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Tunable reversible metallo-hydrogels: a new platform for visual discrimination of biothiols†‡ Weiwei Fang, Cong Liu, Zhengwei Lu, Zheming Sun and Tao Tu*
Received 23rd June 2014, Accepted 14th July 2014 DOI: 10.1039/c4cc04743e www.rsc.org/chemcomm
By using metallo-hydrogel as a new platform, a simple and straightforward selective visual discrimination of cysteine, homocysteine and glutathione from each other as well as from other amino acids has been realized.
Biological thiols, such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), represent the most important and fundamental intracellular bioactive molecules, which are essential for numerous physiological processes and closely associated with various diseases.1 Although they only differ in a single methylene group in the side chain, the biological properties of Cys and Hcy are extremely different.2 As the most abundant non-protein thiol in body, GSH acts as a redox regulator and a key indicator for xenobiotic metabolism, signal transduction and gene regulation, and, thus, behaves extremely different from its precursor Cys.3 Therefore, it is a great challenge to develop a reliable, straightforward and highly selective detection and discrimination methodology for these important biothiols.1 As a kind of ‘‘smart’’ soft materials, stimuli-responsive gels have gained considerable attention due to their broad potential applications in various fields.4–6 In comparison with organo-gels, less studied metallo-gels represent a more facinating matter, due to their additional properties provided by metal ions.7 In consideration of hydrophilic properties of most biologically active compounds, hardly accessible metallo-hydrogels may constitute a potential recognition matrix for amino acids. However, due to poor solubility of the most robust organometallic complexes under aqueous conditions, metallo-hydrogelators have been rarely explored so far. Recently, pincer complexes revealed assembly properties as diverse building blocks in soft materials due to their unique planar coordination geometry.8 Department of Chemistry, Fudan University, 220 Handan Road, 200433, Shanghai, China. E-mail: [email protected]; Fax: +86-21-65102412 † Dedicated to Prof. Li-Xin Dai on the occasion of his 90th birthday. ‡ Electronic supplementary information (ESI) available: 1H NMR, 13C NMR and ESI-MS spectra, temperature-dependent 1H NMR study, X-ray diffraction study, viscoelasticity study, circular dichroism study, and SEM morphologies. See DOI: 10.1039/c4cc04743e
10118 | Chem. Commun., 2014, 50, 10118--10121
Besides their catalytic behaviors,9 we have previously demonstrated several pincer metal complexes as efficient organometallic gelators, which are able to immobilize a variety of organic solvents, ionic liquids and even pure water at extremely low concentrations; we further described their relevance to metallo-gel catalysis, solar cells, molecular switches as well as visual recognition of positional isomers and enantiomers.10 We now report on the assembly properties of structure simple pincer zinc complexes and their applicability in visual recognition of bioactive mercapto amino acids. By stirring 2-furyl terpyridine ligand 110b with ZnCl2 in MeOH at room temperature for 24 h, pincer complex 2 was readily formed in a high yield. Gelation tests revealed that complex 2 hardly dissolved in almost all selected hydrophobic and hydrophilic solvents as well as in water (see ESI,‡ Table S1). The bifunctional alcohol glycerine turned out to be a suitable solvent for gelation at 1 wt%. In order to increase the solubility in water, ligands containing functional groups suitable for hydrogenbonding were selected to substitute the Cl ligands in complex 2. Due to the unique affinity coefficient of sulphur to zinc ions, we chose L-Cys as a promising candidate for this purpose. After substitution for the chloro ligand, L-Cys was efficiently coordinated to Zn2+ via the sulphur atom resulting in an increased solubility of the complex in water. To our delight, a transparent metallogel [2 + Cys]/H2O was obtained after addition of two equiv. of L-Cys to the vial containing 0.4 wt% metallogelator 2 (see ESI,‡ Fig. S2). Addition of one equiv. of additive only resulted in very weak gel formation. Due to their structural similarity with L-Cys, the gelation tests were extended to other mercapto amino acids, L-methionine (Met), L-Hcy and GSH. The addition of two equiv. of L-Hcy also afforded a transparent gel; however, when GSH was involved with 0.4 wt% complex 2, a clear sol was presented instead. An increase of the gelator concentration to 0.5 wt% led to metallo-hydrogel [2 + GSH] formation. As anticipated, precipitates or solutions were obtained with other amino acids (see ESI‡), which not only further confirms the significance of the thiol group but also demonstrates a quick, convenient and straightforward approach
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Fig. 1
Synthesis of terpyridine zinc complex 2 and its role in reversible metallo-hydrogel formation assisted by thiols.
