4`-Rhodamin Derivates as Fluorescent Dyes

Max Planck Society Background
As a result of the evermore-enhanced super resolution microscopic techniques that allow observation of biological processes up to the molecular level in living cells, the demand for fluorescent dyes capable of specific binding to molecules like proteins as labels for visualization aspects is increased.
The search for a suitable fluorescent dye that fulfills all requirements of an ideal candidate for use in biological environments is still challenging:
(i) high photostability
(ii) good brightness – high extinction coefficient and quantum yield
(iii) derivatization possibility of the fluorescent dye molecule
(iv) no unspecific interaction with other biomolecules
(v) high membrane permeability
Several approaches already based on derivatives of rhodamine fluorophores. The most prominent derivates in this area are carbopyronines (CP) or silicone-rhodamine (SiR).
Due to the fact that an equilibrium between their non-fluorescent spirolactone and fluorescent zwitterionic form exists, one can push the preferred form by introducing electron-withdrawing or electron-donating groups into the structure. In this context it should be mentioned that the spirolactone form is the more hydrophobic one and shows better membrane permeability. Several studies attempted to switch the equilibrium towards spirolactone form by introducing the electron-withdrawing groups in the xanthene core or in the benzoic acid substituent. However, all these approaches result in a bulkier core structure and fast altering process of physicochemical properties of the dyes.
Technology Overview
To overcome the aforementioned drawbacks a new class of rhodamine derivates that meets all requirements was developed by researchers Max Planck Institute for Biophysical Chemistry in Göttingen by introducing an amide group in the 4’ position, shifting the equilibrium to the lactone form and hence improving the membrane permeability ().
In such 4’-isomers of rhodamines a superposition of inductive and steric effects and hydrogen bonding due to neighbouring groups in the benzene ring of the fluorophore occurs that provokes a so called neighbouring group effect (NGE). This effect is observed in 4’-isomers of rhodamines only.
Rhodamine 4’-isomers containing NGE show significant NMR chemical shift of the interacting atoms that are covalently bonded and show increased HPLC retention times. Importantly, this results in not so far observed increased cell membrane permeability compared to widely used 5’-isomers or 6’-isomers while keeping all photophysical properties almost unchanged.

Good brightness
Specific binding
No interaction with other biomolecules
Stable against a various of environmental factors: pH, ion concentration, enzyme, tension force, local microenvironment polarity or light
Membrane permeability – no need for injection step
Simply incubate the cells with fluorescent probe

Biomedical applications:

for the labeling of biomolecules like protein or peptide in vitro, in living cells and in living organisms
for sensing oxygen, fluoride or glucose in vitro, in living cells and in living organisms
for drug-target interaction monitoring using fluorescence imaging or NMR
for cell cycle monitoring

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