Isolation and characterization of nitrogenous metabolites from the ascidians Cystodytes sp., Lissoclinum patella, and Botryllus sp.

This dissertation describes the isolation, characterization, and biological evaluation of several nitrogenous metabolites from ascidians. The first chapter reviews marine natural products that have shown promising biological activities or have been instrumental in enhancing our understanding of certain cellular processes. Since the research described is largely based on bioassay guidance to fractionate extracts of marine ascidians into bioactive secondary metabolites, the second chapter covers general approaches used to find bioactive natural products. The subsequent chapters detail the isolation and characterization of several new nitrogenous metabolites using a variety of spectroscopic techniques. The active extract of a Fijian Cystodytes sp. ascidian was fractionated to yield a series of pyridoacridine alkaloids with topoisomerase II (topoII) activity. The structures of these alkaloids were elucidated primarily using nuclear magnetic resonance (NMR) spectroscopic techniques. The pyridoacridine alkaloids were cytotoxic towards the cultured mammalian cell line HCT116; acting by intercalating into deoxyribonucleic acid (DNA) and inhibiting the catalytic activity of topoII. Chapter 3 details the structure elucidation of these alkaloids and the mechanistic and biological experiments undertaken to support this conclusion. A new cyclic peptide, patellamide E, was isolated from the ascidian Lissoclinum patella collected at Pulau Salu, Singapore. Its structure was determined by NMR spectroscopy, and its absolute configuration by acid hydrolysis and analysis of the derivatized constituent amino acids by high performance liquid chromatography (HPLC). The first part of the fourth chapter describes the structure elucidation of patellamide E. The second part of Chapter 4 describes the new cytotoxic cyclic peptides, tawicyclamides A and B, which were isolated from the ascidian Lissoclinum patella collected in the Philippines. The structures of these peptides were determined by NMR spectroscopy, oxidation studies, and tandem mass spectrometry (MS/MS). Their absolute configurations were again determined by HPLC analysis of derivatized constituent amino acids obtained from acid hydrolysis. X-ray crystallography confirmed the structure of tawicyclamide B and showed that the compound assumes an unusual three-dimensional conformation, which was facilitated by a cis-valine-proline amide bond and stabilized by an intramolecular hydrogen bond. Tawicyclamides A and B represent a new family of cyclic octapeptides, possessing thiazole and thiazoline amino acids but lacking the oxazoline ring characteristic of previously reported cyclic peptides from L. patella. NMR data supported the hypothesis that isomerization of the valine-proline amide bond occurred on oxidation of the thiazoline ring to a thiazole. Molecular modeling studies, undertaken to establish the solution structures of tawicyclamide B and dehydrotawicyclamide B, supported the isomerization conjecture. The new bromotyrosine derivatives, botryllamides A-D, which were isolated from a Philippine Botryllus sp. ascidian are discussed in Chapter 5. The structures of these compounds were deduced from NMR and mass spectrometric data.