A three-channel fluorescent probe that distinguishes peroxynitrite from hypochlorite

Article abstract of DOI:10.1021/ja305046u

A novel fluorescent probe for peroxynitrite, PN₆₀₀, was rationally designed on the basis of a unique fluorophore assembly approach. PN₆₀₀ is a green-emitting coumarin derivative. Upon oxidation by peroxynitrite, PN₆₀₀ is transformed into a highly fluorescent red-emitting resorufin derivative via an orange-emitting intermediate. This three-channel signaling capability enables PN₆₀₀ to differentiate peroxynitrite from other reactive oxygen and nitrogen species, including hypochlorite and hydroxyl radical. Moreover, PN₆₀₀ is membrane-permeable and compatible with common TRITC filter sets and displays low cytotoxicity. Therefore, PN₆₀₀ is a promising candidate for in vitro peroxynitrite imaging.

Full text of DOI:10.1021/ja305046u

Communication
pubs.acs.org/JACS
A Three-Channel Fluorescent Probe That Distinguishes Peroxynitrite
from Hypochlorite
Quanjuan Zhang,‡ Zhichuan Zhu,§ Yongli Zheng,∥ Jiagao Cheng,‡,⊥ Na Zhang,# Yi-Tao Long,#
Jing Zheng,*,§ Xuhong Qian,*,†,‡ and Youjun Yang*,†,‡
†State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Meilong Road 130, Shanghai
200237, China
§
⊥
‡Shanghai Key Laboratory of Chemical Biology, Department of Pharmaceutical Science, and Shanghai Key Laboratory of New
Drug Design, School of Pharmacy, East China University of Science and Technology, Meilong Road 130, Shanghai 200237, China
∥School of Chemistry and Chemical Engineering, State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University,
Dongchuan Road 800, Shanghai 200240, China
#Department of Chemistry, East China University of Science and Technology, Meilong Road 130, Shanghai 200237, China
S
* Supporting Information
cially fluorescence-based techniques, are advantageous and have
ABSTRACT: A novel fluorescent probe for peroxynitrite,
PN600, was rationally designed on the basis of a unique
fluorophore assembly approach. PN600 is a green-emitting
coumarin derivative. Upon oxidation by peroxynitrite,
PN600 is transformed into a highly fluorescent red-emitting
resorufin derivative via an orange-emitting intermediate.
This three-channel signaling capability enables PN600 to
differentiate peroxynitrite from other reactive oxygen and
nitrogen species, including hypochlorite and hydroxyl
radical. Moreover, PN600 is membrane-permeable and
compatible with common TRITC filter sets and displays
low cytotoxicity. Therefore, PN600 is a promising candidate
for in vitro peroxynitrite imaging.
become routine practice in the field. Reduced xanthene dyes and
chemiluminescent probes are the earliest luminescent probes for
OONO−. They have been frequently used despite their poor
selectivity.6,7 Recently, a number of small-molecule fluorescent
probes for OONO− [Figure S22 in the Supporting Information
(SI)] have been reported in the literature.8 They are improved in
many aspects compared with the reduced xanthenes and have
found applications in biomedical studies.9 However, optimal
selectivity against highly oxidative species such as hypochlorite
(ClO−) and hydroxyl radicals (HO•) still remains challenging.
Herein we describe a novel small-molecule fluorescent probe,
PN600, that displays high selectivity toward OONO−. It delivers a
sensitive, three-channel fluorescence signal and holds great
potential in cell-imaging applications. Probes that yield signals on
more than one channel are highly desirable since they enable self-
calibration and circumvent complications from fluorophore
photobleaching, uneven probe loading, and fluctuations of the
excitation source.10 To our knowledge, PN600 is the first small-
molecule multichannel probe for OONO−.
eroxynitrite (OONO−) was first discovered as an
1
Pendogenous oxidant in 1990. It is generated via the nearly
diffusion-controlled combination of nitric oxide (NO) and
superoxide radical anion. Further research revealed that it largely
mediates the apparent cytotoxicity of NO.1,2 OONO− can lead to
protein malfunction via two distinct mechanisms: (1) nitration of
the phenol moiety of tyrosine residues and (2) oxidation of a
wide range of substrates, including transition-metal centers,
cysteine, methionine, tryptophan, and histidine. It can also
induce lipid peroxidation and DNA damage.3 Cell apoptosis or
necrosis often results when the cell damage from OONO−
overwhelms the cell repairing capacity. Direct correlations
between OONO− and numerous pathological conditions, such
as cardiovascular diseases, circulatory shock, inflammation,
cancers, stroke, reperfusion injury, neurodegenerative disorders,
and diabetes, have been established.3f Therefore, methods for
detection of cellular OONO− levels are of considerable
significance for both disease diagnosis and exploration of its
diverse pathophysiology.
