Therapeutic Delivery of H2S via COS: Small Molecule and Polymeric Donors with Benign Byproducts

Article abstract of DOI:10.1021/jacs.6b07204

Carbonyl sulfide (COS) is a gas that may play important roles in mammalian and bacterial biology, but its study is limited by a lack of suitable donor molecules. We report here the use of N-thiocarboxyanhydrides (NTAs) as COS donors that release the gas in a sustained manner under biologically relevant conditions with innocuous peptide byproducts. Carbonic anhydrase converts COS into H₂S, allowing NTAs to serve as either COS or H₂S donors, depending on the availability of the enzyme. Analysis of the pseudo-first-order H₂S release rate under biologically relevant conditions revealed a release half-life of 75 min for the small molecule NTA under investigation. A polynorbornene bearing pendant NTAs made by ring-opening metathesis polymerization was also synthesized to generate a polymeric COS/H₂S donor. A half-life of 280 min was measured for the polymeric donor. Endothelial cell proliferation studies revealed an enhanced rate of proliferation for cells treated with the NTA over untreated controls.

Full text of DOI:10.1021/jacs.6b07204

Communication
pubs.acs.org/JACS
Therapeutic Delivery of H S via COS: Small Molecule and Polymeric
2
Donors with Benign Byproducts
Chadwick R. Powell,§ §,†Jeffrey C. Foster, Benjamin Okyere, Michelle H. Theus,
,†
§,†
‡
‡
,
and John B. Matson*
§
Department of Chemistry, Virginia Tech, Blacksburg, Virginia 24061, United States
†
Macromolecules Innovation Institute, Virginia Tech, Blacksburg, Virginia 24061, United States
‡
Department of Biomedical Sciences and Pathobiology, Virginia−Maryland Regional College of Veterinary Medicine, Duck Pond
Drive, Blacksburg, Virginia 24061, United States
*
S Supporting Information
may have enabled COS to serve as a condensing agent in
8
,9
ABSTRACT: Carbonyl sulfide (COS) is a gas that may
play important roles in mammalian and bacterial biology,
but its study is limited by a lack of suitable donor
molecules. We report here the use of N-thiocarboxyanhy-
drides (NTAs) as COS donors that release the gas in a
sustained manner under biologically relevant conditions
with innocuous peptide byproducts. Carbonic anhydrase
prebiotic times for coupling amino acids. Investigation into
the role of COS in biology continues today, with COS-
generating enzymes (thiocyanate hydrolases) identified in
bacteria and COS generation and utilization hypothesized in
10
mammals. The study of COS biology is complicated by its
short half-life in vivo, primarily due to its fast hydrolysis by
carbonic anhydrase (CA). CA is ubiquitous in plants and
mammals and efficiently carries out its primary function of
generating carbonic acid from water and carbon dioxide (CO2).
converts COS into H S, allowing NTAs to serve as either
2
COS or H S donors, depending on the availability of the
2
enzyme. Analysis of the pseudo-first-order H S release rate
2
Because of its structural similarity to CO , COS is also a
2
under biologically relevant conditions revealed a release
half-life of 75 min for the small molecule NTA under
investigation. A polynorbornene bearing pendant NTAs
made by ring-opening metathesis polymerization was also
substrate for CA, which the enzyme rapidly converts into CO
2
5
and H S. In fact, COS itself is relatively nontoxic: its toxicity is
2
11
a result of this conversion into toxic levels of H S.
2
Gasotransmitter donors have become vital tools for studying
synthesized to generate a polymeric COS/H S donor. A
2
the biology of NO, CO, and H S. Donors are typically small
2
half-life of 280 min was measured for the polymeric donor.
Endothelial cell proliferation studies revealed an enhanced
rate of proliferation for cells treated with the NTA over
untreated controls.
molecules that release gasotransmitters under physiologically
3
relevant conditions over a defined period of time. Chemists
strive to synthesize gasotransmitter donors that mimic the
body’s natural signaling through precise exogenous application.
Today, many small molecule donors are available that release
NO, CO, or H S, all triggered by various stimuli with wide-
2
ince nitric oxide (NO) was first proposed to be
ranging half-lives of release. For example, a variety of H2S
donors have been reported in recent years, with release triggers
1
S
endothelium-derived relaxing factor in 1986, an increasing
number of scientists have been drawn to the field of signaling
gases. These short-lived gaseous mediators, classified as
gasotransmitters, exist in most tissues throughout the body
12,13
14−16
17,18
19
including water,
thiols,
light,
and enzymes.
