NEM assisted real-time fluorescence detection of Cys in cytoplasm and mice imaging by a Coumarin probe containing carboxyl group

Article abstract of DOI:10.1016/j.saa.2019.117517

Alterations of the homeostasis balance of cysteine (Cys) are associated with a variety of diseases and cellular functions, and therefore, Cys dynamic real-time living cell intracellular imaging and quantification are important for understanding the pathophysiological processes. Thus, Cys probe that can permeate high efficiently is the first one to be affected. In fact, it is difficult for organic molecular probes to infiltrate cells because of the unique structure of the cell membrane. In this work, we found that probe containing-carboxyl just stagnated in cytomembrane due to carboxyl of probe and amino group of membrane protein forming peptide chains, nevertheless, the addition of NEM, improved membrane permeability by NEM reacting with sulfhydryl of membrane protein, which made probe permeate high efficiently and sequentially real-time detect the Cys in cytoplasm. It is the first time noted that NEM can regulate Cys probe containing-carboxyl for high efficient detection in cytoplasm. Additionally, probe was successfully applied to image Cys in mouse.

Full text of DOI:10.1016/j.saa.2019.117517

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 225 (2020) 117517
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
journal homepage: www.elsevier.com/locate/saa
NEM assisted real-time fluorescence detection of Cys in cytoplasm and
mice imaging by a Coumarin probe containing carboxyl group
Xixi Xie a,b, Fangjun Huo c, Yongkang Yue b, Jianbin Chao c, Caixia Yin a,b,
⁎
a
Department of Chemistry, Xinzhou Teachers University, Xinzhou 034000, Shanxi, China
b
Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Key Laboratory of Materials for Energy Conversion and Storage of Shanxi Province, Institute of Molecular
Science, Shanxi University, Taiyuan 030006, China
c
Research Institute of Applied Chemistry, Shanxi University, Taiyuan 030006, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Alterations of the homeostasis balance of cysteine (Cys) are associated with a variety of diseases and cellular
functions, and therefore, Cys dynamic real-time living cell intracellular imaging and quantification are important
for understanding the pathophysiological processes. Thus, Cys probe that can permeate high efficiently is the first
one to be affected. In fact, it is difficult for organic molecular probes to infiltrate cells because of the unique struc-
ture of the cell membrane. In this work, we found that probe containing-carboxyl just stagnated in cytomem-
brane due to carboxyl of probe and amino group of membrane protein forming peptide chains, nevertheless,
the addition of NEM, improved membrane permeability by NEM reacting with sulfhydryl of membrane protein,
which made probe permeate high efficiently and sequentially real-time detect the Cys in cytoplasm. It is the first
time noted that NEM can regulate Cys probe containing-carboxyl for high efficient detection in cytoplasm. Addi-
tionally, probe was successfully applied to image Cys in mouse.
Received 31 January 2019
Received in revised form 29 July 2019
Accepted 4 September 2019
Available online 5 September 2019
Keywords:
NEM regulated
Detection
Fluorescent probe
Bioimaging
© 2019 Elsevier B.V. All rights reserved.
