Selective Monitoring and Imaging of Eosinophil Peroxidase Activity with a J-Aggregating Probe

Article abstract of DOI:10.1021/jacs.8b07073

The specific detection of eosinophil peroxidase (EPO) activity requires the difficult distinction between hypobromous acid generated by EPO and hypochlorous acid generated by other haloperoxidases. Here we report a fluorogenic probe that is halogenated with high kinetic selectivity (≥1200:1) for HOBr over HOCl. Heavy-atom effects do not quench the dibrominated product because of its self-assembly into emissive J-aggregates that provide a turn-on signal. Applications of this fluorogen to EPO activity assays, dipstick sensors, fluorescence imaging of EPO activity, assays of oxidative stress in cancer cells, and immune response detection in live mice are reported.

Full text of DOI:10.1021/jacs.8b07073

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Article
Selective Monitoring and Imaging of Eosinophil
Peroxidase Activity with a J-Aggregating Probe
Tae-Il Kim, Byunghee Hwang, Boeun Lee, Jeehyeon Bae, and Youngmi Kim
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 29 Aug 2018
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Journal of the American Chemical Society
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Selective Monitoring and Imaging of Eosinophil Peroxidase Activity
with a J-Aggregating Probe
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Tae-Il Kim,† Byunghee Hwang, Boeun Lee, Jeehyeon Bae,* and Youngmi Kim*
,⊥
†,⊥
‡,⊥
,§
,†
†Department of Chemistry and Research Institute of Basic Sciences, Kyung Hee University, 26 Kyungheedae-ro, Dongdae-
mun-gu, Seoul 02447, Korea
‡Department of Life Science, Chung-Ang University, 84 Heukseok-ro, Dongjak-gu, Seoul 06974, Korea
§School of Pharmacy, Chung-Ang University, 84 Heukseok-ro, Dongjak-gu, Seoul 06974, Korea
ABSTRACT: The specific detection of eosinophil peroxidase (EPO) activity requires the difficult distinction between hypobro-
mous acid generated by EPO and hypochlorous acid generated by other haloperoxidases. Here we report a fluorogenic probe that is
halogenated with high kinetic selectivity (≥1200:1) for HOBr over HOCl. Heavy atom effects do not quench the dibrominated
product because of its self-assembly into emissive J-aggregates that provide a turn-on signal. Applications of this fluorogen to EPO
activity assays, dip-stick sensors, fluorescence imaging of EPO activity, assays of oxidative stress in cancer cells, and immune re-
sponse detection in live mice are reported.
4
ic selectivity for HOBr over HOCl (up to 10 :1 for Tyr resi-
INTRODUCTION
30,31
dues).
However, the resulting brominated derivatives are
Eosinophils are granulocytic leukocytes that play critical roles
in host defense against infections, and are involved in the
pathogenesis of asthma and allergic diseases. Moreover,
typically quenched in solution due to heavy atom effects that
increase intersystem crossing and non-radiative deactivation
rates, making this halogenation approach unappealing for the
1,2
32-34
numerous disorders, from autoimmune diseases to Hodgkin's
lymphoma have been associated with abnormally elevated
development of fluorescent probes.
Scheme 1. Schematic illustration of the design of fluores-
cent “turn-on” probes for the detection of EPO activity.
3,4
eosinophil counts. The cytotoxic activity of granulocytes
relies in part on their ability to generate toxic reactive oxygen
–
Previous Work: Commercially available fluorogenic EPO probe
species (ROS), via haloperoxidases that oxidize halides (X ) to
(a)
5
-9
-
hypohalous acids (HOX) using hydrogen peroxide. Myelop-
O
O
O
EPO/H O /Br
O
O
OH
2
2
–
or
eroxidase (MPO), found in neutrophils, mainly converts Cl to
-
MPO/H O /Cl
2
2
1
0,11
NH
2
hypochlorous acid (HOCl, pK
(
a
7.6).
