Optimization of the Azobenzene Scaffold for Reductive Cleavage by Dithionite; Development of an Azobenzene Cleavable Linker for Proteomic Applications

In this paper we conducted an extensive reactivity study to determine the key structural features that favour the dithionite‐triggered reductive cleavage of the azo–arene group. Our stepwise investigation allowed identification of a highly reactive azo–arene structure 25 bearing a carboxylic acid at the ortho position of the electron‐poor arene and an ortho‐O‐alkyl‐resorcinol as the electron‐rich arene. Based on this 2‐(2′‐alkoxy‐4′‐hydroxyphenylazo)benzoic acid (HAZA) scaffold, the orthogonally protected difunctional azo–arene cleavable linker 26 was designed and synthesized. Selective linker deprotection and derivatization was performed by introducing an alkyne reactive group and a biotin affinity tag. This optimized azo–arene cleavable linker led to a total cleavage in less than 10 s with only 1 mM dithionite. Similar results were obtained in biological media. Structural optimization of the azobenzene scaffold for dithionite reductive cleavage led to the development of an efficient azo–arene cleavable linker. This highly reactive compound was designed to be orthogonallydiprotected and easily derivatized by alkyne and biotin. This water‐soluble linker showed a half‐life of < 1 s in biological media and is ready to be used for proteomic applications.