A Catalyst-Free Bioorthogonal Reaction for Malononitrile Addition to Azodicarboxylates

We report a new class of catalyst-free bioorthogonal reactions: malononitrile addition to azodicarboxylates. This reaction proceeds rapidly in organic and aqueous media at ambient temperature without catalysts or additives. Malononitriles can be readily incorporated into biomolecules such as RNA and proteins, and the reaction shows high robustness in complex systems due to its concerted transition state. Importantly, it is compatible with established bioorthogonal reactions (e.g., CuAAC and tetrazine ligation), enabling multiplexed site-specific labeling. These properties make it a valuable tool for biological applications.