A novel 4-hydroxypyrene-based “off–on” fluorescent probe with large Stokes shift for detecting cysteine and its application in living cells

Article abstract of DOI:10.1016/j.tetlet.2019.151467

This paper reports a novel fluorescent probe 4-acrylatepyrene (PYAC) based on 4-hydroxypyrene, which can effectively detect cysteine (Cys). The probe PYAC uses acrylate moiety as a recognition site and has relatively high selectivity and sensitivity for Cys with the detection limit of 0.062 μM. After treatment with Cys, PYAC exhibits “off–on” switching property and large Stokes shift (171 nm). Due to nucleophilic addition and specific intramolecular cyclization, it exhibits higher selectivity for Cys than other amino acids and common ions, including homocysteine (Hcy) and glutathione (GSH) with similar structures to Cys. The recognition mechanism has been characterized by high-performance liquid chromatography (HPLC) and nuclear magnetic resonance spectroscopy (¹H NMR). Anti-interference test and pH influence test display it is suitable for detecting Cys in living cells. Finally, the probe PYAC has been successfully applied to cell imaging with negligible cytotoxicity.

Full text of DOI:10.1016/j.tetlet.2019.151467

Tetrahedron Letters xxx (xxxx) xxx
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Tetrahedron Letters
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A novel 4-hydroxypyrene-based ‘‘off–on” fluorescent probe with large
Stokes shift for detecting cysteine and its application in living cells
⇑
⇑
Wenhao Sun, Xinxue Tang, Jingyang Li, Menglu He, Ran Zhang, Xiang’en Han , Yun Zhao, Zhonghai Ni
School of Chemical Engineering and Technology, China University of Mining and Technology, Xuzhou 221116, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
This paper reports a novel fluorescent probe 4-acrylatepyrene (PYAC) based on 4-hydroxypyrene, which
can effectively detect cysteine (Cys). The probe PYAC uses acrylate moiety as a recognition site and has
Received 26 September 2019
Revised 25 November 2019
Accepted 29 November 2019
Available online xxxx
relatively high selectivity and sensitivity for Cys with the detection limit of 0.062
lM. After treatment
with Cys, PYAC exhibits ‘‘off–on” switching property and large Stokes shift (171 nm). Due to nucleophilic
addition and specific intramolecular cyclization, it exhibits higher selectivity for Cys than other amino
acids and common ions, including homocysteine (Hcy) and glutathione (GSH) with similar structures
to Cys. The recognition mechanism has been characterized by high-performance liquid chromatography
(HPLC) and nuclear magnetic resonance spectroscopy (1H NMR). Anti-interference test and pH influence
test display it is suitable for detecting Cys in living cells. Finally, the probe PYAC has been successfully
applied to cell imaging with negligible cytotoxicity.
Keywords:
4-Acrylatepyrene
Cys detection
Large Stokes shift
Cell imaging
Ó 2019 Elsevier Ltd. All rights reserved.
Introduction
Fluorescent probe does not require expensive equipment com-
pared to the conventional methods described above, so it is a con-
Biothiols play vital roles in human physiological activities,
among which the representative biothiols are cysteine (Cys),
homocysteine (Hcy) and glutathione (GSH). These three biothiols
share similar chemical structures and they all have important
but different roles in physiology [1–6]. For example, abnormal
Cys levels will lead to slow growth, skin lesions, liver injury and
edema, etc. [7]. High serum Hcy levels are associated with hip frac-
ture and cardiovascular disease [8], GSH is an antioxidant that pro-
tects cells from reactive oxygen species (ROS) [9]. Therefore, it is
very important and necessary to develop a method capable of
specifically detecting and distinguishing these three representative
biothiols. In recent years, a variety of assays for biothiols detection
have been developed, which includes high-performance liquid
chromatography (HPLC) [10], titration analysis [11], mass spec-
trometry [12] and potentiometry [13,14]. Although these methods
have various unique advantages, they also have some obvious
shortcomings such as expensive instruments, complicated opera-
tions during pretreatment, and most importantly, these methods
cannot be applied to detect biothiols in living cells. Hence, the flu-
orescent probe capable of solving the above disadvantages has
attracted attention of researchers.
venient method to choose for substance detection and test. More
importantly, most fluorescent probes are capable of detecting tar-
get analytes in living cells with obvious fluorescent phenomenon.
At the same time, a large amount of fluorescent probes show high
selectivity and sensitivity, low detection limit, and strong anti-
interference ability in various detection experiments. Recently,
there have been many reports on the detection of biothiols fluores-
cent probes [15–20]. To date, many fluorescent probes for biothiols
detection have been developed based on different mechanisms
[2,21,22], but most of them have difficulty in distinguishing Cys
from Hcy and GSH because they have similar structure and reactiv-
ity. In the detection of biothiol, the kinetic difference between
acrylate group and Cys, Hcy, GSH conjugate addition/cyclization
reaction can be applied to the targeted detection of Cys. This strat-
egy has been successfully employed to the design and exploitation
of new fluorescent probes for Cys based on some well-known chro-
mophores such as anthraquinone [23], benzothiazole [24], cou-
marin [25], rhodamine [26] and others [27–29], which shows
significant fluorescence changes after treatment with Cys instead
of other biothiols. As one of the most important chromophore of
fluorescent probes, pyrene-based probes have been paid everlast-
ing attentions because they usually exhibit excellent photochemi-
cal and photophysical properties [30]. Up to date, almost all the
reports on pyrene-based probes are 1-substituted derivatives of
pyrene due to that the 1-position is the commonly reactive active
⇑
Corresponding authors.
E-mail addresses: xiangenh@163.com (X. Han), nizhonghai@cumt.edu.cn (Z. Ni).
https://doi.org/10.1016/j.tetlet.2019.151467
0040-4039/Ó 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: W. Sun, X. Tang, J. Li et al., A novel 4-hydroxypyrene-based ‘‘off–on” fluorescent probe with large Stokes shift for detecting cys-
teine and its application in living cells, Tetrahedron Letters, https://doi.org/10.1016/j.tetlet.2019.151467

