The Experts below are selected from a list of 201 Experts worldwide ranked by ideXlab platform
Tomas Hudlicky - One of the best experts on this subject based on the ideXlab platform.
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chemoenzymatic formal total synthesis of ent codeine and other morphinans via nitrone cycloadditions and or radical cyclizations comparison of strategies for control of c 9 c 14 stereogenic centers
Advanced Synthesis & Catalysis, 2014Co-Authors: Mary Ann A Endomaarias, Jason Reed Hudlicky, Razvan Simionescu, Tomas HudlickyAbstract:Formal total syntheses of ent-codeine and other morphinans were accomplished from 1-phenyl-2-acetoxyethane, which was subjected to enzymatic dihydroxylation by toluene dioxygenase overexpressed in Eschericia coli JM109 (pDTG601A). The resulting cis-dihydroarenediol was coupled with a phenol derived from bromoisovanillin and a subsequent Heck reaction was used to establish the C-13 quaternary center. Two strategies were employed to set the C-14 center: nitrone and nitrile oxide cycloadditions to the C-8/C-14 olefin and a radical cyclization of an aldehyde to C-14. Both strategies yielded tetracyclic products that were converted to known intermediates for the synthesis of ent-codeine, ent-Codeinone, and ent-hydrocodone. Experimental and spectral data are provided for all new compounds.
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Chemoenzymatic Formal Total Synthesis of ent‐Codeine and Other Morphinans via Nitrone Cycloadditions and/or Radical Cyclizations. Comparison of Strategies for Control of C‐9/C‐14 Stereogenic Centers
Advanced Synthesis & Catalysis, 2014Co-Authors: Mary Ann A. Endoma-arias, Razvan Simionescu, Jason Reed Hudlicky, Tomas HudlickyAbstract:Formal total syntheses of ent-codeine and other morphinans were accomplished from 1-phenyl-2-acetoxyethane, which was subjected to enzymatic dihydroxylation by toluene dioxygenase overexpressed in Eschericia coli JM109 (pDTG601A). The resulting cis-dihydroarenediol was coupled with a phenol derived from bromoisovanillin and a subsequent Heck reaction was used to establish the C-13 quaternary center. Two strategies were employed to set the C-14 center: nitrone and nitrile oxide cycloadditions to the C-8/C-14 olefin and a radical cyclization of an aldehyde to C-14. Both strategies yielded tetracyclic products that were converted to known intermediates for the synthesis of ent-codeine, ent-Codeinone, and ent-hydrocodone. Experimental and spectral data are provided for all new compounds.
Meinhart H. Zenk - One of the best experts on this subject based on the ideXlab platform.
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purification and properties of Codeinone reductase nadph from papaver somniferum cell cultures and differentiated plants
FEBS Journal, 1995Co-Authors: Rainer Lenz, Meinhart H. ZenkAbstract:Codeinone reductase (NADPH), which catalyzes the stereospecific reduction of (–)Codeinone to (–)codeine, was detected and purified to electrophoretic homogeneity from a cytosolic fraction of Papaver somniferum L. cell cultures. The purification involved ammonium sulfate precipitation (40–80%), affinity chromatography (matrex red A), gel filtration (fractogel TSK HW 55S), affinity chromatography (fractogel TSK AF Blue), ion-exchange chromatography (DEAE-Sephacel) and native PAGE. The purified Codeinone reductase was found to be a monomeric protein of 35±1 kDa that is highly substrate-specific, reducing only the C6 oxo group of Codeinone and morphinone as well as a few analogues. The physiological forward reaction has a pH optimum at 7.0, the reverse reaction at 9.1. The temperature optimum is at 40°C and the isoelectric point (pl) at 4.4. The apparent Km values (forward reaction) for Codeinone and NADPH are 23 μM and 168 μM, respectively. Using capsule tissue of differentiated P. somniferum plants as an enzyme source, two Codeinone reductase (NADPH) isoenzymes were detected and purified to homogeneity. These isoenzymes could not be separated for characterization and showed slightly different kinetic features (Km values: Codeinone 9 μM; NADPH 81 μM) compared with the cell culture enzyme.
