BODIPY-based fluorescent probe for peroxynitrite detection and imaging in living cells

Article abstract of DOI:10.1021/ol900279z

A fluorescent probe, HKGreen-2, has been developed based on a specific reaction between ketone and peroxynitrite (ONOO-). This probe is highly sensitive and selective for the detection of peroxynitrite not only in abiotic but also in biological systems. With this probe, we successfully detected peroxynitrite generated in murine macrophage cells activated by phorbol 12-myristate 13-acetate (PMA), interferon-γ (IFN-γ), and lipopolysaccharide (LPS). This new probe will be a useful tool for studying the roles of peroxynitrite in biological processes.2009 American Chemical Society.

Full text of DOI:10.1021/ol900279z

ORGANIC
LETTERS
2009
Vol. 11, No. 9
1887-1890
BODIPY-Based Fluorescent Probe for
Peroxynitrite Detection and Imaging in
Living Cells
Zhen-Ning Sun,† Hua-Li Wang,† Feng-Qin Liu,‡ Yan Chen,‡ Paul Kwong
Hang Tam,† and Dan Yang*,†
Morningside Laboratory for Chemical Biology, Department of Chemistry, and
Department of Surgery, The UniVersity of Hong Kong, Pokfulam Road,
Hong Kong, People’s Republic of China
yangdan@hku.hk
Received February 9, 2009
ABSTRACT
A fluorescent probe, HKGreen-2, has been developed based on a specific reaction between ketone and peroxynitrite (ONOO-). This probe is
highly sensitive and selective for the detection of peroxynitrite not only in abiotic but also in biological systems. With this probe, we successfully
detected peroxynitrite generated in murine macrophage cells activated by phorbol 12-myristate 13-acetate (PMA), interferon-γ (IFN-γ), and
lipopolysaccharide (LPS). This new probe will be a useful tool for studying the roles of peroxynitrite in biological processes.
Peroxynitrite (ONOO-), a short-lived reactive oxygen species
(ROS) and reactive nitrogen species (RNS), is formed in vivo
from a diffusion-controlled reaction between nitric oxide and
superoxide in a 1:1 stoichiometry (k ) 0.4-1.9 × 1010 M-1
s-1).1 Intracellular peroxynitrite formation is associated with
elevated NO production and its diffusion to sites of super-
oxide formation.1d,2 A well-controlled level of peroxynitrite
is essential for cellular integrity and organ homeostasis.
However, under pathological conditions such as bacterial
infection, lipopolysaccharide (LPS) induces high expression
of inducible nitric oxide synthase (iNOS) in macrophages
and neutrophils to produce large amounts of ONOO- in
addition to other ROS and RNS.3 Accumulating evidence
suggests that aberrant peroxynitrite activities may contribute
to many human diseases including ischemic reperfusion
injury, rheumatoid arthritis, septic shock, multiple sclerosis,
atherosclerosis, stroke, inflammatory bowel disease, cancer,
and several neurodegenerative diseases.4
Unfortunately, attempts to investigate the behavior of
peroxynitrite in biological systems have been hampered by
the difficulty in detecting this highly reactive oxygen species
in vivo. To this end, a number of fluorescent probes have
been ventured for the detection of peroxynitrite in vitro, but
these have proved undependable either because of a lack of
† Morningside Laboratory for Chemical Biology, Department of Chem-
istry.
(3) (a) Forehand, J. R.; Johnston, R. B. J.; Bomalaski, J. S. J. Immunol.
1993, 151, 4918–4925. (b) Fang, F. C. J. Clin. InVest. 1997, 99, 2818–
2825. (c) Muijsers, R. B.; van Den Worm, E.; Folkerts, G.; Beukelman,
C. J.; Koster, A. S.; Postma, D. S.; Nijkamp, F. P. Br. J. Pharmacol. 2000,
130, 932–936. (d) Jacob, A. T.; Ignarro, L. J. J. Biol. Chem. 2001, 51,
47950–47957. (e) Salonen, T.; Sareila, O.; Jalonen, U.; Kankaanranta, H.;
Tuominen, R.; Moilanen, E. Br. J. Pharmacol. 2006, 147, 790–799.
(4) Pacher, P.; Beckman, J. S.; Liaudet, L. Physiol. ReV. 2007, 87, 315–
424.
‡ Department of Surgery.
(1) (a) Beckman, J. S.; Beckman, T. W.; Chen, J.; Marshall, P. A.;
Freeman, B. A. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 1620–1624. (b)
Squadrito, G. L.; Pryor, W. A. Free. Radic. Biol. Med. 1998, 25, 392–403.
(c) Fang, F. C. Nat. ReV. Microbiol. 2004, 2, 820–832. (d) Szabo, C.;
Ischiropoulos, H.; Radi, R. Nat. ReV. Drug DiscoV. 2007, 6, 662–680.
(2) Trujillo, M.; Naviliat, M.; Alvarez, M. N.; Peluffo, G.; Radi, R.
Analusis 2000, 28, 518–527
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10.1021/ol900279z CCC: $40.75
Published on Web 03/30/2009
 2009 American Chemical Society

