Reticuline biosynthesis

The localization of isoquinoline biosynthesis has been investigated at the cellular level in intact poppy plants by using in situ RNA hybridization and immunoflouresence microscopy. The localization of 4 -OMT (reticuline biosynthesis), berberine bridge enzyme (saguinarine biosynthesis), salutaridinol acetyltransferase (morphine biosynthesis), and codeinone reductase (morphine biosynthesis) has been probed. 4 -OMT and salutaridinol acetyltransferase are localized to parenchyma cells, whereas codeinone reductase is localized to laticifer cells in... 

R. Stadler, T.M. Kutchan, S. Loeffler, N. Nagakura, B. Cassels, and M.H. Zenk, Revision of the early steps of reticuline biosynthesis. Tetrahedron Lett. 28 (1987), 1251-1254. 

These results indicate that CjPRlOA is more suitable than CjNCSl for microbial production systems. CjPRlOA has been used as an NCS enzyme in reticuline biosynthesis. 

Stadler R et al (1987) Revision of the early steps of reticuline biosynthesis. Tetrahedron Lett 28 1251-1254... 

The bacterium E. coli and yeast S. cerevisiae have been recently explored as production hosts of plant alkaloids [15, 101, 102]. In both cases, the metabolic engineering efforts in microorganisms entailed the reconstmction of the plant biosynthetic pathways (Fig. 8.12) [101]. To assemble an artificial pathway to achieve (S)-reticuline biosynthesis in E. coli. Micrococcus luteus monoamine oxidase (MAO) was introduced together with Coptis japonica norcoclaurine... 

Reticuline (38), one of the most important intermediates in the biosynthesis of opium alkaloids, has been synthesized in racemic form (Scheme 7) (78). 6-Methoxy-7-benzyloxyisoquinoline (39), prepared from O-benzylisovanillin via a modified Pomeranz-Fritsch isoquinoline synthesis, was treated with benzoyl chloride and potassium cyanide to obtain Reissert compound 40. Alkylation of the anion generated from 40 with 3-benzyloxy-4-methoxybenzyl chloride gave the corresponding 1-substituted Reissert compound 41 which was hydrolyzed in alkaline medium to 1-benzylisoquinoline derivative 42. Quatemarization of 42 with methyl iodide followed by sodium borohydride reduction and debenzylation led to ( )-reticuline (38) in about 25% overall yield from 39. 

The biosynthesis of pavines from (-t-)-reticuline (158) through Route A has also been agreed upon by Barker and Battersby (7/5). They additionally suggested an equilibrium between the iminium ion 160 and the carbinolamine 162 (Scheme 34). 

In order to understand the continuation of the biosynthesis of codeine and morphine from reticuline, the structure for (S)-reticuline can be written as follows ... 

Perhaps the simplest explanation at this stage for the results obtained with dopa is that material which is fed to plants is poorly transported to the appropriate transaminating enzyme that is normally involved in alkaloid biosynthesis. The early pathway to benzylisoquinoline alkaloids that has so far been deduced is illustrated in Scheme 6. The compound (65) is not involved in the biosynthesis of reticuline. For details of the later stages in the biosynthesis of (66)—(71), see refs. 1 and 2. 

The benzylisoquinoline alkaloids are widely distributed in nature and are intermediates in the biosynthesis of alkaloids of this family (2, 3). It is not surprising therefore that several groups (6, 7, 15, 23) have examined their spectra. Among the alkaloids that have been studied are reticuline (26) (7), norlaudanosine (27) (7), laudanosine (28) (6, 15), and the cis- and trans-N-oxides of laudanosine, 29 and 30, respectively (7). The chemical shifts of laudanosine are recorded in Table IV and the structures of the alkaloids may be found in Fig. 4. 

This observation was explained by the assumption that a portion of the protoberberine was formed via norreticuline (96) present in the same incubation mixture and derived from enzymic demethylation of reticuline. Reaction of 96 with an unlabeled one-carbon fragment and subsequent ring closure would then lead to C-8 unlabeled protoberberines. The authors suggest that this one-carbon fragment may be derived from S-adenosyl-methionine, and that the product of its combination with 96 may be converted directly to 91 or 94 without the intermediacy of free reticuline (99). If their assumption is correct, the conversion of norreticuline to the protoberberine alkaloids may not involve the formation of reticuline itself, a suggestion that is at variance with the known intermediacy of reticuline in the biosynthesis of alkaloids of this group. 

