Ratiometric Fluorescent Probe for Vicinal Dithiol-Containing Proteins in Living Cells Designed via Modulating the Intramolecular Charge Transfer-Twisted Intramolecular Charge Transfer Conversion Process

Article abstract of DOI:10.1021/acs.analchem.6b02923

Vicinal dithiol-containing proteins (VDPs) play a significant role in maintaining the cellular redox homeostasis and are implicated in many diseases. To provide new chemical tools for VDPs imaging, we report here a ratiometric fluorescent probe CAsH2 for VDPs using 7-diethylaminiocoumarin as the fluorescent reporter and cyclic 1,3,2-dithiarsenolane as the specific ligand. CAsH2 shows peculiar dual fluorescence emission from the excited intramolecular charge transfer (ICT) and twisted intramolecular charge transfer (TICT) states in aqueous media. However, upon selective binding of protein vicinal dithiols to the trivalent arsenical of CAsH2, the probe was brought from the polar water media into the hydrophobic protein domain, causing the excited state ICT to TICT conversion to be restricted; as a result, an increase from the ICT emission band and a decrease from the TICT emission band were observed simultaneously. The designed probe shows high selectivity toward VDPs over other proteins and biological thiols. Preliminary experiments show that CAsH2 can be used for the ratiometric imaging of endogenous VDPs in living cells. So far as we know, this is a rare example of the ratiometric fluorescent probe designed via modulating the ICT-TICT conversion process, which provides a new way to construct various protein-specific ratiometric fluorescent probes.

Full text of DOI:10.1021/acs.analchem.6b02923

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Article
A Ratiometric Fluorescent Probe for Vicinal Dithiol-Containing Proteins in
Living Cells Designed via Modulating the ICT–TICT Conversion Process
Yuanyuan Wang, Yaogang Zhong, Qin Wang, Xiaofeng Yang, Zheng Li, and Hua Li
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02923 • Publication Date (Web): 20 Sep 2016
Downloaded from http://pubs.acs.org on September 20, 2016
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A Ratiometric Fluorescent Probe for Vicinal Dithiol-Containing
Proteins in Living Cells Designed via Modulating the ICT–TICT
Conversion Process
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Yuanyuan Wang,a † Yaogang Zhong,b † Qin Wang,a XiaoꢀFeng Yang,*,a Zheng Li,b Hua Li*,a, c
aKey Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, College of Chemistry &
Materials Science, Northwest University, Xi’an 710127, P. R. China. Fax: (+) 86ꢀ29ꢀ81535026, Eꢀmail: xfyang@nwu.edu.cn
bCollege of Life Sciences, Northwest University, Xi'an 710069, P. R. China
cCollege of Chemistry and Chemical Engineering, Xi'an Shiyou University, Xi'an 710065, P. R. China.
ABSTRACT: Vicinal dithiolꢀcontaining proteins (VDPs) play a significant role in maintaining the cellular redox homeostasis and
are implicated in many diseases. To provide new chemical tools for VDPs imaging, we report here a ratiometric fluorescent probe
CAsH2 for VDPs using 7ꢀdiethylaminiocoumarin as the fluorescent reporter and cyclic 1,3,2ꢀdithiarsenolane as the specific ligand.
CAsH2 shows peculiar dual fluorescence emission from the excited intramolecular charge transfer (ICT) and twisted
intramolecular charge transfer (TICT) states in aqueous media. However, upon selective binding of protein vicinal dithiols to the
trivalent arsenical of CAsH2, the probe was brought from the polar water media into the hydrophobic protein domain, causing the
excited state ICT to TICT conversion being restricted; as a result, an increase from the ICT emission band and a decrease from the
TICT emission band were observed simultaneously. The designed probe shows high selectivity toward VDPs over other proteins
and biological thiols. Preliminary experiments show that CAsH2 can be used for the ratiometric imaging of endogenous VDPs in
living cells. So far as we know, this is a rare example of ratiometric fluorescent probe designed via modulating the ICT–TICT
conversion process, which provides a new way to construct various proteinꢀspecific ratiometric fluorescent probes.
leading to a nonspecific fluorescent labeling reaction.17
Introduction
Another method adopts 1,3,2ꢀdithiarsenolane as the binding
Native proteins with cysteineꢀsulfhydryls in proximity that
site.20ꢀ22 This approach was initially developed by Griffin et al
can undergo reversible dithiol/disulfide conversions are
referred to as vicinal dithiolꢀcontaining proteins (VDPs).1
to image proteins in living cells based on the specific binding
of two appropriately spaced arsenics within a bisarsenical
VDPs are recognized as key components involved in many
fluorescent probe to two pairs of vicinal thiols in tetracysteine
biological processes through a fine balance between protein
motifs (CCXXCC, where C and X represent Cys and any
vicinal dithiols and disulfides, and play fundamental roles in
the maintenance of redox homeostasis of living systems.2,3
amino acid, respectively) that were genetically fused to the
target protein.23 Later, this strategy was employed by Huang et
VDPs are also associated with the formation and stabilization
al to develop fluorescent probes for VDPs in living cells.20,21
of protein structures during the protein postꢀtranslational
modification.4 Apart from that, VDPs are involved in protein
Unfortunately, unbound probes remaining inside cells will
cause an increase in the background fluorescence and thus
synthesis and functions, and are responsible for many diseases
such as cancer,5,6 stroke, 7 and human immunodeficiency virus
hinder the identification of labeled proteins. To address this
type1(HIVꢀ1)8 and neurological disorders.9ꢀ11 Therefore, the
issue, extensive washing of cells has to be carried out to
remove free probes, which are not readily amenable to realꢀ
development of fluorescence probes for VDPs in biological
time monitoring of VDPs in living cells.
systems is of significant importance.
