Development of a highly sensitive fluorescence probe for hydrogen peroxide

Article abstract of DOI:10.1021/ja203521e

Hydrogen peroxide is believed to play a role in cellular signal transduction by reversible oxidation of proteins. Here, we report the design and synthesis of a novel fluorescence probe for hydrogen peroxide, utilizing a photoinduced electron transfer strategy based on benzil chemistry to control the fluorescence. The practical value of this highly sensitive and selective fluorescence probe, NBzF, was confirmed by its application to imaging of hydrogen peroxide generation in live RAW 264.7 macrophages. NBzF was also employed for live cell imaging of hydrogen peroxide generated as a signaling molecule in A431 human epidermoid carcinoma cells.

Full text of DOI:10.1021/ja203521e

ARTICLE
pubs.acs.org/JACS
Development of a Highly Sensitive Fluorescence Probe for
Hydrogen Peroxide
Masahiro Abo,†,§ Yasuteru Urano,‡ Kenjiro Hanaoka,†,§ Takuya Terai,†,§ Toru Komatsu,†,§ and
Tetsuo Nagano*,†,§
†Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
‡Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
§CREST, JST, Sanbancho-blg, 5 Sanbancho, Chiyoda-ku, Tokyo, 102-0075, Japan
S Supporting Information
b
ABSTRACT: Hydrogen peroxide is believed to play a role in
cellular signal transduction by reversible oxidation of proteins.
Here, we report the design and synthesis of a novel fluorescence
probe for hydrogen peroxide, utilizing a photoinduced electron
transfer strategy based on benzil chemistry to control the
fluorescence. The practical value of this highly sensitive and
selective fluorescence probe, NBzF, was confirmed by its
application to imaging of hydrogen peroxide generation in live
RAW 264.7 macrophages. NBzF was also employed for live cell imaging of hydrogen peroxide generated as a signaling molecule in
A431 human epidermoid carcinoma cells.
’ INTRODUCTION
background levels are generally not satisfactory for biological
applications.16 These probes are also reported to show a fluorescence
increment when they are exposed to nitric oxide, in addition to
hydrogen peroxide. In this manuscript, we describe a new type of
fluorescence probe with high sensitivity and selectivity for hydrogen
peroxide, developed by utilizing the chemical properties of benzil
(R-dibenzoyl) in combination with one of our rational probe design
strategies, i.e., photoinduced electron transfer (PeT).17
As a reactive moiety for hydrogen peroxide, we focused on
benzil, because it has unique reactivity with hydrogen peroxide.
Sawaki et al. have reported that benzil is transformed to benzoic
anhydride through a BaeyerÀVilliger type reaction with hydrogen
peroxide in alkaline organic solvents, and this is followed by hydro-
lysis to give benzoic acid (Scheme 1).18 Though the reported
conditions were far from biological in terms of basicity, solvents,
and concentration of reagents, we expected that the reactivity bet-
ween benzil and hydrogen peroxide could be adjusted by modifica-
tion of the benzene ring of benzil. At the same time, we chose the
photoinduced electron transfer mechanism as a strategy for control-
ling fluorescence, among various possible mechanisms, for the
following reasons. We found by means of cyclic voltammetry that
benzil has a high reduction potential of no less than À1.1 V vs SCE in
acetonitrile. We previously examined the relationship between the
fluorescence quantum efficiency of fluorescein derivatives and the
reduction potential of the fluorescein benzene moiety and found that
fluorescein derivatives whose benzene moiety has a higher reduction
potential than À1.8 V vs SCE show almost no fluorescence owing to
Reactive oxygen species (ROS) are a class of radical or nonradical
oxygen-containing molecules that show high reactivity to biomolec-
ules.1 Excessive ROS generation is involved in the pathogenesis of
many diseases, including cardiovascular disease,2 cancer,3 and neu-
rological disorders.4 However, the physiological level of ROS is
believed to be indispensable in regulating diverse cellular processes,
such as an immune response5 and cell signaling.6 Although genera-
tion of ROS is often simply regarded as representing oxidative stress,
each ROS has unique chemical characteristics in terms of chemical
reactivity and lifetime in aqueous solution and, therefore, may play a
distinct role in biological systems.7 Hydrogen peroxide (H2O2)
exhibits relatively mild reactivity among ROS and has attracted
intense interest recently, because it appears to be involved in signal
transduction by reversible oxidation of proteins, such as phospha-
tases and thioredoxins, in a tightly regulated manner.8À11 For
example, Capasso et al. reported that oxidative inhibition of tyrosine
phosphatase SHP-1 is required for the activation of B cells through a
B cell antigen receptor in vivo.9 Sensitive and selective detection
methods for hydrogen peroxide should be powerful tools for study-
ing such biological phenomena.
