9-vinylanthracene based fluorogens: Synthesis, structure-property relationships and applications

Article abstract of DOI:10.3390/molecules22122148

Fluorescent dyes with aggregation-induced emission (AIE) properties exhibit intensified emission upon aggregation. They are promising candidates to study biomolecules and cellular changes in aqueous environments when aggregation formation occurs. Here, we report a group of 9-position functionalized anthracene derivatives that were conveniently synthesized by the palladium-catalyzed Heck reaction. Using fluorometric analyses, these dyes were confirmed to show AIE behavior upon forming aggregates at high concentrations, in viscous solvents, and when poorly solubilized. Their photophysical properties were then further correlated with their structural features, using density functional theory (DFT) calculation. Finally, we demonstrated their potential applications in monitoring pH changes, quantifying globular proteins, as well as cell imaging with confocal microscopy.

Full text of DOI:10.3390/molecules22122148

molecules
Article
9-Vinylanthracene Based Fluorogens: Synthesis,
Structure-Property Relationships and Applications
Mengjie Liu 1,†, Sawaros Onchaiya 1,†,‡, Lewis Yi Fong Tan 1, Mohammad A. Haghighatbin 1,
Tracey Luu 1, Tze Cin Owyong 1,2, Roozbeh Hushiarian 1, Conor F. Hogan 1, Trevor A. Smith 2
and Yuning Hong 1,
*
ID
1
Department of Chemistry and Physics, La Trobe Institute for Molecular Science, La Trobe University,
Melbourne, VIC 3086, Australia; M.Liu4@latrobe.edu.au (M.L.); sawaros.onc@student.mahidol.ac.th (S.O.);
18768730@students.latrobe.edu.au (L.Y.F.T); M.Haghighatbin@latrobe.edu.au (M.A.H.);
traceyl@student.unimelb.edu.au (T.L.); towyong@student.unimelb.edu.au (T.C.O.);
R.Hushiarian@latrobe.edu.au (R.H.); C.Hogan@latrobe.edu.au (C.F.H.)
School of Chemistry, The University of Melbourne, Parkville, VIC 3010, Australia; trevoras@unimelb.edu.au
Correspondence: Y.Hong@latrobe.edu.au; Tel.: +61-3-9479-2995
These authors contributed equally to this work.
Current address: Department of Chemistry, Faculty of Science, Mahidol University,
Bangkok 10400, Thailand.
2
*
†
‡
Received: 1 September 2017; Accepted: 1 December 2017; Published: 4 December 2017
Abstract: Fluorescent dyes with aggregation-induced emission (AIE) properties exhibit intensified
emission upon aggregation. They are promising candidates to study biomolecules and cellular
changes in aqueous environments when aggregation formation occurs. Here, we report a group
of 9-position functionalized anthracene derivatives that were conveniently synthesized by the
palladium-catalyzed Heck reaction. Using fluorometric analyses, these dyes were confirmed to
show AIE behavior upon forming aggregates at high concentrations, in viscous solvents, and when
poorly solubilized. Their photophysical properties were then further correlated with their structural
features, using density functional theory (DFT) calculation. Finally, we demonstrated their potential
applications in monitoring pH changes, quantifying globular proteins, as well as cell imaging with
confocal microscopy.
Keywords: aggregation-induced emission; anthracene; solvatochromism
1. Introduction
The availability and proper design of fluorescent dyes are the fundamental elements in
optical imaging, enabling highly specific and in vitro and non-invasive in vivo investigation of
biomolecules [
dyes has been a popular research area and fluorescence imaging has become a valuable technique in
both research and clinical settings [ ]. The fluorescence of the conventional dyes can be markedly
1]. Since they were first identified in the 19th century, the development of fluorescent
2–4
disabled when they self-aggregate, which is commonly known as aggregation-caused quenching
(ACQ). This phenomenon is speculated to originate from the aromatic planar structures typically
seen in these dyes, which causes strong
π–π stacking interactions in aggregation and thus switch off
the fluorescence [ ]. Research groups have taken advantage of this ACQ effect in the detection of
5
biomolecules. For example, Doria et al. selectively probed G-quadruplexes by using a naphthalene
derivative, which remained aggregated and quenched in physiological conditions, but became
disaggregated and emissive upon binding to the targets of interest [
6]. However, conjugation of
conventional dyes to biomolecules with multiple labeling sites can readily incur aggregation and
Molecules 2017, 22, 2148; doi:10.3390/molecules22122148
www.mdpi.com/journal/molecules

