Borondipyrromethene-derived Cu2+ sensing chemodosimeter for fast and selective detection

Article abstract of DOI:10.1039/c2ob06980f

Here, we report a new Cu²⁺-selective fluorescent turn-on probe BODIPY-EP, in which the 2-pyridinecarboxylic acid is connected to a 6-hydroxyindole-based BODIPY platform through an ester linkage. The ester bond of BODIPY-EP is selectively hydrol

Full text of DOI:10.1039/c2ob06980f

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PAPER
Borondipyrromethene-derived Cu2+ sensing chemodosimeter for fast and
selective detection†
Chunchang Zhao,* Peng Feng, Jian Cao, Xuzhe Wang, Yang Yang, Yulin Zhang, Jinxing Zhang and
Yanfen Zhang
Received 25th November 2011, Accepted 7th February 2012
DOI: 10.1039/c2ob06980f
Here, we report a new Cu2+-selective fluorescent turn-on probe BODIPY-EP, in which the
2-pyridinecarboxylic acid is connected to a 6-hydroxyindole-based BODIPY platform through an ester
linkage. The ester bond of BODIPY-EP is selectively hydrolyzed by the reaction with Cu2+ under mild
and neutral conditions to generate BODIPY-OH, showing strong characteristic fluorescence of
BODIPY-OH. The favorable features of BODIPY-EP towards Cu2+ include fast response, large
fluorescence enhancement and high selectivity. We further demonstrated that the membrane-permeable
probe reacts with intracellular Cu2+ and exhibits bright fluorescence in living cells.
and specific reaction conditions such as high temperature, acidic
Introduction
or basic environment. Therefore, it is of great interest to design a
new chemodosimeter that can be used to detect Cu2+ rapidly and
Because of continuing concern over copper in the environment
and its critical role in biological systems, development of probes
for selective and sensitive quantification of Cu2+ by fluorescence
techniques has recently emerged as a focal point in the sensing
communities.1 One common approach in the design of fluor-
escent probes is to attach a ligand to a fluorophore to form Cu2+
complexation.2 Chelating of Cu2+ with chemosensors is well
known to induce intrinsic fluorescence quenching due to the
paramagnetic nature of Cu2+, but turn-off signals are usually less
sensitive and offer limited spatial resolution.3 For practical appli-
cations, however, fluorescence probes with fluorescence turn-on
signals in the presence of Cu2+ are superior to those with turn-
off signals. One alternative strategy to achieve fluorescence turn-
on involves the use of reaction-based indicator systems, chemo-
dosimeters, and has attracted a great deal of attention.4–6
Most of the reported chemodosimeters are based on convert-
ing non-emissive molecules to the emissive ones via irreversible
chemical reactions promoted by Cu2+. Excellent examples of
Cu2+-promoted reactions include hydrolysis of esters4 and
amides5 and oxidation of dihydrorosamine, phenothiazine, and
phenol.6 However, many of the reported chemosensors for Cu2+
have limitations such as low Cu2+ selectivity in the presence of
other metal cations, the requirement for long incubation time,
selectively in vitro and in vivo under mild and neutral
conditions.
BODIPY derivatives are among the most widely studied fluor-
escent dyes,7 however, very few investigations have been carried
out on 6-hydroxyindole-based BODIPY, as a scaffold for fluor-
escence probes.8,9 During our study, we found that 6-hydroxyin-
dole-based BODIPY has excellent photophysical properties.
More importantly, it is facile to construct chemodosimeters for
highly selective and sensitive detection of analyte by modifi-
cation of the hydroxyl group in 6-hydroxyindole-based
BODIPY.9 Herein, we report a new Cu2+-selective fluorescent
probe BODIPY-EP (Scheme 1), in which 2-pyridinecarboxylic
acid is connected to a 6-hydroxyindole-based BODIPY platform
through an ester linkage. Interestingly, we found that the ester
bond of BODIPY-EP is selectively hydrolyzed by the reaction
with Cu2+ under mild and neutral conditions to generate BODI-
PY-OH. The favorable features of BODIPY-EP towards Cu2+
include fast response, large fluorescence enhancement and high
selectivity. We further demonstrated that the membrane-per-
meable probe reacts with intracellular Cu2+ and exhibits bright
fluorescence in living systems.
