Endoplasmic Reticulum-Targeted Ratiometric N-Heterocyclic Carbene Borane Probe for Two-Photon Microscopic Imaging of Hypochlorous Acid

Article abstract of DOI:10.1021/acs.analchem.8b03565

The naphthoimidazolium borane 4 is shown to be a selective probe for HOCl over other reactive oxygen species. Unlike other boronate-reactive oxygen species (ROS) fluorogenic probes that are oxidized by HOCl through a nucleophilic borono-Dakin oxidation mechanism, probe 4 is distinguished by its electrophilic oxidation mechanism involving B-H bond cleavage. Two-photon microscopy experiments in living cells and tissues with the probe 4 demonstrate the monitoring of endogenous HOCl generation and changes in HOCl concentrations generated in the endoplasmic reticulum during oxidative stress situations.

Full text of DOI:10.1021/acs.analchem.8b03565

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Article
Endoplasmic Reticulum-Targeted Ratiometric NHC-Borane Probe
for Two-Photon Microscopic Imaging of Hypochlorous Acid
Yenleng Pak, Sang Jun Park, Gyeongok Song, Yubin Yim, Hyuk
Kang, Hwan Myung Kim, Jean Bouffard, and Juyoung Yoon
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b03565 • Publication Date (Web): 10 Oct 2018
Downloaded from http://pubs.acs.org on October 10, 2018
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Analytical Chemistry
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Endoplasmic Reticulum-Targeted Ratiometric NHC-Borane
Probe for Two-Photon Microscopic Imaging of Hypochlorous
Acid
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‡
a
‡b
c
a
c
b,c
Yen Leng Pak , Sang Jun Park , Gyeongok Song , Yubin Yim , Hyuk Kang , Hwan Myung Kim* ,
a
a
Jean Bouffard* , and Juyoung Yoon*
a
Department of Chemistry and Nano Science, Ewha Womans University, Seoul 120ꢀ75003760, Korea
b
Department of Energy Systems Research, Ajou University, Suwon, Gyeonggiꢀdo 443ꢀ749, Korea
Department of Chemistry, Ajou University, Suwon, Gyeonggiꢀdo 443ꢀ749, Korea.
c
ABSTRACT: The naphthoimidazolium borane 4 is shown to be a selective probe for HOCl over other reactive oxygen species.
Unlike other boronateꢀROS fluorogenic probes that are oxidized by HOCl through a nucleophilic boronoꢀDakin oxidation mechaꢀ
nism, probe 4 is distinguished by its electrophilic oxidation mechanism involving B–H bond cleavage. Twoꢀphoton microscopy
experiments in living cells and tissues with the probe 4 demonstrate the monitoring of endogenous HOCl generation and changes in
HOCl
concentrations
generated
in
the
endoplasmic
reticulum
during
oxidative
stress
situations.
In all aerobic living organisms, reactive oxygen species
ROS) are continuously produced, transformed, and conꢀ
on the wellꢀknown ROSꢀmediated oxidation of boronate deꢀ
rivatives to phenols that proceed through a boronoꢀDakin reacꢀ
tion mechanism.
(
sumed. They are generated endogenously and actively particiꢀ
pate as metabolite agents in signaling pathways to support the
Earlier in 2018, we have reported the first use of an Nꢀ
heterocyclic carbene borane (NHCꢀborane) as a ROS probe
1,2
normal function of cells. Oxidative stress, which results
48
from an unbalance between the levels of ROS and antioxidants
inside the cell, causes cell damage and contributes to the aging
specific for HOCl. The reported probe 5 was shown to react
with HOCl through an electrophilic mechanism that proceeds
with a formal hydride abstraction at the borane center, in conꢀ
3,4
process. Their short lifetimes and low concentrations make
their in vivo measurement and the study of their biological
roles more challenging. Among other methods, fluorescence
imaging probes have emerged as powerful measurement tools
to visualize the variation and distribution of these species, with
a popularity owed to their realꢀtime monitoring capabilities,
–
trast with the nucleophilic mechanism for the reaction of OCl
with boronic acids their esters. After oxidative hydrolysis by
HOCl, electrostatic repulsion between the resulting imidazoliꢀ
um salts disfavors the formation of colloidal fluorophore agꢀ
gregates and pyrene excimers.
5
ꢀ20
high sensitivity, and nonꢀinvasive characteristics.
Herein, we report the design and application of a second
NHCꢀboraneꢀbased fluorogenic probe 4 for the specific detecꢀ
tion of HOCl. Unlike its predecessor, its turnꢀon luminescence
signal does not rely on polarity and aggregation effects. Inꢀ
stead, oxidative hydrolysis of the B–H bonds of 4 occurs in the
presence of HOCl to yield the emissive naphthoimidazolium
derivative 3. The latter was chosen on the basis of our earlier
work, which showed in 2015 that 1,3ꢀdimethylꢀ1,3ꢀdihydroꢀ
Notwithstanding earlier advances, there is an unmet need
for analytical methods capable of measuring the concentraꢀ
tions of ROS and other analytes in specifically targeted orgaꢀ
nelles. For fluorescence imaging, this requires probes to be
transported into the specific organelle of a living system, and
21ꢀ24
to be inactive before their localization in that organelle.
The largest organelle, the endoplasmic reticulum (ER) is reꢀ
25,26
sponsible for essential metabolic functions in cells.
