Fluorogenic α-tocopherol analogue for monitoring the antioxidant status within the inner mitochondrial membrane of live cells

Article abstract of DOI:10.1021/ja408227f

We report here the preparation of a lipophilic fluorogenic antioxidant (Mito-Bodipy-TOH) that targets the inner mitochondrial lipid membrane (IMM) and is sensitive to the presence of lipid peroxyl radicals, effective chain carriers in the lipid chain auto

Full text of DOI:10.1021/ja408227f

Article
pubs.acs.org/JACS
Fluorogenic α‑Tocopherol Analogue for Monitoring the Antioxidant
Status within the Inner Mitochondrial Membrane of Live Cells
Katerina Krumova, Lana E. Greene, and Gonzalo Cosa*
Department of Chemistry and Center for Self-Assembled Chemical Structures (CSACS-CRMAA), McGill University, 801
Sherbrooke Street West, Montreal, Quebec H3A 0B8, Canada
S
* Supporting Information
ABSTRACT: We report here the preparation of a lipophilic
fluorogenic antioxidant (Mito-Bodipy-TOH) that targets the
inner mitochondrial lipid membrane (IMM) and is sensitive to
the presence of lipid peroxyl radicals, effective chain carriers in
the lipid chain autoxidation. Mito-Bodipy-TOH enables
monitoring of the antioxidant status, i.e., the antioxidant load
and ability to prevent lipid chain autoxidation, within the inner
mitochondrial membrane of live cells. The new probe consists
of 3 segments: a receptor, a reporter, and a mitochondria-
targeting element, constructed, respectively, from an α-
tocopherol-like chromanol moiety, a BODIPY fluorophore, and a triphenylphosphonium cation (TPP). The chromanol moiety
ensures reactivity akin to that of α-tocopherol, the most potent naturally occurring lipid soluble antioxidant, while the BODIPY
fluorophore and TPP ensure partitioning within the inner mitochondrial membrane. Mechanistic studies conducted either in
homogeneous solution or in liposomes and in the presence of free radical initiators show that the antioxidant activity of Mito-
Bodipy-TOH is on par with that of α-tocopherol. Studies conducted on live fibroblast cells further show the antioxidant
depletion in the presence of methyl viologen (paraquat), a known agent of oxidative stress and source of superoxide radical anion
(and indirectly, a causative of lipid peroxidation) within the mitochondria matrix. We recorded a ca. 8-fold emission
enhancement with Mito-Bodipy-TOH in cells stressed with methyl viologen, whereas no enhancement was observed in control
studies with untreated cells. Our findings underscore the potential of the new fluorogenic antioxidant Mito-Bodipy-TOH to
study the chemical link between antioxidant load, lipid peroxidation and mitochondrial physiology.
Alzheimer’s disease,1,15 amyotropic lateral sclerosis (ALS),16
INTRODUCTION
■
and ataxia.17
The inner mitochondrial membrane is a major site of
Noninvasive imaging methodologies that enable real-time
generation of reactive oxygen species (ROS) produced as
monitoring of the generation, accumulation, and consumption
byproducts of the mitochondrial electron transport chain.
of ROS within the mitochondria of live cells are of paramount
Polyunsaturated fatty acids (PUFA) within the inner
importance to unravel the chemical mechanism behind the
mitochondrial membrane are particularly vulnerable to ROS
cellular pathologies associated to ROS. Significant progress has
been accomplished in this direction in the last 5 years exploiting
elicited oxidative damage.1 PUFA residues may readily undergo
autoxidation, a chain reaction initiated by a ROS such as the
hydroxyl radical and next propagated by lipid peroxyl radicals,
fluorescence imaging with fluorogenic probes that preferentially
target mitochondria and that react specifically with H2O2,18,19
superoxide,20 and highly reactive ROS.21,22 Recently, Murphy
effective chain carriers in the lipid chain autoxidation which are
themselves ROS.2−4 Reactions of peroxyl radicals are slow
and co-workers developed a membrane embedded ratiometric
compared to the time scale of most other free radical reactions
and thus the peroxyl radical is the dominant species present in
the chain.4 Importantly, the action of antioxidants such as α-
tocopherol (TOH), an effective lipid peroxyl radical scavenger
and the most active naturally occurring lipid soluble
antioxidant,2 inhibits lipid chain autoxidation.5
The deleterious effect of ROS in the mitochondrial
membrane, protein, and DNA has been implicated in the
onset of multiple pathologies associated with mitochondrial
dysfunction.6−9 There is a complex relationship between
mitochondrial ROS-mediated damage and a range of neuro-
degenerative disorders10,11 including Parkinson’s disease,12−14
fluorescent sensor (MitoPerOx) that is susceptible to lipid
peroxyl radicals by tagging the fluorescent probe C11-
BODIPY581/591 with a mitochondria-targeting moiety.23 This
probe contains a BODIPY core conjugated via a dienyl link to a
phenyl group. Reaction with peroxyl radicals leads to cleavage
of the dienyl link to give a chromophore with reduced
conjugation characterized by a blue-shifted emission.24
Our own interest lies in determining the antioxidant status
within the inner mitochondrial lipid membrane, i.e., the ability
Received: August 8, 2013
Published: October 10, 2013
© 2013 American Chemical Society
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of the mitochondria to prevent lipid chain autoxidation. A
probe is thus required capable of reporting via, e.g., emission
enhancement of the depletion of α-tocopherol in the inner
mitochondrial membrane and the onset of the lipid chain
autoxidation. Here we describe the preparation, character-
ization, and application to live cell imaging of a lipophilic
fluorogenic α-tocopherol analogue probe Mito-Bodipy-TOH.
