A selective turn-on fluorescent probe for Cd2+ based on a boron difluoride β-dibenzoyl dye and its application in living cells

Article abstract of DOI:10.1039/c3ob40376a

Herein we report the first example of a difluoroboron dibenzoyl based fluorescent probe for Cd²⁺ detection. The probe displays high selectivity and sensitivity toward Cd²⁺ over Zn²⁺ in aqueous solution under physiological conditions. Fluorescence imaging experiments demonstrate its potential application for detecting Cd²⁺ in living cells. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3ob40376a

Organic &
Biomolecular Chemistry
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A selective turn-on fluorescent probe for Cd2+ based on
a boron difluoride β-dibenzoyl dye and its application
in living cells†
Cite this: Org. Biomol. Chem., 2013, 11,
3014
Li Xin,a,b Yu-Zhe Chen,*a Li-Ya Niu,a Li-Zhu Wu,a Chen-Ho Tung,a Qing-Xiao Tong*b
and Qing-Zheng Yanga
Received 21st February 2013,
Accepted 14th March 2013
Herein we report the first example of a difluoroboron dibenzoyl based fluorescent probe for Cd2+ detec-
tion. The probe displays high selectivity and sensitivity toward Cd2+ over Zn2+ in aqueous solution under
physiological conditions. Fluorescence imaging experiments demonstrate its potential application for
detecting Cd2+ in living cells.
DOI: 10.1039/c3ob40376a
www.rsc.org/obc
no fluorescent chemosensors for cation detection based on the
boron difluoride β-dibenzoyl skeleton have been reported so
Introduction
Fluorophores serve as versatile tools for probing environments far.
and living systems. The development of different fluorescent
Environmental pollution caused by cadmium has long
compounds as reporters for biological probes may be suitable been a worldwide public health issue due to the fact that
for specific applications and benefits from the availability of cadmium and its complexes are widely used and are toxic,
structurally diverse fluorescent dyes with different spectro- even at low doses.11 There is evidence of increasing cadmium
scopic properties. Therefore, searching for new fluorescent content in food, which poses severe harm to human health
species as detection tools is still an important issue. Fluor- and environment. Cadmium can accumulate in the human
escent boron difluoride dyes such as “Bodipy” and β-diketone– body and cause pulmonary cancer, renal dysfunction, calcium
boron difluoride complexes are noted for their large molar metabolism disorders and other relevant forms of
absorption coefficient, intense fluorescence, insensitivity to diseases.12–14 Therefore, there is a great need for selective
solvent and pH, and good aqueous stability. Compared to the detection and monitoring of the presence and concentration
commercially available Bodipy, the applications of β-diketone– of cadmium ions in the environment and in biological
boron difluoride dyes are yet to be fully exploited. Studies have systems. Fluorescent chemosensors15 are of great potential for
revealed their promising applications in biological systems,1 tracing cadmium distribution in cells and revealing Cd-
organic light-emitting diodes (OLED),2,3 in vivo detection of carcinogenesis and other biological effects for living systems.
amyloid-β deposit,4 as medium-sensitive two-photon fluor- However, only a few fluorescent sensors for cadmium have
escent probes,5 or as anion sensors with C–H⋯X interactions.6 been reported in living cells so far.16 Furthermore, cadmium
In addition to the common advantages of Bodipy, β-diketone– ions usually exhibit very similar properties to zinc ions, which
boron difluoride dyes have large Stokes shifts, large dipole are the second most abundant transition metal ions in the
moments, large two-photon absorption cross sections and ver- human body. It is still a challenge to develop fluorescent chemo-
satile reactivities,7–10 which made them potential candidates sensors that can discriminate between cadmium and zinc
for constructing fluorescent chemosensors. Despite the boron ions.17 Therefore, it is highly desirable to develop Cd2+-selec-
dye system having been investigated infrequently since 1905, tive sensors that can avoid the interference of Zn2+ and other
biologically relevant metal cations.
