Development of NMR Methodology for Complex Mixture Analysis and Discovery of Antiplasmodial Marine Natural Products

Abstract

One of the challenges in natural product discovery is the re-isolation and identification of known compounds. Dereplication is the process of identifying known compounds, allowing time and effort involved in their isolation and structure elucidation to be saved, thus enabling focus to be directed solely on new compounds. While dereplication is mainly MS-based, the main tool for structure elucidation of natural products is NMR. These techniques are very different to each other, with the only current approach in which they can be correlated is by hyphenated techniques such as LC-NMR-MS, requiring specialist and expensive hardware that is not available in every lab. In this thesis we develop structure and molecular size based NMR methodology for analysis of complex mixtures, such as those encountered in natural product research. We have used Diffusion Ordered Spectroscopy (DOSY), an NMR technique that enables components of a mixture to be distinguished by their diffusion coefficients (D), corresponding to hydrodynamic size. Since DOSY has mostly been used for organometallic and polymer analysis, we have established a calibration curve consisting mostly of natural products that provides a correlation between D to molecular weight (MW), allowing MW prediction from experimental D. Since physicochemical properties such as hydrogen bonding, molar density and molecular shape have been shown to affect D and therefore MW prediction, we have generated two new MW prediction models. The first model takes hydrogen bonding into account by the chemical shift of the most acidic hydrogen bond donor (HBD), and the second model provides a more accurate MW prediction for RP HPLC fractions based on the percentage MeOH the compound elutes it. We have generated predicted D based on structural and chemical properties for a database of 220,817 natural products, allowing dereplication by use of a D filter based on the experimental D in conjunction with structural filters based on NMR data. These models have been validated by the dereplication of a mixture of two sesquiterpenes from the plant Tasmannia xerophila, and the dereplication of three known tropane alkaloids in a complex mixture from Darlingia ferruginea. 3D DOSY methodology was undertaken for the analysis of the D. ferruginea complex mixture, allowing molecular fragments to be constructed from DOSY-COSY data. We have also projected diffusion data derived from DOSY-HSQC data onto an HMBC spectrum to generate complex fragments containing non protonated carbons, and this has provided a powerful tool for dereplication. These molecular fragments allowed dereplication and the annotation of three new tropane alkaloids in a complex mixture. These new compounds, together with other known alkaloids were subsequently isolated, and extensive 2D NMR and ECD data allowed their total absolute structures to be assigned to the three new compounds and the structure revision of two of the known compounds, one previously reported as it is diastereomer and both originally wrongly assigned as their opposite enantiomers. In this thesis we report the chemical investigation of two bryozoan species, members of a marine invertebrate phylum that is one of the most understudied. These species were selected for investigation after their extracts were identified as possessing potent bioactivity during an antiplasmodial screening of 486 marine invertebrate crude extracts collected in Australia as active toward the Plasmodium falciparum parasite, the causative agent of the malaria disease. The bryozoan Orthoscuticella ventricosa has afforded five new β-carboline alkaloids, two of which contained were bis-β-carboline containing an uncommon 1,2-disubstituted cyclobutane moiety. These compounds displayed weak antiplasmodial activity against the chloroquine-sensitive strain (3D7) of the P. falciparum. The bryozoan Amathia lamourouxi has afforded six new brominated alkaloids. Two of these displayed sub-micromolar antiplasmodial activity against both chloroquine-sensitive (3D7) and chloroquine-resistant (Dd2) P. falciparum strains and good selectivity relative to the human cell line, marking these compounds as validated antimalarial hits.