Differential Tuning of the Electron Transfer Parameters in 1,3,5-Triarylpyrazolines: A Rational Design Approach for Optimizing the Contrast Ratio of Fluorescent Probes

Abstract

A large class of cation-responsive fluorescent sensors utilizes a donor−spacer−acceptor (D−A) molecular framework that can modulate the fluorescence emission intensity through a fast photoinduced intramolecular electron transfer (PET) process. The emission enhancement upon binding of the analyte defines the contrast ratio of the probe, a key property that is particularly relevant in fluorescence microscopy imaging applications. Due to their unusual electronic structure, 1,3,5-triarylpyrazoline fluorophores allow for the differential tuning of the excited-state energy ΔE₀₀ and the fluorophore acceptor potential E(A/A⁻), both of which are critical parameters that define the electron transfer (ET) thermodynamics and thus the contrast ratio. By systematically varying the number and attachment positions of fluoro substituents on the fluorophore π-system, ΔE₀₀ can be adjusted over a broad range (0.4 eV) without significantly altering the acceptor potential E(A/A⁻). Experimentally measured D−A coupling and reorganization energies were used to draw a potential map for identifying the optimal ET driving force that is expected to give a maximum fluorescence enhancement for a given change in donor potential upon binding of the analyte. The rational design strategy was tested by optimizing the fluorescence response of a pH-sensitive probe, thus yielding a maximum emission enhancement factor of 400 upon acidification. Furthermore, quantum chemical calculations were used to reproduce the experimental trends of reduction potentials, excited-state energies, and ET driving forces within the framework of linear free energy relationships (LFERs). Such LFERs should be suitable to semiempirically predict ET driving forces with an average unsigned error of 0.03 eV, consequently allowing for the computational prescreening of substituent combinations to best match the donor potential of a given cation receptor. Within the scaffold of the triarylpyrazoline platform, the outlined differential tuning of the electron transfer parameters should be applicable to a broad range of cation receptors for designing PET sensors with maximized contrast ratios.