Synthesis , Characterization , and Self-Assembly of a Tetrathiafulvalene ( TTF ) – Triglycyl Derivative

In this work, we describe the synthesis, characterization, and self-assembly properties of a new tetrathiafulvalene (TTF)–triglycyl low-molecular-weight (LMW) gelator. Supramolecular organogels were obtained in various solvents via a heating–cooling cycle. Critical gelation concentrations (CGC) (range ≈ 5–50 g/L) and thermal gel-to-sol transition temperatures (Tgel) (range ≈ 36–51 ◦C) were determined for each gel. Fourier transform infrared (FT-IR) spectroscopy suggested that the gelator is also aggregated in its solid state via a similar hydrogen-bonding pattern. The fibrillar microstructure and viscoelastic properties of selected gels were demonstrated by means of field-emission electron microscopy (FE-SEM) and rheological measurements. As expected, exposure of a model xerogel to I2 vapor caused the oxidation of the TTF unit as confirmed by UV-vis-NIR analysis. However, FT-IR spectroscopy showed that the oxidation was accompanied with concurrent alteration of the hydrogen-bonded network.


Introduction
Oligonucleotide conjugates carrying aromatic systems are important tools for a large number of applications due to their enhanced hybridization properties and their special fluorescence and chemical properties coming from the aromatic moieties [1][2][3][4][5][6]. Recently, tetrathiafulvalene (TTF) derivatives have been incorporated in oligonucleotides demonstrating the fluorescence-quenching properties of the DNA-TTF conjugates upon hybridization [7], the preservation of the electrochemical properties of TTF [8], an enhanced affinity to complementary sequences [9], and compatibility with the RNA interference mechanism for gene inhibition [10]. In the field of materials chemistry, TTF and its derivatives constitute electron donors that can form charge-transfer (CT) complexes and have been widely studied for the development of electrically conducting materials [11,12]. TTF moiety can be consecutively oxidized to the radical cation TTF + and dication TTF 2+ by either electrochemical or chemical processes. Due to the reversibility of these transformations [13], TTF has become a general building block for the fabrication of switchable functional materials [14][15][16][17], including supramolecular self-assembled nanostructures [18][19][20][21].
Within this context, Jørgensen and co-workers reported in 1994 [54] the first TTF-based LMW gelator bearing bis-arborol units, which was used to fabricate a self-assembling "molecular" nanowire [55]. Since then, a large number of electroactive TTF-based LMW gelators and their gels have been described in the literature [56]. In general, the rational design behind these examples involves the connection of the TTF unit with groups capable of H-bonding, such as urea [57] or amide groups [58][59][60][61][62][63], to form extended structures. The incorporation of other molecular building blocks commonly used to form physical gels [64][65][66][67] or the use of metal-organic interactions [68][69][70] have also been employed to form TTF-based gels. Moreover, the inclusion of hydrophobic aliphatic chains is also employed to facilitate van der Waals interactions and stabilize the supramolecular aggregates [58][59][60][61][62][63][64][65][66][67][68][69][70][71]. If formation of CT complexes takes place, the TTF unit will bear positive charge, which can alter the interaction of neighboring TTF units and influence the intermolecular interactions arising from other groups involved in the self-assembly process. Indeed, the above-mentioned studies have confirmed the possibility of tuning the gel formation by CT interaction and oxidation. In addition, conductive gels have also been fabricated by simple mixing and mechanically grinding neutral TTF and an acceptor (i.e., tetracyanoquinodimethane) in ionic liquids [72]. More recently, gels made of TTF-dipeptide and tripeptide conjugates have been reported [73,74].
In this communication we describe the synthesis, characterization, and gelation properties of a new TTF-triglycyl-based LMW organogelator ( Figure 1). As far as we are aware, a triglycyl peptide has not been used to fabricate TTF-based gelators.

Materials
Unless otherwise noted, all reagents and dry solvents were purchased from commercial suppliers (Sigma-Aldrich, St. Louis, MO, USA, TCI Chemicals, Tokyo, Japan) and used as received.

