A combinatorial approach to the chemical synthesis and biological evaluation of 3,4,5-trisubstiituted furan-2(5H)-ones

Many important natural products contain the furan-2(5H)-one structure. The structure of this molecule lends itself to manipulation using combinatorial techniques due to the presence of more than one site for the attachment of different suhstituents. By developing different reaction schemes at the three sites available for attachment on the furan-2(5H)-one scaffold, combinatorial chemistry techniques can be employed to assemble libraries of novel furan 2(5H)-ones. These libraries can then be entered into various biological screening programmes. This approach will enable a vast diversity or compounds to be examined, in the hope or finding new biologically active Iead structures. The work in this thesis has investigated the potential that combinatorial chemistry has in the quest for new biologically active lead structures based on the furan-2(5H)-one structure. Different reactions were investigated with respect to their suitability for inclusion in a library. Once sets of reactions at the various sites had been established, the viability of these reactions in the assembly of combinatorial libraries was investigated. Purification methods were developed, and the purified products entered into suitable biological screening tests. Results from some of these tests were optimised using structure activity relationships, and the resulting products re-screened. The screening tests performed were for anticancer and antimicrobial activity, cholecystokinin (CCK-B) antagonism and anti-inflammatory activity (in the quest for novel cyclo-oxygenase (COX-2) selective non-steroidal anti-inflammatory drugs). It has been shown that many reactions undergone by the furan-2(5H)-one structure are suitable for the assembly of a combinatorial library. Investigation into the assembly of different libraries has been carried out with initial screening results included. From this work, further investigation into combinatorial library assembly and structure activity relationships of screened reaction products can be undertaken.

Exploration of steroselective cleavage of 4, 5-epoycholestane-3, 6-diols and stero controlled epoxidation of cholest-4-en-3 β, 6β-diol-6-acetate

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An Ins(1,4,5)P3 receptor in Paramecium is associated with the osmoregulatory system

In the ciliate Paramecium, a variety of well characterized processes are regulated by Ca2+, e.g. exocytosis, endocytosis and ciliary beat. Therefore, among protozoa, Paramecium is considered a model organism for Ca2+ signaling, although the molecular identity of the channels responsible for the Ca2+ signals remains largely unknown. We have cloned - for the first time in a protozoan - the full sequence of the gene encoding a putative inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3) receptor from Paramecium tetraurelia cells showing molecular characteristics of higher eukaryotic cells. The homologously expressed Ins(1,4,5)P3-binding domain binds [3H]Ins(1,4,5)P3, whereas antibodies unexpectedly localize this protein to the osmoregulatory system. The level of Ins(1,4,5)P3-receptor expression was reduced, as shown on a transcriptional level and by immuno-staining, by decreasing the concentration of extracellular Ca2+ (Paramecium cells rapidly adjust their Ca2+ level to that in the outside medium). Fluorochromes reveal spontaneous fluctuations in cytosolic Ca2+ levels along the osmoregulatory system and these signals change upon activation of caged Ins(1,4,5)P3. Considering the ongoing expulsion of substantial amounts of Ca2+ by the osmoregulatory system, we propose here that Ins(1,4,5)P3 receptors serve a new function, i.e. a latent, graded reflux of Ca2+ to fine-tune [Ca2+] homeostasis.

Studies on the neurotoxicity of 6,7-dihydroxy-1-methyl-1,2,3,4-tetrahydroisoquinoline (salsolinol) in SH-SY5Y cells

We have studied the hypothesis that 6,7-dihydroxy-1-methyl-1,2,3,4-tetrahydroisoquinoline (salsolinol) is neurotoxic. Salsolinol induced a significant time and dose related inhibition of 3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; thiazoyl blue (MTT) reduction, and increased lactate dehydrogenase release (LDH) release from human SH-SY5Y neuroblastoma cells, at concentrations within the range of 1-methyl-4-phenylpyridinium (MPP+) cytotoxicity, in vitro. Cytotoxicity was not inhibited by the addition of antioxidants, monoamine oxidase inhibitors or imipramine. In confluent monolayers, salsolinol stimulated catecholamine uptake with EC50 values of 17 muM and 11 muM, for noradrenaline and dopamine, respectively. Conversely, at concentrations above 100 muM, salsolinol inhibited the uptake of noradrenaline and dopamine, with IC50 values of 411 muM and 379 muM, respectively. The inhibition of catecholamine uptake corresponded to the increase displacement of [3H]nisoxetine from the uptake 1 site by salsolinol, as the Ki (353 muM) for displacement was similar to the IC50 (411 and 379 muM) for uptake. Salsolinol stimulated catecholamine uptake does not involve the uptake recognition site, or elevation of cAMP, cGMP, or inhibition of protein kinase C. Salsolinol also inhibited both carbachol (1 mM) and K+ (100 mM, Na+ adjusted) evoked released of noradrenaline from SH-SY5Y cells, with IC50 values of 500 muM and 120 muM, respectively. In conclusion, salsolinol appears to be cytotoxic to SH-SY5Y cells, via a mechanism that does not require uptake 1, bioactivation by monoamine oxidase, or membrane based free radical damage. The effects of salsolinol on catecholamine uptake, and the mechanism of toxicity require further investigation.

