Investigations of Azinomycin Biosynthesis and Macular Degenerative Diseases

The production and discovery of natural products has been a beneficial tool that provides a window into the bioorganic chemistry of organisms and offers a template for drug discovery and development. Natural products can be utilized to positively impact the deleterious effects of diseases or they can incur toxicity in certain biological systems. The aim of this work is to elucidate the biosynthetic pathway of the natural product Azinomycin and to evaluate the effects and treatments for lipofuscin accumulation in macular degenerative diseases. Azinomycin A and B are antitumor natural products that are isolated from Streptomyces sahachiroi. Azinomycin A and B form interstrand crosslinks with DNA through their electrophilic epoxide and aziridine [1,2-α] pyrrolidine ring moiety. The biosynthetic pathway for aziridine ring formation was investigated. AziC5 and AziC6, enzymes with a high similarity with transketolase enzymes, are shown to catalyze a two-carbon extension of N-acetyl glutamate semialdehyde into an α, α’ dihydroxy intermediate. Additionally, AziC5 and C6 displayed traditional transketolase properties by demonstrating thiamin dependence. The naphthoate ring on Azinomycin forms noncovalent interactions with DNA that are pivotal for successful cross-linking. 5-methyl-Naphthoic acid is created with a polyketide synthase enzyme, AziB, that contains multiple domains. The acyl carrier protein (ACP) domain of AziB is post-translationally modified by phosphopantetheinyltransferases (PPTase). The optimization of the post-translational modification was achieved through the identification of the most effective PPTase. The thioesterase (TE) domain of AziB hydrolyzes the thioester bond between 5-methyl-naphthoic acid and the ACP domain. The rate of hydrolysis was determined for AziG and a series of AziG mutants to demonstrate which residues are essential for substrate cleavage. The AziG mutants were then employed to investigate their effects on the production of the naphthoate ring. Macular degenerative diseases impact a significant amount of people in the United States. The main contributing factors for these diseases are the accumulation of lipofuscin in the retina and the eye’s inability to degrade them. Vitamin A, a necessary cofactor in the visual cycle, can form dimers, A2E and cycloretinal, in enzymatic and non-enzymatic reactions. The negative effects of macular diseases are hypothesized to decrease with the clearance of accumulated lipofuscin. While the toxicity of A2E has been investigated, the toxicity of cycloretinal has not. Cycloretinal liposomes were synthesized to facilitate introduction into ARPE-19 cells and to evaluate cycloretinal cytotoxicity. In addition, MsP1, a heme-based peroxidase enzyme, was shown to degrade the toxic, all-trans retinal dimers. The degradation products of these dimers were evaluated for toxicity to display MsP1’s efficaciousness as a gene therapy target. The mechanism of formation for the cyclohexadienal moiety in cycloretinal was shown non-enzymatically, with catalytic L-proline, and enzymatically, with the whey protein β-lactoglobulin (BLG). In addition to determining the mechanism of formation, a series of halogenated α, β-unsaturated aldehydes were employed to inhibit cycloterpenal formation. These inhibitors were employed to prevent the dimerization of α, β-unsaturated aldehydes and promote retro-aldol cleavage on a series of aldehydes with hydrophobic side groups. The experiments outlined in this dissertation seek to further our knowledge of the biosynthesis of the azabicycle and naphthoate ring of Azinomycin. In addition, the contributing factors and inhibitors to macular degenerative diseases were investigated to demonstrate the harmful effects of certain natural products. The investigations into natural product formation and degradation could yield beneficial therapeutic agents amongst a wide array of diseases