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A whole cell Escherichia coli biotransformation platform converting thebaine to oripavine and codeine to morphine was demonstrated with industrially applicable yields (∼1.2 × 10 -2 g L -1 h -1 or ∼1.2 × 10 -1 g L -1 h -1 ), improving >13 400-fold upon morphine production in yeast. Mutations enhanced enzyme performance and the use of a purified substrate with rich raw poppy extract expanded applicability.

Systematic screening of morphine pathway intermediates in engineered yeast revealed key biosynthetic enzymes displaying potent feedback inhibition: 3'-hydroxy- N -methylcoclaurine 4'-methyltransferase (4'OMT), which yields ( S )-reticuline, and the coupled salutaridinol-7- O -acetyltransferase (SalAT) and thebaine synthase (THS2) enzyme system that produces thebaine. The addition of deuterated reticuline-d1 to a yeast strain able to convert ( S )-norcoclaurine to ( S )-reticuline showed reduced product accumulation in response to the feeding of all four successive pathway intermediates. Similarly, the addition of deuterated thebaine-d3 to a yeast strain able to convert salutaridine to thebaine showed reduced product accumulation from exogenous salutaridine or salutaridinol. In vitro analysis showed that reticuline is a noncompetitive inhibitor of 4'OMT, whereas thebaine exerts mixed inhibition on SalAT/THS2. In a yeast strain capable of de novo morphine biosynthesis, the addition of reticuline and thebaine resulted in the accumulation of several pathway intermediates. In contrast, morphine had no effect, suggesting that circumventing the interaction of reticuline and thebaine with 4'OMT and SalAT/THS2, respectively, could substantially increase opiate alkaloid titers in engineered yeast.

During our extensive surveillance of opiates in urine specimens of opium users, we noticed the appearance of an unknown peak (compound X) in total ion chromatograms obtained by gas chromatography-mass spectrometry (GC-MS) after enzymatic hydrolysis and trimethylsilyl (TMS) derivatization. We identified the compound X as oripavine. Oripavine was found to be a new and useful putative marker of opium/poppy seed use in differentiation from heroin, pharmaceutical codeine, and pharmaceutical morphine use. The presence of oripavine in the urine of opium users is probably the result of O-demethylation of the opium alkaloid thebaine. Analytical method optimization for GC-MS detection of oripavine in urine was also undertaken. Underivatized oripavine could not be detected by GC-MS, and trials for derivatization of oripavine with acetic anhydride and propionic anhydride were unsuccessful. Trials were successful with bis(trimethylsilyl)trifluoroacetamide/trimethylchlorosilane. It was also disclosed that almost all amounts of oripavine in human urine existed in the unconjugated form; it was absolutely necessary to hydrolyze the conjugate before TMS derivatization of oripavine for its GC-MS analysis. [ABSTRACT FROM AUTHOR]

The search for selective kappa opioid receptor (κOR) agonists with an improved safety profile is an area of interest in opioid research. In this work, a series of m -substituted analogs were designed, synthesized, and assayed, resulting in the identification of compound 6c ( SLL-1206 ) as a κOR agonist with single-digit nanomolar activities. The subtype selectivity of compound 6c appeared to be a consequence of an enormous decrease in the affinity for μOR and δOR, rather than a significant increase in the affinity for κOR, which was not the case for SLL-039 , another selective and potent κOR agonist identified in our previous work. Besides reduced central nervous system effects, SLL-1206 exhibited substantially improved physicochemical and pharmacokinetic properties compared with SLL-039 , with increases of over 20-fold in aqueous solubility and approximately 40-fold in oral bioavailability in rats.

Thebaine synthase 2 (THS2) that can transform (7S)-salutaridinol 7-O-acetate to thebaine catalyzes the final step of thebaine biosynthesis in Papaver somniferum. Here, the crystal structures of THS2 and its complex with thebaine are reported, revealing the interaction network in the substrate-binding pocket. Subsequent docking and QM/MM studies was performed to further explore the catalytic mechanism of THS2. Our results suggest that T105 may abstract the proton of C4-OH from the substrate under the assistance of H89. The resulting C4-O - phenolate anion then attacks the nearby C5, and triggers intramolecular S N 2' syn displacement with the elimination of O-acetyl group. Moreover, the latter S N 2' reaction is the rate-determining step of the whole enzymatic reaction with an overall energy barrier of 18.8 kcal/mol. These findings are of pivotal importance to understand the mechanism of action of thebaine biosynthesis, and would guide enzyme engineering to enhance the production of opiate alkaloids via metabolic engineering., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Exploiting the immunosuppressive, analgesic and highly addictive properties of morphine could increase the success of a bacterial pathogen. Therefore, we performed sequence similarity searches for two morphine biosynthesis demethylases in bacteria. For thebaine 6-O-demethylase and codeine O-demethylase, we found strong alignments to three (Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii) of the six ESKAPE pathogens (Enterococcus faecalis, Staphylococcus aureus, K. pneumoniae, A. baumannii, P. aeruginosa and Enterobacter species) that are commonly associated with drug resistance and nosocomial infections. Expression of the aligned sequence found in P. aeruginosa (NP_252880.1/PA4191) is upregulated in isolates obtained from cystic fibrosis patients. Our findings provide putative mechanistic targets for understanding the role of morphine in pathogenicity.

