A simple and readily available fluorescent turn-on probe for cysteine detection and bioimaging in living cells

Article abstract of DOI:10.1016/j.dyepig.2016.12.011

A new simple and readily available fluorescent probe for cysteine (Cys) was reported. This probe is based on an excited-state intramolecular proton transfer (ESIPT) dye and uses two acrylate moieties as the Cys reaction sites to promote signal changes. Im

Full text of DOI:10.1016/j.dyepig.2016.12.011

Accepted Manuscript
A simple and readily available fluorescent turn-on probe for cysteine detection and
bioimaging in living cells
Haiyan Zhang, Weiyong Feng, Guoqiang Feng
PII:
S0143-7208(16)31151-2
10.1016/j.dyepig.2016.12.011
DYPI 5636
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 10 November 2016
Revised Date: 5 December 2016
Accepted Date: 7 December 2016
Please cite this article as: Zhang H, Feng W, Feng G, A simple and readily available fluorescent turn-
on probe for cysteine detection and bioimaging in living cells, Dyes and Pigments (2017), doi: 10.1016/
j.dyepig.2016.12.011.
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ACCEPTED MANUSCRIPT
A simple and readily available fluorescent turn-on probe for
cysteine detection and bioimaging in living cells
Haiyan Zhanga, Weiyong Fengb and Guoqiang Fengb,*
a School of Chemical and Environmental Engineering, Wuhan Polytechnic University,
68 Xuefu South Road, Wuhan 430023, People's Republic of China
b Key Laboratory of Pesticide and Chemical Biology of Ministry of Education,
College of Chemistry, Central China Normal University, 152 Luoyu Road, Wuhan
430079, People's Republic of China
*Corresponding author. E-mail: gf256@mail.ccnu.edu.cn (G. Feng).
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Graphical Abstract
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Highlights
ꢀ
ꢀ
ꢀ
ꢀ
A new selective fluorescent turn-on probe for detection of Cys was developed.
This probe is readily available with a simple structure.
This probe can be used to develop convenient paper test strips for Cys detection.
This probe can be applied for imaging of Cys in living cells.
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ABSTRACT:
A new simple and readily available fluorescent probe for cysteine (Cys) was reported.
This probe is based on an excited-state intramolecular proton transfer (ESIPT) dye
and uses two acrylate moieties as the Cys reaction sites to promote signal changes.
Importantly, this probe shows rapid fluorescent turn-on signal changes for Cys with
high selectivity and sensitivity over various analytes including the similar structured
homocysteine (Hcy) and glutathione (GSH) in aqueous solution under mild conditions.
The detection limit of this probe for Cys was found to be as low as 0.80 ꢀM, which is
far below the normal content of Cys (30-200 ꢀM) in cells. This probe was also
successfully applied for bioimaging of intracellular Cys in living cells with low
cytotoxicity. Besides, a paper test strip system was developed with this probe for a
convenient detection of Cys.
Keywords: Cysteine; ESIPT; fluorescent turn-on probe; bioimaging
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1. Introduction
Cysteine (Cys), as an important small-molecular-weight amino acid and biothiol,
not only plays essential roles in many significant cellular functions such as in protein
synthesis, detoxification and metabolism [1-2], but is also closely related to many
diseases such as slow growth in children, hair depigmentation, edema, lethargy, liver
damage, loss of muscle and fat, skin lesions, and weakness [3-5]. Thus, specific
detection of Cys in living systems is highly important. Accordingly, the development
of rapid, convenient, and sensitive methods for selective detection of Cys is of great
significance.
In terms of simplicity, high sensitivity and non-invasiveness, fluorescent
detection with fluorescent probes has become a particularly attractive method for Cys
detection. Over the past decade, significant efforts have been made to develop
fluorescent probes for small molecule thiols and a number of fluorescent thiol probes
have been developed [6-11], however, most of them showed high selectivity for Cys
over many other amino acids, but lack selectivity over other biothiols, in particular
homocysteine (Hcy) and glutathione (GSH) due to their similar structures and
reactivity [12-26]. However, since Cys, Hcy and GSH have different biological roles
in living systems, discrimination of Cys from Hcy and GSH is critical in many cases
[27-33]. Pioneered by Strongin’s work, the selective detection of Cys over Hcy and
GSH could be realized by the cyclization of Cys with an aldehyde [34] or acrylate [35]
unit. After that, a number of specific Cys probes were developed, and among them,
using the acrylate group as the reaction site for Cys has become the most popular
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[36-50]. However, most of these developed probes suffered from some drawbacks
such as low efficiency, small Stokes shifts, complicated synthetic work or need of a
high content organic solvent to work. Therefore, development of simple and readily
available fluorescent probes for rapid, selective and sensitive detection of Cys is still
desired.
Extending our interest in developing fluorescent probes for thiols [51-56], we
report herein a rather simple but effective fluorescent probe for Cys (probe 1 in
Scheme 1). This probe is reaction based, which uses a known excited-state
intramolecular proton transfer (ESIPT) dye compound 2 [57] as the fluorophore and
two acrylate moieties as the Cys reaction sites to secure high selectivity for Cys over
Hcy and GSH [56,58-59]. Importantly, this probe has the following merits: (1) it is
simple with low molecular weight and can be easily synthesized from cheap
commercially available materials with good yield; (2) it exhibits near-zero
background fluorescence, and shows a rapid and significant fluorescent turn-on
response for Cys with a large Stokes shifts, allowing a convenient visual and sensitive
detection of Cys with low excitation interference; (3) it shows high selectivity and
sensitivity for Cys over many other analytes including Hcy and GSH; (4) it can be
conveniently used for bioimaging of Cys in living cells. Moreover, since compound 2
is a typical ESIPT dye, which shows strong fluorescence in solid state, this probe can
be used to develop an easy-to-prepare and easy-to-detect paper test strip system for a
convenient detection of Cys.
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Scheme 1. Probe 1 for detection of cysteine
2. Material and methods
2.1. Materials and instrumentation
All reagents and chemicals were purchased from commercial suppliers and used
without further purification. NMR spectra were measured on a Varian Mercury 400 or
1
600 instrument, operating at 400 MHz for H NMR and 600 MHz for 13C NMR.
Coupling constants (J values) are reported in hertz. The low-resolution MS spectra
were performed on a Thermo Scientific DSQ II GC/MS spectrometer. High-resolution
mass spectrometry (HR-MS) spectra were obtained on a Bruker microTOF-Q
instrument. UV-Vis and fluorescence spectra were recorded on an Agilent Cary-100
UV-Vis spectrophotometer and an Agilent Cary Eclipse fluorescence
spectrophotometer, respectively. The pH was measured using a PB-10 digital
pH-meter (Sartorius). HPLC spectra were recorded on an agilent 1100 high
performance liquid chromatography. Cell imaging was performed in an inverted
fluorescence microscopy with a 20×objective lens.
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2.2. Synthesis of compound 2
Compound 2 was synthesized according to the reported procedure [57].
2-aminothiophenol (0.33 ml, 3.12 mmol), 2,4-dihydroxybenzaldehyde (446 mg, 3.16
mmol) and sodium metabisulfite (Na2S2O5, 610 mg) were added to dry DMF (10 mL)
in a 50 mL round bottom flask. After stirring with reflux for 2 h, the reaction mixture
was cooled and added slowly into 200 mL water. The precipitate was filtered off,
washed with water (20 mL ×3) and dried to afford the crude product, which was
further purified by recrystallization from methanol to give 2 as a light yellow solid
(645 mg, yield 85.2%). Mp: 195-197 °C. TLC (silica plate): Rf ~0.48 (petroleum
1
ether:ethyl acetate 1:1, v/v). H NMR (400 MHz, d6-DMSO) δ 11.73 (s, 1H),
10.19 (s, 1H), 8.06 (d, J = 7.8 Hz, 1H), 7.97 (d, J = 8.1 Hz, 1H), 7.90 (d, J = 9.2 Hz,
1H), 7.50 (t, J = 7.6 Hz, 1H), 7.39 (t, J = 7.5 Hz, 1H), 6.41-6.61 (m, 2H). 13C NMR
(150 MHz, d6-DMSO
)
δ
166.24, 161.45, 158.14, 151.33, 133.08, 129.97, 126.27,
+
124.46, 121.76, 121.33, 110.10, 108.48, 102.70. HR-MS (ESI): calcd for C28H21O9
(M + H+): 244.0432; found 244.0416.
