A design of fluorescent probes for superoxide based on a nonredox mechanism

Article abstract of DOI:10.1021/ja047018k

Fluorometric detection of O₂^-? is performed based on desulfonylation of 3 to the corresponding fluoresceins 4 through nucleophilic substitution, and this fluorescing process is quite specific toward O₂^-? over H<

Full text of DOI:10.1021/ja047018k

Published on Web 12/09/2004
A Design of Fluorescent Probes for Superoxide Based on a Nonredox
Mechanism
Hatsuo Maeda,* Kayoko Yamamoto, Yoko Nomura, Iho Kohno, Leila Hafsi, Noritsugu Ueda,
Shoko Yoshida, Masako Fukuda, Yuka Fukuyasu, Yuji Yamauchi, and Norio Itoh
Graduate School of Pharmaceutical Sciences, Osaka UniVersity, 1-6 Yamada-oka, Suita, Osaka 565-0871, Japan
Received May 20, 2004; E-mail: h-maeda@phs.osaka-u.ac.jp
Reactive oxygen species (ROS) such as superoxide (O2-•),
Scheme 1
hydrogen peroxide (H2O2), and hydroxyl radical (HO•) are impor-
tant mediators for the pathological conditions of various diseases.1
Chemiluminescence- and fluorescence-based assays have been
widely used to measure cell-derived O2-•.2 Although less sensitive
-•
than chemiluminescent methods, detecting O2 using fluorescent
probes takes full advantage of benefits provided by fluorescence
microscopy, a microplate reader, and a cell sorter. Hydroethidine
(HE) has been frequently used as a fluorescent probe.2,3 The major
drawback of HE is its poor selectivity toward O2 ,
-• 3b,d,4 which stems
from its oxidative fluorescing mechanism. Although O2-• also works
as a reductant, no fluorescent probe for O2-• based on a reductive
fluorescing mechanism has been reported. This is attributed to the
setback experienced in using spectrophotometric probes such as
cytochrome c and nitroblue tetrazolium. It is well-known that these
-•
measuring O2 generated by the enzymatic reaction of HPX and
-•
probes respond not only to O2 but also to reductases.2b,c,5 Thus,
XO at 37 °C for 10 min. The detection limit was 1.0 pmol (relative
standard deviation, n ) 8; 2.9%); up to 2.0 nmol of HPX, a good
linear calibration curve was obtained with a correlation coefficient
and slope of 0.9983 and 0.80 au/pmol, respectively.
the focus in developing a fluorometric detection method of cell-
-•
derived O2 is to design a fluorescing mechanism for probes
without the recourse of a redox reaction. The resolution of this
issue is of great importance, to impart a high degree of specificity
The absorption spectra observed after reaction of 3 either in the
HPX/XO system or with KO2 coincided with those of the
corresponding fluoresceins 4. Compound 3d (50 µmol) was
subjected to reaction with KO2 (4 equiv) in DMSO-HEPES buffer
(1:1) at room temperature. After 10 min, 3d was completely
consumed, and 2,4-dinitrobenzenesulfonic acid (6) was obtained
as the sole byproduct in 191% HPLC yield. Product analysis with
HPLC was also performed on a reaction mixture of 3d (0.25 µmol)
in the HPX (3 µmol)/XO (65 mU) system. After the enzymatic
reaction was performed in HEPES at 37 °C for 5 h, almost no 3d
remained (about 80% of 3d was consumed in 1 h), and two
byproducts were noted on a chromatogram. One of them was 6
formed in 149% yield, and the other has not yet been identified.
These results suggested that 3 is deprotected by O2-•, leading to
the formation of 4 and 2 equiv of 6. It appeared that either electron-
transfer reaction or nucleophilic substitution by O2-• was responsible
for this transformation of 3. On voltammetry in CH3CN, 3a-3d
exhibited irreversible cathodic waves at -0.60, -0.63, -0.63, and
-0.65 V vs Ag/AgCl, respectively. This small difference in the
reduction potentials was unlikely to provide reasonable explanation
on the observed big difference in their reactivity toward O2-•. Thus,
it was proposed that 3 is transformed to 4 by reaction with O2-• as
a nucleophile rather than a reducing agent, which requires at least
-•
to fluorescent probes for detecting O2 in complex biological
systems.