The isomerization of neopinone to codeinone is a critical step in the biosynthesis of opiate alkaloids in opium poppy. Previously assumed to be spontaneous, the process is in fact catalyzed enzymatically by neopinone isomerase (NISO). Without NISO the primary metabolic products in the plant, in engineered microbes and in vitro are neopine and neomorphine, which are structural isomers of codeine and morphine, respectively. Inclusion of NISO in yeast strains engineered to convert thebaine to natural or semisynthetic opiates dramatically enhances formation of the desired products at the expense of neopine and neomorphine accumulation. Along with thebaine synthase, NISO is the second member of the pathogenesis-related 10 (PR10) protein family recently implicated in the enzymatic catalysis of a presumed spontaneous conversion in morphine biosynthesis.

The biosynthesis path engineering could be very promising for mass production of alkaloids by applying elicitors in the cell suspension culture of Persian poppy (Papaver bracteatum Lindl.). In this work, the effects of different concentrations of methyl jasmonate (MJ) and phloroglucinol (PG) on thebaine and sanguinarine productions in vitro were investigated. Roots as explant and supplementing 3 mg L-1 2,4-Dichlorophenoxyacetic acid with 0.5 mg L-1 Benzyl amino purine to modified MS medium were selected to achieve the most efficient combination for callus induction and production of callus fresh and dry weights. At 48 h after treatment, the addition of PG and MJ individually and in combination together significantly increased both thebaine and sanguinarine contents than the control. The results of high-performance liquid chromatography (HPLC) detection indicated that the highest production rate has been achieved through a synergic effect of two elicitors after 48 h. Results revealed that adding 200 μM of MJ and 100 mg L-1 PG increased thebaine and sanguinarine contents by 56.36 and 107.71-fold than control cells, respectively.

The ultimate step in the formation of thebaine, a pentacyclic opiate alkaloid readily converted to the narcotic analgesics codeine and morphine in the opium poppy, has long been presumed to be a spontaneous reaction. We have detected and purified a novel enzyme from opium poppy latex that is capable of the efficient formation of thebaine from (7S)-salutaridinol 7-O-acetate at the expense of labile hydroxylated byproducts, which are preferentially produced by spontaneous allylic elimination. Remarkably, thebaine synthase (THS), a member of the pathogenesis-related 10 protein (PR10) superfamily, is encoded within a novel gene cluster in the opium poppy genome that also includes genes encoding the four biosynthetic enzymes immediately upstream. THS is a missing component that is crucial to the development of fermentation-based opiate production and dramatically improves thebaine yield in engineered yeast.

The gene actions for yield and its attributes and their inheritance pattern based on five parameter model have been explored in four single crosses (NBIHT-5 × NBIHT-6, NBIHT-5 × NBMHT-1, NBMHT-1 × NBIHT-6 and NBMHT-2 × NBMHT-1) obtained using thebaine rich pure lines of opium poppy (Papaver somniferum L.) for three consecutive generations. All the traits showed nonallelic mode of interaction, however, dominance effect (h) was more pronounced for all the traits except thebaine and papaverine. The dominance × dominance (l) effects were predominant over additive × additive (i) for all traits in all the four crosses except for papaverine. The seed and opium yield, and its contributing traits inherited quantitatively. The fixable gene effects (d) and (i) were lower in magnitude than nonfixable (h) and (l) gene effects. The estimates of heterosis were also higher in comparison to the respective parents which suggested preponderance of dominance gene action for controlling most of the traits. The phenotypic coefficient of variation was marginally higher than those of genotypic coefficient of variation for all the traits. The traits thebaine, narcotine, morphine and opium yield had high heritability coupled with high genetic advance. The leaf number, branches per plant and stem diameter showed positive correlation with opium and seed yields. The selection of plants having large number of leaves, branches and capsules with bigger size would be advantageous to enhance the yield potential.

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.

Opioids are the primary drugs used in Western medicine for pain management and palliative care. Farming of opium poppies remains the sole source of these essential medicines, despite diverse market demands and uncertainty in crop yields due to weather, climate change, and pests. We engineered yeast to produce the selected opioid compounds thebaine and hydrocodone starting from sugar. All work was conducted in a laboratory that is permitted and secured for work with controlled substances. We combined enzyme discovery, enzyme engineering, and pathway and strain optimization to realize full opiate biosynthesis in yeast. The resulting opioid biosynthesis strains required the expression of 21 (thebaine) and 23 (hydrocodone) enzyme activities from plants, mammals, bacteria, and yeast itself. This is a proof of principle, and major hurdles remain before optimization and scale-up could be achieved. Open discussions of options for governing this technology are also needed in order to responsibly realize alternative supplies for these medically relevant compounds., (Copyright © 2015, American Association for the Advancement of Science.)

