A Two-Photon H2O2-Activated CO Photoreleaser

Article abstract of DOI:10.1002/anie.201805806

Carbon monoxide (CO) is proposed as an active pharmaceutical agent with promising pharmaceutical prospects, as it has been involved in multifaceted modulation of diverse physiological and pathological processes. However, questions remain for therapeutic a

Full text of DOI:10.1002/anie.201805806

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Accepted Article
Title: A Two-photon H2O2-activated CO Photoreleaser
Authors: Yong Li, Yingzheng Shu, Muwen Liang, Xilei Xie, Xiaoyun
Jiao, Xu Wang, and Bo Tang
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201805806
Angew. Chem. 10.1002/ange.201805806
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10.1002/anie.201805806
Angewandte Chemie International Edition
A Two-photon H2O2-activated CO Photoreleaser
Yong Li, Yingzheng Shu, Muwen Liang, Xilei Xie, Xiaoyun Jiao, Xu Wang* and Bo Tang*
Abstract: Carbon monoxide (CO) is proposed as an active
pharmaceutical agent with promising pharmic prospect, as it has
been involved in multifaceted modulation of diverse physiological
and pathological processes. However, questions remain for
therapeutic application of inhaled CO attributed to the inherent great
affinity between CO and hemoglobin. Therefore, a robust platform
with function of CO-carrying and controllable releasing, depending
on the local status of organism, is of prominent significance for
effectively avoiding the side effects of CO-inhaling and optimizing
the biological regulation function of CO. Herein, utilizing oxidative
stress biomarker H2O2 as a trigger and combining with photo control
However, the outburst of excessive amounts of ROS, namely
oxidative stress, can induce cell damages, ultimately leading to
various human diseases.[6] Up to date, CO has emerged as a
promising therapeutic agent against various disease modes in
[1,7]
associated with oxidative stress
Thus, ROS may act as a
potential intracellular trigger for precise CO-releasing. Hydrogen
peroxide (H2O2) is regarded as a typical factor responsible for
cellular oxidative damage as it is one of the key contributors in
the redox signaling modulation.[8] Consequently, the fluctuation
of H2O2 may become a modulator for controllable CO releasing.
However, the photo-release of CO are manipulated by
extracellular irradiation only, which fails to be triggered by the
intracellular oxidative stress status.[5] As is known to all,
fluorescence combined with confocal microscope, especially
two-photon imaging technology, demonstrates unique capacity
of visualizing dynamical biological events with high-contrast
spatiotemporal resolution and less phototoxicity.[9] Therefore,
utilizing fluorescent imaging to trace CO-releasing process and
map its physiological effects is of prominent significance for
deeply understanding the biological benefits of CO.
technique, we present
a
two-photon H2O2-activated CO
photoreleaser, FB, featuring capacity of highly sensitive and specific
H2O2 sensing and meanwhile, photocontrollable CO releasing. By
capitalizing on the outstanding features of FB, the H2O2 sensing and
CO photo-releasing were successfully implemented and visualized in
vitro and in vivo. With the aid of FB, the evidence of oxidative stress
associated with H2O2 upon angiotensin II administration was
provided, and the directly visual proof of vasodilation effect of CO
was obtained. Therefore, we anticipate that the superior features of
FB will endow it with potential application for oxidative stress
warning and oxidative stress-mediated CO controlled release.
Carbon monoxide (CO), generated endogenously by enzymatic
heme metabolism in mammals, has widely emerged as crucial
physiological gasotransmitter featuring cytoprotective effect and
maintaining cellular homeostasis, thus endowing it with potential
drug activity. As such, the efficient delivery of CO in the
organism has received much attention.[1] Unfortunately,
attributed to the inherent great affinity between CO and
hemoglobin, multiple uncontrollable issues emerge in direct CO
gas inhalation, such as dose control, targeted delivery, etc. This
often leads to a high level of carboxyhemoglobin and results in
CO poisoning, making it difficult to implement CO’s physiological
and medical effects.[2] Therefore, it is of urgent need to develop
CO carrier aiming at sustained or controlled CO releasing.
Currently, two types of molecules have been employed as CO
releasers, transition metal carbonyl complexes and small
organic molecules.[3] Compared with the potential cytotoxicity
caused by heavy metal, the organic molecular CO releasers with
improved biocompatibility have attracted increasing attention,
especially those triggered in controllable fashions.[4] Recently,
organic photo-triggered CO releasers have demonstrated
appealing profile as it promises precise spatiotemporal control.[5]
In general, normal level of reactive oxygen species (ROS) is
critical for the regulation of diverse physiological processes.
