Cyclic-AMP dependent protein kinase (PKA) is a key intracellular signal transduction kinase that is modulated by Gs- and Gi-coupled GPCRs. Under normal physiological conditions, PKA exists as an inactive holoenzyme made up of two catalytic subunits and two regulatory subunits. Upon cAMP binding to the regulatory subunits, the catalytic subunits (PKACa) are released to perform various downstream phosphorylation events. However, aberrant PKA activation can cause various diseases including Cushing’s Syndrome, which is an endocrine disorder caused by the overproduction of cortisol by the hypothalamus-pituitary-adrenal hormone system. This disorder can be caused by pituitary adenomas that release unregulated amounts of ACTH, adrenal adenomas that release unregulated amounts of cortisol without ACTH stimulation, and ectopic tumors outside the hypothalamus-pituitary-adrenal axis that produce ACTH. In recent genomic studies of patients with ACTH-independent Cushing’s Syndrome, the L205R-PKACamutant has been discovered. Through various studies on the mutant enzyme multiple research groups learned that the single point mutation causes a loss in sensitivity to cAMP signaling, a loss in binding to PKA regulatory subunits, and unregulated phosphorylation of PKACasubstrates, which ultimately leads to the increased cortisol biosynthesis in these patients. The first part of this work describes the enzymology and inhibition studies of known inhibitors against both wt- and L205R-PKACa. Early in the enzymology studies we developed at medium throughput endpoint assay that used Rhodamine-kemptide as the substrate and as a chromophore separating substrate and phosphorylated product using a reverse-phase HPLC method. The analysis of the substrate peptide against both wild-type and mutant enzyme showed a 6-fold decrease in the KMand a 2-fold decrease in kcat, and a similar but lower order of magnitude effect was observed for the studies with ATP. The inhibition studies were performed using the substrate competitive inhibitor PKI(5-24), which showed a 253-fold higher potency towards the wild-type enzyme over the mutant while the ATP-competitive inhibitor was determined to be equipotent. Using this information we used modeling studies to aid in the development of mutant selective functional inhibitors for the substrate-binding pocket. Additionally, we begun to explore the use of Proteolysis Targeting Chimeras, or PROTACs, as another means for targeting the L205R mutant enzyme.

Rhodococcus bacteria have many secondary metabolic pathways that may produce novel natural products. The bacterium Rhodococcus strain KCHXC3 was isolated from a soil sample collected near Kingsport, Tennessee and was found to produce an inhibitory compound active against a broad array of bacterial species, including the Gram negative pathogen Shigella sonnei. The aim of this research is to extract and purify the compound for future structure elucidation. A mixture of compounds from 3 month old agar plates inoculated with strain KCHXC3 was extracted using ethyl acetate. The crude extract was then partially purified utilizing a Sephadex LH-20 column, followed by an analytical NH2 HPLC column. This purification resulted in a dried crystalline-like active compound that is white in color and needle-like in shape. Structural studies such as NMR and GC-MS revealed the presence of aromatic rings in the active compound that appears to be built with amino acids.

The aim of this study was to elucidate the oxidation products of a candidate antimalarial drug, PL69, using a porphyrin system and to determine the accuracy of the oxidation products produced, as compared to what is expected in metabolism. PL69 is a reversed chloroquine (RCQ) that is active against chloroquine resistant malaria. Porphyrin oxidation systems have been shown to mimic in vitro enzymatic metabolism reactions. PL69 and its known metabolite, PL16, were incubated with the porphyrin system, and then the oxidation products were collected and separated by HPLC. The oxidation products were characterized by NMR and mass spectrometry and compared to previous metabolism studies of PL69 with liver microsomes. The results of this research show that this porphyrin system is an acceptable mimic of in vitro metabolism methods for RCQs and provides a good framework for understanding the types of metabolism that will occur in vivo for RCQs.

The control of oxygen delivery to cells in the body is the result of a small group of primary oxygen sensors, one of the most important of which is the hypoxia-inducible transcription factor-1 (HIF-1). Two alpha-ketoglutarate dependent non-heme iron dioxygenases are responsible for the regulation of HIF-1 through hydroxylation of residues on the HIF-1a subunit. One of these enzymes, known as the factor inhibiting HIF-1 (FIH-1) is responsible for hydroxylating residue Asn803 on HIF-1a, preventing the transcription of hypoxia related genes controlled by HIF-1. It was hypothesized that there would be a difference in inhibition of FIH-1 from the other HIF-1 regulating enzyme, the prolyl hydroxylase domain-2 (PHD2), when testing a series of ten small molecule inhibitors. The ten inhibitors chosen fell into three classes: pyrones, pyridines, and catechols. Of these inhibitors, it was found that catechols produced a significant inhibitory difference between PHD2 and FIH, and may provide useful in further inhibitor design and synthesis work.

This dissertation encompasses work related to synthetic methods for the formation of ester linkages in organic compounds, as well as the investigation of the conformational influence of the ester functional group on the flexibility of inter-saccharide linkages, specifically, and the solution phase structure of ester-containing carbohydrate derivatives, in general. Stereoselective reactions are an important part of the field of asymmetric synthesis and an understanding of their underlying mechanistic principles is essential for rational method development. Here, the exploration of a diastereoselective O-acylation reaction on a trans-2-substituted cyclohexanol scaffold is presented, along with possible reasons for the observed reversal of stereoselectivity dependent on the presence or absence of an achiral amine catalyst. In particular, this work establishes a structureactivity relationship with regard to the trans-2-substituent and its role as a chiral auxiliary in the reversal of diastereoselectivity. In the second part, the synthesis of various ester-linked carbohydrate derivatives, and their conformational analysis is presented. Using multidimensional NMR experiments and computational methods, the compounds’ solution-phase structures were established and the effect of the ester functional group on the molecules’ flexibility and three-dimensional (3D) structure was investigated and compared to ether or glycosidic linkages. To aid in this, a novel Karplus equation for the C(sp2)OCH angle in esterlinked carbohydrates was developed on the basis of a model ester-linked carbohydrate. This equation describes the sinusoidal relationship between the C(sp2)OCH dihedral angle and the corresponding 3JCH coupling constant that can be determined from a JHMBC NMR experiment. The insights from this research will be useful in describing the 3D structure of naturally occurring and lab-made ester-linked derivatives of carbohydrates, as well as guiding the de novo-design of carbohydrate based compounds with specific shape constraints for its use as enzyme inhibitors or similar targets. In addition, the above project led to the development of a methodology for the synthesis of symmetrical ester molecules from primary alcohols using a mild oxidative esterification reaction, which proceeds in hydrous solvents using a nitrosyl radical catalyst. The reaction could be performed with a variety of alcohols and the resulting compounds are of interest in the fragrance and flavor industries.

Recently, the compound resveratrol has had media attention as an anti carcinogen. However, the bioavailability of resveratrol is low in the human system due to its hydrophobic nature. Therefore, it must be administered in high dosages to be effective. A plethora of derivatives have been synthesized that have the potential of resveratrol but sadly share low bioavailability. The first effort of this research was an attempt to produce a more hydrophilic ester of resveratrol. Failing this, the final product was synthesized using a glycine derivative to produce 4-[(1E)-2-(3,5-diacetyloxyphenyl)ethenyl]phenyl N-[(1,1-dimethylethoxy)carbonyl]-glycinate.

Conventional methods for diagnosis of human disease are, at times, limited in different regards including time requirement, either experimental or data processing, sensitivity, and selectivity. It is then that a Point of Care Criteria, which considers the true utility and usefulness of the device, is employed to propose new diagnostic devices capable of overcoming the aforementioned shortcomings of conventional tools. Nucleic acid, characterized for its predictable base-pairing nature, is considered to be a highly-selective, yet greatly modifiable device. Its behavior is then described through Boolean Logic, where “true” or “false” outputs are mathematically described as “1” and “0”, respectively. This mathematical approach is then referred to as Logic Gates, where outputs can be predicted based on satisfied environmental conditions. The mechanisms, capable of exhibiting Logic Gate behavior, are described.

Oxaliplatin is one of the three FDA-approved platinum anticancer drugs and considered a third generation drug, discovered after the first generation drug cisplatin and second generation drug carboplatin. It is known to react with proteins and DNA nucleotides in the body. Reaction with DNA occurs primarily at guanosine residues and secondarily at adenine residues for oxaliplatin and other platinum drugs. We have previously studied oxaliplatin and an analog with additional steric hindrance in the amine ligand and found that the analog had different reactivity with methionine. Now, we have prepared oxaliplatin and its three analogs Pt(Me2dach)(ox), Pt(en)(ox) and Pt(Me4en)(ox) and have reacted each platinum compound with both guanine and adenine nucleotides at pH 4 and pH 7 at different molar ratios. These reactions have been characterized by Nuclear Magnetic Resonance (NMR) spectroscopy equipment over time to observe the formation of products and compare them on the basis of their kinetics and binding affinities. NMR has shown that even under the conditions of excess platinum, the dominant products are usually those with two nucleotides coordinated to one platinum center. Reactions are faster at pH 7 than pH 4 due to deprotonation of phosphate group. Reactions of GMP with a platinum center are faster than reaction with AMP because of the chelate formed by the oxalate ligand. The extra methyl groups on the oxaliplatin analogs do not appear to slow down the reactions with nucleotides considerably. The pH generally affects the rate but does not substantially affect the product distribution.

Upregulation of nicotinic acetylcholine receptors (nAChRs) is a well-documented response to chronic nicotine exposure. Nicotinic acetylcholine receptors are pentameric ligand-gated ion channels consisting of alpha (α2-10) and beta (β2-4) subunits. Nicotine, an agonist of nAChRs, alters trafficking and assembly of some subtypes of nAChRs, leading to an increase in expression of high sensitivity receptors on the plasma membrane. These physiological changes in nAChRs are believed to contribute to nicotine addiction, although the mechanism of these processes has not been resolved. Recently, many studies have converged on the idea that nicotine induces upregulation by an intracellular mechanism. In this dissertation, expression levels of nAChRs were quantified upon exposure to nicotine and its primary metabolite, cotinine. A pH sensitive variant of GFP, super ecliptic pHluorin (SEP), was integrated with a nAChR subunit to study expression and trafficking of nAChRs by differentiating intracellular and plasma membrane inserted receptors. In this work, cotinine is shown to increase the number of α4β2 nAChRs within a cell. Cotinine also affects trafficking of α4β2, evident by a redistribution of intracellular receptors and an increase in single vesicle insertion events on the plasma membrane. This work shows both nicotine and cotinine alter the overall assembly of α4β2 to favor the high sensitivity (α4)2(β2)3 version. Since cotinine and nicotine induce similar physiological changes in nAChRs, the metabolite potentially plays a role in the mechanism of nicotine addiction. Although an intracellular mechanism for upregulation has been supported, a shift in assembly to the high sensitivity (α4)2(β2)3 version exclusively in the endoplasmic reticulum has not previously been detected. In order to study organelle specific changes in stoichiometry, a novel method was developed to isolate single nAChRs in nanovesicles derived from native cell membranes. Separation of nanovesicles originating from the endoplasmic reticulum and plasma membrane, encompassing isolated nAChRs, allows precise changes in stoichiometry to be monitored in subcellular regions. In this work, single molecule bleaching steps of green fluorescent protein (GFP) encoded in each alpha subunit of the pentamer are detected. The number of bleaching steps, or transitions to a nonfluorescent state upon continuous excitation, corresponds to the number of GFP-labeled alpha subunits present. Therefore, the stoichiometry can be deduced by detection of two bleaching steps, as in (α4)2(β2)3, or three bleaching steps, seen in (α4)3(β2)2. Using this method on isolated nAChRs, a shift to assembly of high sensitivity (α4)2(β2)3 receptors is detected definitively within the endoplasmic reticulum. In addition, an increase in (α4)2(β2)3 receptors located on the plasma membrane is shown when nicotine is present. This work provides convincing evidence that nicotine acts intracellularly, within the endoplasmic reticulum, to alter stoichiometry of nAChRs.

The development of novel targeted therapeutics for the treatment of cancer remains difficult due to the complex nature of the disease itself as well as the challenges associated with the synthesis of these therapeutics. Impediments to the discovery of novel drug candidates include lack of available starting materials and access to well-developed syntheses which are both convenient and economically feasible. Semicarbazides, for instance, are a critical synthon for the manufacture of numerous biologically important molecules. Historically, convenient methods for the synthesis of semicarbazides and their derivatives did not exist. Recently, a facile and efficient method for the preparation of semicarbazides via their corresponding phenyl carbamates was developed. These phenyl carbamate intermediates may also be used to prepare a wide variety of other derivatives such as substituted ureas as well as the aryl carbamoyl derivatives of 1,3-diazinane-5-carboxamide. While exploring the preparation of the aryl carbamoyl derivatives of 1,3-diazinane-5-carboxamide, it was found that these compounds possess anti-cancer activity against the glioblastoma LN-229 cell line. Intrigued by these results, additional analogues were designed, leading to the development of a small library of chromenopyrimidinedione and pyrimidinequinolinedione compounds as potential anti-cancer agents. Indeed, these two classes of compounds, with many of the derivatives novel, produced a selection of interesting molecules with potent anti-cancer activity against the glioblastoma cell line LN-229 at biologically relevant concentrations. Taken together, these results provide a unique approach not only to the design but also towards the synthesis of novel therapeutics intended for use as anti-cancer agents.