Synthesis, two-photon absorption and aggregation-induced emission properties of multi-branched triphenylamine derivatives based on diketopyrrolopyrrole for bioimaging

Article abstract of DOI:10.1039/c6ra11269b

In this work, three new diketopyrrolopyrrole (DPP)-based multi-branched derivatives (YJ-1, YJ-2 and YJ-3) with triphenylamine, 2,4,6-tri([1,1′-biphenyl]-4-yl)-1,3,5-triazine and 2,2′,2′′-(nitrilotr-is([1,1′-biphenyl]-4′,4-diyl))tris(3-phenylacrylonitrile)

Full text of DOI:10.1039/c6ra11269b

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ARTICLE
Synthesis, two-photon absorption and aggregation-
induced emission properties of multi-branched
triphenylamine derivatives based on
Cite this: DOI: 10.1039/x0xx00000x
diketopyrrolopyrrole for bioimaging
Received 00th January 2012,
Accepted 00th January 2012
a
b
c
a
a
d
b
Ji Yang, Haoqi Tan, Dongyu Li, Tao Jiang, Yuting Gao, Bo Li, Xue Qu,*
a
Jianli Hua*
DOI: 10.1039/x0xx00000x
www.rsc.org/
In this work, three new diketopyrrolopyrrole (DPP)ꢀbased multiꢀbranched derivatives (YJ-1,
YJ-2 and YJ-3) with triphenylamine, 2,4,6ꢀtri([1,1'ꢀbiphenyl]ꢀ4ꢀyl)ꢀ1,3,5ꢀtriazine and 2,2',2''ꢀ
(
nitrilotrꢀis([1,1'ꢀbiphenyl]ꢀ4',4ꢀdiyl))tris(3ꢀphenylacrylonitrile) cores have been designed and
synthesized. Their oneꢀ and twoꢀphoton absorption properties have been investigated. The twoꢀ
photon absorption cross sections (σ) measured by the open aperture Zꢀscan technique are
determined to be 2912, 2016 and 2800 GM for YJ-(1-3), respectively. This result indicates that
donor–acceptor–donor (D–A–D)ꢀtype molecules are benefit to improve σ and their σ data
increase with the better intramolecꢀular charge transfer (ICT). Also, all of the three DPP
derivatives exhibit good aggregationꢀinduced emission (AIE) properties which are very weakly
fluorescent in DMF, but a strong red fluorescent emission in solid state and in the aggregate
state. More importantly, diketopyrrolopyrrole with triꢀphenylamine (YJ-1) was applied for cell
imaging and twoꢀphoton excited fluorescence in vivo imaging of mouse ear.
electronꢀdonor (D) and/or electronꢀacceptor (A) groups designs
including donor–acceptor–donor (DꢀAꢀD)ꢀtype, donorꢀπꢀbridgeꢀ
Introduction
acceptor (DꢀπꢀA)ꢀtype, donor–πꢀbridge–donor (DꢀπꢀD)ꢀtype
The nonlinear optical (NLO) effect of twoꢀphoton absorption
macrocycles, and multiꢀbranched molecules often show large
(
2PA) has attracted widespread attention of the researchers in
4
2
PA cross sections (σ). In addition, a combination of several
recent years. The extremely high intensity of a laser light beam
allows an electronic excited state to be populated via the
simultaneous absorption of two photons. 2PA has applications in
many disparate fields such as 3D data storage and
microfabrication, upconversion lasing, optical power limiting,
pseudolinear subunits into a multidimensional structure is a
familiar approach to assembling new chromophores, allowing
variations in aspects such as the symmetry, length of the
5
, 6
conjugated branches, and nature of the branching moiety.
1
ꢀ3
photodynamic therapy, fluorescent probes, and bioimaging.
Due to the researchers' efforts, a series of new organic materials semiconductor quantum dots (QDs), small organic dyes,
with large 2PA cross sections have been investigated. The fluorescent proteins have been exploited for twoꢀphoton
To date, various fluorescent probes, including inorganic
7
8
9
molecular with extended πꢀconjugated systems functionalized by
fluorescence (2PF) bioimaging. However, these materials have
some drawbacks, such as limited fluorescence stability of organic
a
Key Laboratory for Advanced Materials, Institute of Fine Chemicals and dyes and fluorescent proteins under longꢀterm laser excitation,10
Department of Chemistry, East China University of Science and Technology,
11
potential toxicity and irregular blinking of QDs. To improve
1
30 Meilong Road, Shanghai, 200237, China. Eꢀmail: jlhua@ecust.edu.cn;
the photo bleaching resistance, as well as brightness of organic
Fax:+86ꢀ21ꢀ64250940; Tel: +86ꢀ21ꢀ64250940
b
The State Key Laboratory of Bioreactor Engineering and Key Laboratory dyes, one method is to increase their concentration. Whereas,
for Ultrafine Materials of Ministry of Education. East China University of
most organic dyes suffer from a severe aggregationꢀcaused
quenching (ACQ) effect. Recently, a novel phenomenon of
aggregationꢀinduced emission (AIE) is discovered that the AIE
Science and Technology Shanghai. 200237, China.
12
c
State Key Laboratory of Modern Optical Insrumentations and Center for
Optical and Electromagnetic Research, Zhejiang University (ZJU),
Hangzhou 310058, China.
luminogens are nonemissive in good solvents and become highly
d
Key Laboratory of Polar Materials and Devices, Ministry of Education,
13ꢀ16
emissive in aggregated state.
As far as we know, most studies
East China Normal University, Shanghai 200241, China.
on the AIE fluorophores for bioapplications are focused on the
†
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See tetraphenylethylene (TPE) species with shorter wavelength input
http://www.rsc.org/suppdata/xx/b0/b000000x/
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1
7
and output signals. The longer wavelength of the dyes have
high tissue transparency and low autofluorescence in the long
1
8
wavelength region. Hence, it is still very essential to develop
AIE materials with long wavelength emission and large 2PA
cross sections for biological applications.
