Michael Addition/S,N-Intramolecular Rearrangement Sequence Enables Selective Fluorescence Detection of Cysteine and Homocysteine

Abstract

Acrylate has been widely used as the recognition unit for Cys fluorescent probes. Despite this widespread use, a potential drawback of this probe type is that the ester linkage between the fluorophore and acryloyl recognition unit is liable to be hydrolyzed by abundant esterase in the cytosol, thus affording a high background signal. To solve this problem, we herein put forward a new strategy to construct a selective fluorescent probe for cysteine (Cys)/homocysteine (Hcy) with propynamide as the recognition moiety. The free probe <b>CPA</b> displays weakly fluorescent emission in aqueous media because of the donor-excited photoinduced electron transfer (d-PET) process within the molecule. The Michael addition of Cys (or Hcy) thiols to the conjugated alkyne of <b>CPA</b> gives the expected β-sulfido-α,β-unsaturated amides (<b>1a</b>/<b>1b</b>), which subsequently undergo an intramolecular S,N rearrangement, yielding β-amino-α,β-unsaturated amides (<b>2a</b>/<b>2b</b>) as the final products. The above cascade reaction results in the blockage of d-PET within <b>CPA</b>, thus affording a dramatic fluorescence enhancement at 495 nm. The involvement of the sulfhydryl and the adjacent amino groups in the sensing process renders <b>CPA</b> high selectivity for Cys/Hcy over glutathione as well as other amino acids. The probe has been successfully applied to image Cys in different cell lines. Further, <b>CPA</b> shows two-photon fluorescence properties, and its ability to monitor Cys in deep tissues has been demonstrated by using two-photon microscopy.