A highly selective fluorescent probe for the detection and imaging of peroxynitrite in living cells

Article abstract of DOI:10.1021/ja0603756

We have found a specific reaction between ketone 1 and peroxynitrite (ONOO^-), rather than other reactive oxygen species and reactive nitrogen species generated in the biological system. On the basis of this reaction, we have successfully develo

Full text of DOI:10.1021/ja0603756

Published on Web 04/14/2006
A Highly Selective Fluorescent Probe for the Detection and Imaging of
Peroxynitrite in Living Cells
Dan Yang,*,† Hua-Li Wang,† Zhen-Ning Sun,† Nga-Wai Chung,† and Jian-Gang Shen‡
Department of Chemistry and Open Laboratory of Chemical Biology, and School of Chinese Medicine,
The UniVersity of Hong Kong, Pokfulam Road, Hong Kong, P. R. China
Received January 18, 2006; E-mail: yangdan@hku.hk
Peroxynitrite (ONOO-), a highly reactive oxidant that is gener-
ated through the spontaneous reaction of nitric oxide (NO) and
Scheme 1. Peroxynitrite-Mediated Oxidation of an
Anisole-Derived Ketone via a Dioxirane Intermediate
superoxide (O2•-), has attracted much attention ever since Beckman
and co-workers first proposed its formation in vivo in 1990.1
Peroxynitrite and its secondary metabolites (•NO2, CO3•-, and
•OH) can effect tyrosine nitration2-6 and oxidation of nucleic acids,
sulfhydryls, lipids, and amino acids, all of which may result in
serious damage to living cells.7,8 As a result, the production of
Scheme 2. Reaction of HKGreen-1 with Peroxynitrite
peroxynitrite in biological systems is implicated with a series of
human diseases, including acute and chronic inflammatory pro-
cesses,9 ischemic stroke,10 diabetes,11 sepsis,12 ischemia-reperfu-
sion,13 atherosclerosis,14 and neurodegenerative disorders (ALS, Lou
Gehrig’s disease, and Alzheimer’s disease).15
Unfortunately, the short half-life (<20 ms in a typical physi-
ological environment) and the multiple reaction pathways of
peroxynitrite in biological systems preclude its direct isolation and
unit of the probe would generate a dioxirane that would selectively
detection. To date, no methods are available for the direct detection
oxidize the phenyl ring to afford dienone product and, more
of peroxynitrite with complete specificity and high sensitivity;16
importantly, release the fluorescent molecule. Dichlorofluorescein
indeed, identifying and quantifying peroxynitrite formation neces-
(DCF) is an ideal fluorophore because its excitation and emission
sitates performing tedious and time-consuming control experiments
wavelengths occur in the visible range, it exhibits a high fluores-
using a combination of scavengers and inhibitors. Therefore, there
cence quantum yield, and it has good stability toward oxidants,
especially peroxynitrite. Thus, we synthesized the probe HKGreen-1
(Supporting Information), which possesses a ketone unit linked to
a DCF moiety through an aryl ether linkage. The introduction of
the CH2COOH group not only decreases the fluorescence back-
ground of the probe in cells but also increases the solubility of the
is a need to develop highly sensitive and specific methods for the
detection of peroxynitrite and, more importantly, to allow the direct
analysis of peroxynitrite production in vivo.
Our design concept was inspired by the similar chemical behavior
displayed by ONOO- and peroxymonosulfate (HOOSO3-), the
commercial source of which is Oxone. We demonstrated previously
probe in aqueous solution. We expected that the virtually nonfluo-
that peroxynitrite reacts with activated ketones to form dioxiranes
rescent probe HKGreen-1 (fluorescence quantum yield: Φ ) 0.05
at 0.1 M in pH ) 7.3 buffer solution) would yield the strongly
fluorescent product 3 upon its reaction with peroxynitrite (Scheme
2). In fact, similar to DCF, 3 is a strong fluorophore; it has a
in a manner similar to that by which ketones react with peroxy-
monosulfate.17,18 Furthermore, when the anisole-derived ketone 1
was reacted with peroxynitrite, it formed the same dienone product
(2), albeit in lower conversion and yield (44% yield based on 50%
quantum yield of 0.46 at 0.1 M in buffer solution (pH ) 7.3).
conversion), as did the reaction between 1 and peroxymonosulfate
Initially, we investigated the reactivity of HKGreen-1 toward
(Scheme 1). This result implies that peroxynitrite oxidized ketone
peroxynitrite in an abiotic system. Various amounts of 20 µM
peroxynitrite in 0.1 M sodium hydroxide solution were added to
buffer solutions of HKGreen-1 that were maintained in the dark.
After 15 min, the fluorescence emission of each reaction mixture
1 in situ to form dioxirane intermediate, which subsequently
oxidized the phenyl ring to afford 2.
More promisingly, we found that such reactions did not occur
between ketone 1 and the other reactive oxygen species (ROS) or
was measured using a fluorescence spectrometer. As indicated in
reactive nitrogen species (RNS), such as H2O2, 1O2, •OH, NO, O2
,
•-
Figure 1A, the fluorescence intensity increased gradually upon
increasing in peroxynitrite concentration (e.g., a ca. 7- to 8-fold
enhancement in fluorescence intensity occurred after adding 15
equiv of peroxynitrite to HKGreen-1 solution; Figure 1A).
Moreover, we observed a linear correlation between the emission
intensity and the concentration of peroxynitrite (Figure 1A, inset).
This result demonstrates that, in abiotic systems, our probe
HKGreen-1 could detect peroxynitrite both qualitatively and
quantitatively.
-OCl, and ROO• (i.e., ketone 1 displays considerable specificity
toward peroxynitrite). This phenomenon provides a unique op-
portunity to develop a chemical tool to monitor peroxynitrite in a
specific manner. In this regard, fluorescent probes are well suited
as reagents that allow the cellular chemistry of peroxynitrite to be
examined at the molecular level.
We envisioned that fluorescent probes for peroxynitrite could
be designed by replacing the methoxy group of 1 with a fluorescent
chromophore. Upon treatment with peroxynitrite in situ, the ketone
To investigate the specificity of HKGreen-1 toward peroxyni-
trite, we tested its response toward various oxidative species that
† Department of Chemistry and Open Laboratory of Chemical Biology.
‡ School of Chinese Medicine.
1
•
are present in biological systems, including NO, O2, O2•-, OH,
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10.1021/ja0603756 CCC: $33.50 © 2006 American Chemical Society

