Optimized Fluorescent Probe for Specific Imaging of Glutathione S-Transferases in Living Cells and Mice

Article abstract of DOI:10.1002/asia.202000152

GSTP1 has been considered to be a marker for malignancy in many tissues. However, the existing GST fluorescent probes are unfavorable for in vivo imaging because of the limited emission wavelength or insufficient fluorescence enhancement (six-fold). The limited fluorescence enhancement of GST fluorescent probes is mainly ascribed to the high background signals resulting from the spontaneous reaction between GSH and the probes. In this work, a highly specific GST probe with NIR emission has been successfully developed through optimization of the essential unit of the probe to repress the spontaneous reaction. The novel GST probe exhibits over 100-fold fluorescence enhancement upon incubation with GSTP1/GSH and high selectivity over other potential interference. In addition, the probe has been proved to be capable of tracking endogenous GST in A549 cells. Finally, the in vivo imaging results demonstrate that the probe can be used for effective imaging of endogenous GST activity in subcutaneous tumor mouse with high contrast.

Full text of DOI:10.1002/asia.202000152

DOI: 10.1002/asia.202000152
Full Paper
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Optimized Fluorescent Probe for Specific Imaging of Glutathione
S-Transferases in Living Cells and Mice
Aiguo Song+,*[a, b] Xin Shen+,[a] Tian Feng+,[a, c] Shouchang Gai,[a] Haiqing Wei,[b] Xinxin Li,[b]
and Hui Chen*[a]
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Abstract: GSTP1 has been considered to be a marker for
malignancy in many tissues. However, the existing GST
fluorescent probes are unfavorable for in vivo imaging
because of the limited emission wavelength or insufficient
fluorescence enhancement (six-fold). The limited fluorescence
enhancement of GST fluorescent probes is mainly ascribed to
the high background signals resulting from the spontaneous
reaction between GSH and the probes. In this work, a highly
specific GST probe with NIR emission has been successfully
developed through optimization of the essential unit of the
probe to repress the spontaneous reaction. The novel GST
probe exhibits over 100-fold fluorescence enhancement
upon incubation with GSTP1/GSH and high selectivity over
other potential interference. In addition, the probe has been
proved to be capable of tracking endogenous GST in A549
cells. Finally, the in vivo imaging results demonstrate that the
probe can be used for effective imaging of endogenous GST
activity in subcutaneous tumor mouse with high contrast.
Introduction
total cytosolic protein).[3] The high expression level of GSTP1 in
A549 cells was further confirmed by measuring the enzymatic
activity in cell lysates.[4] In fact, GSTP1 has been considered to
be a marker for malignancy in tissues such as colon, liver, lung,
kidney, stomach, and cervix.[3]
Glutathione S-transferases (GSTs) are traditionally recognized as
phase II detoxification enzymes because of their capability of
catalyzing the conjugation of glutathione (GSH) to numerous
hydrophobic exogenous (chemical carcinogens, pollutants and
even therapeutic agents) and endogenous compounds to form
highly hydrophilic compounds, hence facilitating their excretion
from the body.[1] GSTs are divided into three major families
(cytosolic, mitochondrial and microsomal) according to their
intracellular localization.[1b] The cytosolic family is further
divided into several classes: alpha, mu, omega, pi, sigma, theta,
zeta and omega.[2] The pi isoform (GSTP1) is the predominant
GSTs isozyme overexpressed in a wide range of human tumors.
The steady state expression of glutathione S-transferases (GSTs)
at both the protein and mRNA level in 60 tumor cell lines was
investigated in the National Cancer Institute Drug Screening
Program. In this study, GSTP1 was found to be the predominant
isozyme with highest expression level in A549 cells (2.7% of the
Traditionally, the detection of GSTs has been performed by
monitoring absorption changes of the adduct produced by
GST-catalyzed conjugation reaction between GSH and 2,4-
dinitrochlorobenzene (CDNB).[5] However, this method is limited
to in vitro study, and it is difficult to avoid interference of the
fast spontaneous reaction between GSH and CDNB.
