A highly specific BODIPY-based fluorescent probe for the detection of hypochlorous acid

Article abstract of DOI:10.1021/ol800507m

(Chemical Equation Presented) A fluorescent probe, HKOCl-1, has been successfully developed for the detection of hypochlorous acid on the basis of a specific reaction with p-methoxyphenol. The formation of HOCl has been successfully detected not only in an abiotic system but also in an enzymatic system (myeloperoxidase/H₂O₂/Cl^- system) and in living macrophage cells upon stimulation. This new probe might be used as an efficient tool for probing the roles HOCl plays in biological systems.

Full text of DOI:10.1021/ol800507m

ORGANIC
LETTERS
2008
Vol. 10, No. 11
2171-2174
A Highly Specific BODIPY-Based
Fluorescent Probe for the Detection of
Hypochlorous Acid
Zhen-Ning Sun,† Feng-Qin Liu,‡ Yan Chen,‡ Paul Kwong Hang Tam,‡ and
Dan Yang*,†
Department of Chemistry and Department of Surgery, The UniVersity of Hong Kong,
Pokfulam Road, Hong Kong, P.R. China
yangdan@hku.hk
Received March 10, 2008
ABSTRACT
A fluorescent probe, HKOCl-1, has been successfully developed for the detection of hypochlorous acid on the basis of a specific reaction with
p-methoxyphenol. The formation of HOCl has been successfully detected not only in an abiotic system but also in an enzymatic system
(myeloperoxidase/H2O2/Cl- system) and in living macrophage cells upon stimulation. This new probe might be used as an efficient tool for
probing the roles HOCl plays in biological systems.
Unlike most other reactive oxygen species (ROS) and
reactive nitrogen species (RNS), hypochlorite (OCl-) and
its protonated form hypochlorous acid (HOCl) are encoun-
tered widely in our daily lives. For example, sodium
hypochlorite is frequently used as a disinfectant and a
bleaching agent (common “bleach”). Hypochlorite is a key
microbicidal agent that is used for natural defense because
it behaves as a strong nucleophilic nonradical oxidant; its
efficacy lies in the fact that neither bacteria nor mammalian
cells can neutralize its toxic effectssthey lack the enzymes
required for its catalytic detoxification.1 In living organisms,
hypochlorite is synthesized from hydrogen peroxide and
chloride ions in a chemical reaction catalyzed by the enzyme
myeloperoxidase (MPO), which is localized mainly in
leukocytes, including neutrophils, macrophages, and mono-
cytes.2 MPO is released into the phagolysosomal compart-
ment following stimulation by inflammatory mediators such
as cytokines, antibodies, and the endotoxic bacterial mem-
brane component lipopolysaccharide (LPS). Although hy-
pochlorite functions mainly in the prevention of microor-
ganism invasion, increasing evidence suggests that it is also
involved in several human diseases, including neuron de-
generation,2 cardiovascular diseases,3 and osteoarthritis4
caused by the presence of abnormal levels of MPO.5
Nevertheless, the mechanism of action of hypochlorite in
these diseases is much less clear than that of other ROS and
RNS because of the lack of sensitive and specific probes for
detecting hypochlorite.6 Recently, two red fluorescent probes
for hypochlorous acid were reported by Nagano’s group6c
and Libby’s group,6d but the sensitivity of those fluorescent
probes still needs to be improved. On the other hand, for
(3) (a) Sugiyama, S.; Okada, Y.; Sukhova, G. K.; Virmani, R.; Heinecke,
J. W.; Libby, P. Am. J. Pathol. 2001, 158, 879–891. (b) Sugiyama, S.;
Kugiyama, K.; Aikawa, M.; Nakamura, S.; Ogawa, H.; Libby, P. Arterio-
scler Thromb. Vasc. Biol. 2004, 24, 1309–1314.
† Department of Chemistry.
‡ Department of Surgery.
(4) Steinbeck, M. J.; Nesti, L. J.; Sharkey, P. F.; Parvizi, J. J. Orthop.
Res. 2007, 25, 1128–1135.
(1) Lapenna, D.; Cuccurullo, F. Gen. Pharmacol. 1996, 27, 1145–1147.
(2) Yap, Y. W.; Whiteman, M.; Cheung, N. S. Cell Signal 2007, 19,
219–228.
(5) Hoy, A.; Leininger-Muller, B.; Kutter, D.; Siest, G.; Visvikis, S.
Clin. Chem. Lab. Med. 2002, 40, 2–8.
10.1021/ol800507m CCC: $40.75
Published on Web 05/01/2008
2008 American Chemical Society

