A dual responsive "turn-on" fluorophore for orthogonal selective sensing of biological thiols and hydrogen peroxide

Article abstract of DOI:10.1039/c5tc01465d

Both thiols and hydrogen peroxide (H₂O₂) have great correlations with cancer and other diseases, and hence detection probes for sensing these agents may serve as early diagnostic tools. In this article, we report the development of a

Full text of DOI:10.1039/c5tc01465d

View Article Online
View Journal
Journal of
Materials Chemistry C
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: C. Y. Ang, S. Y.
Tan, S. Wu, Q. Qu, M. F. E. Wong, Z. Luo, P. Li, S. Tamil Selvan and Y. Zhao, J. Mater. Chem. C, 2015, DOI:
1
0.1039/C5TC01465D.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/materialsC

Please do not adjust margins
Journal of Materials Chemistry C
Page 1 of 14
View Article Online
DOI: 10.1039/C5TC01465D
Journal Name
ARTICLE
A Dual Responsive “Turn-On” Fluorophore for Orthogonal
Selective Sensing of Biological Thiols and Hydrogen Peroxide
Received 00th January 20xx,
Accepted 00th January 20xx
a,c,†
a,†
a
a
a
a
Chung Yen Ang, Si Yu Tan, Shaojue Wu, Qiuyu Qu, Mun Fei Eddy Wong, Zhong Luo, Pei-
a
c,d,
a,b,
Zhou Li, Subramanian Tamil Selvan, * and Yanli Zhao
*
DOI: 10.1039/x0xx00000x
Both of thiols and hydrogen peroxide (H
detection probes for sensing these agents may serve as early diagnostic tools. In this article, we report the development of
a dual responsive probe that has the ability to generate two different responses upon reacting with thiols and H in a
highly selective manner. The probe (FLB SSCou) consists of a coumarin unit and a diboron xanthene spiro isobenzofuran
group bridged by a disulfide bond. The detection experiments show that the probe could selectively respond to thiols and
when screening a substrate library containing 20 amino acids, homocysteine, glutathione, dithiothreitol and H
2 2
O ) have great correlations with cancer and other diseases, and hence the
www.rsc.org/
2
O
2
2
H
2
O
2
2 2
O .
The initial off state of the probe was a result of photo-induced electron transfer (PET) from the coumarin group to non-
fluorescing diboron xanthene spiro isobenzofuran group bridged by a disulfide bond. Reductive cleavage of the disulfide
bond leads to the termination of this PET process, thus switching on the fluorescence of the probe. On the other hand, the
oxidation of the diboron group by H
dependent density functional theory calculations were then performed to explain the PET process, and the obtained
results indicate that the PET process occurs from the second excited state (S ) into the first excited state (S ). Finally, the
imaging and detection experiments of the probe on HeLa cancer cells were conducted by means of fluorescence
microscopy and flow cytometry technique. It was observed that the fluorescence of the FLB SSCou probe could be
switched on by endogenous thiols and exogeneous H , demonstrating the applicability of this probe in both extracellular
2 2
O converts the non-fluorescing group into highly fluorescing fluorescein group. Time-
2
1
2
2
O
2
and intracellular environments. The present work exhibits a novel development of a dual responsive probe as contrast to
commonly reported single responsive fluorescence probe, which may inspire future design of multiple responsive
fluorescence probes.
integrated with designed functional groups that would only
interact with the substrate to induce an intended reaction for
the generation of highly fluorescent adducts. Recently, Liu et.
Introduction
Stimuli-responsive fluorescence probes have received a great
deal of attention on account of their special applications for
al. have reported a coumarin based fluorescence probe that
1
-8
has the ability to discriminate glutathione (GSH), cysteine (Cys)
sensing and imaging. Fluorescence-based sensing is cheaper
and more preferred technique over other methods, since their
preparations are relatively easy and the sensing applications
could be realized without the use of sophisticated
1
5
and homocysteine (Hcy) in a simultaneous fashion. In this
work, the fluorescence probe reacts with these three
biological thiol compounds differently and produces
fluorescent adducts with different excitation and emission.
Other than this work, there are various reported examples for
highly selective detections of thiols by means of fluorescence
9
-14
instruments.
Fluorescent probes exhibit interesting
properties, which react with the species of interest in a
chemoselective manner. Hence, these probes are carefully
1
6-28
technique.
However, dual-responsive fluorescence probes
that can respond to two different types of analytes with
different signals have not been commonly reported.
It is well known that the presence of high intracellular
concentration of thiols has a great correlation with certain
2
9-36
types of diseases.
In particular, it was reported that the
intracellular environment of most cancer cell lines expresses
3
7-41
high concentration of GSH.
In view of this fact, various drug
delivery systems were designed for GSH-triggered release of
3
1,42-50
anticancer drugs within cancer cells.
On the other hand,
2 2
H O
was also reported as one of major reactive oxygen
species (ROS) that are capable of inducing oxidative stress to
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
Please do not adjust margins

Please do not adjust margins
Journal of Materials Chemistry C
Page 2 of 14
View Article Online
DOI: 10.1039/C5TC01465D
ARTICLE
Journal Name
2 2 2
Scheme 1. Schematic illustration for the working mechanisms of dual responsive “turn-on” fluorescence probe FLB SSCou when interacting with biological thiols and H O .
5
1-54
physiological environment.
High concentration of
With this design in mind, we conducted this work in a
physiological H
2 2
O was known to associate with various issues systematic manner, beginning with the synthesis of the probe,
such as aging, cancer, Alzheimer’s disease and cardiovascular followed by the studies of its various optical properties, and
5
5-61
disorder.
Herein, we designed a “turn-on” fluorescence probe that is functional theory (DFT). Thereafter, we proceeded to test the
not only responsive to biological thiol compounds and H applicability of the probe for simultaneous imaging of
finally proving the PET mechanism by utilizing the density
2 2
O ,
but also capable of discriminating these two types of analytes intracellular thiol compounds and H
in a highly selective manner by generating different responses. cells.
Moreover, this probe is able to detect these two types of
analytes when they are present simultaneously in the
intracellular environment.
2 2
O in live HeLa cancer
The design of this “turn-on” probe was based upon a
coumarin-containing group that was conjugated to a diboron
xanthene spiro isobenzofuran bridged by a disulfide bond
2
(FLB SSCou in Scheme 1). In the initial form, the probe would
be in its dark state whereby the fluorescence of the coumarin
is quenched by non-fluorescing diboron xanthene spiro
isobenzofuran through the photo-induced electron transfer
(PET) mechanism. The PET mechanism was supported by the
absence of spectral overlapping between the absorption peak
of diboron xanthene spiro isobenzofuran and the emission
peak of coumarin. We thus ruled out the possibility of the
Fӧrster resonance energy transfer (FRET) between the two
moieties in the dark state. Upon reaction with a biological
reducing thiol, the disulfide bond would be cleaved and the
coumarin unit is separated from diboron xanthene spiro
isobenzofuran. This process terminates the electron transfer
from coumarin to diboron xanthene spiro isobenzofuran,
regaining the fluorescence from the coumarin moiety. When
the probe reacts with H
2 2
O , the diboron functional group on
xanthene would be oxidized into corresponding fluorescein
6
2
entity. This process converts the PET mechanism from
coumarin into the FRET mechanism, thereby turning on green
fluorescence under the same excitation of the coumarin Scheme 2. Synthetic scheme for coupling a disulfide bond with a coumarin unit.
moiety. This phenomenon is understandable, since
coumarin/fluorescein is a common FRET pair reported in
6
Results and discussion
3-66
literature.
Hence, the diboron xanthene spiro
2
Synthesis of FLB2SSCou. The synthesis of the FLB SSCou probe
isobenzofuran group serves as not only a quencher for the
coumarin group, but also a secondary probe.
was accomplished in a total of 8 synthetic steps (Schemes 2
2
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 3 of 14
Journal Name
Jou Pr nl e aa ls oe fd Mo an to et r ai ad lj su s Ct hm ea mr g ii sn ts ry C
View Article Online
DOI: 10.1039/C5TC01465D
ARTICLE
and 3). The synthetic strategy begins with the preparation of 7- palladium
diethylamino-3-aminocoumarin ( ) from 4-diethylsalicylihyde bis(pinacolato)diboron, affording compound
through two-step reactions. Firstly, 4-diethylsalicylihyde carboxylic acid group on compound
reacted with methyl-2-nitroacetate to afford a coumarin site with compound to achieve the target probe
compound ( ) by a Knoevenager condensation using piperidine FLB SSCou).
as the amine catalyst in the presence of acetic acid. Thereafter, Spectroscopic properties and optical responses of FLB
the nitro group on compound was reduced to corresponding toward H and biological thiols. After obtaining FLB
amino group on compound in a good yield. The amino group we performed a series of optical studies of this molecule to
of resulted compound was functionalized with an activated investigate its response toward biological thiols and H . We
thiol by reacting with 3-(pyridin-2-yldisulfanyl)propanoic acid first measured the absorption spectra of FLB SSCou before and
through dicyclohexylcarbodiimide (DCC) N- after the introduction of 1.0 mM H and 5.0 mM
hydroxysuccinimide (NHS) coupling, affording compound dithiothreitol (DTT), respectively (Figure 1A). It was observed
With the availability of a pyridin-2-yldisulfanyl group on that all the absorption spectra exhibited a common maximum
compound , it can easily react with the thiol group of 2- peak at the wavelength of 408 nm. However, the absorption
mercaptoethanamine hydrochloride to afford compound spectrum of FLB SSCou after the reaction with H showed a
Thus, compound contains a coumarin moiety and an amino new signal at 497 nm. This new absorption peak coincides with
group bridged by a disulfide bond.
mediated
coupling
reaction
with
2
7. Finally, the
7
was used as the coupling
(or
5
8
1
2
2
SSCou
2
SSCou,
1
2
O
2
2
2
2 2
O
2
(
3)
/
2 2
O
4.
