The chemical similarities and contrasting biochemical properties of piericidin A and ubiquinone are presented and discussed. The former, a penta-substituted pyridine and a metabolite of Streptomyces mobaraensis, is an inhibitor of mitochondrial electron transport, whilst the latter is an electron carrier. A scheme to account for this difference is proposed involving an olefinic linkage of the long hydrocarbon side-chain. This was shown not to be responsible for the inhibitory action of piericidin A, since hydrogenation of the olefinic function did not noticeably reduce inhibitory potency. Piericidin A was labelled with tritium in order to investigate its binding to mitochondria. It was found to be bound unspecifically in a linear manner up to 10 mu moles/mgm. protein, but washing with bovine serum albumen removed most of this piericidin A. However a quantity corresponding to that required for the inhibition of NADH oxidation (0.02 mu moles/mgm. protein) could not be removed in this way. It is concluded that this piericidin A was bound in a site specific manner. This specifically bound inhibitor was recovered unchanged on extraction of the mitochondrial lipids with acetone. Hence it is concluded that the inhibitor is not covalently bound to the mitochondria. A number of analogues of piericidin A were synthesised. None of these compounds approached the potency of piericidin A as inhibitors, although those with lipophilic and phenolic properties were more effective in this respect than those without, suggesting the possible involvement of hydrophobic interactions and of a phenolic function during inhibition. The possible tautomers of the proposed structure of piericidin A are considered in relation to its spectroscopic and chemical properties. Contrary to the behaviour expected from this structure, the molecule appears to exist as a pyridinol, rather than the 1-(H)-4-pyridone tautomer. The evidence for the proposed structure of piericidin A is critically discussed. That for the arrangement of substituents on the pyridine nucleus depends partly on ultraviolet spectroscopic data, and more significantly, on two fragments arising from ozonolysis. The identity of one of these fragments (the 2,4-DlrP of methyl pyruvate) is in doubt, since its properties differ from those of either of two fully characterised isomers of authentic material synthesised specially. It is concluded that piericidin A may be a β-hydroxypyridine, rather than the proposed Ɣ-hydroxypyridine, since the remaining evidence is no longer conclusive. The spectroscopic properties of piericidin A mentioned above also favour this proposal. Two fully substituted β-hydroxypyridines, isomeric with the proposed pyridine nucleus of piericidin A, were synthesised. Neither had properties corresponding to piericidin A. With these and other hydroxypyridines, the comparative studies pertaining to piericidin A were extended, using the techniques of UV, IR H1NMR.and mass spectroscopy. A particularly simple diagnosis of hydroxypyridines by 1H NMR spectroscopy was developed, involving the use of trichloroacetylisocyanate. In addition base catalysed deuterium exchange of protons in the methyl groups of substituted pyridines was studied. As a result of these studies, distinctions can be made between β, and α or Ɣsubstituted methyl- and hydroxy-pyridines. Piericidin A appears to be a β-hydroxypyridine, its most likely structure being 2-alkyl-3,6-dimethoxy-5-hydroxy-4-methylpyridine. It is also concluded from the mass spectrum of piericidin A that there is an olefinic bond between carbon atoms 5 and 6 of the side-chain, rather than between carbon atoms 4 and 5 as published.

No description available.

