A novel fluorescent probe with dual-sites for simultaneously monitoring metabolisms of cysteine in living cells and zebrafishes

Article abstract of DOI:10.1016/j.saa.2020.118602

Understanding cellular metabolism holds immense potential for developing new drugs that regulate metabolic pathways. Two gas signal molecules, SO₂ and H₂S, are the main metabolites from cysteine (Cys) via oxidation and desulfurization pathways, respectively. However, a few fluorescent probes for real-time monitor of the metabolic pathways of cysteine have been reported. To understand metabolic alterations of cysteine, we have rationally designed and prepared a dual-signal fluorescent probe HN, which could differentiate SO₂ and H₂S through two different fluorescence channels simultaneously, along with similar reaction kinetics and both “off-on” fluorescence responses. Probe HN exhibits the potential to monitor the metabolism pathways of cysteine, and the distinguishment of cancer cells from normal cells could be realized. This methodology will promote further understanding of the physiological and pathological roles of cysteine.

Full text of DOI:10.1016/j.saa.2020.118602

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A novel fluorescent probe with dual-sites for simultaneously
monitoring metabolisms of cysteine in living cells and zebrafishes
Guoxing Yin, Ting Yu, Yabing Gan, Li Zhou, Meiling Liu, Youyu
Zhang, Haitao Li, Peng Yin, Shouzhuo Yao
PII:
S1386-1425(20)30581-3
https://doi.org/10.1016/j.saa.2020.118602
SAA 118602
DOI:
Reference:
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date: 13 February 2020
Revised date:
27 May 2020
8 June 2020
Accepted
date:
Please cite this article as: G. Yin, T. Yu, Y. Gan, et al., A novel fluorescent probe
with dual-sites for simultaneously monitoring metabolisms of cysteine in living cells
and zebrafishes, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
(2020), https://doi.org/10.1016/j.saa.2020.118602
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A Novel Fluorescent Probe with Dual-sites for Simultaneously Monitoring
Metabolisms of Cysteine in Living Cells and Zebrafishes
Guoxing Yin,a Ting Yu,*,a Yabing Gan,a Li Zhou,*,b Meiling Liu,a Youyu Zhang,a
Haitao Li,a Peng Yin* a and Shouzhuo Yaoa
a
Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of
Education), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha
410081, China. E-mail: tingyu@hunnu.edu.cn; yinpeng@hunnu.edu.cn
b
Department of Radiation Oncology, State Key Laboratory of Biotherapy and Cancer Center, West
China Hospital, Sichuan University, 610041, China. E-mail: li.zhou@scu.edu.cn
Abstract
Understanding cellular metabolism holds immense potential for developing new drugs that regulate
metabolic pathways. Two gas signal molecules, SO2 and H2S, are the main metabolites from cysteine
(Cys) via oxidation and desulfurization pathways, respectively. However, a few fluorescent probes for
real-time monitor of the metabolic pathways of cysteine have been reported. To understand metabolic
alterations of cysteine, we have rationally designed and prepared a dual-signal fluorescent probe HN,
which could differentiate SO2 and H2S through two different fluorescence channels simultaneously,
along with similar reaction kinetics and both “off-on” fluorescence responses. Probe HN exhibits the
potential to monitor the metabolism pathways of cysteine, and the distinguishment of cancer cells from
normal cells could be realized. This methodology will promote further understanding of the
physiological and pathological roles of cysteine.
Key words: Cysteine, Metabolism pathways, Sulfur dioxide, Hydrogen sulfide, Fluorescent probe

