The determination of thiols based using a probe that utilizes both an absorption red-shift and fluorescence enhancement

Article abstract of DOI:10.1016/j.dyepig.2009.11.011

A pyridylvinylene derivative containing piazselenole displayed high selectivity toward glutathione in the presence of other biorelevant analytes. The compound exhibited a 19?nm red-shift in absorption spectra and ～3-fold fluorescence intensity enhancement; in addition, it was possible to detect micromolar amounts of glutathione quantitatively using both red-shift absorbance and enhanced fluorescence. The mechanism of the reaction between the modified pyridylvinylene derivative and glutathione was confirmed using ESI-MS and absorption/fluorescence spectra.

Full text of DOI:10.1016/j.dyepig.2009.11.011

Dyes and Pigments 86 (2010) 87e92
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Dyes and Pigments
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The determination of thiols based using a probe that utilizes both
an absorption red-shift and fluorescence enhancement
,
b
a
a
c,
**
Baocun Zhu a, Xiaoling Zhang a , Hongying Jia , Yamin Li , Shutang Chen , Sichun Zhang
*
a Department of Chemistry, School of Science, Beijing Institute of Technology, Beijing 100081, People's Republic of China
b Institute of Chemistry, Chinese Academy of Sciences, Beijing 100080, People's Republic of China
c Department of Chemistry, Key Laboratory for Atomic and Molecular Nanosciences of Education Ministry, Tsinghua University, Beijing 100084, People's Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
A pyridylvinylene derivative containing piazselenole displayed high selectivity toward glutathione in the
presence of other biorelevant analytes. The compound exhibited a 19 nm red-shift in absorption spectra
and w3-fold fluorescence intensity enhancement; in addition, it was possible to detect micromolar
amounts of glutathione quantitatively using both red-shift absorbance and enhanced fluorescence. The
mechanism of the reaction between the modified pyridylvinylene derivative and glutathione was
confirmed using ESI-MS and absorption/fluorescence spectra.
Received 7 October 2009
Received in revised form
29 November 2009
Accepted 30 November 2009
Available online 16 December 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Thiol probe
Fluorescence enhancement
Red-shift
Glutathione
Intramolecular charge transfer
Two-channel
1. Introduction
quantitatively detect nanomolar [23,45] or millimolar [52]
concentrations of GSH. It is well-known that the concentration of
Low Mr thiols, such as glutathione (GSH) and cysteine (Cys), play
a crucial role in physiological systems owing to their participation
in reversible redox reactions [1]. GSH is the most abundant cellar
thiol and exists in a redox equilibrium between the reduced (GSH)
and oxidized (GSSG) forms [2]. The ratio of GSH:GSSG is a key
indicator for monitoring cellular oxidative stress and large changes
can lead to some diseases and cancers [3e5]. Hence, the quanti-
tative detection of physiological thiols is very important for
investigating cellular functions. Of the various methods [6e15] for
detecting thiols, great attention has attended the use of fluorescent
probes owing to advantages over other methods, such as high
sensitivity and operational simplicity [16e18]. Recently, attention
has focused on the development of fluorescent probes for thiols,
including maleimide-type [19e24], aldehyde-type [25e31], dithiol
[32e34], 2,4-dinitrobenzenesulfonyl-protected [35e39] probes
and other different response mechanism [2,40e53] probes. Despite
the large number of fluorescent thiols probes, many of them can
GSH is about 2 mM in blood plasma [54], in which context, Yan et al.
[50] and Wang et al. [51] developed two systems based on quantum
dots for the detection of GSH in which GSH was determined by
fluorescence intensity. Recently, Tian et al. have reported many
colorimetric probes based on intramolecular charge transfer (ICT)
mechanism for several analytes, such as mercury [55], pyrophos-
phate (PPi) [56]. However, to the best of our knowledge, few reports
relating to the quantitative detection of GSH at micromolar level
using red-shift absorption and enhanced fluorescence have
appeared, with the exception of a merocyanine-containing che-
modosimeter published by Zhengs et al. [57].
In this paper we describe the synthesis of probe 1, which
comprised an aminophenylpyridylvinylene derivative fluorophore
and
a piazselenole receptor (Scheme 1). Using an amino-
phenylpyridylvinylene derivative as fluorophore offers many
inherent virtues, such as desirable spectroscopic properties and
outstanding intramolecular charge transfer (ICT) [58,59]; the use of
piazselenole as receptor was inspired from previous reports [10,45].
We hypothesize that piazselenole reacts with thiols to provide the
stronger electron-donor, o-phenylenediamine, which induces
changes in two-channel output signals, namely absorption red-shift
and fluorescence enhancement.
* Corresponding author. Tel./fax: þ86 10 88875298.
** Corresponding author.
E-mail addresses: zhangxl@bit.edu.cn (X. Zhang), sczhang@chem.tsinghua.edu.cn
(S. Zhang).
0143-7208/$ e see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.dyepig.2009.11.011

88
B. Zhu et al. / Dyes and Pigments 86 (2010) 87e92
Scheme 1. Synthesis of probe 1 and reference compound 2.
2. Experimental
2.1. General
All reactions were carried out under a nitrogen atmosphere. All
the chemicals used in this paper were obtained from commercial
suppliers. 1H NMR spectra was taken on a Bruker AMX400 spec-
trometer. Chemical shifts (d) were reported in ppm relative to
a Me4Si standard in CDCl3 or DMSO-d6. High-resolution mass data
were measured with fourier transform ion cyclotron resonance
mass spectrometer (APEX IV). Electrospray ionization (ESI) mass
spectra were measured with an LC-MS 2010A (Shimadzu) instru-
ment. Absorption spectra were recorded on TU-1901 UVevis
spectrophotometer. Fluorescence spectra were measured on Hita-
chi F-7000 fluorescence spectrometer. All pH measurements were
made with a Sartorius basic pH-meter PB-10.
2.2. Synthesis of 2-methyl-N-methyl pyridinium iodide
2-Methylpyridine (932.2 mg, 10.01 mmol) and methyl iodide
(4262.9 mg, 30.03 mmol) were dissolved in ethanol (20 mL). After
stirred and refluxed for 4 h under N2 atmosphere, the reaction
mixture was cooled to room temperature and was filtered to give
the product (1695.7 mg) in 72% yield. 1H NMR (400 MHz, DMSO-d6)
d
(*10ꢀ6): 2.82(s, 3H), 4.27(s, 3H), 7.97(t, J ¼ 6.9 Hz, 1H), 8.09(d,
J ¼ 7.9 Hz, 1H), 8.50(t, J ¼ 7.7 Hz, 1H), 9.03(d, J ¼ 6.2 Hz, 1H).
2.3. Synthesis of 5-methyl-2,1,3-benzoselenadiazole
4-Methylbenzene-1,2-diamine (1.2217 g, 10 mmol) and sele-
nium dioxide (1.1096 g, 10 mmol) were ground respectively, and
then mixed in a mortar at room temperature. After 30 min grinding,
the crude products were dissolved in n-hexane, and then filtered.
The solvent was removed under reduced pressure to give the
desired product (1.8133 g, 9.2 mmol, 92% yield). 1H NMR (400 MHz,
CDCl3)
(d, J ¼ 9.2 Hz, 1H).
d
(*10ꢀ6): 2.46(s, 3H), 7.30(d, J ¼ 9.2 Hz, 1H), 7.57(s, 1H), 7.70
Fig. 1. Absorption (a) and fluorescence (c) spectra of 1 (10
mM) in the presence of GSH
(0e12 M), and plot of the absorbance (at 376 nm) (b) and the fluorescence intensity
m
2.4. Synthesis of 2,1,3-benzoselenadiazol-5-carbaldehyde
(at 440 nm) (d) of 1 as a function of the GSH concentration in a mixture of DMF and
water (8:2, v/v) solution. Each spectrum was acquired 30 min after GSH addition at
25 ꢁC. The excitation wavelength was 370 nm. The excitation and emission slit widths
were 2.5 and 5.0 nm.
5-methyl-2,1,3-benzoselenadi-azole (985.8 mg, 5.00 mmol) and
selenium dioxide (1110.8 mg, 10.01 mmol) were dissolved in
methyltoluene (30 mL). After stirring under reflux for 4 h under

B. Zhu et al. / Dyes and Pigments 86 (2010) 87e92
89
a N2 atmosphere, the reaction mixture was cooled to room
temperature and filtered. The filtrate was concentrated and column
chromatographed on silica-gel (elution with chloroform) to give
the product (532.6 mg) in 50% yield. 1H NMR (400 MHz, CDCl3)
1. 1H NMR (400 MHz, DMSO-d6)
d
(*10ꢀ6): 4.45(s, 3H), 7.85(d,
J ¼ 16.0 Hz, 1H), 7.97e8.01(m, 2H), 8.14(d, J ¼ 16.0 Hz, 1H), 8.20(d,
J ¼ 9.4 Hz, 1H), 8.26(s, 1H), 8.59(d, J ¼ 4.2 Hz, 2H), 8.99(d, J ¼ 6.2 Hz,
1H). HRMS (ESI positive) calcd for C14H12N3Se [M]þ 302.01913,
found 302.01860.
d
(*10ꢀ6): 7.93e8.00(m, 2H), 8.34(s, 1H), 10.20(s, 1H).
2.6. Synthesis of reference compound 2
2.5. Synthesis of probe 1
Benzaldehyde (318.4 mg, 3 mmol) and 2-methyl-N-methyl
pyridinium iodide (235.1, 1 mmol) were dissolved in n-butanol
(25 mL). After stirring under reflux for 2 h under N2 atmosphere,
the mixture was cooled to room temperature to afford crude
product after filtration. The pure product was obtained by recrys-
tallization from ethanol. HRMS (ESI positive) calcd for C14H14N [M]þ
196.11208, found 196.11161.
2,1,3-benzoselenadiazol-5-carbaldehyde (253.3 mg, 1.2 mmol)
and 2-methyl-N-methyl pyridinium iodide (235.1, 1 mmol) were
dissolved in absolute ethanol (25 mL). After stirring under reflux
for 45 min under a N2 atmosphere, the reaction mixture was
concentrated under reduced pressure. The residues were added to
a 40:1 mixture of chloroform:methanol (25 mL) and the product
was collected by filtration and washed with 5 ꢂ 3 mL mixture
solvents (chloroform:methanol ¼ 40:1, v/v) to give a pure product
3. Results and discussion
3.1. Spectra titration of 1 with GSH
In this paper, GSH was selected as the representative thiol in the
spectral experiments. The absorption and fluorescence spectra
were determined in a mixture of DMF and water (8:2, v/v) solution.
As shown in Fig. 1a, 1 displayed a major absorption band
centered at 357 nm corresponding to molar absorption coefficient
(
3
) of 2.60 ꢂ 10ꢀ4 Mꢀ1 cmꢀ1. Addition of GSH to the solution of 1
(10 mM) resulted in a remarkable red-shift (19 nm) in the absorp-
tion spectra and a gradual increase of the absorbance at 376 nm.
There was a good linearity between absorbance and concentrations
of GSH in the range of 4e12
was 0.5 M.
mM (Fig. 1b), and the detecting limit
m
In the fluorescence spectra (Fig. 1c), the mixture of DMF and
water (8:2, v/v) solution of 1 exhibited a fluorescence emission
Fig. 2. Absorption (a) and fluorescence (b) spectra of 1 (10
and a variety of biorelevant analytes including Al3þ, Ca2þ, Cd2þ, Cu2þ, Kþ, Mg2þ, Naþ
Zn2þ, H2O2, Fe3þ and ascorbic acid (Vc) (20
M) in a mixture of DMF and water (8:2,
mM) with or without GSH
,
Fig. 3. Absorption (a) and fluorescence (b) responses of 1 (10
mM) to GSH (20 mM) in
m
the absence and presence of biorelevant analytes (20 M) in a mixture of DMF and
m
v/v) solution. Each spectrum was acquired 30 min after various analytes addition at
25 ꢁC. The excitation wavelength was 370 nm. The excitation and emission slit widths
were 2.5 and 5.0 nm.
water (8:2, v/v) solution. Each spectrum was acquired 30 min after various analytes
addition at 25 ꢁC. The excitation wavelength was 370 nm. The excitation and emission
slit widths were 2.5 and 5.0 nm.

90
B. Zhu et al. / Dyes and Pigments 86 (2010) 87e92
peak at 434 nm, with a fluorescence quantum yield of 0.0069
(see Supporting Information). Upon addition of GSH, the maximum
emission peak underwent a red shift to 440 nm and the fluores-
cence quantum yield increased up to 0.0113. There was a good
linearity between the fluorescence intensity and concentrations of
3.3. Spectra of 1 in the presence of other thiols
The reactivity of 1 to other thiols was tested by the absorption
and fluorescence titration experiments (Fig. 4a and b). The results
demonstrated that 1 showed greater response to GSH than other
GSH in the range of 2e12
0.03 M.
mM (Fig. 1d), and the detecting limit was
nonprotein thiols such as thioglycolic acid (TA),
b-mercaptoethyl-
m
amine (MEA), -cysteine (Cys) and dithiothreitol (DTT). This result is
L
Therefore,1 could detect GSH qualitatively and quantitatively by
both absorption and fluorescence spectrometry methods.
in good agreement with the conclusion reported by Tang et al. [45]
and Zhang et al. [10].
3.4. The mechanism of 1 in sensing thiols
3.2. Spectra titration of 1 with biorelevant analytes
To understand the mechanism of 1 in sensing GSH, reference
compound 2 was synthesized. When GSH was added to the solu-
tion of 1 and 2 respectively, the changes of the solution of 2 was
ignored compared with those of the solution of 1 (Fig. 5a and b).
This implied that the reaction of 1 with GSH was attributed to
piazselenole reacting with GSH. The reaction products of 1 with
GSH were subjected to electrospray ionization mass spectral
analyses. The peak at m/z 226 corresponding to the compound 3
was observed (Fig. S6). Additionally, to further demonstrate the
reaction of 1 with GSH by thiol, a mixture of N-ethylmaleimide
(NEM, a known thiol-blocking agent) and GSH was added to the
solution of 1, no obvious changes in the absorption and fluores-
cence spectra was observed (Fig. 6a and b), implying the reaction
of 1 to thiol of GSH. Therefore, a possible mechanism was proposed
as shown in Scheme 2.
For an excellent probe, high selectivity is a matter of necessity.
The specificity of 1 toward GSH was determined by the absorption
and fluorescence titration experiments (Fig. 2a and b). Nearly no
changes was observed in the absorption and fluorescence spectra
with Al3þ, Ca2þ, Cd2þ, Cu2þ, Kþ, Mg2þ, Naþ, Zn2þ, H2O2, Fe3þ and
ascorbic acid (Vc). Next, the absorption and fluorescence responses
of 1 toward GSH in the presence of physiological analytes were
also investigated (Fig. 3a and b). The results showed that 1
possesses high selectivity toward GSH when present with other
analytes.
Fig. 4. Absorption (a) and fluorescence (b) responses of
1 (10 mM) to different
thiols (including TA, MEA, Cys, DTT, and GSH (20 M)) in a mixture of DMF and water
m
Fig. 5. Absorption (a) and fluorescence (b) responses of 1 and 2 (10 mM) to GSH
(8:2, v/v) solution. Each spectrum was acquired 30 min after different thiols addition at
25 ꢁC. The excitation wavelength was 370 nm. The excitation and emission slit widths
were 2.5 and 5.0 nm.
(20 mM) in a mixture of DMF and water (8:2, v/v) solution. Each spectrum was acquired
30 min after GSH addition at 25 ꢁC. The excitation wavelength was 370 nm. The
excitation and emission slit widths were 2.5 and 5.0 nm.

B. Zhu et al. / Dyes and Pigments 86 (2010) 87e92
91
Acknowledgements
This work was supported by the National Nature Science
Foundation of China (No. 20975012) and the 111 Project (B07012).
Appendix. Supplementary data
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.dyepig.2009.11.011.
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