The original intention of the work was to garner evidence for or against the participation of a D process in the substitution reactions of Cr (III) complexes. The kinetics of the aquation Cr(H₂O)5N32+ induced by nitrous acid were studied as an adjunct to stoichiometric experiments using the same reaction, in which the possible five coordinate intermediate, Cr(H₂O)5 3+, was sought. The rapid reaction rate was found to be first-order in [complex], [H+], and [HNO2], and much faster in the presence of chloride ion. High concentrations (2 - 5M) of added anions were used in the stoichiometric experiments in an attempt to trap the intermediate as mon-acidopenta-aquochronium (III) complexes. Low concentrations of such species were, indeed, detected, but the discrimination of the postulated intermediate for the anions and against solvent water was small. The discrimination factors obtained for C1- and Br- were much lower than those reported by another worker during the spontaneous acid hydrolysis of Cr(H2O)5I2+, and indicate that, either the trans-labialising ability of the iodo-ligand was responsible for the incorporation of C1- and Br- in the previous work, or that different intermediates are involved in the spontaneous and induced equations. Though the evidence tends to favour the participation of a highly reactive intermediate in the induced reaction, the data are not totally inconsistent with an interchange mechanism. Due to the inconclusive mature of the results obtained in the competition studies, and the uncertainty that other approaches would provide definitive evidence, attention was turned to an interesting observation that was made during preliminary experiments to the stoichiometric work. Nitrous acid was found to be an efficient catalyst for the aquations of halo-aquochronium (III) complexes in acidic aqueous media. Other oxyacids have little or no effect at low pH values. The kinetics of aquation of Cr(H2O)5Br2+ in the presence of HNO2 were studies in detail. Reaction rates are first-order in [complex] and [HNO2] and are proportional to the reciprocal of [H+] at high acid concentrations. At lower acidities, limiting rate behaviour is indicated. The possibilities of an ion-pair mechanism or direct attack by a nitrosating species on the leaving group were excluded, and transient nitrito-intermediates, formed by the rapid O-nitrosation of an aquo-ligand, are thought to be involved in the catalysis mechanism. Studies on the isomers cis- and trans-Cr(NH3)4(H2O)C12+ showed that the equation of only the cis-isomer is catalysed by HNO2, indicating that a cis-halo-nitritochromium (III) intermediate is labile with respect to loss of halide ion. Possible explanations for this phenomenon are advanced. Preparative work reinforces the conclusions derived from kinetic studies, and several new nitritochromium (III) complexes are reported. The acid hydrolyses of nitritochromium (III) complexes were found to be both rapid and acid catalysed, in contrast to a previous report. The same rate law is applicable to all five complexes studied and contains terms in both [H+] and [H+]2. The rate constant independent of acid concentration appears to be very small. A mechanism involving singly and doubly protonated intermediates is suggested. The reactions are thought to proceed without chromium-oxygen bond cleavage because of their rapid nature, their low ∆H+ values, and by analogy with the formations of nitrito-complexes, which are believed to avoid Cr-O fission. Much of the kinetic work involved in these studies was carried out using the stopped-flow method. Modifications to a Gibson-Milnes stopped-flow spectrophotometer are described which allow the instrument to be used with highly acidic and other corrosive solutions.

This work is the first investigation into the biosynthesis of abscisic acid. Green tomato fruit were injected with a solution of either the potassium salt of (±)-[2- 14 c] mevalonic acid in O.05M phosphate buffer, pH 7.3, containing 10% ethanol, or {±)-[2- 14 C] mevalonic acid in ethanol. Radioactivity was detected in material which co-chromatographed with authentic abscisic acid and the authentic derivatives of abscisic acid. This material exhibited the highly characteristic spectral properties of (+)-abscisic acid. Similar results were obtained when (t)-[2- l4 c] mevalonic acid was supplied to avocado fruit preparations. The results of this work have been published in the Biochemical Journal, Vol.ll2, p.547-548 (1969). Many analogues of abscisic acid have been synthesised at Shell Research Limited, Woodstock Agricultural Research Centre, and their growth inhibitory activity assessed. The strong inhibitory activity of (+)-5-(l',2'~epoxy-2' ,6',6'-trimethylcyclohexyl)-3- methylpenta-cis-2-trans-4-dienoic acid (epoxide) on plant growth was of particular interest since the compound has the same carbon skeleton and similar substitution to abscisic acid. To discover whether this compound was active per se or because it was converted to abscisic acid the (±)-[2- 14 C] labelled material was synthesised and injected, as an ethanol solution, into tomato fruit. Epoxide was converted to abscisic acid in 1.8% yield (3.6% of one enantiomer if only one is utilized) and 15% of the abscisic acid was derived from the precursor. The 2-trans isomer was not converted to abscisic acid. Neither epoxide nor its 2-trans isomer were converted to abscisic acid in boiled tomato fruit. Treatment of epoxide with dilute mineral acid produced the 1',2'-dihydroxy derivative. The 1',2'-dihydroxy derivative was not converted to abscisic acid in tomato fruit. Wright and Hiron (1969) reported that the abscisic acid concentration in wheat leaves increased 40-fold during the first 4 hours of wilting. It was found that this increase was caused by synthesis of abscisic acid rather than by its release from a conjugate or a precursor. This was shown by the increased incorporation of [2- 3 H] mevalonic acid into abscisic acid by wilted wheat shoots in comparison with turgid shoots which had absorbed equal amounts of [2- 3 H] mevalonic acid. When (+)-[2-14C] labelled epoxide was supplied, larger amounts of labelled material were again incorporated into abscisic acid by wilted plants. The incorporation of [14C] epoxide into abscisic acid shows that the carbon skeleton of the epoxide becomes the carbon skeleton of abscisic acid. The fate of the epoxy oxygen was also investigated with wilting wheat because any [18o] containing abscisic acid formed during the experiment would not be diluted by a large pool of endogenous [16o] material. (+)- [1’2’-18o] epoxide was converted to abscisic acid, the oxygen of the l',2'-epoxy group became the tertiary hydroxyl of abscisic acid and the conversion was quantitative. For the [18ᴏ] to be retained the C-l'-oxygen bond must remain intact and epimerization at C-l' is not possible. It follows that only one enantiomer of the epoxide is converted into abscisic acid: that one in which the I' ,2'-epoxy group is on the same side of the six-membered ring as the hydroxyl group in abscisic acid. The results of this work have been published in the Biochemical Journal, VOl.119, p.727-734 (1970). The 1',4'-diols of synthetic abscisic acid were found to inhibit the growth of a number of plants (private communication, Mr. R. Leach and Dr M. Anderson) although they were less potent than abscisic acid. The demonstration of the conversion of the epoxide into (+)-abscisic acid which entails the introduction of a 4'-keto-group, and which by analogy with similar oxidations (Murphy and West, 1969) probably takes place by a hydroxylation, suggested that plants were able to oxidise the 4'-hydroxy group in the l',4'-diols of abscisic acid to a 4' ketone. The abscisic acid produced would then account for the observed growth inhibitory activity of the l',4'-diols. (±)-[2- 14 C]- cis-1’, -l' ,4' and trans-l',4' diols of abscisic acid are both converted to abscisic acid by wilted and turgid wheat shoots. The abscisic acid recovered showed a preponderance of the unnatural (-) enantiomer. The unreacted cis-1’, 4'-diol which was recovered from the tissues also showed an excess of the (-) enantiomer, unreacted trans-I' ,4'-diol which was recovered from the tissue lacked optical activity and is presumed to be a racemic mixture.

The work described in this thesis was concerned primarily with the possible role of quinones in mitochondrial and bacterial reactions. In this respect electron transport and energy-linked reactions were both examined and the major sources of material used for this purpose were beef heart and Escherichia coli. Various approaches were utilized namely, studies with inhibitors, extraction-reactivation experiments, ultraviolet irradiation techniques and quinone deficient mutants. An extensive study was made of the effect of piericidin A, which had been proposed as a ubiquinone analogue, on various bacterial and mitochondrial reactions. It was shown that piericidin was acting at the same site as rotenone in the NADH dehydrogenase region of the respiratory chain of beef heart mitochondria. The possible nature, concentration and specificity of this site was examined. At high concentrations piericidin also inhibited succinate oxidation, possibly due to damage of the mitochondrial membrane system. The effect of piericidin on various other mitochondrial and bacterial reactions was also described. Of particular interest was the inhibition by piericidin of the energy-dependant reduction of NADP+ by NADH in both beef heart submitochondrial particles and small particles derived from E. coli. The ATP-dependant reduction of NADP+ by NADH and the ATP-dependant reduction of NAD+ by succinate catalysed by E. coli small particles were both fully characterised. Extraction techniques were used for studying electron transport and energy-linked reactions in beef heart submitochondrial particles. Reactivation of NADH oxidation, succinate oxidation and the energy-linked reduction of NADP+ by NADH to pentane extracted particles was achieved by the addition of ubiquinone homologues at concentrations equal to those originally present in the particles. The reactions had the same sensitivity to inhibitors of electron transport and oxidative phosphorylation as the normal reactions in unextracted particles. Extraction techniques, ultraviolet irradiation and ubiquinone deficient mutants were used to study the role of quinones in electron ,transport and energy-linked reactions in E. coli. The results obtained were discussed fully in Chapter VI.

In this thesis some chemical aspects of the bile pigment bilirubin have been examined. Bilirubin has been shown to exist as the bislactam tautomer, with an intranolocularly hydrogen bonded structure which is independent of the medium. Possible structures are discussed in Chapter 2. The clinical determination of bilirubin involves treatment of the pigment with diazotised sulphanilic acid (the van den Bergh reaction) when bilirubin is cleaved at the central methylene bridge to form two azopigments. The fate of the central methylene bridge carbon atom was unknown until the present work, but it had been postulated as being liberated as formaldehyde. In Chapter 3, it is shown that formaldehyde is formed during this reaction, and that it can be detected by the product of its reaction with dimedone. Some metal complexes of bilirubin formed in dipolar aprotic solvents are discussed in Chapter 4. A possible structure for the complex with Zn(II), isolated from DMF solution, is suggested. In Chapter 5, a potentially extremely useful method for the synthesis of bilirubin conjugates of defined structures is examined, using substituted aryl triazenes, under mild conditions. This method allows the esterification of a specific hydroxyl group in a polyhydroxylated mono – or oligosaccaride without protection of the other hydroxyl group present. Thus, in this work, using l – alkyl -3-p- tolyltriazenes, the hitherto unknown diethyl, diisopropyl and dibenzyl esters of bilirubin have been prepared and characterised.

The bulk of this thesis is concerned with the calculation of molecular wavefunctions - calculated within a Different Orbitals for Different Spins framework. Initially other important methods of constructing molecular wavefunctions are considered, and the DODS method is discussed in relation to them. Two different DODS methods are considered in detail, namely the Alternant Molecular Orbital method and the Non-Paired Spatial Orbital method. Other workers have previously used these methods and obtained satisfactory wavefunctions. Both methods make good allowance for electron correlation, but for benzene, and several other hydrocarbons, the NPSO method proves superior. Pauncz has stated that, (67), because the NPSO calculations are cumbersome,"one cannot expect to apply this method to larger systems." In this thesis this NPSO method is extended to larger molecules, namely azulene, anthracene and phenanthrene. In all cases the NPSO method proves superior to the AMO method • With azulene, for example, a better ground state energy is obtained using a one-parameter NPSO wavefunction, than with a five-parameter optimised AMO wavefunction. Having found satisfactory ground state wavefunctions for these molecules, and observed that most endorse the suggestions that k - mln 0.25 , the NPSO method was applied to open shell systems, such as benzyl radical, and butadiene negative ion. The results for the benzyl radical are more satisfactory than for other open shell systems. ~he spin density distribution of the benzyl radical was calculated by various approximations, but with limited success. In this context we developed a fully spin projected NPSO method.

Some evidence for the participation of methyl quinones in oxidative phosphorylation is presented and discussed. Although methyl quinones seem to be definitely involved in electron transport in particulate systems, there is no unequivocal evidence that they are actually involved in the coupling reaction. Several hypothetical schemes for oxidative phosphorylation are discussed with particular emphasis on those involving hydro-quinone phosphates. The Vilkas -Lederer scheme and the more recent mechanism proposed by Erickson, Wagner, and Folkers are discussed in detail. An in vitro investigation of the latter proposal is the subject of the present work. Hydrogen isotope exchange in methyl quinones was observed in the presence of carbonate or triethylamine using the techniques of infra-red spectroscopy, NMR spectroscopy, mass spectrometry and scintillation spectrometry. The exchange reactions of duroquinone and 2, 3-dimethyl-J, 4-naphthoquinone together with their derivatives were investigated over a range of pH and at different temperatures. The large primary isotope effect for the reaction indicates that removal of a proton from the methyl group of the quinone is the rate determining step in those reactions 3. involving a quinone methide intermediate and offers an explanation for the negative results of the in vivo hydrogen isotope experiments. The reaction of phosphoric acid and its various anions with both stable and transient quinone methides was investigated. There was no evidence for any nucleophilic addition of a phosphate to a quinone methide, polymerisation of the latter being the only reaction observed. A synthesis of p-hydroxybenzyl phosphate and a study of its behaviour under oxidising conditions were attempted. Production of p-hydroxybenzyl phosphate in situ under acidic or basic conditions resulted in its immediate decomposition to inorganic phosphate and the quinone methide, precluding any observation of oxidative phosphorylation. No evidence for oxidative acylation with the isolable 2, 6-di-t-butyl-4-hydroxybenzyl acetate was obtained.

A short method of purification of P.amagasakiense glucose oxidase has been achieved. Attempts to prepare the apoenzyme were unsuccessful. Inhibition of activity of the holoenzyme by heavy metals was investigated. It was shown that inhibition was due to the metal cations rather than the undissociated metal salts. The effect of bisulphite on the enzyme was shown to be the same as for Aspergillus niger glucose oxidase. From the kinetic experiments with bisulphite, an ionizable group with a pK of 4.2 was found to be involved in the reaction. This pK was assigned to the amino group of adenine of the FAD moiety of the enzyme. Experiments on the binding of halide anions to the enzyme indicated the involvement of an ionizable group with the same pK. Photo-chemical experiments on the enzyme revealed three different phenomena. (1) Photo-oxidation with methylene blue or Rose Bengal as sensitizer, destroyed an ionizable group with pK of 7.2. This was assigned to a histidine residue in the protein. (2) At high pH in the presence of EDTA and with no oxygen present photo-reduction of the enzyme spectrum was obtained. The spectrum of the oxidised enzyme was obtained when air was admitted to the reduced species. (3) At high pH in the presence of EDTA, light at 450 nm wavelength caused the enzyme to lose activity. This activity loss was irreversible with respect to oxygen or glucose. The presence of glucose in the reaction mixture during illumination protected the enzyme from this photo-destruction.

A series of oligomycin resistant mutants has been isolated following u.v. irradiation. The phenotypic and genotypic properties of these mutants, which show high levels of resistance to oligomycin and rutamycin, have been investigated and, on the basis of these results, it has been possible to divide the mutants into two main classes, class I and class II. Class I Mutants of this class show low levels of cross resistance (increases of two-to-four-fold) to various uncouplers such as TTFB, 1799 and chloro CCP, to inhibitors of oxidative phosphorylation like aurovertin and triethyltin, to the inhibitor of electron transport, antimycin A, and protein synthesis inhibitors such as cycloheximide, mikamycin, chloramphenicol and erythromycin. The mutants are apparently, however, resistant to neither DNP nor octylDNP. In a high percentage of the mutants, exposure to low temperatures (20ºC) resulted in the loss of the primary resistance to oligomycin and rutamycin, though the secondary cross resistances were apparently unaffected. No effect of either high or low temperatures on either the fermentable or non-fermentable growth of these mutants were apparent. No alteration in the growth rate of the majority of the strains or in their appearance on electron microscopic examination was detected, though their growth yield is 15 – 20% lower than the wild type strain. Genetic analysis has revealed that this class of mutant clearly differs genotypically as well as phenotypically from the class II mutants and moreover, had indicated that the determinant controlling oligomycin resistance is a nuclear gene. The genetics of the mutants exhibited many anomalous features, which remain to be explained, many of the experiments in this thesis describing, rather than explaining the phenomena observed. Class II The mutants in this class show resistance only to oligomycin and rutamycin. No cold or heat sensitivity of growth was observed and only one mutant in this class shoed any temperature sensitivity with regard to its oligomycin resistance. No alteration in the growth rate, growth yield or mitochondrial morphology of any of the mutants in this class was found. Genetic analysis of these mutants revealed that all the mutants tested showed cytoplasmic inheritance, the genetic determinant being located on the mitochondrial DNA. Allelism tests have demonstrated that genotypically the class is not homogeneous and the mutants were sub-divided into two non-allelic recombination groups. The genetic determinants concerned in each sub-group were confirmed to lie on mDNA.