ESIPT-based fluorescent probe for cysteine sensing with large Stokes shift over homocysteine and glutathione and its application in living cells

Article abstract of DOI:10.1039/c9nj01259a

Cysteine (Cys) is an important amino acid containing a sulfhydryl group, and it is involved in cellular growth and an abnormal level of Cys in vivo is suggestive of many diseases. Hence, development of fluorescent probes for intracellular Cys detection an

Full text of DOI:10.1039/c9nj01259a

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Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
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ESIPT-based fluorescent probe for cysteine sensing with large
Stokes shift over homocysteine and glutathione and its
application in living cells
Received 00th January 20xx,
Accepted 00th January 20xx
Peng Zhanga, Yuzhe Xiaoa, Qian Zhanga, Zixuan Zhanga, Hongwei Yub and Caifeng Dinga*
DOI: 10.1039/x0xx00000x
Cysteine (Cys) is an important amino acid containing a sulfhydryl group, which is involved in cellular growth, and Cys of
abnormal level in vivo is suggestive of many diseases. Hence, development of fluorescent probes for intracellular Cys
detection and imaging is of pivotal importance. So far, a number of fluorescence probes have been developed for Cys
detection successfully. However, many of them could hardly discriminate Cys from Hcy, GSH or other reactive sulfur
species (RSSs) and their emission wavelength of the probes is mainly located in ultraviolet/short region with small Stokes
shifts (<100 nm). Herein, based on the impressive fluorescent properties of ESIPT progress, a new fluorescent probe
named ABT-MVK is synthesized and used to detect Cys. This probe utilizes a typical ESIPT dye (HBT) as the fluorophore and
an acrylate group as ESIPT blocking agent as well as recognition unit. Cleavage of acrylate moiety can be achieved by Cys
rapidly and specifically in aqueous buffer, which leads to restoring ESIPT process and endows the probe with a “Turn ON”
fluorescent detection procedure with large Stokes shifts (ca. 225 nm) for Cys with superior selectivity over various
potential interferences, including Hcy, GSH, Na2S or NaHSO3. The detection limit for Cys was calculated to be as low as 19
nM and intracellular bio-imaging of Cys by this probe was successfully applied in living cells, indicative of great potential
for biological applications.
handy and valid method for Cys sensing and imaging in living
system is crucial.
Introduction
In recent years, detection of RSSs has gained considerable
Intracellular reactive sulfur species (RSSs), including hydrogen
sulfide, thiols, persulfides, polysulfides and S-modified cysteine
adducts such as S-nitrosothiols and sulfenic acids, are generally
considered to be sulfur-containing biomolecules, which play
attention, and available techniques at present for bio-thiols
14, 15
detection include colorimetric method,
electrochemical
16-18
19-21
method,
mass spectrometry
and high performance
liquid chromatography (HPLC) 22-24. However, these detection
methods require the living biosamples to be post-mortem
processed. Thus, they are destructive and not suitable for
monitoring biothiols in native biological environments. Among
the present detection techniques, fluorescent analysis is
particularly attractive, contributed by its facile design, high
essential roles in mediating physiological processes and
1-3
balancing biological systems.
Clinical studies demonstrated
that even though structural frameworks of these RSSs are
similar, they are related to different physiological and
4-8
pathological processes.
Changes in the levels of these RSSs
are linked to many diseases. For example, cysteine (Cys), as an
important amino acid containing sulfhydryl group, plays vital
roles in many significant cellular functions. 9, 10 Cys (30–200 μM)
of normal levels serves as the source of sulfide in protein
25-
sensitivity, low budget, and great feasibility for bioimaging.
32 Although a series of fluorescent schemes have been built up
for fluorescent detection of amino acid and biothiols until now,
33-37
most of them exhibited low selectivity for Cys.
A major
11
synthesis, detoxification and human metabolism. However,
problem for those reported probes is that they could barely
discriminate Cys from other RSSs such as homocysteine (Hcy),
Cys of abnormal levels in living cells is usually associated with
certain diseases including skin lesions, rheumatoid arthritis,
liver damage and Parkinson's disease. 12-13 Hence, developing a
-
glutathione (GSH) and even S2-, HSO3 , as these biothiols
possess similar structures and reactivity. Besides, the higher
38
intracellular level of GSH (1-10 mM) than that of Cys
amplified the difficulty of specific detection for Cys over GSH.
