HKGreen-3: A rhodol-based fluorescent probe for peroxynitrite

Article abstract of DOI:10.1021/ol102182j

A novel fluorescent probe, HKGreen-3, for sensing peroxynitrite is designed on the basis of the rhodol scaffold and a peroxynitrite-specific oxidation reaction. The probe turns out to be highly sensitive and selective for detecting peroxynitrite in both chemical and biological systems.

Full text of DOI:10.1021/ol102182j

ORGANIC
LETTERS
2010
Vol. 12, No. 21
4932-4935
HKGreen-3: A Rhodol-Based Fluorescent
Probe for Peroxynitrite
Tao Peng and Dan Yang*
Morningside Laboratory for Chemical Biology and Department of Chemistry, The
UniVersity of Hong Kong, Pokfulam Road, Hong Kong, People’s Republic of China
yangdan@hku.hk
Received September 12, 2010
ABSTRACT
A novel fluorescent probe, HKGreen-3, for sensing peroxynitrite is designed on the basis of the rhodol scaffold and a peroxynitrite-specific
oxidation reaction. The probe turns out to be highly sensitive and selective for detecting peroxynitrite in both chemical and biological systems.
In the last two decades, the in vivo formation of peroxynitrite
(ONOO-), a short-lived and highly reactive species produced
from the diffusion-controlled reaction of nitric oxide (•NO) and
superoxide (O2•-), has been widely considered as an established
phenomenon since the first paper suggesting that peroxynitrite
could be a potential biological oxidant was published.1 More
and more evidence indicates that peroxynitrite accounts for most
of the cytotoxicity attributed to nitric oxide,2 and its generation
contributes to the pathogenesis of many human diseases such
as stroke, myocardial infarction, chronic heart failure, diabetes,
circulatory shock, chronic inflammatory diseases, cancer, and
neurodegenerative disorders.2,3
However, the biological relevance of peroxynitrite-de-
pendent reactions is still occasionally controversial mainly
due to the difficulty of its direct and unambiguous detection,
which actually is extremely significant to push this field
forward.3,4 In this regard, although a number of peroxynitrite-
responsible fluorescent probes have been developed,5 limited
sensitivity and specificity may seriously hamper their prac-
ticability in the detection of peroxynitrite. Here, we present
a novel rhodol-based fluorescent probe, HKGreen-3, for
highly senstitive and selective detection of peroxynitrite.
Recently, we developed a divergent synthetic route for
rhodol fluorophores, and constructed a rhodol library with
the intention to screen for novel rhodol-based fluorescent
probes.6 We noticed that two members of this library, i.e., 1
and 2, possess distinct fluorescence properties (Figure 1).
The latter displayed strong fluorescence emission (Φ ) 0.67)
in aqueous buffer, while the former surprisingly showed
almost no fluorescence (Φ ) 0.0013). The significant
(4) Radi, R.; Peluffo, G.; Alvarez, M. N.; Naviliat, M.; Cayota, A. Free
Radical Biol. Med. 2001, 30, 463–488.
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Ueno, T.; Urano, Y.; Kojima, H.; Nagano, T. J. Am. Chem. Soc. 2006,
128, 10640–10641. (c) Yang, D.; Wang, H. L.; Sun, Z. N.; Chung, N. W.;
Shen, J. G. J. Am. Chem. Soc. 2006, 128, 6004–6005. (d) Yang, D.; Sun,
Z. N.; Peng, T.; Wang, H. L.; Shen, J. G.; Chen, Y.; Tam, P. K. H. In LiVe
Cell Imaging: Methods and Protocols; Papkovsky, D. B., Ed.; Humana
Press: New York, 2009; Vol. 591, pp 93-103. (e) Sun, Z. N.; Liu, F. Q.;
Chen, Y.; Tam, P. K. H.; Yang, D. Org. Lett. 2008, 10, 2171–2174.
(6) Peng, T.; Yang, D. Org. Lett. 2010, 12, 496–499.
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B. A. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 1620–1624.
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(3) Ferrer-Sueta, G.; Radi, R. ACS Chem. Biol. 2009, 4, 161–177
.
10.1021/ol102182j  2010 American Chemical Society
Published on Web 10/04/2010

