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Naturally-sourced biofunctional peptides have been widely studied for many years. Their clinical potential has become increasingly relevant in so many aspects including antimicrobial and anticancer fields. In this work, short, bioactive helical peptides were analysed that were identified in amphibian skin secretions. Their bioactivity was examined, and several structural alterations were made in order to improve the peptide sequences that had the potential to be used in medicine. In Chapter 3, Temporin-WY2 was isolated from frog skin secretions and then further analogues were designed by changing its amphipathy and a lysine-cluster region was introduced. The in vitro and in vivo antimicrobial activity of the peptides was studied. QUB-1426 and 6K-1426, with potent antimicrobial and anticancer activities both in vitro and in vivo, were regarded as competitive drug candidates. In Chapter 4, the structure-function characteristics of the peptides were studied. This work described a clear relationship between the peptide function and physiochemical parameters including charge, secondary structure, hydrophobicity and amphipathy. 6K-9A with an ideal charge, hydrophobicity and well-defined α-helical structure, was the most potent candidate peptide found here. In Chapter 5, the peptide modification study revolved around a known, moderately functional peptide, aurein 1.2. The introduction of known sequences into the peptide proved an efficient option for short peptides and here, the -IIKK- cell-penetrating region-modified analogues demonstrated outstanding anti-cancer activities, and the KLA-modified analogue, KLA-2, was the most potent antimicrobial agent here.

Mammalian cells are highly sensitive to variations in cues provided by their surroundings. Directed cell migration is key to many physiological processes and there is a need for tissue engineering materials and structures to encourage optimised cell response. This thesis describes the creation of different types of gradients in collagen films and investigates their physicochemical properties and resultant cell behaviour. Crosslinking gradients were formed by gradual immersion of collagen films into different crosslinking solutions. The use of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride with N-hydroxysuccinimide (EDC/NHS) influenced both the mechanical and chemical properties, while crosslinking using genipin altered mechanical behaviour without changing surface chemistry. By adjusting the rate of immersion and solution concentration, gradient shape could be controlled. The effect of crosslinking chemistry was evaluated on uniformly treated films, using tensile testing, atomic force microscopy, and (2,4,6-Trinitrobenzene-1-sulfonic acid). An increase in stiffness from 3.5 to 26.6 MPa was obtained by varying the concentration of EDC/NHS solutions and from 3.5 to 23.9 MPa in the case of genipin. Cell response to mechanical and chemical variations based on crosslinking and peptide dendrimer (IKVAV) attachment was tested using human dermal fibroblasts (HDFs), fibrosarcoma cells (HT1080s) and rat Schwann cells (RSCs). HDFs showed an unfavourable response to high EDC/NHS crosslinking levels, but increased proliferation with increasing genipin crosslinking. The presence of IKVAV dendrimers enhanced RSC adhesion and proliferation. On gradient films, the variation in chemistry and stiffness created by EDC/NHS presented competing effects. The response of HT1080s was dependent on the type and profile of crosslinking gradient. On IKVAV gradients, migration of RSCs towards higher concentrations was promoted, whereas HDF movement was discouraged. This body of work demonstrates, for the first time, the use of this method in forming property gradients in collagen, which can provide cues to achieve directional cell response.

Uranium is a toxic heavy metal, causing biochemical harm to humans and other living organisms if present in significant concentrations. The mechanisms behind this toxicity have been the subject of considerable investigation since the days of the Manhattan Project and remain important today with respect to the safe management of contamination arising from the nuclear energy industry. It is widely acknowledged, however, that there are gaps in understanding, particularly relating to the behaviour of uranium species in complex biological environments and the precise molecular interactions with proteins and other biomolecules. Chapter 1 introduces some fundamental uranium chemistry and photophysics, alongside a brief survey of the current analytical techniques available to monitor uranium in the environment, and of the current understanding with regards to uranium-protein interactions. Chapters 2 and 3 present the successful application of upconverting nanoparticles (UCNPs) to the quantitative detection of aqueous UVIO22+ ions. To the best of our knowledge, this is the first time that such a system has been reliably demonstrated with UCNPs, and likely one of the first UVIO22+ sensors to employ excitation within the near infrared 'biological window'. A brief introduction to the photophysics of upconversion and the wider UCNP field is presented in Chapter 2, alongside results characterising the design and synthesis of suitable nanoparticles. Chapter 3 describes the results of UVIO22+ sensing studies which combine UCNPs with one of three organic dyes: arsenazo-III, Br-PADAP and Br-PAPS. An appendix at the end of this thesis briefly investigates the possibility of luminescence energy transfer between UCNPs and uranyl(VI), in response to literature published during the course of the PhD. Chapters 4 and 5 present complementary studies investigating the interactions of UVIO22+ with a range of de novo coiled coil peptides, synthesised in collaboration with the Peacock group (University of Birmingham). In Chapter 4, the suitability of analytical techniques is assessed with an initial library of peptides; one peptide system (CS1-2) is found to exhibit remarkably bright uranyl emission at room temperature and is therefore subject to further detailed characterisation, with a focus on luminescence spectroscopy. In Chapter 5, two further studies are described, prompted by the results from Chapter 4. The first explores the impact of phosphorylation on peptide-uranyl affinity and luminescence; the second explores the selective complexation of UVIO22+ and Eu3+ within a heterobimetallic coiled coil scaffold, and the possibility of intermetallic energy transfer therein.

One-Carbon Bridge Stapling for use in Peptide-Drug Conjugates Bethany M. Cooper Therapeutic peptides lie in between small molecules and therapeutic proteins in terms of their molecular weight and have many advantageous properties, such as high potency and selectivity. Yet one fundamental shortcoming of therapeutic peptides is their poor stability. Two-component peptide stapling (2C-PS) is an incredibly valuable tool used to increase a peptide's stability and provide a point for further functionalisation. This thesis describes the development of a 2C-PS methodology to mimic a peptides' disulfide bond, and the synthesis of two peptide-drug conjugates (PDCs) using this methodology. 1.0 The Development of a One-Carbon Bridge Peptide Stapling Methodology Disulfide bonds are fundamental in many proteins and peptides; they are responsible for determining the tertiary and secondary structure respectively. Unfortunately these bonds are reversible in a cellular environment; installation of a functionalisable one-carbon bridge provides stability and is irreversible. Four peptides were synthesised, and many reaction conditions utilising 2C-PS were screened with staples bearing either an reactive alkynyl or a 1,1-dichloro motif. The stapling using the latter led to observation of the desired stapled peptide. 2.0 The Development of a Colorectal-Targeting Peptide-Drug Conjugate Peptide-drug conjugates (PDCs) are a class of targeted therapeutics that aim to address adverse events experienced by non-targeting therapeutics. A novel alkynyl bearing staple was synthesised, with stapling yielding a one-carbon bridged colorectal cancer homing peptide. A model reactive oxygen species (ROS) cleavable linker with 7-hydroxycoumarin was analysed using fluorimetry and quencher studies, to confirm hydrogen peroxide dependent release. The same observation was determined for three cytotoxin ROS-cleavable conjugates. Finally, two PDCs were synthesised through a CuAAC reaction with cytotoxin ROS-cleavable linkers. The PDCs were subjected to HPLC hydrogen peroxide cleavage assays that demonstrated rapid cytotoxin release. Cellular assays were used to evaluate the effect of stapled peptides and the PDC on HCT-116 cells. This thesis provides a versatile platform for the development of a novel ROS-cleavable PDC, which can be applied to a range of cancers for specific payload delivery.