1. Introduction
fatty acid molecules covalently on cysteine residues on one side of the
cytoplasmic matrix, inserted between lipid bilayer, a few proteins are
Cell as the basic units of the structure and function of organisms is
inseparable from the existence of cytomembrane [1–5]. Cytomembrane
have irreplaceable and significant physiological functions in biological
systems, which not only enable cells to maintain a stable metabolic in-
tracellular environment, but also regulate and select substance to
entry and exit the cell, specific performance in the following six aspects:
(a) to provide a relatively stable internal environment and improve bi-
ological function; (b) barrier function; (c) selective material transport;
(d) biological function: including that hormone effect, enzymatic reac-
tion, cell recognition, electron transfer, etc.; (e) recognize and transmit
information function; (f) material transport function. Cell membranes
consist of phospholipid bilayers and embedded proteins that pass
through the cell membranes and are adsorbed on the surface of the
cell membranes. Depending on the difficulty of protein separation and
the location of protein distribution in the membrane, membrane pro-
teins can be divided into three categories: exogenous membrane pro-
teins, also known as peripheral membrane proteins, and intrinsic
membrane proteins, also known as integral membrane proteins and
lipid-anchored proteins. Thereinto, integral membrane proteins bind
covalently bound to glycolipids [6]. N-Ethylmalemide (NEM), as a
masking agent for cysteine (Cys), in other words, a covalent modifica-
tion reagent for Cys residues of protein, after using to incubate cells,
Cys residues may form covalent binding with NEM, which breaks the
structure of covalent binding polypeptide formed by Cys residues and
fatty acid molecules on one side of the cytoplasmic matrix of integral
membrane proteins, thus the original structure of the cell membrane
is destroyed and then the permeability of the cell membrane is changes
[7–9].
Cysteine (Cys), the only amino acid with the reducing group (-SH) in
more than score of amino acids that make up proteins, plays a vital role
in maintaining normal physiological and biological processes [10–18].
The total content of Cys in cells is 30–200 μM have been clear [19–21],
elevating and reducing levels of Cys have been associated with various
diseases, for example, rheumatoid arthritis, neurotoxicity, Alzheimer's
disease, Parkinson's disease, slow growth in children and liver damage
[22–31]. Therefore, it is of great value and significant important to de-
tect intracellular even cytoplasm Cys in biochemistry and clinical appli-
cations area.
Previous work, we have designed and synthesized a series of probes
based on coumarin fluorophore. Among them, there was a peculiar
probe containing-carboxyl willfully stayed at cytomembrane when
probe was incubated only with cells. However, subsequent addition of
⁎
Corresponding author at: Department of Chemistry, Xinzhou Teachers University,
Xinzhou 034000, Shanxi, China.
E-mail address: yincx@sxu.edu.cn (C. Yin).
https://doi.org/10.1016/j.saa.2019.117517
1386-1425/© 2019 Elsevier B.V. All rights reserved.

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X. Xie et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 225 (2020) 117517
NEM, probe will exhibit detection effect for Cys in cytoplasm not in cy-
tomembrane [32,33]. To discover this mystery, we obtained another sib-
ling probe to carry out detailed research in this work. The probe showed
very low background fluorescence and highly selectivity to Cys with a
turn-on fluorescent response peaked with 62-fold fluorescent enhance-
ment at 500 nm. It is well known that NEM is a trapping reagent of thiol
species [34–36], which made probe 1 to reversibly detect Cys in our sys-
tem. More important, via the regulation of NEM made probe permeate
high efficiently and sequentially real-time detect the Cys in cytoplasm.
It can be explained that the addition of NEM improved membrane per-
meability by NEM reacting with sulfhydryl of membrane protein.
2.2. Solutions preparation and UV–vis and fluorescence measurements
Probe 1 was prepared with 2 mL DMSO solution at a concentration of
2 mM. Cys and NEM stock solutions (2 mM) were as prepared in deion-
ized water. Other analytes solutions (0.2 M) were prepared by using de-
ionized water, which including amino acids, peptide, metal cations and
anions. And fluorescence measurements were carried out with an exci-
tation wavelength at 440 nm, emission wavelength at 500 nm and a slit
width of 5 nm.
2.3. Cell imaging
2. Materials and methods
The cells were directly incubated with 10 μM probe 1 for 0.5 h. The
cells were sequentially incubated with NEM (1 mM) and probe 1 (10
μM) for 0.5 h each. The cells were sequentially incubated with NEM
(1 mM), Cys (200 μM), and probe 1 (10 μM) for 0.5 h each. The cells
were sequentially incubated with NEM (1 mM), GSH (1 mM), and
probe 1 (10 μM) for 0.5 h each. The cells were sequentially incubated
with NEM (1 mM), Hcy (15 μM), and probe 1 (10 μM) for 0.5 h each. Be-
fore imaging, each step is performed first the cells were washed by PBS
once. Fluorescence images were obtained with the emission was col-
lected at 480–530 nm and the excitation wavelength is at 458 nm by a
Zeiss LSM880 Airyscan confocal laser scanning microscope (Scheme 1).
