Molecular modulated cysteine-selective fluorescent probe.

Biomaterials (2012-08-22)
Hyo Sung Jung, Tuhin Pradhan, Ji Hye Han, Kyung Jun Heo, Joung Hae Lee, Chulhun Kang, Jong Seung Kim
ABSTRACT

We have synthesized a series of coumarins (1-3) that can emit fluorescence in a turn-on manner through a Michael-type reaction with thiol-containing compounds. The only difference among the coumarins is the position of a carboxyl group on its benzene ring moiety near the double-bond conjugated coumarin. Their selectivity for Cys, GSH, and Hcy as well as the associated fluorogenic mechanism were illustrated by fluorescence spectroscopy, DFT calculations, and kinetic studies. All isomers prefer Cys over GSH in the reaction from 48.6 (probe 3) to 111-fold (probe 1) as demonstrated in a second order kinetics. The high selectivity of probe 1 to Cys might be achieved since the ortho carboxyl group on its benzene ring prefers a less negatively charged nucleophile. During intracellular Cys detection using 1, a possible interference by a large amount of GSH in the HepG2 cells was evaluated. The cells were treated with l-buthionine sulfoximine (BSO), an inhibitor of γ-glutamylcysteine synthetase, providing an experimental condition where the cells could not synthesize GSH from Cys or other species. Then, the fluorescence intensity of 1 in HepG2 cells under BSO-H(2)O(2) treatment was strongly enhanced by N-acetylcysteine (NAC), a precursor of Cys, implicating that the fluorescence signal from the cells is mainly associated with changes in intracellular [Cys] rather than that in intracellular [GSH].

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
DL-Buthionine-sulfoximine, ≥99.0% (TLC)
Sigma-Aldrich
Atto 488 maleimide, BioReagent, suitable for fluorescence, ≥90% (coupling to thiols)
Sigma-Aldrich
DL-Buthionine-(S,R)-sulfoximine
Sigma-Aldrich
Atto 565 maleimide, BioReagent, suitable for fluorescence
Sigma-Aldrich
Atto 425 maleimide, BioReagent, suitable for fluorescence, ≥70% (coupling to thiols)
Sigma-Aldrich
Atto 465 maleimide, BioReagent, suitable for fluorescence, ≥90% (coupling to thiols)
Sigma-Aldrich
Atto 495 maleimide, BioReagent, suitable for fluorescence
Sigma-Aldrich
Atto 540 Q maleimide, ≥90% (coupling to thiols)

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