2
W. Sun et al. / Tetrahedron Letters xxx (xxxx) xxx
site of pyrene, which usually emits blue fluorescence. However, the
inactive position-substituted pyrene-based probes are very limited
and should be worthy of much developing, which may exhibit sig-
nificantly different properties.
NMR, 13C NMR and MALDI TOF mass spectrometry techniques
(Figs. S4–S21).
Fluorescence spectra titration
Recently, we designed and synthesized a new pyrene-based 4-
hydroxypyrene with hydroxyl at the inactive 4-position of pyrene
by multistep reactions and found that it emitted strongly yellow
fluorescence with a large Stokes shift, suggesting it can be
employed as a new probe precursor. In this work, a novel 4-
hydroxypyrene-based ‘‘off–on” fluorescent probe PYAC for Cys
with a large Stokes shift has been designed and developed by a
simple reaction between 4-hydroxypyrene and acryloyl chloride.
The probe PYAC has high sensitivity and good selectivity for Cys.
The probe PYAC has been successfully applied to cell imaging
experiments and can detect Cys in living cells with negligible cyto-
toxicity. Therefore, the probe PYAC has great potential for
application.
The dependence of PYAC on the concentration of Cys in the flu-
orescence spectra was investigated. When the probe was titrated
using Cys, a new emission band at 512 nm increased significantly.
Fluorescence reaches maximum when Cys concentration was
increased to 20
ship in the Cys concentration of 2–20
l
M (Fig. 1a). At the same time, the linear relation-
l
M is excellent R2 = 0.99661
(Fig. 1b), this means that the probe PYAC can quantitatively detect
Cys. According to the IUPAC definition, the detection limit of the
probe PYAC is calculated by the following formula: CDL = 3Sa/Kb,
where Sa is the standard deviation of the blank solution and Kb is
the slope of the calibration curve. According to the formula, the
detection limit is calculated to be 6.2 Â 10À8 M. This is much lower
than the normal Cys concentration level (30–200 mM) [32], indi-
cating that the PYAC probe is highly sensitive for detecting Cys
in biological systems.
Results and discussion
Design and synthesis of probe PYAC
Pyrene is a traditional and classical chromophore. Generally, the
reported studies on pyrene-based fluorescent probes focused on its
1-substituted derivatives since 1-position is easily be replaced and
modified. However, the researches on other positions are always
ignored due to the synthetic difficulty. Recently, a new 4-substi-
tuted pyrene-based derivative 4-hydroxypyrene (e) has been syn-
thesized by the following indirectly multistep synthetic strategy
(Scheme 1). Firstly, pyrene is hydrogenated to 1,2,3,6,7,8-hexahy-
dropyrene which can be easily transformed to its 4-bromo-sub-
stitued pyrene-based compound b [31]. Secondly, intermediate b
is transformed to compound c by the typical methoxylation cat-
alyzed by CuI with moderate conversion (51%) but high selectivity
(95%), therefore the material b can be separated to the repeated
reaction and the real yield of this step is very high (ca. 90%).
Thirdly, compound d can be easily prepared by dehydrogenation
oxidation of compound c tin the presence of DDQ with high yield
of 90%. Finally, the expected 4-hydroxylpyrene (e) can be synthe-
sized by the classical demethylation of methoxyl group with nearly
100% yield. The finally probe PYAC is designed and easily synthe-
sized in high yield by the reaction of compound e with acryloyl
chloride in dry THF considering 4-hydroxypyrene unique. All the
intermediates and the final product are fully characterized by 1H
Selectivity and competition studies
The selectivity and anti-interference of fluorescent probes are
important indicators for evaluating their performance. In order to
detect the selectivity of PYAC, the natural amino acids Hcy, GSH,
Ser, His, Ala, Asn, Asp, Gln, Arg, Glu, Met, Phe, Trp, Tyr, Lys
(200
(20
l
l
M), metal ion Na+, Mg2+, K+, Ca2+ (200
l
M) and Cys
M) was separately added to a ACN–PBS buffer solution
M PYAC. As shown in Fig. 2a, sig-
(pH = 7.4, 3:7 v/v) containing 10
l
nificant fluorescence enhancement was observed only at 512 nm
after the addition of Cys. The effect of other amino acids and metal
ions on fluorescence intensity is negligible, including Hcy and GSH.
This indicates that PYAC has good selectivity for Cys. Then, a com-
petitive experiment was conducted to investigate the response of
PYAC to Cys in the presence of other analytes. Add 20
lM Cys to
the above buffer solution to measure the fluorescence, and the
results are shown in Fig. 2b. The black bars indicate the fluores-
cence of PYAC in the presence of various analytes without the
addition of Cys, and the red bars indicate the fluorescence of PYAC
in the presence of various analytes after the addition of Cys. The
results show that the probe PYAC has high selectively to Cys even
in the presence of other amino acids, and there is no adverse
Scheme 1. Synthetic routes of compound PYAC.
Please cite this article as: W. Sun, X. Tang, J. Li et al., A novel 4-hydroxypyrene-based ‘‘off–on” fluorescent probe with large Stokes shift for detecting cys-
teine and its application in living cells, Tetrahedron Letters, https://doi.org/10.1016/j.tetlet.2019.151467