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1. Introduction
Fluorescent probes with multiple reactive sites for multiple analytes have recently attracted lots of
interest for occurrences of many physiological and pathological processes are involved in the
coincidence of multiple chemical events.[1-8] In order to further understand cellular function and the
root causes of diseases, it is therefore crucial to study multiple chemical species simultaneously, and
great challenges are existed for simultaneous sensing of structurally similar targets, which also exhibit
similar chemical properties.[9,10] Cysteine is a semi-essential proteinogenic amino acid with important
functions in redox homeostasis, protein functionality, metabolism, and so on.[11,12] The cellular
concentration of cysteine is maintained within a narrow range through its synthesis and degradation.
There are two main pathways for the metabolism of cysteine, and two main products were yielded:
2-
sulfite (SO3 ) and hydrogen sulfide (H2S).[13] As key intermediates, they have major regulatory
influences on the homeostasis of cysteine in its related functions.[14,15] The oxidation pathway for
cysteine is promoted by cysteine dioxygenase (CDO) and aspartate aminotransferase (AAT), and
pyruvate and sulfite would be yielded.[16,17] While, the desulfuration pathway will release sulfane sulfur
and hydrogen sulfide (H2S), which is mainly catalyzed by several enzymes, such as cystathionine
β-synthase (CBS) and cystathionine γ-lyase (CSE) (Scheme 1a).[17-20] Abnormal metabolism of cysteine
would result in complex diseases including liver damage, Parkinson’s and Alzheimer’s,[21-25] along with
the accumulation of sulfite or H2S in the body.[26] And besides, endogenous sulfite and H2S are also
biomarkers for differentiating cancer cells and normal cells.[27,28] Therefore, it is of great value to
monitor cysteine’s important metabolisms sulfite and H2S simultaneously, and promote deeper
understanding of cellular cysteine’s functions and the root causes for its related diseases.
Fluorescent probes have becoming powerful tools in determining biologically related species from
in vitro to in vivo for they are noninvasive, nondestructive, highly sensitive and relatively simple.[9,29-32]
Though several fluorescent probes for discriminating SO2 and H2S have been reported,[33-35] to the best
of our knowledge, there are few fluorescent probes that could realize the visualization of cysteine’s
metabolisms in real-time. In 2017, Yin’s group reported the first dual-site fluorescent probe for
visualizing cysteine and its oxidative metabolism in A-549 cells.[36] Following the pioneering work,
Zhang and Ye’s groups developed ratiometric and multi-signal mitochondria-targeted fluorescent
probes for visualization of cysteine metabolism,[37,38] respectively. However, these probes could not
simultaneously monitor of the oxidation and desulfurization metabolism pathways for different
concentrations of cysteine from different fluorescence channels. In order to monitor endogenous SO2
and H2S from cysteine to illustrate the two metabolic pathways, the design strategies of fluorescent
probes would benefit from the following criteria: (1) fluorescent probe could simultaneously

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discriminate SO2 and H2S from two emission channels with large differences; (2) similar reaction rate
constants for sensing SO2 and H2S, (3) both “off-on” fluorescence responses towards SO2 and H2S.
Generally, α,β-unsaturated C=C bond was selected as the SO2 reaction site due to its excellent
sensitivity and selectivity,[30,39-42] and the 2,4-dinitrophenyl ether is a typical H2S sensitive reaction site
for H2S (Scheme 1b).[20,43-47] As a proof of concept, based on the two strategies, we have rationally
designed and synthesized a dual-signal fluorescent probe HN to visualize the metabolism pathways of
cysteine in living cells and zebrafishes. This probe could discriminatively detect SO2 and H2S through
two fluorescence channels with large emission distance (66 nm). Similar reaction kinetics and dual
“off-on” fluorescent signals were also obtained. It was successful to apply this novel probe to monitor
the metabolism pathways of cysteine simultaneously in living cells and zebrafishes. This study
provides an efficient method to monitor metabolism pathways of cellular cysteine through the sensing
of SO2 and H2S dynamics and new insights of cellular metabolism and homeostasis were obtained.
Scheme 1. (a) The two main metabolism pathways of cysteine in mammalian cells. (b) Design of a
dual-signal fluorescent probe for monitoring the metabolism pathways of cysteine in real-time.
2. Experimental
2.1. Synthesis of compound 1
Under Argon, fresh distilled DMF (2.8 mL) was added dropwise to POCl3 (2.8 mL) at room
temperature and stirred for 30 minutes to yield
a red solution. Then a portion of
2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinolin-8-ol (3.15 g, 16.64 mmol, dissolved in 10 mL DMF)
was added dropwise to the above solution and a scarlet suspension was yielded. The mixture was
stirred at room temperature for 30 min and 80ºC for 30 min until the reaction was completed, then the
reaction mixture was poured into 150 mL of ice water. NaOH solution (20%) was added to adjust the
pH to 6 and continue stirred for 2 hours, and a large amount of precipitate was obtained. The crude
product was purified by flash column chromatography (PE:EA = 20:1, v/v) to give the product as a
1
brown solid (2.50 g, yield: 69.1%). H NMR (500 MHz, CDCl3) δ 11.80 (s, 1H), 9.36 (s, 1H), 6.82 (s,
1H), 3.27 (m, 4H), 2.66 (t, J = 6.4, 6.4 Hz, 4H), 1.92 (q, J = 6.0, 6.0, 5.9 Hz, 4H). 13C NMR (126 MHz,

