UNCOVERING THE RADIATIVE AND NON-RADIATIVE DECAY PATHWAYS OF N-CYANOINDOLE FLUORESCENT PROBES IN AQUEOUS SOLUTION

Abstract

n-Cyanoindole (n=2-7) fluorescent probes were developed for studying the structure and dynamics of proteins. It has been observed that cyano substitution at the 4-position of the bicyclic ring of indole dramatically increases the relative fluorescence intensity, lifetime, and quantum yield, while functionalizing other positions quenches the fluorescence in aqueous solution. We studied the positional substituent effect on the absorption and fluorescence properties using high-level quantum mechanical methods. In addition, we modeled the important solvation effects found in the interaction between the parent probe, indole, and water solvents using explicit solvation models and molecular dynamics simulations. We have unraveled the important non-radiative decay pathways that govern the fluorescence quenching of the probes in aqueous solution using water cluster models. It was found that upon excited state relaxation, water solvent stabilizes the La(ππ∗) excited state below the Lb(ππ∗) state when substitution takes place on the 6 membered ring causing (4-7)-cyanoindole to fluoresce from the bright La state and 2- and 3-cyanoindole to fluoresce from the dim Lb state. 4-cyanoindole was found to exhibit optimal fluorescence properties because it absorbs to and emits from the S1 excited state and has a high energy barrier in the S1 state potential energy surface along the N-H bond stretch which minimizes access to all non-radiative decay pathways. We also found that explicitly modeling the mutual polarization effects is essential to reproducing the features of the absorption spectrum of indole and the state inversion during fluorescence in aqueous solution. Furthermore, we found that the formation of a cyclic excimer structure results in different photochemical reaction paths such as excited state hydrogen transfer, excited state proton transfer, and excited state proton-coupled electron transfer processes, which if accessed, have the potential to rapidly quench the fluorescence intensity. However, the presence of a high energy barrier traps the 2-, 6-, and 7-cyanoindole-(H2O)1−2 clusters at the S1 state minimum, and thus the fluorescence is quenched due to vibrational relaxation and internal conversion from the S2 state at absorption to the S1 equilibrium geometry at emission.