Self-Immolative Thiocarbamates Provide Access to Triggered H2S Donors and Analyte Replacement Fluorescent Probes

Article abstract of DOI:10.1021/jacs.6b03780

Hydrogen sulfide (H₂S) is an important biological signaling molecule, and chemical tools for H₂S delivery and detection have emerged as important investigative methods. Key challenges in these fields include developing donors that ar

Full text of DOI:10.1021/jacs.6b03780

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Communication
Self-Immolative Thiocarbamates Provide Access to Triggered
H2S Donors and Analyte Replacement Fluorescent Probes
Andrea K Steiger, Sibile Pardue, Christopher G. Kevil, and Michael D Pluth
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b03780 • Publication Date (Web): 24 May 2016
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Andrea K. Steiger,a Sibile Pardue,b Christopher G. Kevil,b and Michael D. Plutha,*
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a Department of Chemistry and Biochemistry, Materials Science Institute, Institute of Molecular Biology. University of Ore-
gon, Eugene, OR 97403. b Department of Pathology. Louisiana State University Health Science Center. Shreveport, LA
71130
Supporting Information Placeholder
and ONOO– play important roles ranging from smooth muscle
ABSTRACT: Hydrogen sulfide (H2S) is an important biological
signaling molecule, and chemical tools for H2S delivery and de-
relaxation to immune response18 and are largely intertwined with
–
reactive oxygen species, such as O2 and H2O2, which are critical
in oxidative stress responses and have been implicated in various
aging mechanisms.19 Similarly, reactive sulfur species, such as
H2S, hydropolysulfides (HSn>1–), and persulfides (RSSH) have
recently garnered interest as important biological signaling mole-
cules with roles in long term potentiation and cardiovascular
health.20 By contrast to their metal ion counterparts, RSONS are
often fleeting in nature and often react irreversibly with cellular
targets. This heightened reactivity has provided chemists with
significant challenges in developing constructs that can release
these reactive molecules under controlled conditions, but have
also provided chemists with different strategies to devise chemical
tools for their detection by engineering reactive groups onto sens-
ing platforms that react selectively albeit irreversibly with the
analyte of interest.21
tection have recently emerged as important investigative methods.
Key challenges in these two related domains include developing
H2S donors that are triggered to release H2S in response to stimu-
li, and developing H2S probes that do not irreversibly consume
H2S. Here we report a new strategy for H2S donation based on the
triggered self-immolation of benzyl thiocarbamates to release
carbonyl sulfide (COS), which is rapidly converted to H2S by
carbonic anhydrase (CA). We leverage the triggered self-
immolative motif to develop a new class of easily-modifiable
donors that can be triggered to release H2S. We also demonstrate
that this approach can be coupled with common H2S-sensing mo-
tifs to generate scaffolds which, upon reaction with H2S, generate
a fluorescence response and also release caged H2S, thus address-
ing challenges of analyte homeostasis in reaction-based probes.
Although small molecule donors and reaction-based probes
have provided significant insights into the roles of RSONS in
biology, key needs remain. For example, engineering donors with
precise but modifiable triggers to enable analyte release in re-
sponse to specific stimuli and developing reaction-based probes
that do not irreversibly consume the analyte would enable new
insights into RSONS biology. Motivated by these needs, as well
as our interest H2S chemistry, we report here a new caged H2S
releasing strategy and provide proof-of-concept applications in
both small-molecule donor and reaction-based probe design. By
leveraging triggerable self-immolative thiocarbamates, we
demonstrate access to easily-modifiable H2S donors that can be
triggered by external stimuli (Figure 1a), and address common
issues of analyte consumption in reaction-based fluorescent
probes (Figure 1b) by developing analyte-replacement reaction-
based platforms (Figure 1c).
The advent of chemical tools to probe and manipulate biochem-
ical processes has revolutionized how biological processes are
investigated.1-3 Spawning from initial investigations into fluores-
cent proteins,4-5 small molecule fluorescent reporters now com-
prise a key pillar of investigative chemical biology with a remark-
able diversity of fluorescent tagging and measurement technolo-
gies.6-7 Recent years have witnessed a significant expansion of
sensor development to include chemical tools for imaging transi-
tion metal, alkali, and alkali earth ions.8-10 Many of these sensing
platforms can provide real-time, quantitative measurements of ion
fluxes due to the reversible interaction of the sensor with the ana-
lyte, thus providing methods for imaging the dynamic process of
metal ion trafficking associated with important signaling events
ranging from Ca2+ sparks during muscle contraction11 to Zn2+
fluxes during mammalian egg fertilization.12 Complementing such
investigative tools are small molecule donors that release caged
analytes with controllable rates.13-16 Such platforms provide pow-
erful methods for controlling levels of specific small molecules,
which often include pro-drugs, metal ions, or small reactive sul-
fur, oxygen, and nitrogen species (RSONS), in different biological
contexts.
