A high signal-to-background ratio H2S-specific fluorescent probe based on nucleophilic substitution and its bioimaging for generation H2S induced by Ca2+ in vivo

Article abstract of DOI:10.1016/j.dyepig.2019.107755

Hydrogen sulfide (H₂S), a new endothelium-derived relaxing factor (EDRF), which plays vital roles in regulating intracellular redoxstatus and other fundamental signaling processes involved in human health and disease. In this work, we designed a fluorescent probe for H₂S successive nucleophilic reaction with high signal-to-background (S/B) ratio. In the probe, we utilized 2-mercaptobenzoic acid as the distinguish reaction site and introduced 2,4-dinitrophenyl to provide appropriate steric hindrance to realize specific response on well-structured H₂S. Other substances sulfur-containing do not disturb the detection of H₂S. The max emission with 197-fold enhancement exhibits high signal-to-background ratio. The detection limit was calculated to be 64 nM. Imaging assays in living cells showed that the probe could penetrate cells membrane easily and allowed to detect endogenous generation H₂S induced by Ca²⁺ mediated cystathionine γ-lyase (CSE). Moreover, the probe was successfully used to detect exogenous H₂S in mice.

Full text of DOI:10.1016/j.dyepig.2019.107755

Accepted Manuscript
A high signal-to-background ratio H S-specific fluorescent probe based on
2
2+
nucleophilic substitution and its bioimaging for generation H S induced by Ca in
2
vivo
Jin Kang, Fangjun Huo, Yishan Yao, Caixia Yin
PII:
S0143-7208(19)31172-6
DOI:
https://doi.org/10.1016/j.dyepig.2019.107755
Article Number: 107755
Reference:
DYPI 107755
To appear in: Dyes and Pigments
Received Date: 22 May 2019
Revised Date: 17 July 2019
Accepted Date: 25 July 2019
Please cite this article as: Kang J, Huo F, Yao Y, Yin C, A high signal-to-background ratio H S-specific
2
fluorescent probe based on nucleophilic substitution and its bioimaging for generation H S induced by
2
2+
Ca in vivo, Dyes and Pigments (2019), doi: https://doi.org/10.1016/j.dyepig.2019.107755.
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Highlights
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1. This probe exhibit high signal-to-background ratio in response to H2S.
2. This probe was used to detect endogenous generation H2S induced by Ca2+
mediated cystathionine γ-lyase (CSE).
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3. This probe was successfully used to detect exogenous hydrogen sulfide in mice.
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Abstract Graphic
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A novel fluorescence H2S probe based on nucleophilic substitution reactions, which
with high signal-to-background ratios. This probe was successfully used to imaging
ex-/endogenous H2S in living cells, and exogenous H2S in mice.
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52 A high signal-to-background ratio H2S-specific fluorescent probe
53 based on nucleophilic substitution and its bioimaging for generation
54 H2S induced by Ca2+ in vivo
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Jin Kang,1a Fangjun Huo, 1b Yishan Yao,1c Caixia Yina,*
a Institute of Molecular Science, Key Laboratory of Materials for Energy Conversion
and Storage of Shanxi Province, Shanxi University, Taiyuan 030006,
b Research Institute of Applied Chemistry, Shanxi University, Taiyuan, 030006, China.
cState Key Laboratory of Tocicology and Medical Countermeasures, Beijing Institute
of pharmacology and Toxicology, No.27 Taiping Road, Haidian District,
Beijing,100850, P. R. China.
*Corresponding author: C.X. Yin, E-mail: yincx@sxu.edu.cn, Tel/Fax:
+86-351-7011022.
64 Abstract Hydrogen sulfide (H2S), a new endothelium-derived relaxing factor
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(EDRF), which plays vital roles in regulating intracellular redoxstatus and other
fundamental signaling processes involved in human health and disease. In this work,
we designed a fluorescent probe for H2S successive nucleophilic reaction with high
signal-to-background (S/B) ratio. In the probe, we utilized 2-mercaptobenzoic acid as