Recently it was found that H2O2 induced deprotection of
monopentafluorobenzenesulfonyl fluoresceins (1) (Scheme 1), but
the corresponding bis-protected derivatives 2 did not react with
H2O2 at all. Thus, we demonstrated that 1 work as novel H2O2
fluorescent probes and that acetylated 1 permeate the cells of green
algae and are transformed by cellular esterases into 1, which then
detect intracellular formation of H2O2.6 On the basis of these
findings, we further investigated what kinds of benzenesulfonyl
groups would be protection groups of choice on designing sulfo-
nylated fluoresceins as H2O2 probes. For this purpose, fluoresceins
doubly protected with various types of benzenesulfonyl groups were
chosen instead of the corresponding mono-protected ones. This is
not only because the former is much more easily synthesized than
the latter but also because, after screening protective groups of
choice, changing their protection mode from bis- to monosulfonyl-
ated types will provide an access to sensitive fluorescent probes.
When reactivity of several bis-sulfonylated fluoresceins toward O2-•
generated by a xanthine oxidase (XO)/hypoxanthine (HPX) system
in the presence or absence of superoxide dismutase (SOD) as well
as H2O2, we found that bis(2,4-dinitrobenzenesulfonyl) fluoresceins
(3) (Scheme 1) function as fluorescent probes not for H2O2 but for
-•
two molecules of O2 for the formation of one molecule of 4
O2-•. This paper proposes a novel fluorescing reaction to design
(Scheme 2).
-•
The reactivity of 3 toward O2-•, other ROS, reducing compounds,
reductases, glutathione (GSH), and esterase was compared. GSH
resulted in large fluorescent augmentation from 3a, 3b, and 3c via
nucleophilic aromatic substitution7 (see Supporting Information),
while 3d provided relatively small response toward GSH in
fluorescent probes for specific detection of O2
.
Enzymatic reaction of 3, XO, and HPX was traced fluorometri-
cally for 10 min, yielding progressive curves (see Supporting
Information). Among these probes, 3d provided the largest fluo-
rescent response to the enzymatic reaction. A 96-well microplate
assay with this probe provided a highly sensitive method for
-•
comparison with that for O2 (Table 1). These results suggested
9
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J. AM. CHEM. SOC. 2005, 127, 68-69
10.1021/ja047018k CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
Scheme 2. Proposed Mechanism for Deprotection of 3 to 4 by
O2-•
responses observed for the stimulated cells were effectively reduced
by addition of SOD (1000 U/mL, 10 µL). This result clearly
indicated that the fluorescent responses observed with 3d on PMA-
-•
stimulated neutrophils arise from O2 released by the cells.
Incremental increases in fluorescence intensity were also observed
for PMA-stimulated neutrophils in the presence of SOD. This was
thought to be due to endogenous thiols and reductases. The
fluorescent augmentation observed for unstimulated neutrophils was
ascribed not only to similar effects by these biological compounds
but also to neutrophil activation by the interaction of the cells with
the surface of the used tissue culture plate, as previously reported.8
In summary, the present work proposes a novel methodology
Table 1. Relative Fluorescent Intensity (RFI) Observed upon
Reaction of 3d with ROS and Other Biological Compounds
compounds
RFIa
compounds
RFIa
blank
10
554
60
11
10
NO• (NOC-5)c
13
13
11
11
104
O2-• (XO/HPX)
O2-• (XO/HPX)/SOD
H2O2
ONOO- (SIN-1)d
ascorbic acid
1,4-hydroquinone
CyP450 reductase/NADPH
-•
for designing fluorescent probes for O2 based on a nonredox
t-BuOOH
-•
mechanism, allowing highly specific detection of O2 over other
NaOCl
12
16
56
91
1O2 (H2O2 + NaOCl)b
diaphorase/NADH
GSH
esterase
36
60
15
ROS. The undesired reactivity of a prototype probe 3d toward thiols
and reductases is thought to come from the 2,4-dinitrobenzene-
sulfonyl functionality. Thus, we expect that more practical fluo-
rescent probes than 3d will be developed by a judicious choice
between bis and mono protection modes as well as the identities
of benzenesulfonyl groups. Further studies along these lines are
currently under way in our laboratory.
HO• (H2O2 + Fe2+
)
Fe2+
a Estimated by setting the observed blank response for each probe as
10. b-d See refs 9, 10, and 11, respectively.
Acknowledgment. This work was supported in part by a Grant-
in-Aid for Scientific Research (B) (15390012) from the Ministry
of Education, Culture, Sports, Science and Technology of Japan,
and by research grants from Shimadzu Science Foundation, Suntory
Institute for Bioorganic Research, and the Mochida Memorial
Foundation for Medical and Pharmaceutical Research.
Supporting Information Available: Detailed experimental pro-
cedures, spectral data of 3, and additional data of fluorometric
measurements with 3. This material is available free of charge via the
Internet at http://pubs.acs.org.
