A multi-signal mitochondria-targeted fluorescent probe for real-time visualization of cysteine metabolism in living cells and animals

Article abstract of DOI:10.1039/c8cc05418e

In this study, we developed a multi-signal mitochondria-targeted fluorescent probe (NIR-Cys) for simultaneous detection of Cys and its metabolite, SO₂. In the design of the probe, the acrylate group and the CC of the coumarin ring were used as the recognizing moiety for Cys and SO₂, respectively. The probe exhibited high sensitivity, excellent specificity, and fast response. NIR-Cys was found to precisely target and visualize Cys metabolism in mitochondria of living cells with a multi-fluorescence signal. This probe is expected to be a useful tool for understanding Cys metabolism.

Full text of DOI:10.1039/c8cc05418e

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A multi-signal mitochondria-targeted fluorescent probe for real-
time visualizing cysteine metabolism in living cells and animals
Xiaopeng Yanga, Wenya Liua, Jun Tanga, Ping Lib, Haibo Wengc*, Yong Yea*, Ming Xiand, Bo Tangb
and Yufen Zhaoa
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
In this study, we developed a multi-signal mitochondria- sensitivity and simplicity, fluorescent probes have been widely used
targeted fluorescent probe (NIR-Cys) for simultaneous detection to monitor biomolecules as well as to visualize metabolic
of Cys and its metabolite, SO2. In the design of the probe, the processes11
. Recently, some fluorescent probes for separate
acrylate group and C=C of coumarin ring were used as the detection of Cys and SO2 have been developed12, but few probes
recognizing moiety for Cys and SO2, respectively. The probe achieved the visualization of Cys metabolism. For example, Yin et al.
exhibited high sensitivity, excellent specificity, and fast response. reported a fluorescent probe for real-time visualizing Cys in living
NIR-Cys was found to precisely target and visualize Cys A549 cells13. But Yin’s probe cannot be used to detect both Cys and
metabolism in mitochondria of living cells with multi-fluorescent SO2. Zhang et al. reported a ratiometric fluorescent probe for SO2
signal. This probe is expected to be a useful tool for understanding detection in hepatic BRL cells. However, Zhang’s probe cannot be
Cys metabolism.
used to detect Cys14. As such, fluorescent probes that can directly
visualize cysteine-SO2 metabolism with high accuracy and resolution
using multi-fluorescence signals are urgently needed. Those probes
will be crucial in understanding the roles of cysteine metabolism in
physiological and pathological conditions.
As the only mercapto (-SH) containing amino acid, cysteine
(Cys) plays crucial roles in a wide variety of physiological and
pathological processes such as protein synthesis, signal
transduction, post-translational modification, diverse redox
chemistry, and metabolism1-3
.
For instance, abnormal
Mitochondria are "power house" and the main source of
concentrations of Cys can lead to various diseases4-7, including liver reactive sulfur species (RSS) and reactive oxygen species (ROS) 15. As
damage, neurotoxicity, hematopoiesis, skin lesions, slowed growth, a vital antioxidant reservoir to regulate ROS homeostasis, Cys is
Alzheimer’s, and Parkinson’s. It has been reported that metabolic always presented in mitochondria. Under oxidative stress, Cys may
pathways of Cys can be divided into two types8. One is the cysteine be converted to SO2. But detailed mechanism and process are still
sulfinate pathway that Cys is oxidized by cysteine dioxygenase unclear. In addition, Cys plays a major role in mitochondrial protein
(CDO) to form cysteinesulfinate, and then enzymatically translates turnover. Therefore, to obtain detailed information on Cys
to β-sulfinylpyruvate, through transamination by aspartate metabolism in mitochondria, the development of fluorescent
aminotransferase
(AAT).
β-Sulfinylpyruvate
spontaneously imaging for real-time observing Cys metabolism in living cells is very
decomposes to SO2 and pyruvate. The other is the desulfuration essential. To the best of our knowledge, fluorescent probes that
metabolism that Cys undergoes 3-mercaptopyruvate pathway in allow direct detection of Cys-SO2 metabolism in mitochondria have
mitochondria without initial oxidation of the sulfur atom (Scheme not been reported.