4
5.
2
2 2
O
5
typical absorption signal of a fluorescein molecule, indicating
that the oxidation of the two boronic acid units on diboron
xanthene spiro isobenzofuran indeed produced corresponding
fluorescein moiety.
(
A) 1
0.9
0
.8
FLB SSCou
FLB2SSCou
2
0.7
DDTTTT
0
0
.6
.5
H O
H2O2
2 2
0.4
0.3
0.2
0.1
0
300
350
400
450
500
550
600
650
Wavelength (nm)
Scheme 3. Synthesis of H
2 2
O responsive diboron xanthene spiro isobenzofuran
(
B)
precursor and FLB SSCou probe.
2
1
40
120
00
In order to obtain H O responsive diboron xanthene spiro
2
2
isobenzofuran unit of the probe, we adopted a two-step
6
synthetic procedure (Scheme 3). It was reported that the
diboron group in molecular probes has the ability to react with
1
7
80
F
FL
DT
H2O
L
B
B
2S
S
SC
S
o
C
u
ou
2
H2O in
a highly selective manner through oxidative
4,68-77
2
60
6
D
T
T
T
deprotection of the boronic ester group.
Thus, it was
40
H
O
2
2
expected that the introduction of the diboron unit could
endow the highly selective detection of H O into the dual
2
20
0
2
2
6
8
responsive probe. In the synthesis, 2 equivalents of benzene-
,2,4-tricarboxylic acid condensed with one equivalent of 3-
bromophenol to form the dibromo xanthene spiro
isobenzofuran derivative . Due to unsymmetrical nature of
1
4
20
470
520
570
620
670
Wavelength (nm)
6
2
Fig 1. (A) UV-Vis and (B) fluorescence spectra of FLB SSCou (50 μM) in the absence and
o
benzene-1,2,4-tricarboxylic acid, the crude product consisted presence of H O (1.0 mM) or DTT (5.0 mM) after overnight incubation at 37 C.
2
2
of a mixture of two isomers in which the carboxylic acid unit Fluorescence spectra were measured under the excitation wavelength of 408 nm. Inset
of Fig 1B shows photoluminescence images of the three samples upon irradiation at
was on either 5- or 6-position of the isobenzofuran site. Based
3
65 nm by a handheld UV lamp. Images from left to right are FLB
2 2
SSCou, FLB SSCou
on the reported procedure, the isomer with carboxylic acid on
the 6-position was isolated by salting out as a pyridine salt in
acetic anhydride/pyridine mixture. Thereafter, the pyridine
salt was neutralized back into the carboxylic acid group. The
after treatment with H , and FLB SSCou after treatment with DTT.
2
O
2
2
We next performed the fluorescence measurements of
SSCou before and after the DTT and H treatments
Figure 1B). The probe without any treatment exhibited a weak
FLB
2
2 2
O
two bromine groups on the xanthene moiety in compound
6
(
were then converted into the pinacolato boronic esters by
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
Journal of Materials Chemistry C
Page 4 of 14
View Article Online
DOI: 10.1039/C5TC01465D
ARTICLE
Journal Name
fluorescence peak at 497 nm (Φ = 0.001). Upon the treatment case in pH 5.5. Similar to the thiol-responsive mechanism,
with DTT, the fluorescence intensity enhanced at the same higher pH could induce the deprotonation of H and thus
wavelength of 497 nm (Φ = 0.168). On the other hand, when increase the initial response rate. On the other hand, lower pH
the probe was treated with H , it showed a fluorescence could increase the hydrolysis rate of the borate group into
2 2
O
2 2
O
enhancement at 525 nm and a fluorescence decrease at 497 corresponding hydroxyl group after the oxidation occurs. This
nm (Φ = 0.101). As abovementioned, the treatment of the explained the unexpected increment in the response rate of
2 2 2 2
probe with H O led to the emergence of a new absorption the probe to H O in pH 5.5.
peak at 497 nm. Thus, a complete spectral overlapping occurs Selectivity and sensitivity studies. Primary advantage of the
between the emission of coumarin unit at 497 nm and the fluorescence probe is its dual responsive property and its
absorbance of the fluorescein moiety at 497 nm. This capability of discriminating reducing thiols and oxidizing H
observation indicates that there is an FRET in the probe upon It is hence important to evaluate the selectivity and sensitivity
the treatment with H . The photographs in the inset of of this probe toward biological thiols and H . Figure 2 shows
Figure 1B are the luminescence images of the probe upon the fluorescence responses of FLB SSCou upon treatments
excitation at 365 nm by a UV lamp before and after the DTT or with various biological agents. As expected, FLB SSCou showed
treatment. Distinctive difference in the fluorescence color positive responses to DTT, GSH, Hcy, Cys and H only,
between the three samples was observed, thus confirming whereas the rest of the amino acids did not induce any
that the probe indeed possesses the capability to show significant responses. Moreover, H exhibited a different
2 2
O .
2
O
2
2 2
O
2
2
H
2
O
2
2 2
O
2 2
O
different responses upon the treatment with thiols and H
2
O
2
.
response as compared with DTT, GSH, Hcy and Cys. This set of
experiments confirmed that only thiol-containing biological
agents have the ability to react with FLB
reductive cleavage of the disulfide bond to enhance the
coumarin fluorescence. On the other hand, H can trigger a
different response upon the reaction with FLB SSCou by
2
SSCou by inducing a
1
18
2 2
O
98
78
58
38
18
2
oxidative deprotection of the boronic ester group into a highly
fluorescent fluorescein derivative. It could be concluded that
the probe has the capability to sense biological thiols and H
without compromising its selectivity.
2 2
O
(A)
220
Blank
mM Hcy
5 mM GSH
5 mM DTT
5 mM Cys
5
1
70
20
1
Biological agents
SSCou (50 μM) toward various amino acids (5.0
mM), biological thiols (5.0 mM), and H (1.0 mM). All emission spectra were
measured under the excitation of 408 nm, and the value of the emission intensity was
obtained at the wavelength of 497 nm (except for H ). The emission intensity for
was obtained at the wavelength of 525 nm. Inset: Fluorescence images of the
SSCou solution after the addition of various additives upon excitation at 365 nm by
a handheld UV lamp. Images from bottom left to top right are for: DTT, GSH, Hcy, Cys,
7
0
Fig 2. Fluorescence responses of FLB
2
2 2
O
20
O
2 2
0
20
40
60
80
100
120
H
O
2 2
Time (mins)
FLB
2
(B)
Ex408nm_Em497nm
Ex408nm_Em525nm
9
7
5
0
0
0
H
2 2
O , Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, and
Val.
We also studied the responsivity of the probe toward thiol
(
DTT) and H
dependent fluorescence response (Figure S1 in ESI). When
comparing the fluorescence responsivity of FLB SSCou to DTT
2 2
O in acidic condition by measuring time
30
2
1
0
in pH 7.4 and 5.5, it was observed that the probe exhibits
faster response to DTT in pH 7.4 than that in pH 5.5, which
may be due to the presence of higher percentage of
0
20
40
60
Time (mins)
80
100
120
2
Fig 3. Time dependent fluorescence response of FLB SSCou (50 μM) (A) in the absence
protonated thiol group on DTT in pH 5.5 than in pH 7.4. The (Blank) and presence of various biological thiol compounds (5.0 mM) as well as (B) by
2 2
co-incubation with H O (1.0 mM). Emission intensity in (A) was obtained at the
protonation of the thiol group on DTT in acidic condition could
decrease its nucleophilicity, and thus has a negative effect in
reducing the disulfide bond of the probe. Interestingly, a
different trend was observed when comparing the responsivity
wavelength of 497 nm under the excitation wavelength of 408 nm. Emission intensity
in (B) was obtained at the wavelengths of 497 and 525 nm under the excitation
wavelength of 408 nm.
of the probe toward H
2 2
O in both pH conditions. Within the
2 2
We then studied the sensitivity of the probe toward H O
first 10 mins, there was higher responsivity of the probe to
and biological thiols. Time-dependent optical responses of the
probe to these agents were performed first. The experiments
2 2
H O in pH 7.4. Then, the rate decreased as compared to the
4
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 5 of 14
Journal Name
were conducted by the co-incubation of FLB
Jou Pr nl e aa ls oe fd Mo an to et r ai ad lj su s Ct hm ea mr g ii sn ts ry C
View Article Online
DOI: 10.1039/C5TC01465D
ARTICLE
2
2 2 2
SSCou (50 μM) fluorescence spectra of FLB SSCou upon treating with H O
and individual biological agent before measuring the emission (Figure 1B). From these observations, it could be further
spectra in a time-dependent basis under the excitation confirmed that the FRET process is indeed present between
wavelength of 408 nm over a period of 120 min (Figure S2 in the coumarin moiety and the fluorescein group on the probe
2 2
ESI). As a control, we also performed similar time-dependent after the reaction with H O . The time dependent decreases in
fluorescence measurement of FLB
biological agent.
