Bioorthogonal ligation with functionalized fluorescent dyes enables visualization of nuclei acids, proteins and metabolites in biological systems. Bright and modular azide- and alkyne-functionalized dyes are therefore needed to expand the fluorescence imaging capabilities of bioorthogonal ligation methods. We describe the concise synthesis of clickable fluorescent dyes based on 2-dicyanomethylene-3-cyano-2,5-dihydrofuran fluorophores and demonstrate their utility for multicolor imaging of azide- and alkyne-modified proteins as well as FRET studies. © 2009 The Royal Society of Chemistry.
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