An excited state intramolecular proton transfer dye based fluorescence turn-on probe for fast detection of thiols and its applications in bioimaging

Article abstract of DOI:10.1016/j.saa.2016.12.031

In this study, a new fluorescent probe 2-(2′-hydroxy-5′-N-maleimide phenyl)-benzothiazole (probe 1), was designed and synthesized by linking the excited state intramolecular proton transfer (ESIPT) fluorophore to the maleimide group for selective detectio

Full text of DOI:10.1016/j.saa.2016.12.031

Accepted Manuscript
An excited state intramolecular proton transfer dye based
fluorescence turn-on probe for fast detection of thiols and its
applications in bioimaging
Yun Zhao, Yuanyuan Xue, Haoyang Li, Ruitao Zhu, Yuehong
Ren, Qinghua Shi, Song Wang, Wei Guo
PII:
S1386-1425(16)30755-7
doi: 10.1016/j.saa.2016.12.031
SAA 14844
DOI:
Reference:
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
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7 December 2016
19 December 2016
Please cite this article as: Yun Zhao, Yuanyuan Xue, Haoyang Li, Ruitao Zhu, Yuehong
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dye based fluorescence turn-on probe for fast detection of thiols and its applications in
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authors. Please check if appropriate. Saa(2016), doi: 10.1016/j.saa.2016.12.031
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ACCEPTED MANUSCRIPT
An excited state intramolecular proton transfer dye based
fluorescence turn-on probe for fast detection of thiols and its
applications in bioimaging
a
c
a
a
a
Yun Zhao *, Yuanyuan Xue , Haoyang Li , Ruitao Zhu ,Yuehong Ren ,
a
a
b
Qinghua Shi , Song Wang , and Wei Guo *
a
Department of Chemistry, Taiyuan Normal University, Jinzhong 030619, China.
School of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006,
b
China.
c
School of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006,
China.
*
Corresponding author. Tel./Fax.: +86 351 2275129; +86 351 7011600;
E-mail address: yunzhao@tynu.edu.cn; guow@sxu.edu.cn
Abstract In this study, a new fluorescent probe 2-(2’-hydroxy-5’-N-maleimide
phenyl)-benzothiazole (probe 1), was designed and synthesized by linking the excited
state intramolecular proton transfer (ESIPT) fluorophore to the maleimide group for
selective detection of thiols in aqueous solution. The fluorescence of probe 1 is
strongly quenched by maleimide group through the photo-induced electron transfer
(
PET) mechanism, but after reaction with thiol, the fluorescence of ESIPT
fluorophore is restored, affording a large Stokes shifts. Upon addition of cysteine
Cys), probe 1 exhibited a fast response time (complete within 30 s) and a high
(
signal-to-noise ratio (up to 23-fold). It showed a high selectivity and excellent
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sensitivity to thiols over other relevant biological species, with a detection limit of

8
3
.78 10 M (S/N=3). Moreover, the probe was successfully applied to the imaging of
thiols in living cells..