The new probe was designed to both mimic the structure and
reactivity of α-tocopherol25−29 and to target the inner
mitochondrial membrane. The photophysical, reactivity, and
live cell studies conducted position Mito-Bodipy-TOH as a
versatile tool to study the relationship between antioxidant
content, ROS elicited oxidative damage, mitochondrial
physiology, and mitochondrial dysfunction.
subsequently utilized by others18,19,38−44 to selectively direct
attached cargo (e.g., fluorescent probes) to the mitochondrial
matrix. The third segment of the probe thus contained a
triphenylphosphonium cation (TPP) appended via an alkyl tail
to the BODIPY dye. The alkyl group additionally increases the
lipophilic character of the fluorogenic probe, facilitating its
insertion into the mitochondrial membrane.
Synthesis of Mitochondria-Targeting Fluorogenic
Antioxidant. Asymmetric BODIPY dyes are traditionally
prepared by condensation of an α-formyl pyrrole and a pyrrole
with a free α-position.45 We rationalized that synthetically it
would be most feasible to first prepare a chromanol-α-formyl
pyrrole adduct (segment A, Scheme 1). Heck coupling was
Scheme 1. (A) Retrosynthetic Analysis of the Mitochondria-
Targeting Fluorogenic Probe; (B) Structures of Fluorogenic
Probes 1 (Nontargeting) and 2 (Targeting) and Fluorescent
Controls 3 (Nontargeting) and 4 (Targeting)
RESULTS AND DISCUSSION
■
Design of Mitochondria-Targeting Fluorogenic Anti-
oxidant. The new probe consists of 3 segments: a receptor, a
reporter, and a mitochondria-targeting element (Figure 1). A
Figure 1. Structure of mitochondria-targeting fluorogenic antioxidant
(Mito-Bodipy-TOH). The three segments are colored to facilitate
visualization.
chromanol ring bearing the chemical structure of the most
potent naturally occurring free radical scavenger α-tocopherol2
was chosen as a receptor. This choice ensured a high specificity
toward lipid peroxyl radicals, effective chain carriers and the
dominant ROS species encountered in lipid membranes under
oxidative stress. Importantly, it further warranted a rapid rate of
reaction of the probe with lipid peroxyl radicals, on par with
that of α-tocopherol (vide infra), characterized by a rate
constant of 3 × 106 M−1 s−1 in homogeneous solution.2
We used a BODIPY fluorophore as the reporter segment.
BODIPY fluorophores have optimal spectroscopic properties
and they are lipophilic. Further, BODIPY structures are
amenable to synthetic modifications to introduce chemical
groups and to tune their redox potentials.25−27 BODIPY dyes
are thus easily tunable to afford exergonic photoinduced
electron transfer (PeT) from the receptor (chromanol)
moiety.25,30 An intramolecular switch based on PeT may thus
render the probe nonemissive. Oxidation of the receptor upon
reaction with peroxyl radicals deactivates PeT and emission in
the reporter segment is restored.25,26,28,29 Our strategy renders
a fluorogenic probe that upon scavenging two peroxyl radicals
(with the concomitant oxidation of the receptor segment)
undergoes a fluorescence enhancement. Reporting is thus
directly related to peroxyl radical scavenging.25−29 A vinyl linker
connecting the chromanol moiety to the BODIPY core helps
minimize the distance between the receptor and reporter
segments, further favoring PeT and providing a lipophilic
substrate.
employed to couple both units via a vinyl linker. We
subsequently assembled the asymmetric BODIPY dye. Here
we envisioned that the pyrrole with a free α-position should
have a functional group to enable the installation of the TPP
moiety. The desired nucleophilic pyrrole was thus prepared
bearing a propyl chloride group (segment B, Scheme 1).
Condensation between the chromanol-α-formyl pyrrole and
segment B was then carried out to obtain the chromanol-
BODIPY precursor 1 (Scheme 1). Mito-Bodipy-TOH 2 was
next obtained upon the nucleophilic substitution of the chloride
in 1 with triphenylphosphine to install the positively charged
phosphonium tag. Control compounds 3 and 4 were
subsequently prepared bearing a methoxy group on the
chromanol ring.
