A coumarin-based fluorescent turn-on probe for detection of biothiols in vitro

Article abstract of DOI:10.1002/bio.2912

A novel fluorescent probe (CA-N) was designed and synthesized for detection of biothiols. CA-N displayed a strong fluorescence in the presence of biothiols with high sensitivity, and the mechanism for detection biothiols was based on the Michael addition reaction of a thiol group to α,β-unsaturated ketones. CA-N showed low detection limit for cysteine (Cys), homocysteine (Hcy), and glutathione (GSH), which were calculated as 3.16, 0.19 and 5.15 μM, respectively. At the same time, CA-N exhibited high selectivity toward biothiols compared with other biological amino acids. In vitro cell experiments proved that CA-N had no cytotoxicity, high cell permeability and could be employed in living cell imaging for biothiols.

Full text of DOI:10.1002/bio.2912

Research article
Received: 25 October 2014,
Revised: 15 January 2015,
Accepted: 03 March 2015
Published online in Wiley Online Library: 29 April 2015
(wileyonlinelibrary.com) DOI 10.1002/bio.2912
A coumarin-based fluorescent turn-on probe
for detection of biothiols in vitro
Mengqiang Liu,a Qian Jiang,a Zhiyun Lu,a Yan Huang,a Yanfei Tanb*
and Qing Jiangb*
ABSTRACT: A novel fluorescent probe (CA-N) was designed and synthesized for detection of biothiols. CA-N displayed a strong
fluorescence in the presence of biothiols with high sensitivity, and the mechanism for detection biothiols was based on the
Michael addition reaction of a thiol group to α,β-unsaturated ketones. CA-N showed low detection limit for cysteine (Cys),
homocysteine (Hcy), and glutathione (GSH), which were calculated as 3.16, 0.19 and 5.15μM, respectively. At the same time,
CA-N exhibited high selectivity toward biothiols compared with other biological amino acids. In vitro cell experiments proved
that CA-N had no cytotoxicity, high cell permeability and could be employed in living cell imaging for biothiols. Copyright ©
2015 John Wiley & Sons, Ltd.
Additional supporting information may be found in the online version of this article at the publisher’s website.
Keywords: fluorescent probe; Michael addition; coumarin; bioimaging
there remains an urgent need for a highly sensitively probe that
could be used in the further application of biotechnology.
Here, we reported a novel coumarin-based derivative fluorescent
probe, namely (E)-7-(diethylamino)-3-(3-(4-nitrophenyl)acryloyl)-2H-
benzo- pyran-2-one (CA-N). CA-N was constructed based on the
Michael addition reaction of thiol to α,β-unsaturated ketones. The
quencher group of nitrobenzene led to the non-fluorescence of
CA-N, although a strong green fluorescence was detected once
the probe reacted with a thiol group. Furthermore, CA-N had high
sensitivity and good biocompatibility, which could be successfully
applied to living cell imaging in vitro. In addition, CA-N could detect
qualitative changes in thiol concentrations in living cells.
Introduction
Biological thiols, such as cysteine (Cys), homocysteine (Hcy) and
glutathione (GSH), play a key role in many physiological processes,
and connections have been found between abnormal levels of
thiols and a number of diseases. A decrease in the level of Cys
may lead to many health problems, such as liver damage, slow
growth, skin lesions, AIDS and oxidative damage (1–4). An abnor-
mal level of Hcy in human plasma has been shown to be correlated
with Alzheimer’s disease, osteoporosis and cardiovascular disease
(5–7). GSH is the most abundant (1–10 mM) intracellular thiol and
has many cellular functions (8). Changes in cellular GSH levels
can also lead to health problems, such as leucocyte loss, heart
problems and cancer (9–11). Therefore, the detection and quanti-
fication of biothiols in biological systems is of great importance.