Scheme 1. The chemical structure of FB and the corresponding proposed CO
photo-releasing mechanism.
Hence, in this work, we engineered
a flavonol-borate-
conjugate, FB, for precise CO-releasing with guiding of H2O2, a
biomarker of intracellular oxidative stress. FB was firstly
employed as a fluorescent sensor for H2O2 via “off-on” behavior
of excited state intramolecular proton transfer (ESIPT) triggered
by borate deprotection.[10] After tracing of H2O2, the activated
flavonol (F) was subsequently irradiated to release CO (Scheme
1).[5c] Therefore, we reasoned that FB could function for both
H2O2 tracing and H2O2-guided CO releasing.
The detailed synthetic procedures are shown in Scheme S1.
With FB in hand, its spectral property was firstly evaluated. As
expected, FB itself exhibited maximal absorbance/emission at
375 nm (ε = 12000 L·mol−1·cm−1)/485 nm (Figure S1). By sharp
contrast, in the presence of H2O2, the solution underwent a
bathochromic shift in the maximal absorption wavelength (405
nm) and presented two fluorescence emission peaks at 480 nm
and 585 nm, respectively, in which the later emission band with
larger stokes shift can be attributed to the recovery of ESIPT
effect due to the liberation of protected hydroxyl group in F. The
photostability of FB and F (with xenon lamp of fluorophotometer,
360 W, slit width: 2 nm/2 nm) were assessed to confirm no
[*]
Y. Li, Y. Shu, M. Liang, Dr. X. Xie, Dr. X. Jiao, Pro. X. Wang, Pro. B.
Tang
College of Chemistry, Chemical Engineering and Materials Science,
Key Laboratory of Molecular and Nano Probes, Ministry of
Education, Collaborative Innovation Center of Functionalized Probes
for Chemical Imaging in Universities of Shandong, Institute of
Molecular and Nano Science, Shandong Normal University, Jinan
250014,
P.
R.
China.
E-mail:
tangb@sdnu.edu.cn,
wangxu@sdnu.edu.cn
Supporting information for this article is given via a link at the end of
the document.
This article is protected by copyright. All rights reserved.

10.1002/anie.201805806
Angewandte Chemie International Edition
photolysis occurred during spectral scanning (Figure S2).
Subsequently, the ratiometric response of FB towards H2O2 was
investigated (Ratio= I585 nm/I485 nm). Gratifyingly, as showed in
Figure 1, FB displayed a gradual increasing in ratio value in the
presence of different concentrations of H2O2, and manifested an
H2O2, satisfactory photostability of FB itself was observed
through the constant fluorescent intensity during irradiation
(Figure S7). In the presence of H2O2, gradually decreasing
fluorescent signals at 585 nm were detected upon the increase
of irradiation time (Figure 2). On the contrary, no obvious ratio
variation of FB solution was detected when it was kept in the
dark upon H2O2 activation (Figure S8). In view of the photolysis
products emiting no fluorescent signal when excited with 405 nm,
these results suggested the effective occurence of the photo-
induced CO-releasing. Meanwhile, as can be seen from Figure
2b, the fluorescent ratio readout sharply attenuated within first
few minutes, indicating a quick CO release during irradiation and
low phototoxcity in living system (Figure S9). These results
suggested that FB can be employed as a potent H2O2-
dependent one-photon-induced CO releaser.
approximately 19-fold ratiometric enhancement upon
5
equivalents of H2O2 addition. The ratio intensity of FB
possessed an outstanding linear relationship (R2 = 0.998) with
H2O2 concentration with a regression equation of Ratio =
0.39062 + 0.1451 [H2O2] (μM). The limit of detection was
calculated as low as 66 nM. In addition, the dependence of
absorption intensity upon reaction time was evaluated. Upon the
addition of 10 equivalents of H2O2, the signal displayed a drastic
increment within 13 min, then leveled off, indicating the high
reactivity of FB towards H2O2 (Figure S3).