This thesis explores aspects of cucurbit[n]uril (CB[n]) chemistry that can be utilised and exploited in the formation of materials suitable for therapeutic peptide delivery and diagnostics. Biomedical applications of CB[n] span simple therapeutic complexation leading to increased solubility and circulation times as well as their incorporation into more complex designs. CB[7] exhibits the strongest binding affinities of the CB[n] family as well as the highest water solubility, making it of high biomedical interest. Therefore, it is the homologue that is investigated throughout this thesis. Chapter 1 introduces the CB[n] family, taking the reader from synthetic functionalisation, an overview of CB[n] utility in biomedical applications such as peptide delivery and analyte detection, ending with a brief description of techniques used to characterise CB[n] complexes. In Chapter 2, isothermal titration calorimetry methodology is developed for the characterisation of ultra-tight CB[7]-guest binary complexes enabling accurate determination of complexes with affinities up to 10¹⁷ M⁻¹. This methodology is then used to assess the binding strength between CB[7] and immunogenic peptide, SIINFEKL, modified with a variety of binding motifs in Chapter 3. In vitro investigations into the effect of binding strength on the bio-activity of SIINFEKL peptides complexed with CB[7] is assessed. The ideal binding motif is dependent on the mechanism of therapeutic action. Functionalisation of CB[n] opens up avenues for its use in more complex architectures. Chapter 4 explores the functionalisation of CB[7] with short thiolated poly(ethylene) glycol (PEG) chains (CB[7]SH) and reports the facile route towards isolation of allyloxy CB[7] species with a definitive number of functional groups. Following these successful results, methodology was applies to CB[8] resulting in an allyloxy CB[8] species, yet to be reported in the literature. These preliminary results are reported in the Appendix. The thiol terminal of CB[7]SH synthesised in Chapter 4 is subsequently used in Chapter 5 to tether CB[7] to the surface of gold nanoparticles (AuNPs) creating a delivery system whose surface chemistry can be readily modified with therapeutic and targeting agents. Preliminary in vitro investigations into the delivery of a pro-apoptotic peptide, (KLAKLAK)₂, are presented as well as a perspective as to where the use of such AuNP-CB[7] structures could be utilised in biomedicine. Chapter 6 investigates an additional CB[7] bioapplication: the self-assembly of AuNP aggregates stabilised with thiolated PEG molecules. Aggregates are shown to disassemble in the presence of common biomolecules found in intracellular and extracellular fluid with timescales that can be tuned depending on the molecular weight of the selected PEG chains. Such aggregates were also shown to be suitable SERS substrates demonstrating a new class of materials that could be developed for in vivo SERS measurements and diagnostics. Chapter 7 ends the thesis with a brief overview as to what has been achieved as well as preliminary results regarding the functionalisation of CB[8], providing a foundation that could be developed in future works. Overall, this thesis focuses on the utilisation of CB[7] in a variety of applications and highlights important design principles that must be considered when employing this unique macrocycle in biomedical applications.

The aims of this thesis were to investigate the immune response to linear peptide fragments derived from 15 distinct proteins encoded by schizophrenia-related genes. An antibody test was developed in-house to detect plasma IgG antibodies against selected target molecules in patients with schizophrenia and control subjects. Six peptide antigens derived from DPYD, MAD1L1, ZNF804A, DRD2, TRANK1 and MMP16 showed an increase in plasma IgG levels and three derived from TSNARE1, TCF4 and VRK2 showed a decrease. As the initial study was conducted with medicated patients with chronic schizophrenia and healthy controls from a European-derived Caucasian population, the initial findings were thus replicated in antipsychotic-naïve patients with first-episode schizophrenia from a Chinese population. The drug-naïve patients showed different results; notably, the significant decrease in plasma IgG levels against TSNARE1 was not observed, showing instead an increase. The inconsistency may result from different ethnicities, B-cell diversity, medication or other confounding factors. To examine the possible mechanism underlying abnormal immune response to these schizophrenia-related peptides, a B-cell model was developed with four immortalized B-cell lines derived from a Caucasian population, 2 cell lines derived from healthy donors and 2 from patients with schizophrenia. These four B-cell lines have proportionally different stages of maturation and differentiation, showing distinct responses to treatment either with target protein-derived antigens or antipsychotic drugs. For example, TRANK1-derived peptide antigen could induce an increase in the proportion of CD83 positive cells and apoptotic cells; antipsychotic treatment generated a dose-dependent reduction in both cell number and viability. Taken together, this PhD work identified novel autoantibodies that might play a role in underlying schizophrenia. A possible mechanism by which abnormal immune responses contribute to the development of schizophrenia was then discussed.

With the rapid development of antibiotic resistance, new types of antimicrobial drugs are urgently needed. A novel AMP, Brevinin-1H, was isolated from skin secretion of Amolops hainanensis (A. hainanensis) with broad spectrum antimicrobial activity, anti-proliferative activity, and strong haemolytic activity. Two analogues were designed based on the secondary structure. Results revealed that the helical structure is critical for antimicrobial activity and haemolytic activity of peptides. The kink structure is important in antimicrobial activity against Gram-negative bacteria. A novel AMP, brevinin-1W peptide, was identified from skin secretion of Amolops wuyiensis (A. wuyiensis) with antimicrobial activity against Gram-positive bacteria, anti-proliferative activity, and strong haemolytic activity. A series of peptides were designed based on brevinin-1W peptide. Results indicated that proline and glycine could both influence the bioactivity of peptide while the proline exhibited stronger impact than the glycine residue and where the kink structure interrupted helical structure, was critical to balance the antimicrobial activity and haemolytic activity. To test the interactions between positive charges and hydrophobicity with bioactivity of peptide, five analogues were designed based on peptide PSN-PC. The hydrophobicity played a more important role in haemolytic activity compared with positive charges. Therefore, the balance between hydrophobicity and positive charges is essential for peptides to balance the cytotoxicity and antimicrobial activity.

