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10.1002/anie.202010951
Angewandte Chemie International Edition
COMMUNICATION
A Platinum-Selective Fluorescent Probe for Real-Time Monitoring
of Cisplatin Accumulation in Mitochondria in Living Cells
Jun Xiang Ong,[a]† Hai Van Le,[a]† Violet Eng Yee Lee,[a, b] and Wee Han Ang*[a, b]
developed several rhodamine-based fluorescent turn-on and
ratiometric probes equipped with dithiocarbamates as recognition
motifs to study the intracellular activation of PtIV carboxylate
prodrug complexes.[9] Leung and co-workers also developed a
BODIPY-based probe for fluorescence imaging of cDDP.[10]
However, these probes lack organelle-targeting abilities and
cannot be applied to study cDDP accumulation in mitochondria.
Herein, we developed the first mitochondria-targeting fluorescent
probe for cDDP to investigate the mitochondria as cellular targets
for cDDP and its PtIV prodrug derivatives, as well as to study
transport pathways for intracellular Pt delivery to mitochondria.
Abstract: Mitochondria have emerged as important target for cisplatin
in cancer therapy. Apart from cisplatin, many Pt complexes based on
various scaffolds have also been developed to target mitochondria.
Yet cellular processing of cisplatin or these mitochondria-targeting Pt
analogues remained unexplored, largely due to a lack of tools capable
of probing these Pt drugs within an intracellular environment. Herein,
we developed the first mitochondria-targeting fluorescent probe for
real-time monitoring of Pt accumulation in mitochondria and applied it
to investigate mitochondria as cellular targets for anticancer Pt drug
candidates in biological systems. We uncovered two distinct pathways
whereby they could be delivered to mitochondria after cell entry.
Mitochondria-
group
targeting
Br-
P+
H3N
S
Br-
S
N
H3N
H3N
Cl
Cl
Pt
Cisplatin (cDDP), together with second-generation platinum-
based drugs carboplatin and oxaliplatin, are amongst the most
effective chemotherapeutic agents for the treatment of testicular,
ovarian, lung and colorectal cancers.[1] Despite its potency and
widespread use in the clinic, there remains considerable gaps in
the understanding of its mechanism of action. It is generally
accepted that cDDP exerts its cytotoxic effect by forming
interstrand and intrastrand crosslinks with nuclear DNA which
inhibit replication and transcription events, triggering apoptosis.
However, the high cytotoxicity of cDDP in enucleated cells raises
the question of non-nuclear targets of Pt drugs.[2] Studies have
also showed that cDDP can exert its cytotoxicity by targeting other
subcellular organelles.[3] Indeed, apoptosis induced by direct
interaction with mitochondria may account for a significant portion
of its antiproliferative activity, with mitochondria DNA being the
likely target.[4] Other studies showed that PtlV prodrugs designed
to deliver cDDP directly to mitochondria promoted cancer cell
death.[5] This had led to a resurgence of interest in diamine Pt
complexes designed to target mitochondria.[6] Yet how cDDP or
these mitochondria-targeting Pt analogues are being processed
intracellularly remain unexplored. In order to determine the role of
mitochondria as biological target for cDDP, we developed a
strategy for monitoring of cDDP to mitochondria in living cells
using fluorescence microscopy.
Exogenous Pt-selective fluorescent probes have emerged as
effective tools for studying the cellular processing of Pt-based
anticancer compounds in biological systems. These exogenous
Pt-selective probes are advantangeous over the direct
conjugation of fluorophores to Pt drugs as modification of Pt
complexes with such bulky organic groups can significantly alter
their drug uptake characteristics and pharmacokinetics, rendering
them different from the parent drugs.[7] New and co-workers
reported a fluorescein-based probe linked to a dithiocarbamic
acid moiety and a rhodamine-based probe incorporating phenyl
isothiocyanate to study the metabolism of PtII drugs.[8] Our group
S
N
O
N
O
Pt
N
P+
S
S
N
N O
S
O
Pt-responsive
motif
EtHN
O
NHEt
EtHN
N+HEt
turn-on
O
non-fluorescent
fluorescence
Figure 1. Design of mitochondria-targeting probe Rho-Mito for organelle-
specific monitoring of cDDP accumulation.
In order to devise an efficient way of incorporating biological
targeting groups onto the rhodamine-based fluorogenic scaffold,
we developed rhodamine dithiocarbamate 2 (Scheme S1) as a
precursor. This scaffold possessed the ability to bind to PtII
species with its dithiocarbamate recognition motif while the free
amine functional group enabled further synthetic modications.
Therefore, amide conjugation with carboxylic acid derivatives of
targeting groups enabled an efficent method to attach these
targeting moieties onto the rhodamine fluorophore. The
triphenylphosphonium (TPP) moiety was selected as
a
mitochondria-targeting functional group due to its chemical
robustness and biocompatibility (Figure 1).[11] The resultant probe
Rho-Mito responded to PtII species via a two-step binding
mechanism culminating in spiro-ring opening.[9] The initial binding
of dithiocarbamate to PtII complexes, including cDDP, resulted in
the displacement of one of the chloride ligands. The thiocarbonyl
group on the dithiocarbamate recognition motif labilised the trans-
ligand via trans-effect, resulting in reaction with the
spirothiophene on rhodamine and triggering spiro-ring opening.
Rhodamine dithiocarbamate scaffold 2 was first prepared by
reacting 1 with mono-piperazinyl dithiocarbamate. The reaction of
2 with 3-(triphenylphosphonium-propionic acid)bromide via amide
coupling yielded Rho-Mito in moderate yields and fully
characterized by NMR and ESI-MS.
We first explored the photophysical properties of Rho-Mito in
the presence of Pt2+ metal ions. Titration experiment of Rho-Mito
with K2
R
R
PtCl4 was carried out in HEPES buffer (10 mM, pH 7.4,
R
R
30 % v/v EtOH). In the absence of Pt2+, free probe presented only
basal absorbance and fluorescence levels. When the probe was
[a]
[b]
[†]
Dr. J. X. Ong, H. V. Le, V. E. Y. Lee, Prof. Dr. W. H. Ang
Department of Chemistry, National University of Singapore, 3
Science Drive 3, Singapore 117543 (Singapore)
E-mail: ang.weehan@nus.edu.sg
incubated with K2
R
R
PtCl4 (0–15 equiv.), an absorption peak at 535
R
R
nm, typical of rhodamine 6G derivatives, was observed with
intensities proportional to added Pt2+ ions (Figure S1). Similarly,
stronger fluorescence emission bands at 565 nm were observed
V. E. Y. Lee, Prof. Dr. W. H. Ang
with gradual addition of K2
R
R
PtCl4 (Figure S2). These observations
R
R
NUS Graduate School of Integrative Sciences and Engineering
Institution, National University of Singapore, 28 Medical Drive,
Singapore 117456 (Singapore)
could be attributed to spiro-ring opening of the spirothiophene
after Pt2+-binding, leading to fluorescence turn-on. Analysis of
These authors contributed equally to this work.
Job’s
plot
indicated
maximum
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