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A rhodamine-based “turn-on” fluorescent probe for Fe3+ in aqueous solution† Shuangzhi Ji,a,b Xiangming Meng,*a Weipeng Ye,a Yan Feng,a Hongting Sheng,a Yulei Cai,a,c Jinsong Liu,*b Xiaofan Zhuc and Qingxiang Guoc A water-soluble “turn-on” fluorescent probe (RD1) for Fe3+ based on rhodamine B was designed and syn-
Received 3rd September 2013, Accepted 28th October 2013
thesized. The fluorescent probe showed “turn-on” fluorescent and colorimetric responses to Fe3+ with a
DOI: 10.1039/c3dt52422a
high selectivity in water containing less than 1% organic cosolvent. Furthermore, bioimaging investigations indicated that the new probe was cell permeable and suitable for monitoring intracellular Fe3+ in living
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cells by confocal microscopy with low cytotoxicity.
Introduction Fe3+ plays a key role in many chemical and biological processes at the cellular level.1,2 Both its deficiency and overdose can induce various diseases, including certain cancers and dysfunction of organs.3,4 The interest in development of real-time Fe3+ monitoring technique is steadily growing in recent years.5–7 Fluorescent probes have become powerful detecting tools in biological and environmental analysis for their real time monitoring capability and high selectivity.8–12 During the last decade, many fluorescent probes for Fe3+ have been reported for the purpose.13–21 Unfortunately, most of the fluorescent probes for Fe3+ are poorly soluble in aqueous solutions, and many of them were of the fluorescence “turn-off” type due to the paramagnetism of Fe3+, which restricted the applications of the probes to biological systems. Development of the “turn-on” fluorescent probe for Fe3+ with a high selectivity and sensitivity in aqueous solution is still a steady challenge. The rhodamine skeleton is an ideal platform for constructing “turn-on” fluorescent probes for metal ions due to the equilibrium between spirocyclic (non-fluorescent) and ringopen forms (highly fluorescent).22–31 Although a few rhodamine-based probes for Fe3+ have been reported,32–39 their
a Department of Chemistry, Anhui University, Hefei 230601, P. R. China. E-mail: [email protected] b School of Pharmacy, Anhui University of Chinese Medicine, Hefei 230031, P. R. China. E-mail: [email protected] c Department of Chemistry, University of Science and Technology, Hefei 230026, P. R. China † Electronic supplementary information (ESI) available: Details on photophysical measurements and cell imaging, NMR, crystallographic data. CCDC 942502. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c3dt52422a
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Scheme 1
The design of RD1 for Fe3+ detection.
application to biological systems is still scarce because of their low selectivity or poor solubility in aqueous solutions.40–42 Herein, we report a “turn-on” fluorescent probe RD1 for Fe3+ in the aqueous solution (Scheme 1). The rhodamine B skeleton was used as the potential fluorophore and chromophore. The 2-picolylamine was chosen as the recognition group to get better selectivity whereas the hydrophilic acylamino group was used to link the rhodamine B group and the 2-picolylamine for the water solubility consideration. The probe RD1 exhibited a “turn-on” fluorescent and colorimetric signal toward Fe3+ with excellent selectivity in a fully aqueous solution. Meanwhile, the probe can image Fe3+ in living cells under confocal laser scanning microscopy.
Experimental section All the reagents were analytical grade and used as received. All solvents were used after appropriate distillation or purification. All reactions were magnetically stirred and monitored by thin layer chromatography (TLC). Flash chromatography (FC) was performed using silica gel 60 (200–300 mesh). Solutions of Fe3+, Cr3+, Hg2+, Fe2+, K+, Mg2+, Mn2+, Co2+, Pb2+, Cu2+, Cd2+, Ni2+, Ca2+ and Zn2+ were prepared from their chloride salts.