1
,4ꢀdiketoꢀ3,6ꢀdiphenylpyrrolo[3,4ꢀc]pyrrole
(DPP)ꢀbased
highꢀperformance materials have been well developed, such as
1
9
20
21ꢀ24
OLEDs, solar cells, fluorescent probes
and twoꢀphoton
2
5
absorption material due to their excellent red and strongly
fluorescent emission and brilliant light, weather, and heat
stability, which could greatly expand the emission wavelength of
dyes. Previously, our group reported DPPꢀbased redꢀemitting
AIE materials with large σ which was used for cell imaging and
2
6
2
7
twoꢀphoton
blood
vasculature
imaging
successfully.
Triarylamine is a traditional luminogen which has been widely
used in optoꢀ and electroactive materials for its good electron
donating and transporting capability, as well as its special
2
8, 29
propeller starburst molecular structure.
In addition, it would
Scheme 2 Synthetic routes to YJ-(1-3) compounds
be a good unit for construction of multiꢀbranched nonꢀplanar
functional materials. Besides, 1,3,5ꢀtriazineꢀbased compounds
show good optical and electrical properties due to their high
electron affinity and symmetrical structure. Recently, our group
has presented multiꢀbranched triphenylamine endꢀcapped DPP
The synthetic routes of YJ-(1-3) are depicted in Scheme 2. The
Suzuki coupling reaction of compound 2 with diisopropyl (4ꢀ
(
bis(4ꢀmethoxyphenyl)amino)phenyl) boronate
3
afforded
compound 4. The important intermediate aldehyde 5 was
synthesized by a Suzuki coupling reaction of compound 4 and (4ꢀ
derivatives
incorporating
an
extended
πꢀdeficient formylphenyl)boronic acid. The tris(4ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀ
phenylacrylonitrile that exhibited good 2PA and AIE dioxaborolanꢀ2ꢀyl)phenyl)amine
properties. To further understand their structureꢀproperties derivative (4 and 6) by the Suzuki reaction gave the target
9
with the respective DPP
2
7
trilateral compounds YJ-1 and YJ-3. The anoxicenvironment and
potassium carbonate are the required parts of the Suzuki
coupling. Finally, the target products YJ-2 was synthesized by
HornerꢀWadsworthꢀEmmons reaction of the aldehydes (5) with
correlation, we connect electronꢀdonating triphenylamine to the
,4,6ꢀpositions of triphenylamine, the triazine and the
2
phenylacrylonitrile via DPP to form three new multibranched
type 2PA derivatives with extended πꢀconjugated systems and
good symmetry (YJ-(1-3), Scheme 1)，respectively. Moreover,
YJ-1 was successfully used for oneꢀphoton laser scanning
confocal microscopy cell imaging and twoꢀphoton vivo
microscopic imaging of ear blood vessels of mice, indicating that
compound YJ-1 has great potential applicability in the
bioapplications.
2
,4,6ꢀtri([1,1'ꢀbiphenyl]ꢀ4ꢀyl)ꢀ1,3,5ꢀtriazine.
The
Hornerꢀ
WadsworthꢀEmmons reaction must use anhydrous reagents and
be under no water environment. All compounds were purified by
column chromatography or recrystallization and characterized by
1
13
H NMR, C NMR and mass spectra (shown in ESI).
Photophysical properties
The Fig. 1 shows the normalized oneꢀphoton absorption of YJ-
(
1-3) in DMF and in dispersion of the aggregate form (90%
ꢀ
5
water) at 1×10 M. Their absorption maxima (λmax) appeared at
19, 511 and 510 nm in dimethyl formamide (DMF),
respectively. It is also noted that the absorption spectra of YJ-(1-
) in DMF/water (90% water in volume) mixtures are all
5
3
broadened and red shifted by 10, 4 and 18 nm, respectively. The
absorption tails extending well into the long wavelength region
further indicated the aggregation of luminogen into particles in
the presence of water, as it is well known that the Mie effect of
3
0
particles causes such levelꢀoff tails in the absorption spectra.
Compared to YJ-1, the absorption maximum of YJ-3 is blue
shifted by 9 nm in DMF. The blue shifted may be caused by the
introduction of the phenylacrylonitrile which block the ICT
process. YJ-2 is blue shifted by 8 nm compared to YJ-1, and it is
possible that YJ-2 has a more twisty structure, which hindered
the intramolecular charge transfer.
Scheme 1 Structures of YJ-(1-3) compounds.
Results and discussion
Synthesis
2
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Fig. 1 Normalized oneꢀphoton absorption of YJ-(1–3) in DMF
solid line) and in dispersion of the nanoaggregate form (90%
(
ꢀ
5
water) (dashed line) at 1×10 M
As expected, the dilute solution YJ-1 in DMF had almost no
fluorescence. But when the nonꢀsolvent water was gradually
added into the solution in DMF, the solution started to emit
fluorescence. After that, with the rising of water fraction in
DMF, the fluorescence intensity of solution increased (Fig.
Fig. 2 (A, B and C) Corresponding emission spectra of
compound YJ- (1-3) in DMF–water mixtures with different water
fractions at 1×10 M. Excitation wavelength: 528 nm, 515nm,
ꢀ
5
2
(A)). When the water content reached to 50%, the fluorescence
intensity of the solution was 19 times larger than that of the
solution without water. The fluorescence peaks are nearly
identical at 643 nm. This phenomenon is normal among the
compounds with AIE properties, which could be caused by the
532nm, respectively. (D) Plot of I/I vs. water content of the
0
solvent mixture, where I is the PL intensity in pure DMF
0
solution. (E) Photographs of YJ-(1-3) in the pure DMF solution
and in 50%, 80%, 70% waterꢀDMF mixture captured under UV
light, respectively.
