Targeted Delivery of Persulfides to the Gut: Effects on the Microbiome

Article abstract of DOI:10.1002/anie.202014052

Persulfides (R?SSH) have been hypothesized as potent redox modulators and signaling compounds. Reported herein is the synthesis, characterization, and in vivo evaluation of a persulfide donor that releases N-acetyl cysteine persulfide (NAC-SSH) in response to the prokaryote-specific enzyme nitroreductase. The donor, termed NDP-NAC, decomposed in response to E. coli nitroreductase, resulting in release of NAC-SSH. NDP-NAC elicited gastroprotective effects in mice that were not observed in animals treated with control compounds incapable of persulfide release or in animals treated with Na₂S. NDP-NAC induced these effects by the upregulation of beneficial small- and medium-chain fatty acids and through increasing growth of Turicibacter sanguinis, a beneficial gut bacterium. It also decreased the populations of Synergistales bacteria, opportunistic pathogens implicated in gastrointestinal infections. This study reveals the possibility of maintaining gut health or treating microbiome-related diseases by the targeted delivery of reactive sulfur species.

Full text of DOI:10.1002/anie.202014052

Angewandte
Research Articles
Chemie
How to cite: Angew. Chem. Int. Ed. 2021, 60, 6061–6067
International Edition: doi.org/10.1002/anie.202014052
German Edition: doi.org/10.1002/ange.202014052
Gasotransmitters
Hot Paper
Targeted Delivery of Persulfides to the Gut: Effects on the Microbiome
Kearsley M. Dillon, Holly A. Morrison, Chadwick R. Powell, Ryan J. Carrazzone,
Veronica M. Ringel-Scaia, Ethan W. Winckler, R. McAlister Council-Troche, Irving C. Allen,*
and John B. Matson*
À
Abstract: Persulfides (R SSH) have been hypothesized as
potent redox modulators and signaling compounds. Reported
herein is the synthesis, characterization, and in vivo evaluation
of a persulfide donor that releases N-acetyl cysteine persulfide
(NAC-SSH) in response to the prokaryote-specific enzyme
nitroreductase. The donor, termed NDP-NAC, decomposed in
response to E. coli nitroreductase, resulting in release of NAC-
SSH. NDP-NAC elicited gastroprotective effects in mice that
were not observed in animals treated with control compounds
incapable of persulfide release or in animals treated with Na2S.
NDP-NAC induced these effects by the upregulation of
beneficial small- and medium-chain fatty acids and through
increasing growth of Turicibacter sanguinis, a beneficial gut
bacterium. It also decreased the populations of Synergistales
bacteria, opportunistic pathogens implicated in gastrointestinal
infections. This study reveals the possibility of maintaining gut
health or treating microbiome-related diseases by the targeted
delivery of reactive sulfur species.
received considerable interest lately because of their role as
likely H2S signaling products, coupled with advances in redox
biology showing that native persulfides carry out specific,
antioxidative physiological functions.[11] Several persulfide-
releasing compounds (termed persulfide donors) release their
payload under a variety of conditions, including changes in
pH,[9d] and addition of fluoride,[12] hydrogen peroxide,[9b] or
esterases.[9a,c] These donors provide insight into persulfide
interactions from a systemic perspective, but it is difficult to
determine the role(s) persulfides may serve in specific areas
of the body due to a lack of persulfide donors with targeting
capabilities.
One particular area where persulfides may play important
physiological and/or pathophysiological roles is in the gut.[13]
The gut microbiome is a promising drug target because many
links have been discovered connecting microbiome health
and composition to a variety of human diseases. The effects of
exogenously delivered H2S in the microbiome are controver-
sial; some studies have shown beneficial effects of H2S in the
regeneration of colon epithelial tissues by decreasing local
inflammation,[14] while others have found harmful effects,
such as inhibiting colonocyte respiration and increasing
inflammation.[15] The discrepancies in the observed outcomes
of exogenously delivered H2S may be a result of inconsistent
delivery methods and concentrations, combined with the use
of H2S donors capable of reaching the bloodstream and
exerting off-target effects. As for other RSS, little is known;
for example, there exist no studies evaluating the effect of
persulfides in the microbiome due to a lack of persulfide
donors specifically activated in the gut.
We sought to address this lack of targeted persulfide
donors and gap in knowledge in how persulfide delivery
affects the gut microbiome by synthesizing a persulfide donor
that responds to the prokaryote enzyme nitroreductase (NR).
