A highly selective ratiometric fluorescent probe for peroxynitrite detection in aqueous media

Article abstract of DOI:10.1246/cl.160213

Herein we developed a fast-responsive fluorescent probe 2-(benzo[d]thiazol-2-yl)-4-methylphenol (HMBT)-Py for ONOO^- detection. HMBT-Py can react with ONOO^- by an activated C=C oxidation cleavage reaction, causing a ratiometric change in the fluorescence spectra. In addition, HMBT-Py showed high selectivity to ONOO^- over other reactive species.

Full text of DOI:10.1246/cl.160213

Received: March 2, 2016 | Accepted: March 18, 2016 | Web Released: March 30, 2016
CL-160213
A Highly Selective Ratiometric Fluorescent Probe for Peroxynitrite Detection in Aqueous Media
Ye-Xin Liao, Zhao-Xuan Yang, Kun Li,* and Xiao-Qi Yu*
Key Laboratory of Green Chemistry and Technology (Ministry of Education), College of Chemistry, Sichuan University,
Chengdu 610064, P. R. China
(
E-mail: kli@scu.edu.cn, xqyu@scu.edu.cn)
Herein we developed a fast-responsive fluorescent probe 2-
¹
(
benzo[d]thiazol-2-yl)-4-methylphenol (HMBT)-Py for ONOO
¹
detection. HMBT-Py can react with ONOO by an activated
C=C oxidation cleavage reaction, causing a ratiometric change
in the fluorescence spectra. In addition, HMBT-Py showed high
¹
selectivity to ONOO over other reactive species.
Keywords: Fluorescence probe
| Peroxynitrite |
C–C double bond cleavage
1
Highly reactive oxygen species (hROS), including O ,
2
¹
1
•
OH, OOH, ONOO , HOCl, and HOBr, can easily cause
oxidative stress (OS), which is an established risk factor because
they can cause huge damage to cells or tissues when they are
Scheme 1. Synthetic routes of target probe and sensing
mechanism.
2
overproduced and mismanaged. As the pKa of peroxynitrite
¹
¹
anion (ONOO ) is 6.8, ONOO is the major form under most
biological conditions, rather than the unstable protonated state
3
¹
ONOO using the oxidative double-bond cleavage reaction.17
On the other hand, pyridine cation can act as the accepted part
in the “donor­acceptor” (D­A) system, which can easily cause
¹
(
ONOOH) at pH 7.40 for instance. Endogenous ONOO is
generated by a combination reaction of nitric oxide (•NO) with
•
¹ 4
superoxide radical (O2
) with a short half-life (ca. 1 s at
5
21
pH 7.40). Due to strong oxidative and nitrative abilities,
ONOO can easily react with a variety of biomolecules (e.g.
bathochromic shift of the emission wavelength, and the new
¹
D­A system might result in a ratiometric phenomenon when it is
2
2
lipid, protein, and nuclear acid), leading to various diseases like
inflammatory and cardiovascular diseases, neurodegeneration,
interrupted. Herein, we introduced a C=C bond linkage, which
is connected with a well-known fluorophore 2-(benzo[d]thiazol-
2-yl)-4-methylphenol (HMBT, compound 1) and N-methylpyr-
idine cation to obtain a target probe HMBT-Py. We expected
6
¹
and cancer. On the other hand, ONOO served as a redox
signaling molecule, modulating the cell signal transduction
7
¹
process. Considering the complex life process in vivo, it is a
that the probe can act as an excellent probe for ONOO
monitoring using the activated C=C to react with ONOO
¹
wonderful choice to conduct in vitro simulation to investigate
¹
the role of ONOO in physiology and pharmacology.
and interrupt the D­A system (Scheme 1). As expected, when
¹
¹
Till now, several detection methods of ONOO monitoring
HMBT-Py reacts with ONOO in aqueous solution, a remark-
8
has been developed, including optical sensing, electrochemical
able ratiometric change is observed (Figure 1).
