Analytica Chimica Acta 826 (2014) 77–83
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A new turn-on fluorescence probe for Zn2+ in aqueous solution and imaging application in living cells Meng-Meng Li a,1, Fang-Wu Wang b,1, Xiao-Yun Wang c , Ting-Ting Zhang a , Yu Xu a , Yu Xiao a , Jun-Ying Miao b, * , Bao-Xiang Zhao a, * a b c
Institute of Organic Chemistry, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, PR China Institute of Developmental Biology, School of Life Science, Shandong University, Jinan 250100, PR China Chemical Technology Academy of Shandong Province, Jinan 250014, PR China
H I G H L I G H T S
G R A P H I C A L A B S T R A C T
A new pyrazoline-based turn-on fluorescence probe toward Zn2+ was synthesized. Upon addition of Zn2+, the fluorescence intensity was enhanced up to 80-fold. The fast response to Zn2+ makes the probe suitable to monitor Zn2+ in living cell.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 February 2014 Received in revised form 28 March 2014 Accepted 1 April 2014 Available online 4 April 2014
We designed and synthesized a new pyrazoline-based turn-on fluorescence probe for Zn2+ by the reaction of chalcone and thiosemicarbazide. The structure of the probe was characterized by IR, NMR and HRMS spectroscopy. The probe (L) exhibits high selectivity and sensitivity for detecting Zn2+ in buffered EtOH/HEPES solution (EtOH/HEPES = 1/1, pH 7.2) with 80-fold fluorescence enhancement, which is superior to previous reports. Job’s plot analysis revealed 1:1 stoichiometry between probe L and Zn2+ ions. The association constant estimated by the Benesi–Hildebrand method and the detection limit were 3.92 103 M1 and 5.2 107 M, respectively. A proposed binding mode was confirmed by 1H NMR titration experiments and density functional theory (DFT) calculations. The probe is cell-permeable and stable at the physiological pH range in biological systems. Because of its fast response to Zn2+, the probe can monitor Zn2+ in living cells. Moreover, the selective binding of L and Zn2+ was reversible with the addition of EDTA in buffered EtOH/HEPES solution and Zn2+ could be imaged in SH-SY5Y neuron cells. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Pyrazoline Fluorescent probe Zinc ion Imaging Neuron cells Reversibility
1. Introduction As the second-most abundant transition metal ion in the human body, Zn2+ plays pivotal roles in physiological processes
* Corresponding authors at: Shandong University, Organic Chemistry, Shanda Nanlu 27, Jinan, Shandong, PR China. Tel.: +86 531 88366425; fax: +86 531 88564464. E-mail addresses: [email protected], [email protected] (B.-X. Zhao). 1 Equal contribution. http://dx.doi.org/10.1016/j.aca.2014.04.001 0003-2670/ ã 2014 Elsevier B.V. All rights reserved.
such as gene transcription, regulation of metalloenzymes, and neural signal transmission [1–5]. Disordered Zn metabolism in biological systems is associated with diseases such as Alzheimer’s disease, diabetes, and epilepsy [6–9]. Exploring Zn2+ fluorescent probes is important, and previous probes have been found to have high sensitivity, detection of metal ions at very low concentration and imaging of Zn2+ in biological samples [10–14]. Fluorescent probes developed for Zn2+ include dipyrromethene boron difluoride (BODIPY) dyes [15–17], quinoline [18,19], bipyridine [20,21], phenoxazinium [22], Schiff-base [23–26], coumarin [27– 30], pyrene [31], rhodamine [32], triazole [33] and other
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Scheme 1. Synthesis of compound 2 (L).
fluorophores [34,35]. However, most of the probes have drawbacks such as poor selectivity and sensitivity, less water solubility, and complicated synthesis, or only work in toxic organiccontaining solutions such as acetonitrile and have poor cell membrane permeability. Recently, a carboxamidoquinoline-based fluorescent Zn2+ probe, with good water solubility, cell membrane permeability, selectivity and sensitivity, was reported; however, the probe produced only about 21-fold enhancement of emission based on the carboxamidoquinoline deprotonation process and ICT [36]. Other new reported probes also show limited increase in fluorescence [37–40]. In addition, some Zn2+ probes are affected by cross interference of Cd2+ owing to their closely related electronic and binding properties [41,42]. Thus, to satisfy practical needs, we need to develop Zn2+ probes with high selectivity, good water solubility, and good tolerance to interference over other metal ions, especially for imaging application in living cells. Pyrazoline derivatives have attracted increasing attention as fluorophores for metal ion probes because of their blue light emission with high fluorescence quantum yield, high fluorescence selectivity and excellent stability [43–46]. Recently, we investigated novel probes for Zn2+ based on pyrazoline [47–50] and successfully used them to monitor Zn2+ ions in cell imaging [49,50]. However, they showed low turn-on response in aqueous
solution. Here, we describe our development of a new fluorescent probe, L, 3-(2-hydroxy-5-methylphenyl)-5-pyridin-3-yl-4,5-dihydro-1H-pyrazole-1-carbothioamide, showing high sensitivity and selectivity for Zn2+ over other metal ions and high turn-on response (80-fold). Furthermore, the probe can be used to image Zn2+ in living cells and has good reversibility. 2. Experimental details 2.1. Reagents Deionized water was used throughout the experiment. All reagents were purchased from commercial suppliers and used without further purification. The salts used in stock aqueous solutions of metal ions were NaNO3, Fe(NO3)39H2O, AgNO3, KNO3, Co(NO3)26H2O, Mg(NO3)26H2O, Ca(NO3)24H2O, Al(NO3)39H2O, Ba(NO3)2, Cr(NO3)39H2O, Ni(NO3)26H2O, Cd(NO3)24H2O, Pb (NO3)2, Cu(NO3)23H2O, Zn(NO3)26H2O and HgCl2. 2.2. Apparatus Thin-layer chromatography involved silica gel 60F254 plates (Merck KGaA). 1H NMR spectra were recorded on a Bruker Avance 300 (300 MHz) spectrometer, with DMSO-6d used as a solvent. 13C NMR spectra were recorded on a Bruker Avance 300 (75 MHz)
Fig. 1. Absorption spectra of probe L (50 mM) in buffered EtOH/HEPES solution (EtOH/HEPES = 1/1, pH 7.2) with Zn2+ (0–50 equiv.). The inset shows the absorbance of L as a function of Zn2+ concentration.