3
0,
variation in the packing mode of the molecules in aggregates.
3
1
The aggregation of particles restricted the intramolecular
rotation (RIR) process, which facilitated the radiative decay of
the excitons, resulting in a strong increase in luminogen
fluorescence. Similar behavior was observed for YJ-2 and YJ-3
in Fig. 2(B and C), verifying that the three luminogens were
AIEꢀactive. The photoꢀluminescence intensity (PL) was very
weak in the pure DMF, but when the water content reached to
8
0% and 70% in volume, YJ-2 and YJ-3 gave the maximum red
luminescence and the peaks located at 651 nm and 672 nm with
0 and 32ꢀ increase, respectively. The fluorescence behavior of
4
YJ-(1-3) is well visualized through fluorescence images of
solution and aggregate dispersions as shown in Fig. 2(E).
YJ-(1-3) are also investigated in the solid state, and their
absolute fluorescence quantum yield (φ ) were 7.30%, 8.32% and
F
6
.08%, respectively (Fig. (3A-3C)). Moreover, to observe the
formation of nanoparticles(NPs), the sizes distribution of YJ-(1-
) NPs were investigated by dynamic light scattering (DLS),
Fig. 3 Emission spectra of YJ-(1-3) (A, B and C) as solid
powders; excitation wavelength: 519 nm for YJ-1, 511 nm for
YJ-2 and 510 nm for YJ-3. Inset: photos of YJ-(1-3) powders
taken under illumination of a UV light (excitation wavelength:
3
indicating that the mean diameters are approximately 64 nm, 404
nm and 103 nm (Fig. (3D-3F)), respectively. Scanning electron
microscopy (SEM) was also performed to study the morphology
of the NPs, suggesting that they are in spherical shape with an
average size of around 50 nm, 350 nm and 70 nm, respectively,
which are slightly smaller than those of DLS due to the shrinking
of samples in the vacuum dry state. All these given evidence
3
65 nm). Particle size distribution and morphology of (D) YJ-1
in DMF/H O (5: 5 v/v) and (E) YJ-2 in DMF/H O (2: 8 v/v) (F)
2
2
YJ-3 in DMF/H
at the concentration of 1×10 M. The scale bar is 500 nm.
O (3: 4 v/v) mixtures studied by DLS and SEM
2
ꢀ
5
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increase in solvent polarity (Fig. 4). Since the three dyes possess
DꢀA structural units, such DꢀA molecules often show
solvatochromic effects, which has been well explained by a
3
2, 33
twisted intramolecular charge transfer (TICT) mechanism.
However, its emission intensity is weakened because of the
susceptibility of the TICT state to various nonradiative
quenching processes.
Two-photon absorption properties
2
PA cross sections of YJ-(1-3) were determined by femtosecond
open aperture Zꢀscan technique. Fig. 5(1a-1c) shows the openꢀ
aperture Zꢀscan data and 2PA coefficient obtained by data fitting.
The σ can be calculated by using the equation of σ = hνβN0,
Fig. 4 (A, B and C) Emission spectra for YJ-(1-3) in different
ꢀ
5
solvents at the concentration of 1×10 M. λ : 528 nm (YJ-1) ,515
where N = N C is the number density of the absorption centers,
0
A
ex
nm (YJ-2) and 532 nm (YJ-3). (D, E and F) Photographs of YJ-
NA is the Avogadro constant and C represents the solute molar
3
4
(1-3) in different solvents under UV light.
concentration and β is the 2PA coefficient. The values of σ for
YJ-(1-3) are 2912, 2016 and 2800 GM at wavelength of 800 nm,
respectively. The values of YJ-1 and YJ-3 are larger than the
YJ-2, because YJ-1 and YJ-3 have better πꢀsystems. The smaller
indicated that the increasing PL intensity was caused by the
increasing aggregation. Thus its AIE nature is adequately
verified.
Meantime, we also investigated their optical properties in the
different solvents, such as toluene (TOL), dichloromethane
2
PA cross section of YJ-3 (2800 GM) arises from the
introduction of a phenylacrylonitrile in the π bridge compared to
YJ-1, which may influence the molecular planarity. The two
photon fluorescence spectrum of the three compounds under
different laser intensities is shown in Fig. 5(2a-2c). As shown in
the inset, the linear dependence of fluorescence intensity on the
square of the excitation intensity confirms that 2PA is the main
(
DCM), chloroform(CHL), tetrahydrofuran (THF) and DMF. As
shown in Fig. S1-S3, the absorption bands of the three
compounds remain almost identical in the above solvents.
However, a significant bathochromic shift of emission band and
the decrease of fluorescence intensities can be observed with the
Fig. 5 (1a–1c) Openꢀaperture Zꢀscan trace of YJ-(1–3) (scattered circle experimental data, straight line theoretic fitted data). (2a–2c)
PF intensities of YJ-(1–3) under different excitation power densities in toluene. Inset: 2PF intensity versus the square of the excitation
power density. (3a–3c) Twoꢀphoton fluorescence emission spectra and 2PA emission images for YJ-(1–3) in DMF and in dispersion of
2
ꢀ
5
the nanoaggregate form (50%, 80% and 70% water) at 1×10 M, excited at 800 nm. YJ-1: 1a–3a; YJ-2: 1b–3b; YJ-3: 1c–3c.
4
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excitation mechanism of the intense fluorescence emission.
