016

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parallel plate geometry at 25 °C. The storage and loss moduli were measured with a frequency sweep test (0.1−100 Hz). NMR Experiment: 1H and 13C NMR measurements were carried out on a Mercury-plus 400 (Varian) at 400 and 100 MHz, respectively. All spectra were recorded in DMSO-d6. Mass spectra were recorded on a Xevo G2 QTOF (Waters) highresolution mass spectrometer. Elemental analyses were performed on vario EL cube V2.0.1 (Elementar) elemental analyzer. Melting point tests were conducted on a DSC-60 (Shimadzu), and the data were uncorrected. Wide-angle X-ray diffraction measurements were performed on xerogels by an Ultima IV Instrument (Rigaku) using Cu Kα (λ = 1.5405 Å) radiation. All gels were freeze-dried to get their xerogels. Gelation Tests. A weighed amount of potential gelator and the organic solvent or water were added into a sealed glass vial to make a total of 1 mL of the mixture and heated to get a clear solution. The solution was then cooled back to room temperature in air. The gelation (G) was confirmed by inverting the glass vial and the solution inside no evident to flow. Systems in which only solution remained until the end of the tests were referred as soluble (S) and others as partial gelation (PG) or insoluble upon heating (I). Fmoc-L-Lys(Fmoc)-L-Glu(OtBu)-OMe (2). To a solution of 1 (5.91 g, 10 mmol) in CH2Cl2 (200 mL) was added EDCI (2.87 g, 15 mmol) and TEA (2 mL) followed by the addition of HOBt (2.03 g, 15 mmol). The reaction mixture was stirred for 2 h at 0 °C and then added dropwise a solution of OtBu-Glu·HCl (3.30 g, 13 mmol) in CH2Cl2 (20 mL). Upon completion of the addition, the reaction mixture was stirred at room temperature for 48 h and subsequently washed with saturated NaCl solution (3 × 100 mL), dried over Na2SO4, filtered, concentrated, and finally purified by chromatography on silica gel (CHCl3:MeOH = 20:1) to give 2 as white solids with yield of 70%; mp = 157.1 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.32 (s, 1H), 7.89 (d, J = 7.0 Hz, 4H), 7.76−7.63 (m, 4H), 7.50 (d, J = 7.7 Hz, 1H), 7.41 (s, 4H), 7.32 (s, 5H), 4.35−4.17 (m, 7H), 4.01 (s, 1H), 3.61 (s, 3H), 2.97 (s, 2H), 2.27 (s, 2H), 1.93 (s, 1H), 1.79 (s, 1H), 1.60 (s, 2H), 1.42−1.21 (m, 13H) ppm. 13C NMR (100 MHz, DMSOd6): δ 172.15, 168.51, 168.01, 157.55, 143.06, 139.90, 137.77, 129.39, 127.75, 121.85, 120.49, 110.19, 80.17, 54.36, 53.66, 49.07, 32.94, 30.94, 29.78, 28.83, 28.21, 22.04 ppm. HR-MS calcd [M+] = 789.3652; obsd [M + 1+] = 790.3689. Elemental analysis: C 69.94, H 6.51, N 5.32; found: C 69.77, H 6.31, N 5.48. OtBu-Cyclo(L-Lys-L-Glu)-NH2 (3). 6 g of 2 (7.61 mmol) was dissolved in 100 mL of cosolvent of piperidine/DMF (v/v = 1:4) and stirred at 60 °C for 3 h. Thereafter the reaction mixture was precipitated into 300 mL of diethyl ether and washed with 100 mL of diethyl ether 5 times. The precipitates were gathered by filtration to give access to 3 as white solids with yield of 80%; mp = 148.1 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.22−7.98 (m, 2H), 3.86 (s, 1H), 3.81 (s, 1H), 3.05 (s, 1H), 2.81 (d, J = 63.8 Hz, 1H), 2.27 (s, 2H), 1.88 (d, J = 22.9 Hz, 2H), 1.65 (s, 2H), 1.36 (d, J = 28.4 Hz, 13H). 13C NMR (100 MHz, DMSO-d6): δ 172.13, 167.78, 161.13, 80.17, 54.33, 53.68, 37.44, 32.91, 30.92, 29.23, 28.78, 28.09, 21.88. HR-MS calcd [M+] = 313.2002; obsd [M + 1+] = 314.2069. Elemental analysis: C 57.49, H 8.68, N 13.41; found: C 57.39, H 8.85, N 13.08. HOOC-Cyclo(L-Lys-L-Glu)-NH2·TFA (4). To a solution of 0.5 g of 3 (1.60 mmol) in CH2Cl2 (6 mL) was added TFA (6 mL). The reaction mixture was stirred for 12 h at room temperature. The reaction mixture gradually turned into an orange solution. The mixture was concentrated under reduced pressure. The residue was suspended in diethyl ether and centrifuged, and finally the resulting wet filter cake was recrystallized from methanol. The pellets were dried under reduced pressure to produce 4 as white solids with yield of 70%; mp = 129.3 °C. 1H NMR (400 MHz, DMSO-d6): δ 12.16 (s, 1H), 8.17 (d, J = 8.1 Hz, 2H), 7.70 (s, 2H), 3.93−3.78 (m, 2H), 2.91 (ddd, J = 20.5, 13.7, 6.3 Hz, 2H), 2.37−2.23 (m, 2H), 1.88 (ddd, J = 20.2, 16.3, 9.6 Hz, 2H), 1.74−1.60 (m, 2H), 1.58−1.20 (m, 4H) ppm. 13C NMR (151 MHz, DMSO-d6): δ 174.33, 168.33, 161.03, 65.14, 54.13, 53.69, 37.38, 32.40, 29.83, 27.12, 21.44 ppm. HR-MS calcd [M+] = 257.1376;