Fluorescence sensor probes have been widely used for detecting
ROS in living cells. For example, reduced fluorescent dyes such as
dichlorodihydrofluorescein are commonly used as fluorescence
probes for ROS. However, they have critical drawbacks for selective
sensing of hydrogen peroxide; namely, they are nonspecific among
ROS and also undergo autoxidation upon light irradiation.12 On the
other hand, some fluorescence probes specific for hydrogen peroxide
have been reported, based on benzenesulfonyl ester or boronate
ester chemistry,13À15 but their reaction rates and fluorescence
Received: April 17, 2011
Published: June 21, 2011
r
2011 American Chemical Society
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Scheme 1. Proposed Reaction Mechanism between Benzil and Hydrogen Peroxide
Scheme 2. Design of Fluorescence Probes for Hydrogen
Peroxide
Evaluation of the Response to Hydrogen Peroxide and
Other Reactive Oxygen Species in Vitro. At the beginning of
our investigation, we synthesized 5-benzoylcarbonylfluorescein
(1) and evaluated its reactivity with hydrogen peroxide by
measuring the fluorescence increment after the reaction. Com-
pound 1 showed a small fluorescence increment after incubation
with 100 μM hydrogen peroxide for 1 h in 0.1 M sodium
phosphate buffer at pH 7.4 (Figure 1). We considered this result
to be promising because it indicated that the reaction of benzil
and hydrogen peroxide could occur under physiological condi-
tions. However, the reactivity of 1 was rather low and further
optimization was needed to obtain a compound exhibiting
sufficient reactivity with hydrogen peroxide to be practically
usable in biological systems. For this purpose, we synthesized
derivatives with various electron-donating or -withdrawing sub-
stituents and evaluated them under the same conditions as those
used for 1. Compound 2, which has a moderately electron-
withdrawing substituent, bromine, showed a larger fluorescence
increment than 1. On the other hand, compound 3 with an
electron-donating methoxy group showed almost no fluores-
cence increment under these conditions. These results led us to
introduce strongly electron-withdrawing substituents, such as
cyano and nitro groups, into benzil. Finally, we found that
derivatives with strongly electron-withdrawing groups, com-
pound 4 with a cyano group and NBzF with a nitro group,
showed a very large fluorescence increment. Further evaluation
was performed with these two compounds. Compound 4 and NBzF
showed a rapid fluorescence increase even at low concentrations of
hydrogen peroxide (Figure 2). By fitting the data with a pseudo-first-
order model (1 μM dye and 1 mM hydrogen peroxide), the reaction
rate constants were obtained as 3.4 Â 10À3 sÀ1 and 4.2 Â 10À3 sÀ1
for 4 and NBzF, respectively, at 37 °C (Table 2). These values are
comparable to those of previously reported fluorescence probes for
hydrogen peroxide based on boronate chemistry.15 Though the
reactivities of 4 and NBzF with hydrogen peroxide were almost the
same, NBzF showed a much higher signal-to-noise ratio than did 4,
because of its extremely low fluorescence quantum efficiency before
reaction with hydrogen peroxide. Indeed, the enhancement of
fluorescence intensity after reaction with hydrogen peroxide was as
high as 150-fold (Figure 3). This fluorescence enhancement ratio is
superior to that of any previously reported fluorescence probe for
hydrogen peroxide. For example, two boronate-based fluorescence
probes for hydrogen peroxide, Peroxy Crimson 1 and Peroxyfluor-3,
are reported to show approximately 40-fold fluorescence enhance-
ment upon addition of hydrogen peroxide.14,15 The generation of
5-carboxyfluorescein after the reaction of NBzF with hydrogen
peroxide was also confirmed by HPLC analysis (Figure 4). Further-
more, we monitored the conversion of NBzF to 5-carboxyfluorescein
(5-CF) and 4-nitrobenzoic acid (4-NO2BA) upon reaction with
fluorescence quenching through the donor-excited photoinduced
electron transfer (d-PeT) process.19 The high reduction potential of
benzil indicates that benzil has a low enough LUMO energy level to
accept an electron from an excited fluorophore and, thus, should
work as a fluorescence quenching moiety in the d-PeT process, if it is
located sufficiently close to the fluorophore. As a fluorophore scaf-
fold, we selected fluorescein, which is widely used for fluorescence
bioimaging because of its biocompatibility and excellent photoche-
mical properties, such as excitation and emission wavelengths in
the visible region and high fluorescence quantum efficiency.20,21
Thus, we designed 5-benzoylcarbonylfluorescein derivatives as can-
didate fluorescence probes for hydrogen peroxide (Scheme 2). The
design is based on the idea that these molecules have low fluores-
cence quantum efficiency, owing to quenching of fluorescence via
the intramolecular d-PeT process, before reaction with hydrogen
peroxide, but are converted to 5-carboxyfluorescein, which is highly
fluorescent, by reaction with hydrogen peroxide.
’ RESULTS AND DISCUSSION
Synthesis and Photochemical Properties of 5-Benzoylcar-
bonylfluoresceins. We synthesized five derivatives with various
electron-donating or -withdrawing substituents on the benzil moiety
(Scheme 3). All of them showed similar photochemical properties
to those of fluorescein, except for low fluorescence quantum
efficiency owing to efficient quenching via the d-PeT process
(Table 1). Among these compounds, NBzF showed extremely
weak fluorescence owing to the presence of the nitro group, which
lowers the LUMO energy level of benzil, and it is considered that the
background fluorescence due to unreacted molecules would be
sufficiently low to permit effective bioimaging.
