Novel fluorescent probe for detecting hydroperoxides with strong emission in the visible range

Article abstract of DOI:10.1016/j.bmcl.2006.02.078

A novel fluorescent probe, a swallow-tailed perylene derivative for detecting hydroperoxides (Spy-HP), containing perylene 3,4,9,10-tetracarboxyl bisimide as the main skeleton in the structure, was developed. Spy-HP reacted rapidly with hydroperoxides such as m-chloroperbenzoic acid (MCPBA) and cumene hydroperoxide to form its oxidized derivative, Spy-HPOx, and emitted an extremely strong fluorescence (Φ ～ 1) in the visible range (λ_ex = 524 nm and λ_em = 535 nm), as the result of canceling the photoinduced electron transfer (PET) effect. The reaction between Spy-HP and hydroperoxides proceeded quantitatively in strict stoichiometry, without being affected by autoxidation or photobleaching. Because of these prominent properties, Spy-HP is expected to be a novel and useful fluorescent probe to 'spy' on hydroperoxides in biosamples.

Full text of DOI:10.1016/j.bmcl.2006.02.078

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2943–2946
Novel fluorescent probe for detecting hydroperoxides with
strong emission in the visible range
Nobuaki Soh,* Tomoyuki Ariyoshi, Tuyoshi Fukaminato, Koji Nakano,
Masahiro Irie and Toshihiko Imato*
Department of Applied Chemistry, Graduate School of Engineering, Kyushu University,
744, Moto-oka, Nishi-ku, Fukuoka 819-0395, Japan
Received 3 November 2005; revised 11 February 2006; accepted 28 February 2006
Available online 22 March 2006
Abstract—A novel fluorescent probe, a swallow-tailed perylene derivative for detecting hydroperoxides (Spy-HP), containing per-
ylene 3,4,9,10-tetracarboxyl bisimide as the main skeleton in the structure, was developed. Spy-HP reacted rapidly with hydroper-
oxides such as m-chloroperbenzoic acid (MCPBA) and cumene hydroperoxide to form its oxidized derivative, Spy-HPOx, and
emitted an extremely strong fluorescence (U ꢀ 1) in the visible range (kex = 524 nm and kem = 535 nm), as the result of canceling
the photoinduced electron transfer (PET) effect. The reaction between Spy-HP and hydroperoxides proceeded quantitatively in strict
stoichiometry, without being affected by autoxidation or photobleaching. Because of these prominent properties, Spy-HP is expected
to be a novel and useful fluorescent probe to ‘spy’ on hydroperoxides in biosamples.
Ó 2006 Elsevier Ltd. All rights reserved.
Lipid peroxidation is generally thought to be an impor-
tant causative factor for some diseases and for aging.1–6
Since large quantities of hydroperoxides are produced in
the first stage of lipid peroxidation, hydroperoxides have
attracted a great deal of considerable attention as a
marker of oxidative stress. Several methods for detecting
hydroperoxides have been reported to date.7–18 Among
them, a method using diphenyl-1-pyrenylphosphine
(DPPP)11–16 appears to be one of the most successful.
Indeed, DPPP has been applied not only to the determi-
nation of various hydroperoxides using an HPLC post-
column detection system,13,14 but also to the monitoring
of lipid peroxidation within cell membranes by utilizing
as a fluorescent probe.15,16 Unfortunately, however, the
both excitation and emission wavelengths of DPPP are
in such a short range (kex = 352 nm and kem = 380 nm)
that detection using DPPP frequently suffers from unde-
sirable effects such as autofluorescence from biomatrices
and damage to living cells.
as the main skeleton of the fluorescent probe. The short
wavelengths of excitation and emission of DPPP are
attributed to the pyrenyl group. If the pyrenyl group
is substituted by a more highly conjugated polycyclic
group, the wavelengths of the probe would be shifted
to longer wavelengths. Although 4-(2-diphenylphosphi-
noethylamino)-7-nitro-2,1,3-benzoxadiazole containing
a benzofurazan skeleton was developed for the detec-
tion of hydroperoxides at longer wavelengths,17 the
excitation wavelength of the reagent would not be suf-
ficiently long (kex = 458 nm) for desirable detection and
the fluorescence quantum yields of the fluorescent
derivative formed after the reaction with hydroperox-
ides would not be sufficiently high (0.44 in acetonitrile
and 0.21 in methanol). Very recently, a fluorescent
probe containing normal perylene was also developed
for determination of hydroperoxides, however, the exci-
tation and emission wavelengths are not in sufficiently
long range (kex = 440 nm and kem = 470 nm).18 On the
other hand, some fluorescent ‘perylene derivatives’ are
known to have excellent fluorescent properties such as
long excitation and emission wavelengths, a high pho-
tochemical stability, a high fluorescence quantum yield,
a low quantum yield of intersystem crossing, and versa-
tile reactivity. Such perylene derivatives would be
promising candidates for use in the design of analytical
fluorescent reagents, especially in designing fluorescent
probes for biosamples. However, perylene derivatives
Basic fluorescent properties of fluorescent probe are
heavily dependent on the fluorophore, that is selected
Keywords: Hydroperoxide; Fluorescent probe; Perylene 3,4,9,10-tetra-
carboxyl bisimide; Photoinduced electron transfer; Lipid peroxidation.
*
Corresponding authors. Tel.: +81 92 802 2891; fax: +81 92 802 2889
(N.S.); tel./fax: +81 92 802 2889 (T.I.); e-mail addresses:
soh@cstf.kyushu-u.ac.jp; imato@cstf.kyushu-u.ac.jp
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.02.078