Lys-L-Lys) was disclosed a safe delivery vehicle for insulin administration via inhalation,18 and its derivatives were also featured by the excellent gelation capability.5,19 Moreover, this cyclic dipeptide after modified by cysteine can selectively gelate chlorinated organic solvents from a mixture with water as well as high efficiently adsorb tested organic dyes from aqueous solutions, offering a possibility for the treatment of industrial wastewater and the recovery of used dyes.4 Similarly, the organogels formed from cyclo(Gly-L-Lys) derivatives were also employed to physically entrap organic dye and drug molecules, and most importantly the hydrogels obtained from these derivatives could be used as candidates for the drug delivery systems and thermoresponsive smart materials.20 Therefore, the rigid structure, preferred intermolecular hydrogen-bonding interactions, and physiological and pharmacological activities make these cyclic dipeptides promising organo- and hydrogelator for diverse biomedical and other high-technological applications. Recently, Ulijn et al. had demonstrated a computational design-oriented approach by screening all 8000 possible tripeptides and provided a set of designing rules for selfassembling sequences of di- and tripeptide-based gelators.21 However, it should be pointed out that the formation of hydrogels from LMWGs is still a very complicated phenomenon. It remains a great challenge for the molecular design and synthesis of new LMWGs or even predicting gelation behaviors from the molecular structure regarding a delicate balance of intermolecular interactions between gelator molecules and between gelator and solvent molecules as well. To better understand the relationship of cyclic dipeptide molecular structure with gelation behaviors, four cyclo(L-Lys-L-Glu) derivatives were synthesized, and the effects of their chemical functionalities, NH2, COOH, and protecting groups Fmoc and OtBu within DKP rings on the gelation behaviors were also assessed in this study. Interestingly, the organogel formed by 3 in acetone was found to undertake a spontaneous chemical reaction between the gelator and solvent molecules providing the color change and increasing gel strength over time. Fortunately, Fmoc monosubstituted 6 bearing a carboxylic acid exhibited an excellent hydrogelation capability showing the potential to be utilized as a candidate injectable hydrogel.22,23

