A highly specific fluorescent probe for hypochlorous acid and its application in imaging microbe-induced hocl production

Article abstract of DOI:10.1021/ja404649m

Oxidative stress induced by reactive oxygen species (ROS) plays crucial roles in a wide range of physiological processes and is also implicated in various diseases, including cancer, chronic inflammatory diseases, and neurodegenerative disorders. Among the various ROS, hypochlorous acid (HOCl) plays as a powerful microbicidal agent in the innate immune system. The regulated production of microbicidal HOCl is required for the host to control the invading microbes. However, as a result of the highly reactive and diffusible nature of HOCl, its uncontrolled production may lead to an adverse effect on host physiology. Because of its biological importance, many efforts have been focused on developing selective fluorescent probes to image ROS. However, it is still challenging to design a fluorescent probe with exclusive selectivity toward a particular member of ROS. In the current work, we designed FBS as a new fluorescent HOCl probe which has high selectivity, sensitivity, and short response time in a broad range of pH. Compared with other sensors, the dual-lock structure of FBS has an advantage of eliminating interferences from other ROS/RNS. Importantly, we further showed that our HOCl probe could be applied for the in vivo imaging of physiological HOCl production in the mucosa of live animals. This probe provides a promising tool for the study of HOCl production.

Full text of DOI:10.1021/ja404649m

Article
pubs.acs.org/JACS
A Highly Specific Fluorescent Probe for Hypochlorous Acid and Its
Application in Imaging Microbe-Induced HOCl Production
†
,§†
‡,§†
†
†
,‡
,†
Qingling Xu,
Kyung-Ah Lee,
Songyi Lee, Kyung Mi Lee, Won-Jae Lee,* and Juyoung Yoon*
†
Department of Chemistry and Nano Science, Global Top 5 Program, Ewha, Womans University, Seoul 120-750, Korea
‡
School of Biological Science and National Creative Research, Initiative Center for Symbiosystem, Seoul National University, Seoul
1-742, Korea
5
*
S Supporting Information
ABSTRACT: Oxidative stress induced by reactive oxygen
species (ROS) plays crucial roles in a wide range of
physiological processes and is also implicated in various
diseases, including cancer, chronic inflammatory diseases, and
neurodegenerative disorders. Among the various ROS,
hypochlorous acid (HOCl) plays as a powerful microbicidal
agent in the innate immune system. The regulated production
of microbicidal HOCl is required for the host to control the
invading microbes. However, as a result of the highly reactive
and diffusible nature of HOCl, its uncontrolled production
may lead to an adverse effect on host physiology. Because of its
biological importance, many efforts have been focused on
developing selective fluorescent probes to image ROS. However, it is still challenging to design a fluorescent probe with exclusive
selectivity toward a particular member of ROS. In the current work, we designed FBS as a new fluorescent HOCl probe which
has high selectivity, sensitivity, and short response time in a broad range of pH. Compared with other sensors, the “dual-lock”
structure of FBS has an advantage of eliminating interferences from other ROS/RNS. Importantly, we further showed that our
HOCl probe could be applied for the in vivo imaging of physiological HOCl production in the mucosa of live animals. This probe
provides a promising tool for the study of HOCl production.
INTRODUCTION
recognition groups and HOCl that give highly fluorescent
products. The reactions include the oxidation reactions of p-
■
(
Reactive oxygen species (ROS) and reactive nitrogen species
RNS) play an essential role in many biological processes such
4a
methoxyphenol to benzoquinone, dibenzoyl hydrazide to
4b
dibenzoyl diimide, rhodamine-hydroxamic acid to rhodamine
as aging and immunity. However, deregulation of ROS
production and/or elimination may cause pathophysiological
consequences such as aging and chronic inflammatory diseases
4
c
4d
1
4
9, thiol/thio ether to sulfonate derivatives, the cleavage of
4e
4f
-aminophenyl, the release of oxazine fluorophore by HOCl,
1
and so on.