Materials
Unless otherwise noted, all reagents and dry solvents were purchased from commercial suppliers (Sigma-Aldrich, St. Louis, MO, USA, TCI Chemicals, Tokyo, Japan) and used as received.

Synthetic Procedures and Characterization of Compounds
{[4 ,5,5 -Tris(butylsulfanyl)-2,2 -bis-1,3-dithiol-4-yl]sulfanyl}propanoic acid (8, 0.95 g, 1.66 mmol) along with N-hydroxysuccinimide (NHS) (Sigma-Aldrich) (0.21 g, 1.83 mmol) were dissolved in anhydrous THF (40 mL) under argon and the solution was cooled at 0 ºC. Then, DCC (0.38 g, 1.83 mmol) was added under argon and the reaction was stirred at 0 • C for 30 min and at RT overnight. The precipitate was filtered out and the solvent was removed under reduced pressure. The resulting N-hydroxysuccinimide ester was used in the next step without further purification. The active ester was dissolved in anhydrous DMF (30 mL). The TFA salt of the deprotected amino compound was dissolved in DMF (30 mL) and N,N-diisopropylethylamine (DIEA) (405 µL, 2.32 mmol) was added. Then, the solution of the active ester was added under argon and the mixture was allowed to react at RT for 30 min. The solvent was removed under reduced pressure and the residue was dissolved in toluene and concentrated to dryness (×3). The crude compound was washed with hexane (5 × 70 mL) and with diethyl ether (5 × 70 mL). The residue was dissolved in DCM (300 mL) and the organic phase was washed with 5% aqueous NaHCO 3 (2 × 75 mL) and saturated aqueous NaCl (75 mL

Preparation and Characterization of Gel Materials
Anhydrous solvents used for gelation tests were purchased from commercial suppliers. Gelation tests were carried out in screw-capped glass vials (4 cm length × 1 cm diameter) having a specific amount of the TTF derivative 14 and the desired solvent (1 mL). The mixture was heated with a standard heat gun until complete dissolution. Then, the clear solution was allowed to cool down to RT. The material was initially classified as gel if it did not flow by turning the vial upside-down. The viscoelastic nature was further confirmed by rheology of a model system.
Critical gelation concentration (CGC) corresponds to the minimum gelator concentration required for gelation. The values were obtained by continuously adding aliquots of solvent (0.02-0.1 mL) into vials having the TTF derivative 14 and performing either the heating or cooling until no gelation was observed. The waiting time used to define the state of the material was~12 h. Concentrations above 200 g/L were not tested.
Thermal gel-to-sol transition temperature (T gel ) values were determined using a calibrated thermoblock at 1 • C/5 min ( Figure S1). The temperature at which the bulk gel started to break was defined as T gel . Due to potential effects of thermal history and hysteresis [75], the apparatus has been previously calibrated with samples measured by differential scanning calorimetry (DSC) [76].
Field emission scanning electron microscopy (FE-SEM) of xerogels was carried out with a Zeiss Merlin, Field Emission Scanning Electron Microscope (accelerating voltage = 10 kV). Xerogels, prepared by freeze-drying the gels, were placed on top of a tin plate and shielded with Pt (40 mA, 30-60 s; film thickness = 5-10 nm). Images were by Servicio General de Apoyo a la Investigación-SAI (University of Zaragoza).
Images under polarized light were obtained using a Wild Makroskop M420 optical microscope equipped with a Canon Power shot A640 camera.
UV-vis spectra were obtained with an Ocean Optics, Flame spectrometer with a DH-2000-BAL light source or on a Varian Cary 50 UV spectrophotometer. Quartz-glass cuvettes of 0.5 cm thickness were used.
Iodine doping was achieved by exposing the xerogel to I 2 vapor for circa 120 min in a sealed glass beaker with I 2 crystals.