to a visual discrimination of Cys, Hcy and GSH from other amino acids via selective gel formation at the first time. In addition, GSH could also be selectively detected out from L-Cys and L-Hcy by their different gelation abilities at the 0.4 wt% concentration. Upon shaking, the [2 + Cys]/H2O gel collapsed to give the corresponding sol, which was immobilized again after resting for 10 min. to reform a stable hydrogel. The sol–gel transition could be repeated several times which indicated thixotropic and self-healing abilities of the gels containing bioactive thiols. With these results in hand, we further explored the possibility of the hydrogel formation by simply stirring the mixture of ligand 1, ZnCl2 and selected thiols directly. Although ligand 1 was insoluble in water, upon gentle heating the mixture (molar ratio of 1 : ZnCl2 : L-Cys is 1 : 1 : 2, at 0.4 wt%) gradually dissolved to form a homogeneous clear aqueous solution, and readily formed a stable hydrogel after resting for an hour (Fig. 1). In the
Fig. 2
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absence of ZnCl2, however, ligand 1 hardly dissolved in water no matter how the mixture of 1 and L-Cys (1 : 2, 0.4 wt%) was treated. This phenomenon obviously revealed that in combination with the thiol group, the Zn2+ ion also plays a crucial role in the gel formation. Similar results were obtained for the mixtures containing ligand 1 and amino acids L-Hcy or GSH. Ethylene diamine tetra-acetate (EDTA) constitutes one of the most important sexadentate ligands, which efficiently chelates Zn2+ ions to form robust complexes.11 As we anticipated, the addition of one equiv. of EDTA to a 0.4 wt% [2 + Cys]/H2O gel resulted in a clear aqueous solution (Fig. 2). Obviously, in the absence of metal chelation, the planar configuration of the terpyridine skeleton, a prerequisite for the molecular assembly, is lost hampering p-stacking and metal–metal interactions between the aromatic rings of the metal-hybrid which are considered to be the crucial contributions for the gel formation. Upon addition of 0.2 equiv. of EDTA, no total collapse of the
Visual discrimination of L-Hcy, L-Cys and GSH via a selective metallo-hydrogel gelation, sol formation and a regelation sequence.
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gel was observed. Therefore, we selected 0.4 equiv. of EDTA to investigate the gelation behaviors of other mercapto gels (0.4 wt% [2 + Hcy] gel and 0.5 wt% [2 + GSH] gel) and similar results were observed (Fig. 2). In combination with their thixotropic and self-healing properties as well as the fact that the mercapto gels are readily accessible by simply stirring the mixture of 1, ZnCl2 and thiols, a potential chemical switch may be envisaged with additional ZnCl2. Therefore, 0.4 equiv. of ZnCl2 was then added to the sol formed by hydrogel [2 + Cys] and EDTA (0.4 equiv.) to further test the possibility of gel reformation. No gel reformation was observed with sols formed by [2 + Cys] and [2 + GSH]. However, when 0.4 equiv. of ZnCl2 was added into the vial containing the collapsed sol of [2 + Hcy]/H2O and EDTA, a striking gel reformation was observed. This observation finally supported the possibility of a reversible transition between the gel and sol, which makes the mercapto metallohydrogels presented here interesting candidates for molecular switches. In addition, the extremely different behaviors of the three mercapto gels in the