Scheme 2. Reaction of HKGreen-3 with Peroxynitrite
Figure 1. The distinct fluorescence property of N-phenylrhodol and
N-methylrhodol. The fluorescence quantum yields were determined
in 50 mM phosphate buffer at pH 8.0, with rhodamine 6G (Φ )
0.76 in water) as reference.
would react with the trifluoromethyl ketone and further lead
to N-dearylation, along with the release of highly fluorescent
compound 2.
fluorescence difference of these two fluorophores (1 vs 2,
Figure 1) suggests a strategy to develop a novel fluorescence
“turn-on” switch, i.e., to convert 1 to 2 by breaking the
covalent bond between the nitrogen and the phenyl ring
through a chemical reaction. The cleavage of the covalent
bond between the nitrogen atom and an anyl ring is certainly
not easy; however, we expected that the strong oxidizing
property of peroxynitrite could make it possible.
Then we assessed the spectroscopic properties of
HKGreen-3 under simulated physiological conditions (0.1
M phosphate buffer, pH 7.4). The probe solution displays
one major absorption peak at 520 nm (Figure S1 in the
Supporting Information), indicating that the probe mainly
exists as the lactone-open form in aqueous media. In the
absence of peroxynitrite, HKGreen-3 exhibits almost neg-
ligible background fluorescence (Φ < 0.0001, Figure 2a) as
Previously, our group reported that peroxynitrite could
react with anisole- or diarylether-derived activated ketones,
3a for example, to provide product 4 (Scheme 1).5c,7 To test
Scheme 1
.
Oxidations of Anisole- and Diarylamine-Derived
Activated Ketones by Peroxynitrite
whether peroxynitrite could break the N-C(phenyl) bond
of diarylamine, we first synthesized a diarylamine-derived
ketone 3b bearing the trifluoromethyl ketone unit (Schemes
1 and S1 in the Supporting Information). When 3b reacted
with peroxynitrite, the same dienone product 4 was isolated
(Scheme 1), implicating that the N-C(phenyl) covalent bond
was indeed cleaved by the oxidation, the mechanism of
which may be similar to that we proposed before.7b
Furthermore, we also found that the reactions of 3b with
other reactive oxygen species (ROS) or reactive nitrogen
species (RNS), such as H2O2, O2, NO, O2•-, ROO•, OH,
and OCl-, could not occur in a similar manner as perox-
ynitrite, suggesting that the diarylamine-derived ketone 3b
reacts with peroxynitrite in a specific way.
1
•
•
Figure 2. (a) Fluorescence response of 5 µM HKGreen-3 to
different amounts of ONOO-. (b) Relative fluorescence intensity
(λem ) 535 nm) of 5 µM HKGreen-3 in the presence of various
ROS and RNS. Data were acquired at 25 °C in 0.1 M phosphate
buffer, pH 7.4, with excitation at 520 nm.
With this information in hand, we next designed and
synthesized a new fluorescent probe, named HKGreen-3,
by incorporating the diarylamine-derived ketone into the
rhodol scaffold (Scheme 2). We expected that peroxynitrie
(7) (a) Yang, D.; Tang, Y. C.; Chen, J.; Wang, X. C.; Bartberger, M. D.;
Houk, K. N.; Olson, L. J. Am. Chem. Soc. 1999, 121, 11976–11983. (b)
expected. The addition of peroxynitrite resulted in dramatic
increases of the fluorescence intensity with a maximum at
Yang, D.; Wong, M. K.; Yan, Z. J. Org. Chem. 2000, 65, 4179–4184
Org. Lett., Vol. 12, No. 21, 2010
.
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535 nm in a dose-dependent manner (Figure 2a). The reaction
of 5 µM HKGreen-3 with 1 equiv of peroxynitrite triggered
a 140-fold fluorescence turn-on response. The fluorescence
increases of HKGreen-3 were not saturated until addition
of more than 15 equiv of peroxynitrite (data not shown). A
linear relationship between the fluorescence intensity and the
peroxynitrite concentration was also observed (Figure S2 in
the Supporting Information). The detection limit of the probe
for peroxynitrite was estimated to be below 50 nM as 50 nM
peroxynitrite could cause a one-fold increase of the fluorescence
of the probe. HPLC and LC-MS analyses established that the
oxidation of HKGreen-3 by peroxynitrite indeed generates
N-methylrhodol 2 as the fluorescent product in 12% yield
(Figures S3 and S4 in the Supporting Information).
esterases.10 RAW 264.7 macrophages were first stimulated
with different reagents, and incubated with HKGreen-3A
(10 µM) for 2 h. Then the cells were monitored using a
confocal fluorescence microscope. Without stimulation no
fluorescence in the cells was observed (Figure 3a), while
Owing to the specific reaction between the diarylamine-
derived ketone and peroxynitrite, HKGreen-3 displays
highly selective response to peroxynitrite. As shown in Figure
2b, most of the biologically relevant ROS and RNS,
including H2O2, 1O2, •NO, O2•-, and ROO•, no matter present
in 1 equiv or 10 equiv, only caused negligible fluorescence
increases of the probe. Moreover, HKGreen-3 shows a >6-
fold higher response toward peroxynitrite (ONOO-) than
toward hydroxyl radical (•OH) or hypochlorite (OCl-).
To demonstrate the ability of HKGreen-3 to image
endogenous peroxynitrite, we chose cultured murine RAW
264.7 macrophages because macrophages are well-known
to generate ROS and RNS in immunological and inflamma-
tory processes.8,9 Considering that the anionic nature of
HKGreen-3 in aqueous media might limit its permeability
through cell membrane, we prepared its acetate derivative,
HKGreen-3A (Scheme 3), anticipating that this more
Figure 3. Confocal fluorescence imaging of live RAW 264.7
Scheme 3. Conversion of Cell Membrane Permeable
HKGreen-3A to HKGreen-3 by Intracellular Esterases
macrophage cells. The cells were treated with various stimulants,
and then stained with HKGreen-3A (green channel) and Hoechst
33342 (blue channel). (a) Cells without stimulation. (b) Cells
stimulated with LPS, IFN-γ, and PMA. (c) Cells pretreated with
TEMPO, and then stimulated with LPS, IFN-γ, and PMA. (d) Cells
pretreated with AG, and then stimulated with LPS, IFN-γ, and
PMA.
strong green fluorescence in the cytoplasm was imaged
(Figure 3b) after treatment with stimulants including li-
popolysaccharide (LPS, 1 µg/mL), interferon-γ (IFN-γ, 50
ng/mL), and phorbol 12-myristate 13-acetate (PMA, 10 nM).
Moreover, we found that the bright fluorescence was
suppressed (Figures 3c and 3d) by pretreatment of the cells
with either a scavenger of superoxide, 2,2,6,6-tetramethyl-
1-piperidinyloxy (TEMPO, 100 µM), or an NO synthase
inhibitor, aminoguanidine (AG, 1 mM).11 Since a NO
synthase inhibitor can hardly attenuate the generation of
H2O2,12 thus that of •OH or OCl-, and a superoxide scavenger
lipophilic derivative would have better permeability and be
converted back to HKGreen-3 by the action of intracellular
(8) Muijsers, R. B. R.; van den Worm, E.; Folkerts, G.; Beukelman,
(10) Kojima, H.; Nakatsubo, N.; Kikuchi, K.; Kawahara, S.; Kirino, Y.;
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.
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Org. Lett., Vol. 12, No. 21, 2010