2.1. Synthesis of probe 1
Probe 1: compound 6 (0.10 mmol, 0.046 g) and 2-cyanoacetic acid
(0.15 mmol, 0.013 g) were dissolved in EtOH (15 mL). 50 μL piperidine
was added and the mixture was stirred at 78 °C refluxed for 4 h (TLC
monitoring process). After cooling to 25 °C, reducing pressure, the red
solid was purified by column chromatography (MeOH:DCM = 1:10),
giving probe
1 ((Z)-2-cyano-3-(4-((E)-3-(7-(4-(ethoxycarbonyl)
piperazin-1-yl)-2-oxo-2H-chromen-3-yl)-3-oxoprop-1-en-1-yl) phe-
nyl) acrylic acid) as a red powder (0.0455 g, 68% yield) (synthetic
route and relevant NMR data see supporting information). 1H NMR
data (600 MHz, DMSO d6): δ 8.65 (s, 1H), 7.97 (t, J = 12.5 Hz, 4H),
7.85 (d, J = 8.2 Hz, 2H), 7.76 (d, J = 9.0 Hz, 1H), 7.71 (d, J = 15.7 Hz,
1H), 7.05 (dd, J = 9.0, 2.0 Hz, 1H), 6.90 (s, 1H), 4.08 (q, J = 7.1 Hz,
2H), 3.58–3.50 (m, 8H), 1.21 (t, J = 7.1 Hz, 3H). 13C NMR data
(151 MHz, DMSO): δ (ppm): 186.3, 160.0, 158.2, 155.3, 155.1, 148.8,
141.4, 132.5, 130.5, 129.3, 126.8, 117.9, 112.1, 109.7, 98.9, 61.4, 46.5,
15.0. ESI-MS: found: m/z 528.1767; Molecular formula: [C29H25N3O7]
+, Calc: 528.5405 (Fig. S5).
2.4. Cell viability
Cytotoxicity was assessed by performing Cell Counting Kit-8 (CCK-
8) assay with the HepG-2 cells. The HepG-2 cells were seeded on 96-
well plates and incubated for 24 h at 37 °C (5% CO2). Different concen-
trations of probes 1 (0, 1, 2, 3, 5, 7, 10 and 20 μM) were then added to
the wells in sequence with 6 replicates per concentration. After incuba-
tion for 5 or 10 h, CCK-8 (100 μL, 10% in serum free culture medium)
was added to each well, and then the plates continue to be incubated
Scheme 1. Possible response mechanism of probe 1 toward Cys: above) the probe 1 reversibility response to Cys in vitro; bottom) the probe 1 response to Cys in cell and in vivo
environment.

X. Xie et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 225 (2020) 117517
3
Scheme 2. Synthesis of probe 1.
for another 1 h. The optical densities of each well at 450 nm were then
measured by a microplate reader (Scheme 2).
2.5. Mice imaging
The bal. a/c mice were fed commercial mice chow in individual
stainless steel cages and left freely wandering in their housing for
2 weeks with 12 h dark/light cycles for acclimatization before the exper-
iment. The mice were in anesthesia by Pentobarbital sodium (100 μL, 0.5
mL/0.03%) subcutaneously injection. The mice were injected in the sub-
cutaneous with 100 μM of probe 1, 2 equal of Cys was slowly injected to
the same location. Then, the fluorescence images were performed at
Equal difference period of time (0.5, 5, 10, 20, 25, 30 min). Images
were carried out on using an excitation laser of 474 nm and emission
was collected 520 5 nm filter.
