Combinatorial Strategy to Identify Fluorescent Probes for Biothiol and Thiophenol Based on Diversified Pyrimidine Moieties and Their Biological Applications

Article abstract of DOI:10.1021/acs.analchem.6b04608

We present a feasible paradigm of developing original fluorescent probes for target biomolecules via combinatorial chemistry. In this developmental program, pyrimidine moieties were investigated and optimized as unique recognition units for thiols for the first time through a parallel synthesis in combination with a rapid screening process. This time-efficient and cost-saving process effectively facilitated the developmental progress and provided detailed structure-reactivity relationships. As a result, Res-Biot and Flu-Pht were identified as optimal fluorescent probes for biothiol and thiophenol, respectively. Their favorable characteristics and superior applicability have been well demonstrated in both chemical and biological contexts. In particular, Res-Biot enables the direct visualization of biothiol fluctuations during oxidative stress and cell apoptosis, indicating its suitability in elucidation of a specific pathophysiological process in both living cells and living animals. Meanwhile, Flu-Pht is competent to visualize thiophenols without the interference from endogenous biothiols in living cells.

Full text of DOI:10.1021/acs.analchem.6b04608

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Article
Combinatorial Strategy to Identify Fluorescent Probes
for Biothiol and Thiophenol Based on Diversified
Pyrimidine Moieties and Their Biological Applications
Xilei Xie, Mengmeng Li, Fuyan Tang, Yong Li, Lei Lei ZHANG, Xiaoyun Jiao, Xu Wang, and Bo Tang
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04608 • Publication Date (Web): 07 Feb 2017
Downloaded from http://pubs.acs.org on February 8, 2017
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Analytical Chemistry
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Combinatorial Strategy to Identify Fluorescent Probes for Biothiol
and Thiophenol Based on Diversified Pyrimidine Moieties and Their
Biological Applications
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†
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‡
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Xilei Xie, Mengmeng Li, Fuyan Tang, Yong Li, Leilei Zhang, Xiaoyun Jiao, Xu Wang,* and Bo
,†
Tang*
†
College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized
Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Eduꢀ
cation, Institute of Molecular and Nano Science, Shandong Normal University, Jinan 250014, P. R. China
‡
Beijing Key Laboratory of Active Substances Discovery and Druggability Evaluation, Institute of Materia Medica, Chinese
Academy of Medical Sciences and Peking Union Medical College, Beijing 10050, China; Eꢀmail: tangb@sdnu.edu.cn;
wangxu@sdnu.edu.cn
ABSTRACT: We present a feasible paradigm of developing original fluorescent probes for target biomolecules via combinatorial
chemistry. In this developmental program, pyrimidine moieties were investigated and optimized as unique recognition units for
thiols for the first time through a parallel synthesis in combination with rapid screening process. This timeꢀefficient and costꢀsaving
process effectively facilitated the developmental progress and provided detailed structure−reactivity relationships. As a result, Resꢀ
Biot and FluꢀPht were identified as optimal fluorescent probes for biothiol and thiophenol respectively. Their favorable characterisꢀ
tics and superior applicability have been well demonstrated in both chemical and biological contexts. In particular, ResꢀBiot enables
the direct visualization of biothiol fluctuations during oxidative stress and cell apoptosis, indicating its suitability in elucidation of a
specific pathophysiological process in both living cells and living animals. Meanwhile, FluꢀPht is competent to visualize thiopheꢀ
nols without the interference from endogenous biothiols in living cells.
1
7−19
Fluorescent probe based assay has emerged as an irreplaceꢀ
able methodology to provide critical insight into function and
mechanism of bioactive molecules due to its high resolution,
operational simplicity, biocompatibility, and noninvasive
drug metabolism, and other pathophysiological processes.
On the other hand, thiophenol serves as a pollutant for the
environment and a poison for living organisms. Exposure to
thiophenol may induce severe kidney and liver injuries, central
1−3
20−22
characteristic. A typical fluorescent probe is constructed by
incorporating the specific recognition unit into a classic fluorꢀ
6
nervous system damage, and even death.
