Radioactive Phosphorylation of Alcohols to Monitor Biocatalytic Diels-Alder Reactions 英文参考文献.docVIP

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Radioactive Phosphorylation of Alcohols to Monitor Biocatalytic Diels-Alder Reactions 英文参考文献.doc

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Radioactive Phosphorylation of Alcohols to Monitor Biocatalytic Diels-Alder Reactions 英文参考文献

RadioactivePhosphorylationofAlcoholstoMonitor BiocatalyticDiels-AlderReactions AlexanderNierth,AndresJa¨schke* InstituteofPharmacyandMolecularBiotechnology(IPMB),HeidelbergUniversity,Heidelberg,Germany Abstract Nature has efficiently adopted phosphorylation for numerous biological key processes, spanning from cell signaling to energystorageandtransmission.Forthebioorganicchemistthenumberofpossiblewaystoattachasinglephosphatefor radioactivelabelingissurprisinglysmall.Herewedescribeaverysimpleandfastone-potsynthesistophosphorylatean alcoholwithphosphoricacidusingtrichloroacetonitrileasactivatingagent.Usingthisprocedure,weefficientlyattached theradioactivephosphorusisotope 32Ptoananthracenediene,whichisasubstratefortheDiels-Alderaseribozyme—an RNAsequencethatcatalyzestheeponymousreaction.Weusedthe 32P-substrateforthemeasurementofRNA-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 32P-labeled reaction components and densitometric analysis of the substrate and productradioactivities,therebyallowingiterativeoptimizationofthedyepositionsforfuturesingle-moleculestudies.The phosphorylationstrategywithtrichloroacetonitrilemaybeapplicableforlabelingnumerousothercompoundsthatcontain alcoholichydroxylgroups. Citation:NierthA,Ja¨schke A(2011)RadioactivePhosphorylationofAlcoholstoMonitorBiocatalyticDiels-AlderReactions.PLoSONE6(6):e21391.doi:10.1371/ journal.pone.0021391 Editor:ShuguangZhang,MassachusettsInstituteofTechnology,UnitedStatesofAmerica ReceivedApril26,2011;AcceptedMay26,2011;PublishedJune22,2011 Copyright: ? 2011 Nierth, Ja¨schke. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourc