Selective detection of Cys and GSH by using one fluorescent probe at two excitation wavelengths

Article abstract of DOI:10.1016/j.tetlet.2020.152462

Selective detection of Cys and GSH by using one fluorescent probe with two different excitation wavelengths is developed based on a naphthyl-coumarin aldehyde probe. Mechanistic studies indicate that the distinctively different fluorescent responses of the probe toward Cys and GSH at the two excitation wavelengths are due to two different reaction pathways that generate different products. The probe has been used to detect Cys and GSH in HeLa cells by using two emission channels.

Full text of DOI:10.1016/j.tetlet.2020.152462

Tetrahedron Letters xxx (xxxx) xxx
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Selective detection of Cys and GSH by using one fluorescent probe
at two excitation wavelengths
Yintang Xiao a,#, Kai Guo a,#, Jun Wei a,#, Xiaowei Gao , Dong Yi , Yang Li , Xiaoqi Yu , Chun Zhang a,
⇑
,
Qin Wang
a
a
a
a
b,
⇑
a,
⇑
Key Laboratory of Medical Electrophysiology, Ministry of Education, School of Pharmacy, Southwest Medical University, Luzhou 646000, China
Key Laboratory of Green Chemistry and Technology, Ministry of Education, College of Chemistry, Sichuan University, Chengdu 610064, China
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Selective detection of Cys and GSH by using one fluorescent probe with two different excitation wave-
lengths is developed based on a naphthyl-coumarin aldehyde probe. Mechanistic studies indicate that
the distinctively different fluorescent responses of the probe toward Cys and GSH at the two excitation
wavelengths are due to two different reaction pathways that generate different products. The probe
has been used to detect Cys and GSH in HeLa cells by using two emission channels.
Ó 2020 Elsevier Ltd. All rights reserved.
Received 15 July 2020
Revised 1 September 2020
Accepted 10 September 2020
Available online xxxx
Keywords:
Selective detection
Fluorescent probe
Amino acids
Mechanistic study
Cell imaging
Introduction
rapid fluorescence response toward Cys or GSH respectively [5].
Intracellular thiols including cysteine (Cys), glutathione (GSH)
and homocysteine (Hcy) play crucial roles in many physiological
Zhang et al reported a dual-emission fluorescent probe for the
detection of H S and GSH by introducing a chlorine on the 4-posi-
2
and pathological processes. Normal levels of Cys (30–200
l
M)
tion of coumarin [6]. Niu et al found a BODIPY-coumarin-based flu-
orescent probe for the selective detection of Cys and GSH based on
the combination of aromatic substitution and dual-fluorophore
fragmentation [7]. Yin et al designed a coumarin-based fluorescent
probe for discriminative detection and cellular imaging of Cys, GSH
and Hcy, and subsequently developed a dual-site fluorescent probe
for visualizing the metabolism of Cys in living cells [8]. Liu et al
maintain the synthesis of GSH (1–10 mM) as well as various pep-
tides and act as the source of sulfide in human metabolism [1]. The
deficiency and excess amounts of Cys and GSH are associated with
diseases including Parkinson’s disease, Alzheimer’s disease, slowed
growth, edema and liver damage [2]. There are extensive studies
conducted to detect these molecules. Although a number of fluo-
rescent probes have been developed for highly selective detection
of Cys, Hcy and GSH from other amino acids in recent years [3],
most of them couldn’t distinguish these biologically significant
biothiols from each other because of their similar structures and
reactivity especially in living cells. Therefore, fluorescent probes
that can selectively detect between Cys and GSH are of great
importance and have attracted particular interest of researchers.
A pioneering study about fluorescence sensing of Cys and GSH
from different emission channels was reported by Guo’s group
developed
a coumarin-fluorophore probe for distinguishing
amongst biothiols, including Cys, Hcy and GSH based on different
cascade reactions [9]. Qian et al reported a fluorescence probe with
coumarin as the fluorophore and maleimide as the receptor for
highly sensitive detection of Cys over GSH or Hcy [10]. Yu et al
reported a new dual-site fluorescent probe for discriminative
respond to Cys through two emission channels and imaging bioth-
iols in living cells [11]. Other groups also developed some specific
probes for these biothiols [12].
[
4]. Liu et al reported highly sensitive coumarin-based probe for
Recently, we reported a new method to determine both the con-
centration and enantiomeric composition of a chiral substrate with
a single fluorescent probe (1) [13]. Mechanistic study on the
BINOL-coumarin-based probe 1 indicated that the distinctively dif-
ferent fluorescent responses of this probe at the two excitation
wavelengths are due to two different reaction pathways,
that is, the 1,2-addition of probe can generate an iminium ion
⇑
Corresponding authors at: School of Pharmacy, Southwest Medical University,
Luzhou 646000, China (Q. Wang).
E-mail addresses: xqyu@scu.edu.cn (X. Yu), zc83good@126.com (C. Zhang),
wq_ring@hotmail.com (Q. Wang).
#
These authors contributed equally to this work.
https://doi.org/10.1016/j.tetlet.2020.152462
0
040-4039/Ó 2020 Elsevier Ltd. All rights reserved.
Please cite this article as: Y. Xiao, K. Guo, J. Wei et al., Selective detection of Cys and GSH by using one fluorescent probe at two excitation wavelengths,
Tetrahedron Letters, https://doi.org/10.1016/j.tetlet.2020.152462

