(E)-2-Styrylanthracene-9,10-dione: A new type of fluorescent probe core and its application in specific mitochondria imaging

Article abstract of DOI:10.1016/j.dyepig.2021.109797

Herein, a new type of fluorescent probe core, (E)-2-styrylanthracene-9,10-dione (EK01), was developed which displayed strong fluorescence quantum yield (Φ = 0.867 in DMF; Φ = 0.561 in acetone; Φ = 0.616 in CH₂Cl₂; Φ = 0.265 in DMSO), good photostability, large stokes shift (90 nm–120 nm) and molar extinction coefficients (0.5875 × 10⁴–0.7609 × 10⁴ mol^?1 L cm^?1). During cell assays and co-localization experiments, EK01 showed excellent cell membrane permeability and low cytotoxicity against MCF-10A (human mammary epithelial cell line) and HT-29 (human colorectal adenocarcinoma cell line). Particularly, we surprisingly discovered that EK01 could selectively aggregate in mitochondria and specific stain it in a green emissive fluorescent form, which means that EK01 could be a real-time specifically monitor of mitochondria in living cells with a high signal-to-noise ratio. Hence a new mitochondria imaging method was established which is incubating EK01 with living cells for 1 h at a final concentration of 6–12 μM, then visualizing under a confocal microscope at 395 nm. It is worth noting that the fluorescence efficiency of EK01 is not outstanding in organisms, it has much stronger fluorescence efficiency in other organic solvent systems (such as DMF, acetone and CH₂Cl₂). Therefore, as a new type of fluorescent core that is easy to synthesis and graft, we believe that (E)-2-styrylanthracene-9,10-diones have the potential to develop a variety of fluorescence platforms applying in different fields.

Full text of DOI:10.1016/j.dyepig.2021.109797

Journal Pre-proof
(E)-2-Styrylanthracene-9,10-dione: A new type of fluorescent probe core and its
application in specific mitochondria imaging
Xu Zhang, Ting-jian Zhang, Zhao-ran Wang, Qiu-yin Wang, Peng-fei Lu, Hai-yang
Zhao, Lin Wang, Fan-hao Meng
PII:
S0143-7208(21)00663-X
DOI:
https://doi.org/10.1016/j.dyepig.2021.109797
DYPI 109797
Reference:
To appear in: Dyes and Pigments
Received Date: 24 July 2021
Revised Date: 24 August 2021
Accepted Date: 6 September 2021
Please cite this article as: Zhang X, Zhang T-j, Wang Z-r, Wang Q-y, Lu P-f, Zhao H-y, Wang L, Meng F-
h, (E)-2-Styrylanthracene-9,10-dione: A new type of fluorescent probe core and its application in specific
mitochondria imaging, Dyes and Pigments (2021), doi: https://doi.org/10.1016/j.dyepig.2021.109797.
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2021 Published by Elsevier Ltd.

Author Contributions:
Xu Zhang: Methodology, Original Draft Writing; Ting-jian Zhang: Conceptualization,
Review and Editing; Zhao-ran Wang: Investigation and Data analysis; Qiu-yin Wang: Validation;
Peng-fei Lu: Synthesis; Hai-yang Zhao: Instrument resources; Lin Wang: Review and Editing;
Fan-hao Meng: Project administration.

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(E)-2-styrylanthracene-9,10-dione: a new type of fluorescent probe
core and its application in specific mitochondria imaging
Xu Zhang a, 1, Ting-jian Zhang a, 1, Zhao-ran Wang , Qiu-yin Wang , Peng-fei Lu , Hai-yang Zhao
a
a
a
b
a
a,
, Lin Wang , Fan-hao Meng *
a
b
School of Pharmacy, China Medical University, 77 Puhe Road, North New Area, Shenyang 110122, China
Teaching Center for Basic Medical Experiment, China Medical University, 77 Puhe Road, North New Area,
Shenyang 110122, China
*Corresponding author.
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E-mail: fhmeng@cmu.edu.cn (F. H. Meng).
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These two authors contributed equally to this work.
