Rational design for multicolor flavone-based fluorophores with aggregation-induced emission enhancement characteristics and applications in mitochondria-imaging

Article abstract of DOI:10.3390/molecules23092290

Fluorophores with aggregation-induced emission enhancement (AIEE) properties have attracted more attention in recent years. In order to realise more valuable applications, the different kinds of AIEE molecules are in serious need of further development. Therefore, a novel flavone-based AIEE system derived from restriction of intramolecular rotation (RIR) was designed and synthesized in this work. The results revealed that six of the compounds showed typical AIEE characteristics, with fluorescence emissions from purple, blue, cyan to green, tunable by changing substituent groups. This flavone-based AIEE system has never been reported before. The AIEE characteristics were investigated by optical spectroscopy, fluorescence photographs, scanning electron microscopy (SEM), fluorescence quantum yields (φ_F) and fluorescence lifetime in the CH₃OH/H₂O mixed solution. Moreover, benefiting from the simple structures and small molecular weight, they could permeate cells faster than current high-molecular-weight AIEE molecules. Furthermore, to examine possible biomedical applications, fluorescence imaging in living A549 lung cells and cell viabilities were examined, and the results displayed that these fluorophores showed good cellular uptake and low cytotoxicity within the experimental concentration range. In addition, these AIEE compounds possessed excellent specificity for mitochondrial targeting and mitochondrial morphological change tracking, besides, they displayed superior photostability, which indicated they are potential candidates for mitochondrial imaging.

Full text of DOI:10.3390/molecules23092290

molecules
Article
Rational Design for Multicolor Flavone-Based
Fluorophores with Aggregation-Induced Emission
Enhancement Characteristics and Applications
in Mitochondria-Imaging
Liyan Liu 1, Yaohui Lei 1, Jianhui Zhang 2, Na Li 1, Fan Zhang 1, Huaqiao Wang 3
and Feng He 1,
*
1
School of Pharmaceutical Science, Sun Yat-sen University, Guangzhou 510006, China;
liuly37@mail2.sysu.edu.cn (L.L.); 13843159413@163.com (Y.L.); 18826072701@163.com (N.L.);
zhang_fan1991@foxmail.com (F.Z.)
School of Chemistry, Sun Yat-sen University, Guangzhou 510275, China; zhangjh@mail.sysu.edu.cn
Department of Anatomy and Neurobiology, Zhongshan School of Medicine, Sun Yat-sen University,
Guangzhou 510080, China; wanghq@mail.sysu.edu.cn
2
3
*
Correspondence: hefeng@mail.sysu.edu.cn; Tel.: +86-203-994-3036
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 16 August 2018; Accepted: 5 September 2018; Published: 7 September 2018
Abstract: Fluorophores with aggregation-induced emission enhancement (AIEE) properties have
attracted more attention in recent years. In order to realise more valuable applications, the different
kinds of AIEE molecules are in serious need of further development. Therefore, a novel flavone-based
AIEE system derived from restriction of intramolecular rotation (RIR) was designed and synthesized
in this work. The results revealed that six of the compounds showed typical AIEE characteristics,
with fluorescence emissions from purple, blue, cyan to green, tunable by changing substituent
groups. This flavone-based AIEE system has never been reported before. The AIEE characteristics
were investigated by optical spectroscopy, fluorescence photographs, scanning electron microscopy
(SEM), fluorescence quantum yields (φF) and fluorescence lifetime in the CH3OH/H2O mixed
solution. Moreover, benefiting from the simple structures and small molecular weight, they
could permeate cells faster than current high-molecular-weight AIEE molecules. Furthermore,
to examine possible biomedical applications, fluorescence imaging in living A549 lung cells and
cell viabilities were examined, and the results displayed that these fluorophores showed good
cellular uptake and low cytotoxicity within the experimental concentration range. In addition, these
AIEE compounds possessed excellent specificity for mitochondrial targeting and mitochondrial
morphological change tracking, besides, they displayed superior photostability, which indicated they
are potential candidates for mitochondrial imaging.
Keywords: AIEE; multicolor; flavone; fluorophores; mitochondria
1. Introduction
Fluorophores have received a great deal of attention in the areas of fundamental photophysics
research and applications in biology, medicine, pharmacy and other fields [
the conventional organic fluorophores frequently suffer from aggregation-caused quenching
(ACQ) [ ], whereby their fluorescence emission becomes weaker when the concentration of
1–4]. Nevertheless,
5
the solution increases, which limits their applications. By contrast, the novel luminescence
phenomenon of aggregation-induced emission (AIE) was first discovered by Tang’s group in
2001 [6]. Inspired by Tang’s group, a lot of new AIE and AIEE molecules were reported, such as
Molecules 2018, 23, 2290; doi:10.3390/molecules23092290
www.mdpi.com/journal/molecules

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2,20-(1,4-phenylene)-bis-(3-(4-butoxyphenyl)acrylonitrile (DBDCS), N,N-bis(salicylidene)-p-phenyl-
enediamine (BSPD), distyreneanthracene (DSA) [ ] and so on. Some mechanisms for the observed
7–9
properties have been put forward, including restriction of intramolecular rotation (RIR), restriction
of intramolecular charge transfer, E-Z isomerization, planarization, J-type aggregate formation and
twisted intramolecular charge transfer (TICT). Among these factors RIR is considered to be the main
cause [10–16].
Sanguinarine was confirmed as an AIEE molecule in our previous study [17], which is coupled
with its large conjugate structure (Figure 1). According to the theory of restriction of intramolecular
rotation (RIR), some primitive AIE(E) molecules were designed by increasing the number of rotatable
benzene rings. These AIE(E) molecules usually have high molecular weights and bulky skeletons,
including a lot of benzene rings and substituents [18–20], which make it difficult for the molecules to
enter cells. In addition, molecules with larger rigid conjugate planes could easily become embedded in
the DNA bases and result in toxicity [21], which indicated that they were mostly non-drug-like and
deviated from Lipinski’s rule of 5 [22]. Conversely, in our work, reducing the conjugated structure can
depress the rigidity of molecules and make a part of molecules easier to rotate, therefore, we broke
the oxygen heterocyclic ring on the basis of sanguinarine, thus obtaining two new AIEE compounds,
chelerythrine and a 13-OH berberine derivative, which represent novel AIEE systems different from the
traditional AIEE compounds [23,24]. Based on these successful cases, a rational strategy by breaking
more rings to facilitate intramolecular rotation to obtain novel AIEE systems was designed. The novel
AIEE system combine small molecular weights and a certain flexibility.
Figure 1.
(a) Chemical structures of sanguinarine, chelerythrine, 13-OH berberine derivative and
flavone derivatives. (b) Synthetic route and chemical structures of flavone derivatives.

Molecules 2018, 23, 2290
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Herein some novel fluorophores with AIEE characteristics are presented, as a series of flavone
derivatives with hydroxy, methyl, methoxy and amino substituents were synthesized (Figure 1) and
their optical properties were subsequently investigated. The results revealed that six of them (5a
,
5b 5c 5d 5e and 5f) were typical AIEE compounds, and represent a novel AIEE system that has
,
,
,
never been reported before. They exhibited scarce fluorescence in an extracellular solvent but became
highly in fluorescent in intracellular aqueous solution, and their fluorescence emission colors upon
UV irradiation were tunable from purple, blue, cyan to green by changing the substituent groups.
Furthermore, these AIEE compounds not only showed very good cellular uptake and low cytotoxicity
at the experimental concentration range, but also possessed excellent specificity for mitochondrial
targeting and mitochondrial morphological change tracking. Furthermore, they displayed superior
photostability. Some AIE(E) molecules were found to act as biological dyes and have been widely
applied in morphological detection and tracking [25–30]. However, most of them suffered from
monotonous color, poor photostability and the need for a high concentration to stain the target, whereas
the AIEE molecules in our work avoid these limitations, which proved that they have potential for
monitoring the morphological changes of mitochondria.
2. Results and Discussion
2.1. Optical Properties and Aggregation-Induced Emission Enhancement Property
The fluorescence characteristics of 5a–j were investigated in CH3OH/H2O mixtures (CH3OH as
a good solvent and H2O as a poor solvent) where the water fraction was changed from 0% to 90%.
In Figure 2b,c,e, as the water fractions changed from 0% to 90%, the PL intensity of the main emission
peak increases gradually. In Figure 2b, when the water fraction is 90%, the PL intensity is enhanced
32-fold more than when it is 0%, and similar results can be found in Figure 2c, where the PL intensity
is enhanced 13-fold. In Figure 2e, the PL intensity increases slowly when the fraction goes from 0%
to 80%, but it suffers from a sudden increase from 80% to 90%, which is nearly 14-fold more than for
0% water. These results show that compounds 5b, 5c and 5e display typical AIEE characteristics [31].
As shown in Figure 2a, the PL intensity of the main emission peak is weak in pure CH3OH solution
and raises swiftly with the increase of water fraction, finally, reaching a maximum at 70%, which
indicates a great AIEE behavior.
Figure 2. Cont.

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Figure 2. PL spectra of (
mixtures with different water fractions (0–90 vol%).
a
)
5a, (
b
)
5b, (
c
)
5c, (
d
)
5d, (e
)
5e, (
f
)
5f. (c = 2.09
×
10−5 M) in CH3OH/H2O
The PL intensity decreases when the fraction goes from 70% to 90%, which as attributed to the
fact the packing mode of the molecules changes from orderly to casual [32]. The results indicate
that compound 5a is an AIEE active compound. In Figure 2d, the PL intensity is raising when the
fraction changes from 0% to 50% but decreases from 50% to 90%, in Figure 2f, the PL intensity reaches
a maximum when the water fraction is 60%, so we guess the attenuation of fluorescence intensity seen
in Figure 2d,f is due to the same reason of Figure 2a, so in conclusion, compounds 5d and 5f show
AIEE properties from the PL spectra. As suggested by the PL spectra in the Supporting Information
(Figure S1a–d), the PL intensity of the main emission peak decreases gradually when the water fraction
changes from 0% to 90% and becomes the smallest at 90%, which indicates an obvious ACQ behavior.
These results reveal that compounds 5g, 5h, 5i and 5j are not active AIEE compounds.
To further study the AIEE behavior of the compounds 5a
spectra were measured in CH3OH/H2O mixtures, where compounds 5a
absorption peaks at 302, 297, 306, 296, 320 and 324 nm in pure CH3OH solution, and when the water
fraction changes to 90%, as shown in Figure 3, the maximum wavelengths of compounds 5a 5b 5c
,
5b
,
5c,
5d
,
5e and 5f, their absorption
,
5b 5c 5d 5e and 5f show
,
,
,
,
,
and 5e show an obvious red shift and the absorption peaks of compounds 5d and 5f disappear, which
indicates the formation of new aggregates in the mixtures [33].
Figure 3. Cont.

