A BODIPY-based colorimetric and fluorometric chemosensor for Hg(II) ions and its application to living cell imaging

Biomolecular

Chemistry

Cite this: Org. Biomol. Chem., 2012, 10, 5410

www.rsc.org/obc

PAPER

A BODIPY-based colorimetric and

fluorometric chemosensor for Hg(

II

) ions

and its application to living cell imaging

†

Mani Vedamalai and Shu-Pao Wu*

Received 20th March 2012, Accepted 30th May 2012 DOI: 10.1039/c2ob25589h

A new monostyryl boron dipyrromethene derivative (MS1) appended with two triazole units indicates the presence of Hg2+among other metal ions with high selectivity by color change and red emission. Upon Hg2+binding, the absorption band of MS1 is blue-shifted by 29 nm due to the inhibition of the

intramolecular charge transfer from the nitrogen to the BODIPY, resulting in a color change from blue to purple. Significant fluorescence enhancement is observed with MS1 in the presence of Hg2+; the metal ions Ag+, Ca2+, Cd2+, Co2+, Cu2+, Fe2+, Fe3+, K+, Mg2+, Mn2+, Ni2+, Pb2+, and Zn2+cause only minor changes in thefluorescence of the system. The apparent association constant (Ka) of Hg2+binding in

MS1 is found to be 1.864 × 105M−1. In addition,fluorescence microscopy experiments show that MS1 can be used as afluorescent probe for detecting Hg2+in living cells.

Introduction

The development of chemosensors for detecting biologically and environmentally important metal ions, such as Cu2+, Zn2+, Hg2+, and Pb2+, has attracted much attention. Mercury is one of the most toxic heavy metal elements and exists in three forms: elemental, inorganic, and organic mercury. Mercury ions have high affinity for thiol groups in proteins, leading to the malfunc-tion of cells and consequently causing many health problems in the brain, kidney, and central nervous system. Its accumulation in the body results in a wide variety of diseases, such as prenatal brain damage; serious cognitive and motion disorders; and Minamata disease.1In order to detect mercury ions in biological and environmental samples, the design of highly selective and sensitive mercury sensors has been an important issue.

In general, several traditional methods2 for the detection of mercury ions in various samples have been developed, including atomic absorption–emission spectroscopy,3 inductively coupled plasma mass spectroscopy (ICPMS),4 and inductively coupled plasma–atomic emission spectrometry (ICP-AES).5 Although these methods are quantitative, most of these methods require expensive instruments and are not good for on-site analysis. Recently, more attention has been focused on the development offluorescent chemosensors for the detection of Hg2+ions.6

Numerous molecular probes using different receptors and fluo-rescent units have been developed for Hg2+ detection. Because Hg2+ is known as a fluorescence quencher due to spin–orbit coupling,7mostfluorescent chemosensors detect Hg2+through a fluorescence quenching. Due to sensitivity concerns, fluorescent chemosensors detecting metal ions using fluorescence enhance-ment are more easily monitored than those using fluorescence quenching. This paper reports on a newly designed monostyryl boron dipyrromethene (BODIPY) based fluorescent enhance-ment Hg2+chemosensor, based on intramolecular charge transfer (ICT). When Hg2+ binds to the chemosensor, it blocks the ICT mechanism, giving rise to a color change and fluorescence enhancement of BODIPY.

In this study, a monostyryl BODIPY-basedfluorescent chemo-sensor (MS1) containing two triazole units was designed for metal ion detection (Scheme 1). MS1 was blue and exhibits weak fluorescence. Binding metal ions to the chemosensor blocks the ICT mechanism and results in a color change and fluo-rescence enhancement of BODIPY. The metal ions Ag+, Ca2+, Cd2+, Co2+, Cu2+, Fe2+, Fe3+, Hg2+, K+, Mg2+, Mn2+, Ni2+, Pb2+, and Zn2+were tested for metal ion binding selectivity with MS1, but Hg2+was the only ion that caused a red emission upon binding with MS1. The fluorescence microscopy experiments also demonstrated that MS1 can be used as afluorescent probe for detecting Hg2+in living cells.

