A BODIPY fluorescence probe modulated by selenoxide spirocyclization reaction for peroxynitrite detection and imaging in living cells

Article abstract of DOI:10.1016/j.dyepig.2012.09.006

We present the synthesis, spectroscopic properties, and live-cell application of a new BODIPY (boron dipyrromethene) fluorescence probe BOD-Se based on selenoxide spirocyclization reaction for peroxynitrite detection. The probe employs BODIPY dye as fluorophore, and is integrated with a chemical peroxynitrite responsive organoselenium functional group. By using reactive diaryl selenide and modulating by intramolecular charge-transfer (ICT) process, the probe is employed for evaluating intracellular peroxynitrite level changes. Different intracellular peroxynitrite levels can be detected with BOD-Se by confocal microscopy experiments using mouse macrophage cell line RAW264.7.

Full text of DOI:10.1016/j.dyepig.2012.09.006

Dyes and Pigments 96 (2013) 383e390
Contents lists available at SciVerse ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
A BODIPY fluorescence probe modulated by selenoxide spirocyclization reaction
for peroxynitrite detection and imaging in living cells
1
1
*
Bingshuai Wang , Fabiao Yu , Peng Li, Xiaofei Sun, Keli Han
State Key Laboratory of Molecular Reaction Dynamics, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, ZHongshan Road 457, Dalian, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 31 July 2012
Received in revised form
11 September 2012
Accepted 12 September 2012
Available online 18 September 2012
We present the synthesis, spectroscopic properties, and live-cell application of a new BODIPY (boron
dipyrromethene) fluorescence probe BOD-Se based on selenoxide spirocyclization reaction for perox-
ynitrite detection. The probe employs BODIPY dye as fluorophore, and is integrated with a chemical
peroxynitrite responsive organoselenium functional group. By using reactive diaryl selenide and
modulating by intramolecular charge-transfer (ICT) process, the probe is employed for evaluating
intracellular peroxynitrite level changes. Different intracellular peroxynitrite levels can be detected with
BOD-Se by confocal microscopy experiments using mouse macrophage cell line RAW264.7.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Fluorescent probes
BODIPY
Selenium
Peroxynitrite
Redox chemistry
Fluorescence imaging
1. Introduction
detection technologies, the non-invasive fluorescence measure-
ments offer advantages, including higher sensitivity, better selec-
Peroxynitrite (ONOOꢀ), the endogenous reaction product
between superoxide (Oꢁ2ꢀ) and nitricoxide (NO), acts as an oxidant
and nitrating agent causing oxidative and nitrosative stress in cells
[1]. Peroxynitrite anion itself is a relatively stable species, but its
conjugated acid (ONOOH, pKa ¼ 6.8) exhibits a half-life of around 1s
at physiological pH and temperature. Both species are strong
oxidizing species in vivo, as they can directly damage a wide array
of intracellular molecules, including DNA, proteins and transition
metal centers, these biological oxidative events will lead to
cardiovascular and neurodegenerative diseases [2e5]. However,
recently, more interests are focused on physiological nitrification
functions of endogenous ONOOꢀ, such as modulating signal
transduction pathways via its nitrosative ability to nitrate tyrosine
residues and thereby influence cellular processes [1,3e6]. There-
fore, the specific and sensitive methods that can be used to detect
the intracellular ONOOꢀ precisely are highly desired.
tivity, and in situ observation. The availability of synthetic
fluorescent probes and the development of powerful new micros-
copy approaches have made fluorescence imaging an essential role
for biomedical science and biology [11,12]. Although a few fluo-
rescent probes for ONOOꢀ have been developed [13e22], there is
still need to hunt for new design concept for the intracellular
ONOOꢀ fluorescent detection because many efforts are rising on
intracellular peroxynitrite research [1e5].
In this paper, we reported the syntheses of fluorescence probe
based on BODIPY (BOD-Se) and its application for peroxynitrite
detection. After reaction with ONOOꢀ, an obvious colour change of
the probe solution was observed by naked eyes along with fluo-
rescence quenching due to an ICT process. Moreover, the probe was
successfully applied to detect peroxynitrite level changes in living
cells.
The traditional testing technologies for ONOOꢀ detection,
including chemiluminescence, electron spin resonance spectrum
(ESR), spectrophotometry, and electrochemical analysis [7e10],
often require sample pretreatment. Compared with other biological
2. Experiments
2.1. Materials
The solution of the probe BOD-Se (acetonitrileꢀ, 1 mmol/L) could
be maintained in refrigerator at 4 ꢂC. The ONOO source used for
chemical and biological experiments was the N-(ethoxycarbonyl)-3-
(4-morpholino)sydnone imine (molsidomine, SIN-10, 1 mmol/mL).
* Corresponding author. Tel.: þ86 411 84379293; fax: þ86 411 84675584.
E-mail address: klhan@dicp.ac.cn (K. Han).
Both authors contribute equally to this work.
1
0143-7208/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.dyepig.2012.09.006

384
B. Wang et al. / Dyes and Pigments 96 (2013) 383e390
NO is generated in form of 3-(Aminopropyl)-1-hydroxy-3-
10 mmol/L with 40 mmol/L PBS buffers (containing 5% acetonitrile),
isopropyl-2-oxo-1-triazene (NOC-5, 50
m
mol/ml). Methyl linoleate
the analytes were added. The mixture was equilibrated for 5 h
before measurement.