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Figure 3. Absorption spectra of compound (
solution and CH3OH/H2O (1:9, v/v) mixture (c = 2.09 × 10 M).
a
)
5a, (
b
)
5b, (
c
)
5c, (
d
)
5d, (
e)
5e and (
f
)
5f in pure CH3OH
−5
The AIEE behavior of the compounds can also be observed from their fluorescence photographs
in CH3OH/H2O mixtures with various water fractions under 365 nm UV excitation (Figure 4). We can
see that compounds 5a–f exhibit emissions with different colors from purple, blue, cyan to green,
because the different substituent groups on the flavonoid nucleus influence the electronic density
distribution of the molecules. By changing the substituents on the compounds, we can successfully
achieve the regulation of the fluorescent colors of these flavone derivatives.
Figure 4. Fluorescence photographs of compounds (
a) 5a, (b) 5b, (c) 5c, (d) 5d, (e) 5e and
(f)
5f in CH3OH/H2O mixtures (with various water fractions) under a 365 nm UV excitation
−5
(c = 2.09 × 10 M).
The fluorescence quantum yields (φF) and fluorescence lifetime of compounds 5a, 5b, 5c, 5d, 5e
and 5f in the CH3OH/H2O mixtures were measured for a quantitative comparison. The fluorescence
quantum yields (φF) were measured by using an integrating sphere. Results are displayed in Table 1,
where the fluorescence quantum yields (φF) of compound 5a in CH3OH, CH3OH/H2O (3:7 v:v)
mixture and CH3OH/H2O (1:9 v:v) mixture are 0.16, 0.23 and 0.19, and the fluorescence lifetimes are
3.15, 3.22 and 3.19 ns, which is in accord with the behavior of the PL intensity spectrum.
Table 1. Quantum yields (φF) and lifetimes (ns) of 5a
(v/v) mixtures (2.09 × 10 M).
,
5b
,
5c
,
5d
,
5e and 5f in different CH3OH/H2O
−5
Compound
Solvents
Quantum Yields (φ )
Time/ns
F
CH3OH
0.16
0.23
0.19
0.03
3.15
3.22
3.19
2.15
5a
CH3OH/H2O (3:7)
CH3OH/H2O (1:9)
CH3OH

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Table 1. Cont.
Compound
Solvents
Quantum Yields (φ )
Time/ns
F
5b
5c
5d
5e
5f
CH3OH/H2O (5:5)
CH3OH/H2O (1:9)
CH3OH
CH3OH/H2O (5:5)
CH3OH/H2O (1:9)
CH3OH
CH3OH/H2O (5:5)
CH3OH/H2O (1:9)
CH3OH
CH3OH/H2O (5:5)
CH3OH/H2O (1:9)
CH3OH
0.09
0.15
0.14
0.22
0.28
0.05
0.10
0.02
0.20
0.27
0.32
0.13
0.18
0.12
2.32
3.10
2.97
3.29
3.34
2.18
2.68
1.67
3.26
3.31
3.40
2.83
3.16
2.79
CH3OH/H2O (4:6)
CH3OH/H2O (1:9)
Similar phenomena occur with compounds 5b−f, where the value of the fluorescence quantum
yields (φF) and fluorescence lifetime change with the changing fluorescence intensity, which leads us
to conclude that compounds 5a−f are typical AIEE molecules.
At present, there are many possible reasons that contribute to AIEE phenomena, but the RIR is
identified as a main cause, so we used high viscosity (CH3OH/ethylene glycol) solvents and SEM
to confirm the AIEE mechanism. As the proportion of ethylene glycol increased, the viscosity of
the solutions increased, which restricts the rotation of the molecules and leads to a fluorescence
enhancement [34]. This speculation is supported by the fluorescence spectra in Figure S2, where the
emission of compounds 5a–f increases significantly in pace with the increase of viscosity, reaching
a maximum when the ethylene glycol fraction is 50%. The SEM results shown in Figure 5 provide
plentiful information to understand the reason for the AIEE effect, as many nanoparticle aggregates
with approximately spherically shapes are formed and, as can be observed from the images, the shape
of the nanoparticles remains spherical but their diameter increases with the increase of water content,
so we can conclude that the formation of large diameter spherical nanoparticles leads to a fluorescence
enhancement in the CH3OH/H2O mixtures. Meanwhile, the shape of nanoparticles changes when the
water fraction is 90%, which indicates that they aggregate into different shapes and new aggregates are
formed [35]. These results correspond to the emission decrease of 5a
Time-dependent fluorescence spectra of 5a are shown in Figure S3, where there are no obvious
changes in fluorescence intensity with increasing irradiation time, showing that compounds 5a are
stable in CH3OH/H2O mixtures, a property that is convenient for their application in other areas.
, 5f and 5d at 90% water fraction.
–
f
–f
Figure 5. SEM images in CH3OH/H2O (5: 5 v:v) mixtures of compounds (
5e, ( 5f. ( ) SEM image in CH3OH/H2O (3:7 v:v) mixtures of 5a. SEM image in CH3OH/H2O (2:8 v:v)
mixtures of compounds ( 5b, ( 5c, ( 5d. SEM image in CH3OH/H2O (4:6 v:v) mixtures of ( 5a, (
5e. SEM image in CH3OH/H2O (1:9 v:v) mixtures of (m) 5a, (n) 5b, (o) 5c, (p) 5d, (q) 5e, (r) 5f.
a) 5a, (b) 5b, (c) 5c, (d) 5d, (e)
f
)
g
h
)
i
)
k
)
i
)
l)