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MATERIALS AND METHODS

Materials. H-L-Glu(OtBu)-Ome·HCl and Fmoc-L-Lys(Fmoc)-OH were purchased from GL Biochem (Shanghai, China) Ltd. All other reagents (available from VAS Chemical Reagent Company, Tianjin, China) were analytical grade and used without further purification unless otherwise noted. Characterizations. Fourier transform infrared spectra were recorded on a IR Trace-100 (Shimadzu) spectrometer. All samples were recorded using the KBr disk technique and scanned between the wavelengths of 4000 and 400 cm−1. Fluorescence spectra were recorded on a Cary Eclipse spectrometer (Varian) with excitation at 265 nm and emission data ranging between 275 and 550 nm. Transmission electron microscopy images were taken on a JEM 1200EX (JEOL) microscope at 120 kV voltages. A drop of dilute solution of gel-phase material was placed on carbon-coated copper grids (300 mesh) and dried by slow evaporation for 2 days. Field emission scanning electron microscopy images were obtained from an S-4800 FE-SEM (Hitachi). The gel samples were prepared by the vacuum freeze-drying method and then coated with gold for 90 s at 5 kV voltages on silicon slices. Oscillatory rheology measurements were performed on a straincontrolled Physica MCR301 rheometer (Anton-Paar) using the B

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Langmuir obsd [M + 1+] = 258.1478. Elemental analysis: C 42.05, H 5.43, N 11.32; found: C 41.94, H 5.17, N 11.33. OtBu-Cyclo(L-Lys-L-Glu)-Fmoc (5). 3 (2.58 g, 8.24 mmol) and NaHCO3 (1.42 g, 16.48 mmol) were dissolved in the mixture solvents of 1,4-dioxane (100 mL) and H2O (100 mL), then added dropwise a solution of Fmoc-OSu (2.63 g, 7.8 mmol) in 1,4-dioxane (60 mL), and stirred for 24 h at room temperature. The solvents were removed, and the residue was washed with H2O (100 mL) and brine (100 mL). After drying and removal of the solvent, the residue was chromatographed (CHCl3:MeOH = 15:1) to give rise to 5 as white solids with yield of 65%; mp = 162.7 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.22−8.06 (m, 2H), 7.87 (dd, J = 17.5, 7.4 Hz, 3H), 7.68 (d, J = 7.3 Hz, 1H), 7.43−7.32 (m, 4H), 7.26 (s, 1H), 4.47−4.11 (m, 3H), 3.86 (s, 1H), 3.81 (s, 1H), 3.04−2.86 (m, 2H), 2.29 (t, J = 10.1 Hz, 2H), 1.89 (d, J = 32.3 Hz, 2H), 1.66 (s, 2H), 1.37 (dd, J = 17.8, 7.9 Hz, 13H) ppm. 13C NMR (100 MHz, DMSO-d6): δ 171.98, 168.03, 157.82, 142.99, 139.84, 137.85, 129.35, 127.61, 121.85, 120.43, 110.20, 80.15, 54.33, 53.69, 32.92, 30.98, 29.77, 28.81, 28.22, 25.68, 22.04 ppm. HR-MS calcd [M+] = 535.2682; obsd [M + 1+] = 536.2752. Elemental analysis: C 67.27, H 6.96, N 7.84; found: C 67.69, H 6.92, N 7.53. HOOC-Cyclo(L-Lys-L-Glu)-Fmoc (6). To a solution of 1 g of 3 (0.72 mmol) in CH2Cl2 (10 mL) was added TFA (10 mL). The reaction mixture was stirred for 12 h at room temperature. The reaction mixture gradually turned into an orange solution. The mixture was concentrated under reduced pressure. The residue was suspended in diethyl ether and centrifuged, and finally the wet filter cake was recrystallized from methanol. The pellets were dried under reduced pressure to give 6 as white solids with yield of 60%; mp = 190.6 °C. 1H NMR (400 MHz, DMSO-d6): δ 12.07 (s, 1H), 8.14 (s, 2H), 7.88 (t, J = 8.7 Hz, 2H), 7.69 (d, J = 7.4 Hz, 2H), 7.41 (t, J = 7.3 Hz, 2H), 7.33 (td, J = 7.4, 1.0 Hz, 2H), 7.25 (t, J = 5.5 Hz, 1H), 4.30 (d, J = 6.9 Hz, 2H), 4.21 (t, J = 6.8 Hz, 1H), 3.88 (d, J = 5.8 Hz, 1H), 3.81 (s, 1H), 3.10−2.88 (m, 2H), 2.39−2.24 (m, 2H), 1.90 (ddd, J = 23.1, 14.3, 6.9 Hz, 2H), 1.67 (s, 2H), 1.45−1.20 (m, 4H) ppm. 13C NMR (151 MHz, DMSO-d6): δ 174.28, 168.51, 168.22, 156.47, 144.43, 141.21, 128.05, 127.44, 125.62, 120.57, 65.65, 54.36, 53.71, 47.25, 32.89, 29.86, 29.58, 28.84, 21.94 ppm. HR-MS calcd [M+] = 479.2056; obsd [M + 1+] = 480.2125. Elemental analysis: C 65.12, H 6.10, N 8.76; found: C 65.21, H 6.53, N 8.81.