Fluorescence imaging is a powerful methodology to detect
enzymatic activities in living cells and animals, being simple,
safe, and sensitive, as well as offering high spatiotemporal
resolution.[6] Consequently, several activatable fluorescent
probes have been designed with a dinitro caging moiety which
reacts with GSH under the catalysis of GSTs to produce strongly
emitting fluorophores.[7,8] However, these GST fluorescent
probes show emission in the visible range, which hinders their
application in vivo. By contrast, near-infrared (NIR) fluorescence
imaging is an accessible tool for non-invasive in vivo visual-
ization of mammalian tissues with decreased autofluorescence,
low light scattering, and high penetration depth.[9] To date,
even though NIR probe has been reported for the detection of
GSTs, the limited fluorescence enhancement (six-fold) still
makes it unfavorable for GST imaging in vivo.[10] Therefore, it is
desirable to develop a novel NIR fluorescent probe with large
fluorescence enhancement for GST activity detection.
[a] Prof. A. Song,+ X. Shen,+ T. Feng,+ S. Gai, Prof. H. Chen
Department of Medicinal Chemistry
School of Pharmacy
Air Force Medical University
Xi’an, 710032 (P. R. China)
E-mail: cchenhui@fmmu.edu.cn
as@nwpu.edu.cn
[b] Prof. A. Song,+ H. Wei, X. Li
Institute of Medical Research
Northwestern Polytechnical University
Xi’an, 710072 (P. R. China)
The limited fluorescence enhancement of GST fluorescent
probes was ascribed to the high background signals resulting
from the spontaneous reaction between GSH and the probes
bearing a 2,4-dinitro caging moiety. We reasoned that highly
specific NIR probe could be achieved by optimizing the caging
moiety (Scheme 1). The specificity could be improved by
replacement of the nitro group on the caging moiety with a
relative weak electron-withdrawing group. Here, we report the
[c] T. Feng+
Department of Chinese Materia Medica and Natural Medicines
School of Pharmacy
Air Force Medical University
Xi’an, 710032 (P. R. China)
[+] Equal contribution.
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/asia.202000152
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Scheme 1. Design of NIR probes for the detection of glutathione S-
transferases.
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discovery a highly stable and specific NIR probe for GST activity
detection in living cells and mice.
Figure 1. Fluorescence enhancement of GSTN, GSTF and GSTC (10 μM in
PBS, pH=7.4) upon incubation with GSH (2 mM) and in the presence and
°
absence of GSTP1 (8 μg/mL) at 37 C. Fluorescence data were collected on
plate reader with excitation at λ=670 nm (bandwidth 12 nm) and emission
at λ=695 nm (bandwidth 12 nm) after 1 h. Error bars are � SD.
Results and Discussion
Synthesis and in vitro evaluation of GST probes
It is necessary to take some essential characteristics into
account to design a NIR GST probe, such as an NIR optical
window, high stability, fast and sensitive response, and good
selectivity. Traditionally, GST probes are designed to be
activated via GST-catalyzed cleavage of sulfonamide bonds to
form a GS conjugate, sulfur dioxide and the corresponding
fluorophores.[8] Structure-activity studies with a variety of
sulfonamides indicate that the electrophilic feature of the
sulfonyl group is critical to design an ideal GST probe with high
stability and selectivity. To optimize the probe structure, differ-
ent electron-withdrawing groups on the sulfonamide moiety
were chosen to investigate the structure-function relationship.
Probes GSTN, GSTF and GSTC can be directly obtained from
hemicyanine under basic conditions though a simple one-step
Figure 2. Uv-vis absorbance of GSTC (10 μM in PBS, pH=7.4) in the absence
°
and presence of GSTP1 and GSH (2 mM) at 37 C. Absorption data were
collected with on photometer after 60 min.
1
reaction (Scheme 1). The probes are fully characterized by H
and 13C NMR and high-resolution mass spectrometry (Figure S2
and S3). Details of the syntheses are described in the
experimental section and the supporting information.
For our initial experiments, we investigated the fluorescence
changes upon incubation of these probes with GSH in PBS
(pH=7.4) using a plate reader. We observed that GSTN and
GSTF were consumed rapidly to produce GSTD in nearly
quantitative yields (Figure 1). By contrast, the reaction between
GSTC and GSH was much slower. Large fluorescence
enhancement was observed when GSTP1 (8 μg/mL) was added
to the above reaction. Obviously, GSTC is more suitable for
specific GST detection since the interference of non-enzymatic
reaction between the probe and GSH is negligible.
