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Appears in Collections:eTheses from Faculty of Natural Sciences legacy departments
Title: Studies related to the biogenesis of bicyclomycin
Author(s): Cain, Robert Orr
Keywords: Dioxopiperazines
Issue Date: 1980
Publisher: University of Stirling
Abstract: The literature concerning the biogenesis of naturally-occurring dioxopiperazines is reviewed. Initially, it was hoped to probe the biogenesis of Bicyclomycin using substrates labelled with stable isotopes. A working hypothesis for the biogenesis of Bicyclomycin is proposed and seemed to indicate that leucine and isoleucine were the principal precursors. To test the validity of this theory [1-13 C] leucine and [ 1-13 C] isoleucine were synthesised by conventional methods. Small scale shake culture fermentations did produce Bicyclomycin but the yield in the case of both Streptomyces aizunensis and S. sapporonensis was very low. The efficiency of Bicyclomycin isolation was shown to be ca. 10%. The production of Bicyclomycin was stimulated by nutrient deprivation (carbohydrate and nitrogen sources) and by the introduction of a thermal shock during fermentation. In the case of S. sapporonensis, thermal shock increased the yield of the crude antibiotic by a factor of five. In general S. aizunensis showed a greater production of the crude antibiotic under shake culture conditions thus reflecting its lower oxygen requirement during fermentation. Large scale fermentation experiments (20L) using S. aizunensis produced a broth with modest antibiotic activity. This was purified by several methods but the final yield of Bicyclomycin was disappointing. S.sapporonensis proved no more efficient than S. aizunensis in large scale fermentations. However, phenylacetamide was isolated as an antibiotic from S. sapporonensis. Limited mutation studies produced one mutant with an enhanced antimicrobial spectrum. The key features of the postulated biogenesis of Bicyclomycin were tested by the synthesis of a target compound 3,6-di-(2'-hydroxybenzyl)-2,5-dihydroxypyrazine. All reactions were tested first on a series of parallel model compounds derived from 3,6-dibenzyl-2,5-dioxopyrazine. A new method for the removal of alkyl groups from ethers of aromatic 1,4-diazines was discovered. These ethers are particularly resistant to cleavage by normal methods but will produce the dealkylated product in high yield when treated with iodotrimethylsilane. The target compound failed to undergo the key photooxygenation step, required by biosynthetic proposals, due to its inherent insolubility in most polar solvents. A second route designed to produce the target compound by way of a substituted 2,5-dichloropyrazine derivative proved only partly successful. The utility and preparation of 2,5-dichloropyrazines was investigated. Several N-oxides of these diazines were produced and the reaction of these compounds with sodium allyloxide was investigated. Thermolysis of one derivative gave a small amount of material whose structure seems to be consistent with a Claisen rearrangement. This is a novel process for 1,4-diazines. Preparation of substrates, for Claisen rearrangement, based on 2,5-diethoxypyrazine met with only limited success. The 13C spectra of most synthetically produced dioxopiperazine and pyrazinederivativesare reported, together with structural assignments for Bicyclomycin itself. It is hoped that these data may be useful in future biosynthetic work.
Type: Thesis or Dissertation

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