High-Affinity and Proteolytically Stable Peptidic Fluorescent NTS₁R Ligands

Labeled ligands for the neurotensin receptor 1 (NTS1R), which is expressed in the CNS, the gastrointestinal tract, and in malignant tumors, are needed to investigate NTS1R-ligand binding and NTS1R expression. Aiming for fluorescence-labeled neurotensin(8-13)-derived NTS1R ligands with high affinity and proteolytic stability, several previous approaches were combined: (1) replacement of Arg8 by an amino-functionalized carbamoylated arginine, allowing conjugation to a fluorophore, (2) Nα-methylation of Arg8 and replacement of Tyr by β,β-dimethyl-l-Tyr11, conferring proteolytic stability, and (3) replacement of Leu13 by trimethylsilyl-Ala, boosting binding affinity. This strategy gave fluorescent NTS1R ligands with unprecedented NTS1R binding affinity (5-TAMRA-labeled ligand 19: Ki 0.14 nM, sulfo-Cy5-labeled probe 21: Ki 0.094 nM) and high stability in human plasma (t1/2 ≫ 48 h). Their suitability for competition binding studies (flow cytometry; 19, 21) and the imaging of NTS1R expression in living cells (confocal microscopy, biomolecular imaging; 19, 21) and tumor tissue (biomolecular imaging; 21) is demonstrated.