FRET-based mitochondria-targetable dual-excitation ratiometric fluorescent probe for monitoring hydrogen sulfide in living cells

Article abstract of DOI:10.1002/asia.201400131

Hydrogen sulfide (H₂S) is connected with various physiological and pathological functions. However, understanding the important functions of H₂S remains challenging, in part because of the lack of tools for detecting endogenous H₂S. Herein, compounds Ratio-H₂S 1/2 are the first FRET-based mitochondrial-targetable dual-excitation ratiometric fluorescent probes for H₂S on the basis of H₂S-promoted thiolysis of dinitrophenyl ether. With the enhancement of H₂S concentration, the excitation peak at λ≈402 nm of the phenolate form of the hydroxycoumarin unit drastically increases, whereas the excitation band centered at λ≈570 nm from rhodamine stays constant and can serve as a reference signal. Thus, the ratios of fluorescence intensities at λ=402 and 570 nm (I₄₀₂/I₅₇₀) exhibit a drastic change from 0.048 in the absence of H₂S to 0.36 in the presence of 180 μM H ₂S; this is a 7.5-fold variation in the excitation ratios. The favorable properties of the probe include the donor and acceptor excitation bands, which exhibit large excitation separations (up to 168 nm separation) and comparable excitation intensities, high sensitivity and selectivity, and function well at physiological pH. In addition, it is demonstrated that the probe can localize in the mitochondria and determine H₂S in living cells. It is expected that this strategy will lead to the development of a wide range of mitochondria-targetable dual-excitation ratiometric probes for other analytes with outstanding spectral features, including large separations between the excitation wavelengths and comparable excitation intensities. Don't panic - FRET! The first Foerster resonance energy transfer (FRET)-based dual-excitation ratiometric fluorescent probe for monitoring H₂S in mitochondrial of living cells is described (see picture). Favorable properties of the probe include large separations of the donor and acceptor excitation bands, comparable excitation intensities, high sensitivity and selectivity, and good function at physiological pH.

Full text of DOI:10.1002/asia.201400131

FULL PAPER
DOI: 10.1002/asia.201400131
FRET-Based Mitochondria-Targetable Dual-Excitation Ratiometric
Fluorescent Probe for Monitoring Hydrogen Sulfide in Living Cells**
Lin Yuan*[a] and Qing-Ping Zuo[b]
Abstract: Hydrogen sulfide (H2S) is
connected with various physiological
and pathological functions. However,
understanding the important functions
of H2S remains challenging, in part be-
cause of the lack of tools for detecting
endogenous H2S. Herein, compounds
Ratio-H2S 1/2 are the first FRET-based
mitochondrial-targetable dual-excita-
tion ratiometric fluorescent probes for
H2S on the basis of H2S-promoted thi-
olysis of dinitrophenyl ether. With the
enhancement of H2S concentration, the
excitation peak at lꢀ402 nm of the
phenolate form of the hydroxycoumar-
in unit drastically increases, whereas
the excitation band centered at
l
tion bands, which exhibit large excita-
tion separations (up to 168 nm separa-
tion) and comparable excitation inten-
sities, high sensitivity and selectivity,
and function well at physiological pH.
In addition, it is demonstrated that the
probe can localize in the mitochondria
and determine H2S in living cells. It is
expected that this strategy will lead to
the development of a wide range of mi-
tochondria-targetable dual-excitation
ratiometric probes for other analytes
with outstanding spectral features, in-
cluding large separations between the
excitation wavelengths and comparable
excitation intensities.
ꢀ570 nm from rhodamine stays con-
stant and can serve as a reference
signal. Thus, the ratios of fluorescence
intensities at l=402 and 570 nm (I402
/
I570) exhibit a drastic change from 0.048
in the absence of H2S to 0.36 in the
presence of 180 mm H2S; this is a 7.5-
fold variation in the excitation ratios.
The favorable properties of the probe
include the donor and acceptor excita-
Keywords: fluorescence
· fluores-
cent probes · FRET · hydrogen sul-
fide · imaging agents
Introduction
with various physiological and pathological functions; how-
ever, many of its underlying molecular events remain un-
known. Therefore, selective detection of H2S in living sys-
tems has received significant attention in recent years.