The
2
Hꢀnaphtho[2,3ꢀd]imidazoleꢀ2ꢀthione (6) is oxidized at sulfur
49
unique oxidizing conditions maintained within the ER, in parꢀ
ticular, are critical for its role in protein folding and disulfide
by HOCl to also give 3. The resulting increased emission at
50 nm established the suitability of the latter as a fluoresꢀ
4
27,28
bond formation.
The specific role of the different ER ROS
cence reporter. The new probe 4 is highly selective for HOCl,
overcomes the aggregation effects of the previously reported
5, and is suitable for the twoꢀphoton microscopic (TPM) imagꢀ
ing of endogenous HOCl generation in living cells and tissues.
Moreover, 4 exhibits outstanding ERꢀtargetability comparable
to that of commercial ER trackers, enabling the monitoring by
TPM of HOCl production within ER in cells under ER stress
conditions.
generated during oxidative protein folding processes and other
signaling mechanisms remains ambiguous, due to the lack of
2
9ꢀ31
ERꢀspecific probes for these species.
Thus, the preparation
of specific probes for ROS located in the ER is of great imꢀ
portance.
Aryl boronic acids and their esters have been broadly used
in the design of fluorescent sensors to detect and screen intraꢀ
32,33
34ꢀ41
cellular ROS,
such as hydrogen peroxide,
hypochlorite
ꢀ 42,43
ꢀ 44ꢀ47
(OCl )
and peroxynitrite (ONOO )
Their design relies
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Scheme 1. a) Synthesis of 4 and b) proposed mechanism
mixture was purified by flash chromatography (SiO
Page 2 of 7
, CH Cl )
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for the reaction of 4 with HOCl.
after evaporation of the volatiles to yield 4 as a white solid
1
(0.12 g, 23 %). H NMR (300 MHz, CDCl ) δ (ppm): 8.05ꢀ
3
8.00 (m, 2H), 7.83 (m, 2H), 7.58ꢀ7.52 (m, 2H), 4.07 (s, 6H),
1
3
1
.74ꢀ1.16 (q, JBꢀH = 90 Hz, 3H). C NMR (CDCl , 62.5 MHz):
3
11
δ 133.3, 130.7, 128.3, 125.8, 107.3, 32.5 (CꢀB not seen).
B
NMR (160.4 MHz, CDCl ): δ ꢀ36.9 (q, J = 90 Hz). FABꢀ
3
BꢀH
+
MS calcd. for C H BN [MꢀH] : 209.1245; found 209.1252.
13
14
2
Fluorescence Studies. A 1 mM stock solution of 4 was
prepared in acetonitrile (CH CN). To prepare a final concenꢀ
3
tration of 10 ꢁM, the stock solution of probe 4 was diluted
0
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with PBS (10 mM, pH = 7.4, 10% CH CN), and then an apꢀ
3
propriate aliquot of ROS or RNS solutions was added before
transfer to quartz cuvettes for spectroscopic measurements.
Samples were excited at 326 nm, with excitation and emission
slit widths of 3.0 nm for all fluorescence experiments.
Generation of ROS or RNS. H O , sodium hypochlorite
2
2
t
(NaOCl), and tertꢀButyl hydroperoxide ( BuOOH) were obꢀ
tained by dilution from commercial aqueous solutions. Hyꢀ
droperoxyl radicals (ROO•) were prepared from 2,2'ꢀazobis(2ꢀ
50
amidinopropane) dihydrochloride. Nitric oxide radical (•NO)
was generated from SNP (sodium nitroferricyanide (III) dihyꢀ
drate). The hydroxyl radical (•OH) was produced from the
reaction between iron (II) chloride (200 ꢁM) and H O (400
2
2
ꢀ
ꢁ
M). ONOO was prepared following reported procedures, and
its concentration was determined by spectrophotometry (ε
ꢀ1
ꢀ1
51
=
1670 M ·cm at 302 nm).
RESULTS AND DISCUSSION
The overall synthetic route of 4 is presented in Scheme 1a.
Specifically, the reaction of the naphthoimidazolium precursor
3
with a borane source (BH in THF) in the presence of a nonꢀ
3
nucleophilic base (NaHMDS) afforded 4 in 23% yield.
The spectral responses of 4 to ROS were examined by using
UVꢀVis absorption and fluorescence emission spectroscopy in
aqueous buffered solution (10 mM PBS containing 10 % of
EXPERIMENTAL SECTION
Materials and Chemicals. Solvents and chemicals used for
synthesis and purification were obtained from commercial
suppliers and used without further purification. Synthetic opꢀ
erations that required an inert atmosphere (where noted) were
conducted under a nitrogen atmosphere. H, B and C NMR
spectra were obtained with 300 MHz and 500 MHz Bruker
Avance spectrometers. Chemical shifts are reported as δ in
CH CN). As presented in Figure S1a, the absorbance band for
3
4
is centered at ca. 322 nm. This band is not shifted but beꢀ
1
11
13
comes broader with hypochromism upon the addition of 100
ꢀ
ꢁ
M OCl . The probe itself emits violetꢀblue light (Φ = 0.42) at
3
61 nm when excited at 326 nm. When the aqueous solution
ꢀ
1
13
of 4 was treated with different concentrations of OCl (0ꢀ100
ꢁM), the emission peak at 361 nm disappeared, and a cyan
fluorescence band at 450 nm grew concomitantly (Figure 1).