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CDCl3) δ 191.66, 159.41, 149.58, 131.25, 113.68, 110.75, 105.35, 50.40, 50.04, 27.32, 21.77, 20.68,
19.75.
2.2. Synthesis of compound 2
To a solution of 8-hydroxy-2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-9-carbaldehyde (1)
(1.80 g, 8.28 mmol) and ethyl 3-oxobutanoate (2.16 g, 16.57 mmol) in 20 mL absolute ethanol was
added 4-5 drops of piperidine. The reaction mixture was stirred at room temperature for 48 h. After
completion of the reaction, the mixture was concentrated to afford the crude product, which was further
1
purified by flash column chromatography to afford the yellow solid (2) (555 mg, 23.4% yield). H
NMR (500 MHz, CDCl3) δ 8.31 (s, 1H), 6.94 (s, 1H), 3.32 (q, J = 5.9, 5.9, 5.8 Hz, 4H), 2.85 (t, J = 6.4,
6.4 Hz, 2H), 2.73 (t, J = 6.3, 6.3 Hz, 2H), 2.64 (s, 3H), 1.95 (m, 4H). 13C NMR (126 MHz, CDCl3) δ
196.16, 161.42, 153.89, 148.91, 147.94, 127.91, 119.69, 114.91, 108.18, 105.72, 50.47, 50.07, 30.77,
27.55, 21.25, 20.29, 20.17.
2.3. Synthesis of compound 3
To
a
solution
of
10-acetyl-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyido[3,2,1-ij]quinolin-11-one (2) (0.50 g, 1.76
mmol) and 4-hydroxybenzaldehyde (539 mg, 4.41 mmol) in 20 mL absolute ethanol was added 2-3
drops of piperidine. The reaction mixture was stirred at 100ºC for 48 h. After completion of the
reaction, the cake was filtered and washed with cold ethanol. The solid was dried under vacuum to give
the red product (3) (0.40 g, 58.5% yield). 1H NMR (500 MHz, DMSO-d6) δ 10.05 (s, 1H), 8.37 (s, 1H),
7.81 (d, J = 15.7 Hz, 1H), 7.56 (m, 3H), 7.19 (s, 1H), 6.83 (d, J = 8.1 Hz, 2H), 3.32 (t, J = 5.7, 5.7 Hz,
4H), 2.70 (m, 4H), 1.86 (m, 4H). 13C NMR (126 MHz, DMSO-d6) δ 185.63, 160.58, 160.34, 153.39,
148.98, 148.26, 142.57, 130.83, 128.23, 126.52, 122.23, 119.86, 116.42, 114.77, 108.13, 105.05, 50.13,
49.61, 27.23, 20.99, 20.02.
2.4. Synthesis of probe HN
To
a
solution
of
(E)-10-(3-(4-hydroxyphenyl)acryloyl)-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quin
olin-11-one (3) (0.20 g, 0.516 mmol) in 20 mL anhydrous acetonitrile was added
1-fluoro-2,4-dinitrobenzene (115mg, 0.619 mmol). The reaction mixture was stirred at room
temperature for 2 min, then a few drops of triethylamine was added. After completion of the reaction,
the cake was filtered and washed with cold acetonitrile. The solid was dried under vacuum and further
purified by flash column chromatography to give the red product (probe HN) (175 mg, 61.2% yield).
1H NMR (500 MHz, CDCl3) δ 8.85 (d, J = 2.8 Hz, 1H), 8.46 (s, 1H), 8.34 (dd, J = 9.0, 2.8 Hz, 1H),
8.18 (d, J = 15.7 Hz, 1H), 7.77 (m, 3H), 7.15 (d, J = 8.2 Hz, 2H), 7.10 (d, J = 9.3 Hz, 1H), 7.01 (s, 1H),

