Orthogonal bioorthogonal chemistries

•Mutually orthogonal bioorthogonal reactions are described.•Current challenges in compatible reaction development are outlined.•Strategies to identify and generate orthogonal reactions are presented.•Recent applications of orthogonal bioorthogonal reactions in cellular imaging and macromolecular assembly are highlighted. Bioorthogonal reactions have long been used to examine individual biomolecules in living systems. Studies of multi-component networks demand not only reliable bioorthogonal chemistries, but also combinations of reactions that can be used in tandem. Such ‘orthogonal bioorthogonal’ transformations have been reported in recent years, and these chemistries are enabling new explorations into biology. This article highlights the development of orthogonal bioorthogonal reactions and their application in multi-target imaging and macromolecule assembly. Methods to tune and control orthogonal reactivity are also discussed, along with prospects for identifying new classes of compatible reactions.