Near-infrared fluorescent 9-phenylethynylpyronin analogues for bioimaging

Article abstract of DOI:10.1021/jo500140y

The syntheses and biological applications of two novel fluorescent 9-phenylethynylpyronin analogues containing either carbon or silicon at the position 10 are reported. Both fluorescent probes exhibited a relatively strong fluorescence in methanol and pho

Full text of DOI:10.1021/jo500140y

Article
pubs.acs.org/joc
Near-Infrared Fluorescent 9‑Phenylethynylpyronin Analogues for
Bioimaging
†,‡
§
†
,†,‡
Tomas Pastierik,†,‡ Peter Sebej, Jirina Medalova, Peter Stacko, and Petr Klan*
̌
̌
́
̌
̌
́
́
†Department of Chemistry and RECETOX, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic
§Department of Animal Physiology and Immunology, Institute of Experimental Biology, Faculty of Science, Masaryk University,
Kotlarska 2, 611 37 Brno, Czech Republic
‡
S
* Supporting Information
ABSTRACT: The syntheses and biological applications of two novel
fluorescent 9-phenylethynylpyronin analogues containing either
carbon or silicon at the position 10 are reported. Both fluorescent
probes exhibited a relatively strong fluorescence in methanol and
phosphate buffer saline in the near-infrared region (705−738 nm)
upon irradiation of either of their absorption maxima in the blue and
red regions. The compounds showed high selectivity toward
mitochondria in myeloma cells in vivo and allowed their visualization
in a favored tissue-transparent window, which makes them promising
NIR fluorescent tags for applications in bioimaging.
pyronin fluorophores presented in this work, has been
claimed.23
In this report, we describe the synthesis and photophysical
properties of the novel 9-(phenylethynyl) C- and Si-pyronin
NIR fluorophores 1a and 1b (Scheme 1). These molecules are
INTRODUCTION
■
Organic fluorescent dyes are a ubiquitous part of molecular and
biological sciences. They are frequently used as chemical
sensors and biosensors and in bioimaging applications.1−4
Their popularity in biosciences has also attracted substantial
attention from the synthetic community.5−8
Scheme 1. Synthesis of 1a,b
Rhodamines are highly favored fluorophores due to their
advantageous photophysical properties, such as high molar
absorption coefficients and fluorescence quantum yields and
good aqueous photostability. Rhodamine 123 and tetrame-
thylrhodamine ethyl ester (TMRE) are probably the most
popular rhodamine derivatives used in flow cytometry and
fluorescence microscopy at the present time. They are
selectively sequestered in active mitochondria and thus often
used as markers of the mitochondrial membrane potential
(MMP).9−11 Monitoring the mitochondrial function is not the
only application of these dyes as a decrease in the MMP is one
of the earliest markers of apoptosis.12 Rhodamine-based
fluorophores are typically excited by a 488-nm argon laser
and emit in the green or red regions, where they do not
interfere with spectroscopic properties of other stains used to
detect cellular parameters in bioimaging applications. Thus, far-
red or near-infrared (NIR) emitting dyes with selective binding
to various cell structures would open new possibilities in
bioimaging.13,14 The first systems, such as Alexa Fluor 633, are
already commercially available.
shown to permeate the cellular membrane allowing the
visualization of mitochondria in vivo with the fluorescence
bathochromically shifted to the near-infrared region in
comparison to the common xanthene- or rhodamine-based
fluorophores.
RESULTS AND DISCUSSION
■
Synthesis. The two 9-phenylethynyl carbo- and silico-
pyronin analogues 1a and 1b were synthesized by nucleophilic
addition of a freshly prepared (phenylethynyl)lithium solution
in THF at −78 °C to the carbonyl group of 2a and 2b,
respectively, in good yields (61 and 83%, respectively; Scheme
1).
From a great variety of xanthene and pyronin analogues
currently known, only a few of them, such as the corresponding
C9-alkyl,15−18 alkenyl,19 cyano,20,21 or carboxy22 derivatives, do
not have aromatic substituents at the position C9. Recently, a
patent describing preparation of 9-phenylethynylxanthene and
pyronin derivatives, structurally analogous to the C- and Si-
Received: January 20, 2014
Published: March 31, 2014
© 2014 American Chemical Society
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Scheme 2. Synthesis of (a) 2a and (b) 2b
The key intermediates 2a and 2b were prepared by different
pathways. The starting 10,10-dimethylcarboxanthen-9-one (2a)
was synthesized analogous to the procedure reported by Hell
and co-workers24 (Scheme 2a). Reductive amination of 3-
bromoaniline, followed by nucleophilic addition of the resulting
3-bromo-N,N-dimethylaniline (3) to acetone, gave 2-(3-
(dimethylamino)phenyl)propan-2-ol (4), which was dehy-
drated to form the synthetic precursor N,N-dimethyl-3-(prop-
1-en-2-yl)aniline (5). Condensation of the two building blocks,
5 and (4-(dimethylamino)phenyl)methanol, in the presence of
a Lewis acid (BCl3 was found to be the most efficient) and
subsequent cyclization of the adduct under dehydrating
conditions followed by oxidation using KMnO4 led to 2a in
31% yield over the three steps. 10,10-Dimethylsilicoxanthen-9-
one 2b was synthesized by a procedure adopted from Nagano
and co-workers (Scheme 2b).25 The key synthetic intermediate,
4,4′-methylenebis(3-bromo-N,N-dimethylaniline) (6), was pre-
pared by condensation of 3 with formaldehyde in 65% yield.
The subsequent ring-closure reaction, a halogen−metal
exchange reaction via a nucleophilic attack of 2,2′-
methylenebis(aryllithium) to dichlorodimethylsilane, and oxi-
dation of the reaction intermediate by KMnO4 gave 2b in 49%
yield over the two steps. The reaction intermediate 7b is
isolable but relatively unstable; it is oxidized by air oxygen to
give 8b (Scheme 2b) which could not be further oxidized to 2b
by KMnO4. The direct synthesis of 2b provided better yields
(49%) compared to a stepwise method (6 → 7b → 2b) in
which the overall yield was 17%. The C- and Si-9H-pyronin
derivatives 8a, b were also prepared by reduction of 2a,b using
LiAlH4 (Scheme 3) as potential fluorescent impurities (see
further in the text).
