A fluorescent sensor for imaging reversible redox cycles in living cells

Article abstract of DOI:10.1021/ja0668973

We report the synthesis, properties, and biological applications of Redoxfluor-1 (RF1), a new fluorescent sensor for imaging reversible oxidation-reduction cycles in aqueous solution and in living cells. RF1 is comprised of a fluorescein reporter coupled to a disulfide/dithiol switch. The green fluorescent sensor features a reversible response to multiple oxidation or reduction events, a >50-fold fluorescence dynamic range, and excitation and emission profiles in the visible region to minimize cellular damage and autofluorescence. Confocal microscopy experiments show that RF1 can visualize multiple cycles of oxidation and reduction within living cells, establishing the potential value of this approach for probing dynamic oxidation biology events in natural systems. Copyright

Full text of DOI:10.1021/ja0668973

Published on Web 03/03/2007
A Fluorescent Sensor for Imaging Reversible Redox Cycles in Living Cells
Evan W. Miller, Shelly X. Bian, and Christopher J. Chang*
Department of Chemistry, UniVersity of California, Berkeley, California 94720
Received September 25, 2006; E-mail: chrischang@berkeley.edu
Scheme 1. Synthesis and Redox Cycling of Redoxfluor-1 (RF1)
Life in aerobic environments requires organisms to maintain strict
control over their internal redox status. At the cellular level, aerobic
respiration poses a particularly unique challenge for living systems,
as the energy-releasing reduction of oxygen to water generates
partially reduced reactive oxygen species (ROS) intermediates that
can exert widely divergent physiological and/or pathological
effects.1 Unregulated production of ROS results in oxidative stress,
and subsequent buildup of free radical damage to proteins, lipids,
and nucleic acids is connected to serious human diseases where
age is a risk factor.2,3 However, emerging evidence suggests that
controlled production of one ROS in particular, hydrogen peroxide,
can mediate cellular signal transduction through reversible oxidation
and reduction of cysteine thiols and other redox-active groups.4-8
The complex, reversible oxidation biology of the cell and its
broad implications in human health and disease provide motivation
for developing new ways to study dynamic redox chemistry in living
systems. In this regard, fluorescence imaging with redox-responsive
chemosensors is a potentially powerful approach to probe various
stages of oxidative signaling, stress, or repair in real time in living
cells. Traditional fluorescent probes for redox activity, including
dichlorodihydrofluorescein or dihydrorhodamine 123, are useful for
cellular studies but can only respond irreversibly to a single initial
oxidation event.9-11 In contrast, fluorophores that can respond
reversibly to changes in oxidation or reduction events would be
much more valuable for visualizing cycles of redox signaling, stress,
or repair and their dynamic interconversion. A few redox-sensitive
fluorescent reporters based on protein12,13 or peptide14,15 scaffolds
have been described, but no small molecules have been reported
to date for imaging reversible oxidation and reduction events in
living biological systems. We now present the synthesis, properties,
and live-cell imaging applications of Redoxfluor-1 (RF1), a new
type of fluorescent sensor for detecting reversible redox cycles in
aqueous solution and in living cells. RF1 features a dual colori-
metric/fluorimetric readout for oxidation-reduction events, a >50-
fold fluorescence dynamic range, and visible wavelength excitation
and emission profiles to minimize cellular damage and autofluo-
rescence. In addition, RF1 can be loaded into living cells and image
multiple, reversible cycles of oxidative stress and reductive repair.
Our design strategy for fluorescence detection of reversible
oxidation-reduction events is inspired by the extensive use of
disulfides as redox reservoirs in biology. We anticipated that
integrating this unit into a fluorescein scaffold would provide a
small-molecule redox reporter with desirable optical properties and
biological compatibility. Along these lines, the synthesis of RF1
proceeds in three steps as shown in Scheme 1. Lithiation of
naphthalene in the presence of TMEDA and quenching with sulfur
affords disulfide 1 according to literature procedures.16 Vilsmeier
formylation of 1 with POCl3/DMF generates 2- and 4-formyl
isomers that are separable by flash column chromatography in 54%
(2a) and 26% (2b) yields, respectively. Acid-catalyzed condensation
of 2b with 2 equiv of resorcinol furnishes RF1 in 12% yield.
Spectroscopic experiments with RF1 were performed under
simulated physiological conditions (20 mM HEPES, pH 7). As
expected, the oxidized probe displays fluorescein-like characteristics
with a strong absorption band centered at 490 nm (ꢀ ) 4.0 × 104
M-1 cm-1) and bright green fluorescence (λem ) 503 nm, Φ )
0.95, Figure 1a). Treatment of RF1 with a variety of mild reductants,
including tris(2-carboxyethyl)phosphine (TCEP), sodium dithionite,
or NaBH4, proceeds smoothly to generate the reduced RF1 probe,
which possesses no absorption features in the visible region and is
nonfluorescent with 490 nm excitation. For example, treatment of
RF1 with 5 equiv of TCEP results in a >50-fold fluorescence
decrease for the dye (Figure 1a). The sensor can be reoxidized by
air or hydrogen peroxide to restore its fluorescent state, and the
reversible oxidation-reduction cycle can be repeated at least 10
times with no loss of dynamic range for the dye. Figure 1b shows
representative kinetics traces for three reversible oxidation-
