Some in vitro reactions of peroxynitrite are examined using 15N CIDNP NMR spectroscopy to elucidate more information about the radical nature of nitration, nitrosation and isomerisation mechanisms. Preliminary work on the nitration of tyrosine with H15NO3 confirms that 3-nitrotyrosine forms predominantly by the same radical mechanism established for other activated aromatic compounds such as 4-methylphenol. Reaction of alkaline [15N] peroxynitrite with biotarget-type molecules such as tyrosine, a tyrosine-containing tripeptide and thiol-containing amino acids allows nuclear polarisation effects to be discerned which are consistent with free and solvent-caged NO2 and HOo or CO3. The phase of the nitrate signal observed during peroxynitrite isomerisation at pH 7.4 is opposite to that observed during reaction with a biotarget-type molecule, suggesting dimerisation of NO2 to N2O4 and its subsequent hydrolysis in addition to in cage geminate pair collapse. Mechanisms in accord with the observations are suggested and their implications and extent to which they concur with established theories are discussed. Kinetic simulations are used to estimate the relative importance of nitryl chloride and peroxynitrite as in vivo reactive nitrogen species. Three furazan 2-oxide (furoxan) derivatives with potent NO- donating activity were synthesised according to literature methods and their decomposition in aqueous solution to yield NO was examined using EFR spectroscopy by spin trapping with Fe2+-N-methyl-D-glucamine dithiocarbamate (Fe2+-MGD). Ammonia, arising from thiol-mediated reductive decomposition pathways, was also detected in quantities up to approximately 8% of the nitrogen- containing decomposition products. A commercially-available enzymatic assay based on reductive amination of 2-oxoglutai-ate using L-glutamate dehydrogenase and NADPH was used. The proportion of furoxan giving rise to these products is estimated and possible mechanisms for their generation are suggested.

The ability of substituted N-hydroxyguanidines to act as nitric oxide donor drugs has been previously established. To further investigate the potential of this class of compound as nitric donor drugs a number of new N-hydroxyguanidines were prepared. The compounds were synthesised and then subjected to a number of chemical and biological tests. A number of the examples prepared were indeed shown to be oxidised under chemical conditions to give nitric oxide. The nitric oxide was tested for using the Greiss test. One example, 1-benzyl-1-methyl-2-hydroxyguanidine showed biological activity and was found to be a vasodilator. In addition an X-ray crystal structure of this compound was obtained which gave a further insight into the conformation of this type compound and related analogues. A number of ureas were also prepared for use in the biological and chemical testing. Under chemical conditions the ureas were not oxidised thus indicating that the hydroxyguanidine functionality was required for nitric oxide generation. O-Substituted N-hydroxyguanidines were also prepared in an effort to examine the possibility of selectively targeting the molecule to the site of action for nitric oxide release. These compounds were found to produce a small amount of nitric oxide under chemical oxidation, therefore confirming their potential as potential nitric oxide donor compounds. Acid catalysed decomposition of O-THP-1,1,-dibenzyl-2- hydroxyguanidine was studied. The reaction was carried out at various pHs and was monitored using HPLC. It was found that the THP protecting group could be removed to give the free N-hydroxyguanidine. More easily removed O-substituents are probably required to produce successful prodrugs. However the reaction only gave a reasonable rate at a very low pH (t1/2 = 122 s at pH 0). A number of O-substituted hydroxylamines were then prepared in an attempt to increase the range of O-substituted N-hydroxyguanidines that could be obtained.