units of parts per million (ppm) vs. Si (CH ) ( H, C) or
4
4
11
BF ·OEt ( B), referenced to the residual solvent. Splitting
3
2
patterns are represented as s (singlet), d (doublet), t (triplet), q
quartet), m (multiplet), and br (broad). Electrospray ionizaꢀ
ꢀ
The UV and emission spectra of 4 after reaction with OCl
(
matched that of 3 (Φ = 0.29) (Figure S1).
tion (ESI) mass spectra were collected on an Agilent G6550A
QꢀTOF mass spectrometer at the Seoul branch of the Korean
Basic Science Institute. A Jeol JMS 700 high resolution mass
spectrometer was used to acquire FAB mass spectra at the
Korea Basic Science Institute (Daegu). UV absorption spectra
were collected on a UVIKON 933 doubleꢀbeam UVꢀvis specꢀ
trometer, and a RFꢀ5301/PC spectrofluorophotometer (Shiꢀ
madzu) was used to record the fluorescence emission spectra.
To explain the difference in the absorption and emission
spectra of 3 and 4, timeꢀdependent density functional theory
(TDꢀDFT) calculations were performed (Table S1 and Figure
S2). Vertical excitation energies for 3 and 4 were calculated to
account for the different absorption maxima in their UVꢀ
visible spectra. Naphthoimidazolium salt 3 shows longer tranꢀ
sition wavelength and lower oscillator strength than probe 4,
which agrees well with its longer absorption maximum and
lower absorbance in solution. Similarly, 3 shows a longer
emission maximum than 4. Both 3 and 4 show higher electron
density of LUMO on the imidazole ring than that of HOMO,
and the excitation can be characterized as a partial charge
transfer excitation from the naphthalene to the imidazole
moiety (Figure S2). Hence, the more apparent charge transfer
character of 3 explains its longer absorption and emission
wavelengths in solution than those of 4.
Synthesis of (1,3-dimethyl-1H-naphtho[2,3-d]imidazol-
ium-2-yl)trihydroborate(4). Sodium bis(trimethylsilyl)amide
(1 M in dry THF, 0.51 mL, 2.79 mmol) was added dropꢀwise
4
9
to a suspension of 3 (0.5 g, 2.53 mmol) in anhydrous THF
10 mL) at ꢀ78˚C. The resulting mixture was stirred for 1h at ꢀ
8˚C. Next, BH ·THF (1 M in THF, 0.24 mL, 2.79 mmol) was
(
7
3
added to the mixture. The resulting solution was stirred overꢀ
night and slowly warmed to ambient temperature. The crude
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Analytical Chemistry
The changes in the ratio between the intensity of the two
3
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emission bands (450 nm for 3 and 361 nm for 4) during the
addition of hypochlorite were recorded (Figure S3). The
F450/F361 ratio increased from 0.05 to 23.41 upon treatment of a
ꢀ
solution of 4 with 100 µM OCl , representing ca. 470ꢀfold
ratiometric signal response. The calculated detection limit of 4
20
ꢀ
for OCl was found to be 3.6 µM (Figure S4). The fluorescence
ꢀ
1
6
3
ratios for 4 before or after the addition of OCl remained stable
F
/
in the 2ꢀ10 pH range (Figure S5). The large changes in ratiꢀ
ometric response were deemed sufficient to apply 4 to the
monitoring of hypochlorite concentrations under physiological
conditions. Time courses experiments were performed on 4 by
recording the fluorescence intensity at 450 nm for 60 min
(Figure S6). The emission signal increased immediately, and
reached a maximum fluorescence at 450 nm within 1 min after
0
5
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F
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ꢀ
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
addition of either 30 ꢁM or 100 ꢁM OCl . The fluorescence
intensities remained largely unchanged over extended moniꢀ
toring time (up to 60 min). The stability of the probe and its
Figure 2. Fluorescence ratio (F450 /F361) of 4 (10 ꢁM) in PBS/
–
response to OCl are particularly suitable to monitor the genꢀ
MeCN (v/v, 9/1) toward 100 ꢁM of different analytes: 1) Blank,
ꢀ
eration of OCl in living systems.
ꢀ
t
ꢀ
2
) OCl , 3) H O , 4) •OH, 5) BuOOH, 6) •NO, 7) ONOO , 8)
2 2
•
ꢀ
1
+
+
2+
2+
ROO•, 9) O , 10) O , 11) Na , 12) K , 13) Ca , 14) Mg , 15)
Zn , 16) Al , 17) Cu , 18) Fe , 19) Fe and 20) Mn .
2
2
300
2+
3+
2+
2+
3+
2+
3
61 nm
48
According to our previous report, HOCl could trigger the
oxidative hydrolysis of NHC boranes by hydride abstraction at
the borane center to result in the formation of imidazolium
salts (Scheme 1b). ESIꢀMS analysis of the reaction mixture of
4 (10 µM) with OCl (100 ꢁM) was carried out to confirm this
hypothesis. The molecular ion attributed to 4 disappeared, and
2
1
00
00
-
ꢀ
1
00 ꢀM OCl
4
50 nm
+
0
was replaced by new a peak of m/z = 197 [M] , consistent with
3
(Figure S7).
