Design and Characterization of a Coumarin-Based Hydrazine Fluorescent Probe for Labeling Biomolecular Carbonyls and Detecting Oxidative Stress in Live Cells

Abstract

Oxidative stress—an imbalance between reactive oxygen species (ROS) and the body's antioxidant defenses—is a key contributor to diseases such as cancer, fibrosis, and neurodegeneration. To study and treat these diseases, it is essential to identify oxidative damage in tissues with tools that offer high spatial resolution, rapid detection and minimal disruption of the existing environment. However, achieving this level of detail is challenging with current technologies, such as PET and MRI scans, which either rely on ionizing radiation or have low sensitivity, respectively. Fluorescent probes offer a safer, higher-resolution, real-time imaging alternative, but many current designs are non-selective and non-specific in their labeling. To address these gaps, we designed, synthesized, and characterized a coumarin-based hydrazine fluorescent probe that forms hydrazones with carbonyls like allysine, a key biomarker of oxidative stress, using bioorthogonal click-like chemistry. Prior research in our lab demonstrated that strategically placing electron-withdrawing and electron-donating groups, along with changing π-conjugation, influences fluorescence properties and shifts absorbance wavelengths—insights that guided the structural design of this probe. Synthesis of the probe was completed in three steps and was characterized using ¹H-NMR, ¹³C-NMR, UV-Vis, and fluorescence spectroscopy. A kinetic assay was conducted to establish the probe’s reactivity and rate of reaction, revealing second-order behavior. This work presents a non-invasive, real-time fluorescent probe for the early detection of oxidative stress, enabling precise disease diagnostics and real-time tracking of therapeutic responses.