Reticuline feeding experiments

Feeding experiments on Thalictrum minus with ( )-[l-14C]-reticuline (444) and with [8 -3H]-thalicarpine (527) showed that both are incorporated into adiantifoline (233) and thalmelatidine (528), supporting the biosynthetic sequence reticuline to thalmelatine to thalicarpine to adiantifoline, thalmelatidine (607). 

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... 

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... 

Corydaline and Ochotensimine.—It has been widely presumed that the alkaloids corydaline (98) and ochotensimine (99) are further structural variants of the ben-zylisoquinoline skeleton cf. refs. 112 and 113). A firm indication that this view is correct for corydaline came from the incorporation of labelled reticuline [as (82)] into the alkaloid.Further evidence for such a route to corydaline and the first evidence on the biosynthesis of ochotensimine (99) have been obtained from feeding experiments with labelled tyrosine and methionine in Corydalis species. 

Analyses of P. orientate from natural habitats showed that the dried latex contained 20% of oripavine (195) and 9% of thebaine (192) (92). Traces of isothebaine (122) were found in plants collected from Khaikhala in northwest Iran. Six different chemotypes of P. orientate were reported (64,93), namely, oripavine, oripavine-thebaine, oripavine-isothebaine, oripavine-aipinigenine, oripavine-thebaine-alpinigenine, and mecambridine-orientalidine-salutari-dine. In all of them, with the exception of one, oripavine was the major alkaloid (82). Feeding experiments in P. orientate with radioactively labeled reticuline and thebaine have demonstrated that oripavine is derived from reticuline and thebaine (93a). Reticuline undergoes racemization in this plant as has been earlier shown for P. somniferum and P. bracteatum. 

The configuration of (76) at C-1 is (5), indicating its derivation from (5)-reticuline. This was confirmed in a feeding experiment with the enantiomers of nor-reticuline (80). 

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). 

The benzophenanthridine skeleton is encountered in approximately 30 alkaloids, principally of the family Papaveraceae (Cordell, 1978a). In contrast to the biosynthesis of protopine alkaloids, phenanthridine alkaloids are synthesized in the cytoplasm (Hartmann, 1991). This type of system arises from a protoberberine precursor by fission of the C-6-N bond and recyclization. The biogenetic sequence leading to chelidonine (80) biosynthesis in Chelidonium majus has been supported by feeding experiments with multiply-labeled (-t-)-reticuline [(5)-reticuline] (20) and with labeled stylopine (79) (Fig. 32.25) (Hutchinson, 1986 Sim ek, 1985 Tanahashi and Zenk, 1988). (5)-Z-V-Methylstylopine and protopine (60) have been shown to be metabolites in this pathway. Reticuline is oxidatively cyclized to ( —)-scoulerine (72). Formation of two methylenedioxy groups results in the formation of stylopine (79) (Hartmann, 1991). 

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). 

Feeding of + )-aryl-3H-reticuline (absolute configuration as in XCVII) and ( —)-aryl-3H-reticuline to H. canadensis in separate experiments resulted in the former being incorporated into berberine fifteen times more efficiently than the (— )-isomer. Corroboration that (+)-reticuline is the true precursor was obtained from the results of administering a mixture of (— )-aryl-3H-reticuline and ( + )-reticuline-3-i4C to H. canadensis. 

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