Selective selenol fluorescent probes: Design, synthesis, structural determinants, and biological applications

Article abstract of DOI:10.1021/ja5099676

Selenium (Se) is an essential micronutrient element, and the biological significance of Se is predominantly dependent on its incorporation as selenocysteine (Sec), the genetically encoded 21st amino acid in protein synthesis, into the active site of selenoproteins, which have broad functions, ranging from redox regulation and anti-inflammation to the production of active thyroid hormones. Compared to its counterpart Cys, there are only limited probes for selective recognition of Sec, and such selectivity is strictly restricted at low pH conditions. We reported herein the design, synthesis, and biological evaluations of a series of potential Sec probes based on the mechanism of nucleophilic aromatic substitution. After the initial screening, the structural determinants for selective recognition of Sec were recapitulated. The follow-up studies identified that probe 19 (Sel-green) responds to Sec and other selenols with more than 100-fold increase of emission in neutral aqueous solution (pH 7.4), while there is no significant interference from the biological thiols, amines, or alcohols. Sel-green was successfully applied to quantify the Sec content in the selenoenzyme thioredoxin reductase and image endogenous Sec in live HepG2 cells. With the aid of Sel-green, we further demonstrated that the cytotoxicity of different selenocompounds is correlated to their ability metabolizing to selenols in cells. To the best of our knowledge, Sel-green is the first selenol probe that works under physiological conditions. The elucidation of the structure-activity relationship for selective recognition of selenols paves the way for further design of novel probes to better understand the pivotal role of Sec as well as selenoproteins in vivo.

Full text of DOI:10.1021/ja5099676

Article
pubs.acs.org/JACS
Selective Selenol Fluorescent Probes: Design, Synthesis, Structural
Determinants, and Biological Applications
Baoxin Zhang, Chunpo Ge, Juan Yao, Yaping Liu, Huichen Xie, and Jianguo Fang*
State Key Laboratory of Applied Organic Chemistry and College of Chemistry and Chemical Engineering, Lanzhou University,
Lanzhou, Gansu 730000, China
S
* Supporting Information
ABSTRACT: Selenium (Se) is an essential micronutrient
element, and the biological significance of Se is predominantly
dependent on its incorporation as selenocysteine (Sec), the
genetically encoded 21st amino acid in protein synthesis, into
the active site of selenoproteins, which have broad functions,
ranging from redox regulation and anti-inflammation to the
production of active thyroid hormones. Compared to its
counterpart Cys, there are only limited probes for selective
recognition of Sec, and such selectivity is strictly restricted at
low pH conditions. We reported herein the design, synthesis,
and biological evaluations of a series of potential Sec probes
based on the mechanism of nucleophilic aromatic substitution.
After the initial screening, the structural determinants for selective recognition of Sec were recapitulated. The follow-up studies
identified that probe 19 (Sel-green) responds to Sec and other selenols with more than 100-fold increase of emission in neutral
aqueous solution (pH 7.4), while there is no significant interference from the biological thiols, amines, or alcohols. Sel-green was
successfully applied to quantify the Sec content in the selenoenzyme thioredoxin reductase and image endogenous Sec in live
HepG2 cells. With the aid of Sel-green, we further demonstrated that the cytotoxicity of different selenocompounds is correlated
to their ability metabolizing to selenols in cells. To the best of our knowledge, Sel-green is the first selenol probe that works
under physiological conditions. The elucidation of the structure−activity relationship for selective recognition of selenols paves
the way for further design of novel probes to better understand the pivotal role of Sec as well as selenoproteins in vivo.
Fluorescence sensing using small molecule probes has been
one of the most powerful and popular tools to visualize the
complicated biological processes. However, designing specific
probes of Sec without suffering from the interference of
biological thiols is a big challenge since the thiols usually
present in high concentration (millimolar levels) in cells and
have the similar chemical properties as Sec. The selenol pKa
value in Sec (∼5.8) is lower than those of the most biological
thiols (∼8.3), which means that under physiological conditions
(pH ∼7.4) the Se in Sec is almost fully present as the selenolate
(R-Se−), while the majority of thiols present as nonionized
form (R-SH). The difference of pKa values opens a window for
selective detection of Sec in vitro by maintaining the reaction
system under acidic condition. Maeda et al. reported the first
fluorescent probe BESThio to discriminate the Sec from its
counterpart Cys at pH 5.8.12 We could also selectively label the
Sec residue in the thioredoxin reductase (TrxR) by biotin-
conjugated iodoacetamide at pH 6.5.13,14 However, all these
assays are not compatible with the biological surroundings
generally having a neutral environment (pH ∼7.4), which
prevents their practical applications in live system. Thus, it is of
INTRODUCTION
■
Selenium (Se) was recognized as an essential micronutrient
element in 1960s.1 Insufficient or excessive intake of Se has
been associated with a number of diseases.2,3 Many different
metabolites of Se, such as hydrogen selenide, selenocysteine
(Sec), selenite, selenophosphate, selenodiglutathione, and
charged Sec-tRNA, are synthesized in animals in the course
of converting inorganic Se to organic forms and vice versa.2,4
Although Se may exist as different forms in vivo, the current
knowledge of the biological significance of Se is predominantly
dependent on its incorporation as the Sec into the active site of
selenoproteins, which have a wide range of function, ranging
from redox signaling and anti-inflammation to the production
of active thyroid hormones.3,5,6 Sec is a cysteine (Cys) analogue
with a selenium-containing selenol group in place of the sulfur-
containing thiol group in Cys and the 21st amino acid in
ribosome-mediated protein synthesis.7,8 Due to the low pKa
value of selenol (pKa ∼5.8),9 the Se in Sec is almost fully
ionized under physiological conditions, which gives it high
reactivity, and consequently Sec is normally essential for the
catalytic efficiencies of selenoproteins.10,11 As the Sec carries
out the majority function of the various Se-containing species in
vivo, it is of high demand to develop reliable and rapid assays
with biocompatibility to determine Sec.
Received: September 27, 2014
© XXXX American Chemical Society
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Scheme 1. Sensing Thiols by the Mechanism of NAS
Figure 1. Chemical structures of probes.
great significance to develop novel probes that could selectively
detect Sec over biological thiols under physiological conditions.
Based on the unique nucleophilicity of the sulfhydryl group,
diverse thiol-selective probes have been rationally developed for
redox systems,26−33 we reported herein the design, synthesis,
and biological evaluations of a series of potential Sec probes
(Figure 1). After an initial screening, the structural determi-
nants for selective recognition of Sec were recapitulated. The
follow-up studies disclosed that compound 19 (Sel-green)
could respond to Sec and other selenols with more than 100-
fold increase of emission in aqueous solution (pH 7.4), while
there is no significant interference from the biological thiols,
amines, or alcohols. Sel-green was successfully applied to
quantify the Sec content in TrxR and image endogenous Sec in
live HepG2 cells. In addition, Sel-green is suitable for
identifying the selenol species metabolized from various
selenocompounds in cells, which helps to clarify the mechanism
underlying the cytotoxicity of different selenocompounds. To
the best of our knowledge, Sel-green is the first selenol probe
that works under physiological conditions. Furthermore,
deciphering the structural determinants for selective selenol
recognition paves the way for design and development of novel
activity-based protein profiling15−19 and thiol sensing.20−22
A
well-elucidated mechanism for sensing thiols is the selective
cleavage of 2,4-dinitrobenzene functionality (a fluorescence
quenching moiety) by thiols via nucleophilic aromatic
substitution (NAS) (Scheme 1).23−25 This strategy utilizes
the strong nucleophilicity of thiolates as well as the good
leaving character of fluorophores. Inspired by this sensing
mechanism and the better nucleophilicity of Sec (mainly
present as selenolate) at neutral pH, we reasoned that we could
adjust the electron density of the fluorescence quenching
moiety and the linkage between the fluorophore and the
quenching moiety. Via such modifications to tune the reactivity
of the molecules, selective Sec probes at neutral pH might be
discovered. As part of our continuing interest in discovering
and developing novel small molecule regulators of cellular
B
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Scheme 2. Synthesis of Ether Series Probes
fluorescent probes with biocompatibility to understand the
pivotal role of Sec and selenoproteins in vivo.
Structural Determinants for Selective Recognition of
Sec. After we have constructed a series of potential probes, we
screened their response to Sec and dithiothreitol (DTT)
(Table 1). As Sec is not stable, we generated it in situ by mixing
equal molar of DTT and selenocystine dimethyl ester
((Sec)2).12,34 The probes were incubated with either DTT
alone or Sec generated from DTT and (Sec)2 for 5 min at room
temperature in 0.1 M phosphate buffer solution (PBS), pH 7.4.
The fluorescence intensity changes were measured and listed in
Table 1. It is clear that the two nitro substitutions are essential,
as removing or replacing them to other groups, such as methyl
or carboxylic acid functionality, completely sacrifices the
response to either DTT or Sec (compounds 2b−2f and 3a−
3c). After introducing the 2,4-dinitrobezene moiety, it appears
that the character of linkages determines the probes’ selectivity.