Morphine, first characterized in opium from the poppy Papaver somniferum, is one of the strongest known analgesics. Endogenous morphine has been identified in several mammalian cells and tissues. The synthetic pathway of morphine in the opium poppy has been elucidated. The presence of common intermediates in plants and mammals suggests that biosynthesis occurs through similar pathways (beginning with the amino acid L-tyrosine), and the pathway has been completely delineated in plants. Some of the enzymes in the mammalian pathway have been identified and characterized. Two of the latter steps in the morphine biosynthesis pathway are demethylation of thebaine at the O(3)- and the O(6)-positions, the latter of which has been difficult to demonstrate. The plant enzymes responsible for both the O(3)-demethylation and the O(6)-demethylation are members of the Fe(II)/α-ketoglutarate-dependent dioxygenase family. Previous studies showed that human cytochrome P450 (P450) 2D6 can catalyze thebaine O(3)-demethylation. We report that demethylation of thebaine at the O(6)-position is selectively catalyzed by human P450s 3A4 and 3A5, with the latter being more efficient, and rat P450 3A2. Our results do not support O(6)-demethylation of thebaine by an Fe(II)/α-ketoglutarate-dependent dioxygenase. In rat brain microsomes, O(6)-demethylation was inhibited by ketoconazole, but not sulfaphenazole, suggesting that P450 3A enzymes are responsible for this activity in the brain. An alternate pathway to morphine, oripavine O(6)-demethylation, was not detected. The major enzymatic steps in mammalian morphine synthesis have now been identified., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Opiates and related molecules are medically essential, but their production via field cultivation of opium poppy Papaver somniferum leads to supply inefficiencies and insecurity. As an alternative production strategy, we developed baker's yeast Saccharomyces cerevisiae as a microbial host for the transformation of opiates. Yeast strains engineered to express heterologous genes from P. somniferum and bacterium Pseudomonas putida M10 convert thebaine to codeine, morphine, hydromorphone, hydrocodone and oxycodone. We discovered a new biosynthetic branch to neopine and neomorphine, which diverted pathway flux from morphine and other target products. We optimized strain titer and specificity by titrating gene copy number, enhancing cosubstrate supply, applying a spatial engineering strategy and performing high-density fermentation, which resulted in total opioid titers up to 131 mg/l. This work is an important step toward total biosynthesis of valuable benzylisoquinoline alkaloid drug molecules and demonstrates the potential for developing a sustainable and secure yeast biomanufacturing platform for opioids.

A major toxicological challenge is distinguishing whether morphine in urine, in the absence of 6-monoacetylmorphine (6-MAM), originates from 'street' heroin use or poppy seed ingestion. Manufacturing byproducts from the synthesis of illicit heroin include those that originate from the reaction of acetic anhydride with the alkaloid impurity, thebaine, which undergoes skeletal rearrangement, resulting in compounds with a 2-(N-methylacetamido)ethyl side-chain. The hypothesis that the tertiary amide in this side-chain is resistant to endogenous hydrolysis was supported from in-vitro experiments; a glucuronide metabolite (designated 'ATM4G') was identified that may be used as a marker of 'street' heroin administration. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis for this metabolite was then performed on selected urine specimens from 22 known heroin users, these being negative on routine testing for 6-MAM by gas chromatography-mass spectrometry (GC-MS), using the generally applied reporting threshold of 10 ng/mL, but positive for the presence of morphine. Peaks corresponding to the retention time for the metabolite marker were clearly observed for 16 of the 22 samples, with variations of the ratios of its three dependent ions being within ± 30% of that produced in vitro. Conversely, 6-MAM was detected in only 3 samples, but at concentrations <1 ng/mL. Such a high frequency for the presence of the metabolite marker in urine, in the absence of 6-MAM, is noteworthy and suggests that detection of this metabolite may offer an important advance in forensic toxicology, allowing the development of a new and more definitive test for heroin abuse and thus a potential solution to the so-called 'poppy seed defense'., (Copyright © 2013 John Wiley & Sons, Ltd.)

In this study, the effect of methyl jasmonate (MJ) and ultrasound (US), individually and in combination with L-tyrosine, on the stimulation of thebaine production in Papaver bracteatum cell suspension cultures was studied. The addition of L-tyrosine did not significantly affect the cell biomass, but significantly increased the thebaine yield of cells compared with the control. The synergistic effects of MJ and L-tyrosine in the combined treatment of 100 μM MJ and 2 mM L-tyrosine increased the thebaine yield of cells up to 84.62 mg L(- 1) at 6 days after treatment. Sonication of the cells for 20 s caused a significant decrease in cell growth and biomass, whereas the thebaine yield increased up to 39.60 mg L(- 1) at 6 days after treatment. The combination of US (10 s) and L-tyrosine feeding (2 mM) significantly increased the production of thebaine in comparison to individual utilisation of 2 mM L-tyrosine and US (10 s).

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.