Molecules 2017, 22, 2148
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therefore annihilate their emission [
7
]. To date, this is an issue that limits the usefulness of the
conventional dyes in biomolecular applications.
Recently, a number of aggregation-induced emission (AIE) dyes have attracted increasing attention
because they show no or weak emission in the solubilized state, but become progressively emissive
when forming aggregates. Unlike “conventional” dyes, AIE-active dyes are typically non-planar
but show a propeller shape, with a stator moiety surrounded by a number of aryl rotors. In a
diluted solution, these rotors are able to freely rotate, thus rendering the dyes non-emissive by
dissipating the energy through non-radiative pathways rather than fluorescence emission. In the
aggregated state where the rotations are markedly constrained, the overall propeller shape prevents
π
–π
stacking formation, and thus the dyes become emissive as the excess energy can now be released
as light [ ]. Symbolic examples of AIE-active dyes include a series of tetraphenylethene (TPE)
5
derivatives comprised of an olefin stator surrounded by four phenyl rotators. They have been
proved valuable in visualization of biomolecules and cellular environments such as mitochondrial
phospholipids [8], nucleic acids [9], tumor proteins [10], and changes in intracellular pH [11],
viscosity [12] and macromolecular crowding conditions [13]. The distyrylanthracene (DSA) derivatives
are another set of typical AIE-active dyes (Figure 1 left). These molecules are structurally non-planar
and therefore emissive in the aggregate state, owing to the steric hindrance induced by their
bulky anthracene core. Several research groups have demonstrated the molecular stacking, crystal
formation and photophysical properties of these dyes with various structural substitution [14
–17],
as well as their applications [18 21]. However, only a few studies have focused on the structural
–
analogues, mono-functionalized anthracene derivatives (Figure 1 right), and their photophysical
properties [22–24]. Additionally, their potential use in biological systems has yet to be demonstrated.
Figure 1. The structures of distyrylanthracene (left) and mono-functionalize anthracene (right).
In this paper, we report a group of 9-position functionalized anthracene derivatives that were
robustly prepared by the well-established palladium-catalyzed Heck reaction. We characterize their
photophysical behaviors systematically to elucidate their structure-property relationships. Significantly,
we were also able to demonstrate their potential applications in sensing pH, detecting proteins of
interest, as well as cell imaging.
2. Results and Discussion
2.1. Dye Synthesis
The synthetic routes towards the desired 9-styrylanthracene-derived fluorophores (dyes 1–3,
Figure 2) and their characterization data are detailed in the Experimental section. In general, the
core structures of these dyes could be conveniently synthesized using the palladium-catalyzed Heck
coupling reaction, which linked the corresponding vinyl moieties to the anthracene system via a C=C
double bond. Importantly, this reaction enabled synthesis of optically pure stereoisomers owing to its
stereo-selectivity towards the trans-configuration. The synthesis of dye 1 was achieved by adapting
the ligand-free Heck reaction strategy reported by Yao et al. [25], where the catalyst system consisted
of Pd(OAc)2 and K3PO4 in dimethylacetamide (DMA) without any triaryl phosphine ligand. On the
other hand, dye
of the ligand PPh3 [22]. The positively charged dye
2
was synthesized following the traditional Heck reaction strategy in the presence
was simply prepared from dye by refluxing
3
2

Molecules 2017, 22, 2148
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in CH3I, followed by ion exchange in the presence of KPF6 as described in the Experimental section.
All products were characterized by 1H- and 13C-NMR and mass spectrometry.
Figure 2. Chemical structures of dyes 1–3.
2.2. Structure-Property Relationships
2.2.1. Absorption and Emission Spectra
As all dyes were found soluble in dimethyl sulfoxide (DMSO), their fluorescence properties were
first measured in this solution. Their absorption profiles were analyzed by normalized absorption
spectra (Figure 3A). Dyes
392 nm, respectively), while dye
at about 420 nm. Subsequently, their emission spectra were measured after exciting the dyes at their
recorded absorption maxima (Figure 3B). Dyes and exhibited emission maxima at 475 and 495 nm,
respectively, with the absolute quantum yields of 0.60 and 0.03, respectively; however, dye showed a
1
and
2
reached maximal excitation at very similar wavelengths (390 and
3
exhibited a slight blue-shift and a second absorption maximum
1
2
3
marked red-shift with the maximum at 618 nm when excited at 420 nm with a quantum yield of lower
than 1% in DMSO solution.
A
B
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
Dye 1
Dye 2
Dye 3
360
420
480
540 400
500
600
700
800
Wavelength (nm)
Wavelength (nm)
Figure 3. (A) Normalized absorption and (B) emission spectra in DMSO. Concentration = 10 µM.
Excitation wavelength: 390 (1), 392 (2) and 420 nm (3).
2.2.2. Effect of Solvent Polarity
We analyzed the absorption and emission profiles of all the dyes in solvents with various polarity.
The results are shown in Figure 4 and Figures S1–S3 in Supplementary Materials. The absorption
maxima of dye 1, obtained from the normalized absorption spectra, ranged from 385 nm to 395 nm
in different solvents (Figure S1A), where water induced a small red-shift to 395 nm and a more
pronounced shoulder at longer wavelengths. Compared with its emission spectra (Figure S1B),
the strongest emission occurred in toluene, the most hydrophobic solvent used in this work. The lowest
emission intensity was observed with water, a highly polar solvent. Compared with the normalized
emission spectra shown in Figure 4A, the emission maxima in different solvents ranged from 470 to
475 nm; as an exception, water caused a marked red-shift to 515 nm.

