The aim of the work presented in this thesis is to investigate novel conducting polymers and copolymers, derived from indole and substituted indoles. Particular emphasis has been given to the study of the electropolymerisation mechanism, the structural characterisation of the deposited layer and possible applications as electrochemical sensors. The redox and morphological properties of the electrochemically deposited polymer layers have been investigated with regard to the steric and electronic effects induced by varying the substituent. The use of substituents to enhance polymer solubility has also been addressed. In particular, a thorough investigation of poly(indole-5-carboxylic acid), poly(5-cyanoindole) and poly(indole) has been undertaken. The major component in the deposited layer has been identified as an asymmetric trimeric unit and has also been identified as the main repeat unit within the polymer. Furthermore, the formation of this trimeric unit is believed to be general to all the indole based conducting polymers studied. The trimeric units and larger oligomers/polymers have been characterised electrochemically and spectroscopically. The polymerisation mechanism has been postulated and has led to the production of a reproducible polymer layer. The electropolymerisation of novel polymers produced by the copolymerisation from a mixture of two substituted monomers has also been investigated. Copolymerisation has resulted in the formation of bifunctional trimer units which have been chromatographically separated and characterised individually by both electrochemical and spectroscopic methods. Potential applications of this type of polymer are as sensors. Several polymers and copolymers have been studied in an aqueous electrolyte, with the electrochemical response being measured as a function of the electrolyte pH. The effect of adding metal ions to the electrolyte medium on the measured potential has also been studied. The data suggest the possible use of these functionalised polymers in simple electrochemical sensor systems.

No description available.

Two approaches for the preparation of some novel functionalised polymers were investigated using a combination of nitrile oxide chemistry and Ring opening Metathesis Polymerisation (ROMP). In the first case, homopolymers of norbornene (NBE) and norbornadiene (NBD) were synthesised and then modified by 1,3-dipolar cycloaddition to nitrile oxides (RCNO) to the poly(di)enes. These reactions were studied at varying molar ratios of the various nitrile oxides (R = Ph, CO₂Et, CO₂Me), which were generated <i>in situ</i> by the thermal dehydrochlorination of the corresponding hydroximoyl chlorides. The second method was via the ROMP of novel isoxazolino norbornenes. Thus, <i>exo</i>-3-phenyl-4,7-methano-3a,4,7,7a-tetrahydrobenzisoxazole and <i>exo</i>-3-oxa-4-aza-ethyl-tricyclo[5.2.1]dec-4-ene-5-carboxylate, were synthesised via the cycloaddition to NBD of the corresponding nitrile oxides, and were subsequently polymerised using {RuCl₂(=CHPh)[P₆H₁₁)₃]₂] 1 and {RuCl₂(=CHPh)[P(C₆H₁₁)₃][IH₂Mes]} 2. Extensive use has been made of monomeric alkenes as model compounds both to test the synthetic pathways and to assist in establishing the structure of products from the equivalent polymer reactions, for this purpose <i>trans</i>-5-decene and cyclopentene were used. The Isoxazolino norbornenes were polymerised with control of molecular weight (M_w and M_n) by reacting <i>exo</i>-3-oxa-4-aza-ethyl-tricyclo[5.2.1]dec-4-ene-5-carboxylic with varying ratios of hex-1-ene as a chain transfer agent (CTA). Thus, [CTA]/[Monomer] of 0.00, 0.5, 0.10, 0.15 and 0.25 yielded oligomers with an average degree of polymerisation av DP, of 554, 157, 16, 8 and 4 (determined by GPC and NMR end group analysis). A sample of an isoxazolino functionalised oligomer (n = 16) was converted into its hydrogenated analogue (83%) by treatment with <i>p</i>-toluenesulfonhydrazide. Having established that isoxazolino-norbornenes were suitable monomers for ROMP, the feasibility of using the analogues isoxazolino compounds was examined. <i>exo-</i>4-Methyl-5<i>^exo</i>-phenyl-3-oxa-4-aza-tricyclo[5.2.1.0^{2,6<i>oxo</i>}]dec-8-ene (70%), was prepared by the cycloaddition of <i>N</i>-methyl-<i>C</i>-phenyl nitrone to NBD. The analogous <i>exo, endo </i>cycloadduct from the cycloaddition of <i>C,N</i>-diphenylnitrone to NBD was also synthesised (32%) and the isoxazolidines polymerised using the initiators 1 and 2. The polymerisation of the <i>N</i>-methyl-<i>C</i>-phenyl and the <i>C-N-</i>diphenyl isoxazolidines with 1 yielded polymers with an av DP of 251 and 234, whereas with 2 the chain length increased to n = 490 and 683 respectively. An increase in polydispersity index (PDI) of 1.84 to 2.37 and 1.54 to 2.09 for was observed on going from 1 to 2.

No description available.

An investigation into the use of a novel N<SUP>PROB*LEM</SUP> protecting group in peptide synthesis is described. The protecting group, 2,2-<i>bis</i>(4'-nitrophenyl)ethoxycarbonyl (Bnpeoc), has been introduced into the full range of amino acids, including those requiring appropriate side chain protection. The Bnpeoc amino acid derivatives have been successfully applied to the solid phase synthesis of a number of peptides from ubiquitin. In addition, the peptides were assembled using a wide range of coupling reagents and conditions. The chemical syntheses of phage λ Cro protein and peptides from its DNA binding region have also been investigated, using both the novel N^PROB*LEM-Bnpeoc protecting group and the established N^PROB*LEM-fluorenylmethoxcarbonyl (Fmoc) protecting group. N.m.r. experiments have indicated that residues corresponding to the DNA recognition α-helix of the Cro protein may adopt a similar conformation in a peptide from that region.

A new family of nucleotides has been designed, synthesised and evaluated for DNA sequencing by synthesis. Each of the nucleotide analogues had a free 3’-OH group and a fluorophore attached to the base through a small peptide linkage that was designed to stop multiple base additions. Analysis by HPLC and MALDI-TOF proved the modified nucleotides were incorporated into a growing DNA strand by a DNA polymerase and that the peptide linker was cleaved with high efficiency after incubation of the extended primer with a protease. Enzymatically cleavable nucleotides were successfully applied to chip-based sequencing by synthesis cycles. The fluorescent emission revealed the identity of the incorporated nucleotide and the removal of the fluorophore by a protease ensured the detection of the next base in the following cycle. Additionally, a practical microwave-mediated solid-phase protocol has been developed for the synthesis of cyanine dyes spanning the visible and the near infrared spectrum to allow the preparation of a series of fluorescent nucleotides for the design of a four colour DNA sequencing technology.

A new method of purification has been developed for peptides synthesised by the Merrifield solid phase methodology. N<SUP>PROB*LEM</SUP>-17-Tetrabenzo(a,c,g,i)fluorenylmethoxycarbonyl glycine (Tbfmoc Gly OH) has been prepared, and coupled <i>via</i> its symmetrical anhydride or its HOBt ester to the N-terminus of a peptide at the final stage of the synthesis. The strong affinity of the Tbfmoc group for graphitised carbon (PGC) allows the retention of Tbfmoc-protected peptides on a column packed with this material, whilst incorrectly terminated sequences and other materials are washed away. Following deprotection of the Tbfmoc group under basic conditions, the free peptide can be eluted from the column. This method has been exemplified by the purification of Ubiquitin (53-76) OH and Ubiquitin (35-76) OH. Preliminary results indicate that the technique may be developed into a form of affinity purification suitable for the resolution of racemates using ligand-exchange chromatography (LEC).

Flavocytochrome <i>c</i>₃ is a fumarate reductase expressed by the marine bacterium <i>Shewanella frigidimarina</i> during anaerobic growth in the presence of fumarate. This soluble periplasmic enzyme is composed of three domains; a flavin domain containing non-covalently bound FAD, a cytochrome domain and a clamp domain. The active site is located in the flavin domain at the interface with the clamp domain. The enzyme catalyses the reduction of fumarate to succinate with a k_cat of 509 ± 15 s^-1 and K_m of 25 ± 2mM at pH 7.2, 25°C. Reduction of fumarate requires hydride transfer from the FAD and protonation by an active site acid. Residues implicated in catalysis have been studied using site directed mutagenesis. Substitution of Arg402 by alanine leads to complete loss of activity whereas neither of the two active site histidines (His504 and His365) is essential for catalysis. The H365A:H504A double mutant enzyme was found to have a k_cat of 0.84 ± 0.05 s^-1 at pH 7.2. Substitution of Arg402 by lysine, histidine or tyrosine led to a fall in k_cat to 0.55 s^-1, 0.091 s^-1 and 0.05 s^-1 respectively. Substrate specificity and inhibition studies have been carried out to probe the active site structure. Fc<i>c</i>₃was unable to catalyse the reduction of alternative enoates to fumarate. However, oxaloacetate, methylsuccinate and 3-nitropropinoate were found to be inhibitors of succinate oxidation with K_i values of 5.3 mM, 1.7 mM, and 0.5 mM respectively.

No description available.

The development of novel protecting group strategies for the 2'-hydroxyl function of ribonucleosides has been reviewed and a new design has been formulated. The design is based upon a two stage deprotection strategy which is compatible with the final steps in RNA synthesis using solid phase methods. Central to this is the intramolecular addition to an acetylenic system leading to an acid-labile enol ether system, collapse of which results in liberation of the 2'-hydroxyl function. A series of propargyl based systems have been synthesised, incorporated and their suitability evaluated for the automated chemical synthesis of oligoribonucleotides using the phosphoramidite approach. Also discussed are the fully automated syntheses of branched oligodeoxynucleotides (ODNs), accomplished using a 5',2'-di(dimethoxytrityl)-protected <I>arabino</I>-uridine derivative as the branching monomer. The synthesis and incorporation of the branching monomer has been described. The affinities of the branched ODNs towards complementary single stranded ODNs were studied using UV thermal denaturing experiments.