Novel fluorescent probes for singlet oxygen

Article abstract of DOI:10.1002/(SICI)1521-3773(19991004)38:19<2899::AID-ANIE2899>3.0.CO;2-M

The first fluorescent chemical traps for ¹O₂ have been developed. DPAXs react specifically with ¹O₂ to yield the corresponding endoperoxides, DPAX-EPs (see scheme; X = H, Cl, F). DPAXs scarcely fluoresce, while DPAX-EPs are strongly fluorescent. Since the fluorescence of these probes is unaffected by H₂O₂, superoxide, and nitric oxide, they are useful for the selective detection of ¹O₂ in biological systems.

Full text of DOI:10.1002/(SICI)1521-3773(19991004)38:19<2899::AID-ANIE2899>3.0.CO;2-M

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[4] The frequently used terms ªtandemº (no time sequence!) or
ªdominoº (table game with 28 divided plates with differing numbers
of points) are less appropriate for sequential reactions that do not
mutually interfere. On the other hand ªcascadesº describe sequential
or stepwise events.
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Novel Fluorescent Probes for Singlet Oxygen**
Naoki Umezawa, Kumi Tanaka, Yasuteru Urano,
Kazuya Kikuchi, Tsunehiko Higuchi, and
Tetsuo Nagano*
Singlet oxygen (1O2), an excited state of molecular oxygen,
has aroused much interest as a chemical and biological
1
oxidant. The chemical reactivity of O2 is well characterized
since 1O2 is useful for organic synthesis and has unique
reactivity.[1] Singlet oxygen is thought to be an important toxic
species in vivo[2] since it can oxidize various kinds of biological
molecules such as DNA, proteins, and lipids, and its reactivity
toward DNA bases has been especially well characterized by
Foote et al.[3] Furthermore, Sies et al. and other researchers
1
have reported that O2 plays a role as an activator of gene
expression.[4]
1
Although many O2 traps have been reported,[5] it is still
difficult to detect 1O2 generated in biological systems because
1
of its short lifetime. The most widely used O2 trap is 9,10-
diphenylanthracene (DPA), which reacts rapidly with 1O2
specifically to form a thermostable endoperoxide at a rate
of k ˆ 1.3  106 m 1 s . The decrease in absorbance at
1 [6]
355 nm is used as a measure of the formation of the
endoperoxide. Many water-soluble DPA derivatives have
been developed,[7] but the quenching of O2 by water means
1
that they are difficult to apply to biological systems. Steinbeck
1
et al. have achieved the direct detection of O2 generation
from phagocytes with DPA by adapting the method to avoid
1O2 quenching.[8]
DPA derivatives are not very sensitive as probes because
the detection is based on the measurement of absorbance.
Hence, we designed and synthesized novel fluorometric
probes for 1O2 in order to improve the sensitivity. In general,
fluorescence measurement is more sensitive, and so is easier
to use in imaging studies, for example, fura-2 is used in Ca2‡
imaging.
[7] Recently, cis-1,2-diacetylethene and 1a were allowed to react in
benzene with analogous result (80% yield): G. Adembri, A. M. Celli,
A. Sega, J. Heterocycl. Chem. 1997, 34, 541.
[8] All new compounds gave correct IR, 1H, 13C NMR, and (highly
1
resolved) mass spectra, for example 3b: IR (KBr): nÄ ˆ 1691 cm
1
ˆ
(C O); H NMR (CDCl3, 300 MHz): d ˆ 8.00 (d, 2H), 7.35 (m, 8H),
4.22 (s, 2H), 3.54 (s, 3H), 3.41 (s, 3H), 2.61 (s, 3H); 13C NMR (CDCl3,
75 MHz): d ˆ 199.12, 165.93, 137.43, 136.91, 133.13, 132.53, 131.33,
130.66 (2C), 128.43 (2C), 128.36 (2C), 128.01 (2C), 127.87, 115.10,
We designed 9-[2-(3-carboxy-9,10-diphenyl)anthryl]-6-hy-
droxy-3H-xanthen-3-one (DPAX-1, Scheme 1) as a suitable
fluorescent probe. We chose fluorescein as a fluorophore
since it has a high fluorescence quantum yield in aqueous
solution and is able to be excited at long wavelength.
Excitation by visible light is preferable for biological appli-
cations as it minimizes cell damage and autofluorescence. We
then fused this fluorophore with the reactive moiety of DPA.
