Unique norbornene based triazole molecule for selective Fe(II) sensing

Abstract

A triazole functionalized norbornene monomer (NFTZ) and its corresponding homopolymer (NFTZH) are synthesized. The unique fluorescence properties of the NFTZ molecule are due to the conjugation of two aromatic rings through an amide bond. The chelating behaviour of the NFTZ monomer is explored through the selective binding of Fe(II) in the presence of other metals. The MALDI-TOF analysis and Job’s plot clearly confirm the 1 : 2 mode of binding of NFTZ with Fe(II). ¹H NMR and IR spectroscopy clearly confirm the unique amide-iminol tautomerisation.

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Fluorimetric quenching techniques have been widely applied as the sensing response in metal ion detection. Mainly, the quenching process can be divided into two kinds of mechanism namely static and dynamic. For example, the static quenching mechanism is observed during the estimation of Fe(III) and inorganic phosphate in blood serum by polyfluorene derivatives.^1,2 On the contrary, the dynamic quenching mechanism is observed while quantifying the molecular oxygen in aqueous and non-aqueous solutions.³ In general static quenching is very common compared to dynamic quenching. In this report, a sensor for Fe(II) with a static quenching mechanism is demonstrated. As the lifetime values are not changed while titrating the probe with Fe(II) ions, it is believed that a static quenching mechanism is followed while sensing.

Iron is one of the most common elements among all metals. In particular, Fe(II) is an essential element to all living organisms owing to its potent redox chemistry and potential to engage in catalytic activity. If the Fe(II) concentration is not measured properly, it can lead to unrestrained oxidative chemistry causing tissue damage and fibrosis to various organs.⁴ Although biological iron is observed frequently as Fe(II) in enzymes and storage proteins, the existence of labile iron within cells is difficult to detect, resulting in high demand for selective sensors for iron.^5–8

Compared to the number of fluorescent sensors for other transition metal ions, Fe(II)-specific fluorescent sensors are quite rare except for very few examples.⁹ Recently, “Calcein” with an EDTA-derived receptor has been reported to detect Fe(II) ions with a ten-fold higher selectivity for Fe(II) over Fe(III).¹⁰

Herein we describe the synthesis and characterization of triazole functionalized norbornene monomer (NFTZ) and its homopolymer (NFTZH). The metal specific terminal chelating properties of the triazole molecule are studied with Fe(II) in the presence of other metals. The quenching of the fluorescence emission is observed as the response. IR, Mass and NMR spectroscopic techniques clearly confirm the binding of Fe(II) to the NFTZ monomer. To the best of our knowledge this is the most efficient method that clearly senses Fe(II) over other metals particularly, Fe(III). exo-Oxabicyclo[2.2.1]hept-5-ene-2,3-dicarboxylic anhydride was prepared following the reported procedure.¹¹ 3-(4-Carboxyphenylcarbamoyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-carboxylic acid was prepared using 4-aminobenzoic acid and acetic anhydride in the presence of sodium acetate in a dimethylformamide solvent. Product formation was confirmed by ¹H NMR, FT-IR and mass spectroscopy techniques. The norbornene functionalized triazole (NFTZ) molecule was prepared by the coupling of nadic acid and 3-amino-1,2,4-triazole-5-thiol using N,N′-dicyclohexylcarbodiimide as a coupling reagent in a dimethylformamide solvent (Scheme 1). Formation of the product was confirmed by ¹H NMR, ¹³C NMR and IR spectroscopy. The appearance of a new proton signal at δ 7.9 (s, 1H) was due to an amide hydrogen which supported the theory that the triazole ring bonded with the –COOH group of nadic acid. The signals at δ 7.5 (s, 2H) & δ 7.2 (s, 2H) were assigned to the aromatic protons while the signals at δ 6.5 (s, 2H) δ 5.3 (s, 2H) & δ 3.2 (s, 2H) were assigned to the oxo-norbornene protons (Fig. S1†). In the ¹³C NMR spectrum, the signal at δ 165 ppm was due to the attachment of the triazole amino group with nadic acid. Furthermore, the presence of signals at δ 151 & δ 148 clearly indicated the formation of the product (Fig. S2†) The stretching frequency at 3244 cm⁻¹ for free carboxylic acid was shifted to 3268 cm⁻¹, which indicated the formation of an amide group as shown in the ESI (Fig. S5†). All the monomers were characterized using a micro-mass spectrometer (Q-TOF), with acetonitrile as the solvent. \The observed mass (m/z) and calculated mass for all the monomers were in good agreement, which confirmed the formation of the product (Fig. 3).

