Simultaneous Visualization of Endogenous Homocysteine, Cysteine, Glutathione, and their Transformation through Different Fluorescence Channels

Article abstract of DOI:10.1002/anie.201813935

Studying numerous biologically important species simultaneously is crucial to understanding cellular functions and the root causes of related diseases. Direct visualization of endogenous biothiols in biological systems is of great value to understanding their biological roles. Herein, a novel multi-signal fluorescent probe was rationally designed and exploited for the simultaneous sensing of homocysteine (Hcy), cysteine (Cys), and glutathione (GSH) using different emission channels. This probe was successfully applied to the simultaneous discrimination between and visualization of endogenous Hcy, Cys, GSH, and their transformation in living cells.

Full text of DOI:10.1002/anie.201813935

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Accepted Article
Title: Simultaneous Visualization of Endogenous Hcy, Cys, GSH and
their Transformation through Different Fluorescence Channels
Authors: Guoxing Yin, Tingting Niu, Ting Yu, Yabing Gan, Xinyu
Sun, Peng Yin, Haimin Chen, Youyu Zhang, Haitao Li, and
Shouzhuo Yao
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201813935
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10.1002/anie.201813935
Angewandte Chemie International Edition
COMMUNICATION
Simultaneous Visualization of Endogenous Hcy, Cys, GSH and
their Transformation through Different Fluorescence Channels
Guoxing Yin,†,§ Tingting Niu,‡,§ Ting Yu,†,§ Yabing Gan,† Xinyu Sun,† Peng Yin,*,† Haimin Chen,*,‡
Youyu Zhang,† Haitao Li*,† and Shouzhuo Yao†
Abstract: Studying biologically important species simultaneously is
crucial to understand cellular functions and the root causes of related
diseases. Direct visualization of endogenous biothiols in biological
systems is of great value to understand their biological roles. Herein,
a novel multi-signal fluorescent probe was rationally designed and
exploited for simultaneous sensing of Hcy, Cys and GSH from
different emission channels. This probe was successfully applied to
the simultaneous discrimination and visualization of endogenous Hcy,
Cys, GSH and their transformation in living cells.
quantify endogenous and exogenous biothiols,[1b, 2b, 7] owing to
their similarities in structures and properties and large differences
in their concentrations, only several fluorescent probes that could
simultaneously detect two or three species of Cys/Hcy/GSH at
one time have been reported (Table S1).[3b, 8] To the best of our
knowledge, there is no fluorescent probe that could
simultaneously differentiate and image endogenous Cys, Hcy and
GSH in living cells, partially due to the low concentration of Hcy.[1b]
Herein, based on our previous report,[9] we rationally designed a
novel multi-signal fluorescent probe with background-free, large
fluorescence enhancement and high quantum yield (QY), which
could simultaneously image intracellular Hcy, Cys, GSH and their
dynamic changes from three different emission channels. This
probe exhibited high selectivity and sensitivity for Hcy, Cys and
GSH with a low detection limit of 0.7, 0.2 and 1 nM, respectively.
This study will greatly improve the understanding of their
generations, relationships, functions and metabolisms in
biological systems, and promote the early diagnosis, intervention
and treatment about their related diseases.
Under the guidance of our previous findings,[9] probe 1,
featured with coumarin as fluorophore with twisted N,N-
diethylamino group, will exhibit high quantum yield, fluorescence
background-free, and good biocompatibility.[10] And ethyl 2-
cyanoacrylate group in the probe would improve its solubility and
cell permeability (Scheme 2).[12] Besides, judging from Guo’s work
and our previous research,[8g, 9] chloro atom in the 4-position of
the coumarin moiety could initially be replaced by thiol group of
biothiols to afford thio-coumarin derivatives, and the following
rearrangement would result in amino/thio-coumarin derivatives.
The cyano group at the α position of the Michael addition acceptor
enables fast reaction kinetics for thiol addition,[7c,7d] and C=C bond
The most abundant intracellular biothiols, homocysteine (Hcy),
cysteine (Cys), and glutathione (GSH), are widely linked to
physiological and pathological processes in complex biological
environments.[1] A lot of research has demonstrated that aberrant
levels of Cys/Hcy/GSH are related to a wide range of human
diseases.[2] As shown in Scheme 1, Hcy, derived from methionine,
is condensed with serine catalyzed by cystathionine β-synthase
(CBS) to generate cystathionine, which is further converted to Cys
by cystathionine γ-lyase (CSE).[3] Cys is one substrate for
glutamate cysteine ligase (GCL) and glutathione synthetase (GS)
to yield GSH.[4] Therefore, Hcy levels are tightly associated with
the levels of Cys and GSH in living systems. Fluctuation in the
concentration of one biothiol would influence another. In order to
elucidate the complicated relationship between Hcy, Cys, GSH
and understand the root causes of their related diseases, it is
greatly challenging and significant to simultaneously differentiate
and quantify endogenous Cys, Hcy and GSH in living cells and
human serum.
