Preparation of the membrane-permeant biarsenicals FlAsH-EDT2 and ReAsH-EDT2 for fluorescent labeling of tetracysteine-tagged proteins - PubMed (original) (raw)

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.

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