Rhodamine-based fluorogenic probe for imaging biological thiol

Article abstract of DOI:10.1016/j.bmcl.2008.03.014

We have developed a new fluorescent probe for biological thiol. The probe was synthesized by the modification of the 2,4-dinitrobenzenesulfonyl group with rhodamine 110. The selective detection of thiol species such as cysteine or glutathione was achieved in biological conditions. Moreover, the probe was successfully applied to the imaging of thiol species in living human cells.

Full text of DOI:10.1016/j.bmcl.2008.03.014

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2246–2249
Rhodamine-based fluorogenic probe for imaging biological thiol
a
a,b
a,
b
a
*
Aya Shibata, Kazuhiro Furukawa, Hiroshi Abe, Satoshi Tsuneda and Yoshihiro Ito
a
Nano Medical Engineering Laboratory Discovery Research Institute, RIKEN 2-1, Hirosawa, Wako-Shi, Saitama 351-0198, Japan
b
Department of Life Science and Medical Bio-Science, Waseda University, 3-4-1 Ohkubo, Shinjuku-ku, Tokyo 169-8555, Japan
Received 8 January 2008; revised 3 March 2008; accepted 6 March 2008
Available online 10 March 2008
Abstract—We have developed a new fluorescent probe for biological thiol. The probe was synthesized by the modification of the 2,4-
dinitrobenzenesulfonyl group with rhodamine 110. The selective detection of thiol species such as cysteine or glutathione was
achieved in biological conditions. Moreover, the probe was successfully applied to the imaging of thiol species in living human cells.
Ó 2008 Elsevier Ltd. All rights reserved.
Cellular thiols play important roles in biological sys-
1
undesired background fluorescence. In this paper, we
add a new fluorescent probe derived from Rhodamine
(Rh) (Scheme 1). Rh dyes are highly fluorescent and
–3
tems.
stress associated with toxic agents and disease. Glutathi-
Changes in their levels are linked to oxidative
1
8a
one is the most abundant cellular thiol. It acts as a scav-
resistant to photo-bleaching. Therefore, they are the
most widely used fluorescent reagents for labeling bio-
enger nucleophile or reducing agent and protects cells
against many carcinogens. Homocysteine is a risk factor
for disorders including cardiovascular diseases and Alz-
8
molecules. There is a series of Rh derivatives with a
wide range of emissions, from 450 to 700 nm. Our design
can be applied to many Rh derivatives and can create
multiple color probes for the detection of biological
thiol.
2
heimer’s disease. The thiol group in cysteine (Cys) res-
idues is involved in three-dimensional structures of
3
proteins through disulfide bond formation. Cys defi-
ciency can cause several health problems. Thus, the
detection of intracellular thiols is very important for
investigating cellular function.
The chemistry of fluorogenic probes involves the nucle-
ophilic attack of the thiol group on the DNB group,
resulting in the cleavage of the sulfone-amide bond,
and then Rh in its open lactone form emits a fluores-
cence signal (Scheme 1). The probe was synthesized in
one step from commercially available Rh 110. The start-
ing material was treated with 2,4-dinitrobenzenesulfonyl
chloride in the presence of KOt-Bu in DMF for 16 h and
Ellman’s reagent is the most widely used reagent for the
4
determination of thiol groups. However, this reagent
can be used only in vitro because its assay is based on
absorption change. Several thiol detection methods,
including sensitive fluorescent probes, have been re-
5
–7
9
ported. Fluorescein derivatives protected by 2,4-dini-
6
trobenzenesulfonyl ester have been reported. The probe
the desired probe was obtained in 10% yield. Mono-
10
DNB-protected Rh was synthesized as a reference.
reacts with biological thiol and offers high fluorescent
intensity with short reaction times. However, the probe
may yield undesired background fluorescence because
the sulfonyl ester is hydrolyzed in biological aqueous
solutions.
The probe and mono-DNB Rh show very low fluores-
cence quantum yields, 0.0007 and 0.003, respectively,
in contrast with the high yield of Rh, 0.645 (Table 1).
This result indicates that the observed fluorescence sig-
nal comes from the Rh after the deprotection of the
bis-DNB group in the probe when sensing biological
thiol.
Recently, fluorescent probes with the 2,4-dinitroben-
7
zenesulfonamide (DNB) group have been reported.
The sulfonamide group resists hydrolysis and yields no
First, to test the fluorescence response, the probe was
incubated in solution with or without Cys in Tris–HCl
buffer (50 mM, pH 7.4). The resulting absorption and
fluorescence spectra are shown in Figure 1. The solution
Keywords: Thiol; Imaging; Fluorescence; Probe; Synthesis.
*
Corresponding author. Tel.: +81 48 467 9303; fax: +81 48 467
300; e-mail: h-abe@riken.jp
9
0
960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.03.014