Scheme 3. Synthesis of 8a,b from 2a,b
Photophysical Properties. The 9-(phenylethynyl)pyronin
analogues 1a,b exhibited a strong absorption in the visible
region (Figures 1 and 2, Table 1). The spectra of methanolic
max
abs
solutions possessed two characteristic absorption bands at λ
(1a) = 472 and 677 nm and λmabasx (1b) = 490 and 712 nm. The
shape and relative intensities of the major absorption bands
were not affected by the dye concentration (Figures S15 and
S28, Supporting Information). The excitation spectra matched
those of absorption (Figures 1 and 2). In contrast, the relative
intensities of the two bands of 1 in phosphate buffer saline
(PBS; pH = 7.4, I = 0.1 mol dm−3) in the region of 600−720
nm changed in the concentration range of 10−7−10−4 mol
dm−3 (Figures S14, S16, and S17 (1a) and S27, S29, and S30
(1b), Supporting Information). The spectra obtained at the
max
abs
lowest concentrations were dominated by the maxima at λ
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solutions. Such hypsochromically shifted bands suggest that H-
aggregates are formed, which have also been observed in the
case of other pyronin derivatives.27,29
max
The NIR emission bands at λ
= 705 and 738 nm in
em
methanol (and 708 and 736 nm in PBS) for 1a and 1b,
respectively, were obtained upon excitation to both absorption
maxima (Table 1; Figures 1 and 2; Figures S13 and S26,
Supporting Information). The position and shape of the
emission maxima were not affected by the dye concentration in
either solvent. The fluorescence quantum yields were found to
be reasonably high in methanol (Φ = 0.07−0.15) and
approximately 1 order of magnitude lower in PBS (Table 1).
Their values were essentially independent of the excitation
wavelength and are comparable to those of commercially
available rhodamine dyes, such as ATTO 725 (Φ = 0.10)30 or
Alexa Fluor 750 (Φ = 0.10),31 which fluoresce in the NIR
region. The fluorescence decay of 1b in a methanolic solution
obeyed a single exponential rate law (τ1b = 1.0 0.3 ns).
Both absorption and emission maxima of 1a,b are shifted
bathochromically compared to the analogues possessing oxygen
at the position 10 as has been reported for a 9-
Figure 1. Absorption (black solid line), normalized emission (λexc
677 nm; red solid line), and excitation (λem = 708 nm; blue dotted
line) spectra of 1a in methanol.
=
(mesitylethynyl)pyronin derivative23 or other pyronin ana-
logues.25,32,33 The difference in λ
between 1a and 1b is
max
em
analogous to that observed for fluorescein and rhodamine
derivatives, possibly reflecting a different stabilization of the
LUMO energies of the two fluorophores.25,32
The optical properties of 1a,b were also determined in a
suspension of living HL-60 cells in the RPMI-1640 media in
connection with our bioimaging studies described further in the
text. The effect of the cell microenvironment was found to be
negligible: both the emission and excitation spectra were nearly
identical to those obtained in PBS. The fluorescence quantum
yields (∼1%) were also similar; however, this value can only be
estimated due to the complexity and heterogeneity of the
system.
Figure 2. Absorption (black solid line), normalized emission (λexc
712 nm; red solid line), and excitation (λem = 738 nm; blue dotted
line) spectra of 1b in methanol.
=
Stability. The chemical stability of 1a,b in the dark was
found to be reasonable in all solvents used (PBS, DMSO, water,
or methanol; see also the Supporting Information). For
example, only ∼2 and 4% of 1a and 1b, respectively,
decomposed in a PBS solution (c ∼ 10−5 mol dm−3) in 12 h
at 20 °C; degradation of the dyes in aq methanol was faster by a
factor of ∼8. High-resolution mass spectrometric analyses of a
solution of 1b in methanol kept in the dark for several days
indicated that 8b and 2b are the degradation products (Figure
S38, Supporting Information). The stability of 1a,b in frozen
DMSO solutions and in a sample containing HL-60 cells in the
RPMI-1640 media was also tested (see further in the text and
the Supporting Information).
Table 1. Absorption and Emission Properties of 1a,b in
Methanol and PBS
absorbance
λmabasx/nm log ε
fluorescence
Φf (λexc/nm)
a
b
dye
solvent
MeOH
λmemax/nm
1a
472
677
476
680
490
712
492
712
4.33
705
705
710
708
738
738
735
736
0.135 0.013 (472)
0.148 0.014 (625)
0.016 0.007 (484)
0.017 0.002 (633)
0.066 0.011 (490)
0.070 0.008 (657)
0.004 0.002 (493)
0.004 0.002 (670)
4.79
4.22
4.60
4.38
4.91
4.45
4.75
c
,d
e
e
PBS
1b
MeOH
c
,d
f
f
PBS
Both 1a and 1b in methanol (c ∼ 10−5 mol dm−3)
decomposed completely upon irradiation with 32 high-energy
LEDs (λem = 653 nm; see the Experimental Section) within 60
min. The major photoproduct was determined to be 9-
methoxypyronin in both cases based on an HR-MS analysis
(Figure S39, Supporting Information), which was also formed
upon irradiation of 9-(1,3-dithian-2-yl)pyronins.16
Fluorescent Impurities. After cautious purification by
chromatography on silica and reversed-phase silica, both
compounds 1a,b still contained a small amount (<1%) of the
corresponding 9H-pyronins 8a,b detectable only by the
fluorescence spectroscopy in PBS solutions but undetectable
by both UV−vis and NMR. Their identification was based on
their comparison with the authentic compounds synthesized
independently from 2a,b (Scheme 3). Compounds 8a,b
a
b
log [ε/mol dm−3 cm−1]. The fluorescence quantum yield (Φf)
values are the averages of five measurements. The standard deviations
and the excitation wavelengths (λexc) are shown. DMSO (1%, v/v)
c
d
was used as a cosolvent due to low solubilities of 1a,b in water. The
absorption characteristics were measured at c < 5 × 10−6 mol dm−3 to
e
ensure that the monomeric form prevails. A PBS/DMSO (39:1, v/v)
mixture was used as a solvent. A PBS/DMSO (999:1, v/v) mixture
f
was used as a solvent.
max
abs
(1a) = 680 nm and λ (1b) = 712 nm. However, the new
maxima at 607 and 652 nm for 1a and 1b, respectively,
appeared at higher dye concentrations (c > 5 × 10−6 mol
dm−3). This behavior is usually associated with dye aggregation
in polar media26−28 and their lower solubilities in aqueous
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exhibited a strong absorption in the visible region; however, in
contrast to 1a,b, they possessed only one absorption band at
max
λ
= 605 and 636 nm, respectively, in methanol. The far-red
abs
emission bands with λmemax = 620 and 648 nm, respectively, found
in methanol solutions were recorded upon excitation at their
absorption maxima (Figure S33 and S36, Supporting
Information).
Bioimaging. In bioimaging applications, there is a need for
light-emitting probes which possess large molar absorption
coefficients, high quantum yields of emission, preferably in the
tissue-transparent window (700−1000 nm),34 and large Stokes
shifts. They should be sufficiently photostable and resistant to
degradation in biological systems. A large Stokes shift can
prevent self-quenching and measurement errors caused by
excitation and scattered light35 and facilitate multichannel
bioimaging.36 Large Stokes shifts (>100 nm) are common for
inorganic phosphorescent heavy-metal complexes37 but rela-
tively rare for small organic monochromophoric fluorescent
probes, such as BODIPY,38,39 cyanines,40 or UV-absorbing 2,3-
naphthalimide41 derivatives. We found that the relatively small
(but typical for rhodamine fluorophores) Stokes shifts of ∼27
nm observed upon irradiation of 1a,b at the major absorption
bands can be overcome by excitation at the second absorption
Figure 3. Overlay images of mouse skeletal myoblast C2C12 stained
with 4′,6-diamidino-2-phenylindole (DAPI; λexc = 405 nm, λem = 460
max
band at λ ∼480 nm, related probably to the extended π-
abs
conjugation of the pyronin chromophore with the 9-phenyl-
ethynyl group, providing the same emission in the NIR region.
