A palette of fluorescent probes with varying emission colors for imaging hydrogen peroxide signaling in living cells

Article abstract of DOI:10.1021/ja1014103

We present a new family of fluorescent probes with varying emission colors for selectively imaging hydrogen peroxide (H₂O₂) generated at physiological cell signaling levels. This structurally homologous series of fluorescein- and rhodol-based reporters relies on a chemospecific boronate-to-phenol switch to respond to H₂O₂ over a panel of biologically relevant reactive oxygen species (ROS) with tunable excitation and emission maxima and sensitivity to endogenously produced H₂O ₂ signals, as shown by studies in RAW264.7 macrophages during the phagocytic respiratory burst and A431 cells in response to EGF stimulation. We further demonstrate the utility of these reagents in multicolor imaging experiments by using one of the new H₂O₂-specific probes, Peroxy Orange 1 (PO1), in conjunction with the green-fluorescent highly reactive oxygen species (hROS) probe, APF. This dual-probe approach allows for selective discrimination between changes in H₂O₂ and hypochlorous acid (HOCl) levels in live RAW264.7 macrophages. Moreover, when macrophages labeled with both PO1 and APF were stimulated to induce an immune response, we discovered three distinct types of phagosomes: those that generated mainly hROS, those that produced mainly H₂O₂, and those that possessed both types of ROS. The ability to monitor multiple ROS fluxes simultaneously using a palette of different colored fluorescent probes opens new opporunities to disentangle the complex contributions of oxidation biology to living systems by molecular imaging.

Full text of DOI:10.1021/ja1014103

Published on Web 04/02/2010
A Palette of Fluorescent Probes with Varying Emission Colors
for Imaging Hydrogen Peroxide Signaling in Living Cells
Bryan C. Dickinson,† Calvin Huynh,† and Christopher J. Chang*,†,‡
Department of Chemistry, Howard Hughes Medical Institute, UniVersity of California,
Berkeley, California 94720
Received February 17, 2010; E-mail: chrischang@berkeley.edu
Abstract: We present a new family of fluorescent probes with varying emission colors for selectively imaging
hydrogen peroxide (H2O2) generated at physiological cell signaling levels. This structurally homologous
series of fluorescein- and rhodol-based reporters relies on a chemospecific boronate-to-phenol switch to
respond to H2O2 over a panel of biologically relevant reactive oxygen species (ROS) with tunable excitation
and emission maxima and sensitivity to endogenously produced H2O2 signals, as shown by studies in
RAW264.7 macrophages during the phagocytic respiratory burst and A431 cells in response to EGF
stimulation. We further demonstrate the utility of these reagents in multicolor imaging experiments by using
one of the new H2O2-specific probes, Peroxy Orange 1 (PO1), in conjunction with the green-fluorescent
highly reactive oxygen species (hROS) probe, APF. This dual-probe approach allows for selective
discrimination between changes in H2O2 and hypochlorous acid (HOCl) levels in live RAW264.7
macrophages. Moreover, when macrophages labeled with both PO1 and APF were stimulated to induce an
immune response, we discovered three distinct types of phagosomes: those that generated mainly hROS, those
that produced mainly H2O2, and those that possessed both types of ROS. The ability to monitor multiple ROS
fluxes simultaneously using a palette of different colored fluorescent probes opens new opporunities to
disentangle the complex contributions of oxidation biology to living systems by molecular imaging.
Introduction
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mammals leads to a condition broadly referred to as oxidative
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Oxidative stress is linked to aging and a host of diseases where
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cell is producing a variety of ROS, where each type exerts
specific downstream biological effects. As the lifetimes and
reactivities of individual classes of ROS can greatly vary,18
studying multiple ROS simultaneously in a single cell is critical
to understanding their discrete roles in complex environments.
Fluorescent probes are well suited to study the generation of
specific types of ROS in biological systems, as optical micros-
copy allows for the use of multiple probes simultaneously in a
single specimen as long as spectral overlap is sufficiently
minimized.23-29 In this regard, several new types of indicators
† Department of Chemistry.
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‡ Howard Hughes Medical Institute.
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5906 J. AM. CHEM. SOC. 2010, 132, 5906–5915
10.1021/ja1014103  2010 American Chemical Society

Fluorescent Probes with Varying Emission Colors
A R T I C L E S
Scheme 1. Boronate-Based H2O2-Specific Fluorescent Indicators
have been reported for the detection of nitric oxide (NO),15,30-36
peroxynitrite,37,38 and nitrative stress,39 as well as superoxide,40-43
singlet oxygen,44,45 ozone,46 H2O2,16,47-65 hypochlorous acid,66,67
highly reactive oxygen species (hROS),68-70 and general redox
events.71-84 In terms of H2O2 detection, we previously described
the design, synthesis, and characterization of Peroxyfluor-1 (PF1,
Scheme 1),51 a novel probe for H2O2 in which two H2O2-
mediated boronate deprotections yield the highly fluorescent dye
molecule fluorescein. Owing to its chemospecific deprotection
mechanism, PF1 and related bisboronate derivatives exhibit high
selectivity for H2O2 over other ROS and can respond to changes
in H2O2 fluxes at oxidative stress levels.53,58,62 To maintain H2O2
specificity while increasing sensitivity to image H2O2 produced
at low signaling levels, we devised the single boronate caged
probes Peroxy Green 1 (PG1, Scheme 1) and Peroxy Crimson
1 (PC1, Scheme 1).16 Green-fluorescent PG1 was capable of
visualizing endogenous H2O2 generation in both A431 cells and
primary neurons upon epidermal growth factor (EGF) stimula-
tion, but red-fluorescent PC1 was not responsive enough to
detect peroxide bursts at these basal levels. Because the most
common types of fluorescent reporters utilize GFP, fluorescein,
or related green-colored fluorophores that overlap and are thus
incompatible with PG1 for multicolor imaging experiments, we
sought to address this shortcoming by expanding the available
palette of colors for chemospecific peroxide imaging at low cell
signaling levels. We now report a new series of monoboronate
probes that respond to H2O2 over a range of rationally tunable
visible excitation and emission wavelengths (Scheme 1). Three
of these derivatives, Peroxyfluor-3 (PF3), Peroxy Yellow 1
(PY1), and Peroxy Orange 1 (PO1), are sensitive enough to
image H2O2 signals produced in RAW 264.7 macrophages and
A431 cells during immune response and growth factor stimula-
tion, respectively. Moreover, dual-color imaging experiments
using PO1 along with the green-fluorescent highly reactive
oxygen species (hROS) probe APF allows for selective dis-
crimination between changes in H2O2 and hypochlorous acid
(HOCl) levels in macrophages, as well as identification of
phagosomes that produce H2O2 and/or hROS during an endog-
enous immune response.
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Design and Synthesis of a Palette of Monoboronate Fluorescent
Probes for Hydrogen Peroxide Based on Fluorescein and Rhodol
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A R T I C L E S
Dickinson et al.
