Morphinan alkaloids biosynthesis

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... 

Whether the laticifer cell is the site of morphinan alkaloid biosynthesis remains controversial. Fairbairn and coworkers (Fairbairn et al. 1968, Fairbairn and Steele 1981) have consistently demonstrated that latex isolated from P. somnifenim transformed labeled tyrosine and L-dopa to morphine. In addition, P. orientale latex exhibited biosynthetic capacity extending over several early steps in the alkaloid biogene-tic pathway (Wilson and Coscia 1975). Enzymes that can rapidly convert tyrosine to dopamine were also detected in P. somnifenim latex (Roberts 1974, Roberts and Antoun 1978). Nevertheless, arguments have been put forward that latex biosynthetic activity is relatively low (Bohm et al. 1972) and most synthesis occurs in cells sur-... 

The biosynthesis of the various isoquinolines is reviewed in two chapters, one devoted solely to the morphinans and the other to the other skeletal types. Attempts to produce morphinan alkaloids by plant cell culture techniques has produced a flurry of activity in several laboratories throu out the world. Four chapters are concerned with plant cell culture techniques for the production of isoquinolines. A series of enzymes which catalyse specific steps in isoquinoline alkaloid biosynthesis have been isolated in recent years and the stage is now set for the isolation and characterization of the enzymes responsible for morphinan alkaloid biosynthesis. The utilization of these enzymes for biotechnological conversions is now envisaged. 

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... 

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. 

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). 

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. 

Enzymes of a secondary biosynthesis are usually localized in different cell types and different subcellular compartments. For instance, three cell types are involved in the biosynthesis of morphinane alkaloids in Papaver, a similar diversity of distribution holds true for the enzymes of terpenoid indole alkaloids in Catharanthus (Facchini... 

Allen RS, Miller JAC, Chitty JA, et al. (2008) Metabolic engineering of morphinan alkaloids by over-expression and RNAi suppression of salutaridinol7-0-acetyltransferase in opium poppy. Plant Biotech J 6 22-30 Allen RS, Millgate AG, Chitty JA, et al. (2004) RNAi-mediated replacement of morphine with the noimarcotic alkaloid reticuline in opium poppy. Nature Biotech 22 1559-1568 AmaimM, Wanner G,ZenkMH (1986) Intracellular compartmentation of two enzymes of berberine biosynthesis in plant cell cultures. Planta 167 310-320... 

Hodges CC, Horn JS, Rapoport H (1977) Morphinan alkaloids in Papaver bracteatum Biosynthesis and fate. Phytochemistry 16 1939-1942... 

In poppy plants morphinan alkaloids are primarily accumulated in latex which in turn is contained in structurally and physiologically specialized cells, laticifers. Roberts et al. (1983 and references therein) demonstrated that 1000 g and supernatant fractions of the latex are required for alkaloid biosynthesis and that a subpopulation of dense organelles of the 1000 g sediment have a function as a storage compartment for alkaloids. Morphinan alkaloid production with poppy cell cultures, therefore, would appear to require the concurrent differentiation of latex vessels. The high levels of alkaloid accumulation would thus appear to be intriguingly correlated with the formation of latex and latex vessels. 

---