Chemical and genetic investigations of marine and terrestrial bacteria towards bioactive natural product discovery

The discovery of novel natural products continues to be critical for the development of new pharmaceuticals. Innovative methods to discover novel natural products can reveal previously overlooked chemical diversity. One such method is genome mining, where sequenced bacterial genomes are assessed for the presence of biosynthetic gene clusters. Another such method is bioassay-guided fractionation. Following either of these approaches, the bacteria must be grown and harvested, and novel natural products must be isolated and characterized. In the first part of this thesis, a nitrogen-NMR guided approach was developed to retrieve genetically predicted natural products from bacterial cultures. Piperazic acid (Piz)-containing natural products were targeted because this unique amino acid is often found in peptidic natural products with biological activity and impressive chemical structures. Piz contains a unique nitrogen-proton NMR correlation targeted through ¹H-¹⁵N HSQC. The unique N-H correlation also gave access to Piz’s diagnostic spin system through ¹H-¹⁵N HSQC-TOCSY NMR experiments. These two ¹⁵N NMR experiments were used to monitor for the presence of peptides containing Piz in culture extracts of genome-mined bacteria. Through the application of these ¹⁵N NMR experiments to guide isolation of Piz natural products, four novel compounds were discovered from Streptomyces incarnatus NRRL 8089. Three of these were isolated and structure’s elucidated as part of this thesis work: dentigerumycin F (4.2), dentigerumycin G (4.1) and incarnatapeptin A (4.3). 4.3 demonstrated a unique bicyclic moiety not previously seen in chemical structures, and a fourth compound, incarnatapeptin B (4.4), has in vitro cytotoxicity. In the second part of the thesis, bioassay-guided fractionation is used to screen a small library of marine bacteria in various assays. Using this method, known natural product molecules were uncovered, along with the discovery of two novel natural products from the marine bacterium Salinispora arenicola RJA3005. These two compounds, 6-(1-(3,5-dihydroxyphenyl)-1-hydroxypropan-2-yl)-4-hydroxy-3-methyl-2H-pyran-2-one (6.1) and N-(3-hydroxy-5-(1-hydroxy-2-(4-hydroxy-3-methyl-2-oxo-2H-pyran-6-yl)propyl)phenyl)acetamide (6.4), were isolated from extracts of wild-type bacteria for the first time. Feeding studies and analysis of ¹³C splitting patterns suggest that these compounds were biosynthesized from bacterium through phosphoenolpyruvate and erythrose precursors. Altogether, this thesis's work develops and harnesses various natural product discovery methods to uncover diverse natural products.Science, Faculty ofChemistry, Department ofGraduat