Our strategy for OONO− detection relies on a rationally
designed oxidative coupling reaction reminiscent of phenol
oxidation. The oxidation of phenol into hydroquinone involves
nucleophilic attack of a cationic intermediate by H2O.11 We
envisioned that oxidation of a phenol with an amino group in
close proximity could lead to predominant aminophenol
formation due to the enhanced nucleophilicity of the amino
group relative to water. Subsequent two-electron oxidation of
aminophenol would give rise to an iminoquinone. On the basis of
this hypothesis, we designed PN600, which bears the aforemen-
tioned structural features for detection of oxidative species and a
coumarin moiety for signal transduction (Figure 1). Importantly,
the coumarin moiety is installed in such a way that a resorufin
derivative (2) is obtained via an oxidation-initiated cascade.
Bathochromic-shifted absorption and emission bands were
expected, generating a multichannel probe.
Immunohistological quantification of cellular nitrotyrosine has
been widely used to estimate the OONO− activity.4 However,
this approach is complicated by the presence of alternative
tyrosine nitration mechanisms and lack of high spatiotemporal
resolution.5 Comparatively, luminescence measurements, espe-
Received: May 24, 2012
Published: September 14, 2012
© 2012 American Chemical Society
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Communication
Figure 1. Detection scheme and normalized excitation/emission spectra
of (A) PN600, (B) 1, and (C) 2. The ε × ϕ of PN600, 1, and 2 were found
to be 9000 × 0.09 in H2O, 17 000 × 0.01 in H2O, and 31 000 × 0.62 in
EtOH, respectively. The values of ε are given in units of cm−1 M−1.
PN600 and intermediate 1 were synthesized and characterized
(see the SI). A solution of the product, 2, which exhibits an
intense purple hue and a red glow under a fluorescent lamp, was
obtained by addition of a large excess (>200 equiv) of ClO− to a
solution of 1 in MeOH. The molecular composition of 2 was
unambiguously confirmed by high-resolution mass spectrometry
([M]+ = m/z 279.0525) . Though the absorption and emission
properties of this solution remained stable for days without
protection from light and oxygen, compound 2 seemed to
undergo reduction upon drying, and therefore, we could not
isolate pure 2 for NMR characterization. The spectral and
photophysical properties of PN600, 1, and 2 are included in Figure
1. The excitation maxima of PN600, 1, and 2 are widely separated
from each other, while their emissions overlay at ca. 600 nm.
Thus, the three species can be monitored simultaneously
through their signature excitation channels at 355, 475, and
575 nm, respectively, via a single fluorescence excitation spectral
scan monitored at the same emission wavelength. In these
spectral studies, we chose to monitor the emission intensity at
620 nm to acquire the entire excitation band of product 2.
Alternatively, 2 could be selectively excited in the presence of
both PN600 and 1 since its maximum excitation wavelength is
much longer.
Figure 2. Fluorescence spectral studies of OONO−, ClO−, and HO•
detection by PN600 (20 μM in 50 mM phosphate buffer at pH 7.4): (1)
Excitation spectra upon addition of varying amounts of (A-1) OONO−,
(B-1) ClO−, and (C-1) HO• with monitoring of the emission at 620 nm.
(2) Changes in the excitation intensities at 355 nm (probe channel), 465
nm (intermediate channel), and 575 nm (product channel) with respect
to the added amounts of (A-2) OONO−, (B-2) ClO−, and (C-2) HO•.
(3) Emission spectra upon addition of varying amounts of (A-3)
OONO−, (B-3) ClO−, and (C-3) HO• with excitation at 550 nm. (4)
Changes in the emission intensity at 595 nm with respect to the added
amounts of (A-4) OONO−, (B-4) ClO−, and (C-4) HO•. Spikes at 310
nm are second-order scattered light.
As shown by the excitation scans monitoring the emission at
620 nm (Figure 2A-1,A-2), addition of an aliquot of OONO−
stock solution led to rapid consumption of probe PN600 and
buildup of the intermediate 1, as indicated by the excitation
intensities at 350 and 467 nm, respectively. The excitation
intensity of the product 2 at 575 nm also increased upon addition
of OONO−; however, the signal enhancement was insignificant
with additions of fewer equivalents. When the OONO−
concentration exceeded 15 equiv (or 300 μM), the signal of
the product started to rise abruptly and eventually leveled off.
Concomitantly, the intermediate signal decreased. This single
experiment clearly exemplifies the multichannel capability of
probe PN600 in OONO− detection. Alternatively, an emission
scan with excitation at 550 nm could be used to monitor the
formation of product selectively (Figure 2A-3,A-4). A
fluorescence turn-on signal at 595 nm was observed to appear
from a dark background. The fluorescence turn-on ratio was
extremely high, and the detection sensitivity was optimal.
Moreover, the ability to be excited efficiently at 550 nm and
emit at 595 nm makes 2 an ideal fluorophore to be coupled with
the widely available TRITC filter set in imaging studies, which
has excitation and emission bandpasses of 555 10 and 600 20
nm, respectively.
disappearance of the probe signal at 350 nm, and the signal
intensity of 1 at 467 nm correspondingly reached a plateau.
However, ClO− could not further oxidize the intermediate to
form the product 2. Essentially no product excitation at 575 nm
was observed with further addition of ClO− up to 30 equiv (or
600 μM). Both ClO− and OONO− are well-known for their high
oxidizing capacities; therefore, it has been challenging to attain
high degree of selectivity for one or the other of these species
when developing fluorescent probes. Herein we have shown that
PN600 can reliably differentiate OONO− from ClO−.
We then examined the selectivity of PN600 against HO•, which
was generated in situ via Fenton chemistry12 by mixing H2O2 and
Fe2+. Although HO• has been found to interfere with OONO−
detection using some benchmark fluorescent probes that also
contain electron-rich aromatic moieties,8b it did not interfere
with PN600 in our case. Addition of up to 30 equiv of Fe(ClO4)2
solution into a probe solution containing a large excess of H2O2
yielded only a 30% decrease of the probe signal intensity (Figure
In a parallel study (Figure 2B), it was found that ClO− could
also induce oxidation of PN600 to form intermediate 1. Addition
of ca. 10 equiv of ClO− (or 200 μM) led to the total
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Communication
19
2C). Only minimal amounts of the intermediate and the product
were observed, and the signal intensities were negligible
compared with those from OONO− oxidation. Attempts to
introduce more Fe(ClO4)2 into the solution resulted in Fe(OH)2
precipitation, which led to intense light scattering.
including DAF18 and NO550
,
are based on this reaction.
However, the amino group in PN600 presumably has reduced
nucleophilicity compared with that in aniline since it is attached
to a rather electron-deficient coumarin core. Thus, hydrolysis of
N2O3 probably outcompetes the N-nitrosation. A higher dose of
NO was not tested because of the low solubility of NO in H2O
(∼1.9 mM ). Upon the addition of a large excess of O2 (ca.
1400 equiv in the form of solid KO2) to the probe solution with
vigorous stirring, the solution fluorescence was measured 5 min
after the oxygen evolution ceased. It was found that the probe
signal was largely diminished with no enhancement of either the
intermediate or product channel. A titration experiment showed
that the UV−vis absorption of the PN600 solution underwent an
insignificant but consistent spectral change (Figure S24). This
suggests that PN600 could react with superoxide via a mechanism
that is still unclear. Nevertheless, neither 1 nor 2 was observed,
and hence, no interferences would result even with an extremely
high dose of superoxide.
The potentials of PN600 for in vitro imaging applications were
examined using human glioma cell line U87. Upon addition of a
PN600 stock solution to the cell culture, the probe was found to
cross the cell membrane readily. The presence of a phenolic
hydroxyl group and an amino group did not raise any
complications in regard to cell permeability, presumably because
of the good separation of their pKa values (ca. 10 for OH and ca.
In aqueous media or cells, OONO− can rearrange into
nontoxic nitrate via H+- or CO2-catalyzed pathways; therefore,
the “production rate” of OONO− in units of μM/min and the
“dosage” in units of μM·min are commonly employed to
complement or replace the use of “steady-state concentration”.13
The physiological steady-state OONO− concentration lies
between nanomolar and low micromolar,14 with the cell
production rate ranging from 0.1−1 μM/min15 to 50−100
μM/min.3e Bolus addition of 600 μM OONO−, which is the
highest concentration employed in our fluorescence titration
study, is equivalent to an exposure of sustained OONO−
production at a rate of 16 μM/min for 1 min or 1.6 μM/min
for 10 min.13,16 Therefore, the seemingly high OONO−
concentration falls within the biorelevant concentration range.
