Resin‐Bound Aminofluorescein for C‐Terminal Labeling of Peptides: High‐Affinity Polarization Probes Binding to Polyproline‐Specific GYF Domains

Abstract

A polymer support for the solid-phase synthesis of C-terminally labeled carboxylic acids has been developed. Fluorophore-labeled peptides were constructed directly on the amino group of resin-bound aminofluorescein. Fmoc-protected aminofluorescein was coupled onto tritylpolystyrene, and the free phenolic hydroxyl positions of the fluorescein were blocked with suitable protecting groups. The mode of attachment was analyzed and found to be selective for the phenoxy ether linkage. The conditions for peptide synthesis on the labeling resin were investigated, and a small library of C-terminally labeled peptides was prepared. The fluorescence quantum yields of C-terminally labeled peptides were determined and indicated the suitability of the compounds for imaging and binding experiments. The obtained peptides were therefore investigated as fluorescence polarization probes. Two different proline-rich binding domains of the GYF family-CD2BP2 and PERQ2-were targeted by peptides labeled either C- or N-terminally. Reversible binding constants were determined by fluorescence polarization measurements and were verified by competition experiments with the corresponding unlabeled peptide. As a second control, the binding constants were measured by NMR titration experiments, recording the HSQC NMR spectra of (15)N-labeled proteins in the presence of the peptide polarization probes. Ligands with higher affinities than all others known previously were identified for both GYF domains. The competition assay with the developed fluorescent probe has a high statistical reliability and can thus be used for screening of GYF domain inhibitors.