This thesis is concerned with the synthesis of acid labile co-polymers. Two polymer systems were examined (1) polyacetals and (2) poly(ortho esters). As for poly(ortho esters), there is a need for better synthetic methods to prepare these polymers more easily without the need of stringent anhydrous conditions, with more broad structural variation, and in a more cost effective manner. Pendent functionalised polyacetals derived from PEG and tyrosine derived monomer diols have been prepared and their structure activity relationships determined. A smaller size alkyl chain on the tyrosine derived monomer diol increased the rate of degradation of these polyacetal libraries. For poly(ortho esters), a first strategy involved the preparation of novel stable orthoester monomers. The key aspect was to embed the orthoester within the monomer while providing orthogonal polymerisation functionality. This synthetic route attempts to address the synthetic limitations for the preparation of existing poly(ortho esters) and it is believed to be the first such example. The stabile symmetrical bicyclic 2.2.2 orthoester monomer molecule derived from the naturally occurring metabolite phenyl acetic acid was used to prepare the new poly(ortho esters). The bicyclic orthoester 2.2.2 ring arrangement provided the monomer with rigidity, therefore enabling a pure solid monomer to be prepared in three synthetic steps. This approach provided a more efficient polymerisation reaction that requires less stringent polymerisation reaction conditions then existing literature examples for preparing poly(ortho esters). The second broad strategy examined the synthesis of a hydrolytically stable precursor poly (oxetane esters), which underwent a pH triggered rearrangement reaction within the polymer mainchain to prepare orthoester moieties in the polymer mainchain. In conclusion these strategies have provided new synthetic examples of preparing the highly degradable acid labile polymers e.g. poly(ortho esters).

A number of groups have been investigated as potential protecting groups for the thiol functionality of cysteine residues during Solid Phase Peptide Synthesis. Of these the 4-picolyl system has been found to be successful. A short, efficient synthesis to the protected amino acid and mild cleavage conditions have been developed. This protected amino acid has been applied in the synthesis of a number of interesting cysteine-containing peptide targets. Deglycosylated human erythropoietin (dhEPO) has been synthesised by SPPS, employing the picolyl group for cysteine protection. Purification of this material has been achieved by gel filtration. The synthetic material has been characterised by amino acid analysis, SDS-PAGE, iso-electric focusing and <i>N</i>-terminal sequencing. Attempts have been made to synthesise deglycosylated human erythropoietin <i>via</i> a fragment coupling technique. All fragments were successfully synthesised and purified and model ligation studies carried out.

We have characterised two biologically important protein complexes. Firstly, we have captured and analysed a complex of the biotin protein ligase (BPL) and the biotinylation domain (BCCPΔ67) of acetyl-CoA carboxylase (ACC) from the hyperthermophile <i>Aquifex aeolicus. </i>The genes encoding both BPL and BCCPΔ67 were overexpressed in <i>E. coli</i> and purified to homogeneity. Isolation of milligram amounts of both recombinant proteins allowed us to perform kinetic analysis of the BPL enzyme using steady-state techniques. Furthermore, a chemically crosslinked complex of BPL and BCCPΔ67 was isolated and a comprehensive mutational study has identified a salt bridge between the two proteins which is important for heterodimerisation. The role of a conserved ‘thumb-like’ motif in BCCPΔ67 was also investigated by mutagenesis. Our results suggest an interaction between the biotin moiety and the ‘thumb’ reduces the propensity of BPL and holo-BCCPΔ67 to heterodimerise. In a separate project we have investigate the relationship between the tertiary structure and biological activity of a novel β-defensin related peptide (Defrl). Defensins are cationic antimicrobial peptides which have a characteristic six cysteine motif and are important components of the innate immune system Defr1 is a polymorphism of mouse β-defensin 8 and contains only 5 cysteines. Against a panel of pathogens, we found that oxidized synthetic Defr1 had significantly higher activity than its reduced form, and the oxidized and reduced forms of its six-cysteine containing analogue (Defrl Y5C). Using non-denaturing gel electrophoresis and high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) we observed Defrl and Defrl Y5C dimers.

The aim of the work described in this thesis was to assess 'Matrix Assisted Laser Desorption/Ionization' (MALDI) Time-of-Flight mass spectrometry as a possible technique for the analysis of synthetic polymers. A compact home-built time-of-flight mass spectrometer of cylindrically symmetrical geometry was used to carry out all the mass spectrometry detailed described in this thesis. A survey of the literature describing the development of matrix assisted laser desorption/ionization spectrometry and also the previous analysis of polymers by laser mass spectrometry was carried out. Experiments comparing the performance of the apparatus with published data on peptides and proteins were carried out, followed by experiments to assess the possibility of analyzing synthetic polymers. Initially polar polymers were investigated, since they could be anlayzed using sample preparations very similar to those developed for peptide and protein analysis. Later investigations were carried out on non-polar polymers such as polystyrene. The attachment of a range of transition metals to a low mass polystyrene was investigated using laser desorption/ionization time-of-flight mass spectrometry, without the use of matrices to increase the ion yield. Systematic investigations into the effects of sample spot composition, i.e. the amounts of matrix, polymer and salt present, were carried out, and used to suggest possible models for the processes leading to the generations of large gas phase ions. The effects of sample spot composition on the size and shape of the polymer molecular weight distributions obtained was also investigated. Liquid polymers such as polysiloxanes and perfluorinated polyethers were investigated using laser desorption/ionization and matrix assisted laser desorption/ionization. Carbon cluster generation from such polymers was investigated, and fullerene and polycyclic aromatic hydrocarbon analysis was also briefly studied. Novel new molecules such as aryl ester dendrimers were investigated, since they could not be successfully analyzed by other mass spectrometric techniques. Single molecular ions were obtained, helping to confirm the expected masses of these molecules. Novel new polymers such as hyperbranched aromatic polyesters were also analyzed, and molecular weight distributions were successfully obtained for a number of samples, showing the utility of MALDI for the analysis of new materials.

Serine and cysteine proteases are potently inhibited by peptide aldehydes. Compared with the plethora of methods available for solution synthesis of peptide aldehydes, there are relatively few methods for the solid phase synthesis of peptide aldehydes. The development of novel linkers for the solid phase synthesis of peptide aldehydes is reported (Scheme 1). When X = N, the aldehyde 4 can be cleaved in mild acid with 97% enantiomeric excess (e.e.). When X = O, base hydrolysis results in aldehyde 4 cleavage with complete epimerisation of the α-stereocentre. The use of <i>Mucor meihei</i> lipase and penicillin acylase generates the aldehyde 4 in 19 and 31% yield respectively, with complete retention of chiral integrity (>99%). The utility of linker 1 was demonstrated by the solid phase synthesis of a tetrapeptide aldehyde.

Flavocytochrome <i>b₂</i> from <i>Saccharomyces cerevisiae</i> is an L-lactate:cytochrome <i>c</i> oxidoreductase. The crystal structure of this homotetrameric enzyme has been solved to 2.4 A (Xia and Mathews, 1990), and many aspects of its function have been investigated by the construction and kinetic analysis of site-directed mutant forms. From inspection of the crystal structure, Arg289 can be seen to interact with Arg376 which is a key residue for formation of the Michaelis complex. In addition Arg289 also forms a hydrogen bond with a water molecule which is itself hydrogen-bonded to a heme propionate. Substitution of this arginine by lysine results in an enzyme with altered kinetic properties. The Arg289(r)Lys mutant flavocytochrome <i>b₂</i> (R289K-<i>b₂</i>) is shown to have a decreased <i>k_cat</i> for ferricyanide-dependent L-lactate dehydrogenation (33.0 ± 3.9 <i>vs</i>. 400 ± 10 s^-1 for the wild-type enzyme) with an increased value of <i>k_cat</i> for L-lactate (1.40 ± 0.20 mM <i>vs</i>. 0.49 ± 0.05 mM for the wild-type enzyme). With cytochrome <i>c</i> as electron acceptor, the <i>k_cat</i> for L-lactate dehydrogenation by R289K-<i>b₂</i> is also decreased by an order of magnitude relative to wild-type enzyme. Pre-steady state kinetics confirm that the rate constant for FMN reduction by L-lactate is significantly decreased (19.6 ± 1.7 s^-1cf. 604 ± 60 s^-1in the wild-type enzyme), leading to the conclusion that substitution of Arg289 with lysine has caused FMN reduction to become the rate-determining step of the catalytic cycle. This is contrary to the situation for the wild-type enzyme, where transfer of an electron from FMN semiquinone to the heme is rate limiting (Daff <i>et al.</i>, 1996a). It is thought that this altered behaviour may be due to structural changes at the active site which affect substrate binding and dehydrogenation. Ligand-binding properties of R289K-<i>b₂</i> were investigated by a series of inhibition experiments which showed significant changes from wild-type behaviour. This was particularly evident with pyruvate, the product of the reaction which displayed a different type of inhibition in R289K-<i>b₂</i>. The crystal structure of R289K-<i>b₂</i> was solved to 2.75 A resolution, and this revealed changes in the position of some active site residues, in particular Arg376, a residue which is important in substrate binding.

No description available.

A procedure has recently been derived which makes it possible to determine the molecular weight of a particle from sedimentation velocity studies alone. Application of this new theory has enabled the molecular weight of both the myosin molecule and the myosin filament isolated from rabbit skeletal muscle to be determined. It has further been demonstrated by exhaustive curve-fitting that myosin A solutions contain no appreciable amounts of dimer at high ionic strength. This is in direct contradiction to the prediction by Godfrey and Harrington (l970a,b) and Harrington and Burke (1972) that a monomer-dimer equilibrium existed in myosin solutions. An analysis has been presented which shows that these latter results may be satisfactorily explained without recourse to a monomer-dimer hypothesis. A detailed analysis of the hydrodynamic properties of synthetic A-filaments has shown that they display a feature not previously demonstrated heretofore; namely that they invariably possess a range of frictional ratios some 75% higher than can be accounted for in terms of the length-equivalent prolate ellipsoids of revolution. On the assumption that preparations of natural A-filaments exhibit similar frictional properties as those formed in vitro, it has been possible to determine the helical symmetry of purified natural A-filaments by measuring their sedimentation coefficient. The sedimentation coefficient close to infinite dilution has been estimated for purified A-filaments by the technique of active enzyme centrifugation yielding a value of 132 3S (in agreement with the earlier but less precise estimate of 136 25S, Trinick, 1973). It was found that native A-filaments contain between 3.15 and 3.40 myosins/14.3nm axial repeat and this is regarded as good evidence in favour of a 3-stranded model for the A-filament.

This project explored the aqueous solution dynamics of the polydimethylaminoethyl methacrylate (PDMAEMA) and polyacrylic acid (PAA) systems using fluorescence spectroscopy techniques. PDMAEMA and PAA were synthesised several times using free radical polymerisation techniques with different fluorophore labels included in synthesis. The resulting polymers were explored with a selection of fluorescence techniques including time resolved anisotropy measurements, fluorescence decay lifetimes and steady state fluorescence studies. Initial solution dynamics of these polymer systems indicate that the PDMAEMA exhibits a tightly coiled structure at high pH values and adopts an uncoiled conformation at low pH values. PAA exhibits a loose coil conformation at particularly low pH values but adopts an open extended conformation from around pH 4.5. The study also shows that the amine groups within PDMAEMA can affect the fluorescence of labels within the polymer. Studies of the polymer samples in the presence of salts show that even small amounts of salts added to either system elicits a change in the polymer systems and these are generally most prominently shown to increase with small salt concentrations, with concentrations of over 1M often having a much lesser effect. It can generally be seen that the addition of salt to either system promotes the coiling of the polymer. In the case of PAA this is a much tighter coil and in PDMAEMA a lowering of the pH at which the polymer exhibits coiling is seen. Studies of systems made up of both polymers show complicated behaviour in the systems with the main effects being shown when the pH of the system is such that one or the other polymer system is ionised and acting in a similar manner to that of the salts.

The aim of the project was to synthesise a novel small peptide containing an ether linkage between two side chains. Computer modelling had shown the structure was likely to form a stable p turn in solution and would therefore be a good candidate to study this class of protein secondary structure. To do this a differentially protected bis-amino acid containing the ether linkage was retrosynthesised to chiral pool synthons L-aspartic acid and L-methionine. Many attempts were then made to differentially protect and manipulate the respective acid and sulfide side chains into synthons that would participate in Williamson ether synthesis. Techniques explored included N-chlorosuccinimide hemithioacetal formation, sulfonium salt displacement, bismuth trichloride and silver salt induced etherifications. Using molecules made during this research we then worked towards making a similarly constrained peptide containing a homolanthionine bridge, a thioether analogue of the anti-tryptic reactive site loop of Bowman Birk Inhibitor, a proteinase inhibitor protein. Chapter 1 contains a review of peptides constrained through bridging of side chains and the effects that this has upon them. Chapters 2 and 3 outline the synthetic steps that were used in the process of synthesising an ether linked bis-amino acid. Subchapter 2.2 shows the synthesis of protected homoserine, a nucleophile for ether reactions. Subchapter 2.3 highlights the difficulty in making differentially protected electrophilic amino acids and coupling reactions between the two synthons. In chapter 3 the protected electrophilic homoserine is coupled with cysteine to make a homolanthionine bridge which is then integrated into a short peptide. Chapter 6 contains the experimental procedures for the reactions carried out and the spectral data for isolated compounds.