110.13, 50.24, 36.53, 31.77, 12.01; HR-MS: calcd. for C22H21NO3:
1
ˆ
347.1521; found: 347.1521. 5: IR (KBr): nÄ ˆ 1695 (C O), 1652 cm
1
ˆ
(C O); H NMR (CDCl3, 300 MHz): d ˆ 8.03 (d, 2H), 7.51 (m, 3H),
7.30 (m, 3H), 7.11 (m, 7H), 4.42 (s, 2H), 2.69 (t, J ˆ 6.05 Hz, 2H), 2.48
(t, J ˆ 6.6 Hz, 2H), 2.14 (quint, J ˆ 6.05 Hz, 2H); 13C NMR (CDCl3,
75 MHz): d ˆ 198.69, 195.12, 144.55, 137.67, 137.51, 132.71, 130.71,
130.31 (2C), 129.07 (3C), 128.48 (2C), 128.39 (2C), 128.19 (2C),
127.92, 127.80 (2C), 127.43, 119.16, 114.19, 38.33, 35.95, 23.70, 23.26;
HR-MS: calcd. for C28H23NO2: 405.1667; found: 405.1698. 11: IR
1
When DPAX reacts with O2 to yield DPAX-endoperoxide
(DPAX-EP) the conjugation between the DPA structure and
xanthene ring is greatly altered, so we expected a change in
fluorescence properties.
ˆ
ˆ
(KBr): nÄ ˆ 3305 (NH, sharp), 1680 (sh, C O), 1658 (C O), 1270,
1
1
ˆ
1218 cm (C S); H NMR (CDCl3/[D6]DMSO ca. 4/1, 300 MHz): d ˆ
8.08 (2NH), 7.52 (d, 2ArH), 7.01 (d, 2ArH), 6.40 (s, 1H), 2.82 (s, 3H);
13C NMR (CDCl3/[D6]DMSO, 75 MHz): d ˆ 180.2, 164.3, 152.7, 141.2,
131.2 (2C), 130.6 (2C), 124.3, 119.6, 101.4, 25.3; HR-MS: calcd. for
C12H10BrN3O2S: 340.9684; found: 340.9687.
[*] Prof. T. Nagano, Dr. N. Umezawa, K. Tanaka, Dr. Y. Urano,
Dr. K. Kikuchi, Dr. T. Higuchi
Graduate School of Pharmaceutical Sciences
The University of Tokyo
[9] SpecTool for Windows, Version 2.1, Chemical Concepts GmbH,
Weinheim, 1994.
7-3-1 Hongo, Bunkyo-ku, 113-0033 (Japan)
Fax : (‡ 81)3-5841-4855
[10] a) G. Kaupp, J. Schmeyers, J. Boy, Eur. J. Chem. 1998, 4, 2467; b) G.
Kaupp, J. Boy, J. Schmeyers, J. Prakt. Chem. 1998, 340, 346.
[11] J. C. J. Bart, G. M. J. Schmidt, Recl. Trav. Chim. Pays-Bas 1978, 97, 231.
[12] V. Bertolasi, P. Gilli, V. Ferretti, G. Gilli, Acta Crystallogr. Sect. B 1998,
54, 50.
E-mail: tlong@mol.f.u-tokyo.ac.jp
[**] This work was supported in part by a research grant from the Ministry
of Education, Science, Sports, and Culture of Japan. N.U. gratefully
acknowledges the Japanese Society for the Promotion of Science for
the JSPS Research Fellowship for Young Scientists.
[13] P. C. Thieme, E. Hädicke, Justus Liebigs Ann. Chem. 1978, 227.
Angew. Chem. Int. Ed. 1999, 38, No. 19
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Table 1. Absorbance and fluorescence properties of DPAXs and DPAX-
EPs.[a]
O
O
1
A [nm]
e [Â104 m 1 cm
]
Em [nm]
Ff
1O2
DPAX-1
DPAX-1-EP
DPAX-2
DPAX-2-EP
DPAX-3
DPAX-3-EP
493
494
507
506
493
494
6.1
7.9
5.7
8.9
7.6
6.7
516
515
524
527
514
515
0.007
0.53
0.006
0.66
0.006
0.70
COOH
COOH
X
X
X
X
HO
O
O
HO
O
O
X=H: DPAX-1-EP
Cl: DPAX-2-EP
F: DPAX-3-EP
X=H: DPAX-1
Cl: DPAX-2
F: DPAX-3
[a] All data were obtained at 208C in 0.1m NaOH (0.1% DMSO).
1
Scheme 1. Reaction of DPAXs with O2.
ance maxima (A), molar absorption coefficients (e), and
emission maxima (Em) were not altered much between
DPAXs and DPAX-EPs. However, the quantum efficiencies
of fluorescence (Ff) were greatly changed: DPAXs them-
selves are almost nonfluorescent and DPAX-EPs are highly
fluorescent. The maximum absorbance and emission of the
chlorinated compound DPAX-2-EP were shifted to longer
wavelengths than the other compounds.
We examined the pH profiles of fluorescence intensity for
the DPAX-EPs (Figure 1). The fluorescence intensities were
greatly decreased under acidic conditions. The pKa values of
DPAX-1-EP, DPAX-2-EP, and DPAX-3-EP were 6.6, 5.7, and
The fluorescence intensity of fluorescein derivatives is
known to be decreased under acidic conditions as a conse-
quence of the protonation of the phenoxide oxygen atom. In
order to stabilize the fluorescence intensity at physiological
pH we incorporated electron-withdrawing groups, Cl (DPAX-
2) and F (DPAX-3), at the 2- and 7-positions of the xanthene
chromophore. This modification lowered the pKa value of the
phenolic oxygen atom.
