(R)-Reticuline

A Regio- and Diastereoselective Anodic Aryl-Aryl Coupling in the Biomimetic Total Synthesis of (-)-Thebaine.[Pubmed:29786941]

Angew Chem Int Ed Engl. 2018 Aug 20;57(34):11055-11059.

The biosynthesis of thebaine is based on the regioselective, intramolecular, oxidative coupling of (R)-Reticuline. For decades, chemists have sought to mimic this coupling by using stoichiometric oxidants. However, all approaches to date have suffered from low yields or the formation of undesired regioisomers. Electrochemistry would represent a sustainable alternative in this respect but all attempts to accomplish an electrochemical synthesis of thebaine have failed so far. Herein, a regio- and diastereoselective anodic coupling of 3',4',5'-trioxygenated laudanosine derivatives is presented, which finally enables electrochemical access to (-)-thebaine.

Total biosynthesis of opiates by stepwise fermentation using engineered Escherichia coli.[Pubmed:26847395]

Nat Commun. 2016 Feb 5;7:10390.

Opiates such as morphine and codeine are mainly obtained by extraction from opium poppies. Fermentative opiate production in microbes has also been investigated, and complete biosynthesis of opiates from a simple carbon source has recently been accomplished in yeast. Here we demonstrate that Escherichia coli serves as an efficient, robust and flexible platform for total opiate synthesis. Thebaine, the most important raw material in opioid preparations, is produced by stepwise culture of four engineered strains at yields of 2.1 mg l(-1) from glycerol, corresponding to a 300-fold increase from recently developed yeast systems. This improvement is presumably due to strong activity of enzymes related to thebaine synthesis from (R)-Reticuline in E. coli. Furthermore, by adding two genes to the thebaine production system, we demonstrate the biosynthesis of hydrocodone, a clinically important opioid. Improvements in opiate production in this E. coli system represent a major step towards the development of alternative opiate production systems.

Modulatory Effects of Eschscholzia californica Alkaloids on Recombinant GABAA Receptors.[Pubmed:26509084]

Biochem Res Int. 2015;2015:617620.

The California poppy (Eschscholzia californica Cham.) contains a variety of natural compounds including several alkaloids found exclusively in this plant. Because of the sedative, anxiolytic, and analgesic effects, this herb is currently sold in pharmacies in many countries. However, our understanding of these biological effects at the molecular level is still lacking. Alkaloids detected in E. californica could be hypothesized to act at GABAA receptors, which are widely expressed in the brain mainly at the inhibitory interneurons. Electrophysiological studies on a recombinant alpha 1 beta 2 gamma 2 GABAA receptor showed no effect of N-methyllaurotetanine at concentrations lower than 30 muM. However, (S)-reticuline behaved as positive allosteric modulator at the alpha 3, alpha 5, and alpha 6 isoforms of GABAA receptors. The depressant properties of aerial parts of E. californica are assigned to chloride-current modulation by (S)-reticuline at the alpha 3 beta 2 gamma 2 and alpha 5 beta 2 gamma 2 GABAA receptors. Interestingly, alpha 1, alpha 3, and alpha 5 were not significantly affected by (R)-Reticuline, 1,2-tetrahydroreticuline, codeine, and morphine-suspected (S)-reticuline metabolites in the rodent brain.

Stereochemical inversion of (S)-reticuline by a cytochrome P450 fusion in opium poppy.[Pubmed:26147354]

Nat Chem Biol. 2015 Sep;11(9):728-32.

The gateway to morphine biosynthesis in opium poppy (Papaver somniferum) is the stereochemical inversion of (S)-reticuline since the enzyme yielding the first committed intermediate salutaridine is specific for (R)-Reticuline. A fusion between a cytochrome P450 (CYP) and an aldo-keto reductase (AKR) catalyzes the S-to-R epimerization of reticuline via 1,2-dehydroreticuline. The reticuline epimerase (REPI) fusion was detected in opium poppy and in Papaver bracteatum, which accumulates thebaine. In contrast, orthologs encoding independent CYP and AKR enzymes catalyzing the respective synthesis and reduction of 1,2-dehydroreticuline were isolated from Papaver rhoeas, which does not accumulate morphinan alkaloids. An ancestral relationship between these enzymes is supported by a conservation of introns in the gene fusions and independent orthologs. Suppression of REPI transcripts using virus-induced gene silencing in opium poppy reduced levels of (R)-Reticuline and morphinan alkaloids and increased the overall abundance of (S)-reticuline and its O-methylated derivatives. Discovery of REPI completes the isolation of genes responsible for known steps of morphine biosynthesis.

Plant science. Morphinan biosynthesis in opium poppy requires a P450-oxidoreductase fusion protein.[Pubmed:26113639]

Science. 2015 Jul 17;349(6245):309-12.