the distinguish reaction site and introduced 2,4-dinitrophenyl to provide appropriate
steric hindrance to realize specific response on well-structured H2S. Other substances
sulfur-containing do not disturb the detection of H2S. The max emission with 197-fold
enhancement exhibits high signal-to-background ratio. The detection limit was
calculated to be 64 nM. Imaging assays in living cells showed that the probe could
penetrate cells membrane easily and allowed to detect endogenous generation H2S
induced by Ca2+ mediated cystathionine γ-lyase (CSE). Moreover, the probe was
successfully used to detect exogenous H2S in mice.
Keywords: H2S-specific; Two nucleophilic; Ca2+ mediated; Vivo
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1. Intruduction
H2S is extensively exist in a wide variety of industries such as oil exploitation,
natural gas well, tunnel and sewage disposal [1-4]. Exposure to high level H2S
atmosphere may cause respiratory symptoms, cardiovascular abnormalities and
neurological disorders [5-7]. H2S was found as the third multifunctional
gasotransmitter along with nitric oxide (NO) and carbon monoxide (CO) since last
decades [8-10]. H2S influences a wide range of physiological and pathophysiological
processes, including its ability to act as a neurotransmitter [11], regulator of blood
pressure [12], immunomodulator [13] or anti-apoptotic agent [14], together with its
great pharmacological potential, such as cardioprotection [15], endogenous
stimulation of angiogenesis [16] and mitochondrial bioenergetics [17]. However,
abnormally H2S concentrations have been significant associated with alzheimer's
disease, down's syndrome, diabetes and liver cirrhosis [18-21]. Physiological effects
of H2S are concentration-dependent, thus, in order to benefit from H2S, the
concentration of H2S must be maintained at the appropriate level. In mammals,
endogenous H2S is synthesized naturally by several enzymes, including cystathionine
γ-lyase (CSE), cystathionine β-synthetase (CBS) and 3-mercaptopyruvate
sulfurtransferase (MST)/cysteine aminotransferase (CAT) [22-25]. Yang et al. reported
that the activity of cystathionine γ-lyase (CSE), an important enzyme responsible for
H2S physiological generation in animals, could be activated by calcium-calmodulin
pathway [26]. However, the mechanism of Ca2+ induced intracellular H2S production
in living cells has not been elucidated. Therefore, developing selective and sensitive
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detection tools for H2S in complex biological systems is important.
Traditional methods of H2S detection, including methylene blue method [27], lead
acetate [28], electrochemical sensors [29], gas chromatography [30] and
monobromobimane derivatization are often limited by poor compatibility with
biosystem, limited temporal resolution or rigorous preparation requirements [31-32].
In contrast, fluorescent probes with highly sensitive, selective, nondestructive are
suitable for detection of this volatile, gaseous molecule with readily available
instruments [33-36]. In the past several years, a number of fluorescent probes for H2S
have been reported [37-41]. However, probes with high signal-to-background (S/B)
ratio are rare. In bio-imaging, the probe with high S/B ratio is benefit for obtaining
more accurate and reliable signals. There are two ways to improve the probe’s S/B
ratio: (1) Increase extent of fluorescence enhancement; (2) Improve selectivity.
Moreover, the application of fluorescent probes to the H2S signaling pathway is still
rare.
The 2,4-dinitrophenyl (DNP) ether moiety has been employed extensively as a
protecting group for tyrosines in peptide synthesis [42]. Direct assemble the DNP
group onto various kinds of fluorophores have yielded a series H2S probes [43-47].
Indeed, biothiols can also remove DNP group, the selectivity of these probes are
unsatisfactory [48-49]. In this work, we employed the DNP derivative
2-((2,4-dinitrophenyl)thio)benzoic acid as the masking group link to the fluorescein
and obtained the probe (Probe 1) (Scheme 1). The probe exhibits high selectivity and
significant fluorescence enhancement up to 197-fold upon the detection of H2S.

