The Thiourea Group Modulates the Fluorescence Emission Decay of Fluorescein-labeled Molecules

We have investigated the spectral properties and emission characteristics of fluorescein-5-thiocarbamoyl-N,N′-caproate (FITC-ACA) to examine the origin of the complex emission decay often observed in fluorescein-labeled molecules. The covalent attachment of fluorescein to ϵ-amino-n-caproic acid does not perturb the prototropic transitions of the chromophore or the general fluorescence characteristics of the various prototropic forms. However, both the monoanion and dianion forms of FITC-ACA are quenched relative to free fluorescein and exhibit a complex emission decay that is described by two discrete lifetimes. The thiourea group that links the chromophore to the caproic acid is shown to modulate the emission properties of the FITC-ACA. We show that the emission decay can also be analyzed using the asymmetric distribution model of Alcala et al. In this analysis, the τL and τu parameters that represent the lower and upper lifetime limits of the distribution reflect the quenched (0 ns) and unquenched lifetimes, respectively. The β parameter that describes the distribution of lifetimes between the two limiting states can be related to the quenching efficiency of the thiourea group and to the structure and dynamics of the FITC-ACA molecule.