9
19
analysis, F magnetic resonance, and electron spin resonance
With HMBT-Py in hand, basic spectral properties of
HMBT-Py were investigated. First of all, fluorescence spectra
of HMBT-Py in different solvents were recorded (Figure S1).
As can be seen, in the investigated solvents (DMSO, DMF,
MeOH, EtOH, THF, MeCN, and PBS buffer (pH 7.40, 10 mM)),
the emission maximum wavelengths were almost similar
(around 660 nm). Although the intensity in PBS buffer was
weak, it is enough to conduct the following experiments in PBS
buffer. Two major peaks (312 and 492 nm) and a side peak
(400 nm) were found in the absorbance spectra (Figure S2). As
1
0,11
(ESR).
Among these methods, fluorescence sensing has
some advantages, such as simplicity, high temporal and spatial
resolutions, and high sensitivity, and it is widely applied in
¹
4,12­18
ONOO detection.
Most of the fluorescent probes for
¹
ONOO are based on the specific oxidation reactions, such as
13
4,14
aryl ether group oxidation, aryl boronate oxidation,
phenol
group oxidation, heteroatom (Se, Te for instance) oxidation,16
15
activated carbon­carbon (C=C) double bond oxidative cleav-
age,1 and others. Although many probes have been reported
7
18
¹
19
¹
for ONOO , most of them have a long response time (>5 min)
or suffer interference from other reactive species. As endoge-
nous ONOO exists in low concentration and has a short
shown in Figure 1, after reacting HMBT-Py with ONOO , the
emission peak (645 nm) disappeared and a new peak centered
at 545 nm was observed. We supposed this hypsochromic shift
should be induced by a disruption of the D­A system in HMBT-
Py molecule after the oxidation cleavage of C=C bond by
¹
lifetime, more sensitive and extremely fast responsive fluores-
cent probes should be developed to meet the demand of
¹
¹
detecting ONOO . Herein, we reported a ratiometric fluorescent
ONOO , which was reported to generate aldehyde product in
1
7
probe for peroxynitrite with fast response time (within 5 min).
The C=C double bond can be oxidized by strong oxidative
the oxidation reaction. Evidences were also found in high-
resolution mass spectrum (HRMS) and TLC results of the
2
0
¹
species to generate various double-bond cleavage products.
reaction solution between HMBT-Py and ONOO . As shown in
¹
Activated C=C was already applied to the recognition of
the ES result in Figure S3, a peak of m/z 268.0432 was found,
Chem. Lett. 2016, 45, 691–693 | doi:10.1246/cl.160213
© 2016 The Chemical Society of Japan | 691

Figure 1. Fluorescent responses of probe HMBT-Py (10 ¯M)
reacted with different concentrations (including: 0, 10, 20, 30,
Figure 2. Time-dependent fluorescence intensity at 545 and
4
0, 50, 60, 70, 80, 90, 100, 110, 130, 150, and 170 ¯M) of
¹
6
45 nm of HMBT-Py (10 ¯M) in the present of ONOO
100 ¯M) in PBS buffer at room temperature. Different time
points including: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 13, 15, 17, 20,
5, and 30 min.
¹
ONOO in PBS buffer after 5 min at room temperature.
ex = 400 nm, slits: 5/5 nm.
(
­
2
which towel matched with the result of compound 2 (calculated
as m/z 268.0438). Moreover, a TLC image of the reaction
¹
solution of HMBT-Py and ONOO was recorded (Figure S4),
in which the Rf value of the new product in the reaction agreed to
that of compound 2 (Rf = 0.49).
After the reaction conditions were confirmed, spectral
¹
responses of HMBT-Py in the presence of ONOO were
¹
recorded. With increasing concentration of ONOO , absorbance
decreased gradually in a broad range from 485 to 600 nm
(Figure S2), and a ratiometric response was observed in the
emission spectra with a weakened emission at 645 nm and an
enhanced emission at 545 nm (Figure 1). As shown in the inset
of Figure 1, the I545/I645 ratio increased gradually to maximum
¹
till the ONOO concentration reached 100 ¯M and finally to
¹
the equilibrium value. Consequently, 100 ¯M ONOO was used
in the following experiment to ensure a complete reaction.