2
SSCou in the absence of any the emission signal at 497 nm correspond to the time
dependent increases in the emission signal at 525 nm, due to
2
The real time fluorescence measurements of FLB SSCou (50 the energy transfer upon the “turn-on” effect. This hence
μM) in the presence of various biological agents (Figure 3A) explained the similarity in the rate changes of the two
showed that DTT could induce the fastest and highest emission intensities.
response rate as compared to other biological agents, reaching
After performing these measurements, the response of the
was
the fluorescence intensity approximately 6 times of higher probe under different concentrations of GSH and H
2 2
O
than the starting fluorescence. Cys and Hcy were the second investigated (Figure 4). The probe showed a good linear
ones with similar response rate. GSH exhibited the slowest response toward GSH and H O at the concentration ranges
2
2
rate as compared to DTT, Cys, and Hcy. This is probably due to from 0.1 to 100 μM and 0.1 to 500 μM, respectively. Based on
larger molecular weight of GSH, and hence, GSH would tend to the linearity within the ranges, we computed the detection
-6
induce higher activation energy during the reaction with the limits of the probe for GSH and H O to be 4.98 × 10 M and
2
2
-5
disulfide bond of the fluorescence probe as compared to 1.37 × 10 M, respectively. The relatively high detection limit
smaller biological thiols. of H O could be attributed to the loss in the efficiency during
A)
2
2
(
120
00
the FRET process from the coumarin group to the fluorescein
moiety. Note that the excitation wavelength used for the
detection of H O remained the same as the coumarin case at
5
.00E-3 M
1.00E-4 M
.00E-4 M
1.00E-5 M
.00E-5 M
1.00E-6 M
.00E-6 M
1
5
2
2
8
6
4
2
0
0
0
0
0
5
408 nm instead of the direct excitation at 497 nm.
5
1.00E-7 M
4
20
470
520
570
620
Wavelength (nm)
(B)
6
5
4
3
2
1
0
0
0
0
0
0
0
1
5
1
5
1
5
1
5
.00E-3 M
.00E-4 M
.00E-4 M
.00E-5 M
.00E-5 M
.00E-6 M
.00E-6 M
.00E-7 M
4
20
470
520
570
620
Wavelength (nm)
Fig 4. Changes in emission spectra of FLB
2
SSCou (50 μM) upon treatment with different
amounts of (A) GSH and (B) H under the excitation wavelength of 408 nm.
2 2
O
2 2 2
In the presence of H O (Figure 3B), FLB SSCou showed the
Fig 5. Calculated frontier molecular orbitals of (A) FLB
2 2
SSCou, (B) FLB SSCou after
fluorescence signals at 497 nm and 525 nm under the reacting with thiols, resulting in the reductive cleavage of the disulfide bond to give a
excitation wavelength of 408 nm. It was found that the coumarin derivative, and (C) FLB SSCou after reacting with H , resulting in the
2
2 2
O
oxidation of the boronic group into the phenoxy group.
emission intensity at 497 nm decreased continuously, whereas
the emission intensity at 525 nm increased correspondingly.
This observation is in line with the result obtained from the
Table 1. TD-DFT/B3LYP/6-31+G(d,p) calculation results of FLB
2
SSCou, FLB
2 2 2 2
SSCou treated with thiol, and FLB SSCou treated with H O .
a
b
Molecule
FLB SSCou
Electronic transition
Excitation wavelength (nm)
394.02
f
Contributed orbitals
HOMO → LUMO
HOMO → LUMO + 1
HOMO → LUMO
HOMO → LUMO
CI coefficient
2
S
S
S
S
0
0
0
0
→ S
→ S
→ S
→ S
1
2
1
1
0.0000
0.6912
0.8414
0.7204
0.70704
0.70002
0.70089
0.69911
377.44 (408)
FLB
2
SSCou + Thiol
388.58 (408)
FLB
2
SSCou + H
2
O
2
447.77 (497)
a
b
Display of selected transition. Oscillator strength.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5
Please do not adjust margins

Please do not adjust margins
Journal of Materials Chemistry C
Page 6 of 14
View Article Online
DOI: 10.1039/C5TC01465D
ARTICLE
Journal Name
Fig 6. Energy level diagrams of (A) FLB
radiative relaxation pathways.
2 2 2 2 2
SSCou, (B) FLB SSCou treated with thiol, and (C) FLB SSCou treated with H O , showing the electronic excitation as well as radiative and non-
Computation studies. Based on experimental observations, it
was speculated that the fluorescence of the initial dark state of calculation with the same basis set on the excited state in
FLB SSCou was quenched by the PET mechanism from the order to have better understanding of electronic transition
Thereafter, we performed time dependent-DFT (TD-DFT)
2
coumarin moiety to the diboron xanthene spiro isobenzofuran pathway during the excitation. The same DFT optimization and
group. To confirm the existence of the PET phenomenon TD-DFT calculation were also performed on the molecule after
within the FLB SSCou probe, computational calculations on the treatment with H O and thiol compounds. The TD-DFT
2 2 2
molecule orbital locations were performed for the ground calculation results showed a lot of useful information,
state and excited state. The ground state density functional including calculated excited wavelength and oscillator strength
theory (DFT) optimization of FLB
environment was first carried out at B3LYP/6-31+G(d,p) basis Base on Kasha law, the fluorescence process is a result of
2
SSCou in aqueous
(f) together with corresponding contributing orbitals (Table 1).
7
8
1
set using Gaussian 09 package. Prior to the reaction with direct relaxation from the 1st excited state (S ) to the ground
8
2, 83
thiol compounds or H
xanthene group adopted spiro conformation with respect to state of FLB
the isobenzofuran group having the lactone ring intact in its
close confirmation. The same optimization calculations on the electronic transition to the first excited state was mainly
probe upon the oxidative reaction with H were then contributed from respective highest occupied molecular
2
O
2
, it was observed that the diboron state (S
0
).
Hence, we paid our attention to the first excited
2
SSCou before and after reacting with thiols and
2 2
H O from the TD-DFT calculation. It was observed that the
2 2
O
performed. Since the reaction was conducted in neutral orbital (HOMO) to lowest unoccupied molecular orbital
conditions, it was expected that the fluorescein group exists in (LUMO) transition. Base on this information, we extracted the
the dianionic state during the calculation process. After the HOMO and LUMO orbital diagrams of these systems (Figure 5).
optimization process, we observed that the fluorescein group After a careful examination of the transition, it was found that
remained its spiro conformation, while the lactone ring was the S
broken into the benzoic derivative. It was well documented oscillator strength of
0
to S
1
transition for FLB
f
2
SSCou was accompanied by the
= 0. This means that the direct
that open ring form of fluorescein would exhibit high excitation from its ground state to its first excited state is
photoluminescence efficiency, whereas the lactone ring form forbidden. The extracted HOMO and LUMO orbital diagrams of
7
9-81
shows no or weak fluorescence.
this molecule reveal that there is a re-distribution in its
6
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 7 of 14
Jou Pr nl e aa ls oe fd Mo an to et r ai ad lj su s Ct hm ea mr g ii sn ts ry C
View Article Online
DOI: 10.1039/C5TC01465D
Journal Name
ARTICLE
electronic density from the coumarin group to the
From the TD-DFT calculation (Figure 6A), we can conclude
SSCou induces an allowed
observed that the lowest allowed transition lies on S to the electron excitation from the HOMO (-5.66 eV) to LUMO + 1 (-
isobenzofuran group during this transition (Figure 5A). We that the photo-excitation of FLB
2
0
2 0 2
2nd excited state (S ) transition with an oscillator strength of f 2.05 eV) orbitals (S → S , f = 0.6912), where there is no re-
=
0.6912. For the cases of FLB SSCou after reacting with thiols distribution of the electronic density. Thereafter, the excited
2
2 2 0 1
and H O , the S to S transition accompanied by a non-zero f electron is transferred to the LUMO (-2.34 eV) of the molecule
value provides a clear indication that the transition is an with a net loss of 0.29 eV worth of energy. The electron
allowed process. The TD-DFT calculation results demonstrate transfer process is evident from the re-distribution of the
that these transitions are contributed by the HOMO to LUMO electron density from the coumarin moiety to the
transition, and a detailed observation on the orbital diagram isobenzofuran group on the orbital diagrams. The electron is
shows no re-distribution of the electronic density during this then re-transferred back to the coumarin group by means of
HOMO to LUMO transition (Figure 5B and 5C). Based on these internal conversion, which is non-radiative in nature. This
observations, we sketched out the orbital energy levels to complete PET cycle explains the initial dark state of the
explain the detailed mechanism of the “turn-on” effect (Figure molecule. By applying the same method of analysis to the
6).
molecule after the reductive cleavage of the disulfide bond
and the oxidation of the diboron group into the phenoxide
5
4
3
2
1
0
0.01%
0.02%
5
4
3
2
1
0
2.88%
3.04%
93.98%
0.04%
5
4
3
2
1
0
19.75%
76.14%
1
1
1
1
1
1
0
0
0
0
0
0
10
10
10
10
10
10
1
1
1
1
1
1
0
0
0
0
0
0
59.72%
40.00%
0.08%
3.96%
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
10
10 10 10 10 10
FITC-A
10 10 10 10 10 10
FITC-A
10
10 10 10 10 10
FITC-A
Fig 7. In vitro bioimaging of HeLa cells. Confocal scanning microscopic images of cells treated with 10 μM FLB
DAPI) channel and (B) FITC channel. (C) Merged image of A and B. Confocal scanning microscopic images of cells treated with 10 μM FLB
treatment with 100 µM H under (D) DAPI channel and (E) FITC channel. (F) Merged image of D and E. Flow cytometry dot-plot diagram of Hela cells (G) without any additives
blank control), (H) after treated with 10 μM FLB SSCou and 20 μM β-CD, and (I) after treated with 10 μM FLB SSCou and 20 μM β-CD followed by post-treatment with 150 μM
2
SSCou and 20 μM β-CD under (A) 4',6-diamidino-2-phenylindole
(
2
SSCou and 20 μM β-CD followed by post-
2 2
O
(
2
2
2 2.
