Remarkable Morphology Transformation from Fiber to Nanotube of a Histidine Organogel in Presence of a Binuclear Iron(III)–Sulfur Complex
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Ferrocene (Fc)–peptide conjugates are an interesting class of compounds that have been explored extensively due to the ability of Fc to act as a scaffold to support peptide secondary structural motifs. Fc–peptide conjugates have also displayed self-assembly and gelation behaviour forming an interesting class of new biomaterials. Here, a Fc–phenylalanine–histidine–OMe conjugate has been synthesized and tested for supramolecular gelation. Interestingly, this compound forms a supramolecular gel in the presence of the binuclear iron–sulfur (Fe–S) cluster and displays interesting properties including redox-responsiveness and morphological changes from fibers to spherical vesicles. The gelation in the presence of the Fe–S cluster has future potential to study prebiotic self-assembly, prebiotic cofactor relationships and biomimetic active sites.
KeywordsOrganogel Ferrocene Peptides Iron–sulfur Supramolecular
Supramolecular gels have attracted a lot of attention over the last several years due to their variety of applications in different fields [1, 2]. They can be studied in materials science, catalysis, drug delivery, environmental studies, as biomimics, and in supramolecular chemistry. The formation of self-assembled gels by low-molecular weight gelators involves non-covalent interactions such as hydrogen bonding, π–π stacking, hydrophobic, and van der Waal interactions [3, 4]. These non-covalent interactions are responsible for forming self-assembled three dimensional nanofibrillar network structures. Hydrogen bonding is stated to be the most significant non-covalent interaction in supramolecular assembly [5, 6, 7]. Gels are also responsive to various external stimuli including heat, light, sonication, and pH [3, 8, 9, 10, 11].
Bio-organometallic ferrocene (Fc) amino acid and peptide conjugates have been explored extensively, largely to study the ability of Fc to act as a scaffold to support peptide secondary structural motifs [12, 13, 14]. More recently, it was discovered that Fc–peptides can also be triggered to form stimuli responsive gels [10, 11, 15, 16]. Many of the reported stable Fc–peptide gels are formed when conjugates have a single podant peptide chain, whereas for di-substituted systems, intramolecular H-bonding interactions between adjacent peptide chains dominate and interfere with effective intermolecular interactions . This subtle interplay between intra- and intermolecular interactions make Fc–peptides an interesting group of conjugates to be studied as small structural and/or environmental changes can influence gelation properties dramatically. The presence of the Fc group also imparts redox responsiveness to Fc–peptide based gels . Fc–peptide derivatives display self-assembly and gelation behaviour, forming a range of nanostructures, including nanofibers, nanotubes, and nanovesicles . For example, Zhang et al. have reported ferrocenoyl phenylalanine as a novel hydrogelator and explored its responsiveness to a series of stimuli including redox reactions, guest–host interactions and pH changes . Fang et al. reported four novel cholesterol-conjugated Fc gelators, which respond to shaking, sonication, heating and redox reactions . In addition, our group has reported multiple Fc–peptide conjugates capable of metal binding and forming organogels [18, 19, 20, 21, 22, 23, 24, 25]. This includes a disubstituted Fc–tryptophan conjugate capable of gel formation and exhibits redox responsiveness .
Histidine is an amino acid that is commonly reported as a metal binding site in metalloproteins and nucleases due to the imidazole ring [26, 27]. Histidine’s stabilizing role in complex formation reactions of short oligopeptides has been well recognized and carefully reviewed. Fc–histidine conjugates have been synthesized and explored as active site mimics and for catalysis however, the addition of prebiotic cofactors have not been explored . Histidine residues have the ability to form discrete metal complexes by coordinating divalent metal ions such as Zn, Cd, and Co . To the best of our knowledge Fc–histidine conjugates have not been widely explored as gelators. Histidine gels without Fc have been reported as organogels , for example Roy et al. recently explored solely a histidine based peptide amphiphile capable of forming a stable hydrogel at physiological pH . They also investigated metal salts inducing variation in self-assembling behaviour of these amphiphiles as a superior strategy to access diverse nanostructures and physical properties utilizing a single gelator design. Combining the metal binding properties of histidine residues and the redox responsiveness of the Fc into one gelator holds promise for the investigation of interesting morphological and physical properties of novel biomimetic gel materials.