Next, the homopolymerization of NFTZ was carried out using a second generation Grubbs’ catalyst at room temperature in a dry dichloromethane solvent and was monitored through NMR (ESI Scheme 2†).¹² New signals were observed at δ 5.4 ppm and δ 4.9 ppm, suggesting the formation of NFTZH. The molecular weight of NFTZH (M_n = 19 000 with PDI 1.2) was determined by gel permeation chromatography (Fig. S3†).

Scheme 1 Synthesis of the triazole functionalized NFTZ monomer.

The unique bluish-white (Fig. S8†) emission from NFTZ was first observed while monitoring the product formation by thin layer chromatography (TLC) under UV light. Both of the compounds, nadic acid and 3-amino-1,2,4-triazole-5-thiol did not show any fluorescence properties. But when both compounds were attached through the coupling reaction, the product showed fluorescence. We hypothesized that it could be because of two aromatic groups attached through an amide bond and therefore the NFTZ created a long conjugative system where electron transfer was possible and hence the molecule was emissive in nature.¹³ The absorbance maximum was found to be 350 nm for NFTZ in THF (Fig. S6†). Because of the transition of electrons from the nitrogen lone pair to the anti-bonding orbital of the norbornene double bond (n to π* transition) we observed an absorption at 350 nm. But as it was not in the visible region, we could not see any colour in the NFTZ monomer. The quantum yield (φ = 0.18) of the compound NFTZ was calculated from its absorption and emission spectra taking quinine sulphate as standard. The optical events generated by inducing alternation into the optical properties of fluorophores through associating metal ions with their binding moieties could be witnessed either by the shift in the wavelength (bathochromic/hypsochromic) or the quenching of the fluorescence intensity. Due to the presence of a chelating functionality (terminal triazole ring), the molecule NFTZ was chosen to study the viability of metal binding, which would facilitate its association with metal ions. Salts of Pb(II), Ba(II), Mn(II), Hg(II), Fe(II), Fe(III), Co(II), Ni(II), Cu(II), and Cd(II) (with concentrations of 0.015 M in water) were titrated with 1 mg ml⁻¹ NFTZ in THF and monitored through fluorescence. The addition of FeSO₄ in water to a solution of NFTZ in THF/water (1 : 1) caused immediate quenching. On the contrary, the addition of the other metals to solutions of NFTZ resulted in little or no effect on NFTZ. Titrating aqueous solutions of Fe(II) metal salts (sulfate) caused large quenching of the fluorescence of NFTZ, about a 90% reduction in the fluorescence intensity occurred, which clearly implied the preferential binding of NFTZ towards Fe(II) (Fig. 1a and b).¹⁴ The instantaneous quenching of NFTZ by Fe(II) was first explained by ¹H NMR spectroscopy which clearly showed that the aromatic region signals at 7–8 ppm as well as the norbornene peak were shifted to the upfield region. Along with that at 1.9 ppm a new signal arose because of unique amide-iminol tautomerisation.¹⁵ As there was equilibrium between –CONH and –C(OH)=N, a little amount of –NH functionality was still there in the reaction medium which was also responsible for binding to Fe(II) and therefore with ¹H NMR spectroscopy a upfield shift was observed (Fig. 2). This binding was further supported by the IR spectra where there was about 30 cm⁻¹ shifting in the position of the –NH stretching frequency which clearly proved that there was a strong binding interaction between NFTZ and Fe(II) (Fig. S5†).

Fig. 1 (a) Emission spectra of NFTZ as a function of increasing Fe(II) concentration. The spectra were collected in THF with an excitation wavelength (Ex) of 350 nm. (b) Selective binding of NFTZ to Fe(II) over other metal ions.

Fig. 2 (a) ¹H NMR spectrum of the NFTZ + Fe(II) complex. (b) ¹H NMR spectrum of the NFTZ monomer. Here the marked peaks were shifted after the addition of Fe(II) to the NFTZ monomer and the appearance of a new signal at 1.9 ppm was due to a hydroxyl group which is absent in the NFTZ NMR spectrum.