gel-reformation also demonstrated the mercapto metallo-hydrogel system as a new platform for visual discrimination of L-Hcy over L-Cys and GSH directly with naked eyes. With these interesting results in hand, a series of experiments were carried out to further account for the origin of the selective visual recognition. Firstly, the gel–sol phase-transition temperatures (Tg) were determined by the ‘‘test-tube-inversion method’’.12 In comparison with the metallo-hydrogel formed by pincer complex 2 with L-Cys (0.4 wt%) and GSH (0.48 wt%), the [2 + Hcy]/H2O gel (0.4 wt%) exhibited a distinctly improved thermo-stability and a higher Tg value (47.0 1C vs. 33.8 and 30 1C, respectively, see ESI‡) was observed. The methylene group in the side chain of L-Hcy may relieve the repulsion caused by molecular bulkiness and favor the molecular assembly. The thermo-stability steadily increased with the gelator concentration as established by the [2 + Hcy]/H2O and [2 + GSH]/H2O gels at
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gelator concentrations varying from 0.37 to 0.67 wt%. Again, the [2 + Hcy]/H2O gels exhibited a higher thermo-stability than those observed for the respective [2 + GSH]/H2O gels at the same concentration, which is also consistent with the results of rheology and circular dichroism (CD) studies (see ESI‡). Temperature-dependent 1H NMR spectroscopy studies were then carried out to investigate the main interactions responsible for gel formation. Upon increasing the temperature, the broad 1H signals of the [2 + Hcy] and [2 + Cys] gels (0.67 wt%) were gradually sharpened and were shifted downfield indicating that hydrogen bonding and p–p stacking interactions significantly contributed to the gel formation, which is also in accordance with the results of X-ray diffraction of the xerogel samples (see ESI‡). In order to investigate the contribution of all functional groups of the gelation process, we extended our studies to mercapto carboxylic acids. When 3-mercaptopropionic acid (3-MPA) was applied, a metallo-hydrogel [2 + 3-MPA] (0.4 wt%) was obtained. Unexpectedly, the addition of 2 equiv. of methyl 3-mercaptopropionate (3-MPE) also resulted in hydrogel formation. The thermo-stability of the resulting [2 + 3-MPE] gel was lower than that of the [2 + 3-MPA] gel as demonstrated by the respective Tg values (42.7 1C vs. 46.4 1C, see ESI‡). Surprisingly, a translucent metallo-hydrogel was formed with butyl mercaptane, although with a lower Tg value (30.8 1C). Taking into account the unsuccessful gelation tests with L-cystine and L-Met, it is obvious to conclude that free SH groups play an essential role in the gelation, and additional hydrogen bonding interactions enforce the gel stability. A combination of complex 2 and benzyl mercaptane provided only precipitates; the steric effect of the phenyl ring may partially hamper the p–p stacking and metal–metal interactions. By scanning electron microscopy (SEM), irregular bulky particles were found in the precipitate formed by complex 2 in water (Fig. 3a); and a network composed of long and thin
Fig. 3 Selected SEM images of: (a) precipitate 2/H2O (0.4 wt%); (b) metallogel 2/glycerin (1 wt%); (c) metallogel [2 + Cys]/H2O (0.4 wt%); (d) metallogel [2 + Hcy]/H2O (0.4 wt%); (e) sol [(2 + Hcy) + EDTA]/H2O formed by addition of 0.4 equiv. of EDTA into the corresponding gel; and (f) the reformed gel by adding 0.4 equiv. of ZnCl2 into the sol [(2 + Hcy) + EDTA]/H2O.
