A cancer cell-specific benzoxadiazole-based fluorescent probe for hydrogen sulfide detection in mitochondria

Article abstract of DOI:10.1039/d0dt03653f

The present work describes the design and biological applications of a novel colorimetric and fluorescence turn-on probe for hydrosulfide detection. The probe was designed to introduce hemicyanine as the fluorescent skeleton and 7-nitro-1,2,3-benzoxadiazole as the recognition site. The optical properties and responses of the probe towards HS-, anions and some biothiols indicate an impressively high selectivity of the probe towards HS- such that it can be effectively used as an indicator for monitoring the level of HS- in living cells. In biological experiments using the probe, the H2S levels are found to be higher in cancer cells than in normal cells. In addition, the probe is shown to specifically and rapidly detect endogenous H2S, which is produced primarily in the mitochondria of cancer cells, as demonstrated by a co-localization experiment using specific trackers for the detection of cellular organelles in pharmacological inhibition or stimulation studies, without any significant cytotoxic effects. Thus, the results of the chemical and biological experiments described herein demonstrate the potential of this novel probe to specifically, safely, and rapidly detect H2S to distinguish cancer cells from normal cells by targeting it specifically in mitochondria. This journal is

Full text of DOI:10.1039/d0dt03653f

Dalton
Transactions
View Article Online
View Journal | View Issue
PAPER
A cancer cell-specific benzoxadiazole-based
fluorescent probe for hydrogen sulfide detection
in mitochondria†
Cite this: Dalton Trans., 2021, 50,
2545
Maxine Mambo Fortibui,a Dae Wui Yoon,b Ja-Yun Lim,c Sohyun Lee,a Minsun Choi,a
a
June Seok Heo,c Jinkwan Kim*b and Jinheung Kim
*
The present work describes the design and biological applications of a novel colorimetric and fluor-
escence turn-on probe for hydrosulfide detection. The probe was designed to introduce hemicyanine as
the fluorescent skeleton and 7-nitro-1,2,3-benzoxadiazole as the recognition site. The optical properties
and responses of the probe towards HS−, anions and some biothiols indicate an impressively high selecti-
vity of the probe towards HS− such that it can be effectively used as an indicator for monitoring the level
of HS− in living cells. In biological experiments using the probe, the H2S levels are found to be higher in
cancer cells than in normal cells. In addition, the probe is shown to specifically and rapidly detect
endogenous H2S, which is produced primarily in the mitochondria of cancer cells, as demonstrated by a
co-localization experiment using specific trackers for the detection of cellular organelles in pharmaco-
logical inhibition or stimulation studies, without any significant cytotoxic effects. Thus, the results of the
chemical and biological experiments described herein demonstrate the potential of this novel probe to
specifically, safely, and rapidly detect H2S to distinguish cancer cells from normal cells by targeting it
specifically in mitochondria.
Received 22nd October 2020,
Accepted 13th January 2021
DOI: 10.1039/d0dt03653f
rsc.li/dalton
brain.7–9 Therefore, the selective detection of H2S in biologi-
cal systems has attracted significant interest over the past few
1. Introduction
Hydrogen sulfide (H2S) can easily be identified by its years, and an understanding of the sensing mechanisms will
pungent smell which is associated with rotten eggs. Since it help to elucidate the biological role of H2S in physiological
has also been identified, along with nitric oxide (NO) and processes.
carbon monoxide (CO), as a gastro/neurotransmitter, the
Various studies aimed at the development of sensors for
physiological level of endogenous H2S has been found to hydrogen sulfide have been performed, including the use of
play important roles in a number of physiological processes, colorimetric methods, electrochemical assays, metal-induced
including relaxation of vascular smooth muscles, inhibition precipitation, and fluorescence techniques.10–12 However, the
of cell apoptosis, and inflammation-induced vasodilation.1–4 fluorescence sensing and imaging technique has attracted the
Furthermore, abnormal levels of H2S have been associated most attention due to various advantages such as simplicity,
with the onset of Alzheimer’s disease and diabetes.5,6 Studies high selectivity, efficiency, and biological applicability. Several
have indicated that H2S appears in the arterial blood during reaction-based fluorescent probes for H2S have been reported:
inhalation at concentrations as low as 50 ppm, is present at nucleophilic addition,13–21 cleavage of leaving groups,22–25
50–160 μM in the central nervous system, and can sometimes reduction of azide and/or nitroso groups,26–34 and copper-ion
even reach a high concentration of 600 μM in the human precipitation.35–39 In this technique, indolium dyes are often
used due to their excellent water solubility, long-wavelength
absorption and fluorescence emission, and relatively large
Stokes shift.40–42 Thus, sensitive fluorescence detection using
indolium probes has also found numerous applications in pH
sensors and cell imaging.43–48
Endogenous H2S is produced by enzymes such as cystathio-
nine-β-synthase (CBS), cystathionine-γ-lyase (CSE), and 3-mer-
captopyruvate sulfurtransferase. A recent study has shown that
CBS and CSE are overexpressed in ovarian cancer cells.49,50 To
aDepartment of Chemistry and Nano Science, Ewha Womans University,
Seoul 03760, Korea. E-mail: jinheung@ewha.ac.kr; Tel: +82-2-3277-4453
bDepartment of Biomedical Laboratory Science, College of Health Science,
Jungwon University, Chung-buk 28024, Republic of Korea. E-mail: jkkim@jwu.ac.kr
cDepartment of Integrated Biomedical and Life Sciences, College of Health Science,
Korea University, Seoul 03722, Republic of Korea
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0dt03653f
This journal is © The Royal Society of Chemistry 2021
Dalton Trans., 2021, 50, 2545–2554 | 2545

View Article Online
Paper
Dalton Transactions
discriminate cancer cells from normal cells, two probes based
on naphthalimide and chromen-2-one have been reported.51,52
However, more examples of cancer cell-specific emission
probes are still in demand for non-destructive and real-time
detection because the quantitative assay on H2S generation
in situ in living cells can be important characteristics for label-
ing of cancer cells.
