Synthesis of γ-Phosphate-Labeled and Doubly Labeled Adenosine Triphosphate Analogs.
Curr Protoc Nucleic Acid Chem
; 60: 13.14.1-13.14.25, 2015 Mar 09.
Article
em En
| MEDLINE
| ID: mdl-25754889
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.
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Base de dados:
MEDLINE
Assunto principal:
Fosfatos
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Coloração e Rotulagem
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Trifosfato de Adenosina
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Nucleosídeos
Idioma:
En
Ano de publicação:
2015
Tipo de documento:
Article