Development of the First Tritiated Tetrazine: Facilitating Tritiation of Proteins

Abstract Tetrazine (Tz)–trans‐cyclooctene (TCO) ligation is an ultra‐fast and highly selective reaction and it is particularly suited to label biomolecules under physiological conditions. As such, a 3H‐Tz based synthon would have wide applications for in vitro/ex vivo assays. In this study, we developed a 3H‐labeled Tz and characterized its potential for application to pretargeted autoradiography. Several strategies were explored to synthesize such a Tz. However, classical approaches such as reductive halogenation failed. For this reason, we designed a Tz containing an aldehyde and explored the possibility of reducing this group with NaBT4. This approach was successful and resulted in [3H]‐(4‐(6‐(pyridin‐2‐yl)‐1,2,4,5‐tetrazin‐3‐yl)phenyl)methan‐t‐ol with a radiochemical yield of 22 %, a radiochemical purity of 96 % and a molar activity of 0.437 GBq/μmol (11.8 Ci/mmol). The compound was successfully applied to pretargeted autoradiography. Thus, we report the synthesis of the first 3H‐labeled Tz and its successful application as a labeling building block.

13 1h-3d - [9] S3 General reduction entry 1 To a solution of Tz 1-6 (10 mg) in ethanol (0.1 mL) was added Pd/C (10 wt.%, 1 mg) at argon atmosphere. Consequently, the reaction mixture was charged with hydrogen while the flask was connected to a vacuum line. The mixture was stirred at room temperature for 24 h and monitored frequently. After 24 hours the temperature was raised to 100 °C, and monitored for 48 h. For all 6 Tzs no reduction was observed, based on LC-MS, TLC and NMR after filtration of the Pd/C, evaporation of the solvent.

General reduction entry 2
To general reduction entry 1, 4 μL trimethylamine was added. For all 6 Tzs no reduction was observed, based on LC-MS, TLC and NMR after filtration of the Pd/C, evaporation of the solvent.

General reduction entry 3
To general reduction entry 1, instead of ethanol, methanol was used. Caution was taken that all glassware and stirring bars were vacuum oven dried before use, and glassware was purged 3 x with argon. For all 6 Tzs no reduction was observed, based on LC-MS, TLC and NMR after filtration of the Pd/C, evaporation of the solvent. Hence, the compounds

General reduction entry 4
To a solution of Tz 1-6 (10 mg) in ethanol (0.1 mL) was added 1 ml of a saturated solution of HCO2NH4 (40-50 mg of HCO2NH4 in MeOH/THF, 2/1). Subsequently, Pd/C (10 wt.%, 1 mg) was added to the flask at argon atmosphere. The mixture was stirred at room temperature for 24 h and monitored frequently. After 24 hours the temperature was raised to 150 °C, and monitored for 48 h. For all 6 Tzs no reduction was observed, based on LC-MS, TLC and NMR after filtration of the Pd/C and drying of the filtrate in vacuo.

General reduction entry 5
To general reduction entry 3, 40 μl of HCl in dioxane (8M) was added. For all 3 Tzs (1, 2, 6) no reduction was observed, based on LC-MS, TLC and NMR after filtration of the Pd/C, evaporation of the solvent.
General reduction entry 6

S4
To general reduction entry 3, 4 μL saturated NaHCO3 was added. THF was used as solvent instead of MeOH. For all 3 Tzs (1, 2, 6) no reduction was observed, based on LC-MS, TLC and NMR after filtration of the Pd/C, evaporation of the solvent.

General reduction entry 7
To general reduction entry 1, instead of ethanol, THF was used. For all 3 Tzs (1, 2, 6) no reduction was observed, based on LC-MS, TLC and NMR after filtration of the Pd/C, evaporation of the solvent.

General reduction entry 8
To general reduction entry 1, instead of Pd/C, 10 wt% PtO2 was used. For all 6 Tzs no reduction was observed, based on LC-MS, TLC and NMR after filtration of the PtO2, evaporation of the solvent.

General reduction entry 9
To general reduction entry 1, instead of Pd/C, 10 wt% Pd(dba)2 was used. For all 6 Tzs no reduction was observed, based on LC-MS, TLC and NMR after filtration of the PtO2, evaporation of the solvent.
Consequently, the reaction mixture was heated until 100 o C. For all 6 Tzs no reduction was observed, based on LC-MS, TLC and NMR after filtration of the PtO2, evaporation of the solvent.

General reduction entry 11
To a solution of 10 mg (1 eq.) Tz 1-6 was added Pd(PPh3)4 (0.10 eq.), HCOONa (0.2 eq.), 100 mL of MeOH. Reaction vial was closed and purged with argon. Consequently, the reaction mixture was heated until 100 o C. For all 11 Tzs no reduction was observed, based on LC-MS and TLC.
Reaction vial was closed and purged with argon. Consequently, the reaction mixture was heated until 100 o C. Consequently, the reaction mixture was charged with hydrogen while the flask was connected to a vacuum line. The mixture was stirred at room temperature for 24 h and monitored frequently. After 24 hours the temperature was raised to 100 °C, and monitored for 48 h. For all 6 Tzs no reduction was observed, based on LC-MS and TLC.

General reduction entry 13
To a solution of Tz 1-6 (10 mg) in deionized water (0.1 mL) was added PdCl2 (0.05 eq.). The reaction mixture was stirred for 10 min. To the reaction mixture was added TMDS (0.4 eq.), dropwise. The mixture was stirred at room temperature for 3 days and monitored frequently.
For all 6 Tz's no reduction was observed, based on LC-MS and TLC.

3-(4-vinylphenyl)-1,2,4,5-tetrazine
To 8 (50 mg, 0.27 mmol), dissolved in 2 mL MeOH was added 2.5 mg of Lindlar catalyst and 25 mg of quinoline. Hydrogen gas contained in a balloon was bubbled into the reaction mixture at room temperature for 3 days. Reaction was monitored by TLC and LC-MS, however after 5 days no conversion was observed yet.