A BODIPY-based probe for the selective detection of hypochlorous acid in living cells

Article abstract of DOI:10.1002/asia.201100025

Stress light up! Methyl thio-BODIPY 2 detects hypochlorous acid on the basis of the selective oxidation of its methylthioether group into the corresponding sulfoxide by the analyte, resulting in a strong fluorescence turn-on signal. Copyright

Full text of DOI:10.1002/asia.201100025

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DOI: 10.1002/asia.201100025
A BODIPY-Based Probe for the Selective Detection of Hypochlorous Acid
in Living Cells
Tae-Il Kim,[a] Seonhwa Park,[b] Yongdoo Choi,*[b] and Youngmi Kim*[a]
Reactive oxygen species (ROS) are essential for a wide
range of biological and pathological events.[1] During infec-
tion and inflammation, the phagocytic leukocytes, including
neutrophils, monocytes, and macrophages generate reactive
oxygen species (ROS) to kill invading bacteria and patho-
gens.[2] Among ROS, hypochlorous acid (HOCl/OClÀ) is a
highly reactive oxygen species produced from hydrogen per-
oxide (H2O2) and chloride ions (ClÀ) by the enzyme myelo-
peroxidase (MPO), which is secreted by activated neutro-
phils.[3] Although hypochlorous acid plays important roles in
the human immune-defense system, overproduction of ROS
in living organism has detrimental effects on biological mol-
ecules, including nucleic acids, lipids, and proteins, resulting
in the inhibition of various protein functions, and contrib-
utes to the progression of numerous human diseases, such as
atherosclerosis, cancer, cardiovascular diseases, and rheuma-
toid arthritis.[4]
Despite its importance in human health and disease, not
as much is known about the mechanism of action and specif-
ic roles of HOCl in living systems in comparison with other
ROS, owing to slower progress in the development of suita-
ble probes. Several fluorescence probes for the detection
and visualization of HOCl in living cells have recently been
developed on the basis of the strong oxidizing properties of
HOCl.[5–7] HOCl-induced oxidation reactions were em-
ployed in the design of fluorescent probes in which the fluo-
rescence properties were regulated by the conversion of the
spirocyclic form of rhodamine fluorophores into their ring-
opened form,[5] or through photoinduced-electron-transfer
processes.[6] To facilitate practical applications of such
probes, next-generation designs should emphasize higher an-
alyte selectivity, limit susceptibility to autooxidation, and
avoid demanding multistep syntheses. Herein, we report the
facile synthesis and properties of a new boron-dipyrrome-
thene (BODIPY) dye bearing a methylthioether group, and
its biological application as a highly sensitive and selective
ꢀturn-onꢁ fluorescent probe for HOCl in living cells. We en-
visioned that the oxidation of thioethers into sulfoxides in
the presence of HOCl could act as a sensing trigger to mod-
ulate the optical properties of molecular probes. We chose a
BODIPY-dye scaffold owing to their desirable features that
include high absorption coefficients, high fluorescence quan-
tum yields, excellent photostability, and versatile chemis-
try.[8] Their tolerance to spontaneous autooxidation and pho-
tobleaching is equally advantageous under bioimaging con-
ditions. Substitution of the BODIPY core at the 2- and 6-po-
sitions is known to greatly affect its photophysical proper-
ties. In particular, the introduction of electron-donating
groups at these positions results in bathochromically shifted
absorption and emission bands, and substitution with heavy
atoms is known to cause marked decrease in fluorescence
quantum yields.[9]
To introduce the desired methylthioether group at these
nucleophilic positions, we relied on the known reaction of
nucleophilic p-systems with activated dimethyl sulfoxide re-
agents.[10] Thus, the methylthio-substituted BODIPY probe
2, is easily prepared in a single step starting from 1 by an
electrophilic substitution reaction according to Scheme 1.
The formation of 2 likely occurs through the reaction of 1
with in-situ generated chlorodimethylsulfonium chloride,
which reacts either as a chlorinating or a thiomethylating
agent, and provides probe 2 in high yield (74%). This
simple synthetic procedure is also applicable to other
BODIPY derivatives (See the Supporting Information).
