Unexpected Discovery of Structure‐dependent Aggregation of N‐Oxide Enables Novel Heme‐detecting Probes

N‐oxides, characterized by a highly polar N + –O − bond, have recently demonstrated rapidly growing applications in biomedicine and material science due to their high solubility and redox activity. However, the chemical and physical properties of the N‐oxide have not been fully studied, limiting its advanced applications. Herein, we report the unprecedented observation that N‐oxide could undergo structure‐dependent aggregation. This observation is initiated by the appearance of dimers and trimers of a model compound, (4‐piperidinophenyl)methanol N‐oxide, in mass spectrometry. More convincingly, when it is conjugated with tetraphenylethylene (TPE) derivatives via the benzyloxy group, 4‐piperidinobenzyl N‐oxide promotes the aggregation and fluorescence emission of the resulting conjugate, though it is highly polar. This observation of aggregation is further confirmed by the morphology study via scanning electron microscopy. Interestingly, no aggregation is observed when N‐oxide is conjugated directly to TPE, indicating that N‐oxide‐induced aggregation is structure‐dependent. Based on these fundamental observations and studies, we develop a novel heme‐targeting probe that can specifically and sensitively detect the level of total heme in the plasma from hemolytic mice to distinguish hemolysis. Altogether, these findings will advance our understanding of structure‐dependent aggregation of the N‐oxide and help to bring new insights into its application in biomedicine and material science.