The TPM images of 4ꢀlabeled Raw 264.7 cells appeared to
heterogeneously emit in the cells. To check the distribution of
the probe at subcellular levels, colocalization experiments of 4
were conducted with endoplasmic reticulum (ER), mitochonꢀ
dria and lysosome commercial organelle trackers using Raw
264.7 cells (Figure 3). The cells were pretreated with 10 ꢁM 4,
and then 1 ꢁM ERꢀTracker Red (or 1 ꢁM Mito/LysoꢀTracker
Red) for 30 min. The TPM image of 4 in Raw 264.7 cells
showed considerable overlap with the oneꢀphoton microscopic
4
00
500
600
Wavelength (nm)
Figure 1. Fluorescence spectra of 4 (10 ꢁM) upon addition of
OCl (0ꢀ100 ꢁM) in PBS/CH CN (v/v, 9/1, 10 mM PBS, pH 7.4).
Inset: Image of a 4 solution before (left) and after (right) addition
of OCl under UV irradiation.
ꢀ
3
ꢀ
(OPM) image of ER Tracker Red. By contrast, the distribution
Selectivity is a major issue in molecular sensing. . The seꢀ
lectivity of 4 toward biological relevant metal ions and
ROS/RNS species including OClꢀ, H2O2, •OH, tBuOOH,
of 4 was weakly correlated with that of mitochondria and lysoꢀ
some trackers. The Pearson's colocalization coefficient (A)
value of 4 with ER tracker is 0.92, which is much higher than
those with mitoꢀtracker red (A = 0.59) and lysoꢀtracker red (A
= 0.34). The result was confirmed by line profile analysis
(Figure 3). Similar results were observed in colocalization
experiments in HeLa and RKO cells between 4 and ER Trackꢀ
er Red (Figure S10).
•
NO, ONOOꢀ, ROO•, O2•ꢀ, and 1O2 were examined. As exꢀ
pected from earlier experiments with NHCꢀboranes,48 4 is
highly selective to OClꢀ over others ROS/RNS and metal ions
ꢀ
shown in Figure 2 Only OCl elicited a ratiometric signal reꢀ
sponse (F450 /F361), and other analytes displayed no interferꢀ
ence.
Generally, fluorescent sensors positioned in the ER region
have 1) a cationic character, 2) a moderate size in conjugated
band numbers (CBN < 40), and 3) an appropriate lipophilicity
Stimulated by the above promising outcomes in vitro, the
aptitude of 4 for sensing endogenous OCl in a ratiometric
ꢀ
52
manner by twoꢀphoton fluorescence microscopy was investiꢀ
gated. Probe 4 itself was first found to have low cytotoxicity to
living HeLa cells through standard MTT assays (Figure S8).
The fluorescence intensity of Raw 264.7 cells incubated with 4
10 ꢁM) showed high photostability during 1 h irradiation at
20 nm wavelength (Figure S9).
(+6 > log Poct > 0). Probe 4 possess a cationic character beꢀ
stowed by its imidazolium ring and a CBN of 16. The lipoꢀ
philicity of 4, determined by a partition experiment between
PBS buffer (10 mM, pH 7.4) and 1ꢀoctanol, was found to be
1.04 ± 0.02 (Table S2), placing it in the proper range for acꢀ
cumulation in the ER.
(
7
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intensity ratios (Fgreen/Fblue) were increased to 3.7 in both the
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Figure 3. TPM (a, e and i), OPM (b, f and j) and merged (c, g and
k) images of Raw 264.7 cells coꢀlabeled with 4 (10 ꢁM) and orꢀ
ganelle trackers (1.0 ꢁM). Line profile of fluorescence intensity
(d, h and l) obtained from corresponding cells images. λex for
TPM and OPM are 720 nm and 552 nm, respectively, and the
corresponding emissions were recorded at 380ꢀ550 nm (4) and
6
00ꢀ650 nm (organelle trackers). Scale bars = 20 ꢁm.
Figure 4. Pseudocolored ratiometric TPM images of Raw 264.7
cells labeled with 4 (10 ꢁM) for 30 min. (a) Control image. Cells
–
Next, to confirm that 4 responds to OCl in living cells, the
ꢀ
1
changes in average emission ratios (Fgreen/Fblue) of 4 following
incubation with NaOCl in Raw 264.7 cells were examined. As
the concentration of NaOCl increased (0ꢀ1 mM), the average
intensity ratios (Fgreen/Fblue) of 4 increased from 3.2 to 6.3
pretreated with (b) Tm (10 ꢁg mL , 16 h), (c) DTT (2 mM, 16 h),
ꢀ
1
ꢀ1
(
d) LPS (100 ng mL , 16 h), IFNꢀγ (50 ng mL , 4 h) and PMA
(10 nM, 30 min), (e) LPS, IFNꢀγ, PMA and 4ꢀABAH (50 ꢁM, 4
h) and (f) LPS, IFNꢀγ, PMA and FFA (50 ꢁM, 4h) and then treatꢀ
ed with 4. (g) Average ratios of Fgreen/Fblue in the TPM images.
(
Figure S11). The applicability of 4 for sensing endogenous
–
OCl in macrophages was then studied. Macrophages were
stimulated by lipopolysaccharides (LPS) and interferon gamꢀ
ma (IFNꢀγ). H O is produced and then converted into OCl by
Asterisks indicate statistical significance (***) for p <
0.001.Excitation wavelength for 4 is 720 nm and TPM images
were obtained at 450ꢀ600 nm (green) and 380ꢀ430 nm (blue).
Scale bars = 20 ꢁm.
