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Browsing Theses by Advisors "Abell, Andrew David"
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Item Open Access Biomimetic synthesis of meroterpenoid natural products using dearomatization strategies(2016) Pepper, Henry Patrick; George, Jonathan; Abell, Andrew David; School of Physical SciencesSynthetic efforts towards various meroterpenoid natural products based on biosynthetic speculation were undertaken in order to gain biosynthetic insight and to develop efficient syntheses of some structurally complex, biologically active compounds. The first total synthesis of the PPAP natural product garcibracteatone was achieved in four linear steps from phloroglucinol (0.6% overall yield). The key biomimetic synthetic step was an oxidative radical cyclization cascade reaction, where four new carbon-carbon bonds, four new carbocyclic rings and five new stereocentres were formed in the one step. The first total synthesis of merochlorin A was achieved in five linear steps from methyl-3,5- dimethoxyphenylacetate (6% overall yield). The key biomimetic synthetic step was a [5 + 2] cycloaddition reaction induced be oxidative dearomatization to form the bicyclo[3.2.1]octane core. The first total synthesis of the napyradiomycin natural product naphthomevalin was achieved in 11 steps from methyl-3,5-dimethoxyphenylacetate (1.4% overall yield). The key biomimetic synthetic step was a thermal α-ketol rearrangement reaction to form the naphthoquinone core of the napyradiomycins. The synthetic naphthomevalin was additionally converted into A80915G via a biomimetic Sɴ2 epoxidation reaction, and into napyradiomycin A1 via a chemoenzymatic reaction.Item Open Access Defining peptide structure with metathesis.(2013) Chua, Krystle Chia Hsien; Abell, Andrew David; Pietsch, Markus; School of Chemistry and PhysicsUnderstanding protein structure and function is central for the development of therapeutics for the treatment of diseases and also novel biocompatible materials. Herein describes studies on the control of peptide structure and function through synthetic modifications, for the synthesis of novel enzyme inhibitors and biomaterials, primarily using olefin metathesis chemistry. Metathesis is chosen for the manipulation of peptide structure in order to induce conformational constraint in novel macrocyclic peptidomimetic inhibitors and to develop novel hydrogel matrices, which are of importance in the advancement of the pharmaceutical and medical industries. The realization that enzymes bind their substrates in an extended β-stranded conformation has led to the development of inhibitors that mimic this bioactive conformation. The controlled organization of secondary structures in peptides by conformational constraint has been utilized to design two novel series of macrocyclic inhibitors, which are constrained by the P₁ and P₃ residues or the P₂ and P₄ residues using ring closing metathesis (RCM). These inhibitors contain a pyrrole group in the peptide backbone, thereby decreasing the peptidic nature of these inhibitors minimising susceptibility to proteolysis, while maintaining the appropriate geometry for inhibitor binding. The corresponding P₁-P₃ and P₁-P₄ acyclic inhibitors are designed and synthesized to provide an insight into the importance of cyclisation on the potency of inhibition against serine and cysteine protease. The macrocyclic and acyclic inhibitors synthesized are assayed against a series of cysteine (calpain and cathepsin) and serine proteases (α-chymotrypsin, human leukocyte elastase and trypsin). These enzyme assays analyse the efficacy of the inhibitors against the enzymes tested. The potency of the inhibitors against the aforementioned proteases provides an insight into the effect of cyclisation, ring size and introduction of aryl groups into the ring system, as well as trends in selectivity between proteases of the same family (calpain vs. cathepsin and α-chymotrypsin vs. HLE and trypsin) and between the cysteine and serine protease families. The ability to mimic the natural environment of structural proteins in wound healing, has led to the development of biocompatible materials, such as hydrogels, through the manipulation of natural peptide structure. The controlled organization of the tertiary structure of naturally occurring peptides is investigated by aqueous metathesis in the synthesis of biocompatible hydrogels derived from gelatin. Novel gelatin-gels are obtained by reacting methacrylate-functionalized gelatin and norbornene dicarboxylic acid in the presence of a catalyst in aqueous media. Optimisation of the hydrogel formation is investigated by; i) varying catalyst utilised and ii) varying ratios of starting gelatin and norbornene dicarboxylic acid. These polymer gels exhibited physical and chemical properties that might be useful in regenerative medicine. Mechanistic studies using MALDI is also performed to provide an insight into the mode of hydrogel formation.Item Open Access Design and synthesis and testing of conformationally constrained peptidomimetics.(2013) Duncan, Joanna Kimberley; Abell, Andrew David; School of Chemistry and PhysicsThis thesis describes the design, synthesis and testing of peptidomimetics pre-organised into bioactive conformations. Chapter One introduces the concept of peptidomimetics, their importance as potential pharmaceuticals. The concept of constraining a compound into a bioactive conformation (α-helix, β-turn or β-strand) by incorporation of a ring or bridge is discussed. The technique of ring closing metathesis as a strategy for cyclisation of peptidomimetics is introduced. Chapter Two surveys β-turn mimics comprised of β-amino acids. The synthesis of novel cyclic peptidomimetics comprised of β-amino acids (cyclised by ring closing metathesis) is presented. Three of the cyclic dipeptides were predicted (through in silico conformational searches) to adopt a β-turn motif. Cyclic scaffolds 2.62, 2.65 and 2.66 were each incorporated into a tri-peptide to give 2.68, 2.69 and 2.70. The propensity of each tri-peptide to adopt a β-turn motif was investigated by 1H NMR. There is strong evidence that 2.70 has a β-turn geometry based on the presence of an intra-molecular hydrogen bond between the i and i+3 residues. Chapter Three introduces cysteine protease calpain II as the primary biological target for this thesis. Calpain is implicated in cataract formation and its inhibition is a logical approach to cataract prevention. Proteases are known to, almost universally, bind substrates and inhibitors in a β-strand conformation. Four macrocycles, designed to be preorganised in a β-strand geometry, were synthesised by ring closing metathesis (compounds 3.02 – 3.05). Macrocycle 3.02 was made to investigate the suitability of an N-terminal 4-fluoro-benzyl-sulfonyl (FBS) in macrocyclic calpain inhibitors. The synthesis of 3.02 was optimised to give the required compound in 33% yield compared to a reported 1% for analogue CAT0811. Diols 3.03 and 3.04 (as a mixture with 3.03 in an 85:15 ratio) were designed to explore possible hydrophilic interactions with the active site of calpain. Macrocycle 3.05 was designed to investigate the relative importance of having an aromatic residue at P₁ for inhibition of calpain, α-chymotrypsin and the 20S proteasome. Chapter Four reports the in vitro testing of macrocycles 3.02, 3.03 and 3.04 against calpain II and discusses these results in the context of the SAR study completed by the Abell group to identify the criteria for the most potent macrocyclic calpain inhibitor. CAT0811 was confirmed as the most potent macrocyclic calpain II inhibitor to date with an IC₅₀ of 0.03 μM. Macrocycle 3.02 had an IC₅₀ of 0.045 μM against calpain II, confirming the suitability of FBS as an N-terminus in these macrocycles. Macrocycle 3.03 had an IC₅₀ of 3.7 μM against calpain II, suggesting a diol substituent is not tolerated by the enzyme at P₁. Diol 3.04 (as a mixture with 3.03 in an 85:15 ratio) was essentially inactive against calpain II (IC50 > 50 μM), presumably as 3.04 has a low propensity to adopt a β-strand conformation. Macrocycle 3.05 had an IC₅₀ of 0.15 μM against calpain II, a Ki of 686 μM against α-chymotrypsin and an IC₅₀ of 1.46 μM against the chymotrypsin-like sub-site of the 20S proteasome. CAT0811 was inactive against α-chymotrypsin and had an IC₅₀ of 1.51 μM against the chymotrypsin-like sub-site of the 20S proteasome. While modification to the P₁ and P₃ positions moderately influenced the selectivity of the macrocycles (comparing 3.05 with CAT0811), a much more dramatic affect was gain by modification of the P₂ residue (as in 4.02). Chapter Five reports the synthesis and in vivo testing of tritiated analogues of CAT0811 and 4.04 by reduction of CAT0811 with NaBT₄ to give macrocycle 5.02, and subsequent oxidation of 5.02 to give 5.03. Compounds 5.02 and 5.03 were separately formulated and administered to sheep from the cataract flock. Liquid scintillation counting was used to get a preliminary outlook on the absorption, distribution and excretion of the macrocycles and to investigate the phenomenon of les crossover