would not affect the production of simple RNS, apparently
it is peroxynitrite, rather than other ROS or RNS, which
induces the strong fluorescence in the macrophages. Simi-
larly, HKGreen-3A was also used for imaging peroxynitrite
in J744.1 macrophages (Figure S5 in the Supporting Infor-
mation). Besides, nuclear staining with Hoechst 33342
(Figure 3) and MTT assays (Figure S6 in the Supporting
Information)13 revealed that HKGreen-3A of concentrations
below 20 µM does not exhibit obvious cytotoxicity.
In summary, we present herein a novel fluorescent probe
HKGreen-3 and its ester derivative HKGreen-3A for the
highly sensitive and selective detection and cellular imaging
of peroxynitrite. These probes were designed on the basis
of a peroxynitrite specific reaction and the rhodol scaffold.
We anticipate that the probe with excellent spectroscopic
properties will find its applications in understanding the
actions of peroxynitrite in biological systems.
Acknowledgment. This work was supported by the
University of Hong Kong, the Hong Kong Research Grants
Council (HKU7060/05P), and Morningside Foundation. We
also thank the Faculty of Medicine at HKU for the Confocal
Microscopy and Live Cell Imaging Facility.
Supporting Information Available: Experimental pro-
cedures and characterization of new compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(12) Domaille, D. W.; Que, E. L.; Chang, C. J. Nat. Chem. Biol. 2008,
4, 168–175.
(13) Mosmann, T. J. Immunol. Methods 1983, 65, 55–64.
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