(MeLH) and 2,20-azobis-(2,4-dimethyl)valeronitrile (AMVN) were
ꢁ
used to produce MeLOOH. Superoxide radicals (Oꢀ2 ) were generated
in DMSO. DMSO was dried with CaH2 and excess CaH2 removed by
centrifugation. Potassium superoxide was subsequently introduced
into the dried DMSO, mixed, and undissolved KO2 removed by
2.4. HPLC analyses
BOD-Se and BOD were separated on an Elite HPLC system
equipped with fluorescence and UVevis absorption detectors.
ꢁ
centrifugation. The Oꢀ2 solution in DMSO was promptly used. tert-
Butylhydroperoxide (t-BuOOH) and cumene hydroperoxide
20
mL of sample was injected into the HPLC system equipped with
(CuOOH) could also use to induce ROS in biological systems. HOCl
a C18 column (Alltech, Kromasil, 250 mm ꢃ 4.6 mm, 5
mm) equili-
was standardized at pH 12 (
prepared by adding HOCl to a small excess of NaBr in water and
3
¼ 350 molꢀ1 Lcmꢀ1). HOBr was
292 nm
brated with CH3CN. The compounds were separated with
CH3CN:water ¼ 90:10 using a flow rate of 1 mL/min. Under those
conditions BOD-Se eluted at 13.5 min, and BOD at 7.7 min. The peak
signals were detected by monitoring the absorption at 567 nm.
ꢁ
standardizing at pH 12 (
3
¼ 332 molꢀ1L cmꢀ1). OH was
329 nm
generated by Fenton reaction between FeII(EDTA) and H2O2 quan-
ꢁ
titively, and FeII(EDTA) concentrations represented OH concentra-
tions. The blue fluorescent Hoechst dyes are cell permeable nucleic
acid stains. The fluorescence of these dyes is very sensitive to DNA
conformation and chromatin state in cells. The regents were
purchased from Invitrogen Corporation, and used according to the
manufacturer’s instructions. All other chemicals were from
commercial sources and of analytical reagent grade, unless indicated
otherwise. RAW264.7 cells (mouse macrophages cell line) were
purchased from the Committee on type Culture Collection of
Chinese Academy of Sciences. Ultrapure water was used throughout.
2.5. Confocal imaging
Florescent images were acquired on Olympus FV1000 confocal
laser-scanning microscope with an objective lens ꢃ 40. The exci-
tation wavelength was 543 nm. Prior to imaging, the medium was
removed. Cell imaging was carried out after washing cells with
40 mmol/L PBS for three times.
2.6. Cell culture
2.2. Instruments
Murine RAW264.7 macrophage cells (ATCC, USA) were main-
tained following protocols provided by the American Type Culture
Collection. Cells were seeded at a density of 1 ꢃ 106 cells mLꢀ1 for
confocal imaging in RPMI 1640 Medium supplemented with 15%
fetal bovine serum (FBS), NaHCO3 (2 g/L), and 1% antibiotics
(penicillin/streptomycin, 100 U/ml). Cultures were maintained at
37 ꢂC under a humidified atmosphere containing 5% CO2. The cells
were subcultured by scraping and seeding on 20/12 mm Petri
dishes according to the instructions from the manufacturer.
Fluorescence spectra were obtained by FluoroMax-4 Spectro-
fluorometer with a Xenon lamp and 1.0-cm quartz cells. Absorption
spectra were measured on Lambda 35 UVevisible spectropho-
tometer (PerkinElmer). 77Se, 1H and 13C NMR spectra were taken on
a Bruker spectrometer. The 1H, 13C, and 77Se chemical shifts are
given in parts per million relative to those of Me4Si, internal CDCl3
in the solvent, and external PhSeSePh, respectively. The fluores-
cence images of cells were taken using an LTE confocal laser
scanning microscope (Olympus FV1000 confocal laser-scanning
microscope) with objective lens (ꢃ40).
2.7. Synthesis and characterization of compounds
The synthetic route for the probe BOD-Se was shown in Figs. 1
2.3. Absorption and fluorescence analysis
and 2.
Absorption and fluorescence spectra were obtained with 10-mm
glass cells. The probe BOD-Se (acetonitrile, 0.10 mL, 1.0 mmol/L)
was added to a 10 mL color comparison tube. After dilution to
2.7.1. Synthesis of 4-(ethylamino)benzaldehyde
A mixture of an 4-fluorobenzaldehyde (12.4 g, 0.1 mol), 60%
aqueous ethylamine solution (50 mL, 0.67 mol), and a stirring bar
Fig. 1. Synthetic procedures of BOD.

B. Wang et al. / Dyes and Pigments 96 (2013) 383e390
385
Fig. 2. Synthetic routes of BOD-Se.
was sealed in a 100 mL autoclave and stirred electromagnetically in
an oil bath at 130 ꢂC for 15 h [23,24]. The mixture was extracted
with ethyl acetate for 3 times (50 mL ꢃ 3), combined organic
extracts and washed by 10% hydrochloric acid twice (50 mL ꢃ 2).
The pH of the aqueous phase was adjusted to 9 by 10% sodium
carbonate solutions. The aqueous phase was extracted with ethyl
acetate for 3 times (30 mL ꢃ 3), combined organic extracts and
dried with NaSO4. Then the solvent was evaporated on a rotary
evaporator until dry. The solid was purified on silica gel chroma-
tography (200e300 mesh) eluted with dichloromethane. The
target product was obtained as light yellow solid (4 g, yield: 30%).
HR-MS (EI) C9H11NO calcd. 149.0841, found [M þ H]þ 149.0840.
microcrystalline solid (18.8 g, 66%). m.p. 68e70 ꢂC. 1H NMR
(400 MHz, CDCl3-D1) (ppm): 7.61e7.59 (d, 6H), 7.26e7.23 (m, 4H).