3. Results and discussion
3.1. The selective response of probe 1 to Cys
The level of probe activity (simple mixture, plasma, in vitro en-
vironment or organism) was depended on the selectivity of the
Fig. 1. (a) Absorption spectra of probe 1 (10 μM) upon addition of Cys (0–450 μM) in PBS–
DMSO buffer (10 mM, pH 7.4, 4:1, v/v) system. (b) Fluorescence spectra of probe 1 (10
μM) upon addition of Cys (0–450 μM) in PBS–DMSO buffer (10 mM, pH 7.4, 4:1, v/v)
system. Working curve of probe 1 to detect Cys obtained by addition of various
concentrations of Cys (0–100 μM) to probe 1 (10 μM). Each spectrum was obtained
3 min after addition. (λem = 500 nm, λex = 440 nm, slit: 5 nm/5 nm).
Fig. 2. Fluorescent (500 nm) of probe 1 (10 μM) upon addition of Cys (20 mM) and NEM
(20 mM). Solvent: 10 mmol/L sodium phosphate buffer, pH 7.4. Cys and NEM
concentrations in the figure are the final concentrations in the cuvette (λem = 500 nm,
λex = 440 nm, slit: 5 nm/5 nm).

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X. Xie et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 225 (2020) 117517
probe. So for a probe, the specific selectivity for a substance is ex-
tremely important. Subsequent the selective experiment of probe
toward Cys was implemented, the fluorescence response of the
probes to various analytes including amino acids and some of ions
in PBS–DMSO buffer (10 mM, pH 7.4, 4:1, v/v) was determined
(Fig.S6). 0.2 mol/L various analytes including amino acids, peptide,
metal cations and anions. Furthermore, analysis of fluorescence en-
hancement at 500 nm (λex = 440 nm) of probe 1 with other amino
acids and some of ions illustrated discriminate ability in the follow-
ing order: Cys N Hcy N GSH ≈ other amino acids ≈ metal cations
and anions. Thus, probe 1 appears to be a relatively high selectivity
probe for Cys.
3.2. Spectral properties of probe 1 toward Cys
Moreover, pH-effect of probe 1 and the probe 1–Cys system was
measured with pH change from 2 to 11 (Fig. S7). The probes itself
displayed inappreciable intensity boost in pace with the pH of the detec-
tion system changing. In the range of pH from 5 to 9, the spectra of the
system indicated strong fluorescent emission at 500 nm (λex
440 nm). The results illustrated that probe 1 could detect Cys could be
realized over normal physiological conditions (pH = 7.4).
Detailed spectral titrations for Cys were carried out. The UV–Visible
spectrum response of probe 1 (10 μM) toward Cys (0–450 μM) was stud-
ied in PBS-DMSO (10 mM, pH 7.4, 4:1, v/v) system. Probe 1 (10 μM) itself
=
Fig. 3. (a) Top: Cell images of HepG-2 cells lines. A: HepG-2 cells incubated with probe 1 (10 μM) for 0.5 h; B: HepG-2 cells pre-treated with 1 mM NEM for 0.5 h, then incubated with probe
1 (10 μM); C, D, E: Cells were pre-treated with 1 mM NEM for 0.5 h, then incubated with 200 μM Cys/15 μM Hcy/1 mM GSH for 0.5 h, then incubated with probe 1(10 μM). Green channel:
λem = 480–530 nm (λex = 458 nm) Scale bar: 20 μm. Bottom: Fluorescence intensity per cells. (b) Cysteine visualization distribution analysis on cytomembrane of cell imaging by probe 1
incubation (from cytomembrane to cytoplasm to cytomembrane or from cytomembrane to cytoplasm to nucleus to cytoplasm to cytomembrane). (c) Time-dependent confocal images of
endogenous Cys in A549 cells with probe 1 (10 μM). Green channel: λem = 480–530 nm (λex = 458 nm) Scale bar: 20 μm.