Eosinophil peroxidase
-
-
COO
COO
–
EPO) instead preferentially oxidizes Br to hypobromorous
acid (HOBr, pK 8.8) despite the 1000-fold excess of Cl over
a
HN
O
−
aminophenyl fluorescein (APF)
fluorescein
strongly fluorescent
–
–
–
12
Br ([Cl ] 100-140 mM, [Br ] 20-100 µM). The strong oxi-
non-fluorescent
dizing and halogenating ability of HOBr contributes to in-
flammation and tissue damage resulting from eosinophilic
disorders.
(b) This Work: Emissive J-aggregate-based fluorogenic EPO probe
O
OCH3
O
OCH3
5
-7
-
O
OCH
3
EPO/H
O /Br
2 2
+
X
X
X
No fluorescent probe for the specific detection of EPO ac-
tivity is currently available. Tremendous strides have been
made in the development of chemically reactive fluorescent
fast
N
F
N
F
N
F
N
F
B
B
N
F
N
F
2 (X= Br)
3 (X= Br)
5
Cl)
(X=
-
B
MPO/H O /Cl
2
2
probes for the selective detection and imaging of H
HOCl (and hence MPO activity),
2
O
2
, or
but the design of probes
to specifically detect HOBr generated by EPO faces important
4
Cl)
Probe 1
(X=
slow
1
3-18
1
9-21
challenges.
HOCl is a more powerful oxidizing agent than
In this paper, we describe the use of BODIPY chromo-
phores that form J-aggregates to solve the HOX selectivity
conundrum in the detection of EPO activity. Probe 1 is rapidly
dibrominated with EPO-generated HOBr with a high kinetic
selectivity over its chlorination with HOCl (Scheme 1b). The
resulting 3 does not suffer from poor photophysical properties
associated with brominated chromophores; on the contrary, it
self-assembles into highly emissive red-shifted J-aggregates to
result in a clean turn-on fluorescence signal. Using this probe,
the selective detection, assaying and imaging of HOBr genera-
tion by EPO without interference from MPO-generated HOCl
are for the first time demonstrated.
HOBr, which, given their similar chemistry, precludes a selec-
2
2
–
tive redox-triggered signal. Other ROS (e.g. H
2
O
2
, ONOO ,
–
·O ) are also known interferences in some redox-based detec-
2
2
3-26
tion schemes.
Indeed, these are issues with the most popu-
lar commercially available fluorescent probe for monitoring
2
7
EPO activity, aminophenyl fluorescein (APF, Scheme 1a).
Though able to detect EPO activity, APF also responds to
MPO-generated HOCl, and other ROS such as the hydroxyl
−
radical (∙OH) or peroxynitrite (ONOO ), often with higher
28,29
sensitivity.
electron-rich aromatic substrates does show appreciable kinet-
ACS Paragon Plus Environment
The electrophilic halogenation of alkenes or

Journal of the American Chemical Society
3
mM acetate buffer, pH 5.0, 0.1% CH CN) at 25 C (Figure 2,
Page 2 of 6
o
RESULTS AND DISCUSSION
1
2
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1
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and Figures S21-22). Upon addition of HOBr, the absorption
and emission peaks of 1 decrease markedly, while the charac-
teristic red-shifted absorption and emission bands of the sus-
pended aggregates of 3 emerge instantly. To the naked eye,
these correspond to a change from pink to purple (Figure 2a,
inset), and under handheld UV lamp irradiation (365 nm) to a
change from orange to red (Figure 2b, inset). Time course
studies revealed that the fluorescence turn-on process is fast (≤
Synthesis. Probe 1 was prepared according to literature pro-
cedures. To confirm the feasibility of the proposed sensing
Scheme 1b, the expected products of the electrophilic bromin-
ation of 1 with HOBr, 2 and 3, were independently synthe-
sized (Scheme S1).
