The development of reagents that can selectively react in complex biological media is an important challenge. Here we show that N1‐alkylation of 1,2,4‐triazines yields the corresponding triazinium salts, which are three orders of magnitude more reactive in reactions with strained alkynes than the parent 1,2,4‐triazines. This powerful bioorthogonal ligation enables efficient modification of peptides and proteins. The positively charged N1‐alkyl triazinium salts exhibit favorable cell permeability, which makes them superior for intracellular fluorescent labeling applications when compared to analogous 1,2,4,5‐tetrazines. Due to their high reactivity, stability, synthetic accessibility and improved water solubility, the new ionic heterodienes represent a valuable addition to the repertoire of existing modern bioorthogonal reagents.

A synthetic strategy to pyrrolo[2,1- f ][1,2,4]triazines is reported. We show that various synthetically easily accessible 1,2,4-triazines can be efficiently alkylated under mild conditions to provide the corresponding 1-alkyl-1,2,4-triazinium salts. These bench-stable salts serve as precursors to triazinium ylides, which react in 1,3-dipolar cycloadditions with electron-poor dipolarophiles to yield polysubstituted pyrrolotriazines in a single step.

The development of reagents that can selectively react in complex biological media is an important challenge. Here we show that N1‐alkylation of 1,2,4‐triazines yields the corresponding triazinium salts, which are three orders of magnitude more reactive in reactions with strained alkynes than the parent 1,2,4‐triazines. This powerful bioorthogonal ligation enables efficient modification of peptides and proteins. The positively charged N1‐alkyl triazinium salts exhibit favorable cell permeability, which makes them superior for intracellular fluorescent labeling applications when compared to analogous 1,2,4,5‐tetrazines. Due to their high reactivity, stability, synthetic accessibility and improved water solubility, the new ionic heterodienes represent a valuable addition to the repertoire of existing modern bioorthogonal reagents.

The development of reagents that can selectively react in complex biological media is an important challenge. Here we show that N1-alkylation of 1,2,4-triazines yields the corresponding triazinium salts, which are three orders of magnitude more reactive in reactions with strained alkynes than the parent 1,2,4-triazines. This powerful bioorthogonal ligation enables efficient modification of peptides and proteins. The positively charged N1-alkyl triazinium salts exhibit favorable cell permeability, which makes them superior for intracellular fluorescent labeling applications when compared to analogous 1,2,4,5-tetrazines. Due to their high reactivity, stability, accessibility and improved water solubility, the new ionic heterodienes represent a valuable addition to the repertoire of existing modern bioorthogonal reagents.