Abstract Background Monoclonal antibodies, engineered protein scaffolds, and peptides are increasingly important in therapy and diagnostics owing to their high specificity and affinity. Recent advances have enabled radiolabelling of such compounds with fluorine-18 in aqueous solution at room temperature via conjugation of 18 Ftetrazines with trans-cyclooctene (TCO) moieties through the inverse electron-demand Diels–Alder (IEDDA) reaction. To simplify fluorine-18 labelling of large molecules, a novel tetrazine: 4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl 6-fluoro- 18 Fnicotinate ( 18 FTzE2), was here developed and synthesized in a single-step procedure directly on a standard QMA cartridge used for trapping 18 Ffluoride. The QMA containing 18 Ffluoride was eluted over 2 min with N , N , N -trimethyl-5-(((4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)oxy)carbonyl)pyridin-2-aminium chloride in acetonitrile, forming 18 FTzE2 instantaneously as it passed through the cartridge. 18 FTzE2 was designed to combine the most favourable features of the previously described tetrazine prosthetic groups, 18 FTzAm and 18 FTzE, specifically, the superior in vivo performance and stability of 18 FTzAm, and the efficient and practical radiosynthesis of 18 FTzE. 18 FTzE2 was evaluated biologically as a reagent for direct labelling of the TCO-conjugated Affibody molecule Z09591, a high affinity marker for PDGFRβ. Results The radiochemical yield for 1 ⁸FTzE2 was 30.4 ± 3% (n = 4), corresponding to an activity yield of 5.8 GBq starting from 20 GBq 1 ⁸Ffluoride, with a total synthesis time of 13 min. 18 FTzE2 demonstrated greater stability in human plasma than the previous ester tetrazine 18 FTzE, but was significantly less stable than 18 FTzAm. The Affibody molecule Z09591 was successfully radiolabelled, giving an activity yield of 410 ± 160 MBq and > 90% radiochemical purity within a total synthesis time of 30 min from 18 Ffluoride. 18 FTzE2-Z09591 exhibited improved plasma stability relative to 18 FTzE-Z09591, though lower than 18 FTzAm-Z09591. 18 FTzE2-Z09591 retained specific blockable binding to PDGFRβ-expressing human and murine tissues as demonstrated by in vitro autoradiography. Biodistribution of 18 FTzE2-Z09591 was rapid, with predominantly renal clearance, and showed improved targeting of PDGFRβ-expressing tissues compared with 18 FTzE-Z09591. Conclusion The novel tetrazine prosthetic group 18 FTzE2 enabled straightforward radiolabelling of Affibody molecule Z09591 without affecting its binding properties. 18 FTzE2-Z09591 showed improved stability and in vivo targeting of PDGFRβ compared with the previously reported tetrazine ester 18 FTzE-Z09591. 18 FTzE2 represents a promising tetrazine prosthetic group for labelling of proteins and peptides with fluorine-18.
Lechi et al. (Mon,) studied this question.