Design strategy of multifunctional and high efficient hydrogen sulfide NIR fluorescent probe and its application in vivo

Article abstract of DOI:10.1016/j.dyepig.2020.108901

With the improvement of organic synthesis technology and the development of fluorescent probe, more and more fluorescent probes with excellent performance and versatility are required, so as to better realize their application value. High sensitivity, high selectivity, targetable and fast response NIR fluorescence probes are still needed for hydrogen sulfide precise detection. In this study, phenol containing 2, 4-dinitro group was selected as a strong electrophilic group to promote the nucleophilic substitution reaction. After the nucleophilic addition reaction between probe and hydrogen sulfide with delicate structure, strong nucleophile, 2, 4-dinitrophenol departure quickly and the NIR fluorophore was released, which showed fluorescence emission at 741 nm, and the probe detection limit for hydrogen sulfide was calculated as 96 nM. Interestingly, because of the positive charge in the hemiocyanine, the probe can locate efficiently in the mitochondria. This fluorescent probe with excellent performance was used to detect the endogenous levels of hydrogen sulfide in vivo. This work provides guidance for the design of multifunctional and high-performance fluorescent probes.

Full text of DOI:10.1016/j.dyepig.2020.108901

Journal Pre-proof
Design strategy of multifunctional and high efficient hydrogen sulfide NIR fluorescent
probe and its application in vivo
Keyan Zhou, Yutao Yang, Tingting Zhou, Ming Jin, Caixia Yin
PII:
S0143-7208(20)31598-9
DOI:
https://doi.org/10.1016/j.dyepig.2020.108901
DYPI 108901
Reference:
To appear in: Dyes and Pigments
Received Date: 21 July 2020
Revised Date: 15 September 2020
Accepted Date: 28 September 2020
Please cite this article as: Zhou K, Yang Y, Zhou T, Jin M, Yin C, Design strategy of multifunctional and
high efficient hydrogen sulfide NIR fluorescent probe and its application in vivo, Dyes and Pigments,
https://doi.org/10.1016/j.dyepig.2020.108901.
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Author contributions
Keyan Zhou carried out synthesis and spectrometry.
Tingting Zhou and Ming Jin carried out cell imaging and mice imaging.
Yutao Yang and Caixia Yin’s idea and writing.

Abstract Graphic

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Design strategy of multifunctional and high efficient hydrogen sulfide NIR
fluorescent probe and its application in vivo
Keyan Zhou, a Yutao Yang, a* Tingting Zhou, a Ming Jin, a Caixia Yinb*
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a Key Laboratory of Medicinal Chemistry, and Molecular Diagnosis of the Ministry of Education,
Key Laboratory of Chemical Biology of Hebei Province, College of Chemistry & Environmental
Science, Key Laboratory of Chemical Biology of Hebei Province, Hebei University, Baoding
071002, China
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*E-mail: yutaoyang2016@163.com.
b Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education,
Institute of Molecular Science, Shanxi University, Taiyuan 030006, China
*E-mail: yincx@sxu.edu.cn.
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Abstract
With the improvement of organic synthesis technology and the development of
fluorescent probe, more and more fluorescent probes with excellent performance and
versatility are required, so as to better realize their application value. High sensitivity,
high selectivity, targetable and fast response NIR fluorescence probes are still needed
for hydrogen sulfide precise detection. In this study, phenol containing 2, 4-dinitro
group was selected as a strong electrophilic group to promote the nucleophilic
substitution reaction. After the nucleophilic addition reaction between probe and
hydrogen sulfide with delicate structure, strong nucleophile, 2, 4-dinitrophenol
departure quickly and the NIR fluorophore was released, which showed fluorescence
emission at 741 nm, and the probe detection limit for hydrogen sulfide was calculated
as 96 nM. Interestingly, because of the positive charge in the hemiocyanine, the probe
can locate efficiently in the mitochondria. This fluorescent probe with excellent
performance was used to detect the endogenous levels of hydrogen sulfide in vivo.
This work provides guidance for the design of multifunctional and high-performance
fluorescent probes.
Keywords: Fluorescent probe; Multifunctional; Efficient; Bioimaging; H2S.
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1. Introduction
Hydrogen sulfide (H2S) is recently regarded as the third gas messenger molecule
in addition to carbon monoxide (CO) and nitric oxide (NO). [1] H2S is not only
widely exists in mammals, including humans, but also plays an important in numerous
pathologic and physiologic processes.[2-5] H2S can be endogenously catalyzed by
enzymes such as cystathionine-γ-lyase (CSE), cystathionine-β-synthase (CBS), and
3-mercapto-sulfurtransferase.[6-11] As an endogenous signal molecule, H2S is
involved in vasodilation, angiogenesis, cell growth, neuromodulation, insulin signal
inhibition, inflammation and other physiological functions.[12-18] Many physical
diseases of diabetes, Alzheimer's disease, cirrhosis of the liver are now widely
accepted as being induced or related to abnormal levels of hydrogen sulfide. Recently,
the potential biological significance of H2S has attracted growing interest. As an
effective detection approach, fluorescent probe has great development potential in
visualizing H2S in biological systems due to the unique advantages of simple
operation, fast response and excellent selectivity for H2S. To date, various red emitted
or NIR fluorescent probes for H2S have been developed, but their emission
wavelengths are under 700 nm. [19-29] In recent years, some NIR fluorescent probes
were reported which the emission wavelengths are beyond 700 nm. In 2018, Ai et al.
developed a NIR fluorescent probe Imazide for H2S and the emission maxima was
733 nm.[30] Wang et al. developed a new fluorescent probe for H2S based on cyanine
dyes and the emission wavelength centered at 830 nm.[31] These probes have made a
perfect effect in detection of mitochondrial H2S in the living cells. However, the
optical properties analysis was performed in the buffer solutions with 90% glycerol or
