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Radioactive Phosphorylation of Alcohols to Monitor Biocatalytic Diels-Alder Reactions

Nature has efficiently adopted phosphorylation for numerous biological key processes, spanning from cell signaling to energy storage and transmission. For the bioorganic chemist the number of possible ways to attach a single phosphate for radioactive labeling is surprisingly small. Here we describe... Full description

Journal Title: PLoS ONE 2011, Vol.6(6)
Main Author: Nierth, Alexander
Other Authors: Jäschke, Andres
Format: Electronic Article Electronic Article
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ID: E-ISSN: 1932-6203 ; DOI: 10.1371/journal.pone.0021391 ; PMCID: 3120863 ; PMID: 21731729
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title: Radioactive Phosphorylation of Alcohols to Monitor Biocatalytic Diels-Alder Reactions
format: Article
creator:
  • Nierth, Alexander
  • Jäschke, Andres
subjects:
  • Research Article
  • Biology
  • Chemistry
ispartof: PLoS ONE, 2011, Vol.6(6)
description: Nature has efficiently adopted phosphorylation for numerous biological key processes, spanning from cell signaling to energy storage and transmission. For the bioorganic chemist the number of possible ways to attach a single phosphate for radioactive labeling is surprisingly small. Here we describe a very simple and fast one-pot synthesis to phosphorylate an alcohol with phosphoric acid using trichloroacetonitrile as activating agent. Using this procedure, we efficiently attached the radioactive phosphorus isotope 32 P to an anthracene diene, which is a substrate for the Diels-Alderase ribozyme—an RNA sequence that catalyzes the eponymous reaction. We used the 32 P-substrate for the measurement of RNA-catalyzed reaction kinetics of several dye-labeled ribozyme variants for which precise optical activity determination (UV/vis, fluorescence) failed due to interference of the attached dyes. The reaction kinetics were analyzed by thin-layer chromatographic separation of the 32 P-labeled reaction components and densitometric analysis of the substrate and product radioactivities, thereby allowing iterative optimization of the dye positions for future single-molecule studies. The phosphorylation strategy with trichloroacetonitrile may be applicable for labeling numerous other compounds that contain alcoholic hydroxyl groups.
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identifier: E-ISSN: 1932-6203 ; DOI: 10.1371/journal.pone.0021391 ; PMCID: 3120863 ; PMID: 21731729
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  • 1932-6203
  • 19326203
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titleRadioactive Phosphorylation of Alcohols to Monitor Biocatalytic Diels-Alder Reactions
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descriptionNature has efficiently adopted phosphorylation for numerous biological key processes, spanning from cell signaling to energy storage and transmission. For the bioorganic chemist the number of possible ways to attach a single phosphate for radioactive labeling is surprisingly small. Here we describe a very simple and fast one-pot synthesis to phosphorylate an alcohol with phosphoric acid using trichloroacetonitrile as activating agent. Using this procedure, we efficiently attached the radioactive phosphorus isotope 32 P to an anthracene diene, which is a substrate for the Diels-Alderase ribozyme—an RNA sequence that catalyzes the eponymous reaction. We used the 32 P-substrate for the measurement of RNA-catalyzed reaction kinetics of several dye-labeled ribozyme variants for which precise optical activity determination (UV/vis, fluorescence) failed due to interference of the attached dyes. The reaction kinetics were analyzed by thin-layer chromatographic separation of the 32 P-labeled reaction components and densitometric analysis of the substrate and product radioactivities, thereby allowing iterative optimization of the dye positions for future single-molecule studies. The phosphorylation strategy with trichloroacetonitrile may be applicable for labeling numerous other compounds that contain alcoholic hydroxyl groups.
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abstractNature has efficiently adopted phosphorylation for numerous biological key processes, spanning from cell signaling to energy storage and transmission. For the bioorganic chemist the number of possible ways to attach a single phosphate for radioactive labeling is surprisingly small. Here we describe a very simple and fast one-pot synthesis to phosphorylate an alcohol with phosphoric acid using trichloroacetonitrile as activating agent. Using this procedure, we efficiently attached the radioactive phosphorus isotope 32 P to an anthracene diene, which is a substrate for the Diels-Alderase ribozyme—an RNA sequence that catalyzes the eponymous reaction. We used the 32 P-substrate for the measurement of RNA-catalyzed reaction kinetics of several dye-labeled ribozyme variants for which precise optical activity determination (UV/vis, fluorescence) failed due to interference of the attached dyes. The reaction kinetics were analyzed by thin-layer chromatographic separation of the 32 P-labeled reaction components and densitometric analysis of the substrate and product radioactivities, thereby allowing iterative optimization of the dye positions for future single-molecule studies. The phosphorylation strategy with trichloroacetonitrile may be applicable for labeling numerous other compounds that contain alcoholic hydroxyl groups.
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pmid21731729
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date2011-06-22