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

Abstract

Hexadentate tris(3,4-hydroxypyridinone) ligands (THP) complex Fe3+ 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 (68Ga). THP-peptide bioconjugates rapidly and quantitatively complex 68Ga at room temperature, neutral pH, and micromolar ligand concentrations, making them amenable to kit-based radiosynthesis of 68Ga PET radiopharmaceuticals. With the aim to produce an N-hydroxysuccinimide-(NHS)-THP reagent for kit-based 68Ga-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 68Ga 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 68Ga 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.

Targeting integrin αvβ6 with gallium-68 tris (hydroxypyridinone) based PET probes

“…Then filtration was followed by evaporation of the solvent to yield 3 (90%) and 4 (89%) as white solids, after recrystallization from isopropyl alcohol. 28 General procedure for the synthesis of THP-glutaric/adipic acid ester (6 16 Synthesis of integrin αvβ6-peptide (8 and 9) and integrin αvβ6-peptide-THP (11 and 12) The precursor linear sequence was synthesized using standard Fmoc solid-phase peptide synthesis approach at peptide synthesizer (Biotage® Initiator + Alstra™). All the Fmoc-…”

“…Then filtration was followed by evaporation of the solvent to yield 3 (90%) and 4 (89%) as white solids, after recrystallization from isopropyl alcohol. 28 General procedure for the synthesis of THP-glutaric/adipic acid ester (6 16 and 7)…”

“…THP-glutaric 6; yield: 85%. 16 Synthesis of integrin αvβ6-peptide (8 and 9) and integrin αvβ6-peptide-THP (11 and 12) The precursor linear sequence was synthesized using standard Fmoc solid-phase peptide synthesis approach at peptide synthesizer (Biotage® Initiator + Alstra™). All the Fmoc-(fluorenylmethyloxycarbonyl)-amino acid residues contained in the sequence were side-chain protected with classical acidlabile protecting groups except from the N-methyl-Lys that was protected with the -Dde: Fmoc-Asp(tBu)-OH -Fmoc-Leu-OH -Fmoc-Ala-OH -Fmoc-Phe-OH -Fmoc-D-Pro-OH -Fmoc-NMe-Lys-Dde-OH -Fmoc-Phe-OH -Fmoc-Arg(Pbf )-OH -Fmoc-Gly-OH.…”

“…By exploiting the N -hydroxysuccinimide (NHS) ester chemistry to generate an easy-to-use kit-based NHS-THP for 68 Ga-labelling and PET imaging, we recently reported an NHS-functionalized THP-derivative that was successfully conjugated to an anti-programmed death-ligand 1 (PD-L1) camelid single-domain antibody (sdAb) and a glucagon-like peptide-1 (GLP-1) targeting peptide. 16 In the present study, the NHS-THP chemistry was further explored by developing a new analogue ( i.e. with a longer linker).…”

“…The detection wavelength was 281 nm and the flow rate was 15 mL min −1 . THP-glutaric 6; yield: 85% 16. THPadipic 7; yield: 90%; 1 H NMR (400 MHz, methanol-d 4 ) δ 6.95 (s, 3H), 4.70 (s, 6H), 3.94 (s, 9H), 3.75-3.70 (m, 4H), 3.24-3.22 (m, 4H), 2.58 (s, 9H), 2.31 (m, 6H), 2.23-2.19 (m, 6H), 1.96 (d, J = 8.6 Hz, 4H), 1.65-1.62 (m, 4H); calculated ESI + [M + H] + : 994.5, [M + 2H] 2+ : 497.7, measured ESI + [M + H] + : 994.5, [M + 2H] 2+ : 497.8.…”