NRs are located specifically in bacteria, sequestered mostly in
the mouth, skin, and intestines in mammals. Pharmacologi-
cally, NRs have been utilized to trigger the release of a variety
of compounds ranging from prodrug systems[16] to chemical
probes used in imaging.[17] For example, Dubikovskaya
developed an NR-responsive caged luciferin probe for use
in gene-directed enzyme prodrug therapy. The probe fluor-
esced exclusively in the presence of NR, yielding a novel
method to view NR transformed cancer cells.[17a] Chakrapani
et al. reported the synthesis and characterization of an NR-
responsive nitric oxide (NO)-releasing prodrug.[16a] The donor
was stable in PBS buffer, but released NO selectively in
response to NR. Chakrapani also synthesized an NR-respon-
sive H2S prodrug by combining p-nitrobenzyl thiol with
a ketone to form a thioketal.[18] The donor released H2S in the
Introduction
Hydrogen sulfide (H2S) has been under investigation as
a biological signaling gas (gasotransmitter) since its physio-
logical signaling capacity was discovered in 1996.[1] To aid in
understanding its (patho)physiological roles, many different
types of donors have been synthesized with a variety of
triggers, including water,[2] nucleophiles,[3] light,[4] and en-
zymes.[5] Some may hold therapeutic value in the form of
prodrugs and drug conjugates.[6] More recently, additional
work has been conducted in the synthesis of compounds that
release a variety of related reactive sulfur species (RSS)
including carbonyl sulfide (COS),[7] sulfur dioxide (SO2),[8]
[9]
À
and persulfides (R SSH), with the goal of understanding
the specific roles of each of these RSS in the greater reactive
species interactome.[10] Of these RSS, persulfides have
[*] H. A. Morrison, V. M. Ringel-Scaia, R. M. Council-Troche, I. C. Allen
Department of Biomedical Sciences and Pathobiology
Virginia-Maryland College of Veterinary Medicine, Virginia Tech
Blacksburg, VA 24061 (USA)
E-mail: icallen@vt.edu
K. M. Dillon, C. R. Powell, R. J. Carrazzone, E. W. Winckler,
J. B. Matson
Department of Chemistry, Virginia Tech Center for Drug Discovery
and Macromolecules Innovation Institute, Virginia Tech
Blacksburg, VA 24061 (USA)
E-mail: jbmatson@vt.edu
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.202014052.
Angew. Chem. Int. Ed. 2021, 60, 6061 –6067
ꢀ 2021 Wiley-VCH GmbH
6061

Angewandte
Research Articles
Chemie
presence of bacteria and mediated cytoplasmic redox poten-
tial upon exposure to H2O2; however, it also significantly
increased antibiotic resistance. Inspired by these studies and
intrigued by the possibility of generating persulfides specif-
ically in the gut, we set out to synthesize and evaluate an NR-
triggered persulfide donor.
Results and Discussion
Prodrug Design and Synthesis
Scheme 2. Synthesis of NDP-NAC. AIBN=azobisisobutyronitrile,
DCE=1,2-dichloroethane.
Using a similar design strategy to a previously reported
H2O2-reponsive persulfide donor,[9b] we synthesized a new
persulfide donor termed NDP-NAC for nitroreductase disul-
fide prodrug–N-acetylcysteine (Scheme 1). NRs utilize a two-
Persulfide Release Kinetics
Due to their inherent instability, persulfides are difficult to
detect directly by high resolution mass spectrometry
(HRMS), so electrophilic trapping reagents such as N-ethyl-
maleimide (NEM), iodoacetamide (IAM),[20] or dinitrofluor-
obenzene (DNFB)[9a] are commonly used. The reaction of
a persulfide with these trapping agents forms a disulfide that
typically can withstand MS ionization conditions, whereas the
persulfide itself mostly disproportionates into H2S and a thiol
or forms trisulfides when directly analyzed.[9e] We attempted
to trap the persulfide using NEM, IAM, and DNFB, and we
found that DNFB worked best in this system.
In the experimental setup, NDP-NAC, DNFB, and
NADH were dissolved in pH 8.0 PBS and added to a vial
with a screwcap lid, and an aliquot was removed for a zero
timepoint (t = 0). Exact procedures for dilution and analysis
are described in the Supporting Information. NR was then
added to the reaction mixture, and aliquots were removed
every 30 min. After a brief workup step to remove excess
NADH, each aliquot was analyzed by analytical HPLC
(Figures 1, S15). The peak corresponding to NDP-NAC
(elution time 4.0 min) decreased in intensity over time as
a new peak (6.1 min) evolved, which we determined corre-
sponded to the trapped persulfide adduct (DNB-NAC, Fig-
ure S10) based on comparison to an authentic sample. After
2 h, NDP-NAC had been consumed, and DNB-NAC re-
mained. The conversion of NDP-NAC into DNB-NAC
indicated that NDP-NAC efficiently released its persulfide
payload in the presence of NR.
Scheme 1. Proposed mechanism of persulfide release from NDP-NAC.