As mentioned, the addition of prebiotic cofactors to Fc–peptide conjugate mimetic models have not been explored. Iron–sulfur (Fe–S) clusters are ubiquitous cofactors composed of iron and inorganic sulfur. They are required for the function of proteins involved in a wide range of activities including electron transport in respiratory chain complexes, regulatory sensing, photosynthesis and DNA repair. They are evolutionary ancient and are present in essentially all organisms including archaea, bacteria, plants, and animals . The high level of evolutionary conservation is consistent with the possibility that Fe–S clusters contributed to the success of early forms of life. Despite their vulnerability to oxidation and degradation, Fe–S clusters are crucial for facilitating enzyme activities in all kingdoms of life because they can bind electron-rich enzymatic substrates, accept or donate single electrons, and stabilize specific protein conformations that are important to the activities of numerous proteins . Constructing enzyme models with the addition of Fe–S clusters present in the system allows the potential to study these enzyme activities and active sites in prebiotic life. Hydrogenase enzymes are a class of enzymes that are reported to achieve electron transfer to or from the active site through a series of Fe–S clusters . One example of a model mimic using a Fc–peptide conjugate and Fe–S cluster was reported by Metzler-Nolte where he constructed a more realistic, peptide-based hydrogenase model covalently attaching a diiron-hexacarbonyl core to two cysteine molecules and are connected via peptide bonds to a Fc moiety .
This study reports in fact the first Fc–histidine gel material of its kind. Changes in the rheological properties were observed as a function of metal ions added, and while we reported on metal sensitive gel materials before, the current paper is the first of its kind that reports metal sensitivity in Fc–histidine gel materials. We synthesize and report the properties of a Fc–Phe–His–OMe (G1) conjugate. This compound was found to form an organogel with self-assembling and thixotropic properties. In addition, this compound gels in the presence of the binuclear Fe–S cluster. This can be used to elucidate a biomimetic material that has potential for examining prebiotic self-assembly and as a prebiotic catalytic model through a gel matrix. Recreating the prebiotic world in the laboratory to investigate these very early reactions and the assemblies and aggregates en route to life and materials is not trivial and have required large assumptions . It was found that the addition also allows for a remarkable morphological transformation. Interestingly, the addition of oxidizing and reducing agents also influences the gel’s morphology as there is a change from a typical nanofiber matrix to round vesicles that are interchangeable with the redox agents.
HCl·H2N–LPhe–OMe, HCl·H2N–LHis–OMe, 6Cl–hydroxybenzotriazole (Cl–HOBt) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDAC·HCl) were purchased from Advanced Automated Peptide Protein Technologies (AAPPTEC). Fc, trifluoroacetic acid (TFA), triethylsilane and triethyl amine (TEA) were purchased from Sigma Aldrich. All the chemicals were used without further purification. Products were purified by column chromatography, on a column packed with silica gel.
2.1 General Synthesis of FcCO–Phe–His–OMe (G1)
The full synthetic scheme of G1 can be found in the SI, Fig. S1. Ferrocene-monocarboxylic (FcCOOH) acid was synthesized according to modified literature , In a 250 mL round bottom flask, 5 mmol of FcCOOH was dissolved in 60 mL of DCM. 10 mmol of EDAC·HCl and 10 mmol of 6Cl–HOBt were added to this solution. This solution was moved to an ice bath with continuous stirring. In another 250 mL round bottom flask, 10 mmol of H2N–(l)Phe–OMe·HCl was dissolved in 50 mL DCM, excess of TEA was added and stirred for 30 min. Excess solvent was evaporated under reduced pressure until a slurry was obtained. This slurry was added to FcCOOH solution and stirred at room temperature for 24 h. After 24 h, the resulting solution was washed with 10% citric acid (30 mL, 3 times), saturated sodium bicarbonate (30 mL, 3 times) and brine solution (30 mL, 3 times), respectively. The resulting organic layer was dried over anhydrous Na2SO4. The resulting solution was filtered and evaporated under reduced pressure and column chromatography was done to purify this mixture to obtain pure FcCO–(l)Phe–OMe.