of the human. Many efforts have been focused on developing
2
3
Among the various ROS, H O and HOCl are closely related
2
2
selective fluorescent probes to image ROS including H O ,
2
2
−
4
5
−
6
•
2−
7
1
8
since MPO converts H O to HOCl. Accordingly, the detection
2 2
OCl , NO, ONOO , O , O . Among the various ROS,
2
of HOCl in the presence of H O and other ROS is critical. For
2
2
hypochlorous acid (HOCl) plays as a powerful microbicidal
agent in the innate immune system. It is generated from H O
H O imaging, arylboronates were heavily employed in the
2
2
2
2
and Cl− by secreted myeloperoxidase (MPO) in vivo in
design of H O sensors for the detection of H O generation in
2 2 2 2
vivo. On the other hand, it is recently reported that
arylboronates can react with peroxynitrite (ONOO ) and
OCl to yield hydroxyl derivatives much faster than does H O
1
3
9
response to inflammatory stimuli. Regulated generation of
hypochlorous acid is required for the host to control the
invading microbes, while produced HOCl can also react with
−
−
2
2
A
1
4
10
using a stopped-flow kinetic technique and HPLC analysis.
amino acids, proteins, cholesterol, and nucleosides Uncon-
trolled production of HOCl derived from phagocytes is
involved in some diseases such as cardiovascular disease and
few boronate-based fluorogenic probes were developed to
monitor ONOO− formation from O and NO. In this
work, “dual-lock” FBS is reported as a selective fluorescent
probe for HOCl. We synthesized FBS and FS (Scheme 1) and
studied their fluorescence responses to various ROS/RNS.
•
2−
•
15
11
inflammatory disease. Synthetic fluorescent probes are among
the most powerful tools for the detection of HOCl due to their
high sensitivities, simple manipulation, and lack of a require-
ment for sophisticated instrumentation. Fluorescent probes
have advantages of facile visualization of intracellular dynamics
−
H O and ONOO can react with arylboronates of FBS to give
2
2
1
2
and high-resolution localization of biomolecules of interest.
The design strategies are based on specific reactions between
Received: May 9, 2013
©
XXXX American Chemical Society
A
dx.doi.org/10.1021/ja404649m | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
a
Scheme 1. Chemical Structure and Synthesis of FBS and FS
EXPERIMENTAL SECTION
■
Materials and Methods. Unless otherwise noted, materials were
1
obtained from Aldrich and were used without further purification. H
NMR and 13C NMR in CDCl were measured on a Bruker AM-300
3
spectrometer with tetramethylsilane (TMS) as internal standard. Mass
spectra were obtained using a JMS-HX 110A/110A tandem mass
spectrometer (JEOL). UV−vis spectra were obtained using a Scinco
3
000 spectrophotometer (1 cm quartz cell) at 25 °C. Fluorescence
spectra were recorded on RF-5301/PC (Shimada) fluorescence
spectrophotometer (1 cm quartz cell) at 25 °C. Deionized water
was used to prepare all aqueous solutions.
a
Reagents and conditions: (i) Lawesson’s reagent, toluene, 90 °C. (ii)
Synthesis of Compound 2. 3′,6′-Dibromofluoran (1.0 g, 2.19
mmol), Lawesson’s reagent (0.89 g, 2.19 mmol), and toluene (45 mL)
were mixed in a 100-mL flask and heated to 90 °C. After stirring for 2
days, the mixture was cooled down, and the solvent was removed
under reduced pressure. The residue was purified by silica gel column
chromatography using CH Cl /hexane (1/4, v:v) as the eluent to get 2
bis(pinacolato)diborane, potassium acetate, 1,4-dioxane, Pd(dppf)-
Cl ·CH Cl , 85 °C. (iii) NaOCl/CH CN/H O or H O /THF/
CH OH. (iv) NaOCl/CH CN/H O.
2
2
2
3
2
2
2
3
3
2
2
2
1
as a white solid (0.11 g, 10.6%). H NMR (CDCl , 300 MHz) δ
3
−
only FS, which is still nonfluorescent. Only OCl can react with
arylboronates and then hydrolyze thiolactone, which can induce
large green fluorescence enhancement. Therefore, a selective
(ppm): 7.91 (d, 1H), 7.58(m, 2H), 7.36 (d, 2H), 7.10−7.16 (m, 3H),
7
1
1
.77 (d, 2H). 13C NMR (CDCl , 75 MHz) δ (ppm): 195.48, 156.15,
3
50.75, 135.00, 130.52, 129.25, 127.70, 127.27, 123.47, 122.75, 121.12,
−1
20.07, 59.46. IR spectrum: 1681 cm (CO). FAB-MS: m/z =
+
“off−on” fluorescence enhancement was observed only in the
472.8849 [M + H] , calc for C H Br O S = 472.8847.