The proposed complex formation was strongly supported by the MALDI-TOF analysis which shows a 1 : 2 binding mode (binding of triazole molecule to iron: NFTZ : Fe(II) = 1:2) (Fig. 3). The calculated m/z value was (2NFTZ + 4Fe(II) + NH₄⁺) 1007.47 (which is the characteristic peak of the Fe(II) isotope) whereas the observed m/z value was 1007.36.¹⁶ MALDI (Matrix-assisted laser desorption/ionization) is a soft ionization technique used in mass spectrometry. In the ionization mechanism, it is hypothesized that the matrix ions, such as, H⁺, Na⁺ and NH₄⁺ normally are transferred to the analyte. As a result when we analysed the mass spectrum we observed the spectrum as an added value of NH₄⁺. The ESI-Mass spectrum further supported our MALDI-TOF analysis data (Fig. S7†). A Job’s plot (a continuous variation method) provided strong confirmation for the stoichiometry of NFTZ/Fe(II).¹⁷ The results confirmed the 1 : 2 binding nature of the NFTZ–Fe(II) complex (Fig. 4b).

Fig. 3 MALDI-TOF analysis of the triazole functionalized NFTZ monomer coordinated with Fe(II) which is clearly showing Fe(II) is coordinated in a 1 : 2 fashion with the triazole monomer.

Fig. 4 (a) Possible binding mode of Fe(II) with the NFTZ monomer along with the amide-iminol tautomeric structure. (b) Job’s plot of the NFTZ monomer and Fe(II). The absorption at 350 nm was plotted against the mole fraction of Fe(II) at a constant total concentration [Fe(II) + NFTZ monomer] of 80 M showing the 1 : 2 binding stoichiometry in 1 : 1 MeOH : H₂O.

It is well-known in the literature that FeSO₄ salt is ESR active.¹⁸ However, it is also well documented that low spin Fe(II) complexes do not give ESR signals.¹⁹ From the ESR experiment, we came to know that when NFTZ formed a complex with Fe(II) the complex did not give a signal in the ESR spectrum. From this experiment it was clear that the NFTZ monomer complexed with Fe(II) (low spin) in a specific structural orientation. To evaluate the sensitivity of this binding system, we determined the Stern–Volmer quenching constant K_sv which was given by the Stern–Volmer equation,⁵ I₀/I = 1 + K_sv[Q]. At low concentration, I₀/I increased linearly as the concentration of the quencher increased. On the basis of the correlation curve, the linear range of the plot was from 1.3 × 10⁻⁷ M to 1.1 × 10⁻⁶ M and the K_sv (quenching constant) was calculated to be 1.5 × 10⁵ M⁻¹ (Fig. S4†).²⁰ The analytical detection limit of the chemosensor, NFTZH, for the detection of Fe(II), determined by standard methods was found to be 1.3 × 10⁻⁷ M (Fig. S9†).

In the case of the homopolymer of NFTZ, it showed the same kind of fluorimetric response in the presence of Fe(II). The homopolymer NFTZH was more useful in the sensing application as there was no need for special equipment we could sense Fe(II) just using polymer-coated paper strips, whereas in the case of the monomer it leached out from the paper.²¹ Therefore, working towards developing paper strips, the homopolymer (NFTZH) was synthesized to demonstrate the versatile utility of our unique sensing system. As a greater terminal chelating functionality (triazole moiety) is present in the case of the polymer, the Stern–Volmer quenching constant (K_sv) of NFTZH is 2.3 × 10⁵ M⁻¹, which is higher than its monomer analogue (NFTZ).

In conclusion, a norbornene functionalized triazole monomer (NFTZ) was synthesized and thoroughly characterized by ¹H, ¹³C and mass spectroscopic analyses. The unusual fluorescence properties of the monomer and polymer were explored through the Fe(II) quenching process. The unique binding mode of NFTZ with Fe(II) was supported by mass and IR spectroscopic analyses. The Job’s plot and ESR analysis further confirmed the nature of the binding and the specific structural orientation of the NFTZ–Fe(II) complex. The ¹H NMR spectra supported the amide-iminol tautomeric structure. The highly sensitive sensing nature of NFTZ and NFTZH suggests the uniqueness of the design.

Acknowledgements

SB thanks CSIR, New Delhi for the fellowship. RS thanks DST, New Delhi for the Ramanujan Fellowship. RS thanks IISER-Kolkata for the infrastructure.

