Fluorescent detection of hydrogen sulfide (H2S) through the formation of pyrene excimers enhances H2S quantification in biochemical systems.

Hydrogen sulfide (H2S) is produced endogenously by several enzymatic pathways and modulates physiological functions in mammals. Quantification of H2S in biochemical systems remains challenging because of the presence of interferents with similar reactivity, particularly thiols. Herein, we present a new quantification method based on the formation of pyrene excimers in solution. We synthesized the probe 2-(maleimido)ethyl 4-pyrenylbutanoate (MEPB) and determined that MEPB reacted with H2S in a two-step reaction to yield the thioether-linked dimer (MEPB)2S, which formed excimers upon excitation, with a broad peak of fluorescence emission centered at 480 nm. In contrast, we found that the products formed with thiols showed peaks at 378 and 398 nm. The difference in emission between the products prevented the interference. Furthermore, we showed that the excimer fluorescence signal yielded a linear response to H2S, with a limit of detection of 54 nM in a fluorometer. Our quantification method with MEPB was successfully applied to follow the reaction of H2S with glutathione disulfide and to quantify the production of H2S from cysteine by Escherichia coli. In conclusion, this method represents an addition to the toolkit of biochemists to quantify H2S specifically and sensitively in biochemical systems.

A Review of Chemical Tools for Studying Small Molecule Persulfides: Detection and Delivery.

Hydrogen sulfide (H2S) has gained significant attention as a potent bioregulator in the redox metabolome, but it is just one of many reactive sulfur species (RSS). Recently, small molecule persulfides (structure RSSH) have emerged as RSS of particular interest due to their enhanced antioxidant abilities compared to H2S and their ability to directly convert protein thiols into protein persulfides, suggesting that persulfides may have distinct physiological functions from H2S. However, persulfides exhibit instability and cross-reactivity that hampers the elucidation of their precise biological roles. As such, chemists have designed chemical tools and techniques to facilitate the study of persulfides under various conditions. These molecules and methods include persulfide trapping reagents and sensors, as well as compounds that degrade in response to various triggers to release persulfides, termed persulfide donors. There now exist a variety of persulfide donor classes, some of which possess tissue-targeting capabilities designed to mimic localized endogenous production of RSS. This Review briefly covers the physicochemical properties of persulfides, the endogenous production of small molecule persulfides, and their reactions with protein thiols and other reactive species. These introductory sections are followed by a discussion of chemical tools used in persulfide chemical biology, with critical analysis of recent advancements in the field and commentary on potential directions for future research.

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

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 Na2 S. 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.

Polymeric persulfide prodrugs: Mitigating oxidative stress through controlled delivery of reactive sulfur species.

Related biologically to the known gasotransmitter hydrogen sulfide (H2S), persulfides (R-SSH) have recently been recognized as native signaling compounds and redox regulators in their own right. Reported here is the synthesis, characterization, and in vitro evaluation of a small molecule persulfide donor and its polymeric counterpart, both of which release N-acetyl cysteine persulfide (NAC-SSH) in response to esterases. The donors, termed EDP-NAC and poly(EDP-NAC), underwent controlled decomposition in response to porcine liver esterase, resulting in pseudo-first-order release half-lives of 1.6 h ± 0.3 h and 36.0 h ± 0.6 h, respectively. In cell experiments, slow-releasing poly(EDP-NAC) rescued H9C2 cardiomyocytes more effectively than EDP-NAC when cells were treated with 5-fluorouricil (5-FU), which induces sustained production of ROS. Neither EDP-NAC nor poly(EDP-NAC) rescued MCF-7 breast cancer cells from 5-FU-induced oxidative stress, suggesting that polymeric persulfide donors could be used as adjuvants to reduce the deleterious cardiotoxic effects of many chemotherapeutics.

Alleviating Cellular Oxidative Stress through Treatment with Superoxide-Triggered Persulfide Prodrugs.

Overproduction of superoxide anion (O2.- ), the primary cellular reactive oxygen species (ROS), is implicated in various human diseases. To reduce cellular oxidative stress caused by overproduction of superoxide, we developed a compound that reacts with O2.- to release a persulfide (RSSH), a type of reactive sulfur species related to the gasotransmitter hydrogen sulfide (H2 S). Termed SOPD-NAC, this persulfide donor reacts specifically with O2.- , decomposing to generate N-acetyl cysteine (NAC) persulfide. To enhance persulfide delivery to cells, we conjugated the SOPD motif to a short, self-assembling peptide (Bz-CFFE-NH2 ) to make a superoxide-responsive, persulfide-donating peptide (SOPD-Pep). Both SOPD-NAC and SOPD-Pep delivered persulfides/H2 S to H9C2 cardiomyocytes and lowered ROS levels as confirmed by quantitative in vitro fluorescence imaging studies. Additional in vitro studies on RAW 264.7 macrophages showed that SOPD-Pep mitigated toxicity induced by phorbol 12-myristate 13-acetate (PMA) more effectively than SOPD-NAC and several control compounds, including common H2 S donors.

The evolving landscape for cellular nitric oxide and hydrogen sulfide delivery systems: A new era of customized medications.

Nitric oxide (NO) and hydrogen sulfide (H2S) are industrial toxins or pollutants; however, both are produced endogenously and have important biological roles in most mammalian tissues. The recognition that these gasotransmitters have a role in physiological and pathophysiological processes has presented opportunities to harness their intracellular effects either through inhibition of their production; or more commonly, through inducing their levels and or delivering them by various modalities. In this review article, we have focused on an array of NO and H2S donors, their hybrids with other established classes of drugs, and the various engineered delivery platforms such a fibers, polymers, nanoparticles, hydrogels, and others. In each case, we have reviewed the rationale for their development.

Self-Immolative Prodrugs: Effective Tools for the Controlled Release of Sulfur Signaling Species.

H2S, persulfides (R-SSH), and related sulfur species have recently received attention due to the pronounced physiological effects they elicit. Delivering sulfur signaling molecules in a controlled manner presents many challenges, as many available donors have short half-lives, lack water solubility, and exhibit poor trigger specificity. Self-immolative prodrugs provide a unique ability to impart stability to H2S precursors and persulfides while allowing for trigger specificity by tuning the functional group installed on the self-immolative linker. This strategy has proven successful in delivering sulfur signaling species under specific conditions and may lead to the further elucidation of persulfide interactions within biological systems, affording effective therapeutics.

Functional N-Substituted N-Thiocarboxyanhydrides as Modular Tools for Constructing H2S Donor Conjugates.

We report a synthetic route toward a family of functional COS/H2S-releasing N-substituted N-thiocarboxyanhydrides (NTAs) with functionalities to accommodate popular conjugation reactions, including olefin cross metathesis, thiol-ene, and copper-catalyzed azide-alkyne cycloaddition. The N-substituted NTAs were attached to small molecules, polymers, and a protein to synthesize novel H2S donors convergently. All conjugates showed sustained H2S release kinetics.

A Persulfide Donor Responsive to Reactive Oxygen Species: Insights into Reactivity and Therapeutic Potential.

Persulfides (RSSH) have been hypothesized as critical components in sulfur-mediated redox cycles and as potential signaling compounds, similar to hydrogen sulfide (H2 S). Hindering the study of persulfides is a lack of persulfide-donor compounds with selective triggers that release discrete persulfide species. Reported here is the synthesis and characterization of a ROS-responsive (ROS=reactive oxygen species), self-immolative persulfide donor. The donor, termed BDP-NAC, showed selectivity towards H2 O2 over other potential oxidative or nucleophilic triggers, resulting in the sustained release of the persulfide of N-acetyl cysteine (NAC) over the course of 2 h, as measured by LCMS. Exposure of H9C2 cardiomyocytes to H2 O2 revealed that BDP-NAC mitigated the effects of a highly oxidative environment in a dose-dependent manner over relevant controls and to a greater degree than common H2 S donors sodium sulfide (Na2 S) and GYY4137. BDP-NAC also rescued cells more effectively than a non-persulfide-releasing control compound in concert with common H2 S donors and thiols.

A review of hydrogen sulfide (H2S) donors: Chemistry and potential therapeutic applications.

Hydrogen sulfide (H2S) is a ubiquitous small gaseous signaling molecule, playing an important role in many physiological processes and joining nitric oxide and carbon monoxide in the group of signaling agents termed gasotransmitters. Endogenous concentrations of H2S are generally low, making it difficult to discern precise biological functions. As such, probing the physiological roles of H2S is aided by exogenous delivery of the gas in cell and animal studies. This need for an exogenous source of H2S provides a unique challenge for chemists to develop chemical tools that facilitate the study of H2S under biological conditions. Compounds that degrade in response to a specific trigger to release H2S, termed H2S donors, include a wide variety of functional groups and delivery systems, some of which mimic the tightly controlled endogenous production in response to specific, biologically relevant conditions. This review examines a variety of H2S donor systems classified by their H2S-releasing trigger as well as their H2S release profiles, byproducts, and potential therapeutic applications.