In the past few years, a number of techniques have been
Experimental
exploited to detect these biological thiols, such as high-
performance liquid chromatography (HPLC) (12,13), Fourier trans-
Materials and methods
Unless otherwise indicated, all materials and solvents were
obtained from commercial suppliers and used without further
purification. The exception was spectrographically pure
dimethylsulfoxide (DMSO) used for the biological evaluation.
form infrared (FTIR) spectroscopy (14), mass spectrometry (MS)
(15,16), surface-enhanced Raman scattering (17), capillary electro-
phoresis (18,19), electrochemistry assay (20,21), UV/vis spectros-
copy (22,23) and fluorescence spectroscopy. Among these
various strategies, fluorescent probes for the detection of biothiols
have been developed quickly because of the method’s ease of op-
eration, high selectivity, sensitivity, low cost, low detection limits
and potential for in vivo imaging (24–26). Therefore, various fluo-
rescent probes for biothiols based on different fluorophores have
been constructed, including rhodamine (27–29), naphthalimide
(30), BODIPY (31,32), fluorescein (33–36), cyanine (37–39), flavone
(40,41) and coumarin (42–50). Of these, coumarin dye is one of
the molecules used most widely in fluorescent probes owing to
its high photostability, large Stokes shift, high cell permeability,
visible emission wavelength and high fluorescence quantum yield
(51,52). However, many of these probes suffer from some limita-
tions, including high-pH aqueous solution, high background fluo-
rescence, low sensitivity in vitro and poor biocompatibility. Thus,
1
Double-distilled water was used in the buffer solutions. H NMR
and 13C NMR spectra were measured using a Bruker AVANCE
ΙΙ-400 MHz spectrometer and TMS as an internal standard.
* Correspondence to: Y. Tan and Q. Jiang, National Engineering Research
Center for Biomaterials, Sichuan University, Chengdu, 610064, China.
E-mail: tanyf@scu.edu.cn; 841523452@qq.com
a
Key Laboratory of Green Chemistry and Technology (Ministry of Education),
College of Chemistry, Sichuan University, Chengdu, 610064, China
b
National Engineering Research Center for Biomaterials, Sichuan University,
Chengdu, 610064, China
Luminescence 2015; 30: 1395–1402
Copyright © 2015 John Wiley & Sons, Ltd.

M. Liu et al.
Fluorescent emission spectra were obtained using a Perkin–Elmer
LS55 fluorescence spectrophotometer. UV/vis absorption spectra
were performed on a Perkin–Elmer Lambda 950 UV/VIS Spectrom-
eter. High-resolution MS were obtained using a Shimadzu LCMS-
IT-TOF. pH was measured with a PHS-3E pH meter. Fluorescence
microscopic images were obtained from laser scanning confocal
microscopy (CLSM; TCS-SP5, Leica, Germany).
Detection limit studies
The detection limit was calculated based on a method described
previously (54,55). The fluorescence intensity of CA-N alone was
measured seven times and the standard deviation of the blank
measurement was obtained. To improve sensitivity, 10μM CA-N
was employed. Under this condition, a good linear working range
between the emission intensity and the Cys concentration with
the range 10–100 μM was observed. The detection limit was
determined as 3σ/k where σ is the standard deviation of the blank
measurement and k is the slope of a plot of fluorescence intensity
against sample concentration.
Synthesis
3-Acetyl-7-(diethylamino)-2H-benzopyran-2-one (2). Compound 2
was synthesized according to a previously reported procedure
(53). 4-Diethylamino salicylaldehyde (10 g, 51.7mmol) and ethyl
acetonacetate (13.5g, 100 mmol) were dissolved in absolute
ethanol (100 mL), and piperidine (0.2mL) was added as a cata-
lyst. After the mixture was refluxed under argon atmosphere
for 6 h, a yellow solid was obtained and collected by filter. The
solid was washed with cool absolute ethanol, crude product
was recrystallized from the absolute ethanol, and a yellow solid
was collected (10.6g, yield: 79%).