To understand the effect of the GST-induced cleavage on
the photophysical properties of the probe, the absorption and
fluorescence emission spectra of GSTC were investigated. GSTC
showed extended absorption regions from the visible to NIR,
with its absorption maxima at 660 nm (Figure 2). Upon
incubation with GSTP1 and GSH, the absorption peak of GSTC
at 660 nm decreased, and a concomitant new absorption peak
appeared at ~670 nm. The fluorescence changes of GSTC in the
absence and presence of GSTP1 were investigated by
fluorescence emission spectroscopy, as depicted in Figure 3. No
significant change was observed in the absence of GSTP1, but a
Figure 3. Fluorescent response of GSTC (10 μM in PBS, pH=7.4) in the
°
absence and presence of GSTP1 and GSH (2 mM) at 37 C. Fluorescence data
were collected with excitation at λ=670 nm on fluorometer after 60 min.
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significant fluorescence enhancement appeared at 695 nm
upon adding GSTP1. The enzymatic activation process of GSTC
by GSTP1 and GSH was analysed by fluorometer and HPLC
(Figure S1), revealing the formation of the expected product
GSTD confirmed by HRMS (Figure S2).
The time-dependent fluorescence intensity of GSTC with
GSH (2 mM) and varied concentrations of GSTP1 was measured
to evaluate the response speed of the probe (Figure 4). As
expected, only subtle fluorescent enhancement was displayed
in the absence of GSTP1. However, a significant fluorescence
enhancement was observed after adding various concentrations
of GSTP1 (2-10 μg/mL). As shown in Figure 4, the time required
to reach the maximum fluorescence intensity was progressively
shortened with increasing GSTP1 concentration.
as shown in Figure S4. A linear regression curve was then fitted
to the normalized fluorescence intensity data, and the point at
which the line crossed the ordinate axis was considered to be
the detection limit (0.037 μg/mL). The quantum yield of GSTD
(Φ=0.564) is much higher than GSTC (Φ=0.011). A linear
relationship (R2 =0.993) was found between the fluorescence
enhancement at 695 nm and the GSTP1 concentration below 2
μg/mL (Figure S5). Meanwhile, the enzyme kinetics of GSTC for
GSTP1 were elucidated through the Michaelis-Menten equation.
Initial reaction velocities at several concentrations of GSTC were
measured for the first 15 minutes (Figure S6). The calculated
values of apparent Michaelis-Menten constant Km and maximum
rate Vmax were 0.39 μM and 0.0357 μM/min, respectively.
To further study the selectivity of GSTC, various potential
interfering species were examined, such as GSH, cysteine,
homocysteine, EtSH, H2S, H2O2, EtNH2, and nitroreductase/NADH
(Figure 5). There were only subtle changes in fluorescence
intensity upon the addition of these substance. Strikingly, when
GSTP1 and GSH were added, significant fluorescence
enhancement was observed. These results demonstrate the
excellent selectivity of GSTC toward GSTP1 over other compet-
itive analytes, which is required in order to achieve accurate
detection under practical applications. An enzyme inhibition
test also demonstrated that the fluorescence enhancement
response of GSTC to GSTP1 arose from the enzyme-catalyzed
reaction. The widely used GST inhibitor ethacrynic acid (EA) was
chosen to pre-treat GSTP1 before incubation with GSH and
GSTC. As expected, the fluorescence intensity was much lower
than that without EA (Figure 5). The observation confirmed that
the enhanced fluorescence response of GSTC to GSTP1 results
from enzyme-promoted cleavage reaction.
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In addition, the detection limit was determined by establish-
ing a plot of normalized fluorescent intensity versus Log[GSTP1]
Figure 4. Time-dependent fluorescence response of GSTC (10 μM in PBS,
pH=7.4) upon incubation with various concentration of GSTP1 and GSH
°
(2 mM) at 37 C. Fluorescence data were collected on plate reader with
excitation at λ=670 nm (bandwidth 12 nm) and emission at λ=695 nm
(bandwidth 12 nm). Error bars are�SD.
