Design and development of a fluorescent probe for monitoring hydrogen peroxide using photoinduced electron transfer

Article abstract of DOI:10.1016/j.bmc.2004.11.023

A novel fluorescent probe, 7-hydroxy-2-oxo-N-(2-(diphenylphosphino)ethyl)- 2H-chromene-3-carboxamide (DPPEA-HC) was developed for use in monitoring hydrogen peroxide (H₂O₂) production. DPPEA-HC, which consists of a diphenylphosphine

Full text of DOI:10.1016/j.bmc.2004.11.023

Bioorganic & Medicinal Chemistry 13 (2005) 1131–1139
Design and development of a fluorescent probe for
monitoring hydrogen peroxide using photoinduced electron transfer
Nobuaki Soh,a Osamu Sakawaki,a Koji Makihara,a Yuka Odo,a Tuyoshi Fukaminato,a
Tsuyoshi Kawai,b Masahiro Iriea and Toshihiko Imatoa,*
aDepartment of Applied Chemistry, Graduate School of Engineering, Kyushu University, 6-10-1, Hakozaki, Higashi-ku,
Fukuoka 812-8581, Japan
bResearch and Education Center for Materials Science, Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma,
Nara 630-0192, Japan
Received 30 October 2004; revised 11 November 2004; accepted 12 November 2004
Available online 2 December 2004
Abstract—A novel fluorescent probe, 7-hydroxy-2-oxo-N-(2-(diphenylphosphino)ethyl)-2H-chromene-3-carboxamide (DPPEA-
HC) was developed for use in monitoring hydrogen peroxide (H2O2) production. DPPEA-HC, which consists of a diphenylphos-
phine moiety and a 7-hydroxycoumarin moiety, reacts with H2O2 to form DPPEA-HC oxide, which is analogous to the reaction
of triphenylphosphine with hydroperoxides such as H2O2 to form triphenylphosphine oxide. Photoinduced electron transfer
(PET) was applied in the design of DPPEA-HC. Since the diphenylphosphine moiety and the 7-hydroxycoumarin moiety would
act as the PET donor and the acceptor, respectively, it would be expected that DPPEA-HC would rationally cancel the PET process
via the formation of DPPEA-HC oxide, based on the calculated energy levels of the donor and the acceptor moieties using the
B3LYP/6-31G*//AM1 method. The fluorescence intensity of DPPEA-HC increased on the addition of a H2O2 solution in
100mM sodium phosphate buffer (pH7.4), as predicted from the energy level calculation and a good correlation between increase
in the fluorescence of DPPEA-HC and the concentration of H2O2 was observed. DPPEA-HC was also fluoresced by H2O2, which
was enzymatically produced in xanthine/xanthine oxidase/superoxide dismutase (XA/XOD/SOD) system. The increase in the fluo-
rescence of DPPEA-HC in the presence of H2O2 immediately ceased on the addition of catalase (CAT), which catalyzes the dispro-
portionation of H2O2. In addition, DPPEA-HC was found to have a much higher selectivity for H2O2 and a greater resistance to
autoxidation than 20,70-dichlorodihydrofluoresein (DCFH). Time-resolved fluorescence measurements of DPPEA-HC and DPPEA-
HC oxide confirmed that the fluorescence off/on switching mechanism of DPPEA-HC is based on the PET on/off control.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
vates potassium channel opening in large cerebral
arteries.6 Although a number of reports have docu-
mented the significance of H2O2 in biological systems,
the mechanism of its action is poorly understood
because of the limited availability of detection methods.
Reactive oxygen species (ROS) are thought to play a key
role not only in pathological conditions and chronic dis-
eases but also in certain normal physiological pro-
cesses.1 Among other ROS, hydrogen peroxide (H2O2)
is vasoactive and has been detected under various
pathophysiological conditions, including inflammation,2
hypoxia-reoxygenation,3 and the deficiency of a co-factor
for nitric oxide (NO) synthesis.4 Recent studies have
suggested that H2O2 functions as an endothelium-de-
rived hyperpolarizing factor5 in small arteries and acti-
Detection methods using fluorescent probes are quite
useful for clarifying the functions of a target biomole-
cule in biological systems because temporal and spatial
information concerning the target molecule can be ob-
tained with a high degree of sensitivity in vivo cellular
systems.7 Therefore, various fluorescent probes for mon-
itoring ROS such as NO,8 singlet oxygen,9 and hydroxyl
radical (ÅOH)10 have been developed in recent years.
