Development of a Pre-assembled Through-Bond Energy Transfer (TBET) Fluorescent Probe for Ratiometric Sensing of Anticancer Platinum(ll) Complexes

Article abstract of DOI:10.1002/asia.202000157

Fluorescence microscopy has emerged as an attractive technique to probe the intracellular processing of Pt-based anticancer compounds. Herein, we reported the first through-bond energy transfer (TBET) fluorescent probe NPR1 designed for sensitive detectio

Full text of DOI:10.1002/asia.202000157

CHEMISTRY
AN ASIAN JOURNAL
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Accepted Article
Title: Development of a Pre-assembled TBET Fluorescent Probe for
Ratiometric Sensing of Anticancer Platinum(ll) Complexes
Authors: Jun Xiang Ong and Wee Han Ang
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Development of a Pre-assembled TBET Fluorescent Probe for
Ratiometric Sensing of Anticancer Platinum(ll) Complexes
Jun Xiang Ong,[a] and Wee Han Ang*[a, b]
[a]
Dr. J. X. Ong, Prof. W. H. Ang
Department of Chemistry
National University of Singapore
3 Science Drive 3, Singapore 117543 (Singapore)
E-mail: ang.weehan@nus.edu.sg
[b]
Prof. W. H. Ang
NUS Graduate School for Integrative Sciences and Engineering
National University of Singapore
28 Medical Drive, Singapore 117456 (Singapore)
Supporting information for this article is given via a link at the end of the document.((Please delete this text if not appropriate))
There is currently a lack of tools capable of visualizing the
uptake and accumulation of Pt-based anticancer compounds
directly in cancer cells that would allow researchers to
investigate how these compounds are processed at the cellular
level. Elemental techniques such as graphite furnace atomic
absorption spectrometry (GF-AAS) and inductively-coupled
plasma mass spectrometry (ICP-MS) have been applied to
quantify Pt content and map cellular localization, but they cannot
be applied to live-cell imaging owning to their destructive
nature.[7] Fluorescence microscopy has emerged as the most
effective and employable tool for probing Pt species at the
cellular level. The general approach has been to conjugate
fluorophores to Pt complexes to track their movement using
fluorescence imaging. Using this strategy, PtII drugs such as
cisplatin and carboplatin have been conjugated with various
fluorophores to probe their intracellular processing in vitro.[8]
Fluorescent ligands have been also attached to axial positions of
PtIV prodrug complexes to monitor the reduction process.[9]
However, modification of PtII/PtIV complexes with such bulky
organic groups can significantly alter their drug uptake
characteristics and pharmacokinetics, rendering them different
from the parent drugs.[10]
Abstract: Fluorescence microscopy has emerged as an attractive
technique to probe the intracellular processing of Pt-based
anticancer compounds. Herein, we reported the first Through-Bond
Energy Transfer (TBET) fluorescent probe NPR1 designed for
sensitive detection and quantitation of PtII complexes. The novel
TBET probe was successfully applied for ratiometric fluorescence
imaging of anticancer PtII complexes such as cisplatin and JM118 in
cells. Capitalizing on the ability of the probe to discriminate between
PtII complexes and their PtIV derivatives, the probe was further
applied to study the activation of PtIV prodrug complexes that are
known to release active PtII species after intracellular reduction.
Introduction
Cisplatin, carboplatin and oxaliplatin constitute an important
class of PtII drugs that are amongst the most widely used and
effective cytotoxic agents for cancer therapy (Figure 1).[1]
However, their efficacies are limited by their high intrinsic
toxicities, debilitating side-effects and occurrences of drug
resistance.[2] To circumvent these limitations, stable octahedral
PtIV prodrug scaffolds, derived from therapeutic square-planar
PtII complexes but with additional axial ligands, are being
developed. These PtIV prodrug complexes are activated via
intracellular reduction to release labile PtII species capable of
exerting their cytotoxic effects. PtIV prodrug complexes have
been proven to be more efficacious than their PtII progenies
while causing less toxicities by virture of their increased aqueous
stabilities.[3] As a result, a significant body of work has been
carried out to create PtIV complexes with highly tuned
properties,[4] capable of targeting specific cancer cells[5] as well
as possessing novel modes of actions.[6] Several notable PtIV
prodrugs that have undergone clinical trials include ormaplatin,
iproplatin and satraplatin (Figure 1).
