Papaver somniferum alkaloid biosynthesis

In the laboratory and associated greenhouse, we gave particular attention to the biosynthesis of the morphine alkaloids.12"14 At first we purchased Papaver somniferum seeds and grew splendid poppies, but they contained no morphine... 

Labelled reticuline was readily incorporated into thebaine 14, codeinone 15a, codeine 15b, and morphine 16 without scrambling of the labels. Salutaridine is not present in detectable quantities in Papaver somniferum. However, when appropriately labelled, it is well incorporated into morphine alkaloids. Salutaridine can be readily reduced to two stereo-isomeric allylic alcohols 17, both of which are converted by mild acid catalysis (17, see arrows) to give thebaine 14. The alkaloids 17, 14, 15a, and 15b were all shown to be precursors of morphine. This was of interest, because the earlier theory of Robinson suggested that unmethylated alkaloids were first assembled and methylation was a terminal stage of biosynthesis. 

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... 

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... 

Figure 7.9 Intercellular and subcellular trafficking in alkaloid biosynthesis. A. Tropane alkaloid biosynthesis in Hyoscyamus muticus. B. Terpenoid indole alkaloid biosynthesis in Catharanthus roseus. C. Trafficking of the berberine bridge enzyme in Papaver somniferum cell cultures.

The benzylisoquinolines are formed from two molecules of fhe aromafic amino acid, tyrosine. In the past ten years, this pathway has been probed at the enzyme and gene level. The recent linking of the phloem-specific expression of tyrosine/Dopa decarboxylase (TYDC) genes with the bios)mthesis of the isoquinoline alkaloids in the opium poppy, Papaver somniferum (Facchini and De Luca, 1994, 1995, 2008 Liscombe and Facchini, 2008), and the association with alkaloid accumulation as part of the plant defence mechanism (Wink, 1993 Facchini et al, 1996) are of particular interest in furthering our knowledge of the location of alkaloid biosynthesis. 

The isoquinoline alkaloids include the analgesics morphine and codeine as well as the antibiotic berberine (Fig. la). Morphine and codeine are two of the most important analgesics used in medicine, and plants remain the main commercial source of the alkaloids (8). Development of plant cell cultures of Eschscholzia califomica, Papaver somniferum, and Coptis japonica has aided in the isolation and cloning of many enzymes involved in the biosynthesis of isoquinoline alkaloids (9). 

Ounaroon A, Decker G, Schmidt J, Lottspeich F, Kutchan TM. (R,S)-reticuline 7-0-methyltransferase and (R,S)- norcoclaurine 6-O-methyltransferase of Papaver somniferum - cDNA cloning and characterization of methyl transfer enzymes of alkaloid biosynthesis in opium poppy. Plant J. 2003 36 808-819. 

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)-... 

Pathak S, Mishra BK, Misra P, Misra P, Jodii VK, Shulda S, et al. High frequency somatic embryogenesis, regeneration and correlation of alkaloid biosynthesis with gene expression in Papaver somniferum. Plant Growth Regul 2012 68(l) 17-25. 

Fig. 2. Alkaloid biosynthetic pathways are associated with a diverse variety of cell types. The tissue-specific localization (shaded) of enzymes and/or gene transcripts are depicted for the biosynthesis of tropane alkaloids in Atropa belladonna and Hyoscyamus niger roots (A), monoterpenoid indole alkaloids in Catharanthus roseus leaves (B), pyrrolizidine alkaloids in Senecio vernalis roots (C), pyrrolizidine alkaloids in Eupatorium cannabinum roots (D), benzyl-isoquinoline alkaloids in Papaver somniferum vascular bundles (E), and protoberberine alkaloids in Thalictrwn flamtm roots (F).

Several studies have reported the production in microorganisms of plant enzymes that are involved in alkaloid biosynthesis [29, 99-102], In this sense, Unterlinner et al. [100] have cloned and expressed in Escherichia coli four cDNAs encoding for different isoforms of the Codeinone reductase NADPH-dependent enzyme isolated from Papaver somniferum. In this report has been investigated the substrate specificity of the enzyme and the structural analysis, being the first report about the cloning and expression of genes of the biosynthetic pathway of morphine [90],... 

The opium poppy (Papaver somniferum L. [Papaveraceae]) latex contains benzylisoquinoline alkaloids and is a widely known source of the analgesic drugs morphine and codeine. The biosynthesis of benzylisoquinoline alkaloids begins with a condensation reaction catalyzed by norcoclaurine synthase of DA [161]. The structural features of benzylisoquinoline alkaloids derived from DA might provide some explanation for the documented affinity of some natural alkaloids of this class, and some synthetic derivatives, for DA receptors [162]. Indeed, the chemical structure of morphine has been used as a template for the development of the PD drug, apomorphine (47). Apomorphine includes a catecholaminergic moiety in its... 

Having looked at the biosynthesis of pyridoxal 5 -phosphate in the previous section, let s now go up a level in complexity by looking at morphine biosynthesis. Morphine, perhaps the oldest and best known of all alkaloids, is obtained from the opium poppy, Papaver somniferum, which has been cultivated for more than 6000 years. Medical uses of the poppy have been known since the early 1500s, when crude extracts, called opium, were used for the relief of pain. Morphine was the first pure compound to be isolated from opium, but its close relative codeine also occurs naturally. Codeine, which is simply the methyl ether of morphine and is converted to morphine in the... 

