Synthesis and properties of fluorescence probe for detection of peroxides in mitochondria

Article abstract of DOI:10.1016/j.bmcl.2010.05.017

In this study, a new type of fluorescence probe, diphenylpyrenylphosphine- conjugated alkyltriphenylphosphonium iodide which was accumulated in mitochondria, has been synthesized. This probe was detected peroxide in living cell. Comparison of the reactivity toward several peroxide indicated that the probe was existed in mitochondrial membrane. Using this probe, generation of peroxide in mitochondria of living cell was successfully visualized.

Full text of DOI:10.1016/j.bmcl.2010.05.017

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3911–3915
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and properties of fluorescence probe for detection of peroxides
in mitochondria
a
a
b
Kosei Shioji a, , Yu Oyama , Kentaro Okuma , Hiroyuki Nakagawa
*
a Department of Chemistry, Faculty of Science, Fukuoka University, Jonan-Ku, Fukuoka 814-0180, Japan
b Department of Earth System Science, Faculty of Science, Fukuoka University, Jonan-Ku, Fukuoka 814-0180, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, a new type of fluorescence probe, diphenylpyrenylphosphine-conjugated alkyltriphenyl-
phosphonium iodide which was accumulated in mitochondria, has been synthesized. This probe was
detected peroxide in living cell. Comparison of the reactivity toward several peroxide indicated that
the probe was existed in mitochondrial membrane. Using this probe, generation of peroxide in mitochon-
dria of living cell was successfully visualized.
Received 29 December 2009
Revised 25 April 2010
Accepted 8 May 2010
Available online 12 May 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Fluorescence probe
Triarylphosphine
Mitochondria
Lipid peroxide
Mitochondria are functionally important subcellular organelles;
they are a major source of reactive oxygen species (ROS) in mam-
malian cells and are major targets of oxidative damage. Such dam-
age to mitochondria has serious consequences such as cell death
caused by the termination of energy generation. A large number
of studies have been conducted on this topic.1 Primarily, the mito-
chondrial respiratory chain produces superoxide anions (O2ÁÀ),
which in turn react to form further reactive oxidants. Types of
damages caused by ROS to mitochondrial components include lipid
peroxidation, protein oxidation, and mitochondrial DNA muta-
tion.2,3 The lipid peroxidation of polyunsaturated fatty acids (PU-
FAs) incorporated in the mitochondrial membrane is one of the
most dangerous oxidative stresses, which induces a mitochondrial
permeability transition.4–6 In order to detect this stress, the devel-
opment of visualizable tools for microscopy is necessary. Although
a rhodamine-based fluorescence probe has been reported for the
selective detection of highly reactive oxygen species, to the best
of our knowledge, no studies have been conducted for the detec-
tion of peroxides in mitochondria.7,8 Triarylphosphines are highly
reactive with peroxides but not with O2ÁÀ. Diphenylpyrenylphos-
phine (DPPP) has potential utility in the detection of peroxides be-
cause phosphine is easily oxidized to yield phosphine oxide, which
has high fluorescence intensity.9 However, as these compounds do
not significantly accumulate within mitochondria, their effective-
ness remains limited. It has been reported that compounds that
conjugate to a triphenylphosphonium cation are preferentially ta-
ken up by mitochondria. Lipophilic cations penetrated through the
lipid bilayer; this is because the positive charge is dispersed over a
large surface area and the potential gradient enables their accumu-
lation into the matrix.10–12 For the detection of lipid peroxides in
mitochondria, we prepare a diarylpyrenylphosphine-conjugated
alkyltriphenylphosphonium moiety as a functional group for bind-
ing the moiety to the mitochondrial membrane. Here, we discuss
the localization of a fluorescence probe to mitochondria by
employing fluorescence microscopy and reactivity; this probe is
used for the detection of several peroxides in the mitochondrial
matrix.
The fluorescence probe [3-(4-phenoxyphenylpyrenylphosphi-
no)propyl]triphenylphosphonium iodide (MitoDPPP) was synthe-
sized through the six steps. [(4-Methoxy)phenyl]phenyl
pyrenylphosphine (1) was prepared from dichlorophenylphosphine
and anysyl magnesium bromide. The phosphine 1 was oxidized to
give corresponding phosphine oxide 2 in 97% yield. The phosphine
oxide 2 was converted to [4-(hydroxyl)phenyl]phenylpyrenylphos-
phine borane (4) via reduction of phosphine oxide 3 and hydrobora-
tion in 75% yield. The phosphine borane was conjugated to
3-iodopropyltriphenylphosphonium iodide (6) followed by depro-
tection with Et2NH to give MitoDPPP in 18% yield (Scheme 1). The
fluorescence intensity of MitoDPPP is low due to the intramolecular
quenching of the phosphinyl moiety.
The oxidation of MitoDPPP with methyl linoleate hydroperoxide
(MeLOOH), cumene hydroperoxide (CumOOH), tert-butyl hydroper-
oxide (t-BHP) and H2O2 proceeded in methanol to give phosphine
oxide (MitoDPPPO), whose fluorescence (kex 350 nm, kem 380 nm)
was 35 times higher than that of MitoDPPP (Scheme 2).
* Corresponding author.
E-mail address: shioji@fukuoka-u.ac.jp (K. Shioji).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.05.017