35
Photophysical Property and Self-Assembly Studies. All
meso-ester-substituted-BODIPYs 1−3 have low solution emis-
sion quantum yields in solution (Φ
F 3
in CH CN = 0.003 (1);
2
s; Figure S23), and HPLC−MS analysis of the reaction mix-
0.004 (2); 0.002 (3); Table S1), as expected for sterically con-
ture confirmed the formation of 3 as a major product (Figure
S39).
3
5
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gested, meso-substituted 1,3,5,7-tetramethyl BODIPYs.
Probe 1 was previously shown to display aggregation-induced
emission enhancement (AIEE) features through J-
3
5
aggregation, evidenced by a large red-shift from 509 to 581
-
1
nm upon aggregation, a small Stokes shift (176 cm ), a nar-
-
1
row emission band (FWHM = 560 cm ), an increased fluores-
7
-1
cence rate constant (from the k
4
F
= 3.3 x 10 s in solution to
8
-1
.4 x 10 s in suspended aggregates), and a head-to-tail ar-
rangement of its transition dipoles (displacement angle θ <
5
4.7°; 33˚ for 1).
The suspended colloidal aggregates of dibromo-BODIPY 3
(
20 µM) in CH
3
CN–water mixtures (f ≥ 90%; 540 ± 10 nm by
w
DLS, Figure 1b) display the same spectroscopic features con-
sistent with J-aggregate formation among meso-substituted
3
5,36
1,3,5,7-tetramethyl BODIPYs.
shifts from 539 nm in CH CN solution to 619 nm in CH
water mixtures (Figure S8). Its fluorescence emission features
Its absorption maximum
3
3
CN–
-
1
a small Stokes shift (52 cm ), a narrow emission band
-
1
(
FWHM = 724 cm ), a 6.3-fold increase in fluorescence rate
Figure 2. Absorption (a) and fluorescence (b) spectra of the self-
assembled 1 (2.5 µM) before (black) and immediately (≤ 2s) after
(red) addition of HOBr (10 µM). Insets: Photographs of 1 (20 µM)
before (A) and after (B) the addition of HOBr (40 µM) under
ambient light (a) and UV light (b). (c) Fluorescence intensity at
616 nm as a function of [HOBr] (0−10 µM). Inset: Ratiometric
(I616/I581) calibration curve. (d) Fluorescence spectra of probe 1
(2.5 µM) in the presence of various biologically relevant species
(5 µM HOBr, 50 µM for other ROS, 1 mM for amino acids and
biothiols, and 1 mg/mL for others). All spectra were obtained
immediately after addition of each analyte to 1 in acetate buffer
7
−1
7 −1
F F
constant (k (sol) = 1.1 × 10 s ; k (agg) = 6.9 × 10 s ; Fig-
ure S20). The characteristic spectroscopic features of J-
aggregates were seen in concentrations of 3 as low as ca. 0.25
µM (Figure S9).
Importantly for a clean turn-on signal, the red emissive (621
nm) aggregates of dibrominated 3 are fully resolved from the
parent probe 1, which self-assembles into orange emissive J-
aggregates showing a sharp emission band at 581 nm (Figures
S2-S3 and Figure 2b). Moreover, aggregates of the monobro-
minated BODIPY 2 in the same CH
3
CN–water mixtures (f ≥
w
90%) do not interfere, displaying absorption and emission
maxima at ca. 567 nm and 580 nm, respectively (Figures S5-
S6).
3
(100 mM, pH 5.0, 0.1% CH CN) at 25 °C. λex = 480 nm.
As shown in Figure 2c, the fluorescence intensity at 616 nm
increases rapidly with increasing HOBr concentration within
the measured range (0−10 µM), levelling off after the addition
of 2 equivalents of HOBr with ca. 22-fold increase in fluores-
cence intensity at 616 nm (Figure S22). A linear relationship
exists between the ratio of the emission intensities (I616/I581
)
2
and the concentration of HOBr in the range of 1−5 µM (R =
0.983) (Figure 2c, inset). The detection limit (3σ/slope) for
HOBr using probe 1 was determined to be 3.8 nM (Figure
S24).
