Selective Mercuration of 2-Hydroxy Nile Red Bull. Korean Chem. Soc. 2010, Vol. 31, No. 12 3539 DOI 10.5012/bkcs.2010.31.12.3539 Selective Mercuration of 2-Hydroxy Nile Red and Its Application towards Chemodosimetric Hg 2+ -selective Signaling Haekyung Lee, Myung Gil Choi, Hyo-Yeon Yu, Sangdoo Ahn, and Suk-Kyu Chang * Department of Chemistry, Chung-Ang University, Seoul 156-756, Korea. * E-mail: skchang@cau.ac.kr Received September 14, 2010, Accepted September 17, 2010 Selective mercuration at the 1,6-positions of 2-hydroxy derivative of Nile Red and its application towards Hg 2+ - selective signaling was investigated. The 2-hydroxy Nile Red exhibited a selective UV-vis absorption and fluorescent signaling behavior towards Hg 2+ ions over common coexisting physiologically important metal ions in aqueous environ- ment. 1 H NMR studies revealed that the mercuration was selectively effected at the 1,6-positions of 2-hydroxy Nile Red, which is quite different from that at the 6,8-positions for the parent Nile Red. Key Words: Hg 2+ signaling, Mercuration, Nile Red, Chemodosimeter, Fluorescence signaling O N N O OH OH NO N OH OH + DMF Reflux 1 OH NO N OH N NaNO 2 HCl Scheme 1. Preparation of the 2-hydroxy derivative of Nile Red 1 300 400 500 600 700 Wavelength (nm) Hg(II) 0.25 0.20 0.15 0.10 0.05 0.00 Absorbance Ni(II), Ca(II), Co(II), Zn(II), Cu(II), Fe(III), Pb(II), Ag(I), Mg(II) Cd(II), K(I), Na(I), 1 only Figure 1. UV-vis spectra of 1 in the presence of various metal ions. [1] = 5.0 × 10 ‒6 M, [M n+ ] = 5.0 × 10 ‒4 M. In acetate buffered (pH 4.7, 10 mM) H2O-MeOH (50:50, v/v). Introduction Selective detection of transition and heavy metal ions is very important in various fields of chemical and biological sciences, as well as in the protection of our environment. 1 Among many important transition metal ions, Hg 2+ ions attract much research interest due to its toxic environmental impact. 2 There are many sophisticated systems for the efficient and selective detection 3 and visualization of Hg 2+ ions. 4 However, a great deal of effort has been continuously devoted toward the construction of de- vices that are able to signal and visualize the presence of Hg 2+ in varying origins. 5 Recently, mercuration of important dyes has been used for the purpose of specific imaging and labeling of proteins. Me- rcuration of fluorescein and resorufin after subsequent trans- metalation with AsCl 3 afforded biarsenical ligands, FlAsH and ReAsH, respectively, which are employed for affinity chro- matography, fluorescence measurements, and localization of tetracysteine-tagged proteins. 6,7 Similarly, mercuration of Nile Red has provided dimercurated derivative for the preparation of biarsenical compound for the imaging of conformational changes of proteins containing tetracysteine motif. 8 Nile Red and related benzophenoxazine derivatives have been utilized as fluorescent dyes for the labeling of biomole- cules. 9 A series of water soluble Nile Red derivatives for the development of fluorescent probes for biotechnology have been prepared accordingly. 10 Chemiluminescent energy-transfer ca- ssettes based on fluorescein and Nile Red 11 and near-IR fluo- rescence probes derived from Nile Red 12 were also reported. Other Nile Red derivatives are currently used as probes for lipid- and drug-binding proteins, 13 tools for hydrophobic cha- racterization of intracellular lipids, 14 and solvatochromic nucleo- side for indicating micropolarity around DNA. 15 During the course of search for a new probe system based on Nile Red derivatives, we found that Nile Red and its 2-hydroxy analogue exhibited quite different mercuration profile. In this paper, we report a selective mercuration of 2-hydroxy Nile Red and its potential for application toward chromogenic and fluorogenic Hg 2+ -selective signaling. The process of the mercuration re- action mode of 2-hydroxy Nile Red could be confirmed by 1 H NMR spectroscopy. Results and Discussion The 2-hydroxy derivative of Nile Red 1 was prepared by the reaction of 5-diethylamino-2-nitrosophenol, which was ob- tained by the reaction of 3-diethylaminophenol with NaNO2, with 1,6-dihydroxynaphthalene following the reported proce- dure (Scheme 1). 16 First, the UV-vis signaling behavior of 1