Bio-orthogonal click-and-release donation of caged carbonyl sulfide (COS) and hydrogen sulfide (H2S)

Article abstract of DOI:10.1039/c6cc09547j

Hydrogen sulfide (H₂S) is an important biomolecule with high therapeutic potential. Here we leverage the inverse-electron demand Diels-Alder (IEDDA) click reaction between a thiocarbamate-functionalized trans-cyclooctene and a tetrazine to deliver carbonyl sulfide (COS), which is quickly converted to H₂S by the uniquitous enzyme carbonic anhydrase (CA), thus providing a new strategy for bio-orthogonal COS/H₂S donation.

Full text of DOI:10.1039/c6cc09547j

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DOI: 10.1039/C6CC09547J
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Bio-orthogonal “Click and Release” Donation of Caged Carbonyl
Sulfide (COS) and Hydrogen Sulfide (H2S)
Andrea K. Steiger,a,† Yang Yangb,†, Maksim Royzen,b,* and Michael D. Plutha,*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Hydrogen sulfide (H2S) is an important biomolecule with high access responsive H2S donors. We demonstrated that self-
therapeutic potential. Here we leverage the inverse-electron immolative thiocarbamates can be triggered to decompose
demand Diels-Alder (IEDDA) click reaction between
a
and release COS, which is rapidly converted to H2S by the
thiocarbamate-functionalized trans-cyclooctene and a tetrazine to ubiquitous enzyme carbonic anhydrase (CA).6 Analogous to the
deliver carbonyl sulfide (COS), which is quickly converted to H2S by broad applications of self-immolative carbamates as delivery
the uniquitous enzyme carbonic anhydrase (CA), thus providing a platforms for prodrugs, fluorophores, and other biologically-
new strategy for bio-orthogonal COS/H2S donation.
relevant payloads, thiocarbamates provide a highly tunable
platform on which the triggering mechanism can be
engineered to initiate self-immolation and COS release by
specific analytes of interest. Since our initial report on caged
COS/H2S release, passive H2S donation from small molecule
and polymeric N-thiocarboxyanhydrides7 as well as responsive
ROS-triggered donors that provide protection against cellular
oxidative stress have been reported.8 Missing from current
COS/H2S donor technologies are platforms activated by bio-
orthogonal triggers to allow precise temporal control for H2S
release. Motivated by this need, we report here the first
example of bio-orthogonal activation of COS/H2S release
through adaptation of the well-developed inverse-electron
demand Diels-Alder (IEDDA) click reaction to release COS/H2S
(Scheme 1).
With the recent addition of hydrogen sulfide (H2S) to the
list of biologically-relevant gasotransmitters,1 significant
efforts have focused on developing H2S donors as powerful
research, and potentially therapeutic, tools.2-3 Endogenous H2S
production occurs primarily from cystathionine-
-lyase (CSE),
cystathionine- -synthase (CBS), and 3-mercaptopyruvate

