Shedding light on the mitochondrial matrix through a functional membrane transporter

Article abstract of DOI:10.1039/c9sc04852a

The first fluorescent probes that are actively channeled into the mitochondrial matrix by a specific mitochondrial membrane transporter in living cells have been developed. The new functional probes (BCT) have a minimalist structural design based on the highly efficient and photostable BODIPY chromophore and carnitine as a biotargeting element. Both units are orthogonally bonded through the common boron atom, thus avoiding the use of complex polyatomic connectors. In contrast to known mitochondria-specific dyes, BCTs selectively label these organelles regardless of their transmembrane potential and in an enantioselective way. The obtained experimental evidence supports carnitine-acylcarnitine translocase (CACT) as the key transporter protein for BCTs, which behave therefore as acylcarnitine biomimetics. This simple structural design can be readily extended to other structurally diverse starting F-BODIPYs to obtain BCTs with varied emission wavelengths along the visible and NIR spectral regions and with multifunctional capabilities. BCTs are the first fluorescent derivatives of carnitine to be used in cell microscopy and stand as promising research tools to explore the role of the carnitine shuttle system in cancer and metabolic diseases. Extension of this approach to other small-molecule mitochondrial transporters is envisaged.

Full text of DOI:10.1039/c9sc04852a

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Shedding light on the mitochondrial matrix
through a functional membrane transporter†
Cite this: DOI: 10.1039/c9sc04852a
a
b
d
*
All publication charges for this article
have been paid for by the Royal Society
of Chemistry
Alberto Blazquez-Moraleja,
Ines Saenz-de-Santa Marıa, b Marıa D. Chiara,
´
´ ´ ´
Delia A´lvarez-Fernandez,a Inmaculada Garcıa-Moreno, c Ruth Prieto-Montero,
´
´
d
d
a
*
Virginia Martınez-Martınez,
Inigo Lopez Arbeloa
and Jose Luis Chiara
´
´
˜
´
The first fluorescent probes that are actively channeled into the mitochondrial matrix by a specific
mitochondrial membrane transporter in living cells have been developed. The new functional probes
(BCT) have a minimalist structural design based on the highly efficient and photostable BODIPY
chromophore and carnitine as a biotargeting element. Both units are orthogonally bonded through the
common boron atom, thus avoiding the use of complex polyatomic connectors. In contrast to known
mitochondria-specific dyes, BCTs selectively label these organelles regardless of their transmembrane
potential and in an enantioselective way. The obtained experimental evidence supports carnitine–
acylcarnitine translocase (CACT) as the key transporter protein for BCTs, which behave therefore as
acylcarnitine biomimetics. This simple structural design can be readily extended to other structurally
diverse starting F-BODIPYs to obtain BCTs with varied emission wavelengths along the visible and NIR
spectral regions and with multifunctional capabilities. BCTs are the first fluorescent derivatives of
carnitine to be used in cell microscopy and stand as promising research tools to explore the role of the
carnitine shuttle system in cancer and metabolic diseases. Extension of this approach to other small-
molecule mitochondrial transporters is envisaged.
Received 25th September 2019
Accepted 8th December 2019
DOI: 10.1039/c9sc04852a
rsc.li/chemical-science
mitochondria have an extraordinary capacity to precisely regu-
late cell chemistry. In addition to cellular respiration, they are
Introduction
involved in many other processes, including calcium homeo-
stasis, production of reactive oxygen species (ROS), the metab-
olism of heme groups, nucleotides, amino acids and
phospholipids, inammation, cell proliferation, and apoptosis,
thus fullling an essential role in cell survival.1 The damage or
deregulation of these mechanisms is implicated in numerous
pathological processes, either directly, as in mitochondrial
genetic diseases, or secondarily in neurodegenerative, inam-
matory, and cardiovascular diseases, as well as in some meta-
bolic syndromes.2
Mitochondria are responsible for the production of energy in
the form of ATP in eukaryotic cells through aerobic respiration.
These highly dynamic organelles present two unique features:
they have their own DNA (mtDNA) and a characteristic double
membrane system separating the mitochondrial matrix from
the cytosol, with an outer and an inner membrane that differ in
shape, composition and physicochemical properties. The inner
membrane is highly impermeable to most molecules and ions
and contains, among others, the proteins involved in oxidative
phosphorylation and proteins of the mitochondrial carrier
system (MCS),1 which regulates the transfer of small molecules
between the matrix and the cytosol. Consequently,
Although great efforts have been devoted to the study of the
biology of mitochondria, we are still far from knowing in detail
many of their functions and basic activities. Molecular imaging3
is currently the most efficient and versatile tool for the study of
subcellular dynamics thanks to its high sensitivity, selectivity
and ease of use, providing very varied information in real time
and in a non-destructive way. A common characteristic of
uorescent probes targeting mitochondria is the presence of
a
´
´
Instituto de Quımica Organica General (IQOG-CSIC), Juan de la Cierva 3, 28006
Madrid, Spain. E-mail: jl.chiara@csic.es
b
´
´
Instituto de Investigacion Sanitaria del Principado de Asturias, Instituto de Oncologıa
del Principado de Asturias (IUOPA), CIBERONC, Universidad de Oviedo, Hospital
Central de Asturias, 33011 Oviedo, Spain. E-mail: mdchiara.uo@uniovi.es
a
lipophilic cation (ammonium or phosphonium)4 that
c
´
´
Instituto Quımica-Fısica “Rocasolano” (IQFR-CSIC), Serrano 119, 28006 Madrid,
promotes the uptake and accumulation of the probe driven by
the high negative potential gradient (DJm) characteristic of
these organelles as a consequence of the oxidative phosphory-
lation process. Impairment of DJm is a hallmark of mito-
chondrial dysfunction5 and, under these circumstances, the
Spain
d
´
´
´
Departamento de Quımica Fısica, Universidad del Paıs Vasco UPV-EHU, Facultad de
´
Ciencia y Tecnologıa, Apartado 644, 48080 Bilbao, Spain
† Electronic supplementary information (ESI) available: Supplementary gures
and tables and copies of the 1H and 13C NMR (1D and 2D) spectra of the new
compounds. See DOI: 10.1039/c9sc04852a
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uorescent labeling with dyes that are exclusively based on this
“passive” electrophoretic diffusion mechanism will dim or even
completely disappear. While the development of mitochondria-
targeted uorescent probes has become one of the most active
areas in chemistry and biology,6 to the best of our knowledge
the design of a functional probe that uses an “active” uptake
mechanism regardless of the transmembrane potential is yet to
be explored. Herein, we address the challenge of developing the
rst uorescent probes that are able to enter mitochondria in
living cells by an active and stereoselective protein-mediated
transport mechanism in preference to the usual passive and
non-specic electrophoretic diffusion process.
