English

• Fluorescent probes are widely investigated for the detection and measurement of ions for their simplicity and high sensitivity of response. Obviously, probes not only provide a means of detection to investigate the process of molecular recognition but also can be used as the architecture for assembling a molecular device.^{[1, 2]} Because of the above advantages, fluorescent probes based on the molecular switches for the detection of relevant ions have attracted numerous attentions.^{[3, 4]}

  As one of most abundant transition metals in the environment and a vital essential element in the human body, iron plays a crucial role in many biological processes.^[5~9] However, iron will go from beneficial to deleterious depending on their concentration.^[10~14] As reported, iron promotes oxidative damages inside the cell and does harm to human body.^{[15, 16]} Even, recent studies show that the cellular toxicity caused by iron has been linked with several serious diseases, such as Alzheimer's and Parkinson's diseases.^[17~19] Therefore, it is of utmost interest to develop highly sensitive and selective probes for Fe³⁺.

  Up to now, different kinds of probes for Fe³⁺ have been designed.^[20~22] However, most of reported fluorescence probes of Fe³⁺ were "turn-off" type due to the paramagnetic nature of Fe³⁺, which is not as sensitive as the fluorescence probe of "turn-on" type. Moreover, some probes can only be used in organic solvents or mixed solvents (organic solvent: water).^[23~29] In addition, many of reported probes have not been applied to detect Fe³⁺ in vitro detection or imaging, which limited their biological application to a large extent. Additionally, poorer selectivity and the unsure interferences from other ions, such as Fe²⁺, Cr³⁺, Cu²⁺ etc., always prevent those probes to be excellent. Therefore, it is still an outstanding challenge to create more new "turn-on" Fe³⁺ probes with good water-solubility which demonstrated more value of practical applications in biological systems.

  Since the pioneering work by Czamik et al., ^[30] the probes based rhodamine have been extensively designed owing to their excellent photophysical properties. In 2011, Du and coworkers have developed a colorimetric probe for the detection of Fe³⁺ by "naked-eye". The probe can be directly applied to environmental Fe³⁺ detection in 100% water medium ^[31]. Gong et al. ^[32] synthesized efficient fluorescent Fe³⁺ probes by linking a conjugated naphthalene chromophore to a rhodamine platform and a lipophilic triphenylphosphonium cation. The probes could monitor mitochondrial Fe³⁺ in living cells sensitively and selectively. It is the equilibrium between the spirolactam and the ring-opened amide that makes rhodamine to be an ideal pattern for designing probes.^[33~36] In order to continue our interests in the design of rhodamine based probes, herein, we described a new "turn-on" Fe³⁺ probe with high selectivity and sensitivity. The probe was synthesized and confirmed. The recognition progress was based on a metal-coordination induced fluorescent enhancement. The probe spectral responses to Fe³⁺ in comparison with various biologically relevant metal ions were demonstrated. Additionally, the probe was further used for detecting Fe³⁺ ions in PC12 cells and the Kunming mouse, which performed remarkable fluorescence imaging.

  1.   Results and discussion

  1.1   Synthesis and optimized structure

  The probe Y was prepared and confirmed by ¹H NMR, ¹³C NMR, IR and MS. The synthetic routes were shown in Scheme 1.

  Scheme 1

  

  Scheme 1.  Synthesis of Y

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  The density functional theory (DFT) quantum calculations for probe Y was performed by using the Gaussian 09 program at the b3lyp/6-31g level. The related data of bond lengths and optimized structure were shown in Table S1 (Supporting Information) and Figure 1.

  Figure 1

  

  Figure 1.  DFT optimized structure of Y

  C, N, S and O atoms are colored in gray, blue, yellow and red, respectively

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  1.2   Fluorescence and absorption spectral responses