Notably, these biothiols play different pivotal biological roles,
a.Key Laboratory of Optic-electric Sensing and Analytical Chemistry for Life Science,
MOE; Shandong Key Laboratory of Biochemical Analysis; Key Laboratory of
Analytical Chemistry for Life Science in Universities of Shandong; College of
Chemistry and Molecular Engineering. Qingdao University of Science and
Technology, Qingdao 266042, PR China. E-mail: dingcaifeng2003@163.com.
b.Qingdao Municipal Center for Disease Control & Prevention, Qingdao 266033, PR
China.
so clear discrimination of Cys from Hcy and GSH would make
39, 40
sense to exploring their complicated function.
In fact, up
to now only a few fluorescent probes were reported to be able
Electronic Supplementary Information (ESI) available: fluorescence, absorption and
NMR spectral profiles. See DOI: 10.1039/x0xx00000x
to detect Cys with improved selectivity over Hcy, GSH, and so
41-46
on.
Nevertheless, many of the reported probes generally
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display some disadvantages such as tedious synthesis routes
and small Stokes shifts (< 100 nm). Therefore, it has been of
focus and difficulty to construct fluorescence probes with
superior properties for the detection of Cys.
View Article Online
DOI: 10.1039/C9NJ01259A
6
ESIPT-active fluorescent dyes have been widely used to
construct fluorescent probes with large Stokes shift. 47-51 2-(2′-
Hydroxyphenyl)-benzothiazoles (HBT), as a typical class of
ESIPT fluorescent dyes, have been utilized as the scaffold to
design fluorescent probes for the detection of many species
with a large Stokes shift. Particularly, extension of the π-
conjugation of HBT at the ortho- or para-position of the phenol
can afford fluorescent probes with red/NIR emission, which
made the probes more suitable for biological imaging. 52, 53
In view of the aforementioned reasons, we report a facile
prepared HBT-based fluorescent probe (ABT-MVK) with large
Stokes shift and high selectivity for Cys sensing in this work, in
which an acrylate group was introduced as the reaction site for
Cys and HBT acted as an ESIPT active group. In order to achieve
the redder emission, conjugation of HBT was prolonged at the
ortho- position of the phenol moiety by Aldol reaction. Notably,
our research found that this probe governed several
impressive sensing properties compared to the reported Cys
probes (Table S1). First and the most important of all, the
probe is highly selective for Cys over other diverse anions,
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Scheme 1. Synthesis route of ABT-MVK.
negligible fluorescence (“off” state) as a result of the
quenching effect by acrylate group. The spectroscopic
response of ABT-MVK (10 μM) to Cys was examined under this
condition. After addition of 40 μM Cys, the absorption
centered at 290 nm decreased accompanied by a new
absorption at 370 nm originating from of n-π transition with an
isosbestic point at 334 nm (Figure 1a). Accordingly, the
fluorescence intensity at 595 nm increased by 73-fold with a
quantum yield of 0.10 (“on” state) when it was excited at 370
nm (Figure 1b), exhibiting a large Stokes shift of ca. 225 nm.
Based on the above principles, a simple visible-light-excitable
ESIPT-based probe for selective “turn-on” detection of Cys in
aqueous solution was thereby developed.
-
cations and amino acids including Hcy, GSH, S2- and HSO3 .
Sensing condition optimization
Second, it offers a rapid (within a few minutes) and sensitive
detection process for Cys in aqueous buffer with a red
fluorescence “turn-on” signal output and a relatively low
detection limit at nanomolar level (19 nM). Third, this probe is
simple with easy and convenient synthesis, readily available
and visible-light-excitable with a large Stokes shift (ca. 225 nm),
and can be used for fluorescence imaging of exogenous and
endogenous Cys in living cells with low cytotoxicity. All these
merits indicate that the new probe appears to be a rather
promising fluorescent probe for Cys.
To construct a sensitive fluorescent probe for Cys, the sensing
conditions usually need to be optimized. The effect of pH on
the emission intensity of ABT-MVK and the reaction solutions
of ABT-MVK toward RSSs was first evaluated. 10 mM PBS
buffer solutions of pH ranging from 5.5 - 9.5 were investigated.
