Improved Metabolic Stability for ¹⁸F PET Probes Rapidly Constructed via Tetrazine trans-Cyclooctene Ligation

The fast kinetics and bioorthogonal nature of the tetrazine trans-cyclooctene (TCO) ligation makes it a unique tool for PET probe construction. In this study, we report the development of an ¹⁸F-labeling system based on a CF₃-substituted diphenyl-s-tetrazine derivative with the aim of maintaining high reactivity while increasing in vivo stability. c­(RGDyK) was tagged by a CF₃-substituted diphenyl-s-tetrazine derivative via EDC-mediated coupling. The resulting tetrazine-RGD conjugate was combined with a ¹⁹F-labeled TCO derivative to give HPLC standards. The analogous ¹⁸F-labeled TCO derivative was combined with the diphenyl-s-tetrazine-RGD at μM concentration. The resulting tracer was subjected to in vivo metabolic stability assessment, and microPET studies in murine U87MG xenograft models. The diphenyl-s-tetrazine-RGD combines with an ¹⁸F-labeled TCO in high yields (>97% decay-corrected on the basis of TCO) using only 4 equiv of tetrazine-RGD relative to the ¹⁸F-labeled TCO (concentration calculated based on product’s specific activity). The radiochemical purity of the ¹⁸F-RGD peptides was >95% and the specific activity was 111 GBq/μmol. Noninvasive microPET experiments demonstrated that ¹⁸F-RGD had integrin-specific tumor uptake in subcutaneous U87MG glioma. In vivo metabolic stability of ¹⁸F-RGD in blood, urine, and major organs showed two major peaks: one corresponded to the Diels–Alder conjugate and the other was identified as the aromatized analog. A CF₃-substituted diphenyl-s-tetrazine displays excellent speed and efficiency in ¹⁸F-PET probe construction, providing nearly quantitative ¹⁸F labeling within minutes at low micromolar concentrations. The resulting conjugates display improved in vivo metabolic stability relative to our previously described system.