In the last two decades, RSONS have emerged as important bi-
oinorganic molecules involved in myriad biological processes,
many of which have been elucidated by utilizing chemical tools
for small molecule detection and delivery. RSONS are involved in
the complex cellular redox landscape and are often involved in
oxidative stress responses, immune responses and signaling path-
ways, as well as other emerging roles.17 For example, NO, HNO,
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Figure 1. (a) Caged donors triggered by different stimuli. (b)
could be efficiently hydrolyzed to H2S by CA. Upon addition of
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Reaction-based probes typically consume the target analyte. (c)
Analyte-replacement reaction-based probes enabled by incorpora-
tion of caged analytes into reaction-based motifs.
COS gas to deoxygenated aqueous buffer (PBS, 1 mM CTAB, pH
7.4) containing CA from bovine erythrocytes, we observed rapid
H2S production using an H2S-responsive electrode. In the absence
of CA, negligible current was observed from COS alone, which is
consistent with slow and pH-dependent, nonenzymatic hydrolysis
in water (Figure S4).33 We also observed a dose-dependent reduc-
tion in H2S production upon addition of the CA inhibitor acetazo-
lamide (AAA),34 which confirmed the enzymatic hydrolysis of
COS by CA (Figure 3).
Development of analyte-replacement sensing platforms requires
two important components: a versatile H2S donation motif that
releases H2S in response to a specific triggering event, and a
method to couple this caged donor to a reaction-based sensing
platform with an optical output. As a proof-of-concept design
toward this objective, we chose to use H2S-mediated azide reduc-
tion for our sensing platform, which has emerged as the most
common method for H2S detection and exhibits high selectivity
for H2S over other RSONs (Figure 2a).22 Although a number of
H2S-donating motifs have been reported and have found utility as
important research tools,23-25 none of these fit the design require-
ment of our approach. To develop an H2S donating motif compat-
ible with our design requirements, we reasoned that common
strategies in drug and fluorophore release, namely the self-
immolative cascade decomposition of para-functionalized benzyl
carbamates (Figure 2b),26, 27, 28 could be modified to enable trig-
gered H2S release. Because self-immolative carbamates release an
amine-containing payload and extrude CO2 as a byproduct, we
reasoned that replacing the carbonyl oxygen with a sulfur atom to
generate a thiocarbamate would result in carbonyl sulfide (COS)
rather than CO2 release (Figure 2c). In a biological environment,
COS is quickly hydrolyzed to H2S and CO2 by carbonic anhy-
drase (CA), which is a ubiquitous enzyme present in plant and
mammalian cells.29-30 The second byproduct of the thiocarbamate
self-immolation is a reactive quinone methide, which rapidly re-
aromatizes upon reaction with available nucleophiles, such as
water or nucleophilic amino acids such as cysteine.31-32 On the
basis of the requirements outlined above, we expected that a
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Figure 3. Conversion of COS to H2S by carbonic anhydrase (CA)
with varying concentrations of CA inhibitor acetazolamide (AAA)
in PBS buffer, pH 7.4.
We next prepared model thiocarbamates to confirm that the pro-
posed decomposition cascade to release COS occurs efficiently
and to demonstrate the biological compatibility of this new H2S
donor motif. We incorporated an azide in the para position of the
benzylthiocarbamate to function as the H2S-responsive trigger for
self-immolation and COS release. To facilitate NMR identifica-
tion of the products, we first prepared azidobenzylthiocarbamate 1
with a p-fluoroaniline payload, and the corresponding carbamate 2
as a control compound (Figure 4a-c). Although 2 should undergo
the same self-immolative decomposition upon azide reduction, it
releases CO2 rather than COS, and thus should not result in H2S
donation upon reaction with CA. To monitor the reactivity of the
model compounds under controlled reaction conditions, we used
tris(2-carboxyethyl)phosphine (TCEP), an azide-reducing agent,
to trigger self-immolation, due to its near-instantaneous reduction
quenched fluorophore could be functionalized with
a p-
azidobenzylthiocarbamate to enable H2S-mediated azide reduction
to form the transient aryl amine intermediate, which would subse-
quently undergo the self-immolative cascade reaction to extrude
COS/H2S and liberate the fluorophore to access an analyte-
replacement
sensing
motif
(Figure
2d).