Scheme 2. Synthesis and Activation of PF2, PF3, and PE1
region by altering oxygen or nitrogen substituents on the 2′
position of the xanthene core (Scheme 1). Schemes 2 and 3
outline synthetic routes to a family of five new monoboronate
probes for chemoselective H2O2 detection. Two of these probes
are based on green methoxyfluorescein (Peroxyfluor-2, PF2) and
fluorescein (Peroxyfluor-3, PF3) platforms, whereas the other
three reporters feature rhodol derivatives made from aminophe-
nol (Peroxy Emerald 1, PE1), diethylaminophenol (Peroxy
Yellow 1, PY1), and julolidine (Peroxy Orange 1, PO1) building
blocks. Briefly, treatment of the parent dyes with N-phenyl
bis(trifluoromethanesulfonamide) affords the corresponding tri-
flate derivatives, and palladium-mediated coupling with bis(pina-
colato)diboron furnishes the final boronate-protected products.
PF3 was then acetylated with acetic anhydride to yield PF3-Ac
to increase cell permeability, where the acetyl functionality
should be removed by nonspecific esterases once inside the
cytoplasm to reveal PF3. We anticipate that this modular
synthetic strategy should be applicable to a wide variety of
scaffolds and note complementary synthetic methods for C-N
and C-O bond formation that have been reported in the recent
literature,85-92 as well as a triphenylphosphonium-capped
piperazine rhodol for mitochondrial targeting.61
Spectroscopic Properties and Responses to Hydrogen Peroxide.
We evaluated the spectral properties and H2O2 responses of the
new monoboronate dyes in aqueous media buffered to physi-
ological pH (20 mM HEPES buffer, pH 7); data are provided
in Table 1. The methoxy and boronate groups on PF2 force it
to adopt a closed lactone form that is nonfluorescent and displays
no absorption features in the visible region. The other four dyes
each feature one prominent absorbance band in the visible region
with weak but measurable fluorescence. The addition of H2O2
triggers a marked increase in fluorescence intensity for all five
probes (Figure 1, S1) with available emission colors spanning
from green to yellow to orange. As expected because of their
common boronate switch, the fluorescein and rhodol reporters
respond with good selectivity to H2O2 over a variety of
biologically relevant ROS, including superoxide, NO, hypochlo-
rite, and tert-butyl hydroperoxide, as well as hydroxyl and
tert-butoxy radicals (Figure 2). Kinetics measurements of the
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Fluorescent Probes with Varying Emission Colors
A R T I C L E S
Scheme 3. Synthesis and Activation of PY1 and PO1
Table 1. Spectroscopic Properties of PF2, PF3, PE1, PY1, and
PO1 Acquired at 25 °C in 20 mM HEPES, pH 7 (Fluorescein
permeable and nontoxic as well as capable of detecting
elevations in H2O2 levels under oxidative stress conditions. The
excitation wavelengths of this series of reagents align well with
the 488 nm (PF2, PF3-Ac), 514 nm (PY1), and 543 nm (PO1)
laser lines commonly used for light microscopy.
Reported in 0.1 M NaOH104
)
Boronate form
Phenol form
λem
λabs
ε
λem
(nm)
λabs
ε
k
(nm) (M-1 cm-1
)
φ
(nm) (M-1 cm-1
)
(nm)
φ
(×10-3 s-1
)
Fluorescence Detection of H2O2 at Signaling Levels upon
Immune or Growth Factor Stimulation. We next tested whether
the monoboronate probes were sensitive enough to detect H2O2
produced at low signaling levels upon physiological stimulation.
Initial experiments focused on macrophages, which fight off
infections by engulfing pathogens and using NADPH oxidase
(Nox) and associated proteins to produce H2O2 and other ROS
to alleviate the threat.12,18 We utilized RAW264.7 macrophages,
which are estimated to produce low micromolar levels of H2O2
upon stimulation with phorbol myristate acetate (PMA),93-95
as a cell line model for H2O2-mediated immune response.
RAW264.7 cells loaded with 10 µM PF2, 5 µM PF3-Ac, 5 µM
PY1, or 5 µM PO1 for 60 min at 37 °C show low levels of
intracellular fluorescence (Figure 4a,e,i,m). Upon stimulation
of probe-loaded cells with PMA for 40 min at 37 °C to induce
an immune response, we observe bright, punctate fluorescent
patterns corresponding to phagocytic vesicles (Figure 4b,f,j,n),
showing that these reporters can visualize endogenous produc-
tion of H2O2 under conditions of immune signaling. We
confirmed cell viability during live-cell imaging using a
combination of brightfield transmission and nuclear staining
experiments (Figures S4, S5).
PF2 n/a
n/a
n/a n/a 475 28 600 511 0.27
4.7(1)
3.8(1)
8.1(1)
3.7(1)
5.2(1)
PF3 454 24 000 521 0.10 492 88 000 515 0.94
PE1 480 16 400 519 0.30 491 51 200 514 0.93
PY1 494 16 200 558 0.01 519 48 900 548 0.12
PO1 507 13 900 574 0.07 540 29 300 565 0.46
pseudo-first-order conditions (5 µM dye, 10 mM H2O2), giving
observed rate constants ranging from k ) 3.7(1) to 8.1(1) ×
10-3 s-1. These data provide further evidence that H2O2-
triggered conversion of boronates to phenols is a robust and
versatile methodology for reaction-based H2O2 detection.
Fluorescence Detection of H2O2 in Living Cells in
Situations of Oxidative Stress. With data establishing that all
five monoboronate reporters selectively respond to H2O2 in
aqueous solution, we turned our attention to assessing their
performance in live-cell imaging assays. Incubation of live A431
cells with 10 µM PF2, 5 µM PF3-Ac, 5 µM PY1, or 5 µM PO1
for 40 min at 37 °C results in low levels of intracellular
fluorescence as determined by scanning confocal microscopy
measurements (Figure 3a,d,g,j), and addition of 100 µM H2O2
to these dye-loaded cells for 20 min at 37 °C triggers increases
in green, yellow, or orange intracellular fluorescence (Figure
3b,e,h,k). PE1 does not show a turn-on response to H2O2 in
cells under similar conditions owing to high background
staining. Further brightfield transmission and nuclear staining
experiments confirm that the cells are viable throughout the
imaging assays (Figures S2, S3). These experiments demonstrate
that four of the probes, PF2, PF3-Ac, PY1, and PO1, are cell-
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Figure 1. Fluorescence turn-on response of 5 µM PF2 (a), PF3 (b), PE1
(c), PY1 (d), or PO1 (e) to H2O2. Data were acquired at 25 °C in 20 mM
HEPES, pH 7, with excitation at λ ) 488 nm for PF2 and PF3, λ ) 490
nm for PE1, λ ) 514 nm for PY1, and λ ) 540 nm for PO1. Emission was
collected between 493 and 750 nm for PF2 and PF3, 495 and 750 nm for
PE1, 520 and 750 nm for PY1, and 545 and 750 nm for PO1. Time points
represent 0, 5, 15, 30, 45, and 60 min after the addition of 100 µM H2O2.
