Fluorescence imaging has been established as a fundamental technology in the life sciences to directly “see” dynamic chemical changes occurring in living organisms. Compared to fluorescent proteins, small organic dyes are less likely to sterically interfere with biological systems, however, background fluorescent signals coming from unattached dyes to a target of interest (TOI) become huge problems in their application. Therefore, fluorogenic dyes, which can turn on their emission upon TOI recognition are required.<br/>Tetrazine exhibits high fluorescent quenching due to its n-π* transition in S1 state. Tetrazine-containing fluorophores are non-emissive, however, once tetrazine is removed by Diels-Alder reactions with strained dienophiles, it shows strong turn-on emission. Since this reaction is classified as a bioorthogonal reaction that proceeds selectively and safely in vivo, tetrazine-containing fluorogenic probes are considered as the desirable candidate for TOI imaging in bio-systems. Especially, red-light (&gt;630 nm) emissive fluorogenic probes are eagerly awaited considering their low phototoxicity and deep penetration depth. However, the quenching ability of tetrazine dramatically decreases once the emission exceeds its absorbance in 400-570 nm, and most reported red-emissive tetrazine fluorogenic probes only show non- or small turn-on emissions up to 50 times.<br/>In my research, tetrazine dyes with strong turn-on luminescence were designed by utilizing an aggregate formation. Molecules with moderate hydrophobicity form nano-aggregate, inside which molecules are packed to each other. Due to this close distance, tetrazine quenching efficiency was enhanced to result in strong turn-on property of the overall system. Here, strong aggregate emission was achieved by choosing aggregation-induced emission luminogens as a fluorophore.<br/>As a result, a series of highly fluorogenic tetrazine probes with up to 2,000 times turn-on efficiency were synthesized. One of them shows 100 times turn-on property even in the red region. By connecting strained dienophiles with TOIs, those dyes successfully stain multiple organelles such as mitochondria, lipid droplets, and plasma membranes with high selectivity and excellent signal-to-noise ratio.

Aggregation-strategy to achieve highly fluorogenic bioorthogonal tetrazine probes

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