Expedient synthesis of a modular phosphate affinity reagent.

Abstract

Isolation and identification of phosphorylated macromolecules is essential for the deconvolution of most biological regulatory networks. Koike and co-workers recently reported the application of a dinuclear zinc-(pyridylmethyl)amine complex to phosphate-specific affinity purifications and gave it the shorthand name "phos-tag". This complex is valuable for studying phosphorylation because it binds selectively to phosphate dianion in the presence of acidic functional groups at physiological pH, and because the binding is largely independent of molecular context. These properties of phos-tag recommend it for applications in phosphoproteomics, metabolomics, and nucleic acid biology. The catch has been that the molecule is difficult to make and prohibitively expensive to buy. Here, we describe an efficient and inexpensive synthesis of a phos-tag derivative with a versatile alkyne handle. The alkyne handle allows for attachment of phos-tag to alkyl azides via the copper(I)-catalyzed azide-alkyne cycloaddition reaction ("click chemistry"). We characterize the phosphate binding behavior of the new phos-tag derivative in a variety of experimental assays, including its conjugation to a fluorescent reporter, to acrylamide gels, and to sepharose chromatography resin. The synthesis we report should enable a broader use of phos-tag for phosphate-related biochemistry, as both an analytical and a preparative reagent.