Under the excitation of 80 fs pulse, 800 nm laser, YJ-(1-3) in
the DMF/water mixtures emit NIR fluorescence with peaks
located at 675 nm ,670 nm and 672 nm (Fig. 5 (3a-3c)),
respectively. The twoꢀphoton excitation fluorescence is slightly
red shifted compared with oneꢀphoton excitation due to
reabsorption of partial emissive fluorescence. The overlap
between oneꢀ and twoꢀphoton excitation fluorescence makes
clear that the emissions resulted from the same excited state,
regardless of the different modes of excitation. It shall be
concentrated on that the studied three compounds have
undergone two photon excitation studies at one wavelength (800
nm). As shown in the inset photos, the 2PF under the excitation
of 800 nm laser was signally intensified by aggregation in
DMF/water mixtures.
Fig. 7 Two-photon excited fluorescence (A) and reconstructed
D image (B) the intravenously injected YJ-1-PEG nanoparticles
in the blood vessel of the ear of a mouse.
3
3
5ꢀ40
cellular uptake of YJ-1 might be the endocytosis process.
The metabolic viability of MGCꢀ803 cells and Hela cells were
further examined by methylthiazolyldiphenyltetrazolium bromide
One-photon cellular imaging and two-photon luminescence vivo
microscopic imaging of ear blood vessels of mice
(
MTT) assays, which revealed high cell viability of near 100%
Due to the good AIE and 2PA properties, high brightness in the
nearꢀinfrared region, and the biggest twoꢀphoton absorption
crossꢀsection (σ) 2912 GM, the YJ-1 was chosen as an example
and their performance was investigated in oneꢀphoton cellular
imaging and twoꢀphoton luminescence vivo microscopic imaging
of ear blood vessels of mice. The cellular imaging in vitro was
studied by confocal laser scanning microscopy (CLSM). In these
experiments, MGCꢀ803 cells are individually incubated in
culture medium with YJ-1 (1×10 M) for 4 h, and then imaged,
as shown in Fig. 6. The nuclei are stained with 2ꢀ(4ꢀ
amidinophenyl)ꢀ6ꢀindolecarbamidine dihydrochloride (DAPI),
which is a fluorescent dye that gives out blue emission, as shown
in Fig. 6(B). Red fluorescence is obviously observed in the
cytoplasm of MGCꢀ803 cells. The appearance of red
fluorescence in the cytoplasm region of the cells surrounding the
nucleus indicates that YJ-1 is successfully endocytosed by cell
lines and accumulated in the entire cell cytoplasm. In Fig. 6(D),
the fluorescence image is the overlaying images of (A), (B) and
ꢀ
5
within 24 h even at a YJ-1 concentration of 1×10 M, which is
1
0ꢀfold higher than that used for imaging (Fig. S4).
To further investigate the ability of YJ-1 for inꢀvivo twoꢀ
41
photon fluorescence imaging, YJ-1-PEG nanoparticles were
synthesized to get stable micelle in order to improve the
biocompatibility in blood vessels. Since samples were
intravenously into mice, the blood vessels of mice were full of
YJ-1-PEG due to blood circulation. Because of the bright 2PF
signals from YJ-1-PEG, as well as low auto fluorescence of
tissue under 800 nm fs excitation, the structure of blood vessels
could be visualized clearly and the signal to noise ratio of the
images was high even at 100ꢁm deep of the mouse ear. As shown
ꢀ
6
(
Fig. 7B) the 3D reconstructed 2PF image of YJ-1-PEG in the
mouse ear, the vascular architecture in the mouse ear was
revealed clearly.
Conclusion
(
C). The cellular imaging of Hela cells with YJ-1 was also
In this work, we have designed and synthesized three new multiꢀ
branched type and DPPꢀbased chromophores (YJ-(1-3)) with
large 2PA cross sections and bright AIE fluorescence in water.
Their σ values were 2912 GM, 2016 GM and 2800 GM,
respectively. Investigation of the 2PA property of these
compounds reveals that their 2PA cross section values increase
with introducing multiꢀbranched molecules and easier ICT
progress around the molecules possible. Moreover, the YJ-1 had
the biggest twoꢀphoton absorption crossꢀsection (σ) among the
three dyes, so we choose the YJ-1 as the fluorescence luminogen
for bioimaging. Cellular imaging results indicated YJ-1 could be
utilized as a fluorescent probe for cellular imaging of 803 and
HeLa cells, where red fluorescence was observed in the
cytoplasm. In addition, 2PF in vivo imaging of mouse ear was
conducted by using YJ-1-PEG as the contrast agents, and the 3D
architecture of blood vessels was vividly reconstructed. The red
emissive AIE nanoparticles with high 2PA efficiency at 800 nm
would be useful for deepꢀtissue functional bioimaging in the
future.
investigated (Fig. 6 (E-H)). The size of YJ-1 in DMSO/Dulbecꢀ
co’s modified Eagle’s medium (DMEM) (1: 99 v/v) mixtures
studied by DLS at concentration of 1×10 M indicated that the
mean diameter is 89 nm while the mean diameters of DMEM
studied by DLS is 28 nm (Fig. S5-S6). The mechanism of
ꢀ
5
Fig. 6 MGCꢀ803 cells (A) and HeLa cells of (E) fluorescence
images of cells nuclei stained by DAPI; (B) and (F) fluorescence
ꢀ
images of MGCꢀ803 cells and Hela cells stained by YJ-1(1×10
6
M), respectively; (C) and (G) brightfield image of MGCꢀ803
cells and Hela cells, respectively; (D) and (H) overlap image of
(A), (B), (C) and (E), (F), (G), respectively.
