Novel fluorescent probe for analysis of hydroperoxides based on boron dipyrromethane fluorophore

Article abstract of DOI:10.1271/bbb.80899

A new reagent, N-[2-(diphenylphosphino)ethyl]-4-(1,3,5,7-tetramethyl-4,4- difluoro-4-bora-3a,4a-diaza-sindacene-8-yl)benzamide (DPPEA-BODIPY), was designed and synthesized for analysis of hydroperoxides. DPPEA-BODIPY fluoresces at low levels in the visibl

Full text of DOI:10.1271/bbb.80899

Biosci. Biotechnol. Biochem., 73 (5), 1215–1217, 2009
Note
Novel Fluorescent Probe for Analysis of Hydroperoxides Based
on Boron Dipyrromethane Fluorophore
y
y
1
Naoko INOUE,1; Yoshio SUZUKI,1; Kenji YOKOYAMA,1;2 and Isao KARUBE
1Research Center of Advanced Bionics, National Institute of Advanced Industrial Science and Technology (AIST),
Katayanagi Institute, Tokyo University of Technology, Tokyo 192-0982, Japan
2Research Center of Advanced Bionics, National Institute of Advanced Industrial Science and Technology (AIST),
Tsukuba Central 4, Tsukuba, Ibaraki 305-8562, Japan
Received December 18, 2008; Accepted February 7, 2009; Online Publication, May 7, 2009
[doi:10.1271/bbb.80899]
A new reagent, N-[2-(diphenylphosphino)ethyl]-4-
(1,3,5,7-tetramethyl-4,4-difluoro-4-bora-3a,4a-diaza-s-
indacene-8-yl)benzamide (DPPEA-BODIPY), was de-
signed and synthesized for analysis of hydroperoxides.
DPPEA-BODIPY fluoresces at low levels in the visible
range (ꢀex=ꢀem = 502 nm/515 nm) and reacts with hy-
droperoxides to produce DPPEA-BODIPY oxide, which
fluoresces at high levels. The fluorescence intensity of
the reaction mixture was observed to be linearly related
to the methyl linoleate hydroperoxide (MeLOOH)
concentration.
dissolved in anhydrous CH2Cl2 (100 ml), and this
mixture was degassed by bubbling a nitrogen gas
through it for 30 min. TFA (one drop) was added to
the solution, which was stirred for 15 h at room
temperature under
a nitrogen atomosphere. DDQ
(5.97 g, 26.26 mmol), dissolved in a mixed solvent of
anhydrous THF (25 ml) and anhydrous CH2Cl2 (25 ml),
was added dropwise over a period of 15 min to the
mixture, which was stirred for 4 h at room temperature.
A saturated aqueous NaHCO3 was added, and the
mixture was extracted with CH2Cl2. The organic layer
was washed with water and brine, dried over Na2SO4,
and concentrated in vacuo. The crude product was
purified by aminopropyl silica gel column chromatog-
raphy using hexane-ethyl acetate to afford 3 (1.71 g,
yield 19.5%), NMR ꢁH (400 MHz, TMS, CDCl3): 1.27
(6H, s, CH3), 2.34 (6H, s, CH3), 3.96 (3H, s, CH3), 5.89
(2H, s, CH), 7.40–7.42 (2H, d, J ¼ 7:9 Hz, benzene),
8.11–8.13 (2H, d, J ¼ 8:0 Hz, benzene), MS m=z: calcd.
for C21H23N2O2 [M + H]þ: 335.18, found: 335.20. 3
(1.6 g, 4.78 mmol) was dissolved in anhydrous toluene
Key words: BODIPY; hydroperoxide; lipid hydroperox-
ide; reagent
Recently, reactive oxygen has attracted attention for
its association with disease. In particular, lipid hydro-
peroxides have been connected to arteriosclerosis,
hyperlipidemia, cancer, and aging.1–5) Several analytical
methods have been proposed for lipid hydroperoxides
and lipid peroxidation.6)
.
Diphenyl-1-pyrenylphophine (DPPP) is used as a
fluorescence probe for the imaging of lipid peroxidation
and the flow injection analysis of lipid hydroperoxide.
However, the excitation and emission wavelengths of
DPPP are in such a UV range (ꢀex=ꢀem = 352 nm/380
nm)7–10) that they damage the biological sample and
suffer interference by autofluorescence from the bio-
logical sample.11) Therefore, a novel reagent that has the
excitation and emission wavelengths in the visible
region is desired.