20
12
2
2
Synthesis of FBS. 2 (0.15 g, 0.32 mmol), bis(pinacolato)diborane
(0.185g, 0.73 mmol), potassium acetate (0.217g, 2.21 mmol), 1,4-
dioxane (10 mL), and Pd(dppf)Cl ·CH Cl (10 mg) were mixed in
presence of HOCl. Our results clearly demonstrate that not
only H O2 but also ONOO and OCl can react with
−
−
2
2
2
2
arylboronate. The high sensitivity and its in vivo compatibility
permit imaging of microbe-induced HOCl production in vivo in
the mucosa of Drosophila. At present, most of the reported
ROS/RNS sensors rely on one kind of recognition group that
can react with the target. The “dual-lock” structure of FBS has
an advantage of eliminating interference from other ROS/RNS.
2
5-mL flask. After N degassing, the mixture was stirred under 85 °C
2
for 36 h. Then the solvent was removed under reduced pressure,
CH Cl was added, the mixture was washed with water three times and
dried over anhydrous MgSO . After the solvent was removed, the
residue was purified by silica gel column chromatography using
CH Cl as the eluent to obtain crude product. It was recrystallized
2
2
4
2
2
from acetonitrile and dried under vacuum to give FBS as a white solid
−
−
Figure 1. (a) Fluorescence spectra changes of FBS with titration of OCl . (b) Fluorescence intensity at 523 nm as a function of added OCl . (c)
−
•
•
2−
•
Fluorescence spectra of FBS before and after addition of various ROS: OCl (20 μM), ROO (1 mM), H O (100 μM), O (25 μM), OH (100
μM). tert-butyl hyperoxide (100 μM), ONOO (22 μM). Insets show the photos of FBS solution without (dark) and with (green) added OCl (20
2
2
−
−
μM). [FBS] = 2 μM, in KH PO buffer (50 mM, pH 7.4), excitation wavelength 498 nm (slit widths: 3 nm/3 nm).
2
4
B
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−
Figure 2. (a) Fluorescence spectra changes of FBS (2 μM) as the titration OCl from 0 to 1.0 μM in KH PO buffer (50 mM, pH 7.4). (b)
2
4
−
Fluorescence intensity at 523 nm as a function of added ClO . Excitation wavelength: 498 nm (slit widths: 3 nm/3 nm).
−
Figure 3. (a) Fluorescence spectra changes of FBS as the titration OCl (0, 3, 6, 9, 12, 15, 18, 21, 24, 27, 30 μM) under pH 5.5 (KH PO buffer, 50
2
4
−
mM). (b) Fluorescence spectra changes of FBS as the titration OCl (0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 μM) under pH 9.3 (K HPO buffer, 50
mM). Insets show fluorescent intensity at 523 nm as a function of added OCl . [FBS] = 2 μM. Excitation wavelength: 498 nm, slit width: 3 nm/3
2
4
−
nm.
1
(
0.072 g, 40%). H NMR (CDCl , 300 MHz) δ (ppm): 7.91 (m, 1H),
300 MHz) δ: 8.01 (m, 1H), 7.56 (m, 2H), 7.21 (m, 1H), 7.06 (d, 2H),
6.54−6.49 (m, 4H).
3
7
2
1
1
.61 (d, 2H), 7.54 (m, 2H), 7.37 (dd, 2H), 7.12 (m, 1H), 6.90 (d,
H), 1.34 (s, 24H). C NMR (CDCl , 75 MHz) δ (ppm): 196.41,
56.95, 150.37, 135.13, 134.74, 129.64, 128.86, 128.44, 127.43, 124.58,
23.37, 123.23, 84.14, 60.49, 24.85. FAB-MS: m/z = 568.2260, [M] ,
13
3
RESULTS AND DISCUSSION
+
■
+
calc for C H B O S = 568.2262; m/z = 569.2343, [M + H] , calc for
Design and Preparation of FBS. In a cellular context, a
variety of ROS (such as HOCl, H O and ONOO ) were
32
34
2
6
−
C H B O S = 569.2340.