Molecules 2017, 22, 2148
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Acetone
Acetonitrile
DCM
1.0
0.8
0.6
0.4
0.2
0.0
DMSO
Ethanol
Ethyl acetate
Methanol
THF
Toluene
water
C
A
B
550
600
650
700
750
800
400 450 500 550 600 650 700
400
450
500
550
600
650
Wavelength (nm)
Wavelength (nm)
Wavelength (nm)
Figure 4. Normalized emission spectra of dyes
Concentration = 10 µM.
1 (A), 2 (B) and 3 (C) in different solvents.
The absorption maxima of dye
2 ranged from 387 nm to 392 nm in different solvents, where
the absorption spectrum in water was markedly broader than those in other solvents (Figure S2A).
Our subsequent experiments demonstrated that this dye formed nanoaggregates in water (ranged
from 500–600 nm, discussed later) with greater size than the wavelength of incident light. This is
expected to cause a Mie scattering effect, which broadens the absorption spectrum. Its emission was
strongest in toluene and weakest in methanol (Figure S2B), where its emission maxima ranged from
480 nm to 500 nm. Similar to the case of dye 1, water caused a slight red shift to 513 nm (Figure 4B).
Dye
Figure S3A). Interestingly, dichloromethane (DCM) caused the most notable red shift. In comparison
with dye and , dye showed two distinctive features. First, it exhibited a dramatic reduction in
3 exhibited absorption maxima ranging from 415 nm to 466 nm in different solvents
(
1
2
3
emission in all solvents (Figure S3B), where the emission was strongest in DMSO and weakest in water.
Second, it exhibited a large red-shift in all solvents, with the emission maxima ranging from 600 nm
to 630 nm. Additionally, some solvents, such as ethyl acetate and toluene, caused a small blue-shift
compared with other solvents (Figure 4C).
The Stokes shift of all dyes as a function of solvent polarity was graphed in the Lippert–Mataga
solvatochromism plot (Figure 5A–C) [26]. It can be observed that, in general, the Stokes shifts of
dyes
2 and 3 are mostly proportional to the solvent polarity, suggesting both dyes possess positive
solvatochromism, whereas the Stokes shift remained almost unchanged in different solvents with the
exception of dye 1 in water.
115
110
105
100
95
190
180
170
160
150
140
A
B
C
120
110
100
90
M
W
W
W
A
D
M
E
AN
AN
D
THF
EA
DCM
A
DCM
E
THF
AN
EA
T
DCM
THF
90
A
T
D
E
EA
M
T
80
85
0.00
0.15
0.20
0.25
0.30
0.35
0.00
0.15
0.20
0.25
0.30
0.35
0.00
0.15
0.20
0.25
0.30
0.35
f
f
f
Figure 5. Lippert–Mataga solvatochromism plot of dyes
1 (A), 2 (B) and 3 (C) in different solvents
as a function of solvent polarity measured by Lippert-Mataga polarity parameter. A = acetone,
AN = acetonitrile, D = DMSO, E = ethanol, EA = ethyl acetate, M = methanol, T = toluene, W = water.

Molecules 2017, 22, 2148
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2.2.3. Effect of Viscosity
We attempted to assess whether our dyes behaved like AIE-active species using viscous solvents,
which were expected to restrict the intramolecular motions of the dye molecules, thus enhancing their
emission intensity [
ethylene glycol/glycerol mixtures, both of which have similar polarity with glycerol being the viscosity
enhancer (Figure 6). It was observed that the emission of dye remained similar until the media
turned highly viscous (glycerol fraction > 75%), while dyes and showed progressively enhanced
emission with the increase of glycerol fraction. It is speculated that, although all these dyes contain
5,27]. Fluorescence measurements were performed on a series of dye solutions in
1
2
3
rotatable moieties, the strong dipole moment within dyes
viscosity changes [12].
2
and
3
can make them more vulnerable to
fG (vol%)
fG (vol%)
fG (vol%)
C
100
75
50
25
0
100
75
50
25
0
100
75
50
25
0
A
B
400 450 500 550 600 650 700 750
450 500 550 600 650 700 750 500 550 600 650 700 750 800
Wavelength (nm)
Wavelength (nm)
Wavelength (nm)
Figure 6. Emission spectra of dyes
1
(
A
),
2
(
B) and
3
(
C) in ethylene glycol/glycerol mixtures, where
fG indicates glycerol fraction. Concentration = 10
µM. Excitation wavelength: 388, 390 and 437 nm,
respectively. Note: in panel C, the peak at approx. 500 nm corresponds to the Raman scattering of
the solvent.
2.2.4. Effect of Concentration and Aggregation
We then attempted to further verify the AIE properties of the dyes by inducing aggregation
with increased concentration and reduced solubilization. We first took photos of their solubilized
and aggregated states under UV light. As illustrated in Figure S4, all dyes are only weakly
emissive when molecularly solubilized in acetone, a solvent with strong dissolving power for organic
molecules. However, intense fluorescence was observed for all dyes in their solid state, where their
concentration is maximized. We then performed serial dilutions to all dyes (solutions in DMSO) and
measured their emission intensity at those resulting concentrations (Figure 7). Basically, all dyes were
non-emissive or only weakly emissive at concentrations below 10−5 M, and then became strongly
fluorescent at higher concentrations. Since aggregation is more likely to occur at higher concentrations,
some conventional dyes such as pyrene experience fluorescence quenching at higher concentrations.
The fluorescence enhancement of these dyes at higher concentrations suggested that all dyes behaved
like AIE-active compounds.
The AIE properties of all dyes were then verified in a series of methanol/water mixtures. Note
that these two solvents possess relatively close polarity so as to minimize the influence of solvent
polarity. We speculated that dyes would become progressively emissive with increasing water fraction.
Strikingly, dye 1 exhibited similar emission until the water fraction increased up to 70%, and then a
sharp reduction in intensity was observed at around 80%. A red shift was also observed at 90% and
100% water (to 500 nm and 520 nm), respectively (Figure 8A,B). Similarly, addition of water did not
intensify the emission of the charged compound dye
3 (Figure 8E,F). On the other hand, dye 2 showed
similar behavior as other AIE-active dyes: it remained weakly emissive until the water percentage
increased up to 80%, thereafter showing a sharp increase in intensity (Figure 8C,D).

