A Dual-Response Fluorescent Probe Reveals the H2O2-Induced H2S Biogenesis through a Cystathionine β-Synthase Pathway

Article abstract of DOI:10.1002/chem.201502832

The two signaling molecules H₂S and H₂O₂ play key roles in maintaining intracellular redox homeostasis. The biological relationship between H₂O₂ and H₂S remains largely unknown in redox biology. In this study, we rationally designed and synthesized single- and dual-response fluorescent probes for detecting both H₂O₂ and H₂S in living cells. The dual-response probe was shown to be capable of mono- and dual-detection of H₂O₂ and H₂S selectively and sensitively. Detailed bioimaging studies based on the probes revealed that both exogenous and endogenous H₂O₂ could induce H₂S biogenesis in living cells. By using gene-knockdown techniques with bioimaging, the H₂S biogenesis was found to be majorly cystathionine β-synthase (CBS)-dependent. Our finding shows the first direct evidence on the biological communication between H₂O₂ (ROS) and H₂S (RSS) in vivo. CBS is responsible: A fluorescent probe with two heads for H₂O₂ and H₂S reveals that both exogenous and endogenous H₂O₂ molecules can induce endogenous H₂S biogenesis on a cystathionine β-synthase (CBS) pathway (see scheme).

Full text of DOI:10.1002/chem.201502832

DOI: 10.1002/chem.201502832
Communication
&
Fluorescent Imaging
A Dual-Response Fluorescent Probe Reveals the H2O2-Induced H2S
Biogenesis through a Cystathionine b-Synthase Pathway
Long Yi,*[a] Lv Wei,[b] Runyu Wang,[b] Changyu Zhang,[a] Jie Zhang,[a] Tianwei Tan,*[a] and
Zhen Xi*[b]
l-cysteine (l-Cys) by cystathionine b-synthase (CBS), cystathio-
Abstract: The two signaling molecules H2S and H2O2 play
nine g-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase
key roles in maintaining intracellular redox homeostasis.
(3MPST)/cysteine aminotransferase (CAT).[5] The endogenous
The biological relationship between H2O2 and H2S remains
H2S level is correlated with numerous diseases, including the
largely unknown in redox biology. In this study, we ration-
symptoms of Alzheimer’s disease, Down syndrome, diabetes,
ally designed and synthesized single- and dual-response
and liver cirrhosis.[5]
fluorescent probes for detecting both H2O2 and H2S in
It is intriguing that the redox atomosphere in the cell should
living cells. The dual-response probe was shown to be ca-
be balanced to maintain the normal cell growth. The bioba-
pable of mono- and dual-detection of H2O2 and H2S selec-
lance proposals include the possible mutual inducement on
tively and sensitively. Detailed bioimaging studies based
the production of ROS and RSS. Many attempts have been ini-
on the probes revealed that both exogenous and endoge-
tiated to understand this redox biobalance in the molecular
nous H2O2 could induce H2S biogenesis in living cells. By
level.[6–8] For example, exogenous H2S has been reported to
using gene-knockdown techniques with bioimaging, the
exert an antioxidant effect against H2O2-induced oxidative
H2S biogenesis was found to be majorly cystathionine b-
stress and senescence in living cells.[6] CBS can be post-transla-
synthase (CBS)-dependent. Our finding shows the first
tionally activated in response to oxidative stress.[8] However,
direct evidence on the biological communication between
this biological regulation’s pertinence to H2S generation is not
H2O2 (ROS) and H2S (RSS) in vivo.
yet clear.[5a] Fluorescent probes should be excellent tools for
real-time monitoring of these ROS and RSS in vivo.[9] To clarify
the intricate biological relationship between H2O2 and H2S, the
Cellular redox balance plays an important role in various phys-
iological processes.[1] Endogenous small molecules are involved
in maintenance of intracellular redox homeostasis including
the reactive oxygen species (ROS) and reactive sulfur species
(RSS). Hydrogen peroxide (H2O2) is the best described signaling
ROS molecule,[2] and its excessive production is implicated with
various diseases including cancer, diabetes, cardiovascular, and
neurodegenerative disorders.[3] Hydrogen sulfide (H2S) as one
of RSS and gasotransmitters has recently been demonstrated
to exert protective effects including preservation of mitochon-
drial function, protection of neurons from oxidative stress, and
inhibition of apoptosis.[4] H2S is proposed to be produced from
development of a fluorescent probe that can selectively sense
both the signaling molecules to give different fluorescent
colors will be highly valuable. The dual-response probe can
avoid inhomogenous intracellular distribution from two differ-
ent probes and can be excitated by a single excitation for ra-
tiometric imaging.
