Authors: N. Hauser
Source: Nuclear Medicine and Biology (article, 2025-11-01)
Field: Chemistry — Organic Chemistry

UV-induced tetrazine labeling of cis-cyclooctene derivatives Introduction: INTRODUCTION Macromolecules, such as nanobodies or peptides, are emerging as new targeting vectors in PET imaging. The most promising radionuclide for labeling these structures is 18F due to its optimal physical properties. However, it is usually impractical to label these compounds directly, due to the harsh conditions required in the labeling and the typically low molar activity of the product.1 Inverse-electron-demand Diels-Alder (iEDDA) reactions with trans-cyclooctenes (TCO) and tetrazines (Tz) are a selective way of introducing radiolabeled moieties into macromolecules on a timescale of minutes.2 While Tz have been continuously improved over the years to offer increased stability combined with high reactivity, TCOs still suffer from stability issues posed by isomerization to the much less active cis-isomer. This has made synthesizing sophisticated TCOs notoriously challenging compounds. To circumvent this issue, we designed an acid- and UV-catalyzed one-pot reaction which can be used to rapidly isomerize, click and oxidize a variety of cis-cyclooctenes (CCO) to their final pyridazine products with cold or 18F-labeled Tzs in a comparable time frame as their TCO counterparts (10 minutes).<br/>Methods: METHODS In this study, we tested cold or 18F-labeled tetrazines and cis-cyclooctene derivatives, with different photosensitizers, solvents, acids, and UV wavelengths. The cold screening was carried out in a UV reactor and the screening of 18F-labeled tetrazines under a standard TLC lamp.The kinetics of the formation of UV- and/or radioactive compounds were monitored via HPLC-MS and radio-HPLC, respectively. Initial cold reaction parameters were incrementally optimized and translated to 18F-labeled tetrazines for further fine-tuning.18F-labeled Tz was standardly produced via SN2 reaction of a nosyl-precursor.3<br/>Results: RESULTS 9 different tetrazines and 4 different cyclooctene derivatives were tested as reactants. Among the tetrazines, 3-(4-(2-fluoroethoxy)phenyl)-1,2,4,5-tetrazine (F-EtO-Tz-H) showed the best results, while of the dienophiles (Z)-cyclooct-5-ene-1,2-diol (CCO(OH)2) and a (Z)‑cycloocteno[d]imidazole derivative (C2CO) performed the best (see Figure A). Cold F-EtO-Tz-H clicked and oxidized with both CCO(OH)2 (80% conversion in 30 min) or C2CO (80% in 60 min) in the presence of methyl benzoate as photosensitizer, 1M acid (HCl and TFA, respectively) and UV light (254 nm) in acetonitrile. Under these conditions [18F]F-EtO-Tz-H (50-80 MBq/mL) gave 95-99% RCC with CCO(OH)2 in 10 min. 88-90% RCC was reached with C2CO with methyl benzoate, while dimethyl isophthalate as photosensitizer achieved 94-95% RCC in 10 min.<br/>Conclusions: CONCLUSIONS We have demonstrated that radiolabeled tetrazines can be clicked to CCOs with a photosensitizer under UV radiation in a comparable time frame and RCC as TCOs. This enables the possibility to utilize more stable precursors making both synthesis and precursor stability more convenient for possible future routine applications. For example, radiolabeling of CCO-modified PSMA and maleimide derivatives with [18F]F-EtO-Tz-H are currently being investigated. Tetrazine ; Isomerization ; Kinetics ; Pyridazine ; Molar ratio ; Chemical kinetics ; Chemical stability Click Chemistry and Applications ; Radiopharmaceutical Chemistry and Applications ; Chemical Reactions and Isotopes