A Ratiometric Near-Infrared Fluorogen for the Real Time Visualization of Intracellular Redox Status during Apoptosis

Article abstract of DOI:10.1002/chem.201700839

Direct monitoring of apoptotic progression is a major step forward for the early assessment of therapeutic efficacy of certain treatments and the accurate evaluation of the spread of a disease. Here, the regulatory role of glutathione (GSH) is explored as a potential biomarker for tracking apoptosis. For this purpose, a near- infrared (NIR) squaraine dye is introduced that is capable of sensing GSH in a ratiometric manner by switching its emission from NIR (690 nm) to visible region (560 nm). The favorable biocompatible attributes of the probe facilitated the real-time monitoring of apoptotic process in line with the conventional apoptotic assay. Furthermore, the robust nature of the probe was utilized for the quantitative estimation of GSH during different stages of apoptosis. Through this study, an easy and reliable method of assaying apoptosis is demonstrated, which can provide valuable insights in translational clinical research.

Full text of DOI:10.1002/chem.201700839

A Journal of
Accepted Article
Title: A Ratiometric Near Infrared Fluorogen for the Real Time
Visualization of Intracellular Redox Status during Apoptosis
Authors: Giridharan Saranya, Palapuravan Anees, Manu M Joseph,
Kaustabh K Maiti, and Ayyappanpillai Ajayaghosh
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201700839
Link to VoR: http://dx.doi.org/10.1002/chem.201700839
Supported by

Chemistry - A European Journal
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COMMUNICATION
A Ratiometric Near Infrared Fluorogen for the Real Time
Visualization of Intracellular Redox Status during Apoptosis
[
a,b]
[a,b]
[a]
[a,b]
Giridharan Saranya, Palapuravan Anees, Manu M. Joseph, Kaustabh K. Maiti,*
and
[
a,b]
Ayyappanpillai Ajayaghosh*
Abstract: Direct monitoring of apoptotic progression is a major step
forward to the early detection of therapeutic efficacy and evaluation
of disease condition. Herein, we explore the regulatory role of
glutathione (GSH) as a potential biomarker for tracking apoptosis.
For this purpose, we introduced a near-infrared (NIR) squaraine dye
that is capable of sensing GSH in a ratiometric manner by switching
its emission from NIR (690 nm) to visible region (560 nm). The
favorable biocompatible attributes of the probe facilitated the real
time monitoring of apoptotic process in line with the conventional
apoptotic assay. Furthermore, the robust nature of the probe was
utilized for the quantitative estimation of GSH during different stages
of apoptosis. Through this study, we demonstrate an easy and
reliable method of assaying apoptosis, which can provide valuable
insights in translational clinical research.
unclear, it has been well established that GSH is an early
[
6,8,9]
hallmark in the apoptotic cascade.
In contrast to other
signaling events which occur during the execution phases or
even later stages, exhaustion of GSH marks the beginning of the
[
6b,8b,10]
cell death phenomena
and can therefore serve as a
promising means for assaying apoptosis right from the initiation
step.
Among numerous detection techniques available for thiol
sensing, fluorescence assay forms one of the most powerful and
convenient tools to visualize the complex biological processes
due to its desirable features in terms of simplicity, low cost and
[
11,12]
high sensitivity.
Moreover, ratiometric probes are superior
over turn-on or turn-off probes as the emission intensity is not
interfered by probe concentration, excitation intensity or other
[
13]
environmental effects. Squaraine dyes are a well-established
class of molecular probes with favorable optical properties of
Apoptosis, a highly organized phenomenon of programmed cell
death, plays an important role in embryonic development and
intense absorption and emission in the visible and near-infrared
[14,15]
(
NIR) regions.
Herein, we report a ratiometric squaraine
[
1]
tissue homeostasis in all organisms. Abnormalities in cell
death regulation can inhibit homeostasis and can lead to several
deadly diseases such as cancer, neurodegenerative and
based fluorescent probe for biothiols which has a great potential
for quantification of thiol level in biological specimens.