BODIPY fluorophores, which show high quantum
yields, hydrophobicity, and large extinction coefficients,
and fluoresce at around 550 nm, are widely used to
measure and to label the constituents of biological
samples.
In this study, for the analysis of hydroperoxides,
we designed and synthesized a new BODIPY reagent
(DPPEA-BODIPY) which utilized a photo-induced
electron transfer mechanism containing a diphenyl
phosphine.
Synthesis of DPPEA-BODIPY. As depicted in
Scheme 1, 2,4-dimethylpyrrole (5 g, 52.55 mol) and
methyl 4-formylbenzoate (4.31 g, 26.25 mmol) were
(65 ml), triethylamine (9.2 ml) and BF3 Et2O (9.2 ml)
were added and the mixture was stirred for 3 h at room
temperature. A saturated aqueous NaHCO3 was added
and the mixture was extracted with CH2Cl2. The organic
layer was washed with brine, dried over Na2SO4,
and concentrated in vacuo. The crude product was
purified by aminopropyl silica gel column chromatog-
raphy using hexane-ethyl acetate to afford 4 (1.76 g,
yield 96.2%). NMR ꢁH (400 MHz, CDCl3): 1.36 (6H,
s, CH3), 2.56 (6H, s, CH3), 3.97 (3H, s, CH3), 5.99 (2H,
s, CH), 7.40–7.42 (2H, d, J ¼ 8:0 Hz, benzene), 8.17–
8.19 (2H, d, J ¼ 8:3 Hz, benzene), MS m=z: calcd. for
C21H21BF2N2O2Na [M + Na]þ: 405.16, found: 405.21.
4 (0.5 g, 1.31 mmol) and potassium carbonate (0.3 g,
2.17 mmol) in THF-water (60 ml–60 ml) was stirred for
10 h at 50 ꢀC for 14 h at room temperature. After
cooling, the mixture was washed with diethyl ether, and
the water phase was extracted with CH2Cl2, dried over
Na2SO4, and concentrated in vacuo. This crude product
5 was used without further purification. MS m=z: calcd.
for C20H18BF2N2O2 [M ꢁ H]ꢁ: 367.14, found: 367.23.
5 (0.48 g), 1-ethyl-3-(3-dimethylaminopropyl)carbodii-
mide hydrochloride (0.89 g, 4.64 mmol), N,N-dimethyl-
y
To whom correspondence should be addressed. Naoko INOUE, Fax: +81-42-637-5379; E-mail: naoko-inoue@aist.go.jp; Yoshio SUZUKI,
Fax: +81-42-637-5379; E-mail: suzuki-yoshio@aist.go.jp

1216
N. INOUE et al.
MeO
O
MeO
O
A
MeO
O
CHO
TFA
BF3 · OEt2
Et3N
DDQ
N
H
CH2Cl2,THF
toluene
CH2Cl2
NHHN
N
HN
P
O
P
2
3
1
HN
O
HN
O
+ L-OOH
+ L-OH
MeO
O
HO
O
P
P
NH2
HN
O
N
DMAP, EDC
MeCN
K2CO3
THF,
(1:1)
H2O
N
N
F
N
N
F
N
N
N
N
B
B
B
B
F2
F2
F
F
N
B
DPPEA-BODIPY
DPPEA-BODIPY oxide
strong fluorescence
F2
weak fluorescence
6
5
4
B
Scheme 1. Synthesis Scheme for DPPEA-BODIPY.
350
300
250
200
150
100
50
4-aminopyridine (0.31 g, 2.54 mmol) and 2-(diphenyl-
phosphino)ethylamine (1 g, 4.36 mmol) were disolved in
anhydrous MeCN (75 ml), which was stirred under a
nitrogen atomosphere in the dark for 30 min at 0 ꢀC and
for an additional 4 d at room temperature. Distilled water
was added, and the mixture was extracted with CH2Cl2.
The organic layer was washed with distilled water and
brine, dried over Na2SO4, and concentrated in vacuo.
The crude product was purified in the dark by silica gel
column chromatography (chloroform) and subsequent
recrystallization under an argon atmosphere from diethyl
ether to afford 6 (160.26 mg, yield 21.1%, from com-
pound 4). NMR ꢁH (400 MHz, CDCl3): 1.34 (s, 6H,
CH3), 2.46–2.50 (2H, t, CH2), 2.56 (6H, s, CH3),
3.67–3.75 (2H, m, CH2), 5.98 (2H, s, CH), 6.30 (1H, s,
NH), 7.32–7.38 (8H, m, benzene), 7.47–7.52 (4H, m,
benzene), 7.70–7.72 (2H, d, J ¼ 8:3 Hz, benzene), MS
m=z: calcd. for C34H33BF2N3OPNa [M + Na]þ: 602.23,
found: 602.11.
