Photocontrolled endogenous reactive oxygen species (ROS) generation

Article abstract of DOI:10.1039/c9cc01747j

A cell-permeable small molecule for light-triggered generation of endogenous reactive oxygen species (ROS) is reported.

Full text of DOI:10.1039/c9cc01747j

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DOI: 10.1039/C9CC01747J
Journal Name
COMMUNICATION
Photocontrolled Endogenous Reactive Oxygen Species (ROS)
Generation
Ajay Kumar Sharma,a Harshit Singha and Harinath Chakrapani*,a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
•
A cell-permeable small molecule for light-triggered generation of menadione, which require bioactivation for O2
endogenous reactive oxygen species (ROS) is reported.
production,20,21 have often been used but at elevated
concentrations that can potentially complicate mechanistic
interpretations. Furthermore, their reactivity with biological
thiols leading to covalent modification is also a major concern.
Hartley and coworkers have developed a mitochondria-specific
ROS generator, MitoPQ, a paraquat derivative.24 This strategy
allows for organelle-specific generation of ROS. Since light as a
tool for activation offers spatiotemporal control,25–33
photocleavable ROS generators are expected to have significant
advantages. Chang and co-workers have reported a light-
triggerable ROS generator that is based on hydroxyquinol.30 This
compound responds to light of wavelength of 305 nm, which is
not desirable. Furthermore, the yield of hydrogen peroxide
from this compound was somewhat diminished. Here, we
report the design, synthesis and evaluation of a light-triggered
endogenous ROS generator.
Oxygen and its reduced forms such as superoxide (O2• ),
hydrogen peroxide (H2O2) and hydroxyl radical are generated
during normal functioning of cells.1 A number of intrinsic
mechanisms have evolved to attenuate these reactive oxygen
species (ROS).2,3 Elevated ROS generated in cells are associated
with numerous pathophysiological conditions including cancer,
inflammation, diabetes and several neurodegenerative
disorders.4–6 ROS is also deployed in immune response to
counter pathogens.7 Elevation of endogenous ROS has been
associated with inhibition of cancer growth both in vitro as well
as in animal models.8–12 For example, piperlongumine has been
shown to enhance ROS within cells13–18 but the precise
mechanism by which this happens is yet unclear and would be
•
critical to determine. Due to its short life, O2 must be
produced in situ by reaction with oxygen and electron donor.19
1e
1e
1e
O2
O2
H2O2
HO
Either small organic molecules that spontaneously react with
Fe2+
SOD
oxygen or enzymatic methods that turnover a substrate to
DNA
Damage
•
generate O2
are frequently used. A combination of
Fe-S
hypoxanthine and xanthine oxidase (X
hypoxanthine is metabolized by XO to produce O2
predominantly produces O2• in the proximity of cells. Any O2
+
XO) where
iron-sulfur
clusters
•
,
•
Figure 1. Endogenous ROS and some of their effects within cells.
that is produced must diffuse across a lipid bilayer to exert its
effects. However, O2 is not highly permeable at neutral pH
Our laboratory has previously reported 1,34 which is a
derivative of juglone, as a ROS generator (Scheme 1). The
proposed mechanism for ROS generation was enolization of the
carbonyl as the first step, followed by generation of a 1,4-diol.
The diolate of this is electron-rich and is known to produce
superoxide. We found that attaching a 4-nitrobenzyl group
resulted in diminished ROS generation.35 When triggered by
nitroreductase, an enzyme that is present in bacteria, we found
enhanced ROS generation.35 Thus, this strategy enabled
triggerable and localization of ROS production.36 Recently, we
reported an esterase-activated ROS generator that was able to
elevate ROS within mammalian cells (Scheme 1).36 Here, we
report the design, synthesis and evaluation of a light-triggerable
ROS generator.
