Academic literature on the topic 'Fields of Research – 250000 Chemical Sciences – 250300 Organic Chemistry'

The iron and manganese superoxide dismutases are a family of metallo-enzymes with highly conserved protein folds, active sites and dimer interfaces. They catalyse the elimination of the cytotoxic free radical superoxide to molecular oxygen and hydrogen peroxide by alternate reduction then oxidation of the activesite with the concomitant transfer of protons from the solvent. There are many key aspects of enzymatic function that lack a structural explanation. The focus of this study is on three crystal structures. The iron-substituted manganese superoxide dismutase from Escherichia coli complexed with azide, a substrate-mimicking inhibitor, was solved to 2.2 Å. This “wrong” metal form shows a binding pattern seen previously in the manganese superoxide dismutase from Thermus thermophilus. Wild-type manganese specific superoxide dismutase from the extremophile Deinococcus radiodurans was solved to 2.0 Å and has an active site reminiscent of other solved manganese superoxide dismutases despite a lack of product inhibition. The azide-inhibited manganese superoxide dismutase from Deinococcus radiodurans was determined to a resolution of 2.0 Å and showed binding of azide, and by inference superoxide, different to that seen in Thermus thermophilus, but reminiscent of that seen in azide-inhibited iron superoxide dismutases. These results indicate that the azide ion, and by inference superoxide, bind to the metal centre of manganese superoxide dismutases in two modes, and transition between the two modes may be entropy dependent. These structures, integrated with knowledge from other structures, known biochemistry and various spectra, provide insight into catalytic function. An outer-sphere mechanism of proton transfer that does not rely on through-peptide proton uptake is proposed and compared to a previously proposed inner-sphere mechanism. This is based on the observation that a water molecule moves into the active site of the manganese superoxide dismutase from Deinococcus radiodurans upon azide binding, providing a Grötthus pathway for rapid proton transfer to the active site from the bulk solvent. Also presented in this study are the partially refined structures of four point mutants (S82T, L83M, L133V, and M164L/L166V) of the manganese superoxide dismutase from Escherichia coli all solved to roughly 2 Å resolution, designed to investigate product inhibition which varies across species.

The immune system often recognises tumour cells and infectious agents from the unique peptides found on their surfaces therefore, synthetic peptides of similar structure can be used as vaccines to stimulate the immune system. Despite the problems associated with proteolysis and delivery to the immune system, peptide-based vaccines have enormous potential due to their ease of synthesis and purification. The aim of this research was to synthesise ligands for mannose receptors (MRs) that are found on human Antigen Presenting Cells (APCs), for use in synthetic vaccines. Carbohydrate bearing antigens are recognised by MRs which play an important role in binding antigens, migration of dendritic cells (DCs) and interaction of DCs with lymphocytes. Hence, incorporation of a sugar residue into a peptide chain can be used to enhance antigen presentation. This thesis describes the synthesis of fluorescein labelled O-mannosylated peptides using either manual or microwave assisted solid phase glycopeptide synthesis (SPGS) on pre-loaded WANG resin. The mannosylated peptides thus prepared can be tested for their ability to bind mannose receptors on human APCs in vitro. In order to prepare compounds that could be analysed in biological screens, a fluorescent label (5(6)-carboxyfluorescein) was introduced into the glycopeptides via the Nα- or the Nε-amino group of the lysine residue. It was found that preparation of the glycopeptide was more facile when the peptide chain was built onto the Nε of Lys (label into Nα) rather than onto the Nα of Lys (label into Nε). In order to overcome problems experienced when introducing more than one glycosylated building block into the peptide chain, a polyethylene glycol (PEG) linker was employed as a sugar carrier. It was found that mono- and dimannosylated building blocks attached to PEG carrier were incorporated more easily into the peptide chain compared to mono- and dimannosylated serine units. Importantly, microwave technology (CEM Liberty microwave peptide synthesiser) was used for SPGS which resulted in improved purity and yields of the glycopeptides thus prepared with a significant reduction in reaction times. The first fifteen glycopeptides prepared in the present study were tested for binding to mannose receptors. Several compounds have shown improved binding to monocytes (bear MRs) in comparison to lymphocytes (do not bear MRs), in the presence of calcium ions. Calcium dependent binding is specific for C type lectin receptor family that MRs belong to. Five remaining glycopeptides are currently undergoing biological evaluation.