of the inhibitors. Previous in vivo trials of CAT0811 have reported that when the formulated inhibitor is administered to the left lens, both lenses are equally observed to have slowing of cataract progression (p < 0.05). Levels of tritium in the treated and untreated lenses were measured. Equal amounts of 5.02 were found in both lenses 48 h after application. This supports our hypothesis that lens crossover of the macrocycles is occurring.Item Open Access Design and synthesis of protein chemical crosslinkers: a modular approach(2018) Downey, Kayla Monique; Pukala, Tara Louise; Abell, Andrew David; School of Physical SciencesThe study of protein structure and interactions is pivotal in understanding the function and malfunction of complex biological systems. The structures of some proteins are unable to be determined using traditional high resolution biophysical techniques, requiring the development of amenable low resolution alternatives. Chemical Crosslinking Mass Spectrometry (CXMS) is one technique which can be used to probe protein structure through the formation of covalent linkages between protein residues. The formation of these links is facilitated by chemical crosslinking reagents. Widespread use of the CXMS technique has been hampered primarily by analytical challenges pertaining to the detection and identification of crosslinked species using Mass Spectrometry (MS). Attempts to mitigate the challenges have been made by modifying the structure of chemical crosslinkers through the addition of functional groups such as affinity tags, isotope labels and cleavable bonds. Crosslinkers combining more than one type of functional group (combination crosslinkers) present the most promising targets for CXMS applications, combining the benefits of each functional group. However, combination crosslinkers are not commercially available, thus necessitating in-house synthesis. Incorporating more than one functionality also results in more complex molecular structures and synthetic processes, making the crosslinkers difficult to adapt to suit a particular experiment. Consequently, the use of combination crosslinkers has been limited to date to a small number of studies. The research presented in this thesis describes the development of a modular chemical crosslinker design and corresponding synthetic protocol for the synthesis of combination crosslinkers. The modular crosslinker structure can be readily modified to include a range of functional groups using a small number of different reactions, including amide coupling and O-alkylation, and commercially available starting materials such as Boc-serine, from a minimum of five synthetic steps. The utility of the synthetic process was validated through the synthesis of a crosslinker containing an alkyne functional group, which can be used to attach a biotin affinity tag through alkyne-azide Huisgen Cyclisation. Synthesis of two custom designed combination crosslinkers utilising alkyne tags and cleavable bonds is also described. The function of the cleavable bonds was established using collision induced dissociation processes within the mass spectrometer. Ensuring that a crosslinker is effective in probing quaternary structure and protein- protein interactions is essential as the investigation of these structures is a major goal of CXMS. Therefore, a crosslinking assay using Staphylococcus aureus biotin protein ligase, which forms homodimers when substrate bound, was also developed using the commercially available crosslinkers Disuccinimidyl Suberate (DSS) and Dithiobis(succinimidyl) Propionate (DSP), to enable the efficacy of crosslinkers synthesised using the modular synthetic protocol to be determined.Item Open Access Design and synthesis of reaction intermediate derivatives as biotin protein ligase inhibitors.(2012) Tieu, William; Abell, Andrew David; Booker, Grant William; School of Chemistry and PhysicsThis thesis reports the development of selective and potent small molecule inhibitors of Staphylococcus aureus biotin protein ligase (SaBPL) using 1,2,3-triazole and phosphodiester linkers as bioisosteric analogues of the phosphoroanhydride linker found in the reaction intermediate biotinyl-5’-AMP 1.03. Chapter one describes the structure and catalytic mechanism of the essential enzyme SaBPL. An overview of reaction intermediate mimics as ligase inhibitors is discussed and the utility of 1,2,3-triazole ring as a bioisosteric analogue is outlined. Chapter two investigates the phosphodiester reaction intermediate mimic biotinol-5’-AMP 1.05 as a potential inhibitor of SaBPL. Two different synthetic approaches towards biotinol-5’-AMP 1.05 were developed with the aim of scaling up the synthesis to enable biological characterisation and animal trials. Assay results indicated biotinol-5’-AMP 1.05 is a potent but a non-selective inhibitor of SaBPL (IC₅₀ = 0.12 ± 0.01 μM). Chapter three investigates the use of 1,2,3-triazole as a bioisostere of the phosphoroanhydride linker of the reaction intermediate biotinyl-5’-AMP 1.03. Both 1,4- triazole 3.25 and 1,5-triazole 3.33 were synthesized from biotin alkyne 3.12 and adenosine azide 3.16 using CuAAC and RuAAC. Optimisation of both CuAAC and RuAAC in the synthesis of 3.25 and 3.33 were also investigated. 1,4-Triazole 3.25 is the first reported selective inhibitor of BPL, inhibiting SaBPL (Ki = 1.83 ± 0.33 μM). Chapter four extends the work described in chapter three with an investigation of 1,2,3- triazole analogues based on triazole 3.25. Structure-activity relationships were developed and a general structure for this novel class of inhibitors was obtained. Triazole 4.01, the lead compound from this class of inhibitors, is a potent and selective inhibitor of SaBPL (Ki = 0.66 ± 0.15 μM). X-ray crystal structure of 4.01 bound to SaBPL illustrated the effective molecular recognition between the 1,2,3-triazole ring and SaBPL and emphasized the 1,2,3-triazole ring as an effective bioisostere of phosphoroanhydride linker. Additionally, a successful in situ click experiment was performed using a library of alkynes/azides fragments and R122G SaBPL mutant enzyme. The mutant enzyme was able to select the appropriate fragments and selectively synthesize the potent 1,4-triazole inhibitor 4.01. Chapter five examines analogues of biotin alkyne 3.12, a precursor to 1,2,3-triazole inhibitors and was found to be a potent inhibitor (SaBPL Ki = 0.30 ± 0.05 μM). Norbiotin alkyne 4.16 was found as highly effective inhibitor against SaBPL (Ki = 0.08 ± 0.01 μM) and an antibacterial agent against methicillin resistant staphylococcus aureus (MIC = 4 - 16 μg/ml). Chapter six extends the work described in chapter four. Using the general structure developed in chapter four, a series of analogues with modifications to the ATP binding component were synthesized and assayed against a SaBPL. Triazole 6.10 containing the privileged scaffold, 2-benzoxazolone, was found as a potent and selective inhibitor against SaBPL (Ki = 0.09 ± 0.02 μM). Chapter seven details the experimental procedures used to synthesize compounds described in chapter 2 – 6.Item Open Access Development of biotin protein ligase inhibitors from Staphylococcus aureus as new antibiotics(2016) Feng, Jiage; Abell, Andrew David; Tieu, William; Booker, Grant William; School of Physical SciencesBiotin protein ligase (BPL) catalyses the ordered reaction of biotin and ATP to give biotinyl-5’-AMP 1.03, which then activates a number of biotin dependent enzymes that are critical to cell survival. Research undertaken in this thesis highlights strategies to selectively inhibit Staphylococcus aureus biotin protein ligase (SaBPL) over the mammalian equivalent using 1,2,3-triazole and acylsulfonamide isosteres to replace the phosphoroanhydride linker found in biotinyl-5’-AMP 1.03. Chapter one describes the structure and catalytic mechanism of the target enzyme SaBPL, along with an overview of chemical analogues of biotin and biotinyl-5’-AMP 1.03 as BPL inhibitors reported to date. Preliminary studies on the utility of a 1,2,3-triazole as a bioisostere of the phosphoroanhydride linker of biotinyl-5’-AMP 1.03 are also discussed. Chapter two further examines 1,2,3-triazole analogues of lead SaBPL bisubstrate inhibitors 1.22 and 1.23. Specific chemical modifications were carried out at the ribose of biotinyl-5’- AMP 1.03, and a new class of purine analogues was developed to mimic the adenine group as in 1.03. In silico docking experiments using our x-ray structure of SaBPL aided in the design of these analogues by predicting optimal binding conformations. A structure activity relationship for the ribose and adenine mimics was developed and this revealed limited improvement in potency against SaBPL on modification at these two sites. Chapter three reports the first examples of truncated 1,2,3-triazole based BPL inhibitors with a 1-benzyl substituent designed to interact with the ribose binding pocket of SaBPL. In silico docking studies using a crystal structure of SaBPL aided in the selection of benzyl groups that present in the ribose-binding pocket of SaBPL. The halogenated benzyl derivatives 3.20, 3.21, 3.23 and 3.24 provided the most potent inhibitors of SaBPL with the respective Kᵢ value of 0.28, 0.6, 0.39 and 1.1 μM. These compounds also inhibited the growth of S. aureus ATCC49775 (MIC = 4 – 16 μg/ml), while possessing low cytotoxicity against HepG2 cells. Chapter four builds upon the active 1,2,3-triazole based inhibitors of SaBPL described in chapter two and three with an investigation at C5 of the triazole ring to generate 1,4,5- trisubstituted 1,2,3-triazoles. A class of 5-iodo 1,2,3-triazoles was synthesised from 1- iodoacetylene 4.02 and azides using CuAAC. Subsequent halogen exchange reaction allowed conversion of iodide to other halogens. 