An Ultrasensitive Cyclization-Based Fluorescent Probe for Imaging Native HOBr in Live Cells and Zebrafish

Article abstract of DOI:10.1002/anie.201606285

Bromine has been reported recently as being the 28^thessential element for human health. HOBr, which is generated in vivo from bromide, is a required factor in the formation of sulfilimine crosslinks in collagen IV. However, to date, no method for the specific detection of native HOBr in vivo has been reported. Herein, we develop a simple small molecular probe for imaging HOBr based on a specific cyclization catalyzed by HOBr. The probe can be easily synthesized in high yield through a Suzuki cross-coupling reaction. The probe exhibits ultrahigh sensitivity at the picomole level, in addition to specificity for HOBr and real-time response. Importantly, without Br^?stimulation, this probe reports native HOBr levels in HepG2 cells. Thus, the probe is a promising new tool for imaging endogenous HOBr and may provide a means for finding new physiological functions of HOBr in living organisms.

Full text of DOI:10.1002/anie.201606285

Angewandte
Communications
Chemie
International Edition: DOI: 10.1002/anie.201606285
German Edition: DOI: 10.1002/ange.201606285
Bioimaging
An Ultrasensitive Cyclization-Based Fluorescent Probe for Imaging
Native HOBr in Live Cells and Zebrafish
Kehua Xu, Dongrui Luan, Xiaoting Wang, Bo Hu, Xiaojun Liu, Fanpeng Kong, and Bo Tang*
th
Abstract: Bromine has been reported recently as being the 28
essential element for human health. HOBr, which is generated
in vivo from bromide, is a required factor in the formation of
sulfilimine crosslinks in collagen IV. However, to date, no
method for the specific detection of native HOBr in vivo has
been reported. Herein, we develop a simple small molecular
probe for imaging HOBr based on a specific cyclization
catalyzed by HOBr. The probe can be easily synthesized in
high yield through a Suzuki cross-coupling reaction. The probe
exhibits ultrahigh sensitivity at the picomole level, in addition
to specificity for HOBr and real-time response. Importantly,
Recently, a number of practical fluorescent probes for HOCl
have been reported, such as two-photon fluorescent probes
[21]
that target mitochondria and lysosomes and an enhanced-
[
22]
PET-based ultrasensitive fluorescent probe. However, to
the best of our knowledge, no fluorescent probe suitable for
the specific detection of HOBr in vivo has been reported,
although the reversible detection of HOBr/Vc (ascorbic acid)
[23,24]
and HOBr/H S were discussed.
Therefore, it would be
2
a challenge to establish a suitable approach for quantifying
endogenous HOBr, because of its low concentration and high
activity. Furthermore, it is notable that previous studies have
not considered the strict distinction between HOBr and
À
without Br stimulation, this probe reports native HOBr levels
À
in HepG2 cells. Thus, the probe is a promising new tool for
imaging endogenous HOBr and may provide a means for
finding new physiological functions of HOBr in living
organisms.
OBr .
Inspired by the latest research by B.G. Hudson et al. on
[13]
the effect of HOBr on crosslink formation in collagen IV,
we have designed a sulfilimine-based fluorescent probe for
the specific detection of HOBr. The simple synthetic strategy
is shown in Scheme 1. The biphenyl probe (BPP) can be
H
ypobromous acid (HOBr) has received a great deal of
attention owing to its strong oxidizing power and antibacterial
[1]
action. The excessive generation and accumulation of HOBr
in vivo is harmful to organisms and its damaging effects are
connected to a wide range of diseases, including rheumatoid
arthritis,
disease,
[
2,3]
[1,4,5]
inflammatory tissue damage,
cardiovascular
[
6–8]
[6]
neurodegenerative conditions, kidney dis-
[
9,10]
[11,12]
Scheme 1. Synthesis of the probe (BPP) and its response to HOBr.
ease,
and cancers.