1A). Although the research on Cys metabolism has received
attention9, simple visualization methods for monitoring the process
To fulfil this gap, herein we report a multi-signal fluorescent
probe for real-time monitoring cysteine-SO2 metabolism in
mitochondria of living cells and animals (Scheme 1B). In the design
of our probe, acrylate group was selected as the recognizing moiety
for Cys. The reaction between Cys and acrylates results a conjugate
addition to produce thioethers, and then an intramolecular
cyclization to release the fluorophore NIR-OH. Meanwhile, sulfite
can add to the C=C of coumarin ring via a unique nucleophilic
addition, and the fluorescence signal should change from red to
green which is dominated by the left benzopyran structure. If Cys
and SO2 co-exist, the response at different wavelengths should be
observed. As expected, the probe showed high sensitivity, excellent
selectivity, and rapid responses for Cys and SO2. It has been
successfully employed in real-time monitoring Cys metabolism in
vivo. As shown in Scheme S1, NIR-Cys was synthesized from
are still lacking. Molecular imaging technology based on fluorescent
probes can enable visualization of biologically relevant species in
cells, tissues and organisms, and can provide useful information
about the biological effects of the analytes10. Owing to their high
a.College of Chemistry and Molecular Engineering, Zhengzhou University,
Zhengzhou 450001, China. E-mail: yeyong03@tsinghua.org.cn
b.College of Chemistry, Key Laboratory of Molecular and Nano Probes, Ministry of
Education, Shandong Normal University, Jinan 250014 , China.
c. School of Life Science, Zhengzhou University, Zhengzhou 450001, China.
d.Department of Chemistry, Washington State University, Pullman, WA 99164, USA.
† Footnotes relating to the title and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
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coumarin in three steps with excellent yields. The probe was fully reaction product was characterized by mass spectrometry. A
characterized by 1H NMR, 13C NMR and HR-MS.
dominant peak at m/z 482.1601 was attributed to NIR-OH. The
proposed sensing mechanism is shown in Scheme S2 based on the
MS analysis (Figure S6) and previous literature17
.
We also found that NIR-Cys could react with HSO3–rapidly (50
–
s) (Figure S7B). After treating with HSO3 (100 µΜ), a new
absorption peak at 470 nm emerged, accompanied with the peaks
at 350 and 620 nm gradually decreased as shown in Figure S8. The
probe NIR-Cys alone displayed weak fluorescence emission at 560
nm. However, the addition of HSO3– into the solution of NIR-Cys led
to a distinct fluorescence increase at 560 nm (Figure S7A). The
–
Scheme 1. (A) Metabolic pathways of Cys in mitochondria. (B) Design of the
fluorescent probe for real-time visualizing Cys-SO2 metabolism.
detection limit for HSO3 based on the 3σ/slope was 11.0 nM
–
(Figure S9). The HSO3 sensing mechanism was also confirmed by
MS as shown in Scheme S3 and Figure S10. A dominant peak at m/z
616.1306 was attributed to NIR-cys-SO3. It should be noted that
although the probe NIR-Cys can reacts with HSO3–, the level of Cys is
much higher than that of HSO3– in living cells4,13. So NIR-Cys can
detect Cys with high selectivity and generate NIR-OH, which can
then monitor the level of SO2 in the process of Cys metabolism.
With the probe in hand, its spectroscopic performance was
evaluated in HEPES buffer (pH= 7.4, 10 mM). As shown in Figure S1,
the probe itself exhibited a central absorption at 570 nm and
emission maximum at 656 nm with weak fluorescence. Upon the
addition of 10 equiv. Cys, a new maximum absorption peaks red
shift to 620 nm, and the fluorescence intensity at 656 nm increased
significantly. Time-dependent responses of NIR-Cys showed that the
reaction between the probe (10 µΜ) and 10 equiv. of Cys
completed within 10 min (Figure 1A, inset). The fluorescent titration
of NIR-Cys toward Cys at low concentrations were conducted and
the detection limit (Figure S2) was calculated to be 30 nM
(according to the 3σ/slope), which appeared to be sensitive than
the reported Cys probes12d,16. The pH effect on fluorescence
property of NIR-Cys (10 µΜ) toward Cys (100 µΜ) was also
investigated. The fluorescence intensities were all obviously
increased at 656 nm in the range of pH 7−10 (Figure S3). Those
results indicated that NIR-Cys was suitable for real-time imaging Cys
in real samples.
NIR-OH is the intermediate produced from the probe (NIR-Cys)
reacting with Cys. We next investigated its spectroscopic properties.
As shown in Figure S11A, the maximum absorption peak was moved
from 620 to 460 nm with the color changing from blue to yellow. A
nucleophilic addition of HSO3- to the C=C of coumarin ring in NIR-OH
took place, and the fluorescence emission of NIR-OH (10 µΜ) at 664
nm gradually decreased while a new emission appeared at 550 nm
(Figure S12). The emission ratio (F550/F664) showed a good linear
relationship to the concentration of HSO3– (R2=0.9923) (Figure S13).
