Versatile probes for the selective detection of vicinal-dithiol-containing proteins: Design, synthesis, and application in living cells

Article abstract of DOI:10.1002/chem.201300567

Endogenous vicinal-dithiol-containing proteins (VDPs) that have two thiol groups close to each other in space play a significant importance in maintaining the cellular redox microenvironment. Approaches to identify VDPs mainly rely on monitoring the different concentration of monothiol and total thiol groups or on indirect labeling of vicinal thiols by using p-aminophenylarsenoxide (PAO). Our previous work has reported the direct labeling of VDPs with a highly selective receptor PAO analogue, which could realize fluorescence detection of VDPs directly in living cells. Herein, we developed a conjugated approach to expand detectable tags to nitrobenzoxadiazole (NBD), fluorescein, naphthalimide, and biotin for the synthesis of a series of probes. Different linkers have also been introduced toward conjugation of VTA2 with these functional tags. These synthesized flexible probes with various features will offer new tools for the potential identification and visualization of vicinal dithiols existing in different regions of VDPs in living cells. These probes are convenient tools for proteomics studies of various disease-related VDPs and for the discovery of new drug targets. Copyright

Full text of DOI:10.1002/chem.201300567

FULL PAPER
DOI: 10.1002/chem.201300567
Versatile Probes for the Selective Detection of Vicinal-Dithiol-Containing
Proteins: Design, Synthesis, and Application in Living Cells
Chusen Huang,[a, b] Qin Yin,[b] Jiangjiang Meng,[b] Weiping Zhu,*[a] Yi Yang,[b]
Xuhong Qian,[a, b] and Yufang Xu*[a, b]
Abstract: Endogenous vicinal-dithiol-
containing proteins (VDPs) that have
two thiol groups close to each other in
space play a significant importance in
maintaining the cellular redox microen-
vironment. Approaches to identify
VDPs mainly rely on monitoring the
different concentration of monothiol
and total thiol groups or on indirect la-
beling of vicinal thiols by using p-ami-
nophenylarsenoxide (PAO). Our previ-
ous work has reported the direct label-
ing of VDPs with a highly selective re-
ceptor PAO analogue, which could re-
alize fluorescence detection of VDPs
directly in living cells. Herein, we de-
have also been introduced toward con-
jugation of VTA2 with these functional
tags. These synthesized flexible probes
with various features will offer new
tools for the potential identification
and visualization of vicinal dithiols ex-
isting in different regions of VDPs in
living cells. These probes are conven-
ient tools for proteomics studies of var-
ious disease-related VDPs and for the
discovery of new drug targets.
veloped
expand detectable tags to nitrobenz-
AHCTUNGTRENNoGUN xadiazole (NBD), fluorescein, naph-
a conjugated approach to
thalimide, and biotin for the synthesis
of a series of probes. Different linkers
Keywords: conjugation
cent probes imaging agents
redox · vicinal dithiols
· fluores-
·
·
Introduction
On the basis that trivalent arsenicals can form high-affini-
ty ring structures with vicinal dithiols, most approaches to
identify VDPs mainly rely on monitoring the different con-
centration of monothiol and total thiol groups through selec-
tive labeling of vicinal thiols with p-aminophenylarsenoxide
(PAO). After blocking monothiols, then vicinal dithiols
were labeled with thiol alkylation agent after the removal of
The homeostasis of a cellular redox environment is one of
the most important foundations of living systems. Among
the important factors associated with redox homeostasis and
signaling, protein thiols are especially attractive for their
direct involvement of many biological processes through
post translational modification.[1] As the reductive form of
the protein thiols, vicinal-dithiol-containing proteins (VDPs)
with two space-closed thiol groups have been responsible
for many diseases such as cancer,[2] diabetes,[3] human immu-
nodeficiency virus type 1 (HIV-1),[4] and neurodegenera-
tion.[5] The development of specific probes to trap and iden-
tify VDPs in biological milieu is of significant value to the
pertinent studies.[6]
ACTHNUTRGNEUNG
PAO.[7] Arsenite(III)-affinity chromatography,[8] biotinylated
conjugates of PAO,[9] and dimaleimide fluorogens[10] have
also been developed to enrich and identify new VDPs and
to make proteomic detection possible.[9,11] Despite these ef-
forts, fluorescent and biotin-conjugated probes are still ur-
gently needed to allow direct, noninvasive imaging of the
dynamics of VDPs in living cells and unveil new VDPs with
various functions that are closely related with different dis-
eases. We have previously designed and conjugated highly
selective receptor 2-p-aminophenyl-1,3,2-dithiarsenolane
(VTA2) to naphthalimide, and realized the selective and
direct detection of VDPs with fluorescence readout.[12] It is
worthy to note that VTA2 is a stable, cell permeable, and
selective ligand for VDPs.
[a] C. Huang, Prof. Dr. W. Zhu, Prof. Dr. X. Qian, Prof. Dr. Y. Xu
Shanghai Key Laboratory of Chemical Biology, School of Pharmacy
East China University of Science and Technology
Meilong Road 130, Shanghai, 200237 (P.R. China)
Fax : (+86)21-64252603
Here we expanded the conjugated approaches to afford
series of chemical probes through attachment of various de-
tectable tags (naphthalimide, nitrobenzoxadiazole (NBD),
fluorescein, and biotin) with different linkers (6-aminocap-
roic acid, succinic acid, and piperazine) to VTA2 (Figure 1).