5-Fluoro-1,2,3-triazole 4.07, the lead compound from this series of inhibitors, proved to be a potent and selective inhibitor of SaBPL (Kᵢ = 0.42 ± 0.06 μM) and it significantly reduced S. aureus growth with no cell growth apparent at 16 μg/mL. Chapter five investigates the use of acylsulfonamide as a bioisostere of the phosphoroanhydride linker as in biotinyl-5’-AMP 1.03. Acylsulfonamide 5.05 was found as the most active and selective inhibitor of SaBPL (Kᵢ = 0.72 x 10⁻³ μM) and MtbBPL (Kᵢ = 0.74 x 10⁻³ μM) reported to date. Antibacterial studies revealed that 5.05 was active against susceptible S. aureus (MIC = 0.5-1.0 μg/mL), methicillin-resistant S. aureus ((MIC = 0.5- 1.0 μg/mL) and Mycobacterial tuberculosis ((MIC = 51 μg/mL). Finally, the x-ray structure 5.05 bound to SaBPL was solved to reveal important molecular interactions critical to the potency of 5.05 and emphasized the acylsulfonamide moiety as an effective bioisostere of phosphoroanhydride linker. Chapter six discusses the use of in situ click chemistry as an alternative approach for the synthesis of 1,2,3-triazoles. The target enzyme SaBPL was directly involved in the selection of its optimum triazole based inhibitor by catalysing the reaction of biotin acetylene and organic azides without copper as a catalyst. The use of high throughput LC/MS provided improved efficiency and sensitivity of detection of triazole-based inhibitors and allowed the in situ approach to be widely applied to BPLs from other bacteria. Chapter seven details the experimental procedures for compounds described in chapter 2 – 6, and the chromatographic analysis of in situ click experiments described in chapter 6.Item Open Access The effects of macrocyclic constraints on electron transfer in peptides(2015) Horsley, John Robert; Abell, Andrew David; Yu, Jingxian; School of Physical SciencesResearch undertaken in this thesis focuses on electron transfer in peptides constrained into either a 3₁₀-helical or a β-strand conformation in order to progress the field of molecular electronics. Chapter One: Natural proteins have evolved to promote electron transfer in many biological processes. However, their complex conformational nature inhibits a thorough investigation, so in order to study electron transfer in proteins, simple peptide models containing redox active moieties present as ideal candidates. Chapter One introduces the importance of secondary structure characteristic to proteins/peptides, and its relevance to electron transfer. The proposed mechanisms responsible for such electron transfer are discussed, along with the various approaches used to further constrain the peptides into their geometric conformations. The methods used to characterize the conformation of all peptides synthesized throughout this thesis are outlined, as are details of the electrochemical techniques used to investigate their electronic properties. A literature review describing several factors that have been shown to influence electron transfer in peptides, and a brief summary of molecular electronics follows. Chapter Two: Two 3₁₀-helical peptides were synthesized, one constrained via a covalent side-chain staple using Huisgencycloaddition, and the other a linear analogue. Both peptides contain a redox active terminal ferrocene moiety, and were separately attached to a single walled carbon nanotube (SWCNT)/gold electrode array for electrochemical analysis. The effect of backbone rigidity imparted by the side-bridge constraint was revealed, which was shown to restrict the necessary torsional motions that lead to facile intramolecular electron transfer along the peptide backbone. High level calculations were used to support the electrochemical observations. Chapter Three: A series of peptides constrained into either a 3₁₀-helix or β-strand conformation were synthesized, each containing a varied number of electron rich alkene side chains. The ability of the alkene(s) to facilitate electron transfer through the peptides by exploiting a hopping mechanism, and thus act as a “stepping stone” was investigated. Ring closing metathesis was used to further rigidify the backbones of a helical and a β-strand peptide via side chain tethers. The ensuing saturated and unsaturated compounds were electrochemically interrogated in order to explore any possible interplay between the effects of the alkene side-chains and backbone rigidity. High level calculations were conducted to verify the observed electrochemical data. Chapter Four: Two β-strand peptides were synthesized, one constrained via a covalent side-chain staple using Huisgen cycloaddition, and the other a linear analogue. Both peptides contain a redox active terminal ferrocene moiety, and were separately attached to a SWCNT/gold electrode array for electrochemical analysis. The charge transfer pathway was determined to be intramolecular by measuring the electron transfer rate at various concentrations of the constrained peptidebound to the electrode. This pathway is analogous to charge transfer through a molecular junction involving a single peptide. Theoretical conductance simulations were then undertaken using two peptide analogues in order to establish a link between the electrochemical observations and conductance measurements through a molecular junction. Chapter Five: Two macrocyclic peptides were synthesized, one constrained into a 3₁₀-helical conformation by linking its i to i+3 residues to form a lactam bridge, and the other constrained into a β- strand geometry via a lactam-bridge tether, linking its i to i+2 residues. These peptides were chosen in order to define the role of the amide bond in a lactam bridge constraint. Direct linear analogues of each were used to establish the effect on electron transfer from a terminal amide bond located in an untethered side-chain. High level calculations were also conducted in order to elucidate the mechanism(s) responsible for electron transfer in each of the linear and macrocyclic helical peptides.Item Open Access Exploring the structure-function relationship of Biotin Protein Ligase from Staphylococcus aureus : implications for selective inhibitor design.(2013) Soares da Costa, Tatiana Pereira; Booker, Grant William; Polyak, Steven William; Abell, Andrew David; School of Molecular and Biomedical ScienceThere is a well-documented need to replenish the antibiotic pipeline with new products to combat the rise of drug resistant bacteria, such as the superbug methicillin resistant Staphylococcus aureus (MRSA). One strategy to combat drug resistance is to identify new chemical classes with novel mechanisms of action and that are not subject to existing resistance mechanisms. As most of the obvious bacterial drug targets with no equivalents in mammals have been well explored, targets with a closely related human homologue represent a new frontier in antibiotic discovery. However, to avoid potential toxicity to the host, these inhibitors must have extremely high selectivity for the bacterial target over the human equivalent. This thesis is focused upon exploiting the ubiquitous enzyme biotin protein ligase (BPL), which is involved in the essential cellular process of attaching biotin onto biotin-dependent enzymes. Due to the pivotal metabolic roles played by biotin-dependent enzymes in bacteria, BPL has been proposed as a promising new antibiotic target. Hence, BPL inhibitors with selectivity for the bacterial isozyme over the human equivalent promise a new class of antibiotic to combat MRSA. The aim of this project was to provide proof of concept data demonstrating that BPL from a pathogen could be selectively targeted for inhibition over the human equivalent. Here I employed a combination of structure-guided drug design and fragment-based approaches to discover novel BPL inhibitors. The X-ray crystal structure of S. aureus BPL (SaBPL) shows two adjacent binding sites for the ligands biotin and ATP, making it an ideal candidate for a fragment-based approach to drug discovery. Although the residues at the biotin-binding site are highly conserved, the nucleotide pocket shows a high degree of variability that can be exploited to create compounds selective towards BPLs from pathogens. The biotin 1,2,3 triazole analogues identified in this work yielded our most potent and selective inhibitor (Ki = 90 nM) [i is subscript] with >1100-fold selectivity for the SaBPL over the human homologue (Chapter 2). The molecular basis for the selectivity was identified using mutagenesis studies with a key arginine residue in the