The detection limit was calculated to be 3.7 nM which far below
endogenous SO2 concentration (0-9.85 µM)3. Time-dependent
emission change studies showed that the emission ratio of NIR-OH
reached the maximum plateau within 90 s upon the addition of
–
HSO3– (Figure 1B). pH effects on the emission ratio of NIR-OH to
HSO3 were evaluated (Figure S14), and the suitable pH range for
–
HSO3 detection was found to be 5-11. These results indicate that
–
NIR-OH could be used for the real-time detection of HSO3 in living
cells.
The responses of NIR-OH toward representative relevant
species were also studied. No significant change was observed in
the (F550/F664) emission ratio after it was treated with other
analytes. Only with the addition of HSO3–, we could observe a
noticeable response (Figure 1B). Furthermore, competitive
experiments demonstrated that competitive species had no
interference on the enhancement of (F550/F664) emission ratio
(Figure S15). These results indicate that NIR-OH has an excellent
Figure 1. (A) The fluorescence intensity of NIR-Cys (10 µM) with Cys (10
equiv.) and other biologically relevant species (10 equiv.). (Including：NIR-
Cys, SO24–, S2O23–, S2O28–, NO3– , NO2– ,Ser, Hcy, 5 mM GSH, ClO–, ClO4– , H2O2, O–2 ,
1O2, ·OH, Na+, Zn2+, Ca2+, Ba2+, Cu2+, Fe2+, Mn2+, Pb2+, Hg2+, Sn2+, Al3+) (B) The
fluorescence intensity of probe NIR-OH (10 µM) with HSO–3 (10 equiv.) and
other biologically relevant species (10 equiv.).(Including：NIR-OH, F–, Cl–, Br–
, I–, CO23–, HCO3– , ClO–, ClO4– , AcO–, PO–4 , HPO4– , H2PO4–, S2O23–, S2O28–, NO3– , NO2–, SO
24–, HS-, SO24–, Cys, Hcy, GSH).
–
selectivity for HSO3 over other biologically relevant species. To
prove the sensing mechanism (Scheme S4), the product of NIR-OH
–
with HSO3 was characterized by HR-MS and the desired peak
(m/z=562.1177) was obtained (Figure S16). 1H NMR titration
experiment further confirmed the nucleophilic addition of sulfite to
the C=C of coumarin ring (Figure S17).
We next evaluated the selectivity and interference of NIR-Cys
for Cys detection. In this study, the probe was incubated with
biologically relevant species such as amino acids (Figure S4),
different ROSs, RSSs and ions (Figure 1A). The probe only showed
slight fluorescence increase at 656 nm in the presence of these
interfering analytes (Figure S5). In contrast, fluorescence intensity
at 656 nm significantly increased upon the addition of Cys (100
µΜ). Furthermore, competitive experiments indicated that other
species had no interference for Cys detection. Thus, the probe has
the potential to detect Cys with excellent selectivity in complex
biological environments. To confirm the sensing mechanism, the
Having demonstrated NIR-Cys and NIR-OH’s sensing ability to
Cys and SO2 in buffers, these two compounds were then applied in
cell imaging. Using the standard CCK-8 assay, NIR-Cys was found to
be non-toxic (or low toxic) for cells (Figure S18). The primary
location where Cys was metabolized to form SO2 is mitochondria.18
We first exploited intracellular location of NIR-Cys. MCF-7 cells were
incubated with NIR-Cys and Mito-Tracker Green FM for co-
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localization experiment (Figure S19). The images of the green and When zebrafish was further treated with NaHSO3 for another 30
red channels overlapped well with a high Pearson’s correlation of min, an obvious fluorescence increase in green channel and
0.9207 and overlap coefficient of 0.9119 demonstrating that NIR- decrease in red channel were observed. These results
Cys stains mainly in mitochondria. This may be due to the charge demonstrated that NIR-Cys could detect Cys and SO2 in live
attraction between NIR-Cys with a positive charge and cellular Zebrafish.
mitochondrial membrane with the negative potential19. The results
indicated that NIR-Cys could potentially imaging Cys and SO2 in
mitochondria. Secondly, we evaluated the potential ability of the
probe to visualize Cys and SO2 in living cells with multi-signal
fluorescence. As shown in Figure 2a, MCF-7 cells only treated with
NIR-Cys exhibited distinct fluorescence in red channel and non-
fluorescence in green channel. When the cells were pretreated with
NEM, and then incubated with NIR-Cys, weak fluorescence in red
channel was noted (Figure 2b). When the cells were pretreated with
increasing dosages of Cys and then incubated with NIR-Cys, a
significant fluorescence increase in red channel was observed
(Figure 2c). These results indicated that NIR-Cys could sense
intracellular and endogenous Cys. As expected, when cells were
pretreated with Cys and NIR-Cys, and then incubated with NaHSO3,
fluorescence in green channel was significantly increased and red
Figure 2. Fluorescent imaging of NIR-Cys responding to Cys or NaHSO3 in
living MCF-7 cells. Cells were incubated with (a) NIR-Cys (10 μM, 30 min); (b)
fluorescence was gradually quenched at the same time (Figure 2d).