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Furthermore, Ca2+ induced H2S production in Hela cells was directly visual
demonstration use the probe.
2. Preparation and characterization of compounds
2.1. Preparation of Probe 1.
The Probe 1 was synthesized from the compound 1 via an ester moiety link to
fluorescein (Scheme 1). The compound 1 was synthesized use the same methods as
previous literature [50].
<Inserted Scheme 1>
Compound 1 (1.0 mmol, 0.32 g) and fluorescein (1.0 mmol, 0.33 g) were dissolved
into 25 mL dichloromethane, added N-(3-dimethylaminopropyl)-N-ethyl carbodi
imide hydrochloride (EDC) (1.0 mmol, 0.19 g) and 4-dimethylaminopyridine (DMAP)
(0.10 mmol, 0.01 g). Surrounded by Ar, the reactant stired overnight (r.t.). Then
solvent was evaporated and resulted residue was subjected to column chromatography.
Probe was obtained as a yellow powder (0.19 g, yield: 30 %).
2.2. Characterization of Probe 1
1H NMR (600 MHz, DMSO-d6) δ 8.88 (d, J = 2.5 Hz, 1H), 8.34 (t, J = 8.0 Hz, 3H),
8.05 (d, J = 7.6 Hz, 1H), 7.85 (d, J = 5.7 Hz, 4H), 7.80 (t, J = 7.5 Hz, 1H), 7.75 (t, J =
7.3 Hz, 1H), 7.37-7.34 (m, 2H), 7.15 (d, J = 9.0 Hz, 2H), 6.96 (d, J = 8.7 Hz, 2H),
6.91 (d, J = 8.7 Hz, 2H). 13C NMR (150 MHz, DMSO-d6) δ 163.4, 151.5, 150.6,
144.9, 144.4, 144.3, 137.5, 134.4, 133.5, 132.1, 131.3, 130.2, 129.9, 129.3, 127.6,
124.9, 123.9, 121.0, 118.3, 116.5, 110.3. ESI-MS m/z: [probe + H]+ Calcd for
635.07604, Found 635.07525; [probe + Na]+ Calcd for 657.05804, Found 657.05683.

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3. Results and Discussion
3.1 Specta properties of Probe 1 response to H2S.
Firstly, we evaluated the UV-vis spectra properties of Probe 1. As shown in Fig. 1,
in 2 mL DMSO : PBS (v/v, 7 : 3, pH = 7.4) solution of Probe 1 (25 µM), gradually
added HS- (0-300 µM), the absorbance at 338 nm increased with concomitant
appearance of absorbance at 475 nm in a short time frame (10 min). Similarly, we also
studied the reaction beween Probe 1 and biothiols (Cys, Hcy and GSH) in UV-vis
spectrometer (Fig. S6 a, b, c). Under the same conditions, the addition of Cys (500
µM) caused a neglectable absorbance enhancement at 338 nm, and Hcy (500
µM)/GSH (500 µM) couldn’t cause obvious changes in UV-vis spectra.
<Inserted Figure 1>
The Probe 1 is considered to be a high S/B ratio probe. On the one hand, high S/B
ratio probe depend on the extent of fluorescence enhancement. As predicted, the
incremental addition of HS- (0-70 µM) to the solution DMSO : PBS (v/v, 7 : 3, pH =
7.4) of Probe 1 (5 µM) resulted in a dramatic enhancement of the emitting band
centered at 538 nm in 10 min (Fig. 2a). Finally, the max emission has 197-fold
enhancement. On the other hand, the high selectivity is very important condition for
high S/B ratio probe. The Probe 1 was treated with HS- and various relevant analyte
(100 eq.) in DMSO : PBS (v/v, 7 : 3, pH = 7.4). As shown in Fig. 2b, the Probe 1 was
highly selective for HS- versus biological relevant thiols or HSO3 . These results
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demonstrate the excellent selectivity of the probe to H2S. These above experiment
results, the emission of Probe 1 has a large enhancement (197-fold) and the high

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selective of the Probe 1 fulfilled the requirements of high S/B ratio probe. Hence, the
probe is a H2S-specific fluorescent probe with high S/B ratio.
<Inserted Figure 2>
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3.2 Working curve and time dependents
Furthermore, a plot of fluorescent intensities at 538 nm versus the concentrations of
H2S showed a good linearity (R2 = 0.991): F-F0 = 94.24c - 293.60 (Fig. 3a). With the
definition: Detection limit = 3σ/k, the detection limit evaluated as 64 nM. In order to
evaluate the response speed of Probe 1 towards H2S, we examined the reactivity of
probe (5 µM) towards the HS- (10 eq.) through time-dependent fluorescence
spectroscopy in DMSO : PBS (v/v, 7 : 3, pH = 7.4). As the Fig. 3b depicted, the
reaction could be completed within 10 min.
<Inserted Figure 3>
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3.3. Proposed mechanism
The proposed mechanism of Probe 1 was depicted in the Scheme 2. The H2S
nucleophilic substitution the carbonyl group of Probe 1, result in the protect group
cleaved and released the fluorescein and compound 2 [51]. Furthermore, the
mechanism was proved by the ESI-MS spectra analysis (Fig. S9). ESI-MS of a
solution mixture of Probe 1 and NaHS exhibited m/z peaks at 331.0611, 198.9816 in
accordance with the fluorescein and compound 2 respectively.
<Inserted Scheme 2>
3.4 Cytotoxicity experiments
HepG2 cells were cultured in 96-well plates. The cell number was determined and a