W. Sun et al. / Tetrahedron Letters xxx (xxxx) xxx
3
Fig. 1. (a) The fluorescence spectrums of probe (10
lM) in the presence of various concentrations of Cys (0–4 equiv) in ACN–PBS buffer solution (pH = 7.4, 3:7 v/v)
(kex = 365 nm, slit: 5 nm/5 nm). Inset: Comparison of fluorescence in the absence of (Cuvette1) and presence of (Cuvette2) Cys (20
l
M) in a solution containing 10 M probe
l
PYAC under 365 nm UV illumination. (b) Plot of the fluorescence intensity of probe at 512 nm as a function of the concentration of Cys in ACN–PBS buffer solution (pH = 7.4,
3:7 v/v) (kex = 365 nm).
Fig. 2. (a) Fluorescence intensity of probe PYAC (10 lM) in ACN–PBS (pH = 7.4, 3:7 v/v) buffer solution in the presence of various analytes. (b) Fluorescence intensity of PYAC
in ACN–PBS buffer solution (pH = 7.4, 3:7 v/v) at 512 nm in the presence of Cys (20 lΜ), 200 lM of other amino acids and cationic (Hcy, GSH, Ser, His, Ala, Asn, Asp, Gln, Arg,
Glu, Met, Phe, Trp, Tyr, Lys, Na+, Mg2+, K+, Ca2+) (black bar). Fluorescence intensity of PYAC in the presence of the above analyte and additional 20
kex = 365 nm, Slit: 5.0 nm/5.0 nm.
lM Cys (red bar).
interference from other species. The above results indicate the
probe PYAC has good selectivity and sensitivity to Cys.
pH effect
Under alkaline aqueous conditions, two Cys molecules are
prone to chemical reactions and produce cysteine, and the acrylate
group is easily decomposed under such conditions. So Cys can only
exist under neutral or slightly acidic conditions, which requires the
probe to remain stable under the same conditions. The effect of pH
on the fluorescence intensity of PYAC (10 lM) in the presence and
absence of Cys has been investigated. As shown in Fig. 3, in the
absence of Cys, there was almost no change in fluorescence inten-
sity from pH 1 to 9, indicating that the probe PYAC is very stable
over a relatively wide pH range. After the addition of 20
lM Cys,
the fluorescence intensity of the probe did not change under strong
acidic conditions but it was significantly enhanced and stable
under neutral conditions. Since the physiological environment of
the human body is weakly alkaline and the probe PYAC works well
under this condition, pH = 7.4 is selected as the experimental
parameter.
Fig. 3. Fluorescence intensity of PYAC with (red dot) or without Cys (black dot) in
solutions with different pH values. Test condition: Cys (20 lM), ACN–PBS (3:7, v/v),
kex = 365 nm, Slit: 5.0 nm/5.0 nm.
Please cite this article as: W. Sun, X. Tang, J. Li et al., A novel 4-hydroxypyrene-based ‘‘off–on” fluorescent probe with large Stokes shift for detecting cys-
teine and its application in living cells, Tetrahedron Letters, https://doi.org/10.1016/j.tetlet.2019.151467

