Electron–Nuclear Hyperfine Coupling Enables Electron Paramagnetic Resonance Detection of Diamagnetic Cu(I) with Trityl Spin Probes

Copper homeostasis is tightly regulated in biological systems, and its dysregulation is implicated in various diseases. In the intracellular reducing environment, Cu(I) is the predominant oxidation state of labile copper; however, its detection remains highly challenging because of strong buffering by endogenous ligands and competition from biologically abundant metal ions. Here, we report an electron paramagnetic resonance (EPR)-based strategy that enables detection of otherwise EPR-silent Cu(I) by converting Cu(I) coordination into a characteristic spectroscopic fingerprint through electron–nuclear hyperfine coupling. A series of Cu(I)-hyperfine spin probes (CHYPs) were developed by tethering stable trityl radicals to Cu(I)-selective ligands through various linkers. Among these probes, CHYP7 formed a 1:1 complex with Cu(I) and displayed a well-resolved four-line EPR signal arising from hyperfine splitting by the copper nucleus (I = 3/2). CHYP7 exhibited high Cu(I) affinity, excellent metal-ion selectivity, and a low detection limit of ∼4 nM (0.2 pmol). A hydrophilic analogue, CHYP8, further enabled quantitative Cu(I) measurement under acidic conditions in the presence of endogenous ligands and competing metal ions. CHYP8 was also applied to measure cellular labile copper pools and monitor copper-level changes following copper supplementation or hypoxia-reoxygenation treatment, as well as to quantify labile copper pools in tissues and serum from mice subjected to ischemia/reperfusion injury. Overall, this work establishes hyperfine-coupling-enabled EPR detection as a new strategy for probing Cu(I) in biological systems and provides a platform for investigating copper homeostasis and copper-associated pathophysiology.