1,6-Elimination may proceed via the hydroxylamine or amine inter-
mediates.
electron reduction mechanism employing nicotinamide ad-
enine dinucleotide (NADH) as a cofactor. The para-posi-
À
À
tioned nitro (R NO2) group is first reduced to a nitroso (R
À
NO) intermediate, then reduced to a hydroxylamine (R
[19]
À
NHOH), and finally to an amine (R NH2) group. In the
Next, we set out to determine the persulfide release half-
1
case of NDP-NAC, we envisioned that this free amine (or
hydroxylamine) could undergo a 1,6-elimination, resulting in
the concomitant release of N-acetylcysteine persulfide (NAC-
SSH) and p-aminobenzyl alcohol (pABA).
life from NDP-NAC using H NMR spectroscopy (Figure 2).
NDP-NAC and NADH were dissolved in a mixture of
[D6]DMSO and deuterated phosphate buffer (dPB) at pD =
8.0 (1:4 v/v). This pD value was chosen to mimic the mildly
basic conditions of the intestines, where most gut bacteria are
located. A zero timepoint was taken, NR was then added to
the NMR tube, and subsequent scans were taken over the
course of several hours. As shown in the zero timepoint
spectrum, a clear doublet (a) corresponding to NDP-NAC
was observed at 7.42 ppm. After the addition of NR, a new
doublet (a’), which we attribute to pABA, was observed at
7.15 ppm. Other signals in the aromatic region were due to
NADH and NAD+, the oxidation product of NADH, includ-
ing the three new signals above 8.6 ppm. By 10.7 h, complete
NDP-NAC was synthesized in four steps (Scheme 2).
First, p-nitrotoluene was brominated using N-bromosuccini-
mide (NBS), yielding p-nitrobenzyl bromide (1). Next,
thiourea was added, and subsequent aminolysis using hexyl-
amine yielded p-nitrobenzyl thiol (2). The resulting thiol was
added to the activated disulfide of NAC (NACpyDS) in
a disulfide exchange reaction to afford the prodrug in a 35%
overall yield with only one column purification step required
throughout the synthesis.
6062 www.angewandte.org
ꢀ 2021 Wiley-VCH GmbH
Angew. Chem. Int. Ed. 2021, 60, 6061 –6067

Angewandte
Research Articles
Chemie
Figure 1. Analytical HPLC chromatogram series depicting persulfide
release from NDP-NAC over time using DNFB as a trapping reagent.
The peak eluting at 4.0 min corresponds to NDP-NAC, while the peak
at 6.1 min corresponds to the persulfide adduct DNB-NAC. Reactions
were performed at 228C in pH 8.0 phosphate buffer (the pH level
stabilized at 7.8) at 100 mM NDP-NAC. The monitoring wavelength
was 282 nm.
Figure 2. 1H NMR analysis of NDP-NAC decomposition kinetics. The
half-life was calculated to be 1.5 h Æ0.3 h (n=3). Reactions were
performed at 228C in pD 8.0 phosphate buffer (the pD level stabilized
at 8.1) at 3.6 mM NDP-NAC.
Effects of NDP-NAC on Mammalian and Bacterial Cells in
Culture
disappearance of NDP-NAC was observed. A decomposition
half-life (t1/2) of 1.5 h Æ 0.3 h (n = 3) was calculated from these
experiments (Figure S16). We and others have observed
similar discrepancies between the half-lives observed in
1H NMR and HPLC experiments previously,[21] and we
attributed them to slower 1,6-elimination rates in organic
solvents relative to aqueous solvent mixtures.[22] Although
replication of the complex gut microbiome environment is not
possible in these NMR and HPLC experiments, taken
together they show that NDP-NAC generates NAC-SSH in
response to nitroreductase over the course of hours under the
mildly basic conditions present in the intestines.
We then aimed to determine the effects of exogenous
delivery of persulfides in the microbiome. However, we first
needed to establish whether NDP-NAC was toxic to human
cells. NDP-NAC cell viability was conducted on H9C2
cardiomyocytes as a representative normal mammalian cell
line (Figure 3A). NDP-NAC was nontoxic up to 400 mM,
inducing proliferation to a small degree at lower levels
compared with untreated controls. Of note, cell viability
greater than 100% in these experiments is likely due to
artifacts of the CCK-8 assay.[23]
Furthermore, we next evaluated the reactivity of NDP-
NAC with Cys to assess the potential for biological reductants
to hamper the ability of the prodrug to release persulfides.
NDP-NAC and Cys (2 equiv) were dissolved in 1:4
[D6]DMSO:pD 8.0 dPB or 1:4 [D6]DMSO:pD 2.5 dPB. In
both cases, no substantial changes to the 1H NMR spectrum of
NDP-NAC were observed after 24 h at room temperature
(Figures S17 and S18), suggesting that NDP-NAC is stable in
the presence of thiols under these conditions.