Notes and references

1. (a) J. R. Lakowicz, Principles of Fluorescence Spectroscopy, Kluwer Academic/Plenum Publishers, New York, 1999; (b) Q. Zhou and T. M. Swager, J. Am. Chem. Soc., 1995, 117, 7017–7018.
2. A. K. Dwivedi, G. Saikia and P. K. Iyer, J. Mater. Chem., 2011, 21, 2502–2507.
3. J. R. Lakowicz and G. Weber, Biochemistry, 1973, 12, 4161–4170.
4. P. H. Proctor, Free Radicals and Human Disease. CRC Handbook of Free Radicals and Antioxidants, 1989, vol. 1, pp. 209–221.
5. R. R. Cricton, S. Wilmet, R. Legssyer and R. J. Ward, Inorg. Biochem., 2002, 91, 9–18.
6. W. Breuer, M. J. J. Ermers, P. Pootrakul, A. Abramov, C. Hershko and Z. I. Cabantchik, Blood, 2001, 97, 792–798.
7. S. D. Lytton, B. Mester, J. Libman, A. Shanzer and Z. I. Cabantchik, Anal. Biochem., 1992, 205, 326–333.
8. (a) Y. Ma, H. D. Groot, Z. Liu, R. C. Hider and F. Petrat, Biochem. J., 2006, 395, 49–55; (b) J.-L. Chen, S.-J. Zhuo, Y.-Q. Wu, F. Fang, L. Li and C.-Q. Zhu, Spectrochim. Acta, Part A, 2006, 63, 438–445.
9. W. Breuer, S. Epsztejn, P. Millgram and I. Z. Cabantchik, Am. J. Physiol., 1995, 268, 1354–1361.
10. J. B. Matson and S. I. Stupp, Chem. Commun., 2011, 47, 7962–7964.
11. V. K. Rao, S. R. Mane, A. Kishore, J. D. Sarma and R. Shunmugam, Biomacromolecules, 2012, 13, 221–230.
12. (a) R. H. Grubbs, Handbook of Metathesis, Wiley-VCH, NewYork, 2003, vol. 3; (b) S. R. Mane, V. N. Rao and R. Shunmugam, ACS Macro Lett., 2012, 1, 482–488; (c) A. E. Madkour, A. H. R. Koch, K. Lienkamp and G. N. Tew, Macromolecules, 2010, 43, 4557–4561.
13. S. Bhattacharya, V. N. Rao, S. Sarkar and R. Shunmugam, Nanoscale, 2012, 4, 6962–6968.
14. H. U. Au-Yeung, J. Chan, T. Chantarojsiri and C. J. Chang, J. Am. Chem. Soc., 2013, 135, 15165–15173.
15. D. P. Fairlie, T. C. Woon, W. A. Wickramasinghe and A. C. Willis, Inorg. Chem., 1994, 33, 6425–6428.
16. (a) J. J. Scepaniak, T. D. Harris, C. S. Vogel, J. Sutter, K. Meyer and J. M. Smith, J. Am. Chem. Soc., 2011, 133, 3824–3827; (b) D. P. Riley, J. A. Stone and D. H. Busch, J. Am. Chem. Soc., 1976, 99, 767–777.
17. J. Hatai, S. Pal and S. Bandyopadhyay, Tetrahedron Lett., 2012, 53, 4357–4360.
18. M. T. Werth, D. M. Kurtz, B. D. Howes and B. H. Huynh, Inorg. Chem., 1989, 28, 1361–1366.
19. (a) M. E. Pascualini, N. V. Di Russo, A. E. Thuijs, A. Ozarowski, S. A. Stoian, K. A. Abboud, G. Christoua and A. S. Veige, Chem. Sci., 2015, 6, 608–612; (b) E. Wong, J. Jeck, M. Grau, A. J. P. White and G. J. P. Britovsek, Catal. Sci. Technol., 2013, 3, 1116–1122.
20. X. Wu, B. Xu, H. Tong and L. Wang, Macromolecules, 2010, 43, 21.
21. S. Bhattacharya, S. Sarkar and R. Shunmugam, J. Mater. Chem. A, 2013, 1, 8398–8405.

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Footnote

† Electronic supplementary information (ESI) available: Detailed synthesis, characterization and other spectroscopic data. See DOI: 10.1039/c5ra11342c

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