<p> Recently, the discovery that taxol (2), a highly modified diterpenoid available from Taxus species, possesses anticancer activity has attracted considerable interest. The synthesis of [1-3H]-geranylgeranyl pyrophosphate(1b), a labelled form of the precursor to the diterpenoids(1a), and its incubation with a cell-free enzyme preparation from Taxus x media densiformis, a species of yew tree, were investigated.</p> <p> The investigation into the biosynthesis of taxol(2) was to be accomplished by monitoring the enzymatic transformations of (1b). The synthesis of 1b was accomplished via a convergent method using geraniol, a ten carbon compound, as the starting material. Essentially two molecules of geraniol were chemically manipulated and condensed to give geranyl geraniol which was oxidized to the corresponding aldehyde and then reduced using sodium borotritide. The radioactive alcohol was subsequently converted to the pyrophosphate (1b) using standard methodology. Cell-free extracts of Taxus x media densiformis were prepared and incubated with [1-3H]-GGPP (1b), the intermediates were extracted, and then analyzed by scintillation counting and radioactive gas chromatography. Identification of these labelled intermediates was attempted in order to provide information about the biosynthesis of 2. (Diagram included in thesis)</p> / Thesis / Master of Science (MSc)

Most gymnosperms produce a pollination drop that captures and transports pollen into the ovule. Pollination drops have other functions. These include influencing pollen germination and pollen tube growth, defending the ovule from pathogens and providing a food reward in insect-pollinated gymnosperms. Mineral and organic molecules, including proteins, are responsible for these additional functions. To date, pollination drops from a handful of conifers and one non-conifer gymnosperm, Welwitschia mirabilis, have been subjected to proteomic analysis. In the present study, tandem mass spectrometry was used to detect proteins in all gymnosperm lineages: cycads (Ceratozamia hildae, Cycas rumphii, Zamia furfuracea); Gnetales (Ephedra compacta, E. distachya, E. foeminea, E. likiangensis, E. minuta, E. monosperma, E. trifurca; Gnetum gnemon; Welwitschia mirabilis); Ginkgo biloba; conifers (Taxus x media). PEAKS 6 DB (Bioinformatics Solutions, Waterloo, ON, Canada) was used to make protein identifications. Proteins were detected in all gymnosperm species analyzed. The numbers of proteins identified varied between samples as follows: one protein in Welwitschia female; nine proteins in Cycas rumphii; 13 proteins on average in Ephedra spp.; 17 proteins in Gnetum gnemon; 38 proteins on average in Zamia furfuracea; 57 proteins in Ginkgo biloba; 61 proteins in Ceratozamia hildae; 63 in Taxus x media; 138 proteins in Welwitschia male. The types of proteins identified varied widely. Proteins involved in carbohydrate modification, e.g. galactosidase, chitinase, glycosyl hydrolase, glucosidase, were present in most gymnosperms. Similarly, defence proteins, e.g. reduction-oxidation proteins, lipid-transfer proteins and thaumatin-like proteins, were identified in many gymnosperms. Gymnosperms that develop a deep pollen chamber as the nucellus degrades, e.g., cycads, Ginkgo, Ephedra, generally contained higher proportions of proteins localized to intracellular spaces. These proteins represent the pollination drop degradome. Gymnosperms that either lack a pollen chamber, e.g. Taxus, or have a shallow pollen chamber, e.g. Gnetum, had greater proportions of extracellular proteins. These proteins represent the pollination drop secretome. Our proteomic analyses support the hypothesis that the pollination drops of all extant gymnosperms constitute complex reproductive secretions. / Graduate