2.3. Synthesis of probe 1
Compound 2 (122 mg, 0.5 mmol), acryloyl chloride (2 mmol) and Et3N (2 mmol)
were mixed in 10 ml of anhydrous CH2Cl2 at 0 °C (ice bath). After stirring for 30 min,
the mixture was allowed to react at room temperature until the reaction was
completed (monitored by TLC). After removal of the white precipitate, the filtrate
was concentrated under reduced pressure, the crude solid product was purified by
recrystallization from CH2Cl2 to afford probe 1 as a white solid (138 mg, yield 80%).
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Mp: 105-106 °C; TLC (silica plate): Rf ~0.6 (petroleum ether:ethyl acetate 1:1, v/v);
1H NMR (400 MHz, CDCl3) δ 8.44 (d, J = 8.7 Hz, 1H), 8.07 (d, J = 8.2 Hz, 1H), 7.91
(d, J = 7.9 Hz, 1H), 7.51 (dt, J = 11.2, 4.0 Hz, 1H), 7.42 (dt, J = 11.2, 4.0 Hz, 1H),
7.22–7.27 (m, 2H), 6.70 (ddd, J = 17.3, 10.4, 1.0 Hz, 2H), 6.51 (dd, J = 17.3, 10.4 Hz,
1H), 6.33 (dd, J = 17.3, 10.4 Hz, 1H), 6.17 (dd, J = 10.4, 0.9 Hz, 1H), 6.08 (dd, J =
10.4, 0.9 Hz, 1H). 13C NMR (150 MHz, CDCl3) δ 163.71, 163.70, 161.56, 152.72,
152.32, 148.54, 135.23, 133.94, 133.39, 130.86, 127.69, 127.50, 126.36, 125.37,
123.89, 123.35, 121.37, 119.78, 117.04. EI-MS: m/z found 351.22 (M+, 40%), 297.11
(M+ - C2H2CO, 37%), 243.07 (M+- C4H4C2O2, 90%). HR-MS Calc. for C19H14NO4S+
(M + H+) 352.0638, found 352.0642.
2.4. Optical studies
Stock solution of probe 1 (1 mM) was prepared in HPLC grade DMSO and used
freshly. Stock solutions (2.5-10 mM) of the analytes including: AcONa, NaCl,
Na2CO3, NaF, Na2HPO4, NaHSO3, NaNO3, Na2S2O3, NaSCN, Na2SO4, Na2S, Cys,
Hcy, GSH, glutamine (Gln), serine (Ser), pyroglutamic acid (Pyr), tryptophane (Trp),
aspartic acid (Asp), alanine (Ala), phenylalanine (Phe), histidine (His), methionine
(Met), threonine (Thr), glycine (Gly), lysine (Lys), glutamic acid (Glu), isoleucine
(Ile), argnine (Arg), tyrosine (Tyr), and leucine (Leu) were prepared in ultrapure
water. For a typical optical study, a probe 1 (10 ꢀM) solution in HEPES buffer (10
mM, pH 7.2, with 10% DMSO, v/v) was prepared. Then 3.0 mL of the solution was
placed in a quartz cuvette which was controlled at 25 °C by a temperature controller.
