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

Abstract

There is increasing attraction of measuring pH in biological studies using nitroxides having pHdependent electron paramagnetic resonance (EPR) spectra. Aiming to improve the spectral sensitivity, a X , of these probes (i.e. the difference between limiting EPR hyperfine splittings (hfs) in their protonated and unprotonated forms), we present here a series of novel linear -carboxy, '-diethoxyphosphoryl nitroxides constructed on an amino acid core and featuring a ( or ')-C−H bond.In buffers the three main hfs (a N , a H and a P ) of their X-band EPR spectra vary reversibly with pH and, when it is inferred from a P or a H titration curves, a 2−4-fold increase in sensitivity is achieved vs reference imidazoline or imidazolidine nitroxides. Lead crystallized carboxylate 10b (pK a ≈ 3.6), which demonstrated low cytotoxicity and good resistance to bioreduction, was applied to probe the stomach acidity in rats. The results pave the way to a novel generation of highly sensitive EPR pH markers.

Exploring the pH-Induced Functional Phase Space of Human Serum Albumin by EPR Spectroscopy

“…From this study it remains yet unsettled to which extent pH-sensitive EPR-active imidazolidine spin labels (IMTSL) [157], or lipid spin probes (IMTSL-PTE) [158] may contribute to a better understanding of HSA's phase space due to their intrinsic capability to act as pH and polarity sensors [159][160][161]. This direct detection of pH-dependent surface potentials of HSA might reveal more precise information about the complex interplay of albumins' charge and shape.…”

An elastase activity reporter for Electronic Paramagnetic Resonance (EPR) and Overhauser-enhanced Magnetic Resonance Imaging (OMRI) as a line-shifting nitroxide

“…Since unpaired electrons are particularly sensitive to the electronic environment some have been designed to display resonance line broadening or shifting to detect various parameters. Hence free radicals have been used for oximetry [7,8], redox status imaging [9,10], pH measurement [11][12][13][14], water content measurement [15] or to report on enzymatic activity [16][17][18]. EPR imaging (EPRI) can be used with these free radicals.…”

Selective On/Off‐Nitroxides as Radical Probes to Investigate Non‐radical Enzymatic Activity by Electron Paramagnetic Resonance

“…This technique requires the use of a nitroxide providing an EPR signal sensitive to this activity. Indeed, free organic radicals are currently used for oximetry, redox status imaging, for pH measurement, and for water content measurement . However, none of these approaches can be employed to investigate nonradical enzymatic activity either by EPR or OMRI.…”

“…Despite suitable enzymatic constants [2] this methods uffers from several drawbacks: incomplete quenching of substrate fluorescence, limited tissue penetration of light, difficult skull imaging or imaging in large animalsa nd three-dimensional images require reconstruction. [6] Thist echnique requires the use of an itroxidep roviding an EPR signal sensitive to this activity.I ndeed, free organic radicals are currently used for oximetry, [7,8] redox status imaging, [9,10] for pH measurement, [11][12][13][14] and for water content measurement. Free radicals such as nitroxides or trityl radicalsa re stable enough in physiological conditions (in vitro and in vivo) to be detected by electronic paramagnetic resonance( EPR).…”