Schiff base derivatives containing thiodiazole: Twisted molecular conformation avoiding π … π stacking promotion aggregation-induced emission enhancement

Article abstract of DOI:10.1016/j.dyepig.2017.05.059

Three novel 2-(4-methoxyphenyl)-1, 3, 4-thiadiazole based Schiff base derivatives (T1-T3) had been synthesized and characterized by¹H NMR,¹³C NMR, MS and IR. The spectroscopic properties of T1-T3 were investigated by UV–vis absorptio

Full text of DOI:10.1016/j.dyepig.2017.05.059

Accepted Manuscript
Schiff base derivatives containing thiodiazole: Twisted molecular conformation
avoiding π … π stacking promotion aggregation-induced emission enhancement
Shun Yao, Xin Zhang, Haiyan Wang, Hui Wang, Xiaoping Gan, Zhichao Wu, Yupeng
Tian, Zhongwen Pan, Hongping Zhou
PII:
S0143-7208(17)30949-X
10.1016/j.dyepig.2017.05.059
DYPI 6021
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 25 April 2017
Revised Date: 28 May 2017
Accepted Date: 31 May 2017
Please cite this article as: Yao S, Zhang X, Wang H, Wang H, Gan X, Wu Z, Tian Y, Pan Z, Zhou H,
Schiff base derivatives containing thiodiazole: Twisted molecular conformation avoiding π … π stacking
promotion aggregation-induced emission enhancement, Dyes and Pigments (2017), doi: 10.1016/
j.dyepig.2017.05.059.
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ACCEPTED MANUSCRIPT
The structure-property relationship of three new AIEE compounds were discussed, which showed the twisted
molecular conformation avoiding π···π stacking facilitated aggregation-induced emission enhancement.

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Schiff base derivatives containing thiodiazole:
twisted molecular conformation avoiding π···π stacking promotion
aggregation-induced emission enhancement
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Shun Yaoa, Xin Zhanga, Haiyan Wanga, Hui Wanga, Xiaoping Ganb, c *, Zhichao Wua, Yupeng Tiana,
Zhongwen Pana, *, Hongping Zhoua, *
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College of Chemistry and Chemical Engineering, Anhui University and Key Labotatory of
Functional Inorganic Materials Chemistry of Anhui Province, Anhui Province Key Laboratory of
Chemistry for Inorganic/Organic Hybrid Functionalized Materials, 230601, Hefei, P.R. China.
b School of Science, Anhui Agricultural University, 230036 Hefei, P. R. China.
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c
Co-operative Innovation Research Center for Weak Signal-Detecting Materials and Devices
Integration, Anhui University, Hefei 230601, P. R. China.
Corresponding author. Fax: +86-551-63861279
E-mail: zhpzhp@263.net
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Abstract
Three novel 2-(4-methoxyphenyl)-1, 3, 4-thiadiazole based Schiff base derivatives
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(T1-T3) had been synthesized and characterized by H NMR, C NMR, MS and IR. The
spectroscopic properties of T1-T3 were investigated by UV–vis absorption and
fluorescence emission spectroscopy, which were coincident with theoretical calculations
that three compounds possessed obvious intramolecular charge transfer (ICT) process.
Their fluorescence emission performance in solution and aggregation state showed that
three compounds all owned obvious aggregation-induced emission enhancement (AIEE)
properties. To further understand AIEE nature, dynamic light scattering (DLS) and
scanning electron microscopy (SEM) were comparatively investigated, which demonstrated
that the small and uniform particles contributed to improve fluorescence emissions. Crystal
structures of T1 and T3 showed that the twisted molecular conformation played an
important role in AIEE. What’s more, T2 and T3 had good biocompatibility, which were
successfully applied in live-cell imaging.
Keywords: Schiff base, aggregation-induced emission enhancement, bio-imaging
32 1. Introduction
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Designing novel chromophores with excellent optical properties in the condensed state
had been a popular research area because of their potential applications in organic
light-emitting diodes, light-emitting liquid crystals, circularly polarized luminescence
(CPL), optical waveguides, and fluorescent sensors, especially fluorescent probe, which
included chemical sensors, biological probes for cellular, bacterial and tissue imaging [1-4].
