Synthesis of triphenylamine (TPA) dimers and applications in cell imaging

Article abstract of DOI:10.1016/j.dyepig.2019.108009

A variety of triphenylamine (TPA) are shown to undergo C–C bond formation using quinone-based chloranil/H⁺ reagent as the metal free oxidative system to afford triphenylamine dimers very conveniently. Then, TPA dimers have been further converte

Full text of DOI:10.1016/j.dyepig.2019.108009

Dyes and Pigments xxx (xxxx) xxx
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: http://www.elsevier.com/locate/dyepig
Synthesis of triphenylamine (TPA) dimers and applications in cell imaging
Yang Yuana, Pei Yina, Tao Wanga, Zengming Yanga, Weidong Yina, Shaoxiong Zhanga,
,
,*
Chunxuan Qib **, Ma Hengchanga
a Key Laboratory of Eco-Environment-Related Polymer Materials Ministry of Education, College of Chemistry and Chemical Engineering, Northwest Normal University,
Lanzhou, 730070, China
b Baoji AIE Research Center, College of Chemistry and Chemical Engineering, Baoji University of Arts and Sciences, Baoji, 721013, China
A R T I C L E I N F O
A B S T R A C T
A variety of triphenylamine (TPA) are shown to undergo C–C bond formation using quinone-based chloranil/Hþ
reagent as the metal free oxidative system to afford triphenylamine dimers very conveniently. Then, TPA dimers
have been further converted to the emission color tunable compounds via simple chemical transformations.
Importantly, we established the primary relationship between chemical structures with the optical behaviors
Keywords:
Oxidative C–C coupling
Triphenylamine
Aggregation-induced-emission (AIE)
Cell imaging
from the aspect of emission (λem) wavelength, fluorescence life time (τ) and aggregation-induced-emission (AIE)
Positive charges
property in this work. The further application of these compounds to cell imaging was fully demonstrated.
1. Introduction
findings, we made a clear understanding on the facts that (1) TPA core is
of very high electron density, which is due to the sp3 hybrid nitrogen
Up to now, numerous typical aggregation-induced-emission (AIE)-
active regents [1] have been developed based on several representative
chemical skeletons such as triphenylamine (TPA) [2] tetraphenylethene
(TPE) [3], hexaphenylsilole (HPS) [4], distyrylanthracene (DSA) [5],
boron diiminates (BDI) [6], and tetraphenylpyrazine (TPP) [7]. As a
very hot research topic, scientific researchers have made great efforts on
the establishment of AIE platform to light up the potential applications
in the fields of bio- [8], material chemistry [9], and also medical science
[10]. Needless to say, that the further exploration of AIEgens with novel
chemical structure is still promising but great challenging. Actually, the
mostly obstacle for the real application is the complicated synthetic
process [11], and too expensive material cost for these AIEgens [12].
Therefore, the available starting materials, the reliable synthetic
methods and the easily functional structures are especially expected.
Meanwhile, Tang and other groups have been discussed the AIE mech-
anism from different point of views [13]. It was generally accepted that
when AIEgens are aggregated, this physical process is able to restrict the
free rotation of C–C bonds. Therefore, non-radiative energy release
could be exhibited, and, radiative energy can be liberated as fluorescent
light. Thus, the typical AIE-active molecules are usually not co-planar
but with the twisted and sigma bond-riched chemical structures [14].
In our previous works, a series of AIE-active molecules based on TPA
[2c,15] and TPE [3d,16] have been prepared. According to these
atom, who is able to donate the p electron to the surrounding benzene
rings. Thus, TPA is particularly prone to take the electrophilic substi-
tution place. Not only that, even a very tiny electron-withdrawing group
–
such as C O could light up the emissions due to the formation of
–
electron donating and accepting (D-A) system. Both of properties are
largely facilitated to the modification of TPA core with a very conve-
nient style [17]. (2) As documented that oxidative C–C coupling of
carbazoles and TPAs possessing various substituents has been demon-
strated in the presence of organic oxidants, such as DDQ or CA/Hþ, for
accessing bicarbazole regioisomers and TPA dimers [18]. Referring from
this simple and feasible method, the homo-coupling of TPA derivatives
by organic oxidants is maybe available to form TPA dimers more
extensively. If possible, it could be obtained a series of more AIE-active
TPA compounds with no co-planar but twisted and sigma bond-riched
chemical structures, which is totally meet the requirements of com-
pounds with AIE property.
Thus, in this paper, we firstly modified TPA core with some active
functional groups, such as -Br, CHO, thiophene, leaving at least one
naked benzene ring for the further oxidative coupling reaction. Then,
employing 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) as the
oxidant in the acid solutions, the prepared TPAs could be generated into
the corresponding dimers (2TPA-2Br, 2TPA-2CHO–2Br, 2TPA-2CHO-
2Thi and 2TPA-4CHO) with high selectivity and yields. In the following
* Corresponding author.
** Corresponding author.
E-mail addresses: qichunxuan@163.com (C. Qi), mahczju@hotmail.com (M. Hengchang).
https://doi.org/10.1016/j.dyepig.2019.108009
Received 9 September 2019; Received in revised form 22 October 2019; Accepted 29 October 2019
Available online 4 November 2019
0143-7208/© 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Yang Yuan, Dyes and Pigments, https://doi.org/10.1016/j.dyepig.2019.108009

Y. Yuan et al.
Dyes and Pigments xxx (xxxx) xxx
steps, Suzuki [19] and typical nucleophilic reactions [20] were applied
to decorate these TPA dimers into pyridine and pyridinium derivatives.
What should be importantly noted that the synthesized TPA dimers can
be structurally elaborated further, if need be, to arrive at a gamut of
functional organic materials of interest and various TPA-based products.
Finally, we obtained a variety of fluorescent materials with tunable
emission color, and the further bio-related applications were fully
extended.
2.5. Fixed cell imaging
HeLa cells were fixed with paraformaldehyde in PBS (4%) for
15 min. Then the fixed cells imaging was the same as Live cell imaging.
2.6. Confocal colocalization
For co-staining with Nile Red, Hoechst 33342 and Mito Tracker Red.
Cells were first stained in the desired concentration by following the
previous staining procedure. Cells were then incubated at 37 �C in 5%
CO2 for 30 min and imaged at ambient temperature in the medium. The
imaging of Hoechst 33342 counterstain followed the similar procedure.
2. Experimental section
2.1. Materials
The final concentration of Hoechst 33342 was 1 μg/mL. Prior to imag-
ing, the cells were rinsed by PBS twice after 10 min. The use of Mito
Tracker Red and Nile Red were the same as the usage of Hoechst 33342.
4-bromotriphenylamine (98%), Triphenylamine (98%), 2-thiophe-
nylboric acid (98%), tetrakis (triphenylphosphine)-palladium(0) (Pd
(PPh3)4, 99%), pyridine-4-boronic acid (99%) were purchased from
aladdin. Methane sulfonic acid (MSA), 2,3-dichloro-5,6-dicyano-1,4-
benzoquinone (DDQ), Phosphorous oxychloride, Iodomethane and
1,4-dimethylpyridinium iodide, Nitrogen with a purity of 99.99% were
provided from commercial source. Other reagents, such as N, N-dime-
thylformamide (DMF), dimethyl sulfoxide (DMSO), dichloromethane
(DCM), tetrahydrofuran (THF), methanol, ethanol, and chloroform were
of analytical grade and was purchased from Energy Chemical Company.
PBS buffer, DMEM (Dulbecco’s Modified Eagle Medium) and fetal
bovine serum (FBS) were purchased from Termo Fisher Scientific
(Shanghai, China). Singlet Oxygen Sensor Green (SOSG), Hoechst
33342, Mito Tracker Red, Nile Red were purchased from Termo Fisher
Scientific (Shanghai, China). MTT was purchased from Tiangen Biotech
(Beijing, China). HeLa cells were obtained from cell culture center of
Institute of Basic Medical Sciences, Chinese Academy of Medical Science
(Beijing, China).
2.7. Cytotoxicity study
3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium
bromide
(MTT) assays were performed to evaluate the cytotoxicity of the AIE-
gens. Cells were planted in 96-well plates at a density of about
10,000 cells per well and grown for 24 h. The AIEgens were added at
different concentrations of 0, 2, 4, 8, 16, 32 and 64 μM after replacing
the medium. The volume fraction of DMSO was below 0.2%. After 24 h
of incubation, 10 μL of MTT solution (5 mg/mL in PBS) was added into
each well. After 4 h of incubation, DMSO was added into each well to
dissolve the precipitates formazan. Finally, the absorption of each well
at 570 nm was recorded via a plate reader. Each trial was performed
with 6 wells parallel.
2.8. Synthesis
2.8.1. Synthesis of diTPA-1 (N4,N4’-bis(4-bromophenyl)-N4,N4’-diphenyl-
[1,10-biphenyl]-4,40-diamine, 2TPA-2Br)
2.2. Instrumentation
A solution of 4-bromotriphenylamine (259.37 mg, 324.21 g/mol,
0.8 mmol) in dry DCM (10 mL) was cooled to 0 �C, and methanesulfonic
acid (MSA, 1 mL) was added slowly at this temperature. After the con-
tents were stirred for a few minutes at 0 �C, 2,3-dichloro-5,6-dicyano-
1,4-benzoquinone (DDQ, 370 mg, 227 g/mol, 1.6 mmol) was added.
