Cell-Penetrating Fluorescent Dyes with Secondary Alcohol Functionalities
Imaging and Microscopy
To address some of the above-stated limitations, hydroxyl groups are introduced into non-allylic and non-benzylic positions of the fluorescent dyes to increase polarity, improve solubility in water and prevent unspecific binding. The structures of some new dyes (emit-ting green and red light) are shown in scheme 1. The free carboxylic acid group in the dyes allows further modification, i. e. conjugation to small molecules (ligand, recognition unit) or to proteins. With these conjugates, not only cytoskeleton proteins but also nuclear components have been stained specifically. In sub-micromolar concentrations – as recommended for imaging – the newly introduces dyes show no evidence of cytotoxicity.
The introduction of electron-withdrawing trifluoromethyl groups and/or hydroxyl groups (dye A in scheme 1) provokes only slight shifts in the absorption and emission spectra compared to the unfunctionalized fluorophores, whereas the equilibrium between the zwitterionic (open) and spirolactone (closed) forms shifts towards the latter, facilitating cell membrane penetration.
Apart from that, substitution of the oxygen atom in the xanthene backbone with a group 14 element atom (Si, Ge, Sn; dye C in scheme 1) leads to significant bathochromic shifts in the absorption and emission spectra. This enables the cross-talk-free two-color detection in living cells, including the STED super-resolution option. For instance, an optical resolution of about 60 nm was achieved by applying dye B and C from scheme 1 and using commercial 775 nm STED laser for the efficient “switching-off” of both dyes.
- improved image quality due to reduced unspecific binding
- adjustable absorption and fluorescence spectra
- cell- and nucleus-permeant fluorescent dyes allow flexible single- and dual-color label-ling in living cells (in vitro or in vivo)
EP patent application filed in September 2016.
- Ref.-No.: 0707-5236-BC (300.3 KiB)
Dr. Bernd Ctortecka, M. Phil.
Phone: +49 89 / 29 09 19-20