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3.36 (q, J = 5.2, 5.2, 5.2 Hz, 4H), 2.89 (t, J = 6.3, 6.3 Hz, 2H), 2.77 (t, J = 6.2, 6.2 Hz, 2H), 1.99 (m,
4H). 13C NMR (126 MHz, CDCl3) δ 186.56, 161.56, 155.83, 154.87, 153.96, 149.17, 149.01, 141.88,
140.70, 139.88, 134.30, 131.00, 129.04, 128.01, 126.49, 122.30, 120.85, 119.92, 119.11, 115.11,
108.77, 105.83, 50.58, 50.18, 27.59, 21.28, 20.32, 20.20. IR (KBr pellet, cm-1): 3029, 2925, 2854, 1718,
1610, 1513, 1438, 1381, 1311 1267, 1192, 1132, 1052, 816, 696. HRMS: m/z [M + H]+ calculated for
C30H24N3O8: 554.1558; measured: 554.1568.
2.5. Cell culture.
HepG2 and L-O2 cells were obtained from China Center for Type Culture Collection (Wuhan,
China). The cells were incubated in Dulbecco’s modified Eagle’s medium (DMEM) with 10% (v/v)
heat-inactivated fetal bovine serum (FBS) (Gibco BRL, Grand Island, NY, USA),
streptomycin-penicillin (100 U/mL, 100 μg/mL) in a humidified incubator of 5% CO2 and 95% air at
37°C.
2.6. Imaging of endogenous and exogenous SO2 and H2S (metabolisms of cysteine) in living cells.
For the detection of SO2 and H2S in cells, HepG2 and L-O2 and were incubated with probe (10 µM)
for 60 min, then imaged after washing by phosphate-buffered saline (PBS) for three times. To detect
exogenous SO2 and H2S, HepG2 and L-O2 cells were pretreated with NEM (0.25 mM) for 30 min,
subsequently incubated with Cys (50, 100, 200, 400, 800, 1000 μM, 60 min) and probe HN (10 μM, 60
min), then washed with phosphate-buffered saline (PBS) for three times. Cells were imaged by a
fluorescence confocal microscope (Leica SP8, Germany). (λex = 458 nm, λem = 480 - 550 nm for the
green channel; and λex = 514 nm, λem = 560 - 650 nm for the red channel).
2.7. Imaging of SO2 and H2S (metabolisms of cysteine) in living zebrafish.
For the detection of metabolisms of cysteine (SO2 and H2S) in vivo, 3-day-old zebrafishes were
prepared. To detect endogenous SO2 and H2S, the zebrafishes incubated with probe (10 μM) for 60 min,
then imaged. Fluorescent Imaging of Cys metabolisms in zebrafishes, zebrafishes were fed with 250
μM NEM in E3 embryo media (15 mM NaCl, 0.5 mM KCl, 1 mM MgSO4, 1 mM CaCl2, 0.15 mM
KH2PO4, 0.05 mM Na2HPO4, 0.7 mM NaHCO3, 10-5% methylene blue; pH 7.5) at 28 °C for 30 min,
then incubated with Cys (600 μM, 60 min), and finally incubated with probe (10 μM) for 60 min. All
the fishes were terminally anaesthetized using MS222, and images were carried out on a confocal
microscope. (Leica SP8, Germany). (λex = 458 nm, λem = 480 - 550 nm for the green channel; and λex
514 nm, λem = 560 - 650 nm for the red channel).
=