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spectrometer, with DMSO-6d used as a solvent and tetramethylsilane (TMS) as an internal standard. Melting points were determined on an XD-4 digital micro-melting-point apparatus. IR spectra were recorded with a VERTEX 70 FT-IR spectrophotometer (Bruker Optics), HRMS spectra were recorded on a QTOF6510 spectrograph (Agilent), and UV–vis spectra were recorded on a U4100 spectrophotometer (Hitachi). Fluorescent measurements were recorded on a PerkinElmer LS-55 luminescence spectrophotometer. All pH measurements involved use of the Model PHS-3C pH meter (Shanghai) at room temperature, about 25 C.
cultivated in flasks for cell adhesion. For the experimental study, cells were grown to 80–90% confluence, harvested with 0.25% trypsin (Sangon Biotech) in phosphate-buffered saline (PBS), and seeded into 24-well plates. Experiments to assay Zn2+ uptake were performed in the same media supplemented with different concentrations of Zn nitrate with pyrithione (Zn ionophore). Cells were rinsed twice with PBS and treated with DMSO-containing L (10 mM, DMSO:water = 1:100) for 20 min at 37 C. After 2 washes with PBS, cells were viewed under an inverted fluorescence microscope (Nikon TE2000-S).
2.3. Procedures
3. Results and discussion
2.3.1. Synthesis of compound 2 (L) The synthetic route of proposed compound 2 is shown in Scheme 1. A mixture of chalcone (1) (0.717 g, 3 mmol), thiosemicarbazide (0.409 g, 4.5 mmol), NaOH (0.368 g, 9 mmol) and ethanol (50 mL) was stirred at reflux temperature for 1 h. After a cooling, water (100 mL) was added to the mixture and hydrochloric acid was added to neutralize it to pH 7. Then the mixture was extracted with dichloromethane, and the combined extraction was dried over anhydrous magnesium sulfate. After filtration, the filtrate was concentrated to give the crude product. Purification by column chromatography (silica gel; 25:1 CH2Cl2:CH3OH) yielded product 2 as a yellow solid. Yield: 47.6%; mp: 230–231 C; IR (KBr, cm1): 3428.1, 3252.7, 1594.5, 1477.3, 1332.1, 1256.1, 819.0, 705.6; 1H NMR (300 MHz, DMSO-6d): 2.22 (s, 3H, CH3), 3.31 (dd, 1H, J = 3.3, 18.4 Hz, 4-Htrans of pyrazoline), 4.00 (dd, 1H, J = 11.5, 18.4 Hz, 4-Hcis of pyrazoline), 5.95 (dd, 1H, J = 3.3, 11.5 Hz, 5-H of pyrazoline), 6.84 (d, 1H, J = 8.2 Hz, benzene H), 7.14 (dd,1H, J = 1.8, 8.2 Hz, benzeneH), 7.35 (dd, 1H, J = 4.8, 7.8 Hz, pyridine H), 7.48 (d, 1H, J = 1.8 Hz, benzene H), 7.50 (d, 1H, J = 2.1 Hz, pyridine H), 8.14 (d, 2H, J = 17.1 Hz, NH2), 8.41 (d, 1H, J = 2.1 Hz, pyridineH), 8.44 (dd, 1H, J = 1.5, 4.8 Hz, pyridine H), 9.52 (s, OH); 13C NMR (75 MHz, DMSO-6d): 175.65, 156.25, 154.42, 148.14, 147.29, 138.25, 132.95, 132.81, 129.51, 128.07, 123.66, 116.71, 115.61, 59.90, 43.78, 19.87; HRMS: calcd for [M + H]+ C16H16N4OS: 313.1123; found: 313.1122. (Fig. S0, ESI)
3.1. UV–vis absorption spectra for probe L The UV–vis absorption spectra for probe L were investigated in buffered EtOH/HEPES solution (EtOH/HEPES = 1/1, pH 7.2). With the addition of excess of 5 equiv. of various metal ions, including Ag+, Al3+, Fe3+, Co2+, Ni2+, Ba2+, Ca2+, Cu2+, Cd2+, Cr3+, K+, Mg2+, Na+, Hg2+, Zn2+, Pb2+, most of the tested metal cations did not induce any distinct change in absorbance at 338 nm, but the absorbance
2.3.2. Cell culture and imaging SH-SY5Y cells were grown in a 1:1 mixture of MEM (Gibco) and F12 (Gibco) medium and 10% supplemental fetal bovine serum (Gibco), at 37 C in a humidified incubator with 5% CO2. Cells were
Fig. 2. Fluorescence intensity changes ((I I0)/I0 of probe L (10 mM) in buffered EtOH/HEPES solution (EtOH/HEPES = 1/1, pH 7.2) with 10 equiv. of metal ions: Al3+, Fe3+, Co2+, Ni2+, Ba2+, Ca2+, Cd2+, Cr3+, K+, Mg2+, Na+, Ag+, Hg2+, Zn2+, Cu2+ ions and blank (excitation wavelength: 390 nm; emission wavelength: 480 nm).
