A benzothiazole-based fluorescent probe for thiol bioimaging Wei Sun a , Wenhua Li a , Jing Li a , Jian Zhang b , Lupei Du a , Minyong Li a,⇑ a Department of Medicinal Chemistry, Key Laboratory of Chemical Biology of Natural Products (MOE), School of Pharmacy, Shandong University, Jinan, Shandong 250012, China b Institute of Immunopharmacology & Immunotherapy, School of Pharmacy, Shandong University, Jinan, Shandong 250012, China article info Article history: Received 20 December 2011 Revised 9 February 2012 Accepted 24 February 2012 Available online 3 March 2012 Keywords: Benzothiazole Fluorescent probe Thiols Cell imaging abstract This study reports a benzothiazole-based fluorescent probe with simple structure for thiols. This probe exhibited high on/off signal ratios and good selectivity toward thiols over other analytes, and was suc- cessfully applied to the imaging of thiols in living cells. Ó 2012 Elsevier Ltd. All rights reserved. Biological thiols/low molecular weight thiols, such as cysteine (Cys), homocysteine (Homo-Cys), and glutathione (GSH), which can regulate the intracellular redox state and higher-order struc- tures of proteins, are active in the catalytic sites of numerous en- zymes, and participate in intracellular signal transduction and gene regulation. 1 Generally, the levels of cellular thiols have been associated to toxic agents and diseases, including slowed growth, leucocyte loss, psoriasis, liver damage, cancer, and AIDS. 2 Conse- quently, selective detection of biological thiols should provide crit- ical insight into pathological and biological sciences. Among the various methods for detecting thiols, fluorescent molecular probes are of great interest because of their simplicity, low detection limit, wide range of dynamic response, and feasibility of intracellular detection. 3 Apart from reasonable physical proper- ties (excitation/emission wavelength, Stokes shift, etc.), high response and good selectivity, an ideal fluorescent probe should be convenient to synthesize, act fast in mild condition, and have high permeability. In the present research, a simple benzothiazole fluorophore was masked with 2,4-dinitrobenzenesulfonyl (DNS), which was studied widely in the detection of thiols. 4 Thiols can release the fluorophore by the nucleophilic cleavage of the 2,4 -dinitrobenzenesulfonyl ester. This simple molecule has most of the desirable features, and thereafter should be particularly applica- ble for the in vitro and in vivo detection of thiols. As depicted in Scheme 1, probe 1 started from commercially available 2-(benzo[d]thiazol-2-yl)phenol and was readily synthe- sized in only one step. 2-(Benzo[d]thiazol-2-yl)phenol was treated with 2,4-dinitrobenzene-sulfonyl chloride to obtain probe 1 in the presence of TEA in DCM with 80% yield. The fluorescent properties of such a probe were evaluated un- der near physiological conditions (0.1 M phosphate buffer, pH 7.4). The absorption at 330 nm of 2-(benzo[d]thiazol-2-yl)phenol was dismissed after the connection of 2,4-dinitrobenzene-sulfonyl, and no fluorescence was found in probe 1. The fluorescent inten- sity change of 1 was initially tested according to the reaction time with thiophenol (PhSH). As demonstrated in Figure 1, the fluores- cent intensity reached the high plateau at 2 min after the addition of PhSH, and almost no change appeared when longer reaction time was examined. To determine the stability of the probe in PBS buffer solution, we treated it under various conditions by detecting the change of its fluorescent intensity (see Supplementary data, Fig. S1). It was found that the probe was essentially stable over a range of 5–9 un- der ambient light or protected from light, with a low level of its hydrolysis at pH 9.0 The sensitivity of probe 1 was then deliberated by fluorescence response toward different concentrations of PhSH. It was examined at 5 min after the addition of PhSH (Fig. 2). Upon the addition of PhSH (40 lM), the fluorescence intensity increases more than 25-fold with the equilibrium constant (log K) being 12.1. And the fluorescence intensity of probe significantly increased as a corre- sponding function of PhSH concentration at more than 1.25 lM. The responses of the probe toward relevant aliphatic thiols, reactive sulfur species, common nucleophiles, and amino acid were considered as well. As shown in Figure 3, probe 1 encourages a significant response to aromatic thiols, including thiophenol (PhSH), 4-chlorothiophenol (4-Cl–PhSH), and 2-aminothiophenol 0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.tetlet.2012.02.098 ⇑ Corresponding author. Tel./fax: +86 531 8838 2076. E-mail address: mli@sdu.edu.cn (M. Li). Tetrahedron Letters 53 (2012) 2332–2335 Contents lists available at SciVerse ScienceDirect Tetrahedron Letters journal homepage: www.elsevier.com/locate/tetlet