In the present work, a novel colorimetric and fluorescence
turn-on probe is developed based on 6,7-benzindole and
4-chloro-7-nitrobenzofuran for hydrogen sulfide detection. The
probe is shown to display absorption and emission properties
that respond selectively to HS− against various potential com-
petitive anions and other biothiol compounds. The results
demonstrate the high selectivity of the probe towards hydrogen
sulfide and, hence, its potential as an indicator for mitochon-
dria targeting, and effective distinguishing of cancer cells from
normal cells by monitoring HS− levels in living cells.
2. Results and discussion
As shown in Scheme 1, a new probe (1) with hemicyanine as
the fluorescent skeleton and 7-nitro-1,2,3-benzoxadiazole
amine as the H2S recognition site was synthesized via a con-
densation reaction of the 6,7-benzindole derivative compound
1a with 4-hydroxybenzaldehyde to give compound 1b, followed
by
a coupling reaction with 4-chloro-7-nitrobenzofuran.
1
Compounds 1a and 1b and probe 1 were characterized by H
and 13C nuclear magnetic resonance (NMR) spectroscopy and
electrospray ionization-mass spectrometry (ESI-MS). The
results are provided in Fig. S1–S5 (ESI†).
A yellow solution of probe 1 was prepared in PBS (sodium
phosphate 10 mM, pH 7.4)/CH3CN (v/v, 99/1), and an absorp-
tion spectrum indicated by a black line in Fig. 1A was
obtained. Here, a broad absorption band is observed at
375–430 nm. The other spectra in Fig. 1A indicate the gradual
appearance of a broad absorption band at 545 nm, along with
the progressive reduction in the bands at 375 and 430 nm,
upon the addition of various concentrations of HS− (up to 10
equiv.), during which the solution of probe 1 was seen to turn
pink (Fig. S6†). In addition, a series of emission spectra were
recorded for a 5 μM solution of probe 1 in the presence of
0–50 μM NaHS, and the resulting gradual increase in the fluo-
rescence intensity is shown in Fig. 1B. No significant emission
Fig. 1 Changes in the absorption and fluorescence spectra of probe 1
with the addition of various concentrations of NaHS: (A) absorption
spectra of a 25 μM solution of probe 1 with the addition of 1–10 equiv.
of NaHS in PBS (10 mM, pH 7.4)/CH3CN (v/v, 99/1); (B) fluorescence
spectra of a 5 μM solution of probe 1 with the addition of 0–10 equiv. of
NaHS. The excitation wavelength is 545 nm.
band is observed in the fluorescence spectrum of the pure
probe 1 solution (black line, Fig. 1B), whereas a significant
enhancement in the bright pink florescence is observed at
570 nm (when excited at 545 nm) upon treatment with HS−.
The detection limit was calculated using the 3σ method to be
0.03 μM, which is well below the reported range of 0.1–5.0 μM
for other probes developed to use H2S-specific reactions. These
results indicate that probe 1 displays a high sensitivity for the
detection of HS− and can be used for the qualitative detection
of even the typically low levels of HS− in biological systems. It
is worth noting that although fluorescent probes with low
detection limits are greatly needed due to the low levels of
endogenous HS− in cells, plasma, and tissues, there are still
few such probes available for in vivo use.
The selectivity of probe 1 for HS− was investigated towards
various anions and sulfur-containing compounds, such as Cl−,
Scheme 1 Synthesis of precursor compounds 1a and 1b, and probe 1.
2546 | Dalton Trans., 2021, 50, 2545–2554
This journal is © The Royal Society of Chemistry 2021

View Article Online
Dalton Transactions
Paper
I−, ClO4−, H2PO4−, HPO42−, HSO3−, I−, NO2−, NO3−, SCN−,
AcO−, S2O42−, SO32−, SO42−, Na+, Ca2+, Mg2+, Al3+, Zn2+, Fe2+,
Hg2+, Cr3+, cysteine (Cys), homocysteine (Hcys) and glutathione
(GSH). These analytes including biothiols triggered no signifi-
cant fluorescence changes of probe 1, as shown in Fig. 2.
Hydrogen sulfide ions only afforded a sharp emission band.
Hence, these results demonstrate that probe 1 has a high
selectivity for HS− over other control analytes at biologically
relevant concentrations. These data imply that HS− can trigger
the cleavage of the amine bond of 7-nitro-1,2,3-benzoxadiazole
(NBD), releasing the fluorescent species 1b (Scheme 2), as
reported in other reaction-based probes.40,53,54
The selectivity of probe 1 towards HS− was investigated in
the presence of various anions and sulfur-containing com-
pounds, including Cl−, I−, ClO4−, H2PO4−, HPO42−, HSO3−, I−,
NO2−, NO3−, SCN−, AcO−, S2O42−, SO32−, SO42−, Na+, Ca2+,
Mg2+, Al3+, Zn2+, Fe2+, Hg2+, Cr3+, cysteine (Cys), homocysteine
(Hcys), and glutathione (GSH). As shown in Fig. 2A, these ana-
lytes and biothiols do not trigger any significant fluorescence
changes in probe 1, with only the presence of hydrogen sulfide
ions giving rise to a sharp emission band. Hence, these results
demonstrate that probe 1 has a high selectivity for HS− over
other control analytes at biologically relevant concentrations.
