Inositol phosphates play a fundamental role in many intracellular processes. Of particular importance is the role of phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] in the protein kinase B (PKB/Akt) signalling pathway. PtdIns(3,4,5)P3 recruits PKB to the cell membrane through binding interactions with its pleckstrin homology (PH) domain. In several human cancers, this signalling pathway is upregulated, resulting in increased cell growth and proliferation. In order to investigate the therapeutic potential of the PtdIns(3,4,5)P3–PH domain binding interaction, it is necessary to develop inositol phosphate-based probes. This DPhil dissertation highlights the synthesis of a number of derivatives of the PtdIns(3,4,5)P3 head group – inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. These derivatives incorporated phosphate isosteres at both the 3- and 5-positions of Ins(1,3,4,5)P4, through the utilisation of novel protection and deprotection strategies. In addition, this dissertation highlights the efficient synthesis of the natural product inositol 1,3-bisphosphate [Ins(1,3)P2] and our work towards the synthesis of inositol pyrophosphate derivatives.

The aim of the work described in this thesis was to explore the synthesis of glycosidase inhibitors and to evaluate their potential as anti-cancer agents. Glycosidases catalyze the fission of glycosidic bonds and are involved in vital biological functions. With regard to their potential for anti-cancer therapy, two glycosidases were identified: α-N-acetyl-galactosaminidase and β-N-acetyl-hexosaminidase. The former has been implicated in causing immunosuppression in advanced cancer patients by negating the effect of the macrophage activating factor (MAF), while the latter is secreted by invading cancer cells and hence associated with metastasis formation. The synthetic focus was on generating piperidine and azetidine iminosugars, carbohydrate mimetics with their endocylic oxygen replaced by nitrogen. Their structural similarity to carbohydrates make iminosugars excellent inhibitors of glycosidases. Following synthesis of a pipecolic amide, its previously reported potent β-N-acetyl-hexosaminidase inhibition was confirmed. This data, along with inhibition profiles of several pyrrolidines, allowed the generation of a molecular model for predicting activity of β-N-acetyl-hexosaminidase inhibitors. The model was used to select azetidines in the D/L-ribo and D-lyxo configuration as suitable candidates to be explored in novel chemical space, leading to the first synthesis of a fully unprotected 3-hydroxy-2-carboxy-azetidine. The potent α-N-acetyl-galactosamindase inhibitor 2-acetamido-1,2-dideoxy-D-galacto-nojirimycin (DGJNAc) was successfully derivatised via N-alkylation. Important structural discoveries with regard to glycosylation of vitamin D₃-binding protein, the precursor of MAF, were made using MALDI mass-spectrometry. By comparing the enzymatic and cellular inhibition of N-alkylated derivatives of DGJNAc and a pyrrolidine the following generalization on iminosugar biodistribution was found: N-butylation promotes uptake into the cell/organelles, while hydrophilic side-chains restrict cellular access. An in vitro assay evaluating cancer cell invasion was devised and β-N-acetyl-hexoamindase inhibitors were shown to retard cell migration, including with the highly metastatic breast cancer cell line MDA-MB-231. Additive effects where found when the iminosugar was combined with a protease inhibitor, suggesting potential for future combination therapy.

This thesis describes supramolecular approaches to porphyrin nanorings. Cyclic porphyrin arrays resemble natural light harvesting systems, and it is of interest to probe the photophysical effects of bending the porphyrin aromatic π-system. A general overview of the synthesis and photophysical properties of porphyrins and their arrays is carried out in Chapter 1. The electronic structure of porphyrins is examined, and how conformational effects in oligomers, such as inter-porphyrin torsional angle and backbone bending influence the π-conjugation pathway. The structures of light harvesting complexes are discussed. Chapter 2 describes the design and synthesis of a complementary 12-armed template designed to coordinate linear porphyrin oligomers in the correct conformation for cyclisation to give a cyclic porphyrin dodecamer. Chapter 3 demonstrates two approaches to a cyclic porphyrin dodecamer ring. Firstly, a classical templating approach using the 12-armed template is described. The limitations of this approach in the quest for larger nanorings are discussed. Vernier templating, which utilises a mismatch in the number of binding sites between a ligand and its receptor is introduced as a general strategy to the synthesis of large nanorings. This is demonstrated by the synthesis of cyclic dodecamer from a linear porphyrin tetramer and a hexadentate template via a figure-of-eight intermediate. The general utility of the Vernier method to large nanorings is explored in Chapter 4 with steps towards the synthesis of a cyclic tetracosamer, consisting of 24 porphyrin subunits. In preliminary experiments, an improved route to the cyclic porphyrin octamer is described. Finally, the photophysical properties of the nanoring series are explored in Chapter 5 as a function of size and conformation. Femtosecond photoluminescence spectroscopy shows that even in cyclic dodecamer, exciton delocalisation over the entire porphyrin backbone occurs on a sub-picosecond timescale, and parallels are drawn with the dynamics of natural light harvesting complexes.