77Se NMR (95 MHz, CDCl3-D1)
(ppm): 463.27.
d
d
2.7.4. Synthesis of 2-(phenylselanyl)benzoic acid
A mixture of 1,2-diphenyldiselane (4 g, 0.013 mol) and hydra-
zine hydrate (80%, 0.4 mL) was stirred in a nitrogen atmosphere,
then added sodium methoxide (1.3 g in 4 g methanol). 10 min later,
o-chlorobenzene nitrile (1.4 g, 0.01 mol) was added in the mixture.
Then the reaction was continued to stir for 10 h at 130 ꢂC [28].
Hydrochloric acid (30 mL, 36.5%) was added into150 mL water. Next
the reaction mixture was poured into the acidic solution. The
aqueous phase was extracted with ether for 3 times (180 mL ꢃ 3),
combined organic extracts and dried with NaCO3. Then the solvent
was evaporated on a rotary evaporator until dry. The solid was
recrystallized with ethanol, affording white solid 2 g, yield 76.9%.
2.7.2. Synthesis of BOD
1,3,5,7-Tetramethyl-8-phenyl-difluorobordiazaindacene
(760 mg, 2.3 mmol) and 4-ethylaminobenzaldehyde (300 mg,
2.1 mmol) were heated at 140 ꢂC for 6 h in a mixture of o-dichlo-
robenzene (20 mL) and piperidine (1 mL) under N2 [25,26]. After
cooling to room temperature, the mixture was evaporated on
a rotary evaporator until dry. The solid was purified on silica gel
chromatography (200e300 mesh) eluted first with dichloro-
methane: petroleum ether ¼ 1:1 (v/v), then eluted with dichloro-
methane, collected the red product. After evaporated the eluent, it
2.7.5. 2-(Phenylselanyl)benzonitrile
(1.5 g, 6 mmol) was dissolve in 16 mL DMSO, after added 4%
NaOH 30 mL, slowly heated the mixture at 100 ꢂC for 6 h. After
cooled the reaction system to 0 ꢂC, the pH of the solution was
adjusted to 2 by hydrochloric acid. The aqueous phase was
extracted with dichloromethane for 3 times (50 mL ꢃ 3), combined
organic extracts and dried with NaSO4. Then the solvent was
evaporated on a rotary evaporator until dry. The solid was recrys-
tallized with ethanol, affording white solid 1 g. Melting point: 203e
afforded green solid 0.25 g. 1H NMR (400 MHz, CDCl3-D1)
d(ppm):
7.50e7.44 (m, 6H), 7.32e7.30 (m, 2H), 7.19 (d, 1H), 6.57 (d, 3H), 5.97
(s,1H), 3.89 (s,1H), 3.22 (q, 2H), 2.59 (s, 3H),1.42 (s, 3H),1.38 (s, 3H),
1.28 (t, 3H). GCeMS (API-ES): m/z C28H28BF2N3 Calcd 455.2, found
455.2.
205 ꢂC. 1H NMR (400 MHz, CDCl3-D1)
8.17 (d, 1H), 7.73-7.71 (d, 2H), 7.49e7.41(m, 3H), 7.26e7.18 (m,
2H), 6.94e9.93 (d, 1H). 13C NMR (100 MHz, CDCl3-D1)
(ppm):
171.85, 141.66, 137.66, 133.48, 132.49, 129.88, 129.32, 129.18, 128.77,
d (ppm): 11.33 (s, 1H), 8.19-
d
2.7.3. Synthesis of 1,2-diphenyldiselane
Under N2, phenylmagnesium bromide is prepared from 32 g
(0.20 mol) of bromobenzene, 4.8 g (0.20 mol) of magnesium, and
106 ml of anhydrous ether [27]. Then 14.5 g (0.184 mol) of
elemental selenium is added in portions at a rate sufficient to
maintain a vigorous reflux. After that the mixture is stirred and
heated at reflux for another 30 min 0.6 ml of water is added to
hydrolyze any excess Grignard reagent, and cooled in an ice bath
while 5 ml of bromine is added dropwise at a rate such that the
ether does not reflux. Finally, 28 ml of ammonium chloride solution
(30%) is added. The mixture is filtered, and the granular precipitate
is washed thoroughly with ether. The combined filtrates are dried
over anhydrous Na2SO4 and then evaporated. Recrystallization
from hexane followed by washing with pentane afforded a yellow
126.15, 124.94. 77Se NMR (95 MHz, CDCl3-D1)
d (ppm): 470.29. LC-
MS (API-ES): m/z C13H10O2Se Calcd 277.9, found [M ꢀ H]ꢀ 276.9.
2.7.6. Synthesis of BOD-Se
2-(phenylselanyl)benzoic acid (0.3 g, 1 mmol) was added into
a solution containing 2 mL thionyl chloride and 10 mL dichloro-
methane, the mixture was stirred at 65 ꢂC for 7 h. Then the solvent
was evaporated on a rotary evaporator until dry. The solid was
dissolved with 10 mL benzene. BOD (0.41 g, 0.9 mmol) was invested
in the reaction system under Ar. The reaction was lasted at 40 ꢂC for
2 h, then added triethylamine (0.5 mL in 10 mL CH2Cl2). The
mixture was continued for 5 h. After cooling to room temperature,
the mixture was evaporated on a rotary evaporator until dry. The

386
B. Wang et al. / Dyes and Pigments 96 (2013) 383e390
solid was purified on silica gel chromatography (200e300 mesh)
eluted first with dichloromethane: petroleum ether ¼ 1:1 (v/v),
then eluted with dichloromethane, collected the red product. After
evaporated the eluent, it afforded red solid (0.57 g, 0.8 mmol). 1H
electron-donor, 4-(ethylamino)benzaldehyde, into the BODIPY core
via Knoevenagel-type reaction. The dye, 3-ethylaminostyryl-
difluoroboron dipyrromethene (BOD), emits faint fluorescence in
aqueous solution because the ICT state can strongly reduce the
fluorescence quantum yields in polar solvents (Fig. 4) [32,38,39].