Fluorescence imaging of GST in A549 cells
The above studies on the optical properties of GSTC in vitro
prove that GSTC is well suited for the detection of GSTP1 under
simulated physiological conditions. Encouraged by the satisfac-
tory fluorescence response of GSTC to GSTP1, we next set out
to evaluate the use of the probe for tracking endogenous GST
activity in living cells. Adenocarcinomic human alveolar basal
epithelial cells (A549 cells) were chosen as cell model because
of their high level of endogenous GST. The MTT assay results
indicated that GSTC exhibited minimal toxicity toward cultured
cell lines (Figure S7). A549 cells show almost no fluorescence
signal in the absence of GSTC, suggesting that the possibility of
interfering with fluorescence is ruled out (Figure 6). In compar-
ison, notable red fluorescence is observed after incubating with
GSTC, indicating that GST is overexpressed in A549 cells. In
addition, a control experiment with N-methyl maleimide (NMM)
was carried out. The A549 cells were pretreated with 100 μM
NMM for 15 min before incubation with 10 μM GSTC for 30 min.
We observed that the fluorescence intensity of these cells
significantly reduced due to the consumption of GSH by the
conjugate reaction between GSH and NMM. To verify that the
obtained fluorescence signal is indeed derived from endoge-
nous GST activity, an inhibitor assay was performed. A549 cells
Figure 5. Fluorescence enhancement of GSTC (10 μM in PBS, pH=7.4) upon
incubation with various species and in the presence and absence of GSTP1
°
(8 μg/mL) at 37 C. EA (100 μM), NTR (8 μg/mL) and 2 mM of the rest of the
species were utilized. Fluorescence data were collected on plate reader with
excitation at λ=670 nm (bandwidth 12 nm) and emission at λ=695 nm
(bandwidth 12 nm) after 1 h. NTR=nitroreductase. EA=ethacrynic acid.
Error bars are�SD.
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A549 cells were grafted into the BALB/c mouse to produce
murine xenograft subcutaneous tumor models. Therefore, to
evaluate the suitability of GSTC to respond to GST in vivo,
BALB/C mice with xenograft A549 cell line tumor were
orthotopically injected with buffer solutions containing GSTC
(100 μM) and then fluorescence images were obtained after
30 min using a small animal in vivo imaging system (IVIS).
Significant fluorescence signal was observed in the tumor
region, indicating that the probe can detect endogenous GST in
tumor-bearing mice. On the other hand, there was almost no
fluorescence in the tumor region of the A549-xenograft mouse
given an intratumoral injection of saline. These results demon-
strate that probe GSTC can be used for effective imaging of
endogenous GST activity in subcutaneous tumor mouse with
high contrast.
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Figure 6. Fluorescent images of A549 cells. (a) Without probe. (b) Incubated
°
with GSTC (10 μM) for 30 min at 37 C. (c) Pre-treated with NMM (100 μM)
°
for 15 min before incubation with GSTC (10 μM) for 30 min at 37 C. (d) Pre-
Conclusion
treated with EA (100 μM) for 15 min before incubation with GSTC (10 μM)
°
for 30 min at 37 C. Fluorescence images (top); Bright-field images (middle);
Merged images (bottom). NMM=N-methyl maleimide. EA=ethacrynic acid.
Error bars are�SD.
In conclusion, an activatable hemicyanine-based fluorescent
probe, GSTC, for the detection of GST activity with near-infrared
emission has been developed successfully. For the detection,
the presence of GST mediates the cleavage of the sulfonamide
linkage in the probe, thereby releasing the NIR chromophore
with NIR fluorescent signals. This highly specific GST probe
exhibits high selectivity over other potential interference with
over 100-fold fluorescence enhancement and a detection limit
of 0.037 μg/mL. GSTC is also successfully applied in evaluating
GST inhibitor. With the low cytotoxicity and great cell mem-
brane permeability, GSTC was successfully applied for bioimag-
ing of endogenous GST in living cells and in vivo. These results
reveal potential clinical value of the NIR fluorescent probe in
the study of the physiological and pathological processes
related to GSTs.
were pre-treated with EA, a GST inhibitor, and subsequently
incubated with GSTC. The strong NIR fluorescence signal was
clearly blocked, illustrating that the distinct signal changes were
triggered by intracellular endogenous GST. The results indicate
that the probe is capable of tracking endogenous GST in living
cells.