In such a case, the gap between the absorption and emission
maxima is very large (233 and 247 nm for 1a,b, respectively),
comparable to that of large pseudo-Stokes shifts reported for
bichromophoric tandem dyes42,43 and fluorescent proteins.44
This also allows utilization of a common 488 nm argon laser as
a source of the excitation light with concomitant detection in
the near-infrared region. Although the molar absorption
coefficients at the band maxima at ∼480 nm (ε ∼25000
mol−1 dm3 cm−1) are lower compared to those of the major
bands by a factor of 3, we have not registered any detrimental
reduction of the fluorescence signal levels in our bioimaging
experiments described in the subsequent paragraph. In
addition, dual band excitation properties of 1a,b offer an
opportunity to selectively identify these fluorescence probes in
a more complex system and in the presence of different
fluorophores.
nm), localized in the cell nuclei (blue fluorescence) and 1 (a: 1a, λexc
=
488 nm; b: 1a, λexc = 670 nm; c: 1b, λexc = 488 nm; d: 1b, λexc = 670
nm). All images were obtained by confocal fluorescence microscopy
using the same emission filter of 700−800 nm (red fluorescence); the
arrows highlight the perinuclear staining; the scale bar is 50 μm.
Information) and fluorescent microscopy analyses, which
implies that their concentrations did not decline markedly
during the experiments. In addition, we aimed to assess the
intracellular localization of the probes by confocal microscopy
that captures cross-sections of observed cells. Compound 1b
stained almost uniformly the whole cytoplasm and nucleus. On
the other hand, 1a had a staining pattern similar to that of the
JC-1 mitochondrial stain (Figure S40, Supporting Informa-
tion); that is, the dye was concentrated mainly in the
perinuclear space where mitochondria are located. Small foci
created by stained mitochondrial clusters were also observed
(Figure 3). The cytotoxicity of 1a,b was evaluated by the flow
cytometry analysis. The extent of apoptotic cell death,
characterized by changes in cell shape and size, was determined
as a decrease in the FSC (forward-light scatter) signal values
(Figure S45, Supporting Information). Our analysis showed
that both dyes were only slightly cytotoxic.
In order to test our novel fluorescent probes for bioimaging,
staining of live cells was studied using confocal fluorescent
microscopy. The excitation of 1a,b by either of the 488 and 670
nm laser beams led to a strong emission in the NIR region
(Figure 3; individual fluorescence images of 1a, b and DAPI are
provided in Figures S41 and S42, Supporting Information).
These excitation wavelengths were chosen to emphasize the
most important spectroscopic feature of both 1a and b, an
identical fluorescence observed upon excitation either of the
major absorption bands in the visible region. Furthermore,
using a NIR emission channel suppressed unwanted cellular
autofluorescence. These absorption wavelengths allowed us to
avoid accidental excitation of the trace impurities 8a,b (see
above).
Staining of cells with both dyes was rapid; the maximal
fluorescence intensity, determined by time-lapse flow cytometry
analyses, was reached in less than 2 min of incubation at 20 °C
at an optimized dye concentration (∼0.01 mg mL−1; Figures
S43 and S44, Supporting Information). The fluorescence
intensity of 1a,b in the cell samples was stable during all flow
cytometric (Figures S43b, S43d, S44b, and S44d, Supporting
CONCLUSIONS
■
Two novel 9-phenylethynyl C- and Si-pyronin analogues 1a,b
were synthesized and their emission properties studied. Both
compounds exhibited a relative strong fluorescence in the NIR
region when irradiated at either of the two major absorption
bands in the visible region. In addition, the compounds showed
high selectivity toward mitochondria in myeloma cells in vivo
and allowed their visualization in a tissue-transparent window.
Characteristic and favorable photophysical properties of 1a,b
can thus give the researcher freedom to choose the excitation
wavelength available for a bioimaging experiment and make
them suitable for multichannel fluorescence microscopy and
multicolor fluorescence applications. Further applications of 9-
phenylethynyl-pyronin dyes are currently under investigation in
our laboratories.
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glutamine (3.3 mmol L−1) was used for the flow cytometry analyses. A
suspension of cells was stained by 1a,b and 8a,b in concentrations of
0−1 mg mL−1. The medians of fluorescence intensities in all the
spectral regions (λexc = 488 nm: λobs(fl) = 530 30 nm (FL-1), λobs(fl)
EXPERIMENTAL SECTION
■
Materials and Methods. Reagents and solvents of the highest
purity available were used as purchased. Acetone and dichloromethane
were dried by standard procedures, kept over activated 3 Å molecular
sieve (8−12 mesh) under dry N2, and freshly distilled before each
experiment. Tetrahydrofuran was dried with sodium, stored over
sodium benzophenone ketyl, and freshly distilled before each
experiment. Phosphate buffer saline was prepared by a 1:10 dilution
of the stock solution of PBS (prepared by direct weighting of NaCl
(80 g), KCl (2.0 g), Na2HPO4·12H2O (23 g), and KH2PO4 (2.4 g)
into a volumetric flask which was filled with distilled water to 1 L).
The synthetic steps were performed under ambient atmosphere
unless stated otherwise. All glassware was oven-dried prior to use when
water- and/or air-sensitive reagents were used. The Schlenk
techniques were utilized when working with air and/or moisture
sensitive chemicals. All column chromatography purification proce-
dures were performed on columns packed with silica gel 60 (40−63
μm).
= 585 40 nm (FL-2) and λobs(fl) = 670 nm long pass (FL-3); λexc
=
630 nm: λobs(fl) = 675 25 nm (FL-4)) together with the time lapse
measurements (0−3 min) were evaluated on a flow cytometer.
A flow cytometry assessment of live HL-60 cells stained with each
individual compound 1a,b and 8a,b (c = 0.01 mg mL−1) showed that
fluorescence of 8 in vivo has a 5-fold to hundred-fold lower intensity
than that of the analogous pyronin 1 (Figures S43 and S44, Supporting
Information), which ensured that no fluorescence interference from
8a,b (<1% dye content) occurred in the study samples.
The microscopic evaluation and cytotoxicity assessment were
performed on mouse skeletal myoblast C2C12 (ATCC) grown on
coverslips in DMEM media, supplemented with 10% fetal bovine
serum, streptomycin (0.1 mg mL−1), penicillin (100 U mL−1), and L-
glutamine (3.3 mM). The cell staining by 1a,b (0.02 mg mL−1) was
performed for 3 min at 20 °C, and then the coverslips were mounted
in glycerol and the specimens observed by a confocal microscope.
Nuclei were prestained by DAPI (the final concentration was 5 μg
mL−1) and mitochondria by JC-1 (0.125 μg mL−1) for 20 min at 37
°C.