Key Laboratory for Advanced Materials and Institute of Fine
Chemicals, East China University of Science & Technology, Shanghai
200237, P.R. China. E-mail: zhaocchang@ecust.edu.cn
†Electronic supplementary information (ESI) available: Copies of emis-
sion spectra, UPLC-MS, HRMS spectra and NMR spectra. See DOI:
10.1039/c2ob06980f
Scheme 1 Structure of BODIPY-EP and the release of BODIPY-OH
by Cu2+ in aqueous solution at room temperature.
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577 nm with an emission quantum yield of ∼0.07. However,
remarkable fluorescence enhancement in the featured emission
of BODIPY-OH at 577 nm (quantum yield ∼0.33) was observed
upon gradual addition of CuCl2, and the fluorescence turns out
to be stable when 3 equiv of Cu2+ was added, higher concen-
tration of Cu2+ does not lead to any noticeable change. Consist-
ently, the emission color of BODIPY-EP solution turned from
dark to bright orange. As shown in Fig. 2a, a linear relationship
is observed between the intensity change and the Cu2+ amount at
concentrations lower than 20 μM. The detection limit for Cu2+
was determined as 1.05 × 10−6 M under the experimental con-
ditions, which is sufficiently low to allow the fluorogenic detec-
tion of micromolar concentrations of Cu2+ in drinking water and
living systems. The experiments of the counter anions’ effect on
the response of BODIPY-EP to Cu2+ were also investigated,
which demonstrated that counter anions have little influence on
the response properties (Fig. S1†).
Results and discussion
Synthesis
Our group has reported a 6-hydroxyindole-based BODIPY with
excellent photophysical properties.9 An important advantage of
this dye is the facile synthesis of derivatives by modification of
the phenol function. Using this strategy, we constructed a chemo-
dosimeter for highly selective and sensitive detection of ben-
zenthiols.9 On the other hand, the synthetic yield of this indole-
based BODIPY is too low for further applications. During our
study we found that another 6-hydroxyindole-based BODIPY,
BODIPY-OH, with phenyl ring in the meso position instead of
H atom, can be easily obtained. BODIPY-OH can be obtained
in a routine BODIPY formation, three-step procedure via con-
densation of 2,4-dimethyl-3-ethylpyrrole with 2-benzoyl-3-
methyl-6-methoxyindole followed by removal of the methoxy
protecting groups (BBr3, CH2Cl2) and boron insertion with
BF3·OEt2 (Scheme 2). The overall yield is 53% for the three
steps. BODIPY-EP was then readily synthesized in one step by
reaction of BODIPY-OH with 2-pyridinecarboxylic acid (PCA)
in the presence of N,N′-diisopropylcarbodiimide (DIPC) and
p-(dimethylamino)pyridinium p-toluenesulfonate (DPTS) in high
yield. The structure was fully characterized by 1H NMR, 13C
NMR, and HRMS analysis.
Kinetics measurements of the fluorescence responses of
BODIPY-EP to Cu2+ were studied by time-dependent
Sensing response of BODIPY-EP to Cu2+
In H2O–DMSO buffer solution (0.05 M Tris-HCl, 50% DMSO,
pH = 7.5), the optical features of BODIPY-EP are characteristics
of the BODIPY platform (Fig. 1a). The main absorption band of
BODIPY-EP, attributed to the 0–0 vibrational band of a strong
S0–S1 transition, is centered at 513 nm. Upon gradual addition
of CuCl2 to a solution of BODIPY-EP in H2O–DMSO buffer
solution at room temperature, the absorption band at 513 nm
decreased and a new band at 546 nm appeared instantly, with a
distinct isosbestic point at 523 nm. The spectral change almost
stops upon addition of 3 equiv of Cu2+ within 20 min. Concomi-
tantly, the color of the solution turned from light pink to reddish
purple. The absorption band at 546 nm is the characteristic
absorption of BODIPY-OH which is red-shifted by 33 nm com-
pared to that of BODIPY-EP. Adding an excess amount of
EDTA to the solution does not show further spectral change,
indicating that BODIPY-EP reacts with Cu2+ irreversibly.