Figure 1. (a) Fluorescence responses of 5 µM RF1 to tris(2-carboxyethyl)-
phosphine (TCEP). Spectra are shown for 0, 1, 2, 3, 4, and 5 equiv of
added TCEP. Spectra were acquired at 25 °C in 20 mM HEPES, pH 7,
with excitation at 450 nm. (b) Fluorescence responses of RF1 to cycles of
oxidation and reduction at 37 °C. A solution of 5 µM RF1 was treated
with 25 µM TCEP reductant and then oxidized with 100 µM H2O2. When
fluorescence returned to starting levels, another 25 µM aliquot of TCEP
was added and the redox cycle was repeated two more times without loss
of fluorescence. Spectra were acquired in 20 mM HEPES, pH 7, with
excitation at 495 nm. Emission intensity was measured at the λmax of 503
nm.
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C O M M U N I C A T I O N S
multiple redox cycling events (Figure 2f). Taken together, the data
show that RF1 can be loaded into living cells and report multiple
oxidation-reduction cycles by a reversible fluorescence response.
The turn-on dynamic range for RF1 oxidation in cells is less than
that in the spectroscopic experiments, presumably due to the
reducing environment of the cell.
In summary, we have presented the synthesis, characterization,
and live-cell imaging applications of RF1, a new redox-sensitive
optical sensor for monitoring reversible oxidation and reduction
events in living systems. RF1 features a reversible change in
fluorescence upon oxidation or reduction, a dual colorimetric/
fluorimetric response, and visible wavelength excitation and emis-
sion profiles. Moreover, the sensor is capable of visualizing multiple
cycles of oxidative stress and reductive repair in living cells.
Experiments are underway to utilize RF1 and related chemical tools
to study the production, propagation, and termination of oxidative
signals in biological systems.
Figure 2. Live-cell imaging of oxidative stress/repair by confocal
microscopy at 37 °C. (a) HEK cells loaded with 5 µM RF1-AM for 30
min. (b) RF1-loaded HEK cells treated with 100 µM H2O2 for 9 min. (c)
RF1-loaded, H2O2-treated cells in panel b after an additional 6 min. (d)
Cells exposed to a second dose of H2O2 (100 µM) for an additional 9 min.
(e) Cells in panel d after an additional 6 min. (f) Brightfield image of live
HEK cells in panels a-e, confirming their viability throughout the
experiment. Scale bar ) 10 µm.
Acknowledgment. We thank the University of California,
Berkeley, the Dreyfus Foundation, the Beckman Foundation, the
Packard Foundation, the American Federation for Aging Research,
the National Science Foundation (CAREER Award CHE-0548245),
and the National Institute of General Medical Sciences (NIH GM
79465) for research support. E.W.M. was supported by a Chemical
Biology Interface Training Grant from the NIH (T32 GM066698)
and a Stauffer fellowship, and S.X.B. was funded by a summer
undergraduate research fellowship from the UC Berkeley Chemical
Biology program. Confocal fluorescence images were acquired at
the Molecular Imaging Center at UC Berkeley. We thank Ann
Fischer at the UC Berkeley Tissue Culture Facility for expert
technical assistance.
reduction cycles mediated by H2O2 and TCEP. Reduction of RF1
by 5 equiv of TCEP occurs promptly upon mixing, whereas
oxidation proceeds more slowly. The observed rate constant for
RF1 reoxidation by H2O2 under pseudo-first-order conditions is kobs
) 4.0(1.1) × 10-2 s-1. The dual colorimetric/fluorimetric response
of RF1 suggests that a simple photoinduced electron-transfer
mechanism is not operable in this redox cycle. Instead, we propose
that reduction of RF1 generates a dihydrofluorescein species from
the disulfide through reduction, internal charge transfer, and
protonation. Along these lines, the 1H NMR of reduced RF1 shows
a new peak at 5.16 ppm, consistent with a methine-type proton on
the quaternary center of a dihydrofluorescein structure. The
reversible response of small-molecule RF1 is complementary to
fiber-optic probes for oxygen and other redox-active analytes.17
With spectroscopic results showing the redox sensitivity and
reversibility of RF1 in hand, we next tested the ability of this new
chemical tool to image reversible redox cycles in living cells. As
initial experiments revealed that RF1 is not membrane permeable,
we prepared the acetoxymethyl (AM) ester derivative for live-cell
labeling. Live HEK 293 cells loaded with 5 µM RF1-AM for 30
min at 37 °C show faint fluorescence, consistent with entry of the
probe into the cells and reduction by the cytosolic environment
(Figure 2a). Washing and treatment of the same RF1-loaded cells
with H2O2 results in a marked increase in intracellular fluorescence
within 5-10 min as the probe senses oxidative stress (Figure 2b).
After 5-10 more min, the observed intracellular fluorescence
decreases to baseline levels as the native reducing environment of
the cells is restored (Figure 2c). Control images on other fields of
cells in the same experiment show that the lack of fluorescence is
not due to RF1 loss or photobleaching. To further demonstrate that
low levels of intracellular fluorescence in panel c of Figure 2 are
not due to photobleaching or loss of dye, addition of a second
aliquot of H2O2 oxidant results in another burst of oxidative stress
and increase in intracellular fluorescence (Figure 2d), which
subsides again after an additional 5-10 min due to restoration of
the reducing cellular environment (Figure 2e). Finally, brightfield
transmission images confirm that the cells are still viable after these
Supporting Information Available: Synthetic and experimental
details (PDF). This material is available free of charge via the Internet
at http://pubs.acs.org.
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