In this report, we present a new fluorescent probe 1 based
on a boron difluoride β-dibenzoyl dye for highly selective
detection of cadmium over zinc ions (Fig. 1). Herein, 2,2′-di-
picolylamine (DPA) is employed as a Cd2+ receptor. DPA has
proven to be a versatile metal chelator with good affinity and
aKey Laboratory of Photochemical Conversion and Optoelectronic Materials,
Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, 29
Zhongguancun East Road, Beijing 100190, China. E-mail: chenyuzhe@mail.ipc.ac.cn
bDepartment of Chemistry, Shantou University, Shantou, Guangdong 515063, China.
high biocompatibility especially for Cd2+ and Zn2+ 18
It is intro-
.
E-mail: qxtong@stu.edu.cn
†Electronic supplementary information (ESI) available: Absorption spectra of 1
upon addition of Cd2+, pH effect on the emission of 1 and Cd–1, calculation of
the binding constant and the detection limit, solvent effect on the fluorescence,
NMR and MS data of 1 and Cd–1. See DOI: 10.1039/c3ob40376a
duced into the boron difluoride β-dibenzoyl fluorescence skele-
ton via an acetyl amine group. A 2-methoxyethoxy group is
introduced to the boron difluoride β-dibenzoyl compound to
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Fig. 1 Fluorescent probe 1.
improve the water-solubility of the fluorescent sensor. We
demonstrate in this contribution that boron difluoride
β-dibenzoyl dyes can be successfully applied in selectively
detecting cations by rational design. As expected, upon
addition of Cd2+, 1 shows a significant fluorescence enhance-
ment in aqueous media. Confocal laser scanning microscopy
(CLSM) experiments demonstrate that 1 could be used as a
fluorescent probe for Cd2+ in living cells.
Results and discussion
Probe 1 was synthesized in six steps as shown in Scheme 1,
with 4-amino ethylbenzoate as the starting material. It was pro-
tected by the di-tert-butyl dicarbonate to form a. Then b, an
important intermediate, was obtained by the Claisen conden-
sation between 1-(4-(2-methoxyethoxy)phenyl)ethanone and a.
After reaction with bromo acetyl bromide and nucleophilic
substitution of the deprotected d with DPA, e was achieved.
The reaction of e with 1.5 equiv. of BF3·OEt2 in CH2Cl2
afforded the desired product 1 in good yield. The intermedi-
Fig. 2 (a) Changes in the fluorescence emission spectra of 1 (5 μM) in CH3CN–
HEPES buffer (1 : 1 v/v, 20 mM, pH 7.4) upon titration of Cd2+ (0–10 μM), λex
=
400 nm. The inset of (a) shows the relative emission intensity (Imax = 464 nm) vs.
the equivalent of Cd2+. (b) Fluorescence intensity change profiles of 1 (5 μM) in
the presence of various cations. Metal ions (1 equiv. relative to 1) were added in
the form of Cd(NO3)2, NiCl2, CoCl2, Zn(NO3)2, AgNO3, CuCl2, FeCl2, Fe(NO3)3,
HgCl2, MnCl2, Pb(NO3)2. Others (30 equiv. relative to 1): KCl, CaCl2, NaCl,
MgSO4.
ates b–e, which would be present in solution as their enol pH 7.4). Fig. 2a shows the emission spectral change of 1
forms, were specified in the experimental parts.19 The struc- (5 μM) upon the addition of Cd2+. The ion-free form of 1 has a
ture of 1 was confirmed by 1H NMR, 13C NMR, and HRMS weak fluorescence peak at 463 nm, while the addition of Cd2+
data. The detailed experimental procedures and characteri- results in an obvious fluorescence enhancement, with no shift-
zations are shown in the ESI.†
ing in the excitation and emission maxima. The fluorescence
The sensing performance of probe 1 towards Cd2+ ions was quantum yield increases from 0.057 for 1 to 0.31 for Cd–1. The
investigated in CH3CN–HEPES buffer (1 : 1, v/v, 20 mM, weak fluorescence of 1 was attributed to the efficient photo-
Scheme 1 Synthesis of probe 1. (i) Dioxane, reflux, 24 h; (ii) 1-(4-(2-methoxyethoxy)phenyl)ethanone, NaH, THF, 0 °C → r.t., 24 h; (iii) trifluoroacetic acid (TFA), 0 °C
→ r.t., 3 h; (iv) bromoacetyl bromide, 4-dimethylaminopyridine (DMAP), CH2Cl2, 0 °C → r.t.; (v) 2,2’-dipicolylamine (DPA), K2CO3, KI, CH3CN, reflux; (vi) boron trifluor-
ide diethyl etherate (BF3·OEt2), CH2Cl2, reflux.
