Deuterated Multi-Color Probes for Reaction-Resolved Mapping of Intracellular Redox Pathways With Magnetic Resonance

Deuterium (²H) offers unique advantages as a nuclear magnetic resonance (NMR) reporter owing to its negligible endogenous biological background and ability to provide chemically distinguishable resonances. In the present study, we propose a chemical-shift encoding concept in which reactions converting the deuterium-bearing carbon from an sp²- to an sp³-hybridized state produce pronounced and predictable ²H NMR chemical-shift changes, enabling distinct reaction outcomes to be encoded as spectroscopically resolvable signatures. Here we introduce deuterated "multi-color" probes (DMCPs), a molecular platform coupling reaction-based molecular recognition with ²H NMR spectroscopy to simultaneously map distinct intracellular redox pathways. DMCPs encode thiol conjugation and enzymatic reduction as spectrally separable ²H NMR signatures, allowing multiplexed detection of glutathione reactivity and NAD(P)H-driven reductive activity within a single acquisition. Applied in living glioblastoma cells, DMCPs reveal parallel glutathione conjugation and NAD(P)H-dependent reduction, with the corresponding reaction-derived signals changing upon glutathione depletion. This method further captures pharmacologically induced redox perturbations, providing a mechanistic framework for evaluating redox-targeting strategies and rationalizing resistance mechanisms in early-stage drug discovery.