Then, the proposed photolysis mechanism for CO-releasing
process was validated. It was reported in previous work that two
compounds were formed in this photolysis reaction. One of
photolysis products, CO, was certified utilizing CO fluorescent
probe ACP-2, which reported by our lab previously (Figure
S10).[7] As shown in Figure S11, fluorescence enhancement of
ACP-2 after irradiation consolidated the releasing of CO. After
that, the solution was analyzed by HRMS. A discriminable mass
signal at m/z 291.0665 (M-H) was presented (Figure S12), which
corresponding to another photolysis product, 3-(benzoyloxy)-2-
naphthoic acid. Collectively, both fluorescent analysis and
HRMS detection provided an evidence for the proposed
mechanism of photodecomposition as displayed in Scheme 1,
which also displayed good consistency with the previous work.[5c]
With data manifesting that FB can serve a bifunctional
platform for H2O2 detection and CO photoreleasing, the imaging
capability of FB was demonstrated. Firstly, the MTT assay was
performed to check the biocompatibility and the results indicated
that these compounds exhibited low cytotoxicity, as the
experimental concentration in cells was far less than the IC50
value (Figure S13). Subsequently, the potential TP excited
capacity was explored. Encouragingly, Both FB and F displayed
high TP absorption cross-section (δ) at 800 nm as 30 GM (1 GM
= 10−50 cm4 s photon−1) and 96 GM, respectively, implying these
two compounds can be effectively excited by TP laser. And the
liver tissue depth imaging assays manifested that FB displayed
a discriminable fluorescent signal at depth up to 153 μm upon
TP excitation (Figure S14). Furthermore, a mass spectra signal
of photolysis product, 3-(benzoyloxy)-2-naphthoic acid, was also
obtained when H2O2-loaded FB solution was exposed to TP
irradiation (Figure S15), thus implying that two-photon can also
effectively induce photolysis of F. Similar to the preceding
verification with one-photon irradiation, the CO-releasing profile
of FB was confirmed in vascular smooth muscle cells (VSMCs)
with TP laser irradiation using ACP-2 (Figure S16). Collectively,
FB can be served as a robust TP fluorescent imaging tool as
well as a H2O2-activated TP-induced CO releaser.
Figure 1. The H2O2 detection capability of FB. (a) Fluorescent response of 10
μM FB to H2O2 (0−50 μM) in PBS buffer solution (50 mM, pH 7.4) with 10%
CH3CN. (b) The linear curve of ratio (585/485 nm) derived from fluorescent
titration.
Next, the specificity of FB was investigated and the results
displayed that no obvious ratio change of FB was triggered after
exposed to various active species which commonly exist in
biological systems, thus implying the high specificity of FB
towards H2O2 (Figure S4). In addition, the influence of pH was
also carried out to confirm that FB can be potentially applied to
trace H2O2 in physiological environment (Figure S5). The
proposed mechanism for detecting H2O2 was verified with high
resolution mass spectra (HRMS) and the result was found a
mass peak at m/z 289.0873 (Figure S6), which was in good
agreement with F as displayed in Scheme 1.
Afterwards, the H2O2 tracing ability of FB was verified in live
cells. Upon phorbol myristate acetate (PMA, a well-defined H2O2
intracellular inducer) treatment, an obvious ratio increment was
obtained (Figure S17), which can be efficiently attenuated by N-
acetyl-L-cysteine (NAC, a well-validated H2O2 scavenger). In
addition, this fluorescent ratio increment exhibited no
discriminable attenuation when the cells were pretreated with L-
NAME (NG-Nitro-L-arginine Methyl Ester Hydrochloride, which
can suppress peroxynitrite up-regulation through Inhibiting nitric
oxide synthase activity). These results suggested that the ratio
enhancement was indeed resulted from up-regulation of H2O2
Figure 2. The H2O2-dependent CO-releasing of FB. Fluorescent intensity
changes of H2O2-loaded FB (10 μM) solution during irradiation (λ= 405 nm,
CEL-HXF300 Xenon lamp with power 15 mW/cm2). (a) and the corresponding
ratio variation (b).
The capacity of the H2O2-activated CO-releasing capability of
FB was performed during light irradiation (λ= 405 nm, CEL-
HXF300 Xenon lamp with power 15 mW/cm2). In the absence of
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10.1002/anie.201805806
Angewandte Chemie International Edition
concentration. Then, the PMA dose-dependent H2O2 fluctuation
was explored. Prior to cells imaging, VSMCs were pretreated
(ROI) during irradiation indicated precisely spatio-temporal
controllable CO release. Taken together, it is credible to draw
with NAC to eliminate the intracellular basal H2O2. Subsequently, the conclusion that FB can be applied as a potent platform for
the cells were exposed to various doses of PMA. As can be
seen from Figure 3 (a-e), fluorescence signal in VSMCs
displayed a significant and PMA dose-dependent ratiometric
change. The data demonstrated that FB was a powerful tool to
map H2O2 fluctuation in live cells.
real-time H2O2 monitoring and H2O2-activated TP-induced CO
release in vivo.
Figure 4. The H2O2 mapping and CO-releasing in vivo. (a-f) The zebrafish was
pretreated with NAC (1.0 mM, 1 h), and then loaded with FB (20 μM, 15 min),
followed by administrated with PMA (2.0 μg/mL). (g-l) The region of interest
(ROI) of the zebrafish (f) was irradiated with 800 nm laser. (m) The ratio
changes of (a-f). (n) The ratio changes of ROI in (g-l). Ratio=F420–510 nm/F520–620
nm. The values are the mean ± s.d. for n = 3, ***p <0.001. N.S. denoted no
significant. Scare bar = 250 μm.
Figure 3. The H2O2 mapping and CO releasing in vitro. (a−e) The NAC (1.0
mM, 1 h) pretreated VSMCs were incubated various concentrations of PMA (0,
0.5, 1.0, 1.5, 2.0 μg/mL) for 3 h and then exposed to FB (20 μM, 15 min). (f−j)
The VSMCs were coincubated with PMA (2.0 μg/mL, 3 h), followed by stained
with FB, and then the cells inside the selected circle were irradiated with two-
photon laser at 800 nm for different time (0, 1, 2, 3, 4 min). (k) The ratio
changes of cells in (a−e). (l) The ratio changes of cells in the circle of (f−j).
Ratio=F420–510 nm/F520–620 nm. The values are the mean ± s.d. for n = 3, **p <
0.01, ***p <0.001. Scale bar = 25 μm (a-e) and 50 μm (f-j).
It was reported that angiotensin II, a potent peptide hormone
vasoconstrictor for hypotension and hypovolemia treatment, can
induce
overproduction of O2
deleterious
effects
on
mitochondria
through
•− [11]
.
However, as one of the key
contributors in modulation of intracellular redox signaling, H2O2
fluctuation in angiotensin-II-induced physiological events is
much less clear. Meanwhile, CO is a well-known promoter for
vasorelaxation through several signal pathways.[12] Nonetheless,
to the best of our knowledge, no straightforward and visualized
evidence of vasodilating capability of CO was demonstrated.
Therefore, encouraged by the excellent behavior of FB in vitro
and in vivo, an attempt was made to simultaneously address the
above two issues by using FB. Firstly, oxidative stress
associated with H2O2 upon angiotensin II treatment and the CO
vasorelaxation effect were evaluated in vitro. Cyclic guanosine
monophosphate (cGMP), a vasodilative biomarker generated
through the activation of soluble guanylyl cyclase (sGC) by
guanosine triphosphate (GTP), was introduced to assess CO
vasodilatation at the cellular level.[2] Upon angiotensin II
treatment, VSMCs exhibited a significant ratio increment which
can be efficiently inhibited by NAC, revealing a H2O2 up-
regulation during drug exposure (Figure S19). Meanwhile, a
sharp up-regulation of cGMP level was observed in both
angiotensin II and FB administrated VSMCs during light
irradiation, demonstrating efficient CO releasing and its
vasodilatation effect (Figure S20). Moreover, it can be found that,
in the absence of angiotensin II, VSMCs treated with FB only
showed no significant cGMP level change during irradiation,
indicating the necessity of oxidative stress for realizing CO
biological effect of FB. Subsequently, the vasodilatation effect of
CO and angiotensin-II-induced H2O2 fluctuation in vivo were
Subsequently, the CO-release ability was investigated in live
cells utilizing TP. The VSMCs were co-incubated with PMA
followed by stained with FB, and then the cells were imaged
during light irradiation. As shown in Figure 3 (f-j), the cells in
given circle were irradiated with TP laser while the other outside
were not. It can be observed that only the cells in the given circle
displayed a remarkable ratiometric signal attenuation whereas
the adjacent unstimulated area presented no obvious variation.
In addition, the VSMCs in the given circle displayed a gradually
attenuated ratio signal upon the increase of irradiation time.
Taken together, the results indicated that H2O2-activated CO-
releasing process can be effectively induced and monitored by
TP laser in precise spatiotemporal controllable fashion.
Having well-established the H2O2 detection ability and the
succedent TP-induced CO-releasing in vitro, the in vivo assays
were carried out in larval zebrafish. Firstly, the H2O2 tracing was
performed. After NAC pretreatment, the increased ratio caused
by PMA administraion was efficiently suppressed, demonstrating
FB was capable of mapping H2O2 fluctuation in zebrafish (Figure
S18). Then, time-dependent ratiometric readout upon PMA
administration was captured. As can be seen from Figure 4 (a-f),
the time-dependent ratiometric signal increment was obtained,
demonstrating the increased H2O2 level. Afterward, the TP-
induced CO release was investigated. As shown in Figure 4 (g-l),
temporal and spatial ratio value changes in the region of interest
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10.1002/anie.201805806
Angewandte Chemie International Edition
mapped in transgenic line of zebrafish, Tg (flila: EGFP), which
vascular endothelium was highlighted with green fluorescent
protein (GFP) (Figure S21). Upon drug administration, the GFP-
labelled zebrafish showed apparent vasoconstriction with
vascular diameter sharply decreasing from 24.5 μm to 16.7 μm,
indicating the vasoconstriction effect of angiotensin II (Figure 5).
Correspondingly, compared with angiotensin-II-free treatment
group (Figure 5a), the obvious fluorescent ratio increment of FB
in zebrafish was observed, thus hinting H2O2 upregulation during
angiotensin II administration. To consolidate this conclusion, the
zebrafish was exposed to NAC after angiotensin II
administration (Figure S22). It was shown that the increased
ratio value was effectively attenuated by NAC. Therefore, it was
credible to draw the conclusion that the H2O2 level was indeed
upregulated during angiotensin II treatment. Then, the
vasodilating capability of CO was explored. The zebrafish
loaded with angiotensin II and FB was irradiated with TP laser to
release CO (Figure 5c). The morphological observation of blood
vessel displayed significant vasodilatation with the vascular
diameter recovering from 16.7 μm to 23.8 μm (Figure 5g), thus
apparently exhibiting the potent hemangiectasis effect of CO. In
sharp contrast, in the absence of FB, the vessel of zebrafish
with angiotensin II administration exhibited negligible
morphological change during irradiation (Figure S23). In addition,
upon NAC treatment after angiotensin II exposure, no vascular
diameter change was observed during irradiation, implying no
vasodilatation occurred without oxidative stress activation of FB
(Figure S22). Taken these results together, the evidence of
oxidative stress associated with H2O2 upon angiotensin II
administration was provided, and the direct visualization of
vasodilation effect of CO was realized.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (21390411, 21535004, 91753111,
21775093, and 21505088).
Keywords: carbon monoxide photo-releasing • oxidative stress •
two-photon control • vasodilatation • fluorescent imaging
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Figure 5. The vasodilatation effect of CO and angiotensin-II-induced H2O2
fluctuation. (a) and (e) Tg zebrafish was soaked with 20 μM FB (15 min). (b)
and (f) Tg zebrafish was exposed to angiotensin II (2 h) after soaked with FB
(15 min). (c) and (g) Tg zebrafish was irradiated with 800 nm laser for 5 min
after treated with FB and angiotensin II. (d) The ratio changes of (a-c), (h) The
blood vessel diameter changes of (e-g). The values are the mean ± s.d. for n =
3, ***p <0.001. Scale bar = 50 μm.
In conclusion, we have presented a new dual functional
platform, FB, for H2O2 sensing and CO releasing. With two-
photon excitation and ratiometric feedbacks, the two-stage
behaviors of FB for H2O2 detection and photo-driven CO
releasing were successfully implemented and monitored in vitro
and in vivo. By capitalizing on the outstanding features of FB,
oxidative stress in association with H2O2 was revealed upon
angiotensin II administration, and the potent vasodilation effect
of CO was mapped straightforwardly. Therefore, we anticipate
that the superior features of FB will endow it with potential
application for oxidative stress warning and CO photo-controlled
release.
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10.1002/anie.201805806
Angewandte Chemie International Edition
Entry for the Table of Contents (Please choose one layout)
Layout 1:
COMMUNICATION
We engineered a two-photon
fluorescent molecule featuring
capacity of highly sensitive and
specific H2O2 sensing and
photo-induced CO releasing.
Yong Li, Yingzheng Shu, Muwen
Liang, Xilei Xie, Xiaoyun Jiao, Xu
Wang* and Bo Tang*
Page No. – Page No.
A Two-photon H2O2-activated
CO Photoreleaser
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