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induced electron transfer (PET) from the amino group to the
fluorophore. Upon binding to Cd2+, the electron-donating
capability of the amino group was reduced effectively, suppres-
sing the PET process and resulting in a considerable increase
in fluorescence. To the best of our knowledge, this is the first
difluoroboron dibenzoyl derivative used as a fluorophore to
detect Cd2+ ions.
The titration experiment showed that 1 forms a 1 : 1 complex
with Cd2+ (Fig. 2a). The ESI-MS of a mixture of 1 and Cd(ClO4)2
also reveals the formation of a 1 : 1 complex through a metal
coordination interaction, where the peak at m/z 812.9 can be
assigned to the species [1 + Cd + ClO4]+ (see ESI†). The binding
constant (K = 5.59 × 105 M−1, R2 = 0.999), which was calculated
via a linear analysis of the fluorescence intensity versus the Cd2+
ion concentration,20,21 suggests a strong binding ability of 1 to
Cd2+ (Fig. 2S†). The detection limit22,23 was estimated to be as
low as 2.19 × 10−7 M (R2 = 0.996) according to the titration
experiment, indicating a high sensitivity of 1 to Cd2+ (Fig. 3S†).
Fig. 4 The fluorescence response of 1 (5 μM) towards Cd2+ and other metal
ions in CH3CN–HEPES buffer (1 : 1 v/v, 20 mM, pH 7.4; λex = 400 nm) at 464 nm
at room temperature. Black bar: other competing metal ions (1 equiv.) were
added to 1. Red bar: Cd2+ (1 equiv.) was added to 1 containing the above metal
ions (100 equiv.).
The titration of 1 with various physiologically and environ-
mentally relevant metal ions showed excellent selectivity to
The binding model of 1 towards Cd2+ was investigated by Cd2+ (Fig. 2b and 4). Only Cd2+ induced a dramatic fluo-
1H NMR in d3-acetonitrile [Fig. 3, 1 and 1–Cd(ClO4)2]. All
rescence enhancement in the emission spectrum. Cations
protons were assigned referring to the literature.24 Compared such as Mg2+, Ca2+, Fe3+, K+, which are abundant in living
to 1, the chemical shifts of Cd–1 have an obvious down field cells, are not able to cause interference with Cd2+. It is worth
noting that 1 shows good selectivity for Cd2+ over Zn2+, which
shift except for Ha (from 11.48 to 9.37). It suggests that the
oxygen atoms of the amide group and the DPA unit participate is the main competitor for cadmium ion chemosensors.
together in binding to a Cd2+ ion, which decreases proton Common heavy and transition metal ions, such as Hg2+, Pb2+
exchange and reduces π-electron density.25–27 The coordination
and Cu2+ induced slight enhancement or even quenching in
of the oxygen atom further enhances the binding capacity of fluorescence. The competition experiment also revealed that 1
the probe to Cd2+, and thus gives excellent selectivity. No reso- showed turn-on fluorescence even in the presence of 100
equivalents of other competing ions with the exception of Cu2+
nance signal changes upon addition of a second equivalent
of Cd2+, which verifies the binding ratio of 1 : 1 as mentioned and Co2+, which limited the turn-on Cd2+ response of 1 as
above.
reported elsewhere28,29 (Fig. 4).
Fig. 3 (a) 1H NMR spectra of 1 in the presence of 1 equiv. of Cd2+ in CD3CN; (b) the proposed binding mode of Cd–1.
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referenced to solvent signals. Mass spectra (EI) were obtained
in the positive ion mode on a Waters GCT premier. HRMS was
measured with a Fourier Transform Ion Cyclotron Resonance
Mass Spectrometer with ESI positive mode. Fluorescence
spectra were recorded at ambient temperature on a Perkin
Elmer LS50B spectrofluorimeter and UV/Vis spectra were
recorded on a Shimadzu UV-1601PC spectrophotometer.
Preparation of 1
Caution: Sodium hydride is potentially explosive and must be
handled with care, shock and high temperatures must be
avoided.
