Bactrocera tryoni Volatiles: Spiroacetal Structure, Synthesis and Biosynthesis

Booth, Yvonne (2007). Bactrocera tryoni Volatiles: Spiroacetal Structure, Synthesis and Biosynthesis PhD Thesis, School of Molecular and Microbial Sciences , University of Queensland.

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Author Booth, Yvonne
Thesis Title Bactrocera tryoni Volatiles: Spiroacetal Structure, Synthesis and Biosynthesis
School, Centre or Institute School of Molecular and Microbial Sciences
Institution University of Queensland
Publication date 2007
Thesis type PhD Thesis
Supervisor Associate Professor James De Voss
Abstract/Summary The Queensland Fruit Fly, Bactrocera tryoni is the most damaging horticultural pest in Australia having in excess of 200 host plants and being found all along the eastern coast. The annual cost to the horticultural industry from this species alone, is estimated to be AUD$500 million, through lost production, population monitoring and control programs. Current control methods are based on a combination of insecticides, bait sprays, chemical lures and the release of sterile males, all of which have inherent problems. Therefore, recent interest has focused on the development of pheromone based control measures, which are more species specific and environmentally benign. However, for this type of approach to become viable a detailed knowledge of pheromone biosynthesis in the target species is essential. This thesis describes the delineation and disruption of biosynthetic pathways to some of the minor spiroacetal components released by B. tryoni. Chapter one provides an overview of previous research into spiroacetal biosynthesis in Bactrocera species and describes the nomenclature and characteristic mass spectrometric fragmentation patterns of spiroacetals, which are referred to throughout this thesis. The suite of spiroacetals produced by female B. tryoni is introduced, as well as those from the closely related fruit fly species B. cucumis, B. cacuminata and B. oleae. The proposed biosynthetic pathways to the major spiroacetal component are summarised for each of these species, with the importance of the cytochromes P450 being highlighted. Chapter two describes an investigation into the biosynthesis of the unusual C12 spiroacetal, 2-ethyl-8-methyl-1,7-dioxaspiro[5.5]undecane and C13 spiroacetal, 2-methyl-8-propyl-1,7-dioxaspiro[5.5]undecane in B. tryoni. The syntheses of over thirty potential precursors, with the majority bearing deuterium labels, are described. This required the development of divergent synthetic routes, which would allow for the introduction of isotopic labelling and characterisation of the deuterated products by 1H and 13C NMR and mass spectrometric analysis. The potential precursors were then administered to female B. tryoni and the level of deuterium incorporation into 2-ethyl-8-methyl-1,7-dioxaspiro[5.5]undecane or 2-methyl-8-propyl-1,7-dioxaspiro[5.5]undecane qualitatively monitored by analysing the volatile fly emissions by SPME coupled GCMS. From the relative incorporation levels and the identification of some of the exceptionally minor spiroacetals, which were also biosynthesised (in particular 2-methyl-8-ethyl-1,7-dioxaspiro[5.6]dodecane), it was ultimately possible to conclude that B. tryoni biosynthesises these minor spiroacetals from fatty acids via 2,6-dioxygenated precursors and presumably by a modified fatty acid β-oxidation pathway. The third chapter describes a detailed investigation into the possibility of P450 mediated hydroxylation of a diketone intermediate rather than a hydroxy ketone being the penultimate biosynthetic step. The synthesis and administration of specifically labelled [2-2H]-2-hydroxydodecan-6-one and [2-2H]-2-hydroxytridecan-6-one provided direct evidence for the former, as the precursors were respectively converted into 2-ethyl-8-methyl-1,7-dioxaspiro[5.5]undecane and 2-methyl-8-propyl-1,7-dioxaspiro[5.5]-undecane with retention of the deuterium label. Even though the alternative pathway could not be ruled out, the former was further supported by the synthesis and administration of 2-hydroxy-2-methyldodecan-6-one which was converted into the non-natural spiroacetal, 2,2-dimethyl-8-ethyl-1,7-dioxaspiro[5.5]undecane. Conversely, the loss of deuterium label from the synthesised and administered mono-oxygenated precursors, [2-2H]dodecan-2-ol and [2-2H]tridecan-2-ol and the lack of incorporation from 2-methyldodecan-2-ol confirmed that the early biosynthetic steps proceed through a C2 ketone, which is in agreement with a modified β-oxidation type pathway. Chapter four describes the identification, synthesis and characterisation of two novel spiroacetals biosynthesised by B. tryoni. The first, 2-methyl-8-ethyl-1,7-dioxaspiro-[5.6]dodecane, is noteworthy not only as the second member of this spiroacetal system but also as its first disubstituted derivative. Furthermore, this spiroacetal, which was determined to have a high enantiomeric excess in favour of the (2S,6R,8S)-isomer in vivo, played a pivotal role in determining that spiroacetal biogenesis in B. tryoni occurred via 2,6-dioxygenated precursors. The second spiroacetal, 2-ethyl-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane is a rare branched chain spiroacetal, which was also determined tentatively to have (2S,6R,8S)-stereochemistry in vivo. However, the biological origin of the branch-position with the required (2S) stereochemistry is still uncertain. The final chapter describes the synthesis of acetylenic compounds based on the highly incorporated potential precursors, which irreversibly bind to and inhibit the action of cytochrome P450 enzymes involved in spiroacetal biosynthesis. Preliminary results from the administration of these inhibitors, followed by precursors to either 2-ethyl-8-methyl-1,7-dioxaspiro[5.5]undecane or 2-methyl-8-propyl-1,7-dioxaspiro[5.5]undecane indicated that two inhibitors resulted in a decrease in production of these spiroacetals in B. tryoni and so show promise for future applications.

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