New Amino-Acid-Based β-Phosphorylated Nitroxides for Probing Acidic pH in Biological Systems by EPR Spectroscopy

There is increasing interest in measuring pH in biological samples by using nitroxides with pH-dependent electron paramagnetic resonance (EPR) spectra. Aiming to improve the spectral sensitivity (Δa_X ) of these probes (i.e., the difference between the EPR hyperfine splitting (hfs) in their protonated and unprotonated forms), we characterized a series of novel linear α-carboxy, α'-diethoxyphosphoryl nitroxides constructed on an amino acid core and featuring an (α or α')-C-H bond. In buffer, the three main hfs (a_N , a_H , and a_P ) of their EPR spectra vary reversibly with pH and, from a_P or a_H titration curves, a two- to fourfold increase in sensitivity was achieved compared to reference imidazoline or imidazolidine nitroxides. The crystallized carboxylate 10 b (pK_a ≈3.6), which demonstrated low cytotoxicity and good resistance to bioreduction, was applied to probe stomach acidity in rats. The results pave the way to a novel generation of highly sensitive EPR pH markers.