Result and discussion

Synthesis of MS1

The synthesis of the fluorescent probe, MS1, is outlined in Scheme 1. Mono formylated dipyrromethane (1) was

†Electronic supplementary information (ESI) available: 1

H and 13C NMR spectra of compounds 2, 3, 4, 5, and MS1; ESI-MS of MS1– Hg2+. See DOI: 10.1039/c2ob25589h

Department of Applied Chemistry, National Chiao Tung University, Hsinchu, Taiwan, Republic of China. E-mail: [email protected]; Fax: +886-3-5723764; Tel: +886-3-5712121-ext56506

synthesized according to the procedure found in the literature.9 Compound 2 was obtained by a Wittig reaction of (4-nitro-benzyl)triphenyl phosphonium bromide and mono formylated dipyrromethane to form a double bond between pyrrole and nitrobenzene. In the next step, compound 2 was transformed into a BODIPY skeleton by a stepwise reaction;first, dipyrromethane was oxidized to form dipyrromethene by DDQ, followed by dipyrromethene conversion into a BODIPY in the presence of boron trifluoride. Further reduction of compound 3 using iron powder gave compound 4. The reaction of compound 4 with propargyl bromide in the presence of potassium carbonate yielded compound 5. MS1 was obtained by treatment of com-pound 5 with picolyl azide under click chemistry conditions. The absorption spectrum of MS1 displays an absorption peak centered at 606 nm with a molar extinction coefficient of 6.2 × 104 M−1 cm−1. The absorption maximum of MS1 has about a 100 nm red shift in comparison to that of the standard

BODIPY dye.8 This red shift was assigned to a substitution of an amino styryl group at the“3” position of the BODIPY group.

Cation sensing selectivity

The sensing ability of MS1 was tested by mixing it with metal ions Ag+, Ca2+, Cd2+, Co2+, Cu2+, Fe2+, Fe3+, Hg2+, K+, Mg2+, Mn2+, Ni2+, Pb2+, and Zn2+. Qualitatively, Hg2+was the only ion that caused a visible color change (from blue to purple) and red fluorescence from MS1 (Fig. 1). Other metal ions led to no sig-nificant change in the fluorescence of MS1. Quantitative absorp-tion andfluorescence spectra of MS1 were taken in the presence of several transition metal ions. Hg2+was the only metal ion that caused a significant red emission (Fig. 2). During Hg2+titration with MS1, the absorption band at 606 nm was shifted to 577 nm (Fig. 2). This caused a visible color change from blue to purple.

Scheme 1 Synthesis of MS1.

During Hg2+ titration with MS1, a new emission band centered at 650 nm formed (Fig. 2). After adding 15 equivalents of Hg2+, the quantum yield of the emission band wasΦ = 0.327, which is 65 fold higher than that of MS1, withΦ = 0.005. These obser-vations indicate that Hg2+is the only metal ion that readily binds with MS1, causing significant fluorescence enhancement and permitting highly selective detection of Hg2+.

To study the influence of other metal ions on Hg2+ binding with MS1, we performed competitive experiments in the pres-ence of Hg2+ (60μM) with other metal ions (150 μM) (Fig. 3).

Fluorescence enhancement caused by the mixture of Hg2+ with most metal ions was similar to that caused by Hg2+ alone. A smaller fluorescence enhancement was observed when Hg2+ was mixed with Co2+or Fe3+. This indicates that only Co2+and Fe3+ compete with Hg2+ for binding with MS1. Most of the other metal ions do not interfere with the binding of MS1 with Hg2+.

