Nucleobase-Specific Quenching of Fluorescent Dyes. 1. Nucleobase One-Electron Redox Potentials and Their Correlation with Static and Dynamic Quenching Efficiencies

TLDR

These findings are relevant to laser‑induced fluorescence DNA sequencing and other dye applications. The study investigates whether photoinduced electron transfer can drive nucleobase‑specific quenching of fluorescent dyes by computing standard free‑energy changes with the Rehm–Weller equation. Electrochemical measurements in aprotic solvents provide the first complete set of one‑electron redox potentials for nucleobases, which are then used to calculate the free‑energy changes for electron transfer. Measured static and dynamic quenching constants reveal a nucleobase‑dependent sequence that correlates with their oxidation and reduction potentials, and the dynamic constants follow Marcus theory, indicating electron transfer as the rate‑limiting step while additional water‑specific energy terms suggest coupled proton transfer and hydrophobic effects, and nucleobase redox potentials also influence ground‑state complex formation.

Abstract

Intermolecular static and dynamic fluorescence quenching constants of eight coumarin derivatives by nucleobase derivatives have been determined in aqueous media. One common sequence of the quenching efficiency has been found for the nucleobases. The feasibility of a photoinduced electron transfer reaction for the nucleobase-specific quenching of fluorescent dyes is investigated by the calculation of the standard free energy changes with the Rehm−Weller equation. A complete set of one-electron redox potential data for the nucleobases are determined electrochemically in aprotic solvents for the first time, which are compared with values obtained by various other methods. Depending on the redox properties of the fluorescent dyes, the sequences of the quenching efficiencies can be rationalized by the orders of electrochemical oxidation potentials (vs NHE) of nucleosides (dG (+1.47 V) < dA < dC ≈ dT < U (≥ +2.39 V)) and reduction potentials (dG (< −2.76 V) < dA < dC < dT < U (−2.07 V)). The correlation between the intermolecular dynamic quenching constants and the standard free energy of photoinduced electron transfer according to the classical Marcus equation indicates that photoinduced electron transfer is the rate-limiting step. However, an additional, water-specific gain of free energy between −0.5 and −0.9 eV shows that additional effects, like a coupled proton transfer and a hydrophobic effect, have to be considered, too. Furthermore, the capability of the nucleobases to form ground state complexes with fluorescent dyes is influenced by their redox potentials. The relevance of these observations to current efforts for DNA sequencing with a detection by laser-induced fluorescence and their application to other dyes are discussed.