Molecules 2017, 22, 2148
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140
120
100
80
875
750
625
500
375
250
125
0
Conc. of Dye (M)
Conc. of Dye (M)
A
B
C
Conc. of Dye (M)
40
10-3
10-4
10-5
10-6
10-7
10-8
10-3
10-4
10-5
10-6
10-7
10-8
10-3
10-4
10-5
10-6
10-7
10-8
30
20
10
0
60
40
20
0
400 450 500 550 600 650 700 750
450 500 550 600 650 700 750
450 500 550 600 650 700 750 800
Wavelength (nm)
Wavelength (nm)
Wavelength (nm)
Figure 7. Emission spectra of dyes
1 (A), 2 (B) and 3 (C) at various concentrations in DMSO. Excitation
wavelength: 390, 392 and 420 nm, respectively.
Figure 8. Emission intensity of dyes
different water fractions. Concentration = 10
437 nm respectively.
1
(
A
,
B
),
2
µ
(
C
,
D
) and
3
(
E
,
F
) in methanol/water mixtures with
ex: 385, 387 and
M. fw and vol% = water fraction. λ

Molecules 2017, 22, 2148
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To search for an explanation for the inconsistency observed with dyes
1
and
3, dynamic light
scattering (DLS) was performed to assess the particle sizes of dyes dispersed in water (Figure S5).
The plot showed that the dispersion of dye formed nanoaggregates with average diameter of 165 nm,
whereas that of dye was averaged at 523 nm. Reliable particle size data could not be obtained for
dye , most likely because aggregation was not formed owing to the positive charge. The influence of
1
2
3
particle sizes will be discussed in more detail later.
2.2.5. Mechanistic Understanding
Absorption and emission spectra revealed a significant bathochromic shift of dye 3 in solution state
when compared to dyes 1 and 2 (Figure 3). To gain insight into the structure–property relationships
of these 9-vinylanthracene derivatives, we performed density functional theory (DFT) calculations
in Gaussian using the B3LYP/6-31G basis set to obtain the optimized molecular structures and the
energy levels of the frontier molecular orbitals (Figure 9). From the optimized structures, the dihedral
angles between the anthracene ring and the central C=C double bond were calculated to be 50.4, 49.4
and 44.8 degrees for dyes
conjugation of dye is the best among these three dyes and thus explains the observed red-shift in
the absorption spectrum. Meanwhile, the band gaps between the highest occupied molecular orbitals
(HOMO) and the lowest unoccupied molecular orbitals (LUMO) of dyes were calculated to be
1–3, respectively. The smallest dihedral angle in dye 3 suggests that the
3
1–3
3.23, 3.21, and 2.45 eV, respectively, which are consistent with our experimental results showing the
absorption maxima of 390, 392 and 429 nm, respectively (Figure 3).
Figure 9. Molecular orbital amplitude plots and the HOMO/LUMO energy levels of
3 (C) calculated using the B3LYP/6-31G basis set.
1 (A); 2 (B) and
The electron clouds of the HOMO and LUMO of dye
anthracene rings and the central C=C double bond, indicating that intramolecular charge transfer (ICT)
processes are unlikely in this molecule [28]. While the HOMO of both dyes and are located on the
vinylanthracene moiety, as for dye , the LUMO of dye has a large portion of electron density located
at the pyridine unit while the LUMO of dye is mostly located at the pyridinium unit (Figure 9).
The difference in the spatial distribution of the HOMOs and LUMOs indicates that the ICT process
is feasible in both these dyes. For dye , the distinct electron distribution of the HOMO and LUMO
suggests a strong push-pull effect with the vinylanthracene as electron donor and the pyridinium as
acceptor [29]. This is in line with our observation of the solvatochromism behaviour of both dyes
and in solvents with different polarity whereas dye is almost inert to the change of solvent polarity
(Figures 4 and 5). The red shift observed in the emission spectrum of dye in water (Figure 4A)
1 are both mostly distributed on the
2
3
1
2
3
3
2
3
1
1
could be attributed to strong intermolecular interactions when the molecules cluster to form dense
nanoaggregates (Figure S5A). To further understand the mechanism, we performed time-resolved
fluorescence measurements (Figure 10 and Table S1). The multi-exponential decay of dye 1 in water
indicates the heterogeneity of aggregates. However, no direct evidence of excimer formation can be
found from the fluorescence decay profile.

Molecules 2017, 22, 2148
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104
103
102
101
100
1 in DMSO
1 in water
IRF
4
6
8
10
12
14
16
18
20
Time (ns)
Figure 10. Fluorescence decay profile of dye 1 in DMSO (red) and water (blue). The black line shows
the instrument response function (IRF) of the measurement system. Dye concentration: 10 µM. λ :
ex
400 nm. λem: 480 nm for DMSO and 520 nm for water.
On the other hand, the large dihedral angles as shown in the optimized structure between the
vinylanthracene and the terminal aromatic rings suggest that none of the dyes is coplanar. Because of
the steric hindrance, the anthracene ring twists out of the central plane. Thus, the conjugation between
anthracene and the central C=C bond is weak, which allows the intramolecular motions of the C-C
bond upon excitation [5]. Such intramolecular motions would serve as a non-radiative decay channel to
deactivate the excited species. As long as such intramolecular motions are restricted, the fluorescence
intensity of the dyes can be enhanced. In viscous medium, such as glycerol, the intramolecular motions
would be hampered and thus more excited species can go through the radiative decay pathway.
Therefore, with the increase of glycerol fraction, the fluorescence of dyes 1–3 is increased (Figure 6).
Conventional ACQ dyes such as pyrene suffer from concentration-caused quenching [
Although when the concentration of pyrene is increased from 10−6 to 10−4 M, the fluorescence
is enhanced, further increasing its concentration results in a decrease of the emission. For dyes 1–3,
5].
increasing the concentration from 10−8 to 10−3 M monotonically intensifies the fluorescence intensity
of the dyes (Figure 7), as occurs with most AIE luminogens. Increasing the dye concentration also led
to the aggregation of the dyes, which, in turn, restricts the intramolecular motions of the dye molecules.
In the solid state, the intramolecular motions are hampered greatly and, as observed, all the dyes
(
1
–
3
) emit strong fluorescence (Figure S4). Usually increasing the water fraction in solvent mixtures
can induce aggregation of hydrophobic dyes; for AIE-active dyes, the fluorescence would increase
along with the increase of water fraction. Dye conformed with the behavior of most AIE dyes: its
2
fluorescence remained low when the water fraction in methanol/water mixture is lower than 85% and
increased dramatically when the water fraction reaches 99% where nanoaggregates form (Figure 8 and
Figure S5). However, for dyes
For dye , although nanoaggregates formed at a high water fraction, the fluorescence was weaker than
in methanol. A red-shift in the spectrum implies strong intermolecular interactions, predominantly on
the anthracene moiety, in the high water fraction and weakened the fluorescence. For dye , due to its
1 and 3, increasing the water fraction does not increase their fluorescence.
1
3
amphiphilic property, addition of water could not induce the aggregate formation of the dye molecules
and we thus were unable to see the increase of its fluorescence.
2.2.6. Comparison with Other Literature Structural Analogs
The basic spectroscopic and photophysical properties of dyes
from representative literature and commercial analogs reported previously (Table 1, structures
shown in Figure S7). Entries and are examples of 9-position mono-functionalised anthracene
derivatives. It can be observed that adding extra bulky conjugations to the phenyl moiety of
1–3 are compared with those
4,
5
6