Fig. 3. (a) Fluorescence emission spectra for probe L (10 mM) with the addition of Zn2+ (0–100 equiv.) in buffered EtOH/HEPES solution (EtOH/HEPES = 1/1, pH 7.2). The inset shows the emission of probe L (10 mM) as a function of Zn2+ concentration. (b) Color change of L with Zn2+ (0, 1, 5, 10 equiv.) (excitation wavelength: 390 nm; emission wavelength: 480 nm).
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Fig. 6. The proposed binding mode of probe L and Zn2+.
Fig. 4. Fluorescence intensity of probe L (10 mM) with 1.0–100 equiv. of Zn2+ (excitation wavelength: 390 nm; emission wavelength: 480 nm).
increased to some extent for Cu2+ (402 nm), Co2+ (428 nm), Ni2+ (402 nm) and Zn2+ (390 nm) (Fig. S1, ESI). The absorption maximum at 390 nm for Zn2+ was associated with color change of the solution from colorless to yellow (Fig. S2, ESI). In addition, the absorption maximum of L at different concentrations appeared at 338 nm, and the extinction coefficient (e) was 2.39 104 M1 cm1 (Figs. S3 and S4, ESI). The UV–vis absorption spectra for L in the presence of different concentrations of Zn2+ ions are in Fig. 1. With increasing concentration of Zn2+ ions, the absorbance of L at 338 nm gradually decreased. Simultaneously, a new absorption band appeared at 390 nm, and absorbance gradually increased with increasing Zn2+ concentration. Moreover, one isobestic point appeared at 364 nm, which indicated the formation of a stable complex with a certain stoichiometric ratio. The red shift of absorption maximum can be ascribed to the coordination of the probe with Zn2+.
with the addition of other cations (Ag+, Al3+, Fe3+, Co2+, Ni2+, Ba2+, Ca2+, Cu2+, Cd2+, Cr3+, K+, Mg2+, Na+, Hg2+, Pb2+). Moreover, with the addition of 10 equiv. of Zn2+, the fluorescence maximum was remarkably enhanced up to 80-fold as compared with the initial fluorescence intensity of probe L. The enhancement was superior compared to that in previous reports [47–50]. In addition, we conducted competition experiments in the presence of Zn2+ and other metal ions under the same solvent (Fig. S5, ESI). The presence of Cd2+, Ba2+, Pb2+, Na+, Mg2+, K+ and Ca2+ did not interfere with the fluorescence response of L to Zn2+. The addition of Cr3+, Fe3+, Hg2+, Al3+ and Ag+ slightly changed the emission of L–Zn2+ at 480 nm. However, some paramagnetic metal ions, such as Co2+, Ni2+, and Cu2+, quenched the fluorescence, which is always encountered with other metal ion probes [22,38,40,51–54]. This observation is probably due to the displacement of Zn2+ by Co2+, Ni2+, and Cu2+. In brief, the probe L had excellent selectivity for Zn2+ over other metal cations in aqueous solution. Fluorescence titration was performed in the presence of different concentrations of Zn2+. From the fluorescence titration profile (Fig. 3a), the addition of Zn2+ increased fluorescence intensity at 480 nm and was saturated with the addition of 80 equiv. of Zn2+. Clear blue fluorescence occurred with irradiation at 365 nm (Fig. 3b), and the fluorescence intensity was enhanced
3.2. Fluorescence spectra of probe L The selectivity of the fluorescence response of probe L to metal ions was investigated in buffered EtOH/HEPES solution. With the addition of Zn2+ ions, fluorescence intensity was significantly induced (Fig. 2), with only weak changes in fluorescence intensity
Fig. 5. Reversibility of L–Zn2+ binding.
Fig. 7. Optimized structures of probe L (a) and L–Zn2+ (b) by calculation.
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with Zn2+ from 0 to 10 equiv. Quantum yield was determined by the relative comparison procedure, with quinine sulfate dehydrate (99.0%) in 0.1 N H2SO4 used as the main standard [55] and was calculated by the following general equation: F = Fs (IAs/IsA) (h2/hs2) [56]. The quantum yield of L in the absence and presence of Zn2+ was 0.0028 and 0.23, respectively, for an 82-fold enhancement in Ff. Moreover, the Job plot showed 1:1 binding stoichiometry for Zn2+ and L (Fig. S6, ESI). Following a Benesi– Hildebrand-type analysis [57], the association constant Ka was determined to be 5.68 103 M1 (Fig. 4). The limit of detection (LOD) was evaluated with 3s bi/m [58] and calculated from the fluorescence titration curves to be 5.0 107 M (Fig. 3a). For practical applications, we investigated the ability of probe L to sense Zn2+ at different pH values (Fig. S7, ESI). The fluorescence of probe L remained constant at 480 nm from pH 5 to 9, whereas the fluorescence intensity of the L–Zn2+ complex increased sharply from pH 5.5 to 7.5 and reached a steady high reading at around pH 7.5. However, the fluorescence of L–Zn2+ decreased with increasing pH from 7.5 to 9.0, which may be due to the formation of Zn(OH)+ or Zn(OH)2 at high pH and thus, reducing the concentration of L– Zn2+ [40]. Therefore, the probe should be used to monitor Zn2+ by covering the physiological pH range in biological systems. In addition, the interaction of L with Zn2+ was completed in a few seconds (Fig. S8, ESI), and the fluorescence intensity of L–Zn2+ was stable in 10 min. To examine the reversibility of the binding of L to Zn2+, we used EDTA as a well-known metal ion chelator in buffered EtOH/HEPES
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solution. The fluorescence intensity of L–Zn2+ (10 mM) at 480 nm was completely quenched by the addition of 1.0 equiv. of EDTA (Fig. 5). The fluorescence intensity increased again with the addition of 1 equiv. Zn2+ to the mixture of L–Zn2+-EDTA (10 mM). Therefore, the selective binding of L and Zn2+ was reversible. 3.3. The proposed binding mechanism of probe L A proposed binding mode is shown in Fig. 6. To understand the binding behavior of probe L with Zn2+, we performed 1H NMR titration experiments. We found partial change in 1H NMR spectra for L without and with 1.0 equiv. Zn2+ in d-DMSO (Fig. S9, ESI). All protons in the pyrazoline ring and aryl showed an upfield or downfield shift due to the formation of the complex. On the basis of the relative intensity of the lines, a complex of probe L with about 0.3 equiv. Zn2+ formed. Of note, the hydroxyl proton was lost when probe L bound to Zn2+. We performed density functional theory (DFT) calculations to verify the binding mechanism of L–Zn2+. With the first principles of DFT, we performed calculations with the CASTEP code [59]. The exchange and correlation interactions were modeled by using the generalized gradient approximation (GGA) with the PW91 function. With the geometric optimization, all forces on atoms converged to 0.05 eV Å1, the maximum ionic displacement was within 0.002 Å, and the total stress tensor was reduced to the order of 0.1 GPa. The calculation results well supported the proposed binding mode (Fig. 7).
