FlAsH-EDT2

FlAsH-EDT₂ is an organoarsenic compound with molecular formula C₂₄H₁₈As₂O₅S₄. Its structure is based around a fluorescein core with two 1,3,2-dithiarsolane substituents. It is used in bioanalytical research as a fluorescent label for visualising proteins in living cells. FlAsH-EDT₂ is an abbreviation for fluorescin arsenical hairpin binder- ethanedithiol, and is a pale yellow or pinkish fluorogenic solid. It has a semi-structural formula (C₂H₄AsS₂)₂-(C₁₃H₅O₃)-C₆H₄COOH, representing the dithiarsolane substituents bound to the hydroxyxanthone core, attached to an ''o''-substituted molecule of benzoic acid.

FlAsH-EDT₂ is used for site-specific labelling, selectively binding to proteins containing the tetracysteine (TC) motif Cys-Cys-Xxx-Xxx-Cys-Cys and becoming fluorescent when bound. It displays non-specific binding to endogenous cysteine-rich proteins, meaning it binds to sites other than the one of interest (CCXXCC). Further optimization of the TC motif has revealed improved FlAsH binding affinity for a CCPGCC motif, and higher quantum yield when the tetracysteine motif is flanked with specific residues (HRWCCPGCCKTF or FLNCCPGCCMEP).

Preparation

FlAsH-EDT₂ can be prepared in three steps from fluorescein (see figure).

Formation of FlAsH-TC adduct

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Many studies show that trivalent arsenic compounds bind to pairs of cysteine residues. This binding is responsible for the toxicity of many arsenic compounds. Binding is reversed by 1,2-ethanedithiol, which binds tightly to arsenic compounds, as shown by the stability of FlAsH-EDT₂. Such strong sulfur-arsenic bond can be, again, regulated by designing a peptide domain that exhibits higher affinity toward the arsenic, such as tetracysteine motif. By modulating the distance between the two pairs of cysteine residues and the space between the arsenic centers of FlAsH-EDT₂, a cooperative and entropically favored dithiol arsenic bond could be achieved.

The binding of FlAsH-EDT₂ is thus subject to equilibration. The FlAsH-peptide adduct formation can be favored in low concentration of EDT (below 10  μM) and be reversed in high concentration of EDT (above 1  mM).

Properties

FlAsH becomes fluorescent upon the binding of tetracysteine motif. It is excited at 508 nm and emits 528 nm, a green-yellow, of free fluorescein. The quantum yield is 0.49 for 250 nM FlAsH is bound to a model tetracysteine-containing peptide in a phosphate-buffered saline at pH 7.4.

Generally, FlAsH-EDT₂ has 0.1-0.6 fluorescence quantum efficiencies with several μM detection limits for diffuse cytosolic tag and 30 - 80 extinction coefficients L mmol⁻¹ cm⁻¹. The FlAsH-peptide complex also has demonstrated fluorescence resonance energy transfer (FRET) from fluorescent proteins, such as from enhanced cyan fluorescent protein (ECFP) of Green Fluorescent Protein (GFP).

Application

FlAsH-EDT₂ enables less toxic and more specific fluorescent labeling that is membrane permeable. The modification of the fluorescein moiety also allows multicolor analysis.TC-FlAsH™ II In-Cell Tetracysteine Tag Detection Kit (Green Fluorescence), for live-cell imaging
/ref> It has been proven to be a good alternative to green fluorescent proteins (GFP) with the advantage that FlAsH-EDT₂ is much smaller (molar mass < 1 kDa) as compared to GFPs (~30 kDa), therefore minimizing the perturbation of activity of the protein under the study.

Use

In the past, FlAsH-EDT₂ has been widely used to study a number of ''in vivo'' cellular events and subcellular structures in animal cells, Ebola virus matrix protein, and protein misfolding. With the electron microscopic imaging, FlAsH-EDT₂ is also used to study the processes of protein trafficking ''in situ''. More recently, it was used in an extended study of plant cells like ''Arabidopsis'' and tobacco.

References

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Biochemistry detection reactions
Fluorescent dyes
Phenols
Benzoic acids
Triarylmethane dyes
Organoarsenic dithiolates
Arsenic heterocycles