A catalyst-free bioorthogonal reaction for malononitrile addition to azodicarboxylates

Developing bioorthogonal reactions that enable fundamental exploration of biological processes remains a key pursuit of chemical biology. We report here malononitrile addition to azodicarboxylate as a distinct class of catalyst-free bioorthogonal reactions. Our findings demonstrate that malononitriles react rapidly with azodicarboxylates in both organic and aqueous environments at ambient temperature, without requiring catalysts, bases, or additives. The facile modification of malononitriles facilitates their incorporation into biomolecules such as RNA and proteins. This malononitrile addition to azodicarboxylate reaction exhibits high robustness in complex biological systems, attributed to its distinct reactivity through a concerted transition state. Furthermore, we find that the malononitrile addition to azodicarboxylate reaction is compatible with other well-established bioorthogonal reactions (such as copper-catalyzed azide-alkyne cycloaddition and tetrazine ligation), enabling integration with click reactions for simultaneous site-specific labeling. These features position it as a valuable tool for various biological applications.