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Stereoselective reduction of Codeinone, the penultimate enzymic step during morphine biosynthesis in Papaver somniferum
Tetrahedron Letters, 1995Co-Authors: Rainer Lenz, Meinhart H. ZenkAbstract:Abstract A highly substrate specific enzyme has been discovered and purified to homogeneity both from cell suspension cultures as well as from capsule tissue of Papaver somniferum which stereoselectively reduces (−)-Codeinone to (−)-codeine and morphinone to morphine at the expense of NADPH.
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The transformation of neopinone to Codeinone in morphine biosynthesis proceeds non-enzymatically
Tetrahedron Letters, 1993Co-Authors: Jürgen Gollwitzer, Rainer Lenz, Norbert Hampp, Meinhart H. ZenkAbstract:Abstract Thebaine after vinylether cleavage yields neopinone which exists in a chemical equilibrium with Codeinone. It is demonstrated that codeine: NADP oxidoreductase in the presence of NADPH shifts this equilibrium quantitatively towards codeine.
Graham G. Skellern - One of the best experts on this subject based on the ideXlab platform.
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The toxicity of opiates and their metabolites in HepG2 cells.
Chemico-biological interactions, 2003Co-Authors: M. Jairaj, David G. Watson, M. Helen Grant, Graham G. SkellernAbstract:The toxicity of codeine (C), Codeinone (CO), morphine (M), oxycodone (OC), pholcodine (P) and pholcodine-N-oxide (P-NOX) was assessed in HepG2 cells by determining cell viability via the measurement of lactate dehydrogenase (LDH) leakage through the membrane, depletion of reduced glutathione (GSH) and measurement of total protein content. Incubation of C, M, OC, P or P-NOX with HepG2 cells resulted in no significant loss of cell viability, depletion of GSH or decreased total protein content. In contrast, with CO there was a marked depletion of GSH with significant differences from control cells (P
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Toxicity of codeine, Codeinone and oxycodone in hep g2 cells: identification of a Codeinone-glutathione conjugate by lC/+ECI-MS/MS
2002Co-Authors: M. Jairaj, David G. Watson, Mary Grant, Graham G. SkellernAbstract:Development of new mass spectrometers and implementation of new analytical methods were the central themes of the conference. This paper focused on the toxicity of codeine, Codeinone and oxycodone in hep g2 cells: identification of a Codeinone-glutathione conjugate by lC/+ECI-MS/MS.
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toxicity of codeine Codeinone and oxycodone in hep g2 cells identification of a Codeinone glutathione conjugate by lc eci ms ms
50th Meeting of the American Society for Mass Spectrometry, 2002Co-Authors: M. Jairaj, David G. Watson, M H Grant, Graham G. SkellernAbstract:Development of new mass spectrometers and implementation of new analytical methods were the central themes of the conference. This paper focused on the toxicity of codeine, Codeinone and oxycodone in hep g2 cells: identification of a Codeinone-glutathione conjugate by lC/+ECI-MS/MS.
Peter J. Facchini - One of the best experts on this subject based on the ideXlab platform.
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Neopinone isomerase is involved in codeine and morphine biosynthesis in opium poppy
Nature Chemical Biology, 2019Co-Authors: Mehran Dastmalchi, Jillian M. Hagel, Limei Chang, Xue Chen, Rongji Chen, Sukanya Ramasamy, Sam Yeaman, Peter J. FacchiniAbstract: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. Neopinone isomerase catalyzes the isomerization of the opiate alkaloid neopinone to Codeinone, driving the biosynthesis of codeine and morphine and preventing accumulation of their isomers neopine and neomorphine.
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Neopinone isomerase is involved in codeine and morphine biosynthesis in opium poppy.
