Cationic imidazolium macrocycles enable array-based, site-selective optical detection of peptide phosphorylation

Simple polycationic imidazolium macrocycles, formed in one step from commercial precursors, show strong affinity for peptide strands with anionic and hydrophobic character, and can be paired with styrylpyridinium dyes in an arrayed format to enable site- and state-selective optical detection of phosphorylation at serine residues. Even though they show affinity for anions in aqueous solution, the cationic macrocyclic hosts do not bind the modified serine phosphate residues themselves, rather they exploit π-stacking interactions with the native peptide backbone and different sidechains, allowing interaction with both modified and unmodified peptides. When combined with cationic indicators that can also bind the peptide targets, a complex equilibrium is established consisting of peptide : host, peptide : dye and dye : host assemblies, as well as peptide : dye : host ternary complexes. These multiple equilibria enable differentiation of minuscule changes in peptide target, differentiating phosphate residues at different sites on otherwise identical peptide strands.