The assumption that CYP2D1 is the corresponding rat cytochrome to human CYP2D6 has been revisited using recombinant proteins in direct enzyme assays. CYP2D1 and 2D2 were incubated with known CYP2D6 substrates, the three morphine precursors thebaine, codeine and (R)-reticuline. Mass spectrometric analysis showed that rat CYP2D2, not 2D1, catalyzed the 3-O-demethylation reaction of thebaine and codeine. In addition, CYP2D2 incubated with (R)-reticuline generated four products corytuberine, pallidine, salutaridine and isoboldine while rat CYP2D1 was completely inactive. This intramolecular phenol-coupling reaction follows the same mechanism as observed for CYP2D6. Michaelis-Menten kinetic parameters revealed high catalytic efficiencies for rat CYP2D2. These findings suggest a critical evaluation of other commonly accepted, however untested, CYP2D1 substrates., (Copyright © 2012. Published by Elsevier B.V.)

Papaverine is one of the earliest opium alkaloids for which a biosynthetic hypothesis was developed on theoretical grounds. Norlaudanosoline (=tetrahydropapaveroline) was claimed as the immediate precursor alkaloid for a multitude of nitrogen containing plant metabolites. This tetrahydroxylated compound was proposed to be fully O-methylated. The resulting tetrahydropapaverine should then aromatize to papaverine. In view of experimental data, this pathway has to be revised. Precursor administration to 8-day-old seedlings of Papaver followed by direct examination of the metabolic fate of the stable-isotope-labeled precursors in the total plant extract, without further purification of the metabolites, led to elucidation of the papaverine pathway in vivo. The central and earliest benzylisoquinoline alkaloid is not the tetraoxygenated norlaudanosoline, but instead the trihydroxylated norcoclaurine that is further converted into (S)-reticuline, the established precursor for poppy alkaloids. The papaverine pathway is opened by the methylation of (S)-reticuline to generate (S)-laudanine. A second methylation at the 3' position of laudanine leads to laudanosine, both known alkaloids from the opium poppy. Subsequent N-demethylation of laudanosine yields the known precursor of papaverine: tetrahydropapaverine. Inspection of the subsequent aromatization reaction established the presence of an intermediate, 1,2-dihydropapaverine, which has been characterized. The final step to papaverine is dehydrogenation of the 1,2-bond, yielding the target compound papaverine. We conclusively show herein that the previously claimed norreticuline does not play a role in the biosynthesis of papaverine., (Copyright 2010 Elsevier Ltd. All rights reserved.)

It has been firmly established that humans excrete a small but steady amount of the isoquinoline alkaloid morphine in their urine. It is unclear whether it is of dietary or endogenous origin. There is no doubt that a simple isoquinoline alkaloid, tetrahydropapaveroline (THP), is found in human and rodent brain as well as in human urine. This suggests a potential biogenetic relationship between both alkaloids. Unlabeled THP or [1,3,4-D(3)]-THP was injected intraperitoneally into mice and the urine was analyzed. This potential precursor was extensively metabolized (96%). Among the metabolites found was the phenol-coupled product salutaridine, the known morphine precursor in the opium poppy plant. Synthetic [7D]-salutaridinol, the biosynthetic reduction product of salutaridine, injected intraperitoneally into live animals led to the formation of [7D]-thebaine, which was excreted in urine. [N-CD(3)]-thebaine was also administered and yielded [N-CD(3)]-morphine and the congeners [N-CD(3)]-codeine and [N-CD(3)]-oripavine in urine. These results show for the first time that live animals have the biosynthetic capability to convert a normal constituent of rodents, THP, to morphine. Morphine and its precursors are normally not found in tissues or organs, presumably due to metabolic breakdown. Hence, only that portion of the isoquinoline alkaloids excreted in urine unmetabolized can be detected. Analysis of urine by high resolution-mass spectrometry proved to be a powerful method for tracking endogenous morphine and its biosynthetic precursors.

The most practical synthetic routes to the preparation of as important pharmaceuticals as oxycodone, naloxone, naltrexone, nalbuphine and buprenorphine have utilized the alkaloid, thebaine, as a starting material. This review intends to focus on chemical transformations of morphinans which resulted in morphinandiene derivatives with well-established and novel pharmacological potencies. These chemical transformations were mainly associated with the formation and substitution of the unique diene structure of the ring C of the morphinan backbone.

We aimed to understand the effects of water stress on the alkaloid production in various developmental stages of poppy plants and the effect of stress on the alkaloids content in the capsules. Three stages of the life cycle of Papaver somniferum L. were selected in our studies: Rosette, Flowering and Lancing developmental stages. Four types of water conditions were examined: Control, Withdrawal of Water, 50% Water Supply and Inundation. The morphological monitoring, results of Relative Water Content and proline content were used as indicators of stress. The result of the measurements in poppy leaves show that the secondary metabolites dramatically respond to these stress conditions. The constant water supply was beneficial for the accumulation of alkaloids in the capsules.

