Coumarin-derived Fe³⁺-selective fluorescent turn-off chemosensors: synthesis, properties, and applications in living cells

Abstract

A novel coumarin-based fluorogenic probe bearing the tris unit (DAT-1) with high selectivity and suitable affinity toward Fe³⁺ over other cations tested was developed in biological systems. The sensing mechanism was studied by quantum calculations. The probe can be applied to the monitoring of Fe³⁺ in aqueous solution with a pH span of 3–8. In addition, biological imaging, membrane permeability and nontoxicity demonstrate that DAT-1 could act as a turn-off fluorescent chemosensor for Fe³⁺ in living cells.

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Introduction

The design and synthesis of high selectivity and sensitivity chemosensors to detect metal ions in the ecological environment and biology have attracted a great deal of attention.¹ A chemosensor shows a significant change in electrical, electronic, magnetic, or optical signal when it binds to a specific guest counterpart.² Current chemosensors have many advantages, including high sensitivity, low cost, easy detection, and suitability as a diagnostic tool for biological purposes.³ As an essential trace element in biological systems, Fe³⁺ plays an important role in living organisms and metabolism. Many enzymatic reactions involving iron are related to oxygen metabolism, electron transfer and the mitochondrial respiratory chain.⁴ Although iron is indispensable for the proper functioning of all living cells, it is disadvantageous when present in excess. Excessive Fe³⁺ in the human body has been found to be related to an increased incidence of certain cancers and the dysfunction of certain organs.⁵ Moreover, hereditary hemochromatosis is characterized by excess iron that causes tissue damage and fibrosis with irreversible damage to various organs.^4a,6 Furthermore, iron homeostasis is an important factor involved in neuroinflammation and the progression of Alzheimer's disease.^4a,7 At the same time, iron deficiency can be as harmful as iron overload, or even more harmful. It is well known that anemia is due to iron deficiency. Therefore, a convenient and rapid method for detecting the concentration of Fe³⁺ in biological samples is of great interest in biological and environmental concerns. Recently, many fluorescent chemosensors for Fe(III)-selective detection were reported and have been used with some success in biological applications.⁸ Nevertheless, a few of them have defects in actual practice such as cross-sensitivity toward other metal cations, long response times, poor water solubility, a narrow pH detection span, a low fluorescence quantum yield in aqueous media, and ligand cytotoxicity.⁹

Of late, coumarin-based chemosensors have been extensively used as fluorescent labeling reagents for their large molar extinction coefficient, relatively long excitation and emission wavelengths and high fluorescence quantum yield.¹⁰ Here, we synthesized a novel coumarin-derived Fe(III)-selective chemo-sensor N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]-7-hydroxycoumarin-3-carboxamide (DAT-1) which is highly selective and sensitive toward Fe³⁺ over other common metal ions such as Na⁺, K⁺, Ca²⁺, Fe²⁺, Mg²⁺, Al³⁺, Co²⁺, Ni²⁺, Ag⁺, Cu²⁺, Zn²⁺, Cr³⁺, Cd²⁺, Mn²⁺, Hg²⁺ and Pb²⁺ in buffered aqueous solutions, and the sensing mechanism was studied by quantum calculations.

Experiment

Ethyl-7-hydroxy-3-carboxylate 2 (ref. 10) was synthesized according to the procedures reported in the literature. 2-Amino-2-(hydroxymethyl)-3-propanediol (tris) was purchased. DAT-1 was prepared by the standard method with a minor modification.¹¹ Compound 2 (234 mg, 1 mmol) was treated with tris (121 mg, 1 mmol) in anhydrous ethanol (5 mL). The reaction was then refluxed for 8 h. The mixture was concentrated under vacuum and the crude product was purified by column chromatography (CH₂Cl₂ : CH₃OH = 10 : 1) to give DAT-1 as a yellow solid (185 mg, yield: 60 %), mp. 239–241 °C. ¹HNMR (400 MHz, DMSO-d₆): δ = 11.09 (s, 1H), 8.96 (s, 1H), 8.81 (s, 1H), 7.81 (d, J = 8.5 Hz, 1H), 6.89 (dd, J = 8.5, 1.7 Hz, 1H), 6.82 (d, J = 1.6 Hz, 1H), 4.86 (s, 3H), 3.65 (s, 6H). ¹³CNMR (400 MHz, DMSO-d₆): δ 169.08, 166.84, 166.39, 161.52, 153.23, 137.19, 119.62, 118.82, 116.19, 106.96, 67.44, 65.30, 45.27, 45.06, 44.86, 44.65, 44.44, 44.23, 44.02. HRMS (ESI): m/z, calcd for (M − H)⁻ 308.0770; found 308.0798 (Scheme 1).

Scheme 1 Synthetic procedure of DAT-1.

Results and discussion

The DAT-1 chemosensor contain a coumarin group used as the fluorescent signal unit and a 2-amino-2-(hydroxymethyl)-1,3-propanediol (tris) acting as a possible recognition unit. Since coumarin derivatives exhibit several advantages such as large Stokes shifts, good photostability and high fluorescence quantum yields (the quantum yield of the system was calculated to be 0.75), they could be used as efficient fluorophores. And the tris which includes three hydroxyl groups and an amide group is expected to serve as a potential binding group.¹¹ Furthermore, the tris group can improve the water solubility of chemosensors due to its hydrophilic properties.

The structure of DAT-1 was confirmed by ¹H NMR, ¹³C NMR, FT-IR, ESI-MS and X-ray analysis (Fig. S7–S10†). The single crystal of DAT-1 was obtained under vapor diffusion of CH₃OH into a solution of water and characterized using X-ray crystallography (Fig. 1).

Fig. 1 View of the structure of DAT-1 with displacement atomic ellipsoids drawn at the 30% probability level. All hydrogen atoms are omitted for clarity.

Fig. 2a shows the change in the fluorescence spectra of DAT-1 upon the addition of Fe³⁺ in the Na₂HPO₄–citric acid buffer solution at pH 4.8. Upon the addition of an increasing amount of Fe³⁺, the emission intensity of DAT-1 at 448 nm gradually decreased. The fluorescence emission intensity were sensitively and proportionately decreased with an increasing concentration of Fe³⁺ as noted from the relationship between the fluorescence emission intensity at 350 nm and Fe³⁺ concentration (Fig. 2b). Therefore, the DAT-1 could be used for the development of an efficient method for Fe³⁺ ion detection. The linear relation of the obtained experimental data for Fe³⁺ detection could be best described as the Stern–Volmer equation. The Stern–Volmer relationship with an R² of 0.996 is given in the following equation (Fig. 2b, inset):

I₀/I = 1 + K_SV[Q]

where I and I₀ are the fluorescence emission intensities of the DAT-1 in the presence and absence of Fe³⁺ respectively. Q is the Fe³⁺ concentration. K_SV is the Stern–Volmer quenching constant and is found to be 3.57 × 10³ M⁻¹. The linear range of Fe³⁺ concentration was from 1 × 10⁻⁵ M to 1 × 10⁻³ M and the detection limit of Fe³⁺ was 3 × 10⁻⁷ M. It can be found that the presented method has wider detection range and higher sensitivity than many of fluorescent chemosensors for Fe³⁺.

Fig. 2 (a) Fluorescence titration spectra (λ_ex = 350 nm) of DAT-1 (20 μM) with the addition of Fe³⁺ (0–200 equiv.) in the 0.2 M Na₂HPO₄–citric acid buffer. (b) Titration data points and the nonlinear least-squares fitting curve. Insert: the Stern–Volmer plot of fluorescence quenching of the DAT-1 by Fe³⁺. R² = 0.996.

There was a remarkable selectivity of DAT-1 for Fe³⁺ over various other metal ions in the competition experiments, which turned out to be applicable in environmental technology (Fig. 3). The fluorescence intensity of the chemosensor DAT-1 was substantially diminished by the addition of Fe³⁺, whereas other cations such as K⁺, Ca²⁺, Fe²⁺, Mg²⁺, Al³⁺, Co²⁺, Ni²⁺, Ag⁺, Cu²⁺, Zn²⁺, Cr³⁺, Cd²⁺, Mn²⁺, Hg²⁺ and Pb²⁺ did not cause any significant changes in the fluorescence emission intensity, even at a concentration of 100 equiv. of guest counterparts. Even with highly concentrated Na⁺, K⁺, Ca⁺, and Mg²⁺ (∼5.0 mM) under physiological conditions,¹² there were no fluorescence changes in DAT-1. The large decrease in fluorescence intensity could be applied to the detection of iron cations by the naked eye (Fig. 4). When the chemosensor (20 μM) was excited at 350 nm in the presence of various cations (100 equiv.) in Na₂HPO₄–citric acid buffer (pH 4.8), hardly any fluorescence response of Fe³⁺ was selectively observed (Fig. 4).