However, only a few fluorescent probes for H2O2 have
been reported. Though 20,70-dichlorodihydrofluorescein
(DCFH) and dihydrorhodamine 123(DHR123) can be
used as a H2O2 probe,11 they tend to react with a variety
Keywords: Hydrogen peroxide; Fluorescent probe; Reactive oxygen
species; Photoinduced electron transfer.
*
Corresponding author. Tel.: +81 92 642 3569; fax: +81 92 642
4134; e-mail: imato@cstf.kyushu-u.ac.jp
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.11.023

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N. Soh et al. / Bioorg. Med. Chem. 13 (2005) 1131–1139
of ROS, and furthermore, the former probe is easily aut-
oxidized and the fluorescence intensity of the probe
spontaneously increases upon exposure to light.12
Therefore, DCFH and DHR123should be considered
as probes for detecting a broad range of oxidizing reac-
tions that may be increased during intracellular oxida-
tive stress. A europium-ion-based luminescent sensing
probe was also reported for detecting H2O2.13 Unfortu-
nately, however, the luminescence is affected by phos-
phate and citrate. If a probe with a higher selectivity
for H2O2 and a better stability against autoxidation
could be developed, the biological roles of H2O2 would
be expected to be clarified greatly.
Scheme 1. Reaction of DPPEA-HC with H2O2.
We wish to report herein on the design and development
of a novel fluorescent probe, 7-hydroxy-2-oxo-N-(2-
(diphenylphosphino)ethyl)-2H-chromene-3-carboxamide
(DPPEA-HC) for monitoring H2O2. For the design of
the proposed probe, a photoinduced electron transfer
(PET) mechanism was utilized because recent stud-
ies8c,9c,14,15 support the effectiveness of the PET-con-
trolled fluorescence off/on switching mechanism for the
rational design of fluorescent probes. The performance
of DPPEA-HC was evaluated with respect to the detec-
tion ability and the selectivity for H2O2. A time-resolved
fluorescence study of the probe was also carried out, to
permit a more comprehensive discussion of the PET-
controlled fluorescence off/on switching mechanism.
4-(2-diphenylphosphinoethylamino)-7-nitro-2,1,3-benz-
oxadiazole was reported as a PET reagent for hydroper-
oxides.15 The reagent quantitatively reacts with tert-butyl
hydroperoxide or cumene hydroperoxide to increase its
fluorescence intensity in organic solvents, however, the
reactivity of the reagent with H2O2 in an aqueous media
has not yet been reported.
For the design of DPPEA-HC, we introduced the ra-
tional method for designing a PET-type fluorescent
probe, which was proposed by Nagano and Urano
et al.8c,9c,14 If DPPEA-HC is divided into two parts,
namely, a diphenylphosphine moiety (donor) and a 7-
hydroxycoumarin moiety (acceptor) (Fig. 1a), a well-
grounded prediction of fluorescence off/on switching
based on the PET process would be possible using the
method. PET is a widely accepted mechanism for fluo-
rescence quenching where an electron transfer from a
PET donor to the excited acceptor diminishes the fluo-
rescence of the acceptor.18 If the highest occupied
molecular orbital (HOMO) energy level of the donor
is sufficiently high for electron transfer to the excited
acceptor, the value of the quantum efficiency of fluores-
cence (/) will be small, that is, fluorescent derivatives
with high / values must have an electron donor moiety
with low HOMO energy levels (Fig. 1b). The HOMO
energy levels of methyldiphenylphosphine (the donor
in DPPEA-HC) and methyldiphenylphosphine oxide
(the donor in DPPEA-HC oxide) were estimated to be
À8.33 and À9.95eV, based on B3LYP/6-31G*//AM1
calculations. Therefore, if the energy level of an acceptor
moiety in the excited state is located between the
HOMO energy levels of methyldiphenylphosphine and
methyldiphenylphosphine oxide, DPPEA-HC would
cancel the PET process when DPPEA-HC reacts with
H2O2 to form DPPEA-HC oxide. For the rational
design of PET-type fluorescent probes with a fluorescein
structure, Nagano and Urano et al. evaluated the fluo-
rescence off/on threshold of fluorescein derivatives by
considering both the calculated HOMO energy levels
of the donor moiety in various fluorescein derivatives
(donor moiety: benzoic acid moiety, acceptor moiety:
