Nitric oxide (NO) has been extensively studied due to its importance as a signaling agent. More recently, the pharmacological benefits of nitroxyl (HNO) in the treatment of cardiovascular disease, cancer, and alcoholism have been discovered.That HNO readily dimerizes complicates analysis and necessitates the use of donors. Diazeniumdiolates (NONOates), which can release either NO or HNO, are particularly attractive in this regard. NONOates from primary amines release HNO at physiological pH, and since the few existing examples have relatively short half-lives, a major research goal was to extend the lifetime range. The effect of amine structure on the lifetimes of ionic primary amine NONOates having the general structure Na(RN(H)[N(O)NO]) was unexpectedly small. This prompted the use of O2-protecting group methodology as an alternate method to stabilize donors toward decomposition. A detailed analysis of the decomposition mechanisms of a representative ionic primary amine NONOate and its O2-protected derivative is presented.NONOates were used as analytical tools to compare several commonly-used methods for detection of HNO. While these methods are used routinely for qualitative analysis of HNO, optimization for quantitative measurements was difficult. To improve method sensitivity, an HPLC assay using the fluorogenic reagent o-phthalaldehyde was developed, which may ultimately allow detection of endogenously-produced HNO.HNO donors such as cyanamide have been utilized in the treatment of alcoholism through the inhibition of aldehyde dehydrogenase (AlDH), which is critical for ethanol metabolism. Cyanamide also releases cyanide, and alternate HNO donors are thus desired for this clinical use. The efficacy of NONOates in the inhibition of AlDH was assayed in purified yeast AlDH and in mouse liver homogenate. However, efficacy was limited in a mouse model, perhaps due to a lack of selective delivery. This drug discovery project provided useful information for the future development of potentially liver-selective HNO-releasing NONOates.Together, these studies demonstrate the utility of NONOates as biomedical research tools, with synthetic modifications allowing for the modulation of decomposition profiles. As analytical tools for the development of HNO detection methods and potential pharmaceuticals in the treatment of alcoholism, NONOates provide convenience and control as donors of NO and HNO.

The reactive nitric oxide species (RNOS) nitroxyl (HNO) has exhibited both beneficial and deleterious biological effects. In particular, HNO autoxidation can lead to harmful modifications of biomolecules, yet the products of HNO/O₂ remain undetermined. A conceivable product is peroxynitrous acid (ONOOH), however a comparison of the chemistry of HNO/O₂ to synthetic peroxynitrite (ONOO⁻) determined that these RNOS have distinct reactive profiles. This study compares the reactivity of HNO and NO⁻ in the presence of O₂ to synthetic ONOO⁻ and the autoxidation of HNO at high pH (NO⁻/O₂) in an effort to shed light on the products of HNO autoxidation. All species exhibited the capacity for two-electron oxidation, but differences between ONOO⁻ and NO⁻/O₂ and HNO/O₂ were observed in terms of one-electron oxidation, hydroxylation, nitration and buffer effects. NO⁻/O₂ exhibited a reactive profile similar to ONOO⁻, suggesting that protonation of ONOO⁻leads to a unique species from the autoxidation of HNO.

Nitric oxide (NO) has been shown to both suppress and promote tumor growth, depending in part on concentration. Exogenous delivery of NO may lead to tumor suppression. Recent studies have proposed ruthenium nitrosyl complexes as catalytic donors of NO in reductive environments. Catalytic donation can provide a long-term, elevated NO flux compared to single use donors. Site-specific delivery is desirable to reduce systemic side effects, such as lowering of blood pressure. Three new ruthenium nitrosyl complexes were synthesized to impart site-specificity through amide coupling to polymers, silica nanoparticles, iron oxide nanoparticles and antibodies. The catalytic activity of new and existing compounds was then assessed. However, upon one-electron reduction of ruthenium nitrosyl complexes, insignificant amounts of NO were detected, suggesting an alternative mechanism than that proposed in prior reports. The mechanism of [Ru(EDTA)NO]²⁻ decay was more thoroughly analyzed. Spectrophotometric decay of [Ru(EDTA)NO]²⁻ indicates that one or multiple nitrogen oxide species are released. Previous studies have suggested a disproportionation mechanism leading to the generation of more highly reduced species such as N₂ and NH₄⁺. Experiments were designed to analyze possible decomposition products such as [Ru(EDTA)NO]⁻ and [Ru(EDTA)H₂O]²⁻. A disproportionation mechanism was determined likely. Decomposition of [Ru(EDTA)NO]²⁻ was also observable following reductive nitrosylation of [Ru(EDTA)H₂O]⁻ in the presence of HNO. The decomposition product, [Ru(EDTA)H₂O]²⁻, was observed through the binding of pyrazine (pz) or dipyridine (bipy) and formation of [Ru(EDTA)pz]²⁻ or [Ru(EDTA)bipy]³⁻. Formation of [Ru(EDTA)bipy]³⁻ or [Ru(EDTA)pz]²⁻ via reductive nitrosylation of [Ru(EDTA)H₂O]⁻ also provides an indirect method of HNO detection that is selective from NO.

Chronic pain is a major health problem worldwide. We have recently demonstrated the analgesic effect of the nitroxyl donor, Angeli's salt (AS) in models of inflammatory pain. In the present study, the acute and chronic analgesic effects of AS was investigated in chronic constriction injury of the sciatic nerve (CCI)-induced neuropathic pain in mice. Acute (7th day after CCI) AS treatment (1 and 3 mg/kg; s.c.) reduced CCI-induced mechanical, but not thermal hyperalgesia. The acute analgesic effect of AS was prevented by treatment with 1H-[1,2, 4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, a soluble guanylate cyclase inhibitor), KT5823 (an inhibitor of protein kinase G [PKG]) or glibenclamide (GLB, an ATP-sensitive potassium channel blocker). Chronic (7-14 days after CCI) treatment with AS (3 mg/kg, s.c.) promoted a sustained reduction of CCI-induced mechanical and thermal hyperalgesia. Acute AS treatment reduced CCI-induced spinal cord allograft inflammatory factor 1 (known as Iba-1), interleukin-1β (IL-1β), and ST2 receptor mRNA expression. Chronic AS treatment reduced CCI-induced spinal cord glial fibrillary acidic protein (GFAP), Iba-1, IL-1β, tumor necrosis factor-α (TNF-α), interleukin-33 (IL-33) and ST2 mRNA expression. Chronic treatment with AS (3 mg/kg, s.c.) did not alter aspartate aminotransferase, alanine aminotransferase, urea or creatinine plasma levels. Together, these results suggest that the acute analgesic effect of AS depends on activating the cGMP/PKG/ATP-sensitive potassium channel signaling pathway. Moreover, chronic AS diminishes CCI-induced mechanical and thermal hyperalgesia by reducing the activation of spinal cord microglia and astrocytes, decreasing TNF-α, IL-1β and IL-33 cytokines expression. This spinal cord immune modulation was more prominent in the chronic treatment with AS. Thus, nitroxyl limits CCI-induced neuropathic pain by reducing spinal cord glial cells activation.

This dissertation focuses on chemical and biological evaluation of diazeniumdiolate based nitrogen oxide releasing prodrugs. Three projects are described. i. Synthesis and biological evaluation of a series of new nitroxyl (HNO) releasing non-steroidal antiinflammatory drugs (NSAIDs) and comparison to related nitric oxide (NO) releasing NSAIDs A series of HNO releasing isopropylamine-based diazeniumdiolate adducts of NSAIDs and the NO releasing diethylamine diazeniumdiolate counterpart were synthesized. The aspirin derivatives were evaluated for antiinflammatory, cardioprotective and anticancer effects. Both prodrugs demonstrated similar antiinflammatory properties to aspirin but significantly lower gastrointestinal ulceration, which is a common side effect of aspirin. The HNO adduct also improved cardiac contractility. The chemotherapeutic potential of the prodrugs was assessed in vitro and in vivo. Both the prodrugs inhibited growth of cultured carcinoma cells without inducing cytotoxicity towards non-tumorogenic cell lines. The higher cytotoxicity of the HNO adduct was in part due to increased production of reactive nitrogen and oxygen species leading to oxidative damage to DNA, inhibition of glyceraldehydes-3-phosphate dehydrogenase and upregulation of signaling pathways leading to caspase-3 mediated induction of apoptosis. The NO adduct is a promising candidate for reduction of metastasis by increasing E-cadherin levels, which influences cellular adhesion. Both derivatives showed significantly reduced angiogenesis in cultured cells and tumor volume in nude mice. ii. Synthesis and characterization of primary amine based cyclic amine diazeniumdiolates and comparison to their acetoxy methyl ester derivatives. A series of HNO releasing cyclic amine diazeniumdiolates were synthesized to expand upon the few examples of primary amine diazeniumdiolates. An ester derivative of cyclopentylamine NONOate was also synthesized, to increase decomposition half-life and to improve HNO production and better cellular uptake. This modification increased its cytotoxicity compared to ionic NONOates. iii. Evaluation of mechanism of action of JS-K. JS-K (O²-(2,4-dinitrophenyl)-1-[(4-ethoxycarbonyl)-piperazin-1-yl]-diazeniumdiolate) has previously been found to be highly cytotoxic in many cancer cell lines compared to ionic diazeniumdiolates. Thus, the role of NO in cytotoxicity of JS-K was explored. A low intracellular NO flux in combination with a lack of any effect on cyclic guanosine monophosphate (cGMP) dependent pathway suggests that NO is not directly responsible for the cytotoxicity of JS-K.

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Phospholamban (PLN) is an integral membrane protein that regulates the sarco(endo)plasmic Ca2+-ATPase (SERCA2a) within the cardiac sarcoplasmic reticulum (CSR). SERCA2a regulates intracellular Ca2+- handling and thus plays a critical role in initiating cardiac contraction and relaxation. It is believed that dysregulation of SERCA2a is a contributing factor in human heart failure patients. Even though there have been substantial advancements in understanding heart failure pharmacological therapies, patient prognosis remains poor. Nitroxyl (HNO), a new candidate heart failure drug therapy, has been shown to enhance overall cardiovascular function in both healthy and failing hearts, at least in part, by increasing Ca²⁺ re-uptake into the CSR. Previous research has shown that activation of SERCA2a by HNO is PLN-dependent; however, the mechanism of action of HNO remains unknown. We propose that HNO, a thiol oxidant, modifies one or more of the three PLN cysteine residues (C36, C41, C46) affecting the regulatory potency of PLN toward SERCA2a. To test this hypothesis, a series of PLN mutants were constructed containing single, double and triple cysteine substitutions. Using the baculovirus expression insect cell system, each PLN cysteine mutant was expressed alone and co-expressed with SERCA2a in insect cells and isolated in cellular endoplasmic reticulum (ER) microsomes. Samples were treated with Angeli's salt (an HNO donor) to determine the role of each PLN cysteine residue in the mechanism of SERCA2a activation by nitroxyl. Using a standard phosphate activity assay and SDS-PAGE/immunoblot techniques, we determined that the PLN cysteine residues at positions 41 and 46 are important in HNO activation of SERCA2a. Both SERCA2a + 41C PLN and SERCA2a + 46C PLN microsomal samples showed a ΔK0.5 of ~0.33 μM and evidence of reversible HNO induced disulfide bond formation. These studies provide important new insight into the mechanism of action of HNO on cardiac SR and thereby help evaluate the drug as a candidate therapy for congestive heart failure. / Ph. D.

Gasotransmitters are endogenous small molecule gases that are freely permeable to membranes and possess biological signaling functions. The three recognized gasotransmitters are carbon monoxide (CO), nitric oxide (NO), and hydrogen sulfide (H2S). H2S is featured in this work, as well as persulfides (RSSH), which also have similar functions to H2S (e.g., angiogenesis) and are the presumed signaling products of H2S but are less studied. Other compounds that are considered potential gasotransmitters include methane, sulfur dioxide, hydrogen cyanide, and nitroxyl (HNO). This dissertation covers compounds that release HNO, which possesses similar functions to NO (cardioprotection and vasodilation) but has been studied much less. While HNO, H2S, and RSSH have vital biological functions, they also have short half-lives in vivo (seconds to minutes), thus necessitating the development of prodrugs, also called donor compounds, that can release these reactive species over a biologically relevant time scale. While donor compounds extend the release rate of such small molecule gases, they do not have the ability to release drugs as slowly and continuously as endogenous gasotransmitter-generating enzymes do. As such, polymeric delivery systems have been developed to extend the release of drugs, and in the case of gasotransmitters this more closely mimics in vivo production of HNO/H2S/RSSH. Polymeric systems have been employed to modulate gasotransmitter delivery to control drug release rate, location, and longevity precisely. H2S has been employed in numerous polymeric systems, as discussed in Chapter 2, but there is a significant gap in the literature focusing on polymeric donors for HNO/RSSH. Researchers need to develop novel materials that demonstrate an extended release of these small molecules to better understand the effects of long-term exogenous delivery of HNO/RSSH/H2S which exist fleetingly in vivo. Therefore, materials that release a continuous, well characterized amount of gasotransmitters are vital for biologists to understand long-term effects of such short-lived gasses. The aim of this dissertation, in part, is to hopefully inspire the development of novel HNO/RSSH/H2S releasing systems. In Chapter 3 we discuss an HNO-donating polymer. Here we demonstrate a simple system derived from polyethylene glycol (PEG) and sulfonated polystyrene (PSS). We synthesized a polymeric version of Piloty's acid — a well-known HNO donor — by converting the PSS into a Piloty's acid motif in a two-step process. We found that by simply changing the block ratio of PEG and PSS, we were able to vary the release rate of HNO over an order of magnitude. In Chapter 4 we focus on the development of depolymerizable H2S donors encapsulated within polymer micelles. We report the synthesis of two classes of monomers, one derived from norbornene and one from acrylates. We anticipate that the results from this study will further direct and impact the study of exogenous H2S-releasing materials. In Chapter 5 we discuss electrospun polymer films made of poly(ethylene oxide terephthalate)/poly(butylene terephthalate) (PEOT/PBT) with embedded RSSH donors. We found that the RSSH donors can rescue cells from H2O2 exposure and do not interfere with angiogenesis in HMVECs. We report the fabrication, characterization, and drug release studies of the polymer fiber mats. Lastly, the appendix included at the end of this dissertation briefly discusses the synthesis of a novel depolymerizable poly(thiourethane) derived from a pyrrole monomer. / Doctor of Philosophy / Polymers, long chains of repeating units, are found everywhere. From biological polymers such as starch or cellulose to commercial plastics that come from crude oil sources, applications of natural and synthetic polymers have grown exponentially over the last century. While most people associate polymers with plastic water bottles or shopping bags, researchers have developed ways to harness the power of these long-chain molecules for drug delivery. Common examples of polymer drug delivery systems include nicotine patches and controlled-release drug capsules such as Allegra D. As scientists push the limits of drug delivery and more advanced materials are developed, the quality of human life could improve drastically. The potential for this industry can be seen by the sheer magnitude of money that has been put toward drug delivery devices – the market is expected to reach $2.2 trillion within the next two years. In this dissertation we highlight three separate projects focusing on the delivery of gasotransmitters from polymer systems. Gasotransmitters are small gases that are produced in the cells of bacteria, plants, fungi, and animals. To be considered a gasotransmitter, the gas has to be able to enter or exit cells freely and also have specific biological functions. There are three currently accepted gasotransmitters: nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S). The first chapter of this work briefly discusses the biological importance of these gases. Each of the aforementioned gasotransmitters possesses key biological functions such as anti-inflammatory properties, protection of the cardiovascular system from injury, and encouraging the growth of new blood cells. Despite their helpful effects, gasotransmitters exist fleetingly in the body, existing for only seconds to minutes before being converted into a different molecule. Therefore, if researchers want to exploit the therapeutic potential of these gases, we must develop small molecules donors of these gasotransmitters, termed prodrugs. A prodrug is a compound that is administered in an inactive form, but upon a triggering event, e.g., a change in pH, or through the body's metabolism of the compound, an active therapeutic is released. Numerous research groups, including ours, have developed and examined the biological effects of gasotransmitter-releasing prodrugs. To push the efficacy of these prodrugs even further, we can incorporate them within polymer drug delivery systems, as is discussed throughout this dissertation. When a drug or a prodrug is used in a drug delivery system, the release rate and location, circulation time, and other critical properties are carefully tuned. In the case of the work reported here, we set out to develop different delivery systems to control the release of H2S (Chapter 4), persulfides (RSSH, Chapter 5), and nitroxyl (HNO, Chapter 3); while not officially recognized as part of the gasotransmitter family, RSSH and HNO are closely related to H2S and NO respectively. Both RSSH and HNO are significantly less studied than the gasotransmitters and developing systems that can help biologists elucidate the effects of RSSH/HNO from H2S/NO could provide a better understanding of how to treat different disease states.

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