the response time of probe to H2S is within 1 h. In 2019, Feng et al. developed a NIR
fluorescent probe for H2S with the signal at 744 nm based nitrobenzoxadiazole ether.
[32] Huang group developed a new probe DBT for detection of H2S with a significant
"turn-on" fluorescence response at 716 nm.[33] Besides, the probes were successfully
applied in imaging H2S in live cells and mice. They did not achieve detection of H2S
in mitochondria as an important organelle of the cell playing a significant role in
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pathophysiology. [34-37] Therefore, it is still need to develop some new types of
fluorescent probes for H2S.
With the above in mind, we focused on development of NIR mitochondrial
targeting fluorescent probe for detection H2S to elucidate the function of hydrogen
sulfide in mitochondria and the association between hydrogen sulfide and related
diseases in mitochondria. It is worth noting that the key to design a
mitochondria-targetable NIR fluorescent probe for H2S is to choose a mitochondrial
targeting group, the near-infrared fluorophore and a specific response site for
detection of H2S. It is generally known that many kinds of cationic dyes, such as
cyanine and rhodamine, possessing a positive charge, which can be well accumulated
in the mitochondria of cells.[38] Thus, we choose the cyanine dye Mito-OH as the
fluorophore, owing to its mitochondrial targeting and NIR emission. In addition, we
used 2,4-dinitrophenyl (DNP) ether for reaction moiety because of its sensitivity and
selectivity for H2S without the interference of biothiols.[39-45] Therefore, in this
work, we designed and synthesis of Mito-DNP with NIR emission for detection of
H2S on the basis of the thiolysis of dinitrophenyl ether via combinatorial chemistry
(Scheme 1). After treatment of H2S, Mito-DNP would occur H2S-triggered thiolysis
of dinitrophenyl ether to release Mito-OH. Moreover, the feasibility of Mito-DNP as
a fluorescent probe to monitor the level of H2S was evaluated in the HepG2 cells and
mice. These results might lead to better understand the roles of H2S in physiological
processes.
2. Experimental
2.1 Reagents and apparatus
Deionized water was used in the whole experiment and the chemicals were of
analytical grade and used directly. The ultraviolet spectra and fluorescence spectra
were measured using the Agilent Technologies UV-visible (Cary 60) and fluorescence
1
spectrophotometer (Cary Eclipse). The spectra of H NMR and 13C NMR were
recorded by BUXI-I NMR spectrometer (Wuhan Zhongke Niu-jin). The mass spectra
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were recorded on ESI mode (Bruker Ultraflex Xtreme MALDI-TOF/TOF). Live cells
imaging was observed with laser confocal microscopy (ZEISS LSM 880). Animal
imaging was taken by the PerkinElmer IVIS Spectrum live animal imaging system.
2.2 Synthesis of Mito-DNP
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The synthesis route of Mito-DNP is summarized in Scheme 1. Fluorophore Mito-OH
was synthesized according to previous research in our laboratory.[46] Mito-OH (0.29
g, 0.5 mmol) and 2,4-dinitrobromobenzene (0.25g, 1 mmol) were dissolved in
dichloromethane (10 mL), then added the K2CO3 (0.21 g, 1.5 mmol) to the above
solution and stirring at 25 for 12 h. Next, after filtration, the solvent was evaporated
to give a crude product and then it was isolated by silica gel chromatography (CH2Cl2:
MeOH=30:1) to obtain a blue-purple solid (0.14 g, 39 %). M.p. >280 . 1H NMR (400
MHz, CDCl3) δ (ppm): 8.82 (d, J = 2.2 Hz, 1H), 8.73 (d, J = 12.0 Hz, 1H, 8.48 (d, J =
7.2 Hz, 1H), 8.22-8.14 (m, 1H), 8.04 (d, J = 8.0 Hz, 1H), 7.98 (d, J = 8.0 Hz, 2H),
7.71 (d, J = 8.6 Hz, 1H), 7.67-7.61 (m, 1H), 7.21 (d, J = 5.8 Hz, 3H), 7.68 (d, J = 8.0
Hz, 1H), 7.69 (d, J = 17.2 Hz, 1H), 4.69 (s, 1H), 4.48 (s, 2H), 2.80-2.72 (m, 4H), 2.04
(s, 6H), 1.93 (s, 2H), 1.60 (d, J = 4.0 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ (ppm):
179.2, 159.1, 155.4, 154.6, 153.7, 145.3, 142.1, 139.7, 138.1, 136.6, 133.0, 131.5,
