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

• Published in: Chembiochem : a European journal of chemical biology Vol. 18; no. 3; pp. 300 - 315
• Main Authors: Thétiot‐Laurent, Sophie, Gosset, Gaëlle, Clément, Jean‐Louis, Cassien, Mathieu, Mercier, Anne, Siri, Didier, Gaudel‐Siri, Anouk, Rockenbauer, Antal, Culcasi, Marcel, Pietri, Sylvia
• Format: Journal Article
• Language: English
• Published: Germany Wiley-VCH Verlag 01-02-2017
• Subjects: A549 Cells; Amino Acids - chemistry; Animals; Cell Survival - drug effects; Chemical Sciences; Crystallography, X-Ray; Electron Spin Resonance Spectroscopy; EPR spectroscopy; Gastric Acid - chemistry; Humans; Hydrogen-Ion Concentration; Magnetic Resonance Spectroscopy; Male; molecular dynamics; Molecular Dynamics Simulation; Nitrogen Oxides - chemistry; Nitrogen Oxides - toxicity; nitroxide; or physical chemistry; Organophosphonates - chemical synthesis; Organophosphonates - chemistry; Phosphorylation; Rats; Rats, Sprague-Dawley; sensors; Stomach - metabolism; stomach acidity; Theoretical and; nitroxide; sensors; molecular dynamics; EPR spectroscopy; stomach acidity
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.201600550

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 (ΔaX) 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 (aN, aH, and aP) of their EPR spectra vary reversibly with pH and, from aP or aH titration curves, a two‐ to fourfold increase in sensitivity was achieved compared to reference imidazoline or imidazolidine nitroxides. The crystallized carboxylate 10 b (pKa ≈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.
McConnell's equation helps EPR pH probing. β‐Phosphorylated nitroxides with a β′‐amino acid core provide enhanced pH sensing in the acidic pH region. To illustrate this, a stable, biocompatible, crystallized model carboxylate resistant to bioreduction was applied to assess rat stomach acidity.