A. Shibata et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2246–2249
2247
O
O
O
H
N
H
N
H
N
NH
O2N
S
O
S
NO2
R-SH
H2N
O
NH O2N
COOH
S
O
O
O
O
NO2
NO2
NO2
O
RS
NO2
SO2
+
O
+
COOH
+
NO2
Probe
no fluorescence
Rhodamine
mono-DNB Rhodamine
strong fluorescence
weak fluorescence
Scheme 1. The reaction mechanism of fluorogenic probe. The DNB group is cleaved through the reaction with thiol and the fluorescence signal
appeared.
a
Table 1. The quantum yields of the compounds
was enhanced about 5800-fold (Fig. 1b). To test the
sensitivity, fluorescence spectra were measured in vari-
ous concentrations of Cys. The concentration limit for
the detection of Cys was 100 lM with 100 nM of probe,
where the signal to background ratio reached 12 times.
To determine the chemical yield in the reaction of the
probe with Cys, the reaction mixture was analyzed by
HPLC (Fig. S1). The treatment of 1 mM Cys gave
mono-DNB Rh and Rh in 14% and 41% yields,
respectively.
Compound
Probe
0.0007
Mono-DNB Rh
0.003
Rh
b
F
U
0.645
a
All measurements were done in sodium phosphate buffer (100 mM,
pH 7.4). Compounds were excited at 490 nm.
b
Quantum yields are determined by using fluorescein (0.85, 0.1 M
NaOH) as a standard.
0
0
0
0
.8
.6
.4
.2
0
a
Selectivity is an important issue in applying the probe to
the detection of biological thiol. Probes should specifi-
cally respond to the thiol and yield no unexpected signal
to other biological substances. To determine the selectiv-
ity, we treated the probe with various biological sub-
stances and measured the fluorescence signals. The
selectivity of the probe for thiol was proved by monitor-
ing the fluorescence emission at 522 nm with biorelevant
analytes under physiological conditions. As shown in
Figure 2, significant fluorescence intensity enhancement
was observed for Cys, phosphorothioate, dithiothreitol,
and glutathione (50- to 200-fold). However, the reaction
with 2-mercaptoethanol (ME) showed only a small in-
3
00
400
500
600
500
400
300
200
100
0
b
Cys
crease in fluorescence (11-fold). The pK value of ME
a
shows a value of 9.5, higher than that of other thiols;
therefore, the reaction with the DNB group should be
1
1
slow. HPLC analysis showed that ME treatment gave
unreacted probe and mono-DNB Rh in 85% and 15%
yields, respectively. As expected, the probe was inactive
to other biological substances such as glycine, ascorbic
acid, or hydrogen peroxide. Likewise, basic amino acid
series such as Lys or Arg showed no signal. The probe
was very stable in biological environments. For exam-
ple, no increase in the fluorescence signal was detected
after the probe was heated for 24 h at 55 °C in 50 mM
Tris buffer (pH 7.4). Thus, we confirmed that the new
probe offers good selectivity to biological thiol and no
undesired fluorescence signals.
5
00
550
600
Figure 1. (a) The absorption spectra of 20 lM probe with (solid line)
and without (dash line) 20 mM Cys in 50 mM Tris–HCl (pH 7.4). (b)
Fluorescence spectra of 100 nM probe with (solid line) and without
(
dash line) 10, 5, 2.5, 1, 0.5, 0.1 mM Cys after incubation at 37 °C for
30 min in 50 mM Tris–HCl (pH 7.4). The fluorescence emission was
monitored at the excitation of 490 nm.
The most important application for this probe is the
monitoring of thiol in living cells. To test this capability,
we tried to image biological thiol in living HeLa cells.
The pictures of brightfield images and fluorescence
images were taken by fluorescence microscope (Fig. 3).
When incubated with the probe (25 lM) for 15 min,
cells showed significant fluorescence signals (Fig. 3d).
The strong signal was localized to cytoplasm. On the
other hand, nuclei showed weak fluorescence signals.
In contrast, when cells were pretreated with the thiol-
without Cys exhibited maximum absorption at 516 nm,
which is derived from the DNB group. When Cys was
added to the probe solution, the maximum absorption
peak was shifted to the shorter wavelength of 498 nm,
resulting from the cleavage of the DNB group
(
Fig. 1a). Fluorescence properties were also examined.
No significant fluorescence with excitation at 490 nm
was observed for the probe without Cys. However, after
the addition of Cys (10 mM) to the solution, a strong
emission appeared around 520 nm and the emission
1
2
blocking reagent N-methylmaleimide and then incu-
bated with the probe in a similar manner, no fluores-