Compounds with the sulfonate connection respond to both Sec
RESULTS AND DISCUSSION
■
Chemical Synthesis. The target probes were composed of
three parts, i.e., fluorophores, electron-deficient quenching
moieties and different linkages (Schemes 2, 3, and 4). The
diverse fluorophores were constructed based on the coumarin
and naphthalimide scaffolds due to their outstanding photo-
physic characters and easy preparation. The coupling of the
fluorophores to the quenching moieties via ether, sulfonamide,
or sulfonate linkage furnishes the final 18 probes (Figure 1).
The straightforward synthetic routes were illustrated in
Schemes 2−4. The original spectra (1H NMR, 13C NMR,
and MS) of the final 18 compounds were included in the
Supporting Information (Figures S3−57).
C
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Scheme 3. Synthesis of Sulfonamide Series Probes
a
>20-fold selectivity of Sec over DTT. However, due to the
intrinsic weak fluorescence of the fluorophores in 9 and 12 in
aqueous solution, we picked up compound 19 for the follow-up
studies. As 19 could be switched on to give green fluorescence,
we termed it here as Sel-green. First, we determined the
absorbance spectra of Sel-green upon addition of Sec. Sel-green
has the maximum absorbance at around 400 nm in PBS buffer.
After addition of Sec, its maximum absorbance is blue-shifted to
∼370 nm (Figure 2A) with an isosbestic point at ∼390 nm,
indicating a clean reaction occurs. The response of Sel-green to
Sec is fast, and the reaction completes within 3 min. The inset
shows the time-dependent changes of absorbance at 401 and
371 nm. Determination of the fluorescence change is shown in
Figure 2B. There is a marginal emission of Sel-green without
Sec (ϕ < 0.001). The emission intensity centered at 502 nm
increased more than 100-fold after addition of Sec when excited
at 370 nm (ϕ = 0.2). The increment of fluorescence intensity is
dependent on the incubation time, and the fluorescence
intensity reaches a plateau after 3 min, consistent with the
results from the absorbance spectra. The inset shows the time-
dependent changes of emission at 502 nm. The fluorescence
emission intensity at varying Sec concentrations (0−1 mM)
was also determined and shown in Figure 2C, which clearly
displays a manner of Sec concentration dependence. Figure 2D
shows the dose-dependent changes of the emission at 502 nm.
The response of Sel-green toward Sec is linear to the
concentrations of the analyte with the range of 0−100 μM
Scheme 4. Synthesis of Sulfonate Series Probes
a
Reagents and conditions: (a) Ethyl acetoacetate (1) or ethyl
trifluoroacetoacetate (4), H3PO4, rt; (b) DCM, TEA, rt; 3a:
R=CH3, R1N, R2=NO2; 3b: R=CH3, R1=NO2, R2H; 3c:
R=CH3, R1H, R2=CH3; 3d: R=CH3, R1=NO2, R2=NO2; 5b:
R=CF3, R1=NO2, R2=NO2.
and DTT almost with the same potency, and thus could not
discriminate the Sec from DTT (compounds 3d and 5b). This
might attribute to the intrinsic higher reactivity of the sulfonate-
bridged compounds. Molecules with ether or sulfonamide
linkages exhibit promising selectivity (compounds 2a, 5a, 9, 12,
14, 16, 19, and 22). (Sec)2 alone gives no response. As several
fluorophores display weak emission in aqueous solution
(compounds 9, 12, and 22), a significant amount of organic
solvents were included in assay buffer for these compounds.
Selective Recognition of Sec by 19. Further analysis of
the data in Table 1 revealed that probes 9, 12, and 19 displayed
D
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a
group give no response to the probe. Vitamin C and inorganic
sulfide are also negative to the probe. Cys, homocysteine
(Hcy), and glutathione (GSH) at high concentration (1 mM
for each) could trivially turn on the fluorescence. However,
compared to the Sec (0.1 mM), this slight increase of
fluorescence is negligible. The inset shows the photo of the
Sel-green solution with Sec (0.1 mM, from 50 μM (Sec)2 and
50 μM DTT), blank, and GSH (1 mM). As a control, DTT
alone (50 μM) gives little response under the same conditions.
The response of other selenocompounds, including Na2SeO3,
Na2Se, and benzenemethaneselenol (BzSeH, in situ generated
from DBDS) was also measured, and only the BzSeH
remarkably switches on the fluorescence. Collectively, Sel-
green could selectively recognize selenols in neutral PBS buffer.
The reaction of Sel-green with Sec was further analyzed by
HPLC. The retention times for Sel-green and the product 18
are 15.66 and 5.05 min, respectively (Figure S1A,B). After
stirring the reaction containing Sel-green (48 mg, 0.1 mmol),
(Sec)2 (100 mg, 0.28 mmol) and DTT (42 mg, 0.28 mmol) in
a DMSO-PBS mixed solvent (1:1, v/v) at room temperature for
1 h, the reaction mixture was extracted and the HPLC profiles
were illustrated in Figure S1C,D. The remaining reactant (Sel-
green) and the product (compound 18) were quantified to be
32.0 and 7.6 mg, respectively. The yield of formation of
compound 18 is 33%. The conversion of Sel-green to the
fluorophore 18 is 95%, indicating a clean conversion of Sel-
green upon reacting with Sec.
Table 1. Response of Probes to Sec and DTT
b
c
Sec
DTT
λex/λem
(nm)
organic
selectivity
d
probes
(F/F0)
(F/F0)
solvent
(Sec/DTT)
2a
2b
2c
2d
2e
2f
16.5
1.1
1.5
0.9
1.2
1.1
1.2
1.3
1.6
1.0
0.9
11.0
2.0
33.2
1.2
323/445 1% DMSO
323/445 1% DMSO
323/445 1% DMSO
323/445 1% DMSO
323/445 1% DMSO
323/445 1% DMSO
323/445 1% DMSO
323/445 1% DMSO
323/445 1% DMSO
323/445 1% DMSO
384/500 1% DMSO
384/500 1% DMSO
11.0
1.2
0.8
0.7
0.8
0.9
1.2
1.3
1.2
0.9
10.0
1.1
20.8
1.0
0.8
0.9
1.2
3a
3b
3c
3d
5a
5b
9
2.0
1.3
1.1
10.1
20.1
37.0
25.0
467/553
40%
DMSO
12
14
16
72.1
8.8
1.5
1.3
2.3
6.2
461/491 40% DMF
48.1
6.8
400/443 1% DMSO
e
25.8
125
465/550
−
11.2
20.2
19 (Sel-
green)
370/502 1% DMSO
22
2.96
1.1
410/500
40%
DMSO
2.7
a
Assays were performed by incubating the probes (10 μM) with Sec or
DTT for 5 min at room temperature. The fluorescence intensity
changes were expressed as F/F0. Sec was generated in situ by mixing
50 μM of (Sec)2 and 50 μM of DTT. The concentration of DTT is 50
b
c
d
Determination of Sec in TrxR. With these results in
hands, we applied the probe Sel-green to analyze the Sec
content in TrxR. The C-terminal extension with the motif -Gly-
Cys-Sec-Gly carrying the essential Sec residue is unique for
mammalian TrxRs. In the reduced TrxR, the Sec residue is
exposed on the surface of the enzyme and easily accessible.35,36
Many small molecule inhibitors are known to interact with the
enzyme by targeting the Sec residue at the active
site.26,28−32,37,38 Thus, quantification of the content of the Sec
residue is of great help to elucidate the mechanism of the
interaction of small molecules with TrxR. The calibration curve
of Sel-green was constructed, and a good linearity was obtained
with the concentration of Sec ranging from 1 to 40 μM (Figure
3A). After addition of the recombinant rat TrxR (final
concentration of 22 μM) and DTT (final concentration of
100 μM), the increase of fluorescence intensity was determined.
According to the calibration curve, the Sec content in TrxR was
calculated to be 16 μM. It should be noted that the
recombinant TrxR used here contains both the full-length
form and truncated form of the enzyme, which lacks the last
two amino acids, i.e., Sec and Gly, at its C-terminus.39,40 The
full-length enzyme is a homodimer and has one Sec residue in
each subunit. The presence of the Sec-deficient truncated TrxR
accounts for the lower Sec content (16 μM) from our result
than the theoretical value (22 μM). We also determined the Sec
content in the U498C TrxR, where the Sec498 was replaced by
Cys. In stark contrast to the TrxR, the mutant enzyme only
gives a background signal (Figure 3B), accentuating the specific
recognition of Sec by Sel-green. Compared to DTT only, the
slight higher fluorescence signal from the mutant enzyme with
DTT could be due to the intrinsic fluorescence from the
mutant enzyme, which contains a flavin molecule in each
subunit. Many proteins bear reactive Cys residues with low pKa
values, such as Trx, Grx, PDI, and papain.41,42 The low pKa Cys
residues are generally created by the defined 3-D structure of
proteins with some spatially neighboring acidic or basic
μM. The probes, except 12 and 16, were dissolved in DMSO to
create stock solutions, so in all assay buffers contain 1% of DMSO.