After equipped with diaryl selenide moiety through amide bond,
the formation of ICT state in BOD can be restrained, and this will
trigger strong fluorescence emission. As shown in Figs. 3 and 5,
upon detected ONOOꢀ, diaryl selenide was oxidized to selenoxide,
the selenoxide would induce the spirocyclization reaction to
release free BOD, at which point the ICT state came back, and the
fluorescence emission was prevented obviously.
NMR (400 MHz, CDCl3-D1) d(ppm): 7.60e7.29 (m, 8H), 7.16e6.96
(m, 12H), 6.55 (s, 1H), 6.03 (s, 1H), 4.10e3.99 (q, 2H), 2.60 (s, 3H),
1.59 (s, 3H),1.42e1.39 (d, 3H),1.27e1.26 (m, 3H). 13C NMR (100 MHz,
CDCl3-D1)
d (ppm): 182.25, 153.79, 143.62, 142.94, 141.93, 141.54,
141.37, 135.62, 132.36, 131.69, 131.62, 130.92, 130.46, 130.13, 129.98,
129.75, 129.50, 129.15, 128.85, 127.62, 123.06, 122.61, 115.21, 115.01,
112.47, 37.83, 23.17, 19.19, 17.36, 13.72. 77Se NMR (95 MHz, CDCl3-
D1)
d (ppm): 444.05. LC-MS (API-ES): m/z C41H36BF2N3OSe Calcd
715.2085, found 715.2094.
3.2. Fluorescence spectra of BOD in different solutions
2.7.7. Characterization of BOD
BOD-Se 1 mmol was dissolved in 0.5 mL of acetonitrile. Subse-
quently, 100 equiv. of SIN-10 was added. After 5 h, BOD was
confirmed by 1H NMR and MS. 1H NMR (400 MHz, CDCl3-D1)
In n-hexane and dichloromethane solution with low polarity,
the fluorescence emission of BOD was rather stronger, however,
when the solvents were changed to PBS buffer solution (40 mmol/L
pH 7.4 containing 5% acetonitrile) with higher polarity, the inten-
sity decreased obviously (Fig. 4). These results exhibited that the
fluorescence emission of BOD was quenched by the charge sepa-
ration at the excited state, in which the high polarity of the solvent
stabilized the charge separation of the ICT process.
d(ppm): 7.51e7.45 (m, 7H), 7.31e7.30 (m, 2H), 7.18 (d, 1H), 6.67 (d,
2H), 6.58 (d, 1H), 5.97 (s, 1H), 3.23 (q, 2H), 2.59 (s, 3H), 1.41 (s, 3H),
1.38 (s, 3H), 1.29 (t, 3H). LC-MS (API-ES): m/z Calcd 455.2, found
[M þ H]þ 456.2.
3. Results and discussion
3.3. Investigation of spectral properties of probe BOD-Se
3.1. Design strategies of fluorescent probe BOD-Se
The absorption and emission spectra of BOD-Se (10
mmol/L)
In our previous work, we mimicked the glutathione peroxidase
(GPx) catalytic cycle and developed a fluorescent probe that can be
used for reversible peroxynitrite detection [29]. Herein, we
employed a new design approach for the selectivedetection of
ONOOꢀ by a synthetic fluorescent probe BOD-Se. Fig. 3 illustrates
our overall strategy for ONOOꢀ detection assays. As a GPx mimics,
diaryl selenide shows the effects on ONOOꢀ-mediated oxidation
reactions, and themselves are oxidize to selenoxide [30]. Selen-
oxide undergoes a spirocyclization reaction to produce the spi-
rodioxyselenurane [31]. We integrated the diaryl selenide into
a BODIPY dye (4,4-difluoro-4-3a,4a-diza-s-indacene, difluoroboron
dipyrromethene) scaffold, for which held good photostability, high
molar absorption coefficients and fluorescence quantum yields,
sharp absorption and fluorescence peaks [32e37]. As shown in
Fig. 3, we attribute the detection mechanism to intramolecular
charge-transfer (ICT) process [32,38,39]. We extend the pushepull
electronic system of the whole molecule by incorporating a strong
were evaluated in an aqueous buffered solution to physiological pH
7.4 (40 mmol/L PBS) containing 5% acetonitrile as co-solvent. BOD-
Se features one prominent optical band at 559 nm
(
3
¼ 7.6 ꢃ 104 molꢀ1L cmꢀ1) and the corresponding fluorescence
emission maximum at 572 nm with strong fluorescence (
F
¼ 0.96).
Here, we used N-(ethoxycarbonyl)-3-(4-morpholino)sydnoneimi-
ne(molsidomine, SIN-10) as the peroxynitrite source. The drug can
provide ONOOꢀ continuously and steadily within a long time in
presence of O2 [40]. Upon addition of 200 mmol/L SIN-10 to the
buffer solution containing BOD-Se, the absorption spectrum of
ONOOꢀ-oxidized probe displays a single major visible absorption
band at 594 nm (
3
¼ 8.9 ꢃ 104 molꢀ1 L cmꢀ1) accompanied with
color changes from red to blue. The isosbestic point is at 567 nm
(Fig. 5a). The fluorescence intensity of BOD-Se decreased by ca.