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Scheme 2. Synthesis of the probe HN.
3. Results and Discussions
3.1 Spectral responses of probe HN toward SO2 and H2S
Firstly, the UV-vis and fluorescent responses of probe HN toward reactive sulfur species
(thiols, NaHSO3 and NaHS) were measured carefully in DMSO/PBS (pH 7.4, 10 mM, v/v, 5/5)
at 30 °C for 30 min. As expected, upon addition of NaHSO3, the initial absorption peak at 499
nm was changed to 470 nm with a 29 nm blue shift, which should be attributed to the addition
of NaHSO3 to the C=C double bond (Fig. S1a, Fig. S2). Correspondingly, a significant
fluorescence turn-on at 515 nm was observed at 460 nm excitation (Fig. 1a, Ф: 0.10 with
fluorescein as reference), while other competitive analytes induced little fluorescence. On the
other hand, for NaHS, a slightly redshifted absorption band and a clear fluorescence
enhancement at 581 nm were observed due to the thiolysis of 2,4-dinitrophenyl ethers induced
by NaHS (Fig. 1b, Fig. S1, S3, Ф: 0.06 with fluorescein as reference). Thus, the probe HN
could differentiate SO2 and H2S from two fluorescence channels with large distance (66 nm)
without interferences from biothiols, for the specific reaction sites for SO2 and H2S were
properly established in the probe HN. More importantly, the sensing of NaHSO3 and NaHS
with probe HN was followed by pseudo-first-order kinetics: kobs = 0.07 min-1 and a half-life of
10.0 min for NaHSO3; kobs = 0.09 min-1 and a half-life of 7.4 min for NaHS based on a global
fitting for the fluorescence growth at 515 and 581 nm, respectively (Fig. 1c, 1d).[48,49] The
results from pH titration have showed that detection of NaHSO3 and NaHS could be realized
over a broad pH range (Fig. S4). All of the results have indicated that probe HN could
simultaneously monitor the two metabolites of cysteine: SO2 and H2S, from two different
emission channels.