Y. Xiao et al.
Tetrahedron Letters xxx (xxxx) xxx
intermediate with extended conjugation to the naphthyl-coumarin
unit leading to the long wavelength emission, while the 1,4-addi-
tion of probe can disrupt the conjugation between the naphthalene
ring and the coumarin unit contributing to the short wavelength
emission [13b]. Inspired by the structure and property of 1, we
have designed a new fluorescent probe by directly coupling a
naphthyl group with a coumarin aldehyde to generate compounds
2
a and 2b. We have discovered that compound 2a can be used to
carry out selective detection of Cys and GSH by exciting at two dif-
ferent wavelengths. Herein, these results are reported.
The naphthyl-coumarin compound 2a was prepared by mixing
the coumarin chloride
3 with 1-bromo-2-(methoxymethoxy)
n
naphthalene 4 in the presence of CuCl and BuLi in DMF and THF
at À78 °C which gave 2a in 10% isolated yield (Scheme 1). Com-
pound 2b without the methoxymethyl group was synthesized
from another synthetic route (Scheme S1d).
The spectroscopic properties of probe 2a were investigated in
PBS/0.5% DMSO solution (pH = 7.4). As shown in Fig. 1a, the UV–
vis spectrum of 2a (black line) has a maximum absorption peak
at 425 nm. When excited at k = 370 nm, 2a showed weak fluores-
cence at 470 nm with low quantum yield (Fig. 1b and Table S1). We
then measured the fluorescence responses of 2a toward 21 com-
mon amino acids (including Hcy) and GSH.
Scheme 1. Synthesis of the naphthyl-coumarin probe 2a.
Fig. 2a gives the fluorescence spectra of 2a with amino acids in
PBS solution. When treated with Cys (20.0 equiv), large fluores-
cence enhancement at k = 470 nm was observed with upon excita-
tion at 370 nm. Under the same conditions, about 4-fold and 3-fold
fluorescence enhancements were observed for Hcy and GSH at
4
70 nm and 468 nm respectively, while the responses of 2a
towards other non-biothoil amino acids were weak. Fig. 2b shows
the fluorescent response of 2a at 470 nm versus the reaction time.
À5
When 2a (1.0 Â 10 M) was treated with Cys (20.0 equiv), the flu-
orescence enhancement reached maximum after 60 min which
remained stable over a prolonged time. At the same time, the
responses of 2a toward Hcy and GSH lagged far behind Cys
(
Fig. 2b). Effect of the concentration of Cys on the fluorescent
À5
Fig. 1. (a) UV À vis spectra of 2a (1.0 Â 10 M in PBS / 0.5% DMSO, pH = 7.4) with
response of 2a was measured (Fig. S2b). After the addition of more
than 20.0 equiv of Cys, the fluorescence enhancement at
k = 470 nm slowed down gradually. The selective fluorescence
response of 2a at 470 nm towards Cys in the presence of the com-
peting analytes was conducted. As shown in Fig. S2c, all of the
competing analytes tested have virtually no influence on the fluo-
rescence detection of Cys except for GSH. GSH might form stable
amino-substituted covalent complexes with 2a.
2
0.0 equiv of Cys and GSH (2a: kmax = 425 nm; 2a + Cys: kmax = 370 nm; 2a + GSH:
max = 485 nm). (b) Fluorescence spectrum of 2a (1.0 Â 10 M in PBS/0.5%DMSO,
À5
k
pH = 7.4, kexc = 370 nm) .
We then examined the emission response of 2a upon addition
of GSH and 21 common amino acids by exciting at a longer wave-
length of 485 nm because of the significant red-shift in its UV–vis
spectrum (green line of Fig. 1a), which means potential application
in the fluorescent imaging in living systems. As shown in Fig. 3a,
the probe and its mixture with 21 common amino acids are almost
non-emissive. Addition of 20.0 equiv of GSH to a solution of 2a eli-
cited a big fluorescence enhancement (ca.500-fold) at 542 nm.
Effect of reaction time on the fluorescent response of 2a toward
À5
Fig. 2. (a) Fluorescence spectra of 2a (1.0 Â 10 M in PBS / 0.5% DMSO, pH = 7.4)
with 20.0 equiv of Cys, GSH, Hcy, Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys,
GSH at 542 nm was measured (Fig. S3a). When the probe 2a
Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val (k = 370 nm, slit: 10/5 nm). (b) Fluorescence
exc
À5
À5
(
1.0 Â 10 M) was treated with GSH (20.0 equiv), the fluorescence
intensity of 2a (1.0 Â 10 M) at 470 nm with Cys, Hcy and GSH (kexc = 370 nm,
slit:10/5nm).
enhancement reached maximum at 90 min. Fig. S3b shows the flu-
orescent response of 2a at 542 nm versus the concentration of GSH.
After addition of 12 equiv of GSH, the fluorescence enhancement
reached saturation. The selective fluorescence response of 2a at
to understand the detection mechanisms of the probe toward Cys
5
42 nm towards GSH in the presence of the competing analytes
and GSH, we studied the reaction of 2a with Cys and GSH by 1
H
was conducted. As shown in Fig. 3b, all of the competing analytes
tested have little influence on the fluorescence detection of GSH
except Cys and Hcy which might consume the probe by reaction.
Thus, the probe 2a not only showed a sensitive response at
NMR and mass spectroscopic analyses. As shown in Fig. S4a, the
reaction of Cys with 2 is complex with the formation of multiple
products. Because 2a exists as a racemic mixture, its reaction with
Cys is not only complicated by various reaction pathways, but also
by the formation of various diastereomeric compounds.
Multiple peaks appeared around d 5.9 suggest the formation of
4
70 nm toward Cys but also had an excellent selectivity toward
GSH at 542 nm without interference of other amino acids. In order
2