Molecules 2017, 22, 2148
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dye
absorption and emission can occur after converting the pyridine moiety into a charged pyridinium
vs. and ). Considering the listed 9,10-bifunctionalised anthracene derivatives ( 15), it appears
that they generally exhibit absorption and emission maxima at longer wavelength in comparison
to their mono-functionalised counterparts ( vs. and 15 vs. ). Electron-donating and weak
electron-withdrawing groups attached to the phenyl moieties of do not incur significant changes to
the spectroscopic profiles ( 13); however, nitrile groups as a strong electron withdrawer can cause
1 (as in entries 4 and 5) caused red-shift in the emission spectra. Marked red-shift in both
(2
3
6
7–
7
1
2
7
8
–
large red-shift in the emission spectra (14). Lastly, most dyes listed show a very low quantum yield
when molecularly solubilised, and this typical AIE phenomenon is consistent with the observations in
dyes 2 and 3.
Table 1. Spectroscopic and photophysical properties of Dyes
reported analogs.
1–3 compared to previously
[a]
Absorbance
PL in Solution
Φ
(%) in
PL
Entry
Compounds
Ref.
λ
max/nm
λ
max/nm
Solution
dye 1 [b]
dye 2 [b]
dye 3 [b]
1
2
3
390
392
385, 420
475
495
618
60
3
<1
This work
t-2-ANE [c],
*
*
4
5
6
387
342, 390
442
490
510
N/A
68
15
N/A
[30]
[31]
[32]
1,4-ASB [d],
“Chromophore 9” [d]
DSA [d],
*
7
8
9
304, 414
307, 415
307, 417
313, 415
555
550
538
567
0.4
0.7
0.5
2.3
BMOSA [d],
*
[33]
B-4-BOSA [d]
B-2-BOSA [d]
10
9,10-MADSA [d]
9,10-PADSA [d]
11
12
~315, ~422
~315, ~422
~550
~550
0.5–0.8
0.5–0.8
[34]
BCSA [e],
CNDSA [d]
BP4VA [d],
*
13
14
15
403
418
410
576
625
584
N/A
25
N/A
[35]
[36]
[37]
*
[a]
[c]
[b]
The chemical structures of all the compounds are shown in Supplementary Information.
solvent = acetonitrile; [d] solvent = THF; [e] solvent = DCM. * = commercially available.
solvent = DMSO;
2.3. Applications
2.3.1. Protonation Effect
In comparison with commonly used electrochemical pH sensors, optical pH sensors possess a
number of advantages including contactless measurement, ease of miniaturization and possibility of
performing concurrent imaging [38]. Based on the well-established understanding that the degree
of protonation may impose significant changes to the absorption/emission [39
pyridine-bearing dye to assess its capability in sensing pH alternation in solutions. Solutions varying
in proton concentration were prepared using trifluoroacetic acid (TFA) as a proton source. As shown
in Figure 11, dye was found to be strongly emissive in neutral and moderately acidic environments,
and then exhibited a sharp reduction in emission up to a 50 mM proton concentration. Similar results
were observed with dye in aqueous media (Figure S6). An exponential relationship between the
emission intensity and proton concentration was established, suggesting that dye may be a suitable
optical pH sensor for conditions ranging from neutral to strong acidic environments.
–41], we selected the
2
2
2
2

Molecules 2017, 22, 2148
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400
300
200
100
A
B
Conc. of TFA (mM)
400
300
200
100
0
0
1.3
2.9
4.6
6.2
32.7
65.3
98.0
130.6
163.3
195.9
228.6
400 450 500 550 600 650 700 750
0
50
100
150
200
250
Wavelength (nm)
+
[H ] (mM)
Figure 11.
(A) Emission spectra of dye 2 in DMSO with increasing concentration of TFA.
Dye concentration = 20 µM; λ 392 nm; (B) emission intensity of dye 2 vs. proton concentration.
ex:
2.3.2. Protein Detection
Fluorometric methods for protein detection and quantification are becoming increasingly used in
research fields, owing to their satisfactory sensitivity, low background noise and wide dynamic ranges.
Protein binding onto AIE-active dyes has been shown to enhance emission intensity [42]. In this
work, we selected dye
solubility than dyes
analysed in a pH 7.0 phosphate buffer. As illustrated in Figure 12, dye
3
for investigation as its positive charge enabled comparatively higher aqueous
and . Its interaction with a model protein, bovine serum albumin (BSA), was
was only weakly emissive
1
2
3
in buffer solution in the absence of BSA. Addition of BSA progressively intensified its fluorescence
emission, with the emission maxima shifting from 620 nm to 575 nm. As BSA contains hydrophobic
binding pockets in its native folded structure, we speculated that the intramolecular rotation of
BSA-bound dye
aggregated state. Meanwhile, the blue-shift of the spectra indicated the transition from a hydrophilic
to hydrophobic environment of the dye molecules. These observations again proved that dye is
3 had been hindered, hence emitting in a manner similar to that observed in an
3
AIE-active and therefore is a valuable candidate for protein fluorescent detection and quantification
in proteomics.
35
Conc. of BSA (M)
100
50
10
1
30
25
20
15
10
5
0
0
500
550
600
650
700
750
800
Wavelength (nm)
Figure 12. Fluorescence emission spectra of dye
3 with various concentrations of BSA in
phosphate-buffered saline (PBS) excited at 420 nm.