H O
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
Please do not adjust margins

Please do not adjust margins
Journal of Materials Chemistry C
Page 8 of 14
View Article Online
DOI: 10.1039/C5TC01465D
ARTICLE
Journal Name
2
2 2
group, chemically induced “turn-on” effect of FLB SSCou could imaging of thiols and H O . We performed the same process of
be well explained. As seen from Figure 6B, the direct transition
from HOMO to LUMO leads to no re-distribution in the
electron density. Based on Kasha law, the absence of any
treatment to the cells before harvesting them for flow
cytometry analysis. The analysis was performed with the
brilliant violet 421 (BV421) channel and FITC channel, and the
intensity of these measurements was then compared with
blank cells, i.e. cells without the treatment of any agent (Figure
1 0
intermediate orbital between S and S states confirms that
the LUMO to HOMO relaxation is emissive. Similarly, we also
did not observe any sign of PET from Figure 6C, thus
concluding the conversion of the emissive FLB SSCou into two
7G). Similar to the fluorescence images, a large percentage of
2
the cells treated with FLB SSCou (10 μM) and β-CD (20 μM)
2
emissive adducts by terminating the PET process.
showed emission signals from the BV421 channel
accompanied by weak emission signals from the FITC channel
In vitro intracellular imaging of thiols and H
evaluate the applicability of the FLB SSCou probe for biological
applications, we conducted the imaging of thiols and H in
2 2
O . In order to
2
(Figure 7H). It was observed that 19.75% of the cell population
2 2
O
occupies the first quadrant of the dot plot, which are much
higher than the cell population (0.01%) on the first quadrant in
the control group (Figure 7G).
intracellular environment. Since it was well documented that
most cancer cells possess high concentration of intracellular
GSH, HeLa cells as the cancer cell model were selected in the
The same flow cytometry experiment was also performed
on FLB SSCou-treated cells with the post-treatment of H O .
2
studies. The biocompatibility of FLB SSCou was first evaluated
using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay. The cell viability results showed that
2
2
2
From the dot plot (Figure 7I), it was observed that the cell
population in the first quadrant (2.88%) greatly reduced, while
having significant population increase in the second quadrant
FLB
in ESI), making it suitable for subsequent biological
applications. Intracellular imaging using FLB SSCou was then
carried out. In this case, the pre-seeded cells were treated
with FLB SSCou (10 μM) and β-cyclodextrin (β-CD, 20 μM) for
4 h prior to the imaging experiments. β-CD used was to
2
SSCou exhibited insignificant toxicity to the cells (Figure S3
(
93.98%). This set of cells have the largest population in the
2
second quadrant as compared to other two cell groups,
indicating that the post-treatment of H O indeed turns on the
2 2
2
green emission of internalized FLB SSCou measured by the
2
2
FITC channel. When comparing the FITC signals from the one-
dimensional histogram plots (Figure S4 in ESI), this group of
cells show the highest FITC signal. Similar to the fluorescence
microscopy images, we did not observe any significant
decrease in the BV421 signal as compared to that of the cells
facilitate the internalization of the probe. Based on the
confocal scanning microscopy images (Figure 7A-7C), strong
blue fluorescence from the cells was observed accompanied by
some negligible green fluorescence signals.
The applicability of FLB
intracellular H was then evaluated. Similarly, the pre-
seeded HeLa cells were treated with FLB SSCou (10 μM) and β-
CD (20 μM) for 24 h before the post-treatment of H for 45
2
SSCou for the imaging of
treated with FLB SSCou and β-CD, once again confirming that
2
2
O
2
the FRET process was terminated by the intracellular thiols.
2
2 2
O
min. As compared to previous images, a great increment in the
green fluorescence of the cells under fluorescein
isothiocyanate (FITC) channel (Figure 7D-7F) was observed. In
Conclusions
In summary, we have developed a novel “turn-on” dual
addition, similar blue fluorescence intensity from the cells with responsive fluorescence probe, FLB
and without the treatment with H
was shown. According to intracellular thiols and H . In contrast to conventional “turn-
the fluorescence studies, it was concluded that the treatment on” fluorescence probes, FLB SSCou is capable of generating
of FLB SSCou with H
resulted in the emergence of a different emission responses when treated with thiols and
fluorescence signal at 525 nm (green emission) accompanied
. In the probe, a non-fluorescent diboronxanthene spiro
2
SSCou, for the detection of
2
O
2
2 2
O
2
2
2 2
O
2 2
H O
by the quenching of the blue emission due to the FRET isobenzofuran and a coumarin unit are bridged by a disulfide
mechanism. The spectroscopic observations seem different to bond. The initial dark state of the probe is due to the PET
the in vitro imaging results that the blue fluorescence process that occurs from the coumarin group into the
remained the same for the cells with and without the post- isobenzofuran group. Upon treatment with thiols, reductive
treatment of H
treatment of FLB
new absorption signal at 497 nm, which overlapped with the unit. On the other hand, upon treatment with H
2
O
2
. As observed from the UV-Vis studies, the cleavage of the disulfide bond terminates this intramolecular
SSCou with H
led to the emergence of a PET process, thus initiating the emission from the coumarin
, the
2
2 2
O
2 2
O
emission of coumarin unit at 497 nm. The presence of the diboron xanthene spiro isobenzofuran group undergoes
disulfide bridge within the molecule allows the FRET process to oxidation into a highly fluorescent fluorescein adduct, whereby
occur especially when the distance between the FRET donor the intramolecular PET process is converted into the
(
coumarin) and the FRET acceptor (fluorescein) is not longer intramolecular FRET process. A series of detection experiments
exist in the intracellular have shown that the probe has the ability to sense thiols and
than 10 nm. Since both GSH and H
2 2
O
environment, thiol-induced reductive cleavage of the disulfide
H
2
O
2
in a highly selective manner against over 20 amino acids.
In order to prove the intramolecular PET mechanism of
SSCou, the TD-DFT calculations have been carried out. A
Flow cytometry experiments were carried out to further different distribution of the electron density between the
confirm the applicability of FLB
SSCou for the intracellular ground state (S ) and the first excited state (S ) has been
bond terminates the FRET process. This explains the presence
of blue emission from the cells even after the H
2
O
2
treatment.
FLB
2
2
0
1
8
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 9 of 14
Journal Name
Jou Pr nl e aa ls oe fd Mo an to et r ai ad lj su s Ct hm ea mr g ii sn ts ry C
View Article Online
DOI: 10.1039/C5TC01465D
ARTICLE
observed, since the emission process is a result of the infrared spectroscopy (FTIR) measurements were performed
electronic relaxation from S to S . Moreover, the transition to on a SHIMADZU IR Prestige-21 spectrophotometer. Melting
the first excited state is a forbidden transition process (f = points of solid compounds were recorded on an OptiMelt
.0000), and instead, the first allowed transition is to the Automated Melting point instrument. High-resolution mass
second excited state (S → S , f = 0.6912), where there is no re- spectrometry (HRMS) was performed on a Waters Q-tof
1
0
0
0
2
distribution of the electron density. Thus, it has been Premier MS. The UV-Vis spectroscopy studies for all optically
confirmed that the PET process occurs during a process where active compounds were performed using a SHIMADZU UV-
the electron first gains its energy to excite into the S
followed by transferring into the S state, since the direct 1.4 mL quartz cuvette. Fluorescence emission spectra were
excitation to the S state is forbidden and there is inadequate recorded on Varian Cary Eclipse fluorescence
2
state 3600 UV-VIS-NIR spectrophotometer with a 10-mm-by-10-mm
1
1
a
energy for the spontaneous transfer.
spectrophotometer equipped with water circulated
temperature controller. Except for time-dependent
2 2
Lastly, the in vitro intracellular imaging of thiols and H O
using FLB
microscopy images show the presence of blue fluorescence were carried out at 37 C using a 10-mm-by-10-mm 1.4 mL
within the intracellular environment of HeLa cells after the fluorescence cuvette. Time-dependent fluorescence
treatment with FLB SSCou, due to the interaction of FLB SSCou spectroscopy analysis was performed with a 10-mm-by-10-mm
with intracellular thiols. On the other hand, the post-treatment 3.5 mL fluorescence cuvette. Unless otherwise stated, all UV-
of H on FLB SSCou-treated cells exhibits the enhancement Vis and fluorescence spectroscopy experiments were
in green fluorescence as compared to the cells without this performed in 0.1 phosphate buffered saline (PBS).
post-treatment, indicating that exogenous H could induce a Spectroscopic studies in acidic conditions were conducted by
turn-on” effect on the probe even in the intracellular dispersing the fluorophores in 0.1 M acetate buffer solution
environment. These results have been further supported by (pH 5.5).
2
SSCou has been conducted. The fluorescence fluorescence spectroscopy analysis, all fluorescence studies
o
2
2
2
O
2
2
X
2 2
O
“
flow cytometry experiments, confirming the reproducibility of
HeLa cells used in this work were obtained from ATCC.
2
the probe for the detection and imaging of intracellular thiols Cells were maintained in a humidified CO (5%) incubator and
and H
the synthesis and applications of
fluorescence probe for the simultaneous detection of thiols streptomycin. Confocal microscopy experiments were
and H . The present research might inspire further conducted on a confocal microscope (Leica TCS SP5, 40× oil
development of “turn-on” fluorescence probes having multiple objective). Absorbance for the MTT cytotoxicity assay was
2
O
2
. All in all, this work has successfully demonstrated proliferated in Dulbecco’s Modified Eagle Medium (DMEM)
a
dual responsive supplement with 10% fetal bovine serum and 1% penicillin
2 2
O
responsive properties.
measured with an Infinite 200 PRO multimode reader. Flow
cytometry experiments were performed on
TM
LSRFortessa X-20 cell analyzer.
a
BD
Experimental section
7
-(Diethylamino)-3-nitro-2H-chromen-2-one (1). The synthesis
of compound
1 was performed based on a reported
General. All chemicals, reagents and solvents were used upon
obtained from commercial sources without further
purifications. Unless otherwise stated, the purification of
compounds by flash chromatography was performed using
Merck Geduran® Si 60 silica gel with particle sizes from 40 to
8
4
procedure. Briefly, to a 50 mL flask containing n-butanol (20
mL) was added with 4-diethylaminosalicyldehyde (0.97 g, 5.0
mmol), methyl 2-nitroacetate (502 μL, 5.15 mmol), piperidine
(
100 μL) and glacial acetic acid (2.5 mL). The mixture was
o
heated to 110 C under stirring condition for 18 h. The
resulting dark brown solution was cooled down under ice bath
when orange crystals were formed in the reaction mixture. The
crystals were isolated by filtration and washed three times
6
3 μm. Column was stacked with the slurry mixture of the
1 13
silica gel and the eluting solvent. All H NMR and C NMR
measurements were performed on a Bruker AMX AV400 MHz
1
spectrophotometer at ambient temperature.