Furthermore, we explored the applicability of the probe in
quantifying GSH imbalance during various stages of apoptosis
[
2]
autoimmune disorders. Therefore, tracking the advancement of
apoptotic events is essential for the sensitive monitoring of
therapeutic responses and evaluation of disease progression. A
variety of strategies have been devoted for screening the
progress of treatment based on certain apoptotic phenotypes
(
Scheme 1). Till date, this could be the first report on a small
molecule based reversible ratiometric fluorescent sensor for
tracking apoptotic events via quantification of intracellular GSH
status.
[
3]
such as caspase activation, phosphatidylserine externalization
[
4]
[5]
and DNA fragmentation.
antioxidant defenses, particularly the intracellular glutathione
More recently, the role of
[
6]
(
GSH) have been found to play a central role in apoptosis.
Although apoptosis is a versatile cellular process which can be
triggered by a variety of pathways, in a general sense, upon
apoptotic induction, GSH, the primary contributor to intracellular
[
7]
redox state, shows a depletion, and promotes the generation
of reactive oxygen species (ROS), which in turn account to the
[
8]
cell death machinery. This implies that stimulation of apoptosis
occurs as a direct consequence of GSH depletion and not
[
8]
directly from ROS induced cell damages. Although the actual
mechanism in the modulation of apoptosis by GSH still remains
[a]
G. Saranya, Dr. P. Anees, Dr. M. M. Joseph, Dr. K. K. Maiti,
Prof. Dr. A. Ajayaghosh
Chemical Sciences and Technology Division
CSIR-National Institute for Interdisciplinary Science and
Technology (CSIR-NIIST)
Thiruvananthapuram 695019 (India)
Scheme 1. Schematic illustration of the Sq dye for the ratiometric sensing of
E-mail: ajayaghosh@niist.res.in
kkmaiti@niist.res.in
GSH during apoptosis.
[b]
G. Saranya, Dr. P. Anees, Dr. K. K. Maiti, Prof. Dr. A.
Ajayaghosh
In this work, we designed a squaraine based fluorogenic
probe (Sq) that reacts rapidly with biothiols, facilitating the
switching of emission profile from NIR to visible region. The dye
was synthesized, purified by column chromatography and
subjected to characterization by NMR and mass spectrometry
Academy of Scientific and Innovative Research (AcSIR)
CSIR-NIIST, Thiruvananthapuram 695019 (India)
[**]
Supporting information for this article is given via a link at the
end of the document.
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COMMUNICATION
(
Supporting Information Figure S1-S3). The photophysical
towards other biologically relevant amino acids, H
(Supporting Information Figure S7). It can be seen that only
GSH, cysteine (Cys) and homocysteine (Hcy) induced
2 2 2
O and H S
properties
acetonitrile/phosphate buffer (25 mM, pH 7.4). Sq exhibited an
absorption maximum at 670 nm and an emission at 690 nm (Φ
0.07 with Nile blue in ethanol as reference, Φ = 0.27). Upon
of
Sq
were
first
examined
in
20%
a
F
fluorescence response while no such detectable signal was
observed even with excess quantities (10 eq.) of other analytes.
These results reveal the potential of Sq for measurement of
intracellular redox state without interference from other
biologically relevant analytes. Moreover, the probe afforded a
reversible recognition with thiols as evident from the interaction
=
R
interaction with biothiols, Sq displayed ratiometric changes in its
absorption and emission characteristics. On addition of GSH,
the absorption maximum at 670 nm decreased with the
simultaneous formation of a new absorption band at 400 nm
(
Figure 1A). Similarly, titration of Sq with GSH resulted in the
formation of a new emission band at 560 nm (Φ = 0.12 with
Rhodamine 6G in ethanol as a reference, Φ = 0.95) with the
concomitant quenching of the NIR emission at 690 nm (Figure
B). To understand more about the emitting species, we have
of Sq with GSH and H
Addition of H to Sq restored the NIR emission of the dye with
simultaneous quenching of the 560 nm emission which could be
repeated for several cycles. This implies that addition of H to
2 2
O (Supporting Information Figure S8).
