Facile fabrication of AIE/AIEE-active fluorescent nanoparticles based on barbituric for cell imaging applications

Article abstract of DOI:10.1039/c7ra03956e

Four barbituric derivatives (1-4), were synthesized with obvious aggregation induced emission (AIE) or aggregation induced emission enhancement (AIEE) behaviors. The optical properties in different states and the mechanisms of the enhanced emission of 1-4 were investigated. We found that there were a large number of globular or blocky nanoparticles with average diameters from 229 to 394 nm in tetrahydrofuran (THF)/water solutions when the water fraction (f_w) was 90%. The single crystal data of 2 and 4 reveal that multiple intermolecular interactions restrict the intramolecular motions, and promote the formation of a herringbone arrangement (4), thus permitting intense emission in the aggregate state. In addition, the frontier molecular orbital energy and band gap calculated by density functional theory (DFT) are consistent with the experimental results. Compound 4 is cell-permeable: upon entering the live cells, the dye molecules can accumulate in the lysosomes and turn on the fluorescence.

Full text of DOI:10.1039/c7ra03956e

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Facile fabrication of AIE/AIEE-active fluorescent
nanoparticles based on barbituric for cell imaging
applications†
Cite this: RSC Adv., 2017, 7, 30229
Kai Li,a Yang Zhang,b Bing Qiao,c Furong Tao,a Tianduo Li,a Yunqiao Ding,a
b
a
*
Françisco M. Raymo
and Yuezhi Cui
Four barbituric derivatives (1–4), were synthesized with obvious aggregation induced emission (AIE) or
aggregation induced emission enhancement (AIEE) behaviors. The optical properties in different states
and the mechanisms of the enhanced emission of 1–4 were investigated. We found that there were
a large number of globular or blocky nanoparticles with average diameters from 229 to 394 nm in
tetrahydrofuran (THF)/water solutions when the water fraction (fw) was 90%. The single crystal data of 2
and 4 reveal that multiple intermolecular interactions restrict the intramolecular motions, and promote
the formation of a herringbone arrangement (4), thus permitting intense emission in the aggregate state.
In addition, the frontier molecular orbital energy and band gap calculated by density functional theory
(DFT) are consistent with the experimental results. Compound 4 is cell-permeable: upon entering the
live cells, the dye molecules can accumulate in the lysosomes and turn on the fluorescence.
Received 7th April 2017
Accepted 5th June 2017
DOI: 10.1039/c7ra03956e
rsc.li/rsc-advances
cell imaging covers the entire visible light range from blue to
red.15–18 However, AIE-luminophores with red and near-infrared
1. Introduction
emission are desired for avoiding possible autouorescence
from biological samples and reducing photocytotoxicity.19–21 To
address these issues, a few examples have been reported to
extend the electronic conjugation of the uorophores, thus
decreasing the energy gap between the electronic ground state
and excited state and consequently tuning the absorption and
emission characters bathochromically.22 Nonetheless, such
a strategy has the disadvantages of tedious synthesis, high
synthetic workload, interchromophoric induced uorescence
quenching, poor photostability and solubility.23 Alternatively,
electron-donating (D) and accepting (A) moieties can be incor-
porated into the design of molecular skeleton. D–p–A molecules
typically form charge-transfer (CT) state with a smaller band gap
in polar solvents, thus shiing the emission to longer wave-
lengths.24 Barbituric acid derivatives display diverse biological
activities, e.g., anesthetic, hypotensive, antibacterial and anti-
tumor activities, and sedative effects.25 And they can be readily
linked with chromophore substrates through extensively-studied,
highly-efficient Knoevenagel reactions, under mild conditions
and with no requirement for a special catalyst.26 Triphenylamine
and barbituric acid derived push-p-pull systems have been re-
ported by Kim et al. to act as dye molecules.27 Shi et al. reported
a type of optical probe for Hg2+ via the formation of a mercury(II)-
barbituric acid coordination complex.28 Zhou et al. synthesized
and evaluated near-infrared probes with barbituric acid acceptors
for in vivo detection of amyloid plaques.29
Most luminescent materials suffer from a severe effect called
aggregation-caused quenching (ACQ), and their luminescence
is quenched in high molecular concentrations or in the aggre-
gation state.1,2 These organic materials exhibit relatively strong
uorescence in dilute solution, while only weak or non-emissive
uorescence is detected in the aggregated states or solid states,
because strong intermolecular interactions facilitate the non-
radiative decay pathway. In contrast, AIE is an opposite
phenomenon to ACQ, and luminogens with AIE characters can
light up in the aggregated states which conventional uo-
rophores can never achieve.
