Straightforward synthesis of bioconjugatable azo dyes. Part 1: Black Hole Quencher-1 (BHQ-1) scaffold

Azo dyes are currently used to quench the fluorescence of energy donors in bioassays through Förster resonance energy transfer (FRET) phenomenon. Common examples of such dark quenchers are DABCYL and the three members of Black Hole Quencher® (BHQ) family. Yet, only carboxylic acid and phosphoramidite derivatives of such azo dyes are presently commercially available. This Letter presents a straightforward synthesis method to novel bioconjugatable quenchers derived from BHQ-1 scaffold and equipped with a reactive group being either azido, terminal alkyne, or maleimide. The potential utility of the ‘clickable’ azido and thiol-reactive derivatives was notably demonstrated through the preparation of the first water-soluble BHQ-1 dye and a FRET-based probe suitable for the detection of urokinase-type plasminogen activator (uPA), a key protease in cancer invasion and metastasis, respectively.