Strain-Promoted Reaction of 1,2,4-Triazines with Bicyclononynes

Strain-promoted inverse electron-demand Diels-Alder cycloaddition (SPIEDAC) reactions between 1,2,4,5-tetrazines and strained dienophiles, such as bicyclononynes, are among the fastest bioorthogonal reactions. However, the synthesis of 1,2,4,5-tetrazines is complex and can involve volatile reagents. 1,2,4-Triazines also undergo cycloaddition reactions with acyclic and unstrained dienophiles at elevated temperatures, but their reaction with strained alkynes has not been described. We postulated that 1,2,4-triazines would react with strained alkynes at low temperatures and therefore provide an alternative to the tetrazine cycloaddition re-action for use in in vitro or in vivo labelling experiments. We describe the synthesis of a 1,2,4-triazin-3-ylalanine derivative fully compatible with the fluorenylmethyloxycarbonyl (Fmoc) strategy for peptide synthesis and demonstrate its reaction with strained bicyclononynes at 37 degrees C with rates comparable to the reaction of azides with the same substrates. The synthetic route to triazinylalanine is readily adaptable to latestage functionalization of other probe molecules, and the 1,2,4-triazine-SPIEDAC therefore has potential as an alternative to tetrazine cycloaddition for applications in cellular and biochemical studies.