A chloroacetate based ratiometric fluorescent probe for cysteine detection in biosystems

Article abstract of DOI:10.1016/j.tetlet.2019.151218

The specific detection of cysteine (Cys) over homocysteine (Hcy), glutathione (GSH) and other amino acids is of great significance for studying its biological functions as well as for the diagnosis of related diseases. Chloroacetyl group was often used as a reaction site for cysteine fluorescent probes for its sensitivity and selectivity. However, high background fluorescence and low stability are common problems encountered by such probes. Here, four chloroacetyl group based fluorescent probes (C1, C2, C3, and H4) was synthesized for a comparative study. We found that the inefficient quenching ability of chloroacetyl group turned into an advantage when connected with a ratiometric fluorophore. With the modification of chloroacetyl group, probe H4 displayed excellent ratiometric property and great selectivity for Cys, the stability was also improved. Additionally, the probe was successfully applied for quantitative detection of Cys in fetal bovine serum and real-time imaging in living HeLa cells with low toxicity.

Full text of DOI:10.1016/j.tetlet.2019.151218

Tetrahedron Letters xxx (xxxx) xxx
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
A chloroacetate based ratiometric fluorescent probe for cysteine
detection in biosystems
Zhengkun Liu a,1, Qianqian Wang a,1, Hao Wang a, Wenting Su a, Shouliang Dong a,b,
⇑
a Institute of Biochemistry and Molecular Biology, School of Life Sciences, Lanzhou University, 222 Tianshui South Road, Lanzhou 730000, China
b Key Laboratory of Preclinical Study for New Drugs of Gansu Province, Lanzhou University, 222 Tianshui South Road, Lanzhou 730000, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The specific detection of cysteine (Cys) over homocysteine (Hcy), glutathione (GSH) and other amino
acids is of great significance for studying its biological functions as well as for the diagnosis of related
diseases. Chloroacetyl group was often used as a reaction site for cysteine fluorescent probes for its sen-
sitivity and selectivity. However, high background fluorescence and low stability are common problems
encountered by such probes. Here, four chloroacetyl group based fluorescent probes (C1, C2, C3, and H4)
was synthesized for a comparative study. We found that the inefficient quenching ability of chloroacetyl
group turned into an advantage when connected with a ratiometric fluorophore. With the modification of
chloroacetyl group, probe H4 displayed excellent ratiometric property and great selectivity for Cys, the
stability was also improved. Additionally, the probe was successfully applied for quantitative detection
of Cys in fetal bovine serum and real-time imaging in living HeLa cells with low toxicity.
Ó 2019 Elsevier Ltd. All rights reserved.
Received 31 July 2019
Revised 25 September 2019
Accepted 26 September 2019
Available online xxxx
Keywords:
Chloroacetyl group
Fluorescent probe
Cysteine
Ratiometric
Introduction
important functional and structural moiety of various peptides or
proteins. The deficiency of Cys would cause many symptoms like
Biological thiols, such as cysteine (Cys), homocysteine (Hcy),
and glutathione (GSH) are widely distributed and play key roles
in the biological systems. In spite of their similar structures, bioth-
iols have different biological functions. In mammalian cells, GSH is
the most abundant intracellular non-protein thiol and plays a key
role in the control of oxidative stress in redox homeostasis [1].
Mitochondrial GSH pool is a critical antioxidant reservoir within
cells and associated with many mitochondria functions, such as
signal transduction, and gene regulation [2]. The fluctuation of
the levels of GSH is very important and has been linked to some
diseases and dysfunction of mitochondrial [3,4]. Hcy is also one
of the important biothiols and has gradually attracted widespread
attention as a biomarker [5]. Clinical trial results have shown that
retarded growth in children, hair depigmentation, lethargy, liver
damage, muscle and fat loss, skin lesions and weakness [18–21],
Furthermore, chronic accumulation of free Cys is related to neuro-
pathic poisoning [22]. The role of Cys in various cancer is also
important yet not completely clear [23,24]. Therefore, the specific
detection of Cys is very important for studying its biological func-
tions and for the diagnosis of related diseases.
