Independent bi-reversible reactions and regulable FRET efficiency achieving real-time visualization of Cys metabolizing into SO2

Article abstract of DOI:10.1039/d0cc04839a

In this work, we synthesized an independent bi-reversible reaction sensorBPCfor simultaneously detecting cysteine (Cys) and sulfur dioxide (SO₂), showing multi-fluorescence signal modes due to the regulable FRET efficiency, and finally achieving real-time process visualization of Cys metabolizing into SO₂in subcellular organelles and tumors.

Full text of DOI:10.1039/d0cc04839a

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Independent bi-reversible reactions and regulable FERT efficiency
achieving Cys metabolism SO2 real-time process visualization
Tao Li, a Fangjun Huo,b Jianbin Chao,b and Caixia Yin*a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
In this work, we synthesized an independent bi-reversible mitochondria, where Cys and SO2 are mainly produced and
reactions sensor BPC for simultaneously detecting cysteine (Cys) stored in eukaryotic cells, will provide a deeper perspective to
and sulfur dioxide (SO2), showing multi-fluorescent signal modes perceive the complex physiological interrelationship between
due to the regulable FRET efficiency, and finally achieving real- them.6 Thus, given the above-mentioned physiological and
time process visualization of Cys metabolism SO2 in subcellular pathological effects of Cys and its metabolite SO2, a simple,
organelle and tumor.
effective and reliable strategy is urgently needed to probe
concentration fluctuation and track its metabolic pathways in
cells and organisms, which is of great significance in preventing
related diseases.
Metabolism refers to the pathway in which organisms
achieve self-renewal by dynamically exchanging substances
and energy with the external environment. However, as one of
them, the biochemical process of substance metabolism of
cysteine (Cys) in vivo has not been well cognized. As a nature
amino acid containing reducing mercapto (-SH) group, Cys is
directly involved in many crucial life processes such as
nutritional intake, antioxidant, protein synthesis, metabolism.1
The concentration unbalance of Cys in organisms is closely
associated with Parkinson’s disease, Alzheimer’s disease and
liver injury.2 Cys mainly is generated in mammalian cells
through three biotransformation pathways: (a) methionine
metabolism, (b) proteolysis, (c) cystine reduction [note that
Cys exists mainly in the state of disulfide bonds in the blood
system due to the high oxidation environment in plasma].3 In
aerobic condition, Cys will be catalyzed to form cysteine
sulfinate by cysteine dioxygenase (CDO), and then
enzymatically converted to β-sulfinylpyruvate by aspartate
To date, as these outstanding features including simple
operation, noninvasiveness and real-time visualization,
fluorescent probes with molecular imagine technology have
become an ideal platform for exploring the distribution of
small biomolecules in living organisms and their corresponding
biological effects.7 In the past decade, great progress has been
made in efforts to develop detection of Cys or SO2 using
fluorescent probe,8 but only a few breakthroughs have been
acquired in evaluating their metabolic relationships. For
example, in 2017, our group firstly reported a turn-on
fluorescent probe with dual-site to visualize the metabolism of
Cys in living cells.3a In 2018, Zhang et al. developed a
ratiometric fluorescent probe to explore the kinetic process of
SO2 in hepatic BRL cells after treating excess exogenous Cys.8c
However, the defects specific to these probes: (a) the inability
to monitor the simultaneous presence of Cys and its
metabolite SO2, (b) long time to detect the analytes, and (c)
insufficient sensitivity to meet the detection requirements of
SO2 as a low concentration of transient metabolite. These
mean that the microscopic physiological process of Cys
metabolism cannot be truly understood and visualized.
Inspired by the above probes and the characteristics of Cys
metabolism, we further came up with a perception: the ideal
probe needs to meet independent bi-reversible reaction sites
and fast response toward analytes.
aminotransferase
decomposed to pyruvate and sulfur dioxide (SO2), which is
(AAT),
eventually
spontaneously
2-
excreted in the urine in the form of its derivatives (SO3
/
-
HSO3 ).4 Interestingly, it has be reported that endogenous SO2
can regulate vascular smooth muscle tone and act as an
antioxidant to protect the redox balance in cells, thereby
improving cardiovascular function.5 Additionally, targeting
a. Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of
Education, Key Laboratory of Materials for Energy Conversion and Storage of
Shanxi Province, Institute of Molecular Science, Shanxi University, Taiyuan
030006, China. E-mail: yincx@sxu.edu.cn
To satisfy the above-mentioned demands, we proposed a
strategy to achieving simultaneously detection of Cys and SO2
by employing the FRET mechanism. To make it a reality, the
coumarin moiety of CP was selected as the energy donor and
the α, β-unsaturated C=C double bond as the recognition site
b. Research Institute of Applied Chemistry, Shanxi University, Taiyuan 030006,
China.
