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Coumarin-naphthol conjugated Schiff base as a “turn-on” fluorescent probe for Cu2+ via selective hydrolysis of imine and its application in live cell imaging
Journal of Photochemistry and Photobiology A: Chemistry 333 (2017) 213–219
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Journal of Photochemistry and Photobiology A: Chemistry journal homepage: www.elsevier.com/locate/jphotochem
Invited feature article
Coumarin-naphthol conjugated Schiff base as a “turn-on” fluorescent probe for Cu2+ via selective hydrolysis of imine and its application in live cell imaging Sisi Wang, Zhen Wang, Yong Yin, Jianguang Luo* , Lingyi Kong* State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People’s Republic of China
A R T I C L E I N F O
Article history: Received 16 August 2016 Received in revised form 23 September 2016 Accepted 11 October 2016 Available online 29 October 2016 Keywords: Fluorescent probe Coumarin-naphthol conjugate Schiff base Imine hydrolysis Copper ion
A B S T R A C T
An off-on fluorescent probe (CuCIN), coumarin-naphthol linked by an imine group, for detecting Cu2+ in DMSO-HEPES buffer (1:99, v/v) solution was successfully developed. Mechanism studies suggested that CuCIN firstly formed a complex with Cu2+ in a 1:2 metal-to-ligand ratio, and then 60-fold fluorescence enhancement was observed when Cu2+ promoted the imine hydrolysis to release the fluorescence compound 7-diethylaminocoumarin-3-aldehyde. This probe displayed desired properties such as high selectivity toward other metal ions, low detection limit (12.7 nM) and low cytotoxicity. Bio-imaging study revealed that the new probe CuCIN could be applied for imaging living cells with good cell permeability. We hope to apply this probe in the biomedical research field for the imaging of disease-relevant Cu2+. ã 2016 Elsevier B.V. All rights reserved.
1. Introduction Divalent copper ion, as the third most abundant transition metals, plays a vital role for most forms of life [1]. Copper ions serve as a structural and catalytic cofactor for many proteins and enzymes including important metabolic factors such as cytochrome c oxidase and copper–zinc superoxide dismutase [2,3]. It is demonstrated that the changes in copper’s cellular homeostasis are connected to serious neurodegenerative diseases, such as Menkes [4], Wilson’s disease [5], familial amyotropic lateral sclerosis, Alzheimer’s disease [6] and prion disease [7]. Therefore, it makes sense to find a sensitive, real-time response tool to analyze the level of copper ion for in vitro and in vivo assays. Conventional analytical methods like atomic absorption spectrometry [8], plasma atomic emission spectrometry [9], mass spectrometry [10], surfaceplasmon resonance [11] are available for the detection of Cu2+. Nonetheless, most of the above mentioned traditional methods are expensive and time-consuming. As a result, fluorescence analysis has emerged as a convenient method for monitoring the biomolecules in living system for their intrinsic sensitivity, high spatial and temporal resolution [12,13]. Most of the reported fluorescent probes for Cu2+ are based on either chelating or chemical reaction. Probes chelating with Cu2+ usually
* Corresponding authors. E-mail addresses: [email protected] (J. Luo), [email protected] (L. Kong). http://dx.doi.org/10.1016/j.jphotochem.2016.10.030 1010-6030/ã 2016 Elsevier B.V. All rights reserved.
induce the intrinsic fluorescence quenching because of the paramagnetic property of Cu2+, which are not as effective as the probes that enhance the emission intensity [14]. Alternatively, fluorescent probes based on reactions have been developed to reach emission enhancement by reacting with Cu2+ to yield fluorescent products which possess little affinity to Cu2+, consequently avoiding the problem of paramagnetic of Cu2+ and satisfying the rational design for fluorescent probes [15]. Some efficient fluorescent probes for Cu2+ based on the highly specific chemical reactions such as hydrolysis [16–21] and oxidation [22– 24] have attracted much attention. However, some of them have shortcomings for quantifying such as slow response [25], cytotoxicity of the ligands, small stoke shift [25], cross-sensitivity toward other metal cations [26]. Consequently, it is necessary to find a fluorescent probe which could overcome these shortcomings. Previously, there are several fluorescent probes based on Cu2 + -promoted hydrolysis of hydrazone [17,18] and lactam [27]. On the other side, some “off-on” fluorescent probes containing C¼N bond with enhanced fluorescence intensity in virtue of forming complex with metal ions have been investigated [28–31]. So far as we know, there is no probe detecting Cu2+ on account of the hydrolysis of the imine group. Hence, it's of our great interest to design a reaction based fluorescent probe for Cu2+ detection, which provided a new mechanism of Cu2+ promoting the imine hydrolysis.
