A Practical Fluorescent Probe for Superoxide
FULL PAPER
orescent probes useful for extra- and intracellularly generat-
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ed O2 , respectively.
This study has also confirmed the utility of a strategy
based on protection–deprotection chemistry for the design
of fluorescent probes, which would be difficult to achieve by
other available methodologies. This strategy can provide
novel fluorescent probes with tunable sensitivities and spe-
cificities by the appropriate selection of fluoresceins and
BES chlorides, rather than by using totally different chemi-
cal structures. The pools of available fluoresceins and BES
chlorides from which one may choose are diverse. This
raises the possibility that this strategy may be more general-
ly applied for the design of fluorescent probes for target
molecules that show specific reactivity in inducing the de-
protection of BES derivatives. Additional studies can be ex-
pected to result in the development of novel fluorescent
probes for other target molecules.
Acknowledgements
This work was supported in part by a Grant-in-Aid for Scientific Re-
search (B) (15390012) from the Ministry of Education, Culture, Sports,
Science and Technology, Japan, and by research grants from the Suntory
Institute for Bioorganic Research, and the Mochida Memorial Founda-
tion for Medical and Pharmaceutical Research.
Figure 5. Phase contrast (b, d, f) and fluorescence images (a, c, e) ob-
tained after incubating human Jurkat T cells loaded with BESSo-AM at
378C for 1 h in the absence (a, b) or the presence (c–f) of 5 mm butyric
acid. (e, f) Cells loaded with Tiron as well as BESSo-AM were used.
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tion with butyric acid. Since Tiron clearly functioned as an
intracellular scavenger of O2 in these experiments, the re-
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sults demonstrate that BESSo-AM is capable of serving as a
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fluorescent probe for the detection of intracellular O2 . This
intracellular assay with BESSo-AM thus revealed that ROS
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.
Conclusion
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We have tested bis- and mono-protected derivatives of 1c
with a number of BES groups, which were selected not only
to eliminate or significantly reduce undesired reactivity of
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the prototype O2 probe 6a, but also to validate our strat-
egy based on protection–deprotection chemistry as a novel
concept for probe design. Of the BES derivatives of 1c that
were examined, the 4,5-dimethoxy-2-nitro-BES derivative
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(BESSo) proved to be the best O2 probe. Thus, compared
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with 6a, BESSo allowed the measurement of O2 with
greater sensitivity, and exhibited greater specificity toward
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O2 relative to GSH and to ROS such as H2O2, NaOCl,
1
ꢀ
C
tBuOOH, O2, NO , and ONOO . The use of BESSo also
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significantly improved specificity toward O2 over Fe2+ as
well as over the reduced forms of CYP reductase and dia-
phorase. In particular, BESSo exhibited no fluorescent re-
sponse to GSH, which is ubiquitous in cells at mm levels.
These features allow BESSo and BESSo-AM to serve as flu-
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Chem. Eur. J. 2007, 13, 1946 – 1954
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