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
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Scheme 1. Design of sensor BPC.
toward Cys (Fig. S5a), which could recover its original state by
introducing
thiol scavenger, N-ethylmaleimide (NEM).9
a
Meanwhile, it was found that the BP showed high sensitivity
for SO2 (Fig. S5b), which was reversible due to the interaction
with formaldehyde (HCHO).10 Coincidentally, the fluorescent
emission wavelength of CP-Cys has a lot of overlap with the
absorbance of BP, which can ensure the FRET effect of the
probe internal (Fig. S5c). In this work, we combined BP with CP
to create an independent bi-reversible reactions sensor BPC
for dynamically tracking SO2 metabolism from Cys (Scheme 1).
As shown in Scheme S2, Cys can attack the α, β-unsaturated
C=C double bond by the nucleophilic addition reaction,
resulting in the red fluorescent signal distinctly enhancement
due to the FRET process from the energy donor CP-Cys to the
acceptor BP. Subsequently, adding the SO2 to the mixture of
BPC-Cys, the π-conjugated system of benzopyrylium unit was
interrupted, causing the red fluorescent signal to decrease due
to the disappearance of the energy acceptor BP and releasing
the fluorescent emission of the energy donor CP-Cys.
Therefore, BPC can achieve the simultaneously detection of
Cys and SO2 through independent bi-reversible reactions and
luminescence regulated by FRET strategy.
To verify our conception, we firstly recorded the fluorescent
spectra of BPC (10 μM) toward Cys. As exhibited in Fig. 1a, the
fluorescence intensity at 500 nm and 637 nm both gradually
increased (excitation wavelength at 430 nm) along with the
addition of Cys (0-40 equiv.), which was due to the
nucleophilic addition reaction caused by Cys to form the
energy donor CP-Cys and the special efficiency of the FRET
mechanism was calculated to be 51.2 % (E = 1 − FDA/FD, Fig.
S5d).11 The detection limit was 2.9 μM based on IUPAC (CDL =
3Sb/m),12 and the equilibrium was obtained after 4 min with
adding different concentrations of Cys (Fig. S1a, b).
Interestingly, we found that the emission peak at 637 nm was
rapidly quenched when Na2SO3 at low concentrations (0-4
equiv.) was added to BPC (Fig. 1b, excitation wavelength at
585 nm), and under the excitation wavelength at 430 nm, the
fluorescent signal at 500 nm had slight increase (Fig. S2c)
showing that the π-conjugated system of benzopyrylium unit
Fig. 1 (a) or (b) The fluorescent spectra of probe BPC (10 μM)
toward Cys (0-40 equiv.) or Na2SO3 (0-4 equiv.), λex = 430 nm,
λem = 500 nm; and λex = 585 nm, λem = 637 nm, slit: 5 nm/10 nm.
(c) The reversible cycle of probe BPC (10 μM) with Cys (40
equiv.) upon addition of NEM (40 equiv.), λex = 430 nm, λem
=
500 nm, slit: 5 nm/10 nm. (d) The fluorescent change of probe
BPC (10 μM) upon the addition of Na2SO3 (4 equiv.) or HCHO (4
equiv.) consecutively, λex = 585 nm, λem = 637 nm, slit: 5 nm/10
nm. Condition: DMSO/PBS solution (10.0 mM, pH = 7.4, 1:9,
v/v). (e) or (f) The fluorescence spectra of BPC (10 μM) upon
the treatment of Cys (40 equiv.) and Na2SO3 (4 equiv.) in turn
or simultaneously, λex = 430 nm, λem = 500 nm and λem = 637
nm, slit: 5 nm/10 nm. Condition: DMSO/PBS solution (10.0 mM,
pH = 7.4, 1:9, v/v).
was interrupted (Fig. S2d). Under this condition, the special
efficiency of the FRET mechanism was 14.5 % (E = 1 − FDA/FD,
Fig. S2e).11 Owing to the quick detection time (20 s) and low
detection limit 0.14 μM, it can perfectly meet the detection
requirements of SO2 as a transient metabolite of Cys in cells
(Fig. S2a, b).
