A chronicle of organic synthesis in water: A trajectory of growth in the intervening quarter century

Tetrazine fluorogenic probes are of high value due to their ability to modulate emission intensity from low to high in response to inverse electron demand Diels-Alder cycloadditions with dienophiles. In this study, a tetrazine substituted BF2-azadipyrromethene probe and a related model tetrazine were investigated for their reactivity with strained alkyne and alkene dienophiles to identify the optimal probe for cellular use. Specifically, the reactivity of 2-deoxy-d-glucose substituted 1-methyl cyclopropene and bicyclononyne was tested, with the latter demonstrating superior reactivity with complete reactions observed within 15 min. Moreover, the tetrazine substituted BF2-azadipyrromethene showed a strong fluorogenic response at a λmax of 715 nm in aqueous solution as the cycloaddition proceeded. The high reaction efficiency and significant 43-fold fluorescence enhancement factor encouraged its translation to bioorthogonal cell imaging. Pre-incubating HeLa Kyoto cells with 2-deoxy-glucose-alkyne dienophile followed by treatment with the BF2-azadipyrromethene fluorogenic probe, allowed dynamic imaging of the bioorthogonal reaction in live cells. Pre-incubation of 2-deoxy-glucose alkyne for 24 h with subsequent fluorogenic cycloaddition indicated that bioorthogonal reaction occurred in subcellular regions such as the mitochondria. This study showed the potential for real-time observation of intra-cellular fluorogenic cycloadditions in the near-infrared spectral region.