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Coumarin is often selected as a signaling moiety due to its high quantum yield, large stokes shift and low cytotoxicity [32]. It is easy to modify the coumarin on its 3-C, 4-C and 7-C positions, introducing electron donating groups and electron accepting groups contributes to produce many strong fluorescent dyes with different wavelength ranges of absorption and fluorescence emission [33–35]. Moreover, naphthol could serve as a molecule which stabilize the Schiff base and there have been some naphtholderivatives developed for detecting copper [36,37]. Herein, we reported a fluorescent probe (CuCIN) for Cu2+ based on coumarinnaphthol conjugated Schiff base which exhibited fluorescence enhancement upon the hydrolysis of the imine group. CuCIN was synthesized in a simple two-step reaction and characterized by 1H NMR, 13C NMR, and HRMS. This probe emerged some advantages for analysis like high selectivity, low detection limit and we successfully used it in bio-imaging of Cu2+ in living cells for its low cytotoxity and good cell permeability. 2. Experimental 2.1. Materials and general methods Unless noted, all the reagents and solvents used in our experiment were analytical grade without further purification, and double-distilled water was used in all experiments. The metal stock solutions were prepared from the salts of AlCl3, CaCl2, CoCl26H2O, CuCl22H2O, FeCl24H2O, FeCl36H2O, KCl, ZnCl2, NaCl, MgCl26H2O, MnCl24H2O, NiCl26H2O, HgCl2, Pb(NO3)2, AgNO3 and CdCl2. A human hepatoblastoma cell line (HepG2) was purchased from Shanghai Institute of Biochemistry & Cell Biology, Chinese Academy of Sciences (Shanghai, China) The 1H NMR (500 MHz) and 13C NMR (125 MHz) spectra were measured on Bruker ACF-500 spectrometer while using TMS as an internal standard. IR (KBr-disc) spectra were recorded by Bruker Tensor 27 spectrometer. MS spectra were measured on a MS Agilent 1100 Series LC/MSD Trap mass spectrometer (ESI–MS) and a Mariner ESI-TOF spectrometer (HRMS), respectively. The pH levels of the stock solution were monitored by PHS-25C Precision pH/mV meter. UV–vis spectra were obtained using a Shimadzu UV2300 spectrophotometer and fluorescence spectra were measured with Perkin LS-55 fluorescence spectrophotometer, respectively. 2.2. Synthesis of CuCIN (1) The solution of 7-diethylaminocoumarin- 3-aldehyde (3) (73.5 mg, 0.30 mmol) in 5 ml hot absolute ethanol was added into the solution of 3-amino-2-naphthol (72.0 mg, 0.45 mmol) in 5 ml absolute ethanol. The mixture was refluxed for 6 h to yield scarlet precipitate. The precipitate was filtrated, washed with hot absolute ethanol three times. After drying under reduced pressure, CuCIN (1) was obtained as scarlet solid (60 mg, 52%).1H NMR (500 MHz, DMSO-d6): dH 9.30 (1H, s, OH), 8.84 (1H, s, C¼CH), 8.75 (1H, s, CH¼N ), 7.81 (1H, d, J = 8.1 Hz, Ar-H), 7.66 (2H, t, J = 9.0 Hz, ArH), 7.60 (1H, s, Ar-H), 7.34 (1H, t, J = 7.4 Hz, Ar-H), 7.26 (1H, t, J = 7.4 Hz, Ar-H), 7.23 (1H, s, Ar-H), 6.83 (1H, d, J = 8.1 Hz, Ar-H), 6.63 (1H, s, Ar-H), 3.51 (4H, q, J = 7.0 Hz, CH2), 1.17 (6H, t, J = 7.0 Hz, CH3). 13C NMR (125 MHz, DMSO-d6): d 161.8, 157.9, 154.8, 152.6, 152.2, 150.9, 142.8, 141.3, 133.5, 132.0, 128.8, 128.1, 126.2, 126.0, 123.6, 116.4, 110.6, 109.9, 108.7, 97.0, 44.9(2C), 12.9(2C). HRMS: [M +H+] calcd. for C24H23N2O3: 387.1703, found 387.1702. 2.3. Cell culture and confocal imaging HepG2 cells were cultured in DMEM (Dulbecco's Modified Eagle Medium) medium supplemented with 10% FCS (Fetal Calf Serum)
at 37 C in a 5% CO2/95% air incubator. Confocal fluorescence imaging studies were performed on a LSM 710 confocal laserscanning microscope (Carl Zeiss Co. Ltd.). The working solution was prepared from 20 mM CuCIN solution in DMSO and then it was diluted with DMEM medium to a final concentration of 20 mM. The cells were incubated with CuCIN for 30 min and washed with HEPES buffer (20 mM) three times, and then incubated with 10 mM CuCl2H2O solution in DMEM medium for 20 min. After another washing with HEPES buffer three times, the cells were in HEPES buffer during the fluorescence imaging. Fluorescence microscopy images of labeled cells were obtained by exciting the probe with a wavelength at 405 nm. 2.4. Cytotoxicity assay The cytotoxicity of the CuCIN to HepG2 cells was obtained through a MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2Htetrazolium bromide) assay. HepG2 cells were seeded into 96well culture plates at 8000 cells per well. Before the treatment with CuCIN, the DMEM (Dulbecco's Modified Eagle Medium) with 10% FCS (Fetal Calf Serum) were removed and replaced with fresh medium. The treated cells were cultured with different concentrations of CuCIN (0, 3, 6, 12.5, 25, 50 mM) at 37 C under 5% CO2/ 95% atmosphere for 24 h, and cells without CuCIN were taken as control. Subsequently, cells were treated with 20 ml MTT (5 mg/ mL) in each well and incubated for 4 h, and then 150 ml DMSO was added to dissolve the form azan crystals. The absorbance at 570 nm was obtained with the microplate reader. The cytotoxicity effect of the CuCIN was calculated through the following equation: Cell viability% = OD570 (sample)/OD570 (control) 100 2.5. Measurement procedure The stock solution of CuCIN (1 mM) was prepared by dissolving it in DMSO, while the stock solution of various metal ions was prepared by dissolving them in double-distilled water. The test solution was prepared by measuring out desired metal ion solution into 10 ml volumetric flask containing CuCIN stock solution and then diluting it by HEPES buffer solution (10 mM, pH = 7.4). The measurements of fluorescence spectra were carried out at the excitation wavelength of 435 nm, while the emission spectra range was 450 to 650 nm and the slit width was 5 nm. 3. Results and discussion A fluorescent probe CuCIN for Cu2+ was designed and synthesized as the route shown in Scheme 1. The new compound CuCIN was synthesized through the nucleophilic addition reaction in absolute ethanol under reflux condition by the two known compounds 7-diethylaminocoumarin (2) and 7-diethylaminocoumarin- 3-aldehyde (3), which were prepared from the starting material 4-diethylamino-salicylaldehyde (In Supporting information) according to the literature [38]. The whole synthetic route was simple and convenient and all the synthesized target compounds were characterized by 1H NMR, 13C NMR and HRMS. 3.1. Titration experiment We investigated the optical properties of probe CuCIN in the absence and the presence of Cu2+. Initially, the UV–visible absorption and fluorescence titrations of the CuCIN were carried out using a solution of 10 mM CuCIN in DMSO/HEPES buffer (1:99 v/v). In Fig. 1a, the test solution showed the adsorption at 345 nm and 452 nm, upon the incremental addition of Cu2+, the
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Scheme 1. Synthesis of CuCIN.