Recently, fluorogenic probes have been developed to
Over the past few years, numerous fluorescent probes for
circumvent this problem.24,25 Generally, these probes exhibit
thiolꢀcontaining compounds have been developed.12ꢀ16
low background fluorescence in a free state and glow only
However, probes that can discriminate VDPs from biothiols
are sparsely reported.17ꢀ22 Currently, two strategies have been
upon association with a prescribed protein. Because the
removal of the unbound probes is unnecessary, realꢀtime
reported for constructing fluorescent probes for VDPs. One
measurements and monitoring of molecular events in living
method employs two spaceꢀclosed maleimide groups as the
cells is possible. In our earlier investigation, we created a
recognition unit, which quenches the probe’s fluorescence
until they both undergo specific thiol addition reaction.17ꢀ19
lightꢀup fluorescent probe for VDPs by adopting a fluorogenic
mechanism based on an environmentꢀsensitive fluorophore.26
However, their application in intracellular labeling is
Despite advances in the lightꢀup fluorescent probes, they tend
interfered by millimolar concentrations of glutathione (GSH)
to be influenced by a variety of factors such as fluctuations in
in cells, which can undergo a similar addition reaction, thus
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excitation intensity, environmental conditions, and the probe
Experimental Section
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concentration. Comparatively, ratiometric fluorescent probes
can reduce the aboveꢀmentioned errors by outputting a
ratiometric signal of the fluorescence intensities at two
emission bands. So far, only one ratiometric fluorescent probe
for imaging VDPs have been developed based on the
fluorescence resonance energy transfer (FRET) mechanism.27
Unfortunately, FRETꢀbased probe is constrained by the
requirement a substantial spectral overlap and favorable
orientations of the transition dipoles for the donor emission
and acceptor absorption bands.28 Obviously, these prerequisite
would restrict the choice of dye pair, thus making the probe
design complicated and lacking versatility. Clearly, the
establishment of new switching strategies for VDPꢀspecific
ratiometric fluorescent probes is highly desired.
Materials and instruments. All chemicals and reagents
were used directly as obtained commercially unless otherwise
stated. Solvents were dried by standard procedures before use.
Double distilled water was used throughout the experiments.
Flash chromatography was performed using Qingdao Ocean
silica gel (200 ꢀ 300 mesh). Human hepatocellular carcinoma
SMMCꢀ7721 cells and murine fibrosarcoma L929 cells were
obtained from Cell Engineering Research Centre and
Department of Cell Biology of Fourth Military Medical
University (China). CAsH1, CAsH2, CAsH3 and compound
6 were dissolved in DMSO as stock solution. Reduced bovine
serum albumin (rBSA) and other reduced form proteins were
prepared and stored according to the literature.17
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Fluorescence measurements were performed on a Perkin
Elmer LSꢀ55 fluorescence spectrometer in 10 × 10 mm quartz
cells. Unless specific noted, the PMT voltage was set at 750 V.
UVꢀvisible spectra were recorded in 1 cm quartz cells with a
Shimadzu UVꢀ2550 spectrophotometer. 1H NMR and 13C
NMR spectra were recorded at 400 and 100 MHz on an
INOVAꢀ400 spectrometer (Varian Unity), respectively.
Chemical shifts are reported in ppm (δ) and are referenced to
residual protic peaks. Highꢀresolution mass spectra were
acquired with a Bruker micrOTOFꢀQ II mass spectrometer
(Bruker Daltonics Corp., USA) in electrospray ionization
(ESI) mode. Fluorescence imaging was performed on an
Olympus FV1000 confocal laser scanning microscope (Japan).
In this study, we describe the rational design of CAsH2 as a
new ratiometric fluorescent probe for VDPs based on the
modulation of ICT to TICT conversion process (Scheme 1). In
the proposed sensing system, 7ꢀdiethylamincoumarin was
selected as the fluorescence reporter and cyclic dithiaarsane as
the specific ligand for VDPs. In aqueous media, the proposed
probe exists both as ICT and TICT excited states upon
photoexcitation, thus a dual fluorescence emission being
observed. Upon selective binding of protein vicinal dithiols to
the trivalent arsenical of CAsH2, the probe was brought from
the polar water media into the hydrophobic protein domain,
which is generally in a lowꢀpolar environment. With the
decrease of environmental polarity around CAsH2, the
formation energy barrier of TICT increases, causing the ICTꢀ
TICT conversion being restricted. Hence, an increase in the
ICT emission band and a decrease in the TICT emission band
should be observed in the fluorescence spectra simultaneously.
Because of the separate emission bands from the ICT and
TICT states, the above fluorescence changes enable us to
develop a ratiometric fluorescent probe for VDPs with a
simple structure. Although TICTꢀbased probes have been
developed for different analytes,29ꢀ32 most of them are operated
on the fluorescence turn–on mode. To the best of our
knowledge, this is the first time that a ratiometric fluorescent
probe with high selectivity for VDPs was designed via
modulating the ICT–TICT state conversion process. The
proposed probe has been successfully applied for visualization
of VDPs in living cells.
Cell Cultures and fluorescence imaging. L929 cells were
seeded in 6ꢀwell culture plates containing sterile coverslips
and were cultured in RPMI 1640 medium supplemented with
10% (v/v) fetal bovine serum (FBS), 100 U mLꢀ1 penicillin
and 100 ꢁg mLꢀ1 streptomycin at 37 oC in a humidity
atmosphere under 5% CO2 for 24 h. The medium was removed
and the adherent cells were washed with PBS buffer (pH 7.4)
three times. After the cells were incubated with 5 ꢁM of
CAsH2 (or compound 6) in serumꢀfree RPMI medium at 37
°C for 20 min, the staining solution was replaced with fresh
PBS to remove the remaining free probe. Cell imaging was
then performed by an Olympus FV1000 confocal laser
scanning microscope immediately. Emission was collected at
430–490 nm for the cyan channel and at 530–590 nm for the
yellow channel (λex = 405 nm). For the control experiment, the
cells were pretreated with dithiothreitol (DTT, 10 mM) for 30
min. After washing with PBS three times, the cells were
further treated with CAsH2 and imaged using the conditions
described above.