Next, the toxicity of NDP-NAC to prokaryotes that are
commonly located in the gut was evaluated (Figure 3B). An
aqueous solution of NDP-NAC at either 1, 10, or 100 mgmLÀ1
was added to molten agar and stirred until homogeneous.
Agar plates were prepared, and then Escherichia coli, Listeria
monocytogenes, and Staphylococcus aureus were then used to
inoculate the plates. After 24 h in culture, NDP-NAC
exhibited no toxicity towards E. coli, a common inhabitant
of the human gut. However, NDP-NAC did elicit a dose-
dependent decrease in viability in L. monocytogenes and
S. aureus, two opportunistic pathogens.
Angew. Chem. Int. Ed. 2021, 60, 6061 –6067
ꢀ 2021 Wiley-VCH GmbH
www.angewandte.org 6063

Angewandte
Research Articles
Chemie
widespread use as a simple, instantaneous H2S donor that
lacks byproducts. After mice (n = 5 with five treatment
groups: NDP-NAC, Benz-NAC, NDP-TE, Na2S, and PBS
vehicle only) were given single daily gavages of NDP-NAC
(100 mL, 100 mgmLÀ1), control compounds (100 mL,
100 mgmLÀ1) or PBS vehicle (100 mL) over 5 d, feces were
harvested and plated on agar. Morphology assessments were
conducted, and individual, morphologically unique colonies
were picked and cultured to logarithmic growth phase in this
biased screening approach. Each specimen was identified to
at least the genera level using matrix-assisted laser desorp-
tion/ionization–time of flight (MALDI-TOF) mass spectrom-
etry.
The types of bacteria discovered in this approach were
quite different in animals treated with NDP-NAC versus all
others (Figure 4B). As expected, animals that received PBS
only were rich in Bacillus species with no unique or non-
Bacillus species. Bacillus species are quite prevalent in
a healthy gut microbiome. Mice treated with Benz-NAC also
elicited no microbiome change, whereas NDP-TE and Na2S
elicited minor changes (two and one unique or non-Bacillus
species, respectively). However, in animals treated with NDP-
NAC, we recovered 8 unique or non-Bacillus species, includ-
ing Enterococcus faecalis, Bacillus cereus, and Staphylococcus
lentus, which are all common probiotic bacteria found in
commercial supplements. While we recognize this prodrug
may react in unintended ways in the complex gut environ-
ment, these findings suggested that NDP-NAC was capable of
regulating the microbiome, warranting a deeper search into
possible effects on metabolism within the gut.
To expand upon the findings associated with the micro-
biome population changes, we also sought to determine the
effects of the altered microbiome on functional and metabolic
processes of biological and physiological relevance by eval-
uating short- and medium-chain fatty acids (SCFAs and
MCFAs) in the fecal specimens using gas chromatography.[24]
SCFA/MCFA analysis revealed mostly minor differences in
metabolites evaluated using NDP-NAC, Benz-NAC, NDP-
TE, and vehicle-treated groups. However, we did observe
a ninefold increase in heptanoic acid production between
NDP-NAC and PBS-treated groups (Figure S19). In the
gastrointestinal tract, heptanoic acid plays key roles in lipid
transport, peroxidation, and metabolism; decreased levels
have been found in patients with Crohnꢀs disease, ulcerative
colitis, and pouchitis.[25] Together, these data suggest that
NDP-NAC may exhibit gastroprotective and anti-inflamma-
tory effects.[26] Mouse blood plasma samples were also
analyzed using ultra-high-performance liquid chromatogra-
phy with tandem mass spectrometry (UPLC-MS/MS). No
trace of NDP-NAC or pABA was detected in any of the six
mouse plasma samples (LOD = 0.1 ngmLÀ1, see the Support-
ing Information). These results suggest that NDP-NAC does
not pass through the intestinal epithelial cell barrier, and its
effects appear to be contained within the gastrointestinal
tract, likely indicating that the prodrug is metabolized directly
by the bacteria or at least in close proximity. Likewise, no
adverse effects were observed in animals treated with NDP-
NAC. In summary, NDP-NAC induced changes in the mouse
microbiome, affecting the types of bacterial species present
Figure 3. Summary of eukaryote and bacteria viability after treatment
with NDP-NAC. A) H9C2 viability after treatment with NDP-NAC at
various concentrations. Viability was quantified using Cell Counting
Kit 8 (CCK-8), comparing treatment groups to an untreated control.
Results are expressed as the mean ÆSEM (n=3, 3–5 wells per
experiment). B) Bacteria viability in response to NDP-NAC at various
concentrations after 24 h culture in agar plates. Viability is reported
with respect to control plates with no NDP-NAC.