–
2
2
the myeloperoxidase enzyme (MPO) when the cells are treated
5
3
with phorbol myristate acetate (PMA). In Figure 4, the averꢀ
age emission ratio (Fgreen/Fblue) of 4 increased to 4.0 in Raw
ꢀ
1
2
64.7 cells preꢀstimulated with LPS (100 ng mL ), IFNꢀγ (50
ꢀ1
ng mL ) and PMA (10 nM). Treatment of the cells with the
MPO inhibitors 4ꢀaminobenzoic acid hydrazide (4ꢀABAH, 50
µM) or flufenamic acid (FFA, 50 µM) resulted in average
intensity ratios (Fgreen/Fblue) undistinguishable from that of the
control, confirming the specificity of the response (Figure 4e
and f). Additionally, Raw 264.7 cells were incubated with
inducers or antagonists of oxidative stress to further confirm
that 4 selectively functions in the ER. Tunicamycin (Tm) is
known to increase oxidative stress of the ER, increasing H O2
2
generation; dithiothreitol (DTT), on the contrary, consumes
54
H O in the ER. When cells were pretreated with Tm (10 ꢁg
2
2
ꢀ
1
mL , Figure 4b), the average intensity ratio (Fgreen/Fblue) of 4
increased to its highest value, 5.8, whereas the average ratio
decreased to 2.7 upon the addition of DTT (2 mM, Figure 4c).
These outcomes reveal that 4 changes directly and mostly in
response to OCl– levels in the ER of live cells, confirming its
Figure 5. Pseudocolored ratiometric TPM images of rat hippoꢀ
campal slices labeled with 4 (100 ꢁM) for 1.5 h. TPM images of
ꢀ1
controls (a, b), and slices preꢀtreated with PMA (10 ng mL , 30
min) (d, e), before loading with 4. (c) Brightꢀfield image of the
CA1 and CA3 regions at the magnification of x10. (f) Average
intensity Fgreen/Fblue ratios in the TPM images. Asterisks indicate
statistical significance (***) for p < 0.001. Excitation wavelength
for 4 was 720 nm, and the TPM images were obtained at 450ꢀ600
nm (green) and 380ꢀ430 nm (blue). Scale bars = 50 ꢁm.
–
suitability for monitoring OCl by twoꢀphoton imaging miꢀ
croscopy.
ꢀ
Finally, the ability of 4 to detect OCl in fresh rat hippocamꢀ
pal slices was examined. In the hippocampal CA1 and CA3
regions, the average intensity ratios (Fgreen/Fblue) in the tissues
incubated with 4 (100 ꢁM) for 1.5 h were 3.3 and 3.1, respecꢀ
tively, values similar to those found in macrophages (Figure
5). By contrast, when hippocampal slices were preꢀincubated
CONCLUSIONS
The fluorogenic naphthoimidazolium borane 4 was successꢀ
fully developed into an ERꢀtargeted probe for the ratiometric
twoꢀphoton imaging of exogenous and endogenous HOCl in
live cells and tissues. Upon oxidation of the B–H bonds by
ꢀ
1
with the stressor PMA (10 ng mL ) for 30 min, the average
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HOCl and subsequent hydrolysis, it is converted to the emisꢀ
sive imidazolium salt 3, giving rise to a turnꢀon response at
450 nm. The probe localizes in the ER, is highly selective for
HOCl over other ROS, functions in a broad range of pH, and
is less susceptible to aggregation effects than previously reꢀ
ported NHCꢀborane probes for HOCl, which relied on the
modulation of pyrene excimer emission. These meritorious
properties portend to the potential value of 4 in the study of
ER signaling and function under oxidative stress.
8. Andina, D.; Leroux, J. C.; Luciani, P. Ratiometric Fluorescent
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Probes for the Detection of Reactive Oxygen Species. Chem. Eur.
J. 2017, 23, 13549ꢀ13573
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Lou, Z.; Li, P.; Han, K. RedoxꢀResponsive Fluorescent Probes
with Different Design Strategies. Acc. Chem. Res. 2015, 48,
1
358ꢀ1368
10. Hu, J. J.; Wong, N. K.; Gu, Q.; Bai, X.; Ye, S.; Yang, D. HKOClꢀ
Series of Green BODIPYꢀBased Fluorescent Probes for Hypoꢀ
2
chlorous Acid Detection and Imaging in Live Cells. Org. Lett.
2014, 16, 3544ꢀ3547
1
1. Yuan, L.; Wang, L.; Agrawalla, B. K.; Park, S. J.; Zhu, H.; Sivaꢀ
raman, B.; Peng, J.; Xu, Q. H.; Chang, Y. T. Development of
Targetable TwoꢀPhoton Fluorescent Probes to Image Hypoꢀ
chlorous Acid in Mitochondria and Lysosome in Live Cell and
Inflamed Mouse Model. J. Am. Chem. Soc. 2015, 137, 5930ꢀ
ASSOCIATED CONTENT
Supporting Information
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The Supporting Information is available free of charge on the
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12. Chen, X.; Lee, K.ꢀA.; Ren, X.; Ryu, J.ꢀC.; Kim, G.; Ryu, J.ꢀH.;
Lee, W.ꢀJ.; Yoon, J. Synthesis of highly HOClꢀselective fluoresꢀ
cent probe and its use for imaging HOCl in cells and organisms.
Nat. Protocols. 2016, 11, 1219ꢀ1228.
experimental details, H NMR, C NMR, ESIꢀMS, UV−Vis, and
fluorescence spectra (Figures S1ꢀS18; Tables S1ꢀS2) (PDF).