3912
K. Shioji et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3911–3915
OMe
O
P
O
P
OMe
OH
1) Pyrene bromide
H2O2
in CH2Cl2
BBr3
PhPCl2
P
in CH2Cl2
2)
MeO
MgBr
in THF
1
3
2
BH3
BH3
O
1) HSiCl3
2) BH3 THF
O
PPh3
I
OH
PPh3
I
.
NaH
DMF
Et2NH
P
P
P
in CHCl3
in toluene
5
+
MitoDPPP
4
I
I
PPh3
6
Scheme 1. Synthesis of MitoDPPP.
MitoDPPP is oxidized by peroxide but not by free radicals such
350
300
250
200
150
100
50
as hydroxyl radicals (OHꢀ) or O2
.
13 The reactivity of MitoDPPP was
ÁÀ
similar to that of diphenylpyrenylphosphine (DPPP).14 The reactiv-
ity of DPPP toward hydroperoxides in homogeneous solution have
been reported to depend on the steric factor around the hydroper-
oxyl group. Nevertheless CumOOH has sterically crowded group,
the reactivity was higher than that of t-BHP. Thus the reactivity
of peroxides in homogeneous solution was not only dominated
by steric factor of the peroxide but also polarity and solubility
(Fig. 1).
On the other hand, in liposome suspension, MitoDPPP is sup-
posed to localize within lipid membranes because of preferential
reaction with lipophilic hydroperoxides such as MeLOOH and Cu-
mOOH. Due to poor lipophilicity of H2O2, the reactivity was lower
than that in methanol. The oxidation of MitoDPPP with H2O2 was
moderately increased by addition of Fe2+ as a catalyst for Fenton
reaction. The hydroxyl radical will be able to oxidize unsaturated
olefin in liposome. The result indicates that the oxidation of lipo-
some was slight in this condition (Fig. 2).
0
0
3
6
9
12
15
Time (min)
Figure 1. Reaction of ROOH with MitoDPPP in methanol; d CumOOH 1.0 mM,
j t-BHP 1.0 mM, h H2O2 1.0 mM, N MeLOOH 1.0 mM, ꢁ control.
250
200
150
100
50
The localization of MitoDPPP was confirmed by comparing the
fluorescent microscopy results of human hepatoma HepG2 cells
obtained by staining MitoRed, a rhodamine-based mitochondrial
dye, with those of the HepG2 cells obtained by staining Mito-
DPPPO. The cells were plated on a glass cover slip that was set in
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with
serum and maintained at 37 °C for 24 h, as described in a previous
study.15 The cells were treated with 1.6
lM of MitoDPPP and
10 nM of MitoRed for 15 min under dark conditions, followed by
washing twice with a DPBS buffer and treatment with 1.0 mM of
t-BHP. The two dyes merged well, which indicated that MitoDPPP
was selectively localized in mitochondria (Fig. 3).
0
0
3
6
9
12
15
Time (min)
Next, we attempted the detection of peroxides in the mitochon-
dria of living cells by employing fluorescence microscopy. The
HepG2 cells were treated with 1.6 lM of MitoDPPP for 15 min un-
Figure 2. Reaction of ROOH with MitoDPPP in liposome; d CumOOH 1.0 mM,
j t-BHP 1.0 mM, h H2O2 1.0 mM, s H2O2 1.0 mM + Fe2+ 10
ꢁ control.
lM, N MeLOOH 10 lM,
der dark conditions, followed by washing twice with the PBS buf-
fer. The cells were subsequently treated with 1.0 mM of
peroxides or 10 lM of MeLOOH for 15 min. A comparison of the
brightness of the cells subjected to peroxide treatment with that
of the untreated cells, where the brightness of both these types
of cells was obtained by fluorescence microscopy, revealed that
after 15 min following peroxide treatment, there was an increase
in the fluorescence intensity of MitoDPPP loaded into the mito-
chondria in the cells. The result revealed the ability of MitoDPPP
to detect peroxides in mitochondria. On the other hand, in the case
of HepG2 cells loaded with MitoDPPP, the fluorescence intensity
did not increase after 15 min. The fluorescence intensities of Mito-
DPPPO, resulting from the oxidation of MitoDPPP loaded into intact
HepG2 cells, were also measured by fluorescence spectroscopy for
evaluating the effectiveness of MeLOOH, CumOOH, t-BHP, and
H2O2 as oxidants. The oxidations of MitoDPPP proceeded by Me-
LOOH, CumOOH, and t-BHP, on the other hand, the oxidation by
H2O2 was slow in comparison with lipophilic peroxide (Fig. 4).
PPh3
O
PPh3
O
O
P
3
I
3
P
I
ROOH ROH
MitoDPPPO
MitoDPPP
Scheme 2. Oxidation of MitoDPPP with peroxide.