We and others have been developing exogenous Pt-selective
fluorescent probes and applying them intracellularly to study the
cellular processing of Pt anticancer compounds. Unlike
conjugated fluorophores, these probes are delinked from their
intended Pt analyte but selectively bind Pt to trigger
fluorogenic response. New and co-workers reported
a
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.[11] Our
group developed a rhodamine-based fluorescent turn-on probe
as well as
a FRET-based ratiometric derivative utilising
dithiocarbamates as recognition motifs to study the intracellular
activation of PtIV carboxylate prodrug complexes in cells.[12] To
date, these aforementioned small-molecule probes are the only
reported fluorescent probes applied intracellularly to study Pt
anticancer compounds. This research space is still largely
unexplored and there is a need to expand the library of these
probes to improve their scope and detection efficiencies.
Recently, there has been a growing interest in ratiometric
fluorescent probes based on Through-Bond Energy Transfer
(TBET). In a TBET system, donor and acceptor fluorophores are
linked via electronically conjugated spacers and energy transfer
occurs through the conjugated bond without the need for
spectral overlap.[13] This unique feature endows TBET
fluorescent probes with high energy transfer efficiencies and
large pseudo-Stokes shifts. The TBET mechanism has been
utilized in the design of rhodamine-based fluorescent probes for
ratiometric sensing of various analytes. In particular, such
chemosensors can be derived from pre-assembled ratiometric
Figure 1. FDA-approved PtII drugs and PtIV prodrugs which was clinically
evaluated.
1
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TBET scaffolds and subsequently derivatized to detect specific
analytes.[14] These TBET precursors serve as versatile platforms
whereby the 2’-carboxy position could readily condense with
amino-containing receptors to generate ratiometric sensors of
various analytes based on the TBET mechanism. Inspired by
this strategy, we fabricated a novel TBET ratiometric dyad from
a naphthalimide-rhodamine platform for selective sensing of PtII.
The TBET dyad was subsequently applied for the ratiometric
fluorescence imaging of PtII drugs as well as to study the
activation of PtIV prodrug complexes in cells. To the best of our
knowledge, this is the first report of a TBET-based ratiometric
sensor for Pt-based anticancer complexes.
4-ethynylaniline which was subsequently reacted with 4-
piperidinylnaphthalic anhydride in an amide condensation
reaction
to
afford
N-(phenylacetylene)-4-
piperidinylnaphthalimide in good yield. N-(phenylacetylene)-4-
piperidinylnaphthalimide was then subjected to Sonogashira
coupling with 5’-Br-rhodamine B to give the naphthalimide-
rhodamine TBET scaffold 1. Treatment of 1 sequentially with
POCl3 and NH2OH yielded the hydroxamate derivative, 2.
Reaction of 2 with excess 1,2-dibromoethane in the presence of
Et3N as the base yielded 3, which was converted to 4 when
treated with NaDDTC. Lastly, 4 was treated with Lawesson’s
reagent in benzene to furnish the target compound NPR1 which
was fully characterized by 1H NMR, 13C{1H} NMR and ESI-MS.
Results and Discussion
Probe Design
The ratiometric TBET dyad comprised a naphthalimide donor
fluorophore tethered to a rhodamine acceptor fluorophore via a
rigid phenylacetylene linker as shown in Figure 2.
Diethyldithiocarbamate (DDTC) was chosen as the recognition
motif which would be attached at the 2’-position of the TBET
platform for the detection of PtII complexes such as cisplatin.
This design was based on the reported Rho-DDTC scaffold
which elicited
a
fluorescence turn-on response upon PtII
In order to engineer a ratiometric response, a
12b]
binding.[12a,
naphthalimide donor moiety was installed on Rho-DDTC to
serve as the donor fluorophore for excitation. The rigid
phenylacetylene linker was essential to allow electronic coupling
between the naphthalimide donor and rhodamine acceptor
fluorophores but at the same time, prevented them from
adopting coplanarity and behaving as an extended conjugated
system. Therefore upon excitation of the naphthalimide donor,
energy would be transferred “through-bond” via the
phenylacetylene linker to the rhodamine B acceptor fluorophore.
Scheme 1. Synthetic route to prepare ratiometric TBET dyad NPR1.
