No description available.

When grown in the presence of the sterol biosynthesis inhibitor, paclobutrazol, suspension cultures of Apium graveolens (celery) accumulate substantial amounts of I 4a-methylsterols, at the expense of 4-demethylsterols. These changes have been correlated with reduced synthesis of phosphatidylcholine (PC) via the CDP-base pathway (Roiph & Goad, 1991). It was subsequently proposed that changes in the membrane sterol composition of plant cells may regulate the activity of CTP: cholinephosphate cytidylyltransferase (CT), the rate-determining enzyme of this pathway (Kinney & Moore, 1989). In preliminary studies, the membrane-associated form of CT in A.graveolens, was found to exhibit optimal activity at pH 7.7, in the presence of 8.0 mM CTP and 3.5 mM Mg2t Microsomal membrane fractions, in which a large proportion of CT activity was found to reside, were analysed in terms of lipid composition. The predominant phospholipid in such membranes, PC, constituted approximately 70% of the total phospholipid content. Other, more minor constituents, included phosphatidylethanolamine (PE), phosphatidylglycerol (PU), phosphatidylinositol (P1), phosphatidylserine (PS), and phosphatidic acid (PA). All phospholipids present in A.graveolens were found to be rich in linoleate (18:2) and palmitate (16:0). Lesser amounts of stearate (18:0), oleate (18:1), and a-linolenate (a- 18:3), were also present. The major phytosterols in microsomes were identified as campesterol, stigmasterol, sitosterol, and isoflicosterol, with trace amounts of cholesterol and 24-methylene cholesterol. The sterol biosynthesis inhibitors, miconazole, terbinafine, fenpropimorph, and tomatidine, proved to be useflul tools in the manipulation of membrane sterol composition in suspension cultures of A.graveolens. Treatment with these inhibitors caused significant alterations in lipid composition with corresponding changes in the activity of membrane-associated CT. Terbinaflne and fenpropimorph caused a large increase in the stigmasterol/sitosterol ratio of cells with a concomitant stimulation of CT activity. The latter compound also resulted in the accumulation of various 9$, 19- cyclopropyl sterols. Similarly, the azasterol inhibitor, tomatidine, resulted in an enhancement of CT activity, but with very lift le change in the stigmasterol/sitosterol ratio of cells. Conversely, miconazole resulted in a decline in the stigmasterol/sitosterol ratio, corresponding to lower membrane-associated CT activity. The latter inhibitor also caused an accumulation of oleoyl residues in the PC fraction of cells, suggesting an inhibition of A 1 2-desaturase activity. Radiolabelling studies with [3IflS-adenosyl-L-methionine revealed a degree of coordinate regulation between the CDP-base and methyltransferase pathways of PC biosynthesis. Consequently, despite changes in CT activity, levels of phospholipid in most inhibitor-treated cultures remained relatively constant. Supplementation of miconazole-treated cultures with free fatty acids partially overcame the cytostatic nature of the azole inhibitor, with a concomitant reactivation of CT. Mono- and diunsaturated fatty acids were found to be the most effective compounds in this respect. The addition of stigmasterol or sitosterol to miconazoletreated cultures also resulted in partial growth restoration and reactivation of membrane-bound CT.

No description available.

No description available.

No description available.

6-Methylsalicylic acid (6-MSA) is one of the oldest known polyketides. It is synthesised in vivo by the polyketide synthase 6-methylsalicylic acid synthase (6-MSAS), a multifunctional enzyme which uses its active sites iteratively. The stereochemistry of the hydroxyl produced from the single ketoreduction, as well as the order of dehydration, cyclisation and aromatisation steps, remain cryptic, despite extensive study. Holo 6-MSAS was heterologously expressed in E. coli and purified in two steps. A non-hydrolysable carba(dethia)malonyl-N-acetylcysteamine analogue was synthesised and used to off-load enzyme-bound intermediates from 6-MSAS. In assays with acetyl-CoA and acetoacetyl-CoA alone, diketide and triketide intermediates were off-loaded and detected by HPLC-HR-ESI-MS. In the presence of NADPH, the off-loaded triketide was reduced by the ketoreductase domain of 6-MSAS. A potential dehydrated intermediate was also observed. The dehydratase domain of 6-MSAS has recently been reassigned as a thioester hydrolase. To test this theory, the catalytic histidine residue in 6-MSAS was mutated to an alanine and the abolition of production of 6-MSA in vivo was observed. Mutated 6-MSAS was still able to produce the shunt product triacetic acid lactone. Incubation of mutated 6-MSAS with acetyl-CoA, malonyl-CoA, NADPH and carba(dethia)malonyl-N-acetylcysteamine saw only the off-loading of diketide and triketide analogues. To investigate the stereochemistry of ketoreduction in 6-MSA biosynthesis, steps were made to synthesise the resolved diastereomeric reduced-triketide CoAs which would be the substrates for the ketoreductase domain. Attempts to phosphopantetheinylate apo 6-MSAS in vitro with three different phosphopantetheinyltransferases were unsuccessful. Limited proteolysis of both holo and apo 6-MSAS found that the apo synthase rapidly lost a C-terminal fragment while holo 6-MSAS was much more stable under the same conditions. Attempts were made to express the acyl carrier protein domain from 6-MSAS to overcome these problems. These experiments represent the first use of the non-hydrolysable analogue methodology in a Type I iterative polyketide synthase and provide a framework for future experiments investigating intermediates in the biosynthesis of 6-MSA.

In part I of this thesis are described some studies toward the total synthesis of tetracycline. Attempts to transform the key aromatic compounds, terrarubein and 6-methylpretetramid into actual or hypothetical biosynthetic intermediates, failed to yield useful non-aromatic products. However, several of the transformations further along the route have been achieved. Thus, the conversion of 12a-deoxy-5a,6-anhydrotetracycline into 7-chloro-5a,6-anhydrotetracycline was successfully carried out. In a different approach it was attempted to convert the synthetic tetracycline derivative 7-chloro-4-dedimethylamino-5a,6-anhydrotetracycline to 7-chloro-5a,6-anhydrotetracycline via a series of bromination-aminatipn experiments. Chromatographic evidence is presented for the formation, in trace amounts, of 7-chloro-5a,6-anhydro-4-epi-tetracycline. Part II is concerned with the study of the possible precursor activity of triacetic acid lactone, a potential polyketomethylene chain intermediate in the biosynthesis of aromatic compounds. (3,5-¹⁴ C) Triacetic acid lactone was fed to P. patulum and labelled griseofulvin was isolated and degraded. It was found that radioactivity is incorporated into griseofulvin a non-specific way. In one strain of the mould used two new metabolites were found as a result of the addition of triacetic acid lactone. Addition of triacetic acid lactone to the mould also causes an unexplained enhancement of metabolite formation. / Science, Faculty of / Chemistry, Department of / Graduate

The in vivo biosynthesis of deoxyribonucleic acid (DNA) from labelled thymidine has been investigated in rat intestinal mucosa. The DNA preparations were fractionated by column chromatography and the fractions were assayed for radioactivity by liquid scintillation counting methods. In the first experiments the DNA was isolated from the intestinal mucosa of rats which had received H³-thymidine 5, 10 or 20 minutes or 24 hours before sacrifice. When the macromolecules were fractionated on ECTEOLA-cellulose the results obtained were inconclusive because no definits pattern of incorporation of radioactivity was observed in the fractions. Chromatography on ECTEOLA-cellulose was considered unsatisfactory, because of the variations in the elution patterns of DNA preparations from experiment to experiment and evidence indicating degradation of DNA during the fractionation procedure. In subsequent experiments fractionation on methylated albumin-kieselguhr (MAK) columns was employed and double labelling experiments were carried out. The animals were injected intravenously with H³-thymidine and 24 hours later with C¹⁴-thymidine. The rats were killed 20 or 40 minutes after the second injection and the double labelled DNA was isolated from the intestinal mucosa. On fractionation by MAK columns reproducible elution patterns were obtained even after storage of the DNA solutions. The main DNA peak was always eluted at the same range of sodium chloride concentration and 95-97 percent of the radioactivity was eluted in this peak. Each subfraction comprising the main peak was examined for H³ and C¹⁴ activity. By studying the H³/C¹⁴ ratios of the fractions newly synthesized material could be compared with older, presumably stabilized DNA. When the animals were exposed to the C¹⁴-labelled thymidine for 40 minutes the H³/C¹⁴ ratios of the subfractions were constant, indicating no metabolic differences between the newly synthesized DNA (C¹⁴-labelled and the "old" (H³-labelled) DNA. However, when the time of exposure to the C¹⁴-labelled precursor in vivo was 20 minutes, the H³ /C¹⁴ ratios of subtractions increased as the sodium chloride concentration of the eluant increased. These results indicated some metabolic differences amongst these fractions. Stepwise enzymatic degradation by snake venom phosphodiesterase of the double labelled DNA preparations, and the main peak obtained after MAK chromatography, indicated the incorporation of thymidine into newly synthesized and "old" DNA occurred well within the chain. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

