Fluorescence and Isotope Effect Analysis of Non-Pauling Hydrogen Bonds in Biologically Relevant Redox Reactions

Non-Pauling hydrogen bonds involving sulfur, such as amide-NH···S(thioether) interactions, are difficult to probe experimentally despite their potential influence on biologically relevant oxidation processes. In this work, we present a fluorescence-isotope strategy for probing non-Pauling hydrogen bonding during thioether oxidation. A naphthalimide-derived fluorescent reporter, FI-Aminde-SPh, was rationally designed to couple three processes within one molecular framework: thioether oxidation, hydrogen-bond transformation, and fluorescence activation. In the initial state, an electron-rich thioether quenches naphthalimide emission through photoinduced electron transfer, while the adjacent amide group forms a weak non-Pauling NH···S interaction. Oxidation of the thioether to sulfoxide suppresses photoinduced electron transfer and converts the weak NH···S interaction into a stronger NH···O═S hydrogen bond, producing a pronounced fluorescence turn-on response. Time-resolved fluorescence analysis in H2O/D2O revealed a measurable kinetic isotope effect for FI-Amide-SPh, whereas the non-hydrogen-bonding reference FI-AP-SPh showed only a minimal isotope effect, supporting the participation of hydrogen-bond reorganization in the oxidation process. This mechanism was further supported by authentic product comparison, NMR analysis, and DFT calculations. In living cells and zebrafish, FI-Amide-SPh enabled fluorescence reporting of hypochlorite-triggered thioether oxidation, demonstrating that the oxidation-coupled readout can operate in complex biological environments. This work establishes fluorescence-coupled isotope-effect analysis as a practical approach for translating weak non-Pauling hydrogen-bond interactions into quantifiable optical and kinetic outputs, providing a useful platform for studying sulfur-centered redox processes.