xanthene ring) and the values of the quantum efficiency
of the fluorescence of the fluorescein derivatives, which
were obtained by experimental fluorometric analysis.9c
2. Results and discussion
2.1. Design of novel fluorescent probe for H2O2 based on
the control of PET process
One of the most important factors in the design of a
fluorescent probe for a target molecule involves the
selection of a specific reaction that can be used as the
basis for selectively detecting the analyte. We concluded
that a reaction of triphenylphosphine with hydroperox-
ides is a suitable reaction that would permit the design
of a novel fluorescent probe for H2O2. Triphenylphos-
phine was reported to react with hydroperoxides such
as H2O2 to produce triphenylphosphine oxide.16 In the
reaction, a lone electron pair of the phosphorus atom
in triphenylphosphine would be lost as the result of
the formation of triphenylphosphine oxide. Therefore,
a phosphine compound, in which an appropriate fluoro-
phore is substituted for a phenyl group of triphenyl
phosphine (i.e., a diphenyl phosphine containing a flu-
orophore) would permit the fluorescence intensity to
be increased when the phosphine compound reacts with
H2O2 because the PET derived from the lone electron
pair of the phosphorus atom to the fluorophore may
be cancelled in the reaction. In fact, diphenyl-1-pyrenyl-
phosphine (DPPP) has been reported to react with
hydroperoxides to form DPPP oxide, resulting in an in-
crease in fluorescence intensity.17 However, DPPP is a
highly hydrophobic compound and, thus, is not suitable
for the analysis of H2O2 in aqueous media. We therefore
designed a more hydrophilic diphenylphosphine deriva-
tive, DPPEA-HC, as a novel fluorescent probe for deter-
mining H2O2 in aqueous media (Scheme 1). Recently,

N. Soh et al. / Bioorg. Med. Chem. 13 (2005) 1131–1139
1133
(a)
(b)
(c)
Figure 1. Design of a novel fluorescent probe for H2O2 based on the control of PET process: (a) the structure of DPPEA-HC divided into two parts,
the PET donor and the acceptor (fluorophore): (b) illustration of the thermodynamic situation for the fluorescence off/on switching based on control
of the PET process: (c) HOMO energy levels of donor (methyldiphenylphosphine, methyldiphenylphosphine oxide), and the energy level of acceptor
(N-ethyl-7-hydroxy-2-oxo-2H-chromene-3-carboxamide) at excited state, obtained from B3LYP/6-31G*//AM1 calculations.
In this study, we evaluated the fluorescence off/on
threshold of the H2O2 probe from the calculated energy
level of the excited acceptor. The energy level of N-ethyl-
7-hydroxy-2-oxo-2H-chromene-3-carboxamide (accep-
tor in DPPEA-HC or DPPEA-HC oxide) in the excited
state was estimated to be À9.23eV, based on B3LYP/6-
31G*//AM1 calculations. Therefore, the HOMO energy
level of methyldiphenylphosphine (À8.33eV), the donor
moiety of DPPEA-HC, is sufficiently high to induce the
PET process to the excited acceptor, on the other hand,
the HOMO energy level of methyldiphenylphosphine
oxide (À9.95eV), the donor moiety of DPPEA-HC
oxide, is sufficiently low to permit cancellation of the
PET process (Fig. 1c). Thus, it would be rationally
expected that DPPEA-HC would react with H2O2 to
cancel PET process and increase the fluorescence
intensity.
2.2. Synthesis of DPPEA-HC and its detection ability for
H2O2
DPPEA-HC and DPPEA-HC oxide were synthesized
from 2-(diphenylphosphino)ethylamine and 7-hydroxy-
coumarin-3-carboxylic acid N-succinimidyl ester, as
shown in Scheme 2. Figure 2 shows fast atom bombard-
ment (FAB) mass spectra of DPPEA-HC, DPPEA-HC
oxide, and DPPEA-HC after reaction with a sufficient
amount of H2O2. The peaks for DPPEA-HC (m/z =
418) and DPPEA-HC oxide (m/z = 434) can be readily
assigned from the calculated values ([M+H]+ = 418 for
DPPEA-HC, 434 for DPPEA-HC oxide), respectively,
and the peak of sample solution of DPPEA-HC, after
reaction with a sufficient amount of H2O2 in aqueous
media (m/z = 434), is in good agreement with that
of DPPEA-HC oxide synthesized. This result clearly

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N. Soh et al. / Bioorg. Med. Chem. 13 (2005) 1131–1139
(a)
(b)
Scheme 2. Synthetic schemes for (a) DPPEA-HC: (b) DPPEA-HC oxide.