Chang et al. developed cell-trappable fluorescent probes for
imaging endogenous H2S production upon VEGF-stimulation,
which was reported to be dependent on NADPH oxidase-de-
rived H2O2 but not exogenous H2O2.[10] We have been interest-
ed in the biodetection of RSS for some time.[11] Our H2S probes
were developed for in situ visualizing cysteine-dependent H2S
biogenesis in living cells.[12] Herein, a series of single- and dual-
response fluorescent probes were developed for live capture
of H2O2 and/or H2S selectively in vivo. Our bioimaging with
RNA interference (RNAi) experiments revealed that both of
exogenous and endogenous H2O2 molecules can induce CBS-
dependent H2S production in living cells, which provides the
direct evidence on the crosstalk between H2O2 (ROS) and H2S
(RSS) in living human cells.
[a] Prof. Dr. L. Yi, C. Zhang, J. Zhang, Prof. Dr. T. Tan
State Key Laboratory of Organic-Inorganic Composites
Beijing University of Chemical Engineering (BUCT)
15 Beisanhuan East Road, Chaoyang District, Beijing 100029 (P. R. China)
E-mail: yilong@mail.buct.edu.cn
twtan@mail.buct.edu.cn
[b] L. Wei,+ R. Wang,+ Prof. Dr. Z. Xi
Department of Chemical Biology
State Key Laboratory of Elemento-Organic Chemistry
National Engineering Research Center of Pesticide (Tianjin)
Collaborative Innovation Center of Chemical Science
and Engineering (Tianjin), Nankai University, Weijin Road 94
Tianjin 300071 (P. R. China)
H2O2-mediated conversion of arylboronates to phenols de-
veloped by the Chang’s group is a general reaction-based
strategy for the design of H2O2 fluorescent probes;[13] and
azido-based fluorescent probes have been developed to image
H2S in living cells.[10,11,14] To fluorescently sense both H2S and
H2O2, we synthesized a dual-response FRET (fluorescence reso-
nance energy transfer) probe 1 that contained a pinacol boro-
E-mail: zhenxi@nankai.edu.cn
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201502832.
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Figure 1. Structures of probes 1–4 and their reactions with H2O2 and/or H2S.
nate moiety at the FRET donor (H2O2 trapper) and an azido
moiety at the FRET acceptor (H2S trapper) (Figure 1). It is noted
that the H2O2 trapper should be at FRET donor, because we
intend to observe the H2O2-induced H2S production by FRET-
based ratiometric imaging. Reaction of probe 1 with H2O2 or/
and H2S leads to a fluorescent turn-on response of FRET donor
or/and acceptor, respectively. Compounds 2–4 were specific
probes for H2S or H2O2. Probe 1 could be conveniently pre-
pared by successive coupling of azido-capped rhodamine 5, pi-
perazyl linker, and coumarin fluorophore 7 (Scheme S1, Sup-
porting Information). Then the arylboronate was installed at
the last step to 8 to produce two isomers, which could be pu-
rified by column chromatography. Probes 1–4 were synthe-
ure 2D), while nearly no emission at 525 nm was observed
with excitation at 488 nm (Figure S6, Supporting Information).
The treatment of 1 with H2S led to turn-on fluorescence at
525 nm with excitation at 488 nm (Figure 2E), but no signifi-
cant change with excitation at 400 nm (Figure S7, Supporting
Information). The titrations of 1 with H2O2 or H2S further sup-
ported the selectivity for both molecules (Figures S8 and S9,
Supporting Information).
