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.