•
and hence, this protocol and other small-molecule based
methods may not be useful for enhancing intracellular ROS. A
number of bioreductively-activated ROS generators are known
and these have been evaluated previously for their cancer
therapeutic potential.20–23 Although triggered by a bioreductive
enzyme, these compounds have little spatiotemporal control
over ROS production. The quaternary amine, paraquat or
a. Department of Chemistry, Indian Institute of Science Education and Research
Pune. Pune 411 008. Maharashtra, India.
† 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
This journal is © The Royal Society of Chemistry 20xx
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Journal Name
O
O
O
OH
O
O
View Article Online
H
H
2O2
DOI: 10.1039/C9CC01747J
Activation
OH
O
2
2O2
O
O
O
O
O
O
OH
H
H
O
O
1
= 4-Nitrobenzyl ;
Bioreduction
Scheme 1. General strategy for generation of compound 1 after photocleavage. Compound 1 generates superoxide during incubation in buffer under ambient aerobic
conditions to produce the quinone 2, which can further generate ROS through redox cycling. The 4-nitrobenzyl derivative is a substrate for nitroreductase while the
pivaloyloxymethyl compound is triggered by esterase. The proposed substrate for photocleavage has a 2-nitroaryl functional group, which is expected to undergo
deprotection to produce the ROS generator 1 (See Table 1).
The 2-nitrobenzyl functional group has been widely used as yield. The desired conjugate 4e was next synthesized by
a protective group for alcohols.26–28,37 Upon irradiation, this reacting 4d with the biotin-alkyne derivative 12 (Figure 2A and
photo-cleavable group releases an alcohol (Scheme 1). This Scheme S2 in ESI).
strategy has been used by Chang and co-workers to release the
1,4-diol.30 The yields of ROS were low possibly due to the poor
A
B
efficiency of cleavage, and perhaps, low ROS generating
capability. Lastly, aqueous solubility and cancer cell uptake may
play a major role in determining the efficiency of ROS
generation within cells. Thus, the ROS generator must have a
synthetic handle to incorporate additional functional groups
(such as biotin).38–40
H
N
O
H
O
O
O
HN
B
O
O
S
H
O
13
H2O2
O
O2N
N
O
NH
N
O
Table 1. Reaction of bromides with 1
O
HO
O
O
O
N
R2
O
O
R2
14
R1
1
R2
4e
R1
, Ag2O
R1
CH2Cl2
Figure 2. A) Structure of Biotinylated adduct 4e B) Reaction of hydrogen peroxide
with coumarin Boronate-ester dye 13. The boronate ester 13 is weakly fluorescent
and upon oxidation, a turn on fluorescence is observed due to the formation of
14.
+
r.t.
O
O
NO2
OH
NO2 Br
NO2
5a-5d
4a-4d
6
Next, the stability of the derivatives was evaluated. In the
dark, we found no evidence for decomposition of these
compounds in pH 7.4 buffer. To evaluate the photocleavage
efficiency, 4a, 4b and 4e were irradiated with 365 nm light and
HPLC analysis revealed complete cleavage within 15 min. This
cleavage resulted in the formation of 1, a known ROS generator
(Figure 3A, figure S2 in ESI). As previously reported, 1, reacts
with oxygen and produces O2• which disproportionates to
Entry
Bromide
R
H
OMe
OMe
OMe
R’
H
OMe
Prod
4a
4b & 6
4c
Yield
55%
30% & 11%
1
2
3
4
5a
5b
5c
5d
O-Propargyl
OCH2CH2N3
-
4d
25%
•
form H2O2,34 another ROS. To validate the generation of O2
The bromides 5a-5b were commercially obtained and
during incubation of 4e, this compound was irradiated and a
independently reacted with 1, resulting into the formation of
compounds 4a and 4b (Table 1, entries 1-2). In the case of the
reaction of the dimethoxy electrophile 5b, the formation of the
C-alkylated product (6) was observed. It is likely that a
phenolate intermediate is formed, which can then react via the
oxygen or the carbon to produce the O-alkylated or C-alkylated
products. We next synthesized the bromide 5c, which has a
propargyl group on it. This alkyne should provide opportunities
to conjugate biotin through the copper-catalyzed alkyne-azide
click reaction. However, under the conditions that we used, we
found no reaction between 5c and 1 (Table 1, entry 3).