Synthesis of TTF-Triglycyl Derivative
The synthesis of the TTF-triglycyl derivative 14 was achieved by assembling the L-threoninol, and triglycine units with the TTF carboxylic derivative 8 (Scheme 1). First, compound 8 was prepared following the procedure described in the literature [10] with slight modifications. The first step consisted in the synthesis of 4,5-bis-(butylthio)-1,3-dithiole-2-thione (3) by alkylation of the zinc complex of bis(1,3-dithiole-2-thione-4,5-dithiolate) tetrabutylammonium salt (1) with 1-bromobutane (2) as described for similar compounds by Simonsen and co-workers [77]. Phosphite-induced coupling of compounds 3 and 4 [77] gave the symmetric TTF derivative 5 in good yield (64%). Selective sequential deprotection of one 2-cyanoethyl group followed by alkylation with 1-bromobutane (2) gave the TTF derivative 6 in very good yield (89%). A second round of sequential deprotection of the second 2-cyanoethyl group followed by alkylation with 3-bromopropionic acid (7)  1-bromobutane (2) gave the TTF derivative 6 in very good yield (89%). A second round of sequential deprotection of the second 2-cyanoethyl group followed by alkylation with 3-bromopropionic acid (7) [78] gave the desired TTF carboxylic derivative 8 in 91% yield. Then Boc-protected triglycine 9 was activated with DCC and 4-nitrophenol (10). Then, the obtained nitrophenyl active ester 11 was reacted with L-threoninol (12) yielding the t-butoxycarbonyl (Boc)-protected triglycine-L-threoninol derivative 13. This compound was treated with a TFA solution in DCM to remove the Boc-amino protecting group yielding the corresponding L-threoninol-triglycine trifluoroacetate salt that was subsequently used without purification for the coupling with compound 8 in the presence of Hünig's base. This reaction was done via classical activation of the carboxyl group with DCC and NHS, affording the desired TTF-based gelator 14 in 24% (isolated yield). Then Boc-protected triglycine 9 was activated with DCC and 4-nitrophenol (10). Then, the obtained nitrophenyl active ester 11 was reacted with L-threoninol (12) yielding the t-butoxycarbonyl (Boc)-protected triglycine-L-threoninol derivative 13. This compound was treated with a TFA solution in DCM to remove the Boc-amino protecting group yielding the corresponding L-threoninol-triglycine trifluoroacetate salt that was subsequently used without purification for the coupling with compound 8 in the presence of Hünig's base. This reaction was done via classical activation of the carboxyl group with DCC and NHS, affording the desired TTF-based gelator 14 in 24% (isolated yield).

Gelation Properties
The gelation ability of TTF-triglycyl derivative 14 was studied for a number of anhydrous solvents of different nature (i.e., apolar, polar aprotic, polar protic) using the standard heating-cooling treatment. Materials that did flow upon inversion of the vial upside-down were initially classified as gels. One of the gels was used later as a model example to demonstrate its viscoelastic nature by oscillatory rheological measurements (see below). Compound 14 was found to be soluble in methanol, ethanol, isopropanol, dimethylformamide, dimethyl sulfoxide, and tetrahydrofuran, whereas it resulted insoluble in diethyl ether, acetonitrile, n-hexane, and cyclohexane. In contrast, opaque gels were obtained in 10 solvents at concentrations varying between 5 ± 1 and 50 ± 10 g/L (Table 1). Table 1. Gelation ability of TTF-triglycyl derivative 14, critical gelation concentration (CGC), gelation time, and gel-to-sol transition temperature (T gel ). 1
These gels remained stable for at least two months when stored in sealed vials at RT. Their color varied from bright orange to dark orange-brown depending on the solvent (Figure 2a). The supramolecular gels showed typical thermoreversibility (Figure 2b), and their opaque appearance suggested the presence of aggregates larger than the wavelength of visible light (λ = 380-780 nm), which was in agreement with electron microscopy (see below). Further investigations were carried out using only some selected gels as representative materials.
As mentioned in the introduction, it is well established that the redox TTF core in this type of LMW also provides both S···S and π···π interactions during molecular aggregation, whereas the peptide chain drives molecular aggregation via hydrogen bonding [73,74]. Comparison of Fourier transform infrared (FT-IR) spectra of solid TTF derivative 14 with that of the xerogel (i.e., prepared by freeze-drying the corresponding organogel) and 14 in solution (i.e., below the CGC) showed only very small frequency shifts (i.e., red-shift ∆ν~5 cm −1 ) for characteristic bands such as C=O stretching (~1641 cm −1 ), N-H amide I (br~3280 cm −1 ), and N-H amide II (bending vibration) (~1553 cm −1 ) ( Figure S2). These values suggest that 14 may also be aggregated in the solid state and in solution via similar intermolecular hydrogen-bonding pattern between amide groups, most likely in the parallel β-sheet conformation [79], as well as van der Waals interactions between the alkyl chains. CGC up to 3-4 times in comparison to anhydrous solvents. Error values were calculated from three randomized experiments. 2 Solvent volume = 1 mL. 3 Xylene was used as mixture of isomers. 4 Partial gel formation was observed. In this case, Tgel value corresponds to the isolated gelled portion.
These gels remained stable for at least two months when stored in sealed vials at RT. Their color varied from bright orange to dark orange-brown depending on the solvent (Figure 2a). The supramolecular gels showed typical thermoreversibility (Figure 2b), and their opaque appearance suggested the presence of aggregates larger than the wavelength of visible light (λ = 380-780 nm), which was in agreement with electron microscopy (see below). Further investigations were carried out using only some selected gels as representative materials.  Table 1); (b) illustration of the thermal sol-gel transition of the gel made of 14 in toluene at the CGC.  Table 1); (b) illustration of the thermal sol-gel transition of the gel made of 14 in toluene at the CGC.