Traditionally, hydrogen sulfide (H2S) was only recognized as
an environmental toxic gas with little or no physiological
significance.[1] However, recent studies suggest that H2S is
a novel gaseous transmitter in cellular signaling pathways
with the unique properties, in addition to nitric oxide (NO)
and carbon monoxide (CO).[2] Endogenous levels of H2S,
which is endogenously produced in mitochondria by en-
zymes through metabolism,[2c] is involved in a vast number
of physiological and pathological processes, such as regula-
tion of vascular function, modulation of blood pressure, re-
duction of ischemia reperfusion injury, and protection of
cells from oxidative stress and vascular injury.[1–5] Moreover,
studies have shown that an abnormal H2S level has also
been associated with various diseases, including stroke, Alz-
heimerꢀs disease, cardiovascular disease, Downꢀs syndrome,
diabetes, and liver cirrhosis.[6–9] In addition, H2S is connected
Recently, the construction of fluorescent probes for H2S
has attracted great attention.[10–12] However, the vast majori-
ty of them respond to H2S with optical signal changes only
in fluorescent intensity. In contrast to intensity-based fluo-
rescent probes, ratiometric fluorescent probes, which display
spectral shifts in the emission or excitation spectrum upon
binding or interacting with the target analytes, can eliminate
most or all environmental effects by self-calibration of two
emission or excitation bands and can also increase the dy-
namic range of fluorescence measurement.[13,14] In addition,
to be practically useful, probes should display two well-sepa-
rated emission or excitation peaks with comparable intensi-
ties, which is highly desirable for determining the emission
intensities and signal ratios with high accuracy and for en-
hancement of the dynamic range of signal ratios. To date,
a few ratiometric fluorescent probes for monitoring H2S in
mitochondria have been reported,[11f–h] although they are
highly desirable for biological imaging of H2S in living cells.
Herein, we describe compounds Ratio-H2S 1/2
(Scheme 1) as new candidates for dual-excitation ratiometric
fluorescent probes for H2S. With the enhancement of H2S
concentration, the excitation peak at lꢀ402 nm of the phe-
nolate form of the hydroxycoumarin unit drastically increas-
es, whereas the excitation band centered at lꢀ570 nm from
rhodamine stays constant and can serve as a reference
[a] Dr. L. Yuan
State Key Laboratory of Chemo/Biosensing and Chemometrics
College of Chemistry and Chemical Engineering
Hunan University, Changsha, Hunan 410082 (P.R. China)
E-mail: lyuan@hnu.edu.cn
[b] Q.-P. Zuo
Department of Pharmacy, The First Hospital of Changsha
Changsha, Hunan (P.R. China)
[**] FRET=Fçrster resonance energy transfer.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.201400131.
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L. Yuan and Q.-P. Zuo
Scheme 1. Synthesis of the ratiometric fluorescence H2S probes Ratio-H2S 1/2.
signal. Thus, the ratios of fluorescence intensities at l=402
and 570 nm (I402/I570) exhibit a drastic change from 0.048 in
the absence of H2S to 0.36 in the presence of 180 mm H2S,
which is a 7.5-fold variation in the excitation ratios. The fa-
vorable properties of the probe include the donor and ac-
ceptor excitation bands, which exhibit large excitation sepa-
rations (up to 168 nm separation) and comparable excitation
intensities, high sensitivity and selectivity, and function well
at physiological pH. In addition, we have demonstrated that
the probe can localize in the mitochondria and be used for
direct visualization of H2S in living cells.
(Scheme 1). 1H and 13C NMR spectroscopy and ESI-MS
were employed to characterize the structures of the product.
With the probes Ratio-H2S 1/2 in hand, we evaluated the
spectral properties in the absence or presence of H2S in
phosphate-buffered saline (PBS; pH 7.8, containing 5%
EtOH, 3 mm cetyltrimethylammonium bromide (CTAB);[16]
Figure 1 and Figures S1–S4 in the Supporting Information).