Due to the unique uorescence properties, luminescent
materials with AIE/AIEE characteristics have attracted consider-
able attention since the discovery of the AIE phenomenon by
Tang in 2001.3,4 AIE/AIEE luminophores have invaluable potential
for sensing and bioimaging applications,5–7 such as optoelec-
tronic devices,8 bio-sensing,9–11 explosive sensors,12 and stimuli
responsive materials.13,14 Up to now, the range of emission
wavelengths of AIE-active molecules that have been developed for
aShandong Provincial Key Laboratory of Fine Chemicals, Qilu University of
Technology, Jinan 250353, P.R. China. E-mail: cyz@qlu.edu.cn; Fax: +86 531
89631760; Tel: +86 531 89631208
bLaboratory for Molecular Photonics, Department of Chemistry, University of Miami,
1301 Memorial Drive, Coral Gables, Florida, 33146-0431, USA
cDepartment of Physics, University of Miami, 1301 Memorial Drive, Coral Gables,
Florida, 33146-0431, USA
The AIE properties of these compounds have not been
investigated except compound 3, and in particular in organism.
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c7ra03956e
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So far, a series of AIE active chromophores have been re- diphenylamino benzaldehyde and N-carbazolyl benzaldehyde
ported and various mechanistic pathways such as conforma- with (1,3-dimethyl) barbituric acid, respectively (Scheme 1).
tional planarization,30 J-aggregate formation,31,32 twisted Then, these luminogens were characterized by 1H NMR (nuclear
intramolecular charge transfer (TICT),33 excited state intra- magnetic resonance spectrometry) and the FTIR (Fourier
molecular proton transfer (ESIPT),34 and restriction of intra- transform infrared spectrometry). Single crystals of compounds
molecular motion (RIM) have been explored to operate AIE 2 and 4 were obtained by slow evaporation of these solutions in
principles by suppressing the intermolecular p–p stacking the n-hexane/CHCl3 solution at room temperature.
interactions.35
2.4.1. Synthesis of 4-(9H-carbazolyl) benzaldehyde (1a). A
Herein, based on the RIM and intramolecular charge trans- mixture of 9H-carbazole (1.7 g, 0.01 mol), and sodium hydroxide
fer (ICT),36 we present a series of AIE/AIEE-active and structur- (1.2 g, 0.03 mol) in dry dimethylformamide (DMF) was heated
ally simple luminogens bearing D–p–A systems. Barbituric acid with stirring. Then 4-uorobenzaldehyde (1.5 g, 0.012 mol) was
ꢀ
is incorporated as the electron-withdrawing group, while tri- added, and the reaction mixture was stirred at 90 C for 24 h.
phenylamine and carbazole moieties are installed as the donor Aer cooling to room temperature, the mixture was poured into
groups because of their electron-rich nature. For these distilled water and extracted with ethyl acetate. The organic
compounds, they exhibit obvious AIE/AIEE behaviors and red layer was dried with anhydrous magnesium sulfate (MgSO4) and
emission. Their mechanisms of enhanced emission behaviors concentrated by vacuum evaporation. The crude product was
can be well explained by intermolecular interaction in their puried by column chromatography using the mixture of
crystal structures. With the D–p–A structures, compound 4 petroleum and dichloromethane (v/v ¼ 15 : 1) as an eluent to
emits at the red visible region of the electromagnetic spectrum get the desired compound as a yellow solid (1.19 g) with a yield
in its aggregate state. Finally, in vivo cell imaging experiments of of 70.1%. 1H NMR (CDCl3) d (ppm): 10.13 (s, 1H), 8.16 (m, 4H),
compound 4 have been performed in Drosophila S2 cells, and 7.81 (d, 2H), 7.52 (d, 2H), 7.45 (t, 2H), 7.34 (t, 2H).
intense intracellular uorescence signal can be detected with
minimal background noise.