Additionally, bolus addition of OONO− stock solution above 1
mM is not uncommon.17
3f
•−
Other common reactive oxygen and nitrogen species,
including hydrogen peroxide (H2O2), singlet oxygen (1O2),
superoxide radical anion (O2•−) and nitric oxide (NO), were
found not to interfere under the same conditions (Figure 3).
+
4.5 for NH3 ) from the physiological pH range (6.9−7.4),
leading to the probe molecule being largely uncharged.
As expected, PN600 (20 μM) did not give any background
signal when imaged with the TRITC filter set (Figure 4B). This is
Figure 4. Live-cell imaging using PN600. Phase contrasts and images of
human glioma cells treated with (A, B) 20 μM PN600, (C, D) 20 μM
PN600 + 200 μM ClO−, and (E, F) 20 μM PN600 + 200 μM SIN-1 are
shown. The scale bar is 50 μm.
highly desirable for imaging applications, since a fluorescence
signal over a dark background renders highest possible contrast
and can be captured in a sensitive and unambiguous manner.
PN600 did not exhibit obvious cytotoxicity at this level within 16
h, as determined by an MTT assay. Addition of ClO− (200 μM)
did not lead to any detectable signal within 12 h (Figure 4D). In
contrast, addition of the OONO− donor 3-morpholinosydnoni-
mine (SIN-1) (200 μM) into cells loaded with the same level of
PN600 resulted in an obvious fluorescence enhancement (Figure
4F). SIN-1 provides a sustained OONO− flow in buffered
aqueous media, making it a donor that well mimics the cell
production of OONO−.20 The maximum OONO− production
rate was assayed to be ca. 1.4% of the added SIN-1
concentration.21 Therefore, the use of 200 μM SIN-1 in cell-
imaging studies is equivalent to treating cells with a sustained
OONO− source of ca. 2.8 μM/min. Again, this is in the
biorelevant range. These experiments proved that PN600 has
great promise for in vitro OONO− imaging applications.
Figure 3. PN600 selectivity studies: (1) Excitation spectra upon addition
of (A-1) H2O2, (B-1) O2, (C-1) NO, and (D-1) O2•−. Emission was
1
monitored at 620 nm. (2) Emission spectra with excitation at 550 nm
•−
upon addition of (A-2) H2O2, (B-2) 1O2, (C-2) NO, and (D-2) O2
.
1
Bolus addition of H2O2 (ca. 17 500 equiv), O2 (ca. 50 equiv),
and NO (ca. 10 equiv) led to essentially no discernible spectral
changes in any of the three channels for probe, intermediate, and
1
product. The O2 was generated by addition of a ClO− stock
solution into a probe solution containing a large excess of H2O2.
NO in an air-saturated solution is oxidized into N2O3, which can
nitrosylate aniline, and many fluorescent probes for NO,
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Communication
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́
In summary, we have developed PN600, a redox-active, low-
molecular-weight fluorescent probe for imaging OONO−.
Notably, PN600 yielded a three-channel fluorescence signal
upon addition of peroxynitrite and a dual-channel signal for
hypochlorite. Other commonly occurring reactive species
involved in cell oxidative burst did not interfere. The chemical
structure of PN600 is compact, with a molecular weight as low as
283 amu, as a result of the use of a novel probe design principle:
the fluorophore for signaling is assembled in situ via a substrate-
triggered chemical reaction. In addition, PN600 is membrane-
permeable and displays low cytotoxicity. With the aid of a routine
fluorescence microscope equipped with a common TRITC filter
set, PN600 selectively detected peroxynitrite in cellular studies.
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ASSOCIATED CONTENT
■
S
* Supporting Information
General methods, synthesis and characterization data, scans of
original 1H/13C NMR spectra and mass spectra, chemical
structures and abbreviations of existing OONO− probes, UV−vis
absorption titration of PN600 by KO2, and cyclic voltammograms
of PN600 and 1. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
youjunyang@ecust.edu.cn; xhqian@ecust.edu.cn
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The work was supported by the State Key Laboratory of
Bioreactor Engineering via the National Special Fund (2060204)
and the Open Funding Project, the Fundamental Research
Funds for the Central Universities (WJ1014005), the Natural
Science Foundation of Shanghai Municipality (11ZR1408800),
and the National Natural Science Foundation of China
(21106043). We thank Prof. Yufang Xu, Prof. Weiping Zhu,
and Prof. Yongfeng Zhou for insightful discussions and Dr. Mark
Lowry, Dr. Michelle A. Ivy, and Prof. Wei Wang for help with
manuscript preparation.
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