Molecules 2018, 23, 2290
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2.2. Electronic Structure
In order to better understand their optical properties, the frontier molecular orbitals and energy
levels of the highest occupied molecular orbitals (HOMOs) and the lowest unoccupied molecular
orbitals (LUMOs) of 5a
In Figure 6, the electron density of the HOMOs of 5b
of the molecules and in 5a 5e 5f they are mainly localized on the A and C rings of the molecules, while
the electron density of the LUMOs of 5a are mainly localized on the whole skeleton of the molecules,
–
f
were calculated by density functional theory using the Gaussian 09 program.
,
5c, 5d are mainly localized on the whole skeleton
,
,
–
f
and the phase positions of the HOMOs and LUMOs are basically symmetrical, so electrons can easily
flow from HOMOs to LUMOs, which illustrates these molecules have a good potential for producing
fluorescence. The energy gaps between the HOMOs and LUMOs for 5a–f are 4.18, 4.51, 4.56, 4.54, 4.36
and 4.26 eV, respectively. These small energy gaps enable a facile electron transit, which indicates these
molecules can be excited easily. The results are well consistent with the strong fluorescence emission
of compounds 5a–f. In addition, the small energy gap can result in a red-shift, which agrees very well
with the absorption spectra measurement results shown in Figure 3.
Figure 6. Frontier molecular orbital amplitude plots and energy levels of the HOMOs and the LUMOs
of 5a–f.

Molecules 2018, 23, 2290
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2.3. Fluorescence Imaging in Living Cells and Cell Viability Assay
To study the applications of compounds 5a
performed with A549 cancer cells. The A549 cells were cultured separately at 37 ◦C for 30 min with
5a (at 10 M concentration). As shown in Figure 7, bright fluorescence with the color of blue, cyan
and green is observed in the cytoplasm of the A549 cells from the CLSM images. These compounds
are therefore cell permeable and display strong fluorescence. The bright fluorescence of 5b is blue,
–f for biological imaging, cell uptake experiments were
–f
µ
–f
that of 5a is cyan and it is green for 5d. The stronger fluorescence signals in the CLSM images indicate
that these AIEE flavone derivatives aggregate in intracellular aqueous solution and show very good
cellular uptake.
Figure 7. CLSM images of A549 cells incubated with (
(10 M) for 30 min with excitation at 405 nm; The bright-field image of A549 cells stained with (
5b, ( 5c, ( 5d, ( 5e, ( 5f. The merged image stained with ( 5a, ( 5b, ( 5c, ( 5d, ( 5e, (
5f. Scale bar: 20 µm.
a
)
5a, (
d
)
5b, (
g
)
5c, (
j
)
5d, (
m
)
5e and (
p
)
)
5f
µ
b
5a,
(
e)
h)
k)
n)
q)
c)
f
)
i
)
l
)
o)
r)
Cell viability, which decides the potential for biological imaging, is an important factor for the
further study of biological applications, so the cytotoxicity of compounds 5a
investigated by using a MTT assay. The A549 cells were cultured at 37 C with different concentrations
–
f
in A549 cells was
◦
(1, 5, 10, 15
µ
M) of compounds 5a
–
f
for 24 h. The results shown in Figure 8 reveal that these compounds
M, the cell
have
show low cytotoxicity in the experimental concentration range, and at a concentration of 10
viability values of all the compounds are more than 90%, which suggests that compounds 5a–f
µ
high potential for live cell imaging applications.

Molecules 2018, 23, 2290
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Figure 8. Cell viabilities of A549 cells treated with different concentrations of (a) 5a, (b) 5b, (c) 5c,
(d) 5d, (e) 5e, (f) 5f for 24 h by MTT assay.
2.4. Mitochondrial Imaging
The fluorescence imaging of compounds 5a, 5b, 5c, 5d, 5e and 5f in Figure 7 indicates that
these compounds can enter the cell membrane and gather in some specific parts of the cell, thus,
co-localization experiments with Mito Tracker Deep Red were performed in order to confirm the
specific location of the aggregation. The A549 cells were separately cultured at 37 ◦C for 30 min with
Mito Tracker Deep Red (100 nM) and 5a–f (10 µM).
Figure 9. Co-localized images of A549 cells incubated with (
a
f
)
)
5a, (
5b, (
e
j
)
)
5b, (
5c, (
i
n
)
)
5c, (
5d, (
5a, (
5e, ( 5f. Scale bar: 20
m
)
5d, (
5e, (
5b, (
q
)
5e and (
5f for 15 min
5c, ( 5d
m.
u) 5f
(10 M) for 30 min and Mito Tracker Red (200 nM) ( 5a, (
µ
b
)
r
)
v)
with excitation at 405 nm; The bright-field image of A549 cells stained with (
5e, ( 5f. The merged image stained with ( 5a, ( 5b, ( 5c, ( 5d, (
c)
g
)
k)
o
)
,
(s)
w)
d)
h
)
l
)
p
)
t
)
x)
µ
As displayed in Figure 9, these compounds stain the mitochondrial region specifically in A549
cells, and the fluorescence signals with different colors from compounds 5a are overlapped well with
the red fluorescence signals from Mito Tracker by colocalization analysis [36]. The reticulum structures
of the mitochondria are clearly visible under the fluorescence of compounds 5a , which indicates
that the fluorescence from these compounds is localized on the mitochondria of the living A549 cells.
The Pearson’s correlation coefficient (Rr; from +1 to 1) which indicates the linear correlation between
–f
–
f
−