In order to understand the binding stoichiometry of MS1– Hg2+ complexes, Job plot experiments were carried out. In Fig. 4, the emission intensity at 650 nm was plotted as a function of the mole fraction of MS1 under a constant total concentration. Maximum emission intensity was reached when the mole frac-tion was 0.5. These results indicate a 1 : 1 ratio for MS1–Hg2+ complexes, in which one Hg2+ ion was bound with one MS1. Further, the formation of 1 : 1 MS1–Hg2+ complex was confirmed using ESI-MS in which the peak at m/z 929.9 indi-cates a 1 : 1 stoichiometry for MS1–Hg2+ complexes (see Fig. S11 in ESI†). The apparent association constant was calcu-lated from Fig. 5 by using nonlinear regression analysis and was found to be 1.864 × 105 M−1. The detection limit of MS1 as a

Fig. 2 Absorption (top) and emission (bottom) changes of chemo-sensor MS1 (4μM) in the presence of various equivalents of Hg2+in acetonitrile–water (v/v = 9 : 1, 2.5 mM Hepes, pH 7.0) solutions. Fig. 1 Colorimetric change (top) andfluorescence change (bottom) of MS1 (4μM) with 60 μM of individual cations.

Fig. 3 Fluorescence response of MS1 (4μM) to the addition of Hg2+ (60μM) or 150 μM of other metal ions (black bars) and to the mixture of other metal ions (150μM) with 60 μM of Hg2+(gray bars) in aceto-nitrile–water (v/v = 9/1, 2.5 mM Hepes, pH 7.0) solutions. The excitation wavelength is 550 nm.

Fig. 4 Job plot of Hg2+_{–MS1 complexes in acetonitrile–water}

(v/v = 9 : 1, 2.5 mM Hepes, pH 7.0) solutions. The monitored wavelength was 650 nm. The total concentration of the sensor and Hg2+ion was 8μM.

fluorescent sensor for the analysis of Hg2+_{was determined from}

the variation offluorescence intensity as a function of the con-centration of Hg2+(see Fig. S12 in the ESI†). It was found that MS1 has a detection limit of 0.226 μM, which allows micro-molar concentrations of Hg2+to be detected.

A pH titration of MS1 was performed to investigate a suitable pH range for Hg2+sensing. As depicted in Fig. 6, the emission intensities of metal-free MS1 were very low. After mixing MS1 with Hg2+, the emission intensity at 650 nm remained a maximum in the pH range of 3.0–7.0. Above pH 7.5, the emission intensity decreased. This indicates poor stability of the MS1–Hg2+complexes at high pH values.

To gain a clearer understanding of the structure of MS1–Hg2+ complexes,1H NMR spectroscopy (Fig. 7) was employed. Hg2+ is a heavy metal ion and can affect the proton signals that are close to Hg2+ binding.9 In the 1H NMR spectra of MS1, the proton (Hl, triazole) signal at 7.75 ppm showed down-field shifts

between Hg2+ and the pyridines. These observations revealed that Hg2+ binding with MS1 was mainly through one amine at the phenyl ring and two nitrogens at two triazole units. Hg2+ also had weak interactions with two nitrogens at pyridine moieties.

Living cell imaging

MS1 was also applied to living cell imaging. For the detection of Hg2+in living cells, HeLa cells were cultured in DMEM sup-plemented with 10% FBS at 37 °C and 5% CO2. Cells were

plated on 14 mm glass coverslips and allowed to adhere for 24 hours. HeLa cells were treated with 2 μM Hg(BF₄)₂ for

30 min and washed with PBS for three times. Then cells were incubated with MS1 (2μM) for 30 min and washed with PBS to remove the remaining sensor. The images of the HeLa cells were obtained using a fluorescence microscope. Fig. 8 shows the images of HeLa cells with MS1 after the treatment of Hg2+. The overlay of fluorescence and bright-field images reveal that the fluorescence signals are localized in the intracellular area, indicating a subcellular distribution of Hg2+ and good cell-membrane permeability of MS1.

Conclusions

In summary, the newfluorescence chemosensor MS1 exhibits a high affinity and selectivity for Hg2+ions over competing metal ions. Fluorescence was significantly enhanced by chemosensor MS1 in the presence of Hg2+, and the addition of Ag+, Ca2+, Cd2+, Co2+, Cu2+, Fe2+, Fe3+, K+, Mg2+, Mn2+, Ni2+, Pb2+, or Zn2+barely affected thefluorescence. This BODIPY-based Hg2+ chemosensor also provides an effective method of Hg2+sensing in living cell imaging.