Results and discussion
Design and synthesis of the new probes
The specic active transport of small molecules by the
membrane proteins of the MCS offers a reasonable, but still
untapped design strategy for new functional probes. To this
end, we chose to address the carnitine system (CS), which
facilitates the transport of long-chain fatty acids through the
mitochondrial membranes into the matrix for b-oxidation.7 A
key protein of the CS is the carnitine/acylcarnitine translocase
(CACT), which is an antiporter carrier that transfers the fatty
acids in the form of acylcarnitines across the inner mitochon-
drial membrane into the matrix, in antiport with carnitine.8
Inherited defects in the CACT gene cause a rare, life-threatening
metabolic disorder in newborns (CACT deciency, also known
as secondary carnitine deciency).9 The non-functioning form
of the transporter prevents the body to use fats to produce
energy, which causes cardiomyopathy, arrhythmias, skeletal
muscle damage, liver problems, hypothermia, neurological
problems, and developmental delay in affected patients, oen
leading to sudden infant death. In addition, recent studies have
established that the CS enzymes are overexpressed in cancer
and promote tumor progression, being thus considered as
attractive anti-tumor therapeutic targets.10
Our functional probes have a modular design (Scheme 1)
based on a BODIPY chromophore and a carnitine molecule as
the biofunctional subunit for mitochondria-specic targeting.
The two subunits are orthogonally linked to the single boron
atom of the BODIPY component through the b-hydroxy acid
grouping of carnitine to render a B-spiro system. In this mini-
malist structural design, the use of complex and long connec-
tors is avoided, thereby simplifying not only the synthetic
protocol but also the size and complexity of the nal probe. The
BODIPY uorophore was selected for the outstanding photo-
physical properties (chemical robustness, strong UV-visible
absorption and high uorescence emission) and well-known
chemical versatility of this group of dyes.11 The presence of
a bulky mesityl substituent at its meso-position signicantly
enhances its photostability and the efficiency of its uorescence
emission when compared to both, related unsubstituted dyes
and free-motion meso-aryl derivatives.12 In addition, its
increased lipophilicity could enhance the specicity of the
probe for lipid-mitochondrial transporters.
Scheme 1 General molecular design of the BODIPY–carnitine BCT
probes and structures of the starting components and final probes
synthetized.
Despite the essential role that carnitine plays in the biology
of eukaryotic cells,13 only a few uorescent derivatives have been
described,14 but, surprisingly, none of them have been previ-
ously used in cell microscopy. Interestingly, since carnitine is
a chiral molecule that is commercially available in both enan-
tiomeric forms as well as the racemate, the corresponding
probes will allow us to study the inuence of stereochemistry on
the specicity of mitochondrial uorescent-labeling. Only the
natural (R)-enantiomer (L-carnitine) is biologically relevant
(Scheme 1).
Scheme 2 Hydrolysis reaction of the carnitine–BODIPY probes (R)-
BCT-1 and (R)-BCT-2.
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Fig. 1 Absorption (height-normalized to molar absorptivity) (A) and fluorescence (height-normalized to quantum yield) (B) spectra of the (R)-
BCT-2 probe in different solvents; chloroform (green), acetone (black), acetonitrile (red), ethanol (blue), and water (grey).
The new BODIPY–carnitine probes included in this work
were readily prepared following our recently described protocol
for the one-step synthesis of O-BODIPYs with a rigid B-spiranic
4,4-diacyloxyl or 4-acyloxyl-4-alkoxyl substitution pattern start-
ing from the corresponding F-BODIPYs (Scheme 1).12 Thus,
a stoichiometric mixture of F-BODIPY 1 or 2 and the corre-
sponding carnitine hydrochloride stereoisomer in anhydrous
MeCN was heated under microwave irradiation (25 ꢀC to 120 ꢀC
gradient over 3.2–3.4 h), in the presence of an excess of TMSCl
(40–50 equiv.) to afford the targeted O-BODIPYs in an almost
quantitative crude yield aer solvent removal (Scheme 1). The
crude samples were readily puried by recrystallization from
CH2Cl2/t-BuOMe to afford compounds (R)-BCT-1, (R)-BCT-2,
and (S)-BCT-2 in 63%, 88%, and 63% yield, respectively. The
structures of BCT-1 and BCT-2 were fully conrmed by multi-
dimensional NMR and HRMS (see Experimental part and ESI†).
Compounds BCT-1 and BCT-2 were insoluble in ether and ester
solvents (Et2O, THF, t-BuOMe, and EtOAc), but readily soluble
in halogenated solvents (CH2Cl2, CHCl3, ClCH2CH2Cl), alcohols
(MeOH, EtOH), and water.
