Investigation of Drug-Induced Hepatotoxicity and Its Remediation Pathway with Reaction-Based Fluorescent Probes

Article abstract of DOI:10.1021/acs.analchem.7b01671

Drug-induced liver injury (DILI) is considered a serious problem related to public health, due to its unpredictability and acute response. The level of peroxynitrite (ONOO^-) generated in liver has long been regarded as a biomarker for the prediction and measurement of DILI. Herein we present two reaction-based fluorescent probes (Naph-ONOO^- and Rhod-ONOO^-) for ONOO^- through a novel and universally applicable mechanism: ONOO^--mediated deprotection of α-keto caged fluorophores. Among them, Rhod-ONOO^- can selectively accumulate and react in mitochondria, one of the main sources of ONOO^-, with a substantial lower nanomolar sensitivity of 43 nM. The superior selectivity and sensitivity of two probes enable real-time imaging of peroxynitrite generation in lipopolysaccharide-stimulated live cells, with a remarkable difference from cells doped with other interfering reactive oxygen species, in either one- or two-photon imaging modes. More importantly, we elucidated the drug-induced hepatotoxicity pathway with Rhod-ONOO^- and revealed that CYP450/CYP2E1-mediated enzymatic metabolism of acetaminophen leads to ONOO^- generation in liver cells. This is the first time to showcase the drug-induced hepatotoxicity pathways by use of a small-molecule fluorescent probe. We hence conclude that fluorescent probes can engender a deeper understanding of reactive species and their pathological revelations. The reaction-based fluorescent probes will be a potentially useful chemical tool to assay drug-induced hepatotoxicity.

Full text of DOI:10.1021/acs.analchem.7b01671

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Article
Investigation of Drug-Induced Hepatotoxicity and Its
Remediation Pathway with Reaction Based Fluorescent Probes
Dan Cheng, Wang Xu, Lin Yuan, and Xiao-Bing Zhang
Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 19 Jun 2017
Downloaded from http://pubs.acs.org on June 20, 2017
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Investigation of Drug-Induced Hepatotoxicity and Its
Remediation Pathway with Reaction Based Fluorescent Probes
†
†,
‖
†
†
Dan Cheng , Wang Xu , Lin Yuan* , Xiaobing Zhang
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†
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical
Engineering, Hunan University, Changsha 410082 (PR China)
‖
Current address: Department of Chemistry, Stanford University
*
Email: lyuan@hnu.edu.cn. Fax: +86ꢀ731ꢀ88821894
Abstract: Drugꢀinduced liver injury (DILI) is considered a serious problem related to public health,
ꢀ
due to its unpredictability and acute response. The level of peroxynitrite (ONOO ) generated in
liver has long been regarded as a biomarker for the prediction and measurement of DILI. Herein we
-
-
ꢀ
present two reaction based fluorescent probes (Naph-ONOO and Rhod-ONOO ) for ONOO
ꢀ
through a novel and universally applicable mechanism: ONOO mediated deprotection of αꢀketo
-
caged fluorophores. Among them, Rhod-ONOO can selectively accumulate and react in
ꢀ
mitochondria, one of the main sources of ONOO , with a substantial lower nanoꢀmolar sensitivity of
43 nM. The superior selectivity and sensitivity of two probes enable realꢀtime imaging of
peroxynitrite generation in LPSꢀstimulated live cells, with a remarkable difference from cells doped
with other interfering ROS, in either oneꢀphoton or twoꢀphoton imaging mode. More importantly,
-
we elucidated the drugꢀinduced hepatotoxicity pathway with Rhod-ONOO , and revealed that
ꢀ
CYP450/CYP2E1ꢀmediated enzymatic metabolism of acetaminophen leads to ONOO generation
in liver cells. This is the first time to showcase the drug induced hepatotoxicity pathways using a
small molecule fluorescent probe. We hence conclude that fluorescent probes can engender a deeper
understanding of reactive species and their pathological revelations. The reaction based fluorescent
probes will be a potentially useful chemical tool to assay drug induced hepatotoxicity.
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Introduction
Drugꢀinduced liver injury (DILI) is the most common concern of acute liver failure,
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,2
accounting for a substantial portion of acute hepatitis. The emergence of DILI is complicated
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and its evaluation is difficult, as its clinical manifestation includes all pathological symptoms
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related to liver. The estimated annual incidence of DILI is between 10 and 15 per 10,000 to
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00,000 persons, a sample size too large to cover during clinical trials. Hence this phenomenon
also widely affects the pharmaceutical industry, leading to drug withdrawal even after released
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into the market. Innovative preclinical hepatotoxicity screening methods could help examine
hepatotoxicity during drug development; however, the development is proved to be complicated
6ꢀ8
because many biomarkers exist during DILI process. The lack of a straightforward, preclinical
screening assay is also an issue.
Epidemiological studies reveal that reactive radicals are the main sideꢀproducts of drug
2
metabolism in liver and led to a range of cell apoptosis and other acute liver damages. For
example, acetaminophen (APAP), a pain killer and fever reducer common found on the market, is
9
wellꢀknown to cause serious hepatotoxicity among certain population. In the liver, APAP
undergoes enzymatic biotransformation and generates reactive radicals, such as peroxynitrite
ꢀ
10,11
(ONOO ), through either oneꢀ or twoꢀelectron oxidation processes.
Since such enzymatic
ꢀ
biotransformation universally exists among a wide range of drugs, ONOO can therefore serve as a
1
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useful biomarker for detecting and studying drugꢀinduced hepatotoxicity. As the adduct of nitric
•ꢀ
ꢀ
2
oxide (NO) and superoxide radical (O ), ONOO is known to react with a wide array of
biomolecules, such as proteins, lipids, and nucleic acids, causing disruption of cellular structure
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and functions and finally leading to cell death.
While generated in liver, elevated levels of
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endogenous ONOO often indicate acute hepatocellular damage. Despite the fact that ONOO
tracing is crucial for both pathological and clinical studies, it has proved to be a tough target to
ꢀ
monitor. The extremely short halfꢀlives of ONOO (less than a second, ∼10 ms) almost precludes
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its detection after extraction, necessitating in situ monitoring upon formation.