Molecules 2017, 22, 2148
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2.3.3. Cell Imaging
All dyes were assessed to determine their potential as luminescent probes for bioimaging.
We stained live HeLa cells with dyes and and fixed the cells before conducting confocal
microscopy bioimaging. The results indicated that all the dyes were capable of entering the cytoplasm,
but only and displayed some nuclear penetration (Figure 13). This could be largely due to the
1,
2
3
1
2
anthracene group, which is able to be taken up by lysosomes and then released into the nucleus along
with lysosomal deoxyribonuclease upon prolonged exposure to light [43]. Additionally, there was
little or no background fluorescence when stained with these three dyes. This further validated
their bioimaging potential as dyes 1, 2 and 3 were able to enter cells whilst exhibiting no off-target
fluorescence. To visualise the intensity of their luminescence, we then used the Fire lookup table (LUT)
colour scheme on the Fiji software (ImageJ2, LOCI, Madison, WI, USA) to compare their linearized
pixel values in the display range (0–255 a.u.). Results revealed that the luminescence intensity of
dyes
Moreover, luminescence was only visible when dye
compared with 1 and 2, which were both excited at a lower laser power (2.0%).
1
and
2
were relatively similar, with dye
3
exhibiting the least intense luminescence (Figure 13C).
3
was excited with a higher laser power (6.0%)
B
A
C
1
2
3
Figure 13. Confocal fluorescence microscopy image of HeLa cells stained with dyes
for 30 min. ( ) Merged image of the fluorescence and bright-field images; ( ) fluorescence image under
excitation of 405 nm laser (2.0% for dyes and , 6.0% for dye ); ( ) the intensity of the signal was
1, 2 and 3 (10 µM)
A
B
1
2
3
C
visualised using the “Fire” lookup table (LUT) colour scheme in Image J2. Linear mapping of pixel
values in the display range indicated by the calibration bar (0–255 a.u.). Scale bar: 15 µm.
Compared with dyes
1 and 2, dye 3 exhibited weaker fluorescence in the cells. We postulated
that the main reason is the hydrophilicity of the dye, which makes it difficult to get through the
hydrophobic interior of the plasma membrane [44]. Furthermore, the low fluorescence quantum

Molecules 2017, 22, 2148
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yield of 3 could be another reason for its weak signal from the cells (Figure S3B). Its overall positive
charge and amphiphilic nature may hinder its ability to form aggregates and enhance its luminescence.
In addition, in contrast to traditional AIE dyes such as TPE, all three compounds experienced significant
and rapid photobleaching upon prolonged laser exposure, most likely due to the oxidation of the
anthracene moiety.
3. Experimental Section
3.1. Materials and General Information
9-Bromoanthracene, styrene and 4-vinylpyridine were purchased from Alfa Aesar (Haverhill,
MA, USA). Pd(OAc)2, PPh3, KPF6, MgSO4, trifluoroacetic acid (TFA) and bovine serum albumin (BSA)
were purchased from Sigma Aldrich (St. Louis, MO, USA). K3PO4 and K2CO3 were purchased from
Chem-supply (Adelaide, SA, Australia). CH3I and all solvents were purchased from Merck (Darmstadt,
Germany). All chemicals were used without further purification.
1H-NMR and 13C-NMR spectra were recorded at 400 MHz using a 400 MHz Bruker AV3HD-400
spectrometer (Billerica, MA, USA) at 298 K. UV-Visible absorption spectra were recorded at room
temperature on a Cary 300 UV-visible spectrophotometer (Agilent Technologies, Inc., Santa Clara,
CA, USA) equipped with a 1.0 cm quartz cell. Fluorescence emission and excitation spectra were
recorded on a Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies, Inc., Santa Clara,
CA, USA) and used 1.0 cm quartz cells. Particle sizes were measured by dynamic light scattering using
Zetasizer Nano S90 (Malvern Instruments Ltd., Malvern, UK). Data were plotted by using Origin 2016
(OriginLab Corp., Northampton, MA, USA).
3.2. Synthesis and Characterization
(E)-9-Styrylanthracene (
1). The synthetic route towards (1) was adopted from the strategies previously
described by Yao et al. [25]. To an oven-dried 25 mL round-bottom flask were added 9-bromoanthracene
(0.3 g, 1.17 mmol), styrene (0.12 g, 1.17 mmol), K3PO4 (0.75 g, 3.51 mmol), Pd(OAc)2 (0.01 g, 0.06 mmol
and dry DMA (10 mL). The mixture was degassed by three freeze-pump-thaw cycles, and then
stirred under N2 at 110 ◦C for 24 h. The mixture was poured into water and extracted with DCM.
The combined organic layer was washed with brine, dried over anhydrous MgSO4 and concentrated
)
in vacuo. Water (6
×
7 mL) was added to the resulting mixture to extract DMA. The crude product
1
was dried in vacuo to give a bright yellow solid (100 mg, isolated yield = 30%). H-NMR (400 MHz,
CDCl3):
δ 6.97 (d, 1H), 7.35–7.41 (m, 1H), 7.44–7.52 (m, 6H), 7.67–7.73 (d, 2H), 7.93 (d, 1H), 7.99–8.06
(m, 2H), 8.34–8.44 (m, 3H). 13C-NMR (400 MHz, CDCl3):
128.04, 128.72, 128.87, 129.77, 131.54, 132.79, 137.35.
δ 124.91, 125.21, 125.49, 126.05, 126.49, 126.63,
(E)-4-(2-(Anthracen-9-yl)vinyl)pyridine (2). The synthetic route towards (2) was adopted from the
strategies previously described by Shih et al. [22]. To an oven-dried 50 mL Schlenk tube were added
9-bromoanthracene (1.0 g, 3.9 mmol), K2CO3 (1.6 g, 11.6 mmol), PPh3 (153 mg, 0.6 mmol) and Pd(OAc)2
(44 mg, 0.2 mmol). After the system was sealed and evacuated, dry DMF (10 mL) and 4-vinylpyridine
(613 mg, 630 µL, 5.8 mmol) were injected. The mixture was degassed by three freeze-pump-thaw cycles,
and then stirred under N2 at 110 ◦C for 24 h. The reaction mixture was poured into a LiCl solution
(5% in 100 mL H2O), and the suspension was extracted with EtOAc. The combined organic layer was
washed by brine, dried over anhydrous MgSO4 and concentrated in vacuo. The crude product was
purified by column chromatography (PET:EtOAc, 4:1 then 1:1) and a bright yellow powder (250 mg,