Fig. 3 Metal ions selectivity of DAT-1 (20 μM) in the Na₂HPO₄–citric acid buffer solution. The black bars represent the fluorescence emission intensity of DAT-1 and 100 equiv. of metal ions. The red bars represent the fluorescence response of DAT-1 to 100 equiv. of Fe³⁺ containing 100 equiv. of other metal ions. λ_ex = 350 nm, λ_em = 448 nm.

Fig. 4 Fluorescent (bottom) and color (top) responses of DAT-1 (20 μM) in Na₂HPO₄–citric acid buffer solutions (0.2 M) upon the addition of (100 equiv.) metal ions in water.

It is well-known that heavy metal ions such as Fe³⁺, Cu²⁺, Cd²⁺, Hg²⁺ and Pb²⁺ tend to quench the luminescence through electron- and/or energy-transfer processes.¹³ Because of the specific structure, the recognition unit of our probe could complex with iron ions selectively. Thus the photoinduced electron transfer (PET) effect on N atom causes fluorescence quenching of coumarin. The fact that our DAT-1 chemosensor can not complex with other ions besides iron ions is attributed to the specific recognition of the recognition unit to iron ions.^13e Therefore, our probe cannot be quenched by other ions without PET effect. To prove the mechanism of fluorescence quenching, we carried out theoretical calculations (Fig. 5; details in Fig. S3†). From the single crystal structure of DAT-1, there are many coordination sites, but it is difficult to determine the structure of coordination between DAT-1 and Fe³⁺. We therefore designed 22 coordinations, considering all possible options except water as ligand. We found, however, that complex-23, with water as ligand, was the optimum structure compared with other complexes. This result agrees with the experimental data reported by other authors.¹¹ Thus we can assume that complex-23 was the best structure in this system. The electron distributions of HOMO, LUMO, and LUMO+4 of complex-23 were calculated by B3LYP/6-31G(d, p) and are shown in Fig. 5. The electronic distribution on LUMO+4 for complex-23 was extended to Fe, indicating the electron transfer from HOMO to LUMO+4. Thus the electrons transfer from DAT-1 to Fe³⁺, making the fluorescence quenching behavior possible. Indeed, it has been demonstrated in the literature that fluorescence quenching of the ligand may occur by the excitation energy transfer from the ligand to the metal d orbital and/or ligand to metal charge transfer (LMCT).^9,14

Fig. 5 The calculated frontier molecular orbitals of complex-23 (a–c) and the best structure of complex-23 (d).

To elaborate upon the mechanism of fluorescence quenching which we speculate takes place by the energy and/or charge transfer model, fluorescence lifetime testing was conducted. The fluorescence decay behavior in the presence of Fe³⁺ is shown in Fig. 6, and the exponential fit results are summarized in Table 1. In the absence of Fe³⁺, the fluorescence decays single exponentially by a 3.68 ns time constant, which is the lifetime of the S1 state of free DAT-1. The time constant (τ) of the lifetime decay component drops off when Fe³⁺ is added.

Fig. 6 Time-resolved fluorescence of DAT-1 with an excitation at 405 nm in the absence (black) and in the presence of 1 equiv. (green), 5 equiv. (blue), 25 equiv. (magenta) and 50 equiv. (red) of Fe³⁺ ions.

Table 1 Fluorescence decay time constants of 1 in the presence of Fe³⁺ ions

Fe^3+ (equiv.)	τ (ns)	Fe^3+ (equiv.)	τ (ns)
0	3.68	25	2.76
1	3.60	50	2.30
5	3.42

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For biological applications of the chemosensor, the sensing should operate in a wide range of pH. Fig. 7 shows that in aqueous solution the suitable pH range for Fe³⁺ determination is pH 3–8 where the “on–off” fluorescence can be operated by the iron ion binding. Consequently, our present Fe³⁺-selective receptor would be an ideal chemosensor for monitoring Fe³⁺.