specificity toward peroxynitrite or incompatibility with living
cells and more complex natural environments.5
With this modified moiety, we designed a new probe,
HKGreen-2, that would be suitable for peroxynitrite detec-
tion based on a photoinduced electron transfer (PET)
mechanism (Figure 1b and c).7 We first investigated the
reactivity of HKGreen-2 toward peroxynitrite in an abiotic
system. As indicated in Figure 2a, the probe is nonfluorescent
Our overall strategy is to develop more sensitive fluores-
cent probes on the basis of a specific reaction for peroxyni-
trite discovered by our group. We previously developed a
fluorescent probe, HKGreen-1, which can detect peroxyni-
trite selectively.6 The poor yield of the reaction between
HKGreen-1 and peroxynitrite, however, limits the probe’s
ability to detect peroxynitrite in vivo. As shown in Figure
1a, only 21% yield (based on 80% conversion) of the desired
Figure 1. (a) Model reactions of 1a and 1b toward peroxynitrite.
(b) Reaction between HKGreen-2 and peroxynitrite. (c) HOMO
and LUMO energies of BODIPY units (fluorophore) and the top
units of both HKGreen-2 and its oxidation product. The values
were obtained from semiempirical PM3 calculation.
compound 2 is obtained in the model reaction between
peroxynitrite and phenoxyphenyl-derived ketone 1a. We
reasoned that the poor yield of this reaction was due to the
difficulty in the cleavage of the aryl-oxygen bond. Thus, we
modified compound 1a by removing the aryl group to
improve the reaction yield. Gratifyingly, ketone 1b gave a
better yield (52% yield based on 48% conversion) in its
reaction with peroxynitrite.
Figure 2. (a) Fluorescence emission spectra of HKGreen-2
(10 µM) in 0.1 M potassium phosphate buffer (pH 7.4), recorded
0.5 h after reaction with various amounts of ONOO- (ranging from
0 to 7 equiv). (Insert) Linear correlation between the emission
intensity and the concentration of peroxynitrite. (b) Real-time
detection of ONOO-. The real-time fluorescence emission spectrum
of HKGreen-2 (10 µM) in 0.1 M potassium phosphate buffer (pH
7.4) was recorded during addition of 1 equiv of ONOO- (repeating
three times). (c) Fluorescence intensity of HKGreen-2 (10 µM) in
various ROS and RNS-generating systems (described in the
Supporting Information).
(5) (a) Setsukinai, K.; Urano, Y.; Kakinuma, K.; Majima, H. J.; Nagano,
T. J. Biol. Chem. 2003, 278, 3170–3175. (b) Ueno, T.; Urano, Y.; Kojima,
H.; Nagano, T. J. Am. Chem. Soc. 2006, 128, 10640–10641.
(6) (a) Yang, D.; Wong, M. K.; Yan, Z. J. Org. Chem. 2000, 65, 4179–
4184. (b) Yang, D.; Wang, H. L.; Sun, Z. N.; Chung, N. W.; Shen, J. G.
J. Am. Chem. Soc. 2006, 128, 6004–6005.
(7) (a) Kollmannsberger, M.; Gareis, T.; Heinl, S.; Breu, J.; Daub, J.
Angew. Chem., Int. Ed. 1997, 36, 1333–1335. (b) Gabe, Y.; Urano, Y.;
Kikuchi, K.; Kojima, H.; Nagano, T. J. Am. Chem. Soc. 2004, 126, 3357–
3367. (c) Ueno, T.; Urano, Y.; Setsukinai, K.; Takakusa, H.; Kojima, H.;
Kikuchi, K.; Ohkubo, K.; Fukuzumi, S.; Nagano, T. J. Am. Chem. Soc.
2004, 126, 14079–14085. (d) Ziessel, R.; Ulrich, G.; Harriman, A. New
J. Chem. 2007, 31, 496–501. (e) Loudet, A.; Burgess, K. Chem. ReV. 2007,
107, 4891–4932. (f) Ulrich, G.; Ziessel, R.; Harriman, A. Angew. Chem.,
Int. Ed. 2008, 47, 1184–1201.
prior to its reaction with peroxynitrite; intensity of the
fluorescence signal rises dramatically with increasing per-
oxynitrite concentration. Upon treatment with 7 equiv of
peroxynitrite, HKGreen-2 exhibits a 69-fold surge in
fluorescence intensitysa much higher sensitivity than that
displayed by our previous probe, HKGreen-1. A linear
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Org. Lett., Vol. 11, No. 9, 2009

