A BODIPY-Based Highly Selective Fluorescent Chemosensor for Hg2+ Ions and Its Application in Living Cell Imaging

DOI: 10.1002/ejoc.201101623

A BODIPY-Based Highly Selective Fluorescent Chemosensor for Hg

2+

_Ions

and Its Application in Living Cell Imaging

Mani Vedamalai

[a]

_{and Shu-Pao Wu*}

[a]

Keywords: Sensors / Mercury / Fluorescence / Imaging agents

A new boron–dipyrromethene (BODIPY) derivative (FS1) containing two triazole units exhibits an enhanced fluores-cence in the presence of Hg2+_{ions and a high selectivity for}

Hg2+_{ions over competing metal ions in methanol: Ag}+_{, Ca}2+_,

Cd2+_{, Co}2+_{, Cu}2+_{, Fe}2+_{, Fe}3+_{, K}+_{, Mg}2+_{, Mn}2+_{, Ni}2+_{, Pb}2+_{, and}

Introduction

Mercury is one of the most toxic heavy metal elements.

[1]

It exists in three different forms: Inorganic mercury,

alkyl-mercury, and elemental mercury. Mercury contamination

occurs through various processes, such as the combustion

of fossil fuels, mining, and solid-waste incineration.

Mer-cury ions show a high affinity for thiol groups in proteins,

leading to the malfunction of cells and consequently

caus-ing many health problems in the brain, kidney, and central

nervous system. Its accumulation in the body can

contrib-ute to the development of a wide variety of diseases, such

as prenatal brain damage, serious cognitive and motion

dis-orders, and Minamata disease.

[2]

_{Owing to the extreme}

tox-icity of mercury, the United States Environmental

Protec-tion Agency (EPA) established the standard for the

maxi-mum allowed level of mercury in dietary and environmental

sources to be 2 ppb (10 n

m).

Numerous methods

[3]

_{for the detection of mercury ions}

in various samples have been proposed, including atomic

absorption/emission spectroscopy,

[4]

_{inductively coupled}

plasma-mass spectroscopy (ICPMS),

[5]

_{inductively coupled}

plasma-atomic emission spectrometry (ICP-AES),

[6]

_and

voltammetry.

[7]

_{Most of these methods require expensive}

in-struments and are not suitable for performing assays. Over

the past decade more attention has been focused on the

development of fluorescent chemosensors for the detection

of Hg

2+

_ions.

[8]

[a] Department of Applied Chemistry, National Chiao Tung University,

Hsinchu, Taiwan, Republic of China Fax: +886-3-5723764

E-mail: [email protected]

Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/ejoc.201101623.

Zn2+ _{produced only minor changes in the fluorescence of}

FS1. The apparent dissociation constant (Kd) of FS1–Hg2+

was found to be 62μM. Moreover, fluorescence microscopy experiments showed that FS1 can be used as a fluorescent probe for detecting Hg2+_{ions in living cells.}

Because Hg

2+

_{is known to be a fluorescence quencher,}

most fluorescent chemosensors detect Hg

2+

_{by fluorescence}

quenching through spin–orbit coupling.

[9]

_{Owing to their}

sensitivity, fluorescent chemosensors that detect metal ions

by fluorescence enhancement are more easily monitored

than those that operate by fluorescence quenching. This

pa-per reports on a newly designed BODIPY-based fluorescent

enhancement chemosensor for Hg

2+

_{based on}

photoin-duced electron transfer (PET). The binding of Hg

2+

_{to the}

chemosensor blocks the PET mechanism and greatly

en-hances the fluorescence of BODIPY.

In this work we designed a BODIPY-based fluorescent

chemosensor for metal-ion detection. Two parts make up

the chemosensor FS1: A BODIPY moiety as reporter and

two triazole units that chelate the metal ion (Scheme 1).

