Developing bioorthogonal probes to span a spectrum of reactivities

Bioorthogonal chemistries enable researchers to interrogate biomolecules in living systems. These reactions are highly selective and biocompatible, and can be performed in many complex environments. However, like any organic transformation, there is no perfect bioorthogonal reaction. Choosing the 'best fit' for a desired application is crucial. Correspondingly, there must be a variety of chemistries-spanning a range of rates and other features - to choose from. Over the past few years, considerable strides have been made towards not only expanding the number of bioorthogonal chemistries but also fine-tuning existing reactions for particular applications. In this Review, we highlight recent advances in the development of bioorthogonal reactions, focusing on how principles of physical organic chemistry have guided probe design. The continued expansion of this toolset will provide precisely tuned reagents for manipulating bonds in distinct environments.