  In this study, the sensing properties of probe Y were investigated by UV-Vis and fluorescence measurements in Tris-HCl aqueous buffer solution. It was found that the probe Y displayed sensitive color change from colorless to pink via rhodamine lactam ring opening in presence of Fe³⁺, which could be easily detected by the naked eye. The addition of other metal ions (including Ag⁺, Al³⁺, Ba²⁺, Co²⁺, Cu²⁺, Ca²⁺, Cd²⁺, Fe²⁺, Cr³⁺, Hg²⁺, K⁺, Li⁺, Mg²⁺, Mn²⁺, Na⁺, Ni²⁺, Pb²⁺ and Zn²⁺) to the solution of Y showed no clear color change (Figure 2). Upon addition of Fe³⁺, probe Y exhibited a new sharp absorption peak at 562 nm, and a remarkable fluorescence emission peak at 586 nm, which suggested the opening of the rhodamine-spirolactam ring. However, other metal ions did not induce any obvious spectral response. Furthermore, the competition experiments of Fe³⁺ (15 μmol/L) mixed with 2.0 equiv. of the above-mentioned metal ions (30 μmol/L) were also conducted. No significant variation was observed in fluorescence intensity (Figure 3). Even Cr³⁺ and Fe²⁺, as the common competing ions for Fe³⁺, in this experiment conditions, there were no obvious effect. These facts were indicative of a high selectivity of Y toward Fe³⁺ over other competitive ions.

  Figure 2

  

  Figure 2.  Fluorescence spectra (a) and UV-vis spectra (b) of Y upon addition of different ions in Tris-HCl aqueous buffer solution

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  Figure 3

  

  Figure 3.  Fluorescent intensity of Y upon addition of Fe³⁺ in the presence of competing ions

  The competing ions: 1, Ag⁺; 2, Al³⁺; 3, Ba²⁺; 4, Ca²⁺; 5, Cd²⁺; 6, Co²⁺; 7, Cu²⁺; 8, Fe²⁺; 9, Cr³⁺; 10, Hg²⁺; 11, K⁺; 12, Li⁺; 13, Mg²⁺; 14, Mn²⁺; 15, Na⁺; 16, Ni²⁺; 17, Pb²⁺; 18, Zn²⁺; 19, Fe³⁺

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  For practical applicability, the optimal pH condition of probe was evaluated. No obvious fluorescence for free probe was observed when pH > 5 (Figure S1, see Supporting Information). After the addition of Fe³⁺ between pH 5 and 8, there was an obvious fluorescence emission. The results indicated that probe could be used in some environmental and physiological regions.

  Figure 4 exhibited the UV-Vis and fluorescence spectral variation of probe with the different mount of Fe³⁺. The free Y, as expected, exhibited almost no absorption near 500 nm, indicating that Y existed as a spirocyclic form. Upon the addition of increasing concentrations of the Fe³⁺, a new absorption at 562 nm appeared with increasing intensity, which clearly suggesting the formation of the ring-opened form of Y due to Fe³⁺ binding. The titration curve (a plot of fluorescent intensity vs. Fe³⁺ concentration) increased linearly and nearly plateaued at 1:1 ratio (probe: Fe³⁺), suggesting the formation of a 1:1 Fe³⁺-probe complex. The association constant (K_a) of Y to Fe³⁺ according to the 1:1 binding mode by non-linear fitting of the spectrometric titration curve was obtained as 8.77×10⁻⁴ mol⁻¹•L. Based on the definition of detection limit, the limit of detection for Fe³⁺ was up to 0.25 μmol/L (Figure S2, see Supporting Information). Addition of ethylenediamine to the solution of Y and Fe³⁺ decreased the fluorescence intensity, which indicated the reversibility of recognition process (Figure 5). This result indicated that compound Y could be easily recovered for repeating employ. All these facts illustrated that probe Y could be applied as a fine fluorescent probe for Fe³⁺.

  Figure 4

  

  Figure 4.  Changes in fluorescence spectra (a) and (b) UV-Vis absorption spectra of Y with various amounts of Fe³⁺

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  Figure 5

  

  Figure 5.  Fluorescence intensity changes of Y upon the addition of each equiv. of ethylenediamine with the presence of Fe³⁺.

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  1.3   Proposed binding mode

  To determine the stoichiometry of the ferric-ligand complex, the Job's plot for fluorescence measurement was applied. Keeping the sum of the initial concentration of Y and Fe³⁺ at 20 μmol/L, the molar ratio of Fe³⁺ was changed. As shown in Figure 6, the binding mode was 1:1. The absorption at 500~600 nm is a clear symbol of the opening of rhodamine spirolactam.^[32] The fluorescence enhancement was attributed to the Fe³⁺ induced ring-opened formation of rhodamine spirolactam. According to the familiar binding sites of rhodamine-based Fe³⁺ probes, Y was most likely to chelate with Fe³⁺ via its oxygen, sulphur and nitrogen atoms in a special pattern (Eq. 1).