Fc–CO–(l)Phe–OMe was dissolved in MeOH and 1 N NaOH (20 mL) was added. The reaction mixture was stirred and the progress of saponification was monitored by thin layer chromatography. After 10 h methanol was removed under vacuum, the residue was taken in 50 mL of water, washed with diethyl ether (2 × 50 mL). Then the pH of the aqueous layer was adjusted to 2 using 1 M HCl and it was extracted with ethyl acetate (3 × 50 mL). The extracts were dried over anhydrous Na2SO4, and evaporated in vacuum to yield as a yellow solid Fc–CO–(l)Phe–OH.
In a 250 mL round bottom flask, 3.0 mmol of Fc–CO–(l)Phe–OH was dissolved in 50 mL of DCM by continuous stirring. Once the entire solid dissolved the mixture was cooled to 0 °C in an ice bath. To the cooled mixture, 6 mmol of EDAC·HCl was added following the addition of 6 mmol of 6Cl–HOBt. In the same flask an excess of TEA was added to neutralize the HCl. In another 250 mL round bottom flask, 6 mmol of H2N–(l)His(Trt)–OMe·HCl was dissolved in 25 mL DCM, excess of TEA was added and stirred for 30 min. Excess solvent was evaporated under reduced pressure until a slurry was obtained. This slurry was added to Fc–CO–(l)Phe–OH solution and stirred for 24 h. The resulting solution washed with saturated 10% citric acid (30 mL, 3 times), saturated sodium bicarbonate (30 mL, 3 times) and brine solution (30 mL, 3 times) respectively. The resulting organic layer was dried using anhydrous Na2SO4 and evaporated under reduced pressure. Column chromatography was done to purify this mixture to obtain a pure compound, Fc–CO–(l)Phe–(l)His(Trt)–OMe.
2.2 mmol of Fc–CO–(l)Phe–(l)His(Trt)–OMe was dissolved in DCM and 2.25 mmol of triethylsilane was added. The reaction mixture was stirred under N2 atmosphere. After 15 min, 2 mL of TFA was added to the reaction mixture. After 1 h, the TFA was removed under vacuum. The reaction product was diluted with 3–4 mL water and washed with diethyl ether. DCM was added to the reaction mixture and the clear water part then neutralized by NH4OH solution. The orange product was pulled in DCM layer and dried over anhydrous Na2SO4. Column chromatography was done to purify this mixture to obtain a pure compound, Fc–CO–(l)Phe–(l)His–OMe (G1). 1H NMR (500 MHz, CDCl3): δ 7.38–7.07 (m, 10H, Aromatic Phe), 6.66–6.65 (d, 1H, NH Phe, J = 8.0), 6.43–6.42 (d, 1H, NH Leu, J = 8.0), 5.95–5.93 (d, 1H, NH Phe, J = 6.0), 4.74–4.31 (m, 7H, 3αH and 4H Cp ring which is directly attached to peptide segment), 3.95 (s, 5H, another Cp ring which is not directly attached to peptide segment), 3.71 (s, 3H, OCH3), 3.23–2.96 (m, 4H, βCH2 Phe), 1.61–1.45 (m, 3H, βCH2 and γCH Leu), 0.90–0.87 (t, 6H, 2CH3, Leu, J = 6.75); 13C NMR (126 MHz, CDCl3) δ 138.18, 135.12, 129.19, 129.14, 128.51, 128.47, 126.62, 75.54, 70.39, 70.35, 70.34, 69.52, 69.48, 68.60, 68.57, 67.86, 67.73, 54.54, 54.44, 52.70, 51.71, 36.96, 36.86, 28.61; MS–(ESI) m/z 529.1528 [M+H]+. Yield 1.4 mmol, 63%.
2.2 Nuclear Magnetic Resonance (NMR)
All liquid-phase NMR studies were carried out on a Bruker spectrometer 500 MHz 300 K using CDCl3 maintaining the concentration 10 mM. Chemical shifts (δ) were reported in ppm for all 1D-HNMR. Data processed in MestReNova software.
2.3 Mass Spectrometry
All experiments were performed on Agilent Technologies 6530 Accurate-Mass Q-TOF LC/MS spectrometer.
A small piece of gel sample (10 mM) was placed on Peltier controlled rheology plate (20 mm parallel plate geometry maintaining 500 μm gap) and rheological measurements were carried out using a TA Instruments DHR-1 rheometer. Temperature was kept at 25 °C for all experiments. For frequency sweep experiments, the gels were scanned in the range of frequency 0.1–100 rad s−1.
