Arylboronate ester based diazeniumdiolates (BORO/NO), a class of hydrogen peroxide inducible nitric oxide (NO) donors

Article abstract of DOI:10.1021/ol5010643

Here, we report the design, synthesis, and evaluation of arylboronate ester based diazeniumdiolates (BORO/NO), a class of nitric oxide (NO) donors activated by hydrogen peroxide (H₂O₂), a reactive oxygen species (ROS), to generate NO. We provide evidence for the NO donors' ability to permeate bacteria to produce NO when exposed to H₂O₂ supporting possible applications for BORO/NO to study molecular mechanisms of NO generation in response to elevated ROS.

Full text of DOI:10.1021/ol5010643

Letter
pubs.acs.org/OrgLett
Arylboronate Ester Based Diazeniumdiolates (BORO/NO), a Class of
Hydrogen Peroxide Inducible Nitric Oxide (NO) Donors
Allimuthu T. Dharmaraja,† Govindan Ravikumar,† and Harinath Chakrapani*
Indian Institute of Science Education and Research Pune, Dr. Homi Bhabha Road, Pashan, Pune 411 008, Maharashtra, India
S
* Supporting Information
ABSTRACT: Here, we report the design, synthesis, and evaluation of
arylboronate ester based diazeniumdiolates (BORO/NO), a class of nitric
oxide (NO) donors activated by hydrogen peroxide (H2O2), a reactive oxygen
species (ROS), to generate NO. We provide evidence for the NO donors’
ability to permeate bacteria to produce NO when exposed to H2O2 supporting
possible applications for BORO/NO to study molecular mechanisms of NO
generation in response to elevated ROS.
anions can, however, be derivatized into “protected” forms,
which can be activated in the presence of a specific metabolic
trigger to generate NO.24,25 Numerous methodolo-
gies11,12,26−33 for cleavage of otherwise stable diazeniumdiolate
derivatives are known, but to our knowledge a nitric oxide
donor that is selectively activated by ROS is not available.
Boronate ester protecting groups are highly specific to
cleavage by H2O2 and have been extensively used in
imaging34−37 as well as in drug delivery.38−41 For mechanistic
studies with bacteria, elevated H2O2 is frequently used to
simulate conditions of oxidative stress.2,42 Arylboronate ester
based diazeniumdiolates (BORO/NO), a class of diazenium-
diolate derivatives that are attached to a pinacolboronate ester
through a self-immolative aryl linker, were considered as H2O2
activated NO donors (Scheme 1). Reaction of BORO/NO with
H2O2 should produce the phenolate intermediate I, which
could rearrange to produce the diazeniumdiolate anion, which
in pH 7.4 releases NO (Scheme 1.).
lthough reactive oxygen species (ROS) such as hydrogen
peroxide (H2O2) mediate numerous physiological pro-
A
cesses,1 elevated levels of ROS can cause oxidative stress
leading to cell death.2−6 Similarly, nitric oxide (NO), a reactive
nitrogen species (RNS), has multifarious functions and effects
in cells, but increased NO has been associated with nitrosative
stress and growth inhibition.7−10 Both ROS and RNS are
generated during immune response to counter infectious
pathogens, presumably due to their ability to synergize11,12
leading to increased damaging effects.13−15 However, due to its
antioxidant capability,16,17 NO has also been implicated in
protecting bacteria from oxidative stress and may hence
contribute to bacterial drug resistance.18−20 Thus, the precise
effects of NO when generated in the presence of elevated ROS
are yet to be completely understood.
Due to its diverse roles, NO for use in biological studies must
be generated in a controlled manner and preferably in response
to a stimulus.21,22 A number of nitric oxide donors are available,
but among these, sodium salts of diazeniumdiolates (such as
DEA/NO 1a, Scheme 1) are reliable sources of NO.23
However, these NO donor salts lack specificity and would
generate NO in the absence of H2O2 as well. Diazeniumdiolate
Following a reported procedure, compound 5 was
synthesized by bromination of 443,44 (Figure 1) and reacted
with DEA/NO 1a to obtain 2a (Scheme 2). A similar
procedure was used to synthesize 2b−2d by the reaction of
Scheme 1. BORO/NO, H2O2 Activated NO Donors
Figure 1. Structure of compounds 4−7 and 3a−3d.
Received: March 11, 2014
Published: May 5, 2014
© 2014 American Chemical Society
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Letter
Scheme 2. Synthesis of 4-BORO/NO Derivatives
the bromide 5 with the corresponding diazeniumdiolate salts,
1b−1d (Scheme 2).45 In order to study possible differences in
1,4- versus 1,2-elimination38 of the phenolate intermediate
generated during hydrogen peroxide activated NO release from
this class of compounds, derivatives 1a, 1b, and 1d were reacted
with the corresponding 2-(pinacol boronate ester)benzyl
bromides to produce 3a−3d (Figure 1).
The 4-BORO/NO derivative 2a was incubated in pH 7.4
buffer, and no evidence for NO production in the headspace
was found as determined by a chemiluminescence-based assay
that is selective for NO (Figure 2a). In the presence of H2O2,
Figure 3. Solution phase nitric oxide analysis using a chemilumi-
nescence detector: (a) Calibration curve generated using sodium
nitrite; (b) Representative spectrum when a solution of 2a in H2O2
was analyzed; (c) Nitric oxide analysis of BORO/NO derivatives and
4, 6, and 7; (d) NO generated in solution when incubated with various
biologically relevant species. Aliquots from the solution phase of the
reaction vial were injected into a chemiluminescene-based nitric oxide
analyzer using argon as the carrier gas containing a reducing mixture.
Under these conditions, values of NO reported are NO + nitrite.
NAcCys: N-acetyl cysteine; GSH: glutathione; TEMPO: 2,2,6,6-
tetramethylpiperidinyloxy; Fe2+; TBHP: tert-butyl hydroperoxide;
OCl−: sodium hypochlorite; O2−•: generated using hypoxanthine/
xanthine oxidase; HO• was generated using H2O2 and Fe(II) mixed at
a 1:10 ratio; here, DTPA was not added.
incubation of 2a in the presence of H2O2 in buffer (Figure
3b; see Supporting Information Figure S3). When 2a (25 μM)
was incubated with varying equivalents of H2O2, we found a
dose-dependent increase in nitric oxide produced after 10 min
(Figure 2c; see Supporting Information, Table S6) and 10
equiv of H2O2 were chosen for further studies.
A time course of headspace NO produced during incubation
of 2a (25 μM) in the presence of H2O2 (10 equiv) showed a
gradual increase of NO during 10 min, and the yield of NO was
nearly 12 μM (Figure 2d). When the other analogues 2b−2d
and 3a−3d were tested, we found nitric oxide production only
when exposed to H2O2 and the yields of NO (see Supporting
Information, Table S1 and Figure 3c) were comparable or
lower than the yield of NO produced during incubation of 2a
under similar conditions. A time course of NO production
during incubation of the 2-BORO/NO derivative 3a in the
presence of H2O2 showed no major difference in the rate of
NO release possibly because 1,2- and 1,4-eliminations occurred
at comparable rates (Figure 2d).
Compound 4 (Figure 1) which contained the boronate ester
but no capability to generate NO did not produce significant
levels of NO during incubation with H2O2 (Figure 3c). Next,
the diethylamino derivative 6 and benzyl derivative of DEA/
NO, 7 (Figure 1), were prepared using reported procedures
(see Supporting Information). During incubation with H2O2
(10 equiv), as expected, we found no evidence for NO
generation by these compounds suggesting the requirement for
the boronate ester functionality as well as the diazeniumdiolate
for NO production (Figure 3c).
Figure 2. Nitric oxide analysis in the headspacea using a
chemiluminescence detector: (a) Traces for 2a alone, in the presence
of H2O2 with and without a NO scavenger;b (b) Calibration curve
generated using NO gas; (c) NO generated by 2a with increasing
equivalents of H2O2; (d) Time course of NO generated during
a
incubation of 2a and 3a in the presence of 10 equiv H2O2.
Experiments were conducted in 10 mM pH 7.4 phosphate buffer
containing 100 μM of diethylene triamine pentaacetic acid (DTPA), a
metal ion chelating agent at 37 °C. Reaction mixtures from headspace
of reaction vial were injected into a chemiluminescene-based nitric
b
oxide analyzer using argon as the carrier gas. Arrow indicates
injection of sample.
we found significant levels of NO produced (Figure 2a). When
2a was incubated in the presence of H2O2 and (2-(4-
carboxyphenyl)-4,4,5,5-tetramethyl imidazoline-1-oxyl-3-oxide
(c-PTIO), a scavenger for NO, we found nearly complete
abrogation of signal attributable to NO in the headspace
confirming NO generation by 2a (Figure 2a). A calibration
curve with nitric oxide gas was generated using authentic NO
gas (linear regression analysis coefficient, R2 = 0.998, Figure
2b).
Next, hydrogen peroxide induced NO generation in solution
was analyzed. First, a calibration curve with sodium nitrite in a
reducing mixture, which converts nitrite to NO, was generated
(linear regression analysis coefficient, R2 = 0.999, Figure 3a).
We found evidence for nitric oxide production during
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Letter
Having established that BORO/NO derivatives were capable
of generating NO when triggered by H2O2, a number of
biologically relevant nucleophiles, reductants, and oxidants were
utilized to study the selectivity of activation of 2a to produce
NO (Figure 3d). The NO donor 2a was found to be highly
specific toward activation by H2O2 and did not generate
significant levels of NO in the presence of biologically relevant
thiols, metal ions, antioxidants, and other common ROS
(Figure 3d). The observed specificity is consistent with
previously published data on reactivity of boronate esters
toward H2O2.36,46
In order to study if BORO/NO was capable of generating
NO in bacteria, Escherichia coli (E. coli) cells were incubated
with 2a (25 μM). The ability of this compound to enhance NO
was studied by measuring extracellular nitrite using a Griess
assay. We found negligible NO during incubation of 2a alone,
but under similar conditions, when 2a was cotreated with
H2O2, we found a significant increase in extracellular nitrite, a
marker for increased NO (Figure 4a). The amount of nitrite
bacteria with 2a resulted in increased nitrite (see Supporting
Information) in the presence of hydrogen peroxide (Figure 4c).
Taken together, we report BORO/NO, a novel class of cell
permeable H2O2 activated NO donors. Antibiotic resistance is
emerging as a major global health problem with millions
affected each year, and the role of NO in bacteria developing
drug resistance remains unclear. NO has been reported to
synergize with silver(I) sulfadiazine, an oxidative stress inducing
antimicrobial, against a host of bacteria including S. aureus
suggesting the possible use of NO in combination therapy. In
contrast, NO generated by bacterial nitric oxide synthase
(bNOS) has been proposed as a possible cellular antioxidant
that protects MRSA from oxidative stress,18−20 and specific
bNOS inhibitors are in development as possible drug
candidates.48 Thus, molecular mechanisms of NO-derived
protective effects (or otherwise)16,17 remain to be elucidated
and it is anticipated that BORO/NO will be useful in
deciphering such mechanisms.42 The use of BORO/NO for
localized delivery of NO to cancers,24,25 which are known to
have elevated ROS as a phenotype, and other applications are
currently under investigation.24,25,49,50
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthesis, characterization data, assay protocols and associated
data. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: harinath@iiserpune.ac.in.
Author Contributions
Figure 4. (a) Extracellular nitrite in E. coli after 1 h; (b) Intracellular
NO from 2a in E. coli; (c) Extracellular nitrite release upon incubation
of 2a in the presence of Gram-positive and Gram-negative bacteria.
†These authors contributed equally.
Notes
The authors declare no competing financial interest.
generated was diminished when cotreated with c-PTIO, a
scavenger for NO. Compounds 4, 6, and 7, which did not
generate NO upon reaction with H2O2 in buffer (Figure 3c),
were incapable of enhancing nitrite when incubated with
bacteria and H2O2 (Figure 4a).
ACKNOWLEDGMENTS
■
The authors thank IISER Pune and the Department of
Biotechnology, Ministry of Science and Technology, India
(DBT, BT/PR6798/MED/29/636/2012) for financial support.
A.T.D. and G.R. acknowledge research fellowships from
Council for Scientific and Industrial Research (CSIR).
Diazeniumdiolate salts were a gift from Dr. Larry Keefer and
Dr. Joseph Saavedra (Chemical Biology Laboratory, National
Cancer Institute).
We next studied the ability of 2a to enhance intracellular NO
levels in E. coli in the presence of elevated levels of H2O2.47
Bacteria were incubated with 2a (100 μM) for 30 min followed
by centrifugation of the bacterial suspension to aspirate out any
excess 2a in the medium. The collected bacterial pellet was
resuspended with buffer and plated in a 96-well microplate, and
cells were incubated with and without added H2O2 at 37 °C.
Any NO that is produced must be due to 2a that has permeated
bacteria. After 30 min, Griess reagent was added and we found
significantly increased NO in bacteria treated with H2O2 in
comparison with untreated cells (Figure 4b). Under similar
conditions, when bacterial cells were treated with H2O2 and c-
PTIO, a NO scavenger, we found diminished nitrite supporting
the intermediacy of NO during decomposition of 2a (Figure
4b). Taken together, our data support 2a as a cell permeable
hydrogen peroxide activated NO donor.
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