title: Synthesis of γ-Phosphate-Labeled and Doubly Labeled Adenosine Triphosphate Analogs.
abstract: This unit describes the synthesis of γ-phosphate-labeled and doubly labeled adenosine triphosphate (ATP) analogs and their characterization using the phosphodiesterase I from Crotalus adamanteus (snake venom phosphodiesterase; SVPD). In the key step of the synthesis, ATP or an ATP analog, bearing a linker containing a trifluoroacetamide group attached to the nucleoside, are modified with an azide-containing linker at the terminal phosphate using an alkylation reaction. Subsequently, different labels are introduced to the linkers by transformation of one functional group to an amine and coupling to an N-hydroxysuccinimide ester. Specifically, the Staudinger reaction of the azide is employed as a straightforward means to obtain an amine in the presence of various labels. Furthermore, the fluorescence characteristics of a fluorogenic, doubly labeled ATP analog are investigated following enzymatic cleavage by SVPD.
authors.affiliation: Department of Chemistry and Konstanz Research School Chemical Biology, University of Konstanz, Konstanz, Germany.
authors.forename: Stephan M
authors.initials: SM
authors.lastname: Hacker
authors.affiliation: Department of Chemistry and Konstanz Research School Chemical Biology, University of Konstanz, Konstanz, Germany.
authors.forename: Moritz
authors.initials: M
authors.lastname: Welter
authors.affiliation: Department of Chemistry and Konstanz Research School Chemical Biology, University of Konstanz, Konstanz, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Marx
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000138:chemical synthesis* / Q000737:chemistry; D001386:Azides / Q000737:chemistry; D005453:Fluorescence; D009705:Nucleosides / Q000138:chemical synthesis / Q000737:chemistry*; D010710:Phosphates / Q000737:chemistry*; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry; D013194:Staining and Labeling; D013388:Succinimides
chemical_list: D001386:Azides; D009705:Nucleosides; D010710:Phosphates; D013388:Succinimides; D000255:Adenosine Triphosphate; D010727:Phosphoric Diester Hydrolases; C001426:N-hydroxysuccinimide
keywords: Förster resonance energy transfer; Staudinger reaction; alkylation reaction; enzyme probes; modified nucleotides