Endoplasmic reticulum-targetable fluorescent probe for visualizing HClO in EC1 cells

Article abstract of DOI:10.1016/j.tetlet.2020.152301

The endoplasmic reticulum is the largest organelle in the cell. Studies have shown that the concentration of HClO is related to many diseases. A new type of probe R1 has positioned to the endoplasmic reticulum. The probe shows good positioning effect, excellent selectivity and high sensitivity, excellent anti-interference, and can quickly reach the response platform. The obvious color change can be used for naked eye observation. Due to its low cytotoxicity, probe R1 was successfully used for EC1 cells imaging under a laser scanning confocal microscope.

Full text of DOI:10.1016/j.tetlet.2020.152301

Journal Pre-proofs
Endoplasmic reticulum-targetable fluorescent probe for visualizing HClO in
EC1 cells
Jianfei Liu, Zhiyao Zhai, Huawei Niu, Yongru Zhang, Xiangzhi Song, Panke
Zhang, Yong Ye
PII:
S0040-4039(20)30768-1
DOI:
Reference:
https://doi.org/10.1016/j.tetlet.2020.152301
TETL 152301
To appear in:
Tetrahedron Letters
Received Date:
Revised Date:
Accepted Date:
6 June 2020
24 July 2020
26 July 2020
Please cite this article as: Liu, J., Zhai, Z., Niu, H., Zhang, Y., Song, X., Zhang, P., Ye, Y., Endoplasmic
reticulum-targetable fluorescent probe for visualizing HClO in EC1 cells, Tetrahedron Letters (2020), doi:
https://doi.org/10.1016/j.tetlet.2020.152301
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1
Tetrahedron Letters
journal homepage: www.elsevier.com
Endoplasmic reticulum-targetable fluorescent probe for visualizing HClO in EC1
cells
Jianfei Liua, Zhiyao Zhaia, Huawei Niua, Yongru Zhanga, Xiangzhi Songb, Panke Zhanga*, Yong Yea
a College of Chemistry, Zhengzhou University, 100 Kexue Avenue, Zhengzhou, Henan 450001, PR China. E-mail: pkzhang@zzu.edu.cn
b College of Chemistry & Chemical Engineering, Central South University, Changsha, Hunan, 410083, China
ARTICLE INFO
ABSTRACT
The endoplasmic reticulum is the largest organelle in the cell. Studies have shown that the
concentration of HClO is related to many diseases. A new type of probe R1 has positioned to the
endoplasmic reticulum. The probe shows good positioning effect, excellent selectivity and high
sensitivity, excellent anti-interference, and can quickly reach the response platform. The obvious
color change can be used for naked eye observation. Due to its low cytotoxicity, probe R1 was
successfully used for EC1 cells imaging under a laser scanning confocal microscope.
Article history:
Received
Received in revised form
Accepted
Available online
Keywords:
fluorescent probe
EC1 cells
2009 Elsevier Ltd. All rights reserved.
Endoplasmic reticulum
HClO
Although some fluorescent probes for HClO were reported[16-
18], they suffered from co-organic solvent or slow response speed.
To address this, here, we developed a simple and effective probe R1,
which consisted of a fluorophore, p-toluenesulfonamide and a HClO-
1. Introduction
Reactive oxygen species and reactive nitrogen species play
important roles in biological pathways and stress processes [1-3].
Hypochlorous acid (HClO) is one of the most important reactive
oxygen species produced by peroxides and chloride ions catalyzed
by bone marrow oxidase in active organisms [4,5]. It is an extremely
important germicidal oxidant in the immune system and also widely
used as a bleach and disinfectant for drinking water in daily life [6,7].
In the physiological environment, the normal concentration of HClO
is beneficial to the human body and is necessary to maintain the
normal function of living cells [8]. However, excessive production of
HClO may cause tissue damage and many diseases such as arthritis,
kidney disease, lung injury, cardiovascular disease, asthma and even
cancer [9-13]. Most of the developed HClO detection methods such
as iodometric method, colorimetric and polarographic methods have
limitations on organisms [14,15]. Therefore, new fluorescent probes
need to be developed for the selective and sensitive detection of
HClO [16-20]. The fluorescent probes can be used for biological
analysis and labeling [21-24]. And they have become the basis for
sensing biological molecules in living systems due to their low cost,
simplicity, excellent selectivity, high sensitivity, and real-time
analysis [25,26].
responsible
site
dimethylthiocarbamate
(DMTC).
p-
Toluenesulfonamide is typical ER targetable group[32-34]. The
targeting mechanism maybe due to its good binding to lots of protein
in ER. In the presence of HClO, the DMTC part of R1 was oxidized,
and intramolecular charge transfer (ICT) was turned on, then
fluorescence emission appeared. As expected, this probe can target
ER and sensitively measure HClO in PBS buffer. After adding HClO,
it will show a very rapid response in 30 S. When reaching the
response platform, the fluorescence intensity increased by 12 times.
And co-localization experiments also showed that R1 has a good
positioning ability for endoplasmic reticulum and good
biocompatibility.
2. EXPERIMENTAL SECTION
Apparatus, chemical and reagents. The absorption spectra
were precisely gauged employing the UV-2102 dual beam UV/Vis
spectrophotometer. We used a F-4500 FL spectrophotometer with a
10 mm quartz cuvette for fluorescent spectroscopy record. Its
excitation and emission wavelength band pass was set to 5.0 nm. The
pH was surveyed using a DAPU PHS-3C instrument. Employing
TMS as an inner standard, nuclear magnetic resonance spectra were
recorded on a Bruker DTX-400 spectrometer. Mass spectrometry
was performed with high-performance liquid chromatography Q-T
HR-MS. Reagents and materials gained from commercial suppliers
were used directly. The water used in the experiment was fresh
double distilled water.
The endoplasmic reticulum (ER) is the largest organelle in cell
and plays an important role in the synthesis of biological proteins.
ER homeostasis can be disturbed by misfolded and unfolded protein
accumulation in the ER cavity. This condition is called ER stress.
When misregulated, the accumulation of HClO may cause oxidative
damage to cellular proteins. It can even trigger apoptosis by cleaving
caspase-12 in the ER [27-30], leading to apoptotic neurodegenerative
and cardiovascular diseases, metabolic diseases and cancers [31].
Therefore, it is of great significance to develop an effective HClO
analysis method in ER.
The synthesis of probe R1. Compound 1 was synthesized by
1
reported methods [35]. H NMR (400 MHz, DMSO-d6): δ 12.35 (s,

