Synergistic Experimental and Computational Investigation of the Bioorthogonal Reactivity of Substituted Aryltetrazines

Tetrazines (Tz) have been applied as bioorthogonal agents for various biomedical applications, including pretargeted imaging approaches. In radioimmunoimaging, pretargeting increases the target-to-background ratio while simultaneously reducing the radiation burden. We have recently reported a strategy to directly F-18-label highly reactive tetrazines based on a 3-(3-fluorophenyl)-Tz core structure. Herein, we report a kinetic study on this versatile scaffold. A library of 40 different tetrazines was prepared, fully characterized, and investigated with an emphasis on second-order rate constants for the reaction with trans-cyclooctene (TCO). Our results reveal the effects of various substitution patterns and moreover demonstrate the importance of measuring reactivities in the solvent of interest, as dick rates in different solvents do not necessarily correlate well. In particular, we report that tetrazines modified in the 2-position of the phenyl substituent show high intrinsic reactivity toward TCO, which is diminished in aqueous systems by unfavorable solvent effects. The obtained results enable the prediction of the bioorthogonal reactivity and thereby facilitate the development of the next generation of substituted aryltetrazines for in vivo applications.

Automated summary

Tetrazines have wide applications in biomedical field, including pretargeted imaging. In this study, a kinetic investigation on different tetrazine compounds reacting with trans-cyclooctene (TCO) was conducted. The results reveal that the reactivity of tetrazines in different solvents may not correlate well and tetrazines modified in the 2-position of the phenyl substituent show high intrinsic reactivity towards TCO, which is diminished in aqueous systems. These findings enable the prediction of bioorthogonal reactivity and facilitate the development of substituted aryltetrazines for in vivo applications.