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Scheme 3. Synthesis of 5-Benzoylcarbonylfluorescein Derivativesa
a Reagents and conditions: (a) ethynylbenzenes, PdCl2(PPh3)2, triethylamine, THF; (b) (i) PdCl2, DMSO, (ii) K2CO3, MeOH for 1À4 or (i) K2CO3,
MeOH (ii) PdCl2, DMSO for NBzF.
superoxide (O2À•), hydroxyl radical (•OH), hypochlorite (ÀOCl),
Table 1. Photochemical Properties of 1À4 and NBzF in 0.1
nitric oxide (•NO), or singlet oxygen (1O2). Small fluorescence
M Sodium Phosphate Buffer at pH 7.4
enhancements were observed with peroxynitrite (ONOOÀ) andtert-
butyl hydroperoxide (TBHP), which afforded 5-carboxyfluorescein
Fluorescence
Absorbance
max
Extinction
Emission
max
quantum
efficiency
Φfl
as a product (Figure S9, Supporting Information). Compared with
previously reported fluorescence probes utilizing benzenesulfonyl
ester or boronate ester chemistry, the selectivity of NBzF among ROS
is unique, because it is unresponsive to nitric oxide. We also inves-
tigated the fluorescence response of compounds 1À4to various ROS
(Figure S11, Supporting Information). The selectivity of 4was similar
to that of NBzF; it showed a large fluorescence increment with hydro-
gen peroxide and small increments with peroxynitrite and tert-butyl
hydroperoxide. Compared with 4 and NBzF, compound 2 showed
small fluorescence increments with hydrogen peroxide and perox-
ynitrite, exhibiting relatively poor selectivity for hydrogen peroxide.
Compounds 1and 3showed very small fluorescence changes, regard-
less of the ROS that was added. Taking the reaction mechanism into
account, it is reasonable that the chemical reactivities of peroxynitrite
and tert-butyl hydroperoxide are similar to that of hydrogen peroxide,
because all of them have a hydroperoxide structure. However, the
concentration of peroxynitrite in living organisms is thought to be far
lower than that of hydrogen peroxide, because peroxynitrite has a very
short lifetime under physiological conditions owing to its high reac-
tivity with cellular antioxidants.7 In the case of the fluorescence incre-
ment due to tert-butyl hydroperoxide, the rate of 5-carboxyfluorescein
formation was approximately 7 times slower than that with hydrogen
peroxide. This result indicates that NBzF is potentially responsive to
alkyl hydroperoxides present in living organisms, such as lipid hydro-
peroxides. However, the concentration of lipid hydroperoxides in
plasma and erythrocytes of healthy human subjects is reported to be
less than 1 μM, and the interference from lipid hydroperoxides at this
level of concentration would be minimal.23 In addition, NBzF may
not react readily with lipid hydroperoxides in living cells, because
NBzF is distributed mainly in the cytosolic environment owing to
its hydrophilic fluorescein scaffold, while alkyl peroxides exist in
the hydrophobic membrane environment.24 Thus, although NBzF
showed relatively small fluorescence enhancements in response to
peroxynitrite and tert-butyl hydroperoxide in vitro, we expected that it
would have a high selectivity for hydrogen peroxide in living or-
coefficient ε
(MÀ1 cmÀ1
Compound
(nm)
)
(nm)
1
494
493
494
493
495
7.8 Â 104
7.8 Â 104
6.9 Â 104
7.2 Â 104
6.8 Â 104
520
518
518
518
519
0.023
0.022
0.026
0.060
0.004
2
3
4
NBzF
Figure 1. Fluorescence increment of the compounds after reaction with
hydrogen peroxide. Compounds (10 μM) were incubated with hydrogen
peroxide (100 μM) at 37 °C for 1 h in 0.1 M sodium phosphate buffer at pH
7.4. σp is the para substituent constant of the Hammett equation.22
Fluorescence increase at 520 nm was measured with excitation at 490 nm.
hydrogen peroxide by means of NMR spectroscopy (Figures 5 and
S12, Supporting Information). The reaction was performed with
2 mM NBzF and 20 mM hydrogen peroxide in DMSO-d6 containing
20% 0.1 M sodium phosphate buffer at pH 7.4. No major product
other than 5-carboxyfluorescein and 4-nitrobenzoic acid was detected
after incubation for 30 min. In the NMR spectra of the reaction
mixture, signals of a benzoic anhydride intermediate were not
observed at any time point, which indicates that the intermediate
would be hydrolyzed immediately under such neutral aqueous
conditions. We further examined the reactivity of NBzF toward
various ROS by means of fluorescence spectrometry (Figure 6).
NBzF did not show any fluorescence enhancement in response to
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Figure 2. Fluorescence response of4 and NBzF upon addition of hydrogen peroxide. The indicated concentration of hydrogen peroxide was added at 60 s, and
the mixture was incubated at 37 °C in 0.1 M sodium phosphate buffer at pH 7.4. Fluorescence intensity at 520 nm was measured with excitation at 490 nm.
Live Cell Imaging of Hydrogen Peroxide Generated in
A431 Human Epidermoid Carcinoma Cells by Using NBzF.