More importantly, the probe 1 (2.5 µM) displayed excellent
selectivity toward HOBr over other biologically relevant mol-
ecules (Figure 2d). Among reactive oxygen species (ROS),
only HOBr promotes a dramatic enhancement in emission at
616 nm; no significant change in the emission spectra of 1
Figure 1. (a) Absorption (dotted lines) and emission (solid lines)
spectra of 3 (20 µM) in CH
CH CN:H O (red, 100 mM acetate buffer, pH 5.0). λex = 480 nm.
Insets: Photographs of each solution (A: CH CN, B: CH CN/H
0.1:99.9 (v/v)) under irradiation at 365 nm. (b) Size distribution
of aggregates of 3 by DLS (540 ± 10 nm). Inset: SEM images
3
CN (black) and 0.1:99.9 (v/v)
3
2
3
3
2
O
−
−
=
2 2 2
occurs in the presence of HOCl/OCl , H O , superoxide (∙O ),
t
hydroxyl (∙OH) or tert-butoxy radicals (∙O Bu), tert-butyl hy-
droperoxide (t-BuOOH), or peroxynitrite (ONOO ), even at
−
(
490 ± 30 nm) of aggregates of 3. [3] = 20 µM.
higher concentrations (5 µM for HOBr and 50 µM for others).
HOBr Sensing in Solution. The sensory response of probe
–
Probe 1 can react with a large excess of HOCl/OCl (≥ 200
1
toward HOBr was first evaluated in aqueous solution (100
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µM) to give the dichlorinated product 5 (Figures S26 and S40).
1
2
3
4
5
6
7
8
9
However, the chlorination reaction is not kinetically competi-
tive with the bromination reaction. HPLC−MS analysis con-
firmed that the reaction of probe 1 (2.5 µM) in the presence of
both HOBr (12.5 µM) and HOCl (up to 400 µM) only lead to
the formation of 3 (Figure S41). Furthermore, in the absence
of any HOBr, false positive responses elicited by HOCl do not
take place within biologically relevant concentrations ([HOCl]
3
7-39
≤
100 µM; Figures S26-S27).
The robustness of this assay
–
against the interference of HOCl/OCl results from a 1200-
fold kinetic selectivity for HOBr over HOCl in the halogena-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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tion reactions of the BODIPY dye (Figure S28).
In addition, probe 1 is not affected by the presence of amino
acids with nucleophilic side chains (Cys, His, Lys), biothiols
(GSH, HCy), or common hydrolytic enzymes (esterase, tryp-
sin, lipase, lysozyme). (Figure 2d). Moreover, the self-
assembled J-aggregates of both 1 and 3 remained stable in the
presence of proteins (myoglobin, hemoglobin, bovine serum
albumin) and lipids (cholesterol, oleic acid, phosphatidylcho-
line) (Figure S16), and in a pH range from 4 to 9 (Figure S17).
The fluorescence response of probe 1 toward HOBr is never-
theless pH-dependent. The greater response under acidic con-
ditions (pH < 6) is consistent with the proposed electrophilic
halogenation pathway (Figure S25).
Figure 3. Absorption (a) and fluorescence (b) spectra of the self-
assembled 1 upon treatment with EPO (0−150 ng/mL) in 100 mM
acetate buffer (pH 5.0, 0.1% CH
and 100 µM KBr. Inset: Ratiometric (I616/I581) calibration
curve ([EPO]: 0−125 ng/mL). (c) Fluorescence response (I616/I581
3
CN, 25 °C) containing 50 µM
2 2
H O
)
of probe 1 in various conditions. All spectra were obtained imme-
diately after addition of 1 to each assay solution, and fluorescence
intensities at 581 nm and 616 nm were recorded. [1] = 2.5 µM. λex
EPO Activity Assays in Solution. Incubation of 1 (2.5 µM)
with EPO (0.5−150 ng/mL) in acetate buffer solution (100
=
480 nm.