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(b)
(a)
probe HN
HN + NaHSO3
HN + NaHS
HN + Cys
probe HN
HN + NaHSO3
500
400
300
200
100
0
5000
4000
3000
2000
1000
0
HN + NaHS
HN + Hcy
HN + Cys
HN + GSH
HN + Hcy
HN + GSH
500
550
600
650
550
600
650
700
750
Wavelength (nm)
Wavelength (nm)
5000
(d)
probe HN
500
HN + NaHS
HN + NaHSO3
400
(c)
5000
4000
3000
2000
1000
0
probe HN
500
400
300
200
100
0
HN
HN
+
+
NaHS
4000
3000
2000
1000
0
NaHSO3
300
200
100
0
0
5
10
15
20
25
30
0
5
10
15
20
25
30
Time (min)
Time (min)
500
550
600
650
550
600
650
700
750
Wavelength (nm)
Wavelength (nm)
Fig. 1. (a-b) Fluorescence spectra of probe HN (10 µM) upon addition of 10 equiv. of Cys, Hcy, GSH,
NaHS and NaHSO3 (100 µM) for 30 min. Inset: The inset photos show color changes of probe HN in
the absence and presence of (a) NaHSO3, (b) NaHS under UV lamp at 365 nm. (c-d) Time-dependent
fluorescence spectra of probe HN (10 µM) in the presence of 10 equiv. of (c) NaHSO3, (d) NaHS. Inset:
the corresponding time-dependent fluorescence intensity changes at (c) 515 nm, (d) 581 nm.
Conditions: DMSO-PBS (pH 7.4, 10 mM, v/v, 5/5), Temperature: 30 °C. (a, c) λex = 460 nm, (b, d) λex
= 500 nm. Slit (nm): 2.5/5.
3.2. Selectivity and sensitivity of probe HN for SO2 and H2S
Next, we assessed the selectivity of probe HN over other biologically related or environmentally
important species, such as Cys (0.2 mM), GSH (2.5 mM), and Hcy (0.1 mM) in DMSO-PBS (pH 7.4,
10 mM, v/v, 5/5) at 30°C for 60 min. Encouragingly, except NaHSO3 and NaHS, addition of other
analytes resulted in no remarkable fluorescence enhancement at 515 and 581 nm excited at 460 and
500 nm, respectively (Fig. S5, S6). Therefore, probe HN could detect NaHSO3 and NaHS with high
selectivity even in the existence of high concentrations of thiols. The fluorescence enhancement at 515
-
nm and 581 nm showed good linear relationship (R2 > 0.99) to the concentration of HSO3 and HS- (Fig.
-
S7, S8), and the detection limit were calculated to be as low as 20 and 120 nM for HSO3 and HS-
based on IUPAC definition (LOD = 3σ/S), respectively. These results clearly demonstrated this novel
dual-function probes could simultaneously discriminate two analytes from two emission channels with
large distance (> 50 nm), along with excellent chemical stability under physiological conditions.
3.3. Mechanism of probe HN with SO2 and H2S.

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As shown in scheme 1b, to verify whether the spectroscopic differences in the responses of probe
HN toward NaHSO3 and NaHS were caused by the different sensing mechanisms as designed. The
compound 3 and HN-SO2 were easily isolated and characterized well by NMR analyses (Figs.
S29-S32). The product 3-SO2 (Ф: 0.58 with fluorescein as reference) was also successfully obtained
from the reaction between compound 3 with NaHSO3 (Figs. S33-S35). The absorption and
fluorescence spectra of isolated products were well studied (Fig. S1), which were consistent with the
proposed mechanism. In addition, theoretical calculations showed that the product 3 from the reaction
between HN and H2S exhibited a bigger energy gap between the HOMO (highest occupied molecular
orbital) and LUMO (lowest unoccupied molecular orbital) (3.40 eV) than that of HN (2.41 eV) (Fig.
S16), which indicated that compound 3 will be more difficult to react with SO2 to afford the compound
3-SO2, which further supports the sensing mechanism of probe HN with both “off-on” fluorescence
response toward SO2 and H2S. Thus, probe HN could simultaneously react with SO2 and H2S to afford
different spectroscopic properties of compound 3 and HN-SO2, respectively.
The responses of compound 3 toward representative relevant species were also performed. Almost
no significant changes were observed in the time-dependent absorption spectra and fluorescence
intensities (515 nm) after it was treated with 20 equiv. of reactive sulfur species (Cys, GSH and Hcy et
al.) within a short time (Fig. S9). Meanwhile, it was clear that using different excitations the mixture of
NaHSO3, NaHS, Cys, Hcy and GSH could elicit dramatic fluorescence enhancement within their
specific channels (Fig. S10-S12). Furthermore, in presence of H2S, HN-SO2 was transformed to
product 3-SO2 very slowly (Fig. S13, S14). To confirm the bioavailability of probe HN, we have also
examined their photostability under the same conditions. Solutions of probe HN, HN-SO2 and
compound 3 were continuously irradiated via a Xe-lamp at 460 and 500 nm wavelengths, respectively.
It was observed that all compounds were stable after continuous irradiation for 2h (> 95% of the initial
values for compound HN-SO2 and compound 3) (Fig. S15). Therefore, these experiments well
demonstrated that probe HN could be applied to simultaneously sense SO2 and H2S from two
fluorescence channels with continuous fluorescence signal retention, and it would be an excellent tool
for monitoring the metabolism pathways of cysteine in vivo in real-time.
3.4. Fluorescent Imaging of SO2 and H2S in Living Cells.
Encouraging by the aforementioned results, the practicality of probe HN for imaging metabolites of
cysteine was evaluated in biological environment. Firstly, probe HN was found to be low toxic for
living cells, which was evaluated by the standard CCK-8 assay (Fig. S17). After pretreated with
N-ethylmaleimide (NEM, scavenger of SH compounds), Cells exhibited no fluorescence in both green
and red channels after incubation with probe HN for 2 h(Fig. S18). However, when treatment with