Recently, B.G. Hudson et al.
found that HOBr plays key roles in the formation of
[
13]
sulfilimine crosslinks in collagen IV. Collagen IV scaffolds
are critical for the formation and function of basement
directly synthesized using commercially available o-bromoa-
niline and o-(methylthio)-phenylboronic acid in high yield. In
the presence of HOBr, a rapid cyclization reaction occurs
between the amino group and S-methyl group of the probe
molecule, thus generating a product with a red-shifted
emission. This red-shift is extremely important for the
method because it ensures that the fluorescence of the
unreacted probe will not interfere with the measurement. Our
probe was easily synthesized in 85% yield, and the maximum
excitation and emission wavelengths of the probe are 375 and
435 nm, respectively, whereas those of the reaction product
are 480 and 525 nm. A rapid response to HOBr was observed
for the probe with high selectivity and ultrasensitivity, and the
proposed reaction mechanism is shown in Scheme 2. Impor-
[14–16]
membranes (BMs) in animals.
In vivo, HOBr is gener-
À
ated from the peroxidation of the bromide anion (Br ) with
hydrogen peroxide, a reaction catalyzed by a heme perox-
[17]
idase,
peroxidase (EPO).
such as myeloperoxidase (MPO) or eosinophil
[18,19]
To better elucidate the physiological
and pathological functions of HOBr, it is necessary to develop
suitable probes for endogenous HOBr.
À
The blood and plasma levels of Br are far lower than that
À
[20]
of chloride anion (Cl ) by approximately 1000-fold, which
leads to a relatively lower concentration of HOBr than HOCl.
[*] Prof. K. Xu, Dr. D. Luan, Dr. X. Wang, Dr. B. Hu, Dr. X. Liu, Dr. F. Kong,
Prof. B. Tang
À
College of Chemistry, Chemical Engineering and Materials Science,
Collaborative Innovation Center of Functionalized Probes for Chem-
ical Imaging in Universities of Shandong, Key Laboratory of
Molecular and Nano Probes, Ministry of Education, Shandong
Provincial Key Laboratory of Clean Production of Fine Chemicals,
Shandong Normal University
tantly, neither Br nor H O could affect the fluorescence
2
2
intensity of the probe in simulated physiological conditions,
À
À
but HepG2 cells and zebrafish treated with Br , Br /H O , or
2
2
HOBr showed different intensities of fluorescence emission,
À
which revealed a relationship between HOBr and Br /H O
2
2
in vivo. Thus, this probe should be a promising tool for
quantifying endogenous HOBr and could provide a means of
finding new functions of HOBr and aid in studying the
Jinan 250014 (P.R. China)
E-mail: tangb@sdnu.edu.cn
Supporting information for this article can be found under:
http://dx.doi.org/10.1002/anie.201606285.
À
À
interconversion of Br , Br /H O , and HOBr in living
2
2
Angew. Chem. Int. Ed. 2016, 55, 1 – 5
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1
These are not the final page numbers!

Angewandte
Communications
Chemie
from the reaction of BPP with HOBr does not change over
a wide pH range, and the changes are especially negligible
between pH values of 6.0 and 8.0. The responses of the probe
(5 mm) to various concentrations of HOBr were measured at
pH 7.4 (Figure 2a,b), and a linear relationship was observed
between the fluorescence intensity and HOBr concentrations
Scheme 2. The proposed mechanism for the response of BPP to
HOBr.
organisms. We expect that BPP will be widely used as
a commercial reagent.
The probe was synthesized as shown in Scheme 1, and the
structures of BPP and the reaction product of BPP with
1
HOBr were fully characterized by HRMS, H NMR, and
1
3
C NMR, and a titration experiment of BPP with HOBr
1
based on H NMR was performed (see Supporting Informa-
tion, Section S2, S3 and S14). The fluorescence properties of
the probe and reaction product were also investigated, as
shown in Figure 1a. A large red shift in the maximum
emission wavelength (ca. 100 nm) was observed under
simulated physiological conditions, which improves the
detection sensitivity.
Figure 2. a) Fluorescence spectra of the probe (5 mm) toward various
concentrations of HOBr (0–20 mm) in PBS (10 mm, pH 7.4, 0.5%
CH CN as a cosolvent). Slit widths: 10/ 10 nm. b) Fluorescence
3
spectra of the probe (5 mm) with various concentrations of HOBr (0–
100 nm) in PBS (10 mm, pH 7.4). Slit widths: 20/ 20 nm. c) The linear
relationship between fluorescence intensity and HOBr concentrations
in the range from 0 to 100 nM. d) Time course of the fluorescence
intensities of 5 mm BPP with 20 mm HOBr in PBS (10 mm, pH 7.4).
