DOI: 10.1002/chem.201301164 9-(4-Dimethylaminophenyl)benzo[b]quinolizinium: A Near-Infrared Fluorophore for the Multicolor Analysis of Proteins and Nucleic Acids in Living Cells Roberta Bortolozzi, [a] Heiko Ihmels,* [b] Laura Thomas, [b] Maoqun Tian, [b] and Giampietro Viola* [a] The visualization of cell components or processes within cells is an essential task in bioanalytical chemistry. [1] Specifi- cally, the fluorimetric detection of biomacromolecules has developed as a key technique in this research area, [2] mainly because emission spectroscopy is a highly sensitive and straightforward method with relatively few demands on the equipment. As a consequence, several fluorescent probes have been established that enable the selective detection of cells or cellular components. [2–4] For example, DNA–fluoro- phore conjugates, [5] peptide-based molecular beacons, [6] groove-binding cyanine dyes, [7] and light-cleavable caged dyes [8] were shown to operate as DNA stains in live cells. Similarly, it was demonstrated that appropriately substituted metallointercalators have a high propensity to bind to cellu- lar DNA and thus enable its fluorimetric detection, [2a, c, 9] or, in some cases, the detection of other cell components. [10, 11] The same principle was applied to detect RNA in nucleoli and the cytoplasm with a 2,7-carbazole derivative. [12] Fur- thermore, exciton-controlled hybridization-sensitive fluores- cent oligonucleotide (ECHO) probes allow multicolor RNA imaging in cells. [13] Recently, a chemosensor has been pre- sented that enables the fluorimetric differentiation of quad- ruplex DNA from other nucleic acids in cells. [14] Moreover, it has been shown that the fluorimetric discrimination of re- gions with different polarities may be accomplished in cells with quinoxaline derivatives. [15] Along these lines, the use of near-infrared (NIR, 650–900 nm) fluorescent probes is ad- vantageous for biological applications, because NIR fluoro- phores exhibit low or almost no phototoxicity, relatively deep penetration into tissue, and negligible interference with the autofluorescence of cells. [16, 17] We have shown recently that benzo[b]quinolizinium de- rivatives may be functionalized such that the fluorescence is quenched by different independent deactivation pathways and that the association with biomacromolecules results in light-up effects and shifts in the emission energy. [18] In some cases, separate deactivation channels enable the indepen- ACHTUNGTRENNUNGdent or even simultaneous detection of DNA and metal ions with one chemosensor. [19] In this context, we synthesized 9-(4-dimethylamino)benzo[b]quinolizinium (1a), which ex- hibits a very low emission quantum yield, [20] presumably caused by deactivation of the excited state by torsional re- laxation and photoinduced electron transfer (PET) or charge shift (CS). Notably, some structural features of the derivative 1a resemble aminophenylpyridinium derivatives such as 2, [21] which have been used as fluorescent probes in nerve membranes, [22] and Thioflavin T (3), which has been applied for fluorimetric analysis of amyloid fibril forma- tion. [23] Therefore, we proposed that the derivative 1a may represent a complementary fluorimetric tool for the selec- tive analysis of biomacromolecules, especially considering our experience with the benzo[b]quinolizinium ion as a ligand for DNA and proteins. [18, 24] Herein, we demonstrate that different physiologically relevant host systems are stained with the chemosensor 1a, most remarkably with dif- ferent emission wavelengths and intensities. In addition, we show that, due to these properties, the chemosensor 1a rep- resents one of the rare examples of probes that stain cells with multicolored fluorescence. In aqueous solution, the benzo[b]quinolizinium derivative 1a is essentially nonfluorescent (F FL = 1.0  10 3 in BPE buffer; see experimental section). However, we have shown already that the emission intensity increases significantly upon protonation, with a blueshifted fluorescence maxi- mum. [20] The emission also increases with increasing viscosi- ty of the solution (Figure 1, also see the Supporting Infor- [a] Dr. R. Bortolozzi, Dr. G. Viola Author names in alphabetical order Dipartimento di Salute della Donna e del Bambino Laboratorio di Oncoematologia University of Padova, via Giustiniani 3, I-35128 Padova (Italy) E-mail : giampietro.viola.1@unipd.it [b] Prof. Dr. H. Ihmels, L. Thomas, Dr. M. Tian Department Chemie–Biologie Universität Siegen Adolf-Reichwein-Strasse 2, 57068 Siegen (Germany) E-mail : ihmels@chemie.uni-siegen.de Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201301164. Chem. Eur. J. 2013, 00,0–0 # 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim These are not the final page numbers! ÞÞ &1& COMMUNICATION