In Part I, hypotheses for the biogenesis of Erythrina alkaloids are discussed. Di-(β-3,4-dihydroxyphenyl)-ethylamine the theoretical precursor predicted by the biogenetic theory, was prepared and ring closure to the erythrinane ring system by oxidative coupling was attempted under various conditions. Consequently, the biogenesis of the Erythrina alkaloids was re-examined and a new proposal is advanced for the biosynthesis of these alkaloids. Synthetic routes to a hypothetical precursor, proposed here for the first time as a potential intermediate, are described. The biogenetic-type synthesis of the spiro-amine ring system present in the Erythrina alkaloids was achieved by oxidative coupling of the blocked diphenolic precursor, as predicted by the proposed biosynthetic scheme. Oxidation of di-(β-3-hydroxy-4-methoxyphenyl)-ethylamine by alkaline potassium ferricyanide afforded 3,15-dimethoxy-16-hydroxy-2-oxo-erythrina-1(6),3-diene in 15% yield. Reduction of the latter by sodium borohydride gave 3,15-dimethoxy-2,16-dihydroxyerythrina-l(6),3-diene. Acetylation of the dienone yielded 3,15-dimethoxy-16-acetoxy-2-oxoerythrina-1(6),3-diene. The total biogenetic-type synthesis of erysodine is therefore but two steps from completion. The results as a whole confirm the hypothesis that Erythrina alkaloids are produced in Nature by oxidative coupling of diphenols. They also demonstrate the directing role of the protective groups in the phenolic precursor. The evidence allows a biosynthetic pathway for the aromatic Erythrina alkaloids to be considered, and the mechanism for the ring closure process is discussed. The isotopically labelled precursor 3-hydroxy-4-methoxy-N-(3-hydroxy-4-methoxyphen[1-¹⁴C]ethyl)-phenethylamine was prepared to test the biosynthetic hypothesis in the plant. Feeding experiments are in progress. In Part II, the biogenesis of cholesterol and methods for functionalising inert methyl groups are reviewed, and a new theoretical approach to removal of the 14α-methyl group from lanosterol is described. The removal of this methyl group in vitro could not be achieved, but a series of interesting compounds was obtained. Evidence for the structures of these compounds is presented. Thus, photosensitized oxygenation of dihydrolanosteryl acetate in the presence of para-nitrobenzenesulphonyl chloride yielded 3β-acetoxylanosta-7,9(ll)-diene, 3β-acetoxylanost-8-ene-7-one and 3β-acetoxylanost-8-ene-7a-hydroperoxide. In addition a compound having an ambiguous structure and designated as IP1 was obtained. The dibromo-derivative of the latter is 33-acetoxy-7a,lla-dibromolanostane-8 a,9α-epoxide, the structure of which was determined by X-ray crystallographic study. A working structure for compound IP1 based on the physical and chemical evidence is discussed. / Science, Faculty of / Chemistry, Department of / Graduate

Part I of the thesis describes four investigations of some of the neutral components of bark extractives. The petroleum ether extract of grand fir [Abies grandis (Dougl.) Lindl.] was found to contain two triterpene lactones. The first compound, cyclo-grandisolide, was shown by chemical and spectroscopic considerations and confirmed by X-ray analysis to be (2 3R)-3a-methoxy-9,19-cyclo-9β-lanost-24-ene-26 ,23-lactone (38) . The second component, epi-cyclograndisolide, was isomeric with the first and was assigned as (23S)-3α-methoxy-9,19-cyclo-9 β- lanost-24-ene-26,23-lactone (43). In the second investigation, three triterpenes of the chloroform extract of Pacific silver fir [A. amabilis (Dougl.) Forbes] were examined. The main component, abieslactone, was known and had been assigned as (23R)-3α-methoxylanosta-9(11),24—diene-26,23-lactone (30). Chemical and spectroscopic evidence is considered which indicates that assignment to be incorrect and abieslactone is tentatively re-assigned as (23R)-3a-methoxy-9β-lanosta-7,24-diene-26,23-lactone (81). A minor component, AA₃ was assigned on the basis of methylation studies as 3-desmethylabieslactone or (23R)-3α-hydroxy-9β-lanosta-7,24-diene-26,23-lactone (83). Oxidation of AA₃ gave a ketone identical to the second minor component, AA₂, which is then (23R)-3-oxo-9β-lanosta-7,24-diene-26,23-lactone (82). The third investigation concerns the structure of W₄, a triterpene ketone from the petroleum ether extract of Western white spruce [Picea glauca (Moench) Voss. var. albertiana (S. Brown) Sarg.]. The structure tentatively assigned on the basis of spectroscopic evidence is 3β-methoxy-8α-serrat-13-en-21-one (91). The fourth investigation was a chemosystematic study of the petroleum ether extract of Engelmann spruce [P. engelmannii Parry]. The presence of methoxyserratene derivatives known to be present in other members of the same genus were not detected in the present investigation. Part II of the thesis describes synthetic endeavors leading to possible bio-intermediates of indole alkaloids and the biosynthetic evaluation of one synthetic compound. Condensation of 3-ethylpyridine with 2-carboethoxy-3(β-chloroethyl)indole (60) followed by reduction gave N-[β{3(2-hydroxymethylindolyl)}ethyl]-3-ethy1-1,2,5,6-tetrahydropyridine (64). The benzoxymethyl derivative 65 of compound 64 was treated with potassium cyanide to give the cyanomethyl derivative 66 which could be hydroxyzed to N-[β{3(2-carbomethoxymethylindolyl)}ethyl]-3-ethyl-1,2,5,6-tetrahydropyridine (67). Alkylation of the compound with methyl formate followed by reduction of the resulating enol, gave 16,17-dihydrosecodin-17-ol (69). This compound was shown to be not, or very slightly, incorporated into the alkaloids of Vinca rosea L. plants. Attempts to oxidize the tetrahydropyridine 64 with mercuric acetate under various conditions failed to give detectable amounts of the corresponding pyridinium salt. In another synthetic sequence, condensation of the tryptophyl derivative 60 with 3-acetylpyridine ethylene ketal followed by the same sequence of reduction and homologation as employed before gave N-[β{3(2-carbomethoxy-methylindolyl)}ethyl]-3-acetyl-l,2,5,6-tetrahydropyridine (82). Attempts to oxidize 82 with mercurous acetate followed by hydrogenation failed to give the desired N-[β{3(2-carbomethoxymethylindolyl)}ethyl]-3-acetyl-l,4,5,6-tetrahydropyridine (83). In a second attempt to synthesize 83, the pyridinium chloride salt 84 from the condensation of 3-acetylpyridine with the tryptophyl derivative 60, was hydrogenated to N-[β{3(2-carboethoxyindolyl) }ethyl]-3-acetyl-l,4,5, 6-tetrahydropyridine (85). Reduction of 85 under a variety of conditions gave major amounts of N-[β{3(2-hydroxymethylindolyl)}ethyl]-3-acetylpiperi-dine (86) with only trace amounts of N-[β{3(2-hydroxymethylindoiy 1)}ethy1]-3-acety1-1,4,5,6-tetrahydropyridine (87) containing the necessary vinylogous amide chromophore. In a third approach to the synthesis of 83, methyl indole-2-carboxylate (88) was reduced and homologated as before to give methyl indole-2-acetate (92). Treatment of 92 with ethylene oxide and stannic.chloride gave methyl 3(β.-hydroxyethyl)indole-2-acetate (93). Treatment of the tryptophyl bromide derivative 94, produced by the action of phosphorous tribromide on hydrogenated to the vinylogous amide 83. More conveniently, treatment of 93 in 3-acetylpyridine with phosphorous tribromide and immediate hydrogenation gave 83 in better yield. / Science, Faculty of / Chemistry, Department of / Graduate