Kinetic analysis
The time-dependent response of CA-N (10 μM) with Cys, Hcy
and GSH (100 eq.) in DMSO/phosphate buffer (1: 1 v/v, pH7.4)
exhibited pseudo-first-order reaction conditions at room temper-
ature, and the fluorescence intensity at 494 nm was recorded.
As described in the literature (56,57), the pseudo-first-order rate
constant for the reaction was determined by fitting the fluores-
cence intensities of CA-N to following the pseudo-first-order
equation:
1H NMR (400 MHz, CDCl3) δ (ppm): 8.44 (s, 1H), 7.41 (d, J = 8.8Hz,
1H), 6.64 (dd, J = 8.8Hz, J =2.0 Hz, 1H), 6.48 (d, J = 1.6Hz, 1H), 3.49
(q, 4H), 2.68 (s, 3H), 1.26 (t, J = 7.2Hz, 6H). m.p. 151–153 °C.
ꢀ
ꢁ
.
¼ ꢀ k’t
ðFmax ꢀFtÞ
ln
Fmax
(E)-7-(diethylamino)-3-(3-(4-nitrophenyl)acryloyl)-2H-benzopyran-2-
one (CA-N). Compound
2
(100 mg, 0.39 mmol) and 4-
nitrobenzaldehyde (89.2 mg, 0.59 mmol) were dissolved in absolute
ethanol (10 mL) and piperidine (0.1 mL) was added as a catalyst. Af-
ter the mixture had been refluxed under argon atmosphere for
24 h, the solvents were removed by distillation under reduced
pressure and the red solid was collected. The crude solid was ex-
tracted with dichloromethane and the solvent was then removed.
The crude solid was further purified by column chromatography
on silica gel to give a red solid. After recrystallization in absolute
ethanol, and drying under vacuum, a red solid was acquired
(45.9 mg, yield: 30%).
Where Ft and Fmax are the fluorescence intensities at 494 nm at
time t and the maximum value obtained after the reaction was
completed. k’ is the pseudo-first-order rate constant.
Cell experiments
Cytotoxicity assay. For the cell cytotoxicity assay, MG63 cells
were seeded in 96-well plates at a concentration of 1 × 104
cells/well for 24 h, then different concentrations of CA-N (0, 5,
10 and 20 μM final concentration) were added to the wells and
the cells were cultured for another 24 h. Cytotoxicity was deter-
mined by MTT colorimetric method using five parallel wells at
each time point. Cells were incubated with 5 mg/mL of MTT for
4 h at 37 °C, the supernatant was then discarded, and DMSO
(200 μL/well) was added to dissolve the formazan; the absor-
bance was then measured at 570 nm with a microplate reader
(BioRad, model 550).
1H NMR (400MHz, CDCl3) δ (ppm): 8.58 (s, 1H), 8.31 (d,
J =16.0 Hz, 2H), 8.26 (d, J = 8.8 Hz, 1H), 7.82 (d, J =8.8 Hz, 2H), 7.81
(d, J =16.0 Hz, 1H), 7.46 (d, J = 9.2 Hz, 1H), 6.67–6.64 (dd, J = 8.8Hz,
2.4 Hz, 1H), 6.51 (d, J = 2.0Hz, 1H), 3.51 (q, 4H), 1.29 (t, J = 7.2Hz,
6H). 13C NMR (100 MHz, CDCl3) δ (ppm): 184.9, 160.0, 157.9, 152.4,
148.2, 147.3, 140.6, 138.60, 131.1, 128.1, 128.0, 123.0, 114.9, 109.1,
107.7, 95.7, 44.2, 11.5. HR-MS (ESI): m/z [M+Na]+ 415.1265, calcd.:
415.1264. m.p. 227–228 °C.