Keywords ESIPT; PET; Fluorescent probe; Thiols; Cell imaging
1
. Introduction
Thiols, such as cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) play
crucial roles in in physiological and pathological processes. For instance, a deficiency
of Cys causes various health problems, such as retarded growth, lethargy, liver
damage, skin lesions, etc. [13] At elevated levels in plasma, Hcy is a risk factor for
cardiovascular disease, Alzheimer's disease [4]. GSH is played a crucial role in
mammalian and eukaryotic cells [5, 6]. Thus, the development of an effective method
for the detection and quantification of thiols is of great importance in physiological
media for academic research and clinical applications [7, 8]. Many techniques
including, high-performance liquid chromatography [9, 10], electrochemical methods
[
11], liquid chromatography-electrospray tandem mass spectrometry [12], and mass
mpectroscopy [13, 14] have been developed for the detection of thiols. Among the
various detection techniques, fluorescencebased method has become a popular
approach for detection of thiols due to its high sensitivity, high temporal and spatial
resolution, and noninvasiveness [1517]. In particular, reaction-based fluorescent
probes have been rapidly developed in recent years due to their high selectivity with
large spectroscopic changes [1820]. To date, a large number of fluorescent probes
for thiols have been successfully developed by utilizing the strong nucleophilicity of
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the thiol group to the specific reaction sites, such as an alkene or alkyne group with an
electron withdrawing group[2123], sulfonamide[24], sulfonate ester[25, 26],
selenium-nitrogen bond[27, 28], disulfide bond[29, 30], nitroolefin moiety[31] and
other[3238]. Among the reactive sites, maleimides are known to react fairly
selectively with thiols via a Michael addition reaction [35]. Using this general
approach, maleimide derivatives based on triphenylamine [39], chromenoquinoline
[
40], naphthalimide[4143], fluorescein[44], dansyl amide[45], coumarin[46, 47], and
boron-dipyrromethene [48] have been developed as thiols probes. However, there are
still some limitations in these fluorescence probes, including a small Stock’s shifts, a
long response time, and their poor water-solubility. Fluorescent probes with larger
Stokes shifts and a rapid response time can diminish self-quenching and light
scattering interferences, and enhance the detection sensitivity in complicated
biosystems. Thus, it is of considerable interest to design and develop new fluorescent
probes for thiols [4951].
It is well known that dyes based on ESIPT mechanism have a large Stokes shifts
compared to the normal fluorophores, which can be avoided the self-absorption, the
inner filter effect, and improved the fluorescence analysis[5254]. According to these,
we successfully synthesized a new fluorescent probe 1 for thiols, combining
2
-(2'-hydroxyphenyl)-benzothiazole (HBT) derivative which are known to exhibit an
excited state intramolecular proton transfer (ESIPT) from the phenol form to the keto
form results in a large Stokes shift as the fluorophore platform, and a maleimide
group as the reactive site for thiols (Schemes 1 and 2). This is significantly important
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to enhance the Stock’s shifts and sensitivity toward thiols. The probe 1 exhibits a
highly selectivity toward Cys with a larger Stokes shift and a very rapid response time
in an aqueous medium.
Scheme 1 The chemical structure and synthetic routes to the probe 1.
2
. Experimental
2
.1. Materials and Methods
Amino acids, 5-aminosalicylic acid, 2-aminophenol, polyphosphoric acid, maleic
anhydride, and N-ethylmaleimide were purchased from Aladdin Reagents. The
solutions were purchased from Shanghai Experiment Reagent Co., Ltd (Shanhai,
China). All chemicals were used without further purification. All reactions were
magnetically stirred and monitored by thin layer chromatography (TLC). Flash
chromatography was performed using Qingdao Haiyang silica gel (200300 mesh).
The solutions of amino acids and reduced glutathione (GSH) were prepared in
deionized water. The solution of Nethylmaleimide was prepared in EtOH.
Absorption spectra were recorded using a Varian Carry 4000 spectrometer.
Fluorescence spectra were obtained using a Hitachi F-7000 Fluorescence
1
Spectrophotometer, the slit width of 1.0 nm and matched quartz cells. The H NMR
1
3
and C NMR spectra were collected on a BRUKER AVANCE III HD 600 MHz
spectrometer at 600 MHz and 150 MHz NMR spectrometer, respectively. The
following abbreviations were used to explain the multiplicities: s=singlet; d=doublet;
t=triplet; q=quartet; m=multiplet; br=broad. Mass spectra were obtained using a
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Thermo Scientific Q Exactive LC-MS/MS. The fluorescence images were obtained
through EVOS® FLAuto Imaging System.
