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 (Delta a(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 alpha-carboxy, alpha'-diethoxyphosphoryl nitroxides constructed on an amino acid core and featuring an (alpha or alpha')-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 10b (pK(a) approximate to 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.