Immediately, the color of the reaction mixture turned blue-green to blue
depending on the substrate. The progress of the reaction was monitored
by TLC regularly. As soon as the starting material disappeared
completely, as noticed by TLC, the saturated NaHCO3 solution was
carefully added under ice-cold conditions. The whole of the reaction
contents was transferred into a separating funnel, and the organic
portion was extracted into chloroform (3 � 10 mL). The combined
organic layer was washed once again with saturated sodium bicarbonate
solution. The aqueous portion appeared orange to red in color, and the
organic portion remained colorless. The organic layer was then washed
with brine (saturated sodium chloride) solution, dried over anhydrous
Na2SO4, filtered, and dried under vacuum. The crude product was pu-
rified over a silica gel column with the mixture petroleum ether and
ethyl acetate (500: 1 by volume), The pure products isolated was white
solid (0.256 g, yield: 99%), known compound [18a]. Rf ¼ 0.79 (15: 1,
petroleum ether/ethyl acetate). 1H NMR (600 MHz, CDCl3) δ (TMS,
ppm): 7.44 (d, J ¼ 8.6 Hz, 2H), 7.34 (d, J ¼ 8.8 Hz, 2H), 7.29 - 7.25 (m,
2H), 7.10 (dd, J ¼ 8.2, 2.5 Hz, 4H), 7.05 (t, J ¼ 7.4 Hz, 1H), 6.98 (d,
J ¼ 8.8 Hz, 2H) (Fig. S1). 13C NMR (151 MHz, CDCl3) δ (TMS, ppm):
147.20, 146.84, 146.33, 135.11, 132.18, 129.40, 127.45, 125.28,
124.53, 124.28, 123.38, 114.96 (Fig. S2). HRMS (ESI) m/z: [M þ H]þ
calculated for [C36H27Br2N2]þ ¼ 645.0541; found 645.0536 (Fig. S43).
1H NMR and 13C NMR spectra were recorded on MERCURY spec-
trometer at 25 �C, and all NMR spectra were referenced to the solvent.
Mass spectrometric (MS) data were carried out using Thermoscientific Q
Exactive instruments. UV–visible absorption spectra (UV) were recorded
on a TU-1901 spectrometer. Fluorescence spectra and fluorescence
quantum yields were measured using a FluoroSENS-9003 Fluorescence
Spectrophotometer. Fluorescence lifetimes were measured with Hama-
matsu Quantaurus-Tau C1136 at room temperature. Fourier transform
infrared (FT-IR) spectra were recorded on a DIGIL FTS3000 spectro-
photometer using KBr tablets. Confocal laser scanning microscope
(CLSM) characterization was conducted with a confocal laser scanning
biological microscope (LSM 710, Zeiss, Germany). The absorbance for
MTT analysis was recorded on a microplate reader (Thermo Fisher, USA)
at a wavelength of 570 nm. Automated cell counter (Countess II, Invi-
trogen) was employed for cell counting.
2.3. Cell culture
Cell lines were cultured in the DMEM containing 10% FBS and an-
tibiotics (100 units/mL penicillin and 100 mg/mL streptomycin) in a 5%
CO2 humidity incubator at 37 �C.
2.4. Live cell imaging
Cells were grown in a 35 mm Petri dish with a coverslip at 37 �C. The
live cells were incubated with certain dye at certain concentration for
certain time (by adding 10
μ
L of a stock solution in DMSO solution to
2.8.2. Synthesis of diTPA-2 (4,4’-([1,10-biphenyl]-4,40-diylbis((4-
bromophenyl)azanediyl))dibenzaldehyde, 2TPA-2CHO–2Br) [18,25]
4-Bromotriphenylamine (3.24 g, 324.21 g/mol, 10 mmol) was dis-
solved in DMF (15 mL) and placed in a 100 mL flask. Phosphorous
1 mL of cell culture medium). After incubation with AIEgen, the washing
procedure was not conducted. The AIEgen-labelled cells were mounted
and imaged using a laser scanning confocal microscope.
2

Y. Yuan et al.
Dyes and Pigments xxx (xxxx) xxx
oxychloride (15 mL) was added dropwise to it in an ice bath, and the
reaction mixture was stirred for 10 min at 0 �C. Then, the mixture was
refluxed at 80 �C for 4 h under a N2 atmosphere. After this, the reaction
was quenched with cold water (100 mL) and yellow solid was precipi-
tated. The crude product was purified over a silica gel column with the
mixture petroleum ether, dichloromethane and ethyl acetate (500: 20: 1
by volume), obtain 4-((4-bromophenyl)(phenyl)amino)benzaldehyde
(TPA–CHO–Br) as a yellow solid (2.782 g, yield: 79%) known com-
pound. Rf ¼ 0.60 (5: 1, petroleum ether/ethyl acetate). IR (KBr, cmÀ 1):
3053, 2821, 2744, 1685, 1587, 1487, 1323, 1292, 1269, 1222, 1159
(Fig. S41). 1H NMR (600 MHz, CDCl3) δ (TMS, ppm): 9.82 (s, 1H), 7.69
(d, J ¼ 8.8 Hz, 2H), 7.43 (d, J ¼ 8.8 Hz, 2H), 7.34 (dd, J ¼ 8.3, 7.6 Hz,
2H), 7.18 (t, J ¼ 7.4 Hz, 1H), 7.14 (dd, J ¼ 8.4, 0.9 Hz, 2H), 7.03 (dd,
J ¼ 8.7, 6.6 Hz, 4H) (Fig. S3). 13C NMR (151 MHz, CDCl3) δ (TMS, ppm):
190.37, 152.83, 145.82, 145.35, 132.75, 131.30, 129.85, 129.64,
127.39, 126.24, 125.37, 119.90, 117.69 (Fig. S4). Then, A solution of
TPA–CHO–Br (281.78 mg, 352.22 g/mol, 0.8 mmol) in dry DCM (10 mL)
was cooled to 0 �C, and MSA (1 mL) was added slowly at this tempera-
ture. After the contents were stirred for a few minutes at 0 �C, DDQ
(370 mg, 227 g/mol, 1.6 mmol) was added. Immediately, the color of
the reaction mixture turned blue-green to blue depending on the sub-
strate. The progress of the reaction was monitored by TLC regularly. As
soon as the starting material disappeared completely, as noticed by TLC,
the saturated NaHCO3 solution was carefully added under ice-cold
conditions. The whole of the reaction contents was transferred into a
separating funnel, and the organic portion was extracted into chloro-
form (3 � 10 mL). The combined organic layer was washed once again
with saturated sodium bicarbonate solution. The aqueous portion
appeared orange to red in color, and the organic portion remained
colorless. The organic layer was then washed with brine (saturated so-
dium chloride) solution, dried over anhydrous Na2SO4, filtered, and
dried under vacuum. The crude product was purified over a silica gel
column with the mixture petroleum ether, dichloromethane and ethyl
acetate (100: 5: 1 by volume). The pure products isolated was yellow
solid (0.278 g, yield: 99%), unknown compound. Rf ¼ 0.50 (2: 1, pe-
troleum ether/ethyl acetate). IR (KBr, cmÀ 1): 2922, 2825, 2744, 1692,
1593, 1469, 1317, 1273 (Fig. S41). 1H NMR (600 MHz, CDCl3) δ (TMS,
ppm): 9.85 (s, 1H), 7.72 (d, J ¼ 8.7 Hz, 2H), 7.55 (d, J ¼ 8.6 Hz, 2H),
7.46 (d, J ¼ 8.8 Hz, 2H), 7.20 (d, J ¼ 8.5 Hz, 2H), 7.09 (t, J ¼ 8.7 Hz, 4H)
(Fig. S5). 13C NMR (151 MHz, CDCl3) δ (TMS, ppm): 190.33, 152.62,
145.21, 136.83, 132.85, 131.32, 129.97, 128.09, 127.48, 126.15,
120.36, 117.91 (Fig. S6). HRMS (ESI) m/z: [M þ Na] calculated for
[C38H26Br2N2NaO2] ¼ 723.0259; found 723.0253 (Fig. S44).
the reaction was monitored by TLC regularly. As soon as the starting
material disappeared completely, as noticed by TLC, the saturated
NaHCO3 solution was carefully added under ice-cold conditions. The
whole of the reaction contents was transferred into a separating funnel,
and the organic portion was extracted into chloroform (3 � 10 mL). The
combined organic layer was washed once again with saturated sodium
bicarbonate solution. The aqueous portion appeared orange to red in
color, and the organic portion remained colorless. The organic layer was
then washed with brine (saturated sodium chloride) solution, dried over
anhydrous Na2SO4, filtered, and dried under vacuum. The crude residue
was purified by silica gel column chromatography using hexane-ethyl
acetate mixtures as the eluent. The pure products isolated was yellow
solid (0.109 g, yield: 50%), known compound [18]. Rf ¼ 0.50 (5: 1, pe-
troleum ether/ethyl acetate). 1H NMR (400 MHz, CDCl3) δ (TMS, ppm):
9.83 (s, 2H), 7.71 (d, J ¼ 8.4 Hz, 4H), 7.55 (d, J ¼ 8.3 Hz, 4H), 7.37 (t,
J ¼ 7.6 Hz, 4H), 7.22 (t, J ¼ 7.1 Hz, 10H), 7.09 (d, J ¼ 8.3 Hz, 4H)
(Fig. S9). 13C NMR (151 MHz, CDCl3) δ (TMS, ppm): 190.35, 153.13,
146.06, 145.43, 136.65, 131.28, 129.77, 129.41, 127.93, 126.36,
126.17, 125.23, 119.79 (Fig. S10). HRMS (ESI) m/z: [M þ Na] calcu-
lated for [C38H28N2NaO2] ¼ 567.2048; found 567.2043 (Fig. S45).
