Isonitrile induced bioorthogonal activation of fluorophores and mutually orthogonal cleavage in live cells.

Fluorophores with different emission wavelengths were efficiently quenched by a tert -butyl terminated tetrazylmethyl group and activated by an isonitrile-tetrazine click-to-release reaction. Nucleic acid templated chemistry significantly accelerated this bioorthogonal cleavage. Moreover, two mutually orthogonal fluorogenic cleavage reactions were simultaneously conducted in live cells for the first time.