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.[1] 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
Names
Other names
Fluorescein Arsenical Hairpin Binder; Lumio green
Identifiers
3D model (JSmol)
ChEBI
ChEMBL
ChemSpider
UNII
  • InChI=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)
    Key: KCPRYVGBEBFLIG-UHFFFAOYSA-N
  • c1ccc(c(c1)c2c3ccc(c(c3oc-4c(c(=O)ccc24)[As]5SCCS5)[As]6SCCS6)O)C(=O)O
  • OC(=O)C1=C(C=CC=C1)C1=C2C=CC(=O)C([As]3SCCS3)=C2OC2=C([As]3SCCS3)C(O)=CC=C12
Properties
C24H18As2O5S4
Molar mass 664.49 g·mol−1
Appearance Solid
Melting point 169 to 172 °C (336 to 342 °F; 442 to 445 K)
Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa).
Infobox references

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,[2] and higher quantum yield when the tetracysteine motif is flanked with specific residues (HRWCCPGCCKTF or FLNCCPGCCMEP).[3]

Preparation
Step 1: HgO 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).[1]

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.[4] Binding is reversed by 1,2-ethanedithiol, which binds tightly to arsenic compounds, as shown by the stability of FlAsH-EDT2.[5] 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.[6]

Formation of FlAsH-TC adduct

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).[6]

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.[6]

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).[7]

Application

FlAsH-EDT2 enables less toxic and more specific fluorescent labeling that is membrane permeable.[8] The modification of the fluorescein moiety also allows multicolor analysis.[9] 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.[1][10]

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.[11] More recently, it was used in an extended study of plant cells like Arabidopsis and tobacco.[12]

References
  1. Adams, Stephen R.; Tsien, Roger Y. (2008). "Preparation of the membrane-permeant biarsenicals FlAsH-EDT2 and ReAsH-EDT2 for fluorescent labeling of tetracysteine-tagged proteins". Nat. Protoc. 3 (9): 1527–1534. doi:10.1038/nprot.2008.144. PMC 2843588. PMID 18772880.
  2. Adams, Stephen R.; Campbell, Robert E.; Gross, Larry A.; Martin, Brent R.; Walkup, Grant K.; Yao, Yong; Llopis, Juan; Tsien, Roger Y. (2002). "New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications". Journal of the American Chemical Society. 124 (21): 6063–6076. doi:10.1021/ja017687n. PMID 12022841.
  3. Martin, Brent R.; Giepmans, Ben N.G.; Adams, Stephen R.; Tsien, Roger Y. (2005). "Mammalian cell–based optimization of the biarsenical-binding tetracysteine motif for improved fluorescence and affinity". Nature Biotechnology. 23 (10): 1308–1314. doi:10.1038/nbt1136. PMID 16155565. S2CID 16456334.
  4. Kalef, Edna; Gitler, Carlos (1994). "Purification of Vicinal Dithiol-containing Proteins by Arsenical-Based Affinity Chromatography". In Sies, Helmut (ed.). Oxygen Radicals in Biological Systems, Part C. Methods in Enzymology. Vol. 233. Academic Press. pp. 395–403. doi:10.1016/S0076-6879(94)33046-8. ISBN 9780080883465. PMID 8015475.
  5. Whittaker, Victor P. (1947). "An Experimental Investigation of the 'Ring Hypothesis' of Arsenical Toxicity". Biochem. J. 41 (1): 56–62. doi:10.1042/bj0410056. PMC 1258423. PMID 16748119.
  6. Griffin, B. Albert; Adams, Stephen R.; Tsien, Roger Y. (1998). "Specific Covalent Labeling of Recombinant Protein Molecules Inside Live Cells". Science. 281 (5374): 269–272. Bibcode:1998Sci...281..269G. doi:10.1126/science.281.5374.269. PMID 9657724.
  7. Adams, Stephen R.; Campbell, Robert E.; Gross, Larry A.; Martin, Brent R.; Walkup, Grant K.; Yao, Yong; Llopis, Juan; Tsien, Roger Y. (2002). "New Biarsenical Ligands and Tetracysteine Motifs for Protein Labeling in Vitro and in Vivo: Synthesis and Biological Applications". J. Am. Chem. Soc. 124 (21): 6063–6076. doi:10.1021/ja017687n. PMID 12022841.
  8. Hoffmann, Carsten; Gaietta, Guido; Zürn, Alexander; Adams, Stephen R.; Terrillon, Sonia; Ellisman, Mark H.; Tsien, Roger Y.; Lohse, Martin J. (2010). "Fluorescent labeling of tetracysteine-tagged proteins in intact cells". Nature Protocols. 5 (10): 1666–1677. doi:10.1038/nprot.2010.129. PMC 3086663. PMID 20885379.
  9. TC-FlAsH™ II In-Cell Tetracysteine Tag Detection Kit (Green Fluorescence), for live-cell imaging
  10. Griffin, B. Albert; Adams, Stephen R.; Jones, Jay; Tsien, Roger Y. (2000). "Fluorescent labeling of recombinant proteins in living cells with FlAsH". In Thorner, Jeremy; Emr, Scott D.; Abelson, John N. (eds.). Applications of Chimeric Genes and Hybrid Proteins, Part B: Cell Biology and Physiology. Methods in Enzymology. Vol. 327. Academic Press. pp. 565–578. doi:10.1016/S0076-6879(00)27302-3. ISBN 9780080496825. PMID 11045009.
  11. Gaietta, Guido; Deerinck, Thomas J.; Adams, Stephen R.; Bouwer, James; Tour, Oded; Laird, Dale W.; Sosinsky, Gina E.; Tsien, Roger Y.; Ellisman, Mark H. (2002). "Multicolor and Electron Microscopic Imaging of Connexin Trafficking". Science. 296 (5567): 503–507. Bibcode:2002Sci...296..503G. doi:10.1126/science.1068793. PMID 11964472. S2CID 16397816.
  12. Estévez, José M.; Somerville, Chris (2006). "FlAsH-based live-cell fluorescent imaging of synthetic peptides expressed in Arabidopsis and tobacco". BioTechniques. 41 (5): 569–574. doi:10.2144/000112264. PMID 17140113.
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