Recently, hydrogen peroxide has emerged as an important signaling
molecule for cellular signal transduction following stimulation of
cells, e.g., with cytokines, growth factors, and hormones.26 For
example, A431 human epidermoid carcinoma cells are thought to
utilize hydrogen peroxide as a signal transducer following epidermal
growth factor (EGF) stimulation.27 It is reported that EGF-stimu-
lated A431 cells transiently generate hydrogen peroxide by activating
Nox, leading to inhibition of protein-tyrosine phosphatase 1B by
reversible oxidation of the protein and thus strengthening the
phosphorylation signal from the EGF receptor.28 So, we investigated
whether NBzF could enable visualization of hydrogen peroxide
generated as a signaling molecule in A431 cells. Cultured A431 cells
were stained with 5 μM NBzFDA for 10 min at 37 °C and then
stimulated with 500 ng/mL of EGF. Images were taken for 30 min by
conventional fluorescence microscopy. Time-lapse imaging revealed
that A431 cells stimulated with EGF showed a rapid intracellular
fluorescence increment, within 20 min, while control cells showed
only a slight fluorescence change (Figure 8). This time scale of
fluorescence increment is consistent with the literature; it was
reported that EGF stimulation induces temporary production of
hydrogen peroxide in A431 cells.27 The fluorescence increment was
suppressed by inhibitors of Nox and hydrogen peroxide, supporting
the idea that NBzF can detect endogenous hydrogen peroxide in
A431 cells (Figure 9). The role of hydrogen peroxide as a signaling
molecule has recently attracted enormous interest in biological
studies. Our present results suggest that fluorescence imaging with
NBzF would offer a powerful detection technique for biological
investigations of the involvement of hydrogen peroxide in cellular
signal transduction, because NBzF has a much higher reactivity
toward hydrogen peroxide as compared with dichlorodihydrofluor-
escein, the most commonly used indicator for ROS, including
hydrogen peroxide (Figure S13, Supporting Information).
Table 2. Observed Reaction Rate Constants (k) of 4 and
NBzF with Hydrogen Peroxide at 25 and 37 °Ca
k ( Â 10À3 sÀ1
)
Compound
25 °C
37 °C
4
1.8
3.2
3.4
4.2
NBzF
a The reaction was performed with 1 μM compound and 1 mM hydrogen
peroxide in 0.1 M sodium phosphate buffer at pH 7.4.
ganisms. Furthermore, it is possible to distinguish hydrogen peroxide
from peroxynitrite by using combinations of inhibitors.
Application of NBzF to Live Cell Imaging Using RAW 264.7
Macrophages. To confirm the practical usefulness of NBzF, we
applied diacetylated NBzF (NBzFDA), a membrane-permeable pre-
cursor that is hydrolyzed to NBzF by intracellular esterases, for the
imaging of hydrogen peroxide generation in living RAW 264.7
macrophages. These macrophages produce superoxide upon stimula-
tion with phorbol-12-myristate-13-acetate (PMA),25 and it is well-
known that superoxide is degraded to hydrogen peroxide by super-
oxide dismutase or by spontaneous dismutation. In this experiment,
RAW 264.7 macrophages were stimulated with 1 μg/mL PMA for
20 min and then loaded with NBzFDA for 20 min. PMA-stimulated
RAW 264.7 macrophages formed endosomes, which displayed high
levels of fluorescence intensity, while macrophages not stimulated with
PMA showed only weak intracellular fluorescence (Figure 7A,B). The
fluorescence increment in the endosomes was suppressed by pre-
treatment with apocynin (5 mM), which is an inhibitor of NADPH
oxidase (Nox), or by ebselen (5 μM), which is a mimic of glutathione
peroxidase (Figure 7C,D). L-NAME (5 mM), which inhibits the
production of nitric oxide by nitric oxide synthase and the subsequent
conversion of nitric oxide to peroxynitrite, had little effect on the
fluorescence intensity (Figure 7E). Figure 7F shows the averaged
fluorescence intensities of the endosomes in the presence or absence
of the inhibitors. None of the inhibitors had much influence on the
morphological change of the macrophages following PMA stimula-
tion, as judged from bright-field images. Taken together, these results
indicate that NBzF is able to visualize endogenous hydrogen peroxide
generation in RAW 264.7 macrophages. The background fluorescence
was low, and we confirmed that NBzF showed little fluorescence
enhancement due to spontaneous decomposition or autoxidation
upon light irradiation under the conditions of this experiment.
’ CONCLUSION
We designed 5-benzoylcarbonylfluorescein derivatives as can-
didate fluorescence probes for the detection of hydrogen per-
oxide by utilizing the unique chemical reactivity of benzil and
hydrogen peroxide, together with the d-PeT mechanism to
control the fluorescence. Among the compounds synthesized,
NBzF afforded the highly sensitive detection of hydrogen per-
oxide, owing to its extremely low background fluorescence, with a
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Figure 3. Absorbance and fluorescence spectra of NBzF (10 μM) after incubation with the indicated concentration of hydrogen peroxide. The reaction
was performed at 37 °C for 60 min in 0.1 M sodium phosphate buffer at pH 7.4. Fluorescence spectra were measured with excitation at 490 nm.
Figure 4. HPLC chromatograms of NBzF (10 μM) before and after
reaction with hydrogen peroxide. The reaction was performed with 100 μM
hydrogen peroxide at 25 °C for 50 min in 0.1 M sodium phosphate buffer
at pH 7.4. The chromatogram of 5-carboxyfluorescein (5-CF,
10 μM) is also shown. Elution was done with a linear gradient (eluent,
0 min, 8% CH3CN/0.1% TFA aq. ∼20 min, 80% CH3CN/0.1% TFA
aq.; flow rate = 1.0 mL/min). Absorbance at 490 nm was detected.
