Modulating Photostability and Mitochondria Selectivity in Far-Red/NIR Emitting Coumarin Fluorophores through Replacement of Pyridinium by Pyrimidinium

Article abstract of DOI:10.1021/acs.joc.0c00570

Mitochondrial dysfunction has been associated with several human pathological conditions, including cancer, aging, and neurodegenerative diseases. Thus, the availability of selective fluorescent probes for mitochondria could play an important role in the future for monitoring cellular functions and disease progression. In this work, we have studied how the photophysical properties and subcellular accumulation of nonconventional coumarin-based COUPY fluorophores can be fine-tuned through replacement of the para-pyridinium moiety with several heterocycles. Among them, ortho,para-pyrimidinium substitution provided novel fluorophores with suitable photophysical properties for bioimaging applications, including emission in the far-red to NIR region, large Stokes' shifts, and high photostability. Furthermore, the compounds exhibited excellent cell membrane permeability in living cells and a higher selectivity for mitochondria compared with the parent COUPY fluorophores. Overall, these results provided useful insights into the development of novel mitochondria-targeted fluorescent probes based on small organic molecules, since higher selectivity for this organelle can be achieved through the replacement of conventional N-alkylated pyridinium moieties by the corresponding N-alkylated-ortho,para-pyrimidinium counterparts.

Full text of DOI:10.1021/acs.joc.0c00570

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Article
Modulating Photostability and Mitochondria Selectivity in Far-Red/
NIR Emitting Coumarin Fluorophores through Replacement of
Pyridinium by Pyrimidinium
́
Anna Rovira, Miriam Pujals, Albert Gandioso, Marta Lopez-Corrales, Manel Bosch,
and Vicente Marchan*
́
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ABSTRACT: Mitochondrial dysfunction has been associated with
several human pathological conditions, including cancer, aging, and
neurodegenerative diseases. Thus, the availability of selective
fluorescent probes for mitochondria could play an important role
in the future for monitoring cellular functions and disease
progression. In this work, we have studied how the photophysical
properties and subcellular accumulation of nonconventional
coumarin-based COUPY fluorophores can be fine-tuned through replacement of the para-pyridinium moiety with several
heterocycles. Among them, ortho,para-pyrimidinium substitution provided novel fluorophores with suitable photophysical properties
for bioimaging applications, including emission in the far-red to NIR region, large Stokes’ shifts, and high photostability.
Furthermore, the compounds exhibited excellent cell membrane permeability in living cells and a higher selectivity for mitochondria
compared with the parent COUPY fluorophores. Overall, these results provided useful insights into the development of novel
mitochondria-targeted fluorescent probes based on small organic molecules, since higher selectivity for this organelle can be achieved
through the replacement of conventional N-alkylated pyridinium moieties by the corresponding N-alkylated-ortho,para-
pyrimidinium counterparts.
homeostasis.4 Mitochondrial dysfunction has been associated
with a wide range of human pathological conditions, such as
INTRODUCTION
■
Fluorescence imaging in combination with organic fluoro-
phores has become a powerful tool for understanding
biological events at a molecular level. In this context, the use
of fluorescent probes with operability in the less energetic far-
cancer disease, aging, and metabolic and neurodegenerative
diseases.5 Thus, the availability of fluorescent dyes that can
selectively stain mitochondria opens the door to monitoring
cellular functions and disease progression by studying
mitochondrial morphology and mitophagy.6 Among fluores-
cent mitochondrial probes described to date, some of them
intrinsically target this organelle such as some cyanine
derivatives (e.g., IR-780 and MHI-148) and some rhodamines
(Rhodamine 123).7 Another approach to achieve mitochondria
selectivity consists of incorporating lipophilic positively
charged moieties such as triphenylphosphonium or pyridinium
groups, which exploit the negative potential across the outer
and inner mitochondrial membrane.8 In this context,
anticancer mitochondria-targeted fluorescent molecules are
considered attractive theranostic agents.9 Similarly, mitochon-
dria-targeted photocages based on organic chromophores
red and near-infrared (NIR) region of the electromagnetic
spectrum offers several appealing features for in vivo imaging
applications, such as increased tissue penetration depth and
minimal autofluorescence interference from natively occurring
biomolecules.1 For this reason, recent efforts have been
devoted to the development of novel organic chromophores
operating in the phototherapeutic window with optimal
photophysical (e.g., long-wavelength absorption and emission,
brightness, large Stokes’ shifts, and photostability) and
physicochemical (e.g., aqueous solubility, good cell perme-
ability, and target specificity) properties. Ideally, such
fluorescent compounds should be based on chemically stable,
low molecular weight scaffolds amenable to smart and simple
structural modifications to fine-tune the abovementioned
properties and to facilitate conjugation to targeting ligands.2
Besides biocompatibility, an ideal fluorophore should permit
the interrogation of intracellular architectures and dynamics
without disturbing and compromising the integrity of the
cellular target.3
Received: March 3, 2020
Published: April 2, 2020
Mitochondria are involved in many key cellular processes,
including ATP synthesis, calcium signaling, and redox
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Scheme 1. Rational design of novel COUPY fluorophores
Scheme 2. Synthesis of COUPY scaffolds (6−9) and the corresponding N-methylated dyes (10−13)
provide a powerful method for releasing bioactive compounds
within this organelle.10
can be red-shifted through the incorporation of strong
electron-withdrawing groups like CF3 either at the 4-position
or via N-alkylation of the pyridine heterocycle,11 while
photostability can be increased through replacement of the
electron-donating N,N-dialkyl groups (e.g., NMe2 or NEt2) at
position 7 with azetidine.12 In addition, conjugatable versions
of COUPY dyes can be easily obtained by incorporation of
suitable functional groups (e.g., carboxylic acid, amino, azide,
or alkyne) via N-alkylation of the pyridine moiety. Such
COUPY derivatives allowed us to label receptor-binding
peptides on a solid phase by using efficient click chemistry
methodologies13 and to develop novel photosensitizers for
photodynamic anticancer therapy via conjugation to cyclo-
metalated Ir(III) complexes.14
On the basis of all these precedents, in this work we focused
on investigating how the modification of the pyridine moiety in
COUPY chromophores could influence the photophysical
properties and subcellular accumulation of the compounds.
With this idea in mind, herein we describe the synthesis and
characterization of four analogues (compounds 10−13,
Scheme 1) of our original COUPY dyes (4 and 5).
Surprisingly, the replacement of the para-pyridine heterocycle
by ortho-pyridine (10) or ortho,ortho-pyrimidine (11) had a
negative effect on the spectroscopic properties of the
compounds since both absorption and emission were blue-
In our laboratory, we have recently described a new class of
coumarin-based chromophores in which the carbonyl group of
the electron-withdrawing lactone in conventional coumarin 1
was replaced by the cyano(4-pyridine)methylene moiety (e.g.,
compounds 2 and 3; see Scheme 1) with the aim of increasing
the push-pull character of the π-delocalized system.11 N-
Alkylation of the pyridine heterocycle provides low molecular
weight fluorophores, nicknamed COUPYs, with several
attractive characteristics, including emission in the far-red/
NIR region, large Stokes’ shifts, and good brightness.11
Moreover, COUPY dyes (e.g., see compounds 4 and 5 in
Scheme 1 as representative examples) exhibited moderate to
good aqueous solubility and excellent cell membrane
permeability and accumulated preferentially in two specific
cellular compartments, the mitochondria and nucleoli. After
the discovery of this promising fluorescent platform, we have
initiated a systematic study to unveil the principles governing
the structure-photophysical property relationships (SPPR) of
COUPY dyes, which are necessary to design new fluorescent
probes as per need. Compared with conventional coumarins
(e.g., 1), the molecular framework of COUPY scaffolds offers
several advantages for carrying out a systematic SPPR study.
