Analytical & Biological Chemistry Research Facility - Masters by Research Theses
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Item Design and use of novel metal catalysts in organic synthesis(University College Cork, 2023) Fitzgerald, Deirbhile; Maguire, Anita; Collins, Stuart; Irish Research CouncilThis project focuses on the design and synthesis of novel dirhodium carboxylate catalysts, and their precursor ligands, for use as enantioselective catalysts in transformations of α-diazocarbonyl compounds. Chapter One focuses on the literature background over the past thirty years in transition metal-catalysed asymmetric transformations of α-diazocarbonyl compounds. This overview aims to give an insight into the use of both copper and rhodium-based catalysts in intramolecular and intermolecular C-H insertion reactions, cyclopropanation and desymmetrisation reactions. The evolution of dirhodium carboxylate design since the 1980s is explored. This summary provides insight into their use as enantioselective catalysts in asymmetric transformations of α-diazocarbonyl compounds, thus providing context for this work. Chapter Two describes the design and synthesis of novel, enantiopure dirhodium carboxylate complexes, drawing inspiration from the rhodium (S)-mandelate skeleton. Initially, two enantiopure rhodium carboxylates were synthesised, incorporating carboxylic acid ligands bearing a fenchyl chiral auxiliary. Synthesis of carboxylic acid ligands bearing a bulky adamantyloxy group or a tert-butoxy group in place of the fenchyl substituent was investigated to simplify the stereochemical features, with just one stereogenic centre in each ligand. Two target carboxylic acids were obtained in racemic form, one bearing an adamantyloxy group and the other bearing a tert-butoxy group adjacent to the carboxylic acid moiety. Resolution of these carboxylic acids was explored through formation of diastereomeric salts or enzymatic kinetic resolution; while enantioenrichment was observed, further work is required to obtain the ligands in an enantiopure form for catalyst preparation. Chapter Three describes the synthesis of both racemic mandelamide and enantiopure (S)-mandelamide. In addition, a pair of diastereomerically pure mandelamides with α-methylbenzylamine were synthesised and characterised. The mandelamides were prepared to enable exploration of their crystal landscape in collaboration with another research group in UCC. Chapter Four contains the full experimental details and spectroscopic and analytical characterisation of all compounds synthesised in this project, while details of chiral stationary phase HPLC analysis are included in Appendix 1.Item Interrogating regulated intramembrane proteolysis of IGF-1R, and its role in cell migration(University College Cork, 2023) O'Donoghue, Jordan Christopher; McCarthy, Justin V.; O'Connor, Rosemary; University College CorkThe IGF-1R has long been implicated in numerous malignant characteristics of cancers including enhanced proliferation, insensitivity to apoptosis, chemoresistance, increased migratory and invasive capacity, epithelial-to-mesenchymal transition, and metabolic augmentation. IGF-1R, much like other receptor tyrosine kinases, has been characterised as a substrate for gamma-secretase-mediated regulated intramembrane proteolysis (RIP). This proteolytic pathway begins with ectodomain shedding, mediated by sheddases, which produces a soluble fragment in the extracellular space. Following ectodomain shedding, a membrane anchored C-terminal fragment (CTF) is produced which is then cleaved by gamma-secretase at the internal juxtamembrane region to produce a soluble intracellular domain (ICD). Classically, RIP was thought to be a means by which cells would terminate receptor signalling, however contemporary research indicates a more nuanced role. It is now understood that the fragments generated through RIP can retain signalling of the full-form receptors and, in some instances, may acquire novel functionality. Although the RIP of IGF-1R has been outlined, the underlying dynamics of this pathway and the consequences of such remain to be elucidated. With this in mind, we endeavoured to identify the regulatory mechanisms underlying IGF-1R RIP and subsequently aimed to identify potential functions of the fragments produced. Firstly, we generated a GFP-tagged K1003 IGF-1R point mutant, termed a kinase dead mutant, which lacks the ability to catalyse trans-autophosphorylation of the IGF-1R kinase domains thus preventing downstream signal transduction. Trans-autophosphorylation is a critical step in the activation of receptor tyrosine kinases during which the kinase domain of one intracellular domain catalyses the phosphorylation of tyrosine residues within the other intracellular domain. With this tool, we validated the cleavage of IGF-1R and the production of its CTF via transient transfection of HEK293T cells with plasmids expressing either wild-type or K1003R kinase dead mutant forms of IGF-1R C-terminally tagged with GFP. We subsequently identified receptor kinase activation as a potential driver of IGF-1R RIP and determined that ligand stimulation significantly catalyses the accumulation of IGF-1R CTF. This was accomplished via transient transfection of HEK293T cells with plasmids expressing the aforementioned IGF-1R forms. Following this, we elucidated clathrin-mediated endocytosis as a probable regulatory pre-requisite step for IGF-1R RIP by gamma-secretase. Furthermore, our data indicates that ectodomain shedding of IGF-1R is likely catalysed in a metalloprotease dependent fashion, consistent with other receptor tyrosine kinases. We also confirmed that the CTF can undergo nuclear translocation in a clathrin-mediated endocytosis dependent manner, consistent with the translocation of the holoreceptor. Utilising wound healing assays, conducted in Hela cells, our data indicates that gamma-secretase inhibition by DAPT (a small molecule gamma-secretase inhibitor) alone does not antagonise IGF-1-induced cell migration. Similarly, the administration of Pitstop alone does not impinge upon IGF-1 induced cell migration. Interestingly, the administration of batimistat (a broad spectrum metalloprotease inhibitor) significantly suppresses cell migration in the presence of IGF-1. Collectively, our data provides a framework for the construction of the IGF-1R RIP cascade. Following ligand-binding it appears that IGF-1R undergoes metalloprotease-dependent ectodomain shedding leading to loss of the extracellular alpha-chains. The CTF, which remains in the plasma membrane, then undergoes clathrin-mediated endocytosis and packaging into early endosomes. Following internalisation, the CTF is cleaved by gamma-secretase to generate a soluble ICD. Our data also demonstrates that IGF-1R CTF undergoes nuclear translocation in a clathrin-mediated endocytosis dependent fashion. Lastly, data gathered from our wound healing assays indicates that IGF-1R ectodomain shedding is likely a required event for the efficient IGF-1-induced migration of cells, possibly through the proteolytic augmentation of focal adhesion complexes containing IGF-1R.Item Computational analysis and partialsynthesis of resolvin analogues(University College Cork, 2022-06-02) Daly, Kevin; O'Sullivan, Tim; Irish Research CouncilAn introduction to the role of resolvins in inflammation is outlined in Chapter 1. In addition, a literature survey of the structure-activity relationships of resolvins, protectins and maresins is also included. Chapter 2 briefly outlines the aims and objectives of this project. Chapter 3 focuses on the computational analysis of the outlined virtual library of Resolvin D2 analogues. The key physicochemical properties of each candidate are calculated and unsuitable candidates are eliminated using standard screening protocols. Chapter 4 describes the attempted synthesis of a key intermediate for the preparation of an aromatic resolvin analogue. This attempted synthesis encompasses a monosilylation of a diol, a selective oxidation of an alcohol to an aldehyde, a Wittig olefination and, finally, a comprehensive trial of acetal deprotection methods for a 1,2 dioxane acetal in the presence of a silyl ether. Chapter 5 summarises the overall findings of this project and outlines possible future avenues for exploration. The full computational data set and experimental procedures, including spectroscopic data, are detailed in Chapter 6.Item The crystal landscape and cocrystallization of primary aromatic sulfonamides(University College Cork, 2020-07) Downey, John D.; Lawrence, Simon; Science Foundation IrelandThis thesis is focused on primary benzenesulfonamides. They have been structurally characterised and analysed for common structural motifs present in the solid state. A systematic investigation of their cocrystallization with a range of neutral, zwitterionic and ionic coformers has been carried, with a strong focus on coformers that have the potential for sulfonamide drug development. Chapter One discusses and illustrates the literature for the solid-state and supramolecular chemistry of benzenesulfonamides; topics covered include non-covalent interactions, synthons, crystal engineering, cocrystallization, eutectic compositions, solvates, biochemistry of sulfa drugs and crystal polymorphism. Chapter Two delves into the crystal landscape of the sulfonamide functional group. In this chapter we critically assess several simple primary aromatic sulfonamides identifying the different motifs present in their crystalline structures. Chapter Three focuses on the design and cocrystallization of sulfonamides with a range of neutral, zwitterion and ionic coformers. Initial screening was complemented by the solid-state characterization of the products obtained. Chapter Four outlines the future work that could be implemented to advance the library of supramolecular synthons for the sulfonamide functional group, in addition to furthering the improvement of sulfonamide drug development whether by using cocrystallization, solvate formation or by eutectic compositions.