Fluorescent probes have long been considered as ideal candidates for biological studies [1, 2] because the detection process is non-sample-destructing and less cell-damaging,and the instrumentation required for fluorescent detection is relatively simple,the selectivity and sensitivity is high,and the dynamic range is broad. Fluorescent detection in combination with microscopy can provide results in real time with spatial resolution and is amenable to high throughput methodology . Since Czarnik  firstly reported the spiro ring-opening reaction of xanthene derivatives,this pioneering work has stimulated the development of a wide range of probes utilized for metal ions , biological relevant ions and the recognition and detection of biomolecules . Among the most attractive functional groups,the Schiff base moiety has demonstrated to be an ideal receptor with significantly improved sensitivity,and selectivity as well as fluorescent properties and has proven to be highly suitable for the detection of heavy and transition metal ions at physiological pH,as well as thiols and reactive oxygen species (ROS) .
Due to the crucial roles in a wide scope of biological processes, the ROS have gained significant scientific interest . As playing critical roles in water treatment and in the immune system ,hypochlorous acid (HOCl),usually existing as the hypochlorite ion (OCl-) at physiological pH,have especially received a considerable amount of clinical,commercial,and academic attention. The importance of hypochlorite is reflected by the fact that abnormal levels of hypochlorite have been implicated in a series of human diseases,such as cardiovascular diseases,damage of human red blood cells,neuron degeneration,lung injury,kidney disease,and cancer . Therefore,the design and synthesis of highly selective and sensitive functionalized fluorescent probes with Schiff base function based on xanthene derivatives that are capable of detecting hypochlorite in biological systems is of particular importance .
Herein we report the design and biological application of a series of efficient fluorescent probes 1-3 with fluorescein as the fluorophore and a Schiff base moiety as the receptor. The probes displayed remarkable selectivity and anti-interference performance to hypochlorite in MeOH/H2O (4:6,v/v) solution with an extremely low detection limit at physiological pH range,thus enabling facile imaging of hypochlorite in living cells. 2. Experimental
Absorbance spectra were performed on a Shimadzu UV-1700 spectrophotometer. Fluorescent spectra measurements were performed on a Hitachi F-4500 fluorescent spectrophotometer.X-ray crystal data were collected on the Bruker Smart APEX II CCD diffractometer. Elemental analyses were measured on a Vario EL III analyzer. IR spectra were acquired on the Bruker Tensor 27 spectrometer in KBr disks. Mass spectra were performed on the Bruker micrOTOF-Q II ESI-Q-TOF LC/MS/MS Spectrometer. NMR spectra were recorded on the Varian Inova-400 MHz spectrometer (at 400 MHz for 1H and 100 MHz for 13C). The chemicals and reagents were obtained from Sigma-Aldrich Co.,LLC. Analytical thin layer chromatography was performed using Merck 60 GF254 silica gel coated glass plates. Silica gel (0.200-0.500 mm,60A,J&K Scientific Ltd.) was used for column chromatography.
Synthesis of the probes: Fluorescein hydrazide was synthesized according to Ref. . Fluorescein hydrazine (3.46 g,0.01 mol) was then dissolved in 50 mL ethanol. The cinnamyl aldehydes (0.01 mol) were added in 30 min. The reaction mixture was refluxed for 12 h. After cooling to the room temperature,the solvent was evaporated and then washed with anhydrous ether to obtain a white or light yellow powder.
Probe 1: White powder. mp: 289-290°C. Anal. Calcd. for C29H20N2O4: H 4.38,C 75.64,N 6.08. Found: H 4.36,C 75.69,N 6.09. 1H NMR (400 MHz,DMSO-d6,TMS): δ9.92 (s,2H),8.93 (d,1H, J= 9.1 Hz),7.88 (d,1H,J= 7.2 Hz),7.58 (dd,4H,J= 14.4,7.3 Hz), 7.31 (dd,3H,J= 15.0,7.5 Hz),7.04 (d,1H,J= 7.0 Hz),6.95 (d,1H, J= 16.0 Hz),6.76 (dd,1H,J= 15.9,9.2 Hz),6.64 (s,2H),6.49 (dd,4H, J= 21.3,8.6 Hz). 13C NMR (100 MHz,DMSO-d6,TMS): δ 163.9, 158.5,153.0,151.9,151.2,140.4,135.6,133.9,129.0,128.7,128.4, 128.0,127.3,126.2,123.6,123.2,112.4,110.0,102.6,65.2. MS (ESI):m/z461.1489 [M+H]+,Calcd. for C29H20N2O4: 460.1423.
