Labeling of Highly Reactive Tetrazines using [¹⁸F]SuFEx

Abstract

Pretargeted imaging is an emerging technique to study the in vivo biodistribution of nanomedicines. Currently, the tetrazine ligation is considered the most promising bioorthogonal reaction for pretargeting. Recently, Zheng et al. described an ultrafast late-stage radiolabeling of tetrazines based on sulfur ¹⁸F-fluoride exchange click chemistry ([¹⁸F]SuFEx). However, bispyridyl and H-tetrazines—the most promising structures for in vivo pretargeted applications—cannot be labeled using the proposed reaction conditions as they lead to decomposition of the tetrazine core. Here, we report improved conditions, exploiting basic preconditioning conditions for the quaternary methyl ammonium (QMA) cartridge and the use of low basic anions that allow ¹⁸F-labeling of bispyridyl and H-tetrazines using SuFEx. This strategy resulted in fast and efficient radiolabeling of highly reactive tetrazines with radiochemical conversions of up to 85% and radiochemical purity above 95%. This opens up the possibility to use SuFEx to ¹⁸F-label tetrazines, which are suitable for in vivo pretargeted imaging.

Key words

fluorine-18 - SuFEx - tetrazine - radiochemistry - bioorthogonal chemistry

• References

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• 26 General procedure for manual synthesis: To the dry [¹⁸F]fluoride was added a mixture of the selected compound (0.0099 mmol) in dry MeCN/DMF (0.5 mL). The reaction was left at room temperature, and quenched with H₂O (0.1 mL) after 5 min. Aliquots of the reaction were taken after 1 and 5 min. The aliquots were added to H₂O + 0.1% TFA (0.1 mL) and analyzed by radio-HPLC.
• 27 General procedure for automated synthesis: A solution of either the selected tetrazine (0.5 μmol) in dry MeCN (0.5 mL) or in dry DMF (0.5 mL) was added to the reaction vial containing the dried [¹⁸F]Bu₄NF (5.0–6.0 GBq), after being cooled with compressed air to room temperature for 8 min. The reaction was left at room temperature for 30 sec, before being immediately quenched with 0.3 mL of H₂O + 0.1 % TFA. The crude reaction was then purified via semi-preparative HPLC. Formulation of the product was obtained by diluting the collected fraction with Milli-Q H₂O (1:20) and trapping the resulting solution on a C18 solid-phase extraction cartridge. Subsequently, the C18 solid-phase extraction cartridge was washed with Milli-Q H₂O (7 mL), and the compound was eluted with EtOH (1 mL), followed by concentration under continuous N₂ flow at r.t. The resulting dry [¹⁸F]-tetrazine was dissolved in phosphate buffer, resulting in an activity concentration of 100 MBq/mL.