Synthesis of FS. H O (0.5 mL, 28% in water) was added to FBS
0.128 g, 0.225 mmol) solution in THF (4 mL) and CH OH (2 mL),
the mixture was stirred overnight. After reaction, solvents were
removed under reduced pressure, and the residue was purified by silica
gel column chromatography using CH Cl /CH OH (90/1, v:v) as the
eluent to get crude product as a white solid (0.045g, 57.4%). H NMR
CD OD, 300 MHz) δ (ppm): 7.85 (m, 1H), 7.62 (m, 2H), 7.17 (m,
H), 6.67 (d, 2H), 6.55 (d, 2H), 6.46 (q, 2H). C NMR (CDCl , 75
3
MHz) δ (ppm): 198.26, 159.97, 158.87, 153.18, 136.60, 135.95,
30.97, 129.90, 128.50, 123.63, 114.14, 113.53, 103.38, 62.86. FAB-
MS: m/z = 348.0451 [M] , calc for C H O S = 348.0456; m/z =
49.0536 [M + H] , calc for C H O S = 349.0535.
Reaction of FBS and NaClO To Give FS. NaClO (3 mM in
water, 30 mL) was added to FBS (10 mg, 0.0176 mmol) solution in
CH CN (17 mL) slowly under stirring. After the solvents were
removed under reduced pressure, the residue was purified on silica gel
column chromatography using CH Cl /CH OH (90/1, v:v) as the
eluent to get the product FS (3.0 mg, 48.9%).
The Reaction of FS and NaClO. NaClO solution (30 mL, 1 mM)
was added to the solution of FS (10 mg, 0.029 mmol) in CH CN (28
mL) slowly and stirred for 30 min. Then the solvents were removed
under reduced pressure, and the residue was purified on silica gel
column chromatography using CH Cl /CH OH (5/1, v:v) as the
3
2
35
2
6
2
2
2
2
simultaneously generated in response to physiological stimuli.
Therefore, the in vivo applications of arylboronates-based
sensors are limited due to lack of specificity toward a single
species of ROS. To develop a specific HOCl sensor without
noticeable activity toward other ROS including H O and
(
3
2
2
3
1
2
2
−
ONOO , we designed and synthesized a nonfluorescent
xanthenone derivative which combined with boronic esters
and thiolactone (FBS, Scheme 1). The reaction between 3′,6′-
(
1
3
13
16
dibromofluoran and Lawesson’s reagent yielded thiolactone
compound 2. The following Miyaura borylation reaction with
bis(pinacolato)diborane gave FBS. Although boronic esters can
react with the three ROS, the product is expected to be the low-
fluorescent FS. We expected that thiolactone of FS could be
oxidized by HOCl and give the high fluorescent product
fluorescein. The “dual-lock” structure of FBS is expected to
exclude interferences from almost all other molecules. The
detailed experimental procedures are explained in the
1
+
20
12
4
+
3
20 13 4
3
2
2
3
1
13
Experimental Section and H and C NMR spectra are
3
reported in the Supporting Information (SI).
Selectivity and Sensitivity of FBS for HOCl. To examine
the sensitivity and selectivity of FBS to HOCl, we measured the
fluorescence spectra of FBS with HOCl and other ROS/RNS.
2
2
3
1
eluent to get the product (0.32 mg). H NMR (CD OD, with NaOH,
3
C
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−
Figure 4. (a) Fluorescence spectra changes of FS as the titration of OCl : 0, 0.5, 1, 2, 4, 5, 6, 8, 10, 16, and 19 μM. (b) Fluorescence spectra of FBS
−
•
•
before and after addition of OCl (19 μM) and other ROS for 30 min, NO (1 mM), ROO (1 mM), H O (200 μM), tert-butyl hyperoxide (200
2
2
−
μM) and ONOO (22 μM). [FS] = 2 μM, in KH PO buffer (50 mM, pH 7.4), excitation wavelength: 498 nm (slits width: 3 nm/3 nm).