Characterization of Organogels
In general, relatively low gel-to-sol transition temperatures (T gel ) were determined for all gels ranging from 36 to 51 • C (±3) ( Table 1). As typically observed with most supramolecular gels, T gel gradually increased with increasing gelator concentration due to the generation of denser networks (Figure 3). Taking the gels prepared in toluene and ethyl acetate as representative systems, a plateau region was reached before the gels collapsed into partial and inhomogeneous gels that constantly lose solvent over time. The increment of the T gel until the plateau region was~39 • C for toluene and 25 • C for ethyl acetate with respect to the initial values obtained at their CGC. Linear Ln-Ln plot (Figure 3, inset) using the percentage increases of T gel showed that the percentage increment of T gel was~1.1-fold higher for the gel made in ethyl acetate. Further research is necessary to understand the reasons behind such differences.
Appl. Sci. 2018, 8, x FOR PEER REVIEW 9 of 16 As mentioned in the introduction, it is well established that the redox TTF core in this type of LMW also provides both S···S and π···π interactions during molecular aggregation, whereas the peptide chain drives molecular aggregation via hydrogen bonding [73,74]. Comparison of Fourier transform infrared (FT-IR) spectra of solid TTF derivative 14 with that of the xerogel (i.e., prepared by freeze-drying the corresponding organogel) and 14 in solution (i.e., below the CGC) showed only very small frequency shifts (i.e., red-shift Δν ~ 5 cm −1 ) for characteristic bands such as C=O stretching (~1641 cm −1 ), N-H amide I (br ~ 3280 cm −1 ), and N-H amide II (bending vibration) (~1553 cm −1 ) ( Figure S2). These values suggest that 14 may also be aggregated in the solid state and in solution via similar intermolecular hydrogen-bonding pattern between amide groups, most likely in the parallel β-sheet conformation [79], as well as van der Waals interactions between the alkyl chains.