Recently, compared to conventional analytical methods,[5]
multi-signal fluorescent probes together with confocal
fluorescence imaging techniques are widely applied to multicolor
imaging due to their simultaneous and discriminative monitor of
multiple analytes or dynamic processes for specific analyte as
well as the virtues of fluorescent methods, such as noninvasive,
relative low cost, high sensitivity and high selectivity.[6] Though a
lot of fluorescent probes have been developed to sense and
breakage would be occured to form the intermediate of
12]
imine/iminium for amino addition (Scheme S2).[8j,
Thus,
reactions between probe 1 with Cys, Hcy and GSH could yield
different products with distinct signal outputs. And the
simultaneous discrimination of Hcy, Cys and GSH from different
fluorescence channels could be realized.
[*]
G. Yin, T. Yu, Y. Gan, X. Sun, Dr. P, Yin, Prof. Y. Zhang. Prof. H. Li,
Prof. S. Yao.
Key Laboratory of Chemical Biology and Traditional Chinese
Medicine Research (Ministry of Education), Key Laboratory of the
Assembly and Application of Organic Functional Molecules of
Hunan Province, Hunan Normal University, Changsha, 410081,
China
E-mail: yinpeng@hunnu.edu.cn
haitao-li@hunnu.edu.cn
T. Niu, Prof. H. Chen.
Scheme 1. The compact relationship between Hcy, Cys and GSH.
Key Laboratory of Marine Biotechnology of Zhejiang Province,
Ningbo University, Ningbo, Zhejiang 315211, PR China
E-mail: chenhaimin@nbu.edu.cn
Initially, probe
1 was easily synthesized and well-
[§]
These authors contributed equally to this work.
characterized (Scheme S1, Figures S23-S30). Its sensing
behavior towards biothiols was examined through UV-vis and
fluorescence spectra in DMSO-PBS (pH 7.4, 10 mM, v/v, 4/6) at
Supporting information for this article is given via a link at the end of
the document.
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10.1002/anie.201813935
Angewandte Chemie International Edition
COMMUNICATION
room temperature. As shown in Figure 1a, upon addition of
Cys/Hcy/GSH, the initial absorption peak of probe 1 at 527 nm
was decreased dramatically. For Hcy, two new peaks at 461 and
excellent selectivity of probe 1 for the simultaneous discrimination
of Hcy, Cys, and GSH. In addition, based on thiol specific
addition/rearrangement-cyclization reactions, probe 1 was able to
preclude the cross-talk caused by sulfur-containing protein and
other reactive sulfur species such as SO2, H2S2 and H2S, which
are existed in living cells (Figure S9). Therefore, probe 1 exhibited
superiorities over other fluorescent probes.[2d]
378 nm were emerged. Correspondingly,
a
significant
fluorescence turn-on at 467 nm with 457-fold fluorescence
increment was observed (λex = 375 nm) (Figure 1b). For Cys, an
increasing absorption peak at 397 nm was found. And a large
fluorescence turn-on with 740-fold increment at 503 nm was
observed when excited at 400 nm (Figure 1). Besides, for GSH,
Next, fluorescence titration experiments were conducted
carefully with three different channels. As shown in Figure 2, the
dose-dependent fluorescence enhancement of probe 1 exhibited
good linearity with the concentration of Hcy/Cys in the range from
0 to 30 μM, and the detection limits were calculated to be as low
as 0.7 and 0.2 nM based on S/N = 3, respectively. The
fluorescence intensities were linearly proportional to the amount
of GSH from 0 to 10 μM and 10 to 25 μM with low detection limit
(1 nM). Probe 1 was also suitable to be applied to the mixture of
Cys/Hcy/GSH with different concentrations (Figures S10-S12).