2
1H), 8.63 (dd, J = 8.3, 1.2 Hz, 1H), 8.50 (dd, J = 7.5, 1.2 Hz, 1H),
Cell culture. EC1 cells were cultured in 1640 Petri dish and
four seasons serum. 1% streptomycin, 10% fetal bovine serum (FBS),
and 1% penicillin were added to the Petri dish. The cells were then
placed in a cell incubator containing 5% CO2 and cultured at 37 °C.
One day before imaging, the cells were digested into confocal dishes
for subsequent experiments.
8.42 (d, J = 8.5 Hz, 1H), 7.81 (dd, J = 8.3, 7.3 Hz, 1H), 7.22 (d, J =
8.1 Hz, 1H); 13C NMR (100 MHz, DMSO-d6): 161.94, 161.88,
160.81, 136.18, 133.55, 132.37, 130.66, 126.53, 123.03, 119.13,
110.96, 110.96. (Figures S1−S2).
Synthesis of Compound 2: Compound 1 (1.070 g, 5 mmol) and N-
(2-amino-ethyl)-4-methyl-benzenesulfonamide (1.605 g, 7.5 mmol)
were dissolved in 60 mL of ethanol under nitrogen, and heated at
80 °C overnight. After the reaction was completed, the solvent was
removed under reduced pressure. The residue was purified by
column chromatography (dichloromethane : methanol = 30 : 1).
Compound 2 was obtained as a tan solid in 51% yield. 1H NMR
(400 MHz, DMSO-d6): δ 11.83 (s, 1H), 8.55 (d, J = 8.4 Hz, 1H),
8.54 (d, J = 7.2 Hz, 1H), 8.45 (d, J = 8.1 Hz, 1H), 7.79 (dt, J = 12.5,
7.0 Hz, 2H), 7.74 (d, J = 7.2 Hz, 2H), 7.70 (d, J = 7.4 Hz, 2H), 7.60
(d, J = 8.3 Hz, 1H), 4.09 (t, J = 6.4 Hz, 2H), 3.06 (q, J = 6.1 Hz, 2H),
2.26 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ 164.17, 163.45,
160.67, 142.86, 138.06, 133.86, 131.42, 129.89, 129.67, 129.23,
126.80, 125.88, 122.74, 122.20, 113.02, 110.27, 40.63, 39.46, 21.30.
(Figures S3−S4).
Cytotoxicity test. EC1 cells were prepared into a density of 106
cell / mL with Petri dish , and then the cells were seeded into 96-well
plates. 100 μL of medium was added to each well and placed in a
cell culture incubator (5% CO2 / 95% Air). Medium culture, after
attachment is completed, adding a series of probe solutions (0 μM,
2.5 μM, 4 μM, 6 μM, 8 μM, 10 μM) to 96-well plates, and placing
them in the cell culture incubator (5% CO2 / 95% Air) continuely
incubating for 24 h, aspirating the culture medium from the 96-well
plate, adding 100 μL of culture medium, and then adding 10 μL of
CCK-8 solution to each well, and continuely in the cell incubator
(5 % CO2 / 95% Air) for 1-4 h, and then taking out culture medium
under the absorbance at 450 nm on a microplate reader.
Cells imaging. EC1 cells were plated on Petri dish for 24 hours.
Cells were washed using PBS before the experiments. In order to
know the subcellular position of R1, the cells were stained with
endoplasmic reticulum sensor, mitochondrial staining sensor, and
lysosomal staining sensor for 30 minutes. Cells imaging was
obtained by Leica TCS SP8 confocal microscope.
Synthesis of R1: Compound
2 (410 mg, 1 mmol) and
dimethylthiocarbamoyl chloride (618 mg, 5.0 mmol) were dissolved
in 10 mL of anhydrous dichloromethane, followed by the addition of
400 uL of N, N-diisopropyl ethylamine. The mixture was stirred at
room temperature for 4 h, and the dichloromethane was removed
under reduced pressure. Purification by column chromatography
(dichloromethane) gave a pale-yellow solid R1, Yield 43%. 1H NMR
(400 MHz, DMSO-d6): 8.47 (dd, 2H, J=8.0 Hz, 4.8 Hz), 8.28 (d, 1H,
J=8.4 Hz), 7.88 (t, 1H, J=8.0 Hz), 7.78 (t, 1H, J=6.4 Hz), 7.59 (dd,
3H, J=8.0 Hz, 5.6 Hz), 7.25 (d, 2H, J=8.0 Hz), 4.12 (t, 2H, J=6.4 Hz),
3.51 (s, 3H), 3.44 (s, 3H), 3.09 (d, 2H, J=6.4 Hz), 2.26 (s, 3H). 13C
NMR (100 MHz, DMSO-d6): 190.6, 168.6, 168.1, 159.6, 147.7,
142.8, 136.2, 136.2, 134.7, 133.8, 133.6, 132.7, 130.8, 127.7, 126.6,
3. CONSEQUENCE AND DISCUSSION
The synthetic route of R1 was shown in Scheme 1. The title
compound was prepared through three steps and was well
characterized by 1H NMR, 13C NMR and ESI-HRMS (Fig. S5–S7).
First, the R1 kinetics was measured (Fig. 1a). After adding 10
equivalents of HClO, the response was very quick within 30 seconds.
We analyzed the selectivity of fluorescent probe R1 to HClO, other
amino acids and inorganic salts (Fig. 1b). It can be seen that the
strong fluorescence appeared at 550 nm, after adding 10 equivalents
of HClO. The fluorescence intensity was increased by 11.6 times.
After adding other amino acids and inorganic salts, the fluorescence
intensity hardly changed. Therefore, the fluorescent probe R1 has
high selectivity. In addition, a competitive experiment (Fig.1c) was
carried out. After adding 10 eq. of HClO in the presence of other
amino acids and inorganic salts, it was gratifyed that the fluorescence
intensity of R1 was significantly enhanced. Therefore, the
fluorescent probe R1 has excellent anti-interference with other
amino acids and inorganic salts. A comparison of the reported probes
with the probe R1 was list in Table. S1. It can be found that the
performance of the synthesized probe was good.
+
125.1, 44.1, 48.3, 26.1. HR-MS: m/z calcd for C24H24N3O5S2 [M]+:
498.1152, found: 498.1150.
O
S
NH
O
O
O
O
O
O
O
1) NaN3, DMSO
NH2
2) PPh3, HCl, NaOH
EtOH
3) NaNO2, H2SO4
4) H2O
OH
Br
1
S
O
H
O
N
O
S
N
O
N
N
S
H
Cl
S
N
O
N
HO
O
O
O
O
R1
2
In order to study the sensitivity of fluorescent probe R1 to
HClO in PBS buffer solution, we measured the fluorescence titration
experiment (Fig.2a). As can be seen from the figure, when the
content of HClO was gradually increased, the fluorescence intensity
at 550 nm was gradually enhanced. As shown in Fig.2b, the
fluorescence intensity of the probe had a good linear relationship
with HClO (0.1-0.35 eq.), and the correlation coefficient was as high
as 0.9971 and with a detection limit of 12.59 nM (S/N=3), which was
better than other methods for detecting HClO in endoplasmic
reticulum. The quantum yield (Ф) of Y1 and the probe R1 are 0.12
and 0.014 respectively using coumarin 6 as a reference.
Scheme 1. Synthesis of probe R1
Optical studies. A stock solution of R1 (1 mM) was dissolved
with DMSO. Stock solutions (1-10 mM) of other analytes,
containing amino acids (for instance, Hcy, GSH, Cys), and
corresponding salts (such as KI, KBr, NaCl, NaF, NaHSO3,Na2SO4,
Na2S2O5, Na2S2O8, Na2CO3, NaHCO3, K3PO4, NaNO3, NaNO2, Ac-,
NaHS, ONOO-, K+, Na+, H2O2, O2-, NO., HClO) were prepared in
ultra-pure H2O. For optical studies, 3.0 mL of R1 (10 μM) in PBS
(pH=7.4, 10 mM) buffer solution was decanted in a quartz cuvette.
The UV or fluorescent spectra was measured after adjunction of
analytes. The excitation voltage is set to 700V. For the reaction
kinetics of R1 with HClO, 10 mM of HClO was incubated with 1
mM of R1 in PBS at 37 °C. Next, fluorescent intensity was recorded
at a different time point.
The UV-selective spectrum of probe R1 (Fig.S8) showed that
absorption of R1 at 450 nm is only 0.03 after adding other amino
acids and inorganic salts. The absorption of R1 at 450 nm was
enhanced after the addition of HClO. It indicated that probe R1 can
specifically recognize HClO. We also tested the UV titration
spectrum of probe R1 (Fig. S9). As the concentration of HClO
increased, the UV absorbance also increased.