These results further indicate that the protection of the
Fig. 3 Fluorescence emission intensity at 464 nm for a 10 μM solution
of probe 1 after treatment with 100 μM NaHS in the presence of various
anions and biothiols (50 μM) at 25 °C in PBS (10 mM, pH 7.4)/CH3CN
(v/v, 99/1). λex = 545 nm. The incubation time was 10 min. Black bars: 1
+ analyte; red bars: 1 + analyte + HS−; analytes: (1) none, (2) Cl−, (3) I−,
(4) ClO4−, (5) H2PO4−, (6) HPO42−, (7) HSO3−, (8) NO2−, (9) NO3−, (10)
SCN−, (11) AcO−, (12) S2O42−, (13) SO32−, (14) SO42−, (15) Cys, (16) Hcys,
(17) GSH, (18) Na+, (19) Ca2+, (20) Mg2+, (21) Al3+, (22) Zn2+, (23) Fe2+
(24) Hg2+, (25) Cr3+, and (26) HS−.
,
Fig. 2 Fluorescence analysis of a 5 μM probe 1 solution in PBS (10 mM, pH 7.4)/CH3CN (v/v, 99/1) after 20 min of incubation with HS− (50 μM) in
the presence of various anions including biothiols (10 equiv.): (A) fluorescence spectra; (B) bar chart representation of the fluorescence intensities (I
− I0) at 570 nm. I0 is the intensity with probe 1 alone and I is that of 1 in the presence of analytes. Analytes: (1) probe 1, (2) Cl−, (3) I−, (4) ClO4−, (5)
H2PO4−, (6) HPO42−, (7) HSO3−, (8) NO2−, (9) NO3−, (10) SCN−, (11) AcO−, (12) S2O42−, (13) SO32−, (14) SO42−, (15) Cys, (16) Hcys, (17) GSH, (18) Na+,
(19) Ca2+, (20) Mg2+, (21) Al3+, (22) Zn2+, (23) Fe2+, (24) Hg2+, (25) Cr3+, and (26) HS−.
Scheme 2 Mechanism of HS− sensing by probe 1.
This journal is © The Royal Society of Chemistry 2021
Dalton Trans., 2021, 50, 2545–2554 | 2547

View Article Online
Paper
Dalton Transactions
hydroxy group of the cyanine dye by 7-nitro-1,2,3-benzoxadia-
zole (NBD) could be specifically enabled by cleavage of the
amine bond in 7-nitro-1,2,3-benzoxadiazole (NBD) in the pres-
ence of HS−, thus releasing the fluorescent species 1b, as
shown in Scheme 2 and as reported for other probes.40,53,54
The emission band of probe 1 upon addition of HS− well
matched with the spectrum of compound 1b. This type of clea-
vage mechanism by HS− has previously been reported for
some HS− chemosensors.40,53,54 The formation of fluorophore
1b after the cleavage was also confirmed by ESI-MS. The probe
1 solution treated with HS− afforded the signal of [1b − I−]+ at
m/z = 328.25 (Fig. S7†).
In addition, interference studies were performed to eluci-
date how the fluorescence response of probe 1 towards HS− is
influenced by the coexistence of other potential inhibitors
Fig. 4 Changes in the fluorescence intensity of a 5 μM solution of
probe 1 with 50 μM NaHS according to the pH of the buffer solution
(PBS/CH3CN: 99/1).
−
such as Cl−, Br−, I−, H2PO4−, HPO42−, HSO3−, NO2−, NO3
,
SCN−, AcO−, Cys, Hcys, and GSH (Fig. 3). Competitive assays
showed that the coexistence of these compounds did not
change the emission intensity at 464 nm.
The time-dependent fluorescence behavior of probe 1 in the
presence of HS− was then examined by monitoring the
changes in the fluorescence intensity at 464 nm as a function
of time. The results, which are provided in Fig. S8,† reveal an
immediate increase in the fluorescence signal upon the
addition of 10 equiv. of HS− to probe 1, to reach a plateau
within 7 min at room temperature. By contrast, in the absence
of HS−, no increase in the fluorescence of the probe 1 solution
is observed.
The potential effects of pH upon the fluorescence signal of
probe 1 were then investigated in the presence of HS−, as
shown in Fig. 4. Here, a remarkable enhancement in the fluo-
rescence is observed when the probe is treated with HS−
within the pH range of 7.0–10.0. This result indicates the suit-
Fig. 5 Changes in the fluorescence spectrum of a 5 μM solution of
probe 1 in the presence of various amounts of rotten egg extract
(20–80 μL) in PBS (10 mM, pH 7.4)/CH3CN (v/v, 99/1).
Fig. 6 Comparative fluorescence intensity of probe 1 (compound) in various cancer cell lines (Huh7, MDA231, and U87MG) and a normal cell line
(H9C2).
2548 | Dalton Trans., 2021, 50, 2545–2554
This journal is © The Royal Society of Chemistry 2021

View Article Online
Dalton Transactions
Paper
ability of probe 1 for detecting HS− in a relatively wide range of
pH values including the physiological range.
