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.

Pretargeting is a powerful nuclear imaging strategy to achieve enhanced imaging contrast for nanomedicines and reduce the radiation burden to healthy tissue. Pretargeting is based on bioorthogonal chemistry. The most...

Pretargeting is a promising nuclear imaging technique that allows for the usage of antibodies (Abs) with enhanced imaging contrast and reduced patient radiation burden. It is based on bioorthogonal chemistry with the tetrazine ligation—a reaction between trans-cyclooctenes (TCOs) and tetrazines (Tzs)—currently being the most popular reaction due to its high selectivity and reactivity. As Abs can be designed to bind specifically to currently ‘undruggable’ targets such as protein isoforms or oligomers, which play a crucial role in neurodegenerative diseases, pretargeted imaging beyond the BBB is highly sought after, but has not been achieved yet. A challenge in this respect is that large molecules such as Abs show poor brain uptake. Uptake can be increased by receptor mediated transcytosis; however, it is largely unknown if the achieved brain concentrations are sufficient for pretargeted imaging. In this study, we investigated whether the required concentrations are feasible to reach. As a model Ab, we used the bispecific anti-amyloid beta (Aβ) anti-transferrin receptor (TfR) Ab 3D6scFv8D3 and conjugated it to a different amount of TCOs per Ab and tested different concentrations in vitro. With this model in hand, we estimated the minimum required TCO concentration to achieve a suitable contrast between the high and low binding regions. The estimation was carried out using pretargeted autoradiography on brain sections of an Alzheimer’s disease mouse model. Biodistribution studies in wild-type (WT) mice were used to correlate how different TCO/Ab ratios alter the brain uptake. Pretargeted autoradiography showed that increasing the number of TCOs as well as increasing the TCO-Ab concentration increased the imaging contrast. A minimum brain concentration of TCOs for pretargeting purposes was determined to be 10.7 pmol/g in vitro. Biodistribution studies in WT mice showed a brain uptake of 1.1% ID/g using TCO-3D6scFv8D3 with 6.8 TCO/Ab. According to our estimations using the optimal parameters, pretargeted imaging beyond the BBB is not a utopia. Necessary brain TCO concentrations can be reached and are in the same order of magnitude as required to achieve sufficient contrast. This work gives a first estimate that pretargeted imaging is indeed possible with antibodies. This could allow the imaging of currently ‘undruggable’ targets and therefore be crucial to monitor (e.g., therapies for intractable neurodegenerative diseases).

Aliphatic nucleophilic substitution (S_N2) with [¹⁸F]fluoride is the most widely applied method to prepare ¹⁸F-labeled positron emission tomography (PET) tracers. Strongly basic conditions commonly used during ¹⁸F-labeling procedures inherently limit or prohibit labeling of base-sensitive scaffolds. The high basicity stems from the tradition to trap [¹⁸F]fluoride on anion exchange cartridges and elute it afterwards with basic anions. This sequence is used to facilitate the transfer of [¹⁸F]fluoride from an aqueous to an aprotic organic, polar reaction medium, which is beneficial for S_N2 reactions. Furthermore, this sequence also removes cationic radioactive contaminations from cyclotron-irradiated [¹⁸O]water from which [¹⁸F]fluoride is produced. In this study, we developed an efficient elution procedure resulting in low basicity that permits S_N2 ¹⁸F-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. Four PET tracers and three synthons could be radiolabeled in substantially higher RCYs (up to 2.5-fold), even from lower precursor amounts, using this procedure. 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 ¹⁸F-labeling procedures. Labeling succeeded in RCYs of up to 20%. We believe that our findings facilitate PET tracer development by opening the path towards simple and direct S_N2 ¹⁸F-fluorination of base-sensitive substrates.

Aliphatic nucleophilic substitution (S_N2) with [¹⁸F]fluoride is the most widely applied method to prepare ¹⁸F-labeled positron emission tomography (PET) tracers. Strongly basic conditions commonly used during ¹⁸F-labeling procedures inherently limit or prohibit labeling of base-sensitive scaffolds. The high basicity stems from the tradition to trap [¹⁸F]fluoride on anion exchange cartridges and elute it afterwards with basic anions. This sequence is used to facilitate the transfer of [¹⁸F]fluoride from an aqueous to an aprotic organic, polar reaction medium, which is beneficial for S_N2 reactions. Furthermore, this sequence also removes cationic radioactive contaminations from cyclotron-irradiated [¹⁸O]water from which [¹⁸F]fluoride is produced. In this study, we developed an efficient elution procedure resulting in low basicity that permits S_N2 ¹⁸F-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. Four PET tracers and three synthons could be radiolabeled in substantially higher RCYs (up to 2.5-fold), even from lower precursor amounts, using this procedure. 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 ¹⁸F-labeling procedures. Labeling succeeded in RCYs of up to 20%. We believe that our findings facilitate PET tracer development by opening the path towards simple and direct S_N2 ¹⁸F-fluorination of base-sensitive substrates.