A benzothiazole-based fluorescent probe for thiol bioimaging

Article abstract of DOI:10.1016/j.tetlet.2012.02.098

This study reports a benzothiazole-based fluorescent probe with simple structure for thiols. This probe exhibited high on/off signal ratios and good selectivity toward thiols over other analytes, and was successfully applied to the imaging of thiols in living cells.

Full text of DOI:10.1016/j.tetlet.2012.02.098

Tetrahedron Letters 53 (2012) 2332–2335
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
A benzothiazole-based fluorescent probe for thiol bioimaging
Wei Sun a, Wenhua Li a, Jing Li a, Jian Zhang b, Lupei Du a, Minyong Li a,
⇑
a Department of Medicinal Chemistry, Key Laboratory of Chemical Biology of Natural Products (MOE), School of Pharmacy, Shandong University, Jinan, Shandong 250012, China
b Institute of Immunopharmacology & Immunotherapy, School of Pharmacy, Shandong University, Jinan, Shandong 250012, China
a r t i c l e i n f o
a b s t r a c t
Article history:
This study reports a benzothiazole-based fluorescent probe with simple structure for thiols. This probe
exhibited high on/off signal ratios and good selectivity toward thiols over other analytes, and was suc-
cessfully applied to the imaging of thiols in living cells.
Received 20 December 2011
Revised 9 February 2012
Accepted 24 February 2012
Available online 3 March 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Benzothiazole
Fluorescent probe
Thiols
Cell imaging
Biological thiols/low molecular weight thiols, such as cysteine
(Cys), homocysteine (Homo-Cys), and glutathione (GSH), which
can regulate the intracellular redox state and higher-order struc-
tures of proteins, are active in the catalytic sites of numerous en-
zymes, and participate in intracellular signal transduction and
gene regulation.1 Generally, the levels of cellular thiols have been
associated to toxic agents and diseases, including slowed growth,
leucocyte loss, psoriasis, liver damage, cancer, and AIDS.2 Conse-
quently, selective detection of biological thiols should provide crit-
ical insight into pathological and biological sciences.
Among the various methods for detecting thiols, fluorescent
molecular probes are of great interest because of their simplicity,
low detection limit, wide range of dynamic response, and feasibility
of intracellular detection.3 Apart from reasonable physical proper-
ties (excitation/emission wavelength, Stokes shift, etc.), high
response and good selectivity, an ideal fluorescent probe should
be convenient to synthesize, act fast in mild condition, and have
high permeability. In the present research, a simple benzothiazole
fluorophore was masked with 2,4-dinitrobenzenesulfonyl (DNS),
which was studied widely in the detection of thiols.4 Thiols can
release the fluorophore by the nucleophilic cleavage of the 2,4
-dinitrobenzenesulfonyl ester. This simple molecule has most of
the desirable features, and thereafter should be particularly applica-
ble for the in vitro and in vivo detection of thiols.
with 2,4-dinitrobenzene-sulfonyl chloride to obtain probe 1 in the
presence of TEA in DCM with 80% yield.
The fluorescent properties of such a probe were evaluated un-
der near physiological conditions (0.1 M phosphate buffer, pH
7.4). The absorption at 330 nm of 2-(benzo[d]thiazol-2-yl)phenol
was dismissed after the connection of 2,4-dinitrobenzene-sulfonyl,
and no fluorescence was found in probe 1. The fluorescent inten-
sity change of 1 was initially tested according to the reaction time
with thiophenol (PhSH). As demonstrated in Figure 1, the fluores-
cent intensity reached the high plateau at 2 min after the addition
of PhSH, and almost no change appeared when longer reaction
time was examined.
To determine the stability of the probe in PBS buffer solution,
we treated it under various conditions by detecting the change of
its fluorescent intensity (see Supplementary data, Fig. S1). It was
found that the probe was essentially stable over a range of 5–9 un-
der ambient light or protected from light, with a low level of its
hydrolysis at pH 9.0
The sensitivity of probe 1 was then deliberated by fluorescence
response toward different concentrations of PhSH. It was examined
at 5 min after the addition of PhSH (Fig. 2). Upon the addition of
PhSH (40 lM), the fluorescence intensity increases more than
25-fold with the equilibrium constant (logK) being 12.1. And the
fluorescence intensity of probe significantly increased as a corre-
As depicted in Scheme 1, probe 1 started from commercially
available 2-(benzo[d]thiazol-2-yl)phenol and was readily synthe-
sized in only one step. 2-(Benzo[d]thiazol-2-yl)phenol was treated
sponding function of PhSH concentration at more than 1.25 lM.
The responses of the probe toward relevant aliphatic thiols,
reactive sulfur species, common nucleophiles, and amino acid were
considered as well. As shown in Figure 3, probe 1 encourages a
significant response to aromatic thiols, including thiophenol
(PhSH), 4-chlorothiophenol (4-Cl–PhSH), and 2-aminothiophenol
⇑
Corresponding author. Tel./fax: +86 531 8838 2076.
E-mail address: mli@sdu.edu.cn (M. Li).
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2012.02.098