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Experimental section
A mixture of 3 (738 mg, 1.2 mmol), 2 (434 mg, 1.0 mmol), and
Pd(PPh3)4 (21 mg, 0.02 mmol) was dissolved in 15 mL THF
under argon atmosphere. Aqueous potassium carbonate solution
Materials and measurements
(
°
2 M, 5 mL) was added to the reaction solution and stirred at 40
C for 3 h. After cooling to room temperature, the reaction
Tetrahydrofuran (THF) was preꢀdried over 4 °A molecular sieves
and distilled under argon atmosphere from sodium benzophenone
ketyl immediately prior to use. N, NꢀDimethyl formamide (DMF)
and dichloromethane (DCM) were refluxed with calcium hydride
mixture was extracted by diꢀchloromethane (3 × 15 mL). The
combined organic layer was washed with water and dried over
Na2SO4. After removal of the solvent, the crude product was
purified by column chromatography on silica gel (eluent:
DCM/petroleum ether (3:1, v/v) to obtain 4 (270 mg, 32% yield)
4
2
and distilled before use. Compound 1, compound 2, compound
4
3
17
9
and compound 10 were prepared according to previous
literature protocols. All other reagents, proteins and fetal bovine
serum were purchased from SigmaꢀAldrich and used as received.
The solutions for analytical studies were prepared with deionized
water treated with a MilliꢀQ System (Billerica, MA, USA).
1
as a red solid. H NMR (400 MHz, Chloroformꢀd) δ 7.89 (d, J =
8
7
3
(
1
.2 Hz, 2H), 7.74ꢀ7.64 (m, 6H), 7.47 (d, J = 8.8 Hz, 2H), 7.14ꢀ
.09 (m, 4H), 6.99 (d, J = 8.2 Hz, 2H), 6.89ꢀ6.84 (m, 4H), 3.83ꢀ
.80 (m, 6H), 3.79ꢀ3.68 (m, 4H), 1.28ꢀ1.21 (m, 12H), 0.94ꢀ0.74
1
3
m, 10H); C NMR (101 MHz, Chloroformꢀd) δ 162.64, 156.19,
49.01, 146.37, 143.58, 140.48, 132.14, 131.06, 130.14, 129.28,
Instruments
1
13
H and C NMR spectra were recorded on a Bruker AMꢀ400 127.59, 126.97, 126.46, 120.10, 114.80, 109.39, 55.50, 31.24,
spectrometer using chloroformꢀd (CDCl3) as solvent and 29.41, 26.42, 22.50, 14.00; MALDIꢀTOF: [M ] calcd for
+
tetramethylsilane (δ=0) as internal reference. The UV/vis spectra
were recorded on a VarianꢀCary 500 spectrophotometer with 2
C H52BrN O : 837.3141, found: 837.3142.
50 3 4
nm resolution at room temperature. The fluorescence spectra Synthesis of 4'-(4-(4'-(bis(4-methoxyphenyl)amino)-[1,1'-bip-
henyl]-4-yl)-2,5-di-hexyl-3,6-dioxo-2,3,5,6-tetrahydropyrrolo-
3,4-c]pyrrol-1-yl)-[1,1'-biphenyl]-4-carba-ldehyde (5)
were taken on a VarianꢀCary fluorescence spectrophotometer.
Timeꢀresolved fluorescence measurements in this study were
performed using the Edinburgh OB 900ꢀL timeꢀcorrelated single
photon counting system (TCSPC). Emission was collected at
right angle with respect to the pump. A temper deconvolution
from the system response function, a temporal resolution of ~30
ps can be reliably obtained. The 2PA cross sections of YJ-(1–3)
were measured by femtosecond openꢀaperture Zꢀscan technique
[
A mixture of 4 (839 mg, 1.0 mmol), (4ꢀformylphenyl)boronic
acid (300 mg, 2.0 mmol), and Pd(PPh ) (21 mg, 0.02 mmol) was
3
4
dissolved in 15 mL THF under argon atmosphere. Aqueous
potassium carbonate solution (2 M, 5 mL) was added to the
reaction solution and stirred at 60 °C for 12 h. After cooling to
room temperature, the reaction mixture was extracted by DCM (3
4
4
according to a previously described method.
Twoꢀphoton
×
15 mL). The combined organic layer was washed with water
excited fluorescence (2PF) was excited by fs pulses with
different intensities at a wavelength of 800 nm. The repetition
rate of the laser pulses was 250 kHz, and the pulse duration was
and dried over Na2SO4. After removal of the solvent, the crude
product was purified by column chromatography on silica gel
(
7
1
eluent: DCM/petroleum ether (5:1, v/v) to obtain 5 (650 mg,
5% yield) as a red solid. H NMR (400 MHz, Chloroformꢀd) δ:
0.02 (s, 1H), 7.91 (dd, J = 16.7, 8.1 Hz, 4H), 7.83 (d, J = 8.3
8
0 fs. The measurement was performed with a fixed scattering
1
angle of 90°. The size of the nanoparticles was determined by an
ALVꢀ5000 laser light scattering spectrometer (DLS). The SEM
micrographs were obtained on a JEOL JSMꢀ6360 scanning
electron microscope (SEM). The cell imaging experiments were
carried out with an Olympus FV1000 laser scanning confocal
microscope and a 60 × oil immersion objective lens.
Hz, 2H), 7.78ꢀ7.71 (m, 4H), 7.64 (d, J = 8.2 Hz, 2H), 7.43ꢀ7.38
(
(
(
m, 2H), 7.05 (d, J = 8.8 Hz, 4H), 6.92 (d, J = 8.8 Hz, 2H), 6.80
d, J = 8.8 Hz, 4H), 3.81ꢀ3.69 (m, 10H), 1.54ꢀ1.52 (m, 4H), 1.19
t, J = 9.7 Hz, 16H), 0.76 (t, J = 6.4 Hz, 6H); C NMR (101
13
MHz, Chloroformꢀd) δ: 191.81, 162.79, 156.20, 148.94, 146.96,
1
1
1
45.83, 143.59, 141.93, 140.45, 135.66, 131.03, 130.40, 129.39,
29.30, 128.34, 127.73, 127.57, 127.00, 126.48, 125.89, 120.03,
Synthesis of 3,6-bis(4-bromophenyl)-2,5-dihexyl-2,5-dihydro-
pyrrolo[3,4-c]pyrrole-1,4-dione (3)
14.80, 110.31, 109.55, 67.99, 55.50, 31.26, 26.45, 25.63, 22.51,
+
To a suspension of 1 (500 mg, 1.1 mmol) in 10 mL N,Nꢀ 14.01; HRMS (ESI) (m/z): [M ] calcd for C57
H N O : 863.4298,
57 3 5
found: 863.4296.