Reactions are not complete at these time points. Full turn-on response of
each probe is shown in Figure S1.
Figure 2. Fluorescence responses of 5 µM PF2 (a), PF3 (b), PE1 (c), PY1
(d), and PO1 (e) 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 200 µM NO and 100 µM for all other ROS. Data
were acquired at 25 °C in 20 mM HEPES, pH 7, with excitation at λ )
488 nm for PF2 and PF3, λ ) 490 nm for PE1, λ ) 514 nm for PY1, and
λ ) 540 nm for PO1. Emission was collected between 493 and 750 nm for
PF2 and PF3, 495 and 750 nm for PE1, 520 and 750 nm for PY1, and 545
and 750 nm for PO1. Time points represent 0, 5, 15, 30, 45, and 60 min
after the addition of 100 µM H2O2. Reactions are not complete at these
time points.
We then sought to expand the utility of these fluorescent
indicators to image H2O2 signaling in nonphagocytic systems.
In particular, mounting data reveal that Nox proteins used in
phagocytic ROS killing are also expressed in a wide variety of
nonphagocytic cell types throughout the body and that these
enzyme complexes can be activated by ligands such as growth
factors and cytokines. We previously showed that A431 cells,
which express high levels of epidermal growth factor receptor
(EGFr), respond to epidermal growth factor (EGF) stimulation
by producing H2O2 via a Nox/PI3K pathway.16 Along these
lines, A431 cells loaded with 5 µM PF3-Ac, 5 µM PY1, or 5
µM PO1 for 60 min at 37 °C display modest intracellular
fluorescence (Figure 5a,d,g). Treatment of the dye-loaded cells
with 500 ng/mL EGF ligand for 40 min at 37 °C triggers a
modest turn-on enhancement in bright green, yellow, or orange
intracellular fluorescence compared to unstimulated cells (Figure
5b,e,h) via Nox-generated H2O2. Brightfield transmission and
nuclear staining measurements confirm that the cells are viable
throughout the imaging experiments (Figures S6, S7). Taken
together, PF3-Ac, PY1, and PO1 offer a set of green, yellow,
and orange dyes that are capable of detecting endogenous bursts
of H2O2 produced for physiological signaling purposes.
Simultaneous, Dual-Color Imaging of H2O2 and hROS in
Living Cells with Complementary Chemoselective Fluorescent
Probes. Our next goal was to exploit the utility of the newly
developed H2O2-specific fluorescent indicators with varying
emission profiles to image multiple analytes/processes in living
cells. In particular, because a given ROS can undergo an array
of primary and secondary reactions to produce other classes of
ROS molecules, we envisioned using a dual-probe approach to
simultaneously visualize H2O2 and an additional important
downstream product of cellular H2O2 chemistry. We focused
on macrophages as a cell model system, as H2O2 produced by
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Figure 3. Confocal fluorescence images of H2O2 in live A431 cells under
oxidative stress with PF2, PF3-Ac, PY1, and PO1. A431 cells incubated
with 10 µM PF2 for 40 min at 37 °C (a). A431 cells incubated with 10 µM
PF2 for 40 min at 37 °C with 100 µM H2O2 added for the final 20 min (b)
and quantification (c). A431 cells incubated with 5 µM PF3-Ac for 40 min
at 37 °C (d). A431 cells incubated with 5 µM PF3-Ac for 40 min at 37 °C
with 100 µM H2O2 added for the final 20 min (e) and quantification (f).
A431 cells incubated with 5 µM PY1 for 40 min at 37 °C (g). A431 cells
incubated with 5 µM PY1 for 40 min at 37 °C with 100 µM H2O2 added
for the final 20 min (h) and quantification (i). A431 cells incubated with 5
µM PO1 for 40 min at 37 °C (j). A431 cells incubated with 5 µM PO1 for
40 min at 37 °C with 100 µM H2O2 added for the final 20 min (k) and
quantification (l). Data were normalized to controls, and statistical analyses
were performed with a two-tailed Student’s t-test (n ) 4). **P e 0.005
and error bars are ( s.e.m.
Figure 4. Confocal fluorescence images of PMA-induced H2O2 production
in live RAW264.7 macrophages with PF2, PF3-Ac, PY1, and PO1.
Macrophages incubated with 10 µM PF2 for 60 min at 37 °C (a).
Macrophages incubated with 10 µM PF2 for 60 min at 37 °C with 1 µg/
mL PMA added for the final 40 min (b) with a brightfield overlay (c) and
quantification (d). Macrophages incubated with 5 µM PF3-Ac for 60 min
at 37 °C (e). Macrophages incubated with 5 µM PF3-Ac for 60 min at 37
°C with 1 µg/mL PMA added for the final 40 min (f) with a brightfield
overlay (g) and quantification (h). Macrophages incubated with 5 µM PY1
for 60 min at 37 °C (i). Macrophages incubated with 5 µM PY1 for 60 min
at 37 °C with 1 µg/mL PMA added for the final 40 min (j) with a brightfield
overlay (k) and quantification (l). Macrophages incubated with 5 µM PO1
for 60 min at 37 °C (m). Macrophages incubated with 5 µM PO1 for 60
min at 37 °C with 1 µg/mL PMA added for the final 40 min (n) with a
brightfield overlay (o) and quantification (p). Data were normalized to
controls, and statistical analyses were performed with a two-tailed Student’s
t-test (n ) 4). **P e 0.005 and error bars are ( s.e.m.
Nox complexes in these cell types can be converted to HOCl
by reaction with heme-dependent myeloperoxidase enzymes.96-99
any change in orange emission from the PO1 dye (Figure 6e,f).
In all cases, brightfield transmission and nuclear staining
measurements confirm reasonable cell viability through the
duration of the imaging assays (Figure S8); however the
exogenous ROS treatments can cause some toxicity. This set
of experiments establishes not only that fluorescence signals
arising from green APF and orange PO1 probes can be
distinguished by optical microscopy but also that each reporter
maintains its chemical selectivity in the presence of one another
in living systems.
We then moved on to study endogenously produced ROS
bursts using this dual-probe imaging approach. APF- and PO1-
loaded macrophages stimulated with 1 µg/mL PMA for 20 min
display bright, punctate patterns of green and/or orange fluo-
rescence localized to phagosomes, where ROS are generated
in response to immune insult (Figures 6g,h, S8). As shown in
Figure 7 and Figure S9, we discovered three classes of
phagosomes that are characterized by different relative ratios
of H2O2 and hROS fluxes. Specifically, some phagosomes
display only PO1-derived orange fluorescence, indicating that
H2O2 production dominates (Figure 7a,b,c,d), whereas other
phagosomes show only APF-derived green fluorescence, indi-
cating that most of the phagocytic burst results in formation of
hROS (Figure 7e,f,g,h). A third distinct class of phagosomes
possesses both green and orange fluorescence signals (Figure
7i,j,k,l) resulting from a combination of phagocytic increases
For dual-color imaging experiments, we selected APF,68
a
4-aminophenyl-fluorescein reporter developed by Nagano and
co-workers that responds selectively to HOCl and other highly
reactive oxygen species (hROS) by an increase in green
fluorescence (488 nm excitation, 520 nm emission), along with
the H2O2-selective probe PO1 that responds in a complementary
orange optical window (543 nm excitation, 565 nm emission).
