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. 2008;3(9):1527-34.
doi: 10.1038/nprot.2008.144.

Preparation of the membrane-permeant biarsenicals FlAsH-EDT2 and ReAsH-EDT2 for fluorescent labeling of tetracysteine-tagged proteins

Stephen R Adams  1 , Roger Y Tsien
Affiliations

Preparation of the membrane-permeant biarsenicals FlAsH-EDT2 and ReAsH-EDT2 for fluorescent labeling of tetracysteine-tagged proteins

Stephen R Adams et al. Nat Protoc. 2008.

Abstract

The membrane-permeant fluorogenic biarsenicals FlAsH-EDT(2) and ReAsH-EDT(2) can be prepared in good yields by a straightforward two-step procedure from the inexpensive precursor dyes fluorescein and resorufin, respectively. Handling of toxic reagents such as arsenic trichloride is minimized so the synthesis can be carried out in a typical chemistry laboratory, usually taking about 2-3 d. A wide range of other biarsenical reagents and intermediates that also bind to tetracysteine-tagged (CysCysProGlyCysCys) proteins can be prepared similarly using this general procedure.

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Figures

Figure 1
Figure 1
Synthesis of FlAsH-EDT2 and ReAsH-EDT2; (a) HgO, TFA (b) AsCl3, Pd(OAc)2, DIEA, NMP (c) EDT, aqueous acetone.
Figure 2
Figure 2
Structures of some other useful biarsenicals reagents and intermediates. CrAsH-EDT2 (refs 4, 11, 17), fluorogenic biarsenicals and intermediate for CaG FlAsH and immobilized biarsenicals; sFlAsH-EDT2 (ref 4) membrane-impermeable fluorogenic biarsenicals; F2-FlAsH (ref 18), photostable fluorogenic biarsenicals; CHoXAsH-EDT2 (refs 4, 17), aggregator for BA-GFP; SpLAsH (ref, colorless and nonfluorescent biarsenicals for targeting other dyes e.g. Alexa594; CaG FlAsH (ref 11), targetable low-affinity Ca2+ indicator; AsCy3 (ref 20), fluorogenic biarsenical based on cyanine dye.
Figure 3
Figure 3
Typical time courses for the reaction of FlAsH-EDT2 (green trace) and ReAsH-EDT2 (red trace) with the tetracysteine peptide, FLNCCPGCCMEP (added at 700 s).
Figure 4
Figure 4
Absorbance (red trace) and fluorescence emission (blue trace) of FlAsH complex with FLNCCPGCCMEP.
Figure 5
Figure 5
Absorbance (red trace) and fluorescence emission (blue trace) of ReAsH complex with FLNCCPGCCMEP.
See this image and copyright information in PMC

References

    1. Tsien RY. The green fluorescent protein. Annu. Rev Biochem. 1998;67:509–544. - PubMed
    1. Giepmans BN, Adams SR, Ellisman MH, Tsien RY. The fluorescent toolbox for assessing protein location and function. Science. 2006;312:217–224. - PubMed
    1. Griffin BA, Adams SR, Tsien RY. Specific covalent labeling of recombinant protein molecules inside live cells. Science. 1998;281:269–272. - PubMed
    1. Adams SR, et al. New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications. J. Am. Chem. Soc. 2002;124:6063–6076. - PubMed
    1. Martin BR, Giepmans BN, Adams SR, Tsien RY. Mammalian cell-based optimization of the biarsenical-binding tetracysteine motif for improved fluorescence and affinity. Nat. Biotechnol. 2005;23:1308–1314. - PubMed

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