Dimethylformamide (DMF) was added sodium hydride (108 mg,
.4 mmol) with stirring at 0 °C. Then the mixture was warmed to
4
Synthesis of 3-(4'-(4-(4'-(bis(4-methoxyphenyl)amino)-[1,1'-
biphenyl]-4-yl)-2,5-dihexyl-3,6-dioxo-2,3,5,6-tetrahydropyrr-
olo[3,4-c]pyrrol-1-yl)-[1,1'-biphenyl]-4-yl)-2-(4-bromophen-
yl)acrylonitrile (6)
room temperature (rt) and stirred for another 1.5 h. Then 1ꢀ
bromohexane (0.64 mL, 4.4 mmol) was added dropwise. The
mixture was stirred overnight at rt. After completion of the
reaction, the mixture was poured into ice water, filtered, and then
washed with water and hexane. The filter cake was dried under
A
mixture of
5
(864 mg, 1.0 mmol), 2ꢀ(4ꢀbromꢀ
vacuum. The resulting crude product was purified by column ophenyl)acetonitrile (392 mg, 2.0 mmol), and Potassium tertꢀ
chromatography on silica gel (eluent: DCM/petroleum ether (1: butylate (KTB) (448 mg, 4.0 mmol) was dissolved in 20 mL
1
, v/v) to obtain 3 (310 mg, 45.01% yield) as an orangeꢀred solid. ethanol under argon atmosphere. The mixture was stirred at 50
C for 12 h. After cooling to room temperature, the reaction
Synthesis of 3-(4'-(bis(4-methoxyphenyl)amino)-[1,1'-biphen-yl]- mixture was removed under vacuum. Then the mixture was
°
4
-yl)-6-(4-brom-ophenyl)-2,5-dihexyl-2,5-dihydropyrrolo-[3,4-
extracted by DCM (10 mL) and water (3 × 5 mL). The combined
organic layer was washed with water and dried over Na SO .
c]pyrrole-1,4-dione (4)
2
4
6
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012

Page 7 of 10
Journal Name
RSC Advances
View Article Online
ART69ICLE
DOI: 10.1039/C6RA112 B
After removal of the solvent, the crude product was purified by × 15 mL). The combined organic layer was washed with water
column chromatography on silica gel (eluent: DCM/petroleum and dried over Na2SO4. After removal of the solvent, the crude
ether (15:1, v/v) to obtain 6 (860 mg, 83% yield) as a red solid. product was purified by column chromatography on silica gel
1
H NMR (400 MHz, Chloroformꢀd) δ: 7.93 (d, J = 8.1 Hz, 2H), (eluent: DCM/petroleum ether (15:1, v/v) to obtain YJ-1 (200
1
7
1
7
3
.87 (d, J = 8.2 Hz, 2H), 7.83 (d, J = 8.1 Hz, 2H), 7.71 (dd, J = mg, 55% yield) as a red solid. H NMR (400 MHz, Chloroformꢀ
4.0, 8.1 Hz, 4H), 7.62 (d, J = 7.9 Hz, 2H), 7.55ꢀ7.37 (m, 8H), d) δ: 7.80 (d, J = 8.1 Hz, 6H), 7.73 (d, J = 8.0 Hz, 6H), 7.59 (d, J
.19 (s, 1H), 7.03 (d, J = 10.9 Hz, 4H), 6.80 (d, J = 8.0 Hz, 4H), = 8.1 Hz, 6H), 7.48 (dd, J = 15.1, 8.2 Hz, 12H), 7.32 (d, J = 8.3
1
3
.89ꢀ3.62 (m, 10H), 1.20ꢀ1.15 (m, 16H), 0.80ꢀ0.74 (m, 6H);
C
Hz, 6H), 7.22ꢀ7.15 (m, 6H), 7.01 (d, J = 8.5 Hz, 12H), 6.92ꢀ6.84
NMR (101 MHz, Chloroformꢀd) δ: 162.81, 156.17, 148.94, (m, 6H), 6.77 (d, J = 8.9 Hz, 12H), 3.73 (s, 30H), 1.17 (q, J =
1
3
1
1
1
1
48.72, 147.19, 143.51, 142.07, 141.73, 140.48, 133.18, 132.27, 10.6, 8.6 Hz, 48H), 0.75 (q, J = 6.9, 5.4 Hz, 18H); C NMR (101
31.09, 130.05, 129.39, 129.30, 127.89, 127.57, 127.46, 127.36, MHz, Chloroformꢀd) δ: 161.69, 155.05, 150.48, 147.70, 147.32,
26.97, 126.47, 125.93, 123.55, 120.09, 117.73, 114.80, 110.54, 146.73, 145.94, 139.47, 134.72, 133.42, 130.16, 128.43, 128.27,
10.17, 109.57, 55.52, 42.07, 31.27, 29.73, 26.47, 22.52, 14.02; 126.98, 126.49, 125.87, 125.56, 125.17, 124.49, 123.48, 119.07,
HRMS (ESI) (m/z): [M ] calcd for C H BrN O : 1040.3876, 113.74, 108.63, 108.42, 54.43, 33.19, 30.22, 29.28, 25.41, 21.46,
+
6
5
61
4
4
+
found: 1040.3895.
12.95; MALDIꢀTOF: [M ] calcd for C168H168N O : 2517.2843,
10 12
found: 2517.2878.