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HEPES buffer solutions (20 mM, pH 7.0) were prepared in water. 1H NMR spectra were recorded on a Bruker-400 MHz spectrometer and 13C NMR spectra recorded on a 100 MHz spectrometer. UV-vis spectra were recorded on a Techcomp UV1000 spectrophotometer. Fluorescence responses were recorded on a FL2500 spectrofluorometer. MS spectra were recorded on a Bruker autoflex III MALDI-TOF mass spectrometer. Synthesis of compound 1 As shown in Scheme 2, compound 1 was prepared according to the literature method.43 1H NMR (400 MHz, DMSO-d6, ppm): δ: 1.15 (12H, t, J = 7.02 Hz), 3.34 (8H, q, J = 7.00 Hz), 3.61 (2H, s), 6.45–6.29 (6H, m), 7.10 (1H, m), 7.45 (2H, m), 7.93 (1H m). 13C NMR (100 MHz, DMSO-d6, ppm), δ: 12.41, 43.64, 64.71, 97.35, 105.44, 107.73, 122.12, 123.47, 127.68, 132.36, 148.06, 151.85, 152.99, 165.26. MS(MALDI-TOF) calculated for [M + H]+ 458.26, found 458.25. Synthesis of compound 2 A mixture of compound 1 (1.36 g, 3 mmol), chloroacetyl chloride (0.5 g, 4.5 mmol) and dichloromethane (30 mL) was cooled with ice bath. Then Et3N (0.76 g, 7.5 mmol) was dissolved in 5 mL dichloromethane and added dropwise to the solution with vigorous stirring. After the addition, the ice bath was kept for about 30 min, and the color of the solution changed from pink to brown. The solvent was removed under reduced pressure to give the crude product. The target product was recrystallized in ethyl acetate to give 1.2 g of 2 in 75% yield. 1 H NMR (400 MHz, DMSO-d6, ppm), δ: 1.17 (12H, t, J = 7.03 Hz), 3.34 (8H, q, J = 7.02 Hz), 3.61 (2H, s), 6.30 (2H, m), 6.44 (4H, dd, J = 16.14 Hz), 7.11 (1H, t, J = 8.26 Hz), 7.45 (2H, m), 7.94 (1H, q, J = 5.78 Hz). 13C NMR (100 MHz, DMSO-d6, ppm): δ: 12.42, 14.04, 20.71, 40.66, 43.63, 59.72, 65.05, 97.03, 103.97, 107.62, 122.63, 123.82, 128.19, 128.45, 129.13, 133.33, 148.37, 151.81, 152.97, 163.41, 164.63, 170.31. MS(MALDI-TOF) calculated for [M + H]+ 533.22, found 533.20.
Synthesis of compound RD1 The mixture of 2 (0.53 g, 1 mmol), 2-picolylamine (0.324 g, 3 mmol), KI (0.5 mg, 0.003 mmol) and 20 mL acetonitrile was heated to 80 °C for 8 h and cooled to room temperature. Then the reaction mixture was poured into distilled water and extracted by ethyl acetate (3 × 15 mL). The organic phase was combined, dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude product was purified by column chromatography by using a mixed eluent EtOAc–PE (1 : 3) to give 0.36 g of RD1 in 60% yield. 1H NMR (400 MHz, DMSO-d6, ppm), δ: 1.10 (12H, t, J = 6.9 Hz), 3.13 (2H, s), 3.34 (8H, dd, J = 6.8 Hz,), 3.56 (2H, s), 6.46–6.31 (4H, m), 6.57 (2H, d, J = 8.6 Hz), 7.08 (1H, d, J = 7.3 Hz), 7.31–7.21 (2H, m), 7.60 (2H, p, J = 7.3 Hz), 7.74 (1H, t, J = 7.7 Hz), 7.88 (1H, d, J = 7.0 Hz), 8.48 (1H, d, J = 4.6 Hz), 9.61 (1H, s). 13C NMR (100 MHz, DMSO-d6, ppm): δ: 12.38, 38.80, 39.01, 39.22, 39.42, 39.63, 39.84, 40.05, 43.57, 49.77, 53.22, 65.00, 96.88, 104.09, 107.50, 121.79, 122.58, 123.85, 128.59, 129.27, 133.20, 136.30, 148.27, 148.70, 151.73, 152.98, 159.44, 163.49, 169.41. MS (MALDI-TOF) calculated for [M + H]+ 605.42, found 605.46. X-ray crystallography X-ray diffraction data of single crystals were collected using a Siemens Smart 1000 CCD diffractometer. The determination of unit cell parameters and data collections were performed with Mo Kα radiation (λ = 0.71073 Å). Unit cell dimensions were obtained with least-squares refinements, and all structures were solved by direct methods using SHELXS-97.44 The other non-hydrogen atoms were located in successive difference Fourier syntheses. The final refinement was performed by using the full-matrix least-squares method with anisotropic thermal parameters for non-hydrogen atoms on F2. The hydrogen atoms were added theoretically and rode on the concerned atoms. Crystallographic data reported in this contribution have been deposited with the Cambridge Crystallographic Data Center, CCDC 942502 for RD1 (ESI†). Preparation of fluorescent titration The inorganic salt was dissolved in distilled water to afford 10 mM aqueous solution. The 1 mM stock solution of RD1 was prepared in absolute methanol. All the measurements were carried out according to the following procedure. To 10 mL volumetric flask containing 100 μL of the solution of RD1, different amounts (10 μL–400 μL) of metal ions were added directly with a micropipette, then diluted with buffered ( pH 7.0, 20 mM HEPES) solution. Fluorescence measurements were carried out with excitation and emission slit widths of 10 and 10 nm and the PMT voltage and the excitation wavelength were 400 V and 400 nm, respectively. Cell culture and fluorescence microscopy imaging
Scheme 2
Synthesis of RD1.
