Abstract
Investigation of the biological roles of inorganic polyphosphate has been facilitated by our previous development of a carbodiimide-based method for covalently coupling primary amine-containing molecules to the terminal phosphates of polyphosphate. We now extend that approach by optimizing the reaction conditions and using readily available “bridging molecules” containing a primary amine and an additional reactive moiety, including another primary amine, a thiol or a click chemistry reagent such as dibenzocyclooctyne. This two-step labeling method is used to covalently attach commercially available derivatives of biotin, peptide epitope tags, and fluorescent dyes to the terminal phosphates of polyphosphate. Additionally, we report three facile methods for purifying conjugated polyphosphate from excess reactants.
Highlights
Polyphosphate is a linear polymer of inorganic orthophosphates that is widespread in biology [1]
We previously reported a method for covalently coupling the terminal phosphates of polyP to primary amine-containing molecules via phosphoramidate linkages, in a reaction mediated by the water-soluble carbodiimide, N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide (EDAC) [6]
That including Ca2+ in EDAC-mediated labeling reactions increases labeling efficiency by promoting chain cleavage and increasing the number of polyP ends that can react with primary amines
Summary
Polyphosphate (polyP) is a linear polymer of inorganic orthophosphates that is widespread in biology [1]. Recent years have seen rapidly increasing interest in understanding the biological roles of this polymer in both lower organisms and mammals [2,3,4,5]. Progress in this area has been hampered by a dearth of methods and tools for manipulating and analyzing polyP. We previously reported a method for covalently coupling the terminal phosphates of polyP to primary amine-containing molecules via phosphoramidate linkages, in a reaction mediated by the water-soluble carbodiimide, N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide (EDAC) [6]. We report optimized reaction conditions for attaching bridging molecules
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