In Vivo Effects of NDP-NAC on the Mouse Microbiome
With these results in hand, we set out to investigate effects
on general bacteria populations elicited by NDP-NAC and
various controls in the mouse microbiome. Two compounds
that are similar in structure to NDP-NAC were synthesized as
control compounds for use in these studies (Figure 4A).
Benz-NAC, which is analogous to NDP-NAC but lacks the
nitro group, was prepared as a control compound that could
not release a persulfide but was otherwise structurally similar.
NDP-TE, which possesses a thioether linkage instead of
a disulfide linkage, was synthesized as a second control
compound that could undergo decomposition in response to
nitroreductases but would release a thiol (NAC) instead of
a persulfide. Despite potentially low bioavailability due to the
oral gavage method used for delivery, Na2S was utilized as
a model H2S donor control in these experiments due to its
Figure 4. Summary of structural analogues of NDP-NAC used in
MALDI-TOF. A) Chemical structures NDP-NAC and controls Benz-
NAC (lacking nitro group) and NPD-TE (a thioether in place of
a disulfide) used in MALDI-TOF analysis. B) Broad identification of
non-Bacillus species present in the mouse gastrointestinal microbiome
upon treatment with NDP-NAC or various controls using biased
sampling and MALDI-TOF.
6064 www.angewandte.org
ꢀ 2021 Wiley-VCH GmbH
Angew. Chem. Int. Ed. 2021, 60, 6061 –6067

Angewandte
Research Articles
Chemie
and the production of metabolites that were not observed in
clostridium, eubacterium, clostridiales, lachnoclostridium,
either of the control molecules or Na2S.
pseudoflavonifractor, and acutalibacter species in the micro-
biome isolated from the vehicle-treated animals. Conversely,
the microbiome from the NDP-NAC-treated animals had
a significant increase in members of the Turicibacter genus,
specifically Turicibacter sanguinis species. Intriguingly, T.
sanguinis has recently been identified as a highly relevant
gut bacterium that naturally expresses a neurotransmitter
sodium symporter related protein homologous to mammalian
SERT, which is a host 5-HT (serotonin) transporter.[27]
T. sanguinis blooms in the GI tract can alter a broad range
of host genes impacting multiple biochemical pathways,
including increases in those important for lipid and steroid
metabolism and decreasing systemic triglyceride levels and
inguinal adipocyte size.[27] In addition to T. sanguinis being
increased in the NDP-NAC-treated animals, additional meta-
stats analysis was conducted, which relies on a nonparametric
t-test for detecting differentially abundant features in meta-
genomic studies. Results revealed a significant decrease in the
relative abundance of Synergistales bacterium in NDP-NAC-
treated animals (Figure 5C). Synergistales bacteria are a re-
cently identified family of anaerobic, Gram-negative, oppor-
tunistic pathogens that are implicated in the formation and
progression of cysts, abscesses, periodontal disease, and
gastrointestinal infections.[28]
Building upon these findings, we next repeated our animal
studies (same administration method, dose, and numbers of
animals, but only using NDP-NAC and PBS-vehicle treat-
ment groups) and utilized a more robust, nonbiased screening
approach to better define microbiome population differences
between the NDP-NAC-treated and untreated animals. Feces
were harvested from mice at the end of the 5 day study, with
collection directly from the colon at necropsy; tissue scraping
was conducted to also recover adherent populations. Fecal
specimens were flash frozen upon sterile collection, followed
by nucleic acid extraction for shotgun metagenomics analysis.
Consistent with the biased screening assessments noted
above, treatment with NDP-NAC resulted in changes in
microbiome populations, with significant differences noted
between the PBS-vehicle- and NDP-NAC-treated animals.
Sample clustering with taxonomic abundance revealed that
the majority of the PBS vehicle-treated animals clustered
together (Figure 5A). However, treatment with NDP-NAC
generated two distinct groups that were significantly different
in composition at the Phylum level compared to the PBS
vehicle (Figure 5B). Further assessments by LEfSe (linear
discriminant analysis effect size) analysis revealed that the
differences between the PBS-vehicle- and NDP-NAC-treated
animals were due to decreased members of the genus
Pseudoflavonifractor and at the species level decreased
Finally, to gain additional insight into the microbial
metabolic processes impacted by NDP-NAC, we also utilized
Figure 5. Sample clustering with taxonomic abundance. A) Sample clustering analysis based on Bray–Curtis distance highlighting the similarity of
samples from each animal. The distance was calculated according to relative taxonomic abundance, and the data shown were generated by
combining the clustering results and relative abundances of different samples at the phylum level. B) LEfSe analysis identifying genomic features
(taxa) characterizing the significant differences between the microbiome populations among treatment groups. The histogram of the LDA scores
shows the species whose LDA scores are larger than the set threshold (4 set by default). C) Metastats analysis demonstrating a significant
decrease in the relative abundance of Synergistales bacterium in mice treated with NDP-NAC. *p<0.05.