The UV-Vis or fluorescent spectra were then recorded after addition of an analyte.
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2.5. Imaging of Cys in living cells
HeLa cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)
including 10% FBS (Fetal Bovine Serum) according to our published procedure
[51-57]. In the experiment of cell imaging, living cells were incubated with 5 ꢀM of
probe 1 (with 0.1% DMSO, v/v) at 37 °C for 30 min and washed with PBS for three
times, and then imaged immediately. As a control experiment, HeLa cells were
pre-cultured with NEM (500 ꢀM) for 30 min at 37 °C to remove the intracellular
biothiols. After washed three times with PBS buffer, the cells was then incubated with
5 ꢀM of probe 1 for 30 min at 37 °C and imaged immediately. In the imaging of
exogenous Cys, HeLa cells were pre-treated with NEM (500 ꢀM) for 30 min at 37 °C,
and washed three times with PBS. Then HeLa cells were treated with probe 1 (5 ꢀM)
for 30 min at 37 °C, washed three times with PBS. Finally, the resulting HeLa cells
were incubated with Cys (50 ꢀM) for 30 min at 37 °C. The imaging of cells was then
carried out after washing with PBS buffer.
3. Results and discussion
3.1. Probe design and synthesis
The probe 1 was designed using compound 2 as the fluorophore and two acrylate
moieties as the reaction sites. Compound 2 was selected because it is a well-known
ESIPT fluorophore of small molecular weight with two hydroxyl groups [57]. This
fluorophore can be readily prepared, and the two hydroxyl groups can be easily
protected by acrylate moieties to afford a double acrylate-functionalized probe.
Acrylate moiety has been reported to be a good reaction site for development of
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fluorescent Cys probes with an addition-cyclization reaction mechanism, in which the
reaction with Cys generally showed faster reaction kinetics than Hcy and GSH [35-50,
54]. Particularly, double acrylate-functionalized probes can make the difference of the
reaction kinetics between these biothiols even more obvious, thus guarantee them to
achieve a very high selectivity for Cys over Hcy and GSH [58-59]. We recently used
this approach and reported a readily available naphthofluorescein-based fluorescent
probe for selective detection of Cys [56]. Based on these works, probe 1 was expected
to be a new highly selective fluorescent probe for Cys. Thus, probe 1 was prepared in
the lab for investigation.
Probe 1 can be easily prepared from the readily available compound 2 with
acryloyl chloride in dry DCM under basic conditions (Scheme 2). The synthetic
process for both 2 and probe 1 are simple, as they can be isolated and purified by
recrystallization in good yield without using column chromatography. The structure
of products was well confirmed by NMR and mass analyses, which can be found in
the experimental section and the supplementary data.
O
O
CHO
O
HO
OH
SH
O
S
N
S
N
Na2S2O5
Cl
+
OH
O
DCM,Et3N
80%
DMF
85%
NH2
OH
probe 1
2
Scheme 2. The synthesis of compound 2 and probe 1.
3.2. Fluorescent detection of Cys with probe 1
The sensing ability of probe 1 for Cys was investigated in HEPES buffer (10 mM,
pH 7.2, with 10% DMSO, v/v) at 25 °C. Initial studies showed that addition of 10
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equiv of Cys to the probe 1 (10 ꢀM) solution resulted in rapid and distinct optical
responses. As shown in Fig. 1, the probe 1 solution shows strong absorption at 303
nm and is almost non fluorescent (Ф = 0.0008, using quinine sulfate as reference).