Indeed, we have found that absorption and emission maxima
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shifted. By contrast, the incorporation of ortho,para-pyrimidine
(12 and 13) led to highly photostable fluorophores with
improved photophysical properties compared with that of the
parent compounds, including emission in the far-red to NIR
region and large Stokes’s shifts. Furthermore, high cell
permeability was retained in both ortho,para-pyrimidine-
containing dyes and a higher selectivity for mitochondria of
HeLa cells was achieved. Overall, these results provided new
insights into the design and optimization of mitochondria-
targeted fluorescent probes.
exchange cross-peaks between the resonances of E and Z
rotamers were observed in the 2D NOESY spectrum of
coumarin 8. In the case of coumarins 6, 8, and 9, the two
rotamers were present in a ∼60:40 ratio according to the
integration of the 1H NMR spectrum, the E rotamer being the
major species. This is in contrast with the para-pyridine-
containing coumarins 2 and 3 in which the E/Z ratio was
∼90:10. Surprinsingly, the Z rotamer was the major species in
solution (95%) in the COUPY scaffold containing the
ortho,ortho-pyrimidine heterocycle (7), which was confirmed
by the existence of a NOE cross-peak between the proton at
position 3 of the coumarin moiety and the proton at the ortho
position of the pyrimidine ring (Figure S3). It is worth noting
that in all of the COUPY scaffolds the chemical shift of H3
appears at higher δ in the Z isomer (e.g., 8.18 ppm in 7 and
8.26 ppm in 8) than in the E isomer (e.g., 6.70 ppm in 7 and
6.75 ppm in 8).
Next, we synthesized the corresponding N-methylated
pyridinium (10) and pyrimidinium (11−13) dyes by reaction
with methyl trifluoromethanesulfonate in DCM at room
temperature.11 The four new coumarin derivatives were
isolated after silica column chromatography as reddish-orange
(10 and 11), purple (12), and dark blue (13) solids (yields
64−80%), and their purity was assessed by reversed-phase
HPLC (Figure S1). Characterization was carried out by HR
ESI-MS and 1D and 2D NMR spectroscopy. As expected,
methylation occurred at the less sterically hindered nitrogen in
the ortho,para-pyrimidine derivatives (12 and 13) according to
NOESY NMR characterization, and in all cases the E rotamer
was the major species in solution (Figures S6−S9).
Photophysical Characterization of COUPY Fluoro-
phores. Having at hand COUPY dyes 10−13, we investigated
the effect of replacing the para-pyridine moiety in the parent
fluorophores (4 and 5) by the ortho-pyridine or ortho,ortho- or
ortho,para-pyrimidine on the spectroscopic and photophysical
properties of the compounds with the aim of establishing new
SPPR. Although water or phosphate buffered saline (PBS) is
often used to evaluate the usefulness of a new fluorophore
within a spectroscopy cuvette, the heterogeneity and complex-
ity of the cellular environment cannot be accurately described
by using a simple aqueous buffer.16 For this reason, we decided
to register the UV−vis absorption and emission spectra in four
solvents of different polarities (PBS buffer pH 7.4, EtOH,
ACN, and DCM; Figure 2 and Figures S10−S17) to get some
RESULTS AND DISCUSSION
■
Design, Synthesis, and Characterization of COUPY
Dyes. COUPY dyes 10−13 were synthesized by following our
previously described methodology for the parent dyes (4 and
5), which is based on the reaction of a thiocoumarin precursor
with suitable heteroarylacetonitrile derivatives followed by N-
methylation of the pyridine or pyrimidine moieties (Scheme
2). First, thiocoumarin 1815 was reacted with 2-(pyridin-2-
yl)acetonitrile (14), 2-(pyrimidin-2-yl)acetonitrile (15), or 2-
(pyrimidin-4-yl)acetonitrile (16) in the presence of NaH
followed by AgNO3 treatment, which afforded COUPY
scaffolds 6, 7, and 8, respectively, after purification by silica
column chromatography with moderate to good yields (51−
82%). Similarly, coumarin 9 was obtained by condensation of
thiocoumarin 1911 with 16 (yield 75%). All the compounds
were fully characterized by reversed-phase high-performance
liquid chromatography (HPLC) (Figure S1), HR ESI-MS, and
1H, 19F (only in the case of 9), and 13C NMR.
It is worth noting that 1H-1H NOESY experiments (Figure 1
and Figures S2−S5) account for the existence of two species in
equilibrium in solution because of the rotation around the
exocyclic CC bond, which reproduces the behavior
previously found in the parent COUPY scaffolds (compounds
2 and 3, Scheme 1).11 As shown in Figure 1, chemical
Figure 1. (A) Structures of the E and Z rotamers of coumarin 8. (B)
Expansion of the 2D NOESY spectrum (tm = 500 ms, 25 °C) of 8 in
DMSO-d6 showing NOE cross-peaks and exchange cross-peaks
between rotamer resonances of the same sign as the diagonal.
Figure 2. Comparison of the normalized absorption (solid lines) and
fluorescence (dotted lines) spectra of COUPY dyes (compounds 4,5,
12, and 13) in EtOH.
C
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Table 1. Photophysical Data of the Coumarin Derivatives 4 and 5 and 10−13 in Different Solvents
a
b
c
d
e
compd.
X, Y, Z
solvent
λabs (nm)
ε (M−1 cm−1
)
λem (nm)
Stokes shift (nm)
ΦF
4
CH, CH, N
PBS
545
552
548
569
502
515
505
543
525
528
526
551
557
558
557
575
568
568
569
600
600
598
597
624
34,000
59,000
75,000
67,000
25,000
31,000
26,000
36,000
37,000
59,000
23,000
45,000
56,000
58,000
50,000
73,000
14,000
20,000
47,000
34,000
35,000
58,000
42,000
60,000
604
603
609
607
583
592
589
594
561
564
575
565
628
615
621
618
660
648
668
657
673
667
674
663
59
51
61
38
81
77
84
51
36
36
49
14
71
57
64
43
92
80
99
57
73
69
77
39
0.12
0.45
0.18
0.70
0.01
0.02
0.02
0.20
0.01
0.11
0.02
0.02
0.01
0.13
0.10
0.11
0.02
0.05
0.02
0.05
0.02
0.08
0.10
0.41
EtOH
ACN
DCM
PBS
EtOH
ACN
DCM
PBS
EtOH
ACN
DCM
PBS
EtOH
ACN
DCM
PBS
10
11
12
5
N, CH, CH
N, N, CH
N, CH, N
CH, CH, N
N, CH, N
EtOH
ACN
DCM
PBS
EtOH
ACN
13
DCM
a
b
c
Wavelength of the absorption maximum. Molar absorption coefficient at λmax. Wavelength of the emission maximum upon excitation at a
d
e
wavelength 20 nm below λmax. Stokes’ shift. Fluorescence quantum yields (ΦF) were measured by a comparative method using cresyl violet in
ethanol (ΦF;Ref = 0.54) as a reference for compounds 4, 5, 12, and 13. Fluorescein dissolved in aqueous sodium hydroxide (0.1 M; ΦF;Ref = 0.92)
was used as a reference in the case of compounds 10 and 11.17
insights into the photophysical behavior of the compounds in
polar and less-polar environments. As shown in Table 1, the
photophysical properties of coumarins 10−13 were compared
with those of the parent compounds 4 and 511 to facilitate the
establishment of SPPR.
absorption maxima was blue-shifted with increasing polarity of
the solvent (e.g., compare the λabs of 10−13 in DCM with the
corresponding values in aprotic (ACN) and protic (EtOH)
polar solvents). Moreover, the molar absorption coefficients of
the two derivatives containing the ortho,para-pyrimidine
moiety were similar to or even higher than those of their
respective parent dyes, especially in the 4-CF3 analogue (e.g.,
compare ε = 58 mM−1 cm−1 for 13 and ε = 20 mM−1 cm−1 for
5 in EtOH).