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serial dilution on Probe 1 was performed in culture medium (without serum),
incubated for 5 or 10 h. Subsequently, CCK-8 (10 % in serum free culture medium)
was added to each well, which was washed with PBS two times and the plate was
incubated for another 1 h. Then measure optical densities at 450 nm. Cytotoxicity
assay demonstrating the Probe 1 was benign to cells and has the potential to be used
in biological applications (Fig. S8).
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3.5. Imaging of living cells
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The HepG2 cells incubated with Probe 1 (5 µM) only for 20 min showed no
fluorescence in the green channel (Fig. 4 a1). In contrast, treatment of Probe 1-loaded
cells with 20 µM NaHS for 20 min triggered an obvious increased in green
fluorescence signal (Fig. 4 b1).
<Inserted Figure 4>
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Encouraged by its good sensing performances for H2S in vitro, we applied Probe 1
to image the endogenously generated H2S by exogenous compound stimulation.
S-Nitroso-N-acetyl-DL-penicillamine (SNP) was used to stimulate the generation of
endogenous H2S in HepG2 cells. As we expected, after incubation with 10 µM SNP
for 1 h, the fluorescence intensities from the green channel clearly increased, which
are similar to that of the addition of exogenous NaHS (Fig. 5 d1),. These results
indicated that Probe 1 capable of visualization of endogenous H2S generation in
HepG2 cells. These cell experiments showed the Probe 1 can thus be used to imaging
ex/endogenous H2S in living cells.
<Inserted Figure 5>

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3.6. Imaging the Ca2+-dependent H2S
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Ca2+ is the second messenger and involved in a variety of intracellular signaling
pathways. According to reported, that calcium-calmodulin regulating the activity of
cystathionine γ-lyase (CSE) responsible for H2S physiological generation in animals.
To demonstrate the mechanism of Ca2+ induced intracellular H2S production in cells,
we conducted imaging experiments with Probe 1 in Hela cells under conditions of
both negative and positive controls. No fluorescent signal was observed in green
channel when the cells were pre-incubated with DL-propargylglycine (PAG, which
suppresses CSE) along with Probe 1 and Ca2+. In contrast, obvious fluorescent was
observed when the cells were pre-incubated with Ca2+ and Probe 1, or cells
pre-incubated with aminooxyacetic acid (AOAA, a potent inhibitor of CBS) along
with Probe 1 and Ca2+. This indicated that CSE contributes to the observed H2S
generation upon Ca2+ stimulation.
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<Inserted Figure 6>
3.7. Imaging of living mice
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Furthermore, we applied Probe 1 imaging in mice. The Kunming mice were fed
commercial mouse chow in individual cages and left to freely wander in their housing
for two weeks with 12 h dark/light cycles for acclimatization before the experiment. A
living animal imaging system is used in imaging the mice. After all the preparatory
work was completed, the mice were under anesthesia with 100 mL pentobarbital
intraperitoneal injection. As Fig. 7 shows, the mice were injected with 50 mM of

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Probe 1 in the abdomen, HS- was carefully injected into the same location. Then, the
fluorescence images were recorded at different periods of time (0, 5, 10, 15 min).
These results displayed that Probe 1 could visualize the endogenous H2S in vivo.
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<Inserted Figure 7>
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4. Conclusions
In conclusion, we prepared a fluorescein-based H2S-specific probe Probe 1 with
high S/B ratio. The results of UV-vis spectrum and fluorescence emission spectrum
studies showed that Probe 1 had good selectivity for H2S and having a 64 nM
detection limit for H2S. Kinetics studies showed that the response can be completed in
10 min. Furthermore, this probe was used to directly image Ca2+-dependent H2S
production, and in vivo imaging.
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5. Acknowledgments
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We thank the National Natural Science Foundation of China (No. 21775096,
21302223), Shanxi Province "1331 project" key innovation team construction plan
cultivation team (2018-CT-1), Shanxi Province Foundation for Returness (2017-026),
Shanxi Province Science Foundation for Youths (201701D221061), Scientific
Instrument Center of Shanxi University (201512).
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[45] Liu HY, Zhao M, Qiao QL, Lang HJ, Xu JZ, Xu ZC. Fluorescein-derived
fluorescent probe for cellular hydrogen sulfide imaging. Chinese Chem. Lett
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[51] Chen W, Rosser EW, Matsunaga T, Pacheco A, Akaike T, Xian M. The