Fluorescein was used as an internal standard (peak: 10 min).
good reaction rate and high specificity. We confirmed the
practical value of NBzF by using it to detect endogenous
hydrogen peroxide generation in living RAW 264.7 macrophages
and A431 human epidermoid carcinoma cells. This fluorescence
probe offers the highest fluorescence enhancement ratio cur-
rently available for hydrogen peroxide detection and should be
Figure 5. Monitoring of reaction between NBzF and hydrogen peroxide by
means of NMR spectroscopy. The reaction was performed with 2 mM NBzF
and 20 mM hydrogen peroxide at 25 °C for the indicated time period in
DMSO-d6 containing 20% 0.1 M sodium phosphate buffer (PBS) at pH 7.4.
practically useful as a tool for the study of redox biology. Though
indicators for hydrogen peroxide utilizing fluorescent proteins
have also been reported,29 our chemical probe provides an
alternative methodology with advantages such as ease of use
and high selectivity among ROS. Further, NBzF should be
suitable for cell-based high-throughput screening of inhibitors
’ EXPERIMENTAL SECTION
of ROS-producing enzymes, such as NADPH oxidase, providing
high screening reliability owing to its high signal-to-noise ratio.30
The design strategy employed here should be applicable to not
only fluorescein derivatives but also other fluorophores, such as
rhodamines, boron dipyrromethenes (BODIPYs), and calceins,
which would allow control of excitation and emission wave-
lengths, leakage from cells, and localization to organelles.31
Materials. General chemicals were of the best grade available, supplied
by Tokyo Chemical Industries, Wako Pure Chemical, Daiichi Chemical Co.,
or Aldrich Chemical Co., and were used without further purification. All
solvents were used after appropriate distillation or purification.
Instruments. NMR spectra were recorded on a JEOL JNM-LA300
instrument or a JEOL JNM-LA 400. Mass spectra were measured with a
JEOL JMS-T 100LC AccuToF. UVÀvisible spectra were obtained on a
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JASCO V-550. Fluorescence spectroscopic studies were performed on a
JASCO FP-6500.
ROS Generating System. The concentration of H2O2 was
determined from the absorption at 240 nm (ε = 43.6 MÀ1 cmÀ1). To
a solution of dye (10 μM) in 0.1 M sodium phosphate buffer at pH
7.4 containing 0.1% DMF as a cosolvent, H2O2 was added at 37 °C, and the
spectrum was measured 60 min later. The concentration of ONOOÀ was
determined from the absorption at 302 nm (ε = 1670 MÀ1 cmÀ1) in 0.1 N
NaOH aq. To a solution of dye (10 μM) in 0.1 M sodium phosphate buffer
at pH 7.4 containing 0.1% DMF as a cosolvent, ONOOÀ was added at
25 °C, and the spectrum was measured 5 min later. The concentration of
ÀOCl was determined from the absorption at 292 nm (ε = 350 MÀ1 cmÀ1).
To a solution of dye (10 μM) in 0.1 M sodium phosphate buffer at pH
7.4 containing 0.1% DMF as a cosolvent, ÀOCl was added at 25 °C, and the
spectrum was measured 5 min later. •OH was generated at 25 °C by means
of the Fenton reaction with H2O2 at the concentration indicated in
Figure 2D and a 10-fold excess of Fe(ClO4)2. tert-Butyl hydroperoxide
was added at 37 °C to a solution of dye (10 μM) in 0.1 M sodium phosphate
buffer at pH 7.4 containing 0.1% DMF as a cosolvent, and the spectrum was
measured 60 min later. Nitric oxide was generated from NOC 13 and singlet
oxygen was generated from EP-1, which was added at 37 °C to a solution of
dye (10 μM) in 0.1 M sodium phosphate buffer at pH 7.4 containing 0.1%
DMF as a cosolvent; the spectrum was measured 30 min later.
HPX/Xanthine Oxidase System. Xanthine oxidase (x.o.) from
bovine milk, superoxide dismutase (SOD) from bovine erythrocytes,
and catalase from bovine liver were purchased from Sigma. Hypox-
anthine (HPX) was dissolved in Milli-Q water as a 1.0 mM solution. The
generation of superoxide was determined by measuring the reduction of
cytochrome c (ε550 = 19 500 MÀ1 cmÀ1). HPX was added at 25 °C to a
solution of dye (10 μM), xanthine oxidase (23.6 munit/L), and catalase
(25 μg protein/mL), and the spectrum was measured 20 min later.
Cell Culture. RAW 264.7 macrophages and A431 cell line were
purchased from the RIKEN cell bank. The cells were grown in DMEM
(GIBCO) containing 10% heat-inactivated fetal bovine serum
(GIBCO), 100 U/mL of penicillin, and 100 μg/mL streptomycin at
37 °C in humidified air containing 5% CO2.
HPLC Analysis. HPLC analysis was performed on an Inertsil ODS-
3 column (4.6 mm  250 mm; GL Sciences Inc.) using an HPLC system
composed of a pump (PU-980, JASCO) and a detector (MD-2015 or
FP-2025, JASCO). Preparative HPLC was performed on an HPLC
system composed of a pump (PU-2080, JASCO) and a detector (MD-
2015, JASCO), with an Inertsil ODS-3 column (10.0 mm mm  250
mm; GL Sciences Inc.).
Fluorometric Analysis. Relative fluorescence quantum efficien-
cies of dyes were obtained by comparing the area under the emission
spectrum of the test sample excited at 490 nm with that of a solution of
fluorescein in 0.1 N NaOH, which has a quantum efficiency of 0.85.
Fluorescence Microscopy. Confocal fluorescence imaging stud-
ies were performed with a TCS SP5 (Leica Microsystems) equipped
with a 63Â oil-immersion objective lens and a white-light laser, using a
Leica Application Suite Advanced Fluorescence (LAS-AF). NBzF-loaded
cells were excited at 495 nm, and emission was collected between 505
and 540 nm. PMT; Gain, 700 V; Offset 0%. Conventional fluorescence
images were captured using an Olympus IX 71 equipped with a cooled
CCF camera (Coolsnap HQ, Olympus) and a xenon lamp (AH2RX-T,
Olympus) with a BP-470-490 excitation filter and a BA-510-550
emission filter.