Molecules 2018, 23, 2290
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two variables, is used to quantify the staining region overlap between compounds 5a
–
f
and MT.
The coefficients are 0.96, 0.95, 0.95, 0.97, 0.98 and 0.95 for 5a–f, respectively (Figure S4), indicating the
specific targeting ability of these AIEE active flavone derivatives on mitochondria.
2.5. Mitochondrial Morphological Change
According to the literature, mitochondria continuously oxidize substrates and preserve a proton
gradient across the phospholipid bimolecular layer with a large membrane potential (∆Ψm) of around
−
180 mV [37]. To further research the ability of these AIEE flavone derivatives, morphological change
tracking experiments were carried out. Carbonyl cyanide m-chlorophenylhydrazone (CCCP), which
is known as an uncoupler that causes the quick acidification of mitochondria and malfunction of
ATP synthase, thus bringing about the decrease of the mitochondrial ∆Ψm [38], was used to treat the
living A549 cells before staining. Compound 5e was selected as a representative of these active AIEE
flavone derivatives for the staining experiments because of its strong fluorescence emission. As shown
in Figure 10, after staining by CCCP, the reticulum-like mitochondria were transformed into small
and dispersed fragments step by step, which was observed clearly with the blue fluorescence of 5e
Compound 5e showed outstanding photostability during this process. Mitochondrial morphological
changes are involved in the early stages of apoptosis and are considered an irreversible process, so 5e
has potential for apoptosis research. In conclusion, compound 5e showed excellent ability for tracking
mitochondrial morphological changes, which suggested these active AIEE flavone derivatives are
potential candidates for mitochondrial research.
.
Figure 10. CLSM images of A549 cells treated with CCCP (20
increasing scanning time (0, 15, 30 min). Scale bar: 20 µm.
µM) stained with 5e (10 µM) with
3. Materials and Methods
3.1. Materials and Instruments
All the regents and analytical grade solvents in this paper were obtained commercially and
used without further purification unless indicated. 20-Hydroxyacetophenone, benzoyl chloride,
20-hydroxy-50-methoxyacetophenone, 20-hydroxy-40-methoxyacetophenone and 20,40-dihydroxy-
acetophenone were obtained from Macklin (Shanghai, China). 3,4-Dimethoxybenzoylchoride,
0
0
4-methoxybenzoylchoride and 2 -hydroxy-5 -methylacetophenone were obtained from Sigma-Aldrich
(Shanghai, China), Pyridine and potassium hydroxide (KOH) were from Aladdin (Shanghai,
1
China). Pyridine was distilled from anhydrous potassium hydroxide (KOH) before use. H-NMR
(400 MHz) and 13C-NMR (101 MHz) spectra were obtained on an Avance III 400 MHz spectrometer
(Bruker, Karlsruhe, Germany) in CDCl3. The progress of reactions was checked by analytical
thin layer chromatography (TLC). The mass spectra were obtained on a LCMS-IT-TOF mass
spectrometer (Shimadzu, kyoto, Japan). Photoluminescence (PL) spectra and absolute PL quantum
yields were obtianed on an FLS920 spectrophotometer (Edinburgh Instruments, Edinburgh, UK).
The fluorescence lifetimes were obtained with an FLS 980 spectrometer (Edinburgh Instruments,
Edinburgh, UK). Ultraviolet (UV) absorption spectra were collected on a UV-2600 spectrometer
(Shimadzu, Kyoto, Japan). Scanning electron microscope (SEM) images were obtained on a Zeiss
Merlin emission scanning electron microscope (Zeiss Co., Oberkochen, Germany). The human