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development of fluorescent probes for visualizing intracellular hydrogen
polysulfides. Angew. Chem. Int. Ed 2015;54:13961-13965.
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Figure captions
Scheme 1 Synthesis of Probe 1.
Figure 1 UV-vis responses of Probe 1 (25 µM) in DMSO : PBS (v/v, 7 : 3, pH = 7.4)
solution, added HS- (0-300 µM).
Figure 2 (a) fluorescent responses of Probe 1 (5 µM) toward HS-; Inset: Visual
fluorescence change photograph for Probe 1 only and upon addition of NaHS under
illumination with a 365 nm UV lamp; (b) the selective of Probe 1 (5 µM) towards HS-
and various relevant analyte (100 eq.), λex = 512 nm, slit: 5 nm/5 nm.
Figure 3 (a) The working curve of Probe 1 (5 µM) in the presence of various
concentrations of HS-(0-70 µM); (b) Time dependent of Probe 1 (5 µM) to HS- (10
eq.). λex = 512 nm, slit: 5 nm/5 nm.
Scheme 2 The mechanism of the Probe 1 responsing to HS-.
Figure 4 Confocal fluorescence imaging the H2S in HepG2 cells of Probe 1 20 min:
(a1-a3) Incubated with the Probe 1 only: (a1) green channel; (a2) brightfield image;
(a3) Overlay. (b1-b3) Incubated with the Probe 1 20 min, then treat with HS- 20 min:
(b1) green channel; (b2) brighfield image; (b3) Overlay. Excitation at 488 nm, the
green channel was set at 545 ± 15 nm scale bar = 20 µm.
Figure 5. Confocal fluorescence imaging the endogenously generated H2S in HepG2
cells of Probe 1: (c1-c3) Incubated with the Probe 1 only: (c1) green channel; (c2)
brightfield image; (c3) Overlay. (d1-d3) Incubated with the SNP for 1 h, and then
incubated with Probe 1 for 20 min: (d1) green channel; (d2) brighfield image; (d3)
Overlay. Excitation at 488 nm, the green channel was set at 545 ± 15 nm, scale bar =

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20 µm.
Figure 6. CLSM images of Ca2+ regulated H2S production pathways. (e1-e3)
Incubated with the Probe 1 only: (e1) green channel; (e2) brightfield image; (e3)
Overlay. (f1-f3) Incubated with CaCl2 (200 µM, 1 h), then incubated with Probe 1 20
min: (f1) green channel; (f2) brighfield image; (f3) Overlay. (g1-g3) pre-incubated
with AOAA (100 µM, 3 h), incubated with CaCl2 (200 µM, 1 h), then incubated with
Probe 1 20 min: (g1) green channel; (g2) brighfield image; (g3) Overlay. (h1-h3)
pre-incubated with PAG (100 µM, 3 h), incubated with CaCl2 (200 µM, 1 h), then
incubated with Probe 1 20 min: (h1) green channel; (h2) brighfield image; (h3)
Overlay. Excitation at 488 nm, the green channel was set at 545 ± 15 nm scale bar =
20 µm.
Figure 7. The in vivo imaging of H2S in a nude mice model of Probe 1 (20 µM). (a)
Control group; (b, c, d) First injected 40 µM Probe 1 solution, then injected 50 µM
HS- solution for 5, 10, 15 min. Excitation at 475 nm, the green channel was collected
at 520±5 nm.