The emission maxima of coumarins 10 and 11 were also
blue-shifted both in polar and less-polar solvents with respect
coumarin 4 (e.g., λem = 564 nm for 11 and λem = 603 nm for 4
in EtOH), which reproduces the effect of replacing para-
pyridine by ortho-pyridine or ortho,ortho-pyrimidine moieties
on the compounds’ absorption maxima. By contrast, the
emission maxima of COUPY dyes containing the ortho,para-
pyrimidine moiety was red-shifted with respect to that of their
parent compounds (11−24 nm in 12 and 6−19 nm in 13),
especially in polar solvents. As a consequence, COUPY dyes
12 and 13 showed emission in the far-red to NIR region
(Figure 2 and Figures S10−S17), the emission maximum in
polar media being particularly appealing in the case of the 4-
CF3 fluorophore (λem = 673 nm for 13 in PBS). Interestingly,
coumarin 12 exhibited larger Stokes’ shifts than its parent dye
All the new compounds exhibited an intense absorption
band in the visible region of the electromagnetic spectrum,
with the absorption maxima ranging from 502 nm (10) to 600
nm (13) in aqueous solution (PBS pH 7.4) and from 543 nm
(10) to 624 nm (13) in DCM. To our surprise, the absorption
maximum of coumarin 10 was significantly blue-shifted with
respect the reference compound 4 (e.g., compare λabs = 515
and 543 nm for 10 and λabs = 552 and 569 nm for 4 in EtOH
and DCM, respectively). Although not as pronounced as in the
case of 10, a similar trend was found in ortho,ortho-pyrimidine-
containing coumarin (11) since its absorption maximum was
also blue-shifted (18−24 nm depending on the solvent) with
respect 4. By contrast, a slight red shift was found in the
absorption maximum of the ortho,para-pyrimidine-containing
coumarin 12 (12 nm in PBS and 6 nm in DCM). This red-shift
was considerably larger in the 4-CF3 analogue (e.g., compare
λabs = 600 nm for 13 and λabs = 568 nm for 5 in PBS). As
previously observed with the first generation of COUPY
dyes,11,12 10−13 showed negative solvatochromism since the
D
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4 in all the solvents evaluated, while the replacement of para-
pyridine by ortho,para-pyrimidine in 5 led to slightly lower
values. Nevertheless, the Stokes’ shifts of 12 and 13 in polar
solvents are sufficiently large (e.g., 71 and 73 nm in PBS,
respectively) to avoid the light reabsorption problems typically
found in bioimaging applications.1
maxima were red-shifted with respect to that of the parent
compounds, especially in polar protic solvents. Indeed, the
incorporation of the second nitrogen atom in the pyridine
moiety in 5 caused a red shift of the absorption (32 nm) and
emission (12 nm) bands in PBS pH 7.4 in the resulting
coumarin 13 when compared with the parent compound. Very
interestingly, the emission of 13 was extended beyond 750 nm
(Figure 2), which would facilitate in vivo imaging. Although
the incorporation of the ortho,para-pyrimidine moiety caused a
slight decrease in the fluorescence quantum yields of the
compounds (e.g., compare ΦF of 4 and 12), the combination
of this heterocycle with the CF3 group at position 4 seems to
compensate for this phenomenon, leading to moderate ΦF
values in protic and aprotic polar solvents (0.08−0.13) in the
case of compound 13.
Fluorescence Imaging of COUPY Dyes in Living Cells.
Taking into account the large photostability and the photo-
physical properties of the two ortho,para-pyrimidine-containing
COUPY dyes (12 and 13), we decided to evaluate their
usefulness as fluorescent probes in a more realistic situation
(e.g., in living cells). As previously stated, the heterogeneous
environment of an organic fluorophore within a cell or within a
specific cellular organelle might be considerably different than
the homogeneity of a solution in a spectroscopy cuvette. In
fact, the presence of biomolecules such as proteins and lipids
or the interaction with the components of cellular membranes
might provide the fluorescent probe with an environment less
hydrophilic than expected, thereby modifying the key
parameters for bioimaging applications such as brightness.
The cellular uptake of 12 and 13 was first investigated in
living HeLa cells (2 μM, 30 min incubation) by using confocal
microscopy and compared with that of the para-pyridine-
containing coumarins (compounds 4 and 5, respectively).
Irradiation was carried out with a yellow light laser (λex = 561
nm) in the case of 4-CH3 coumarins (4 and 12), while the
higher red-shift absorption of the 4-CF3 compounds (5 and
13) allowed the use of a red one (λex = 633 nm). As shown in
Figure 4, in all cases the fluorescence signal was clearly
observed inside the cell, which confirms that the excellent
cellular uptake of the parent COUPY dyes was retained after
replacement of pyridine with pyrimidine. In addition, it is
worth noting that no cell toxicity was observed during these
studies. The overall pattern of staining (but not the relative
fluorescence intensity between organelles; see below for a
discussion) of pyrimidine-containing coumarins (12 and 13)
was similar to that found with the parent fluorophores (4 and
5), suggesting accumulation in mitochondria, nucleoli and, to a
lesser extent, in intracellular vesicles, mostly lysosomes.
Colocalization experiments with two specific markers for
labeling mitochondria (MitoTracker Green FM, MTG) and
lysosomes (LysoTracker Green FM, LTG) confirmed the
subcellular localization of the compounds.
As shown in Table 1, compounds 10 and 11 exhibited very
weak fluorescence in all the solvents investigated. By contrast,
fluorescence quantum yields for coumarin 13 were much
higher than those of the parent compound 5, especially in less-
polar solvents (e.g., 0.41 and 0.05 in DCM, respectively). This
tendency was reversed in the case of the 4-CH3 analogue since
the ΦF for 12 was smaller compared with that of 4 (e.g., 0.11
and 0.70 in DCM, respectively). Both ortho,para-pyrimidine-
containing compounds exhibited moderate fluorescence
quantum yields in polar protic solvents (ΦF = 0.13 for 12
and ΦF = 0.08 for 13 in EtOH).
Finally, the photostability of the most promising coumarins
(12 and 13) was investigated in PBS buffer (pH 7.4) under
green LED irradiation (Figure 3 and Figure S18). As shown in
Figure 3. Fluorescence bleaching of COUPY dyes (4, 5, 12, and 13)
in PBS buffer pH 7.4 (5 μM) irradiated with green light (505 nm, 100
mW/cm2).
Figure 3, the replacement of pyridine in coumarin 4 with
ortho,para-pyrimidine had a positive effect on the photo-
stability of the resulting fluorophore (12). By contrast,
coumarin 13 was found less photostable than its parent
compound 5 and coumarin 12, which indicates that
replacement of the CH3 group at the 4-position with CF3 in
ortho,para-pyrimidine-containing COUPY dyes does not lead
to an improvement of the overall photostability of the
compounds. Nevertheless, it is worth noting that the two
new pyrimidine-containing COUPY dyes were found photo-
stable up to light fluences larger than 1000 J/cm2 (12) and 200
J/cm2 (13), which are more than 50- and 10-fold, respectively,
higher than those typically used in imaging experiments with
living cells.
As shown in Figure 5, the distribution of the fluorescence
emission of the compounds was similar to that of MitoTracker
Green FM, which confirmed accumulation into the mitochon-
dria. Pearson’s and Manders’ (M1 and M2) coefficients were
used to measure the degree of colocalization.11,12,18 On the
one hand, Pearson’s coefficients of 0.86 (12) and 0.87 (13)
confirmed a clear correlation between the coumarin signals and
MTG (Pearson’s coefficients ranging from −1 to +1, +1 being
the indicator of a perfect match). Such coefficients were higher
than those obtained for the parent COUPY dyes (0.65 for 4
and 0.73 for 5), indicating a better correlation between
In summary, all these observations allowed us to establish
some SPPR. First, replacement of para-pyridine in COUPY
dyes with ortho-pyridine or ortho,ortho-pyrimidine moieties had
a negative effect on the spectroscopic properties of the
fluorophores since both absorption and emission maxima were
blue-shifted. Moreover, compounds 10 and 11 were found to
be weakly fluorescent and their extinction coefficients were
smaller than those of the parent coumarin 4. By contrast, the
photophysical properties of COUPY dyes were clearly
improved when para-pyridine was replaced with ortho,para-
pyrimidine. On the one hand, both absorption and emission
E
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Figure 4. Cellular uptake of COUPY fluorophores (A) 4, (B) 5, (C) 12, and (D) 13. Single confocal planes of HeLa cells incubated with the
compounds (2 μM, 30 min, 37 °C). White arrows point out mitochondria, white arrowheads nucleoli, and yellow arrowheads stained vesicles. Scale
bar: 20 μm. All images are at the same scale as (A) and color coded using the Fire lookup table from Fiji (intensity calibration bar is showed in
(B)).
