The design of a solid phase peptide synthesis linker, which on cleavage with trifluoroacetic acid leaves the peptide C-terminus with a hydroxy ester functionality, is described herein. The enhanced rate of hydrolysis of hydroxy esters and carbamates under basic conditions has been investigated with a view to utilising this effect in the orthogonal protection of amino acid side chains during the syntheses of large peptides. The development of protecting groups for both acid and amine side chains has been investigated in order that the syntheses of proteins via chemical fragment coupling can proceed with a high degree of fidelity. The base hydrolysis of various esters and carbamates has been studied, as has the stability of these compounds to acidic conditions. The syntheses of a number of protecting group candidates based on tris(hydroxymethyl) derivatives has been undertaken and conditions for their removal optimised. The syntheses of various test compounds and small peptides utilising these types of protecting groups has been completed. The synthesis, purification and deprotection of a 32 residue peptide, with one lysine protected as the tris(hydroxymethyl)nitromethyl carbamate is also reported. The synthesis of a section of the CD4 binding region of the HIV coat protein Gp120 has been investigated. Various methods of cysteine protection have been studied and the difficulties associated with solubility and protecting group removal highlighted.

The chemical synthesis of the 184aa anti-angiogenic peptide, endostatin, was undertaken. Preparation <i>via</i> the stepwise of two large fragments approximately 90aa in length was attempted but was unsuccessful. A method that would allow the efficient, sequential coupling of several small fragments was required. To this end, the segment coupling of minimally protected fragments <i>via</i> transfer active ester condensation (TAEC), a technique recently developed within this research group, was investigated. The fragments for synthesis were provisionally selected based on hydrophobicity and potential coupling sites. These peptide fragments were then optimised for stepwise solid phase peptide synthesis (SPPS), using the Fmoc strategy. Peptides containing two types of C-terminal functionality - hydrazides and semicarbazides - were prepared. An alternative strategy for the synthesis of the Wang resin-based hydrazide linker, first proposed by Wang and Merrifield in 1969, was developed to facilitate this process. Peptide fragments of up to 20aa in length were successfully coupled using TAEC. A novel approach to the protection of arginine side chains was also investigated. This target was based on the dibenzocycloheptenyl system, a species which was originally deigned for use as a linker in the preparation of peptide amides. Recent work by Noda involved a derivative of this linker, which has proven to be significantly more acid labile than current arginine protection. Concurrent work on an improved design led to an alternative system - the dimethoxy-suberyl compound - being proposed. The synthesis of the target molecule was achieved in seven steps; key steps involved a Perkin reaction and an intramolecular Friedel Krafts acylation. Coupling to arginine was undertaken but proved unsuccessful.

The viability of extending the present methodology designed for the solid phase synthesis of peptides has been investigated. Using the catalytic domain of stromelysin (SCD. 173 residues) as the model system, a number of different factors affecting the preparation and purification of chemically synthesised proteins are examined. The purification SCD, prepared using stepwise solid phase synthesis is described. Following characterisation, it is evident that protein of the correct primary sequence has been prepared, furthermore, preliminary studies centred on the enzymatic activity of SCD indicate that active protease has been isolated. However, comparison of the conformational and biophysical properties of chemically synthesised SCD with the recombinant counterpart, suggests that there are problems associated with the folding of the synthetic SCD. The construction of chemically synthesised SCD <I>via</I> convergent protein synthesis is also described. Two different coupling strategies involving classical and azide fragment condensation are examined where it has been highlighted that the overall success of each fragment coupling is greatly dependent on the peptide length and sequence. As well as comparing methods for the preparation and coupling of fully and minimally protected peptides, general procedures for both solution and solid phase fragment coupling are discussed. A novel strategy for the convergent synthesis of peptides and proteins has been investigated. In this total chemical synthesis, two minimally protected peptides are joined through unique, mutually reactive functional groups, yielding a peptide analogue with a thioether replacement for the native peptide bond at the site of ligation. A general route to C-terminal sulfhydryl and N-terminal haloacetylated peptides is presented, accompanied with results of the preliminary ligation studies.

Flavocytochrome <I>c</I> from the Gram-negative bacterium <I>Shewanella</I> <I>putrefaciens</I> is a soluble, periplasmic fumarate reductase induced under anaerobic conditions. In order to study this protein the entire structural gene was cloned and sequenced, along with some surrounding sequence. Analysis of the cloned gene indiciates that flavocytochrome <I>c </I>consists of 571 amino acids preceded by a putative periplasmic signal sequence of 25 amino acids. Flavocytochrome <I>c</I> appears to consist of two domains: -a cytochrome domain consisting of approximately the first 117 amino acids containing the four <I>c</I>-type haem binding motifs (CxxCH), which shows some similarity to a tetrahaem cytochrome from the purple phototrophic bacterium <I>H-1-R </I>-a flavin domain comprising the remainder of the protein which shows significant similarity to the flavoprotein subunits of the family containing fumarate reductases and succinate dehydrogenases from other organisms. For example, it shows 26% identity to the flavoprotein subunit of <I>Escherichia coli</I> fumarate reductase at the amino acid level, including all the active site residues which have been identified. Three additional open reading frames have been identified in the regions flanking the <I>fcc</I> gene. ORF2, is situated -400bp downstream of <I>fcc</I> and reads in the same direction as the <I>fcc</I> gene. The deduced amino acid sequence of this open reading frame is 135 amino acid residues in length and shows some limited sequence similarity for FrdD, one of the two subunits of <I>E. coli</I> fumarate reductase which serves to anchor the enzyme to the membrane and is involved in electron transport to the catalytic domain. Since flavocytochrome <I>c</I> is a soluble enzyme, the product of ORF2 does not appear to be a membrane anchor for this enzyme, however a role in electron transport to flavocytochrome <I>c </I>has not been ruled out. ORFI and ORF3 both read in the opposite direction from flavocytochrome <I>c </I>and neither is completely contained on the cloned fragment. Searching of the databases has revealed no apparent homologues for either of these reading frames.

The preparation and evaluation of a number of carboxylic acid protecting groups is reported. The preparation of aspartic acid side chain esters of 9-anthracenylmethanol, 1-(9'-anthracenyl0-3-methylbutan-3-o1, 1-4(4'-fluorophenyl)-2-methylpropan-2-o1, dibenzosuberol and 4-methyl-2,6,7-trioxobicyclo[2.2.2]octane are described. The conversion of these into suitably protected N<SUP>α</SUP>- Fmoc derivatives for the solid phase synthesis of peptides has been carried out and the comparison with established protection strategies is discussed. Model compounds of these derivatives have been prepared which have enabled an evaluation to be carried out of the stability of these systems to acidic and basic hydrolysis.

No description available.

No description available.

No description available.

Although a considerable amount of circumstantial evidence exists which indicates the involvement of Ca<SUP>2</SUP>&43 in the regulation of phytochrome-mediated responses (Bossen <i>et al</i>., 1988; Viner <i>et al</i>., 1988; Roux, 1986), the exact nature of this involvement has remained unclear. Etiolated wheat leaf protoplasts swell in response to red light (Bossen <i>et al</i>., 1988) by a mechanism which is phytochrome-mediated and is related to leaf growth and unrolling (Zhou <i>et al</i>., 1990). In this study fluorescent Ca<SUP>2</SUP>+ indicators have been used in conjunction with Laser Scanning Confocal Microscopy to measure changes in [Ca<SUP>2</SUP>+ ]<SUB>i</SUB> following red light irradiation of these protoplasts. In most cases (n = 18 of 23), an increase in [Ca<SUP>2</SUP>+ ]<SUB>i</SUB> of approximately 400-700nM was observed, followed by a decrease to below resting level. This response preceded the physiological response of protoplast swelling, although the timing of the [Ca<SUP>2</SUP>+ ]<SUB>i</SUB> response varied between protoplasts. Transient increases in [Ca<SUP>2</SUP>+ ]<SUB>i</SUB> initiated by photoactivation of caged Ca<SUP>2</SUP>+ or caged InsP<SUB>3</SUB> mimicked the swelling response, and photoactivation of Diazo-2, a caged Ca<SUP>2</SUP>+ scavenger, within these protoplasts prevented the swelling response. Data is also presented here which suggests that cAMP, released from caged cAMP, can also induce an increase in protoplast volume, possibly by causing an increase in [Ca<SUP>2</SUP>+ ]<SUB>i</SUB>. These data support the hypothesis that phytochrome signals are transduced through Ca<SUP>2</SUP>+ and suggest a possible role for cAMP in signal transduction within plant cells.

No description available.