cell imaging applications, it is important to develop green
fluorescent probes for hypochlorous acid. Here, we report a
green fluorescent probe for highly specific and sensitive
detection of hypochlorous acid and its application in cell
imaging.
Our approach is to find a specific reaction for hypochlorites
one that does not proceed in the presence of other ROS, such
as peroxynitrite, hydroxyl radical, and H2O2. Because hy-
pochlorite is a strong oxidant in biological systems, we
investigated its reactivity toward p-methoxyphenol. We found
that 1 equiv of NaOCl oxidized p-methoxyphenol to ben-
zoquinone (Scheme 1). Gratifyingly, we found that p-
oxidation, the HOMO energy levels of the benzoquinone
moiety (-10.9 eV) is lower than that of the BODIPY unit.8
Therefore, the PET process is prohibited and product 2
should be fluorescent.
We first investigated the reactivity of HKOCl-1 toward
hypochlorite in an abiotic chemical system. As indicated in
Figure 1a, the probe was nonfluorescent prior to its reaction
Scheme 1. Formation of Benzoquinone from p-Methoxyphenol
methoxyphenol was stable toward most other common ROS
and RNS. For ONOO-, the nitration product was formed,
but the conversion was very low (<5%). Thus, the oxidation
of p-methoxyphenol to 1 is specific for hypochlorite.
With this specific reaction for hypochlorite in hand, we
designed a probe, HKOCl-1, that would function based on
a photoinduced electron transfer (PET) mechanism.7 Scheme
2 displays a proposed mechanism for the reaction between
Scheme 2. Reaction of Probe HKOCl-1 with HOCl
Figure 1. (a) Fluorescence spectra of HKOCl-1 (final concentra-
tion: 10 µM) in 0.1 M potassium phosphate buffer (pH 7.5) recorded
2 min after the addition of NaOCl (ranging from 0 to 8 µM). The
fluorescence intensity was determined with excitation at 520 nm.
Inset: Fluorescence intensity (Ex@520 nm, Em@541 nm) plotted
against the concentration of NaOCl. (b) Fluorescence spectra of
HKOCl-1 (final concentration: 10 µM) with the addition of NaOCl
(final concentration: 10 µM) in 0.10 M sodium phosphate buffer
at various pH values (Ex@520 nm, Em@541 nm).
HKOCl-1 and hypochlorite. Before the reaction with hy-
pochlorite, the HOMO energy level (-8.71 eV) of the
p-methoxyphenol moiety is higher than that of the BODIPY
unit (-9.14 eV);8 hence, the fluorescence of HKOCl-1 is
quenched through a PET process (Φfl < 0.01). After
with hypochlorite; the fluorescence signal appeared and
increased dramatically upon increasing the hypochlorite
concentration. A linear correlation existed between the
emission intensity and the concentration of hypochlorite
within the range from 3 to 8 µM (Figure 1a, inset). After
treatment with 1 equiv of hypochlorite, the solution of
HKOCl-1 exhibited a 1079-fold increase in its fluorescence
intensity at pH 7.5. This result suggested that HKOCl-1 is
by far the most sensitive probe for the detection of hypochlo-
rite in abiotic systems.6c,d Moreover, we confirmed the
formation of compound 2 through an ESI-MS measurement.8
We noticed that HKOCl-1 showed pH-dependence in the
detection of hypochlorite. As shown in Figure 1b, the
fluorescence increase was significantly higher at pH 5-7.5
(6) (a) Bizyukin, A.; Korkina, L.; Velichkovskii, B. Bull. Exp. Biol. Med.
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(8) See the Supporting Information.
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Org. Lett., Vol. 10, No. 11, 2008