adsorption band at 345 nm decreased while the adsorption band at 452 nm increased gradually. These changes were complemented by clear isosbestic point at 382 nm. The observed UV spectra change was associated with the hydrolysis reaction upon the addition of Cu2+. By gradually adding Cu2+, the fluorescence intensity at the wavelength of 502 nm(lex = 435 nm) increased step by step and reached the maximum when the Cu2+ was 0.5 equiv. Then the
fluorescence changed from colorless to green obviously. As shown in Fig. 1b, the emission intensity at 502 nm associated with the CuCIN was low probably attributed to a decay process of the C¼N bond cis-trans isomerization as reported[39]. The emission intensity was enhanced about 60-fold when 0.5 equiv. of Cu2+ was added. Moreover, the good relationship of emission intensity against Cu2+ from 0 to 0.5 equiv. was observed from the Fig.S1. The detection limitation (CDL = 3Sb/m) was calculated as 0.82 mg/L (12.7 nM), which is much lower than the World Health Organization recommended level (2.0 mg/L) in drinking water[42]. The detection limitation of other Cu2+ probes were exhibited in Table. S1. Taken together, these data proved that the new probe was a highly sensitive sensor to detect Cu2+ in a quantitative manner. 3.2. Selectivity study To obtain the specific selectivity property of the CuCIN toward various metal ions, the influence of other metal ions such as Na+, K+, Pb2+, Co2+, Al3+, Hg2+, Mg2+, Ni2+, Cd2+, Ca2+, Fe3+, Fe2+, Mn2+, Hg2+, Ag+ and Zn2+ were measured. There was no obvious fluorescence response toward the aforementioned metal ions as shown in Fig. 2a. As we known, the ability of a probe to analyze the target over other metal ions is important, so we further investigated the interference of other metal ions to detection Cu2+. As shown in Fig. 2b, the CuCIN could distinguish the Cu2+ from other metal ion related to the environment and biological condition. Colorimetric sensing by naked eye to observe the high selectivity toward various metal ions was also shown in Fig. 3. 3.3. The pH effect It is very important for a fluorescent probe to be stable at the biological pH environment, so we investigate the pH effect on the CuCIN. As shown in Fig.S2, the CuCIN was stable and weak fluorescent over all the pH range (pH = 5–11). On the other hand, toward adding Cu2+ into the solution, strong fluorescence emission enhanced at the pH under 6 to 9. It showed that the CuCIN could be applicable at complex biological environment 3.4. Kinetics
Fig. 1. (a) Absorption spectra and (b) Fluorescence spectra (lex = 435 nm) of CuCIN (10 mM) in the presence of increasing amount of Cu2+ in HEPES buffer (10 mM, pH 7.4, containing 1% DMSO) after the incubation with Cu2+ for 20 min at 25 C.
To better study the reaction of the CuCIN in the presence of Cu2+, we examined the kinetic of CuCIN and Cu2+ by the response time of CuCIN toward Cu2+. After CuCIN incubated with Cu2+ in HEPES buffer, the fluorescence intensity at 502 nm (lex = 435 nm) increased gradually and saturated at 20 min and then kept almost invariant (as shown in Fig. 4a). The hydrolysis kinetics were studied from the fluorescence spectra and the rate constant k was calculated by the equation as followed: ln((Fmax-F)/Fmax) = kt, as result, k = 0.104 min1(as shown in Fig. 4b). The medium constant
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Fig. 2. (a) Fluorescence spectra of CuCIN (10 mM) to Cu2+ (1 equiv.) and other metal ions (1 equiv.) in HEPES buffer (10 mM, pH 7.4, containing 1% DMSO) after the incubation for 20 min at 25 C (lex = 435 nm). (b) Fluorescence intensity of CuCIN to various metal ions in HEPES buffer (10 mM, pH 7.4, containing 1% DMSO) after the incubation for 20 min at 25 C (lex = 435 nm). The black bars represent the emission of CuCIN in the presence of various metal ions (2 equiv.) to CuCIN. The red bars represent the emission that occurs upon subsequent addition of Cu2+ (2 equiv.) to the solution. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 4. (a) Time dependent fluorescence spectra changes of probe CuCIN (10 mM) with Cu2+ (0.5 equiv.) in HEPES buffer (10 mM, pH 7.4, containing 1% DMSO) at 25 C (lex = 435 nm). (b) Curves fitted with first-order reaction dynamics (ln ((Fmax-F)/ Fmax) = kt) and rate constant (k) was calculated: k = 0.104 min1 ([Cu2+] = 10 mM).
Fig. 3. Photograph of CuCIN (10 mM) to various metal ions under UV lamp (lex = 365 nm).
Fig. 5. Job’s plot of the complex formed by [Cu2+]/[Cu2+] + [CuCIN], the total concentration of CuCIN and Cu2+ was 10 mM (lex = 435 nm).
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Scheme 2. Sensing mechanism of CuCIN based on Cu2+ catalyzed hydrolysis reaction.
k = 0.104 min1 indicated the reaction activation energy might be relatively high. 3.5. Job’s plot For further determination the stiochiometric between Cu2+ and CuCIN, Job’s plot analysis was also used. Total concentration of CuCIN and Cu2+ was 10 mM. As shown in Fig. 5, the maximum fluorescence intensity exhibited at around 0.33 mol fractions that indicated a 2:1 stiochiometric between CuCIN and Cu2+. Thus, a possible coordination mode for CuCIN with Cu2+ was proposed in
Scheme 2. And the association constant (Ka) of CuCIN with Cu2+ was also determined using the Benesi–Hildebrand equation as follows: 1 1 1 þ ¼ F F min ka ðF max F min Þ½Cu2þ F max F min F and Fmin represent the fluorescent intensity of the CuCIN at moderate concentration and none of Cu2+, respectively. Fmax is the saturated fluorescent intensity of CuCIN in the presence of excess amount of Cu2+. The association constant was calculated to be 1.74 105 M1.
Fig. 6. 1H NMR spectra of (a) CuCIN in DMSO-d6, (b) CuCIN in DMSO-d6 + 1 equiv. Cu2+ in H2O, (c) the standard 7-diethylaminocoumarin-3-aldehyde in DMSO-d6.