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. 

Papaverine.—The biosynthesis of the simple benzylisoquinoline papaverine (89) is known to proceed via nor-reticuline (48) and tetrahydropapaverine (88).71 Dehydrogenation of the latter affords papaverine, and examination of the stereochemistry of the processes involved has led to the conclusion72 that loss of the proton at C-3 [in nor-reticuline (48)] is stereospecific (loss of the pro-S hydrogen atom) but removal of the C-4 proton is essentially non-stereospecific. These observations are perhaps best explained if enzyme-catalysed oxidation of (88) occurs to give (90), subsequent non-stereospecific imine-enamine isomerization occurring without enzyme participation to give (91). A further amine to imine oxidation then occurs to give papaverine (89).72... 

The stereochemical removal of hydrogen from positions 3 and 4 of the isoquinoline ring during aromatization of papaverine during biosynthesis has been studied by incorporation experiments, using stereospecifically labelled [3H]nor-reticulines.24... 

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... 

The biosynthesis of magnoflorine (47) and laurifoline (53) has been studied, and the specific incorporation [of ( )-nor-reticuline (54)1 and (+)-(5 )-reticuline (55) has been demonstrated.66 This work is described in greater detail elsewhere in this volume. 

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... 

The systemic transport of pathway intermediates or end-products might also occur in BA biosynthesis. Several BA biosynthetic enzymes are preferentially active in certain organs. Transcripts encoding various TYDC isoforms were most abundant in roots, or roots and stems.3 NCS was also most active in roots and stems of opium poppy,112 whereas CYP80B1 transcripts were most abundant in stems followed by roots, leaves, and floral tissues.148 STS and SOR, which convert (R)-reticuline to salutaridinol, exhibited abundant activity in roots and shoots.40,41 In contrast, COR... 

Several BA biosynthetic enzymes also occur in subcellular locations other than the cytosol. Of the five non-cytosolic enzymes involved in the conversion of (5)-reticuline to dihydrosanguinarine, four - BBE, CFS, SPS, and MSH - were localized to a membrane fraction with a unique density (8 = 1.14 g mL 1),25 26,29 38 while one (PPH) was associated with a membrane fraction of the same density as the ER (8 = 1.11 g mL 1).155 STS, which is involved in morphine biosynthesis, was also localized to a microsomal fraction with a density (8 = 1.14 g mL 1) higher than expected for the ER.40 With the exception of BBE, all of these non-cytosolic enzymes are P450-dependent and, as such, must be integral proteins of the ER, or ER-derived compartments.110... 

Other important mammalian benzylisoquinolines which thus far have escaped detection in mammals are (5)-reticuline and its (R) enantiomer TIQ 121b, shown in Fig. 29. The latter compound is required for the biosynthesis of morphinandienones in the opium poppy, and its possible role in biosynthesis of mammalian morphine is discussed in Section IV. 

Is (/ )-reticuline, which is required for the synthesis of morphine in plants (272,273) and formed from the (5 )-enantiomer in benzyliso-quinoline-producing plants (274) by an oxidation-reduction sequence (257), also the crucial intermediate in the biosynthesis of mammalian morphine Confirmation that it is must await its detection. Conversion of... 

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). 

Figure 2.6 Biosynthesis of berberine from (5)-reticuline in Berberis species and Coptis japonica. BBE, berberine bridge enzyme SCMT, (5)-scoulerine-9-0-methyltransferase CAS, (5)-canadine synthase STOX, tetrahydroberberine oxidase COX, enzyme found in Coptis japonica.

Figure 2.8 Biosynthesis of morphine via the conversion of (S)-reticuline to (R)-reticuline, salutaridine and thebaine. SalS, salutaridine synthase SaIR, salutaridine reductase THS, thebaine synthase COR, codeinone reductase.

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). 

Stadler, R. and Zenk, M.H. (1990) A revision of the generally accepted pathway for the biosynthesis of tetrahydroisoquinoline alkaloid reticuline. Liebigs Ann. Chem., 555-62. 

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