confirms that DPPEA-HC reacted with H2O2 to yield
DPPEA-HC oxide in aqueous media.
mutase (XA/XOD/SOD) system20 xanthine oxidase
(XOD) catalyzes the incorporation of oxygen (derived
from water) into xanthine (XA) to yield uric acid, super-
oxide anion (O2ÅÀ) and H2O2. Superoxide dismutase
DPPEA-HC was next applied to the detection of H2O2
in 100mM sodium phosphate buffer (pH7.4). As ex-
pected from the previous section, the fluorescence inten-
sity of DPPEA-HC increased with an increase in the
concentration of added H2O2, and was saturated at con-
centrations higher than 50lM, where 99% of DPPEA-
HC is calculated to be reacted with H2O2 to form
DPPEA-HC oxide from the fluorescence intensity of
DPPEA-HC oxide, which was synthesized in this work
(Fig. 3). Figure 4 shows the time courses for the fluores-
cence intensity of DPPEA-HC after the addition of
various concentrations of H2O2. In this case, the
fluorescence intensity of DPPEA-HC was normalized
by the intensity before the addition of H2O2. The fluo-
rescence intensity of DPPEA-HC increased as a function
of H2O2 concentration. From the results shown in Fig-
ures 3and 4 , DPPEA-HC was able to detect lM level
of H2O2.
ÅÀ
(SOD) catalizes the disproportionation of O2 to yield
H2O2.21 Thus, H2O2 is generated enzymatically in the
XA/XOD/SOD system. As shown in Figure 6, the fluo-
rescence intensity of a DPPEA-HC solution in the pres-
ence of XA and SOD increased on the addition of XOD
(XA/XOD/SOD system) as well as the case, in which
H2O2 was added directly to the DPPEA-HC solution
(Figs. 3and 4 ). In addition, the increase in fluorescence
was distinctly suppressed on the addition of CAT (XA/
XOD/SOD/CAT system) as well as the case shown in
Figure 5. The results, showing that DPPEA-HC func-
tioned, even in an enzymatic system, strongly support
the conclusion that the probe would be applicable in
biological systems.
Table 1 shows the increment of the fluorescence intensity
of DPPEA-HC in various ROS generating systems. The
values were calculated by subtracting the experimental
values of fluorescence intensities of DPPEA-HC before
the addition of these ROS from those of DPPEA-HC
after the addition of the ROS. The data for DCFH,
taken from literature,10c are also listed in Table 1, for
comparison. As shown in Table 1, DPPEA-HC has a
much higher selectivity for H2O2 than DCFH. It is
particularly noteworthy that the fluorescence of
DPPEA-HC was not influenced significantly by peroxy-
nitrite (ONOOÀ), ÅOH, and alkylperoxyl radical
(ROOÅ), while the fluorescence of DCFH was greatly
increased in the presence of these ROS. In the ONOOÀ
generating system, a commercial ONOOÀ solution, in
which excess H2O2 is removed with manganese dioxide,
To confirm that the fluorescence changes of DPPEA-HC
were caused by H2O2, catalase (CAT), which acts as a
scavenger of H2O2 by catalyzing the disproportionation
of H2O2 into water and molecular oxygen,19 was added
to a solution of DPPEA-HC, in which the reaction of
DPPEA-HC with H2O2 was proceeding. As can be seen
from Figure 5, the increase in fluorescence intensity was
completely suppressed by the addition of CAT. This re-
sult confirms that the fluorescence changes in DPPEA-
HC shown in Figures 3and 4 are due to H2O2.
DPPEA-HC was also applied to the detection of H2O2
generated in a xanthine/xanthine oxidase/superoxide dis-

N. Soh et al. / Bioorg. Med. Chem. 13 (2005) 1131–1139
1135
(a)
(a)
(b)
(b)
(c)
Figure 3. (a) Emission spectra (representative data are shown): (b)
fluorescence intensity at 449nm (data are the mean SD, n = 3) for
DPPEA-HC at 37ꢁC in 100mM sodium phosphate buffer (pH7.4) in
the presence of various concentration of H2O2 ranging from 0 to 50lM
with excitation at 396nm. The emission spectra were obtained 30min
after the addition of H2O2 to a 5lM DPPEA-HC solution under
aerobic conditions. The denoted values for H2O2 concentration are
cumulative concentrations.