Angew. Chem. Int. Ed. 2021, 60, 6061 –6067
ꢀ 2021 Wiley-VCH GmbH
www.angewandte.org 6065

Angewandte
Research Articles
Chemie
shotgun metagenomics and to assemble protein sequence Conclusion
data to predict microbial protein function. This analysis
identified changes in only three functions following NDP-
NAC treatment: genetic information processing/translation,
lipid metabolism, and endocrine and metabolic diseases
(Figure 6). The increased translation and lipid metabolism
In summary, NDP-NAC, a nitroreductase-responsive
persulfide prodrug, which to our knowledge represents the
first persulfide donor with targeting capabilities, released
persulfides in the presence of microbial nitroreductase with
a half-life on the order of hours. NDP-NAC significantly
impacted the composition and function of the host micro-
biome in mice treated with the prodrug compared with mice
treated with non-persulfide-releasing control compounds and
Na2S, an H2S donor. Changes elicited by NDP-NAC created
favorable niches for the expansion of a limited repertoire of
bacteria species, including Turicibacter sanguinis, that appear
to have robust host effects impacting metabolism and overall
gastrointestinal health. Conversely, NDP-NAC also demon-
strated clear antimicrobial effects on a diverse range of
opportunistic and pathogenic bacteria in the gut. This
includes limiting Synergistales bacteria, which are associated
with a variety of human health issues. Thus, NDP-NAC
enhances several health-promoting bacteria while limiting the
growth of various pathogenic bacteria. This work adds to the
body of knowledge in the rapidly growing field of gut
microbiome health, showing that controlled delivery of RSS
to the gut may be useful for modulating the gastrointestinal
microbiome during disease states, shifting the balance from
dysbiosis to homeostasis.
Acknowledgements
This research was supported by the National Institutes of
Health (R01GM123508), the Virginia Tech Center for Drug
Discovery, and the Dreyfus Foundation. We thank Dr. Mehdi
Ashraf-Khorassani for assistance with LCMS, Dr. Kristin
Eden for her animal model expertise, and Elizabeth Smiley
for fatty acid analysis. The authors also acknowledge Novo-
gene Co., Ltd. for their genome sequencing and bioinformat-
ics work.
Conflict of interest
Figure 6. Metastats analysis of functional differences. Three functions,
whose abundances were significantly different between the PBS-
vehicle- and NDP-NAC-treatment groups, were identified from shotgun
metagenomic analyses: A) genetic information processing/translation,
B) lipid metabolism, C) endocrine and metabolic diseases. *p<0.05.
The authors declare no conflict of interest.
Keywords: drug delivery ·inflammation ·microbiome ·
oxidative stress ·stimuli responsiveness
differences are highly consistent with the findings associated
with the increased Turicibacter sanguinis levels (Figure 5B) as
this bacterium significantly impacts lipid metabolism, trigly-
ceride levels, and host genetic functions.[27] These findings are
also consistent with the observed changes in heptanoic acid
(Figure S19), which is critical for lipid transport and metab-
olism. We also found that in contrast to previous reports on
H2S delivery to the gut,[18] genetic markers for antibiotic
resistance did not increase in the NDP-NAC treatment group
(Figures S23, S24, and S25).
[1] K. Abe, H. Kimura, J. Neurosci. 1996, 16, 1066 –1071.
[2] a) L. Li, M. Whiteman, Y. Y. Guan, K. L. Neo, Y. Cheng, S. W.
Lee, Y. Zhao, R. Baskar, C.-H. Tan, P. K. Moore, Circulation
2008, 117, 2351; b) L. A. D. Nicolau, R. O. Silva, S. R. B.
Damasceno, N. S. Carvalho, N. R. D. Costa, K. S. Arag¼o,
A. L. R. Barbosa, P. M. G. Soares, M. H. L. P. Souza, J. V. R.
Medeiros, Braz. J. Med. Biol. Res. 2013, 46, 708 –714; c) W.
Zhao, J. Zhang, Y. Lu, R. Wang, EMBO J. 2001, 20, 6008 –6016;
d) J. Kang, Z. Li, C. L. Organ, C.-M. Park, C.-t. Yang, A.
Pacheco, D. Wang, D. J. Lefer, M. Xian, J. Am. Chem. Soc. 2016,
138, 6336 –6339.
6066 www.angewandte.org
ꢀ 2021 Wiley-VCH GmbH
Angew. Chem. Int. Ed. 2021, 60, 6061 –6067

Angewandte
Research Articles
Chemie
[3] a) Y. Zhao, H. Wang, M. Xian, J. Am. Chem. Soc. 2011, 133, 15 – [15] M. Beaumont, M. Andriamihaja, A. Lan, N. Khodorova, M.