However, the frequently reported aggregation-caused quenching fluorescence (ACQ)
occurred in common fluorescent molecules under high concentration solution or condensed
state had greatly limited their potential applications. Fortunately, in 2001, Tang and
co-workers firstly discovered a novel luminescence phenomenon defined as
aggregation-induced emission (AIE), which could overcome the defect of ACQ [5-7]. In
2002, Park et al. also reported aggregation-induced emission enhancement (AIEE)
materials were similar to AIE materials. The most popular AIE/AIEE mechanism at the
single molecular level involved the restriction of intramolecular rotation (RIR) [8-10], viz,
the σ bonds connecting the periphery phenyl rings and the core structure were able to rotate
freely in dilute solution, which consumed the excited state energy and hence quenched the
emission. However, in an aggregated state, such as nanoparticles or solid thin
films/powders, the molecules were in a crowded neighborhood and these intramolecular
motions were restricted, which diminished the non-radiative decay of the excited state
energy and consequently induced strong emission [11]. According to the RIR mechanism,
various excellent chromophores with aggregation-induced emission or enhanced emission
(AIE/AIEE) properties had been developed, including tetraphenylethene (TPE),
hexaphenylsilole (HPS), 1-methyl-pentaphenylsilole (MPPS), 1, 1-dimethyl-2, 3, 4,
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5-tetraphenylsilole
(DMTPS),
distyreneanthracene
(DSA),
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3-dicyano-5,
6-diphenylpyrazine, organoboron complexes and salicylaldehyde azine (SAA), and so on,
which were widely applied in the novel luminogenic material [12-13]. These reported
AIE/AIEE molecules effectively promoted the application of fluorescent probe in
biological domain, while the new AIEE molecules still need further development，
especially thiodiazole derivatives applied to luminescent material were rarely reported.
Thiodiazole derivatives had the vast potentials and demands in disinfection, weedicide,
and sterilization due to it possessed a variety of biological activities [14]. Moreover,
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thiodiazole molecule was also a highly conjugated aromatic heterocyclic which could be
applied in optical materials. Triphenylamine had been widely used in opto- and electro-
active materials due to its good electron-donating and hole-transporting capabilities, as well
as the special propeller starburst molecular structure [15-16]. Schiff base compounds
usually displayed superior AIEE property due to the isomerization/rotation of C=N group,
and the intramolecular hydrogen bond which restricted the intermolecular rotation
process[17-19].
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Considering the factors mentioned above, a series of AIEE luminogenic compounds
were developed by a simple preparation and purification at lower cost and shown in
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Scheme 1. The spectroscopic properties, DLS, SEM and crystal structure (T1 and T3)
were researched in detail to elucidate the mechanism of enhanced fluorescence in the
aggregated state, furthermore, T2 and T3 were successfully applied in live-cell imaging.
13 2. Experimental
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2.1 Materials and instruments
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Chemicals were purchased and used as received. Every solvent was purified as
conventional methods beforehand. IR spectra were recorded with a Fourier transform
infrared microscopy (VERTEX 80+HYPERION2000) in the 4000 - 400 cm-1 region.
Melting points (uncorrected) were determined on a XT4 MP apparatus (Taike Corp.,
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Beijing, China). H and 13C NMR were recorded on 400 MHz and 100 MHz NMR
instruments using DMSO or CDCl3 as solvent. Chemical shifts were reported in parts per
million (ppm) relative to internal TMS (0 ppm) and coupling constants in Hz. Splitting
patterns were described as singlet (s), doublet (d), triplet (t), quartet (q). The mass spectra
were obtained on a Bruker Autoflex III smart beam mass spectrometer. The X-ray
diffraction measurements were performed on a CCD area detector using graphite
monochromated MoKa radiation (λ = 0.71069 (Å) at 298 (2) K. The non-hydrogen atoms
were refined anisotropically and hydrogen atoms were introduced geometrically.
Calculations were performed with SHELXTL-97 program package. Crystallographic data
have been deposited with the Cambridge Crystallographic Data Centre as supplementary
publication no. CCDC: 1498598 (for T1), 1498596 (for T3). DLS measurements were
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conducted on a Malvern Zetasizer Nano ZS90 size analyzer. SEM images were obtained
using a Zeiss MERLIN Compact. The one-photon absorption (OPA) spectra were recorded
on the UV-3600 spectrophotometer and one-photon emission fluorescence (OPEF) spectra
measurements were performed using a Hitachi F-7000 fluorescence spectrophotometer. The
quartz cuvettes used were of 1 cm path length. The slit pass width of emission spectra: 10
nm, Voltage: 500 V.