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7402 Cells were cultured in DMEM supplemented with 10% FCS, penicillin (100 μg mL−1), and streptomycin (100 μg mL−1) at 37 °C under a humidified atmosphere with 5% CO2 and 95% air.45 Cytotoxicity assays show that RD1 is safe enough for
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confocal microscopy at low concentrations, so that the cells were incubated with 15 μM RD1 at 37 °C under 5% CO2 for 30 min, washed once and bathed in DMEM containing no FCS prior to imaging. Then 30 μM Fe3+ was added into the growth medium for 0.5 h at 37 °C, and washed 3 times with PBS buffer. Then, the cells were imaged on a Zeiss LSM 510 Meta NLO confocal microscope.
Results and discussion As shown in Scheme 2, RD1 was easily synthesized via three steps from the readily available rhodamine B with an overall yield of 40.5%. The structures of RD1 and the intermediates were all confirmed by 1H NMR and 13C NMR. The single crystal of RD1 was grown from diethyl ether–petroleum ether solution. The crystal structure diagrams are shown in Fig. 1. The details of the crystallographic data are listed in Table 1. UV-vis and fluorescence spectra responses
Paper Table 1
X-ray crystallography data for RD1
Compound
RD1
Chemical formula Formula mass Crystal system Space group a/Å b/Å c/Å α/° β/° γ/° Unit cell volume/Å Temperature/K Z Number of reflections measured Number of independent reflections Rint Final R1 values (I > 2σ(I)) Final wR(F 2) values (I > 2σ(I)) Final R1 values (all data) Final wR(F 2) values (all data) Goodness of fit on F 2
C36H40N6O3 604.74 Triclinic ˉ P1 12.0273(5) 12.0667(4) 12.6247(4) 100.282(3) 110.275(3) 102.003(3) 1617.22(10) 290 2 16 241 5147 0.0206 0.0450 0.1246 0.0482 0.1282 1.025
Absorption and fluorescence spectra were performed in methanol–water (1/99) buffer ( pH 7.0, 20 mM HEPES, 50 mM NaNO3). As shown in Fig. 2, the solution of RD1 in water was colorless and exhibited no absorption above 500 nm in a UV-vis spectrum, which was ascribed to the spirolactam form of RD1. On addition of Fe3+, the solution turned from colorless to deep red immediately (in less than 1 second) (Fig. 2 inset). A new and strong absorption centered at 568 nm appeared and was enhanced with the addition of Fe3+, suggesting that the ring-open formation of RD1 should result from Fe3+ binding. Such a dramatic color change ensured the RD1 as a sensitive “turn-on” and colorimetric probe for Fe3+. RD1 showed almost no fluorescent emission upon excitation at 530 nm. Upon the addition of Fe3+, the fluorescence
Fig. 2 The UV-vis absorption titration spectra of RD1 (10 μM) with Fe3+ (0–2.4 equiv.) in methanol–water (1/99) buffer ( pH 7.0, 20 mM HEPES, 50 mM NaNO3). Inset: digital photographs of RD1 and RD1 in the presence of equivalent amount of Fe3+ under normal light.
Fig. 1 Molecular structure of RD1 (ORTEP drawings of RD1 showing thermal ellipsoids at 30% probability level). Selected bond distances [Å] and angles [°]: O2–C28 1.2171(19), O3–C29 1.210(2), N3–C28 1.3813(17), N3–C7 1.4976(18), N4–C29 1.359(2), N5–C30 1.446(3), N5–C31 1.466(3), N6–C36 1.337(4), N6–C32 1.339(3), C28–N3–N4 122.59(12), N4–N3–C7 120.82(11), C29–N4–N3 120.09(13), N3–N4–H4 118.6(13), C30–N5–H5 105.0(16), C31–N5–H5 107.4(16), N5–C31–C32 114.38(16), N6–C32–C31 115.0(2).
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intensity at 586 nm was enhanced dramatically (Fig. 3). The emission intensity reached its maximum after the addition of 2.2 equivalent of Fe3+, and the intensity increased about 40-fold. According to the reported method,46,47 the association constant (Ka) of RD1 for Fe3+ was calculated to be 7.52 × 104 M−1. The quantum yield of RD1 displayed the enhancement of ca. 10-fold (from Φfree = 0.026 to ΦFe3+ = 0.273) upon binding to Fe3+. Job’s plot analysis indicates a 1 : 1 binding model between the probe and Fe3+ (Fig. S1 ESI†). Another solid evidence for the 1 : 1 binding model between the probe and Fe3+ came from the MALDI-TOF MS analysis. As shown in Fig. 4, RD1 has a peak at m/z = 605.46. Upon the addition of Fe3+, a peak at m/z = 696.47 ([RD1 + FeCl]2+) appeared along with the disappearance of the peak at m/z = 605.46. The observations indicated that RD1 bound with Fe3+ in a 1 : 1 binding
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Fig. 3 Fluorescence emission spectra of RD1 (10 μM) with the excitation at 530 nm, upon the titration of Fe3+ (0–2.4 equiv.) in methanol– water (1/99) buffer ( pH 7.0, 20 mM HEPES, 50 mM NaNO3). Inset: changes of emission intensity at 586 nm (λex = 530 nm).
Fig. 5 (a) Fluorescence spectra of RD1 (10 μM) upon addition of Fe3+ and other metal ions (λex = 530 nm). (b) Fluorescence intensity at 586 nm of RD1 (10 μM) upon addition of various metal ions (black bars: RD1 with other metals, red: RD1 with Fe3+ and other metals). Experimental conditions: methanol–water (1/99) buffer ( pH 7.0, 20 mM HEPES, 50 mM NaNO3), 0.40 mM of K+(1), Ca2+(2), Mg2+(3) and 20 μM for Co2+(4), Ni2+(5), Fe2+(6), Cd2+(7), Mn2+(8), Pb2+(9), Zn2+(10), Cu2+(11), Hg2+(12), Cr3+(13), Fe3+(14) (λex = 530 nm).
Fig. 4 (a) MALDI-TOF MS spectrum of RD1 (DCTB as a matrix). (b) MALDI-TOF MS spectrum of RD1-Fe3+ complex (DCTB as a matrix).
stoichiometry. When EDTA was added to the solution of the complex RD1-Fe3+, the fluorescence was switched off gradually. Further excess addition of Fe3+ could recover the fluorescence of the solution (Fig. S2 ESI†). These results indicated that the fluorescent response of RD1 to Fe3+ was caused by the spiroring opening of the rhodamine B group. Ions selectivity and pH stability Metal ion selectivity studies were performed in HEPES buffer. As shown in Fig. 5, K+, Ca2+ and Mg2+, which are abundant in living cells, exerted a negligible effect on the fluorescence of RD1 even at higher concentrations (40 times of the Fe3+). The data also show that Mg2+, Pb2+, Zn2+, Hg2+, Co2+, Cd2+, Mn2+,
1586 | Dalton Trans., 2014, 43, 1583–1588
Fig. 6 Top: color of RD1 with different metal ions. Bottom: fluorescence (λex = 365 nm) change upon addition of different metal ions.
Hg2+, Fe2+, Ni2+ and Cu2+ caused negligible response to the fluorescence of RD1, while Cr3+ has a slight effect. The color change of RD1 with different metal ions is shown in Fig. 6. Among the metal ions tested, only Fe3+ caused dramatic changes both in color and in fluorescence. Therefore, RD1 can serve as a colorimetric and “turn-on” fluorescent probe for Fe3+ with remarkable selectivity.
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Furthermore, pH effect on fluorescence of RD1 was investigated in water. As shown in Fig. S3 (ESI†), the solution showed a strong and apparent fluorescence band at 586 nm when the pH value is lower than 6 and increased with the decrease of the solution pH, which signified the ring opening of spirolactam in RD1. Fortunately, in the biological relevant pH range (e.g. 6–9), RD1 always kept non-fluorescent, indicating that the probe could be used under common environmental and physiological conditions.
Cell cytotoxicity and fluorescence microscopy imaging Cell cytotoxicity assays were conducted in 7402 cells to test the cytotoxicity of RD1. As shown in Fig. S4 (ESI†), MTT assay demonstrated that the cell viability remains more than 90% after treating with 10 μM RD1 for 24 h. The result indicated that RD1 has almost no cytotoxicity for long term incubation at low concentration and should be safe when used for fluorescence microscopy imaging. With the above data in hand, we then applied RD1 in the cell imaging of Fe3+ by laser scanning confocal microscopy. 7402 Cells were cultured as the sample, and stained with RD1 within 30 min and washed by PBS buffer, no fluorescence was detected (Fig. 7a). After further incubation with FeCl3 for another 2.5 h at 37 °C, a red fluorescence appeared from the intracellular region (Fig. 7b). A bright field image of 7402 cells treated with RD1 and Fe3+ confirmed that the cells were viable throughout the imaging experiments (Fig. 7c). The results indicated that the RD1 was cell permeable and could be used for imaging of Fe3+ in living cells.
Fig. 7 (a) Fluorescence image of 7402 cells labeled with 15 μM RD1 after 30 min at 37 °C of incubation, washed with PBS buffer. (b) Fluorescence image of 7402 cells labeled treatment with RD1 and then 30 μM FeCl3 aqueous solution for 2.5 h at 37 °C. (c) Bright-field image of 7402 cells. (d) The overlay of (a)–(c).
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Conclusion In summary, we have developed a new rhodamine-based fluorescent probe RD1 which showed real-time and “turn-on” fluorescent and colorimetric responses to Fe3+ with a high selectivity in the aqueous solution. Compared with rhodamine-based probes for Fe3+ previously reported, the amount of organic cosolvent in detecting media was greatly reduced to less than 1%. In addition, the confocal fluorescence imaging confirmed that RD1 is cell permeable and can be used for monitoring intracellular Fe3+ in living cells with low cytotoxicity. This work was supported by NSFC (21102001, 21102002, 21372005 and 21272223), Natural Science Foundation of Education Department of Anhui Province (KJ2010A028, KJ2011A018), 211 Project of Anhui University, and Doctor Research Start-up Fund of Anhui University for supporting the research.