2.8.4. Synthesis of diTPA-4 (4,4’-([1,10-biphenyl]-4,40-diylbis((4-
(thiophen-2-yl)phenyl)azanediyl))dibenzaldehyde, 2TPA-2CHO-2Thi)
[18,25]
TPA–CHO–Br (352.22 mg, 352.22 g/mol, 1 mmol), 2-thiophe-
nylboric acid (159.94 mg, 127.95 g/mol, 1.25 mmol), tetrakis (triphe-
nylphosphine)-palladium(0) (57.75 mg, 1155 g/mol, 5% equiv.) and
potassium carbonate (138.21 mg, 138.21 g/mol, 1 mmol). Then the
mixture of THF (25 mL) and MeOH (25 mL) were added. And then the
mixture was refluxed at 120 �C for 36 h under a N2 atmosphere. The
reaction mixture was concentrated by rotary evaporation. Then, 4-
(phenyl(4-(thiophen-2-yl)phenyl)amino)benzaldehyde (TPA–CHO–Thi)
was purified by column chromatography on silica gel (200–300 mesh)
with a mixture of petroleum ether and ethyl acetate as an eluent (20: 1
by volume), obtaining a yellow solid (0.284 g, 80% yield). Rf ¼ 0.58 (5:
1, petroleum ether/ethyl acetate). 1H NMR (600 MHz, CDCl3) δ (TMS,
ppm): 9.83 (s, 1H), 7.70 (d, J ¼ 8.7 Hz, 2H), 7.57 (d, J ¼ 8.6 Hz, 2H),
7.36 (dd, J ¼ 8.4, 7.5 Hz, 2H), 7.29 - 7.26 (m, 2H), 7.22 - 7.14 (m, 5H),
7.09 - 7.06 (m, 3H) (Fig. S11). 13C NMR (151 MHz, CDCl3) δ (TMS, ppm):
190.36, 153.02, 145.97, 145.41, 143.60, 131.29, 131.02, 129.79,
129.45, 128.08, 127.10, 126.32, 126.15, 125.25, 124.74, 122.96,
119.84 (Fig. S12). Then, A solution of TPA–CHO–Thi (284.36 mg,
355.45 g/mol, 0.8 mmol) in dry DCM (10 mL) was cooled to 0 �C, and
MSA (1 mL) was added slowly at this temperature. After the contents
were stirred for a few minutes at 0 �C, DDQ (370 mg, 227 g/mol,
1.6 mmol) was added. Immediately, the color of the reaction mixture
turned blue-green to blue depending on the substrate. The progress of
the reaction was monitored by TLC regularly. As soon as the starting
material disappeared completely, as noticed by TLC, the saturated
NaHCO3 solution was carefully added under ice-cold conditions. The
whole of the reaction contents was transferred into a separating funnel,
and the organic portion was extracted into chloroform (3 � 10 mL). The
combined organic layer was washed once again with saturated sodium
bicarbonate solution. The aqueous portion appeared orange to red in
color, and the organic portion remained colorless. The organic layer was
then washed with brine (saturated sodium chloride) solution, dried over
anhydrous Na2SO4, filtered, and dried under vacuum. The crude product
was purified over a silica gel column with the mixture petroleum ether/
ethyl acetate (40:1 by volume), The pure products isolated was yellow
solid (0.261 g, yield: 92%), unknown compound. Rf ¼ 0.65 (2: 1, pe-
troleum ether/ethyl acetate). 1H NMR (600 MHz, CDCl3) δ (TMS, ppm):
9.83 (s, 1H), 7.70 (d, J ¼ 8.7 Hz, 2H), 7.57 (d, J ¼ 8.6 Hz, 2H), 7.36 (dd,
J ¼ 8.4, 7.5 Hz, 2H), 7.29 - 7.26 (m, 2H), 7.22 - 7.14 (m, 5H), 7.09 - 7.06
(m, 3H) (Fig. S13). 13C NMR (151 MHz, CDCl3) δ (TMS, ppm): 190.36,
153.02, 145.97, 145.41, 143.60, 131.29, 131.02, 129.79, 129.45,
128.08, 127.10, 126.32, 126.15, 125.25, 124.74, 122.96, 119.84
2.8.3. Synthesis of diTPA-3 (4,4’-([1,10-biphenyl]-4,40-diylbis
(phenylazanediyl))dibenzaldehyde, 2TPA-2CHO) [18,25]
Triphenylamine (4.91 g, 245.32 g/mol, 20 mmol) was dissolved in
DMF (16 mL) and placed in a 100 mL flask. Phosphorous oxychloride
(16 mL) was added dropwise to it in an ice bath, and the reaction
mixture was stirred for 10 min at 0 �C. Then, the mixture was refluxed at
80 �C for 4 h under a N2 atmosphere. After this, the reaction was
quenched with cold water (100 mL) and yellow solid was precipitated.
The crude product was purified over a silica gel column with the mixture
petroleum ether and ethyl acetate (40: 1 by volume), obtain 4-(diphe-
nylamino)benzaldehyde (TPA-CHO) as a yellow solid (4.318 g, yield:
79%), known compound [25]. Rf ¼ 0.63 (5: 1, petroleum ether/ethyl
acetate). 1H NMR (600 MHz, CDCl3) δ (TMS, ppm): 9.81 (s, 1H), 7.67 (d,
J ¼ 8.8 Hz, 2H), 7.34 (dd, J ¼ 8.5, 7.5 Hz, 4H), 7.20 - 7.14 (m, 6H), 7.01
(d, J ¼ 8.7 Hz, 2H) (Fig. S7). 13C NMR (151 MHz, CDCl3) δ (TMS, ppm):
190.37, 153.33, 146.14, 131.27, 129.70, 129.10, 126.29, 125.08,
119.33 (Fig. S8). Then,
a solution of TPA-CHO (218.66 mg,
273.33 g/mol, 0.8 mmol) in dry DCM (10 mL) was cooled to 0 �C, and
MSA (1 mL) was added slowly at this temperature. After the contents
were stirred for a few minutes at 0 �C, DDQ (370 mg, 227 g/mol,
1.6 mmol) was added. Immediately, the color of the reaction mixture
turned blue-green to blue depending on the substrate. The progress of
3

Y. Yuan et al.
Dyes and Pigments xxx (xxxx) xxx
(Fig. S14). HRMS (ESI) m/z: [M
þ
Na] calculated for
J ¼ 8.8 Hz, 2H) (Fig. S19). 13C NMR (151 MHz, CDCl3) δ (TMS, ppm):
150.12, 148.78, 146.84, 132.19, 129.46, 129.40, 127.66, 127.47,
125.30, 125.09, 124.86, 124.55, 124.36, 124.26, 123.74, 123.40,
123.12, 120.84, 109.99 (Fig. S20). HRMS (ESI) m/z: [M þ H]þ calcu-
lated for [C41H31BrN3]þ ¼ 644.1701; found 644.1704 (Fig. S48). diTPA-
7 was purified by column chromatography on silica gel (200–300 mesh)
with a mixture of petroleum ether and ethyl acetate as an eluent (5: 1 by
volume), obtaining a yellow solid (2.056 g, 80% yield), unknown com-
pound. Rf ¼ 0.50 (10:1, ethyl acetate/methanol). 1H NMR (600 MHz,
CDCl3) δ (TMS, ppm): 8.62 (d, J ¼ 6.0 Hz, 4H), 7.54 (d, J ¼ 8.6 Hz, 4H),
7.50 (d, J ¼ 8.5 Hz, 4H), 7.48 (d, J ¼ 6.1 Hz, 4H), 7.32 (t, J ¼ 7.9 Hz,
4H), 7.19 (d, J ¼ 8.5 Hz, 12H), 7.10 (t, J ¼ 7.4 Hz, 2H) (Fig. S21). 13C
NMR (151 MHz, CDCl3) δ (TMS, ppm): 150.21, 148.73, 147.55, 147.09,
146.27, 135.41, 131.22, 129.47, 127.67, 127.53, 125.10, 124.83),
123.76, 123.17, 120.85 (Fig. S22). HRMS (ESI) m/z: [M þ H]þ calcu-
lated for [C46H35N4]þ ¼ 643.2862; found 643.2856 (Fig. S49).
[C46H33N2NaO2S2] ¼ 731.1803; found 731.1797 (Fig. S46).
2.8.5. Synthesis of diTPA-5 (4,40,400,4000-([1,10-biphenyl]-4,40-diylbis
(azanetriyl))tetrabenzaldehyde, 2TPA-4CHO) [18,25]
Triphenylamine (4.91 g, 245.32 g/mol, 20 mmol) was dissolved in
DMF (16 mL) and placed in a 100 mL flask. Phosphorous oxychloride
(16 mL) was added dropwise to it in an ice bath, and the reaction
mixture was stirred for 10 min at 0 �C. Then, the mixture was refluxed at
80 �C for 10 h under a N2 atmosphere. After this, the reaction was
quenched with cold water (100 mL) and yellow solid was precipitated.