Photophysical properties of NPR1
The photophysical properties of NPR1 in CH3CN/HEPES buffer
(v/v=7:3, 5 mM, pH 7.4) were investigated. In the absence of
Pt2+, NPR1 displayed a broad absorption band centered at 418
nm attributed to the naphthalimide moiety. With the addition of
K2PtCl4 (0–8 eq.), a new absorption band centered at 565 nm
appeared gradually, accompanied by a colour change from
yellow to pink, corresponding to the rhodamine acceptor (Figure
3a and Figure S1). Upon excitation of the free NPR1 at 420 nm,
an emission band centered at 545 nm was observed. There was
no energy transfer in the free probe as the rhodamine acceptor
was in the closed-ring form and therefore only emission from the
naphthalimide donor was observed.
Figure 2. Proposed TBET probe for ratiometric sensing of cisplatin.
In keeping with our previous findings, we postulated that the
activation of NPR1 would take place through a two-step
mechanism culminating in spiro-ring opening.[12] The initial
binding of DDTC to cisplatin and its structural analogues would
result in the displacement of one of the ammine or chloride
ligands. The thiocarbonyl group on the DDTC recognition motif
would labilise the trans-ligand on Pt via trans-effect, promoting
reaction with the spirothiophene motif and triggering spiro-ring
opening. This would allow energy to be transferred from the
donor to the rhodamine B acceptor when NPR1 is excited,
Upon addition of K2PtCl4 (0–8 eq.), the intensity of the
emission band at 545 nm decreased in intensity and
simultaneously a new emission band at 595 nm increased in
intensity (Figure 3b). This observation was ascribed to increased
TBET from naphthalimide donor to rhodamine acceptor following
spiro-ring opening of the spirothiophene induced by Pt2+. At the
same time, the emission colour of the solution turned from
yellowish-green to orange-red (Figure 3a). The ratio of emission
intensities of rhodamine to naphthalimide (I595/I545) varied from
0.7 in the absence of Pt2+ to 20.9 in the presence of 8
equivalents of Pt2+, corresponding to 30-fold enhancement in the
ratios. NPR1 also showed a linear relationship between the
emission ratios with increasing concentrations of Pt2+ suggesting
that it was potentially useful for quantitative determination of Pt2+.
The energy transfer efficiency of NPR1 was calculated to be as
resulting in emission from rhodamine
B fluorophore (red
emission). In the absence of spiro-ring opening, however, TBET
would not occur and only emission from the donor naphthalimide
fluorophore (green emission) would be observed upon NPR1
excitation. By measuring in parallel these emission intensities
from the donor and acceptor fluorophores, PtII species could be
ratiometrically quantitated.
Synthesis and Characterization
The naphthalimide-rhodamine ratiometric TBET probe was
synthesized as shown in Scheme 1. The naphthalimide donor
was synthesized from 4-iodoaniline (Scheme S1). 4-iodoaniline
was first treated with trimethylsilylacetylene under Sonogashira
coupling conditions to afford 4-[(trimethylsilyl)ethynyl]aniline.
Deprotection of 4-[(trimethylsilyl)ethynyl]aniline with K2CO3 gave
2
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Figure 3. (a) Colour change (top) and fluorescence change (bottom) before and after addition of K2PtCl4 to NPR1. (b) Fluorescence spectra of NRP1 (20 μM) in
response to the presence of Pt2+ (0–8 eq.) in CH3CN/HEPES buffer (v/v=7:3, 5 mM, pH 7.4) (λex= 420 nm). Inset: Linear plot of emission ratios (I595/I545) against
Pt2+ concentrations (0–160 μM). (c) Fluorescence spectra of NPR1 (20 μM) in response to cisplatin (0–14 eq.) in CH3CN/HEPES buffer (v/v=7:3, 5 mM, pH 7.4).
