Towards Engineering Pathways for the Synthesis of Analgesics and Antitussives<i><sup>a</sup></i>

Bruce, Neil C.; Caswell, Deborah; French, Christopher E.; Hailes, A M; Long, Marianne T.; Willey, David L.

doi:10.1111/j.1749-6632.1994.tb47380.x

Cited by 14 publications

(5 citation statements)

References 28 publications

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“…reductase system has been applied to the biocatalytic production of the medically useful semisynthetic opiates hydromorphone and hydrocodone [9][10][11].…”

Section: Figure 1 Reaction Catalysed By Morphine Dehydrogenasementioning

confidence: 99%

Mechanistic studies of morphine dehydrogenase and stabilization against covalent inactivation

Walker¹,

French²,

Rathbone

et al. 2000

Biochemical Journal

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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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“…reductase system has been applied to the biocatalytic production of the medically useful semisynthetic opiates hydromorphone and hydrocodone [9][10][11].…”

Section: Figure 1 Reaction Catalysed By Morphine Dehydrogenasementioning

confidence: 99%

Mechanistic studies of morphine dehydrogenase and stabilization against covalent inactivation

Walker¹,

French²,

Rathbone

et al. 2000

Biochemical Journal

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“…Specimens obtained more than 72 h after the last heroin/morphine dose was taken are especially likely to fall into these categories . A step such as β- d -glucuronidase hydrolysis will always be required to determine morphine-3-glucuronide with a morphine dehydrogenase-based method, as morphine dehydrogenase has no activity against conjugated morphine and partly because morphine dehydrogenase has an apparent K m for codeine ∼10-fold less than that for morphine …”

Section: Resultsmentioning

confidence: 99%

“…Twelve compounds described by Syva as “being members of a specific assay class” give a positive response to the EMIT dau opiate test, although sensitivity to each compound is different. The specificity of the luminescence assay is conferred by the enzyme morphine dehydrogenase, which has activity against morphine, codeine, codeine N -oxide, nalorphine, and ethylmorphine .…”

Section: Discussionmentioning

confidence: 99%

Bioluminescent Assay for Heroin and Its Metabolites

1996

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Illicit heroin is trafficked as a solid particulate drug, while heroin abuse is monitored by testing urine samples for its principal metabolites, morphine and morphine-3-glucuronide. Two novel bacterial enzymes were used in the development of a linked-enzyme assay for heroin and its metabolites: heroin esterase, which converts heroin to morphine, and morphine dehydrogenase, which oxidizes morphine to morphinone with the concomitant reduction of NADP+. A bioluminescent assay involving heroin esterase, morphine dehydrogenase, and the bacterial luciferase from Vibrio harveyi was developed and shown to be sensitive to 89 ng/mL heroin and 2.0 ng/mL morphine. Excellent correlation with the results from 83 authentic samples submitted for urine drug screening at a hospital laboratory was obtained. The bioluminescent assay exhibited greater specificity and speed than current immunological screening methods. A novel format of the bioluminescent assay involving immobilized enzymes was sensitive to 101 ng (250 pmol) of heroin and responded well to particulate heroin. This form of the test was sensitive enough to respond to one or two typical particles of illicit heroin.

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“…Early attempts to prepare novel alkaloid analogs using microorganisms were limited to exploring the range of transformations available, relying on fortuitous side reactions due to enzymes with broad substrate specificities. More recently, work has concentrated on the degradation of morphine alkaloids by soil bacteria and the use of specific enzymes that initiate the metabolism of morphine for the synthesis of existing clinically useful opiate drugs and new analogs (1).…”

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confidence: 99%

Transformations of morphine alkaloids by Pseudomonas putida M10

Long

Hailes

Kirby

et al. 1995

Appl Environ Microbiol

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The oxidation of morphine by washed-cell incubations of Pseudomonas putida M10 gave rise to a large number of transformation products including hydromorphone (dihydromorphinone), 14␤-hydroxymorphine, 14␤-hydroxymorphinone, and dihydromorphine. Similarly, in incubations with oxymorphone (14␤-hydroxydihydromorphinone) as substrate, the major transformation product was identified as oxymorphol (14␤-hydroxydihydromorphine). The identities of all these biological products were confirmed by mass spectrometry and 1 H nuclear magnetic resonance spectroscopy. This is the first report describing structural evidence for the biological synthesis of 14␤-hydroxymorphine and 14␤-hydroxymorphinone. These products have applications as intermediates in the synthesis of semisynthetic opiate drugs.

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Towards Engineering Pathways for the Synthesis of Analgesics and Antitussives^a

Cited by 14 publications

References 28 publications

Mechanistic studies of morphine dehydrogenase and stabilization against covalent inactivation

Mechanistic studies of morphine dehydrogenase and stabilization against covalent inactivation

Bioluminescent Assay for Heroin and Its Metabolites

Transformations of morphine alkaloids by Pseudomonas putida M10

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Towards Engineering Pathways for the Synthesis of Analgesics and Antitussivesa

Cited by 14 publications

References 28 publications

Mechanistic studies of morphine dehydrogenase and stabilization against covalent inactivation

Mechanistic studies of morphine dehydrogenase and stabilization against covalent inactivation

Bioluminescent Assay for Heroin and Its Metabolites

Transformations of morphine alkaloids by Pseudomonas putida M10

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Product

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Towards Engineering Pathways for the Synthesis of Analgesics and Antitussives^a