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2.2.1 Synthesis of 1
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p-Anisic acid (3.04 g, 20 mmol) and thiosemicarbazide (1.82 g, 20 mmol) were
dissolved in 30 mL of phosphorus oxychloride，the reaction mixture was refluxed at 85 0C
for 10 h. After the reaction finished, the mixture was poured into ice water slowly and then
saturate NaOH solution was added to adjust the pH value to 8.0 under vigorous stirring.
The mixture was extracted with ethyl acetate three times and the organic layer was washed
with brine and dried over anhydrous sodium sulfate. After solvent evaporation, the crude
product was purified by recrystallization from anhydrous ethanol to get 3.15 g white
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powder, yield: 76.09%. Melting point: 188.1-189.5 C. FT-IR (KBr, Disc, cm−1): 3409.28
(m, νNH2), 3377.72 (m,νNH2), 3304.97 (w), 3105.35 (m), 1647.15 (m), 1608.48 (m, νbenzene),
1578.80 (w), 1511.49 (s, νbenzene), 1465.51(s), 1304.97 (w), 1267.34 (w), 1246.65 (s, νOCH3),
1174.54 (m, νOCH3), 1128.97 (w), 1052.67 (w), 1031.98 (m), 978.88 (w), 829.22 (m),
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658.86 (w), 520.48 (w). H NMR (DMSO-d6, 400 MHz, ppm) δ: 7.69-7.67 (d, J = 8.0 Hz,
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2H), 7.28 (s, 2H, NH2), 7.03-7.01 (d, J = 8.0, 2H), 3.80 (s, 3H, OCH3). C NMR (100
MHz, DMSO) δ 167.85, 160.24， 156.25, 127.77, 123.61, 144.46，55.27. MS (ESI) m/z:
found, [M+H]+, 208.0542; molecular formula C9H9N3OS requires [M+H]+, 208.0544.
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2.2.2 Synthesis of 2-4
The compounds 2, 3 and 4 [20-21] were prepared according to the reported method.
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2.2.3 Synthesis of T1-T3
Scheme 1 Preparation of compounds T1, T2, T3
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T1 (0.271 g, 1 mmol) and compound 1 (0.248 g, 1.2 mmol) were dissolved in 15 mL of
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anhydrous alcohol, the reaction mixture was refluxed at 70 C for 12 h. After the reaction
finished, the solution was cooled to room temperature, the solid was filtered and washed
two times with anhydrous ethanol to give 0.37 g green solid, yield: 80.43 %. Melting point:
217.4-218.5 °C. FT- IR (KBr, Disc, cm−1): 1601.19 (s, νbenzene), 1582.00 (m), 1558.11 (m),
1514.46 (s, νbenzene), 1445.05 (s), 1417.20 (m), 1360.58 (m), 1337.06 (m), 1312.98 (w),
1252.84 (m), 1224.75 (m, νOCH3), 1178.97 (m, νOCH3), 1159.28 (m, νOCH3), 825.46 (m),
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737.66 (m), 718.23 (m). H NMR (DMSO-d6, 400 MHz, ppm) δ: 9.20 (s, 1H, CH=N),
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8.38-8.36 (d, J = 8.0 Hz, 2H), 8.30-8.28 (d, J = 8.0 Hz, 2H), 7.98-7.91 (q, 4H), 7.58-7.56 (d,
J = 8.0 Hz, 2H), 7.51-7.48 (t, 2H), 7.37-7.34 (t, 2H), 7.16-7.14 (d, J = 8.0 Hz, 2H), 3.85(s,
3H, OCH3). 13C NMR (100 MHz, CDCl3) δ 172.83, 167.39, 165.10, 162.03, 142.39, 140.16,
133.11, 131.72, 129.25, 126.89, 126.27, 123.95, 123.25, 120.74, 120.49, 114.59, 109.86,
55.50.MS (ESI) m/z: found, [M+H]+, 461.1427; molecular formula C28H20N4OS requires
[M+H]+, 461.1436.
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T2 and T3 were obtained by following the similar procedure of T1.
