Dynamic bioorthogonal imaging using a tetrazine NIR-AZA fluorogenic probe

Abstract

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.