Scheme 1. Synthetic Route of Cyclic Dipeptides from Coupling L-Lysine with L-Glutamic Acid

Table 1. Results of Gelation Testing and MGCs (mg/mL) of Gelators in Various Organic Solvents and Water at Room Temperaturea

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RESULTS AND DISCUSSION Synthesis. The synthetic approach of cyclo(L-Lys-L-Glu) derivatives 3−6 is outlined in Scheme 1. First the dipeptide 2 was prepared from the coupling reaction of 1 with H-LGlu(OtBu)-OMe·HCl in the presence of EDCI/HOBT. As a result, the cyclic dipeptide 3 was obtained by the one-pot deprotection and cyclization reaction of 2 in a cosolvent of piperidine/DMF. An asymmetrically tBuO- and Fmoc-substituted 5 was synthesized from the protection of 3 using FmocOSu. Subsquently 6 bearing a carboxylic acid and Fmoc group was produced by deprotecting OtBu in TFA. 4 without any protecting group was also obtained in the same way. These cyclic depeptides were characterized by 1H/13C NMR as well as high-resolution mass spectrometric and elemental analyses. Gelation Properties. The ability of the resulting cyclo(LLys-L-Glu) derivatives 3−6 to gelate organic solvents and water was eventually determined by inverting the vials upside down. The gelation testing results and the minimum gelation concentration (MGC, mg/mL) data are summarized in Table 1. As is well-known, an optimized balance of hydrophilicity to lipophilicity within the molecular structure is the prerequisite for a LMWG to gelate organic solvents and water. However, it remains a great challeng to predict whether or not a small molecule is a LMWG.24 As shown in Table 1, the resulting DKPs 3−6 presented the diverse gelation abilities in a wide

solvents

3

4

5

6

MeOH EtOH isopropanol n-butanol n-hexanol benzene toulene xylol p-xylene o-dichlorobenzene CH2Cl2 1,2-dichloroethane CHCl3 sym-tetrachloroethane EtOAc acetone MeCN 1.4-dioxane THF DMSO DMF H2O PBS