Results and Discussion
The rational design of the dual-excitation ratiometric fluo-
rescent probes Ratio-H2S 1/2 is based on careful considera-
tions. Recently, we introduced a Fçrster resonance energy
transfer (FRET)-based molecular strategy (coumarin–rhoda-
mine FRET platform) for the rational design of dual-excita-
tion fluorescent probes with the two excitation bands, which
showed large excitation separations and comparable excita-
tion intensities.[15] Thus, coumarin–rhodamine FRET dyes
are suitable as platforms for the development of dual-excita-
tion ratiometric fluorescent probes. In this work, water-solu-
ble coumarin–rhodamine FRET dyes 3a/b with an optically
tunable hydroxyl group were synthesized as the platform for
the construction of new dual-excitation ratiometric fluores-
cent H2S probes Ratio-H2S 1/2. Furthermore, the H2S-pro-
moted thiolysis of dinitrophenyl ether was employed for the
design of the probes Ratio-H2S 1/2.[12e,f] Based on the struc-
ture–excitation property relationship of dyes 3a/b,[15] we en-
visioned that probes Ratio-H2S 1/2 and dyes 3a/b would dis-
play distinct excitation intensity profiles. This may serve as
the basis for ratiometric fluorescence sensing of H2S in
living cells. The compounds Ratio-H2S 1/2 were readily pre-
pared by reaction of compounds 3a/b with 2,4-dinitrofluoro-
benzene under basic conditions in moderate yield
Figure 1. Fluorescence excitation spectral changes of Ratio-H2S 1 (3 mm)
in the presence of increasing concentrations of NaHS (0–50 equiv) moni-
tored at l=590 nm. The inset shows the ratiometric response (I402/I570) to
varying concentrations of NaHS (0–60 equiv) monitored at l=590 nm.
Each spectrum was acquired 30 min after H2S addition at 258C.
Figure 1 shows the changes in the dual-excitation spectra of
probe Ratio-H2S 1 to various H2S concentrations, monitored
at the rhodamine emission. The probe displayed a strong ex-
citation band at lꢀ570 nm and almost no excitation band at
lꢀ402 nm. Upon excitation of probe Ratio-H2S 1 at l=
402 nm, only a slight fluorescence emission band of rhoda-
mine (lꢀ590 nm) was observed (Figure S2 in the Support-
ing Information);, however, upon excitation of probe Ratio-
H2S 1 at l=560 nm, a strong fluorescence emission at l
ꢀ590 nm was observed (Figure S3 in the Supporting Infor-
mation). Because the absorption and excitation wavelengths
of the hydroxycoumarin moieties were blueshifted after al-
kylation, non-FRET was observed upon excitation at l=
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L. Yuan and Q.-P. Zuo
402 nm (Scheme S1 in the Supporting Information). With in-
creased NaHS concentration, the excitation peak at l
ꢀ402 nm, which was ascribed to the phenolate form of the
hydroxycoumarin unit, was formed and gradually increased
upon monitoring at l=590 nm. In addition, upon excitation
at l=402 nm, the fluorescence emission band of rhodamine
at lꢀ590 nm gradually increased (Figure S2 in the Support-
ing Information). As the dinitrophenyl ether group in hy-
droxycoumarin moieties was deprotected and the absorb-
ance at the excitation wavelength of the phenolate form of
the hydroxycoumarin moieties increased (Figure S1 in the
Supporting Information), FRET took place from hydroxy-
coumarin to rhodamine upon excitation at l=402 nm
(Scheme S1 in the Supporting Information). In contrast, the
excitation band centered at lꢀ570 nm, which is attributed
to the rhodamine moiety, is almost unchanged and can serve
as a reference signal. Thus, the ratios of fluorescence intensi-
ties at l=402 and 570 nm (I402/I570) exhibited a drastic
change from 0.048 in the absence of H2S to 0.36 in the pres-
ence of 500 mm H2S (Figure 1a), which was a 7.5-fold varia-
tion in the excitation ratios. In addition, probe Ratio-H2S
1 showed a good linearity between the ratios (I402/I570) and
concentrations of NaHS in the range from 1 to 50 mm (Fig-
ure S4 in the Supporting Information) with a detection limit
0.5 mm (signal to noise (S/N)=3).
under pseudo-first-order conditions give an observed rate
constant of kobs =0.069 minÀ1 (Figure S8 in the Supporting
Information). In addition, the excitation ratio at l=402 and
570 nm (I402/I570) of probe Ratio-H2S 1 is stable over a pH
region of 4–9 (Figure S8 in the Supporting Information).