Scheme 1. Proposed mechanism for the ratiometric
fluorescent sensing of VDPs with CAsH2
To test colocalization with the mitochondria, SMMCꢀ7721
cells (which were cultured using the same procedure for L929
cells) were coꢀstained with 2 ꢁM of CAsH2 and 0.5 µM of
MitoꢀTracker deep red for 30 min and then fluorescence image
were acquired by confocal microscopy. Emissions were
collected at 430–490 nm for CAsH2 (λex = 405 nm) and at
655–755 nm for MitoꢀTracker deep red (λex = 640 nm). ImageJ
software was used for analysis of the images.
Results and Discussion
Probe design. In our continuing efforts to develop
fluorescent probes for VDPs, we have developed a fluorogenic
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Scheme 2. Synthesis of probe CAsH1, CAsH2, CAsH3 and compound 6 a
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O
S
As
S
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O
HN
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S
As
S
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a
NH2
+
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COOH
PAO-EDT
1
CAsH1
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As
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N
COOH
5
N
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O
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d
e
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6
aReagents and conditions: (a) EDC, HOBt, CH2Cl2, room temperature, 4 h; (b) 6ꢀaminohexanoic acid, DSC, DMAP, DMF, room
temperature, 4 h; (c) PAOꢀEDT, HATU, DIPEA, DMF, room temperature, overnight; (d) diethyl malonate, piperidine, C2H5OH,
reflux, 5 h; (e) 6ꢀaminohexanoic acid, DSC, DMAP, DMF, room temperature, 4 h; (f) PAOꢀEDT, HATU, DIPEA, DMF, room
temperature, overnight; (g) HATU, DIPEA, DMF, room temperature, overnight.
probe for specific detection of VDPs in living cells. However,
the intensityꢀbased probe is inferior to ratiometric one, as the
former is vulnerable to variations in the assay environment,
thus posing potential problems for their use in quantitative
measurements of VDPs in biological systems. Therefore,
ratiometric fluorescent probes for VDPs are highly desired.
because it can discriminate VDPs from monothiols proteins
through the specific interchange of 1,2ꢀethanedithiol (EDT) in
cyclic dithiaarsanes with vicinal dithiols in proteins.20
With the reasoning described above, we designed probe
CAsH1 and CAsH2 by coupling 7ꢀdiethylaminocoumarinꢀ3ꢀ
carboxylic acid with 2ꢀ(4ꢀaminophenyl)ꢀ1,3,2ꢀdithiarsolane
(PAOꢀEDT). We envisioned that the binding of the probe to
VDPs would bring it into the hydrophobic pocket of the
protein, which is generally less polar than the bulk aqueous
phase and would greatly affect the ICT and TICT emission,
thus resulting in ratiometric fluorescent signals output.
Scheme 2 shows the synthesis routes of CAsH1 and CAsH2,
as well as its control probe CAsH3 and compound 6. The
It is wellꢀknown that 7ꢀdialkylaminiocoumarins are
solvatochromic dyes, which exhibit dim fluorescence in polar
solvents but become brightly fluorescent in lowꢀpolar
solvents.33 The marked change of the emission yields of these
coumarin derivatives is interpreted in terms of a charge
stabilizing influence by solvent or the microenvironment of
the dye, which tends to affect the energy barrier for torsional
motion leading to the TICT state.34ꢀ36 Such control on the
charge distribution in the coumarins and hence their optical
properties have been widely studied.37ꢀ40 It is thus expected
that solvatochromic coumarins will be ideal fluorophores to
probe the target proteins by incorporating with a proteinꢀ
specific ligand. On the other hand, 2ꢀ(4ꢀaminophenyl)ꢀ1,3,2ꢀ
dithiarsolane (PAOꢀEDT) was selected as the recognition unit
detailed
synthetic
procedures
and
spectroscopic
characterization of these compounds are provided in the
Supporting Information.
With the probe in hand, we initially study its optical
properties in aqueous media. Surprisingly, compared with
common aminocoumarin dyes, it was observed that CAsH2
features a broad emission peak at 550 nm with a blueꢀshifted
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shoulder at 475 nm (Figure 1). This dual emission can be
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fd(vol%)
rationalized by the wellꢀaccepted ICT–TICT conversion
process in aminocoumarins.41,42 Upon photoexcitation, CAsH2
initially forms an ICT state with a partial charge transfer. In
highly polar media, the diethylamino group subsequently
undergoes twisting which makes the donor orbital
perpendicular to the acceptor orbital. This results in a full
charge separation and in the formation of the TICT state.
Based on the above facts, it is assumed that the observed dual
emission of CAsH2 in aqueous solution is attributed to the
two excited states ICT and TICT, respectively.
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To prove the above hypothesis, we studied the optical
characteristics of CAsH2 by measuring its absorption and
emission spectra in the mixture solutions of water and 1,4ꢀ
dioxane with different polarities. As the proportion 1,4ꢀ
dioxane of in the binary mixture (1,4ꢀdioxane – water)
increased from 0% to 99%, corresponding to a decrease in
solution polarity (ꢂf: 0.32 → 0.08),43 the absorption maxima
of CAsH2 shifted from 430 to 408 nm, and only small
changes were observed in the absorbance in all of these
solutions with different polarities (Figure S1). In contrast, the
fluorescence band of CAsH2 at 475 nm was found to shift to
440
480
520
560
600
640
Wavelength (nm)
Figure 1. Fluorescence spectra (λex = 415 nm) of CAsH2 (1 ꢁM)
in water/1,4ꢀdioxane mixtures with different volume fractions of
1,4ꢀdioxane (fd). HV = 650 V.