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Fig. 7. Confocal fluorescence images of HepG2 cells, (a, b and c) images of cells after incubated with CuCIN (20 mM) for 30 min; (d, e and f) images of cells after incubated with CuCIN (20 mM) for 30 min and subsequently with Cu2+ (0.5 equiv.) for 20 min; (a and d) green channel image (lex = 405 nm); (b and e) bright field image; (c) overlay image of a and b; (f) overlay image of d and e. Scale bar: 50 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3.6. The sensing mechanism There have been some “turn-on” fluorescent probes containing C¼N bond for detecting Cu2+ based on chelating copper ions to prevent the C¼N isomerization[29,40] or promoting hydrolysis the hydrazone[17] to release the fluorescent compound. However, no mechanism based on the imine hydrolysis in the presence of Cu2+ was revealed. Therefore, we illuminated the probe CuCIN sensing Cu2+ mechanism using the combination of 1H NMR, IR, MS and UV– vis spectroscopic methods. Firstly, we verified the complex of copper ion and CuCIN by FTIR spectrum, and the stiochiometric complexation of CuCIN Cu2+ was evaluated by the Job’s plot data. As shown in Fig.S3, a shift could be observed of the naphtholic hydroxyl group from 3426 cm1 (free CuCIN) to3383 cm1(CuCIN-Cu2+), the C¼O group of the free CuCIN(1702 cm1) almost disappeared and the C¼N group changed from 1615 cm1 to 1577 cm1. All the change of the FT-IR spectra of CuCIN in the absence and presence of Cu2+ revealed that the naphtholic hydroxyl, ester carbonyl and C¼N bond was involved in the metal coordination. To gain more information of the kind of reaction that CuCIN underwent in presence of copper ion, the 1H NMR titration experiment was carried out. When 1 equiv. Cu2+ was added to the probe solution in DMSO-d6, the singlet at dH 8.84 ppm assigned to the proton of CH¼N group disappeared, along with appearance of the singlet at dH 9.89 ppm attributed to the proton of aldehyde moiety as shown in Fig. 6. Additionally, all proton signals of 3 (Fig. 6c) were detected in the 1H NMR of this solution (Fig. 6b). On the other hand, when the solution of CuCIN was combined with Cu2+, a peak corresponding to [3+H+] appeared at m/z = 246.04 in the ESI–MS spectra further demonstrated the hydrolysis of imine group into aldehyde (Fig.S4). UV–vis spectra of the CuCIN solution in the presence of Cu2+ were compared with the solution of compound 3, and the spectra of the two solutions were nearly identical as shown in Fig.S5. Overall, the products were just confirmed to contain compound 3. Therefore, the sensing mechanism of the probe can be proposed. Initially, the ester carbonyl, naphtholic hydroxyl and
the N atom of CuCIN were chelated with Cu2+. The coordinate bond between Cu2+ and N atom led to the electronic cloud distribution of the N atom apart from the C atom, as a result, the bond energy of C¼N bond was weakened. Sequentially, the imine sequentially was hydrolyzed to release the aldehyde (compound 3) as shown in Scheme 2. The solution of probe CuCIN with almost no fluorescence transferred to that of the corresponding aldehyde derivative (compound 3) with strong green fluorescence due to the Cu2+ promoted C¼N hydrolysis reaction of CuCIN. 3.7. Laser scanning confocal fluorescent imaging It is reported that the hepatocytes are important in copper metabolism, therefore we tested the Cu2+ sensing ability of probe CuCIN in copper overloaded HepG2 cells[41]. Furthermore, to ensure the bio-imaging in a safe condition, we investigated the cytotoxicity of the probe by MTT with the CuCIN concentration ranging from 3 mM to 50 mM, and the result was shown in Fig.S6. The results of MTT indicated that the cell viability was 62.4% versus the control group when incubated with 50 mM compound CuCIN for 24 h. It proved that the compound CuCIN did not show any significant cytotoxicity toward HepG2 cells. When HepG2 cells were incubated with CuCIN (20 mM) for 30 min at 37 C in HEPES buffer, there was negligible intracellular fluorescence upon excitation at 405 nm. When cells were incubated with Cu2+ (10 mM) for another 20 min, intense fluorescence emerged in the green channel was observed in Fig. 7, showing that CuCIN was cellpermeable and could be efficiently used for imaging of Cu2+ in living cells. 4. Conclusions In summary, a coumarin-naphthol conjugated Schiff base fluorescent probe based Cu2+-promoted imine hydrolysis mechanism was designed and synthesized, which showed a remarkable fluorescence enhancement toward Cu2+ with high selectivity and low detection. In addition, the probe was successful applied for
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bio-imaging which demonstrated the potential value in living system. Acknowledgements This research work was funded by the Key Project of National Natural Science Foundation of China (No. 81430092), Ph.D. Programs Foundation of Ministry of Education of China (20120096130002), the Program for Changjiang Scholars and Innovative Research Team in University (IRT_15R63) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). 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. jphotochem.2016.10.030. References [1] D.L. Huffman, T.V. O'Halloran, Function, structure, and mechanism of intracellular copper trafficking proteins, Annu. Rev. Biochem 70 (2001) 677–701. [2] S. Lutsenko, Human copper homeostasis: a network of interconnected pathways, Curr. Opin. Chem. Biol. 14 (2010) 211–217. [3] S. Puig, D.J. Thiele, Molecular mechanisms of copper uptake and distribution, Curr. Opin. Chem. Biol. 6 (2002) 171–180. [4] E. Madsen, J.D. Gitlin, Copper and iron disorders of the brain, Annu. Rev. Neurosci. 30 (2007) 317–337. [5] K.J. Barnham, C.L. Masters, A.I. Bush, Neurodegenerative diseases and oxidative stress, Nat. Rev. Drug Discov. 3 (2004) 205–214. [6] E. Gaggelli, H. Kozlowski, D. Valensin, G. Valensin, Copper homeostasis and neurodegenerative disorders (Alzheimer's, prion, and Parkinson's diseases and amyotrophic lateral sclerosis), Chem. Rev. 106 (2006) 1995–2044. [7] D.R. Brown, H. Kozlowski, Biological inorganic and bioinorganic chemistry of neurodegeneration based on prion and Alzheimer diseases, Dalton Trans. 190 (2004) 7–17. [8] M.-S. Chan, S.