Fig. 5 Confocal fluorescence of living HeLa cells: (a) fluorescence image of
HeLa cells incubated with 1 (5 μM); (b) fluorescence image of HeLa cells incu-
bated with 1 after adding Cd2+ (25 μM).
Synthesis of a. A solution of 4-amino ethylbenzoate (3 g,
18.2 mmol) and di-tert-butyl dicarbonate (6.3 g, 29 mmol) in
1,4-dioxane (67 mL) was stirred at 80 °C for 24 h. The reaction
was cooled to room temperature and then evaporated to
dryness. The residue was purified by column chromatography
on silica gel with petroleum ether : ethyl acetate (1 : 3) as the
eluent to give a (4.34 g, yield: 90%). 1H NMR (CDCl3, 400 MHz)
δ (ppm): 7.98 (2H, d, J = 8.8 Hz), 7.43 (2H, d, J = 8.4 Hz), 6.73
(1H, br), 4.37 (2H, q, J = 7.2 Hz), 1.51 (9H, s), 1.37 (3H, t, J =
7.2 Hz).
The influence of pH on the emission intensity was studied
from pH 5.82 to 7.85 (Fig. 4S†). Probe 1 exhibited intense fluo-
rescence at pH 5.82 due to the protonation of the amino group
under acidic conditions, and decreasing intensity above pH
6.17, which was attributed to the PET from the amino to the
difluoroboron dibenzoyl fluorophore. A weaker pH effect was
observed for the complex Cd–1 in the pH range of 6.17–7.85.
Therefore, the physiological pH would be suitable for Cd2+
detection.
To further demonstrate the practical application of the
probe, we carried out fluorescence imaging experiments in
living cells. HeLa cells incubated with 1 (5 μM) at 37 °C for
15 min showed almost no fluorescence (Fig. 5a). Treatment of
HeLa cells with Cd2+ (25 μM) for 10 min and subsequent treat-
ment of cells with 1 (5 μM) for another 15 min showed an
obvious blue fluorescence (Fig. 5b). The results suggest that 1
can penetrate the cell membrane and can potentially be used
for imaging of Cd2+ in living cells and in vivo.
Synthesis of 1-(4-(2-methoxyethoxy)phenyl)ethanone. 4-
Hydroxyacetophenone (1.8 g, 13.2 mmol), 1-bromo-2-methoxy-
ethane (2.4 g, 17.1 mmol) and K2CO3 (4.15 g, 30 mmol) in
DMF (40 mL) were heated at 90 °C for 24 h. The mixture was
then cooled, diluted with water and extracted with CH2Cl2. The
organic phase was dried over MgSO4, and the solvent was evap-
orated. The residue was purified by column chromatography
on silica gel with petroleum ether : ethyl acetate (1 : 10) as the
eluent to give 1-(4-(2-methoxyethoxy)phenyl)ethanone as a
1
white solid (2.32 g, yield: 90.7%). H NMR (CDCl3, 400 MHz) δ
(ppm): 7.92 (2H, d, J = 9.2 Hz), 6.95 (2H, d, J = 8.8 Hz), 4.17
(2H, t, J = 4.4 Hz), 3.76 (2H, t, J = 4.4 Hz), 3.44 (3H, s), 2.54
(3H, s).
Conclusions
Synthesis of b. 1-(4-(2-Methoxyethoxy)phenyl)ethanone
(1.8 g, 15 mmol) was dissolved in 100 mL dry THF, then the
mixture was cooled to 0 °C under N2. Sodium hydride was
added to the mixture slowly. After 15 min, a (1.35 g, 9 mmol)
was added. Then the mixture was stirred for 8 h at room temp-
erature. The reaction was quenched with water, and extracted
with ethyl acetate. The organic phase was dried over Na2SO4,
and the solvent was evaporated to dryness. The residue was
purified by column chromatography on silica gel with pet-
roleum ether : ethyl acetate (1 : 4) as the eluent to give b in its
enol form as a pale yellow powder (1.56 g, yield: 42%). 1H
NMR (CDCl3, 400 MHz) δ (ppm): 17.08 (1H, s), 7.94 (4H, m),
7.48 (2H, d, J = 8.8 Hz), 7.00 (2H, d, J = 8.8 Hz), 6.76 (1H, s),
6.73 (1H, s), 4.19 (2H, t, J = 4.8 Hz), 3.78 (2H, t, J = 4.8 Hz),
3.46 (3H, s), 1.53 (9H, s).