3
We next tested the pH suitability of probe R1. As shown in Fig.
3, the probe was insensitive to pH in the range of pH 3-6. In the
range of 7-13, when 10 eq. of HClO was added, the fluorescence
intensity at 550 nm was obviously enhanced, indicating that R1 was
widely tolerated to pH. Since the normal physiological environment
of the human was neutral, the probe R1 can be well applied to the
endoplasmic reticulum of the cell, which was also useful for further
cell imaging.
(PBS 10 mM, pH = 7.4 λex= 450 nm, slit: 5 nm).
Fig. 2. (a) Fluorescent emission spectra of R1 (10.0 μM) in the presence of ClO- (0–10
equiv.). (b) Linear plot of the fluorescence intensity at 550 nm against NaClO
concentrations in the PBS buffer (10 mM, pH = 7.4 λex= 450 nm, slit: 5 nm).
Fig. 3. The fluorescent intensity (at 550 nm) of R1 (10 μM) in the PBS buffer under
different pH (3–13) in the absence and presence of NaOCl (10 equiv.) (PBS 10 mM, pH
= 7.4 λex=450 nm, slit: 5 nm).
Fig. 1. (a) Fluorescent kinetics of R1 (10 µM). Fluorescence was calendared at 550 nm.
(b) The fluorescent intensity of R1 (10 μM) with HClO (10 equiv.) and other various
analytes (10 equiv.). (c) The fluorescent intensity of R1 (10 μM) at 550 nm changes
upon the addition of various analytes (100 μM) in the presence of ClO− (100 μM).
According to the reported literature [36,37], we gave a possible
response mechanism (shown in Scheme 2). Under the addition of
HClO, the thiourethane moiety in the probe R1 underwent S-
oxidation to release SO2, then compound R1 was hydrolyzed to the
compound Y1, and the ICT was turned on. So the fluorescence