Bioimaging for biothiols in living cells
Preparation of solutions for fluorescence and UV/vis
absorption
MG63 cells were seeded on 35 mm glass-bottomed dishes at a
density of 1 × 105 cells/dish in culture medium and incubated over-
night in preparation for live cell imaging. The cells were divided
into four groups: (1) no added dye or biothiols; (2) treatment with
10 μM of CA-N in a serum-free medium for 30 min and washed
twice with prewarmed PBS; (3) treatment with 1.0 mM N-
ethylmaleimide (NEM, a thiol-blocking reagent) for 30min to de-
crease the concentration of cellular biothiols, then washed twice
with prewarmed PBS buffer, followed by addition of CA-N for
30 min; (4) pretreatment with NEM for 30 min, followed by addition
of Cys (100 μM) and CA-N for 30 min and washed twice with
prewarmed PBS. The four groups were observed under CLSM
using the excitation channel (λex = 405 nm).
A stock solution of CA-N (1.5mM) was prepared in DMSO. Stock
solution of Cys, Hcy, GSH (30 mM) and other amino acids
(10 mM) including Ala, Arg, Asp, Glu, Gly, His, Leu, Lys, Phe, Pro,
Ser, Tle, Thr, Trp, Tyr and Val were dissolved in double-distilled wa-
ter, and solutions (DMSO/aqueous buffer, 1: 1 v/v, pH 1.0–11.0)
with different pH values were prepared. In a typical experiment,
20 μL of the CA-N stock solution was added to 3 mL
DMSO/phosphate buffer (1: 1 v/v, pH7.4) and then an appropriate
buffer of each amino acids was added. Fluorescence and absorp-
tion spectra were recorded after the addition of amino acids at
room temperature.
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Coumarin-based probe for the detection of biothiols
Results and discussion
Design and synthesis of CA-N
To obtain a fluorescent probe that selectively detected thiols for
potential biological applications, the α,β-unsaturated ketones
moiety was chosen as the Michael addition reaction recognition
unit, and diethylaminocoumarin was selected as the fluorophore.
The synthetic route for probe CA-N is shown in Scheme 1.
Compound 2 was synthesized and used as a reference compound
to reveal the Michael addition reaction mechanism. The structure
of CA-N was confirmed by 1H NMR, 13C NMR and high-resolution MS.
UV/vis absorption and fluorescence studies with thiols
The absorption and fluorescence emission spectra of CA-N in the
absence and presence of Cys in DMSO/phosphate buffer
(1: 1 v/v, pH 7.4) at room temperature were examined and are
recorded in Fig. 1. Because of use of the nitrobenzene group as
the quencher moiety, CA-N alone did not exhibit fluorescence,
although upon addition of Cys (100 eq.), the fluorescence spectra
changed dramatically, and a strong emission peak was observed
at 494 nm. Furthermore, in the absorption spectrum, addition of
Cys caused a clear hypsochromic shift (~20 nm) from 472 to
452 nm. The changes in the absorption spectrum and fluorescence
intensity of CA-N in the presence of Cys were attributed to the con-
jugate addition of thiol to a carbon–carbon double bond through
Michael addition. When CA-N reacted with thiols, it was able to
break the carbon–carbon double bond, the quencher was broken
and the fluorescence recovered (Scheme 2). Thus, CA-N could be
used as a fluorescent turn-on probe for thiols.
Figure 1. Normalized absorption spectrum of CA-N (■) and upon addition of 100 eq.
Cys (●). Normalized fluorescence spectra of CA-N (▲) and upon addition of 100 eq.
Cys (▼). Conditions: CA-N in the absence and presence of Cys in DMSO/phosphate
buffer (1: 1 v/v, pH 7.4) at room temperature (λex = 400 nm).
biological thiols. Meanwhile, these data suggested that CA-N could
be applied to the quantitative detection of biothiols.
pH-dependent studies
In order to understand the pH-dependent effects of physiological
media, CA-N solutions at different pH values (1.0–11.0) in the
presence and absence of Cys, Hcy and GSH were investigated. As
shown in Fig. 4, CA-N alone showed weak fluorescence only, its
intensity did not change at different pH values (1.0–11.0), which
suggested that CA-N was not affected by pH. By contrast, in the
presence of Cys, the fluorescence intensity showed a large en-
hancement when the pH was > 5.0, and the fluorescence intensity
reached a maximum at pH8.5. At the same time, in the presence of
Hcy or GSH, the fluorescence intensity reached a maximum value
at pH8.5 and 9.0, respectively. These results might be due to the
influence of the pKa value of aliphatic thiols on the reaction. The
pKa value of aliphatic thiols was ~ 8.5 (58,59)._ENREF_47 These
results indicate that CA-N was sensitive enough to detect thiols
under neutral to alkaline conditions.
Detection limit studies
To evaluate the detection limit of CA-N for Cys, Hcy and GSH in
solution, CA-N was treated with various concentrations of Cys,
Hcy or GSH in DMSO/phosphate buffer (1: 1 v/v, pH7.4) at room
temperature. The results show that the fluorescence intensity of
CA-N gradually increased with increasing of Cys concentration
(0–100 eq.) (Fig. 2). Simultaneously, under the same conditions,
when Hcy or GSH (0–100 eq.) was added to CA-N solution, similar
results were obtained (Fig. S1).
The results of the titration experiment indicated a good linear
relationship between the fluorescence intensity and Cys concen-
tration within the range 10–100 μM (Fig. 3). Under these condi-
tions, the detection limit for Cys was calculated as 3.16 μM.
Under the same conditions, the detection limits for Hcy and GSH
were 0.19 and 5.15 μM, respectively (Table 1 and Fig. S2). The
low detection limit showed that CA-N was highly sensitive to
Kinetic analysis
The time-dependent fluorescence intensity of CA-N (10 μM) with
Cys, Hcy and GSH (100 eq.) in DMSO/phosphate buffer (1: 1 v/v,
pH7.4) was recorded. As shown in Fig. 5, the fluorescence intensity
reached a maximum after ~ 35 min in the presence Cys and Hcy.
However, in the presence of GSH, the fluorescence intensity
Scheme 1. Synthetic route for probe CA-N.
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M. Liu et al.
Scheme 2. The design concept of a fluorescent turn-on probe (CA-N) for thiols.
Figure 2. Fluorescent titrations of probe CA-N (10 μM) upon addition of various con-
centrations of Cys (0–100 eq.) in DMSO/phosphate buffer (1: 1 v/v, pH 7.4) after 35 min
at room temperature (λex = 400 nm).
Figure 4. Fluorescence intensity of CA-N (10 μM) at 494 nm before and after addition
of Cys, Hcy and GSH (0–100 eq.) at different pH values in DMSO/phosphate buffer for
35 min at room temperature (λex = 400 nm).
Figure 5. Time-dependent fluorescence intensity of CA-N (10 μM) at 494 nm upon
addition of Cys, Hcy and GSH (100 eq.) in DMSO/phosphate buffer (1: 1 v/v, pH 7.4)
at room temperature (λex = 400 nm).
Figure 3. Linear plot of CA-N (10 μM) in the presence of Cys (10–100 μM) against
fluorescence intensity at 494 nm (λex = 400 nm).
Table 2. Pseudo-first-order rate constants and lifetime of CA-N
to Cys, Hcy, and GSH
Table 1. Detection limit of the probe to Cys, Hcy and GSH
Number
Thiol
Kinetic constant;
Life time; t1/2
(min)
Number Thiol
σ
k
Detection limit (μM)
k′ (min–1)
1
2
3
Cys
Hcy
GSH
0.7157 6.79 × 105
0.7157 1.14 × 106
0.7157 4.17 × 105
3.16
0.19
5.15
1
2
3
Cys
Hcy
GSH
0.106
0.114
0.020
6.54
6.10
34.55
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Coumarin-based probe for the detection of biothiols
fluorescence intensity at 494 nm was recorded and the results
are shown in Fig. 6. We found that CA-N displayed weak fluores-
cence in the presence of Ala, Arg, Asp, Glu, Gly, His, Leu, Lys, Phe,
Pro, Ser, Tle, Thr, Trp, Tyr and Val. However, under the same condi-
tions, when thiol-containing analytes (Cys, Hcy and GSH) were
added to CA-N, a great enhancement of the fluorescence intensity
was observed. The data suggest that CA-N had a high selectivity
for thiol-containing molecules.