In conclusion, we have designed and synthesized a difluoro-
boron dibenzoyl-based fluorescent probe 1 for Cd2+ based on a
PET mechanism. Probe 1 displayed high selectivity and sensi-
tivity towards Cd2+ with significantly enhanced fluorescence
intensity due to the formation of a 1 : 1 metal–ligand complex.
It can distinguish Cd2+ from other metal ions, especially from
Zn2+ in aqueous solution. This is the first example of metal
ion detection with difluoroboron dibenzoyl derivatives as
fluorophores. The living cell image experiments further
demonstrate its value in the practical applications of biological
systems. Efforts to employ difluoroboron dibenzoyl derivatives
as fluorophores to detect various metal ions and to further
improve their water-solubility are underway.
Synthesis of c. The solution of b (0.7 g, 1.7 mmol) in
dichloromethane (40 mL) was added slowly to TFA (6 mL) at
0 °C. The mixture was stirred for 3 h at room temperature and
Experimental section
General information
All the reagents and solvents are of commercial quality and evaporated to dryness. The residue was redissolved in 10 mL of
used without further purification. 1H and 13C NMR spectra 2 M NaOH, extracted with CH2Cl2, dried over anhydrous K2CO3
were recorded on an Advance Bruker 400 M spectrometer and and evaporated to obtain a yellow solid. The residue was
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purified by column chromatography on silica gel with pet- Fluorescence quantum yield of 1
roleum ether : ethyl acetate (3 : 1) as the eluent to give c as its
Quantum yields of 1 were determined using quinine sulfate in
enol form as a yellow powder (0.32 g, yield: 60%). 1H NMR
(CDCl3, 400 MHz) δ (ppm): 17.23 (1H, s), 7.95 (2H, d, J =
8.8 Hz), 7.86 (2H, d, J = 8.8 Hz), 7.01 (2H, d, J = 8.8 Hz), 6.72
(1H, s), 6.70 (2H, m), 4.20 (2H, t, J = 4.4 Hz), 4.13 (2H, s), 3.80
(2H, t, J = 4.4 Hz), 3.48 (3H, s).
0.1 N H2SO4 as standards according to a published method.30
The quantum yields were calculated according to the following
equation:
Φ ¼ ΦS ꢀ I=IS ꢀ ODS=OD ꢀ η2=ηS
ð1Þ
2
Synthesis of d. A solution of bromo acetyl bromide (0.2 g,
1 mmol) in dry CH2Cl2 (80 mL) was added dropwise to a solu-
tion of c (0.2 g, 0.64 mmol) and DMAP (0.014 g, 0.12 mmol) in
dry CH2Cl2 (20 mL) stirred in an ice bath. After 6 h at room
temperature, the mixture was diluted with water and extracted
with CH2Cl2, dried over anhydrous MgSO4 and evaporated.
The residue was purified by column chromatography on silica
gel with petroleum ether : dichloromethane (1 : 1) as the eluent
to afford d in its enol form as a pale yellow powder (0.17 g,
in which Φ is the quantum yield, I is the integrated intensity, η
is the refractive index (ηH O = 1.333 was used here), OD is the
2
optical density. The subscript S refers to the standard.
Cell culture and fluorescence imaging
HeLa cells were cultured in culture media (DMEM/F12 sup-
plemented with 10% FBS, 50 unit mL−1 penicillin, and 50 μg
mL−1 of streptomycin) at 37 °C under a humidified atmos-
phere containing 5% CO2 for 24 h. The cells were treated and
incubated with 25 μM of Cd(NO3)2 at 37 °C under 5% CO2 for
10 min, and washed three times with phosphate buffered
saline (PBS). Then 5 μM of 1 were added to the above cells,
incubated for another 15 min at 37 °C under 5% CO2, and
washed three times with PBS. For the control experiment, the
cells were incubated with 5 μM of 1 in culture media for
15 min. Confocal fluorescence imaging was performed with a
Nikon A1R multiphoton microscope with a 60× oil-immersion
objective lens. Fluorescence was excited at 405 nm with a Si
laser and emission was collected by a 425–475 nm band pass
filter.
