TCO-NHS axial isomer

• TCO-NHS axial isomer [T4456]
• Triethylamine [T0424]
• Biotin-PEG3-Amine [B3172]
• 3,6-Di(2-pyridyl)-1,2,4,5-tetrazine [D3640]
• Acetonitrile
• Water

TCO-NHS axial isomer (5.0 mg, 0.018 mmol) was dissolved in acetonitrile (500 µL), and then triethylamine (3.0 µL, 0.022 mmol), and biotin-PEG3-amine (9.2 mg, 0.022 mmol) were added sequentially. The mixture was stirred at room temperature for 30 min. After confirming the disappearance of the TCO-NHS axial isomer by UPLC-MS, this reaction mixture was used as solution (1) for the next reaction. 3,6-Di(2-pyridyl)-1,2,4,5-tetrazine (4.5 mg, 0.019 mmol) was dissolved in a 1:1 acetonitrile:water solution (1.0 mL) to prepare the tetrazine solution. 30 µL of solution (1) and 60 µL of the tetrazine solution were withdrawn and mixed. Immediately after mixing, the purple-red color originating from the tetrazine solution disappeared, and a yellow transparent solution was obtained. UPLC-MS analysis confirmed the disappearance of 1 and the quantitative formation of the cycloadduct 2, the target compound.

• The reaction was monitored by UPLC-MS.
• As a preliminary step, UPLC-MS measurements were performed on the blank and each reagent, respectively.
• The cycloaddition reaction proceeded very rapidly; immediately after mixing, the reddish-purple color derived from the tetrazine derivative disappeared, and the solution turned yellow and transparent, indicating completion of the reaction.
• No new peaks were observed other than the cycloadduct 2 in the second step.

Cycloadduct 2

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

• Fast and Sensitive Pretargeted Labeling of Cancer Cells through a Tetrazine/trans-Cyclooctene Cycloaddition
  • N. K. Devaraj, R. Upadhyay, J. B. Haun, S. A. Hilderbrand, R. Weissleder, Angew. Chem. Int. Ed. 2009, 48, 7013.

• 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.