H
two-
with ice cold n-butanol before drying under vacuo to afford
1
dimension correlation spectroscopy (COSY) spectrum of
FLB SSCou was measured with a Bruker AVIII 400 MHz NMR
BBFO probe spectrophotometer at ambient temperature.
o
as a red-orange crystal (0.37 g, 28%). M.P.: 198.6–199.3 C.
2
-1
FTIR ꢀꢁ /cm : 2980.02, 2935.66, 1743.65, 1631.78, 1591.27,
544.98, 1477.47, 1427.32, 1327.03, 1188.15, 823.60, 767.67.
2
1
1
Signals from H NMR spectra were reported as chemical shift
in parts per million (ppm) calibrated using either deuterated
1
H NMR (400 MHz, CDCl
3
): δ 8.72 (s, 1H), 7.44 (d, J = 9.08 Hz,
1
3
H), 6.70 (dd, J = 9.09 & 2.37 Hz, 1H), 6.48 (d, J = 2.22 Hz, 1H),
3 6
solvent (7.26 ppm for CDCl and 2.54 ppm for DMSO-d ) or
1
3
.50 (q, J = 7.16 Hz, 4H), 1.27 (t, J = 7.16 Hz, 6H). C NMR (400
): δ 158.90, 154.69, 153.60, 143.47, 132.69, 127.03,
11.26, 106.35, 96.96, 45.66, 12.57. HRMS (H O/MeOH): m/z:
downfield from tetramethylsilane (0.00 ppm). Numbers of
equivalent resonance protons are reported as nH and
multiplicities are recorded as s (singlet), d (doublet), t (triplet),
q (quartet), dd (doublet of doublet), ddd (doublet of doublet of
doublet) and m (multiplet). Corresponding coupling constants
MHz, CHCl
3
1
2
+
calcd: 263.1032, found: 263.1038 [M+H] .
3
2
-Amino-7-(diethylamino)-2H-chromen-2-one (2). Compound
8
5
was synthesized based on a reported procedure. To 25 mL
(
J value) for these multiplicities are reported in Hz. Signals
1
3
flask containing 37% HCl (5 mL) was added with stannous
chloride (1.35 g, 7.12 mmol) under stirring condition.
from C NMR spectra were reported as chemical shift in parts
per million (ppm) calibrated using deuterated solvent (7.26
Thereafter, compound
1 (0.25 g, 0.95 mmol) was added slowly
3 6
ppm for CDCl and 2.54 ppm for DMSO-d ). Fourier transform
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 9
Please do not adjust margins

Please do not adjust margins
Journal of Materials Chemistry C
Page 10 of 14
View Article Online
DOI: 10.1039/C5TC01465D
ARTICLE
Journal Name
into the stirring solution and the resulting mixture was stirred 1627.92, 1539.20, 1444.68, 1261.45, 1192.01, 1132.21, 763.81.
1
for 4 h before pouring it into crushed ice (~20 g) with aqueous
3
H NMR (400 MHz, CDCl ): δ 8.59 (s, 1H), 8.54 (d, J = 4.83 Hz,
NaOH (5 M) in an ice bath. The resulting suspension was 1H), 8.15 (s, 1H), 7.63 (m, 2H), 7.11 (m, 1H), 6.62 (dd, J = 8.81
saturated with NaCl before extracting three times with diethyl & 2.46 Hz, 1H), 6.50 (d, J = 2.39 Hz, 1H), 3.40 (q, J = 7.10 Hz,
ether. The organic layer was then washed with brine and dried 4H), 3.17 (t, J = 6.71 Hz, 2H), 2.85 (t, J = 6.75 Hz, 2H), 1.20 (t, J =
1
3
over Na
to afford
2
SO
4 3
before the solvent was removed under pressure 7.07 Hz, 6H). C NMR (400 MHz, CHCl ): δ 169.74, 159.57,
o
as an orange solid (0.17 g, 77%). M.P.: 89.6-90.3 C. 152.83, 150.05, 149.59, 137.09, 128.81, 126.23, 122.64,
2
-
1
FTIR ꢀꢁ /cm : 3400.50, 3338.78, 2970.38, 1695.43, 1624.06, 121.08, 120.27, 118.83, 109.75, 108.30, 105.76, 97.48, 44.85,
591.27, 1523.76, 1134.14, 813.96, 798.53. H NMR (400 MHz, 36.85, 34.59, 12.57. HRMS (H O/MeOH): m/z: calcd: 430.1259,
2
1
1
+
CDCl
3
): δ 7.11 (d, J = 8.68 Hz, 1H), 6.70 (s, 1H), 6.57 (dd, J = found: 430.1249 [M+H] .
8
.69 & 2.51 Hz, 1H), 6.53 (d, J = 2.44 Hz, 1H), 3.37 (q, J = 7.08 3-((2-Aminoethyl)disulfanyl)-N-(7-(diethylamino)-2-oxo-2H-
1
Hz, 4H), 1.18 (t, J = 7.07 Hz, 6H). C NMR (400 MHz, CHCl
3
3
): δ chromen-3-yl)propanamide HCl salt (5). In a 25 mL round
1
1
2
3
60.33, 151.63, 147.50, 127.52, 125.95, 114.46, 109.68, bottom flask was loaded with compound 4 (0.22 g, 0.50 mmol)
09.33, 98.03, 44.64, 12.50. HRMS (H
+
33.1290, found: 233.1296 [M+H] .
2
O/MeOH): m/z: calcd: and acetic acid (30 μL) in methanol (5 mL). The stirring solution
was then added with mercaptoethanamine hydrochloride
-(Pyridin-2-yldisulfanyl)propanoic acid (3). Compound
8
3 was (0.05 g, 0.48 mmol). The reaction was allowed to react for 24 h
6
synthesized based on a reported procedure. To a stirring and then the solvent was removed by rotary evaporation. The
solution of 2,2’-dipyridyl disulfide (3.75 g, 17 mmol) in ethanol residue was re-dissolved in a small amount of methanol, which
(10 mL) was added with acetic acid (0.4 mL). Thereafter, 3- was poured into diethyl ether (100 mL) to induce the
mercaptopropionic acid (0.90 g, 8.47 mmol) in ethanol (5 mL) precipitation. The solid was isolated by filtration and then re-
was dropwise added into the above solution, and the solution dissolved in a small amount of methanol before re-
turned yellow gradually. The mixture was stirred for 20 h precipitating in diethyl ether (100 mL). The process was
before ethanol was removed by rotary evaporation. The repeated for two more times until the supernatant turned
resulting yellow syrup was dissolved in CH
2
Cl
2
/ethanol (3:2, 10 colorless after the precipitation. The resulting yellow solid was
(0.15 g, 67%). FTIR
2 2
Cl /ethanol (3:2). The column was flushed with ꢀꢁ /cm : 3327.21, 2970.38, 1734.01, 1707.00, 1670.35, 1602.85,
mL) and loaded into a neutral alumina column, equilibrated dried under vacuo to afford compound
5
-1
with CH
CH Cl /ethanol (3:2) until the yellow fraction was completely 1535.34, 1411.89, 1261.45, 1130.29, 823.60, 763.81. H NMR
eluted out of the column. Thereafter, the column was flushed (400 MHz, DMSO-d ): δ 9.64 (s, 1H), 8.45 (s, 1H), 8.30 (broad s,
with CH Cl /ethanol (3:2) containing 4% acetic acid to elute the 1H), 7.46 (d, J = 8.80 Hz, 1H), 6.73 (dd, J = 9.0 & 2.20 Hz, 1H),
1
2
2
6
2
2
target product out of the column. The solvent was removed by 6.56 (d, J = 8.81 2.0 Hz, 1H), 3.44 (q, J = 6.80 Hz, 7.5H,
rotary evaporation, and the remaining liquid was re-dissolved overlapped with HDO), 3.13 (t, J = 6.40 Hz, 2H), 3.04 (t, J = 6.00
1
3
C
in CH
acid completely. The organic layer was washed with brine and NMR (400 MHz, DMSO): δ 170.68, 158.69, 153.17, 149.68,
dried over Na SO before the solvent was removed by rotary 129.25, 128.22, 118.95, 109.91, 107.88, 97.08, 44.45, 38.20,
evaporation. Finally, the residue was dried under vacuo to 36.07, 34.39, 34.02, 12.79. HRMS (H O/MeOH): m/z: calcd:
2
Cl
2
and washed three times with water to remove acetic Hz, 4H), 2.90 (t, J = 7.20 Hz, 2H), 1.15 (t, J = 6.80 Hz, 6H).
2
4
2
o
+
afford
3 as a white solid (1.42 g, 78%). M.P.: 61.2-62.6 C. FTIR 408.1416, found: 408.1432 [M-Cl] .
-1
ꢀ
ꢁ
/cm : 3300-2400 (Broad), 2912.51, 1728.22, 1583.56, 3',6'-Dibromo-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-
1
446.61, 1423.47, 1188.15, 1002.98, 765.74, 715.59. H NMR 6-carboxylic acid (6). 3-Bromophenol (27.64 g, 159.80 mmol)
1
(
400 MHz, CDCl
3
): δ 10.21 (broad s, 1H), 8.47 (m, 1H), 7.66 (m, and benzene-1,2,4-tricarboxylic acid (16.80 g, 79.90 mmol)
2
H), 7.13 (ddd, J = 6.58 & 4.85 & 1.85 Hz, 1H), 3.04 (t, J = 6.95 were charged into a 3-neck flask equipped with a reflux
1
Hz, 2H), 2.79 (t, J = 6.94, 2H). C NMR (400 MHz, CHCl
3
3
): δ condenser. Gaseous exchange was then performed, cycling
176.16, 159.53, 149.44, 137.65, 121.27, 120.50, 34.04, 33.74. between N and vacuo for at least three times. Thereafter,
2
HRMS (H
+
M+H] .