Probe 12 was dissolved in DMF to create a stock solution. Due to the
fluorophores of probes 9, 12, and 22 have very faint fluorescence in
aqueous solution, 40% of DMSO, or DMF was introduced to facilitate
the screening. Probe 16 is soluble in water, and the stock solution of
16 was prepared in water. There is no organic solvent in the assay
buffer.
e
(R = 0.9967, inset in Figure 2D). The experimental detection
limit is 0.5 μM, which displays a ∼1.4-fold increase of the
fluorescence intensity with good reproducibility. The theoreti-
cal detection limit was calculated to be 62 nM with the
equation of detection limit = 3σ/k, where σ is the standard
deviation of blank measurement, and k is the slope between the
fold of fluorescence increase versus the Sec concentration.
The pH-dependent fluorescence change of Sel-green in
recognition of Sec and Cys is shown in Figure 3A. The
emission intensity increases as the pH rises and reaches a
maximal value at ∼7.4. This attributes to that the higher pH
facilitates the ionization of Sec to selenolate and hence
promotes the NAS reaction to release the fluorophore. As the
pH further increases from 7.4 to 9.0, the emission intensity
decreases, which could be due to the instability of the lactone
moiety in the fluorophore and the nonspecific reaction between
Sec and Sel-green at higher pH. Sel-green exhibits stable
response to Sec at the pH range of 6.0 to 7.7, indicating that it
is suitable for applying under physiological environment. The
response of Cys toward Sel-green has the similar trend as that
of Sec, nevertheless with much less significance. The dynamic
change of the fluorescence upon the reaction of Sel-green with
Sec and Cys is shown in Figure 3B. Compared to the fast
response of Sec, Cys gives a pretty sluggish reaction with Sel-
green.
We then determined the selectivity of Sel-green toward the
Sec. As shown in Figure 3C, amino acids without a sulfhydryl
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Figure 2. Response of Sel-green to Sec. (A). Time-dependent absorbance spectra of Sel-green toward Sec. Sel-green (10 μM) was incubated with
DTT (50 μM) and (Sec)2 (50 μM) at room temperature in PBS, pH 7.4. The absorbance spectra were scanned every 1 min for 5 min. The arrows
indicate the change of the spectra as the function of time. The inset shows the time-dependent changes of absorbance at 401 and 371 nm. (B). Time-
dependent emission spectra of Sel-green toward Sec. Sel-green (10 μM) was incubated with DTT (50 μM) and (Sec)2 (50 μM) at room
temperature in PBS, pH 7.4. The emission spectra (λex = 370 nm) were recorded every 1 min for 5 min. The arrow indicates the change of the
spectra as the function of time. The inset shows the time-dependent changes of emission at 502 nm. (C). Dose-dependent emission spectra of Sel-
green toward Sec. Sel-green (10 μM) was incubated with increasing concentrations of DTT and (Sec)2 at room temperature in PBS, pH 7.4. The
emission spectra (λex = 370 nm) were recorded after 2 min. The arrow indicates the change of the spectra as the function of concentration. (D).
Plotting the fold of fluorescence increase (F/F0) at 502 nm (λex = 370 nm) versus the concentrations of Sec. The experimental conditions are the
same as those in (C). The inset shows F/F0 is linear to the concentrations of Sec in the range of 0.5−100 μM. Data were expressed as mean
standard deviation (SD) of three experiments.
residues. We further determined the response of Sel-green
toward these proteins. As shown in Figure 4B, none of the
tested proteins give significant response toward Sel-green. This
observation is probably due to that Sel-green has little
accessibility to the reactive Cys residues as they are usually
buried within proteins to prevent their inactivation by
electrophiles.
Se is an essential element to humans. However, the overdose
intake of Se or selenium-containing compounds is toxic. The
toxicity of the selenocompounds is, at least in part, due to the
metabolism of them to selenols, which react with cellular thiols
to generate ROS via redox cycling.2,44 We then applied the
probe to study the metabolism of different selenocompounds,
including (Sec)2, SeO2, Na2SeO3, Na2SeO4, DBDS, BPS, and
BPSO, in cells. Except Na2SeO4, BPS and BPSO, all other
selenocompounds significantly induce the green fluorescence in
HepG2 cells at different time points (Figure 6), indicating the
generation of selenol species. The similar results were also
observed in HeLa cells (Figure S2). The fluorescence pictures
were acquired under the same conditions. The slight high
background in some pictures is probably due to the leakage of
the fluorophore from the cells into the culture medium. Next,
we determined the cytotoxicity of these selenocompounds to
the HepG2 cells and HeLa cells. As shown in Figure 7, only the
compounds which could be metabolized to selenol species
cause significantly cytotoxicity. Thus, our results provide
evidence to support that the cytotoxicity of selenocompounds
is related to their ability to metabolize to selenol species.
One of the outstanding functions of Sec is that it is a key
residue in several redox enzymes, such as glutathione
peroxidase and TrxR.36,45 These redox enzymes regulate the
diverse cellular redox signaling pathways, which are involved in
cell proliferation, differentiation, and death. Together with
thiols, Sec is increasingly recognized as an important
Exogenous and Endogenous Sec Imaging in Live
Cells. The live cell imaging was performed in HepG2 cells. As
the physiological concentration of Sec is low in cells, we first
determine if Sel-green could respond to the exogenous Sec.
After addition of (Sec)2 to the cultured HepG2 cells, a bright
green fluorescence signal appears (Figure 5D,E), while there is
no response in the control cells (Figure 5A,C). Another control
experiment with the thiol probe BESThio indicates that the
cellular thiols do not interfere with the detection of Sec by Sel-
green (Figure 5B). This exciting observation motivates us to
further determine the endogenous Sec in the cells. Sodium
selenite (Na2SeO3) is a precursor of Sec biosynthesis, and
supplement of cells with sodium selenite could significantly
increase the Sec level in cells.43 After the cells were stimulated
with sodium selenite for 12 h, the notable fluorescence
appeared (Figure 5G). A short time treatment (1h) gives
weak but reproducible signal (Figure 5F). These results
demonstrated that the probe Sel-green is suitable for imaging
Sec in live cells.
F
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Figure 3. (A) Influence of pH on the response of Sel-green to Sec and Cys. The fold of fluorescence increase at 502 nm was determined after mixing
Sel-green with Sec or Cys for 2 min in 0.1 M PBS buffers with the pH range from 5.0 to 9.0. Sec was generated from 50 μM DTT and 50 μM (Sec)2.
(B) Kinetics of the fluorescence change upon Sel-green responding to Sec and Cys. The fold of fluorescence increase at 502 nm was determined after
mixing Sel-green with Sec or Cys at the indicated times in the PBS buffer, pH 7.4. The error bars for Cys were omitted for clarity, and the SD for
each data point is less than 10%. (C) Selective recognition of Sec by Sel-green. The fold of fluorescence increase at 502 nm was determined after
mixing Sel-green with various analytes for 2 min in the PBS buffer, pH 7.4. Sec was generated from 50 μM DTT and 50 μM (Sec)2, and BzSeH was
from 50 μM DTT and 50 μM DBDS. The concentration of other analytes is 1 mM. The photo of the inset was taken under UV illumination (365
nm). Data were expressed as mean SD of three experiments.
Figure 4. Response of Sel-green to selenoprotein TrxR and other proteins. (A). Calibration curve of response of Sel-green to Sec. (B). Response of
Sel-green to TrxR, U498C TrxR, PDI, Grx, and Trx. The concentrations of the probe, DTT, and proteins are 50, 100, and 22 μM, respectively. The
fluorescence intensity was read after incubation of the probe with the analytes at room temperature for 2 min. Data were expressed as mean SD of
three experiments.
component of the cellular redox network.5 The literature is
replete with the studies on the fluorescent probes of thiols,
which were elegantly summarized in the recent reviews.20−22
However, the selective probes for Sec are quite limited, and the
application of such probes was only restricted to the
nonphysiological conditions.12−14 There is no Sec probe that
can be used in neutral pH and live cells prior to this report.
Based on the NAS sensing mechanism for thiols and the
stronger nucleophilicity of selenols, we rationally synthesized a
series of potential probes with varying reactivity. After careful
evaluation, we discovered that Sel-green could selectively
recognize selenols under physiological conditions. The
potential disadvantage of Sel-green is its low sensitivity for
cell imaging, which might limit its application in vivo due to the
low amount of endogenous selenols. As illustrated in Figure 5,
the cellular fluorescence intensity is lower than that from the
thiol probe BESThio (Figure 5B) but is significantly higher
than that from the background (Figure 5C). Our SAR studies
provide the structural determinants for selective recognition of
selenols under physiological conditions, which will guide
further development of novel probes with improved properties
to understand the pivotal role of selenols as well as
selenoproteins in vivo.
The mechanism of selective turn-on the fluorescence of Sel-
green by Sec is illustrated in Scheme 5. The lower pKa value of
the selenol group renders Sec predominantly present as the
selenolate form (R-Se−) at neutral pH. However, due to the
higher pKa value of thiols, the majority of the sulfhydryl group
in a thiol is un-ionized and present as a neutral form (R-SH).