1
of azides. In each case, H, 13C{1H}, and 19F NMR spectroscopy
was used to monitor the reaction after reduction of the model
complexes by TCEP. Consistent with our design hypothesis, we
observed the disappearance of the benzylic peak, loss of the thio-
carbonyl carbon peak, and formation of new resonances upon
self-immolation by NMR spectroscopy (Figure S1-S3). All such
changes were observed within 5 minutes of TCEP addition, con-
firming the rapid self-immolation of the scaffold upon reduction,
and were consistent with COS release from the thiocarbamate
scaffold upon azide reduction.
Having confirmed that CA rapidly catalyzes the hydrolysis of
COS, we next investigated the H2S-donating ability of model
compounds 1 and 2 under identical conditions. Monitoring thio-
carbamate 1 in buffer containing CA did not result in H2S for-
mation, confirming that the thiocarbamates do not react directly
with CA and that aryl azides are stable in the presence of CA
(Figure S5). Upon injection of TCEP, however, rapid release of
H2S was observed, indicating that reduction of the azide to an
amine is essential to trigger self-immolation and COS release.
Additionally, repetition of the experiment with added AAA signif-
icantly reduced the rate of H2S production, confirming that unin-
hibited CA is required for significant H2S production from the
triggered thiocarbamate scaffold (Figure 4d). Finally, the analo-
gous carbamate (2) was investigated under identical conditions,
and as expected no H2S was produced upon addition of TCEP,
confirming that the sulfur-containing thiocarbamate is required for
H2S formation. In total, these experiments demonstrate the validi-
ty of using the thiocarbamate group as a triggerable source of H2S
release in aqueous solution, which we expect will prove fruitful
Figure 2. Established strategies for using (a) H2S-mediated azide
reduction in H2S sensing, and (b) self-immolative carbamates to
deliver an amine-bound payload, such as a fluorescent dye. Incor-
poration of self-immolative thiocarbamates enables access to (c)
triggered H2S donors, and (d) analyte replacement reaction-based
probes.
To confirm that the COS could serve as a potential source of H2S
donation, we first established that independently prepared COS
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for researchers interested in the pharmacological and physiologi-
cal roles of sulfide donating
molecules.23-24
demonstrate that the thiocarbamate group could be appended to
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common fluorophore motifs and efficiently quench the fluores-
cence. We chose to use methylrhodol (MeRho)38 as the fluoro-
phore due to its high quantum yield and single fluorogenic amine,
which could be readily converted into the desired thiocarbamate.
Since the azide-functionalized scaffold would be triggered by H2S
to release both MeRho and COS, this would function as a fluores-
cent H2S probe that would replenish sulfide through the release of
COS. To access the desired scaffold, we treated MeRho with thio-
carbonyldiimidazole (TCDI) and NEt3 in DMF to afford methyl-
rhodol isothiocyanate (MeRho-NCS) in 60% yield. Subsequent
treatment with 4-azidobenzyl alcohol and NaH afforded the
methylrhodol thiocarbamate azide (MeRho-TCA) in 35% yield
(Figure 5a). We note that one benefit of this simple synthetic
route is that almost any fluorophore containing a fluorogenic ni-
trogen can readily be functionalized with the benzylazide thiocar-
bamate group, thus providing access to a diverse library of fluoro-
phores.
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With a sulfide-replenishing H2S probe in hand, we investigated
the fluorescence response upon addition of sulfide. Treatment of
MeRho-TCA with 50 equiv. of NaSH in aqueous buffer (PBS, 1
mM CTAB, pH 7.4) resulted in a 65-fold fluorescence turn-on
over 90 minutes (Figure 5b). Additionally, we confirmed that the
MeRho-TCA scaffold was selective for HS– over other RSONs,
Figure 4. (a,b) Synthesis of model thiocarbamates and carba-
mates. (c) Model compounds. (d) H2S release from 1 after reduc-
tion by TCEP in the presence of CA, under identical conditions
with the addition of AAA (2.5 µM,), and from carbamate 2. (e)
Quantification of total sulfide in whole mouse blood after treat-
ment 25 M 3 and 4 after 30 min of incubation time in the pres-
ence of excess TCEP.