Fig. 7 Variation of fluorescence spectra of DAT-1 (20 μM) in aqueous solution (Na₂HPO₄–citric acid buffer) (0.2 M) with and without Fe³⁺ (100 equiv.) ion as a function of pH. Fluorescence intensity at 448 nm.

The ability of biosensing molecules to selectively monitor guest species in living cells is of great importance for biological applications. First, we selected breast carcinoma cell lines (MCF-7) to investigate the toxicity of DAT-1. The toxicity data were obtained by performing 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assays as shown in Fig. 8. The cells were cultured in RPMI 1640 (Gibco) and supplemented with 10% fetal bovine serum (FBS; Gibco) at 37 °C in a humidified atmosphere of 5% CO₂. For all experiments, cells were harvested from subconfluent cultures by the use of trypsin and were resuspended in fresh complete medium before plating. In vitro cytotoxicity was assessed by using MTT reduction assays. In a typical procedure, about 2000 cells were plated in 96-well plates for 24 h to allow the cells to attach, and then incubated with DAT-1 (20 μM) in 5% CO₂ at 37 °C. At the end of the incubation time, MTT solution (10 μL, diluted in culture medium to a final concentration of 1 mg mL⁻¹) was added, and then the mixture was incubated for another 4 h. Finally, the incubation solution was removed and dimethyl sulfoxide (DMSO, 150 μL) was added to each well. The absorbance of MTT was determined (λ = 490, 630 nm) by using a microplate reader (BioTek, ELX808). The cell viability obtained was expressed as a percentage relative to the control. The viability of untreated cells was assumed to be 100%. When the concentration of chemosensor DAT-1 was 5 μM, more than 99% of the MCF-7 cells were alive. Even when the concentration of DAT-1 increased to 100 μM, there were still about 87% of the MCF-7 cells alive. From the results of cytotoxicity experiments, the chemosensor DAT-1 shows low toxicity even at a high concentration of 100 μM particles and an incubation time of 24 h, which means that DAT-1 possesses high biocompatibility.

Fig. 8 Percentage of MCF-7 cell viability remaining after cell treatment with DAT-1 (untreated cells were considered to have 100% survival). Cell viability was assayed by the MTT method (values: mean ± standard deviation).¹⁵

To determine the cell permeability of DAT-1, MCF-7 cells were incubated with DAT-1 (20 μM) for 25–30 min at 37 °C, and washed with PBS to remove the remaining compound DAT-1. The results are shown in Fig. 9b. One can clearly observe significant confocal imaging changes of the medium upon addition of FeCl₃ for 20 min (25, 50, and 100 equiv., respectively) at 37 °C. MCF-7 cells incubated with DAT-1 initially display a strong fluorescence image, but the fluorescence image immediately becomes faint in the presence of Fe³⁺ (Fig. 9c–e). Thus, the chemosensor DAT-1 can be a suitable fluorescence chemosensing probe for Fe³⁺ detection in biological systems.

Fig. 9 Confocal fluorescence images of Fe³⁺ in MCF-7 cells (Olympus FV1000, 40× objective lens). (a) Bright-field transmission image of MCF-7 cells. (b) Fluorescence image of MCF-7 cells incubated with DAT-1 (20 μM). Further incubated with addition of various concentrations of FeCl₃ [(c) 25, (d) 50, and (e) 100 equiv., respectively]. (f) Return of intracellular Fe³⁺ to the resting level was achieved by addition of EDTA (2 mM).

Conclusion

In summary, this study has reported the synthesis, properties, and cellular applications of a novel chemosensor DAT-1 for Fe³⁺ sensing. This chemosensor exhibits high sensitivity and selectivity toward Fe³⁺ in aqueous solution. We simulated the mechanism of fluorescence quenching by quantum calculations. In terms of good optical properties, fine water solubility, excellent membrane permeability and non-toxicity, DAT-1 could be one of the most important chemosensors for the detection of Fe(III) in living cells.

Acknowledgements

We are very grateful for the support of this work from the National Natural Science Foundation of China (no. 21072158 and 20802056), the Special Foundation of the Education Committee of Shaanxi Province (no. 12JK0580), the French Chinese Foundation for Science and Applications (FFCSA) and the China Scholars Council (CSC).

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Footnote

† Electronic supplementary information (ESI) available. CCDC 939460. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c3ra44843f

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