130.4, 129.7, 128.9, 128.7, 128.2, 127.5, 126.9, 126.6, 122.4, 121.9, 120.1, 116.7,
115.5, 111.6, 108.1, 105.5, 52.7, 42.2, 33.6, 29.6, 27.6, 20.1, 13.5. HRMS (ESI, m/z):
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+
Calcd for C37H32N3O6 ([M]+) 614.2286, found: 614.2284.
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<Inserted Scheme 1>
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2.3 General preparation for optical measurements
The stock solution of Mito-DNP and various analytes were prepared in DMSO
and deionized water respectively. All spectra were measured in DMF-PBS (10 mM,
pH 7.4, v:v =1:1) buffer solution. For the measurements, excitation wavelength was
680 nm, the slit widths were 5 nm and the voltage was fixed at 600V.
2.4 Cell culture and imaging
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The HepG2 cells were grown in DMEM medium supplemented with 10 % FBS, 1 %
of penicillin at 37 °C under 5 % CO2. For imaging of H2S in living cells, HepG2 cells
were incubated with 10 μM of Mito-DNP for 30 min, and then imaged. Meanwhile,
for imaging of exogenous H2S in live cells, after being pretreated with Na2S (20, 50,
100 μM) for 30 min, following by 10 μM of Mito-DNP. For imaging of endogenous
H2S, three groups of experiments were carried out. The cells were pretreated with an
inhibitor of H2S production by cystathionine γ-lyase propargylglycine (PAG), cysteine
(Cys) or PAG and Cys, then were hatched with Mito-DNP (10 μM) subsequently.
2.5 Imaging of H2S in vivo
BALB/c Nude Mice were purchased from Beijing Spaifu Biotechnology Co., Ltd.
and utilized as biological models. The mice were split into four groups. As the control
group, the mice were injected with only Mito-DNP, the other three groups of mice
were injected with Na2S, Cys or Cys and PAG respectively, following by injection
with Mito-DNP. All injections were performed intraperitoneally, and images were
recorded by the PerkinElmer IVIS Spectrum live animal imaging system.
3. Results and discussion
3.1. Response of Mito-DNP to H2S
The sensing properties of Mito-DNP for H2S were initially investigated. The
absorption and fluorescence emission spectra were recorded in PBS buffer (10 mM,
pH 7.4) with 50 % DMF. Figure 1 depicted that the probe Mito-DNP exhibited an
absorbance peak at around 609 nm and a fluorescence emission at 741 nm with
addition of H2S. Herein, it will helpful for further fluorescence imaging of H2S in
organisms. Subsequently, the absorption and fluorescence titration of Mito-DNP
towards H2S were studied (Figure 2). In the absorbance spectrum, the probe
Mito-DNP (10 μM) showed maximum absorption peak at 609 nm. When the Na2S
(100 μM, the source for H2S) was added, two new absorption peaks at 718 nm and
380 nm significantly increased, while the absorption peak at 609 nm gradually
decreased. Corresponding to the fluorescence spectrum, the free probe Mito-DNP had
no fluorescence, however, the fluorescence intensity of Mito-DNP remarkably
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increased at 741 nm after adding 100 μM of Na2S due to the thiolysis of dinitrophenyl
ether and the Mito-OH to release. The fluorescence intensity of 741 nm reached
saturation and increased nearly by 20-fold. In addition, the detection limit of
Mito-DNP for H2S was calculated as 0.096 μM by S/N =3 method. [47] Moreover,
the kinetic analysis was also investigated and the fluorescence intensity stabilized in 5
min after adding of H2S, indicating that Mito-DNP has a fast response to H2S (Figure
S1). Futhermore, such large emission wavelengths of Mito-DNP were superior to
some reported probes for H2S (Table S1) and it might be an excellent candidate for
detecting H2S in biological systems. Beyond that, the effects of pH value on the
fluorescence response of Mito-DNP to H2S was also studied and discussed. The free
probe Mito-DNP was no fluorescence in the pH value of 3-11, but obvious
fluorescence enhancement of Mito-DNP at 741nm in the pH value of 6-9 with H2S