Thus, it is a
ꢀ
grand and urgent challenge in modern pharmaceutical industry to monitor ONOO in real time.
In this work, we investigated the production and remediation pathways of drug induced
hepatotoxicity in live cells using a newly designed, reaction based fluorescent probe. Since the
emergence of fluorescence imaging technique, it has not only greatly enhanced the efficiency of
preclinical studies, but also enabled a nonꢀinvasive, powerful and clinically translatable way for
18ꢀ26
tracing the progression of drugs’ action and evaluating the extent of damages in realꢀtime.
So
ꢀ
27ꢀ38
ꢀ
far, there are a number of fluorescent probes for ONOO ,
most of which rely on ONOO
mediated oxidation that releases ketone end groups. Although some of these probes have been
39ꢀ41
used for biomedical studies,
there are very limited examples targeting drugꢀinduced
42,43
ꢀ
hepatotoxicity.
Our probe exploits an brand new reaction: ONOO mediated deprotection of
ꢀ
αꢀketoamide, in which an ONOO ꢀtriggered nucleophilic attack at the αꢀcarbonyl group leads to
the dissociation of diꢀketo group. Hence by linking a fluorophore and a quencher with the
ꢀ
αꢀketoamide group, we formulate a selective and sensitive fluorescent turnꢀon probe for ONOO .
ꢀ
As far as we know, this is not only a demonstration of new reaction based ONOO fluorescent
probe, but also a novel study of drug induced hepatotoxicity pathways using small molecule
fluorescent probes.
Experimental Section
Materials and General Experimental Methods. For details, see the Supporting Information.
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Synthesis of Naph-ONOO /Rhod-ONOO . Compounds Naph-NH /Rhod-NH was prepared by
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reported method.
HATU (38.0 mg, 0.1 mmol) and triethylamine (2 µL) in 1.5 mL anhydrous dichloromethane was
stirred for 20 min, then compound Naph-NH (27.0 mg, 0.1 mmol) or Rhod-NH (48.6 mg, 0.1
A mixture of compound 4ꢀnitrophenylglyoxylic acid (19.5 mg, 0.1 mmol),
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mmol) was added under argon atmosphere. The mixture was stirred at room temperature for 2
hours. Then the solvent was removed under vacuum condition, and the mixture was purified by
-
column chromatography on silica gel with (CH
2
Cl
2
/EtOH=60:1) to offer Naph-ONOO as a
-
yellow solid (14.5 mg, 32.5%) or Rhod-ONOO as a red solid (8.4 mg, 12.7%). For
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1
Naph-ONOO : H NMR (400 MHz, CDCl
3
) δ 9.91 (s, 1H), 8.71ꢀ8.69 (5H), 8.40 (d, J = 7.9 Hz,
2
2
1
H), 8.32 (d, J = 8.3 Hz, 1H), 7.88 (t, J = 7.5 Hz, 1H), 4.45 (t, J = 5.1 Hz, 2H), 3.75 (t, J = 5.1 Hz,
1
3
H), 3.39 (s, 3H). C NMR (100 MHz, CDCl ) δ 184.3, 162.9, 156.9, 136.1, 135.6, 131.8, 131.2,
3
17 3 7
30.7, 126.4, 124.7, 122.7, 122.6, 118.6, 117.6, 68.6, 57.8, 38.4. HRMS (EI) C23H N O (M),
-
1
calculated for 447.1057, found 447.1066. For Rhod-ONOO : H NMR (400 MHz, MeOD +
CDCl ) δ 8.26 (d, J = 8.8 Hz, 2H), 8.02 (d, J = 7.8 Hz, 1H), 7.98 (d, J = 8.8 Hz, 2H), 7.82 (t, J =
2.1 Hz, 1H), 7.76 (dd, J = 8.0, 4.9 Hz, 1H), 7.70 (t, J = 7.4 Hz, 1H), 7.27 (d, J = 8.7 Hz, 1H), 7.20
d, J = 7.5 Hz, 1H), 6.71 (d, J = 8.7 Hz, 1H), 6.57 (d, J = 8.8 Hz, 1H), 6.53 – 6.46 (2H), 3.42 (t, J
3
(
1
3
= 7.0 Hz, 4H), 1.19 (t, J = 6.8 Hz, 6H). C NMR (100 MHz, MeOD+CDCl
53.1, 152.1, 150.1, 148.2, 147.1, 139.7, 134.9, 131.6, 129.7, 128.9, 128.7, 128.3, 127.6, 124.6,
124.1, 122.9, 115.6, 115.4, 115.4, 108.8, 108.0, 97.2, 78.2, 77.9, 77.5, 44.1, 11.2. HRMS (ESI)
3
) δ 170.4, 169.4,
1
+
+
32 26 3 7
C H N O (M ), calculated for 564.1779, found 564.1776.
Fluorescence microscopic imaging of APAP-induced cell damage experiments. HepG2 cells
were pretreated with APAP (0 ꢀ 500 ꢁM, respectively) for 12 hours, and then incubated with 5.0
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M Naph-ONOO or Rhod-ONOO for 40 min. Cell imaging was carried out after washing the
cells with PBS for three times.
The remediation of hepatoxicity in living cells. HepG2 cells were treated with 200 ꢁM GSH for
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hour, or 20 ꢁM transꢀ1,2ꢀdichloroethylene (tꢀ1,2ꢀDCE) for 2 hours or 160 ꢁM
ꢀaminobenzotriazole (1ꢀABT) for 12 hours before drug treatment, respectively. Then the cells
-
were treated with APAP (250 ꢁM) for 12 hours, and then incubated with 5.0 ꢁM Rhod-ONOO
for 40 min before imaging.
Results and Discussion
-
Design and synthesis of fluorescent probes for ONOO . It is possible to design reaction based
ꢀ
fluorescent probes for ONOO owing to its strong nucleophilicity and oxidation capacity. Previous
ꢀ
literature has reported that αꢀketo esters can react with ONOO smoothly under physiological
27
conditions (Scheme 1A). Thus, we propose that deprotecting the αꢀketo caged fluorophore may
ꢀ
generate a specific turnꢀon probe for ONOO . In fact, the protection/deprotection approach
46ꢀ51
represents a new research focus for developing selective probes in biological systems.
