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The biological role of choline (I), and the metabolic pathways involved in the synthesis and degradation of lecithin (II) and other phosphatides, have been discussed. Reference has been made to current ideas on the properties of phospholipids, both in cell membranes and in isolation, and to the physical methods required for their evaluation. The published literature on the biological properties of choline analogues has been reviewed. Although the earliest investigations, mainly by Hunt and Renshaw, were concerned solely with pharmacological properties, the observation by Best et al. of such consequences of choline deficiency as fatty liver and renal haemorrhage stimulated the search for substances which could effectively replace choline in the diet, and. these lipotropic compounds included the phosphorus (III) and arsenic (IV) analogues. Welch claimed to have isolated the arsenic analogue of lecithin from the tissues of rats fed a choline-deficient arsenocholine (lV)-supplemented diet, but his evidence was not conclusive by modern standards. Moreover, knowledge of the lipotropic role of phosphocholine (III) was confined solely to the fact that its administration allayed the symptoms of choline deficiency. It was therefore decided to undertake a thorough chemical and biological investigation of this compound. The tri-n-butylphosphonium analogue (V) was used in all exploratory synthetic work, however, for reasons of convenience and economy. The chemical investigation involved the synthesis of the phosphorus analogues of those derivatives of choline involved in the cytidine pathway to lecithin, namely phosphorylphosphocholine (VI), cytidine-5andprime;-diphosphate phosphocholine (CDP-phosphocholine) (VII) and phosphatidyl-phosphocholine ("phospholecithin") (VIII). Of these, the synthesis of phosphorylphosphocholine proved the most difficult, as the standard methods of choline phosphorylation were unsuccessful when applied to the phosphorus analogue. Use was eventually made of a novel phosphorylating agent, a complex (IX) formed by methylphosphorodichloridate (X) in pyridine, discovered, by chance, by Smart and Catlin. Thus both phosphocholine and its tri-n-butyl homologue were phosphorylated in fair yield (31-40%) in a single-stage process. The synthesis of CDP-choline, in 24% yield, was a straightforward adaption of the method of Kennedy, and involved the condensation of phosphorylphosphocholine (VI) with, cytidine-5andprime;-monophosphate in the presence of dicyclohexylcarbodiimide (XI). Well-established methods of lecithin synthesis were considered unsuitable for the phosphonium compound, but a recent method of Aneja and Chadha, involving the condensation of choline and phosphatidic acid (XII) in the presence of triisopropylbenzenesulphonyl chloride (XIII) proved to be satisfactory, and in this way both 1,2-distearoyl-DL-phosphatidylphosphocholine (VIII, R ?(CH₂)₁₆CH₃) and its tri-n-butyl homologue were prepared in reasonable yields (33-35%). Some of the properties of these synthetic "phospholecithins" were compared with those of distearoylphosphatidylcholine; thus the phosphorus analogues were found to exhibit lower transition temperatures, and remarkably lower melting points (ca. 135°), than the nitrogen compound (m.p. 233°). Both the trimethylphosphonium and trimethylammonium compounds appeared to form similar spherical or ellipsoidal single-bilayer liposomes in aqueous dispersion, but those of the tri-n-butylphosphonium compound were unusually long and thin, like collapsed vesicles; this is possibly due to the less hydrophilic nature of the tri-n-butylphosphonium group. Both trimethyl compounds were attacked to a similar extent by phospholipase C, while the enzyme showed a far lower affinity for the tri-n-butylphosphonium lecithin. Since the phosphorus-31 nucleus has a spin of andfrac12;, and couples with the proton, both proton magnetic resonance (PMR) and ³¹P nuclear magnetic resonance (³¹P-NMR) studies of the phosphonium analogues were found to be of interest. Thus the methyl proton signal in the PMR spectrum of phosphocholine (III) is a doublet with a coupling constant of ca. 14.5 Hz, and is characteristic of all the trimethylphosphonium derivatives. This property later proved to be a useful method of structure confirmation when phosphatidylphosphocholine (VTII), synthesized in vivo, was isolated from rat tissues. As phosphorylphosphocholine (VI) and its derivatives were probably the first compounds to be prepared containing both a phosphonium and a phosphate group, they provided a unique opportunity for the simultaneous study of two very different phosphorus groups by ³¹P-NMR. The use of ³H-labelled choline and ¹⁴C-labelled phosphocholine