(Figure S2). Thus, Mito-DNP could function at physiological pH.
<Inserted Figure 1>
<Inserted Figure 2>
3.2 The Selectivity of probe Mito-DNP
Selectivity is an essential factor for fluorescent probe. Thus, the specific selectivity
of Mito-DNP for H2S was demonstrated. We performed interference analysis of
fluorescence spectra (Figure 3). Under the same conditions, some analytes including
−
2−
−
−
H2S, H2O2, various anions (F−, SCN−, I−, Br−, HSO3 , SO42−, CO3 , NO3 , NO2 , Cl−,
−
N3 , ClO−), biothiols (Hcy, Cys, GSH) and various cations (K+, Ba2+, Ca2+, Mg2+, Al3+,
Cu2+) were added into the PBS-DMF solution containing 10 μM Mito-DNP
respectively. None of these species led to significant fluorescence response and only
H2S induced obvious fluorescent turn-on changes. The results showed that the probe
did not react with any one of these analytes. In addition, the competitive experiments
were also performed and the results showed that the probe Mito-DNP has the
anti-interference ability for the detection of H2S (Figure S3). Therefore, it
demonstrated that Mito-DNP has a high selectivity for H2S, confirming that the
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dinitrophenyl ether can selectively react with H2S in the biological systems.
<Inserted Figure 3>
3.3 Proposed mechanism
The proposed sensing mechanism of Mito-DNP for H2S was shown in Scheme 2.
The nucleophilic HS− attacked directly on the dinitrophenyl ether of Mito-DNP and
thereby led to a rapid release of the fluorescent Mito-OH. The proposed mechanism
was further confirmed by analyzing mass spectra changes of Mito-DNP before and
after the addition of Na2S. Upon addition of Na2S to the chromatographic methanol
solution containing Mito-DNP and then was subjected to HRMS analysis. The results
showed a peak at 448.2274, which could be ascribed to the released Mito-OH ([M]+,
calcd m/z=448.2271) (Figure S4). It revealed that H2S triggered thiolysis of
dinitrophenyl ether of Mito-DNP to release Mito-OH indeed happened and proved
the mechanism we speculated.
<Inserted Scheme 2>
3.4 Cellular H2S Analysis
Encouraged by above excellent performance of probe Mito-DNP, the capability of
probe Mito-DNP for imaging H2S in biological systems was explored. Initially, the
cytotoxicity of probe Mito-DNP was determined by the MTT assay. The results
suggested that the probe has negligible toxicity to HepG2 within 20 μM (Figure S5).
The cellular distribution of Mito-DNP was further measured whether Mito-DNP
could detect H2S in living cells and localize in mitochondria by commercial
mitochondria labeling agent Mito-Tracker Green. Figure 4 indicated that Mito-DNP
could easily penetrate cell membrane and aggregate in mitochondria and calculated
the overlap coefficient is about 0.963.
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To further investigate the potential capability of Mito-DNP for quantitative
detection of H2S in HepG2 cells. First, incubation of the HepG2 cells with Mito-DNP
showed weak fluorescence. However, the HepG2 cells were pretreated with Na2S (20,
50, 100 μM), then incubated with Mito-DNP, the enhanced fluorescence was
observed (Figure 5a), indicating that Mito-DNP could image exogenous H2S in live
cells with outstanding performance. Figure 5b depicted that the cells were hatched
with only Mito-DNP (10 μM) and showed weak fluorescence. When cells were
pre-incubated with PAG for 30 min, following by Mito-DNP (10 μM), the
fluorescence signal decreased. However, the HepG2 cells were treated with Cys (an
inductor of endogenous H2S), a stronger fluorescence has been observed. Furthermore,
when the HepG2 cells were incubated with PAG and Cys, then treated with
Mito-DNP, the fluorescence was inhibited to some extent. It exhibited that
Mito-DNP could mornitor H2S fluctuations in living cells. All the above results
demonstrated that Mito-DNP could be used to track H2S in cells.
<Inserted Figure 4>
<Inserted Figure 5>
3.5 Living Animals H2S analysis
With the above-mentioned results, the capability of Mito-DNP to image H2S in