Nature chemical biology, 2019Co-Authors: Mehran Dastmalchi, Jillian M. Hagel, Limei Chang, Xue Chen, Rongji Chen, Sukanya Ramasamy, Sam Yeaman, Peter J. FacchiniAbstract: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.
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Codeinone reductase isoforms with differential stability, efficiency and product selectivity in opium poppy.
The Plant journal : for cell and molecular biology, 2018Co-Authors: Mehran Dastmalchi, Limei Chang, Miguel A. Torres, Peter J. FacchiniAbstract:Codeinone reductase (COR) catalyzes the reversible NADPH-dependent reduction of Codeinone to codeine as the penultimate step of morphine biosynthesis in opium poppy (Papaver somniferum). It also irreversibly reduces neopinone, which forms by spontaneous isomerization in aqueous solution from Codeinone, to neopine. In a parallel pathway involving 3-O-demethylated analogs, COR converts morphinone to morphine, and neomorphinone to neomorphine. Similar to neopine, the formation of neomorphine by COR is irreversible. Neopine is a minor substrate for codeine O-demethylase (CODM), yielding morphine. In the plant, neopine levels are low and neomorphine has not been detected. Silencing of CODM leads to accumulation of upstream metabolites, such as codeine and thebaine, but does not result in a shift towards higher relative concentrations of neopine, suggesting a mechanism in the plant for limiting neopine production. In yeast (Saccharomyces cerevisiae) engineered to produce opiate alkaloids, the catalytic properties of COR lead to accumulation of neopine and neomorphine as major products. An isoform (COR-B) was isolated from opium poppy chemotype Bea's Choice that showed higher catalytic activity than previously characterized CORs, and it yielded mostly neopine in vitro and in engineered yeast. Five catalytically distinct COR isoforms (COR1.1-1.4 and COR-B) were used to determine sequence-function relationships that influence product selectivity. Biochemical characterization and site-directed mutagenesis of native COR isoforms identified four residues (V25, K41, F129 and W279) that affected protein stability, reaction velocity, and product selectivity and output. Improvement of COR performance coupled with an ability to guide pathway flux is necessary to facilitate commercial production of opiate alkaloids in engineered microorganisms.
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Khat unigenes representing enzymes putatively involved in ephedrine alkaloid biosynthesis.
2015Co-Authors: Ryan A. Groves, Jillian M. Hagel, Ye Zhang, Korey Kilpatrick, Asaf Levy, Frédéric Marsolais, Efraim Lewinsohn, Christoph W. Sensen, Peter J. FacchiniAbstract:Each unigene is assigned an identifier, which corresponds to a database ID in the CED-Trinity library. Percent amino acid identity between unigenes and queries is provided. Abbreviations: AAO4, aromatic aldehyde oxidase 4; Am, Antirrhinum majus; AHAS, acetohydroxyacid synthase; ArAT, aromatic amino acid transaminase; At, Arabidopsis thaliana; BALDH, benzaldehyde dehydrogenase; BDH, benzaldehyde dehydrogenase; BL, benzoate CoA-ligase; BZO, benzoyloxyglucosinolate; CHD, cinnamoyl-CoA hydratase-dehydrogenase; 4CL, 4-coumaroyl-CoA ligase; Ce, Catha edulis; Cm, Cucumis melo; COR, Codeinone reductase; Ds, Datura stramonium; Ec, Eschscholzia californica; Es, Ephedra sinica; KAT, 3-ketoacyl-CoA thiolasae; Ps, Papaver somniferum; PAL, L-phenylalanine ammonia lyase; PDC, pyruvate decarboxylase; Ph, Petunia x hybrida; PPA-AT, prephenate aminotransferase; RED, reductase; SanR, sanguinarine reductase; TA, transaminase; ThDPC, thiamin diphosphate-dependent carboligase; TR, tropinone reducase.Khat unigenes representing enzymes putatively involved in ephedrine alkaloid biosynthesis.
Neil C. Bruce - One of the best experts on this subject based on the ideXlab platform.
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Crystal Structure of Bacterial Morphinone Reductase and Properties of the C191A Mutant Enzyme.
