Abstract The development of new bioorthogonal reactions with mutual orthogonality to classic bioorthogonal reactions such as the strain‐promoted azide–alkyne click reaction and the inverse‐electron‐demand Diels–Alder reaction is of great importance in providing chemical tools for multiplex labelling of live cells. Here we report the first anionic cycloaddend‐promoted bioorthogonal cycloaddition reaction between phenanthrene‐9,10‐dione and furan‐2(3H)‐one derivatives, where the high polarity of water is exploited to stabilize the highly electron‐rich anionic cycloaddend. The reaction is metal‐ and strain‐free, which proceeds rapidly in aqueous solution and on live cells with a second‐order rate constant up to 119?M?1?s?1. The combined utilization of this reaction together with the two other widely used bioorthogonal reactions allows for mutually orthogonal labelling of three types of proteins or three groups of living cells in one batch without cross‐talking. Such results highlight the great potential for multiplex labelling of different biomolecules in live cells.
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