Morphine is a plant (opium poppy)-derived alkaloid and one of the strongest known analgesic compounds. Studies from several laboratories have suggested that animal and human tissue or fluids contain trace amounts of morphine. Its origin in mammals has been believed to be of dietary origin. Here, we address the question of whether morphine is of endogenous origin or derived from exogenous sources. Benzylisoquinoline alkaloids present in human neuroblastoma cells (SH-SY5Y) and human pancreas carcinoma cells (DAN-G) were identified by GC/tandem MS (MS/MS) as norlaudanosoline (DAN-G), reticuline (DAN-G and SH-SY5Y), and morphine (10 nM, SH-SY5Y). The stereochemistry of reticuline was determined to be 1-(S). Growth of the SH-SY5Y cell line in the presence of (18)O(2) led to the [(18)O]-labeled morphine that had the molecular weight 4 mass units higher than if grown in (16)O(2), indicating the presence of two atoms of (18)O per molecule of morphine. Growth of DAN-G cells in an (18)O(2) atmosphere yielded norlaudanosoline and (S)-reticuline, both labeled at only two of the four oxygen atoms. This result clearly demonstrates that all three alkaloids are of biosynthetic origin and suggests that norlaudanosoline and (S)-reticuline are endogenous precursors of morphine. Feeding of [ring-(13)C(6)]-tyramine, [1-(13)C, N-(13)CH(3)]-(S)-reticuline and [N-CD(3)]-thebaine to the neuroblastoma cells led each to the position-specific labeling of morphine, as established by GC/MS/MS. Without doubt, human cells can produce the alkaloid morphine. The studies presented here serve as a platform for the exploration of the function of "endogenous morphine" in the neurosciences and immunosciences.

The opium poppy is a source of the pharmaceuticals codeine, morphine and their derived analgesics. Here we describe the initial characterization of the poppy mutant known as top1 (for 'thebaine oripavine poppy 1'), which accumulates the morphine and codeine precursors thebaine and oripavine and does not complete their biosynthesis into morphine and codeine. The original discovery of top1 stimulated a re-engineering of the opioid industry in the island state of Tasmania, which grows over 40% of the world's licit opiates, in order to produce thebaine and oripavine efficiently from morphine-free poppy crops to provide precursors for highly effective analgesics and for treatment of opioid addiction.

The biotransformation of thionorphine (N-cyclopropylmethyl-7alpha-[(s)-1-hydroxy-1-methyl-3-(2thiophene)-propyl]-6,14-endo-ethano tetrahydrooripavine), a new analgesic, was in-vestigated in rats. The results of metabolite analysis by liquid chromatography/electrospray ionization tandem mass spectrometry with positive ion mode, in which a mobile phase of 10 mM ammonium acetate (pH 3.0)/acetonitrile (25/75) was used, suggested that thionorphine is biotransformed to two potentially active metabolites, the N-dealkylated thionorphine (M-I) and the oxidized thionorphine (M-II), and subsequently form conjugates with glucuronic acid of both thionorphine and the metabolites., (Copyright 2004 John Wiley & Sons, Ltd.)

A novel regiospecific N- to O-methyl transfer reaction has been characterised in the biotransformation of an N-CD3-thebaine derivative with the fungus Cunninghamella echinulata NRRL 1384.

The photooxidation of thebaine (3) with a sun lamp affords two main products: hydrodibenzofuran 10 (major) and benzofuran 11 (minor). The latter compound becomes predominant if a Hg lamp is used instead of a sun lamp. The formation of 10 proceeds via an endoperoxide intermediate that undergoes oxidation at the nitrogen atom. Protection of the nitrogen either by protonation or quaternization prevents its oxidation and thus the photooxidation of 3 in acid media constitutes a one-pot procedure for the preparation of 14-hydroxycodeinone 35. Photooxidation of the thebaine ammonium salt 31 allows the isolation in good yields of the corresponding to thebaine endoperoxide 32. The selective protection and reduction of the keto, aldehyde, and olefinic groups of hydrodibenzofuran 10 allowed the preparation of several diol and keto alcohol derivatives. This is the first report on the use of photooxidation to functionalize thebaine at C(6) and C(14) and also the first on the isolation of opioid endoperoxides.

This study examines the possibility that oripavine opioid receptor agonists bind equally to both high and low affinity states of the mu-opioid receptor. Studies were performed in C6 cells expressing mu- or delta-opioid receptors; high and low agonist affinity states of the receptors were defined by the absence and presence, respectively of Na+ ions and the GTP analog Gpp(NH)p. At the mu-opioid receptor dihydroetorphine and etorphine were full agonists, buprenorphine had moderate efficacy while diprenorphine was an antagonist. At the delta-opioid receptor, dihydroetorphine, etorphine, and diprenorphine had moderate efficacy while buprenorphine was an antagonist. The binding affinities of the oripavines at the mu-opioid receptor decreased only one to 2-fold in the presence of NaCl and Gpp(NH)p. In contrast, decreases in oripavine affinity at the delta-opioid receptor correlated with delta-opioid receptor efficacy. The ability of oripavine agonists to bind with high affinity to the low agonist affinity state of the nu-opioid receptor may explain the high potencies of these compounds in vivo.