In vivo fluorescence Imaging of GST in A549 tumor-bearing
mouse model
Besides the living cell study, the ability of probe GSTC for
in vivo imaging of GST activity in subcutaneous tumor mouse
models was examined as well (Figure 7). Since the emission of Experimental Section
the probe is in the NIR region, it causes only slight light damage
to biological samples. In particular, it could penetrate deeper
tissue with weak background fluorescence interference. These
merits make GSTC well suited for in vivo imaging experiments.
Materials and methods
All chemicals were purchased from Energy-Chemical unless other-
wise stated. GSTP1 was obtained from Sigma-Aldrich. Fetal Bovine
Serum (FBS), PBS and Dulbecco’s Modified Eagle Medium (DMEM)
were purchased from Gibco. A549 cells were purchased from
Procell Life Science & Technology Co., Ltd. Nuclear magnetic
resonance (NMR) spectra were recorded on a Bruker AVANCE 400
spectrometer. Mass spectra (MS) was measured on a Bruker
Autoflex MALDI-TOF mass spectrometer (Germany). Flash column
chromatography was performed using silica gel (particle size 200–
300 mesh). Absorption spectra were obtained on a Hitachi U3310
fluorometer. Fluorescence spectra were recorded on a Hitachi
F4500 fluorometer. Kinetic experiments were performed in a
Thermo Scientific Varioskan LUX 3020–265 microplate reader.
Fluorescent images of A549 cells were obtained with Nikon Eclipse
Ti microscope. Fluorescence imaging of mice were conducted by
IVIS Lumina small animal optical in vivo imaging system (USA).
Figure 7. Fluorescence imaging of tumor-bearing mice. The mice were
intratumorally injected with 100 μL GSTC (100 μM) or saline. Fluorescence
images were recorded after 30 min.
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Synthesis of the GSTC
Conflict of Interest
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To a mixture of GSTD (20 mg, 0.039 mmol) and DIPEA (8.32 μL,
0.047 mmol) in anhydrous DCM (3 mL), 2-nitro-4-cynobenzenesul-
fonyl chloride (11.6 mg, 0.047 mmol) was added. The solution was
stirred at room temperature for 5 h before removing the volatile
species on rotavapor. The residue was purified on silica column
chromatography (DCM:MeOH=20:1 as eluent) to give the GSTC
The authors declare no conflict of interest.
Keywords: Fluorescence · Probe · GSTP1 · Imaging · tumor
1
(13.8 mg, 49% yield). H NMR (400 MHz, DMSO-d6): δ=8.58 (s, 1H;
[1] a) J. D. Hayes, J. U. Flanagan, I. R. Jowsey, Annu. Rev. Pharmacol. Toxicol.
2005, 45, 51–58; b) G. Di Pietro, L. A. V. Magno, F. Rios-Santos, Expert
Opin. Drug Metab. Toxicol. 2010, 6, 153–170; c) B. Wu, D. Dong, Trends
Pharmacol. Sci. 2012, 33, 656–668; d) P. C. Babbitt, Proc. Natl. Acad. Sci.
USA 2000, 97, 10298–10300.
[2] a) R. N. Armstrong, Chem. Res. Toxicol. 1997, 10, 2–18; b) R. N.
Armstrong, Curr. Opin. Chem. Biol. 1998, 2, 618–623.
[3] a) K. D. Tew, A. Monks, L. Barone, D. Rosser, G. Akerman, J. A. Montali,
J. B. Wheatley, D. E. Schmidt, Mol. Pharmacol. 1996, 50, 149–159; b) T.
Simic, A. Savic-radojevic, M. Pljesa-Ercegovac, M. Matic, J. Mimic-Oka,
Nat. Rev. Urol. 2009, 6, 281–289; c) D. Wu, J. Ni, P. Graham, J. Beretov, P.
Cozzi, M. Willcox, V. Wasinger, B. Walsh, Y. Li, Crit. Rev. Oncol. Hematol.