¹
Next, the reaction kinetics of HMBT-Py (10 ¯M) with ONOO
(
100 ¯M) in PBS buffer was investigated (Figure 2 and
¹
Figures S5, S6, and S7). After the addition of ONOO , ether
absorbance or fluorescence emission spectra change imme-
diately, and no obvious changes were observed after 5 min.
Hence, 5 min was set as the final reaction time in the following
experiment.
Figure 3. Selectivity of HMBT-Py (10 ¯M) to different
reactive species (ROS, RNS, and RSS include: 100 ¯M for
¹
¹
t
1
•¹
2¹
2¹
ONOO , ClO , NO, BuOO•, HO•, O2, O2 , SO3 , and S
1
,
t
¹
¹
mM for H2O2, BuOOH, NO2 , NO3 , Cys, Hcy, and GSH) in
PBS (pH 7.40, 10 mM) at room temperature.
High selectivity is an important criterion for an excellent
probe. Up to date, most ONOO probes were still interfered by
¹
other ROS such as HClO and H2O2. Highly selective fluorescent
probes for ONOO recognition are in high demand. Hence,
our disappointment, the fluorescence intensity ratio (Igreen/Ired)
showed no obvious difference in the absence and presence of
3-morpholinosydnonimine (SIN-1, a common ONOO donor,
¹
¹
to demonstrate the selectivity of HMBT-Py, the reactions of
different reactive species, including ROS and some typical
reactive nitrogen species (RNS) or reactive sulfur species (RSS)
Figures 4q and 4r), indicating HMBT-Py could not image
¹
ONOO in living cells. As was reported, positively charged
¹
1
•¹
t
t
(
including ClO , H2O2, HO•, O2, O2 , BuOOH, BuOO•,
compounds could easily bind to negatively charged biomole-
¹
¹
2¹
2¹
23
NO, NO2 , NO3 , SO3 , S , Cys, Hcy, and GSH) to probe
cules like proteins and nucleic acid, we suspected that those
HMBT-Py were investigated in aqueous solution (Figure 3).
As is shown, except ONOO , the other species could not
negatively charged biomolecule in cells would complex with
HMBT-Py due to the positive charge of the pyridine salt, which
might further hinder the sensing process.
¹
cause any remarkable change demonstrating high selectivity of
¹
HMBT-Py to ONOO over other reactive species in aqueous
In conclusion, we have developed a water-soluble ratio-
¹
condition.
metric fluorescent probe HMBT-Py for ONOO monitoring.
Finally, we investigated the response ability of HMBT-Py
towards ONOO in living HeLa and A549 cells (Figure 4). To
HMBT-Py exhibited high reactivity and selectivity towards
ONOO over other reactive species in aqueous condition.
¹
¹
692 | Chem. Lett. 2016, 45, 691–693 | doi:10.1246/cl.160213
© 2016 The Chemical Society of Japan

Figueiredo, E. Aikawa, J. McCarthy, R. Weissleder, S. A.
Hilderbrand, J. Am. Chem. Soc. 2009, 131, 15739.
9
1
a) Y. Wang, Z. Chen, Talanta 2010, 82, 534. b) J. S. Cortés, S. G.
Granados, A. A. Ordaz, J. A. L. Jiménez, S. Griveau, F. Bedioui,
Electroanalysis 2007, 19, 61. c) W. C. A. Koh, J. I. Son, E. S.
Choe, Y.-B. Shim, Anal. Chem. 2010, 82, 10075.
0 K. J. Bruemmer, S. Merrikhihaghi, C. T. Lollar, S. N. S. Morris,
J. H. Bauer, A. R. Lippert, Chem. Commun. 2014, 50, 12311.
11 a) R. M. Gatti, B. Alvarez, J. Vasquez-Vivar, R. Radi, O.