1
yield: 60%). H NMR (CDCl3, 400 MHz) δ (ppm): 17.01 (1H, s),
8.35 (1H, s), 7.97 (4H, m), 7.69 (2H, m), 7.01 (2H, d, J = 8.0 Hz),
6.76 (1H, s), 4.20 (2H, t, J = 4.8 Hz), 4.05 (2H, s), 3.74 (2H, t, J =
4.8 Hz), 3.47 (3H, s).
Synthesis of e. A suspension of d (0.1 g, 0.23 mmol), DPA
(72 mg, 0.36 mmol), K2CO3 (0.1 g, 0.72 mmol) and KI (15 mg,
0.09 mmol) in 40 mL of acetonitrile was refluxed for 24 h
under N2. After evaporation of the acetonitrile, the residue was
diluted with water and extracted with CH2Cl2, dried over anhy-
drous Na2SO4 and evaporated to obtain a viscous yellow liquid.
The residue was purified by column chromatography on silica
gel with 1 : 1 methanol : dichloromethane as the eluent to
give e in its enol form as a yellow oil (63 mg, yield: 50%).
1H NMR (CDCl3, 400 MHz) δ (ppm): 17.10 (s, 1H), 11.27
(s, 1H), 8.63 (2H, d, J = 4.4 Hz), 7.95 (6H, m), 7.62 (2H, dt, J =
7.8 Hz, 1.6 Hz), 7.28 (2H, d, J = 7.6 Hz), 7.19 (2H, dd, J =
5.6 Hz, 1.6 Hz), 7.01 (2H, d, J = 8.8 Hz), 6.77 (1H, s), 4.20
(2H, t, J = 4.8 Hz), 3.95 (4H, s), 3.79 (2H, t, J = 4.8 Hz), 3.51
(2H, s), 3.46 (3H, s). MS (EI): calcd for [M]+ 552.24, found
552.23.
Synthesis of 1. BF3·OEt2 (86 mg, 0.6 mmol) was added to a
solution of dry e (0.2 g, 0.4 mmol) in dry CH2Cl2 (80 mL). The
mixture was refluxed for 3 h under N2. After cooling to room
temperature, the mixture was diluted with water and extracted
with CH2Cl2, dried over anhydrous MgSO4 and evaporated to
obtain a pale yellow viscous liquid. The residue was purified
by column chromatography on silica gel with 40 : 1
dichloromethane : methanol as the eluent to give 1 as a yellow
powder (14 mg, yield: 60%). 1H NMR (CDCl3, 400 MHz)
δ (ppm): 11.48 (1H, s), 8.64 (2H, d, J = 4.8 Hz), 8.15 (4H, dd, J =
7.2 Hz, 2.0 Hz), 8.00 (2H, d, J = 8.8 Hz), 7.64 (2H, dt, J = 7.6 Hz,
1.6 Hz), 7.29 (2H, d, J = 6.0 Hz), 7.21 (2H, dd, J = 4.8 Hz,
2.4 Hz), 7.07 (2H, d, J = 3.2 Hz), 7.05 (1H, s), 4.24 (2H, t, J =
4.8 Hz), 3.97 (4H, s), 3.80 (2H, t, J = 4.8 Hz), 3.55 (2H, s), 3.47
(3H, s). 13C NMR (CDCl3, 100 MHz) δ (ppm): 181.21, 180.77,
170.94, 164.79, 157.99, 149.56, 145.15, 137.00, 131.47, 130.41,
126.95, 124.79, 123.54, 122.91, 119.46, 115.28, 92.04, 70.83,
67.98, 60.53, 59.44, 59.15. MS (EI): calculated for [M]+ 600.24,
found for 600.38. HRMS (ESI) m/z calcd for C32H32BF2N4O5
(MH+), 601.2436; found 601.2432.
Acknowledgements
We are grateful for financial support from the 973 Program
(2013CB933800, 2013CB834803), the National Natural Science
Foundation of China (21222210, 91027041, 51273108), and the
Chinese Academy of Sciences (100 Talents Program). We thank
Prof. P. Wang (TIPC, CAS) for providing HeLa cells and Dr
M. Zheng (TIPC, CAS) for her help in confocal laser scanning
microscopy.
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