The development of a fluorescence turn-on sensor for cysteine, glutathione and other biothiols. A kinetic study

Article abstract of DOI:10.1016/j.tetlet.2011.09.137

Two fluorescence probes for the detection of cysteine (Cys), glutathione (GSH) and other biothiols, such as homocysteine (Hcy) and cysteinyl-glycine (Cys-Gly), were developed. These molecular probes are coumarin-based derivatives containing a chalcone-like moiety that reacts with biothiols through a Michael addition reaction, leading to strong fluorescence enhancements. The reactivity of the tested biothiols toward both probes (ChC1 and ChC2) follows the order Cys > GSH > Hcy > Cys-Gly, ChC1 being less reactive than ChC2. Possible interference with other amino acids was assessed. ChC1 and ChC2 display a highly selective fluorescence enhancement with thiols, allowing these probes to be used for fluorimetric thiol determination in SH-SY5Y cells.

Full text of DOI:10.1016/j.tetlet.2011.09.137

Tetrahedron Letters 52 (2011) 6606–6609
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
The development of a fluorescence turn-on sensor for cysteine, glutathione
and other biothiols. A kinetic study
Olimpo García-Beltrán a,d, Natalia Mena b, Edwin G. Pérez c,e, Bruce K. Cassels a,c, Marco T. Nuñez b,c
,
e,
Francisca Werlinger e, Daniel Zavala e, Margarita E. Aliaga e, , Paulina Pavez
⇑
⇑
a Department of Chemistry, Faculty of Sciences, University of Chile, Santiago, Chile
b Department of Biology, Faculty of Sciences, University of Chile, Santiago, Chile
c Institute for Cell Dynamics and Biotechnology, University of Chile, Santiago, Chile
d Departamento de Ciencias Químicas, Facultad de Ciencias Exactas, Universidad Andrés Bello, Avenida República 275, Piso 3, Santiago, Chile
e Facultad de Química, Pontificia Universidad Católica de Chile, Av. Vicuña Mackenna 4860, Santiago 6094411, Chile
a r t i c l e i n f o
a b s t r a c t
Article history:
Two fluorescence probes for the detection of cysteine (Cys), glutathione (GSH) and other biothiols, such
as homocysteine (Hcy) and cysteinyl-glycine (Cys-Gly), were developed. These molecular probes are
coumarin-based derivatives containing a chalcone-like moiety that reacts with biothiols through a
Michael addition reaction, leading to strong fluorescence enhancements. The reactivity of the tested bio-
thiols toward both probes (ChC1 and ChC2) follows the order Cys > GSH > Hcy > Cys-Gly, ChC1 being less
reactive than ChC2. Possible interference with other amino acids was assessed. ChC1 and ChC2 display a
highly selective fluorescence enhancement with thiols, allowing these probes to be used for fluorimetric
thiol determination in SH-SY5Y cells.
Received 22 August 2011
Revised 22 September 2011
Accepted 29 September 2011
Available online 6 October 2011
Keywords:
Biothiols
Coumarin-based derivatives
Fluorescence probes
Michael addition
Ó 2011 Elsevier Ltd. All rights reserved.
Biothiols (RSH) are compounds involved in many essential cel-
lular functions.1–4 Among these, the tripeptide glutathione (GSH),
The probes ChC1 and ChC2 were readily synthesized in four
steps from commercial precursors (Scheme 1; Supplementary
data). We then proceeded to study the fluorescence response of
these probes to 2-mercaptoethanol (ME), as a model compound
for biothiols (Figs. S3(A) and S4; Supplementary data). Figure
S3(B) shows the turn-on fluorescence of ChC1 upon addition of
GSH (1 mM/HEPES buffer). The formation of the ChC1-GSH adduct
is apparently complete within 50 min.
c
-glutamyl-L-cysteinyl-glycine, is the most abundant low-molecu-
lar-mass non-protein thiol compound in cells.1 Other low molecu-
lar mass thiols that contribute to the intracellular thiol pool are
precursors for the synthesis of GSH: the aminoacids homocysteine
(Hcy) and cysteine (Cys); and the first product of GSH degradation;
the dipeptide cysteinyl-glycine (Cys-Gly).1,3 Diverse reports have
demonstrated that abnormal levels of these biothiols can cause a
number of health problems.5–7 Thus, development of optical
probes for their detection has been an active research area in re-
cent years.8 Among the available techniques to detect and quantify
biothiols (Cys, Hcy and GSH) fluorescence methods have been
extensively pursued due to their simplicity, sensitivity, and
versatility.8
ChC1, excited at 340 nm, emitted with kmax = 430 nm and quan-
tum yield
U = 0.00083. Upon addition of 10 equiv of Cys or GSH the
quantum yield increased to 0.0013 and 0.0010, respectively, with
no band shift. A similar experiment with ChC2 (kmax = 435 nm,
U
= 0.0014) led to the increase of the quantum yield to 0.0022
and 0.0016, respectively.
That these changes are due to a Michael addition was corrobo-
rated by 1H NMR spectroscopy of the probes in the absence or pres-
ence of ME. As shown in Figure 1, upon addition of ME the vinyl
proton resonances (Hb and Ha at d 7.80 and 7.74 ppm, respectively)
disappeared, and concurrently two new triplets, assigned to the
thioether methylene protons Hb and Ha, emerged at d 4.74 and
4.46 ppm, respectively, a result that is consistent with the forma-
tion of the ChC1–ME adduct.
In this work, we focus on the interaction between these biothiols
and coumarin-based compounds containing a chalcone-like moiety.
Specifically, we describe here (E)-3-cinnamoyl-7-methoxy-2H-
chromen-2-one (ChC1) and (E)-3-(3-(2-hydroxyphenyl)acryloyl)-
7-methoxy-2H-chromen-2-one (ChC2) as fluorescence probes for
the detection of Cys, Hcy, GSH and, for the first time, Cys-Gly, med-
iated by a Michael addition reaction (Scheme 1).
The selectivity of ChC1 and ChC2 toward biothiols was investi-
gated by incubating these probes with different species including
non-sulfur amino acids (Asp, Lys, Ser, Hys, Pro, Phe, and Val). As
shown in Figure 2 for the case of ChC1, none of these compounds
⇑
Corresponding authors. Tel.: +56 2 3544742; fax: +56 2 3544744.
E-mail addresses: mealiaga@uc.cl (M.E. Aliaga), ppavezg@uc.cl (P. Pavez).
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.09.137

O. García-Beltrán et al. / Tetrahedron Letters 52 (2011) 6606–6609
6607
R
O
O
O
S
RSH
MeO
O
O
X
MeO
O
X
ChC1 X=H
Strong Fluorescence
Weak Fluorescence
ChC2 X=OH
Scheme 1. Reaction of ChC1 and ChC2 with tested biothiols (RSH).
A
Hc
O
O
O
Hb
Ha
MeO
B
ppm
Figure 1. Change in the partial 1H NMR spectrum of ChC1 (20 mM) upon addition of ME (1.8 equiv) in DMSO-d6 at 25 °C. (A) ChC1 only; (B) ChC1 + ME, (10 min).
Table 1
150
Values of pKa for the sulfhydryl group of biothiols and kN values for the reactions of
these thiols with ChC1 and ChC2
120
90
60
30
0
Biothiols
pKa
kN/sÀ1 MÀ1
ChC1
ChC2
Cysteinylglycine (Cys-Gly)
Cysteine (Cys)
Homocysteine (Hcy)
Glutathione (GSH)
7.00
8.10
8.25
8.72
0.34 0.02
5.17 0.31
0.49 0.04
1.56 0.22
20.14 1.67
60.03 2.34
20.92 1.14
47.48 1.91
constants were calculated. Table 1 shows a comparison of the nucle-
ophilic rate constants (kN) for the reactions of ChC1 and ChC2 with
the tested biothiols. It can be seen that in both cases the reactivity
increases in the sequence Cys > GSH > Hcy P Cys-Gly. This order
is in agreement with the basicity of the sulfhydryl group of each
thiol (pKa values). The only exception is for Cys which, being one
of the less basic thiols, is the most reactive compound. However, a
similar result has been found very recently by other authors.8i,j
Interestingly, we found that GSH is more reactive than Hcy in
the Michael addition to ChC1 and ChC2, in contrast to other probes
which have proved to be less reactive toward GSH in comparison
with Hcy.8i,j However, a very recent paper describes a coumarin-
substituted chalcone probe that reacts efficiently, via activation
by an intramolecular hydrogen bond, with GSH and Cys but only
slightly with non-thiol natural amino acids.9 In addition, a compar-
ison of the nucleophilic rate constants (kN) for the reactions with
all four biothiols (Table 1) shows that under our experimental con-
ditions ChC2 is 12 to 60-fold more reactive toward thiolate ions
than ChC1.
Figure 2. Fluorescence responses of ChC1 (20
amino acids (2 mM), GSH (2 mM) or GSH plus NEM (500
l
M; kex 340 nm) in the presence of
M).
l
interferes to any obvious extent with the detection of biothiols,
even at a 100:1 molar ratio with respect to the probe. In addition,
when GSH is co-incubated with N-ethylmaleimide (NEM), a sulfhy-
dryl alkylating reagent, the fluorescence response of ChC1 de-
creases considerably.
We then studied the kinetic profiles of the reactions of ChC1 and
ChC2 with the tested biothiols under pseudo-first-order conditions
(large excesses of the thiols). The observed rate constants (kobsd) ob-
tained at 30.0 °C are shown in the Supplementary data (TableS1 and
S2). For the purpose of comparison the relative second-order rate

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O. García-Beltrán et al. / Tetrahedron Letters 52 (2011) 6606–6609
(A)
(B)
4000
3500
3000
5000
**
**
4500
4000
3500
**p<0,01
Control
NAC
Control
Cys
Figure 3. The ChC1 fluorescence in SH-SY5Y cells increased by treatment with NAC or Cys. (A) The cells were treated for 12 h with NAC (30 mM), washed, and then incubated
for 20 min with ChC1 (5 M) and the fluorescence was measured using a microplate fluorescence reader and by epifluorescence microscopy. (B) The cells were incubated for
20 min with ChC1 (5 M) and then Cys (5 mM) was added and the fluorescence was measured after 5 min using a microplate fluorescence reader and by epifluorescence
l
l
microscopy. Data represent mean SEM; n = 6, P < 0.01.
We propose that the higher reactivity of ChC2 than ChC1 toward
thiolate ions might be a consequence of proton donation by the
hydroxy group of ChC2 to the vinyl carbon neighboring the
carbonyl group, thus making the reaction site more prone to attack
by the thiolate. While this manuscript was in preparation, it was
reported that (E)-7-(diethylamino)-3-(3-(2-hydroxyphenyl)acry-
loyl)-2H-chromen-2-one, which bears an o-hydroxyl group in the
same relative position as ChC2, showed an only threefold enhance-
ment of its reaction rate with GSH as compared to (E)-3-cinnamoyl-
7-diethylamino-2H-chromen-2-one, the corresponding analogue of
ChC1.10 Those authors concluded that the participation of the
hydroxyl group in this Michael addition was unlikely and therefore
the presence of this group made little difference. In our hands, ChC2
proved to be remarkably more reactive than ChC1, particularly to-
ward Cys-Gly, Hcy, and GSH, warranting future studies of the
detailed reaction mechanism.
A practical application of ChC1 and ChC2 was to evaluate the
effect of N-acetylcysteine (NAC), an agent capable of stimulating
the synthesis of glutathione,11 on the fluorescence of both probes
in human neuroblastoma SH-SY5Y cells. This cell line was treated
for 12 h with NAC (30 mM) after which the cells were washed
and incubated with the probes. The fluorescence was measured
using a microplate fluorescence reader and by epifluorescence
microscopy (Figs. 3A and 4A). The results showed that the fluores-
cence was significantly increased after NAC treatment. Similar
results were observed when the cells were loaded with the probes
and Cys (5 mM) was subsequently added, incubating for 5 min
(Figs. 3B and 4B). The data represent mean SEM; n = 6, P < 0.01.
In conclusion, we have prepared and characterized two new
fluorescence turn-on sensors (ChC1 and ChC2) that exhibit a highly
selective response to biothiols both in vitro and in vivo. Unexpect-
edly, introduction of an ortho-hydroxyl group on the cinnamoyl
(A)
(B)
5500
5000
4500
4000
3500
**
**
5000
4500
**p<0,01
Control
NAC
Cys
Control
Figure 4. The ChC2 fluorescence in SH-SY5Y cells increased by treatment with NAC or Cys. (A) The cells were treated for 12 h with NAC (30 mM) and then incubated for
20 min with ChC2 (5
20 min with ChC2 (5
l
l
M) and the fluorescence was measured using a microplate fluorescence reader and by epifluorescence microscopy. (B) The cells were incubated for
M) and then Cys (5 mM) was added and the fluorescence was measured after 5 min using a microplate fluorescence reader and by epifluorescence
microscopy. Data represent mean SEM; n = 6, P < 0.01.

O. García-Beltrán et al. / Tetrahedron Letters 52 (2011) 6606–6609
6609
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Acknowledgments
This work was supported by FONDECYT (Grants #11090115
and 1100640), and the Millennium Scientific Initiative (Grant
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2011.09.137.
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