2
.2. Synthesis and characterization
The synthetic routes to compounds 1 and 2 are shown in Scheme 1. The starting
material 2 was prepared according to the reported procedure. Compound 2 was
prepared by a simple one-step procedure in good yield from the reaction of
2
-aminophenol and 5-aminosalicylic acid in polyphosphoric acid (PPA) as a catalyst
55, 56]. Condensation of 2 with maleic anhydride in refluxing glacial acetic acid
afforded the desired product 1 as a yellow solid. The structures of 1 and 2 were
[
1
13
characterized by ESIMS, H and CNMR and the corresponding spectra are
shown in Supplementary information.
2
.3. Typical procedure for thiols determination
Double distilled water was used to prepare all aqueous solutions. All the
measurements were made according to the following procedure. PBS buffer solution
10 mM, pH 7.4) 2 mL and the stock solution of probe 1 (2.0 mM) 2 μL were mixed
(
in a 1 cm path quartz cuvette, followed by addition of an appropriate volume of thiols
sample solution. The reaction solution was mixed and kept 30 s at room temperature,
which was then transferred to measure UV-vis absorbance within the wavelength
range of 250∼450 nm or fluorescence intensity/spectrum with λex/em = 370/460 nm,
and the excitation slit widths of 5 nm and the emission slit widths of 10 nm. In the
meantime, a blank solution of probe 1 was prepared and measured under the identical
conditions for stable and photostable.
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2
.4. Fluorescence imaging in living cells
The HeLa cell line was provided by Key Laboratory of Chemical Biology and
Molecular Engineering of Ministry of Education. Cells were grown in RPMI 1640
medium supplemented with 10% FBS (Fetal Bovine Serum) and 1% antibiotics at 37
o
C in humidified environment of 5% CO . Fluorescence imaging were acquired
2
through an EVOS® FL Auto Cell Imaging System with a 40× oil immersion objective
lens. Emission was collected at DAPI light channel (λ = 357 nm, λ = 447 nm).
ex
em
2
.5 Theoretical calculation methods
Geometry of probe 1 and probe 1 + Cys were first optimized by the DFT method at
the B3LYP/6-31g* level, then optimized geometries were used time dependent DFT
TDDFT) calculations for understanding the electron excitation process. All these
calculations were performed with Gaussian 09 [57].
(
3
. Results and discussion
3
.1. The sensing properties of probe 1 towards Cys
As the representative of thiols, Cys was used to examine the sensing properties of
probe 1. Firstly, the solubility of probe 1 in aqueous solution was examined by UV
absorption spectra with the increased concentration of probe 1. As shown in Fig. S1,
the absorbance intensity was closely related with the concentration of probe 1 from 0
2
to 12 μM with a good linear relationship (R =0.998) in PBS buffer solution,
suggesting that the solubility of probe 1 in aqueous solution was 12 μM. At the same
time, the molar absorption coefficient of probe 1 at 340nm was measured as ɛ=18900
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
1
1
M cm using the Beer-Lambert Law. In addition, the stability of probe 1 was
investigated using fluorescence spectrum on the aqueous probe solution. After stated
in the PBS solution for 24 hours, the relative fluorescence intensity of probe 1 has no
apparent change (Fig. S2a). Moreover, the photostability of probe 1 was also
evaluated by continuous irradiation for 20 minutes using a 150 W Xe lamp as the light
source, also the relative fluorescence intensity of probe 1 has no apparent change (Fig.
S2b), indicating that probe 1 is sufficient photostability in aqueous solution.