We then confirmed the reversibility of BPC toward Cys or
SO2, respectively. Fortunately, upon addition of NEM in the
presence of Cys, the fluorescent emission peak of BPC-Cys at
500 could be decreased to its original state and recovered by
introducing Cys into the solution again (Fig. 1c), which was
consistent with our idea that NEM can capture Cys from the
compound BPC-Cys to release free BPC. According to the
previous literature reported, HCHO as an electrophile can
effectively react with SO2.10 After adding the HCHO, the
fluorescent intensity of BPC-SO2 at 637 nm gradually increased
to near the initial level of BPC and further quenched by adding
equivalent Na2SO3 (Fig 1d). Consequently, the probe BPC can
be considered as an
2 | J. Name., 2012, 00, 1-3
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Fig. 2 Real-time visualizing Cys metabolism in cells using BPC: (a) the time-dependented images of BPC (10 μM) incubated HeLa
cells after adding Cys (400 μM) every 4 min (from 0 to 28 min); (b) Corresponding average fluorescence intensities of red and
green channel in every 4 min (from 0 to 28 min). Red channel, λem = 610-670 nm and Green channel, λem = 470-530 nm, λex = 458
nm. Scale bar: 10 μm.
ideal candidate to achieve the reversible detection of Cys or feasibility of this conjecture. We initially explored the
SO2, individually.
cytotoxicity of BPC by MTT assay (Fig. S10). It should be noted
Inspired by the above data, we wondered if BPC could be that HeLa cells incubated with BPC (0-10 μM) for 12 h still
applied more profoundly to investigate Cys-SO2 metabolism. As show high survival rates. Since BPC with a positively charged
described in Fig. 1e, the fluorescence peaks of BPC at 500 nm part, we guessed that it could locate the mitochondria. As
and 637 nm initially enhanced after the treatment with Cys shown in Fig. S11, HeLa cells were sequentially incubated with
due to this special FRET mechanism. Subsequently, upon the BPC (10 μM) and MitoTracker Green (0.2 μM). The fluorescent
addition of Na2SO3 to the mixture of BPC-Cys, the fluorescence signal of red and green channels shown synchronous tendency
peak at 637 nm disappeared promptly while a sharp increase and the Pearson’s co-localization coefficient of 0.88, which
at 500 nm simultaneously, which was due to the well revealed the BPC can serve as the mitochondrial targeting
disappearance of the energy acceptor BP and the release of tool.
the fluorescence of the energy donor CP-Cys. The 137 nm
We next conducted the fluorescent imaging of exogenous
fluorescent blue-shift indicated the disruption of the FRET SO2 (Na2SO3). To demonstrate that BPC can still function as
strategy from CP-Cys to BP and the formation of reaction FRET in cellular level, we used a single wavelength to excite
product (BPC-Cys-SO2). Because Cys and SO2 coexist in different channels. After HeLa cells treated with 10 μM BPC for
complicated physiological system, we added them 20 min, then removed the residue, adding the Na2SO3 (40 μM)
synchronously. As explicated in Fig. 1f, this situation was the red fluorescence signal gradually declined from 0 to 12 min
consistent with the above consequence, just consuming 4 min (Fig. S12). To confirm the above-mentioned reversibility,
to reach equilibrium, meaning that BPC can take on the task of adding HCHO (40 μM) into above system, the fluorescent
simultaneously detecting Cys and SO2.