Referring to the results shown in Figure 2, for ABT-MVK only,
the emission intensity at 595 nm did not change within the
investigated pH range, indicating that ABT-MVK showed
relative stability under the experimental pH conditions. While
ABT-MVK was treated with 40 μM Cys, significant fluorescent
enhancement at 595 nm could be observed in the pH range of
7.0-9.0. When it was applied to other biothiols (Hcy or GSH) of
the same concentration, the fluorescence of ABT-MVK hardly
Results and discussion
Design and synthesis of probe ABT-MVK
5000
To design a feasible fluorescence probe for detecting Cys
selectively with larger Stokes shift, the traditional ESIPT active
group HBT was employed as fluorophore framework, to which
an acrylate group was introduced as the binding site, and its
conjugation was prolonged at the ortho- position of the phenol
by Aldol reaction to achieve a red emission. The probe was
synthesized by simple reaction of HBT-MVK and acryloyl
chloride in the presence of triethylamine in CH2Cl2 (Scheme 1),
it was fully characterized by standard spectroscopic techniques
0.15
(a)
(b)
ABT-MVK
ABT-MVK+Cys
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0.10
0.05
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including H NMR, 13C NMR and HR-MS. It is noteworthy that
the preparation could be achieved readily and tedious
purification procedures are avoided.
The optical property of the ESIPT-based fluorescent probe
ABT-MVK and the feasibility study was first investigated. As
shown in Figure 1, 10 μM ABT-MVK in PBS buffer (10 mM, pH
8.0) shows a maximum absorption at 290 nm and emits
320 400 480 560 480
Wavelength / nm
560
640
Fig. 1. Absorption (a) and fluorescence (b) spectra of ABT-MVK
without (gray line) and with (cyan line) 40 μM Cys in 10 mM
PBS buffer of pH 8.0. [ABT-MVK] = 10 μM, λex = 370 nm.
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Cys over Hcy, GSH, Na2S and NaHSO3, indicatingViietws AprtoictleeOnntliinael
usage for selective Cys detection in biosystems.
probe
Cys
Hcy
DOI: 10.1039/C9NJ01259A
Recognition performance of ABT-MVK for Cys
GSH
Response of ABT-MVK towards Cys of varying concentrations
was investigated in 10 mM PBS buffer pH 8.0. ABT-MVK alone
was almost no emission because of the quenchment of
acrylate group. However, upon increasing concentration of Cys
from 0 to 40 μM, a sensitive fluorescent response toward Cys
was observed (Figure 4a), by a rapid enhancement of the red
emission with a maximum wavelength at 595 nm. When 15
μM Cys was added, the fluorescence intensity of ABT-MVK at
595 nm increased by ca. 73-fold comparing with that of the
original ABT-MVK solution (Figure 4a inset). It is
understandable that Cys could effectively eliminate the acrylic
ester moiety in ABT-MVK to generate the chromophore HBT-
MVK with red emission based on ESIPT process. Job plots
showed that ABT-MVK to Cys stoichiometry was 1:1 (Figure S6),
which also confirmed the abovementioned reaction
mechanism. The fluorescence intensity at 595 nm increased
linearly with Cys concentration over 0 to 15 μM (y=309.6x-2.06,
R2=0.995, Figure 4a inset and Figure S7), 54, 55 giving a detection
limit of 19 nM in terms of 3σ/k, in which σ is the standard
deviation of 11 blank measurements and k is the linear slope
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5.5
6.0
6.5
7.0
7.5
pH
8.0
8.5
9.0
9.5
Fig. 2. Plots of intensity of ABT-MVK at 595 nm without (black
squares) or with 40 μM Cys (red dots), Hcy (blue triangles) and
GSH (gray triangles) versus solution pH from 5.5 to 9.5
incubated for 10 min. Solution pH was tuned by 10 mM
phosphate buffer. [ABT-MVK] = 10 μM, λex = 370 nm.
changed. These results implied that ABT-MVK could be applied
to detect Cys in physiological circumstances with high
selectivity.