Aqueous stability
To dene the viability and traceability of the new functional
dyes as mitochondria labels, it is important to analyze their
long-term water stability since the new probes contain two
potentially labile B–O bonds connecting the uorophore to the
carnitine moiety. We measured the time evolution of 15 mM
solutions of (R)-BCT-1 and (R)-BCT-2 in D2O at 25 ꢀC using 1H
NMR to establish not only their chemical stability but also the
degradation products (see Fig. S1 in the ESI†). Both
compounds hydrolyzed to free carnitine and the correspond-
ing 4,4-dihydroxy-BODIPY15 derivatives 3 and 4 (Scheme 2).
Interestingly, while (R)-BCT-1 was quickly hydrolyzed (t1/2 ¼ 10
min), its more hindered tetramethylated analog (R)-BCT-2
showed a superior aqueous stability (t1/2 ¼ 6 h), which enables
its use as a uorescent probe in live cell imaging. Formation of
free carnitine and the boron acid BODIPY derivatives 3 and 4
at a physiological pH range may, in fact, be advantageous for
cell staining purposes. Thus, the liberated (R)-carnitine will be
directly incorporated into the mitochondrial metabolism,
Fig. 2 Dose-dependent cellular distribution of (R)-BCT-1 and (R)-BCT-2 compounds. Representative fluorescence images of live SCC38 cells
incubated with the indicated concentrations of (R)-BCT-1 and (R)-BCT-2 compounds for 30 min before washing and microscopic analysis under
the same acquisition conditions. Scale bars, 10 mm.
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while the boron acid group of the hydrolyzed BODIPY will Reversible formation of these boron ester derivatives will
likely form covalent bonds with polyhydroxylated biomole- immobilize the probe within the mitochondrion, impeding its
cules, such as mitochondrial glycolipids and glycoproteins, diffusion out of the organelle even in instances when the DJm
generating cyclic boron esters as already described for some transmembrane potential is eliminated, as experimentally
BODIPYs16 and boronic acid-based saccharide sensors.17 observed (see below).
Fig. 3 Colocalization of (R)-BCT-2 and MitoTracker Red CMXRos fluorescence signals in SCC38 cells. (A–C) Representative fluorescence
images of SCC38 cells co-labelled with 50 nM (R)-BCT-2 and 50 nM MitoTracker Red CMXRos for 30 minutes before microscopic analysis.
Magnified images of the areas indicated in panels A and B are shown to better visualize the staining of tubular (A) and spherical (B) mitochondria.
(D) Graphic shows the fluorescence intensity profiles of the region of interest indicated with a white line in panel C. Scale bars, 10 mm.
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information for cell imaging. No aggregation band corre-
sponding to either H- or J-aggregates could be detected in the
UV-visible spectra at a concentration of 10ꢁ4 M. Since the new
probes have been designed for live cell uorescent imaging,
Photophysical characterization
We studied the UV-vis absorption and uorescence emission
spectra of BCT-1 and BCT-2 to provide fundamental
Fig. 4 Mitochondria staining by (R)-BCT-2 is time-dependent. HeLa or SCC38 cells were incubated with either 50 nM (R)-BCT-2 or 50 nM
MitoTracker Red CMXRos for the indicated times before cell washing and analysis by flow cytometry (A) or microscopy (B). Scale bars, 10 mm.
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their photophysical properties were initially analyzed in water showed similar photophysical features even by signicantly
(see Fig. 1, and Table S1 in ESI†). Following the expected decreasing the solvent polarity from water to chloroform (Fig. 1
behavior, the photophysical signatures were signicantly and Table S1†).
dependent on the degree of alkylation of the BODIPY chromo-
Interestingly, probe (R)-BCT-2 exhibited also a high photo-
phore, which provides enough steric hindrance to prevent the stability, which is of outmost importance on building smarter
deleterious effect that free rotation of the 8-mesityl group could uorescent probes. The slow hydrolysis of (R)-BCT-2 in water
have on the uorescence response.12 In fact, probe (R)-BCT-2 solution, observed in the previous 1H NMR study, did not result
outstands by a high molar absorption coefficient (3.8 ꢂ in a signicant reduction of the overall uorescent emission
104 Mꢁ1 cmꢁ1 at 499 nm) in water and an efficient uorescent (see Fig. S2, and Table S2 in ESI†). In fact, its uorescence
emission peaked at 511 nm with a quantum yield higher than quantum yield remained unchanged aer 7 hours. However,
80%. This behavior is related to the low probability of non- due to the slow oligomerization of the hydrolysis product 4 and
radiative processes due to the rigid and constrained molec- the observed consequent precipitation of the resultant oligo-
ular structure resultant from the rings graed at the meso mers, the quantum yield gradually dropped aer 24 hours,
position and at the boron atom, together with the 1,3,5,7-tet- although still remained at a suitable 65%. Very similar results
rametylation of the chromophore scaffold. Supporting our were also obtained in PBS solution at pH 6.0, 7.4, and 8.0 and in
initial hypothesis, probe (R)-BCT-2 stands out as a highly culture medium (see Table S3 and Fig. S3 in ESI†). Compared to
effective uorescent dye regardless of solvent nature since it pure water, only a slight decrease of the uorescence quantum
Fig. 5 Mitochondrial labelling by (R)-BCT-2 does not depend on DJm. (A) Microscopic analysis of SCC38 cells pretreated or not with FCCP
before co-labelling with 20 nM (R)-BCT-2 and 0.5 nM MitoTracker. Scale bars, 10 mm. (B) Graphic shows the fluorescence intensity profiles of the
regions of interest indicated with white lines in right panels. (C) Data represent the mean fluorescence intensity (MFI) of labelled cells from flow
cytometry analysis of SCC38 cells incubated in the presence (+FCCP) or absence (ꢁFCCP) of 60 mM FCCP for 3 h, before labelling with either
20 nM (R)-BCT-2 or 0.5 nM Mitotracker.