S G(4) G(2) G(3) G(2) G(0.5) G(0.5) G(0.5) G(0.5) G(0.3) G(2) G(1) G(2) G(1) G(1) G(2) G(1) G(2) G(1) S S S S

S S S G(5) G(5) I I I I I I I I I I I I I I S S S S

S S PG G(4) G(3) G(0.3) G(0.3) G(0.3) G(0.3) G(0.3) G(2) G(2) S S G(3) PG G(3) S S S S I I

S S PG G(3) G(3) I I I I G(3) G(0.5) G(0.3) G(1) G(0.3) I I I S S S S I G(1)

a

G: gelation; PG: partial gelation; S: soluble (>5 mg/mL); I: insoluble upon heating.

range of polar and apolar solvents. The cyclic dipeptide 4 is precipitated in chlorinated and aromatic solvents but well C

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Figure 1. (a) Macroscopic visualization of organogel formed by 3 in acetone and (b) hydrogel formed by 6 in PBS.

Figure 2. TEM (left) and SEM (right) images of organogels: (a) and (b) organogel 3 in n-butanol and o-dichlorobenzene; (c) and (d) organogel 4 in n-butanol and n-hexanol; (e) and (f) organogel 5 in n-butanol and o-dichlorobenzene; (g) and (h) organogel 6 in n-butanol and odichlorobenzene. Scale bars = 1 μm.

definitely attributed to the resulting Schiff base ([M + 1]+), clearly indicating that part of −NH2 are transformed into the CN structure with time. Meanwhile, the FT-IR spectrum (Figure S2) of this red aged xerogel depicts a new peak at 1623 cm−1, assigned to the characteristic νCN streching vibration,25 also confirming the spontaneous reaction occurred between 3 and acetone. Because of the strong hydrophilicity of pendent NH2 and the relatively poor hydrophobicity of OtBu26 in 3, a Fmoc protecting group was introduced to replace OtBu tuning the balance of hydrophobicity to hydrophilicity to gain access to a hydrogelator. Obviously, 5 is insoluble in water but soluble in most aliphatic and aromatic organic solvents with varying gelation abilities. However, after deprotecting OtBu in TFA, the resulting 6 bearing a carboxylic acid and Fmoc can not only form organogels in most selected alcohols and chlorinated

dissolved in water because of the presence of hydrophilic −COOH and −NH2 groups, whereas in n-butanol and nhexanol it undergoes stable thermoreversible gelation processes. In comparison, an OtBu protecting group imparts 3 a strong gelling capability to form thermoreversible gels in most organic solvents, such as alcohols, and chlorinated and aromatic solvents. Accidentally, a visual color change of gel formed from 3 in acetone was observed, and as depicted Figure 1a, the color was gradually changed from translucent white to yellow to orange to red in 1 week and finally to dark red. This is because the pendent amine group in 3 nucleophilically adds to the carbonyl group of acetone to form a Schiff base CN structure leading to the visualized color shift. This spontaneous transition was verified by the high resolution mass spectrum (MS) of red xerogel aged for 10 days (Figure S1). The peak at 314.2079 corresponds to 3 ([M + 1]+), and the peak at 354.2392 is D

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Figure 3. TEM images of the formation of hydrogel process, from turbid viscous solution to the semitransparent hydrogel. Scale bars = 1 μm.

Figure 4. Viscoelastic behaviors of 3 (a), 4 (b), 5 (c), and 6 (d) organogels formed in n-butanol at MGC. Frequency sweep using a constant strain of 0.1% from 0.1 to 100 Hz at 25 °C.

solvents but also generate a hydrogel in phosphate buffer saline (PBS). Interestingly, this hydrogelation process was accompanied by a change in the transparency from a turbid viscous solution to a semitransparent hydrogel within 1 min as illustrated in Figure 1b. The results clearly indicated that the gelation behaviors can be realized by altering either the hydrophilic functionalized groups 3 or the hydrophobic protecting groups of the cyclic dipeptide gelators. Aggregation Morphology Studies. As mentioned above, the cyclic dipeptides 3−6 gave access to translucent or opaque organogels in alcohols and chlorinated solvents and transparent organogels in aromatic solvents. Therefore, n-butanol, nhexanol, and o-dichlorobenzene were selected to shed light on the microstructures of the resultant gels, respectively. The interior morphologies were observed by TEM without staining, and the corresponding freeze-dried xerogels were monitored by SEM. As shown in Figure 2, the aggregates of the transparent organogels in o-dichlorobenzene indicated that nanofibers are formed of ca. 10−20 nm in the width and up to several micrometers in the length and seem to stack on top of each other at intersections to yield a meshed structure (Figure

