Alkaloid biosynthesis in Croton flavens

The biosynthesis of the morphinandienone alkaloids norsinoacutine, sinoacutine and flavinantine has been studied using 1-³ H-sinoacutine, 1-³H-norsinoacutine, 1-³H-norsinoacutinols, l-[S-methyl-¹⁴C]-methionine, glycine-2-¹⁴C, 1-³H-8,14-dihydronorsalutaridine, 1-³ H-8,14-dihydrosalutaridine, 1-³H-sinomenine, 1-³H-isosinomenine, (±)-[2-¹⁴C]phenylalanine, (±)-[N-methyl-¹⁴C]orientaline and (±)-[N-methyl-¹⁴C]reticuline.     

Biosynthesis of N-methylphenethylamine in Dolichothele sphaerica

Living Dolichothele sphaerica metabolized 1.91% of administered [2-¹⁴Clphenylalanine to N- methylphenethylamine. Phenethylamine, the presumable intermediate in this biosynthetic conversion, was detected in an extract of the cactus in very low concentrations. The addition of carrier to the extract allowed the isolation of radiolabelled phenethylamine and the establishment of its probable involvement in N- methylphenethylamine formation. This is the first report of N- methylphenethylamine biosynthesis, of phenyl- alanine serving as an efficient precursor to cactus alkaloids, and of the occurrence of phenethylamine in the Cactaceae.     

δ-N-Methylornithine: a natural constituent of Atropa belladonna

δ-N-Methylornithine, a tropane alkaloid precursor, is shown for the first time to be a natural plant constituent; it was isolated in radioactive form after feeding [5-¹⁴C]- and [5-³H]ornithine to Atropa belladonna. This finding supports the deduced role of δ-N-methylornithine in tropane alkaloid biosynthesis.     

Normetanephrine and octopamine involvement in normacromerine biosynthesis in Coryphantha macromeris var. runyonii

Administration of [7-³H]normetanephrine to living Coryphantha macromeris var. runyonii resulted in the formation of labeled normacromerine     

Biosynthesis of alpinigenine by way of tetrahydroprotoberberine and protopine intermediates

In the biosynthesis of the benzazepine alkaloid alpinigenine a N-methylation step followed by hydroxylation α to nitrogen has now been shown more conclusively to be involved in the transformation of a N-heterocyclic ring system. After feeding Papaver bracteatum plants both the precursors (±)-tetrahydropalmatine-[8,13,14-³H] and (±)-tetrahydropalmatine methiodide-[8,13,14-³H;8-⁴C] an identical mode of abstraction of tritium was observed including a complete loss of the isotope from C-14. The next member in the biogenetic chain, muramine-[8-¹⁴C], was incorporated into alpinigenine very efficiently. Furthermore, using structurally different precursors not utilized for normal alkaloid formation, e.g. 2′-hydroxymethyl-laudanosine-[¹⁴CH₂OH], 13-hydroxymuramine-[8-¹⁴C], the specificity of alkaloid metabolism was examined in the whole plant. Tracer dilution technique was applied to confirm the occurrence in the plant of three established intermediates. Chemical syntheses of four of the alkaloids used during these investigations were developed.     

Biosynthesis of Camptotheca acuminata alkaloids

Tracer feeding experiments with Camptotheca acuminata plants show that [1′-¹⁴C]L-tryptophan, [Ar-³H₄]L-tryptophan, [Ar-³H₄,1′-¹⁴C]tryptophan, [1′-¹⁴C]-tryptamine, [2-¹⁴C]DL-mevalonate, and [2-¹⁴C]geraniol-[2-¹⁴C]nerol are incorporated into camptothecin. Direct stem injection of the labeled precursors into C. acuminata plants resulted in a substantial increase in the activity of isolated Camptotheca alkaloids as compared to root feeding of the same tracer.     

Biosynthetic conversion of α-methylbutyric acid to tiglic acid in Datura meteloides

The administration of RS-α-methylbutyric-[1-¹⁴C] acid to Datura meteloides plants resulted in the formation of radioactive meteloidine. A systematic degradation indicated that essentially all the activity was located at C-1 of the tiglic acid moiety of the alkaloid.     