In addition, the sensing of HS− in a food sample was tested
by preparing a solution using a rotten egg. After the solution
was added to a 5 μM solution of probe 1 in PBS/CH3CN, the
resulting samples were incubated for 5 min, and the emission
spectra were then recorded, as shown in Fig. 5. Here, a pro-
gressive increase in the emission intensity is observed with the
increasing amount of extraction solution.
The ability of probe 1 to detect endogenously produced H2S
in living cells was then evaluated. Previous studies have indi-
cated that cystathionine-β-synthase (CBS) and cystathionine-
γ-lyase (CSE), which are responsible for the endogenous pro-
duction of H2S, are overexpressed in cancer cells.1,2 Therefore,
a cancer cell line with high levels of endogenous H2S was first
selected in order to test the potential of probe 1 to detect intra-
cellular H2S without any manipulation. As a control, H9C2
cells were used because of their low expression of CSE. The
results presented in Fig. 6 indicate that Huh7, MDA231, and
U87MG cells are strongly fluorescent after pretreatment with a
Fig. 7 Cytotoxicity test of probe 1: 5 × 103 Huh7 cells were treated with
vehicle, 1, 5, 10, 20, or 40 μM of probe 1 followed by incubation for 24 h.
The optical density was measured at 450 nm. All experiments were con-
ducted in triplicate.
Fig. 8 (A) Time-dependent fluorescence intensities of Huh7 cancer cells obtained at 0, 5, 10, 30, and 60 min following treatment with a 20 µM
solution of probe 1 (indicated as compound). (B) The fluorescence intensity of probe 1 at different times.
Fig. 9 (A) Confocal microscopy fluorescence images of H9C2 normal cells treated with a 20 μM solution of probe 1 after pretreatment with various
concentrations of NaHS (vehicle, 40, or 80 µM), respectively. (B) The relative fluorescence intensity of probe 1 at different H2S concentrations.
This journal is © The Royal Society of Chemistry 2021
Dalton Trans., 2021, 50, 2545–2554 | 2549

View Article Online
Paper
Dalton Transactions
5.0 μM solution of probe 1. By contrast, normal H9C2 cells are loaded with various concentrations of probe 1 (0–40 mM) and
seen to remain non-fluorescent after pretreatment with probe incubated for 24 h, thus indicating that the probe has low cyto-
1 under the same conditions. Among the tested cancer cells, toxicity and can be used for potential applications in living cell
the Huh7 cells showed the strongest fluorescence signals after culture models.
treatment with probe 1; hence, the Huh7 cells were used for
The time-dependent intracellular uptake of probe 1 in the
further experiments including a dose- and time-dependent Huh7 cells was then further investigated by measuring the
test, co-localization experiments, a cytotoxicity test, and a intracellular fluorescence at various times from 0 to 60 min, as
pharmacological inhibition or stimulation study.s
shown in Fig. 8. Thus, a red fluorescence is almost immedi-
The biocompatibility of probe 1 was also evaluated by the ately observed during the first 5 min, and the maximum inten-
MTT assay for the viability of the Huh7 cells, as shown in sity is reached by 60 min. This indicates that the probe is an
Fig. 7. Compared to the vehicle at any dose of probe 1, no sig- effective molecular tool for the rapid detection of H2S in living
nificant decrease in viability is observed when the cells are cells.
Fig. 10 (A) Confocal microscopy images of Huh7 cells treated with probe 1 after the pretreatment with AOAA (vehicle, 100, and 200 µM, respect-
ively). (B) The relative fluorescence intensity.
Fig. 11 Co-localization experiments: (left-to-right) confocal microscopy images of the Huh7 cells with a commercial tracker, with probe 1, the
overlaid images, and intensity profile (green and red channels) of the overlay; (top row) Mito-Tracker, (middle row) ER-Tracker, and (bottom row)
LYSO-Tracker. The green fluorescence of each tracker was obtained under 488 nm excitation, and the emission was collected at 500–600 nm. The
red fluorescence of probe 1 was obtained by excitation at 555 nm and collected at 580–700 nm.
2550 | Dalton Trans., 2021, 50, 2545–2554
This journal is © The Royal Society of Chemistry 2021

View Article Online
Dalton Transactions
Paper
To determine whether the response of probe 1 is pro- system. Unless otherwise stated, all solvents and reagents were
portional to the level of H2S in living cells, the H9C2 cells were commercially available and used without further purification.
pre-incubated with various concentrations of H2S (0, 40, or Doubly distilled water was used throughout all experiments.
80 µM) for 1 h to increase the intracellular levels of H2S before 1H NMR and 13C NMR spectra were recorded using a Varian
treating them with probe 1, as shown in Fig. 9. Here, the fluo- 500 MHz (Bruker, Germany) with tetramethylsilane (TMS) as
rescence intensity is seen to increase with increasing concen- an internal standard. Mass spectra were collected in the ESI
tration of H2S.
mode. UV-Visible and emission spectra were collected using a
Aminooxyacetic acid (AOAA) is a well-known inhibitor for PerkinElmer model Lambda 2S UV/Vis and SHIMADZU
endogenous-H2S-producing enzymes CBS and CSE. In the RF6000 Spectro Fluorophotometer, respectively, with a 1 cm
present work, the pretreatment with AOAA was found to dose- standard quartz cell.
dependently reduce the H2S fluorescence intensity of probe 1 in
the presence of Huh7 cells. Moreover, the results of stimulation
Synthesis of compound 1a
and inhibition experiments presented in Fig. 10 further demon- 1,1,2-Trimethyl-1H-benz[e]indole (6.23 mmol, 1 equiv.) was dis-
strate the high specificity of this probe for H2S in living cells.