Particularly, it is almost non-fluorescent in PBS buffer solution, in which a PET
process took place from the electron-donating maleimide groups to the
electron-accepting the HBT derivative. The addition of Cys, however, a dramatic
fluorescence intensity increase was observed in a few seconds centered at 460 nm
(Fig. 1), which was in agreement with a typical PET process. The PET quenching
process in probe 1 could be rationalized by density function theory (DFT) and time
dependent DFT (TDDFT) calculations at the B3LYP/6-31g* level [40, 58]. As shown
in Fig. 2 and Table 1, the HOMO of probe 1 was localized on HBT moiety but the
LUMO was located on maleimide part, and according to TDDFT calculations, the
S →S of probe 1 was a forbidden transition due to oscillator strength ( f=0.0016)
0
1
based on the principle of Kasha’s rule [59]. Therefore, the S of probe 1 would be a
1
dark state, the presence of maleimide part induced the non-radiative transitions, and
thus probe 1 was non-fluorescen. In contrast, the allowed S →S transition (f=0.468)
0
1
in the corresponding probe 1 + Cys indicated that the species could be fluorescent.
The results were in agreement with the experimental studies, which could further
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prove the sensing mechanism.
Fig. 1 Changes in absorption spectra (a) and fluorescence spectrum (b) of 1 (2 μM)
measured with and without 3 μM Cys in a PBS buffer solution (10 mM, pH 7.4,
containing 0.1% DMSO).
Fig. 2 View of the frontier molecular orbitals (MOs) of probe 1 and generated from
DFT B3LYP/6-31g* geometry optimization.
Table 1. Selected electronic excitation energies (eV) and oscillator strengths (f),
configurations of the low-lying excited states of probe 1 and probe 1+Cys, calculated
by TDDFT//B3LYP/6-31g*, based on the optimized ground state geometries.
Encouraged by these results, the fluorescence titration studies of probe 1 (2 μM)
towards different amounts of Cys were performed in a PBS buffer solution (10 mM,
pH 7.4, containing 0.1% DMSO). As shown in Fig. 3, the free probe 1 was almost no
fluorescent (Φ = 0.015, with quinine sulfate as a reference) [60]. Upon treatment with
increasing concentrations of Cys, the fluorescence intensity of probe 1 at 460 nm
gradually increased, and reached saturation when the amount of Cys was more than 3
μM. In this case, a ca. 23fold fluorescence enhancement was observed. Moreover, a
linear relationship with the Cys concentration from 0 to 3.0 μM could be obtained
(
Fig. 3b). Based on the definition by IUPAC (C = 3 Sb/m) [61], the detection limit
DL

8
was found to be 3.78 10 M. Upon addition of GSH to probe 1, a similar
phenomenon on fluorescence spectra could be observed (Fig. S3).
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Fig. 3 (a) Changes in fluorescence emission spectrum of probe 1 (2 μM) measured
with various amounts of Cys in a PBS buffer solution (10 mM, pH 7.4, containing 0.1%
DMSO); (b) Fluorescence titration profile (at 460 nm) vs. concentration of Cys in
solution for probe 1.
3
.2 The selectivity of probe 1 for Cys.
Next, we examined the fluorescence spectral changes of probe 1 (2 μM) towards
natural amino acids, including thiols with mercapto groups (GSH, Hcy and Cys), and
those without mercapto groups (Lalanine (Ala), Larginine (Arg), Lasparagine
(Asp), Lglutamine (Gln), Lglutamic acid (Glu), Lglycine (gly), Lhistidine (His),
Lisoleucine (Ile), Lleucine (Leu), Llysine (Lys), Lmethionine (Met),
Lphenylalanine (Phe), Lproline (Pro), Lserine (Ser), Lthreonine (Thr),
Ltryptophan (Trp), Ltyrosine (Tyr), and Lvaline (Val) ). As shown in Fig. 4a, only
thiols with mercapto groups caused a significant enhancement in fluorescence
intensity and the other amino acid samples exhibited no noticeable increase of the
fluorescence signal. At the same time, all the competing amino acids did not interfere
the detection of Cys (Fig. 4b). In addition, the large fluorescence change of probe 1
induced by Cys was also observable by the naked eye. When probe 1 was excited at
3
65 nm using a UV lamp in the presence of 2.0 equiv. of various amino acids, only
Cys caused a strong green fluorescence (Fig. 4c). These results indicated that probe 1
is highly selectivity towards thiols over other amino acids.