Live Cell Imaging of RAW 264.7 Cells and Statistical Analysis.
RAW 264.7 cells were planted at 5 Â 104 cells/mL on noncoated glass-
bottomed dishes and cultured in DMEM containing 10% heat-inactivated
fetal bovine serum (GIBCO), 100 U/mL of penicillin, and 100 μg/mL
streptomycin at 37 °C in humidified air containing 5% CO2 for 2 days.
The cells were treated with inhibitors in HBSS for 20 min, activated with
Figure 6. Response of NBzF to various ROS. Fluorescence intensities
at 520 nm with excitation at 490 nm are shown. For details; see Figure
S1ÀS8, Supporting Information.
Figure 7. Live cell imaging of RAW 264.7 macrophages stimulated by PMA. RAW 264.7 cells were stimulated for 20 min in the presence or absence of
inhibitors, and NBzFDA was loaded for 20 min. (A) Without PMA stimulation. (B) PMA (1 μg/mL) stimulation only. (C) PMA stimulation with 5 mM
apocynin. (D) PMA stimulation with 5 μM ebselen. (E) PMA stimulation with 5 mM L-NAME. (F) Fluorescence intensities of endosomes were
averaged. Statistical analyses were performed with Student’s t-test (n = 4). *P < 0.01, **P < 0.001 and error bars are ( s.d.
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Figure 8. Time-lapse imaging of A431 cells stimulated by EGF. EGF stimulation started at 0 min. Scale bars are 50 μm.
Figure 9. Live cell imaging of A431 cells with inhibitors. NBzFDA (5 μM) and inhibitors were loaded on A431 cells at 37 °C for 10 min, and then cells
were stimulated by 500 ng/mL of EGF for 30 min. Scale bars are 50 μm.
1 μg/mL of PMA, and stained with NBzFDA for 20 min at 37 °C in
humidified air containing 5% CO2. The cells were washed by HBSS once,
and images were taken. For statistical analysis, fluorescence images were
taken every ca. 1 μmalongthezaxis, affording 10 images over each cell body
in a field. Fluorescence intensities of endosomes in all 10 images per cell
were averaged.
1H, J = 7.98, 0.64 Hz), 7.11 (dd, 2H, J = 1.83, 0.73 Hz), 6.84 (m, 4H).
13C NMR (75 MHz, CDCl3) δ 168.8, 168.2, 152.1, 151.5, 138.2, 131.8,
128.9, 128.5, 128.0, 126.6, 125.8, 124.1, 122.3, 117.8, 116.1, 110.5, 91.9,
87.3, 81.8, 76.6, 21.1. HRMS (ESI+) Calcd for [M + H], 517.12873;
Found, 517.12477 (À3.95 mmu).
6, 7, 8, and 9 were similarly prepared from 4-bromoethynylbenzene,
4-methoxyethynylbenzene, 4-cyanoethynylbenzene, and 4-nitroethynyl-
benzene in 57%, 91%, 91%, and 78% yield, respectively.
Live Cell Imaging of A431 Cells. A431 cells were plated at
5 Â 104 cells/mL on PLL-coated glass-bottomed dishes and cultured in
DMEM containing 10% heat-inactivated fetal bovine serum (GIBCO),
100 U/mL of penicillin, and 100 μg/mL streptomycin at 37 °C in
humidified air containing 5% CO2 for 2 days. The cells were cultured in
DMEM without FBS for a day before the imaging experiment. The
medium was replaced with 2 mL of DPBS containing 0.1 g/L CaCl2 and
0.1 g/L MgCl2•6H2O, and then 5 μM NBzFDA and inhibitor were
loaded into the cells at 37 °C for 10 min. Cells were stimulated with 500
ng/mL EGF, and images were taken by conventional microscopy at
37 °C over 30 min.
6: 1H NMR (300 MHz, CDCl3) δ 8.15 (dd, 1H, J = 1.47, 0.73 Hz),
7.80 (dd, 1H, J = 7.89, 1.47 Hz) 7.53 (dt, 2H, J = 8.74, 2.11 Hz) 7.42 (dt,
2H, J = 8.74, 2.02 Hz), 7.18 (dd, 1H, J = 8.07, 1.01 Hz), 7.11 (dd, 2H, J =
1.74, 1.01 Hz), 6.83 (m, 4H), 2.32 (s, 6H). 13C NMR (75 MHz, CDCl3) δ
168.6, 168.0, 152.1, 151.4, 138.1, 133.1, 128.8, 127.9, 126.5, 125.3, 124.1,
123.2, 121.1, 117.8, 115.8, 110.4, 90.7, 88.3, 81.6, 21.0. HRMS (ESI+) Calcd
for [M + H], 595.03924; Found, 595.03543 (À3.81 mmu).