Figure 5. Colocalization studies with COUPY dyes 12 (top) and 13 (bottom) and MitoTracker Green FM. Single confocal plane of HeLa cells
incubated with 12 or 13 (2 μM, red) and MitoTracker Green FM (0.1 μM, green). (A, D) Overlay of the two staining. (B, E) coumarin 12 and 13
signal, respectively. (C, F) MitoTracker Green FM signal. Scale bar: 10 μm. All images are at the same scale as (A).
pyrimidine-containing coumarins and MTG. On the other
hand, the Manders’ coefficients (which ranges from 0 to 1,
determining the intensities of one channel colocalizing with the
other) also confirmed that 12 and 13 were mainly placed in the
mitochondria. The degree of colocalization of 12 over MTG
(M1 coefficient) was 0.40, whereas that of MTG over 12 (M2
coefficient) was 0.73. These values indicate that there is more
MTG signal colocalizing with 12 than 12 colocalizing with
MTG. The localization of the coumarin probe in other
organelles such as nucleoli and intracellular vesicles accounts
for the differences in both Manders’ coefficients. It is worth
noting that smaller values for M1 (0.28) and M2 (0.68) were
F
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Figure 6. Colocalization studies with COUPY dyes and LysoTracker Green FM. Single confocal plane of HeLa cells incubated with coumarins 12
and 13 (2 μM, red) and LysoTracker Green FM (0.2 μM, green). (A, D) Overlay of the two staining. (B, E) coumarins 12 and 13 signal. (C, F)
LysoTracker Green FM staining. White arrowheads point out COUPY dye vesicles colocalized with LysoTracker staining. Scale bar: 10 μm. All
images are at the same scale as (A).
obtained in the case of the parent coumarin 4, which indicates
that the signal from the fluorophore that colocalizes with MTG
is higher in the case of pyrimidine-containing coumarin (12)
than in the pyridine analogue (4), thereby suggesting a higher
preference for mitochondria. Although not as pronounced, a
similar trend was found for coumarins 13 (M1 = 0.19 and M2 =
0.86) and 5 (M1 = 0.18 and M2 = 0.76).
observation. Indeed, the mean intensity for mitochondria and
nucleoli was quite similar in the parent coumarins 4 and 5,
whereas the mean intensity for nucleoli staining was
considerably reduced in case of the pyrimidine analogues 12
and 13. These results are in line with the higher M1 coefficients
measured in colocalization experiments with MTG and
indicate that replacement of para-pyridine by ortho,para-
pyrimidine in COUPY dyes caused an increase in the
selectivity of the compounds toward mitochondria.
Finally, the photostability of coumarins 12 and 13 was
evaluated in a more realistic situation and compared with that
of the parent compounds (4 and 5, respectively). For this
purpose, living HeLa cells were incubated at 37 °C with the
compounds and irradiated continuously with the laser beam of
the confocal microscope (λex = 561 nm at 4.32 μW for 4 and
12, and λex = 633 nm at 10.2 μW for 5 and 13) and images
were acquired with an interval time of 0.42 s for 4.5 min. As
shown in Figures S19 and S20, the overall fluorescence signal
was significantly decreased in all the cases, In good agreement
with the photobleaching studies (Figure 3), the ortho,para-
containing coumarin 12 was found to be slightly more
photostable than its parent compound (4), while this trend
was found to be opposite in the case of the 4-CF3-containing
compounds. Nevertheless, it is worth noting that the laser
power used in the photobleaching experiments was much
higher than the one used during the conventional observation
(0.3 and 1.7 μW with 561 and 633 nm lasers, respectively),
which confirms a good photostability and applicability of these
molecules for imaging living cells by using fluorescence
microscopy.
As previously observed with the parent fluorophores,12
colocalization experiments with LTG (Figure 6) confirmed
that most of the fluorescence observed in intracellular vesicles
along the cytoplasm was associated with lysosome accumu-
lation (Pearson’s coefficients being equal to 0.52 (12) and 0.49
(13) on average). The smaller Mander’s coefficients for the
colocalization of the compounds over LTG (M1 = 0.02 for 12
and 13) and of LTG over the coumarins (M2 = 0.45 for 12 and
0.62 for 13) in the colocalization experiment with LTG
compared with MTG might be explained by a reduced affinity
of COUPY dyes for lysosome and/or by lower abundance of
this organelle in cells compared with mitochondria.
Very interestingly, the nucleoli inside the nuclei was much
less intensely stained in compounds 12 and 13 than in the
parent coumarins: compare in Figure 4 panels (C) (12) and
(D) (13) with panels (A) (4) and (B) (5), respectively. As
shown in Figure 7, measurement of the mean fluorescence
intensity both in the mitochondria and nucleoli confirmed this
CONCLUSIONS
■
In summary, in this manuscript we have investigated how the
photophysical properties and subcellular accumulation of
coumarin-based COUPY fluorophores can be fine-tuned
through replacement of the para-pyridine moiety with different
heterocycles (ortho-pyridine and ortho,ortho- or ortho,para-
pyrimidine), with the aim of establishing new SPPR. COUPY
Figure 7. Graph of COUPY dyes 4, 5, 12, and 13 vs mean intensity
(Arbitrary Units) at the mitochondria and nucleoli.
G
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Article
Hz), 7.29 (1H, t, J = 5.1 Hz), 4.11 (s, 2H). 13C{1H} NMR (101 MHz,
CDCl3, δ (ppm)): 161.4, 158.0, 120.3, 116.1, 28.6. LRMS (ESI-TOF)
(m/z): [M + H]+ Calcd for C6H6N3, 120.06; found, 119.90.
2-(Pyrimidin-4-yl)acetonitrile (16). A modified method was
followed to synthesize compound 1619
(E)-N,N-Dimethyl-2-(pyrimidin-4-yl)ethen-1-amine (17). 4-Meth-
ylpyrimidine (10 g, 106 mmol) and 1,1-dimethoxy-N,N-dimethylme-
thanamine (38 g, 319 mmol) were dissolved in 50 mL of anhydrous
DMF. The solution was heated at 140 °C with a hot plate magnetic
stirrer for 24 h. The crude product was evaporated under reduced
pressure to provide 17 as brown oil, which was used without further
purification. TLC: Rf (DCM/MeOH 9:1) 0.50. 1H NMR (400 MHz,
CDCl3, δ (ppm)): 8.73 (1H, s), 8.21 (1H, d, J = 5.6 Hz), 7.76 (1H, d,
J = 13 Hz), 6.71 (1H, dd, J = 5.6, 1.6 Hz), 4.99 (1H, d, J = 13 Hz),
2.95 (6H, s).
2-(Pyrimidin-4-yl)acetonitrile (16). A solution of hydroxylamine-
O-sulfonic acid (7.31 g, 57.5 mmol) in Milli-Q water (100 mL) was
added to the crude product 17, and the reaction mixture was heated at
50 °C with a hot plate magnetic stirrer for 45 min. Then, the mixture
was cooled at 0 °C and saturated NaHCO3 was added until basic pH,
and the aqueous phase was extracted with ethyl acetate (6 × 200 mL).
The combined organic phases were dried over anhydrous Na2SO4,
filtered, and evaporated under reduced pressure. The brown oil was
purified by column chromatography (silica gel, 0−25% ethyl acetate
in DCM) to give 1.46 g of a yellow solid (yield 11%). TLC: Rf (ethyl
acetate/DCM 1:1) 0.34. 1H NMR (400 MHz, CDCl3, δ (ppm)): 9.20
(1H, s), 8.80 (1H, d, J = 5.2 Hz), 7.51 (1H, d, J = 5.2 Hz), 3.94 (2H,
s). 13C{1H} NMR (101 MHz, CDCl3, δ (ppm)): 159.3, 159.2, 158.3,
119.7, 115.5, 26.5. LRMS (ESI-TOF) (m/z): [M + H]+ Calcd for
C6H6N3, 120.06; found, 119.90.
dyes 10−13 were easily obtained from cheap, commercially
available compounds in three linear synthetic steps, the
reaction of thiocoumarins with suitable heteroarylacetonitrile
derivatives followed by N-methylation of the pyridine or
pyrimidine moieties being the key steps. Very interestingly, the
photophysical properties of the new fluorophores were
strongly influenced by these modifications. On the one hand,
the replacement of the para-pyridine moiety with ortho-
pyridine (10) or ortho,ortho-pyrimidine (11) had a negative
effect on the spectroscopic properties of the dyes since both
absorption and emission were blue-shifted, leading to weakly
fluorescent compounds. On the other hand, the absorption and
emission maxima of ortho,para-pyrimidine-containing fluoro-
phores (12−13) were red-shifted with respect to the parent
compounds, particularly in polar solvents. Besides emission in
the far-red to NIR region, the newly synthesized fluorophores
exhibited other appropriate characteristics for bioimaging
applications such as large Stokes’ shifts and high photostability.