A fluorescence titration experiment was used to assess the
ability of BODIPY-EP to detect Cu2+ at room temperature
(Fig. 1b). Free BODIPY-EP shows weak fluorescence at about
Fig. 1 Spectra of BODIPY-EP (5 × 10−6 M) in the presence of differ-
ent concentrations of CuCl2 (0, 0.15, 0.30, 0.45, 0.60, 0.75, 0.90, 1.05,
1.20, 1.35, 1.50, 1.65, 1.80, 2.25, 3.00 equiv) in H2O–DMSO buffer sol-
ution (0.05 M Tris-HCl, 50% DMSO, pH = 7.5). (a) Absorption spectra.
Insets: visible color change of BODIPY-EP upon additions of CuCl2 (4
equiv). (b) Fluorescence titration spectra (λex = 523 nm). Inset: fluor-
escence color change of BODIPY-EP upon additions of CuCl2 (6
equiv) on excitation at 365 nm using a handheld UV lamp. Each measur-
ing was conducted after 10 min of mixing.
Scheme 2 Synthesis of BODIPY-EP.
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sensing mechanism can be proposed as the Cu2+-induced selec-
tive hydrolysis of BODIPY-EP to release BODIPY-OH
(Scheme 1). This mechanism is confirmed by identifying the
same mass of BODIPY-EP + Cu2+ as that of BODIPY-OH
(Fig. S4†). In UPLC-Mass spectra, BODIPY-EP gave a reten-
tion time at 3.63 min, and peak of [M − H]− with 508.2032.
Likewise, BODIPY-EP + Cu2+ gave a retention time at
3.43 min, and peak of [M − H]− with 403.1841, identical to that
1
of BODIPY-OH. H-NMR spectrometry also revealed the iden-
tity of the fluorescent product to be BODIPY-OH (Fig. S5†).
Absorption and fluorescence spectra titration of BODIPY-EP
(5 μM) in the presence of 0–30 μM Cu2+ also demonstrated that
Cu2+-promoted hydrolysis was a stoichiometric reaction rather
than a Cu2+-catalyzed reaction, because Cu2+–PCA chelate pro-
duced by the hydrolysis reaction inhibited the reactivity of the
Cu2+. This was further confirmed by adding Cu2+ ions to the sol-
ution of BODIPY-EP in the presence of excess 2-pyridinecar-
boxylic acid (PCA, 1000 equiv), where no apparent fluorescence
changes were observed (Fig. S6–S7†). The inhibition of Cu2+–
PCA chelate clearly demonstrated that the coordination of Cu2+
to pyridinecarboxylate ester is requisite for hydrolysis of the
probe, supporting the mechanism proposed above.
Selectivity studies of BODIPY-EP toward Cu2+
The selectivity of BODIPY-EP towards Cu2+ over relevant
cations was then evaluated by measuring the changes in the
optical spectra upon addition of excess amount of various
cations with an assay time of 10 min. The unique absorption
change with appearance of the characteristic absorption of
BODIPY-OH was observed only by the addition of CuCl2,
which can be ascribed to the Cu2+-promoted hydrolysis of BOD-
IPY-EP to give BODIPY-OH. By contrast, no noticeable
change in the UV spectra was noted with other cations such as
K+, Na+, Ca2+, Ag+, Zn2+, Hg2+, Cd2+, Co2+, Fe2+, Fe3+, Ni2+,
Mn2+, Pb2+ (Fig. 3a).
Fig. 2 (a) Fluorescence change of BODIPY-EP (5 μM) in intensity at
577 nm as a function of Cu2+ concentration. Error bars represent the
standard deviation of three measurements. Each measuring was con-
ducted after 10 min of mixing. (b) Kinetics of fluorescence enhancement
profile of BODIPY-EP at 577 nm in the absence and presence of Cu2+
.
