Exclusively Chemoselective S‑Acylation for Peptide Radiolabeling Using [¹⁸F]Fluoronicotinic Acid 4‑Nitrophenyl Ester...

Sulfhydryl functionality is useful for the chemoselective and site-specific conjugation of biomolecules for medical diagnosis and therapeutic purposes. Several types of thiol-reactive conjugation chemistry have been developed, including a maleimide-based addition reaction. Our previous study showed that activated 6-[¹⁸F]-fluoronicotinic acid ([¹⁸F]-FNA) ester chemoselectively forms an S-acylated product with peptide ACooP (H-ACGLSGLGVA-NH₂) bearing both a free amino group and a sulfhydryl group for positron emission tomography applications. The aim of this work is to better understand the chemoselectivity of S-acylation and explore its potential use for site-specific peptide radiolabeling. Accordingly, three new peptide variants of the decapeptide ACooP were designed as model sequences bearing both free amino and sulfhydryl functionalities, with the cysteine residue located at different positions in the sequences. The peptide variants C@3 (H-AGCLSGLGVA-NH₂), C@4 (H-AGLCSGLGVA-NH₂), and C@5 (H-AGLSCGLGVA-NH₂) were conjugated with FNA to prepare the corresponding nonradioactive reference compounds. The conjugated products were characterized using one- and two-dimensional nuclear magnetic resonance analysis and high-resolution mass spectrometry. Peptide radiolabeling tests were performed using [¹⁸F]-FNA 4-nitrophenyl ester as the prosthetic group at pH 8.6 and 7.4. The radiolabeled products were S-acylated compounds with high or exclusive chemoselectivity (>95%) in all three cases. To demonstrate the utility of this type of chemoselective S-acylation for radiotracer preparation, [¹⁸F]-FNA-S-C@5 was prepared with a radiochemical purity of 97.0% ± 0.8 (n = 3) and a decay-corrected radiochemical yield of 16.1% ± 3.5. A batch size of the end product with hundreds of MBq was easily achieved, which is sufficient for PET imaging applications. [¹⁸F]-FNA-S-C@5 showed limited in vitro stability in rat plasma, with intact tracer observed at 13.1% ± 4.2 (n = 3) after 15 min of incubation. In conclusion, chemoselective S-acylation-based peptide radiolabeling was achieved using [¹⁸F]-FNA 4-nitrophenyl ester, and this reaction holds promise for highly chemoselective and site-specific radiolabeling of other types of biomolecules.