Brochmann-Hanssen E, Cheng CY (1984) Biosynthesis of a narcotic antagonist. Conversion of N-allylnorreticuline to N-allylnormorphine in Papaver somniferum. J Nat Prod 47 175-176 Brochmann-Hanssen E, Furuya T (1964) A new opium alkaloid. Isolation and characterization of ( )-l-(3 -hydroxy-4 -methoxybenzyl)-2-methyl-6-methoxy-7-hydroxy-l,2,3,4-tetrahydro-isoquinoline [( )-reticuline]. Planta Med 12 328-333 Brochmann-Hanssen E, Nielsen B (1965) (+)-Reticuhne - a new opium alkaloid. Tetrahedron Lett 1271-1274... 

Brochmann-Hanssen E, Okamoto Y (1980) Biosynthesis of opium alkaloids. Substrate specificity and aberrant biosynthesis. Attempted detection of oripavine in Papaver somniferum. JNat Prod 43 731-735... 

Brochmann-Hanssen E, Chen CH, Chen CR, Chiang HC (1975) Opium alkaloids, part XVI. The biosynthesis of 1-benzylisoquinolines in Papaver somniferum. Preferred and secondary pathways stereochemical aspects. J Chem Soc Perkin Trans 1 1931-1937 Brochmann-Hanssen E, Cheng CY, Chiang HC (1982) Biosynthesis of opium alkaloids The effects of structural modification of reticuline on racemization and bio transformation. J Nat Prod 45 629-634... 

Horn JS, Paul AG, Rapoport H (1978) Biosynthetic conversion of thebaine to codeinone. Mechanism of ketone formation from enol ether in vivo. J Am Chem Soc 100 1895-1898 Kametani T, Ihara M, Honda T (1970) The alkaloids of Corydalis pallida var. tenuis (Yatabe) and the structures of pallidine and kikemanine. J Chem Soc (C) 1060—1064 Kirby GW, Massey SR, Steinreich P (1972) Biosynthesis of unnatural morphine derivatives in Papaver somniferum. J Chem Soc Perkin Trans 1 1642-1647 Kleinschmidt G, Mothes K (1959) Physiology and biosynthesis of alkaloids in Papaver somniferum. Z Naturforsch 14b 52-56... 

Rapoport H, Stermitz FR, Baker DR (1960) The biosynthesis of opium alkaloids. I. The interrelationship among morphine, codeine and thebaine. J Am Chem Soc 82 2765-2772 Stermitz FR, Rapoport H (1961) The biosynthesis of opium alkaloids. Alkaloid interconversion in Papaver somniferum andP. orientale. J Am Chem Soc 83 4045-4050 Stuart KL, Teetz V, Franck B (1969) Alkaloid biosynthesis in Croton flavens. Chem Commun 333... 

Bohm H, Franke J (1982) Accumulation and excretion of alkaloids by Macleaya microcarpa cell cultures. I. Experiments on solid medium. Biochem Physiol Pflanz 177 345-356 Bohm H, Olesch B, Schultze CH (1972) Further investigations on the biosynthesis of alkaloids in isolated latex from opium poppy, Papaver somniferum L. Biochem Physiol Pflanz 163 126-136... 

Aberrant Biosynthesis of Unnatural Alkaloids.—Kirby S has demonstrated that the opium poppy Papaver somniferum) is capable of carrying out transformations of unnatural precursors to unnatural alkaloids. The 0natural conversion of codeine into morphine by feeding to the plant a mixture of a [2- H]codeine derivative and [N-methyl- C]codeine. One unnatural transformation which was more efficient than the natural one was the conversion of dihydrodeoxycoddne (10) into di-hydrodeoxymorphine (11) (Scheme 4). Kirby concluded that neither the 6-hydroxy-group nor the 7,8-double bond in codeine is important for binding to the enzyme responsible for demethylation of the 3-methoxy-group of codeine. 

With a morphine biosynthetic gene in hand, we believed we could begin to address the question why only P. somniferum produces morphine, while other Papaver species such as P. rhoeas, P. orientale, and P. bracteatum do not. Unexpectedly, we found that the codeinone reductase transcript was present to some degree in all four species investigated. A review of the literature revealed no alkaloids reported in P. rhoeas for which codeinone reductase should participate in the synthesis. Similarly, P. orientale accumulates the alternate morphine biosynthetic precursor oripavine, but codeinone reductase is not involved in the biosynthesis of oripavine, acting instead after this alkaloid along the biosynthetic pathway to morphine.22 P. bracteatum produces the morphine precursor thebaine as a major alkaloid. As for oripavine in P. orientale, codeinone reductase would act in P. bracteatum after thebaine formation on the pathway to morphine. It appears, therefore, that the reason that P. rhoeas, P. orientale, and P. bracteatum do not produce morphine is not related to the absence of the transcript of the morphine biosynthesis-specific gene codeinone reductase. The expression of codeinone reductase may simply be an evolutionary remnant in these species. 

---