Probe 2: White powder. mp: 277-278°C. Anal. Calcd. for C30H22N2O4: H 4.67,C 75.94,N 5.90. Found: H 4.66,C 75.99,N 5.89. 1H NMR (400 MHz,DMSO-d6,TMS):δ9.90 (s,2H),8.85 (s,1H),7.90 (d,1H,J= 7.3 Hz),7.69-7.55 (m,2H),7.37 (d,4H,J= 6.5 Hz),7.28 (s, 1H),7.13 (d,1H,J= 7.3 Hz),6.67 (d,3H,J= 18.6 Hz),6.46 (s,4H), 1.80 (s,3H). 13C NMR (100 MHz,DMSO-d6,TMS): d163.3,158.6, 154.5,152.4,150.2,137.8,136.1,134.8,133.8,129.4,129.1,128.4, 128.1,127.8,123.9,123.2,112.3,110.4,102.5,65.4,11.9. MS (ESI): m/z475.1652 [M+H]+,Calcd. for C30H22N2O4: 474.1582.
Probe 3: Yellow powder. mp: 284-285°C. Anal. Calcd. for C29H19BrN2O4: H 3.55,C 64.58,N 5.19. Found: H 3.54,C 64.59,N 5.21. 1H NMR (400 MHz,DMSO-d6,TMS):δ9.91 (s,2H),9.08 (s,1H), 7.92 (d,1H,J= 7.2 Hz),7.77 (d,2H,J= 7.1 Hz),7.67-7.56 (m,3H), 7.40 (d,3H,J= 7.6 Hz),7.15 (d,1H,J= 7.3 Hz),6.64 (s,2H),6.47 (s, 4H). 13C NMR (100 MHz,DMSO-d6,TMS): δ163.6,158.6,152.4, 150.3,148.9,139.0,134.5,134.2,129.8,129.3,129.0,128.4,128.0, 124.0,123.4,120.6,112.3,110.1,102.5,65.8. MS (ESI): m/z 537.0463 [M-H]-,Calcd. for C29H19BrN2O4: 538.0528.
Synthesis of the product of probe 1 upon addition of OCl-: Probe 1(0.46 g,1 mmol) was dissolved in 20 mL ethanol. The 1.0 mL of 30% NaOCl solution was then added. The reaction mixture was refluxed for 1 h. After cooling to the room temperature,the mixture was filtrated and solvent evaporated under reduced pressure and the resulting powder was purified by column chromatography on silica gel (eluent: ethyl acetate only) yielding an orange powder. 1H NMR (400 MHz,DMSO-d6,TMS): δ7.80 (d, 1H,J= 8.2 Hz),7.54 (s,3H),7.30 (d,4H,J= 7.4 Hz),6.81 (d,2H, J= 7.9 Hz),6.10 (s,1H),6.01 (s,1H). MS (ESI):m/z333.0757 [M+H]+, Calcd. for C20H12O5: 332.0685.
General procedure: The stock solutions of probes were prepared at 500mmol/L concentration. The solutions of ions,including LiCl, NaCl,KCl,BaCl2,MgCl2,CaCl2,CdCl2,MnCl2,CoCl2,ZnCl2,NiCl2, CuCl2,HgCl2,PbCl2,CrCl3,FeCl3,AlCl3,SnCl4,NaNO3,Na2CO3, NaHCO3,Na2SO4,Na3PO4,PO2HPO4,NaH2PO4,NaOAc,H2O2,and NaOCl,were also prepared at 500mmol/L in MeOH/H2O (4:6,v/v). The fluorescence intensity was recorded at 533 nm with an excitation wavelength at 490 nm. The absorption data were recorded at 517 nm.