However, after addition of Cys, the absorption at 303 nm of the probe 1 solution
gradually decreased against time, accompanied by a gradually enhanced absorption
band at 333 nm with an isosbestic point at around 316 nm. Meanwhile, significant
fluorescence turn-on signal changes at 446 nm were observed, which leads to the
solution showing strong blue-green fluorescence (Ф = 0.025, using quinine sulfate as
reference). Kinetic study under this condition showed that the reaction of probe 1 with
10 equiv of Cys completed at about 30 min and produced about 30-fold fluorescence
enhancement, and the observed pseudo-first-order rate constant kobs for the reaction
was found to be about 0.12 min-1 (Fig. S1). In addition, the amount of DMSO can be
reduced and even in pure HEPES buffer, fluorescent detection of Cys with probe 1 is
still efficient, but showing less fluorescence enhancement (Fig. S2). Moreover, the
fluorescence of probe 1 itself is stable in the tested pH range (1–11) while probe 1
shows significant fluorescence enhancement for Cys over a wide pH range and the
best working pHs for probe 1 are around the physiological pH region (Fig. S3),
indicating probe 1 is highly favorable for biological applications. Overall, all these
results indicate that probe 1 can be used as a very promising fluorescent turn-on
sensor for rapid detection of Cys in aqueous solution under mild conditions.
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(a)
(b)
400
0-30 min
0-30 min
0.2
0.1
0.0
300
200
100
0
250
300
350
Wavelength (nm)
400
450
350
400
450
500
550
Wavelength (nm)
Fig. 1. (a) UV‐ vis spectra changes and (b) fluorescent spectra changes of probe 1 (10
µM) against time in the presence of Cys (100 µM) in HEPES buffer (10 mM, pH 7.2,
with 10% DMSO, v/v) at 25 °C. Emission color changes are inserted. For fluorescent
measurement, λex = 333 nm, dex = dem = 5 nm.
3.3. The selectivity
Encouraged by the above results, the selectivity of probe 1 for Cys was then
investigated. As shown in Fig. 2, upon addition of various analytes under the same
conditions, in sharp contrast to Cys, other amino acids such as Gln, Ser, Pyr, Trp, Asp,
Ala, Phe, His, Met, Thr, Gly, Lys, Glu, Ile, Arg, Tyr, and Leu, and other potential
−
−
2−
anionic competitors such as AcO−, Cl−, CO32−, F−, HPO42−, HSO3 , NO3 , S2O3
,
SCN−, SO42− and S2− showed almost no changes to the fluorescent spectra of probe 1.
Although Hcy and GSH showed some changes, they are far less than that of Cys even
they were used at higher concentrations (Fig. S4). This result indicates that probe 1
has high selectivity for Cys over these analytes including Hcy and GSH. This
selective detection of Cys can be conveniently observed by the naked-eye under a
portable 365 nm light (Fig. S5). Moreover, detection of Cys using probe 1 in the
presence of various analytes was effective (Fig. S6). It is worth noting that probe 1
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showed small changes to millimolar level of GSH, which is important for Cys
detection in living systems, because as mentioned before, the abundant GSH in living
cells could be the main interference during detection of Cys. Clearly, all these results
showed that probe 1 has high selectivity for Cys.
(b)
(a)
Cys
400
300
200
100
0
400
300
200
100
0
GSH
Hcy
others
2
4
6
8 10 12 14 16 18 20 22 24 26 28 30 32
350
400
450
500
550
600
Wavelength (nm)
Fig. 2. (a) Fluorescent spectra responses of probe 1 (10 µM) to various analytes. (b)
Fluorescent intensity responses of probe 1 (10 µM) at 446 nm for various analytes
−
(1‐ 32 represent: 1. none, 2. AcO−, 3. Cl−, 4. CO32−, 5. F−, 6. HPO42−, 7. HSO3 , 8.
NO3 , 9. S2O32−, 10. SCN−, 11. SO42−, 12. S2−, 13. Gln, 14. Ser, 15. Pyr, 16. Trp, 17.
−
Asp, 18. Ala, 19. Phe, 20. His, 21. Met, 22. Thr, 23. Gly, 24. Lys, 25. Glu, 26. Ile, 27.