Voltage: 600 V. Slit widths: 10/10 nm. l /l =480/525 nm.
ex em
from 0 to 100 nm (Figure 2c). The regression equation was
F = 0.48 + 2.64[HOBr] nm, with a linear coefficient of 0.9960.
The detection limit (3S/m) was determined to be 17 pM,
which is sufficient to image endogenous HOBr (20–100 mm in
plasma ). Obviously, the ultrasensitivity of the probe is due
to the fast cyclization of the amino group and S-methyl group
of BPP catalyzed by HOBr to generate a high-quantum-yield
Figure 1. a) Excitation and emission spectra of the probe (black) and
the product of the probe with HOBr (red). b) The fluorescence
response of the probe (5 mm) toward HOBr (20 mm) or NaOBr
(
500 mm) in ultrapure water. Slit widths: 10/10 nm. l /l =480/
[27]
ex em
5
25 nm.
product (F = 0.31, see Supporting Information, Section S10)
F
Previous reports have suggested that the molecular
with a large red-shift in emission. The large red-shift in
emission wavelength greatly improves the signal-to-noise
ratio of the detection signal. Finally, the reaction kinetics of
BPP with HOBr in phosphate-buffered saline (PBS, 10 mm,
pH 7.4) was analyzed. As shown in Figure 2d, the method
allows the real-time determination of HOBr, which is
important for accurately quantifying highly active species.
Next, we studied the selectivity of the probe to HOBr. The
potential interfering species were classified into two groups:
highly active oxidizing species and active reducing species.
Figure 3a shows that the probe did not display a significant
fluorescence increase in the presence of other reactive oxygen
mechanism during the HOCl oxidation of sulfide-type
amino acids is an electrophilic addition reaction triggered
+
À
[25,26]
by chloronium ions (Cl ) rather than OCl.
Additionally,
+
B.G. Hudson et al. proposed that Br from HOBr catalyzes
the coupling reaction of an amino group with an S-methyl
group to form a S=N bond in collagen IV.[ Herein, we first
13]
tested the responses of the probe to HOBr and NaOBr in
ultrapure water. As shown in Figure 1b, our probe is able to
À
recognize HOBr but not OBr, which suggests that the
proposed HOBr detection mechanism given in Scheme 2 for
our probe is reasonable. Next, the response of BPP to HOBr
was tested under different conditions, including pH values
ranging from 2.0 to 12.0 (Supporting Information, Figure S1)
and BPP concentrations ranging from 1 nm to 20 mm (Sup-
porting Information, Figure S2). The fluorescence resulting
À
À
species (ROS), including H O , t-BuOOH, ONOO , O ,
2
2
2
1
O , COH, NO, or even HOCl. It was interesting for us to see
2
this difference between HOBr and HOCl in the coupled
reaction of the amino group and S-methyl group (Supporting
2
www.angewandte.org
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2016, 55, 1 – 5
These are not the final page numbers!

Angewandte
Communications
Chemie
Figure 4. Confocal fluorescence imaging of intracellular HOBr in
Figure 3. a) Fluorescence intensity of the probe after adding active
oxidizing species. Black bars show the addition of one of these
interferents to a solution of 5 mm BPP. The red bars represent the
addition of both HOBr and one interferent to the probe solution.
HepG2 cells. a) HepG2 cells incubated with BPP (50 mm) for 30 min;
b) HepG2 cells first incubated with BPP (50 mm) for 30 min and then
incubated with NaBr (100 mm) for 30 min; c) same as (a) and then
incubated with NaBr (100 mm) and NAC (10 mm) for 30 min; d) same
as (a) and then incubated with HOBr (100 mm) for 30 min. e)–h)
Brightfield images of (a)–(d). i) Normalized fluorescence intensity of
cells in panels (a)–(d). The cell images shown are representative
À
À
1
5
00 mm each for H O , t-BuOOH, ONOO , O , O , COH, and NO;
2
2
2
2
2
00 mm for HOCl; and 20 mm for HOBr. b) Fluorescence intensity of
the reaction product of the probe with HOBr after adding active
reducing species. 500 mm for GSH; 300 mm each for Cys and Hcy;
(n=10 fields of cells). Fluorescence images were acquired using
1
1
50 mm for Vc; 200 mm for DTT; and 80 mm for H S. Slit widths: 10/
2
a confocal microscope with 488 nm excitation and 500–600 nm
collection. Data are presented as the mean Æ SEM; ***p<0.001
versus control group cells. The results are representative of three
independent experiments. Scale bar=25 mm.
