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This thesis is regarded with the biomimetic total synthesis of phenolic natural products and describes two different projects. Chapter 1 introduces phenolic natural products, the most important biosynthetic pathways for their formation and some examples of relevant biomimetic syntheses. A short introduction to dimeric and pseudo-dimeric natural products can also be found in this chapter. Specific introductions can be found at the start of Chapters 2 and 3. Chapter 2 describes the total synthesis of a dimeric thymol derivative isolated from Arnica sachalinensis (which we have named “thymarnicol”). Inspired by the biosynthesis proposed by Passreiter and co-workers, we tested and confirmed the feasibility of a key hetero-Diels–Alder dimerisation step. During our investigations, we gained significant new insights into the origin and reactivity of thymarnicol. The final oxidative cyclisation has been found to occur spontaneously upon exposure to visible light in air. Chapter 3 discusses our efforts to develop a divergent biomimetic synthetic strategy towards a family of prenylated phenylpropanoid natural products isolated from Illicium genus plants. Our first biomimetic approach revealed the chemical instability of our proposed key intermediates. Therefore, a revised approach was trialled, allowing the total synthesis of a small set of natural products and related structures. We envisage that this strategy could be exploited by accessing a large number of members of this family of compounds.

This thesis describes the total synthesis of the polyketide derived marine natural product (-)-maurenone (14) and synthetic studies of a model system for the marine polyketides, the spiculoic acids (20, 22-24). A biomimetic approach involving cyclisation of linear polyketide precursors to install the complex chemical frameworks was employed. Maurenone is a polypropionate derived metabolite isolated from pulmonate molluscs collected off the coast of Costa Rica. While structural assignment following isolation revealed a relatively uncommon tetra-substituted dihydropyrone moiety the only stereochemical information deduced was the trans-relative relationship between the C8 and C9 protons. The total synthesis of a series of eight stereoisomeric putative structures was achieved in order to assign the stereochemistry of (-)-maurenone (14), as that depicted above. A time and cost efficient strategy was developed utilising common intermediates providing access to the eight stereoisomeric structures in a convergent manner. Six key fragments, four aldehydes (109) and two ketones (110), were synthesised using highly diastereoselective syn- and anti-boron aldol reactions and were coupled using a lithium-mediated aldol reaction. Trifluoroacetic acid-promoted cyclisation/dehydration enabled installation the ƒ×-dihydropyrone ring. All eight isomers of one enantiomeric series were synthesised by coupling two ketones with each of four aldehydes. By comparison of the NMR data for the eight isomers with that reported for the natural product, the relative stereochemistry was established as shown. The (-)-enantiomer of maurenone was synthesised in nine linear steps (13 % overall yield) from (R)-2-benzylpentan-3-one ((R)-40) and (R)-2-benzoyloxypentan-3-one ((R)-39). The spiculoic acid family of polyketide derived natural products, isolated from plakortis sponges, possess a unique [4.3.0]-bicyclic core which is proposed to be formed via an enzyme catalysed Intramolecular Diels-Alder (IMDA) cycloaddition reaction of linear polyene precursors 25. Model linear precursors (114), possessing various olefin geometries at C2 and both stereochemical orientations of the C5 stereocentre, were synthesised in order to examine stereoselectivity of the thermally induced IMDA cycloaddition reaction. The two alternative C4-C6 stereotriads of the linear precursors 114 were achieved by employing highly diastereoselective substrate-controlled aldol reactions; an anti-boron aldol reaction, controlled by the facial preference of (R)-2-benzoyloxypentan-3-one ((R)-39), and a syn-titanium aldol reaction, under the control of chiral N-acylthiazolidinethione ((R)-43a). The diene and dienophile moieties were installed using either standard Wittig, H.W.E. or ¡§modified¡¨ Julia olefination reactions. A thorough stereochemical assignment of the cycloadducts of the thermally induced IMDA reaction of each linear precursor was accomplished employing 2D NMR techniques. Comparison of the stereochemistry of each of the cycloadducts with the spiculoic acids revealed that the linear precursor (2E,5S)-114 produced a cycloadduct 232 with stereochemistry analogous to the natural products in 94 % diastereoselectivity. Thus, a synthetic approach to the spiculoic acids via synthesis of a linear precursor 285 possessing a TBS ether at C5 in the S configuration was proposed. Unfortunately, problems encountered in the synthesis of the proposed linear precursors to the spiculoic acids ultimately prevented the total synthesis from being achieved.