The experiment was carried out in H2O–DMSO buffer solution (0.05 M
Tris-HCl, 50% DMSO, pH = 7.5) at room temperature. λex = 523 nm.
The fluorescence response of BODIPY-EP with various
cations and its selectivity for Cu2+ are shown in Fig. 3b. The
weak fluorescence of BODIPY-EP was only marginally
increased upon addition of representative cations such as K+,
Na+, Ca2+, Ag+, Zn2+, Hg2+, Cd2+, Co2+, Fe2+, Fe3+, Ni2+, Mn2+,
Pb2+. Only when Cu2+ was added was the unique fluorescent
characteristic of BODIPY-OH detected. It is noted that the
unique absorbance and fluorescence bands resulting from the
addition of the Cu2+ ions were not influenced by other cations.
We further examined the fluorescence response of BODIPY-EP
toward Cu2+ in the presence of other potentially competing
cations (Fig. 4). The titration of Cu2+ and BODIPY-EP in the
presence of various metal ions was conducted, and the exper-
imental results indicate that most of the relevant metal ions only
display minimum interference. The selectivity was also con-
ducted at an elevated temperature (37 °C), and no noticeable
changes in UV and emission spectra were observed upon
addition of the competitive cations. These results indicate the
excellent selectivity of BODIPY-EP towards Cu2+ over the
other competitive cations.
fluorescence spectra (λex = 523 nm, λem = 577 nm). Fig. 2b and
S2† display the representative kinetics plots for BODIPY-EP in
the presence of different amounts of Cu2+. The fluorescence inten-
sity increase displays in a concentration dependent fashion.
Higher concentrations of Cu2+ afforded a quicker and more dra-
matic response. Observed rate constants for Cu2+-mediated depro-
tection of BODIPY-EP are in the range of 0.06–0.37 min−1
.
Notably, a drastic characteristic of BODIPY-EP is the rapid reac-
tion with excess of Cu2+. As shown in Fig. 2b, BODIPY-EP
(5 μm) exhibited no observable changes in the emission intensities
at 577 nm in the absence of Cu2+ within 2 hours. By contrast, a
pronounced enhancement at 577 nm was noted even after 1 min
upon the addition of 6 equiv of Cu2+ and the enhancement
reached equilibrium within 10 min, indicative of a fast reaction of
BODIPY-EP with Cu2+ to release BODIPY-OH. The Cu2+-
induced fluorescence enhancement of BODIPY-EP occurs in a
pH range from 5 to 8 (Fig. S3†), although the reaction time
is much longer in an acid environment, suggesting that
BODIPY-EP enables Cu2+ detection at a physiological pH range.
The UV–vis and fluorescence spectra of the BODIPY-EP–
Cu2+ system are characteristic of BODIPY-OH. Therefore, the
A variety of other transition metal ions also display affinities,
but these ions did not elicit the fluorescence response of probe
BODIPY-EP within an assay time of 10 min (Fig. 3), which
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Fig. 5 (a) Fluorescence microscopy image of HEK293A cells pre-
treated with Cu2+ (50 μM) for 30 min, washed with PBS, and then incu-
bated with BODIPY-EP (10 μM) for another 30 min at room tempera-
ture. (b) Bright-field image of the cells shown in (a). (c) Fluorescence
image of cells incubated with 10 μM BODIPY-EP for 30 min.
by different metal ions. For instance, solutions containing BOD-
IPY-EP (5 μM) and Zn2+ (30 μM) in buffer solutions were
allowed to react for 2 hours to reach equilibrium (Fig. S8†).
However, Cu2+ ion promotes the hydrolysis at rates much greater
than other metal ions, only several minutes are needed. At the
same time, the fluorescence enhancement is much stronger than
that of BODIPY-EP + other cations.