Y. Xiao et al.
Tetrahedron Letters xxx (xxxx) xxx
Fig. 3. (a) Fluorescence spectra of 2a (1.0 Â 10À5 M in PBS / 0.5% DMSO, pH = 7.4)
with 20.0 equiv of GSH and 21 common amino acid respectively (kexc = 485 nm, slit:
2
0/5nm). (b) The selective fluorescence response of 2a at 542 nm towards GSH in
the presence of the competing analytes (Aa: Cys, Hcy, Ala, Arg, Asn, Asp, Gln, Glu,
Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val).
the diastereomeric products like 5 (Scheme 2), with a methine pro-
ton (H
is supported by the significant blue-shift for the UV absorption of
a from 425 nm to 370 nm upon reaction with Cys (blue line of
a
) in the newly formed thiazolidine rings (Fig. S4b) [14]. This
Scheme 2. Proposed thiazolidine product structures 5a, b for the reaction of 2a
with Cys.
2
Fig. 1a) since formation of a thiazolidine reduces the conjugation
of the coumarin unit of 2a. The ESI-MS spectrum in Fig. S4f gives
+
a peak at m/z = 507.2 for the Cys + 2a system [M + H] consistent
with the formation of 5a (calcd for 5a + H: 507.2).
Intramolecular hydrogen bonding as shown in 5a and 5b in
Scheme 2 might increase the structure rigidity of the compound,
leading to the enhanced fluorescence at 470 nm. Conformer search
of 5a and 5b was carried out by Avogadro (Fig. S4c and S4d). The
conformation of 5a with one hydrogen bond is easier to form than
that of 5b with two hydrogens based on potential steric hindrance
between coumarin unit and naphthol plane bonded by CAC, as
well as its subsequent extension of the thiazolidine and methoxy-
methoxy group respetively. The four diastereomers of 5a were
optimized by using Gaussian09D01. As shown in Fig. S4c, there is
little energy difference for the four structures, and the diastere-
omer of 5a from (S)-2a and L-Cys is the optimized structure among
the lowest energy ones by frequency analysis.
The reaction of 2a with GSH gave one major product together
with the disappearance of the aldehyde group as shown by the
H NMR spectra and COSY spectrum in Fig. 4 and Fig. S5a. A minor
Fig. 4. 1H NMR spectra of the reaction of probe 2a (2.0 mM in 475
with GSH (190 mM in 25 L D O) from 10 to 80 min.
6
lL DMSO d )
1
l
2
product was observed to still contain an aldehyde signal. When
excess amount of GSH (5 equiv) was added to the solution of probe
2
a, the aldehyde proton signal of 2a gradually disappeared with
the quick appearance of an imine proton signal at 8.55. The signal
of –OCH O- (5.09 ppm) in the probe 2a was also shifted downfield
2
to 5.25 ppm, which might be attributed to generate a macrocyclic
iminium ion product 6 with intramolecular hydrogen bonds.
Using the same conformer search method as done for 5,
revealed that it is difficult for 6 to form two hydrogen bonds
between the two oxygen atoms of the MOM-protected naphthol