The Journal of biological chemistry, 2002Co-Authors: Terez Barna, Neil C. Bruce, Nigel S. Scrutton, Hanan L. Messiha, Carlo Petosa, Peter C. E. MoodyAbstract:Abstract The crystal structure of the NADH-dependent bacterial flavoenzyme morphinone reductase (MR) has been determined at 2.2-A resolution in complex with the oxidizing substrate Codeinone. The structure reveals a dimeric enzyme comprising two 8-fold β/α barrel domains, each bound to FMN, and a subunit folding topology and mode of flavin-binding similar to that found in Old Yellow Enzyme (OYE) and pentaerythritol tetranitrate (PETN) reductase. The subunit interface of MR is formed by interactions from an N-terminal β strand and helices 2 and 8 of the barrel domain and is different to that seen in OYE. The active site structures of MR, OYE, and PETN reductase are highly conserved reflecting the ability of these enzymes to catalyze “generic” reactions such as the reduction of 2-cyclohexenone. A region of polypeptide presumed to define the reducing coenzyme specificity is identified by comparison of the MR structure (NADH-dependent) with that of PETN reductase (NADPH-dependent). The active site acid identified in OYE (Tyr-196) and conserved in PETN reductase (Tyr-186) is replaced by Cys-191 in MR. Mutagenesis studies have established that Cys-191 does not act as a crucial acid in the mechanism of reduction of the olefinic bond found in 2-cyclohexenone and Codeinone.
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Mechanistic studies of morphine dehydrogenase and stabilization against covalent inactivation.
Biochemical Journal, 2000Co-Authors: Edward H. Walker, Christopher E. French, Deborah A. Rathbone, Neil C. BruceAbstract:Morphine dehydrogenase (MDH) of Pseudomonas putida M10 catalyses the NADP(+)-dependent oxidation of morphine and codeine to morphinone and Codeinone. This enzyme forms the basis of a sensitive detection and assay method for heroin metabolites and a biotransformation process for production of hydromorphone and hydrocodone. To improve these processes we have undertaken a thorough examination of the kinetic mechanism of MDH. Sequence comparisons indicated that MDH belongs within the aldose reductase enzyme family. MDH was shown to be specific for the pro-R hydrogen of NADPH. In steady-state kinetic studies, product inhibition patterns suggested that MDH follows a Theorell-Chance mechanism for Codeinone reduction at pH 7, and a non-Theorell-Chance sequential ordered mechanism for codeine oxidation at pH 9.5. Residues corresponding to the catalytically important Tyr-48, Lys-77 and Asp-43 of aldose reductase were modified by site-directed mutagenesis, resulting in substantial loss of activity consistent with a catalytic role for these residues. Loss of activity of MDH in the presence of the reaction product morphinone was found to be due to the formation of a covalent adduct with Cys-80; alteration of Cys-80 to serine resulted in an enzyme with greatly enhanced stability.
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Transformations of codeine to important semisynthetic opiate derivatives by Pseudomonas putida m10
Fems Microbiology Letters, 1999Co-Authors: Diane L. Lister, Deborah A. Rathbone, Gayatri Kanungo, Neil C. BruceAbstract:A biotransformation mixture which contained codeine and washed cells of Pseudomonas putida M10 gave rise to a number of transformation products that are of clinical importance which included hydrocodone, dihydrocodeine and 14β-hydroxycodeine. Incubations with the same organism and Codeinone gave rise to 14β-hydroxyCodeinone and 14β-hydroxycodeine. Cell-free extracts and membrane fractions of P. putida M10 were shown to catalyse the 14β-hydroxylation of Codeinone. In addition, the potent analgesic oxycodone was shown to be produced from 14β-hydroxyCodeinone.
