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 ³ H-Tz based synthon would have wide applications for in vitro/ex vivo assays. In this study, we developed a ³ H-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 [³ H]-(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 ³ H-labeled Tz and its successful application as a labeling building block.

Keywords: autoradiography; bioorthogonal chemistry; pre-targeting; tetrazine ligation; tritiation.

Figure 1

A) The tetrazine‐TCO ligation. B) Previously reported tetrazines were labeled with fluorine‐18, carbon‐11, copper‐64, gallium‐68, scandium‐44 (pet) and indium‐111 (SPECT) radionuclides. This study reports the first synthesis of a ³H‐labeled Tz.

Scheme 1

A) Reduction of different iodine‐substituted tetrazines with H₂. Reagents and conditions: a) For further details please check Table S1 in the SI. B) Reduction of alkene group in 3‐(4‐(allyloxy)phenyl)‐1,2,4,5‐tetrazine 7 and alkyne group in 3‐(4‐ethynylphenyl)‐1,2,4,5‐tetrazine 8. Reaction conditions: b) Pd/C, H₂, R.T. to 100 °C, EtOH, 72 h; or Pd/C, H₂, R.T. to 100 °C, TEA, EtOH, 72 h; or Pd/C, H₂, HCO₂NH₄, R.T. to 100 °C, MeOH/THF, 72 h; c) Lindlars catalyst, quinoline, H₂ (g), R.T. 72 h. C) Alternative reduction of bispyridyl tetrazine. Reagents and conditions: d) Pd/C, H₂, R.T., EtOH, 72 h; 0 % or Pd/C, H₂, R.T. to 100 °C, TEA, EtOH, 72 h; or Pd/C, H₂, HCO₂NH₄, R.T. to 100 °C, MeOH/THF, 72 h; e) NaNO₂, AcOH, H₂O, rt, 10 min.

Scheme 2

Reagents and conditions. a) Ethylene glycol, PTSA, toluene, reflux, 18 h; b) 2‐pyridinecarbonitrile, NH₂NH₂ ⋅ H₂O, S₈, EtOH, 90 °C, 12 h; c) H₂O, DMSO, MW 110 °C, 1 h; d) NaBH₄ or NaBD₄, MeOH/CH₂Cl₂, rt, 5 min; e) NaNO₂, AcOH, H₂O, rt, 10 min.

Figure 2

A) Tritiation of 12: a) NaBT₄, MeOH, CH₂Cl_2, rt, 5 min. b) NaNO₂, AcOH, H₂O, rt, 10 min. B) HPLC chromatograms of the final product [³H]16.

Figure 3

Results of the pretargeted autoradiography. A) CTX/CB ratio of [³H]16 binding. Control=AD Brain, 0 μg/mL mAb‐TCO. AD low=AD brain, 0.006 μg/mL mAb‐TCO. AD High=AD brain, 0.06 μg/mL mAb‐TCO. WT low=WT brain, 0.006 μg/mL mAb‐TCO. WT High=WT brain, 0.06 μg/mL mAb‐TCO. Standard deviation calculated ANOVA. B) Autoradiographic image, showing the image contrast between AD High, AD control and WT low.