and the hydrogen of NAH from GSH (Fig. S5b in SI). As shown in
Scheme 3, the optimized structure of 6 with the lowest energy gave
three intramolecular hydrogen bonds. The mass spectrum of the
reaction mixtures is consistent with the formation of 6 as the
Scheme 3. Proposed product 6 for the reaction of 2a with GSH.
+
major product with a [M + H] signal at m/z = 693.4 (calcd for
6
+ H: 693.2) as shown in Fig. S5e.
10
lM (Fig. S6a). The confocal fluorescence imaging experiments
When compound 2b without the methoxymethyl group was
used to interact with 21 common amino acids and GSH, no signif-
icant fluorescence enhancement and selectivity were observed
were conducted with HeLa cells in the pH 7.4 HBSS system (Hanks’
2
+
2+
Balanced Salt Solution with Ca , Mg ) in the presence of probe 2a
(Fig. S6b). As shown in Fig. 5, the HeLa cells precultured with
0.05 mM NEM (N-ethyl maleimide, a biological thiol quencher)
(
Fig. S5f). This supports the formation of intramolecular hydrogen
bonding from the reaction of 2a with Cys and GSH as shown in
Schemes 2 and 3.
To evaluate the applicability of probe 2a in biological systems,
we measured the CCK-8 assay with HeLa cells, and the results
showed minimal cytotoxicity of probe 2a at a concentration of
and then 6 lM probe 2a in HBSS buffer displayed dim emission
in both the blue and green channels (A and C) as the control proce-
dures. Further incubation with exogenous Cys induced distinct flu-
orescence emission in the two emission channels (B and D). As for
GSH, these cells displayed strong fluorescence emission in the
3

Y. Xiao et al.
Tetrahedron Letters xxx (xxxx) xxx
cells by using two emission channels respectively at two different
excitation wavelengths. Thus, 2a is a promising fluorescent probe
for application in cell study.
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.
Acknowledgments
This work was supported by the Sichuan Science and Technol-
ogy Program (2019JDTD0016) and the State Scholarship Fund
(
201708510004) from China Scholarship Council.
Fig. 5. Confocal fluorescence image of HeLa cells. The white bar in the right corner
Appendix A. Supplementary data
shows 25
l
m: Using the control procedures, the cells were preincubated with
0
.05 mM NEM for 30 min and further incubated with HBSS of pH 7.4 in the presence
l
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.tetlet.2020.152462.
of 6
M probe 2a for 60 min (A, C); The cells were preincubated with 0.05 mM NEM
M Cys in HBSS of pH 7.4 for 60 min, and
probe 2a for 60 min (B, D); The cells were
preincubated with 0.05 mM NEM for 30 min and then incubated with 60
GSH in HBSS of pH 7.4 for 60 min, and further incubated with 6 M probe 2a for
0 min (E, F).
for 30 min and then incubated with 60
l
further incubated with
6
lM
lM
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