4
emission increased. In order to prove the possibility of the above
tested in EC1 cells for toxicity
recognition mechanism, the HR-MS of R1 with HClO was measured.
After adding HClO to the test system of probe R1, the mixture was
tested by high resolution mass spectrometry (Fig. S10). New peak
[M + H]+ 411.1010 was found, indicating that compound Y1
(theoretical value [M + H]+ = 411.1009) was formed after R1
responding to HClO.
Fig. 5. Co-localization experiment of R1 (10.0 μM) with various organelle specific
markers in EC1 cells. The panels (b, f and j) showed the fluorescence imaging of R1
(λex=488 nm, λem=510-570 nm). The panels (c, g and k) showed the images of ER
Tracker (1.0 μM) (λex=552 nm,λem=538-640 nm), Lyso-Tracker (500.0 nM) (λex=552nm,
λem=538-620 nm) and Mito-Tracker (500.0 nM) (λex=638 nm, λem=608-670nm),
respectively. The panels (d, h and l) showed the images of b + c, f + g, and j + k merging,
respectively.Scale bar = 10 μm
Scheme 2. The proposed sensing mechanism of R1 for detecting ClO− based on ICT.
Before imaging application of R1 in cells, the cytotoxicity assay
of probe R1 was determined (Fig.4). After incubating EC1 cells with
different concentrations of probe R1 for 24 h, it can be seen that the
cells survival rate was as high as 86% as the concentration of the 10
μM of the probe. The probe R1 showed very low toxicity to cells in
the range of 0-10 μM.
In order to determine whether R1 can be localized to the
endoplasmic reticulum, a cell co-localization experiment was
performed (Fig.5). From Fig.5, it can be seen that the probe showed
green fluorescence after aligning with HClO. Commercially
positioned endoplasmic reticulum dyes (Fig. 5c), mitochondrial dyes
(Fig. 5k), and the localized lysosomal dyes (Fig. 5g) showed red
fluorescence. The overlap coefficient between probe R1 and the
endoplasmic reticulum dye was as high as 0.8052, while the overlap
coefficient between probe R1 and mitochondrial dye was 0.2091,
and the lysosomal dye overlap coefficient was 0.0280, indicating that
the probe R1 can locate the endoplasmic reticulum well. It may
provides a good tool for us to further reveal the relationship between
the endoplasmic reticulum and active oxygen.
Fig.6. Confocal fluorescence images of ClO− with R1 in EC1 cells. (b) EC1 cells
pretreated R1 (10 μM) for 30 min. (d) EC1 cells were pretreated with probe R1(10 μM)
and then incubated with HClO (100 μM) for 30 min. (a) and (c): bright field images of (b)
and (d), correspondingly.Scale bar =25 μm
Next, we further performed R1 cell bioimaging experiments,
and allowing the cells to incubate with 10 μM R1 for 30 min. It can
be seen from Fig. 6b that no obvious fluorescence appeared in the
green channel. And after adding 10 eq. of ClO-, obvious green
fluorescence appeared (Fig. 6d). Thus, the cell membrane
permeability of R1 was confirmed, and biofilming of HClO in a
biological sample was possible.
4. Conclusion
Fig. 4. Different R1 concentrations ( 2.5 μM, 4 μM, 6 μM, 8 μM, and 10 μM) were
In conclusion, we synthesized a probe that located the

5
[33] M. Yang, J. Fan, J. Zhang, J. Du, X. Peng, Chem. Sci. 9 (2018) 6758-
6764
endoplasmic reticulum to recognize HClO. After adding HClO, the
probe can quickly reach the response platform. The probe has good
selectivity, excellent anti-interference, and good cell penetration.
Furthermore, it can be well imaged in cells, and positioned to the
endoplasmic reticulum.
[34] H. Xiao, C. Wu, P. Li, W. Gao, W. Zhang, W. Zhang, L. Tong, B. Tang,
Chem. Sci. 8 (2017) 7025-7030
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(2016) 8579–8585.
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Declaration of competing interest
[37] D. Ji, G. Li, S. Zhang, M. Zhu, C. Li, R. Qiao, Sensor. Actuat. B- Chem.
259 (2018) 816-824.
There are no conflicts to declare.
Acknowledgements
This work was financially supported by the National Science
Foundation of China (No. 21572209).
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://...
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6
Graphical abstract
A ER-targetable probe R1 for HOCl was
synthesized. R1 shows excellent selectivity and
high sensitivity, excellent anti-interference, and can
quickly reach the response platform.

7
1. A new ER-targetable ICT-based fluorescent
probe R1 for HClO was developed.
2. Probe have rapid response (30 s), big Stock's
shift and a wide range of pH(7-13).
3. The imaging in EC1 cells demonstrated its
value of practical application.

8
Declaration of interests
We submit this paper which title is "
Endoplasmic reticulum-targetable
fluorescent probe for visualizing HClO in
EC1 cells " and declare that we have no
known competing financial interests or
personal relationships that could have
appeared to influence the work reported in
this paper.