Synthesis and characterization of the first fluorescent nonpeptide NPY Y1 receptor antagonist

Cyanine-5-labelled neuropeptide Y (NPY) was demonstrated to be an ideal universal fluorescent ligand for the combined investigation of NPY Y(1), Y(2) and Y(5) receptors. With respect to improved stability, detection of receptor subtypes in cells and tissues, and prevention of receptor internalization, small nonpeptidic fluorescent antagonists should be superior. Here we present a set of four fluorescent nonpeptide NPY Y(1) receptor (Y(1)R) antagonists. The highest affinity was obtained by labelling an N(G)-(6-aminohexanoyl)argininamide derived from the Y(1)R antagonist BIBP 3226, with Py-1, a small pyrylium dye. The fluorescent pyridinium-type Y(1)R antagonist, compound 4 had K(i) values of 29 nM and 2.7 nM, which were determined by radioligand binding and flow cytometry under equilibrium conditions, respectively; 4 had a K(b) value of 0.6 nM (Ca(2+) assay). The large Stoke's shift (541 vs. 615 nm) in buffer (PBS, pH 7.4) in the presence of 1% BSA and the red emission (quantum yield 56%) are advantageous with respect to the signal-to-noise ratio. The new probe was successfully used in fluorescence-based binding experiments evaluated by flow cytometry and confocal microscopy; this demonstrates the potential of pyrylium dyes for the preparation of fluorescent ligands that are applicable for the study of G protein-coupled receptors on living cells.