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exogenous NaHSO3 (Fig. 2A-B) and NaHS (Fig. 2C-D) in the NEM-pretreated HepG2 cells significant
fluorescence in the green and red channel were observed after incubated with HN for 10 min,
respectively. Application of probe HN for simultaneous visualization of endogenous SO2 and H2S in
cancer cells (HepG2) and normal cells (L-O2) were then conducted. The L-O2 cells were exhibited
bright red and weak green fluorescence after stained with HN for 60 min simultaneously (Fig. 3A1-B1),
while HepG2 cells were shown brighter green fluorescence (Fig. S19, A1-B1), which indicated that
different levels of sulfite and H2S are existed in the normal cells and cancer cells. Therefore, probe HN
could be applied to image the endogenous and exogenous SO2 and H2S through two different
fluorescence channels with high selectivity and high sensitivity. And the discrimination of normal and
cancer cells could be realized through the monitor of the metabolites of cysteine.
Fig. 2. Confocal fluorescence images of SO2 and H2S in HepG2 cells. A-D). Cells were pretreated with
NEM (0.25 mM, 30 min), then treated with probe HN (10 μM, 30 min) and NaHSO3 (A-B), NaHS
(C-D) (250 μM) for 10, 30, 60, and 90 min, respectively, then imaged. (λex = 458 nm, λem = 480 - 550
nm for the green channel; and λex = 514 nm, λem = 560 - 650 nm for the red channel). Scale bar: 50 μm.
3.5. Fluorescence imaging of metabolism pathways of cysteine in living cells and zebrafish
Subsequently, based on its efficient sensing capability for SO2 and H2S in living cells, probe HN was
applied to monitor the metabolism pathways of cysteine. L-O2 cells were first treated with NEM (250
µM) for 60 min, and then treated with different concentration of Cys (50, 100, 200, 400, 800 and 1000
μM) for another 60 min. As showed in Fig. 3, L-O2 cells were exhibited brighter red fluorescence after
incubated low concentration of Cys (50 μM, Fig. 3A2-B2). However, fluorescence in green channel

