Rational engineering of a recognition group to construct a two-photon reaction-based fluorescent probe for rapid and selective sensing of cysteine†

Abstract

It is highly required to rationally design fluorescent probes via a molecular engineering strategy with desired analytical performance for applications in sensing and imaging. Reaction-based fluorescent probes for highly selective sensing of cysteine (Cys) are mainly based on the participation of Cys in reactions such as, addition–cyclization with acrylates, cyclization with aldehydes, coordination displacement, Michael addition reactions, and cleavage reactions. Cys-triggered reactions with the O atoms of ether bonds has also been used to construct reaction-based fluorescent probes based on the substitution of the ether with the nucleophilic thiolate of Cys. However, many of the developed probes still suffer from long response times, interference from homocysteine (Hcy) and glutathione (GSH), high background fluorescence, and a lack of two-photon absorption (TPA) properties. Herein, we successfully design a Cys-sensitive two-photon fluorescent probe (F-BTD) using a donor–acceptor–donor (D–A–D) type π-extended benzothiadiazole framework as the fluorophore, with nitrobenzofuran (NBD) as the recognition unit. The proposed F-BTD probe displays some advantages over other probes including a rapid response time, high selectivity, low background fluorescence, and two-photon imaging capability. The F-BTD probe is applied to the two-photon fluorescence imaging of endogenous and exogenous Cys in HeLa cells with satisfactory results. For comparison, commonly used biothiol recognition groups including 2,4-dinitrobezensulfonyl and 2,4-dinitrophenyl are also used to construct S-BTD and N-BTD probes, respectively. The response mechanism of F-BTD to Cys is studied in detail through kinetic studies and transition-state analysis. This study may provide an example of how to design fluorescent probes with desired analytical performance by considering the recognition group as an important index of the design.