Inverse electron‐demand Diels–Alder cycloadditions (iEDDAC) between tetrazines and strained alkenes/alkynes have emerged as essential tools for studying and manipulating biomolecules. A light‐triggered version of iEDDAC (photo‐iEDDAC) is presented that confers spatio‐temporal control to bioorthogonal labeling in vitro and in cellulo. A cyclopropenone‐caged dibenzoannulated bicyclo[6.1.0]nonyne probe (photo‐DMBO) was designed that is unreactive towards tetrazines before light‐activation, but engages in iEDDAC after irradiation at 365 nm. Aminoacyl tRNA synthetase/tRNA pairs were discovered for efficient site‐specific incorporation of tetrazine‐containing amino acids into proteins in living cells. In situ light activation of photo‐DMBO conjugates allows labeling of tetrazine‐modified proteins in living E. coli. This allows proteins in living cells to be modified in a spatio‐temporally controlled manner and may be extended to photo‐induced and site‐specific protein labeling in animals.

Inverse electron-demand Diels-Alder cycloadditions (iEDDAC)b etween tetrazines and strained alkenes/ alkynes have emerged as essential tools for studying and manipulating biomolecules.Alight-triggered version of iED-DAC( photo-iEDDAC)i sp resented that confers spatiotemporal control to bioorthogonal labeling in vitro and in cellulo.Acyclopropenone-caged dibenzoannulated bicyclo-[6.1.0]nonyne probe (photo-DMBO) was designed that is unreactive towards tetrazines before light-activation, but engages in iEDDAC after irradiation at 365 nm. Aminoacyl tRNAsynthetase/tRNApairs were discovered for efficient sitespecific incorporation of tetrazine-containing amino acids into proteins in living cells.Insitu light activation of photo-DMBO conjugates allows labeling of tetrazine-modified proteins in living E. coli. This allows proteins in living cells to be modified in aspatio-temporally controlled manner and may be extended to photo-induced and site-specific protein labeling in animals.