Abstract
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Herein, a new type of fluorescent probe core, (E)-2-styrylanthracene-9,10-dione (EK01), was developed which
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displayed strong fluorescence quantum yield (Φ = 0.867 in DMF; Φ = 0.561 in acetone; Φ = 0.616 in CH Cl ; Φ =
0.265 in DMSO), good photostability, large stokes shift (90 nm to 120 nm) and molar extinction coefficients (0.5875
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× 10 - 0.7609 × 10 mol ·L·cm ). During cell assays and co-localization experiments, EK01 showed excellent cell
membrane permeability and low cytotoxicity against MCF-10A (human mammary epithelial cell line) and HT-29
(human colorectal adenocarcinoma cell line). Particularly, we surprisingly discovered that EK01 could selectively
aggregate in mitochondria and specific stain it in a green emissive fluorescent form, which means that EK01 could
be a real-time specifically monitor of mitochondria in living cells with a high signal-to-noise ratio. Hence a new
mitochondria imaging method was established which is incubating EK01 with living cells for 1 h at a final
concentration of 6 ~ 12 μM, then visualizing under a confocal microscope at 395 nm. It is worth noting that the
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fluorescence efficiency of EK01 is not outstanding in organisms, it has much stronger fluorescence efficiency in
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other organic solvent systems (such as DMF, acetone and CH Cl ). Therefore, as a new type of fluorescent core that
is easy to synthesis and graft, we believe that (E)-2-styrylanthracene-9,10-diones have the potential to develop a
variety of fluorescence platforms applying in different fields.
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Keywords: Fluorescent probe; (E)-2-Styrylanthracene-9,10-dione; Mitochondrial imaging.
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1. Introduction
Organic small molecule fluorescent probes have become valuable tools in a variety of related
research fields, including physics, chemistry and life science, and played important roles in food
safety detection, environmental monitoring, medical diagnosis and pharmacological and
toxicological analyses, due to their high sensitivity and selectivity, tunable excitation and emission
wavelengths, and customizable functionalization structure modification [1-5]. In recent years,
fluorescent probes based on xanthene, coumarin, and BODIPY are currently used, some of which
are commercially available [6-10]. But to meet the demands of fluorescent probes for changing
rapidly bioanalytical applications, research and development of more new fluorescent probes with
good fluorescence spectrum characteristics is still the key and core of the development of
fluorescence analysis technology.
Traditional bio-detection methods include radioisotope labeling, microscopy, and
electrochemistry, but the radioisotope labeling method has the risk of radiation and with the arrival
of the half-life, the detection signal will be greatly reduced; the microscopy observation method
cannot be effective for the observation of specific biological activity due to the limitation of
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magnification; and electrochemical monitoring easily damages biological samples. As the result of
these drawbacks, the traditional bio-detection methods have been greatly limited in practical
applications. Compared with traditional bio-detection methods, fluorescence analysis technology
has become one of the most important methods of biological detection now and had great practical
application because of its advantages such as non-hazard, rapid and intuitive analysis, high
sensitivity, and friendly operating environment [11-14]. Due to its advantages, fluorescence analysis
technology can be applied not only to mice, but also to biological tissues or living cells to perform
cell fluorescence coloring, to realize in-situ detection and real-time monitoring for the content and
spatial distribution of the measured substance in the biological tissues or cells [15-17]. Ultimately,
fluorescence imaging technology can achieve the visual observation of cell morphology, structure,
and life activity.
Anthraquinone is a traditional fluorescent core [6,7]. In our previous research, we accidentally
found that some anthraquinone synthetic intermediates possess explicit fluorescent phenomenon
[18]. Driven by the curiosity, subsequently, we screened an in-house chemical library of
anthraquinones and fortunately identified (E)-2-styrylanthracene-9,10-dione (EK01). EK01
exhibited strong fluorescence under an ultraviolet lamp at the excitation wavelength of 312 nm
(Fig.S1), obviously demonstrating that it’s a new type of fluorescent core with a unique chemical
structure. In the present work, we described the synthesis method, spectrophotometric properties,
cytotoxicity and fluorescence quenching of EK01, especially, its application in specific
mitochondria imaging in living cells was developed.