Molecules 2018, 23, 2290
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lung cancer A549 cell line was obtained from laboratory animal center of Sun Yat-sen University.
Commercially available RPMI Medium Modified, Dulbecco’s modified Eagle’s Medium (DMEM),
0.25% trypsin solution, phosphate buffered saline (PBS), the Mito Tracker Red assay kit (MT)
and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) were obtained from
Thermo-Fisher Biochemical Products (Beijing, China). Fluorescent images were examined on an
OLYMPUS FV3000 laser scanning confocal microscope (Zeiss Co., Oberkochen, Germany) and cell
viabilities were analyzed by a Flex Station 3 microplate reader (Molecular Devices, silicon valley, USA).
3.2. Synthesis of Flavone Derivatives
The structures and synthesis route of the flavone derivatives are exhibited in Figure 1. This route
involves three steps. The reaction involved in the first step was a modified Schotten-Baumann reaction,
then, a Baker-Venkataraman rearrangement and cyclization catalyzed by acid were involved in the
next steps [39,40]. The final products 5a–j were obtained in good yield.
3.2.1. Synthesis of Compounds 3a–j
Compounds 1a–j (0.9 mL), compounds 2a–j (1 mL) and dried and distilled pyridine (2.5 mL)
were put into in a 50 mL round bottomed flask equipped with a reflux condenser, then the mixture
was stirred about 30 min at 55 ◦C, and then poured into hydrochloric acid (60 mL, 1 mol/L) mixed
with 25 g of crushed ice, then stirred until solids were formed. After that the solids were filtered from
the solution and washed by 2.5 mL methanol and 2.5 mL water, to give, after recrystallization from
methanol and water, compounds 3a–j.
3.2.2. Synthesis of Compounds 4a–j
Compounds 3a–j (0.01 mol), dried and distilled pyridine (9 mL) and powdered KOH (0.03 mol)
were placed a round bottomed flask equipped with a reflux condenser and stirred for about 15 min at
50 ◦C. After the reaction, the mixture was allowed to cool to room temperature, then, aqueous acetic
acid solution (12.5 mL, 10% concentration) were added into the mixture, giving compounds 4a–j after
the solids were formed and filtered.
3.2.3. Synthesis of Compounds 5a–j
Compounds 4a–j (1.8 g) glycerol triacetate (10 mL) and concentrated sulfuric acid (0.4 mL) were
placed in a round bottomed flask equipped with a reflux condenser, stirred about 1 h at 100 ◦C and
then the mixture was poured into a beaker containing 50 g of crushed ice, where solids were formed
after stirring and then filtered from the solution, Next the solids were washed with water until the acid
was removed. Finally, the solids were purified and separated by column chromatography eluting with
a mixed petroleum ether and ethyl acetate solvent until the final products 5a–j were obtained.
◦
1
6-Methoxyflavone (5a). Yield: 78%; white solid, mp.: 163–165 C; H-NMR (CDCl3)
1H), 7.52 (t, J = 7.6 Hz, 2H), 7.51–7.39 (m, 1H), 7.24 (d, J = 3.1 Hz, 1H), 7.22 (d, J = 3.1 Hz, 2H), 7.19 (s,
1H), 6.79 (s, 1H), 3.85 (s, 3H); 13C-NMR (CDCl3)
177.27, 162.12, 155.98, 150.04, 130.83, 130.46, 127.99,
δ 7.93–7.80 (m,
δ
127.99, 125.20, 125.20, 123.52, 122.76, 118.48, 105.79, 103.81, 54.90. MS (ESI-MS) m/z calcd for C16H12O3
[M + H]+: 253.26; found: 253.28.
◦
1
6-Methylflavone (5b). Yield: 74%; white solid, mp.: 119–122 C; H-NMR (CDCl3)
7.90–7.83 (m, 1H), 7.46 (dd, J = 5.1, 2.2 Hz,2H), 7.44 (d, J = 1.9 Hz, 1H), 7.42 (s, 1H), 7.40 (s, 1H), 7.19 (s,
1H), 6.79 (s, 1H), 2.41 (s, 3H); 13C-NMR (CDCl3)
177.54, 162.25, 153.53, 134.19, 133.97, 130.88, 130.48,
δ 7.96 (s, 1H),
δ
127.99, 127.99, 125.24, 125.24, 124.03, 122.58, 116.82, 106.40, 19.91. MS (ESI-MS) m/z calcd for C16H12O2
[M + H]+: 237.26; found: 237.27.
7-Methoxyflavone (5c). Yield: 57%; slight yellow solid, mp.: 110–112 ◦C; 1H-NMR (CDCl3)
J = 8.8, 3.5 Hz, 1H), 7.90–7.78 (m, 1H), 7.60–7.53 (m, 1H), 7.48–7.48 (m, 1H), 7.49–7.46 (m, 1H), 7.46–7.43
δ 8.08 (dd,