FS1 exhibits weak fluorescence due to quenching by

photo-induced electron transfer from the lone-pair electrons on

the nitrogen atom attached to the phenyl group. The

bind-ing of metal ions to the chemosensor blocks the PET

mechanism and results in considerable fluorescence

en-hancement of BODIPY. The metal ions Ag

+

_{, Ca}

2+

_{, Cd}

2+

_,

Co

2+

_{, Cu}

2+

_{, Fe}

2+

_{, Fe}

3+

_{, Hg}

2+

_{, K}

+

_{, Mg}

2+

_{, Mn}

2+

_{, Ni}

2+

_{, Pb}

2+

_,

and Zn

2+

_{were tested for metal-ion-binding selectivity with}

FS1, but Hg

2+

_{was the only ion that caused green emission}

upon binding with FS1.

Results and Discussion

Synthesis of FS1

The synthesis of FS1 is outlined in Scheme 1. Aniline

was treated with propargyl bromide in the presence of

K

2

CO

3

to afford compound 1. Compound 2 was obtained

Scheme 1. Synthesis of FS1.

DMF at 80 °C. Treatment of compound 2 with excess

pyrrole in the presence of TFA under nitrogen yielded the

corresponding dipyrromethane 3. In the next step,

com-pound 3 was oxidized with DDQ to yield the corresponding

dipyrromethene, which was transformed into the BODIPY

skeleton 4 in the presence of BF

3

under N

2

. Treatment of

compound 4 with picolyl azide yielded FS1 under click

chemistry conditions. FS1 has an absorbance maximum at

493 nm, assigned to the S

0

씮S

1

transition of the BODIPY

chromophore,

[10]

_{and a molar extinction coefficient of}

3.83

⫻ 10

4

_m

–1

_cm

–1

_{. FS1 displays weak fluorescence with a}

quantum yield of Φ = 0.002, because photoinduced electron

transfer from the aromatic amine group to the BODIPY

moiety takes place.

Figure 1. Color (top) and fluorescence (bottom) changes in FS1 (30 μm) upon the addition of various metal ions (60 μm) in methanol.

Cation-Sensing Selectivity

The sensing ability of FS1 was tested by mixing it with

the metal ions Ag

+

_{, Ca}

2+

_{, Cd}

2+

_{, Co}

2+

_{, Cu}

2+

_{, Fe}

2+

_{, Fe}

3+

_,

Hg

2+

_{, K}

+

_{, Mg}

2+

_{, Mn}

2+

_{, Ni}

2+

_{, Pb}

2+

_{, and Zn}

2+

_.

Qualita-tively, Hg

2+

_{was the only ion that caused a change in color}

(from red to yellow) of the FS1 solution and green

fluores-cence from FS1 (Figure 1). Other metal ions had no

signifi-cant effect on the fluorescence of FS1. Quantitative

fluores-cence spectra of FS1 were recorded in the presence of

sev-eral transition-metal ions. Hg

2+

_{was the only metal ion that}

caused significant green emission (Figure 2). During the

ti-tration of FS1 against Hg

2+

_{, a new emission band centered}

at 520 nm was observed (Figure 3). After the addition of

4 equiv. of Hg

2+

_{, the emission intensity reached a}

maxi-mum. The quantum yield of the emission band was Φ =

0.035, which is 17-fold higher than that of FS1, with Φ =

0.002. These results indicate that Hg

2+

_{is the only metal ion}

of those studied that readily binds to FS1, causing

signifi-cant fluorescence enhancement and permitting the highly

selective detection of Hg

2+

_.

Figure 2. Fluorescence response of FS1 (30 μm) to various metal cations (30 μm) in methanol. The excitation wavelength was 492 nm.

To study the influence of other metal ions on the binding

of Hg

2+

_{to FS1, we performed competitive experiments}

with Hg

2+

_{(150 μ}

_{m) and other metal ions (150 μm;}

mix-Figure 3. Fluorescence response of FS1 (30 μm) to various equiva-lents of Hg2+_{in methanol. The excitation wavelength was 492 nm.}

tures of Hg

2+

_{with most metal ions was similar to that}

caused by Hg

2+

_{alone. Reduced fluorescence enhancement}

was observed when Hg

2+

_{was mixed with Co}

2+

_{or Fe}

3+

_.

