Fluorescent probes that light-up upon reaction with complementary bioorthogonal reagents are superior tools for no-wash fluorogenic bioimaging applications. In this work, a thorough study is presented on a set of seventeen structurally diverse coumarin-tetrazine probes that produce fluorescent dyes with exceptional turn-on ratios when reacted with trans-cyclooctene (TCO) and bicyclononyne (BCN) dienophiles. In general, formation of the fully aromatic pyridazine-containing dyes resulting from the reaction with BCN was found superior in terms of fluorogenicity. However, evaluation of the probes in cellular imaging experiments revealed that other factors, such as reaction kinetics and good cell permeability, prevail over the fluorescence turn-on properties. The best compound identified in this study showed excellent performance in live cell-labeling experiments and enabled no-wash fluorogenic imaging on a timescale of seconds.

• Keywords
• click chemistry, coumarins, fluorescence, live cell bioimaging, tetrazines,
• MeSH
• Fluorescent Dyes analysis   MeSH
• Heterocyclic Compounds analysis   MeSH
• Microscopy, Confocal MeSH
• Coumarins analysis   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Optical Imaging * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Fluorescent Dyes MeSH
• Heterocyclic Compounds MeSH
• Coumarins MeSH

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