Morphinans biosynthesis

There would appear, from Scheme 4, to be many opportunities to modify the genetic material that creates the enzymes responsible for the biosynthesis of the morphinans. Blocking the specific methylation of norlaudanosine to (—) retuciline would be one worthwhile target, perhaps coupled with promoting the aromatization and methylation sequences leading from norlaudanosine to papaverine. 

S)-Reticuline is a branch-point intermediate in the biosynthesis of most BAs. Most work has focused on branch pathways leading to the benzophenanthridine (e.g., sanguinarine), protoberberine (e.g., berberine), and morphinan (e.g., morphine and codeine) alkaloids.19 Most enzymes involved have been isolated, many have been purified, and four corresponding cDNAs have been cloned.19 The first committed step in benzophenanthridine and protoberberine alkaloid biosynthesis involves the conversion of (S)-reticuline to (5)-scoulerine by the berberine bridge enzyme (BBE) (Fig.7.2). BBE was purified from Berberis beaniana,20 corresponding cDNAs were cloned from E. californica and B. stolonifera,21 22 and BBE genes have been isolated from P. somniferum and E. californica.23,24... 

Conversion of (S)-reticuline to its ( )-epimer is the first committed step in morphinan alkaloid biosynthesis in certain species. 1,2-Dehydroreticuline reductase catalyzes the stereospecific reduction of 1,2-dehydroreticuline to (7 )-reticuline.39 Intramolecular carbon-carbon phenol coupling of (if)-reticuline by the P450-dependent enzyme salutaridine synthase (STS) results in the formation of salutaridine.40 The cytosolic enzyme, salutaridine NADPH 7-oxidoreductase (SOR), found in Papaver bracteatum and P. somniferum, reduces salutaridine to (7S)-salutaridinol.41 Conversion of (7S)-salutaridinol into thebaine requires closure of an oxide bridge between C-4 and C-5 by acetyl coenzyme A salutaridinol-7-0-acetyltransferase (SAT). The enzyme was purified from opium poppy cultures and the corresponding gene recently isolated (Fig.7.2).42,43 In the last steps of morphine... 

S)-Reticuline is also the precursor for the biosynthesis of benzophenanthri-dine (e.g. sanguinarine, marcarpine), protoberberine, berberine, palmatine) and morphinan alkaloids (morphine, codeine) (see next few paragraphs). 

The role of reticuline as an intermediate in the biosynthesis of the mor-phinan alkaloids (Fig. 2.8) was demonstrated by the isolation both of (S)-and (f )-reticuline from the opium poppy. An excess of the (S)-reticuline over the (f )-isomer was found in opium (poppy latex) obtained from the mature plant, in contrast to the roughly equal amounts of these two isomers that occur in poppy seedlings. Both isomers were found to be incorporated into morphine, the major alkaloid isolated from opium, although incorporation of the (f )-isomer was slightly more efficient. (f )-Reticuline is firmly established in P. somniferum as the precursor of the morphinan-type alkaloids (Loefer and Zenk, 1990). (S)-Reticuline, however, is the central intermediate in isoquinoline alkaloid biosynthesis. It has been postulated that (R)-reticuline is formed from (S)-reticuline by isomerization. This inversion of configuration can be explained by the intermediate formation of the 1,2-dehydroreticulinium ion originating from (S)-reticuline, followed by stereospecific reduction to yield the (R) counterpart. The 1,2-dehydroreticulinium ion is efficiently incorporated into opium alkaloids and its role as a precursor of the morphinan-t)q)e alkaloids has been unequivocally established (De-Eknamkul and Zenk, 1990, 1992). 

The cytochrome P450 responsible for the oxidation of (S)-N-methylcoclaurine to (S)-3 -hydroxy-N-methylcocluarine has been overexpressed in opium poppy plants, and morphinan alkaloid production in the latex is increased subsequently to 4.5 times the level in wild-type plants (58). Additionally, suppression of this enzyme resulted in a decrease in morphinan alkaloids to 16% of the wild-type level. Notably, analysis of a variety of biosynthetic gene transcript levels in these experiments supports the hypothesis that this P450 enzyme plays a regulatory role in the biosynthesis of benzylisoquinoline alkaloids. Collectively, these studies highlight that the complex metabolic networks found in plants are not redirected easily or predictably in all cases. 