Gasotransmitter donors can be covalently attached to
20,21
drugs,
which extends the capacities of existing therapeutics,
2
and play a critical role in cellular signaling and homeostasis. To
or to polymers, providing localized release. Multiple types of
meet the definition of a gasotransmitter, a gas must be
enzymatically generated and regulated, have specific molecular
targets and physiological functions, and be freely permeable to
NO, CO, and H S-releasing polymers have been reported
2
22−24
recently.
In contrast, COS donors have received little
attention, which has impeded studies on COS biology or
3
cellular membranes. Three species are currently classified as
delivery of H S via COS release.
2
gasotransmitters: NO, carbon monoxide (CO), and hydrogen
To address this gap, we set out to design a molecule that
could readily generate COS in a controlled fashion, easily attach
to a polymer scaffold, and conveniently decompose to yield
biologically innocuous byproducts. We envisioned that this
COS-releasing small molecule and the resulting polymer could
serve as either COS donors (in the absence of CA) or as H2S
donors (in the presence of CA) to facilitate biological studies.
There exist a handful of methods to generate COS, including
sulfide (H S). However, evidence suggests that a variety of
2
other gases may be involved in cellular processes. Gases
currently under investigation include ammonia, methane, sulfur
4
−6
dioxide, and carbonyl sulfide (COS).
Of these, COS is
perhaps the least studied in biological systems, in large part due
to a lack of suitable donor molecules.
COS is the most abundant sulfur compound in the
atmosphere (500 ppt), generated naturally from emissions
7
from hot springs, trees, and volcanoes. Despite its low overall
Received: July 12, 2016
atmospheric concentration, high concentrations near volcanoes
©
XXXX American Chemical Society
A
DOI: 10.1021/jacs.6b07204
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
from potassium thiocyanate and sulfuric acid in the presence of
min (Figure S17). Further studies of H S release using an H S-
selective electrochemical probe gave a peaking time of 45 min
(Figure 1A). Additional release studies performed in complete
2
2
7
water and a recently published metal-catalyzed reaction of CO
25
and elemental sulfur. Although useful for generating solutions
of the gas, these methods are not capable of generating COS in
a biological environment. In the lone example of COS
generation in a biological system, Pluth and co-workers
recently reported on a class of turn-on fluorescent H S sensors
2
that regenerate the detected H S through a decomposition
2
26
reaction involving COS. Considering our requirements for
sustained release under biologically relevant conditions and
nontoxic byproducts, we envisioned that N-thiocarboxyanhy-
drides (NTAs) might provide a suitable platform from which to
build small molecule and polymeric COS donors.
NTAs were first reported in 1971 by Hirschmann et al. as an
alternative to N-carboxyanhydrides (NCAs) for use in the
solution-phase synthesis of oligopeptides. More recently,
Figure 1. (A) H
NTA1 (10 μM) in the presence of glycine (1 mM) with 300 nM CA
black curve) or without CA (gray curve) in PBS buffer (pH = 7.4).
S-selective electrochemical probe release curves for
2
27
(
NTAs have been synthesized from N-alkyl amino acids and
28
(B) Representative fluorescence spectra (λex = 340 nm) exhibiting an
polymerized to afford polypeptoids. Because the ring-opening
polymerization of NTAs occurs with loss of COS, we
envisioned that NTAs could be suitable COS donors upon
ring-opening by a biological nucleophile such as an amine. After
increase in intensity over time as H S reduces the nonemissive Da-N
2
3
probe into fluorescent Da-NH . (C) First-order kinetic plot of Da-
2
NH generation over time, as measured by the change in fluorescence
2
emission intensity at 535 nm. Line represents the pseudo-first-order
[
−kt]
COS release, conversion into the known gasotransmitter H S is
2
kinetic fit using the equation: conv. = 1 − e
.
expected to occur by one of two routes: (1) the rapid,
enzymatic conversion by CA; or (2) hydrolysis, a relatively
slower process.
endothelial cell media exhibited a faster release profile and a
quicker return to baseline (Figure S19). No measurable release
was observed in the absence of CA. Although a half-life cannot
be determined using this electrochemical method due to
oxidation and volatilization of the gas, it is a reliable way to
To test our hypothesis that NTAs could be used as COS/
H S donors, we began by synthesizing the NTA derivative of
2
sarcosine (NTA1) through the route shown in Scheme 1. With
measure instantaneous H S concentration.