Augusto, Arch. Biochem. Biophys. 1998, 349, 36. b) S.
Bartesaghi, V. Valez, M. Trujillo, G. Peluffo, N. Romero, H.
Zhang, B. Kalyanaraman, R. Radi, Biochemistry 2006, 45, 6813.
2 a) P. Chen, X. Zhou, H. Shen, N. M. Andoy, E. Choudhary, K.-S.
Han, G. Liu, W. Meng, Chem. Soc. Rev. 2010, 39, 4560. b) Z.-Y.
Wu, J.-N. Cui, X.-H. Qian, T.-Y. Liu, Chin. Chem. Lett. 2013, 24,
1
1
1
359. c) W.-Z. Zhang, Z.-B. Du, B. Song, Z.-Q. Ye, J.-L. Yuan,
Chin. Chem. Lett. 2015, 26, 1465.
3 a) D. Yang, H.-L. Wang, Z.-N. Sun, N.-W. Chung, J.-G. Shen,
J. Am. Chem. Soc. 2006, 128, 6004. b) Z.-N. Sun, H.-L. Wang,
F.-Q. Liu, Y. Chen, P. K. H. Tam, D. Yang, Org. Lett. 2009, 11,
1887. c) J. Ma, J. Wu, W. Liu, P. Wang, Z. Fan, Spectrochim.
Acta, Part A 2012, 94, 340.
4 a) A. Sikora, J. Zielonka, M. Lopez, J. Joseph, B. Kalyanaraman,
Free Radical Biol. Med. 2009, 47, 1401. b) J. Kim, J. Park, H.
Lee, Y. Choi, Y. Kim, Chem. Commun. 2014, 50, 9353. c) X. Sun,
Q. Xu, G. Kim, S. E. Flower, J. P. Lowe, J. Yoon, J. S. Fossey, X.
Qian, S. D. Bull, T. D. James, Chem. Sci. 2014, 5, 3368. d) J.
Zhou, Y. Li, J. Shen, Q. Li, R. Wang, Y. Xu, X. Qian, RSC Adv.
Figure 4. HeLa cells or A549 cells were incubated with
HMBT-Py (5 ¯M) for 30 min (HeLa: a­d, A549: e­h), then
incubated with 100 ¯M SIN-1 (HeLa: i­l, A549: m­p). Images of
green channel (525­565 nm, ex@405 nm) and red channel (625­
2
014, 4, 51589.
1
5 a) T. Peng, N.-K. Wong, X. Chen, Y.-K. Chan, D. H.-H. Ho, Z.
Sun, J. J. Hu, J. Shen, H. El-Nezami, D. Yang, J. Am. Chem. Soc.
2
Jiang, Y.-M. Lu, M.-H. Liao, W.-F. Ye, N.-N. Lu, F. Han, Y.-Z.
Hu, Y.-H. Hu, J. Am. Chem. Soc. 2015, 137, 12296. c) H. Zhang,
J. Liu, Y.-Q. Sun, Y. Huo, Y. Li, W. Liu, X. Wu, N. Zhu, Y. Shi,
W. Guo, Chem. Commun. 2015, 51, 2721.
014, 136, 11728. b) X. Li, R.-R. Tao, L.-J. Hong, J. Cheng, Q.
665 nm, ex@405 nm) were collected. Ratios of green channel
and red channel (Igreen/Ired) before and after incubating 100 ¯M
SIN-1 in HeLa cells (q) or A549 cells (r) were calculated. The
scale bar in the images (white bar) stands for 10 ¯m.
1
1
6 a) F. Yu, P. Li, G. Li, G. Zhao, T. Chu, K. Han, J. Am. Chem. Soc.
2011, 133, 11030. b) F. Yu, P. Li, B. Wang, K. Han, J. Am. Chem.
This work was financially supported by the National
Program on Key Basic Research Project of China (973 Program,
Nos. 2012CB720603) and the National Science Foundation of
China (Nos. 21572147, 21232005, and J1310008).