To accomplish the Heck coupling, we first prepared the
chromanol alkene 6 and the iodo-formyl pyrrole 7 utilizing
previously reported procedures (Scheme 2).29,46,47 We further
protected the pyrrole with a methoxymethyl (MOM) group to
yield compound 8. Traditional protecting groups used for
pyrroles such as tert-butyloxycarbonyl (Boc) and benzoyl (Bz)
groups were not amenable to our synthetic conditions as they
were easily deprotected under the high temperatures (100 °C)
required for of the Heck reaction (Scheme 2).
We utilized a largely delocalized positively charged segment
for preferential accumulation of the probe in the inner
mitochondrial membrane.31−33 We resorted to the use of
phosphonium groups, established by Murphy23,34−37 and
We subsequently proceeded with the Heck coupling of
compounds 6 and 8. Standard Heck coupling conditions
involving triethylamine and Pd(OAc)2 in dimethylformamide
(DMF) at 100 °C led exclusively to deiodination of pyrrole 8.
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Having prepared all the necessary segments to assemble the
BODIPY precursor, we proceeded with their condensation.
Pyrrole 17 was subjected to a condensation reaction with
pyrrole 11 in the presence of phosphorus oxychloride (Scheme
4). Complexation of the so formed dipyrromethene with BF3·
OEt and diisopropylethylamine yielded the chormanol-
BODIPY adduct 1.
Scheme 2. Synthesis of Segment A
Scheme 4. Synthesis of Mitochondria-Targeting Fluorogenic
Probe Mito-Bodipy-TOH (2) and Its Precursor 1
The final step in the synthesis of the new mitochondria-
targeting fluorogenic antioxidant 2 was the nucleophilic
substitution of the chloride in the lipophilic tail of the BODIPY
dye with triphenylphosphene to install the positively charged
phosphonium tag (Scheme 4). The substitution was facilitated
by the use of sodium iodide.
Examining the mechanism of the reaction we concluded that
the loss of iodine was possibly due to the base used. We
modified the conditions employing potassium acetate,
tetrabutylammonium bromide, and 10% Pd(PPh3)4 (Scheme
2).48 The reaction was heated to 100 °C for 24 h. The coupling
yielded the desired chromanol-pyrrole adduct 9 in 68% yield.
Deiodination was still observed but only in 30% yield.
To subject compound 9 to a condensation with the pyrrole
bearing a free α-position to form the BODIPY fluorophore, a
removal of both protecting groups (MOM on the pyrrole and
methoxy on the chromanol) was necessary. MOM deprotection
proved to be challenging as most of the product decomposed
under the acidic conditions used (1 M HCl or 1 M
trifluoroacetic acid). Only 10% yield was achieved under
these conditions. We thus developed an alternative two-step
deprotection approach (Scheme 2). In the first step, addition of
2 equiv of BBr3 at −10 °C removed both the methoxy group on
the phenol and the methoxy part of the MOM protection to
yield 10. Compound 10 is a hemiaminal, which was hydrolyzed
to formaldehyde and fully deprotected pyrrole 11 following
treatment with 1 M NaOH.49 Maintaining the right temper-
ature throughout the reaction of the formation of 10 was
crucial. An increase in the temperature led to decomposition of
the intermediate 10.
Control compounds 3 and 4 were next prepared bearing the
methoxy group on the chromanol ring (Scheme 5). Starting
Scheme 5. Synthesis of Fluorescent Mitochondria-Targeting
Control Compound 4 and Its Precursor 3
from compound 9, it was partially deprotected using 1 equiv of
BBr3, where only the pyrrole protecting MOM-group was
removed to yield compound 12. The same synthetic
procedures described above and outlined in Scheme 5 were
next employed to obtain 3 and 4.
Spectroscopic Properties of the Fluorogenic Antiox-
idant and Analogues. Table 1 lists the absorption and
emission maxima, emission quantum yields, and absorption
extinction coefficients determined in acetonitrile for com-
pounds 1−4 (see also Figure 2). Compounds 1 and 2 displayed
We next proceeded with the preparation of the pyrrole with a
free α-position (segment B). The synthesis of compound 17
was accomplished following previously developed procedures
(Scheme 3).50,51
Table 1. Photophysical Properties of Dyes 1−4 in
Scheme 3. Synthesis of Segment B
a
Acetonitrile at Room Temperature
Abs λmax (nm)
Em λmax (nm)
Φf
ε × 103 (M−1 cm−1
)
1
2
3
4
536
537
536
536
582
572
585
582
0.04
0.02
0.30
0.23
34
34
34
34
a
Quantum yields were measured using PM605 in acetonitrile as a
standard (Φst = 0.72).30
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Figure 3. Structures of α-tocopherol and PMHC.
antioxidants. The relative antioxidant activities of the
antioxidants were obtained from the analysis of the initial
slope of a plot of ln[(I∞ − It)/(I∞ − Io)] vs ln[1 − t/τ]. Studies
in acetonitrile show that peroxyl radicals react with the same
rate constant with 1, 2, TOH and PMHC compounds (Table
S1). The inherent chemical reactivity of the chromanol ring in
1 and 2 is thus on par with the reactivity of α-tocopherol and
PMHC and is not diminished by its coupling to the BODIPY
segment or the presence of the TPP segment (Figure S2A in
the Supporting Information section and discussion therein).