The crude product was purified over a silica gel column with the mixture
petroleum ether and ethyl acetate (40: 1 by volume), as an eluent to
obtain 4,40-(phenylazanediyl)dibenzaldehyde (TPA-2CHO) as a yellow
solid (4.761 g, yield: 79%), known compound [25]. Rf ¼ 0.38 (5:1, pe-
troleum ether/ethyl acetate). IR (KBr, cmÀ 1): 3062, 3036, 2803, 2732,
1691, 1585, 1493, 1329, 1282, 1275, 1216, 1165 (Fig. S42). 1H NMR
(600 MHz, CDCl3) δ (TMS, ppm): 9.88 (s, 2H), 7.77 (d, J ¼ 8.7 Hz, 4H),
7.39 (t, J ¼ 7.9 Hz, 2H), 7.25 (t, J ¼ 7.4 Hz, 1H), 7.17 (dd, J ¼ 7.9,
5.9 Hz, 6H) (Fig. S15). 13C NMR (151 MHz, CDCl3) δ (TMS, ppm):
190.46, 151.98, 145.48, 131.27, 130.12, 127.03, 126.23, 122.74
(Fig. S16). Then, a solution of TPA-2CHO (241.07 mg, 301.34 g/mol,
0.8 mmol) in dry DCM (10 mL) was cooled to 0 �C, and MSA (1 mL) was
added slowly at this temperature. After the contents were stirred for a
few minutes at 0 �C, DDQ (370 mg, 227 g/mol, 1.6 mmol) was added.
Immediately, the color of the reaction mixture turned blue-green to blue
depending on the substrate. The progress of the reaction was monitored
by TLC regularly. As soon as the starting material disappeared
completely, as noticed by TLC, the saturated NaHCO3 solution was
carefully added under ice-cold conditions. The whole of the reaction
contents was transferred into a separating funnel, and the organic
portion was extracted into chloroform (3 � 10 mL). The combined
organic layer was washed once again with saturated sodium bicarbonate
solution. The aqueous portion appeared orange to red in color, and the
organic portion remained colorless. The organic layer was then washed
with brine (saturated sodium chloride) solution, dried over anhydrous
Na2SO4, filtered, and dried under vacuum. The crude product was pu-
rified over a silica gel column with the mixture petroleum ether and
ethyl acetate (10: 1 by volume) The pure products isolated was yellow
solid (0.235 g, yield: 98%), unknown compound. Rf ¼ 0.78 (1: 2, pe-
troleum ether/ethyl acetate). IR (KBr, cmÀ 1): 3036, 2808, 2724, 1694,
1589, 1493, 1321, 1276, 1215, 1162 (Fig. S42). 1H NMR (400 MHz,
CDCl3) δ (TMS, ppm): 9.92 (s, 4H), 7.84 - 7.79 (m, 8H), 7.62 (d,
J ¼ 8.6 Hz, 4H), 7.27 (d, J ¼ 3.2 Hz, 6H), 7.25 (d, J ¼ 3.6 Hz, 6H)
(Fig. S17). 13C NMR (151 MHz, CDCl3) δ (TMS, ppm): 190.39, 151.82,
144.97, 137.53, 131.54, 131.33, 128.43, 127.03, 123.03 (Fig. S18).
HRMS (ESI) m/z: [M þ Na] calculated for [C40H28NaN2O4] ¼ 623.1947;
found 623.1941 (Fig. S47).
2.8.7. Synthesis of diTPA-8 (4-((4-bromophenyl)(4’-((4-formylphenyl)
(4-(pyridin-4-yl)phenyl)amino)-[1,10-biphenyl]-4-yl)amino)benzaldehyde,
2TPA-2CHO–Br-Py) and diTPA-9 (4,4’-([1,10-biphenyl]-4,40-diylbis((4-
(pyridin-4-yl)phenyl)azanediyl))dibenzaldehyde, 2TPA-2CHO-2Py) [19]
diTPA-2 (2.81 g, 702.43 g/mol, 4 mmol), pyridine-4-boronic acid
(1.23 g, 122.92 g/mol, 10 mmol), tetrakis (triphenylphosphine)-palla-
dium(0) (231 mg, 1155 g/mol, 5% equiv.) and potassium carbonate
(552.84 mg, 138.21 g/mol, 4 mmol). Then the mixture of THF (25 mL)
and MeOH (25 mL) were added. And then the mixture was refluxed at
120 �C for 36 h under a N2 atmosphere. The reaction mixture was
concentrated by rotary evaporation. Then, diTPA-8 was purified by
column chromatography on silica gel (200–300 mesh) with a mixture of
petroleum ether and ethyl acetate as an eluent (20: 1 by volume),
obtaining an orange solid (2.662 g, 95% yield), unknown compound. IR
(KBr, cmÀ 1): 3033, 2852, 2732, 1690, 1592, 1489, 1320, 1273, 1219,
1163 (Fig. S41). Rf ¼ 0.37 (1:2, petroleum ether/ethyl acetate). 1H NMR
(400 MHz, CDCl3) δ (TMS, ppm): 9.89 - 9.82 (m, 2H), 8.67 (d, J ¼ 5.7 Hz,
2H), 7.74 (dd, J ¼ 11.9, 8.7 Hz, 4H), 7.63 (d, J ¼ 8.4 Hz, 2H), 7.60 - 7.55
(m, 4H), 7.49 (dd, J ¼ 16.3, 7.4 Hz, 4H), 7.32 - 7.27 (m, 3H), 7.19 (dd,
J ¼ 14.9, 8.5 Hz, 5H), 7.13 - 7.05 (m, 4H) (Fig. S23). 13C NMR (151 MHz,
CDCl3) δ (TMS, ppm): 190.34, 150.32, 147.12, 145.89, 144.69, 132.85,
131.32, 129.66, 128.21, 128.12, 128.10, 127.48, 127.24, 126.89,
126.44, 126.22, 126.15, 125.82, 121.11, 120.37 (Fig. S24). HRMS (ESI)
m/z: [M þ H]þ calculated for [C43H31BrN3O2]þ ¼ 700.1600; found
700.1583 (Fig. S50). diTPA-9 was purified by column chromatography
on silica gel (200–300 mesh) with a mixture of petroleum ether and
ethyl acetate as an eluent (2: 1 by volume), obtaining an orange solid
(2.655 g, 95% yield), unknown compound. IR (KBr, cmÀ 1): 3033, 2832,
2726, 1690, 1591, 1490, 1322, 1288, 1275, 1221, 1163 (Fig. S41).
Rf ¼ 0.63 (10: 1, ethyl acetate/methanol). 1H NMR (600 MHz, CDCl3) δ
(TMS, ppm):9.87 (s, 1H), 8.67 (d, J ¼ 5.8 Hz, 2H), 7.76 (d, J ¼ 8.6 Hz,
2H), 7.63 (d, J ¼ 8.5 Hz, 2H), 7.60 (d, J ¼ 8.5 Hz, 2H), 7.51 (d,
J ¼ 5.9 Hz, 2H), 7.30 (d, J ¼ 8.5 Hz, 2H), 7.27 (d, J ¼ 8.5 Hz, 2H), 7.18
(d, J ¼ 8.6 Hz, 2H) (Fig. S25). 13C NMR (151 MHz, CDCl3) δ (TMS,
ppm):190.37, 152.63, 150.34, 150.29, 145.29, 131.33, 128.22, 128.14,
126.45, 125.83, 121.12, 120.91, 113.71, 109.98, 109.96, 106.10
2.8.6. Synthesis of diTPA-6 (N4-(4-bromophenyl)-N4,N4’-diphenyl-N4’-(4-
(pyridin-4-yl)phenyl)-[1,10-biphenyl]-4,40-diamine, 2TPA-Br-Py) and
diTPA-7 (N4,N4’-diphenyl-N4,N4’-bis(4-(pyridin-4-yl)phenyl)-[1,10-
biphenyl]-4,40-diamine, 2TPA-2Py) [19]
diTPA-1 (5.17 g, 646.41 g/mol, 8 mmol), pyridine-4-boronic acid
(2.46 g, 122.92 g/mol, 20 mmol), tetrakis (triphenylphosphine)-palla-
dium(0) (0.462 g, 1155 g/mol, 5% equiv.) and potassium carbonate
(1.105 g, 138.21 g/mol, 8 mmol). Then the mixture of THF (50 mL) and
MeOH (50 mL) were added. And then the mixture was refluxed at 120 �C
for 36 h under a N2 atmosphere. The reaction mixture was concentrated
by rotary evaporation. Then, diTPA-6 was purified by column chroma-
tography on silica gel (200–300 mesh) with a mixture of petroleum ether
and ethyl acetate as an eluent (20: 1 by volume), obtaining a yellow solid
(1.993 g, 75% yield), unknown compound. Rf ¼ 0.50 (5: 1, petroleum
ether/ethyl acetate). 1H NMR (400 MHz, CDCl3) δ (TMS, ppm): 8.62 (d,
J ¼ 5.8 Hz, 2H), 7.54 (d, J ¼ 8.6 Hz, 2H), 7.50 - 7.45 (m, 6H), 7.38 - 7.27
(m, 6H), 7.19 (d, J ¼ 7.4 Hz, 6H), 7.15 - 7.08 (m, 6H), 6.99 (d,
(Fig. S26). HRMS (ESI) m/z: [M
þ
H]þ calculated for
[C48H35N4O2]þ ¼ 699.2760; found 699.2755 (Fig. S51).
2.8.8. Synthesis of diTPA-10 (4-(4-((4’-((4-bromophenyl)(phenyl)
amino)-[1,10-biphenyl]-4-yl)(phenyl)amino)phenyl)-1-methylpyridin-1-ium
iodide, 2TPA-2Br-1)
diTPA-6 (644.64 mg, 644.64 g/mol, 1 mmol), Iodomethane
(425.85 mg, 141.94 g/mol, 3 mmol) and THF (20 mL). Then, the
mixture was stirred at 90 �C for 36 h. After that, the reaction mixture was
precipitated from THF (dry), and washed using THF and n-hexane
several times and dried to give the iodide salt of the product as an orange
solid (0.653 g, 85% yield), unknown compound. 1H NMR (600 MHz,
CDCl3) δ (TMS, ppm): 9.03 (d, J ¼ 6.7 Hz, 1H), 8.07 (d, J ¼ 6.8 Hz, 1H),
4

Y. Yuan et al.
Dyes and Pigments xxx (xxxx) xxx
7.67 (d, J ¼ 8.9 Hz, 1H), 7.53 (d, J ¼ 8.5 Hz, 1H), 7.46 (d, J ¼ 8.5 Hz,
1H), 7.40 - 7.31 (m, 2H), 7.29 - 7.26 (m, 1H), 7.20 (d, J ¼ 7.4 Hz, 2H),
7.13 - 7.09 (m, 3H), 7.09 - 7.01 (m, 1H), 6.98 (d, J ¼ 8.8 Hz, 1H)
(Fig. S27). 13C NMR (151 MHz, CDCl3) δ (TMS, ppm): 155.03, 152.17,
145.67, 144.68, 132.23, 129.87, 129.44, 129.07, 127.87, 127.63,
126.32, 126.27, 125.43, 124.66, 124.06, 123.71, 123.54, 122.65,
120.53, 48.07 (Fig. S28).
and the solution was refluxed for 30 h. Upon cooling to room tempera-
ture, the red precipitated solid was filtered, washed with cold ethanol,
and dried to give the iodide salt of the product as a red solid (0.939 g,
yield: 75%), unknown compound. 1H NMR (600 MHz, DMSO) δ (TMS,
ppm): 8.87 (d, J ¼ 5.1 Hz, 2H), 8.23 (d, J ¼ 5.2 Hz, 2H), 8.04 (d,
J ¼ 16.0 Hz, 1H), 7.73 (d, J ¼ 6.3 Hz, 4H), 7.51 - 7.46 (m, 2H), 7.41 (d,
J ¼ 16.1 Hz, 2H), 7.21 (dd, J ¼ 26.5, 7.8 Hz, 4H), 7.11 (d, J ¼ 8.1 Hz,
2H), 4.30 (s, 3H) (Fig. S35).13C NMR (151 MHz, DMSO) δ (TMS, ppm):
161.38, 157.94, 154.11, 151.04, 149.50, 146.93, 146.23, 145.37,
135.15, 134.90, 133.88, 130.80, 130.42, 130.08, 128.13, 126.86,
109.99, 51.91 (Fig. S36).
2.8.9. Synthesis of diTPA-11 (4,4’-(([1,10-biphenyl]-4,40-diylbis
(phenylazanediyl))bis(4,1-phenylene))bis(1-methylpyridin-1-ium) iodide,
2TPA-2Br-2)
diTPA-7 (642.79 mg, 642.79 g/mol, 1 mmol), Iodomethane
(567.76 mg, 141.94 g/mol, 4 mmol) and THF (20 mL). Then, the
mixture was stirred at 90 �C for 48 h. After that, the reaction mixture was
precipitated from THF (dry), and washed using THF and n-hexane
several times and dried to give the iodide salt of the product as an orange
solid (0.788 g, 85% yield), unknown compound. 1H NMR (400 MHz,
DMSO) δ (TMS, ppm): 8.88 (d, J ¼ 6.6 Hz, 4H), 8.38 (d, J ¼ 6.7 Hz, 4H),
8.04 (d, J ¼ 8.7 Hz, 4H), 7.73 (d, J ¼ 8.4 Hz, 4H), 7.46 (t, J ¼ 7.7 Hz,
4H), 7.28 - 7.21 (m, 10H), 7.06 (d, J ¼ 8.7 Hz, 4H) (Fig. S29). 13C NMR
(151 MHz, DMSO) δ (TMS, ppm): 153.70, 151.32, 146.01, 145.44,
136.06, 130.54, 129.92, 128.28, 126.58, 126.26, 125.89, 125.27,
122.74, 120.69, 55.29 (Fig. S30).
2.8.13. Synthesis of diTPA-15 (4,4’-((1E,10E)-(([1,10-biphenyl]-4,40-
diylbis((4-(thiophen-2-yl)phenyl)azanediyl))bis(4,1-phenylene))bis
(ethene-2,1-diyl))bis(1-methylpyridin-1-ium) iodide, 2TPA-2VPyr-2Thi)
[20]
diTPA-4 (907.38 mg, 708.89 g/mol, 1.28 mmol) and 1,4-dimethyl-
pyridinium iodide (272.68 mg, 235.07 g/mol, 1.16 mmol) were dis-
solved in dry ethanol (15 mL). A single drop of piperidine was added,
and the solution was refluxed for 30 h. Upon cooling to room tempera-
ture, the red precipitated solid was filtered, washed with cold ethanol,
and dried to give the iodide salt of the product as a red solid (1.17 g,
yield: 80%), unknown compound. 1H NMR (600 MHz, DMSO) δ (TMS,
ppm): 8.89 (d, J ¼ 7.5 Hz, 2H), 8.80 (d, J ¼ 3.8 Hz, 2H), 8.14 (d,
J ¼ 3.9 Hz, 2H), 7.96 (d, J ¼ 16.5 Hz, 1H), 7.64 (d, J ¼ 6.9 Hz, 4H), 7.38
(d, J ¼ 6.2 Hz, 2H), 7.32 (d, J ¼ 16.4 Hz, 1H), 7.17 (s, 1H), 7.14 (d,
2.8.10. Synthesis of diTPA-12 (4-(4-((4’-((4-bromophenyl)(4-
formylphenyl)amino)-[1,10-biphenyl]-4-yl)(4-formylphenyl)amino)
phenyl)-1-methylpyridin-1-ium iodide, 2TPA-2CHO–2Br-1)
J ¼ 6.5 Hz, 4H), 6.99 (d, J ¼ 7.1 Hz, 2H), 4.21 (s, 3H) (Fig. S37).13
C
diTPA-8 (700.62 mg, 700.62 g/mol, 1 mmol), Iodomethane
(425.85 mg, 141.94 g/mol, 3 mmol) and THF (20 mL). Then, the
mixture was stirred at 90 �C for 36 h. After that, the reaction mixture was
precipitated from THF (dry), and washed using THF and n-hexane
several times and dried to give the iodide salt of the product as an orange
solid (0.716 g, 85% yield), unknown compound. IR (KBr, cmÀ 1): 3033,
1689, 1641, 1590, 1491, 1323, 1286, 1220, 1169 (Fig. S41).1H NMR
(400 MHz, CDCl3) δ (TMS, ppm): 9.94 - 9.82 (m, 2H), 9.10 (d, J ¼ 6.3 Hz,
2H), 8.17 (d, J ¼ 5.7 Hz, 2H), 7.85 - 7.70 (m, 6H), 7.63 - 7.54 (m, 4H),
7.49 - 7.45 (m, 2H), 7.30 (d, J ¼ 8.5 Hz, 4H), 7.22 (dd, J ¼ 10.4, 1.1 Hz,
5H), 7.12 - 7.07 (m, 3H), 4.61 (s, 3H) (Fig. S31).13C NMR (151 MHz,
CDCl3) δ (TMS, ppm): 190.44, 145.20, 145.10, 132.87, 131.66, 131.39,
131.35, 129.82, 129.35, 128.46, 128.20, 127.51, 127.04, 126.11,
123.78, 123.74, 123.64, 123.11, 123.09, 120.44, 48.35 (Fig. S32).
NMR (151 MHz, DMSO) δ (TMS, ppm): 187.21, 183.68, 169.21, 166.90,
165.97, 165.41, 153.22, 146.52, 145.84, 145.26, 140.87, 135.41,
130.37, 130.15, 128.04, 126.02, 125.54, 123.39, 121.71, 121.16,
109.99, 43.93 (Fig. S38).
2.8.14. Synthesis of diTPA-16 (4,40,400,4000-((1E,10E,100E,1000E)-(([1,10-
biphenyl]-4,40-diylbis(azanetriyl))tetrakis(benzene-4,1-diyl))tetrakis
(ethene-2,1-diyl))tetrakis(1-methylpyridin-1-ium) iodide, 2TPA-4Vpyr)
[20]
diTPA-5 (768.84 mg, 600.66 g/mol, 1.28 mmol) and 1,4-dimethyl-
pyridinium iodide (272.68 mg, 235.07 g/mol, 1.16 mmol) were dis-
solved in dry ethanol (15 mL). A single drop of piperidine was added,
and the solution was refluxed for 60 h. Upon cooling to room tempera-
ture, the red precipitated solid was filtered, washed with cold ethanol,
and dried to give the iodide salt of the product as a red solid (1.128 g,
yield: 60%), unknown compound. IR (KBr, cmÀ 1): 3033, 1641, 1618,
1586, 1508, 1423, 1321, 1175 (Fig. S42).1H NMR (600 MHz, CD3OD) δ
(TMS, ppm): 8.67 (d, J ¼ 6.6 Hz, 8H), 8.12 (d, J ¼ 7.0 Hz, 8H), 7.90 (s,
8H), 7.74 - 7.71 (m, 8H), 7.35 (d, J ¼ 16.1 Hz, 4H), 7.27 (d, J ¼ 8.5 Hz,
4H), 7.20 (d, J ¼ 8.7 Hz, 8H), 4.30 (s, 12H) (Fig. S39).13C NMR
(151 MHz, DMSO) δ (TMS, ppm): 153.06, 146.13, 145.37, 130.43,
130.23, 129.89, 129.85, 126.47, 125.66, 123.89, 123.59, 122.13,
120.11, 47.24 (Fig. S40).
