Spectral characterization of fluorescent 5-iodoacetamidotetramethylrhodamine and its N-acetylcysteine derivative

This study describes a spectroscopic characterization of the fluorescent probe 5-iodoacetamidotetramethylrhodamine and its N-acetylcysteine derivative in various solvents. The monomeric character and the purity of the two compounds was established in ethanol, ethanol-water, water, dimethylformamide, dimethyl sulfoxide, glycerol, polyvinylalcohol and nitrocellulose films, using a combination of absorption and fluorescence spectroscopy and mass spectrometry. In all the polar solvents studied here, the probes were present in the open form only and thus, from the spectral parameters, the natural lifetime and Forster distance in those solvents could be calculated. Fluorescence lifetimes were determined and the homogeneity of probe distribution in both stretched and unstretched polymer films was checked. For this, a combination of confocal microscopy and polarized fluorescence experiments was used. Finally, applying angle-resolved fluorescence depolarization for immobilized molecules in uniaxially stretched polymer films, the orientation of both rhodamines in stretched polyvinylalcohol films was examined and the orientation of the transition dipole moments within the molecular frame was determined. There were no differences between the 5-isomer and its N-acetylcysteine derivative, but a strong environmental dependence of most optical properties was observed. This dependence was especially intriguing for the difference angle between the absorption and emission dipole moment. The implications of our results and suggestions for future research are discussed.