Meanwhile, to further test the Cys selectivity of CA-N in the
presence of other analytes, CA-N (10 μM) was added to Cys (10 eq.)
in the presence of various amino acids (10 eq.) in DMSO/phosphate
buffer. None of analytes measured had an effect on the fluorescence
detection of Cys (Fig. S7). Therefore, at physiological pH, CA-N could
be highly selective for biothiols, even in the presence of these rele-
vant amino acids in a biological system.
Figure 6. Fluorescence intensity of CA-N (10 μM) at 494 nm in the presence of vari-
ous amino acids (10 eq.) in DMSO/phosphate buffer (1: 1 v/v, pH 7.4) for 35 min at
room temperature (λex = 400 nm). 1, Cys; 2, Hcy; 3, GSH; 4, Ala; 5, Arg; 6, Asp; 7, Glu;
8, Gly; 9, His; 10, Leu; 11, Lys; 12, Phe; 13, Pro; 14, Ser; 15, Tle; 16, Thr; 17, Trp; 18, Tyr;
19, Val; 20, blank.
Mechanism of CA-N for detecting Cys
From the fluorescence spectral studies, CA-N shows a selective
response to biothiols. CA-N was able to react through conjugate
addition with a thiol group of Cys and form a CA-N–Cys adduct.
In order to provide evidence of the reaction mechanism, the
fluorescence spectra of adduct CA-N–Cys and compound 2 in
DMSO/phosphate buffer at room temperature were recorded.
Compound 2 had a similar structure to the diethylaminocoumarin
fluorophore with CA-N–Cys adduct and served as a reference com-
pound. We found that CA-N and compound 2 had almost the
same fluorescence spectra (Fig. S8). Meanwhile, 1H NMR spectros-
copy studies also provided evidence of CA-N in the absence or
presence of Cys. As shown in Fig. 7, the alkene proton at around
8.08 ppm (Ha) disappeared, and a new peak emerged at around
4.58 ppm (Ha′); there was another alkene proton at around
7.76 ppm (Hb), and in the presence of Cys, two new peaks
emerged at 3.71 ppm (Hb′) and 3.32 ppm (Hb′′). These results proved
that CA-N could react with the thiol group of Cys to form a
carbon–carbon double bond via Michael addition reaction. Fur-
thermore, MS analysis of CA-N with Cys revealed formation of a
CA-N–Cys adduct. The MS displayed peaks at m/z 415.1254 and
536.1466, corresponding to [CA-N + Na] and [CA-N–Cys + Na], re-
spectively (Fig. S9). Taken together, these results consistently
reached a maximum after ~ 90 min (Fig. S3). CA-N exhibited much
higher reactivity toward Cys and Hcy than towards GSH, in agree-
ment previous reports (55,60). This may be explained by steric
hindrance effects on the thiol 1,4-addition reaction.
Meanwhile, the kinetic profiles of the CA-N reaction with Cys,
Hcy and GSH were calculated from the fluorescence intensity at
494 nm. The pseudo-first-order rate constant k’ was determined fol-
lowing the pseudo-first-order equation. The pseudo-first-order rate
constants for Cys, Hcy and GSH were determined as k’ = 0.106,
0.114 and 0.020 min-1, respectively; the corresponding half-life
(t1/2) values were 6.45, 6.10 and 34.55min, respectively (Table 2
and Figs. S4–6). These results further demonstrated that CA-N
had high reactivity for Cys and Hcy compared with GSH.