AUTHOR INFORMATION
Corresponding Author
1
3. Chen, S.; Lu, J.; Sun, C.; Ma, H. A highly specific ferroceneꢀ
based fluorescent probe for hypochlorous acid and its application
to cell imaging. Analyst. 2010, 135, 577ꢀ582.
*
*
*
Eꢀmail: kimhm@ajou.ac.kr. Phone: +82 (031) 219ꢀ2609
Eꢀmail: bouffard@ewha.ac.kr. Phone: +82 (02) 3277ꢀ3427
Eꢀmail: jyoon@ewha.ac.kr. Phone: +82 (02) 3277ꢀ2400
1
4. Kenmoku, S.; Urano, Y.; Kojima, H.; Nagano, T. Development of
a Highly Specific RhodamineꢀBased Fluorescence Probe for Hyꢀ
pochlorous Acid and Its Application to RealꢀTime Imaging of
Phagocytosis. J. Am. Chem. Soc. 2007, 129, 7313ꢀ7318.
ORCID
15. Chen, G.; Song, F.; Wang, J.; Yang, Z.; Sun, S.; Fan, J.; Qiang,
X.; Wang, X.; Dou, B.; Peng, X. FRET spectral unmixing: a ratiꢀ
ometric fluorescent nanoprobe for hypochlorite. Chem. Commun.
2012, 48, 2949ꢀ2951.
Hwan Myung Kim: 0000ꢀ0002ꢀ4112ꢀ9009
Jean Bouffard: 0000ꢀ0003ꢀ2281ꢀ2088
Juyoung Yoon: 0000ꢀ0002ꢀ1728ꢀ3970
1
6. Wang, B.; Chen, D.; Kambam, S.; Wang, F.; Wang, Y.; Zhang,
W.; Yin, J.; Chen, H.; Chen, X. A highly specific fluorescent
probe for hypochlorite based on fluorescein derivative and its enꢀ
dogenous imaging in living cells. Dyes Pigments. 2015, 120, 22ꢀ
Author Contributions
‡
These authors contributed equally
Notes
2
9.
The authors declare no competing financial interest.
1
7. Srikun, D.; Miller, E. W.; Domaille, D. W.; Chang, C. J. An ICTꢀ
Based Approach to Ratiometric Fluorescence Imaging of Hydroꢀ
gen Peroxide Produced in Living Cells. J. Am. Chem. Soc. 2008,
130, 4596ꢀ4597.
ACKNOWLEDGMENT
We thank the National Research Foundation of Korea for the
financial
support
(2012R1A3A2048814
to H. M.
to
J.
Y.;
and
18. Lou, Z.; Li, P.; Panab, Q.; Han, K. A reversible fluorescent probe
for detecting hypochloric acid in living cells and animals: utilizꢀ
ing a novel strategy for effectively modulating the fluorescence of
selenide and selenoxide. Chem. Commun., 2013, 49, 2445ꢀ2447.
19. Zhang, W.; Liu, W.; Li, P.; Kang, J.; Wang, J.; Wang, H.; Tang,
B. Reversible twoꢀphoton fluorescent probe for imaging of hypoꢀ
chlous acid in live cells and in vivo. Chem. Commun., 2015, 51,
2
2
016R1E1A1A02920873
018R1D1A1B07043568 to J. B). We also thank the Western
K.;
Seoul (LCꢀMS) and Daegu (FAB) branches of the Korea Basic
Science Institute for mass spectrometric data.
REFERENCES
1
0150ꢀ10153.
1
.
Winterbourn, C. C. Reconciling the chemistry and biology of
reactive oxygen species. Nat. Chem. Biol. 2008, 4, 278ꢀ286.
Breckwoldt, M. O.; Chen, J. W.; Stangenberg, L.; Aikawa, E.
Rodriguez, E.; Qiu, S.; Moskowitz, M. A.; Weissleder, R. Trackꢀ
ing the inflammatory response in stroke in vivo by sensing the
enzyme myeloperoxidase. Proc. Natl. Acad. Sci. U. S. A. 2008,
2
0. Li, P.; Zhang, W.; Li, K.; Liu, X.; Xiao, H.; Zhang, W.; Tang, B.
Mitochondriaꢀtargeted reactionꢀbased twoꢀphoton fluorescent
probe for imaging of superoxide anion in live cells and in vivo.
Anal. Chem. 2013, 85, 9877ꢀ9881
2
.
2
1. Xu, W.; Zeng, Z.; Jiang, JꢀH.; Chang, YꢀT.; Yuan, L. Discerning
the Chemistry in Individual organelles with SmallꢀMolecule Fluoꢀ
rescent Proves. Angew. Chem. Int. Ed. 2016, 55, 13658ꢀ13699.
1
05, 18584ꢀ18589.
3
.
Barnham, K. J.; Masters, C. L.; Bush, A. I. Neurodegenerative
22. Zhu, H.; Fan, J.; Du, J.; Peng, X. Fluorescent Probes for Sensing
diseases and oxidative stress. Nat. Rev. Drug Discov. 2004, 3,
and Imaging within Specific Cellular Organelles. Acc. Chem. Res.
2
05ꢀ214.
2
016, 49, 2115ꢀ2126.
