A targetable fluorescent probe for imaging hydrogen peroxide in the mitochondria of living cells

Article abstract of DOI:10.1021/ja802355u

We present the design, synthesis, and biological applications of mitochondria peroxy yellow 1 (MitoPY1), a new type of bifunctional fluorescent probe for imaging hydrogen peroxide levels within the mitochondria of living cells. MitoPY1 combines a chemoselective boronate-based switch and a mitochondrial-targeting phosphonium moiety for detection of hydrogen peroxide localized to cellular mitochondria. Confocal microscopy and flow cytometry experiments in a variety of mammalian cell types show that MitoPY1 can visualize localized changes in mitochondrial hydrogen peroxide concentrations generated by situations of oxidative stress. Copyright

Full text of DOI:10.1021/ja802355u

Published on Web 07/08/2008
A Targetable Fluorescent Probe for Imaging Hydrogen Peroxide in the
Mitochondria of Living Cells
Bryan C. Dickinson and Christopher J. Chang*
Department of Chemistry, UniVersity of California, Berkeley, California 94720
Received April 2, 2008; E-mail: chrischang@berkeley.edu
Scheme 1. Synthesis and Activation of MitoPY1
Hydrogen peroxide (H2O2) is an increasingly recognized small-
molecule mediator of physiology, aging, and disease in living
organisms.1–6 In this regard, aberrant production or accumulation
of H2O2 within cellular mitochondria over time due to environ-
mental stress(es) and/or genetic mutation(s) is connected to serious
diseases where age is a risk factor, including cancer7 and neuro-
degenerative Alzheimer’s, Parkinson’s, and Huntington’s diseases.8,9
Indeed, overexpression and mitochondrial targeting of catalase, a
peroxide-detoxifying enzyme, can increase life span in mouse
models.10 On the other hand, newer data suggest that controlled
bursts of mitochondrial H2O2 can also serve beneficial roles for
cell survival, growth, differentiation, and maintenance.3–6
New imaging methods that allow visualization of localized
production and accumulation of mitochondrial H2O2 in living
samples are potentially useful for disentangling the complex
contributions of this reactive oxygen species (ROS) to both healthy
and diseased states. Synthetic fluorescent H2O2 indicators that can
be targeted to precise subcellular locations offer one approach to
this goal and do not require transfection like their protein
counterparts,11,12 but traditional ROS indicators such as dihydro-
rhodamine (DHR) are uncharged and hence not preferentially
localized in cells before oxidation.13 In addition, DHR and related
dyes are not specific for H2O2 over other ROS. Accordingly,
mitochondrial-targeted small molecules for detection of specific
ROS remain rare,13,14 and none of the probes reported to date are
selective for H2O2. We now report the synthesis and applications
of mitochondria peroxy yellow 1 (MitoPY1), a new type of
fluorophore for imaging mitochondrial H2O2 in living cells with
ROS and spatial specificity.
Our overall strategy for fluorescence imaging of mitochondrial
H2O2 in living systems is to create bifunctional dyes that contain
both a peroxide-responsive element and a mitochondrial-targeting
moiety. For the latter purpose, we were inspired by the use of
phosphonium head groups by Murphy and others to deliver
antioxidants, electrophiles, and EPR and optical probes to mito-
chondria, as these and related lipophilic cations selectively ac-
cumulate in this organelle due to proton gradient considerations.14–16
In addition, we sought a modular synthetic route that would allow
facile introduction of a phosphonium or any other desired targeting
group after installation of the boronate switch, which circumvents
potential complications arising from sensitive functionalities that
are incompatible with palladium-catalyzed Miyaura-Suzuki reac-
tions typically used to introduce the H2O2-cleavable boronate cage.
Both of these design criteria can be met by the approach outlined
in Scheme 1 for the synthesis of MitoPY1. The ability to append
additional groups postboronation offers a host of opportunities for
generating new multifunctional H2O2 imaging probes.
fluorescence increase by its conversion to MitoPY1ox, which
possesses one major absorption band at 510 nm (ꢀ ) 22 300 M-1
cm-1) and enhanced emission (λem ) 528 nm, Φ ) 0.405). Kinetics
measurements of the H2O2-mediated boronate deprotection were
performed under pseudo-first-order conditions (5 µM dye, 10 mM
H2O2), giving an observed rate constant of k ) 2.0(1) × 10-3 s-1
.
Figure 1 shows the relative turn-on fluorescence responses of
MitoPY1 to a panel of biologically relevant ROS. Owing to its
chemospecific boronate switch,17,18 the probe is selective for H2O2
over ROS like superoxide, nitric oxide, and hydroxyl radical.
MitoPY1 was then tested for its ability to both target the
mitochondria and respond to H2O2 in living biological systems.
Cervical cancer HeLa cells loaded with 5 µM MitoPY1 for 1 h at
37 °C show faint but measurable levels of fluorescence in discrete
Figure 1. Fluorescence responses of 5 µM MitoPY1 to various reactive
oxygen species (ROS). Bars represent relative responses at 0, 5, 15, 30,
45, and 60 min after addition of each ROS. Data shown are for 10 mM
O2-, 200 µM NO, and 100 µM for all other ROS. Data were acquired at
25 °C in 20 mM HEPES, pH 7, with excitation λ ) 503 nm and emission
collected between 510 and 750 nm.
MitoPY1 features two major visible region absorptions (λabs
)
)
489 nm, ꢀ ) 14 300 M-1 cm-1; 510 nm, ꢀ ) 14 200 M-1 cm-1
and a weak emission (λem ) 540 nm, Φ ) 0.019, Figure S1) in 20
mM HEPES, pH 7. Reaction of MitoPY1 with H2O2 triggers a
9
9638 J. AM. CHEM. SOC. 2008, 130, 9638–9639
10.1021/ja802355u CCC: $40.75
2008 American Chemical Society