transferase (3-MST), and the slow production of H2S exerts
protective effects throughout the body.1 Although convenient,
inorganic sulfide salts (NaSH and Na2S) provide a large,
instantaneous bolus of H2S, and sulfide oxidation often occurs
rapidly after administration.4 These limitations suggest that
more efficacious donors should either more closely mimic
slower enzymatic production rates or be stable until triggered
to release H2S in response to specific stimuli. Available
synthetic slow-release donors have already made major
impacts in H2S research, and several small molecule H2S
donors have already entered clinical trials.5 Despite this
promise, providing temporal control over H2S release remains
a
major challenge, and there is significant interest in
developing synthetic H2S donors that are activated by well-
defined triggering mechanisms that enable on-demand H2S
release.
Aligned with this need, we recently pioneered the use of
carbonyl sulfide (COS)-releasing molecules as a strategy to
Scheme 1. IEDDA reaction of thiocarbamate-functionalized TCO
tetrazine to generate COS/H2S.
1 with
a.Materials Science Institute, Institute of Molecular Biology. Department of
Chemistry and Biochemistry. University of Oregon. Eugene, OR 97403 USA. Email:
pluth@uoregon.edu
Department of Chemistry, University at Albany, SUNY, 1400 Washington Avenue,
Albany, NY 12222 USA. Email: mroyzen@albany.edu
† These authors contributed equally
The IEDDA reaction between a trans-cyclooctene (TCO) and
a tetrazine is a proven platform for bio-orthogonal click
reactions in living systems.9-11 In addition to providing an
important biocompatible bond-forming tool, the IEDDA
reaction has also been adapted for targeted drug release by
b
Electronic Supplementary Information (ESI) available: Synthetic and experimental
details. See DOI: 10.1039/x0xx00000x
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using functionalized benzylic carbamates, which can be system in aqueous buffer at physiological pH (PBS, pH 7.4)
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DOI: 10.1039/C6CC09547J
triggered to undergo self-immolative decomposition following using an H2S-selective electrode (Figure 2). Non-enzymatic
the click reaction and subsequently release the attached drug, background
as well as CO2 as a byproduct.12-14 We envisioned that a similar
strategy could be leveraged to develop a fully bio-orthogonal
COS/H2S releasing platform by using a benzylic thiocarbamate-
functionalized TCO (Scheme 1). The initial IEDDA click reaction
would
dihydropyridazine,
generate
the
which
thiocarbamate-functionalized
after tautomerization,
deprotonation, and rearomatization can extrude COS, BnNH2,
and the cyclooctylpyridazine product. To test this hypothesis,
we prepared TCO
isothiocyanate in the presence of NaH.13 In parallel, we
prepared the analogous carbamate-functionalized TCO
which undergoes the same IEDDA reaction but releases CO2
rather than COS (Scheme 2). Both TCO and are isolated as
1 by treating (E)-cyclooct-2-enol with benzyl
2
,
1
2
the axial isomer, which is estimated to be significantly more
reactive than the analogous equatorial isomer.15 Importantly,
this design strategy provides simple synthetic access to both
the thiocarbamate donor and key carbamate control
compounds. Additionally, click-and-release CO donors were
recently reported utilizing an intramolecular Diels-Alder
reaction, thus supporting the validity of our approach for
accessing biorthogonal gasotransmitter release.16
1
Figure 1. (a) H NMR spectra of the reaction of
1 and tetrazine (b) ESI-
MS of reaction products, confirming self-immolation. (c) MS
confirmation of COS formation.
hydrolysis of COS to H2S is very slow at physiological pH, but is
rapid in the presence of carbonic anhydrase (CA). Using
biologically-relevant CA concentrations (25 µg/mL) we first
monitored TCO
1 alone and confirmed that H2S is not released
spontaneously in the presence of CA. As anticipated, the bis-
isopropyl-1,2,4,5-tetrazine alone also failed to produce an H2S
response. We next monitored H2S release from TCO
with varying concentrations of tetrazine (5 – 25 equiv.) and
observed increased H2S production in the presence of excess
1 (50 µM)
Scheme 2. Synthesis of thiocarbamate-functionalized COS/H2S donor
TCO
TCO
1 and the associated carbamate-functionalized control compound
2
.
tetrazine. Using a calibration curve, we measured 12
M H2S
release from 50 M TCO with 25 equiv. of tetrazine, resulting

1
To confirm that the IEDDA reaction would initiate self-
immolative decomposition of the thiocarbamate moiety, we
monitored the reaction of and 2 equiv. of bis-isopropyl-
1,2,4,5-tetrazine in wet methanol-d4 by H NMR spectroscopy
(Figure 1a). Within 5 minutes of tetrazine addition, we
observed disappearance of the alkene peaks (5.3-5.7 ppm),
indicative of cycloaddition. New resonances corresponding to
in an H2S release efficiency of approximately 25%. As
additional confirmation of the importance of CA for H2S
formation, we performed identical reactions in the presence of
1
1
acetazolamide (AAA, 2.5 µM),
Furthermore, use of the control compound TCO
a
known CA inhibitor.
in the
2
presence of excess tetrazine failed to generate COS/H2S.
Together, these data confirm that the IEDDA click reaction is
necessary to generate COS, and that uninhibited CA is required
for efficient conversion of this released COS to H2S at
physiological pH.
BnNH2 were subsequently observed at 4.25 ppm, while
1
continues to decompose over 24 hours. Due to the complexity
of this reaction and the different potential intermediates that
could be formed en-route to COS extrusion, we also monitored
product formation by mass spectrometry. Consistent with our
design hypothesis we observed the re-aromatized IEDDA
product (M+H+ 247.239), BnNH2 (M+H+ 108.091), and COS
(M+H+ 61.044) (Figure 1b,c) using direct analysis in real time
mass spectrometry (DART-MS). Taken together, these data
indicate that addition of the tetrazine to
1 results in the
expected click reaction and initiates self-immolation of the
thiocarbamate moiety, thus producing COS.
Having confirmed the fidelity of the IEDDA reaction, we
next investigated click-and-release H2S-donation from this
2 | Chem. Commun., 2016, 00, 1-3
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Figure 2. H2S release profiles from TCO
1 (50 µM) with 5-25 equiv of
the IEDDA reaction can be improved through strVaiteewgAircticclehOonilcinee
of the tetrazine. These, as well as other modifications to the
tetrazine in the presence of CA (25 µg/mL) in buffer (PBS, pH 7.4).
DOI: 10.1039/C6CC09547J
thiocarbamate scaffold are expected to provide much more
efficient H2S release from future click and release scaffolds.
In summary, we have reported the first example of
COS/H2S donors activated by a bio-orthogonal trigger, which
provides a significant step toward developing controllable H2S
donors with high temporal resolution. Given the novelty of this
bio-orthogonal reaction in the field of sulfide donation, as well
as the significant impact that similar click strategies have
provided to adjacent fields in chemical biology, we anticipate
that future optimization of this system will result in fast and
highly targeted method for H2S donation.
This material is based upon work supported by the
National Science Foundation Graduate Research Fellowship
Program under Grant No. 1309047. Any opinions, findings, and
conclusions or recommendations expressed in this material
are those of the author(s) and do not necessarily reflect the
views of the National Science Foundation. Support was also
provided by the Sloan Foundation and Dreyfus Foundation (to
MDP); and the Research Foundation of the New York State (to
MR).
To demonstrate the basic biological compatibility of the
reaction, we also investigated H2S release from TCO
1 (50 µM)
with the tetrazine (500 µM) in complex media (Figure 3). For
these experiments, we chose to use whole sheep and bovine
blood due to the presence of CA. Using sheep blood and
plasma, diluted 1:1 in PBS (pH 7.4) with no additional CA
added, a similar H2S release profile was observed using an H2S-
selective electrode. Additionally, H2S production was also
observed in diluted whole bovine blood, although the process
was slower. These experiments confirm that bio-orthogonal
click-and-release strategy has significant potential within a
biological environment and endogenous CA levels are
sufficient to allow for H2S donation from the released COS.
Additionally, we confirmed the cellular compatibility of TCO
1
using the CCK-8 cell viability assay, which indicated that
concentrations up to 100 µM of TCO
neuroblastoma cells (see SI).
1 are not cytotoxic in N2A
Notes and references
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TOC Image
The inverse-electron demand Diels-Alder (IEDDA) click reaction
between thiocarbamate-functionalized trans-cyclooctenes and
tetrazines provides a new strategy for bio-orthogonal COS/H2S
delivery.
4 | Chem. Commun., 2016, 00, 1-3
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