(λex= 420 nm). Inset: Calibration curve showing range (0–160 μM) of linear dependence. The slop was used for the calculation of the detection limit of cisplatin.
high as 91 % which is expected of a TBET dyad (Figure S2).[14b]
Job’s plot analysis also revealed that NPR1 binds to Pt2+ in a 1:1
ratio (Figure S3). Time-dependent fluorescence response
studies revealed that the fluorescence intensity ratios increased
linearly over time in the presence of Pt2+ and remained constant
after 150 min (Figure S4).
pyriplatin and carboplatin (Figure 4 and Figure S10). The binding
of these PtII complexes to the probe induced spiro-ring opening
of the spirothiophene leading to TBET turn-on, which allowed for
ratiometric sensing of these PtII drugs. On the other hand, PtII
complexes containing chelating ammine ligands such as
oxaliplatin and Pt(en)Cl2 resulted in no activation of the probe
due to the inability of the dithiocarbamate recognition motif to
displace the stable ammine chelate.[12] As expected, NPR1 was
able to differentiate between PtII and PtIV metal centers by virtue
of their chemical reactivity. PtIV complexes such as cP-(OH)2,
cP-(OBz)2, cP-(OAc)2 and satraplatin, derived from cisplatin and
JM118 pharmacophores, did not result in activation of the probe.
Since PtIV prodrug complexes undergo intracellular reduction to
release active PtII species, NPR1 would be an ideal tool to study
Selectivity and pH response
Fluorescence measurements of the TBET probe with various
metal ions (3 eq.) revealed excellent selectivity for Pt2+. NPR1
did not give any observable response in the presence of
biologically-relevant metal ions such as Na+, K+, Mg2+, Cu2+, Ni2+,
Zn2+, Fe2+ and Fe3+ (Figure S5 and Figure S6). In addition, the
colour of the solutions remained yellow indicating that the probe
was not activated in the presence of other metal ions (Figure S7). the uptake and activation of PtIV prodrugs.
NPR1 was stable over a wide pH range 3–11 as variations in pH
did not alter the fluorescence ratio (I595/I545) of the free probe,
indicating the closed-ring form of the spirothiophene was
retained (Figure S8). The activation of the probe in the presence
of Pt2+ was pH-dependent. Fluorescence turn-on responses
(I595/I545) increased as the pH of the solutions decreased, with
significant activation of the probe observed in the pH range 3–10.
Since intracellular pH typically ranged from 5–8, NPR1 was
expected to function adequately in a cellular environment with
negligible background fluorescence.
Ratiometric detection of cisplatin
Having ascertained that ratiometric detection of Pt2+ metal ion
was feasible, the ability of NPR1 to perform ratiometric detection
of anticancer PtII complexes such as cisplatin was investigated.
NPR1 was titrated with cisplatin (0–14 eq.) in CH3CN/HEPES
buffer (v/v=7:3, 5 mM, pH 7.4) and fluorescence measurements
were recorded. As shown in Figure 3c, titration of NPR1 with
cisplatin resulted in good ratiometric response, in keeping with
Pt2+. A good linear working range was observed from the
addition of 0–160 μM of cisplatin in the titration experiment
signifying its potential for quantitative determination of cisplatin.
The detection limit (3σ/slope)[15] for cisplatin was determined to
be 202 nM which was sufficiently low enough to detect
micromolar doses of cisplatin typically administered. The
selectivity of the probe towards cisplatin was high and
competitive experiments showed that the presence of other
metal ions did not interfere with detection of cisplatin (Figure S9).
The high sensitivity and selectivity makes NPR1 a good
candidate for the ratiometric sensing of PtII anticancer
compounds in biological systems.
Figure 4. (Top) Chemical structures of various PtII and PtIV complexes.
(Bottom) Fluorescence spectra of NPR1 (20 μM) after treatment with various
PtII and PtIV complexes (5 eq.) in CH3CN/HEPES buffer (v/v=7:3, 5 mM, pH
7.4) (λex= 420 nm).
Differentiation of PtII from PtIV complexes
Apart from cisplatin, activation of NPR1 was notable in several
other PtII anticancer compounds such as JM118, picoplatin,
3
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Fluorescence imaging of NPR1 in vitro
that NPR1 was cell-permeable and could be applied for
ratiometric fluorescence imaging of anticancer PtII complexes
such as cisplatin. Capitalizing on the ability of NPR1 to
differentiate between PtIV prodrug complexes and their reduced
PtII products, we successfully applied the probe to monitor the
intracellular activation of several PtIV complexes in vitro. We
believed that this novel TBET probe will serve as an excellent
tool for researchers to acquire a better understanding of how
PtII/PtIV complexes are processed at the cellular level.