Detection of Cu2+ in living HEK293A cells
We next assessed the ability of our probe for fluorescence
imaging of Cu2+ in living HEK293A cells (Fig. 5). HEK293A
cells incubated with 10 μM of BODIPY-EP for up to 120 min at
37 °C show negligible intracellular fluorescence image.
However, if cells were pre-treated with Cu2+ in the culture
medium for 30 min, washed with phosphate buffered saline to
remove extracellular Cu2+ and further incubated with 10 μM
BODIPY-EP for 30 min, a bright fluorescence image was then
observed from these cells. These results demonstrate that
BODIPY-EP is cell membrane permeable and can be used as a
possible sensor to detect Cu2+ within living cells.
Fig. 3 (a) Absorption and (b) fluorescence spectra (λex = 523 nm) of
BODIPY-EP (5 μM) in the absence and presence of 6 equiv of various
cations in H2O–DMSO buffer solution (0.05 M Tris-HCl, 50% DMSO,
pH = 7.5). The assay time was 10 min.
Conclusions
In conclusion, a borondipyrromethene-derived fluorescence turn-
on probe for Cu2+, BODIPY-EP, was developed. This probe was
constructed by the modification of the hydroxyl group in 6-
hydroxyindole-based BODIPY through an ester linkage to 2-pyr-
idinecarboxylate. The ester bond is selectively hydrolyzed by the
reaction with Cu2+ under mild and neutral conditions. Therefore,
BODIPY-EP displays favorable features towards Cu2+, includ-
ing: (1) fast response to Cu2+ under mild and neutral conditions
within several minutes, enabling Cu2+ detection rapidly; (2)
remarkable fluorescence enhancement in the featured emission
of BODIPY-OH at 577 nm (quantum yield increased from
∼0.07 to ∼0.33) upon addition of Cu2+; (3) high selectivity
based on reaction time, due to Cu2+ ion promoting the hydrolysis
at rates much greater than other metal ions. Importantly, the
probe is membrane-permeable and can react with intracellular
Cu2+, displaying bright fluorescence in living systems.
Fig. 4 Fluorescence response to 6 equiv of Cu2+ in the presence of 6
equiv of other cations (λex = 523 nm). (a) Cu2+, (b) K+, (c) Na+, (d)
Ca2+, (e) Ag+, (f) Zn2+, (g) Hg2+, (h) Cd2+, (i) Co2+, ( j) Fe2+, (k) Fe3+
(l) Ni2+, (m) Mn2+, (n) Pb2+. The assay time was 10 min.
,
The probe constructed here has absorption and emission wave-
lengths in the visible range. In contrast, near-infrared (NIR) flu-
orescent sensors are advantageous due to less photodamage,
minimum fluorescence background, and less light scattering by
NIR light. Given the fact that the anion form of BODIPY-OH is
highly fluorescent around 650 nm (falling in NIR region),8
allows the probe to exhibit the high selectivity for Cu2+. This
high selectivity could be attributed to reaction time and con-
ditions required for the hydrolysis of pyridinecarboxylate ester
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construction of a chemodosimeter with release of the anion form
via a reaction may open an avenue for the development of NIR
fluorescent sensors. As modulation of the pKa of BODIPY-OH
is a key point to get the brightest fluorescence emission of the
phenolate species in aqueous media under physiological con-
ditions, further study to reduce the pKa of the phenol and to
improve the water-solubility10 of these BODIPY derivatives is
currently underway.
removed in vacuo, and the residual solid was purified by flash
chromatography (silica gel) to afford 446 mg (88%) of
1
BODIPY-EP. H NMR (400 MHz, CDCl3) δ 1.05–1.01 (t, 3H),
1.40 (s, 3H), 1.62 (s, 3H), 2.40–2.34 (m, 2H), 2.69 (s, 3H),
6.97–6.94 (dd, 1H), 7.36–7.34 (m, 2H), 7.50–7.48 (d, 1H),
7.57–7.55 (m, 4H), 7.64 (s, 1H), 7.95–7.90 (m, 1H), 8.32–8.30
(d, 1H), 8.87–8.86 (d, 1H). 13C NMR (100 MHz, CDCl3) δ
164.07, 162.76, 150.50, 149.08, 146.59, 143.42, 141.76, 141.04,
136.93, 136.16, 135.84, 133.94, 132.76, 129.88, 129.70, 128.61,
128.36, 127.77, 127.24, 127.10, 126.28, 124.79, 120.66, 114.46,
106.33, 21.95, 16.14, 13.06, 11.28, 10.13. HRMS (ESI-TOF) m/
z calcd for C30H25BF2N3O2 [M − H]−: 508.2008, found
508.2015.
Experimental section
General methods
All chemical reagents and solvents for synthesis were purchased
from commercial suppliers and were used without further purifi-
cation. Dichloromethane was dried with CaH2 and distilled
immediately prior to use. All moisture-sensitive reactions were
carried out under an atmosphere of argon. 1H NMR and 13C
NMR spectra were recorded on a Bruker AV-400 spectrometer
with chemical shifts reported in ppm (in CDCl3, TMS as internal
standard) at room temperature. Mass spectra were measured on a
HP 1100 LC-MS spectrometer.
Acknowledgements
We gratefully acknowledge the financial support by the National
Science Foundation of China (grant no.: 20902021, 21002013),
the Scientific Research Foundation for the Returned Overseas
Chinese Scholars (State Education Ministry).
Notes and references
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Synthesis
Synthesis of BODIPY-OH. To a solution of 2-benzoyl-3-
methyl-6-methoxyindole (1 g, 3.76 mmol) in CH2Cl2 (60 mL)
was added POCl3 (1.05 mL, 11.28 mmol) at 0 °C, the resulted
solution was stirred for another 5 min, followed by the addition
of 2,4-dimethyl-3-ethylpyrrole. The resulting mixture was
warmed to room temperature and stirred for 3 days, cooled to
0 °C, neutralized with saturated Na2CO3, and washed with H2O.
The organic phase was dried with Na2SO4, and the solvent was
removed to give dark oil. To the resulting oil in anhydrous
CH2Cl2 was added BBr3 (3.48 mL, 37.6 mmol) at −15 °C. The
reaction mixture was further stirred for 1 hour at −15 °C,
warmed to room temperature, quenched with H2O, extracted
with CH2Cl2, and washed with H2O. The combined organic
extracts were dried with Na2SO4, and Na2SO4 was removed by
filtration. Then Et3N (5.6 mL) was added to the solution at room
temperature, and the resulting mixture was stirred for 5 min,
cooled to 0 °C, followed by addition of BF3·OEt2, and stirred for
another 30 min. The reaction mixture was extracted with
CH2Cl2, washed with H2O, dried over Na2SO4, and the solvent
was removed in vacuo. The crude product was purified by flash
chromatography (silica gel, eluent: CH2Cl2 then EtOAc–CH2Cl2
1 : 10) to afford 800 mg (53%) BODIPY-OH: 1H NMR
(400 MHz, CDCl3) δ 1.03 (t, 3H), 1.40 (s, 3H), 1.60 (s, 3H),
2.35–2.41 (q, 2H), 2.69 (s, 3H), 6.61–6.65 (dd, 1H), 7.1 (m,
1H), 7.34–7.37 (m, 3H), 7.54–7.56 (m, 3H); 13C NMR
(100 MHz, CDCl3) δ 161.8, 157.7, 146.5, 141.6, 141.5, 136.8,
135.3, 133.3, 132.7, 129.3, 129.1, 128.3, 127.0, 122.7, 112.4,
98.7, 17.2, 14.2, 13.4, 12.2, 11.3; HRMS (ESI-TOF) m/z calcd
for C24H22BF2N2O [M − H]−: 403.1793. Found: 403.1825.
Synthesis of BODIPY-EP. A mixture of BODIPY-OH
(404 mg, 1.0 mmol), 2-pyridinecarboxylic acid (127 mg,
1.03 mmol), DPTS (780 mg, 2.5 mmol), and DIPC (315 mg,
2.5 mmol) in CH2Cl2 were refluxed overnight. The solvent was
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