Characterization of Organogels
In general, relatively low gel-to-sol transition temperatures (Tgel) were determined for all gels ranging from 36 to 51 °C (±3) ( Table 1). As typically observed with most supramolecular gels, Tgel gradually increased with increasing gelator concentration due to the generation of denser networks (Figure 3). Taking the gels prepared in toluene and ethyl acetate as representative systems, a plateau region was reached before the gels collapsed into partial and inhomogeneous gels that constantly lose solvent over time. The increment of the Tgel until the plateau region was ~39 °C for toluene and ~25 °C for ethyl acetate with respect to the initial values obtained at their CGC. Linear Ln-Ln plot (Figure 3, inset) using the percentage increases of Tgel showed that the percentage increment of Tgel was ~1.1-fold higher for the gel made in ethyl acetate. Further research is necessary to understand the reasons behind such differences.  Dynamic rheological experiments (Figure 4) for the model gel made of 14 in toluene confirmed its viscoelastic nature. Within the linear regime established by dynamic frequency sweep (DFS) and dynamic strain sweep (DFS) measurements, the storage modulus G was found to be one order of magnitude higher than the loss modulus G with low frequency dependency (i.e., G ≈ 8.9 ± 0.5 kPa, G ≈ 0.75 ± 0.1 kPa) (Figure 3a). Moreover, the gels were brittle in nature, which was confirmed by its destruction at low frequency and~7% of strain (Figure 4a). Finally, dynamic time sweep (DTS) measurements at 0.1% strain and 1 Hz frequency confirmed the stability of the gel over time at RT (Figure 4b).
Appl. Sci. 2018, 8, x FOR PEER REVIEW 10 of 16 Dynamic rheological experiments (Figure 4) for the model gel made of 14 in toluene confirmed its viscoelastic nature. Within the linear regime established by dynamic frequency sweep (DFS) and dynamic strain sweep (DFS) measurements, the storage modulus G′′ was found to be one order of magnitude higher than the loss modulus G′′ with low frequency dependency (i.e., G′ ≈ 8.9 ± 0.5 kPa, G′′ ≈ 0.75 ± 0.1 kPa) (Figure 3a). Moreover, the gels were brittle in nature, which was confirmed by its destruction at low frequency and ~7% of strain (Figure 4a). Finally, dynamic time sweep (DTS) measurements at 0.1% strain and 1 Hz frequency confirmed the stability of the gel over time at RT (Figure 4b).
(a) (b) Morphological studies of selected organogels were conducted by field emission scanning electron microscopy (FE-SEM) of the corresponding xerogels ( Figure 5). In general, fibrillar networks with high aspect ratio were observed for several organogels made of 14. As observed with other supramolecular self-assembled gels, interactions between solvent-aggregate, solvent-gelator, and gelator-gelator molecules are also regulated by the solvent nature and have a major effect on the microstructure of the materials. For instance, dense and entangled leaf-like structures were observed for the material obtained in ethyl acetate (Figure 5a), whereas the samples prepared in benzonitrile Morphological studies of selected organogels were conducted by field emission scanning electron microscopy (FE-SEM) of the corresponding xerogels ( Figure 5). In general, fibrillar networks with high aspect ratio were observed for several organogels made of 14. As observed with other supramolecular self-assembled gels, interactions between solvent-aggregate, solvent-gelator, and gelator-gelator molecules are also regulated by the solvent nature and have a major effect on the microstructure of the materials. For instance, dense and entangled leaf-like structures were observed for the material obtained in ethyl acetate (Figure 5a), whereas the samples prepared in benzonitrile showed a smooth surface with fibrillar structures of~0.5-1 µm in diameter (Figure 5b). A highly dense and porous network made of fibers up to~1 µm in diameter was observed for the gel made in 1,4-dioxane (Figure 5c). Surfaces made of smaller fibers of~50-100 nm in diameter resembling a mountainous valley were observed for the specimens prepared in toluene (Figure 5d). However, fibrillar-globular structures with diameters between~100 and 500 nm were distinguished when the solvent used for the preparation of the gel was dichloromethane (Figure 5e). Although we systematically took pictures of the bulk samples at different magnifications and locations on the grid in order to identify possible artifacts in the microstructures, it should be considered that such artifacts can be produced during the preparation and visualization of the samples and, therefore, the conclusions derived from these images should not be oversold. Furthermore, the typical anisotropic growth in this type of materials, suggesting well-ordered molecular packing, is also evidenced by the observation of birefringent domains under polarized light (Figure 5f), which is important for optical applications [80]. showed a smooth surface with fibrillar structures of ~0.5-1 µm in diameter (Figure 5b). A highly dense and porous network made of fibers up to ~1 µm in diameter was observed for the gel made in 1,4-dioxane (Figure 5c). Surfaces made of smaller fibers of ~50-100 nm in diameter resembling a mountainous valley were observed for the specimens prepared in toluene (Figure 5d). However, fibrillar-globular structures with diameters between ~100 and 500 nm were distinguished when the solvent used for the preparation of the gel was dichloromethane (Figure 5e). Although we systematically took pictures of the bulk samples at different magnifications and locations on the grid in order to identify possible artifacts in the microstructures, it should be considered that such artifacts can be produced during the preparation and visualization of the samples and, therefore, the conclusions derived from these images should not be oversold. Furthermore, the typical anisotropic growth in this type of materials, suggesting well-ordered molecular packing, is also evidenced by the observation of birefringent domains under polarized light (Figure 5f), which is important for optical applications [80].