T2: yellow powder, 0.35 g, yield: 75.76 %. Melting point: 184.6-185.7 0C. FT- IR (KBr,
Disc, cm−1): 1606.39 (m, νC=N), 1582.88 (s), 1552.78 (m), 1509.19 (m, νbenzene), 1490.09 (m,
νbenzene), 1442.73 (m), 1332.67 (m), 1301.00 (m), 1284.38 (m), 1257.11 (m), 1170.88 (s,
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νOCH3), 829.85 (w), 700.67 (w). H NMR (DMSO-d6, 400 MHz, ppm) δ: 8.83 (s, 1H,
CH=N), 7.91-7.88 (q, 4H), 7.45-7.41 (t, J = 8.0, 4H), 7.26-7.21 (q, 6H), 7.12-7.10 (d, J = 8
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Hz, 2H), 6.92-6.90 (d, J = 8Hz, 2H), 3.85 (s, 3H, OCH3). C NMR (100 MHz, DMSO) δ
173.37, 167.44, 164.88, 161.49, 152.32, 145.54, 131.83, 130.00, 128.86, 126.33, 125.44,
122.71, 118.58, 114.84, 55.47. MS (ESI) m/z: found, [M+H]+, 463.1584; molecular
formula C28H22N4OS requires [M+H]+, 463.1592.
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T3: orange solid, 0.48 g, yield: 70.69 %. Melting point: 239.3-240.6 C. FT- IR (KBr,
Disc, cm−1): 1608.27 (m, νbenzene), 1587.27 (s), 1505.43 (s, νbenzene), 1444.61 (m), 1413.57
(w), 1325.46 (m), 1305.39 (m), 1287.21 (m), 1256.48 (s), 1168.06 (s, νOCH3), 832.15 (w).
1H NMR (DMSO-d6, 400 MHz, ppm) δ: 8.95(s, 2H, CH=N), 8.03-8.01 (d, J = 8.0, 4H),
7.94-7.91 (d, J = 12 Hz, 4H),7.52-7.49 (t, 2H), 7.36-7.32 (t, J = 8.0Hz, 1H), 7.28-7.20(q,
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6H), 7.14-7.12 (d, J = 8.0 Hz, 4H), 3.86(s, 6H, OCH3). C NMR (100 MHz, CDCl3) δ
173.37, 166.64, 165.22, 161.89, 151.20, 145.50, 131.70, 130.11, 129.32, 129.17, 126.96,
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126.16, 123.38, 123.02, 114.52, 55.47. MS (ESI) m/z: found, [M+H]+, 680.1889; molecular
formula C38H29N7O2S2 requires [M+H]+, 680.1902.
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3. Results and discussion
3.1 Spectroscopic properties
Fig. 1 Absorption (a) and fluorescence spectra (b) of T2 in five organic solvents with different polarities
at a concentration of 1×10-5 mol L-1.
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The absorption and fluorescence spectra were depicted in Fig. 1 and Fig. S2-S3, the
corresponding spectroscopic data were collected in Table. S1. Taking T2 as an example, it
could be seen from Fig. 1a that T2 had two absorption peaks locted at ~293 nm and ~425
nm, respectively, the former was assigned to the π-π* electronic transition caused by the
triphenylamine core whereas the latter was likely ascribed to intramolecular charge transfer
between the triphenylamine core and the terminal group. Fig. S2a-3a showed T1 and T3
had similar feature in absorption spectra compared with T2. The fluorescence spectrum of
T2 (Fig. 1b) exhibited one emission peak located from 528 nm to 568 nm in different
polarity solvents, which was assigned to the ICT emission [22]. As shown in Table. S1 and
Fig. 1b the fluorescence emission of T2 was obvious red-shifted with increasing solvent
polarity, Fig. S2b-3b presented T1 and T3 had the similar phenomenon compared with T2.
Large stokes shifts were observed for the three compounds in five solvents due to strong
solvent-solute dipole-dipole interactions, a manifestation of the large dipole moment and
orientational polarizability. An increased dipole–dipole interaction between the solute and
solvent generate a lower energy level. Stokes shift were approximately proportional to the
orientational polarizability. To discuss the effect of solvents on the fluorescence spectrum,
Lippert-Mataga plots for T1-T3 were given in Fig. S4 and the calculation dates were
collected in Table S2 [23-24].
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in which ∆ν = νabs –νem stands for Stokes shift, νabs and νem are absorption and emission
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(cm-1), h is the Planck constant, c is the velocity of light in vacuum, a is the Onsager radius
and b is a constant. ∆f is the orientation polarizability, µe and µg are the dipole moments of
the emissive and ground states, respectively, and ε0 is the permittivity of the vacuum. (µe -
µg)2 is proportional to the slope of the Lippert-Mataga plot. Fig. S4 showed the slopes of
the fitting line for compounds T1-T3 were as high as 17268, 10643 and 7066, respectively.