Upon excitation with specific wavelength, each specie from the
mixture of Hcy/Cys/GSH could elicit dramatic fluorescence
enhancement from specific channel. Furthermore, after
sequentially reacted with Hcy/Cys/GSH, probe 1 exhibited
significant fluorescence turn-on in the blue, green and red
fluorescence channels, respectively (Figures S13). Therefore,
probe 1 has the potential to simultaneously discriminate Hcy, Cys,
GSH and monitor their transformation from three different
emission channels in vitro and in complex biological environments.
the initial absorption peak was decreased and
a slight
bathochromic shift was displayed. A new peak was found at 502
nm with a 25 nm blue shift, and the fluorescence spectra for probe
with GSH demonstrated a 115-fold fluorescence enhancement at
568 nm (λex = 500 nm) (Figure 1). Compared to our previous
work,[9] probe 1 exhibited more favorable sensing properties, such
as improved water solubility, minimal fluorescence background
and high quantum yield (Table S2), which are beneficial for
fluorescence imaging of endogenous Cys/Hcy/GSH with different
concentrations.
Scheme 2. The comparison of probe BCC and probe 1.
3200
2400
1600
800
0
2500
2000
1500
1000
500
(b)
(a)
35 μM
0 μM
y = 993.8x + 10.1
R2 = 0.997
0
0.0
0.5
1.0
1.5
CHcy/C1
2.0
2.5
400
(c)
450
450
450
500
550
600
650
700
700
700
Wavelength (nm)
5000
4000
3000
2000
1000
0
5000
4000
3000
2000
1000
0
(d)
35 μM
Figure 1. (a) Absorption spectra of probe 1 (10 µM) upon addition of 10 equiv.
of biothiols (100 µM) at room temperature for 20 min. (b) The corresponding
y = 1559.9x + 11.1
R2 = 0.998
fluorescence spectra of probe 1 to 10 equiv. of Hcy (λex = 375 nm), Cys (λex
=
0 μM
400 nm), and GSH (λex = 500 nm). Condition: DMSO-PBS (pH 7.4, 10 mM, v/v,
4/6). The inset photos show the color changes of probe 1 (10 µM) in the
presence of 10 equiv. of Hcy, Cys and GSH under UV lamp at 365 nm,
respectively.
400
(e)
500
550
600
650
0.0
(f)
0.5
1.0
1.5
CCys/C1
2.0
2.5
3.0
Wavelength (nm)
2000
1600
1200
800
400
0
2000
1500
1000
500
0
y = 465.2x + 783.1
R2 = 0.984
30 μM
In the following, results from time-dependent UV-vis and
fluorescence spectra studies were further demonstrated that
probe 1 could selectively discriminate Hcy, Cys and GSH from
blue, green and yellow fluorescence channels, respectively
(Figures S1-S7). The signals for Hcy, Cys and GSH could reach
a plateau within a short time (15 min), which is beneficial for
practical applications. The selectivity of probe 1 over other
biologically related species were further assessed, including
various amino acids, sulfur-containing compounds (Figure S9).
Multi-signal outputs of probe 1 were only observed for Hcy, Cys
and GSH when excited at 375, 400 and 500 nm, respectively, but
there is no obvious fluorescence change to a series of other
biologically related species. These results demonstrated the
0 μM
y = 1117.6x + 17.3
R2 = 0.997
400
500
550
600
650
0.0
0.5
1.0
CGSH/C1
1.5
2.0
2.5
Wavelength (nm)
Figure 2. Fluorescence intensity spectra of 1 (10 μM) in the presence of (a) 0
to 35 μM Hcy excited at 375 nm, (c) 0 to 35 μM Cys excited at 400 nm and (e)
0 to 30 μM GSH excited at 500 nm, respectively. The linear changes of the
fluorescence intensity of probe 1 at 467, 503 and 568 nm and as a function of
(b) Hcy, (d) Cys and (f) GSH concentration, respectively. Condition: DMSO-PBS
(pH 7.4, 10 mM, v/v, 4/6) at room temperature for 20 min. Slit (nm): 2.5/2.5.
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10.1002/anie.201813935
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In order to understand the reaction mechanism between
probe 1 and Cys/Hcy/GSH, several control compounds (C5-C9)
were synthesized (Scheme S2), and characterized (Figures S31–
S40). Judging from the LC-MS and HRMS spectra from the
reactions between probe 1 with Hcy/Cys/GSH and absorption
spectra of these control compounds (Figures S8 and S14-S16),
reasonable reaction mechanisms were proposed for probe 1 with
Hcy/Cys/GSH (Scheme S2).[11] The isolation of these products
was failed, which may be due to their instabilities. Further
mechanism studies are still investigated in our lab.
Subsequently, simultaneous visualizations of endogenous
Hcy, Cys and GSH in living cells using probe 1 were conducted.