0 nm. l /l =480/525 nm.
ex em
Information, Scheme S1 and Figure S3). Figure 3b shows that
the fluorescence responses of the probe to HOBr were hardly
affected by the presence of H S, vitamin C (Vc), dithiothreitol
2
(
DTT), GSH, Cys, or Hcy, which confirmed that the reaction
product of BPP and HOBr is stable in complex environments.
Additionally, the responses of BPP toward metal ions and
amino acids were examined under the same conditions
(
Supporting Information, Figures S4 and S5). In short, the
probe possesses excellent selectivity for HOBr over possible
interfering species derived from living cells, which suggests
that it would be useful for imaging HOBr in vitro and in vivo.
To test the ability of the probe to image HOBr in
biological systems, we first studied the cytotoxicity of the
probe and the photostability of the reaction product. The
MTT assays were performed in HepG2 cells (Supporting
Information, Figure S6). The IC50 value was 711.20 mm,
suggesting that the probe has low toxicity towards living
cells. Photostability tests showed that the reaction product of
BPP with HOBr in HepG2 cells is highly resistant to
photobleaching (Supporting Information, Figure S7). After
confirming the sensitivity, selectivity, cytotoxicity, and photo-
stability of the probe or product, we explored the potential of
BPP for imaging HOBr in vitro. Probe-treated HepG2 cells
were divided into four groups and incubated in Dulbeccoꢀs
modified Eagleꢀs medium (DMEM) containing PBS, NaBr,
NaBr and NAC (N-acetyl-l-cysteine, a scavenger of HOBr),
Figure 5. Fluorescence imaging of native HOBr. a) HL-7702 cells
incubated with NAC (10 mm) for 30 min and then with BPP (50 mm) for
30 min; b) HL-7702 cells incubated with probe (50 mm) for 30 min; c)–
d) bright-field images of HL-7702 cells; e) HepG2 cells incubated with
NAC (10 mm) for 30 min and then with BPP (50 mm) for 30 min;
f) HepG2 cells incubated with probe (50 mm) for 30 min; g)–h) bright-
field images of HepG2 cells. i) Normalized fluorescence intensity of
cells in panels (a), (b), (e), and (f). The provided images of cells are
representative (n=10 fields of cells). Fluorescence images were
acquired at 488 nm excitation and 500–600 nm emission wavelengths.
Data are presented as the mean Æ SEM; ***p<0.001 versus group
(b) cells. Results are representative of three independent experiments.
Scale bar=25 mm.
cence-inhibiting effect by NAC was observed in HepG2 and
HL-7702 cells, further supporting the idea that the fluores-
cence of untreated cells incubated with BPP arises from the
action of native HOBr. Additionally, a close relationship
or HOBr. After 30 min, fluorescence images were obtained
À
(
Figure 4). The results show that the Br -stimulated cells and
the HOBr-treated cells displayed strong fluorescence and the
fluorescence enhancement can be largely inhibited by NAC.
This demonstrates that BPP is capable of monitoring the
generation of endogenous HOBr in HepG2 cells. Moreover,
a weak fluorescence signal was observed in HepG2 cells
treated with PBS only (Figure 4a,i), which suggests that
native HOBr could also be monitored. Subsequently, imaging
of native HOBr was carried out by pretreating cells with NAC
and comparing them to untreated cells (Figure 5). The results
show that a brighter fluorescence signal was detected from
HepG2 cells than from HL-7702 cells, and a similar fluores-
À
between Br , H O and HOBr was verified when probe-
2
2
À
À
treated cells were incubated with Br , Br and H O , or
2
2
HOBr, as shown in the Supporting Information, Figure S8.
The result shows that HOBr can be generated by the
À
peroxidation of bromide anion (Br ) with hydrogen peroxide
in living cells.