2017, 118, 15–26.
[4] Y. Fujikawa, Y. Urano, T. Komatsu, K. Hanaoka, H. Kojima, T. Terai, H.
Inoue, T. Nagano, J. Am. Chem. Soc. 2008, 130, 14533–14543.
[5] a) W. H. Habig, M. J. Pabst, W. B. Jakoby, J. Biol. Chem. 1974, 249, 7130–
7139; b) M. A. Vargo, L. Nguyen, R. F. Colman, Biochemistry 2004, 43,
3327–3335; c) S. A. Misquitta, R. F. Colman, Biochemistry 2005, 44, 8608–
8619.
[6] a) L. Yuan, W. Lin, K. Zheng, L. He, W. Huang, Chem. Soc. Rev. 2013, 42,
622–661; b) J. Chan, S. C. Dodani, C. J. Chang, Nat. Chem. 2012, 4, 973–
984; c) A. Song, Y. Cheng, J. Xie, N. Banaei, J. Rao, Chem. Sci. 2017, 8,
7669–7674; d) J. Zhou, H. Ma, Chem. Sci. 2016, 7, 6309–6315; e) J.-L.
Zhu, Z. Xu, Y. Yang, L. Xu, Chem. Commun. 2019, 55, 6629–6671; f) S. H.
Alamudi, Y.-T. Chang, Chem. Commun. 2018, 54, 13641–13653; g) D. G.
Hoch, D. Abegg, A. Adibekian, Chem. Commun. 2018, 54, 4501–4512;
h) F. Yu, X. Han, L. Chen, Chem. Commun. 2014, 50, 12234–12249; i) S.-H.
Park, N. Kwon, J.-H. Lee, J. Yoon, I. Shin, Chem. Soc. Rev. 2020, 49, 143–
179.
ArH), 8.38 (d, J=14.4 Hz, 1H; ArH), 8.13 (d, J=8.2 Hz, 1H; ArH), 8.04
(d, J=8.2 Hz, 1H; ArH), 7.75 (d, J=7.4 Hz, 1H; ArH), 7.50 (dd, J=
19.9, 10.5 Hz, 3H; ArH), 7.37 (t, J=7.3 Hz, 1H; ArH), 7.32 (d, J=
8.5 Hz, 1H; alkene-H), 6.80 (d, J=8.6 Hz, 1H; alkene-H), 6.69 (s, 1H;
ArH), 6.25 (d, J=14.5 Hz, 1H; ArH), 3.73 (s, 3H; CH3), 2.63 (dt, J=
22.9, 6.0 Hz, 4H; CH2), 1.77 (s, 2H; CH2), 1.71 (s, 6H; CH3); 13C NMR
(101 MHz, DMSO-d6): δ=174.56, 162.01, 156.02, 154.63, 148.36,
142.67, 142.07, 142.05, 141.81, 141.15, 137.28, 135.13, 130.47,
128.59, 128.18, 127.18, 125.53, 123.65, 122.52, 122.35, 116.56,
114.00, 113.80, 111.79, 103.87, 101.11, 49.21, 31.62, 28.04, 27.65,
23.60, 20.14; HRMS: m/z calcd for [C33H29N4O5S]+: 593.1853; found:
593.1815.
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Fluorescence imaging in living cells
A549 cells were cultured in DMEM (Dulbecco’s modified Eagle’s
medium) supplemented with 10% fetal calf serum (FBS) and
penicillin/streptomycin mixture (penicillin 100 UmLÀ 1, streptomycin
100 μgmLÀ 1). The cells were cultured in a saturated humidity
°
incubator at a temperature of 37 C and 5% CO2. A549 cells were
seeded into culture dishes for laser confocal microscopy with a
concentration of 5×103 cells/well. After incubation for 24 h, the
cells were pre-treated with NMM or EA before incubation with
GSTC solution (10 μM) in DMEM. Excessive GSTC was washed
before obtaining the fluorescent images of A549 cells on Nikon
Eclipse Ti microscope.
[7] a) J. Lu, Y. Song, W. Shi, X. Li, H. Ma, Chin. J. Chem. 2013, 31, 472–478;
b) L. Qin, X. He, L. Chen, Y. Zhang, ACS Appl. Mater. Interfaces 2015, 7,
5965–5971.
