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David M. Perrin - One of the best experts on this subject based on the ideXlab platform.
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Meeting key synthetic challenges in amanitin synthesis with a new cytotoxic analog: 5′-hydroxy-6′-deoxy-amanitin
Chemical Science, 2020Co-Authors: Alla Pryyma, Kaveh Matinkhoo, Antonio A. W. L. Wong, David M. PerrinAbstract:Appreciating the need to access synthetic analogs of amanitin, here we report the synthesis of 5′-hydroxy-6′-deoxy-amanitin, a novel, rationally-designed bioactive analog and constitutional isomer of α-amanitin, that is anticipated to be used as a payload for antibody drug conjugates. In completing this synthesis, we meet the challenge of diastereoselective sulfoxidation by presenting two high-yielding and diastereoselective sulfoxidation approaches to afford the more toxic (R)-sulfoxide.
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Synthesis of the Death-Cap Mushroom Toxin α-Amanitin.
Journal of the American Chemical Society, 2018Co-Authors: Kaveh Matinkhoo, Alla Pryyma, Mihajlo Todorovic, Brian O. Patrick, David M. PerrinAbstract:α-Amanitin is an extremely toxic bicyclic octapeptide isolated from the death-cap mushroom, Amanita phalloides. As a potent inhibitor of RNA polymerase II, α-amanitin is toxic to eukaryotic cells. Recent interest in α-amanitin arises from its promise as a payload for antibody–drug conjugates. For over 60 years, A. phalloides has been the only source of α-amanitin. Here we report a synthesis of α-amanitin, which surmounts the key challenges for installing the 6-hydroxy-tryptathionine sulfoxide bridge, enantioselective synthesis of (2S,3R,4R)-4,5-dihydroxy-isoleucine, and diastereoselective sulfoxidation.
Fernando Ramos - One of the best experts on this subject based on the ideXlab platform.
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development optimization and application of an analytical methodology by ultra performance liquid chromatography tandem mass spectrometry for determination of Amanitins in urine and liver samples
Analytica Chimica Acta, 2013Co-Authors: Marta Leite, Andreia Freitas, Anabela Marisa Azul, Jorge Barbosa, Saul C Costa, Fernando RamosAbstract:Abstract Amanitins, highly toxic cyclopeptides isolated from various Amanita species, are the most potent poisons accounting for the hazardous effects on intestinal epithelium cells and hepatocytes, and probably the sole cause of fatal human poisoning. The present study was focused on the development, optimization and application of an analytical methodology by ultra performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS), following urine and liver sample preparation by protein precipitation with organic solvents, and solid phase extraction (SPE) procedure, for the determination of the amatoxins, α- and β-amanitin. Linearity, detection and quantification limits, selectivity, sensitivity, intra and inter-assay precision and recovery were studied, in order to guarantee reliability in the analytical results. The developed method proved to be specific and selective, with LOD (Limit of Detection) values for α- and β-amanitin of 0.22 and 0.20 ng mL −1 in urine and 10.9 and 9.7 ng g −1 in liver, respectively. LOQ (Limit of Quantification) values ranged from 0.46 to 0.57 ng mL −1 in urine and 12.3–14.7 ng g −1 in tissue, for both Amanitins. Linearity, in the range of 10.0–200.0 ng mL −1 or ng g −1 , shows that coefficients of correlation were greater than 0.997 for α-amanitin and 0.993 for β-amanitin. Precision was checked at three levels during three consecutive days with intra-day and inter-day coefficients of variation not greater than 15.2%. The extraction recovery presents good results for the concentrations analyzed, with values ranging from 90.2 to 112.9% for both matrices. Thus, the proposed analytical method is innovative, presents a high potential in the identification, detection and determination of α- and β-Amanitins in urine and tissue samples, as well as in other biological samples, such as kidney and mushrooms.
Akira Ishii - One of the best experts on this subject based on the ideXlab platform.
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Simple analysis of α-amanitin and β-amanitin in human plasma by liquid chromatography-mass spectrometry
Forensic Toxicology, 2010Co-Authors: Tanahashi Masakazu, Rina Kaneko, Yukari Hirata, Makoto Hamajima, Tetsuya Arinobu, Tadashi Ogawa, Akira IshiiAbstract:A number of reports are available in the literature that describe liquid chromatography-mass spectrometry (LC-MS) and LC-tandem mass spectrometry (LC-MS-MS) analysis of Amanitins, very toxic mushroom toxins, in biological samples. However, the extractive pretreatment methods and LC separation column materials vary remarkably according to the different reports. This communication presents a very simple and suffi ciently sensitive method for LC-MS analysis of Amanitins. A plasma sample was diluted with distilled water and buffer solution, and applied to a Discovery DSC 18 column (500 mg packing material), followed by washing with distilled water and elution with methanol. The extract, after evaporation and reconstitution in mobile phase solution, was subjected to LC-MS analysis with a conventional octadecyl LC separation column. The selected ion monitoring of α-amanitin and β-amanitin at m/z 919–921 and m/z 920–922, respectively, gave symmetrical peaks and good separation of both amanitin peaks. Using an external calibration method, linearity, detection limits, recovery rates, and precision were tested; they were all satisfactory. To our knowledge, the present method gives the simplest LC-MS analysis for Amanitins among those so far reported. We recommend the method for use in actual forensic and clinical toxicological analysis of Amanitins in biological samples.