We investigated the photophysical properties of the new
methylthio-BODIPY 2 in various solvents (See the Support-
[a] T.-I. Kim,+ Prof. Y. Kim
Department of Chemistry
Dankook University
126 Jukjeon-dong, Yongin-si, Gyeonggi-do 448-701 (Korea)
Fax : (+82)31-8005-3148
E-mail: youngmi@dankook.ac.kr
[b] S. Park,+ Dr. Y. Choi
Molecular Imaging & Therapy Branch
National Cancer Center
323 Ilsan-ro, Goyang-si, Gyeonggi-do 410-769 (Korea)
E-mail: ydchoi@ncc.re.kr
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.201100025.
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Chem. Asian J. 2011, 6, 1358 – 1361

range whereas the absorption spectra of probe 2 are slightly
red-shifted under acidic conditions (pH 3-6).
First, we measured the sensory response of probe 2
toward NaOCl in HEPES buffer (10 mm, pH 7.4, 0.2%
DMSO). As shown in Figure 1, the activation of probe 2
Scheme 1. Synthesis of methylthio-BODIPY 2. DMSO=dimethyl sulfox-
ide.
ing Information). Probe 2 shows an absorption maximum at
a wavelength comparable to that of precursor 1, but exhibits
a broad red-emission band with an unusually large Stokes
shift. In aqueous solution, probe 2 displays absorption and
emission maxima at 519 nm and 650 nm, respectively. The
emission spectrum of 2 is affected by solvent polarity (see
the Supporting Information, Table S1), which implies a
degree of internal charge transfer character associated with
these electronic transitions. As expected, probe 2 presents a
reduced fluorescence-quantum yield (FF 0.09) relative to
the parent BODIPY 1 (FF 0.62).[11]
As the introduction of the electron-donating methyl-
thioether substituent at the 2-position dramatically reduces
the quantum yield and red-shifts its emission spectra, oxida-
tion of the thioether into the corresponding electron-with-
drawing sulfoxide was predicted to restore the quantum
yield of the fluorophore and blue-shift its emission spectra,
thus providing a dark-field turn-on signal in the presence of
HOCl. To confirm this hypothesis, the expected oxidation
product 3 was prepared by oxidation of 2 and fully charac-
terized (see the Supporting Information). Gratifyingly, con-
version into the sulfoxide was accompanied by hypsochro-
mic shifts in the absorption (labs 505 nm) and emission (lem
525 nm) maxima that were evident to the naked eye
(Scheme 2). Moreover, 3 exhibits an excellent fluorescence
quantum yield (FF 0.82) in aqueous solution, as anticipated.
Figure 1. Fluorescence turn-on response of 2 (5 mm) to NaOCl (50 mm).
The spectra were obtained every 10 min (0–60 min) after the addition of
NaOCl. All data were obtained in HEPES buffer (10 mm, pH 7.4, 0.2%
DMSO) at 258C. Excited at 465 nm. Inset: A linear correlation between
emission intensity and concentrations of NaOCl. The emission intensity
of probe 2 (5 mm) at 525 nm was determined after incubation at 258C for
1 h in presence of NaOCl.
after addition of NaOCl results in a decrease of emission in-
tensity at 650 nm, together with the instant growth of the
emission band of 3 at 525 nm. A remarkable increase in the
fluorescence intensity was observed, depending on the con-
centration and reaction time (see the Supporting Informa-
tion). Upon treatment with 10 equivalents of NaOCl, the so-
lution of probe 2 already exhibited an approximately 88-fold
increase in its fluorescence intensity within only 10 minutes.
A linear correlation between the emission intensity and the
concentration of NaOCl was also found (Figure 1, inset).
Figure 2 compares the reactivity of probe 2 toward vari-
ous ROS. Probe 2 showed a positive response only in the
Scheme 2. Proposed sensing mode of probe 2.
To evaluate the practical utility of probe 2 in the detec-
tion of HOCl (pKa =7.6), the pH dependence of the photo-
physical properties of 2 and 3 was investigated in a series of
buffers with different pH values ranging from 3 to 9. These
results (see the Supporting Information, Figure S4) indicate
that the emission spectra of both compounds 2 and 3 are es-
sentially pH-insensitive across this biologically relevant pH
Figure 2. Relative fluorescence responses of 2 (5 mm) to various ROS
(25 mm NaOCl, 25 mm H2O2, and 200 mm for others). Bars represent rela-
tive responses at 0, 30, and 60 min after the addition of each ROS. Emis-
sion signals was recorded at 525 nm (excited at 465 nm). All data were
obtained in HEPES buffer (10 mm, pH 7.4, 0.2% DMSO) at 258C.