2.8.11. Synthesis of diTPA-13 (4’-(([1,10-biphenyl]-4,40-diylbis((4-
formylphenyl)azanediyl))bis(4,1-phenylene))bis(1-methylpyridin-1-ium)
iodide, 2TPA-2CHO–2Br-2)
diTPA-9 (698.81 mg, 698.81 g/mol, 1 mmol), Iodomethane
(425.85 mg, 141.94 g/mol, 3 mmol) and THF (20 mL). Then, the
mixture was stirred at 90 �C for 36 h. After that, the reaction mixture was
precipitated from THF (dry), and washed using THF and n-hexane
several times. An orange solid powder was obtained by filtration, and
dried to give the iodide salt of the product (0.835 g, 85% yield), un-
known compound. IR (KBr, cmÀ 1): 3033, 1686, 1641, 1589, 1493, 1327,
1283, 1220, 1184, 1166 (Fig. S41).1H NMR (400 MHz, DMSO) δ (TMS,
ppm): 9.90 (s, 2H), 8.95 (d, J ¼ 6.7 Hz, 4H), 8.46 (d, J ¼ 6.8 Hz, 4H),
8.12 (d, J ¼ 8.7 Hz, 4H), 7.88 (d, J ¼ 8.6 Hz, 4H), 7.81 (d, J ¼ 8.5 Hz,
4H), 7.32 (d, J ¼ 8.5 Hz, 8H), 7.22 (d, J ¼ 8.5 Hz, 4H), 4.31 (s, 6H)
(Fig. S33).13C NMR (151 MHz, DMSO) δ (TMS, ppm): 191.57, 153.59,
151.87, 149.89, 145.80, 144.99, 136.98, 131.81, 131.35, 130.19,
128.71, 128.44, 127.36, 124.60, 123.54, 122.56, 55.34 (Fig. S34).
3. Results and discussion
3.1. Synthesis of TPA dimers by oxidizing homo-coupling reaction [18]
Very recently, Venkatakrishnan’ group [18a] reported a very effi-
cient method to prepare bicarbazoles by oxidizing homo-coupling re-
actions via DDQ&CH3SO3H. It was demonstrated that metal-free process
being of the effective advantages over the traditional methods, such as
versatile starting materials’ availability, mild reaction conditions (room
temperate in DCM), high yields (up to 99%) and unique regioselectivity
(3-30 coupling) even within very short reaction period. Inspired from this
research finding, we readily applied this methodology to the formation
of TPA dimers or diTPAs due to the electron-rich property, which was
almost the same reason shared by carbazole compounds. Therefore, we
conveniently obtained TPA dimers as follows: diTPA-1, diTPA-2,
2.8.12. Synthesis of diTPA-14 (4,4’-((1E,10E)-(([1,10-biphenyl]-4,40-
diylbis(phenylazanediyl))bis(4,1-phenylene))bis(ethene-2,1-diyl))bis(1-
methylpyridin-1-ium) iodide, 2TPA-2Vpyr) [20]
diTPA-3 (679.14 mg, 544.64 g/mol, 1.28 mmol) and 1,4-dimethyl-
pyridinium iodide (272.68 mg, 235.07 g/mol, 1.16 mmol) were dis-
solved in dry ethanol (15 mL). A single drop of piperidine was added,
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Dyes and Pigments xxx (xxxx) xxx
diTPA-3, diTPA-4 and diTPA-5 (Scheme 1). What we noted that this
method is even more reliable to produce TPA dimers than bicarbazoles.
Four of them have been yielded almost quantitatively and even with
several grams’ scale. The other way, for the purpose of decorating TPA
dimers further, bromo- and formyl groups substituted TPA dimers were
preferably synthesized in our works.
cell staining fluorescent materials, the positive charges were especially
indispensable for organic dyes, which was involved in the cell trans-
membrane process and the electrostatic interaction toward to the
negative bio-molecules, such as DNA, RNA and ATP. Thus, the diverse
chemical structures with different amount of positive charges have been
realized by only single step.
3.2. Synthesis of pyridine-substituted TPA dimers by Suzuki cross-
coupling reaction [19]
3.4. Synthesis of vinylpyridinium-substituted TPA dimers by nucleophilic
addition reaction [20]
Undoubtedly, due to the presence of very active bromine substituent
in the compounds of diTPA-1 and diTPA-2, a comprehensive organic
reaction could be applied to synthesize a variety of chemicals with
different purposes. Thereof, in our present works, we employed 4-pyri-
dine phenylboric acid as the cross-coupling partners to result in the
pyridine decorated products by Suzuki reaction (Scheme 2). This was
mainly because of that the electron D-A system formation within mol-
ecules’ frameworks is a more powerful method to produce fluorescent
materials with desirable optical behaviors, such as longer emission
wavelength, and higher fluorescence quantum yield as well. Thus, four
compounds (diTPA-6–9) with pyridine as the electron accepting groups
were achieved. And also, the different electron D-A patterns were suc-
cessfully constructed, including of A-D-D, A-D-D-A, A-A-D-D-A, and A-A-
D-D-A-A (A: pyridyl, formyl group, D: the entire triphenylamine moiety)
systems, which were benefited to make a clear understanding on the
primary relationships between chemical structures with optical
performances.
It was well knowed that the electron D-π-A system formation within
molecules’ framework is much expected, which could greatly shift the
emission wavelength into more red region. Therefore, due to the com-
pounds diTPA-3, diTPA-4, and diTPA-5 in hand, by using 4-methyl
pyridine salt as the nucleophilic reagent, and fluxing it with diTPA-3
diTPA-4, and diTPA-5 respectively, we successfully obtained the prod-
ucts of diTPA-14, 15 and diTPA-16 with good yields and also perfect
purities. It was displayed from Scheme 4 that both of products are
constructed by A-π-D-π-A patterns and equipped with two and four
positive charges respectively. In this work, the different positive charges
guided transmembrane process and targeted cell imaging are key
research purposes in our present study.
Thereof, starting from the synthetic steps of oxidizing homo-coupling
reactions, then Suzuki cross-coupling reactions, to Menshutkin reactions
and nucleophilic addition reactions, a small TPA dimers’ library with
various functional groups were obtained. And, we established a platform
to study the primary relationship between chemical structures with the
corresponding optical behaviors, which was definitely different from the
reported works.
3.3. Synthesis of pyridinium-substituted TPA dimers by Menshutkin
reaction
3.5. The fluorescence performances of all diTPA derivatives in solid state
and in organic solvents
Based on our previous research works, we were conscious of that
pyridinium could be obtained very easily by Menshutkin reaction. Not
only that, upon forming those cationic compounds, the emission wave-
length and fluorescence quantum yield have been enhanced drastically,
which was due to the stronger electron accepting capability of pyr-
idinium than that of the precursors of pyridines. Thus, diTPA-6–9 were
transformed into the corresponding pyridinium products diTPA-10–13
in the presence of CH3I by Menshutkin reaction (Scheme 3). Under a
small modified condition, diTPA-10–13 could be collected from THF
solvent by simple filtrating with almost quantitative yields and perfect
purities. Very importantly, for the purpose of being used as the targeted-
With all TPA dimers in hand, we evaluated their fluorescence per-
formances in solid state. The corresponding maximum emission (λem
)
wavelength (Fig. S56, Fig. S57), and fluorescence life time (τ) (Fig. S54,
Fig. S55) were depicted in Fig. 1. Thereof, we also illustrated a detailed
structure information and the corresponding electron distribution
models, such as D-A and D-π-A. In general speaking, D-A systems
exhibited the average emission (λem) wavelengths from 492 to 595 nm
but great fluorescence quantum yields. And, D- -A systems were of the
longer emission (λem) wavelengths above 600 nm and more decreased
π
Scheme 1. The chemical structure of TPA dimers of diTPA-1~5.
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Y. Yuan et al.
Dyes and Pigments xxx (xxxx) xxx
Scheme 2. The chemical structure of TPA dimers of diTPA-6~9.
Scheme 3. The chemical structure of TPA dimers of diTPA-10–13.
Scheme 4. The chemical structure of TPA dimers of diTPA-14–16.
fluorescence quantum yields. Thus, these new compounds will be of
great application potentials for the different research purposes. Taking
the starting compound diTPA-1 as example, after converting it into
pyridine substituted dimers of diTPA-6 by Suzuki reaction, the emission
wavelength was red-shifted nearly 30 nm, which is due to the D-A sys-
tem formation. Afterwards, transforming diTPA-6 into pyridinium sub-
stance of diTPA-10 by Menshutkin reaction, the emission wavelength
was red-shifted almost 120 nm, accompanying by the elevated D-A
reliable methods consecutively. The bottom-to-top synthetic evolution
process could supply a more convenient method to produce the more
efficient fluorescence compounds’ library with tunable emission colors
in the future. In another way, all of TPA dimers were typical as the no co-
planar but twisted and sigma bond-riched chemical structures, that met
the requirements of AIE property compounds. Correspondingly, all of
these compounds demonstrated AIE behavior in solid state and in so-
lutions (Fig. S74).