The coenzyme B12-dependent enzyme, glutamate mutase (E. C. 5.4.99.1), catalyses the reversible carbon-skeleton rearrangement of (2S)-glutamic acid to (25.35)-3-methylaspartic acid. Glutamate mutase is the first enzyme on the mesaconate pathway. A variety of glutamate and 3-methylaspartate analogues (which also include isotopically labelled molecules), were synthesised as molecular probes of the enzyme. Synthesis of stereospecifically labelled 3-ethylaspartic acid: (2S,3S)-[3'-C2H3], and (2S,3S)-[C2H2C2H3]-ethylaspartic acids were constructed using appropriately labelled iodoethane. (2S,3S)-2-Bromo-3-methylsuccinic acid was synthesised via the diazotization of (2S,3S)-3-methylaspartic acid, in the presence of bromide ion. (2S)-Methylsuccinic acid was synthesised by the catalytic hydrogenation of (2S,3S)-2-bromo-3-methylsucdnic acid. Biological studies of the synthesised compounds (including the labelled isotopomers) displayed no activity against glutamate mutase. 3-Methylaspartate ammonia-lyase, the second enzyme in the mesaconate pathway, catalyses the deamination of (2S,3S)-3-methylaspartic acid to mesaconic acid. A range of 1-substituted cyclopropane 1,2-dicarboxylic acids were synthesised using short efficient routes and were found to be good to potent inhibitors of 3-methylaspartase. X-ray crystallographic studies have determined the absolute stereochemistry. The mode of action of the most potent inhibitor, (1S,2S)-1-methylcyclopropane 1,2-dicarboxylic acid (20 mumol dm-3), is consistent with it acting as a transition state analogue for the central substrate deamination reaction catalysed by the enzyme. beta-Amino acids are constituents of many biologically active peptides. A general procedure for the synthesis of alpha-substituted-beta-amino acids has been developed. The synthesis involves a Baylis-Hillman amine catalysed conversion of methyl acrylate, with an appropriate aldehyde, to give the alpha-(hydroxyalkyl) acrylate. Bromination and subsequent azide displacement furnishes the azido alkene, which is catalytically hydrogenated, to furnish the beta-amino ester.

The question under examination is whether there are various 5-HT receptor types present in the hearts of different species of molluscan bivalves. In order to examine this question, the actions of 5-HT and some analogues on the hearts of several species of bivalve molluscs were investigated in two different preparations. The effects of 5-HT and analogues were examined on isolated whole ventricle preparations and on the unitary currents of patch-clamped myocytes from some of the same species. The 5-HT receptor activated in Mya arenaria and Mercenaria mercenaria myocytes produced a decrease in unitary current activity across the membrane, which correlated with the effect seen by elevation of cAMP intracellularly. This fits well with the excitation of the whole hearts by 5-HT, and the earlier literature. In Geukensia demissa the presence of another 5-HT receptor was observed which when activated also causes an increase in K+ unitary current activity; which was not mimicked by elevation of intracellular cAMP. This also correlated with some of the mixed effects of 5-HT on the whole hearts. However, 5-CT and 5-MEOT caused a decrease in K+ unitary current activity; which also correlated with the inhibition of the whole hearts by 5-CT and 5-MEOT. The effects observed with 5-HT, 5-CT, 5-MEOT in Geukensia demissa appear to be linked to the activation of an intracellular pathway independent of cAMP. The results described from the whole heart and the myocytes experiments suggest that there are in fact more than one conformation of 5-HT receptor present in the hearts of bivalves.

The mechanism of action of some conventional (sodium nitroprusside, NP and S-nitroso-N-acetyl penicillamine, SNAP) and novel (iron-sulphur cluster nitrosyls) nitric oxide (NO) 'donor' drugs has been studied. Experiments were performed on segments of internally-perfused, rat isolated tail artery. Bolus injections (10μl) of SNAP and NP produce fully reversible (transient or T-type) vasodilator responses. Injections of Roussin's black salt (RBS), an iron-sulphur cluster containing 7 ligated nitrosyl groups, and a related cubane-like iron-sulphur cluster with 4 ligated nitrosyls (CUB), produce conventional T-type responses at doses below a critical threshold concentration (Dt). However, both compounds generate extraordinarily long- lasting (sustained or S-type) responses when the injected dose >Dt: these comprise an initial, rapid vasodilation, followed by partial recovery only. The resulting 'plateau' of reduced tone persists for several hours. Histochemical and X-ray microprobe analyses demonstrate that the plateau is due to slow release of NO from RBS or CUB taken up into the endothelium. RBS and CUB are also shown to be photosensitive: vasodilator responses to both compounds are potentiated by light. A study has also been made of the mechanism of vascular smooth muscle (VSM) photorelaxation. The phenomenon is shown to be due to release of NO from a photodegradable molecular store, which probably exists in the form of nitrosothiol(s), located in VSM cells. The store can be depleted by exposure to laser light, and then regenerated in the dark. The repriming process is dependent on basal release of NO: it is prevented by known inhibitors of NO synthase and by haemoglobin. Prior treatment of vessels with ethacrynic acid, a thiol alkylating agent, also prevents repriming. The therapeutic implications of these findings are discussed.

In the first part of this thesis (chapters 1-8) the structure/activity (S/A) correlation studies on a class of anti-cancer drugs based on flavone acetic acid (FAA) by means of computer modelling techniques are reported. In particular, semiempirical and ab-initio quantum mechanical calculations have been performed on ten FAAs whose expeirmental anti-cancer activity was known. The results show that some calculated properties such as bond lengths, atomic charges, energies of the HOMO and the atomic orbitals involved in its formation, correlate with the anti-tumour activity. The correlations found were then used on another 38 molecules analogous to FAA whose anti-cancer activity had also been measured and of the 21 active molecules, 20 were predicted to be active by these SA correlations (95% success rate). From this study it also emerged that the pyrone ring may be directly involved in the anti-tumour mode of action of the FAA and it is suggested that vitamin-K may also play a role. The second part (chapter 9) is a study of the dependence of the molecular electrostatic potential on the basis set. From this study it emerged that GEOSMALL and MINI-1 minimal basis sets produce MEPs that are more similar to those obtained with the 6-31G** basis set than the MEP obtained with the STO-NG basis set. GEOSMALL and MINI-1 also give better energies and better properties than STO-NG and their use is recommended when properties of large organic molecules are of interest. Also, from this study it emerged that the use of Mulliken charges for the calculation of the MEP with the point charge approximation is not advisable for it may lead to very different pictures of the electrostatic potential calculated directly from the wave function.

Thermodynamic parameters have been obtained for the complexes formed between protons, first Transition Series metal ions and the asparaginate ion in aqueous solution at 25°C in an ionic background of 3.00 M (sodium) perchlorate. The metal ions under consideration are manganese(II), iron(II), cobalt(II), nickel(II), copper(II) and zinc(II). The techniques employed were potentiometry to obtain the formation constants (and hence ΔG°) for the complexes, and aqueous titration calorimetry to obtain the corresponding ΔH° (and hence Δs°) values. The results show that iron, cobalt, nickel and zinc are capable of binding up to three asparaginate ligands, whereas for manganese and copper the maximum number of bound ligands is two. All the complexes reported one simple AnB species: no hydroxy, protonated or polynuclear species were detected. The results are used (l) to discuss an unusual "homologue" effect whereby the trends among homologues for bonding to protons and to metal ions are opposite to each other, this apparently being a repercussion of solvation differences between homologues and (2) to show that quite a large proportion of asparaginate in blood plasma may be complexed to Zn(II), Fe(II), and Co(II), as well as the expected Cu(II). The techniques of potentiometry and calorimetry are further employed to determine the thermodynamic parameters for three ternary systems, one, copper(II)-histidinate-threoninate, which is known to exist in vivo, and two others, copper(II)-asparaginate-histidinate and copper(II)-asparaginate-threoninate, which have not yet been detected. Structures are suggested for the complexes Cu.asn.his,H+, Cu.asn.his.thr. , Cu.asn.thr. Cu.his.thr. and Cu.his.thr.H+ . In these complexes histidinate is tridentate to Cu(II), threoninate is bidentate and asparaginate is intermediate between bi and tridentate. The site of proton attachment in the protonated complexes is the primary amine site of the histidinate ligand. Throughout the work extensive use was made of computer programs, all of which are described in the chapter entitled "Computational Aspects".

Section A; Insoluble elastin was isolated from bovine aorta and ligaraentum nuchae by the use of guanidine and dithiothreitol in conjunction with collagenase, purified by affinity chromatography. The preparations were free from carbohydrate and exhibited amino acid compositions similar to that of alkali-purified elastin, with the exception of the concentration of some polar amino acid residues. N-terminal analyses indicated a very low level of polypeptide chain damage in the preparations. Upon mild alkaline treatment glycine was selectively liberated as amino-end group. Elastin from ligamentum nuchae was examined by electron microscopy, X-ray diffraction, optical polarisation analysis and dynamometry. It is suggested that elastin fibrils consist of a lateral array of primary filaments. Section B: The microfibrillar component of adult bovine ligamentum nuchae was isolated by solubilisation in guanidine-dithiothreitol followed by S-carboxymethylation and treatment with collagenase. The amino acid composition of the material was at variance to those previously reported for similar preparations. N-terminal analysis revealed glycine as the only end-group, at a concentration of 65.9 moles/106g of protein. This value, corresponding to a molecular weight of about 15,000 daltons, was in good agreement with the results of sedimentation equilibrium analyses.

No description available.

No description available.