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Y. Choi, Y. Kim et al.
COMMUNICATION
presence of NaOCl and H2O2, whereas no significant fluo-
rescence signal at 525 nm was observed in presence of other
ROS, such as tert-butyl hydroperoxide (TBHP), superoxide
À
(O2 ), hydroxyl radical (·OH), peroxynitrite (ONOOÀ), and
tert-butoxy radical (·OtBu). The response of probe 2 also
shows selectivity greater than 10:1 in favor of HOCl over
H2O2.[12] LC-MS analysis of the test solutions confirmed that
the major product of the reaction of probe 2 with either
NaOCl or H2O2 was the expected oxidation product 3.
Hence, spectral changes observed in the presence of NaOCl
or H2O2 origin from the oxidation of the methylthioether 2
into the sulfoxide 3 (Figure 3; also see the Supporting Infor-
mation).
Figure 4. Relative confocal fluorescence images of living macrophages
(RAW264.7) under different conditions with probe 2. a) Macrophages
were incubated with 5 mm probe 2 for 1 h at 378C and then imaged. b)
Macrophage cells loaded with Probe 2 were incubated with 100 mm
NaOCl for 20 min. c) Macrophage cells were stimulated with 1 mgmLÀ1
LPS for 24 h, further stimulated with 1 mgmLÀ1 PMA for 1 h, and incu-
bated with 5 mm probe 2 for 1 h at 378C before being imaged. d) MPO in-
hibitor, 4-ABAH (final concentration=100 mm) was co-incubated during
PMA stimulation; the other procedures were the same. (top: fluores-
cence images, middle: bright-field images, bottom: merged images, Ex=
488 nm, Em=505–550 nm).
which showed a 5-fold signal increase within 20 minutes in
the presence of 100 mm HOCl, compares favorably with the
only other probe for which comparable experimental cell-
imaging data in RAW264.7 macrophages have been report-
Figure 3. HPLC chromatograms of probe 2 a) without ROS treatment,
c) after reaction with NaOCl for 60 min at 258C, d) after reaction with
H2O2 for 60 min at 258C, and b) sulfoxide 3 only. The samples were ana-
lyzed by LC-MS with a linear gradient elution (eluent A/B=20:80, A:
deionised water with 1% formic acid, B: acetonitrile, flow rate
0.3 mLminÀ1). The MW of the retention time at 10.0 min was 405.2,
which corresponds to [M+H]+ for probe 2 and MW of the retention time
at 3.8 min is 421.1, which corresponds to [M+H]+ for sulfoxide 3. [2]=
5 mm, [NaOCl]=[H2O2]=50 mm.
ed,
a
4-aminophenyl fluorescein reporter,[7b] for which
Chang and co-workers have found less than a 2-fold signal
increase within 40 minutes upon treatment with 100 mm
HOCl.[14]
Cell-viability assays confirm that probe 2 shows low cyto-
toxicity to macrophage cells over a concentration range
from 5 to 50 mm (see the Supporting Information). Having
established that probe 2 is cell-permeable, non-toxic, and
able to measure HOCl levels in biological systems, we eval-
uated the potential utility of probe 2 for monitoring endoge-
nously generated-HOCl at low signaling levels upon physio-
logical stimulation. Macrophages and other phagocytic cells
are known to produce low micromolar levels of H2O2 or
other ROS during phagocytosis, or when stimulated by
agents such as lipopolysaccharide (LPS) and phorbol myris-
tate acetate (PMA).[15] Up to 80% of H2O2 generated is
converted into HOCl/OClÀ in a reaction catalyzed by the
myeloperoxidase (MPO) enzyme, which is localized in phag-
ocytic leukocytes.[16] In addition, the production of MPO in
the macrophage cells can be further induced upon stimula-
tion.[16] RAW264.7 macrophage cells were incubated with
stimulants to induce an immune response.
