3,6-Di(2-pyridyl)-1,2,4,5-tetrazine

Used Chemicals

• TCO-PEG3-Biotin axial isomer [T4236]
• 3,6-Di(2-pyridyl)-1,2,4,5-tetrazine [D3640]
• Acetonitrile

Procedure

TCO-PEG3-biotin axial isomer (1) (5.9 mg, 0.01 mmol) was dissolved in 1:1 acetonitrile: water (1.4 mL) to prepare the 7.5 mmol/L solution (colorless). Next, 3,6-di(2-pyridyl)-1,2,4,5-tetrazine (2) (5.0 mg, 0.02 mmol) was dissolved in 1:1 solution of acetonitrile: water (1.4 mL) to prepare the 5.0 mmol/L solution (reddish-purple). 75 µL of each of the solutions were mixed together. During mixing, the reddish-purple color from 2 disappeared and the color of the solution became clear yellow, and UPLC-MS analysis confirmed the disappearance of 2 and the quantitative formation of the cycloaddition product 3.

Experimenter’s Comments

• The reaction mixture was monitored by UPLC-MS.
• As a preliminary step, UPLC-MS measurements of 1, 2, and blank were performed first.
• This reaction proceeded very rapidly, and the reddish-purple color from 2 disappeared immediately after mixing, changing to a clear yellow solution, and the reaction was completed.
• In this reaction, none of the new peaks except 3 were observed.

Analytical Data

Cycloaddition Compound 3

HRMS (ESI-TOF); m/z calced for C₃₉H₅₅N₈O₇S (M+H), 779.97; found 779.9.

Lead Reference

• Tetrazine Ligation: Fast Bioconjugation Based on Inverse-Electron-Demand Diels-Alder Reactivity
  • M. L. Blackman, M. Royzen, J. M. Fox, J. Am. Chem. Soc. 2008, 130, 13518.

References

• Benzannulated Isobenzofurans
  • J. Moursounidis, D. Wege, Aust. J. Chem. Soc. 1998, 41, 235.