Biosynthesis of tropic acid: Feeding experiments with cinnamoyltropine and littorine

The administration of cinnamoyl-[2-¹⁴C]-tropine-[N-methyl-¹⁴C] to Datura stramonium plants resulted in the formation of labeled atropine and scopolamine. However the atropine was found to have almost all its radioactivity located on the N-methyl group of the alkaloid, indicating that the administered ester had undergone hydrolysis in the plant affording tropine and cinnamic acid, the latter not being utilized for the biosynthesis of tropic acid. Dual labeled RS-littorine (3β-(2-hydroxy-3-phenylpropionyloxy-[1-¹⁴C]-tropane-[3β-³H]) was also fed to D. stramonium and radioactive atropine was obtained. However the drastic change in the ³H:¹⁴C ratio found in the atropine indicated that the littorine was not converted directly to the alkaloid, and it is suggested that the littorine is hydrolysed _{in vivo} to tropine and phenyl-lactic acid, the latter undergoing rearrangement to tropic acid prior to esterification with tropine.     

Synthesis of [3-14C]-2′-methylreticuline and its incorporation into alkaloid fractions of Papaver somniferum

[3-¹⁴C]-2′-Methylreticuline has been synthesized by standard methods. This modified opium alkaloid precursor is efficiently incorporated by aberrant biosynthesis into alkaloid fractions of Papaver somniferum, particularly into a highly purified codeine fraction.     

Control of methionine synthesis by lysine in Lemna minor

When Lemna minor was cultured in the presence of 0.25 mM l-lysine, the concentration of free methionine and formyl and methyl tetrahydrofolate (THFA) were decreased. l-lysine, l-homoserine, l-threonine and l-methionine at concentrations up to 8 mM did not affect N¹⁰-formyl THFA synthetase (E.C. 6.3.4.3) and N⁵,N¹⁰-methylene THFA reductase (E.C. 1.1.1.68). In contrast, serine hydroxymethyltransferase (E.C. 2.1.2.1) activity was inhibited by lysine. This inhibition gave a sigmoidal curve when plotted for a range of l-lysine or THFA concentrations. Exogenous lysine also reduced the incorporation of glycine [¹⁴C] and serine [3-¹⁴C] into free and protein methionine. Lysine, which is known to control synthesis of homocysteine in L. minor, may also regulate production of C-1 units for methionine synthesis by inhibition of serine hydroxymethyltransferase.     

The function of the pentose phosphate pathway in Phaseolus mungo hypocotyls

The metabolism of d-gluconate-[1-¹⁴C] and -[6-¹⁴C] by segments from etiolated hypocotyls of Phaseolus mungo has been studied. The release of ¹⁴CO₂ from gluconate-[1-¹⁴C] was greater than that from gluconate-[6-¹⁴C] in all parts of hypocotyls examined. Incorporation of the radioactivity from gluconate-[6-¹⁴C] into RNA, lignin and aromatic amino acid fractions was greater in the upper (younger) part of the hypocotyls. Incorporation into sugars was greater in the lower (more mature) parts.     

Furanocoumarin biosynthesis in Ruta graveolens cell cultures

The biosynthetic routes to four linear furanocoumarins—psoralen, xanthotoxin, bergapten. isopimpinellin-co-occurring in Ruta graveolens cell cultures have been investigated with six ¹⁴C-labelled compounds. Mevalonic acid was only poorly incorporated, in contrast to umbelliferone. In support of previous suggestions, 7-demethylsuberosin and (±)-marmesin were very good precursors of the linear furanocoumarins. 7-O-Prenylumbelliferone also was fairly well utilized, but this was probably owing to a prior ether cleavage yielding umbelliferone. Psoralen was well incorporated into bergapten and xanthotoxin, but not into the dimethoxylated isopimpinellin. Differences exist between the organized plant and its cell culture in terms of metabolic products and, by implication, precursor utilization. S(+)-Marmesin was obtained in small quantity from an acid-hydrolysable conjugate present in the culture medium. Syntheses of [2-¹⁴C]7-demethylsuberosin, [2-¹⁴C]osthenol, [2-¹⁴C]7-O-prenylumbelliferone, [3-¹⁴C] (±)-marmesin, and [3-¹⁴C]psoralen are described, as well as an improved method for separation of furanocoumarin mixtures by TLC and GLC.     

Biosynthesis of azetidine-2-carboxylic acid in Convallaria majalis

Convallaria majalis plants were fed dl-methionine-[1-¹⁴C]. [1-¹⁴C, 4-³H], and [1-¹⁴C, 2-³H], S-adenosyl-l-methionine-[1-¹⁴C], and dl-homoserine-[1-¹⁴C], resulting in the formation of labeled azetidine-2-carboxylic acid (A-2-C). The complete retention of tritium relative to carbon-14 in the feeding experiment involving methionine-[1-¹⁴C, 4-³H] indicates that aspartic acid or aspartic-β-semialdehyde are not intermediates between methionine and A-2-C. However, since the A-2-C derived from methionine-[1-¹⁴C, 2-³H] had lost 95% of the tritium relative to the C-14, it is not considered that methionine or its S-adenosyl derivative are the immediate precursors of A-2-C. Our data and that of others is consistent with the intermediate formation of γ-amino-α-ketobutyric acid which on cyclization yields 1-azetine-2-carboxylic acid, A-2-C then being formed on reduction.     

Biosynthesis of the 3-benzylchroman-4-one eucomin in Eucomis bicolor

Feeding experiments have demonstrated the specific incorporation of radioactivity from dl-phenylalanine-[1-¹⁴C], l-phenylalanine-[U-¹⁴C], sodium acetate-[2-¹⁴C] and l-methionine-[methyl-¹⁴C] into the 3-benzylchroman-4-one eucomin in Eucomis bicolor. The labelling patterns indicate that eucomin is biosynthesized by the addition of a carbon atom derived from methionine onto a C₁₅ chalcone-type skeleton. Radioactivity from 2′,4′,4-trihydroxy-6′-methoxychalcone-[methyl-¹⁴C] and 2′,4′-dihydroxy-4,6′-dimethoxychalcone-[6′-methyl-¹⁴C] was incorporated into eucomin, the latter compound being the better precursor, demonstrating the feasibility that 2′-methoxychalcones are biosynthetic precursors of the “homoisoflavonoids”. Possible biosynthetic relationships in this class of compounds are discussed.     

In vitro synthesis of mycolic acids by the fluffy layer fraction of Bacterionema matruchotii

Biosynthetic activity for mycolic acid occurred in the fluffy layer fraction but not in the 5000g supernatant of Bacterionema matruchotii. With [1-¹⁴C]palmitic acid as precursor for the in vitro system, the predominant product was identified as C_32:0 mycolic acid by radio-gas-liquid chromatographie (radio-GLC) and gas chromatographic/mass spectroscopic analyses; if [1-¹⁴C]stearic acid was used, two major radioactive peaks appeared on GLC: one corresponding to the peak of (C_34:0 + C_34:1) mycolic acids and the other to (C_36:0 + C_36:1) mycolic acids. By pyrolysis/radio-GLC analysis, C_32:0 mycolic acid synthesized by [1-¹⁴C]palmitic acid was pyrolyzed at 300 °C to form palmitaldehyde (the mero moiety) and methyl palmitate (the branch moiety). The pH optimum for the incorporation of [1-¹⁴C]palmitate into bacterionema mycolic acids was 6.4 and the reaction required a divalent cation. The in vitro system utilized myristic, palmitic, stearic and oleic acids (probably via their activated forms) well as precursors, among which myristic and palmitic acids were more effective than the rest. Avidin showed no effect on the biosynthesis of mycolic acid from ¹⁴C-palmitate whereas cerulenin, a specific inhibitor of β-ketoacyl synthetase in de novo fatty acid synthesis, inhibited the reaction at a relatively higher concentration. Thin-layer chromatographic analysis of lipids extracted from the reacting mixture without alkaline hydrolysis showed that both exogenous [1-¹⁴] fatty acid and synthesized mycolic acids were bound to an unknown compound by an alkali-labile linkage and this association seemed to occur prior to the condensation of two molecules of fatty acid.     

Metabolism of nicotine in Nicotiana glauca

A mixture of (?)-nicotine-[2′-³H] and (±)-nicotine-[2′-¹⁴C] was administered to Nicotiana glauca plants for 3 days, resulting in the formation of radioactive nornicotine (49·5% incorporation) and myosmine (2·05% incorporation). Negligible activity was detected in anabasine, cotinine, or 3-acetylpyridine, the last two compounds being added as carriers to the harvested plants. The radioactive nornicotine consisted of 48% (?)-nornicotine-[2′-¹⁴C,³H] and 52% (+)-nornicotine-[2′-¹⁴C]. Thus if (+)-nornicotine is formed from (?)-nicotine the transformation must involve loss of the hydrogen from C-2′. Myosmine is presumably formed from nicotine via nornicotine. However by feeding myosmine-[2′-¹⁴C] to N. glauca it was shown that the dehydrogenation is not reversible, no activity being detected in nornicotine. Nicotinic acid (0·14% incorporation) was a metabolite of myosmine-[2′-¹⁴C]. Essentially all the activity of the nicotinic acid was located on its carboxyl group, indicating that myosmine was a direct precursor.     

The involvement of sucrose,glucose and other metabolites in the synthesis of triterpenes and dopa in the laticifers of Euphorbia lathyris

A quantitative triterpene analysis was made of latex stem tissue of Euphorbia lathyris. Young plants seedlings of E. lathyris were incubated with various labelled precursors. Incorporation into triterpenes was obtained from [2-¹⁴C]mevalonic acid, [1-¹⁴C]acetate, [3-¹⁴C]pyruvate, [U-¹⁴C]sucrose, [U-¹⁴C]glucose, [U-¹⁴C]xylose, [U-¹⁴C]glyoxylate, [2,3-¹⁴C]succinic acid, [1-¹⁴C]glycerol [U-¹⁴C]serine. Both sugars tyrosine appeared to be effective precursors in DOPA synthesis inside the laticifers. Exogenously supplied mevalonic acid was only involved in triterpene synthesis outside the laticifers. GC-RC of triterpenes synthesized from [U-¹⁴C]glucose revealed the origin of these compounds in the latex. The labelled triterpenes obtained after incorporation of the other mentioned labelled precursors were only partly synthesized in the laticifers. For quantitative data on latex triterpene synthesis seedlings were incubated with [U-¹⁴C]sucrose, [U-¹⁴C]glucose, [U-¹⁴C]xylose [1-¹⁴C]acetate in the presence of increasing amounts of unlabelled substrate. From the amount of ¹⁴C incorporated into the triterpenes the amount of substrate directly involved in triterpene synthesis was calculated, as was the absolute triterpene yield. Sucrose showed the highest triterpene yield, equivalent to the daily increase of the triterpene content of growing seedlings. The possible significance of the other precursors in triterpene synthesis in the laticifers is discussed.     

Biosynthesis of the 6-oxygenated isoflavone afrormosin in Onobrychis viciifolia

dl-Phenylalanine-[2-¹⁴C], 2′,4′,4-trihydroxychalcone-[carbonyl-¹⁴C] and formononetin-[Me-¹⁴C] were all good precursors of afrormosin (7-hydroxy-6,4′-dimethoxyisoflavone) in Onobrychis viciifolia seedlings. This demonstrates that 6-oxygenation may be a late process in the biosynthesis of isoflavones.     

The metabolism of anatabine to α,β-dipyridyl in Nicotiana species

α,β-Dipyridyl isolated from Nicotiana tabacum plants which had been fed anatabine-[2′-¹⁴C, ¹³C], and then allowed to dry in air for 20 days was radioactive (82% specific incorporation)An examination of its ¹³C NMR spectra established that it was enriched only at C-2, indicative of its direct formation from anatabineThe labelled anatabine was also fed to Nglauca and Nglutinosa plants, which were extracted immediately after harvestingIn these experiments no radioactive α,β-dipyridyl was detected, suggesting that α,β-dipyridyl is an artifact produced by the oxidation of anatabine in the drying leaves of tobaccoAnabasine isolated from the Nicotiana species which had been fed anatabine-[2′- ¹⁴C, ¹³C] was unlabelled, indicating that none of this alkaloid is formed by the reduction of anatabine.     

Pathways of carbohydrate oxidation in leaves of Pisum sativum and Triticum aestivum

The aim of this work was to establish the pathways of carbohydrate oxidation used in the dark by leaves of Pisum sativum and Triticum aestivum. Segments of young and mature leaves of pea released the carbons of glucose-[¹⁴C] as ¹⁴CO₂ in the order 3,4 > 1 > 2 > 6 whereas in segments of young and mature leaves of wheat the order was 3,4 > 1 > 6 > 2. The detailed labelling of the constituents of mature leaves of wheat by glucose-[1-¹⁴C], -[2-¹⁴C], -[3,4-¹⁴C], and -[6-¹⁴C] was determined and showed that the high yield of CO₂ from C-6 relative to that from C-2 was due to release of C-6 during pentan synthesis. Estimates were made of the maximum catalytic activities of phosphofructokinase and glucose-6-phosphate dehydrogenase in pea and wheat leaves of three ages. The results of all the above investigations strongly indicate that both pea and wheat leaves in the dark oxidize carbohydrate via glycolysis and the pentose phosphate pathway with the latter accounting for no more than a third of the total. No evidence was obtained of any major change in the relative activities of the two pathways during the development of either type of leaf.