17; b) J. C. Foster, C. R. Powell, S. C. Radzinski, J. B. Matson,
Org. Lett. 2014, 16, 1558 –1561.
Audebert, J.-M. Blouin, M. Grauso, L. Lancha, P.-H. Benetti, R.
Benamouzig, D. TomØ, F. Bouillaud, A.-M. Davila, F. Blachier,
Free Radical Biol. Med. 2016, 93, 155 –164.
[4] a) N. Fukushima, N. Ieda, K. Sasakura, T. Nagano, K. Hanaoka,
T. Suzuki, N. Miyata, H. Nakagawa, Chem. Commun. 2014, 50,
587 –589; b) N. O. Devarie-Baez, P. E. Bagdon, B. Peng, Y.
Zhao, C.-M. Park, M. Xian, Org. Lett. 2013, 15, 2786 –2789.
[5] a) Y. Zheng, B. Yu, K. Ji, Z. Pan, V. Chittavong, B. Wang, Angew.
Chem. Int. Ed. 2016, 55, 4514 –4518; Angew. Chem. 2016, 128,
4590 –4594; b) P. Chauhan, P. Bora, G. Ravikumar, S. Jos, H.
Chakrapani, Org. Lett. 2017, 19, 62 –65; c) Z. Li, C. L. Organ, Y.
Zheng, B. Wang, D. J. Lefer, Circulation 2016, 134, 17903.
[6] C. R. Powell, K. M. Dillon, J. B. Matson, Biochem. Pharmacol.
2018, 149, 110 –123.
[7] a) C. R. Powell, J. C. Foster, B. Okyere, M. H. Theus, J. B.
Matson, J. Am. Chem. Soc. 2016, 138, 13477 –13480; b) A. K.
Steiger, S. Pardue, C. G. Kevil, M. D. Pluth, J. Am. Chem. Soc.
2016, 138, 7256 –7259; c) C. R. Powell, J. C. Foster, S. N. Swilley,
K. Kaur, S. J. Scannelli, D. Troya, J. B. Matson, Polym. Chem.
2019, 10, 2991 –2995.
[16] a) K. Sharma, K. Sengupta, H. Chakrapani, Bioorg. Med. Chem.
Lett. 2013, 23, 5964 –5967; b) P. F. Searle, M.-J. Chen, L. Hu,
P. R. Race, A. L. Lovering, J. I. Grove, C. Guise, M. Jaberipour,
N. D. James, V. Mautner, L. S. Young, D. J. Kerr, A. Mountain,
S. A. White, E. I. Hyde, Clin. Exp. Pharmacol. Physiol. 2004, 31,
811 –816.
[17] a) A. G. Vorobyeva, M. Stanton, A. Godinat, K. B. Lund, G. G.
Karateev, K. P. Francis, E. Allen, J. G. Gelovani, E. McCormack,
M. Tangney, E. A. Dubikovskaya, PLoS One 2015, 10, e0131037;
b) D. Zhu, L. Xue, G. Li, H. Jiang, Sens. Actuators B 2016, 222,
419 –424.
[18] P. Shukla, V. S. Khodade, M. SharathChandra, P. Chauhan, S.
Mishra, S. Siddaramappa, E. P. Bulagonda, A. Singh, H.
Chakrapani, Chem. Sci. 2017, 8, 4967 –4972.
[19] A.-F. Miller, T. J. Park, L. K. Ferguson, W. Pitsawong, S. A.
Bommarius, Molecules 2018, 23, 211.
[8] a) W. Wang, X. Ji, Z. Du, B. Wang, Chem. Commun. 2017, 53,
1370 –1373; b) J. J. Day, Z. Yang, W. Chen, A. Pacheco, M. Xian,
ACS Chem. Biol. 2016, 11, 1647 –1651; c) S. R. Malwal, D.
Sriram, P. Yogeeswari, V. B. Konkimalla, H. Chakrapani, J. Med.
Chem. 2012, 55, 553 –557.
[20] T. Chatterji, K. Keerthi, K. S. Gates, Bioorg. Med. Chem. Lett.
2005, 15, 3921 –3924.
[21] K. M. Dillon, C. R. Powell, J. B. Matson, Synlett 2019, 30, 525 –
531.
[22] K. M. Schmid, L. Jensen, S. T. Phillips, J. Org. Chem. 2012, 77,
4363 –4374.
[9] a) Y. Zheng, B. Yu, Z. Li, Z. Yuan, L. Chelsea, K. Rishi, S. Wang,
J. Lefer David, B. Wang, Angew. Chem. Int. Ed. 2017, 56, 11749 – [23] J. Lin, M. Akiyama, I. Bica, F. T. Long, C. F. Henderson, R. N.