Experimental section

General

All reagents were obtained from commercial sources and used as received without further purification. UV-vis spectra were recorded on an Agilent 8453 UV-vis spectrometer. Fluorescence spectra were recorded in a Hitachi F-4500 spectrometer.1H and

13_{C NMR spectra were recorded on a Bruker DRX-300 NMR}

Spectrometer, Varian AS500 Unity Innova Spectrometer and Varian VNMRS 600 NMR Spectrometer.

Fig. 6 Fluorescence intensity (650 nm) of MS1 (4μM) (■), and after addition of Hg2+ (60 μM) ( ) in an acetonitrile–water (v/v = 9 : 1, 2.5 mM buffer) solution as a function of different pH values. The exci-tation wavelength was 550 nm.

Fig. 5 Benesi-Hildebrand plot of the Hg2+–MS1 complexes in aceto-nitrile–water (v/v = 9 : 1, 2.5 mM Hepes, pH 7.0) solutions. The moni-tored emission wavelength was 650 nm.

Synthesis

Synthesis of 1-formyl-5-phenyldippyromethane (1). Com-pound 1 was obtained in modest yield by treating 5-phenyl-dipyrromethane with benzoyl chloride and DMF under dry N2.10

Synthesis of 1-[2-(4-Nitro-phenyl)-vinyl]-5-phenyl-4,6-dipyrro-methane (2). Potassium tert-butoxide (281 mg, 2.5 mmol) was added to a solution of (4-nitrobenzyl)triphenyl phos-phonium bromide (1.002 g, 2.1 mmol) in dry THF (30 mL). The

solution was stirred at room temperature for 30 min. Compound 1 (500.2 mg, 2 mmol) dissolved in dry THF (10 mL) was added dropwise to the mixture. The reaction mixture was heated at 66 °C for 12 h. Then solvents were removed under reduced pressure, and the crude product was purified by on column chromatography (hexane–ethyl acetate, 5 : 1) to give a compound 2 as a red solid. Yield: 70%, 517 mg. Melting point 163–164 °C.

1_{H NMR (CD}

3OD):δ = 8.14 (d, J = 9 Hz, 2H), 7.57 (d, J =

9 Hz, 2H), 7.17–7.31 (m, 6H), 6.80 (d, J = 16.5 Hz, 1H), 6.67 (dd, J = 1.5 Hz, 2.7 Hz, 1H), 6.33 (d, J = 3.6 Hz, 1H), 6.00

Fig. 7 1H NMR spectra of MS1 (5 mM) in the presence of different concentrations of Hg2+in CD3CN.

(t, J = 3.0 Hz, 1H), 5.75 (d, J = 3.3 Hz, 1H), 5.73 (dd, J = 1.8 Hz, 2.1 Hz, 1H), 5.45 (s, 1H). 13C NMR (CD3OD): δ =

146.9, 146.7, 144.2, 139.0, 133.9, 131.4, 129.6, 129.2, 127.5, 126.7, 125.3, 125.0, 120.2, 118.2, 112.8, 110.3, 108.1, 107.8, 45.5. MS(FAB): m/z = 369. HRMS (FAB): calcd for C23H19N3O4: 369.1477; found 369.1481.

Synthesis of compound 3. 2,3-Dichloro-5,6-dicyano-1,4-benzo-quinone (DDQ; 318 mg, 1.4 mmol) dissolved in CH2Cl2

(50 mL) was added to a solution of compound 2 (443 mg, 1.2 mmol) in CH2Cl2(100 mL) under nitrogen, and the mixture

