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Chikungunya virus (CHIKV) is a re-emerging arbovirus in the alphavirus genus. Upon infection, it can cause severe joint pain that can last years in some patients, significantly affecting their quality of life. Currently, there are no vaccines or anti-viral therapies available against CHIKV. Its spread to the Americas from the eastern continents has substantially increased the count of the infected by millions. Thus, there is an urgent need to identify therapeutic targets for CHIKV treatment. A potential point of intervention is the sphingosine-1-phosphate (S1P) pathway. Conversion of sphingosine to S1P is catalyzed by Sphingosine kinases (SKs), which we previously showed to be crucial pro-viral host factor during CHIKV infection. In this study, we screened inhibitors of SKs and identified a novel potent inhibitor of CHIKV infection—SLL3071511. We showed that the pre-treatment of cells with SLL3071511 in vitro effectively inhibited CHIKV infection with an EC50 value of 2.91 µM under both prophylactic and therapeutic modes, significantly decreasing the viral gene expression and release of viral particles. Our studies suggest that targeting SKs is a viable approach for controlling CHIKV replication. Published version

Organoboron derivatives represent an important class of compounds due to the versatility of the carbon-boron bond in a variety of chemical reactions. Boron-containing compounds have garnered increasing attention as synthetic intermediates and medicinal agents. Therefore, the introduction of carbon-boron bonds to organic molecules continues to be an important field of study. This dissertation describes novel methodology for the regio- and stereo-selective introduction of carbon-boron bonds to generate β-borylacrylonitrile and 1-boryl-1,3-enyne products. Propiolonitriles are intriguing research targets due to the electron-withdrawing nature of the cyano group on the adjacent alkyne. In this dissertation, we developed a phosphine-catalyzed regio- and stereo-selective hydroboration of propiolonitriles to generate novel β-borylacrylonitriles in up to 89% yield and 97:3 (E)-selectivity. These products were converted to the corresponding postassium 1,2-vinylcyanotrifluoroborate salts and demonstrated applications in oxidation and Suzuki-Miyaura cross-coupling reactions. Interestingly, 31P and 13C NMR studies suggest that this hydroboration reaction proceeds in a 1,2-phosphine addition pathway instead of a canonical 1,4-conjugate addition pathway. We also developed a transition metal-free cis hydroboration of 1,3-diyne substrates. In the presence of catalytic amounts of tri-n-butylphosphie and the unsymmetric diboron reagent pinBBdan, 1-boryl-1,3-enyne products were generated in up to 63% and >99:1 (Z)-selectivity. These 1,8-diaminonaphthalene products can be converted to the corresponding pinacolboranes or trifluoroborate salts. They also demonstrated applications in protodeboronation and Suzuki-Miyaura cross-coupling reactions. We propose that this hydroboration occurs via a nucleophilic boron addition mechanism. Doctor of Philosophy Incorporating boron into organic molecules provides access to a variety of otherwise difficult chemical reactions. Therefore, our laboratory seeks to develop new methods for synthesizing organoboron compounds. A major goal of our work is to develop transition metal-free reactions due to the expense and environmental impact of transition-metal mediated methodology. This dissertation reports two new methods for installing boron to organic molecules without the use of transition metals. The reported reactions utilize mild conditions to selectively generate functionalized products, and applications of these products are demonstrated. Chapter 1 describes a hydroboration reaction of propiolonitrile derivatives to afford (E)-β-borylacrylonitrile products. Notably, this reaction proceeds via a unique mechanism, contrasting that of similar reported reactions. Chapter 2 reports a transition metal-free hydroboration of 1,3-diynes to afford (Z)-1-boryl-1,3-enyne products. These products are structurally similar to relevant molecules in medicinal, polymer, and synthetic chemistry.

Malaria, a disease caused by the parasite Plasmodium, continues to be one of the deadliest diseases worldwide. The WHO reported over 627,000 deaths in 2020, and over 1 billion people are at risk of infection. Even though Artemisinin-based Combination Therapies (ACT) are the current standard of care for malaria, the emergence of drug resistance generates a constant need to develop and synthesize new drugs. Tetrahydro-β-carboline acid (THβC) 1-(2,4-dichlorophenyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-2-ium-3-carboxylate (MMV008138) has promising antimalarial properties; it was discovered by screening the Malaria Box with the so-called IPP Rescue assay. This assay identified MMV008138 as an inhibitor of the MEP pathway, which produces essential isoprenoid precursors (IPP and DMAPP) in the malaria parasite P. falciparum (EC50 250 ± 70 nM, IPP rescue 100% @ 2.5 μM). Subsequent investigation revealed that (1R,3S)-configuration and 2',4'-dihalogen substitution were critical for the activity of this compound, and that substitution of the non-aromatic ring was not tolerated. To search for new antimalarial structures, our collaborator Dr. Max Totrov constructed a generalized 3D pharmacophore-based on MMV008138 and 92 of its analogs and used it for a virtual ligand screen (VLS) of the 13K compound hit set from which MMV008138 had been selected. This exercise identified TCMDC-140230, a THβC, 1-(3,4-dichlorophenyl)-8-methyl-N-(2-(methylamino)ethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxamide (undefined stereochemistry) reported having nearly the same potency of MMV008138. Synthesis of the stereoisomers of compound TCMDC-140230 was accomplished via Pictet-Spengler reaction of (S)- and (R)-7-methyl tryptophan methyl ester and 3,4-dichlorobenzaldehyde. The individual stereoisomeric esters were converted to the corresponding amides, but none of the stereoisomers of TCMDC-140230 were potent antimalarials (IC50 = 1,300 – 3,700 nM). However, a significant amount of oxidized byproduct 1-(3,4-dichlorophenyl)-8-methyl-N-(2-(methylamino)ethyl)-9H-pyrido[3,4-b]indole-3-carboxamide (MMV1803522) was observed in the synthesis of (1S,3S)- and (1R,3R)-TCDMC-140230. This achiral β-carboline amide (PRC1584, IC50 = 108 ± 7 nM) proved more potent towards P. falciparum than MMV008138 and its toxicity was not reversed by co-application of IPP. Thus, the antimalarial target of MMV1803522 is distinct from that of MMV008138. Most importantly, MMV1803522 at 40 mg/kg/day (oral) cured P. berghei malaria infection in mice. The lead compound also was found to have a good safety profile. Medicines for Malaria Venture (MMV) has expressed interest in this compound which is now also known as MMV1803522. The results from these biological assays gave the insight to develop new analogs that have better asexual blood stage inhibition potency. Extensive structure-activity relationship studies were conducted by synthesizing analogs of the compound MMV1803522. The studies were mainly focused on analyzing the effect of aliphatic substitutions, how well the potency can be improved with different D-ring substitutions, and amide substitutions. In addition to this structural optimization, several metabolism studies were also conducted on this new lead compound. The potency study results of C1 alkyl-substituted analogs of MMV1803522 showed that aromatic substitutions are required at C1 for maintaining good inhibition potency. The heteroaryl substituents at C1 were found to be slightly less potent than the lead compound MMV1803522. Synthesis of analogs without C8 methyl group as in lead compound showed an EC50 < 100 nM is possible with a C8 hydrogen substitution. Most noteworthy is 3,4,5-trichlotophenyl-bearing compound 3.20a, which had an EC50 of 54 ± 8 nM. This compound is twice as potent as MMV1803522. Equipotent analogs to MMV1803522 were also synthesized with different amide substituents. The metabolism studies showed low solubility for compounds having an EC50 less than or close to 100 nM. Unfortunately, the intrinsic clearance rate of several selected compounds was found to be higher than MMV1803522. These results left us with scope for the development of new analog compounds. The emerging structure-activity relationship within this scaffold and outline of remaining challenges to improve potency sub-100 nM without compromising moderate solubility and good metabolic stability are in progress. Doctor of Philosophy Malaria is a global health problem that causes significant sickness and death annually in the developing world. The emergence of resistant parasite strains of malaria massively challenges efforts to eliminate this threat. To control the spread of malaria, there is a continuous need for the development of new antimalarial drugs that ideally offer a single-dose cure and new mechanism of action. One such promising target, called, Methyl Erythrytol Phosphate (MEP) pathway which produces IPP and DMAPP, are important isoprenoid precursors required in living beings. A compound MMV008138 was identified from a collection of compounds that exhibited antimalarial activity, the so-called "Malaria Box", and this compound was further analyzed for several biological assays. Unfortunately, MMV008138 was unsuccessful Since it was found toxic in mice when ingested orally. The efforts to develop structurally similar analogs of MMV008138 resulted in the accidental discovery of a compound that inhibits the parasites' growth much better than the former compound. This compound has a similar molecular structure to MMV008138, and the Medicines for Malaria organization (MMV) has designated it as MMV1803522. The newly obtained compound and its analogs were investigated and found to have promising potency to inhibit the growth of the malarial parasite Plasmodium falciparum. Multiple biological assays were conducted and found that even though MMV1803522 is toxic to malarial parasites, it does not show toxicity to other cells. The studies in mice showed that it was not toxic orally. Also, it was found to be non-toxic towards several mammalian cell lines. The development of structurally similar analogs can help in improving the potency of the compound, make a better orally bioavailable compound, and improve oral efficacy. Analyzing these results will help to determine the mechanism of action of the compound.

There are immense efforts to install the trifluoromethyl group (CF3) into organic molecules since it significantly affects reactivity, as well as the biological and pharmaceutical properties of the molecule. The α-trifluoromethylacrylates are useful synthetic intermediates for the synthesis of trifluoromethyl-bearing nucleosides or peptides. However, methods to prepare such derivatives are scarce and suffer narrow substrate scope, lack of diastereoselectivity, use of costly reagents, and lack of user‐friendliness. Chapter 2 discusses the synthesis of α‐trifluoromethylacrylates from converting shelf-stable α‐trifluoroborylacrylates via a stereoretentive radical trifluoromethylation with inexpensive reagents NaSO2CF3 and TBHP with copper catalysis at room temperature. Under these conditions, a wide substrate scope afforded the (E)‐diastereomer exclusively in moderate to good yield. The reaction products' utility is demonstrated in synthesizing phenyl‐4H‐pyran, a potent and selective class of IKCa channel blockers. Branching out from metal-catalyzed reactions, Chapter 3 and 4 discusses metal-free organo-catalytic reactions which are more economical and greener. Boron-containing compounds are important in organic chemistry due to the different transformations that can be performed to afford useful synthetic intermediates and pharmaceuticals. Chapter 3 elaborates on the Borylation of 1,3-butadiynes, which are molecules that are widely used to synthesize biologically active molecules, polymers, and supramolecular tools under phosphine-catalyzed conditions. The reaction proceeds with both regio- and stereo-selectivity, and the boryl group is installed on the beta carbon to the alkyne in a trans fashion as established from 2D NMR and X-ray crystallography. The reaction products are converted into useful intermediates under Suzuki Miyuara, protodeborylation, etc. 31P NMR tudies were conducted to understand the mechanism of the reaction, which is supported by the DFT calculations revealing that phosphine attack is the most crucial step. A similar strategy was utilized with pinacol borane and tri-n-butyl phosphine catalyst to achieve borylation on ynamides (Chapter 4). A wide variety of ynamides underwent hydroboration to install the boron group in a regio- and stereoselective fashion. Under radical trifluoromethylating conditions, the boryl moeity was converted into the trifluoromethyl group to afford β-CF3 enamides. 31P NMR studies were conducted to elucidate the mechanism. Finally, chapter 5 provides insight into the synthesis of 1,2,4-Oxadiazoles which are privileged scaffolds in medicinal chemistry. The developed reaction occurs under metal-free conditions using sodium carbonate to couple N-hydroxylbenzenecarbonimodyl chlorides with nitriles. Doctor of Philosophy Installing fluorine in organic molecules significantly improves their pharmaceutical properties and the trifluoromethyl (CF3) group is one such group. Chapter 2 describes a simple, cost-effective method to synthesize trifluoromethylated compounds with readily available reagents under copper-catalyzed conditions, whereas in Chapters 3 and 4, a metal-free method is discussed. In comparison with metal catalysts, organo catalysts are less toxic, readily available, avoid contamination, and are more economic. Boron is used for different transformations in organic chemistry and for making medicinally relevant molecules. An organo-catalytic method to install boron on various substrates and potential applications of such molecules as synthetic intermediates are discussed. 1,2,4-Oxadizoles are molecules that are considered privileged scaffolds imparting pharmaceutical properties. Finally, in Chapter 5, a procedure to synthesize 1,2,4-Oxadiazoles under mild reaction conditions and readily available reagents are discussed.

Doctor of Philosophy White blood cells comprise a significant portion of the body's natural defense mechanisms. In healthy individuals, these white blood cells identify and destroy foreign materials and organisms. However, in patients with multiple sclerosis, immune cells can become sensitized to protein fragments lining the myelin sheath of neurons. These autoreactive immune cells recognize the body's natural neuronal proteins as antigens. Damage exerted by autoreactive cells leads to the development of neurological impairments (i.e., fatigue, muscle weakness, and slurred speech) as nerve impulses are disrupted before reaching their target. First-line treatment of multiple sclerosis often centers on the administration of immunosuppressive drugs to curtail the progression of the disease and mitigate immune cell-directed demyelination. A driving factor in white blood cell localization is the lipid sphingosine-1-phosphate (S1P). Concentrations of S1P are often not static in the body, with different tissue types and fluids possessing variable levels. Immune cells, and lymphocytes in particular, use this natural S1P gradient to dictate their movement within the body. Lymphocytes will track with the S1P gradient, going from areas of lower S1P concentration (lymph tissue) to areas of higher S1P concentration where synthetic enzyme expression is upregulated (multiple sclerosis lesions). Consequently, the development of drugs that can alter this S1P gradient represents an ideal avenue to achieve immunosuppression. One key mediator of S1P release is the transmembrane transport protein Spinster homolog 2 (Spns2). This protein directs the secretion of intracellular S1P into the extracellular space and is necessary for lymphocytes to enter circulation. However, little effort has been devoted to the development of Spns2 inhibitors. As such, the inhibition of this protein represents a novel and underexplored target for the treatment of autoimmune disorders. In this disclosure, the structures of several highly potent Spns2 inhibitors are revealed. The work around these structures led to the discovery of 4.11i. This compound proved highly potent in biological assays and animal models. Mice treated with 4.11i experienced a reduction in circulating lymphocyte counts and demonstrated less symptom manifestation in multiple sclerosis disease models.

Organoboron compounds are viewed as a crucial intermediate for a wide variety of reactions. The most notorious reaction that exemplifies the capability of organoboron reagents is the Suzuki-Miyaura cross-coupling reaction, which is used to generate carbon-carbon bonds under mild conditions. Because of the versatility of organoboron reagents, methods to selectively install boron remains crucial. Boron containing compounds have recently garnered significant interest in the medicinal chemistry field. Boron's unique properties allows the development of potential new boron-based drugs targeting novel signaling pathways with great efficacy. This dissertation describes the use of a diboron reagent to install boron on the electron withdrawing allenoate scaffold as well as on disubstituted 1,3-diynes. Lastly, this dissertation will cover the preliminary anti-fungal activity of a novel oxaborole scaffold. We investigated the borylation of the electron withdrawing allenoate scaffold. Reports in the literature were scarce and limited in the scope of the work or required the use of less available diboron reagents. We developed a method for the addition of the diboron reagent B2pin2 and a copper chloride catalyst at 60 °C to generate the (Z)-β-borylen-oate in an 18 – 81% yield. A diverse substrate scope was produced, with the reaction being very tolerable with both electron donating and electron withdrawing functional groups attached to the phenyl ring with yields ranging from 29 – 81%. To our delight as well, straight alkyl chains maintained the respective Z stereoselectivity while having yields range from 46 – 60%. During the reaction, activation of the diboron reagent using the copper catalyst in methanol then undergoes boryl-cupration that can subsequently be protonated to form the Z-product. The steric effect of the activated boron complex and the allenoate drives the stereoselectivity of the reaction. To continue the borylation of unique scaffolds, we developed a selective cis phosphinoboration of 1,3-diynes. In this reaction, a catalytic amount of tributyl phosphine and a diphenyl(4,4,5,5-tetramethyl-1-3-2-dioxaborolan-2-yl)phosphane is used to generate the corresponding cis 1,2-phosphinoboronate in yields ranging from 18 – 75%. The reaction is performed in dichloromethane at 40 °C. Substrates bearing an electron donating group on the phenyl ring resulted in the need for a longer reaction time and decreased yields (18 – 39%), while substrates bearing an electron withdrawing groups resulted in increased yields (55 – 72%). The phosphinoboration reaction was also tolerable towards heterocycles (64%) and alkyl groups (34 – 53%). This reaction is able to attach both boron and phosphorous simultaneously without the use of a transition metal. Mild oxidation using iodine resulted in oxidation of the phosphorous. The resulting product interestingly generated a B-O-P heterocycle. In medicinal chemistry, new boron containing scaffolds have shown promising preliminary anti-fungal activity. The oxaborole scaffold is widely seen as a privileged scaffold due to the unique ability of boron to behave as a pharmacophore. The five-membered ring in the oxaborole scaffold also enhances the Lewis acidity of the boron. Our group has previously identified a synthetic route in obtaining a novel 3-monosubstituted oxaborole scaffold. Herein, we have developed a small library of compounds that were tested against a variety of fungal strains. Assaying the library at 25 μg/mL identified multiple hits that allowed the development of a preliminary structure activity relationship profile. Compounds containing electron withdrawing groups on the phenyl ring demonstrated higher anti-fungal activity. This phenomenon is explained due to the change in Lewis acidity of the compound. Incorporating electron withdrawing groups increases the overall Lewis acidity of the oxaborole scaffold, and therefore allowing for stronger covalent interactions in the active site. Increasing the length of the scaffold resulted in a drastic loss in activity, suggesting a smaller scaffold is a necessity. Compounds containing a 4-fluoro, 4-chloro, 4-trifluoromethoxy, and 4-tetrafluoromethoxy were all compounds that consistently observed below 30% cell survival in the candida albicans, aspergillus niger, metarhizium anisopliiae, aspergillus flavus, penicillum chrysogenum, and saccharomyces cerevisiae fungal assays. To further explore the promising potential of the new scaffold, minimum inhibitory concentrations for our lead compounds will be conducted in the future. Doctor of Philosophy Boron-containing compounds are prevalent in a multitude of chemical reactions. Due to the versatility of organoborons in chemical transformations, the development for new chemical reactions that install boron is vital. Of great importance in the installation is the ability to perform the reactions under mild conditions and low cost under environmentally friendly fashion. Boron-containing drugs are also a unique scaffold due to the ability boron has in its ability to act as a drug. Boron is able to covalently bind to molecules in the active site, creating an "anchor" that can then therefore deliver the respective therapeutic effect. This dissertation discusses two reactions that install boron in a selective fashion on challenging substrates. The first chapter discusses the installation of boron on a challenging allene scaffold. The focus of the installation is to maintain selectivity of where the boron ends up on the resulting product which we were successful in. The following chapter discusses the installation of both boron and phosphorous in a one-step fashion. Previous methods would require more steps, harsher conditions, and lower overall yields while we can now circumnavigate these challenges in our new method. The third part of my dissertation will discuss the discovery of a novel boron-containing drug scaffold that has promising anti-fungal activity. Anti-fungal drugs are usually difficult to come by, allowing for our scaffold to shine in a challenging field. We have identified multiple hits in our preliminary assays and we can show that manipulating the potential reactivity of the boron can result in greater or lesser anti-fungal activity.

Mitochondrial uncouplers have the capacity of passively shuttling protons from the mitochondrial intermembrane space to the mitochondrial matrix, independent of ATP synthase. This results in the disruption of oxidative phosphorylation and increased rate of metabolism as a counter action from the mitochondria. Therefore, small molecule mitochondrial uncouplers have potential for the treatment of obesity, diabetes, non-alcoholic fatty liver disease (NAFLD), neurodegenerative disorders, amongst others. A one-pot method for the synthesis of 1H-imidazo[4,5-b]pyrazines from [1,2,5]oxadiazolo[3,4-b]pyrazines is herein disclosed. In the presence of Fe, Yb(OTf)3, and the desired electrophile partner, in situ reduction of the oxadiazole fragment followed by cyclization afforded imidazolopyrazines in moderate to good yields. The selection of different orthoesters as electrophiles also allowed functionalization on the 2-position of the imidazole ring. This new method was used to synthesize 1H-imidazo[4,5-b]pyrazines to perform structure-activity relationship studies. Thus, a library of 75 compounds was synthesized and characterized for mitochondrial uncoupling activity. The biological activity of the compounds was demonstrated in oxygen consumption rate assays affording potent mitochondrial uncouplers. The method was further applied to the synthesis of 5-alkoxy-2-(trifluoromethyl)-1H-imidazo[4,5-b]pyrazin-6-amines, with over 50 derivatives synthesized. A structure-activity relationship study was performed using a variety of substituents to fine-tune the scaffold's potency. The installation of a methoxy group at the 5-position of the scaffold resulted in the discovery of compound 4.3.20, which exhibited the best activity with an EC50 of 3.6 ± 0.4 μM in rat L6 myoblasts and a half-life of 4.4 h in mice. Compound 4.3.20 displayed potential as an anti-obesity agent in a mouse model with an effective dose of 50 mg kg-1 without changes in food intake or lean mass. Tissue distribution studies revealed predominance in the liver and both white and brown adipose tissue. In addition, 4.3.20 improved serum markers of insulin sensitivity and hyperlipidemia such as insulin, glucose, triglycerides, cholesterol, and HOMA-IR. Taken together, compound 4.3.20 and related mitochondrial uncouplers show promise for further development in the treatment of obesity and other diseases. Doctor of Philosophy The mitochondria, which is an organelle within our cells, is where all the nutrients ingested in the form of food are metabolized, and either used for energy or stored as fat if they are not used. The latter is the main cause of obesity, carrying with it a myriad other comorbidities, such as high blood pressure, heart disease, diabetes, certain types of cancer. Obesity has become a great concern with an incidence of 42% in the US. Mitochondrial uncouplers are molecules that target the mitochondria with a mechanism of action of converting some of the energy ingested in the form of nutrients to be lost as heat instead of being stored as fat. The potential result is a regulated form of weight-loss. Herein, we developed a method for the synthesis of a novel mitochondrial uncoupler scaffold and disclose the mitochondrial uncoupler activity of over 150 molecules. In particular, compound 4.3.20 was tested in an obesity mouse model and was shown to induce fat loss with mice fed a high fat diet. Our investigations support potential use of mitochondrial uncouplers as a mechanism for the treatment and prevention of obesity and other metabolic diseases.

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.