10120 | Chem. Commun., 2014, 50, 10118--10121
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fibers (100–200 mm long and 1–2 mm wide, Fig. 3b) was observed for gel 2/glycerin (1 wt%). Interestingly, in the case of 0.4 wt% metallogel [2 + Cys]/H2O, snowflake-shaped networks containing a dense porous substructure were observed, which efficiently immobilized water molecules leading to the gel formation (Fig. 3c). Morphologies consisting of dense porous networks were also presented in 0.4 wt% metallogel [2 + Hcy]/H2O (Fig. 3d). Lotus-like networks with a porous substructure were found in 0.5 wt% metallo-gel [2 + GSH]/H2O (Fig. S26, ESI‡). Similar metallo-gels, which were formed from complex 2 and NH2-free mercapto molecules (3-MPA, 3-MPE and butyl mercaptane) in water, also revealed thick and dense nanoparticles with a porous substructure (see ESI‡). However, diverse types of morphologies (irregular crystalline particles, sheets and fibers) were encountered for the precipitates or sols obtained from complex 2 and other amino acids (see ESI‡). As expected, sols obtained by adding 0.4 equiv. of EDTA into metallo-gels revealed distinctly different morphologies: thick and bulky rhombic crystalline rods (several mm long and wide) were observed for sols [(2 + Cys) + EDTA]/H2O and [(2 + Hcy) + EDTA]/H2O, and irregular crystalline particles for [(2 + GSH) + EDTA]/H2O (Fig. 3e, see ESI‡). Surprisingly, networks of dense nano-fibers with porous substructures were observed in the reformed gel [(2 + Hcy) + EDTA + ZnCl2]/H2O (Fig. 3f). In contrast, precipitates prepared by adding 0.4 equiv. of ZnCl2 to the [(2 + Cys) + EDTA]/H2O and [(2 + GSH) + EDTA]/H2O sols revealed irregular particles consisting of dense nanofibers without porous substructures, which may be exploited in a selective gel reformation and visual discrimination of Hcy over Cys and GSH by using metallo-hydrogels based on zinc complex 2 as a new platform. In summary, a simple and straightforward protocol for visual discrimination of mercapto amino acids (L-Cys, L-Hcy and GSH) from various amino acids has been realized via selective metallohydrogel formation by using pincer zinc complex 2 and the corresponding amino acids. In combination with the self-healing properties of the mercapto metallo-hydrogels and the strong coordination ability of EDTA to Zn2+ ions, a more challenging visual recognition of Hcy over Cys and GSH has been achieved via selective gel reformation which also offers a perspective for potential application towards the chemo-switch. In light of this strikingly selective molecular recognition on the macro-scale, further studies on the applicability of complex 2 in cell imaging and diagnosis of the diseases relative with L-Cys, L-Hcy and GSH are under investigation in our laboratory.
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Financial support from the National Natural Science Foundation of China (No. 21172045 and 91127041), the Changjiang Scholars and Innovative Research Team in University (IRT1117), the doctoral fund of Ministry of Education of China (20130071110032) and Shanghai Shuguang and Pujiang Programs and Department of Chemistry, Fudan University is gratefully acknowledged.
Notes and references 1 (a) H. S. Jung, X. Chen, J. S. Kim and J. Yoon, Chem. Soc. Rev., 2013, 42, 6019; (b) X. Chen, Y. Zhou, X. Peng and J. Yoon, Chem. Soc. Rev., 2010, 39, 2120. 2 (a) S. Shahrokhian, Anal. Chem., 2001, 73, 5972; (b) K. S. McCully, Nat. Med., 1996, 2, 386; (c) Homocysteine in Health and Disease, ed. R. Carmel and D. W. Jacobsen, Cambridge University Press, Cambridge, UK, 2001. 3 L. Yi, H. Li, L. Sun, L. Liu, C. Zhang and Z. Xi, Angew. Chem., Int. Ed., 2009, 48, 4034. 4 (a) X. Yan, D. Xu, X. Chi, J. Chen, S. Dong, X. Ding, Y. Yu and F. Huang, Adv. Mater., 2012, 24, 362; (b) S. Dong, B. Zheng, D. Xu, X. Yan, M. Zhang and F. Huang, Adv. Mater., 2012, 24, 3191; (c) M. Zhang, D. Xu, X. Yan, J. Chen, S. Dong, B. Zheng and F. Huang, Angew. Chem., Int. Ed., 2012, 51, 7011. 5 (a) J. de Jong, B. L. Feringa and J. van Esch, Responsive molecular gels, in Molecular Switches, ed. B. L. Feringa and W. R. Browne, Wiley-VCH, 2nd edn, 2011, p. 517; (b) Y. Qiu, P. Chen, P. Guo, Y. Li and M. Liu, Adv. Mater., 2008, 20, 2908. 6 (a) T. Tu, W. Fang and Z. Sun, Adv. Mater., 2013, 25, 5304; (b) M.-O. M. Piepenbrock, G. O. Lloyd, N. Clarke and J. W. Steed, Chem. Rev., 2010, 110, 1960. 7 (a) A. Y.-Y. Tam and V. W.