–
system formation. Notably, the smallest
π bridge of C C bond played
–
important role for the long wavelength emission materials formation.
This slight chemical change red-shifted the maximum emission wave-
length from 580 to 640 nm in the cases of diTPA-11 and diTPA-14.
Meanwhile, we also concerned that the quantity of positive charges
demonstrates no obvious influence on the emission performance regard
to emission wavelength and fluorescence life time. For instances, diTPA-
14 was equipped with two positive charges, but four to diTPA-16.
However, their emission wavelengths were almost located the same
region at 640 nm. From this research point, the excessive decorating by
vinyl pyridine salts were seem not largely necessary. However, diTPA-
16 was very water soluble, this property was definitely benefited to
more wide applications in both organic solvent and aqueous phase.
Additionally, thiophene ring has been recognized as very electron
donating group, and commonly used to prepare the fluorescence com-
pounds as well. However, in our research field, it could be almost
neglected to the presence of thiophene rings within the TPA dimers’
framework. For instances, diTPA-14 (absence of thiophene ring) and
diTPA-15 (presence of two thiophene rings) displayed the almost same
emission wavelengths (640, 635 nm respectively) and the less difference
of fluorescence life time (1.30, 1.38 ns respectively). We successfully
prepared various TPA dimers with versatile functional groups by very
3.6. The toxicity evaluation of TPA dimers
It was very necessary that the fluorescent materials are of better
biocompatibility toward target biosubstrates. Thus, the metabolic
viability of HeLa cells and after incubating with TPA dimers was
determined through methylthiazolyldiphenyl tetrazolium bromide
(MTT) assays [21]. The results demonstrated that the cell viabilities still
remain above 80% after a 24 h incubation with these TPA dimers
respectively at different concentrations, indicating that the these TPA
derivatives are of low cytotoxicity within the testing window. In the
ongoing cell staining experiment, we decided the working concentration
of all TPA dimers as 40 mМ (Fig. 2).
3.7. The cells’ staining by TPA dimers of diTPA-3 (2TPA-2CHO), 4
(2TPA-2CHO-2Thi), 5 (2TPA-4CHO) and 9 (2TPA-2CHO-2Py)
Initially, we figured out diTPA-3 (2TPA-2CHO), 4 (2TPA-2CHO-
2Thi), 5 (2TPA-4CHO) and 9 (2TPA-2CHO-2Py) as the probes to the cell
imaging application, which was due to their different emission colors
and the neutral or non-positive charges’ property. Fig. 3 displayed the
fluorescence image of the living and the fixed HeLa cells incubated with
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Fig. 1. The detailed structure information with the corresponding optical behaviors.
Fig. 2. Cell viability of HeLa cells stained with different concentrations of diTPA-12 (left) and diTPA-16 (right) for 24 h.
diTPA-5 (2TPA-4CHO) for 60 min at temperature of 37 �C. Obviously,
the bright blue fluorescence images were observed inside the cells,
indicating that probes successfully penetrate the cell membrane and
enrich into specific cell organelles under physiological conditions. In
order to assess the specificity of lipid droplet-target or not, we costained
cells with Nile Red [22]. Nile Red is known as to localize and quantitate
lipids, particularly neutral lipid droplets within cells. Fig. 3 indicated
that the staining patterns with perfect overlap by diTPA-5 (2TPA-4CHO)
and Nile Red without staining other parts of the cells, even in both cases
of living and fixed cells. Therefore, the specificity as an lipid droplet
staining dye of diTPA-5 (2TPA-4CHO) is even better than that of Nile
Red, which is due to the more readily preparation and low cost as well.
And, these results also demonstrated the fact that the exogenous sub-
stances toward mammalian cells with highly hydrophobic nature are of
better affinity to lipid droplets.
It was well known that among organelles, lipid droplets uniquely
constitute a hydrophobic phase in the aqueous environment of the
cytosol. Their hydrophobic core of neutral lipids stores metabolic energy
and membrane components [23]. Additionally, lipid droplets are
implicated in a number of other cellular functions, ranging from protein
storage and degradation to viral replication. Therefore, the lipid
droplets-targeted fluorescent probes are much required for the impor-
tant research interests. In our present study, the lipid droplets-targeted
and emission color tunable probes are especially realized simulta-
neously due to our very reliable synthetic methods. For instances,
diTPA-3 (2TPA-2CHO), 4 (2TPA-2CHO-2Thi), 9 (2TPA-2CHO-2Py)
were also detected as lipid droplets-targeted probes with green emission
color (Fig. 4). Therefore, we termed diTPA-5 (2TPA-4CHO) as the
Lipi-Blue, and diTPA-3 (2TPA-2CHO),
4 (2TPA-2CHO-2Thi), 9
(2TPA-2CHO-2Py) as the Lipi-Green. The fluorescence properties of all
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Fig. 3. Co-staining HeLa cells by diTPA-5 (left), Nile Red (middle) and merge (top: live cell, bottom: dead cell, merge: diTPA-5, Nile Red and Bright field). (For
interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
of them are perfectly fitted to common filter sets in fluorescence mi-
croscopy for the different research purposes.
self-reporter to monitor the cell apoptosis process. In another way, living
or health and apoptotic cells are different from many aspects, such as the
cell membrane potentials, and the related enhancement of cell perme-
ability, and degradation of nuclear DNA. However, there were few re-
ports to make clear understanding on the differentiation of living or
health and apoptotic cells by fluorescence imaging method. With a very
sharp contrast, we synthesized various fluorescent compounds with
tunable emission colors in hand, which of them definitely are able to
establish a platform for us, making a primary relationship between
different quantity of positive charges with targeted cell imaging in the
both living and fixed cell lines. Initially, we carried out the cell staining
by diTPA-11 (2TPA-2Br-2, 2 positive charges), diTPA-12 (2TPA-2-
CHO–2Br-1, 1 positive charges), diTPA-14 (2TPA-2Vpyr, 2 positive
charges), diTPA-16 (2TPA-4Vpyr, 4 positive charges) for the living
cells, the captured images were displayed in Fig. 5. Very differently from
3.8. The cells’ staining by TPA dimers of diTPA-11 (2TPA-2Br-2), 12
(2TPA-2CHO–2Br-1), 14 (2TPA-2Vpyr) and 16 (2TPA-4Vpyr)
According to the previous publication from Tang [14c] and our
groups [2c], it was conscious of that positive charges are usually play
decisive role for the targeted cell imaging. For the representative ex-
amples, TPE-4EPþ [14a] equipped with four positive charges, was re-
ported as mitochondria specific probe in the early stage apoptotic cells,
which was due to its high affinity to negatively charged mitochondrial
membrane. Easily it can be translocated from mitochondria to nucleus
gradually because of the photosensitized 1O2 generation, and leading to
the cell apoptosis. So TPE-4EPþ could be employed as in situ
Fig. 4. Co-staining HeLa cells by diTPA-3, 4, 9 (top) and Nile Red (bottom). (For interpretation of the references to color in this figure legend, the reader is referred to
the Web version of this article.)
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Fig. 5. The living cells’ staining by diTPA-11, 12, 14 and 16.
the previous publications, our probes were demonstrated to less
depended on the quantity of positive charges, and also less affinity to-
wards the living cells if the incubating time was less than 5 h. However,
the cell membrane targeted staining was totally achieved for all probes
when the incubating time lasted more than 5 h. Once again, our probes
are emissive from the green and red channels. Correspondingly, we
termed diTPA-11 (2TPA-2Br-2) and 12 (2TPA-2CHO–2Br-1) as the
Memb-Green, and diTPA-14 (2TPA-2Vpyr) and 9 (2TPA-2CHO-2Py) as
the Memb-Red. Especially, what should be pointed out that diTPA-12
(2TPA-2CHO–2Br-1) is of only mono-charge, but seems to have better
affinity with cell membrane, which is postulated from two aspects that
(1) the well-matched electrostatic attraction between probes with
phospholipid layers is much demanded for the cell membranes staining.
(2) the halogen atoms are also necessary for the stronger interactions
between probes with phospholipids due to the halogen bonding occur-
ring. This interesting, but very challenging research topic is undergoing
further investigation in our group.
with more positively charged pyridinium moieties could dock on the
surfaces and in the cavities of the negatively charged DNA via electro-
static interactions, leading to the cell nucleolus-targeted staining with
enhanced emission intensity. The in vitro DNA detection experiments
also verified these results totally (Fig. S75, Fig. S76).
From both of research results regard to the living and fixed cells’
staining by our cationic probes, we can conclude that (1) different
quantity of positive charges are of much less influences on the targeted
staining for the living and fixed cells respectively. For living cells, cell
membranes are targeted but required for longer incubating time; for
fixed cells, mitochondria is targeted within shorter incubating time.