Soc. 2013, 135, 7674. c) K. Xu, H. Chen, J. Tian, B. Ding, Y. Xie,
M. Qiang, B. Tang, Chem. Commun. 2011, 47, 9468.
7 a) J.-T. Hou, J. Yang, K. Li, Y.-X. Liao, K.-K. Yu, Y.-M. Xie,
X.-Q. Yu, Chem. Commun. 2014, 50, 9947. b) A. J. Shuhendler,
K. Pu, L. Cui, J. P. Uetrecht, J. Rao, Nat. Biotechnol. 2014, 32,
Supporting Information is available on http://dx.doi.org/
373. c) X. Zhou, Y. Kwon, G. Kim, J.-H. Ryu, J. Yoon, Biosens.
1
0.1246/cl.160213.
Bioelectron. 2015, 64, 285.
1
8 a) J.-C. Huang, D.-J. Li, J.-C. Diao, J. Hou, J.-L. Yuan, G.-L. Zou,
Talanta 2007, 72, 1283. b) Q. Zhang, Z. Zhu, Y. Zheng, J. Cheng,
N. Zhang, Y.-T. Long, J. Zheng, X. Qian, Y. Yang, J. Am. Chem.
Soc. 2012, 134, 18479. c) K.-K. Lin, S.-C. Wu, K.-M. Hsu, C.-H.
Hung, W.-F. Liaw, Y.-M. Wang, Org. Lett. 2013, 15, 4242.
References
1
a) Y. You, W. Nam, Chem. Sci. 2014, 5, 4123. b) Y.-Y. Huang,
M.-J. Wang, Z. Yang, M.-Y. She, S. Wang, P. Liu, J.-L. Li, Z. Shi,
Chin. Chem. Lett. 2014, 25, 1077.
2
3
4
5
6
B. C. Dickinson, C. J. Chang, Nat. Chem. Biol. 2011, 7, 504.
R. Radi, J. Biol. Chem. 2013, 288, 26464.
F. Yu, P. Song, P. Li, B. Wang, K. Han, Analyst 2012, 137, 3740.
N. Ashki, K. C. Hayes, F. Bao, Neuroscience 2008, 156, 107.
a) C. Szabó, H. Ischiropoulos, R. Radi, Nat. Rev. Drug Discovery
19 T. Ueno, Y. Urano, H. Kojima, T. Nagano, J. Am. Chem. Soc.
2006, 128, 10640.
20 a) S. Pennathur, D. Maitra, J. Byun, I. Sliskovic, I. Abdulhamid,
G. M. Saed, M. P. Diamond, H. M. Abu-Soud, Free Radical Biol.
Med. 2010, 49, 205. b) J. Park, H. Kim, Y. Choi, Y. Kim, Analyst
2013, 138, 3368.
21 N. Karton-Lifshin, L. Albertazzi, M. Bendikov, P. S. Baran, D.
Shabat, J. Am. Chem. Soc. 2012, 134, 20412.
22 M. H. Lee, J. S. Kim, J. L. Sessler, Chem. Soc. Rev. 2015, 44,
4185.
23 G. Han, D. Kim, Y. Park, J. Bouffard, Y. Kim, Angew. Chem., Int.
Ed. 2015, 54, 3912.
2
007, 6, 662. b) J. P. Eiserich, M. Hristova, C. E. Cross, A. D.
Jones, B. A. Freeman, B. Halliwell, A. van der Vliet, Nature
998, 391, 393. c) P. Pacher, J. S. Beckman, L. Liaudet, Physiol.
1
Rev. 2007, 87, 315.
7
8
T. Okamoto, T. Akaike, T. Sawa, Y. Miyamoto, A. van der Vliet,
H. Maeda, J. Biol. Chem. 2001, 276, 29596.
P. Panizzi, M. Nahrendorf, M. Wildgruber, P. Waterman, J.-L.
Chem. Lett. 2016, 45, 691–693 | doi:10.1246/cl.160213
© 2016 The Chemical Society of Japan | 693