7: 1H NMR (300 MHz, CDCl3) δ 8.13 (dd, 1H, J = 1.47, 0.72 Hz),
7.78 (dd, 1H, J = 7.86, 1.47 Hz), 7.50 (dd, 2H, J = 6.75, 2.01 Hz), 7.15
(dd, 1H, J = 7.86, 0.54 Hz), 7.10 (dd, 2H, J = 2.01, 0.75 Hz), 6.91 (dd,
2H, J = 6.96, 2.19 Hz), 6.85 (m, 2H), 6.84 (m, 2H), 3.85 (s, 3H), 2.32
(s, 6H). 13C NMR (75 MHz, CDCl3) δ 168.8, 168.3, 160.2, 152.1, 151.6,
151.6, 138.0, 133.3, 128.9, 127.8, 126.6, 126.2, 124.0, 117.8, 116.1, 114.4,
114.2, 110.4, 92.1, 86.2, 81.7, 55.3, 21.1. HRMS (ESI+) Calcd for
[M + H], 547.13929; Found, 547.13710 (À2.19 mmu).
Synthesis of 5-Iodofluorescein. 5-Iodofluorescein diacetate was
prepared by a known method.32
Synthesis of 5. 5-Iodofluorescein (206 mg, 0.380 mmol), ethynyl-
benzene (0.050 mL, 0.455 mmol), PdCl2(PPh3)2 (17.5 mg, 0.0250
mmol), and CuI (5.18 mg, 0.0272 mmol) were mixed in 10 mL of dry
THF, and 0.25 mL of triethylamine was added. The mixture was stirred
at 25 °C for 10 h under an Ar atmosphere. THF was evaporated and the
mixture was purified by column chromatography over silica gel using
dichloromethane as the eluent, affording the product (150 mg, 76%) as a
brown solid. 1H NMR (300 MHz, CDCl3) δ 8.16 (d, 1H, J = 0.73 Hz),
7.81 (dd, 1H, J = 8.07, 1.47 Hz), 7.51 (m, 2H), 7.40 (m, 3H), 7.18 (dd,
8: 1H NMR (300 MHz, CDCl3) δ 8.18 (d, 1H, J = 0.54 Hz), 7.82 (dd,
1H, J = 8.07, 1.47 Hz), 7.68 (d, 2H, J = 7.89 Hz), 7.64 (d, 2H, J = 7.86
Hz), 7.20 (d, 1H, J = 8.04 Hz), 7.11 (m, 2H), 6.84 (m, 4H), 2.32 (s, 6H).
13C NMR (75 MHz, CDCl3) δ 168.7, 168.0, 152.7, 152.2, 151.4, 138.3,
132.2, 132.1, 128.8, 128.3, 127.1, 126.6, 124.7, 124.3, 118.2, 117.8, 115.8,
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ARTICLE
112.2, 110.5, 91.3, 89.8, 81.8, 21.0. HRMS (ESI+) Calcd for [M + H],
542.12398; Found, 542.12316 (À0.82 mmu).
125.1, 124.0, 112.7, 108.5, 102.3. HRMS(ESIÀ) Calcd for [M À H],
508.06686; Found, 508.07060 (3.74 mmu).
9: 1H NMR (300 MHz, CDCl3) δ 8.25 (dt, 2H, J = 8.79, 2.11 Hz),
8.20 (dd, 1H, J = 1.28, 0.79 Hz), 7.84 (dd, 1H, J = 8.07, 1.47 Hz), 7.72 (dt,
2H, J = 8.79, 2.11 Hz), 7.22 (dd, 1H, J = 7.98, 0.64 Hz), 7.11 (t, 2H, J = 1.38
Hz), 6.85 (m, 4H). 13C NMR (75 MHz, CDCl3) δ 168.8, 167.9, 152.9,
152.2, 151.5, 147.5, 138.3, 132.5, 129.1, 128.8, 128.4, 126.7, 124.6, 124.4,
123.8, 117.9, 115.8, 110.5, 92.1, 89.6, 81.8, 21.1. HRMS (ESI+) Calcd for
[M + Na], 584.09575; Found, 584.09197 (À3.78 mmu).
Synthesis of NBzFDA. 9 (20.1 mg, 0.0358 mmol) was dissolved in
1 mL of DMSO, and PdCl2 (0.65 mg, 3.67 μmol) was added. The
mixture was stirred at 145 °C for 4 h under an Ar atmosphere and
purified by preparative HPLC to afford NBzFDA (11.0 mg, 52%) as a
slightly yellow solid. 1H NMR (300 MHz, DMSO-d6) δ 8.60 (s, 1H),
8.44 (m, 1H), 8.42 (m, 2H), 8.30 (d, 2H, J = 8.79 Hz), 7.69 (d, 1H,
J = 8.04 Hz), 7.32 (d, 2H, J = 1.47 Hz), 7.00 (d, 2H, J = 8.76 Hz), 6.97 (dd,
2H, J = 8.79, 2.22 Hz), 2.30 (s, 6H). 13C NMR (75 MHz, DMSO-d6)
190.5, 190.2, 168.7, 157.0, 152.3, 150.8, 137.0, 134.2, 131.7, 129.3, 127.3,
126.3, 125.2, 124.0, 118.7, 115.2, 110.6, 81.4, 20.8. HRMS (ESI+) Calcd
for [M + Na], 616.08558; Found, 616.08239 (À3.18 mmu).
Synthesis of 1. 5 (51.6 mg, 0.100 mmol) was dissolved in 2 mL of
DMSO, and PdCl2 (4.95 mg, 0.0279 mmol) was added to the solution.