Upon the addition of sulfite, the excitation intensity ratio of
probe Ratio-H2S 1 at I402/I570 shows clear changes under dif-
ferent pH values from 5.5 to 9, and displays a clear response
toward NaHS in the physiological pH region (7.0–8.0; Fig-
ure S9 in the Supporting Information). The results show that
Ratio-H2S 1 is suitable for application under physiological
conditions.
We further examined the selectivity of the probe Ratio-
H2S 1/2. As exhibited in Figure 2 and Figure S10 in the Sup-
porting Information, the introduction of GSH (5 mm) and
cysteine (0.5 mm) to probe Ratio-H2S 1 resulted in a slight
The absorption changes of the probe in the presence of
H2S (Figure S1 in the Supporting Information) are in good
agreement with the ratiometric response in the fluorescence
excitation spectra. We confirmed that the product generated
by the H2S reaction with Ratio-H2S 1 was compound 3a by
MS analysis (Figure S5 in the Supporting Information),
which was in accordance with the H2S-promoted thiolysis of
dinitrophenyl ether group.[12e,f] Similar H2S-induced changes
in the excitation spectra were also observed for probe
Ratio-H2S 2 (Figure S6 in the Supporting Information).
The time course of the fluorescence excitation ratios of
probe Ratio-H2S 1 at l=402 and 570 nm (I402/I570) in the ab-
sence or presence of H2S is shown in Figure S7 in the Sup-
porting Information. In the absence of H2S, probe Ratio-
H2S 1 exhibited no visible variations in the ratios of excita-
tion intensities at l=402 and 570 nm (I402/I570 =0.048); this
suggested that probe Ratio-H2S 1 was stable under the assay
conditions and not converted into deprotected compound
3a. However, upon addition of H2S at room temperature,
a marked increase in the ratio was observed within seconds,
and the ratio essentially reached a maximum in 20 min,
which was indicative of rapid deprotection of protected
probe Ratio-H2S 1 to give deprotected compound 3a, as an-
ticipated. With a few exceptions,[10j,11g,j,12a] tens of minutes or
hours are required for assays of many reported fluorescent
H2S probes; thus, probe Ratio-H2S 1 is faster or comparable
with most reported fluorescent H2S probes.[10–12] Because
H2S is a highly reactive species and poised to air oxidation,
probe Ratio-H2S 1 can react with NaHS within seconds,
which indicates that probe Ratio-H2S 1 may be able to trap
H2S in real biological systems. Kinetics measurements of the
reaction of probe Ratio-H2S 1 (3 mm) with NaHS (200 mm)
Figure 2. Fluorescence response of probe Ratio-H2S 1 (3m) to various
species (0.5 mm NO2À, N3À, HCO3À, K+, Na+, Ca2+, Zn2+, Mg2+, ClÀ,
Cys, FÀ, BrÀ, and IÀ; 5 mm glutathione (GSH); 150 mm NaHS and other
analytes tested) in PBS (pH 7.8, containing 5% EtOH and 3 mm CTAB).
0: free Ratio-H2S 1; 1: HCO3À; 2: N3À; 3: ClOÀ; 4: tBuOOH; 5: SO32À; 6:
S2O32À; 7: SCNÀ; 8: NO2À; 9: H2O2; 10: NO; 11: O2À; 12: FÀ; 13: ClÀ; 14:
BrÀ; 15: IÀ; 16: lipoic acid; 17: Ca2+; 18: Mg2+; 19: Zn2+; 20: K+; 21:
Na+; 22: Fe3+; 23: GSH; 24: Cys; 25: NaHS; 26: NaHS+GSH; 27:
NaHS+Cys.
increase of the excitation ratio (I402/I570); however, H2S in-
duced a significant increase in the excitation ratio (I402/I570).