In vivo and in vitro binding studies with natural thebaine and its enantiomer, (+)-thebaine were conducted to elucidate further their interactions with the opioid system. (-)-Thebaine a key intermediate in the biosynthesis of morphine in the poppy plant (Papaver somniferum) and in mammalian tissue, was poorly effective antinociceptively in mice at doses to 30 mg/kg. Its principal behavioral manifestation was lethal convulsions. Naltrindole, at doses of 1 and 10 mg/kg did not block either the convulsions or lethal effects, suggesting that the delta-opioid receptor system was not involved in this action. Surprisingly, the dextrorotatory isomer exhibited significant antinociceptive activity in the tail-flick [ED50 = 8.9 (3.4-22.1) mg/kg], hot-plate [ED50 = 22.9 (10.9-48.1) mg/kg] and phenylquinone [ED50) = 1.9 (1.6-9.5) mg/kg] assays. Studies with opioid receptor-subtype antagonists, beta-funaltrexamine, nor-binaltorphimine and naltrindole, indicated that antinociception was associated with mu- and delta-opioid receptors. Results of displacement experiments supported the in vivo data. Significant competition for [3H]diprenorphine binding with both isomers for cloned mu- and delta-opioid receptors was observed. However, (-)-thebaine was more effective at the delta-opioid receptor (Ki = 1.02+/-0.01 microM) whereas (+)-thebaine was more effective at the mu-opioid receptor ( Ki = 2.75+/-0.01 microM). Opioid-induced antinociception associated with unnatural thebaine raises the possibility of additional mu- and delta-opioid receptor sites.

Acetyl coenzyme A:salutaridinol-7-O-acetyltransferase, a highly substrate-specific enzyme, has been purified nearly 3,000-fold to homogeneity from Papaver somniferum plant cell suspension cultures. Purification was achieved by fractionated ammonium sulfate precipitation, dye-ligand affinity chromatography on matrex red A, gel filtration, ion exchange chromatography on Mono Q and a second dye-ligand affinity chromatography on fractogel TSK AF Blue. The purified enzyme was a single polypeptide with an M(r) = 50,000 displaying an isoelectric point of 4.8, a pH optimum between pH 6 and 9 and a temperature optimum at 47 degrees C. The Km values for the substrate salutaridinol and the co-substrate acetyl co-enzyme A were 7 and 46 microM, respectively. Salutaridinol-7-O-acetyltransferase catalyzes the stoichiometric transfer of the acetyl group from acetyl coenzyme A to the 7-OH group of salutaridinol yielding salutaridinol-7-O-acetate, which is a new intermediate in morphine biosynthesis. Salutaridinol-7-O-acetate undergoes a subsequent spontaneous allylic elimination at pH 8-9, leading to the formation of thebaine (1), the first morphinan alkaloid with the complete pentacyclic ring system, or at pH 7 leading to dibenz[d,f]azonine alkaloids that contain a nine-membered ring. Acetylation and subsequent allylic elimination is a new enzymic mechanism in alkaloid biosynthesis, which in the poppy plant can transform one precursor into alkaloids possessing markedly different ring systems, depending on the reaction pH.

This review surveys the discovery of endogenous alkaloids in mammals. The formation of morphine in mammalian brain was assumed in 1970. The existence of morphine was demonstrated by radioimmunoassay. Identification of morphine was performed by spectroscopic methods. The isolation of mammalian morphine raises the question of biosynthesis. Recently, it has been shown that the biosynthetic pathway is similar to that that exists in poppy.

Alkylation of sarcosine with 4-chloro-nitrobenzo-2-oxa-1,3-diazole (NBD-chloride) furnished a fluorescent tag that was coupled with a tetrahydrothebaine derivative and beta-naltrexamine, respectively, to yield the fluorescent opioids 7 alpha-(1R)-1-hydroxy-1-methyl-3-(4-hydroxyphenyl)-propyl]-6,14- endoethenotetrahydrothebaine NBD-sarcosinate (ASM-5-10) and N-cyclopropylmethyl-3-hydroxy-14 beta-hydroxy-6 beta-(NBD sarcosinyl)-amino-epoxymorphinan (ASM-5-67). The fluorescence intensity of the novel opioids allowed their detection at subnanomolar concentrations, and was dependent on the polarity of the solvent. Maximum quantum yield was obtained in ethyl acetate and ethanol, and minimal fluorescence in heptane and water. Compounds ASM-5-10 and ASM-5-67 displaced the opioid receptor binding of [3H]Tyr-D-Ala-Gly-(Me)Phe-Gly-ol in monkey brain membranes with IC50 values of 8.4 and 1.5 nM, respectively. Whereas ASM-5-67 bound to mu, delta, and kappa receptors with comparable affinities, ASM-5-10 was mu-selective, with selectivity indices (ratio of respective IC50 values) of 0.04 for both mu/delta and mu/kappa. The sodium response ratio in binding revealed a pronounced agonist property of ASM-5-10. Both opioids were lipophilic, with octanol-water partition coefficients (log Papp) of 2.8 (ASM-5-10) and 1.0 (ASM-5-67). ASM-5-10 exhibited particularly strong membrane retention that was not reversible by four washes. Their favorable characteristics in fluorescence, receptor binding, and membrane interaction make these newly developed ligands useful molecular probes to study opioid receptor mechanisms.