The
protection groups could play a crucial role in modulating fluorescence “offꢀon” status.
Accordingly, we employed phenylglyoxylic acid to protect the amine group in the αꢀketo caged
fluorophore. The αꢀketoaminde protection of the amino group on the fluorophores weakens their
πꢀconjugation via combined effects of internal charge transfer and photoꢀinduced electron transfer.
The result is the low fluorescence emission of the fluorophore scaffolds. Upon reaction with
ꢀ
ONOO , the probe 1) undergoes a nucleophilic attack at aꢀketo position, forming an intermediate;
2) releases NO species by intramolecular cyclization and generates highly reactive epoxide; 3)
2
proceeds to intramolecular rearrangement to yield an anhydride, which hydrolyzes to dissociate
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into a carboxylic acid and amino group (Scheme 1B).
Based on the known mechanism, we incorporated the aꢀketoaminde group into two prominent
chromophores, rhodamine and amino naphthalimide. They are linked with phenylglyoxylic units
bearing simple electronꢀdonating or withdrawing substituents, such as hydrogen, methoxy group,
and nitro group at the paraꢀposition of the phenyl ring of phenylglyoxylic acid (Scheme 1C).
These fluorescent probes were prepared through simple oneꢀstep reactions (for synthetic and
characterization details, see the Supporting Information).
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Scheme 1. (A) The proposed reaction mechanism of αꢀketo esters with ONOO . (B) The proposed reaction
ꢀ
ꢀ
mechanism of αꢀketo caged fluorophore with ONOO . (C) Design of new fluorescent probes for ONOO .
-
To verify our proposed mechanism, we chose Naph-ONOO as the pilot compound and
-
analyzed the reaction mixture of Naph-ONOO and peroxynitrite. Apart from the final major
2
product Naph-NH with a mass of 269.2, we surprisingly found another peak at m/z = 418.2 (Figure
S1). We propose that another intramolecular rearrangement may exist in the reaction process.
ꢀ
ONOO engages in a nucleophilic addition at αꢀketo and forms a dioxirane, which quickly
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undergoes rearrangement to result in a reactive anhydride species. After anhydride forms, it is
prone to release carbon dioxide and render a reaction intermediates that is stable enough to be
captured by mass spectrometry: the amide product Naph-1. Then under aqueous condition the
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2 .
amide hydrolyzes to produce the final product, Naph-NH (Scheme S2) The capture of the
intermediate can partially attribute to the slow kinetics of amide hydrolysis through reactions with
external water molecules.
-
ꢀ
Spectral response of the probes to ONOO . We first examined their reactivity with ONOO at
physiological buffer conditions (PBS buffer solution, 25 mM, 1% DMF, pH 7.4). As shown in
Figure S2, Naph-H and Naph-OCH
3
displayed minor fluorescence enhancement after reaction
ꢀ
-
with ONOO while Naph-ONOO exhibited a more substantial fluorescence change within 0.5
-
ꢀ
hour, which indicates Naph-ONOO reacted faster with ONOO than Naph-H and Naph-OCH ,
3
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showing a kinectic advantage. We propose that the nitro group of Naph-ONOO can serve as a
strong electronꢀwithdrawing substituent, which enhances the electrophilicity of the para carbonyl
3
group, as compared to the hydrogen atom of Naph-H and the methoxy group of Naph-OCH ,
ꢀ
respectively. Accordingly, we have also developed another ONOO probe with potential
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mitochondriaꢀtargetable capacity based on rhodamine fluorophore, Rhod-ONOO , which has the
-
same reaction site as Naph-ONOO (Scheme 1C).
ꢀ
-
In addition, after reaction with ONOO , the absorption maxima of Naph-ONOO shifts from
350 nm to 382 nm, whereas a green emission band at 546 nm appears concomitantly (Figure S3
ꢀ
and 1A). Linearity is observed between fluorescence intensity and ONOO concentration (Figure
-
ꢀ
S4). The fluorescence and absorbance spectra of Rhod-ONOO at a range of ONOO
ꢀ
concentrations were also recorded, shown in Figure 1C and S5. With an increasing ONOO
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concentration (0ꢀ16 ꢁM), the fluorescence intensity of Rhod-ONOO at 558 nm also increases to
more than 14ꢀfold. The fluorescent color also gradually changes from colorless to orange emission
(
Figure 1C, inset), a consequence of rhodamine recovery. The detection limits of both probes are
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reaching 43 nM (Figure S4 and S6). The superior sensitivity of both probes ensures their usage in
ꢀ
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monitoring trace amounts of intracellular ONOO . Additionally, Naph-ONOO and Rhod-ONOO
display weak fluorescence emission at pH 4.1–9.1, and exhibit dramatic fluorescence
ꢀ
enhancement to ONOO in pH 7.0–9.0, which are well consistent with the physiological pH range
(
Figures S7 and S8).
To measure the selectivity of Naph-ONOO and Rhod-ONOO , we examined both probes
with a range of biologically relevant metabolites, including reactive oxygen species (ROS) (H O ,
-
-
2
2
•ꢀ
ꢀ
HOCl, O
2
, •OH, BuOO•), reactive sulphur species (RSS) (H
2
S, H
2
S
2
, HSO
3
, Cys, GSH), reactive
ꢀ
ꢀ
2ꢀ
nitrogen species (RNS) (HNO, NO, NO
2
, NO
3
) and bioꢀrelevant anions and cations (SO
4
,
ꢀ
+
+
2+
2+
2+
3
CH COO , Na , K , Mg , Fe and Cu ) (Figure 1B and D). No detectable emission increase was
noted even when the probes were preꢀincubated with a panel of 100 µM physiological analytes (1
ꢀ
mM for GSH). As a contrast, remarkable fluorescence enhancement was triggered by ONOO . A
more intense study was carried out to examine the potential disturbance of hydrogen peroxide
52,53
(H
2
2
O ), a known ROS reacting with αꢀketo compounds.