To obtain the appropriate reaction time of ABT-MVK and
Cys, time-dependent fluorescence of ABT-MVK (10 μM) with 4
equivalents Cys was investigated by measuring fluorescence
intensity changes at 595 nm every 1 minute. As shown in
Figure 3, fluorescence intensity at 595 nm increased
immediately and levelled off within 10 minutes after Cys was
added (Figure 3a). However, free ABT-MVK (10 μM) emission
was negligible during the same observation time (Figure S5).
The results suggest that ABT-MVK could rapidly respond to Cys.
In the vivid contrast against Cys, under the same conditions,
fluorescence changes of ABT-MVK upon addition of other RSS
(Hcy, GSH, Na2S or NaHSO3) are almost negligible within
observation time in 24 minutes (Figure 3b). As a result, it is
affirmed that ABT-MVK shows a highly selective response to
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10 12 14
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[Cys] / M
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Wavelength / nm
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(a)
(b)
24 min
0 min
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Cys
Hcy
GSH
Na2S
Cys
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GSH
Na2S
NaHSO3
NaHSO3
480
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5
10 15 20 25
Time / min
0
10
20
30
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Concentration / 
Wavelength / nm
Fig. 4. (a) Fluorescence spectra of ABT-MVK in 10 mM PBS
buffer of pH 8.0 in the presence of increasing concentration of
Cys from 0 to 40 μM. (b) Plots of intensity at 595 nm of ABT-
MVK versus RSSs concentration from 0 to 40 μM. [ABT-MVK] =
10 μM, λex = 370 nm.
Fig. 3. (a) Time-dependent fluorescence spectra of ABT-MVK
incubated from 0 to 24 min upon addition of 40 μM Cys in 10
mM PBS buffer (pH =8.0). (b) Intensity of ABT-MVK at 595 nm
after addition 40 μM RSSs from 0 to 24 min in 10 mM PBS
buffer (pH =8.0). [ABT-MVK] = 10 μM, λex = 370 nm.
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of the dependence of intensity of ABT-MVK at 595 nm versus proton signals in aromatic region are consistentVwiewitAhrtictlheaOtnlionef
Cys concentrations.56 The above results indicate that ABT-MVK HBT-MVK. The above results supported tDhOeI:p1r0o.1p0o39se/Cd9NreJa01c2t5io9nA
can be readily applied for quantitative detection of Cys with mechanism between ABT-MVK and Cys.
high sensitivity in aqueous medium.
The selectivity and competition studies
Proposed mechanism of ABT-MVK with Cys
To examine the high selectivity of ABT-MVK toward Cys, the
The fluorescent enhancement of ABT-MVK caused by Cys was effect of other biologically relevant analytes was then
mainly due to the eliminated acrylic ester moiety. The reaction evaluated. The fluorescence spectrum of HBT-MVK was
between biothiols and acrylate group has been well studied so measured in the presence of various anions (F-, Cl-, I-, AcO-,
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-
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far.
Referring to these reported literatures and our NO3 , CO3 , S2- and HSO3 as shown in Figure 6a), cations (Na+,
experimental results, proposed detection mechanism of probe K+, Mg2+, Ca2+, Ni2+, Zn2+, Co2+, Pb2+, Fe3+, Al3+ and Cu2+ as
ABT-MVK for Cys was shown in Scheme 2. The reaction first shown in Figure 6b) and amino acids (Ala, Asp, Gln, Glu, Gly,
underwent by a conjugate addition of Cys to α, β-unsaturated His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Val, Hcy, GSH and Cys as
carbonyl group of ABT-MVK to generate the corresponding shown in Figure 6c). It was found that only addition Cys
thioether, a transient intermediate which could subsequently influenced the emission of ABT-MVK. Excess amount (10
proceed an intramolecular cyclisation to afford the free equivalents) of those foreign compounds did not change the
chromophore HBT-MVK. As shown in the above experiments, fluorescence of ABT-MVK at 595 nm, further confirming the
ABT-MVK exhibits high selectivity for Cys over GSH and Hcy. It high selectivity to Cys. Moreover, these results clearly showed
could be illustrated by the fact that Cys exhibits faster reaction that ABT-MVK was highly selective for Cys over Hcy, GSH, Na2S
kinetics than GSH and Hcy in the process of intramolecular and NaHSO3, which deserves considerable attention, because
cyclisation, because the formation of seven-membered ring so far highly selective fluorescent probes for Cys are relatively
product was more favorable in kinetics. To prove the rare, as mentioned above. As for the sensing strategy present
speculation, HR-MS spectrometry analysis was displayed, herein, the Michael addition/cyclisation procedure between
signal at m/z 310.0886 (calcd. 310.0896) for compound HBT- acrylic ester and Cys generally exhibited faster reaction
61-64
MVK ([C18H15NO2S+H]+) in the mixture of ABT-MVK/Cys was kinetics than Hcy and GSH.