225-fold with a red shift of the emission maximum to 680 nm. This
phenomenon was consistent with the ICT signaling mechanism as
expected. However, the ICT effect in BOD was so distinguished that
Fig. 3. The design strategy, structure, and detection mechanism of BOD-Se.

B. Wang et al. / Dyes and Pigments 96 (2013) 383e390
387
selenoxide would induce the spirocyclization reaction to release
free BOD. The oxidative product was also tested by 1H NMR and
mass spectrometry. We also employed High-Performance Liquid
Chromatography (HPLC) to monitor the reaction process between
ONOOꢀ and probe BOD-Se. Fig. 6 showed the HPLC chromatogram
signals of the reaction between BOD-Se (10
(derived from 200 mol/L SIN-10) during 5 h. In the presence of
m
mol/L) and ONOOꢀ
m
different times, the intensity of the peak BOD-Se (eluted at
13.5 min) decreased and oxidation products peaks eluted at 7.7 min
emerged. These results indicated that BOD-Se could detect ONOOꢀ
in water solution by HPLC.
3.4. Effects of pH values on fluorescence intensity
Fig. 4. The fluorescence spectra of BOD (10
mmol/L) in different solutions.
lex ¼ 594 nm, fluorescence acquisition from 580 to 800 nm.
We investigated the effect of pH on the fluorescence emission.
The probe BOD-Se itself had nearly no effect on fluorescence
intensity by the pH of the mediums within the range from 1.0 to 9.0.
However, the fluorescence of the oxidized product BOD showed
strong dependent on pH. The fluorescence of BOD began to quench
at pH above 2.0. We attributed the phenomenon of the fluorescence
decreasing to the deprotonation of the ammonia group increasing
the electron-donating ability of the lone pair electrons in ammonia
group and strengthening the ICT effect. The pKa value of protonated
BOD was determined to be 2.46 (Fig. 7). These spectral properties
suggested that ICT was regulated in the molecule successfully. As
shown in Fig. 7, the pH of the medium had hardly any effect on the
the electronic excited state would hinder the fluorescence emission
almost completely (Fig. 5b). This specific chemical reaction could
complete within 5 h (Fig. S1), therefore, in the later tests, we chose
5 h this point in time. In aqueous buffered solution, BOD only
provided a fluorescence quantum yield of
F
¼ 0.05. Therefore, the
steady-state fluorescence spectra of BOD-Se varied obviously than
that of BOD in visual. In order to indicate the trend of fluorescence
changes at 680 nm, the fluorescence spectra were normalized to
the BOD-Se maximum emission (Fig. 5b Insert). As shown in Fig. 5b
Insert, the emission maximum at 680 nm was increasing gradually.
The spectral changes confirmed our detection mechanism that the
oxidized probe (BOD) over the pH range from 6.8 to 7.8.
DF ¼ F0 ꢀ F,
where F0 is fluorescence intensity of the probe BOD-Se, and F is the
maximum fluorescence intensity of BOD at different pH, respec-
tively. Therefore, the probe was expected to work well under
physiological conditions.
3.5. Linear relationship between fluorescence intensity and ONOOꢀ
concentrations
In order to explore the ability of BOD-Se in the determination
of ONOOꢀ concentration in water solution, the probe was treated
with SIN-10 under varies concentrations for 5 h (Fig. 8). The final
concentration of BOD-Se was maintained at 10
concentrations of SIN-10 varied from 0 to 200
m
mol/L, while the
mol/L. For the
m
easy and accurate analysis, a linear relationship between fluo-
rescence signal and target analyte is always important. Now we
got a standard curve between emission intensity (F572 nm) and
ONOOꢀ concentrations. The regression equation was F572
¼ ꢀ515 ꢃ [ONOOꢀ]
m
mol/L þ 100,772, with r ¼ 0.994.
nm
Fig. 5. Time-dependent absorption and fluorescence spectra of probe BOD-Se
(10 mmol/L) upon treatment with molsidomine (SIN-10, 200 m
mol/L) at 37 ꢂC in PBS
buffer solution (40 mmol/L, pH 7.4, 5% acetonitrile). Spectra were acquired at 0, 0.5, 1,
1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 h after incubation of the probe with SIN-10. a) BOD-Se
displayed a major absorption band centered at 559 nm. Upon reaction with SIN-10,
Fig. 6. Monitoring the reaction process between BOD-Se and ONOOꢀ by HPLC anal-
yses. The concentrations of BOD-Se was 10
mol/L, ONOOꢀ was derived from
200
mol/L SIN-10 at 37 ꢂC in PBS buffer solution (40 mmol/L, pH 7.4, 5% acetonitrile).
HPLC chromatogram signals were acquired at 0, 1, 2.5, 4, 5 h after incubation of the
probe with SIN-10. BOD-Se eluted at 13.5 min, BOD eluted at 7.7 min with
acetonitrile:water ¼ 90 : 10 using UVeVis detector at 567 nm.
a
new absorption peak emerged at around 594 nm with an isosbestic point at
m
567 nm. b) Corresponding fluorescence emission spectra respond to SIN-10,
lex ¼ 567 nm, fluorescence acquisition from 525 to 800 nm. Inset: normalized fluo-
rescence spectra for ONOOꢀ detection to the emission maximum of BOD-Se in PBS
solution.
m

388
B. Wang et al. / Dyes and Pigments 96 (2013) 383e390
Fig. 7. The effect of pH value on the fluorescence intensity of the probe in 40 mmol/L
PBS. Fluorescence pH titrations were performed in the solution at BOD-Se and BOD all
Fig. 9. Fluorescence response of 10
(ROS). Bars represent fluorescence intensity at 0, 1, 2, 3, 4, and 5 h after addition of
various ROS. 1, SIN-10 (200 mol/L); 2, NO (200 mol/L); 3, H2O2 (200 mol/L); 4,
methyl linoleate hydroperoxide (MeLOOH, 200 mol/L); 5, tert-butyl hydroperoxide (t-
BuOOH, 200 mol/L); 6, cumene hydroperoxide (CuOOH, 200
mol/L); 7, ꢁOH
(200 mol/L).
mol/L); 8, ClOꢀ (200 mol/L); 9, BrOꢀ (200 mol/L); 10, Oꢁ2ꢀ (200
mmol/L BOD-Se to various reactive oxygen species
at concentration of 10
m
mol/L, respectively (lem ¼ 567 nm).