This indicates that only Co

2+

_{and Fe}

3+

_{compete with Hg}

2+

for binding with FS1. Most of the other metal ions do not

interfere with the binding of FS1 to Hg

2+

_.

Figure 4. Fluorescence response of FS1 (30 μm) to Hg2+_{(150 μm)}

and other metal ions (150 μm; black bars) and to mixtures of Hg2+

(150 μ_{m) with other metal ions (150 μm; gray bars) in methanol.}

To determine the binding stoichiometry of the FS1–Hg

2+

complex, the emission intensity of FS1 at 520 nm was

plot-ted as a function of the molar fraction of FS1 under a

con-stant total concentration. The resulting Job plot is shown

in Figure 5. The maximum emission intensity was reached

when the molar fraction was 0.5, which indicates a 1:1 ratio

for the FS1–Hg

2+

_{complex, that is, one Hg}

2+

_{ion binds to}

one molecule of FS1. Furthermore, the formation of a 1:1

FS1–Hg

2+

_{complex was confirmed by ESI-MS, in which the}

peak at m/z = 828.1 indicates a 1:1 stoichiometry for the

FS1–Hg

2+

_{complex (see Figure S9 in the Supporting}

Infor-mation). The apparent dissociation constant was calculated

from Figure 3 by using a nonlinear regression analysis; a

value of 62.1

⫾5.7 μm was determined (see Figure S10 in

the Supporting Information). The detection limit of FS1 as

a fluorescent sensor for the analysis of Hg

2+

_was

deter-mined from the variation of fluorescence intensity as a

function of the concentration of Hg

2+

_{(see Figure S12 in}

the Supporting Information). It was found that FS1 has a

detection limit of 2.8 μ

m, which allows micromolar

concen-trations of Hg

2+

_{to be detected.}

Figure 5. Job plot of FS1–Hg2+_{complexes in methanol. The}

solu-tions were monitored at a wavelength of 520 nm. The total concen-tration of the sensor and Hg2+_{ion was 250 μm.}

To gain a clearer understanding of the structure of the

FS1–Hg

2+

_complex,

1

_{H NMR spectroscopy (Figure 6) was}

employed. Hg

2+

_{is a heavy metal ion and can affect the}

proton signals that are close to the Hg

2+

_{binding site.}

[11]

The

1

_{H NMR spectra of FS1 recorded with increasing}

amounts of Hg

2+

_{show that the proton (H}

g

, triazole) signal

at δ = 7.8 ppm is shifted downfield as Hg

2+

_{is added. This}

indicates that Hg

2+

_{binds to FS1 mainly through the}

nitro-gen atom in the triazole ring. The proton signals H

e

and

H

d

are shifted upfield upon addition of Hg

2+

. This also

indicates that Hg

2+

_{binds through the amino group}

at-tached to the phenyl ring. The proton signals from H

i

, H

j

,

H

k

, and H

l

on the pyridine ring are slightly affected by the

binding of Hg

2+

_{. These observations show that Hg}

2+

_binds

to FS1 through an amino group, two nitrogen atoms of two

triazole units, and two pyridine nitrogen atoms.

A pH titration of FS1 was carried out to investigate a

suitable pH range for Hg

2+

_{sensing. As depicted in Figure 7,}

the emission intensities of metal-free FS1 are very low at all

pH values. After mixing FS1 with Hg

2+

_{, the emission}

inten-sity at 520 nm is markedly higher at pH = 5.0 and is a

maxi-mum in the pH range 5.0–10.0. At pH

⬎ 10, the emission

intensity decreases. This indicates poor stability of the FS1–

Hg

2+

_{complexes at high pH values. At pH}

_{⬍ 5, the emission}

intensity is also lower due to the protonation of the amino

groups, which prevents the formation of the FS1–Hg

2+

complex.