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N. Soh et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2943–2946
have not been extensively examined as a skeleton for
fluorescent probes in spite of their excellent fluorescent
properties, while many other fluorophores such as fluo-
rescein derivatives are widely used for this purpose. In
this paper, we wish to report on a novel perylene-based
fluorescent probe, a swallow-tailed perylene derivative
for detecting hydroperoxides (Spy-HP), which reacts
with hydroperoxides and emits a strong fluorescence
in the visible range. As the perylene fluorophore in
the main skeleton of the probe, perylene 3,4,9,10-tetra-
carboxyl bisimide was selected because the fluorophore
has prominent properties such as sufficiently long exci-
tation and emission wavelengths, a high fluorescence
quantum yield (ꢀ1), and high molar coefficient for
the extinction maximum (88,000).19 Furthermore, the
fluorophore can be excited using frequently used laser
and the fluorophore is useful for single-molecule fluo-
rescence-based studies because of such excellent
properties.20,21
of the fluorescence quantum yield of Spy-HPOx (ꢀ1)
was nearly equal to that of perylene 3,4,9,10-tetracarb-
oxyl bisimide itself, it appeared likely that the PET effect
would be cancelled in Spy-HPOx. Similar results were
obtained for other organic solvents, such as methanol
and CH2Cl2.
To evaluate the applicability of Spy-HP as a fluorescent
probe for hydroperoxides, Spy-HP (1 lM) was treated
with various concentrations (50, 100, 200, 400, 800 nM
and 1–4 lM) of m-chloroperbenzoic acid (MCPBA),
which is a lipid-soluble hydroperoxide and was used as
a representative hydroperoxide in this study. Figure 1a
shows the fluorescence spectra of Spy-HP, after a
5 min incubation with MCPBA in ethanol at 37 °C. As
expected, the fluorescence intensity of Spy-HP increased
with an increase in the concentration of the hydroperox-
ide and a good linear relationship was observed between
the fluorescence intensity at 535 nm and the concentra-
tion of the hydroperoxide in the range of 0–1 lM, as
can be seen from Figure 1b. The fluorescence intensity
was saturated at concentrations higher than 1 lM and
a sharp breakpoint was observed at a concentration of
1 lM. Taking into consideration the fact that the con-
centration of the Spy-HP solution before the addition
of hydroperoxide was 1 lM, the result would be quite
reasonable and strongly suggests that Spy-HP reacts
with the hydroperoxide rapidly at a stoichiometric mo-
lar ratio of 1:1 strictly without being affected by autox-
idation or photobleaching. Spy-HP also increased the
fluorescence intensity upon the addition of cumene
hydroperoxide. The results indicate that Spy-HP reacted
with hydroperoxides quantitatively and Spy-HP would,
therefore, be useful for the determination of
hydroperoxides.
The structure of Spy-HP and its detection mechanism
for hydroperoxides are shown in Scheme 1. Spy-HP
was synthesized by coupling N-(1-hexylheptyl)perylene-
3,4,9,10-tetracarboxyl-3,4-anhydride-9,10-imide and 4-
(diphenylphosphino)benzeneamine.22 As shown in
Scheme 1, we expected that Spy-HP would react with
hydroperoxides to form an oxidized derivative (Spy-
HPOx) and, as a result, emit a strong fluorescence based
on cancellation of photoinduced electron transfer (PET)
effect23 from the donor moiety (triphenylphosphine moi-
ety) to the acceptor moiety (perylene moiety).
The fluorescent properties of Spy-HP and Spy-HPOx
were initially investigated. The excitation and emission
wavelengths of both Spy-HP and Spy-HPOx were found
to be sufficiently long (kex = 524 nm and kem = 535 nm in
ethanol). The fluorescence of Spy-HP was efficiently
suppressed due to the PET effect (U ꢀ 0.05 in ethanol),
on the other hand, Spy-HPOx emitted an extremely
strong fluorescence (U ꢀ 1 in ethanol). Since the value
Figure 2 shows fluorescent images of solutions contain-
ing Spy-HP (sample a: 10 lM Spy-HP), Spy-HPOx
(sample b: 10 lM Spy-HPOx), and Spy-HP with hydro-
peroxide (sample c: 10 lM Spy-HP and 20 lM
Scheme 1. Chemical structure of Spy-HP and a schematic illustration of the mechanism of detection of hydroperoxides using the probe.

N. Soh et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2943–2946
2945
100
50
0
a
a
b
c
1000 nM
800 nM
400 nM
200 nM
100 nM
50 nM
0 nM
Concentration of hydroperoxide
500
550
600
650
Wavelength / nm
b
Figure 2. Photographs of CH2Cl2 solutions of Spy-HP (sample a:
10 lM Spy-HP), Spy-HPOx (sample b: 10 lM Spy-HPOx), and Spy-
HP treated with hydroperoxide (sample c: 10 lM Spy-HP and 20 lM
MCPBA). Photographs were obtained using a mercury–xenon lamp.
10
10
prototype of fluorescent probes, having perylene-deriva-
tive skeletons with prominent fluorescent properties.
Although many fluorescent probes have been reported
to date, most were designed and developed to function
in the cytoplasm. Different from ordinary fluorescent
probes, highly hydrophobic fluorescent probes based on
a perylene-derivative skeleton are promising candidates
for use as new fluorescent probes, which could be local-
ized to monitor biomolecules or bioprocesses in hydro-
phobic regions of living cells such as cell membranes.
F
F
5
0
F
F
5
0
200
400
600
800
1000
Concentration of hydroperoxide / nM
0
0
1
2
3
4
5
Concentration of hydroperoxide / μM
Figure 1. (a) Emission spectra for Spy-HP at 37 °C in ethanol in the
presence of hydroperoxide (MCPBA) at various concentrations. The
emission spectra were obtained 5 min after the addition of the
hydroperoxide to the Spy-HP (1 lM) under aerobic conditions. (b)
Relationship between the concentration of the hydroperoxide and the
ratio of the fluorescence intensity. F0 and F denote the fluorescence
intensities at 535 nm before and after the addition of the hydroper-
oxide, respectively.
Acknowledgments
This work was supported by Industrial Technology
Research Grant Program in 2005 (Grant No.
05A01507a to N.S.) from New Energy and Industrial
Technology Development Organization (NEDO) of
Japan, and by the Iketani Science and Technology
Foundation (Grant No. 0171030A to N.S.).
MCPBA). A mercury–xenon lamp (Moritex, MUV-
202U) was used to acquire the images. As seen from
Figure 2, it can be clearly confirmed with the naked
eye that weakly fluorescent Spy-HP reacts with hydro-
peroxide to give strongly fluorescent Spy-HPOx, owing
to the extremely high fluorescence quantum yields (ꢀ1)
of Spy-HPOx and the efficient PET effect induced in
Spy-HP.
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In conclusion, we report on the successful development of
a novel fluorescent probe for monitoring hydroperoxides,
Spy-HP, containing perylene 3,4,9,10-tetracarboxyl bisi-
mide as the main skeleton in the structure. Spy-HP react-
ed quantitatively with hydroperoxides to form its
oxidized derivative, Spy-HPOx, which emits a strong
fluorescence in the visible range, without being affected
by autoxidation or photobleaching. Due to these unique
properties, Spy-HP is expected to be a novel and useful
fluorescent probe to ‘spy’ on hydroperoxides, particularly
in biosamples. Furthermore, Spy-HP is also the first

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N. Soh et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2943–2946
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1.9 (m, 2H, CH2CHN), 2.1–2.3 (m, 2H, CH2CHN), 5.15–
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