Ireland, Martin James; (1993) The biosynthesis of terpenoids in tissue culture: Syntheses of lethal metabolites and toxicity studies. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The work described can be conveniently divided into four related but distinct sections. Part One describes a set of experiments that follow on from a previous study of the incorporation of 1-14C-Isopentenyl pyrophosphate (1-14C-IPP) into terpenoids by cell-free extracts from cultures of Lavandula angustifolia. Of the total incorporations (ca. 5%) most (70%) of the label was present in the farnesols. The addition of NADP caused an increase of incorporation into the sesquiterpenoid hydrocarbons caryophyllene (13%) and humulene (30%), with a concomitant decrease of incorporation into the farnesols. By using enriched cell-fractions the site of sesquiterpenoid biosynthesis was found to be associated with the microsomal fraction. Part Two describes a series of experiments carried out on cell-suspension cultures of Pelargonium fragrans. A statistically reliable and novel method of estimating cell-viability was developed to study the toxicities of some common terpenoids as such toxicity may account for the lack of accumulation of terpenoids in culture. All compounds were toxic (in the range 1-5 mmol.dm-3) and the toxicity (LD50) was greatest during the exponential-period of culture-growth. The cultures could however, be habituated to the terpenoids over a number of subcultures. The inclusion of a surfactant in the culture-medium lowered the toxicity of the terpenoids and therefore provided a model storage mechanism (sink) for these compounds in a single-phase culture. The polyethoxylate-surfactants were found to be the most suitable for this purpose. Two subsidiary studies deal with the toxicity of some fluorinated compounds to tissue cultures and the reactions of exogenous terpenoids with the culture medium. Part Three describes the syntheses of five fluorinated monoterpenoids. Two fluorinated linalools (4-fluoro- and 9-fluoro-) were prepared by treatment of the respective fluoro-6-methyl-hept-5-en-2-ones with vinylmagnesium bromide. A number of methods of introducing fluorine into 6-methyl-hept-5-en-2-one were attempted; the most successful method involved fluorination of the trimethylsilyl-enol ethers using N-fluoro-pyridinium triflate. Linaloyl, neryl and geranyl fluorides were prepared by treatment of the corresponding chlorides with anhydrous tetrabutylammonium bifluoride. 19F-, 1H- and 13C-nuclear magnetic resonance and mass spectrometry were used to characterise the products. Some unexpected results are discussed in detail. Part Four describes studies that were used to interpret the fragmentation patterns in the mass spectra of three monoterpenoid acetates occurring in the oil of L. angustifolia that was studied in Part One. Linaloyl, neryl and geranyl acetates all showed identical electron-impact mass spectra. A combination of linked-scanning and deuterium-labelling experiments were used in order to characterise the fragmentation patterns. Other methods of ionisation (fast atom bombardment and chemical ionisation in the positive and negative modes) were also used to confirm the patterns.

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