From colchicine-treated seeds of Papaver bracteatum Lindl. some poppy plants were obtained that developed capsules richer in thebaine than the controls. The individual poppies were analyzed for capsule thebaine content annually for eight successive years, the results revealing significant year-to-year differences. One of the poppies, X7, (ca 5% thebaine) developed capsules consisting partly of polyploid tissue during the first and second year. This plant was propagated vegetatively to give a series (the X7 series) of new P. bracteatum plants. The capsule thebaine content of these individuals differed markedly the first two years, whereupon the alkaloid production decreased and appeared to level out and reach a value still clearly higher than the controls (mean values 2.3% and 1.3%, respectively). From seeds of four of the thebaine-rich poppies of the X7 series, four new series of P. bracteatum plants were obtained. The capsule thebaine level of these was significantly lower than that of the mother plants.

1. Codeine O-demethylation to morphine is mediated by cytochrome P450 IID1 (rat), or P450 IID6 (man), and exhibits genetic polymorphism. Thebaine is a precursor in the formation of endogenous morphine and codeine in man, being O-demethylated to oripavine. 2. The objective of the present study was to ascertain whether the O-demethylation of thebaine to oripavine was mediated by cytochrome P450 IID1 in rat liver microsomes. 3. Thebaine O-demethylation showed strain differences in female Sprague-Dawley (SD) and female Dark-Agouti (DA) rats, which serve as a model for the human debrisoquine/sparteine metabolism phenotypes. 4. The total intrinsic clearance of thebaine to oripavine was high (19.7 ml/h per mg protein) in SD rats, indicating that oripavine is a major metabolite of thebaine. A 3-fold lower intrinsic clearance was observed in DA rats (6.7 ml/h per mg protein). 5. Thebaine O-demethylation was inhibited by quinine and known substrates of cytochrome P450 IID1/P450 IID6, supporting the major involvement of cytochrome P450 IID1 in oripavine formation in rats.

Isothiocyanate and alpha-methylene-gamma-lactone analogues of 6,14-endo-ethenotetrahydrothebaine and -oripavine were prepared with the electrophilic groups being located at C-19 in the C-7 alpha-side chain. Isothiocyanates were prepared in the N-Me and N-CPM (N-cyclopropylmethyl) series, both as the phenols and 3-O-methyl ethers from the diastereomeric amines formed from reductive amination of thevinone (2) and N-(cyclopropylmethyl)northevinone (13). Although addition of the organozinc reagent from methyl alpha-bromomethacrylate to 25 failed, addition to 3-O-protected aldehydes 27 and 35 produced, after subsequent deprotection, alpha-methylene-gamma-lactones 29 and 37, respectively. In the opioid receptor displacement assays against [3H]bremazocine as the radiolabeled ligand, the phenolic compounds were most potent with N-CPM isothiocyanates 20 and 21 showing IC50s of 0.32 and 0.76 nM, respectively, and N-CPM alpha-methylene-gamma-lactone 37 having an IC50 = 1.0 nM. Compound 37 showed irreversible effects in the binding assay which were mu-selective, as demonstrated by analogous experiments using [3H]DAGO, and naloxone was found to protect against the irreversible effects. This observation suggests that a receptor-bound nucleophile is located at a position where it can readily reach the alpha-methylene group of lactone 37.

A strain of Pseudomonas putida was isolated by selective enrichment with morphine that was capable of utilising morphine as a primary source of carbon and energy for growth. Experiments with whole cells showed that both morphine and codeine, but not thebaine, could be utilised. A novel NADP-dependent dehydrogenase, morphine dehydrogenase, was purified from crude cell extracts and was shown to be capable of oxidising morphine and codeine to morphinone and codeinone, respectively. This NADP-dependent morphine dehydrogenase was not observed in any other species of pseudomonads examined and was quite distinct from the beta-hydroxysteroid dehydrogenase found in Pseudomonas testosteroni, which had previously been shown to have activity against morphine.

Thebaine, an intermediate of morphine biosynthesis in the poppy plant, Papaver somniferum, was transformed to oripavine, codeine, and morphine by rat liver, kidney, and brain microsomes in the presence of an NADPH-generating system. The formation of morphine, codeine, and oripavine was identified by a specific RIA, HPLC, and GCMS. Thebaine also gave rise to four other compounds, which for the moment are unidentified. NADH dramatically increased the formation of both codeine and morphine when used together with an NADPH-generating system, especially in liver microsomes. NADPH is essential in the formation of oripavine from thebaine and morphine from codeine, while NADH is critical in the conversion of thebaine to codeine and from oripavine to morphine. Carbon monoxide or SKF 525A inhibited the conversion, indicating a role of cytochrome P-450. These results provide evidence for the enzymatic in vitro conversion by mammalian tissues of thebaine to morphine. The pathway is similar to that which exists in plants.

Syntheses of affinity reagents for opiate receptors based on the fentanyl, endo-ethenotetrahydrooripavine, and etonitazene carbon-nitrogen skeletons are described. The isothiocyanate, bromoacetamido, and methylfumaramido alkylating functions were employed in these compounds, some of which had previously been shown to be mu specific (12, BIT) and delta specific (8, FIT and 19, FAO) in vitro. Antinociceptive activity of the title compounds was determined in the mouse hot-plate test, which revealed that certain compounds in each class showed morphine-like activity. The binding EC50 values against [3H]Dalamid for opiate receptors in NG108-15 (delta receptors) and rat brain membranes (mu + delta receptors) are also reported. With this type of experiment, it was possible to independently measure the apparent affinity of the etonitazene congeners 12-14 for the mu and delta receptors.