The yield of DPPEA-BODIPY was 4%, and the
structure of DPPEA-BODIPY was characterized by
mass spectrometry (Waters ZQ, Nihon Waters, Tokyo)
and FT-NMR (Bruker AV400M Spectrometer, Bruker
BioSpin, Tsukuba, Japan). To prevent air oxidation,
DPPEA-BODIPY was stored at ꢁ20 ꢀC in the dark, and
was stable for two months.
DPPEA-BODIPY oxide was synthesized in the
oxidation manner in the literature.10) DPPEA-BODIPY
oxide: NMR ꢁH (400 MHz, CDCl3): 1.35 (6H, s, CH3),
2.56 (6H, s, CH3), 2.63–2.68 (2H, m, CH2), 3.87–3.95
(2H, m, CH2), 5.98 (2H, s, CH), 7.35–7.37 (2H, d, J ¼
8:0 Hz, benzene), 7.48–7.57 (6H, m, benzene), 7.75–
7.80 (4H, m, benzene), 7.93–7.95 (2H, d, J ¼ 8:0 Hz,
benzene), 8.12 (1H, s, NH), MS m=z: calcd. for
C34H33BF2N3O2PNa[M + Na]þ: 618.23, found: 618.15.
The reaction of DPPEA-BODIPY with lipid hydro-
peroxides and its excitation and emission spectra are
shown in Fig. 1. DPPEA-BODIPY was estimated to
fluoresce at low levels for the spacer between a
phosphine and fluorophore. The oxidant was expected
to fluoresce at high levels due to the hindrance of
photo-induced electron transfer like other phosphine
reagents.12)
1
2
0
400
450
500
Wavelength (nm)
550
600
Fig. 1. Reaction of DPPEA-BODIPY with Hydroperoxide (A),
Excitation Spectrum (1) and Emission Spectrum (2) of DPPEA-
BODIPY in Chloroform (B).
The excitation fluorescence spectrum of DPPEA-
BODIPY was measured in chloroform. The excitation
and emission wavelengths of both DPPEA-BODIPY and
its oxidant produced by the reaction between DPPEA-
BODIPY and hydrogen peroxide were approximately
500 nm (ꢀex=ꢀem = 502 nm/515 nm, Fig. 1), the same
as the other BODIPY reagents. The concentration of
quenching was also measured with DPPEA-BODIPY
and DPPEA-BODIPY oxide.
Lipid hydroperoxide analysis. The lipid hydroperox-
ide of the hydroperoxide sample was synthesized by air
oxidation of methyl linoleate. Its concentration was
quantified by the DPPP method,7) which was used to
evaluate the validity of lipid hydroperoxide analysis of
DPPEA-BODIPY. 2,6-Di-tert-butyl-p-cresol (BHT) was
dissolved at chloroform in 3 mg per 10 ml, and this was
used for the stock solution and the reaction solvent. The
DPPP method was modified with DPPPEA-BODIPY,
which was applied in MeLOOH concentration analysis.
In test tubes, 50 ml of 55.23 mM (2.76 nmol) DPPEA-
BODIPY was added to 100 ml of various concentrations
(0–61.76 mM) of MeLOOH. The test tubes were sealed
tightly with screw caps, protected from light, and
reacted at 37 ꢀC for 60 min. The reaction mixture was
cooled in an ice bath, and 3 ml of chloroform was added

Novel Fluorescent Probe for Analysis of Hydroperoxides
1217
BODIPY disappeared completely, and DPPEA-BODIPY
oxide was produced (Fig. 2B).
A
275
270
265
260
255
250
245
These results indicate that DPPEA-BODIPY reacts
with hydroperoxides quantitatively to produce DPPEA-
BODIPY oxide. This oxide fluoresces in the visible
range, which was able to use for the reagent of the
hydroperoxide determination.
Recently, the sensor has been studied for the
analytical methods. In the sensor, an optical chemical
sensor such as a sensing film is a relatively simple
analytical method that has several advantages, such as
rapidity and ease of preparation and procedure.13) The
hydrophobic probe is suitable for the sensing film
sensor, and DPPEA-BODIPY is a hydrophobic probe
based on BODIPY.