solved in 15 mL of acetonitrile. Iodomethane (9.34 mmol, 1.5
Co-localization experiments were then performed to ident- equiv.) was added to the solution, and the resulting mixture
ify the subcellular location of the H2S signal of probe 1. was refluxed at 80 °C overnight. Then, the precipitate was col-
Commercially available specific trackers of cell organelles lected by filtration and washed with diethyl ether to afford
including the mitochondria (Mito), endoplasmic reticulum compound 1a as a gray solid without further purification
(ER), and lysosomes (LYSO) were used for this experiment. The (2.05 g, 94% yield). 1H NMR (400 MHz, DMSO-d6, ppm) 8.39
confocal microscopy images in Fig. 11 indicate a good overlap (d, J = 8.4 Hz, 1H), 8.31 (d, J = 8.8 Hz, 1H), 8.23 (d, J = 8.4 Hz,
of the green fluorescence of the Mito-tracker and the red fluo- 1H), 8.12 (d, J = 9.2 Hz, 1H), 7.80 (t, J = 7.2 Hz, 1H), 7.73 (t, J =
rescence of probe 1. Moreover, the variations in the intensity 7.6 Hz, 1H), 4.11 (s, 3H), 2.88 (s, 3H), 1.76 (s, 6H).
of the green and red fluorescence signals show a tendency
Synthesis of compound 1b
toward synchrony. However, poor synchrony is observed
between the green channels of the ER-tracker, or those of the To a mixture of compound 1b (5 mmol, 1 equiv.) and p-hydro-
LYSO-tracker, and the red channels of probe 1, thus suggesting xybenzaldehyde (6 mmol, 1.2 equiv.) in 30 mL of ethanol were
that H2S is mainly accumulated in the mitochondria. The lipo- added 2 drops of piperidine. Then the reaction solution was
philic cations of probe 1 with delocalized positive charge effec- refluxed for 18 h. After cooling down to room temperature, the
tively penetrate the hydrophobic barriers of the phospholipid precipitate was filtered and washed with ethyl ether to afford a
1
layer to target mitochondria.
red solid 1b (1.76 g, 78% yield). H NMR (400 MHz, DMSO-d6)
10.81 (s, 1H), 8.47–8.40 (m, 2H), 8.28 (d, J = 8.8 Hz, 1H), 8.21
(d, J = 8.0 Hz, 1H), 8.15 (d, J = 8.4 Hz, 2H), 8.07 (d, J = 8.0 Hz,
1H), 7.80 (t, J = 9.2 Hz, 1H), 7.71 (t, J = 8.0 Hz, 1H), 7.51 (d, J =
16.0 Hz, 1H), 6.98 (d, J = 8.0 Hz, 2H), 4.22 (s, 3H), 2.00 (s, 6H).
3. Conclusions
The synthesis and evaluation of a 6,7-benzindole-based fluo-
rescent probe for the detection of biological H2S was
Synthesis of probe 1
described. This cleavage reaction-based probe was shown to be Compound 1b (0.25 g 0.76 mmol) and 4-chloro-7-nitrobenzo-
highly selective for the detection of H2S and could be used for furan (0.15 g 0.76 mmol) were dissolved in acetonitrile
imaging H2S in living cells. The fluorescence imaging results (20 mL). To this mixture was added 100 μL of triethylamine,
imply that the probe is able to specifically assay H2S gene- and the resulting solution was refluxed for 8 h. The reaction
ration in cancer cells. Also, endogenous H2S was shown to be mixture was cooled to room temperature, filtered and washed
primarily produced in the mitochondria and was rapidly and with acetonitrile, and then it was further recrystallized in
specifically detected by the probe without any cytotoxicity. ethanol to obtain probe 1 as a red solid (150 mg 60% yield).
More sensitive and responsive probes for the fluorescence 1H NMR (500 MHz, DMSO-d6): 8.72 (d, 1H), 8.57 (d, 1H), 8.44
imaging of H2S are being actively pursued for utilization in the (m, 4H), 8.26 (d, 2H), 8.15 (d, 1H), 8.07 (d, 1H), 8.07 (d, 1H),
study of endogenous H2S production in living cells in order to 7.75 (d, 1H), 7.51 (t, 1H), 4.34 (s, 3H), 2.03 (s, 6H). 13C NMR
contribute to distinguishing cancer cells from normal cells (500 MHz, DMSO-d6): 183.24, 157.17, 153.51, 152.07, 150.60,
and an understanding of the resulting physiological and 146.17, 145.02, 140.21, 139.17, 135.52, 134.01, 133.59, 131.86,
pathological processes.
130.59, 129.01, 128.19, 127.03, 124.11, 123.77, 121.53, 116.78,
113.87, 112.70, 54.32, 35.99, 25.66. ESI-MS: calcd for [M]+
491.52, found 491.42.
=
4. Experimental section
Absorption and fluorescence measurements
Materials and instrumentation
A stock solution of probe 1 (1.0 mM) was prepared in aceto-
All chemicals were obtained commercially without further nitrile. All stock solutions (1.0 mM) of analytes, HS−, Cl−, Br−,
purification. Water was purified with a MilliQ purification I−, ClO4−, H2PO4−, HPO42−, HSO3−, NO2−, NO3−, SCN−, AcO−,
This journal is © The Royal Society of Chemistry 2021
Dalton Trans., 2021, 50, 2545–2554 | 2551

View Article Online
Paper
Dalton Transactions
S2O42−, SO32−, SO42−, Cys, Hcys, and GSH, were prepared in H2S stimulation and pharmacologic inhibition assay with AOAA
distilled water. All measurements were carried out in a mixture
Huh7 cells were seeded at a density of 1 × 105 cells per well on
of water and CH3CN (v/v, 80/20). The samples were excited at
400 nm.