Among various detection methods, fluorescent probes have
emerged as the most common imaging strategy due to its simplic-
ity, high sensitivity and non-destructive advantages [25]. In the
past ten years, a large number of fluorescent probes for Cys detec-
tion have been developed based on different mechanisms [25,26].
Many response groups such as aldehyde group [27], acrylate [28],
maleimide [29], 7-nitrobenzofurazan (NBD) [30] and various metal
ions [31,32] were used to design probes for Cys. Chloroacetyl group
was also employed for its great selectivity (Scheme 1). But com-
pare to widely used acrylate, the same nucleophilic addition-
cyclization type reaction group, the application of chloroacetyl
group for Cys detection was relatively fewer. This may be caused
by some disadvantages of chloroacetyl group, such as inefficient
quenching ability which caused high background fluorescence,
low stability and relatively slow reactivity [33–37]. To solve these
problems, four chloroacetyl group based probes, C1, C2, C3, and H4,
with different connecting structures and fluorophores were
high concentrations of Hcy in serum or plasma (mild: 15–30
lΜ,
moderate: 30–100 Μ) are predictors of cardio-
lΜ, severe: ꢀ100
l
vascular disease and independent risk factors. Elevated plasma
homocysteine levels were found in Alzheimer’s disease [6], Parkin-
son’s disease [7,8], cancer [9–13], diabetes [14–16]. HIV-infected
patients found a decrease in plasma homocysteine concentration
[17]. Cys as a semi-essential amino acid and biothiol, is a very
⇑
Corresponding author.
E-mail address: dongsl@lzu.edu.cn (S. Dong).
These authors contributed equally to this work.
1
https://doi.org/10.1016/j.tetlet.2019.151218
0040-4039/Ó 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Z. Liu, Q. Wang, H. Wang et al., A chloroacetate based ratiometric fluorescent probe for cysteine detection in biosystems, Tetra-
hedron Letters, https://doi.org/10.1016/j.tetlet.2019.151218

2
Z. Liu et al. / Tetrahedron Letters xxx (xxxx) xxx
Scheme 1. Proposed response mechanism of chloroacetate-based probes to Cys.
designed and synthesised. The stability, reactivity and spectral
properties of the four probes were thoroughly investigated. We
found that the inefficient quenching ability of chloroacetyl group
turned into an advantage when connected with a ratiometric fluo-
rophore (H4) and with the addition of cetyltrimethylammonium
bromide (CTAB), the stability and reactivity of H4 were greatly
improved. Additionally, probe H4 was successfully applied for
detecting and real-time imaging of Cys in fetal bovine serum and
living HeLa cells with low toxicity.
HMBA and HEBA displayed different reactivity and stability when
deprotection [39]. So, we employed both two groups for C2 and
C3 design to achieve the completely quenching effect. With probes
C1, C2, and C3 in hand, we first investigated the quenching ability
of chloroacetyl group (C1) and the new designed mask groups of C2
and C3. As depicted in Fig. 2A, the high background fluorescence
was observed in C1 probably due to the inefficient quenching abil-
ity of chloroacetyl group mentioned above. Comparatively only
weak background fluorescence was detected in C2 and C3, this
result indicated that connection with HMBA and HEBA greatly
improved the quenching ability of chloroacetyl group. The
improved quench effects may be caused by electron-withdrawing
effect of ester bond [40] and the reduced rigidity of probes struc-
ture [41]. C2 and C3 also showed great selectivity for Cys over
Hcy and GSH (Fig. 2B, 2C). We then further studied the kinetic
properties of C2 and C3 upon reaction with three biothiols, unfor-
tunately, we found that the stabilities of C2 and C3 were greatly
influenced by temperature and polar solvents. In the biocompati-
ble condition of 37 °C and 1% DMSO/PBS (V/V), C2 and C3 were
completely hydrolyzed within 2 h which perhaps caused by two
unstable ester bonds. So C1, C2 and C3 were not suitable for biolog-
ical detection.