These probes with various properties will be used to identify
the diversities of vicinal dithiols in VDPs in living cells[13]
and present an alternative perspective for drug design.[13b] In
particular, the fluorescence probes can enable the detection
E-mail: wpzhu@ecust.edu.cn
yfxu@ecust.edu.cn
[b] C. Huang, Q. Yin, J. Meng, Prof. Dr. Y. Yang, Prof. Dr. X. Qian,
Prof. Dr. Y. Xu
State Key Laboratory of Bioreactor Engineering, School of Pharmacy
East China University of Science and Technology
Meilong Road 130, Shanghai, 200237 (P.R. China)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201300567.
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Figure 1. Design strategy of probes for direct detection of VDPs.
of VDPs with direct observation of fluorescence both on gel
electrophoresis and in living cells.
Results and Discussion
Design strategy of probes for VDPs: VDPs have received
considerable attention in the past decade.[14] However, it is
difficult to determine protein vicinal dithiols in situ due to
the reversibility between vicinal dithiols and disulfides
within living cells and during sample processing. An ideal
detection method for protein vicinal dithiols should be
rapid, specific, effective, and in situ, avoiding transfer or loss
of the modification signal in the process. Since vicinal dithiol
can form a stable, five-membered ring with PAO,[15] and
monothiols of proteins have much lower affinity,[16] PAO has
been widely used in its specific interaction with vicinal thiols
on proteins. Most strategies have been implemented in the
selective tracing of VDPs by using PAO to block vicinal di-
thiols following thiol alkylation agents to label other thiols.
Finally, the vicinal dithiols were labeled after the removal of
PAO.[7] To simultaneously monitor the VDPs and their dy-
namic response in living cells, especially the role of VDPs in
the regulation of biological process, a fluorescent probe was
a better choice since the detection signal of labeled-VDPs
can be visualized directly.
In our previous report, we have developed the stable and
specific vicinal dithiol ligand VTA2, which can selectively
interact with vicinal thiols on proteins. By conjugation of
the naphthalimide fluorophore with VTA2 to form S3
(Scheme 1), the endogenous VDPs can be visualized directly
on SDS-PAGE and in living cells through the direct fluores-
cence readout. Herein, we now expand the conjugated strat-
egies to afford probes with different properties for the
direct labeling of endogenous VDPs utilizing various func-
tional tags including NBD, fluorescein, and biotin (S1, S2,
and S4, Scheme 1, for the detailed synthesis see the Support-
ing Information). Different linkers (6-aminocaproic acid,
succinic acid, and piperazine) were also introduced for the
conjugation of VTA2 with these tags. For the identification
and selective tracing of VDPs in living cells, the control
probes (SC3, SC1, and SC2, Scheme 1, detailed synthetic
procedures were described in the Supporting Information)
without trivalent arsenical were also synthesized. S4 was
prepared for potential application in proteomic analysis of
VDPs in cell lysis. A mechanism in which S1, S2, S3, and S4
can selectively label VDPs through the specific interchange
Scheme 1. Molecular structures of target and control probes for VDPs.
of 1,2-ethanedithiol (EDT) in cyclic dithiaarsanes of probes
with vicinal dithiols on proteins has been elucidated in
detail.[12] The detailed synthetic design of probes and their
potential application in the labeling of diversities of vicinal
dithiols in various VDPs according to the different proper-
ties of probes are discussed in the following sections.
Synthetic approaches for synthesis of probes for VDPs: In
our initial synthetic approaches, the starting material PAO
was used directly to conjugate with different tags through
various coupling reagents. However, PAO exists as hy-
drate,[17] which makes the purification of PAO-based deriva-
tives difficult on a silica chromatographic column. Although
PAO is a popular specific ligand for protein vicinal dithiols,
it is easily oxidized[18] and no longer exists as a monomer
over time,[17] which means the freshly synthesized PAO can
only be used in one conjugation reaction. All these factors
hinder the direct coupling reaction of PAO with tags and re-
sults in low product yields during the synthesis of target
probes. Another major consideration was that PAO will lose
the specific affinity for vicinal diththiols during oxidation of
trivalent arsenic in PAO to pentavalent arsenic. The stable
crystal structure of tolylarsenic 2,3-dimercaptopropanolate
complex[15] and biarsenical fluorescent analogues[19] promot-
ed us to explore a relatively facile synthetic strategy involv-
ing protection of PAO with 1,2-ethanedithiol (EDT) to first
obtain the stable receptor VTA2. Next, different tags can
then be conjugated with VTA2. Our previous report has re-
vealed that VTA2 was not only more stable but also a
highly selective receptor for vicinal thiols in proteins. VTA2
can selectively discriminate vicinal dithiols from other forms
of thiols through the interchange of 1,2-ethanedithiol (EDT)
in cyclic dithiaarsanes with vicinal dithiols in proteins.
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Next, we explored the coupling reactions of VTA2 with
different tags through various linkers (Scheme 1). The bio-
Similarly, treatment of succinic anhydride with VTA2 gave
VTA6 (Scheme 2) in nearly quantitative yield. These two
flexible VTA2 analogues with one free amino and carboxyl
group, respectively, can be easily conjugated to various tags.