-W. Yam, Chem. Soc. Rev., 2013, 42, 1540; (b) F. Fages, Angew. Chem., Int. Ed., 2006, 45, 1680. ´, V. Noponen, E. Kolehmainen and E. Sieva ¨nen, 8 (a) H. Svobodova RSC Adv., 2012, 2, 4985; (b) A. Winter, S. Hoeppener, G. R. Newkome and U. S. Schubert, Adv. Mater., 2011, 23, 3484; (c) M. Albrecht and G. van Koten, Angew. Chem., Int. Ed., 2001, 40, 3750. 9 (a) M. Xu, X. Li, Z. Sun and T. Tu, Chem. Commun., 2013, 49, 11539; ¨tz, Chem. Commun., (b) T. Tu, H. Mao, C. Herbert, M. Xu and K. H. Do 2010, 46, 7796; (c) T. Tu, X. Feng, Z. Wang and X. Liu, Dalton Trans., ¨tz, Adv. 2010, 39, 10598; (d) T. Tu, J. Malineni, X. Bao and K. H. Do ¨tz, Synth. Catal., 2009, 351, 1029; (e) T. Tu, J. Malineni and K. H. Do Adv. Synth. Catal., 2008, 350, 1791; ( f ) Z. Wang, X. Feng, W. Fang and T. Tu, Synlett, 2011, 7, 951. 10 (a) W. Fang, X. Liu, Z. Lu and T. Tu, Chem. Commun., 2014, 50, 3313; (b) W. Fang, Z. Sun and T. Tu, J. Phys. Chem. C, 2013, 117, 25185; ¨tz, Angew. Chem., Int. (c) T. Tu, W. Fang, X. Bao, X. Li and K. H. Do Ed., 2011, 50, 6601; (d) T. Tu, X. Bao, W. Assenmacher, H. Peterlik, ¨tz, Chem. – Eur. J., 2009, 15, 1853; (e) T. Tu, J. Daniels and K. H. Do ¨tz, W. Assenmacher, H. Peterlik, G. Schnakenburg and K. H. Do Angew. Chem., Int. Ed., 2008, 47, 7127; ( f ) T. Tu, W. Assenmacher, ¨tz, Angew. Chem., H. Peterlik, R. Weisbarth, M. Nieger and K. H. Do Int. Ed., 2007, 46, 6368. 11 Z. Elbowicz-Waniewska, Pol. Tyg. Lek., 1967, 22, 924. 12 K. Murata, M. Aoki, T. Suzuki, T. Harada, H. Kawabata, T. Komori, F. Ohseto, K. Ueda and S. Shinkai, J. Am. Chem. Soc., 1994, 116, 6664.
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Tunable reversible metallo-hydrogels: a new platform for visual discrimination of biothiols†‡ Weiwei Fang, Cong Liu, Zhengwei Lu, Zheming Sun and Tao Tu*
Received 23rd June 2014, Accepted 14th July 2014 DOI: 10.1039/c4cc04743e www.rsc.org/chemcomm
By using metallo-hydrogel as a new platform, a simple and straightforward selective visual discrimination of cysteine, homocysteine and glutathione from each other as well as from other amino acids has been realized.
Biological thiols, such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), represent the most important and fundamental intracellular bioactive molecules, which are essential for numerous physiological processes and closely associated with various diseases.1 Although they only differ in a single methylene group in the side chain, the biological properties of Cys and Hcy are extremely different.2 As the most abundant non-protein thiol in body, GSH acts as a redox regulator and a key indicator for xenobiotic metabolism, signal transduction and gene regulation, and, thus, behaves extremely different from its precursor Cys.3 Therefore, it is a great challenge to develop a reliable, straightforward and highly selective detection and discrimination methodology for these important biothiols.1 As a kind of ‘‘smart’’ soft materials, stimuli-responsive gels have gained considerable attention due to their broad potential applications in various fields.4–6 In comparison with organo-gels, less studied metallo-gels represent a more facinating matter, due to their additional properties provided by metal ions.7 In consideration of hydrophilic properties of most biologically active compounds, hardly accessible metallo-hydrogels may constitute a potential recognition matrix for amino acids. However, due to poor solubility of the most robust organometallic complexes under aqueous conditions, metallo-hydrogelators have been rarely explored so far. Recently, pincer complexes revealed assembly properties as diverse building blocks in soft materials due to their unique planar coordination geometry.8 Department of Chemistry, Fudan University, 220 Handan Road, 200433, Shanghai, China. E-mail: [email protected]; Fax: +86-21-65102412 † Dedicated to Prof. Li-Xin Dai on the occasion of his 90th birthday. ‡ Electronic supplementary information (ESI) available: 1H NMR, 13C NMR and ESI-MS spectra, temperature-dependent 1H NMR study, X-ray diffraction study, viscoelasticity study, circular dichroism study, and SEM morphologies. See DOI: 10.1039/c4cc04743e
10118 | Chem. Commun., 2014, 50, 10118--10121