X. Xie et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 225 (2020) 117517
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Fig. 3 (continued).
featured two absorption peaks at 312 nm and 403 nm in an aqueous
buffer solution of PBS-DMSO (10 mM, pH 7.4, 4:1, v/v) (Fig. 1a). With
adding Cys (450 μM), the both absorption peak decreased and an obvious
new band at 262 nm appeared with isosbestic point at 270 nm forming,
which implied new compounds generated in the system. Fig. 1b showed
the fluorescence spectra change of probe 1 (10 μM) upon adding Cys
(0–450 μM) in PBS-DMSO (10 mM, pH 7.4, 4:1, v/v).
Probe 1 itself had non-fluorescent emission, until the addition of
Cys (450 μM) induced a turn-on emission at peaked 500 nm with a
61-fold enhancement in above system. The fluorescence intensity
was in good agreement with Cys concentration in the linear range
(R2 = 0.9988). Based on the detection limit defined by IUPAC (CDL
= 3Sb/m), the corresponding of probe 1 for Cys was 1.22 μM from
10 blank solutions.
presence of 10 eq. of Cys. The fluorescent intensity of the above system
was balanced and kept within 200 s (Fig. S8), indicating that this probe
has the potential for real-time detecting Cys in vivo.
3.4. Reversibility test in vitro
In order to further test probe 1 whether has the potential for real-
time detecting Cys in vivo and whether NEM enable the probe to revers-
ibly detect Cys in vitro, the reversibility test of probe 1-Cys system was
carried out: the fluorescence emission intensity at 500 nm (λex
=
440 nm) were monitored upon addition of Cys followed by addition of
NEM (Fig. S9). The result displayed that the intensity at 500 nm of the
system enhanced immediately after addition of Cys, and addition of
NEM subsequently 6 times resulted in decreased rapidly and step by
step of the fluorescence intensity, then restored by adding Cys,
confirming reversibility. The reversible response was estimated by the
fluorescence response of the probe 1-Cys system under the action of
NEM. NEM could effectively terminate the fluorescent emission of
probe 1-Cys system and which could be regained by subsequent
3.3. Time-dependence in the detection process of Cys
The kinetic study experiment of probe 1 to Cys was implemented
that monitored fluorescence spectra of the probe system was in the

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X. Xie et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 225 (2020) 117517
addition of Cys (Fig. 2). The reversible cycling tests–that is to say, three
times addition of Cys and subsequent NEM added all induced consistent
fluorescent signal changes, which promoted probe 1 as a potential effec-
tive tool for real-time determination of Cys content in vitro. It was ver-
ified that NEM reduced the fluorescence intensity of the probe 1-Cys
system by consuming Cys.
HepG-2 cells incubated with probe 1 showed obvious green fluores-
cence on cytomembrane (Fig. 3a (A) and Fig. 3b), indicating that probe 1
could answer Cys on the cytomembrane. While the cells were sequen-
tially incubated with NEM and probe 1, the obvious strong fluorescent
in HepG-2 cells cytoplasm or not on cytomembrane or none-
fluorescence in cells cytoplasm (Fig. 3a), indicating the role of NEM
here was not to eliminate thiols in the cytoplasm as usual, and Cys con-
tent in cytoplasm is much higher than that of Cys on cytomembrane. Be-
sides, the exogenous Cys result in that a more obvious fluorescence
increase in green channel. The above imaging results of this probe
were consistent with the imaging results of that probe in the previous
work. Comparing fluorescence intensity in the Green Channel of four
images of B/C/D/E in Fig. 3(a), showed that probe 1 can specific detect
Cys, this was consistent with spectral data results, also verified the fol-
lowing conjecture: probably, NEM does not eliminate the thiols (GSH/
Hcy/Cys) in the cytoplasm, but only improved membrane permeability
by NEM reacting with sulfydryl of membrane protein which made
probe permeate high efficiently and detect the Cys in cytoplasm. It is
the first time improved membrane permeability by NEM reacting with
sulfydryl of membrane protein which made probe permeate high effi-
ciently and detect the Cys in cytoplasm. It is the first time noted that
NEM can regulate Cys probe containing-carboxyl for high efficient de-
tection in cytoplasm. Further experiments to reveal the above conclu-
sion are on-going in our lab.