Effect of Iodine-Doping
Iodine-doping of the fibrous xerogel derived from a model gel made in DCM confirmed the oxidation of the TTF moiety in the gelator preserving the self-assembled network structure. UV-vis-NIR spectrum of the xerogel after exposure to I2 vapor showed new absorption bands centered at approximately 360, 500, and 850 nm ( Figure 6). The absorption bands at 360 and 500 nm are typically ascribed to intramolecular electronic transitions involving the TTF cation radical, whereas the broad band located at 850 nm indicates the formation of a full CT state between cation radicals of self-stacked TTF units (i.e., TTF + I -). The increase in the absorbance above 600 nm is in agreement with the formation of a partial mixed-valence CT state between TTF-containing neutral and cation radicals (i.e., (TTF)(I)n with n < 1) [58,73]. Unfortunately, the intermolecular hydrogen-bonded network seemed to be altered during the oxidation as shown by visible changes in the intensity and position of the IR absorption bands associated to N-H and C=O stretching vibrations of the amides ( Figure S3). Unfortunately, although iodine can create the necessary unfilled states required for conduction, the instability of the fibrillar network prevented us from obtaining reproducible conductivity values. As observed with other similar gels, preliminary experiments also showed a gel-to-sol transition after exposure of the wet gel to iodine vapors.

Effect of Iodine-Doping
Iodine-doping of the fibrous xerogel derived from a model gel made in DCM confirmed the oxidation of the TTF moiety in the gelator preserving the self-assembled network structure. UV-vis-NIR spectrum of the xerogel after exposure to I 2 vapor showed new absorption bands centered at approximately 360, 500, and 850 nm ( Figure 6). The absorption bands at 360 and 500 nm are typically ascribed to intramolecular electronic transitions involving the TTF cation radical, whereas the broad band located at 850 nm indicates the formation of a full CT state between cation radicals of self-stacked TTF units (i.e., TTF + I − ). The increase in the absorbance above 600 nm is in agreement with the formation of a partial mixed-valence CT state between TTF-containing neutral and cation radicals (i.e., (TTF)(I) n with n < 1) [58,73]. Unfortunately, the intermolecular hydrogen-bonded network seemed to be altered during the oxidation as shown by visible changes in the intensity and position of the IR absorption bands associated to N-H and C=O stretching vibrations of the amides ( Figure S3). Unfortunately, although iodine can create the necessary unfilled states required for conduction, the instability of the fibrillar network prevented us from obtaining reproducible conductivity values. As observed with other similar gels, preliminary experiments also showed a gel-to-sol transition after exposure of the wet gel to iodine vapors.

Conclusions
In conclusion, TTF-triglycyl derivative 14 was synthesized and fully characterized by standard techniques. This compound was found to act as a LMW gelator for a series of organic solvents after a heating-cooling cycle. Therefore, the presence of aromatic rings on the peptide linked to the TTF unit is not a requirement for the formation of supramolecular gels using TTF-peptide conjugates. The critical gelation concentrations ranged from ~5 to 50 g/L, and the thermal gel-to-sol transition temperatures varied from ~36 to 51 °C. FE-SEM imaging of the xerogels, prepared by freeze-drying the organogels, showed fibrillar microstructures with distinctive features depending on the solvent used to prepare the gels. Moreover, the viscoelastic and brittle nature of a model gel system was also supported by dynamic rheological experiments. Finally, UV-vis-NIR analysis of a model xerogel confirmed the oxidation of the TTF unit upon exposure to I2 vapor. Nevertheless, FT-IR spectroscopy showed that the oxidation was accompanied with concurrent alteration of the intermolecular hydrogen-bonded network.
Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, NMR spectra, FT-IR spectra, Figure S1