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Scheme 1
COOH
F
TEA, 80oC
SH
S
+
2-propanol
COOH
O2N
NO2
O2N
NO2
Compound 1
O
O
COOH
HO
O
O
NO2
S
EDC ,DMAP
+
O
S
O
O2N
NO2
HO
O
OH
NO2
O
Probe 1
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Figure 1
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Figure 2
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Figure 3
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Scheme 2
HS-
HO
O
O
HO
O
O
NO2
SH
O
S
+
O
+
O
-
CO2
O2N
O2N
NO2
SH
O
S-
Probe 1
Compound 2
Fluorescein
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Figure 4
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Figure 5
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Figure 6
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Figure 7
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Supporting Information
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495 Table S1: Compare of reported fluorescent hydrogen sulfide probes in
recent years.
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497 I Material and Methods
498 II (Fig.S1-S5): Copies of NMR and ESI-MS of related compounds.
499 III(Fig. S6-S9): Spectroscopic studies.
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Table S1:
Properties of the reported fluorescent hydrogen sulfide probes in recent years.
probes
S/B ratio
60
detection limit
10.5 nM
response time
2.5 min
Ref.
[1]
--
--
18 nM
50 nM
30 min
<1 min
[2]
[3]
40
120 nM
15 min
[4]
200
--
90 nM
29 nM
30 nM
64 nM
15 min
5 min
[5]
[6]
[7]
--
60 min
10 min
This work
(probe 1)
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[1] Lin L, Zeng XQ, Shen YN, Zhu HL, Qian Y. An ultrasensitive fluorescent probe for rapid
determination of thiophenols. Talanta 2017;165:321-325.
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[2] Qiao Z, Zhang HY, Wang KW, Zhang YR. A highly sensitive and responsive fluorescent probe
based on 6-azidechroman dye for detection and imaging of hydrogen sulfde in cells. Talanta
2019;195:850-856.
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[3] Yang Y, Feng Y, Jiang Y, Qiu FZ, Wang YZ, Song XR, Tang XL, Zhang GL, Liu WS. A
coumarin-based colorimetric fluorescent probe for rapid response and highly sensitive detection of

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hydrogen sulfde in living cells. Talanta 2019;197:122-129.
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[4] Ma C, Wei C, Li XY, Zheng XY, Chen B, Wang M, Zhang PZ, Li XL. A mitochondria-targeted
dual-reactable fluorescent probe for fast detection of H2S in living cells. Dyes and Pigments 2019;
162:624-631.
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[5] Ismail I, Wang DW, Wang ZH, Wang D, Zhang CY, Yi L, Xi Z. A julolidine-fused
coumarin-NBD dyad for highly selective and sensitive detection of H2S in biological samples.
Dyes and Pigments 2019;163:700-706.
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[6] Chen HH, Gong XY, Liu XW, Li Z, Zhang JJ, Yang XF. A nitroso-based fluorogenic probe for
rapid detection of hydrogen sulfide in living cells. Sensors Actuators: B. Chemical
2019;281:542-548.
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[7] Huang XT, Liu HY, Zhang JW, Xiao BR, Wu FX, Zhang YY, Tan Y, Jiang YY. A novel
near-infrared fluorescent hydrogen sulfide probe for live cell and tissue imaging. New J. Chem.,
2019;43:6848-6855.
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I.
Materials and apparatus.
Reagents with analytical grades were purchased from commercial and used without
further purification.
UV-vis spectra were taken on a HITACHI U-3900 spectrophotometer and
1
fluorescence spectra were recorded using a HITACHI F-7000 spectrophotometer. H
NMR and 13C NMR experiments were measured by a Bruker AVANCE-600 MHz
spectrometer (Bruker, Billerica, MA). Electrospray ionization (ESI) mass spectra were

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acquired using an AB Triple TOF 5600plus System (AB SCIEX, Framingham, USA).
The cell imaging experiments used Zeiss LSM880 CLSM.
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Imaging Experiments
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HepG2 cells and HeLa cells were cultured in dulbecco’s modified eagle’s medium
(DMEM, Gibco) in an atmosphere of 5% CO2 and 95% air at 37 °C. Before the
CLSM imaging, the cells were plated on 14 mm glass coverslips and were incubated
overnight. The cells were washed with PBS and then incubated with Probe 1 in
DMSO/PBS (0.5 %, v/v) for 3 h at 37 °C. After washing three times, the cells were
subjected to CLSM imaging.
Imaging procedures were conducted with adult nude mice under general anesthesia
by injection of sodium pentobarbital (0.5 mL/0.03%). Images were taken using an
excitation of 475 nm and emission was collected 520±5 nm.
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Ⅱ: Copies of NMR and ESI-MS of related compounds
Fig. S1
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Figure S1. The 1H NMR (600 MHz) spectra of the compound 1 in DMSO-d6.
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Figure S2. The 1H NMR (600 MHz) spectra of the Probe 1 in DMSO-d6.
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Fig. S3