Synthesis of 3,6-Bis(dimethylamino)-10,10-dimethylanthra-
cen-9(10H)-one (2a). 3-Bromo-N,N-dimethylaniline (3). Aqueous
H2SO4 (3 M, 56 mL) was added to a stirred solution of aq
formaldehyde (37%, 20.8 mL, 279 mmol) in tetrahydrofuran (250
mL), and the mixture was cooled to 0 °C and stirred for another 10
min. 3-Bromoaniline (16 g, 93 mmol) was then added dropwise within
10 min, and the reaction mixture was stirred for an additional 10 min.
Solid NaBH4 (14.1 g, 372 mmol) was slowly added portionwise within
30 min while the temperature was maintained at 0 °C. The resulting
mixture was allowed to warm and stirred for 1 h at 20 °C. Saturated aq
NaHCO3 (350 mL) was then added, and the reaction mixture was
extracted with CH2Cl2 (3 × 100 mL). The combined organic layers
were dried over anhydrous MgSO4 and filtered, and solvents were
evaporated under reduced pressure. The crude product was of
sufficient purity for the next step. Yield: 18.5 g (98%). Yellowish liquid.
1H NMR (300 MHz, CDCl3): δ (ppm) 2.98 (s, 6H), 6.68 (dd, 1H, J1
The NMR spectra (Supporting Information) were recorded on 300
and 500 MHz spectrometers in chloroform-d, dimethylsulfoxide-d6,
methanol-d4, and water-d2. The signals in H and 13C NMR spectra
1
were referenced45 to the residual peak of a deuterated solvent. The
deuterated solvents except water-d2 were kept over activated 3 Å
molecular sieve (8−12 mesh) under dry N2. The mass spectra were
recorded on a GC-coupled (a 30 m DB-XLB column) spectrometer in
a positive mode with EI (70 eV) or FAB. A mass spectrometer
equipped with a N2 laser (λ = 337 nm) with output set to ∼3 mW was
used for MALDI analyses; a mass detector was calibrated with red
phosphorus. The UV−vis spectra were obtained with matched 0.1, 1.0,
and 5.0 cm quartz cells. IR spectra were obtained on a FT
spectrometer using ATR or KBr pellets. The exact masses were
obtained using a triple quadrupole electrospray ionization mass
spectrometer in positive or negative modes. Melting points were
obtained using a noncalibrated Kofler’s hot stage melting point
apparatus. The solution pH values were determined using a glass
electrode calibrated with certified buffer solutions at pH = 4, 7, or 10.
Fluorescence was measured on an automated luminescence
spectrometer in 1.0 cm quartz fluorescence cuvettes at 26
1 °C;
the sample concentration was set to keep the absorbance below 0.1 at
λmax; each sample was measured five times, and the corrected spectra
were averaged. In some cases (cell suspensions), the dye concentration
was set to 0.01 mg mL−1 (A(λmax) ∼ 0.5) and the fluorescence was
measured in a front-face mode to eliminate reabsorption of the
emitted light. A front-face mode was also employed to determine the
concentration dependence of the emission spectra. Emission and
excitation spectra are normalized; they were corrected using standard
correction files. Fluorescence quantum yields were determined as the
absolute values in 1.0 cm quartz fluorescence cuvettes at 26 1 °C
using an integration sphere. For each sample, the quantum yield was
measured five times, and the values were averaged. A nanosecond flash
lamp (filled with H2) was used for measurements of the fluorescence
lifetimes. The data obtained were reconvoluted from the correspond-
ing measured decay curves and an instrumental response function.
Thermal Stability. The chemical stability of 1a,b (c ∼1 × 10−5
mol dm−3, A = 1.0 0.2 at λmabasx) in methanol, DMSO, water, and PBS
(pH = 7.4, I = 0.1 mol dm−3) solutions in the dark at 20 °C was tested
using UV−vis spectroscopy.
= 8.7 Hz, J2 = 1.9 Hz), 6.88−6.90 (m, 2H), 7.14 (t, 1H, J = 8.0 Hz)
(Figure S1, Supporting Information). 13C NMR (75.5 MHz, CDCl3):
δ (ppm) 40.5, 111.1, 115.3, 119.3, 123.6, 130.4, 151.9 (Figure S2,
Supporting Information). FTIR (KBr, cm−1): 2985, 2937, 2885, 2804,
1595, 1554, 1498, 1443, 1354, 1230, 1180, 1095, 1066, 983, 957, 829,
783, 758, 681, 660, 440. MS (EI): 202 (7), 201 (90), 200 (100), 199
(95), 198 (98), 185 (10), 155 (10), 118 (30), 104 (20), 91 (10), 77
(20), 63 (10), 50 (1), 42 (12) 27(3). This compound has also been
characterized elsewhere.46
2-(3-(Dimethylamino)phenyl)propan-2-ol (4). n-Butyllithium (2.5
M solution in cyclohexane, 4.00 mL, 10.0 mmol) was added dropwise
to a solution of 3-bromo-N,N-dimethylaniline (3, 2.00 g, 10.0 mmol)
in dry tetrahydrofuran (40 mL) in a Schlenk tube during 15 min at
−78 °C under dry N2 atmosphere. The reaction mixture was stirred
for 15 min, and dry acetone (0.734 mL, 10.0 mmol) was added in two
portions at −78 °C. The stirring continued for another 15 min at this
temperature, and the temperature was subsequently allowed to rise to
20 °C. The reaction was quenched by dropwise addition of aq HCl
(10 mL, 2 M), and the solution was neutralized to pH 7 with satd aq
NaHCO3 (∼ 25 mL). The reaction mixture was then extracted with
dichloromethane (3 × 50 mL). The combined organic layers were
dried over anhydrous MgSO4 and filtered, and the solvents were
removed under reduced pressure. Column chromatography (ethyl
acetate/n-hexane, 1:5, v/v) gave the pure product. Yield: 1.32 g (74%).
Irradiation Experiments. A solution of 1 (c ∼ 10−5 mol dm−3, A
(at λmabasx) = 1.0) in nondried methanol (3 mL) in a matched 1.0 cm
quartz cuvette was irradiated at 20 °C using a homemade light source
equipped with 32 high-energy LEDs emitting at λem = 653 11 nm
(power dissipation of 1 LED: 100 mW; the bandwidth at half-height
=22 nm; its spectrum is shown in Figure S39b, Supporting
Information) until 1 disappeared. The reaction progress was
monitored by UV−vis spectrometry using a diode-array spectrometer,
and the final reaction mixture was further analyzed by HRMS.