2
O/MeOH): m/z: calcd: 216.0157, found: 216.0153 methanesulfonic acid (60 mL) was injected into the flask and
o
the reaction was heated at 140 C for 72 h. The reaction was
[
N-(7-(Diethylamino)-2-oxo-2H-chromen-3-yl)-3-(pyridin-2-
yldisulfanyl)propanamide (4). To a stirring solution of ice water (400 mL). The precipitate was then collected by
compound (0.24 g, 1.10 mmol) in THF (5 mL) was added with filtration and dried in vacuo. The resulting brown solid was
cooled down to ambient temperature before pouring it into
3
dicyclohexyl carbodiimide (0.23 g, 1.10 mmol) and N- dissolved in acetic anhydride/pyridine mixture (3:1, 160 mL) to
hydroxysuccinimide (0.12 g, 1.07 mmol). The reaction was induce the precipitation. The white precipitate was isolated
stirred for 2 h with the formation of white precipitate. and the red filtrate was discarded. The white precipitate was
Compound
2 (0.23 g, 1.00 mmol) was then added, and the washed with acetic anhydride/pyridine mixture (2:1, 60 mL)
resulting solution was refluxed for 24 h. The solid was removed for six times to ensure complete removal of any pink stain. The
by filtration, and the filtrate was concentrated by rotary resulting white solid was then washed with aqueous HCl (1 M,
evaporation. The obtained residual was purified by column 100 mL) for three times followed by washing with water for
chromatography eluted with 1:1 hexane/ethyl acetate to three times. The solid was dried under vacuo to afford
o
as a yellow solid (0.16 g, 37%). M.P.: 130.8-131.9 C. compound
afford
4
6
as a brownish white solid (9.14 g, 23%). M.P.:
-1
-1
o
FTIR ꢀꢁ /cm : 3338.78, 2972.31, 2929.87, 1699.29, 1668.43, 89.6-90.3 C (decomposition temperature). FTIR ꢀꢁ /cm : 3300-
1
0
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 11 of 14
Journal Name
Jou Pr nl e aa ls oe fd Mo an to et r ai ad lj su s Ct hm ea mr g ii sn ts ry C
View Article Online
DOI: 10.1039/C5TC01465D
ARTICLE
2
400 (broad), 2974.23, 2766.80, 1720.50, 1593.20, 1479.40, (1:1) to afford compound
1
8
as a yellow solid (30 mg, 13%).
-1
o
1230.58, 1074.35, 921.97, 817.82, 754.17. H NMR (400 MHz, M.P.: 140.2-141.7 C. FTIR ꢀꢁ /cm : 3313.71, 2976.16, 2926.01,
DMSO-d
6
): δ 8.61 (s, 1H), 8.30 (dd, J = 8.01, 1.26, 1H), 8.21 (d, J 2908.65, 1772.58, 1676.14, 1654.92, 1608.63, 1560.41,
=
=
7.97 Hz, 1H), 7.89 (s, 1H), 7.73 (d, J = 1.90 Hz, 2H), 7.36 (dd, J 1533.41, 1508.33, 1406.11, 1357.89, 1141.86, 1087.85, 974.05,
1
3
1
8.51 & 1.92 Hz, 2H), 6.91 (d, J = 8.52 Hz, 2H). C NMR (400 854.47, 825.53, 692.44. H NMR (400 MHz, CDCl
): δ 168.01, 166.40, 152.65, 151.20, 149.79, 1H), 8.09 (s, 2H), 8.02 (s, 1H), 7.72 (s, 2H), 7.54 (s, 1H), 7.40 (d,
38.25, 136.96, 131.95, 130.49, 129.03, 128.14, 126.17, J = 7.84 Hz, 2H). 7.14 (d, J = 8.63 Hz, 1H), 6.82 (d, J = 7.75 Hz,
3
): δ 8.38 (s,
MHz, DMSO-d
6
1
1
5
3
3
25.17, 124.49, 81.26. HRMS (H
+
00.8973 found: 500.8986 [M+H] .
2
O/MeOH): m/z: calcd: 2H), 6.62-6.59 (m, 1H), 6.49 (s, 1H), 3.71 (q, J = 5.91 Hz, 2H),
3.45-3.38 (m, 4H), 3.00 (t, J = 6.56 Hz, 2H), 2.86 (t, J = 6.05 Hz,
-Oxo-3',6'-bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylic acid (7)
2H), 2.76 (t, J = 6.55 Hz, 2H), 1.33 (s, 24H), 1.23 (t, J = 10.85 Hz,
1
6H). C NMR (400 MHz, CDCl
3
.
3
): δ 169.67, 168.81, 165.88,
To a oven-dried 3-neck flask was charged with compound
2.00 g, 4.00 mmol),
bis(diphenylphosphino)ferrocene]dichloro palladium
6
159.55, 154.47, 152.76, 150.44, 149.65, 140.95, 129.49,
(
[1,1’- 129.02, 128.71, 127.63, 126.83, 126.30, 125.57, 123.70,
(II) 122.66, 120.70, 118.42, 109.75, 108.00, 97.40, 84.25, 82.19,
dichloromethane adduct (0.98 g, 1.20 mmol), potassium 44.78, 39.03, 37.88, 37.30, 33.92, 33.50, 24.83. HRMS
+
acetate (3.94 g, 40.10 mmol) and bis(pinacolato)diboron (4.00 (H
g, 15.80 mmol). The flask was evacuated before back filling Quantum yield measurements. Relative quantum yields of
with N gas. The process was repeated twice before anhydrous various compounds were measured by the following formula
2
O/MeOH): m/z: calcd: 972.3542, found: 972.3682 [M+H] .
2
DMF (50 mL) was injected into the flask. The mixture was using fluorescein as the standard reference (Φstd = 0.85 in 1 N
o
stirred at room temperature for 5 min, and then stirred at 80 C NaOH).
87
ꢊ ꢋꢌꢍꢎꢏꢐꢑꢒꢓꢔ 1 − 10ꢗꢘꢙꢍ
ꢚꢛꢜ
for 12 h. Thereafter, DMF was distilled off under reduced
pressure and the resulting black mass was suspended in ethyl
Φꢂꢃꢄꢅꢆꢇ = Φꢂꢈꢉ
ꢊ ꢋꢌꢍꢕꢖꢓꢔ 1 − 10ꢗꢘꢙꢍꢚꢝꢞꢟꢠꢡ
acetate (~30 mL). The mixture was washed three times with where Φ represents the quantum yield, ∫ Fl dλ represents the
aqueous HCl (0.1 M) and once with water. The organic layer integrated area in fluorescence spectra, and Abs represents
was subjected to filtration to remove any insoluble solid, and the absorbance at the λmax of sample (sample) and standard
2 4
the filtrate was washed with brine and dried over Na SO to (std).
remove any trace of water. The solvent was then removed by Detection limit measurements. Detection limit measurements
rotary evaporation. The obtained crude product was purified of FLB SSCou with respect to GSH and H O were performed
2
2 2
2
by flash column chromatography equilibrated with hexane, based on a reported procedure. The measurements were
2
eluting with hexane/ethyl acetate mixture (gradient from carried out by incubating the analyte (various concentrations)
5
o
0:50 to 10:90). The solvent was removed under pressure to with the probe in PBS at 37 C overnight followed by measuring
obtain brown slurry, and the slurry was added with Et
solid was then obtained by vacuum filtration and washed with plotted against the concentrations to obtain a linear plot, and
Et O to afford compound as a white solid (0.52 g, 22%). M.P.: the gradient (k) of the plot was then obtained. The maximum
2
O. The the emission spectra. The maximum emission intensities were
2
7
o
-1
2
3
1
65.0-265.8 C (decomposition temperature). FTIR ꢀꢁ /cm : emission intensity of FLB SSCou was measured five times, and
2
600-2400 (broad), 3145.90, 2980.02, 1737.86, 1406.11, the standard deviation (σ) of these measurements was
1
359.82, 1143.79, 1087.85, 974.05, 854.45, 825.53, 688.59. H obtained. Lastly, detection limit was measured using the
NMR (400 MHz, DMSO-d
6
): δ 8.28 (dd, J = 8.04 & 1.05 Hz, 1H), following formula.
3
ꢭ
ꢬ
8
7
.22 (d, J = 8.02 Hz, 1H), 7.71 (s, 1H), 7.65 (s, 2H), 7.44 (d, J =
ꢢꢣꢤꢣꢥꢤꢦꢧꢨꢩꢪꢦꢫꢦꢤ =ꢩ
1
3
.84 Hz, 2H), 6.98 (d, J = 7.85 Hz, 2H), 1.35 (s, 24H). C NMR
): δ 168.31. 166.30, 153.62, 150.29,
38.24, 131.79, 130.00, 128.56, 128.02, 126.18, 124.78,
23.31, 121.15, 84.68, 81.64, 25.08. HRMS (H O/MeOH): m/z:
Computation studies. Theoretical calculations on various
adducts were performed using the Gaussian 09 package. DFT
optimizations of the adducts were performed using the basis
set of B3LYP/6-31++G(d,p). Water as solvent was simulated
with the polarizable continuum model (PCM) using the integral
equation formalism variant (IEFPCM) during the calculation
process. The optimized structures were used to proceed with
the TD-DFT calculation using the same basis set and solvent
simulation.
