N(G)-Functionalized Arginines as Arginine Substitutes: Preparation and Biological Characterization of Novel-Type Fluorescence and Radiolabelled Peptidic Receptor Ligands

• Applicant: Privatdozent Dr. Max Keller
• Subject Area: Pharmacy
• Term: since 2013
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 233221909

Radio- and fluorescence labeling of bioactive peptides gives comparatively convenient access to molecular tools, which play an important role in pharmaceutical, biochemical and medical research. Proteinogenic amino acids, allowing an efficient and diverse conjugation of peptides, are lysine, cysteine and unmasked N-terminal amino acids. However, the modification of peptides usually affects their biological activity and many peptides are devoid of lysine, cysteine or a free N-terminus. Therefore, a new, widely applicable labeling strategy for peptides, based on the replacement of arginine by an amino-functionalized, NG-carbamoylated arginine, was introduced within the original previous project. The modified, functionalized arginine enables a versatile conjugation (labeling) of the peptides, thereby retaining their bioactivity. This was demonstrated for arginine-containing ligands of neurotensin, angiotensin II und neuropeptide Y (NPY) receptors. During the follow-up project, the aforementioned labeling strategy was extended by the introduction of an alkyne-functionalized arginine building block. The latter enables, after incorporation into a peptide, a regioselective “bioorthogonal” conjugation based on “click chemistry” even if the peptide contains, e.g. lysine residues. In particular, the application of the new labeling strategy was broadened by preparing and characterizing new highly potent fluorescence or radiolabeled ligands for neurotensin receptors (NTS1R, NTS2R), the angiotensin II receptor type 1 and the NPY Y4 receptor (Y4R), as well as potent and stable NTS1R PET ligands (PET = positron emission tomography). The submitted proposal is aiming at in vivo studies with the aforementioned PET ligands (18F or 68Ga labeled) in immunodeficient, tumor-bearing nude mice (Cerenkov imaging, biodistribution studies, PET scans) in order to explore the suitability of these PET ligands for in vivo imaging of NTS1R positive tumors such as prostate, pancreas and colon carcinoma (permissions for these studies were granted). Moreover, the synthesis of dually labeled (radioactive (tritium) + fluorescence) NTS1R ligands, using the aforementioned labeling strategy, which is based on modified arginine residues, is planned. Such combined tritium and fluorescence labeled ligands, in conjunction with their “cold”, physicochemically identical analogs (only fluorescently labeled), represent special molecular tools, which will be used for the systematic comparison of radiochemical and fluorescence-based techniques. Finally, based on the recent finding that cyclization of linear hexapeptidic Y4R ligands via carbamoylated arginines results in Y4R ligands with very high affinity (Ki < 0.1 nM), new fluorescently labeled Y4R ligands will be prepared and characterized as molecular tools.

• DFG Programme: Research Grants