The nucleophilic attack of the probe Sel-green by selenolate,
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Figure 5. Imaging Sec in live HepG2 cells. The cells were treated with vehicle (A), the thiol probe BESThio (20 μM, B), and Sel-green (20 μM, C)
for 30 min, and the bright-field images (top panel) and fluorescence images (bottom panel) were acquired. HepG2 cells were pretreated with (Sec)2
(5 μM) for 1 h (D) or 12 h (E) and Na2SeO3 (5 μM) for 1 h (F) or 12 h (G), followed by incubation with Sel-green (20 μM) for additional 30 min.
The bright-field images (top panel) and fluorescence images (bottom panel) were acquired. The representative pictures were shown from three
experiments. Scale bar: 15 μm.
Figure 6. Imaging the metabolites of different selenocompounds in live HepG2 cells. The cells were pretreated with 5 μM of (Sec)2, SeO2, Na2SeO3,
Na2SeO4, DBDS, BPS, and BPSO for 1, 6, or 12 h, followed by incubation with Sel-green (20 μM) for additional 30 min. The bright-field images
(top panel) and fluorescence images (bottom panel) were acquired. The representative pictures were shown from three experiments. Scale bar: 15
μm.
which is supposed to be a stronger nucleophile than thiols,
would be favorable. Subsequently, the quenched fluorophore
was released to trigger on the fluorescence. Several probes with
sulfonamide linkage have been reported to selective recognize
thiolphenols at neutral pH.24,46−49 The thiophenols have pKa
values of ∼6.5, which are significantly lower than those of most
aliphatic thiols. Thus, the selectivity of these probes toward
thiolphenols could follow the same mechanism as that of our
probe Sel-green to Sec. However, compared to Sec,
thiolphenols are not naturally present in biological systems.
We believe that the selective detection of Sec and other selenols
is of high physiological significance.
CONCLUSIONS
■
In summary, we have rationally designed and prepared a series
of potential selenol probes. After an initial screening, the
structural determinants for selective recognition of selenol were
delineated. The follow-up studies identified that Sel-green (19)
could selectively respond to Sec and other selenols with more
than 100-fold increase of emission in neutral aqueous solution
(pH 7.4), while there is little interference from the biological
thiols, amines, or alcohols. Sel-green was able to determine the
selenol content in selenoprotein TrxR, while there is little
response of the probe to the mutant enzyme U498C TrxR,
demonstrating the specific recognition of Sec by the probe.
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Figure 7. Cytotoxicity of selenocompouds. HepG2 (A) and HeLa (B) cells were treated with varying concentrations of different selenocompounds
for 24 h, and the cell viability was determined by the MTT assay. Data were expressed the mean values from three experiments. The error bars were
omitted for clarity, and the SD is <10% for each data point.
were carried out using a LUMINA fluorescence spectrofluorometer
(Thermo Scientific) at room temperature. MS spectra were recorded
Scheme 5. Proposed Mechanism for Selective Recognition of
Sec over Thiols
on Trace DSQ GC-MS spectrometer or Bruker Daltonics esquire 6000
mass spectrometer. HRMS was obtained on Orbitrap Elite (Thermo
Scientific). The quantum yields (ϕ) of Sel-green with and without Sec
were determined on FLS920 spectrometer (Edinburgh Instruments,
U.K.) with λex = 370 nm. 1H and 13C NMR spectra were recorded on
Bruker Advance 400, and tetramethylsilane (TMS) was used as a
reference. (Sec)2 was synthesized according to the literature52 in our
laboratory.
Synthesis of Compound 1 (7-Hydroxy-4-methylcoumarin,
Scheme 2).53 3-Hydroxylphenol (11 g, 100 mmol) and ethyl
acetoacetate (13 mL, 100 mmol) were added to concentrated
phosphoric acid (52 mL, 85%). After stirring at room temperature
for 12 h, the reaction mixture was poured into 150 mL water. The
crude product was collected by filtration and purified by recrystalliza-
1
tion in ethanol to afford 1 as white crystal (19.3 g, yield 80%). H
NMR (400 MHz, DMSO-d6) δ: 10.54 (s, 1H), 7.59 (d, J = 8.4 Hz,
1H), 6.81 (dd, J = 8.4, 2.0 Hz, 1H), 6.70 (d, J = 2.4 Hz, 1H), 6.12 (s,
1H), 2.35 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ: 161.21, 160.34,
154.88, 153.31, 126.36, 112.84, 112.02, 110.31, 102.24, 18.08; mp:
189−190 °C. ESI-MS (m/z): [M + H]+ 177.1.
Finally, Sel-green was successfully applied to image the
endogenous Sec in live HepG2 cells and disclose the
involvement of selenol species for the cytotoxicity of
selenocompounds. To the best of our knowledge, Sel-green is
the first selective selenol probe that works under physiological
conditions. Furthermore, deciphering the SAR of the selective
selenol recognition paves the way for further development of
novel fluorescent probes to understand the pivotal role of Sec
as well as selenoproteins in vivo.
Synthesis of Compound 4 (7-Hydroxy-4-trifluoromethylcoumar-
in, Scheme 2).53 3-Hydroxylphenol (11 g, 100 mmol) and ethyl
trifluoroacetoacetate (15 mL, 100 mmol) were added to concentrated
phosphoric acid (52 mL, 85%). After stirring at room temperature for
12 h, the reaction mixture was poured into 150 mL water. The crude
product was collected by filtration and purified by recrystallization in
ethanol to afford 4 as white crystal (19.3 g, yield 80%). 1H NMR (400
MHz, DMSO-d6) δ: 10.96 (s, 1H), 7.55 (m, 1H), 6.91 (dd, J = 8.8, 2.4
Hz, 1H), 6.83 (t, 1H), 6.76 (d, J = 4.8 Hz, 1H); 13C NMR (100 MHz,
DMSO-d6) δ: 162.29, 158.82, 155.93, 140.33, 140.01, 139.70, 139.38,
126.00, 123.14, 120.40, 114.01, 105.15, 103.14; mp: 186−188 °C. ESI-
MS (m/z): [M − H]+ 228.9.
Synthesis of Compounds 2a−2f and 5a. Compound 1 or 4 (0.6
mmol), substituted fluorobenzenes (1.0 mmol), and anhydrous
potassium carbonate (414 mg, 3 mmol) were added to a three-necked
flask under Ar atmosphere, and then anhydrous DMF (5 mL) was
injected into the reaction flask with a syringe. The mixture was stirred
at 80 °C until the starting material disappeared. The solvent was
removed under reduced pressure, and the resulting residue was
purified by chromatography on silica gel to give compound 2a−2f and
5a.
EXPERIMENTAL SECTION
■
Materials and Instruments. The recombinant rat TrxR was
essentially prepared as described40 and is a gift from Prof. Arne
Holmgren at Karolinska Institute, Sweden. The recombinant U498C
TrxR mutant (Sec → Cys) was produced as described.10,28 The
HepG2 and HeLa cells were obtained from the Shanghai Institute of
Biochemistry and Cell Biology, Chinese Academy of Sciences.
Dulbecco’s modified Eagle’s medium (DMEM), GSH, dimethyl
sulfoxide (DMSO), and dibenzyldiselenide (DBDS) were obtained
from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS)
was obtained from Sijiqing (Hangzhou, China). Papain and protein
disulfide isomerase (PDI) were from Sangon Biotech (Shanghai,
China). Thioredoxin (Trx) and glutaredoxin (Grx) from E. coli were
from IMCO (Stockholm, Sweden, www.imcocorp.se). Penicillin and
streptomycin were obtained from Sangon (Shanghai, China).
Benzylphenylselenide (BPS) and benzylphenylselenoxide (BPSO)
were synthesized according to the published refs 50 and 51. The
thiol probe BESThio was synthesized according to the literature.12,25
All other reagents were of analytical grade and were purchased from
commercial supplies. Absorption spectra were recorded on UV−vis
spectrometer evolution 200 (Thermo Scientific). Fluorescence studies
2a (7-(2,4-Dinitrophenoxy)-4-methylcoumarin, Scheme 2). 1H
NMR (400 MHz, DMSO-d6) δ: 8.95 (d, J = 2.8 Hz, 1H), 8.52 (dd, J =
8.8, 2.4 Hz, 1H), 7.93 (d, J = 8.8 Hz, 1H), 7.43 (d, J = 9.2 Hz, 1H),
7.38 (d, J = 2.0 Hz, 1H), 7.29 (dd, J = 8.4, 2.0 Hz, 1H), 6.43 (s, 1H),
2.46 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ: 159.93, 157.12,
154.73, 154.02, 153.38, 142.81, 140.43, 130.22, 128.14, 122.47, 121.51,
117.81, 116.30, 114.08, 108.16; mp: 186−187 °C; EI-MS (m/z) (%):
342(M+, 85), 175(79), 147(100).