Molecules 2017, 22, 2148
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1
isolated yield = 23%) was collected. H-NMR (400 MHz, CDCl3):
δ
6.92 (d, 1H), 7.48–7.57 (m, 6H),
8.02–8.07 (m, 2H), 8.16 (dd, 1H), 8.26–8.32 (m, 2H), 8.46 (s, 1H), 8.71 (s, 2H). 13C-NMR (400 MHz,
CDCl3):
δ 121.03, 125.33, 125.52, 125.96, 127.36, 128.87, 129.61, 130.00, 131.26, 131.43, 134.78, 144.56,
150.24. ESI-MS: m/z 282.06, calcd. 281.36.
(E)-4-(2-(Anthracen-9-yl)vinyl)-1-methylpyridin-1-ium hexafluorophosphate (3). To a 100 mL round-bottom
flask were added compound 2 (230 mg, 0.8 mmol), acetone (5 mL) and iodomethane (2 mL). The mixture
was refluxed at 50 ◦C for 1 hour. The precipitate was collected by filtration, washed by acetone and
air dried. This intermediate compound (30) was collected as a pale orange powder in quantitative
1
yield. H-NMR (400 MHz, DMSO-d6):
δ
4.34 (s, 3H), 7.34 (d, 1H), 7.55–7.67 (m, 4H), 8.15–8.22 (m, 2H),
47.59,
8.35–8.41 (m, 2H), 8.51 (d, 2H), 8.72 (s, 1H), 8.91–9.03 (m, 3H). 13C-NMR (400 MHz, DMSO-d6):
δ
124.63, 125.76, 126.14, 127.21, 128.90, 129.41, 129.51, 130.42, 131.41, 132.53, 137.95, 145.75, 152.29.
To a 50 mL round-bottom flask were added 30 (120 mg, 0.3 mmol), acetone (25 mL) and saturated
KPF6 solution (5 mL). The resulting solution was stirred at room temperature for 1 hour. After the
solvent was dried in vacuo, the solid was collected by filtration, washed with H2O and dried in the
1
oven. The product was obtained as a deep orange powder (105 mg, isolated yield = 84%). H-NMR
(400 MHz, DMSO-d6):
(
δ
4.34 (s, 3H), 7.33 (d, 1H), 7.57–7.65 (m, 4H), 8.15–8.20 (m, 2H), 8.35–8.41
47.56, 124.63,
m, 2H), 8.50 (d, 2H), 8.72 (s, 1H), 8.91–9.01 (m, 3H). 13C-NMR (400 MHz, DMSO-d6):
δ
125.74, 126.14, 127.21, 128.91, 129.42, 129.51, 130.42, 131.42, 132.53, 137.94, 145.75, 152.30. ESI-MS: m/z
296.06 (anthracene-pyridinium), 144.88 (PF6−), calcd. 296.39 (anthracene-pyridinium), 144.96 (PF6−).
3.3. Sample Preparation for Spectroscopy Measurement
All stock solutions of dyes were prepared in 1 mM in DMSO and kept at room temperature in
dark. BSA stock solution was prepared in PBS. The total concentration of all stock solutions was 1 mM.
For UV-vis absorption measurement, the background of solvent alone was subtracted. For fluorescence
measurement, the excitation and emission slits were fixed at 5 nm for all experiments. Scan speed was
set at medium and the curves presented were the average of 3 times measurement in every experiment.
The working concentration of dyes was 10 µM unless specified elsewhere.
3.4. Quantum Yield Measurements
Absolute quantum yield measurements were performed using a Quanta-Phi HORIBA Scientific
6 in. diameter integrating sphere (HORIBA Scientific, Edison, NJ, USA) at room temperature
(
22 ± 2 ◦C) Photoexcitation was with a 450 W arc-xenon lamp and the emission was directed toward a
nitrogen cooled Symphony II xenon CCD (Model SII-1LS-256-06, HORIBA Scientific, Edison, NJ, USA)
CCD via optical fibers. FluorEssence v3.5 software (HORIBA, Edison, NJ, USA) was used to calculate
the quantum yields in a 4-curve analysis mode using the following equation:
Photons out
Photons in
(Ec − Ea)/A
La − Lc
Φp =
=

