Diversification of polyphosphate end-labeling via bridging molecules

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 react...

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Bibliographic Details
Published in:PloS one 2020-08, Vol.15 (8), p.e0237849-e0237849
Main Authors: Baker, Catherine J, Smith, Stephanie A, Morrissey, James H
Format: Article
Language:English
Subjects:
Acids
Affinity labeling
Amines
Biology and Life Sciences
Biotin
Carbodiimide
Chemical properties
Chemical synthesis
Chromatography
Coupling (molecular)
Efficiency
Epitopes
Fluorescent dyes
Fluorescent indicators
Labeling
Labelling
Methods
Peptides
Phosphates
Physical Sciences
Polymers
Polyphosphates
Reagents
Research and analysis methods
Structure
Tandem affinity purification
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Summary: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.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0237849