than at other pH values. Considering the pKa of HOCl is
7.6, we concluded that HKOCl-1 detects HOCl rather than
OCl-, similar to the behavior of fluorescent probes reported
by other groups.6c,d Because our probe HKOCl-1 exhibits
high sensitivity toward HOCl, it can be used for the detection
of HOCl at a wider pH range (pH 4-9) than previously
reported probes.
fluorescence intensity increased dramatically upon the ad-
dition of HKOCl-1 whereas almost no fluorescence was
detected in the control experiment (in the absence of H2O2).
Because HKOCl-1 does not respond to H2O2, the observed
fluorescence increase must be related to the formation of
HOCl; i.e., this system allows the detection of the production
of HOCl in the enzymatic system.
Next we compared the reactivity of HKOCl-1 toward
various ROS and RNS, including H2O2, 1O2, NO, O2•-, •OH,
HOCl, ONOO-, and alkylperoxyl radicals (ROO•), added
independently to the HKOCl-1 solution. The changes in
fluorescence intensity before and after the addition of ROS
and RNS (Figure 2a) indicated that fluorescence augmenta-
A cytotoxicity assay exhibited that HKOCl-1 was nontoxic
to macrophages when its concentration was below 80 µM.8
Therefore, we applied our HKOCl-1 probe to macrophages
for fluorescence imaging of HOCl in cells.
The murine macrophage cell line RAW264.7 produces
MPO upon stimulation.10 In addition, exposure of macroph-
ages to stimuli such as LPS/IFN-γ (interferon-γ)11 and
phorbol myristate acetate (PMA)12 will also activate the
generation of other ROS and RNS. In this experiment, we
used RAW264.7 as a model to test whether we could use
HKOCl-1 (20 µM) to detect HOCl generated by an MPO/
H2O2/Cl- system under stimulation. We observed no obvious
fluorescence in the cells prior to treatment with the stimulants
(Figure 3a). It is noteworthy that fluorescent cells appeared
Figure 3. Images of RAW 264.7 macrophages (lower: phase-
contrast images; upper: fluorescence imaging) treated with
various stimulants and then incubated with HKOCl-1 (20 µM)
for 1 h: (a) control; (b) LPS (1 µg/mL) and IFN-γ (50 ng/mL) for
4 h, then PMA (10 nM) for 0.5 h; (c) TEMPO (100 µM), LPS (1
µg/mL), and IFN-γ (50 ng/mL) for 4 h, then PMA (10 nM) for
0.5 h.
Figure 2. (a) Fluorescence intensity of HKOCl-1 (final concentra-
tion: 10 µM) in various ROS- and RNS-generating systems. (see
Supporting Information for details). (b) Application of HKOCl-1
to an MPO/H2O2/Cl- system. HKOCl-1 (final concentration: 10
µM) was added to sodium phosphate buffer (0.1 M, pH 7.4)
containing MPO (1 U/100 mL) and NaCl (150 mM) at 37 °C. Lower
line: Background fluorescence in the absence of H2O2 (blank).
Upper line: H2O2 added (final concentration: 5 µM).
after the stimulation with LPS/IFN-γ followed by PMA
(Figure 3b). However, after the cells were pretreated with
2,2,6,6-tetramethylpiperidinooxy (TEMPO), a radical that can
scavenge superoxide (the precursor to HOCl),11 much weaker
fluorescence was observed upon stimulation. Our results
tion occurred only upon reaction with HOCl; i.e., HKOCl-1
exhibits excellent selectivity toward HOCl among the various
ROS and RNS in abiotic systems. In fact, this high selectivity
toward HOCl was also observed over a wide pH range (pH
4-9).8
Because hypochlorite in living organisms is synthesized
predominantly from hydrogen peroxide and chloride ions in
a reaction catalyzed by the enzyme MPO,9 we applied
HKOCl-1 to an MPO/H2O2/Cl- system (Figure 2b). The
(9) Hidalgo, E.; Bartolome, R.; Dominguez, C. Chem. Biol. Interact.
2002, 139, 265–282.
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N.; Yotsumoto, S.; Aratani, Y.; Ohno, N.; Petty, H. R. J. Immunol. 2006,
176, 5033–5040.
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Org. Lett., Vol. 10, No. 11, 2008
2173

confirm for the first time that macrophages do indeed produce
HOCl upon stimulation-and that this process can be visual-
ized using HKOCl-1.
Acknowledgment. This work was supported by the
University of Hong Kong and the Hong Kong Research
Grants Council (HKU7060/05P). Z.-N.S. is a recipient of a
University Postdoctoral Fellowship.
In conclusion, on the basis of a specific reaction for
hypochlorite and exploiting a PET mechanism, we have
successfully developed a new BODIPY-type green fluorescent
probe, HKOCl-1, which is highly sensitive and specific for the
detection of HOCl in buffer-based and enzyme-containing
systems. Most importantly, HKOCl-1 can be employed to
image the production of HOCl selectively in living macrophage
cells; use of this efficient probe might allow an evaluation of
the roles HOCl plays in biological systems.
Supporting Information Available: Synthesis, experi-
mental details, and characterization of HKOCl-1. This
material is available free of charge via the Internet at
http://pubs.acs.org.
OL800507M
2174
Org. Lett., Vol. 10, No. 11, 2008