For the time course experiment, NaHS (5 μM) was incu-
bated with 5 μM probe 1 at room temperature, and the fluo-
35 mm glass bottom confocal dishes. After incubation for
24 h, the cells were pre-incubated with different concentrations
of H2S (0, 40, and 80 µM). AOAA, a biological inhibitor of the
endogenous H2S-producing enzymes, was used at different
rescence intensity was measured with time. For the inter-
concentrations (0, 100, and 200 µM) for pre-treating the Huh7
ference experiments, NaHS solutions coexisting with other
cells at 1 h before treating with 40 µM probe 1. After additional
interference species (50 µM) were prepared and then mixed
24 h incubation, the cells were washed two times with cold
with the probe solution in water and CH3CN (v/v, 80/20). After
PBS and fixed with 4% paraformaldehyde for 15 min. Then,
10 min, the fluorescence spectra of the resulting solutions
the corresponding confocal fluorescence images were recorded
to determine the intracellular fluorescence intensity.
were obtained by using 400 nm excitation.
Food test
Co-localization experiment of probe 1 with a specific tracker of
A sample solution of a rotten egg was prepared according to
the following procedure. A hole was made in a fresh egg using
a needle, and the egg was kept at room temperature for 2
weeks to facilitate the rotting process. Then, the egg was
cracked into a breaker, to which 30 ml of deionized water was
added and mixed. The mixture was shaken well and ultra-soni-
cated for 15 min before it was filtered with a 0.45 mm syringe
membrane filter. The filtrate was diluted to 100 mL. Aliquots
of the sample solution were directly added to probe 1 (10 μM)
in a mixture of water and CH3CN.
cellular organelle
Mito-Tracker™ Green, ER-Tracker™ Green, and LYSO-Tracker™
Green (Thermo Fisher Scientific Inc., Waltham, Massachusetts,
US) were used to determine the intracellular localization of
probe 1. Fluorescence images were obtained using a confocal
laser scanning microscope. Huh7 (1 × 105) cells were seeded on
35 mm glass bottomed confocal dishes (SPL Life Science).
When the cell density reached about 80% of confluency, the
cells were washed two times with PBS and incubated with
20 μM probe 1 for subsequent 24 h. Then, the cells were washed
with PBS two times again and incubated in FBS-low (2.5%)
media containing 100 nM organelle trackers for 40 min. Finally,
the cells were washed with PBS twice. The red probe fluo-
rescence channel was excited at 555 nm with a laser, and its
emission was collected with a 580–700 nm long pass filter. All
green tracker fluorescences were excited at 488 nm with a laser
and emission was observed with a 500–560 nm band-pass filter.
Cell culture
Huh7, H9C2, MDA-MB-231 and U-87 MG cell lines were
obtained from Korea Cell Line Bank and used for this experi-
ment. Huh7, H9C2, MDA-MB-231 and U-87 MG cells were
seeded at a density of 1 × 105 cells per well on 35 mm glass
bottom confocal dishes (SPL Life Science, Kyongki, Korea),
and the cells were cultured in high glucose DMEM (Gibco™,
USA) supplemented with 10% Fetal Bovine Serum (FBS) (Cell
Signaling Technology, Danvers, Massachusetts, USA) and 1%
Penicillin/Streptomycin (Hyclone, Marlborough, MA, USA)
under 5% carbon dioxide gas at 37 °C. Prior to the cell viability
assay, evaluation of the intracellular uptake of probe 1, and
pharmacologic inhibition test, the Huh7 cells were seeded at a
density of 1 × 105 cells per well on a 35 mm glass bottom con-
focal dish (SPL Life Science, Korea).
Time-dependent intracellular uptake of probe 1
Huh7 cells (1 × 105) were seeded on 35 mm−1 confocal culture
dishes (SPL Life Science) and incubated for 24 h. Then, all cell
lines were treated with 20 μM probe, and the intracellular fluo-
rescence intensities were evaluated using a confocal laser scan-
ning microscope at intervals of 10 min from 0 to 60 min.
Cell viability assay
Confocal laser scanning microscopy fluorescence imaging
The Quanti-Max™ WST-8 Cell Viability Assay Kit (Biomax,
Korea) based on the use of a water-soluble tetrazolium dye was
used to evaluate the cell viability. Huh7 cells were seeded at a
density of 5 × 103 cells per well on a 96-well clear bottom plate
and treated with different concentrations of probe 1 (1, 5, 10,
20, and 40 μM) for 24 h. Next, 10 μl of Quanti-Max™ reagent
was used to treat the cells and they were incubated for 2 h. The
optical density was observed at a wavelength of 450 nm using a
microplate reader (Sense, Hidex, Finland). All experiments
were replicated three times to obtain the average values.