2. Experimental section
2.1. Synthesis
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The synthetic route of EK01 was outlined in Scheme 1. Using phthalimide and toluene as
starting materials, 2-(4-methylbenzoyl)benzoic acid (1) was synthesized via a Friedel-Crafts
acylation, which was further cyclized to afford 2-methylanthracene-9,10-dione (2). Bromination of
intermediate 2 provided 2-(bromomethyl)anthracene-9,10-dione (3), which then treated with
triphenyl phosphorus to yield 2-((triphenyl-λ5-phosphanylidene)methyl)anthracene-9,10-dione (4).
Finally, EK01 was successfully synthesized via a classical Wittig reaction. According to the high
1
coupling constant of double bond hydrogens (J = 16.3 Hz) in H NMR spectrum, the double bond
configuration of EK01 was identified as E-form (or trans-form). The synthetic procedure of
intermediates 1, 2 and 3 had been described in our previous work [18].
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Scheme 1 Synthetic route of EK01. Reagents and conditions: (i) toluene, anhydrous AlCl3, 50°C, 4 h; (ii) conc.
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2 4 4
H SO , 100℃ , 1 h; (iii) N-bromosuccinimide, benzoyl peroxide, CCl , refluxed for 24 h Pd/C; (iv)
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triphenylphosphine, CH
2
Cl
2
3 2 2
, refluxed for 6 h; (v) benzaldehyde, (Et) N, CH Cl , room temperature for 12 h.
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2.1.1. Preparation of 2-((triphenyl-λ -phosphanylidene)methyl)anthracene-9,10-dione (4)
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A mixture of 3 (0.5 g, 1.7 mmol) and triphenylphosphine (0.65 g, 2.5 mmol) in CH
2
Cl (30 mL)
2
was stirred and refluxed for 6 h. The reactive mixture which had been taken out and spin-dried in
ethyl acetate (40 mL) was refluxed at 60℃ for 30 min, then cooled, filtered and dried to obtain 4
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(0.42 g, 45.16%) as a yellow powder which was used directly in the next step.
2.1.2. Synthesis of (E)-2-styrylanthracene-9,10-dione (EK01)
A mixture of 4 (0.42 g, 0.83 mmol), benzaldehyde (0.075 g, 0.71 mmol) and triethylamine
(0.17 g, 1.7 mmol) in CH
2
Cl (6 mL) was stirred at room temperature for 12 h. The reactive mixture
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was added in H
2
O (30 mL) and CH
2
2
Cl (30 mL) and the organic layer was extracted, vacuum
distillation and silica gel column chromatography (ethyl acetate: petroleum ether = 1:20) to obtain
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EK01 (0.133 g, 50.53%) as a yellow powder. H NMR (600 MHz, DMSO-d
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) δ 8.30 (s, 1H), 8.23
– 8.14 (m, 3H), 8.10 (d, J = 7.9 Hz, 1H), 7.91 (m, 2H), 7.70 (d, J = 7.4 Hz, 2H), 7.54 (d, J = 16.3
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Hz, 1H), 7.47 (d, J = 16.5 Hz, 1H), 7.41 (t, J = 7.3 Hz, 2H), 7.33 (t, J = 7.1 Hz, 1H) [19]. C NMR
(150 MHz, DMSO-d ) δ 182.54, 181.92, 143.14, 136.36, 134.59, 134.41, 133.54, 133.17, 133.08,
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132.77, 131.62, 131.52, 131.44, 128.81, 128.62, 127.48, 127.16, 126.76, 126.70, 124.57. HRMS
+
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(ESI) m/z [M+H] calcd. for C22
H
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O : 311.10720, found: 311.10516.
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2.2. Spectrophotometric Measurements
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The UV-vis measurements (UV-2100 spectrophotometer, UNICO) were performed in different
solvents (CH
Cl
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, CCl
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, DMSO, DMF, THF, CH CN, MeOH, acetone, ethyl acetate, petroleum
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ether) with the concentration of 64.4 μM. The fluorescence measurements (F-7100 Fluorescence
Spectrophotometer, HITACHI) were performed in the above-mentioned solvents with a
concentration of 6.29 μM. The fluorescence of the probe in DMF-H
2
O and DMSO-H O with
2
different proportions was measured. The absorption spectra were recorded at 350-475 nm. The
fluorescent emission spectra were recorded at 475-750 nm with an excitation wavelength at 395 nm.
The viscosities of DMF-H
2
O and DMSO-H O with different proportions were measured at the same
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temperature as the relevant fluorescence spectra.