NPR1 was applied for ratiometric fluorescence imaging of Pt-
based anticancer complexes in biological systems. HeLa cells
were treated with cisplatin (30 μM) for 3 h, fixed with 4 %
paraformaldehyde, and further incubated with NPR1 (20 μM) for
4 h at 37 °C. The cells stained with probe only displayed strong
green intracellular fluorescence of naphthalimide and negligible
red fluorescence of rhodamine B indicating the cell membrane
was permeable to NPR1. The incubation of cisplatin-treated
cells with the probe exhibited significant increase in red
fluorescence accompanied by partial decrease in green
fluorescence (Figure 5). These results were consistent with the
emission profile of NPR1 in the presence of cisplatin in aqueous
solutions. The emission profile indicated that cisplatin was
distributed within the cytoplasm. Based on time-course
experiments, maximum emission intensities occurred within 3-4
h treatment after which free cisplatin taken up was bound to
other biomolecules. Similar trend was observed when the cells
were treated with JM118 (30 μM) (Figure S11).
Experimental Section
Materials and instrumentation
All reagents were purchased from commercial vendors and used
without further purification. 5’-Br-Rhodamine B was synthesized
according to literature methods.[16] Cisplatin,[17] Pt(en)Cl2,[18]
[4a]
picoplatin,[19] pyriplatin,[20] cP-(OH)2,[4a] cP-(OBz)2
(OAc)2
and cP-
[4a]
were prepared in accordance with literature
procedures. Satraplatin, JM118, oxaliplatin and carboplatin were
1
purchased from commercial vendors. H NMR and 13C{1H} NMR
spectra were recorded on a Bruker Advance 300 MHz and 500
MHz model. Chemical shifts are reported in parts per million
relative to residual solvent peaks. Electro-spray ionization mass
spectrometry spectra were obtained on a Thermo Finnigan MAT
ESI-MS system. UV–vis spectra were recorded on a Shimadzu
UV-1800 UV–vis spectrophotometer. Emission fluorescence
spectra were recorded on a JOBIN YVON Fluorolog by HORIBA
with iHR320 detector. Fluorescence images were captured with
Olympus FluoView FV1000 (Olympus, Japan) laser scanning
confocal microscope, with 488 nm laser as the excitation source.
Synthesis of 4-piperidinylnaphthalic anhydride
4-bromonaphthalic anhydride (300 mg, 1.08 mmol) and
piperidine (0.197 mL,
2
mmol) were refluxed in 2-
methoxyethanol for 6 h. After the reaction, solvent was removed
and the crude residue purified by column chromatography
(100 % CH2Cl2) to give the product as orange solid. Yield: 260
mg (85.8%, Rf = 0.5). 1H NMR (300 MHz, CDCl3): δ 8.57 (1H, d),
8.49 (1H, d), 8.44 (1H, d), 7.71 (1H, t), 7.20 (1H, d), 3.30 (4H, t),
1.90 (4H, m), 1.76 (2H, q).
Figure 5. Fluorescence images of HeLa cells treated with cisplatin and PtIV
complex (30 μM). a) Untreated (probe only), and exposed to b) cisplatin and c)
cP-(OBz)2 for 3 h, fixed with 4 % paraformaldehyde and further incubation with
NPR1 (20 μM) for 4 h at 37 °C.
The excellent ratiometric response of NPR1 in the presence of
cisplatin and JM118 in cells prompted us to investigate the
ability of NPR1 to monitor the intracellular activation of PtIV
prodrug complexes in biological systems, specifically those that
are based on the cisplatin and JM118 templates. HeLa cells
were treated with cP-(OBz)2 (30 μM), which contained the
cisplatin drug motif, for 3 h and further incubated with NPR1 (20
μM) for 4 h at 37 °C. As shown in Figure 5, cP-(OBz)2-treated
cells displayed fluorescence turn-on in the red channel indicating
the fast conversion of cP-(OBz)2 to the active PtII species. A
similar trend was observed for satraplatin (30 μM), which
underwent intracellular reduction to release JM118 (Figure S11).
Taken together, the results showed that NPR1 can be applied to
monitor the intracellular reduction of PtIV prodrug complexes to
further our understanding on how this new class of PtIV prodrugs
were being processed intracellularly.
Synthesis of 4-[(trimethylsilyl)ethynyl]aniline
4-iodoaniline (1 g, 4.56 mmol), PdCl2(PPh3)2 (88 mg, 0.13 mmol)
and CuI (22 mg, 0.12 mmol) were added in a round-bottom flask
followed by evacuation/flushing with N2 for three times. Et3N (20
mL) was added and stirred for 30 min. Trimethylsilylacetylene
(0.78 mL, 5.5 mmol) was subsequently added and the reaction
mixture was heated at 70 °C for 12 h. Water (20 mL) was added
to the reaction mixture and extracted with EtOAc (3x 25 mL).