The absolute value for the rate constant of reaction of the new
probe with peroxyl radicals may thus be positioned in the order
of ca. 3 × 103 M−1 s−1, the value reported for α-tocopherol
under similar conditions.52,53
Control experiments were further conducted with the
fluorescent analogue 3 in the presence of the competing
antioxidant PMHC (Figure S2B). No emission enhancement
was observed for compound 3, where PeT is not operational,
and where the methoxy protected phenol cannot undergo H-
abstraction by peroxyl radicals (Figure S2B). A linear drop in
emission intensity with time was recorded once PMHC is
consumed (Figure S2B). This result is consistent with the onset
of radical-mediated BODIPY degradation.
Figure 2. Normalized absorption and emission spectra obtained in
acetonitrile for compounds 1−4. Plots are offset with respect to each
other in the ordinate axis to facilitate comparison.
an emission quantum yield 10-fold lower than that of
counterparts 3 and 4 that bear a methoxy-protected phenol.
Whereas the former two compounds can readily undergo
intramolecular PeT from the chromanol moiety to the
BODIPY fluorophore,25,26,29 the introduction of the methoxy
group in 3 and 4 increases the oxidation potential of the
chromane moiety deactivating the intramolecular PeT. Emissive
compounds 3 and 4 thus provide for exquisite controls in cell
studies with fluorogenic compounds 1 (lipophilic yet not
organelle-specific) and 2 (mitochondria-targeting), respectively
(vide infra).
A 6-fold increase in fluorescence intensity with time was
observed for 1 or 2 when embedded in Egg PC lipid
membranes upon exposure to hydrophilic (ABAP source) or
lipophilic (MeO-AMVN) peroxyl radicals. Competitive kinetic
studies conducted on liposome suspensions show that Mito-
Bodipy-TOH and α-tocopherol have the same rate constant for
reaction with peroxyl radicals. In stark contrast, PMHC
outcompetes the new probes for reaction with peroxyl radicals
(Figure S4 and discussion therein). Mobility and to a lesser
extent physical accessibility account for the larger relative
antioxidant activity of tocopherol analogues bearing short
(PMHC) over long (α-tocopherol) aliphatic tails, but that
otherwise have the same inherent chemical reactivity.29,54
Importantly, our competing kinetic studies not only pinpoint
the similar antioxidant activity of α-tocopherol and Mito-
Bodipy-TOH, but further underscore that Mito-Bodipy-TOH is
embedded within the membrane much like α-tocopherol.
Monitoring Antioxidant Load in the Mitochondria of
Living Cells. We initially conducted fluorescence colocaliza-
tion studies to ensure that the probe localizes in mitochondria.
Studies were conducted involving either the fluorogenic probe
Mito-Bodipy-TOH (2) or its mitochondria targeting (4) or
nontargeting fluorescent analogue (3), and MitoTracker Deep
Red (a commercially available mitochondrial indicator). As a
model cell line we used NIH 3T3 mouse embryo fibroblast
cells. Cells were simultaneously stained with a 1 μM solution of
Mito-Bodipy-TOH and a 200 nM solution of MitoTracker
Deep Red at 37 °C for 5 min. Upon selective excitation of
Mito-Bodipy-TOH at 488 nm, the cells exhibited extremely
faint, yet detectable, fluorescence (500−620 nm) in discrete
subcellular locations as determined by confocal microscopy
(Figure 4A1). Similar images were obtained upon selective
excitation of MitoTracker Deep Red at 633 nm and emission
collection from 650 to 740 nm (Figure 4A2). The
Peroxyl Radical Scavenging Activity of Mito-Bodipy-
TOH. To investigate the sensitivity and reactivity of 1 and 2
toward peroxyl radicals in homogeneous (acetonitrile solution)
and microheterogeneous media (liposome suspension), these
fluorogenic antioxidants were subjected to reaction with peroxyl
radicals generated via thermolysis of azo-initiators in oxygen
equilibrated solutions. The analysis of the intensity-time
trajectories we recorded provided a measure of the sensitivity
(fluorescence enhancement) and reactivity/antioxidant activity
of the new probes when experiments were conducted with
competing antioxidants. The experimental procedures and
analysis used have been previously reported by us.25,29 Three
different azo-initiators, two generating lipophilic peroxyl
radicals (AMBN and MeO-AMVN)) and one a hydrophilic
peroxyl radical (ABAP), were used to examine the reactivity
and sensitivity of the probes (Figure S1).
