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Browsing Theses by Advisors "Abell, Andrew"
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Item Open Access Controlling Peptide Structure and Function(2020) Yeoh, Yuan Qi; Abell, Andrew; Yu, Jingxian; School of Physical Sciences : ChemistryThe function of a protein or peptide is governed by its unique secondary structure and intrinsic dynamic properties. Hence, an ability to control secondary structure and an understanding of its dynamic behavior would allow for the regulation of protein function. However, proteins exhibit conformational complexity, which impedes a thorough investigation of the relationship between structure and function. Thus, model peptides present as ideal candidates for this purpose. Research undertaken in this thesis aims to control the secondary structure and hence, peptide activity, in addition to probing the intrinsic dynamic properties corresponding to specific structural changes within peptides. This research deepens our understanding of the these fundamental behaviors potentially transcending to proteins, which may provide important insights into their functions.Chapter one provides an overall introduction to the importance of secondary structure in proteins/peptides, and the structural dynamic behavior that governs protein function. Various strategies are introduced to control the secondary structure and antibacterial activity of peptides, along with the single-molecule junction technique used to probe the structural dynamic properties of peptides. Chapter two presents the photopharmacological approach to regulate secondary structure, and hence, antibacterial activity of a series of gramicidin S peptide mimetics through the photochemical control of a component azobenzene photoswitch. Detailed 1H NMR spectroscopy and density functional theory (DFT) calculations were used to define the secondary structure in the cis and trans configurations of the peptides, and both isomers of all peptide mimetics were assayed against S. aureus. Notably, peptides 2a and 2b were found to exhibit a four-fold difference between the cis-enriched and trans-enriched photostationary states. This study revealed a clear relationship between well-defined secondary structure, amphiphilicity, and optimal antibacterial activity. Chapter three describes the design and synthesis of three short photoswitchable tetrapeptides based on a known synthetic antibacterial. Each peptide contains an azobenzene photoswitch incorporated into either the N-terminal side chain, C-terminal side chain, or the C-terminus, to allow reversible switching between the cis and trans configurations. The C-terminus azobenzene (3) exhibited the most potent antibacterial activity against S. aureus, with an MIC of 1 μg/mL. A net positive charge, hydrophobicity, position of the azobenzene, secondary structure, and amphiphilicity were all found to be crucial to antibacterial activity. Chapter four reports a prodrug based on a known antibacterial compound to target S. aureus and E. coli under reductive conditions. The prodrug was synthesized by masking the N-terminus and side chain amines of a component lysine residue as 4-nitrobenzyl carbamates, while activation of the prodrug was achieved by removing the protecting groups under conditions that mimic hypoxia. Antibacterial susceptibility assays confirmed the liberation of the active antibacterial agent from the inactive prodrug to kill S. aureus and E. coli. Chapter five investigates the structural dynamic properties of a single-peptide using the single-molecule junction technique characterized by electrical conductance. The nanodevice was fabricated using a photoswitchable peptide containing an azobenzene photoswitch to provide both a well-defined secondary structure, and an intrinsically disordered structure. Real-time conductance measurements revealed three distinct states for each isomer, with molecular dynamics simulations showing each state corresponds to a specific range of hydrogen bond lengths within the cis isomer, and specific dihedral angles in the trans isomer. This study provides previously undisclosed insights into the structural dynamic behavior of peptides, which may well be applicable to proteins.Item Open Access The Design, Synthesis, and Evaluation of a New Class of Antibiotic(2022) Stachura, Damian; Abell, Andrew; Avery, Thomas; School of Physics, Chemistry and Earth SciencesBiotin protein ligase (BPL) is a ubiquitous enzyme that catalyzes the conjugation of biotin and ATP to give biotinyl-5’-AMP 1.02, a key intermediate in the activation of biotin dependent enzymes that are crucial to the survival of all cells. Inhibition of this enzyme critically damages bacteria and thus presents an important strategy to develop a new class of antibiotic. Described in this thesis is the design, synthesis, and biological assay of potent inhibitors of Staphylococcus aureus biotin protein ligase (SaBPL) with a 1,2,3-triazole or sulfonamide based isostere. Chapter One highlights the structure and catalytic mechanism of the target enzyme SaBPL. A summary of the chemistry and in vitro biology of known 1,2,3-triazole-based and sulfonamidebased inhibitors, designed to mimic the natural intermediate biotinyl-5’-AMP 1.02, is also discussed. Chapter Two presents work on the development of a new N1-diphenylmethyl-triazole scaffold to replace the adenosine of biotinyl-5’-AMP 1.02. A series of N1-diphenylmethyl-triazole analogues were designed and prepared, with the guidance of in silico docking, to maximise interactions with the active site of SaBPL. This manifested in significant in vitro potency improvements over previous lead triazole-based inhibitors, where 12 and 13, with imidazole and aminopyridine groups respectively, being the most potent inhibitors reported to date for this chemotype (Ki = 6.01 ± 1.01 and 8.43 ± 0.73 nM). Triazole 12 also demonstrated the best antimicrobial activity reported to date for the triazole-based SaBPL inhibitors against S. aureus ATCC 49775, exhibiting a minimum inhibitory concentration (MIC) of 1 μg/mL. Chapter Three presents a series of potent N1-diphenylmethyl-triazole-based analogues targeted to exploit crucial hydrogen bond interactions within the adenine binding site of SaBPL. The inhibitory activity against SaBPL strongly correlated with the in silico docking, particularly analogues that were proposed to hydrogen bond to Asn212 and Ser128 within the adenine binding site. For example, the butanamide substituent of 14 was predicted by docking to hydrogen bond these key amino acids, which manifested in a Ki value of 10.2 ± 2.4 nM. Chapter Four describes the design, detailed characterisation by NMR, and biological assay of a new 1,2,3-triazole-based inhibitor design. Inclusion of a carbonyl at the C10 position of a benzyl-triazole-based SaBPL inhibitor (1.07) gave analogue 4.02, which was shown by docking to hydrogen bond Lys187 within the phosphate binding region. This presumed hydrogen bonding interaction reflected the enhanced Ki value exhibited by 4.02 against SaBPL, relative to triazole 1.07 (Ki = 0.12 ± 0.01 vs 0.25 ± 0.03 μM, respectively). Chapter Five is concerned with the optimisation of the acidity of central NH comprising the sulfonyl linker of the highly effective sulfonamide based SaBPL inhibitors. Acidity of the central NH has been proposed to influence SaBPL inhibitory activity, and based on this, a series of sulfonylurea and sulfonylcarbamate linked analogues were prepared. The relative central sulfonyl NH acidity of these compounds was assessed by 1H NMR, with inhibitor activity against SaBPL shown to correlate with the relative acidity of the central NH. In particular, the acidic sulfonylcarbamate analogue (5.76) exhibited an excellent Ki value of 10.3 ± 3.8 nM, whilst also demonstrating potent whole cell activity against S. aureus ATCC 49775 (MIC = 4 μg/mL). Chapter Six reports details of the biological protocols, docking method, and experimental synthetic procedures for compounds described throughout Chapters, Four, and Five.Item Open Access Development of New Antibiotics Based on Biotin Biology(2019) Lee, Kwang Jun; Abell, Andrew; George, Jonathan; School of Physical SciencesPARTIAL ABSTRACT: Biotin is an essential vitamin that is required for growth and pathogenicity of bacteria. Biotin protein ligase (BPL) catalyses the ordered reaction of biotin and ATP to give biotinyl-5̕´-MP, which then activates a number of biotin dependent enzymes that are critical to cell survival. Dethiobiotin synthase (DTBS) is the sole enzyme responsible for catalysing a key step in biotin biosynthesis, namely the carboxylation of 7,8-diaminopelargonic acid (DAPA), closing the ureido ring to form dethiobiotin in a reaction requiring a nucleotide triphosphate. Research undertaken in this thesis highlights strategies to selectively inhibit Staphylococcus aureus biotin protein ligase (SaBPL) using 1,2,3-triazole and sulfonamide isosteres to replace the phosphoroanhydride linker found in biotinyl-5´-AMP. In addition, this thesis discusses the design and development of inhibitors targeting Mycobacterium tuberculosis dethiobiotin synthase (MtbDTBS).Item Open Access