ExTzBox: A Glowing Cyclophane for Live-Cell Imaging

The ideal fluorescent probe for live-cell
imaging is bright and
non-cytotoxic and can be delivered easily into the living cells in
an efficient manner. The design of synthetic fluorophores having all
three of these properties, however, has proved to be challenging.
Here, we introduce a simple, yet effective, strategy based on well-established
chemistry for designing a new class of fluorescent probes for live-cell
imaging. A box-like hybrid cyclophane, namely ExTzBox·4X (6·4X, X = PF6−, Cl−), has
been synthesized by connecting an extended viologen (ExBIPY) and a
dipyridyl thiazolothiazole (TzBIPY) unit in an end-to-end fashion
with two p-xylylene linkers. Photophysical studies
show that 6·4Cl has a quantum yield ΦF = 1.00. Furthermore, unlike its ExBIPY2+ and TzBIPY2+ building units, 6·4Cl is non-cytotoxic
to RAW 264.7 macrophages, even with a loading concentration as high
as 100 μM, presumably on account of its rigid box-like structure
which prevents its intercalation into DNA and may inhibit other interactions
with it. After gaining an understanding of the toxicity profile of 6·4Cl, we employed it in live-cell imaging. Confocal
microscopy has demonstrated that 64+ is taken
up by the RAW 264.7 macrophages, allowing the cells to glow brightly
with blue laser excitation, without any hint of photobleaching or
disruption of normal cell behavior under the imaging conditions. By
contrast, the acyclic reference compound Me2TzBIPY·2Cl (4·2Cl)
shows very little fluorescence inside the cells, which is quenched
completely under the same imaging conditions. In vitro cell investigations
underscore the significance of using highly fluorescent box-like rigid
cyclophanes for live-cell imaging.