NHS-Functionalized THP Derivative for Efficient Synthesis of Kit-Based Precursors for (68)Ga Labeled PET Probes

Hexadentate tris(3,4-hydroxypyridinone) ligands (THP) complex Fe(3+) at very low iron concentrations and their high affinities for oxophilic trivalent metal ions have led to their development for new applications as bifunctional chelators for the radiometal gallium-68 ((68)Ga). THP-peptide bioconjugates rapidly and quantitatively complex (68)Ga at room temperature, neutral pH, and micromolar ligand concentrations, making them amenable to kit-based radiosynthesis of (68)Ga PET radiopharmaceuticals. With the aim to produce an N-hydroxysuccinimide-(NHS)-THP reagent for kit-based (68)Ga-labeling and PET imaging, THP-derivatives were designed and synthesized to exploit the advantages of NHS chemistry for coupling with peptides, proteins, and antibodies. The more stable five-carbon atoms linker product was selected for a proof-of-concept conjugation and radiolabeling study with an anti-programmed death ligand 1 (PD-L1) camelid single domain antibody (sdAb) under mild conditions and further evaluated for site-specific amide bond formation with a synthesized glucagon-like peptide-1 (GLP-1) targeting peptide using solid-phase synthesis. The obtained THP-GLP-1 conjugate was tested for its (68)Ga chelating ability, demonstrating to be a promising candidate for the detection and monitoring of GLP-1 aberrant malignancies. The obtained sdAb-THP conjugate was radiolabeled with (68)Ga under mild conditions, providing sufficient labeling yields after 5 min, demonstrating that the novel NHS-THP bifunctional chelator can be widely used to easily conjugate the THP moiety to different targeting molecules (e.g., antibodies, anticalins, or peptides) under mild conditions, paving the way to the synthesis of different imaging probes with all the advantages of THP radiochemistry.