Combined Inductive and Dispersion Effects Enhance Bioorthogonal Reactivity of Tetrazines Toward Isonitriles

Inverse-electron demand cycloaddition reactions of tetrazines are widely used in bioorthogonal chemistry, but the opposing effects of substituents on reactivity and stability make optimizing tetrazines for chemical biology applications challenging. Building on the discovery that bulky substituents can unexpectedly enhance both the stability of tetrazines and their reactivity toward isonitriles, we hypothesized that substituents that are both bulky and electron-withdrawing could yield tetrazines with desirable properties. We synthesized a series of tetrazines designed to explore these kinetic properties. A novel computational method quantifying intermolecular atomic contributions to dispersion forces supported the analysis substituent effects on tetrazine reactivity. Study results indicate that tetrazine reactivity is governed by an interplay of frontier-orbital levels, dispersion forces, and conformational preferences. These insights were apparent in the form of a "bromo effect," where bromine atoms enhanced tetrazine reactivity by influencing frontier-orbital levels and increasing dispersion forces. Notably, 3,6-bis(2-bromopropan-2-yl)-1,2,4,5-tetrazine exhibited a ∼80-fold increase in reactivity to isonitriles compared to dimethyltetrazine with high orthogonality to other dienophiles. The 2-bromoprop-2-yl group could also be incorporated into asymmetric tetrazines for tuning reactivity properties. In addition to introducing novel tetrazines for bioorthogonal chemistry, this work provides valuable insights into the role of dispersion forces in the transition states of cycloaddition reactions.