Phosphate oxoanions and phosphorylated biomolecules (such as nucleotides, lipids, and proteins) play key roles in a wide range of biological processes. The ability to selectively detect these ions in the presence of each other has numerous applications in biochemistry and biomedicine. However, receptors and sensors that can discriminate between polyphosphate species with high selectivity and in biologically relevant conditions are rare.
In this Account, we show how peptides (both cyclic and linear) can be used to position two zinc(II)dipicolylamine [Zn(II)DPA] binding sites in an appropriate arrangement to provide selective binding of pyrophosphate (PPi) in the presence of other polyphosphate species, including ATP, and in complex media such as cell growth buffer.
The use of peptide scaffolds to position the Zn(II)DPA anion binding sites allowed the synthesis of small receptor libraries in which the arrangement of the two binding sites could be subtly altered to evaluate the factors affecting both binding selectivity and affinity for PPi. We altered a number of structural elements including peptide length, cyclic peptide ring size, amino acid composition, the positioning of the binding sites with respect to one another, and the relative stereochemistry of the peptides. Backbone modified cyclic peptides based on the Lissoclinum class of natural products were initially employed to provide an added degree of preorganization to the receptors, although it was subsequently found that short, flexible bis[Zn(II)DPA]-functionalized linear peptides are also effective scaffolds for selective pyrophosphate recognition.
The peptidic receptors were successfully employed for the detection of PPi in aqueous media by indicator displacement assays using both colorimetric and fluorescent indicators, with the best compounds able to bind to PPi selectively in both cell growth media and artificial urine and also allow the accurate determination of PPi concentrations in physiologically relevant ranges (micromolar concentrations) in these complex media. Improved pyrophosphate selectivity was observed upon increasing the complexity of the media from HEPES buffer to cell growth media (Krebs saline).
Pyrophosphate sensors in which a fluorescent indicator was covalently attached to either a linear or cyclic peptide scaffold through a flexible linker were then constructed. When the Zn(II)DPA binding sites and the indicator were suitably placed with respect to one another on the peptide scaffold, these ‘intramolecular indicator displacement assays’ showed improved selectivity for PPi over other polyphosphate anions, such as ATP, when compared to the intermolecular indicator displacement assays. This observation provides the basis for the design and application of future PPi sensors in biochemistry and biomedicine.