Thiophene-based dyes for probing membranes

Abstract

We report the synthesis of four new cationic dipolar push-pull dyes, together with an evaluation of their photophysical and photobiological characteristics pertinent for imaging membranes by fluorescence and second harmonic generation. All four dyes consist of an N,N-diethylaniline electron-donor conjugated to a pyridinium electron-acceptor via a thiophene bridge, with either vinylene (-CH=CH-) or ethynylene 10 (-CC-) linking groups, and with either singly-charged or doubly-charged pyridinium terminals. The absorption and fluorescence behavior of these dyes was compared to a commercially available fluorescent membrane stain, the styryl dye FM4-64. The hyperpolarizabilities of all dyes were compared using hyper-Rayleigh scattering at 800 nm. Cellular uptake, localization, toxicity and phototoxicity were evaluated using tissue cell cultures (HeLa, SK-OV-3 and MDA-231). Replacing the central alkene bridge 15 of FM4-64 with a thiophene does not substantially change the absorption, fluorescence or hyperpolarizability, whereas changing the vinylene-links to ethynylenes shifts the absorption and fluorescence to shorter wavelengths, and reduces the hyperpolarizability by about a factor of two. SHG and fluorescence imaging experiments in live cells showed that the doubly-charged thiophene dyes localize in plasma membranes, and exhibit lower internalization rates compared to FM4-64, resulting in 20 less signal from the cell cytosol. At a typical imaging concentration of 1 µM, the doubly-charged dyes showed no significant light or dark toxicity, whereas the singly-charged dyes are phototoxic even at 0.5 µM, and toxic in the dark at concentrations above 5 µM. The doubly-charged dyes showed phototoxicity at concentrations greater than 10 µM, although they do not generate singlet oxygen, indicating that the phototoxicity is type I rather than type II. Our data demonstrate that the doubly-charged thiophene dyes 25 are more effective than FM4-64 as nonlinear optical imaging agents for live cells.

Visualising the membrane viscosity of porcine eye lens cells using molecular rotors

“…We used three previously reported fluorescence sensors of lipid organisation (Chart 1): a pyrene-based dye (PA), 13 di-4-ANEPPDHQ 30 and molecular rotor 2 , which was previously shown to selectively stain the plasma membranes of eukaryotic cells 24,31 and is characterised by equal partitioning of the L d and L o phases of model lipid bilayers. We recently used the phasor analysis 32 of the fluorescence decays of 2 which indicated that 2 detects both L o and L d domains in the plasma membranes of live eukaryotic cells.…”

“…The synthesis of 1a , 16 1b , 23 2   31 and PA 13 has been reported previously, while di-4-ANEPPDHQ was obtained from Thermo Fisher Scientific. All solvents used were spectroscopic grade.…”

Mechanosensitive Oligodithienothiophenes: Transmembrane Anion Transport Along Chalcogen‐Bonding Cascades

“…Mechanical planarization of the twisted push‐pull probe 3 in the ground state causes the excitation maxima to shift to the red. This differs conceptually from other membrane probes that operate by deplanarization (molecular rotors), polarization (solvatochromism) and electron or proton transfer in the excited state and thus report in emission rather than excitation . The resulting flipper probes 3 are important because they provide access to the so far elusive imaging of forces such as membrane tension in living cells (unpublished).…”

Twisted Push‐Pull Probes with Turn‐On Sulfide Donors

“…The application of this more complex lesson from nature to fluorescent probes has received little attention so far. However, a combination of planarization and polarization in the ground state appeared most intriguing, particularly with regard to fluorescent probes that could report on key characteristics of biomembranes such as fluidity, potential as well as the so far elusive membrane tension …”