was stirred for 1 h. It was then treated with Et3N (3.0 mL) and

BF3·OEt2 (4.0 mL) for 3 h. The solvent was evaporated under

reduced pressure, and the crude product was purified by column chromatography (ethyl acetate–hexane, 1 : 10) to give compound 3 as a pink solid. Yield 78%, 388.6 mg. Melting point 265–266 °C.1H NMR (CD2Cl2): δ = 8.24 (d, J = 8.7 Hz, 2H), 7.87 (s, 1H), 7.83 (d, J = 16.8 Hz, 1H), 7.76 (d, J = 9 Hz, 2H), 7.52–7.61 (m, 5H), 7.43 (d, J = 16.5 Hz, 1H), 7.04 (d, J = 4.5 Hz, 1H), 7.00 (d, J = 4.8 Hz, 1H), 6.90 (d, J = 3.9 Hz, 1H), 6.58 (d, J = 2.1 Hz, 1H).13C NMR (CD2Cl2):δ = 155.6, 148.1, 148.0, 145.0, 142.7, 142.6, 137.4, 135.2, 134.2, 132.5, 130.95, 130.90, 130.3, 128.8, 128.4, 124.5, 123.0, 118.5, 117.8. MS (EI): m/z (%) = 415 (100.0), 414 (30.1), 349 (8.4), 347 (10.4), 291 (5.1), 174 (4.8); HRMS (EI): calcd for C23H16BF2N3O4

415.1304; found: 415.1303.

Synthesis of compound 4. Iron powder (803.5 mg, 14.4 mmol) and water (4 mL) were added to a solution of com-pound 3 (373.6 mg, 0.9 mmol) in methanol (12 mL). It was then treated with HCl in methanol (6 mL, 0.5 mol L−1). The reaction mixture was heated at 80 °C for 6 h. The reaction mixture was cooled to room temperature, and concentrated at reduced pressure. The crude product was purified by column chromato-graphy (ethyl acetate–hexane, 1 : 3) to give a blue solid. Yield 80%, 277.3 mg). Melting point 238–239 °C. 1H NMR (CD2Cl2): δ = 7.69 (s, 1H), 7.48–7.57 (m, 6H), 7.47 (d, J =

Synthesis of compound 5. Propargyl bromide (0.174 mL, 80% solution in toluene, 1.6 mmol) and potassium carbonate (276.4 mg, 2 mmol) were added to a solution of compound 4 (269.6 mg, 0.7 mmol) in acetone (5 mL). The reaction mixture was refluxed for two days. The solvent was evaporated under reduced pressure, and the crude product was purified by column chromatography (ethyl acetate–hexane, 1 : 5) to give compound 5 as a violet solid. Yield 87%, 281.5 mg. Melting point 156–157 °C. 1H NMR (CD3CN): δ = 7.76 (s, 1H), 7.49–7.67 (m, 9H), 7.16 (d, J = 4.8 Hz, 1H), 7.05 (d, J = 8.0 Hz, 1H), 7.01 (d, J = 9.0 Hz, 2H), 6.76 (d, J = 3.9 Hz, 1H), 6.53 (dd, J = 2.1 Hz, 3.9 Hz, 1H), 4.25 (d, J = 2.4 Hz, 4H), 2.56 (t, J = 2.4 Hz, 2H).13C NMR (CD3CN):δ = 160.4, 149.7, 142.0, 141.8, 139.0, 137.9, 134.9, 134.5, 133.9, 131.4, 131.1, 130.3, 129.3, 127.2, 119.5, 118.3, 117.3, 115.4, 115.3, 79.9, 74.0, 40.8. MS (ESI): m/z = 462.1 [M + H]+; HRMS (ESI): calcd C29H22BF2N3 [M + H]+462.1953; found 462.1944.

Synthesis of MS1. Picolyl azide (160.9 mg, 1.2 mmol), CuSO4·5H2O, (15.0 mg, 10 mol%), and sodium ascorbate

(30.0 mg, 20 mol%) were added to a solution of compound 5 (277.3 mg, 0.6 mmol) in THF–H2O (7 : 3, v/v; 15 mL) under

nitrogen. The solution was stirred at room temperature for 12 h. A saturated ammonium chloride solution (20 mL) was added to the reaction mixture, and the organic phase was extracted with dichloromethane (100 mL, 3×). The combined organic extracts were dried with anhydrous MgSO4. The solvent was evaporated

under reduced pressure, and the crude product was purified by column chromatography (dichloromethane–methanol, 20 : 1) to give compound MS1 as a dark violet solid. Yield 71%, 311.1 mg. Melting point 94–95 °C.1H NMR (CD3CN):δ = 8.50 (d, J = 5.0 Hz, 2H), 7.76 (s, 2H), 7.69–7.72 (m, 3H), 7.40–7.59 (m, 9H), 7.26 (dd, J = 5.0 Hz, 7.0 Hz, 2H), 7.14 (d, J = 8.0 Hz, 2H), 7.06 (d, J = 5.0 Hz, 1H), 6.93–6.96 (m, 3H), 6.68 (d, J = 4.0 Hz, 1H), 6.48 (dd, J = 2.5 Hz, 3.8 Hz, 1H), 5.47 (s, 4H), 4.75 (s, 4H). 13C NMR (CD3CN): δ = 161.6, 156.6, 151.4, 151.2, 146.2, 143.2, 141.9, 139.0, 138.8, 138.7, 135.7, 135.1, 134.6, 132.1, 131.7, 131.3, 130.0, 127.2, 126.2, 125.2, 124.8, 123.7, 120.3, 118.9, 117.7, 114.9, 56.6, 47.6. MS (ESI): m/z = 730.2 [M + H]+. HRMS (ESI): calcd for C41H34BF2N11

[M + H]+730.3138; found 730.3146.

Determination of the binding stoichiometry and the

apparent dissociation constants for the binding of

Hg(

) to MS1

The binding stoichiometry of MS1–Hg2+ complexes was deter-mined from a Job plot.11 The fluorescence intensity at 650 nm

Fig. 8 Hg2+-treated HeLa cell images. (Top left) Brightfield image; (Top right)fluorescence image; and (Bottom) merged image.

was plotted against the molar fraction of MS1 with a total con-centration of the sensor and Hg2+ion of 8.0μM. The molar frac-tion at maximum emission intensity represents the binding stoichiometry of the MS1–Hg2+ complexes. The maximum emission intensity was reached at a molar fraction of 0.5 (In Fig. 4). This result indicates that chemosensor MS1 forms a 1 : 1 complex with Hg2+. The apparent association constant (Ka) of

MS1–Hg2+complexes was determined by the Benesi-Hildebrand eqn (1)12,13

1=ðF  F0Þ ¼ 1=fKa ðFmax F0Þ  ½Hg2þg

þ 1=ðFmax F0Þ; ð1Þ

where F is the fluorescence intensity at 650 nm at any given Hg2+ concentration, F0 is the fluorescence intensity at 650 nm

in the absence of Hg2+, and Fmax is the maxima fluorescence

intensity at 650 nm in the presence of Hg2+ in solution. The association constant Kawas evaluated graphically by

plot-ting 1/(F − F0) against 1/[Hg2+]. Data were linearly fitted

according to eqn (1) and the Ka value was obtained from the

slope and intercept of the line.

Cell culture

The cell line HeLa was provided by the Food Industry Research and Development Institute (Taiwan). The HeLa cells were grown in DMEM (Dulbecco’s modified Eagle’s medium) supplemented with 10% FBS (fetal bovine serum) at 37 °C and 5% CO2. Cells

were plated on 14 mm glass coverslips and allowed to adhere for 24 hours.

Fluorescence imaging

Experiments to assess Hg2+uptake were performed in PBS with 2 μM Hg(ClO4)2. Treat the cells with 4 μL of 1 mM metal

ions (final concentration: 2 μM) dissolved in sterilized PBS ( pH 7.4) and incubated for 30 min at 37 °C. The treated cells was washed PBS (3 × 2 mL) to remove remaining metal ions. Culture media (2 mL) was added to the cell culture, which was treated with a 10 mM solution of chemosensor MS1 (4μL; final concentration: 2 μM) dissolved in DMSO. The samples were incubated at 37 °C for 30 min. The culture media was removed, and the treated cells were washed with PBS (3 × 2 mL) before observation. Fluorescence imaging was performed with a ZEISS Axio Scope A1 Fluorescence Microscope. Cells loaded with MS1 were excited at 545 nm using a lamp (Hg 50 W). Emission Filter was 570 nm.

Acknowledgements

We gratefully acknowledge thefinancial support of the National Science Council (ROC) and National Chiao Tung University.

Notes and references

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