Synthesis of γ-Phosphate-Labeled and Doubly Labeled Adenosine Triphosphate Analogs

Stephan M. Hacker, Moritz Welter, Andreas Marx
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引用次数: 2

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. © 2015 by John Wiley & Sons, Inc.

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γ-磷酸标记和双标记三磷酸腺苷类似物的合成
本单元描述了γ-磷酸标记和双标记三磷酸腺苷(ATP)类似物的合成,并利用Crotalus adamanteus(蛇毒磷酸二酯酶)中的磷酸二酯酶I对其进行了表征;SVPD)。在合成的关键步骤中,ATP或ATP类似物通过烷基化反应,在末端的磷酸基上用含叠氮化物的连接剂修饰ATP或ATP类似物,其连接剂含有与核苷相连的三氟乙酰胺基团。随后,通过将一个官能团转化为胺并偶联到n -羟基琥珀酰亚胺酯,将不同的标签引入到连接剂中。具体地说,叠氮化物的Staudinger反应被用作在各种标签存在下获得胺的直接手段。此外,研究了双标记ATP类似物在SVPD酶切后的荧光特性。©2015 by John Wiley &儿子,Inc。
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Current Protocols in Nucleic Acid Chemistry
Current Protocols in Nucleic Acid Chemistry Chemistry-Organic Chemistry
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期刊介绍: Published in association with International Society for Nucleosides, Nucleotides & Nucleic Acids (IS3NA) , Current Protocols in Nucleic Acid Chemistry is equally valuable for biotech, pharmaceutical, and academic labs. It is the resource for designing and running successful research projects in the rapidly growing and changing field of nucleic acid, nucleotide, and nucleoside research.
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