I
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2b (Methyl 4-(4-methyl-2-oxo-2H-chromen-7-yloxy)-3-nitroben-
Hz, 1H), 6.42 (d, J = 1.6 Hz, 1H), 2.42 (s, 3H), 2.40 (s, 3H); 13C
NMR (100 MHz, DMSO-d6) δ: 159.61, 153.73, 152.97, 151.14,
146.60, 135.68, 133.59, 131.41, 130.77, 128.72, 127.42, 119.17, 118.52,
114.91, 110.70, 21.59, 18.51; mp: 92−95 °C; ESI-MS (m/z): [M +
H]+ 331.2.
1
zoate, Scheme 2). H NMR (400 MHz, DMSO-d6) δ: 8.56 (s, 1H),
8.22 (d, J = 7.2 Hz, 1H), 7.88 (dd, J = 8.8, 4.0 Hz, 1H), 7.36 (dd, J =
8.4, 3.6 Hz, 1H), 7.27 (s, 1H), 7.20 (d, J = 8.0 Hz, 1H), 6.38 (s, 1H),
3.90 (s, 3H), 2.43 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ:
164.51, 159.98, 157.79, 154.73, 153.39, 152.63, 140.91, 135.99, 127.98,
127.39, 126.09, 121.74, 117.29, 115.83, 113.81, 197.51, 53.20, 18.62;
mp: 164−165 °C; ESI-MS(m/z): [M + H]+ 356.3.
3d (4-Methyl-2-oxo-2H-chromen-7-yl-2,4-dinitrobenzenesulfo-
1
nate, Scheme 4). H NMR (400 MHz, DMSO-d6) δ: 9.12 (s, 1H),
8.63 (d, J = 8.0 Hz, 1H), 8.32 (d, J = 8.4 Hz, 1H), 7.85 (d, J = 8.4 Hz,
1H), 7.32 (s, 1H), 7.23 (d, J = 8.0 Hz, 1H), 6.44 (s, 1H), 2.41 (s, 3H);
13C NMR (100 MHz, DMSO-d6) δ: 159.52, 153.92, 152.90, 152.02,
150.37, 148.57, 134.10, 131.01, 128.09, 127.86, 121.69, 119.95, 118.54,
115.35, 110.85, 18.57; mp: 187−188 °C; ESI-MS (m/z): [M + H]+
407.2.
1
2c (7-(4-Nitrophenoxy)-4-methylcoumarin, Scheme 2). H NMR
(400 MHz, CDCl3) δ: 8.29 (d, J = 9.2 Hz, 2H), 7.66 (d, J = 8.0 Hz,
1H), 7.15 (d, J = 9.2 Hz, 2H), 7.04 (d, J = 8.4 Hz, 1H), 7.02 (d, J = 2.4
Hz, 1H), 6.28 (s, 1H), 2.46 (s, 3H); 13C NMR (100 MHz, CDCl3) δ:
161.86, 160.00, 157.79, 154.77, 153.37, 143.47, 127.89, 126.66, 119.01,
117.21, 116.48, 113.72, 108.13, 18.60; mp: 156−158 °C; ESI-MS (m/
z): [M + H]+ 298.3.
5b (4-Trifluoromethyl-2-oxo-2H-chromen-7-yl-2,4-dinitrobenze-
1
nesulfonate, Scheme 4). H NMR (400 MHz, DMSO-d6) δ: 9.12
(s, 1H), 8.64 (t, 1H), 8.35 (d, J = 8.8 Hz, 1H), 7.80 (d, J = 8.4 Hz,
1H),7.54 (s, 1H), 7.32 (t, 1H), 7.16 (s, 1H); 13C NMR (100 MHz,
DMSO-d6) δ: 158.21, 154.99, 152.09, 150.91, 148.54, 134.05, 130.97,
128.18, 127.21, 121.76, 120.50, 119.42, 118.83, 118.77, 113.62, 111.96;
mp: 125−126 °C; ESI-MS (m/z): [M + H]+ 461.2.
2d (4-(4-Methyl-2-oxo-2H-chromen-7-yloxy)-3-nitrobenzoic acid,
Scheme 2). 1H NMR (400 MHz, DMSO-d6) δ: 13.56 (s, 1H), 8.55 (d,
J = 1.6 Hz, 1H), 8.22 (dd, J = 8.8, 2.0 Hz, 1H), 7.87 (d, J = 8.8 Hz,
1H), 7.35 (d, J = 8.8 Hz, 1H), 7.26 (d, J = 2.4 Hz, 1H), 7.20 (dd, J =
8.8, 2.0 Hz, 1H), 6.38 (s, 1H), 2.44 (s, 3H); 13C NMR (100 MHz,
DMSO-d6) δ: 165.57, 160.02, 158.07, 154.75, 153.41, 152.20, 140.97,
136.18, 127.94, 127.64, 127.46, 121.87, 117.15, 115.65, 113.73, 102.27,
18.63; mp: 230−232 °C; ESI-MS (m/z): [M − H]+ 340.1.
2e (7-Phenoxy-4-methylcoumarin, Scheme 2). 1H NMR (400
MHz, CDCl3) δ: 7.56 (d, J = 8.8 Hz, 1H), 7.44 (t, 2H), 7.25 (t, 1H),
7.10 (d, J = 8.0 Hz, 2H), 6.97 (dd, J = 8.8, 2.4 Hz, 1H), 6.87 (d, J = 2.4
Hz, 1H), 6.19 (s, 1H), 2.42 (s, 3H); 13C NMR (100 MHz, CDCl3) δ:
160.56, 160.20, 155.38, 154.80, 153.53, 130.78, 127.52, 125.29, 120.28,
115.47, 114.46, 112.74, 105.33, 18.56; mp: 59−60 °C; ESI-MS (m/z):
[M + H]+ 253.2.
Synthesis of Compound 8 (4-Hydroxy-n-butyl-1,8-naphthali-
mide, Scheme 2). A mixture of 7 (2.08 g, 7.3 mmol), which was
prepared according to the literature from 4-bromo-1,8-naphthalic
anhydride via two steps,54 and 57% (v/v) hydroiodic acid (100 mL)
was refluxed under argon for 10 h. After cooling, the resulting mixture
was filtered, then the crude product was washed with water to afford
yellow-green solid. The crude product was purified by column
chromatography on silica gel to afford compound 8 as a pale yellow
1
solid (0.88 g, yield 45%). H NMR (400 MHz, DMSO-d6) δ: 11.83
(s,1H), 8.54 (dd, J = 8.4, 1.2 Hz, 1H), 8.48 (dd, J = 7.2, 1.2 Hz, 1H),
8.36 (d, J = 8.4 Hz, 1H), 7.78 (d, J = 7.6 Hz, 1H), 7.16 (d, J = 8.4 Hz,
1H), 4.03 (t, 2H), 1.63 (m, 2H), 1.38 (m, 2H), 0.93 (t, 3H); 13C
NMR (100 MHz, DMSO-d6) δ: 163.64, 162.96, 160.20, 133.51,
131.09, 129.16, 128.85, 125.59, 122.36, 121.81, 112.61, 109.94, 29.72,
19.77, 13.68; mp: 165−166 °C. ESI-MS (m/z): [M − H]+ 268.5.
Synthesis of Compound 9 (4-(2,4-Dinitrophenoxy)-n-butyl-1,8-
naphthalimide, Scheme 2).55 2,4-Dinitrofluorobenzene (186 mg, 1.0
mmol), compound 8 (134 mg, 0.5 mmol), and anhydrous potassium
carbonate (414 mg, 3 mmol) were added to a three-necked flask under
Ar atmosphere, and anhydrous DMF (5 mL) was injected into the
reaction flask with a syringe. The mixture was stirred at 80 °C until the
starting material disappeared. The solvent was removed under reduced
pressure, and the resulting residue was purified by chromatography on
1
2f (7-(2-Nitrophenoxy)-4-methylcoumarin, Scheme 2). H NMR
(400 MHz, CDCl3) δ: 8.16 (t, 1H), 7.80 (m, 2H), 7.52 (t, 1H),
7.39(d, J = 8.0 Hz, 1H), 7.04 (t, 2H), 6.33 (s, 1H), 2.41 (s, 3H); 13C
NMR (100 MHz, CDCl3) δ: 160.09, 159.53, 154.74, 153.46, 147.89,
142.01, 135.97, 127.71, 126.45, 123.28, 116.22, 114.29, 113.21, 105.56,
102.57, 18.59; mp: 132−134 °C; ESI-MS(m/z): [M + H]+ 298.3.
5a (7-(2,4-Dinitrophenoxy)-4-trifluoromethylcoumarin, Scheme
2). 1H NMR (400 MHz, DMSO-d6) δ: 8.96 (d, J = 2.8 Hz, 1H), 8.63
(dd, J = 9.2, 2.8 Hz, 1H), 7.92 (dd, J = 8.8, 1.6 Hz, 1H), 7.66 (d, J =
9.2 Hz, 1H), 7.38 (d, J = 2.8 Hz, 1H), 7.36 (d, J = 2.0 Hz, 1H), 6.94 (s,
1H); 13C NMR (100 MHz, DMSO-d6) δ: 158.58, 158.25, 155.82,
153.28, 143.36, 140.83, 130.37, 127.38, 122.54, 122.43, 116.90, 116.80,
110.98, 108.54; mp: 162−163 °C, EI-MS(m/z) (%): 396(M+, 40),
229(33), 167(100).
1
silica gel to give compound 9 (217 mg, yield 50%). H NMR (400
MHz, CDCl3) δ: 8.99 (d, J = 2.8 Hz, 1H), 8.71 (dd, J = 7.6, 1.2 Hz,
1H), 8.60 (d, J = 8.0 Hz, 1H), 8.49 (d, J = 1.2 Hz, 1H), 8.47 (dd, J =
9.2, 2.8 Hz, 1H), 7.87 (m,1H), 7.25 (dd, J = 8.0, 1.2 Hz, 2H), 4.21 (t,
2H), 1.76 (m, 2H), 1.50 (m, 2H), 1.00 (t, 3H); 13C NMR (100 MHz,
CDCl3) δ: 163.66, 163.05, 155.21, 153.90, 143.11, 140.52, 132.34,
131.82, 129.71, 129.26, 127.86, 127.54, 124.06, 122.99, 122.38, 121.05,
120.19, 114.40, 40.30, 30.10, 20.30, 13.79; mp: 179−180 °C. EI-
MS(m/z) (%): 435(M+, 3), 268(100), 44(40).
Synthesis of Compounds 3a−3d and 5b. The substituted
benzenesulfonyl chloride (1.0 mmol) in 5 mL of CH2Cl2 was slowly
added to the solution of compound 1 or 4 (1.2 mmol) and Et3N (200
mg, 2 mmol) in 20 mL of CH2Cl2. The mixture was stirred overnight
at room temperature. After the reaction was completed, the mixture
was extracted with CH2Cl2 and dried with anhydrous Na2SO4. After
removing the solvent under reduced pressure, the residue was purified
by flash column chromatography to give compound 3a−3d and 5b.
3a (4-Methyl-2-oxo-2H-chromen-7-yl-4-nitrobenzenesulfonate,
Synthesis of Compound 11 (7-Hydroxy-3-benzo[d]thiazol-2-
ylcoumarin, Scheme 2). 2,4-Dihydroxybenzaldehyde (345.3 mg, 2.5
mmol) and compound 10 (435.55 mg, 2.55 mmol), which was
prepared according to the literature23,56 from 2-aminobenzenethiol,
were dissolved in 5 mL ethanol, and five drops of piperidine were
added. The mixture was stirred at room temperature overnight. After
filtration, the yellow solid was treated with 10% hydrochloric acid. The
suspended solution was stirred at 130 °C overnight, and the resulting
yellow residue was collected by filtration, washed with water, dried
under reduced vacuum, and then purified by silica gel column
1
Scheme 4). H NMR (400 MHz, DMSO-d6) δ: 8.48 (m, 2H), 8.22
(m, 2H), 7.82 (d, J = 8.8 Hz, 1H), 7.26 (d, J = 2.4 Hz, 1H), 7.14 (dd, J
= 8.8, 2.4 Hz, 1H), 6.45 (d, J = 1.2 Hz, 1H), 2.41 (s, 3H); 13C NMR
(100 MHz, DMSO-d6) δ: 159.59, 153.83, 152.96, 151.61, 150.69,
139.45, 130.55, 127.67, 125.56, 119.60, 118.60, 115.15, 110.92, 18.54;
mp: 184−185 °C; ESI-MS (m/z): [M + H]+ 362.3.
3b (4-Methyl-2-oxo-2H-chromen-7-yl-2-nitrobenzenesulfonate,
1
Scheme 4). H NMR (400 MHz, DMSO-d6) δ: 8.24 (d, J = 8.0 Hz,
1H), 8.11 (d, J = 7.2 Hz, 1H), 8.06 (d, J = 8.0 Hz, 1H), 7.92 (t, 1H),
7.83 (d, J = 8.8 Hz, 1H), 7.26 (s, 1H), 7.17 (d, J = 8.0 Hz, 1H), 6.43
(s, 1H), 2.39 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ: 159.54,
153.85, 152.92, 150.57, 148.34, 137.76, 133.70, 132.30, 127.76, 126.23,
126.06, 119.72, 118.40, 115.22, 110.78, 18.53; mp: 181−182 °C; ESI-
MS (m/z): [M + H]+ 362.2.
1
chromatography to afford compound 11 (515 mg, yield 70%). H
NMR (400 MHz, DMSO-d6) δ: 11.13 (s, 1H), 9.13 (s, 1H), 8.14 (d, J
= 7.6 Hz, 1H), 8.03 (d, J = 8.0 Hz, 1H), 7.90 (d, J = 8.4 Hz, 1H), 7.55
(m, 1H), 7.45 (t, 1H), 6.93 (dd, J = 8.8, 2.4 Hz, 1H), 6.86 (d, J = 2.0
Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ: 163.45, 160.38, 159.75,
155.73, 151.95, 142.63, 135.59, 131.90, 126.45, 124.95, 122.14, 122.06,
114.51, 114.43, 111.40, 102.01; mp: >300 °C. ESI-MS (m/z): [M +
H]+ 296.0.
3c (4-Methyl-2-oxo-2H-chromen-7-yl-4-methylbenzenesulfo-
1
nate, Scheme 4). H NMR (400 MHz, DMSO-d6) δ: 7.80(t, 3H),
7.50 (d, J = 8.0 Hz, 2H), 7.13 (d, J = 2.4 Hz, 1H), 7.07 (dd, J = 8.8, 2.4
J
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Synthesis of Compound 12 (7-(2,4-Dinitrophenoxy)-3-benzo[d]-
thiazol-2-ylcoumarin, Scheme 2).23 Compound 11 (59 mg, 0.2
mmol), 2,4-dinitrofluorobenzene (111.6 mg, 0.6 mmol), and
anhydrous potassium carbonate (276 mg, 2 mmol) were added to a
three-necked flask under Ar atmosphere, and anhydrous DMF (3 mL)
was injected into the reaction flask with a syringe. The mixture was
stirred at 80 °C for 8 h. The solvent was removed under reduced
pressure, and the resulting residue was purified by chromatography on
J = 8.4, 2.0 Hz, 1H), 8.41 (d, J = 8.8 Hz, 1H), 8.30 (d, J = 8.8 Hz, 1H),
6.69 (d, J = 8.8 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ: 150.89,
141.95, 136.01, 131.47, 126.01, 119.26, 107.70; mp: 172−173 °C; ESI-
MS (m/z): [M + H]+ 409.1.
Synthesis of Compound 18 (7-Diethylamino-3-aminocoumarin,
Scheme 3).46 SnCl2·2H2O (19.4 g, 85.81 mmol) and 60 mL of 37%
HCl were added to a 100 mL flask. Next, compound 17 (3.0 g, 11.45
mmol), which was prepared according to the literature47 from 4-
(diethylamino)salicylaldehyde, was added portion-wisely, and the
resultant solution was further stirred at room temperature for 6 h.
Then, a solution of 5 M NaOH was employed to neutralize the
excessive acid, followed by extraction with ethyl acetate. The organic
layer was dried with anhydrous Na2SO4 and evaporated to dryness.
The crude product was purified by silica gel chromatography to afford
desired product (1.27 g, yield 48%). 1H NMR (400 MHz, DMSO-d6)
δ: 7.19 (d, J = 8.8 Hz, 1H), 6.68 (s,1H), 6.61 (dd, J = 8.8, 2.4 Hz, 1H),
6.48 (d, J = 2.4 Hz, 1H), 4.99 (s, 2H), 3.36 (m, 4H), 1.09 (t, 6H); 13C
NMR (100 MHz, DMSO-d6) δ: 159.68, 150.84, 146.77, 129.14,
126.08, 111.55, 110.26, 109.66, 97.68, 44.19, 12.71; mp: 85−87 °C.
ESI-MS (m/z): [M + H]+ 233.4.
1
silica gel to give compound 12 (64.5 mg, yield 70%). H NMR (400
MHz, DMSO-d6) δ: 9.31 (s, 1H), 8.97 (d, J = 2.8 Hz, 1H), 8.57 (dd, J
= 9.2, 2.8 Hz, 1H), 8.24 (t, 2H), 8.11 (d, J = 8.0 Hz, 1H), 7.62 (t,
1H),7.54(m, 3H), 7.39 (dd, J = 8.4, 2.4 Hz, 1H); 13C NMR (100
MHz, DMSO-d6) δ: 160.12, 159.64, 158.88, 155.28, 153.46, 152.39,
143.35, 141.96, 140.84, 136.37, 132.91, 130.40, 127.19, 125.93, 122.97,
122.72, 122.57, 122.44, 118.94, 117.05, 116.84, 107.45; mp: 248−250
°C. ESI-MS(m/z): [M + H]+ 462.3.
Synthesis of Compound 13 (Ethyl-7-hydroxy-2-oxo-2H-chro-
mene-3-carboxylate, Scheme 2).57 Diethyl malonate (6.96 g, 43.45
mmol), 2,4-dihydroxybenzaldehyde (6.0 g, 43.44 mmol), and
piperidine (1 mL) were dissolved in ethanol (75 mL), and the
resulting solution was heated under reflux for 4 h. After cooling, the
precipitate was collected by filtration. The crude product was
recrystallized from ethanol to afford the compound 13 as white
solid (7.12 g, yield 70%). 1H NMR (400 MHz, DMSO-d6) δ: 11.06 (s,
1H), 8.66 (s, 1H), 7.76 (d, J = 8.4 Hz, 1H), 6.85 (dd, J = 8.4, 2.4 Hz,
1H), 6.72 (d, J = 2.4 Hz, 1H), 4.28 (q, 2H), 1.31 (t, 3H); 13C NMR
(100 MHz, DMSO-d6) δ: 164.02, 162.91, 157.07, 156.35, 149.35,
132.05, 113.96, 112.09, 110.39, 101.76, 60.76, 14.09; mp: 170−171 °C.
ESI-MS(m/z): [M − H]+ 233.1.
Synthesis of Compound 14 (Ethyl-7-(2,4-dinitrophenoxy)-2-oxo-
2H-chromene-3-carboxylate, Scheme 2). Compound 13 (117 mg,
0.5 mmol), 2,4-dinitrofluorobenzene (186 mg, 1.0 mmol), and
anhydrous potassium carbonate (414 mg, 3 mmol) were added to a
three-necked flask under N2 atmosphere, and then anhydrous DMF (5
mL) was injected into the reaction flask with a syringe. The mixture
was stirred at 80 °C until the starting material disappeared. The
solvent was removed under reduced pressure, and the resulting residue
was purified by chromatography on silica gel to give compound 14
(120 mg, yield 60%). 1H NMR (400 MHz, DMSO-d6) δ: 8.95 (d, J =
2.8 Hz, 1H), 8.81 (s, 1H), 8.56 (dd, J = 9.2, 2.8 Hz, 1H), 8.06 (d, J =
8.8 Hz, 1H), 7.54 (d, J = 9.2 Hz, 1H), 7.36 (d, J = 2.0 Hz, 1H), 7.29
(dd, J = 8.0, 2.4 Hz, 1H), 4.33 (q, 2H), 1.33 (t, 3H); 13C NMR (100
MHz, DMSO-d6) δ: 162.99, 159.69, 156.62, 156.15, 153.23, 148.79,
143.49, 140.96, 133.06, 130.41, 122.74, 122.57, 116.90, 116.50, 115.74,
107.11, 61.72, 14.55; mp: 184−185 °C; ESI-MS(m/z): [M + H]+
401.0.
Synthesis of Compound 15 (4-Amino-7-nitrobenzofurazan,
Scheme 3).24 To a solution of 4-chloro-7-nitrobenzofurazan (204
mg, 1 mmol) in MeOH (20 mL) was added ammonium hydroxide (4
mL, 30% in water) at room temperature. The reaction was stirred at
room temperature for 24 h under nitrogen atmosphere. The solvent
was evaporated in vacuo, and then the crude product was purified by
silica gel chromatography to afford the product as a dark-green solid
(108 mg, yield 60%). 1H NMR (400 MHz, DMSO-d6) δ: 8.87 (s, 1H),
8.51 (d, J = 8.8 Hz, 1H), 6.41 (d, J = 8.8 Hz, 1H); 13C NMR (100
MHz, DMSO-d6) δ: 147.78, 144.79, 144.58, 138.46, 103.13; mp: 236−
237 °C. ESI-MS (m/z): [M + H]+ 181.1.
Synthesis of Compound 19 (N-(7-(Diethylamino)-2-oxo-2H-
chromen-3-yl)-2,4-dinitrobenzenesulfonamide, Scheme 3).47 2,4-
Dinitrobenzenesulfonyl chloride (319 mg, 1.2 mmol) in 6 mL of
CH2Cl2 was slowly added to the solution of 18 (232 mg, 1 mmol) and
Et3N (101 mg, 1 mmol) in 10 mL of CH2Cl2. The mixture was stirred
overnight at room temperature. After the reaction was completed, the
mixture was extracted with CH2Cl2 and dried over anhydrous Na2SO4.
After removing the solvent under reduced pressure, the residue was
purified by flash column chromatography to give the title compound as
1
a red solid (147 mg, yield 32%). H NMR (400 MHz, DMSO-d6) δ:
10.56 (s, 1H), 8.86 (d, J = 2.0 Hz, 1H), 8.64 (dd, J = 8.8, 2.0 Hz, 1H),
8.32 (d, J = 8.4 Hz, 1H), 7.82 (s,1H), 7.51 (d, J = 9.2 Hz, 1H), 6.75
(dd, J = 8.8, 2.0 Hz, 1H), 6.50 (d, J = 2.0 Hz, 1H), 3.45 (q, 4H), 1.27
(t, 6H); 13C NMR (100 MHz, DMSO-d6) δ: 159.70, 155.53, 151.15,
150.21, 147.83, 140.93, 138.30, 132.53, 130.25, 127.36, 120.12, 114.43,
109.86, 107.56, 96.74, 44.51, 12.69; mp: 172−174 °C; ESI-MS (m/z):
[M + H]+ 463.1; HRMS (m/z) [M + H]+ calcd for 463.0918, found
463.0907.
Synthesis of Compound 20 (7-Diethylamino-3-(4-nitrophenyl)-
coumarin, Scheme 3).23 2, 4-Dihydroxybenzaldehyde (345.3 mg, 2.5
mmol) and 4-nitrobenzyl cyanide (413 mg, 2.55 mmol) were dissolved
in 5 mL ethanol, and five drops of piperidine were then added. The
mixture was stirred at room temperature overnight. After filtration, the
yellow solid was treated with 10% hydrochloric acid. The suspended
solution was stirred at 130 °C overnight, and the resulting yellow
residue was collected by filtration, washed with water, dried under
reduced vacuum, and then purified by silica gel column chromatog-
1
raphy to afford the compound (420 mg, yield 50%). H NMR (400
MHz,CDCl3) δ: 8.26 (dd, J = 7.2, 2.0 Hz, 2H), 7.92 (dd, J = 7.2, 2.0
Hz, 2H), 7.82 (s, 1H), 8.26 (d, J = 9.2 Hz, 1H), 6.64 (dd, J = 8.8, 2.4
Hz, 1H), 6.54 (d, J = 2.0 Hz, 1H), 3.48 (q, 4H), 1.26 (t, 6H); EI-MS
(m/z) (%): 338 (M+, 4), 193 (28), 44 (100).
Synthesis of Compound 21 (7-Diethylamino-3-(4-aminophenyl)-
coumarin, Scheme 3). SnCl2·2H2O (19.4 g, 85.81 mmol) and 60 mL
of 37% HCl were added to a 100 mL round-bottomed flask. Next,
compound 20 (3.9 g, 11.45 mmol) was added portion-wisely, and the
resultant solution was refluxed for 6 h. Then, a solution of 5 M NaOH
was employed to neutralize the excessive acid, followed by extraction
with ethyl acetate. The organic layer was dried with anhydrous Na2SO4
and evaporated to dryness. The crude product was purified by silica gel
Synthesis of Compound 16 (2,4-Dinitro-N-(7-nitrobenzo[c]-
[1,2,5]oxadiazol-4-yl)benzenesulfonamide, Scheme 3).24 To a
solution of 15 (17 mg, 0.094 mmol) in anhydrous THF (2 mL)
was added NaH (3.4 mg, 0.141 mmol) at 0 °C. After stirring for 15
min, 2,4-dinitrobenzensufonyl chloride (38 mg, 0.141 mmol) in 1.0
mL of anhydrous THF was added dropwisely to the reaction mixture.
The reaction was warmed up to room temperature and stirred for
another 1 h. Then the reaction mixture was quenched with saturated
NaHCO3 solution and extracted with ethyl acetate. The combined
organic layer were washed with brine, dried with magnesium sulfate
and evaporated in vacuo. The crude product was purified by silica gel
chromatography affording desired product as an orange solid (24 mg,
yield 62%). 1H NMR (400 MHz, DMSO-d6) δ: 8.76 (s, 1H), 8.51 (dd,
1
chromatography to afford desired product (1.27 g, yield 48%). H
NMR (400 MHz, DMSO-d6) δ: 7.61 (s, 1H), 7.54 (m, 2H), 7.29 (t,
1H), 6.74 (m, 2H), 6.59 (dd, J = 8.8, 2.4 Hz, 1H), 6.53 (d, J = 2.4 Hz,
1H), 3.75 (s, 2H), 3.44 (q, 4H), 1.23 (t, 6H); 13C NMR (100 MHz,
DMSO-d6) δ: 161.95, 155.80, 150.04, 146.18, 138.53, 129.29, 128.50,
126.02, 121,14, 114.82, 109.38, 108.81, 97.22, 44.80, 12.48; mp: 178−
180 °C. ESI-MS (m/z): [M + H]+ 309.0.
Synthesis of Compound 22 (N-(4-(7-Diethylamino-2-oxo-2H-
chromen-3-yl)phenyl)-2,4-dinitrobenzenesulfonamide, Scheme 3).