Expanding on our cuvette-based studies, we also investigated
H2S release from model thiocarbamates in whole mouse blood to
expand on the efficacy of H2S release from thiocarbamates in
biologically-relevant contexts. Although murine systems provide
a convenient model, mice have among the lowest CA levels in
mammals, with murine blood only containing about 15% of the
CA present in human blood,35 and thus represent a challenging
target for sulfide release mediated by CA. To quantify total sul-
fide levels, we used the monobromobimane (mBB) method which
allows for the analytical measurement of different sulfide pools
and is compatible with many types of biological samples.36 Meas-
urement of the total sulfide, which includes free sulfide as well as
bound sulfane-sulfur, revealed background levels of 8 M, which
are higher than total sulfide levels commonly observed and re-
2–
by measuring the fluorescence response to Cys, GSH, Hcy, S2O3
, SO32–, SO42–, H2O2, and NO (Figure 5c). As expected, the MeR-
ho-TCA scaffold exhibited excellent selectivity for sulfide over
other RSONs, demonstrating that the thiocarbamate linker group
did not erode the selectivity of the azide trigger, and also estab-
lishing that the MeRho-TCA scaffold can function as a viable H2S
reporter. Because MeRho-TCA releases H2S upon reaction with
H2S, we note that one consequence of this analyte replacement
approach is that the resultant fluorescence response is not directly
proportional to the initial H2S concentration. Additionally, in
isolated systems, two equiv. of HS– are required for complete
azide reduction, suggesting that the first-generation analyte-
replacement scaffolds only replace one half of the consumed sul-
fide.39 It is also possible, however, that in biological media one
equiv. of a thiol may play a role in H2S-mediated azide reduction,
which remains a question for future investigations. In the present
system, preliminary mechanistic investigations indicate that H2S-
mediated azide reduction is the rate-limiting step of the self-
immolative process, and that the subsequent release of COS and
hydrolysis by CA to form H2S is rapid. Taken together, these data
highlight the potential of this strategy to access analyte-
replacement, reaction-based fluorescent scaffolds.
ported in plasma, but are consistent with the high sulfane-sulfur
36
content in red blood cells.37,
We prepared thiocarbamate 3,
In summary, we have outlined and demonstrated a new strategy
for triggered H2S release based on self-immolative thiocarba-
mates. Importantly, this strategy provides solutions to key chal-
lenges associated with both H2S delivery and detection. Thiocar-
bamate-based H2S donors provide a new, versatile, and readily
modifiable platform for developing new H2S donor motifs that
can be triggered by endogenous or biorthogonal triggers. Similar-
ly, this same H2S donation strategy can be coupled to fluorescent
probe development to access reaction-based fluorescence report-
ers that replace the analyte that has been consumed by the detec-
tion event. In a broader context, we expect that the self-
immolative thiocarbamate donors will find significant utility as a
potential platform for academic and potentially therapeutic H2S
donors. Moreover, we anticipate that similar strategies can be
applied to other RSONS reaction-based sensing methods to pro-
vide access to analyte-replacement sensing strategies for reaction-
based sensing motifs.
which lacks the azide trigger, to confirm that the thiocarbamate
group was stable in whole blood and did not release COS without
activation of the trigger group, and compared results obtained
with this model compound with azide-functionalized 4. Total
sulfide levels were measured for each compound, as well as the
control, after 30 minutes of incubation with excess TCEP (Figure
4e). Consistent with our expected results, only samples containing
donor 4 with the azide trigger increased total sulfide levels in
blood (p ≤ 0.0001). These results establish the stability of the
thiocarbamate in biological milieu and confirm that endogenous
CA in murine blood, even though significantly lower than in most
other biological environments,35 is sufficient to hydrolyze the
COS released from thiocarbamates after the self-immolation cas-
cade is triggered, highlighting the efficacy of this H2S-releasing
strategy in biological environments.
Having confirmed the viability of triggered H2S release with
the model compounds, we next applied this design to incorporate
a fluorophore to access an H2S-responsive fluorescent probe that
releases H2S upon H2S detection. Our primary goal was to
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Figure 5. (a) Synthesis of MeRho-TCA. (b) Fluorescence re-
sponse of MeRho-TCA to H2S. Inset shows integrated fluores-
cence over time by comparison to MeRho-TCA in the absence of
NaSH. (c) Selectivity of MeRho-TCA for H2S over other
RSONs. Conditions: 5 µM probe, 250 µM RSONs unless noted
otherwise, in PBS buffer, 1 mM CTAB, pH 7.4, 37 °C. λex = 476
nm, λem = 480-650 nm.
Experimental details, H2S release profiles, spectra. This material
is available free of charge via the Internet at http://pubs.acs.org.
We thank Mr. Keenan Woods and Mr. Matthew Hartle for prelim-
inary experiments with COS gas and Prof. James Prell for acquir-
ing MS data. Research reported here was supported by the NIH
(R01GM113030 to MDP; HL113303 to CGK) and the Sloan
Foundation (to MDP). The NMR facilities at the University of
Oregon are supported by the NSF (CHE-1427987) and the Bio-
molecular Mass Spectrometry Core at Oregon State University is
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The authors have filed a provisional patent on thiocarbamate-
based and other COS-related donation strategies, including those
outlined within this manuscript.
pluth@uoregon.edu
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