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yield and lifetime was observed in PBS and in culture medium. studies were performed only with (R)-BCT-2, which produced
Based on these results, we conclude that hydrolysis, small pH a clear green uorescence signal in a dose-dependent manner
changes, or the presence of inorganic salts (CaCl2$2H2O, and had better stability in aqueous solution. The lowest
Fe(NO3)3$9H2O, MgSO4$7H2O, KCl, NaHCO3, NaCl, NaH2PO3- concentration (50 nM) was found optimal to obtain a bright
$2H2O) and other culture medium components (amino acids, staining of the cells without causing any observed cytotoxicity.
vitamins, D-glucose, fetal bovine serum) in millimolar concen- Localization of (R)-BCT-2 in live cells was studied by co-staining
trations do not signicantly affect the uorescence performance experiments with commercially available MitoTracker Red
of the probe. In addition, its ability to covalently attach to CMXRos (hereaer simply called MitoTracker Red). The cells
mitochondrial glycoproteins and glycolipids should avoid were coincubated with (R)-BCT-2 (50 nM) and MitoTracker Red
subsequent leakage and unwanted diffusion, excretion or (50 nM) at 37 ꢀC for 30 min. As shown in Fig. 3, the green
metabolism. Leakage of dyes non-covalently bonded to cellular uorescence from (R)-BCT-2 colocalized well with the red uo-
organelles is one of the major challenges in uorescence rescence from the MitoTracker dye, with a Pearson's correlation
labeling since it induces a signicant decrease in the uores- coefficient of 0.82 (95% CI ¼ 0.77–0.86; p < 0.0001). As observed
cent emission and, consequently, in the experimental accuracy. with the MitoTracker dye, (R)-BCT-2 stained spherical and
In this regard, the rational design of probe (R)-BCT-2 should tubular mitochondria (Fig. 3). These data revealed that (R)-BCT-
prevent signal lost and enhance measurement reliability 2 acts as an organelle-specic probe that targets mitochondria
impelling label traceability.
and is retained within the organelle like the commercial Mito-
tracker dye. Mitochondrial staining with (R)-BCT-2 was time-
dependent, as observed by ow cytometry (Fig. 4), in sharp
contrast with MitoTracker Red that gave an almost instant
uorescent labeling of these organelles. Although this time
lapse may be the result of a slower uptake of (R)-BCT-2 by the
cells as compared to MitoTracker, it can also be a rst indica-
tion of the possible involvement of an active transport system in
the mitochondrial internalization of the carnitine-based probe.
The staining was independent of the cell type, since similar data
Live cell imaging
To assess the mitochondrial staining ability of the new probes,
live SCC38 cells (derived from human larynx squamous cell
carcinoma) were incubated with different concentrations of
just-prepared aqueous solutions of (R)-BCT-1 and (R)-BCT-2 (50,
100 and 500 nM) (Fig. 2). As expected from the photophysical
studies, compound (R)-BCT-1 gave weaker cell staining than (R)-
BCT-2 at all concentrations assayed. Thus, all subsequent
Fig. 6 Mechanistic hypothesis for the transport (and fate) of BCT probes into the mitochondrial matrix via the carnitine shuttle system.
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were obtained in HeLa cells derived from human cervical cancer
Considered in concert, the above divergent observations for
(see Fig. S4 in ESI,† and Fig. 4 below). To conrm that the both types of dyes provide support for the notion that (R)-BCT-2
presence of the covalently attached carnitine was essential for is internalized into mitochondria via an active transport system
mitochondria localization, we performed control experiments irrespective of DJm, in contrast to MitoTracker and similar
with 24 h-old aqueous solutions of (R)-BCT-2. Only a very weak cationic dyes that are rapidly taken up into the negatively
and diffuse cell staining (see Fig. S5 in ESI†) was observed using charged organelle by passive electrophoretically-driven diffu-
these solutions, which mainly contained the hydrolyzed probe sion. A compelling candidate for the active transport of (R)-BCT-
(i.e., 4,4-dihydroxy-BODIPY derivative 4 and free carnitine; see 2 into mitochondria is the CS (Fig. 6), as initially designed. In
Scheme 2).
the CS, fatty acids are rst activated by the acyl-CoA synthetase
The B-chelated covalent linkage of carnitine to the BODIPY (ACSL) in the cytosol to acyl-CoA thioesters, which cross the
scaffold in our probes renders cationic dyes, which may also outer mitochondrial membrane through the voltage-dependent
simultaneously target mitochondria via their electrostatic anion channel (VDAC), but do not cross the inner mitochon-
interaction with the negative electric potential of healthy drial membrane due to the lack of a specic transporter. Fatty
mitochondria. To elucidate the impact of the mitochondrial acyl-CoAs are trans-esteried to acylcarnitines by the carnitine
transmembrane potential (DJm) on the subcellular distribution palmitoyltransferase-1a (CPT-1a), an integral outer mitochon-
of (R)-BCT-2, staining was analyzed by cell microscopy and ow drial membrane enzyme, and are then released into the inter-
cytometry in response to carbonyl cyanide-4-(triuoromethoxy) membrane space. Acylcarnitines are transported across the
phenylhydrazone (FCCP), a protonophore used to decrease inner mitochondrial membrane by the carnitine/acylcarnitine
DJm by depolarization of the mitochondrial membrane. translocase (CACT), which mediates the antiport reaction
Whereas FCCP-treated cells (60 mM FCCP for 3 h, at 37 ꢀC) allowing entry of acylcarnitines into the mitochondrial matrix
exhibited highly attenuated mitochondrial uorescence aer and exit of free carnitine.8,19 Finally, the enzyme carnitine
incubation with MitoTracker Red, a dye relying on DJm for palmitoyltransferase-2 (CPT-2) catalyzes the trans-esterication
mitochondrial internalization, compound (R)-BCT-2 showed in of the acyl group from carnitine to CoA in the mitochondrial
fact an increase in uorescence intensity upon loss of DJm, matrix affording the substrates for b-oxidation. Thus, the
while still remaining within the depolarized mitochondria transport function of CACT is crucial for the b-oxidation
(Fig. 5). As described for other dyes,18 this increase of the uo- pathway.