A significant, ca. 10-fold, fluorescence enhancement was
recorded with time following reaction of 1 and 2 with peroxyl
radicals generated from AMBN in homogeneous media. The
enhancement is consistent with the deactivation of the
intramolecular PeT process upon oxidation of the receptor
segment, and provides a magnitude for the sensitivity of the
new probe toward ROS.
The reactivity of 1 and 2 toward peroxyl radicals was
determined in competitive kinetic studies performed in the
presence of either α-tocopherol or 2,2,5,7,8-pentamethyl-6-
hydroxychroman (PMHC), an α-tocopherol analogue lacking
the phytyl chain (Figure 3).29 Briefly, the new probe was used
as a signal carrier in competitive kinetic studies in the presence
of TOH and PMHC. In our assay, we used a microplate reader
to monitor over time the emission intensity enhancement of
membrane-embedded Mito-Bodipy-TOH upon scavenging
peroxyl radicals when alone or in the presence of competing
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Figure 4. Confocal fluorescence images of live NIH 3T3 mouse embryo fibroblast cells acquired with 100× magnification objective. Cells were
incubated with 1 μM probes 2, 3, and 4, together with 200 nM MitoTracker Deep Red at 37 °C for 5 min in DMEM media supplemented with 10%
FBS. Images display emission intensities collected in optical windows between 500 and 620 nm (for probes 2, 3, and 4) upon excitation at 488 nm
(125 μW laser power) and between 650 and 740 nm (for MitoTracker Deep Red) upon excitation at 633 nm (160 μW laser power): (A1) probe 2
(Mito-B-v-TOH); (A2) MitoTracker Deep Red; (A3) overlay of 1 and 2; (A4) bright field image; (B1) probe 4; (B2) MitoTracker Deep Red; (B3)
overlay of 1 and 2; (B4) bright field image; (C1) probe 3; (C2) MitoTracker Deep Red; (C3) overlay of 1 and 2 (C4) bright field image.
Cytofluorograms are displayed in panels A5−C5, demonstrating the extent of the colocalization for each probe (2, 3, and 4) with MitoTracker Deep
Red. Scale bar is 10 μm.
colocalization of both dyes confirmed that Mito-Bodipy-TOH
is localized in the mitochondria of live cells (Figure 4A3). We
calculated the overlap coefficients for Mito-Bodipy-TOH and
MitoTracker Deep Red according to Manders, a method that
allows for reliable estimation of close proximity localization.55
We obtained Manders’ colocalization coefficients of M1 =
0.999 and M2 = 0.998, which further confirms the
colocalization of the two molecules.
A 3D visualization of the cells enabled by stacks of confocal
images further confirmed the colocalization of both dyes. A
bright field image in turn revealed that cells are viable during
the experiments (Figure 4A4). The fluorescent analogue 4 also
showed colocalization with MitoTracker Deep Red under the
same imaging conditions (Figure 4B1−4). We calculated
Manders’ colocalization coefficients of M1 = 0.984 and M2 =
0.723 for compound 4 and MitoTracker Deep Red. No
colocalization was observed with MitoTracker Deep Red in
control experiments using nontargeting fluorescent analogue 3
(Figure 4C1−4) with Mander’s colocalization coefficients of
M1 = 0.11 and M2 = 0.01. Taken together, these results
establish that the lipophilic fluorogenic antioxidant Mito-
Bodipy-TOH (2) and its fluorescent analogue 4 accumulate
preferentially in the inner mitochondrial membrane due to the
lipophilic cationic segment (phosphonium tag) and overall
lipophilic moiety.
(scavenging of chromanol moieties) in live cell studies, see
Figure 5. Methyl viologen (MV2+) was used as a source of ROS
in our experiments.56,57 MV2+ is transported into mitochondria,
where once in the matrix and in the presence of intracellular
electron donors such as flavin-containing oxidoreductase
enzymes,58,59 it catalyzes the reduction of molecular oxygen
to water yielding various ROS intermediates along the way.
Specifically, MV2+ undergoes reduction to a radical cation that
may be subsequently oxidized back to MV2+ upon reaction with
molecular oxygen, yielding superoxide radical anion. A series of
subsequent electron transfer and proton uptake reactions may
lead to the formation of various intermediate ROS species such
as hydrogen peroxide and hydroxyl radical56 that ultimately lead
to lipid chain autoxidation in the absence of antioxidants.
