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Bioconjugatable Azo-Based Dark-Quencher Dyes: Synthesis and Application to Protease-Activatable Far-Red Fluorescent Probes

Authors

  • Arnaud Chevalier,

    1. Université de Rouen, Laboratory COBRA UMR 6014 & FR 3038, IRCOF, rue Lucien Tesnière, 76821 Mont-Saint-Aignan (France), Fax: (+33) 2-35-52-29-71
    2. CNRS Délégation Normandie, 14 Rue Alfred Kastler, 14052 Caen Cedex (France)
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  • Dr. Cédrik Massif,

    1. Université de Rouen, Laboratory COBRA UMR 6014 & FR 3038, IRCOF, rue Lucien Tesnière, 76821 Mont-Saint-Aignan (France), Fax: (+33) 2-35-52-29-71
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  • Prof. Pierre-Yves Renard,

    Corresponding author
    1. Université de Rouen, Laboratory COBRA UMR 6014 & FR 3038, IRCOF, rue Lucien Tesnière, 76821 Mont-Saint-Aignan (France), Fax: (+33) 2-35-52-29-71
    2. CNRS Délégation Normandie, 14 Rue Alfred Kastler, 14052 Caen Cedex (France)
    3. INSA de Rouen, Avenue de l'Université, 76800 St Etienne du Rouvray (France)
    • Université de Rouen, Laboratory COBRA UMR 6014 & FR 3038, IRCOF, rue Lucien Tesnière, 76821 Mont-Saint-Aignan (France), Fax: (+33) 2-35-52-29-71
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  • Dr. Anthony Romieu

    Corresponding author
    1. Université de Rouen, Laboratory COBRA UMR 6014 & FR 3038, IRCOF, rue Lucien Tesnière, 76821 Mont-Saint-Aignan (France), Fax: (+33) 2-35-52-29-71
    2. CNRS Délégation Normandie, 14 Rue Alfred Kastler, 14052 Caen Cedex (France)
    3. INSA de Rouen, Avenue de l'Université, 76800 St Etienne du Rouvray (France)
    • Université de Rouen, Laboratory COBRA UMR 6014 & FR 3038, IRCOF, rue Lucien Tesnière, 76821 Mont-Saint-Aignan (France), Fax: (+33) 2-35-52-29-71
    Search for more papers by this author

Abstract

We describe the efficient synthesis and one-step derivatization of novel, nonfluorescent azo dyes based on the Black Hole Quencher-3 (BHQ-3) scaffold. These dyes were equipped with various reactive and/or bioconjugatable groups (azido, α-iodoacetyl, ketone, terminal alkyne, vicinal diol). The azido derivative was found to be highly reactive in the context of copper-catalyzed azide–alkyne cycloaddition (CuAAC) reactions and allowed easy synthetic access to the first water-soluble (sulfonated derivative) and aldehyde-modified BHQ-3 dyes, the direct preparation of which failed by means of conventional azo-coupling reactions. The aldehyde- and α-iodoacetyl-containing fluorescence quenchers were readily conjugated to aminooxy- and cysteine-containing peptides by the formation of a stable oxime or thioether linkage, respectively. Further fluorescent labeling of the resultant peptide conjugates with red- or far-red-emitting rhodamine or cyanine dyes through sequential and/or one-pot bioconjugations, led to novel Förster resonance energy transfer (FRET) based probes suitable for the in vivo detection and imaging of urokinase plasminogen activator, a key protease in cancer invasion and metastasis.

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