Esterase-Activated Perthiocarbonate Persulfide Donors Provide Insights into Persulfide Persistence and Stability.

Persulfides (RSSH) are important reactive sulfur species (RSS) that are intertwined with the biological functions of hydrogen sulfide (H2S). The direct study of persulfides is difficult, however, due to their both nucleophilic and electrophilic character, which leads to the generation of an equilibrium of different RSS. To investigate the effects of persulfides directly, especially in biological systems, persulfide donors are needed to generate persulfides in situ. Here, we report the synthesis of esterase-activated perthiocarbonate persulfide donors and investigate the effects of structural modifications on persulfide release. Although steric bulk of the ester did not significantly alter persulfide release kinetics, increased steric bulk of the thiol increased the persulfide release rate. In addition, we found that the steric bulk and identity of the thiol significantly impact persulfide persistence. Further mechanistic investigations into different competing reaction pathways from perthiocarbonates revealed that multiple RSS can be delivered (i.e., H2S, COS, or RSSH) depending on the persulfide donor structure and activator identity.

Progress toward colorimetric and fluorescent detection of carbonyl sulfide.

We report here that a fluorescent benzobisimidazolium salt (TBBI) can be used for the fluorescent and colorimetric detection of carbonyl sulfide (COS) over related heterocumulenes including CO2 and CS2 in wet MeCN. The reaction between TBBI and COS in the presence of fluoride yields a highly fluorescent (λem = 354 nm) and colored product (λmax = 321, 621 nm), that is readily observed by the naked eye. We view these results as a first step toward developing activity-based probes for COS detection.

H2S donors with optical responses.

Reactive sulfur species, including hydrogen sulfide (H2S), are important biological mediators and play key roles in different pathophysiological conditions. Small molecules that release H2S on demand, often referred to as "H2S donors," constitute a key investigative tool for H2S-related research. A significant challenge, however, is correlating the rate of H2S release from such donors in complex systems with biological outcomes, because release rates are commonly perturbed by different biological environments. In this chapter, we outline an approach to use H2S donors that provide a fluorescent response upon H2S release to address this problem. These compounds leverage the intermediate release of carbonyl sulfide (COS), which is quickly converted to H2S by the endogenous enzyme carbonic anhydrase (CA), to provide activatable donors with an optical response. The described donors are activated by biological thiols and provide a fluorescence response that correlates directly with H2S delivery, which allows for delivered H2S levels to be measured in real time by fluorescence techniques.

Use of Dithiasuccinoyl-Caged Amines Enables COS/H2 S Release Lacking Electrophilic Byproducts.

The enzymatic conversion of carbonyl sulfide (COS) to hydrogen sulfide (H2 S) by carbonic anhydrase has been used to develop self-immolating thiocarbamates as COS-based H2 S donors to further elucidate the impact of reactive sulfur species in biology. The high modularity of this approach has provided a library of COS-based H2 S donors that can be activated by specific stimuli. A common limitation, however, is that many such donors result in the formation of an electrophilic quinone methide byproduct during donor activation. As a mild alternative, we demonstrate here that dithiasuccinoyl groups can function as COS/H2 S donor motifs, and that these groups release two equivalents of COS/H2 S and uncage an amine payload under physiologically relevant conditions. Additionally, we demonstrate that COS/H2 S release from this donor motif can be altered by electronic modulation and alkyl substitution. These insights are further supported by DFT investigations, which reveal that aryl and alkyl thiocarbamates release COS with significantly different activation energies.

Activatable Small-Molecule Hydrogen Sulfide Donors.