Fig. 4 (a) The fluorescence spectra of probe 1 (2 μM) upon addition of 4 μM of Cys
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and various other amino acid in a PBS buffer solution (10 mM, pH 7.4, containing 0.1%
DMSO); (b) Fluorescence response of probe 1 (2 μM) to 3μM of Cys in the solution
containing 10μM of various amino acid; (c) Fluorescence images of probe 1 (2 μM) to
various amino acids (4 μM) in PBS buffer (10 mM, pH 7.4, containing 0.1% DMSO)
on excitation at 365 nm using a UV lamp.
3
.3 Influences of time and pH
We also carried out the effect of reaction time on the fluorescence emission of the
system (Fig. 5). The reaction of probe 1 with Cys reached equilibrium within 60
seconds after the addition of 3μM of Cys, indicating that probe reacted rapidly with
Cys under the experimental conditions. In addition, the probe 1 was stable in a pH
region of 2.0-12.0, and displayed good fluorescence response toward Cys in the
physiological pH range, which was favorable in practical application (Fig. S4). As
described above, probe 1 displayed excellent analytical property comparing with
some other fluorescent probes of recent reports for the detection of Cys. The
comparison data are listed in Table 2, indicating that probe 1 is promising for practical
analysis.
Fig. 5 Timedependent fluorescence intensity changes of probe 1 (2 μM) at 460 nm,
upon addition of 3 μM of Cys. Conditions: PBS buffer (10 mM, pH 7.4, containing
o
0
.1% DMSO) at 25 C.
Table 2. Comparison of probe 1 for the detection of thiols
3
.4 Proposed mechanism
1
0

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To confirm the reaction mechanism, we first performed NMR studies of probe 1 in
1
the presence of 2mercaptoethanol (ME). Compared with the H NMR spectrogram
of probe 1, the signal of original proton(―CH=CH―) at 7.190 ppm disappeared and
appearance of new signals at 4.5-3.3 ppm which were supposed to arise due to the
Michael addition of ME to the electron-deficient alkene groups in probe 1 (Fig.
S5)[62]. We also studied the interaction of substance 2 and Cys using UV-vis
absorption. As shown in Fig S6, 2 did not react with Cys. We reasoned that the
plausible mechanism of the interaction between probe 1 and Cys was presented in
Scheme 2. Further, we performed the HRMS experiment on probe 1 treated with Cys
+
(Fig. 6), where in the peak at m/z = 444.0673 corresponding to [probe 1+Cys + H]
(444.0682 calc.) was clearly observed. These results provide further evidence for the
proposed mechanism.
Scheme 2 The proposed sensing mechanism of probe 1 toward Cys.
Fig. 6 HRMS charts of probe 1 treated with Cys. The peak (m/z) at 444.0673
+
corresponds to [probe 1 + Cys + H] ( Calcd for: 444.0682).
3
.5 Fluorescence imaging for living cells
To value the practical utilities, we further measured the permeability and the
monitoring of probe 1 toward Cys in living cells using an EVOS® FLAuto Cell
Imaging System. The optical window at the DAPI channel (345447 nm) was chosen
as a signal output. As shown in Fig. 6, the HeLa cells were found to have almost no
11

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fluorescence in the channel (Figure 7A). When the cells were incubated with probe 1
(
5μM) for 30 min at 37◦C, they showed weak fluorescence as a result of the fact that
probe 1 binds the cellular biothiols (Figure 7B). When the cells were pretreated with
00μM Cys and then incubated with probe 1 (5μM), they gave a stronger fluorescence
Figure 7C). When the cells were pretreated with Nethylmaleimide (NEM, 1.0mM, a
1
(
thiol blocking reagent) and then incubated with probe 1 (5μM), a remarkable decrease
in fluorescence intensity was observed (Figure 7D). These results suggested that
probe 1 was good cell-membrane permeability and responsive to intracellular thiols.