rescence signal could be ascribed to “unquenching” of the
Due to the lipophilic nature of the BODIPY subunit, (R)-BCT-
loaded probe due to its reduced concentration inside the 2 can be reasonably regarded as a biomimetic of acylcarnitines,
organelle caused by the suppression of the electrophoretic being thus able to travel through the CS in their place. To test if
passive pathway.
CACT was in fact involved in the channeling of (R)-BCT-2 into
Fig. 7 Inhibition of mitochondria staining with (R)-BCT-2 in the presence of increasing concentrations of (R)-palmitoylcarnitine. SCC38 cells
were co-incubated with the indicated concentrations of (R)-palmitoylcarnitine and 50 nM (R)-BCT-2 (A) or 50 nM MitoTracker (B) prior to
analysis by flow cytometry. Data represent the mean fluorescence intensity (MFI) of labelled cells (upper panels) and representative images of
flow cytometry profiles (lower panels).
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the mitochondrial matrix, we rst studied the effect of (R)-pal- unnatural (S)-enantiomer of carnitine (Fig. 8). For this, the cells
mitoylcarnitine, a natural CACT substrate,20 on the staining of were coincubated with (R)-BCT-2, (S)-BCT-2, or MitoTracker Red
the cells by this probe. Flow cytometry analysis of live SCC38 (all at 50 nM) and increasing concentrations of the inhibitor (50,
cells coincubated with (R)-BCT-2 (50 nM) and increasing 100, 200, 500 nM). In the case of (R)-BCT-2, which contains the
concentrations of (R)-palmitoylcarnitine showed
a dose- natural enantiomer of carnitine, ow cytometry analysis
dependent reduction of the uorescence intensity, as expected showed a strong reduction of the mean uorescence intensity
for a substrate inhibition process (Fig. 7). In contrast, a parallel even at the lowest inhibitor concentration used (Fig. 8A).
slight increase of uorescence was observed in the case of Interestingly, (S)-BCT-2, which contains the unnatural enan-
MitoTracker Red under the same experimental conditions. In tiomer of carnitine, exhibited only a marginal reduction of the
order to further examine the biomimetic hypothesis, we carried mean uorescence intensity with increasing inhibitor concen-
out a similar experiment using (S)-decanoylcarnitine, a known trations (Fig. 8B). In contrast, MitoTracker Red displayed an
CACT-specic inhibitor (IC50 ꢃ 5 mM)20 derived from the increase of uorescence at intermediate inhibitor
Fig. 8 Inhibition of mitochondria staining in the presence of increasing concentrations of (S)-decanoylcarnitine. SCC38 cells were co-incubated
with the indicated concentrations of (S)-decanoylcarnitine and 50 nM solutions of (R)-BCT-2 (A), (S)-BCT-2 (B), or MitoTracker (C), prior to
analysis by flow cytometry. Data represent the mean fluorescence intensity (MFI) of labelled cells (left panels) and representative images of flow
cytometry profiles (right panels).
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concentrations, as in the experiment with (R)-palmitoylcarni- example of enantioselective staining of cell organelles in live
tine, which slightly dimmed only at the highest inhibitor cells.21 To further conrm the stereoselectivity of our chiral
concentration used (Fig. 8C).
carnitine-based probes, we compared the cell staining of both
The inhibition experiments with (S)-decanoylcarnitine enantiomers at different concentrations under identical exper-
uncovered an additional important observation. In the absence imental conditions in the absence of inhibitors. Flow cytometry
of inhibitor, the uorescence staining with the “natural” evaluation of these staining experiments clearly conrmed
carnitine probe (R)-BCT-2 was much brighter than with its a considerably brighter mean uorescence intensity when the
enantiomer (S)-BCT-2. At increasing inhibitor concentrations, SCC38 cells were loaded with (R)-BCT-2 that when using the (S)-
when the activity of CACT was mostly suppressed and the BCT-2 probe, at all concentrations tested (Fig. 9). All the
residual uptake mechanism is basically the electrophoretic observed activities of our probes parallel the known stereo-
diffusion, the two intensities leveled. This is an exceptional specicities shown by CACT for the corresponding
Fig. 9 Flow cytometry analysis of SCC38 cells labelled with increasing concentrations of (R)-BCT-2 or (S)-BCT-2. The cells were incubated with
the indicated concentrations of the probes for 30 min before flow cytometry analysis. (A) Representative images of flow cytometry profiles. (B)
Data represent the mean fluorescence intensity of labelled cells.
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enantiomeric palmitoylcanitines.19a,20 Overall, these observa- stage for the development of novel functional probes and drugs
tions substantiate the view that (R)-BCT-2 behaves as a substrate targeted to CACT and other small-molecule mitochondrial
of CACT, which actively channels the probe through the mito- transporters for the study of the biology and pathology of this
chondrial inner membrane into the matrix, where it suffers fascinating organelle.‡
hydrolysis to carnitine and boron acid derivative 4. Carnitine is
then back-transported out of the matrix into the cytosol by
CACT, while 4 is likely retained by covalent conjugation to
Experimental section
General methods for synthesis and chemical characterization
glycolipids and glycoproteins in the matrix, as already
mentioned. It is also possible that, once in the matrix, the
hydrolyzed probe is not able to cross the impermeable inner
membrane back to the cytosol. In any case, our probe does not
need to have a reactive functional group that covalently attach it
to proteins in mitochondria to prevent its diffusion out of the
organelle, in contrast to Mitotracker and related mitochondria-
specic dyes. As already commented above, the cationic char-
acter of the BCT probes allows the coexistence of the active and
stereoselective protein-mediated transport and the passive and
non-specic electrophoretic uptake of the probes, which may
explain the residual staining observed in the presence of high
concentrations of the acylcarnitine substrate or inhibitor.
