Positron Emission Tomography (PET) is a non-invasive molecular imaging method that relies on radiolabeled molecules and is routinely used for clinical diagnosis, treatment monitoring and drug development. The key advantages of PET over other molecular imaging techniques are its quantitativity, high sensitivity, superior resolution and low radiation dose for patients. Therefore, the development of new PET tracers is essential, especially for the emergence of new treatment forms. Monoclonal antibodies (mAbs) are particularly promising vectors for diagnostic imaging. This is because of their high target specificity and low non-displaceable binding. Recently, it became possible to use these vectors also for targets within the brain. Penetration of the blood-brain-barrier (BBB) can be achieved by several methods such as active transport, for example by utilizing the transferrin transporter, or by opening the BBB, for example by using focused ultrasound (FUS) based strategies. Despite these advances, the use of mAbs for diagnostic brain imaging is still in its infancy as imaging is hindered by the slow pharmacokinetics of mAbs. Although there are long-lived radionuclides compatible with these pharmacokinetics, respective radionuclides provide inferior image quality and result in high radiation burden for the patient. Pretargeting allows to combine slow-circulating targeting vectors with short-lived PET radionuclides (Figure ). In this approach, a tagged mAb is injected first and given time to accumulate at target-rich sites and eliminated from blood circulation. Then, a radiolabeled small molecule (effector molecule) is injected, which possesses fast pharmacokinetics and can rapidly and selectively react with the tag of the beforehand administered mAb. The use of trans-cyclooctene (TCO) derivatives as chemical tags and 1,2,4,5tetrazine (Tz) derivatives as effector molecules has become state-of-the-art for pretargeted PET imaging due to the ultra-fast kinetics of the inverse electron demand Diels-Alder (IEDDA) "click" cycloaddition between these two structures, its bioorthogonality and compatibility with multiple scaffolds. Figure . A) Pretargeting concept beyond the BBB. In a first step, a BBB-penetrating TCO-tagged mAb is injected. The mAb is actively transported over the BBB and binds to its target. In a second step, a 18 F-radiolabeled Tz is injected. The Tz clicks with the TCO-tagged mAbs thus imaging 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 18 F-Tzs were then tested for imaging contrast with in vitro autoradiography. Finally, compounds were injected in mice to evaluate the brain uptake. Metabolism was then evaluated for the best compounds.