mM, pH 5.0, 50 µM H
2
O
2
, 100 µM KBr, 0.1% CH
3
CN) at
Dip-Stick Sensors. The EPO activity assay could finally be
2
5 °C resulted in spectral changes consistent with the for-
simplified by taking advantage of bright emission of the ag-
gregates of 1 and 3 in the solid-state. As shown in Figure 4,
paper strips coated with 1 can be used as ‘dip-sticks’ to esti-
mate the concentration of EPO in aqueous samples. The avail-
ability of simplified and rapid EPO assays is especially ap-
pealing for point-of-care measurements using nasal mucus or
bronchoalveolar lavage fluids in cases of allergies, asthma, or
mation of emissive J-aggregates of dibromo-BODIPY 3 (Fig-
ure 3a and 3b). The characteristic red-shifted absorption and
emission bands (λabs.max = 613 nm, λem.max = 616 nm) grow
instantly (≤ 2s, Figure S32), and the latter increases linearly
2
(
R = 0.9957) with EPO concentrations ranging from 0 to 125
ng/mL (Figure 3b inset). The major reaction product was un-
ambiguously identified as dibromo-BODIPY 3 by HPLC–MS
analysis of the assay solution (Figure S42). Probe 1 can detect
EPO activity as low as 0.09 ng/mL (3σ/slope, Figure 3b inset
and Figure S33), which is much lower than the median EPO
serum levels of either healthy subjects (7.2 ng/mL) or severely
4
1,45
related eosinophilic disorders.
4
1,42
asthmatic patients (22.9 ng/mL).
The generation of 3 was
optimal at pH 5, but there was substantial production of 3 even
at pH 7.4 (Figure S35).
The enhancement of fluorescence intensity at 616 nm was
suppressed in a dose-dependent manner in the presence of a
Figure 4. Photographs of color (a) and fluorescence (b) responses
of probe 1-coated paper strips following a 15 s dip in solutions of
known EPO inhibitor, 4,4’-diaminodiphenylsulfone (dapsone)
43
-
-
-
(
(
Figure S37) or the HOBr scavenger N-acetyl-cysteine (NAC)
MPO/H
2 2 2 2
O /Cl (left) or EPO/H O /Br (right). [Cl ] = 100 mM,
44
-
Figure S38). These results confirm that the formation of 3 is
[H ] = 50 µM, [MPO] =150 ng/mL; [Br ] = 100 µM, [H O ] =
2
O
2
2 2
driven by EPO-dependent HOBr generation.
50 µM, [EPO] = 5, 10, and 20 ng/mL. The fluorescence response
images were taken under UV irradiation (365 nm).
Control experiments established that a positive response is
−
only observed in the simultaneous presence of EPO, Br , and
Fluorescence Imaging of HOBr Generation by EPO.
H
2
O
2
in the analytical sample (Figure 3c). The HOCl-
Probe 1 is suitable for the confocal fluorescence imaging of
−
−
generating combination of MPO, H
2
O
2
and Cl system did not
EPO/H O /Br (HOBr) in living RAW264.7 macrophage cells,
2
2
elicit a response from probe 1 (entry 7); nevertheless, their
presence did not interfere with the EPO activity assay (entries
9-10). Considering the similarities between the structures and
functions EPO and MPO, and that of their respective outputs
HOBr and HOCl, the selective detection of EPO over MPO
with 1 is notable. To our knowledge, probe 1 is the first small
molecule fluorescent “turn-on” probe that can selectively de-
tect EPO activity over that of MPO.
and shows no cytotoxicity up to 100 µM (Figure S46). Cells
incubated with probe 1 (10 µM) alone showed strong yellow
channel fluorescence (570–590 nm, λex 488 nm), but none was
observed from the red channel (600–620 nm, Figure 5a, #1-2).