Fluorescent
probes will be powerful promotion to comprehensively eluciꢀ
date the biological function of biothiol and the toxic mechaꢀ
nism of thiophenol.
4
5
ophore platform, such as cyanine, fluorescein, and resorufin,
etc. Generally, the recognition unit mainly accounts for the
7−13
reactivity and specificity of fluorescent probes.
Even nowꢀ
Biothiol and thiophenol, especially their corresponding
deprotonated thiolate anions, can serve as strong nucleophiles
to initiate nucleophilic aromatic substitution (S Ar). Several
fluorescent probes for biothiol and thiophenol have been conꢀ
structed by taking advantage of this unique chemical feaꢀ
At the same time, we noticed that pyrimidine moiety
has the potential to function as a novel recognition unit for
and biocomꢀ
However, it has never been investigated to deꢀ
sign fluorescent probes. Thus, we speculated that incorporatꢀ
ing pyrimidine moiety onto a fluorophore platform might genꢀ
adays, confined by chemists’ limited ability to predict and
simulate using theoretical calculations, rational design of origꢀ
inal recognition units still remains an enormous challenge,
especially when there is no definite structural requirements for
a target analyte of interest. Combinatorial chemistry, characꢀ
terized by parallel synthesis and highꢀthroughput screening,
has been demonstrated an efficacious methodology in drug
discovery and development and can partially overcome the
N
23−33
ture.
34−36
biothiol/thiophenol due to its electrophilicity
patibility.
37,38
1
4−16
limitation of rational design strategy.
Thereby, it is exꢀ
pected that combinatorial strategy will facilitate the discovery
and optimization of original recognition units so as to conꢀ
struct novel fluorescent probes with improved performance.
erate
a novel fluorescent probe candidate for biothiꢀ
ol/thiophenol. Besides, the reactivity and specificity profiles of
the probe candidate can be easily modulated by regulating the
electronic characteristic of the pyrimidine moiety. Hence a
probe library can be created by altering substituents on the
pyrimidine ring, which provides great potential to generate
ideal fluorescent probes with suitable reactivity and high specꢀ
ificity towards biothiol and thiophenol.
To this end, thiols were selected as the target analytes of inꢀ
terest to develop novel probes via combinatorial methodology.
Thiols, mainly comprising biothiol and thiophenol, are both of
considerable biological significance. The former refers to bioꢀ
active aliphatic thiols, mainly consists of glutathione (GSH),
cysteine (Cys), and homocysteine (Hcy). Biothiols are extenꢀ
sively involved in redox homeostasis, signal transduction,
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To test the above hypothesis, we implemented a developꢀ (dd, J = 8.7, 2.0 Hz, 1H), 6.86 (dd, J = 9.8, 1.2 Hz, 1H), 6.31
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mental program to identify novel reactionꢀbased fluorescent
probes for biothiol and thiophenol based on pyrimidine chemꢀ
istry via a combinatorial strategy. To accelerate the developꢀ
mental progress, probe candidates were prepared by parallel
synthesis and crude products were directly screened by fluoꢀ
rescence measurement without elaborate purification. In conꢀ
sequence, ResꢀBiot and FluꢀPht were identified as fluorescent
probes for biothiol and thiophenol respectively. Their practical
utility in chemical and biological contexts was well demonꢀ
strated. Particularly, ResꢀBiot enabled the direct visualization
of endogenous biothiol fluctuations during oxidative stress and
cell apoptosis in both living cells and living animals. At the
same time, FluꢀPht demonstrated its capability of visualizing
thiophenol without the interference from endogenous biothiols
in living cells.
(d, J = 1.2 Hz, 1H). C NMR (75 MHz, DMSOꢀd ): δ 185.60,
6
165.63, 156.78, 155.41, 154.69, 149.37, 148.24, 144.29,
139.92, 135.10, 134.69, 131.18, 119.22, 109.69, 106.09.
+
+
HRMS (ESI): calculated for C H N O (M+H ) 337.0567,
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found 337.0506.
FluꢀPht was synthesized from fluorescein and 4ꢀ
chloropyrimidine according to the above general procedure.
The obtained crude product was further purified by column
chromatography on silica gel (dichloromethane/methanol,
1
00:1 v/v) to afford a light yellow solid (22 mg, 45% yield).