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Fluorescence quantum yields (Φ) of EK01 in a variety of solvents were measured by using
quinine sulfate dihydrate in 0.1 mol/L sulfuric acid solution (Φ = 0.54) as standard. The
measurements of fluorescence quantum yields have been made using the “comparative method”,
which the standard equation is given below [20,21].
2
푘ꢀ
휂
ꢀ
Φ = Φ (푘ꢁꢂ) ꢃ ꢄ ꢅ퐴 ≤ 0.05ꢆ
2
푥
푆푇
휂
ꢁꢂ
In the formula, ΦST is the fluorescence quantum yield of the standard substance, k
are the slopes of the linear fitting line of the test substance and the standard substance, respectively,
and η and ηST are the refractive indices of the test substance and the standard substance,
X
and kST
X
respectively.
2.3. Cell cytotoxicity
The cell cytotoxicity was detected by MTT assay on MCF-10A cells (human mammary
epithelial cell line) and HT-29 cells (human colorectal adenocarcinoma cell line). The cells were
cultured in a 96-well plate for 24 h and then incubated with different concentrations of EK01 (0 -
50 μM) for 24 h. Then, 10 μL MTT (10 mg/mL) was added and incubated for 4 h at 37°C. The
nutrient solution was removed slowly and 100 μL DMSO was added into the 96-well plate which
finally was shaken for 10 min. The absorbance intensities at 490 nm were measured by the
microplate reader. All the experiments were repeated three times, and the data were expressed as the
percentage of control cells.
2.4. Cell imaging assay
To demonstrate the fluorescence imaging capability of EK01, HT-29 cells were used for cell
imaging under a confocal laser scanning microscope. The cells were seeded in a glass-bottom petri
dish and incubated with 5% CO
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at 37°C for 24 h. The probe was then dissolved in DMSO at 3.22
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μmol/mL and diluted with a DMEM medium to obtain the desired concentration. The final
concentration of DMSO in the medium was less than 1%.
3. Results and discussion
3.1. UV-vis Absorption and Fluorescence Emission
3.1.1. Optical properties of EK01
The absorption spectra and emission spectra of EK01 in the different solvents were shown in
Fig. 1 and the optical properties were tabulated in Table1. The UV-vis absorption of EK01 exhibited
the absorption maximum in the range of 380 – 399 nm with molar extinction coefficients (ε) at
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around 0.5875 × 10 - 0.7609 × 10 mol ·L·cm . The absorption maximum displayed obvious red-
shift in polar solvents, such as DMSO (at 399 nm) and DMF (at 395 nm), compared with in non-
polar solvent petroleum ether (at 380 nm). Besides, EK01 displayed strong fluorescence quantum
yield in a variety of solvents as displayed in Table 1. The emission wavelengths were ranging from
477.4 nm to 529.6 nm. It was worth pointing out that EK01 possesses large stokes shift (91.4 –
149.6 nm) in all the tested solvents. In addition, the photostability assay revealed that the
fluorescence intensity of EK01 only weakly reduced over a long irradiation time of 24 h, suggesting
that EK01 has high photostability (Fig.S5).
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Fig. 1 (a) The absorption spectra and (b) fluorescence emission spectra of EK01 in the different solvents.
Table 1 optical properties of EK01 in the different solvents.
λ
max(exc)ab
395
εc
λ
max(em)a
506
Intensity(λem
2051
521
)
Stokes shift
111
Φ
Solvent
DMF
7391
7203
6031
7234
6281
7313
5875
6015
7609
5906
0.867
0.265
0.602
0.561
0.616
NA d
NA
DMSO
THF
399
517
118
386
477
1083
3376
4050
26
91
Acetone
386
495
109
95
CH
2
3
Cl
2
389
484
CH
OH
389
507
118
Petroleum ether
CCl
380
530
10
150
NA
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383
NA
NA
NA
Acetonitrile
Ethyl acetate
389
501
2279
2383
112
0.767
0.343
383
479
96
aλ values in nm. bλmax(exc) for direct excitation of the fluorescence. ( mol ·L·cm ). experimental data cannot
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be measured.