The large slopes verified large dipole moment changes for these compounds, the large
values of µe - µg indicated that the molecule in the excited state had an extremely polar
structure.
3.2 Electronic Structure
Fig. 2 Electron density distributions of the frontier molecular orbitals of compounds T1-T3
To understand the relationship between the optical properties and the electronic structure,
the highest occupied and the lowest unoccupied molecular orbitals (HOMO and LUMO)
were obtained by Gaussian 03 program and the B3LYP/6-31G basis sets based on crystal
structure (T1 and T3) and optimized structure (T2), the results were similar to all of which
based on optimized structure. Most of the electron clouds of the HOMO were evenly
distributed in 9-phenyl-carbazole and triphenylamine part, while the majority of electron
clouds of the LUMO were transferred and evenly distributed in the Schiff base (C=N) and
1, 3, 4-thiadiazole moieties, which revealed that absorption and emission of the three
compounds originated from the ICT transitions [25]. It can be seen that the energy gaps
calculated of the chromophores followed the order ∆E(T3) < ∆E(T2) < ∆E(T1) from Fig. 2,
while the linear absorption wavelength increased in the order T3 > T2 > T1 in five solution,
which were well consistent with experiments.
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3.3 Aggregation-induced emission enhancement
Fig. 3 Absorption (a) and fluorescence spectra (b) of T2 in ethanol/water mixtures with different fw at a
concentration of 1×10-5 mol L-1. The inset depicts the changes of fluorescence intensity with different
fw .
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Considering the three compounds were insoluble in water but soluble in organic solvents,
we determined the absorption and emission spectra of the T1-T3 in organic/organic-water
mixtures with different water fractions (fw, the volume percentage of water in organic-water
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mixtures, that can adjust the solvent polarity subtly), which were revealed by the images in
Fig. 3 and Fig. S5-S6. We used T2 as example to discuss the AIEE property. The
absorption and fluorescence spectra of T2 were investigated in mixtures of ethanol/water
with various water volume fractions (fw). Fig. 3a showed T2 had a large absorption peaks at
430 nm, meanwhile, when fw ≥ 70%, the absorption spectra started to show level-off tails in
the long wavelength region caused by the Mie scattering effect, which indicated the
formation of nanoaggregates. Similar effects of T1 and T3 were observed in Fig. S5a-S6a.
The fluorescence spectra of T2 in the ethanol/water mixture with different water contents
were shown in Fig. 3b, which exhibited a strong AIEE activity. When fw ≤ 60%, the
fluorescence (FL) intensity primarily decreased with increasing fw, which was due to the
increase of the solvent polarity and then transform to the twisted intramolecular charge
transfer (TICT) state [26-27]. The light emission was invigorated from fw ≥ 70% and
reached its maximum value at 90% water content, which was 4.2-fold higher than that in
pure ethanol solution, accompanying a small red-shift in whole spectra. The increased in
FL intensity can be attributed to the AIEE effect. We can also clearly see that the
fluorescence spectra of T3 in ethanol/water mixtures had similar trend with that of T2 (Fig.
S6b). Fig. S5b showed that T1 had two emission peaks in dense THF solution, located at
λem1 (~450 nm) and λem2 (~500 nm), which had a slightly difference compared with that of
T2 and T3. As fw increased, λem1 disappeared gradually, the emission located at λem2
increased correspondingly. The FL intensity was very weak accompanying with the slow
increase as fw from 0% to 60%, and a significant enhancement of fluorescence was observed
when fw > 70% accompanying a small red-shift in the spectrum. It was noteworthy that the
fluorescence intensity of fw = 70% lower than that of fw = 60%, this phenomenon was often
observed in some compounds with AIEE properties [28-30], but the reasons remained
unclear. There were two possible explanations for this phenomenon, first, after the
aggregation, only the molecules on the surface of the nanoparticles emitted light and
contributed to the fluorescence intensity upon excitation, leading to a decrease in
fluorescence intensity. However, the restriction of intramolecular rotations of the aromatic
rings in the aggregation state could enhanced light emission. The net outcome of these
antagonistic processes depended on which process played a predominant role in affecting
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the fluorescence behavior of the aggregated molecules. Second, when water was added, the
solute molecules can aggregate into two kinds of nanoparticle suspensions: crystal particles
and amorphous particles. The former leaded to an enhancement in the fluorescence
intensity, while the latter leaded to a reduction in intensity. However, it was difficult to
control the formation of nanoparticles in high water content. Thus, the measured
fluorescence intensity often showed no regularity in high water.