Engineering artificial cells is currently an emerging area of research that involves constructing mimics of biological cells. These biomimetic cellular systems hold tremendous promise for the different biomedical applications (diagnostics, therapy, tissue engineering, gene transfection, bioactive coatings) as well as aspects of synthetic biology. A key architectural principle of the cell is a multicompartmentalized assembly, which is one of the features of biological cells that enable the performance of multiple complex biochemical reactions within confined environments. For this purpose, this study demonstrates novel artificial cells, not only presenting organelle mimics but also incorporating various stimuli for regulating enzymatic cascade reactions within the artificial cell and for controlled simultaneous and/or subsequent release of the encapsulated (therapeutic) molecules. To successfully fabricate the multifunctional polymeric multicompartmentalized systems as artificial cells aimed for, in the first step a hollow capsule as biomimetic cellular membrane was developed to simulate a key characteristic of functional artificial cells for the selective uptake and release of (bio)molecules and particles for intra- and intercellular signaling processes. Herein using LbL technique which involved alternate deposition of oppositely charged polyelectrolytes on silica template via electrostatic interaction, the pH and temperature dual-responsive and photo-crosslinked hollow capsule was fabricated and they can be used for the subsequent post-encapsulation process of protein-like macromolecules (≤ 11 nm) and their controllable release triggered by external stimuli for mimicking the controllable bio-inspired functions of cell membranes. The reversible temperature and pH dual-response ability of the hollow capsules has been analyzed. The uptake and release properties of the resulting hollow capsules with different degree of photo-crosslinking for cargos have been further investigated at various temperatures (25, 37 or 45°C) and pH (5.5 or 7.4) of the solution. Next, the design of the polymersomal subcompartmens as organelle mimics, which divide the interior of the multicompartmentalized systems into subcompartments and can stably encapsulate fragile hydrophobic and hydrophilic cargo, e.g., enzymes in order to conduct encapsulated catalysis-resembling cell organelles, was also an important subject. The fabrication of these subcompartments was starting with the synthesis of suitably end-group block copolymers to realize the enzyme-loaded, multifunctional, pH-responsive, photo-crosslinked and post-labelled polymersomes decorated with adamantane groups. The pH sensitivity and various enzymatic reactions of the established multifunctional Ada-polymersomes have been investigated. Based on the above concepts, a bottom-up approach was developed to assemble a structural and functional eukaryotic cell mimics, including “membrane-associated” multicompartmentalized system (MS1) and “free-floating” multicompartmentalized system (MS2), by loading pH-sensitive Ada-polymersomes inside the multifunctional cell membrane. The creation of these multicompartmentalized systems was based on the assembly of enzyme-loaded Ada-polymersomes as organelle mimics onto sacrificial particle templates by host-guest interaction, followed by the LbL deposition of temperature-responsive and photo-crosslinkable PMA(β-CD)/[PAH/PNMD]3 multilayers and outer protective capping PAH/PMA(PEG) bilayer as biomimetic cellular membrane. Upon photo-crosslinking the polymer biomimetic membrane and dissolution of the particle templates, multicompartmentalized systems were obtained. Spatial position of the subcompartments can be controlled using non-covalent host-guest concept, which yielded multicompartmentalized systems containing “membrane-associated” and “free-floating” subunits. Moreover, the metabolism mimicry of multicompartmentalized systems by performing multiple successive two-enzyme cascade reactions in the cells and the multiple parallel reactions by using a third enzyme for deactivating the reaction product and interfering the cascade reaction have been investigated. In conclusion, these multicompartmentalized systems, combining the advantages of both pH-responsive Ada-polymersomes as organelle mimics and multifunctional hollow capsule as biomimetic cellular membrane, present new opportunities for the development of functional cell mimics. The presented studies highlight crucial aspects for the successful applications of such cell mimics for diagnostics, tissue engineering, as nanoreactors, as carriers for multiple drug delivery with controlled release profiles, or as therapeutic artificial cells.