The competitive experiments
found, that matched well with the theoretical simulation were also implemented to investigate the fluorescent behavior
(Figure S8). of ABT-MVK with Cys in the presence of various species
1
Moreover, H NMR spectra of ABT-MVK, ABT-MVK-Cys mentioned above (Figures S9-S11). The results indicated that
reaction solution and HBT-MVK were further examined to Cys can be well detected even in the presence of other anions,
verify the abovementioned mechanism (Figure 5). As shown in cations and amino acids except Cu2+. The fluorescence
Figure 5, after the reaction of HBT-MVK with Cys, the quenching induced by Cu2+ may be due to the coordination of
characteristic proton signals of acrylate group of HBT-MVK copper ions with the product (HBT-MVK) of ABT-MVK and
(signals j, l, k) at 6.64, 6.35 and 6.68 ppm disappear and the
cysteine. Fortunately, fluorescent response to Cu2+ can be
Scheme 2. Proposed response mechanism for ABT-MVK to Cys.
Fig. 6. Fluorescence intensity of ABT-MVK at 595 nm in 10 mM
PBS buffer of pH 8.0 in the presence of 100 μM anions (a, free,
F-, Cl-, I-, AcO-, NO3 , CO3 , S2-, HSO3 and Cys), cations (b, free,
Na+, K+, Mg2+, Ca2+, Ni2+,Zn2+, Co2+, Pb2+, Fe3+, Al3+, Cu2+ and Cys)
and amino acids (c, 1-18: Free, Ala, Asp, Gln, Glu, Gly, His, Ile,
Leu, Lys, Met, Pro, Ser, Thr, Val, Hcy, GSH and Cys). Inset:
Photographs of before mentioned solutions taken under the
irradiation of 365 nm UV light. [ABT-MVK] = 10 μM, λex = 370
nm.
-
2-
-
1
Fig. 5. Partial H NMR (500 MHz) spectra of (a) ABT-MVK (5.0
mM), (b) ABT-MVK with 1.5 equivalents of Cys and (c) HBT-
MVK measured in DMSO-d6. Inset: A partially enlarged view of
1H NMR (500 MHz) spectra. [ABT-MVK] = [HBT-MVK] = 5.0 mM.
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masked upon the usage of EDTA as a coadditive (Figure S12).
These above results indicated that ABT-MVK could selectively
distinguish Cys from GSH, Hcy and other biologically relevant
analytes.
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DOI: 10.1039/C9NJ01259A
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Cytotoxicity assay and fluorescence imaging in living cells
For intracellular fluorescent imaging, cytotoxicity of the probe
is a critical consideration. Thus, we evaluated the cell
cytotoxicity of ABT-MVK by CCK8 analysis for HeLa cells
afterward. As shown in Figure S13, HeLa cells viability
remained above 80% after being incubated with 2-50 μM of
ABT-MVK for 2 h, which suggested that the probe exhibited
limited cytotoxicity to living cells under the experimental
conditions, and hold great potential for biological applications.
Based on the above research, the practical applications of ABT-
MVK for fluorescent imaging of intracellular Cys in living cells
was investigated. As revealed in Figure 7, when HeLa cells
were incubated with 10 μM ABT-MVK for 30 min, the cells
started to show bright red fluorescence (A1). However, when
cells were pretreated with 1.0 mM N-ethylmaleimide (NEM),
which is a well known thiol trapping reagent, before cultured
with probe ABT-MVK, little fluorescence was seen in the
observation window (B1). As another control experiment, after
HeLa cells were treated with NEM, exogenous Cys (100 μM),
and ABT-MVK for 30 min sequentially, the red fluorescence
was also observed (C1). In contrast, when HeLa cells went
similar treatment scheme with NEM, Hcy (100 μM) or GSH
(100 μM) followed by incubation with ABT-MVK for another 30
min respectively, red fluorescence can hardly be visualized (D1
and E1). The results demonstrate that ABT-MVK possesses
good membrane permeability and is able to detect
intracellular Cys in living cells.