2
4
Figure 5. Detection of DUOX-dependent HOCl induction in the intestinal epithelia of Drosophila. Nuclear staining of midgut cells was performed
with DAPI (blue). Representative confocal microscopic images of dissected guts from different genotypes in the presence or absence of oral
−/−
7
ingestion of bacterial extract. The genotypes of the flies used in this study were as follows: Cont (Da-GAL4/+); PLCβ
(norpA ); DUOX-
knockdown (KD) (UAS-DUOX-RNAi/+; Da-GAL4/+); DUOX-KD + DUOX (UAS-DUOX-RNAi/UAS-DUOX; Da-GAL4/+).
−
As shown in Figure 1a, FBS was not fluorescent at all. When
HOCl was titrated from 0 to 80 μM, the fluorescence emission
increased remarkably and quickly. This can be attributed to the
production of fluorescein by the reaction between FBS and
HOCl (Scheme 1), which was confirmed by NMR and FAB-
Mass. The fluorescence emissions as well as UV−vis
absorptions (SI Figure S1) are red-shifted during the titration.
1c). ONOO of less than 22 μM could induce a very limited
increase in fluorescence, but then the fluorescence decreased at
higher concentrations (SI, Figure S3). To find out the
minimum concentration of HOCl that can induce fluorescence,
titration of HOCl from 0 to 1.0 μM was carried out (Figure 2).
HOCl concentration as low as 0.2 μM can be detected. In
short, FBS is a good HOCl sensor with high sensitivity,
selectivity, and short response time.
17
This may be attributed to the chlorination of fluorescein. As
the concentration of HOCl increased to 100 μM, a small
fluorescence decrease was observed. (SI Figure S2). The
fluorescence intensity at 523 nm as a function of HOCl
concentration was recorded, and a nearly linear relationship in
the range of 0−20 μM was obtained (Figure 1b). Strong green
fluorescence can be observed by the naked eye (Figure 1c). In
The Effect of pH. We also studied the performances of FBS
in acidic or basic solutions with added HOCl. As shown in
Figure 3, strong fluorescence enhancement was observed
during titration of OCl to FBS solution of pH 5.5 and pH
9.3. These results clearly explain that this probe can be used in a
broad range of pH.
−
•
•
−
•
•
contrast, other ROS (H O , NO , O , OH, ROO , t-
Fluorescent Response of FS to ROS/RNS. The reactions
of FBS with H O and HOCl were carried out respectively, and
2
2
2
BuOOH) of higher concentration could not induce measurable
2
2
fluorescence changes even after incubation of 30 min (Figure
the main product was found to be FS. The fluorescent
D
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Journal of the American Chemical Society
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1
performance of FS and its response to ROS/RNS are critical
for the success of our strategy. As shown in Figure 4, FS has
very weak fluorescence itself. After the titration of OCl from 0
infrared spectrum of compound 2, H NMR and 13C NMR
spectra of all compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
−
to 19 μM, strong fluorescence enhancement was observed. We
•
•
then incubated FS with other ROS/RNS: NO (1 mM), ROO
1 mM), H O (200 μM), tert-butyl hyperoxide (200 μM), and
AUTHOR INFORMATION
Corresponding Author
jyoon@ewha.ac.kr (J.Y.); lwj@snu.ac.kr (W.-J.L.)
■
(
2
2
−
ONOO (22 μM) but failed to induce obvious fluorescence
changes. Although FS was also found to have high selectivity
for HOCl, FS showed background fluorescence, relatively
stronger than that of FBS, and low stability after long-time
storage in solution which is a drawback. Interestingly, we found
Author Contributions
Q.X. and K.A.L. contributed equally to this work.
§†
Notes
−
that added OCl failed to react with FBS in HEPES buffer (20
The authors declare no competing financial interest.
mM, pH 7.4), as no fluorescence enhancement can be
observed. It is probably due to the fact that OCl reacted
with HEPES. Therefore, the use of HEPES buffer is suggested
to be avoided for ROS detection.
−
ACKNOWLEDGMENTS
■
This research was supported by the National Creative Research
Initiative programs from the National Research Foundation of
Korea (NRF) grant funded by the Korea government (MSIP)
No. 2012R1A3A2048814 to J.Y. and No. 2006-0050687 to
W.-J.L.).