C O M M U N I C A T I O N S
Figure 2. Confocal fluorescence images of live HeLa cells with increases in mitochondrial H2O2 levels visualized using MitoPY1. Images displayed represent
emission intensities collected in optical windows between 527 and 601 nm upon excitation at 510 nm for MitoPY1. HeLa cells incubated with 5 µM
MitoPY1 for 60 min at 37 °C and imaged with MitoPY1 (a), MitoTracker Red and Hoechst (overlay, b), and MitoPY1 with MitoTracker Red (overlay, c).
HeLa cells incubated with 5 µM MitoPY1 for 60 min at 37 °C with 100 µM H2O2 added for the final 40 min and imaged with MitoPY1 (d), MitoTracker
Red and Hoechst (overlay, e), MitoPY1 and MitoTracker Red (overlay, f), and brightfield (g) with 20 µm scale bar. HeLa cells incubated with 5 µM
MitoPY1 for 60 min at 37 °C and imaged with MitoPY1 (h), MitoTracker Red and Hoechst (overlay, i), and MitoPY1 with MitoTracker Red (overlay, j).
HeLa cells incubated for 24 h with 1 mM paraquat, then washed and incubated with 5 µM MitoPY1 for 60 min at 37 °C and imaged with MitoPY1 (k),
MitoTracker Red and Hoechst (overlay, l), MitoPY1 and MitoTracker Red (overlay, m), and brightfield (n) with 20 µm scale bar.
subcellular locations as determined by confocal microscopy (Figure
2a). Co-staining experiments with 50 nM MitoTracker Deep Red,
a commercially available mitochondrial indicator (Figure 2b,c), or
500 nM LysoTracker Red, a lysosomal indicator (Figures S4-S7),
establish that the observed fluorescence from MitoPY1 is localized
to the mitochondria of these live cells. Addition of 100 µM H2O2
to HeLa cells loaded with MitoPY1 display a marked localized
increase in fluorescence compared to control cells (Figure 2d-f).
Again, co-staining with MitoTracker confirms that the dye is
retained in the mitochondria and detects localized rises in H2O2
concentrations. Brightfield measurements and nuclear staining with
Hoechst 33342 indicate that the cells are viable throughout the
imaging experiments (Figures 2b,e,g). In addition, control experi-
ments using a probe lacking the phosphonium targeting moiety
(ContPY1, Figures S9-S12) or the oxidized probe (MitoPY1ox,
Figures S13-S18) confirm that only MitoPY1 targets the mito-
chondria, and complementary flow cytometry experiments (Figure
S8) provide supporting data over a larger population of cells.
Finally, analogous experiments in Cos-7, HEK293, and CHO.K1
cell lines give similar results and expand the scope of the probe
(Figures S5-S7). Taken together, these data establish that MitoPY1
is targeted to cellular mitochondria, where it can respond to
localized changes in H2O2 levels in living samples.
Finally, we sought to utilize MitoPY1 to visualize endogenous
production of H2O2 in the mitochondria of living cells. To this end,
we treated HeLa cells with paraquat, a small-molecule inducer of
oxidative stress that produces Parkinson’s-like phenotypes.19 The
images in Figure 2h-n show clear increases in mitochondrial-
localized H2O2 levels detected with MitoPY1 within cells that had
been exposed to 1 mM paraquat compared to control cells (IC50 of
paraquat in HeLa cells is 1.02 mM).20 These data indicate that
MitoPY1 is sensitive enough to detect local mitochondrial H2O2
elevations associated with oxidative stress in this Parkinson’s model.
To close, we have presented the synthesis, properties, and