2b,f,h). The microstructure of the opaque organogels obtained from 3 in n-butanol was described like a micron-sized core− shell structure. Upon careful inspection of the images, the gelator was inclined to form prickly fiber aggregations with a floppy surrounding layer formed by entangled fibers (Figure 2a). 4 was self-assembled into stubbly fibers in n-butanol (Figure 2c) and a thin ribbon-like structure in n-hexanol (Figure 2d). Whereas, Figure 2e presents the formation of a spherulitic fiber network of 5 in n-butanol through the nucleation and growth of fibers, but no core was constructed in its organogel in o-dichlorobenzene. As for 6, its aggregates in n-butanol and o-dichlorobenzene featured a highly ordered pore net-like structure with a pore of about 1 μm in the diameter (Figure 2g). Besides a change in the transparency was visible by the naked eye during the formation of hydrogel from 6 in PBS, the micromorphologies from stubbly rod-like aggregates to nanofibrillar geometry with a diameter of about 10 nm were also evidenced (Figure 3). The results clearly demonstrated that the nature of solvents from which the gels come plays a vital role in regulating the microstructures of the gel 3D networks. The E

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Figure 5. (a) Viscoelastic behaviors of organogels formed by 3 in acetone at MGC as a function of standing time. (b) Viscoelastic behaviors of hydrogel formed from 6 at MGC. Frequency sweep using a constant strain of 0.1% from 0.1 to 100 Hz at 25 °C.

Figure 6. FT-IR spectra of 3 (a), 4 (b), 5 (c), and 6 (d) in solution, gel, and solid states.

thinner the fiber diameter is, the more transparent the gel becomes.19 Rheological Measurements. The viscoelastic properties of the organogels obtained from 3−6 in n-butanol at their MGCs were tested at 25 °C, respectively. As can be seen in the insets of Figure 4, the elastic modulus (G′) of these gels gave a long plateau at a low strain range and began to sharply decrease. Therefore, γ0 was restricted to 0.1% for the rheological testing in this study. It was found that the G′ is an order of magnitude higher than the loss modulus G″ at all the testing frequencies. This behavior is characteristic of true gels containing a static network structure, meaning that they could not deform under the pressure of its own weight.19 Especially, G′ and G″ at high frequencies are always slightly higher than those at low frequencies, pointing to the slow

rearrangement of organogelator molecules in gels at high stress.14 Furthermore, G′ and G″ values of 3 become larger than 4, and this also holds true for 5 against 6 as well, showing a significant role that OtBu plays in the improvement of gel mechanical properties. For example, at ω = 10 Hz, the G′ of 3 and 5 reach 4.9 × 103 and 2.3 × 104 Pa, whereas those of the 4 and 6 only attain 7.3 × 102 and 3.1 × 103 Pa, respectively. Besides, the organogel formed from 5 has the highest G′ and G″ in the range of testing frequencies, which suggested that 5 as an organogelator is stronger than others due to the synergetic interactions of hydrogen bonding between amide groups of the DKP ring, the π−π stacking interactions between Fmoc groups, and hydrophobic interactions of OtBu3 as well. Therefore, the mechanical properties of the gels are a result of the different microstructure of the gelators.27 F

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Figure 7. Fluorescence emission spectra of organogel of 5 (a) and 6 (b) in solution and gel states (λexcitation = 265 nm).