Engineering of Light-Emitting Nanoporous Photonic Crystal Structures(2024) Gunenthiran, Satyathiran; Santos, Abel; Alwahabi, Zeyad; Abell, Andrew; School of Chemical EngineeringConventional laser systems have limitations that restrict their applications including large operating size, high costs and complexity in design. Thus, there is a widespread demand for alternative laser systems that can address these limitations for a range of technologies and applications. Current progress in laser technology is enabling the development of cost-competitive, miniaturised, and highly integrable systems harnessing a range of optical phenomena that have broad applicability across different disciplines, including energy, sensing, environment, security, and aerospace. In this context, this thesis presents the development of a palette of composite lasing platforms based on nanoporous anodic alumina photonic crystals (NAA–PCs). These nanoporous PC structures are engineered through electrochemical oxidation (anodisation) of aluminium and their porosity tailored to harness distinct light–matter interactions. The inner surface of the nanopores of NAA–PCs is functionalised with distinct light-emitting gain media. These include the fluorophore rhodamine B (RhoB) coupled with a surfactant (sodium dodecyl sulphate (SDS) and dendrimers (PAMAM). Chemical modification of NAA–PCs is performed through micellar solubilisation. NAA–PCs with functionalised light-emitting gain media were demonstrated to produce cavity-free lasing through the mechanisms of slow photons and random lasing under stimulated optical pumping conditions. The combinational effects of structure of NAA–PCs and gain media towards lasing performance were analysed in detail. This thesis demonstrated that: (i) NAA–PCs provide a highly versatile platform material to harness light–matter interactions for light-emitting applications; (ii) engineering the structure of NAA–PCs enables a highly controllable approach to tune lasing from a range of gain media; (iii) the gain medium critically determines the features of the lasing emission from NAA–PCs; (iv) the combination of NAA–PCs with precisely engineered gain media enables the realisation of innovative lasing platforms using slow photon and random lasing as lasing mechanisms. The studies completed in this thesis advance both fundamental understanding and applied knowledge on the lasing performance of light-emitting NAA–PCs with optimised structural and chemical properties. These advanced materials could potentially be integrated into fully functional and marketable lasing systems to address some of the challenges faced by conventional laser systems and improve their applications across various fields.Item Open Access Engineering the Iontronic Properties of Nanoporous Anodic Alumina and Its Applications(2025) Wang, Juan; Santos, Abel; Law, Cheryl Suwen; Abell, Andrew; School of Chemical EngineeringSolid-state electronics have experienced an unprecedented development in the past decades in terms of their computation capacity, from basic calculation to memorising, and their integration in smart devices to support the raise of artificial intelligence. However, the solid-state electronics have intrinsic constraints to satisfy the increasing requirements of these emerging technologies. Conventional solid-state electronics suffer from source to drain leakage, quantum effects, high driving operation voltage and Joule heat generation. In contrast, biological information transduction system based on ionic transmitters are much more energy efficient and versatile. Synthetic iontronic systems bridge solid-state electronics and biological structures, providing an alternative platform for generating and transmitting signals based on the control and distribution of ions when these flow through synthetic nanochannels. This thesis presents the development of iontronic systems based on nanoporous anodic alumina (NAA) by harnessing its intrinsic iontronic properties. The unique characteristics of this platform materials are finely tuned through structural engineering and chemical modification for specific applications, including sensing and energy generation. NAA is an ideal platform for iontronic systems due to its ease of surface functionalisation, tunable pore size, high pore density and excellent mechanical stability. More importantly, NAA exhibits an inherent ionic current rectifying effect due to its barrier oxide layer (BOL). The hemispherical BOL closing the bottom tips of the nanopores in blind-hole NAA membranes features a gradient distribution of vacancies, which is generated from the diffusion of electrolytic species during fabrication. The inhomogeneous distribution of vacancies across the BOL is responsible for the ionic current rectification properties of NAA, which can be exploited for applications such as sensing and blue energy generation. The four main outcomes from this thesis are: (i) tuning of intrinsic iontronic properties of NAA by tailoring the properties of its BOL through anodisation; (ii) unravelling the mechanisms of ionic rectification properties of NAA membranes with distinct BOL features through selective chemical etching; (iii) harnessing the intrinsic iontronic properties of NAA for ultrasensitive detection of heavy metal ions; and (iv) understanding of electroporation and use of the unique features of electroporated membranes for osmotic energy generation. The work completed in this thesis advances both fundamental understanding and applied knowledge on the intrinsic iontronic properties of NAA featuring optimised geometric and chemical properties. This provides new avenues for developing cutting-edge iontronic technologies based on NAA structures for applications across multiple disciplines such as energy conversion, sensing and nanofluidics.Item Open Access Fluorescent sensor development through surface functionalisation(2021) Capon, Patrick Keith; Abell, Andrew; Purdey, Malcolm; School of Physical SciencesThe research undertaken in this thesis is concerned with the attachment of fluorophores to both optical fibres and nanodiamonds to provide sensing devices with application to biological sensing. Fluorescent sensors are commonly used to detect biological analytes through a change in fluorescence, a concept introduced in Chapter 1. However, the successful application of fluorescent sensors to biological systems is inhibited by a number of challenges, including delivery of the fluorophore to the measurement site, photobleaching, and fluorophore cell permeability. Chapter 1 introduces optical fibres and nanodiamonds as solid supports for attachment of fluorophores to overcome these challenges. Chapter 2 presents a new fibre functionalisation architecture for dual imaging and sensing within a single optical fibre probe. The fluorescent pH sensor, 5(6)carboxy-SNARF2, was reacted with the N-terminus of a silk-binding peptide to form an amide bond and provide the peptide SBP-SNARF. A fluorescence polarisation assay showed this peptide to bind with a Kd of 36 μM when added to aqueous silk fibroin protein. Fibre probes were prepared by dip-coating the fibre tip into the silk and peptide mixture, which provided a uniform silk coating (determined by scanning electron microscopy) that was stable to repeated washing in water and did not affect the imaging light emitted. This allows concurrent optical coherence tomography (OCT) imaging and pH sensing, which was demonstrated in an in vitro fertilisation (IVF) setting. Specifically, the probe detected a change in pH of 0.04 in cumulus-oocyte complexes after metabolic stimulation with CoCl2 to produce lactic acid, with the distance between the probe tip and the oocyte monitored by simultaneous OCT acquisitions. Notably, OCT imaging of an ovary revealed the presence/absence of an oocyte within an ovarian follicle, an important first step toward improving IVF patient outcomes by limiting the number of follicle punctures required to collect oocytes. Collectively, these results demonstrate the utility of the new fibre coating system to enable simultaneous OCT imaging and sensing, which provides significant insight into complex biological systems. Chapter 3 reports the detection of AlexaFluor-532 tagged streptavidin through its binding to D-biotin, which was reacted with the N-terminus of a silk-binding peptide to form the complex SBP-Biotin. This complex then adheres to a silk-coated fibre tip to provide an optical fibre probe for streptavidin, which is stable to repeated washing and long-term exposure to water. The probes were prepared by two methods that either distribute the SBP-Biotin throughout the silk fibroin matrix, or isolate the SBP-Biotin to the exterior of the silk layer. Only probes with surface bound SBP-Biotin successfully detected streptavidin, with a fluorescence-based detection limit of 15 mg/mL. Atomic absorption spectroscopy revealed that silk coating formation was inhibited by a lithium concentration of 200 ppm, however reduction to less than 20 ppm by dialysis re-enabled fibre coating. Together, Chapters 2 and 3 demonstrate that judicious preparation of optical fibre probes provides an opportunity for a wide array of fibre-based sensors using the silk fibroin and silk-binding peptide-based fibre functionalisation architecture described herein. Chapter 4 explores functionalisation of carboxylic acid laden nanodiamonds (NDs) through amide bond formation. This was first probed using an analytical high pressure liquid chromatography system to quantify the amount of 4-fluorobenzylamine attached to NDs. However, it was found that 4-fluorobenzylamine did not bind to NDs. Next, 1H nuclear magnetic resonance spectroscopy was employed to determine that the amount of diamino-PEG molecules attached to the NDs varied between 0 and 0.2 mmol/g. This indicated an inconsistent yield from the amidation reactions, which was investigated in more detail across ten separate amidation reactions between 4-cyanobenzylamine and NDs. However, none of these reactions resulted in an observable loading of 4-cyanobenzylamine, and it was concluded that amide bond formation is not an effective strategy for ND functionalisation. Chapter 5 presents four carbon-binding peptides (1-DLC, 2-CN, 3-DF and 4-GF) for ND functionalisation. Retention of each peptide on NDs was assessed by colourimetric assay and their presence confirmed through infrared spectroscopy and thermogravimetric analysis. 1-DLC was found to be the most well-retained peptide, at 87% and 35% on detonation and high-pressure high-temperature NDs respectively. This interaction was determined to be predominantly electrostatic, while 2-CN bound through non-polar interactions. Both 1-DLC and 2-CN coatings improved the colloidal stability of detonation NDs in aqueous solution, however neither peptide affected the colloidal stability of high-pressure, high-temperature NDs. This study provides a new, highly adaptable approach to functionalise NDs using carbon-binding peptides. Chapter 6 presents the synthesis of 4-aminobutanenitrile, an important synthetic intermediate for neurological disorder therapeutics, including Parkinson’s and Alzheimer’s diseases. Preparation of 4-aminobutanenitrile by Co(II) catalysed reduction, or a one-pot Staudinger reduction, of 4-azidobutanenitrile was low yielding. The reported Staudinger reaction was investigated through 1H-NMR analysis to reveal formation of the iminophosphorane intermediate after 22 h at rt, and increasing the temperature to 40 °C promoted hydrolysis of this intermediate to the desired amine. The Staudinger reduction was performed using pyridine solvent in place of THF, with water added 3 h after reaction initiation. These conditions gave rise to 4-aminobutanenitrile in 69% yield and 94% purity (calculated by qHNMR) without chromatography. However, 4-aminobutanenitrile was found to be unstable at rt, and cyclised to 2-aminopyrroline over several days. This was circumvented by preparation of the hydrochloride salt, which was shown to be stable at rt. Hence, 4-aminobutanitrile is best stored as the corresponding hydrochloride salt.Item Open Access Harnessing Light–Matter Interactions in Nanoporous Photonic Crystal Structures for Gas Sensing(2025) Tran, Nhu Khoa; Santos, Abel; Abell, Andrew; Stanley, Nathan; Law, Cheryl Suwen; School of Chemical EngineeringAdvances in sensing technologies have revolutionised the way gases are detected and monitored, enabling the development of tools that cater to the growing need for precise, real-time data in various applications. Among these, optical gas sensors have emerged as a promising solution by offering high sensitivity, molecular-level selectivity, compact designs, and non-intrusive sensing capabilities, while also addressing key limitations of conventional sensors, including long-term signal drift, cross-sensitivity to non-target analytes, and the need for frequent calibration. Additionally, optical gas sensors developed based on nanoporous materials open avenues for integration into lab-on-a-chip devices, providing a transformative potential for miniaturised, portable gas sensing solutions. As the demand for cost-competitive, accurate, and highly-integrable sensing systems grows, the development of next-generation gas sensors is vital to meet the requirements of diverse applications, from large-scale industrial systems to portable personal monitoring devices. In this context, this thesis explores the development of optical gas sensors based on nanoporous anodic alumina (NAA), focusing on the implementation of NAA-based photonic crystals (NAA–PCs) and functional materials. The fabrication of these NAA-based optical sensors was achieved through the structural engineering of NAA–PCs using electrochemical oxidation (i.e., anodisation) of aluminium. The porosity of these structures was tailored using pulse-like anodisation strategies to optimise optical properties and efficiently harness distinct forms of light–matter interactions at the nanoscale. The sensing characteristics of NAA optical sensing systems, in terms of selectivity and sensitivity, were further engineered by functionalising these porous platforms with different functional molecules. Various optical sensing mechanisms—including refractive index shifts, resonance band modulation, and random lasing—were systematically investigated and demonstrated through real-time detection of volatile organic compounds (VOCs) and other gases and vapours. The ability of NAA optical sensors to discriminate between different gases and vapours was also investigated by analysing the dynamic optical signal responses associated with each gas or vapor. The work completed in this thesis demonstrated that: (i) NAA–PCs are versatile and compact platforms to harness distinct forms of light–matter interactions for gas sensing applications; (ii) engineering the structure and porosity of NAA–PCs via anodisation enables precise tuning of optical properties to optimise light–matter interactions, resulting in tailored optical signals and structures with unique multiple outputs; (iii) functionalisation of NAA–PCs with diverse sensing materials, such as quantum dots and analyte-specific chemical modifiers, enhances the selectivity through tailored interaction of the sensing platform with target gases; and (iv) the optical signals produced by NAA-based sensors can be analysed to identify the fingerprints of different gases based on their unique physical and chemical interactions with the sensing platform. The findings from this work establish a framework for the precise design of NAA-based optical sensors, advancing both the fundamental understanding and applied knowledge of the gas sensing performance of NAA–PCs with optimised geometrical, chemical, and optical properties. These advancements unlock the transformative potential of the developed optical gas sensing systems to evolve into fully functional and marketable analytical tools for real-world applications in environmental monitoring, industrial safety, and medical diagnostics.Item Open Access Harnessing P450 enzymes as biocatalysts for selective C-H bond hydroxylation(2018) Lee, Joel Hoong Zhang; Bell, Stephen; Abell, Andrew; School of Physical Sciences : ChemistryThe cytochrome P450 enzyme, CYP101B1 from Novosphingobhium aromaticivorans can catalyse the highly efficient and regioselective oxidation of norisoprenoids. However, it has lower affinity towards hydrophobic substrates. Site-saturation mutagenesis of its Histidine85 (H85) residue was carried out as the equivalent tyrosine residue in the sequence of the closely related P450cam enzyme (CYP101A1 from a Pseudomonas species) provides hydrophilic interactions with camphor. Mutagenesis of the H85 residue to a phenylalanine residue (H85F) has been reported to increase the enzyme's affnity towards small hydrophobic molecules. Eleven mutants that differ at position 85 were successfully produced and these were incorporated into two separate plasmid vectors for enzyme purification and whole-cell studies. Whole-cell oxidation of the CYP101B1 mutants with various substrates that include norisoprenoids, terpenoids, and hydrophobic molecules of varying sizes were carried out to screen if the mutants have activity towards these substrates. A number of the mutants such as H85V, H85S, H85G and H85I displayed increased product formation and altered product selectivity but most were not as effective as the WT or the H85F mutant in oxidising hydrophobic molecules. Purification of both the H85A and H85G mutants was also carried out and ferrous- CO assays showed they were functional P450 enzymes. In vitro studies with these two mutants demonstrated they have poorer binding affnity and oxidation activity towards phenylcyclohexane when compared to the WT enzyme and the H85F variant. A mutant library of rationally designed P450cam variants which have been created to increase the affinity and oxidation activity towards the monoterpene, α-pinene, was tested with other monoterpenoids and related compounds. The mutations in this library were incorporated at residues in the active site. The library was screened using a whole-cell system with five substrates of similar size and chemical functionality to camphor and pinene. These were fenchone, fenchyl acetate, isophorone, 1,8-cineole and 1,4-cineole. The screening of these mutants displayed higher selectivity for the oxidations of these substrates compared to the WT. The metabolites were produced in larger quantities for isolation and identification. Fenchone and fenchyl acetate oxidation by different mutants generated a mixture of the C5, C6 and C7 hydroxylation products with the exo face being preferred. Isophorone oxidation by the mutants F87W-Y96FL244A- V247L (WFAL) and F87W-Y96F-L244A (WFA) gave selective formation of (4R)-hydroxyisophorone. This product is valuable as a avouring agent. Several of the mutants also selectively oxidised 1,8- and 1,4-cineole at different C-H bonds. A desaturation product was observed at the isopropyl group during 1,4-cineole oxidation. Concurrent studies of isophorone and cineole oxidation by mutants of another enzyme (P450BM3) also gave selective transformation of both substrates with high yields in whole-cell turnover systems. The whole-cell oxidation of isophorone and cineole by these P450cam and P450BM3 mutants were compared to determine if oxidation activity of the two enzymes were similar. For the isophorone turnovers, the P450cam mutants resulted in greater product yields over the P450BM3 mutants, whereby most if not all substrate added was converted to product. In contrast, cineole oxidation by the P450BM3 mutants gave higher yields over the P450cam mutants. As well as oxidising norisoprenoids, CYP101B1 displayed efficient and selective oxidation of monoterpenoid acetates. P450BM3 mutants have also been developed to oxidise terpenes and norisoprenoids. A selection of avour and fragrance compounds with structures similar to norisoprenoids and monoterpenoid acetates were screened both in vitro and in vivo with CYP101B1 and P450BM3-A74G/F87V/L188Q (GVQ). CYP101B1 displayed selective oxidation towards norisoprenoids such as δ- and α- damascone. P450BM3-GVQ also showed selective oxidation for acetate compounds such as cuminyl acetate and verdyl acetate. Larger scale turnovers with these two P450 enzymes were carried out and the metabolites were identified. CYP101B1 oxidises norisoprenoids at the ring C3/C4 position and this was preserved in the oxidation of δ- and α-damascone. α-Damascone oxidation by CYP101B1 formed both C3 hydroxy diastereomers and a further oxidation product at the same position. δ-Damascone oxidation occurred across its ring C3/C4 alkene to form the epoxide. P450BM3-GVQ oxidised both cuminyl and verdyl acetate at regions distant from the acetate group. Cuminyl acetate oxidation by this mutant also generated a desaturation product at the isopropyl group.Item Open Access Inhibition of serine and cysteine proteases by peptidomimetic inhibitors(2017) Schumann, Nicholas; Abell, Andrew; George, Jonathan; School of Physical SciencesProteases are responsible for the hydrolysis of proteins and peptides and have been implicated in the development of various diseases. Herein describes the design and synthesis of reversible peptidomimetic inhibitors of serine and cysteine protease for the control of protease activity. Proteases recognise substrates’ secondary peptide structure which conforms to a saw-tooth arrangement of amino acids, known as an extended β-strand geometry. This property has led to the development of potent inhibitors which mimic this conformation. Chapter two discusses defining β-strand geometry in inhibitors, while maintaining key substituents necessary for recognition by protease hosts, by the replacement of N-terminal residues with heterocyclic constraints. The inclusion of a heterocycle both enforces backbone β-strand conformation while increasing inhibitor bioavailability and metabolic stability. A series of peptidomimetic inhibitors containing heterocycles, including pyrrole, furan, thiophene and pyridine were synthesised, and the associated inhibitory activities against a model serine protease, α-Chymotrypsin, determined in proteolytic assays. Of the series of heterocycles, pyrrole was determined to be the optimum heterocycle for inclusion in inhibitors of this class. Extension of this concept of constraint was investigated in the approach towards heterocycle-containing macrocycles constructed by ring-closing metathesis for the inhibition of cysteine Cathepsin proteases, where the introduction of a macrocyclic tether couples with the heterocyclic constraint to define the desired backbone β-strand geometry. Pyrrole-containing macrocycle 2.47 was constructed by ring-closing metathesis, but was found to be unstable to hydrogenation conditions. A similar pyridine-containing macrocycle 2.61 was successfully synthesised and hydrogenated, but was unable to be functionalised with appropriate C-terminal residues due to its poor solubility profile. Chapter three details the design and synthesis of a series of peptidomimetic inhibitors of the serine protease Hip1 which has been implicated with a host innate immune response pathway of Mycobacterium tuberculosis. Described is the synthesis of a library of tripeptides containing an electrophilic boronic ester at the C-terminus for reversible covalent attachment to the Hip1 active site and the associated inhibitory assay data. A lead compound, 3.23, which has exceptional potency of inhibition (low nM activity) was discovered, from which highly potent derivatives featuring a C-terminal aldehyde 3.44, α-keto heterocycle 3.49, and α-keto ester 3.58 were established. Further refinement of these inhibitors presents an opportunity for the development of therapeutics for the treatment of tuberculosis.Item Open Access Investigations into the Divergent, Biogenically Inspired Synthesis of Structurally Related Natural Products(2019) Markwell-Heys, Adrian Webster; Abell, Andrew; George, Jonathan; School of Physical Sciences : ChemistryNatural products are pivotal in the development of new pharmaceutical agents as they may possess novel mechanisms of actions, inherent to their structural framework. Such intriguing metabolites are of interest to various fields of research, such as pharmacology, biochemistry and total synthetic chemistry. Perhaps the most compelling synthetic method is found with the biomimetic approach, which in addition to affording the natural product of interest in an efficient manner, such strategies may also offer insight into a given metabolite’s biosynthetic origins. This thesis will detail our investigations into the biogenic origins of two structural related families of natural products via a biomimetic total synthetic approach. The total synthesis of the structurally related marine natural products from Aka coralliphaga, has been achieved via a biogenically inspired divergent approach. This divergent strategy detailed siphonodictyal B as the biogenic precursor to liphagal, corallidictyals A – D and siphonodictyals B1 – B3. We report the successful total synthesis and stereochemical reassignment of siphonodictyal B, in accordance with our proposal. Additionally, the total synthesis of liphagal and the corallidictyals A – D was achieved directly fromour confirmed reassigned configuration of siphonodictyal B. We propose these transformations of siphonodictyal B to liphagal and the corallidictyals, detailed within this work, are representative of biosynthetic reactions that occur within the host organism, Aka coralliphaga. Progress towards the biogenically inspired, total synthesis of virgatolide B has been made. Our method sought to afford virgatolide B via a hetero-Diels-Alder reaction between a Z-exocyclic enol ether dienophile and an o-QM, generated in situ from an analogue of pestaphthalide A. Synthesis of the key biogenic precursors, that would in our opinion be representative of those that may occur in nature, was been achieved. However, upon investigating various thermal and basic conditions, synthesis of virgatolide B could not be achieved. Despite our failed attempts at synthesising virgatolide B, we still assert that the virgatolides A – C are biosynthesised in nature via a divergent, [4 + 2] cycloaddition of an appropriate Z-exocyclic enol ether with an o-QM derived from either of the co-isolated pestaphthalides A or B.Item Open Access Mass Spectrometric Methods for the Analysis of Chemically Modified Proteins(2021) Stevens, Katherine Grace; Pukala, Tara; Abell, Andrew; Trim, Paul; School of Physical Sciences : ChemistryMass spectrometry has critical roles in analytical chemistry, biomedical research and the diagnosis and treatment of human disease. Its ability to unambiguously identify and quantify a diverse range of biomolecules, from small molecule metabolites to large intact protein complexes in complex biological matrices, has solidified this technique’s place in the clinical chemists’ laboratory. However, mass spectrometry’s wider adoption by biochemists and clinicians is hindered by the inherent volume and complexity of the data it can generate from a biological context, and the subsequent level of specialist equipment and knowledge required to interpret results in a reliable and meaningful way. It is therefore apparent that a potential solution to this issue is to develop new methodologies, which adapt simple biochemical techniques into mass spectrometry sample preparation workflows that can be performed by biochemists using standard laboratory equipment. This thesis begins by reviewing some recent advances in clinical mass spectrometry and the current limitations of this technology. In doing so, we establish the importance of hyphenated techniques, which merge the concept of immunoassays with mass spectrometric detection to enable its adoption in high-throughput clinical and biomedical research applications. Many of these new approaches are only possible because of the development of efficient biocompatible chemical reactions, which facilitate the covalent modification of antibodies to improve their analysis via mass spectrometry. The concept of chemical labelling with mass tags, or synthetic linkers that fragment during ionisation and/or other stages of gas-phase analysis, has been widely explored as a strategy for detecting large heterogeneous biomolecules, such as antibodies. Chapter 2 explores the design, synthesis and application of an ultra-violet-cleavable linker for labelling intact antibodies to simplify their detection via matrix-assisted laser desorption/ionisation mass spectrometry. The novel aspect of this project is the utilisation of copper-catalysed azide-alkyne cycloaddition, or copper click chemistry, to enable straightforward modification of the detected photodissociation product, thereby offering the potential for multiplexed analysis of biomolecules. Despite the established utility of the copper click reaction, there are several limitations for its application in biological contexts. We therefore sought to identify alternative routes for modifying mass tags. Chapter 3 explores the utilisation of Diels-Alder cycloaddition chemistry for synthesising mass-tagged biomolecules, which conveniently facilitate efficient gas-phase fragmentation, even with the absence of a photocleavable moiety. The ability to easily detect proteins within biological specimens is incredibly valuable for diagnostics; however, the aetiology of many diseases involves modifications to proteins that occur after biosynthesis, or post-translational modifications. Chapter 4 explores the application of bottom-up proteomics methods to identify amino acid modifications to equine heart myoglobin in response to a model for oxidative stress, and characterise the products of its labelling with a novel fluorophore. Whereas the ability to analyse primary protein sequences and post-translational modifications is undoubtedly valuable, the function of many proteins is closely linked to their three-dimensional higher order structures, including transient interactions with other biomolecules. Reagents known as chemical crosslinkers, can be utilised to stabilise interactions between nearby amino acids prior to bottom-up proteomic analysis, thereby retaining tertiary and quaternary protein structural information. Chapter 5 describes some of the recent efforts from our research group to develop efficient strategies for synthesising and applying chemical cross-linkers with improved features for the purification and identification of cross-linked proteins. This thesis therefore explores various potential implementations of mass spectrometry for investigating the dynamic and complex roles of proteins in the context of human disease, from altered expression levels to post-translational modifications and protein-protein interactions.Item Open Access Mass spectrometry-based structural insights into protein assemblies(2020) Sanders, Henry Michael; Pukala, Tara; Abell, Andrew; Roberts, Blaine; School of Physical Sciences : ChemistryComplications with protein homeostasis, genetic mutations or post-translational modifications can lead to deficits in the correct folding, and therefore function, of proteins. These misfolding events are often associated with protein aggregation via amorphous and fibrillar pathways. Protein fibrils have long been associated with a range of neurodegenerative diseases, such as Alzheimer’s and Parkinson’s diseases, through the formation of fibrillar plaques that are deposited in diseased regions of the brain. Current understanding of misfolding diseases point to low molecular weight oligomers en route to fibril formation, instead of fibrils themselves, as the pathological species. More specifically, the relationship between lipid membranes and misfolded oligomers plays a role fibril kinetics and may be a vital component of disease aetiology via membrane disruption or leakage by oligomeric pore formation. To date, there are no effective therapies associated with neurodegenerative misfolding diseases, despite great advances in our understandings. To treat diseases such as these we require a greater understand of the mechanisms at play. Traditional structure determination techniques such as X-Ray crystallography and nuclear magnetic resonance (NMR) spectroscopy are often not amenable to the study of misfolding proteins given their transient nature, therefore new structure determination techniques must be exploited. This thesis utilised and developed a combined biophysical approach, with a focus on mass spectrometry (MS), to better understand protein misfolding and aggregation. Protein quality control systems like chaperones combat misfolding but these can fail, leading to disease. The mechanisms of molecular chaperones depend in part on protein morphology and can interact differently with the same protein if it undergoes different structural stresses. In this work, ion-mobility mass spectrometry (IM-MS) was utilised to understand the protective mechanisms of a molecular chaperone, β-casein (β-CN), against the aggregation of α-lactalbumin (α-LA). α-LA is capable of both fibrillar and amorphous aggregation, both of which were inhibited by β-CN at substoichiometric concentrations. During amorphous aggregation, analytical size exclusion revealed α-LA formed stable, high molecular weight complex with β-CN. IM-MS coupled with collision-induced dissociation (CIU) described transient structural interactions wherein β-CN stabilised α-LA, as well as reduced conformational heterogeneity while in the gas-phase. Overall, this data demonstrates the practicality of biophysical techniques, particularly IM-MS with CIU, to explore the interactions between misfolding proteins and chaperones. α-LA is a powerful model for protein misfolding, but it is not associated with disease. Amyloid beta 40 (Aβ40) and α-synuclein (α-syn) proteins are implicated in Alzheimer’s and Parkinsons’s diseases, respectively. Here these intrinsically disordered proteins were examined to better understand their interaction with the cellular membrane and its effects on fibril inhibition by small molecules, EGCG and resveratrol. Large unilamellar vesicles (LUVs) were used to model the cellular membrane, and it was revealed that they both increased the rate of fibrillar aggregation and decreased the effectiveness of the known fibril inhibitors. Using oligomer specific immunoblotting and IM-MS it was shown that EGCG and resveratrol work through differing mechanisms, wherein resveratrol targeted the elongation phase of aggregation while EGCG targeted the nucleation phase. IM-MS showed EGCG to preference binding to more compact forms of monomeric Aβ40 and α-syn, while LUVs influenced conformational changes indicative of nucleation. These observations combined to form a more detailed mechanistic insight into the protein-lipid-inhibitor relationship and highlighted that current approaches toward drug design may be misguided if the effects of lipid membranes are not properly considered. The transient nature of misfolding proteins can make structural determination complicated using traditional high-resolution structure determination techniques like X-ray crystallography and NMR spectroscopy. While useful, native- and IM-MS cannot offer specific detail in terms of tertiary structure and subunit architecture. Cross-linking MS (XL-MS) has emerged as a valuable complementary tool for uncovering structural information from proteins. While cross-linkers are available commercially, the rapid emergence of new analytical strategies means specific features or combinations of features are not readily available, and the highly variable nature of proteins means the optimum cross-linking regent may be system specific. To remedy this and enable to a wide range of cross-linker chemistries, a modular synthetic protocol was designed to allow for the incorporation custom reactive groups, spacer-arms, enrichment motifs and other features. The protocol was used to develop a small library containing 8 unique cross-linkers, each containing different features. The reagents were optimised using model peptides to monitor efficiency, labile spacer-arm fragmentation and derivatisation with enrichment and dye motifs. The wide applicability and straightforward synthesis of the modular protocol aims to remedy the lacking diversity in available cross-linkers for broad uptake in XL-MS workflows. Overall, the work in this thesis aims to highlight and expand MS-based techniques for protein structure determination. The presented examples showcase that investigating transient proteins require adaptable techniques like native MS, IM-MS and XL-MS. The combination of these and other biophysical techniques provide a toolset capable of handling a great range of protein structures deemed too complicated for traditional structure determination techniques.Item Open Access Optimising photopharmacology for future clinical applications(2024) Palasis, Kathryn Angela; Abell, Andrew; McLaughlin, Robert; School of Physics, Chemistry and Earth Sciences : ChemistryThe research undertaken in this thesis is focused on addressing some of the key challenges associated with the field of photopharmacology, in order to help progress it toward a clinical application. The concept of a “magic bullet” for treatment of diseases is explored in Chapter 1, and photopharmacology is presented as a promising approach to the development of more selective drugs. The physical and chemical properties of the photoswitches azobenzene and spiropyran are discussed in detail, and the major challenges involved in developing photopharmaceuticals are identified. Two of the most significant challenges are explored by the work in this thesis; (1) maximising the difference in biological activity between isomeric states, and (2) delivering light deep inside the body for local activation of a photoswitchable drug. Chapter 2 investigates the effect of 3D structure of a photoswitchable drug on its ability to modulate biological affinity toward a target. Here, two different classes of photoswitchable drugs – small molecule and peptide-based – are designed and synthesised as inhibitors of the same protease: trypsin. The best small molecule inhibitor showed a 3.4-fold difference in biological activity between isomers, while the best peptidic inhibitor showed a difference of >5-fold. Molecular modelling and docking show this is likely due to a large change in the structural positioning of key binding residues in the peptides upon isomerisation, which is not as prominent in the small molecules. Therefore, this is an important consideration in the design of new photopharmaceuticals with a maximised difference in activity between isomers. Chapter 3 is focused on optimising the photostationary state in spiropyran derivatives upon irradiation, to aid development of more efficient photoswitches and hence improved photopharmaceuticals. Derivatives of the common spiropyran 6-nitro BIPS were synthesised, with functional groups of various electron donating/withdrawing character substituted at the 5-indoline position. Following UV irradiation, 1H NMR was used to determine the degree of ring opening for each analogue, switching from the spiropyran isomer to the protonated merocyanine isomer. The electronic effects of the substituent were found to correlate with the degree of ring opening, such that the compound with the most electron donating group at the 5-indoline position showed the greatest proportion of protonated merocyanine in the irradiated sample. These results can help to inform the development of photopharmaceuticals based on spiropyran, with an optimised photostationary state in the ring opened direction. Chapter 4 explores one of the most significant challenges facing the clinical progress of photopharmacology – delivering light efficiently, and safely, deep inside the body for local activation of photoswitchable drugs. Here, an optical fibre is presented as a potential tool to address this issue. Light delivered by an optical fibre is shown to activate an anticancer photoswitchable drug, which is characterised by its dose-response against colon cancer cell line HCT-116. The two-fold difference in activity between isomeric states demonstrates optical fibres are a feasible tool for light delivery to activate a photoswitchable anticancer drug, and hence could be used in a clinical setting for photoswitchable chemotherapy.Item Open Access Probing a Promiscuous Binding Pocket of the Proteasome(2019) Turner, Dion Joel Lammas; Abell, Andrew; Avery, Thomas; School of Physical SciencesThe proteasome is a multi-catalytic protease complex responsible for the degradation of unneeded, damaged or misfolded proteins. The proteasome is a validated target for the treatment of haematological malignancies such as multiple myeloma and mantle cell lymphoma, as demonstrated by the FDA approved proteasome inhibitors bortezomib, carfilzomib and ixazomib. These inhibitors, especially bortezomib, suffer from poor specificity and relatively high prevalence of resistance, therefore new inhibitors should be designed such that these characteristics improve. This thesis details probing of the relatively unexplored primed site binding channel of the β5 subunit of the proteasome with P2 extended proteasome inhibitors. This work indicates the primed site binding channel as a promiscuous target for interaction which may aid in increasing the specificity of proteasome inhibitors Chapter 1 introduces the structure and activity of the proteasome and its implications and relevance to human diseases. Inhibition of the proteasome by small molecule inhibitors is discussed, including the main classes, exemplary inhibitors, their mechanisms and applications. The primed site binding channel is then identified via examination of the crystal structure of the proteasome as a pocket which provides potential for new inhibitor-enzyme interactions. Chapter 2 details the design, synthesis and evaluation of inhibitors 2.01-2.04 which probe the extent of the promiscuity of the primed site binding channel. The collection of published inhibitors which are known to, or are likely to, occupy the primed binding sites demonstrate the primed site binding channel as promiscuous regarding the substituents it accepts. The P2 residue of bortezomib was identified as providing an access point to the primed binding sites. Imidazolyl and phenyl substituents were demonstrated to be accommodated by the primed site binding channel, with greater potency found for longer extensions into the pocket, or inhibitors with a phenyl substituent within the pocket. Chapter 3 describes further probing of the primed site binding channel with the azobenzene-containing proteasome inhibitor 3.01, which can be converted between cis- and trans-enriched isomeric states using light. The azobenzene substituent was placed at the P2 position of a bortezomib-inspired inhibitor and allowed probing of the primed binding sites with greater conformation predictability. Remarkably, despite significant change in conformation between the cis and trans isomers, there is little difference between the low nanomolar range potencies of the isomeric states. This further indicates the significant promiscuity of the primed site binding channel. Chapter 4 presents the evaluation of inhibitors 2.01-2.04 and the thermally adapted state of 3.01 alongside bortezomib against bovine α-chymotrypsin to examine the specificity of such inhibitors. Primed site-occupying inhibitors 2.01 and 2.04 demonstrate more than 2.5 times greater specificity towards the β5 subunit of the proteasome over α-chymotrypsin. This result indicates occupying the primed site binding channel as an effective strategy of improving proteasome inhibitor specificity, which may be critical in improving upon the currently available proteasomeItem Open Access Progress Towards the Biomimetic Total Synthesis of Meroterpenoid Natural Products(2021) Sassnink, Stefania Alessandra; George, Jonathan; Abell, Andrew; School of Physical Sciences : ChemistryThis thesis outlines synthetic efforts towards three different natural product families. Norascyronones A, B and C are complex polycyclic polyprenylated acylphloroglucinols (PPAPs) isolated from Hypericum ascyron in 2019. A total synthesis of norascyronone C was achieved in 8 steps from commercially available 3-ethoxy-2-cyclohexanone. With norascyronone C in hand, the biosynthetically-inspired radical cascade reaction to norascyronone A was explored. We discovered intriguing reactivity under radical oxidation conditions. A similar synthetic approach was taken in the efforts of synthesising a structurally related (not yet published) natural product. These synthetic efforts led to the synthesis of a precursor which we propose to be an “undiscovered natural product”. Investigation of the key radical cyclisation gave access to several structurally unique side products. The furaquinocin and neomarinone natural product family is a class of meroterpenoid natural products isolated from marine actinomycete bacteria. We proposed that the dihydrobenzofuran motif could be installed by aromatic Claisen rearrangement followed by intramolecular cyclisation of a geranyl or farnesyl side chain. A synthetic model system was developed to investigate this biomimetic theory. Several rearranged intermediates with fascinating structures have been made through three different synthetic routes. Furobinordentatin, furobiclausarin and claudimerines-A and -B are dimeric pyranocoumarin natural products isolated from Citrus plants. We proposed and investigated two possible pathways to access these dimeric natural products from simpler precursor molecules, nordentatin and clausarin. Nordentatin was successfully synthesised in 5 steps from commercially available phloroglucinol. With the synthetic precursor in hand, the oxidative dimerisation was attempted. However, even after an extensive screening of reaction conditions we were unable to access the dimeric natural products.Item Open Access Proteins as Therapeutic Targets in Ovulation, Cancer and Antibiotic Resistance(2022) Becker, Rouven; Abell, Andrew; Russell, Darryl; School of Physics, Chemistry and Earth SciencesThe research undertaken in this thesis presents studies on the design, synthesis and evaluation of potential inhibitors, aiming to achieve a therapeutic response in ovulation (Chapters 2 and 3), cancer and diet-induced diabetes (Chapter 4) and antibiotic resistance (Chapter 5). Proteins play an important role in every aspect of human life and as such, are ideal targets to interrupt disease progression, a concept introduced in Chapter 1. Furthermore, Chapter 1 presents three proteins with relevance in ovulation (N-cadherin), cancer and diabetes (mammalian target of rapamycin) and antibiotic resistance (D-Alanine-D-Alanine Ligase) as biological targets for the development of new therapeutic agents. Strategies to progress drug development towards the effective inhibition of these proteins are described, as well as techniques to evaluate the activity of potential inhibitors against such proteins in vitro and in vivo. Chapter 2 details the effect of known N-cadherin inhibitors CRS-066 and LCRF-0006, and analogues thereof, on endocrine response, and oocyte maturation in ovarian follicles and ovulation. Analysis of these inhibitors in xCELLigence real-time adhesion assays revealed CRS-066 inhibited the adhesive capacity of ovulating cumulus oocyte complexes (COCs) in low micromolar drug concentrations. In spheroid formation assays, using cells which overexpress extracellular N-cadherin, CRS-066 reduced formation of cell spheroids. Further analysis of CRS-066 and LCRF-0006 revealed an ability to disrupt cumulus expansion and oocyte meiotic maturation in vitro. Finally, in vivo evaluation in mouse models showed that CRS-066 effectively blocks ovulation. These combined results emphasise the importance of N-cadherin function in ovarian granulosa cell differentiation and ovulation, highlighting the potential of N-cadherin inhibitors for development of non-hormonal contraceptives. The further development of CRS-066 toward this application is discussed in Chapter 3. Chapter 3 presents the design and synthesis of a series of analogues of the known N-cadherin inhibitor CRS-066. The 4-aminopiperidine core was replaced with a selection of bioisoteric heterocycles to give 15 analogues, to investigate structure activity relationships and improve drug potency towards the inhibition of N-cadherin. Of these analogues, five displayed an ability to inhibit spheroid formation with similar potency (0.3-1.0 μM), to CRS-066 (0.3 μM) in N-cadherin-specific spheroid formation assays. Further biological evaluation revealed the ability of the 1,4-diazepane-containing analogue to completely inhibit COC expansion and cause 100% arrest of oocyte maturation at the germinal vesical stage at 1 μM concentration. Analysis of this most active analogue in xCELLigence real-time adhesion assays showed decreased N-cadherin-mediated adhesiveness of preovulatory COCs with an IC50 of 5.3 μM, equipotent to CRS-066 (IC50 = 4.0 μM). Collectively, these data identify 1,4-diazepane as an important structural motif for the development of N-cadherin inhibitors targeting ovulation. Strategies to achieve a therapeutic response in cancer and diabetes are addressed in Chapter 4. The presented research explores the use of bone-targeting conjugates to achieve tissuespecific inhibition of mTOR activity in osteoclasts and breast cancer cells, for the treatment of breast cancer-induced osteolysis and diabetes. The design and synthesis of two conjugates containing literature mTOR inhibitors (Everolimus and AZD8055) bound to bone-homing polyaspartic acid oligopeptides via a 3-maleimidopropionic acid linker is described. The active inhibitor was designed to be liberated from the inactive conjugate by esterase activity on the bone surface, to allow systemic administration without showing dose-limiting off-target effects. The initial inactivity of the intact Everolimus- (EVAC6) and AZD8055-conjugates (AZAC6), as well as the activity of the mTOR inhibitors after time-dependent liberation from the conjugate over 6 h, was demonstrated by Western blot analysis. AZAC6 was further analysed in cell proliferation assays, where the conjugate showed similar anti-proliferative activity as AZD8055 after 24 h. Evaluation of AZAC6 in mouse serum stability assays revealed 29% of the intact conjugate recovered from serum after 6 h, verifying sufficient conjugate stability to enable further in vivo investigation. An osteolytic breast cancer mouse model revealed increased activity of AZAC6 compared to AZD8055, however both the unmodified inhibitor and the conjugate failed to significantly reduce tumour burden and osteolytic bone damage over a 10- day treatment compared to vehicle-only control mice. Chapter 5 presents the structure-guided design, synthesis and evaluation of acylsulfamide bisubstrate inhibitors targeting the D-Ala-D-Ala ligase from Staphylococcus aureus (SaDdl), as potential new antibiotic agents. An X-ray co-crystal structure of ATP and D-Ala-D-Ala dipeptide bound to SaDdl is presented, to aid a structure-guided inhibitor design through molecular docking. A versatile 4-step synthesis gave 14 bisubstrate inhibitors. The most active compound showed moderate activity against SaDdl (70% remaining enzyme activity; IC50 = 1.7 mM), compared to the only FDA-approved Ddl inhibitor (D-cycloserin, 35% remaining enzyme activity; IC50 = 0.1 mM)). This paves the way for the development of linked bisubstrate SaDdl inhibitors with increased potency.Item Restricted Small Molecules Targeting Novel Mycobacterium tuberculosis Drug Targets(2023) Schumann, Nicholas; Abell, Andrew; Fallon, Thomas; School of Physics, Chemistry and Earth SciencesTuberculosis is the leading cause of fatality among all bacterial infections worldwide. The emergence of Mycobacterium tuberculosis (Mtb, the causative agent of tuberculosis) strains resistant to first and second line drugs is rapidly reducing the number of antibiotics available for clinical use, resulting in a desperate need for the discovery of new therapeutic agents. One important strategy is to target novel pathways for which there are no pre-existing resistance mechanisms. This thesis highlights strategies to selectively inhibit two novel Mtb drug targets: hydrolase important for pathogenicity 1 (Hip1), a serine protease critical for the virulence of the bacterium, and Mycobacterium tuberculosis dethiobiotin synthase (MtDTBS), a protein involved in the biosynthesis of biotin, a vitamin essential for the growth and pathogenicity of Mtb. Chapter One describes the structure and specificity of the target enzymes Hip1 and MtDTBS. Preliminary studies on the use of peptidomimetic α-keto esters as inhibitors of Hip1 and small molecule cyclopentyl fragments as binders of MtDTBS are also discussed. Chapter Two reports a library of compounds derived from the potent inhibitor 1.04 (Ki = 309 ± 15 pM) with reduced MW and or increased aqueous solubility. The reported library includes inhibitors with truncated sequences that contain either an N-terminal Cbz or pyrazine group. Additionally, a tripeptide derivative of 1.04 containing a 5-memebered lactam (a cyclic derivative of Gln) at P1 is also reported. The truncated inhibitors exhibit reduced inhibitory activity against Hip1 relative to 1.04, indicating that a tripeptide sequence is essential for potent inhibition, as evidenced by the tripeptide containing the Gln-lactam at P1 which showed comparable inhibitory potency (Ki = 126 ± 7 pM) relative to the parent compound 1.04. Chapter Three describes the structural characterisation of the lead tripeptide inhibitors containing either Leu (1.04) or Gln-Lac at P1, bound to Hip1, as determined by X-ray crystallography. Chapter Four focuses on the structure-guided chemical optimisation of the hit compound 2 (identified by in silico screening; KD = 3.4 ± 0.4 mM) targeting the DAPA binding site of MtDTBS. Attaching an acidic group to the pposition of the aromatic ring of the scaffold, as well as an additional carboxy group on the cyclopentyl ring, significantly improved binding affinity. Further optimisation led to tetrazole 7a as a particularly tight binder (KD = 57 ± 5 nM) and moderately potent inhibitor (Ki = 5 ± 1 μM) of MtDTBS. Chapter Five reports a new series of small molecule binders of MtDTBS derived from the lead compound 7a. Various tetrazoles were designed to investigate the role of charge delocalisation on binding to MtDTBS, while analogues containing related carboxylic acid bioisosteres were prepared to determine what other functionalities (aside from a tetrazole) at the p-position of the aromatic ring can improve binding. Methylated tetrazoles and the analogous benzyl tetrazole (of 7a) showed diminished binding affinity (KD = >100 μM) relative to 7a (KD = 57 ± 5 nM), while the deoxygenated derivative showed comparable activity (KD = 69 ± 4 nM), suggesting that delocalisation of a negative