Many researchers have so far investigated tissue culture of P. somniferum [131, ref. cited therein], and most cultured P. somniferum cells, either as callus or cell suspensions, readily produced sanguinarine and its analogs [130-137], but rarely, if even, produce morphinan alkaloids [138]. Kamo et al. [139], Schuchmann and Wellmann [134], and Yoshikawa and Furuya [140] reported the production of morphinan alkaloids in redifferentiated organs, either shoots or somatic embryos, and their results emphasize the importance of the degree of cell differentiation for the biosynthesis of morphinan alkaloids. 

Similar to proaporphine bases which are intermediates in the biosynthesis of the aporphine alkaloids, the promorphinanes, having a cyclohexadienone ring D, form an intermediate in the biosynthesis of the morphinane alkaloids. They also arise from benzyltetrahydroisoquino-line bases by phenolic oxidation. The first known alkaloid of this group was salutaridine (36) (first isolated from Groton salwtaris) (see Scheme 3) which in all probability is identical with the already known floripavine... 

The group of morphinane alkaloids has already been discussed in detail in previous Volumes of The Alkaloids (Vol. II, Chapter 8-1 Vol. II, Chapter 8-II Vol. VI, Chapter 7). Furthermore, there appeared a monograph 62) and two reviews 134, 182) on this problem, and the chemistry of the stereoisomeric sinomenine was dealt with in a book 188). Therefore, in this chapter only a tabulation (Table VIII) of the known bases and the biosynthesis of the morphinane alkaloids in the genus Papaver are given. 

The biosynthesis of cularine alkaloids (239) by phenolic oxidation also proceeds via the dienone compound (Scheme 4) similar to the aporphine or morphinane alkaloids. 

Although in P. somniferum and Sinomenium acutum the biosynthesis of the morphinane bases can be carried out with (+)-reticuline as starting material, these bases and also protopine (101a) cannot be obtained biosyn-thetically in satisfactory yield from (+)-tembetarine chloride (reticuline methochloride). Similar results were obtained with D. spectabilis (683). Important intermediates of the biosynthesis between tetrahydroprotober-berine and protopine alkaloids are the compounds of the dihydroprotopine type (102), which were prepared in high yield by partial synthesis with cyanogen bromide (von Braun method) (684). The biosynthesis of pro topine alkaloids is also discussed in Section III,N. 

Fig. 4. Biosynthesis of the morphinan alkaloids thebaine (192), codeine (198), and morphine (204).

Morphinan Alkaloids.—Extensive research on the biosynthesis of morphine (51) and related alkaloids in Papaver somniferum has allowed a detailed description of the pathway from the amino-acid tyrosine through reticuline (44), thebaine (46), and codeine (50) to morphine (51) (Scheme The incorporation of (R)-... 

The conversion of reticuline (44) into morphinan alkaloids, which occurs with loss of tritium from C-1 in P. somniferum (see above)," has been observed also for the formation of thebaine (46) in P. bracteatum, a plant which produces this alkaloid but not codeine or morphine. Radioactive 1,2-dehydroreticuline (47) labelled both reticuline (44) and thebaine (46), whilst radioactive reticuline again labelled thebaine (46). ° Codeinone (48) and codeine (50) are biosynthetic intermediates between thebaine (46) and morphine (51) in P. somniferum, and it was shown that (48) was efficiently reduced to (50) in P. bracteatumf It is apparent that alkaloid biosynthesis in the two plants is similar, with the important difference that in P. bracteatum the enzymes which effect demethylation of (46) are missing, and so biosynthesis goes no further than thebaine (46). 

Despite the detection of COR and other known morphinan branch pathway enzymes in opium poppy cell cultures (63-65,67), morphine and codeine are not produced. The inability of cultured cells to accumulate morphine might reflect the absence of other relevant enzymes or structural proteins that preclude the formation of a requisite metabolic channel. Nevertheless, the efficient operation of the sang-uinarine pathway indicates that cultured opium poppy cells possess the essential regulatory, eellular, and metabolic components to support the biosynthesis and... 

As described in the previous section, morphinan alkaloids are produced by the para, ort/zo -coupling of a biradical derived from (S)-reticuline. During biosynthesis, (S)-reticuline is transformed into (i .)-reticuline via a 1,2-dehy-droderivative, which is then used in the formation of the morphinan alkaloids. 

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