We then investigated the fluorescence response of 1 by ad-
dition of H2O2 followed by H2S. As shown in Figure 2F, addition
of H2O2 to 1 triggered a large fluorescence enhancement at
460 nm with excitation at 400 nm. After further addition of
H2S, a significant enhancement at 525 nm was observed. The
decreased signal at 460 nm should be due to the FRET effect
from donor to acceptor, and the FRET product has been identi-
fied by HRMS (see the Supporting Information). The fluores-
cent change (color) of 1 upon tandem treatment with H2O2
and H2S can also be observed by the naked eye under a UV
lamp (Figure 2G). Moreover, the time-dependent fluorescence
response of 1 by addition of both H2O2 and H2S was tested
(Figure S10, Supporting Information). The results indicated that
probe 1 can respond to the both signal molecules simultane-
ously to give different emission peaks. These results imply that
1 is a single-wavelength-exited dual-responsive probe, which
can be used for detection of both H2O2 and H2S simultaneous-
ly. The fluorescence response of 1 by addition of H2S followed
by H2O2 also gave dual-emission peaks (Figure S11, Supporting
Information), though the treatment of 1 with H2S could not
lead to obvious turn-on fluorescence with excitation at 400 nm
(Figure S7 and S11, Supporting Information). Therefore, the
stimulant H2O2 should react with the FRET donor part of the
dual-response probe for dual-emission detection.
1
sized and characterized by H and 13C NMR spectroscopy and
HRMS (see the Supporting Information for detailed informa-
tion).
Firstly, the UV/Vis absorption spectra were examined for
probes 1–3 and their reactions with H2O2 and/or H2S (Figure 2
and Figures S1 and S2, Supporting Information). After treat-
ment with H2O2, the probe 3 exhibited a time-dependent in-
creased absorption at 405 nm (Figure S1, Supporting Informa-
tion); while no obvious absorption change was observed for
that of H2S (using Na2S as an equivalent). We also observed
the time-dependent absorption change at 500 nm for probe 2
through treatment with H2S, but not with H2O2 (Figure S2, Sup-
porting Information). These results indicated that the boro-
nate-based and azide-based reaction sites have good selectivi-
ty toward H2O2 and H2S, respectively. As expected, the probe
1 as a combination cassette of 2 and 3 displayed absorption
changes at both 405 and 500 nm upon treatment with tandem
H2O2 and H2S (Figure 2A–C).
Probes 1–4 were further characterized by a series of fluores-
cent tests. Single-response probes 2–4 displayed a significant
fluorescent turn-on response upon treatment with their specif-
ic activator (H2O2 or H2S) in PBS buffer (Figures S3–S5, Support-
ing Information). For dual-response probe 1, H2O2 elicited in-
tense emission at 460 nm with excitation at 400 nm (Fig-
To examine the selectivity, probe 1 was incubated with dif-
ferent ROS and RSS including NaOCl, tert-butyl hydroperoxide
(TBHP), glutathione (GSH), homocysteine (Hcy), and Cys (Fig-
ure 2H,I). Subsequently the fluorescence signal was measured
using different emission channels. The experimental results in-
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Figure 2. Fluorescence response of the probe 1 toward H2O2 and/or H2S. Time-dependent absorption spectra of 1 (10 mm) in the presence of A) 1 mm H2O2 or
B) 1 mm Na2S or C) 1 mm H2O2 followed by 2 mm Na2S. Time-dependent emission spectra of 1 (1 mm) upon reaction with D) 200 mm H2O2 or E) 200 mm H2S or
F) 100 mm H2O2 followed by 200 mm H2S. The excitation wavelengths were 400, 488, 400 nm for (D–F), respectively. G) Photographs under UV lamp (365 nm)
of 1 in the absence (lane 1) or presence of H2O2 (lane 2) or H2O2 followed by H2S (lane 3). H,I) Relative fluorescence responses of 1 (1 mm) to various biological-
ly relevant species (GSH, 5 mm; Cys, 1 mm; Hcy, 1 mm; Na2S, 100 mm; FeCl3, 100 mm; TBHP, 100 mm; NaClO, 100 mm; H2O2, 100 mm) in PBS (pH 7.4) with H) exci-
tation at 400 nm and emission at 460 nm or I) excitation at 488 nm and emission at 520 nm.
dicated that 1 can be used to selectively detect H2O2 and/or
H2S at different emission wavelengths, making it a potential
valuable tool for dual-color visualization of both H2O2 and H2S.