Fig.4 Particle size distribution and SEM images of T2 in the ethanol/water mixtures with different fw at a
concentration of 1×10-5 mol L-1: (a, a1) in ethanol/water (20/80, v/v); (b, b1) in ethanol/water (10/90,
v/v); (c, c1) in ethanol/water (5/95, v/v) .
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To further elucidate the influence of morphology and particle size on AIEE properties,
SEM and DLS studies were supplemented. As shown in Fig. 4, the size distribution of T2
were 241.2 nm (fw = 80%), 135.3 nm (fw = 90%) and 164.0 nm (fw = 95%) respectively,
which was conducted by DLS in solvent mixtures with high water ratios. SEM was used to
observe the aggregation, which were also well certified the nano-level aggregation of T2 at
the same water fractions as DLS. The results illustrated that the enhanced emission was
related to the small and uniform particles, the reason may be that the weak intermolecular
interactions could work efficiently under ordered aggregation condition. Fig. S7-S8 showed
that T1 and T3 had the similar size distribution influence on AIEE properties in mixture
solution.
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3.4 Mechanisms of emission enhancement
Crystal structure was most important for us to understand the optoelectronic properties
and molecular packing, which will help deeply our understanding of luminescence
processes and guided us to develop new AIEE systems. Single crystals of T1 and T3 were
obtained by slow evaporation from the solutions in dichloromethane / acetonitrile at room
temperature. The crystal structures of T1 and T3 were shown in Fig. 5 and Fig. S9. Their
crystallographic dates were summarized in Table. S3. The ORTEP diagrams of T1 and T3
with the N, O and S atom numbering schemes and some of the packing interactions in the
crystals were depicted in Fig. 5a and Fig. S9a.
As shown in Fig. 5b and Fig. S9b, the dihedral angles between the plane of the 1, 3,
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4-thiadiazole ring and the adjacent benzene ring are 5.85° for T1 and 7.98° for T3, which
indicated that the T1 and T3 had good planarity. For compound T1, we could see that the
∠
C-H···N hydrogen bonds (d = 2.494 Å C18-H18···N3 = 173.66°) and C-H···π weak
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interactions (C8-H8···π, d = 2.894 Å) played significant role in the growth of 1D chain,
then the 2D layer structure formed through C-H···N hydrogen bonds (d = 2.724 Å
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C5-H5···N4 = 134.89°) (Fig. 5c-5d). However, the vertical distances between the
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terminal benzene ring and double bonds (C=N) was 3.454 Å and the slip angles was 33.90°
in the neighboring molecules, which indicated the molecule had π···π packing interactions
(Fig. 5e), this was why T1 only possessed weak FL intensity in aggregation state.
For compound T3, every two molecules were packed into a small unit, the adjacent three
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small units were stacked together to form the 1D chain structure via three kinds of C-H···
O
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(d = 2.521 Å, C50-H50-O1 = 147.22°, d = 2.501 Å, C5-H5-O2 =
hydrogen bonds
145.04° and d = 2.664 Å C1-H1-O3 = 151.02°
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and two kinds of C-H···π interactions
(C54-H54···π, d = 2.813 Å and C24-H24···π, d = 2.842 Å) (Fig. S9c). Furthermore, the
two 1D chain structure were connected to form the 2D layer structure through C-H···π
(C33-H33···π, d = 2.820 Å) interactions (Fig. S9d). The results illustrated that twisted
molecular conformation could avoid π···π stacking and boost aggregation induced emission
enhancement.
Fig. 5 Crystal structure of T1, (a) ORTEP diagram of T1, (b) The dihedral angles of T1, (c) The 1D
linear structure of T1, (d) The 2D linear structure of T1,(e)The π···π stacking.