Fig. 8. Fluorescence microscopy of living SH-SY5Y cells. Cells of control group were incubated with 2.5 mM probe L for 20 min at 37 C Cells of the experimental group were exposed to 30 mM pyrithione with 1, 5 and 25 mM extracellular Zn2+ for 45 min. After 2 rinses with PBS, cells were treated with 2.5 mM probe L for 20 min at 37 C. (A) (a) Bright-field, (b) fluorescent, (c) overlay, and (d) fluorescent images after EDTA treatment and (B) quantitation. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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3.4. Biological applications of probe L To investigate the potential biological application of probe L in living cells, we performed intracellular Zn2+ imaging of SH-SY5Y neuron cells by fluorescence microscopy. After incubation with DMSO-containing L (2.5 mM, DMSO:water = 1:100) for 20 min at 37 C, cells showed faint fluorescence. However, fluorescence became visible in the cytoplasm when exogenous Zn2+ (1, 5, 25 mM) was introduced in the presence of the Zn-selective ionophore (30 mM) pyrithione (2-mercaptopyridine N-oxide), for visual evidence of probe L readily entering cells and information on the intracellular existence of Zn2+ (Fig. 8). Moreover, with increasing concentration of Zn2+, fluorescence intensity was enhanced concentration-dependently. In addition, fluorescence intensity was greatly decreased with the addition of 5 mM EDTA for 30 min. Thus, probe L has potential for biological applications. 4. Conclusions In summary, we developed a highly sensitive and selective probe for Zn2+ ions in aqueous media. Fluorescence intensity was remarkably enhanced up to 80-fold with the addition of Zn2+. The addition of EDTA could quench the fluorescence of the L– Zn2+ complex, which indicates that probe L for Zn2+ can be reversible. In addition, the probe was successfully used to image Zn2+ in SH-SY5Y neuron cells, with excellent water solubility and high sensing ability under physiological pH. The fluorescent image was reversed by adding an appropriate amount of EDTA. Therefore, probe L can be used to selectively monitor Zn2+ in living neuron cells. Acknowledgments This study was supported by the 973 Program (2010CB933504) and National Natural Science Foundation of China (91313303 and J1103515). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aca.2014.04.001. References [1] T.V. O’Halloran, Transition metals in control of gene expression, Science 26 (1993) 1715–1725. [2] P. Jiang, Z. Guo, Fluorescent detection of zinc in biological systems: recent development on the design of chemosensors and biosensors, Coordination Chemistry Reviews 248 (2004) 205–229. [3] D.Y. Sasaki, S. Singh, J.D. Cox, P.I. Pohl, Fluorescence detection of nitrogen dioxide with perylene/PMMA thin films, Sensors and Actuators B 72 (2001) 51–55. [4] C.J. Frederickson, A.I. Bush, The neurobiology of zinc in health and disease, Biometals 14 (2001) 53–66. [5] K.H. Falchuk, The molecular basis for the role of zinc in developmental biology, Molecular and Cellular Biochemistry 188 (1998) 41–48. [6] A.B. Chausmer, Zinc, insulin and diabetes, Journal of the American College of Nutrition 17 (1998) 109–115. [7] A.I. Bush, The metallobiology of Alzheimer’s disease, Trends in Neurosciences 26 (2003) 207–214. [8] J.L. Smith, S. Xiong, W.R. Markesbery, M.A. Lovell, Altered expression of zinc transporters-4 and -6 in mild cognitive impairment, early and late Alzheimer’s disease brain, Neuroscience 140 (2006) 879–888. [9] C.J. Frederickson, J.Y. Koh, A.I. Bush, The neurobiology of zinc in health and disease, Nature Reviews Neuroscience 6 (2005) 449–462. [10] J. Li, W. Ling, G. Min, Y. Gui, R.Y. Wang, Fluorescence turn-on of easily prepared fluorescein derivatives by zinc cation in water and living cells, Sensors and Actuators B 156 (2011) 825–831. [11] B. Valeur, I. Leray, Design principles of fluorescent molecular sensors for cation recognition, Coordination Chemistry Reviews 205 (2000) 3–40. [12] L. Prodi, F. Bolletta, M. Montalti, N. Zaccheroni, Luminescent chemosensors for transition metal ions, Coordination Chemistry Reviews 205 (2000) 59–83.