Finally, we explored the potential of the probe as an
in vivo imaging tool. In these experiments, different devel-
opmental stages of zebrafish embryos were used. Briefly, 48,
72, and 120 hpf (hours postfertilization) zebrafish embryos
Angew. Chem. Int. Ed. 2016, 55, 1 – 5
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
3
These are not the final page numbers!

Angewandte
Communications
Chemie
were each divided into two groups, each having 10 embryos.
The embryos in a control group were only fed the probe for
[
[
1] A. Lane, J. Tan, C. Hawkins, A. Heather, M. Davies, Biochem. J.
010, 430, 161 – 169.
2] J. J. Hu, N. K. Wong, S. Ye, X. Chen, M. Y. Lu, A. Q. Zhao, Y.
2
3
0 min, and the embryos in the other group were pretreated
with NaBr or NAC for 30 min, and then fed with the probe for
0 min. NAC was used to eliminate endogenous HOBr.
Guo, A. C. Ma, A. Y. Leung, J. Shen, D. Yang, J. Am. Chem. Soc.
3
2015, 137, 6837 – 6843.
Fluorescence images were collected using scanning confocal
microscopy, as shown in Figure 6. The zebrafish embryos that
were fed Br emitted brighter fluorescence than those that
[3] D. L. Longo, S. L. Archer, N. Engl. J. Med. 2013, 369, 2236 –
2251.
[4] M. J. Ceko, K. Hummitzsch, N. Hatzirodos, W. Bonner, S. A.
James, J. K. Kirby, R. J. Rodgers, H. H. Harris, Metallomics 2015,
À
7, 756 – 765.
[
[
[
[
5] J. C. van Dalen, W. M. Whitehouse, C. C. Winterbourn, J. A.
Kettle, Biochem. J. 1997, 327, 487 – 492.
6] Y. W. Yap, M. Whiteman, N. S. Cheung, Cell. Signalling 2007, 19,
219 – 228.
7] S. Sugiyama, Y. Okada, G. K. Sukhova, R. Virmani, J. W.
Heinecke, P. Libby, Am. J. Pathol. 2001, 158, 879 – 891.
8] M. J. Steinbeck, L. J. Nesti, P. F. Sharkey, J. Parvizi, J. Orthop.
Res. 2007, 25, 1128 – 1135.
Figure 6. In vivo fluorescence imaging of HOBr in zebrafish at three
different developmental stages. a) 48 hpf (hours past fertilization),
b) 72 hpf, c) 120 hpf and d) elimination of endogenous HOBr with
NAC. In all cases, the embryos in the control group were only fed the
probe for 30 min, whereas the embryos in the other group were
[9] K. L. Brown, C. Darris, K. L. Rose, O. A. Sanchez, H. Madu, J.
Avance, N. Brooks, M. Zhang, A. Fogo, R. Harris, Diabetes 2015,
64, 2242 – 2253.
[10] V. M. Monnier, Diabetes 2015, 64, 1910 – 1911.
[11] R. A. Cairns, I. S. Harris, T. W. Mak, Nat. Rev. Cancer 2011, 11,
85 – 95.
À
pretreated with Br (100 mm) or NAC (10 mm) for 30 min and then fed
with the probe for 30 min. The embryo images shown are representa-
tive (n=10 fields of embryos). The results are representative of three
independent experiments. Scale bar=250 mm.
[12] C. Gorrini, I. S. Harris, T. W. Mak, Nat. Rev. Drug Discovery
2013, 12, 931 – 947.
[13] A. S. McCall, C. F. Cummings, G. Bhave, R. Vanacore, A. Page-
McCaw, B. G. Hudson, Cell 2014, 157, 1380 – 1392.
[
14] R. Vanacore, A. J. Ham, M. Voehler, C. R. Sanders, T. P.
Conrads, T. D. Veenstra, K. B. Sharpless, P. E. Dawson, B. G.
Hudson, Science 2009, 325, 1230 – 1234.
À
were not fed Br , while those embryos pretreated with NAC
displayed the lowest fluorescence. These results further
confirmed that HOBr can be generated from Br in living
organisms and the native HOBr can be measured by BPP.
Therefore, we believe that the probe is a promising tool for
monitoring changes in HOBr levels in vivo.
À
[15] K. Kꢁhn, Matrix Biol. 1995, 14, 439 – 445.