Besides their catalytic behaviors,9 we have previously demonstrated several pincer metal complexes as efficient organometallic gelators, which are able to immobilize a variety of organic solvents, ionic liquids and even pure water at extremely low concentrations; we further described their relevance to metallo-gel catalysis, solar cells, molecular switches as well as visual recognition of positional isomers and enantiomers.10 We now report on the assembly properties of structure simple pincer zinc complexes and their applicability in visual recognition of bioactive mercapto amino acids. By stirring 2-furyl terpyridine ligand 110b with ZnCl2 in MeOH at room temperature for 24 h, pincer complex 2 was readily formed in a high yield. Gelation tests revealed that complex 2 hardly dissolved in almost all selected hydrophobic and hydrophilic solvents as well as in water (see ESI,‡ Table S1). The bifunctional alcohol glycerine turned out to be a suitable solvent for gelation at 1 wt%. In order to increase the solubility in water, ligands containing functional groups suitable for hydrogenbonding were selected to substitute the Cl ligands in complex 2. Due to the unique affinity coefficient of sulphur to zinc ions, we chose L-Cys as a promising candidate for this purpose. After substitution for the chloro ligand, L-Cys was efficiently coordinated to Zn2+ via the sulphur atom resulting in an increased solubility of the complex in water. To our delight, a transparent metallogel [2 + Cys]/H2O was obtained after addition of two equiv. of L-Cys to the vial containing 0.4 wt% metallogelator 2 (see ESI,‡ Fig. S2). Addition of one equiv. of additive only resulted in very weak gel formation. Due to their structural similarity with L-Cys, the gelation tests were extended to other mercapto amino acids, L-methionine (Met), L-Hcy and GSH. The addition of two equiv. of L-Hcy also afforded a transparent gel; however, when GSH was involved with 0.4 wt% complex 2, a clear sol was presented instead. An increase of the gelator concentration to 0.5 wt% led to metallo-hydrogel [2 + GSH] formation. As anticipated, precipitates or solutions were obtained with other amino acids (see ESI‡), which not only further confirms the significance of the thiol group but also demonstrates a quick, convenient and straightforward approach
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Fig. 1
Synthesis of terpyridine zinc complex 2 and its role in reversible metallo-hydrogel formation assisted by thiols.
to a visual discrimination of Cys, Hcy and GSH from other amino acids via selective gel formation at the first time. In addition, GSH could also be selectively detected out from L-Cys and L-Hcy by their different gelation abilities at the 0.4 wt% concentration. Upon shaking, the [2 + Cys]/H2O gel collapsed to give the corresponding sol, which was immobilized again after resting for 10 min. to reform a stable hydrogel. The sol–gel transition could be repeated several times which indicated thixotropic and self-healing abilities of the gels containing bioactive thiols. With these results in hand, we further explored the possibility of the hydrogel formation by simply stirring the mixture of ligand 1, ZnCl2 and selected thiols directly. Although ligand 1 was insoluble in water, upon gentle heating the mixture (molar ratio of 1 : ZnCl2 : L-Cys is 1 : 1 : 2, at 0.4 wt%) gradually dissolved to form a homogeneous clear aqueous solution, and readily formed a stable hydrogel after resting for an hour (Fig. 1). In the
Fig. 2
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absence of ZnCl2, however, ligand 1 hardly dissolved in water no matter how the mixture of 1 and L-Cys (1 : 2, 0.4 wt%) was treated. This phenomenon obviously revealed that in combination with the thiol group, the Zn2+ ion also plays a crucial role in the gel formation. Similar results were obtained for the mixtures containing ligand 1 and amino acids L-Hcy or GSH. Ethylene diamine tetra-acetate (EDTA) constitutes one of the most important sexadentate ligands, which efficiently chelates Zn2+ ions to form robust complexes.11 As we anticipated, the addition of one equiv. of EDTA to a 0.4 wt% [2 + Cys]/H2O gel resulted in a clear aqueous solution (Fig. 2). Obviously, in the absence of metal chelation, the planar configuration of the terpyridine skeleton, a prerequisite for the molecular assembly, is lost hampering p-stacking and metal–metal interactions between the aromatic rings of the metal-hybrid which are considered to be the crucial contributions for the gel formation. Upon addition of 0.2 equiv. of EDTA, no total collapse of the
Visual discrimination of L-Hcy, L-Cys and GSH via a selective metallo-hydrogel gelation, sol formation and a regelation sequence.