F
2 2
O
R
2
O
2
1
Sq-GSH adduct causes the oxidation of GSH to its oxidized form,
glutathione disulphide (GSSG) which thereby releases the
molecule free for further sensing of GSH. We then examined the
response velocity of Sq towards GSH by monitoring the
emission intensity at 560 nm as a function of time under the
pseudo first order conditions. The time course of fluorescence
response of Sq in presence of GSH is shown in Figure 1D. It is
noteworthy that the reaction was completed within 50 seconds
recorded the excitation spectra of Sq and Sq-GSH adduct as
shown in Supporting Information Figure S4. The ratiometric
sensing mechanism underlying the above changes in absorption
and emission spectra is based on the chemical activation of the
initially dormant fluorophore through a conjugation break via
Michael addition reaction of thiol to the cyclobutene ring of the
squaraine moiety. The chemical reaction of thiol towards Sq was
confirmed by ESI mass spectrometry. The mass spectrum of Sq
in presence of hexanethiol showed a peak at m/z 584.3312
corresponding to the mass of Sq-hexanethiol adduct (Supporting
Information Figure S5).
-
2
-1
(kobs = 7.0 × 10 s ) implying the fast response of the probe
towards GSH and thereby demonstrating its capability for real
time detection of thiols in biological specimens (Supporting
Information Figure S9).
Figure 1. (A) UV-Vis absorption and (B) emission spectral changes of Sq with
increasing concentrations of GSH (0-4 eq.). Inset: photographs showing the
visual color (A) and fluorescence (B) changes of Sq in the absence (left) and
in the presence (right) of GSH. Fluorescence changes were detected under
UV illumination at 365 nm. (C) Linear plot showing the fluorescence intensity
of Sq at 560 nm as
a function of incremental concentrations of GSH,
normalized between the minimum fluorescence intensity, recorded at zero eq.
-
8
of GSH, and the maximum fluorescence intensity, recorded at [GSH] = 6 x 10
M. (D) Time dependent fluorescence enhancement profile of Sq (λem @ 560
nm) in presence of 100 eq. of GSH. All experiments were performed using 2
µM Sq in 20% acetonitrile/ phosphate buffer (25 mM, pH 7.4). The emission
spectra were obtained by excitation at 400 nm.
Figure 2. (A) Fluorescence images of Sq labeled HepG2 cells either untreated
(
a) or pre-treated with (b) LPA (500 µM for 24h), (c) LPA (500 µM for 24h) +
NEM (200 µM for 30 min) and (d) NEM (200 µM for 30 min) before labeling
with Sq. Scale bar corresponds to 30 µm. Green channel at 540-580 nm and
red channel at 640-700 nm. (B) Average intensity ratios from ratio images of A.
The linear response of the probe towards GSH has been
studied over a wide range of concentration varying from 5.0 ×
-
7
-4
1
0 – 5.0 × 10 M as indicated in Supporting Information Figure
Photostability of the probe is an important criterion for
imaging application. Exposure of the molecule at 385 nm over a
period of 15 minutes revealed that Sq is photostable (Supporting
Information Figure S10). Encouraged by the outstanding
features of the probe, we next investigated the suitability of the
probe for evaluation of redox imbalance in biological systems.