Activation of the bromide by silver ions is an important step in
the substitution reaction. It is likely that the alkyne coordinates
with the silver ions and competes with activation of the
bromide. We therefore revised our strategy and we first
synthesized the bromide 5d, which has an azide that can be
utilized for orthogonal conjugation (Scheme S1 in ESI) and this
was reacted with 1 and gave the corresponding azide 4d in 25%
dihydroethidium (DHE, see ESI) assay was used. The DHE dye
reacts with O2 and other ROS (H2O2, ȮH) to form 2-hydroxy
•
ethidium(2-OH-E+) and ethidium (E+) respectively (Scheme S4 in
ESI), which can be detected by HPLC analysis.41 When
incubated, 4e in the presence of light, under the assay
conditions HPLC analysis revealed a peak at 29.5 min, which
corresponds to 2-hydroxy ethidium (2-OH-E+). This signal
diminished when solution was treated with superoxide
dismutase, a known quencher of O2•. When this experiment
was conducted in the dark, no peak for 2-OH-E+ was observed
(Figure 3B). This data supports the generation of O2• by 4e only
in the presence of light. This assay was also performed with 4a
and 4b and similar results were obtained (Figure S3 in ESI).
The radical anion, O2• disproportionates to form H2O2. We
next assessed the production of H2O2 using two independent
assays. First, the oxidation of fluorogenic boronic acids/esters
has been frequently used to detect the presence of hydrogen
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peroxide.42,43 The non-fluorescent coumarin based dye 13
COMMUNICATION
Further evaluation of intracellular ROS was alVsioewdAortniclee Obnylinae
DOI: 10.1039/C9CC01747J
reacts with H2O2 to form umbelliferone derivative 14, a highly cell permeable weakly fluorescent ROS responsive dye, 2',7'-
fluorescent molecule (Figure 2B).
dichlorodihydrofluorescein diacetate (H2DCF-DA), which upon
reaction with ROS forms a highly florescent molecule, 2’,7’-
dichlorodihydrofluorescein (DCF) and fluorescence intensity of
this can be measured in well plate reader. This assay was
performed in A549 cells and it was found that cells treated with
4e had enhanced ROS level in the presence of light when
compared with a similar treatment in the dark (Figure 4B & 4C).
In addition, ROS enhancement was not observed when cells
without 4e was irradiated, suggesting that 365 nm UV light
alone does not enhance the ROS level in cells. This study
suggests that 4e is cell permeable and elevate the ROS level only
after irradiation with light.
Figure 3. Photolysis and ROS analysis of 4e; A) HPLC traces of 4e (50 µM) in the
presence and absence of Light, Light irradiation was performed by 365 nm light
for 15 min with the 30 mW/cm2 intensity. B) HPLC traces of DHE assay for
superoxide detection, 4e (25 µm) was irradiated with 30 mW/cm2 365 nm light for
15 min followed by addition of DHE and incubated for 1 h, as a control, SOD was
used for quenching the superoxide; C) Hydrogen peroxide detection using dye the
coumarin-boronate ester 13 as a probe. (excitation 320 nm; emission 460 nm); D)
Hydrogen peroxide detection using Amplex red assay, 4e (25 µM) was irradiated
in 365 nm light for 15 min and incubated for 2 h, (cat.= Catalase enzyme). Data
represent the mean±s.d. for 3 technical replicates per group.
Figure 4. Cellular assays; A) Extracellular Hydrogen peroxide detection by Amplex
red assay (excitation 550 nm; emission 590 nm; *** = p <0.0001); B & C)
Intracellular ROS detection using H2DCF-DA as a probe; Scale bar = 200 m and ns
= not significant. D) Cell viability assay using A549 cells with 10 µM compounds
and E) Growth inhibition curve after irradiation with 4e and IC50 was found to be
5.8 µM. Data represent the mean ± s.e.m. for 3 technical replicates per group.
Irradiated and non-irradiated samples of 4e were
independently incubated with dye 13 and fluorescence
enhancement was found only with the irradiated sample (Figure
3C). This fluorescence signal was diminished in the presence of
catalase, a known quencher of H2O2. This study was also
performed with compounds 4a and 4b and as expected,
fluorescence enhancement was observed in irradiated samples
(Figure S4 in ESI). These results suggest that the production of
H2O2 by 4e occurred only in the presence of light. Next, an
Amplex Red assay was used to infer the production of H2O2.
Again, irradiated and non-irradiated samples of 4e were
independently treated with Amplex Red solution containing
horse reddish peroxidase (HRP) enzyme and found that
enhanced fluorescence signal was observed only in the
irradiated sample. This signal was diminished in the presence of
catalase (Figure 3D). Taken together, our results support the
ability of 4e to undergo photocleavage to produce ROS.
Next, this compound was evaluated as an endogenous ROS
generator in cells after activation with light. First, generation of
extracellular H2O2 was assessed by coumarin based dye 13 and
Amplex Red in lung carcinoma cell line, A549 cells and it was
found that 4e was able to generate H2O2 extracellularly only in
the presence of light. However, no ROS enhancement was
observed by cells only in the presence of light and cells treated
with 4e in dark (Figure 4A, Figure S5 in ESI).
Elevated levels of ROS can damage essential biomolecules
leading to oxidative stress, which in turn can result in cell death.
In order to test the hypothesis, cell viability assay was
conducted using A549 and DLD-1 cell lines. Under the assay
conditions, in the presence of 4e, no significant inhibition of
growth was observed in dark even at 50 M (Figure S7 & S9 in
ESI). When this experiment was conducted with cells that were
irradiated (30 mW/cm2 intense 365 nm light, 5 min), nearly
complete inhibition of growth of these cells was observed at 10
µM (Figure 4D, Figure S8 in ESI). The inhibitory concentration
50% (IC50) of 4e in the presence of light was found to be 5.8 µM
(Figure 4E). Similarly, in DLD-1 cells, no significant inhibition was
observed in the dark while potent inhibitory effects were
observed during irradiation (IC50 = 5.3 µM) (Figure S8-S9 in ESI).
Recently, the ROS generating capability of 1 was compared
with various other probes. A major drawback of several ROS
generators is the inherent reactivity with thiols. For example,
menadione and Juglone reacts with glutathione (Figure S10D in
ESI). However, with 1 and the oxidized form of 1, i.e. 2, we found
no evidence for reaction with glutathione even after incubation
for several hours (Figure S10 in ESI). The reason for negligible
reactivity with glutathione might be the absence of α, β-
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21 D. M. Frank, P. K. Arora, J. L. Blumer and L. M. Sayre, Biochem. Biophys.
unsaturated site in 1 whereas this site could be sterically
inaccessible in 2. Hence, this ROS generator has advantages
over other ROS generators such as menadione since it allows us
to study the effects of ROS without complications associated
with covalent modification. Furthermore, the improved
aqueous solubility of 4e presents opportunities for wide
applicability of this compound as a ROS generator.
Taken together, our results suggest the use of the
compound for photolabile “turn on” ROS generation and
overexpression of biotin receptors in cancer cells may provide
an opportunity to selectively target tumour.38–40 As a cancer
therapeutic, singlet oxygen has been used;44 however,
examples of the use of other biological ROS are fewer.45 The
ability to conjugate this ROS generator with directing groups
enhances the versatility of this tool.
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DOI: 10.1039/C9CC01747J
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This work was supported by the Department of Biotechnology,
India (Grant number: BT/PR17514/BRB/10/1615/2017) and the
Council for Scientific and Industrial Research (Fellowship). The
authors thank Ms. Preeti Chauhan, IISER Pune for help with
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There are no conflicts to declare.
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