A mitochondria-targeted protonophoric uncoupler derived from fluorescein

Article abstract of DOI:10.1039/c4cc04996a

Linking decyl-triphenyl-phosphonium to fluorescein yields a fluorescent probe that accumulates in energized mitochondria, facilitates proton transfer across membranes and stimulates mitochondrial respiration. This features a mitochondria-targeted uncouple

Full text of DOI:10.1039/c4cc04996a

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A mitochondria-targeted protonophoric
uncoupler derived from fluorescein†
Cite this:DOI: 10.1039/c4cc04996a
Stepan S. Denisov,ab Elena A. Kotova,a Egor Y. Plotnikov,a Artur A. Tikhonov,b
Dmitry B. Zorov,a Galina A. Korshunovaa and Yuri N. Antonenko*a
Received 3rd July 2014,
Accepted 16th October 2014
DOI: 10.1039/c4cc04996a
www.rsc.org/chemcomm
Linking decyl-triphenyl-phosphonium to fluorescein yields a fluor-
Earlier attempts were made to render uncouplers specific to
escent probe that accumulates in energized mitochondria, facilitates mitochondria by their conjugation to the triphenylphosphonium
proton transfer across membranes and stimulates mitochondrial (TPP) cation preferentially accumulating in the negatively charged
respiration. This features a mitochondria-targeted uncoupler, being interior of mitochondria. Unfortunately, the obtained compounds
of potential interest for therapeutic use against oxidative stress- either lacked uncoupling activity,5 or showed rather weak activity
related diseases.
which was mediated by the mitochondrial carrier adenine
nucleotide translocase (ANT).6 To combine three essential
An electrochemical proton gradient across energy-coupling (either requirements such as protonophoric activity, intense fluores-
mitochondrial or photosynthetic) membranes resulting from oxida- cence and mitochondria targeting, we designed here a com-
tive or photosynthetic electron transfer is employed to drive ATP pound containing a TPP moiety attached to fluorescein via an
synthesis. This fundamental principle of bioenergetics was validated alkyl linker (mitofluorescein, mitoFluo), taking into account
primarily by using uncouplers – agents capable of dissipating the that the use of lipophilic cations such as alkylTPP has enabled
proton gradient and electrical potential difference by facilitating targeting of a great variety of molecules to mitochondria.7 The
transmembrane proton movement thereby leading to uncoupling of rationale of the design is based on our previous study, where we
electron transfer and phosphorylation. The protonophoric uncouplers observed the submicromolar uncoupling activity of dodecyl and
have attracted much attention not only from the mechanistic point of octyl fluorescein esters, brightly fluorescing compounds, which
view, but also as promising agents potentially curing obesity. Of note, did not accumulate in mitochondria in an energy-dependent
the anti-obesity use of the uncoupler dinitrophenol (DNP), later manner.8 The uncoupling activity of fluorescein alkyl esters
dismissed because of deaths caused by occasional overdosing,1 depended non-monotonically on the alkyl chain (CnH2n+1
)
was demonstrated very early along with its protonophoric activity.2 length with an optimum at n = 8 (n – the number of carbon
Recently, limited uncoupling, i.e. a reduction of proton-motive force to atoms). Here we chose an alkyl linker with n = 10, because
a level being still sufficient for maintaining ATP synthesis but conjugation with positively charged TPP was expected to reduce
diminishing reactive oxygen species (ROS) production,3 was found the hydrophobicity of the chimeric compound.
to be beneficial, in particular by protecting living organisms from a
The synthesis of mitoFluo, 10-[2-(3-hydroxy-6-oxo-xanthen-9-
variety of pathologies associated with oxidative stress.4 On the way to yl)benzoyl]oxydecyl-triphenyl-phosphonium bromide, involved
therapeutic use of mild uncouplers, it is of importance to carefully quaternization of triphenylphosphine and then synthesis of
examine their interaction with different cell compartments and fluorescein ester by reaction with the resulting 10-bromdecyl-
tissues, for which brightly fluorescent compounds as reporters of triphenyl-phosphonium bromide (Scheme S1, ESI†).
their intracellular localization could be useful. On the other hand,
Most of the conventional uncouplers are weak acids with a
direct targeting to mitochondria could reduce the therapeutic pKa close to physiological pH, which are able to permeate the
concentration and side effects of uncouplers.
membrane both in charged and neutral forms.1,9 These substances
usually show maximal activity at pH values higher than pKa due to a
lower membrane permeability of a negatively charged form than
that of a neutral form. Being also a weak acid with a pKa of 6.9, as
measured by fluorescence titration (Fig. S1, inset, ESI†), mitoFluo
could be expected to possess protonophoric activity under physio-
logical conditions. Actually, the addition of mitoFluo to aqueous
a Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State
University, Leninskie Gory 1, Moscow 119991, Russia.
E-mail: antonen@genebee.msu.ru
b Faculty of Chemistry, Lomonosov Moscow State University, Moscow 119991,
Russia
† Electronic supplementary information (ESI) available: Materials, experimental
details, characterization data, and biological assays. See DOI: 10.1039/c4cc04996a bathing solutions at both sides of a planar bilayer lipid membrane
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Fig. 1 Response of the transmembrane electrical current to mitoFluo. (A) Changes in the electrical current across planar BLM formed from the
Escherichia coli polar lipid extract at the applied voltage of 50 mV upon successive additions of mitoFluo. (B) pH dependence of mitoFluo-mediated
electrical current across planar BLM formed from DPhyPC at the applied voltage of 50 mV (mitoFluo concentration 400 nM).