The piperazine was conjugated to succinic acid to form the
piperazine alkyl ether linker, which was another important
linker because the nitro atoms of piperazine can form a hy-
drogen bond with amino acid in some region of the protein.
Additionally, this piperazine moiety can give rigidity to the
flexible linker and modulate the balance between the rigidi-
ty and the flexibility of the probe.[20]
An alternative approach was the development of deriva-
tives of the fluorophores with sites that can be conveniently
conjugated with VTA2. Fluorescein was first activated with
N-hydroxysuccinimide (NHS). After the byproduct (dicyclo-
hexylurea) was filtered off, the filtrate can be treated with
anhydrous piperazine to directly synthesize F2 (Scheme 2).
This one-pot reaction can afford F2 without further purifica-
tion of the fluorescein-activated derivative (F1, Scheme 2)
by using column chromatography and thus avoiding the in-
stability of the active intermediate F1 (see the detailed syn-
thesis in the Supporting Information). By coupling NBD
with 6-aminocaproic acid in basic conditions, the NBD de-
rivative (N1, Scheme 2, see the detailed synthesis in the Sup-
porting Information) was also obtained. The free amino
group of F2 (or free carboxyl of N1) can also aid the further
conjugation of fluorescein (or NBD) with VTA2 and its ana-
logues conveniently. 1,8-Napthalic anhydride was another
derivative of the fluorophore with easy derivation. In our
previous report, VTA4 can be attached to the anhydride
group in ethanol heated at reflux. Most importantly, differ-
ent groups, such as biocompatible diglycol amine, poly(ethy-
lene glycol), can be conveniently attached to the 4- and 5-
positions of 1,8-naphtalimide, which will make the target
probe more suitable for different applications in the biologi-
cal milieu.
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
detailed information in Scheme S2 and the Supporting Infor-
mation), which was better than the multistep methodology.
This versatile coupling strategy was also involved in the con-
jugation of VTA2 with NBD, 5(6)-fluorescein, and 1,8-naph-
thalic anhydride under various conditions in our previous
effort (see the detailed synthetic route in Scheme S1, the
Supporting Information). However, thin-layer chromatogra-
phy (TLC) and NMR analyses showed no products were ob-
served, which may due to the more rigid structures of target
probes just within NBD (fluorescein or 1,8-naphthalic anhy-
dride) and VTA2 (detailed molecular structures in
Scheme S1, the Supporting Information). Another major
factor that promotes us to introduce conjugated linkers was
that the rigid structures of designed probes (structures in
Scheme S1, the Supporting Information) may not enter into
some target regions of VDPs and cannot interact with vici-
nal thiols effectively, which may cause a decrease in the la-
beling efficiency. Thus, different linkers were introduced to
allow the efficient coupling of VTA2 with various functional
tags to afford different flexible probes. One strategy for ac-
complishing this involved two flexible linkers, 6-aminocapro-
ic acid and succinic acid, which were introduced at the same
amino site of VTA2. 6-aminocaproic acid can be easily con-
jugated to VTA2 in three steps to form VTA4 (Scheme 2).
The spectroscopic properties of S1, S2, and S3: For the spe-
cific trapping of endogenous VDPs, the probe must be selec-
tive, stable, and biocompatible.[21] Our investigation began
with analyzing the effect of pH on the UV/Vis and fluores-
cence spectra of S1 and S2. As shown in Figure 2a, c, and e
(detailed information in Figure S1, the Supporting Informa-
tion), the intensity of the UV/Vis and fluorescence spectra
of S1, S2, and S3 was relatively stable at physiological condi-
tions. Thus, phosphate-buffered saline (10 mm PBS, pH 7.4,
1% DMSO) was taken as the test solution in the following
sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) and cell-imaging assay. S1, S2, and S3 dis-
played suitable excitation and emission wavelength (495/
553, 500/523, and 460/538 nm, respectively, Figure 2b, d, and
f) in PBS buffer. These fluorescence properties of S1, S2,
and S3 enable them to label VDPs in living cells. Moreover,
the fluorescence intensity of S1, S2, and S3 changes at differ-
ent pH values. S1 can work in a broad pH range without a
change of its fluorescence intensity. The suitable pH range
for S2 was above 7.4, whereas the fluorescence intensity of
Scheme 2. Approaches for synthesis of different probes for VDPs. a) Ap-
proaches for synthesis of VTA2 and its analogues. b) An alternative
approach for the synthesis of probes through derivatization of the
fluorophore.
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W. Zhu, Y. Xu et al.
labeled rTrx (Figure 3). All
these results indicated the high
selectivity of S1 (or S2) for vici-
nal dithiol in rTrx.
MTT assay: Next, to confirm
the potential availability of S1
and S2 in the in situ imaging of
VDPs, cytotoxicity assays were
performed to evaluate the bio-
compatibility of these two
probes. As shown by the Cell-
Titer 96 AQueous non-radio-
AHCTUNGTRENGNaUN ctive cell proliferation assay,
minimal cytotoxicity at the con-
centration of 0–25 mm was de-
tected (Figure 4), suggesting the
high biocompatibility of S1 and
S2. This result promoted our
further investigation on live-cell
labeling of endogenous VDPs
by using this chemical probe-
based fluorescent technology.