As shown in Figure S9, the
2 2
fluorescence titration experiments show that both probes resist the interference of H O with a broad
concentration range from 0 to 1000 µM. The superior selectivity can be attributed to the much
ꢀ
stronger nucleophilicity of ONOO than H O , and thus the diꢀketo linker could resist H O . These
2
2
2
2
-
-
ꢀ
results demonstrate that Naph-ONOO and Rhod-ONOO are feasible for ONOO detection in
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ꢀ
Figure 1. Fluorescence spectra of (A) Naph-ONOO and (C) Rhod-ONOO (5 ꢁM) upon addition of ONOO
(
0ꢀ 25 µM and 0ꢀ16 µM, respectively) in PBS buffer solution (25 mM, 1% DMF, pH 7.4); Fluorescence intensity
-
-
of (B) Naph-ONOO and (D) Rhod-ONOO (5 ꢁΜ) in PBS buffer solution (25 mM, 1% DMF, pH 7.4) toward
ꢀ
various analytes: ONOO (25 µM and 15 µM, respectively); other analytes (100 µM, 1 mM for GSH). 1, blank; 2,
•ꢀ
2
ꢀ
ꢀ
ꢀ
H
2
O
2
; 3, HOCl; 4, H
2
S; 5, Cys; 6, GSH; 7, H
2
S
2
; 8, O
; 9, HSO
3
; 10, NO
2
; 11, HNO; 12, NO; 13, NO
3
; 14,
ꢀ
+
+
2+
2+
2+
2ꢀ
ꢀ
CH
3
COO ; 15, BuOO•; 16, •OH; 17, Na ; 18, K ; 19, Mg ; 20, Fe ; 21, Cu ; 22, SO
4
; 23, ONOO . The mixture
was kept for 40 min at room temperature before the fluorescence intensity of the probe solution was recorded.
Excitation wavelength was 450 nm (A) and 500 nm (C), respectively.
-
Fluorescence imaging of exogenous/endogenous ONOO generation in living cells.
Encouraged by the above promising results in vitro, we further carried on with cellular bioꢀimaging
ꢀ
of exogenous/endogenous ONOO generation. Cytotoxicity measurements of both probes
-
-
demonstrate that Naph-ONOO and Rhod-ONOO present minimal toxic effects on cell viability
(Figure S10). To evaluate the efficacy of these probes on monitoring endogenously generated
ꢀ
ONOO , we stimulated the cells with bacterial endotoxin lipopolysaccharide (LPS) and
proꢀinflammatory cytokine interferonꢀgamma (IFNꢀγ). These agents are known to trigger cellular
ꢀ
54
apoptosis and release ROS/RNS, including ONOO . To further validate the specificity of our
probes, H O and HOCl, both generated in large amount during apoptosis, were selected as the
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representative interfering ROS in our studies. HepG2 cells were first stained with the fluorescent
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probes (Naph-ONOO and Rhod-ONOO ), then treated with the stimulating reagents or control.
As shown in Figure 2, when the cells were treated with control (PBS), they emitted negligible
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fluorescence signals. Similarly, H O and HOClꢀtreated cells did not exhibit any marked
fluorescence enhancement after incubation. Whereas as a sharp contrast, significant fluorescent
increase was observed when cells were stimulated with LPS/IFNꢀγ, indicating that the increased
ꢀ
signal was primarily due to the generated ONOO during the stimulation. Furthermore, when the
cells were preꢀincubated with superoxide scavenger (2,2,6,6ꢀtetramethylpiperidineꢀNꢀoxyl,
ꢀ
TEMPO) or nitric oxide synthase inhibitor (aminoguanidine, AG), the generation of ONOO was
significantly reduced, as shown by the decreased fluorescence signals in HepG2 cells (Figure 2).
These results evidence that our probes can specifically monitor low level of endogenously
ꢀ
generated ONOO , without the interference from other ROS in living systems. Remarkably,
because of the broad twoꢀphoton absorption action cross section exhibited by naphthalimide
-
fluorophore (26 GM at 800 nm excitation) (Figure S11), fluorescent imaging for Naph-ONOO
-
was also conducted under twoꢀphoton irradiation. Naph-ONOO thus can be applied for
deepꢀtissue imaging of peroxynitrite.
ꢀ
Apart from the qualitative detection of ONOO , the quantitation or semiꢀquantitation is even
ꢀ
more desired in screening assays. We next sought to semiꢀquantify the ONOO generation using our
probes. The HepG2 cells were incubated with both probes (5 ꢁM) and the fluorescence intensities
-
were recorded under either twoꢀphoton (TP) irradiation (800 nm, for Naph-ONOO ) or oneꢀphoton
-
(OP) irradiation (488 nm, for Rhod-ONOO ) (Figure 3A and S12). Compared to the nonꢀstimulated
cells that exhibit very weak fluorescence, the preꢀstimulated cells show a doseꢀdependent
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fluorescence increase with different concentrations of LPS/IFNꢀγ (Figure 3A). Fluorescence
intensity measurements indicate dynamic response range of the probes, as well as the potential to
ꢀ
use fluorescence spectroscopy to conduct ONOO quantitation. By recording the realꢀtime
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peroxynitrite generation process in LPSꢀstimulated live cells, we confirmed that our probes can be
ꢀ
used to monitor ONOO for multiple purposes, including clinical research (Figure 3B and S13,
S14). As a supporting test, we also demonstrated that these probes could be employed to image
ꢀ
ꢀ
exogenous ONOO by treating with 3ꢀmorpholinosydnonimine hydrochloride (SINꢀ1), an ONOO
-
-
generator (Figure S15 and S16). Importantly, Naph-ONOO and Rhod-ONOO can be applied to
ꢀ
image ONOO in other types of cell lines, such as HeLa cells and A549 cells (Figure S17 and S18).
ꢀ
These features are very important to develop the fluorescent probes further into potent ONOO
assays and clinical trials.