In contrast, other biologically relevant analytes, RSS (lipoic
acid, SO32À, S2O32À, SCNÀ), ROS/RNS (ClOÀ, tBuOOH,
À
À
NO2 , H2O2, NO, O2 ), and metal ions and anions (Ca2+
,
À
À
K+, Na+, Fe3+, Zn2+, Mg2+, HCO3 , N3 , FÀ, ClÀ, BrÀ, and
IÀ), tested elicited no visible changes in the excitation ratio
(I402/I570). In addition, probe Ratio-H2S 1 also displayed
a large excitation ratio (I402/I570) response upon the addition
of 150 mm NaHS in the presence of GSH (5 mm) or Cys
(0.5 mm; Figure 2). Similar selectivity was also observed for
probe Ratio-H2S 2 (Figure S11 in the Supporting Informa-
tion). These data demonstrate that probes Ratio-H2S 1/2
have a high selectivity for H2S over various biologically rele-
vant analytes tested. In addition, probes Ratio-H2S 1/2 still
display high selectivity for H2S over thiols (Cys and GSH)
in the absence of CTAB (Figure S12 in the Supporting Infor-
mation).
The favorable fluorescence properties, appropriate amphi-
pathicity, and low cytotoxicity (Figure S13 in the Supporting
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L. Yuan and Q.-P. Zuo
Information) of probe Ratio-H2S 1 prompted us to test its
potential use for H2S ratiometric imaging in living cells. It
was reported that positively charged dyes were inclined to
localize in the mitochondria.[11g,h,17] In addition, H2S was re-
cently shown to be produced in mitochondria through me-
tabolism.[2c] Therefore, it is necessary to investigate whether
Ratio-H2S 1 can localize in the mitochondria. MCF-7 cells
were simultaneously stained with probe Ratio-H2S 1 and
MitoTracker Green at 378C for 20 min. The colocalization
confirmed that probe Ratio-H2S 1 was mainly localized in
the mitochondria of live cells (Figure 3).
Figure 4. Bright-field and dual-excitation confocal fluorescence images of
MCF-7 cells: a) bright-field image of MCF-7 cells only incubated with
probe Ratio-H2S 1 (3 mm) for 10 min; b) fluorescence image of a) excited
at lꢀ405 nm and an emission scan range of l=580–650 nm; c) fluores-
cence image of a) excited at lꢀ559 nm and an emission scan range of
l=580–650 nm; d) bright-field image of MCF-7 cells pretreated with
NaHS (100 mm) for 30 min at 378C, washed with PBS to remove the re-
maining NaHS, and further incubated with probe Ratio-H2S 1 (3 mm) for
10 min; e) fluorescence image of d) excited at lꢀ405 nm and an emission
scan range of l=580–650 nm; f) fluorescence image of d) excited at l
ꢀ559 nm and an emission scan range of l=580–650 nm. Scale bar:
10 mm. (For a color version, see Figure S15 in the Supporting Informa-
tion.)
Figure 3. Bright field (a) and confocal fluorescence (b–c) images of
MCF-7 cells. Cells were incubated with 3 mm Ratio-H2S 1 and 200 nm Mi-
toTracker Green at 378C for 20 min in Dulbeccoꢀs modified Eagle
medium (DMEM) media supplemented with 10% fetal bovine serum
(FBS): b) MitoTracker Green with excitation at l=488 nm and a scan
range of l=520–550 nm; c) probe Ratio-H2S 1 with excitation at l=
559 nm and a scan range of l=580–650 nm; d) overlay of b) and c);
e) overlay of a), b), and c); f) colocalization coefficient (Pearsonꢀs coeffi-
cient) of Ratio-H2S 1 and MitoTracker Blue of 0.90. Scale bar: 10 mm.
(For a color version, see Figure S14 in the Supporting Information.)