Bioimaging Experiments. The leukemic cell line HL-60 (ATCC)
cultivated in RPMI1640 media supplemented with 10% fetal bovine
serum, streptomycin (0.1 mg mL−1), penicillin (100 U mL−1), and L-
Colorless crystals. Mp: 72−74 °C (lit.47 mp 72−73 °C). H NMR
1
(300 MHz, CDCl3): δ (ppm) 1.61 (s, 6H), 1.89 (s, 1H, −OH), 2.99
(s, 6H), 6.66 (ddd, 1H, J1 = 8.3 Hz, J2 = 2.6 Hz, J3 = 0.6 Hz), 6.83
(ddd, 1H, J1 = 7.7 Hz, J2 = 1.6 Hz, J3 = 0.8 Hz), 6.96−6.97 (m, 1H),
7.24 (t, 1H, J = 8.0 Hz) (Figure S3, Supporting Information). 13C
NMR (75.5 MHz, CDCl3): δ (ppm) 31.9, 40.9, 72.9, 109.0, 111.3,
113.1, 129.1, 150.4, 150.9 (Figure S4, Supporting Information). FTIR
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(KBr, cm−1): 3344 (br), 2974, 2935, 2872, 2845, 2804, 1603, 1576,
1493, 1433, 1402, 1375, 1348, 1230, 1182, 1138, 1088, 1061, 995, 955,
881, 862, 793, 775, 702, 679, 565, 484. MS (EI): 179 (83), 178 (12),
164 (16), 162 (13), 148 (3), 134 (4), 122 (100), 120 (28), 107 (18),
91 (8), 81 (4), 77 (12), 63 (7), 43 (22). This compound has also been
characterized elsewhere.48
Powdered potassium permanganate (6.80 g, 43.0 mmol) was added
portionwise to a solution of the above-described crude product (20.5
mmol) in acetone (150 mL) at 0 °C over 2 h. The reaction mixture
was stirred for another 1 h (the reaction progress was monitored by
TLC). When no starting material was observed, the reaction mixture
was filtered through a short pad of silica gel to remove MnO2, and the
pad was thoroughly washed with chloroform (1.4 L). The filtrate was
collected, and the solvents were evaporated to give a green crystalline
powder. Column chromatography (n-hexane/ethyl acetate/chloro-
form, 5:1:1, v/v) gave the pure product. Yield: 1.90 g (31%). Yellowish
N,N-Dimethyl-3-(prop-1-en-2-yl)aniline (5). A suspension of 2-(3-
(dimethylamino)phenyl)propan-2-ol (4, 1.24 g, 6.92 mmol) and
potassium hydrogen sulfate (0.942 g, 6.92 mmol) in xylenes (7 mL)
was stirred in a pressure tube at 150 °C for 1 h. The reaction mixture
was cooled to 20 °C, water (20 mL) was added, and the mixture was
stirred until an inorganic precipitate was dissolved. The pH of the
reaction mixture was adjusted to ∼8 by dropwise addition of aq NaOH
(20%, w/w), and the organic material was extracted with dichloro-
methane (3 × 50 mL). The combined organic layers were dried over
anhydrous MgSO4 and filtered, and the solvents were removed under
reduced pressure to give a brown oil. Column chromatography (ethyl
acetate/n-hexane, 1:50, stepwise changed to 1:5, v/v) gave the pure
1
crystalline solid. Mp: 233−234.5 °C. H NMR (300 MHz, CDCl3): δ
(ppm) 1.73 (s, 6H), 3.10 (s, 12H), 6.73−6.80 (m, 4H), 8.28 (dd, 2H,
J1 = 8.3 Hz, J2 = 0.6 Hz) (Figure S9, Supporting Information). 13C
NMR (75.5 MHz, CDCl3): δ (ppm) 33.9, 38.4, 40.3, 108.0, 111.0,
120.2, 129.3, 152.5, 153.3, 181.3 (Figure S10, Supporting Informa-
tion). FTIR (KBr, cm−1): 3078, 2981, 2968, 2935, 2910, 2808, 1595,
1520, 1444, 1433, 1367, 1333, 1217, 1078, 966, 929, 850, 831, 781,
694, 582, 509. MS (EI): 308 (95), 293 (100), 277 (14), 265 (13), 250
(9), 221 (13), 178 (12), 140 (22), 132 (26), 95 (16), 81 (24), 69 (45),
41 (30). HRMS (APCI+): calcd for C20H25N2O (M + H+) 309.1961,
found 309.1967.
1
product. Yield: 803 mg (72%). Colorless oil. H NMR (300 MHz,
CDCl3): δ (ppm) 2.18 (s, 3H), 2.99 (s, 6H), 5.08 (s, 1H), 5.36 (s,
1H), 6.68−6.74 (m, 1H), 6.84−6.89 (m, 2H), 7.23 (t, 1H, J = 8.2 Hz)
(Figure S5, Supporting Information). 13C NMR (75.5 MHz, CDCl3):
δ (ppm) 22.3, 41.0, 110.4, 112.3, 112.3, 114.7, 129.1, 142.6, 144.6,
150.9 (Figure S6, Supporting Information). FTIR (KBr, cm−1) = 3084,
2970, 2945, 2914, 2885, 2843, 2800, 1599, 1574, 1498, 1433, 1350,
1230, 1182, 1120, 1061, 995, 970, 889, 849, 779, 725, 685, 548, 523,
442. MS (EI): 162 (8), 161 (82), 160 (100), 144 (7), 130 (5), 118
(6), 115 (13), 102 (4), 91 (9), 77 (7), 63 (4), 51 (3), 42 (3). This
compound has also been characterized elsewhere.49,50
N-(7-(Dimethylamino)-9,9-dimethyl-10-(phenylethynyl)-
anthracen-2(9H)-ylidene)-N-methylmethanaminium Chloride (1a).
n-Butyllithium (2.2 M solution in cyclohexane, 1.4 mL, 3.08 mmol)
was added dropwise to a solution of phenylacetylene (0.37 mL, 3.39
mmol) in dry tetrahydrofuran (10 mL) in a Schlenk tube under dry N2
atmosphere at −78 °C. The reaction mixture was stirred for 30 min,
and a solution of 3,6-bis(dimethylamino)-10,10-dimethylanthracen-
9(10H)-one (2a, 190 mg, 0.616 mmol) in dry tetrahydrofuran (10
mL) was subsequently added dropwise. The reaction mixture was
stirred at −78 °C for 1 h and then stirred overnight while the
temperature was allowed to rise to 20 °C. The reaction was quenched
by satd aq NH4Cl (3 mL), and aq HCl (2 M, 0.7 mL) was
subsequently added dropwise to the reaction mixture. After being
stirred for 5 min, the mixture was extracted with dichloromethane (10
mL). Green solid precipitated in a separatory funnel. The organic layer
was carefully separated and discarded. An aqueous layer and the solid
were then washed with dichloromethane (3 × 10 mL) to dissolve the
precipitate. The organic layers were combined and dried over
anhydrous MgSO4. The solvents were removed under reduced
pressure to give the pure title product. Yield 160 mg (61%). Dark
green solid. Mp: 231−233 °C. 1H NMR (300 MHz, CDCl3): δ (ppm)
1.81 (s, 6H), 3.48 (s, 12H), 6.98 (dd, 2H, J1 = 9.4 Hz, J2 = 2.5 Hz),
7.13 (d, 2H, J = 2.5 Hz), 7.48−7.58 (m, 3H), 7.78 (dd, 2H, J1 = 7.7
Hz, J2 = 1.8 Hz), 8.28 (d, 2H, J = 9.4 Hz) (Figure S11, Supporting
Information). 13C NMR (75.5 MHz, CDCl3): δ (ppm) 34.7, 41.47,
41.52, 86.5, 111.0, 112.8, 113.7, 121.1, 121.5, 129.2. 131.4, 132.9,
136.8, 144.0, 155.8, 156.5 (Figure S12, Supporting Information). FTIR
(KBr, cm−1): 2359, 2180, 1577, 1386, 1320, 1262, 1219, 1157, 1054,
952, 906, 844, 772, 690. MS (MALDI): 393 (100), 378 (31), 293
(14). UV−vis (CH3OH), (c = 1.5 × 10−5 mol dm−3): λmax/nm (log ε/
M−1 cm−1) = 445 (4.04), 472 (4.33), 625 (4.34), 677 (4.79) (Figure
S13, Supporting Information). UV−vis (PBS buffer), (c ∼ 1 × 10−5
mol dm−3): λmax/nm (A/a.u.) = 452 (0.66), 475 (0.96), 609 (1.00),
631 (0.94). Fluorescence (CH3OH, A(λmax (excitation)) ≤ 0.1): λmax
(emission) = 705 nm (Figure S13, Supporting Information). HRMS
(APCI+): calcd for C28H29N2 (M+) 393.2325, found 393.2315.