Living Cell Imaging

FS1 was also used for living cell imaging. For the

detec-tion of Hg

2+

_{in living cells, HeLa cells were treated with}

20 µ

m Hg(BF

4

)

2

for 30 min and washed with

phosphate-buffered saline (PBS) three times. Then the cells were

incu-bated with FS1 (20 µ

m) for 30 min and washed with PBS to

Figure 6.1_{H NMR spectra of FS1 (5 mm) in the presence of different concentrations of Hg}2+_{in CD} 3CN.

Figure 7. Fluorescence intensity (520 nm) of free FS1 (30 μm; 䊊) and of FS1 after the addition of Hg2+ _{(150 μm; 䊏) in methanol/}

water solution (9:1, v/v; 1 m_{m buffer) as a function of the pH. The} excitation wavelength was 492 nm. Buffer solutions: pH = 1–2, KCl/HCl; pH = 2.5–4, KHP/HCl; pH = 4.5–6, KHP/NaOH; pH = 6.5–12 HEPES.

Figure 8. Hg2+_{-treated HeLa cell images. Top left: bright-field}

remove any remaining sensor. Images of the HeLa cells

were obtained with a fluorescence microscope. Figure 8

shows the images of the HeLa cells with FS1 after treatment

of Hg

2+

_{. The overlapping of the fluorescence and}

bright-field images reveals that the fluorescence signals are

local-ized in the intracellular area, which indicates a subcellular

distribution of Hg

2+

_{and good permeability of the cell}

membrane of FS1.

Conclusions

The new fluorescence chemosensor FS1 exhibits a high

affinity and selectivity for Hg

2+

_{ions over competing metal}

ions. The fluorescence of FS1 was significantly enhanced in

the presence of Hg

2+

_{, and the addition of Ag}

+

_{, Ca}

2+

_{, Cd}

2+

_,

Co

2+

_{, Cu}

2+

_{, Fe}

2+

_{, Fe}

3+

_{, K}

+

_{, Mg}

2+

_{, Mn}

2+

_{, Ni}

2+

_{, Pb}

2+

_{, or}

Zn

2+

_{barely affected the fluorescence. This BODIPY-based}

Hg

2+

_{chemosensor is also an effective method for Hg}

2+

sensing in living cell imaging.

Experimental Section

General: All solvents and reagents were obtained from commercial sources and used as received without further purification. UV/Vis spectra were recorded with an Agilent 8453 UV/Vis spectrometer. Fluorescence spectra were recorded with a Hitachi F-4500 spec-trometer.1_{H and}13_{C NMR spectra were recorded with a Bruker}

DRX-300 NMR spectrometer.

Synthesis of N,N-Diprop-2-ynylaniline (1): Compound 1 was ob-tained in modest yield by treating aniline with propargyl bromide in the presence of potassium carbonate.[12]

Synthesis of 4-(Diprop-2-ynylamino)benzaldehyde (2): Phosphorus oxychloride (306.7 mg, 2 mmol) was added dropwise to a solution of compound 1 (338.2 mg, 2 mmol) in DMF (2 mL), and the mix-ture was then heated at 90 °C for 3 h. After cooling to ambient temperature, the reaction mixture was dissolved in CH2Cl2

(100 mL) and washed with a dilute sodium hydrogen carbonate solution. The solvent was evaporated under reduced pressure, and the crude product was purified by column chromatography (ethyl acetate/hexane, 1:10) to give compound 2 as a white solid. Yield: 354.7 mg (90 %); m.p. 54–55 °C.1_{H NMR (CDCl} 3): δ = 9.82 (s, 1 H), 7.81 (d, J = 8.7 Hz, 2 H), 6.96 (d, J = 8.7 Hz, 2 H), 4.22 (d, J = 2.4 Hz, 4 H), 2.30 (t, J = 2.4 Hz, 2 H) ppm.13_{C NMR (CDCl} 3): δ = 191.0, 152.2, 132.2, 127.9, 113.7, 78.7, 73.6, 40.5 ppm. MS (EI): m/z (%) = 197 (100.0), 172 (13.86), 167 (58.54), 132 (19.59). HRMS (EI): calcd. for C13H11NO 197.0841; found 197.0838.

Synthesis of 4-[Bis(1H-pyrrol-2-yl)methyl]-N,N-diprop-2-ynylaniline (3): Trifluoroacetic acid (TFA, 0.1 mL) was added to a solution of compound 2 (296 mg, 1.5 mmol) in pyrrole (2 mL). The solution was stirred under nitrogen at room temperature for 4 h, and the reaction was then quenched with 0.1m sodium hydroxide. The or-ganic phase was extracted with ethyl acetate and dried with anhy-drous MgSO4. The solvent was evaporated under reduced pressure,

and the crude product was purified by column chromatography (ethyl acetate/hexane, 3:10) to give compound 3 as a yellowish vis-cous liquid. Yield: 389.9 mg (83 %).1_{H NMR (CDCl}

3): δ = 7.91 (s, 2 H), 7.13 (d, J = 8.7 Hz, 2 H), 6.91 (d, J = 8.7 Hz, 2 H), 6.67– 6.69 (m, 2 H), 6.15 (q, J = 2.7 Hz, 2 H), 5.91–5.94 (m, 2 H), 5.42 (s, 1 H), 4.11 (d, J = 2.4 Hz, 4 H), 2.25 (t, J = 2.4 Hz,2 H) ppm. 13_{C NMR (CDCl} 3): δ = 146.4, 133.1, 132.8, 129.0, 117.0, 115.6, 108.1, 106.9, 79.1, 72.8, 42.9, 40.4 ppm. MS (FAB): m/z = 313. HRMS (FAB): calcd. for C21H19N3313.1579; found 313.1578.

Synthesis of 4,4-Difluoro-8-[4-(N,N-diprop-2-ynylamino)phenyl]-4-bora-3a,4a-diaza-s-indacene (4): 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone (DDQ; 318 mg, 1.4 mmol) dissolved in CH2Cl2

(50 mL) was added to a solution of compound 3 (376 mg, 1.2 mmol) in CH2Cl2 (100 mL) under nitrogen, and the mixture

was stirred for 3 h. It was then treated with Et3N (4 mL) and

BF3·OEt2(5 mL) for 12 h. The solvent was evaporated under

re-duced pressure, and the crude product was purified by column chromatography (ethyl acetate/hexane, 1:10) to give compound 4 as an orange solid. Yield: 258.6 mg (60 %); m.p. 226–227 °C. 1_H

NMR (CDCl3): δ = 7.90 (s, 2 H), 7.58 (d, J = 8.7 Hz, 2 H), 7.02–

7.05 (m, 4 H), 6.55 (d, J = 2.4 Hz, 2 H), 4.24 (d, J = 2.4 Hz, 4 H), 2.33 (t, J = 2.4 Hz, 2 H) ppm.13_{C NMR: δ = 149.7, 147.8, 142.6,}

134.6, 132.6, 131.1, 124.2, 118.0, 113.7, 78.4, 73.1, 40.2 ppm. MS (FAB): m/z = 359. HRMS (FAB): calcd. for C21H16BF2N3

359.1405; found 359.1407.

Synthesis of 4,4-Difluoro-8-[4-(N,N-bis{[1-(pyridin-2-ylmethyl)-1H- 1,2,3-triazol-4-yl]methyl}amino)phenyl]-4-bora-3a,4a-diaza-s-indacene (FS1): Picolyl azide (180 mg, 1.34 mmol), CuSO4·5H2O,