-D. Huang, Direct determination of cadmium and copper in seawater using a transversely heated graphite furnace atomic absorption spectrometer with Zeeman-effect background corrector, Talanta 51 (2000) 373–380. [9] P. Fodor, B.D. Zs, Uncertainty in environmental ICP-AES measurements, Microchem. J. 51 (1995) 151–158. [10] J. Wu, E.A. Boyle, Low blank preconcentration technique for the determination of lead, copper, and cadmium in small-volume seawater samples by isotope dilution ICPMS, Anal. Chem. 69 (1997) 2464–2470. [11] J. Homola, S.S. Yee, G. Gauglitz, Surface plasmon resonance sensors: review, Sens. Actuators B 54 (1999) 3–15. [12] Y. Ding, Y. Tang, W. Zhu, Y. Xie, Fluorescent and colorimetric ion probes based on conjugated oligopyrroles, Chem. Soc. Rev. 44 (2015) 1101–1112. [13] C.Y. Lai, B.G. Trewyn, D.M. Jeftinija, K. Jeftinija, S. Xu, S. Jeftinija, et al., A mesoporous silica nanosphere-based carrier system with chemically removable CdS nanoparticle caps for stimuli-responsive controlled release of neurotransmitters and drug molecules, J. Am. Chem. Soc. 125 (2003) 4451– 4459. [14] J. Liu, Y. Lu, A DNAzyme catalytic beacon sensor for paramagnetic Cu2+ ions in aqueous solution with high sensitivity and selectivity, J. Am. Chem. Soc. 129 (2007) 9838–9839. [15] S. Pal, N. Chatterjee, P.K. Bharadwaj, Selectively sensing first-row transition metal ions through fluorescence enhancement, RSC Adv. 4 (2014) 26585– 26620. [16] X.-X. Hu, X.-L. Zheng, X.-X. Fan, Y.-T. Su, X.-Q. Zhan, H. Zheng, Semicarbazidebased naphthalimide as a highly selective and sensitive colorimetric and turnon fluorescent chemodosimeter for Cu2+, Sens. Actuators, B 227 (2016) 191– 197. [17] M.H. Kim, H.H. Jang, S. Yi, S.-K. Chang, M.S. Han, Coumarin-derivative-based off-on catalytic chemodosimeter for Cu2+ ions, Chem. Commun. 483 (2009) 8– 40.
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Journal of Photochemistry and Photobiology A: Chemistry journal homepage: www.elsevier.com/locate/jphotochem
Invited feature article
Coumarin-naphthol conjugated Schiff base as a “turn-on” fluorescent probe for Cu2+ via selective hydrolysis of imine and its application in live cell imaging Sisi Wang, Zhen Wang, Yong Yin, Jianguang Luo* , Lingyi Kong* State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People’s Republic of China
A R T I C L E I N F O
Article history: Received 16 August 2016 Received in revised form 23 September 2016 Accepted 11 October 2016 Available online 29 October 2016 Keywords: Fluorescent probe Coumarin-naphthol conjugate Schiff base Imine hydrolysis Copper ion
A B S T R A C T
An off-on fluorescent probe (CuCIN), coumarin-naphthol linked by an imine group, for detecting Cu2+ in DMSO-HEPES buffer (1:99, v/v) solution was successfully developed. Mechanism studies suggested that CuCIN firstly formed a complex with Cu2+ in a 1:2 metal-to-ligand ratio, and then 60-fold fluorescence enhancement was observed when Cu2+ promoted the imine hydrolysis to release the fluorescence compound 7-diethylaminocoumarin-3-aldehyde. This probe displayed desired properties such as high selectivity toward other metal ions, low detection limit (12.7 nM) and low cytotoxicity. Bio-imaging study revealed that the new probe CuCIN could be applied for imaging living cells with good cell permeability. We hope to apply this probe in the biomedical research field for the imaging of disease-relevant Cu2+. ã 2016 Elsevier B.V. All rights reserved.
1. Introduction Divalent copper ion, as the third most abundant transition metals, plays a vital role for most forms of life [1]. Copper ions serve as a structural and catalytic cofactor for many proteins and enzymes including important metabolic factors such as cytochrome c oxidase and copper–zinc superoxide dismutase [2,3]. It is demonstrated that the changes in copper’s cellular homeostasis are connected to serious neurodegenerative diseases, such as Menkes [4], Wilson’s disease [5], familial amyotropic lateral sclerosis, Alzheimer’s disease [6] and prion disease [7]. Therefore, it makes sense to find a sensitive, real-time response tool to analyze the level of copper ion for in vitro and in vivo assays. Conventional analytical methods like atomic absorption spectrometry [8], plasma atomic emission spectrometry [9], mass spectrometry [10], surfaceplasmon resonance [11] are available for the detection of Cu2+. Nonetheless, most of the above mentioned traditional methods are expensive and time-consuming. As a result, fluorescence analysis has emerged as a convenient method for monitoring the biomolecules in living system for their intrinsic sensitivity, high spatial and temporal resolution [12,13]. Most of the reported fluorescent probes for Cu2+ are based on either chelating or chemical reaction. Probes chelating with Cu2+ usually
* Corresponding authors. E-mail addresses: [email protected] (J. Luo), [email protected] (L. Kong). http://dx.doi.org/10.1016/j.jphotochem.2016.10.030 1010-6030/ã 2016 Elsevier B.V. All rights reserved.
induce the intrinsic fluorescence quenching because of the paramagnetic property of Cu2+, which are not as effective as the probes that enhance the emission intensity [14]. Alternatively, fluorescent probes based on reactions have been developed to reach emission enhancement by reacting with Cu2+ to yield fluorescent products which possess little affinity to Cu2+, consequently avoiding the problem of paramagnetic of Cu2+ and satisfying the rational design for fluorescent probes [15]. Some efficient fluorescent probes for Cu2+ based on the highly specific chemical reactions such as hydrolysis [16–21] and oxidation [22– 24] have attracted much attention. However, some of them have shortcomings for quantifying such as slow response [25], cytotoxicity of the ligands, small stoke shift [25], cross-sensitivity toward other metal cations [26]. Consequently, it is necessary to find a fluorescent probe which could overcome these shortcomings. Previously, there are several fluorescent probes based on Cu2 + -promoted hydrolysis of hydrazone [17,18] and lactam [27]. On the other side, some “off-on” fluorescent probes containing C¼N bond with enhanced fluorescence intensity in virtue of forming complex with metal ions have been investigated [28–31]. So far as we know, there is no probe detecting Cu2+ on account of the hydrolysis of the imine group. Hence, it's of our great interest to design a reaction based fluorescent probe for Cu2+ detection, which provided a new mechanism of Cu2+ promoting the imine hydrolysis.