4
W. Sun et al. / Tetrahedron Letters xxx (xxxx) xxx
Fig. 5. Toxicity test for HeLa cells at different concentrations of probe PYAC.
Fig. 4. HPLC chromatogram of probe PYAC, compound e, PYAC + Cys (sample A:
PYAC only, sample B: PYAC + 2 equiv Cys, sample C: PYAC + 5 equiv Cys, sample D:
compound e only).
Cell imaging experiment
Recognition mechanism
The above test results indicate that the probe was capable of
detecting Cys in cells, so cell imaging experiments are ready to per-
form. Firstly, the toxicity of the probe to cells was tested using the
To investigate the detection mechanism, probe PYAC, com-
pound e, and the reaction product of probe PYAC with Cys were
analyzed by HPLC (Fig. 4). A new peak with a retention time of
4.7 min appeared when 2 equiv of Cys was added to the probe
(Fig. 4B). When 5 equiv of Cys were added, the peak at 9.9 min
completely disappeared, leaving only a peak at 4.7 min (Fig. 4C).
This indicates that the probe PYAC is converted to compound e
upon detection of Cys. At the same time, PYAC and Cys were added
to acetonitrile–H2O and stirred at room temperature for 2 h, then
the product is named as product 1. The product 1 was isolated
by column chromatography and then characterized by 1H NMR
(Fig. S22). The result showed that the product 1 was the compound
e. Then, by comparing the 1H NMR spectrum of compound e in
pure DMSO and DMSO–water mixed solution, it is found that the
protons on the hydroxyl group will dissociate in the presence of
water (Fig. S3). Based on the above experiments and related liter-
atures in recent years [33–36], the detection mechanism of probe
PYAC was proposed as shown in Scheme 2.
MTT method. Probes at concentrations of 0
10 M, 20 M, and 30 M were separately added to HeLa cells
for incubation, and survival was analyzed one day later. Even with
probe concentrations as high as 30 M, cell viability was still very
lM, 1 lm, 5 lM,
l
l
l
l
high, indicating that the probe is less toxic to cells (Fig. 5). Then,
cell imaging experiment was performed, and fluorescence of the
probe itself was extremely weak, but after the probe was added
to HeLa cells, the cells showed fluorescence. In the control group,
the cells was treated with NEM at 100
lM and 300 lM, respec-
tively, and then added the probe PYAC. After 1 h of incubation,
the cells were photographed. N-ethylmaleimide (NEM) is a widely
used thiol-blocking agent, cells treated with NEM at a concentra-
tion of 100
lM were still found to have weak fluorescence. Cells
treated with NEM at a concentration of 300
lM showed no fluores-
cence (Fig. 6). This indicates that the probe is capable of detecting
Cys in living cells.
Scheme 2. Reaction mechanism of probe PYAC for detecting Cys.
Please cite this article as: W. Sun, X. Tang, J. Li et al., A novel 4-hydroxypyrene-based ‘‘off–on” fluorescent probe with large Stokes shift for detecting cys-
teine and its application in living cells, Tetrahedron Letters, https://doi.org/10.1016/j.tetlet.2019.151467

W. Sun et al. / Tetrahedron Letters xxx (xxxx) xxx
5
Fig. 6. Confocal microscopy images of Hela cells under different treatments. (a) Hela cells were incubated with probe PYAC (10
lM) for 1 h. (b) Hela cells were incubated with
NEM (100
lM) for 1 h and then incubated with probe PYAC (10 lM) for 1 h. (c) Hela cells were incubated with NEM (300 lM) for 1 h and then incubated with probe PYAC
(10 M) for 1 h. kex = 488 nm, images were collected from green (500–550 nm) channel.
l
Conclusion
In this paper, a new 4-hydroxypyrene-based fluorescent probe
PYAC has been designed and synthesized with the recognizing site
at the inactive 4-position of pyrene, which exhibits excellent ‘‘off–
on” switching property and large Stokes shift (171 nm) after treat-
ment with the representative biothiol Cys. The probe PYAC shows
high selectivity and sensitivity for Cys, and the detection limit of
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.tetlet.2019.151467.
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Please cite this article as: W. Sun, X. Tang, J. Li et al., A novel 4-hydroxypyrene-based ‘‘off–on” fluorescent probe with large Stokes shift for detecting cys-
teine and its application in living cells, Tetrahedron Letters, https://doi.org/10.1016/j.tetlet.2019.151467