2248
A. Shibata et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2246–2249
40
30
20
10
0
blank 3’-thio dT DTT
L-Cys
ME
GSH
L-Gly
L-Arg L-Lys H O
Vc
2
2
Figure 2. The fluorescence intensity of 1 lM probe with 1 mM thiols in 50 mM Tris–HCl (pH 7.4). After the addition of thiol, the solution was
0
0
0
incubated at 37 °C for 30 min. The fluorescence emission at 522 nm was monitored by excitation at 490 nm. 3 -Thio dT, 3 -phosphorothioate-2 -
deoxythymidine; DTT, dithiothreitol; GSH, glutathione; L-Gly, L-glysine; L-Arg, L-arginine; L-Lys, L-lysine; Vc, ascorbic acid.
Figure 3. The fluorescence image of HeLa cells: (a and b) the control image of cells pretreated with N-methylmaleimide (1 mM) for 60 min at 37 °C
and then incubated with probe (25 lM) for 15 min at 37 °C; (c and d) the image of cells incubated with probe (25 lM) for 15 min at 37 °C; (a and c)
brightfield image; (b and d) fluorescence image. Microscope settings were as follows: excitation: 470/40 bandpass filter; emission: 525/50 bandpass
filter; exposure time 300 ms.
cence signal was observed (Fig. 3b). The results demon-
strate that the probe penetrates cell membranes and
images the changes in thiol levels of living cells.
References and notes
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In conclusion, we developed a novel fluorescent probe
that yields a signal responsive to thiols. The probe was
successfully applied to the imaging of biological thiol
in living cells.
2
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Acknowledgments
H.A. was financially supported by the Riken Directors’
Fund, MEXT (Ministry of Education, Culture, Sports,
Science, and Technology), and NEDO (New Energy
and Industrial Technology Development Organization).
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2249
1
9. Probe; H NMR (400 MHz, CDCl
(
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3
/CD
3
OD): d 8.47–8.46
(d, 2H, J = 2.2), 8.37–8.34 (dd, 2H, J = 2.2, 11.0), 8.19–
8.17 (d, 2H, J = 8.8), 7.91–7.89 (d, 1H, J = 7.1), 7.61–7.53
(m, 2H), 7.04–7.01 (m, 3H), 6.74–6.72 (dd, 2H, J = 2.2,
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1
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11.0), 6.53–6.51 (d, 2H, J = 8.5); C NMR (99.5 MHz,
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CDCl /CD OD) d 168.97, 151.38, 149.84, 148.06, 137.38,
135.28, 133.07, 130.11, 129.16, 127.12, 126.62, 125.99,
125.10, 123.58, 120.31, 116.52, 115.68, 108.70; HRMS
3
3
(
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(ESI) m/z calcd for C32
found 789.0176.
H
17
N
6
O
15
S
2
(M–H) 789.0193;
1
10. Mono-DNB Rh; H NMR (400 MHz, CDCl
6
7
8
3
/CD OD): d
3
8.48–8.47 (d, 1H, J = 2.2), 8.43–8.40 (dd, 1H, J = 2.2, 6.6),
8.31–8.29 (d, 1H, J = 8.8), 8.05–8.02 (m, 1H), 7.64–7.61
(m, 2H), 7.15–7.13 (m, 3H), 6.85–6.82 (dd, 2H, J = 2.2,
6.8), 6.78–6.72 (m, 2H), 6.57–6.56 (d, 1H, J = 2.2), 6.49–
1
3
6.46 (dd, 1H, J = 2.2, 6.6); C NMR (99.5 MHz, CDCl
3
/
CD OD) d 170.41, 154.34, 153.64, 152.49, 148.70, 148.01,
3
140.82, 132.86, 131.95, 129.87, 129.45, 129.11, 126.49,
125.87, 125.59, 119.33, 119.19, 114.26, 113.31, 107.06,
99.63; HRMS (ESI) m/z calcd for C26H N O S (M–H)
15 4 9
559.0560; found 559.0569.
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