2.4.2. Synthesis of 4-(diphenylamino)-benzaldehyde (1b).
2 ml of dry DMF (25.7 mmol) was added dropwise to 5 ml of
phosphorus oxychloride (54 mmol) maintained at 0 ꢀC. Aer the
solution was stirred for 15 min, triphenylamine (1 g, 4 mmol)
was added to the above Vilsmeier reagent, and the mixture was
2. Experimental section
2.1. Materials
ꢀ
heated to 45 C for an additional 2.5 h. Aer cooling, the clear
4-Fluorobenzaldehyde (Jiu Ding Chemistry, 97%), triphenyl-
amine (Aladdin, 99%), carbazole (energy, 98%), N,N-dimethy-
lamino benzaldehyde (Jiu Ding Chemistry, 99%), barbituric
acid (Aladdin, 98%), 1,3-dimethyl-barbituric acid (Aladdin,
99%), were used without further purication. DMF was gener-
red solution was added dropwise to ice water with stirring, and
the resulting mixture was allowed to stand for 2 h. The crude
product was collected by ltration, washed with water, and
dissolved in dichloromethane. The organic solution was
washed with H2O (150 ml) and dried over MgSO4. Aer the
solvent was evaporated off, the residue was recrystallized from
ethyl acetate to afford aldehyde as a pale yellow crystal with
˚
ally dried by molecular sieves (4 A), sodium sands and CaH2
successively, and distilled prior to use. Schneider's Drosophila
line 2 [D. mel. (2), SL2] (ATCC® CRL-1963™) cell line (Drosophila
S2 cell) was purchased from ATCC.
1
a yield of 0.78 g (80%). H NMR (CDCl3) d (ppm): 9.82 (s, 1H),
7.69 (d, 2H), 7.35 (t, 4H), 7.19 (m, 6H), 7.02 (m, 2H).
2.4.3. Synthesis of 5-(N-carbazole styryl)-barbituric acid (1).
A mixture of barbituric acid (0.128 g, 1 mmol) and N-carbazolyl
benzaldehyde (0.274 g, 0.7 mmol) in acetic (15 ml), acetic
anhydride (15 ml) and benzene (5 ml) was added to dissolve the
undissolved N-carbazolyl benzaldehyde, then the mixture was
reuxed for 2 h. The reaction was cooled to room temperature
and the solid particles were ltered. The product was then
puried by recrystallization with acetic acid to give 0.30 g with
75% yield. 1H NMR (DMSO) d (ppm): 11.43 (s, 1H), 11.30 (s, 1H),
8.43 (d, 2H), 8.39 (s, 1H), 8.26 (d, 2H) 7.78 (d, 2H), 7.46 (m, 2H),
7.34 (m, 4H).
2.2. Characterizations
Absorption spectra were recorded using a UV-2500 spectrom-
eter. The uorescence spectra were measured with an F-4600
uorescence spectrophotometer. The luminescence quantum
yields were determined relative to Coumarin 307 in ethanol
solution as a quantum yield standard (F ¼ 0.56). Confocal Laser
Scanning Microscope (CLSM) experiments were carried out with
Leica SP5.
2.3. Computation
2.4.4. Synthesis of 5-(4-dimethylamino styrene)-1,3-
The geometric and electronic structures of 4 compounds were
optimized by density functional theory (DFT) at the B3LYP level.
The HOMO and LUMO energy levels are predicated by 6-31G(d)
basis set. The compositions of molecular orbits were analyzed
using the Gauss View 5.0 program.
dimethyl-barbituric acid (2).
A mixture of 1,3-dimethyl-
barbituric acid (0.78 g, 5 mmol) and N,N-dimethylamino benz-
aldehyde (0.745 g, 5 mmol) in acetic (5 ml) and acetic anhydride
(5 ml) was reuxed for 2 h. The reaction was cooled to room
temperature and the solid particles were ltered. The product
was then puried by recrystallization with acetic acid to give
2.4. Synthesis of compounds
1
0.97 g with 68% yield. H NMR (CDCl3) d (ppm): 8.45 (s, 1H),
The target luminogens were synthesized by the Knoevenagel 8.40 (d, 2H), 6.76 (d, 2H), 3.41 (d, 6H), 3.17 (s, 6H).
condensation of N,N-dimethylamino benzaldehyde, N,N-
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Scheme 1 Synthetic routes to 1–4.