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H NMR (400 MHz, CDCl ): δ 8.80 (s, 2H), 8.63 (d, J = 5.6
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Hz, 2H), 8.06 (d, J = 7.5 Hz, 1H), 7.73 (t, J = 7.3 Hz, 1H),
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.67 (t, J = 7.4 Hz, 1H), 7.29 (d, J = 7.6 Hz, 1H), 7.16 (s, 2H),
.98 (d, J = 5.6 Hz, 2H), 6.90 (s, 4H). C NMR (100 MHz,
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CDCl ): δ 169.13, 168.65, 158.98, 158.80, 153.84, 152.74,
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52.08, 135.44, 130.30, 129.47, 126.51, 125.46, 124.28,
17.84, 116.67, 110.38, 1+09.00, 8+1.84. HRMS (ESI):
EXPERIMENTAL SECTION
Materials and Instruments. Unless otherwise stated, all
reagents and solvents were obtained from commercial suppliꢀ
ers and used without further purification. Sartorius ultrapure
water (18.2 Mꢁ cm) was used throughout the experiments.
HRMS analysis was operated on a Bruker MaXis UHRꢀTOF
calculated for C H N O
489.1158.
(M+H ) 489.1193, found
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RESULTS AND DISCUSSION
Design, Synthesis, and Screening of the Fluorescent
Probe Library. The pyrimidine moiety is expected to funcꢀ
tion as a novel recognition unit for biothiol/thiophenol. The πꢀ
electron density of pyrimidine ring is decreased to a large exꢀ
tent, which facilitates the S Ar pathway. Therefore, pyrimiꢀ
dine is expected to possess potential reactivity to nucleophile
1
13
system. H NMR and C NMR spectra were taken on a
Bruker Advance 300 MHz or 400 MHz spectrometer using
tetramethylsilane as internal standard. The pH values were
measured with a Model pHꢀ3c digital pH meter. Fluorescence
spectra were recorded by a FLSꢀ980 fluorescence spectromeꢀ
ter equipped with a xenon lamp and 1.0ꢀcm quartz cells at the
slits of 2.0/2.0 nm. Cell imaging was taken with a Zeiss LSM
N
34−36
thiols.
Besides, pyrimidine ring can be modified by diꢀ
verse substituents to adjust its electronic characteristic. Acꢀ
cordingly, the chemical profile of the probe candidate can be
easily manipulated, which provides great potential to generate
ideal fluorescent probes with optimized reactivity and speciꢀ
ficity. Additionally, cytosine, thymine, and uracil, these three
pyrimidine derivatives are nucleobases of living organisms,
implying the excellent biocompatibility of pyrimidine moieꢀ
8
(
80 confocal laser scanning microscope with an objective lens
×20). In vivo imaging was carried out on IVIS Lumina III
system with a metal halide lamp (150 W).
Parallel Synthesis of the Fluorescent Probe Library.
Resorufin (21.3 mg, 0.1 mmol) or fluorescein (33.2 mg, 0.1
mmol) was dissolved in 1.5 mL of DMF, 1.0 mmol of pyrimiꢀ
37,38
dine
reagent
(2ꢀchloropyrimidine,
2ꢀchloroꢀ4ꢀ
ty.
(
5
trifluoromethyl)pyrimidine, 4ꢀchloropyrimidine, or 2ꢀchloroꢀ
ꢀnitropyrimidine) and K CO (27.6 mg, 0.2 mmol) were addꢀ
2 3
As for the fluorophore platform, commercially available
resorufin and fluorescein are selected due to their outstanding
photophysical characteristics and excellent biocompatibility.