DPAX-1, DPAX-2, and DPAX-3 were synthesized accord-
ing to Scheme 2. The corresponding endoperoxides were
synthesized with chemically generated O2 (MoO4 /H2O2).
The fluorescence parameters are shown in Table 1; these
values were obtained under basic conditions, as the com-
pounds are highly fluorescent when deprotonated. Absorb-
1
2
O
O
COOCH3
COOCH3
b, c
a
O
+
O
O
O
O
O
O
C6H5
H
C6H5
O
COOCH3
COOCH3
COOCH3
COOCH3
d
O
+
O
O
Figure 1. Effect of pH on the fluorescence intensity of DPAX-EPs. DPAX-
EPs (1 mm; 0.1% DMSO as a cosolvent) were added to sodium phosphate
solution (0.1m) adjusted to various pH values. The fluorescence intensity of
the DPAX-EPs was measured at 515, 530, and 515 nm with excitation at
&
H
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
O
O
O
*
^
:
495, 505, and 494 nm, respectively. : DPAX-1-EP, : DPAX-2-EP,
COOCH3
COOCH3
DPAX-3-EP. F ˆ fluorescence intensity (in arbitrary units).
f –h
e
5.3, respectively. In contrast to the rapid decrease of
fluorescence intensity of DPAX-1-EP below pH 8, the
intensity was stable above pH 7 in the cases of DPAX-2-EP
and DPAX-3-EP. These profiles showed that DPAX-2 and
DPAX-3 should be useful as probes under neutral condi-
tions.
C6H5
C6H5
O
X
i
O
+
2
Then, we tried to detect chemical 1O2 production using
COOH
HO
OH
O
X
X
2
DPAX-2. The MoO4 /H2O2 system was again used as a
chemical source of 1O2. The reaction was performed at
HO
O
O
2
pH 10.5, since the MoO4 /H2O2 system only works effectively
X=H: DPAX-1
Cl: DPAX-2
F: DPAX-3
under basic conditions.[9] Hydrogen peroxide (final concen-
tration: 20 mm) was added to a buffer solution of DPAX-2
2
Scheme 2. Synthesis of DPAXs. a) THF, room temperature, 46%; b) H2O,
room temperature, 98%; c) CH3I, Cs2CO3, acetone, room temperature,
56%; d) CHCl3, reflux, quantitative yield; e) H2SO4, CH2Cl2, reflux, 41%;
f) dioxane, KOH, CH3OH, reflux, 91%; g) HCl, H2O, room temperature,
quantitative yield; h) Ac2O, reflux, 97%; i) CH3SO3H, 80 ± 858C (DPAX-1
(43%) and DPAX-2 (59%)) or 1508C (DPAX-3 (63%)).
(10 mm; 0.1% DMSO) and MoO4 (1 mm) every hour; the
2
reaction of MoO4 with H2O2 is slow and excess H2O2 inhibits
the reaction. The excitation and emission spectra of DPAX-2
are shown in Figure 2. Each spectrum was measured one hour
after the addition of H2O2. The fluorescence intensity
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and DPAX-2DA, however, cells were stained similarly in
both cases. This observation means that DPAX-2 itself is
membrane-permeable.
Received: April 19, 1999 [Z13290IE]
German version: Angew. Chem. 1999, 111, 3076 ± 3079
Keywords: dyes ´ endoperoxides ´ fluorescence spectrosco-
py ´ singlet oxygen
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Figure 2. a) Excitation spectra recorded at 530 nm and b) emission spectra
1
recorded at 505 nm of DPAX-2 in the reaction with O2 generated from a
2
MoO4 /H2O2 system. The reaction was performed at 258C in 0.1m sodium
phosphate buffer at pH 10.5 containing 0.1 mm EDTA.
increased with the generation of 1O2 in a dose-dependent
manner. An increase in fluorescence was also observed at
pH 7.4 using 3-(4-methyl-1-naphthyl)propionic acid endoper-
1
oxide (EP-1) as a O2 source.[10] These results showed that
DPAX-2 is a useful probe in both basic and neutral aqueous
solutions. In addition, we confirmed the production of DPAX-
2-EP in these reactions by HPLC. The fluorescence intensity
did not change upon reaction with H2O2, superoxide, and
nitric oxide, which are reported to be produced in biological
samples. The specificity of DPAX-2 for 1O2 is therefore
confirmed.
The detection of 1O2 in biological samples was also
investigated. For this we prepared DPAX-2 diacetate
(DPAX-2DA), which is thought to be more permeable to
the cell. DPAX-2DA will be hydrolyzed by intracellular
esterases to generate DPAX-2. We have tried both DPAX-2
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