(100 mL) and imaged. All images were recorded with a Leica SP5
confocal laser scanning microscope (CLSM).37
2.4.5. Synthesis of 5-(4-diphenylamino styrene)-barbituric
acid (3). A mixture of barbituric acid (0.384 g, 3 mmol) and 4-
(diphenylamino) benzaldehyde (0.447 g, 3 mmol) in acetic (5
ml) and acetic anhydride (5 ml) was reuxed for 2 h. The reac-
tion was cooled to room temperature and the solid particles
were ltered. The product was then puried by recrystallization
with acetic acid to give 0.87 g with 78% yield. 1H NMR (DMSO)
d (ppm): 11.21 (s, 1H), 11.07 (s, 1H), 8.28 (d, 2H), 8.15 (s, 1H),
7.44 (t, 4H), 7.25 (m, 6H), 6.76 (d, 2H).
For the colocalization experiments, a PBS solution (50%, v/v)
of 4 (0.1 mM) was added to the cells for 2 hour, and washed
three times with PBS (100 mL). Then PBS solutions (50%, v/v) of
40,6-diamidino-2-phenylindole (DAPI) or Lysotracker Blue (10
mM) were added to the cells for 30 min and washed three times
with PBS (100 mL). The uorescence of DAPI or Lysotracker Blue
was recorded in blue channels (lEx ¼ 405 nm, lEm ¼ 420–470
nm), the uorescence of compound 4 was recorded in red
channel (lEx ¼ 488 nm, lEm ¼ 500–600 nm).
2.4.6. Synthesis of 5-(4-diphenylamino styrene)-1,3-
dimethyl-barbituric acid (4).
A mixture of 1,3-dimethyl-
barbituric acid (0.163 g, 1 mmol) and 4-(diphenylamino) benz-
aldehyde (0.276 g, 1 mmol) in acetic (5 ml) and acetic anhydride
(5 ml) was reuxed for 2 h. The reaction was cooled to room
temperature and the solid particles were ltered. The product
was then puried by recrystallization with acetic acid to give
3. Results and discussion
3.1. Optical properties
Optical properties of the compounds 1–4 were investigated by
UV-vis and uorescence spectroscopy. All the compounds
demonstrate good solubility in typical organic solvents such as
THF, DMF, chloroform, and DMSO, but they are hardly soluble
in water. The UV-vis absorption spectra of compounds 1–4 are
presented in Fig. 1.
1
0.27 g with 70% yield. H NMR (CDCl3) d (ppm): 8.44 (s, 1H),
8.24 (d, 2H), 7.38 (t, 4H), 7.23 (m, 6H), 6.95 (d, 2H), 3.40 (d, 6H).
2.5. Cell culture and imaging
Drosophila melanogaster S2 cells were cultured in Shields and
As shown in Fig. 1, they exhibit quite similar absorption
Sang M3 Insect Medium (39.4 g Lꢁ1) with fetal bovine serum spectra in THF. The absorption bands ranging between 270 and
(15%, v/v), penicillin–streptomycin solution (1% v/v at a nal 350 nm are assigned to p–p* electronic transition of the
concentration of 100k units per L penicillin and 100 mg Lꢁ1 respective benzene ring, carbazole and diphenylamine units.
streptomycin) and KHCO3 (0.5 g Lꢁ1) and incubated at 22 C. The molecules exhibit strong absorption peaks at 412, 454, 454,
ꢀ
The cells were seeded in glass-bottom plates at a density of 5 ꢂ and 458 nm for 1–4, respectively, which can be attributed to
4
ꢀ
10 cells per mL and incubated for 30 min at 22 C. A DMSO intramolecular charge transfer (ICT) from the carbazole,
solution of 4 (10 mM) was diluted with phosphate buffered dimethylamino and diphenylamine units to the (1,3-dimethyl)
saline (PBS) to a nal concentration of 0.1 mM. The cultured barbiturate moiety.38 Density functional theory (DFT) calcula-
cells were incubated with a PBS solution (50%, v/v) of tions also conrmed the ICT possibilities of these compounds
compound 4 (0.1 mM) for 2 hour, washed three times with PBS (Fig. 9).