Fig. 7 A) Fluorescence image of Cys in HeLa cells using probe 1.A) HeLa cells only;
B) HeLa cells incubated with probe 1 (5 μM, 30 min); C) HeLa cells pre-incubated
with Cys (100 μM, 30 min), and then treated with probe (5 μM, 30 min); D) HeLa
cells preincubated with NEM (1 mM, 30 min), and then treated with probe 1 (5 μM,
3
0 min). Emission was collected at DAPI Light Cube (excited at 357 nm). Scale bar:
2
0 μm.
Conclusions
In summary, a new “turnon” fluorescence probe 1 for thiols was exploited by
coupling the reactionbased strategy with ESIPT and PET mechanism. The probe can
highly selectively detect Cys with a fast fluorescence offon response and an
extremely low detection limit. Preliminary fluorescence imaging experiments in cells
indicate its potential to probe biothiols chemistry in biological systems.
Acknowledgements
We would like to thank the Scientific and Technologial Innovation Programs of
1
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Higher Education Institutions in Shanxi (No. 2015173) for financial support.
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Biographies
Yun Zhao received the Ph. D degree from Shanxi University in 2013. Now, he is a
teacher of Department of Chemistry, Taiyuan Normal University. His research
interests include organic synthesis and fluorescent probes.
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Yuanyuan Xue received the Master degree from Shanxi University in 2009. Now, she
is an engineer of Shanxi Analytical Science Academy. Her research interests include
detection and analysis.
Haoyang Li is Professor. Department of Chemistry, Taiyuan Normal University. Her
current research interests are bioorganics, sensors and interaction of fluorescent
material.
Ruitao Zhu is an associate professor in the Department of Chemistry, Taiyuan
Normal University. His current research interests are chemosensors and interactions
of organic macromolecules with biological molecules.
Qinghua Shi is an assistant engineer in the Department of Chemistry of Taiyuan
Normal University. His major is organic chemistry. His current research interests are
calculation chemistry.
Song Wang is an assistant engineer in the Department of Chemistry of Taiyuan
Normal University. His major is organic chemistry. His current research interests are
synthesis chemistry.
Yuehong Ren is Professor, Master. Department of Chemistry, Taiyuan Normal
University. His current research interests are bioorganics, sensors and interaction of
organic macromolecules with sugar.
Wei Guo received the PhD degree from Naikai University in 2002. In the same
year,he joined the School of Chemistry and Chemical Engineering in Shanxi
University. Hebecame professor in 2006. His research interests include
supramolecular chemistry,organic synthesis and medicine synthesis.
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Fig. 1 Changes in absorption spectra (a) and fluorescence spectrum (b) of 1 (2 μM)
measured with and without 3 μM Cys in a PBS buffer solution (10 mM, pH 7.4,
containing 0.1% DMSO).
Fig. 2 View of the frontier molecular orbitals (MOs) of probe 1 and generated from
DFT B3LYP/6-31g* geometry optimization.
Fig. 3 (a) Changes in fluorescence emission spectrum of probe 1 (2 μM) measured
with various amounts of Cys in a PBS buffer solution (10 mM, pH 7.4, containing 0.1%
DMSO); (b) Fluorescence titration profile (at 460 nm) vs. concentration of Cys in
solution for probe 1.
Fig. 4 (a) The fluorescence spectra of probe 1 (2 μM) upon addition of 4 μM of Cys
and various other amino acid in a PBS buffer solution (10 mM, pH 7.4, containing 0.1%
DMSO); (b) Fluorescence response of probe 1 (2 μM) to 3μM of Cys in the solution
containing 10μM of various amino acid; (c) Fluorescence images of probe 1 (2 μM) to
various amino acids (4 μM) in PBS buffer (10 mM, pH 7.4, containing 0.1% DMSO)
on excitation at 365 nm using a UV lamp.