intensity in red channel gradually increased with time. This
The sensing mechanism of BPC for detecting Cys and SO2 phenomenon suggested that BPC can in situ real-time
was described in Scheme S2. The corresponding reaction reversible imaging of exogenous SO2 in living cells. Related
products were proved by ESI-MS characterization. The highest literature reported that high level of Cys can stimulate CDO
peak at m/z 735.35436 represented BPC in Fig. S6. Obviously, activity,13 then SO2 was metabolized. Subsequently, we tried to
there was a main peak at m/z 856.37438 corresponding to the real-time visualize Cys metabolism in cells using BPC. HeLa
BPC-Cys product (Fig. S7). As described in Fig. S8, it can be cells were incubated with BPC (10 μM) in turn and Cys (400 μM)
ascribed the dominant peak at m/z 815.31147 to the deserved for imaging. As is described in Fig. 2, the time-dependent
mixture of BPC-SO2. The final product BPC-Cys-SO2 was m/z imaging showed a gradual increase of fluorescence signals in
936.33250 (Fig. S9). These conclusive evidences signified that the red and green channels from 0 to 12 min, indicating that
the principles for BPC responding Cys or SO2 all conformed Cys entered the cells and reacted with BPC. Then, the red
with our previous conception.
fluorescence suddenly decreased while the green fluorescence
Therefore, the above series of in vitro spectral response kept rising, meaning the endogenous SO2 also generated and
results demonstrated that the probe BPC may have potential be detected. From the above phenomena, BPC possesses
to become an effective and reliable tool in tracking Cys multi-fluorescence modes to investigate and visualize the
metabolic pathways in cells and organisms. Then we process for SO2 metabolism from Cys in mitochondria of living
conducted biological imaging experiments to evaluate the cells.
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Conflicts of interest
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DOI: 10.1039/D0CC04839A
There are no conflicts to declare.
Notes and references
1.
(a) K. G. Reddie and K. S. Carroll, Curr. Opin. Chem. Biol.,
2008, 12, 746-754. (b) E. Weerapana, C. Wang, G. M.
Simon, F. Richter, S. Khare, M. B. D. Dillon, D. A.
Bachovchin, K. Mowen, D. Baker and B. F. Cravatt, Nature,
2010, 468, 790-795.
2.
(a) M. T. Heafield, S. Fearn, G. B. Steventon, R. H. Waring,
A. C. Williams and S. G. Sturman, Neurosci. Lett., 1990,
110, 216-220. (b) L. Y.Niu, Y. S. Guan, Y. Z. Chen, L. Z. Wu,
C. H. Tung and Q. Z. Yang, Chem. Commun., 2013, 49,
1294-1296
Fig. 3 The metabolic imaging of Cys (400 μM) using BPC in
HeLa tumor-bearing nude mice at different time, Red channel:
3.
4.
(a) Y. Yue, F. Huo, P. Ning, Y. Zhang, J. Chao, X. Meng and
C. Yin, J. Am. Chem. Soc., 2017, 139, 3181-3185. (b) J. Liu,
M. X. Liu, H. X. Zhang, X. H. Wei, J. J. Wang, M. Xian and
W. Guo, Chem. Sci., 2019, 10, 10065-10071.
(a) T. Ubuka, J. Ohta, R. Akagi, Y. Hosaki, Y. Ishimoto, S.
Kiguchi, T. Ikeda and K. Ishino, Amino Acids, 1992, 3, 243-
252. (b) L. M. Luo, S. Chen, H. F. Jin, C. S. Tang and J. B.
Du, Biochem. Biophys. Res. Commun., 2011, 415, 61-67. (c)
D. P. Li, Z. Y. Wang, X. J. Cao, J. Cui, X. Wang, H. Z. Cui, J. Y.
Miao and B. X. Zhao, Chem. Commun., 2016, 52, 2760-
2763
(a) G. Chen, W. Zhou, C. Y. Zhao, Y. X. Liu, T. Chen, Y. L.
Li and Tang, B. Anal. Chem., 2018, 90, 12442-12448. (b) W.
Zhang, F, Huo and C. Yin, Org. Lett., 2019, 21, 5277-5280.
P. Gao, W. Pan, N. Li and B. Tang, Chem. Sci., 2019, 10,
6035-6071.
(a) M. M. Xing, K. N. Wang, X. W. Wu, S. Y. Ma, D. X. Cao,
R. F. Guan and Z. Q. Liu, Chem. Commun., 2019, 55,
14980-14983; (b) D. X. Cao, Z. Q. Liu, P. Verwilst, S. Koo, P.