−
By contrast, upon treatment with exogenous EPO/H
2
O
2
/Br ,
red channel luminescence increased as the signals for the yel-
low channel diminished (Figure 5a, #6-7) within the cells in
proportion to the added EPO (Figure S47). The red fluores-
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Journal of the American Chemical Society
Page 4 of 6
cence signal was observed within 10 min and remained stable
for at least 90 min (Figure S48).
increases in fluorescence intensity ratios (I616/I581) by 1.5−2
fold, relative to their respective normal cells. The responses
were further amplified (4−7 fold) in cancer cells sensitized to
oxidative stress by the mitochondrial electron-transport-chain
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
In control experiments, co-treatment of the cells with the
ROS scavenger N-acetylcysteine (NAC, Figure 5a, #17) or the
4
8
blocker antimycin A (AntA).
EPO inhibitor dapsone (Figure S49) inhibited the response of
−
probe 1 toward EPO/H
2
O
2
/Br . In addition, no red fluores-
Eosinophil Immune Response Detection in Live Mice.
The utility of probe 1 was further extended to the detection of
EPO-generated HOBr in response to a bacterial infection in a
mouse model (Figure 6). Acute infection in a BALB/c mouse
cence was observed in the probe 1-loaded cells incubated with
MPO (up to 600 ng/mL), H (50 µM), and KCl (100 mM)
Figure 5a, #12, and Figure S50). Co-staining experiments
2
O
2
(
4
9
with the DNA-binding dye DAPI indicate that the probe did
not co-localize in the nuclei, but instead accumulated in the
cytoplasm of the cells. Taken together, these results indicate
that probe 1 is taken up by the macrophage cells efficiently,
and that the internalized probe is selectively activated by the
EPO-generated HOBr.
was induced by intraperitoneal (i.p.) injection of Salmonella.
Although the mice did not show any visible sign of disease at
72 h post-inoculation, a significant increase in the numbers of
eosinophils in Salmonella-infected mice was confirmed (Fig-
ure 6a). The plasma and granulocytes were then separated
from the whole blood, and treated with probe 1. No noticeable
increase in fluorescence intensity ratios (I616/I581) was observed
in the plasma and granulocyte mixture obtained from uninfect-
ed mice. By contrast, a 3.5-fold increase (I616/I581) was ob-
served in the samples from Salmonella-infected mice (Figure
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
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
b). Probe 1-coated paper strips could also be used to detect
increased EPO activity in the same samples (Figure S52).
These results demonstrate the applicability of probe 1 in the
detection of eosinophil immune response elicited by early-
stage bacterial infections in live mammals.
Figure 5. (a) Confocal fluorescence images of probe 1 (10 µM)-
loaded living macrophage cells (RAW264.7) treated with differ-
ent conditions. The cells were incubated with 1 for 30 min at 37
o
C and then imaged (#1-5); 1-loaded cells were incubated with
KBr (100 µM), H (50 µM) and EPO (150 ng/mL) for 30 min
either in the absence (#6-10) or the presence of 10 mM NAC
#16-20); 1-loaded cells were incubated with KCl (100 mM),
(50 µM) and MPO (600 ng/mL) for 30 min (#11-15). The
2
O
2
Figure 6. (a) Above: Mice experiment schedule; Below: Levels of
eosinophils and neutrophils in Salmonella-infected mice (red bars)
relative to uninfected mice as control (black bars). (b) Fluores-
cence response (I616/I581) of probe 1 (25 µM) in the absence (gray)