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A rhodamine-based “turn-on” fluorescent probe for Fe3+ in aqueous solution† Shuangzhi Ji,a,b Xiangming Meng,*a Weipeng Ye,a Yan Feng,a Hongting Sheng,a Yulei Cai,a,c Jinsong Liu,*b Xiaofan Zhuc and Qingxiang Guoc A water-soluble “turn-on” fluorescent probe (RD1) for Fe3+ based on rhodamine B was designed and syn-
Received 3rd September 2013, Accepted 28th October 2013
thesized. The fluorescent probe showed “turn-on” fluorescent and colorimetric responses to Fe3+ with a
DOI: 10.1039/c3dt52422a
high selectivity in water containing less than 1% organic cosolvent. Furthermore, bioimaging investigations indicated that the new probe was cell permeable and suitable for monitoring intracellular Fe3+ in living
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cells by confocal microscopy with low cytotoxicity.
Introduction Fe3+ plays a key role in many chemical and biological processes at the cellular level.1,2 Both its deficiency and overdose can induce various diseases, including certain cancers and dysfunction of organs.3,4 The interest in development of real-time Fe3+ monitoring technique is steadily growing in recent years.5–7 Fluorescent probes have become powerful detecting tools in biological and environmental analysis for their real time monitoring capability and high selectivity.8–12 During the last decade, many fluorescent probes for Fe3+ have been reported for the purpose.13–21 Unfortunately, most of the fluorescent probes for Fe3+ are poorly soluble in aqueous solutions, and many of them were of the fluorescence “turn-off” type due to the paramagnetism of Fe3+, which restricted the applications of the probes to biological systems. Development of the “turn-on” fluorescent probe for Fe3+ with a high selectivity and sensitivity in aqueous solution is still a steady challenge. The rhodamine skeleton is an ideal platform for constructing “turn-on” fluorescent probes for metal ions due to the equilibrium between spirocyclic (non-fluorescent) and ringopen forms (highly fluorescent).22–31 Although a few rhodamine-based probes for Fe3+ have been reported,32–39 their
a Department of Chemistry, Anhui University, Hefei 230601, P. R. China. E-mail: [email protected] b School of Pharmacy, Anhui University of Chinese Medicine, Hefei 230031, P. R. China. E-mail: [email protected] c Department of Chemistry, University of Science and Technology, Hefei 230026, P. R. China † Electronic supplementary information (ESI) available: Details on photophysical measurements and cell imaging, NMR, crystallographic data. CCDC 942502. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c3dt52422a
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Scheme 1
The design of RD1 for Fe3+ detection.
application to biological systems is still scarce because of their low selectivity or poor solubility in aqueous solutions.40–42 Herein, we report a “turn-on” fluorescent probe RD1 for Fe3+ in the aqueous solution (Scheme 1). The rhodamine B skeleton was used as the potential fluorophore and chromophore. The 2-picolylamine was chosen as the recognition group to get better selectivity whereas the hydrophilic acylamino group was used to link the rhodamine B group and the 2-picolylamine for the water solubility consideration. The probe RD1 exhibited a “turn-on” fluorescent and colorimetric signal toward Fe3+ with excellent selectivity in a fully aqueous solution. Meanwhile, the probe can image Fe3+ in living cells under confocal laser scanning microscopy.
Experimental section All the reagents were analytical grade and used as received. All solvents were used after appropriate distillation or purification. All reactions were magnetically stirred and monitored by thin layer chromatography (TLC). Flash chromatography (FC) was performed using silica gel 60 (200–300 mesh). Solutions of Fe3+, Cr3+, Hg2+, Fe2+, K+, Mg2+, Mn2+, Co2+, Pb2+, Cu2+, Cd2+, Ni2+, Ca2+ and Zn2+ were prepared from their chloride salts.
