Hydrogen sulfide (H2S) is an important biomolecule for its role in mediating redox homeostasis and signaling biological processes. The study of biological sulfide is currently impeded by a lack of tools available that adequately address the questions currently facing the field. The most pressing of these questions are: how does H2S signal biological processes. To produce tools for studying H2S, chemiluminescent scaffolds were designed to study both H2S producing enzymes and directly measure free H2S. Additionally, small molecule organic persulfides were synthesized and characterized in order to study the properties and reactivity of H2S signaling species. By creating methods to directly measure biological H2S and creating model systems to investigate the active signaling species, the biological reactivity of H2S can be better understood. The luminescent methods for detecting H2S were developed in order to avoid photodecomposition inherent with fluorescent methods while still providing a spectroscopic readout for performing measurements in cells. D-cysteine concentrations can be measured using luciferin bioluminescence, and utilized to back out the H2S producing activity of DAO. Free H2S was measured using luminol derived chemiluminescence. The luminol scaffolds were studied in depth to determine what makes an H2S probe selective for H2S in order to inform the design of future H2S probes. Sulfide signaling processes were investigated using organic persulfide model systems. We found that under reducing conditions persulfides liberate free H2S, and that under basic conditions they decompose. The decomposition pathway is governed by substitution at the -carbon, which dictates the steric accessibility of the inner sulfur atom to act as an electrophile. Persulifdes do not react with acids, and are easily tagged by electrophiles to form disulfides. Persulfides are sufficiently reducing to generate NO from nitrite, facilitating cross-talk between multiple signaling species. This cross talk is mediated by formation of perthionitrite, which may function as an independent signaling species.

Hydrogen sulfide (H2S) has been recognized as a biological signaling molecule for over twenty years now. Since these important findings emerged, many collaborative projects among chemists, biologists, and clinicians have demonstrated the physiological roles and potential therapeutic benefits of exogenous H2S delivery. As our understanding of the active roles H2S plays in biological systems has increased, so has the desire to investigate other related sulfur species (i.e. persulfides, R–SSH) for their physiological interactions with H2S and potential therapeutic efficacy. This recent interest in persulfides has stimulated a flurry of research in the field and created a new set of scientific problems to solve and opportunities to improve our understanding of persulfides in a biological context. With this surge of interest in persulfides, chemists set out to synthesize and characterize a variety of stimuli-responsive compounds that release persulfides under specific, biologically relevant conditions. In order to better understand persulfide reactivity and biological activity, and provide several prodrug platforms that respond to a variety of stimuli, this dissertation describes four persulfide-releasing prodrug systems, a pyrene-based fluorescent probe that measures H2S release in the presence of thiols, and efforts toward a peptide-based system for the release of H2S from a peptide thioacid (C(O)SH). The first four systems described utilize the well-known 1,6-benzyl elimination reaction (sometimes called self-immolation) to trigger release of a persulfide from a small molecule, polymeric, or peptide-based prodrug platform. Importantly, the first self-immolative small molecule persulfide prodrug (termed BDP-NAC) was designed to respond to reactive oxygen species (ROS). Specifically, BDP-NAC utilized a para-positioned boronic acid pinacol ester functionality to selectively react with H2O2, yielding N-acetylcysteine persulfide (NAC-SSH) and p-hydroxybenzyl alcohol as a byproduct. BDP-NAC showed trigger specificity towards H2O2, as determined by the use of a structurally analogous fluorescent probe (termed BDP-fluor). The prodrug also exhibited antioxidant properties in vitro, and served as the first example in the literature of a self-immolative persulfide donor. The second group of donors, self-immolative small molecule and peptide-based persulfide prodrugs (termed SOPD-Pep and SOPD-NAC), were designed to be responsive to superoxide (O2∙–), the primary precursor to all other ROS. In this work, the advantages of attaching small molecule persulfide donors to peptides were explored. In vitro experiments showed that SOPD-Pep mitigated toxicity induced by phorbol 12-myristate 13-acetate (PMA) more effectively than its small molecule counterpart SOPD-NAC and several common H2S donors. It is proposed that peptide scaffolds offer increased cellular uptake due to their nanoscale size, allowing for better antioxidant activity, as confirmed by fluorescence microscopy. The third section of this dissertation compares an esterase-responsive small molecule to an analogous polymeric persulfide releasing prodrug (termed EDP-NAC and polyEDP-NAC) and their abilities to decrease oxidative stress in response to immediate (H2O2) and sustained (5-fluorouracil, 5-FU) forms of ROS. Persulfide release half-lives were characterized using 1H NMR spectroscopy and showed over one order of magnitude difference between EDP-NAC and polyEDP-NAC. In vitro evaluation of the donors showed polyEDP-NAC was better suited to combat sustained production of ROS induced by 5-FU, whereas EDP-NAC was better suited to combat immediately available ROS from H2O2. These discrepancies in antioxidant activity between the two donors were deemed to be a result of their different persulfide release half-lives, indicating that scientists must take these factors into consideration when designing R–SSH prodrugs for specific disease indications. The fourth donor, NDP-NAC, responded to the bacteria-specific enzyme nitroreductase to release its persulfide payload. 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 Na2S. 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. Lastly, two appendices are provided in this dissertation that briefly describe the synthesis of a pyrene-based H2S sensor and efforts toward a readily accessible peptide-based thioacids as H2S donors. / Doctor of Philosophy / Hydrogen sulfide (H2S), produced naturally in hydrothermal vents and as a byproduct of industrial processes, has historically been known for its potent smell and toxicity. However, the recent discovery of H2S as a naturally-produced signaling molecule (termed gasotransmitter) in mammals has changed the way scientists view this malodorous gas. Our understanding of the biological roles and production of H2S is still growing, and recent research has suggested various links between changes in H2S concentrations in the body and a variety of disease states, including Alzheimer's, cardiovascular disease, and inflammation. Because of this link between various diseases and alterations in natural H2S production, collaborative efforts among chemists, biologists, and pharmacologists have demonstrated the usefulness of therapeutics that contain H2S-donating moieties, in an effort to alleviate these disease conditions. Persulfides (R-SSH), biological signaling molecules related to H2S, have emerged as critical species in sulfur signaling because of the similar observed antioxidative effects compared to H2S. This dissertation focuses on the synthesis and characterization of several compounds that release persulfides in response to specific stimuli (called persulfide donors). The first donor system described here releases persulfides in response to hydrogen peroxide (H2O2), a major cellular oxidant, and reduces oxidative stress in response to H2O2. The second donor system responds to superoxide (O2∙–), a precursor oxidant to H2O2 in cells, to release persulfides. Specifically, two variants of these donors, a small molecule and a peptide-based donor, exhibited antioxidant activity in response to O2∙–, but to varying degrees based on differences in cellular uptake of small molecules and self-assembled peptide nanostructures. The third donor system compares persulfide release from a small molecule and polymeric scaffold, both of which release persulfides in response to esterase enzymes. A large persulfide release half-life range was observed between the two donor systems, and antioxidant activity in response to H2O2 also varied based on the source and timescale of oxidant (H2O2 versus 5-fluorouracil). The fourth section of this dissertation focuses on a persulfide donor that responds to the bacterial enzyme nitroreductase. This donor increased levels of beneficial bacteria and short and medium chain fatty acids in murine models, while simultaneously decreasing levels of a niche subset of harmful bacteria. Taken together, these persulfide donor systems exhibit the strong reducing ability of persulfides in a biological context, showcasing the potential for therapeutic efficacy and avenues for more advanced donors to be synthesized in the future.