Therefore, for the purpose of targeted cells staining in our studies, the
multiple steps’ synthesis is possibly not much necessary. (2) Impor-
tantly, our cationic probes display the potential application to discrim-
inating the health and the apoptotic cells due to the totally different
organelle-targeted staining for the living and fixed cells. In order to
further demonstrate the translocation phenomenon from cell mem-
branes to mitochondria accompanying by the process of cell apoptosis,
we performed the living cells’ staining experiments by diTPA-12 (2TPA-
2CHO–2Br-1) and diTPA-16 (2TPA-4Vpyr), and then upon exposing
them under UV light continuously. It was captured from Fig. 7 that the
different cell translocation is visible. After exposing for 20 h, diTPA-12
(2TPA-2CHO–2Br-1) (Memb-Green) was gradually translocated from
membranes into mitochondria; diTPA-16 (2TPA-4Vpyr) (Memb-Red)
also followed such process. This fact can be largely referred to the
photosensitized 1O2 generation, which is due to the presence of pyr-
idinium segments within the framework of Memb-Green and -Red.
Therefore, our probes can be employed the self-reporters for the early
and late cell apoptosis by green and red channels alternatively. Thereof,
In the following research steps, we are anxious to make sense of the
cell staining behaviors regard to the fixed cells. From Fig. 6, we can see
that diTPA-11 (2TPA-2Br-2), diTPA-12 (2TPA-2CHO–2Br-1), and
diTPA-14 (2TPA-2Vpyr), diTPA-16 (2TPA-4Vpyr) are located into
mitochondria clearly within 60 min, which was largely due to the cell
membrane potentials’ changes, and leading to the enhancement of cell
permeability when cells were fixed by paraformaldehyde. Especially, we
found that diTPA-16 (2TPA-4Vpyr), who carries four positive charges,
is enriched nucleolus more specifically, but not in the cell nucleus
outside nucleolus. As we know that DNA, one of the most abundant
components in the nucleus, especially in nucleolus, exhibits high affinity
for cationic molecules by electrostatic force. Therefore, the molecules
Fig. 6. The fixed cells’ staining by diTPA-11 (top, merge: diTPA-11 and Bright field), 12, 14 and 16 (right: partial enlargement of diTPA-16, the yellow dotted marks
the nucleolus). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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Fig. 7. In situ cell staining experiments with diTPA-12 (top) and diTPA-16 (bottom) at different times under UV light.
we should declare from Fig. 7 that the images’ resolution ratio is not
much high but still distinguishable, but these static processes are
captured without any other disturbances only by UV light exposure.
Thus, these results are still more reliable.
reliable and useful methods consecutively, including of oxidative C–C
coupling reaction, Suzuki reaction, Menshutkin reaction, and typical
nucleophilic addition reaction. Especially, for the research interests
being used as fluorescent materials in the field of bio-related applica-
tions, we mainly followed the lines to construct electron D-A, and D-π-A
system within the molecular structures, accompanying by the formation
of zwitterionic compounds with different amount of positive charges.
Thereof, we fully discussed the primary relationship between the
chemical structures with optical behaviors from the aspects of emission
3.9. The co-staining cells by diTPA-12 (2TPA-2CHO–2Br-1) and 16
(2TPA-4Vpyr) with Mito tracker red and Hoechst 33342
It was verified from Fig. 5 that Memb-Green and -Red are more
difficult to interact with living cells within incubating time less than 5 h,
which is largely agreed with the most publications. However, both of
them are very readily come cross the cell membranes, and stain with
mitochondria specifically in the case of fixed cells. Meanwhile, two
important reasons have been concluded, such as 1O2 generation and
noxiousness. Very interestingly, during our careful studies, we found
that co-staining with some commercially available dyes is greatly
helpful for Memb-Green and -Red to penetrate into organelles simulta-
neously. Therefore, diTPA-12 (2TPA-2CHO–2Br-1) and 16 (2TPA-
4Vpyr) were employed again to co-stain with Mito Tracker Red [24] and
Hoechst 33342 respectively. What should be pointed out that these ex-
periments are performed with living cells but no-wash process with
incubating for 60 min (not 5 h mentioned in Fig. 5). It can be seen from
Fig. 8 that diTPA-12 (2TPA-2CHO–2Br-1) and 16 (2TPA-4Vpyr) are
largely mitochondria targeted with almost negligible noise ratio. This
very interesting phenomenon is seldom to seen in the previous findings.
Notably, these results are also totally incompatible with that observed in
Fig. 5. Thus, we were conscious of that the cooperating interactions
between exogenous substrates are existed. Another way, as we postu-
lated that when Mito Tracker Red and Hoechst 33342 are penetrating
phospholipids layers, which probably results in some damages to the
phospholipids layers or something else. Finally, diTPA-12 (2TPA-2-
CHO–2Br-1) and 16 (2TPA-4Vpyr) were demonstrated very sensitively
to probe these subtle changes, leading to the penetration of cell mem-
branes and enriching into mitochondria. Therefore, mitochondria could
be obviously visualized with the bright emission colors. Partially, the
enhanced emission colors and the almost negligible noise ratio are
contributed to the AIE property of our probes. This interesting, but very
challenging research topic is undergoing further investigation in our
group.
(λem) wavelength, fluorescence life time (τ) and aggregation-induced-
emission (AIE) property, which was definitely different from the re-
ported works. Importantly, we employed these emission color tunable
TPA dimers into living and fixed cell’s staining. Especially, for the
neutral TPA dimers, all of them were detected as lipid droplet-targeted
probes in both cases of living and fixed cells. However, for the zwitter-
ionic TPA dimers, the mitochondria-targeted staining was realized to-
ward the fixed cells; but in the case of living cell’s staining, some great
challenges occurred to us. Notably, we found in the co-staining experi-
mental that a cooperating interaction between exogenous substrates are
really presented. As we stated that such phenomenon is seldom to see in
Fig. 8. Co-staining living cells by diTPA-12, 16 with Mito Tracker Red and
Hoechst 33342 (merge (top): diTPA-12, Mito Tracker Red and Bright field,
merge (bottom): diTPA-16, Hoechst 33342 and Bright field). (For interpretation
of the references to color in this figure legend, the reader is referred to the Web
version of this article.)
4. Conclusions
In this work, we synthesized a variety of TPA dimers via several
11

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Dyes and Pigments xxx (xxxx) xxx
Fig. 9. Summary of research results toward TPA dimers.
[3] (a) Chen C, Song ZG, Zheng XY, He ZK, Liu B, Huang XH, Kong DL, Ding D,
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2016;12:6528–35.(c) Gu XG, Kwok RTK, Lam JWY, Tang BZ. AIEgens for
biological process monitoring and disease theranostics. Biomaterials 2017;146:
115–35.(d) Ma HC, Qi CX, Cheng C, Yang ZM, Cao HY, ang ZW, Tong JH, Yao YQ,
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the previous reports, but is very interesting. We will devote much efforts
to make clear sense of such interactions between these exogenous sub-
strates in the process of cell staining, this challenge is probably much
benefited to have a deep understanding on some important cell life
processes in the future study. Meanwhile, we clearly summarized the
research results in Fig. 9. In general, our important findings are very
useful to design and synthesize probes with improved cellular uptake
capability and significantly enhanced cell targetability, which is defi-
nitely benefited to the study on the membrane activity, cellular
functions.
[4] (a) Zhao GN, Tang B, Dong YQ, Xie WH, Tang BZ. A unique fluorescence response
of hexaphenylsilole to methyl parathion hydrolase: a new signal generating system
for the enzyme label. J Mater Chem B 2014;2:5093–9.(b) Nicol A, Kwok RTK,
Chen CP, Zhao WJ, Chen M, Qu AJ, Tang BZ. Ultrafast delivery of aggregation-
induced emission nanoparticles and pure organic phosphorescent nanocrystals by
saponin encapsulation. J Am Chem Soc 2017;139:14792–9.
Declaration of competing interest
There are no conflicts to declare.
Acknowledgments
[5] (a) Wang LJ, Xu B, Zhang JB, Dong YJ, Wen SP, Zhang HY, Tian WT. Theoretical
investigation of electronic structure and charge transport property of 9, 10-dis-
tyrylanthracene (DSA) derivatives with high solid-state luminescent efficiency.
Phys Chem Chem Phys 2013;15:2449–58.(b) Wang ZL, Ma K, Xu B, Li X, Tian WJ.
A highly sensitive “turn-on” fluorescent probe for bovine serum albumin protein
detection and quantification based on AIE-active distyrylanthracene derivative. Sci
China Chem 2013;56:1234–8.(c) Li X, Xu B, Lu HG, Wang ZL, Zhang B, Zhang Y,
Dong YJ, Ma K, Wen SP, Tian WJ. Label-free fluorescence turn-on detection of Pb2þ
based on AIE-active quaternary ammonium salt of 9, 10-distyrylanthracene. Anal
Methods 2013;5:438–41.
This work is supported by the National Natural Science Foundation
of China (nos. 21764012). We thank the Key Laboratory of Eco-
Environment-Related Polymer Materials (Northwest Normal Univer-
sity) and the Ministry of Education Scholars Innovation Team (IRT
1177) for financial support. We also thank Prof. Anjun, Qin, Dr. Rong,
Hu from SCUT (South China University of Technology).
[6] (a) Yoshii R, Hirose A, Tanaka K, Chujo Y. Functionalization of boron diiminates
with unique optical properties: multicolor tuning of crystallization-induced
emission and introduction into the main chain of conjugated polymers. J Am Chem
Soc 2014;136:18131–9.(b) Yoshii R, Hirose A, Tanaka K, Chujo Y. Boron diiminate
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enhancement characteristics. Chem Eur J 2014;20:8320–4.(c) Hirose A, Tanaka K,
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Qin AJ, Tang BZ. Tetraphenylpyrazine-based AIEgens: facile preparation and
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Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.dyepig.2019.108009.
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