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 biorthogonal 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 reaction 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 Fmoc strategy for peptide synthesis and demonstrate its reaction with strained bicyclononynes at 37 °C with rates comparable to the reaction of azides with the same substrates. The synthetic route to triazinylalanine is readily adaptable to late-stage 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.