Protopine alkaloids precursors

In addition to the pathway leading to the protoberberines, a second one leading to the protopine alkaloids has now been elucidated at the enzyme level utilizing plant cell cultures as the enzyme source. Since the protopines are regarded as the biosynthetic precursors of the benzazepine and benzophenanthri-dine alkaloids (refs. 36-39) these now can be the target of further enzymatic investigations. 

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 protoberberine alkaloids (5-75) play important roles as precursors in the biosynthesis of a variety of related isoquinoline alkaloids such as protopine, phthalideisoquinoline, spirobenzylisoquinoline, rhoeadine, inde-nobenzazepine, secoberbine, and benzo[c]phenanthridine alkaloids. Chemical transformations of protoberberines to these alkaloids are particularly interesting and exciting from the biogenetic viewpoint and further from ready availability of starting protoberberines in nature or synthesis. 

The probability63 that (69) lies along the pathway to protoberberine and derived alkaloids, between scoulerine (65) and stylopine (70), has been supported by the observation that tritiated (69) is a precursor for protopine (73), and also for corynoline (78).64 Evidence previously obtained for the intermediacy of the metho-salt of stylopine [as (71)] in the biosynthesis of chelidonine (62)63 and protopine (73)63,65 has been affirmed, and it is apparently the a-form (71) and not the /3-form that is involved. [The authors are mistaken in assuming that ( —)-stylopine has the R-configuration at C-14 cf. ref. 63]. 

Another important route stems from the formation of the N-methylated moieties of the (S)-tetrahydroprotoberberines, which serve as precursors for the protopine, benzophenanthridine, tetrahydrobenzazepines (rhoeadines) and spirobenzylisoquinoline alkaloids (Kutchan and Zenk, 1993 Liscombe and Facchini, 2008 Ziegler and Facchini, 2008) (Fig. 2.7). 

P450 enzyme, protopine 6-hydroxylase, to yield an intermediate that rearranges to dihydrosanguinarine (40). This intermediate also serves as the precursor to the benzo(c)phenanthridine alkaloid macarpine (Fig. la). The copper-dependent oxidase dihy-drobenzophenanthridine oxidase, which has been purified (41, 42), then catalyzes the formation of sanguinarine from dihydrosanguinarine. 

Additional results are that (-)-(5)-scoulerine (68) is a precursor for san-guinarine (77) and chelerythrine (78) and labelled (-)-(5)-stylopine (71) is incorporated into coptisine (72). [N-methyl8- H]Stylopine methochloride [as (70)] afforded chelidonine (76) and protopine (73) essentially without change in isotope ratio and must thus be considered as implicated on the pathway to both alkaloids. (The same conclusion for protopine has been reached independently. ) On the other hand radioactive nandinine (79) failed to label chelidonine, stylopine, or protopine, suggesting that the alternative (69) may be an intermediate instead. In agreement with other work (5)-reticuline (67) and (5)-scoulerine (68) were much better precursors than the i -isomers for protopine (73), and incorporation of (68) resulted in complete loss of tritium label from C-14. Allocryptopine (81) was well labelled by radioactive isocorypalmine (82) (which also serves as a precursor for narcotine see below). The combined results lead to the pathways shown in Scheme 3. 

Opitim contains 10-25% alkaloids, and the main constituent is morphine. Other than morphine, more than 25 alkaloids are known, and among them are other morphinan-type alkaloids (codeine and thebaine), benzyl-isoquinoline-type alkaloids (papaverine and noscapine), and protopine-type alkaloids (protopine). The biosynthetic precursor of all of these alkaloids is phenylalanine. 

Berberine or protoberberine alkaloids [such as stylopine (79)] may be converted into alkaloids of the protopine type (60) (Fig. 32.24) (Geissman and Crout, 1969 Hartmann, 1991). Highly specific microsomal cytochrome P-450-de-pendent enzymes from cells of Eschscholtzia californica (and other species of the Papaveraceae and Fumariaceae) introduce two methylenedioxy bridges into (5)-scoulerine (72) to form (5)-stylopine (79), which is, in turn, an important precursor of the protopine, phthalideisoquinoline, and benzophenanthridine groups (Hartmann, 1991). 

Reticuline is a precursor of sinomenine, via sinocutine, in Sino-menium acutum (115). It is also a precursor of berberine (LXI) in Hydrastis canadensis L. and of protopine (LXII) in Dicentra spectabilis (L.) Lem. (116). In the case of the former base the i T-methyl group of reticuline is used to form the berberine carbon (carbon atom 8) of the tetracyclic alkaloid and with the latter base it is found as the same carbon atom in the heterocyclic ring of protopine. By feeding multiple-labeled (+ )-reticuline, as indicated in LXIII, to Chelidonium majus L., it was shown that it is a precursor of chelidonine (LXIV), (— )-stylopine (LXV), and protopine (LXII), and that the labeled atoms are found in the places expected from current biogenetic theories (117). 

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