C O M M U N I C A T I O N S
the neuronal cells was induced after treatment with SIN-1 (10 µM)
(Figure 2B), but not with either SNAP (Figure 2C) or xanthine/
xanthine oxidase (Figure 2D). Thus, we conclude that HKGreen-1
can be used for the selective detection of peroxynitrite produced
in cultured cells.
In summary, we have developed a new fluorescent probe
HKGreen-1 that is effective for the highly selective detection of
peroxynitrite in living cells. We anticipate that this simple, sensitive
fluorescent probe will be of great benefit for biomedical researchers
investigating the effects of peroxynitrite in biological systems.
Figure 1. (A) Fluorescence response of 20 µM HKGreen-1 to ONOO-.
Spectra were acquired in 0.1 M potassium phosphate buffer at pH 7.3 (λex
) 490 nm). Inset: A linear correlation between emission intensity and
concentrations of ONOO-. (B) Fluorescence intensity of HKGreen-1 (20
µM) in various ROS/RNS (300 µM) generating systems at 25 °C for 1 h.
The fluorescence intensity was determined at 521 nm with excitation at
490 nm (slit width 2.5 nm).
Acknowledgment. We thank The University of Hong Kong,
Hong Kong Research Grants Council (HKU7060/05P and HKU7495/
04M), the Area of Excellence Scheme (AoE/P-10-01) established
under the University Grants Committee (HKSAR), and Bristol-
Myers Squibb Foundation (in the form of the Unrestricted Grant
in Synthetic Organic Chemistry to D.Y.) for financial support. We
dedicate this work to Professor Ronald Breslow (Columbia
University) on the occasion of his 75th birthday.
Supporting Information Available: Experimental details on the
synthesis and characterization of HKGreen-1. This material is available
free of charge via the Internet at http://pubs.acs.org.
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Figure 2. Fluorescence imaging of primary cultured neuronal cells. Left:
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ROO•, -OCl, and ONOO-. Figure 1B indicates that a 7- to 8-fold
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