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Coumarin is often selected as a signaling moiety due to its high quantum yield, large stokes shift and low cytotoxicity [32]. It is easy to modify the coumarin on its 3-C, 4-C and 7-C positions, introducing electron donating groups and electron accepting groups contributes to produce many strong fluorescent dyes with different wavelength ranges of absorption and fluorescence emission [33–35]. Moreover, naphthol could serve as a molecule which stabilize the Schiff base and there have been some naphtholderivatives developed for detecting copper [36,37]. Herein, we reported a fluorescent probe (CuCIN) for Cu2+ based on coumarinnaphthol conjugated Schiff base which exhibited fluorescence enhancement upon the hydrolysis of the imine group. CuCIN was synthesized in a simple two-step reaction and characterized by 1H NMR, 13C NMR, and HRMS. This probe emerged some advantages for analysis like high selectivity, low detection limit and we successfully used it in bio-imaging of Cu2+ in living cells for its low cytotoxity and good cell permeability. 2. Experimental 2.1. Materials and general methods Unless noted, all the reagents and solvents used in our experiment were analytical grade without further purification, and double-distilled water was used in all experiments. The metal stock solutions were prepared from the salts of AlCl3, CaCl2, CoCl26H2O, CuCl22H2O, FeCl24H2O, FeCl36H2O, KCl, ZnCl2, NaCl, MgCl26H2O, MnCl24H2O, NiCl26H2O, HgCl2, Pb(NO3)2, AgNO3 and CdCl2. A human hepatoblastoma cell line (HepG2) was purchased from Shanghai Institute of Biochemistry & Cell Biology, Chinese Academy of Sciences (Shanghai, China) The 1H NMR (500 MHz) and 13C NMR (125 MHz) spectra were measured on Bruker ACF-500 spectrometer while using TMS as an internal standard. IR (KBr-disc) spectra were recorded by Bruker Tensor 27 spectrometer. MS spectra were measured on a MS Agilent 1100 Series LC/MSD Trap mass spectrometer (ESI–MS) and a Mariner ESI-TOF spectrometer (HRMS), respectively. The pH levels of the stock solution were monitored by PHS-25C Precision pH/mV meter. UV–vis spectra were obtained using a Shimadzu UV2300 spectrophotometer and fluorescence spectra were measured with Perkin LS-55 fluorescence spectrophotometer, respectively. 2.2. Synthesis of CuCIN (1) The solution of 7-diethylaminocoumarin- 3-aldehyde (3) (73.5 mg, 0.30 mmol) in 5 ml hot absolute ethanol was added into the solution of 3-amino-2-naphthol (72.0 mg, 0.45 mmol) in 5 ml absolute ethanol. The mixture was refluxed for 6 h to yield scarlet precipitate. The precipitate was filtrated, washed with hot absolute ethanol three times. After drying under reduced pressure, CuCIN (1) was obtained as scarlet solid (60 mg, 52%).1H NMR (500 MHz, DMSO-d6): dH 9.30 (1H, s, OH), 8.84 (1H, s, C¼CH), 8.75 (1H, s, CH¼N ), 7.81 (1H, d, J = 8.1 Hz, Ar-H), 7.66 (2H, t, J = 9.0 Hz, ArH), 7.60 (1H, s, Ar-H), 7.34 (1H, t, J = 7.4 Hz, Ar-H), 7.26 (1H, t, J = 7.4 Hz, Ar-H), 7.23 (1H, s, Ar-H), 6.83 (1H, d, J = 8.1 Hz, Ar-H), 6.63 (1H, s, Ar-H), 3.51 (4H, q, J = 7.0 Hz, CH2), 1.17 (6H, t, J = 7.0 Hz, CH3). 13C NMR (125 MHz, DMSO-d6): d 161.8, 157.9, 154.8, 152.6, 152.2, 150.9, 142.8, 141.3, 133.5, 132.0, 128.8, 128.1, 126.2, 126.0, 123.6, 116.4, 110.6, 109.9, 108.7, 97.0, 44.9(2C), 12.9(2C). HRMS: [M +H+] calcd. for C24H23N2O3: 387.1703, found 387.1702. 2.3. Cell culture and confocal imaging HepG2 cells were cultured in DMEM (Dulbecco's Modified Eagle Medium) medium supplemented with 10% FCS (Fetal Calf Serum)
at 37 C in a 5% CO2/95% air incubator. Confocal fluorescence imaging studies were performed on a LSM 710 confocal laserscanning microscope (Carl Zeiss Co. Ltd.). The working solution was prepared from 20 mM CuCIN solution in DMSO and then it was diluted with DMEM medium to a final concentration of 20 mM. The cells were incubated with CuCIN for 30 min and washed with HEPES buffer (20 mM) three times, and then incubated with 10 mM CuCl2H2O solution in DMEM medium for 20 min. After another washing with HEPES buffer three times, the cells were in HEPES buffer during the fluorescence imaging. Fluorescence microscopy images of labeled cells were obtained by exciting the probe with a wavelength at 405 nm. 2.4. Cytotoxicity assay The cytotoxicity of the CuCIN to HepG2 cells was obtained through a MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2Htetrazolium bromide) assay. HepG2 cells were seeded into 96well culture plates at 8000 cells per well. Before the treatment with CuCIN, the DMEM (Dulbecco's Modified Eagle Medium) with 10% FCS (Fetal Calf Serum) were removed and replaced with fresh medium. The treated cells were cultured with different concentrations of CuCIN (0, 3, 6, 12.5, 25, 50 mM) at 37 C under 5% CO2/ 95% atmosphere for 24 h, and cells without CuCIN were taken as control. Subsequently, cells were treated with 20 ml MTT (5 mg/ mL) in each well and incubated for 4 h, and then 150 ml DMSO was added to dissolve the form azan crystals. The absorbance at 570 nm was obtained with the microplate reader. The cytotoxicity effect of the CuCIN was calculated through the following equation: Cell viability% = OD570 (sample)/OD570 (control) 100 2.5. Measurement procedure The stock solution of CuCIN (1 mM) was prepared by dissolving it in DMSO, while the stock solution of various metal ions was prepared by dissolving them in double-distilled water. The test solution was prepared by measuring out desired metal ion solution into 10 ml volumetric flask containing CuCIN stock solution and then diluting it by HEPES buffer solution (10 mM, pH = 7.4). The measurements of fluorescence spectra were carried out at the excitation wavelength of 435 nm, while the emission spectra range was 450 to 650 nm and the slit width was 5 nm. 3. Results and discussion A fluorescent probe CuCIN for Cu2+ was designed and synthesized as the route shown in Scheme 1. The new compound CuCIN was synthesized through the nucleophilic addition reaction in absolute ethanol under reflux condition by the two known compounds 7-diethylaminocoumarin (2) and 7-diethylaminocoumarin- 3-aldehyde (3), which were prepared from the starting material 4-diethylamino-salicylaldehyde (In Supporting information) according to the literature [38]. The whole synthetic route was simple and convenient and all the synthesized target compounds were characterized by 1H NMR, 13C NMR and HRMS. 3.1. Titration experiment We investigated the optical properties of probe CuCIN in the absence and the presence of Cu2+. Initially, the UV–visible absorption and fluorescence titrations of the CuCIN were carried out using a solution of 10 mM CuCIN in DMSO/HEPES buffer (1:99 v/v). In Fig. 1a, the test solution showed the adsorption at 345 nm and 452 nm, upon the incremental addition of Cu2+, the
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Scheme 1. Synthesis of CuCIN.