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HEPES buffer solutions (20 mM, pH 7.0) were prepared in water. 1H NMR spectra were recorded on a Bruker-400 MHz spectrometer and 13C NMR spectra recorded on a 100 MHz spectrometer. UV-vis spectra were recorded on a Techcomp UV1000 spectrophotometer. Fluorescence responses were recorded on a FL2500 spectrofluorometer. MS spectra were recorded on a Bruker autoflex III MALDI-TOF mass spectrometer. Synthesis of compound 1 As shown in Scheme 2, compound 1 was prepared according to the literature method.43 1H NMR (400 MHz, DMSO-d6, ppm): δ: 1.15 (12H, t, J = 7.02 Hz), 3.34 (8H, q, J = 7.00 Hz), 3.61 (2H, s), 6.45–6.29 (6H, m), 7.10 (1H, m), 7.45 (2H, m), 7.93 (1H m). 13C NMR (100 MHz, DMSO-d6, ppm), δ: 12.41, 43.64, 64.71, 97.35, 105.44, 107.73, 122.12, 123.47, 127.68, 132.36, 148.06, 151.85, 152.99, 165.26. MS(MALDI-TOF) calculated for [M + H]+ 458.26, found 458.25. Synthesis of compound 2 A mixture of compound 1 (1.36 g, 3 mmol), chloroacetyl chloride (0.5 g, 4.5 mmol) and dichloromethane (30 mL) was cooled with ice bath. Then Et3N (0.76 g, 7.5 mmol) was dissolved in 5 mL dichloromethane and added dropwise to the solution with vigorous stirring. After the addition, the ice bath was kept for about 30 min, and the color of the solution changed from pink to brown. The solvent was removed under reduced pressure to give the crude product. The target product was recrystallized in ethyl acetate to give 1.2 g of 2 in 75% yield. 1 H NMR (400 MHz, DMSO-d6, ppm), δ: 1.17 (12H, t, J = 7.03 Hz), 3.34 (8H, q, J = 7.02 Hz), 3.61 (2H, s), 6.30 (2H, m), 6.44 (4H, dd, J = 16.14 Hz), 7.11 (1H, t, J = 8.26 Hz), 7.45 (2H, m), 7.94 (1H, q, J = 5.78 Hz). 13C NMR (100 MHz, DMSO-d6, ppm): δ: 12.42, 14.04, 20.71, 40.66, 43.63, 59.72, 65.05, 97.03, 103.97, 107.62, 122.63, 123.82, 128.19, 128.45, 129.13, 133.33, 148.37, 151.81, 152.97, 163.41, 164.63, 170.31. MS(MALDI-TOF) calculated for [M + H]+ 533.22, found 533.20.
Synthesis of compound RD1 The mixture of 2 (0.53 g, 1 mmol), 2-picolylamine (0.324 g, 3 mmol), KI (0.5 mg, 0.003 mmol) and 20 mL acetonitrile was heated to 80 °C for 8 h and cooled to room temperature. Then the reaction mixture was poured into distilled water and extracted by ethyl acetate (3 × 15 mL). The organic phase was combined, dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude product was purified by column chromatography by using a mixed eluent EtOAc–PE (1 : 3) to give 0.36 g of RD1 in 60% yield. 1H NMR (400 MHz, DMSO-d6, ppm), δ: 1.10 (12H, t, J = 6.9 Hz), 3.13 (2H, s), 3.34 (8H, dd, J = 6.8 Hz,), 3.56 (2H, s), 6.46–6.31 (4H, m), 6.57 (2H, d, J = 8.6 Hz), 7.08 (1H, d, J = 7.3 Hz), 7.31–7.21 (2H, m), 7.60 (2H, p, J = 7.3 Hz), 7.74 (1H, t, J = 7.7 Hz), 7.88 (1H, d, J = 7.0 Hz), 8.48 (1H, d, J = 4.6 Hz), 9.61 (1H, s). 13C NMR (100 MHz, DMSO-d6, ppm): δ: 12.38, 38.80, 39.01, 39.22, 39.42, 39.63, 39.84, 40.05, 43.57, 49.77, 53.22, 65.00, 96.88, 104.09, 107.50, 121.79, 122.58, 123.85, 128.59, 129.27, 133.20, 136.30, 148.27, 148.70, 151.73, 152.98, 159.44, 163.49, 169.41. MS (MALDI-TOF) calculated for [M + H]+ 605.42, found 605.46. X-ray crystallography X-ray diffraction data of single crystals were collected using a Siemens Smart 1000 CCD diffractometer. The determination of unit cell parameters and data collections were performed with Mo Kα radiation (λ = 0.71073 Å). Unit cell dimensions were obtained with least-squares refinements, and all structures were solved by direct methods using SHELXS-97.44 The other non-hydrogen atoms were located in successive difference Fourier syntheses. The final refinement was performed by using the full-matrix least-squares method with anisotropic thermal parameters for non-hydrogen atoms on F2. The hydrogen atoms were added theoretically and rode on the concerned atoms. Crystallographic data reported in this contribution have been deposited with the Cambridge Crystallographic Data Center, CCDC 942502 for RD1 (ESI†). Preparation of fluorescent titration The inorganic salt was dissolved in distilled water to afford 10 mM aqueous solution. The 1 mM stock solution of RD1 was prepared in absolute methanol. All the measurements were carried out according to the following procedure. To 10 mL volumetric flask containing 100 μL of the solution of RD1, different amounts (10 μL–400 μL) of metal ions were added directly with a micropipette, then diluted with buffered ( pH 7.0, 20 mM HEPES) solution. Fluorescence measurements were carried out with excitation and emission slit widths of 10 and 10 nm and the PMT voltage and the excitation wavelength were 400 V and 400 nm, respectively. Cell culture and fluorescence microscopy imaging
Scheme 2
Synthesis of RD1.