(400 MHz, DMSO-d
6
1
1
2
+
calcd: 596.2504, found: 596.2534 [M+H] .
N-(2-((3-((7-(Diethylamino)-2-oxo-2H-chromen-3-yl)amino)-3-
oxopropyl)disulfanyl)ethyl)-3-oxo-3',6'-bis(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)-3H-spiro[isobenzofuran-
1,9'-xanthene]-6-carboxamide (8) or FLB
2
SSCou. To a stirring
solution of compound 7 (0.14 g, 0.23 mmol) in THF (5 mL) was
Confocal scanning microscopy experiments. HeLa cells were
first seeded on a SPL 200350 coverglass-bottom dish at a
5
seeding density of 2 × 10 cells (2 mL) per well. The cells were
added with N-hydroxysuccinimide (0.03 g, 0.25 mmol) and
dicyclohexylcarbodiimide (0.05 g, 0.25 mmol). The solution
was stirred for 3 h when white precipitate of dicyclohexylurea
incubated overnight prior to the addition of FLB SSCou (10
2
was formed. Thereafter, compound
5 (0.10 g, 0.23 mmol) and
μM) and β-CD (20 μM) solution. The cells were then incubated
overnight with the samples followed by the removal of the
medium. The cells were washed three times with PBS and
finally added with fresh medium (2 mL). For the post-
Et N (70 μL, 0.50 mmol) were added to the above solution for
3
refluxing overnight. The solvent was removed by rotary
evaporation and the residue was purified by flash
chromatography, eluting with ethyl acetate/hexane mixture
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 11
Please do not adjust margins

Please do not adjust margins
Journal of Materials Chemistry C
Page 12 of 14
View Article Online
DOI: 10.1039/C5TC01465D
ARTICLE
Journal Name
2 2 2 2
treatment with H O , H O
was added into the cells right after 11. R. W. Sinkeldam, N. J. Greco and Y. Tor, Chem. Rev., 2010, 110,
2
579-2619.
the medium replacement to achieve a concentration of 150
o
μM. All the cells were incubated at 37 C for another 20 min
12. M. Schäferling, Angew. Chem. Int. Ed., 2012, 51, 3532-3554.
3. H. Kobayashi, M. Ogawa, R. Alford, P. L. Choyke and Y. Urano,
Chem. Rev., 2009, 110, 2620-2640.
1
1
1
prior to washing the cells for 3 times with PBS and then fixing
with 4% formaldehyde. The cells were washed once with PBS
again before imaging.
4. X. Li, X. Gao, W. Shi and H. Ma, Chem. Rev., 2013, 114, 590-
6
59.
Flow cytometry experiments. HeLa cells were first seeded on
5
a 6-well plate at a seeding density of 2 × 10 cells (2 mL) per
5. J. Liu, Y. Q. Sun, Y. Y. Huo, H. X. Zhang, L. F. Wang, P. Zhang, D.
Song, Y. W. Shi and W. Guo, J. Am. Chem. Soc., 2014, 136, 574-
well. The cells were incubated overnight prior to the addition
5
77.
of FLB
2
SSCou (10 μM) and β-CD (20 μM) solution. The cells 16. H. S. Jung, X. Q. Chen, J. S. Kim and J. Yoon, Chem. Soc. Rev.,
2013, 42, 6019-6031.
were then incubated with the samples overnight before the
removal of the medium. The cells were washed three times 17. H. Guo, Y. Jing, X. Yuan, S. Ji, J. Zhao, X. Li and Y. Kan, Org.
Biomol. Chem., 2011, 9, 3844-3853.
with PBS and finally added with trypsin (500 μL) to induce cell
1
1
2
2
8. S. Sreejith, K. P. Divya and A. Ajayaghosh, Angew. Chem. Int.
Ed., 2008, 47, 7883-7887.
9. S.-T. Huang, K.-N. Ting and K.-L. Wang, Anal. Chim. Acta, 2008,
detachment. Thereafter, fresh medium (500 μL) was added to
quench off trypisin before harvesting the cells. The cells were
isolated by low speed centrifugation (2000 rpm, 3 min), and
the obtained cell pellets were washed two times with PBS and
finally re-suspended in PBS (1 mL). For the post-treatment with
6
20, 120-126.
0. L. Yi, H. Y. Li, L. Sun, L. L. Liu, C. H. Zhang and Z. Xi, Angew.
Chem. Int. Ed., 2009, 48, 4034-4037.
1. B. Tang, Y. Xing, P. Li, N. Zhang, F. Yu and G. Yang, J. Am. Chem.
Soc., 2007, 129, 11666-11667.
H
2
O
2
2 2
, H O was added into the cells right after the PBS
suspension so as to achieve a concentration of 150 μM. All the
o
cells were incubated at 37 C for another 45 min prior to flow 22. L. L. Long, L. P. Zhou, L. Wang, S. C. Meng, A. H. Gong, F. Y. Du
cytometry experiments. Flow cytometry experiments were
performed at the gated population of 10,000 events.
and C. Zhang, Org. Biomol. Chem., 2013, 11, 8214-8220.
23. G.-J. Kim, K. Lee, H. Kwon and H.-J. Kim, Org. Lett., 2011, 13,
2
799-2801.
2
2
2
4. T. Zou, C. T. Lum, S. S.-Y. Chui and C.-M. Che, Angew. Chem.
Int. Ed., 2013, 52, 2930-2933.
5. L.-Y. Niu, Y.-S. Guan, Y.-Z. Chen, L.-Z. Wu, C.-H. Tung and Q.-Z.
Yang, J. Am. Chem. Soc., 2012, 134, 18928-18931.
6. F. Wang, Z. Guo, X. Li, X. Li and C. Zhao, Chem. Eur. J., 2014, 20,
11471-11478.
Acknowledgements
This work is supported by the National Research Foundation
(NRF), Prime Minister’s Office, Singapore under its NRF
Fellowship (NRF2009NRF-RF001-015), and Campus for
Research Excellence and Technological Enterprise (CREATE) 27. H. Zhang, C. Zhang, R. Liu, L. Yi and H. Sun, Chem. Commun.,
Programme–Singapore Peking University Research Centre for a
Sustainable Low-Carbon Future, as well as the NTU-A*Star
Silicon Technologies Centre of Excellence under the program
grant No. 112 351 0003.
2015, 51, 2029-2032.
8. A. R. Sarkar, C. H. Heo, E. Kim, H. W. Lee, H. Singh, J. J. Kim, H.
Kang, C. Kang and H. M. Kim, Chem. Commun., 2015, 51, 2407-
2
2
410.
2
3
9. N. Sato, S. Iwata, K. Nakamura, T. Hori, K. Mori and J. Yodoi, J.
Immunol., 1995, 154, 3194-3203.
0. S. Zhang, C. N. Ong and H. M. Shen, Cancer Lett., 2004, 208,
Notes and References
1
43-153.
1
2
3
4
5
6
7
8
9
1
.
.
.
.
.
.
.
.
.
Y. Yang, Q. Zhao, W. Feng and F. Li, Chem. Rev., 2012, 113, 192- 31. C. Y. Ang, S. Y. Tan and Y. Zhao, Org. Biomol. Chem., 2014, 12,
70.
4776-4806.
Y. Zhou, J. F. Zhang and J. Yoon, Chem. Rev., 2014, 114, 5511- 32. R. H. Schirmer, J. G. Muller and R. L. Krauthsiegel, Angew.
571.
Chem. Int. Ed. Engl., 1995, 34, 141-154.
J. Chan, S. C. Dodani and C. J. Chang, Nat. Chem., 2012, 4, 973- 33. S. Seshadri, A. Beiser, J. Selhub, P. F. Jacques, I. H. Rosenberg,
2
5
9
84.
X. Chen, Y. Zhou, X. J. Peng and J. Yoon, Chem. Soc. Rev., 2010,
9, 2120-2135.
M. E. Jun, B. Roy and K. H. Ahn, Chem. Commun., 2011, 47,
583-7601.
R. B. D'Agostino, P. W. F. Wilson and P. A. Wolf, N. Engl. J.
Med., 2002, 346, 476-483.
34. L. A. Herzenberg, S. C. De Rosa, J. G. Dubs, M. Roederer, M. T.
Anderson, S. W. Ela, S. C. Deresinski and L. A. Herzenberg, Proc.
Natl. Acad. Sci. U.S.A., 1997, 94, 1967-1972.
3
7
D. Kim, H. G. Ryu and K. H. Ahn, Org. Biomol. Chem., 2014, 12, 35. K. Yuan, Y. Liu, H.-N. Chen, L. Zhang, J. Lan, W. Gao, Q. Dou, E.
550-4566.
C. Nice and C. Huang, Proteomics, 2015, 15, 287-299.
S. Ziegler, V. Pries, C. Hedberg and H. Waldmann, Angew. 36. C. R. Schlegel, M. L. Georgiou, M. B. Misterek, S. Stocker, E. R.
4
Chem. Int. Ed., 2013, 52, 2744-2792.
M. H. Lee, J. S. Kim and J. L. Sessler, Chem. Soc. Rev., 2015, DOI:
Chater, C. E. Munro, O. E. Pardo, M. J. Seckl and A. P. Costa-
Pereira, Cell Death Dis., 2015, 6, e1671.
1
0.1039/C4CS00280F.
K. P. Carter, A. M. Young and A. E. Palmer, Chem. Rev., 2014,
14, 4564-4601.
0. J. Wu, W. Liu, J. Ge, H. Zhang and P. Wang, Chem. Soc. Rev.,
011, 40, 3483-3495.
37. G. K. Balendiran, R. Dabur and D. Fraser, Cell Biochem. Funct.,
2004, 22, 343-352.
38. H. Aziz, R. Gaafar, A. Bahnassy and A. Helal, Lung Cancer, 2012,
77, Supplement 1, S21.