The goal of this project was to synthesise potential flavour compounds combinatorially and identify key components for further investigation as flavourants in dairy products. This thesis describes the design and synthesis of libraries of ketones and γ-lactones that will be evaluated for flavour potential. Gas chromatography-mass spectrometry (GCMS), the Fox, and gas chromatography-olfactometry (GC-O) were used throughout this study. Ketones were synthesised individually via a two-step sequence: a Grignard reaction followed by the oxidation of the resulting alcohol in Chapter 2. Some compounds selected from the Fox analysis were assessed by GC-O. The analysis gave promising results for aromatic and cyclopropyl ketones and a library of cyclopropyl ketones was prepared. Individual racemic lactones were synthesised via a two-step sequence: the Linstead modification of the Knoevenagel reaction and subsequent lactonisation in Chapter 3. Libraries of racemic γ-lactones (C8-C12), including α-substituted γ-lactones, were produced combinatorially. Further, synthesis of a library of γ-thionolactones was achieved by treatment of a library of γ-lactones with Lawesson's reagent. The libraries were analysed by GC-O. A (R)-dodecalactone was synthesised from L-glutamic acid and the (S)-enantiomer was synthesised by the same sequence from D-glutamic acid in Chapter 4. Asymmetric syntheses of both enantiomeric series of γ-lactones utilizing the Sharpless asymmetric dihydroxylation reaction was employed to give the libraries in Chapter 5. Libraries of a-substituted and β-substituted γ-lactones were synthesised combinatorially and analysed by GC-O.

Content removed due to copyright restrictions: E I-Sherbiny, I.M., Lins, R.l , Abdel-Bary, E.M., Harding, D.R.K. Preparation, characterization, swelling and in vitro drug release behaviour of poly[Nacryloylglycine- chitosan] interpolymeric pH and thermally-responsive hydrogels. Eur. Polym. J. 41 (2005) 2584. E I-Sherbiny, I.M., Abdel-Bary, E.M., Harding, D. R.K. Preparation and swelling study of a pH-dependent interpolymeric hydrogel based on chitosan for controlled drug release. Inl. J. Polym. Mater. 55 (2006) 789. El-Sherbiny, I.M., Abdel-Bary, E.M., Harding, D. R. K. Swelling characteristics and in-vitro drug release study with pH and thermo-sensitive hydro gels based on modified chitosan. J. Appl. Polym. Sci. 102 (2006) 977. Abdelaal, M.Y., Abdel-Razik, E . A , Abdel-Bary, E.M., El-Sherbiny, I.M. Study on chitosan-poly(vinyl alcohol) interpolymeric pH-responsive hydrogel films for controlled drug delivery. J. Appl. Polym. Sci. (2006) in press. El-Sherbiny, I. M., Abdel-Bary, E.M., Harding, D. R. K. In-vitro investigation of new biodegradable pH-responsive hydrogel beads for oral delivery of protein drugs in the small intestine. New Zealand Institute of Chemistry Conference (NZIC), (2006) Rotorua, New Zealand, 2-6 December.<br>Controlled drug release enhances the safety, efficacy and reliability of drug therapy. Regulation of the drug release rate results in a reduction in the frequency of drug administration and should encourage patients to comply with dosing instructions. Hydrogels are crosslinked, three-dimensional hydrophilic polymers, which swell without dissolving when brought into contact with water or other biological fluids. The number of polymers suitable for the controlled release of viable therapeutics is quite limited because of inherent toxicity or lack of certain properties such as biodegradability. In this thesis, chitosan was chosen as the base polymer for the development of new hydrogels that can be tailored for use in the site-specific delivery of drugs to the gastrointestinal tract. Chitosan is a non-toxic and biodegradable polymer obtained through the alkaline deacetylation of natural chitin. The interesting characteristics of chitosan make it an ideal candidate for use in controlled drug release formulations. However, chitosan exhibits some shortcomings such as hydrophobicity and a high pH-dependency for its physical properties. Hence, it is very difficult to control drug release with chitosan itself because of the various pH values of the internal organs of the human body. This may negatively affect the human body because of drug under- or over-release. In a structured programme, some new chitosan-based hydrogels have been prepared for controlled drug release investigations by applying three main approaches to overcome the shortcomings of chitosan. The first approach was the incorporation of chitosan into interpenetrating polymer network hydrogels with either a hydrophilic polymer or with hydrophilic monomers treated to bring about in situ copolymerization in the presence of chitosan and a suitable crosslinking agent. The second approach was the chemical modification of chitosan by grafting of a suitable vinyl macromer such as poly(ethylene glycol)-diacrylate, then crosslinking this modified chitosan. The equilibrium swelling studies were carried out for the hydrogels prepared using these two approaches at 37 °C at pH 2.1 (simulated gastric fluid, SGF) and at pH 7.4 (simulated intestinal fluid, SIF). The swelling results showed a pH-responsive nature of these hydrogels. They attained higher swelling values in SGF than in SIF. 5-Fluorouracil (5-FU), an anti cancer drug, was entrapped as a model drug in all the hydrogels prepared using these two approaches. The in-vitro drug release studies were carried out at 37 °C in SGF and SIF. From the preliminary investigations of the prepared hydrogels, they may be customized and used to expand the utilization of these systems in drug delivery applications. In the third approach, chitosan was modified in such a fashion that the hydrogels produced were also pH-responsive but attained limited swelling in SGF and higher swelling in SIF. Hence, the resulting hydrogels could be tailored for utilization for intestine-targeted delivery of peptide and protein drugs with a potential protection of the drugs from the harsh acidity of the stomach. In this third approach the ionotropic gelation was used for the preparation of the hydrogels based on the modified chitosan with another natural polymer (sodium alginate) in the presence of a divalent ion. Bovine serum albumin (BSA) was entrapped as a model protein drug and the in-vitro drug release profiles were established at 37 °C in SGF and SIF. The results showed promising release profiles of BSA. However, this hydrogel study requires more effort to limit the swelling and consequently the loss of drug in the SGF, to act as an excellent candidate for intestine-specific delivery of peptide and protein drugs.

Bovine ß-lactoglobulin (ß-Lg) has been studied extensively but there is no clear identification of its biological function. Hydrophobic molecules have been observed binding into the hydrophobic calyx of ß-Lg. By comparison with other members of lipocalin family, it is probable that ß-Lg plays a role of transport of ligands, as ligands also bind into the central cavity of lipocalins. The structurally similar MUP is a pheromone-binding protein; therefore, it is possible that ß-Lg may also fulfil a similar role. This study has begun to test this hypothesis by investigating the interactions between bovine ß-Lg and several small volatile molecules (2-sec-4,5-dihydrothiazole, 3-methyl-2-butenal, 3-methyl-2-buten-1-ol and phenylacetic acid). The interactions between the volatile molecules and ß-Lg were studied by both two-dimensional NMR spectroscopy and X-ray crystallographic methods. TOCSY spectra were recorded for ß-Lg and the complex between ß-Lg and the ligands. The observed chemical shifts in the HN-Ha region are sensitive to the proximity of ligands, and hence chemical shift changes on ligand binding provide information on possible binding sites. It appears that several amino acids with hydrophobic sidechains are affected by interaction with volatile molecules at pH 2.0. The X-ray crystallographic study at pH 8.5 showed that the potential ligand, 2-sec-4,5-dihydrothiazole, may have decomposed into a linear 2-methyl-butanol. The refined structure (R=0.281, Rfree=0.354 for reflections to 2.6 Å resolution) reveals that the potential ligand may bind to the central cavity in a manner similar to the binding of 12-bromodecanoic acid to ß-Lg.

Milk is provided to new borns as their first food source and it contains essential nutrients, vitamins and other beneficial components, such as enzymes and antibodies that are required for rapid growth and development of the new born and for sustained growth over time. Milk contains two main types of proteins; casein proteins and whey proteins. Although casein proteins account for up to 80% of the proteins found in bovine milk, it is the whey protein that has become of high interest because of its bioactive content. Whey, a very watery mixture of lactose, proteins, minerals and trace amounts of fat, is formed from milk when the milk is coagulated and/or the casein proteins are removed from the milk. Bovine whey protein, including both the acidic and basic fractions (low and high isoelectric point, respectively), has previously been studied in vitro (cell based) and in vivo (using rats) for its impact on bone to determine if it can help improve bone mineral density and help reduce the risk of developing bone diseases, such as osteoporosis. Bone is constantly undergoing a remodelling process of being dissolved and reformed and the two main cell types responsible for this bone remodelling process are mature osteoclasts, which dissolve (resorb) bone, and osteoblasts, which reform the bone. Prior work has shown that acidic protein fractions derived from different sources of whey protein concentrate (WPC) have both in vivo and in vitro activity on bone, particularly anti-resorptive properties. However, the component(s) which confer activity have not yet been identified. In this thesis, work was undertaken to better understand the analytical composition of three types of WPC (cheese, mineral acid and lactic acid) and their associated acidic protein fractions and relate this to bone activity in the hope of identifying where the activity lies. Bone activity was assessed using in vitro screening with osteoblast cells (MC3T3-E1) and osteoclast cells (RAW 264.7). Comparison of the cell-based bone activity of the parent WPCs and corresponding acidic fractions indicated that the acidic fractions derived from both mineral acid and lactic WPC were superior in their ability to inhibit osteoclast development. Although compositional data was complex and definitive correlations with both bone bioactivities could not be made, it appeared that elements common to both the acidic fractions were a higher proportion of GLYCAM-1 and bone sialoprotein-1 (osteopontin). Further studies to more closely investigate the bone bioactivity of the acidic fractions are warranted.

3-Deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P) synthase and 3-deoxy-Dmanno- octulosonate 8-phosphate synthase (KDO8P) synthase are two enzymes that catalyse very similar reactions. DAH7P synthase is the first enzyme of the shikimate pathway and catalyses the condensation reaction between the four-carbon sugar erythrose 4-phosphate (E4P) 1 and the three-carbon sugar phosphoenolpyruvate (PEP) 2 to give the seven-carbon sugar DAH7P 3. KDO8P synthase catalyses a similar condensation reaction between the five-carbon sugar arabinose 5-phosphate (A5P) 8 and PEP 2 to give the eight-carbon sugar KDO8P 9. Early mechanistic studies have shown the reaction mechanisms of these two enzymes to be very similar and structural and phylogenic analysis has suggested that the two enzymes share a common ancestor. However, there are differences between the two enzymes that have not been explained by the current literature. Whereas all DAH7P synthases require a divalent metal ion for activity, there exists both metallo and non-metallo KDO8P synthases. As well as this, there is the difference in substrate specificity. The natural substrate of KDO8P synthase, A5P, is one carbon longer and has the opposite C2 stereochemistry to E4P, the natural substrate of DAH7P synthase. This study investigates the role of the C2 and C3 hydroxyl groups of E4P and A5P in the enzyme catalysed reactions. The E4P analogues 2-deoxyE4P 38 and 3-deoxyE4P 39 have been synthesised from [beta]-hydroxy-[gamma]-butyrolactone and malic acid respectively. The two analogues were tested as substrates for DAH7P synthase from a variety of organisms, including N. meningitidis, the purification and characterisation of which was carried out during the course of these studies. It was found that both analogues were substrates for DAH7P synthase. 2-DeoxyE4P was found to be the best alternative substrate for DAH7P synthase to date. The analogous study was carried out on KDO8P synthase from N. meningitidis with 2- deoxyR5P 34 and 3-deoxyA5P 40. It was found that removal of the C2 and C3 hydroxyl groups of A5P was much more catastrophic for the KDO8P synthase catalysed reaction. Commercially available 2-deoxyR5P was found to be a very poor substrate, whereas 3-deoxyA5P, which was prepared according to a literature procedure was not a substrate. The difference in substrate specificities of DAH7P synthase and KDO8P synthase is consistent with the hypothesis that despite their similarities, these two related enzymes have different mechanisms. The key step for DAH7P synthase appears to be coordination of the E4P carbonyl to the divalent metal. The metal appears to play a less important role in the KDO8P synthase reaction and the key step is the correct orientation of A5P in the active site.