Hydrodabcyl: A Superior Hydrophilic Alternative to the Dark Fluorescence Quencher Dabcyl

Abstract

Dark fluorescence quenchers are nonfluorescent dyes that can modulate the fluorescence signal of an appropriate fluorophore donor in a distance-dependent manner. Dark quenchers are extensively used in many biomolecular analytical applications, such as studies with fluorogenic protease substrates or nucleic acids probes. A very popular dark fluorescence quencher is dabcyl, which is a hydrophobic azobenzene derivative. However, its insolubility in water may constitute a major drawback, especially during the investigation of biochemical systems whose natural solvent is water. We designed and synthesized a new azobenzene-based dark quencher with excellent solubility in aqueous media, which represents a superior alternative to the much-used dabcyl. The advantage of hydrodabcyl over dabcyl is exemplarily demonstrated for the cleavage of the fluorogenic substrate hydrodabcyl-Ser-Phe-EDANS by the proteases thermolysin and papain.

Efficient Synthesis of a Wide‐Range Absorbing Azaphthalocyanine Dark Quencher and Its Application to Dual‐Labeled Oligonucleotide Probes for Quantitative Real‐Time Polymerase Chain Reactions

“…Quenching the fluorescence of af luorescent probe by different processesi safundamentalm echanism for an umber of different assays used in biochemistry,a nalytical chemistry,b iology, and genetica nalysis. Fluorescence quenching is routinely used for monitoringt he enzymatic cleavage of peptides [1,2] and oligonucleotide probes, [3] in real-time polymerase chain reactions (PCR), [4] in the detection of specific targets, [5,6] and in observations of changes in the cellular environment. [7,8] Atypicalo ligonucleotide probeh as two dyes connected to ap roperly designed sequence-the reporter( fluorescent dye)a nd the quencher.T wo mechanisms arer esponsible for the quenching of fluorescencee mitted by the reporter,i .e.,t he dynamic and static quenching.…”

“…Several novel dark quenchers were, therefore, synthesized in the last decade with the aim of broadening their absorption spectra (e.g., multipath quenchers, trimers of different dyes, or polymers) . At the same time, only limited structural variation exists in the quenchers because most of the used or studied quenchers are azo dyes, derivatives of rhodamine, or cyanine dyes . Only a few novel structural motifs are currently under development—e.g., BODIPY and the antraquinone dyes.…”

Stimuli-Responsive Supramolecular Coaggregation and Disaggregation of Host–Guest Conjugates Having a Disulfide Linkage

“…Despite the presence of a hydrophobic macrocyclic skeleton and a dabsyl moiety, compounds 1a and 2a were fairly soluble in water in neutral pH ranges owing to the three peripheral polar side chains. It is well known that dabsyl chromophores − have an intense and broad visible absorption. Actually, both compounds 1a and 2a showed UV–vis absorption spectra between 350 and 600 nm in aqueous 2-[4-(2-hydroxyethyl)-1-piperazinyl]­ethanesulfonic acid (HEPES) buffer (0.01 M, pH 7.4, 0.15 M with NaCl).…”

Chemoselective Attachment of the Water-Soluble Dark Quencher Hydrodabcyl to Amino Groups in Peptides and Preservation of Its Spectroscopic Properties over a Wide pH Range

“…The use of water-soluble derivatives of dabcyl can help to overcome these issues. In a previous publication, 6 we reported the synthesis of the water-soluble derivative hydrodabcyl [4-(2′,6′-dihydroxy-4′-dimethylaminophenylazo)-2-hydroxybenzoic acid, 2] and its purification by sequential cycles of precipitation and centrifugation to achieve purity, enabling its direct use in spectroscopic applications. For most purposes, however, hydrodabcyl has to be linked to target molecules.…”