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SHORT COMMUNICATION for Forensic Toxicology Simple analysis of α−amanitin and β−amanitin in human plasma by LC−MS
2010Co-Authors: Tanahashi Masakazu, Rina Kaneko, Yukari Hirata, Makoto Hamajima, Tadashi Ogawa, Akira Ishii, M. Tanahashi, Yoshihiro HirataAbstract:Although some reports dealing with liquid chromatography (LC)– mass spectrometry (MS) (−MS) analysis of Amanitins, very toxic mushroom toxins, in biological samples are available, the extractive pretreatment methods and also LC separation column materials remarkably vary according to the different reports. In this communication, we present a very simple and sufficiently sensitive method for LC−MS analysis of Amanitins. A plasma sample was diluted with distilled water and buffer solution, and applied to a Discovery DSC 18 column (500 mg packing material), followed by washing with distilled water and elution with methanol. The extract, after evaporation and reconstitution in mobile phase solution, was subjected to LC−MS analysis with the conventional octadecyl LC separation column. The selected ion monitoring of α−amanitin and β−amanitin at m/z 919−921 and m/z 920−922, respectively, gave symmetrical peaks and good separation of both amanitin peaks. Using the external calibration method, linearity, detection limits, recovery rates and precision were tested; they were all satisfactory. To our knowledge, the present method seems to be the simpliest LC−MS analysis for Amanitins among those so far reported. We can recommend it to be widely used in actual forensic and clinical toxicological analysis of Amanitins in biological samples.
Olivier Bensaude - One of the best experts on this subject based on the ideXlab platform.
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In vivo degradation of RNA polymerase II largest subunit triggered by alpha-amanitin.
Nucleic acids research, 1996Co-Authors: Van Trung Nguyen, Federico Giannoni, Marie-françoise Dubois, Sook-jae Seo, Marc Vigneron, Claude Kedinger, Olivier BensaudeAbstract:Alpha-Amanitin is a well-known specific inhibitor of RNA polymerase II (RNAPII) in vitro and in vivo. It is a cyclic octapeptide which binds with high affinity to the largest subunit of RNAPII, RPB1. We have found that in murine fibroblasts exposure to alpha-amanitin triggered degradation of the RPB1 subunit, while other RNAPII subunits, RPB5 and RPB8, remained almost unaffected. Transcriptional inhibition in alpha-amanitin-treated cells was slow and closely followed the disappearance of RPB1. The degradation rate of RPB1 was alpha-amanitin dose dependent and was not a consequence of transcriptional arrest. Alpha-Amanitin-promoted degradation of RPB1 was prevented in cells exposed to actinomycin D, another transcriptional inhibitor. Epitope-tagged recombinant human RPB1 subunits were expressed in mouse fibroblasts. In cells exposed to alpha-amanitin the wild-type recombinant subunit was degraded like the endogenous protein, but a mutated alpha-amanitin-resistant subunit remained unaffected. Hence, alpha-amanitin did not activate a proteolytic system, but instead its binding to mRPB1 likely represented a signal for degradation. Thus, in contrast to other inhibitors, such as actinomycin D or 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole, which reversibly act on transcription, inhibition by alpha-amanitin cannot be but an irreversible process because of the destruction of RNAPII.
Kaveh Matinkhoo - One of the best experts on this subject based on the ideXlab platform.
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Meeting key synthetic challenges in amanitin synthesis with a new cytotoxic analog: 5′-hydroxy-6′-deoxy-amanitin
Chemical Science, 2020Co-Authors: Alla Pryyma, Kaveh Matinkhoo, Antonio A. W. L. Wong, David M. PerrinAbstract:Appreciating the need to access synthetic analogs of amanitin, here we report the synthesis of 5′-hydroxy-6′-deoxy-amanitin, a novel, rationally-designed bioactive analog and constitutional isomer of α-amanitin, that is anticipated to be used as a payload for antibody drug conjugates. In completing this synthesis, we meet the challenge of diastereoselective sulfoxidation by presenting two high-yielding and diastereoselective sulfoxidation approaches to afford the more toxic (R)-sulfoxide.
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Synthesis of the Death-Cap Mushroom Toxin α-Amanitin.
Journal of the American Chemical Society, 2018Co-Authors: Kaveh Matinkhoo, Alla Pryyma, Mihajlo Todorovic, Brian O. Patrick, David M. PerrinAbstract:α-Amanitin is an extremely toxic bicyclic octapeptide isolated from the death-cap mushroom, Amanita phalloides. As a potent inhibitor of RNA polymerase II, α-amanitin is toxic to eukaryotic cells. Recent interest in α-amanitin arises from its promise as a payload for antibody–drug conjugates. For over 60 years, A. phalloides has been the only source of α-amanitin. Here we report a synthesis of α-amanitin, which surmounts the key challenges for installing the 6-hydroxy-tryptathionine sulfoxide bridge, enantioselective synthesis of (2S,3R,4R)-4,5-dihydroxy-isoleucine, and diastereoselective sulfoxidation.