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7402 Cells were cultured in DMEM supplemented with 10% FCS, penicillin (100 μg mL−1), and streptomycin (100 μg mL−1) at 37 °C under a humidified atmosphere with 5% CO2 and 95% air.45 Cytotoxicity assays show that RD1 is safe enough for
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confocal microscopy at low concentrations, so that the cells were incubated with 15 μM RD1 at 37 °C under 5% CO2 for 30 min, washed once and bathed in DMEM containing no FCS prior to imaging. Then 30 μM Fe3+ was added into the growth medium for 0.5 h at 37 °C, and washed 3 times with PBS buffer. Then, the cells were imaged on a Zeiss LSM 510 Meta NLO confocal microscope.
Results and discussion As shown in Scheme 2, RD1 was easily synthesized via three steps from the readily available rhodamine B with an overall yield of 40.5%. The structures of RD1 and the intermediates were all confirmed by 1H NMR and 13C NMR. The single crystal of RD1 was grown from diethyl ether–petroleum ether solution. The crystal structure diagrams are shown in Fig. 1. The details of the crystallographic data are listed in Table 1. UV-vis and fluorescence spectra responses
Paper Table 1
X-ray crystallography data for RD1
Compound
RD1
Chemical formula Formula mass Crystal system Space group a/Å b/Å c/Å α/° β/° γ/° Unit cell volume/Å Temperature/K Z Number of reflections measured Number of independent reflections Rint Final R1 values (I > 2σ(I)) Final wR(F 2) values (I > 2σ(I)) Final R1 values (all data) Final wR(F 2) values (all data) Goodness of fit on F 2
C36H40N6O3 604.74 Triclinic ˉ P1 12.0273(5) 12.0667(4) 12.6247(4) 100.282(3) 110.275(3) 102.003(3) 1617.22(10) 290 2 16 241 5147 0.0206 0.0450 0.1246 0.0482 0.1282 1.025
Absorption and fluorescence spectra were performed in methanol–water (1/99) buffer ( pH 7.0, 20 mM HEPES, 50 mM NaNO3). As shown in Fig. 2, the solution of RD1 in water was colorless and exhibited no absorption above 500 nm in a UV-vis spectrum, which was ascribed to the spirolactam form of RD1. On addition of Fe3+, the solution turned from colorless to deep red immediately (in less than 1 second) (Fig. 2 inset). A new and strong absorption centered at 568 nm appeared and was enhanced with the addition of Fe3+, suggesting that the ring-open formation of RD1 should result from Fe3+ binding. Such a dramatic color change ensured the RD1 as a sensitive “turn-on” and colorimetric probe for Fe3+. RD1 showed almost no fluorescent emission upon excitation at 530 nm. Upon the addition of Fe3+, the fluorescence
Fig. 2 The UV-vis absorption titration spectra of RD1 (10 μM) with Fe3+ (0–2.4 equiv.) in methanol–water (1/99) buffer ( pH 7.0, 20 mM HEPES, 50 mM NaNO3). Inset: digital photographs of RD1 and RD1 in the presence of equivalent amount of Fe3+ under normal light.
Fig. 1 Molecular structure of RD1 (ORTEP drawings of RD1 showing thermal ellipsoids at 30% probability level). Selected bond distances [Å] and angles [°]: O2–C28 1.2171(19), O3–C29 1.210(2), N3–C28 1.3813(17), N3–C7 1.4976(18), N4–C29 1.359(2), N5–C30 1.446(3), N5–C31 1.466(3), N6–C36 1.337(4), N6–C32 1.339(3), C28–N3–N4 122.59(12), N4–N3–C7 120.82(11), C29–N4–N3 120.09(13), N3–N4–H4 118.6(13), C30–N5–H5 105.0(16), C31–N5–H5 107.4(16), N5–C31–C32 114.38(16), N6–C32–C31 115.0(2).
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intensity at 586 nm was enhanced dramatically (Fig. 3). The emission intensity reached its maximum after the addition of 2.2 equivalent of Fe3+, and the intensity increased about 40-fold. According to the reported method,46,47 the association constant (Ka) of RD1 for Fe3+ was calculated to be 7.52 × 104 M−1. The quantum yield of RD1 displayed the enhancement of ca. 10-fold (from Φfree = 0.026 to ΦFe3+ = 0.273) upon binding to Fe3+. Job’s plot analysis indicates a 1 : 1 binding model between the probe and Fe3+ (Fig. S1 ESI†). Another solid evidence for the 1 : 1 binding model between the probe and Fe3+ came from the MALDI-TOF MS analysis. As shown in Fig. 4, RD1 has a peak at m/z = 605.46. Upon the addition of Fe3+, a peak at m/z = 696.47 ([RD1 + FeCl]2+) appeared along with the disappearance of the peak at m/z = 605.46. The observations indicated that RD1 bound with Fe3+ in a 1 : 1 binding
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Fig. 3 Fluorescence emission spectra of RD1 (10 μM) with the excitation at 530 nm, upon the titration of Fe3+ (0–2.4 equiv.) in methanol– water (1/99) buffer ( pH 7.0, 20 mM HEPES, 50 mM NaNO3). Inset: changes of emission intensity at 586 nm (λex = 530 nm).