The reaction mixture was stirred at 105 °C for 12 h under an Ar
atmosphere. Then, 2 mL of methanol and K2CO3 (100 mg) were added,
and the mixture was stirredat25 °C for 10 min. Purification bypreparative
HPLC afforded 1 (19.2 mg, 41%) as a yellow solid. 1H NMR (300 MHz,
CD3OD) δ 8.53 (s, 1H), 8.35 (dd, 1H, J = 8.07 Hz), 8.05 (d, 2H, J = 7.70
Hz), 7.76 (t, 1H, J = 7.43 Hz), 7.61 (t, 2H, J = 7.70 Hz), 7.43 (d, 1H,
J = 7.89 Hz), 6.73 (d, 2H, J = 2.02 Hz), 6.69 (d, 2H, J = 8.99 Hz), 6.58 (dd,
2H, J = 8.62, 2.20 Hz). 13C NMR (75 MHz, CD3OD) δ 194.9, 193.9,
169.6, 162.4, 154.5, 136.9, 136.5, 136.0, 134.0, 131.1, 130.5, 130.4, 129.4,
128.1, 127.0, 114.4, 110.9, 103.6. HRMS (ESIÀ) Calcd for [M À H],
463.08178; Found, 463.07858 (À3.20 mmu).
’ ASSOCIATED CONTENT
S
Supporting Information. Detailed absorbance and fluor-
b
escence changes of NBzF upon addition of various ROS; HPLC
analysis of the reaction product of NBzF with various ROS;
Fluorescence response of 1À4 to various ROS; NMR spectral
analysis of the reaction of NBzF and hydrogen peroxide; Kinetic
traces of NBzF and hydrogen peroxide; Full information of ref 9.
This material is available free of charge via the Internet at http://
pubs.acs.org.
2, 3, and 4 were similarly prepared from 6, 7, and 8 in 57%, 41%, and
48% yield, respectively.
2: 1H NMR (300 MHz, CD3OD) δ 8.43 (d, 1H, J = 0.75 Hz), 8.26
(dd, 1H, J = 7.86, 1.47 Hz), 7.87 (d, 2H, J = 8.61 Hz), 7.70 (d, 2H, J =
8.61 Hz), 7.31 (d, 1H, J = 8.04 Hz), 6.60 (d, 2H, J = 2.22 Hz), 6.54
(d, 2H, J = 8.61 Hz), 6.45 (dd, 2H, J = 8.79, 2.40 Hz). 13C NMR (100 MHz,
DMSO-d6) δ 192.1, 191.7, 167.5, 159.8, 157.8, 151.7, 136.4, 133.8,
132.5, 132.0, 131.1, 130.1, 129.3, 127.0, 126.6, 125.3, 112.8, 108.5, 102.3,
83.5. HRMS (ESIÀ) Calcd for [M À H], 540.99229; Found, 540.98767
(À4.62 mmu).
’ AUTHOR INFORMATION
Corresponding Author
tlong@mol.f.u-tokyo.ac.jp
’ ACKNOWLEDGMENT
3: 1H NMR (300 MHz, DMSO-d6) δ 10.2 (s, 2H), 8.34 (s, 1H), 8.27
(dd, 1H, J = 8.07, 1.29 Hz), 8.00 (d, 2H, J = 8.97 Hz), 7.49 (d, 1H, J =
8.04 Hz), 7.16 (d, 2H, J = 8.79 Hz), 6.70 (d, 2H, J = 2.19 Hz), 6.65 (d,
2H, J = 8.61 Hz), 6.53 (dd, 2H, J = 8.61, 2.19 Hz), 3.89 (s, 3H). 13C
NMR (100 MHz, DMSO-d6) δ 193.0, 192.0, 167.4, 165.18, 159.8, 151.8,
136.1, 134.1, 132.6, 129.3, 127.2, 126.1, 125.4, 124.9, 114.9, 112.7, 108.5,
102.3, 55.9. HRMS (ESIÀ) Calcd for [M À H], 493.09234; Found,
493.08791 (À4.43 mmu).
This work was supported in part by a Grant-in-Aid for JSPS
Fellows (to M.A.), by a Grant-in-Aid for Specially Promoted
Research No. 22000006 to T.N., by the Ministry of Education,
Culture, Sports, Science and Technology of Japan (Grant Nos.
19205021 and 20117003 to Y.U., 20689001 and 21659204 to K.H.,
and 21750135 to T.T.), and by the Industrial Technology Research
Grant Program in 2009 for the New Energy and Industrial Techno-
logy Development Organization (NEDO) of Japan (to T.T.). K.H.
was also supported by the Sankyo Foundation of Life Science.
4: 1H NMR (300 MHz, DMSO-d6) δ 10.1 (s, 2H), 8.50 (m, 1H), 8.37
(d, 1H, J = 8.07 Hz), 8.20 (d, 2H, J = 8.25 Hz), 8.10 (d, 2H, J = 8.07 Hz),
7.51 (d, 1H, J = 8.04 Hz), 6.69 (d, 2H, J = 1.65 Hz), 6.63 (d, 2H, J = 8.61
Hz), 6.54 (dd, 2H, J = 8.79, 1.65 Hz). 13C NMR (100 MHz, DMSO-d6)
δ 191.2, 190.7, 167.5, 159.8, 157.8, 151.8, 136.7, 135.5, 133.8, 133.0,
130.8, 129.3, 127.0, 126.9, 125.1, 118.0, 116.8, 112.8, 108.5, 102.3.
HRMS (ESIÀ) Calcd for [M À H], 488.07703; Found, 488.07359
(À3.43 mmu).
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