DF ¼ F0 ꢀ F, where F0 is
m
m
m
fluorescence intensity of the probe BOD-Se, and F is the maximum fluorescence
intensity of BOD at different pH, respectively. pH values: 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.8,
3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.4, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4,
7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8, 9.0.
m
m
m
m
m
m
m
words, BOD-Se can selectively respond to ONOOꢀ and can avoid
a host of other relevant ROS. These Selective experiment results
elaborate that the selective oxidation of BOD-Se to BOD can be used
for fluorescence detection of ONOOꢀ under simulated physiological
conditions.
Standard errors for slope and intercept were 18 and 3095,
respectively. We also obtained a linear relationship between the
normalized fluorescence intensity (F680
)
and ONOOꢀ
was F680
mol/L e 0.106, with r ¼ 0.996. Standard
nm
concentrations,
the
regression
equation
¼ 0.005 ꢃ [ONOOꢀ]
m
nm
errors for slope and intercept were 0.0002 and 0.028, respec-
tively. The limit ONOOꢀ detection was to be 5
mmol/L. All these
3.7. Fluorescence imaging of endogenous ONOOꢀ in RAW264.7 cells
spectroscopic properties of BOD-Se demonstrate that our probe
BOD-Se can respond to ONOOꢀ under simulated physiological
conditions.
With spectroscopic data establishing that BOD-Se can selec-
tively respond to ONOOꢀ in aqueous solution, the diaryl selenide
dye was next tested in living biological samples using confocal
microscope under physiological conditions. We chose the mouse
macrophage cell line RAW264.7 as a model cell line, because the
macrophage cells activate the generation of reactive oxygen and
nitrogen species after exposure to lipopolysaccharide (LPS)/inter-
3.6. Selectivity of fluorescent response to peroxynitrite
To study the specificity of BOD-Se toward peroxynitrite, an
important procedure was carried out to determine whether reac-
tive oxygen species (ROS) other than ONOOꢀ could potentially
introduce the fluorescent signal changes. As demonstrated in Fig. 8,
our probe BOD-Se can dynamically respond to ONOOꢀ level
changes in aqueous media (PBS buffer at pH 7.4). The relevant ROS,
including H2O2, tert-butyl hydroperoxide (t-BuOOH), cumene
feron-
RAW264.7 cells were stimulated by various reagents, incubated
with BOD-Se (10 mol/L) for 20 min, and then washed three times
with PBS buffer before imaging. Live RAW264.7 cells loaded with
10
mol/L BOD-Se for 20 min at 37 ꢂC showed levels of background
fluorescence from latent probes (Fig. 10a). After addition of
100 mol/L SIN-10 to BOD-Se-loaded cells for 5 h, there triggered
g (IFN-g) and phorbol 12-myristate 13-acetate (PMA) [41].
m
m
ꢁ
hydroperoxide (CuOOH), OH, ClOꢀ, and BrOꢀ undergo limited
m
fluorescence responses. However, the intensity of the fluorescence
changes is far weaker than that caused by ONOOꢀ (Fig. 9). In other
clear decrease in intracellular fluorescence because the probe
detected oxidative stress caused by SIN-10 (Fig. 10b). Faint fluo-
rescence was also observed after treatment with LPS (1
mg/mL) and
IFN- (50 ng/mL) for 4 h followed by additional stimulation with
g
PMA (10 nmol/L) for 0.5 h (Fig. 10c). When the cells described in
Fig. 10c were pretreated with aminoguanidine (AG), an inhibitor of
nitric oxide synthase [42], much stronger fluorescence was detec-
ted in the stimulated cells (Fig. 10d). Dye-loaded cells in Fig. S2
lasted cultured for 6 h showed a strong intracellular fluorescence.
The experiment demonstrates that the low levels of intracellular
fluorescence of Fig. 10b and 10c were not due to photobleaching or
dye leakage. These results indicate that BOD-Se can be used to
visualize ONOOꢀ in cells. Next, we examined the subcellular loca-
tions of endogenous peroxynitrite in RAW264.7 cells using confocal
fluorescence microscopy. By co-staining with cell permeable
nucleic acid stains, Hoechst dye, and overlaying with brightfield
images, we confirmed that the probe was retained in the cytoplasm
(Fig. 10eeh). Brightfield transmission measurements (see SI) after
incubation with both BOD-Se and various reagents along with the
nuclear stain Hoescht dye [43] revealed that the cells are viable
throughout the imaging experiments.
Fig. 8. The linear relationship between the fluorescence intensity at 572 nm and
ONOOꢀ. The probe BOD-Se (10 mol/L) was treated with SIN-10 at 37 ꢂC in PBS buffer
m
solution (40 mmol/L, pH 7.4, 5% acetonitrile) for 5 h. SIN-10: 0, 10, 20, 30, 40, 60, 80,
100, 120, 140, 160, 180, 200 M. Insert: Relationship between the normalized fluo-
rescence intensity at 680 nm and ONOOꢀ
m
.