charge over the tetrazole and aryl groups of 7a is critical for tight binding to MtDTBS. Analogues containing bioisosteres displayed weak binding affinities (KD = ≥ 150 μM) to MtDTBS and this is attributed to the bioisosteres being too large to bind to the narrow terminus of the DAPA-pocket, as elucidated by X-ray crystallography. Analogues containing a phosphonate moiety designed to bind to the P-loop of MtDTBS were found to have weaker binding affinity relative to 7a (KD = 1.4 ± 0.03 and 1.4 ± 0.08), and this was deemed to be due to the bulky phosphonate ethyl esters binding away from the P-loop, as revealed by the co-crystal structure of one of these compounds bound to MtDTBS.Item Open Access A study on side chain linked peptides, toward the development of talin inhibitors using β3 integrin peptide analogues(2018) Keeling, Kelly Lee; Abell, Andrew; Wegener, Kate; George, Jonathan; School of Physical SciencesThis thesis discusses the design and production of peptides with side chain linkers that are intended to bind to the F3 domain of talin. The talin F3 domain was targeted as it is involved in the activation of integrin membrane proteins present in platelets. The over activation of these integrins can result in clotting within the blood vessels causing heart disease, however, current medication targeting integrin have negative side effects. The design and synthesis of short peptides based on the sequence of the β3 integrin tail that binds to the F3 domain of talin is presented. The binding affinity of peptides to the talin F3 domain was tested using NMR titrations to reveal the ideal location for the linker in the production of potential therapeutics which target integrin activation. Side chain linked peptides with high helical content have previously been shown to improve binding affinity. This drove investigation of side chain constrained peptides to increase their helical content, and thus, their binding affinity to talin F3 domain and cellular uptake. It is demonstrated that side chain linkers are effective in stabilising the helical structure of the short peptides. When incorporated in the β3 integrin sequence in specific locations, lactam linkers improved binding affinity of these peptides to the talin F3 domain. Additionally, all-hydrocarbon and triazole linkers enhanced the peptide’s cellular uptake when compared to the native peptide of this sequence. The position and type of side chain linkers were investigated. The result of which showed that the position of the linker had a significant impact on the binding affinity to talin. The lactam linker between residues in positions 725 and 729 created a peptide (7) with the highest binding affinity. The cell penetration of peptides with different linker types was tested using NIH 3T3 mouse cells, and HEK298 cells. A number of side chain linkers were tested with the triazole linker producing the most α-helical peptide, and the all-hydrocarbon linker producing peptides with the greatest cellular uptake.Item Open Access Synthetic, Biomimetic and Chemoenzymatic Studies of Meroterpenoid Natural Products(2020) Murray, Lauren Ashleigh Marie; George, Jonathan; Abell, Andrew; School of Physical Sciences : ChemistryMeroterpenoid natural products are interesting bioactive molecules produced by both terrestrial and marine organisms in nature. In Streptomyces bacteria, these natural products are derived from the polyketide 1,3,6,8-tetrahydroxynaphthalene (THN), giving a structurally complex group of antibiotic molecules. The biological activity and interesting scaffolds of these natural products has inspired many attempts at their chemical synthesis. This thesis will highlight how biosynthetic studies of these compounds has inspired their biomimetic and chemoenzymatic syntheses, gaining insight into their biosynthesis. The naphterpins and marinones are two families of naphthoquinone-based meroterpenoids, isolated form Streptomyces bacteria. Biosynthetic speculation inspired the first total syntheses of two members from the naphterpin and marinone families, 7-demethylnaphterpin and debromomarinone, mimicking the entire proposed biosynthetic pathway. In validation of this biogenetic hypothesis, proposed biosynthetic intermediates from these biomimetic total syntheses were used in chemoenzymatic studies. These studies stimulated the discovery of multiple enzymes responsible for interconverting several of the proposed biosynthetic intermediates within these natural product biosyntheses, therefore confirming the biosynthetic proposal. Following these biomimetic total syntheses, a concise and divergent strategy for the synthesis of more members from the naphterpin and marinone families was developed. This approach utilised a plethora of pericyclic reactions, enabling the efficient synthesis of six meroterpenoid natural products, in the absence of protecting group strategies. This synthetic approach facilitated the first total synthesis of naphterpin in five steps from 2,5-dimethoxyphenol, alongside similar syntheses of 7-demethylnaphterpin and debromomarinone. Late-stage oxidation and bromination reactions were also investigated, resulting in the first total syntheses of naphterpins B and C, and isomarinoneItem Open Access Targeting PCNA with Cell and Nuclear Permeable p21-derived Peptides(2021) Chav, Theresa; Abell, Andrew; Bruning, John; School of Physical Sciences : ChemistryThe Proliferating Cell Nuclear Antigen (PCNA) is a sliding clamp protein essential for DNA replication and repair and is upregulated in a large number of cancers. This work centres on targeting PCNA with peptides derived from a segment of the p21(WAF/CIP1) protein, termed p21(139-160) (¹³⁹GRKRRQTSMTDFYHSKRRLIFS¹⁶⁰), that is known to inhibit PCNA. A potential therapeutic must be cell permeable and translocate to the nucleus to reach PCNA, thus defining the aims of Chapter Two and Three. Furthermore, the p21(139-160) sequence was used to develop a peptide-based fluorescent sensor for PCNA which is discussed in Chapter Four. Previous work in our lab identified the truncated p21-derived peptide, p21(143-155) (¹⁴³RQTSMTDFYHSK¹⁵⁵), termed P1 as a lead inhibitor of PCNA due to its short length and high affinity for PCNA. However, P1 was found to be impermeable to breast cancer cells (T47D). In Chapter Two, peptides P2 (¹⁴⁰RKRRQTSMTDFYHSK¹⁵⁵), P3 (¹⁴³RQTSMTDFYHSKRR¹⁵⁷) and P4 (¹⁴⁰RKRRQTSMTDFYHSKRR¹⁵⁷) with additional residues from the longer and cell-permeable p21(139-160) were tagged with fluorescein and administered to breast cancer cells to determine if the added residues facilitate cell permeability. This revealed modest cell permeability of P2 and P4, whereas P3 showed no cell entry. P4 displayed the most intracellular accumulation, which indicated extension of the P1 sequence at both termini facilitated cell permeability. Chapter Three presents studies on conjugating Nuclear Localisation Sequence (NLS) peptides; Tat(48-57) (N1F), SV40(126-132) (N2F), cMyc(320-328), (N3F) and R6W3 (N4F) to P1 to provide a series of linear peptide conjugates termed P1b-N1F, P1b-N2F, P1b-N3F and P1b-N4F, respectively. P1b-N1F and P1b-N3F displayed modest cell permeability to breast cancer cells. P1b-N2F displayed cell and nuclear permeability, which suggests the N2F imparted both cell and nuclear entry. The NLS peptides were also conjugated to a macrocyclic bimane analogue of P1, P1c. This gave a series of macrocyclic bimane peptide conjugates P1c-N1F, P1c-N2F, P1c-N3F and P1c-N4F. The SV40(126-132) tagged macrocyclic peptide, P1c-N2F, showed nuclear entry. Additionally, the control peptide, P1bimF, which contains a bimane linker and fluorescein tag but no NLS peptide, was also nuclear permeable. In contrast, analogues of P1bimF and P1c-N2F, without the fluorescein tag (P1bim and P1c-N2), were only cell permeable, highlighting the effect the fluorescein tag has in altering nuclear uptake, in this instance. This work presents P1b-N2F, P1bimF and P1c-N2F as three nuclear permeable peptide leads towards the development of a viable pre-clinical anti-cancer therapeutic that targets PCNA. Chapter Four details the development of a p21-derived fluorescent sensor for PCNA. The solvatochromic fluorophore, 4-dimethylaminophthalimide (4-DMAP) was introduced at positions 147, 150 or 151 in a p21(141-155) (¹⁴¹KRRQTSMTDFYHSKR¹⁵⁵) scaffold to provide three fluorescent sensor peptides termed B1, B2 and B3, respectively. The 151-substituted peptide, B3, exhibited the largest fluorescence response in the presence of PCNA, with a 7.9-fold change. The binding affinity of all peptides for PCNA were determined by Surface Plasmon Reasonance (SPR) with only B3 binding specifically to PCNA with a KD of 1.28 µM. B1 and B2 largely interacted non-specifically with PCNA, suggesting insertion of the 4-DMAP fluorophore at 147 or 150 disrupts PCNA binding. This work demonstrates that incorporation of a solvatochromic fluorophore is most favourable at position 151 for a p21(141-155) scaffold, which facilitates effective PCNA binding and a resultant ‘turn on’ fluorescence response. B3 presents a promising lead for further development of a fluorescent PCNA sensor to measure levels of cell proliferation.