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are shown in Fig. 3, and the changes of FL peak depict the
variation tendency of uorescence intensity with different fw.
We can see from the photographs that in pure THF with low
concentration, compounds 2 and 4 are nonemissive, while 1
and 3 show weak emission in dilute pure THF (Fig. 4). In Fig. 3,
the data indicate that all chromophores exhibit different uo-
rescent behaviors in mixed solvents with different fw. The FL
intensities of 2 and 4 in pure THF are so low that only weak
signals are recorded with maxima at 542.8 and 633.4 nm,
respectively (Fig. 3b and d). Then they start to increase when the
fw is increased to 70% and 60%, respectively. The solutions emit
orange and red light with lEm at 603.4 and 627.2 nm at fw
¼
90%, respectively. The FL intensity of 2 enhances 23.6-fold and
the uorescence quantum yield (FF) increases from 0.4% to
8.2% when fw increases from 0% to 90%. While the FL intensity
of 4 enhances 42.2-fold (FF: 0.1% to 3.2%). These data
demonstrate 2 and 4 are AIE-active molecules.
Fig. 1 UV-visible absorption spectra of 1–4 (24 mM) in THF solution.
The compounds 1 and 3 display a different uorescent
behavior from 2 and 4 in mixed solvents with different fw. In pure
THF, 1 and 3 show weak emission at 592.6 and 619.4 nm (Table
1). The emission of 3 is obviously red-shied (26.8 nm)
compared with 1, because the diphenylamino group is a much
stronger electrondonor than the carbazole group, resulting in an
increase in the HOMO energy.41 And the subsequent theoretical
calculations support this speculation (Fig. 9). The FL intensity of
1 and 3 gradually decreases when fw < 70% which may be caused
by ICT.42 When fw > 70, the FL intensity increases rapidly. The FL
intensity of 1 increases 3.3-fold and the FF increases from 5.4%
to 11.4% when fw increases from 0% to 90%, while the FL
intensity of 3 enhances 6.5-fold (FF: 1.8% to 7.4%). These date
demonstrate that 1 and 3 are AIEE-active molecules.
It is also noted that the emission peak varies with fw (Fig. 3,
right). The emission peak of 2 is red-shied from 542.8 to
603.4 nm. The large red-shi may be caused by p–p staking
when the 2 aggregate. However, compound 3 exhibits a more
complicated tendency with the increase of fw. The emission
peak of 3 red-shis from 619.4 nm to 648.4 nm when fw
increases from 0% to 70%. Furtherly, it blue-shis to 624.4 nm
as fw increase to 90%. When fw is no more than 70%, molecule 3
is still completely dissolved in the mixtures, and increased fw
enhances the polarity of mixture, generating red-shi emission
due to the ICT effect. As fw reaches 70%, due to the decreased
solvating power, molecule 3 starts to aggregate, resulting in less
We investigated the emission of the four compounds in
different states. As shown in Table 1, in pure THF with low
concentration, these compounds are almost molecularly iso-
lated without any interactions between the adjacent molecules.
All the molecules exhibit distorted conformation due to the
intramolecular steric hindrance.39 The optimized geometric
structure of 1 show twisted conformation with a dihedral angle
of 56.63ꢀ (Fig. S17†). Thus the molecules are not in good
conjugation state, which contribute to the shortest emission
wavelength (Fig. 2).