2.5 Transmission Electron Microscopy
The morphologies of all organogels have been studied by using transmission electron microscopy (TEM) at room temperature (25 °C). 10 mM, 1 mL organogel was prepared freshly for each gelators in toluene (Tol). 20 µL of these gel samples was further diluted to 1 mL of Tol and 5 µL of these dilute solution was placed on a TEM grid (300 mesh size Cu grid) coated with a carbon film. The grid was allowed to dry by slow evaporation in air, and then allowed to dry separately in a vacuum overnight. Images were taken using a Hitachi 7500 transmission EM. The fiber widths were calculated by using ImageJ software.
2.6 Fourier Transform Infrared Spectroscopy (FTIR)
A drop of gel sample was allowed to dry in air and FTIR spectra were recorded using a Bruker ALPHA FTIR spectrometer equipped with a diamond ATR.
2.7 UV–Vis Spectroscopy
Ultraviolet–visible (UV–vis) absorption spectra were acquired on Agilent Technologies Cary 60 UV–vis spectrophotometer in 1 cm quartz cuvette cell at 20 °C. Samples were studied at concentrations of 0.33 mM in the acetonitrile (ACN). The metal salts were added in 0.1 equiv. portions to the gelator solution.
2.8 Gelation Properties
The compound was taken in a glass vial and heated gently to dissolve in organic solvents. After dissolving and cooling to room temperature to form organogels. Gel melting temperature determination was performed by heating gels in an oil bath fitted with a thermometer at a heating rate of 2 °C 5 min−1 until the gel melted. The melting has been confirmed by vial inversion method. The calculated error range in Tgel determination was found to be ± 1 °C.
3 Results and Discussion
3.1 Organogel Formation
3.2 Metal-Binding Studies and Its Viscoelastic Properties
3.3 Redox-Responsiveness Behaviour of Fc–Phe–His–OH
The morphology of the self-assembled gel obtained from gelator G1 was studied by TEM. The organogel formed a cross-linked fibrous network (Fig. 6b) in mixed solvent system (ACN:Tol 1:2) which is characteristic of supramolecular gels. Average width of the nanofibers obtained from Tol/ACN gel is 28 (± 7) nm at MGC. Remarkably, after oxidation of the self-assembled gel, a vesicular morphology (Fig. 6c, d) was obtained from the blue solution. From the TEM images it is evident that the large vesicles were capable of encapsulating smaller vesicles. The diameters of the vesicles ranged from 1050 to 200 nm. The thickness of the side-wall of the large vesicles appeared to be 110 nm. However, after reduction, the recovered compound formed organogel and the fibrous morphology appeared similar to the organogel before oxidation (Fig. 6e). The average width of the nanofibers appeared 16 (± 3) nm. Moreover, the metallogel obtained from the gelator G1 in presence of the binuclear iron complex C2 showed a nanotubular morphology (Fig. S11) which is remarkably different from any of the organogels. The average diameter of these nano-tubes appeared 24 (± 6) nm.
In this work a Fc–peptide conjugate was synthesized and explored for supramolecular gel formation. It was found that this compound formed an organogel in a Tol/ACN mixture with a nanofiber morphology. Interestingly, due to the histidine amino acid residue, this gel was able to self-assemble in the presence of an Fe–S cluster and coordinate to different metals. Incorporating the Fc moiety imparted redox responsiveness, where this gel was responsive to redox agents. Upon addition of an oxidizing agent the supramolecular state was disrupted and reverted the gel into a sol state, while having an effect on the morphology by turning the fibers to spheres. Upon the addition of a reducing agent, the gel can be reformed with a fiber morphology. Combining the metal binding properties of histidine residues and the redox responsiveness of the Fc into one gelator holds promise for interesting morphological and physical properties characterization of novel gel materials. In addition, the gelation in the presence of the Fe–S cluster has future potential to study prebiotic self-assembly, prebiotic cofactor relationships and biomimetic active sites.
We appreciate and gratefully acknowledge the funding from the National Sciences and Engineering Research Council (NSERC) and the University of Toronto.
- 24.N. Falcone, H.-B. Kraatz, Chapter 3: ferrocene peptide-based supramolecular gels: current trends and applications, in Advances in Bioorganometallic Chemistry (2019), pp. 57–74. https://doi.org/10.1016/B978-0-12-814197-7.00003-0 CrossRefGoogle Scholar