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Fig. 8. Confocal microscopy fluorescence images of (A) HeLa
cells incubated with ABT-MVK (10 μM) for 30 min; (B) HeLa
cells pretreated with H2O2 (200 μM) for 30 min and then
incubated with ABT-MVK (10 μM) for 30 min; (C) HeLa cells
pretreated with Cys (100 μM) for 30 min and then incubated
with ABT-MVK (10 μM) for 30 min. Top: bright field, middle:
fluorescent field, bottom: merged field. λex = 405 nm, λem
550-650 nm. Scale bar: 50 μm.
=
the change of Cys concentration in living cells was
demonstrated by altering the redox balance. When the HeLa
cells were pretreated with 200 μM H2O2 for 30 min before
incubation with ABT-MVK (10 μM), there was a dramatic
decrease observed in red fluorescence (Figure 8B) compared
with those without H2O2 pretreatment (Figure 8A). However,
cells pretreated with exogenous 100 μM cysteine rather than
H2O2 showed the strongest red fluorescence (Figure 8C). Such
changes in fluorescence acted as response to the decrease of
intracellular Cys concentration arising from the Cys oxidation
by H2O2. Therefore, these findings suggested that ABT-MVK
probe could be used as a tool to track the level of intracellular
Cys.
As the intracellular Cys level is known to associate with
oxidative stress, the investigation of the probe in response to
Conclusions
In summary, we have developed a new type of ESIPT-based
fluorescent probe, ABT-MVK, for detecting Cys. The probe was
easily synthesized by using a traditional ESIPT unit HBT as
fluorophore, the conjugation of which was prolonged by Aldol
reaction to achieve a red emission, and an acrylate group was
introduced as a binding site for Cys. The probe itself was
almost non-fluorescent (“off” state) due to the quenching
effect from acrylate group. When Cys was introduced, the
fluorescence enhancement degree of ABT-MVK estimated at
595 nm was more than 73-fold (“on” state), which arose from
the Michael addition/cyclisation sequence mechanism and
Fig. 7. Confocal microscopy fluorescence images of Cys in living
HeLa cells. (A) HeLa cells incubated with ABT-MVK (10 μM) for
30 min. (B) HeLa cells pretreated with NEM (1.0 mM) for 30
min and then incubated with ABT-MVK (10 μM) for 30 min. (C,
D, E) HeLa cells pretreated with NEM (1.0 mM) for 30 min and
then incubated with Cys, Hcy, and GSH (100 μM) for 30 min,
respectively, and finally incubated with ABT-MVK (10 μM) for
30 min. Top: bright field, middle: fluorescent field, bottom:
merged field. λex = 405 nm, λem = 550-650 nm. Scale bar: 50 μm.
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exhibited a large Stokes shift of ca. 225 nm upon excitation at dropped slowly, and the contents were heated at 50 °C with
View Article Online
370 nm. Most importantly, the probe showed a rapid, sensitive stirring for another 4 h, after whichDOcIo: o10le.1d039t/oC9Nr.Jt0. 12a5n9dA
and selective fluorescent sensing proposal for Cys in aqueous neutralized by 3 N of HCl, the precipitate was filtered and
buffer over various potential interferences including Hcy, GSH, washed with water 3 times. At last purification of the obtained
Na2S and NaHSO3. In addition, this probe possessed low power on a silica gel column (CH2Cl2 and MeOH 80 : 1, v/v)
1
cytotoxicity and well-behaved membrane permeability, furnished the pure product (0.280 g) in 60% yield. H NMR
allowing it to be applied for detecting Cys in living cells. As a (500 MHz, DMSO-d6) δ (ppm) 12.96 (s, 1H), 8.20 (d, J = 8.0 Hz,
result, this fluorescent probe would be practically useful and 1H), 8.11 (d, J = 8.1 Hz, 1H), 7.86 (d, J = 16.4 Hz, 1H), 7.77 (s,
greatly beneficial to investigate and monitor Cys in living 1H), 7.73 (s, 1H), 7.59 (t, J = 7.6 Hz, 1H), 7.51 (t, J = 7.5 Hz, 1H),
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biological systems.