Arg, 28. Tyr, 29. Leu, 30. Hcy, 31. GSH, 32. Cys. Cys and GSH was used 100 ꢀM
and 1mM, respectively, others were used 200 µM). Each spectrum was collected after
30 min of mixing each analyte with probe 1 in HEPES buffer (10 mM, pH 7.2,
containing 10% DMSO, v/v) at 25°C. λex = 333 nm, dex = dem = 5 nm.
The optical changes of probe 1 toward Cys suggested that the reaction produced
the fluorescent compound 2 (Fig. S7). To prove this, TLC, HPLC and HR-MS
analyses of the reaction mixture were performed. The results of TLC and HPLC
analyses with the reference compound 2 clearly indicated that the reaction produced 2
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(Fig. S8-9). This was further proved by the HR-MS analysis of the reaction mixture,
in which the peak at 244.0425 can be assigned to 2 + H+ (Fig. S10). With this
information and according to the conjugate addition-cyclization reaction process
recently reported for acrylate-functionalized fluorescent Cys probes [35-50], the
sensing mechanism of probe 1 for Cys is most likely the process as proposed in
Scheme S1. The high selectivity of probe 1 for Cys over Hcy and GSH can be
explained by the fact that the double acrylate-functionalized probe 1 showed much
faster reaction kinetics in a dual conjugate addition-cyclization reaction process for
Cys over Hcy and GSH to release compound 2, which results in much faster optical
changes [56,59]. This was proved by kinetic experiments as shown in Fig. S11.
3.4. The sensitivity
After figuring out the high selectivity of probe 1 for Cys, we turned our attention
to its sensitivity. As shown in Fig. 3, upon addition of different concentrations of Cys,
the fluorescence enhancement of probe 1 for Cys showed a dose-dependent response
until reached saturation after addition of 10 equiv of Cys. Importantly, the fluorescent
intensity changes of the probe 1 solution at 446 nm was found linearly increased when
Cys was progressively added from 0 to 100 ꢀM (R2 = 0.9975, Fig. 4b), which gives a
detection limit of probe 1 for Cys to be about 0.80 ꢀM (based on signal-to-noise ratio
S/N = 3). This value is much lower than the reported intracellular concentration of
Cys (30–200 ꢀM), indicating that probe 1 has sufficient sensitivity to detect Cys
found in biological systems. In addition, the wide linear range of probe 1 for Cys is
highly favorable for quantitative detection of Cys.
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(b)
y = a + b*
No Weigh
436.9672
Equation
Weight
Residual
Sum of
(a)
400
300
200
100
0
400
200
0
Cys
0-200 µM
400
300
200
100
0
Squares
0.99749
Adj. R-Squar
Value Standard E
Intercept 8.451
Slope
3.3033
0.0583
B
B
3.855
0
50
100 150 200
[Cys]
µM
0
20
40
[Cys]
60
80
100
350
400
450
500
550
600
µ
M
Wavelength (nm)
Fig. 3. (a) Fluorescence spectra changes of probe 1 (10 µM) upon addition of
different concentrations of Cys (0, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150,
170 and 200 µM) in HEPES buffer (10 mM, pH 7.2, with 10% DMSO, v/v) at 25 °C.
Each spectrum was obtained 30 min after Cys addition. λex = 333 nm, dex = dem = 5
nm. The plot of fluorescence intensity at 446 nm as a function of Cys concentrations
was inserted. (b) Linear relationship of the fluorescence intensity at 446 nm as a
function of concentration from 0 to 100 ꢀM. Data were fitted by an equation: y =
8.451 + 3.855× [Cys] with R2 = 0.9975.