Figure 4. Confocal fluorescence images of live J774.1 macrophage
Figure 3. Fluorescence imaging of macrophage cells (left: fluo-
cells incubated with HKGreen-2 and MitoTracker Red CMXRos.
(a-d) Controls; (e-h) treated with LPS (1 µg/mL) and IFN-a˜ (50
ng/mL) for 4 h then PMA (10 nM) for 0.5 h. (a) HKGreen-2. (b)
MitoTracker Red CMXRos. (c) HKGreen-2 and MitoTracker Red
CMXRos (overlay). (d) Brightfield, HKGreen-2 and MitoTracker
Red CMXRos (overlay). (e) HKGreen-2. (f) MitoTracker Red
CMXRos. (g) HKGreen-2 and MitoTracker Red CMXRos (over-
lay). (h) Brightfield, HKGreen-2 and MitoTracker Red CMXRos
(overlay).
rescence imaging; right: phase contrast imaging). Macrophage cells
were treated with various stimulants and then incubated with
HKGreen-2 (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) AG
(1 mM), LPS (1 µg/mL), and IFN-γ (50 ng/mL) for 4 h then PMA
(10 nM) for 0.5 h. (d) TEMPO (100 µM), LPS (1 µg/mL), and
IFN-γ (50 ng/mL) for 4 h then PMA (10 nM) for 0.5 h.
correlation exists between the emission intensity and the
concentration of peroxynitrite when the concentration of
ONOO- ranges from 0 to 15 µM (Figure 2a insert graph).
The real-time emission spectrum shows rapid increases
(within 2 s) in fluorescence signal after each addition of
peroxynitrite (Figure 2b). Moreover, we have confirmed the
formation of compound 3 through an ESI-MS measurement.8
Therefore, HKGreen-2 can detect peroxynitrite in an abiotic
system in terms of a dose-dependent increase of fluorescence.
While HKGreen-2 is highly sensitive to peroxynitrite, it
is important to investigate its selectivity toward various ROS
and RNS. As shown in Figure 2c, HKGreen-2 exhibits a
21-fold increase in fluorescence intensity upon reaction with
peroxynitrite, whereas a much weaker response is observed
vent.8 These results suggest that, in an abiotic system,
HKGreen-2 displays high selectivity toward peroxynitrite
over other ROS and RNS.
Our ultimate goal is to use HKGreen-2 to detect endog-
enous ONOO- production in cells under physiological
conditions. We chose the murine macrophage cell line J774.1
as a testing model, because exposure of macrophage cells
to stimuli such as LPS/IFN-γ (interferon-γ) and phorbol
myristate acetate (PMA) activates the generation of ROS and
RNS.3c,e We stimulated J774.1 macrophage cells with various
reagents, incubated the cells with HKGreen-2 (20 µM) for
1 h, and then washed them three times with PBS buffer
(Figure 3). We observed no fluorescence in the absence of
a stimulant (Figure 3a) but strong fluorescence after treatment
with LPS (1 µg/mL) and interferon-γ (IFN-γ; 50 ng/mL)
for 4 h followed by additional stimulation with phorbol 12-
myristate 13-acetate (PMA; 10 nM) for 30 min (Figure 3b).
However, after the cells were pretreated with aminoguanidine
•
1
with other ROS and RNS such as HOCl, OH, H2O2, O2,
-
-
NO2 , NO3 , NO, O2•- and ROO•. The fluorescence response
•
toward OH can be significantly suppressed when 0.1%
DMSO (a known scavenger of OH) was used as a cosol-
•
(8) See Supporting Information.
Org. Lett., Vol. 11, No. 9, 2009
1889