K. Shioji et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3911–3915
3913
Figure 3. Localization of MitoDPPP in mitochondria in HepG2 cells. MitoDPPP and MitoRed were loaded into HepG2 cells. (a) Fluorescent image of MitoDPPPO formed by
oxidation of MitoDPPP with t-BHP; (b) fluorescent image of MitoRed; (c) brightfield image; (d) merged image.
12
16
14
12
10
8
10
8
6
4
6
4
2
2
0
H2O2
control
t-BHP
CumOOH MeLOOH
0
H2O2
control
H2O2+3AT t-BHP
CumOOH MeLOOH
Figure 5. Reactivity of MitoDPPP with various peroxides in isolated mitochondria
at 37 °C. Isolated mitochondria from 2.0 Â 105 cells loaded MitoDPPP was treated
Figure 4. Reactivity of MitoDPPP with various peroxides in HepG2 cells at 37 °C.
HepG2 cells loaded MitoDPPP was treated 1.0 mM of peroxide (MeLOOH: 10 M).
l
10 lM of peroxide (MeLOOH: 1.0 lM). The figure shows relative fluorescence
The figure shows relative fluorescence intensity (F/F0 measured at 380 nm with
intensity (F/F0 measured at 380 nm with excitation at 350 nm), after the reaction
excitation at 350 nm), after the reaction with peroxides for 15 min.
with peroxides for 15 min.
The tendency of the reactivity is similar to the reaction in liposome
solution. Cells have quenching systems for H2O2. Catalase is one of
enzyme in the system. In order to judging whether catalase would
be acted as a scavenger for H2O2, the oxidation of HepG2 cells load-
ing MitoDPPP was carried out by H2O2 in the presence of 3-amin-
otriazol (3-AT) as an inhibitor of catalase. The fluorescence
intensity was not so increased by addition of 3-AT (10 mM). The
difference between their efficiencies as oxidants was caused by
the difference in their membrane permeability. It was reported
that t-BHP was a membrane-permeable reagent for inducing cell
death through apoptosis and necrosis.16,17 These phenomena are
thought to be mediated by the generation of free radicals and by
lipid peroxidation. The difference in the present results of the oxi-
dation efficiencies of H2O2 and other peroxide originated from the
difference in their membrane permeability. The efficiencies of
these peroxides in isolated mitochondria18 were similar to those
of intact cell and liposome solution (Fig. 5). These results suggest
that MitoDPPP accumulates in mitochondrial membrane.
It has been reported that lipophilic triarylphosphines that accu-
mulate in a cell membrane do not detect H2O2.19 When MitoDPPP
loaded HepG2 cells were washed with PBS buffer containing 5%
DMSO solution, the fluorescence intensity of some cells were in-
creased by the oxidation with H2O2 (Fig. 6). DMSO is a widely used
agent in cell biology. It is well known as a cryoprotectant and a
permeability enhancing agent.20,21 On the basis of the difference
in the reactivity for peroxide, at least, diphenylpyrenylphosphinoyl
moiety of MitoDPPP is buried into the mitochondrial lipid bilayer,
and the oxidation with H2O2 promotes by DMSO.
Finally, we attempted to detect lipid peroxide generating in
cells. The MitoDPPP loaded cell was treated with glutamate
(100 lM), which inhibit cystine transport leading to both reduc-
tion of intraceller cystine and GSH resulting that lipid peroxidation
of membrane was caused by intraceller H2O2.22 When the HepG2
cells were incubated with glutamate solution for 20 h, the fluores-
cence intensity was increased by oxidation with peroxide (Fig. 7b).
After incubating with glutamate for 2 h, the increasing of intensity