3.5. The potential applications of probe 1
The potential applications of probe 1 were also investigated. Since compound 2
shows bright fluorescence in solid state (Fig. S12), a paper test strip system with
probe 1 should be good for a convenient detection of Cys. Thus, ready to use paper
test strips were first made simply by wetting neutral filter paper with a probe 1
solution (1 M in DCM) and then dried in air. As shown in Fig. S13, when a Cys
solution (200 ꢀM) was spotted to this test paper with a glass capillary, rapid and
distinct emission color changes can be observed under a portable 365 nm light. In
contrast, other analytes did not cause any detectable changes except that Hcy and
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GSH showed slight changes; however, they are obvious difference to the changes
caused by Cys. This result indicates that probe
1 can be used to develop an
easy-to-prepare and easy-to-detect paper test strip system for a convenient detection
of Cys.
Fig. 4. Fluorescent imaging of Cys with probe
field images. A: blank HeLa cells; B: HeLa cells were treated with probe
30 min; C: HeLa cells were pre-incubated with NEM (500 ꢀM) for 30 min and then
treated with probe (5 ꢀM) for 30 min; D: HeLa cells were pre-incubated with NEM
(500 ꢀM) for 30 min and then treated with Cys (50 ꢀM) for 30 min and followed by
1
in living cells. Top (A-D): bright
1
(5 ꢀM) for
1
treating with probe
respectively.
1 for 30 min. Bottom: A1-D1 are fluorescence images of A-D,
The application of probe
investigated. As shown in Fig. 4, HeLa cells did not show fluorescence (A1), however,
when cells were incubated with probe , they started to show strong blue green
1 for bioimaging of intracellular Cys was also
1
fluorescence (B1). As a control experiment, when cells were pre-treated with
N-ethylmaleimide (NEM), a known thiol trapping reagent [15], and followed by
incubation with probe
1, the cells showed no fluorescence (C1). As another control,
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after HeLa cells were pretreated with NEM, with the addition of Cys (50 ꢀM) and
then incubated with probe
(D1). These results indicate that probe
to image intracellular and exogenous Cys. The cytotoxicity of probe
investigated by MTT assays and the results showed that probe is of low cytotoxicity
holds
1
, the strong blue green fluorescence can be observed again
has good membrane permeability and is able
was also
1
1
1
to cultured cells (Fig. S14). Therefore, all of the results indicate that probe
great potential for applications involving bioimaging of Cys.
4. Conclusions
1
In summary, we developed a new fluorescent probe for Cys. This probe has very
simple structure and can be easily prepared. More importantly, it shows rapid, highly
selective and sensitive fluorescent turn-on signal changes for Cys in aqueous solution
under mild conditions. In addition, it can be applied to develop a cheap and effective
paper test strip system for Cys detection and can be applied for imaging of both
intracellular and exogenous Cys in living cells. Based on these merits, this probe can
be used as a new valuable research tool for applications involving Cys detection.
Acknowledgment
This work was financially supported by National Natural Science Foundation of
China (Grant Nos. 21472066 and 21672080), the Natural Science Foundation of
Hubei Province (No. 2014CFA042) and the self-determined research funds of CCNU
from the colleges’ basic research and operation of MOE (CCNU16A02028).
Appendix
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For the structure characterizations of the probe and additional data, please refer
to Supplementary data.
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Supplementary Data
for
A simple and readily available fluorescent turn-on probe for
cysteine detection and bioimaging in living cells
Haiyan Zhanga, Weiyong Fengb and Guoqiang Fengb,*
a School of Chemical and Environmental Engineering, Wuhan Polytechnic University,
68 Xuefu South Road, Wuhan 430023, People's Republic of China
bKey Laboratory of Pesticide and Chemical Biology of Ministry of Education,
College of Chemistry, Central China Normal University, 152 Luoyu Road, Wuhan
430079, People's Republic of China
*Corresponding author. E-mail: gf256@mail.ccnu.edu.cn (G. Feng).
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1. Structure characterizations for compound 2 and probe 1
1 H-NMR spectrum of compound
2 in d6-DMSO
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13C-NMR spectrum of compound
2 in d6-DMSO
HR-MS spectrum of compound
2
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1 H-NMR spectrum of probe
1 in CDCl3
13C-NMR spectrum of probe
1 in CDCl3
29