the emission spectroscopy of the 5 organogel, possibly due to the presence of an extensive J-aggregate with fluorenyl rings,11 implying that multiple fluorenyl rings are stacked more efficiently and moving less freely in the gel state.9 Moreover, a broad shoulder peak centered at 380 nm in the emission spectroscopy of the organogel and hydrogel of 6 is believed to be a portion of the parallel arrangement between fluorenyl groups.3 As described above, the hydrogen bonds are indeed the major driving forces for the gelation processes of all the gelators 3−6, and moreover, the π−π interactions driven by fluorenyl rings also play an indispensable role in building up macromolecule-like aggregates of 5 and 6 in organic solvents and water. XRD Studies. To further understand the molecular arrangements and the orientation of the gelator molecules in the gel state, the organogels of four cyclic dipeptides prepared in n-butanol were dried by a freeze-drying method and then tested by using XRD equipment (Figure S3). In fact, these xerogels created the sponge-like configurations instead of the typical crystals. To highlight the relationship between the physical gelation properties and molecular packing of these gelator molecules appears to be challenging.29 However, the xerogels formed by 3 and 4 exhibited the specific XRD patterns with sharp reflections as outlined in Figure S3a,b. Especially, the diffraction pattern of the xerogel 3 gave periodical diffraction peaks, indicating that 3 enables to self-assemble into a lamellar organization with a interlayer distance 20.82 Å corresponding to the (100) plane.29,30 However, the patterns of 5 and 6 displayed extensive broadening diffraction peaks, which correspond to the disordered structure formed in n-butanol. This is maybe due to the fact that 3 and 4 simply create a layered structure by sequential intermolecular hydrogen bonds between N−H and CO in the 2,5-DKP rings,30 whereas 5 and 6 generate organogels by both hydrogen bonding and π−π stacking of fluorenyl rings, in which the strong π−π interactions are partly interposed by the sequential intermolecular hydrogen bonds of DKP rings leading to the unordered molecular packing and broadening diffraction peaks in the xerogels formed in n-butanol. Moreover, the XRD measurements were also carried out on the xerogels freshly prepared and dried from the organogel formed by 3 in acetone (Figure S4). After aging for 10 days, the organogel was dried and its xerogel yielded the sharper peaks than the freshly prepared one, implying a more ordered structure of gelator molecules.23

Oscillatory rheological assay was also performed to trace the dynamic transition of 3 in acetone with time (Figure 5a). The G′ and G″ values become increasingly higher as a function of standing time, from 2.1 × 104 and 4.1 × 103 Pa to 1.3 × 105 and 2.7 × 104 Pa at ω = 10 Hz in 20 days, respectively. This observation suggested that the formation of Schiff base between 3 and acetone leads to a more rigid and “solid-like” material5 with the progress of time. As the sol-to-gel or gel-to-sol transformations are well-known, whereas the time dependent gel-to-gel transformations are rarely reported in the literature, so the gel-to-gel or self-reinforcement transformation induced by 3 in acetone would provide a hint at a study toward the mechanism of the gel-to-gel transformation of LMWGs. Meanwhile, even though the hydrogel resulted from 6 in PBS presents relatively lower G′ and G″ as compared with the organogel fromed in n-butanol (Figure 5b), the G′ still exceeds the G″ at all frequencies, displaying an elastic rather than viscous material. Determination of Driving Forces in Self-Assembly. FTIR spectroscopy is widespread employed to gain insight into the hydrogen bonds between the gelators in the gel state.6,28 In Figure 6, the νN−H signals of all the cyclic dipeptide derivatives in DMSO solution appear at about 3515−3500 cm−1, and amide carbonyl stretching (N−CO, amide I) and N−H bending (amide II) band appeared at around 1687−1682 and 1500−1510 cm−1, respectively, indicating the occurrence of a non-hydrogen-bond in them. In the gel state, a sharp peak at 1670−1677 cm−1 is ascribed to the amide I stretching frequency, and the peaks at 1520−1538 and at 3320−3200 cm−1 correspond to the amide II and N−H stretching frequencies of the gelator, respectively. Moreover, the carboxylate (O−CO) band at 1725 cm−1 was seen in xerogels. These observations suggested that all the N−H and N−CO are hydrogen bonded in the assembled gel state.28 On the other hand, the corresponding peaks were observable in the hydrogel of 6, which indicated that a hydrogen bonded network structure is also formed in the hydrogel state. Fluorescence spectroscopy is broadly used to investigate into the π−π interactions between the fluorenyl groups in the solution and gel phases.3,27 As shown in Figure 7, a distinct single fluorescence peak was observed at ∼326 nm for 5 and 6 in DMF solution. This peak is indicative of the Fmoc group of gelators in the monomeric state in the solution. It is red-shifted to 340 nm in the gel phase, suggesting the antiparallel packing between fluorenyl groups in the gel phase. Noticeably, a pronounced peak with a maximum at about 460 nm appears in G