(BLM) caused an increase in membrane proton conductance As seen in Fig. 2A, the addition of 200 nM mitoFluo to the
(Fig. 1A) with a maximum at pH 3 (Fig. 1B), as detected by suspension of isolated rat liver mitochondria (RLM) energized by
recording the electrical current across BLM under voltage- succinate resulted in significant acceleration of mitochondria
clamp conditions. In view of the known bell-shaped pH dependence respiration. The mitoFluo-induced increase in the respiration
of the activity of conventional protonophores with the optimum pH rate was ANT-independent, as shown by its insensitivity to the
depending on the limiting step of proton transfer across BLM,9 the ANT inhibitor atractyloside (Fig. 2A, ii). Concentration depen-
above data allowed us to conclude that mitoFluo-mediated proton dence of the mitoFluo effect on the respiration rate (Fig. 2B)
conductance is limited by membrane permeability for the protonated appeared to be non-monotonic: it reached maximum at a mito-
cationic form of the compound. Thus, the proton transfer cycle is Fluo concentration of 1 mM, which was followed by a modest
suggested to involve deprotonation of mitoFluo and movement of gradual decline of the effect upon further increase in concen-
the zwitter ion through the membrane, followed by protonation tration, in contrast to an abrupt drop in the uncoupling activity
and back transfer of the cation, the latter being the rate limiting of fluorescein octyl ester (C8-FL). Such a gap between the
step (Scheme 1). In support of this idea, proton selectivity of uncoupling and inhibiting concentrations of mitoFluo, not
mitoFluo-mediated permeability of BLM was demonstrated by characteristic of known protonophores,10 could be associated
measuring current–voltage dependencies under symmetrical and with its self-limiting potential-dependent behavior.5,6,11 Simulta-
asymmetrical conditions (Fig. S3, ESI†).
neous recording of RLM respiration using a Clark oxygen
The finding of mitoFluo protonophoric activity on BLM electrode and that of membrane potential using a tetraphenyl-
encouraged us to examine its effect on mitochondria respiration. phosphonium (TPP+)-selective electrode (Fig. S4, ESI†) demon-
strated the applicability of the latter for monitoring the
concentration of mitoFluo in the medium (showing mitoFluo
uptake by mitochondria upon energization with its subsequent
partial release upon the addition of the potent conventional
uncoupler carbonyl cyanide m-chloro phenyl hydrazone (CCCP)
and oxygen exhaustion) and also correspondence between the
signals of the TPP+-electrode in the presence of mitoFluo and
concomitant changes in the respiration rate.
Bright fluorescence of mitoFluo (Fig. S1, ESI†) enabled us to
supplement the data on the macroscopic energy-dependent uptake
of mitoFluo from the medium into mitochondria (Fig. S4, ESI†) by
fluorescence correlation spectroscopy (FCS) measurements of mito-
Fluo accumulation inside single mitochondria (Fig. 2C). Energiza-
tion of mitochondria with succinate resulted in the appearance
of high-amplitude peaks in the fluorescence intensity traces
recorded using an FCS set-up which reflected mitoFluo binding
to mitochondria. Subsequent addition of CCCP leading to complete
deenergization of mitochondria markedly (but not completely)
suppressed the high-amplitude fluorescence bursts, thereby
showing a partial release of mitoFluo from mitochondria. A similar
energy-dependent behavior of the fraction of high-amplitude peaks
was observed in our previous FCS study with tetramethylrhodamine
ethyl ester (TMRE),12 the widely used mitochondrial marker.13
Scheme 1 Schematic representation of mitoFluo accumulation and
uncoupling activity.
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Fig. 2 Responses of isolated rat liver mitochondria to mitoFluo. (A) Stimulation of RLM respiration by mitoFluo, as evidenced by the mitoFluo-induced
increase in the rate of succinate-driven mitochondrial oxygen consumption both in the absence (i, ii – black curves) and presence (ii – grey curve) of
20 mM atractyloside. (B) Dependence of the mitochondrial respiration rate on the concentration of mitoFluo and C8-FL. (C) Energy-dependent
accumulation of mitoFluo in RLM, as monitored by mitoFluo fluorescence intensity traces recorded using the FCS set-up in the presence of RLM (black
curve), successively supplemented with 5 mM succinate (red curve) and 1 mM FCCP (blue curve). Inset: corresponding dependencies of the number of
peaks with the fluorescence intensity F exceeding the threshold F0, n(F 4 F0), on the value of F0. (D) Confocal fluorescence microscopy of primary kidney
tubule cells loaded with mitoFluo (i, green), TMRE (ii, red) and overlay showing co-localization as integral yellow color (iii). Phase contrast microscopy of
the cells loaded with mitoFluo (iv).
Confocal fluorescence microscopy of primary kidney tubule
This work was financially supported by the Russian
cell culture revealed significant colocalization of mitoFluo and Science Foundation (No. 14-24-00107). We are very grateful to
TMRE fluorescence, thus demonstrating the accumulation of Professor Vladimir Skulachev for stimulating discussion and
mitoFluo in mitochondria (Fig. 2D). By contrast, according to Drs Ljudmila Khailova and Tatyana Rokitskaya for their assistance
ref. 14 the conjugate of TPP with 6-carboxyfluorescein, being with experiments.
predominantly anionic at physiological pH, failed to accumu-
late in mitochondria. Measurements of mitoFluo cytotoxicity
on L929 fibrosarcoma cells showed a decrease in cell viability
starting from 2 mM mitoFluo (Fig. S6, ESI†).
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fluorescent probe that proved to be an effective mitochondria-
targeted protonophore, promising for use both in uncoupling and
fluorescence imaging16 of mitochondria in cells.
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