BPL active site necessary for selective binding. Importantly, the biotin triazole inhibitors showed in vivo cytotoxicity against S. aureus, but not cultured mammalian cells (Chapter 2). In an attempt to identify new chemical scaffolds with improved ligand efficiency for chemical development, a series of analogues based on the natural ligand biotin were also designed and tested for enzyme inhibition and antimicrobial activities against clinically relevant strains of S. aureus (Chapter 3). This approach resulted in highly potent compounds (Ki < 100 nM) [i is subscript] with antibacterial activity against MRSA strains (MIC = 2 – 16 μg/mL). Whilst only moderate selectivity over the human enzyme (10 - 20 fold) was observed, the biotin analogues provided a suitable chemical scaffold with high ligand efficiency for further chemical development. One of the compounds identified was biotin acetylene, which forms a long lived complex with SaBPL and is a precursor for in situ click reactions. This target-guided approach to drug discovery relies on the ability of the enzyme to choose its own inhibitors from a range of acetylene and azide building blocks to form specific triazole products. Since a class of biotin-triazole molecules had already been identified as selective inhibitors of SaBPL, we reasoned that this enzyme would provide an ideal candidate for performing in situ click approach to inhibitor discovery. In this work, a protocol for the BPL-catalyzed in situ click reaction was optimized to select the optimum triazole-based inhibitor using biotin acetylene as an anchor molecule to recruit complimentary fragments that could bind in the peripheral ATP pocket (Chapter 4). The in situ reaction was shown to be improved by the use of a SaBPL mutant that promoted diffusion of the triazole product from the active site following synthesis. This novel approach improved efficiency and ease of detection (Chapter 4). Apart from drug discovery, this thesis also focuses on enzymatic characterization of SaBPL and highlighting the key differences between SaBPL and the human homologue. The structure of human BPL is yet to be reported, so structure-function studies were performed to elucidate new information about the bacterial and human enzymes. The oligomeric state of SaBPL was investigated using analytical ultracentrifugation in its apo form and in the presence of ligands (Chapter 5). Unlike human BPL, SaBPL was shown to dimerize in solution. A single amino acid in SaBPL, Phe123, was identified to have a dual key role in dimer formation and inhibitor binding (Chapter 5). One of the major roadblocks to obtaining crystals of the full-length human enzyme is the low yield of protein obtained from recombinant expression and purification. In this thesis, an alternative approach is described that could be used to increase our chances of obtaining structural data about the human BPL. I created a ‘humanized’ chimeric protein in which all seven residues in the nucleotide pocket of SaBPL that are not conserved with the human BPL were mutated to their human equivalents. This ‘humanized’ protein exhibited similar kinetic and inhibition properties to the human enzyme (Chapter 6). Crystal trials have commenced to help direct future drug development efforts. Further studies on the human BPL enzyme will also be described, including the dissection of the binding mechanism using surface plasmon resonance (Chapter 7). The N-terminal domain of this enzyme was shown to play a role in stabilizing the complex between the enzyme and the biotin domain substrate, providing the first molecular explanation for human BPL-deficient patients that do not respond to biotin therapy. In summary, this work demonstrates for the first time that BPL from the clinically important pathogen Staphylococcus aureus can be selectively inhibited. A provisional patent has been filed for the biotin 1,2,3 triazole molecules I have identified. These discoveries will enable further development of a new class of antibiotics.Item Open Access Hydrogen peroxide sensing for reproductive health(2016) Purdey, Malcolm Stuart; Abell, Andrew David; Monro, Tanya Mary; Carver, John Adrian; School of Physical SciencesThe research presented in this thesis details the synthesis, surface functionalisation and photochemical studies of fluorescent probes for the detection of hydrogen peroxide (H₂O₂) in reproductive health. Chapter 1: H₂O₂ is an important reactive oxygen species (ROS) that is detrimental to the health of spermatozoa and embryos. Fluorescent probes are commonly used for the detection of ROS and here examples with different mechanisms of detection are examined; such as turn-on probes, turn-off probes, Förster resonance energy transfer (FRET)-based and photoinduced electron transfer (PET)-based probes. Specific reference is given to the aryl boronate and benzil classes of probe, which show good selectivity for H₂O₂ over other ROS. The attachment of the fluorescent probe to an optical fibre as a non-invasive sensing platform is discussed. This then allows sensing in a sensitive biological environment, such as an embryo, without exposure to the probe in solution. Fibre tip sensors and microstructured optical fibre-based sensors are discussed for use in such biological environments. Finally, a summary is provided detailing the objectives of this thesis and the chapters in which these are addressed. Chapter 2: Three aryl boronate probes [peroxyfluor-1 (PF1), carboxy peroxyfluor-1 (CPF1) and a novel probe 2(2-ethoxyethoxy)ethoxy peroxyfluor-1 (EEPF1)] were synthesised for use in the detection of H₂O₂ in human spermatozoa. The activity and selectivity of these probes was then compared to three commonly used commercial probes, 2’,7’-dichlorohydrofluorescein diacetate (DCFH), dihydroethidium (DHE) and MitoSOX red (MSR). PF1 and EEPF1 were found to be effective at detecting H₂O₂ and peroxynitrite (ONOO⁻) produced by spermatozoa when stimulated with menadione or 4-hydroxynonenal. Flow cytometry was used to demonstrate that EEPF1 is more effective at detecting ROS in spermatozoa compared to DCFH, DHE and MSR. Furthermore, EEPF1 distinguished poorly motile sperm from motile sperm as revealed by an enhanced production of ROS. Chapter 3: A fibre-tip based probe constructed by encapsulating CPF1-NHS in a polyacrylamide matrix is reported for the detection of H₂O₂. This non-invasive platform avoids the need to introduce an organic fluorophore into a sensitive cell such as an embryo as discussed above. A number of derivatives of PF1 were investigated, with carboxylated fluorophore CPF1 proving to be the easiest to synthesise and characterise. CPF1 was functionalised to glass slides using layer-by-layer deposition of polyelectrolytes. This functionalised surface showed a fluorescent response to H₂O₂ comparable to solution-based measurements. Three surface functionalisation methods were then investigated for attachment to an optical fibre tip, specifically polyelectrolyte deposition, silane monolayer formation, and light-catalysed polymerisation of acrylamide. The most effective method of functionalisation was found to be light-catalysed formation of a polyacrylamide matrix with the CPF1 embedded. These polyacrylamide fibre tip probes were then guided into microdroplets of bovine in vitro fertilisation (IVF) media using a micromanipulator. This was visualised under an optical microscope to detect the controlled release of H₂O₂. This fibre probe is thus compatible with imaging techniques used in IVF research laboratories. Chapter 4: This chapter presents the development of a single optical fibre tip probe capable of detecting both the concentration of H₂O₂ and the pH of the associated solution. The sensor was constructed by embedding two fluorophores [CPF1 and seminaphtharhodafluor-2 (SNARF2) for H₂O₂ and pH detection respectively] on the tip of an optical fibre using the previous developed polyacrylamide matrix methodology. The functionalised fibre probes reproducibly sensed pH with a resolution of 0.1 pH units. The probe also accurately detected H₂O₂ over a biologically significant concentration range, of 50-100 μM. This study revealed the importance of simultaneous detection of H₂O₂ and pH, where changes in pH were shown to affect the fluorescent response of CPF1. This new fibre probe offers potential for noninvasive detection of pH and H₂O₂ in biological environments using a single optical fibre. Chapter 5: Two new cell-permeable boron-dipyrromethene (BODIPY) based fluorescent probes for the detection of H₂O₂ were designed and synthesised. The aryl boronate peroxyBODIPY-1 (PB1) gave rise to a decrease in fluorescence on reaction with H₂O₂, while the fluorescence of the benzil-based nitrobenzoylBODIPY (NbzB) probe increased on reaction with H₂O₂. The benzil probe NbzB exhibited a high degree of selectivity for H₂O₂ over other ROS. The aryl boronate PB1 showed a greater change in fluorescence on reaction with H₂O₂ compared to NbzB, and PB1 also detected H₂O₂ in bovine oocytes under oxidative stress. These results suggest that aryl boronates (i.e. PB1) and benzils (i.e. NbzB) have use in biological environments requiring