Besides, their fluorescence OFF/ON can be easily switched by
masking or exposing the hydroxyl group of these two fluoroꢀ
phores. Incorporating diversely substituted pyrimidine moieꢀ
ties onto resorufin or fluorescein via ether linkage generates a
novel probe library for thiols. We envisioned that fluorescence
emission of probe candidates would be quenched by hydroxyl
protection. Once thiolyzed by biothiol or thiophenol, the
fluorophore would be released and fluorescence emission reꢀ
stored. On the basis of the above consideration, we proposed a
diversityꢀoriented probe library consisting of eight candidates
ed. The reaction mixture was stirred at 70 °C. Chemical conꢀ
version was monitored by TLC analysis. After resorufin or
fluorescein was completely consumed, the above mixture was
added into 70 mL of ethyl acetate, and washed with 50 mL of
saturated NaCl solution three times. The organic solvent was
evaporated under reduced pressure. The crude product was
obtained and confirmed by HRMS analysis, and then screened
directly without further purification.
5,6
Rapid Screening of Probe Candidates. The above crude
products were dissolved in DMSO and diluted to 40 ꢂM with
0 mM PBS buffer solution, and then incubated with GSH
5
(
500 ꢂM) or thiophenol (500 ꢂM) at 37 °C for 60 min. For
(Figure
1).
Coupling
of
pyrimidinꢀ2ꢀyl,
4ꢀ
resorufin analogs, the fluorescence intensity was measured at
λex/em = 560/585 nm. For fluorescein analogs, the fluorescence
intensity was measured at λex/em = 460/519 nm.
(trifluoromethyl)pyrimidinꢀ2ꢀyl, pyrimidinꢀ4ꢀyl, and 5ꢀ
nitropyrimidinꢀ2ꢀyl moieties with resorufin furnishes probe
Resꢀ1−Resꢀ4 respectively. Similarly, integrating the four pyꢀ
rimidine moieties onto fluorescein platform generates probe
Fluꢀ1−Fluꢀ4.
Preparation and Characterization of Res-Biot and Flu-
Pht. ResꢀBiot was synthesized from resorufin and 2ꢀchloroꢀ5ꢀ
nitropyrimidine according to the above general procedure. The
obtained crude product was further purified by column chroꢀ
matography on silica gel (dichloromethane/methanol, 100:1
1
v/v) to afford a yellow solid (28 mg, 83% yield). H NMR
(
1
400 MHz, DMSOꢀd ): δ 9.50 (s, 2H), 7.96 (d, J = 8.7 Hz,
6
H), 7.61 (d, J = 2.0 Hz, 1H), 7.58 (d, J = 9.9 Hz, 1H), 7.42
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Analytical Chemistry
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Figure 1. Chemical structures of designed fluorescent probe candidates.
The probe candidates were prepared via oneꢀstep parallel
synthesis shown in Scheme S1. Excess pyrimidine reagents
were added to ensure that the resorufin/fluorescein was comꢀ
pletely consumed. Inside the reaction system, there were mainꢀ
ly the newly generated probe candidates and remained pyrimiꢀ
dine reagents, which was confirmed by TLC analysis. The
reagents were always nonꢀfluorescent due to its short conjuꢀ
gated structure. Considering there was no other substance exꢀ
cept the probe candidates to induce fluorescence variation in
the reaction system, the crude products were directly evaluated
for their reactivity and specificity by fluorescence measureꢀ
respectively, and selected for extensive evaluation in both
chemical and biological contexts. These two probes were reꢀ
1
newedly prepared and characterized by HRMS, H NMR, and
13
C NMR.
Fluorescence Response and Reaction Mechanism. The
fluorescence spectra variation of ResꢀBiot toward GSH was
depicted in Figure 2a. As can be seen, ResꢀBiot presented
nearly no fluorescence emission at 585 nm in the absence of
GSH. Upon introduction of GSH, the maximum fluorescence
intensity at 585 nm increased and exhibited an excellent linear
2
correlation (R = 0.994) with the GSH concentrations in range
39
ment without elaborate purification. GSH is the most abunꢀ
of 0 to 20 ꢂM (Figure 2a, insert). The limit of detection (LOD)
value was calculated to be as low as 0.29 ꢂM based on S/N = 3.
The fluorescence spectra of ResꢀBiot toward GSH were identiꢀ
cal to those of authentic resorufin, which was ascribed to the
release of resorufin fluorophore by thiolysis of GSH. The reacꢀ
tion mechanism between ResꢀBiot and GSH was further conꢀ
firmed by HRMS analysis. After incubation, the peaks at m/z
212.0387 and 429.0881 were both detected out in the reaction
mixture (Figure S2), which agreed well with resorufin and the
pyrimidine derivative respectively. The result testified the proꢀ
posed reaction pathway shown in Scheme 1a. Additionally, we
also investigated the reaction mechanism of ResꢀBiot for Cys
and Hcy. HRMS spectra (Figure S3 and S4) indicated that
ResꢀBiot was thiolyzed to resorufin (m/z 212.0368 or 212.0376)
and the corresponding pyrimidine derivative (m/z 243.0208 or
dant and comprises about 90% of nonꢀprotein biothiols inside
40
the cell, thereby selected as the representative biothiol in the
experiments. After incubation with GSH/thiophenol, Resꢀ1,
Resꢀ2, Fluꢀ1, and Fluꢀ2 could not be lighted on by GSH or
thiophenol (Figure S1a, S1b, S1e, and S1f), inferring pyrimꢀ
idinꢀ2ꢀyl moiety and its trifluoromethyl analog were chemicalꢀ
ly stable toward GSH and thiophenol. To the contrary, pyrimꢀ
idinꢀ4ꢀyl moiety presented moderate reactivity. When it was
installed onto fluorescein, the resultant probe (Fluꢀ3, also
called FluꢀPht) specifically responded to thiophenol (Figure
S1g), while the corresponding resorufinꢀbased counterpart
(Resꢀ3) displayed no response toward thiophenol (Figure S1c),
corroborating the close relationship between the reactivity of a
probe and its fluorophore platform. Furthermore, fluorescence
of Resꢀ4 (also called ResꢀBiot) and Fluꢀ4 was turned on by
both GSH and thiophenol (Figure S1d and S1h), revealing that
strong electronꢀwithdrawing substituent on pyrimidine ring
enhanced the reactivity of the probe.
2
57.0376), and the reaction pathway (Scheme S2) was similar
to that proposed by GSH.
Thereby, structure−reactivity relationships were outlined as
follows. First, despite possessing analogical structure, pyrimꢀ
idinꢀ4ꢀyl moiety presents higher reactivity than pyrimidinꢀ2ꢀyl.
Second, the reactivity of a probe mainly depends on its recogꢀ
nition unit, and is partially related to its fluorophore in certain
case. Third, strong electronꢀwithdrawing substituent (e.g. nitro
group) is preferred to enhance the reactivity of a thiol probe,
but probably reduces the specificity. This parallel synthesis in
combination with rapid screening process, characterized by
timeꢀefficient and costꢀsaving, effectively facilitated the deꢀ
velopmental progress and provided detailed strucꢀ
ture−reactivity relationships which would be valuable for furꢀ
ther thiol probe design.
Figure 2. (a) Emission spectra (excited at 560 nm) of 10 ꢂM Resꢀ
Biot toward varied concentrations of GSH (0−100 ꢂM) in HEPES
buffer (20 mM, pH 7.4) at 37 °C for 30 min, insert: linear curve
derived from fluorescence titration. (b) Emission spectra (excited
at 460 nm) of 10 ꢂM FluꢀPht toward varied concentrations of
thiophenol (0−100 ꢂM) in PBS buffer (50 mM, pH 7.4) at 37 °C
for 2 h, insert: linear curve derived from fluorescence titration.
Compared to Fluꢀ4, ResꢀBiot was preferred due to its red
fluorescence emission. It is worth noting that ResꢀBiot unaꢀ
voidably responds to thiophenol due to its higher nucleoꢀ
The same investigation was performed on the response of
FluꢀPht toward thiophenol. Briefly, the emission maximum at
519 nm increased gradually with the increasing concentration
4
1
philicity than that of biothiol. However, interference from
thiophenol can be excluded in living system because it is an
exogenous species. Therefore, ResꢀBiot and FluꢀPht were
identified as fluorescent probes for biothiol and thiophenol
2
of thiophenol (Figure 2b). There was excellent linearity (R =
0.992) between the fluorescence intensity at 519 nm and thioꢀ
phenol concentrations in range of 0 to 100 ꢂM (Figure 2b,
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insert). The LOD value was found to be 1.8 ꢂM. Additionally, cal system encouraged us to investigate their practical utilities
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the fluorescence spectra of FluꢀPht in the presence of thiopheꢀ
nol were identical to those of original fluorescein, suggesting
the liberation of fluorescein fluorophore by thiolysis of thioꢀ
phenol. The HRMS analysis results (Figure S5) validated the
proposed reaction pathway in Scheme 1b.
