radioactive phosphorylation of alcohols to monitor biocatalytic diels-alder reactions放射性的磷酸化醇监控biocatalytic diels-alder反应.pdfVIP

Radioactive Phosphorylation of Alcohols to Monitor Biocatalytic Diels-Alder Reactions ¨ Alexander Nierth, Andres Jaschke* Institute of Pharmacy and Molecular Biotechnology (IPMB), Heidelberg University, Heidelberg, Germany Abstract 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 32P to an anthracene diene, which is a substrate for the Diels-Alderase ribozyme—an RNA sequence that catalyzes the eponymous reaction. We used the 32P-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 32P-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. ¨ Citation: Nierth A, Jaschke A (2011) Radioactive Phosphorylation of Alcohols to Monitor Biocatalytic Diels-Alder Reactions. PLoS ONE 6(6): e21391. doi:10.137