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

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.

Guiding Principles in The Design of Ligand-Targeted Nanomedicines

“…In this construct, the fluorescent dye only dequenches when the DNA probe hybridizes with a complementary DNA or RNA within the target cell [210,211]. Related 'protease activatable fluorophores' similarly exploit one or more tumor-specific proteolytic enzymes to cleave a peptide bridge linking a quencher to a fluorescent dye [212][213][214], thereby activating fluorescence emission. A second major difference in the fundamental principles governing the design of NPs for imaging and therapeutic applications lies in their relative need for deep tissue penetration.…”

Constructing a FRET-based molecular chemodosimeter for cysteine over homocysteine and glutathione by naphthalimide and phenazine derivatives

“…57 Phenazine was rst reduced by Na 2 S 2 O 4 , then alkylated by butyl iodide followed by formylation through the Vilsmeier-Haack reaction. Cyanoacidic acid was then introduced by the Knoevenagel reaction.…”

Sonophore labeled RGD: a targeted contrast agent for optoacoustic imaging

“…However, these dyes mainly emit absorbed energy in the form of fluorescence, making only a partial amount of the energy available for thermoelastic expansion, which results in the generation of a weaker optoacoustic signal. Therefore, a group of dyes called dark quenchers, so far mainly used to quench fluorescence of other dyes in the study of biomolecule dynamics [11], [12], [13], is highly promising for the design of contrast agents in optoacoustic imaging. They emit absorbed energy mainly as heat, by a non-radiative conversion of the light energy, which is accompanied by the formation of acoustic waves.…”