Furthermore, coumarins 12 and 13 retained the excellent cell
permeability of COUPY dyes but exhibited a higher preference
for mitochondria. Overall, these results provide useful
indications for the development of novel mitochondria-
targeted fluorescent probes based on small organic compounds
since we have demonstrated that the selectivity for this
organelle can be improved through the replacement of the N-
alkylated pyridinium moieties by the corresponding N-
alkylated ortho,para-pyrimidinium counterparts.
Synthesis of COUPY Scaffolds (6−9). 2-(Cyano(2-pyridine)-
methylene)-7-(N,N-diethylamino)-4-methyl-coumarin (6). 7-(N,N-
Diethylamino)-4-methyl-2-thiocoumarin 18 (0.25 g, 1.01 mmol), 2-
(pyridin-2-yl)acetonitrile 14 (0.24 g, 2.02 mmol), and NaH (60%
dispersion in mineral oil, 0.36 g, 9 mmol) were dissolved in anhydrous
acetonitrile (15 mL) and the mixture was stirred for 2 h under an
argon atmosphere at room temperature and protected from light.
Then, AgNO3 (0.34 g, 2.02 mmol) was added and the resulting
mixture was stirred for 2 h under Ar at room temperature. The crude
product was evaporated under reduced pressure and purified by
column chromatography (silica gel, 0−25% ethyl acetate in DCM) to
give 240 mg of an orange solid (yield 72%). TLC: Rf (ethyl acetate/
EXPERIMENTAL SECTION
■
Materials and Methods. Unless otherwise stated, common
chemicals and solvents (HPLC grade or reagent grade quality) were
purchased from commercial sources and used without further
purification. Aluminum plates coated with a 0.2 mm-thick layer of
silica gel 60 F254 were used for thin-layer chromatography (TLC)
analysis, whereas flash column chromatography purification was
carried out using silica gel 60 (230−400 mesh). Reversed-phase
HPLC analyses were carried out on a Jupiter Proteo C18 column (250
× 4.6 mm, 90 Å 4 μm, flow rate: 1 mL/min) using linear gradients of
0.1% formic acid in H2O (A) and 0.1% formic acid in ACN (B).
NMR spectra were recorded at 25 °C in a 400 MHz spectrometer
using the deuterated solvent as an internal deuterium lock. The
residual protic signal of chloroform or DMSO was used as a reference
in 1H and 13C NMR spectra recorded in CDCl3 or DMSO-d6,
respectively. Chemical shifts are reported in parts per million (ppm)
in the δ scale, coupling constants in Hz, and multiplicity as follows: s
(singlet), d (doublet), t (triplet), q (quartet), qt (quintuplet), m
(multiplet), dd (doublet of doublets), dq (doublet of quartets), br
(broad signal), etc. The proton signals of the E and Z rotamers were
1
DCM 1:1) 0.90. H NMR (400 MHz, DMSO-d6, δ (ppm)): (E + Z
rotamers) 8.56 (1H, m), 8.13 (0.6H, dt, J = 8.2, 0.8 Hz), 7.82 (1.4H,
m), 7.46 (1.4H, m), 7.18 (1H, m), 6.70 (1H, dd, J = 9.0, 2.4 Hz), 6.65
(1.2H, m), 6.42 (0.4H, d, J = 2.4 Hz), 3.46 (4H, q, J = 7.2 Hz,), 2.35
(1.8H, d, J = 0.8 Hz), 2.32 (1.2H, d, J = 0.8 Hz), 1.14 (6H, m).
13C{1H} NMR (101 MHz, DMSO-d6, δ (ppm)): (E + Z rotamers)
163.2, 161.8, 154.0, 153.8, 152.7, 152.0, 150.6, 150.5, 149.0, 148.8,
144.6, 144.0, 137.1, 136.8, 126.0, 125.9, 121.8, 121.2, 120.7, 120.2,
119.6, 119.3, 110.8, 110.4, 109.8, 109.2, 109.1, 96.7, 96.1, 85.3, 81.0,
44.0, 43.8, 18.5, 18.0, 12.4. HRMS (ESI-TOF) (m/z): [M + H]+
Calcd for C21H22N3O, 332.1763; found, 332.1759. Analytical HPLC
(30−100% B in 30 min, formic acid additive): Rt = 15.2 min.
2-(Cyano(2-pyrimidine)methylene)-7-(N,N-diethylamino)-4-
methyl-coumarin (7). 7-(N,N-Diethylamino)-4-methyl-2-thiocou-
marin 18 (0.25 g, 1.01 mmol), 2-(pyrimidin-2-yl)acetonitrile 15
(0.24 g, 2.02 mmol), and NaH (60% dispersion in mineral oil, 0.36 g,
9 mmol) were dissolved in anhydrous acetonitrile (15 mL) and the
mixture was stirred for 2 h under an argon atmosphere at room
temperature and protected from light. Then, AgNO3 (0.34 g, 2.02
mmol) was added and the resulting mixture was stirred for 2 h under
Ar at room temperature. The crude product was evaporated under
reduced pressure and purified by column chromatography (silica gel,
0−10% ethyl acetate in DCM) to give 171 mg of an orange solid
1
identified by simple inspection of the H spectrum and the rotamer
ratio was calculated by peak integration. 2D-NOESY spectra were
acquired in DMSO-d6 with a mixing time of 500 ms, either at 298 K
or 350 K. Electrospray ionization mass spectra (ESI-MS) were
recorded on an instrument equipped with a single quadrupole
detector coupled to an HPLC, and high-resolution (HR) ESI-MS on a
LC/MS-TOF instrument.
Synthesis of Pyrimidin-Acetonitrile Derivatives. 2-(Pyrimi-
din-2-yl)acetonitrile (15). A solution of KCN (0.63 g, 9.70 mmol) in
DMSO (5 mL) was slowly added to a solution of 2-(chloromethyl)-
pyrimidine (0.5 g, 3.89 mmol) in DMSO (4 mL). The reaction
mixture was stirred overnight at 35 °C with a hot plate magnetic
stirrer. After addition of 50 mL of K2CO3 (10%), the aqueous phase
was extracted with diethyl ether (10 × 30 mL). The combined organic
fractions were dried over anhydrous Na2SO4, filtered, and evaporated
under reduced pressure. The crude product was purified by column
chromatography (silica gel, 0−15% ethyl acetate in DCM) to give 84
mg of colorless oil (yield 19%). TLC: Rf (ethyl acetate/DCM 1:1)
1
(yield 51%). TLC: Rf (ethyl acetate/DCM 1:1) 0.63. H NMR (400
MHz, DMSO-d6, δ (ppm)): (major rotamer) 8.74 (2H, d, J = 4.8
Hz), 8.18 (1H, s), 7.53 (1H. d, J = 9.0 Hz), 7.17 (1H, t, J = 4.8 Hz),
6.76 (1H, dd, J = 9.0, 2.4 Hz), 6.46 (1H, d, J = 2.4 Hz), 3.47 (4H, q, J
= 7.2 Hz), 2.39 (3H, s), 1.15 (6H, t, J = 6.9 Hz). 13C{1H} NMR (101
1
0.44. H NMR (400 MHz, CDCl3, δ (ppm)): 8.74 (2H, d, J = 5.1
H
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Article
MHz, DMSO-d6, δ (ppm)): (major rotamer) 166.6, 162.9, 157.0,
154.1, 150.9, 146.6, 126.3, 118.4, 116.6, 110.5, 109.9, 109.7, 96.0,
82.5, 44.1, 18.7, 12.4. HRMS (ESI-TOF) (m/z): [M + H]+ Calcd for
C20H21N4O, 333.1715; found, 333.1710. Analytical HPLC (30−100%
B in 30 min, formic acid additive): Rt = 21.2 min.