(AG), an inhibitor of nitric oxide synthase,3c much weaker
fluorescence was observed in stimulated cells (Figure 3c).
In principle, the amount of •OH produced in stimulated cells
should not be affected by the presence of AG. Thus, the much
weaker fluorescence in Figure 3c indicated that the strong
fluorescence in Figure 3b should be induced by peroxynitrite
nitrite level in the cytoplasm. Again, by costaining with
MitoTracker Red CMXRos and overlaying with brightfield
images, we found that the green fluorescence is retained in
the cytoplasm (Figures 4f-h). These data indicate that
HKGreen-2 can be used for imaging intracellular peroxy-
nitrite selectively in biological systems.
In summary, on the basis of a specific reaction of
peroxynitrite and a PeT mechanism, we have developed a
new BODIPY-type fluorescent probe, HKGreen-2, which
is highly sensitive for the specific detection of peroxynitrite
in abiotic and living-cell systems. This probe will be a useful
tool for elucidating the role of ONOO- in various biological
processes.
•
rather than OH. Furthermore, after pretreatment of cells
with 2,2,6,6-tetramethyl-piperidinooxy (TEMPO), a scav-
enger of superoxide,3c almost no fluorescence was ob-
served in stimulated cells (Figure 3d). These results reveal
that HKGreen-2 can be used to visualize peroxynitrite
selectively in biological systems.
Finally, we sought to utilize HKGreen-2 to probe the
subcellular locations of endogenous peroxynitrite in J774.1
macrophage cells by using confocal fluorescence microscopy.
Very weak green fluorescence in the cytoplasm of macroph-
age cells was observed when the cells were loaded with
HKGreen-2 (20 µM) (Figure 4a). Costaining with Mi-
toTracker Red CMXRos (50 nM), a commercially available
mitochondrial fluorescent probe, showed the location of
active mitochondria in the cytoplasm of these living cells
(Figures 4b-d). After treating the cells with the same
stimulants as we used in Figure 3b, we observed much
stronger green fluorescence (Figure 4e) compared to the
control (Figure 4a), corresponding to an increase of peroxy-
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. We also thank the
Faculty of Medicine at HKU for the Confocal Microscopy
and Live Cell Imaging Facility.
Supporting Information Available: Synthesis, experi-
mental details, and characterization of HKGreen-2. This
material is available free of charge via the Internet at
http://pubs.acs.org.
OL900279Z
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Org. Lett., Vol. 11, No. 9, 2009