Morphinan alkaloids from the opium poppy are used for pain relief. The direction of metabolites to morphinan biosynthesis requires isomerization of (S)- to (R)-Reticuline. Characterization of high-reticuline poppy mutants revealed a genetic locus, designated STORR [(S)- to (R)-Reticuline] that encodes both cytochrome P450 and oxidoreductase modules, the latter belonging to the aldo-keto reductase family. Metabolite analysis of mutant alleles and heterologous expression demonstrate that the P450 module is responsible for the conversion of (S)-reticuline to 1,2-dehydroreticuline, whereas the oxidoreductase module converts 1,2-dehydroreticuline to (R)-Reticuline rather than functioning as a P450 redox partner. Proteomic analysis confirmed that these two modules are contained on a single polypeptide in vivo. This modular assembly implies a selection pressure favoring substrate channeling. The fusion protein STORR may enable microbial-based morphinan production.

Organocatalytic enantioselective Pictet-Spengler approach to biologically relevant 1-benzyl-1,2,3,4-tetrahydroisoquinoline alkaloids.[Pubmed:25909585]

J Org Chem. 2015 May 15;80(10):5125-32.

A general procedure for the synthesis of 1-benzyl-1,2,3,4-tetrahydroisoquinolines was developed, based on organocatalytic, regio- and enantioselective Pictet-Spengler reactions (86-92% ee) of N-(o-nitrophenylsulfenyl)-2-arylethylamines with arylacetaldehydes. The presence of the o-nitrophenylsulfenyl group, together with the MOM-protection in the catechol part of the tetrahydroisoquinoline ring system, appeared to be a productive combination. To demonstrate the versatility of this approach, 10 biologically and pharmaceutically relevant alkaloids were prepared using (R)-TRIP as the chiral catalyst: (R)-norcoclaurine, (R)-coclaurine, (R)-norreticuline, (R)-Reticuline, (R)-trimemetoquinol, (R)-armepavine, (R)-norprotosinomenine, (R)-protosinomenine, (R)-laudanosine, and (R)-5-methoxylaudanosine.

Synthesis of Morphinan Alkaloids in Saccharomyces cerevisiae.[Pubmed:25905794]

PLoS One. 2015 Apr 23;10(4):e0124459.

Morphinan alkaloids are the most powerful narcotic analgesics currently used to treat moderate to severe and chronic pain. The feasibility of morphinan synthesis in recombinant Saccharomyces cerevisiae starting from the precursor (R,S)-norlaudanosoline was investigated. Chiral analysis of the reticuline produced by the expression of opium poppy methyltransferases showed strict enantioselectivity for (S)-reticuline starting from (R,S)-norlaudanosoline. In addition, the P. somniferum enzymes salutaridine synthase (PsSAS), salutaridine reductase (PsSAR) and salutaridinol acetyltransferase (PsSAT) were functionally co-expressed in S. cerevisiae and optimization of the pH conditions allowed for productive spontaneous rearrangement of salutaridinol-7-O-acetate and synthesis of thebaine from (R)-Reticuline. Finally, we reconstituted a 7-gene pathway for the production of codeine and morphine from (R)-Reticuline. Yeast cell feeding assays using (R)-Reticuline, salutaridine or codeine as substrates showed that all enzymes were functionally co-expressed in yeast and that activity of salutaridine reductase and codeine-O-demethylase likely limit flux to morphine synthesis. The results of this study describe a significant advance for the synthesis of morphinans in S. cerevisiae and pave the way for their complete synthesis in recombinant microbes.

Morphine biosynthesis in opium poppy involves two cell types: sieve elements and laticifers.[Pubmed:24104569]

Plant Cell. 2013 Oct;25(10):4110-22.

Immunofluorescence labeling and shotgun proteomics were used to establish the cell type-specific localization of morphine biosynthesis in opium poppy (Papaver somniferum). Polyclonal antibodies for each of six enzymes involved in converting (R)-Reticuline to morphine detected corresponding antigens in sieve elements of the phloem, as described previously for all upstream enzymes transforming (S)-norcoclaurine to (S)-reticuline. Validated shotgun proteomics performed on whole-stem and latex total protein extracts generated 2031 and 830 distinct protein families, respectively. Proteins corresponding to nine morphine biosynthetic enzymes were represented in the whole stem, whereas only four of the final five pathway enzymes were detected in the latex. Salutaridine synthase was detected in the whole stem, but not in the latex subproteome. The final three enzymes converting thebaine to morphine were among the most abundant active latex proteins despite a limited occurrence in laticifers suggested by immunofluorescence labeling. Multiple charge isoforms of two key O-demethylases in the latex were revealed by two-dimensional immunoblot analysis. Salutaridine biosynthesis appears to occur only in sieve elements, whereas conversion of thebaine to morphine is predominant in adjacent laticifers, which contain morphine-rich latex. Complementary use of immunofluorescence labeling and shotgun proteomics has substantially resolved the cellular localization of morphine biosynthesis in opium poppy.