3914
K. Shioji et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3911–3915
Figure 6. Distribution map of brightness of HepG2 cells loading MitoDPPP: (a) washing with DPBS buffer prior to treat with H2O2; (b) washing with DPBS buffer containing 5%
DMSO prior to treat with H2O2.
Figure 7. Distribution map of brightness of HepG2 cells loading MitoDPPP: (a) control (loading 1.6
l
M of MitoDPPP for 15 min, washing twice with DPBS buffer, incubating
for 20 h in dark); (b) treatment with glutamate (100
for 2 h with 5% DMSO washing before measuring.
lM) for 20 h; (c) treatment with glutamate for 2 h with addition of 3-AT before measuring; (d) treatment with glutamate
was too small to detect by microscope. In the presence of 3-AT, the
fluorescence intensity did not also increase (Fig. 7c). However,
washing with PBS buffer containing 5% DMSO after glutamate
treatment, the intensity was increased significantly (Fig. 7d). The
difference suggests that MitoDPPP is difficult to contact with
H2O2 produced in mitochondria without DMSO treatment. Thus,
oxidation of MitoDPPP in mitochondrial membrane might be dom-
inated by the formation of lipid peroxide of the membrane.
In summary, we have synthesized a fluorescent probe, localized
to mitochondria, for the selective detection of peroxides that are
permeable in membrane and cause cell death. Observations made
using this probe have revealed that lipophilic peroxide effectively
penetrates the mitochondrial lipid bilayer. H2O2 in mitochondria
sensitive fluorescence probe (MitoPY1) has been repoted.23 The
probe has hydrophilic moiety as a functional group. In contrast,
due to the high hydrophobicity of functional group in our probe,
the affinity of the moiety to mitochondrial membrane is higher
than that of MitoPY1. Since the present probe is inactive toward
H2O2 in mitochondria, lipid peroxides can be distinguished from
H2O2.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.05.017.
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