The development of highly selective and fast biocompatible reactions for ligation and cleavage has paved the way for new diagnostic and therapeutic applications of pretargeted in vivo chemistry. The concept of bioorthogonal pretargeting has attracted considerable interest, in particular for the targeted delivery of radionuclides and drugs. In nuclear medicine, pretargeting can provide increased target-to-background ratios at early time-points compared to traditional approaches. This reduces the radiation burden to healthy tissue and, depending on the selected radionuclide, enables better imaging contrast or higher therapeutic efficiency. Moreover, bioorthogonally triggered cleavage of pretargeted antibody–drug conjugates represents an emerging strategy to achieve controlled release and locally increased drug concentrations. The toolbox of bioorthogonal reactions has significantly expanded in the past decade, with the tetrazine ligation being the fastest and one of the most versatile in vivo chemistries. Progress in the field, however, relies heavily on the development and evaluation of (radio)labeled compounds, preventing the use of compound libraries for systematic studies. The rational design of tetrazine probes and triggers has thus been impeded by the limited understanding of the impact of structural parameters on the in vivo ligation performance. In this work, we describe the development of a pretargeted blocking assay that allows for the investigation of the in vivo fate of a structurally diverse library of 45 unlabeled tetrazines and their capability to reach and react with pretargeted trans -cyclooctene (TCO)-modified antibodies in tumor-bearing mice. This study enabled us to assess the correlation of click reactivity and lipophilicity of tetrazines with their in vivo performance. In particular, high rate constants (>50 000 M –1 s –1 ) for the reaction with TCO and low calculated log D 7.4 values (below −3) of the tetrazine were identified as strong indicators for successful pretargeting. Radiolabeling gave access to a set of selected 18 F-labeled tetrazines, including highly reactive scaffolds, which were used in pretargeted PET imaging studies to confirm the results from the blocking study. These insights thus enable the rational design of tetrazine probes for in vivo application and will thereby assist the clinical translation of bioorthogonal pretargeting.

Pretargeted imaging of nanomedicines have attracted considerable interest because it has the potential to increase imaging contrast while reducing radiation burden to healthy tissue. Currently, the tetrazine ligation is the fastest bioorthogonal reaction for this strategy and, consequently, the state-of-art choice for in vivo chemistry. We have recently identified key properties for tetrazines in pretargeting. We have also developed a method to 18 F-label reactive tetrazines using an aliphatic nucleophilic substitution strategy. Here, we combined this knowledge and developed an 18 F-labeled tetrazine for pretargeted imaging. In order to develop this ligand, a small SAR study was performed. The most promising compound was selected for labeling and subsequent positronemission-tomography in vivo imaging. Radiolabeling was achieved in satisfactory yields, molar activities, and high radiochemical purities. [ 18 F]15 displayed favorable pharmacokinetics and remarkable target-to-background ratiosas early as 1 h post injection. We believe that this agent could be a promising candidate for translation into clinical studies.

Aliphatic nucleophilic substitution (S N 2) with [ 18 F]fluoride is the most widely applied method to prepare 18 F-labeled positron emission tomography (PET) tracers. Strong basic conditions commonly used during 18 F-labeling procedures inherently limit or prohibit labeling of base-sensitive scaffolds. The high basicity stems from the tradition to trap [ 18 F]fluoride on anion exchange cartridges and elute it afterward with basic anions. This sequence is used to facilitate the transfer of [ 18 F]fluoride from an aqueous to an aprotic organic, polar reaction medium, which is beneficial for S N 2 reactions. Furthermore, this sequence also removes cationic radioactive contaminations from cyclotron-irradiated [ 18 O]water from which [ 18 F]fluoride is produced. In this study, we developed an efficient elution procedure resulting in low basicity that permits S N 2 18 F-labeling of base-sensitive scaffolds. Extensive screening of trapping and elution conditions (>1000 experiments) and studying their influence on the radiochemical yield (RCY) allowed us to identify a suitable procedure for this. Using this procedure, four PET tracers and three synthons could be radiolabeled in substantially higher RCYs (up to 2.5-fold) compared to those of previously published procedures, even from lower precursor amounts. Encouraged by these results, we applied our low-basicity method to the radiolabeling of highly base-sensitive tetrazines, which cannot be labeled using state-of-art direct aliphatic 18 F-labeling procedures. Labeling succeeded in RCYs of up to 20%. We believe that our findings facilitate PET tracer development by opening the path toward simple and direct S N 2 18 F fluorination of base-sensitive substrates.