(3H, s), 3.46 (4H, q, J = 7.2 Hz), 2.43 (3H, s), 1.15 (6H, t, J = 7.2
Hz). 13C{1H} NMR (101 MHz, 77 °C, DMSO-d6, δ (ppm)): 165.6,
153.9, 151.2, 149.3, 147.8, 147.1, 144.6, 130.5, 126.2, 125.3, 120.5 (q,
J = 323 Hz, Tf), 116.5, 110.3, 109.4, 108.0, 96.2, 71.0, 46.0, 43.7, 17.6,
12.0. 19F NMR (376 MHz, DMSO-d6) δ (ppm): −77.8 (3F, s).
HRMS (ESI-TOF) (m/z): [M]+ Calcd for C22H24N3O, 346.1914;
found, 346.1911. Analytical HPLC (30−100% B in 30 min, formic
acid additive): Rt = 5.5 min.
2-(Cyano(4-pyrimidine)methylene)-7-(N,N-diethylamino)-4-
methyl-coumarin (8). 7-(N,N-Diethylamino)-4-methyl-2-thiocou-
marin 18 (0.25 g, 1.01 mmol), 2-(pyrimidin-4-yl)acetonitrile 16
(0.24 g, 2.02 mmol), and NaH (60% dispersion in mineral oil, 0.36 g,
9 mmol) were dissolved in anhydrous acetonitrile (15 mL) and the
mixture was stirred for 2 h under an argon atmosphere at room
temperature and protected from light. Then, AgNO3 (0.34 g, 2.02
mmol) was added and the resulting mixture was stirred for 2 h under
Ar at room temperature. The crude product was evaporated under
reduced pressure and purified by column chromatography (silica gel,
0−25% ethyl acetate in DCM) to give 275 mg of an orange solid
2-(Cyano(1-methyl(2-pyrimidin-1-ium))methylene)-7-(N,N-di-
ethylamino)-4-methyl-coumarin triflate (11). Methyl trifluorome-
thanesulfonate (47.5 μL, 0.42 mmol) was added to a solution of 2-
(cyano(2-pyrimidine)methylene)-7-(N,N-diethylamino)-4-methyl-
coumarin 7 (70 mg, 0.21 mmol) in DCM (30 mL) under an argon
atmosphere. The mixture was stirred for 4 h at room temperature and
protected from light. Then, an additional amount of methyl
trifluoromethanesulfonate (47.5 μL, 0.42 mmol) was added to the
reaction mixture and was stirred for 2 h. The reaction mixture was
evaporated under reduced pressure and purified by column
chromatography (silica gel, 0−4% MeOH in DCM) to give 66 mg
of an orange solid (yield 64%). TLC: Rf (DCM/MeOH 9:1) 0.31. 1H
NMR (400 MHz, DMSO-d6, δ (ppm)): 9.28 (1H, dd, J = 4.2, 2.0
Hz), 9.16 (1H, dd, J = 6.6, 1.8 Hz), 7.91 (1H, dd, J = 6.4, 5.6 Hz,
1H), 7.70 (1H, d, J = 9.2 Hz), 7.06 (1H, s), 6.94 (1H, dd, J = 9.2, 2.8
Hz), 6.70 (1H, d, J = 2.8 Hz), 4.14 (3H, s), 3.50 (4H, q, J = 7.2 Hz),
2.49 (3H, s), 1.15 (6H, t, J = 7.2 Hz). 13C{1H} NMR (101 MHz,
DMSO-d6, δ (ppm)): 162.9157.6, 154.7, 154.6, 152.5, 151.8, 127.1,
120.7 (q, J = 323 Hz, Tf), 118.7, 116.8, 111.6, 110.4, 109.2, 96.0, 73.4,
46.5, 44.2, 18.5, 12.3. 19F NMR (376 MHz, DMSO-d6) δ (ppm):
−77.8 (3F, s). HRMS (ESI-TOF) (m/z): [M]+ Calcd for
C21H23N4O, 347.1866; found, 347.1865. Analytical HPLC (30−
100% B in 30 min, formic acid additive): Rt = 5.1 min.
2-(Cyano(1-methyl(4-pyrimidin-1-ium))methylene)-7-(N,N-di-
ethylamino)-4-methyl-coumarin triflate (12). Methyl trifluorome-
thanesulfonate (47.5 μL, 0.42 mmol) was added to a solution of 2-
(cyano(4-pyrimidine)methylene)-7-(N,N-diethylamino)-4-methyl-
coumarin 8 (70 mg, 0.21 mmol) in DCM (30 mL) under an argon
atmosphere. The mixture was stirred for 4 h at room temperature and
protected from light. The reaction mixture was evaporated under
reduced pressure and purified by column chromatography (silica gel,
0−2% MeOH in DCM) to give 83 mg of a violet solid (yield 80%).
TLC: Rf (DCM/MeOH 9:1) 0.28. 1H NMR (400 MHz, 77 °C,
DMSO-d6, δ (ppm)): 9.11 (1H, m), 8.53 (1H, dd, J = 7.6, 2.0 Hz),
8.00 (1H, d, J = 7.6 Hz), 7.84 (1H, d, J = 9.2 Hz), 7.60 (1H, br s),
7.09 (1H, dd, J = 9.2, 2.4 Hz), 6.89 (1H, d, J = 2.4 Hz), 4.02 (3H, s),
3.60 (4H, q, J = 7.2 Hz), 2.63 (3H, s), 1.23 (6H, t, J = 7.2 Hz).
13C{1H} NMR (101 MHz, DMSO-d6, δ (ppm)): 167.3, 163.4, 155.6,
1
(yield 82%). TLC: Rf (ethyl acetate/DCM 1:1) 0.50. H NMR (400
MHz, DMSO-d6, δ (ppm)): (E + Z rotamers) 9.04 (1H, m), 8.69
(0.6H, d, J = 5.6 Hz), 8.62 (0.4H, d, J = 5.6 Hz), 8.26 (0.4H, d, J = 0.8
Hz), 8.18 (0.6H, dd, J = 5.6, 1.6 Hz), 7.59 (0.4H, d, J = 9.2 Hz), 7.56
(0.6H, d, J = 8.8 Hz), 7.42 (0.4H, dd, J = 5.6, 1.6 Hz), 6.81 (1.6H,
m), 6.75 (0.6H, d, J = 0.8 Hz), 6.51 (0.4H, d, J = 2.8 Hz), 3.49 (4H,
m), 2.43 (1.2H, d, J = 1.2 Hz),2.42 (1.7H, d, J = 1.2 Hz), 1.16 (6H, t,
J = 7.0 Hz). 13C{1H} NMR (101 MHz, DMSO-d6, δ (ppm)): (E + Z
rotamers) 165.5, 165.2, 160.3, 159.1, 158.1, 157.6, 156.9, 156.7,
154.2, 151.2, 151.0, 148.5, 147.6, 126.5, 126.4, 118.6, 118.4, 117.5,
116.9, 116.7, 110.6, 110.4, 110.3, 110.2, 109.2, 96.6, 95.8, 82.8, 78.5,
44.1, 43.9 18.8, 18.2, 12.4, 12.3. HRMS (ESI-TOF) (m/z): [M + H]+
Calcd for C20H21N4O, 333.1715; found, 333.1709. Analytical HPLC
(30−100% B in 30 min, formic acid additive): Rt = 17.5 min.
2-(Cyano(4-pyrimidine)methylene)-7-(N,N-dimethylamino)-4-tri-
fluoromethyl-coumarin (9). 7-(N,N-Dimethylamino)-4-trifluoro-
methyl-2-thiocoumarin 19 (0.25 g, 0.92 mmol), 2-(pyrimidin −4-
yl)acetonitrile 16 (0.22 g, 1.83 mmol), and NaH (60% dispersion in
mineral oil, 0.33 g, 8.25 mmol) were dissolved in anhydrous
acetonitrile (15 mL) and the mixture was stirred for 2 h under an
argon atmosphere at room temperature and protected from light.