In Vivo Imaging of Physiological HOCl Production
Using FBS. To test whether FBS can be used as a specific
fluorescent sensor for the detection of physiological HOCl
production in vivo, we applied FBS in the Drosophila gut
(
17
system, a well-known HOCl producing organ. In Drosophila,
gut epithelia produce HOCl as a microbicidal agent via DUOX,
a member of the NADPH oxidase family, in response to
REFERENCES
■
(
1) (a) Rhee, S. G. Science 2006, 312, 1882. (b) Cui, Y.; Lu, Z.; Bai,
L.; Shi, Z.; Zhao, W.; Zhao, B. L. Eur. J. Cancer 2007, 43, 2590.
2) Chen, X.; Tian, X.; Shin, I.; Yoon, J. Chem. Soc. Rev. 2011, 40,
4783.
(3) (a) Lo, L.-C.; Chu, C.-Y. Chem. Commun. 2003, 2728. (b) Chang,
M. C. Y.; Pralle, A.; Isacoff, E. Y.; Chang, C. J. J. Am. Chem. Soc. 2004,
26, 15392. (c) He, F.; Tang, Y.; Yu, M.; Wang, S.; Li, Y.; Zhu, D. Adv.
1
8
bacterial challenge. To initiate physiological HOCl produc-
tion, the flies were subjected to oral ingestion of bacterial
extracts. FBS was subsequently introduced to the gut by oral
ingestion to image bacterial-induced HOCl production in situ.
As shown in Figure 5, the gut of the wide-type Drosophila
shows green fluorescence following treatment of bacterial
extracts. In contrast, there is no detectable fluorescence without
the treatment. Recently it was found that phospholipase C-β
(
1
Funct. Mater. 2006, 16, 91. (d) Wolfbeis, O. S.; Durkop, A.; Wu, M.;
̈
Lin, Z. Angew. Chem., Int. Ed. 2002, 41, 4495. (e) Lo, L.-C.; Chu, C.-Y.
Chem. Commun. 2003, 2728. (f) Zhang, T.; Fan, H.; Liu, G.; Jiang, J.;
Zhou, J.; Jin, Q. Chem. Commun. 2008, 5414. (g) Dickinson, B. C.;
Huynh, C.; Chang, C. J. J. Am. Chem. Soc. 2010, 132, 5906.
(
PLCβ) signaling is required for DUOX activity to produce
19
microbicidal ROS. As expected, no bacterial-induced
fluorescence can be observed in the absence of PLCβ signaling
pathway (in the gut of PLCβ mutant flies) as well as in the
knockdown (KD) of DUOX expression (in the gut of DUOX-
KD flies). Furthermore, normal level of bacteria-induced HOCl
production was restored when DUOX-KD flies were rescued by
overexpressing Drosophila DUOX. Taken together, these data
indicated that FBS can detect PLCβ-DUOX-dependent HOCl
production in vivo in response to a physiological signaling such
as bacterial challenge.
(
h) Karton-Lifshin, N.; Segal, E.; Omer, L.; Portnoy, M.; Satchi-
Fainaro, R.; Shabat, D. J. Am. Chem. Soc. 2011, 133, 10960. (i) Yu, F.;
Li, P.; Zhao, G.; Chu, T.; Han, K. J. Am. Chem. Soc. 2011, 133, 11030.
(j) Yuan, L.; Lin, W.; Xie, Y.; Chen, B.; Zhu, S. J. Am. Chem. Soc. 2012,
134, 1305. (k) Yuan, L.; Lin, W.; Zhao, S.; Gao, W.; Chen, B.; He, L.;
Zhu, S. J. Am. Chem. Soc. 2012, 134, 13510.
(4) (a) Chen, X.; Lee, K.-A.; Ha, E.-M.; Lee, K. M.; Seo, Y. Y.; Choi,
H. K.; Kim, H. N.; Kim, M. J.; Cho, C.-S.; Lee, S. Y.; Lee, W.-J.; Yoon,
J. Chem. Commun. 2011, 47, 4373. (b) Koide, Y.; Urano, Y.; Hanaoka,
K.; Terai, T.; Nagano, T. J. Am. Chem. Soc. 2011, 133, 5680. (c) Zhang,
W.; Guo, C.; Liu, L.; Qin, J.; Yang, C. Biomol. Chem. 2011, 9, 5560.
(
d) Cheng, X.; Jia, H.; Long, T.; Feng, J.; Qin, J.; Li, Z. Chem.