biological applications of MitoPY1, a new targeted fluorescent probe
that can selectively detect H2O2 in the mitochondria of living cells.
Our data show that MitoPY1 is capable of imaging changes in the
levels of H2O2 within the mitochondria of a variety of mammalian
cell lines, as well as H2O2 elevations caused by an oxidative stress
model of Parkinson’s disease. In addition to applying MitoPY1 and
related chemical tools for studies of mitochondrial redox biology,
we anticipate that this modular probe scaffold should prove useful
for creating new multifunctional probes for targeting, activation,
and detection in living systems and are actively pursuing these
possibilities.
Acknowledgment. We thank the Beckman, Packard, and Sloan
Foundations, and the NIH (GM 79465) for providing funding for
this work. B.C.D. thanks the NIH Chemical Biology Graduate
Program (T32 GM066698) for support. We thank Holly Aaron
(UCB Molecular Imaging Center) and Ann Fischer (UCB Tissue
Culture Facility) for expert technical assistance.
Supporting Information Available: Synthetic and experimental
details (PDF). This material is available free of charge via the Internet
at http://pubs.acs.org.
References
(1) Rhee, S. G. Science 2006, 312, 1882–1883.
(2) Stone, J. R.; Yang, S. Antioxid. Redox Signal. 2006, 8, 243–270.
(3) Veal, E. A.; Day, A. M.; Morgan, B. A. Mol. Cell 2007, 26, 1–14.
(4) D’Autre´aux, B.; Toledano, M. B. Nat. ReV. Mol. Cell Biol. 2007, 8, 813–
824.
(5) Giorgio, M.; Trinei, M.; Migliaccio, E.; Pelicci, P. G. Nat. ReV. Mol. Cell
Biol. 2007, 8, 722–728.
(6) Poole, L. B.; Nelson, K. J. Curr. Opin. Chem. Biol. 2008, 12, 18–24.
(7) Finkel, T.; Serrano, M.; Blasco, M. A. Nature 2007, 448, 767–774.
(8) Barnham, K. J.; Masters, C. L.; Bush, A. I. Nat. ReV. Drug DiscoVery 2004,
3, 205–214.
(9) Lin, M. T.; Beal, M. F. Nature 2006, 443, 787–795.
(10) Schriner, S. E.; Jinford, N. J.; Martin, G. M.; Treuting, P.; Ogburn, C. E.;
Emond, M.; Coskun, P. E.; Ladiges, W.; Wolf, N.; Van Remmen, H.;
Wallace, D. C.; Rabinovitch, P. S. Science 2005, 308, 1909–1911.
(11) Belousov, V. V.; Fradkov, A. F.; Lukyanov, K. A.; Staroverov, D. B.;
Shakhbazov, K. S.; Terskikh, A. V.; Lukyanov, S. Nat. Methods 2006, 3,
281–286.
(12) Hanson, G. T.; Aggeler, R.; Oglesbee, D.; Cannon, M.; Capaldi, R. A.;
Tsien, R. Y.; Remington, S. J. Biol. Chem. 2004, 279, 13044–13053.
(13) Koide, Y.; Urano, Y.; Kenmoku, S.; Kojima, H.; Nagano, T. J. Am. Chem.
Soc. 2007, 129, 10324–10325.
(14) Robinson, K. M.; Janes, M. S.; Pehar, M.; Monette, J. S.; Ross, M. F.;
Hagen, T. M.; Murphy, M. P.; Beckman, J. S. Proc. Natl. Acad. Sci. U.S.A.
2006, 103, 15038–15043.
(15) Murphy, M. P.; Smith, R. A. Annu. ReV. Pharmacol. Toxicol. 2007, 47,
629–656.
(16) Hardy, M.; Chalier, F.; Ouari, O.; Finet, J.; Rockenbauer, A.; Kalyanaraman,
B.; Tordo, P. Chem. Commun. 2007, 1083–1085.
(17) Chang, M. C. Y.; Pralle, A.; Isacoff, E. Y.; Chang, C. J. J. Am. Chem.
Soc. 2004, 126, 15392–15393.
(18) Miller, E. W.; Tulyathan, O.; Isacoff, E. Y.; Chang, C. J. Nat. Chem. Biol.
2007, 3, 263–267.
(19) McCormack, A. L.; Thiruchelvam, M.; Manning-Bog, A. B.; Thiffault, C.;
Langtson, J. W.; Cory-Clechta, D. A.; Di Monte, D. A. Neurobiol. Dis.
2002, 10, 119–127.
(20) Park, S. Y.; Choi, J. EnViron. Int. 2007, 33, 817–822.
JA802355U
9
J. AM. CHEM. SOC. VOL. 130, NO. 30, 2008 9639