B. Wang et al. / Dyes and Pigments 96 (2013) 383e390
389
Fig. 10. Confocal fluorescence images of ONOOꢀ induced oxidative stress in RAW264.7 cells. Macrophage cells were incubated with BOD-Se (10
m
mol/L) for 20 min and then treated
with various stimulants at 37 ꢂC for next 5 h. (a,e,i) Controls. (b,f,j) Cells treated with 100
mmol/L SIN-10 for 15 min (c,g,k) LPS (1
mg/mL) and IFN-g (50 ng/mL) for 4 h then PMA
(10 nmol/L) for 0.5 h. (d,h,l) AG (1 mmol/L), LPS (1 mg/mL), and IFN-g (50 ng/mL) for 4 h then PMA (10 nmol/L) for 0.5 h. (eeh) overlay of images showing fluorescence from BOD-Se
and Hoescht dye; (iel) overlay of bright-field, BOD-Se, and Hoescht dye images.
4. Conclusions
peroxynitrite detection. BOD-Se is equipped with a chemospecific
ONOOꢀ switch to modulate ICT process with a marked red-to-blue
color change in absorption. The probe exhibits high sensitivity and
selectivity in detecting peroxynitrite oxidation under physiological
conditions in aqueous solution. Confocal microscopy imaging using
In summary, we have presented the synthesis, spectroscopic
properties, and live-cell application of a new type of BODIPY fluo-
rescence probe based on selenoxide spirocyclization reaction for

390
B. Wang et al. / Dyes and Pigments 96 (2013) 383e390
[20] Zielonka J, Sikora A, Joseph J, Kalyanaraman B. Peroxynitrite is the major
species formed from different flux ratios of co-generated nitric oxide and
superoxide: direct reaction with boronate-based fluorescent probe. J Biol
Chem 2010;285:14210e6.
RAW264.7 cell lines show that BOD-Se is able to sense different
intracellular ONOOꢀ level changes.
[21] Yu FB, Song P, Li P, Wang BS, Han KL. A fluorescent probe directly detect
peroxynitrite based on boronate oxidation and its applications for fluores-
cence imaging in living cells. Analyst 2012;137:3740e9.
Acknowledgements
[22] Ma JJ, Wu JS, Liu WM, Wang PF, Fan ZY. Ruthenium(II) complex-based fluo-
rescent sensor for peroxynitrite. Spectrochim Acta Part A 2012;94:340e5.
[23] Wang QC, Qu DH, Ren J, Xu LH, Liu MY, Tian H. New benzo[e]indolinium
cyanine dyes with two different fluorescence wavelengths. Dyes Pigments
2003;59:163e72.
This work was supported by NSFC (20833008).
Appendix A. Supplementary material
[24] Jiao J, Zhang XR, Chang NH, Wang J, Wei JF, Shi XY, et al. A facile and practical
copper powder-catalyzed, organic solvent- and ligand-free Ullmann amina-
tion of aryl halides. J Org Chem 2011;76:1180e3.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.dyepig.2012.09.006.
[25] Peng XJ, Du JJ, Fan JL, Wang JY, Wu YK, Zhao JZ, et al. A selective fluorescent
sensor for imaging Cd2þ in living cells. J Am Chem Soc 2007;129:1500e1.
[26] Rurack K, Kollmannsberger M, Daub J. Molecular switching in the near
infrared (NIR) with a functionalized boron-dipyrromethene dye. Angew Chem
Int Ed 2001;40:385e7.
[27] Reich HJ, Cohen ML, Clark PS. Reagents for synthesis of organoselenium
compounds: diphenyl diselenide and benzeneselenenyl chloride. Org Synth
1979;59:141.
[28] Henriksen L, Stuhr-Hansen N. Rapid and precise preparation of reactive
benzeneselenolate solutions by reduction of diphenyl diselenide with
hydrazineesodium methanolate. J Chem Soc Perkin Trans 1999;1:1915e6.
[29] Yu FB, Li P, Li GY, Zhao GJ, Chu TS, Han KL. A near-IR reversible fluorescent
probe modulated by selenium for monitoring peroxynitrite and imaging in
living cells. J Am Chem Soc 2011;133:11030e3.
[30] Mukherjee AJ, Zade SS, Singh HB, Sunoj RB. Organoselenium chemistry: role of
intramolecular interactions. Chem Rev 2010;110:4357e416.
[31] Sarma BK, Manna D, Minoura M, Mugesh G. Synthesis, structure, spirocycli-
zation mechanism, and glutathione peroxidase-like antioxidant activity of
stable spirodiazaselenurane and spirodiazatellurane. J Am Chem Soc 2010;
132:5364e74.
References
[1] Ducrocq C, Blanchard B, Pignatelli B, Ohshima H. Peroxynitrite: an endogenous
oxidizing and nitrating agent. Cell Mol Life Sci 1999;55:1068e77.
[2] Radi R, Cassina A, Hodara R, Quijano C, Castro L. Peroxynitrite reactions and
formation in mitochondria. Free Radic Biol Med 2002;33:1451e64.
[3] Pacher P, Beckman JS, Liaudet L. Nitric oxide and peroxynitrite in health and
disease. Physiol Rev 2007;87:315e424.
[4] Ferrer-Sueta G, Radi R. Chemical biology of peroxynitrite: kinetics, diffusion,
and radicals. ACS Chem Biol 2009;4:161e77.
[5] Szabó C, Ischiropoulos H, Radi R. Peroxynitrite: biochemistry, pathophysiology
and development of therapeutics. Nat Rev Drug Discov 2007;6:662e80.
[6] Liaudet L, Vassalli G, Pacher P. Role of peroxynitrite in the redox regulation of
cell signal transduction pathways. Front Biosci 2009;14:4809e14.