adsorption band at 345 nm decreased while the adsorption band at 452 nm increased gradually. These changes were complemented by clear isosbestic point at 382 nm. The observed UV spectra change was associated with the hydrolysis reaction upon the addition of Cu2+. By gradually adding Cu2+, the fluorescence intensity at the wavelength of 502 nm(lex = 435 nm) increased step by step and reached the maximum when the Cu2+ was 0.5 equiv. Then the
fluorescence changed from colorless to green obviously. As shown in Fig. 1b, the emission intensity at 502 nm associated with the CuCIN was low probably attributed to a decay process of the C¼N bond cis-trans isomerization as reported[39]. The emission intensity was enhanced about 60-fold when 0.5 equiv. of Cu2+ was added. Moreover, the good relationship of emission intensity against Cu2+ from 0 to 0.5 equiv. was observed from the Fig.S1. The detection limitation (CDL = 3Sb/m) was calculated as 0.82 mg/L (12.7 nM), which is much lower than the World Health Organization recommended level (2.0 mg/L) in drinking water[42]. The detection limitation of other Cu2+ probes were exhibited in Table. S1. Taken together, these data proved that the new probe was a highly sensitive sensor to detect Cu2+ in a quantitative manner. 3.2. Selectivity study To obtain the specific selectivity property of the CuCIN toward various metal ions, the influence of other metal ions such as Na+, K+, Pb2+, Co2+, Al3+, Hg2+, Mg2+, Ni2+, Cd2+, Ca2+, Fe3+, Fe2+, Mn2+, Hg2+, Ag+ and Zn2+ were measured. There was no obvious fluorescence response toward the aforementioned metal ions as shown in Fig. 2a. As we known, the ability of a probe to analyze the target over other metal ions is important, so we further investigated the interference of other metal ions to detection Cu2+. As shown in Fig. 2b, the CuCIN could distinguish the Cu2+ from other metal ion related to the environment and biological condition. Colorimetric sensing by naked eye to observe the high selectivity toward various metal ions was also shown in Fig. 3. 3.3. The pH effect It is very important for a fluorescent probe to be stable at the biological pH environment, so we investigate the pH effect on the CuCIN. As shown in Fig.S2, the CuCIN was stable and weak fluorescent over all the pH range (pH = 5–11). On the other hand, toward adding Cu2+ into the solution, strong fluorescence emission enhanced at the pH under 6 to 9. It showed that the CuCIN could be applicable at complex biological environment 3.4. Kinetics
Fig. 1. (a) Absorption spectra and (b) Fluorescence spectra (lex = 435 nm) of CuCIN (10 mM) in the presence of increasing amount of Cu2+ in HEPES buffer (10 mM, pH 7.4, containing 1% DMSO) after the incubation with Cu2+ for 20 min at 25 C.
To better study the reaction of the CuCIN in the presence of Cu2+, we examined the kinetic of CuCIN and Cu2+ by the response time of CuCIN toward Cu2+. After CuCIN incubated with Cu2+ in HEPES buffer, the fluorescence intensity at 502 nm (lex = 435 nm) increased gradually and saturated at 20 min and then kept almost invariant (as shown in Fig. 4a). The hydrolysis kinetics were studied from the fluorescence spectra and the rate constant k was calculated by the equation as followed: ln((Fmax-F)/Fmax) = kt, as result, k = 0.104 min1(as shown in Fig. 4b). The medium constant
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Fig. 2. (a) Fluorescence spectra of CuCIN (10 mM) to Cu2+ (1 equiv.) and other metal ions (1 equiv.) in HEPES buffer (10 mM, pH 7.4, containing 1% DMSO) after the incubation for 20 min at 25 C (lex = 435 nm). (b) Fluorescence intensity of CuCIN to various metal ions in HEPES buffer (10 mM, pH 7.4, containing 1% DMSO) after the incubation for 20 min at 25 C (lex = 435 nm). The black bars represent the emission of CuCIN in the presence of various metal ions (2 equiv.) to CuCIN. The red bars represent the emission that occurs upon subsequent addition of Cu2+ (2 equiv.) to the solution. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 4. (a) Time dependent fluorescence spectra changes of probe CuCIN (10 mM) with Cu2+ (0.5 equiv.) in HEPES buffer (10 mM, pH 7.4, containing 1% DMSO) at 25 C (lex = 435 nm). (b) Curves fitted with first-order reaction dynamics (ln ((Fmax-F)/ Fmax) = kt) and rate constant (k) was calculated: k = 0.104 min1 ([Cu2+] = 10 mM).
Fig. 3. Photograph of CuCIN (10 mM) to various metal ions under UV lamp (lex = 365 nm).
Fig. 5. Job’s plot of the complex formed by [Cu2+]/[Cu2+] + [CuCIN], the total concentration of CuCIN and Cu2+ was 10 mM (lex = 435 nm).
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Scheme 2. Sensing mechanism of CuCIN based on Cu2+ catalyzed hydrolysis reaction.
k = 0.104 min1 indicated the reaction activation energy might be relatively high. 3.5. Job’s plot For further determination the stiochiometric between Cu2+ and CuCIN, Job’s plot analysis was also used. Total concentration of CuCIN and Cu2+ was 10 mM. As shown in Fig. 5, the maximum fluorescence intensity exhibited at around 0.33 mol fractions that indicated a 2:1 stiochiometric between CuCIN and Cu2+. Thus, a possible coordination mode for CuCIN with Cu2+ was proposed in
Scheme 2. And the association constant (Ka) of CuCIN with Cu2+ was also determined using the Benesi–Hildebrand equation as follows: 1 1 1 þ ¼ F F min ka ðF max F min Þ½Cu2þ F max F min F and Fmin represent the fluorescent intensity of the CuCIN at moderate concentration and none of Cu2+, respectively. Fmax is the saturated fluorescent intensity of CuCIN in the presence of excess amount of Cu2+. The association constant was calculated to be 1.74 105 M1.
Fig. 6. 1H NMR spectra of (a) CuCIN in DMSO-d6, (b) CuCIN in DMSO-d6 + 1 equiv. Cu2+ in H2O, (c) the standard 7-diethylaminocoumarin-3-aldehyde in DMSO-d6.
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Fig. 7. Confocal fluorescence images of HepG2 cells, (a, b and c) images of cells after incubated with CuCIN (20 mM) for 30 min; (d, e and f) images of cells after incubated with CuCIN (20 mM) for 30 min and subsequently with Cu2+ (0.5 equiv.) for 20 min; (a and d) green channel image (lex = 405 nm); (b and e) bright field image; (c) overlay image of a and b; (f) overlay image of d and e. Scale bar: 50 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3.6. The sensing mechanism There have been some “turn-on” fluorescent probes containing C¼N bond for detecting Cu2+ based on chelating copper ions to prevent the C¼N isomerization[29,40] or promoting hydrolysis the hydrazone[17] to release the fluorescent compound. However, no mechanism based on the imine hydrolysis in the presence of Cu2+ was revealed. Therefore, we illuminated the probe CuCIN sensing Cu2+ mechanism using the combination of 1H NMR, IR, MS and UV– vis spectroscopic methods. Firstly, we verified the complex of copper ion and CuCIN by FTIR spectrum, and the stiochiometric complexation of CuCIN Cu2+ was evaluated by the Job’s plot data. As shown in Fig.S3, a shift could be observed of the naphtholic hydroxyl group from 3426 cm1 (free CuCIN) to3383 cm1(CuCIN-Cu2+), the C¼O group of the free CuCIN(1702 cm1) almost disappeared and the C¼N group changed from 1615 cm1 to 1577 cm1. All the change of the FT-IR spectra of CuCIN in the absence and presence of Cu2+ revealed that the naphtholic hydroxyl, ester carbonyl and C¼N bond was involved in the metal coordination. To gain more information of the kind of reaction that CuCIN underwent in presence of copper ion, the 1H NMR titration experiment was carried out. When 1 equiv. Cu2+ was added to the probe solution in DMSO-d6, the singlet at dH 8.84 ppm assigned to the proton of CH¼N group disappeared, along with appearance of the singlet at dH 9.89 ppm attributed to the proton of aldehyde moiety as shown in Fig. 6. Additionally, all proton signals of 3 (Fig. 6c) were detected in the 1H NMR of this solution (Fig. 6b). On the other hand, when the solution of CuCIN was combined with Cu2+, a peak corresponding to [3+H+] appeared at m/z = 246.04 in the ESI–MS spectra further demonstrated the hydrolysis of imine group into aldehyde (Fig.S4). UV–vis spectra of the CuCIN solution in the presence of Cu2+ were compared with the solution of compound 3, and the spectra of the two solutions were nearly identical as shown in Fig.S5. Overall, the products were just confirmed to contain compound 3. Therefore, the sensing mechanism of the probe can be proposed. Initially, the ester carbonyl, naphtholic hydroxyl and
the N atom of CuCIN were chelated with Cu2+. The coordinate bond between Cu2+ and N atom led to the electronic cloud distribution of the N atom apart from the C atom, as a result, the bond energy of C¼N bond was weakened. Sequentially, the imine sequentially was hydrolyzed to release the aldehyde (compound 3) as shown in Scheme 2. The solution of probe CuCIN with almost no fluorescence transferred to that of the corresponding aldehyde derivative (compound 3) with strong green fluorescence due to the Cu2+ promoted C¼N hydrolysis reaction of CuCIN. 3.7. Laser scanning confocal fluorescent imaging It is reported that the hepatocytes are important in copper metabolism, therefore we tested the Cu2+ sensing ability of probe CuCIN in copper overloaded HepG2 cells[41]. Furthermore, to ensure the bio-imaging in a safe condition, we investigated the cytotoxicity of the probe by MTT with the CuCIN concentration ranging from 3 mM to 50 mM, and the result was shown in Fig.S6. The results of MTT indicated that the cell viability was 62.4% versus the control group when incubated with 50 mM compound CuCIN for 24 h. It proved that the compound CuCIN did not show any significant cytotoxicity toward HepG2 cells. When HepG2 cells were incubated with CuCIN (20 mM) for 30 min at 37 C in HEPES buffer, there was negligible intracellular fluorescence upon excitation at 405 nm. When cells were incubated with Cu2+ (10 mM) for another 20 min, intense fluorescence emerged in the green channel was observed in Fig. 7, showing that CuCIN was cellpermeable and could be efficiently used for imaging of Cu2+ in living cells. 4. Conclusions In summary, a coumarin-naphthol conjugated Schiff base fluorescent probe based Cu2+-promoted imine hydrolysis mechanism was designed and synthesized, which showed a remarkable fluorescence enhancement toward Cu2+ with high selectivity and low detection. In addition, the probe was successful applied for
S. Wang et al. / Journal of Photochemistry and Photobiology A: Chemistry 333 (2017) 213–219
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