DOI: 10.1021/acs.langmuir.6b01059 Langmuir XXXX, XXX, XXX−XXX

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CONCLUSION We synthesized four cyclo(L-Lys-L-Glu) derivatives having different pendent groups. They enabled to gelate a wide variety of organic solvents. At the same time, the gelator bearing a carboxylic acid and Fmoc group displayed a robust hydrogelation capability in PBS with the potential to be used as an injectable hydrogel. In addition, the morphological observations and FTIR and fluorescence spectroscopic and rheological measurements suggested that the π−π stacking of fluorenyl rings and hydrogen-bonding interactions are responsible for the formation of both organo- and hydrogels. The XRD studies showed the existence of interdigitated bilayers in the hydroxerogel. This research work will help to elucidate the relationship between the structure and gelation properties of cyclic dipeptide derivatives as LMWGs.

As outlined in Figure 8, a high-resolution XRD pattern of the dried hydrogel of 6 with strong well-resolved peaks was

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.6b01059. Figure S1: mass spectrum of gelator 3 (a) and red aged xerogel in acetone (b); Figure S2: FT-IR spectra of fresh xerogel and red xerogel aged for 10 days; Figure S3: XRD patterns of xerogel of 3 (a), 4 (b), 5 (c), and 6 (d) in nbutanol; Figure S4: XRD patterns of fresh gel and red aged gel in acetone of 3; additional chemical characterization data for cyclic dipeptides 2−6, including 1H NMR and 13C NMR (PDF)

Figure 8. Calculated and observed XRD patterns of xerogel prepared from the hydrogel of 6 (2 wt % in PBS).

fortunately obtained in this study. An investigation was carried out into a possible molecular packing model, and the cell parameters were calculated as follows: a = 14.93 Å, b = 53.08 Å, c = 5.65 Å, β = 97.04°, and space group P2 based on the observed XRD pattern by using Jade 6.0. Then the generated best structure was refined via Reitveld refinement with energy by using Reflex plus module in Materials Studio, and a high quality fit was acquired between the calculated and observed powder patterns. Moreover, this xerogel gives a small second-order diffraction with a d-spacing ratio of 1:1/2:1/3 (26.24, 13.12, and 8.78 Å), also indicating a lamellar structure.5,31 Taking account these data, the arrangement of 6 in the hydrogel state was proposed and is outlined in Figure 9 by Materials Studio. The folded

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]; Tel 86-10-68912650; Fax 86-1068912650 (Z.F.). Funding

This work was financially supported by the National Natural Science Foundation of China (No. 21174018). Notes

The authors declare no competing financial interest.

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REFERENCES

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Figure 9. Perspective projection of molecular packing of 6, viewed perpendicular to the oc plane, Z = 3 (left). The wire-frame model of 6 arranged in PBS (right).

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DOI: 10.1021/acs.langmuir.6b01059 Langmuir XXXX, XXX, XXX−XXX