Receptor for anionic pyrene derivatives provides the basis for new biomembrane assays

This study describes a new receptor cyclen 1 capable of strong selective binding of pyrene-based anionic dyes under near-physiological conditions. This receptor comprises four naphthylthiourea groups tethered to a cyclen core via an ester linkage. The complexation behavior of cyclen 1 receptor is characterized by a series of (1)H NMR, microcalorimetry, UV-vis, and fluorometry experiments. The relevance of structural features of this receptor to its recognition function is assessed using control compounds that lack some of the groups found in cyclen 1. The specificity of cyclen 1 toward pyrene-based dyes is assessed through experiments using dyes with different molecular organization. The most important finding was the ability of cyclen 1 to bind efficiently to a pH-sensitive dye pyranine, a dye that is commonly used in various biomembrane assays. The high affinity of cyclen 1 to pyranine, its impermeability to the lipid bilayer membrane, fast kinetics of binding, and ability to quench the pyranine's fluorescence were used as a basis for a new membrane leakage assay. This membrane leakage assay is fully compatible with the commonly applied pH-stat transport assay, and therefore it allows for differentiation of the ion transport and nonselective leakage mechanisms within a single set of experiments. The ability of cyclen 1 to quench the fluorescence of pyranine also finds limited applicability to the detection of endovesiculation.