Proton and carbon-13 nuclear magnetic resonance (1H NMR,
13C NMR) spectra were recorded on a Bruker Avance III-400 (400
and 100 MHz, respectively) or a Varian System 500 (500 and 125
MHz, respectively) spectrometers. Chemical shis are expressed
in parts per million (d scale) downeld from tetramethylsilane
and are referenced to residual peaks of the deuterated NMR
solvent used. Data are presented as follows: chemical shi,
multiplicity (s ¼ singlet, d ¼ doublet, t ¼ triplet, m ¼ multiplet
and/or multiple resonances, b ¼ broad), integration, coupling
constants in Hertz (Hz), and assignment. Proton and carbon-13
assignments are based on gCOSY, gNOESY, gHSQC, and
gHMBC correlation experiments. FT-IR spectra were recorded in
a PerkinElmer Spectrum One instrument. Thin layer chroma-
tography (TLC) was performed with Merck Silica Gel 60 F254
plates. Chromatograms were visualized using UV light (254 nm
or 365 nm). Column chromatography was performed on a 971-
FP Flash Purication System from Agilent Technologies using
SF Si35 silica cartridges. High-resolution mass spectra (HRMS)
were recorded on an Agilent 6520 Q-TOF instrument with an ESI
source. Anhydrous solvents were prepared according to stan-
dard methods by distillation over drying agents or via elution
through a PureSolv™ column drying system from Innovative
Technology, Inc. All other solvents were of HPLC grade and were
used as provided. Microwave irradiation experiments were
performed under magnetic stirring with a single-mode Anton
Parr Monowave 300 reactor, using standard Pyrex tubes (10 mL
capacity) sealed with a PTFE-lined rubber septum. (R)-Palmi-
toylcarnitine was obtained from commercial sources (Merck),
and (S)-decanoylcarnitine was synthetized from (S)-carnitine
(Carbosynth) following a described procedure.22
Conclusions
We have described the rst uorescent probes that are actively
channeled into the mitochondrial matrix by a specic mito-
chondrial membrane transporter in living cells. Our BCT
functional probes have a minimalist structural design based on
the highly efficient and photostable BODIPY chromophore and
carnitine as a sacricial biotargeting unit. Both components are
orthogonally bonded through the common boron atom forming
a B-spiro system, thus avoiding the use of complex polyatomic
connectors. In contrast to most commonly used mitochondria-
specic dyes, BCT probes enter mitochondria in a time-
dependent way, regardless of the mitochondrial trans-
membrane potential. We have obtained convincing experi-
mental evidence supporting the carnitine–acylcarnitine
translocase (CACT) as the key transporter protein for BCTs.
Thus, (1) mitochondrial staining is competitively inhibited in
the presence of the CACT natural substrate (R)-palmitoylcarni-
tine or the CACT specic inhibitor (S)-decanoylcarnitine in
a dose-dependent manner; and (2) staining is enantioselective,
increasing in the order (S)-BCT-2 < (R)-BCT-2, which parallels
the known stereochemical preference of CACT for acylcarnitine
derivatives. (R)-BCT-2 behaves therefore as an acylcarnitine
biomimetic in which the lipophilic BODIPY system plays the
role of the fatty acid part of the natural substrate. Our simple
structural design of a functional carnitine-based mitochondrial
probe could be readily extended to other structurally diverse
starting F-BODIPYs to obtain BCTs with varied wavelength
emission along the visible and NIR spectrum and with multi-
functional capabilities. BCTs are the rst uorescent derivatives
of carnitine to be used in cell microscopy and stand as prom-
ising research tools to investigate the role of the CS in cancer
metabolic rewiring and related alterations in metabolic
diseases for the development of new therapies. Although no
Synthesis of (R)-BCT-1
To a suspension of (R)-carnitine (20 mg, 0.100 mmol) in
practical applications of the new dyes have been explored in this anhydrous MeCN (5 mL), was added F-BODIPY 1 (30 mg, 0.097
preliminary research, we believe that our approach sets the mmol) and trimethylsilyl chloride (491 mL, 3.87 mmol). The
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stirred reaction mixture was heated under microwave irradia- CH3600 and CH3700), 3.26 (1H, s, H400), 2.87 (1H, br d, J ¼ 14.4 Hz,
tion using the following step-wise gradient: 30 min at 80 ꢀC, H200), 2.54 (3H, s, CH35), 2.47 (1H, br d, J ¼ 14.4 Hz, H200), 2.39