The molecules of 2 in the crystal state are arranged so
regularly that the rotatable single bonds are locked due to the
multiple intermolecular interactions (Fig. 6). These interactions
help to further solidify the molecular conformation in the
aggregated state, and they block the nonradiative pathways.40
3.2. AIE/AIEE properties
Emission spectra of 1–4 in THF and THF/water media were
recorded to facilitate the aggregation due to their water insol-
uble characters (Fig. 3). Therefore, the luminogens are able to
aggregate in the aqueous mixtures with high water fractions
(fw). Mixture of THF and water was used for preparation of
nanoaggregates by increasing the percentage of water in the
THF solution. Their uorescence (FL) spectra with different fw
Table 1 Optical properties of compounds 1–4
Monomer/THF
Aggregatesd
lsolid (nm)
a
c
labs (nm)
lEmb (nm)
Stokes shi
FF (%)
lEm (nm)
FF (%)
Powder
Crystal
1
2
3
4
412.0
454.0
454.0
458.0
592.6
542.8
619.4
633.4
180.6
88.8
162.4
175.4
5.4
0.4
1.8
0.1
612.0
603.4
624.4
627.2
11.4
8.2
7.4
3.2
597.0
620.0
637.6
608.0
—
621.0
—
582.0
a
b
c
The maxima absorption peak. The maxima emission peak, in dilute THF solution (24 mM). Fluorescence quantum yields were determined
relative to Coumarin 307 in ethanol solution as quantum yield standard (F ¼ 0.56). d In mixed water/THF solution with fw ¼ 90%.
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Fig. 2 Normalized FL spectra and photographs of (a) 1, (b) 2, (c) 3 and (d) 4 in different state (THF solution, solid powders, crystal).
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Fig. 3 FL spectra of (a) 1, (b) 2, (c) 3 and (d) 4 in THF/H2O mixtures with different water fraction (fw) (left); excitation wavelength ¼ 450 nm,
398 nm, 510 nm, 460 nm. Plot of relative FL peak location (blue) and emission intensity (red) of 1–4 versus the water fraction in the aqueous
mixtures (right).
polar micro-environment for the luminogens because of self- electron microscopy (SEM) and dynamic light scattering
wrapping. Thus reversely giving blue-shied emissions.43,44 (DLS) (Fig. 5), which show the presence of spherical and blocky
Compounds 1 and 4 behave similarly to 3.
The morphology and size of the nanoaggregates in the THF/
H2O solvent with different fw were detected by scanning the nanoaggregates for 1–4 are 394.2, 229.3, 382.4 and 346 nm,
aggregates.
As shown in Fig. 5, at fw ¼ 90%, the average diameters (d) of
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Fig. 4 Photographs of (a) 1, (b) 2, (c) 3 and (d) 4 at fw ¼ 0% to 90% under 365 nm UV light illumination.
respectively. The size of nanoaggregates decrease gradually with
increasing fw (Table S1†), due to the rapidly decreased solvating
power of the mixed solvent (THF/H2O).43 These data indicate
that the enhanced emission of compounds is related to the
formation of nanoaggregates.
3.4. Mechanisms of enhanced emission
As discussed above, compounds 1–4 are practically nonemissive
when molecularly dissolved in solvents due to the active intra-
molecular rotations and vibrations. The intermolecular inter-
actions are mainly reected in restriction of intramolecular
motions and some specic molecular stacking modes, such as J-
aggregation and herringbone stacking,45,46 which can restrain
3.3. Single crystal structure
Single crystal X-ray diffraction (SXRD) analysis can provide the radiative decay process and promotes the enhanced emis-
a direct insight into the molecular conformation and the sion behavior. On the contrary, the H-aggregation, where
packing structure of a compound. Compound 2 adopts planar molecules are arranged in face-to-face type to form excimers or
construction, and it has good planarity (Fig. 6b). The dihedral exciplexes, can promote the nonradiative deactivation process.47
angles of 4 between the terminal unit and the phenyl ring are
59.50ꢀ and 87.23ꢀ, respectively, which indicates that compound and adopt a twisted conformation due to the steric hindrances
4 shows a large torsion (Fig. 7b).
in the molecules which decreases the p-conjugation. The
In diluted solutions, the molecules 1–4 are almost isolated
As shown in Fig. 6b, in crystal 2, the C–H/O interaction intramolecular motions such as rotations, vibrations, stretch-
between carbanyl group and methyl group links the adjacent ing, will consume the energy of excited state and suppress the
molecules into one-dimensional linear chain, and the distance radiative decay process. While in the aggregated state, there are
˚
of C–H/O interaction are 2.581 A. Then, the adjacent chains are numerous intermolecular interactions between the hydrogen
further linked into two-dimensional network structures through and oxygen, carbon or nitrogen atoms; besides, some p–p
˚
C–H/O interactions (the distances are 2.548 and 2.665 A, stacking or C–H/p interactions exist in the aggregate struc-
respectively) between carbanyl groups and methyl groups, C– tures. These interactions lead to the planarity and stability of
˚
H/H–C (d ¼ 2.310 A) and two C–H/p interactions (d ¼ 2.896 molecular conformation. Under the effect of intermolecular
˚
A). These interactions make the molecular conformations stable. interactions, intramolecular vibrations or rotations are locked,
As depicted in Fig. 7, the molecule 4 adapts a twisted and the molecular conformations are more rigid and planar.