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REDUCTIVE AND OXIDATIVE HALF-REACTIONS OF MORPHINONE REDUCTASE FROM PSEUDOMONAS PUTIDA M10 : A KINETIC AND THERMODYNAMIC ANALYSIS
Biochemistry, 1998Co-Authors: Daniel H. Craig, Neil C. Bruce, Peter C. E. Moody, Nigel S. ScruttonAbstract:The reaction of morphinone reductase (MR) with the physiological reductant NADH and the oxidizing substrate Codeinone has been studied by multiple and single wavelength stopped-flow spectroscopy. Reduction of the enzyme with NADH proceeds in two kinetically resolvable steps. In the first step, the oxidized enzyme forms a charge-transfer intermediate with NADH. The charge-transfer complex is characterized by an increase in absorbance at long wavelength (540 to 650 nm), and its rate of formation is dependent on substrate concentration and is controlled by a second-order rate constant of 4. 8 x 10(5) M-1 s-1 at pH 7.0 and 5 degrees C. In the second step, the enzyme-bound flavin is reduced to the dihydroflavin form. The rate of flavin reduction (23.4 s-1 at pH 7.0 and 5 degrees C) is independent of substrate concentration and is observed as a monophasic decrease in absorbance at 462 nm. The oxidative half-reaction proceeds in three kinetically resolvable steps. The first is due to the formation of a reduced enzyme-Codeinone charge-transfer complex and is observed at long wavelength (about 650 nm). The rate of charge-transfer complex formation is dependent on Codeinone concentration and is controlled by a second-order rate constant of 11.5 x 10(3) M-1 s-1 at pH 7.0 and 5 degrees C. The second step represents flavin reoxidation and is observed at 462 (absorption increase) and 650 nm (absorption decrease) and progresses with a rate (about 45 s-1) which is independent of Codeinone concentration. The third step is observed as a further small increase in absorbance at 462 nm and proceeds with a rate of about 2.5 s-1. This step most likely represents hydrocodone release from the oxidized enzyme. Analysis of the temperature dependence of the reductive half-reaction has enabled calculation of the entropic and enthalpic contributions for charge-transfer formation, charge-transfer decay (yielding free enzyme and substrate), and electron transfer to the enzyme-bound FMN, and the construction of a partial energy profile for the reaction catalyzed by MR. The reaction scheme and redox properties of MR are compared with those described previously for the closely related flavoprotein, old yellow enzyme. Although common features are identified, there are notable differences in the kinetic and redox properties of the two enzymes.
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Purification and characterization of morphinone reductase from Pseudomonas putida M10
Biochemical Journal, 1994Co-Authors: Christopher E. French, Neil C. BruceAbstract:The NADH-dependent morphinone reductase from Pseudomonas putida M10 catalyses the reduction of morphinone and Codeinone to hydromorphone and hydrocodone respectively. Morphinone reductase was purified from crude cell extracts to apparent homogeneity in a single affinity-chromatography step using Mimetic Yellow 2. The purified enzyme was a dimeric flavoprotein with two identical subunits of M(r) 41,100, binding non-covalently one molecule of FMN per subunit. The N-terminal sequence was PDTSFSNPGLFTPLQ. Morphinone reductase was active against morphinone, Codeinone, neopinone and 2-cyclohexen-1-one, but not against morphine, codeine or isocodeine. The apparent Km values for Codeinone and 2-cyclohexen-1-one were 0.26 mM and 5.5 mM respectively. The steroids progesterone and cortisone were potent competitive inhibitors; the apparent K1 for cortisone was 35 microM. The pH optimum for Codeinone reduction was 8.0 in phosphate buffer. No reverse reaction could be detected, and NADPH could not be used as a reducing substrate in place of NADH. Morphinone reductase activity was strongly inhibited by 0.01 mM CuSO4 and p-hydroxymercuribenzoate, suggesting the presence of a vital thiol group. Steady-state kinetic studies suggested a Ping Pong (substituted enzyme) kinetic mechanism; however, product-inhibition patterns were inconsistent with a classical Ping Pong mechanism. Morphinone reductase may, like several other flavoprotein dehydrogenases, operate by a hybrid two-site Ping Pong mechanism.