Discussion of the potential la-
beling of various VDPs with
different probes in living cells:
Living cells are rich in VDPs,
but the microenvironments that
the VDPs exist in differ. Many
VDPs are enzymes the active
sites of which are vicinal
thiols.[22] However, the active
cores of these enzymes are dif-
Figure 2. Influence of pH on the fluorescence intensity of a) S1, c) S2, and e) S3 in water solution. Normalized
absorption (left) and fluorescence spectra (right) of b) S1, d) S2, and f) S3 in PBS buffer (10 mm PBS, pH 7.4,
1% DMSO).
S3 remained stable at pH 5–8. All these fluorescence prop-
erties of the three probes make them suitable for labeling
VDPs at a different pH range.
Selectivity of S1 and S2 for VDPs by using SDS-PAGE:
The specific assay of S1 and S2 for vicinal dithiols in pro-
teins was conducted by SDS-PAGE. We used the reduced
form of Trx (rTrx, see the detailed amino acid sequence in
our reported literature[12]) as a typical VDP that only con-
tains one pair of vicinal thiols. A mono-thiol mutant of thio-
redoxin (Trx-M, with C69G mutation) and oxidized thiore-
doxin (oTrx, the vicinal dithiol of oTrx was oxidized by
H2O2 immediately) were taken as the control model protein.
These different forms of Trx were incubated with S1 and S2,
respectively, and then separated by electrophoresis. A fluo-
rescent band was observed only in the lane loaded with rTrx
and S1 (or S2), whereas lanes loaded with oTrx or Trx-M ex-
hibited no fluorescence signal (Figure 3). By contrast, these
forms of Trx were observed on the subsequent Coomassie
Brilliant Blue (CBB) staining, which demonstrated that the
fluorescent band corresponded to the formation of a probe-
Figure 3. Fluorescent imaging of VDPs with S1 and S2 by using SDS-
PAGE. oTrx: thioredoxin (Trx) oxidized immediately by hydrogen perox-
ide (H2O2); Trx-M: reduced thioredoxin mutation (Trx C69G); rTrx: re-
duced thioredoxin; CBB: Coomassie Brilliant Blue.
ferent according to their functional diversities, which indi-
cates that the microenvironments of vicinal dithiols may
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Figure 5. a) The logP values of different probes (S1: 1.84Æ0.03, S2:
0.24Æ0.04, S3: 0.81Æ0.01, S4: 2.45Æ0.37). b) Zeta potentials of different
probes in PBS buffer (10 mm, pH 7.4, 1% DMSO).The data were the
average of more than three measurements. Error bars: ÆS.E.M.
Figure 4. MTT assay for cell toxicity of S1 and S2. Cell viability assay in
the presence of different concentrations of a) S1 and b) S2. The data
were the average of quadruplicate measurements. Error bars: ÆS.E.M.
The image was plotted according to the Promega CellTiter 96 AQueous
non-radioactive cell proliferation assay.
meable fluorophore at physiological conditions.[25] To realize
the live-cell labeling of VDPs, the fluorescein was linked to
VTA2 through a relatively hydrophobic linker (piperazine
alkyl ether linker, Scheme 1) to increase the cell-permeabili-
ty of S2. Although the logP value of S2 was increased, its
value was still relatively lower than other probes (0.24Æ
0.04, Figure 5a). However, this increased hydrophilicity of
S2 makes it more prone to label vicinal dithiols in the hy-
drophilic region of VDPs relative to other probes. The mid-
range logP value of S3 (0.81Æ0.03, in contrast to S1 and S2,
Figure 5a) may make it unbiased in the labeling of VDPs.
By comparing to the potential application in selective la-
beling of various VDPs depending on the hydrophobic (or
hydrophilic) property of probes, the different electrostatic
potentials of probes also can be utilized to trace VDPs with
different isoelectric point (pI) in living cells. As shown in
Figure 5b, different electrostatic potentials of these probes
in PBS buffer can be observed. The lowest electrostatic po-
tential of S3 was ascribed to the attachment of two diglycol-
amine to the 4 and 5 positions of naphthalimide. This fea-
ture of S3 may make it more prone to label VDPs with posi-
tive electrostatic potential in living cells. In addition, all the
fluorescent probes with different electrostatic potentials
may also be used to label various vicinal dithiols in VDPs
with different isoelectric points (pI). In conclusion, diverse
vicinal thiols in various VDPs may be labeled by these se-
lective probes according to their different properties.
differ between different types of VDPs and the nature of
the microenvironments. Thus, the development of different
probes for selective labeling of these different vicinal di-
thiols in the various enzymes was a great challenge. Here,
we discuss the potential application of our probes in the la-
beling of these various vicinal dithiols in VDPs. The logP
values of target probes and their controls were estimated by
using the shake-flask method. As shown in Figure 5a, probe
S1 was a more hydrophobic fluorescence probe compared
with S2 and S3, which makes it more cell-permeable and ac-
cessible to hydrophobic regions of VDPs. An interesting
characteristic of S1 (with NBD fluorophore) was its sensitiv-
ity to the environment.[23] The fluorescence quantum yield
of S1 increased from 0.02 to 0.30 after addition of 0.5%
Tween to the PBS buffer (Table S1, detailed photoproperties
of other probes are shown in Table S1 in the Supporting In-
formation) because the surfactant Tween can form a hydro-
phobic microenvironment for S1. This was also identified by
a small blue-shift in absorbance and significant increase in
fluorescence intensity of S1 with the addition of 0.5%
Tween to PBS (Figure S2, the Supporting Information). The
increase of fluorescence intensity of S1 with decreasing po-
larity of the medium can make it suitable for labeling of vi-
cinal dithiols existing in the hydrophobic regions of VDPs
(such as the vicinal dithiol in hydrophobic core of glucosa-
mine-6-phosphate deaminase).[24] Fluorescein is a cell-imper-
Fluorescent labeling of VDPs in live cells: Firstly, we per-
formed the fluorescent labeling of endogenous VDPs in
living cells using S1, S2, and S3 through fluorescence micro-
scopy. Chang liver cells were incubated with the probe for
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W. Zhu, Y. Xu et al.
30 min at 378C and then washed to remove the free probe
before the fluorescence signal was collected. As shown in
Figure 6, cells treated with S1 (or S2, S3) showed a strong
cell permeability of S2 may be still relatively lower than S1
(or S3), which will hinder the labeling efficiency of S2 for
endogenous VDPs in living cells. On the other hand, the hy-
drophilic characteristics of S2 may make it more suitable for
labeling of hydrophilic VDPs; this needs to be further inves-
tigated in our experiments.
Next, the application of S3 for subcellular labeling VDPs
was explored in Chang liver cells by using confocal micro-
scopy. As shown in Figure 7a, the S3 labeled fluorescence
Figure 7. Application of S3 for labeling subcellular VDPs in Chang liver
cells. Triple staining of endoplasmic reticulum (ER), mitochondria and
VDPs with ER-tracker Blue-White DPX, Mito-Tracker Deep Red and
S3 (5 mm). a) Fluorescence signal of S3, Mito-Tracker Deep Red and ER-
tracker Blue-White DPX labeled, respectively. b) Co-localization of S3
with Mito-Tracker Deep Red and ER-tracker Blue-White DPX. S3/Mito-
chondria: overlay image of S3 and Mito-Tracker Deep Red; S3/ER: over-
lay image of S3 and ER-tracker Blue-White DPX; S3/Mitochondria/ER:
overlay image of S3, Mito-Tracker Deep Red and ER-tracker Blue-
White DPX. Scale bar=10 mm. Images were taken with an Olympus
FV1000+IX481 confocal laser scanning microscopy by using UPLSAPO
100ꢁ oil objective (NA: 1.40).
Figure 6. In situ imaging of VDPs in Chang liver cells. Fluorescent
images of cells labeled with S1 (5 mm; scale bar=50 mm), control probe
SC1 (5 mm; scale bar=100 mm), S2 (5 mm; scale bar=50 mm), control
probe SC2 (5 mm; scale bar=50 mm), S3 (5 mm; scale bar=50 mm), and
control probe SC3 (5 mm; scale bar=100 mm). Images were taken with an
Olympus IX71 microscope equipped with a 20ꢁ objective.
signal displayed punctate pattern that mainly concentrated
in a small, eccentric perinuclear zone, which was similar as
Mito-Tracker Deep Red-labeled signal. The overlay image
of S3 and Mito-Tracker Deep Red showed convincing
yellow fluorescence in punctate pattern (Figure 7b). In con-
trast, the merged fluorescence signal demonstrated less coin-
cidental staining of the ER with S3 and ER-tracker Blue-
White DPX. The overlay image of S3, Mito-Tracker Deep
Red, and ER-tracker Blue-White DPX also implies the co-
localization of the VDPs with mitochondria (Figure 7b),
which was explained in detail in our previous report.[12]
Moreover, enlarged fluorescent images of S1-labeled cells
exhibited a clear fluorescence signal (Figure 8), which needs
to be further studied through proteomic analysis by using
confocal microscopy and SDS-PAGE analysis. Taken togeth-
er, these results suggest that S1, S2, and S3 are highly selec-
tive fluorescent probes for VDPs and can realize the in situ
imaging of VDPs in living cells. Subsequent studies will
focus on further investigation of these fluorescence probes
for the selective labeling of various vicinal thiols in VDPs
fluorescence signal. In contrast, only a background signal
was observed from cells treated with the control probe
(SC1, SC2, or SC3). These results suggested that S1, S2, and
S3 can be used in the selective labeling of endogenous
VDPs in living cells, which was consistent with the SDS-
PAGE results. These results also confirmed that the specific
binding of S1 (or S2, S3) to VDPs in living cells occurred
through the interaction of vicinal thiols on VDPs with reac-
tive cyclic dithiaarsanes in S1, S2, and S3.
It is interesting that the fluorescence intensity of S2-treat-
ed cells was lower than that of S1- (or S3)-treated cells
under the same conditions. We ascribed this phenomenon to
the low cell permeability of fluorescein at physiological con-
dition.[25] After the fluorescein was linked to VTA2 through
a
relatively hydrophobic piperazine alkyl ether linker
(Scheme 1), the cell-permeability of S2 might be increased
(see the logP value in Figure 5a). This feature may enable
S2 to label intracellular VDPs in living cells. However, the
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Probes for Vicinal-Dithiol-Containing Proteins
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a HP 1100 LC-MS spectrometer. Melting points were determined by an
X-6 micro-melting point apparatus and were uncorrected. IR spectra
were recorded on a Nicolet Nexus 770 spectrometer. All pH measure-
ments were performed by using a Sartorius basic pH-Meter PB-20. Fluo-
rescence spectra were determined using a Varian Cary Eclipse fluores-
cence spectrometer. Absorption spectra were determined by a Varian
Cary 100 UV/Vis spectrophotometer.
Synthesis of target probes: The detailed syntheses of VTA2, VTA4,
VTA6, F2, and other related compounds are described in the Supporting
Information according to published procedures.[12]
Figure 8. In-situ-imaging of VDPs in Chang liver cells with S1 (5 mm).
Images were taken with an Olympus IX71 microscope at 10ꢁ, 20ꢁ and
40ꢁ magnification, respectively. Scale bar: 100, 50, 10 mm, respectively.
N-[4-(1,3,2-Dithiarsolan-2-yl)phenyl]-6-[(7-nitrobenzo[c]ACTHNUTRGNE[UNG 1,2,5]oxadiazol-
4-yl)amino]hexanamide (S1): Et3N (55 mL, 0.393 mmol) was added to a
solution of VTA4 (122 mg, 0.328 mmol) in dry dichloromethane, followed
by addition of 4-chloro-7-nitrobenzo[c]ACTHNUGTRNEUNG[1,2,5]-oxadiazole (NBD-Cl,78 mg,
0.393 mmol) in dry dichloromethane (or another procedure was that
VTA4 was dissolved in dry acetonitrile, added to potassium carbonate,
then NBD-Cl in dry acetonitrile was added dropwise to the reaction mix-
ture) at room temperature under an Ar atmosphere overnight. TLC
showed that the reaction was complete, and then the solvent was re-
moved under reduced pressure. The crude product was purified by chro-
matography on a silica gel column (CH2Cl2/methanol, 25:1, v/v) to give
S1 as a brown powder (58 mg, 33% yield). S1 was also can be prepared
through the conjugation of NBD derivative N1 with VTA2 in a relatively
high yield (detailed procedures was described in the Supporting Informa-
tion). 1H NMR (400 MHz, CDCl3): d=8.50 (d, J=8.8 Hz, 1H), 7.63 (d,
J=8.0 Hz, 2H), 7.54 (d, J=8.0 Hz, 2H), 7.21 (s, 1H), 6.43 (br, 1H), 6.20
(d, J=8.8 Hz, 1H), 3.56 (q, J=6.4 Hz, 2H), 3.41–3.35 (m, 2H), 3.22–3.15
(m, 2H), 2.46 (t, J=7.2 Hz, 2H), 1.93–1.82 (m, 4H), 1.64–1.56 ppm (m,
2H); 13C NMR (100 MHz, CDCl3): d=170.7, 144.2, 143.9, 139.0, 138.5,
136.4, 131.6, 123.9, 119.4, 98.5, 43.5, 41.8, 37.0, 28.0, 26.2, 24.3 ppm; IR
according to the features of the probes (such as the fluores-
cence response of the probes at different pH values, differ-
ent logP values, and electrostatic potentials, etc.) in living
cells. Meanwhile, these probes afford convenient tools for
the ongoing proteomics study of various disease-related
VDPs.
Conclusion
In summary, we have expanded the conjugation approaches
to afford a series of chemical probes through attachment of
various functional tags (naphthalimide, NBD, fluorescein,
and biotin) to different linkers (6-aminocaproic acid, succin-
ic acid and piperazine) attached to VTA2. These different
linkers were introduced to make the synthesis more facile.
Most importantly, the property of the probes (such as logP
value, rigidity and biocompatibility, etc.) can be tuned
through these linkers. Additionally, the different characteris-
tics of the fluorophores (different pH response, hydropho-
bicity, etc.) make the probes suitable for potential labeling
of a variety of vicinal dithiols in VDPs. These new probes
will be utilized to label various types of VDPs in different
microenvironments in living cells and to identify new endog-
enous VDPs in subsequent proteomics analyses. Especially,
the fluorescence characteristics of probes S1 and S2 may
make in situ imaging of vicinal thiols existing in different re-
gions of VDPs in living cells possible through direct fluores-
cence readout. This versatile chemical strategy provides an
alternative to antibody-based methods and affords a rapid,
effective and convenient tool for the in situ imaging of
VDPs and the ongoing proteomics study of various disease-
related VDPs.
(KBr): n˜ =3334, 3269, 2940, 1667, 1596, 1513, 1294, 1246, 1181 cmÀ1
;
HRMS (ES+): m/z calcd for C20H22N5O4NaS2As: 558.0227 [M++Na];
found: 558.0222.
Alternative approach for the synthesis of S1: HATU (76 mg, 0.2 mmol),
VTA2 (51 mg, 0.2 mmol) and DIPEA (35 mL, 0.2 mmol) were added to a
solution of N1 (58 mg, 0.2 mmol) in anhydrous DMF (10 mL). The mix-
ture was stirred at room temperature for about 12 h until TLC showed
that the reaction was complete. The crude product was obtained after the
solvent was removed under reduced pressure and then it was purified by
chromatography on a silica gel column (CH2Cl2: methanol, 25:1, v/v) to
afford brown powder (70 mg, 65% yield). 1H NMR (400 MHz, CDCl3):
d=8.50 (d, J=8.8 Hz, 1H), 7.63 (d, J=8.0 Hz, 2H), 7.54 (d, J=8.0 Hz,
2H), 7.21 (s, 1H), 6.43 (br, 1H), 6.20 (d, J=8.8 Hz, 1H), 3.56 (q, J=
6.4 Hz, 2H), 3.41–3.35 (m, 2H), 3.22–3.15 (m, 2H), 2.46 (t, J=7.2 Hz,
2H), 1.93–1.82 (m, 4H), 1.64–1.56 (m, 2H); 13C NMR (100 MHz,CDCl3):
d=170.7, 144.2, 143.9, 139.0, 138.5, 136.4, 131.6, 123.9, 119.4, 98.5, 43.5,
41.8, 37.0, 28.0, 26.2, 24.3 ppm; IR (KBr): 3334, 3269, 2940, 1667, 1596,
1513, 1294, 1246, 1181 cmÀ1
C20H22N5O4NaS2As: 558.0227 [M++Na]; found: 558.0222.
6-[(7-Nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino]-N-phenylhexanamide
(SC1): Compound SC1 was prepared by the reaction of VTAC2 with 4-
chloro-7-nitrobenzo[c][1,2,5]- oxadiazole (NBD-Cl) to give a dark red
;
HRMS (ES+): m/z calcd for
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
solid (0.23 g, 88% yield). The procedure was the similar as synthesis of
1
S1. H NMR (400 MHz, [D6]DMSO): d=9.85 (s, 1H), 9.58 (br, 1H), 8.49
(d, J=8.8 Hz, 2H), 7.55 (d, J=7.6 Hz, 2H), 7.28–7.24 (m, 2H), 7.02–6.98
(m, 1H), 6.41 (d, J=7.2 Hz, 1H), 3.49–3.46 (m, 2H), 2.31 (t, J=7.2 Hz,
2H), 1.75–1.60 (m, 4H), 1.45–1.37 ppm (m, 2H); 13C NMR (100 MHz,
[D6]DMSO): d=171.5, 139.7, 138.4, 134.9, 129.0, 123.3, 119.4, 111.1, 99.6,
43.6, 36.7, 27.9, 26.5, 25.2 ppm; MS (ESI): m/z (%): 370 (100) [M++H].
Experimental Section
N-[4-(1,3,2-Dithiarsolan-2-yl)phenyl]-5-{(3aS,4S,6aR)-2-oxohexahydro-
Materials and apparatus: All chemical reagents and solvents were pur-
chased from Sigma–Aldrich and were used without further purification
except for THF and N,N-dimethylformamide (DMF), which were puri-
fied according to the literature.[26] Thin-layer chromatography (TLC) was
performed on silica gel plates. Column chromatography was performed
by using silica gel (Hailang, Qingdao) 200–300 mesh. 1H and 13C NMR
spectra were recorded employing a Bruker AV-400 spectrometer with
chemical shifts expressed in parts per million (Me4Si as internal stand-
ard). Electrospray ionization (ESI) mass spectrometry was performed in
1H-thieno
ACHTUNGTRENNUNG
hexahydro-1H-thieno
AHCTUNGTRENNUNG
244 mg, 1.0 mmol) and hydroxybenzotriazole (HOBt, 27 mg, 0.2 mmol)
were suspended in anhydrous DMF (10 mL), and heated until a clear sol-
ution was obtained. Then the reaction mixture was cooled to room tem-
perature and a solution of DCC in CH2Cl2 (2.7 mL of a 1.0m solution in
CH2Cl2, 2.7 mmol) was added dropwise and the mixture remained stirring
at room temperature for another 3 h. VTA2 (311 mg, 1.2 mmol) and
Chem. Eur. J. 2013, 19, 7739 – 7747
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
7745

W. Zhu, Y. Xu et al.
DMAP (12 mg, 0.1 mmol) were added, and the mixture was warmed to
608C and it continued to stir for 4 h. Then the mixture was cooled to
room temperature and it continued to stir for 24 h. TLC showed that the
reaction was complete. Then, the solvent was removed under reduced
pressure. The residue was dissolved in CH2Cl2 (80 mL), washed with
water (40 mL), ice-cold HCl solution (5%, 40 mL), and saturated
NaHCO3 (40 mLꢁ2), brine (40 mL). The organic layer was taken to dry-
ness under reduced pressure. The crude product was purified by chroma-
tography on a silica gel column (petroleum ether: EtOAc, 2:1, v/v) to
give S4 as a white powder (350 mg, 72% yield). 1H NMR (400 MHz,
[D6]DMSO):d=9.97 (s, 1H), 7.61 (d, J=8.4 Hz, 2H), 7.55 (d, J=8.4 Hz,
2H), 6.42 (s, 1H), 6.35 (s, 1H),4.32–4.29 (m, 1H), 4.14–4.12 (m, 1H),
3.38–3.32 (m, 2H), 3.19–3.09 (m, 3H), 2.82 (dd, J1 =12.4 Hz and J2 =
5.2 Hz, 1H), 2.57 (d, J=12.4 Hz, 1H), 2.30 (t, J=7.2 Hz, 2H), 1.64–1.58
(m, 4H), 1.52–1.45 (m, 1H), 1.42–1.33 ppm (m, 2H); 13C NMR
(100 MHz, [D6]DMSO):d=171.8, 163.1, 140.6, 137.3, 131.7, 119.2, 61.5,
59.6, 55.8, 41.8, 36.6, 28.6, 28.5, 25.5 ppm; IR (KBr): n˜ =3334, 3269, 1657,
DPBS (containing 1% DMSO, v/v) at 378C. After 30 min for S1,SC1, S2,
SC2, S3, and SC3, respectively, the cells were washed three times to
remove unbound probes before in situ imaging with a fluorescence mi-
croscope (Olympus IX71, magnification 10ꢁ, 20ꢁ and 40ꢁ) with excita-
tion by 488 nm laser, and 500–550 nm emission light was collected for S1,
SC1, S2, SC2, S3, and SC3.
Co-localization of S3 with ER-tracker and Mito-tracker: For triple-stain-
ing of ER, mitochondria, and S3, the cells were first labeled by using
Mito-tracker Deep Red (50 nm), then S3 (5 mm) in DPBS with 1%
DMSO for 30 min at 378C, then finally ER-tracker Blue-White DPX
(100 nm). To exclude the interference to the fluorescent signal, the fol-
lowing emission/excitation wavelengths were used: excitation of ER-
tracker Blue-White DPX at 405 nm, 425–490 nm emission light was col-
lected. Excitation of S3 at 488 nm, 500–600 nm emission light was collect-
ed. Excitation of Mito-Tracker Deep red 635 nm, >650 nm emission light
was collected.
1596, 1513, 1294, 1246, 1181 cmÀ1
; HRMS (ES+): m/z calcd for
C18H24N3O2NaS3As: 508.0114 [M++Na]; found: 508.0145.
N-[4-(1,3,2-Dithiarsolan-2-yl)phenyl]-4-{4-[2-(6-hydroxy-3-oxo-3H-xan-
ACHTUNGTRENNUNGthen-9-yl)benzoyl]piperazin-1-yl}-4-oxobutanamide (S2): HATU (76 mg,
0.2 mmol), VTA6 (72 mg, 0.2 mmol), and DIPEA (35 mL, 0.2 mmol) were
added to a solution of F2 (80 mg, 0.2 mmol) in anhydrous DMF (10 mL).
The mixture was stirred at room temperature for about 12 h until TLC
showed that the reaction was complete. The crude product was obtained
after the solvent was removed under reduced pressure. Then, the crude
material was purified by chromatography on a silica gel column (CH2Cl2/
methanol, 20:1, v/v) to afford a red solid (46 mg, 31% yield). 1H NMR
(400 MHz,[D6]DMSO): d=10.25 (brs, 1H), 7.61 (br, 6H), 7.55–7.53 (m,
3H), 7.39 (s, 1H), 8.61 (d, J=6.8 Hz, 2H), 6.09 (d, J=8.0 Hz, 2H), 5.99
(s, 1H), 3.28–3.15 (m, 12H), 2.56–2.54 (m, 2H), 2.03–1.97 ppm (m, 2H);
13C NMR (100 MHz,[D6]DMSO): d=157.6, 148.5, 140.8, 137.1, 135.9,
132.6, 131.7, 130.8, 130.4, 130.3, 129.6, 129.1, 123.5, 119.0, 103.5, 44.9,
41.7, 31.7, 29.5, 29.2, 22.5 ppm; HRMS (ES+): m/z calcd for
C36H34N3O6NaS2As: 765.0880 [M++Na]; found 765.0890.
Acknowledgements
We thank the financial support from National Natural Science Founda-
tion of China (Grants 21076077, 21236002), the National Basic Research
Program of China (973 Program, 2013CB733700, 2010CB126100), the
National High Technology Research and Development Program of China
(863 Program, 2011AA10A207), Shanghai Pujiang Program and the Fun-
damental Research Funds for the Central Universities, The Innovative
Program of Shanghai Normal University (SK201331), the Science and
Technology Innovation Foundation for College Students (B-7062-12-
001114).
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N-phenylbutanamide (SC2): SC2 was prepared by reaction of VTAC6
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SDS-PAGE and fluorescence imaging of gels: The selectivity of probes
was verified by 15% SDS-PAGE. Samples were labeled in the PBS
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was performed immediately. The gel was imaged by Carestream in vivo
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Cell culture: Chang liver cells were obtained from American Type Cul-
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Eagle’s medium (DMEM, high glucose medium) supplemented with
10% fetal bovine serum (FBS) and typically passaged with sub-cultiva-
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midified incubator at 378C.
MTT assay: Chang liver cells were seeded to Coring 96 well plate the
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Cell labeling and in situ imaging: The Chang liver cells seeded to 35 mm
glass bottom dishes the day before imaging to get the cell density about
80%. The cells were labeled with 5 mm of S1/SC1/S2/SC2/S3/SC3 in
7746
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Received: February 13, 2013
Published online: April 16, 2013
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