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(m, 1H), 7.37–7.24 (m, 1H), 6.99–6.88 (m, 1H), 6.79 (s, 1H), 3.87 (s, 3H); 13C-NMR (CDCl3)
δ
176.91,
163.20, 162.04, 157.01, 130.84, 130.41, 128.35, 128.35, 126.04, 126.04, 125.16, 116.81, 113.43, 106.53, 99.42,
54.85. MS (ESI-MS) m/z calcd for C16H12O3 [M + H]+: 253.26; found: 253.24.
◦
1
7-Hydroxyflavone (5d) Yield: 59%; slight yellow solid, mp.: 244–246 C; H-NMR (CDCl3)
(dd, J = 29.6, 8.2 Hz, 3H), 7.89–7.58 (m, 3H), 7.48 (s, 1H), 7.24 (dd, J = 8.6, 1.7 Hz, 1H), 6.78 (s, 1H);
13C-NMR (CDCl3)
176.76, 163.39, 162.76, 155.79, 153.95, 133.12, 130.64, 129.32, 128.09, 127.76, 126.19,
125.30, 118.58, 110.29, 106.71; MS (ESI-MS) m/z calcd for C15H10O3 [M + H]+: 239.12; found: 239.16.
δ 8.20
δ
40-Methoxyflavone (5e). Yield: 71%; white solid, mp.: 161–163 ◦C; 1H-NMR (CDCl3)
8.23 (d, J = 7.9
Hz, 1H), 7.90 (d, J = 8.8 Hz, 2H), 7.69 (t, J = 7.7 Hz, 1H), 7.56 (d, J = 8.4 Hz, 1H), 7.42 (t, J = 7.5 Hz, 1H),
7.03 (d, J = 8.8 Hz, 2H), 6.77 (s, 1H), 3.90 (s, 3H); 13C-NMR (CDCl3)
178.37, 163.48, 162.46, 156.21,
δ
δ
133.60, 128.04, 128.04, 125.68, 125.11, 124.05, 123.92, 117.97, 114.49, 114.49, 106.17, 55.52; MS (ESI-MS)
m/z calcd for C16H12O3 [M + H]+: 253.26; found: 253.28.
6,40-Methoxyflavone (5f). Yield: 75%; white solid, mp.: 185–188 ◦C; 1H-NMR (CDCl3)
1H), 7.53 (d, J = 3.1 Hz, 2H), 7.43 (d, J = 9.1 Hz, 1H), 7.23 (d, J = 3.1 Hz, 1H), 7.21–7.19 (m, 1H), 7.01–6.91
(m, 1H), 6.73 (s, 1H), 3.83 (d, J = 8.3 Hz, 6H); 13C-NMR (CDCl3)
178.24, 163.21, 162.33, 156.91, 150.99,
δ 7.88–7.76 (m,
δ
127.93, 127.93, 124.52, 124.11, 123.53, 119.38, 114.44, 114.44, 105.47, 104.89, 55.93, 55.50; MS (ESI-MS)
m/z calcd for C17H14O4 [M + H]+: 283.29; found: 283.21.
◦
Flavone (5g). Yield: 81%; white solid, mp: 95–97 C; 1H-NMR (CDCl3)
(m, 2H), 7.71 (t, J = 7.8 Hz, 1H), 7.63–7.49 (m, 4H), 7.43 (t, J = 7.5 Hz, 1H), 6.84 (s, 1H); 13C-NMR (CDCl3)
177.40, 162.41, 155.25, 132.76, 130.76, 130.68, 128.03, 128.03, 125.28, 124.69, 124.22, 124.22, 122.93,
117.07, 106.56. MS (ESI-MS) m/z calcd for C15H10O2 [M + H]+: 223.24; found: 223.12.
δ 8.25(d, J = 7.9 Hz, 1H), 7.99–7.90
δ
◦
6-Aminoflavone (5h). Yield: 66%; slight yellow solid, mp: 196–200 C; 1H-NMR (CDCl3)
δ 7.87–7.84 (m,
1H), 7.83 (dd, J = 6.3, 3.1 Hz, 1H), 7.45 (d, J = 2.0 Hz, 1H), 7.43 (dd, J = 7.3, 3.2 Hz, 1H), 7.36–7.33 (m,
1H), 7.33 (s, 1H), 7.00 (d, J = 2.8 Hz, 1H), 6.97 (d, J = 2.9 Hz, 1H), 6.70 (s, 1H), 3.83 (s, 2H); 13C-NMR
(CDCl3)
δ 177.48, 161.99, 148.95, 143.10, 131.09, 130.31, 127.95, 127.95, 125.19, 125.19, 123.72, 121.21,
118.04, 106.91, 105.67; MS (ESI-MS) m/z calcd for C15H11O2N [M + H]+: 238.25; found: 238.27.
0
0
◦
3 ,4 -Methoxyflavone (5i). Yield: 72%; white solid, mp: 154–155 C; 1H-NMR (CDCl3)
1.6 Hz, 1H), 7.69–7.59 (m, 1H), 7.52 (dd, J = 8.3, 3.0 Hz, 2H), 7.37 (d, J = 7.2 Hz, 1H), 7.34 (d, J = 2.1 Hz,
1H), 6.93 (d, J = 8.5 Hz, 1H), 6.77 (s, 1H), 3.93 (s, 3H), 3.91 (s, 3H); 13C-NMR (CDCl3)
177.34, 162.35,
δ 8.17 (dd, J = 7.9,
δ
155.16, 151.08, 148.28, 132.5, 124.64, 124.12, 123.22, 122.92, 119.00, 116.96, 110.16, 107.85, 105.45, 55.07,
55.07; MS (ESI-MS) m/z calcd for C17H14O4 [M + H]+: 283.29; found: 283.19.
◦
1
6,30,40-Methoxyflavone (5j). Yield: 69%; white solid, mp.: 183–185 C; H-NMR (CDCl3)
δ 7.53 (dd,
J = 6.3, 2.6 Hz, 1H), 7.50 (d, J = 2.1 Hz, 1H), 7.46 (d, J = 9.1 Hz, 1H), 7.33 (d, J = 2.1 Hz, 1H), 7.23 (dd,
J = 9.1, 3.1 Hz, 1H), 6.92 (d, J = 8.5 Hz, 1H), 6.79 (s, 1H), 3.92 (s, 3H), 3.90 (s, 3H), 3.85 (s, 3H); 13C-NMR
(CDCl3)
δ 178.20, 163.20, 156.97, 152.03, 151.00, 149.29, 124.42, 123.61, 123.61, 119.95, 119.40, 111.18,
108.84, 105.76, 104.87, 56.09, 55.94, 55.94; MS (ESI-MS) m/z calcd for C18H16O5 [M + H]+: 313.32; found:
313.33.
3.3. Preparation for UV–Vis Spectra, Pl Spectra and SEM Measurements
The flavone derivatives were prepared as a series of stock methanol solutions with a concentration
of 10−4 M. Then different volumes of methanol and water were added cautiously under vigorous
stirring, the water fractions changed from 0 to 90 vol% while the final concentration was kept at
10−5 M. UV-vis absorption spectra and PL spectra compounds 5a
–j were measured immediately at
room temperature with the excitation wavelengths 360, 346, 305, 396, 310, 360, 310, 354, 365, 363 nm
respectively in the CH3OH/H2O mixtures. The absolute PL quantum yields (φF) of 5a in CH3OH,
CH3OH/H2O (3:7, v/v) mixture, CH3OH/H2O (1:9, v/v) mixture, 5b in CH3OH, CH3OH/H2O
(5:5, v/v) mixture, CH3OH/H2O (1:9, v/v) mixture, 5c in CH3OH, CH3OH/H2O (5:5, v/v) mixture,