The organic layers were combined, washed with brine (50 mL)
and dried over Na2SO4. The solvent was removed and the crude
residue purified by column chromatography (1:3 v/v
EtOAc:hexane) to give the product as yellow solid. Yield: 750
1
mg (87.7%, Rf = 0.43). H NMR (300 MHz, CDCl3): δ 7.27 (2H,
d), 6.58 (2H, d), 3.71 (2H, br), 0.23 (9H, s).
Synthesis of 4-ethynylaniline
4-[(trimethylsilyl)ethynyl]aniline (750 mg, 4 mmol) was dissolved
in CH2Cl2/MeOH (1:1) (30 mL), followed by the addition of K2CO3
(2.82 g, 20.4 mmol). The reaction mixture was stirred at r.t. for
12 h. Water (30 mL) was added to quench the reaction and
extracted with CH2Cl2 (3x 20 mL). The organic layers were
combined, washed with brine (50 mL) and dried over Na2SO4.
The solvent was removed and the crude residue purified by
column chromatography (1:3 v/v EtOAc:hexane) to give the
product as yellow solid. Yield: 330 mg (70.5%, Rf = 0.33). 1H
NMR (300 MHz, CDCl3): δ 7.30 (2H, d), 6.59 (2H, d), 3.82 (2H,
br), 2.96 (1H, s).
Conclusion
In summary, a novel TBET fluorescent probe NPR1 based on
the
naphthalimide-rhodamine
dyad
conjugated to
a
dithiocarbamate recognition motif was successfully synthesized
for sensing of Pt-based anticancer compounds. Excellent
ratiometric fluorescence turn-on responses were exhibited for a
panel of PtII anticancer drugs. The probe also possessed high
selectivity and was stable over a wide pH range making it
compatible with physiological conditions. In vitro studies showed
4
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Synthesis of
N-(phenylacetylene)-4-piperidinylnaphtha-
Synthesis of NPR1
limide
4 (30 mg, 0.030 mmol) and Lawesson's reagent (12 mg, 0.030
mmol) were suspended in benzene (5 mL) and refluxed for 3 h.
CH2Cl2 (10 mL) was added and the reaction mixture was
washed with water (2x 10 mL). The organic portion was then
dried over Na2SO4. The solvent was removed and the crude
residue was purified by column chromatography (1:2 v/v
EtOAc:hexane) to give the product as yellow solid. Yield: 20 mg
4-ethynylaniline (224.2 mg, 1.91 mmol), 4-piperidinylnaphthalic
anhydride (490 mg, 1.74 mmol) and Zn(OAc)2 (319.4 mg, 1.74
mmol) were refluxed in pyridine (10 mL) for 48 h. After the
reaction, the solvent was removed and the crude residue
purified by column chromatography (3:1 v/v CH2Cl2:hexane) to
give the product as yellow solid. Yield: 512 mg (77.6%, Rf =
1
1
0.41). H NMR (300 MHz, CDCl3): δ 8.61 (1H, d), 8.53 (1H, d),
(64.9%, Rf = 0.70). H NMR (500 MHz, CDCl3): δ 8.59 (1H, d),
8.48 (1H, d), 7.72 (1H, m), 7.65 (2H, d), 7.28 (2H, d), 7.23 (1H,
d), 3.28 (4H, t), 3.13 (1H, s), 1.93 (4H, m), 1.76 (2H, m).
8.51 (1H, d), 8.44 (1H, d), 7.90 (1H, d), 7.70 (1H, t), 7.58-7.60
(3H, m), 7.25-7.27 (2H, m), 7.20 (1H, d), 6.85 (2H, m), 6.35 (4H,
m), 4.48 (2H, t), 4.05 (2H, q), 3.74 (4H, m), 3.25-3.36 (12H, m),
1.90 (4H, m), 1.74 (2H, m), 1.20-1.29 (18H, m). 13C NMR (75
MHz, CDCl3): δ 12.27, 13.22, 25.01, 26.89, 30.37, 36.89, 45.21,
47.45, 50.29, 55.22, 73.67, 90.26, 115.47, 116.33, 121.96,
123.77, 126.11, 126.41, 127.05, 129.61, 130.85, 131.29, 131.83,
132.18, 133.10, 133.86, 134.35, 136.45, 152.29, 158.43, 164.75,
165.28, 196.03. ESI-MS: m/z 1027.2 [M+H]+. Anal. Calcd. for
C60H62N6O4S3: C 70.15, H 6.08, N 8.18. Found: C 70.31, H 6.42,
N 8.37.