Neither the colorimetric nor the electrochemical assay were
2
a
Scheme 1. Synthesis of NTA1
suitable to quantify the H S release rate in the absence of CA
2
because COS hydrolysis is sufficiently slow to limit the use of
these methods. To make a comparison between the H S release
2
rates with and without CA, additional analysis of the H2S
release profile of NTA1 was performed using the azide
derivative of dansyl sulfonamide developed by Wang et al.
a
Conditions: (i) NaOH, H O, 98% yield; (ii) CH Cl , 0 °C, 53%
31
2
2
2
yield.
The dansyl azide (Da-N ) probe exhibits pronounced
3
−
selectivity for the hydrosulfide anion (HS ) over other anions
28
modifications of literature procedures, we were able to obtain
NTA1 in two steps with minimal purification. Although COS
release from NTAs during ring-opening polymerization has
been hypothesized, direct detection of COS generation from
ring-opening of NTAs has not been reported. To identify COS
evolution, NTA1 was added to an aqueous solution of glycine
in an airtight vial. GC−MS analysis of samples taken from the
vial headspace after 5 min revealed the presence of COS (m/z
and reactive species commonly found in biological assays with a
response time on the order of seconds. The probe acts through
−
a turn-on fluorescence mechanism whereby HS reduces the
nonfluorescent azide in solution to give the corresponding
fluorescent amine (Da-NH ). Although Da-NH is a known CA
2
2
32
inhibitor, we used the initial rates of Da-N reduction by
3
NTA1 to compare the rates of H S production in the presence
2
and absence of CA.
=
60). A small peak at m/z = 34 was also observed, which is
Fluorescence assays were performed under similar conditions
to those described above for the methylene blue and
electrochemical probe experiments, monitoring the increase
in emission intensity at 535 nm over 10 h (Figure 1B). Pseudo-
consistent with slow hydrolysis of COS into H S (Figures S14
and 15). Confirmation of the expected dipeptide byproduct
upon nucleophilic attack by glycine was realized by LC-MS.
2
2
9
(
Figure S16).
first-order analysis of Da-NH production in the first 60 min of
2
After confirming COS release, we next measured the H2S
the experiment in the presence of CA gave an H S release half-
2
release kinetics of NTA1 both in the presence and absence of
CA. Currently, there are no simple and reliable methods for
measuring the rate COS release in solution. However, the fast
life of 120 min (Figure 1C). The discrepancy between the half-
lives based on methylene blue and fluorescence is likely due to
some inhibition of CA by Da-NH . Performing the assay in the
2
conversion of COS into H S in the presence of CA allows for
absence of CA gave a release half-life of 990 min, in excellent
agreement with previously measured COS hydrolysis rates in
2
indirect quantification of COS release kinetics using methods
29
commonly employed to detect and quantify H S. To evaluate
aqueous media.
2
the rate of H S evolution, a colorimetric assay known
Next, we set about preparing an NTA-containing polymer for
2
30
commonly as the methylene blue assay was chosen. In the
presence of 300 nM CA and 1 mM glycine (biologically
relevant values) in PBS buffer (pH = 7.4), the pseudo-first-
potential use in COS or H S-releasing films, hydrogels,
2
nanoparticles, or other materials that could serve to localize
gas delivery. A polymerization technique was needed that
would be compatible with the reactive NTA moiety. Ring-
order release half-life of H S for NTA1 was measured to be 75
2
B
DOI: 10.1021/jacs.6b07204
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Journal of the American Chemical Society
Communication
opening metathesis polymerization (ROMP) is typically
performed under mild conditions (room temperature, no
potential nucleophiles or radical initiators), proceeds rapidly to
33
high conversions, and is highly functional group tolerant.
Therefore, we designed a synthesis to couple a ROMP-active
norbornene to a COS donor. ROMP monomer NB-NTA was
prepared similarly to NTA1 in three steps starting from
iminodiacetic acid (Scheme 2A).
Scheme 2. Synthesis of Norbornene-NTA Monomer and
Copolymer polyNTA1
a
Figure 2. (A) H S-selective electrochemical probe release curves for
polyNTA1 (10 μM in NTA) in the presence of glycine (100 μM) with
2
3
00 nM CA in PBS buffer (pH = 7.4). Release was sustained out to 20
h. (B) Absorbance spectra from the methylene blue assay. The
increase in intensity over time corresponds to in situ generation of
methylene blue from H S. (C) First-order kinetic plot derived from
2
the methylene blue data, measured by an increase in absorbance at 676
nm. Line represents the pseudo-first order kinetic fit using the
[
−kt]
equation: conv. = 1 − e
.
proliferation is an important first step in angiogenesis, the
formation of new blood vessels from existing vessels, which is
vital for several biological processes, most notably wound
3
4
healing. H S promotes angiogenesis; therefore, H S donors
2
2
35
may be useful as wound healing agents. Experiments were
performed by treating endothelial cells with NTA1, Na S (an
2
H S source used as a positive control), or the Sar-Gly dipeptide
2
byproduct for 24 h, followed by measuring the percentage of
BrdU positive cells in each treatment group. No CA enzyme
was added to any of the treatment groups because the
concentration of endogenous CA, which is widely distributed in
mammalian cells, is sufficient to quickly convert COS into
11
H S. A significant increase in endothelial cell proliferation was
2
observed for both NTA1 and Na S at 100 μM (Figure 3)
2
a
Conditions: (i) NaOH, H O, 52% yield; (ii) EDC, pyridine, CH Cl ,
2
2
2
5
2% yield; (iii) CH Cl , 0 °C, 50% yield.
2
2
Polymerizations of NB-NTA were carried out in CH Cl2
2
initiated by Grubbs’ third generation catalyst (H IMes)
2
(
pyr) (Cl) RuCHPh (G3). The resulting homopolymer
2
2
was insoluble in water, so a poly(ethylene glycol) (PEG)
norbornene comonomer was introduced into the feed at a 9:1
ratio with respect to NB-NTA (Scheme 2B). The polymer-
ization yielded a water-soluble random copolymer (polyNTA1)
with a number-average molecular weight (M ) of 37 kDa and a
n
dispersity of 1.06 (Figure S13).
Figure 3. Endothelial cell proliferation data showing the ratio of
proliferating cells in each treatment group. Cells were treated for 24 h
in serum-free media, and quantification was performed by counting the
number of BrdU /Dapi cells (n = 7−8 for each treatment group). *
indicates p < 0.05, *** indicates p < 0.001 relative to untreated
control. Error bars represent standard error of the mean.
H S release from copolymer polyNTA1 was analyzed using
2
the H S-selective electrochemical probe (Figure 2A). A rapid
2
+
+
initial release was observed, followed by a plateau after
approximately 2 h and sustained release out to at least 20 h.
Additionally, the methylene blue assay (Figure 2B) revealed an
H S release half-life of 280 min (Figure 2C), a 3-fold increase
2
over the small molecule NTA1. This result is consistent with
similar gasotransmitter-releasing copolymers in which the
release of the polymeric donor is sustained over a longer
compared with the untreated control group. No increase was
observed for the Sar-Gly byproduct. Caspase activity was also
measured for NTA1 to assess apoptosis, revealing no significant
activity up to 100 μM (Figure S20). We also evaluated the
effect of polyNTA1 on endothelial cell proliferation (Figure
S21). No increase in proliferation was observed, which may be
23
period than the corresponding small molecules. We speculate
that this phenomenon may be due to the steric crowding
imparted by the polymer backbone as well as the pendant PEG
chains in polyNTA1.
a result of the slower rate of H S generation from the polymeric
2
To test the ability of NTA1 and polyNTA1 to deliver H S in
a biological system, we assessed their efficacy in promoting
proliferation of brain-derived endothelial cells. Endothelial cell
donor.
2
In summary, NTA1 and polyNTA1 were synthesized to
afford donor compounds that released COS in a sustained
C
DOI: 10.1021/jacs.6b07204
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Journal of the American Chemical Society
Communication
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.6b07204.
■
*
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AUTHOR INFORMATION
Corresponding Author
jbmatson@vt.edu
■
*
Author Contributions
The paper was written through contributions of all authors. All
authors have given approval to the final version of the paper.
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Funding
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This work was supported by the NSF (DMR-1454754 to
J.B.M.), the Virginia Tech Institute for Critical Technologies
and Applied Science (JFC12-256 to J.B.M.), and the National
Institutes of Health (NS096281 and NS081623 to M.H.T.). We
also thank 3M for support of this work through a Non-Tenured
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(
Notes
The authors declare no competing financial interest.
(
ACKNOWLEDGMENTS
We thank Kyle Arrington and Dr. Mehdi Ashraf-Khorassani for
helpful discussions, and Jose
■
(
́
́
Rodriguez-Corrales, Alex Hawes
(
and Prof Jatinder Josan for instrumental assistance. We also
thank the VT Initiative for Maximizing Student Diversity
(34) Papapetropoulos, A.; Pyriochou, A.; Altaany, Z.; Yang, G.;
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