Figure 2. FAB mass spectra of (a) DPPEA-HC: (b) DPPEA-HC
oxide: (c) DPPEA-HC, after reaction with a sufficient amount of
H2O2.
DPPEA-HC may be therefore deducted if such an NO
fluorescent probe is applied together with DPPEA-HC
for the imaging of H2O2. Moreover, the interference
was used. In the ÅOH generating system, the molar ratio
of Fe(II):H2O2 in the Fenton reagent is 1:1. Therefore, it
would be expected that insignificant amount of H2O2
exists in both systems and the results in these systems
would reflect the effects of ONOOÀ and ÅOH themselves.
In addition, the fluorescence of DPPEA-HC increased
negligibly due to autoxidation upon illumination,
whereas DCFH was extensively autoxidized, resulting
in a dramatic increase in fluorescence intensity. Unex-
ÅÀ
from O2 may be also differentiated from H2O2, for
example, by utilizing SOD, which is thought to internal-
ize in living cells through endocytosis.22 Thus, DPPEA-
HC appears to be superior to DCFH, an ordinary probe
for H2O2, from the standpoint of both selectivity to
H2O2 against other ROS and resistance to autoxidation.
Thus, we believe that DPPEA-HC represents an attrac-
tive novel fluorescent probe for detecting H2O2.
ÅÀ
pectedly, DPPEA-HC fluoresced with O2 and NO as
with H2O2. Although the reason for this is not clear at
this stage, it is possible that DPPEA-HC reacted with
these radicals as well as H2O2 to form DPPEA-HC
oxide. However, concerning NO, since many fluorescent
probes that perform well have been reported,8 interfer-
ence from NO for the biological imaging of H2O2 using
2.3. Fluorescence parameters of DPPEA-HC and PET-
controlled fluorescence off/on switching mechanism
In this section, the fluorescence parameters of DPPEA-
HC were evaluated, to permit the PET-controlled fluo-
rescence off/on switching mechanism of DPPEA-HC

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N. Soh et al. / Bioorg. Med. Chem. 13 (2005) 1131–1139
Figure 6. Time courses for the relative fluorescence intensity (F/F0) of
DPPEA-HC in the enzymatic XA/XOD/SOD system and the XA/
XOD/SOD/CAT system at 37ꢁC in 100mM sodium phosphate buffer
(pH7.4). In the XA/XOD/SOD system, XOD (final 0.05units/mL) was
added to a 5lM DPPEA-HC solution with 100lM XA and 100units/
mL SOD. In the XA/XOD/SOD/CAT system, CAT (final 90units/mL)
was further added after the addition of the same concentration of
XOD. The fluorescence intensity (F0: initial fluorescence intensity, F:
fluorescence intensity) was determined at 449nm with excitation at
396nm. The added points of XOD and CAT were indicated by arrows.
Figure 4. Time courses of relative fluorescence intensity (F/F0) of
DPPEA-HC depending on H2O2 at 37ꢁC in 100mM sodium
phosphate buffer (pH7.4). A solution of H2O2 (final 0–50lM) was
added at the point indicated by an arrow. F0 and F denote the
fluorescence intensity at 449nm before and after the addition of H2O2,
respectively, with excitation at 396nm.
Table 1. Fluorescence increase in DPPEA-HC after reaction with
various ROS
ROS
DPPEA-HC
DCFHi
a
H2O2
ONOOÀb
ÀOClc
137
11
190
6600
86
<1
8
ÅOHd
7400
710
67
ROOÅe
46
ÅÀf
O2
126
126
27
NOg
150
2000
Autoxidationh
Various ROS were generated in a DPPEA-HC solution (final 5lM,
0.2% DMSO as a co-solvent) and the mixture was stirred at 37ꢁC for
60min. The fluorescence intensity was determined at 449nm with
excitation at 396nm.
Figure 5. Effect of catalase (CAT) on fluorescence changes in DPPEA-
HC. CAT (final 90units/mL) was added to 100mM sodium phosphate
buffer (pH7.4), which contains 5lM DPPEA-HC and 50lM H2O2.