CONCLUSIONS
■
Commun. 2011, 47, 11978. (e) Guo, T.; Cui, L.; Shen, J.; Wang, R.;
In summary, we have developed a novel “dual-lock” fluorescent
HOCl probe, FBS, bearing boronic esters and thiolactone. Its
reaction with HOCl produces fluorescein as a product, which
shows strong green fluorescence. FBS shows high selectivity for
Zhu, W.; Xu, Y.; Qian, X. Chem. Commun. 2013, 49, 1862.
(5) (a) Lim, M. H.; Lippard, S. J. Acc. Chem. Res. 2007, 40, 41.
(b) Gabe, Y.; Urano, Y.; Kikuchi, K.; Kojima, H.; Nagano, T. J. Am.
Chem. Soc. 2004, 126, 3357. (c) Sun, C.; Shi, W.; Song, Y.; Chen, W.;
Ma, H. Chem. Commun. 2011, 47, 8638. (d) Meineke, P.; Rauen, U.;
de Groot, H.; Korth, H.-G.; Sustmann, R. Biol. Chem. 2000, 381, 575.
−
HOCl over H O , ONOO , and other ROS/RNS. H O and
2
2
2
2
ONOO− can convert FBS only to FS, which is still
nonfluorescent, on the other hand, only HOCl can convert
FBS to fluorescein. FBS can be used in neutral, acidic, and basic
solutions. As a proof-of-principle, bacteria-induced HOCl
generation was successfully visualized by FBS in the mucosa
of live animals. We believe this “dual-lock” probe provides a
promising tool for in vivo HOCl imaging.
(e) Yang, Y.; Seidlits, S. K.; Adams, M. M.; Lynch, V. M.; Schmidt, C.
E.; Anslyn, E. V.; Shear, J, B. J. Am. Chem. Soc. 2010, 132, 13114.
(f) Yu, H.; Xiao, Y.; Jin, L. J. Am. Chem. Soc. 2012, 134, 17486.
(6) (a) Peng, T.; Yang, D. Org. Lett. 2010, 12, 4932. (b) 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. (c) Yu, F.; Li, P.; Li, G.; Zhao, G.; Chu,
T.; Han, K. J. Am. Chem. Soc. 2011, 133, 11030. (d) Tian, J.; Chen, H.;
Zhuo, L.; Xie, Y.; Li, N.; Tang, B. Chem.Eur. J. 2011, 17, 6626.
ASSOCIATED CONTENT
Supporting Information
■
(7) (a) Kundu, K.; Knight, S. F.; Willett, N.; Lee, S.; Taylor, W. R.;
*
S
Murthy, N. Angew. Chem., Int. Ed. 2009, 48, 299. (b) Maeda, H.;
Yamamoto, K.; Nomura, Y.; Kohno, I.; Hafsi, L.; Ueda, N.; Yoshida, S.;
Fukuda, M.; Fukuyasu, Y.; Yamauchi, Y.; Itoh, N. J. Am. Chem. Soc.
2005, 127, 68. (c) Sekiya, M.; Umezawa, K.; Sato, A.; Citterio, D.;
Suzuki, K. Chem. Commun. 2009, 3047.
Generation of ROS/RNS, intestinal HOCl detection in live
animals, absorbance spectra changes of FBS with added HOC−l,
fluorescence spectra changes of FBS as time with added OCl−
of 100 μM, fluorescence changes of FBS with added ONOO ,
E
dx.doi.org/10.1021/ja404649m | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
(
8) (a) Tanaka, K.; Miura, T.; Umezawa, N.; Urano, Y.; Kikuchi, K.;
Higuchi, T.; Nagano, T. J. Am. Chem. Soc. 2001, 123, 2530. (b) Tan,
M.; Song, B.; Wang, G.; Yuan, J. Free Radical Biol. Med. 2006, 40,
1
2
644. (c) Song, B.; Wang, G.; Tan, M.; Yuan, J. J. Am. Chem. Soc.
006, 128, 13442. (d) Naito, K.; Tachikawa, T.; Cui, S.-C.; Sugimoto,
A.; Fujitsuka, M.; Majima, T. J. Am. Chem. Soc. 2006, 128, 16430.
9) Winterbourn, C. C.; Hampton, M. B.; Livesey, J. H.; Kettle, A. J.