We further designed and synthesized H4 through a directly
linking between chloroacetyl group and 2-(20-hydroxy-30-methox-
yphenyl)benzothiazole (HMBT), Scheme 2. HMBT is a widely used
fluorophore for its small size, good photostability and relative bio-
logical compatibility [42]. Besides, HMBT can undergo an excited-
state intramolecular photon transfer (ESIPT) process upon pho-
toexcitation, resulting in dual emission bands which originate from
its enol and keto tautomeric forms [43]. The detailed information
of synthesis for H4, characteristic spectrums of the intermediates
and final products can be seen in ESI. The absorption and fluores-
cence emission spectra of H4 in the absence and presence of cys-
teine were shown in Fig. S1, as also the HMBT. In addition, the
absolute quantum yield of H4 in 320–420 nm were measured to
be 0.49%. After addition of cysteine the emission redshifted to
450–600 nm and the absolute quantum yield was 0.48% compared
to 0.61% of HMBT. H4 displayed strong fluorescence at 372 nm
which was in accordance with the emission band from enol forms
Results and discussion
As shown in Fig. 1, C1 was constructed by a directly connection
of chloroacetyl group with coumarin, while C2 and C3 were con-
nected by 2-(hydroxymethyl)benzoic acid (HMBA) or 2-(2-hydrox-
yethyl)benzoic acid (HEBA) group for better quenching effect. The
detailed information of synthesis for C1, C2, and C3 was shown in
ESI, the intermediates and final products were confirmed by 1H
NMR, 13C NMR and mass spectrometry. Proposed response mecha-
nisms of C1, C2, and C3 for Cys detection were shown in
Scheme S1. For C2 and C3, after the addition-cyclization release
of chloroacetyl group, the HMBA and HEBA group both can
undergo another cascaded intramolecular cyclization reaction to
generate the highly fluorescent coumarin. These groups were ini-
tially designed for the protecting of carbohydrates [38,39] and
Fig. 1. The structure of probes C1, C2, and C3.
Fig. 2. (A) Fluorescence intensity changes of the probes C1, C2, and C3 (all 10
intensity ratios F/F0 of 10 M probes C2 and C3 after addition of Cys, Hcy and GSH 100
pH = 7.4) at 20 °C, kex = 327 nm, kem = 454 nm, slits (10, 10).
l
M) before and after treatment with 100
lM Cys for 2 h; (B, C) Time-dependent fluorescence
l
lM respectively. All experiments carried in 50% ACN/PBS (V/V) buffer (50 mM,
Please cite this article as: Z. Liu, Q. Wang, H. Wang et al., A chloroacetate based ratiometric fluorescent probe for cysteine detection in biosystems, Tetra-
hedron Letters, https://doi.org/10.1016/j.tetlet.2019.151218

Z. Liu et al. / Tetrahedron Letters xxx (xxxx) xxx
3
Scheme 2. The structure of probe H4, HMBT and proposed response mechanism of H4 to Cys.
of HMBT. This phenomenon was different with many other HMBT
based probes, because the enol-like emission was usually
quenched and only showed slightly changes after reacted with
analytes [44–46]. Thus the inefficient quenching ability of chloroa-
cetyl group turned into an advantage when connected with HMBT.
As expected, after treatment with Cys, the enol-like emission of H4
declined and new emission at 482 nm appeared (Fig. 3A). The pro-
posed response mechanism was also confirmed by mass and the
Fig. 3. (A, C) Time-dependent fluorescence intensity of 10
l
M H4 upon addition of 500
lM Cys; (B, D) Time-dependent fluorescence intensity ratios at F482 nm/F372
of
nm
10 M probes H4 after addition of Cys, Hcy and GSH, 500 M respectively. All experiments in A and B were carried in 50% ACN/PBS (V/V) buffer (50 mM, pH = 7.4) at 37 °C, All
l
l
experiments in C and D were carried in 1% ACN/PBS (V/V) buffer (50 mM, pH = 7.4) with addition of 1 mM CTAB at 37 °C. kex = 310 nm, kem = 372 nm, 482 nm, slits (10, 10).
