FlAsH-EDT2
{{DISPLAYTITLE:FlAsH-EDT2}}
{{Chembox
| ImageFile = FlAsH-EDT2.svg
| ImageAlt =
| IUPACName =
| OtherNames = Fluorescein Arsenical Hairpin Binder; Lumio green
|Section1={{Chembox Identifiers
| CASNo = 212118-77-9
| UNII_Ref = {{fdacite|correct|FDA}}
| UNII = Z8CH38Y9L3
| PubChem = 2763100
| ChemSpiderID = 2043798
| ChEBI = 52107
| ChEMBL = 454668
| SMILES1 = c1ccc(c(c1)c2c3ccc(c(c3oc-4c(c(=O)ccc24)[As]5SCCS5)[As]6SCCS6)O)C(=O)O
| StdInChI = 1S/C24H18As2O5S4/c27-17-7-5-15-19(13-3-1-2-4-14(13)24(29)30)16-6-8-18(28)21(26-34-11-12-35-26)23(16)31-22(15)20(17)25-32-9-10-33-25/h1-8,27H,9-12H2,(H,29,30)
| StdInChIKey = KCPRYVGBEBFLIG-UHFFFAOYSA-N
| SMILES2 = OC(=O)C1=C(C=CC=C1)C1=C2C=CC(=O)C([As]3SCCS3)=C2OC2=C([As]3SCCS3)C(O)=CC=C12}}
|Section2={{Chembox Properties
| C=24 | H=18 | As=2 | O=5 | S=4
| Appearance = Solid
| Density =
| MeltingPtC = 169 to 172
| MeltingPt_notes =
| BoilingPt =
| Solubility = }}
|Section3={{Chembox Hazards
| MainHazards =
| FlashPt =
| AutoignitionPt =
}}
}}
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.{{cite journal | doi = 10.1038/nprot.2008.144 | title = Preparation of the membrane-permeant biarsenicals FlAsH-EDT2 and ReAsH-EDT2 for fluorescent labeling of tetracysteine-tagged proteins | year = 2008 | last1 = Adams | first1 = Stephen R. | last2 = Tsien | first2 = Roger Y. | author-link2 = Roger Y. Tsien | journal = Nat. Protoc. | volume = 3 | issue = 9 | pages = 1527–1534 | pmid = 18772880 | pmc = 2843588}} 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,{{cite journal | last1 = Adams | first1 = Stephen R. | last2 = Campbell | first2 = Robert E. | last3 = Gross | first3 = Larry A. | last4 = Martin | first4 = Brent R. | last5 = Walkup | first5 = Grant K. | last6 = Yao | first6 = Yong | last7 = Llopis | first7 = Juan | last8 = Tsien | first8 = Roger Y. | year = 2002 | title = New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications | journal = Journal of the American Chemical Society | volume = 124 | issue = 21| pages = 6063–6076 | doi=10.1021/ja017687n| pmid = 12022841 }} and higher quantum yield when the tetracysteine motif is flanked with specific residues (HRWCCPGCCKTF or FLNCCPGCCMEP).{{cite journal | last1 = Martin | first1 = Brent R. | last2 = Giepmans | first2 = Ben N.G. | last3 = Adams | first3 = Stephen R. | last4 = Tsien | first4 = Roger Y. | year = 2005 | title = Mammalian cell–based optimization of the biarsenical-binding tetracysteine motif for improved fluorescence and affinity | journal = Nature Biotechnology | volume = 23 | issue = 10| pages = 1308–1314 | doi=10.1038/nbt1136| pmid = 16155565 | s2cid = 16456334 }}
Preparation
File:FlAsH preparation.png in TFA; Step 2: AsCl3, followed by Pd(OAc)2 and DIEA; Step 3: H2EDT in aqueous acetone.]]
FlAsH-EDT2 can be prepared in three steps from fluorescein (see figure).