Initial experiments sought to test the ROS specificities of these
dyes in a dual-probe/dual-color imaging mode. To this end,
RAW264.7 macrophages loaded simultaneously with 5 µM APF
and 5 µM PO1 for 50 min at 37 °C display low levels of both
green and orange fluorescence (Figure 6a,b). In contrast, APF-
and PO1-stained cells incubated with 50 µM H2O2 for 20 min
exhibit a selective increase in intracellular orange fluorescence
from PO1 without any change in green emission from the APF
probe (Figure 6c,d). Moreover, APF- and PO1-loaded cells
incubated with 100 µM HOCl for 20 min give a selective turn-
on increase in intracellular green fluorescence from APF without
(96) Hampton, M. B.; Kettle, A. J.; Winterbourn, C. C. Blood 1998, 92,
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Figure 5. Confocal fluorescence images of EGF-induced H2O2 in live A431
cells with PF3-Ac, PY1, and PO1. A431 cells incubated with 5 µM PF3-
Ac for 60 min at 37 °C (a). A431 cells incubated with 5 µM PF3-Ac for 60
min at 37 °C with 500 ng/mL EGF added for the final 40 min (b) and
quantification (c). A431 cells incubated with 5 µM PY1 for 60 min at 37
°C (d). A431 cells incubated with 5 µM PY1 for 60 min at 37 °C with 500
ng/mL EGF added for the final 40 min (e) and quantification (f). A431
cells incubated with 5 µM PO1 for 60 min at 37 °C (g). A431 cells incubated
with 5 µM PO1 for 60 min at 37 °C with 500 ng/mL EGF added for the
final 20 min (h) and quantification (i). Data were normalized to controls,
and statistical analyses were performed with a two-tailed Student’s t-test
(n ) 4). *P e 0.05; **P e 0.005 and error bars are ( s.e.m.
Figure 7. Confocal fluorescence images of different types of phagosomes
in live RAW264.7 macrophages distinguished by PO1 and APF. A
macrophage producing mostly H2O2 as shown by the PO1 signal (a), APF
signal (b), overlay (c), and brightfield (d). A macrophage producing mostly
hROS as shown by the PO1 signal (e), APF signal (f), overlay (g), and
brightfield (h). A macrophage producing a mixture of H2O2 phagosomes,
hROS phagosomes, and H2O2 and hROS phagosomes. as shown by the
PO1 signal (i), APF signal (j), overlay (k), and brightfield (l). 10 µm scale
bar shown.
in both H2O2 and hROS. These data establish that the dual-
probe approach can reveal chemical differences through simul-
taneous imaging of discrete classes of ROS molecules.
microscopy. All five probes in this series give a selective turn-
on fluorescence response to H2O2 in aqueous solution buffered
to physiological pH with minimal interference from other
biologically relevant ROS. Moreover, PF2, PF3-Ac, PY1, and
PO1 are capable of visualizing changes in H2O2 levels in living
cells in situations of oxidative stress, and PF3-Ac, PY1, and
PO1 can be used to image low levels of H2O2 produced for
signaling purposes upon phagocytic immune or nonphagocytic
growth factor stimulation in live samples. Owing to the
expanded color palette of H2O2-selective probes with sensitivity
Concluding Remarks
To close, we have described the synthesis, properties, and
biological applications of a new class of monoboronate fluo-
rescent probes for imaging H2O2 produced at low signaling
levels with a range of varying excitation and emission colors.
By rationally tuning substitutents on a homologous xanthene
core, we have developed boronate-caged fluorescein and rhodol
derivatives for H2O2 detection with excitation profiles that match
well with common 488, 514, and 543 nm lines used for light
Figure 6. Confocal fluorescence images of PMA-induced ROS production in live RAW264.7 macrophages with PO1 and APF simultaneously. Macrophages
incubated with 5 µM PO1 and 5 µM APF for 40 min at 37 °C and imaged for PO1 (a) and APF (b). Macrophages incubated with 5 µM PO1 and 5 µM APF
for 40 min at 37 °C with 50 µM H2O2 added for the final 20 min and imaged for PO1 (c) and APF (d). Macrophages incubated with 5 µM PO1 and 5 µM
APF for 40 min at 37 °C with 100 µM HOCl added for the final 20 min and imaged for PO1 (e) and APF (f). Macrophages incubated with 5 µM PO1 and
5 µM APF for 40 min at 37 °C with 1 µg/mL PMA added for the final 20 min and imaged for PO1 (g) and APF (h). Quantification of a-h (i). Data were
normalized to controls, and statistical analyses were performed with a two-tailed Student’s t-test (n ) 4). **P e 0.005 and error bars are ( s.e.m.
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A R T I C L E S
(1H, dd, J ) 2.4, 8.8 Hz), 3.89 (3H, s). 13C NMR ((CD3)2O, 100
MHz): δ 169.2, 162.9, 153.6, 152.9, 152.8, 151.1, 136.6, 131.6,
131.4, 130.1, 127.3, 125.9, 125.0, 121.5, 118.1, 113.6, 111.9, 111.5.
101.73, 56.24. 19F NMR ((CD3)2O, 376.5 MHz): δ -73.25. HR-
FABMS: calculated for [M+] 479.0412, found 479.0426.
to low cell signaling levels, we were able to combine one of
our newly synthesized H2O2 probes, PO1, with a complementary
green-fluorescent reporter for hROS, APF, for selective dis-
crimination between changes in H2O2 and HOCl levels in live
RAW264.7 macrophages by a dual-probe/dual-color imaging
approach. Further, we observed that PMA-stimulated macroph-
ages were shown to respond with three discrete types of
phagosomes via PO1/APF imaging: those that produced pri-
marily hROS and selectively turned on APF during the
phagocytic burst, those that primarily generated H2O2 and caused
an increase in PO1 fluorescence, and those that deprotected both
PO1 and APF probes and, therefore, possessed an elevated
mixture of both H2O2 and hROS during immune signaling. By
establishing selective H2O2-responsive probes with varying
emission colors that can be used at low signaling levels, this
work greatly expands our ability to study peroxide biology,
particularly in conjunction with other reporters for cellular
analytes and reaction processes. We are currently pursuing such
opportunities in a variety of physiological and disease models,
with particular interest in brain and immune systems.