Crystal growth and conditions: White single crystals of the probes were obtained at room temperature from a mixed solution of CH2Cl2-CH3CN by slow evaporation and then mounted on the goniometer of a single crystal diffractometer. The crystal data have been collected at 296 K by using MoKaradiation (l= 0.710713 Å ) and the φ/ω scan mode and Analyzed for Lorentz and polarization effect (SADABS). The structure was solved using the direct method and refined by full-matrix least-squares fitting onF2by SHELX-97.
Fluorescent imaging: Human Osteosarcoma MG-63 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS,at 37°C in a humidified atmosphere of 5% CO2and 95% air. The cells were then cultured for 2 h until they plated on glass-bottomed dishes. The growth medium was then removed and the cells were washed with DMEM without FBS and incubated with 20umol/L of the probes for 4 h at 37°C,washed three times with PBS and imaged. Then the cells were supplemented with 200mmol/L NaOCl in the growth medium for 4 h at 37°C and imaged . 3. Results and discussion
The probes were prepared by the synthetic route outlined in Scheme 1. The facile formylation reaction of fluorescein and hydrazine hydrate gave fluorescein hydrazide,which was further treated with cinnamyl aldehydes in EtOH solution at reflux temperature to yield the target probes. The probes were characterized by X-ray single crystal analysis in combination with elemental analysis,IR,NMR,and MS spectra. The X-ray crystal structural investigation clearly showed the special Schiff base functional group (Fig. 1). The structural differences between the probes are a result of the different substitution groups,which were further investigated to have obvious effects on the fluorescent properties.
We commenced our investigation by first verification of the sensing media. The combination of MeOH/H2O (4:6,v/v) was proved to be highly efficient (Figs. S1-S3 in Supporting information). The selective properties of the probes were then examined under simulated physiological conditions (PBS buffer,pH 7.4) based on the investigation of pH responses using the solutions with pH values from 1.7 to 13.0 (Figs. S4-S6 in Supporting information). The probes exhibited similar high selectivity toward OCl-(Fig. 2, red bars,Fig.S7-S9 in Supporting information) among the various testing ions including Li+,Na+,K+,Mg2+,Ba2+, Ca2+,Mn2+,Ni2+,Hg2+,Co2+,Zn2+,Pb2+,Cu2+,Cd2+,Fe3+,Al3+, Cr3+,Sn4+,SO42-,Cl-,CO32-,HCO3-,NO3-,OAc-,PO43-,HPO42-,H2PO4-,H2O2,and OCl-.By successively adding OCl-to the solutions of each probes in the presence of other ions,a series of competition experiments were investigated. The results revealed that the addition of the commonly co-existent ions did not significantly result in interference,although Fe3+induced a slight fluorescent quenching (Fig. 2, cation 10,green bars).
|Fig. 2. Fluorescence intensity changes of the probes (20 umol/L) upon addition of various ions (10 equiv.) in MeOH/H2O (4:6,v/v) solution. Red bars represent the fluorescence responses of the probes to the ions of interest: 1. blank,2. Li+,3.Na+,4.K+,5.Mg2+,6.Ca2+,7.Al3+,8.Cr3+,9.Mn2+,10. Fe3+,11. Co2+,12. Ni2+,13. Cu2+,14. Zn2+,15. Cd2+,16. Sn4+,17. Ba2+,18. Hg2+ ,19. Pb2+,20. Cl-,21.SO42-,22. NO3-,23. PO43-,24. HPO42-,25. H2PO4-,26. CO32-,27. HCO3-,28. OAc-,29. H2O2,30. OCl-. The green bars represent the subsequent addition of 200mmol/L OCl- to the above solutions.|
Absorption and fluorescent titrations were then conducted in MeOH/H2O solution (4:6,v/v,PBS buffer). As expected,the probe solution was colorless without OCl-,while the obvious color changed from colorless to yellow occurred upon addition of OCl-with the absorbance at 517 nm for all probes (Figs. S13-S18 in Supporting information). On the other hand,the fluorescent emission wavelength of the probes all appeared at 533 nm upon addition of OCl-,indicating the occurrence of the ring-opening process which was supposed to be induced by the irreversible OCl-promoted oxidation and hydrolyzation reaction in formation of the ring-opened product of fluorescein according to Ref. . The mechanism was also supported by the 1H NMR spectra in which the product displayed significant differences from probe 1,but quite close similarity to fluorescein (Fig. S31 in Supporting information). In addition,the mass spectral data,upon addition of OCl-manifested the peaks at 330.0757 ([M+H]+ ,probe 1 upon addition of OCl-,Fig. S32 in Supporting information),330.0744 ([M+H]+,probe 2 upon addition of OCl-,Fig. S33 in Supporting information),and 330.0753 ([M+H]+,probe 3 upon addition of OCl-,Fig. S34 in Supporting information) which also produced powerful proofs for the predicted product of fluorescein (C20H12O5, Calcd. 332.0685).