Then, AgNO3 (0.31 g, 1.83 mmol) was added and the resulting
mixture was stirred for 2 h under Ar at room temperature. The crude
product was evaporated under reduced pressure and purified by
column chromatography (silica gel, 0−10% ethyl acetate in DCM) to
give 248 mg of a red solid (yield 75%). TLC: Rf (ethyl acetate/DCM
1
1:1) 0.30 and 0.64. H NMR (400 MHz, DMSO-d6, δ (ppm)): (E +
Z rotamers) 9.17 (0.55H, d, J = 1.2 Hz), 9.15 (0.45H, d, J = 1.2 Hz),
8.83 (0.55H, d, J = 5.6 Hz), 8.78 (0.45H, d, J = 5.6 Hz), 8.74 (0.45H,
s), 8.28 (0.55H, dd, J = 5.6, 1.6 Hz), 7.56 (0.45H, dd, J = 5.6, 1.6 Hz),
7.44 (1H, m), 7.03 (0.55H, s), 7.00 (0.55H, d, J = 2.8 Hz), 6.87 (1H,
m), 6.64 (0.45H, d, J = 2.4 Hz), 3.13 (3.3H, s), 3.11 (2.7H, s).
13C{1H} NMR (101 MHz, DMSO-d6, δ (ppm)): (E + Z rotamers)
155.5, 152.7, 152.2, 147.5, 127.5, 120.7 (q, J = 323 Hz, Tf), 117.4,
114.8, 113.1, 111.5, 110.7, 95.9, 44.4, 42.5, 18.9, 12.4. 19F NMR (376
MHz, DMSO-d6) δ (ppm): −77.8 (3F, s). HRMS (ESI-TOF) (m/z):
[M]+ Calcd for C21H23N4O 347.1866; found, 347.1866. Analytical
HPLC (30−100% B in 30 min, formic acid additive): Rt = 5.4 min.
2-(Cyano(1-methyl(4-pyrimidin-1-ium))methylene)-7-(N,N-dime-
thylamino)-4-trifluoromethyl-coumarin triflate (13). Methyl tri-
fluoromethanesulfonate (47.3 μL, 0.40 mmol) was added to a solution
of 2-(cyano(4-pyrimidine)methylene)-7-(N,N-dimethylamino)-4-tri-
fluoromethyl-coumarin 9 (70 mg, 0.20 mmol) in DCM (30 mL)
under an argon atmosphere. The mixture was stirred for 4 h at room
temperature and protected from light. The reaction mixture was
evaporated under reduced pressure and purified by column
chromatography (silica gel, 0−2% MeOH in DCM) to give 75 mg
163.3, 162.6, 158.7, 158.3, 157.9, 157.7, 157.6, 154.6, 154.5, 153.7,
153.6, 125.3, 118.8, 117.9, 117.4, 116.8, 111.0, 110.9, 110.8, 110.7,
110.7, 110.5, 110.4, 110.4, 103.5, 102.5, 98.1, 97.4, 89.7, 89.5, 85.2,
39.7. 19F NMR (376 MHz, DMSO-d6) δ (ppm): (E + Z rotamers)
-63.0 (1.35F, s), −63. (1.65F, s). HRMS (ESI-TOF) (m/z): [M +
H]+ Calcd for C18H14F3N4O, 359.1114; found, 359.1112. Analytical
HPLC (30−100% B in 30 min, formic acid additive): Rt = 22.8 min.
Synthesis of COUPY Fluorophores (10−13). 2-(Cyano(1-
methyl(2-pyridin-1-ium))methylene)-7-(N,N-diethylamino)-4-
methyl-coumarin triflate (10). Methyl trifluoromethanesulfonate
(47.5 μL, 0.42 mmol) was added to a solution of 2-(cyano(2-
pyridine)methylene)-7-(N,N-diethylamino)-4-methyl-coumarin 6 (70
mg, 0.21 mmol) in DCM (30 mL) under an argon atmosphere. The
mixture was stirred for 4 h at room temperature and protected from
light. The reaction mixture was evaporated under reduced pressure
and purified by column chromatography (silica gel, 0−3% MeOH in
DCM) to give 77 mg of a red solid (yield 74%). TLC: Rf (DCM/
MeOH 9:1) 0.35. 1H NMR (400 MHz, 77 °C, DMSO-d6, δ (ppm)):
9.02 (1H, d, J = 6.4 Hz), 8.53 (1H, td, J = 7.6, 1.2 Hz), 8.26 (1H, d, J
= 8.0 Hz), 8.00 (1H, td, J = 7.6, 1.2 Hz), 7.59 (1H, d, J = 9.2 Hz),
6.83 (1H, dd, J = 9.2, 2.4 Hz), 6.62 (1H, br s), 6.51 (1H, br s), 4.28
1
of a blue solid (yield 72%). TLC: Rf (DCM/MeOH 9:1) 0.48. H
NMR (400 MHz, 77 °C, DMSO-d6, δ (ppm)): 9.40 (1H, s), 8.81
(1H, dd, J = 7.4, 1.8 Hz), 8.21 (1H, br s), 7.65 (1H, dq, J = 9.2, 2.0
Hz), 7.13 (1H, dd, J = 9.2, 2.6 Hz), 7.01 (1H, br s), 4.13 (3H, s), 3.22
(6H, s). 13C{1H} NMR (101 MHz, 77 °C DMSO-d6, δ (ppm)):
166.8, 163.2, 158.8, 156.0, 155.4, 154.5, 153.1, 152.4, 136.7 (q, J = 32
Hz), 125.4, 124.9, 121.5 (q, J = 277 Hz), 120.5 (q, J = 323 Hz), 116.6,
115.6, 113.3, 109.7, 108.4, 107.7, 101.5, 97.7, 97.2, 43.0, 40.0. 19F
NMR (376 MHz, DMSO-d6) δ (ppm): −63.1 (3F, s), −77.8 (3F, s).
HRMS (ESI-TOF) (m/z): [M]+ Calcd for C19H16F3N4O 373.1271;
I
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Article
found, 373.1274. Analytical HPLC (30−100% B in 30 min, formic
acid additive): Rt = 5.1 min.
Bleaching experiments were performed at 37 °C by continuous
image acquisition with an interval time of 0.42 s for 4.5 min.
Coumarins 4 and 12 were bleached using the 561 nm laser at 4.32
μW and coumarins 5 and 13 were bleached with the 633 nm laser at
10.2 μW.
Photophysical Characterization of the Compounds. Absorp-
tion spectra were recorded on a Jasco V-730 spectrophotometer at
room temperature. Molar absorption coefficients (ε) were determined
by direct application of the Beer−Lambert law, using solutions of the
compounds in each solvent with concentrations ranging from 10−6 to
10−5 M. Emission spectra were measured on a Photon Technology
International (PTI) fluorimeter. Fluorescence quantum yields (ΦF)
were measured by a comparative method using Cresyl Violet in
ethanol (ΦF;Ref = 0.54) as a reference for compounds 4, 5, 12, and 13.
Image processing and analysis was performed using Fiji.20
Intensity Measurement. The nuclei and coumarin channels were
processed by median filtering (radius = 2), Gaussian filtering (sigma =
2), and background subtraction (rolling ball radius = 300). The mean
intensity in the nucleoli was measured in the maximum intensity
projection of the processed coumarin image after manually drawing
ROIs around each nucleoli. The intensity measurement in
mitochondria needed further processing. First, the nuclei were
segmented using the Intermodes algorithm,21 and the resulting binary
image was processed by filling holes and opening operations. Then
the binary image of the nuclei was subtracted to the processed
coumarin image to get rid of any nuclear signal. After the subtraction,
the coumarin staining channel was projected with the maxim intensity
and the mitochondria were segmented using the Phansalkar algorithm
(radius = 5).22 The binary image of the mitochondria was used as a
mask to obtain the coumarin signal in the mitochondria and then
measure its mean intensity.
Fluorescein dissolved in aqueous sodium hydroxide (0.1 M; ΦF;Ref
=
0.92) was used as a reference in the case of compounds 10 and 11.17
Then, optically matched solutions of the compounds and CV were
prepared and fluorescence spectra were recorded. The absorbance of
the sample and reference solutions was set below 0.1 at the excitation
wavelength and ΦF was calculated using the following eq 1:
2
i η
y
z
z
z
AreaSample
AreaRef
j
Sample
j
j
ΦF;Sample
=
×
× ΦF;ref
j
z
j
j
z
z
ηRef
(1)
k
{
Bleaching Analysis. Images were first processed by median filtering
(radius = 1) and background subtraction (rolling ball radius = 50).
