In-vivo feeding experiment

In vivo feeding experiments with singly and doubly labeled strictosidine (33) in C. roseus shoots afforded labeled ajmalicine (39), serpentine (40), vindoline (3), and catharanthine (4). Vincoside (85, page 37) was not incorporated into the alkaloids, suggesting that it was biologically inert 188). Brown and co-workers 190) conducted somewhat parallel studies examining the precursor relationship of strictosidine in C. roseus. Incorporation into tetrahydroalstonine (75), ajmalicine (39), catharanthine (4), and vindoline (3) was observed. 

These findings have been corroborated by in vivo feeding experiments with the fungus using deuterium-labeled acetate as precursor (37). CyA samples isolated from fermentations supplemented with either H,)- or (Z- C, H jacetate were analyzed... 

Whichever way the problem was approached, a completely conclusive experiment that would settle, unambiguously, the question of nonenzymatic condensation of j9-ketoacids with imines under the conditions of in vivo feeding experiments in Sedum was not forthcoming. We eventually decided to interpret the results of the feeding experiments with [1,2- 2] acetate described above as evidence that N-methylpelletierine and N-methylallosedridine were formed according to the classical scheme of Robinson (Path Cl). This interpretation suggests that the acetate derived C3 units of cocaine and the pelletierine alka-... 

The most obvious metabolic alteration of norlaudanosoline is 0-methylation. A series of elegant experiments were summarized by Davis et al. (1982), who showed that norlaudanosoline is methylated in the presence of SAM and rat liver enzymes (ca-techol-O-methyltransferase), yielding a mixture of monomethylated norlaudanoso-lines. Interestingly, (S)-norlaudanosoline yielded 79% 6-0-methylnorlaudanosoline and 14% of the 7-0-methyl derivative, while the (R)-isomer gave only 26% 7-0-me-thyl- and 68% of the 6-0-methylnorlaudanosoline (analysis by GC-MS and HPLC). In animals the site of enzymatic 0-methylation is clearly dictated by the stereoiso-meric form of the alkaloid substrate. In plants, it has been amply documented that reticuline is the branch point for a multitude of different isoquinoline alkaloid types. This means that norlaudanosoline must be transformed to reticuline by three methylation reactions, two 0-methylations (position 6 and 4 ) and one N-methyl-ation. Since norreticuline has been undoubtedly demonstrated to be a precursor of reticuline in vivo, one has to assume that 0-methylation precedes N-methylation. This is also in agreement with in vivo feeding experiments (Brochmann-Hanssen et al. 1975). 

In addition to the inhibitory effect of cinnamic acid derivatives on the PAL activity, they generally lower the amount of extractable PAL. This has been studied in (a) feeding experiments, (fo) experiments in which the hydroxy-cinnamic acids were converted in vivo into other products, and (u) experiments in which the accumulation of cinnamic acid derivatives was prevented by in-vivo inhibition of PAL activity. 

Several types of data based on feeding experiments, isolation of intermediates and in vivo NMR experiments, indicate that norcoclaurine (23) serves as a precursor to both coclaurine (24) and (S)-reticuline (20) (Stadler et al., 1987, 1989), and that (5)-coclaurine (24) serves as a specific precursor to other classes of benzylisoquinoline alkaloids, such as the protoberberines, benzophenanthridines, and morphinandie-nones, as well as for pavine and benzophenanthridine alkaloids in intact plants (Stadler et al., 1987, 1989). The five enzymes involved in the conversion of dopamine and 4-hydroxyphenylacetaldehyde (19) to (S)-reticuline (20) have been elucidated (Frenzel and Zenk, 1990b). 

C-, 0-, and A -methylations are encountered frequently in the biosynthesis of secondary metabolites. All appear to involve nucleophilic substitution on the 5-methyl group of 5-adenosyl-L-methionine (7.59) (Scheme 1.12). In biosynthetic feeding experiments it is normal to use [m / /- C]methionine [as (7.55)] as a precursor for methyl groups reaction in vivo with ATP and Mg affords (7.59). Particu-... 

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. 

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