Peroxyfluor-2, PF2 (2). Compound 1 (150 mg, 0.31 mmol),
bis(pinacolato)diboron (120 mg, 0.47 mmol), palladium acetate (63
mg, 0.10 mmol), and cyclo-hexyl JohnPhos (66 mg, 0.20 mmol)
were added to a glass vial in an inert atmosphere glovebox and
dissolved in 3 mL of dioxane. Diisopropylethamine (0.31 mL, 1.79
mmol) was then added the vial, and the contents were stirred at
room temperature overnight. The contents were then brought out
of the box and dried under reduced pressure. Purification by column
chromatography (2:1 hexanes/ethyl acetate) afforded PF2 as a white
1
solid (103 mg, 73%). H NMR ((CD3)2O, 400 MHz): δ 8.02 (1H,
d J ) 7.6 Hz), 7.79 (1H, t, J ) 6.8 Hz), 7.74 (1H, t, J ) 7.2 Hz),
7.66 (1H, s), 7.46 (1H, d, J ) 8.0 Hz), 7.26 (1H, d, J ) 7.6 Hz),
6.90 (1H, d, J ) 4.0 Hz), 6.89 (1H, s), 6.78 (1H, d, J ) 8.8 Hz),
6.72 (1H, dd, J ) 2.4, 8.8 Hz), 3.86 (3H, s), 1.35 (12H, s). 13C
NMR ((CD3)2O, 100 MHz): δ 169.5, 162.6, 154.1, 153.2, 151.6,
136.3, 131.0, 130.4, 130.0, 128.3, 127.2, 125.7, 124.9, 123.8, 123.1,
112.8, 112.1, 106.7, 85.1, 82.7, 56.1, 25.5. HR-FABMS: calculated
for [M+] 457.1822, found 457.1831.
Experimental Section
Fluorescein Monotriflate (3). Fluorescein (2.0 g, 5.8 mmol) and
N-phenyl bis(trifluoromethanesulfonamide) (2.1 g, 5.8 mmol) were
added to a dry Schlenk flask and flushed with nitrogen. The reaction
contents were dissolved in 15 mL of DMF, and diisopropylethyl-
amine (3.8 mL, 23.1 mmol) was added. After 48 h of stirring of
the contents at room temperature, the reaction mixture was acidified
with hydrochloric acid, extracted into ethyl acetate, and evaporated
under reduced pressure. Purification by column chromatography
(1:1 hexanes/ethyl acetate) afforded 3 as a pale yellow solid (1.7
Synthetic Materials and Methods. All reactions were carried
out under a dry nitrogen atmosphere. 2-(4-Diethylamino-2-hy-
droxybenzoyl)benzoic acid,100 8-hydroxy-9-o-carboxybenzoylju-
lolidine,101 rhodol,102 and methoxyfluorescein103 were synthesized
according to literature methods. Silica gel P60 (SiliCycle) was used
for column chromatography. Analytical thin layer chromatography
was performed using SiliCycle 60 F254 silica gel (precoated sheets,
0.25 mm thick). All chemicals were purchased from Sigma-Aldrich
1
g, 63%). H NMR ((CD3)2O, 400 MHz): δ 8.01 (1H, d, J ) 8.0
1
(St. Louis, MO) and used as received. H NMR, 13C NMR, and
Hz), 7.81 (1H, dt, J ) 1.2, 7.6 Hz), 7.75 (1H, dt, J ) 1.2, 7.6 Hz),
7.50 (1H, d, J ) 2.4 Hz). 7.36 (1H, d, J ) 7.6 Hz), 7.21 (1H, dd,
J ) 2.4, 8.8 Hz), 7.09 (1H, d, J ) 8.8 Hz), 6.81 (1H, d J ) 2.4
31P NMR spectra were collected in CDCl3, (CD3)2O, or 9:1 CDCl3/
CD3OD (Cambridge Isotope Laboratories, Cambridge, MA) at 25
°C on a Bruker AV-300, AVQ-400, or DRX-500 spectrometer at
the College of Chemistry NMR Facility at the University of
California, Berkeley. All chemical shifts are reported in the standard
δ notation of parts per million using the peak of residual solvent
proton signals as an internal reference. High-resolution mass spectral
analyses were carried out at the College of Chemistry Mass
Spectrometry Facility at the University of California, Berkeley.
Low-resolution mass spectral analyses were carried out on an
Agilent 6130 LC/MS system (Santa Clara, CA). Microwave
reactions were performed using a CEM Intelligent Explorer/
Discover (Matthews, NC).
Hz), 6.72 (1H, d, J ) 8.8 Hz), 6.68 (1H, dd, J ) 2.4, 8.8 Hz). 13
C
NMR ((CD3)2O, 100 MHz): δ 168.2, 159.7, 152.6, 152.0, 151.8,
150.0, 135.5, 130.5, 130.3, 129.3, 126.3, 124.8, 124.0, 120.5, 117.0,
113.2, 110.4, 110.0, 102.5, 81.1. 19F NMR ((CD3)2O, 376.5 MHz):
δ -73.21. HR-FABMS: calculated for [M+] 465.0256, found
465.0243.
Peroxyfluor-3, PF3 (4). Compound 3 (200 mg, 0.43 mmol),
bis(pinacolato)diboron (111 mg, 0.430 mmol), Pd(dppf)Cl2 ·CH2Cl2
(106 mg, 0.130 mmol), potassium acetate (127 mg, 1.29 mmol),
and 10 mL of toluene were added to a dry pressure tube in an inert
atmosphere glovebox. The pressure tube was then brought out of
the box and microwave-heated for 2 h at 110 °C. After the reaction
mixture was cooled to room temperature, the contents of the
pressure flask were washed into a round-bottom flask with dichlo-
romethane and evaporated to dryness. Purification by column
chromatography (1:1 hexanes/ethyl acetate) delivered 4 as a yellow
Methoxyfluorescein Triflate (1). Methoxyfluorescein (500 mg,
1.45 mmol) and N-phenyl bis(trifluoromethanesulfonamide) (2.06
g, 5.78 mmol) were added to a vial and dissolved in 10 mL of
dimethylformamide. Diisopropylethylamine (1.22 mL, 7.37 mmol)
was then added, and the reaction stirred for 120 min at room
temperature. The reaction was then extracted into dichloromethane,
washed three times with water, and dried under reduced pressure.
Purification by column chromatography (2:1 hexanes/ethyl acetate)
yielded 1 as a white solid (440 mg, 63%). 1H NMR ((CD3)2O, 400
MHz): δ 8.0 (1H, d, J ) 7.6 Hz), 7.84 (1H, dt, J ) 1.2, 7.2 Hz),
7.78 (1H, dt, J ) 1.2, 7.2 Hz), 7.54 (1H, D, J ) 2.4 Hz), 7.38 (1H,
d, J ) 7.6 Hz), 7.25 (1H, dd, J ) 2.4, 8.8 Hz), 7.13 (1H, d, J )
8.8 Hz), 7.94 (1H, d, J ) 2.4 Hz), 6.82 (1H, d, J ) 8.8 Hz), 6.78
1
solid (105 mg, 55%). H NMR ((CD3)2O, 400 MHz): δ 8.00 (1H,
d, J ) 7.6 Hz), 7.76 (1H, t, J ) 6.8 Hz), 7.70 (1H, t, J ) 6.8 Hz),
7.64 (1H, s), 7.43 (1H, d, J ) 7.6 Hz), 7.24 (1H, d, J ) 7.6 Hz),
6.86 (1H, d, J ) 8.0 Hz), 6.80 (1H, d, J ) 2.0 Hz), 6.69 (1H, d,
J ) 8.4 Hz), 6.64 (1H, dd, J ) 2.0, 8.4 Hz), 1.32 (12H, s). 13C
NMR ((CD3)2O, 100 MHz): δ 168.7, 159.7, 153.2, 152.2, 151.4,
150.7, 135.3, 130.0, 129.3, 129.1, 127.4, 126.3, 124.7, 123.9, 122.9,
122.2, 112.7, 110.1, 102.6, 84.1, 82.1, 24.3. HR-FABMS: calculated
for [M+] 443.1676, found 443.1666.
