Highly selective fluorescence turn-on probe for glutathione

Article abstract of DOI:10.1016/j.tetlet.2011.05.086

An activated coumarin-3-phenyl enone (1) by an intramolecular hydrogen bond has shown a selective and ratiometric response toward GSH and Cys over other natural amino acids through the Michael addition reaction of a thiol group to 1. When GSH was added to

Full text of DOI:10.1016/j.tetlet.2011.05.086

Tetrahedron Letters 52 (2011) 3902–3904
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Highly selective fluorescence turn-on probe for glutathione
Soo-Yeon Lim a, Sanghee Lee b, Seung Bum Park b,c, , Hae-Jo Kim a,
⇑
⇑
a Department of Chemistry, Hankuk University of Foreign Studies, Yongin 449-791, Republic of Korea
b Department of Chemistry, Seoul National University, Seoul 151-742, Republic of Korea
c Department of Biophysics and Chemical Biology, Seoul National University, Seoul 151-742, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
An activated coumarin-3-phenyl enone (1) by an intramolecular hydrogen bond has shown a selective
and ratiometric response toward GSH and Cys over other natural amino acids through the Michael addi-
tion reaction of a thiol group to 1. When GSH was added to 1, a prominent color change together with a
fluorescence turn-on property was observed so that submillimolar GSH was detectable.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 26 April 2011
Revised 16 May 2011
Accepted 18 May 2011
Available online 25 May 2011
Keywords:
Fluorescence
GSH
Hydrogen bond
Michael addition
Biothiols such as cysteine (Cys), homocysteine (Hcy), and
c
-
Probe 2 without an o-hydroxyl group was also synthesized as a
control compound (see the Supplementary data).
glutamylcysteinylglycine (GSH) are involved in a myriad of vital
cellular processes, including redox homeostasis1 and cellular
growth.2 GSH is the most abundant cellular thiol whose concentra-
tion ranges from 1.0 to 15 mM.3 It serves many cellular functions,
including the maintenance of intracellular redox activity, xenobi-
otic metabolism, intra-cellular signal transduction, and gene regu-
lation.4 Moreover, abnormal levels of GSH are known to be
implicated in human diseases, such as leucocyte loss, psoriasis,
liver damage, cancer, and AIDS.5 Although a number of probes have
been developed on the basis of the changes in the optical intensity
upon binding with GSH, they cannot provide quantitative informa-
tion about changes in GSH concentrations due to artifacts associ-
ated with probe concentration and the environment. Therefore, it
is of great scientific interest and intense activity to develop a ratio-
metric probe. Herein we report a conjugated enone type of Michael
acceptor (1), which exhibits a highly selective and ratiometric
response to GSH both in vitro and in vivo.
Recently, we reported optical probes with an o-hydroxy car-
bonyl group, which could be applied for sensing a reactive nucleo-
phile such as cyanide or thiol-containing amino acid.6 For the
ratiometric probe, a coumarin derivative (1) was conjugated to
enone possessing a hydroxyl group (Scheme 1), where the couma-
rin moiety was designed to function as a signaling unit, the conju-
gated enone as a reaction unit, and the o-hydroxyl group as an
activation unit through the resonance-assisted hydrogen bond.7
The reaction of 1 with a thiol is observable by 1H NMR spectros-
copy (Fig. 1). Upon the addition of ME as a model compound of
biothiol, the phenolic proton (Ha at d 12.6 ppm) of 1 of the reso-
nance-assisted hydrogen bond was significantly upfield shifted to
Hb at d 11.7 ppm due to the plausible Michael addition reaction.
High resolution mass spectral analysis of the resulting mixture
has shown corroborative evidence for the product (1ÀME) forma-
tion at m/z obsd 518.2004 (calcd 518.2001 for C30H32NO5S, Fig. S8,
Supplementary data).
As expected, the UV–vis spectra exhibit a ratiometric change
when 1 is treated with GSH. While 1 shows an absorbance maxi-
mum at 434 nm (
triggers a hypsochromic shift (DA = 30 nm) to kmax 404 nm with
e
= 3.81 Â 104 MÀ1 cmÀ1), the 1-GSH conjugate
an apparent isosbestic point at 417 nm (Fig. 2). Time-dependent
UV–vis spectra of 1 (10 M) were monitored in the presence of
l
excess GSH (20 mM) in DMF/HEPES (3:1, 0.10 M, pH 7.4). The for-
mation of 1-GSH was complete within 2 h with a rate constant of
kobs = 9.8 Â 10À5 sÀ1 under the pseudo first-order reaction condi-
tions, whereas the formation rate of 2-GSH was too slow to mea-
sure (Fig. S2). The kinetic experiments indicate that the hydroxyl
group of 1 plays a significant role for the rate acceleration through
an intramolecular hydrogen bond.
The fluorescence spectra of 1 undergo a dramatic increase (F/Fo
22 at kmax 480 nm) upon the addition of GSH in DMF/HEPES (3:1,
0.10 M, pH 7.4) (Fig. 3). Similarly, Cys also induces a significant
fluorescence turn-on of 1 (F/Fo 16). The other natural amino acids
with neutral, basic, or acidic side chains, however, do not induce
any significant fluorescence changes (F/Fo <1.1). Competition
⇑
Corresponding authors. Tel.: +82 31 330 4703, fax: +82 31 330 4566 (H.-J.K.).
E-mail addresses: sbpark@snu.ac.kr (S.B. Park), haejkim@hufs.ac.kr (H.-J. Kim).
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.05.086

S.-Y. Lim et al. / Tetrahedron Letters 52 (2011) 3902–3904
Signaling/Reaction/Activation
3903
Et2N
O
O
Et2N
O
O
R-CH2SH
b
H
O
RCH2S
O
O
O
a
1
H
Weak Fluorescence
1-thiol
Strong Fluorescence
R =
CHCO2
NH3
-CH2OH
Cys
ME
Scheme 1. Reaction of 1 with a thiol.
150
C
GSH
A
100
Cys
B
A
50
1 and other AA
0
420
470
520
AA AA + GSH
570
620
25
Ha
Hb
B
20
13
12
11 ppm
15
Figure 1. Partial 1H NMR spectra of 1 (20 mM) upon the addition of ME (3 equiv) in
DMSO-d6. (A) 1, (B) 1+ME, 1 d, (C) 1+ME, 7 days.
10
5
1.8
0
0.6
Host GSH Pro Ala Val Phe His Ser Asp Asn Lys Arg
1.2
0.6
0
Figure 3. (A) Fluorescence responses of 1 (10 lM, kex 417 nm) in the presence of
amino acids (AA) and GSH (20 mM) in DMF/HEPES (3:1, 0.10 M, pH 7.4). (B) The
competitive assay of 1 AA by GSH.
0
2400 4800 7200 9600
Time/sec
0.4
0.2
0
To evaluate the detection limit of GSH in solution, the fluores-
cence spectra of 1 (10 lM) were measured by the incremental
addition of GSH in DMF/HEPES (3:1, 0.10 M, pH 7.4). The lower
limit of detection was 0.18 mM at F/F0 = 3 (Fig. S5), which indicates
probe 1 is operable well below the cellular GSH levels. A Job’s plot
was carried out8 and the binding stoichiometry between 1 and GSH
was observed as 1:1 (Fig. S6).
The change of 1 into 1-GSH was observable by the naked eye
(Fig. 4). Upon the addition of GSH, the solution color of 1 was chan-
ged from yellow to colorless, whereas no significant changes were
observed with other amino acids (Pro, Ala, His, Ser, Asp, Asn, Lys).
The solution of 1-GSH adduct also displayed a strong blue fluores-
cence under a portable UV–vis spectroscope (kex 365 nm).
Probe 1 was applied for cellular imaging of GSH (Fig. 5),9 the most
abundant cellular biothiol.10 For the detection of cellular GSH level,
250
350
450
550
/nm
Figure 2. Time-dependent UV–vis spectra of 1 (10 lM) upon the addition with GSH
(20 mM) in DMF/HEPES buffer (3:1, v/v, 0.10 M, pH 7.4). Inset: its kinetics.
HeLa cells were incubated with 1 (20
twice with PBS before imaging with fluorescent microscopy. When
lM) for 30 min and washed
experiments have shown a consistent selectivity of 1 toward GSH.
The fluorescence of 1-AA was restored to be as large as that of 1-
GSH when GSH was added to the mixtures of 1 and other amino
acid (AA). From the fluorescence experiments, it becomes readily
apparent that probe 1 selectively reacts with GSH and gives rise
to profound fluorescence turn-on changes.
the cells are treated with 250 lM lipoic acid for 48 h to boost cellular
GSH level,11 the intensity of 1 significantly enhanced to 2-fold higher
than the control. In contrast, fluorescence signal of 1 decreased upon
treatment with 500 l
M N-ethylmaleimide, scavenger of GSH12 for

3904
S.-Y. Lim et al. / Tetrahedron Letters 52 (2011) 3902–3904
Acknowledgment
This study was partly supported by National Research Founda-
tion of Korea (NRF) and the WCU program of the NRF funded by the
Korean Ministry of Education, Science, and Technology (MEST). S.
Lee is grateful for the fellowships awarded by the BK21 Program
and the Seoul Science Fellowship.
Supplementary data
Supplementary data (experimental procedures) associated with
this article can be found, in the online version, at doi:10.1016/
j.tetlet.2011.05.086.
Figure 4. Naked-eye (A) and fluorescence (B) images (kex 365 nm) of 1 (10 lM in
DMF/HEPES, 3:1, 0.10 M, pH 7.4) upon the addition of amino acids (2000 equiv).
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Figure 5. Fluorescence microscopic images of HeLa cells, incubated with 20
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30 min due to partial sequestering of GSH. Therefore, probe 1 can
visualize the cellular GSH level effectively.
In conclusion, we prepared a highly activated fluorescent probe
(1) by an intramolecular hydrogen bond. Probe 1 exhibits a highly
selective fluorescence response to GSH both in vitro and in vivo.
The fluorescence turn-on response of 1 allows us to discriminate
the biothiols including GSH and Cys from other natural amino
acids by naked eye.
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