6.96 (d, J = 16.4 Hz, 1H), 2.34 (d, 6H).
Synthesis of ABT-MVK. Compound HBT-MVK (0.155 g, 0.5
mmol) was dissolved in CH2Cl2 (30 mL) with 100 μL TEA and
stirred at 0 °C. Acryloyl chloride (100 μL) was added slowly
though syringe. After that, the reaction solution was stirred at
r. t. for another 4 h. Then, the solution was concentrated
under reduced pressure. After being purified by the silica gel
chromatography (CH2Cl2/MeOH, 100 : 1, v/v), compound ABT-
Experimental section
Reagents and instruments
All reagents and solvents utilized in organic syntheses were
purchased from Aladdin reagent (Shanghai) co., Ltd and were
used as received. Cervix carcinoma (HeLa cells) were provided
by FDCC (Shanghai, China). Buffer solutions for spectral
investigation herein were prepared by triple-distilled water.
Silica gel plates were used for thin layer chromatography
(TLC) and silica gel of 200-300 mesh was applied for column
chromatography. 1H NMR and 13C NMR spectra were recorded
by Bruker AV500 or AV600 NMR spectrometer using DMSO-d6
as solvent. Chemical shifts were reported in parts per million
(ppm) downfield from tetramethylsilane (TMS, as internal
standards, δ=0 ppm). High-resolution mass spectra (HRMS)
1
MVK was obtained as a light yellow solid (0.138 g, 76%). H
NMR (600 MHz, DMSO-d6) δ (ppm) 8.18 (d, J = 7.7 Hz, 2H), 8.05
(d, J = 8.0 Hz, 1H), 7.97 (s, 1H), 7.60 – 7.57 (m, 1H), 7.52 – 7.49
(m, 1H), 7.47 (d, J = 16.2 Hz, 1H), 7.00 (d, J = 16.2 Hz, 1H), 6.72
– 6.68 (m, 1H), 6.67 – 6.62 (m, 1H), 6.35 (dd, J = 10.0, 1.5 Hz,
1H), 2.49 (s, 3H), 2.32 (s, 3H); 13C NMR (151 MHz, DMSO-d6): δ
(ppm) δ 197.48, 164.01, 161.41, 152.16, 144.29, 136.82,
135.80, 134.95, 134.35, 131.93, 130.56, 129.78, 128.51, 126.96,
126.72, 126.32, 125.82, 122.89, 122.10, 27.97, 20.23. ESI-MS
m/z: [M+Na]+ Calcd for C21H17NO3SNa+ 386.0821; Found
386.0811.
were measured by
a
FT-ICR-MS mass spectrometer.
Absorption spectra were obtained on a UH5300 UV-Vis
spectrophotometer. Fluorescence spectral measurements
were operated on a Hitachi F-4600 spectrophotometer, and
the excitation and emission slits were both set at 5 nm. All the
spectra were measured using a 1-cm quartz cell. Cellular
imaging by laser scanning confocal microscopy was carried out
on Leica SP5 Point Scanning Confocal. Additionally, the
cytotoxic effect of the probe was examined by Cell Counting
Kit-8 (CCK-8, purchased from Sigma-Aldrich and used as
received) assay using Bio-Tek ELX800 microplate reader.
General procedures for spectra measurements
The stock solutions of ABT-MVK (5.0 mM) and N-
ethylmaleimide (NEM, 100 mM) were prepared in HPLC-grade
DMSO and stored at 4 °C. Stock solutions of 10 mM RSSs,
anions (sodium salts), cations (chloride salts) and amino acids
were prepared by direct dissolution in deionized water.
Absorption and fluorescence measurements were carried out
using the stock solution of ABT-MVK in DMSO, which was
diluted by 10 mM PBS buffer of required pH, to afford the
desired concentration of ABT-MVK at 10 μM, to which Cys or
other guest species were added.
Synthesis of the probe
Synthesis of HBT. 2-(2-Hydroxyphenyl)-benzothiazole (HBT)
was synthesized according to the literature. 65
Detection limit
Synthesis of HBTA. 0.432
g
(2.0 mmol) 2-(2-
The detection limit of Cys (3σ/k) was calculated based on the
linear relationship between the intensity at 595 nm and its
concentration, where σ is the standard deviation of the blank
measurements (n=11) and k is the slope of the linear
dependence of intensity versus Cys concentration.
Hydroxyphenyl)-benzothiazole (HBT) and 0.840 g (6.0 mmol)
hexamethylenetetramine were dissolved in 20 mL TFA. The
contents were heated at reflux with stirring for 24 h after
which the heating was discontinued and allowed to cool to
room temperature. The solution was neutralized by 1 N of
NaOH, and the precipitate generated was filtered and washed
with water. And then purification of the residue on a silica gel
column (CH2Cl2 and MeOH 100 : 1, v/v) furnished the pure
Cell cytotoxicity study
To study the cytotoxicity of the probe, HeLa cells were seeded
at 6×104 cells per well in 96-well plates and incubated for 24 h
before the treatment, followed by exposure with different
concentrations (2-50 μM) of probe ABT-MVK for 4 h. After
being washed with Dulbecco's Phosphate Buffered Saline
(DPBS), the cells were incubated for further several hours, and
then the cytotoxic effects of ABT-MVK were determined using
Cell Counting kit-8 (CCK-8) assays.
1
product (0.404 g) in 82% yield. H NMR (500 MHz, DMSO-d6) δ
(ppm) 12.75 (s, 1H), 10.32 (s, 1H), 8.20 (d, J = 7.9 Hz, 2H), 8.11
(d, J = 8.1 Hz, 1H), 7.72 (d, J = 1.3 Hz, 1H), 7.58 (t, J = 7.6 Hz,
1H), 7.50 (t, J = 7.5 Hz, 1H), 2.38 (s, 3H).
Synthesis of HBT-MVK. 0.400 g (1.5 mmol) HBTA was
dissolved in 20 mL acetone and the solution was heated at
50 °C. Then 2 mL of 10 % sodium hydroxide solution was
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Cell culture and fluorescent imaging
HeLa cells were seeded in high-glucose DMEM supplemented
with 10 % fetal bovine serum, 1 % streptomycin and 1 %
penicillin. Cultures were maintained in a humidified incubator
at 37 °C, in 5 % CO2 / 95 % atmosphere. For intracellular Cys
imaging, the cells were incubated with 10 μM ABT-MVK (with
0.2% DMSO, v/v) for 30 min at 37 °C and washed three times
with PBS buffer for imaging immediately. For NEM-treated
experiments, HeLa cells were pretreated with 1.0 mM NEM for
30 min at 37 °C, washed three times with PBS buffer, and
incubated with 10 μM ABT-MVK (or incubated with 100 μM
Cys, Hcy or GSH for 30 min prior to addition of ABT-MVK) for
30 min at 37 °C. The cell imaging was carried out after they
were washed with PBS. For H2O2-treated experiments, HeLa
cells were pretreated with 200 μM H2O2 for 30 min at 37 °C,
washed with PBS buffer, and incubated with 10 μM ABT-MVK
for 30 min. Cell imaging was carried out after washing cells
with PBS. Fluorescence imaging of HeLa cells was performed
on Leica SP5 Point Scanning Confocal fluorescence microscope
with excitation wavelength fixed at 405 nm and fluorescence
wavelengths in the range of 550 - 650 nm.
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25 J. X. Hong, W. Y. Feng, G. Q. Feng. Sen. Actuat. B Chem., 2018,
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Conflicts of interest
There are no conflicts to declare.
Acknowledgements
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1014–1020.
This work was supported by the National Natural Science
Foundation of China (21375070, 21422504), the Fund Project
for Shangdong Key R&D Program (2017GGX20121), and the
support from Key Laboratory of Spectrochemical Analysis &
Instrumentation (Xiamen University), Ministry of Education
(SCAI1702, SCAI1703).
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An HBT-based fluorescent probe for Cys with large Stokes shift and high selectivity
was developed due to ESIPT process.