Phytochemical profiling and anti-microbial studies of Australian marine and terrestrial organisms: the search for new natural product derived anti-biotic and anti-fungal agents

This thesis describes the identification of sixty five secondary metabolites from thirteen separate organisms (three terrestrial and ten marine specimens). A total of twenty two new and forty two known secondary metabolites, as well as one new natural product derivative were identified by chemical profiling utilising HPLC-NMR and HPLC-MS together with traditional off-line isolation strategies. Of the new compounds identified, fourteen displayed antibiotic activity. For nine of the compounds identified the 2D NMR characterisation is now reported for the first time in addition to five carbon and one proton NMR chemical shift reassignments. The single X-ray crystallographic structures for three of the compounds identified were also reported. Five previously reported compounds are now reported as natural products for the first time. HPLC-NMR was vital in the identification of an unstable meroditerpenoid that could not be isolated by off-line approaches. This PhD study also resulted in the first report of the acquisition of a gHMBCAD NMR spectrum obtained in the stop-flow HPLC-NMR mode, without pre-concentration, microcoils or cryogenically cooled probes. Chapters 1-3 provide an introduction to natural products, isolation techniques as applied to natural products, an explanation and examples of dereplication strategies, an outline of the common NMR experiments used for natural product identification and a review of the recent advancements made in HPLC-NMR as well as its application to natural product identification. Chapter 4 details the limit of detection determined for five key NMR experiments in HPLC-NMR, which were found to be within the 700 ng to 1 mg range, typical of that observed in natural product crude extracts. HPLC-NMR was subsequently successfully applied to a natural product crude extract for which complete 2D NMR data could be obtained for the major component. Chapter 5 outlines the constituents identified from the flowers and bulbs of the Australian plant, Macropidia fuliginosa. The flowers and bulbs produced very different chemistry, and a total of sixteen compounds were identified, including six new and ten previously reported compounds. The single X-ray crystal structure for three compounds is also presented. Chapter 6 describes the identification of eleven compounds including two new phenylphenalenones and two new chromenes from the bulbs of the Australian plant Haemodorum spicatum. Chapter 7 summarises the identification of three fatty acids from the yellow fungus Leucocoprinus birnbaumii. The positions of the double bonds were secured by ozonolysis methods. Chapter 8 outlines the dereplication of compounds from seven marine algae. Compounds were dereplicated using HPLC-NMR and HPLC-MS data in conjunction with the MarinLit database. Sixteen compounds were dereplicated including four new compounds. Chapter 9 highlights the identification of four new and nine known meroditerpenoids from the brown alga Sargassum paradoxum. HPLC-NMR was vital for the identification of an unstable meroditerpenoid that oxidised during off-line isolation. Chapter 10 describes the dereplication and identification of phloroglucinols from two Cystophora species, including four new and eight known phloroglucinols. HPLC-NMR and HPLC-NMR was able to rapidly establish that both marine algae contained the same structure class of compounds. Chapter 11 summaries the relative configuration of elatenyne which was proposed on the basis of 1D nOe NMR and chemical derivatisation methods

Surface water dependent properties of sulphur-rich molybdenum sulfides: electrolyteless gas phase water splitting

Sulfur-rich molybdenum sulfides are an emerging class of inorganic coordination polymers that are predominantly utilized for their superior catalytic properties. Here we investigate surface water dependent properties of sulfur-rich MoSx (x = 32/3) and its interaction with water vapor. We report that MoSx is a highly hygroscopic semiconductor, which can reversibly bind up to 0.9 H2O molecule per Mo. The presence of surface water is found to have a profound influence on the semiconductor's properties, modulating the material's photoluminescence by over 1 order of magnitude, in transition from dry to moist ambient. Furthermore, the conductivity of a MoSx-based moisture sensor is modulated in excess of 2 orders of magnitude for 30% increase in humidity. As the core application, we utilize the discovered properties of MoSx to develop an electrolyteless water splitting photocatalyst that relies entirely on the hygroscopic nature of MoSx as the water source. The catalyst is formulated as an ink that can be coated onto insulating substrates, such as glass, leading to efficient hydrogen and oxygen evolution from water vapor. The concept has the potential to be widely adopted for future solar fuel production

Recent advancements in HPLC-NMR and applications for natural product profiling and identification

This review provides a summary of the recent advances in high pressure liquid chromatography (HPLC) coupled to nuclear magnetic resonance (NMR) spectroscopy. Significant improvements have included the miniaturization of NMR components as well as the use of cryogenically cooled probes. Further hyphenated applications such as HPLC-SPE-NMR, column trapping and HPLC-NMR-MS are also discussed. Finally, an overview of the application of these methodologies to the profiling and dereplication of natural products is provided

Limit of detection studies for application to natural product identification using high performance liquid chromatography coupled to nuclear magnetic resonance spectroscopy

In the pursuit of new natural products, the demand to rapidly identify compounds present, in ever decreasing amounts, in complex crude extracts has become a limiting factor. Despite improvements in HPLC-NMR hardware and pulse sequences, no extensive limit of detection (LOD) investigations have been reported for the acquisition of 20 NMR spectroscopic experiments acquired through HPLC-NMR. In this study the LOD for five key 1D and 2D NMR spectroscopic experiments have been established, using two reference compounds, including the on-flow (WET 1D proton), stop-flow (WET1D proton), gCOSY, HSQCAD and gHMBCAD NMR experiments. The LOD for all of the NMR experiments were within the range of 700 ng to 1 mg for the set of fixed experimental parameters implemented. For principle components in a complex multi-component mixture, this would allow for in situ compound identification. HPLC-NMR analysis was employed to investigate the principle components present in a marine brown alga crude extract, Cystophora subfarcinata

HPLC-NMR and HPLC-MS Profiling and Bioassay-Guided Identification of Secondary Metabolites from the Australian Plant Haemodorum spicatum

Phytochemical dereplication was undertaken on the bioactive crude CH2Cl2 extract of the bulbs of the Australian plant Haemodorum spicatum employing HPLC-NMR and HPLC-MS methodologies. Subsequent bioassay-guided isolation resulted in the identification of two new phenylphenalenones [haemoxiphidone (8) and haemodoronol (17)] and two new chromenes [haemodordione (13) and haemodordiol (16)], together with seven previously described compounds. Antimicrobial testing showed that the compounds displayed selective antibacterial activity. Most noteworthy were the activities displayed by several of the compounds against multi-drug-resistant Pseudomonas aeruginosa

HPLC-NMR and HPLC-MS investigation of antimicrobial constituents in Cystophora monilifera and Cystophora subfarcinata

The crude dichloromethane extracts of the marine brown algae Cystophora monilifera and Cystophora subfarcinata were subjected to phytochemical profiling. This enabled the structures of both new and known phloroglucinols to be dereplicated and proposed using HPLC-NMR and HPLC-MS. Subsequent isolation confirmed the presence of four new and eight previously reported compounds. Five of the isolated phloroglucinols displayed selective antimicrobial activity

Rapid dereplication and identification of the bioactive constituents from the fungus leucocoprinus birnbaumii

A series of fatty acids were rapidly dereplicated and partially identified from the flowerpot fungus, Leucocoprinus birnbaumii using HPLC-NMR and HPLC-MS. Subsequent off-line isolation unequivocally established the structures, and anti-microbial testing concluded that the fatty acids displayed moderate but selective anti-microbial activity. This represents the first report of these compounds occurring in this particular terrestrial fungus

Dereplication and chemotaxonomical studies of marine algae of the Ochrophyta and Rhodophyta phyla

Dereplication and chemotaxonomic studies of six marine algae of the Ochrophyta and one of the Rhodophyta phyla resulted in the detection of 22 separate compounds. All 16 secondary metabolites, including four new compounds (16-19), could be rapidly dereplicated using HPLC-NMR and HPLC-MS methodologies in conjunction with the MarinLit database. This study highlights the advantages of using NMR data (acquired via HPLC-NMR) for database searching and for the overall dereplication of natural products

Phytochemical investigation of the constituents derived from the Australian plant macropidia fuliginosa

A phytochemical study of the flowers and bulbs derived from the Australian plant Macropidia fuliginosa, involving hyphenated spectroscopic methodologies (HPLC-NMR and HPLC-MS), together with conventional isolation strategies, resulted in the identification of 16 constituents (1, 2, 4-17) representative of six different structural classes. Six new compounds (12-17) were identified from the bulbs of the plant. The isolated compounds were assessed for antimicrobial activity, and compound 8 was found to be more potent against OH P. aeruginosa than ampicillin

Absolute configuration determination of retroflexanone using the advanced mosher method and application of HPLC-NMR

The absolute configuration of retroflexanone (1) and a closely related phlorogluinol (2) was established using the advanced Mosher method and by application of HPLC-NMR. HPLC-NMR permitted a small scale Mosher method analysis to be carried out on these unstable phloroglucinols