The sensing and reacting mechanisms of a fluorescent probe for cysteine: a detailed theoretical exploration

Abstract This study established a multiscale theoretical framework integrating the polarizable continuum model (PCM), time-dependent density functional theory (TD-DFT), and thermal vibration correlation function (TVCF) to explore the photophysical properties and fluorescence quantum yield of the 4F-2CN fluorescent probe. Theoretical results show that 4F-2CN emits fluorescence in the ultraviolet region with a quantum yield ≤ 0.5%, resulting in undetectable visible emission. By contrast, its cysteine reaction product (4F-2CN-Cys) exhibits visible fluorescence with high quantum yield; notably, the value calculated via the CAM-B3LYP method matches experimental data, verifying 4F-2CN’s validity as a cysteine probe. 4F-2CN-Cys also displays a markedly enhanced near-infrared (NIR) two-photon absorption (TPA) cross-section, enabling deep-tissue imaging applications. Potential energy surface analysis confirms ion-mediated pathway B as the dominant reaction route due to its lower activation energy. Molecular design simulations suggest substituting fluorine in 4F-2CN with chlorine or bromine can improve single-photon and two-photon absorption/emission properties, offering practical strategies for developing next-generation NIR two-photon fluorescent probes. This work provides critical theoretical insights into probe-cysteine interaction mechanisms and molecular design optimization.