J. Biol. Chem. 2006, 281, 39860.
10) (a) Domigan, N. M.; Charlton, T. S.; Duncan, M. W.;
Winterbourn, C. C.; Kettle, A. J. J. Biol. Chem. 1995, 16542.
b) Hazen, S. L.; Hsu, F. F.; Duffin, K.; Heinecke, J. W. J. Biol.
(
(
(
Chem. 1996, 271, 23080. (c) Wynalda, K. M.; Murphy, R. C. Chem.
Res. Toxicol. 2010, 23, 1293.
(
11) (a) Yap, Y. W.; Whiteman, M.; Cheung, N. S. Cell Signal 2007,
1
9, 219. (b) Sugiyama, S.; Okada, Y.; Sukhova, G. K.; Virmani, R.;
Heinecke, J. W.; Libby, P. Am. J. Pathol. 2001, 158, 879. (c) Steinbeck,
M. J.; Nesti, L. J.; Sharkey, P. F.; Parvizi, J. J. Orthop. Res. 2007, 25,
1
(
128.
12) (a) Peng, X.; Wu, T.; Fan, J.; Wang, J.; Zhang, S.; Song, F.; Sun,
S. Angew. Chem., Int. Ed. 2011, 50, 4180. (b) Kim, H. N.; Guo, Z.; Zhu,
W.; Yoon, J.; Tian, H. Chem. Soc. Rev. 2011, 40, 79.
(
13) (a) Dickinson, B. C.; Peltier, J.; Stone, D.; Schaffer, D. V.;
Chang, C. J. Nat. Chem. Biol. 2011, 7, 106. (b) Cocheme, H. M.; Quin,
́
C.; McQuaker, S. J.; Cabreiro, F.; Logan, A.; Prime, T. A.; Abakumova,
I.; Patel, J. V.; Fearnley, I. M.; James, A. M.; Porteous, C. M.; Smith, R.
A.; Saeed, S.; Carre,
Partridge, L.; Murphy, M. P. Cell Metab. 2011, 13, 340.
14) Sikora, A.; Zielonka, J.; Lopez, M.; Joseph, J.; Kalyanaraman, B.
Free Radical Biol. Med. 2009, 47, 1401.
15) (a) Zielonka, J.; Sikora, A.; Joseph, J.; Kalyanaraman, B. J. Biol.
́
J. E.; Singer, M.; Gems, D.; Hartley, R. C.;
(
(
Chem. 2010, 285, 14210. (b) Zielonka, J.; Zielonka, M.; Sikora, A.;
Adamus, J.; Joseph, J.; Hardy, M.; Ouari, O.; Dranka, B. P.;
Kalyanaraman, B. J. Biol. Chem. 2012, 287, 2984.
(
16) Woodroofe, C. C.; Lim, M. H.; Bu, W.; Lippard, S. J.
Tetrahedron 2005, 61, 3097.
17) Hursts, J. K.; Albrich, J. M.; Green, T. R.; Rosena, H.; Klebanoff,
S. J. Biol. Chem. 1984, 259, 4812.
18) (a) Lee, W.-J. Sci. Signaling 2008, 1, pe24. (b) Leto, T. L.;
Geiszt, M. Antioxid. Redox Signaling 2006, 8, 1549.
19) (a) Ha, E.-M.; Lee, K.-A.; Seo, Y. Y.; Kim, S.-H.; Lim, J.-H.; Oh,
(
(
(
B.-H.; Kim, J.; Lee, W.-J. Nat. Immunol. 2009, 10, 949. (b) Ha, E.-M.;
Oh, C.-T.; Bae, Y. S.; Lee, W.-J. Science 2005, 310, 847. (c) Ha, E.-M.;
Lee, K.-A.; Park, S. H.; Kim, S.-H.; Nam, H.-J.; Lee, H.-Y.; Kang, D.;
Lee, W.-J. Dev. Cell 2009, 16, 386. (d) Lee, K.-A.; Kim, S.-H.; Kim, E.-
K.; Ha, E.-M.; You, H.; Kim, B.; Kim, M.-J.; Kwon, Y.; Ryu, J.-H.; Lee,
W.-J. Cell 2013, 153, 797.
F
dx.doi.org/10.1021/ja404649m | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX