Radioactive feeding experiments

In 1990, Nakamura et al. reported for the first time the complete biosynthetic origin of the whole carbon skeleton of carbazomycin B isolated from lower plants (378). Based on feeding experiments with and C-labeled compounds, followed by measurement of radioactivity and C-NMR spectroscopy, it was shown that L-tryptophan (408) contributes to C-3 and C-4 of the hexasubstituted aromatic ring, in addition to the indole ring, indicating tryptophan as the progenitor of carbazomycin B (261), in contrast to Chakraborty s proposal of 2-methylcarbazole. The indole part of L-tryptophan (408) is formed by incorporation of two carbons from phosphor-ibosyl diphosphate (430), with loss of the anthraniiic acid (397) carboxyl. The... 

Papaver Alkaloids.—Biosynthesis of morphine (36) occurs, in Papaver somniferum, through reticuline (33) by way of thebaine (35). The sequence from (35) to (36) involves, inter alia, two O-demethylations, with that at the methoxy-group at C-6 occurring first.1,2 Confirmation that the other methoxy-group is not demethylated first in this Papaver species obtains from the failure to detect oripavine (37), which is found in other Papaver species, as a natural constituent of P. somniferum. The experiment involved attempted isolation of radioactive (37), using inactive alkaloid as carrier, following a feeding experiment with radioactive reticuline (33).37... 

The biosynthesis of GAs was extensively surveyed during the 1960-70s by feeding experiments using Gibberella fujikuroi and radioactive precursors.199 200 The studies on the biosynthesis of GAs in plants were led by Graebe and coworkers.201-203 Recent research progress in the biosynthesis of GAs is described in Section 4.02.3.4. 

The demonstration166 that the side-chains of 24-methylenecycloartanol (96), 24-methylenelophenol, and campesterol incorporated two deuterium atoms, whilst those of 24-ethylidenecycloartanol, stigmasterol, and sitosterol contained a maximum of four deuterium atoms when biosynthesized by cultures of barley embryo in the presence of [Me-2H3]methionine, provides further evidence for the intermediacy of 24-methylene- and 24-ethylidene-compounds in the biosynthesis of C28 and C29 phytosterols. The barley system was also able to convert labelled 24-ethylidenelophenol into radioactive sitosterol efficiently. These results, and those obtained from feeding experiments with [2-14C,(4R)-4-3Hi]MVA, are consistent with a pathway (Scheme 9) to stigmasterol (97) involving isomerization of (98) to a... 

The furan ring and the side-chain of dehydrotremetone (121) from Eupatorium rugosum are derived from the mevalonate pathway.387 Cell-free preparations from the plant incorporated [1-14C]IPP more efficiently (0.1% incorporation) into (121) than either [1-14C]DMAPP or [5-3H]MVA, and a radioactive intermediate, possibly 4-hydroxy-3-(isopenten-2-yl)acetophenone, was isolated388 from these feeding experiments which was efficiently (1.12% incorporation) and preferentially incorporated into dehydrotremetone by the same preparation. 

A biosynthetic scheme for lyoniresinol, a syringyl lignan from Lyonia ovalifolia var. elliptica, has been proposed after feeding experiments with radioactively labelled precursors. Two moieties of sinapyl alcohol were... 

Classical feeding experiments with both stable and radioactive isotopic labels (7) enabled the biosynthetic origin of the polyethers to be elucidated and for a general stereochemical model to be proposed (1). More recent work on this class of compounds has focused on a genetic approach, and unusual and interesting genes specific to polyether biosynthesis have been isolated from these clusters. 

Preliminary characterization of some of the other intermediates involved in the biosynthesis of berberine in H. canadensis has been reported. Both the isomeric ( + )-norreticulines XCIXa and XCIXb are efficiently incorporated into berberine indicating that each is methylated to reticuline. Evidence for postreticuline intermediates is less equivocal. (+ )-Tetrahydroberberubine-12-3H (C) was only poorly incorporated (0.0064%) into berberine, and although ( + )-tetrahydroberberine was efficiently incorporated (8.9%), the ease with which it undergoes autoxidation to berberine makes interpretation of the result difficult. However, (— )-tetrahydroberberine [(— )-canadine], having the same absolute configuration as (4- )-reticuline, does occur in H. canadensis, and its isolation from a feeding experiment with labeled reticuline showed it to be radioactive (0.035% incorporation). Nevertheless, the demonstration... 

An extensive use of radioactive tracer technique permits a deeper insight into the formation of the tropane skeleton in vivo. Hyoscyamine-(methyl-i C) (76), atropine- C, tropic acid-i C (77), and succindi-aldehyde-2,3-i4C (78) were synthesized. The last was not incorporated at all by D. stramonium seedlings (78), while the former feeding experiments with ornithine-a-i4C proved its incorporation into hyoscyamine (79) hjD. stramonium (see Volume VI, pp. 172-173). More recently two independent teams (80, 81) reported that this incorporation takes place asymmetrically. 

Differences in incorporation of radioactivity from the C-1 and C-2 carbons of propionate into cholesterol and bile acids by biliary fistula rats have been demonstrated. It appears that incorporation of propionate via HCO3" is not the major pathway. Feeding experiments with sodium [ C]bicarbonate produced no labelled cholesterol or bile acids. 

Anderson et al. (103) carried out feeding experiments with cell cultures of A. altissima and L-[/Me7Ay/e/je- C]methionine and obtained radioactive 14. They supposed, therefore, that L-methionine would become S-adenosylmethionine and associate with transmethylation of 10 to produce 11. In accordance with growth of the cell culture, radioactive 11 gradually increases hence, they stated that the O-methyltransferase acts at the last stage of synthesis. 

To validate the model predictions, we performed radioactive tracer experiments in one of our commercial plants during normal production. The Ar tracer was injected in the air feed at different positions. Fourteen detectors on the reactor wall yielded a tracking of the gas tracer, from which among other things the gas rise velocities and the liquid circulation patterns could be determined. 

Robinson s original suggestion that the natural pyrrolizidine bases are derived in vivo from two molecules of ornithine (149) has been supported by studies using C-labeled compounds. Ornithine has been shown to be a specific precursor for the pyrrolizidine nucleus present in a variety of alkaloids. The feeding experiments carried out to date are listed in Table I. Three groups of workers have obtained labeled alkaloids after feeding C-labeled ornithines.Hydrolysis of the alkaloid to yield the free base, retronecine (127), has established that almost all the radioactivity is in the base portion. It is likely that ornithine is incorporated into retronecine after decarboxylation to putrescine (150) [Eq. (40)] since labeled putrescine is also specifically incorporated into retronecine (Table I). 

Feeding experiments with labelled pelletierine (15) have been carried out on cernuine and lycopodine. [4- H,2, 6- C2]Pelletierine gave radioactive cemuine... 

Further experiments with threonine and isoleucine have been carried out in Senecio magnificus Both [2- " C]- and [6- " C]-isoleucine were incorporated into senecic acid (21) and degradation gave a pattern of incorporation consistent with biosynthesis from two isoleucine units (Scheme 5). [ /- " CJ-L-Threonine was equally well incorporated into senecic acid, which was shown to contain essentially no activity at C-2 and C-8, consistent with the way in which threonine condenses with pyruvic acid to give isoleucine the route from threonine to senecic acid is illustrated (Scheme 6). In these three feeding experiments the incorporation of radioactivity into the main alkaloidal component of this plant, retronecine, was very low. 

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