Synthesis and Biosynthesis of Quinolizidine Alkaloids With Enzyme Work

McClintock, Carol Ann Lindsay (1994) Synthesis and Biosynthesis of Quinolizidine Alkaloids With Enzyme Work. PhD thesis, University of Glasgow.

Abstract

The research presented in this thesis can be divided into two main areas: a) the biosynthesis of quinolizidine alkaloids; b) enzyme work involving the synthesis and testing of diamines and polyamines as substrates and inhibitors of pea seedling diamine oxidase, and experiments involving the feeding of N-alklycadaverines to transformed root cultures of Nicotiana rustica and Datura stramonium. Biosynthesis of Quinolizidine Alkaloids Previous work has revealed that cadaverine is a good precursor of quinolizidine alkaloids. (R)- and (S)-[1-2H]Cadaverines were synthesised by a known route and isolated as their dihydrochloride salts. Samples of these labelled precursors were fed with a radioactive tracer, by the wick method, to Sophora microphylla plants in order to study the biosynthesis of matrine. After ten to fourteen days the plants were macerated and the alkaloids extracted. The alkaliod extract contained matrine, anagyrine, N-methylcytisine and cytisine. Levels of incorporation, established by 2H and NMR spectroscopy. were comparable to those obtained in a similar experiment by a previous researcher. Attempts were made to synthesise (R)- and (S)-[2-2H]cadaverine dihydrochlorides by the route devised by a previous researcher. Problems were experienced in the early stages of the synthesis and repeated attempts were made to obtaine pure pentane-1,2,5-triol for use in the later stages of the synthesis. Various methods were attempted with varying degrees of success. An alternative synthesis, avoiding the need for extraction and purification of triol from the aluminium complex is proposed. The Synthesis and Testing of Diamines and Polyamines as Substrates and Inhibitors of Diamine Oxidase. Diamine oxidase catalyses the oxidative deamination of diamines to their corresponding aminoaldehydes. Cadaverine and putrescine have been shown to be the best substrates of diamine oxidase. The cadaverine and putrescine analogues N-methylcadaverine and N-methylputrescine have also been shown to be good substrates. A series of N-alkylcadaverines were synthesised by a new, improved route and were tested as substrates of diamine oxidase. Also tested were a series of N-alkylputrescines. The assay used in the testing was Stoner's spectrophotomeric assay which involves the measurement of hydrogen peroxide produced as a byproduct of the enzymic reaction. From this assay KM and Vmax values were obtained for the oxidation of these substrates using diamine oxidase. The Km is a measure of the strength of the enzyme-substrate complex and determines the binding efficiency of the substrate to the enzyme. The Vmax is the maximal rate and is related to the turnover number of an enzyme. Analysis of results provided information on the steric constraints of the active site and showed that as the steric bulk of the alkyl group increases the Vmax decreases. Several of these compounds were also tested as inhibitors of diamine oxidase, and Ki values obtained The Ki is the dissociation constant of the enzyme-inhibitor complex. Most of the compounds did inhibit the diamine oxidase catalysed reaction. The best inhibitors of the compounds tested were N-cyclohexylcadaverine and N-benzylputrescine, both of which were shown to be apparent competitive inhibitors. A series of N-ethylpolyamines were also synthesised and tested as inhibitiors and substrates of diamine oxidase using the same assay as before. Since N-ethylpolyamines can interfere with polyamine metabolisism and may have useful biological activity, selected compounds were tested by other workers for biological activity against cancer and fungi. Two compounds were also used to confirm the identity of certain natural compounds extracted by other workers from plants and fungi. The synthesised compounds proved to be the same as the natural compounds. The Feeding of N-Alkylcadaverines to Transformed Root Cultures of N. rustica and D. stramonium. It has been established that feeding certain modified precursors of a natural precursor to biological systems may result in new or modified alkaloids being produced. Transformed root cultures of Nicotiana rustica have been shown to produce mainly nicotine, with smaller quantities of anabasine, nomicotine and anatabine. Datura stramonium produces hygrine and hyoscamine. N-Alkylcadaverines were fed to both species with the aim of discovering whether any new alkaloid analogues were formed. No new alkaloid analogues appeared to be produced from the compounds fed to D. stramonium. N-Ethylanabasine appeared to be a new alkaloid analogue formed in response to the feeding of N-ethylcadaverine to N. rustica. The N-propylcadaverine feed to N. rustica appeared to stimulate the formation of the new alkaloid analogue, N-propylanabasine.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: D J Robins
• Keywords: Organic chemistry
• Date of Award: 1994
• Depositing User: Enlighten Team
• Unique ID: glathesis:1994-75658
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 19 Nov 2019 19:00
• Last Modified: 19 Nov 2019 19:00
• URI: https://theses.gla.ac.uk/id/eprint/75658