Ratiometric quantitation of redox status with a molecular Fe₂ magnetic resonance probe

We demonstrate the ability of a molecular Fe₂ complex to enable magnetic resonance (MR)-based ratiometric quantitation of redox status, namely through redox-dependent paramagnetic chemical exchange saturation transfer (PARACEST). Metalation of a tetra(carboxamide) ligand with Fe^II and/or Fe^III in the presence of etidronate ion affords analogous FeII2, Fe^IIFe^III, and FeIII2 complexes. Both FeII2 and Fe^IIFe^III complexes give highly-shifted, sharp, and non-overlapping NMR spectra, with multiple resonances for each complex corresponding to exchangeable carboxamide protons. These protons can be selectively irradiated to give CEST peaks at 74 and 83 ppm vs. H₂O for the Fe^IIFe^III complex and at 29, 40 and 68 ppm for the FeII2 complex. The CEST spectra obtained from a series of samples containing mixtures of FeII2 and Fe^IIFe^III are correlated with independently-determined open-circuit potentials to construct a Nernstian calibration curve of potential vs. CEST peak intensity ratio. In addition, averaged intensities of phantom images collected on a 9.4 T MRI scanner show analogous Nernstian behavior. Finally, both the FeII2 and Fe^IIFe^III forms of the complex are stable to millimolar concentrations of H₂PO₄^-/HPO₄^2-, CO₃^2-, SO₄^2-, CH₃COO^-, and Ca²⁺ ions, and the FeIII2 form is air-stable in aqueous buffer and shows >80% viability in melanoma cells at millimolar concentration. The stability suggests the possible application of this or related complexes for in vivo studies. To our knowledge, this concentration-independent method based on a single Fe₂ probe provides the first example of MR-based ratiometric quantitation of redox environment.