  Figure 6

  

  Figure 6.  Job's plot of Y and Fe³⁺ (the total concentration was 20 μmol/L)

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  1.4   Cytotoxicity and fluorescence imaging

  Then, Y was taken to conduct a MTT assay on PC12 cells with different concentrations (0, 6.25, 12.5, 20, 50, 100 μmol/L) to evaluate cytotoxicity. The cellular viability estimated was about 95% with 20 μmol/L Y (Figure 7), exhibiting lower toxicity to cells and being suitable for bioimaging.

  Figure 7

  

  Figure 7.  Cell viability was quantified by the MTT assay.

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  To explore more biological application, the experiment about cell permeability was examined in vivo. As monitored by fluorescence microscopy, incubation of PC12 cells with Y (20 μmol/L) for 1 h at 37 ℃ gave almost no intracellular fluorescence (Figure 8a). As shown in Figure 8b, a red fluorescence increase was observed from the intracellular region after further incubation with Fe³⁺ (20 μmol/L). A bright field image of PC12 cells treated with Y and Fe³⁺ implied that the cells were viable throughout the experiments (Figure 8c). The results indicated that the Y was cell permeable and could be used for imaging of Fe³⁺ in vitro.

  Figure 8

  

  Figure 8.  Fluorescence images in PC12 cells

  (a) Cells incubated with 20 μmol/L probe Y for 1 h. (b) Then further incubated with 20 μmol/L Fe³⁺ for 1 h. (c) Brightfield image of cells shown in panel, confirming their viability. (d) The overlay image of (b) and (c)

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  We then evaluated the suitability of the probe for imaging Fe³⁺ in Kunming mouse body. Pictures were taken under the imaging system. As shown in Figure 9a, there was little fluorescence when the mouse was only treated with Y. Obviously, a large fluorescence signal was captured after the mouse was injected with Y and Fe³⁺ (Figure 9b). The result established that Y was a desired probe for imaging Fe³⁺ in the living mouse.

  Figure 9

  

  Figure 9.  Fluorescent images of probe and Fe³⁺ in vivo

  (a) Probe was injected in the intraperitoneal (i.p.) cavity of mouse; (b) Then followed by i.p. injection of Fe³⁺. Ten minutes after probe injection, the mouse was imaged

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  2.   Conclusion

  In summary, a new fluorescent probe Y was synthesized and the recognition properties for Fe³⁺ were evaluated. It specifically responded to Fe³⁺ with high selectivity, sensitivity and excellent photophysical properties. Further studies demonstrated that the probe Y could be employed for bio-imaging toward Fe³⁺ not only in living cells but also in living animals. We anticipate that the design method may be applicable for other types of fluorescent probes and the probe will find more interesting applications as sensing and labeling agent in Fe³⁺-related biological and medical studies.

  3.   Experimental section

  3.1   Apparatus reagents and chemicals

  The fluorescence spectra were measured on a HITACHI F-4500 fluorescence spectrophotometer. A Shimadzu UV-1700 spectrophotometer was used for absorption spectra. NMR spectra were performed on a Varian INOVA-400 MHz spectrometer with TMS as internal standard. Mass spectra were measured with a Bruker microTOF Spectroscopy. Results of cytotoxicity were analyzed in a Spectra max190-Molecular Devices. Bioimaging was performed on an Olympus FV1000 confocal microscopy.

  The related chemicals were commercially available and used without further purification. The solutions of metal ions were performed from their nitrate or chloride salts. And double distilled water was used throughout the experiments.

  3.2   Synthesis of probe Y

  Compounds 1 and 2 were synthesized with high yield according to literature.^{[37, 38]}

  To a 10 mL of flask, phenyl isothiocyanate (0.12 mL, 1.0 mmol) was dissolved in 1.5 mL of DMF. Then a solution of compound 2 (0.27 g, 0.5 mmol) in DMF (1.5 mL) was added dropwise with vigorous stirring at room temperature for 6 h. Then the mixture was cooled down. Water was then added to the mixture and the aqueous was extracted with dichloromethane. The organic layer was dried over anhydrous MgSO₄ and filtered. The crude product was purified by silica gel column chromatography to afford pure probe Y (white solid) in 63.0% yield. m.p. 194~196 ℃. ¹H NMR (CDCl₃, 400 Hz) δ: 1.14 (t, J＝8 Hz, 12H), 3.29 (q, J＝8 Hz, 8H), 4.12 (q, J＝8 Hz, 1H), 6.18 (dd, J＝4, 8 Hz, 2H), 6.26 (d, J＝8 Hz, 1H), 6.34 (d, J＝4 Hz, 2H), 6.45 (d, J＝8 Hz, 1H), 6.58 (d, J＝8 Hz, 2H), 7.04 (d, J＝8 Hz, 1H), 7.27 (m, 1H), 7.34 (t, J＝10 Hz, 1H), 7.42 (m, 4H), 7.79 (d, J＝8 Hz, 2H), 7.90 (d, J＝4 Hz, 1H), 8.65 (s, 1H); ¹³C NMR (CDCl₃, 100 MHz) δ: 12.6, 14.1, 20.6, 26.9, 44.2, 60.6, 66.7, 98.0, 105.8, 107.7, 108.3, 109.6, 123.6, 123.7, 125.8, 126.0, 127.9, 128.2, 128.3, 128.6, 133.8, 139.4, 139.9, 148.3, 148.8, 150.7, 152.3, 154.0, 167.9, 178.5; IR (KBr) ν: 3414, 2971, 1712, 1612, 1520, 1434, 1356, 1330, 1302, 1258, 1223, 1189, 1152, 1118, 1077, 822, 785, 751, 696, 654 cm^－1. Anal. calcd for C₄₀H₄₀N₆O₂S: C 71.87, H 5.98, N 12.57, S 4.80; found C 71.92, H 5.90, N 12.55, S 4.79. MALDI-TOF MS calcd for C₄₀H₄₁N₆O₂S (M⁺+1) 669.3006, found 669.2984.

  3.3   Preparation of solutions for absorption and fluorescence measurements

  Stock solution of Probe Y (2.0×10⁻⁴ mol•L⁻¹): in a 100 mL of flask, 33.40 mg of Y was dissolved in ethanol, and then diluted to the mark with water. Before spectroscopic measurements, Y was then diluted to the corresponding concentrations.

  The solutions of metal ions including Fe³⁺ were performed from their nitrate or chloride salts.

  All samples were dissolved in Tris-HCl aqueous buffer solution. Before determination, the solutions were shaken for 10 s and waited for 20 min. Excitation wavelength was 550 nm. The bandpasses were both set at 5.0 nm.

  Supporting Information The related experimental preparations and ¹H NMR, ¹³C NMR and MS spectra. The Supporting Information is available free of charge via the Internet at http://sioc-journal.cn.

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• 

  Scheme 1  Synthesis of Y

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  Figure 1  DFT optimized structure of Y

  C, N, S and O atoms are colored in gray, blue, yellow and red, respectively

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  Figure 2  Fluorescence spectra (a) and UV-vis spectra (b) of Y upon addition of different ions in Tris-HCl aqueous buffer solution

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  Figure 3  Fluorescent intensity of Y upon addition of Fe³⁺ in the presence of competing ions

  The competing ions: 1, Ag⁺; 2, Al³⁺; 3, Ba²⁺; 4, Ca²⁺; 5, Cd²⁺; 6, Co²⁺; 7, Cu²⁺; 8, Fe²⁺; 9, Cr³⁺; 10, Hg²⁺; 11, K⁺; 12, Li⁺; 13, Mg²⁺; 14, Mn²⁺; 15, Na⁺; 16, Ni²⁺; 17, Pb²⁺; 18, Zn²⁺; 19, Fe³⁺

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  Figure 4  Changes in fluorescence spectra (a) and (b) UV-Vis absorption spectra of Y with various amounts of Fe³⁺

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  Figure 5  Fluorescence intensity changes of Y upon the addition of each equiv. of ethylenediamine with the presence of Fe³⁺.

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  Figure 6  Job's plot of Y and Fe³⁺ (the total concentration was 20 μmol/L)

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  Figure 7  Cell viability was quantified by the MTT assay.

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  Figure 8  Fluorescence images in PC12 cells

  (a) Cells incubated with 20 μmol/L probe Y for 1 h. (b) Then further incubated with 20 μmol/L Fe³⁺ for 1 h. (c) Brightfield image of cells shown in panel, confirming their viability. (d) The overlay image of (b) and (c)

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  Figure 9  Fluorescent images of probe and Fe³⁺ in vivo

  (a) Probe was injected in the intraperitoneal (i.p.) cavity of mouse; (b) Then followed by i.p. injection of Fe³⁺. Ten minutes after probe injection, the mouse was imaged

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•