in living cells. Prior to being applied in cell imaging, MTT
assay was first performed on HepG2 cells to assess their bioꢀ
compatibility (Figure S7). The IC50 values of ResꢀBiot and
FluꢀPht were up to 200 ꢂM and 256 ꢂM respectively, inferring
their low cytotoxicity and superior practical capability in bioꢀ
logical samples.
Scheme 1. Proposed Reaction Mechanism of (a) Res-Biot
and (b) Flu-Pht
ResꢀBiot was employed to visualize the endogenous biothiꢀ
ols in Hela cells. Phorbol 12ꢀmyristate 13ꢀacetate (PMA) is a
wellꢀestablished protein kinase C activator to trigger cellular
42
oxidative stress. LꢀButhionine sulfoximine (BSO) serves as
an inhibitor of γꢀglutamylcysteine synthetase to decline GSH
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level and finally results in cell apoptosis. These two widely
approved reagents were applied to induce intracellular biothiol
fluctuation. As expected, after incubation with ResꢀBiot, bright
red fluorescence was observed inside the cells (Figure 4a),
indicating the cellꢀpermeability of ResꢀBiot and the abundant
contents of intracellular biothiols. Pretreating the cells with a
The effect of pH value was estimated, revealing that Resꢀ
Biot and FluꢀPht exhibited higher OFF/ON ratio toward their
own target analytes in the pH 6.0−8.0 (Figure S6). This was
well consistent with the physiological pH range, indicating
their potential application in living systems.
44
thiolꢀspecific scavenger, Nꢀethylmaleimide (NEM), the fluoꢀ
rescence signal was obviously suppressed (Figure 4b), conꢀ
firming the specific fluorescence response to cellular biothiols.
Furthermore, a pronounced fluorescence decrease was obꢀ
served when cells were pretreated with PMA (Figure 4c) or
BSO (Figure 4d), indicating the downꢀregulated biothiol level
during the oxidative stress and cell apoptosis process. These
outcomes collectively demonstrated that ResꢀBiot was capable
of visualizing endogenous biothiol fluctuation during a specifꢀ
ic pathophysiological process in living cells.
Specificity Investigation. Next, the specificity of ResꢀBiot
and FluꢀPht was evaluated by screening their fluorescence
response to biologically relevant species under physiological
conditions. As shown in Figure 3a, ResꢀBiot displayed signifiꢀ
cant fluorescence increments only in the present of biothiols
(GSH, Cys, and Hcy). Negligible fluorescence changes were
induced by potential competing species, including other natuꢀ
ral animo acids (Gly, Met, and Lys), reactive oxygen and niꢀ
−
•
1
trogen species (H O , ClO , OH, O , and NO), and metal ions
2
2
2
+
2+
2+
(
K , Fe , and Zn ).
Figure 4. Fluorescence images of endogenous biothiols in Hela
cells. Cells were treated with (a) vehicle, (b) 1 mM NEM, (c) 1
ꢂg/mL PMA, or (d) 100 ꢂM BSO for 30 min followed by incubaꢀ
tion with 10 ꢂM ResꢀBiot for 20 min. (e−h) Second row shows the
corresponding brightꢀfield images for the first row. The images
were acquired using a confocal microscope with 543ꢀnm excitaꢀ
tion and 560−620ꢀnm collection. Scale bar: 20 ꢂm.