Figure 1. Time course for the change in fluorescence intensity observed
with 3d for PMA-stimulated or unstimulated human neutrophils (1 × 105
cells/well). The data points are the mean ( standard deviation (n ) 8).
that 3a, 3b, and 3c would be useless as O2-• probes. This is thought
to be because GSH generally exists at a relatively high concentration
in cells. As summarized in Table 1, 3d provided highly specific
References
(1) Halliwell, B.; Gutteridge, J. Free Radicals in Biology and Medicine, 3rd
ed.; Clarendon Press: Oxford, 1999.
(2) For selected late reviews, see: (a) Nakano, M. Cell. Mol. Neurobiol. 1998,
18, 565-579. (b) Murrant, C. L.; Reid, M. B. Microsc. Res. Tech. 2001,
55, 236-248. (c) Tarpey, M. M.; Fridovich, I. Circ. Res. 2001, 89, 224-
236. (d) Mu¨nzel, T.; Afanas’ev, I. B.; Klescchyov, A. L.; Harrison, D. G.
Arterioscler., Thromb., Vasc. Biol. 2002, 22, 1761-1768. (e) Esposti, M.
D. Methods 2002, 26, 335-340.
-•
fluorescent response toward O2 over other ROS. The response
observed in a Fenton system is not ascribed to HO• because Fe2+
itself caused deprotection of 3d. Ascorbic acid, 1,4-dihydroquinone,
and esterase induced only negligible response from 3d. Fluorescent
augmentation was also observed with the reduced forms of XO,
cytochrome P450 (CyP450) reductase and diaphorase, as did GSH
and Fe2+. Among these reducing systems, CyP450 reductase-
NADPH induced the largest response from 3d, 19% of that observed
for O2-•. However, half of this response was thought to be a result
of the generation of O2-• in the enzyme system, since SOD reduced
the fluorescence augmentation observed for CyP450 reductase/
NADPH by 50%. Thus, among the present probes, 3d is the best
fluorescent probe for detecting O2-• with relatively high specificity.
The applicability of 3d as a probe for a fluorescence-based assay
(3) For selected references, see: (a) Rothe, G.; Valet, G. J. Leukoc. Biol.
1990, 47, 440-448. (b) Carter, W. O.; Narayanan, P. K.; Robinson, J. P.
J. Leukocyte Biol. 1994, 55, 253-258. (c) Bindokas, V. P.; Jorda´n, J.;
Lee, C. C.; Miller, R. J. J. Neurosci. 1996, 16, 1324-1336. (d) Al-Mehdi,
A. B.; Shuman, H.; Fisher, A. B. Am. J. Pysiol. 1997, 272, L294-L300.
(4) Benov, L.; Sztejnberg, L.; Fridovich, I. Free Radical Biol. Med. 1998,
25, 826-831.
(5) Beauchamp, C.; Fridovich, I. Anal. Biochem. 1971, 44, 276-287.
(6) Maeda, H.; Fukuyasu, Y.; Yoshida, S.; Fukuda, M.; Saeki, K.; Matsuno,
H.; Yamauchi, Y.; Yoshida, K.; Hirata, K.; Miyamoto, Y. Angew. Chem.,
Int. Ed. 2004, 43, 2389-2391.
(7) Fukuyama, T.; Cheung, M.; Jow, C.-K.; Hidai, Y.; Kan, T. Tetrahedron
Lett. 1997, 38, 5831-5834.
(8) Mohanty, J. G.; Jaffe, J. S.; Schulman, E. S.; Raible, D. G. J. Immunol.
Methods 1997, 202, 133-141.
-•
(9) For example, see: Kajiwara, T.; Kearns, D. R. J. Am. Chem. Soc. 1973,
95, 5886-5890. (b) Held, A. M.; Halko, D. J.; Hurst, J. K. J. Am. Chem.
Soc. 1978, 100, 5732-5740.
of cell-derived O2 was demonstrated by experiments using
neutrophils stimulated with phorbol myristate acetate (PMA). A
cell suspension (1.0 × 106 cells/mL, 100 µL) was incubated at 37
°C with 3d (25 µM, 50 µL) in the presence or absence of PMA
(0.64 µM, 50 µL). As shown in Figure 1, the assay with 3d provided
much larger fluorescence augmentation at each measurement point
for PMA-stimulated neutrophils than unstimulated cells. The
(10) Feelisch, M. J. CardioVasc. Pharmacol. 1991, 17 (Suppl. 3), 25-33.
(11) Hrabie, J. H.; Klose, J. R.; Wink, D. A.; Keefer, L. K. J. Org. Chem.
1993, 58, 1472-1476.
(12) Sun, W.-C.; Gee, K. R.; Klaubert, D. H.; Haungland, R. P. J. Org. Chem.
1997, 62, 6469-6475.
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