This thesis describes the development of nitro-Mannich/hydroamination cascade reactions for the synthesis of N-heterocycles, which are important motifs found in a variety of biologically active natural products and pharmaceuticals, such as atorvastatin (Lipitor®). Chapter 2 outlines the development of an efficient synthesis of 2,5-disubstituted pyrroles using a nitro-Mannich/hydroamination cascade. Starting from easily prepared N-protected imines and nitroalkyne substrates, a compatible combination of KOtBu (10 mol%) and AuCl3 (5 mol%) was used to afford the desired pyrrole products, after an alkene isomerisation/HNO2 elimination reaction sequence. Chapter 3 describes the extension of this methodology to the diastereo- and enantioselective synthesis of 1,2,3,4-tetrahydropyridine derivatives using a nitroalkyne substrate with an extended carbon chain. The sequential addition of a bifunctional Brønsted base/H-bond donor organocatalyst and a gold complex was found to facilitate the desired cascade reaction affording substituted 1,2,3,4-tetrahydropyridine products. We then established that highly substituted pyrrolidine compounds could be prepared by replacing the nitroalkyne substrate with a nitroallene substrate (Chapter 4). The combination of KOtBu (5 mol%) and a gold catalyst derived from Au(PPh3)Cl (10 mol%) and AgSbF6 (20 mol%) was found to give an efficient diastereoselective synthesis of pyrrolidine derivatives after an additional nitro group epimerisation step. In addition, the nitro-Mannich/hydroamination cascade using nitroallene substrates was developed into an enantioselective variant using the previously employed bifunctional Brønsted base/H-bond donor organocatalyst. This afforded enantioenriched pyrrolidine derivatives.

Increasing antibiotic resistance has created a need to develop rapid and reliable methods to identify bacteria and provide pertinent information to ensure suitable antibiotics or sugar therapeutics can be chosen for treatment. Carbohydrate structures attached to proteins on host cell surfaces provide a binding point for many pathogens, including bacteria. These structures can be mimicked using single monosaccharides glycosylated to alpha-hemolysin (alpha-HL). Alpha-HL is a beta-barrel pore-forming toxin secreted by Staphylococcus aureus that forms an SDS stable heptamer, which can be expressed by coupled in vitro transcription and translation and purified by polyacrylamide gel electrophoresis. The purified heptamers can be reconstituted into planar lipid bilayers and studied at the single channel level. Through single channel recordings the effects of sugar-linker lengths, different glycans and the interaction between the ‘Glycopore’ and sugar binding molecules can be studied. The glycopore, therefore, acts as a scaffold for analysing protein-sugar interactions. Studies in this thesis have focused on the synthesis of carbohydrates for site-selective protein glycosylation; cloning and in vitro transcription translation of alpha-HL monomers; and glycosylation and oligomerisation of alpha-HL to form glycopores suitable for lectin-binding studies. Lectins DC-SIGN and FimH have been expressed in Escherichia coli and these lectins as well as others have been screened using alpha-HL glycopores. The glycopores have also been investigated with bacteria in serum in a controlled molecule-specific manner using single-channel electrical recording. In this work glycosylated alpha-HL-monomers have been found to form stable heptamers which can be formed by oligomerisation on red blood cell membranes. The purified glycopores were reconstituted into planar lipid bilayers and studied at the single-channel level. Through single-channel recordings an optimised glycopore has been shown to be effective in distinguishing lectins alone and in a mixture and has afforded qualitative and quantitative information about the binding interactions between carbohydrates and sugar binding proteins. Furthermore, the glycopore has been used to sense bacteria which may provide an insight into modes of bacterial infection. In addition, a multivalent glycopore has been formed which has proved preliminary information about the effects of multivalency in lectin binding. The design and synthesis of non-beta-lactam antibiotic candidates and their evaluation has also been carried out.

Iminosugars, carbohydrate analogues in which nitrogen replaces the endocyclic oxygen, have attracted much interest due to their biological activity. Iminosugars inhibit carbohydrate-processing enzymes, thereby affecting many biological processes. Several iminosugars are licensed drugs, with many more compounds undergoing clinical trials. The main subject of this thesis is the synthesis and evaluation of novel iminosugars, particularly the effects of structural modifications on the biological activity of these compounds. Chapter 1 describes the role of carbohydrate-processing enzymes in the body, and explores the therapeutic applications of iminosugars that arise from their activity against these enzymes. Examples of substituted iminosugars are reviewed, and the effects of substituents on enzyme inhibition are described. Chapter 2 concerns methyl-branched swainsonine derivatives. Swainsonine has shown potential as a cancer treatment through its inhibition of α-mannosidase. The synthesis of (6R)- and (6S)-C-methyl D-swainsonine is described; both compounds were potent and selective α-mannosidase inhibitors (IC₅₀ 3.8 μM, 14 μM). Although less active than the parent compound, their selectivity for Golgi mannosidase over lysosomal mannosidase may be more important than the absolute value against the model enzyme. Chapter 3 describes the synthesis of a 2-C-methyl L-fucose derivative. A diastereoselective Kiliani reaction allowed the formation of a single lactone bearing a new quaternary centre. The utility of this intermediate in accessing di-branched iminosugars was explored; however, attempts to introduce nitrogen to the lactone lacked the necessary stereoselectivity. Chapter 4 relates to the synthesis of pyrrolidine iminosugars, specifically methyl amides. Two enantiomeric dihydroxyproline amides were synthesised; the D-proline derivative was a potent β-N-acetylhexosaminidase inhibitor (IC₅₀ values of up to 3.6 μM), but the L-enantiomer was completely inactive. Inhibition of N-acetylhexosaminidases is relevant to the treatment of cancer and lysosomal storage diseases, and this work contributed to a wider project investigating the effects of altered stereochemistry on the biological activity of pyrrolidine amides.

Analogues of Antibacterial Natural Products Elizabeth Anne Heaviside, St Catherine’s College, University of Oxford DPhil Thesis, Trinity Term 2012 This thesis is concerned with the synthesis and biological evaluation of structural mimics for the natural products 16-methyloxazolomycin and lemonomycin which display potent biological activity including antibacterial and antitumour activity. Chapter 1 explores methods and approaches to the discovery of new antibacterial drugs and the challenges faced in this respect. It also gives an overview of the properties of the natural products investigated in the following chapters and summarises previous synthetic approaches to these molecules published in the scientific literature. Chapter 2 describes the work carried out towards the synthesis of the diazabicyclo[3.2.1]octane unit of the tetrahydroisoquinoline antitumour antibiotic lemonomycin. The intended retrosynthesis of the natural product led to a 2,5-disubstituted pyrrolidine bearing a 1ʹ-amino functional group; a series of routes were explored for the synthesis of this unit. Using (S)-pyroglutamic acid, strategies using Eschenmoser and thiolactim ether coupling reactions were investigated. A sequence based on the formation of a pyrrolidine ring from the cyclisation of an appropriately substituted oxime ether derived from L-phenylalanine was then implemented but a competing Beckmann rearrangement/Grob fragmentation prevented access to the desired heterocycle. Preliminary investigations were also carried out on the modification of cyclic imines derived from oxime ethers which did not undergo Beckmann rearrangement. Chapter 3 describes the synthesis of a library of densely functionalised tetramic acid and pyroglutamate mimics for the right-hand fragment of 16-methyloxazolomycin, and their coupling with a gem-dimethylamide unit mimicking the middle fragment of the natural product. Tetramates were accessed through the Dieckmann cyclisation of N-acyloxazolidines and were derivatised with various alkyl halides. The pyroglutamates were accessed via the highly diastereoselective aldol cyclisation of N-acyloxazolidines formed by the amide coupling of a threonine derived oxazolidine and β-keto-acids. A series of β-keto-acids were synthesised through the acylation and subsequent ring-opening/decarboxylation reaction of Meldrum’s acid. The formation of right-hand/middle fragment adducts was explored using cycloaddition, alkylation and Sonogashira chemistry before a Wittig protocol led to the formation of adducts (E)- and (Z)- 402 and 403. Biological evaluation of the compounds synthesised in this chapter was carried out using both broth and hole-plate bioassays and active compounds were identified. Of particular note was that the Wittig adducts displayed a higher level of activity against Gram-negative E. coli than either the pyroglutamate or amide motifs alone.

In bacteria, protein synthesis can occur tightly coupled to transcription. In eukaryotes, it is believed that translation occurs solely in the cytoplasm; I test whether some occurs in nuclei and find: (1) L-azidohomoalanine (Aha) – a methionine analogue (detected by microscopy after attaching a fluorescent tag using ‘click’ chemistry) – is incorporated within 5 s into nuclei in a process sensitive to the translation inhibitor, anisomycin. (2) Puromycin – another inhibitor that end-labels nascent peptides (detected by immuno-fluorescence) – is similarly incorporated in a manner sensitive to a transcriptional inhibitor. (3) CD2 – a non-nuclear protein – is found in nuclei close to the nascent RNA that encodes it (detected by combining indirect immuno-labelling with RNA fluorescence in situ hybridization using intronic probes); faulty (nascent) RNA is destroyed by a quality-control mechanism sensitive to translational inhibitors. I conclude that substantial translation occurs in the nucleus, with some being closely coupled to transcription and the associated proof-reading. Moreover, most peptides made in both the nucleus and cytoplasm are degraded soon after they are made with half-lives of about one minute. I also collaborated on two additional projects: the purification of mega-complexes (transcription ‘factories’) containing RNA polymerases I, II, or III (I used immuno-fluorescence to confirm that each contained the expected constituents), and the demonstration that some ‘factories’ specialize in transcribing genes responding to tumour necrosis factor α – a cytokine that signals through NFκB (I used RNA fluorescence in situ hybridization coupled with immuno-labelling to show active NFκB is found in factories transcribing responsive genes).

<strong>Chapter 1</strong> Provides an overview of drug discovery with particular emphasis on library selection and hit identification methods using virtual based approaches. <strong>Chapter 2</strong> Gives an outline of the bone morphogenetic protein (BMP) signalling pathway and literature BMP pathway modulators. The association between the regulation of BMP pathway and cardiomyogenesis is also described. <strong>Chapter 3</strong> Describes the use of ligand based virtual screening to discover small molecule activators of the BMP signalling pathway. A robust cell based BMP responsive gene activity reporter assay was developed to test the libraries of small molecules selected. Hit molecules from the screen were synthesised to validate activity. It was found that a group of known histone deacetylase (HDAC) inhibitors displayed most promising activity. These were evaluated in a secondary assay measuring the expression of two BMP pathway regulated genes, hepcidin and Id1, using reverse transcription polymerase chain reaction (RT-PCR). 188 was discovered to increase expression of both BMP-responsive genes. <strong>Chapter 4</strong> Provides an overview of existing cannabinoid receptor (CBR) modulating molecules and their connection to progression of atherosclerosis. <strong>Chapter 5</strong> Outlines the identification and optimisation of selective small molecule agonists acting at the cannabinoid 2 receptor (CB₂R). Ligand based virtual screen was undertaken and promising hits were synthesised to allow structure activity relationship (SAR) to be developed around the hit molecule providing further information of the functional groups tolerated at the active site. Subsequent studies led to the investigation and optimisation of physicochemical properties around 236 leading to the development of a suitable compound for in vivo testing. Finally, a CB₂R selective compound with favourable physicochemical properties was evaluated in vivo in a murine inflammation model and displayed reduced recruitment of monocytes to the site of inflammation.

Chemical modification of proteins is critical for many areas of biochemistry and medicine. Several methods for site-selective protein modification are reported in this Thesis that are useful in accessing both natural and artificial protein architectures. Multiple, complementary methods for the conversion of cysteine to dehydroalanine are described. Dehydroalanine is used as a general precursor to several post-translational modifications and glycosylation, polyprenylation, phosphorylation, and lysine methylation and acetylation are all accessible. These modifications and their mimics were explored on multiple proteins, including histone proteins. Unnatural modifications were also explored. The first examples of olefin metathesis and Suzuki-Miyaura cross-coupling on protein substrates are reported. Allyl sulfides were discovered to be remarkably reactive substrates in olefin metathesis, allowing use of this reaction in water and on proteins. For Suzuki-Miyaura cross-coupling, a new catalyst is described that is fully compatible with proteins. Both olefin metathesis and cross-coupling allow the formation of carbon-carbon bonds on proteins. The prospects of these transformations in chemical biology are discussed. Finally, a novel strategy is reported for the installation of natural, unnatural, and post-translationally modified amino acid residues on proteins. This technology relies on addition of carbon radicals to dehydroalanine. This method of "chemical mutagenesis" is anticipated to complement standard genetic manipulation of protein structure.