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gel was observed. Therefore, we selected 0.4 equiv. of EDTA to investigate the gelation behaviors of other mercapto gels (0.4 wt% [2 + Hcy] gel and 0.5 wt% [2 + GSH] gel) and similar results were observed (Fig. 2). In combination with their thixotropic and self-healing properties as well as the fact that the mercapto gels are readily accessible by simply stirring the mixture of 1, ZnCl2 and thiols, a potential chemical switch may be envisaged with additional ZnCl2. Therefore, 0.4 equiv. of ZnCl2 was then added to the sol formed by hydrogel [2 + Cys] and EDTA (0.4 equiv.) to further test the possibility of gel reformation. No gel reformation was observed with sols formed by [2 + Cys] and [2 + GSH]. However, when 0.4 equiv. of ZnCl2 was added into the vial containing the collapsed sol of [2 + Hcy]/H2O and EDTA, a striking gel reformation was observed. This observation finally supported the possibility of a reversible transition between the gel and sol, which makes the mercapto metallohydrogels presented here interesting candidates for molecular switches. In addition, the extremely different behaviors of the three mercapto gels in the gel-reformation also demonstrated the mercapto metallo-hydrogel system as a new platform for visual discrimination of L-Hcy over L-Cys and GSH directly with naked eyes. With these interesting results in hand, a series of experiments were carried out to further account for the origin of the selective visual recognition. Firstly, the gel–sol phase-transition temperatures (Tg) were determined by the ‘‘test-tube-inversion method’’.12 In comparison with the metallo-hydrogel formed by pincer complex 2 with L-Cys (0.4 wt%) and GSH (0.48 wt%), the [2 + Hcy]/H2O gel (0.4 wt%) exhibited a distinctly improved thermo-stability and a higher Tg value (47.0 1C vs. 33.8 and 30 1C, respectively, see ESI‡) was observed. The methylene group in the side chain of L-Hcy may relieve the repulsion caused by molecular bulkiness and favor the molecular assembly. The thermo-stability steadily increased with the gelator concentration as established by the [2 + Hcy]/H2O and [2 + GSH]/H2O gels at
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gelator concentrations varying from 0.37 to 0.67 wt%. Again, the [2 + Hcy]/H2O gels exhibited a higher thermo-stability than those observed for the respective [2 + GSH]/H2O gels at the same concentration, which is also consistent with the results of rheology and circular dichroism (CD) studies (see ESI‡). Temperature-dependent 1H NMR spectroscopy studies were then carried out to investigate the main interactions responsible for gel formation. Upon increasing the temperature, the broad 1H signals of the [2 + Hcy] and [2 + Cys] gels (0.67 wt%) were gradually sharpened and were shifted downfield indicating that hydrogen bonding and p–p stacking interactions significantly contributed to the gel formation, which is also in accordance with the results of X-ray diffraction of the xerogel samples (see ESI‡). In order to investigate the contribution of all functional groups of the gelation process, we extended our studies to mercapto carboxylic acids. When 3-mercaptopropionic acid (3-MPA) was applied, a metallo-hydrogel [2 + 3-MPA] (0.4 wt%) was obtained. Unexpectedly, the addition of 2 equiv. of methyl 3-mercaptopropionate (3-MPE) also resulted in hydrogel formation. The thermo-stability of the resulting [2 + 3-MPE] gel was lower than that of the [2 + 3-MPA] gel as demonstrated by the respective Tg values (42.7 1C vs. 46.4 1C, see ESI‡). Surprisingly, a translucent metallo-hydrogel was formed with butyl mercaptane, although with a lower Tg value (30.8 1C). Taking into account the unsuccessful gelation tests with L-cystine and L-Met, it is obvious to conclude that free SH groups play an essential role in the gelation, and additional hydrogen bonding interactions enforce the gel stability. A combination of complex 2 and benzyl mercaptane provided only precipitates; the steric effect of the phenyl ring may partially hamper the p–p stacking and metal–metal interactions. By scanning electron microscopy (SEM), irregular bulky particles were found in the precipitate formed by complex 2 in water (Fig. 3a); and a network composed of long and thin
Fig. 3 Selected SEM images of: (a) precipitate 2/H2O (0.4 wt%); (b) metallogel 2/glycerin (1 wt%); (c) metallogel [2 + Cys]/H2O (0.4 wt%); (d) metallogel [2 + Hcy]/H2O (0.4 wt%); (e) sol [(2 + Hcy) + EDTA]/H2O formed by addition of 0.4 equiv. of EDTA into the corresponding gel; and (f) the reformed gel by adding 0.4 equiv. of ZnCl2 into the sol [(2 + Hcy) + EDTA]/H2O.