Conclusions
We developed Ratio-H2S 1/2 as the first FRET-based dual-
excitation ratiometric fluorescent probes for H2S. Upon ad-
dition of H2S, the excitation band at lꢀ402 nm of the hy-
droxycoumarin unit drastically increased, whereas the exci-
tation band centered at lꢀ570 nm from rhodamine stayed
constant and served as a reference signal; thus resulting in
the ratiometric detection of H2S. Favorable properties of the
probe include the donor and acceptor excitation bands,
which exhibited large excitation separations and comparable
excitation intensities, high sensitivity and selectivity. In addi-
tion, we demonstrated that the probe could localize in the
mitochondria and determined H2S in living cells. It is ex-
pected that this strategy will lead to the development of
a wide range of mitochondria-targetable dual-excitation ra-
tiometric probes for other analytes with outstanding spectral
features, including large separations between the excitation
wavelengths and comparable excitation intensities.
To preliminarily illustrate the utility of the novel probes
for fluorescence imaging in living cells, MCF-7 cells incubat-
ed with Ratio-H2S 1 for 10 min at 378C provide slight fluo-
rescence (Figure 4b) when excited at l=405 nm. However,
when the cells were pretreated with NaHS (100 mm) for
30 min and then further treated with Ratio-H2S 1, there was
a strong fluorescence (Figure 4e) when excited at l=
405 nm. In contrast, when excited at l=559 nm, the bright-
ness of the red fluorescence was almost unchanged in the
absence or presence of H2S (Figure 4c and f). These data
are in good agreement with H2S-induced variations of the
emission intensity of probe Ratio-H2S 1, as illustrated in Fig-
ures S2 and S3 in the Supporting Information. The ratiomet-
ric fluorescence images are shown in Figure S16 in the Sup-
porting Information. Thus, the results indicate that probe
Ratio-H2S 1 is cell membrane permeable and may be em-
ployed for ratiometric fluorescence imaging of H2S in living
cells.
Experimental Section
Materials and Instruments
Unless otherwise stated, all reagents were purchased from commercial
suppliers and used without further purification. Solvents were purified by
standard methods prior to use. Low-resolution mass spectra were per-
formed by using an LCQ Advantage ion trap mass spectrometer from
Thermo Finnigan or Agilent 1100 HPLC/MSD spectrometer. High-reso-
lution mass spectrometry (HRMS) analyses were performed on a Finni-
gan MAT 95 XP spectrometer. NMR spectra were recorded on an
INOVA-400 spectrometer, with tetramethylsilane (TMS) as an internal
standard. Electronic absorption spectra were obtained on a LabTech UV
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L. Yuan and Q.-P. Zuo
Power spectrometer. Photoluminescent spectra were recorded with a HI-
TACHI F4600 fluorescence spectrophotometer. Imaging of the cells was
performed with an Olympus FV1000 confocal microscope. The pH meas-
urements were performed on a Mettler-Toledo Delta 320 pH meter. TLC
analyses were performed on silica gel plates and column chromatography
was conducted over silica gel (mesh 200–300), both of which were ob-
tained from Qingdao Ocean Chemicals.
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Synthesis of Ratio-H2S 1/2
Compound 4 (44.5 mg, 0.24 mmol) and Cs2CO3 (78.6 mg, 0.24 mmol)
were added to a solution of 3a/b (108.0 mg, 0.12 mmol) in anhydrous
CH2Cl2 (6 mL) at room temperature, and then the reaction mixture was
stirred at room temperature for 6 h under a nitrogen atmosphere. The
solvent was removed under reduced pressure and the resulting residue
was subjected to column chromatography on silica (CH2Cl2 to CH2Cl2/
CH3OH=9: 1) to yield Ratio-H2S 1/2 as a red powder.