(16.8 mg, 10 mol-%), and sodium ascorbate (26.6 mg, 20 mol-%) were added to a solution of compound 4 (240.6 mg, 0.67 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 (10 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 (dichlo-romethane/methanol, 6:1) to give compound FS1 as a dark-red so-lid. Yield: 320.0 mg (76 %); m.p. 145–146 °C.1_{H NMR (CD} 3CN): δ = 8.50 (d, J = 4.2 Hz, 2 H), 7.84 (s, 2 H), 7.79 (s, 2 H), 7.72 (dt, J= 1.8, 7.5 Hz, 2 H), 7.53 (d, J = 9.0 Hz, 2 H), 7.26 (dd, J = 4.8, 7.5 Hz, 2 H), 7.18 (d, J = 7.8 Hz, 2 H), 7.09 (d, J = 8.7 Hz, 2 H), 7.03 (d, J = 3.9 Hz, 2 H), 6.59 (dd, J = 2.1, 4.1 Hz, 2 H), 5.58 (s, 4 H), 4.81 (s, 4 H) ppm. 13_{C NMR (CD} 3CN): δ = 155.7, 151.8, 150.4, 149.2, 145.2, 142.5, 138.0, 134.9, 134.0, 131.8, 124.4, 124.0, 122.9, 122.8, 118.7, 113.4, 55.8, 46.8 ppm. MS (ESI): m/z = 628.3 [M + H]+_{. HRMS (ESI): calcd. for C}

33H29BF2N11 [M + H]+

628.2669; found 628.2678.

Determination of the Binding Stoichiometry and the Apparent Disso-ciation Constants for the Binding of HgII_{to FS1: The binding} stoi-chiometry of the FS1–Hg2+_{complex was determined from a Job}

plot. The fluorescence intensity at 520 nm was plotted against the molar fraction of FS1 with a total concentration of the sensor and Hg2+_{ion of 250 μm. The molar fraction at maximum emission}

in-tensity represents the binding stoichiometry of the FS1–Hg2+

com-plex. The maximum emission intensity was reached at a molar frac-tion of 0.5 (Figure 4). This result indicates that chemosensor FS1 forms a 1:1 complex with Hg2+_{. The apparent dissociation constant}

(Kd) was calculated by nonlinear regression analysis.[13]The plot

was fitted with normalized fluorescence emission intensity against the concentration of the Hg2+ _{ion according to Equation (1) in}

which F is the fluorescence intensity at 520 nm at any given Hg2+

concentration, Fminis the fluorescence intensity at 520 nm in the

absence of Hg2+_{, F}

max is the maximum fluorescence intensity at

520 nm in the presence of Hg2+_{, n is the number of Hg}2+ _ions

bound per probe molecule, and Kdis the dissociation constant: n

= 1 according to the Job plot.

Cell Culture: The cell line HeLa was provided by the Food Industry Research and Development Institute (Taiwan). The HeLa cells were grown in DMEM supplemented with 10 % FBS at 37 °C and 5 % CO2. Cells were plated on 14 mm glass coverslips and allowed

to adhere for 24 h.

Fluorescence Imaging: HeLa cells were cultured in Dulbecco’s mo-died Eagle’s medium (DMEM) supplemented with 10 % fetal bo-vine serum (FBS) at 37 °C under an atmosphere of 5 % CO2. Cells

were plated on 14 mm glass coverslips and allowed to adhere for 24 h. Experiments to assess the Hg2+_{uptake were performed in}

phosphate-buffered saline (PBS) with 20 µm Hg(BF4)2. The cells

were treated with 10 mm solutions of Hg(BF4)2(2 µL; final

concen-tration: 20 µm) dissolved in sterilized PBS (pH = 7.4) and incubated at 37 °C for 30 min. The treated cells were washed with PBS (3⫻2 mL) to remove remaining metal ions. Culture medium (2 mL) was added to the cell culture, which was then treated with a 10 mm solution of chemosensor FS1 (2 µL; final concentration: 20 µm) dissolved in DMSO. The samples were incubated at 37 °C for 30 min. The culture medium 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 FS1 were excited at 480 nm by using a 50 W Hg lamp. An emission filter of 535 nm was used.

Supporting Information (see footnote on the first page of this arti-cle): 1_{H and}13_{C NMR spectra of compounds 2, 3, 4, and FS1;}

ESI-MS of FS1–Hg2+_.

Acknowledgments

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

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Received: November 10, 2011 Published Online: January 4, 2012