Synthesis of 1
5’-Br-Rhodamine
B
(390.1
mg,
0.75
mmol),
N-
(phenylacetylene)-4-piperidinylnaphthalimide (285.3 mg, 0.75
mmol), PdCl2(PPh3)2 (53.9 mg, 0.077 mmol), PPh3 (40.0 mg,
0.154 mmol) and CuI (7.5 mg, 0.039 mmol) in THF/Et3N (4:1)
(40 mL) were refluxed for 18 h under N2. The solvent was
removed and the crude residue purified by column
chromatography (95:5 v/v CH2Cl2:MeOH) to give the product as
1
red solid. Yield: 280 mg (45.5% Rf = 0.32). H NMR (500 MHz,
CDCl3): δ 8.58 (1H, d), 8.51 (1H, d), 8.45 (1H, d), 8.05 (1H, d),
7.70-7.75 (2H, m), 7.61 (2H, d), 7.37 (1H, d), 7.28 (2H, d), 7.19
(1H, d), 6.79 (2H, d), 6.48-6.53 (4H, m), 3.43 (8H, q), 3.26 (4H,
t), 1.89 (4H, m), 1.74 (2H, m), 1.21 (12H, t). ESI-MS: m/z 821.5
[M+H]+.
Fluorescence studies
NPR1 was dissolved in DMSO to obtain 1 mM stock solution.
K2PtCl4 and Ptll complexes were prepared as 1 mM stock
solutions in H2O. PtlV complexes were prepared as 1 mM stock
solutions in DMSO. Fluorescence experiments were carried out
by incubating NPR1 dissolved in CH3CN/HEPES buffer (v/v=7:3,
5 mM, pH 7.4) with various amounts of Pt2+ or Pt complexes at
25 °C and 37 °C respectively for 4 h. The detection limit for
cisplatin was calculated based on fluorescence titration
experiment. Fluorescence spectrum of the dyad was measured
10 times to obtain standard deviations (σ) of the blank
measurements and the slope was obtained from the plot of
intensity ratio against the concentrations of cisplatin. Job’s plot
was carried out with a total [NPR1] and [Pt2+] concentration of 50
μM by varying the mole fraction of Pt2+ from 0 to 1. NaCl, KCl,
MgCl2, NiCl2, CuSO4, ZnCl2, FeSO4 and FeCl3 were used as the
source of metal ions. Selectivity of the dyad towards various
metal ions was carried out by incubating the dyad with metal
ions at 25 °C. Competitive assays were carried out by incubating
the dyad together with cisplatin and other metal ions at 37 °C.
pH response studies of the dyad was measured from pH 3–11.6
by substituting buffers of different pH while keeping the final
buffer concentrations constant. pH 3–6 were measured using
200 mM AcOH/AcO- buffer, pH 7.4 was measured using 50 mM
HEPES buffer and pH 8.4–11.6 were measured with 100 mM
Synthesis of 2
1 (280 mg, 0.34 mmol) in 1, 2-dichloroethane (5 mL) was added
POCl3 (0.4 mL) dropwise over 3 min. The reaction mixture was
refluxed for 5 h. The solvent was evaporated to give the crude
acid chloride as violet-red oil. The crude acid chloride was
dissolved in CH3CN (5 mL) and added drop-wise to a THF/H2O
(5:1) (6 mL) mixture containing hydroxylamine hydrochloride
(0.380 g, 5.5 mmol) and Et3N (1.2 mL, 8.7 mmol). The reaction
mixture was stirred at r.t. for 12 h. The reaction mixture was
extracted with CH2Cl2 (3x 10 mL). The organic layers were
combined and dried over Na2SO4. The solvent was removed and
the crude residue purified by column chromatography (3:1 v/v
EtOAc:hexane) to give the product as yellow solid. Yield: 60 mg
1
(21.1% Rf = 0.50). H NMR (500 MHz, CDCl3): δ 8.59 (1H, d),
8.52 (1H, d), 8.44 (1H, d), 7.85 (1H, d), 7.70 (1H, t), 7.59 (2H, d),
7.26-7.58 (3H, m), 7.20 (1H, d), 6.57 (2H, m), 6.45 (2H, s), 6.34
(2H, m), 3.36 (8H, q), 3.26 (4H, t), 1.90 (4H, m), 1.74 (2H, m),
1.19 (12H, t). ESI-MS: m/z 836.3 [M+H]+.
-
Synthesis of 4
H+/HPO4 buffer.
2 (60 mg, 0.072 mmol) was dissolved in DMF (2 mL) to which
Et3N (0.098 mL, 0.72 mmol) and 1, 2-dibromoethane (0.124 mL,
1.44 mmol) were added. The reaction mixture was then heated
at 60 °C for 4 h. The solvent was removed and CH2Cl2 (15 mL)
was added to the residue. The organic layer was washed with
water (10 mL) and NaHCO3 (10 mL), and dried over Na2SO4.
The solvent was removed and the crude residue purified by
column chromatography (1:2 v/v EtOAc:hexane) to give the
intermediate compound 3 as yellow solid. Yield: 32 mg (47.8%
Rf = 0.48). 3 (32 mg, 0.034 mmol) was dissolved in DMF (1 mL)
and NaDDTC (16.2 mg, 0.070 mmol) was added. The reaction
mixture was then heated at 60 °C for 12 h. The solvent was
removed and CH2Cl2 (15 mL) was added to the residue. The
organic layer was washed with water (10 mL) and brine (10 mL),
and dried over Na2SO4. The solvent was removed and the crude
residue purified by column chromatography (1:2 v/v
EtOAc:hexane) to give the product as yellow solid. Yield: 30 mg
Ratiometric fluorescence imaging of Pt-based drugs
HeLa cells were cultured in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 10 % FBS and 1 % antibiotics at
37 °C with 5 % CO2. Cells were seeded on cover slips in 6-well
plates at a density of 20 x 104 cells per mL and incubated
overnight. The cells were washed with PBS buffer twice and
incubated with PtII or PtIV complexes (30 μM) for 3 h at 37 °C.
Subsequently, the cells were washed with PBS buffer thrice,
fixed with 4% paraformaldehyde for 15 min and then washed
thrice with PBS buffer. The treated cells were then incubated
with NPR1 (20 μM) for 4 h at 37 °C and washed thrice with PBS
buffer. The control experiments were prepared by incubating the
fixed cells with probe only at 37 °C for 4 h and washed with PBS
buffer thrice. The cover slips were mounted onto glass slides in
a mounting medium and fluorescence images were acquired
with Olympus FluoView FV1000 (Olympus, Japan) laser
scanning confocal microscope, with 488 nm laser as the
1
(88.2%, Rf = 0.33). H NMR (500 MHz, CDCl3): δ 8.59 (1H, d),
8.52 (1H, d), 8.44 (1H, d), 7.92 (1H, d), 7.70 (1H, t), 7.65 (1H, m), excitation source.
7.59 (2H, d), 7.26-7.28 (3H, m), 7.20 (1H, d), 6.59 (2H, m), 6.35-
6.40 (4H, m), 3.98 (4H, m), 3.73 (2H, q), 3.35-3.42 (10H, m),
3.26 (4H, t), 1.90 (4H, m), 1.74 (2H, m), 1.19-1.29 (18H, m). ESI-
MS: m/z 1011.3 [M+H]+.
5
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10.1002/asia.202000157
Chemistry - An Asian Journal
FULL PAPER
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The authors gratefully acknowledge financial support from
Ministry of Education and the National University of Singapore
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Conflict of Interest
[14]
The authors declare no conflict of interest.
Keywords: Platinum Drugs • Cisplatin • Fluorescent Probes •
Ratiometric • Through Bond Energy Transfer
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Entry for the Table of Contents
Pt(II)-selective TBET probe: A novel TBET-based fluorescent probe was developed for ratiometric sensing of anticancer platinum(II)
drugs. By exploiting the difference in chemical reactivity of platinum(IV) and platinum(II) complexes, the probe was applied to monitor
the intracellular activation of platinum(IV) prodrug complexes in biological systems.
7
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