[8] a) P. Hulbert, S. Yakub, J. Pharm. Pharmacol. 1983, 35, 384–386; b) R.
Nauen, N. Stumpf, Anal. Biochem. 2002, 303, 194–198; c) R. Svensson, C.
Gren, A.-S. Johansson, B. Mannervik, R. Morgenstern, Anal. Biochem.
2002, 311, 171–178; d) J. Zhang, A. Shibata, M Ito, Satoshi Shuto, Y. Ito,
B Mannervik, H. Abe, R. Morgenstern, J. Am. Chem. 2011, 133, 14109–
14119; e) J. Zhang, Z. Jin, X. Hu, H. Meng, J. Li, X. Zhang, H. Liu, T. Deng,
S. Yao, L. Feng, Anal. Chem. 2017, 89, 8097–8103; f) A. Song, T. Feng, X.
Shen, S. Gai, Y. Zhai, H. Chen, Chem. Commun. 2019, 55, 7219–7222.
[9] a) Y. Ning, S. Cheng, J.-X. Wang, Y.-W. Liu, W. Feng, F. Li, J.-L. Zhang,
Chem. Sci. 2019, 10, 4227–4235; b) P. Li, J. Wang, X. Wang, Q. Ding, X.
Bai, Y. Zhang, D. Su, W. Zhang, W. Zhang, B. Tang, Chem. Sci. 2019, 10,
2805–2810; c) R. Wang, K. Dong, G. Xu, B. Shi, T. Zhu, P. Shi, Z. Guo, W.-
H. Zhu, C. Zhao, Chem. Sci. 2019, 10, 2785–2790; d) H. Li, Y. Li, Q. Yao, J.
Fan, W. Sun, S. Long, K. Shao, J. Du, J. Wang, X. Peng, Chem. Sci. 2019,
10, 1619–1625; e) Y. Lv, C. Dan, D. Su, M. Chen, B.-C. Yin, L. Yuan, X.-B.
Zhang, Chem. Sci. 2018, 9, 7606–7613; f) Y. Liu, L. Teng, L. Chen, H. Ma,
H.-W. Liu, X.-B. Zhang, Chem. Sci. 2018, 9, 5347–5353; g) W. Chen, S. Xu,
J. J. Day, D. Wang, M. Xian, Angew. Chem. Int. Ed. 2017, 56, 16611–
16615; Angew. Chem. 2017, 129, 16838–16842.
Fluorescence imaging in mice
All animal operations were conducted in accordance with the
regulations issued by the Ethical Committee of Northwest Poly-
technical University. Six week-old female BALB/C nude mice were
subcutaneously injected with A549 cells to establish subcutaneous
tumor model and separated into two groups. The first group of
mice were orthotopically injected 100 μL saline, whereas the
second group of mice were orthotopically injected 100 μL GSTC
solution (100 μM in PBS). All the mice were anesthetized with
isoflurane (2% in oxygen) during the fluorescence imaging experi-
ments on a small animal optical in vivo imaging system.
Acknowledgements
[10] N. He, S. Bai, Y. Huang, Y. Xing, L. Chen, F. Yu, C. Lv, Anal. Chem. 2019,
91, 5424–5432.
This work was supported by the Fundamental Research Funds for
the Central Universities of China (31020180QD107 and
3102019YX01004) and the National Natural Science Foundation of
China (81573343).
Manuscript received: February 5, 2020
Revised manuscript received: March 14, 2020
Version of record online: March 30, 2020
Chem Asian J. 2020, 15, 1–6
www.chemasianj.org
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© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Prof. A. Song*, X. Shen, T. Feng, S.
Gai, H. Wei, X. Li, Prof. H. Chen*
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Optimized Fluorescent Probe for
Specific Imaging of Glutathione
S-Transferases in Living Cells and
Mice
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A specific GST probe with NIR
emission has been developed
through optimization of the essential
unit of the probe. The novel GST
probe exhibits high selectivity toward
GSTP1/GSH with >100-fold
fluorescence enhancement. Besides,
the probe has been proved to be
capable of tracking endogenous GST
in A549 cells and mice.