Figure 3. (a) Fluorescence responses of ResꢀBiot (10 ꢂM) toward
various species (100 ꢂM): (1) blank, (2) GSH, (3) Cys, (4) Hcy,
−
•
1
(
5) Gly, (6) Met, (7) Lys, (8) H O , (9) ClO , (10) OH, (11) O ,
2
2
2
+
2+
2+
(12) NO, (13) K , (14) Fe , (15) Zn , λ
= 560/585 nm. (b)
ex/em
Fluorescence responses of FluꢀPht (10 ꢂM) toward various species
(
(
100 ꢂM): (1) blank, (2) thiophenol, (3) GSH, (4) Cys, (5) Hcy,
6) Gly, (7) phenol, (8) aniline, (9) NaN , (10) KI, (11) H O , (12)
3
2
2
Similar experiments were performed on FluꢀPht toward thiꢀ
ophenol in HepG2 cells. Cells treated with FluꢀPht exhibited
almost no fluorescence signal (Figure 5a), revealing that enꢀ
dogenous biothiols could not illuminate the fluorescence of
FluꢀPht and there was no endogenous thiophenol inside the
cells. When pretreated with thiophenol, a significant fluoresꢀ
cence enhancement was observed (Figure 5b), demonstrating
the capability of discriminating thiophenol from endogenous
biothiols in living cells.
−
•
1
ClO , (13) OH, (14) O , (15) NO, λ
= 460/519 nm.
2
ex/em
The fluorescence of FluꢀPht was exclusively triggered on by
thiophenol, while the biothiols (GSH, Cys, and Hcy) induced
no observable fluorescence signal enhancements, demonstratꢀ
ing its capability of discriminating thiophenol from biothiol.
Additionally, FluꢀPht presented excellent immunity to interferꢀ
ence from other potential interfering substances, including
some nucleophilic species (Gly, phenol, aniline, NaN , and KI),
3
−
•
1
reactive oxygen and nitrogen species (H O , ClO , OH, O ,
2
2
2
and NO) (Figure 3b). Above all, these results collectively indiꢀ
cated the high specificity of ResꢀBiot and FluꢀPht toward their
own target analytes.
Fluorescence Imaging in Living Cells. The aforemenꢀ
tioned favorable properties of ResꢀBiot and FluꢀPht in chemiꢀ
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Analytical Chemistry
the first time that pyrimidine functionality is exploited to conꢀ
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struct optical probes for thiols. The present work evidently
exhibited the advantage of combinatorial strategy in fluoresꢀ
cent probe exploitation and showed that in principle, pyrimiꢀ
dine moiety can be installed onto alternative nearꢀinfrared or
twoꢀphoton fluorophores to generate nextꢀgeneration fluoresꢀ
cent sensors for thiols with improved performance.
ASSOCIATED CONTENT
Supporting Information
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The Supporting Information is available free of charge on the
ACS Publications website.
Figure 5. Fluorescence images of thiophenol in HepG2 cells.
Cells were treated with (a) vehicle or (b) 50 ꢂM thiophenol for 20
min followed by incubation with 5 ꢂM FluꢀPht for 20 min. (c, d)
Second row shows the corresponding brightꢀfield images for the
first row. The images were acquired using a confocal microscope
with 488ꢀnm excitation and 500−560ꢀnm collection. Scale bar: 20
ꢂm.
Experimental details, supplementary data, synthesis and characterꢀ
ization of compounds (PDF)
AUTHOR INFORMATION
Corresponding Author
Visualizing Biothiols in Animal Models. Inspired by the
red fluorescence emission of ResꢀBiot, we subsequently exꢀ
plored its feasibility of visualizing endogenous biothiols durꢀ
*Eꢀmail: tangb@sdnu.edu.cn; wangxu@sdnu.edu.cn
*Fax: +86ꢀ531ꢀ86180017
Notes
4
5
ing LPSꢀinduced inflammation response and BSOꢀinduced
apoptotic process in living animals. As shown in Figure 6, the
mouse treated with ResꢀBiot emitted high fluorescence intensiꢀ
ty (Figure 6a). By contrast, the fluorescence was apparently
attenuated in the mouse pretreated with NEM (Figure 6b),
which was attributed to the specific biothiol depletion by NEM.
At the same time, fluorescence emission was efficiently
blocked in the mouse pretreated with LPS (Figure 6c) or BSO
The authors declare no competing financial interest.
ACKNOWLEDGMENT
This work was supported by 973 Program (2013CB933800) and
National Natural Science Foundation of China (21390411,
21535004, 21375080, and 21505088).
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