Le but de ce travail de doctorat est l’étude chimique et biosynthétique de familles d’alcaloïdes guanidiniques d’origine marine provenant d’éponges de Méditerranée.Le travail est divisé en trois parties successives : 1) l’isolement d’alcaloïdes produits par des éponges marines de l’ordre des Poeciloscerida; 2) l’élucidation de la biosynthèse de la crambescine C1 par des études in vivo d’incorporation de précurseurs marqués au 14C; 3) la synthèse biomimétique de la crambescine A2 448 et de dérivés proches.La famille des alcaloïdes guanidiniques cycliques des crambescines est au coeur de la thèse, ces substances naturelles sont produites par l’éponge incrustante Crambe crambe. Ces alcaloïdes ont été découverts dans les années 1990 et ont suscité beaucoup d’intérêt pour leurs propriétés biologiques et écologiques et leurs synthèses totales. Par contre, leur biosynthèse était encore inconnue à ce jour. La seule synthèse biomimétique disponible était basée sur une hypothèse d’origine polyacétique. Les hypothèses récentes de nos groupes ont permis de mettre en avant une origine mixte: la partie cyclique guanidinique proviendrait d’un pyrrolidinium issu de l’arginine et d’un précurseur “céto-acide” proche des acides gras. Sur la base de cette hypothèse, nous avons mis au point une expérience d’incorporation qui a ensuite inspirée une voie de synthèse biomimétique pour l’accès aux crambescines et dérivés. Les premières conclusions quant à l’origine biosynthétique de ces molécules sont les faits les plus marquants de ce travail. Nous apportons une meilleure compréhension de la biochimie des alcaloïdes guanidiniques marins de structures complexes. / This thesis aims at the study of the chemical and biogenetic origin of specialized guanidine-alkaloid metabolites produced by sponges from the Mediterranean Sea.The work is divided into three main parts: 1) isolation of alkaloids produced by sponges of the Poeciloscerida order; 2) biosynthesis of crambescin C1 by in-vivo 14C-feeding experiments with Crambe crambe sponge; 3) biomimetic synthesis of crambescin A2 448 and derivatives. The main focus of the thesis will be the family of cyclic-guanidine alkaloids "crambescins", produced by the red incrusting sponge Crambe crambe.These alkaloids were discovered in the early 90s and despite the large interest on their biological activities, ecological roles, and synthesis, their biosynthesis is still unknown.The only available biomimetic synthesis of crambescins was based on a fully polyketide origin hypothesis. Recently our groups suggested an alternative biosynthetic hypothesis in which the guanidine-core would be originated from a condensation between a guanidinated pyrrolidinium derived from arginine and a beta-keto fatty acid. Based on this hypothesis, we designed a biosynthesis experiment that inspired a biomimetic synthesis route to access the crambescins and derivatives. The insights from these studies are the first experimental conclusions about the biosynthesis of crambescins. Finally, this work leads to a better comprehension of the biochemistry involved in guanidine marine alkaloids of complex structures.

Engineering artificial cells is currently an emerging area of research that involves constructing mimics of biological cells. These biomimetic cellular systems hold tremendous promise for the different biomedical applications (diagnostics, therapy, tissue engineering, gene transfection, bioactive coatings) as well as aspects of synthetic biology. A key architectural principle of the cell is a multicompartmentalized assembly, which is one of the features of biological cells that enable the performance of multiple complex biochemical reactions within confined environments. For this purpose, this study demonstrates novel artificial cells, not only presenting organelle mimics but also incorporating various stimuli for regulating enzymatic cascade reactions within the artificial cell and for controlled simultaneous and/or subsequent release of the encapsulated (therapeutic) molecules. To successfully fabricate the multifunctional polymeric multicompartmentalized systems as artificial cells aimed for, in the first step a hollow capsule as biomimetic cellular membrane was developed to simulate a key characteristic of functional artificial cells for the selective uptake and release of (bio)molecules and particles for intra- and intercellular signaling processes. Herein using LbL technique which involved alternate deposition of oppositely charged polyelectrolytes on silica template via electrostatic interaction, the pH and temperature dual-responsive and photo-crosslinked hollow capsule was fabricated and they can be used for the subsequent post-encapsulation process of protein-like macromolecules (≤ 11 nm) and their controllable release triggered by external stimuli for mimicking the controllable bio-inspired functions of cell membranes. The reversible temperature and pH dual-response ability of the hollow capsules has been analyzed. The uptake and release properties of the resulting hollow capsules with different degree of photo-crosslinking for cargos have been further investigated at various temperatures (25, 37 or 45°C) and pH (5.5 or 7.4) of the solution. Next, the design of the polymersomal subcompartmens as organelle mimics, which divide the interior of the multicompartmentalized systems into subcompartments and can stably encapsulate fragile hydrophobic and hydrophilic cargo, e.g., enzymes in order to conduct encapsulated catalysis-resembling cell organelles, was also an important subject. The fabrication of these subcompartments was starting with the synthesis of suitably end-group block copolymers to realize the enzyme-loaded, multifunctional, pH-responsive, photo-crosslinked and post-labelled polymersomes decorated with adamantane groups. The pH sensitivity and various enzymatic reactions of the established multifunctional Ada-polymersomes have been investigated. Based on the above concepts, a bottom-up approach was developed to assemble a structural and functional eukaryotic cell mimics, including “membrane-associated” multicompartmentalized system (MS1) and “free-floating” multicompartmentalized system (MS2), by loading pH-sensitive Ada-polymersomes inside the multifunctional cell membrane. The creation of these multicompartmentalized systems was based on the assembly of enzyme-loaded Ada-polymersomes as organelle mimics onto sacrificial particle templates by host-guest interaction, followed by the LbL deposition of temperature-responsive and photo-crosslinkable PMA(β-CD)/[PAH/PNMD]3 multilayers and outer protective capping PAH/PMA(PEG) bilayer as biomimetic cellular membrane. Upon photo-crosslinking the polymer biomimetic membrane and dissolution of the particle templates, multicompartmentalized systems were obtained. Spatial position of the subcompartments can be controlled using non-covalent host-guest concept, which yielded multicompartmentalized systems containing “membrane-associated” and “free-floating” subunits. Moreover, the metabolism mimicry of multicompartmentalized systems by performing multiple successive two-enzyme cascade reactions in the cells and the multiple parallel reactions by using a third enzyme for deactivating the reaction product and interfering the cascade reaction have been investigated. In conclusion, these multicompartmentalized systems, combining the advantages of both pH-responsive Ada-polymersomes as organelle mimics and multifunctional hollow capsule as biomimetic cellular membrane, present new opportunities for the development of functional cell mimics. The presented studies highlight crucial aspects for the successful applications of such cell mimics for diagnostics, tissue engineering, as nanoreactors, as carriers for multiple drug delivery with controlled release profiles, or as therapeutic artificial cells.

The development of a vasculature within a tissue-engineered construct is one of the largest hurdles to successful bone regeneration. This thesis investigates methods to increase vasculature of such transplanted constructs, based on in vivo transplant studies and in vitro analysis of cell behaviors. A syngeneic mouse model in immunocompetent mice was developed and analyzed for both osteogenesis and hematopoiesis. This study demonstrates that syngeneic bone marrow stromal cells (BMSCs) are not rejected by the host, provided the strain of mice is sufficiently inbred. Additionally, an effective protocol was developed for the isolation of endothelial cells (ECs) from the bone marrow of mice. Two different sets of materials for this study were analyzed, both collagen based, and the GelfoamTM scaffold was found to possess advantages over synthesized collagen or collagen/hydroxyapatite composites, although only for mouse and not human bone transplantation. In order to gain rapid and integrated vasculature formation within the transplant, attempts were made to increase both (de novo) vasculogenesis and angiogenesis (ingrowth) from the surrounding tissue. For the former, transplant studies were combined with in vitro osteogenic calcification studies. Direct co-culture of the BMSCs and ECs increased osteogenic calcification and was monitored by using both alizarin red S quantification and quantitative polymerase chain reaction. Angiogenesis (as assessed by cell migration) was studied by various motility and chemotaxis assays in vitro, as well as through use of a directed in vivo angiogenesis assay. Growth factors, particularly TGF-β1 and BMP-4, were found to increase cell movement in these systems. In conclusion, we show that although much work remains to be done in order to increase the vasculature in bone transplants, systematic combination of in vivo and in vitro assays can elucidate the nature behind this crucial process in this context.

In the post-genomic era, recognition of the importance of sugars is increasing in biological research. For the precise analysis of their functions, homogeneous materials are required. Chemical synthesis is a powerful tool for preparation of homogeneous oligosaccharides and glycoconjugates. Glycosidases are potent catalysts for this purpose because they realize high stereo- and regio- selectivities under conditions benign to biomolecules without repetitive protection/deprotection procedures. A glycosynthase is an aritificial enzyme which is derived from a glycosidase and is devised for glycosylation reaction. To suppress the mechanistically inherent oligomerization side reaction of this class of biocatalysts, a glycosidase with plastic substrate recognition was engineered to afford the first α-mannosynthase. This novel biocatalyst showed low occurrence of oligomerized products as designed and was applied to prepare a wide range of oligosaccharides. Glycosidases are also valuable tools for glycan engineering of glycoconjugates, which is a pivotal issue in the development of pharmaceutical agents, including immunoglobulin G (IgG)-based drugs. EndoS, an endo-β-N-acetylglucosaminidase from Streptococcus pyogenes, natively cleaves N-glycans on IgG specifically. When the latent glycosylation activity of this enzyme was applied, the N-glycan remodelling of full-length IgG was successfully achieved for the first time and a highly pure glycoform was obtained using the chemically synthesized oxazoline tetrasaccharide as glycosyl donor. This biocatalytic reaction allows development of a novel type of antibody-drug conjugates (ADCs) in which drug molecules are linked to N-glycans site-specifically. For this purpose, glycans with bioorthogonal reaction handles were synthesized and conjugated to IgG. A model reaction using a dye compound as reaction partner worked successfully and the synthetic method for this newly designed ADC was validated. Glycan trimming of glycoproteins expressed from Pichia pastoris was performed using exoglycosidases to derive homogeneous glycoform. Jack Bean α-mannosidase (JBM) trimmed native N-glycans down to the core trisaccharide structure but some of the glycoforms were discovered to be resistant to the JBM activity. Enzymatic analyses using exoglycosidases suggested that the JBM-resistant factor was likely to be β-mannoside. In summary, this work advanced application of modified glycosidases for preparation of oligosaccharides and also demonstrated biocatalytic utility of glycosidases to produce biologically relevant glycoconjugates with homogeneous glycoforms.

A synthetic investigation on the chemistry of cyclotryptamine derived natural products, with a particular focus on the synthesis of the trimeric-alkaloid, hodgkinsine. Methodology has been developed to tackle this complex natural product which utilises a desymmetrization approach; this strategy hinges on the development and applications of asymmetric C-N bond forming reactions. Chapter one examines elements of symmetry in natural products, looking in particular at the synthesis of compounds which contain cyclotryptamine functionality. Chapter two contains a brief review of enantioselective desymmetrization paying attention, if possible, on its application in the synthesis of natural products. In the remaining chapters we discuss our own progress and results in our pursuit of an efficient enantioselective total synthesis of hodgkinsine.

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.