Significance: Hydrogen sulfide (H2S) is an important biological signaling molecule involved in many physiological processes. These diverse roles have led researchers to develop contemporary methods to deliver H2S under physiologically relevant conditions and in response to various stimuli. Recent Advances: Different small-molecule donors have been developed that release H2S under various conditions. Key examples include donors activated in response to hydrolysis, to endogenous species, such as thiols, reactive oxygen species, and enzymes, and to external stimuli, such as photoactivation and bio-orthogonal chemistry. In addition, an alternative approach to release H2S has utilized the catalyzed hydrolysis of carbonyl sulfide (COS) by carbonic anhydrase to generate libraries of activatable COS-based H2S donors. Critical Issues: Small-molecule H2S donors provide important research and pharmacological tools to perturb H2S levels. Key needs, both in the development and in the use of such donors, include access to new donors that respond to specific stimuli as well as donors with well-defined control compounds that allow for clear delineation of the impact of H2S delivery from other donor byproducts. Future Directions: The abundance of reported small-molecule H2S donors provides biologists and physiologists with a chemical toolbox to ask key biological questions and to develop H2S-related therapeutic interventions. Further investigation into different releasing efficiencies in biological contexts and a clear understanding of biological responses to donors that release H2S gradually (e.g., hours to days) versus donors that generate H2S quickly (e.g., seconds to minutes) is needed.

Development and Application of Carbonyl Sulfide-Based Donors for H2S Delivery.

In addition to nitric oxide and carbon monoxide, hydrogen sulfide (H2S) has been recently recognized as an important biological signaling molecule with implications in a wide variety of processes, including vasodilation, cytoprotection, and neuromodulation. In parallel to the growing number of reports highlighting the biological impact of H2S, interest in developing H2S donors as both research tools and potential therapeutics has led to the growth of different H2S-releasing strategies. Many H2S investigations in model systems use direct inhalation of H2S gas or aqueous solutions of NaSH or Na2S; however, such systems do not mimic endogenous H2S production. This stark contrast drives the need to develop better sources of caged H2S. To address these limitations, different small organosulfur donor compounds have been prepared that release H2S in the presence of specific activators or triggers. Such compounds, however, often lack suitable  control compounds, which limits the use of these compounds in probing the effects of H2S directly. To address these needs, our group has pioneered the development of carbonyl sulfide (COS) releasing compounds as a new class of H2S donor motifs. Inspired by a commonly used carbamate prodrug scaffold, our approach utilizes self-immolative thiocarbamates to access controlled release of COS, which is rapidly converted to H2S by the ubiquitous enzyme carbonic anhydrase (CA). In addition, this design enables access to key control compounds that release CO2/H2O rather than COS/H2S, which enables delineation of the effects of COS/H2S from the organic donor byproducts. In this Account, we highlight a library of first-generation COS/H2S donors based on self-immolative thiocarbamates developed in our lab and also highlight challenges related to H2S donor development. We showcase the release of COS in the presence of specific triggers and activators, including biological thiols and bio-orthogonal reactants for targeted applications. We also demonstrate the design and development of a series of H2O2/reactive oxygen species (ROS)-triggered donors and show that such compounds can be activated by endogenous levels of ROS production. Utilizing approaches in bio-orthogonal activation, we establish that donors functionalized with an o-nitrobenzyl photocage can enable access to light-activated donors. Similar to endogenous production by cysteine catabolism, we also prepared a cysteine-selective COS donor activated by a Strongin ligation mechanism. In efforts to help delineate potential differences in the chemical biology of COS and H2S, we also report a simple esterase-activated donor, which demonstrated fast COS-releasing kinetics and inhibition of mitochondrial respiration in BEAS-2B cells. Additional investigations revealed that COS release rates and cytotoxicity correlated directly within this series of compounds with different ester motifs. In more recent and applied applications of this H2S donation strategy, we also highlight the development of donors that generate either a colorimetric or fluorescent optical response upon COS release. Overall, the work described in this Account outlines the development and initial application of a new class of H2S donors, which we anticipate will help to advance our understanding of the rapidly emerging chemical biology of H2S and COS.

Dithioesters: simple, tunable, cysteine-selective H2S donors.

Dithioesters have a rich history in polymer chemistry for RAFT polymerizations and are readily accessible through different synthetic methods. Here we demonstrate that the dithioester functional group is a tunable motif that releases H2S upon reaction with cysteine and that structural and electronic modifications enable the rate of cysteine-mediated H2S release to be modified. In addition, we use (bis)phenyl dithioester to carry out kinetic and mechanistic investigations, which demonstrate that the initial attack by cysteine is the rate-limiting step of the reaction. These insights are further supported by complementary DFT calculations. We anticipate that the results from these investigations will allow for the further development of dithioesters as important chemical motifs for studying H2S chemical biology.

Fluorogenic hydrogen sulfide (H2S) donors based on sulfenyl thiocarbonates enable H2S tracking and quantification.

Hydrogen sulfide (H2S) is an important cellular signaling molecule that exhibits promising protective effects. Although a number of triggerable H2S donors have been developed, spatiotemporal feedback from H2S release in biological systems remains a key challenge in H2S donor development. Herein we report the synthesis, evaluation, and application of caged sulfenyl thiocarbonates as new fluorescent H2S donors. These molecules rely on thiol cleavage of sulfenyl thiocarbonates to release carbonyl sulfide (COS), which is quickly converted to H2S by carbonic anhydrase (CA). This approach is a new strategy in H2S release and does not release electrophilic byproducts common from COS-based H2S releasing motifs. Importantly, the release of COS/H2S is accompanied by the release of a fluorescent reporter, which enables the real-time tracking of H2S by fluorescence spectroscopy or microscopy. Dependent on the choice of fluorophore, either one or two equivalents of H2S can be released, thus allowing for the dynamic range of the fluorescent donors to be tuned. We demonstrate that the fluorescence response correlates directly with quantified H2S release and also demonstrate the live-cell compatibility of these donors. Furthermore, these fluorescent donors exhibit anti-inflammatory effects in RAW 264.7 cells, indicating their potential application as new H2S-releasing therapeutics. Taken together, sulfenyl thiocarbonates provide a new platform for H2S donation and readily enable fluorescent tracking of H2S delivery in complex environments.

Effects of sulfane sulfur content in benzyl polysulfides on thiol-triggered H2S release and cell proliferation.

Investigations into hydrogen sulfide (H2S) signaling pathways have demonstrated both the generation and importance of persulfides, which are reactive sulfur species that contain both reduced and oxidized sulfur. These observations have led researchers to suggest that oxidized sulfur species, including sulfane sulfur (S0), are responsible for many of the physiological phenomena initially attributed to H2S. A common method of introducing S0 to biological systems is the administration of organic polysulfides, such as diallyl trisulfide (DATS). However, prior reports have demonstrated that commercially-available DATS often contains a mixture of polysulfides, and furthermore a lack of structure-activity relationships for organic polysulfides has limited our overall understanding of different polysulfides and their function in biological systems. Advancing our interests in the chemical biology of reactive sulfur species including H2S and S0, we report here our investigations into the rates and quantities of H2S release from a series of synthetic, pure benzyl polysulfides, ranging from monosulfide to tetrasulfide. We demonstrate that H2S is only released from the trisulfide and tetrasulfide, and that this release requires thiol-mediated reduction in the presence of cysteine or reduced glutathione. Additionally, we demonstrate the different effects of trisulfides and tetrasulfides on cell proliferation in murine epithelial bEnd.3 cells.

Thionoesters: A Native Chemical Ligation-Inspired Approach to Cysteine-Triggered H2S Donors.

Native chemical ligation (NCL) is a simple, widely used, and powerful synthetic tool to ligate N-terminal cysteine residues and C-terminal α-thioesters via a thermodynamically stable amide bond. Building on this well-established reactivity, as well as advancing our interests in the chemical biology of reactive sulfur species including hydrogen sulfide (H2S), we hypothesized that thionoesters, which are constitutional isomers of thioesters, would undergo a similar NCL reaction in the presence of cysteine to release H2S under physiological conditions. Herein, we report mechanistic and kinetic investigations into cysteine-mediated H2S release from thionoesters. We found that this reaction proceeds with high H2S-releasing efficiency (∼80%) and with a rate constant (9.1 ± 0.3 M-1 s-1) comparable to that for copper-catalyzed azide-alkyne cycloadditions (CuAAC). Additionally, we found that the final product of the reaction of cysteine with thionoesters results in the formation of a stable dihydrothiazole, which is an iron-binding motif commonly found in siderophores produced by bacteria during periods of nutrient deprivation.

S Marks the Spot: Linking the Antioxidant Activity of N-Acetyl Cysteine to H2S and Sulfane Sulfur Species.

N-Acetyl cysteine (NAC) is commonly used as an antioxidant and cytoprotectant, yet a broadly applicable mechanism of these activities has remained elusive. In this issue of Cell Chemical Biology, Ezerina et al. (2018) report an alternative mechanism for NAC cytoprotection and antioxidant activity by demonstrating that NAC treatment increases sulfane sulfur production via intermediate H2S generation.

Bis(aryl) Tetrasulfides as Cathode Materials for Rechargeable Lithium Batteries.

An organotetrasulfide consists of a linear chain of four sulfur atoms that could accept up to 6 e- in reduction reactions, thus providing a promising high-capacity electrode material. Herein, we study three bis(aryl) tetrasulfides as cathode materials in lithium batteries. Each tetrasulfide exhibits two major voltage regions in the discharge. The high voltage slope region is governed by the formation of persulfides and thiolates, and the low voltage plateau region is due to the formation of Li2 S2 /Li2 S. Based on theoretical calculations and spectroscopic analysis, three reduction reaction processes are revealed, and the discharge products are identified. Lithium half cells with tetrasulfide catholytes deliver high specific capacities over 200 cycles. The effects of the functional groups on the electrochemical characteristics of tetrasulfides are investigated, which provides guidance for developing optimum aryl polysulfides as cathode materials for high energy lithium batteries.

Applications of Synthetic Organic Tetrasulfides as H2S Donors.

In an effort to expand the availability of simple polysulfides for H2S donation, we report here the synthesis and H2S release profiles of bis(aryl) and bis(alkyl) tetrasulfides. The tetrasulfide donors release H2S in a first-order dependence on reduced glutathione (GSH) and release more H2S than the commonly used trisulfide DATS.

A Bright Fluorescent Probe for H2S Enables Analyte-Responsive, 3D Imaging in Live Zebrafish Using Light Sheet Fluorescence Microscopy.

Hydrogen sulfide (H2S) is a critical gaseous signaling molecule emerging at the center of a rich field of chemical and biological research. As our understanding of the complexity of physiological H2S in signaling pathways evolves, advanced chemical and technological investigative tools are required to make sense of this interconnectivity. Toward this goal, we have developed an azide-functionalized O-methylrhodol fluorophore, MeRho-Az, which exhibits a rapid >1000-fold fluorescence response when treated with H2S, is selective for H2S over other biological analytes, and has a detection limit of 86 nM. Additionally, the MeRho-Az scaffold is less susceptible to photoactivation than other commonly used azide-based systems, increasing its potential application in imaging experiments. To demonstrate the efficacy of this probe for H2S detection, we demonstrate the ability of MeRho-Az to detect differences in H2S levels in C6 cells and those treated with AOAA, a common inhibitor of enzymatic H2S synthesis. Expanding the use of MeRho-Az to complex and heterogeneous biological settings, we used MeRho-Az in combination with light sheet fluorescence microscopy (LSFM) to visualize H2S in the intestinal tract of live zebrafish. This application provides the first demonstration of analyte-responsive 3D imaging with LSFM, highlighting the utility of combining new probes and live imaging methods for investigating chemical signaling in complex multicellular systems.