and the presence of plasma and granulocytes from uninfected
(
2 2
H O
cells were co-stained with the blue fluorescent DNA binding dye
DAPI. (Top to bottom) Fluorescence images from yellow channel
(
=
Ex = 488 nm, Em = 570-590 nm), red channel (Ex = 488 nm, Em
600-620 nm), blue channel (Ex = 405 nm, Em = 400–520 nm),
(black) and Salmonella-infected mice (red). The fluorescence
intensity ratios (I616/I581) were normalized to that of uninfected
mice. Five mice per group were used for analysis. **p < 0.01,
bright-field, and merged images from the four channels. (b) Com-
parative fluorescence intensity ratios (I616/I581) of probe 1 meas-
ured using a multi-plate reader in cancer (HCT116, A549), normal
***p < 0.005.
(CCD-18Co, MRC-5), and AntA-stimulated (red) cell lysates,
CONCLUSION
before (black) and after (blue) conversion of H by EPO/KBr.
2
O
2
In summary, differences in the rates of electrophilic aromatic
halogenation of probe 1, and in the photophysical properties of
the resulting halogenated BODIPY dyes (e.g. 3) were exploit-
ed to allow for the selective detection, assaying and imaging
of HOBr generation by EPO without interference from the
corresponding HOCl production by MPO. The probe’s re-
sponse is fast (≤ 2s), highly sensitive (LOD: 0.09 ng/mL for
EPO), and benefits from the advantageous photophysical
properties that results from the self-assembly of both 1 and 3
in J-aggregates (narrow, non-overlapping bright emission
bands). These features make probe 1 uniquely suited for inves-
tigating delicate changes in EPO activity and HOBr levels as
pathological biomarkers.
Fluorescence Assays of Oxidative Stress in Cancer Cells.
Cancer cells are under increased oxidative stress due to altered
metabolic activity and mitochondrial dysfunction, resulting in
4
6,47
higher steady-state H
Although probe 1 does not respond to H
2
O
2
levels compared to normal cells.
, its catalytic con-
2
O
2
−
version to HOBr by EPO/Br elicits a response that is suffi-
ciently sensitive to discriminate between the oxidative stress
levels found in cancer and normal cells (Figure 5b). The fluo-
rescence response (I616/I581) was measured in human colon and
lung cancer cell (HCT116 and A549) lysates incubated with
probe 1, and compared with those of respective normal CCD-
18Co colon cell and MRC-5 lung cell lysates (Figure 5b). In
−
the presence of added EPO/Br , cancer cells lysates showed
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20) Product selectivity for HOBr over HOCl, see: Xu, K.; Luan, D.;
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
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AUTHOR INFORMATION
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24) Liu, S.-R.; Wu, S.-P. Org. Lett. 2013, 15, 878−881.
25) Manjare, S. T.; Kim, S.; Heo, W. D.; Churchill, D. G. Org. Lett.
Corresponding Authors
*E-mail: jeehyeon@cau.ac.kr. Fax: +82 2-861-7338 (J.B.).
2
014, 16, 410−412.
26) Qu, Z.; Ding, J.; Zhao, M.; Li, P. J. Photochem. Photobiol. A
2015, 299, 1−8.
27) Flemmig, J.; Zschaler, J.; Remmler, J.; Arnhold, J. J. Biol. Chem.
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*E-mail: youngmi.kim@khu.ac.kr. Fax: +82 2-961-0443 (Y.K.).
(
Author Contributions
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012, 287, 27913−27923.
⊥T.-I.K., B.H. and B.L. contributed equally to this work.
(28) Setsukinai, K.; Urano, Y.; Kakinuma, K.; Majima, H. J.; Nagano,
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Notes
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29) Gomes, A.; Fernandes, E.; Lima, J. L.F.C. J. Biochem. Biophys.
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Methods 2005, 65, 45−80.
(30) Pattison, D. I.; Davies, M. J. Biochemistry 2004, 43, 4799−4809.
ACKNOWLEDGMENT
This research was supported by National Research Foundation of
Korea (NRF) grants funded by the Korean government (MSIP)
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chem. 2015, 146, 61−68.
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(NRF-2018R1D1A1B07044894, NRF-2017M3D9A1073769 and
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