0
0
2
20
4
40
6
60
8 (nmol)
80 (µM)
Concentration of MeLOOH
B
602.18
On the other hand, the sensor fabricated on the
microelectromechanical system (MEMS) has been stud-
ied for the compact analytical system,14,15) and the probe
of long excitation and emission wavelength needs for
the down-sized analytical instrument.
100
1
Hence this new fluorescent molecular probe will be
used to investigate the quantity of hydroperoxides, and it
is expected to be useful in the development of analytical
methods such as sensors, which is in progress.
0
400
500
600
m/z
700
800
Acknowledgments
This work was supported by The Highly Integrated,
Complex MEMS Production Technology Development
Project (project code, P06022) of the New Energy
and Industrial Technology Development Organization
(NEDO) of Japan.
618.15
100
2
References
1) Kinoshita M, Oikawa S, Hayasaka K, Sekikawa A, Nagashima
T, Toyota T, and Miyazawa T, Clin. Chem., 46, 822–828
(2000).
0
400
500
600
m/z
700
800
2) Kumaraguruparana R, Kabalimoorthyb J, and Naginia S, Clin.
Biochem., 38, 154–158 (2005).
3) Rasheed MH, Beevi SS, and Geetha A, Oral Oncol., 43, 333–
338 (2007).
Fig. 2. Relationship between the Concentration of Methyl Linoleate
Hydroperoxide and Fluorescence Intensity (A), ESI Mass Spectra of
DPPEA-BODIPY (2.65 nmol) after Reaction with 0 nmol Methyl
Linoleate Hydroperoxide (1) and DPPEA-BODIPY (2.65 nmol) after
Reaction with 2.76 nmol Methyl Linoleate Hydroperoxide (2) (B).
4) Jurek D, Udilova N, Jozkowicz A, Nohl H, Marian B, and
Schulte-Hermann R, FASEB J., 19, 97–99 (2005).
5) Spiteller G, Exp. Gerontol., 36, 1425–1457 (2001).
6) Gray JI, J. Am. Oil Chem. Soc., 55, 539–546 (1978).
7) Akasaka K, Suzuki T, Ohrui H, and Meguro H, Anal. Lett., 20,
797–807 (1987).
to the mixture. The fluorescence intensity at 515 nm
(ex 502 nm) was measured.
8) Akasaka K, Ohrui H, and Meguro H, Anal. Lett., 21, 965–975
(1988).
Figure 2A shows the relation between the intensity of
fluorescence and the quantity of hydroperoxide. The
fluorescence intensity of the reaction mixture showed
a linear relationship with the MeLOOH concentration
(0–2.76 nmol:0–27.6 mM).
The reaction of the MeLOOH and DPPEA-BODIPY
was examined by mass spectrometry to confirm that the
high fluorescence was produced by DPPEA-BODIPY
oxide. Mass spectrometric results were obtained for two
reaction mixtures where 52.9 mM DPPEA-BODIPY
(2.65 nmol) reacted with MeLOOH (0 mM and 27.6 mM
(2.76 nmol)). Then they were reacted at 37 ꢀC for 60 min
by the DPPP modified method. In the 0 mM reaction
mixture, DPPEA-BODIPY oxide was not produced. On
the other hand, in the 27.6 mM reaction mixture, DPPEA-
9) Okimoto Y, Watanabe A, Nikia E, Yamashita T, and Noguchi
N, FEBS Lett., 474, 137–140 (2000).
10) Akasaka K, Suzuki T, Ohrui H, and Meguro H, Anal. Lett., 20,
731–745 (1987).
11) Hirano T, Kikuchi K, Urano Y, and Nagano T, J. Am. Chem.
Soc., 124, 6555–6562 (2002).
12) Soh N, Sakawaki O, Makihara K, Odo Y, Fukaminato T, Kawai
T, Irie M, and Imato T, Bioorg. Med. Chem., 13, 1131–1139
(2005).
13) Khezri B, Amini MK, and Firooz AR, Anal. Bioanal. Chem.,
390, 1943–1950 (2008).
14) Shukla S, Zhang P, Cho HJ, Ludwig L, and Seal S, Int. J.
Hydrogen Energy, 33, 470–475 (2008).
15) Lee JA, Hwang S, Kwak J, Park SI, Lee SS, and Lee KC, Sens.
Actuators B Chem., 129, 372–379 (2008).