The emission intensity reached its maximum when 10 equiv. of OCl-was added. The fluorescent quantum efficiency were calculated to be 0.22 (probe 1),0.15 (probe 2) and 0.31 (probe 3),respectively,by using fluorescein as the standard. Furthermore, the linear dependence of the fluorescence intensity on the concentration of OCl-in the range of 0-200mmol/L (Figs. S10- S12),and the lower detection limit of 2mmol/L also provided powerful evidence for the possibility of the use of the probes in quantitative detection of OCl-(Fig. 3). Although the R groups caused no changes in absorption and emission wavelengths,the fluorescence intensity and absorbance were greatly affected by different substituent groups. It is clear that the effect of a heavy atom induced a significant enhancement in the fluorescent intensity and absorbance,while the alkyl groups weakened both the intensity and the absorbance,thus making probe 3 superior to the other ones. The investigation of the effect of the R group provides significant contributions in understanding the function of the substituent group in the receptor moiety,thus contributing to the utilization in the screening of effective recognition moieties in the design and application of probes.
|Fig. 3. Fluorescent titration of probe 1(a),probe 2 (b) and probe 3 (c) in MeOH/H2O (4:6,v/v) solution (20 umol/L) in the presence of OCl- ions of different concentrations (0- 10 equiv.). Inset: changes of the fluorescence intensities at 533 nm as the function of OCl-concentration.|
To further evaluate the potential application of the probes for tracking of OCl-in living cells,fluorescent imaging for OCl-was carried out in combination with confocal laser scanning microscopy. No intracellular fluorescence was observed (Fig. 4a) when cultured Human Osteosarcoma MG-63 cells were incubated with the probes in the medium for 4 h at 37°C. In contrast,significant fluorescence increases were monitored when the cells were supplemented with 10 equiv. of OCl-at 37°C and then loaded with the probes for 4 h in the same growth media (Fig. 4b). Moreover,the cells were proved to be viable throughout the imaging experiments (Fig. 4c) and the fluorescence signals were observed to be localized in the cytosol (Fig. 4d). This preliminary investigation suggested that the probes are cell membrane permeable and can be efficiently used for sensing of OCl-in living cells.
|Fig. 4. (1-3a) Fluorescent images of Human Osteosarcoma MG-63 cells incubated with 20mmol/L probes for 4 h at 37°C. (1-3b) Cells supplemented with 10 equiv. of OCl-at 37°C and then loaded with the probes for 4 h in the same growth media. (1-3c) Bright field images of the cells incubated with OCl-and the probes. The overlay image of (b) and (c) is shown in (d).|
In summary,we have demonstrated a series of novel probes with special Schiff base functional group for direct detection of OCl-in which switchable fluorescence was achieved from the irreversible OCl--promoted oxidation and hydrolyzation reaction in the formation of the ring-opened product,fluorescein. The probes showed remarkable fluorescence enhancement in response to OCl-with significantly high sensitivity and selectivity. The probes have been proven to be well suitable for tracking of OCl-in living cells,thus providing ideal candidates in understanding the basis of the biological importance of OCl-in physiological progresses. Acknowledgments
The work was supported by National Natural Science Foundation of China (Nos. 21272184,20972124,21143010 and J1210057), the Shaanxi Provincial Natural Science Fund Project (No. 2012JQ2007),the Northwest University Science Foundation for Postgraduate Students (Nos. YZZ12030,YZZ13042),and the Chinese National Innovation Experiment Program for University Students (No. 201210697011). 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.cclet.2014.05.011.
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