Fibroblast cells were cultured in media deprived of growth
factors and supplemented with 1 mM MV2+ for 4 h. Following
the removal of the MV2+ solution, the cells were incubated at
37 °C with 1 μM Mito-Bodipy-TOH in media. After 5 min, the
cells were washed and the solution was replaced with media.
Control cells were cultured in media with growth factors and
no MV2+ under otherwise identical conditions. Both the healthy
and stressed cells were imaged by confocal microscopy under
the same imaging conditions (Figure 5). Whereas no emission
enhancement was recorded in healthy cells (Figure 5A), we
recorded a ca. 8-fold emission enhancement for Mito-Bodipy-
TOH in stressed cells (Figure 5B). Clearly, the ongoing
production of superoxide radial anion catalyzed by MV2+ within
We next tested for the ability of Mito-Bodipy-TOH to report
production of lipid peroxyl radicals and antioxidant depletion
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Figure 5. Detection of ROS with mitochondria-targeting fluorogenic probe Mito-Bodipy-TOH (2) in live NIH 3T3 mouse embryo fibroblast cells.
Cells were incubated with 1 μM Mito-Bodipy-TOH and 200 nM MitoTracker Deep Red at 37 °C for 5 min. Emission intensities were recorded 15
min after incubation with the dyes and collected in optical windows between 500 and 620 nm (for probes 2 and 4) upon excitation at 488 nm (125
μW laser power) and between 650 and 740 nm (for MitoTracker Deep Red) upon excitation at 633 nm (160 μW laser power). The panels show
(left to right) confocal fluorescence images of 1 μM Mito-Bodipy-TOH, 200 nM MitoTracker Deep Red, overlay of the first two images, and bright
field image of (A) fibroblast cells in the presence of 10% FBS, (B) fibroblast cells under oxidative stress conditions (deprived of growth factors and
additionally stressed with 1 mM MV2+ for 4 h prior to imaging), (C) fibroblast cells in the presence of 10% FBS incubated with 1 μM control
mitochondria-targeting fluorescent probe 4. Scale bar is 20 μm.
the mitochondria matrix results in the production of lipid
peroxyl radicals, among other ROS, which are readily scavenged
by Mito-Bodipy-TOH, triggering its oxidation and concomitant
emission enhancement.
The intensity recorded in control studies with fluorescent
analogue 4 and healthy cells was ca. 7-fold higher than the one
observed with the fluorogenic analogue Mito-Bodipy-TOH in
healthy cells under otherwise identical conditions (Figure 5C),
and similar to the intensity recorded for Mito-Bodipy-TOH in
cells treated with MV2+.
Altogether, our results confirm that the mitochondria-
targeting fluorogenic antioxidant readily reports via emission
enhancement the generation of lipid peroxyl radicals, and the
depletion of antioxidants and thus the onset25,26,29 of the lipid
chain autoxidation in the inner mitochondrial membrane.
In closing, we note that Mito-Bodipy-TOH is complemen-
tary in its action to lipophilic probes such as MitoPerOx,23
derived from C11-BODIPY581/591.24 In the presence of
peroxyl radicals, the former undergoes H-atom abstraction from
its chromanol moiety with a rate constant comparable to that of
α-tocopherol. Mito-Bodipy-TOH thus reports the antioxidant
consumption, and the onset of lipid chain autoxidation.60,61 In
turn, C11-BODIPY581/591 and related probes rely on a slower
reaction involving the breaking of the chromophore dienyl link
upon reaction with lipid peroxyl radicals.23,62 This reaction
takes place once antioxidants have been consumed and lipid
peroxyl radicals become the effective chain carriers in the lipid
chain autoxidation.63 Both probes thus target different stages of
the membrane peroxidation process. Importantly, small
concentrations of Mito-Bodipy-TOH should be employed in
order to avoid changes in the antioxidant status of the live cell.
The preferential accumulation of the fluorogenic antioxidant
within the mitochondria might otherwise result in an enhanced
protection of the mitochondrial function from oxidative
damage.36,37
We also note that the intensity discrimination between the
probe and its oxidized form may be exploited toward
developing a ratiometric analysis based on fluorescence
lifetimes. We are currently exploring experiments conducted
in a fluorescence lifetime imaging microscope setup toward
monitoring the ratio of oxidized to pristine Mito-Bodipy-TOH
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absorption for the standard Ast, integrated emission Ix and solvent
refractive index nx for the unknown Φx vs the product of absorption
for the unknown Ax, integrated emission Ist and solvent refractive index
nst for the standard.
based on their characteristic fluorescence lifetimes (long and
short, respectively).