Synthesis of tris(4-bromophenyl)amine (8)
Synthesis of 6,6',6''-((((1,3,5-triazine-2,4,6-triyl)tris(benzene-4,1-
diyl))tris(e-thene-2,1-diyl))tris([1,1'-biphenyl]-4',4-diyl))-tris(3-
To a doubleꢀneck round bottom flask (250 mL) which equipped
with mechanical stirrer were charged triphenylamine (3 g, 12.23
mmol) and DMF (100 mL). A DMF (20 mL) solution of Nꢀ
bromosuccinimide (9.79 g, 55.03 mmol) was dropwisely added to
the reaction flask. After addition of the DMF solution, the
mixture was kept stirring for 1 h. The reaction was quenched
with water and extracted with ethyl acetate (100 mL × 3). The
(4'-(bis(4-methoxyphenyl)amino)-[1,1'-biphenyl]-4-yl)-2,5-
dihexyl-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione)(YJ-2)
In a 100 mL roundꢀbottom flask, 5 (361 mg, 0.42 mmol), 10 (71
mg, 0.1 mmol), potassium tertꢀbutoxide (117 mg, 1.05 mmol),
1
8ꢀcrownꢀ6 (10 mg, 0.04 mmol) and 50 mL DCM were added
under argon atmosphere. After stirring at 45 °C for 6 h, the
mixture was poured into distilled water and extracted with DCM
and water. The combined organic phases were dried over
anhydrous MgSO4 and concentrated using a rotary evaporator.
The crude product was purified by column chromatography on
organic layer was washed by NH Cl solution, then separated and
4
dried over anhydrous MgSO4. The solution was filtered and
evaporated to remove the solvent. The crude product was
purified by column chromatography on silica gel (eluent:
1
petroleum) to obtain 8 (5.1 g, 87%yield) as a white solid. H
silica gel (eluent: DCM/petroleum ether (20:1, v/v) to obtain YJ-
NMR (400 MHz, Chloroformꢀd) δ: 7.38 (d, J = 8.4 Hz, 6H), 6.95
1
2
(185 mg, 61% yield) as a red solid. H NMR (400 MHz,
(
d, J = 8.5 Hz, 6H).
Chloroformꢀd) δ: 8.31 (d, J = 7.7 Hz, 6H), 7.63 (d, J = 7.7 Hz,
6
6
8
H), 7.54 (d, J = 7.8 Hz, 6H), 7.41ꢀ7.22 (m, 30H), 7.21ꢀ7.14 (m,
H), 7.00ꢀ6.88 (m, 18H), 6.80 (d, J = 8.2 Hz, 6H), 6.73 (d, J =
Synthesis of 4,4',4"-Tris(pinacolatoborane)phenylamine) (9)
A mixture of 8 (1.93 g, 4.0 mmol), bis(pinacolato)diboron (3.75
.6 Hz, 12H), 3.71 (s, 30H), 1.20ꢀ1.06 (m, 48H), 0.73 (t, J = 6.6
g,
palladium(II) chloride (147 mg, 0.2 mmol)and potassium acetate
7 g, 71.33 mmol) was dissolved in 55 mL dioxane under argon
3.69
mmol),
[1,1'ꢀBis(diphenylꢀphosphino)ferrocene]ꢀ
13
Hz, 18H); C NMR (101 MHz, Chloroformꢀd) δ: 169.17,
1
1
1
3
61.41, 161.30, 154.95, 150.49, 147.49, 147.38, 146.54, 141.43,
40.84, 139.49, 134.73, 130.16, 128.42, 127.22, 126.42, 125.82,
25.19, 124.69, 124.50, 119.07, 113.73, 108.34, 108.01, 54.41,
(
atmosphere. The mixture was stirred at 85 °C for 12 h. After
cooling to room temperature, the reaction mixture was removed
under vacuum. Then the mixture was extracted by dichloꢀ
romethane (30 mL) and water (3 × 15 mL). The combined
+
3.20, 30.21, 29.29, 25.42, 21.48, 12.96; MALDIꢀTOF: [M ]
calcd for C195H186N O : 2887.4313, found: 2887.4312.
12
12
organic layer was washed with water and dried over Na SO .
2
4
Synthesis of 2,2',2''-(nitrilotris([1,1'-biphenyl]-4',4-diyl))-tris(3-
After removal of the solvent, the crude product was purified by (4'-(4-(4'-(bis(4-methoxyphenyl)amino)-[1,1'-biphenyl]-4-yl)-2,5-
column chromatography on silica gel (eluent: DCM/petroleum dihexyl-3,6-dioxo-2,3,5,6-tetrahydropyrrolo-[3,4-c]-pyrrol-1-yl)-
ether (1:2, v/v) to obtain 9 (500 mg, 20% yield) as a white solid. [1,1'-biphenyl]-4-yl)acrylonitrile) (YJ-3)
1
H NMR (400 MHz, Chloroformꢀd) δ: 7.68 (d, J = 8.1 Hz, 6H),
A mixture of 6 (261 mg, 0.25 mmol), 9 (39 mg, 0.06 mmol), and
7
.07 (d, J = 8.0 Hz, 6H), 1.34 (s, 36H).
