Selective Selenol Fluorescent Probes: Design, Synthesis, Structural Determinants, and Biological Applications Baoxin Zhang, Chunpo Ge, Juan Yao, Yaping Liu, Huichen Xie, and Jianguo Fang* State Key Laboratory of Applied Organic Chemistry and College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou, Gansu 730000, China * S Supporting Information ABSTRACT: Selenium (Se) is an essential micronutrient element, and the biological significance of Se is predominantly dependent on its incorporation as selenocysteine (Sec), the genetically encoded 21st amino acid in protein synthesis, into the active site of selenoproteins, which have broad functions, ranging from redox regulation and anti-inflammation to the production of active thyroid hormones. Compared to its counterpart Cys, there are only limited probes for selective recognition of Sec, and such selectivity is strictly restricted at low pH conditions. We reported herein the design, synthesis, and biological evaluations of a series of potential Sec probes based on the mechanism of nucleophilic aromatic substitution. After the initial screening, the structural determinants for selective recognition of Sec were recapitulated. The follow-up studies identified that probe 19 (Sel-green) responds to Sec and other selenols with more than 100-fold increase of emission in neutral aqueous solution (pH 7.4), while there is no significant interference from the biological thiols, amines, or alcohols. Sel-green was successfully applied to quantify the Sec content in the selenoenzyme thioredoxin reductase and image endogenous Sec in live HepG2 cells. With the aid of Sel-green, we further demonstrated that the cytotoxicity of different selenocompounds is correlated to their ability metabolizing to selenols in cells. To the best of our knowledge, Sel-green is the first selenol probe that works under physiological conditions. The elucidation of the structure-activity relationship for selective recognition of selenols paves the way for further design of novel probes to better understand the pivotal role of Sec as well as selenoproteins in vivo. ■ INTRODUCTION Selenium (Se) was recognized as an essential micronutrient element in 1960s. 1 Insufficient or excessive intake of Se has been associated with a number of diseases. 2,3 Many different metabolites of Se, such as hydrogen selenide, selenocysteine (Sec), selenite, selenophosphate, selenodiglutathione, and charged Sec-tRNA, are synthesized in animals in the course of converting inorganic Se to organic forms and vice versa. 2,4 Although Se may exist as different forms in vivo, the current knowledge of the biological significance of Se is predominantly dependent on its incorporation as the Sec into the active site of selenoproteins, which have a wide range of function, ranging from redox signaling and anti-inflammation to the production of active thyroid hormones. 3,5,6 Sec is a cysteine (Cys) analogue with a selenium-containing selenol group in place of the sulfur- containing thiol group in Cys and the 21st amino acid in ribosome-mediated protein synthesis. 7,8 Due to the low pK a value of selenol (pK a ∼5.8), 9 the Se in Sec is almost fully ionized under physiological conditions, which gives it high reactivity, and consequently Sec is normally essential for the catalytic efficiencies of selenoproteins. 10,11 As the Sec carries out the majority function of the various Se-containing species in vivo, it is of high demand to develop reliable and rapid assays with biocompatibility to determine Sec. Fluorescence sensing using small molecule probes has been one of the most powerful and popular tools to visualize the complicated biological processes. However, designing specific probes of Sec without suffering from the interference of biological thiols is a big challenge since the thiols usually present in high concentration (millimolar levels) in cells and have the similar chemical properties as Sec. The selenol pK a value in Sec (∼5.8) is lower than those of the most biological thiols (∼8.3), which means that under physiological conditions (pH ∼7.4) the Se in Sec is almost fully present as the selenolate (R-Se - ), while the majority of thiols present as nonionized form (R-SH). The difference of pK a values opens a window for selective detection of Sec in vitro by maintaining the reaction system under acidic condition. Maeda et al. reported the first fluorescent probe BESThio to discriminate the Sec from its counterpart Cys at pH 5.8. 12 We could also selectively label the Sec residue in the thioredoxin reductase (TrxR) by biotin- conjugated iodoacetamide at pH 6.5. 13,14 However, all these assays are not compatible with the biological surroundings generally having a neutral environment (pH ∼7.4), which prevents their practical applications in live system. Thus, it is of Received: September 27, 2014 Published: January 5, 2015 Article pubs.acs.org/JACS © 2015 American Chemical Society 757 DOI: 10.1021/ja5099676 J. Am. Chem. Soc. 2015, 137, 757-769