This work describes the development of phenyl diazenyl piperidine triazene derivatives that can be activated to release aryl diazonium ions for labeling of proteins using light. These probes show marked bench stability at room temperature and can be photoisomerized via low-intensity UVA irradiation at physiological pH. Upon isomerization, the triazenes are rendered more basic and readily protonate to release reactive aryl diazonium ions. It was discovered that the intensity and duration of the UV light was essential to the observed diazonium ion reactivity in competition with the traditionally observed photolytic radical pathways. The combination of their synthetic efficiency coupled with their overall stability makes triazenes an attractive candidate for use in bioconjugation applications. Bioorthogonal handles on the triazenes are used to demonstrate the ease by which proteins can be modified.

Triazenes can easily be synthesized from various anilines via diazotization and conjugation with secondary amines, including piperidine. Previous literature has noted that such triazene formation can act as a protecting mechanism for aryl diazoniums that can be subsequently released in highly acidic conditions. Previous work in our own lab pertaining to triazabutadienes (TBDs) has shown analogous reactivity, whereas aryl diazoniums can be released under various conditions including low pH, or by irradiation with UV light. Such initiated release shows potential advantages for studying various biological systems. In this work we show that a class of phenylazo‐piperidine triazenes have similar potential utility to be used as protected diazoniums for biological study. We were able to easily synthesize a small library of triazenes with various electronic properties including those with benzene, p‐nitrobenzene, p‐benzamide, and m‐methoxy substituents, among others. Subsequent studies on these compounds showed that the electronics of the functional groups alters the stability and subsequent reactivity of the respective triazenes allowing for different rates of aryl diazonium release. We have seen that decreasing electron density within the aryl ring leads to more stable, and less reactive triazenes, whereas increasing the electron donating potential of these substituents increases reactivity. Furthermore, we have shown that these triazenes are capable of releasing their diazonium species in neutral pH via irradiation with 350 nm UV light, a phenomenon that has not been previously reported to our knowledge. Lastly, we report the ability for these triazenes to label proteins in situ using UV irradiation and display the utility of a novel coumarin triazine that has analogous reactivity to previously reported coumarin triazabutadienes developed in our lab. Support or Funding Information NSF CAREER Award