Chemistry - Masters by Research Theses

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Differential regulation of the phosphorylated and dephosphorylated forms of the ubiquitin-conjugating enzyme Ubc6e
(University College Cork, 2024) Burns, Stephanie; Fleming, John V (Eoin)
The ubiquitin proteasome system (UPS) is a key stress response employed by cells to remove damaged and misfolded proteins that accumulate in the endoplasmic reticulum (ER) during energy deprivation, hypoxia, or viral infection. Ubc6e is an ER-localised ubiquitin-conjugating enzyme that plays an important role in the proteasomal degradation of misfolded proteins and can be phosphorylated at serine residue 184. Recent studies have demonstrated that Ubc6e is co-localised with p62/sequestrosome, which is a cargo receptor that delivers ubiquitinated cargo to autophagosomes for degradation. To further investigate the possible role that Ubc6e may play in autophagy, we aimed to investigate any protein-protein interactions between p62 and Ubc6e and we also aimed to investigate the effect of autophagic-related proteins, p62 and ULK1 on Ubc6e. From our research, we saw that Ubc6e levels are reduced when co-expressed with proteins that promote autophagy. Specifically for the autophagy inducer ULK1, we saw differential regulation between the phosphorylated and dephosphorylated forms of Ubc6e, suggesting that the S184 phosphorylation may protect the protein from autophagic degradation. Degradation of the dephosphorylated form, on the other hand, did not depend on S184 phosphorylation, or the catalytic activity of Ubc6e. Neither was it dependent absolutely on ER localisation. Finally, although ULK1 co-expression did not appear to specifically alter the ER/ perinuclear localisation of Ubc6e, it was noted that amino-terminal tagging with GFP led to a ULK1-related localisation of Ubc6e to intracellular vesicles. Together our results suggest that Ubc6e does play a role in autophagy however it is not mediated by the direct interaction with p62.
Enhancing the potential of hydrolases in enantioselective synthesis
(University College Cork, 2024) Torley, Rachel; Maguire, Anita; Collins, Stuart; Synthesis and Solid State Pharmaceutical Centre; Thermo Fisher Scientific
This interdisciplinary research project focuses on the utilisation of a novel hydrolase of marine origin as a biocatalyst in asymmetric synthesis, bringing together synthetic organic chemistry and molecular biology. The primary objective of this research was to investigate the effect of histidine tags on the activity and selectivity of this biocatalyst. Chapter 1 summarises literature reports of the applications of biocatalysts in asymmetric processes and, specifically, the reported effects of histidine tags on enzyme activity. Chapter 2 describes the synthesis and use of ester substrates for biocatalysis screening against three variants of the novel marine esterase, esterase 26D. The enzyme variants included the wild-type, non-histidine tagged strain, and two histidine tagged enzymes (C-terminal and N-terminal tagged). Overall, the investigation showed that the histidine tag had no detectable effect on the selectivity or activity of the esterase 26D. An important outcome of the work was development of a standard procedure to be undertaken at the outset of future investigations of novel biocatalysts to definitively establish whether the histidine tag impacts on the enzyme activity. Chapter 3 details the expression of the novel esterase, esterase 26D, variants. The non-histidine tag variant was cloned into E. coli where it was subsequently expressed alongside the readily available histidine tagged counterparts. Additionally, variants of other enzymes, esterase 32C and 51C, were cloned and expressed to provide another set of enzymes that could be screened for activity in the future. Lastly, Chapter 4 contains the full experimental details and spectroscopic characterisation of the compounds synthesised in the work, the biocatalysis studies, along with the general methods employed for generating the desired enzymes.
Rhodium catalysed 1,4-addition of N-Boc-indol-3-ylboronic acid to α,β-unsaturated carbonyls
(University College Cork, 2024) O'Driscoll, Conor C.; McGlacken, Gerard; Irish Research Council for Science, Engineering and Technology
In recent years, work within the McGlacken group has focused on the asymmetric conjugate additions of indole boronic acids. The work presented in this thesis is concerned with the exploration of the asymmetric rhodium-catalysed conjugate addition of indol-3-ylboronic acids to α,β-unsaturated carbonyl compounds. Optimisation studies towards the asymmetric addition of N-Boc-indol-3-ylboronic acid to 2-cyclohexen-1-one are described herein. With the optimised reaction conditions, a variety of substrates was explored and the synthesis of a number of novel compounds is described. Modest yields of up to 39% were achieved, with excellent enantioselectivities of up to 96%.
Evaluation of dynamic light scattering as a technique for the routine monitoring of therapeutic proteins on stability
(University College Cork, 2023) O'Kennedy, Laura; Moore, Eric; Crowley; Lowney, Declan; Crowley, Stephen
Throughout the course of biotherapeutic development and manufacturing, the detection and quantification of protein aggregates and particles is critical to ensure patient safety. Size Exclusion Chromatography (SEC) is one of the most widely used techniques for the separation of proteins and allows for the detection and quantification of aggregates. In terms of aggregate detection at drug substance (DS) and drug product (DP) release, as well as for stability testing, the ICH guidelines reference SEC as an example. However, it is acknowledged that new analytical techniques are continuously being developed and can be used where appropriate. Dynamic Light Scattering (DLS) is one such technique, that has been described on several occasions as an early indicator of protein aggregation. Some benefits of DLS over other aggregation-monitoring techniques, is its ability to analyse proteins in their native environment, its fast analysis time, and its ability to detect a wide particle size range. Additionally, for high molecular weight species (HMWS) detection, DLS is an exceptionally sensitive technique. This sensitivity and level of detection are particularly important in the biopharmaceutical industry as the presence of even a small number of aggregates can significantly increase upon long-term storage. However, this sensitivity can also be a weakness, as scattering from traces of other large particulates can interfere with results, and overall, the quantification of results can be difficult. Consequently, the technique is typically used in a complementary fashion with more widely used techniques such as SEC. The capabilities and applications of DLS have been improved over time with the invention and incorporation of e.g. multi angle DLS (MADLS), extended size range analysis and diffusion interaction parameter (kD) analysis, into the one instrument and software. The aim of this research was to explore the possible benefits and limitations associated with DLS, in order to determine if the technique has the potential to be used more widely for testing protein therapeutics on stability. A range of biotherapeutics including monoclonal, bispecific and trispecific antibodies were tested, that varied in concentration from 2 mg/mL to 160 mg/mL. Multiple stressed samples were included in the test panel to explore the techniques’ ability to detect large particles and aggregates. The particle size and size distribution results were compared to those generated from SEC, to explore whether DLS could give additional insight into the molecules stability. It was found that the technique showed great sensitivity and, in some cases, detected an increase in large particles where SEC failed to do so. These results were then substantiated by looking at data from sub-visible particle analysis via light obscuration. The zetasizer instrument, used to measure DLS, can also perform various other measurements that are indicators of colloidal and/or thermal stability. These include electrophoretic light scattering (ELS), extended size range analysis, particle concentration, kD determination, osmotic second virial coefficient (B22) determination and molecular weight (Mw) measurements. These measurements were performed to determine their respective abilities to provide additional insight into therapeutic protein stability. For most of the measurements however, the results and trends weren’t as consistent and as reliable as the particle size and particle distribution measurements. Further evaluation of these measurements, protein concentration optimisation and additional knowledge of the sample properties would be needed to provide more accurate results. However, overall, DLS is seen to be a highly sensitive and fast analysis tool for the determination of particle size and can provide additional insight into molecule stability when used in conjunction with SEC.
Design and use of novel metal catalysts in organic synthesis
(University College Cork, 2023) Fitzgerald, Deirbhile; Maguire, Anita; Collins, Stuart; Irish Research Council
This 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.

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