-
-
Figure 2. Pseudoꢀcolor fluorescence images of probes Naph-ONOO (A) and Rhod-ONOO (B) in HepG2 cells
under different conditions by confocal fluorescence images. First column, cells were incubated with probe
-
-
Naph-ONOO or Rhod-ONOO (5 ꢁM, 40 min), respectively, then imaged; secondꢀthird column, cells were
-
-
pretreated with probe Naph-ONOO or Rhod-ONOO (5 ꢁM, 40 min), respectively, subsequently incubated with
H O (100 ꢁM) (second column) or NaOCl (100 ꢁM) (third column) for 30 min, then imaged; forth column, cells
2
2
were preꢀstimulated with LPS (1 ꢁg/mL) and IFNꢀγ (50 ng/mL) for 12 hours, subsequently incubated with probe
-
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Naph-ONOO or Rhod-ONOO (5 ꢁM, 40 min), respectively, then imaged; fifthꢀsixth column, cells pretreated
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ꢀ
with O
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scavenger TEMPO (300 ꢁM) (fifth column) or NOS inhibitor AG (5 mM) (sixth column) during
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stimulation with LPS (1 ꢁg/mL)/IFNꢀγ (50 ng/mL) for 12 hours, subsequently incubated with probe Naph-ONOO
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or Rhod-ONOO (5 ꢁM, 40 min), respectively, then imaged. The fluorescence images were captured from the green
channel of 500ꢀ570 nm with an excitation at 405 and 488 nm, respectively. First row: brightꢀfield images. Second
row: the fluorescence images of green channel collected at 500ꢀ570 nm. Scale bar: 20 ꢁm.
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ꢀ
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Figure 3. (A) Pseudoꢀcolor fluorescence images of ONOO in HepG2 cells incubated with only Naph-ONOO and
-
Rhod-ONOO (5.0 ꢁM) (a) or coꢀincubated with different concentrations of LPS and IFNꢀγ (bꢀc). First column,
-
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cells were incubated with probe Naph-ONOO or Rhod-ONOO (5 ꢁM, 40 min), then imaged; second and third
.
ꢀ1
.
ꢀ1
.
ꢀ1
.
ꢀ1
column, cells were preꢀstimulated with LPS/IFNꢀγ (1 ꢁg mL /50 ng mL or 3 ꢁg mL /150 ng mL ) for 12 hours,
-
-
subsequently incubated with probe Naph-ONOO or Rhod-ONOO (5 ꢁM, 40 min), then imaged. (d) Average
intensity in aꢀc. Data are expressed as mean ± SD of three experiments. Excitation wavelength was 405 and 488 nm
for the first and second row, respectively. The emission band was at 500ꢀ570 nm. Scale bar = 20 ꢁm. (B) Realꢀtime
-
-
images of LPSꢀstimulated live cells. HepG2 cells were pretreated with Naph-ONOO or Rhod-ONOO (10 ꢁM)
-
for 20 min, and then with LPS (1 mg/mL, 50 µL). Excitation wavelength: 800 nm for Naph-ONOO and 488 nm
-
-
-
for Rhod-ONOO . The emission band was at 500ꢀ560 nm for Naph-ONOO and 500ꢀ570 nm for Rhod-ONOO .
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Scale bar = 20 ꢁm.
Drug induced hepatotoxicity and remediation. To understand the cellular generation
mechanism of peroxynitrite and to elaborate the drugꢀinduced hepatotoxicity phenomenon, we
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ꢀ
proceeded to examine the ONOO induced by commercially available drugs that are known to
cause liver injury. It is reported that mitochondrial oxidative stress is a key source of ROS/RNS
51
production. Thus to examine the localization of our probes at subcellular levels, the cells were
ꢀ
-
preꢀtreated with SINꢀ1, the ONOO generator, then incubated with Rhod-ONOO and subsequently
incubated with MitoꢀTracker Red or LysoꢀTracker Red. As shown in Figure 4, the fluorescence of
-
Rhod-ONOO accumulates within mitochondria and overlaps well with the signals of
MitoꢀTracker Red (overlap coefficient is 0.89) but not with LysoꢀTracker (Figure 4). The positively
charged rhodamine fluorophore may be the main driving force of mitochondrial localization. On
-
the other hand, Naph-ONOO is mainly dispersed within the cytoplasm. Hence it is not overlapping
-
with either MitoꢀTracker or LysoꢀTracker (Figure S19). We thus conclude that Rhod-ONOO
should be more suitable for drugꢀinduced hepatotoxicity measurements.
-
Figure 4. Intracellular localization of Rhod-ONOO in HepG2 cells. Images of HepG2 cells preꢀtreated with
-
SINꢀ1 (100 ꢁM, 2 hours ), then incubated with 5 ꢁM Rhod-ONOO for 40 min and subsequently 1 ꢁM
MitoꢀTracker Red (or 1 ꢁM LysoꢀTracker Red) for 10 min. Red channel: MitoꢀTracker Red (λex= 635 nm, λem
=
6
00ꢀ675 nm) and LysoꢀTracker Red fluorescence (λ = 559 nm, λ = 585ꢀ620 nm); green channel: probe
ex
em
fluorescence (λex = 488 nm, λem = 500ꢀ570 nm); yellow: merged signal. Line profile: Intensity profile of the white
line in image overlap. Scale bar: 10 ꢁm.