Synthesis of 3,7-Bis(N,N-dimethylamino)-5,5-dimethyldi-
benzo[b,e]silin-10(5H)-one (2b). 4,4′-Methylene-bis(3-bromo-
N,N-dimethylaniline) (6). Aqueous formaldehyde (37%, 3.26 mL,
43.5 mmol) was added dropwise to a solution of 3-bromo-N,N-
dimethylaniline (3, 17.5 g, 87 mmol) in glacial acetic acid (200 mL)
over 15 min. The reaction mixture was then stirred at 90 °C for 1 h.
Acetic acid was evaporated under reduced pressure, and the residual
acid was neutralized with satd aq NaHCO3. The obtained residue was
extracted with dichloromethane (3 × 50 mL). The combined organic
layers were washed with water (2 × 50 mL) and brine (50 mL), dried
over MgSO4, and filtered. The solvents were removed under reduced
pressure, and the resulting red viscous oil was chromatographed (ethyl
acetate/n-hexane, 1:30, v/v) to give the pure product. Yield: 11.7 g
(4-(Dimethylamino)phenyl)methanol. Lithium aluminum hydride
(22.9 g, 0.60 mol) in dry tetrahydrofuran (500 mL) was added
dropwise to a solution of 4-(dimethylamino)benzaldehyde (45 g, 0.30
mol) in dry tetrahydrofuran (60 mL) at 0 °C. The mixture was stirred
for 3 h, and then water (23 mL), aq NaOH (23 mL, 15%, w/w), and
water (70 mL) were added slowly in sequence (caution: hydrogen is
evolving during this procedure). The mixture was stirred for another
30 min at 0 °C and filtered through a thin pad of silica. The filtrate was
extracted with dichloromethane (3 × 50 mL). Mixed organic layers
were dried over anhydrous MgSO4 and filtered, and the solvents were
removed under reduced pressure to give the title product. No further
purification was necessary. Yield: 43.5 g (95%). Yellowish viscous oil,
slowly crystallizing in a freezer. Mp <25 °C (lit.51 mp 22−24 °C). H
1
NMR (300 MHz, CDCl3): δ (ppm) 2.90 (s, 1H, −OH), 2.97 (s, 6H),
4.55 (s, 2H), 6.77 (d, 2H, J = 8.8 Hz), 7.26 (d, 2H, J = 8.8 Hz) (Figure
S7, Supporting Information). 13C NMR (75.5 MHz, CDCl3): δ (ppm)
40.7, 64.9, 112.8, 128.5, 129.3, 150.2 (Figure S8, Supporting
Information). FTIR (neat on a Teflon foil, cm−1): 3379, 2862, 2800,
1612, 1568, 1522, 1477, 1444, 1344, 1230, 1186, 1163, 1130, 1016,
944, 804, 739, 692, 563, 511. MS (EI): 151 (80), 134 (100), 120 (10),
107 (10), 91 (4), 77 (7). This compound has also been characterized
elsewhere.52,53
3,6-Bis(dimethylamino)-10,10-dimethylanthracen-9(10H)-one
(2a). Boron trichloride (1 M solution in dichloromethane, 23.5 mL,
23.5 mmol) was added dropwise to a solution of (4-(dimethylamino)-
phenyl)methanol (3.09 g, 20.5 mmol) and N,N-dimethyl-3-(prop-1-
en-2-yl)aniline (5, 3.30 g, 20.5 mmol) in dry dichloromethane (100
mL) at 0 °C under dry N2 atmosphere over 15 min. The reaction
mixture was stirred overnight and was allowed to warm to 20 °C.
Polyphosphoric acid (≥83% phosphate (as P2O5), 50 g) was then
added to the reaction mixture. The mixture was warmed to 40 °C, and
dichloromethane was allowed to slowly evaporate through a thick
cannula under slow flow of N2. The reaction mixture was then heated
to 130 °C, and the viscous material was stirred for an additional 3 h.
The reaction mixture was allowed to cool to 20 °C, poured onto ice
(0.5 kg) in a beaker, neutralized with cold aq NaOH (20%, w/w), and
extracted with dichloromethane (4 × 100 mL). The combined organic
layers were concentrated under reduced pressure to ∼100 mL and
washed with aq Na2S2O3 (25 mL, 10%). The organic layer was then
separated, and the solvent was removed under reduced pressure to
give a yellow viscous residue (6.80 g).
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(65%). White crystalline solid. Mp: 105−107 °C (lit.54,55 mp 100−105
(∼ 1.2 L). Mixed filtrates were concentrated under reduced pressure.
Column chromatography (n-hexane/chloroform/ethyl acetate, 5:1:1,
v/v) provided the pure product. Yield: 2.31 g (49%). Slightly greenish
1
°C). H NMR (300 MHz, CDCl3): δ (ppm) 2.95 (s, 12H), 4.07 (s,
2H), 6.63 (dd, 2H, J1 = 8.6 Hz, J2 = 2.6 Hz), 6.91 (d, 2H, J = 8.6 Hz),
7.00 (d, 2H, J = 2.6 Hz) (Figure S18, Supporting Information). 13C
NMR (75.5 MHz, CDCl3): δ (ppm) 40.0, 40.6, 112.0, 116.4, 125.8,
127.2, 130.9, 150.2 (Figure S19, Supporting Information). FTIR (KBr,
cm−1): 2896, 2807, 1608, 1543, 1508, 1442, 1360, 1325, 1232, 1174,
1066, 1021, 958, 906, 829, 805, 796, 736, 669, 669. MS (EI): 414 (48),
412 (100), 410 (51), 395 (4), 368 (4), 333 (10), 331 (12), 317 (6),
315 (6), 288 (8), 286 (8), 272 (2), 252 (63), 251 (71), 237 (24), 235
(21), 214 (25), 212 (26), 208 (53), 192 (11), 165 (31), 152 (9), 132
(28), 125 (27), 104 (18), 89 (12). This compound has also been
characterized elsewhere.25
1
solid. Mp: 276.3−278.5 °C. H NMR (300 MHz, CDCl3): δ (ppm)
0.48 (s, 6H, ((CH3)2Si<)), 3.10 (s, 12H), 6.81 (d, 2H, J = 2.8 Hz),
6.85 (dd, 2H, J1 = 8.9 Hz, J2 = 2.8 Hz), 8.41 (d, 2H, J = 8.9 Hz)
(Figure S22, Supporting Information). 13C NMR (75.5 MHz, CDCl3):
δ (ppm) −0.8 ((CH3)2Si<), 40.2, 113.4, 114.5, 129.9, 131.8, 140.7,
151.7, 185.5 (Figure S23, Supporting Information). FTIR (KBr,
cm−1): 1583, 1504, 1364, 1299, 1179, 1150, 1064, 925, 843, 767, 574.
MS (EI): 324 (100), 309 (99), 294 (11), 293 (32), 277 (4), 265 (4),
161 (5), 148 (26), 132 (7), 119 (2). HRMS (APCI+): calcd for
C19H25N2OSi (M + H+) 325.1731, found 325.1732. This compound
has also been characterized elsewhere.25
N3,N3,N7,N7,5,5-Hexamethyl-5,10-dihydrodibenzo[b,e]siline-3,7-
diamine (7b). This compound was prepared from 4,4′-methylenebis-
(3-bromo-N,N-dimethylaniline) (6, 120 mg, 0.29 mmol) according to
a known procedure.32 Column chromatography (ethyl acetate/n-
hexane 1:20 or 1:10, v/v) afforded a relatively pure product (Figures
S20 and S21, Supporting Information). Yield: 64.8 mg (72%). Slightly
N-(7-(Dimethylamino)-5,5-dimethyl-10-(phenylethynyl)dibenzo-
[b,e]silin-3(5H)-ylidene)-N-methylmethanaminium Chloride (1b). n-
Butyllithium (2.5 M solution in hexanes, 0.62 mL, 1.54 mmol) was
added dropwise to a solution of phenylacetylene (0.186 mL, 1.70
mmol) in dry tetrahydrofuran (5 mL) in a Schlenk tube at −78 °C
under dry N2 atmosphere (Scheme 1). The mixture was stirred for 30
min, and a solution of 3,7-bis(N,N-dimethylamino)-5,5-dimethyldi-
benzo[b,e]silin-10(5H)-one (2b, 100 mg, 0.31 mmol) in dry
tetrahydrofuran (10 mL) was added dropwise. The reaction mixture
was stirred at −78 °C for 30 min and then stirred overnight while the
temperature was allowed to rise to 20 °C. The reaction was quenched
by dropwise addition of aq HCl (3 mL, 2 M), and the mixture was
extracted with dichloromethane (3 × 20 mL). The combined organic
extracts were dried over anhydrous MgSO4, and the solvents were
removed under reduced pressure to give an oily crude product.
Column chromatography (methanol/chloroform, 1:20 to 1:8, v/v)
gave the pure crystalline product. Yield: 114 mg (83%). Dark green
microcrystalline powder. Mp: 221−223 °C. 1H NMR (300 MHz,
CD3OD): δ (ppm) 0.56 (s, 6H, ((CH3)2Si<), 3.38 (s, 12H), 7.07 (dd,
2H, J1 = 9.5 Hz, J2 = 2.8 Hz), 7.25 (d, 2H, J = 2.8 Hz), 7.50−7.59 (m,
3H), 7.76−7.81 (m, 2H), 8.52 (d, 2H, J = 9.5 Hz) (Figure S24,
Supporting Information). 13C NMR (75.5 MHz, CD3OD): δ (ppm)
−1.0 ((CH3)2Si<), 41.1, 90.5, 113.2, 116.0, 121.6, 122.7, 129.8, 130.2,
132.4, 133.7, 141.7, 147.2, 149.0, 155.8. (Figure S25, Supporting
Information). FTIR (KBr, cm−1): 2937, 2885, 2179, 1577, 1491, 1379,
1317, 1263, 1219, 1180, 1157, 1057, 955, 908, 845, 835, 806, 773, 752,
688, 646, 524. MS (EI): 409 (6), 368 (35), 353 (5), 324 (7), 309 (12),
255 (15), 236 (26), 152 (17), 135 (21), 123 (19), 111 (38), 97 (67),
83 (71), 57 (100). UV−vis (CH3OH), (c = 1.0 × 10−5 mol dm−3):
λmax/nm (log ε/M−1 cm−1) 462 (4.08), 490 (4.38), 660 (4.57), 712
(4.91) (Figure S26, Supporting Information). UV−vis (PBS buffer), (c
∼ 1 × 10−5 mol dm−3): λmax/nm (rel A/a.u.) = 469 (0.72), 492 (1.00),
646 (0.83), 709 (0.56). Fluorescence (CH3OH, A(λmax (excitation)) ≤
0.1): λmax (emission) = 738 nm (Figure S26, Supporting Information).
HRMS (APCI+): calcd for C27H29N2Si (M+) 409.2095, found
409.2097.
Caution: Addition of a molar excess of n-BuLi provides favorably N-
(10-butyl-7-(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-3(5H)-yli-
dene)-N-methylmethanaminium chloride (9b) as a side product
(HRMS (APCI+): calcd for C23H33N2Si (M+) 365.2408, found
365.2407). We found it inseparable from the title product 1b by
column chromatography on silica (Scheme 4). Therefore, special
attention should be taken to avoid unreacted n-BuLi in the reaction
mixture prior to the ketone addition.
9H-Pyronin Analogues. General Procedure. Lithium aluminum
hydride (69 mg, 1.85 mmol) was added to a solution of the
corresponding xanthenone (2, 0.62 mmol) in dry THF (20 mL) in
one portion, and the reaction mixture was stirred at 25 °C for 12 h
(Scheme 3). Aqueous HCl (3 mL, 2 M) was then added dropwise to
the reaction mixture, the solution was saturated with solid NaCl, and
the mixture was extracted with dichloromethane (6 × 15 mL). The
combined organic extracts were dried over anhydrous MgSO4 and
filtered, and solvents were removed under reduced pressure to give the
product.
1
yellowish crystalline solid. Mp: 120−123 °C. H NMR (300 MHz,
CDCl3): δ (ppm) 0.51 (s, 6H, ((CH3)2Si<), 2.98 (s, 12H), 4.01 (s,
2H), 6.79 (dd, 2H, J1 = 8.4 Hz, J2 = 2.8 Hz), 7.05 (d, 2H, J = 2.8 Hz),
7.24 (d, 2H, J = 8.5 Hz) (Figure S20, Supporting Information). 13C
NMR (75.5 MHz, CDCl3): δ (ppm) −2.56 ((CH3)2Si<), 39.4, 41.4,
114.4, 117.8, 128.7, 129.6, 136.3, 148.9 (Figure S21, Supporting
Information). FTIR (KBr, cm−1): 1583, 1520, 1490, 1299, 1247, 1222,
1175, 814, 771, 669, 650, 579, 527. MS (EI): 310 (100), 309 (80), 295
(36), 293 (18), 266 (77), 254 (19), 253 (16), 251 (10), 210 (8), 208
(8), 154 (20), 147 (16), 139 (14), 126 (13). HRMS (APCI+): calcd
for C19H27N2Si (M + H+) 311.1938, found 311.1945.