We further investigated two-photon fluorescence imaging of NIR-
Cys in living cells (Figure S20). The two-photon fluorescence imaging
was consistent with the one photon fluorescence imaging. These
results demonstrated that the probe was effective for imaging Cys
and SO2 in cellular environments.
NEM (500 μM, 30 min), subsequently incubated with NIR-Cys (10 μM, 30
min); (c) Cys (100 μM, 30 min), subsequently incubated with NIR-Cys (10
μM, 30 min); then incubated with NaHSO3 (100 μM, 30 min) (d). (green
channel of 500-590 nm, red channel of 610-700 nm)
We next tried to visualize Cys metabolism in zebrafish using
NIR-Cys (Figure 3). Zebrafish was pretreated with Cys for 30 min
which should accelerate Cys metabolism to produce SO2. NIR-Cys
was then loaded. A notable fluorescence decrease in red channel
and a dramatic fluorescence increase in green channel were found
in the following 120 min. It is the first example imaging Cys
metabolism in living animals.
Next, we evaluated the application of NIR-OH for imaging SO2
in biological environment. As shown in Figure S21, when MGC-803
cells were incubated with NIR-OH (10 µΜ), strong fluorescence
emission in red channel was observed and almost non-fluorescence
in green channel was found. However, upon the addition of 100 µΜ
NaHSO3, red fluorescence was almost quenched and green
fluorescence was distinctly increased. To further real-timely
–
visualizing HSO3 of NIR-OH in living cells, time-dependent confocal
images were conducted (Figure S22). The MCF-7 cells were
pretreated with NIR-OH, and then incubated with NaHSO3. The
fluorescence emission enhanced in green channel was very obvious,
and emission in red channel almost fully quenched. Above results
illustrate that NIR-OH is an efficient candidate for imaging SO2 in
living cell.
20
Previous literature9b,
reported that CDO's activity can be
enhanced by high levels of Cys. This causes quick generation of SO2.
We thus wondered if NIR-Cys could monitor Cys metabolism in real
time. As shown in Figure S23, time-dependent fluorescence
increase in green channel and decrease in red channel were
observed (0-240 min). The real time imaging process was also
recorded with video (provided in SI). The results demonstrated that
cysteine sulfinate pathway was induced by CDO. NIR-Cys was
capable of visualizing the metabolism of Cys by multi-fluorescence
signal in mitochondria of living cells.
Figure 3. Confocal fluorescence images of zebrafish incubated with 10 mM
of cysteine for 30 min and then treated with NIR-Cys (10 μM) for different
times. (green channel of 500-590 nm with excitation 488 nm, red channel of
610-700 nm at 552 nm)
Because the fluorescence emission of NIR-Cys for Cys located
in NIR region, we further examined its capability for visualizing Cys
metabolism in Kunming mice. As shown in Figure 4, three mice (~20
g) were separately treated with PBS buffer (100 μL, left), NEM (100
μL, 1 mM, middle) or Cys (100 μL, 100 μM, right) via intraperitoneal
injection. Then these animals were subcutaneously injected with
NIR-Cys (100 μL, 10 μM). The fluorescence emission of left mouse
was less significant than the emission of right mouse that was
exogenous injected with Cys. Moreover, fluorescence signals in
both animals reached maximum in 30 min. In contrast, negligible
fluorescence emission was observed in middle mouse. We also
validated NIR-OH’s response to SO2 in mice. The right mouse was
Zebrafish is a highly valuable model for in vivo imaging21. We
then examined the feasibility of visualizing Cys metabolism by NIR-
Cys in zebrafish. As shown in Figure S24, zebrafish incubated with
only NIR-Cys displayed red fluorescence and almost no fluorescence
in green channel. In the negative control experiment, the NEM-
pretreated zebrafish performed a much dimed fluorescence in both
green and red channels. When zebrafish was pretreated with Cys
and then incubated with NIR-Cys, strong fluorescence in red
channel and non-fluorescence in green channel were observed.
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