Cells (1.0 × 105) were seeded on 35 mm glass bottom confocal
dishes and incubated for 24 h. Then, the cells were treated
with probe 1 at a concentration of 5 to 40 µM for 2 h. DMSO-
treated cells acted as a vehicle control. The cells were washed
two times with cold PBS, and fluorescence images were
obtained using a confocal laser scanning microscope (CLSM,
Carl-Zeiss LSM 700 Exciter, Germany). All fluorescence images
were obtained using a confocal laser scanning microscope and
the intracellular fluorescence intensities from all experiments
were recorded under the same conditions. Green (Tracker)
fluorescence excited at 488 nm with an emission was observed
Statistical analysis
using a BP (band pass) 500–600 nm filter. The red fluorescence Data are presented as mean standard deviations. Statistical
of probe 1 was excited at 555 nm and its emission was col- significance was determined by one-way analysis of variance
lected with a 580–700 nm filter.
with post hoc Bonferroni corrections. All statistical analyses
2552 | Dalton Trans., 2021, 50, 2545–2554
This journal is © The Royal Society of Chemistry 2021

View Article Online
Dalton Transactions
Paper
were performed using IBM SPSS version 20.0 (SPSS; Chicago, 17 C. R. Liu, B. Peng, S. Li, C. M. Park, A. R. Whorton and
IL, USA). A p-value of less than 0.05 was considered as statisti-
cally significant.
M. Xian, Org. Lett., 2012, 14, 2184–2187.
18 Y. Chen, C. Zhu, Z. Yang, J. Chen, Y. He, Y. Jiao, W. He,
L. Qiu, J. Cen and Z. Guo, Angew. Chem., Int. Ed., 2013, 52,
1688–1691.
Conflicts of interest
19 J. Zhang, Y. Q. Sun, J. Liu, Y. Shi and W. Guo, Chem.
Commun., 2013, 49, 11305–11307.
20 X. Wang, J. Sun, W. Zhang, X. Ma, J. Lv and B. Tang, Chem.
Sci., 2013, 4, 2551–2556.
There are no conflicts to declare.
21 H. D. Li, Q. C. Yao, J. L. Fan, N. Jiang, J. Y. Wang, J. Xia and
X. J. Peng, Chem. Commun., 2015, 52, 16225–16228.
22 E. Karakusş, M. Ucuncu and M. Emrullahoğlu, Anal.
Chem., 2016, 88, 1039–1043.
23 C. Zhang, L. Wei, C. Wei, J. Zhang, R. Wang, Z. Xi and
L. Yi, Chem. Commun., 2015, 51, 7505–7508.
24 T. Liu, Z. Xu, D. R. Spring and J. Cui, Org. Lett., 2013, 15,
2310–2313.
Acknowledgements
This work was supported by the National Research
Foundation (NRF) grant funded by the Korean government
(NRF-2017R1A5A1015365, 2019R1A2C1007278), and the
“Next Generation Carbon Upcycling Project” (Project No.
2017M1A2A2046740) through the NRF funded by the Ministry
of Science and ICT.
25 X. Cao, W. Lin, K. Zheng and L. He, Chem. Commun., 2012,
48, 10529–10531.
26 A. R. Lippert, E. J. New and C. J. Chang, J. Am. Chem. Soc.,
2011, 133, 10078–10080.
References
1 L. Li, P. Rose and P. K. Moore, Annu. Rev. Pharmacol. 27 V. S. Lin, A. R. Lippert and C. J. Chang, Proc. Natl. Acad.
Toxicol., 2011, 51, 169–187.
Sci. U. S. A., 2013, 110, 7131–7135.
2 G. Yang, L. Wu and R. Wang, FASEB J., 2006, 20, 553–555.
3 G. Yang, L. Wu, B. Jiang, W. Yang, J. Qi, K. Cao, Q. Meng,
A. K. Mustafa, W. Mu, S. Zhang, S. H. Snyder and R. Wang,
Science, 2008, 322, 587–590.
4 K. Eto, T. Asada, K. Arima, T. Makifuchi and H. Kimura,
Biochem. Biophys. Res. Commun., 2002, 293, 1485–1485.
5 W. Yang, G. Yang, X. Jia, L. Wu and R. J. Wang, Physiology,
2005, 569, 519–531.
28 W. Sun, J. Fan, C. Hu, J. Cao, H. Zhang, X. Xiong, J. Wang,
S. Cui, S. Sun and X. Peng, Chem. Commun., 2013, 49,
3890–3892.
29 Z. Wu, Z. Li, L. Yang, J. Han and S. Han, Chem. Commun.,
2012, 48, 10120–10122.
30 C. S. Lim, S. K. Das, S. Y. Yang, E. S. Kim, H. J. Chun and
B. R. Cho, Anal. Chem., 2013, 85, 9288–9295.
31 S. Wang, S. Xu, G. Hu, X. Bai, T. D. James and L. Wang,
Anal. Chem., 2016, 88, 1434–1439.
6 L. Li, M. Bhatia, Y. Z. Zhu, Y. C. Zhu, R. D. Ramnath,
Z. J. Wang, F. B. Anuar, M. Whiteman, M. Salto-Tellez and 32 M. D. Hammers, M. J. Taormina, M. M. Cerda,
P. K. Moore, FASEB J., 2005, 19, 1196–1198.
L. A. Montoya, D. T. Seidenkranz, R. Parthasarathy and
7 A. Papapetropoulos, A. Pyriochou, Z. Altaany, G. Yang,
M. D. Pluth, J. Am. Chem. Soc., 2015, 137, 10216–10223.
A. Marazioti, Z. Zhou, M. G. Jeschke, L. K. Branski, 33 L. Wei, Z. Zhu, Y. Li, L. Yi and Z. Xi, Chem. Commun., 2015,
D. N. Herndon, R. Wang and C. Szabo, Proc. Natl. Acad.
Sci. U. S. A., 2009, 106, 21972–21977.