higher sensitivity or selectivity to H₂O₂. Chapter 6: The research discussed here extends the solution-based and fibre tip experiments to the detection of H₂O₂ in biological environments. Detection of H₂O₂ within cells is often frustrated by autofluorescence in the green emission region. Contrastingly, the red emission region in biological systems shows a lower autofluorescence background signal. Therefore a redemitting fluorescent probe for H₂O₂, naphthoperoxyfluor-1 (NPF1), was synthesised. However, when incubated with H₂O₂ in cuvette, NPF1 showed a greater than 20-fold reduced fluorescent response to H₂O₂ compared with CPF1. This poor sensitivity suggests that NPF1 should not be used for the detection of H₂O₂, but rather fluorophores with a greater fluorescent response should be utilised (e.g. CPF1). A reversible optical fibre-based sensor for H₂O₂ was then explored by attaching a reversible fluorescent probe for ROS (nicotinamide coumarin redox sensor 3, NCR3) to an optical fibre tip. The sensor was constructed using light-catalysed polymerisation to give a polymer matrix on the tip containing NCR3. This allowed the fibre tip to be reversibly oxidised by H₂O₂ and reduced by NaCNBH₃. The sensor exhibited good reversibility over at least seven cycles of oxidation and reduction, with consistent fluorescent ratios of its maxima at 500 and 635 nm. However, its fluorescence intensity decreased over time, suggesting that NCR3 leached from the polymer into the buffer solution. This nevertheless represents the first example of a reversible fibre sensor for ROS and is as such an important first step towards a reusable optical fibre probe for H₂O₂.Item Open Access New peptide-based templates constrained into a β-strand by Huisgen cycloaddition.(2012) Pehere, Ashok D.; Abell, Andrew David; School of Chemistry and PhysicsChapter One introduces the concept of peptide 'secondary structure' with an emphasis on β-strand geometry in macrocycles. This structural design is crucial for targeting different proteases. The significance of the macrocylic β-strand ‘bioactive’ conformation is discussed in detail. In particular the exploitation of the conformationally constrained peptidomimetic macrocylic backbone, which is constrained by a number of synthetic approaches to lock the ‘bioactive’ conformation in place. Chapter Two describes simple and scalable methodology for the preparation of N-Cbz protected amino acids by reaction with Cbz-Cl which uses a mixture of aqueous sodium carbonate and sodium bicarbonate to maintain the appropriate pH. This method proceeds without the formation of by-products. The method is extended to large scale preparation of an intermediate zofenopril, an ACE inhibitor. Chapter Three describes new peptidic templates constrained into a β-strand geometry by linking acetylene and azide containing P₁ and P₃ residues of a tripeptide by Huisgen cycloaddition. The conformations of the macrocycles are defined by NMR studies and those that best define a β-strand are shown to be potent inhibitors of the protease calpain. The β-strand templates presented and defined here are prepared under optimized conditions and should be suitable for targeting a range of proteases and other applications requiring such geometry. Chapter four describes a new approach to non-covalent peptide-based nanotubular or rodlike structures, whereby the monomeric units are preorganised into a β-strand geometry that templates the formation of an extended and unusual parallel β-sheet rod-like structure. The conformational constraint is introduced by Huisgen cycloaddition to give a triazolebased macrocycle, with the resulting self-assembled structures stabilized by a well-defined series of intermolecular hydrogen bonds. Chapter Five the 26S proteasome has emerged over the past decade as an attractive therapeutic target in the treatment of cancers. Here, we report new tripeptide aldehydes that are highly specific for the chymotrypsin-like catalytic activity of the proteasome. These new CT-L specific proteasome inhibitors demonstrated high potency and specificity for cancer cells, with therapeutic windows superior to those observed for benchmark proteasome inhibitors, MG132 and Bortezomib. Constraining the peptide backbone into the β-strand geometry was associated with decreased activity in vitro and reduced anticancer activity, suggesting that the proteasome prefers to bind a conformationally flexible ligand. Using these new proteasome inhibitors, we show that the presence of an intact p53 pathway significantly enhances cytotoxic activity, thus suggesting that this tumor suppressor is a critical downstream mediator of cell death following proteasomal inhibition. Chapter Six peptide derived protease inhibitors represent an important class of compounds with the potential to treat a wide range of serious medical conditions. Herein we describe the synthesis of a series of triazole containing macrocylic protease inhibitors preorganised in a β-strand conformation and evaluate their selectivity and potency against a panel of protease inhibitors. A series of acyclic azido-alkyne-based aldehydes is also evaluated for comparison. The macrocyclic peptidomimetics showed considerable activity towards Calpain II, Cathepsin L and S and the 26S proteasome chymotrypsin-like activity. Importantly, the first examples of potent and selective inhibitors of Cathepsin S were identified and shown to adopt a well-defined β-strand geometry by NMR, X-ray and molecular docking studies. Chapter Seven describes simple and efficient methodology for the selective acylation and alkylation of biotin at its 3′-nitrogen. This methodology is used to prepare of other biotin derivatives.Item Open Access Peptidomimetic protease inhibitors: activity and mechanism of inhibition(2015) Zhang, Xiaozhou; Abell, Andrew David; George, Jonathan; School of Physical SciencesThe study of protein mechanism and function is central to the development of biosensing tools and therapeutics for the treatment of diseases. This thesis describes an NMR and X-ray crystallography-based characterisation of the mechanism by which a macrocyclic peptidomimetic, the backbone of which is constrained into a β-strand conformation, inhibits α-chymotrypsin. This allowed the development of new peptidomimetic inhibitors that target the 26S proteasome and also inhibitors the activity of which can be modulated photochemically. This then provides a basis for biosensing and therapeutic applications. Chapter one introduces the structures and mechanism of serine, cysteine and threonine proteases, and discusses how theses proteases universally bind ligands in an extended β-strand conformation. In addition, this chapter details the strengths and limitations of current peptidomimetic inhibitors of α- chymotrypsin, calpains and the 26S proteasome and their implications in the treatment of human diseases. Chapter two describes optimisation of the synthesis of two macrocyclic peptidic aldehyde inhibitors 2.12 and 2.13 that target cysteine proteases and α-chymotrypsin, respectively. This allowed the preparation of an analogue of 2.13 containing a ¹³C label in the aldehyde, which was used to confirm the mechanism of inhibition of α-chymotrypsin by ¹³C NMR spectroscopy. This confirmed the formation of a stable hemiacetal intermediate upon the binding of 2.13 with α-chymotrypsin. X-ray crystallography of a complex of 2.13 bound to α-chymotrypsin revealed that the backbone adopts a stable β-strand conformation as per its design. The binding of 2.13 to α-chymotrypsin is further stabilised by the oxyanion hole near the S₁ subsite and multiple hydrogen bonding interactions. Chapter three details the development of new acyclic proteasome inhibitors 3.05-3.08 containing a peptidomimetic backbone and a C-terminal boronate. All analogues showed selectivity for the chymotrypsin-like subunit of the 26S proteasome with IC₅₀ values in the low nanomolar range. Compound 3.08, with an IC₅₀ of 13 nM, was 2-fold more active than the anti-myeloma therapeutics bortezomib and carfilzomib. This inhibitor is more cytotoxic against a range of solid tumour cells and has a larger therapeutic window compared to existing FDA approved drugs. Chapter four presents a new approach to the regulation of the activity of α- chymotrypsin using a new spiropyran-based moiety that can be reversibly switched between an ‘on’ (SP isomer) and ‘off’ (MC isomer) state photochemically. This is demonstrated in solution and also when attached to a microstructured optical fibre (MOF), as a first step to the development of a biosensor. The most active analogue in this series displayed a Kᵢ of 115 nM in solution. The active SP isomer of an analogue 4.07 with a C-terminal Weinreb amide was significantly more active than the corresponding MC isomer both in solution and on fibre.Item Open Access Photoswitchable sensors: reversible ion detection using optical fibres(2017) Stubing, Daniel Brian; Abell, Andrew David; Heng, Sabrina; Monro, Tanya Mary; School of Physical SciencesIn studying and diagnosing cellular systems and diseases, the ability to accurately detect and monitor the concentrations and fluctuations of metal ions is of particular importance. Fluorescent photoswitchable sensors provide a means to reversibly detect metal ions in solution. This class of sensors uses a light stimulus to chemically switch between two distinct species, one that can bind to an analyte of choice and one that cannot bind. This then provides sensors that can be turned off at will, allowing the sensor to be reset and used again at a different time point. This thesis investigates the design, synthesis and metal ion selectivity of a series of photoswitchable sensors. These sensors contain a spiropyran core unit with differing ion binding domains, such as an aza-crown ether, providing ion specificity, as well as a free carboxyl group that allows for attachment to a solid support. A discussion on the choice of this photoswitchable moiety and subsequent design and synthesis as a new metal ion sensor is presented in Chapters 2, and 3 and 4, respectively. These photoswitchable sensor molecules were then used within a microstructured optical fibre (MOF) sensing platform. Suspended core microstructured optical fibres provide a biologically suitable platform that provides a very sensitive means to sense in nanolitre volumes of sample. Covalently attaching these photoswitchable sensors to the light guiding core, via APTES silanization, provided a reversible sensing system capable of detecting picomolar concentrations of metal ions, such as Ca²⁺ in a biological sample while not contaminating the sample. The MOF not only provided a means to detect a fluorescence signal, it also allowed for repetitive on/off photocycling of the photoswitch, both in solution and attached to the surface.Item Open Access Sensing in biological systems(2018) Sylvia, Georgina Maree; Abell, Andrew David; Heng, Sabrina; George, Jonathan; School of Physical SciencesMetal ions are critical to a range of mammalian cell functions, including cell signalling, proliferation, differentiation and death. As such, changes in metal ion homeostasis can have a significant effect on cell health. Fluorescent chemosensors, in combination with specialised imaging technologies, provide a useful tool to study the role of metal ions in cellular processes as they enable detection in and around cells with spatial and temporal resolution. Commercially available and literature fluorescent probes, with core structures based on traditional fluorophores (e.g. fluorescein, coumarin and rhodamine) have been used extensively to study the relationship between cellular metal ion dynamics and disease. However, one drawback of current sensors is the lack of reusability. Photoswitchable molecules present as an alternative core unit for the development of fluorescent metal ion sensors, with the potential for reversible analyte binding. The photochromic spiropyran molecule is of particular interest in our research group, as the core structure can be readily functionalised for analyte selectivity, surface attachment, and tuning of the photochromic properties. Photo-controlled switching between the weakly fluorescent spiropyran (SP) and highly fluorescent merocyanine (MC) isomers occurs on irradiation with UV light or in the presence of the target metal ion, while visible light reverses the isomerisation (see Figure 1). This thesis describes the functionalisation of the spiropyran molecule in order to improve fluorescence intensity (Chapter 2), develop selective metal ion sensors for Mg²⁺ (Chapter 3) and investigate structure-metal ion selectivity relationships (Chapter 4). Chapter 1: Chapter 1 gives a broad introduction to fluorescent sensors and presents a literature review highlighting the use of photoswitchable spiropyrans as effective sensors for biologically relevant metal ions relevant such as calcium (Ca²⁺) and zinc (Zn²⁺), as well as the alkali metals sodium (Na⁺), potassium (K⁺) and lithium (Li⁺). Chapter 2: In Chapter 2, we took a unique approach to Ca²⁺ sensing with a rationally designed sensor which possess all the desirable characteristics (brightness, photostability and red fluorescence emission) of both the traditional and spiropyran-type sensor. This was accomplished by combining a traditional fluorophore (pyrene) with a photoswitch (spiropyran) with a Ca²⁺-selective ionophore. The rationally designed, pyrene-spiropyran hybrid Ca²⁺ sensor (Py-1) displays enhanced fluorescence intensity compared to a standalone spiropyran analogue. Importantly, Py-1 retains the characteristic red emission profile of the spiropyran, while fibre-based photostability studies show the sensor is stable after multiple cycles of photoswitching, without any sign of photodegradation. Such properties are of real advantage for cell-based sensing applications. An interesting observation is that, Py-1 presents with two excitation options; direct green excitation (532 nm) of the photoswitch for a red emission, and UV excitation (344 nm) of the component pyrene, which gives rise to distinct blue and red emissions. This proof-of-concept hybrid sensing system presents as a general approach to brighter spiropyran-based sensors. Chapter 3: Magnesium ions (Mg²⁺) play an important role in mammalian cell function; however, relatively little is known about the mechanisms of Mg²⁺ regulation in disease states. An advance in this field would come from the development of selective, reversible fluorescent sensors, capable of repeated measurements. To this end, Chapter 3 details the development of the first rationally designed, spiropyran-based fluorescent Mg²⁺ sensors. The most promising analogue, sensor 1, exhibits 2-fold fluorescence enhancement factor and 3-fold higher binding affinity for Mg²⁺ (Kd 6.0 µM) [d subscript] over Ca²⁺ (Kd 18.7 µM) [d subscript]. Incorporation of spiropyran-based sensors into optical fibre sensing platforms has been shown to yield significant signal-to-background changes with minimal sample volumes, a real advance in biological sensing that enables measurement on subcellular-scale samples. In order to demonstrate sensor compatibility within the light intense microenvironment of an optical fibre, photoswitching and photostability of 1 within a suspended core optical fibre (SCF) was subsequently explored, revealing reversible Mg²⁺ binding with improved photostability compared to the non- photoswitchable Rhodamine B fluorophore. The spiropyran-based sensors reported here highlight untapped opportunities for a new class of photoswitchable Mg²⁺ probe and present a first step in the development of a light-controlled, reversible dip-sensor for Mg²⁺. Chapter 4: In Chapter 4, the influence of multiple chelating groups on calcium ion (Ca²⁺) selectivity are explored with a series of spiropyran-based sensors incorporating both C8' and N1- indole metal ion binding domains. The sensors possess N1-indole functionalisation in the form of hydroxyethyl (SP-1), ethoxycarbonylbutyl (SP-2) and carboxybutyl (SP-3) groups, while all three sensors incorporate a 1-aza-15-crown-5 ionophore at the C8' position. Absorbance and fluorescence characterisation of metal ion binding revealed that in particular, sensor SP-3 gave excellent Ca²⁺-selectivity, improved dissociation constant (K𝖽 MC(SP-3)-Ca²⁺ = 22 µM) [d subscript] and quantum yield of fluorescence (Φ MC(SP-3)-Ca²⁺ = 0.37), compared to the other sensors. These results suggest the carboxybutyl N1-indole functionality of SP-3 may play a role in stabilizing Ca²⁺ in the 1-aza-15-crown-5 ionophore, promoting metal-induced isomerisation to the MC(SP-3)-Ca²⁺ complex and thus a bright, Ca²⁺-selective, red fluorescence signal. Appendix A: One sensor from the selectivity study in Chapter 4 (labelled ‘SHL’) was subsequently utilized with collaborators in a biological application, to study lithium ‘hot-spots’ in living colon cancer cells. The results show ion binding to the sensor intracellularly is dependent on exogenous Li⁺ transport into the cell, and repeated cycles of photoswitching gave reproducible changes in fluorescence, demonstrating the ability of the sensor to reversibly photoswitch in living cells. Furthermore, ‘hot-spots’ of Li⁺-SHL binding induced fluorescence were observed at the leading edges of migrating cells, which correlates with ion movement through aquaporin transmembrane channels. These results suggest that the aquaporin-1 (AQP1) ion channel could be a novel candidate for therapeutic interventions to manage metastasis in AQP1-dependent cancers. Appendix B: The themes of tailored selectivity and signal enhancement developed in this thesis are further explored in Appendix B, where we report on improved sensitivity in a nanoporous anodic alumina (NAA) sensing platform, targeted towards the detection of analytes in biological media. Sensing on an NAA platform utilises reflectometric interference spectroscopy (RIFS), where the amount of light-based signal is proportional to the degree of conformational change of the surface. This work describes a range of Au³⁺ selective binding molecules, in a series of combined surface attachment strategies in order to improve fundamental knowledge of surface-engineering in these nanoporous materials. The most sensitive functional molecules from sensing approaches (i) and (ii) were combined into a third sensing strategy whereby the nanoporous platforms are functionalised on both the top and inner surfaces concurrently. Engineering of the surface according to this approach resulted in an additive enhancement in sensitivity of up to 5-fold compared to previously reported systems.Item Open Access A study on the interactions of synthetic IGF-II analogues with the type 1 IGF and insulin receptors.(2014) Cottam, Jade Misty; Abell, Andrew David; Pukala, Tara Louise; Forbes, Briony Evelyn; Scanlon, Denis B.; School of Chemistry and PhysicsInsulin-like growth factor II (IGF-II) is a unique regulatory peptide containing 67 residues and three disulfide bonds. It binds with high affinity to three receptors, the insulin receptor (IR), the type 1 insulin-like growth factor receptor (IGF-1R) and the We 2 insulin-like growth factor receptor (IGF-2R). Binding of IGF-II to these receptors signals mitogenic responses, such as cell proliferation, differentiation and migration. The interactions of IGF-II with the IR and IGF-1R have recently been identified as potential therapeutic targets for the treatment of cancer. Thus, an increased understanding of the interactions of IGF-II with the IGF-1R and the IR-A is required for the improved design and development of potential anticancer therapeutics. A crystal structure of IGF-II bound to either the IGF-1R or the IR-A has not been reported. Thus, the precise location of IGF-II within the receptor binding pocket remains undefined. A fluorescence resonance energy transfer (FRET) approach was proposed to investigate the binding location and orientation of IGF-II within the IGF-1R. Two fluorescent IGF-II analogues, the Fl9Cou IGF-II and F28Cou IGF-II proteins, were synthesised for use in the desired FRET studies. These FRET experiments first required the synthesis of an appropriate coumarin-based probe for incorporation into IGF-II. The synthesis of a range of fluorescent coumaryl amino acids is described in Chapter 2, and an analysis of the spectroscopic properties of these coumaryl amino acids is also detailed. Site-specific incorporation of the coumarin-based probe into IGF-II was then undertaken. Three complementary methods were used for the preparation of the desired fluorescent IGFII analogues. Chapter 3 describes the use of the nonsense suppression methodology for the expression of the novel Fl9Cou IGF-II protein. This was followed by an improved chemical synthesis of the Fl9Cou IGF-II protein using a linear solid phase peptide synthesis (SPPS) approach and is detailed in Chapter 4. A robust native chemical ligation approach was developed in Chapter 5, which allowed for the facile incorporation of the coumarin-based probe at various locations within the IGF-II protein. Chapter 5 also details the synthesis of the native IGF-II, Fl9Cou IGF-II and F28Cou IGF-II proteins. The biological activity of the resultant IGF-II analogues was evaluated by competition binding assays. The fluorescent IGFII analogues bind with low nanomolar affinity to the IR and IGF-1R, and as such were deemed suitable for use in the desired FRET-based experiments. The FRET-based investigation into the binding interactions of the native IGF-II, Fl9Cou IGFII and F28Cou IGF-II proteins to the IGF-1R is described in Chapter 6. FRET interactions were observed for both the Fl9Cou IGF-II and F28Cou IGF-II proteins. The results show the fluorophore binds in close proximity to Trp residues within the IGF-1R receptor and suggest the location of IGF-II binding within the IGF-1R is consistent with what is proposed in the literature. These experiments provide a basis for further investigations for determining the precise binding location and orientation of IGF-II within the IGF-1R.Item Open Access The synthesis of bimane constrained peptides and their fluorescent and structural properties(2017) Horsfall, Aimee Jade; Abell, Andrew David; Heng, Sabrina; School of Physical SciencesAberrant protein-protein interactions often result in disease, and as such, effective protein-protein interaction inhibitors are needed to mitigate the disease state. These interaction interfaces often involve secondary structural motifs, for example, an α-helix or β-sheet. Small molecule drugs are not well suited to inhibit protein-protein interactions however constrained peptides, have shown to have great therapeutic potential. Short peptides display little secondary structure in aqueous solution and as such, peptide sequences derived from a protein-protein interaction interface for use as a protein-protein interaction inhibitor, must be constrained into the native secondary structure. This can be achieved by installing a linker between the side-chains of two appropriately spaced amino-acids in the sequence. Many different linker chemistries have been designed and implemented with good biological results. However, these constrained peptide therapeutics are still restricted by traditional small molecule drug hurdles including cell permeability, protease degradation and the ability to visualise and track a molecule intracellularly. Linkers such as the all-hydrocarbon metathesis linker have shown great promise in reducing protease degradation and increasing cell permeability, however a fluorescent tag is still necessary to visualise a drug candidate. Here, a bimane linker is proposed as a new peptide linker to help overcome these limitations. Dibromobimane is reacted with thiol-containing amino-acid side chains to introduce a new fluorescent constraint in a series of model peptides. The reaction conditions with dibromobimane are optimised in solution to reveal that a buffered system is required for the cyclisation to occur efficiently. Optimal reaction conditions, determined by monitoring the increase of the fluorescent product, were 0.5 mg/ml peptide in 10 mM PBS with one equivalent of dibromobimane. The reaction was shown to be facile and versatile; in this thesis an array of peptides with varied sequence length, constraint length and amino-acid composition were cyclised under the same conditions, all reaching reaction completion in under 30 minutes. Additionally, these same conditions were applied successfully to react monobromobimane with series of short peptides. Cyclisation on reaction with dibromobimane, was also demonstrated on-resin with similar efficiency. The fluorescent properties of the resultant peptides were then explored to reveal that pH does not affect the observed fluorescence however a longer peptide length resulted in greater fluorescence intensity. Furthermore, acyclic mono-bimane-functionalised peptides displayed lower fluorescence intensity than the bimane-cyclised counterparts. The fluorescence of the bimane cyclised peptide could be detected as low as 10 nM on a plate reader, which is expected to further improve on a more sensitive instrument. The secondary structure of a series of tri- and penta-peptides were investigated through CD and NMR techniques. It was deduced that the bimane linker can induce β-strand like structure in an i-i+2 constrained peptide; in contrast an i-i+4 constrained pentapeptide with homocysteine in the 1 and 5 positions results in a 3₁₀ helical like structure. β-alanine containing analogues of these peptides were also synthesised and showed minimal structure.This work outlines the synthesis of macrocyclic peptides containing a peptide constraint, in the form of a fluorescent bimane, both in solution and on-resin to produce cyclised peptides. The fluorescent properties of the resultant peptides have been shown to be biologically compatible with great fluorescence sensitivity. Furthermore, different secondary structure can be introduced by simply alterations of the constraint length from i-i+2 to i-i+4. This work provides a foundation on which to design new fluorescent bimane-cyclised peptide-based protein-protein interaction inhibitors.Item Open Access Synthesis of glutamate mimics as neuropathic pain modulating agents.(2010) Stanley, Nathan John; Taylor, Dennis Kenwyn; Abell, Andrew David; Irvine, Rod; School of Chemistry and PhysicsAs part of the vital search towards improved therapeutic agents for the treatment of neuropathic pain, the central nervous system ubiquitous glutamate receptors have become a major focus of research. As such, the discovery of glutamate receptor ligands with improved potency and selectivity has been an important area of study for many decades, though there is still much knowledge to be gained. Outlined herein are the syntheses towards a series of potentially biologically active 3’-cycloalkyl-substituted carboxycyclopropylglycine analogues. These syntheses utilize novel synthetic chemistry to construct the cyclopropane core with all required stereochemistry. As a consequence of this work, two new cycloalkylcarboxycycloproplyglycine analogues were successfully synthesized, utilizing the reaction of 1,2-dioxines with protected phosphonates in a 20% overall yield for one diastereoisomer. Secondly, the syntheses of a series of 1,4- and 1,5-substituted 1,2,3-triazole amino acids as a new class of potential glutamate receptor ligands. Briefly, a series of six 1,4- and 1,5-triazole amino acids were successfully synthesized utilizing both copper (I) and ruthenium-catalysed cycloaddition of functionalized azides and alkynes. Furthermore, contained within Chapter 4 are the details and results of in vitro binding assays used in screening for possible active compounds. As an example, in vitro drug screening at NMDA, kainate and AMPA ionotropic glutamate receptor subtypes revealed activity of triazole amino acid 48 with an EC₅₀ value of 49 μ M at AMPA receptors. Also, drug screening at metabotropic glutamate receptor subtypes 1, 2 and 4 revealed potent agonist activity of cyclopropane amino acid 44a at mGluR2 with an EC₅₀ value of 0.05 μ M. Cyclopropane amino acid 44a was thus selected for further testing in vivo in a rodent model of neuropathic pain. The results indicated that cyclopropane amino acid 44a significantly and dose-dependently decreased mechanical allodynia, one of the symptoms of neuropathic pain. It was suggested that this effect was due to activation of mGlu2 and 3 receptors located on both neuronal and glial cells within the dorsal horn of the spinal cord. Lastly, in an effort to rationalize the in vitro binding data, the newly synthesized cyclopropane and triazole amino acids were docked in silico into the NMDA, AMPA, mGluR1 and mGluR3 receptors available as x-ray crystal structures. Only limited data was obtained regarding the mGluR1 and mGluR3 dockings. However, AMPA receptor docking of the new in vitro active triazole amino acids 45 and 48 revealed positive docking interactions in agreement with those seen for the endogenous ligand, glutamate and the selective agonist AMPA. The docking of these new compounds was also computed to be highly energetically favourable, thus suggesting plausible binding modes.Item Open Access Utilising CYP199A4 from Rhodopseudomonas palustris HaA2 for biocatalysis and mechanistic studies(2016) Chao, Rebecca; Bell, Stephen G.; Abell, Andrew David; School of Physical SciencesThe cytochrome P450 enzyme CYP199A4 from Rhodopseudomonas palustris strain HaA2 is highly specific for the regioselective oxidation of para-substituted benzoic acids. A selection of these compounds was tested with the enzyme with the aim of investigating the mechanism of different P450-catalysed reactions. These studies revealed that the binding affinity and oxidative activity of CYP199A4 is influenced by the substituent at the para-position, and that to the enzyme’s known oxidative activities (demethylation, hydroxylation, heteroatom oxidation and desaturation) can be added alkene epoxidation, alkyne oxidation and aldehyde oxidation. The active oxidants involved in these CYP199A4-catalysed oxidations were investigated using two active site mutants at the conserved acid-alcohol pair, T252A CYP199A4 [CYP199A4 subscript] and D251N CYP199A4 [CYP199A4 subscript], which should disrupt different steps of the catalytic cycle. There was a general increase in hydrogen peroxide uncoupling in the T252A CYP199A4 [CYP199A4 subscript] mutant but significant levels of product formation were observed with each substrate. The D251N mutation reduced the activity of the enzyme dramatically in all but one case, suggesting that this mutation interferes with proton delivery as expected. The elevated rate of 4-ethynylbenzoic acid oxidation by T252A CYP199A4 [CYP199A4 subscript] when compared to the wild-type enzyme suggested the involvement of Cpd 0 in alkyne oxidation, while a reduction in activity with 4-methoxybenzoic acid implicated Cpd I in demethylation. Additionally, the notable increase in product formation and coupling efficiency of D251N CYP199A4 [CYP199A4 subscript] with 4-formylbenzoic acid suggested the involvement of the peroxo-anion in aldehyde oxidation. Larger cinnamic acids and closely related substrates were also investigated with CYP199A4. The binding affinity and oxidative activity of the enzyme decreased in the order 4-methoxybenzoic acid > 4-methoxycinnamic acid > 3-(4- methoxyphenyl)propionic acid > 4-methoxyphenylacetic acid, highlighting its selectivity for a planar, benzoic acid- or cinnamic acid-like framework. The exclusive oxidation of cinnamic acids and related derivatives at the para-position further demonstrated the high regioselectivity of CYP199A4. While CYP199A4 exhibited low oxidation activity towards para-methoxy substituted benzene derivatives, considerably higher levels of activity reminiscent of the demethylation of 4-methoxybenzoic acid were observed for the Ser244 → Asp244 (S244D) mutant of CYP199A4. The exclusive demethylation of the para-methoxy substituted benzenes by S244D revealed that the regioselectivity of CYP199A4 oxidation is maintained in this mutant. The regioselectivity of the S244D mutant was further investigated using a selection of methyl- and ethyl-substituted derivatives. The methyl analogues were exclusively oxidised at the para-position to a single α-hydroxylation product. α-Hydroxylation and Cα [α subscript] -Cᵦ desaturation products were generated in the turnovers of the ethyl derivatives. The alcohol was formed with high stereoselectivity. The electronic properties of the ethyl substrates were found to influence the ratio of hydroxylation/desaturation product, with the more electron donating substrates giving rise to a greater proportion of the latter. This suggested the involvement of a cationic intermediate in CYP199A4- catalysed desaturation.Item Open Access β-strand mimicry as the basis for a universal approach to protease inhibition.(2011) Jones, Seth Adam; Abell, Andrew David; School of Chemistry and PhysicsThis thesis describes the design, preparation, and testing of a range of protease inhibitors. Chapter One introduces the concept of peptidomimetics, and discusses how proteases almost universally bind their ligands in a β-strand conformation. The idea of constraining a compound into a biologically active conformation by the introduction of a ring or bridge is discussed. The technique of ring closing metathesis as a strategy for macrocyclisation is introduced. The chapter also discusses calpain and HIV proteases and their structures and implications in human disease. Chapter Two surveys the acyclic calpain inhibitors reported in the literature. A series of N-heterocyclic peptidic calpain inhibitors were docked in silico into an ovine m-calpain homology model using Glide, which revealed that compounds 2.60 – 2.67 all adopted a β-strand conformation upon binding. The modelling revealed low energy conformations of 2.60, 2.61 and 2.66 not in a β-strand geometry. The synthesis and testing of these inhibitors is described, with 2.63 displaying an IC₅₀ of 40 nM against m-calpain in an in vitro assay. Chapter Three describes the design and synthesis of the β-strand mimic macrocycle 3.8, which was prepared using ring closing metathesis. The chapter also describes the design of a number of calpain and HIV protease inhibitors that incorporate 3.8. Each inhibitor is designed to bind and inhibit a specific protease target. Chapter Four describes the synthesis and testing of a series of macrocyclic calpain and proteasome 20S inhibitors. The preparation of the aldehydes 3.9 and 3.10 by elaboration of the macrocycle 3.8 is described. As well, the preparation of 3.10 from the N-capped 4-fluorosulphonyl diene 4.4 is described. The most potent macrocycle in the series was 3.10, which displays an IC₅₀ against m-calpain of 2000 nM, and an IC₅₀ against the chymotrypsin like activity of proteasome 20S of 2 nM. Chapter Five describes the synthesis of a series of building blocks, and their use in the attempted preparation of the potential HIV protease inhibitor 3.12a, as well as the successful preparation of the potential HIV protease inhibitors 3.11 and 3.12b. Preliminary studies testing the biological activity of compounds 3.11, 3.12b and 5.21 found that they displayed a percentage inhibition of HIV-1 subtype B protease of 86, 63, and 26%, respectively. The Ki of 3.11 against HIV-1 subtype B protease was also determined to be 62 nM. The activity of 3.11 against HIV-1 protease establishes that the common macrocyclic core 3.8 can be incorporated into inhibitors of both calpain, and HIV-1 protease. Chapter Six describes the preparation of a key macrocycle by cross-metathesis. The preparation of 6.4 by cross-metathesis of the olefins 6.5 and 6.24 is described, as well as the elaboration of 6.4 to give the macrocycle 6.1. A systematic study of the cross-metathesis of the olefins 6.5, 6.6, 6.23 and 6.24 is described. Their percentage conversion to 6.4 was calculated using high performance liquid chromatography analysis. The highest conversion to 6.4 was found to be 60%, from the cross metathesis of an equimolar mixture of 6.6 and 6.23. Chapter Seven describes a multi-gram synthesis of the potent macrocyclic calpain inhibitor CAT0811. The key step in the synthesis is the base induced macrocyclisation of the iodopeptide 7.10 to give 7.6. The macrocycle 7.6 was also prepared by macrolactamisation of the pseudopeptide 7.9. The synthesis was found to be scalable, affordable and efficient, and removes the need for Grubbs’ 2nd generation catalyst (II).