Fig. 5 Timedependent fluorescence intensity changes of probe 1 (2 μM) at 460 nm,
upon addition of 3 μM of Cys. Conditions: PBS buffer (10 mM, pH 7.4, containing
o
0
.1% DMSO) at 25 C.
Fig. 6 HRMS charts of probe 1 treated with Cys. The peak (m/z) at 444.0673
+
corresponds to [probe 1 + Cys + H] ( Calcd for: 444.0682).
Fig. 7 A) Fluorescence image of Cys in HeLa cells using probe 1.A) HeLa cells only;
B) HeLa cells incubated with probe 1 (5 μM, 30 min); C) HeLa cells pre-incubated
with Cys (100 μM, 30 min), and then treated with probe (5 μM, 30 min); D) HeLa
cells preincubated with NEM (1 mM, 30 min), and then treated with probe 1 (5 μM,
3
0 min). Emission was collected at DAPI Light Cube (excited at 357 nm). Scale bar:
2
0 μm.
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Fig. 1 Changes in absorption spectra (a) and fluorescence spectrum (b) of probe 1 (2
μM) measured with and without 3 μM Cys in a PBS buffer solution (10 mM, pH 7.4,
containing 0.1% DMSO).
Fig. 2 View of the frontier molecular orbitals (MOs) of probe 1 and generated from
DFT B3LYP/6-31g* geometry optimization.
Fig. 3 (a) Changes in fluorescence emission spectrum of probe 1 (2 μM) measured
with various amounts of Cys in a PBS buffer solution (10 mM, pH 7.4, containing 0.1%
DMSO); (b) Fluorescence titration profile (at 460 nm) vs. concentration of Cys in
solution for probe 1.
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Fig. 4 (a) The fluorescence spectra of probe 1 (2 μM) upon addition of 4 μM of Cys
and various other amino acid in a PBS buffer solution (10 mM, pH 7.4, containing 0.1%
DMSO); (b) Fluorescence response of probe 1 (2 μM) to 3μM of Cys in the solution
containing 10μM of various amino acid; (c) Fluorescence images of probe 1 (2 μM) to
various amino acids (4 μM) in PBS buffer (10 mM, pH 7.4, containing 0.1% DMSO)
on excitation at 365 nm using a UV lamp.
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Fig. 5 Timedependent fluorescence intensity changes of probe 1 (2 μM) at 460 nm,
upon addition of 3 μM of Cys. Conditions: PBS buffer (10 mM, pH 7.4, containing
o
0
.1% DMSO) at 25 C.
Fig. 6 HRMS charts of probe 1 treated with Cys. The peak (m/z) at 444.0673
+
corresponds to [probe 1 + Cys + H] ( Calcd for: 444.0682).
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Fig. 7 A) Fluorescence image of Cys in HeLa cells using probe 1.A) HeLa cells only;
B) HeLa cells incubated with probe 1 (5 μM, 30 min); C) HeLa cells pre-incubated
with Cys (100 μM, 30 min), and then treated with probe (5 μM, 30 min); D) HeLa
cells preincubated with NEM (1 mM, 30 min), and then treated with probe 1 (5 μM,
3
0 min). Emission was collected at DAPI Light Cube (excited at 357 nm). Scale bar:
2
0 μm.
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Scheme 1 The chemical structure and synthetic routes to the probe 1.
Scheme 2 The proposed sensing mechanism of probe 1 toward Cys.
Scheme 1 The chemical structure and synthetic routes to the probe 1.
Scheme 2 The proposed sensing mechanism of probe 1 toward Cys.
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Table 1. Selected electronic excitation energies (eV) and oscillator strengths (f),
configurations of the low-lying excited states of probe 1 and probe 1+Cys, calculated
by TDDFT//B3LYP/6-31g*, based on the optimized ground state geometries.
Table 2. Comparison of probe 1 for the detection of thiols
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