Straightforward synthesis of bioconjugatable azo dyes. Part 2: Black Hole Quencher-2 (BHQ-2) and BlackBerry Quencher 650 (BBQ-650) scaffolds

[Display omitted] •Synthesis of novel bioconjugatable azo-based dark quencher dyes.•Post-synthetic formylation/sulfonation of azo dyes by ‘click’ chemistry.•One-step synthesis of a chemically convertible BlackBerry Quencher dye.•Urokinase sensing using a FRET-based peptide probe. A further extension of the efficient synthetic methodology described in Part 1, to the aromatic bis-diazo scaffold of Black Hole Quencher-2 dye is presented. Bioconjugatable derivatives bearing either azido, terminal alkyne, or maleimide reactive group were easily obtained as well as the free-phenol form of BlackBerry® Quencher 650 (BBQ-650®) initially developed by Berry & Associates, Inc. Company. The efficient conjugation ability of azido- and maleimide-quenchers was demonstrated through the facile preparation of the first water-soluble and formylated BHQ-2 dyes and a FRET-based probe suitable for the in vitro/in cellulo detection of a cancer-associated protease namely urokinase-type plasminogen activator.