We next applied probe 2 to the imaging of HOCl in living
RAW264.7 macrophages. Confocal microscopic images of
probe-2-loaded macrophages that were treated with exoge-
nous NaOCl for 20 minutes at 378C showed an increase in
green fluorescence inside living cells whereas macrophages
untreated with NaOCl showed negligible intracellular fluo-
rescence (Figure 4a,b). The fluorescence intensity within the
cells increased linearly with the concentration of added
NaOCl (see the Supporting Information, Figure S17 and
S18). In the case of cells treated with 100 mm NaOCl, a
more-than 5-fold increase in fluorescence intensity was ob-
served compared to the control cells. The lower analytical
responses observed for cell imaging experiments (5-fold)
over solution assays (up to ca. 1000-fold) reflect the difficul-
ty in the permeation of hypochlorous acid through the cell
membrane given that the HOCl/ÀOCl couple is, to a large
extent, present in its anionic form at physiologically relevant
pH.[13] In spite of these fundamental limitations, probe 2,
When LPS- and PMA-activated macrophage cells were
incubated with probe 2, a 2.2-fold increase of fluorescence
intensity was observed in the cells, whereas no obvious fluo-
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Chem. Asian J. 2011, 6, 1358 – 1361

BODIPY-Based Probes
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rescence was observed in the cells untreated with the stimu-
lants (Figure 4c). Upon treatment with a MPO inhibitor[17]
during stimulation, no obvious increase of fluorescence
signal in the cells was observed (Figure 4d). These experi-
ments demonstrate that probe 2 enables the visualization of
both exogenous and endogenous HOCl in living cells.
In summary, we have shown that methylthio-BODIPY 2
is easily prepared from readily accessible precursors and
constitutes a highly sensitive and selective fluorescence
turn-on probe for HOCl in living cells. HOCl-selective
probe
2 joins the expanding toolbox of ROS-specific
probes[18] that can be applied to the challenging yet critically
important elucidation of the roles of individual ROS in bio-
logical and pathological processes.
[10] H.-J. Liu, J. M. Nyangulu, Tetrahedron Lett. 1988, 29, 5467–5470.
[11] Quantum yields (FF) of probe 2 and parent BODIPY 1 in various
solvents are presented in the Supporting Information, Table S1.
[12] In preparative organic syntheses, the oxidation of thioethers into
sulfoxides can be accomplished with many oxidizing agents, includ-
ing peracids or hydrogen peroxide in the presence of various cata-
lysts. Organic peroxides, such as tert-butyl hydroperoxide have also
been widely used as stoichiometric oxidants in such transition-
metal-catalyzed reactions. However, in the absence of catalysts, the
reaction of hydrogen peroxides or alkyl hydroperoxides is sluggish
in aqueous solutions at room temperature and physiological pH. By
contrast, HOCl shows sufficient oxidizing power under these condi-
tions for the oxidation to proceed, which helps rationalize the ob-
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[13] A slow internalization of hypochlorite is further supported by com-
parison with the unionized H2O2 (pKa =11.6). Whilst cells treated
with 100 mm HOCl conserve a healthy morphology indicative of low
internalized concentrations of the ROS, treatment with only 25 mm
of the more cell-permeable H2O2 already causes significant cytotox-
icity. Consequently, longer reaction times and/or higher concentra-
tions of the analyte were required to compensate for the slow kinet-
ics of internalization of hypochlorite into the cells.
Experimental Section
Full experimental details and characterization data are given in the Sup-
porting Information.
Synthesis of Compound 2
Dimethyl sulfoxide (1.8 mL, 22.5 mmol) and triethylamine (1 drop) were
added to a stirred solution of 1 (100 mg, 0.31 mmol) in dichloromethane
(1 mL) at room temperature. The resulting solution was cooled to 08C in
an ice-water bath. Phosphorus oxychloride (40 mL, 0.46 mmol) was added
dropwise to the solution via syringe, and the contents were stirred for
3 min at 08C. The solution rapidly turned from orange to dark-red. Fol-
lowing the removal of the solvent under reduced pressure, an excess of
water (30 mL) was added to the reaction mixture. The crude red precipi-
tate obtained by filtration was purified by column chromatography on
silica gel using progressively more polar eluent, 10:1 to 5:1 hexanes/ethyl
acetate, to afford 2 (92.5 mg, 74%).
Acknowledgements
This research was supported by a grant from the Fundamental R&D Pro-
gram for Core Technology of Materials funded by the Ministry of Knowl-
edge Economy of the Republic of Korea and by a Korea Research Foun-
dation (KRF) grant funded by the Korea Government (MEST) (no.
20100011160).
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Keywords: bioimaging
· bodipy · fluorescent probes ·
hypochlorus acid · reactive oxygen species
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Received: January 14, 2011
Published online: April 5, 2011
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ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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