The fluorescence intensity (F0: initial fluorescence intensity, F:
fluorescence intensity) was determined at 449nm with excitation at
396nm. The added points of H2O2 and CAT were indicated by arrows.
a H2O2 (final 50lM) was added to 100mM sodium phosphate buffer
(pH7.4).
b ONOOÀ (final 1.5lM) was added to 100mM sodium phosphate
buffer (pH7.4).
c NaOCl (final 1.5lM) was added to 100mM sodium phosphate buffer
(pH7.4).
that we designed at the first stage to be discussed. The
absorbance and fluorescence properties of DPPEA-HC
and DPPEA-HC oxide are summarized in Table 2.
The formation of DPPEA-HC oxide from DPPEA-HC
led to little change in the absorbance maxima but in-
creased the value for the quantum efficiency of fluores-
cence, /, a characteristic feature of PET-type probes.
The increase in with the formation of DPPEA-HC oxide
strongly supports the hypothesis that the fluorescence
off/on switching of DPPEA-HC is based on the PET
mechanism. Figure 7 shows time-resolved fluorescence
decay curves for DPPEA-HC and DPPEA-HC oxide.
The lifetimes of DPPEA-HC and DPPEA-HC oxide,
as evaluated from the slopes of the decay curves in Fig-
ure 7, were determined to be 2.8 and 3.8ns, respectively.
d Ferrous perchlorate and H2O2 (final 50lM, each) were added to
10mM Tris–HCl buffer (pH7.4).
e 2,20-Azobis(2-amidinopropane)dihydrochloride (final 50lM) was
added to 100mM sodium phosphate buffer (pH7.4).
f KO2 (final 50lM) was added to 100mM sodium phosphate buffer
(pH7.4).
g NOC7 (final 50lM) was added to 100mM sodium phosphate buffer
(pH7.4).
h A dye solution was placed under a fluorescent lamp for 2.5h.
i Fluorescence increase of DCHF was also listed in Table 1 for refer-
ence. The data for DCFH were extracted from Ref. 10c. A part of the
experimental conditions for DCFH are different from that for
DPPEA-HC, for example, the slit width (2.5nm for both excitation
and emission were used) in fluorometric analysis, concentrations of
fluorescent probe and ROS (basically twice the concentrations were
used, such as 10lM dye (DCFH), 100lM H2O2, or 3lM ONOOÀ).

N. Soh et al. / Bioorg. Med. Chem. 13 (2005) 1131–1139
1137
Table 2. Absorbance and fluorescence properties of DPPEA-HC and
DPPEA-HC oxidea
where / and /ref denote the quantum efficiencies of fluo-
rescence of DPPEA-HC and DPPEA-HC oxide, respec-
tively, and kref = 1/sref, which was determined to be
2.6 · 108 sÀ1. The ket value calculated using Eq. 2 was
determined to be 1.1 · 108 sÀ1, a value that is almost
the same as the ket value (9.4 · 107 sÀ1) determined from
Eq. 1. This result indicates that the obtained ket values
represent reasonable rate constants for the forward elec-
tron transfer in DPPEA-HC.
Compound
Absorbance Extinction Emission
maximum
(nm)
Relative
coefficient maximum quantum
(·104 MÀ1 (nm)
cmÀ1
yieldb
0.82
)
DPPEA-.H4C 4033 449
DPPEA-HC 405
oxide
0.58
3.1
446
a All data were measured in 100mM sodium phosphate buffer at pH7.4
(0.2% DMSO as a cosolvent).
b Quantum yield of fluorescence was determined using that of 7-
hydroxycoumarin (0.76) in 100mM sodium phosphate buffer at
pH7.4 as a standard.
3. Conclusion
In conclusion, we report on the design and development
of a novel fluorescent probe for the detection of H2O2 by
utilizing the fluorescence off/on switching mechanism,
based on the PET on/off control. The fluorescence inten-
sity of DPPEA-HC increased with an increase in the
concentration of H2O2, as expected from the calculated
energy levels of the donor and acceptor moieties in
DPPEA-HC and DPPEA-HC oxide using the B3LYP/
6-31*//AM1 method. DPPEA-HC was superior to
DCFH in terms of both selectivity for H2O2 and resis-
tance to autoxidation, therefore, DPPEA-HC would
be expected to be a novel and useful fluorescent probe
for H2O2. The biological imaging of H2O2 in living cells
with DPPEA-HC would be achieved by a microinjection
method or by converting DPPEA-HC to ester deriva-
tives, which would be able to permeate through the cell
membrane. We hope that our report will lead to a better
understanding of biological roles of H2O2 in the future.