4. Finkel, T.; Serrano, M.; Blasco, M. A. The common biology of
2
2
3. Satori, C. P.; Henderson, M. M.; Krautkramer, E. A.; Kostal, V.;
Distefano, M. M.; Arriaga, E. A. Bioanalysis of Eukaryotic Orgaꢀ
nelles. Chem. Rev. 2013, 113, 2733ꢀ2811.
cancer and ageing. Nature. 2007, 448, 767ꢀ774.
5
.
Chen, X.; Wang, F.; Hyun, J.; Wei, T.; Qiang, J.; Ren, X.; Shin, I.;
Yoon, J. Recent progress in the development of fluorescent, luꢀ
minescent and colorimetric probes for detection of reactive oxyꢀ
gen and nitrogen species. Chem. Soc. Rev. 2016, 45, 2976ꢀ3016
,
4. Zielonka, J.; Joseph, J.; Sikora, A.; Hardy M.; Ouari, O.;
VasquezꢀVivar, J.; Cheng, G.; Lopez, M.; Kalyanaraman, B. Miꢀ
tochondriaꢀTargeted TriphenylphosphoniumꢀBased Compounds:
Syntheses, Mechanisms of Action, and Therapeutic and Diagnosꢀ
tic Applications. Chem. Rev. 2017, 117, 10043ꢀ10120.
5. Ron, D.; Walter, P. Signal integration in the endoplasmic reticuꢀ
lum unfolded protein response. Nature Reviews Molecular Cell
Biology. 2007, 8, 516ꢀ529.
6. Chen, X.; Tian, X.; Shin, I.; Yoon, J. Fluorescent and luminescent
probes for detection of reactive oxygen and nitrogen species.
Chem. Soc. Rev. 2011, 40, 4783ꢀ4804
2
7
.
Ren, M.; Zhou, K.; He, L.; Lin, W. Mitochondria and lysosomeꢀ
targetable fluorescent probes for HOCl: recent advance and perꢀ
spectives. J. Mater. Chem. B. 2018, 6, 1716ꢀ1733
ACS Paragon Plus Environment

Analytical Chemistry
Page 6 of 7
2
2
6. Mandl, J.; Meszaros, T.; Banhegyi, G.; Hunyady, L.; Csala, M.
Endoplasmic reticulum: nutrient sensor in physiology and patholꢀ
ogy. Trends Endocrinol. Metab. 2009, 20, 194ꢀ201.
7. Gao, C.; Tian, Y.; Zhang, R.; Jing, J.; Zhang, X. Endoplasmic
ReticulumꢀDirected Ratiometric Fluorescent Probe for Quantitive
Detection of Basal H2O2. Anal. Chem. 2017, 89, 12945ꢀ12950.
42. Xu, Q.; Lee, K.ꢀA.; Lee, S.; Lee, K. M.; Lee, W.ꢀJ.; Yoon, J. A
Highly Specific Fluorescent Probe for Hypochlorous Acid and Its
Application in Imaging MicrobeꢀInduced HOCl Production. J.
Am. Chem. Soc. 2013, 135, 9944ꢀ9949.
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
43. Wang, Q.; Liu, C.; Chang, J.; Lu, Y.; He, S.; Zhao, L.; Zeng, X.
Novel water soluble styrylquinolinium boronic acid as
a
28. Schröder, M.; Kaufman, R. J. THE MAMMALIAN UNFOLDED
ratiometric reagent for the rapid detection of hypochlorite ion.
Dyes and Pig. 2013, 99, 733ꢀ739.
PROTEIN RESPONSE. Annual Review Biochemistry. 2005, 75,
7
39ꢀ789.
44. Sikora, J. Zielonka, M. Lopez, J. Joseph, Kalyanaraman, B. Diꢀ
rect oxidation of boronates by peroxynitrite : mechanism and imꢀ
plications in fluorescence imaging of peroxynitrite. Free Rad.
Biol. Med. 2009, 47, 1401ꢀ1407;
45. Zielonka, J.; Sikora, A.; Joseph, J.; Kalyanaraman, B. Peroxyniꢀ
trite is the major species formed from different flux ratios of coꢀ
generated nitiric oxide and superoxide : direct reason with boroꢀ
nateꢀbased fluorescent probe. J. Biol. Chem. 2010, 285, 14210ꢀ
14216.
46. Yu, F.; Song, P.; Li, P.; Wang, B.; Han, K. A fluorescent probe
directly detect peroxynitrite based on boronate oxidation and its
applications for fluorescence imaging in living cells. Analyst.
2012, 137, 3740ꢀ3749.
47. Sun, Z.; Xu, Q.; Kim, G.; Flower, S. E.; Lowe, J. P.; Yoon, J.;
Fossey, J. S.; Qian, X.; Bull, S. D.; James, T. D. A waterꢀsoluble
boronateꢀbased fluorescent probe for the selective detection of peꢀ
roxinitrite and imaging in living cells. Chem. Sci. 2014, 5, 3368ꢀ
3373.
48. Pak, Y. L.; Park, S. J.; Wu, D.; Cheon, B.; Kim, H. W.; Bouffard.
J.; Yoon, J. NꢀHeterocyclic Carbene Boranes as Reactive Oxygen
SpeciesꢀResponsive Materials: Application to the TwoꢀPhoton
Imaging of Hypochlorous Acid in Living Cells and Tissues. Anꢀ
gew. Chem. Int. Ed. 2018, 57, 1567ꢀ1571.