Measurements of stress-optic coefficient in polymer optical fibers

We have systematically measured the differential stress-optic coefficient, ?C, in a number of poly(methyl methacrylate) (PMMA) fibers drawn with different stress, ranging from 2 up to 27 MPa. ?C was determined in transverse illumination by measuring the dependence of birefringence on additional axial stress applied to the fiber. Our results show that ?C in PMMA fibers has a negative sign and ranges from -4.5 to -4.5×10-12 Pa-1, depending on the drawing stress. Increase of the drawing stress results in greater initial fiber birefringence and lower ?C.

The activity of pH membrane disruptive pseudopeptides and their subcellular fate in mammalian cells cultured in-vitro

This thesis describes investigations upon pseudopeptides which were conducted to improve our understanding of the fate of synthetic macromolecules in cells and to develop approaches to influence that fate. The low uptake of molecules across the external cellular membrane is the principal barrier against effective delivery of therapeutic products to within the cell structure. In nature, disruption of this membrane by amphiphilic peptides plays a central role in the pathogenesis by bacterial and toxin infections. These amphiphilic peptides contain both hydrophobic and weakly charged hydrophilic amino acid residues and upon activation they become integrated into the lipid bilayers of the extracellular or endosomal membranes. The architectures of the pseudopeptides described here were designed to display similar pH dependent membrane rupturing activity to that of peptides derived from the influenza virus hemagglutinin HA-2. This HA protein promotes fusion of the influenza virus envelope with the cell endosome membrane due to a change in conformation in response to the acidic pH of the endosome lumen (pH 5.0-6.0). The pseudopeptides were obtained by the copolymerisation of L-lysine and L-lysine ethyl-ester with various dicarboxylic acid moieties. In this way a linear polyamide comprising of alternating pendant carboxylic acids and pendant hydrophobic moieties was made. At physiological pH (pH 7.4), electrostatic repulsion of pendant anionic carboxyl groups along the polymer backbone is sufficient to overcome the intramolecular association of the hydrophobic groups resulting in an extended conformation. At low pH (typically pH 4.8) loss of charge results in increased intramolecular hydrophobic association and the polymer chain collapses to a compact conformation, leading to precipitation of the polymer. Consequently, a conformation dependent functional property could be made to respond to small changes in the environmental pH. Pseudopepides were investigated for their cytoxicity towards a well known cell line, namely C26 (colorectal adenocarcinoma) and were shown through the use of a cell viability assay, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) to be well tolerated by C26 cells over a range of concentrations (2-500,μg/ml) at physiological pH (pH 7.4). A modified version of a shorter 30-minute coupled enzymatic assay, the LDH (lactate dehydrogenase) assay was used to evaluate the ability of the pseudopeptides to disrupt the membrane of two different cell lines (COS-1; African green monkey, kidney and A2780; human ovarian carcinoma) at low pH (pH 5.5). The cell membrane disruption property of the pseudopeptides was successfully demonstrated for COS-I and A2780 cell lines at this pH (pH 5.5). A variety of cell lines were chosen owing to limited availability and to compare the cytotoxic action of these pH responsive psudopeptides towards normal and tumorogenic cell lines. To investigate the intracellular delivery of one of the pseudopeptides, poly (L-lysine iso-phthalamide) and its subcellular location, a Cy3 bisamine fluorophore was conjugated into its backbone, at ratios of dye:lysine of 1:20, 1:30, 1:40, 1:60 and 1:80. Native polyacrylacrylamide gel electrophoresis (PAGE) and high voltage paper electrophoresis (HVPE) studies of the polydyes were conducted and provided evidence that that the Cy3 bisamine fluorophore was conjugated into the backbone of the polymer, poly (L-lysine iso-phthalamide). The subcellular fate of the fluorescentlylabelled "polydye" (hereafter PD20) was monitored by laser scanning confocal microscopy (LSCM) in CHO (Chinese hamster ovary) cells cultured in-vitro at various pH values (pH 7.4 and 5.0). LSCM images depicting time-dependent internalisation of PD20 indicated that PD20 traversed the extracellular membrane of CHO cells cultured in-vitro within ten minutes and migrated towards the endosomal regions where the pH is in the region of 5.0 to 6.0. Nuclear localisation of PD20 was demonstrated in a subpopulation of CHO cells. A further study was completed in CHO and HepG2 (hepatocellular carcinoma) cells cultured in-vitro using a lower molecular weight polymer to demonstrate that the molecular weight of "polydye" could be tailored to attain nuclear trafficking in cells. Prospective use of this technology encompasses a method of delivering a payload into a living cell based upon the hypercoiling nature of the pseudopeptides studied in this thesis and has led to a patent application (GB0228525.2; 20(2).

Synthesis and characterisation of vinyl block copolymers

A study has been made of the anionic polymerisation of methyl methacrylate using butyllithium and polystyryl lithium as initiators and the effects of lithium chloride and aluminium alkyls on the molecular weight and molecular weight distributions. Diblock copolymers of styrene-b-methyl methacrylate were synthesised at -78oC in THF in the presence of lithium chloride, and at ambient temperatures in toluene in the presence of aluminium alkyls. Studies in the presence of lithium chloride showed that the polymerisation was difficult to control; there was no conclusive evidence of a living system and the polydispersity indices were between 1.5 and 3. However, using relatively apolar solvents, in the presence of aluminium alkyls, homopolymerisation of methyl methacrylate showed characteristics of a living polymerisation. An investigation of the effects of the structures of the lithium and aluminium alkyls on the efficiency of initiation showed that a t-butyllithium/triisobutylaluminium initiating system exhibited an efficiency of 80%, compared with lower efficiencies (typically 30%) for systems based on butyllithium/triethylaluminium.The polydispersity index was found to decrease from ∼2.2 to ∼1.5 when butyllithium was replaced by t-butyllithium. The efficiency of the initiator was found to be solely dependent on the size of the alkyl group of the aluminium component, whereas the polydispersity index was found to be solely dependent on the size of the alkyl group on the lithium component. The aluminium alkyl is thought to be co-ordinated to the ester carbonyl groups of both the monomer and polymer. There is a critical degree of polymerisation, at which point the rate of polymerisation decreases, which probably relates to a change in structure of the active chain end. Characterisation of poly(styrene )-b-poly(4-vinylpyridine) and poly(styrene)-b-poly(4-vinylpyridine methyl iodide) diblock copolymers using static light scattering techniques, showed the formation of star-shaped 'reverse' micelles when placed in toluene. Temperature effects on micellization behaviour are only exhibited for the unquaternised micelles, which showed characterisically lower aggregation numbers than their quaternised counterparts. A suitable solvent was not obtained for characterisation of the styrene-b-methyl methacrylate diblock copolymers synthesized.

Autonomous volume transitions of a polybase triblock copolymer gel in a chemically driven pH-oscillator

A pH-responsive ABA triblock copolymer, comprising poly(methyl methacrylate)-b/ock-poly(2-(diethylamino)ethyl methacrylate)-block-poly(methyl methacrylate) [PMMA-b-PDEA-b-PMMA], has been cast Into thin films with a well-defined microstructure. Small Angle X-ray Scattering (SAXS) and Atomic Force Microscopy (AFM) studies confirm that this copolymer forms a hydrogel consisting of PMMA spheres embedded within a polybase PDEA matrix, with the PMMA domains acting as physical cross-links. The hydrogel has a pH-reversible coil-globule transition at around pH 4.5. This responsive physical property was exploited by immersing a sample of copolymer hydrogel in an aqueous solution containing a cyclic pH-oscillating reaction, whereby the pH was continuously oscillated above and below the transition pH so as to induce autonomous volume transitions. The changes in microscopic and macroscopic length scales correlate closely during (de)swelling cycles, with affine behaviour occurring over five orders of magnitude. Copyright © 2007 WILEY-VCH Verlag GmbH & Co. KGaA,.

Reactive processing of polymers:effect of in situ compatibilisation on characteristics of blends of polyethylene terephthalate and ethylene-propylene rubber

Ethylene-propylene rubber (EPR) functionalised with glycidyl methacrylate (GMA) (f-EPR) during melt processing in the presence of a co-monomer, such as trimethylolpropane triacrylate (Tris), was used to promote compatibilisation in blends of polyethylene terephthalate (PET) and f-EPR, and their characteristics were compared with those of PET/f-EPR reactive blends in which the f-EPR was functionalised with GMA via a conventional free radical melt reaction (in the absence of a co-monomer). Binary blends of PETand f-EPR (with two types of f-EPR prepared either in presence or absence of the co-monomer) with various compositions (80/20, 60/40 and 50/50 w/w%) were prepared in an internal mixer. The blends were evaluated by their rheology (from changes in torque during melt processing and blending reflecting melt viscosity, and their melt flow rate), morphology scanning electron microscopy (SEM), dynamic mechanical properties (DMA), Fourier transform infrared (FTIR) analysis, and solubility (Molau) test. The reactive blends (PET/f-EPR) showed a marked increase in their melt viscosities in comparison with the corresponding physical (PET/EPR) blends (higher torque during melt blending), the extent of which depended on the amount of homopolymerised GMA (poly-GMA) present and the level of GMA grafting in the f-EPR. This increase was accounted for by, most probably, the occurrence of a reaction between the epoxy groups of GMA and the hydroxyl/carboxyl end groups of PET. Morphological examination by SEM showed a large improvement of phase dispersion, indicating reduced interfacial tension and compatibilisation, in both reactive blends, but with the Tris-GMA-based blends showing an even finer morphology (these blends are characterised by absence of poly-GMA and presence of higher level of grafted GMA in its f-EPR component by comparison to the conventional GMA-based blends). Examination of the DMA for the reactive blends at different compositions showed that in both cases there was a smaller separation between the glass transition temperatures compared to their position in the corresponding physical blends, which pointed to some interaction or chemical reaction between f-EPR and PET. The DMA results also showed that the shifts in the Tgs of the Tris-GMA-based blends were slightly higher than for the conventional GMA-blends. However, the overall tendency of the Tgs to approach each other in each case was found not to be significantly different (e.g. in a 60/40 ratio the former blend shifted by up to 4.5 °C in each direction whereas in the latter blend the shifts were about 3 °C). These results would suggest that in these blends the SEM and DMA analyses are probing uncorrelatable morphological details. The evidence for the formation of in situ graft copolymer between the f-EPR and PET during reactive blending was clearly illustrated from analysis by FTIR of the separated phases from the Tris-GMA-based reactive blends, and the positive Molau test pointed out to graft copolymerisation in the interface. A mechanism for the formation of the interfacial reaction during the reactive blending process is proposed.

Unravelling the photodegradation mechanisms of a low bandgap polymer by combining experimental and modeling approaches

Large-scale introduction of Organic Solar Cells (OSCs) onto the market is currently limited by their poor stability in light and air, factors present in normal working conditions for these devices. Thus, great efforts have to be undertaken to understand the photodegradation mechanisms of their organic materials in order to find solutions that mitigate these effects. This study reports on the elucidation of the photodegradation mechanisms occurring in a low bandgap polymer, namely, Si-PCPDTBT (poly[(4,4′-bis(2-ethylhexyl)dithieno[3,2-b:2′,3′-d]silole)-2,6-diyl-alt-(4,7-bis(2-thienyl)-2,1,3-benzothiadiazole)-5,5′-diyl]). Complementary analytical techniques (AFM, HS-SPME-GC-MS, UV-vis and IR spectroscopy) have been employed to monitor the modification of the chemical structure of the polymer upon photooxidative aging and the subsequent consequences on its architecture and nanomechanical properties. Furthermore, these different characterization techniques have been combined with a theoretical approach based on quantum chemistry to elucidate the evolution of the polymer alkyl side chains and backbone throughout exposure. Si-PCPDTBT is shown to be more stable against photooxidation than the commonly studied p-type polymers P3HT and PCDTBT, while modeling demonstrated the benefits of using silicon as a bridging atom in terms of photostability. (Figure Presented).

Synthesis and application of novel probes for the detection of cysteine sulphenic acids

Cysteine is a thiol containing amino acid that readily undergoes oxidation by reactive oxygen species (ROS) to form sulphenic (R-SOH) sulphinic (RSO2H) and sulphonic (RSO3H) acids. Thiol modifications of cysteine have been implicated as modulators of cellular processes and represent significant biological modifications that occur during oxidative stress and cell signalling. However, the different oxidation states are difficult to monitor in a physiological setting due to the limited availability of experimental tools. Therefore it is of interest to synthesise and use a chemical probe that selectively recognises the reversible oxidation state of cysteine sulphenic acid to understand more about oxidative signalling. The aim of this thesis was to investigate a synthetic approach for novel fluorescent probe synthesis, for the specific detection of cysteine sulphenic acids by fluorescence spectroscopy and confocal microscopy. N-[2-(Anthracen-2-ylamino)-2-oxoethyl]-3,5-dioxocyclohexanecarboxamide was synthesised in a multistep synthesis and characterised by nuclear magnetic resonance spectroscopy. The optimisation of conditions needed for sulphenic acid formation in a purified protein using human serum albumin (HSA) and the commercially available biotin tagged probe 3-(2,4-dioxocyclohexyl)propyl-5-((3aR,6S,6aS)-hexahydro-2-oxo-1H-thieno[3,4-d]imidazol-6-yl)pentanoate (DCP-Bio1) were identified. This approach was extended to detect sulphenic acids in Jurkat T cells and CD4+ T cells pre- and post-stimulus. Buthionine sulfoximine (BSO) was used to manipulate the endogenous antioxidant glutathione (GSH) in human CD4+ T cells. Then the surface protein thiol levels and sulphenic acid formation was examined. T cells were also activated by the lectin phytohaemagglutinin-L (PHA-L) and formation of sulphenic acid was investigated using SDS-PAGE, western blotting and confocal microscopy. Resting Jurkat cells have two prominent protein bands that have sulphenic acid modifications whereas resting CD4+ T cells have an additional band present. When cells were treated with BSO the number of bands increased whereas activation reduced the number of proteins that were modified. The identities of the protein bands containing sulphenic acids were explored by mass spectrometry. Cysteine oxidation was observed in redox, metabolic and cytoskeletal proteins. In summary, a novel fluorescent probe for detection of cysteine sulphenic acids has been synthesised alongside a model system that introduces cysteine sulphenic acid in primary T cells. This probe has potential application in the subcellular localisation of cysteine oxidation during T cell signalling.

Identification of the degradation mechanisms of organic solar cells:active layer and interfacial layers

Organic Solar Cells (OSCs) represent a photovoltaic technology with multiple interesting application properties. However, the establishment of this technology into the market is subject to the achievement of operational lifetimes appropriate to their application purposes. Thus, comprehensive understanding of the degradation mechanisms occurring in OSCs is mandatory in both selecting more intrinsically stable components and/or device architectures and implementing strategies that mitigate the encountered stability issues. Inverted devices can suffer from mechanical stress and delamination at the interface between the active layer, e.g. poly(3-hexylthiophene):[6,6]-phenyl-C61-butyric acid methyl ester (P3HT:PCBM), and the hole transport layer, e.g. poly(3,4-ethylenedioxythiophene):poly(p-styrene sulfonate) (PEDOT:PSS). This work proposes the incorporation of a thin adhesive interlayer, consisting of a diblock copolymer composed of a P3HT block and a thermally-triggerable, alkyl-protected PSS block. In this context, the synthesis of poly(neopentyl p-styrene sulfonate) (PNSS) with controlled molar mass and low dispersity (Ð ≤ 1.50) via Reversible Addition-Fragmentation chain Transfer (RAFT) polymerisation has been extensively studied. Subsequently, Atomic Force Microscopy (AFM) was explored to characterise the thermal deprotection of P3HT-b-PNSS thin layers to yield amphiphilic P3HT-b-PSS, indicating that surface deprotection prior to thermal treatment could occur. Finally, structural variation of the alkyl protecting group in PSS allowed reducing the thermal treatment duration from 3 hours (P3HT-b-PNSS) to 45 minutes for the poly(isobutyl p-styrene sulfonate) (PiBSS) analogous copolymer. Another critical issue regarding the stability of OSCs is the sunlight-driven chemical degradation of the active layer. In the study herein, the combination of experimental techniques and theoretical calculations has allowed identification of the structural weaknesses of poly[(4,4’- bis(2-ethylhexyl) dithieno [3,2-b:2’,3’-d]silole)-2,6-diyl-alt-(4,7-bis(2-thienyl)-2,1,3-benzothiadiazole)-5,5’-diyl], Si-PCPDTBT, upon photochemical treatment in air. Additionally, the study of the relative photodegradation rates in air of a series of polymers with systematically modified backbones and/or alkyl side chains has shown no direct correlation between chemical structure and stability. It is proposed instead that photostability is highly dependent on the crystalline character of the deposited films. Furthermore, it was verified that photostability of blends based on these polymers is dictated by the (de)stabilising effect that [6,6]-phenyl-C61-butyric acid methyl ester (PCBM) has over each polymer. Finally, a multiscale analysis on the degradation of solar cells based on poly[4,4' bis(2- ethylhexyl) dithieno[3,2-b:2',3'-d]silole)-2,6-diyl-alt-[2,5 bis(3 tetradecylthiophen 2-yl)thiazole[5,4-d]thiazole)-1,8-diyl] and PCBM, indicated that by judicious selection of device layers, architectures, and encapsulation materials, operational lifetimes up to 3.3 years with no efficiency losses can be successfully achieved.