Next, to confirm the longer emission band of CAsH2 is
from the TICT state, we prepared probe CAsH3, where unlike
the probe CAsH2, the N,Nꢀdiethylamino group is substituted
by a bridged julolidinyl structure which makes the rotation of
the diethylamino group with respect to the coumarin skeleton
unavailable; as a result, TICT state formation would be
absolutely forbidden. The emission spectra of CAsH2 and
CAsH3 in aqueous media were shown Figure S3. Contrary to
the dual emission of CASH2, CAsH3 exhibits one major
emission band from the ICT state (λem = 495 nm). It is thus
evident that the second redꢀshifted emission band (λem = 550
nm) of CAsH2 is attributed to the TICT state, since no such
emission is observed for CAsH3.
440 nm, concomitant with
a
significantly increased
fluorescence intensity (110ꢀfold increase). Obviously, this
emission band originates from the ICT process, which
generally shows a positive response to the environmental
polarity similarly to classical solvatochromic dyes.
Meanwhile, it was observed that the longer emission band at
550 nm is very sensitive to the solution polarity and the
emission band completely vanished even in 10% 1,4ꢀdioxane
aqueous solution (Figure 1), which indicates that this emission
band is indeed contributed by the TICT state. The quenching
of TICT emission can be explained as follows: with the
decrease of solution polarity, the formation energy barrier of
TICT state increases; as a result, TICT process is prohibited.
To get more information about the effect of solution polarity
on the emission behavior of CAsH2, we further test the
fluorescence spectra of CAsH2 in the mixture solutions of
water and ethanol with different polarities. Because the
polarity of ethanol is higher than that of 1,4ꢀdioxane (dielectric
constant at 20 oC: ethanol, ɛ = 25.3; 1,4ꢀdioxane, ɛ = 2.219),44
it was observed that when the ethanol fraction (fe, by
volume%) in the H2O/ethanol mixture is increased from 5 to
30%, the intensity of the TICT emission band of CAsH2 at
550 nm decreases markedly, while the ICT band at 475 nm
undergoes a distinct increment simultaneously. When fe is
30%, the TICT peak (550 nm) in the longer wavelength region
disappears, and the ICT emission (475 nm) becomes the only
peak observed in the fluorescence spectrum (Figure S2). As a
result, the ratio of ICT and TICT emission intensities
(IICT/ITICT) can be used as a polarity indicator. It was also
observed from Figure S2 that the emission peak at 475 nm
further increased with decreasing the polarity of the media (fe
> 30%), which is caused by reducing the charge transfer
between the fluorophore and its surrounding media.
Despite the wide acceptance of TICT process in
aminocoumarin derivatives, fluorescence emission from the
TICT states in these dyes are scarcely observed. To further
ascertain the contribution of the emissive TICT in CAsH2,
compound 6 was prepared and compared its optical properties
with that of CAsH2. The molecular structures of CAsH2 and
compound 6 differ in that the former bears a cyclic
dithiaarsane (Scheme 2). As shown in Figure S4, the
fluorescence spectra of compound 6 showed a maximum
emission at 475 nm. In the case of CAsH2, however, a redꢀ
shifted new emission band (λmax = 550 nm) was observed,
which has been proved to be contributed by the TICT state.
Thus, it can be concluded that the 5ꢀmembered dithiarsolane
ring is exclusively responsible for the emissive TICT state of
CAsH2. However, the complexity of the photophysical pattern
requires further investigation to provide a detailed explanation.
Based on the above results, it is shown that CAsH2 affords
peculiar dual fluorescence emissions from the ICT and TICT
excited states in highly polar solvent, their populations
depending on the environmental polarity. Moreover, the ICT
and TICT emissions of CAsH2 show completely opposite
polarityꢀdependence, and a hypochromatic shift of ~ 110 nm
was observed with decreasing the polarity of the media. The
above characteristic makes CAsH2 favorable for the dualꢀ
emission ratiometric sensing the polarity of its immediate
environment. Thus, we assumed that CAsH2 would serve as a
ratiometric fluorescent probe for VDPs. The selective binding
of protein vicinal dithiols to the trivalent arsenical of CAsH2
would bring the probe from the polar water media to the
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oC. Inset: time course of fluorescence intensity of CAsH2 in the
presence of rBSA.
hydrophobic protein domains with low polarity, which would
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result in the enhancement of ICT emission channel at the
expense of TICT emission band. Therefore, ratiometric
sensing of VDPs might be realized.
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Next, the sensing behavior of CAsH2 toward rBSA was
examined with absorption and fluorescence spectra. As shown
in Figure S8, the absorption spectrum of CAsH2 displays a
strong ICT band at 440 nm. Upon incremental addition of
rBSA, this band blueꢀshifted to 428 nm, concomitant with a
gradual increase in absorbance signal. The above results are
consistent with the solvatochromic properties of environmentꢀ
sensitive dyes. Furthermore, free CAsH2 (1 µM) shows two
emission bands centered at 475 and 550 nm (λex = 415 nm),
which can be assigned as the ICT and TICT emission bands,
respectively. When increasing concentrations of rBSA (0 – 1.8
ꢁM) were introduced, the fluorescent spectrum of CAsH2
exhibited significant changes (Figure 3). It was observed that
the emission band at 475 nm undergoes a distinct increase and
the emission maximum blueꢀshifted to 468 nm, while the band
at 550 nm exhibits an obvious decrease simultaneously.