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3.1.2. Viscosity Dependence Study
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The effect of viscosity on the fluorescence emission of EK01 was investigated. As depicted in
Fig. 2, the fluorescence intensity increased with increasing organic phase volume fraction separately
in DMF/H
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O and DMSO/H O mixed solvents. To study the relationship of viscosity and
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fluorescence intensity, the fluorescence emission spectra at 505.6 nm (DMF/H O) and 516.8 nm
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(DMSO/H O) were measured at room temperature, meanwhile, the corresponding viscosities of
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EK01 in the two mixed solvent systems were tested. The values of both the fluorescence intensity
and the viscosity were converted into natural logarithmic form. As shown in Fig. 2b, the
corresponding plots of ln (fluorescence intensity) – ln (viscosity) exhibited great linear relationship
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(Adj.R = 0.9901 in DMF/H
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O, Adj.R = 0.9901 in DMSO/H O). The results suggested that EK01
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could act as a viscosity indicator.
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Fig. 2 The fluorescence emission spectra of EK01 (a) in DMSO/H O mixtures and (b) in DMF/H O mixtures; Plot
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of natural logarithm of the fluorescence intensity and the natural logarithm of the viscosity (c) in DMSO/H O
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mixtures and (d) in DMF/H O mixtures.
3.1.3. Fluorescence Quenching mechanism
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During spectroscopy experiments, it had found that EK01 has much weaker fluorescence in
protic solvents (such as methanol and water) than in non-protic solvents (such as DMF, DMSO and
CH
2
2
Cl ). Meanwhile, when a small amount of water (less than 2%) existed, EK01 showed a rapid
decrease in fluorescence intensity in both DMF and DMSO solutions (as depicted in Fig. 3). This
phenomenon is difficult to explain by viscosity dependence as mentioned above, but is consistent
with intramolecular charge transfer (ICT) mechanism. It is known that ICT fluorescent probes are
shown to be environmentally sensitive due to the formation of ITC excited states, which means that
in hydrophobic, the probes exhibit strong fluorescence, however, in highly polar solvents, the probes
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have only weak fluorescence [22,23]. EK01, as a typical ITC fluorescent probe with large conjugate
system, we speculated that its fluorescence quenching mechanism in the presence of a small amount
of H O may be caused by the vibrational deactivation via hydrogen bond [22]. As shown in Fig. 4,
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the water molecules could form hydrogen bonds with the carbonyl groups of the anthraquinone core,
so that producing a strong solvation effect. This opens a way for fluorescence inactivation. The
similar fluorescence quenching channel was also reported on some UV stabilizers [22].
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Fig. 3 The change of fluorescence intensity depended on H O proportion. (a) in DMF/H O mixtures and (b) in
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DMSO/H O mixtures.
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Fig. 4 Hydrogen-bond donation to EK01 from protic solvents.
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3.2. Cytotoxicity Assay
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Cytotoxicity was evaluated by the standard MTT assay in the two types of cell lines (MCF-
10A healthy cells line and HT-29 cancer cells line) to quantify cell viability after treated with
different concentrations of the probe. Fig. 5 clearly indicated that the viability of both cell lines
decreased along with the increase of the three relatively low concentrations (1.57, 3.145, 6.29 μM).
Nevertheless, the viabilities were more than 85% in both MCF-10A cells and HT-29 cells after 24
h of incubation with EK01, which means that EK01 only possesses a slight cytotoxic effect on cells.
Interestingly, the cell survival rate increased at high concentrations of 25.16 μM and 50.32 μM. This
phenomenon can be explained by the physical aggregation of EK01 at high concentrations, resulting
in a decrease in dissolved EK01. In summary, EK01 is a safe cell staining agent, which will few
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affect the survival of cells under staining conditions.
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Fig. 5 MTT assay of (a) HT-29 cell and (b) MCF-10Awere treated in the presence of EK01 (1.57, 3.145, 6.29, 12.58,
25.16, 50.32 μM) and incubated for 24 h.
