C O M M U N I C A T I O N S
imaging of intracellular H
2 2
O . This fluorescein-based reagent
2 2
features excellent selectivity for H O over competing cellular ROS,
a large dynamic response range owing to its dual colorimetric/
fluorometric detection mechanism, and long-wavelength visible
excitation and emission profiles to minimize cell and tissue damage
while avoiding interfering autofluorescence from native cellular
species. Furthermore, we have demonstrated the value of this probe
by measuring changes in intracellular [H O ] within living mam-
2 2
malian cells. Current efforts are directed toward applying PF1 and
related tools for studying the oxidation biology of living systems.
Acknowledgment. We thank the University of California,
Berkeley, for startup funds and the Camille and Henry Dreyfus
Foundation for a New Faculty Award.
Supporting Information Available: Synthetic and experimental
details. This material is available free of charge via the Internet at http://
pubs.acs.org.
References
Figure 1. (A) Fluorescence response of 5 µM PF1 to 100 µM H2O2. The
dotted and solid line spectra were recorded before and after H2O2 addition,
respectively. Spectra were acquired in 20 mM HEPES, pH 7 (λexc ) 450
nm). (B) Fluorescence responses of 5 µM PF1 to various ROS (10 mM
(1) Halliwell, B.; Gutteridge, J. M. C. Free Radicals in Biology and Medicine,
3rd ed.; Clarendon Press: Oxford, UK, 1999.
(
2) Finkel, T. Curr. Opin. Cell Biol. 2003, 15, 247-254.
(3) Ohshima, H.; Tatemichi, M.; Sawa, T. Arch. Biochem. Biophys. 2003,
417, 3-11.
-
•
•
t
O2 , 100 µM for all other ROS). OH and O Bu were generated by reaction
(
4) Shah, A. M.; Channon, K. M. Heart 2004, 90, 486-487.
2
+
of Fe with H2O2 or tert-butyl hydroperoxide (TBHP), respectively. NO
was delivered using S-nitrosocysteine (SNOC). Spectra were acquired in
(5) Barnham, K. J.; Masters, C. L.; Bush, A. I. Nat. ReV. Drug DiscoVery
2004, 3, 205-214.
(
(
6) Wood, Z. A.; Poole, L. B.; Karplus, P. A. Science 2003, 300, 650-653.
7) Woo, H. A.; Chae, H. Z.; Hwang, S. C.; Yang, K.-S.; Kang, S. W.; Kim,
K.; Rhee, S. G. Science 2003, 300, 653-656.
2
0 mM HEPES, pH 7, and all data were obtained after incubation with the
appropriate ROS at 25 °C for 1 h. Collected emission was integrated between
60 and 700 nm (λexc ) 450 nm).
4
(
8) Budanov, A. V.; Slabina, A. A.; Feinstein, E.; Koonin, E. V.; Chumakov,
P. M. Science 2004, 304, 596-600.
(
9) Guyton, K. Z.; Liu, Y.; Gorospe, M.; Xu, Q.; Holbrook, N. J. J. Biol.
Chem. 1996, 271, 4138-4142.
(
10) Schmidt, K. N.; Amstad, P.; Cerutti, P.; Baeuerle, P. A. Chem. Biol. 1995,
2, 13-22.
(
11) Negre-Salvayre, A.; Aug e´ , N.; Duval, C.; Robbesyn, F.; Thiers, J.-C.;
Nazzal, D.; Benoist, H.; Salvayre, R. Methods Enzymol. 2002, 352, 62-
71.
(
(
(
12) Hempel, S. L.; Buettner, G. R.; O’Malley, Y. Q.; Wessels, D. A.; Flaherty,
D. M. Free Rad. Biol. Med. 1999, 27, 146-159.
13) Haugland, R. P. Handbook of Fluorescent Probes and Research Products,
9th ed.; Molecular Probes: Eugene, OR, 2002.
Figure 2. Confocal fluorescence and phase contrast images of live HEK
cells. (A) Fluorescence image of HEK cells incubated with 5 µM PF1 for
14) Akasaka, K.; Suzuki, T.; Ohrui, H.; Meguro, H. Anal. Lett. 1987, 20,
731-745.
5
min at 25 °C. (B) Fluorescence image of PF1-stained HEK cells treated
(15) Onoda, M.; Uchiyama, S.; Endo, A.; Tokuyama, H.; Santa, T.; Imai, K.
with 100 µM H2O2 for 5 min at 25 °C. (C) Brightfield image of live HEK
cells after H2O2 addition to confirm viability. Scale bar ) 30 µm.
Org. Lett. 2003, 5, 1459-1461.
(
16) Wolfbeis, O. S.; D u¨ rkop, A.; Wu, M.; Lin, Z. Angew. Chem., Int. Ed.
2002, 41, 4495-4498.
(
17) Setsukinai, K.; Urano, Y.; Kakinuma, K.; Majima, H. J.; Nagano, T. J.
Biol. Chem. 2003, 278, 3170-3175.
and brightfield transmission measurements after PF1 incubation and
H O addition (Figure 2C) confirm that the cells are viable
2 2
throughout the imaging experiments. These data establish that PF1
is membrane-permeable and can respond to micromolar changes
(
(
18) Lo, L.-C.; Chu, C.-Y. Chem. Commun. 2003, 2728-2729.
19) Maeda, H.; Fukuyasu, Y.; Yoshida, S.; Fukuda, M.; Saeki, K.; Matsuno,
H.; Yamauchi, Y.; Yoshida, K.; Hirata, K.; Miyamoto, K. Angew. Chem.,
Int. Ed. 2004, 43, 2389-2391.
(
20) Kuivila, H. G.; Armour, A. G. J. Am. Chem. Soc. 1957, 79, 5659-5662.
in H
2
O
2
concentrations within living cells. In addition, subsequent
(21) The corresponding 3′,6′-difluoro-, 3′,6′-dichloro-, and 3′,6′-dibromofluoran
analogues have been reported previously: Gronowska, J.; Dabkowska-
Naskret, H. Pol. J. Chem. 1981, 55, 2151-2163.
experiments show that fluorescence responses to 100 nM H
readily detectable in vitro.
2 2
O are
(22) Ishiyama, T.; Murata, M.; Miyaura, N. J. Org. Chem. 1995, 60,
7508-7510.
To close, we have presented the synthesis, properties, and
biological applications of PF1, a new type of probe for optical
JA0441716
J. AM. CHEM. SOC.
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