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Overdose of APAP is known to cause liver injury through enzymatic biotransformation that
ꢀ
12
ꢀ
could result in the overproduction of ONOO . Thus, efficient tracking of ONOO during drug
induced liver injury will benefit the deeper understanding of the hepatotoxicity mechanism. We
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ꢀ
first assessed the production of ONOO with APAP. Cells were treated with APAP at a range of
-
increasing concentrations (0ꢀ500 ꢁM) for 12 hours, then incubated with Rhod-ONOO (5 ꢁM) for
4
0 min. Apparently, the reaction based fluorescent probe is capable of trapping even trace amount
ꢀ
of labile ONOO generated through APAPꢀinduced hepatotoxicity, as shown by the increased
fluorescence signals (Figure 5A and B). The doseꢀdependent fluorescence intensities not only
ꢀ
prove that ONOO generation is correlated with APAP concentrations in cells, but also
-
demonstrated the sensitivity of Rhod-ONOO . On the other hand, negligible fluorescence signals
-
are observed with Naph-ONOO (Figure 5C and D). Because the colocalization experiments
-
55
determine that Naph-ONOO accumulates mainly in cytoplasm (Figure S19), these results
ꢀ
suggest that ONOO is mainly produced from mitochondrial oxidative stress during APAP drug
ꢀ
stimulation. Furthermore, to test whether our probe can monitor ONOO generation in real time,
we recorded the fluorescence images at different time points. We first incubated the cells with
-
Rhod-ONOO , then incubated with APAP, and imaged the cells at a set of times from 2 min to
120 min. As shown in Figure 5E and S20, fluorescence signals can already be seen from 80 min
onwards, as compared to the negligible signals from cells without APAP stimulation (Figure S20).
ꢀ
In addition, we also evaluated the level of ONOO endogenously generated by other known DILI
drugs (e.g. tetracycline and phenytoin). The results show that a significant reduced fluorescence
ꢀ
intensity were observed comparing with APAP (Figure S21), demonstrating low level of ONOO
generation in the presence of tetracycline or phenytoin.
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ꢀ
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Figure 5. Pseudoꢀcolor fluorescence images of ONOO in HepG2 cells incubated with only probes Rhod-ONOO
-
(
A) and Naph-ONOO (C) (5.0 ꢁM) or coꢀincubated with different concentrations of APAP (0ꢀ500 ꢀM) for 12
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hours. First column, the HepG2 cells were treated with 5 ꢁM Rhod-ONOO or Naph-ONOO ; secondꢀfifth column,
the HepG2 cells were preꢀtreated with different concentrations of APAP (50, 100, 250, 500 ꢀM) for 12 hours, and
-
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then treated with 5 ꢁM probe Rhod-ONOO or Naph-ONOO for 40 min. (B) and (D): Average intensity in Figure 5
(
(
A) and (C), respectively. Data are expressed as mean ± SD of three experiments. Excitation wavelength was 488 nm
A) and 405 nm (C). The emission band was at 500ꢀ570 nm. Scale bar = 20 ꢁm. (E) Realꢀtime images of
-
APAPꢀstimulated live cells. HepG2 cells were pretreated with Rhod-ONOO (10 ꢁM) for 20 min, and then with
APAP (250 ꢀM). Excitation wavelength was 488 nm. The emission band was at 500ꢀ570 nm. Scale bar = 20 ꢁm.
Understanding the molecular pathway of drug induced hepatotoxicity can help researchers
examine and ultimately avoid this phenomenon. Among the broad range of enzymes that could
interact with drugs, cytochrome P450 families of oxidases are involved in xenobiotics metabolism
in human bodies. The proposed mechanism for APAP induced hepatotoxicity involves
CYP2E1ꢀregulated oxidation of APAP to an iminoquinone, Nꢀacetylparaquinonimine (NAPQI),
which binds directly to proteins and causes the dysfunction of mitochondrial reactive species
4
production (Figure 6A). We hence predict that inhibiting either CYP2E1 enzyme or the whole
ꢀ
CYP450 oxidase family should reduce ONOO generation. Indeed, when the cells were
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preꢀincubated with transꢀ1,2ꢀdichloroethylene (tꢀ1,2ꢀDCE; a specific inhibitor of CYP2E1 )
during stimulation with APAP, downregulation of enzymatic activities significantly decreased the
-
fluorescence intensities of Rhod-ONOO , as compared to the strong emission from control cells
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with only APAP stimulation. 1ꢀAminobenzotriazole (1ꢀABT; an inhibitor of CYP450 enzymes ),
which can universally inhibit the CYP450 family oxidase, perform similarly to tꢀ1,2ꢀDCE by
downregulating the enzyme activity (Figure 6B and C). These results corroborated the enzymatic
pathways associated with APAPꢀinduced hepatotoxicity for the first time with a small molecule
fluorescent probe.
In addition, GSH is a potent antioxidant and nucleophilic scavenger of reactive metabolites (i.e.,
57
NAPQI and ROSꢀRNS ). We predict that adding GSH into the system should also quench the
-
fluorescence of Rhod-ONOO . Indeed, observable fluorescence decrease was monitored with
addition of GSH. However, the decrease was not as marked as the addition of enzyme inhibitors
(Figure 6B and C). We proposed that it may due to the closer role of enzymatic pathways
associated with APAPꢀinduced hepatotoxicity than GSH. As a contrast, isoniazid (antiꢀtuberculosis
agent, INH), another commercially available drug known to cause hepatotoxicity, was treated
concomitantly to the cells with APAP. The fluorescence imaging exhibits that higher
ꢀ
concentrations of INH indeed induce generation of more ONOO , as verified by our probe (Figure
S22), and reveals the synergistic hepatotoxicity of combination therapy with APAP and INH.
These results not only prove that our reaction based probe is very suitable for sensitive detection
ꢀ
of ONOO in situ, but also point out a potential pathway to elucidate the drugꢀinduced
hepatotoxicity process.
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Figure 6. Fluorescent imaging remediation of APAPꢀinduced hepatotoxicity with enzyme inhibitors and antioxidant
scavengers in living HepG2 cells. (A) Mechanism of APAPꢀinduced toxicity, with effects of inhibition by GSH,
1ꢀABT and tꢀ1, 2ꢀDCE. (B) Representative images of cells receiving from left to right: Blank, 250 ꢀM APAP alone,
-
and 250 ꢀM APAP with GSH (200 ꢀM), 1ꢀABT (160 ꢀM), or tꢀ1,2ꢀDCE (20 ꢀM), followed by Rhod-ONOO (5
ꢀM). (C) Average intensity in (B). Data are expressed as mean ± SD of three experiments. Excitation wavelength at
4
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Conclusion
Drugꢀinduced hepatotoxicity has long affected public health, while its underlying molecular
mechanism still remaining unsolved. With the newly designed, reaction based fluorescent probes,
ꢀ
we have tried to investigate how ONOO is generated and metabolized during the hepatotoxicity
process. Based on the sensitive, biorthogonal deprotection of a-ketoamide, our probes enable the
bioimaging and realꢀtime monitoring of in situ generated peroxynitrite species. The selective
ꢀ
ONOO mediated nucleophilic reaction also ensures high specificity over other biological ROS and
RNS. Remarkably, the reaction motif can be equipped with a range of fluorophores with diverse
ꢀ
optical properties, as demonstrated by twoꢀphoton imaging of ONOO with a naphthalimide probe
-
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Naph-ONOO . The mitochondrial accumulation of another probe, Rhod-ONOO , reveals that
peroxynitrite is mainly generated during drug induced oxidative stress within mitochondria. These
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features allow us to investigate deeper into the molecular mechanism of drugꢀinduced hepatoxicity.