conformation due to the steric hindrance between the diphe- These motions are suppressed greatly due to the spatial
nylamino unit and the 1,3-dimethylbarbiturate moiety. And the constraint, which blocks the nonradiative decay pathway and
adjacent molecules are connected via intermolecular C–H/O promotes the radiative decay of the excited state.33
interactions into one-dimensional linear bands, the distances
The restriction of intramolecular motion (RIM) induced by
of C–H/O interactions are 2.531 A and 3.211 A, respectively. As the molecular aggregation can be considered as the mechanism
shown in Fig. 7c, the adjacent molecules in crystal 4 are of enhanced emission for 1–3. Furthermore, the molecule 4 has
˚
˚
˚
herringbone stacking by C–H/O (d ¼ 2.564 A), C–H/p (d ¼ a herringbone stacking conformation in crystal. Thus the
˚
˚
2.762 A), C]O/C and C]O/N interactions (d ¼ 2.973 A, enhanced emission of 4 was attributed to the synergetic effects
˚
˚
3.048 A, and 3.155 A). The crystal also possesses a p–p inter- of the herringbone stacking formation and RIM. Therefore, the
action between two terminal phenyl of adjacent molecules and phenomenon of uorescence enhancement can be ascribed as
˚
the distance is 3.384 A in Fig. 7d. These interactions make their the formation of nanoaggregates of molecules 1–4 at high fw
conguration stable.40
according to the SEM and DLS results.
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Fig. 5 SEM images and particle size distributions of (a) 1, (b) 2, (c) 3 and (d) 4 in THF/H2O mixtures (fw ¼ 90%) at concentrations of 24 mM.
in the white-led channel, which suggest its non-cytotoxic
feature. The excellent biocompatibility of compound 4 moti-
vated us to further investigate their application in cell
imaging. The time scanning spectra of compound 4 at fw ¼ 0.9
were shown in Fig. S18.† As can be seen, the uorescence
3.5. In vivo imaging
Before exploring the application for live cell imaging, we
examined the biocompatibility of compound 4. Comparing the
morphologies of S2 cells with (Fig. 8a) and without (Fig. S19†)
the presence of compound 4, they are almost identical shape
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Fig. 6 ORTEP (thermal ellipsoids at the 50% probability level) diagram (a), one-dimensional structure (b), two-dimensional network structure (c)
and three-dimensional structure (d) of 2.
Fig. 7 ORTEP (thermal ellipsoids at the 50% probability level) diagram (a), one-dimensional structure (b), herringbone stacking structure (c) and
p–p interactions(d) of 4.
intensity hardly changed in 1800 s. These data showed that the internalized into the cells, and red uorescence can be detected
nanoparticles exhibited excellent stability under light intracellularly with minimal background autouorescence
irradiation.
signals. Presumably, compound 4 can be internalized into the
Compound 4 was then assessed for its capability to cell endosome compartments and eventually move to the lysosome
imaging by confocal laser scanning microscopy (CLSM). Aer compartment of the cells within 2 hours. Indeed, the water
incubation with Drosophila S2 cells with 4, compound 4 can be insoluble character of compound 4 facilitates the passive cell
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Intracellular co-localization experiment was performed
with compound 4 and 4,6-diamino-2-phenylindole (DAPI).
DAPI is a nucleus-targeting orescence probe, and accumu-
lates selectively in the nucleus structure of cells. In the co-
localization experiment, intense blue uorescence can be
detected in nucleus of the S2 cell, while no red uorescence
can be detected in the same region, suggesting that the
compound 4 is unable to pass the nuclear membrane
(Fig. 8f). The images show intense uorescence was detected
exclusively in the cytoplasm part of the Drosophila S2 cell,
which might because of the molecule aggregation in the cells.