Meanwhile, it was observed that the emission color of CAsH2
turned from pale green to cyan in the presence of rBSA when
illuminated by a handꢀheld UV lamp at 365 nm (inset of
Figure 3). The above changes in emission spectra suggest that
location of the probe was changed from the polar aqueous
environment to the lowꢀpolar protein interior. Another
possible explanation is that the interior of protein might be too
rigid to twist the probe in the excited state, thus the relaxation
from the highly fluorescent ICT state to a weakly fluorescent
TICT state being hampered. The ratio of emission intensities
at 468 and 550 nm (I468/I550) with the addition of rBSA varies
from 0.33 to 24.5, a ca. 74ꢀfold emission ratio change.
Furthermore, the emission ratios (I468/I550) were plotted as a
function of rBSA concentration and a typical calibration graph
was obtained as shown in Figure 4. The I468/I550 value was
linearly related to rBSA concentration up to 1.2 ꢁM with a
detection limit of 2.6 nM (3δ). These results demonstrate that
CAsH2 can detect rBSA quantitatively.
Optical response of CAsH2 toward VDPs. Inspired by the
ratiometric fluorescence response of CAsH2 toward the
environmental polarity, we then examined the sensing of
probe CAsH2 toward VDPs in phosphate buffer solution (20
mM, pH 7.4, containing 1% DMSO). We selected rBSA as the
model protein because it contains eight vicinal Cys pairs after
BSA is reduced with tris(2ꢀcarboxyethyl)phosphine (TCEP).17
In our initial attempt to develop ratiometric fluorescent probe
for VDPs, we constructed CAsH1 and examined its sensing
behavior toward rBSA (Figure S5). Although CAsH1 shows a
desirable ratiometric fluorescence response toward rBSA, its
slow binding kinetics (> 60 min) makes its unsuitable for
monitoring the dynamic changes of VDPs inside living cells.
We envisioned that the slow response of CAsH1 toward rBSA
might be due to the steric hindrance arising from the
fluorophore adjacent the active site. In an advance, this issue
was addressed by placing a flexible linker (6ꢀaminocaproic
acid) between the coumarin fluorophore and PAOꢀEDT (to
form CAsH2).
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The time course of CAsH2 in the presence of rBSA was
then examined by fluorescence spectroscopy. It can be
observed from Figure 2 that the fluorescence emission band at
468 nm increases dramatically upon addition of rBSA,
concomitant with a decrease of TICT emission band at 550
nm. The emission ratio (I468/I550) of the sensing system affords
an initial fast, followed by a gradual increase in the sensing
process, while the free probe exhibited no noticeable emission
ratio changes under identical conditions (Figure S6). The
emission ratio of the sensing system essentially reached a
maximum after 20 min, and thereafter remained almost
unchanged. Compared with CAsH1 and the previous reported
probe, the present probe CAsH2 affords a fast response to
rBSA, which makes it suitable for monitoring the dynamic
changes of VDPs in situ. In addition, the reversibility of the
present fluorescent sensing system was also proved by adding
a large amount of EDT to the solution of CAsH2ꢀrBSA
complex (Figure S7).20
600
Blank
rBSA
480
360
240
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750
rBSA
468 nm
550 nm
750
600
450
600
300
150
450
0
0
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Time (min)
450
500
550
600
650
300
150
0
Wavelength (nm)
Figure 3. Fluorescence spectra of CAsH2 (1 ꢁM) upon addition of
increasing concentrations of rBSA (0 – 1.8 ꢁM) in phosphate
buffer (pH 7.4, 20 mM, containing 1% DMSO) for 20 min at 37
oC. λex = 415 nm. HV = 700 V. Inset: images of CAsH2 alone and
in the presence of rBSA exposed to a UV lamp at 365 nm.
450
500
550
600
650
Wavelength (nm)
Figure 2. Timeꢀdependent fluorescence spectral (λex = 415 nm)
changes of CAsH2 (1 ꢁM) in the presence of rBSA (0.6 ꢁM) in
phosphate buffer (20 mM, pH 7.4, containing 1% DMSO) at 37
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BSA
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Figure 4. Linear plot of the emission ratio (I468/I550) against rBSA
concentration when using CAsH2 (1 ꢁM).
(b)
Furthermore, to test the generalization of the probe, other
reduced forms of proteins (human serum albumin, ovalbumin,
lysozyme, pepsin and trypsin) were screened under the same
conditions, and it was observed that reduced human serum
albumin (rHSA) affords a fluorescence response similar to
rBSA, while reduced forms of ovalbumin and lysozyme
affords a relatively small fluorescence changes (Figure S9).
This is apparently due to different VDPs having different
reactivities with CAsH2. It was further observed that reduced
pepsin and trypsin afford almost no changes in emission ratio.
This can be explained by the fact that pepsin and trypsin
contain almost no hydrophobic pockets as in rBSA.45
To verify our hypothesis that the fluorescence change comes
from the binding reaction between CAsH2 and VDPs and
provide a better understanding of the above detecting
mechanism, the following experiments were carried out. First,
the reference compound 6 was treated with rBSA, however, it
shows negligible fluorescence increase (increased by 26%) at
480 nm (Figure S10). The above experiments prove that the 5ꢀ
membered dithiarsolane ring in CAsH2 is involved in the
binding reaction and the fluorescence change is indeed due to
the selective binding of CAsH2 with vicinal dithiols in
proteins, while not the nonspecific interaction between the
fluorophore and proteins. Next, the sensing behavior of
CAsH3 toward rBSA was examined. As shown in Figure S11,
CAsH3 displays a moderate increase (~ 3ꢀfold increase) in
fluorescence intensity with the addition of rBSA, accompanied
by a blueꢀshift of emission maxima from 495 to 488 nm. The
above variations in fluorescence spectrum are analogous to
that of CAsH2 at its ICT emission band. As the diethylamino
group is rigidized by the julolidinyl structure, the fluorescence
enhancement of CAsH3 is apparently ascribed to the lowꢀ
polar environment of protein interior, which reduces the
charge transfer between the fluorophore and polar media, thus
exhibiting a pronounced fluorescence enhancement at ICT
band. However, due to a slight blueꢀshift in emission maxima
(~ 7 nm), only fluorescence ‘turnꢀon” mode is applicable for
rBSA detection in terms of CAsH3. The data convincingly
demonstrates the participation of TICT in the signaling event
of CAsH2 toward rBSA.
