We are exploring bioorthogonal reactions as tools for addressing problems in biological imaging and bottom up synthetic biology. Recently we have developed and implemented catalytic protocols to synthesize functional 1,2,4,5-tetrazines, either from conveniently obtained precursors or using newly described tetrazine building blocks. These approaches have increased the diversity of accessible tetrazines and have led to the discovery of highly quenched tetrazine fluorophores which can increase &gt;100-fold in fluorescence intensity upon reaction with strained dienophiles such as <i>trans</i>-cyclooctene (TCO). We foresee that these probes will become highly useful for cellular imaging applications, by mitigating signals from background probe uptake. Additionally, we have introduced the use of small dienophiles, namely methylcyclopropenes, as stable yet reactive tetrazine ligation partners. These compounds have enabled the use of tetrazine cycloadditions for imaging and labeling applications that require the use of low molecular weight probes. We have implemented cyclopropenes to track the distribution of lipid analogs in live mammalian cells using fluorogenic tetrazine imaging probes. Additionally, we have recently synthesized sugar analogs containing methyl-cyclopropenes, enabling metabolic imaging of glycan synthesis in live cells. Importantly, this technique can be done sequentially with classic “click” chemistry approaches, enabling multicolor imaging of 2 different metabolically incorporated molecules. We are currently implementing the new tetrazines and methyl-cyclopropenes to various cellular imaging applications, such as pretargeted imaging of antibody targeted cancer biomarkers and the fluorescent detection of specific oligonucleotide sequences in live cells.

Advancing tetrazine ligations to develop fluorogenic probes for cellular imaging

249th National Meeting (2015)

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