Formation of FlAsH-TC adduct
Many studies show that trivalent arsenic compounds bind to pairs of cysteine residues. This binding is responsible for the toxicity of many arsenic compounds.{{cite book | last1 = Kalef | first1 = Edna | last2 = Gitler | first2 = Carlos | year = 1994 | chapter = Purification of Vicinal Dithiol-containing Proteins by Arsenical-Based Affinity Chromatography | series = Methods in Enzymology | volume = 233 | pages = 395–403 | doi = 10.1016/S0076-6879(94)33046-8 | title = Oxygen Radicals in Biological Systems, Part C | isbn = 9780080883465 | editor-first = Helmut | editor-last = Sies | publisher = Academic Press| pmid = 8015475 }} Binding is reversed by 1,2-ethanedithiol, which binds tightly to arsenic compounds, as shown by the stability of FlAsH-EDT2.{{cite journal | last = Whittaker | first = Victor P. | author-link = Victor P. Whittaker | year = 1947 | title = An Experimental Investigation of the 'Ring Hypothesis' of Arsenical Toxicity | journal = Biochem. J. | volume = 41 | issue = 1 | pages = 56–62 | doi=10.1042/bj0410056| pmc = 1258423 | pmid=16748119}} 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.{{cite journal | last1 = Griffin | first1 = B. Albert | last2 = Adams | first2 = Stephen R. | last3 = Tsien | first3 = Roger Y. | author-link3 = Roger Y. Tsien | year = 1998 | title = Specific Covalent Labeling of Recombinant Protein Molecules Inside Live Cells | journal = Science | volume = 281 | issue = 5374 | pages = 269–272 | doi = 10.1126/science.281.5374.269 | pmid = 9657724| bibcode = 1998Sci...281..269G }}
File:Formation of FlAsH-TC adduct.png
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).{{cite journal | last1 = Adams | first1 = Stephen R. | last2 = Campbell | first2 = Robert E. | last3 = Gross | first3 = Larry A. | last4 = Martin | first4 = Brent R. | last5 = Walkup | first5 = Grant K. | last6 = Yao | first6 = Yong | last7 = Llopis | first7 = Juan | last8 = Tsien | first8 = Roger Y. | author-link8 = Roger Y. Tsien | year = 2002 | title = New Biarsenical Ligands and Tetracysteine Motifs for Protein Labeling in Vitro and in Vivo: Synthesis and Biological Applications | journal = J. Am. Chem. Soc. | volume = 124 | issue = 21| pages = 6063–6076 | doi = 10.1021/ja017687n| pmid = 12022841 }}
Application
FlAsH-EDT2 enables less toxic and more specific fluorescent labeling that is membrane permeable.{{cite journal | last1 = Hoffmann | first1 = Carsten | last2 = Gaietta | first2 = Guido | last3 = Zürn | first3 = Alexander | last4 = Adams | first4 = Stephen R. | last5 = Terrillon | first5 = Sonia | last6 = Ellisman | first6 = Mark H. | last7 = Tsien | first7 = Roger Y. | last8 = Lohse | first8 = Martin J. | year = 2010 | title = Fluorescent labeling of tetracysteine-tagged proteins in intact cells | journal = Nature Protocols | volume = 5 | issue = 10| pages = 1666–1677 | doi=10.1038/nprot.2010.129| pmid = 20885379 | pmc = 3086663 }} The modification of the fluorescein moiety also allows multicolor analysis.[https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cellular-imaging/high-content-screening/flash-and-reash.html TC-FlAsH™ II In-Cell Tetracysteine Tag Detection Kit (Green Fluorescence), for live-cell imaging] 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.{{cite book | first1 = B. Albert | last1 = Griffin | first2 = Stephen R. | last2 = Adams | first3 = Jay | last3 = Jones | first4 = Roger Y. | last4 = Tsien | author-link4 = Roger Y. Tsien | year = 2000 | chapter = Fluorescent labeling of recombinant proteins in living cells with FlAsH | pages = 565–578 | title = Applications of Chimeric Genes and Hybrid Proteins, Part B: Cell Biology and Physiology | editor1-first = Jeremy | editor1-last = Thorner | editor2-first = Scott D. | editor2-last = Emr | editor-link2 = Scott D. Emr | editor3-first = John N. |editor3-last = Abelson | editor-link3 = John Abelson | series = Methods in Enzymology | volume = 327 | isbn = 9780080496825 | publisher = Academic Press | doi = 10.1016/S0076-6879(00)27302-3| pmid = 11045009 }}
=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.{{cite journal | last1 = Gaietta | first1 = Guido | first2 = Thomas J. | last2 = Deerinck | first3 = Stephen R. | last3 = Adams | first4 = James | last4 = Bouwer | first5 = Oded | last5 = Tour | first6 = Dale W. | last6 = Laird | first7 = Gina E. | last7 = Sosinsky |first8 = Roger Y. | last8 = Tsien | author-link8 = Roger Y. Tsien | first9 = Mark H. | last9 = Ellisman | year = 2002 | title = Multicolor and Electron Microscopic Imaging of Connexin Trafficking | journal = Science | volume = 296 | issue = 5567 | pages = 503–507 | doi = 10.1126/science.1068793 | pmid = 11964472| bibcode = 2002Sci...296..503G | s2cid = 16397816 }} More recently, it was used in an extended study of plant cells like Arabidopsis and tobacco.{{cite journal | last1 = Estévez | first1 = José M. | last2 = Somerville | first2 = Chris | year = 2006 | title = FlAsH-based live-cell fluorescent imaging of synthetic peptides expressed in Arabidopsis and tobacco | journal = BioTechniques | volume = 41 | issue = 5 | pages = 569–574 | doi = 10.2144/000112264| pmid = 17140113 | doi-access = free }}