Pd(PPh ) (5 mg, 0.001 mmol) was dissolved in methylbenzene
3
4
(
21 mL) and ethanol (7 mL) under argon atmosphere. Aqueous
Synthesis of 6,6',6''-(nitrilotris([1,1'-biphenyl]-4',4-diyl))tris-(3-
4'-(bis(4-metho-xyphenyl)amino)-[1,1'-biphenyl]-4-yl)-2,5-
dihexyl-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione) (YJ-1)
potassium carbonate solution (2 M, 5 mL) was added to the
reaction solution and stirred at 105 °C for 72 h. After cooling to
room temperature, the reaction mixture was extracted by DCM (3
(
A mixture of 4 (470 mg, 0.56 mmol), 9 (89 mg, 0.14 mmol), and
×
15 mL). The combined organic layer was washed with water
Pd(PPh ) (5 mg, 0.001 mmol) was dissolved in methylbenzene
3
4
and dried over Na2SO4. After removal of the solvent, the crude
product was purified by column chromatography on silica gel
(
21 mL) and ethanol (7mL) under argon atmosphere. Aqueous
potassium carbonate solution (2 M, 5 mL) was added to the
reaction solution and stirred at 105 °C for 72 h. After cooling to
room temperature, the reaction mixture was extracted by DCM (3
(
eluent: DCM/petroleum ether (20:1, v/v) to obtain YJ-3 (115
1
mg, 59% yield) as a red solid. H NMR (400 MHz, Chloroformꢀ
d) δ: 7.91 (d, J = 7.9 Hz, 6H), 7.83 (d, J = 8.0 Hz, 6H), 7.72 (dd,
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 7

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ARTICLE
DOI: 10.1039/C6RA1126
Journal Na9Bme
J = 22.2, 8.1 Hz, 18H), 7.65ꢀ7.55 (m, 18H), 7.54ꢀ7.45 (m, 12H), were housed in cages in groups (5 mice per cage) and fed with
7
1
1
.36 (d, J = 8.6 Hz, 6H), 7.22 (d, J = 8.6 Hz, 6H), 7.10 (dd, J = standard mouse chow and water. The cages were maintained in a
8.0, 8.8 Hz, 12H), 6.94 (d, J = 8.4 Hz, 3H), 6.85 (t, J = 10.3 Hz, room with controlled temperature (25 61 uC) and a 12 h
2H), 3.81 (d, J = 3.6 Hz, 30H), 1.23 (d, J = 22.0 Hz, 48H), 0.81 light/dark cycle. The protocol of animal experiments was
1
3
(
d, J = 6.9 Hz, 18H); C NMR (101 MHz, Chloroformꢀd) δ: approved by the Institutional Ethical Committee of Animal
1
1
1
2
61.55, 155.02, 147.76, 146.19, 139.49, 130.13, 128.88, 128.49, Experimentation of Zhejiang University in China, and the
28.36, 126.83, 126.46, 126.07, 125.86, 125.25, 124.87, 123.43, experiments were performed strictly according to governmental
19.06, 113.75, 108.70, 108.13, 54.44, 30.20, 28.68, 28.10, and international guidelines on animal experimentation.
+
5.40, 21.46, 12.94; MALDIꢀTOF: [M ] calcd for According to requirements for Biosafety and Animal Ethics, all
C213H195N O : 3128.5115, found: 3128.5300.
efforts were made to minimize the number of animals used and
their suffering.
13
12
Cell Culture
Acknowledgements
All cells were purchased from the American Type Culture
Collection (ATCC). cells were grown in DMEM supplemented
For financial support of this research, we thank the National
Basic Research 973 Program (2013CB733700), and
NSFC/China (21421004, 21172073, 21372082, 21572062,
with 10% fetal bovine serum (FBS) and 100 U/mL penicillin and
2
1
00 ꢁg/mL streptomycin and cultured in 75 cm tissue culture
flasks in a humidified 5% CO environmental incubator at 37 °C
2
9
1233207 and 51573047).
and cultivated at 80% confluency.
The cultured cells were harvested with 0.05/0.02% trypsin
Notes and references
/
EDTA, centrifuged at 800 rpm for 4 min and resuspended in the
culture medium, followed by cellꢀcounting using
haemocytometer.
a
1
2
.
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Cytotoxicity Assay by MTT
MGCꢀ803 and Hela Cells were seeded in 96ꢀwell plates with a
confluence of about 8000 cells/well. After incubation for 24 h at
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4
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7 °C, 200 µL fresh culture medium containing YJ-1
nanoparticles with the concentrations 10 µM, was added into the
different cell plates. After another 24 h incubation, 30 µL MTT
solution (5 mg/mL) were added to each well and the plates were
incubated for another 4 h at 37 °C. Than the medium was
removed and the yielded purple formazan crystals inside cells
were dissolved by DMSO and OD values were measured at 492
nm using a a microplate reader (SPECTRAmax 384, Molecular
Devices, USA) .
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medium with YJ-1 (10 M). Then, the fixed cells were attained
with 5 ꢁg/mL of DAPI for 15 min for nuclei staining. Cell
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(
CLSM, Nikon, Japan).
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5
4
00 ꢁL of YJ-1 in chloroform solution (2 mg/mL) was added to
00 ꢁL of DSPEꢀmPEG5000 in chloroform solution (10 mg/mL).
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vacuum in a rotary evaporator at 35 °C to remove the
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Animals
Female white mice (ICR line) were obtained from the Laboratory
Animal Center of Zhejiang University (Hangzhou, China). Mice
8
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012

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Journal Name
RSC Advances
View Article Online
ART69ICLE
DOI: 10.1039/C6RA112 B
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RSC Advances
Page 10 of 10
View Article Online
DOI: 10.1039/C6RA11269B
Graphical Abstract
Synthesis, two-photon absorption and
aggregation-induced emission properties of
multi-branched triphenylamine deriva
tives based on diketopyrrolopyrrole for
bioimaging
Ji Yang, Haoqi Tan, Dongyu Li, Tao Jiang, Yuting
Gao, Bo Li, Xue Qu,* and Jianli Hua*
Three new diketopyrrolopyrrole (DPP)-based
multi-branched derivatives (YJ-1-3) with AIE and
2
PA properties were designed and synthesized.
YJ-1 containing triphenylamine donor was applied
for cell imaging and two-photon excited
fluorescence in vivo imaging of mouse ear.