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) N^α-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: K_i 0.14 nM, sulfo-Cy5-labeled probe 21: K_i 0.094 nM) and high stability in human plasma (t_1/2 ≫ 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.