Figure 5. (a) Fluorescence spectra (λex = 415 nm) of CAsH2 (1
ꢁM) with the addition of various species in phosphate buffer
(pH 7.4, 20 mM, containing 1% DMSO) for 20 min at 37 C.
The testing species are methionine, Cys, GSH, Hcy, cystine,
DTT ascorbic acid (1 mM of each), lysozyme, ovabumin,
pepsin, trypsin, αꢀchymotrypsin, BSA, rBSA (0.6 ꢁM of each),
respectively. (b) Fluorescence intensity ratio (I468/I550) of
CAsH2 with the addition of various species.
o
Selectivity studies. The selectivity of CAsH2 was studied
by incubating the probe with some biologically relevant
species in phosphate buffer. As shown in Figure 5, no
significant fluorescence changes were observed when CAsH2
was mixed with some amino acids (methionine, Cys,
homocysteine (Hcy), cystine), GSH, ascorbic acid and DTT (1
mM of each), proteins (lysozyme, ovabumin, pepsin, trypsin,
αꢀchymotrypsin, BSA, 0.6 µM of each). In contrast, a dramatic
ratiometric fluorescence change was obtained upon the
addition of rBSA. It is worth noting that BSA (monothiol
protein) affords a very small fluorescence change under
identical conditions. This can be explained by the fact that
monothiol arsines formed between BSA and CAsH2 are
acyclic compounds which are unstable and spontaneously
decompose to the starting materials.46,47 Thus, only VDPs can
bind to CAsH2 with high affinity. It the case of DTT,
although it contains vicinal dithiols, it induces no fluorescence
changes under identical conditions. This is apparently because
DTT is a small molecule and contains no hydrophobic pocket
in its structure. The above results suggest the pronounced
specificity of the probe toward VDPs.
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channel (430–490 nm); middle: the emission was collected at
yellow channel (530–590 nm); right: brightꢀfield image. λex = 405
nm. Scale bar: 40 ꢁm.
Imaging of endogenous VDPs in live cells. Encouraged by
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the aboveꢀmentioned outcome, we then examined the
capability of CAsH2 for VDPs ratiometric fluorescence
imaging in living cells. In our experiments, L929 cells were
selected to demonstrate the application of live cell imaging of
VDPs with CAsH2. Initially, the cytotoxicity of CAsH2 was
evaluated by a standard MTT assay. Though inorganic arsenic
is associated with high cytotoxicity, organic arsenicals are
reported to be more stable and less toxic owing to enhanced
rates of excretion.48 As shown in Figure S12, CAsH2 has no
marked toxicity to L929 cells below 10 ꢁM. Therefore, 5 ꢁM
of CAsH2 was selected for further imaging experiments in
live cells. The dualꢀchannel fluorescence images recorded at
430ꢀ490 nm and 530ꢀ590 nm with excitation at 405 nm were
shown in Figure 6. Incubation of L929 cells with CAsH2 for
30 min provided a strong fluorescence in the cyan channel
(Figure 6a) and a weak fluorescence in the yellow channel
(Figure 6b). By contrast, in a control experiment, compound 6
was used for cell stain and it affords almost no fluorescence
emission at the two emission bands (Figure 6 d and e). The
above results indicate the selective binding of CAsH2 to
endogenous VDPs inside living cells. Furthermore, the cells
were pretreated with DTT (DTT is introduced as a reductant
stimulation and can lead to an increase in the levels of
endogenous VDPs22) for 30 min, and further incubated with
CAsH2 for 30 min, eliciting an obvious fluorescence increase
in the cyan channel (Figure 6g) but essentially no fluorescence
in the yellow channel (Figure 6h). The semiquantitative
calculation of ratio of cyan channel to yellow channel was
further conducted, and it was observed that the ratio of DTTꢀ
treated cells is higher than that of the control cells (Figure
S13). The above variations are consistent with the VDPsꢀ
induced fluorescence spectral changes of the probe. Thus,
these data demonstrate that CAsH2 can be applied for
ratiometric tracking of endogenous VDPs levels in living cells,
which is of great importance to clarify the physiological roles
of VDPs in living systems.
It was reported that there are abundant VDPs distributed in
the mitochondria of cells, which make
a significant
contribution to the responses of mitochondria to both oxidative
damage and redox signaling.49 Therefore, we further
investigate the subcellular localization of VDPs in SMMCꢀ
7721 cells by fluorescence colocalization experiments. The
cells were incubated with CAsH2 and MitoꢀTracker Deep Red
(a commercially available mitochondrial localizing dye)
simultaneously for 30 min, and then the cells were imaged by
confocal microscopy. As shown in Figure 7, there is an
obvious overlap between fluorescence signal from CASH2
and MitoꢀTracker Deep Red (the Pearson’s correlation
coefficient between cyan and red fluorescence images was
calculated to 0.82), implying that CAsH2ꢀlabeled VDPs are
mainly distributed in the mitochondria of these live cells. The
above experiments prove that there are abundant VDPs within
mitochondria, which is in accordance with the previous
studies.22,49
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a
b
c
Figure 7. Colocalization experiments of CAsH2 and MitoꢀTracker
Deep Red in SMMCꢀ7721 cells. (a) CAsH2 (2 ꢁM) stain, λex
=
405 nm, collected 430ꢀ 490 nm. (b) MitoꢀTracker Deep Red (0.5
ꢁM) stain, λex = 635 nm, collected 655ꢀ 755 nm. (c) Merged
image of (a) and (b). Scale bar, 10 ꢁm.
