The unifying theme of this thesis is the exploitation of the reactivity of aryliodonium salts as electrophile transfer reagents. In the first part of the thesis, diaryliodonium salts are employed as arylation reagents for the enantioselective copper-catalysed arylative semipinacol rearrangement (SPR) of various tertiary allylic alcohols. This cascade reaction is a rare example of asymmetrically activating SPR using carbon electrophiles. Different substrate classes - including dihydropyran, indene and dihydronaphthalene moieties - are converted to enantioenriched beta-aryl spirocyclic ketones in excellent yields and enantioselectivities, and often as a single diastereomer. These are in turn useful functional handles for transformations into other moieties, including further rearrangements via Baeyer-Villiger oxidation. In the second part of this thesis, a two-step process for the macrocyclisation of native peptides via a non-natural linkage is developed. This study exploits previous work conducted in the group on the use of aryliodonium salts as methionine-selective diazoacetate transfer reagents. The functionalised methionine is in turn used for an intramolecular rhodium-catalysed insertion into tryptophan. Eventual translation onto solid-phase enables facile access into various macrocyclic peptides.

No description available.

Peptides are a promising class of therapeutic candidates due to their high specificity and affinity for cellular protein targets. However, peptides are susceptible to protease degradation and are typically not cell-permeable. In efforts to design more effective peptide drug discovery systems, investigators have discovered that incorporation of non-canonical amino acids (ncAAs) and macrocyclization overcome these limitations, making peptides more drug-like. In this work, we exploit the promiscuity of wild-type aminoacyl-tRNA synthetases (aaRSs) to ‘mischarge’ ncAAs onto tRNA and ribosomally incorporate them into peptides using a cell-free translation system. We have demonstrated the ability to incorporate five ncAAs into a single peptide with near-wild type yield and fidelity. We also demonstrated the in situ incorporation of ncAAs containing azide and alkyne functionalities, enabling the use of CuAAC (click chemistry) to generate triazole-bridged cyclic peptides. When combined with bisalkylation of peptides containing two cysteines via an α,α’-dibromo-m-xylene linker, we created bicyclic peptides which are structurally similar to the highly bioactive knotted peptide natural products. Biological display methods, such as mRNA display, are powerful peptide discovery tools based on their ability to generate libraries of >1014 unique peptides. We combined our ability to incorporate ncAAs with our bicyclization technique adapted for use with mRNA display to create knotted peptide library scaffolds. We performed side-by-side monocyclic and bicyclic in vitro selections against a model protein (streptavidin). Both selections resulted in peptides with mid-nM affinity, and the bicyclic selection yielded a peptide with remarkable protease resistance. We used a new library that enables the generation of a diverse collection of linear, monocyclic and bicyclic scaffolds in one pot, increasing the likelihood of target-ligand conformational alignment. We performed a second selection against streptavidin and revealed a nearly unanimous preference for linear peptides containing an HPQ motif, a known streptavidin-binding sequence. However, when we used these libraries for in vitro selection against a biological target, DNA repair protein XRCC4, we did not observe convergence. In summary, we have developed a novel technique for production of bicyclic peptide libraries. These highly-constrained protease-stable scaffolds can be used as platforms to identify high affinity, drug-like ligands using mRNA display.

<p>This thesis examines the isolation and characterisation (biological and chemical) of polypeptides from plants. A fractionation protocol was developed and applied on 100 plant materials with the aim of isolating highly purified polypeptide fractions from small amounts of plant materials. The polypeptide fractions were analysed and evaluated for peptide content and biological activities. A multitarget functional bioassay was optimised as a method for detecting substances interacting with the inflammatory process of activated neutrophil granulocytes. In this assay, the neutrophil was challenged with an inflammatory mediator, <i>N</i>-formyl methionyl-leucyl-phenylalanine (fMLP), or with platelet activating factor (PAF), to induce exocytotic release of the enzyme elastase, which then was quantified by photometric determination of the product p-nitroanilide (pNA) formed from a chromogenic substrate for elastase. Of the tested extracts, 41% inhibited pNA formation more than 60%, and 3% stimulated formation.</p><p>Phoratoxin B and four new peptides, phoratoxins C-F, were isolated from <i>Phoradendron tomentosum</i>. In addition, the cardiac glycoside digitoxin was isolated from <i>Digitalis purpurea</i>. All these substances expressed cytotoxicity and a neutrophil challenging activity.</p><p>Phoratoxins C-F were similar to earlier described phoratoxins A and B, which belong to the group of thionins. All the peptides were evaluated for cytotoxicity in a human cell line panel. Phoratoxin C was the most potent towards the cell lines (mean IC₅₀: 160 nM), and was therefore investigated further on tumour cells from patients. Correlation analysis of the log IC₅₀ values indicated a mechanism of action different from clinically used archetypal cytotoxic drugs. Phoratoxin C also showed selective toxicity to the solid tumours when compared to the haematological cancer types. The phoratoxin C was 18 times more potent towards the solid tumour samples from breast cancer cells (87 nM) compared to the tested haematological malignancies.</p><p>The structure-activity relationship concerning cytotoxicity was evaluated for digitoxin and related cardiac glycosides. Digitoxin was shown to be potent, with the average IC₅₀ 37 nM being within the therapeutic concentration used for cardiac congestion (13-45 nM). Digitoxin expressed selective toxicity towards solid tumours from patients compared to haematological malignancies.</p>

This thesis examines the isolation and characterisation (biological and chemical) of polypeptides from plants. A fractionation protocol was developed and applied on 100 plant materials with the aim of isolating highly purified polypeptide fractions from small amounts of plant materials. The polypeptide fractions were analysed and evaluated for peptide content and biological activities. A multitarget functional bioassay was optimised as a method for detecting substances interacting with the inflammatory process of activated neutrophil granulocytes. In this assay, the neutrophil was challenged with an inflammatory mediator, N-formyl methionyl-leucyl-phenylalanine (fMLP), or with platelet activating factor (PAF), to induce exocytotic release of the enzyme elastase, which then was quantified by photometric determination of the product p-nitroanilide (pNA) formed from a chromogenic substrate for elastase. Of the tested extracts, 41% inhibited pNA formation more than 60%, and 3% stimulated formation. Phoratoxin B and four new peptides, phoratoxins C-F, were isolated from Phoradendron tomentosum. In addition, the cardiac glycoside digitoxin was isolated from Digitalis purpurea. All these substances expressed cytotoxicity and a neutrophil challenging activity. Phoratoxins C-F were similar to earlier described phoratoxins A and B, which belong to the group of thionins. All the peptides were evaluated for cytotoxicity in a human cell line panel. Phoratoxin C was the most potent towards the cell lines (mean IC50: 160 nM), and was therefore investigated further on tumour cells from patients. Correlation analysis of the log IC50 values indicated a mechanism of action different from clinically used archetypal cytotoxic drugs. Phoratoxin C also showed selective toxicity to the solid tumours when compared to the haematological cancer types. The phoratoxin C was 18 times more potent towards the solid tumour samples from breast cancer cells (87 nM) compared to the tested haematological malignancies. The structure-activity relationship concerning cytotoxicity was evaluated for digitoxin and related cardiac glycosides. Digitoxin was shown to be potent, with the average IC50 37 nM being within the therapeutic concentration used for cardiac congestion (13-45 nM). Digitoxin expressed selective toxicity towards solid tumours from patients compared to haematological malignancies.

No description available.