The aggregated state tends to prevent uorescence quench-
ing resulted from non-radiative decay caused in aqueous
environment, which is actual a common phenomenon in AIE-
based bioimaging probes.48
3.6. Theoretical calculation
To have a better insight into their optical properties, we
calculated frontier molecular orbitals by using the DFT at
B3LYP/6-31G(d) basis set. The optimized geometries and
HOMO/LUMO plots of 1–4 are illustrated in Fig. 9. The
geometry optimized structures of 1–4 show highly twisted
conformation, which are favorable for active intramolecular
rotations of multiple phenyls and carbazole groups in solu-
tions, thereby powerfully dissipating the excitons energy and
making them nonemissive or weak emissive in solvents. The
theoretical calculated HOMO levels of 1–4 are ꢁ5.61, ꢁ5.53,
ꢁ5.47, and ꢁ5.44 eV, respectively. The energy gaps for 1–4 are
2.89, 3.27, 2.98, and 3.01 eV, respectively. And the gaps esti-
mated by absorption maxima of 1–4 are 3.81, 4.18, 4.12 and
4.10 eV, respectively. Therefore, the calculated values agree
well with the test ones.
Fig. 8 (a–c) CLSM of Drosophila S2 cells incubated with compound 4
(50 mM). (a)White-field channel, (b) fluorescence channel, (c) merged
channel; (d–f) co-localization experiment of Drosophila S2 cells
incubated with compound 4 (red channel) and DAPI (blue channel)
and the merged images (f) (inset is an enlarged view of (f)); (g–i) co-
localization experiment of Drosophila S2 cells incubated with
compound 4 (red channel) and Lysotracker Blue (blue channel) and
the merged image (i). Scale bar ¼ 20 mm; inset scale bar ¼ 10 mm.
uptake and accumulation of the molecule into the lysosome.
Indeed, co-localization experiments of compound 4 with Lyso-
tracker Blue suggested they moved into the same intracellular
space – lysosomes (Fig. 8g–i).
Fig. 9 Energy levels of HOMO and LUMO, energy gaps, and electron cloud distributions of molecules 1–4 calculated by the B3LYP/6-31G(d)
program. (HOMO ¼ the highest occupied molecular orbital energy; LUMO ¼ the lowest unoccupied molecular orbital energy; Eg ¼ energy gap
between HOMO and LUMO levels.)
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with scale regulated, phase-switchable, polymorphism-
dependent, and amplied spontaneous emission
properties, J. Phys. Chem. Lett., 2016, 7, 1697–1702.
9 X. Q. Zhang, X. Y. Zhang, B. Yang, J. F. Hui, M. Y. Liu,
Z. G. Chi, et al., Novel biocompatible cross-linked
uorescent polymeric nanoparticles based on an AIE
monomer, J. Mater. Chem. C, 2014, 2, 816–820.
10 P. J. Parrott, N. Y. Tan, R. R. Hu, J. Zeitler, B. Z. Tang and
P. Emma, Direct evidence to support the restriction of
intramolecular rotation hypothesis for the mechanism of
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1, 251–258.
4. Conclusions
In summary, we report four luminogens 1–4 with typical AIE/
AIEE characteristics. Their emission wavelength changed with
different fw, and their powders emit orange (597 nm and 608
nm) and red (620 nm and 637 nm) light. UV-vis absorption
spectra and theoretical calculation reveal the ICT possibilities of
1–4 from the donor units to the acceptor units. And we analyze
the mechanisms of enhanced emission of these compounds are
RIM. Furthermore, the theoretically calculated results agree
with the measured optical properties. With the advantages of
excellent biocompatibility and simplicity, compound 4 repre-
sents an excellent probe for Drosophila S2 cells imaging. Once
entering lysosomes, the intramolecular motion of compound 4
will be restricted and the emission from compound 4 will be
turned on.
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Acknowledgements
This work was nancially supported by the National Natural
Science Foundation of China (No. 21276149, 21376125) and the
Program for Scientic Research Innovation Team in Colleges
and Universities of Shandong Province.
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