Conclusion
In summary, CAsH2 has been designed as a ratiometric
fluorescent probe for VDPs in living cells, where 1,3,2ꢀ
dithiarsenolane group functions not only as a recognition unit
but also as an initiator of TICT emission. Comparison of
structural analogues of CAsH2 in their fluorescent properties
suggests that the sensing mechanism for VDPs occurs through
the modulation of ICTꢀTICT conversion process. The
designed probe shows high selectivity toward VDPs over other
proteins and biological thiols and proves to be able to imaging
of VDPs in living cells. Compared with the existing FRETꢀ
based probe, the present probe requires no dye pair in
proximity to provide a signal and can be prepared via simple
synthetic steps. As far as we know, this is a rare example of
ICTꢀTICT based ratiometric fluorescence sensing system, and
we believe that the present study paves the way to construct
new proteinꢀspecific ratiometric fluorescent probes.
Figure 6. Confocal fluorescence images of L929 cells treated with
CAsH2 or compound 6 (both 5 ꢁM). (a – c), The cells was stained
by CAsH2; (d – f), the cells was stained by compound 6; (g – i),
the cells were preincubated with DTT (10 mM) followed by
incubation with CAsH2. Left: the emission was collected at cyan
ASSOCIATED CONTENT
Supporting Information
Experimental supplementary methods for chemical synthesis and
characterization of compounds, more experimental results and
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(22) Fu, N.; Su, D.; Cort, J. R.; Chen, B.; Xiong, Y.; Qian, W.ꢀJ.;
Konopka, A. E.; Bigelow, D. J.; Squier, T. C. J. Am. Chem. Soc.
2013, 135, 3567ꢀ3575.
figures as noted in text. This material is available free of charge
via the Internet at http://pubs.acs.org.
1
2
3
4
5
6
7
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9
(23) Griffin, B. A.; Adams, S. R.; Tsien, R. Y. Science 1998, 281,
269ꢀ272.
(24) Zhuang, Y. D.; Chiang, P. Y.; Wang, C. W.; Tan, K. T.
Angew. Chem. Int. Ed. 2013, 52, 8124ꢀ8128.
(25) Hori, Y.; Norinobu, T.; Sato, M.; Arita, K.; Shirakawa, M.;
Kikuchi, K. J. Am. Chem. Soc. 2013, 135, 12360ꢀ12365.
(26) Wang, Y.; Yang, X.ꢀF.; Zhong, Y.; Gong, X.; Li, Z.; Li, H.
Chem. Sci. 2016, 7, 518ꢀ524.
(27) Huang, C.; Jia, T.; Tang, M.; Yin, Q.; Zhu, W.; Zhang, C.;
Yang, Y.; Jia, N.; Xu, Y.; Qian, X. J. Am. Chem. Soc. 2014, 136,
14237ꢀ14244.
AUTHOR INFORMATION
Corresponding Author
* Eꢀmail: xfyang@nwu.edu.cn; huali@nwu.edu.cn;
Author Contributions
Y.W. and Y.Z. contributed equally to the present work.
Notes
The authors declare no competing financial interest.
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44
45
46
47
48
49
50
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52
53
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56
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58
59
60
(28) Lakowicz, J. R. Principles of fluorescence spectroscopy, 3rd
ed.; Springer: New York, 2006, p 443ꢀ475.
(29) Ren, M.; Deng, B.; Kong, X.; Zhou, K.; Liu, K.; Xu, G.; Lin,
W. Chem. Commun. 2016, 52, 6415ꢀ6418.
(30) Reja, S. I.; Khan, I. A.; Bhalla, V.; Kumar, M. Chem.
Commun. 2016, 52, 1182ꢀ1185.
(31) Zhu, L.; Xu, J.; Sun, Z.; Fu, B.; Qin, C.; Zeng, L.; Hu, X.
Chem. Commun. 2015, 51, 1154ꢀ1156.
(32) Li, Q.; Peng, M.; Li, H.; Zhong, C.; Zhang, L.; Cheng, X.;
Peng, X.; Wang, Q.; Qin, J.; Li, Z. Org. Lett. 2012, 14, 2094ꢀ2097.
(33) Loving, G. S.; Sainlos, M.; Imperiali, B. Trends Biotechnol.
2010, 28, 73ꢀ83.
(34) Van Gompel, J. A.; Schuster, G. B. J. Phys. Chem. 1989, 93,
1292ꢀ1295.
(35) Raju B, B.; Varadarajan, T. J. Phys. Chem. 1994, 98, 8903ꢀ
8905.
(36) Jones, G.; Jackson, W. R.; Choi, C. Y.; Bergmark, W. R. J.
Phys. Chem. 1985, 89, 294ꢀ300.
(37) Verma, S.; Ghosh, H. N. J. Phys. Chem. C 2014, 118, 10661ꢀ
10669.
(38) Dana, J.; Debnath, T.; Maity, P.; Ghosh, H. N. J. Phys. Chem.
C 2015, 119, 2046ꢀ2052.
(39) Signore, G.; Nifosì, R.; Albertazzi, L.; Storti, B.; Bizzarri, R.
J. Am. Chem. Soc. 2010, 132, 1276ꢀ1288.
(40) Barooah, N.; Mohanty, J.; Pal, H.; Bhasikuttan, A. C. Org.
Biomol. Chem. 2012, 10, 5055ꢀ5062.
(41) Debnath, T.; Dana, J.; Maity, P.; Lobo, H.; Shankarling, G. S.;
Ghosh, H. N. Chem. Eur. J. 2015, 21, 5704ꢀ5708.
