Fig. 1. A) The concept of pretargeting beyond the BBB. In the first step, a BBB-penetrating TCO-tagged mAb is injected. The mAb is actively transported over the BBB and binds to its target. In the second step, a ¹⁸F-radiolabeled Tz is injected. The Tz clicks to the TCO-tagged mAbs, thus enabling the imaging of the selected target. B) Comparison between pretargeted and conventional imaging C) strategy and workflow of this study to develop BBB permeable Tz imaging agent that is able to click in vivo to targets within the brain. The starting point was a Tz probe developed for cancer pretargeting. A library was designed based on this scaffold to have optimal parameters to cross the BBB and click in vivo. All the designed molecules were synthesized to evaluate labeling feasibility and in vitro stability. The ¹⁸F-Tzs were then tested for imaging contrast with in vitro autoradiography. Finally, compounds were injected into rats to evaluate the brain uptake. Metabolism was then evaluated for the best compounds.