3.5. Proposed mechanism
1H NMR and ESI-MS experiments were performed to clear the sens-
ing mechanism of probe 1 toward Cys. The 1H NMR data displayed that
the bimodal signals at 8.00, and 7.71 part to α, β-unsaturated ketone
double bonds disappeared, and new signals appeared at 4.50/3.70/
3.40 (Fig. S10). To further testify that the response site of Cys with
probe 1 is the double bond of α, β-unsaturated ketone, the 1H NMR ti-
tration experiment of 2-mercaptoethanol (a Cys analogue) with probe
1 was implement (Fig. S11). The results showed that the site of the re-
action with 2-mercaptoethanol is α, β-unsaturated ketone double
bonds (the bimodal peaks of 8.00 and 7.71 are weakened), the proton
e and f of probe 1 shift from 8.00, 7.71 to 4.49 and 3.63. Additionally,
ESI-MS (Fig. S12) data of the reaction mixture of probe 1 with Cys
displayed signal at 649.1962 which matched the structure [1-Cys
+ H]+ and other fragments (m/z = 671.1781, [1-Cys + Na]+). Further,
the fluorescent emission of the reaction mixture of probe 1 with 2-
mercaptoethanol matched the Cys added probe 1 system very well
(Fig. S13). These results provided strong support, wherein fluorescence
signal enhancement of probe 1 responses Cys by nucleophilic addition
reaction of –SH of Cys with α, β-unsaturated ketone double bonds of
the probe caused.
3.7. Fluorescence imaging in mice
Next, we investigated the utility of probe 1 in mice. The mice were
subcutaneously injected with probe 1 (20 μL 0.1 mM) and succedent
Cys (20 μL, 0.2 mM) solution at different times after subcutaneous injec-
tion at the right rear side. The fluorescent intensity of the mouse en-
hances inch by inch over time and reaches maximum value in 20 min,
was maintained by the body later (Fig. 4c-h).
3.6. Fluorescence imaging in living cells
To evaluate the capability of NEM that regulate probe 1 to come
true high efficient detect Cys in cytoplasm. Herein, HepG-2 cells
were chosen for the confocal fluorescence imaging. Cell Counting
Kit-8 (CCK-8) was carried out to evaluate the cytotoxicity of probe
1. The result revealed that low cytotoxicity (83.9% viability) and
good biocompatibility of probe 1 (20 μM) to HepG-2 cells at 37 °C
for 5/10 h (Fig. S14), implied the probe has the potential to be used
on living cells and mouse.
4. Conclusions
In summary, probe 1 can be recycled three times or more in the pres-
ence of NEM in vitro, indicating that this probe could reflect Cys dynam-
ics in the detection system on line. In this work, we found that probe
containing-carboxyl just stagnated in cytomembrane due to carboxyl
Fig. 4. Fluorescence images of probe 1 responding to exogenous Cys in mice. (a) Mice without any treatment, (b) 100 μM of probe 1 was subcutaneously injected only, (c-h) Representative
fluorescence images (pseudocolor) of living mice treated with probe 1 (20 μL, 0.2 mM) and succedent Cys (20 μL, 0.2 mM) solution at different times (0, 0.5, 5, 10, 20, 25, 30 min) after
subcutaneous injection. Images were taken using an excitation laser of 474 nm and an emission filter of 515–525 nm.

X. Xie et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 225 (2020) 117517
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We thank the National Natural Science Foundation of China (Nos.
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