35 min at 100 ꢀC, 40 min at 120 ꢀC, 1.5 h at 150 ꢀC. The solvent (3H, s, CH33), 2.34 (3H, s, CH340), 2.08 (3H, s, CH320/CH360), 1.98
was removed at reduced pressure, the crude was dissolved in (3H, s, CH360/CH320), 1.39 (3H, s, CH37), 1.38 (3H, s, CH31). 13C
a minimum amount of CH2Cl2 and tert-butyl methyl ether was NMR (100 MHz, CDCl3): d ¼ 168.8 (C100), 155.7 (C3), 154.1 (C5),
slowly added until precipitation of the product started. It was 143.8 (C8a), 143.5 (C7a), 142.5 (C8), 139.2 (C40), 135.0 (C20/C60),
then kept at 4 ꢀC overnight and the resulting crystalline product 133.9 (C60/C20), 131.5 (C1), 131.4 (C7), 130.8 (C10), 129.6 (C30/
was isolated by ltration and washing with additional tert-butyl C50), 129.2 (C50/C30), 122.5 (C6), 122.5 (C2), 69.5 (C400), 62.3
methyl ether to afford pure (R)-BCT-1 as dark red crystals (C300), 55.1 (C500, C600 and C700), 37.1 (C200), 21.4 (CH340), 19.8
(26 mg, 63% yield). FT-IR (KBr): n ¼ 1720 (s), 1560 (vs), 1411 (s), (CH320/CH360), 19.4 (CH360/CH320), 17.4 (CH35), 15.7 (CH33),
1384 (vs), 1258 (vs), 1109 (vs), 1071 (vs) cmꢁ1. 1H NMR (CDCl3, 13.9 (CH37), 13.7 (CH31). HRMS (ESI+) m/z: calcd. for
+
400 MHz): d ¼ 8.14 (1H, s, H5), 7.83 (1H, s, H3), 6.95 (2H, s, H30
and H50), 6.70 (1H, br d, J ¼ 4.8 Hz H1/H7), 6.69 (1H, br d, J ¼
4.8 Hz H7/H1) 6.50 (1H, m, H2/H6), 6.47 (1H, m, H6/H2), 4.99
(1H, br m, H300), 4.33 (1H, br d, J ¼ 13.5 Hz, H400), 3.56 (1H, br m,
H400), 3.41 (9H, s, CH3500, CH3600 and CH3700), 2.98 (1H, br d, J ¼
16.3 Hz, H200), 2.67 (1H, br m, H200), 2.36 (3H, s, CH340), 2.05
(6H, s, CH320 and CH360). 13C NMR (100 MHz, CDCl3): d ¼ 168.8
(C100), 148.0 (C8), 145.3 (C3), 144.4 (C5), 139.2 (C40), 136.4 (C20/
C60), 135.9 (C60/C20), 135.5 (C7a/C8a), 135.5 (C8a/C7a), 131.1
(C7), 130.8 (C1), 129.5 (C10), 128.5 (C30/C50), 128.3 (C50/C30),
119.2 (C6), 119.2 (C2), 69.5 (C400), 63.1 (C300), 55.0 (C500, C600 and
C700), 37.0 (C200), 21.3 (CH340), 20.1 (CH320/CH360), 20.0 (CH360/
CH320). HRMS (ESI+) m/z: calcd. for C25H31BN3O3+: 432.2458
[M]+; found: 432.2453.
C
29H39BN3O3 : 488.3084 [M]+; found: 488.3092.
Synthesis of (S)-BCT-2
Synthesis of (R)-BCT-2
To a suspension of (S)-carnitine (14.5 mg, 0.072 mmol) in
anhydrous MeCN (4 mL) was added F-BODIPY 2 (32.3 mg, 0.089
mmol) and trimethylsilyl chloride (466 mL, 3.67 mmol). The stirred
reaction mixture was heated under microwave irradiation using
the following step-wise gradient: 35 min at 80 ꢀC, 50 min at 100 ꢀC,
2 h at 120 ꢀC. The solvent was removed at reduced pressure, the
crude was dissolved in a minimum amount of CH2Cl2 and tert-
butyl methyl ether was slowly added until precipitation of the
product started. It was then kept at 4 ꢀC overnight and the
resulting crystalline product was isolated by ltration and
washing with additional tert-butyl methyl ether to afford pure (S)-
BCT-2 as dark red crystals (23.5 mg, 63%). As expected, the spec-
tral data are identical to those of (R)-BCT-2. HRMS (ESI+) m/z:
+
calcd. for C29H39BN3O3 : 488.3084 [M]+; found: 488.3089.
To a suspension of (R)-carnitine (14.5 mg, 0.072 mmol) in
anhydrous MeCN (5 mL) was added F-BODIPY 2 (32 mg, 0.088
mmol) and trimethylsilyl chloride (466 mL, 3.67 mmol). The
stirred reaction mixture was heated under microwave irradia-
tion using the following step-wise gradient: 35 min at 80 ꢀC,
50 min at 100 ꢀC, 2 h at 120 ꢀC. The solvent was removed at
reduced pressure, the crude was dissolved in a minimum
amount of CH2Cl2 and tert-butyl methyl ether was slowly added
until precipitation of the product started. It was then kept at
4 ꢀC overnight and the resulting crystalline product was isolated
by ltration and washing with additional tert-butyl methyl ether
to afford pure (R)-BCT-2 as dark red crystals (32 mg, 88%). FT-IR
(KBr): n ¼ 1721 (s), 1546 (vs), 1505 (vs), 1470 (s), 1295 (vs), 1187
(s), 1156 (vs) cmꢁ1. 1H NMR (CDCl3, 400 MHz): d ¼ 6.97 (1H, s,
1H NMR study of the aqueous stability of (R)-BCT-1 and (R)-
BCT-2
A just-prepared solution of each probe in D2O (15 mm) at 25 ꢀC
was immediately placed in the NMR instrument probe set at
25 C and H NMR spectra were automatically acquired at the
different time intervals shown in Fig. S1 (ESI†). The spectra in
each stack were automatically processed and normalized to the
residual proton signal of the solvent using the MestReNova
soware.