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Peroxyfluor-3 Acetate, PF3-Ac (5). Compound 4 (33 mg, 0.080
mmol) and cesium carbonate (121 mg, 0.370 mmol) were added
to a dry Schlenk tube and dissolved in 3 mL of dry acetonitrile.
Acetic anhydride (14 µL, 0.15 mmol) was added, and the reaction
stirred at room temperature for 30 min. The contents of the reaction
were evaporated under reduced pressure. Purification by column
chromatography (3:1 hexanes/ethyl acetate) afforded 5 as a clear
film (16 mg, 43%). 1H NMR (CDCl3/10% CD3OD, 400 MHz): 8.04
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(1H, d, J ) 6.8 Hz), 7.75 (1H, s), 6.64 (2H, quintet, J ) 7.2 Hz),
4.45 (1H, d, J ) 7.6 Hz), 7.14 (1H, d, J ) 7.2 Hz), 7.08 (1H, d,
J ) 2.0 Hz), 6.82 - 6.88 (2H, m), 6.80 (1H, dd, J ) 2.0, 8.4 Hz),
2.32 (1H, s), 1.35 (12H, s). HR-FABMS: calculated for [M+]
485.1772, found 485.1780.
2.4, 8.8 Hz), 6.84 (1H, d, J ) 8.8 Hz), 6.54 (1H, d, J ) 9.2 Hz),
6.44 (1H, d, J ) 2.4 Hz), 6.36 (1H, dd, J ) 2.4, 8.8 Hz), 3.31 (4
H, q, J ) 7.2 Hz), 1.11 (6H, t, J ) 7.2 Hz). 13C NMR (CDCl3/
10% CD3OD, 100 MHz): δ 169.5, 152.5, 152.4, 152.4, 149.8, 149.8,
135.3, 130.0, 129.2, 128.7, 126.7, 125.0, 124.0, 123.4, 120.2, 116.1,
113.8, 110.3, 109.0, 104.1, 97.4, 83.1, 44.4, 12.3. 19F NMR (CDCl3/
10% CD3OD, 376.5 MHz): δ -72.05. HR-FABMS: calculated for
[M+] 520.1042, found 520.1036.
Peroxy Yellow 1, PY1 (10). Compound 9 (200 mg, 0.38 mmol),
bis(pinacolato)diboron (94 mg, 0.38 mmol), Pd(dppf)Cl2 ·CH2Cl2
(94 mg, 0.12 mmol), potassium acetate (113 mg, 1.16 mmol), and
3 mL of toluene were added to a dry pressure tube in an inert
atmosphere glovebox. The pressure tube was then brought out of
the box and microwave-heated for 2 h at 110 °C. After cooling the
reaction mixture to room temperature, the contents of the pressure
flask were washed into a round-bottom flask with dichloromethane
and evaporated to dryness. Purification by column chromatography
(4:1 hexanes/ethyl acetate) delivered PY1 as a light tan solid (41
mg, 22%). 1H NMR (CDCl3/10% CD3OD, 400 MHz): δ 7.98 (1H,
d, J ) 7.6 Hz), 7.69 (1H, s), 7.62 (1H, t, J ) 7.2 Hz), 7.58 (1H,
t, J ) 7.2 Hz), 7.35 (1H, d, J ) 7.6 Hz), 7.13 (1H, d, J ) 7.2 Hz),
6.73 (1H, d, J ) 8.0 Hz), 6.55 (1H, d, J ) 9.2 Hz), 6.433 (1H, s),
6.34 (1H, d, J ) 8.8 Hz) 3.33 (4H, q, J ) 7.2 Hz), 1.31 (12H, s),
1.14 (6H, t, J ) 7.2 Hz). 13C NMR (CDCl3/10% CD3OD, 100
MHz): δ 170.1, 153.2, 152.8, 151.1, 134.9, 129.6, 128.8, 128.8,
127.2, 126.7, 124.9, 124.0, 123.9, 121.8, 84.2, 44.5, 12.4. HR-
FABMS: calculated for [M+] 498.2452, found 498.2441.
Aniline Rhodol Triflate (6). Rhodol (150 mg, 0.46 mmol) and
N-phenyl bis(trifluoromethanesulfonamide) (250 mg, 0.69 mmol)
were added to a glass vial and dissolved in 10 mL of acetonitrile.
Diisopropylethylamine (0.23 mL, 1.4 mmol) was added, and the
reaction stirred at room temperature for 72 h. The product was then
extracted into ethyl acetate, washed once with water, and dried
under reduced pressure. Purification by column chromatography
(1:1 hexanes/ethyl acetate) yielded 6 as a white solid (49 mg, 23%).
1H NMR ((CD3)2O, 400 MHz): δ 7.99 (1H, d, J ) 7.6 Hz), 7.80
(1H, t, J ) 7.6 Hz), 7.73 (1H, t, J ) 7.2 Hz), 7.46 (1H, d, J ) 2.0
Hz), 7.33 (1H, d, J ) 7.6 Hz), 7.17 (1H, dd, J ) 2.0, 8.8 Hz), 7.04
(1H, d, J ) 8.8 Hz), 6.60 (1H, s), 6.52 (1H, d, J ) 8.8 Hz), 6.48
(1H, d, J ) 8.4 Hz). 13C NMR ((CD3)2O, 100 MHz): δ 168.4, 152.6,
152.2, 152.0, 149.9, 135.4, 130.4, 130.1, 128.7, 126.7, 124.6, 124.0,
120.8, 118.7 (q, J ) 318 Hz), 116.6, 112.0, 110.3, 106.1, 99.8,
81.9. 19F NMR (Acetone, 376.5 MHz): δ -73.217. Low-res MS:
464.1.
Peroxy Emerald 1, PE1 (7). 6 (49 mg, 0.11 mmol), bis(pina-
colato)diboron (27 mg, 0.11 mmol), Pd(dppf)Cl2 ·CH2Cl2 (26 mg,
0.03 mmol), potassium acetate (31 mg, 0.32 mmol), and 3 mL of
toluene were added to a dry pressure tube in an inert atmosphere
glovebox. The pressure tube was then brought out of the box and
microwave-heated for 4 h at 110 °C. After the reaction cooled to
room temperature, the reaction contents were washed into a round-
bottom flask with dichloromethane and methanol and dried under
reduced pressure. Purification by column chromatography (1:1
hexanes/ethyl acetate) yielded PE1 as a light orange solid (7.0 mg,
15%). 1H NMR (Acetone, 400 MHz): δ 7.99 (1H, d, J ) 7.6 Hz),
7.78 (1H, t, J ) 7.6 Hz), 7.72 (1H, t, J ) 7.6 Hz), 7.62 (1H, s),
7.41 (1H, d, J ) 7.6 Hz), 7.26 (1H, d, J ) 7.6 Hz), 6.83 (1H, d,
J ) 7.6 Hz), 6.60 (1H, d, J ) 2.0 Hz), 6.52 (1H, d, J ) 8.8 Hz),
6.45 (1H, dd, J ) 2.0, 8.8 Hz), 1.34 (12H, s). 13C NMR (Acetone,
100 MHz): δ 169.6, 154.2, 153.3, 152.1, 151.9, 136.1, 130.8, 129.9,
129.6, 128.3, 127.7, 125.4, 124.9, 123.8, 123.4, 112.4, 107.4, 100.9,
85.0, 83.7, 74.9, 25.2. HR-FABMS: calculated for [M+] 442.1831,
found 442.1820.
Julolidine Rhodol (11). Synthesis adopted from literature.101
8-Hydroxy-9-o-carboxybenzoyljulolidine (3.23 g, 9.56 mmol) and
resorcinol (1.05 g, 9.56 mmol) were added to a heavy-walled
pressure flask and dissolved in 20 mL of methane sulfonic acid.
The reaction contents were heated to 90 °C for 3 h, then cooled to
room temperature, and basified with aqueous sodium hydroxide.
The mixture was then extrated into ethyl acetate and evaporated
under reduced pressure. Purification by column chromatography
(4.5:4.5:2 dichloromethane/ethyl acetate/methanol) delivered 11 as
a maroon solid (2.5 g, 63% yield). 1H NMR (CDCl3 /10% CD3OD,
400 MHz): δ 8.01 (1H, d, J ) 7.2 Hz), 7.46 (2H, quartet, J ) 8.8
Hz), 7.02 (1H, d, J ) 7.2 Hz), 6.92 (1H, d, J ) 9.2 Hz), 6.64 (1H,
s), 6.58 (1H, s), 6.49 (1H, d, J ) 7.6 Hz), 3.33 (4H, quintet, J )
6.0 Hz), 2.86 (2H, d, J ) 6.4 Hz), 2.50-2.58 (2H, m), 1.72-1.99
(4H, m). 13C NMR (CDCl3/10% CD3OD, 100 MHz): δ 174.6, 167.8,
153.9, 148.1, 146.4, 134.0, 126.9, 125.9, 125.8, 125.3, 125.0, 122.9,
118.5, 117.4, 109.7, 108.7, 100.9, 99.0, 46.7, 46.2, 23.4, 16.7, 15.8.
HR-FABMS: calculated for [M+] 412.1549, found 412.1555.
Julolidine Rhodol Triflate (12). Compound 11 (500 mg, 1.2
mmol) and N-phenyl bis(trifluoromethanesulfonamide) (868 mg,
2.4 mmol) were added to a vial and flushed with nitrogen. The
reaction contents were dissolved in 5 mL of DMF, and diisopro-
pylethylamine (805 µL, 4.9 mmol) was added. After 20 min of
stirring at room temperature, the product was extracted into ethyl
acetate, washed three times with water, and evaporated under
reduced pressure. Purification by column chromatography (1:1
hexanes/ethyl acetate) afforded 12 as a light red solid (224 mg,
34%). 1H NMR ((CD3)2O, 400 MHz): δ 7.98 (1H, d, J ) 7.6 Hz),
7.79 (1H, t, J ) 7.2 Hz), 7.73 (1H, t, J ) 7.6 Hz), 7.52 (1H, d, J
) 2.8 Hz), 7.31 (1H, d, J ) 7.6 Hz), 7.15 (1H dd, J ) 2.4, 8.8
Hz), 7.00 (1H, d, J ) 8.8 Hz), 6.19 (1H, s), 3.20 (2H, t, J ) 6.0
Hz), 3.15 (2H, t, J ) 6.0 Hz), 2.91 (2H, t, J ) 6.8 Hz), 2.41-2.59
(2H, m), 1.92-2.00 (2H, m), 1.76-1.86 (2H, m). 13C NMR
((CD3)2O/10% CD3OD, 100 MHz): δ 168.3, 152.5, 152.4, 149.9,
147.3, 144.7, 135.2, 130.3, 130.0, 126.9, 124.6, 124.5, 124.1, 120.7,
118.5, 116.3, 110.4, 106.8, 104.5, 82.6, 49.4, 48.9, 21.4, 20.8, 20.7,
19.9. 19F NMR ((CD3)2O, 376.5 MHz): δ -73.284. HR-FABMS:
calculated for [M+] 544.1042, found 544.1036.
Diethylamino Rhodol (8). Synthesis adopted from literature.101
2-(4-Diethylamino-2-hydroxybenzoyl)benzoic acid (1.26 g, 4.00
mmol) and resorcinol (443 g, 4.0 mmol) were added to a heavy-
walled pressure flask and dissolved in 15 mL of trifluoroacetic acid.
The reaction contents were heated to 90 °C for 12 h, then cooled
to room temperature, and evaporated to dryness. Purification by
column chromatography (4.5:4.5:1 dichloromethane/ethyl acetate/
methanol) delivered 8 as a red-brown solid (1.2 g, 77% yield). 1H
NMR (CDCl3/10% CD3OD, 400 MHz): δ 8.19 (1H, d, J ) 7.2
Hz), 7.59 (2H, quartet, J ) 7.2 Hz), 7.11 (1H, d, J ) 7.2 Hz),
6.86-6.95 (3H, m), 6.68 (2H, dd, J ) 2.0, 9.2 Hz), 6.64 (1H, d, J
) 2.0 Hz), 3.44 (4H, q, J ) 7.2 Hz), 1.16 (6H, t, J ) 7.2 Hz). 13
C
NMR (CDCl3/10% CD3OD, 100 MHz): δ 163.5, 153.4, 152.4,
150.9, 128.9, 127.3, 126.7, 126.2, 126.0, 124.4, 113.2, 110.0, 109.7,
108.7, 98.6, 92.3, 41.8, 8.3. HR-FABMS: calculated for [M+]
388.1549, found 388.1546.