Please cite this article as: Z. Liu, Q. Wang, H. Wang et al., A chloroacetate based ratiometric fluorescent probe for cysteine detection in biosystems, Tetra-
hedron Letters, https://doi.org/10.1016/j.tetlet.2019.151218

4
Z. Liu et al. / Tetrahedron Letters xxx (xxxx) xxx
Fig. 4. (A) Concentration-dependent fluorescence spectra of H4 (10
l
M) upon addition of Cys (0–500
l
M) and the linear relationship between the fluorescence intensity
and concentrations of Cys (0–20 M) in 1%
M) at different pH values in
M) and FBS (0–500 L). A, C and D all in 50%
ratios at F482 nm/F372 and Cys (0–50 M); (B) Linear relationship between the fluorescence intensity ratios at F482 nm/F372
l
l
nm
nm
ACN/PBS (V/V) buffer (50 mM, pH = 7.4) with 1 mM CTAB at 37 °C; (C) Fluorescence intensity ratios (F482 nm/F372 nm) changes of the probe H4 (10
the absence or presence of Cys; (D) Linear relationship between the fluorescence intensity ratios at F482 nm/F372
l
of H4 (10
l
l
nm
ACN/PBS (V/V) buffer (50 mM, pH = 7.4) at 37 °C, kex = 310 nm, kem = 372 nm, 482 nm, slits (10, 10).
Table 1
pleted within 40 mins, the sensitivity of H4 for Hcy was also
slightly improved. But considering the relatively low content of
Hcy in biosystem, the selectivity of H4 for Cys would not be
affected (Fig. 3D).
We then evaluated the sensitivity of H4 for Cys. Concentration-
dependent studies showed an excellent linearity between
Determination of Cys in fetal bovine serum sample.a
Added Cys (
lM)
Measured (lM)
Recovery (%)
RSD (n = 3, %)
0
5
10
20
21.02
26.11
31.79
37.76
–
2.7
3.1
3.3
3.7
100.36
102.48
92.04
fluorescence intensity ratios at F482 nm/F372 nm and Cys (0–50
in 50% ACN/PBS (V/V) buffer, with a calculated detection limit of
1.01 M (Fig. 4A). Besides, with the addition of CTAB in 1% ACN/
PBS (V/V) buffer, the detection limit can as low as 0.4 M (Fig. 4B).
lM)
a
Conditions: 10 lM H4 in PBS buffer (50 mM, pH = 7.4, 50% ACN), incubated for
2 h, kex = 310 nm, kem = 372 nm/482 nm.
l
l
These results showed that H4 has enough sensitivity for Cys
detection in biological systems. Furthermore, the excellent linear-
ity equipped probe H4 with the ability of quantitative detection of
Cys.
formation of thiomorpholinone was clearly showed in mass spec-
trum, calculated for 161.01, found m/z 160.01 [MꢁH]ꢁ (Fig. S4).
Next we investigated the fluorescence sensing behavior of H4
toward biothiols. Because of the low solubility of H4 in PBS, we
first chose 50% ACN/PBS as reaction solvent. As shown in Fig. 3B,
although H4 showed great selectivity to Cys over Hcy and GSH,
the reaction rate was slow and it spent more than 2 h to complete
the detection. The addition of surfactants such as CTAB were usu-
ally applied to improve the solubility and increase the kinetics of
probes’ reaction in aqueous solution [47,48]. With the addition of
CTAB, H4 can easily dissolved in 1% ACN/PBS and showed great sta-
bility, the keto emission of H4 was also greatly enhanced (Fig. 3C).