8 K. Abe and H. Kimura, J. Neurosci., 1996, 16, 1066–1071.
51, 10463–10466.
34 S. Yang, Y. Qi, C. Liu, Y. Wang, Y. Zhao, L. Wang, J. Li,
W. Tan and R. Yang, Anal. Chem., 2014, 86, 7508–7515.
9 X. Gu, C. Liu, Y. C. Zhu and Y. Z. Zhu, Tetrahedron Lett., 35 K. Sasakura, K. Hanaoka, N. Shibuya, Y. Mikami,
2011, 52, 5000–5003.
10 D. G. Searcy and M. A. Peterson, Anal. Biochem., 2004, 324,
269–275.
Y. Kimura, T. Komatsu, T. Ueno, T. Terai, H. Kimura and
T. Nagano, J. Am. Chem. Soc., 2011, 133, 18003–18005.
36 X. Qu, C. Li, H. Chen, J. Mack, Z. Guo and Z. Shen, Chem.
Commun., 2013, 49, 7510–7512.
11 M. Ishigami, K. Hiraki, K. Umemura, Y. Ogasawara, K. Ishii
and H. Kimura, Antioxid. Redox Signaling, 2008, 11, 205–214. 37 M. Sun, H. Yu, H. Li, H. Xu, D. Huang and S. Wang, Inorg.
12 L. Yang, X. Li, J. B. Yang, Y. Qu and J. L. Hua, ACS Appl.
Mater. Interfaces, 2013, 5, 1317–1326.
13 Y. Qian, J. Karpus, O. Kabil, S. Y. Zhang, H. L. Zhu, R. Banerjee,
J. Zhao and C. He, Nat. Commun., 2011, 2, 495–511.
14 C. R. Liu, J. Pan, S. Li, Y. Zhao, L. Y. Wu, C. E. Berkman,
Chem., 2015, 54, 3766–3772.
38 C. Kar, M. D. Adhikari, A. Ramesh and G. Das, Inorg.
Chem., 2013, 52, 743–752.
39 Z. Hai, Y. Bao, Q. Miao, X. Yi and G. Liang, Anal. Chem.,
2015, 87, 2678–2684.
A. R. Whorton and M. Xian, Angew. Chem., Int. Ed., 2011, 40 D. H. Yu, F. H. Huang, S. S. Ding and G. Q. Feng, Anal.
50, 10327–10329. Chem., 2014, 86, 8835–8841.
15 Z. Xu, L. Xu, J. Zhou, Y. F. Xu, W. P. Zhu and X. H. Qian, 41 J. A. Richard, M. Massonneau, P. Y. Renard and A. Romieu,
Chem. Commun., 2012, 48, 10871–10873. Org. Lett., 2008, 10, 4175–4178.
16 Y. Qian, L. Zhang, S. Ding, X. Deng, C. He, X. E. Zheng, 42 L. Yang, X. Li, Y. Qu, W. Qu, X. Zhang, Y. Hang, H. Ågren
H. L. Zhu and J. Zhao, Chem. Sci., 2012, 3, 2920–2923.
and J. Hua, Sens. Actuators, B, 2014, 203, 833–847.
This journal is © The Royal Society of Chemistry 2021
Dalton Trans., 2021, 50, 2545–2554 | 2553

View Article Online
Paper
Dalton Transactions
43 Q. Lin, X. Liu, T. B. Wei and Y. M. Zhang, Chem. – Asian J., 49 Y. Kushida, T. Nagano and K. Hanaoka, Analyst, 2015, 140,
2013, 8, 3015–3021. 685–695.
44 H. H. Wang, L. Xue, Z. J. Fang, G. P. Li and H. Jiang, J. New 50 B. Dong, X. Song, X. Kong, C. Wang, Y. Tang, Y. Liu and
Chem., 2010, 34, 1239–1242. W. Lin, Adv. Mater., 2016, 28, 8755–8759.
45 J. Chao, Y. Liu, J. Sun, L. Fan, Y. Zhang, H. Tong and Z. Li, 51 N. Velusamy, A. Binoy, K. N. Bobba, D. Nedungadi, N. Mishra
Sens. Actuators, B, 2015, 221, 427–433. and S. Bhuniya, Chem. Commun., 2017, 53, 8802–8805.
46 W. Niu, L. Fan, M. Nan, Z. Li, D. Lu, M. S. Wong, S. Shuang 52 X. Song, B. Dong, X. Kong, C. Wang, N. Zhang and W. Lin,
and C. Dong, Anal. Chem., 2015, 87, 278. RSC Adv., 2017, 7, 15817–15822.
47 Y. H. Li, Y. J. Wang, S. Yang, Y. R. Zhao, L. Yuan, J. Zheng 53 N. Dufton, J. Natividad, E. F. Verdu and J. L. Wallace, Sci.
and R. H. Yang, Anal. Chem., 2015, 87, 2495–2503. Rep., 2012, 2, 499–509.
48 X. Tian, Z. Li, C. Lau and J. Lu, Anal. Chem., 2015, 87, 54 Y. Pak, J. Li, K. C. Ko, G. Kim, J. Y. Lee and J. Yoon, Anal.
11325–11331.
Chem., 2017, 87, 11325–11331.
2554 | Dalton Trans., 2021, 50, 2545–2554
This journal is © The Royal Society of Chemistry 2021