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Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
A new turn-on fluorescence probe for Zn2+ in aqueous solution and imaging application in living cells Meng-Meng Li a,1, Fang-Wu Wang b,1, Xiao-Yun Wang c , Ting-Ting Zhang a , Yu Xu a , Yu Xiao a , Jun-Ying Miao b, * , Bao-Xiang Zhao a, * a b c
Institute of Organic Chemistry, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, PR China Institute of Developmental Biology, School of Life Science, Shandong University, Jinan 250100, PR China Chemical Technology Academy of Shandong Province, Jinan 250014, PR China
H I G H L I G H T S
G R A P H I C A L A B S T R A C T
A new pyrazoline-based turn-on fluorescence probe toward Zn2+ was synthesized. Upon addition of Zn2+, the fluorescence intensity was enhanced up to 80-fold. The fast response to Zn2+ makes the probe suitable to monitor Zn2+ in living cell.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 February 2014 Received in revised form 28 March 2014 Accepted 1 April 2014 Available online 4 April 2014
We designed and synthesized a new pyrazoline-based turn-on fluorescence probe for Zn2+ by the reaction of chalcone and thiosemicarbazide. The structure of the probe was characterized by IR, NMR and HRMS spectroscopy. The probe (L) exhibits high selectivity and sensitivity for detecting Zn2+ in buffered EtOH/HEPES solution (EtOH/HEPES = 1/1, pH 7.2) with 80-fold fluorescence enhancement, which is superior to previous reports. Job’s plot analysis revealed 1:1 stoichiometry between probe L and Zn2+ ions. The association constant estimated by the Benesi–Hildebrand method and the detection limit were 3.92 103 M1 and 5.2 107 M, respectively. A proposed binding mode was confirmed by 1H NMR titration experiments and density functional theory (DFT) calculations. The probe is cell-permeable and stable at the physiological pH range in biological systems. Because of its fast response to Zn2+, the probe can monitor Zn2+ in living cells. Moreover, the selective binding of L and Zn2+ was reversible with the addition of EDTA in buffered EtOH/HEPES solution and Zn2+ could be imaged in SH-SY5Y neuron cells. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Pyrazoline Fluorescent probe Zinc ion Imaging Neuron cells Reversibility
1. Introduction As the second-most abundant transition metal ion in the human body, Zn2+ plays pivotal roles in physiological processes
* Corresponding authors at: Shandong University, Organic Chemistry, Shanda Nanlu 27, Jinan, Shandong, PR China. Tel.: +86 531 88366425; fax: +86 531 88564464. E-mail addresses: [email protected], [email protected] (B.-X. Zhao). 1 Equal contribution. http://dx.doi.org/10.1016/j.aca.2014.04.001 0003-2670/ ã 2014 Elsevier B.V. All rights reserved.
such as gene transcription, regulation of metalloenzymes, and neural signal transmission [1–5]. Disordered Zn metabolism in biological systems is associated with diseases such as Alzheimer’s disease, diabetes, and epilepsy [6–9]. Exploring Zn2+ fluorescent probes is important, and previous probes have been found to have high sensitivity, detection of metal ions at very low concentration and imaging of Zn2+ in biological samples [10–14]. Fluorescent probes developed for Zn2+ include dipyrromethene boron difluoride (BODIPY) dyes [15–17], quinoline [18,19], bipyridine [20,21], phenoxazinium [22], Schiff-base [23–26], coumarin [27– 30], pyrene [31], rhodamine [32], triazole [33] and other
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Scheme 1. Synthesis of compound 2 (L).
fluorophores [34,35]. However, most of the probes have drawbacks such as poor selectivity and sensitivity, less water solubility, and complicated synthesis, or only work in toxic organiccontaining solutions such as acetonitrile and have poor cell membrane permeability. Recently, a carboxamidoquinoline-based fluorescent Zn2+ probe, with good water solubility, cell membrane permeability, selectivity and sensitivity, was reported; however, the probe produced only about 21-fold enhancement of emission based on the carboxamidoquinoline deprotonation process and ICT [36]. Other new reported probes also show limited increase in fluorescence [37–40]. In addition, some Zn2+ probes are affected by cross interference of Cd2+ owing to their closely related electronic and binding properties [41,42]. Thus, to satisfy practical needs, we need to develop Zn2+ probes with high selectivity, good water solubility, and good tolerance to interference over other metal ions, especially for imaging application in living cells. Pyrazoline derivatives have attracted increasing attention as fluorophores for metal ion probes because of their blue light emission with high fluorescence quantum yield, high fluorescence selectivity and excellent stability [43–46]. Recently, we investigated novel probes for Zn2+ based on pyrazoline [47–50] and successfully used them to monitor Zn2+ ions in cell imaging [49,50]. However, they showed low turn-on response in aqueous
solution. Here, we describe our development of a new fluorescent probe, L, 3-(2-hydroxy-5-methylphenyl)-5-pyridin-3-yl-4,5-dihydro-1H-pyrazole-1-carbothioamide, showing high sensitivity and selectivity for Zn2+ over other metal ions and high turn-on response (80-fold). Furthermore, the probe can be used to image Zn2+ in living cells and has good reversibility. 2. Experimental details 2.1. Reagents Deionized water was used throughout the experiment. All reagents were purchased from commercial suppliers and used without further purification. The salts used in stock aqueous solutions of metal ions were NaNO3, Fe(NO3)39H2O, AgNO3, KNO3, Co(NO3)26H2O, Mg(NO3)26H2O, Ca(NO3)24H2O, Al(NO3)39H2O, Ba(NO3)2, Cr(NO3)39H2O, Ni(NO3)26H2O, Cd(NO3)24H2O, Pb (NO3)2, Cu(NO3)23H2O, Zn(NO3)26H2O and HgCl2. 2.2. Apparatus Thin-layer chromatography involved silica gel 60F254 plates (Merck KGaA). 1H NMR spectra were recorded on a Bruker Avance 300 (300 MHz) spectrometer, with DMSO-6d used as a solvent. 13C NMR spectra were recorded on a Bruker Avance 300 (75 MHz)
Fig. 1. Absorption spectra of probe L (50 mM) in buffered EtOH/HEPES solution (EtOH/HEPES = 1/1, pH 7.2) with Zn2+ (0–50 equiv.). The inset shows the absorbance of L as a function of Zn2+ concentration.
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spectrometer, with DMSO-6d used as a solvent and tetramethylsilane (TMS) as an internal standard. Melting points were determined on an XD-4 digital micro-melting-point apparatus. IR spectra were recorded with a VERTEX 70 FT-IR spectrophotometer (Bruker Optics), HRMS spectra were recorded on a QTOF6510 spectrograph (Agilent), and UV–vis spectra were recorded on a U4100 spectrophotometer (Hitachi). Fluorescent measurements were recorded on a PerkinElmer LS-55 luminescence spectrophotometer. All pH measurements involved use of the Model PHS-3C pH meter (Shanghai) at room temperature, about 25 C.
cultivated in flasks for cell adhesion. For the experimental study, cells were grown to 80–90% confluence, harvested with 0.25% trypsin (Sangon Biotech) in phosphate-buffered saline (PBS), and seeded into 24-well plates. Experiments to assay Zn2+ uptake were performed in the same media supplemented with different concentrations of Zn nitrate with pyrithione (Zn ionophore). Cells were rinsed twice with PBS and treated with DMSO-containing L (10 mM, DMSO:water = 1:100) for 20 min at 37 C. After 2 washes with PBS, cells were viewed under an inverted fluorescence microscope (Nikon TE2000-S).
2.3. Procedures
3. Results and discussion
2.3.1. Synthesis of compound 2 (L) The synthetic route of proposed compound 2 is shown in Scheme 1. A mixture of chalcone (1) (0.717 g, 3 mmol), thiosemicarbazide (0.409 g, 4.5 mmol), NaOH (0.368 g, 9 mmol) and ethanol (50 mL) was stirred at reflux temperature for 1 h. After a cooling, water (100 mL) was added to the mixture and hydrochloric acid was added to neutralize it to pH 7. Then the mixture was extracted with dichloromethane, and the combined extraction was dried over anhydrous magnesium sulfate. After filtration, the filtrate was concentrated to give the crude product. Purification by column chromatography (silica gel; 25:1 CH2Cl2:CH3OH) yielded product 2 as a yellow solid. Yield: 47.6%; mp: 230–231 C; IR (KBr, cm1): 3428.1, 3252.7, 1594.5, 1477.3, 1332.1, 1256.1, 819.0, 705.6; 1H NMR (300 MHz, DMSO-6d): 2.22 (s, 3H, CH3), 3.31 (dd, 1H, J = 3.3, 18.4 Hz, 4-Htrans of pyrazoline), 4.00 (dd, 1H, J = 11.5, 18.4 Hz, 4-Hcis of pyrazoline), 5.95 (dd, 1H, J = 3.3, 11.5 Hz, 5-H of pyrazoline), 6.84 (d, 1H, J = 8.2 Hz, benzene H), 7.14 (dd,1H, J = 1.8, 8.2 Hz, benzeneH), 7.35 (dd, 1H, J = 4.8, 7.8 Hz, pyridine H), 7.48 (d, 1H, J = 1.8 Hz, benzene H), 7.50 (d, 1H, J = 2.1 Hz, pyridine H), 8.14 (d, 2H, J = 17.1 Hz, NH2), 8.41 (d, 1H, J = 2.1 Hz, pyridineH), 8.44 (dd, 1H, J = 1.5, 4.8 Hz, pyridine H), 9.52 (s, OH); 13C NMR (75 MHz, DMSO-6d): 175.65, 156.25, 154.42, 148.14, 147.29, 138.25, 132.95, 132.81, 129.51, 128.07, 123.66, 116.71, 115.61, 59.90, 43.78, 19.87; HRMS: calcd for [M + H]+ C16H16N4OS: 313.1123; found: 313.1122. (Fig. S0, ESI)
3.1. UV–vis absorption spectra for probe L The UV–vis absorption spectra for probe L were investigated in buffered EtOH/HEPES solution (EtOH/HEPES = 1/1, pH 7.2). With the addition of excess of 5 equiv. of various metal ions, including Ag+, Al3+, Fe3+, Co2+, Ni2+, Ba2+, Ca2+, Cu2+, Cd2+, Cr3+, K+, Mg2+, Na+, Hg2+, Zn2+, Pb2+, most of the tested metal cations did not induce any distinct change in absorbance at 338 nm, but the absorbance
2.3.2. Cell culture and imaging SH-SY5Y cells were grown in a 1:1 mixture of MEM (Gibco) and F12 (Gibco) medium and 10% supplemental fetal bovine serum (Gibco), at 37 C in a humidified incubator with 5% CO2. Cells were
Fig. 2. Fluorescence intensity changes ((I I0)/I0 of probe L (10 mM) in buffered EtOH/HEPES solution (EtOH/HEPES = 1/1, pH 7.2) with 10 equiv. of metal ions: Al3+, Fe3+, Co2+, Ni2+, Ba2+, Ca2+, Cd2+, Cr3+, K+, Mg2+, Na+, Ag+, Hg2+, Zn2+, Cu2+ ions and blank (excitation wavelength: 390 nm; emission wavelength: 480 nm).
Fig. 3. (a) Fluorescence emission spectra for probe L (10 mM) with the addition of Zn2+ (0–100 equiv.) in buffered EtOH/HEPES solution (EtOH/HEPES = 1/1, pH 7.2). The inset shows the emission of probe L (10 mM) as a function of Zn2+ concentration. (b) Color change of L with Zn2+ (0, 1, 5, 10 equiv.) (excitation wavelength: 390 nm; emission wavelength: 480 nm).
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Fig. 6. The proposed binding mode of probe L and Zn2+.
Fig. 4. Fluorescence intensity of probe L (10 mM) with 1.0–100 equiv. of Zn2+ (excitation wavelength: 390 nm; emission wavelength: 480 nm).
increased to some extent for Cu2+ (402 nm), Co2+ (428 nm), Ni2+ (402 nm) and Zn2+ (390 nm) (Fig. S1, ESI). The absorption maximum at 390 nm for Zn2+ was associated with color change of the solution from colorless to yellow (Fig. S2, ESI). In addition, the absorption maximum of L at different concentrations appeared at 338 nm, and the extinction coefficient (e) was 2.39 104 M1 cm1 (Figs. S3 and S4, ESI). The UV–vis absorption spectra for L in the presence of different concentrations of Zn2+ ions are in Fig. 1. With increasing concentration of Zn2+ ions, the absorbance of L at 338 nm gradually decreased. Simultaneously, a new absorption band appeared at 390 nm, and absorbance gradually increased with increasing Zn2+ concentration. Moreover, one isobestic point appeared at 364 nm, which indicated the formation of a stable complex with a certain stoichiometric ratio. The red shift of absorption maximum can be ascribed to the coordination of the probe with Zn2+.
with the addition of other cations (Ag+, Al3+, Fe3+, Co2+, Ni2+, Ba2+, Ca2+, Cu2+, Cd2+, Cr3+, K+, Mg2+, Na+, Hg2+, Pb2+). Moreover, with the addition of 10 equiv. of Zn2+, the fluorescence maximum was remarkably enhanced up to 80-fold as compared with the initial fluorescence intensity of probe L. The enhancement was superior compared to that in previous reports [47–50]. In addition, we conducted competition experiments in the presence of Zn2+ and other metal ions under the same solvent (Fig. S5, ESI). The presence of Cd2+, Ba2+, Pb2+, Na+, Mg2+, K+ and Ca2+ did not interfere with the fluorescence response of L to Zn2+. The addition of Cr3+, Fe3+, Hg2+, Al3+ and Ag+ slightly changed the emission of L–Zn2+ at 480 nm. However, some paramagnetic metal ions, such as Co2+, Ni2+, and Cu2+, quenched the fluorescence, which is always encountered with other metal ion probes [22,38,40,51–54]. This observation is probably due to the displacement of Zn2+ by Co2+, Ni2+, and Cu2+. In brief, the probe L had excellent selectivity for Zn2+ over other metal cations in aqueous solution. Fluorescence titration was performed in the presence of different concentrations of Zn2+. From the fluorescence titration profile (Fig. 3a), the addition of Zn2+ increased fluorescence intensity at 480 nm and was saturated with the addition of 80 equiv. of Zn2+. Clear blue fluorescence occurred with irradiation at 365 nm (Fig. 3b), and the fluorescence intensity was enhanced
3.2. Fluorescence spectra of probe L The selectivity of the fluorescence response of probe L to metal ions was investigated in buffered EtOH/HEPES solution. With the addition of Zn2+ ions, fluorescence intensity was significantly induced (Fig. 2), with only weak changes in fluorescence intensity
Fig. 5. Reversibility of L–Zn2+ binding.
Fig. 7. Optimized structures of probe L (a) and L–Zn2+ (b) by calculation.
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with Zn2+ from 0 to 10 equiv. Quantum yield was determined by the relative comparison procedure, with quinine sulfate dehydrate (99.0%) in 0.1 N H2SO4 used as the main standard [55] and was calculated by the following general equation: F = Fs (IAs/IsA) (h2/hs2) [56]. The quantum yield of L in the absence and presence of Zn2+ was 0.0028 and 0.23, respectively, for an 82-fold enhancement in Ff. Moreover, the Job plot showed 1:1 binding stoichiometry for Zn2+ and L (Fig. S6, ESI). Following a Benesi– Hildebrand-type analysis [57], the association constant Ka was determined to be 5.68 103 M1 (Fig. 4). The limit of detection (LOD) was evaluated with 3s bi/m [58] and calculated from the fluorescence titration curves to be 5.0 107 M (Fig. 3a). For practical applications, we investigated the ability of probe L to sense Zn2+ at different pH values (Fig. S7, ESI). The fluorescence of probe L remained constant at 480 nm from pH 5 to 9, whereas the fluorescence intensity of the L–Zn2+ complex increased sharply from pH 5.5 to 7.5 and reached a steady high reading at around pH 7.5. However, the fluorescence of L–Zn2+ decreased with increasing pH from 7.5 to 9.0, which may be due to the formation of Zn(OH)+ or Zn(OH)2 at high pH and thus, reducing the concentration of L– Zn2+ [40]. Therefore, the probe should be used to monitor Zn2+ by covering the physiological pH range in biological systems. In addition, the interaction of L with Zn2+ was completed in a few seconds (Fig. S8, ESI), and the fluorescence intensity of L–Zn2+ was stable in 10 min. To examine the reversibility of the binding of L to Zn2+, we used EDTA as a well-known metal ion chelator in buffered EtOH/HEPES
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solution. The fluorescence intensity of L–Zn2+ (10 mM) at 480 nm was completely quenched by the addition of 1.0 equiv. of EDTA (Fig. 5). The fluorescence intensity increased again with the addition of 1 equiv. Zn2+ to the mixture of L–Zn2+-EDTA (10 mM). Therefore, the selective binding of L and Zn2+ was reversible. 3.3. The proposed binding mechanism of probe L A proposed binding mode is shown in Fig. 6. To understand the binding behavior of probe L with Zn2+, we performed 1H NMR titration experiments. We found partial change in 1H NMR spectra for L without and with 1.0 equiv. Zn2+ in d-DMSO (Fig. S9, ESI). All protons in the pyrazoline ring and aryl showed an upfield or downfield shift due to the formation of the complex. On the basis of the relative intensity of the lines, a complex of probe L with about 0.3 equiv. Zn2+ formed. Of note, the hydroxyl proton was lost when probe L bound to Zn2+. We performed density functional theory (DFT) calculations to verify the binding mechanism of L–Zn2+. With the first principles of DFT, we performed calculations with the CASTEP code [59]. The exchange and correlation interactions were modeled by using the generalized gradient approximation (GGA) with the PW91 function. With the geometric optimization, all forces on atoms converged to 0.05 eV Å1, the maximum ionic displacement was within 0.002 Å, and the total stress tensor was reduced to the order of 0.1 GPa. The calculation results well supported the proposed binding mode (Fig. 7).
Fig. 8. Fluorescence microscopy of living SH-SY5Y cells. Cells of control group were incubated with 2.5 mM probe L for 20 min at 37 C Cells of the experimental group were exposed to 30 mM pyrithione with 1, 5 and 25 mM extracellular Zn2+ for 45 min. After 2 rinses with PBS, cells were treated with 2.5 mM probe L for 20 min at 37 C. (A) (a) Bright-field, (b) fluorescent, (c) overlay, and (d) fluorescent images after EDTA treatment and (B) quantitation. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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3.4. Biological applications of probe L To investigate the potential biological application of probe L in living cells, we performed intracellular Zn2+ imaging of SH-SY5Y neuron cells by fluorescence microscopy. After incubation with DMSO-containing L (2.5 mM, DMSO:water = 1:100) for 20 min at 37 C, cells showed faint fluorescence. However, fluorescence became visible in the cytoplasm when exogenous Zn2+ (1, 5, 25 mM) was introduced in the presence of the Zn-selective ionophore (30 mM) pyrithione (2-mercaptopyridine N-oxide), for visual evidence of probe L readily entering cells and information on the intracellular existence of Zn2+ (Fig. 8). Moreover, with increasing concentration of Zn2+, fluorescence intensity was enhanced concentration-dependently. In addition, fluorescence intensity was greatly decreased with the addition of 5 mM EDTA for 30 min. Thus, probe L has potential for biological applications. 4. Conclusions In summary, we developed a highly sensitive and selective probe for Zn2+ ions in aqueous media. Fluorescence intensity was remarkably enhanced up to 80-fold with the addition of Zn2+. The addition of EDTA could quench the fluorescence of the L– Zn2+ complex, which indicates that probe L for Zn2+ can be reversible. In addition, the probe was successfully used to image Zn2+ in SH-SY5Y neuron cells, with excellent water solubility and high sensing ability under physiological pH. The fluorescent image was reversed by adding an appropriate amount of EDTA. Therefore, probe L can be used to selectively monitor Zn2+ in living neuron cells. Acknowledgments This study was supported by the 973 Program (2010CB933504) and National Natural Science Foundation of China (91313303 and J1103515). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aca.2014.04.001. References [1] T.V. O’Halloran, Transition metals in control of gene expression, Science 26 (1993) 1715–1725. [2] P. Jiang, Z. Guo, Fluorescent detection of zinc in biological systems: recent development on the design of chemosensors and biosensors, Coordination Chemistry Reviews 248 (2004) 205–229. [3] D.Y. Sasaki, S. Singh, J.D. Cox, P.I. Pohl, Fluorescence detection of nitrogen dioxide with perylene/PMMA thin films, Sensors and Actuators B 72 (2001) 51–55. [4] C.J. Frederickson, A.I. Bush, The neurobiology of zinc in health and disease, Biometals 14 (2001) 53–66. [5] K.H. Falchuk, The molecular basis for the role of zinc in developmental biology, Molecular and Cellular Biochemistry 188 (1998) 41–48. [6] A.B. Chausmer, Zinc, insulin and diabetes, Journal of the American College of Nutrition 17 (1998) 109–115. [7] A.I. Bush, The metallobiology of Alzheimer’s disease, Trends in Neurosciences 26 (2003) 207–214. [8] J.L. Smith, S. Xiong, W.R. Markesbery, M.A. Lovell, Altered expression of zinc transporters-4 and -6 in mild cognitive impairment, early and late Alzheimer’s disease brain, Neuroscience 140 (2006) 879–888. [9] C.J. Frederickson, J.Y. Koh, A.I. Bush, The neurobiology of zinc in health and disease, Nature Reviews Neuroscience 6 (2005) 449–462. [10] J. Li, W. Ling, G. Min, Y. Gui, R.Y. Wang, Fluorescence turn-on of easily prepared fluorescein derivatives by zinc cation in water and living cells, Sensors and Actuators B 156 (2011) 825–831. [11] B. Valeur, I. Leray, Design principles of fluorescent molecular sensors for cation recognition, Coordination Chemistry Reviews 205 (2000) 3–40. [12] L. Prodi, F. Bolletta, M. Montalti, N. Zaccheroni, Luminescent chemosensors for transition metal ions, Coordination Chemistry Reviews 205 (2000) 59–83.
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