2,4-Dinitrobenzenesulfonyl chloride (319 mg, 1.2 mmol) in 6 mL of
K
DOI: 10.1021/ja5099676
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Journal of the American Chemical Society
Article
CH2Cl2 was slowly added to the solution of 21 (308 mg, 1 mmol) and
Et3N (101 mg, 1 mmol) in 20 mL of CH2Cl2. The mixture was stirred
overnight at room temperature. After the reaction was completed, the
mixture was extracted with CH2Cl2 and dried with anhydrous Na2SO4.
After removing the solvent under reduced pressure, the residue was
purified by flash column chromatography to give the title compound as
calculated amount of (Sec)2 and then 5 μL of DTT (1 mM) was added
in a final volume of 50 μL. The fluorescence intensity was read after a
2 min incubation at room temperature. The calibration curve was
constructed by plotting the folds of fluorescence change (λex = 370
nm, λem = 502 nm) versus the concentrations of Sec. For the detection
of the response of Sel-green to different proteins, all proteins were first
adjusted to the concentration of 36 μM in PBS. DTT (1 mM, 5 μL)
were incubated with a blank (30 μL TE buffer) or different proteins
(30 μL) together with 10 μL PBS for 5 min at room temperature, and
the probe (5 μL, 500 μM) was added to a final volume of 50 μL, and
further incubating for 2 min. The fold of fluorescence change was
measured. The protein concentrations of TrxR and U498C TrxR were
calculated by measuring the absorbance of flavin at 460 nm (ε = 11.3
mM−1 cm−1),10 while others were calculated based on their stocks
from vendors.
1
a red solid (215 mg, yield 40%). H NMR (400 MHz, DMSO-d6) δ:
11.17(s,1H), 8.89 (d, J = 2.4 Hz, 1H), 8.63 (dd, J = 8.8, 2.4 Hz, 1H),
8.27 (d, J = 8.8 Hz, 1H), 8.02 (s, 1H), 7.66 (d, J = 8.4 Hz, 2H), 7.48
(d, J = 8.8 Hz, 1H), 7.19 (d, J = 8.8 Hz, 2H), 6.74 (dd, J = 9.2, 2.4 Hz,
1H), 6.54 (d, J = 2.4 Hz, 1H), 3.46 (q, 4H), 1.14 (s, 6H); 13C NMR
(100 MHz, DMSO-d6) δ: 160.71, 156.09, 150.83, 150.42, 148.23,
141.23, 136.71, 135.47, 132.84, 131.94, 129.96, 129.30, 127.62, 120.83,
120.74, 118.10, 109.53, 108.71, 96.45, 44.43, 12.66; mp: 238−240 °C;
ESI-MS (m/z): [M − H]+ 537.14.
Imaging Sec in live HepG2 Cells. HepG2 cells were cultured in
DMEM supplemented with 10% FBS, 2 mM glutamine, and 100
units/mL penicillin/streptomycin and maintained in an atmosphere of
5% CO2 at 37 °C. The cells were seeded in 12-well plates at 2 × 104
cells per well in 1 mL growth medium and incubated at 37 °C for 24 h,
and then the cells were exposed to (Sec)2 (5 μM) for 1 or 12 h. After
washing with PBS three times to remove the remaining (Sec)2, the
cells were further incubated with Sel-green (20 μM) for 30 min at 37
°C. After washing the cells with PBS three times, the fluorescence
images were acquired with Floid cell imaging station (life technology).
To induce the endogenous Sec, the HepG2 cells were exposed to
sodium selenite (5 μM) for 1h or 12 h, and then the cells were further
incubated with the probe (20 μM) for 30 min at 37 °C. After washing
the cells with PBS three times, the fluorescence images were acquired
with Floid cell imaging station.
Fluorescence Imaging of Selenol Species from Selenocompounds
in Cells. The HepG2 and HeLa cells were seeded in 24-well plates at 1
× 104 cells per well in 0.5 mL growth medium and incubated at 37 °C
for 24 h, and then the cells were exposed to different
selenocompounds (5 μM) for 1, 6, and 12 h. After washing with
PBS three times to remove the remaining selenocompounds in
medium, the cells were further incubated with the probe Sel-green (20
μM) for 30 min at 37 °C. After washing the cells with PBS three times,
the bright-field and fluorescence images were acquired with Floid cell
imaging station (Life Technology).
Cytotoxicity Assay. The cytotoxicity of different selenocompounds
was determined by the MTT assay. Cells (5 × 103) were incubated
with varying concentrations of the compounds in triplicate in a 96-well
plate at 37 °C in a final volume of 100 μL. At the end of the treatment
(20 h), 10 μL of MTT (5 mg/mL) was added to each well and
incubated for an additional 4 h at 37 °C. An extraction buffer (100 μL,
10% SDS, 5% isobutanol, 0.1% HCl) was added, and the cells were
incubated overnight at 37 °C. The absorbance was measured at 570
nm on Multiskan GO (Thermo Scientific). The cell viability was
expressed as the percentage of the control (cells without drug
treatment).
UV−vis and Fluorescence Spectroscopy. UV−vis spectra were
acquired from UV−vis spectrometer evolution 200 (Thermo
Scientific). Fluorescence spectroscopic studies were performed with
a LUMINA fluorescence spectrophotometer (Thermo Scientific). The
slit width was 2.5 nm for both excitation and emission. For absorption
or fluorescence measurements, compounds were dissolved in DMSO
or double distilled water to obtain stock solutions. These stock
solutions were diluted with PBS to the desired concentration.
Screening of the Probes. DTT (10 mM, 10 μL), (Sec)2 (10 mM,
10 μL), probe (1 mM, 20 μL), and PBS were mixed to a final volume
of 2000 μL. After incubating for 5 min at room temperature, the
fluorescence intensity was measured. The (Sec)2 was omitted for
determining the response of the probe to DTT.
Response of Sel-Green to Sec. The time-dependent absorbance
spectra and fluorescence spectra (λex = 370 nm) were acquired with
the following procedures: Sel-green (10 μM) was incubated with DTT
(50 μM) and (Sec)2 (50 μM) at room temperature in PBS, pH 7.4.
The spectra were scanned every 1 min for 5 min. To acquire the
emission spectra of Sel-green toward different concentrations of Sec,
Sel-green (10 μM) was incubated with increasing concentrations of
DTT and (Sec)2 at room temperature in PBS, pH 7.4. The emission
spectra (λex =370 nm) were recorded after 2 min. The selectivity of
Sel-green toward different analytes was studied as the following
procedures. The fold of fluorescence increase at 502 nm (λex =370
nm) was determined after mixing Sel-green with various analytes for 2
min in PBS buffer, pH 7.4. Sec and BzSeH were generated from 50
μM (Sec)2 and 50 μMDBDS in the presence of 50 μM DTT,
respectively, while the concentration of other analytes was 1 mM.
Kinetic Response of Sel-Green to Sec and Cys. Sel-green (10 μM)
was incubated with DTT (50 μM) and (Sec)2 (50 μM) or different
concentrations of Cys at room temperature in PBS, pH 7.4. The
emission intensity at 502 nm (λex =370 nm) was measured every 30 s
for 10 min. The fold of fluorescence increase (F/F0) was normalized to
the base fluorescence intensity of Sel-green.
pH-Dependent Fluorescence Response of Sel-Green to Sec and
Cys. The fold of fluorescence increase at 502 nm (λex =370 nm) was
determined after mixing Sel-green (10 μM) with DTT (50 μM) and
(Sec)2 (50 μM) or Cys (1 mM) at room temperature for 2 min in
different 0.1 M phosphate buffers (pH 5−9).
ASSOCIATED CONTENT
■
S
* Supporting Information
HPLC Analyses of the Reaction Between Sel-Green and Sec. Sel-
green (48 mg, 0.1 mmol) was incubated with (Sec)2 (100 mg, 0.28
mmol) and DTT (42 mg, 0.28 mmol) in a DMSO-PBS mixed solvent
(1:1, v/v) at room temperature for 1 h. The reaction mixture was dried
under reduced pressure, and the residue was extracted with DCM. The
combined solvent was removed under vacuum, and the residue was
reconstituted in methanol. All samples were passed through a 0.22 μm
filter, and 10 μL of sample was loaded onto the Waters symmetry C18,
reversed-phase column (3.5 μm, 4.6 × 75 mm) on a Waters 1525
binary HPLC system. The mixture of methanol and water (7:3, v/v)
was used as eluent at the flow rate of 1.0 mL min−1. The detection
wavelength for Sel-green was set at 392 nm and for the fluorophore 18
was set at 337 nm.
The original spectra (1H NMR, 13C NMR, and MS) of the final
18 compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
fangjg@lzu.edu.cn
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Response of Sel-Green to TrxR and Other Proteins. The detection
of Sec was performed at room temperature in PBS, pH 7.4. Sel-green
(5 μL, 500 μM) was added to PBS buffer, followed by the addition of a
The financial supports from the Lanzhou University (the
Fundamental Research Funds for the Central Universities,
L
DOI: 10.1021/ja5099676
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
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Institute) for the recombinant rat TrxR.
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DOI: 10.1021/ja5099676
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