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fibers (100–200 mm long and 1–2 mm wide, Fig. 3b) was observed for gel 2/glycerin (1 wt%). Interestingly, in the case of 0.4 wt% metallogel [2 + Cys]/H2O, snowflake-shaped networks containing a dense porous substructure were observed, which efficiently immobilized water molecules leading to the gel formation (Fig. 3c). Morphologies consisting of dense porous networks were also presented in 0.4 wt% metallogel [2 + Hcy]/H2O (Fig. 3d). Lotus-like networks with a porous substructure were found in 0.5 wt% metallo-gel [2 + GSH]/H2O (Fig. S26, ESI‡). Similar metallo-gels, which were formed from complex 2 and NH2-free mercapto molecules (3-MPA, 3-MPE and butyl mercaptane) in water, also revealed thick and dense nanoparticles with a porous substructure (see ESI‡). However, diverse types of morphologies (irregular crystalline particles, sheets and fibers) were encountered for the precipitates or sols obtained from complex 2 and other amino acids (see ESI‡). As expected, sols obtained by adding 0.4 equiv. of EDTA into metallo-gels revealed distinctly different morphologies: thick and bulky rhombic crystalline rods (several mm long and wide) were observed for sols [(2 + Cys) + EDTA]/H2O and [(2 + Hcy) + EDTA]/H2O, and irregular crystalline particles for [(2 + GSH) + EDTA]/H2O (Fig. 3e, see ESI‡). Surprisingly, networks of dense nano-fibers with porous substructures were observed in the reformed gel [(2 + Hcy) + EDTA + ZnCl2]/H2O (Fig. 3f). In contrast, precipitates prepared by adding 0.4 equiv. of ZnCl2 to the [(2 + Cys) + EDTA]/H2O and [(2 + GSH) + EDTA]/H2O sols revealed irregular particles consisting of dense nanofibers without porous substructures, which may be exploited in a selective gel reformation and visual discrimination of Hcy over Cys and GSH by using metallo-hydrogels based on zinc complex 2 as a new platform. In summary, a simple and straightforward protocol for visual discrimination of mercapto amino acids (L-Cys, L-Hcy and GSH) from various amino acids has been realized via selective metallohydrogel formation by using pincer zinc complex 2 and the corresponding amino acids. In combination with the self-healing properties of the mercapto metallo-hydrogels and the strong coordination ability of EDTA to Zn2+ ions, a more challenging visual recognition of Hcy over Cys and GSH has been achieved via selective gel reformation which also offers a perspective for potential application towards the chemo-switch. In light of this strikingly selective molecular recognition on the macro-scale, further studies on the applicability of complex 2 in cell imaging and diagnosis of the diseases relative with L-Cys, L-Hcy and GSH are under investigation in our laboratory.
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Financial support from the National Natural Science Foundation of China (No. 21172045 and 91127041), the Changjiang Scholars and Innovative Research Team in University (IRT1117), the doctoral fund of Ministry of Education of China (20130071110032) and Shanghai Shuguang and Pujiang Programs and Department of Chemistry, Fudan University is gratefully acknowledged.
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