animals was also evaluated. For the experiment, we selected the four-week old
athymic nude mice for the research. The mice were divided into four groups and
induced by intraperitoneal injection. For the control group, the mouse was
anesthetized by urethane, then injected with probe Mito-DNP (Figure 6A). In a
second group, the mouse was pre-treated with Cys and injected with Mito-DNP in the
same place, the higher fluorescence intensity was noticed compared with the control
group (Figure 6B). Similarly, after injection of Cys, then the mouse were treated PAG
and Mito-DNP, the fluorescence intensity weakened significantly (Figure 6C).
However, the mice was injected with Na2S and Mito-DNP, the obvious fluorescence
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was also observed (Figure 6D). Experimental data clearly indicated that Mito-DNP
could detect exogenous and endogenous H2S in mice.
<Inserted Figure 6>
4. Conclusion
In conclusion, we designed and developed a new NIR fluorescent probe Mito-DNP
on the basis of thiolysis of dinitrophenyl ether (DNP) for selective detection of H2S
over various analytes and biothiols via turn-on fluorescence emission. Due to lower
detection limit (96 nM) and application in a wide pH range, the probe Mito -DNP
could be specifically triggered by endogenous H2S in HepG2 cells. Moreover, the
probe is also suitable for tracking endogenous and exogenous of H2S in mice.
Therefore, probe Mito-DNP is helpful for detection of H2S in organisms and has the
application of a large space in the disease diagnosis.
Acknowledgments
This work was supported by the Natural Science Foundation of China (21807021),
the Hebei Province Natural Science Foundation (B2020201074) advanced talents
incubation program of the hebei university and the Priority Strategy Project of Key
Laboratory of Medicinal Chemistry and Molecular Diagnosis of Ministry of
Education (ts2019004).
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Scheme 1 (a) (a) Design and (b) synthesis route of Mito-DNP.
Scheme 2 Proposed detection mechanism.
Figure 1 The UV-vis absorption and fluorescence spectra of Mito-DNP in the
presence of H2S (10 equiv.) in PBS buffer with 50 % DMF, excited with 680 nm.
Figure 2 (a) The UV-vis absorption and (b) fluorescence spectral changes of
Mito-DNP (10 μM) upon addition of H2S (0-10 equiv.), excited at 680 nm.
Figure 3 (a) Fluorescence spectral of probe Mito-DNP (10 μM) with various analytes
(200 μM of each unless otherwise stated) in the DMF-PBS buffer (10 mM, pH 7.4, v:
v = 1: 1), such as (1) none, (2) F−, (3) SCN−, (4) I−, (5) Br−, (6) NO3 , (7) HSO3 , (8)
−
−
−
2−
2−
−
NO2 , (9) SO4 , (10) CO3 , (11) ClO−, (12) Cl−, (13) H2O2, (14), N3 , (15) K+, (16)
Ba2+, (17) Ca2+, (18)Mg2+, (19)Al3+, (20)Cu2+, (21) Hcy, (22) Cys, (23) 1mM GSH,
(24) 100μM Na2S. (b) The changes of corresponding fluorescence intensity at 741 nm,
excited at 680 nm.
Figure 4 Intracellular localization of Mito-DNP in living cells. (A) HepG2 cells were
incubated with 10 μM of Mito Tracker Green (λex = 488 nm, λem = 490−624 nm). (B)
HepG2 cells were incubated with 20 μM of Na2S and following by 10 μM of
Mito-DNP (λex = 633 nm, λem = 638−747 nm). (C)The merged images. (D) The image
of bright-field. (E) The corresponding intensity profiles. (F) colocalization coefficient
of MitoTracker green and Mito-DNP. Scale bar: 10 μm.
Figure 5 (a) Images of exogenous H2S in living cells. (A-D) The HepG2 cells were
pretreated with Na2S (0, 20, 50, 100 μM), following by Mito-DNP (10 μM). (E) The
corresponding fluorescence intensity. (b) Images of endogenous H2S in live cells.
(A-D) The cells were respectively pretreated with none, PAG (200 μM), Cys (100
μM), Cys (100 μM) and PAG (200 μM), then incubated with Mito-DNP (10 μM). (E)
The corresponding fluorescence intensity. (λex = 633 nm, λem = 638−747 nm), Scale
bar: 10 μm.
Figure 6 Images of H2S in BALB/c Nude Mice. (A) The mice incubated with
Mito-DNP (0.2mM, 100μL) for 30min, (B) The mice preincubated with Mito-DNP
15