[7] Chen SL, Jian L, Lang HQ. Optimization of peroxynitrite-luminol
chemiluminescence system for detecting peroxynitrite in cell culture solu-
tion exposed to carbon disulphide. Luminescence 2003;18:249e53.
[8] Bian ZY, Guo XQ, Zhao YB, Du JO. Probing the hydroxyl radical-mediated
reactivity of peroxynitrite by a spin-labeling fluorophore. Anal Sci 2005;21:
553e9.
[32] Boens N, Leen V, Dehaen W. Fluorescent indicators based on BODIPY. Chem
Soc Rev 2012;41:1130e72.
[33] Ulrich G, Ziessel R, Harriman A. The chemistry of fluorescent bodipy dyes:
versatility unsurpassed. Angew Chem Int Ed 2008;47:1184e201.
[34] Guo HM, Jing YY, Yuan XL, Ji SM, Zhao JZ, Li XH, et al. Highly selective fluo-
rescent OFF-ON thiol probes based on dyads of BODIPY and potent intra-
molecular electron sink 2,4-dinitrobenzenesulfonyl subunits. Org Biomol
Chem 2011;9:3844e53.
[9] Daiber A, Oelze M, August M, Wendt M, Sydow K, Wieboldt H, et al. Detection
of superoxide and peroxynitrite in model systems and mitochondria by the
luminol analogue L-012. Free Radic Res 2004;38:259e69.
[10] Xue J, Ying XY, Chen JS, Xian YZ, Jin LT, Jin JY. Amperometricultramicrosensors
for peroxynitrite detection and its application toward single myocardial cells.
Anal Chem 2000;72:5313e21.
[35] Chen YH, Zhao JZ, Guo HM, Xie LJ. Geometry relaxation-induced large stokes
shift in red-emitting borondipyrromethenes (BODIPY) and applications in
fluorescent thiol probes. J Org Chem 2012;77:2192e206.
[11] Wang R, Yu CW, Yu FB, Chen LX. Molecular fluorescent probes for monitoring
pH changes in living cells. In: TrAC Trends Anal Chem, vol. 29; 2010. p.
1004e13.
[12] Ueno T, Nagano T. Fluorescent probes for sensing and imaging. Nat Methods
2011;8:642e5.
[13] Ueno T, Urano Y, Setsukinai K, Takakusa H, Kojima H, Kikuchi K, et al. Rational
principles for modulating fluorescence properties of fluorescein. J Am Chem
Soc 2004;126:14079e85.
[14] Ueno T, Urano Y, Kojima H, Nagano T. Mechanism-based molecular design of
highly selective fluorescence probes for nitrative stress. J Am Chem Soc 2006;
128:10640e1.
[15] Yang D, Wang HL, Sun ZN, Chung NW, Shen JG. A highly selective fluorescent
probe for the detection and imaging of peroxynitrite in living cells. J Am Chem
Soc 2006;128:6004e5.
[16] Sun ZN, Wang HL, Liu FQ, Chen Y, Tam PK, Yang D. BODIPY-based fluorescent
probe for peroxynitrite detection and imaging in living cells. Org Lett 2009;
11:1887e90.
[17] Oushiki D, Kojima H, Terai T, Arita M, Hanaoka K, Urano Y, et al. Development
and application of a near-infrared fluorescence probe for oxidative stress
based on differential reactivity of linked cyanine dyes. J Am Chem Soc 2010;
132:2795e801.
[18] Peng T, Yang D. HKGreen-3: a rhodol-based fluorescent probe for peroxyni-
trite. Org Lett 2010;12:4932e5.
[36] Pahari P, Chaturvedi S. Determination of best-fit potential parameters for
a
reactive force field using
a genetic algorithm. J Mol Model 2012;18:
1049e61.
[37] Zhang ZY, Xu B, Su JH, Shen LP, Xie YS, Tian H. Color-tunable solid-state
emission of 2,20-biindenyl-based fluorophores. Angew Chem Int Ed 2011;
50:11654e7.
[38] de Silva AP, Gunaratne HQ, Gunnlaugsson T, Huxley AJ, McCoy CP,
Rademacher JT, et al. Signaling recognition events with fluorescent sensors
and switches. Chem Rev 1997;97:1515e66.
[39] Baruah M, Qin W, Flors C, Hofkens J, Vallée R, Beljonne D, et al. Solvent and pH
dependent fluorescent properties of
a
dimethylaminostyryl bor-
ondipyrromethene dye in solution. J Phys Chem A 2006;110:5998e6009.
[40] Bassenge E, Kukovetz WR. Molsidomine. Cardiovasc Drug Rev 1984;2:
177e91.
[41] Iovine NM, Pursnani S, Voldman A, Wasserman G, Blaser MJ, Weinrauch Y.
Reactive nitrogen species contribute to innate host defense against
campylobacter jejuni. Infect Immun 2008;76:986e93.
[42] Muijsers RB, Van Den Worm E, Folkerts G, Beukelman CJ, Koster AS,
Postma DS, et al. Apocynin inhibits peroxynitrite formation by murine
macrophages. Br J Pharmacol 2000;130:932e6.
[43] Dickinson BC, Peltier J, Stone D, Schaffer DV, Chang CJ. Nox2 redox
signaling maintains essential cell populations in the brain. Nat Chem Biol
2011;7:106e12.
[19] Xu KH, Chen HC, Tian JW, Ding BY, Xie YX, Qiang MM, et al. A near-infrared
reversible fluorescent probe for peroxynitrite and imaging of redox cycles in
living cells. Chem Commun 2011;47:9468e70.