1
2
1
2
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 13 of 14
Journal Name
Jou Pr nl e aa ls oe fd Mo an to et r ai ad lj su s Ct hm ea mr g ii sn ts ry C
View Article Online
DOI: 10.1039/C5TC01465D
ARTICLE
39. C. S. Morrow, C. Peklak-Scott, B. Bishwokarma, T. E. Kute, P. K. 70. D. Srikun, E. W. Miller, D. W. Domaille and C. J. Chang, J. Am.
Smitherman and A. J. Townsend, Mol. Pharmacol., 2006, 69,
499-1505.
0. N. Shao, J. Jin, H. Wang, J. Zheng, R. Yang, W. Chan and Z. Abliz,
J. Am. Chem. Soc., 2009, 132, 725-736.
Chem. Soc., 2008, 130, 4596-4597.
71. A. E. Albers, B. C. Dickinson, E. W. Miller and C. J. Chang,
Bioorg. Med. Chem. Lett., 2008, 18, 5948-5950.
72. D. Srikun, A. E. Albers, C. I. Nam, A. T. Iavarone and C. J. Chang,
J. Am. Chem. Soc., 2010, 132, 4455-4465.
1
4
4
1. K. D. Tew, Pigment Cell Melanoma Res., 2011, 24, 1078-1079.
4
2. R. Hong, G. Han, J. M. Fernández, B.-j. Kim, N. S. Forbes and V. 73. B. C. Dickinson, C. Huynh and C. J. Chang, J. Am. Chem. Soc.,
M. Rotello, J. Am. Chem. Soc., 2006, 128, 1078-1079. 2010, 132, 5906-5915.
3. X. Wang, X. Cai, J. Hu, N. Shao, F. Wang, Q. Zhang, J. Xiao and Y. 74. A. R. Lippert, T. Gschneidtner and C. J. Chang, Chem. Commun.,
Cheng, J. Am. Chem. Soc., 2013, 135, 9805-9810. 2010, 46, 7510-7512.
4. H. Kim, S. Kim, C. Park, H. Lee, H. J. Park and C. Kim, Adv. 75. C. Chung, D. Srikun, C. S. Lim, C. J. Chang and B. R. Cho, Chem.
Mater., 2010, 22, 4280-4283. Commun., 2011, 47, 9618-9620.
5. Q. Zhang, F. Liu, K. T. Nguyen, X. Ma, X. J. Wang, B. G. Xing and 76. Bryan C. Dickinson, Y. Tang, Z. Chang and Christopher J. Chang,
Y. L. Zhao, Adv. Funct. Mater., 2012, 22, 5144-5156. Chem. Biol., 2011, 18, 943-948.
6. J. Zhang, Z.-F. Yuan, Y. Wang, W.-H. Chen, G.-F. Luo, S.-X. 77. V. Carroll, B. W. Michel, J. Blecha, H. VanBrocklin, K. Keshari, D.
4
4
4
4
Cheng, R.-X. Zhuo and X.-Z. Zhang, J. Am. Chem. Soc., 2013,
35, 5068-5073.
Wilson and C. J. Chang, J. Am. Chem. Soc., 2014, 136, 14742-
14745.
1
47. Q. Zhang, X. Wang, P.-Z. Li, K. T. Nguyen, X.-J. Wang, Z. Luo, H. 78. M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A.
Zhang, N. S. Tan and Y. Zhao, Adv. Funct. Mater., 2013, 24,
450-2461.
8. S. Y. Tan, C. Y. Ang, P. Li, Q. M. Yap and Y. Zhao, Chem. Eur. J.,
014, 20, 11276-11282.
9. X. Ma, K. T. Nguyen, P. Borah, C. Y. Ang and Y. Zhao, Adv.
Healthc. Mater., 2012, 1, 690-697.
0. S. Y. Tan, C. Y. Ang, Z. Luo, P. Li, K. T. Nguyen and Y. Zhao,
Chem. Eur. J., 2015, 21, 6123-6131.
1. C. Nathan and A. Cunningham-Bussel, Nat. Rev. Immunol.,
Robb, J. R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G.
A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H. P. Hratchian, A.
F. Izmaylov, J. Bloino, G. Zheng, J. L. Sonnenberg, M. Hada, M.
Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T.
Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J. A., Jr.
Montgomery, J. E. Peralta, F. Ogliaro, M. Bearpark, J. J. Heyd, E.
Brothers, K. N. Kudin, V. N. Staroverov, R. Kobayashi, J.
Normand, K. Raghavachari, A. Rendell, J. C. Burant, S. S.
Iyengar, J. Tomasi, M. Cossi, N. Rega, J. M. Millam, M. Klene, J.
E. Knox, J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R.
Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C.
Pomelli, J. W. Ochterski, R. L. Martin, K. Morokuma, V. G.
Zakrzewski, G. A. Voth, P. Salvador, J. J. Dannenberg, S.
Dapprich, A. D. Daniels, Ö. Farkas, J. B. Foresman, J. V. Ortiz, J.
Cioslowski and D. J. Fox, Gaussian 09, Revision A.02, Gaussian,
Inc., Wallingford CT, 2009.
2
4
4
5
5
2
2013, 13, 349-361.
5
5
5
2. T. Finkel, Curr. Opin. Cell Biol., 2003, 15, 247-254.
3. S. G. Rhee, Science, 2006, 312, 1882-1883.
4. H. Yagi, J. Tan and R. S. Tuan, J. Cell. Biochem., 2013, 114,
1163-1173.
5
5
5
5
5. E. W. Miller, O. Tulyathan, E. Y. Isacoff and C. J. Chang, Nat.
Chem. Biol., 2007, 3, 263-267.
6. T. P. Szatrowski and C. F. Nathan, Cancer Res., 1991, 51, 794- 79. V. R. Batistela, J. da Costa Cedran, H. P. Moisés de Oliveira, I. S.
798.
Scarminio, L. T. Ueno, A. Eduardo da Hora Machado and N.
Hioka, Dyes Pigments, 2010, 86, 15-24.
80. D. P. Murale, H. Kim, W. S. Choi and D. G. Churchill, RSC Adv.,
2014, 4, 5289-5292.
7. R. S. Sohal, H.-H. Ku, S. Agarwal, M. J. Forster and H. Lal, Mech.
Ageing Dev., 1994, 74, 121-133.
8. L. Park, P. Zhou, R. Pitstick, C. Capone, J. Anrather, E. H. Norris,
L. Younkin, S. Younkin, G. Carlson, B. S. McEwen and C. 81. M. Ali, P. Dutta and S. Pandey, J. Phys. Chem. B, 2010, 114,
Iadecola, Proc. Natl. Acad. Sci. U.S.A., 2008, 105, 1347-1352.
9. A. M. Shah and K. M. Channon, Heart, 2004, 90, 486-487.
0. G. C. Van de Bittner, E. A. Dubikovskaya, C. R. Bertozzi and C. J.
Chang, Proc. Natl. Acad. Sci. U.S.A., 2010, 107, 21316-21321.
15042-15051.
5
6
82. B. Valeur, Molecular Fluorescence: Principles and Applications,
Wiley-VCH, Weinheim, Germany, 2001.
83. Y. Kanaoka, Angew. Chem. Int. Ed. Engl., 1977, 16, 137-147.
6
6
6
6
6
6
6
6
6
1. S. D. Lim, C. Sun, J. D. Lambeth, F. Marshall, M. Amin, L. Chung, 84. Y. Zhou, K. Chu, H. Zhen, Y. Fang and C. Yao, Spectrochim. Acta
J. A. Petros and R. S. Arnold, Prostate, 2005, 62, 200-207. Mol. Biomol. Spectros., 2013, 106, 197-202.
2. A. R. Lippert, G. C. Van de Bittner and C. J. Chang, Acc. Chem. 85. N. Gagey, M. Emond, P. Neveu, C. Benbrahim, B. Goetz, I.
Res., 2011, 44, 793-804.
3. J. Fan, M. Hu, P. Zhan and X. Peng, Chem. Soc. Rev., 2013, 42,
Aujard, J.-B. Baudin and L. Jullien, Org. Lett., 2008, 10, 2341-
2344.
2
9-43.
4. A. E. Albers, V. S. Okreglak and C. J. Chang, J. Am. Chem. Soc.,
006, 128, 9640-9641.
86. A. Janshoff, K.-P. S. Dancil, C. Steinem, D. P. Greiner, V. S. Y.
Lin, C. Gurtner, K. Motesharei, M. J. Sailor and M. R. Ghadiri, J.
Am. Chem. Soc., 1998, 120, 12108-12116.
2
5. H. Takakusa, K. Kikuchi, Y. Urano, H. Kojima and T. Nagano, 87. T. Miura, Y. Urano, K. Tanaka, T. Nagano, K. Ohkubo and S.
Chem. Eur. J., 2003, 9, 1479-1485.
Fukuzumi, J. Am. Chem. Soc., 2003, 125, 8666-8671.
6. G. Zheng, Y.-M. Guo and W.-H. Li, J. Am. Chem. Soc., 2007, 129,
1
0616-10617.
7. D. Srikun, A. E. Albers and C. J. Chang, Chem. Sci., 2011, 2,
156-1165.
1
8. M. C. Y. Chang, A. Pralle, E. Y. Isacoff and C. J. Chang, J. Am.
Chem. Soc., 2004, 126, 15392-15393.
9. E. W. Miller, A. E. Albers, A. Pralle, E. Y. Isacoff and C. J. Chang,
J. Am. Chem. Soc., 2005, 127, 16652-16659.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 13
Please do not adjust margins

Please do not adjust margins
Journal of Materials Chemistry C
Page 14 of 14
View Article Online
DOI: 10.1039/C5TC01465D
ARTICLE
Journal Name
TOC Figure
A dual responsive probe capable of generating two different
responses upon reacting with thiols and H
selective manner was developed.
2 2
O in a highly
1
4
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins