Sodium borohydride and thiol mediated nitrite release from nitroaromatic antibiotics

Article abstract of DOI:10.1016/j.bmcl.2021.128245

Nitroaromatic antibiotics are used to treat a variety of bacterial and parasitic infections. These prodrugs require reductive bioactivation for activity, which provides a pathway for the release of nitrogen oxide species such as nitric oxide, nitrite, and/or nitroxyl. Using sodium borohydride and 2-aminoethanol as model reductants, this work examines release of nitrogen oxide species from various nitroaromatic compounds through several characterization methods. Specifically, 4- and 5-nitroimidazoles reproducibly generate higher amounts of nitrite (not nitric oxide or nitroxyl) than 2-nitroimidazoles during the reaction of model hydride donors or thiols. Mass spectrometric analysis shows clean formation of products resulting from nucleophile addition and nitro group loss. 2-Nitrofurans generate nitrite upon addition of sodium borohydride or 2-aminoethanethiol, but these complex reactions do not produce clean organic products. A mechanism that includes nucleophile addition to the carbon βto the nitro group to generate a nitronate anion followed by protonation and nitrous acid elimination explains the observed products and labeling studies. These systematic studies give a better understanding of the release mechanisms of nitrogen oxide species from these compounds allowing for the design of more efficient therapeutics.

Full text of DOI:10.1016/j.bmcl.2021.128245

Bioorg. Med. Chem. Lett. 48 (2021) 128245
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Sodium borohydride and thiol mediated nitrite release from
nitroaromatic antibiotics
Allison M. Rice, Allison Faig, David E. Wolff, S. Bruce King*
Wake Forest University, Department of Chemistry, Winston-Salem, NC 27101, United States of America
A R T I C L E I N F O
A B S T R A C T
Keywords:
Nitroaromatic antibiotics are used to treat a variety of bacterial and parasitic infections. These prodrugs require
reductive bioactivation for activity, which provides a pathway for the release of nitrogen oxide species such as
nitric oxide, nitrite, and/or nitroxyl. Using sodium borohydride and 2-aminoethanol as model reductants, this
work examines release of nitrogen oxide species from various nitroaromatic compounds through several char-
acterization methods. Specifically, 4- and 5-nitroimidazoles reproducibly generate higher amounts of nitrite (not
nitric oxide or nitroxyl) than 2-nitroimidazoles during the reaction of model hydride donors or thiols. Mass
spectrometric analysis shows clean formation of products resulting from nucleophile addition and nitro group
loss. 2-Nitrofurans generate nitrite upon addition of sodium borohydride or 2-aminoethanethiol, but these
complex reactions do not produce clean organic products. A mechanism that includes nucleophile addition to the
carbon βto the nitro group to generate a nitronate anion followed by protonation and nitrous acid elimination
explains the observed products and labeling studies. These systematic studies give a better understanding of the
release mechanisms of nitrogen oxide species from these compounds allowing for the design of more efficient
therapeutics.
Nitroaromatic antibiotics
PA-824
Metronidazole
Nitrite
Reactive nitrogen species (RNS)
Nitroaromatic antibiotics, possess a long history of therapeutic use
for the treatment of anaerobic bacterial and parasitic infections.1–4
Metronidazole, a well-known and cost effective nitroaromatic antibiotic
on the list of the World Health Organizations (WHO) 50 Essential
Medicines, has been used since the 1950′s to treat a variety of ailments
including Heliobacter pylori infections, trichomoniasis, giardiasis, and
amoebiasis.5–7 PA-824 (Pretomanid) was recently approved by the Food
and Drug Administration (FDA) as a combination treatment of highly
resistant TB.8,9 PA-824 also shows activity against various parasites and
these nitro-containing drugs hold promise as treatments for many
neglected tropical diseases, such as leishmaniasis.3,10,11 However, the
absence of clear and complete molecular mechanisms of action and their
associated toxicities have limited development of nitroaromatics as drug
candidates despite their biological activity.
products may also generate reactive oxygen species (ROS) or trigger the
unraveling of the aromatic ring to yield reactive organic com-
pounds.12–15 These reactive species likely play important roles in the
observed biological activity of these antibiotics.12,16 Reductive bio-
activation also provides the opportunity for the conversion of the
organic nitro group (N oxidation state = +3) to reactive nitrogen species
(RNS) including nitrite (NOꢀ2 ), nitric oxide (NO), and nitroxyl (HNO).
Nitric oxide, in particular, demonstrates activity against a number of
micro-organisms and many NO-based approaches towards infectious
disease treatment have been explored.2,3,17–19 Isolated examples of
nitroaromatic antibiotics generating RNS upon reduction exist and have
been recently reviewed.3,16 For example, metronidazole directly reacts
with thiols to release NOꢀ2 through nitro group substitution by the
thiol.20 Reaction of metronidazole with thiols and iron generates NO as
judged by electron paramagnetic resonance (EPR) spectroscopy.21 Pa-
tients taking metronidazole metabolize it to acetamide and an oxamic
acid derivative accounting for only two of the initial three N atoms of the
drug.3,16 The biological activity of PA-824 depends upon its ability to
release NO or other RNS upon deazaflavin-dependent nitroreductase
(F420H2-dependent Ddn) catalyzed hydride transfer.22
Nitroaromatic antibiotics act as prodrugs and require reductive
bioactivation for activity. Nitroreductase (NTR, Type I, oxygen insen-
sitive and Type II, oxygen sensitive) catalyzed nitro group reduction
results in the formation of reduced nitrogen species including nitro
radical anions, nitroso compounds, hydroxylamines or amines depend-
ing on the substrate, organism, and specific NTR. The formation of these
* Corresponding author.
E-mail address: kingsb@wfu.edu (S.B. King).
https://doi.org/10.1016/j.bmcl.2021.128245
Received 21 May 2021; Received in revised form 24 June 2021; Accepted 30 June 2021
Available online 7 July 2021
0960-894X/© 2021 Elsevier Ltd. All rights reserved.

A.M. Rice et al.
Bioorganic & Medicinal Chemistry Letters 48 (2021) 128245
These nitroaromatic antibiotics clearly release biologically active
chromatographic (GC) headspace detection of nitrous oxide (N2O)
provides evidence for the formation of HNO, which rapidly dimerizes
and dehydrates to yield N2O.29 Electrospray ionization mass spectro-
metric (ESI-MS) analysis of the reaction mixtures reveal the presence of
organic based derivatives of the nitroaromatics (Figures S11-S23). The
Supporting Information provides further details for these procedures.
RNS upon reduction under some conditions.16 Given the known bio-
logical activity of NO2ꢀ , NO, and HNO,23–27 this chemistry may provide a
powerful design element toward new therapeutics for bacterial and
parasitic infections. As such, we have examined the ability of nitro-
aromatic antibiotics including metronidazole, PA-824, nitrofurantoin,
furaltadone, benznidazole, and nitrofurazone, as well as isoniazid (a
non-nitroaromatic control antibiotic) (Figure 1) to release RNS during
model reduction with a hydride donor or thiol.
Reactions with sodium borohydride (NaBH4)
In this report, we systematically examine and compare these clini-
cally important agents as RNS sources upon reaction with hydride do-
nors and thiols using various spectroscopic and analytical techniques
that allow for the identification of the RNS and organic product. Such
results give insight toward the potential mechanisms of and the struc-
tural features that support RNS release in these nitroaromatic
antibiotics.
The F420H2-dependent Ddn catalyzed reduction of PA-824 generates
three organic products; the des-nitro derivative (1), the cyclic lactam
(2), and 3 that may form from further reduction of an unstable hy-
droxylamine (Scheme 1).22 These products form from the apparent
transfer of hydride (-H) to the antibiotic that also results in the release of
NOꢀ2 and/or NO.22 NaBH4 thus provides a model hydride source to
mimic this enzymatic reduction and treatment of PA-824 with NaBH4
generates NOꢀ2 and 2 as the major organic product.22
Results/Discussion
Table 1 summarizes the formation of NOꢀ2 and nitrous oxide upon
treatment of the above nitroaromatic antibiotics with NaBH4 in a
mixture of buffer (e.g. PBS buffer, 100 mM, pH = 7.4)/ethanol at 37 ◦C
for one day. Metronidazole, a 5-nitroimidazole, shows the highest
amount (~45%) of NOꢀ2 formation consistent with its ability to release
NOꢀ2 upon reaction with thiols (vide infra).20 In general, measurement of
NOꢀ2 using the Griess assay or the NOA gave similar results for all
compounds except PA-824, which gave lower amounts in the Griess
assay suggesting the electron-rich aromatic ring of PA-824 may interfere
with the assay (Table 1). For the Griess assay experiments, a 96 well
plate and a UV–vis plate reader was used to rapidly screen multiple
antibiotics and conditions at one time for NOꢀ2 release. PA-824, a 4-
nitroimidazole, generates NOꢀ2 (28%), but benznidazole, a 2-nitroimida-
zole, produced less than 5% NOꢀ2 . The lack of NO2ꢀ formation from
benznidazole appears consistent with previous work showing the
reduction of 2-nitroimidazoles yields glyoxal and guanidine de-
rivatives.14 The 2-nitrofuran derivatives all gave similar amounts of NO2ꢀ
(10–15%) upon NaBH4 reduction (Table 1). Headspace NOA detection
of NO (using buffer (e.g. PBS buffer, 100 mM, pH = 7.4) in the NOA
reaction chamber) showed no significant NO formation during these
reactions. GC measurements were used to probe N2O formation as evi-
dence of HNO release, as HNO dimerizes and dehydrates to give N2O.
While the 2-nitrofuran derivatives formed the most N2O, none of these
reductions produced more than 10% N2O.
For studies of the NaBH4 and thiol mediated nitrogen oxide release
from various nitroaromatic antibiotics, commercially available metro-
nidazole, nitrofurantoin, furaltadone, benznidazole, nitrofurazone, and
isoniazid were used as purchased (Figures S1-S7). PA-824 was synthe-
sized according to a literature procedure (Figure S8-S10).28 Determi-
nation of NOꢀ2 was accomplished using the spectrophotometric Griess
assay and a commercial Nitric Oxide Analyzer (NOA), which relies on
highly sensitive ozone-chemiluminescence technology, with a reaction
chamber solution of KI/acetic acid.29,30 Nitric oxide detection was
accomplished using the NOA and a non-reducing buffer solution (e.g.
PBS buffer, 100 mM, pH = 7.4) in the reaction chamber. Gas
Control experiments showed no NOꢀ2 or N2O release by the Griess
Assay, NOA, or GC from these nitroaromatic antibiotics in the absence of
NaBH4. Further GC controls show the headspace of a solution of NaNO2
and NaBH4 in EtOH does not contain N2O eliminating the borohydride
reduction of NOꢀ2 as an HNO source. Treatment of isoniazid, a non-
nitroaromatic containing antibiotic as a control, with NaBH4 does not
produce NOꢀ2 , NO or N2O.
In addition, after reduction with NaBH4, ESI-MS data was obtained
for each sample to assess the loss of the nitro group and the formation of
a reduced product. The reaction of metronidazole with NaBH4 showed a
prominent peak with a mass/charge (m/z) ratio = 127 (Figure S11),
indicative of the loss of the nitro group and the formation of 2-(2-
Figure 1. Nitroaromatic antibiotics used for sodium borohydride (NaBH4) and
thiol mediated RNS release. Isoniazid was used as a non-nitroaromatic con-
trol antibiotic.
Scheme 1. PA-824 reduction products 1–3 with F420-H2-Ddn.22.
2

A.M. Rice et al.
Bioorganic & Medicinal Chemistry Letters 48 (2021) 128245
NaBH4 shows a m/z = 331, which suggests the formation of the lactam
(2, Scheme 1 and Figure S12) and is consistent with the reported NaBH4
reduction of PA-824 under similar conditions.22 This ESI-MS analysis
shows some remaining PA-824, but no evidence of the des-nitro product
(1) (m/z = 315) (Figure S12).22 The NaBH4 reduction of the 2- nitro-
furan, nitrofurantoin, yields a mixture of the des-nitro product (m/z =
194), starting material (m/z = 239), and evidence of a diazo dimer (m/z
= 413) (Figure S13). The mass spectrum of the NaBH4 reduction of
nitrofurantoin is much less defined as those of the reactions of metro-
nidazole and PA-824. Treatment of nitrofurantoin with NaBH4 on a
preparative scale also gave the diazo dimer as evidenced by ESI-MS (m/
z = 413) (Scheme 3). Direct two-electron reduction of the nitro group to
form the nitroso compound, which could dimerize and undergo further
reduction, provides a possible mechanism for diazo dimer formation
(Scheme 3).
Table 1
Nitrogen oxide release results for nitroaromatic antibiotics treated with NaBH4.
Nitroaromatic
Antibiotics
Nitro Group
Position
Griess
Assay
(NOꢀ2 )
NOA
GC
(NOꢀ2 )
(HNO)
Metronidazole
PA-824
5
45%
10%
8%
43%
28%
9%
3%
2%
8%
2%
6%
3%
—
4
Nitrofurantoin
Furaltadone
Nitrofurazone
Benznidazole
Isoniazid
2
2
14%
13%
4%
9%
2
12%
1%
2
—
—
—
methyl-1H-imidazol-1-yl)ethan-1-ol, and only a small peak for metro-
nidazole itself (m/z = 172). Given this clean reaction profile and the
large amount of NOꢀ2 released during the NaBH4 reduction of metroni-
dazole, further mechanistic studies on this reaction were performed. The
use of deuterated reducing agents and deuterated solvents identified the
position of hydride addition (Figures S24-S30). Reduction of metroni-
dazole on a preparative scale gave 2-(2-methyl-1H-imidazol-1-yl)ethan-
The other nitrofurans (e.g. furaltadone and nitrofurazone) also
demonstrated poorly defined ESI-MS that showed mostly starting ma-
terial (Figures S14-S15). ESI-MS analysis of the reduction of benznida-
zole with NaBH4 did not show the formation of any products, with the
starting material (m/z = 361) being the prominent species (Figure S16).
1-ol in 62% yield, which was characterized by H NMR and 13C NMR
1
spectroscopy and through comparison to a commercial standard
(Scheme 2 and Figures S26 and S27).
Reactions with thiols
The 1H NMR spectra of this material shows two aromatic resonances
at δ = 6.7 and 6.9 that have been assigned to the protons attached to
carbons 4 and 5, respectively (Figure S26). Treatment of metronidazole
with sodium borodeuteride (NaBD4) in ethanol led to the formation of 2-
(2-methyl-1H-imidazol-1-yl-4-d)ethan-1-ol (18%) (Scheme 2) and
unreacted metronidazole (82%) as confirmed by 1H NMR spectroscopy
(Figure S28). The 1H NMR spectrum showed the intensity of the more
upfield aromatic signal (δ = 6.7 ppm) to be dramatically decreased
(Figure S28) suggesting deuterium incorporation at C-4 of the imidazole
ring system. Treatment of metronidazole with NaBH4 in deuterated
methanol (CD3OD) gave the 2-(2-methyl-1H-imidazol-1-yl-5-d)ethan-1-
ol with a dramatic reduction in the intensity of the most deshielded
aromatic signal (δ = 6.9 ppm, Figure S29, S30). The decreased proton
signal implies deuterium incorporation into the imidazole, however at
C-5 (Scheme 2). These results are consistent with and similar to previous
results for NaBH4/NaBD4 reduction of PA-824, which forms 1, 2 (major
product) and NOꢀ2 .22 The major peak from the reduction of PA-824 with
Similar to the reaction of PA-824 with NaBH4, the treatment of
metronidazole with 2-aminoethanethiol yields 4-[(2-aminoethyl)thio]-
2-methylimidazole-1-ethanol and nitrous acid (nitrite) (Scheme 4).20
This study confirmed the release of nitrogen oxides from metroni-
dazole and structurally similar compounds upon nucleophilic substitu-
tion of the nitro group of 5-nitroimidazoles by thiols.20 Under more basic
conditions, an isomeric imidazole product forms and a plausible mech-
anism may be direct thiol addition to the nitroimidazole forming a
Meisenheimer-type complex that generates nitrous acid and restores
aromaticity upon protonation.20 These results clearly show direct ni-
trogen oxide release from metronidazole and supports the use of 2-ami-
noethanethiol as a model reductant for nitrogen oxide release from
nitroaromatic antibiotics. In these experiments, the aforementioned
nitroaromatic antibiotics were treated with excess 2-aminoethanethiol
in buffer (e.g. PBS buffer, 100 mM, pH = 7.4) at 37 ◦C for one day
(Scheme 4), followed by nitrogen oxide release measurements utilizing
the Griess Assay, NOA, and GC.
Table 2 displays the results for the release of nitrogen oxides upon
thiol addition to the nitroaromatic antibiotics. As expected, metroni-
dazole shows robust amounts (36–48%) of NOꢀ2 formation.20 Interest-
ingly, the addition of thiol to PA-824, a 4-nitroimidazole, liberates
smaller but reproducible amounts of NOꢀ2 indicating a potentially new
mechanism for nitrogen oxide release from this drug. As before, benz-
nidazole only produced 1% NOꢀ2 under these conditions, consistent with
its poor RNS release profile.14 The 2-nitrofuran derivatives, nitro-
furantoin and nitrofurazone both generated significant amounts of NO2ꢀ
(~45%) similar to that seen with metronidazole, but furaltadone only
gave ~ 10% NO2. Headspace NOA detection of NO (using buffer (e.g.
PBS buffer, 100 mM, pH = 7.4) in the NOA reaction chamber) showed no
Scheme 2. Metronidazole reactions with NaBH4 and NaBD4.
Scheme 3. Reduction of nitrofurantoin leading to a diazo-dimer product.
3

A.M. Rice et al.
Bioorganic & Medicinal Chemistry Letters 48 (2021) 128245
adduct with loss of the nitro group (Figure S18). These results contrast
older work that indicates metronidazole does not generate NOꢀ2 or GSH
adducts in rat and human liver homongenates.34 Despite the promising
ESI-MS results with GSH and metronidazole, difficulties arose during the
assessment of NOꢀ2 release from these reactions using the Griess assay or
NOA as GSH appeared to interfere with NOꢀ2 detection giving low and
irreproducible results. This interference may be related the differences
in pKa’s for 2-aminoethanethiol and GSH, the buffer composition and
strength and the expected decrease in pH upon release of HNO2. The
reactivity of GSH with RNS (NO, NOꢀ2 , HNO) is complex and may
complicate these results.35,36 For example, the formation of NOꢀ2 under
slightly acidic conditions in the presence of excess GSH could generate S-
nitrosoglutathione (GSNO) thus masking NOꢀ2 detection.37 Further work
will clarify NOꢀ2 /NO release from the GSH reactions as well as GSNO
production, but the ESI-MS clearly shows the ability of this important
biological thiol to directly react with metronidazole.
Scheme 4. Metronidazole reaction with 2-aminoethanethiol.
Table 2
Nitrogen oxide release results for nitroaromatic antibiotics treated with 2-
aminoethanethiol.
Nitroaromatic
Antibiotics
Nitro Group
Position
Griess
Assay
(NOꢀ2 )
NOA
GC
(NOꢀ2 )
(HNO)
Scheme 5 (with metronidazole) depicts a possible mechanism for
NOꢀ2 release in these reactions where hydride ion or thiol adds to the
carbon β to the nitro group to generate a nitronate anion that is pro-
tonated by the solvent. Elimination of nitrite/nitrous acid from this
species forms the observed products and a reactive nitrogen species
(HNO2/NOꢀ2 ) and the isotopic labeling experiments support this
pathway. Nucleophile addition to the carbon β to the nitro group as
opposed to other sites allows for resonance stabilization of the resulting
anion. Addition at other sites, forming a Meisenheimer-type complex
that generates nitrous acid and restores aromaticity upon protonation as
previously proposed,20 would account for isomeric addition products. 4-
and 5-Substituted imidazoles appear particularly suited to this reactivity
compared to 2-substituted imidazoles or 2-nitrofurans. Scheme 5 pro-
vides a mechanistic framework for consideration in the design of 4- and
5-nitroimidazoles capable of NOꢀ2 release.
Metronidazole
PA-824
5
36%
7%
48%
17%
41%
9%
2%
2%
2%
2%
3%
2%
—
4
Nitrofurantoin
Furaltadone
Nitrofurazone
Benznidazole
Isoniazid
2
48%
9%
2
2
48%
1%
44%
1%
2
—
—
—
significant NO formation during these reactions. GC measurements were
used to probe N2O formation as evidence of HNO release as before. None
of these compounds under these conditions produced more than 3%
N2O. Low N2O amounts in these experiments could indicate the lack of
HNO formation or result from the reaction of HNO with excess thiol to
generate the corresponding disulfide and hydroxylamine.31 Control ex-
periments showed no NOꢀ2 or N2O release by the Griess Assay, NOA, or
GC from these nitroaromatic antibiotics in the absence of thiol. Treat-
ment of isoniazid with 2-aminoethanethiol does not produce NOꢀ2 , NO or
N2O.
While not observed as predominant products in the true reaction,
Scheme 5 also outlines the potential mechanism of NO and HNO for-
mation. Further one electron reduction of nitrite, especially under acidic
conditions, would yield NO.16 Hydrolysis of the nitronate (Nef Reac-
tion)38 gives a dihydroxy nitroso compound (Scheme 5) that would
decompose to lactam and HNO. While the lactam product 2 was
observed during the NaBH4 reduction of PA-824 (Scheme 1), N2O as
evidence of HNO, was not detected. Given the reactivity of HNO, with
the absence of N2O formation, could indicate HNO trapping by another
species (RSH, NaBH4). In these simple studies, NOꢀ2 appears to be the
major RNS formed.
ESI-MS measurements provides evidence that 2-aminoethanethiol
adds to each of the nitroaromatic antibiotics with the loss of the nitro
group, however some reactions provided much cleaner more efficiently
ionized spectra than others (Figures S17-S23). Metronidazole showed a
prominent peak at m/z = 202, with a small peak for metronidazole itself
(m/z = 172), indicative of a thiol adduct (98%) and nitro group loss, as
expected (Figure S17).20 While some PA-824 remains upon treatment
with 2-aminoethanethiol as judged by ESI-MS (m/z = 360), this reaction
mixture also showed a thiol adduct with loss of the nitro group at m/z =
390, supporting the notion of thiol-mediated NOꢀ2 release (Figure S19).
ESI-MS experiments with nitrofurantoin revealed a prominent peak at
m/z = 283 indicative of thiol addition/nitro group loss, as well as
starting material (m/z = 239) (Figure S20). Although these results are
promising for nitrofurantoin, the known bioactivation of nitrofurantoin
remains poorly understood and speculation remains whether it produces
RNS rather than ROS.32,33 The reaction of furaltadone with 2-aminoe-
thanethiol, which was poorly ionized, also showed starting material
(m/z = 325), with very little evidence of the thiol adduct (m/z = 355),
which corresponds to the small amounts of NOꢀ2 measured (Figure S21).
While nitrofurazone did not show strong evidence of the thiol adduct
(m/z = 229), the starting material peak (m/z = 199) is not pronounced
by ESI-MS, which corresponds with the significant NOꢀ2 release
measured (Figure S22). For benznidazole, the starting material (m/z =
361) is the most prominent peak in the ESI-MS, which supports the lack
of NOꢀ2 release as evident in both the Griess Assay and NOA experiments
(Figure S23).
Conclusion
These systematic studies show release of nitrogen oxides species
from various nitroaromatic antibiotics upon reaction with NaBH4 or
thiols. Specifically, 4- and 5-nitroimidazoles reproducibly generate
higher amounts of NOꢀ2 (not NO or HNO) during the reaction of model
hydride donors or thiols. Mass spectrometric analysis shows clean for-
mation of products resulting from nucleophile addition and nitro group
loss. Of particular interest is the formation of NOꢀ2 from the NaBH4
Metronidazole was also treated with the more biologically relevant
thiol, glutathione (GSH) using the same reaction conditions developed
with 2-aminoethanethiol (vide supra). ESI-MS formation of a thiol adduct
for metronidazole-GSH showed the best evidence of a reaction, with a
prominent peak (m/z = 432) corresponding to a metronidazole-GSH
Scheme 5. Possible mechanism for NOꢀ2 release from metronidazole.
4

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Bioorganic & Medicinal Chemistry Letters 48 (2021) 128245
13 McClelland RA, Panicucci R, Rauth AM. Products of the Reductions of 2-
Nitroimidazoles. J. Am. Chem. Soc. 1987;109:4308–4314. https://doi.org/10.1021/
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reduction of metronidazole and the reaction of 2-aminoethanethiol with
PA-824, which reveal new pathways of RNS release from these clinically
used drugs. Scheme 5 illustrates a simple nucleophilic addition-nitrous
acid (nitrite) elimination that accounts for these reactions. While 2-ni-
trofurans generate NOꢀ2 upon addition of NaBH4 or 2-aminoethane
thiol, these reactions appear much more complex and do not produce
a clean organic product. In future studies, other nitroaromatic com-
pounds can be examined as well as other reductants, including nitro-
reductases or other reducing enzymes. The reaction of GSH with
metronidazole signals that GSH and other biologically relevant low
molecular weight (LMW) thiols, such as bacillithiol (BSH) and mycothiol
(MSH),39–41 may react with these compounds. The release of NOꢀ2 , NO,
and HNO could incite the continued development and attention to the
potential for controlled bioactivation through the design of nitro-
aromatic antibiotics by organism-specific reducing systems, which can
lead to the delivery of biologically active nitrogen oxide agents with
high activity and minimal toxicity.
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Declaration of Competing Interest
22 Singh R, Manjunatha U, Boshoff HIM, et al. PA-824 Kills Nonreplicating
Mycobacterium Tuberculosis by Intracellular NO Release. Science. 2008;322:
1392–1395. https://doi.org/10.1126/science.1164571.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
23 Methods in Nitric Oxide Research; Feelisch, M., Stamler, J. S., Eds.; John Wiley and
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This work was financially supported by the Center for Molecular
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Appendix A. Supplementary data
27 Blood AB. The Medicinal Chemistry of Nitrite as a Source of Nitric Oxide Signaling.
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28 Marsini MA, Reider PJ, Sorensen EJ. A Concise and Convergent Synthesis of PA-824.
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30 Miranda KM, Espey MG, Wink DA. A Rapid, Simple Spectrophotometric Method for
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