Non-fluorescent azo dyes are extensively employed in advanced optical bioprobes, particularly those utilizing the Förster resonance energy transfer (FRET) mechanism. These probes find widespread application in protease activity assays, nucleic acid hybridization, and real-time PCRs, effectively quenching the fluorescence of energy donors. The unique photochemical properties of these dyes, such as the isomerization of the -N=N- moiety upon UV-vis irradiation, render them weakly or non-fluorescent, thereby enabling their use as fluorescence quenchers. Common examples of such quenchers include DABCYL and members of the Black Hole Quencher (BHQ) family, covering the spectrum from 480 nm to the near-infrared (NIR) region. Among them, BHQ-3, with a maximal absorbance at 672 nm, effectively overlaps the emission of the pentamethine cyanine (Cy5) fluorophore. Consequently, the Cy5/BHQ-3 FRET pair has been employed in various protease activity assays. Its effectiveness in deep tissue imaging is notable due to Cy5's far-red excitation and emission wavelengths (Exc. 650 nm, Em. 670 nm).

Following our research interests in developing nanoconstructed FRET-labeled peptide probes for real-time monitoring of proteases activity in therapy and medical imaging, we present the synthesis and characterization of the fluorescence quencher BHQ-3. It was synthesized via an azo-coupling reaction between an aryl-phenazonium salt and an N,N-substituted aniline functionalized with a primary amine, enabling its further conjugation to the C-terminal of a specific peptide via amide bond formation. Additionally, we investigated the quenching mechanism of the Cy5/BHQ-3 FRET pair through photometric and fluorimetric studies.