Thesis
Studies on natural products : resistance modifying agents, antibacterials and structure elucidation
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- Awarding institution
- University of Strathclyde
- Date of award
- 2021
- Thesis identifier
- T16184
- Qualification Level
- Qualification Name
- Department, School or Faculty
- Abstract
- This thesis describes research starting in 1999 on three areas of natural product science, namely bacterial resistance modifying agents, antibacterials and structure elucidation of natural products. Plants produce an array of structurally-complex and diverse chemical scaffolds and whilst there is an expanding volume of published literature on structure elucidation, there remains a need to understand why these compounds are produced and how they function in terms of biological activity. That can only be properly realised by a full and determined attempt at structure elucidation. This is an important concept as molecular structure describes and precedes function. The chirality and functional group chemistry of natural products defines the way in which a compound specifically binds to a receptor, protein or drug target. My independent research career started with studies on the ability of plant extracts and phytochemicals to modulate the activity of antibiotics that are substrates for bacterial multidrug efflux. These investigations are described in the first section, “Natural Product Resistance Modifying Agents”. Studies were, in the first instance, simple assays to look at potentiation and synergy of extracts and pure phytochemicals to potentiate the activity of antibiotics against resistant bacteria. This research evolved to study efflux inhibition, where we learnt much from the collaborations with Professors Piddock (Birmingham), Kaatz (Wayne State) and Bhakta (Birkbeck). Latterly, we were inspired by the highly imaginative and creative work of Dr Paul Stapleton (UCL), to study the plasmid transfer inhibitory effects of natural products; the rationale being that plasmids carry antibiotic-resistance genes and virulence factors. Inhibition of transfer could result in a reduction in the spread of antibiotic resistance and a reduction in pathogenicity. The second section of this thesis describes antibacterial natural products that were evaluated against clinically-relevant species of bacteria, in the main Gram-positive organisms such as Staphylococcus aureus and its methicillin- (MRSA) and multidrug-resistant variants and Mycobacterium tuberculosis, the causative agent of tuberculosis, which still continues to affect millions of people globally and for which antibiotic resistance is considerable. The papers described in this section detail the extraction of the plant and the bioassay-guided isolation of the active compounds, which were then subjected to structure elucidation, using in the majority of cases, Nuclear Magnetic Resonance (NMR) spectroscopy, High-Resolution Mass Spectrometry, and Infrared and Ultraviolet-Visible Spectroscopy. Natural products from the acylphloroglucinol, terpenoid, polyacetylene, alkaloid and sulphide classes are well represented in these publications with some of these antibacterial natural products displaying minimum inhibitory concentrations (MIC) values of less than 1 mg/L against MRSA and Mycobacterium tuberculosis strains. These activity levels approach those of existing clinically used antibiotics and this highlights the value of plant natural products as a resource for antibacterial templates. Mechanistic studies have also been conducted on selected compounds, for example the natural products from Hypericum acmosepalum were found to inhibit ATP- dependent MurE ligase, a key enzyme involved in bacterial cell wall biosynthesis. Other examples included the main component of cinnamon (Cinnamomum zeylanicum), an ancient medicinal material cited in the Bible in Exodus, which has been used in antiquity as an anti-infective substance. The main compound from this medicinal material is trans-cinnamaldehyde, a simple phenylpropanoid which has been shown to inhibit Acetyl-CoA Carboxylase, a pivotal enzyme that catalyses the first committed step in fatty acid biosynthesis in all animals, plants and bacteria. In collaboration with the marine natural product chemist Professor Vassilios Roussis, we have also been able to characterise the antibacterial activities of marine plants, particularly compounds of the diterpene class that display promising levels of antibacterial activity against MRSA and S. aureus strains. Work on the antibacterial properties of Cannabis sativa showed that some of the main cannabinoids display excellent potency towards drug-resistant variants of S. aureus and support the ancient medicinal usage of Cannabis as an anti-infective and wound healing preparation. The acylphloroglucinol class of plant natural products are also noteworthy, particularly from Hypericum and Mediterranean medicinal plant species such as Myrtle (Myrtus communis), again with MIC values reaching 1 mg/L against pathogenic bacteria. We synthesised some of these acylphloroglucinols and made analogues and not surprisingly, were unable to improve the activity as nature really is the best chemist of all. The final section describes early and continuing research into the isolation and structure elucidation of natural products from plants and microbes. The rationale for this research is manifold: training for isolation to understand the medicinal use of a plant or microbe, chemotaxonomic investigations, the ecological relevance of phytochemicals in plants that are halophytic and xerophytic and in some cases just plain academic curiosity. These studies use classical phytochemical techniques to isolate and determine the structures of the species of investigation and where possible, absolute stereochemistry is undertaken. It should be noted however that isolation can be exceptionally challenging and frustrating. This can be due to the paucity of biomass, low concentrations of compounds, complexity of the resulting natural product mixtures and finally a lack of chemical stability of the products. All of these issues need to be faced before structure determination can even be attempted. A word of caution is therefore needed to the young natural product chemist embarking on their first isolation project. However, words of encouragement are also needed: the isolation of new, chemically complex and exquisitely biologically active molecules is a beautiful endeavour and exceptionally rewarding on many levels.
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