Molecules 2018, 23, 2290
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CH3OH/H2O (1:9, v/v) mixture, 5d in CH3OH, CH3OH/H2O (5:5, v/v) mixture, CH3OH/H2O (1:9,
v/v) mixture, 5e in CH3OH, CH3OH/H2O (5:5, v/v) mixture, CH3OH/H2O (1:9, v/v) mixture and
5f in CH3OH, CH3OH/H2O (4:6,v/v) mixture, CH3OH/H2O(1:9, v/v) mixture were recorded on an
integrating sphere and the fluorescence lifetimes of 5a
the same water fractions change as the absolute PL quantum yields. Solutions of 5a
–
f
were obtained on a FLS 980 spectrometer with
with the same
–f
water fraction change of fluorescence lifetime were placed dropwise on a silicon wafer respectively and
evaporated over 12 h at room temperature. After completely drying, metal spraying was performed
for SEM imaging [41,42].
3.4. Preparation for Ethylene Glycol (EG) Measurement
The flavone derivatives were dissolved in methanol solutions with a concentration of 10−4 M.
Then different volumes of methanol and EG were added cautiously with the EG fractions changed
from 0 to 50 vol% and the final concentration was kept at 10−5 M. As the EG fractions changed the
viscosity of the solution changed [43]. After stirring, PL spectra of 5a–f with different EG fractions
were measured immediately at room temperature using a FLS 980 spectrometer.
3.5. Theoretical Calculations
The highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital
(LUMO) of 5a–f were calculated by density functional theory (DFT) using the Gaussian 09 program at
the B3LYP/6-31G* level.
3.6. Cell Viability Evaluation
A549 lung cancer cells were seeded in 96-well microplates at a density of 9
and maintained overnight in RPMI Medium Modified (RPMI-1640) media, containing 10% fetal bovine
×
103 cells per well
serum (FBS), 10 mg/mL of streptomycin and 10 mg/mL penicillin. After 24 h of incubation in an
◦
incubator with 5% CO2 at 37 C, treated with various concentrations of flavone derivatives (1
−
15
L 0.5% MTT (1 mg/mL in phosphate
buffered saline [PBS]) was added to each well, then, incubated for 4 h at 37 ◦C. After 4 h the medium
was removed, 150 L DMSO was added to per well and shaken slowly for 10 min. Absorbance of
µM)
at 37 ◦C for 24 h. Then the medium was poured out and 20
µ
µ
plates were then monitored by a microplate reader (Molecular Devices) at 490 nm.
3.7. Cell Imaging
A549 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS), then
seeded in a glass bottom dish with a density of 1
×
105 cells per dish and incubated in an incubator
with 5% CO2 for 24 h. Flavone derivatives (1000
µ
L, 10
µM) were added to the dish and incubated
◦
at 37 C for 30 min, the medium was later removed, and the cells were washed three times with
phosphate buffered saline (PBS) buffer. Then the cell imaging of these compounds were observed on a
FV3000 laser scanning confocal microscope (Zeiss) using an oil immersion lens (60
NA 1.4) within a bandwidth of 460 560 nm. A549 cells were cultured for 24 h with the same way the
above mentioned then flavone derivatives (10 M) and 200 nM Mito Tracker Deep Red (a commercially
×
magnification,
−
µ
available mitochondrial dye) were added to the dish and incubated for 30 min for colocalization
experiments. Thirty min later, the solutions were removed and cells were washed with PBS three times.
Last, cell imaging of these flavone derivatives were obtained on a confocal laser scanning microscope.
4. Conclusions
In summary, a novel AIEE system involving flavone-based derivatives was developed based
on our previous study of AIEE molecules. They showed obvious AIEE characteristics, which were
confirmed by their optical spectra, fluorescence photographs, fluorescence quantum yields (φF) and
fluorescence lifetime. These compounds exhibit various fluorescence emissions ranging from purple,

Molecules 2018, 23, 2290
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blue, cyan to green as the substituent groups change. These different colors offer great possibilities
for their application in other aspects. Moreover, these AIEE compounds have excellent specificity
for mitochondrial targeting and morphological change tracking, and they display low cytotoxicity
at the experimental concentration range, which indicates their potential application prospects in
mitochondrial imaging. Because of these excellent properties, further studies on the other applications
such as their use as multifunctional probes and in vivo imaging are easy to carry out. Further efforts
to discover more multipurpose AIEE fluorophores and their applications in living cell imaging and
in vivo imaging are in progress.
Supplementary Materials: The following are available online. Figure S1: title PL spectra of 5g, 5h, 5i, 5j in
CH3OH/H2O mixtures, Figure S2: PL spectra of 5a, 5b, 5c, 5d, 5e and 5f in CH3OH/ethylene glycol mixtures,
Figure S3: Time-dependent fluorescence spectra of 5a, 5b, 5c, 5d, 5e and 5f in CH3OH/H2O mixtures, Figure S4:
1
Pearson’s correlation coefficient of 5a
,
5b
,
5c
,
5d
,
5e and 5f in A549 cells, Figure S5: H, 13C-NMR spectra and
ESI-MS analysis for compounds 5a–5j.
Author Contributions: Conceptualization, F.H.; Synthesis and data collection, L.L., Y.L., J.Z. and N.L.;
Computations, H.W. and F.Z.; Writing-Original Draft Preparation, L.L.; Writing-Review & Editing, F.H., Y.L., J.Z.,
F.Z., N.L. and H.W.
Funding: This research was funded by the National Natural Science Foundation of China [Grant number:
81774099 and 81173577], the National Key R&D Program of China [2017YFC1701100] and the Natural Science
Foundation of Guangdong [8451008901000380].
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 5a–5j are available from the authors.
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