CONCLUSIONS
■
Here we have described a convergent methodology for the
preparation of BODIPY-based fluorogenic antioxidants and,
more generally, functionalized BODIPY dyes. Using this
methodology, we have accomplished the synthesis of a 3-
segment lipophilic fluorogenic antioxidant that targets the inner
mitochondrial lipid membrane of live cells. Mechanistic studies
in the presence of free radical sources conducted both in
homogeneous and microheterogeneous systems for the new
probe herein reported position its antioxidant activity on par
with of α-tocopherol, the most potent naturally occurring lipid
soluble antioxidant. The probe is thus characterized by a rapid
rate of reaction with lipid peroxyl radicals. Both the sensitivity
and the reactivity toward lipid peroxyl radicals within the inner
mitochondrial membrane are unique attributes of the new
probe Mito-Bodipy-TOH.
ϕ
Ast × Ix × nx2 =
× Ax × Ist × ns2t
x
ϕ
st
Steady State Fluorescence Studies. A Cary Eclipse Spectropho-
tometer with temperature controller was utilized to measure the
emission intensity profiles of (A) 9.5 μM compounds 1 and 2 and (B)
9.0 μM α-tocopherol or PMHC solutions with 0.5 μM compounds 1
and 2 in 3 mL of acetonitrile. Fluorescence was recorded at 570 nm
upon exciting at 510 nm using 2.5 nm excitation and 2.5 (for case A)
or 5 nm (for case B) emission slits. A total of 2.875 mL of acetonitrile
solutions containing compounds 1 or 2 with or without α-tocopherol
or PMHC was incubated for 10 min at 37 °C before 125 μL of AMBN
2.028 M in acetonitrile was added to each cuvette for final AMBN
concentration of 84.5 mM. The emission intensity was followed at 2.5
s intervals for 4000 s.
Liposome Preparation and Microplate Assays. Assays were
conducted following a previously reported procedure.29
Given the location of the electron transport chain, the inner
mitochondrial membrane is highly susceptible to the chemical
activity of ROS. Polyunsaturated fatty acids (PUFA) within the
inner mitochondrial membrane are particularly vulnerable to
oxidative modifications that result in the lipid chain
peroxidation. Here we have shown via a suite of imaging
studies conducted on live cells that Mito-Bodipy-TOH enables
real-time monitoring of lipid peroxyl radical generation and
antioxidant depletion within the inner mitochondrial mem-
brane. Our findings underscore the potential of the fluorogenic
antioxidant Mito-Bodipy-TOH to investigate the relationship
between antioxidant load, onset of the lipid chain autoxidation
and mitochondrial physiology and mithochondrial dysfunction.
Cell Preparation and Staining. NIH 3T3 mouse embryo
fibroblast cells were cultured in Dulbecco’s Modified Eagle Medium
(DMEM) containing high glucose with L-glutamine, phenol red, and
sodium pyruvate (Gibco), supplemented with 10% fetal bovine serum
(FBS) and 1% penicillin-streptomycin. Cells were maintained at 37 °C
(5% CO2) in a humidified atmosphere. Cells were trypsinized and split
1/40 once a week when confluence of cells was reached. For
fluorescence microscopy, cells were passed, split 1/5, plated on 35 mm
glass imaging dish (World Precision Instruments, Inc.) coated with
fibronectin (1 μg/mL), and cultured in DMEM containing growth
factors 1 day prior to imaging. For all experiments, solutions of dyes
were prepared as follows: 60 μM MitoTracker Deep Red (Molecular
Probes) or 300 μM of 2, 3, and 4 stock solutions in DMSO were
prepared. Ten microliters of each stock solution was added to 3 mL of
Dulbecco’s Modified Eagle Medium (DMEM) containing high glucose
with L-glutamine, without phenol red, sodium pyruvate (Gibco), or
HEPES. Cells were incubated with the DMEM solutions with final
concentrations of 200 nM MitoTracker Deep Red and 1 μM of either
2, 3, or 4 for 5 min at 37 °C (5% CO2) in a humidified atmosphere.
Following incubation, the DMEM solutions were removed and
exchanged with DMEM containing high glucose, L-glutamine, and
10% fetal bovine serum (FBS), without phenol red, or sodium
pyruvate.
Cells Treated with Methyl Viologen. NIH 3T3 mouse embryo
fibroblast cells were prepared as described above. The DMEM growth
media was removed 4 h prior to imaging and substituted with 1.5 mL
of DMEM containing 1 mM MV2+ (methyl viologen) and glucose with
L-glutamine, without phenol red, sodium pyruvate (Gibco), or HEPES.
After 4 h, the MV2+ solution was removed, cells were washed with
Dulbecco’s Phosphate-Buffered Saline (DPBS) and stained as
described above, and the media was exchanged to DMEM containing
high glucose with L-glutamine, without phenol red, sodium pyruvate
(Gibco), or HEPES.