11753; Angew. Chem. 2017, 129, 11911 –11915; b) C. R. Powell,
K. M. Dillon, Y. Wang, R. J. Carrazzone, J. B. Matson, Angew.
Chem. Int. Ed. 2018, 57, 6324 –6328; Angew. Chem. 2018, 130,
6432 –6436; c) Z. Yuan, Y. Zheng, B. Yu, S. Wang, X. Yang, B.
Wang, Org. Lett. 2018, 20, 6364 –6367; d) I. Artaud, E. Galardon,
ChemBioChem 2014, 15, 2361 –2364; e) V. S. Khodade, J. P.
Toscano, J. Am. Chem. Soc. 2018, 140, 17333 –17337.
[10] M. M. Cortese-Krott, A. Koning, G. G. C. Kuhnle, P. Nagy, C. L.
Bianco, A. Pasch, D. A. Wink, J. M. Fukuto, A. A. Jackson, H.
van Goor, K. R. Olson, M. Feelisch, Antioxid. Redox Signaling
2017, 27, 684 –712.
[11] a) S. Kasamatsu, A. Nishimura, M. Morita, T. Matsunaga, H.
Abdul Hamid, T. Akaike, Molecules 2016, 21, 1721; b) P. K.
Yadav, M. Martinov, V. Vitvitsky, J. Seravalli, R. Wedmann,
M. R. Filipovic, R. Banerjee, J. Am. Chem. Soc. 2016, 138, 289 –
299; c) E. Cuevasanta, M. N. Mçller, B. Alvarez, Arch. Biochem.
Biophys. 2017, 617, 9 –25.
[12] J. Kang, S. Xu, N. Radford Miles, W. Zhang, S. K. Shane, J.
Day Jacob, M. Xian, Angew. Chem. Int. Ed. 2018, 57, 5893 –
5897; Angew. Chem. 2018, 130, 5995 –5999.
[13] a) L. L. Barton, N. L. Ritz, G. D. Fauque, H. C. Lin, Dig. Dis. Sci.
2017, 62, 2241 –2257; b) F. Carbonero, A. C. Benefiel, A. H.
Alizadeh-Ghamsari, H. R. Gaskins, Front. Physiol. 2012, 3, 448 –
448.
Goddu, V. Suarez, B. Baker, T. Ida, Y. Shinkai, P. Nagy, T.
Akaike, J. M. Fukuto, Y. Kumagai, Chem. Res. Toxicol. 2019, 32,
447 –455.
[24] V. M. Ringel-Scaia, Y. Qin, C. A. Thomas, K. E. Huie, D. K.
McDaniel, K. Eden, P. A. Wade, I. C. Allen, J. Innate Immun.
2019, 11, 416 –431.
[25] V. De Preter, K. Machiels, M. Joossens, I. Arijs, C. Matthys, S.
Vermeire, P. Rutgeerts, K. Verbeke, Gut 2015, 64, 447.
[26] a) K. M. Maslowski, A. T. Vieira, A. Ng, J. Kranich, F. Sierro, D.
Yu, H. C. Schilter, M. S. Rolph, F. Mackay, D. Artis, R. J. Xavier,
M. M. Teixeira, C. R. Mackay, Nature 2009, 461, 1282 –1286;
b) M. Wlodarska, A. D. Kostic, R. J. Xavier, Cell Host Microbe
2015, 17, 577 –591; c) Y. I. Kim, Nutr. Rev. 1998, 56, 17 –24.
[27] T. C. Fung, H. E. Vuong, C. D. G. Luna, G. N. Pronovost, A. A.
Aleksandrova, N. G. Riley, A. Vavilina, J. McGinn, T. Rendon,
L. R. Forrest, E. Y. Hsiao, Nat. Microbiol. 2019, 4, 2064 –2073.
[28] a) E. Jumas-Bilak, J.-P. Carlier, H. Jean-Pierre, D. Citron, K.
Bernard, A. Damay, B. Gay, C. Teyssier, J. Campos, H. March-
andin, Int. J. Syst. Evol. Microbiol. 2007, 57, 2743 –2748; b) H.-P.
Horz, D. M. Citron, Y. A. Warren, E. J. C. Goldstein, G. Con-
rads, J. Clin. Microbiol. 2006, 44, 2914 –2920; c) S. R. Vartou-
kian, R. M. Palmer, W. G. Wade, Anaerobe 2007, 13, 99 –106.
[14] J. L. Wallace, J.-P. Motta, A. G. Buret, Am. J. Physiol. Gastro-
intest. Liver Physiol. 2018, 314, G143 –G149.
Manuscript received: October 19, 2020
Version of record online: January 29, 2021
Angew. Chem. Int. Ed. 2021, 60, 6061 –6067
ꢀ 2021 Wiley-VCH GmbH
www.angewandte.org 6067