Fluorogenic bioorthogonal reactions enable visualization of biomolecules with excellent signal-to-noise ratio. A bicyclononyne-tetrazine ligation that produces fluorescent pyridazine products has been developed. In stark contrast to previous approaches, the formation of the dye is an inherent result of the chemical reaction and no additional fluorophores are needed in the reagents. The crucial structural elements that determine dye formation are electron-donating groups present in the starting tetrazine unit. The newly formed pyridazine fluorophores show interesting photophysical properties the fluorescence intensity increase in the reaction can reach an excellent 900-fold. Model imaging experiments demonstrate the application potential of this new fluorogenic bioorthogonal reaction.

• Keywords
• Diels-Alder reactions, cyclic alkynes, cycloaddition reactions, fluorogenic probes, tetrazines,
• MeSH
• Cycloaddition Reaction MeSH
• Fluorescent Dyes chemistry   MeSH
• Heterocyclic Compounds, 1-Ring chemistry   MeSH
• Microscopy, Confocal MeSH
• Humans MeSH
• Bridged Bicyclo Compounds chemistry   MeSH
• Cell Line, Tumor MeSH
• Pyridazines chemistry   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Fluorescent Dyes MeSH
• Heterocyclic Compounds, 1-Ring MeSH
• Bridged Bicyclo Compounds MeSH
• Pyridazines MeSH

Inverse-electron-demand Diels-Alder (iEDDA) cycloaddition between 1,2,4,5-tetrazines and strained dienophiles belongs among the most popular bioconjugation reactions. In addition to its fast kinetics, this cycloaddition can be tailored to produce fluorescent products from non-fluorescent starting materials. Here we show that even the reaction intermediates formed in iEDDA cycloaddition can lead to the formation of new types of fluorophores. The influence of various substituents on their photophysical properties and the generality of the approach with use of various trans-cyclooctene derivatives were studied. Model bioimaging experiments demonstrate the application potential of fluorogenic iEDDA cycloaddition.

• Keywords
• bioorthogonal chemistry, click chemistry, cycloaddition, heterocycles, tetrazines,
• MeSH
• Cycloaddition Reaction MeSH
• Cyclooctanes chemistry   MeSH
• Fluorescent Dyes chemical synthesis   chemistry   MeSH
• Microscopy, Fluorescence methods   MeSH
• HeLa Cells MeSH
• Heterocyclic Compounds, 2-Ring chemical synthesis   chemistry   MeSH
• Heterocyclic Compounds, 1-Ring chemistry   MeSH
• Microscopy, Confocal methods   MeSH
• Humans MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cyclooctanes MeSH
• Fluorescent Dyes MeSH
• Heterocyclic Compounds, 2-Ring MeSH
• Heterocyclic Compounds, 1-Ring MeSH