Selective studies
In order to check the selective response of CA-N toward biothiols
over other amino acids, various amino acids were separately
added to CA-N in DMSO/phosphate buffer for 35 min, and the
Figure 7. (Upper) A proposed mechanism of the reaction between CA-N and Cys. (Lower) 1H NMR spectra changed of CA-N in the absence (A) or presence (B) of Cys in DMSO-d6/
D2O (4: 1, v/v) at room temperature.
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M. Liu et al.
Figure 8. Confocal Laser microscope images of MG63 cells. (A–C) Control group. (D–F) MG63 cells incubated with CA-N (10 μM) for 30 min at 37 °C. (G– I) Cells pretreated with
NEM (1.0 mM) for 30 min and then incubated with CA-N (10 μM) for 30 min at 37 °C. ( J–L) Cells pretreated with NEM (1.0 mM) for 30 min and tzhen added to Cys (100 μM) and
incubated with CA-N (10 μM) for 30 min at 37 °C. (Left) Bright channel, (middle) fluorescence channel (right) overlay. (M) Fluorescence intensity of each group.
revealed the reaction of CA-N with Cys and the formation of the
CA-N–Cys adduct.
these results proved that CA-N could be used as an excellent fluo-
rescent probe for biothiol imaging in living cells, and the alteration
of fluorescence intensity indicates changes in the concentration of
cellular biothiols.
Fluorescent imaging in MG63 cells
To investigate the permeability of cells and clarify whether CA-N
was sensitive enough to detect biothiols in living cells, the cytotox-
icity of CA-N was evaluated using MTT assays. The results showed
that cells grew very well even with 20 μM CA-N at 37 °C for 24 h
and no significant difference was observed between the groups.
The results indicated no cytotoxicity of CA-N (Fig. S10).
Furthermore, cell-imaging experiments with CA-N were carried
out and observed under CLSM. As shown in Fig. 8, MG63 cells with-
out added CA-N did not show any fluorescence (Fig. 8B), whereas
after incubation with CA-N at 37 °C for 30 min, significant green
fluorescence was observed in the cytoplasm of these cells (Fig. 8E),
which proved that CA-N was capable of permeating cells and
reacting with intracellular biothiols.
Conclusions
We designed and synthesized a novel coumarin-based fluorescent
probe CA-N, which exhibited high sensitivity and selectivity
toward biothiols (Cys, Hcy, GSH) over other natural amino acids.
1
Fluorescence spectra, H NMR and MS studies demonstrated the
mechanism by which thiols combined with the α,β-unsaturated
ketone of CA-N via the Michael addition reaction. In addition,
in vitro cell imaging found that CA-N could easily penetrate cell
membranes, react with biothiols in the cytoplasm, and show
strong green fluorescence. Our results prove that CA-N is useful
for imaging biothiols in living cells.
To further determine whether CA-N could highlight changes in
the concentration of biothiols in living cells, one group was pre-
treated with 1.0mM NEM for 30 min to eliminate any biothiols in
the cells; only weak fluorescence was observed in these cells after
incubation with CA-N (Fig. 8H). However, strong green fluores-
cence was again observed, when NEM-treated cells were added
with Cys (100 μM) and incubated with CA-N for another 30 min
(Fig. 8K). According to Image J™ software, the fluorescence inten-
sity value of each group was obtained (Fig. 8M). Taken together,
Acknowledgements
The authors acknowledge the financial support for this work by Re-
search Fund for the Doctoral Program of Higher Education of China
(20130181110089), the National Natural Science Foundation of
China (NSFC)(Grants No.21372168) and National Key Technology
R&D Program (2012BAI42G01).
Electronic Supplementary Information (ESI) are available.
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Luminescence 2015; 30: 1395–1402