W. Sun et al. / Tetrahedron Letters 53 (2012) 2332–2335
2333
NO2
O2N
SO2Cl
NO2
O
S
R
O
S
O
HO
HO
O2N
R-SH
NO2
S
N
S
N
S
N
+
+
SO2
NO2
Probe 1
NO2
S R
R-SH
O2N
O
S
O
O
S
N
Scheme 1. Synthesis of probe 1.
weak responses, which are 1/2–1/3 those of aromatic thiols. The
aniline and other active sulfur species, including sodium hydrosul-
fide (NaHS, a well-known donor of H2S), the sulfide anion and
bisulfite, which can also cleave benzenesulfonamide,5 induced
weaker responses (which are 1/3–1/4 those of aliphatic thiols),
while the other analytes including common nucleophile (KI), ami-
35
30
25
20
15
10
5
no acids (L-Gly and L-Glu) did not result in a significant fluorescent
response. Considering the possible reaction of DNS-based fluores-
cent probes with ROS,6 the fluorescence response to ROS of the
probe was also detected. In brief, this probe has no response to
KO2 and H2O2.
The implementation of the probe on fluorescent imaging was fi-
nally explored in living cells. After incubated with probe 1 (40 lM)
for 30 min at 37 °C, human prostate cancer cell line (PC-3) was ob-
served by fluorescence microscopy. As shown in Figure 4a, probe 1
can penetrate cell membrane to react with intracellular thiols,
resulting in strong fluorescence emission. Furthermore, PC-3 cells
0
0
100
200
300
400
500
were pretreated with 200
0.5 h to reduce the concentration level of biological thiols, and then
they were incubated with the probe 1 (40 M) for another 30 min.
lM N-methylmaleimide (NEM) for
Time (s)
l
Figure 1. Time course of the interaction of probe 1 (20
(pH 7.4, 0.2% DMSO as a cosolvent). The concentration of thiophenol is 20 lM.
Fluorescence intensity was recorded at 460 nm with kex = 340 nm.
l
M) with thiophenol in PBS
Distinct decrease of fluorescence response in PC-3 cells was ob-
served (Fig. 4c and e). These interesting results demonstrated that
probe 1 could be used for fluorescent imaging of thiols in living
cells.
200
Additionally, we detected the fluorescent response of 1 toward
different ratios of commercial rabbit plasma pretreated with tri-
phenylphosphine which can reduce the disulfides to free thiols.7
As depicted in Figure S2, the fluorescence intensity undertook linear
relationship with the concentration of plasma. Such evidence indi-
cates that probe 1 could detect free thiols in plasma quantitatively.
In conclusion, this report describes a selective benzothiazole-
based fluorescent probe with small size and easy synthetic handling
for the detection of thiols in cellulo. This probe has significant re-
sponses to thiophenols. Aliphatic thiols can also induce slight re-
sponses for this probe (1/2–1/3 those of aromatic thiols), while
the other competitive analytes exhibit weak or no response. Fur-
thermore, probe 1 is satisfactory for determining cellular thiols,
which is substantiated by control experiments (with N-methylma-
leimide) to be specific toward thiols in living cells. Consequently,
the ramifications of this study suggest that this probe may offer a
quick, simple, and selective way for the detection of thiols in vitro
and vivo.
40 μΜ
20 μΜ
10 μΜ
5 μΜ
2.5 μΜ
1.25 μΜ
0
180
160
140
120
100
80
60
40
20
0
450
500
550
600
Wavelength (nm)
Figure 2. Concentration-dependent emission intensity changes of probe 1 at room
temperature: Experiments were conducted in 0.1 M phosphate buffer, pH 7.4. The
emission spectra were obtained at 5 min after the addition of PhSH to a 20 lM
Acknowledgments
solution of probe 1 with kex = 340 nm.
The present work was financed by grants from Shandong Natu-
ral Science Foundation (No. JQ201019) and Independent Innova-
tion Foundation of Shandong University, IIFSDU (No. 2010JQ005).
(2-NH2–PhSH). In this case, aliphatic thiols, including
L-glutathione
(GSH), -cysteine (Cys), and thioglycolic acid (TGA), can also induce
L

2334
W. Sun et al. / Tetrahedron Letters 53 (2012) 2332–2335
40
30
20
10
0
5 min
10 min
20 min
30 min
Na2S NaHSO3 KI
Aniline H2O2 KO2
Gly
blank
NaSH
Glu
PhSH Cl-PhSH NH2-PhSH GSH Cys
TGA
Analytes
Figure 3. Fluorescence responses of probe 1 (20
lM) to different analytes. Relative florescence intensities of probe before and after incubation with analytes (25 lM) were
acquired in 0.1 M phosphate buffer (0.2% DMSO), pH 7.4, and all data were obtained at 450 nm (kex = 340 nm).
a
b
d
c
e
15
10
5
0
probe 1
probe 1 + NEM
Figure 4. Fluorescence microscopic images of live PC-3 cells. Cells were incubated with probe 1 (40 lM) for 30 min at 37 °C before imaging. (a) Is the fluorescence image of
cells incubated with probe 1; (b) is the corresponding bright field image of (a); (c) represents the fluorescence image of cells incubated with probe 1, after preincubation with
NEM (200 M) for 30 min; (d) represents the corresponding bright field image of (c); (e) shows the mean fluorescent photons and relative ratio of images (a) and (c) processed
l
by ImageJ software.
Supplementary data
References and notes
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A.; Choi, Y. B.; Takahashi, H.; Zhang, D. X.; Li, W. Z.; Godzik, A.; Bankston, L. A.
Trends Neurosci. 2002, 25, 474–480.
Supplementary data associated with this article can be found, in the
online version, at doi:10.1016/j.tetlet.2012.02.098. These data include
MOL files and InChiKeys of the most important compounds described
in this article.

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