Figure 7. Time-resolved fluorescence decay curves for DPPEA-HC
and DPPEA-HC oxide (5lM, respectively) at room temperature in
100mM sodium phosphate buffer (pH7.4) under excitation with
362nm light.
4. Experimental
4.1. Materials
2-(Diphenylphosphino)ethylamine, 7-hydroxycoumarin-
3-carboxylic acid N-succinimidyl ester, and 7-hydroxy-
coumarin-3-carboxylic acid were purchased from Fluka.
Hydrogen peroxide and sodium hypochlorite solution
were purchased from Kishida Chemical Co. Ltd. Xan-
thine (XA), xanthine oxidase (XOD), superoxide dismu-
tase (SOD), and catalase (CAT) were purchased from
Sigma. Peroxynitrite solution, 1-hydroxy-2-oxo-3-(N-
The longer lifetime of DPPEA-HC oxide than that of
DPPEA-HC indicates that the PET process was sup-
pressed in the case of DPPEA-HC oxide, different from
DPPEA-HC. The lifetime of DPPEA-HC after the addi-
tion of a sufficient amount of H2O2 to the aqueous solu-
tion (5lM DPPEA-HC, 3mM H2O2) was determined to
be 3.7ns, similar to that of DPPEA-HC oxide (3.8ns).
This result clearly demonstrates that the fluorescence
off/on switching of DPPEA-HC triggered by the addi-
tion of H2O2 is due to the PET on/off switching
mechanism.
methyl-3-aminopropyl)-3-methyl-1-triazene
(NOC7)
were purchased from Dojindo Laboratories. Potassium
superoxide (KO2) was purchased from Acros. 2,2-Azo-
bis(2-amidinopropane)dihydrochloride were purchased
from Wako Pure Chemical Industries Co. Ltd. Other
materials were of the highest grade available and used
without further purification.
The rate constant for the forward electron transfer (ket)
in DPPEA-HC from donor to acceptor (see Fig. 1b, left)
can be calculated using the following equation23
ket ¼ 1=s À 1=sref
ð1Þ
4.2. Instruments
where s and sref denote the fluorescence lifetime of
DPPEA-HC and DPPEA-HC oxide, respectively. The
ket value in DPPEA-HC was determined to be
9.4 · 107 sÀ1 from Eq. 1 by using the fluorescence life-
times of DPPEA-HC and DPPEA-HC oxide. On the
other hand, the ket value for DPPEA-HC can be also
calculated using the following equation14,23
1H NMR spectra were recorded on an AC-250P spec-
trometer (Bruker). Mass spectra were obtained with a
JMS mate II (JEOL). UV–vis spectra were obtained
on a V-560 UV/Vis spectrophotometer (JASCO). Stea-
dy-state fluorescence spectroscopic studies were per-
formed on a RF-5300PC (Shimadzu). Fluorescence
lifetimes were measured with a time-resolved spectroflu-
orometer (Hamamatsu Photonics C4334-01, C5094, and
/ref =/ ¼ 1 þ ket=kref
ð2Þ

1138
N. Soh et al. / Bioorg. Med. Chem. 13 (2005) 1131–1139
C4792) excited with a pulsed N2 laser and a laser system
(Laser Photonics, Inc., LN-203C and LD2S).
Relative quantum efficiencies of fluorescence of
DPPEA-HC and DPPEA-HC oxide were obtained by
comparing the area of the emission spectrum for the test
sample excited at 366nm in 100mM sodium phosphate
buffer (pH7.4) with that for a solution of 7-hydroxy-
coumarin excited at the same wavelength, which has a
quantum efficiency of 0.76 according to the literature.24
The quantum efficiencies of fluorescence were obtained
from multiple measurements (n = 3) with the following
equation
4.3. Synthesis of DPPEA-HC
2-(Diphenylphosphino)ethylamine (52.0mg, 0.23mmol)
and 7-hydroxycoumarin-3-carboxylic acid N-succinim-
idyl ester (63.9mg, 0.21mmol) were dissolved in
0.3mL of absolute DMF under a N2 atmosphere, and
the solution was stirred at room temperature for 3h.