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was significantly increased along with the increasement of the concentration of Cys. Fluorescence
imaging of metabolites of cysteine were also performed in HepG2 cells (Fig. S19), and similar results
were obtained. Moreover, sensing of metabolism pathways of cysteine in zebrafish with probe HN was
conducted. After pretreated with NEM, zebrafish was illustrated no fluorescence in both the green and
red channels after incubated with probe HN (Fig. 4, A1−C1). However, endogenous SO2 and H2S in
zebrafish could be sensed simultaneously from two different fluorescence channels (Fig. 4, A2, B2).
After further treated with Cys, significant fluorescence enhancement in the green channels could be
observed, which reflected more endogenous bisulfite/sulfite (Fig. 4, A2−A7, Fig. S20) were released.
The results demonstrated that probe HN could monitor H2S and SO2 in real-time, which are produced
from Cys metabolism in living cells and zebrafish. And the differences in fluorescence intensities
indicated that desulfuration pathway would be dominate when cysteine is deficient, whereas oxidative
metabolism would be the main pathway when cysteine is in excess (Fig. 3B; Fig. S20).[13-15]
Green channel
Red channel
B)12
9
6
3
0
Endogenous50
100
200
400
800
1000
Cys (μM)
Fig. 3. Confocal fluorescence images of metabolisms of cysteine in L-O2 cells. Cells were incubated
with probe HN (10 μM) for 60 min, then imaged (A1-B1). Cells were pretreated with NEM (250 μM,
30 min), then incubated with 50, 100, 200, 400, 800, 1000 μM Cys for 60 min, respectively, and finally
incubated with probe (10 μM) for 60 min (A2-B7). (B) The intensity of green and red channel in Fig.
A1-B7. (λex = 458 nm, λem = 480 - 550 nm for the green channel; and λex = 514 nm, λem = 560 - 650 nm
for the red channel). Scale bar: 50 μm. The fluorescence intensity was calculated by Leica LAS X

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program. Scale bar: 50 μm.
Fig. 4. Confocal fluorescence images of metabolisms of cysteine in zebrafishes. Zebrafish was
pretreated with NEM (250 μM, 30 min), then incubated with probe HN (10 μM) for 60 min, and finally
imaged (A1-C1). Zebrafish was incubated with probe HN (10 μM) for 60 min, then imaged (A2-C2).
Zebrafish was pretreated with NEM (250 μM, 30 min), then incubated with Cys (100, 200, 400, 800
and 1000 μM, respectively, 60 min), and finally incubated with probe (10 μM) for 60 min, then imaged
(A3-A7). (λex = 458 nm, λem = 480 - 550 nm for the green channel; and λex = 514 nm, λem = 560 - 650
nm for the red channel). Scale bar: 0.5 mm.
4. Conclusion
In conclusion, we have rationally developed a dual-signal fluorescent probe for monitoring the
metabolism pathways of cysteine in real-time. For the simultaneous detection of NaHSO3 and NaHS,
the probe HN was featured with large emission differences (66 nm), similar reaction rate constants and
sustained fluorescence signals. This probe was successfully applied to the simultaneous imaging SO2
and H2S from different emission channels. Importantly, probe HN could be used to monitor the
metabolism pathways of cysteine. Pathways of desulfurization or oxidation are mainly depended on the
concentrations of cysteine. This methodology will provide better understanding for the physiological
and pathological roles of cysteine, and further promote the diagnosis and treatment of diseases, which
are related to cysteine and its metabolites.
Conflicts of interest
The authors declare no competing financial interest.

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Acknowledgements
This work was supported by the funding from the National Natural Science Foundation of China
(Grant No. 21877035, 21977028 and 11905150); Research Foundation of Education Bureau of Hunan
Province (No. 18B004) and Sichuan Science and Technology Program (2018HH0099).
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Credit Author Statement
Guoxing Yin: Conceptualization, Writing - original draft, Methodology, Investigation, Formal analysis.
Yabing Gan and Meiling Liu: Investigation, Validation.
Youyu Zhang, Haitao Li and Shouzhuo Yao: Writing - Review & Editing.
Ting Yu, Li Zhou and Peng Yin: Supervision, conceptualization, funding acquisition, project
administration.

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Declaration of interests
■ The authors declare that they have no known competing financial interests or
personal relationships that could have appeared to influence the work reported in
this paper.
■The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests:
Declarations of interest: none

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Graphical abstract

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Highlights


A novel fluorescent probe for simultaneously monitoring metabolisms of
cysteine was developed.
The probe could simultaneously differentiate SO2 and H2S through two different
fluorescence channels with similar reaction kinetic and both “off-on”
fluorescence responses.

The probe was successfully applied to monitor and visualize the metabolism
pathways of cysteine in living cells and zebrafishes in real time.