S6. The sensitivity of the probe was then investigated by the
fluorescence response of Sq with increasing concentrations of
GSH. A typical calibration plot showed a linear increase in the
fluorescence intensity of Sq with GSH concentration with a
correlation coefficient of 0.984, achieving a detection limit as low
as 2 nM (Figure 1C). The selectivity of the probe was evaluated
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[
5c, 7d]
Prior to cell tests, an 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay was performed to
quantify the cytotoxicity of the probe (Supporting Information
Figure S11). In the MTT assay, HepG2 and 3T3L1 cells were
treated with varying concentrations of the probe (1 µM to 10 M)
for 24 h. The results reveal that the probe possessed high cell
viabilities among all the tested concentrations suggesting that
the probe can be safely applied to culture cells under standard
experimental conditions. Owing to the favorable biocompatible
characteristics of Sq, our next focus was to evaluate the
intracellular uptake and distribution of Sq in cancer cells using
fluorescence microscopy. Incubation of HepG2 cells with Sq
produced significant green fluorescence in the extranuclear
environment suggestive of the excellent membrane permeability
and intracellular localization of the probe. Co-staining studies
with Hoechst 33342 revealed that fluorescence signals from Sq
is mainly localized in the cytosolic regions with much little or no
fluorescence from the nuclei (Supporting Information Figure
S12). These results make Sq a suitable candidate for the
estimation of cytoplasmic GSH concentrations inside the cells.
Next, the thiol specificity of the probe in cellular environment
was demonstrated with HepG2 cells in the presence and
absence of a GSH blocking agent, N-ethylmaleimide (NEM) and
GSH inducing agent α-lipoic acid (LPA). As shown in Figure 2A,
the cells displayed strong green fluorescence and weak red
fluorescence on incubation with Sq. When the cells were
pretreated with LPA for 24 h, a significant enhancement in the
green fluorescence was observed with much less emission from
the red channel. Contrarily, cells pretreated with LPA followed by
treatment with NEM generated weak green fluorescence and
strong red emission. Finally, cells pretreated with NEM alone
produced a remarkable fluorescence in the red channel with
much weaker signals from the green region. The corresponding
ratiometric pixel intensity ratios (G/R) obtained from the ratio
images is shown in Figure 2B.
cancer cells when compared to normal cells,
we intended to
detect the thiol levels in a cancer and a normal cell which were
chosen to be the HepG2 and 3T3L1 cells respectively. HepG2
cells incubated with Sq for 4 h exhibited a strong green emission
and a weak red emission indicative of high levels of GSH in
cancer cells (Figure 3A and Supporting Information Figure
S13A). Contrarily, 3T3L1 cells showed a dramatic decrease in
the fluorescence intensity in the green channel with a substantial
amplification in the red channel well correlating with the reduced
levels of GSH in normal cells (Figure 3B and Supporting
Information Figure S13B). As shown in Figure 3C, the ratio of
fluorescence pixel intensities at 560 and 690 nm (G/R) of Sq
incubated with HepG2 and 3T3L1 cells were found to be 1.95
and 0.15 respectively, accounting for a 13 fold increase in the
thiol level in HepG2 cells which demonstrates the successful
discrimination of cancer cells from normal cells. Although
cysteine and homocysteine have similar reactivities, their
presence at relatively low concentrations inside the cells when
compared to GSH levels, will not introduce much error in our
measurements. Furthermore, we exploited Sq for the
quantitative assessment of GSH in various living cells and
compared those with a commercial GSH assay kit. Lysates from
eight different cells with varying amounts of GSH were subjected
to quantitative evaluation by monitoring the Sq emission at 560
nm and 690 nm. The GSH contents were then estimated from
the value of I560/I690 using a calibration graph (Supporting
Information Figure S14). Figure 3D shows a comparative plot of
the GSH concentrations in different cell extracts using our probe
and those obtained by the standard kit, which reveals a
substantially good agreement between the two results.
Depletion of GSH levels is a common feature of the
apoptotic signaling cascade and the degree of GSH reduction is
directly related to the progression of apoptosis. Thus it was
reasonable to anticipate that the intracellular redox state,
primarily controlled by GSH, could be used to assay apoptosis.
Figure 3. Fluorescence imaging of (A) HepG2 and (B) 3T3L1 cells with Sq
dye (10 µM). Green channel at 540−580 nm; red channel at 640−700 nm.
Ratio images generated from green/red channel using Matlab software. Scale
bar corresponds to 30 µm. (C) Average intensity ratios from ratio images of A
and B. (D) Quantification of intracellular thiol form cell lysates using Sq dye
and commercial GSH assay kit. Data are the mean standard deviation of three
independent experiments.
Figure 4. Real time fluorescence imaging of HepG2 cells with Sq dye (10 µM)
after apoptotic induction with paclitaxel (10 µΜ) from 0 to 6 h. (A) represents
green channel at 540−580 nm, (B) represents red channel at 640−700 nm and
(
C) represents the ratio images generated from green/red channel using
Matlab software. Scale bar corresponds to 30 µm. (D) Average intensity ratios
obtained from ratio images of C. (E) Quantification of intracellular GSH form
apoptotic cell lysates using Sq dye. Data are the mean standard deviation of
three independent experiments.
The practical applicability of the probe was illustrated by
evaluating its potential for visualizing endogenous thiols in live
cells. Since there is a marked increase in the GSH levels in
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COMMUNICATION
To explore this possibility, we aimed to investigate the cellular Acknowledgements
redox changes during the different stages of apoptosis. HepG2
cells were induced to undergo apoptosis by a well-known
anticancer drug, paclitaxel and ratiometric changes in the GSH
levels of Sq labeled cells were recorded at 0, 2, 4 and 6 hours
respectively (Figure 4A-D). The high levels of GSH indicated by
A.A. is grateful to SERB, Department of Science and
Technology, Govt. of India, for a J. C. Bose National Fellowship
(
SERB Order No. SB/S2/JCB-11/2014). K.K.M. wish to thank
CSIR network project CSC-0134, BSC-0112 and DBT
BT/PR14698/NNT/28/832/2015), Govt. of India for research
(
an intense green emission and a weak red emission during the
funding. S.G. and P.A. thank UGC and CSIR for research
fellowships, respectively. M.M.J. acknowledges KBC-KSCSTE,
Govt. of Kerala for the postdoctoral research fellowship.
th
0
hour showed a noticeable drop in the green channel and a
nd
dramatic enhancement in the red channel at the 2 hour,
resulting in approximately 1.9 fold decline in the emission ratio.
The emission ratio further exhibited a drop by 1.98 fold during
Keywords: apoptosis • fluorescence imaging • squaraines •
th
the 4 hour resulting in a significant enhancement in the red
glutathione • cancer
channel and a noticeable decrease in the green channel. At the
th
6
hour, no significant changes in the red or green emission was
[
[
1]
2]
R. S. Hotchkiss, A. Strasser, J. E. McDunn, P. E. Swanson, New Engl.
J. Med. 2009, 361, 1570–1583.
observed and the ratio of emission intensities reached more or
th
less the same value as that observed in the 4 hour. These
a) D. A. Carson, J. M. Ribeiro, Lancet 1993, 341, 1251–1254; b) L. A.
Herzenberg, S. C. De Rosa, J. G. Dubs, M. Roederer, M. T. Anderson,
S. W. Ela, S. C. Deresinski, Proc. Natl. Acad. Sci. USA 1997, 94,
results underpin the fact that GSH extrusion occurs at the onset
of apoptosis, declines at a faster rate during the initial stages
and attains a stable level, causing an increase in the level of
reactive oxygen species and thereby facilitating efficient cell
death. To reaffirm this point, we have monitored the ROS
generation in cells using dichlorofluorescein diacetate (DCFDA)
assay for the same time intervals and found that GSH depletion
causes an obvious increase in the level of ROS as shown in
Supporting Information Figure S15. These changes in the GSH
levels were found to be well correlating with the apoptotic
progression as evidenced by the conventional apoptotic assay
using acridine orange-ethidium bromide staining (Supporting
Information Figure S16). In order to gain a quantitative picture of
the apoptotic process, we further monitored the emission
responses of Sq towards apoptosis induced HepG2 cell lysates
during different intervals between 0-8 h (Figure 4E). At the onset,
HepG2 cells possessed high levels of GSH amounting to about
1
967–1972; c) H. Refsum, P. M. Ueland, O. Nygard, S. E. Vollset,
Annu. Rev. Med. 1998, 49, 31–6; d) A. Haunstetter, S. Izumo, Circ. Res.
998, 82, 1111–1129; e) S. Seshadri, A. Beiser, J. Selhub, P. F.
1
Jacques, I. H. Rosenberg, R. B. D’. Agostino, P. W. F. Wilson, New
Engl. J. Med. 2002, 346, 476–483.
[
3]
a) Y. Wu, D. Xing, S. Luo, Y. Tang, Q. Chen, Cancer Lett. 2006, 235,
2
39–247; b) L. E. Edgington, A. B. Berger, G. Blum, V. E. Albrow, M. G.
Paulick, N. Lineberry, M. Bogyo, Nat. Med. 2009, 15, 967–973; c) K.
Boeneman, B. C. Mei, A. M. Dennis, G. Bao, J. R. Deschamps, H.
Mattouss, I. L. Medintz, J. Am. Chem. Soc. 2009, 131, 3828–3829; d) N.
Dai, J. Guo, Y. N. Teo, E. T. Kool, Angew. Chem. Int. Ed. 2011, 50,
5
105–5109; Angew. Chem. 2011, 123, 5211–5215; e) Q. D. Nguyen, I.
Lavdas, J. Gubbins, G. Smith, R. Fortt, L. S. Carroll, M. A. Graham, E.
O. Aboagye, Clin. Cancer Res. 2013, 19, 3914–3924.
[
[
[
4]
5]
6]
a) A. Petrovsky, E. Schellenberger, L. Josephson, R. Weissleder, A.
Bogdanov, Cancer Res. 2003, 63, 1936–1942; b) G. A. F. van Tilborg,
W. J. M. Mulder, P. T. K. Chin, G. Storm, C. P. Reutelingsperger, K.
Nicolay, G. J. Strijkers, Bioconjugate Chem. 2006, 17, 865–868.
a) Y. A. Loannou, F. W. Chen, Nucl. Acids Res. 1996, 24, 992–993; b) P.
R. Walker, J. Leblanc, B. Smith, S. Pandey, M. Sikorska, Methods 1999,
5
.30 ± 0.1 mM which decreased to 2.26 ± 0.2 mM after two
hours. In another two hour, it dropped down to 1.26 ± 0.2 mM
th
th
and the GSH level during the 6 and 8 hours turned out to be
.02 ± 0.3 mM and 0.96 ± 0.15 mM respectively. These results
1
7, 329–338; c) D. Matassov, T. Kagan, J. Leblanc, M. Sikorska, Z.
1
Zakeri, Mol Biol. 2004, 282, 1–17.
were also validated by the commercial GSH assay kit,
reaffirming the reliability of our probe in estimating the
intracellular GSH changes during apoptosis (Supporting
Information Table S1). Collectively, these results imply the
immense potential of Sq for the ratiometric detection of
apoptosis on both qualitative and quantitative grounds.
a) P. Marchetti, D. Decaudin, A. Macho, N. Zamzami, T. Hirsch, S. A.
Susin, G. Kroemer, Eur. J. Immunol. 1997, 27, 289–296; b) R. N. T.
Coffey, R. W. G. Watson, N. J. Hegarty, A. O’ Neill, N. Gibbons, H. R.
Brady, J. M. Fitzpatrick, Cancer 2000, 88, 2092–2104; c) M. L. Circu, T.
Y. Aw, Free Radic Res. 2008, 42, 689–706.
[
[
7]
8]
C. Hwang, A. J. Sinskey, H. F. Lodish, Science 1992, 257, 1496–1502.
a) R. Franco, M. I. Panayiotidis, J. A. Cidlowski, J. Biol. Chem. 2007,
In conclusion, we have successfully developed a squaraine
based fluorescent sensor for the quantitative ratiometric
fluorescence imaging of GSH, leading to the visualization of the
redox process during apoptosis. The favorable attributes of high
biocompatibility, excellent membrane permeability and fast
response inspired us to utilize the Sq dye for detecting the
intracellular GSH concentrations in live cells and cell extracts.
The probe afforded a promising strategy for discriminating
cancer cells from normal cells via the investigation of cellular
redox status. Further applicability of the probe was elucidated by
developing an easy and reliable method for the on-demand
apoptotic progression assay in real-time by probing the role of
GSH during various time spans of apoptosis on both semi-
quantitative and quantitative grounds. These results open before
us a new non-invasive pathway for diagnosing apoptosis, which
is of prime importance in drug discovery and the effective follow-
up of treatment responses.
2
82, 30452–30465; b) A. L. Ortega, S. Mena, J. M. Estrela, Cancers
011, 3, 1285–1310; c) J. S. Armstrong, K. K. Steinauer, B. Hornung, J.
2
M. Irish, P. Lecane, G. W. Birrell, D. M. Peehl, S. J. Knox, Cell Death
Differ. 2002, 9, 252-263.
[
[
[
9]
a) A. Fernandez, J. Kiefer, L. Fosdick, J. McConkey, J. Immunol. 1995,
155, 5133–5139; b) R. Franco, O. J. Schoneveld, A. Pappa, M. I.
Panayiotidis, Arch. Physiol. Biochem. 2007, 113, 234–258.
10] C. Tormos, F. J. Chaves, M. J. Garcia, F. Garrido, R. Jover, J. E.
O’Connor, A. Iradi, A. Oltra, M. R. Oliva, G. T. Saez, Cancer Lett. 2004,
2
08, 103–113.
11] a) M. Zhang, M. Yu, F. Li, M. Zhu, M. Li, Y. Gao, L. Li, Z. Liu, J. Zhang,
D. Zhang, T. Yi, C. Huang, J. Am. Chem. Soc. 2007, 129,10322–
1
0323; b) S. Sreejith, K. P. Divya, A. Ajayaghosh, Angew. Chem., Int.
Ed. 2008, 47, 7883–7887; Angew. Chem. 2008, 120, 8001–8005; c) D.
G. Cho, J. L. Sessler, Chem. Soc. Rev. 2009, 38, 1647–1662; d) L. Yi,
H. Li, L. Sun, L. Liu, C. Zhang, Z. Xi, Angew. Chem., Int. Ed. 2009, 48,
4
034–4037; Angew. Chem. 2009, 121, 4094–4097; e) M. H. Lee, Z.
Yang, C.W. Lim, Y. H. Lee, S. Dongbang, C. Kang, J. S. Kim, Chem.
Rev. 2013, 113, 5071–5109; f) H. S. Jung, X. Chen, J. S. Kim, J. Yoon,
Chem Soc. Rev. 2013, 42, 6019–6031; g) C. Yin, F. Huo, J. Zhang, R.
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201700839
COMMUNICATION
Martinez-Manez, Y. Yang, H. Lv, S. Li, Chem. Soc. Rev. 2013, 42,
6
032–6059.
[
12] a) A. P. de Silva, H. Q. N. Gunaratne, T. A. Gunnlaugsson, T. M. Huxley,
C. P. McCoy, J. T. Rademacher, T. E. Rice, Chem. Rev. 1997, 97,
1
515–1566; b) V. Hong, A. K. Alexander, M. G. Finn, J. Am. Chem. Soc.
009, 131, 9986–9994; c) J. Wu, W. Liu, J. Ge, H. Zhang, P. F. Wang,
2
Chem. Soc. Rev. 2011, 40, 3483–3495; d) D. Zhai, S. C. Lee, S. W.
Yun, Y. T. Chang, Chem. Commun. 2013, 49, 7207–7209; e) G. K.
Such, Y. Yan, A. P. R. Johnston, S. T. Gunawan, F. Caruso, Adv.
Mater. 2015, 27, 2278–2297; f) Y. Liu, Y. Tian, Y. Tian, Y. Wang, W.
Yang Adv. Mater. 2015, 27, 7156–7160; g) A. Fernandez, M. Vendrell,
Chem. Soc. Rev. 2016, 45, 1182–1196.
[
13] a) D. Srikun, E. W. Miller, D. W. Domaille, C. J. Chang, J. Am. Chem.
Soc. 2008, 130, 4596-4597; b) A. P. Demchenko, J. Fluoresc. 2010, 20,
1
099–1128.
[
14] a) A. Ajayaghosh, Acc. Chem. Res. 2005, 38, 449–459; b) H. S.
Hewage, E. V. Anslyn, J. Am. Chem. Soc. 2009, 131, 13099–13106; c)
S. Sreejith, X. Ma, Y. Zhao, J. Am. Chem. Soc. 2012, 134, 17346–
1
7349; d) Y. Zhang, X. Yue, B. Kim, S. Yao, M. V. Bondar, K. D.
Belfield, ACS Appl. Mater. Interfaces 2013, 5, 8710–8717; e) F.-P. Gao,
Y.-X. Lin, L.-L. Li, U. Mayerhöffer, P. Spenst, J.-G. Su, J.-Y. Li, F.
Würthner, H. Wang, Biomaterials 2014, 35, 1004–1014; f) P. Anees, S.
Sreejith, A. Ajayaghosh, J. Am. Chem. Soc. 2014, 136, 13233–13239.
g) I. A. Karpenko, M. Collot, L. Richert, C. Valencia, P. Villa, Y. Mely, M.
Hibert, D. Bonnet, A. S. Klymchenko, J. Am. Chem. Soc. 2015, 137,
4
05–412.ꢀ
[
15] a) J. V. Ros-Lis, B. Garcia, D. Jimenez, R. Martinez-Manez, F.
Sancenon. J. Soto, F. Gonzalvo, M. C. Valldecabres, J. Am. Chem. Soc.
2
004, 119, 5624–5627; b) J. R. Johnson, N. Fu, E. Arunkumar, W. M.
Leevy, S. T. Gammon, D. Piwnica-Worms, B. D. Smith, Angew. Chem.
Int. Ed. 2007, 46, 5528–5531; Angew. Chem. 2007, 119, 5624–5627; c)
Y. Xu, B. Li, L. Xiao, W. Li, C. Zhang, S. Sun, Y. Pang, Chem. Comm.
2
013, 49, 7732–7734; d) D. Zhang, Y.-X. Zhao, Z.-Y. Qiao, U.
Mayerhoffer, P. Spenst, X.-J. Li, F. Würthner, H. Wang, Bioconjugate
Chem. 2014, 25, 2021–2029; e) P. Anees, J. Joseph, S. Sreejith, N. V.
Menon, Y. Kang, S. W.-K. Yu, A. Ajayaghosh, Y. Zhao, Chem. Sci.
2
016, 7, 4110–4116; f) P. Anees, K. V. Sudheesh, P. Jayamurthy, A. R.
Chandrika, R. V. Omkumar, A. Ajayaghosh Chem. Sci. 2016, 7, 6808–
6
814; g) W. Sun, S. Guo, C. Hu, J. Fan, X. Peng, Chem. Rev. 2016,
16, 7768–7817.ꢀ
1
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Chemistry - A European Journal
10.1002/chem.201700839
COMMUNICATION
COMMUNICATION
A small molecule based ratiometric
fluorescent probe was designed for
the detection of intracellular redox
status which served as a reliable
assay for the on-demand real-time
probing of GSH fluctuations during
apoptosis.
G.Saranya, Dr. P. Anees, Dr. M. M.
Joseph, Dr. K. K. Maiti,* and
Prof. Dr. A. Ajayaghosh*
Page No. – Page No.
A Ratiometric Near Infrared
Fluorogen for the Real Time
Visualization of Intracellular Redox
Status during Apoptosis
This article is protected by copyright. All rights reserved.