This thesis examines a range of bioactive peptides from the frog skin secretions and scorpion venoms. In chapter 3, studies focused on a novel brevinin-1 type peptide, brevinin-1GHd, found in frog Hylarana guentheri, skin secretion. Functional screening revealed that brevinin-1GHd exhibited potent antimicrobial, antibiofilm, and anticancer efficacy without inducing obvious haemolysis and cytotoxicity at working concentrations, which made it promising to be further developed as an alternative novel antibiotic agent or used as a template to design some derivatives with improved functions. In chapter 4, a novel phylloxin type peptide, PL-PB1, was identified from the skin secretion of the frog, Phyllomedusa baltea. Besides studying its potential applications, seven derivatives were designed and subjected to a set of experiments to study the structure-activity relationship. In chapter 5, a novel non-disulphide-bridge peptide (NDBP), AaHAP1, was characterised from scorpion Androctonus australis Hector venom. To improve its therapeutic potential, five analogues were designed based on previous modification experience to acquire improved functions. The identification and modification of these naturally-occurring antimicrobial peptide could provide valuable experience for subsequent peptides design. The modified peptides such as 3W, KK and RV5, with obviously improved bioactivity are promising to be further developed as excellent candidates to fight with the fast- growing drug resistance strains.

Antimicrobial resistance is a growing threat that will have profound effects on global health and the economy. In order to avoid a worst-case scenario, there is a need to develop structurally distinct classes of antibiotics. Herein, the synthesis of cyclic lipopeptides based on the tridecaptins is reported. In particular, analogues where residues D-Trp5 and Phe9 were substituted for D-Cys and Cys respectively were synthesized and cross-linked with various benzylic linkers of various sizes. Ortho-xylyl, meta-xylyl and para-xylyl cross-linked Oct-TriA1 maintained strong activity against Escherichia coli and other ESKAPE pathogens (including carbapenem-resistant Acinetobacter baumannii). Interestingly, these analogues were resistant to enzymatic degradation in the presence of recently reported D-stereoselective peptidases. Secondly, brevicidine represents a promising candidate for further research and development and this ultimately relies on a synthetic route to acquire sufficient material. The first reported synthesis of brevicidine is reported within this thesis. Briefly, an on-resin approach was used to pre-form the sensitive macrocyclic ring with a modified Yamaguchi esterification. Subsequent Fmoc-SPPS and global cleavage yielded the desired peptide in impressively high yields. With a synthetic route in hand, early structure-activity relationship studies were commenced. Subtle alterations did not ablate the antimicrobial activity while theoretically stabilizing the peptide towards in vivo hydrolysis. Finally, attempts were made to synthesize various lipid tails to install on the depsipeptide globomycin. The natural and non-allo versions of linear globomycin were synthesized and tested using a recently developed fluorescence resonance energy transfer (FRET) assay developed by the Caffrey group.

Biopharmaceuticals, including proteins and peptides, are becoming increasingly important as therapeutic agents. However, the clinical use of protein and peptide therapeutics is still restricted due to undesirable properties such as their ability to self-assemble and aggregate. These properties not only influence production processes and storage but can also have adverse immunogenic effects within humans. To enhance their use, it is of great importance to understand and be able to manipulate their aggregation behaviour. In vivo, protein- and peptide-based drugs suffer from different problems, such as short lifetimes and low stability. Lipidation of peptides is widely established as a means of increasing stability in vivo. However, relatively little is known about the effect of lipidation on peptide self-assembly and other aggregation phenomena in vitro. The aim of this study is to develop a better understanding of the mechanism of aggregation and amyloid fibrillation of the therapeutic peptide GLP-1 and chemically modified forms of GLP-1. GLP-1, a metabolic hormone, has the ability to decrease blood sugar levels in a glucose-dependent manner by enhancing the secretion of insulin. Previous measurements of GLP-1 aggregation revealed that, at certain pH values, unusual behaviour is observed that has established that the standard nucleation-polymerization mechanism is insufficient to fully describe the reaction under these conditions. To study the aggregation mechanism of GLP-1 and chemically modified forms of the peptide, aggregation kinetics were measured over a wide range of different conditions. In addition, many biophysical techniques, such as AFM, SEM, far-UV CD, FT-IR, λmax, ANS, ex situ ThT and DLS, were employed to probe the structure, size and properties of species in solution during aggregation. The influence of amidation at the C-terminus of GLP-1 on physical stability was assessed and significant differences to GLP-1 were found. The study of this relatively small modification generates a better understanding of the interdependence of net charge, solubility and secondary structure on the aggregation kinetics. The results of these studies also provide further evidence that peptides belonging to the GLP-1 family can form off-pathway oligomeric species that have a significant impact on the aggregation kinetics. Two lipidated analogues of C-terminally amidated GLP-1 (Am-GLP-1) were also studied and the results analysed in detail and compared to those obtained for GLP-1 and Am-GLP-1. Both lipidated peptides show a strong, nearly switch-like, pH dependence. Surprisingly, it was also shown that the amidation of the C- terminus had a bigger influence on the secondary structure of the peptides in comparison to the lipidation.

Peptides are useful modulators of protein-protein interactions (PPIs) and cellular membranes, both of which are traditionally challenging to target using small molecules. Often therapeutic peptides suffer from issues including poor proteolytic stability. Two-component peptide stapling can improve stability and enable facile peptide functionalisation. This thesis describes the development of functionalised stapled peptides for two biological applications. 1. The development of stapled peptides as chemical tools to investigate PPIs in human platelets Platelets are a vital, anuclear component of blood. The Bcl-2 family of proteins are known to be key mediators of apoptosis in nucleated cells, however the role of each Bcl-2 protein in platelet apoptosis and activation is unknown. Recently, stapled peptides were deemed useful chemical tools for studying PPIs in platelets. In this section, three polyarginine-functionalised stapled peptides were developed as Bcl-2 PPI inhibitors. These novel chemical tools are anticipated to provide valuable insight into the roles of Bcl-2 PPIs in platelet modulation, which could ultimately lead to new therapeutic targets. 2. The development of cleavable stapled peptide-drug conjugates to target Pseudomonas aeruginosa Antimicrobial resistance (AMR) is a major healthcare threat, and Gram-negative bacteria such as Pseudomonas aeruginosa pose a significant therapeutic challenge. Antimicrobial peptides disrupt bacterial membranes, however functionalised two-component stapled antimicrobial peptides (STAMPs) are underexplored. This section describes the development of a STAMP-drug conjugate, constructed by functionalisation of the STAMP staple with a small molecule antibiotic, attached via a β-lactamase-cleavable motif. The resulting conjugate combines two mechanisms of action, which is a validated strategy for overcoming AMR. To this end, two novel, unfunctionalised STAMPs were identified that exhibited good minimum inhibitory concentrations against P. aeruginosa (64 μg/mL) and selectivity over a Gram-positive strain, from a panel of seven novel STAMPs. Subsequently, a tractable synthesis of the proposed functionalised STAMP was investigated. It is hypothesised that the cleavable STAMP-drug conjugate will enable infection-controlled drug-release and dual-targeting of P. aeruginosa.