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3.3. Imaging Assay
Firstly, we interestingly observed that EK01 possessed apparent green fluorescence staining
phenomenon for HT-29 cells under an inverted fluorescence microscope (Fig.S6). In order to obtain
higher resolution images and to better delineate the area around the subcellular structure of HT-29
cells, we proceeded to co-localization experiments with EK01 and other commercial organelles
dyes under a confocal microscopy. The preliminary studied surprisingly found that EK01 seemed
to be extremely specific for mitochondria, but had no response to other organelles, such as nucleus,
cell membrane and endoplasm (Fig. S7). To accurately confirm the mitochondria localization of
EK01, co-localization investigations of EK01 and a commercial deep-red mitochondrial tracker
(CMXRos) were performed. As shown in Fig. 6c, f, i, l, the merged images clearly indicated that
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the green staining images of EK01 and the red staining images of CMXRos perfectly coincided,
forming merged orange patterns. These results, clearly demonstrated that EK01 is a specific
mitochondria probe for living cell imaging, and its specificity and imaging ability for mitochondria
are equivalent to CMXRos. Besides, Fig.6b, e, h, k showed that the fluorescence intensity revealed
a concentration-dependent increase in the concentration range of 6.29 ~ 25.16 μM. When the
concentration of EK01 reached 6.29 μM, a good green visual rendering effect can be achieved,
meeting the needs of dyeing (Fig. 6b). Meanwhile, the resolution of the image will not be
significantly improved when the concentration is higher than 25.16 μM. In short, co-localization
assay proved that EK01 is a green specific mitochondria probe for living cells, and its staining
concentration is recommended between 6 ~ 12 μM.
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Fig. 6 Confocal fluorescence images of HT-29 cells stained with CMXRos (3 μM) and EK01 (6.29 μM, 12.58 μM,
25.16 μM, 50.32 μM). Column 1: CMXRos (a, d, g, j; red channel: λex = 579 nm, λem =599 nm). Column 2: EK01
(b, e, h, k; green channel: λex = 395 nm, λem =500 nm). Column 3: Merged images (c, f, i, l). Scar bar: 10 μm.
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In the following study, we investigated the influence of incubation time for the stain. Two time
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points of 30 min and 1 h were set, and the result was illustrated in Fig. 7. As shown in Fig. 7c, d, g,
h, the staining results of 30 min and 1 h were all excellent at highly concentrations of 25.16 μM and
50.32 μM, implying that these concentrations may already be higher than the optimum concentration
required for dyeing. Furthermore, as shown in Fig. 7a, b, e, f, the fluorescence intensities emerged
an obviously time-dependent improvement at relatively low concentrations of 6.29 μM and 12.58
μM. After 30 min of incubation, the fluorescence intensities at 6.29 μM and 12.58 μM were both
too weak to image, correspondingly, the imaging results were excellent after 1 h staining under same
concentrations. The time-dependent stain manner suggested that EK01 needed to take a certain time
to stain or specific accumulate in mitochondria.
In summary, the staining result of 1 h was better than that of 30 min at low concentrations. And
after 1h incubation time, the increase in concentration had little effect on the outcome of staining
for cells. Finally, based on the cytotoxicity and imaging assays, an EK01 based special mitochondria
imaging method was suggested which is incubation of EK01 with living cells for 1 h at a final
concentration of 6 ~ 12 μM.
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Fig. 7 Confocal fluorescence images in HT-29 cells stained with EK01 at the four concentrations for 30 min and 1h
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incubation time.
4. Conclusions
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In summary, a novel type of fluorescent probe core, (E)-2-styrylanthracene-9,10-dione (EK01),
was discovered. A series of advantages including strong fluorescence quantum yield, good
photostability, large stokes shift, high molar extinction coefficients and well biocompatibility,
implies its potential to be widely applied in numerous fields. The facile and highly efficient synthetic
method based on a Wittig reaction ensures the reliable access of EK01 and its derivatization. In
addition, using EK01 as a green staining agent for living cells, a new specific mitochondria imaging
method was established with high signal-to-noise ratio and low-cytotoxicity. Other applications of
EK01 and its derivatives are under exploration.
Declaration of competing interests
The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (Grant No.
81803355) and Young Talents Program of China Medical University.
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Highlights
1
. A novel fluorescent probe core (EK01) was developed for specific mitochondria
imaging in living cells.
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. The probe shows massive stokes shift and high fluorescence quantum yield.
. The probe shows excellent cell membrane permeability and low cytotoxicity.
. The probe could real-time specifically monitor mitochondria for a long time.

Conflict of Interest
The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.