Studies on the Stability and Stabilization of trans- Cyclooctenes through Radical Inhibition and Silver (I) Metal Complexation

Conformationally strained trans-cyclooctenes (TCOs) engage in bioorthogonal reactions with tetrazines with second order rate constants that can exceed 10⁶ M^-1s^-1. The goal of this study was to provide insight into the stability of TCO reagents and to develop methods for stabilizing TCO reagents for long-term storage. The radical inhibitor Trolox suppresses TCO isomerization under high thiol concentrations and TCO shelf-life can be greatly extended by protecting them as stable Ag(I) metal complexes. ¹H NMR studies show that Ag-complexation is thermodynamically favorable but the kinetics of dissociation are very rapid, and TCO•AgNO₃ complexes are immediately dissociated upon addition of NaCl which is present in high concentration in cell media. The AgNO₃ complex of a highly reactive s-TCO-TAMRA conjugate was shown to label a protein-tetrazine conjugate in live cells with faster kinetics and similar labeling yield relative to a 'traditional' TCO-TAMRA conjugate.