49. Xu, Q.; Heo, C. H.; Kim, G.; Lee, H. W.; Kim, H. M.; Yoon, J.
Development of imidazolineꢀ2ꢀthiones based twoꢀphoton fluoresꢀ
cence probes for imaging hypochlorite generation in a coꢀculture
system. Angew. Chem. Int. Ed. 2015, 54, 4890ꢀ4894.
50. Güçlü, K.; Kibrislioglu, G.; Özyürek, M.; Apak, M. Development
of a Fluorescent Probe for Measurement of Peroxyl Radical
Scavenging Activity in Biological Samples. J. Agric. Food
Chem., 2014, 62, 1839ꢀ1845.
51. Halliwell. P, Evans. M, Whiteman. Assessment of peroxynitrite
scavengers in vitro. Methods in Enzymology. 1999, 301, 333ꢀ342.
52. Colston, J.; Horobin, R, RashidꢀDoubell, F.; Pediani, J.; Johal, K.
K. Why fluorescent probes for endoplasmic reticulum are selecꢀ
tive: an experimental and QSARꢀmodelling study. Biotech. Histoꢀ
chem., 2003, 78, 323ꢀ332.
2
3
9. Tu, B. P.; Weissman, J. S. Oxidative protein folding in eukaryꢀ
otes: mechanisms and consequences. J. Cell Biol. 2004, 164, 341ꢀ
346.
0. Shimizu, Y.; Hendershot, l. M. Oxidative Folding: Cellular Stratꢀ
egies for Dealing with the Resultant Equimolar Production of Reꢀ
active Oxygen Species. Antioxidants & Redox Signalling. 2009,
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
1
1, 2317ꢀ2331.
31. Dickinson, B. C.; Chang, C. J. Chemistry and biology of reactive
oxygen species in signaling or stress responses. Nat Chem Biol.
2
011, 7, 504ꢀ511.
3
2. Guo, Z.; Shin, I.; Yoon, J. Recognition and sensing of various
species using boronic acid derivatives. Chem. Commun. 2012, 48,
5
956ꢀ5967.
33. Zielonka, J.; Sikora, A.; Hardy, M.; Joseph, J., Dranka, B. P.;
Kalyanaraman, B. Boronate Probes as Diagnostic Tools for Real
Time Monitoring of Peroxynitrite and Hydroperoxides. Chem Res
Toxicol. 2012. 25, 1793ꢀ1799.
3
3
3
3
4. Lo, L.ꢀC.; Chu, C.ꢀY. Developement of highly seletive and
sensitive probes for hydrogen peroxide. Chem. Commun. 2003,
2728ꢀ2729.
5. Chang, M.; Pralle, A.; Isacoff, E. Y.; Chang, C. J. A Selective,
CellꢀPermeable Optical Probe for Hydrogen Peroxide in Living
Cells. J. Am. Chem. Soc. 2004, 126, 15392ꢀ15393.
6. Miller, E. W.; Albers, A. E.; Pralle, A.; Isacoff, E. Y.; Chang, C.
J. BoronateꢀBased Fluorescent Probes for Imaging Cellular
Hydrogen Peroxide. J. Am. Chem. Soc. 2005, 127, 16652ꢀ16659.
7. KartonꢀLifshin, N.; Segal, E.; Omer, L.; Portnoy, M.; Satchiꢀ
Fainaro, R.; Shabat, D. A Unique Pradigm for a TurnꢀON Nearꢀ
Infrared CyanineꢀBased Probe: Noninvasive Intravital Optical
Imaging of Hydrogen Peroxide. J. Am. Chem. Soc. 2011, 133,
1
0960ꢀ10965.
3
8. Du, L.; Li, M.; Zheng, S.; Wang, B. Rational Design of a
Fluorescent Hydrogen Peroxide Probe Based on Umbelliferone
Fluorophore. Tetrahedron Lett. 2008, 49, 3045ꢀ3048.
39. Michalski, R.; Zielonka, J.; Gapys, E.; Marcinek, A.; Joseph, J.;
Kalyanaraman, B. Realꢀtime Measurements of Amino Acid and
Protein Hydroperoxides Using Coumarin Boronic Acid. J. Biol.
Chem. 2014, 289, 22536ꢀ22553.
53. Spalteholz, H.; Panasenko, O. M.; Arnhold, J. Formation of reacꢀ
tive halide species by myeloperoxidase and eosinophil peroxiꢀ
dase. Arch. Biochem. Biophys. 2006, 445, 225ꢀ234.
54. Wu, R.ꢀF.; Ma, Z.; Liu, Z.; Terada, L. S. Nox4ꢀderived H2O2
mediates endoplasmic reticulum signaling through local Ras actiꢀ
vation. Mol. Cell. Biol. 2010, 30, 3553ꢀ3568.
4
0. Hanna, R. D.; Naro, Y.; Dieters, A.; Floreancig, P. E. Alcohol,
Aldehyde, and Ketone Liberation and Intracellular Cargo Release
through PeroxideꢀMediated αꢀBoryl Ether Fragmentation. J. Am.
Chem. Soc. 2016, 138, 13353ꢀ13360.
1. Li, H.; Yao, Q.; Fan, J.; Du, J.; Wang, J.; Peng, X. A twoꢀphoton
NIRꢀtoꢀNIR fluorescent probe for imaging hydrogen peroxide in
living cells. Biosens. Bioelectron. 2017, 94, 536ꢀ543.
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