Jangjili, J. S. Kim and W. Y. Lin, Chem. Rev., 2019, 119,
10403-10519; (c) L. Y. Niu, Y. S. Guan, Y. Z. Chen, L. Z. Wu,
C. H. Tung and Q. Z. Yang, J. Am. Chem. Soc., 2012, 134,
18928-18931.
λem = 610-670 nm; and Green channel: λem =470-530 nm, λex
=
430 nm.
To further explore the application in biological aspects, we
established a mouse tumor biological model for visualizing the
Cys metabolism. The tumor areas in mice with HeLa cells, as
described in Fig. 3, displayed the weak red and green
fluorescent signals after injection BPC (20 μM). When
exogenous Cys (400 μM) was added to this tumor areas, the
fluorescence signal in red channel reached the maximum after
30 min, followed by declined over time. This phenomenon may
be due to the high level of SO2 produced through by the
enzymaticly metabolic reaction of Cys in tumor areas, resulting
in red fluorescence decreased.
In summary, we firstly synthesized an independent bi-
reversible reactions sensor BPC for precisely dynamic real-time
process visualization of Cys metabolism SO2. Based on the
special FRET mechanism, BPC can emit completely different
fluorescent signal patterns with increased or decreased red
fluorescence corresponding to Cys and SO2. Furthermore,
when coexist of both, a red-to-green signal shift occurred. It is
these superior properties that have led to the successful
application of BPC in subcellular organelle and living mice to
real-time visualizing the biological process of Cys metabolizing
SO2. We expect that probe BPC can be a dynamic monitoring
tool with medical value in preventing related diseases caused
by metabolic disorders of Cys using fluorescence technology.
5.
6.
7.
8.
(a) B.J. Wang, R. J. Liu, J. G. Fang, Y. W. Wang and Y. Peng,
Chem. Commun., 2019, 55, 11762-11765 (b) L. J. Tang, P.
He, X. M. Yan, J. Z. Sun, K. L. Zhong, S. H. Hou and Y. J.
Bian, Sens. Actuators B: Chem., 2017, 247, 421-427; (c) B.
Y. Xu, H. B. Zhou, Q. S. Mei, W. Tang, Y. L. Sun, M. P. Gao,
C. L. Zhang, S. S. Deng and Y. Zhang, Anal. Chem., 2018, 90,
2686-2691.
9.
J. W. Chen, X. Q. Jiang, S. L. Carroll, J. Huang and J. Wang,
Org. Lett., 2015, 17, 5978-5981.
We thank the National Natural Science Foundation of China
10.
11.
12.
Y. Y. Ma, W. J. Gao, L. L. Zhu, Y. P. Zhao and W. Y. Lin,
Chem. Commun., 2019, 55, 11263-11266.
K. E. Sapsford, L. Berti and I. L. Medintz, Angew. Chem., Int.
Ed. 2006, 45, 4562−4588.
(a) H. L. Zhang, P. F. Xu, X.T. Zhang, X. Z. Cao, W. X. Han, M.
H. Liu, X. H. Liu and W. B. Zang, Chin. Chem. Lett., 2020, 31,
1083-1086. (b) Y. Yan, X. D. Zhang, X. F. Zhang, N. Li, H. Z.
Man, L. C. Chen and Y. Xiao, Chin. Chem. Lett., 2020, 31,
1091-1094.
M. H. Stipanuk, J. J. E. Dominy, J.-I. Lee and R. M. Coloso, J.
Nutr., 2006, 136, 1652S-1659S.
(No. 21775096),
Shanxi Province"1331 project" key
innovation team construction plan cultivation team (2018-CT-
1), 2018 Xiangyuan County Solid Waste Comprehensive
Utilization Science and Technology Project (2018XYSDJS-05),
the Shanxi Province Foundation for Selected (No. 2019),
Innovative Talents of Higher Education Institutions of Shanxi,
Scientific and Technological Innovation Programs of Higher
Education Institutions in Shanxi (2019L0031), Key R&D
Program of Shanxi Province (201903D421069), the Shanxi
Province Science Foundation (No. 201901D111015), China
Institute for Radiation Production and Scientific Instrument
Center of Shanxi University (201512).
13.
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