(42) Debnath, T.; Maity, P.; Lobo, H.; Singh, B.; Shankarling, G.
S.; Ghosh, H. N. Chem. Eur. J. 2014, 20, 3510ꢀ3519.
(43) Nad, S.; Kumbhakar, M.; Pal, H. J. Phys. Chem. A 2003, 107,
4808ꢀ4816.
(44) Dean, J. A. Lange's Handbook of Chemistry, 14th ed.;
McGrawꢀHill: New York, 1992.
(45) Hong, Y.; Feng, C.; Yu, Y.; Liu, J.; Lam, J. W. Y.; Luo, K.
Q.; Tang, B. Z. Anal. Chem. 2010, 82, 7035ꢀ7043.
(46) Loiseau, P. M.; Lubert, P.; Wolf, J.ꢀG. Antimicrob. Agents
Chemother. 2000, 44, 2954ꢀ2961.
ACKNOWLEDGMENT
This research was supported by the National Natural Science
Foundation of China (nos. 21475105, 21275117, 21375105) and
the Education Department (no. 12JK0581) of Shaanxi Province of
China.
REFERENCES
(1) Gitler, C.; Zarmi, B.; Kalef, E. Anal. Biochem. 1997, 252, 48ꢀ
55.
(2) Maron, B. A.; Tang, S.ꢀS.; Loscalzo, J. Antioxid. Redox Signal.
2013, 18, 270ꢀ287.
(3) Ying, J.; Clavreul, N.; Sethuraman, M.; Adachi, T.; Cohen, R.
A. Free Radical Biol. Med. 2007, 43, 1099ꢀ1108.
(4) Borges, C. R.; Lake, D. F. Antioxid. Redox Signal. 2014, 21,
392ꢀ395.
(5) Valko, M.; Rhodes, C.; Moncol, J.; Izakovic, M.; Mazur, M.
Chem. Biol. Interact. 2006, 160, 1ꢀ40.
(6) Yang, Y.; Song, Y.; Loscalzo, J. Proc. Natl. Acad. Sci. 2007,
104, 10813ꢀ10817.
(7) Alexandrova, M. L.; Bochev, P. G. Free Radical Biol. Med.
2005, 39, 297ꢀ316.
(8) Matthias, L. J.; Hogg, P. J. Antioxid. Redox Signal. 2003, 5,
133ꢀ138.
(9) Bánhegyi, G.; Mandl, J.; Csala, M. J. Neurochem. 2008, 107,
20ꢀ34.
(10) Smeyne, M.; Smeyne, R. J. Free Radical Biol. Med. 2013, 62,
13ꢀ25.
(11) GarciaꢀGarcia, A.; ZavalaꢀFlores, L.; RodriguezꢀRocha, H.;
Franco, R. Antioxid. Redox Signal. 2012, 17, 1764ꢀ1784.
(12) Niu, L.ꢀY.; Chen, Y.ꢀZ.; Zheng, H.ꢀR.; Wu, L.ꢀZ.; Tung, C.ꢀ
H.; Yang, Q.ꢀZ. Chem. Soc. Rev. 2015, 44, 6143ꢀ6160.
(13) Yin, C.; Huo, F.; Zhang, J.; MartinezꢀManez, R.; Yang, Y.;
Lv, H.; Li, S. Chem. Soc. Rev. 2013, 42, 6032ꢀ6059.
(14) Jung, H. S.; Chen, X.; Kim, J. S.; Yoon, J. Chem. Soc. Rev.
2013, 42, 6019ꢀ6031.
(15) Chen, H.; Tang, Y.; Lin, W. TrAC, Trends Anal. Chem. 2016,
76, 166ꢀ181.
(16) Chen, H.; Tang, Y.; Ren, M.; Lin, W. Chem. Sci. 2016, 7,
1896ꢀ1903.
(17) Pan, X.; Liang, Z.; Li, J.; Wang, S.; Kong, F.; Xu, K.; Tang,
B. Chem. Eur. J. 2015, 21, 2117ꢀ2122.
(47) Liu, Y.; Duan, D.; Yao, J.; Zhang, B.; Peng, S.; Ma, H.; Song,
Y.; Fang, J. J. Med. Chem. 2014, 57, 5203ꢀ5211.
(48) Wilson, P.; Anastasaki, A.; Owen, M. R.; Kempe, K.;
Haddleton, D. M.; Mann, S. K.; Johnston, A. P.; Quinn, J. F.;
Whittaker, M. R.; Hogg, P. J. J. Am. Chem. Soc. 2015, 137, 4215ꢀ
4222.
(49) Requejo, R.; Chouchani, E. T.; James, A. M.; Prime, T. A.;
Lilley, K. S.; Fearnley, I. M.; Murphy, M. P. Arch. Biochem. Biophys.
2010, 504, 228ꢀ235.
(18) Girouard, S.; Houle, M.ꢀH.; Grandbois, A.; Keillor, J. W.;
Michnick, S. W. J. Am. Chem. Soc. 2005, 127, 559ꢀ566.
(19) Chen, Y.; Clouthier, C. M.; Tsao, K.; Strmiskova, M.;
Lachance, H.; Keillor, J. W. Angew. Chem. Int. Ed. 2014, 53, 13785ꢀ
13788.
(20) Huang, C.; Yin, Q.; Zhu, W.; Yang, Y.; Wang, X.; Qian, X.;
Xu, Y. Angew. Chem. Int. Ed. 2011, 50, 7551ꢀ7556.
(21) Huang, C.; Yin, Q.; Meng, J.; Zhu, W.; Yang, Y.; Qian, X.;
Xu, Y. Chem. Eur. J. 2013, 19, 7739ꢀ7747.
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