provided a means of investigating the relative affinity of the enzymes of the cytidine pathway for phosphonium compounds. Thus it was found that phosphocholine was at least as acceptable as choline to the enzyme choline phosphokinase: ATP + choline → phosphorylcholine + ADP and was even the preferred substrate in the initial stages of competitive incubations near enzyme-saturation level. Phosphorylcholine-cytidyl-transferase: CTP + phosphorylcholine and ? CDP-choline + pyrophosphate was far more specific, and initial rates of conversion of the phosphorylated bases to the CDP derivatives indicated a threefold preference for the natural substrate. In the case of phosphorylcholine-diglyceride-transferase: CDP-choline + diglyceride and ? lecithin + CMP both phosphorus and nitrogen substrates were equally acceptable in the forward reaction, but phosphatidylphosphocholine, once formed, appeared far less susceptible to degradation by the reverse reaction. The in vitro investigation was extended to cell culture, using the P815Y line of neoplastic mouse mast cell. The cells showed similar growth characteristics in choline-supplemented and in phosphocholine-supplemented media, and extraction of lecithin from cells grown in the latter led to the conclusion that both bases were equally acceptable; radioactivity readings showed similar choline/phosphocholine ratios in the medium and in the extracted lecithin. Moreover, measurement of the relative areas of phosphate and phosphonium peaks in the ³¹P-NMR spectrum of the extract confirmed this finding. Finally, an in vivo study was undertaken using choline-deficient weanling rats. One group of animals was provided with a choline-free ¹⁴C-phosphocholine-supplemented diet ("P-diet"), while a control group received food containing a full complement of choline (N-diet); the two analogues were provided in equimolecular quantities to their respective groups. Both diets contained ³H-labelled choline of negligible molarity and high specific activity. The animals were killed, and their livers, kidneys, lungs and brains removed, after a period of 7 weeks. Neutral phospholipid was extracted, from the organs, and preparative thin-layer chromatography (TLC) of these extracts led to the isolation of biosynthesized phosphatidylphosphocholine (VIII), free from phosphatidylcholine and identified by NMR and ³¹P-NMR; the latter gave a spectrum bearing two signals of equal intensity, from phosphonium and phosphate. Measurement of relative peak areas in the ³¹P-NMR spectra of some of the extracts gave a measure of the level of phosphocholine incorporation, varying from 33% in lung lecithin to 6% in kidney sphingomyelin. Although the effect of lecithin already present in the tissues at the start of the experiment was unknown, it was suggested that much of the phosphatidylcholine in the P-diet rats originated from sequential methylation of phosphatidylethanolamine, a route well-established in the liver but claimed to be of little significance in other tissues. It was also suggested that sphingomyelin was being synthesized mainly by an indirect route, from CDP-choline formed reversibly from this methylation-derived phosphatidylcholine, and not by a direct pathway involving CDP-phosphocholine. Additional signals in the ³¹P-NMR spectra of extracts from P-rats (but not N-rats) were attributed to phosphobetaine (XIV) produced by oxidation of phosphocholine in the liver, and trimethylphosphine oxide (XV) in the kidney, originating from phosphocholine oxidised by the bacteria of the intestinal tract. Similar liver oxidation of arsenocholine had been observed by Mann et al., while the oxidation of choline to trimethylamine oxide in the intestine, and its excretion in the urine, was first reported by Davies. Comparison of ³H/¹⁴C ratios in the P-diet itself and in the phospholipid extracts gave a possible indication of the differing preferences of some enzyme systems for choline and phosphocholine, although it was necessary to make several basic assumptions. Comparison of the relative fatty acid composition in the lecithin and "phospholecithin" of the four organs investigated showed little difference between the two in liver, lung and brain, but a profound difference in the kidney. Thus renal phosphatidylphosphocholine contained greatly elevated levels of stearic and polyunsaturated acids, and it was suggested that this might be due to a need to maintain a critical optimum membrane fluidity, since it had already been noted that a change from ammonium to phosphonium produced a lecithin with a lover transition temperature. Thus a change from palmitic to stearic acid increased the transition temperature, while a change from oleic and linoleic acids to polyunsaturates increased the membrane stability.

The chemotaxonomy and the biosynthesis of tetranortriterpenes is briefly reviewed in chapter one and the partial syntheses of some of these products is described. The work described in the first part of chapter two was directed towards a synthesis of the γ-lactone ring system in the side chain of flindissol, while that in the latter part involved studies of the allylic oxidation in ring D of some apotirucallol derivatives. The acid constituents of Manila elemi resin were separated and methyl 3α-acetoxytirucalla-8,24-dien-21-oate (1) was then converted to 3β-acetoxytirucall-8,24-dien-21-oic acid. Bromination of this acid resulted in low yields of the dibromide and an explanation for this was put forward. The action of ammonia on the acyl chloride of 3α-acetoxytirucalla-8,24-dien-21-oic acid followed by reaction with lead tetra-acetate and iodine led to formation of 3α-acetoxy(21-24)cyclotirucalla-7,9(11),24-trien-21-one. Reaction of 3α-acetoxytirucall-8-en-21-amide with lead tetra-acetate and iodine resulted in formation of 3α-acetoxytirucalla-7,9(11)-dien-21-isocyanate and explanations for these results are given. Ozonolysis was used extensively during this work and the mechanism of ozonolysis is discussed. Treatment of a mixture of methyl 3α-acetoxytirucall-7 and 8-en-21-oate with ozone gave three products, methyl 3α-acetoxy-7α,8α-epoxytirucallan-21-oate (2), methyl 3α-acetoxy-7-oxotirucall-8-en-21-oate and methyl 3α-acetoxy-7,11-dioxotirucall-8-en-21-oate. The 7α,8α-epoxide (2) was rearranged with boron trifluoride and acetylation of the product gave methyl 3α,7α-diacetoxyapotirucall-14-en-21-oate (3). A crude mixture of dibromo elemi acid methyl esters was ozonised and reductive work up gave four products, (13αH)ursan-3,12-dione, methyl 3,7-dioxotirucalla-8,24-dien-21-oate, methyl 3,7, 11-trioxotirucalla-8,24-dien-21-oate and methyl 7α-bromo-3,15-dioxo-(14αH)apotirucall-24-en-21-oate (4). Similarly ozonolysis of a crude mixture of methyl 3α-acetoxy-24,25-dibromo-tirucall-7 and 8-en-21-oate and reductive work up gave a mixture of seven products. Methyl 3β-acetoxy-12-oxo-(13βH)ursan-28-oate, methyl 3α-acetoxy-7α,8α-epoxytirucall-24-en-21-oate (5), methyl 3α-acetoxy-7α-bromo-15-oxo-(14αH)apotirucall-24-en-21-oate (6), methyl 3β-acetoxy-11-oxours-12-en-28-oate (7), methyl 3α-acetoxy-7,11-dioxotirucalla-8,24-dien-21-oate, methyl 3α-acetoxy-7-oxotirucalla-8,24-dien-21-oate and methyl 3α-acetoxy-14β,15β-epoxy-7α-hydroxyapotirucall-24-en-21-oate (8). The structure and mechanism of formation of (7) and (8) is discussed. The crude 7α,8α-epoxide (5) was rearranged with boron trifluoride and acetylation of the product gave methyl 3α,7α-diacetoxyapotirucalla-14,24-dien-21-oate (9). The structure and mechanism of formation of the two 7α-bromides (4) and (6) is discussed. Reaction of methyl 3α-acetoxy-24,25-dibromotirucall-8-en-21-oate with tetramethylammonium acetate gave methyl 3α-acetoxy-24-bromotirucalla-8,24-dien-21-oate which was reduced to 24-bromotirucalla-8,24-dien-3α,21-diol and reduction of this diol gave tirucall-8-en-3α,21-diol. Oxidation of (1) with iodine and iodic acid in aqueous dioxan and acetylation of the products gave methyl 3α-acetoxy-24andxi;,25-epoxytirucall-8-en-21-oate and methyl 3α,24andxi;-diacetoxy-25-hydroxytirucall-8-en-21-oate which was hydrolysed to methyl 3α,24,25-trihydroxytirucall-8-en-21-oate. Bromination of (9) gave methyl 3α,7α-diacetoxy-24,25-dibromoapotirucall-14-en-21-oate (10) in 38% yield. Oxidation of this dibromide with selenium dioxide gave what is thought to be the 14,15-diol and the mechanism of oxidation with selenium dioxide is discussed. Treatment of (3) with bispyridinechromium oxide and also with aqueous N-bromosuccinimide gave methyl 3α,7α-diacetoxy-16-oxoapotirucall-14-en-21-oate in good yield. Similarly oxidation of (10) with bispyridinechromium oxide gave the 14-en-16-one in good yield but after denomination, methyl 3α,7α-diacetoxy-16-oxoapotirucall-14,24-dien-21-oate decomposed on attempted purification. Deoxygenation of havanensin triacetate with a zinc-copper couple deoxyhavanensin triacetate in good yield but attempts to oxidise this alkene with either selenium dioxide or bispyridinechromium oxide were unsuccessful. Oxidation of this alkene with aqueous chromic acid gave isophotodeoxyhavanensin triacetate. The literature of the chemistry of some pentacyclic triterpene-12-ketones is discussed in chapter three together with the structures of some new 12-ones and their o.r.d. curves and mass spectra. The triterpene lupeol was used as a model system to construct the 1α,3α-diacerate group common to the meliacins and this work is described in chapter four. Lupeol benzoate was firstly converted to lupan-3-one. Treatment of lupan-3-one hydrazone with lead tetra-acetate gave 5(4→3)-abeolup-3-ene and the mechanism of this reaction is discussed. Bromination of lupan-3-one gave a mixture of 2(α and β)-bromolupan-3-ones which was dehydrobrominated to lup-1-en-3-one. Epoxidation of this enone with hydrogen peroxide gave 1α,2α-epoxylupan-3-one which on rearrangement with hydrazine gave lup-2-en-1α-ol. Epoxidation of this alcohol gave 2α,3α-epoxylupan-1α-ol which was reduced with lithium in ethylamine to lupan-1α,3α-diol. The yield of this diol was greater by this route than by reduction of 1α,2α-epoxylupan-3-one. The o.r.d. of some lupan-3-ones is also discussed. The structure of a new diterpene furan and an attempt to synthesise this product from sclareol is described in chapter five. Oxidation of sclareol with chromic acid in acetic acid gave norambreinolide and 8α-acetoxy-13,14,15,16-tetranorlabdan-12-oic acid. Methanolysis of norambreinolide gave a 1:1 mixture of starting material and methyl 8α-hydroxy-13,14,15,16-tetranorlabdan-12-oate and a synthetic route via this ester was not pursued. Reduction of norambreinolide gave 13,14,15,16-tetranorlabdan-8α,12-diol which gave 8α-acetoxy-13,14,15,16-tetranorlabdan-12-yl acetate on acetylation. Dehydration of this acetate with phosphoryl chloride gave a mixture of 13,14,15,16-tetranorlabd-7 and 8(17)-en-12-yl acetates. Hydrolysis of this mixture and oxidation of the alcohols with silver carbonate gave a mixture of 13,14,15,16-tetranorlabd-7 and 8(17)-en-12-als. Reformatsky condensation of these aldehydes with ethyl α-bromoacetate gave a mixture of ethyl 12-hydroxy-15,16-dinorlabd-7 and 8(17)-en-14-oates. Treatment of this mixture of hydroxyesters with o-monoperphthalic acid gave pure ethyl 12andxi;-hydroxy-15,16-dinorlabd-8(17)-en-14-oate, together with ethyl 7α, 8α-epoxy-12andxi;-hydroxy-15,16-dinorlabdan-14-oate. The C-12 epimers of these products were separated and their spectra and the configuration of these epimers at C-12 is discussed. Oxidation of ethyl 12andxi;-hydroxy-15,16-dinorlabd-8(17)-en-14-oate gave ethyl 12-oxo-15,16-dinorlabd-8(17)-en-14-oate. Reaction of this β-keto-ester with 1,2-dichloroethyl ethyl ether and aqueous sodium hydroxide gave only traces of a furan while in triethylamine, a mixture of products was obtained. From these results it was concluded that a different approach to synthesis of the furan ring may be necessary.

More than five hundred polyacetylenes have now been isolated from plants and fungi. Bu'Lock has proposed that polyynes originate in vivo from oleate (1) via crepenynate (2).Those steps of polyacetylene biogenesis that have been established through study of the incorporation of labelled compounds into natural systems are summarised in Part I of this thesis. This section also includes a review of natural acetylenes which do not obviously evolve from oleate (1). Polyyne aldehydes (3) have often been utilised in syntheses of polyacetylenes, even though they are unstable in the condensed phase. Hexa-2,4-diynal (3; R = Me) was shown not only to polymerise but also to be converted into a C₁₁ aldehyde of structure (4) (Part II). 3-Phenylprop-2-ynal 'dimerised' similarly on heating at 130° into both the stereoisomers of aldehyde (5). A scheme for this reaction of α,β-acetylenic aldehydes is proposed (Scheme A; R³ = H) which is consistent with other evidence, e.g. use of labelled compounds and the small yield (3%) of cross-dimer (6; R¹ = Ph.Candequiv;C; R² = Me.Candequiv;C; R³ = Me) obtained from a mixture of 5-phenylpenta-2,4-diynal and hepta-3,5-diyn-2-one. The ester (7) is a likely precursor of some short-chain polyacetylenes in line with Bu'Lock's hypothesis, and was to be prepared with radioactive label in the proximal half. Radioactive compounds which were to be isolated after administering this ester to a suitable fungus might throw light on the mode of chain shortening. Various routes suitable for the synthesis of a tritium-labelled proximal half are investigated in Part III of this thesis, for the C₁₈ chain was to be built up by condensation of dehydromatricarianal and the Grignard reagent of an andomega;-iodoacetal (Scheme B). However, neither of the acetals (8) and (9) nor even the ether (10) would form a useful Grignard reagent. Possible reasons for this phenomenon, which prevented the synthesis of the ester (7), are discussed. A systematic search for polyacetylenes in previously untested plant families is in progress in this department. As part of this, examinations of two species of the Lobeliaceae, Lobelia syphilitica L. and Lobelia cardinalis L., and their hybrid Lobelia andtimes; vedariensis were carried out (Part IV). The last two contained the new (6-R,7-R) triol (11), but apart from this, only traces of other polyacetylenes were detected. Wyerone (12) is a natural acetylene, with antifungal properties, that is produced by the broad bean, Vicia faba L. It is of biosynthetic interest as present concepts of polyacetylene biogenesis (Bu'Lock's hypothesis) would not easily accommodate the presence of a cis-butenyl end group in such an acetylene. [1-¹⁴C] Acetate and the doubly-labelled fatty acid esters, oleate (1) and crepenynate (2) [tritium at C(9) and C(10), carbon-14 at C(9), C(10) or C(l)] were incorporated into sprouting beans. The isolations of active wyerone (12) and the closely related metabolites (13-15) are described in part V. The incorporations were positive (ca. 0.04%), and tritium isotope effects were observed. Possible pathways for wyerone biosynthesis are discussed.

The reported work on the reactions between indoles and arylsulphonyl azides was extended with a view to identifying any intermediates involved and establishing the relationships between the products formed. The effect of varying the structures of the indoles on these products has also been investigated. Compounds of type (I) appear to be key intermediates in the reactions of alkyl indoles with azides. Compound (I) has recently been prepared . The suggested intermediacy of this compound in the reaction between tosyl azide and 9-methyltetrahydrocarbazole was proved, the reaction of (I) with p-chlorobenzenesulphonyl azide yielding (II). As an extension of the work on tetrahydrocarbazoles, the reactions of 5,6,7,8,9,10-hexahydrocyclohept[b]indole, 6,7,8,9,10,11-hexahydro-5H-cyclooct[b]indole and their N-methyl derivatives have been examined. From the reactions of the N-H compounds have been isolated the products (III, n = 5, Z = Ts), (III, n = 6, Z = Ts) and (III, n = 6, Z = Cbs). The action of heat on (III, n = 6, Z = Ts) has been investigated and the products (IV) and (V) isolated. In acetic acid solution (III, n = 6, Z = Ts) was oxidised to (VI); a plausible mechanism for this oxidation was proposed. On heating, (VI) isomerised to (VII), and on treatment with base (VI) added water, giving (VIII). 5,6,7,8,9,10-Hexahydro-5-methylcyclohept[b]indole gave four products on treatment with tosyl azide, (IX-XII, n = 4). The structures of the latter two products .were proved by degradation to the compounds (XIII) and (XIV), and synthesis of these degradation products. The homologous compounds (IX-XII, n = 5) were obtained when 6,7,8,9,10,11-hexahydro-5-methyl-5H-cyclooct[b]indole was treated with tosyl azide. The stereochemistry of the compounds (XI, n = 4) and (XI, n = 5) has been determined and the transformation of (X, n = 5) into (XII, n = 5) on heating was demonstrated. Rapoport has reported the high reactivity of indoles with structures of the type (XV), this reactivity being due to a high degree of strain in the molecule. It was of interest, therefore, to extend the present work by synthesising the compounds (XV, n = 4) and (XV, n - 5) and investigating their reactions with arylsulphonyl azides. The former yielded the products (XVI), (XVII), (XVIII, n = 3) and (XIX), depending on the conditions used. From the reaction of p-nitrobenzenesulphonyl azide in methanol a further product (XX) was obtained. The effect of heat on the compounds (XVI) and (XX) has been examined, both yielding (XXI). (XV, n = 5) reacted with azides to give the compounds (XXII) as major product. Also obtained were compounds (XVIII, n = 4) and from the reaction with p-nitrobenzenesulphonyl azide (XXIII) was also isolated. Since this was the first time that compounds of the type of structure (XXII) had been obtained in good yield, the chemistry of these products was studied in some detail. A number of interesting rearrangements were observed. Treatment with base gave the compounds (XXIV, R = NZ) and (XXIV, R = O), while the effect of acid was to produce the ion (XXV). This was not isolated, products resulting from the addition of hydroxylic solvents to the molecule being the only compounds obtained. A similar rearrangement occurred on heating (XXII) in hydroxylic solvents. The rearrangement in acidic solvent was studied by means of n.m.r. spectroscopy. The reactions of 2-alkylthioindoles with azides were investigated, the major products being the azobisindoles (XXVI). Prom the reaction of 1-methyl-2-methylthioindole compound (XXVII) was also obtained; the formation of this compound involves a 1,2-shift of the alkylthio group. 2-Indolinethiones gave two products on treatment with arylsulphonyl azides, compounds (XXVIII) and the 1,2,3-thiadiazoles (XXIX). Finally, the reported work on 1,2,3,4-tetrahydro-2,5-dimethyl-5H-pyrido[4,3-b]indole was repeated and a further product (XXX) was obtained.

One of the most important considerations in peptide synthesis is the avoidance of loss of optical activity at the asymmetric centres of the amino-acids when these are condensed together. This racemisation is undesirable not only because it limits the yield of required product but also because the presence of even traces of racemates or disaster- eoisomers may inhibit crystallisation of a peptide, thus rendering purification more difficult. The most commonly employed amino protecting group, the benzyloxycarbonyl group, confers a remarkable optical stability upon the amino-acid which it protects, and the observation that a benzyloxycarbonyl amino-acid may be coupled without danger of racemisation under conditions which may cause considerable loss of activity when the benzyloxycarbonyldi- peptide, so formed, is coupled further, led to a much greater awareness of the problem. Thus, in 1955, North and Young initiated a study of coupling reactions from the point of view of racemisation, using the condensation of acetyl- <u>L</u>-leucine with glycine ester as a model reaction. They found that the azide procedure was remarkably free from the danger of racemisation but other procedures, including those which have been postulated to involve amino-activation, may result in considerable loss of activity.