Through enzyme inhibitors and reactive metabolite scavengers, we for the first time imaged the
ꢀ
ONOO generation by CYP450 family oxidaseꢀmediated xenobiotics metabolism. This study can be
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pushed further to pursue other enzymes or machinery associated with ONOO generation. A potent
fluorescence assay can also be formulated to monitor drugꢀinduced hepatotoxicity in preꢀclinical
studies.
ACKNOWLEDGMENT
This work was financially supported by National Science Fund for Excellent Young Scholars
(
Grant 21622504), NSFC (Grant 21302050), the Foundation for Innovative Research Groups of
NSFC (Grant 21521063), the Hunan University Fund for Multidisciplinary Developing
2015JCA04), and the Foundation of Hunan Educational Committee (Grant YB2016B037).
(
Supporting Information
Experimental details for chemical synthesis of all compounds, supplementary photophysical
characterization of probes, and imaging methods and data. This material is available free of charge
via the Internet at http://pubs.acs.org.
References
(1) Zimmerman, H. J., Clin. Liver Dis. 2000, 4, 73–96.
(2) Yuan, L.; Kaplowitz, N., Clin. Liver Dis. 2013, 17, 507–518.
(3) Chalasani, N.; Fontana, R. J.; Bonkovsky, H. L.; Watkins, P. B.; Davern, T.; Serrano, J.; Yang, H.;
Rochon, J. Gastroenterology, 2008, 135, 1924–1934.
(4) Holt, M.; Ju, C. Handb. Exp. Pharmacol. 2010, 196, 3–27.
(5) Xu, J. J.; Diaz, D.; O'Brien, P. J. Chem. Biol. Interact. 2004, 150, 115–128.
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(
(
(
(
6) Dimasi, J. A. Clin. Pharmacol. Ther. 2001, 69, 297–307.
7) Kola, I.; Landis, J. Nat. Rev. Drug Discov. 2004, 3, 711–715.
8) Tengowski, M. W.; Kotyk, J. J. Prog. Drug Res. 2005, 62, 257–278.
9) Hinson, J.; Roberts, D.; James, L., in Adverse Drug Reactions, Springer, Berlin, 2010, 196, 369–
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4
05.
(10) Bertolini, A.; Ferrari, A.; Ottani, A.; Guerzoni, S.; Tacchi, R.; Leone, S. CNS Drug Rev. 2006, 12,
2
50–275.
(11) Walsh, J. S.; Miwa, G. T. Annu. Rev. Pharmacol. Toxicol. 2011, 51, 145–167.
(12) Cover, C.; Mansouri, A.; Knight, T. R.; Bajt, M. L.; Lemasters, J. J.; Pessayre, D.; Jaeschke, H. J.
Pharmacol. Exp. Ther. 2005, 315, 879–887.
(
(
(
13) Radi, R. J. Am.Chem. Soc. 2013, 288, 26464–26472.
14) Nagano, T. J. Clin. Biochem. Nutr. 2009, 45, 111–124.
15) Mohar, I.; Stamper, B. D.; Rademacher, P. M.; White, C. C.; Nelson, S. D.; Kavanagh, T. J. Redox
Biology 2014, 2, 377–387.
(16) Ducrocq, C.; Blanchard, B.; Pignatelli, B.; Ohshima, H. Cell. Mol. Life Sci. 1999, 55, 1068–1077.
(17) Masumoto, H.; Kissner, R.; Koppenol, W. H.; Sies, H. FEBS Lett. 1996, 398, 179–182.
(18) Zhang, H.; Liu, R.; Liu, J.; Li, L.; Wang, P.; Yao, S. Q.; Xu, Z.; Sun, H. Chem. Sci. 2016, 7, 256–
2
60.
(19) Lee, M. H.; Kim, J. S.; Sessler, J. L. Chem. Soc. Rev. 2015, 44, 4185–4191.
(20) Ueno, T.; Nagano, T. Nat. Methods. 2011, 8, 642–645.
(21) Wan, Q. Q.; Chen, S. M.; Shi, W.; Li, L. H.; Ma, H. M. Angew. Chem. Int. Ed. 2014, 53, 10916–
1
0920.
19 / 23
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Analytical Chemistry
Page 20 of 23
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
(
(
(
(
(
22) Lin, V. S.; Chen, W.; Xian, M.; Chang, C. J. Chem. Soc. Rev. 2015, 44, 4596–4618.
23) Yang, X.; Guo, Y.; Strongin, R. M. Angew. Chem. Int. Ed. 2011, 50, 10690–10693.
24) Ferna ´ndez, A.; Vendrell, M. Chem. Soc. Rev. 2016, 45, 1182–1196.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
25) Yu, F.; Li, P.; Wang, B.; Han, K. J. Am. Chem. Soc. 2013, 135, 7674–7680.
26) Li, J.; Yim, D.; Jang, WꢀD.; Yoon, J. Chem. Soc. Rev. 2017, 46, 2437
–2458.
27) 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.
(28) Yang, D.; Wang, H. L.; Sun, Z. N.; Chung, N. W.; Shen, J. G. J. Am. Chem. Soc. 2006, 128, 6004–
6
005.
(29) Sun, Z. N.; Wang, H. L.; Liu, F. Q.; Chen, Y.; Tam, P. K. H.; Yang, D. Org. Lett. 2009, 11, 1887–
1
890.