EXPERIMENTAL SECTION
■
Abbreviations. 2,2′-Azobis-(2-methylpropionamidine) dihydro-
chloride (ABAP); 2, 2′-azobis-(2-methylbutyronitrile) (AMBN);
2,2′-azobis(4-methoxy-2.4-dimethyl valeronitrile) (MeO-AMVN);
benzoyl group (Bz); boron-dipyrromethene (4,4-difluoro-4-bora-
3a,4a-diaza-s-indacene) (BODIPY); tert-butyloxycarbonyl group
(Boc); dimethylformamide (DMF); dimethyl sulfoxide (DMSO);
Dulbecco’s Modified Eagle Medium (DMEM); fetal bovine serum
(FBS); 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES);
L-α-phosphocholine (EggPC); methoxymethyl group (MOM); N,N′-
dimethyl-4,4′-bipyridinium dichloride (Methyl Viologen) (MV2+);
2,2,5,7,8-pentamethyl-6-chromanol (PMHC); α-tocopherol (TOH).
Materials. 2,2′-Azobis(4-methoxy-2.4-dimethyl valeronitrile)
(MeO-AMVN) and 2,2′-azobis(2-methylbutyronitrile) (AMBN)
were supplied by Wako Pure Chemical Industries, Ltd. L-α-
Phosphocholine (EggPC) was obtained from Avanti Polar Lipids,
(Alabaster, AL). All other chemicals were purchased from Sigma-
Aldrich (Oakville, Ontario, Canada) and were used without further
purification. Water was purified by a Millipore Milli-Q system.
Instrumentation. Absorption and emission spectra were recorded
on a Cary 5000 UV−vis-NIR and Cary Eclipse Fluorescence
Fluorescence Imaging Experiments. Confocal fluorescence
imaging studies were performed using a Zeiss LSM 710 laser scanning
microscope equipped with either a C-Apochromat 40×/1.20 W Korr
M27 water-immersion objective lens or a Plan-Apochromat 100×/1.40
oil-immersion objective lens. Cells stained with either dyes 2, 3, or 4
and MitoTracker Deep Red were excited with the 488 nm (125 μW)
laser output of a cw Ar+ laser (for imaging 2, 3, or 4) and with the 633
nm (160 μW) laser output of a cw HeNe laser (MitoTracker Deep
Red). An MBS 488/543/633 dichrioc beam splitter was utilized.
Emission was collected using a META detector between 500 and 620
nm and 650−740 nm, respectively, using sequential scans.
1
Spectrophotomeres, using 1 cm × 1 cm quartz cuvettes. H NMR
and 13C NMR spectra were recorded on a Varian VNMRS 500
instrument at 500 and 126 MHz, respectively. ESI mass spectra were
measured on a Thermo Scientific Exactive Orbitrap.
Fluorescence Quantum Yields. PM605 in acetonitrile (Φst
=
0.72) was used as a standard to calculate the emission quantum yields
of the new compounds (Φx). The absorbance at 510 nm of solutions
of PM605 in acetonitrile and the dye of interest in acetonitrile at five
different concentrations were recorded. Emission spectra were
recorded for all solutions using excitation and emission slits of 2.5
nm upon excitation at 510 nm. Relative quantum efficiencies with
respect to the standard were obtained from the slope of the product of
Calculations for cell fluorescence. All quantifications were
performed using ImageJ software following a protocol previously
published.64,65 We used the equation below to calculate the corrected
total cell fluorescence (CTCF):
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Journal of the American Chemical Society
Article
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CTCF = IntDensity − (Cell Area × BkgFluorescence)
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̀
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∑i Ri,coloc
∑i Ri
∑i Gi,coloc
∑i Gi
M1 =
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i.e., M1 is the sum of the intensities of red pixels that have a green
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ASSOCIATED CONTENT
■
S
* Supporting Information
Kinetic studies in homogeneous and microheterogeneous
1
media, synthesis, IR, H NMR and 13C NMR spectra for all
new compounds and precursors. This material is available free
of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
(27) Khatchadourian, A.; Krumova, K.; Boridy, S.; Ngo, A. T.;
Maysinger, D.; Cosa, G. Biochemistry 2009, 48, 5658.
(28) Krumova, K.; Friedland, S.; Cosa, G. J. Am. Chem. Soc. 2013,
135, 1625.
(29) Krumova, K.; Freidland, S.; Cosa, G. J. Am. Chem. Soc. 2012,
134, 10102.
■
Corresponding Author
gonzalo.cosa@mcgill.ca
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
G.C. is grateful to the Natural Sciences and Engineering
Research Council (NSERC) and Canadian Foundation for
Innovation (CFI) for funding. K.K. is thankful to the Drug
Discovery and Training Program (CIHR) for postgraduate
scholarships; L.E.G. is thankful to Vanier Canada for a
postgraduate scholarship. We are also grateful to Prof. Siegfried
Hekimi and Ms. Ying Cheng at McGill Biology for the use of
their facilities. Imaging and image processing/analysis for this
manuscript were performed in the McGill University Life
Sciences Complex Imaging Facility.
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