In this solvent condition, the reaction of H4 with Cys was com-
The selectivity of H4 for Cys over other amino acids was also
evaluated. Significant fluorescent emission (Fig. S2) was induced
only upon addition of Cys, indicating great selectivity of H4 toward
Cys. The pH effect of H4 in the presence or absence of Cys was also
investigated (Fig. 4C). The fluorescence spectra showed no
significant change for the free probe over a wide pH range of
3.0–11.0, indicating that H4 is very stable in a relatively wide pH
range. On the other hand, upon treatment with Cys, there was a
progressive fluorescence enhancement between pH 3.0 and 9.0,
this may be partially caused by enhanced reactivity of H4 under
alkaline conditions, but stronger alkali seems to suppressed the
Please cite this article as: Z. Liu, Q. Wang, H. Wang et al., A chloroacetate based ratiometric fluorescent probe for cysteine detection in biosystems, Tetra-
hedron Letters, https://doi.org/10.1016/j.tetlet.2019.151218

Z. Liu et al. / Tetrahedron Letters xxx (xxxx) xxx
5
Fig. 5. Fluorescence images of HeLa cells. (A, D) Cells incubated with only H4 (20
l
M) for 10 min. (B, E) Cells incubated with NEM (200
lM) for 30 min before with H4
(20
lM) for 10 min. (C, F) Cells incubated first with NEM (200
lM) for 30 min, then Cys (200 lM) for 30 min and finally H4 (20 lM) for another 10 min. 37 °C, green channel:
Ex = 426–446 nm, Em = 460–500 nm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
keto-emission of H4 [49]. These results indicated that H4 could
serve as a satisfactory Cys sensing probe under physiological pH
conditions.
back for its application in living system imaging and a near-infra-
red ratiometric fluorophore is needed.
Before the applications of H4 for Cys detection in biosystems,
standard methyl thiazolyl tetrazolium (MTT) assay was carried
out to evaluate its cytotoxicity. After incubation with H4 for 24 h,
no significant reduction in cell viability can be observed with the
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
probe at concentrations of 5–20 lM (Fig. S3), indicating low cyto-
toxicity of H4. Then the detection of Cys in diluted (5%) reduced
fetal bovine serum (FBS) was carried out. A concentration-depen-
dent fluorescence intensity ratio increase (F482 nm/F372 nm) was
observed with excellent linearity characteristics (Fig. 4D). By using
the standard addition method, the amount of Cys in the FBS sample
was determined to be 384.5 lM with satisfactory recovery
(Table 1). The results suggested that the H4 can be applied to the
quantitative detection of Cys in serum samples.
We next evaluated the capability of the probe H4 for Cys imag-
ing in HeLa cells. As exhibited in Fig. 5, when Hela cells were incu-
Acknowledgements
The authors want to thank Mr. Feiyun Gao (School of Basic Med-
ical Sciences, Lanzhou University) for the Mass spectra, Mr. Shunxi
Li (Northwest Normal University, Gansu, China) for the NMR spec-
tra, Core Facility of School of Life Sciences (Lanzhou University) for
the imaging and the Central Laboratory (the First Hospital, Lanzhou
University) for the cell experiments.
bated with H4 (20 lM) for 10 min, a strong green fluorescence
Appendix A. Supplementary data
enhancement was observed implying the long-wavelength keto
emission of H4. In contrast, when Hela cells were pre-treated with
1 mM NEM (N-ethylmaleimide, a commonly used intracellular
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.tetlet.2019.151218.
thiol scavenger) before treated with H4 (20
intensity was decreased, but a strong fluorescence was recovered
when 200 M Cys was added after treated with NEM, confirming
lM), the fluorescence
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Please cite this article as: Z. Liu, Q. Wang, H. Wang et al., A chloroacetate based ratiometric fluorescent probe for cysteine detection in biosystems, Tetra-
hedron Letters, https://doi.org/10.1016/j.tetlet.2019.151218