(30) Peng, T.; Yang, D. Org. Lett. 2010, 12, 4932–4935.
(31) Chang, M. C. Y.; Pralle, A.; Isacoff, E. Y.; Chang, C. J. J. Am. Chem. Soc. 2004, 126, 15392–
1
5393.
(32) Oushiki, D.; Kojima, H.; Terai, T.; Arita, M.; Hanaoka, K.; Urano, Y.; Nagano, T. J. Am. Chem.
Soc. 2010, 132, 2795–2801.
(33) Zhou, M.; Diwu, Z.; PanchukꢀVoloshina, N.; Haugland, R. P. Anal. Biochem. 1997, 253, 162–168.
(34) Setsukinai, K. I.; Urano, Y.; Kikuchi, K.; Higuchi, T.; Nagano, T. J. Chem. Soc., Perkin Trans.
2000, 2, 2453–2457.
(35) Wardman, P. Free Radical Biol. Med. 2007, 43, 995–1022.
(36) Shepherd, J.; Hilderbrand, S. A.; Waterman, P.; Heinecke, J. W.; Weissleder, R.; Libby, P. Chem.
Biol. 2007, 14, 1221–1231.
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Page 21 of 23
Analytical Chemistry
1
2
3
4
5
6
7
8
9
(
37) Panizzi, P.; Nahrendorf, M.; Wildgruber, M.; Waterman, P.; Figueiredo, J. L.; Aikawa, E.;
McCarthy, J.; Weissleder, R.; Hilderbrand, S. A. J. Am. Chem. Soc. 2009, 131, 15739–15744.
(38) Zhang, Q.; Zhu, Z.; Zheng, Y.; Cheng, J.; Zhang, N.; Long, YꢀT.; Zheng, J.; Qian, X.; Yang, Y. J.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Am. Chem. Soc. 2012, 134, 18479–18482.
(39) Peng, T.; Wong, N. K.; Chen, X.; Chan, Y. K.; Ho, D. H.; Sun, Z.; Hu, J. J.; Shen, J.; ElꢀNezami,
H.; Yang, D. J. Am. Chem. Soc. 2014, 136, 11728–11734.
(40) Cheng, D.; Pan, Y.; Wang, L.; Zeng, Z. B.; Yuan, L.; Zhang, X. B.; Chang, Y. T. J. Am. Chem. Soc.
2
017, 139, 285–292.
(41) Peng, T.; Chen, X.; Gao, L.; Zhang, T.; Wang, W.; Shen, J.; Yang, D. Chem. Sci. 2016, 7, 5407–
5
413.
(42) He, X.; Li, L.; Fang, Y.; Shi, W.; Lia, X.; Ma, H. Chem. Sci. 2017, 8, 3479–3483 .
(43) Peng, J.; Samanta, A.; Zeng, X.; Han, S.; Wang, L.; Su, D.; Loong, D. T. B.; Kang, NꢀY.; Park, SꢀJ.;
Homayoun All, A.; Jiang, W.; Yuan, L.; Liu, X.; Chang, YꢀT. Angew. Chem. Int. Ed. 2017, 56,
4
165−4169.
(44) Montoya, L. A.; Pluth, M. D. Chem. Commun. 2012, 48, 4767–4769.
(45) Sakabe, M.; Asanuma, D.; Kamiya, M.; Iwatate, R. J.; Hanaoka, K.; Terai, T.; Nagano, T.; Urano,
Y. J. Am. Chem. Soc. 2013, 135, 409−414.
(46) Sun, Q.; Yang, SꢀH.; Wu, L.; Dong, QꢀJ.; Yang, WꢀC.; Yang, GꢀF. Anal. Chem. 2016, 88,
6084−6091.
(47) Areti, S.; Verma, S. K.; Bellare, J.; Rao, C. P. Anal. Chem. 2016, 88, 7259−7267.
(48) Feng, S.; Liu, D.; Feng, W.; Feng, G. Anal. Chem. 2017, 89, 3754–3760.
(49) Li, M.; Feng, W.; Zhai, Q.; Feng, G. Biosens. Bioelectron. 2017, 87, 894–900.
21 / 23
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Page 22 of 23
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2
3
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5
6
7
8
9
1
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5
5
5
5
5
5
5
5
5
6
(
50) Zhang, L.; Zhu, H.; Zhao, CꢀC.; Gu, XꢀF. Chin. Chem. Lett. 2017, 28, 218–221.
(51) Xu, W.; Zeng, Z. B.; Jiang, JꢀH.; Chang, YꢀT.; Yuan, L. Angew. Chem. Int. Ed. 2016, 55, 13658–
1
3699.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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3
4
5
6
7
8
9
0
(52) Xie, X.; Yang, X.; Wu, T.; Li, Y.; Li, M.; Tan, Q.; Wang, X.; Tang, B. Anal. Chem. 2016, 88, 8019–
8
025.
(53) Abo, M.; Urano, Y.; Hanaoka, K.; Terai, T.; Komatsu, T.; Nagano, T. J. Am. Chem. Soc. 2011, 133,
1
0629–10637.
(54) Knight, T. R.; Kurtz, A.; Bajt, M. L.; Hinson, J. A.; Jaeschke, H. Toxicol. Sci. 2001, 62, 212–220.
(55) Srikun, D.; Miller, E. W.; Domaille, D. W.; Chang, C. J. J. Am. Chem. Soc. 2008, 130, 4596–4597.
(56) Jackson, T. E.; Lilly, P. D.; Recio, L.; Schlosser, P. M; Medinsky, M. A. Toxicol. Sci. 2000, 55,
2
66–273.
(57) James, L. P.; McCullough, S. S.; Lamps, L.W.; Hinson, J. A. Toxicol. Sci. 2003, 75, 458–467.
(58) Similar result was reported during the preparation of this manuscript: Li, Y.; Xie, X.; Yang, X.; Li,
M.; Jiao, X.; Sun, Y.; Wang, X.; Tang, B. Chem. Sci. 2017, 8, 4006–4011.
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