The diminished incretin effect is a pathological characteristic of type 2 diabetes mellitus (T2DM), caused by reduced secretion of glucagon-like-peptide-1 (GLP-1) and blunted biological actions of glucose-dependent insulinotropic polypeptide (GIP). Presently, clinically approved GLP-1 analogues, targeting the GLP-1 arm of the incretin axis, help to overcome the diminished GLP-1 secretion. However, currently there is no clinically approved therapeutic agent to potentiate the biological actions of GIP. Furthermore, established pharmacological agents currently fail to replicate the benefits of bariatric surgery, which along with increasing prevalence increases both health and socio-economic burdens. Likewise, due to the multifactorial characteristics, current monotherapies fail to elicit persistent effective management. Therefore, novel, more-effective treatment paradigms are required to address these unmet needs. The gut hormone xenin has been reported to potentiate biological actions of GIP, with truncated analogues of xenin displaying more potent antidiabetic effects. This thesis characterised the GIP-potentiating effects of xenin-based therapies, along with their therapeutic applicability for diabetes and/or obesity. This included xenin-8-Gln, as part of a hybrid agent- (DAla2)GIP/xenin-8-Gln, multiple novel xenin hexapeptides, as well as TNP-470, an angiogenesis inhibitor shown to increase xenin concentrations. Importantly, in all therapies assessed, there was clear augmentation of GIP action, resulting in enhanced metabolic effects. (DAla2)GIP/xenin-8-Gln administered in combination with exendin-4 demonstrated metabolic benefits that were persistent in nature, which is highly favourable in such a progressive disease. Further characterisation of truncated xenin peptides showed that ψ-xenin-6 was the most potent truncated hexapeptide assessed, with GIP-potentiating and satiety effects shown, along with biological effects evident up to 8 hours. Furthermore, repeated administration of ψ-xenin-6 in combination with sitagliptin further augmented the efficacy of sitagliptin, with effects more prominent and rapid with combined modulation. Additionally, beneficial efficacy of sitagliptin was augmented through combined administration with TNP-470, displaying improvements in glucose tolerance, insulin sensitivity and significant anti-obesity effects. Together, these findings provide reinforcing evidence for development of novel xenin-based therapies, which act to potentiate biological actions of GIP, and the need for combinational therapeutic approaches to target multiple pathways to help alleviate hyperglycaemia in T2DM, with potential for translation to the clinic.

Structural Dynamics of Amorphous Solid-State Macromolecular Materials at Terahertz Frequencies Talia Amira Shmool The aim of this thesis is to investigate the ability of terahertz time-domain spectroscopy (THz-TDS) to study complex amorphous solid-state systems. THz-TDS is a vibrational optical spectroscopy technique which can probe the intermolecular librations and vibrations of materials. Previous work has explored the use of THz-TDS in probing crystalline systems and amorphous small organic molecules, and uniquely this thesis focuses on studying amorphous macromolecular systems. In addition to contributing to the literature and increasing the understanding of the fundamental properties of amorphous macromolecular solid-state systems, this thesis provides a framework and methodology for assessing the stability of amorphous biological systems using THz-TDS. This thesis is organised as follows: Chapter 1 includes an introduction to terahertz spectroscopy and the theoretical framework relevant for understanding the behaviour of the biological systems investigated in the following chapters. Chapter 2 first provides a detailed explanation to THz-TDS data acquisition and analysis. Then, sample preparation methods for THz-TDS experiments are described and formulation fabrication is outlined. Finally, the methodologies used for each experiment conducted in this thesis are detailed. Chapter 3 introduces a qualitative physical interpretation of the dynamic motions of polymers that contribute to the β- and α-relaxation processes identified by THz-TDS and dynamic mechanical analysis data. These processes are then related to the combined contributions of temperature and free volume effects. Chapter 4 investigates the interactions between peptides and polymers at terahertz frequencies, with an emphasis on the molecular mobility behaviour leading to the glass transition temperature of lyophilised polymeric microspheres. Chapter 5 utilises THz-TDS to study the vibrational dynamic properties of lyophilised protein formulations complemented with Fourier-transform infrared (FTIR), circular dichroism (CD), and solid-state nuclear magnetic resonance (ssNMR) spectroscopy experiments to further investigate the structural changes observed for the formulations. Chapter 6 explores the extent to which THz-TDS can be used to track thermally induced conformational changes of spray-dried formulations. Extending this work, Chapter 7 examines a series of spray-dried formulations and connects the THz-TDS results to the trends observed in the storage stability study conducted for these formulations. Finally, Chapter 8 summarises the findings and contributions of this thesis and considers future experiments which would be interesting to perform as an extension of this work.

Signal transduction is one of the most fundamental biochemical processes that occurs in living organisms and involves the transmission of molecular signals from a cell's exterior to its interior. Foldamers of 2-aminoisobutyric acid (Aib) have shown potential in the creation of a transducer of structural information across nm distances, through a variety of covalent and non-covalent interactions both in solution and aqueous environment. This thesis details the advancement of these helical systems towards a functioning synthetic GPCR mimic capable of signal transduction. Foldamers containing ZnII-recognition sites at the N-terminus were used to recognise chiral carboxylates that switch on signal transduction and conformation control. A C-terminal glycinamide spectroscopic reporter was used to detect this conformation control in solution. A [ZnII(BQPA)]-binding site was pivotal for the creation of a foldamer as a dual NMR and CD probe which cannot only sense helical screw-sense preferences but also sense enantioselectivity of the binding carboxylate. An attempt to activate this functioning foldamer by the binding of a messenger, produced from a desymmetrisation reaction, was also described. An alternative C-terminal reporter was also assessed, not only in solution but in artificial membranes. A highly sensitive bis(pyrene) reporter was exploited for the fluorometric detection of induced screw-sense preferences in covalently controlled Aib foldamers. The effect of phospholipid chirality on foldamer conformation was also explored through the synthesis and incorporation of enantiomeric foldamers bearing the bis(pyrene) reporter. Finally, the synthesis of a ruthenium transfer hydrogenation catalyst and its attachment to Aib foldamers was explored aiming to create a foldamer that can initiate remote catalysis once activated.

Tandem-repeat (TR) domains occur in approximately one third of all proteins, yet these domains are less well understood compared with their globular counterparts. TR proteins act as molecular scaffolds that facilitate protein-protein interactions (PPIs) in diverse biological contexts. In disease pathologies such as cancers, misfolded, mutated or deregulated TR function is often the cause. However, targeting tandem-repeat proteins is challenging due to their flat and large interaction interfaces. In this thesis, I have explored the development of peptide-based inhibitors of PPIs involving two targets, Tankyrase (TNKS) the Anaphase Promoting Complex/Cyclosome (APC/C-Cdc20). TNKS belong to the poly-ADP-ribose polymerases (PARP) family of enzymes that PARylate their substrates, leading to ubiquitination and subsequent degradation. TNKS bind their protein substrates through an ankyrin-repeat domain comprising five so-called Ankyrin Repeat Clusters (ARCs). TNKS is of particular therapeutic interest in cancers due to its PARylation of Axin1, a subunit of the β-catenin destruction complex. We investigated the design of peptide inhibitors of TNKS ARC domains through a structure guided approach. We first attempted to expand the range of biophysical assays to assess ligand binding to TNKS ARCs. We then designed and tested a set of linear and chemically constrained peptides for their ability to bind to the ARCs. The APC/C is an E3 ubiquitin ligase that has broad substrate-specificity via its two WD40-domain co-activators, Cdc20 and Cdh1, which recognise three peptide motifs (degrons) on its substrates: the D-box, the KEN box and the ABBA motif. The regulation of mitotic exit is tightly controlled by modulating the levels of Cdc20 and Cdh1 through different stages of the cell cycle. Cdc20 overexpression is associated with many cancers. Currently, no specific inhibitors of the APC/C-Cdc20 exist in the clinic or in clinical trials. We designed a series of synthetic peptides based on the D-box to bind to Cdc20, and thereby modulate APC/C activity. Peptides were tested using surface plasmon resonance and differential scanning fluorimetry assays using recombinant Cdc20. Furthermore, D-box peptides were able to bind exogenous Cdc20 in mammalian cell lysates using a Cellular Thermal Shift Assay (CETSA). Lastly, I determined the co-crystal structures of three of the highest affinity D-box peptides bound to Cdc20, providing high-resolution insights into the binding interface, which should aid in the further development of Cdc20 inhibitors.

Type 2 diabetes mellitus (T2DM) is a leading non-communicable disease with increasing health, socio-economic implications and with pharmacological agents that fail to replicate the success of bariatric surgery. This thesis aims to evaluate the therapeutic potential of customised gastrointestinal (GIT)-derived peptide hormones, namely glucose-dependent insulinotropic polypeptide (GIP), xenin and neurotensin (NT) in the hope of advancing the therapeutic repertoire. GIP has important glucoselowering endocrine and exocrine actions that become impaired in T2DM. Xenin is cosecreted with GIP and known to potentiate its biological actions. Thus, the biological and therapeutic potential of twice daily administration of a previously characterised GIP/xenin hybrid, (DAla2 )GIP/xenin-8-Gln, both alone and in combination with exendin-4 was assessed in high fat fed and db/db mouse models of T2DM. In HFF mice, treatment with (DAla2 )GIP-xenin-8-Gln in combination with exendin-4 was the most effective therapeutic strategy, although (DAla2 )GIP-xenin-8-Gln alone induced notable benefits in this model. Interestingly, in the db/db model, (DAla2 )GIP-xenin-8- Gln alone was much less efficacious than combined treatment with exendin-4, most likely linked to the disease severity and notable beta cell dysfunction. Further to this, NT has several antidiabetic actions and is known to facilitate fatty acid absorption. Xenin is structurally related to NT, with similar biological actions, thought to be partially mediated through NT receptors. Preliminary in vitro studies revealed that the novel acetyl-neurotensin(8-13)-xenin-8-Gln hybrid had antidiabetic attributes that warranted further in vivo assessment. Twice daily administration of acetylneurotensin(8-13)-xenin-8-Gln in combination with exendin-4 to HFF mice had positive glucose-lowering and insulinotropic effects with beneficial actions on lipid metabolism. Interestingly, GIP action has been linked to the exacerbation of obesity and T2DM, increasing insulin resistance and fat deposition. Thus, postulation that inhibiting GIP action could potentially halt the progression of obesity-related diabetes. However, to date there is no definitively characterised peptide-based GIP antagonist. Manipulation of the amino acid sequence, with N- and C- termini truncation, yielded Pro3 (3-30)GIP as a notable GIP receptor antagonistic that merits further testing. Overall, these data show that modified GIT peptides possess notable therapeutic efficacy with potential for translation to human T2DM and obesity.

Anti-apoptotic proteins play a fundamental role in cell survival. Under physiological conditions, such proteins trigger apoptosis in defective or damaged cells only; under pathological conditions, however, they can be dysregulated allowing the cells to survive despite being harmful. Considering the importance of anti-apoptotic proteins in many physio-pathological roles, their specific inhibition is an attractive strategy to develop safe therapeutics. This thesis describes the inhibition of two classes of anti-apoptotic proteins: 1) Inhibition of the anti-apoptotic protein CK2 to develop novel anti-cancer molecules targeting pockets outside the well-conserved ATP-binding site: -Using a Fragment-Based-Drug-Discovery (FBDD) approach twelve small molecule inhibitors of CK2 were developed. The lead molecule, 3l, inhibited the catalytic activity of CK2α by binding in the cryptic αD pocket with a Kd of 4 μM. 3l stopped proliferation of colorectal cancer cells with a GI50 of 10 μM and presented improved drug-like properties and selectivity compared to previously reported inhibitors. Remarkably, 3l has the potential to be developed into a potent and selective anticancer drug. -Using a combination of rational-based approach and peptide stapling, twenty-two conformationally-constrained peptides were generated to target the protein-protein interaction (PPI) of CK2 and affect its function. The lead peptide, P7-F1C5, presented a novel, highly-functionalised constraint that allowed the molecule to become cell-permeable, exert its anti-proliferative activity in cancerous cells, and to become resistant to serum proteases. P7-F1C5 is the first macromolecule reported in the literature that binds to CK2α with sub-micromolar affinity (Kd 150 nM), and that can act as a chemical probe for targeting the PPI of CK2. 2) Inhibition of the anti-apoptotic Bcl-2 proteins to dissect their role in platelet activation and apoptosis. Bcl-2 proteins regulate cell lifespan; however, their role in non-nucleated platelets is not fully understood. The elucidation of these pathways in platelets is crucial to the development of selective anti-platelet therapeutics. To this end, this thesis describes the development and the first application of twenty-seven BH3-only peptides in human platelets highlighting how peptides can provide an alternative to conventional methodologies to study PPIs in platelets. The most promising peptide, P9-F5C5, engaged the anti-apoptotic protein Bcl-xL with 26 nM affinity and reviled a new role for the protein Bim in platelet activation.

Rheumatoid arthritis (RA) is a chronic joint disease of unknown aetiology. The autoimmune nature of RA is underlined by abundant generation of rheumatoid factor (RF) autoantibodies to IgG1 Fc, and anti-citrullinated protein antibodies (ACPA) to citrullinated autoantigens such as fibrinogen. Although RA pathogenesis has not been elucidated, genetic predisposition, environmental insults and viral pathogens are considered contributory factors. Human endogenous retrovirus K10 (HERV-K10) is one such virus as it retained the capacity to produce viral particles in RA synovium. This study set out to explore how HERV-K10 Gag matrix region could contribute to RA pathogenesis and perpetuation, with particular emphasis on its ability to mimic host autoantigens. We showed that Gag region exhibits high levels of sequence and structural homology to IgG1 Fc and it could provide a key epitope important for auto-reactivity in RA. Analysis of how HERV-K10 may evoke immune responses in RA was broadened by investigation of serological cross-reactivity of novel anti-K10 polyclonal antibody (PAbMAG) with IgG1 Fc. We showed that PAbMAG cross-reacted with linear and conformational epitopes on IgG1 Fc. In a further development, we showed a significantly elevated mean IgG response to HERV-K10 epitopes in serum samples from RA patients when compared to other arthritides. These data suggest that molecular mimicry between viral and host proteins has the potential to lead to antigen-driven high-affinity RF IgG immunological reactivity in RA. Finally, we broadened our study of mimicry in RA by the investigation of citrullinated autoantigens. Structural studies demonstrated high levels of homology between citrullinated fibrinogen, IgG1 Fc and HERV. We further explored how protein citrullination affects the cross-reactivity of autoantibody responses in RA. These experiments revealed that generation of neoepitopes through citrullination of HERV-K10 and autoantigens IgG1 Fc and fibrinogen enhanced the reactivity of RA sera to these targets. Moreover, we showed that RF autoantibodies could mediate responses to a classical ACPA target fibrinogen, only when it is citrullinated, in the absence of ACPAs. These data provide a new insight into the initiation and propagation of immunological responses in RA and how viral/host molecular mimics and citrullination could modulate serum cross-reactivity profiles in RA.

The N-methyl-D-aspartate (NMDA) receptors (NMDAR) are a subtype of ionotropic glutamate receptors that are highly expressed in the central nervous system (CNS) and are involved in the excitatory synaptic transmission and synaptic plasticity that underpin many critical CNS functions. NMDAR dysfunction has been implicated in Alzheimer's disease, neuropathic pain, schizophrenia, and depression, among others. Most non-selective NMDAR antagonists (e.g. ketamine) have undesirable side-effects that restrict their clinical utility for relieving symptoms of neuropathic pain and treatment-resistant depression, but indirect modulators of NMDAR function offer the potential to have reduced side-effects relative to non-selective antagonists. One approach is to inhibit the production of the NMDAR co-agonist, D-serine, which is produced endogenously by conversion of L-serine to D-serine by the enzyme serine racemase (SR). Inhibitors of SR that reduce the production of D-serine may therefore have therapeutic benefits in disorders associated with NMDAR hyperfunction. In this thesis, human SR(hSR) was expressed in a bacterial host and isolated to a high degree of purity (>90%). The crystal structure of SR was solved in-house using the purified protein, to a resolution of 2.2Å (in complex with malonate) and 1.9Å (holoenzyme). To measure SR activity, a coupled biochemical assay was optimised, in which produced D-serine is degraded by D-amino acid oxidase (DAO) and the resulting H2O2 is quantified by a chemiluminescence reaction with HRP and luminol to produce light. Assay parameters such as Kmand IC50 were determined, and following further optimisation to enable medium-through put screening, a single-point screen of 3000 fragments was performed to identify novel inhibitory hit matter. A final list of 61 specific hits was collated of fragments that did not exhibit nonspecific inhibition in a DAO counter screen, and demonstrated IC50 values against SR approximately 1mM or below. These specific hits were characterised with biophysical methods adapted for SR (thermal shift, microscale thermophoresis) to determine binding affinities and structure stabilisation. Finally, via crystal soaking, the crystal structure of SR bound to an inhibitory fragment (KD=960μM) was revealed to a resolution of 1.8Å.

This thesis uses neutron and x-ray reflectivity to measure the interfacial structures of three molecular components associated with bacteria. Firstly, the way in which the membrane targeting sequence of a cell division protein interacts with monolayer models for the inner leaflet of the inner membrane of bacteria was measured at the air-water interface. Secondly, the influence of lipopolysaccharide on a monolayer model for the outer leaflet of the outer membrane of Gram-negative bacteria was measured at the air-water interface, as well as how this lipopolysaccharide interacts with an antimicrobial peptide. Finally, the structure of a layer of protein found at the surface of a Gram-positive biofilm was measured at the air-water interface. Binding of the membrane targeting sequence of the MinD protein (MinD-mts) to the inner leaflet of the cytoplasmic membrane is thought to be key for bacterial cell division. Modelling this membrane as a monolayer at the air-water interface, it was found that the insertion of the MinD-mts increased with decreasing lateral pressure within the monolayer, as well as with increasing unsaturation of the lipid components, and the incorporation of cardiolipin into the monolayer. Lipopolysaccharide (LPS) is the major component of Gram-negative outer membranes, such as Escherichia coli, and can be considered as having three structural components: lipid A, a core oligosaccharide, and a variable polysaccharide chain. By incorporating LPS into a model membrane at the air-water interface, it was found that the polysaccharide chain undergoes conformational changes depending on the area per molecule. The effect of the antimicrobial peptide Pexiganan on the structure of this LPS layer was also determined, and was found to insert into the polysaccharide chain layer, but have no impact on the conformation of the chains. In nature, many bacteria live within a biofilm structure. A critical component of the Gram-positive Bacillus subtilis biofilm is a surface active amphipathic protein called BslA, which gives rise to the formation of the highly hydrophobic surface of the biofilm. The kinetics of this film formation, its thickness, and the lateral packing of the protein at the air-water interface, were measured using both neutron and x-ray reflectivity. It was found that a native BslA protein consistently formed the same structural film, whilst the structure of films formed by mutant proteins depended on the conditions under which the film was formed.

The evolution of drug resistant pathogens creates the need for the introduction of new antimicrobial drugs. Peptaibols, a class of naturally occurring peptides, contain large amounts of alpha aminoisobutyric acid (Aib). They are known to exhibit their antimicrobial activity by perturbing the membranes of pathogens. However, a comprehensive model of action for these peptides has not yet been identified. Aib residues support the formation of 310-helix conformation and it is thought that this secondary structure is important for their antimicrobial activity. It is possible to design small synthetic peptides, known as foldamers, endowed with specific properties; in particular, Aib-rich foldamers are used as a model for the understanding of the folding and membrane interaction of the naturally occurring species. The aim of this thesis is to investigate the conformational preference of monodisperse Aib-oligomers as well as understanding their interaction with bilayer membranes. A large set of spectroscopic techniques have been used to establish the conformation of Aib-rich foldamers both in solution and when bound to membranes. In particular: Raman, Raman Optical Activity (ROA), Infrared (IR), Vibrational Circular Dichroism (VCD), Linear Dichroism (LD) and Neutron Scattering (NR) were employed to provide new structural insights. These vibrational analysis (VA) and vibrational optical analysis (VOA) investigations in solution were focused on the identification of spectral features for 310-helix conformation, particularly with Raman and Raman Optical Activity spectroscopies. Spectroscopic markers for this conformation in the amide I region were successfully identified. Moreover, it is known that chiral Aib-rich peptides can show a right or left handed screw-preference based on the primary sequence. VOA studies successfully distinguished between peptides with opposite helicity. VCD, ROA, LD and NR of Aib-foldamers bound to membranes were shown to be useful for identification of conformational preferences of the peptides within the membrane as well as for determining their orientation in the bilayer, and ultimately the effect of the peptides on the membrane structure.

Ribosomal synthesized and post translational modified peptide natural products have attracted a lot of interest in the past decade. Backbone cyclization of the translated linear peptides is generally catalysed by specific enzymes giving them peptidase resistance, thermodynamic stability and various other physiological activities. These features have made backbone cyclic peptide to become an attractive resource for drug discovery. Here, we described the synthesis of linear peptides containing natural and unnatural residues and its biosynthetic mechanism to generate man-made cyclic peptides. In this thesis we used SPPS to make short and medium linear peptide chains, we purified them using HPLC, and analysed them using MS. We incorporated unnatural residues such as homocysteine, homoserine, aminoalanine, propargyl glycine and the substrates were subjected to different enzymatic reaction such as prenylation, heterocyclization and macrocyclization modification reactions to generate small macrocycles (4-6 residues), prenylated linear peptides, and patellamime analogues. The final products were analysed using LC-MS. In our results, we verified that kawaguchipeptin (kgp) gene cluster is responsible for the production of kawaguchipeptins through heterologous expression of the kgp gene cluster in Escherichia coli. The KgpF prenyltransferase was overexpressed and was shown to prenylate C-3 of Trp residues in both linear and cyclic peptides in vitro. We also found out that PatGmac can macrocyclise short peptides (4-6 residues) to generate small macrocyclic peptides. We also tested the flexibility of OscGmac using unnatural amino acid residues such as pseudoprolines and pipecolic acid that can mimic the heterocyle incorporated as the final residue in the natural product. Our results show that OscGmac recognises pseudoprolines before AYD(G) to process a linear peptide.

Recent approaches to constraining peptide sequences into more structurally-defined α- helical secondary structures, so-called peptide stapling, are discussed. Stapled peptides are a class of therapeutics that have been shown to more effectively target protein-protein interactions, which are harder to target using a classical small-molecule therapeutic approach. Stapling a peptide constrains it into a well-defined secondary structure. This more accurately mimics the protein-protein interaction making the peptide a more viable therapeutic. Starting from the p53-MDM2 interaction, a protein-protein interaction with important implications in cell health, a known peptidyl inhibitor of this interaction was stapled and analysed for increased α-helicity. This was achieved by using monomers that utilise the copper (I) alkyne azide cycloaddition as a cross-linking methodology, which has been less well researched in the context of peptide stapling. The viability of a novel stapled peptomer inhibitor approach, accomplished using a new, optimised monomer synthesis, is investigated. Additionally, the synthesis of a ligand series designed for use in the copper(I) alkyne azide cycloaddition is also discussed.

Bacteria have adapted to CAMP insult in many ways, this study has focused on two resistance mechanism for CAMPs, firstly the putatative potassium proton antiporter RosB has been shown not to efflux potassium with treatment of polymyxin B. The mechanism of resistance of RosB in Yersinia enterococcus was proposed to be a lowering of internal pH from a potassium efflux and a proton influx. The RosB homologue from Vibrio paraheamolyticus is able to efflux potassium with electrophile treatment, but unlike the potassium proton antiporter KefKC this potassium transport is not associated with CAMP resistance. The lack of resistance is likely to be due to an absence of intracellular pH regulation seen with the Vibrio RosB. The RosB homologue from E. coli YbaL has been shown to increase resistance to the electrophile NEM in high potassium media, but YbaL does not transport potassium when NEM is present. This suggests that YbaL is transporting another ion. The mechanism of ion transport for RosB is based on the sodium antiporter NhaA and NapA, this is due to similarities in the ion selection motif. There are differences between the Vibrio and Yersina homologue which would suggest that the Yersinia homologue is not transporting potassium. This second CAMP resistance mechanism studied was the ABC transporter Sap, with the focus of work done on the periplasmic substrate binding protein SapA. SapA binds the peptide within the periplasm and delivers it to the membrane domain for uptake in to the cell. It was not possible to detect any peptide binding to the SapA homologues from Klebsiella pneumonia or E. coli. Finally CAMPs purification was attempted under taken through Ni-NTA and chitin chromatography to produce human antimicrobial peptides. It was possible to 12 produce the human antimicrobial peptide HBD2 with thioredoxin fusion complex via Ni-NTA chromatography.

Ligand-observed NMR spectroscopy is frequently employed in early-stage drug discovery, often as an initial screen to narrow the field of potential drug-like molecules. However, its use is limited to this early stage. More information regarding binding mode can be extracted from these experiments via quantification, and this should help extend the remit of these experiments beyond simple screening functions. Initially, it was shown that the amount of signal that could be produced from an STD NMR experiment could be dramatically increased by careful consideration of the selective saturation pulse. By systematically shortening the Gaussian pulse and positioning it at specific offset positions, it was shown that these dramatic increases in signal are genuine and need not result in false positives. Quantitative STD NMR spectroscopy as applied to Hsp90 and a series of small fragment ligands provided evidence to suggest that the precise inter-atomic distances between a protein and ligand within a crystal structure correlate with both initial rates of STD build up, and T1-adjusted STD values. This precise correlation has implications for chemotype clustering and initial binding mode selection, something which should be useful in the absence of a crystal structure. Taking the same quantitative principles and applying to LOGSY experiments elucidated another, discrete property of protein-ligand binding. Examining the ‘LOGSY difference’ signal for protons of a ligand allows us to see what protons are in close proximity to conserved, bound water at the protein-ligand binding interface. This is fundamentally different to the information gained from STD experiments. Applying the insights to a protein of a different nature, Ras, it was shown that quantitative STD can be applied to proteins of both different size and structure. Furthermore, more evidence was acquired to suggest that conserved, bound water in the binding site really is responsible for generating LOGSY signal. In the absence of these molecules, as in Ras, proximity of a proton to an exchangeable tends to dominate. In addition we were able to show that these quantitative methods can be used together to help eliminate incorrect computationally generated docking poses. The work presented in this thesis provides evidence for the advantages of STD and LOGSY NMR spectroscopy in fragment-based drug discovery. The information that can be extracted from relatively simple ligand-observed NMR experiments should be used to provide more evidence at an earlier stage of the drug discovery process, hopefully reducing late-stage attrition and helping us get to the therapeutic drug molecules we need a little more quickly.

Dysfunctional or over and under expressed enzymes play a crucial role in a variety of diseases. A tool that can identify dis-regulated enzymes in individual patients would be beneficial and would allow personalised treatment. For this purpose, a 10,000 membered ‘spit-and-mix’ PNA-encoded peptide library with a cell penetrating peptide was synthesised and interrogated with K562 cell lysate and intact K562 cells. This allowed the specific enzyme activity pattern for ABL tyrosine kinase from both inside a cell and a lysate to be obtained. Hybridisation of this library with a DNA-microarray resulted in bio-fouling by the cell lysate, thereby preventing analysis of the phosphorylation pattern. To allow extraction and purification of the peptide library from the cell lysates, a His-tag was incorporated into the library, and enabled successful library analysis. In addition to this 10,000 member library, a focused 100 PNA-encoded peptide library was synthesised. The library included peptide sequences known to be phosphorylated by specific tyrosine kinases deregulated in acute lymphoblastic leukaemia (ALL) with a PNA-tag complementary to a DNA microarray. Different ligation methods to conjugate the peptides to PNA-tags were screened – this included amide coupling, copper catalysed azide–alkyne cycloaddition, strain promoted azide–alkyne cycloaddition and Diels–Alder cycloaddition. The inverse electron demand Diels–Alder cycloaddition between a tetrazine and norbornene was chosen as the preferred ligation method, and the reaction conditions optimised. To purify the library from cell lysate, a His-tag was again coupled to each member using the strain promoted azide–alkyne cycloaddition. To test the tetrazine ligation, fluorescence in situ hybridisation (FISH) was used in cells, whereby a fluorophore was ligated onto a tetrazine–conjugated PNA probe. This was hybridised onto an mRNA in fixed cells. Results indicated that the ligation needed further optimisation.

One of the requisites for a growth factor and a biomaterial in tissue engineering, cell therapy and regenerative medicine is the ability to control cell fate. Cells exist in a complex micro-environment consisting of extra-cellular matrix, growth factors, together with adjacent cells. Stem cell culture and modulation remains a challenge due to insufficient, undefined and costly culture systems. This thesis describes synthetic approaches that can modulate stem cell fate by the identification of new synthetic substrates for the growth of cancer, embryonic stem cells and potential short peptide sequences that can mimic the biological functions of the native cytokine used to culture stem cells. Glioma cancers exist as a heterogeneous population of cancer stem cells and cancer progenies. Scale up and spin coating of a polyurethane and polyacrylate polymers was done on agarose for the enrichment of the cancer stem cell population from glioma cells. A polyurethane, synthesised from poly(tetramethylene glycol) and 1,3-bis(isocyanatomethyl)cyclohexane spin (PU10) coated on an agarose surface, was identified to have a higher affinity for the cancer stem cell population over its progenies. By using this polymer to study the mechanism of the cancer stem cell adhesion, two niche components i.e. galectin, transferrin that are enriched by the polymer that contributed to the growth of the cancer stem cells were identified. A synthetic hydrogel (HG21) was identified as substrate for the culture of mouse embryonic stem cells (mESC) as a replacement for gelatin. mESCs were cultured on the hydrogel under undefined and defined conditions. Under both culture conditions, mESC pluripotency and naïve phenotype markers were verified. Marker profiles by immunostaining (Oct-4, Nanog), flow cytometry (SSEA-1) and qPCR (14 gene markers) of mESC grown on the hydrogel were comparable to gelatin, while enabling thermo-detachment for enzyme free passaging of mESC. To identify alternative substances to the cytokines used in stem cell culture, a microarray system was developed. The microarray system was developed initially with adhesion cellulose peptides printed onto polyacrylamide coated microscope slides. These slides were then screened for interaction with human embryonic stem cells (hESCs). After successful development of the cell based cellulose peptide microarray system, overlapping 25-mer peptides based on of basic fibroblast growth factor were synthesised, printed onto the same type of slide and screened with hESC. The screen identified “hit” peptides, which could potentially mimic the biological effects of the native cytokine on hESCs. These “hit” peptides were scaled up and tested in solution with hESC. In the linear form the peptides were not sufficient to sustain pluripotency and further optimisation is needed.

N-carboxy-α-amino acid anhydrides (NCAs) are amino acid derivatives discovered by Leuch in 1906. Ever since, they have found invaluable use in the preparation of peptide drugs and polypeptides. NCAs are generally synthesised using phosgene; however, due to the hazardous nature of this chemical, safer method of synthesis using protected amino acids in the presence of activating reagents such as P2O4, T3P, HBTU and BOP-Cl were investigated, where a protonated oxazolone intermediate is dealkylated to generate the corresponding amino acid NCA. After the successful synthesis of a variety of NCAs, starting from Boc-protected amino acids and using T3P, we attempted to work out a new method of sequential peptide synthesis using NCAs in a controlled way. The challenge involved in the use of NCAs in the synthesis of peptides is to prevent decarboxylation of the intermediate products, which lead to the generation of unwanted peptide oligomers. An elegant way to prevent the decarboxylation was studied in this work, which consists of the stabilisation of the carbamate intermediates using an appropriate receptor. We prepared several carbamate receptors and tested them in model reactions using amines and NCAs under various conditions. Despites partial success in limiting the amount of oligomerisation, we were never able to prevent it completely, and could not established conditions to prepare peptides in good purity.

A novel class of potential positron emission tomography (PET) radiotracers for imaging aminopeptidase N (also known as APN or CD13) and cannabinoid type 1 (CB1) receptors were designed and synthesised with an efficient chemical strategy. Both targets have remarkable diagnostic and therapeutic potential, in fact the CD13 receptors are over-expressed during tumour angiogenesis and the CB1 receptors are highly expressed in the brain playing important functions in several pathophysiological processes. The target compounds were obtained by means of oxime-bio-conjugation between fluoro-deoxy-carbohydrates, used as prosthetic groups, and hydroxylamine-functionalised cyclic NGR (asparagine-glycine-arginine) motif sequences for CD13 receptor and rimonabant-type pyrazoles for the CB1 receptor. In particular, aminooxy-cyclic NGR peptides were conjugated with the novel prosthetic group 5-FDR (5-fluoro-5-deoxy-D-ribose) and the aminooxy- pyrazole-type cannabinoid molecules were conjugated with both 5-FDR and with FDG (2-fluoro-2-deoxy-D-glucose). 5-FDR proved to be superior to FDG, as the bioconjugation reaction occurred in milder conditions (room temperature vs 100 °C) and at faster rate. Furthermore, we observed that the rate of the oxime bond formation depends on the solubility of the aminooxy-functionalized core used. In fact, the bioconjugation with hydrophilic cyclic aminooxy-NGR peptides was faster than in the case of lipophilic aminooxy-pyrazoles (10 min vs 20-30 min). The receptor affinity is decreased in the case of the CB1 receptors after conjugation with the fluoro-carbohydrates. This is not observed with the conjugated NGR peptides, which maintain similar affinity for the CD13 receptor compared with the unconjugated NGR. In conclusion, we have developed an efficient strategy for the synthesis of a novel class of CD13 ligands, which may be also produced in radiofluorinated form, and explored a novel bioconjugation strategy for CB1 receptor ligands. Both may have important applications in the development of PET tracers.