Fig. 5 (a) Fluorescence spectra of RD1 (10 μM) upon addition of Fe3+ and other metal ions (λex = 530 nm). (b) Fluorescence intensity at 586 nm of RD1 (10 μM) upon addition of various metal ions (black bars: RD1 with other metals, red: RD1 with Fe3+ and other metals). Experimental conditions: methanol–water (1/99) buffer ( pH 7.0, 20 mM HEPES, 50 mM NaNO3), 0.40 mM of K+(1), Ca2+(2), Mg2+(3) and 20 μM for Co2+(4), Ni2+(5), Fe2+(6), Cd2+(7), Mn2+(8), Pb2+(9), Zn2+(10), Cu2+(11), Hg2+(12), Cr3+(13), Fe3+(14) (λex = 530 nm).
Fig. 4 (a) MALDI-TOF MS spectrum of RD1 (DCTB as a matrix). (b) MALDI-TOF MS spectrum of RD1-Fe3+ complex (DCTB as a matrix).
stoichiometry. When EDTA was added to the solution of the complex RD1-Fe3+, the fluorescence was switched off gradually. Further excess addition of Fe3+ could recover the fluorescence of the solution (Fig. S2 ESI†). These results indicated that the fluorescent response of RD1 to Fe3+ was caused by the spiroring opening of the rhodamine B group. Ions selectivity and pH stability Metal ion selectivity studies were performed in HEPES buffer. As shown in Fig. 5, K+, Ca2+ and Mg2+, which are abundant in living cells, exerted a negligible effect on the fluorescence of RD1 even at higher concentrations (40 times of the Fe3+). The data also show that Mg2+, Pb2+, Zn2+, Hg2+, Co2+, Cd2+, Mn2+,
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Fig. 6 Top: color of RD1 with different metal ions. Bottom: fluorescence (λex = 365 nm) change upon addition of different metal ions.
Hg2+, Fe2+, Ni2+ and Cu2+ caused negligible response to the fluorescence of RD1, while Cr3+ has a slight effect. The color change of RD1 with different metal ions is shown in Fig. 6. Among the metal ions tested, only Fe3+ caused dramatic changes both in color and in fluorescence. Therefore, RD1 can serve as a colorimetric and “turn-on” fluorescent probe for Fe3+ with remarkable selectivity.
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Furthermore, pH effect on fluorescence of RD1 was investigated in water. As shown in Fig. S3 (ESI†), the solution showed a strong and apparent fluorescence band at 586 nm when the pH value is lower than 6 and increased with the decrease of the solution pH, which signified the ring opening of spirolactam in RD1. Fortunately, in the biological relevant pH range (e.g. 6–9), RD1 always kept non-fluorescent, indicating that the probe could be used under common environmental and physiological conditions.
Cell cytotoxicity and fluorescence microscopy imaging Cell cytotoxicity assays were conducted in 7402 cells to test the cytotoxicity of RD1. As shown in Fig. S4 (ESI†), MTT assay demonstrated that the cell viability remains more than 90% after treating with 10 μM RD1 for 24 h. The result indicated that RD1 has almost no cytotoxicity for long term incubation at low concentration and should be safe when used for fluorescence microscopy imaging. With the above data in hand, we then applied RD1 in the cell imaging of Fe3+ by laser scanning confocal microscopy. 7402 Cells were cultured as the sample, and stained with RD1 within 30 min and washed by PBS buffer, no fluorescence was detected (Fig. 7a). After further incubation with FeCl3 for another 2.5 h at 37 °C, a red fluorescence appeared from the intracellular region (Fig. 7b). A bright field image of 7402 cells treated with RD1 and Fe3+ confirmed that the cells were viable throughout the imaging experiments (Fig. 7c). The results indicated that the RD1 was cell permeable and could be used for imaging of Fe3+ in living cells.
Fig. 7 (a) Fluorescence image of 7402 cells labeled with 15 μM RD1 after 30 min at 37 °C of incubation, washed with PBS buffer. (b) Fluorescence image of 7402 cells labeled treatment with RD1 and then 30 μM FeCl3 aqueous solution for 2.5 h at 37 °C. (c) Bright-field image of 7402 cells. (d) The overlay of (a)–(c).
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Conclusion In summary, we have developed a new rhodamine-based fluorescent probe RD1 which showed real-time and “turn-on” fluorescent and colorimetric responses to Fe3+ with a high selectivity in the aqueous solution. Compared with rhodamine-based probes for Fe3+ previously reported, the amount of organic cosolvent in detecting media was greatly reduced to less than 1%. In addition, the confocal fluorescence imaging confirmed that RD1 is cell permeable and can be used for monitoring intracellular Fe3+ in living cells with low cytotoxicity. This work was supported by NSFC (21102001, 21102002, 21372005 and 21272223), Natural Science Foundation of Education Department of Anhui Province (KJ2010A028, KJ2011A018), 211 Project of Anhui University, and Doctor Research Start-up Fund of Anhui University for supporting the research.
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