1
ꢀ
Photophysical characterization
H30/H50), 6.95 (1H, s, H50/H30), 5.99 (1H, s, H6), 5.97 (1H, s, H2), The photophysical properties for all the compounds were
4.55 (1H, br s, H300), 4.17 (1H, br s, H400), 3.32 (9H, br s, CH3500, measured in different solvents at low concentrations (approx. 5
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a CO2 incubator, and a ZEISS ApoTome.2 structured illumina-
tion system. ApoTome.2 allows acquisition of optical sections of
the uorescent sample by moving the appropriate grid into the
beam path and calculating the optical section from three
images with different grid positions without time lag. Acquisi-
tion and processing of images were conducted using the Zen
(Carl Zeiss) soware. For direct comparison of uorescence
intensities between the two BCT probes (Fig. 2), microscope
settings were the followings: 420 ms exposure time with a 0.79
mm depth in focus, 1.17 mm section thickness and maximum
intensity of the HXP 120 C lighting unit. Images in Fig. 3–5 were
acquired with the maximum intensity of the HXP 120 C lighting
unit and exposure times of 420 ms for (R)-BCT-2 and 20 ms for
MitoTracker.
ꢂ 10ꢁ6 M) in 1 cm optical path cuvettes. The UV/vis absorption
spectra were recorded on a Varian dual beam spectrometer
(CARY 7000) in transmittance mode. Emission spectra were
recorded in right-angle conguration on a spectrouorimeter
Edinburgh Instruments (FLSP920 model) with a xenon ash
lamp 450 W as the excitation source. The uorescence spectra
were corrected from the wavelength dependence of the detector
sensibility. Fluorescence quantum yield (F) was obtained
using as reference PM546 (F ¼ 0.87) in ethanol. The uores-
cence lifetime decay curves were measured with the time
correlated single-photon counting technique in the same
spectrouorimeter using
a multichannel plate detector
(Hamamatsu R38094-50) with picosecond time-resolution.
Fluorescence decay curves were monitored at the maximum
emission wavelength aer excitation by means of a Fianium
Supercontinuum laser with 150 ps full width at half maximum
(FWHM) pulses. The uorescence lifetime (s) was obtained aer
deconvolution of the instrumental response signal from the
recorded decay curves by means of an iterative method. The
goodness of the exponential t was controlled by statistical
parameters (chi square, c2, and analysis of the residuals).
Flow cytometry
To evaluate the cellular labeling efficiency of BCT functional
probes, SCC38 cells (1 ꢂ 106 cells suspended in PBS) were
incubated for 5, 15 or 30 min at 37 ꢀC with 50 nM BCT probes or
50 nM MitoTracker in the presence or absence of 100, 250, or
500 nM (R)-palmitoylcarnitine or 50, 100, 200, 500 nM (S)-dec-
anoylcarnitine as indicated in gure legends. Cells were
subsequently washed three times with PBS to remove uores-
cent probes, resuspended in PBS, and ltered through a 40 mm
cell strainer. Unlabeled cells and cells exposed to culture
medium containing 0.002% DMSO for 30 min (the same solu-
tion composition and time used in the treatment with the
different uorescent probes) served as negative controls.
Labelled cells were then counted by using appropriate gates and
controls by employing a BD FACSAria™ IIu (BD Biosciences,
San Jose, CA) ow cytometer and the FlowJo v.10.6 soware.
Cell culture
The established human squamous cell carcinoma (SCC38)-
derived cell line was kindly provided by Dr R Grenman
(University Central Hospital, Turku, Finland). SCC38 and HeLa
cells were grown in DMEM supplemented with 10% fetal bovine
serum, 100 units per mL penicillin, 200 mg mLꢁ1 streptomycin,
2 mmol Lꢁ1 L-glutamine and 100 mmol Lꢁ1 nonessential amino
acids. Cell lines were periodically tested for human pathogens
and mycoplasma infection. All methods were carried out in
accordance with the approved guidelines of our institution.
Conflicts of interest
Fluorescent cell labeling
There are no conicts to declare.
Cells (50 ꢂ 103) were plated on black 24 well plates with at and
clear bottom suitable for uorescence-based imaging of living
cells (Ibidi GmbH) 24 hours before labeling. (R)-BCT-1 and (R)-
BCT-2 probes were diluted in H2O or DMSO, respectively, to
a stock concentration of 500 mM, 50 mM or 20 mM. Adhered
living cells were incubated with 500 nM, 50 nM, or 20 nM of
probes, as indicated in gure legends, in DMEM without
supplements for 30 minutes at 37 ꢀC. Subsequently, the probes
were removed and the cells were washed with PBS (3 ꢂ 5 min).
Supplemented DMEM was then added to cells prior to micros-
copy analysis. Where indicated, mitochondria were labeled by
co-incubation of cells with the probes and 50 nM MitoTracker®
Red CMXRos (Thermo Fisher).
Acknowledgements
We gratefully acknowledge the Spanish, Agencia Estatal de
´
Investigacion (AEI), and European Regional Development Fund
(ERDF) (projects MAT2017-83856-C3-1-P, MAT2017-83856-C3-3-
P, and MAT2015-68837-REDT), Gobierno Vasco (IT912-16), and
the Instituto de Salud Carlos III-Fondo de Investigacion Sani-
taria (CIBERONC and PI17/01901) for nancial support. A. B.-M.
and R. P. M. thank, AEI and ERDF, and UPV-EHU for a FPI
predoctoral contract and predoctoral fellowship, respectively.
´
Notes and references
‡ The new BCT probes were patented in Spain in 2017 by authors of this paper
Live-cell microscopy
(patent application PCT/ES2018/070477).
Microscopy imaging was performed on a Cell Observer equip-
ment composed of a Zeiss AxioObserver Z1 wide eld inverted
uorescence microscope (Carl Zeiss, Germany) with a Plan-
Apochromat 40X/1.3 (NA ¼ 1.3, working distance ¼ 0.21 mm)
or Plan-Apochromat 63X/1.4 (NA ¼ 1.4, working distance ¼ 0.19
mm) oil lens objective, a camera (AxioCam MRm; Carl Zeiss),
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