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3.5 Cell Imaging
To explore the application in biology for T1-T3, the bio-imaging experiments were
carried out by confocal laser scanning microscopy using HepG2 cells as an example. The
tested compounds were dissolved in DMSO and then serially diluted in complete culture
medium. The excitation wavelength was fixed at their maximum absorption wavelength in
microscopy imaging. From Fig.6 a2-a3, we can see that the bright-yellow fluorescence
mainly focused on the cytoplasm for T2 and T3, which confirmed that the two compounds
both had good biocompatibility and could enter into the cells and aggregate in the
cytoplasm successfully. But, any fluorescence in live-cell for T1 could not observed
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although we had tried many times (Fig. 6a1), which could be attributed the weak FL
intensity caused by the particular molecular structure
Fig. 6 (a1 - a3) Fluorescence image of HepG2 cells with T1-T3 after 15min of incubation and washed
with PBS buffer. (b1 - b3) Bright-field images, (c1 - c3) Merged images
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4. Conclusion
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5
In summary, three AIEE-active compounds have been designed and synthesized by
subtle structure adjustment. Their fluorescence emission performance in ethanol/water or
THF/water solutions were obviously different, DLS and SEM images of the compounds
revealed that the small and uniform particles were in favor of fluorescence emissions.
Crystal structure demonstrated that the structural variations had a great influence on
photophysical properties, molecular packing, electronic structure, and aggregation-induced
fluorescence properties. Furthermore, T2 and T3 were successfully applied to label the
cytoplasm in live cells, which could offer a new direction for developing biological probe
based on AIEE luminophores.
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13 Acknowledgements
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This work was supported by General Program of National Natural Science Foundation of
China (51472002, 51432001), Science and Technology Plan of Anhui Province
(1604b0602016), the Ministry of Education of China, Higher Education Revitalization Plan
Talent Project of Anhui Province (2013) and Open Foundation of Co-operative Innovation
Research Center for Weak Signal-Detecting Materials and Device Integration of Anhui
University (Y01008411).
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Figure captions
Scheme 1 Preparation of compounds T1, T2, T3.
Fig. 1 Absorption (a) and fluorescence spectra (b) of T2 in five organic solvents with
different polarities at a concentration of 1×10-5 mol L-1.
Fig. 2 Electron density distributions of the frontier molecular orbitals of compounds
T1-T3.
Fig. 3 Absorption (a) and fluorescence spectra (b) of T2 in ethanol/water mixtures
with different fw at a concentration of 1×10-5 mol L-1. The inset depicts the changes of
fluorescence intensity with different fw .
Fig.4 Particle size distribution and SEM images of T2 in the ethanol/water mixtures
with different fw at a concentration of 1×10-5 mol L-1: (a, a1) in ethanol/water (20/80,
v/v); (b, b1) in ethanol/water (10/90, v/v); (c, c1) in ethanol/water (5/95, v/v).
Fig. 5 Crystal structure of T1, (a) ORTEP diagram of T1, (b) The dihedral angles of
T1, (c) The 1D linear structure of T1, (d) The 2D linear structure of T1,(e)The weak
π···π stacking.
Fig. 6 (a1 - a3) Fluorescence image of HepG2 cells with T1-T3 after 15min of
incubation and washed with PBS buffer. (b1 - b3) Bright-field images, (c1 - c3)
Merged images.

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Scheme 1 Preparation of compounds T1, T2, T3
Fig. 1 Absorption (a) and fluorescence spectra (b) of T2 in five organic solvents with
different polarities at a concentration of 1×10-5 mol L-1.

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Fig. 2 Electron density distributions of the frontier molecular orbitals of compounds
T1-T3
Fig. 3 Absorption (a) and fluorescence spectra (b) of T2 in ethanol/water mixtures
with different fw at a concentration of 1×10-5 mol L-1. The inset depicts the changes of
fluorescence intensity with different fw .

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Fig.4 Particle size distribution and SEM images of T2 in the ethanol/water mixtures
with different fw at a concentration of 1×10-5 mol L-1: (a, a1) in ethanol/water (20/80,
v/v); (b, b1) in ethanol/water (10/90, v/v); (c, c1) in ethanol/water (5/95, v/v) .

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Fig. 5 Crystal structure of T1, (a) ORTEP diagram of T1, (b) The dihedral angles of
T1, (c) The 1D linear structure of T1, (d) The 2D linear structure of T1,(e)The weak
π···π stacking.
Fig. 6 (a1 - a3) Fluorescence image of HepG2 cells with T1-T3 after 15min of
incubation and washed with PBS buffer. (b1 - b3) Bright-field images, (c1 - c3)
Merged images

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Highlights
‹
‹
‹
The structures of T1 and T3 were determined by single crystal X-ray diffraction
analysis.
The twisted molecular conformation facilitated aggregation-induced emission
enhancement.
T2 and T3 were applied in live cell imaging, which highlighted their practical
performance.