To investigate the biological relationship between H2O2 and
H2S, human embryonic kidney 293 cells (HEK293) were selected
as the model biological system, because endogenous H2S
could be produced from the cells.[12,15] Firstly, we tested the in-
tracecullar fluorescence in the presence of probes and exoge-
nous H2S or H2O2. The addition of both 1 and H2S resulted in
a significant fluorescence increase at the green channel but
not at the blue channel (Figure 3B), indicating that probe
1 could be used to image H2S in living cells. When the living
cells were treated with H2O2, fluorescent turn-on signals at
both the blue and green channels were observed, implying
that 1) H2O2 can enter into cells to trigger blue fluorescence of
probe 1 and 2) H2O2 can directly induce endogenous produc-
tion of H2S from the living cells. The single-response probe 2
was further employed for quantification of the H2O2-induced
H2S production (Figure 3C and Figure S12, Supporting Informa-
tion). The results indicated that higher concentration of exoge-
nous H2O2 could trigger stronger green fluorescence inside the
living cells, implying more endogenous H2S molecules were
produced from the living cells. It is well known that living or-
ganisms have evolved complicated molecular systems to
combat oxidative pressure, including antioxidant metalloen-
zymes, an array of thiol-based redox couples and high concen-
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The turn-on response at the
blue channel should be attribut-
ed to endogenous H2O2 produc-
tion, while the turn-on signal at
the green channel could be due
to endogenous H2S production.
These results, for the first time,
directly indicated that endoge-
nous H2O2 can induce endoge-
nous H2S in living cells. To fur-
ther verify this point, inhibition
experiments were performed by
using Propargylglycine (PPG), an
analogue of Cys as a known in-
hibitor for CBS and CSE.[12] The
PMA-induced green fluorescence
was obviously decreased with
addition of inhibitors, while the
average fluorescence intensities
at the blue channel are similar
(Figure S14, Supporting Informa-
tion). These results implied that
PPG could inhibit PMA-induced
H2S biogenesis but not for H2O2
biogenesis.
Figure 3. Exogenous H2O2 can induce endogenous H2S production in living cells. A) Schematic drawing of exoge-
nous H2O2 stimulation leads to H2S production in cells. B) HEK293 cells were treated with 1, 1+Na2S, and
1+H2O2, respectively, and the imaging was taken immediately after media exchange with PBS buffer. Emission
was collected at the blue (425–480 nm) and green channels (500–600 nm) with 405 and 488 nm excitation, re-
spectively. C) HEK293 cells were preincubated with probe 2 for 30 min and then with H2O2 (0–250 mm) for 1 h.
Data are expressed as the average fluorescence intensity of each image versus H2O2 concentration.
trations of GSH (1–10 mm) (RSS).[16] Herein, the observation of
endogenous production of H2S from living human cells by
exogenous H2O2 stimulation should help to further understand
the intracellular redox homeostasis.
In order to further analyze the PMA-induced endogenous
H2S production, ratiometric imaging of FRET-donor excitation-
only was performed (Figure S15, Supporting Information). The
results indicated that 1) endogenous H2S could be produced
through PMA stimulation; 2) such endogenous H2S production
could also be suppressed by enzyme inactivation using the
Encouraged by the above results, we further tested whether
endogenous H2O2 can induce H2S biogenesis in living human
cells. To demonstrate the useful-
ness of probe 1 for detection of
endogenous H2S, 200 mm GSH
was added to the cells, which re-
sulted in an obviously enhanced
fluorescence at green channel
(Figure 4B). The increased fluo-
rescence signal could be attrib-
uted to endogenous generation
of H2S from GSH in living cells.[17]
When cells pretreated by probe
4 were co-incubated with phor-
bol-12-myristate-13-acetate
(PMA), an inducer for endoge-
nous
H2O2
production,[18]
a marked red fluorescence in-
crease was observed (Figure S13,
Supporting Information), imply-
ing the PMA-induced endoge-
nous H2O2 production. While
cells were co-incubated with
Figure 4. Images of PMA-induced endogenous H2O2 and H2S in living cells. A) Schematic drawing indicates that
PMA can induce biogenesis of both H2O2 and H2S inside cells, and the endogenous production of H2S can be in-
PMA and probe 1, a fluorescence hibited by PPG. B) Dual-channel imaging of cells in the presence of probe 1 and stimulants as indicated on the
left side. Cells were preincubated with DL-PPG (50 mgmLÀ1) for 20 min or not, and then incubated with PMA
increase at both blue and green
(1 mgmLÀ1) and probe 1 (1 mm) for 60 min without media exchange. After being washed with PBS, cells were used
channels was observed (Fig-
for imaging immediately. Emission was collected at the blue (425–480 nm) and green channels (500–600 nm) with
405 and 488 nm excitation, respectively. C) The average fluorescence intensity from the blue and green channels
by addition of exogenous H2O2. of probe 1 in the absence or presence of stimulants.
ure 4B), just like the observation
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PPG inhibitor; and 3) endogenous H2O2 can induce enzymatic
H2S production in living cells. Furthermore, because PMA can
trigger endogenous H2O2 production during cell signaling to
result in an inflammatory response,[18] the PMA-induced H2S
production could help in the understanding of the anti-inflam-
matory role of H2S-releasing drugs at a molecular level. H2S-re-
leasing drugs may serve as a treatment for PMA- and oxida-
tive-induced inflammation.
we also treated living cells with a high concentration Na2S or
Cys and found no obvious H2O2-triggering fluorescence based
on probe 4 (Figure S18, Supporting Information).
In summary, based on two selective chemical reactions,
a series of single- and dual-response fluorescent probes were
rationally designed and synthesized to investigate the biologi-
cal relationship between H2O2 and H2S. The endogenous pro-
duction of H2O2 and H2S can be imaged simultaneously with
a dual-response fluorescent probe. Our finding shows the first
direct evidence on the crosstalk between H2O2 (ROS) and H2S
(RSS) in vivo and the H2O2-induced H2S production was found
to be CBS-dependent. Consideration of the irreversible reactivi-
ty of the dual-response probe 1, the development of dual-de-
tection probes based on reversible reactions is still needed. It
is well known that H2S could exert its biological action through
modulation of [Ca2+] and intracellular pH as well as interacting
with other signaling molecules.[20] The development of dual-
and multiresponse fluorescent probes should serve as invalua-
ble tools to investigate the possible link between H2S and
other biological molecules in vivo at the molecular level.
The H2S biogenesis pathways include CBS, CSE, and 3MPST/
CAT.[5] By combining the sequence-specific gene-knockdown
technology (RNAi) with bioimaging, we seek to gain a better
understanding about the pathway for the H2O2-induced H2S
biogenesis. CBS, CSE, and 3MPST genes were specifically si-
lenced by using three designed siRNA sequences with the effi-
ciency of above 80% (Figure S16, Supporting Information).[19]
Subsequently, these gene-silencing cells were used for bio-
imaging studies with probe 1. No significant increase of green
fluorescence was observed in the PMA treatment series in CBS-
knockdown cells, implying no endogenous H2S production
during PMA stimulation. But for CSE- or 3-MPST-knockdown
cells, still significant green fluorescence signals were observed
(Figure 5A and Figure S17, Supporting Information). These re-
sults implied that H2O2-induced H2S biogenesis may largely
depend on the CBS pathway. Consideration of the substrate of
CBS in vivo, l-Cys should be involved in the H2O2-induced H2S
production under oxidative stress (Figure 5B). Apart from that,
Acknowledgements
This work was supported by the MOST (2010CB126102,
2013CB733600), NSFC (21332004, 21390202, 21402007,
21572019), 111 project (B14004) and the Fundamental Re-
search Funds for the Central Universities (YS1401).
Keywords: fluorescent imaging
·
fluorescent probes
·
hydrogen peroxide · hydrogen sulfide · redox homeostasis
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Published online on September 4, 2015
Chem. Eur. J. 2015, 21, 15167 – 15172
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15172
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