Note: This compound is readily oxidized to the corresponding
pyronin 8b under ambient atmosphere (Scheme 2b). This hampers
characterization of a pure compound (especially using 13C NMR).
Therefore, it was used immediately in the next step without any
further purification.
3,7-Bis(N,N-dimethylamino)-5,5-dimethyldibenzo[b,e]silin-
10(5H)-one (2b). Two methods for preparation of this compound
were carried out.
Method A. Inspired by a procedure published by Hell and co-
workers.24 Powdered potassium permanganate (81 mg, 0.51 mmol)
was added portionwise to a solution of N3,N3,N7,N7,5,5-hexamethyl-
5,10-dihydrodibenzo[b,e]siline-3,7-diamine (7b, 57.9 mg, 0.186
mmol) in acetone (30 mL) at 0 °C over 2 h, and the reaction
mixture was stirred for 1 h at 0 °C. The reaction progress was
monitored by TLC. Another portion of powdered potassium
permanganate (81 mg, 0.51 mmol) was then added over 2 h at 0
°C, and the reaction mixture was stirred overnight at 20 °C.
Dichloromethane (20 mL) was then added, the mixture was filtered,
and the solvents were removed under reduced pressure. Column
chromatography (ethyl acetate/n-hexane, 1:5, v/v) gave a pure
product. Yield: 13.9 mg (23%). Beige crystalline solid.
Method B. Adopted according to procedures by Nagano et al.32 and
Hell et al.24 n-Butyllithium (2.5 M solution in cyclohexane, 15.1 mL,
37.8 mmol) was added dropwise within 15 min to a solution of 4,4′-
methylenebis(3-bromo-N,N-dimethylaniline) (6, 6.0 g, 14.6 mmol) in
dry tetrahydrofuran (100 mL) at −78 °C under dry N2 atmosphere.
The reaction mixture was stirred for 15 min. Dichlorodimethylsilane
(2.3 mL, 18.9 mmol) was then added dropwise over a period of 15
min. The reaction mixture was stirred overnight, while the temperature
was allowed to rise to 20 °C. The reaction mixture was quenched by
dropwise addition of aq HCl (30 mL, 2 M) and subsequently
neutralized with satd aq NaHCO3 (100 mL). The mixture was
extracted with CH2Cl2 (3 × 50 mL) and dried over MgSO4, and the
solvents were removed under reduced pressure. The obtained crude
solid mixture was diluted in acetone (100 mL) and cooled to 0 °C, and
potassium permanganate (6.90 g, 43.7 mmol) was added portionwise
over the period of 2 h. The reaction mixture was then allowed to warm
to 20 °C. When no starting material was observed (usually after an
additional 3 h; TLC) manganese(IV) oxide was filtered off through a
pad of silica gel, and the solid was thoroughly washed with chloroform
Note: 9H-Pyronin analogues are very soluble in water but only
slightly soluble in ethyl acetate and other polar water-immiscible
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Notes
Scheme 4. Nucleophilic Addition of n-Butyllithium on 3,7-
Bis(N,N-dimethylamino)-5,5-dimethyldibenzo[b,e]silin-
10(5H)-one (2b)
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Support for this work was provided by the Czech Science
Foundation (13-25775S), the Czech Ministry of Education
(LO1214) (P.K.), Masaryk University (MUNI/A/0975/2009;
J.M.), and the project “Employment of Best Young Scientists
for International Cooperation Empowerment” (CZ.1.07/
2.3.00/30.0037) cofinanced by the European Social Fund and
̌
the state budget of the Czech Republic. (P.S.). We express our
́
́ ̌ ́ ̌
Maier, Miroslava Bittova, and Lubomir Prokes
thanks to Lukas
for their help with the mass spectrometry and NMR. Jan
Krausko and L’ubica Krauskova are acknowledged for their help
with the fluorescence and UV measurements. The Institute for
Biophysics, Academy of Sciences of the Czech Republic, v.v.i., is
acknowledged for access to the confocal fluorescence micro-
solvents. The compounds decompose on silica. Excess reducing agent
is important for obtaining a sufficiently pure product 8.
́
́
N-(7-(Dimethylamino)-9,9-dimethylanthracen-2(9H)-ylidene)-N-
methylmethanaminium Chloride (8a). The compound was prepared
from 3,6-bis(dimethylamino)-10,10-dimethylanthracen-9(10H)-one
(2a) according to a general procedure. Yield: 185 mg (87%). Blue
1
powder with a metal gloss. Mp: 147−150 °C. H NMR (500 MHz,
̌
̌
scope, and Ludmila Sebejova, Tomas Solomek, and Jakob Wirz
are acknowledged for fruitful discussions.
́
́
̌
CD3OD): δ (ppm) 1.72 (s, 6H), 3.37 (s, 12H), 7.00 (dd, 2H, J1 = 9.0
Hz, J2 = 2.4 Hz), 7.19 (d, 2H, J = 2.4 Hz), 7.72 (d, 2H, J = 9.1 Hz),
8.13 (s, 1H) (Figure S31, Supporting Information). 13C NMR (125
MHz, CD3OD): δ (ppm) 34.2, 41.1, 43.3, 112.3, 114.2, 122.2, 140.3,
155.40, 155.44, 158.7. (Figure S32, Supporting Information). FTIR
(neat, cm−1) = 2963, 2923, 2863, 2805, 1594, 1560, 1515, 1461, 1425,
1330, 1285, 1157, 1016, 888, 797, 698, 522, 469. MS (MALDI):
293.25 (85), 278.22 (100), 222.17 (9). UV−vis (CH3OH), (c = 2.3 ×
10−5 mol dm−3): λmax/nm (log ε/mol dm−3 cm−1) = 605 (4.75)
(Figure S33, Supporting Information). UV−vis (PBS buffer), (c ∼ 2.5
× 10−5 mol dm−3): λmax/nm (log ε/M−1 cm−1) = 604 (4.74).
Fluorescence (CH3OH, A(λmax (excitation)) ≤ 0.1): λmax (emission) =
620 nm (Figure S33, Supporting Information). Fluorescence (PBS
buffer, A(λmax (excitation)) ≤ 0.1): λmax (emission) = 621 nm. HRMS
(APCI+): calcd for C20H25N2 (M+) 293.2012, found 293.2008. This
compound has also been characterized elsewhere.56,57
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N-(7-(Dimethylamino)-5,5-dimethyldibenzo[b,e]silin-3(5H)-yli-
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1
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compound has also been characterized elsewhere.58
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ASSOCIATED CONTENT
* Supporting Information
■
S
NMR, UV−vis, and fluorescence data; optimization of the
parameters for in vivo applications; images of cells stained by
the fluorescent dyes. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: klan@sci.muni.cz. Tel: +420-54949-4856. Fax: +420-
54949-2443.
■
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