[
16] A. L. Fidler, R. M. Vanacore, S. V. Chetyrkin, V. K. Pedchenko,
G. Bhave, V. P. Yin, C. L. Stothers, K. L. Rose, W. H. McDonald,
T. A. Clark, D. B. Borza, R. E. Steele, M. T. Ivy, J. K. Hudson,
B. G. Hudson, Proc. Natl. Acad. Sci. USA 2014, 111, 331 – 336.
[
13]
In conclusion, we have developed an ultrasensitive
fluorescence probe for detecting hypobromous acid in vitro
and in vivo. The probe is easily synthesized in high yield and
exhibits a real-time response to HOBr with specificity and
ultrasensitivity (picomole level). The imaging of endogenous
HOBr in HepG2 cells and zebrafish can be performed using
the probe. Moreover, the probe reports native HOBr levels in
HepG2 cells without bromine anion stimulation. Therefore,
this probe is a promising tool for monitoring changes in HOBr
levels in vivo and paves the way for acquiring new insight into
the physiological action of HOBr.
[17] W. Wu, Y. Chen, A. D’Avignon, S. L. Hazen, Biochemistry 1999,
8, 3538 – 3548.
3
[
[
18] C. J. van Dalen, A. J. Kettle, Biochem. J. 2001, 358, 233 – 239.
19] P. G. Furtmꢁller, U. Burner, G. Regelsberger, C. Obinger,
Biochemistry 2000, 39, 15578 – 15584.
[
20] V. F. Ximenes, N. H. Morgon, A. R. de Souza, J. Inorg. Biochem.
2015, 146, 61 – 68.
[21] L. Yuan, L. Wang, B. K. Agrawalla, S. J. Park, H. Zhu, B.
Sivaraman, J. Peng, Q. H. Xu, Y. T. Chang, J. Am. Chem. Soc.
2
015, 137, 5930 – 5938.
22] H. Zhu, J. Fan, J. Wang, H. Mu, X. Peng, J. Am. Chem. Soc. 2014,
36, 12820 – 12823.
23] F. Yu, P. Song, P. Li, B. Wang, K. Han, Chem. Commun. 2012, 48,
735 – 7737.
[
[
[
1
7
24] B. Wang, P. Li, F. Yu, J. Chen, Z. Qu, K. Han, Chem. Commun.
2013, 49, 5790 – 5792.
Acknowledgements
[
[
25] P. Nagy, M. T. Ashby, Chem. Res. Toxicol. 2005, 18, 919 – 923.
26] X. L. Armesto, M. Canle, M. I. Fernꢂndez, M. V. Garcia, J. A.
Santaballa, Tetrahedron 2000, 56, 1103 – 1109.
This work was supported by the 973 Program
(
2013CB933800) and the National Natural Science Founda-
tion of China (21535004, 21227005, 21390411, 21275092,
1575081, and 21507075).
[
27] J. P. Gaut, G. C. Yeh, H. D. Tran, J. Byun, J. P. Henderson, G. M.
Richter, M. L. Brennan, A. J. Lusis, A. Belaaouaj, R. S. Hotch-
kiss, J. W. Heinecke, Proc. Natl. Acad. Sci. USA 2001, 98, 11961 –
2
11966.
Keywords: bioimaging · cyclization reactions ·
fluorescent probes · hypobromous acid
Received: June 28, 2016
Revised: August 3, 2016
Published online: && &&, &&&&
4
www.angewandte.org
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2016, 55, 1 – 5
These are not the final page numbers!

Angewandte
Communications
Chemie
Communications
Bioimaging
A small molecular probe for imaging
HOBr based on a specific cyclization
catalyzed by HOBr was developed. The
probe can be easily synthesized in high
yield through a Suzuki cross-coupling
reaction and exhibits ultrahigh sensitivity
and specificity for HOBr in real-time. It
was used to measure native HOBr levels
in HepG2 cells and may provide a means
for finding new physiological functions of
HOBr.
K. Xu, D. Luan, X. Wang, B. Hu, X. Liu,
F. Kong, B. Tang*
&&&& — &&&&
An Ultrasensitive Cyclization-Based
Fluorescent Probe for Imaging Native
HOBr in Live Cells and Zebrafish
Angew. Chem. Int. Ed. 2016, 55, 1 – 5
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!