Improved Metabolic Stability for <sup>18</sup>F PET Probes Rapidly Constructed via Tetrazine <i>trans</i>-Cyclooctene Ligation

The fast kinetics and bioorthogonal nature of the tetrazine <i>trans</i>-cyclooctene (TCO) ligation makes it a unique tool for PET probe construction. In this study, we report the development of an <sup>18</sup>F-labeling system based on a CF<sub>3</sub>-substituted diphenyl-<i>s</i>-tetrazine derivative with the aim of maintaining high reactivity while increasing in vivo stability. c­(RGDyK) was tagged by a CF<sub>3</sub>-substituted diphenyl-<i>s</i>-tetrazine derivative via EDC-mediated coupling. The resulting tetrazine-RGD conjugate was combined with a <sup>19</sup>F-labeled TCO derivative to give HPLC standards. The analogous <sup>18</sup>F-labeled TCO derivative was combined with the diphenyl-<i>s</i>-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-<i>s</i>-tetrazine-RGD combines with an <sup>18</sup>F-labeled TCO in high yields (>97% decay-corrected on the basis of TCO) using only 4 equiv of tetrazine-RGD relative to the <sup>18</sup>F-labeled TCO (concentration calculated based on product’s specific activity). The radiochemical purity of the <sup>18</sup>F-RGD peptides was >95% and the specific activity was 111 GBq/μmol. Noninvasive microPET experiments demonstrated that <sup>18</sup>F-RGD had integrin-specific tumor uptake in subcutaneous U87MG glioma. In vivo metabolic stability of <sup>18</sup>F-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 CF<sub>3</sub>-substituted diphenyl-<i>s</i>-tetrazine displays excellent speed and efficiency in <sup>18</sup>F-PET probe construction, providing nearly quantitative <sup>18</sup>F labeling within minutes at low micromolar concentrations. The resulting conjugates display improved in vivo metabolic stability relative to our previously described system.