Bioorthogonal Cycloadditions with Sub‐Millisecond Intermediates

Tetrazine‐ and sydnone‐based click reactions have emerged as important bioconjugation strategies with fast kinetics and N₂ or CO₂ as the only byproduct. Mechanistic studies of these reactions have focused on the initial rate‐determining cycloaddition steps. The subsequent N₂ or CO₂ release from the bicyclic intermediates has been approached mainly through computational studies, which have predicted lifetimes of femtoseconds. In the present study, bioorthogonal cycloadditions involving N₂ or CO₂ extrusion have been examined experimentally at the single‐molecule level by using a protein nanoreactor. At the resolution of this approach, the reactions appeared to occur in a single step, which places an upper limit on the lifetimes of the intermediates of about 80 μs, which is consistent with the computational work.