Condensation of the Grignard reagent derive from 2-[4-(allyloxy)phenyl]ethyl bromide (4b) with 7 alpha-acetyl-6,14-endo-ethenotetrahydrothebaine (5) furnished the (R) tertiary carbinol, 7, which upon methoxymercuration followed by treatment with the KBr gave the bromomercurio compound 10 (Hybromet). The corresponding N-cyclopropylmethyl analogue, 11, was prepared also. The bromomercurio compound, 1, and the mercaptobenzothiazole derivative, 3, gave allyl phenyl ether when treated with BAL at room temperature. Similar treatment of 10 with BAL gave 7 in high yield. Binding studies using rat brain homogenates indicated that 7, 13, and 14 have moderately high affinities for mu rather than delta binding sites. Although much weaker, 10 showed preferential mu binding also. These results along with the fact that 10 reacted smoothly with sulfhydryl groups suggest that Hybromet would be a suitable ligand for use in affinity chromatography.

A p-isothiocyanatophenylacetylamino moiety at the C7alpha position on an endoethenooripavine (NIH 10358) was found to irreversibly bind to both the mu and delta opioid receptors in rat brain membrane preparations. However, the same endoethenooripavine nucleus with a p-methylfumaroylaminophenylacetylamino moiety at that position (NIH 10364) binds specifically and irreversibly to only mu opioid receptors. Hypotheses are being developed concerning receptor subclass alkylation specificity. For example, it has now been found that the delta receptor irreversible agonist, FIT, can interact with both mu and delta receptors in a reversible manner.

The series of experiments reported in this paper examined the spectrum of subtypes of opioid receptors alkylated in vitro by N-cyclopropylmethyl-7 alpha-methylfumaramido-6,14- endoethenotetrahydronororipavine (NIH10236) and four optical isomers of the methylfumaramidophenethyl derivatives of 3-methylfentanyl. Pretreatment of membranes with NIH10236 resulted in a wash-resistant inhibition of the binding of [3H]6 beta-fluoro-6-desoxyoxymorphone (mu binding sites), the binding of [3H][D-ala2,D-leu5]-enkephalin (both the higher and lower affinity delta binding sites) and was without effect on kappa binding sites labelled with [3H]bremazocine. All four potential alkylating derivatives of 3-methylfentanyl were inactive. Pretreatment of membranes with 1 microM of the reversible ligands, (+)-cis-3-methylfentanyl, but not its enantiomer, inhibited the binding of [3H]6 beta-fluoro-6-desoxyoxymorphone and the binding of [3H][D-ala2,D-leu5]enkephalin to the lower affinity binding sites by over 90%. This phenomenon is termed "pseudo-irreversible inhibition." Incubation of pretreated membranes for 60 min at 37 degrees C, in the presence of 200 mM NaCl and 50 microM GppNHp, only partially reversed the masking of opioid receptors by (+)-cis-3-methylfentanyl. For in vivo experiments, membranes were prepared 18-24 hr after the intracerebroventricular administration of 80 and 50 micrograms of NIH10236. This resulted in decreased labelling of mu binding sites, lower affinity [3H][D-ala2,D-leu5]enkephalin binding sites, as well as kappa binding sites, labelled by [3H]U69,593 and [3H]bremazocine. There was no apparent alteration in the higher affinity [3H][D-ala2,D-leu5]enkephalin binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

Standard samples of pure (+)-salutaridine and (-)-thebaine required to study the mammalian origin of morphine, were prepared from (+)-6-demethylsalutaridine by published procedures and were characterized by CD spectra and physical data. Reductive N-methylation of (-)-northebaine afforded (-)-thebaine, and when 13C-labeled formalin was used, (-)-thebaine with a 13C label on the N-methyl carbon atom resulted. The latter represents a model procedure to prepare ultimately N-14CH3-labeled (-)-thebaine and 14C-labeled congeners.

The in vivo opiate receptor binding of three putative delta-selective opioid antagonists, the irreversible oripavine ligand NIH 10236 and two enkephalin analogs, ICI 154,129 and ICI 174,866, was studied in rat brain. Following i.c.v. injection, potent and apparently irreversible opiate receptor binding was observed for NIH 10236 with similar affinity for mu, delta and kappa sites. However, opiate receptor binding was not evident either after s.c. injection of NIH 10236 or ICI 154,129, or following i.c.v. administration of ICI 154,129 or ICI 174,864. The receptor sites that mediate the pharmacological effects of these drugs should be reevaluated.

The dependence potential of thebaine is at least partially attributed to oripavine which is one of the principal metabolites of thebaine. The analgesic potency of oripavine in mice is found to be much higher than that of thebaine and comparable to morphine. The reinforcing effect of this substance also appears to be more potent than thebaine. In rats the physical dependence potential of oripavine at a dose of 4 mg/kg is almost comparable to that of morphine at 0.5 mg/kg. Studies carried out on monkeys show that oripavine possesses weak morphine-antagonist properties. Further pharmacological studies of the metabolites of thebaine are recommended.