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Ratio-H2S 1: Yield: 69 mg, 64%; 1H NMR (400 MHz, CD3OD): d=1.25
(t, J=6.8 Hz, 12H), 3.50-3.83 (16H), 6.92 (s, 2H), 7.01 (2H), 7.13 (d, J=
7.6 Hz, 1H), 7.14 (s, 1H), 7.35 (2H), 7.38 (d, J=9.2 Hz, 1H), 7.52–7.54
(2H), 7.69–7.71 (2H), 7.77 (d, J=9.2 Hz, 1H), 8.12 (s, 1H), 8.45 (dd, J=
9.2, 2.4 Hz, 1H), 8.85 ppm (d, J=2.4 Hz, 1H); 13C NMR (100 MHz,
CD3OD): d=13.8, 24.7, 31.7, 47.9, 44.2, 98.5, 109.1, 115.4, 116.6, 118.3,
124.1, 124.2, 125.4, 129.8, 131.5, 132.2, 133.4, 133.9, 136.4, 139.3, 143.3,
145.4, 145.8, 155.6, 157.9, 158.2, 158.6, 160.5, 161.1, 166.9, 172.7 ppm; MS
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+
(ESI): 865.3 [M]+; HRMS (ESI): m/z calcd for C48H45N6O10 [M]+:
865.3192; found: 865.3197.
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Ratio-H2S 2: Yield: 64 mg, 59; 1H NMR (400 MHz, CD3OD): d=1.24 (t,
J=7.2 Hz, 12H), 3.18 (1H), 3.33-3.55 (7H), 3.63 (q, J=7.2 Hz, 12H),
6.91 (2H), 6.98–7.03 (2H), 7.12 (s, 2H), 7.18–7.25 (2H), 7.38 (t, J=
8.4 Hz, 1H), 7.46 (s, 1H), 7.62–7.72 (4H), 8.04 (d, J=7.6 Hz, 1H), 8.45
(t, J=7.2 Hz, 1H), 8.85 ppm (s, 1H); 13C NMR (100 MHz, CD3OD): d=
13.9, 24.0, 43.7, 47.9, 98.4, 109.0, 115.8, 116.5, 118.1, 118.2, 124.1, 125.2,
130.0, 131.5, 132.4, 132.8, 133.4, 134.2, 137.5, 143.3, 145.3, 145.9, 155.6,
157.9, 158.2, 161.2, 166.7, 170.6 ppm; MS (ESI): 865.3 [M]+; HRMS
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+
(ESI): m/z calcd for C48H45N6O10 [M]+: 865.3192; found: 865.3186.
MCF-7 Cell Culture and Imaging
MCF-7 cells were obtained from Xiangya Hospital and cultured in
DMEM supplemented with 10% FBS in an atmosphere of 5% CO2 and
95% air at 378C. For imaging studies, the cells were plated and allowed
to adhere for 24 h. Immediately before the experiments, the cells were
washed with PBS, followed by incubation with probe Ratio-H2S 1 (3 mm)
for 10 min at 378C (in PBS containing 0.5% EtOH). For intracellular
H2S imaging, the cells were incubated with NaHS (100 mm) in PBS at
378C for 30 min. After removal of excess NaHS and washing with PBS,
the cells were incubated with probe Ratio-H2S 1 (3 mm) for 10 min at
378C (in PBS containing 0.5% EtOH). Cell imaging was performed after
washing the cells three times with PBS.
Cytotoxicity Assays
MCF-7 cells were cultured in DMEM containing 10% FBS supplement-
ed with 100 UmLÀ1 of penicillin and 100 mgmLÀ1 streptomycin in an at-
mosphere of 5% CO2 and 95% air at 378C. The cells were seeded into
96-well plates and then 1.0, 2.0, 5.0, 10.0, or 15.0 mm (final concentration)
of Ratio-H2S 1 (99.9% DMEM and 0.1% DMSO) was added (n=6).
Subsequently, the cells were incubated at 378C in an atmosphere of 5%
CO2 and 95% air for 24 h. An untreated assay with DMEM (n=6) was
also performed under the same conditions.
Acknowledgements
This work was financially supported by the NSFC (21302050), the Hunan
Provincial Natural Science Foundation of China (grant no. 14JJ2047),
and the Young Teachersꢀ Growth Plan of Hunan University.
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Received: January 28, 2014
Revised: February 17, 2014
Published online: April 1, 2014
Chem. Asian J. 2014, 9, 1544 – 1549
1549
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim