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This thesis describes the isolation and structure elucidation of ten novel secondary metabolites from one fungus (Pycnoporus cinnabarinus), four lichens (Chrysothrix xanthina, Candelaria concolor, Ramalina glaucescens and Xanthoria parietina), three algae (Plocamium mertensii, Laurencia filiformis and Laurencia elata), two plants (Haemodorum simplex and Dianella callicarpa) and one sponge (Dactylospongia sp). The structures of these isolated compounds were elucidated by a combination of spectroscopic and chemical methods. This thesis also reports two new crystal structures, the identification of two new methylsilylated derivatives as well as the isolation of thirty seven previously reported compounds in which the complete structural assignment by one and two dimensional nuclear magnetic resonance spectroscopy (NMR) has been carried out on known compounds with incomplete or no NMR spectroscopic data. Furthermore, detailed spectroscopic analyses resulted in the re assignment of 1H and 13C chemical shifts for several previously isolated natural products. The biological screening (antimicrobial, antiviral and antitumor assays) of crude extracts and isolated natural products has also been presented. The application of chemical profiling techniques including GCxGC and high pressure liquid chromatography-nuclear magnetic resonance (HPLC-NMR) were utilised to assist with the natural product dereplication process (chemical profiling), monitor chemical degradations in situ and to identify the presence of new natural products and artefacts. In total, fifteen separate terrestrial and marine organisms were investigated.

The natural resources offer a unique opportunity for the discovery of active compounds, due to the complexity and biodiversity of their chemical structures. Natural resources have been used as medicines throughout human history. Saudi Arabia’s natural resources, for instance its terrestrial medicinal plants and the Red Sea sponges, have not been extensively investigated with regard to their biological activities. To better identify the diversity of compounds with bioactive potential, new techniques are also necessary in order to improve the drug discovery path. This study comprises three sections. The first section examines Juniperus phoenicea (Arar), Anastatica hierochuntica (Kaff Maryam) and Citrullus colocynthis (Hanzal); these herbal plants were screened for potential bioactivity using a newly developed pipeline based on a high-content screening technique. We report a new cell-based high-throughput phenotypic screening for the bio-prospecting of unknown natural products from Saudi Arabian plants, in order to reveal their biological activities. The second section investigates Avicennia marina plants, screened for reverse transcriptase anti-HIV bioactivity using biochemical assay. Image-based high-content screening with a set of cellular stains was used to investigate the phenotypic results of toxicity and cell cycle arrest. The third section considers the isolation of Actinomycetes from Red Sea Sponges. Actinomycetes bacterial isolates were tested for bioactivity against West Nile Virus NS3 Protease. Analytical chemical techniques such as liquid chromatography–mass spectrometry (LC-MS), gas chromatography–mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) were used to gain more understanding of the possible chemical compounds responsible for this bioactivity. Overall, the aim of this work is to investigate the potential bioactive effect of several Saudi Arabian plants and Red Sea sponges against cancer cells and viral infections. Our study demonstrates the efficiency of the newly developed pipeline using cell-based phenotypic screening. Anti-cancer potential activity was detected in Juniperus phoenicea. Bioactive potential against the reverse transcriptase enzyme of HIV virus was confirmed in Avicennia marina leaves. The organic extracts of Actinomycetes bacterial isolates were found active against West Nile Virus NS3 Protease. Here, promising starting point for the potential of drug discovery of plants and marine organism of Saudi Arabia.

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The yeast Saccharomyces cerevisiae is a major chemical production platform in the biotechnological industry. It is also increasingly being used as a whole cell biosensor. One method of developing such whole cell biosensors in yeast is by exploiting its mating pathway, which is normally induced by secreted pheromones leading to downstream expression of various genes. Functional expression of different recognition elements or receptors and their coupling to the yeast mating pathway can enable sensing of a variety of ligands. In this work, we have engineered a yeast strain to functionally express a heterologous human olfactory receptor gene which can be coupled to the pheromone signaling pathway, allowing yeast to detect medium chain length fatty acids, alcohols and aldehydes for the first time. Functionally expressing heterologous olfactory receptors in yeast is a challenging task because no definitive method exists on how to express such receptors on the yeast cell surface and couple them to the downstream signaling pathway. We explore in this work how the yeast cell can selectively respond to two activating ligands via two different receptors. We also demonstrate in this work that a synthetic transcription factor can substitute for the native transcription factor in the yeast mating pathway. We believe our biosensor will not only have various uses as a versatile sensor but also aid in the design of synthetic genetic circuits.

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Chemical Characterization of Pseudognaphalium obtusifolium by Gas Chromatography – Mass Spectrometry (GC-MS) to Assess Potential Therapeutic Phytochemicals and Toxicological Concerns Using Simulated Use Conditions By Regina Ballentine Virginia Commonwealth University, 2019 Director: Sarah C. Rutan, Professor, Department of Chemistry Currently, there is an increasing demand for natural therapies and herbal products to treat various ailments. It is generally believed that natural therapies have fewer side-effects than traditional western medicine; however, they are often used in different strengths and formulations without consistency of the levels of target compounds or knowledge about toxicity. Due to this growing trend, a comprehensive chemical evaluation of plants used for medicinal purposes is necessary. Pseudognaphalium obtusifolium is a plant that has been used historically by Native Americans as an herbal medicine. It is a flowering plant belonging to the Asteraceae family indigenous to the Eastern United States. There are documented accounts of the Native Americans using the herb therapeutically. Reportedly, they used the plant to prepare tea and as filler for bedding. Additionally, they smoked the plant material. To date, there has been little research published on the chemical composition of this plant. Thus, the objective of this work was to conduct a chemical survey of P. obtusifolium using methodologies that would simulate the three historical routes of administration (tea, bedding material, and smoke inhalation). To determine the types of compounds that may be found in the plant, initial experiments using pressurized solvent extraction (PSE) with an ethanolic solvent were performed followed by analysis using gas chromatography – mass spectrometry (GC-MS) in scan mode. This extraction technique enabled a broad range of compounds to be identified. For the analysis of the tea, the leaves and the flowers were ground and analyzed separately. The “tea” simulation was then performed using a water extraction which was then back extracted into dichloromethane for GC-MS analysis in Selected Ion Monitoring (SIM) mode. Seventeen target compounds (terpenes, terpinoids, flavanoids, etc.) were quantified using this method. A bedding material simulation was performed using headspace solid phase micro-extraction (HS-SPME) to collect the volatile and/or semi-volatile components of the headspace. The compounds collected on the SPME fiber were then analyzed by GC-MS in scan and SIM modes to qualitatively and quantitatively determine the types of chemical compounds (most of which were terpenes) that may be off-gassed from bedding material. This analysis compared levels of compounds in two different crop years and four terpene compounds were quantified. To simulate smoking of the plant material, the leaves and flowers were fashioned into smoking articles. Sample collection was performed by a smoking machine and smoke condensate was collected. The smoke condensate was then analyzed by GC-MS in scan mode. As combustion and pyrolysis of plant material are known to produce toxic products, specific potentially harmful compounds were investigated and quantified. This chemical analysis of P. obtusifolium identified target compounds that can be found in the three simulated usage forms. Identification of these compounds gives insight on why the Native Americans may have used P. obtusifolium as an herbal medicine. Among the detected compounds, there were many unknowns. Elucidating these unknown compounds will be important in the effort to understand the full chemical profile of this plant.

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Separation technologies have occupied a central role in the current practices of analytical methods used for drug analysis today. As the emphasis in contemporary drug analysis shifts towards ultra-trace concentrations, the contribution from unwanted matrix interferences takes on greater significance. In order to single out a trace substance with confidence from a rapidly expanding list of drug compounds (and their metabolites) in real complex specimens, analytical technologies must evolve to keep up with such trends. Today, the task of unambiguous identification in forensic toxicology still relies heavily upon chromatographic methods based on mass spectrometric detection, in particular GC-MS in electron ionisation (EI) mode. Although the combined informing power of (EI) GC-MS has served faithfully in a myriad of drug application studies to date, we may ask if (EI) GC-MS will remain competitive in meeting the impending needs of ultra-trace drug analysis in the fut ure? To what extent of reliability can sample clean-up strategies be used in ultra-trace analysis without risking the loss of important analytes of interest? The increasing use of tandem mass spectrometry with one-dimensional (1D) chromatographic techniques (e.g. GC-MS/MS) at its simplest, considers that single-column chromatographic analysis with mass spectrometry alone is not sufficient in providing unambiguous confirmation of the identity of any given peak, particularly when there are peak-overlap. Where the mass spectra of the individual overlapping peaks are highly similar, confounding interpretation of their identities may arise. By introducing an additional resolution element in the chromatographic domain of a 1D chromatographic system, the informing power of the analytical system can also be effectively raised by the boost in resolving power from two chromatographic elements. Thus this thesis sets out to address the analytical challenges of modern drug analysis through the application of high resolut ion comprehensive two-dimensional gas chromatography (GC„eGC) to a series of representative drug studies of relevance to forensic sciences.