The reaction mixture was evaporated and the crude
compound was purified by thin layer chromatography
on silica gel 60 plates (Merck) eluting with CHCl3/
Absstandard
Â
Abssample
R½F sampleꢀ
/
¼ /standard
Â
sample
R½F standard
ꢀ
MeOH = 85/15,
and
extracting
with
CHCl3/
where Abs and F denote the absorbance and fluores-
cence intensity, respectively, and R[F] denotes the peak
area of the fluorescence spectra, calculated by summa-
tion of the fluorescence intensity.
MeOH = 60/40. The purification process was carried
out by two times to afford a yellow powder (25mg, yield
28%). H NMR (DMSO-d6): d 2.39 (t, 2H, J = 7.5Hz);
3.36–3.48 (m, 3H); 6.73 (s, 1H); 6.82 (d, 1H,
J = 8.3Hz); 7.26–7.47 (m, 10H); 7.76 (d, 1H,
J = 8.6Hz); 8.72 (s, 1H); 8.78 (t, 1H, J = 5.42Hz).
HRMS (FAB+): calcd for [M+H]+, 418.1208; found,
418.1208. Mp: 167–169ꢁC.
1
In time-resolved fluorescence studies, the pulse width
was 600ps and the wavelengths were 337.1nm (N2
laser), 396nm (dye laser for DPPEA-HC and DPPEA-
HC oxide). The decay curves were analyzed by single-
exponential fitting after deconvolution of the excitation
light pulse profile. The goodness of the fit was judged
with the reduced v2 value (<1.2 in all cases), the
randomness of the residuals, and the autocorrelation
function.
4.4. Synthesis of DPPEA-HC oxide
2-(Diphenylphosphino)ethylamine (38.1mg, 0.17mmol)
was dissolved in 0.2mL of MeOH, then 100lL of an
H2O2 solution (1mmol) was added, and the reaction
mixture was stirred at room temperature for 1h. The
resulting mixture was evaporated and dissolved in
0.3mL of absolute DMF, and 7-hydroxycoumarin-3-
carboxylic acid N-succinimidyl ester (31.4mg, 0.1mmol)
was then added. The reaction mixture was stirred at
room temperature for 3h. The mixture was evaporated
and the crude compound was purified by thin layer
chromatography on silica gel 60 plates (Merck) eluting
with CHCl3/MeOH = 85/15, and extracting with
CHCl3/MeOH = 60/40 to afford a yellowish brown pow-
der (20.3mg, yield 45%). 1H NMR (DMSO-d6): d 2.69
(t, 2H, J = 10.4Hz); 3.39–3.62 (m, 3H); 6.72 (s, 1H);
6.82 (d, 1H, J = 8.6Hz); 7.42–7.50 (m, 6H); 7.59–7.77
(m, 5H); 8.67 (s, 1H); 8.78 (t, 1H, J = 5.3Hz). HRMS
(FAB+): calcd for [M+H]+, 434.1157; found, 434.1155.
Mp: 109–111ꢁC.
4.7. Computational methods
All geometry optimizations were performed using
PM3,25 which is available in the molecular orbital pack-
age WinMOPAC3.0 by Fujitsu Ltd. The results of the
calculation were recalculated using a hybrid density
functional theory (B3LYP)26 with the 6-31G* basis set
as implemented in Gaussian 98W.27 Calculations using
AM128 in WinMOPAC3.0 were subsequently carried
out where the optimized structures obtained by the cal-
culations using Gaussian 98W were maintained. In the
AM1 calculation, the CIS calculation method29 was
used for the evaluation of the energy value at the excited
state.
4.5. Mass spectrometric analysis
Acknowledgements
3-Nitrobenzyl alcohol was used as the matrix. A powder
of DPPEA-HC or DPPEA-HC oxide was used in the
measurement. A sample, 1mg of DPPEA-HC dissolved
in 10lL of DMSO in the presence of 1M H2O2, was
used, in order to confirm that DPPEA-HC reacts with
H2O2 to form DPPEA-HC oxide.
We wish to thank Mr. Taisuke Iseda for the measure-
ments of mass spectra. This work was supported by a
research grant from the Ministry of Education, Science,
Sports and Culture of Japan (grant number; 16023249
to N.S.).
4.6. Fluorometric analysis
References and notes
In steady-state fluorescence spectroscopic studies, the
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DPPEA-HC and DPPEA-HC oxide were dissolved in
DMSO to produce 5mM stock solutions for each com-
pound. These solutions were then diluted to the required
concentration for the subsequent measurements. The
content of DMSO in each solution was 0.2% (v/v).
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