Then, an ROI was manually drawn around each cell and in the
background to measure the mean intensity along time. Intensity
normalization was performed using the following eq 2:
where AreaSample and AreaRef are the integrated fluorescence for the
sample and the reference and ηSample and ηRef are the refractive index
of the sample and reference solutions, respectively. The uncertainty in
the experimental value of ΦF has been estimated to be approximately
10%.
Photostability studies were performed by monitoring fluorescence
bleaching of a 5 μM PBS solution (pH 7.4) of the compounds at 37
°C irradiated with a high power 505 nm LED (100 mW/cm2).
Fluorescence intensity values were recorded at t = 0 (F0) and after
different irradiation times (F).
Cell Culture and Treatments. HeLa cells were maintained in
DMEM (Dulbecco’s Modified Eagle Medium) containing high
glucose (4.5 g/L) and supplemented with 10% fetal calf serum
(FCS) and 50 U/mL penicillin−streptomycin. For cellular uptake
experiments and posterior observation under a microscope, cells were
seeded on glass-bottom dishes (P35G-1.5-14-C, Mattek). The cells
were incubated for 30 min at 37 °C with COUPY dyes (4, 5, 12, or
13, 2 μM) in supplemented DMEM, 24 hours after cell seeding.
Then, the cells were washed three times with DPBS (Dulbecco’s
phosphate buffered saline, pH 7.0−7.3) to remove the excess
fluorophores and kept in low glucose DMEM without phenol red
for fluorescence imaging.
Cell(t) − Backg (t)
Cell(0) − Backg (0)
Normalized intensity =
(2)
where Cell(t) is the mean intensity in a cell at t time, Cell(0) is the
mean intensity in that cell at the beginning of the experiment,
Backg(t) is the mean intensity in the background at t time, and
Backg(0) is the mean intensity in the background at the beginning of
the experiment. After intensity normalization, the time at which the
intensity dropped to half the initial one (t50) was obtained.
Differences between the different t50 of coumarin 4 and 12 were
tested with Student’s t-test.
Colocalizations Coefficients. The MitoTracker or LysoTracker
and coumarin channels were processed by median filtering (radius =
1), Gaussian filtering (sigma = 1), and background subtraction
(rolling ball radius = 300). Colocalization coefficients were measured
using the JaCoP plugin17 on the different stacks of images (n = 4)
with each stack containing 3−5 cells. The threshold for the coumarin
channel was set to include the signal in the mitochondria, nucleoli,
and vesicles. The threshold for the MitoTracker or LysoTracker
channels was set to specifically select mitochondria and lysosomes,
respectively.
For colocalization experiments with MitoTracker Green FM, HeLa
cells were treated with COUPY dyes (2 μM) and MitoTracker Green
FM (0.1 μM) for 30 min at 37 °C in nonsupplemented DMEM. After
removal of the medium and washing three times with DPBS, cells
were kept in low glucose DMEM without phenol red for fluorescence
imaging.
For colocalization experiments with LysoTracker Green FM and
Hoechst 33342, HeLa cells were treated with COUPY dyes (2 μM)
and LysoTracker Green FM (0.2 μM) for 30 min at 37 °C in
nonsupplemented DMEM. After removal of the medium and washing
three times with DPBS, the cells were incubated for 10 min at 37 °C
with Hoechst 33342 (1 μg/mL) in supplemented DMEM. Finally, the
cells were washed and kept in low glucose DMEM without phenol red
for fluorescence imaging.
Fluorescence Imaging. All microscopy observations were
performed using a Zeiss LSM 880 confocal microscope equipped
with a 405 nm laser diode, an argon-ion laser, a 561 nm laser, and a
633 nm laser. The microscope was also equipped with a Heating
Insert P S (Pecon) and a 5% CO2 providing system. Cells were
observed at 37 °C using a 63 × 1.2 glycerol immersion objective.
Coumarins 4 and 12 were excited using the 561 nm laser and detected
from 570 to 670 nm. Coumarins 5 and 13 were excited using the 633
nm laser and detected from 643 to 758 nm. In colocalization studies,
MitoTracker Green FM and LysoTracker Green were observed using
the 488 nm laser line of the argon-ion laser, whereas the 405 nm laser
diode was used for observing Hoechst 33342.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.joc.0c00570.
Copies of HPLC traces and UV−vis absorption and
fluorescence emission spectra of the compounds;
additional fluorescence imaging studies; and 1D NMR
(1H, 13C, and 19F), MS, and selected 2D NMR spectra
(PDF).
AUTHOR INFORMATION
■
Corresponding Author
́
́
̀
Vicente Marchan − Departament de Quımica Inorganica i
̀
́
́
̀
Organica, Seccio de Quımica Organica, IBUB, Universitat de
Barcelona, E-08028 Barcelona, Spain; orcid.org/0000-
0002-1905-2156; Email: vmarchan@ub.edu
J
https://dx.doi.org/10.1021/acs.joc.0c00570
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The Journal of Organic Chemistry
Authors
pubs.acs.org/joc
Article
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Preferential accumulation of the near infrared heptamethine dye IR-
780 in the mitochondria of drug-resistant lung cancer cells.
Biomaterials 2014, 35, 4116−4124. (b) Yang, X.; Shi, C.; Tong, R.;
Qian, W.; Zhau, H. E.; Wang, R.; Zhu, G.; Cheng, J.; Yang, V. W.;
Cheng, T.; Henary, M.; Strekowski, L.; Chung, L. W. K. Near IR
Heptamethine Cyanine Dye-Mediated Cancer Imaging. Clin. Cancer
Res. 2010, 16, 2833−2844. (c) Johnson, L. V.; Walsh, M. L.; Chen, L.
B. Localization of mitochondria in living cells with rhodamine 123.
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Development of fluorescent mitochondria probe based on 1,2-
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468. (d) Zielonka, J.; Joseph, J.; Sikora, A.; Hardy, M.; Ouari, O.;
Vasquez-Vivar, J.; Cheng, G.; Lopez, M.; Kalyanaraman, B.
Mitochondria-Targeted Triphenylphosphonium-Based Compounds:
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́
̀
̀
Anna Rovira − Departament de Quımica Inorganica i Organica,
́
́
̀
Seccio de Quımica Organica, IBUB, Universitat de Barcelona,
E-08028 Barcelona, Spain
́
̀
Miriam Pujals − Departament de Quımica Inorganica i
̀
́
́
̀
Organica, Seccio de Quımica Organica, IBUB, Universitat de
Barcelona, E-08028 Barcelona, Spain
́
̀
Albert Gandioso − Departament de Quımica Inorganica i
̀
́
́
̀
Organica, Seccio de Quımica Organica, IBUB, Universitat de
Barcelona, E-08028 Barcelona, Spain
́
́
̀
Marta Lopez-Corrales − Departament de Quımica Inorganica i
̀
́
́
̀
Organica, Seccio de Quımica Organica, IBUB, Universitat de
Barcelona, E-08028 Barcelona, Spain
̀
̀
Manel Bosch − Unitat de Microscopia Optica Avancada,
̨
́
̀
Centres Cientıfics i Tecnologics, Universitat de Barcelona, E-
08028 Barcelona, Spain
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.joc.0c00570
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by funds from the Spanish
Government (MCIU/AEI/FEDER, UE; grant CTQ2017-
84779-R) and the Generalitat de Catalunya (2017 DI 072).
The authors acknowledge the helpful assistance of Dr.
́
́
Francisco Cardenas (NMR) and Dr. Irene Fernandez and
Laura Ortiz (MS) from CCiTUB. A.R. and A.G. were recipient
fellows of the University of Barcelona.
(9) (a) Wu, S.; Cao, Q.; Wang, X.; Cheng, K.; Cheng, Z. Design,
synthesis and biological evaluation of mitochondria targeting
theranostic agents. Chem. Commun. 2014, 50, 8919−8922. (b) Luo,
S.; Tan, X.; Fang, S.; Wang, Y.; Liu, T.; Wang, X.; Yuan, Y.; Sun, H.;
Qi, Q.; Shi, C. Mitochondria-Targeted Small-Molecule Fluorophores
for Dual Modal Cancer Phototherapy. Adv. Funct. Mater. 2016, 26,
2826−2835. (c) Gao, P.; Pan, W.; Li, N.; Tang, B. Fluorescent probes
for organelle-targeted bioactive species imaging. Chem. Sci. 2019, 10,
6035−6071.
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