Molecules 2017, 22, 2148
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where Ec is the integrated luminescence of the sample resulted by direct excitation, Ea is the integrated
luminescence of the blank, La is the integrated excitation from the blank and Lc is the integrated
excitation from the sample, and A is the area balance factor obtained from the multiplication of the
CCD integration time. Spectral measurements were averaged from at least three replicates.
3.5. Particle Size Measurements
As part of the experiment, 20
water and kept in a 15 mL Falcon tube at room temperature. The total concentration was around
10 M. The particle size distribution was measured via Zetasizer Nano S90 (Malvern Instruments Ltd.,
µL of stock solution of dye 1 and 2 was added in 2 mL Milli-Q
µ
Malvern, UK).
3.6. Time-Resolved Fluorescence Measurements
Fluorescence decay measurements were performed by the time-correlated single-photon counting
technique using the frequency doubled output (400 nm) of a mode-locked and cavity-dumped
Titanium: sapphire laser (Coherent Mira 900f). The repetition rate of the laser pulses was reduced to
5.4 MHz (APE PulseSwitch, Angewandte Physik & Elektronik GmbH, Berlin, Germany). Emission was
collected under magic angle conditions, spectrally selected (480 nm or 520 nm) using a Jobin Yvon H20
monochromator (Longjumeau Cédex, France) and detected with a microchannel plate photomultiplier
(Eldy EM1-132-1, St. Petersburg, Russia). Data were acquired using a Becker and Hickl SPC-150 TCSPC
card (Becker & Hickl GmbH, Berlin, Germany) and SPCM software (version 9.6, Becker & Hickl GmbH,
Berlin, German), and analysed using the FAST (Fluorescence Analysis Software Technology) software
(version 3.1, Edinburgh Instruments, Livingston, UK).
3.7. Cell Culture and Imaging
3.7.1. Cell Culture
HeLa (human cervical cancer) cell line was purchased from American Type Culture Collection
(ATCC® CCL2
™) and used within 40 passages of purchase. The cells were cultured in Dulbecco’s
modified Eagles Medium (DMEM) (HyClone®) with 10% fetal calf serum (FCS) (Thermo Fisher
Scientific Inc., Waltham, MA, USA) and Penicillin-Streptomycin (P/S) (Thermo Fisher Scientific Inc.),
and incubated at 5% CO2, 37 ◦C.
Dyes 1, 2 and 3 were stored as stock solutions at 1 mM in dimethyl sulfoxide (DMSO), purchased
from Sigma-Aldrich Co.
3.7.2. Cell Staining and Confocal Imaging
Pre-sterilized
(Sigma-Aldrich Co., St. Louis, MO, USA) for 2 h prior to cell seeding to promote cell adherence. Cells
µ
-Slide 8 Well (Ibidi®) slides were coated with 1 mL/25 cm2 of Poly-L-Lysine
were then seeded at a density of 7
×
105 cells per well and incubated in cell culture medium for 48 h
at 5% CO2 and 37 ◦C. Afterwards, cells were first fixed in 4% paraformaldehyde solution (PFA) in
Phosphate Buffered Saline (PBS) (HyClone®) at 37 ◦C for 5 min to further promote cell adherence.
Prior to cell staining, stock solutions of 1, 2 and 3
1 mM in DMSO were diluted in DMEM (HyClone®)
without FCS and PBS to a working concentration of 50 µM.
Following washing with PBS, the cells were then stained with 50 µM of dyes 1, 2 or 3 at room
temperature for 30 min. After a further wash with PBS, the stained and fixed cells were then stored in
cold PBS.
Imaging was performed on the slides using the LSM 780 confocal microscope (Zeiss, Jena,
Germany), equipped with an AxioObserver Z1 inverted microscope, 63
(NA 1.4), 405 nm diode laser (30 mW), and a multi-anode PMT (32 elements). Luminescence spectrum
was registered from single confocal sections (pinhole set to 1 Airy unit), in 427–529 nm range, using
×
oil immersion objective lens

Molecules 2017, 22, 2148
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405 nm (dye
1
and
2
—2.0% power, dye
3—6.0% power). Images were processed using the free Fiji
software (ImageJ2, LOCI, Madison, WI, USA).
4. Conclusions
In summary, we have synthesized three fluorescent dyes derived from the 9-position
functionalized anthracene moieties using a simple and robust Pd-catalyzed Heck reaction. We then
demonstrated their enhanced emission in the aggregated state using standard fluorometric techniques,
and established their structure–property relationships using DFT calculations. Additionally, the results
of further experiments described here suggest the potential of these dyes in such applications as pH
sensing, protein detection and quantification, and, for the first time, in fluorescence live cell imaging.
Supplementary Materials: Supplementary materials are available online.
Acknowledgments: We thank Kaye Truscott from La Trobe University for providing the HeLa cell line,
Wallace Wong from University of Melbourne for technical support and helpful discussion and La Trobe Institute
for Molecular Science (LIMS) Bioimaging Facility for the access of confocal microscopes. Y.H. is thankful for the
support of the Bruce Stone Fellowship from La Trobe University. S.O. is thankful for the support of the Foreign
Academic Exchange Program Scholarship for Mahidol University Undergraduate Students 2017.
Author Contributions: Y.H. conceived and designed the experiments; M.L., S.O., L.Y.F.T, M.A.H., T.L., T.C.O.
and T.A.S. performed the experiments; M.L., M.A.H., R.H., T.A.S., C.F.H. and Y.H. analyzed the data; M.L., S.O.,
M.A.H., R.H. and Y.H. wrote the manuscript with feedback from the other authors.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds dyes 1–3 are available from the authors.
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