Diethylamino Rhodol Triflate (9). Compound 8 (500 mg, 1.39
mmol) and N-phenyl bis(trifluoromethanesulfonamide) (745 mg,
2.09 mmol) were added to a vial and flushed with nitrogen. The
reaction contents were dissolved in 5 mL of DMF, and diisopro-
pylethylamine (691 µL, 4.18 mmol) was added. After 10 min of
stirring at room temperature, the product was extracted into ethyl
acetate, washed three times with water, and evaporated under
reduced pressure. Purification by column chromatography (3:1
hexanes/ethyl acetate) afforded 9 as a light pink solid (440 mg,
61%). 1H NMR (CDCl3/10% CD3OD, 400 MHz): δ 7.99 (1H, d, J
) 7.6 Hz), 7.65 (1H, t, J ) 7.2 Hz), 7.60 (1H, t, J ) 7.2 Hz), 7.20
(1H, d, J ) 2.4 Hz), 7.17 (1H, d, J ) 7.6 Hz), 6.90 (1H, dd, J )
Peroxy Orange 1, PO1 (13). Compound 12 (100 mg, 0.18
mmol), bis(pinacolato)diboron (47 mg, 0.18 mmol), Pd(dppf)Cl2 ·
CH2Cl2 (45 mg, 0.05 mmol), potassium acetate (55 mg, 0.55 mmol),
and 5 mL of toluene were added to a dry pressure tube in an inert
9
5914 J. AM. CHEM. SOC. VOL. 132, NO. 16, 2010

Fluorescent Probes with Varying Emission Colors
A R T I C L E S
atmosphere glovebox. The pressure tube was then brought out of
the box and microwave-heated for 2 h at 110 °C. After cooling the
reaction mixture to room temperature, the contents of the pressure
flask were washed into a round-bottom flask with dichloromethane
and evaporated to dryness. Purification by column chromatography
(4.5:4.5:1 dichloromethane/ethyl acetate/methanol) delivered PO1
DMSO), or EGF (100 µg/mL stock in Millipore water) was added
by bath application to the cell culture media. The cells were then
kept in an incubator (37 °C, 5% CO2) during the course of all
experiments.
Fluorescence Imaging Experiments. Confocal fluorescence
imaging studies were performed with a Zeiss LSM510 NLO
Axiovert 200 laser scanning microscope and a 40× or 63×
Achroplan IR water-immersion objective lens. Excitation of PF2-
and PF3-loaded cells at 488 nm was carried out with an Ar laser,
and emission was collected using a META detector between
495 and 581 nm. Excitation of PY1-loaded cells at 514 nm was
carried out with an Ar laser, and emission was collected using a
META detector between 516 and 581 nm. Excitation of PO1-loaded
cells at 543 nm was carried out with a HeNe laser, and emission
was collected using a META detector between 548 and 613 nm.
Excitation of Hoechst 33342 was carried out using a MaiTai two-
photon laser at 780-nm pulses (mode-locked Ti:sapphire laser,
Tsunami Spectra Physics), and emission was collected between 452
and 537 nm. Excitation of PO1- and APF-loaded cells at 488 and
543 nm was carried out with Ar and HeNe lasers, respectively,
and emission was collected using a META detector at 495-538
and 548-602 nm, respectively, using sequential scans. Image
analysis was performed in Image J.
1
as light red solid (36 mg, 38%). H NMR (CDCl3/10% CD3OD,
400 MHz): δ 7.95 (1H, d, J ) 7.2 Hz), 7.40 (1H, s), 7.58 (1H, t,
J ) 7.2 Hz), 7.54 (1H, t, J ) 7.2 Hz), 7.33 (1H, d, J ) 8.0 Hz),
7.11 (1H, d, J ) 7.6 Hz), 6.69 (1H, d, J ) 7.6 Hz), 6.10 (1H, s),
3.10 (2H, t, J ) 5.2 Hz), 3.06 (2H, t, J ) 5.2 Hz), 2.87 (2H, t, J
) 6.4 Hz), 2.37-2.54 (2H, m), 1.91-1.99 (2H, m), 1.79 (2H, t, J
) 5.2 Hz), 1.28 (12H, s). 13C NMR (CDCl3/10% CD3OD, 100
MHz): δ 170.1, 153.2, 151.1, 147.8, 144.7, 134.9, 129.5, 128.6,
127.1, 126.8, 124.8, 124.6, 124.0, 123.5, 121.6, 117.8, 107.2, 104.5,
85.1, 84.1, 74.9, 49.7, 49.3, 27.3, 24.8, 21.6, 21.1, 20.9. HR-
FABMS: calculated for [M+] 522.2456, found 522.2452.
Spectroscopic Materials and Methods. Millipore water was
used to prepare all aqueous solutions. All spectroscopic measure-
ments were performed in 20 mM HEPES buffer, pH 7. Absorption
spectra were recorded on a Varian Cary 50 spectrophotometer
(Walnut Creek, CA), and fluorescence spectra were recorded on a
Photon Technology International Quanta Master 4 L-format scan-
ning spectrofluorometer (Lawrenceville, NJ) equipped with an LPS-
220B 75-W xenon lamp and power supply, A-1010B lamp housing
with integrated igniter, switchable 814 photon-counting/analog
photomultiplier detection unit, and MD5020 motor driver. Samples
for absorption and emission measurements were contained in 1-cm
× 1-cm quartz cuvettes (1.5-mL volume, Starna, Atascadero, CA).
Fluorescence quantum yields were determined by reference to
fluorescein in 0.1 M NaOH (Φ ) 0.94) or rhodamine B in water
(Φ ) 0.31).
Preparation and Staining of Cell Cultures. RAW264.7 mac-
rophages were cultured in Dulbecco’s Modified Eagle Medium
(DMEM) containing high glucose with GlutaMAX (Invitrogen,
Carlsbad, CA) and supplemented with 10% Fetal Bovine Serum
(FBS, Hyclone). A431 cells were cultured in DMEM plus GlutaMAX
supplemented with 10% FBS. Cells were split 1/30 twice a week.
Cells were passed and plated on 18- mm glass coverslips coated
with poly-L-lysine 2 days before imaging (50 µg/mL, Sigma, St.
Louis, MO). A431 cells that were to be used for EGF stimulation
experiments were serum-starved in DMEM alone 18 h before
imaging. For all experiments, solutions of dyes (from 5 mM stocks
in DMSO) were made in DPBS with calcium chloride and
magnesium chloride (Sigma). H2O2 (100 mM stock solution in
Millipore water), HOCl (100 mM stock solution in Millipore water),
phorbol myristate acetate (PMA, 1 mg/mL stock solution in
Acknowledgment. We thank the Packard and Sloan Founda-
tions, the Hellman Faculty Fund (UC Berkeley), Amgen, Astra
Zeneca, Novartis, and the National Institute of General Medical
Sciences (NIH GM 79465) for funding this work. C.J.C. is an
Investigator with the Howard Hughes Medical Institute. B.C.D.
thanks the NIH Chemical Biology Graduate Program (T32
GM066698) for support, and C.H. thanks the Department of
Chemistry at UC Berkeley for a Summer Undergraduate Research
award. We thank Holly Aaron (UCB Molecular Imaging Center)
and Ann Fischer (UCB Tissue Culture Facility) for expert technical
assistance and helpful discussions.
Note Added after ASAP Publication. The formula for the
reagent used to treat the parent dye was incorrect in Schemes 2
and 3 in the version of this article published ASAP April 2, 2010.
The corrected version was reposted April 7, 2010.
Supporting Information Available: Additional imaging data.
This material is available free of charge via the Internet at http://
pubs.acs.org.
JA1014103
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J. AM. CHEM. SOC. VOL. 132, NO. 16, 2010 5915