Concentration of total homocysteine is normally below 15 uM and
free homocysteine is minimal,[1b] so there is no report about
fluorescence imaging of endogenous Hcy in living cells. Featured
with high quantum yield (QY1-Hcy = 0.13), large fluorescence
enhancement (457 fold for Hcy) and high sensitivity (LOD = 0.7
nM for Hcy), probe 1 would be suitable to monitor endogenous
Hcy in living cells. In the advantage of low toxicity and good cell
permeability of probe 1 (Figure S17), pretreated with N-
ethylmaleimide (NEM, scavenger of SH compounds), almost no
fluorescence could be observed after incubated with probe 1
(Figure S18, A1-A3). While, after subsequently treated with Hcy,
Cys and GSH respectively, blue, green and red fluorescences
were observed from three different emission channels (Figure
S18, B-D, and Figure S19), which demonstrated that fluorescence
imaging of Hcy, Cys and GSH simultaneously could be achieved
with high selectivity. After cells were only incubated with probe 1,
blue, green and red fluorescence images were obtained for the
monitor of cellular Hcy, Cys and GSH in different living cells,
respectively (Figure 3). These results exhibit excellent
perspective for the simultaneous and quantitative fluorescence
analysis of these important biothiols in living cells and human
serum.
Dynamic fluorescence imaging for biothiols was further con-
ducted. NEM was firstly used to eliminate SH-containing com-
pounds and proteins in living cells. When only incubated with
probe 1 in living cells, almost no fluorescence was observed from
three emission channels up to 120 min (Figure S20). However,
after incubated with exogenous Hcy and probe 1, blue and green
fluorescence could be observed from different channels after 30
min. Green fluorescence was weakened along with the prolonged
incubation time. While red fluorescence will be appeared after
incubated for 60 min, then weakened (Figure 4). Meanwhile, when
cells were incubated with exogenous Cys and probe 1,
fluorescence changes could be observed from green and red
fluorescence channels (Figure S21). These results demonstrated
the transformation for Hcy, Cys and GSH was proceeded. And
dynamic fluorescence images of Hcy/Cys/GSH were successful
for the first time. In addition, it was found that this probe could be
used to detect Hcy and Cys in algal cells. Pyropia haitanensis was
taken as the experimental object. When it was stimulated by
different stresses, different antioxidant states within the cells
could be reflected from fluorescence images, but the red
fluorescence was disturbed due to the effect of algal
phycoerythrin (Figure S22).
Figure 4. Confocal fluorescence images of Hcy in BEL-7402. (A-E) Cells were
pretreated with NEM (0.25 mM, 30 min), then treated with probe (5 μM, 30 min),
Hcy (100 μM) for 10, 30, 60, 90 and 120 min respectively, then imaged. (λex
=
405 nm, λem = 421 - 475 nm for the blue channel; λex = 458 nm, λem = 500 - 550
nm for the green channel; and λex = 543 nm, λem = 552 - 617 nm for the red
channel). Scale bar: 10 μm.
Figure 3. (A) BEL-7402, (B) L-02 cells and (C) Raw 264.7 were incubated with
probe 1 for 30 min, then imaged. (A4-C4) represent the bright field images of
A1-C1, respectively. (λex = 405 nm, λem = 421 - 475 nm for the blue channel; λex
= 458 nm, λem = 500 - 550 nm for the green channel; and λex = 543 nm, λem =552
- 617 nm for the red channel). Scale bar: 20 μm.
In conclusion, a novel multi-signal fluorescent probe 1 has
successfully designed and synthesized. Based on different
reactions between probe 1 with Cys/Hcy/GSH, simultaneous
discrimination and imaging of endogenous Hcy, Cys, GSH and
their transformation via three emission channels in different living
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10.1002/anie.201813935
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Acknowledgements
This work was supported by the National Natural Science
Foundation of China (Grant No. 21877035, 31872540 and
21675051); Opening Fund of Key Laboratory of Chemical Biology
and Traditional Chinese Medicine Research (Ministry of
Education), Hunan Normal University; Science and Technology
Planning Project of Hunan Province (2018TP1017); K.C. Wong
Magna Fund in Ningbo University and Ningbo Programs for
Science and Technology Development (2017C110026).
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Keywords: endogenous biothiols • transformation • fluorescent
probe • simultaneous sensing
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COMMUNICATION
COMMUNICATION
Guoxing Yin, Tingting Niu, Ting Yu,
Yabing Gan, Xinyu Sun, Peng Yin,*
Haimin Chen,* Youyu Zhang, Haitao Li*
and Shouzhuo Yao
A multi-signal fluorescent probe was
exploited for simultaneously
differentiating and sensing of
endogenous homocysteine, cysteine,
glutathione and their transformation in
living cells.
Page No. – Page No.
Simultaneous Visualization of
Endogenous Hcy, Cys, GSH and their
Transformation through Different
Fluorescence Channels
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