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437
438
439
440
441
(0.2mM, 100μL) for 30min after injection of Cys (2mM,100μL) for 30min, (C) The
mice preincubated with Mito-DNP (0.2mM, 100μL) for 30 min after injection of PAG
(2mM, 200μL) and Cys (2mM,100μL) for 30 min, (D) The image of the mice
incubated with Mito-DNP (0.2mM, 100μL) for 30min after injection of Na2S (2 mM,
100μL). excited at 680 nm.
Scheme 1
442
443
(a)
444
445
(b)
446
Scheme 2
447
16

448
Figure 1
449
450
Figure 2
451
452
(a)
453
454
(b)
17

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Figure 3
457
458
(a)
459
460
(b)
461
Figure 4
18

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Figure 5
464
465
(a)
19

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468
(b)
Figure 6
469
20

Highlights
1. A multifunctional and high efficient hydrogen sulfide NIR
fluorescent probe was developed.
2. Strong electrophilic group was selected to promote the
nucleophilic substitution reaction
3. The probe demonstrated excellent performance in the
detection hydrogen sulfide in vivo.

Supporting Information
Figure captions
Figure S1: Reaction time profile of the probe Mito-DNP and towards Na2S.
Figure S2: Choice of pH range for the measurements.
Figure S3: Competing experiments.
Figure S4: HRMS spectra.
Figure S5: MTT assay.
Figure S6: NMR spectra.
Table S1 Comparison of fluorescent probes for H2S.
1

Figure S1: Reaction time profile of the probe Mito-DNP and towards Na2S.
Figure S1: Reaction time profile of the probe Mito-DNP (10 µM) and towards Na2S
(100 μM) at 741 nm ( λEx = 680 nm, λEm = 741 nm, slit: 5 nm/5 nm).
Figure S2: Choice of pH range for the measurements.
Figure S2: Effect of pH on the fluorescence intensity of Mito-DNP (10 µM) in DMF-
PBS buffer (10mM, pH7.4, v:v=1:1). Concentration of sodium sulfide: (a) 0 µM, (b)
100 µM.
2

Figure S3 Competing experiments.
Figure S3 Fluorescence intensity (λem= 680 nm) changes of the probe Mito-DNP (10
µM) in DMF- PBS buffer (10mM, pH7.4, v:v=1:1) solution upon addition of various
species.
Figure S4: MTT assay
3

Figure S4: MTT assay to determine the survival rate of different concentrations of
Mito-DNP (a-f：0µM、2µM、5µM、10 µM、15µM、20µM) on HepG2 cells for 12
h.
Figure S5: HRMS spectra
HRMS of Mito-DNP.
HRMS of Mito-OH.
Figure S6: NMR spectra
4

1H NMR and 13C NMR of probe Mito-DNP in CDCl3.
Table S1 Comparison of fluorescent probes for H2S.
λex/λem
Detection
Detection
Response
Ref
probe
5

(nm)
medium
PBS/CH3CN
=4:1 (v/v)
solution
limit
time
2 h
410/541
0.70 μM
1
2
550/690
PBS buffer
solution
0.28 μM
3 min
(10mM PBS,
CTAB 4 mM,
pH 7.4).
512/538
510/650
DMSO: PBS
(v/v, 7 : 3,
pH=7.4)
64 nM
600 s
60 s
3
4
solution
PBS buffer
solution (pH
7.4)
1.08 nM
410/575
543/660
BR buffer
solution
11.2 nM
15 min
5
6
(H2O;
pH=7.42, 40
mM)
DMF/H2O
(3:7, v/v, PBS
10 mM, pH =
7.4)
3.09 μM
170 min
N
CN
O
CN
CN
O
NO2
O2N
470/556
488/565
EtOH/H2O
medium (2
mL, pH ~ 7,
1:1, v/v)
50 nM
60 s
7
8
PBS buffer
(20 mM, pH
7.4)
120 nM
20 min
448/522
buffer
PBS-DMSO
(9:1 v/v, pH
7.4)
2.55 μM
55 s
9
6