New Amino-Acid-Based ß-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 (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.

CyDEPMPOs: a class of stable cyclic DEPMPO derivatives with improved properties as mechanistic markers of stereoselective hydroxyl radical adduct formation in biological systems.

The cis/trans diastereoisomeric composition of hydroxyl radical adducts to chiral cyclic nitrones can be used to approach mechanisms of free radical formation in biological systems. Such determination is greatly simplified when both diastereoisomers have ESR spectra with at least two non-overlapping lines. To achieve this prerequisite, a series of DEPMPO-derived spin traps bearing one unsubstituted or alkyl-substituted 2-oxo-1,3,2-dioxaphosphorinane ring were synthesized and their structures were confirmed by X-ray diffraction, (1)H, (13)C and (31)P NMR. These CyDEPMPOs nitrones showed variable lipophilicities and LD(50) values on murine fibroblasts compatible with a safe use in biological spin trapping. All CyDEPMPOs formed persistent spin adducts with a series of free radicals, including superoxide and hydroxyl (i.e., CyDEPMPOs-OH) and the in vitro half-life times of these two latter were at least as extended as those of parent DEPMPO. Using four methods of CyDEPMPOs-OH formation, the cis-CyDEPMPOs-OH percentage was found significantly varied with substitution on the P-containing ring and, more interestingly, with the generating system.

Absence of tumor suppressor tumor protein 53-induced nuclear protein 1 (TP53INP1) sensitizes mouse thymocytes and embryonic fibroblasts to redox-driven apoptosis.

The p53-transcriptional target TP53INP1 is a potent stress-response protein promoting p53 activity. We previously showed that ectopic overexpression of TP53INP1 facilitates cell cycle arrest as well as cell death. Here we report a study investigating cell death in mice deficient for TP53INP1. Surprisingly, we found enhanced stress-induced apoptosis in TP53INP1-deficient cells. This observation is underpinned in different cell types in vivo (thymocytes) and in vitro (thymocytes and MEFs), following different types of injury inducing either p53-dependent or -independent cell death. Nevertheless, absence of TP53INP1 is unable to overcome impaired cell death of p53-deficient thymocytes. Stress-induced ROS production is enhanced in the absence of TP53INP1, and antioxidant NAC complementation abolishes increased sensitivity to apoptosis of TP53INP1-deficient cells. Furthermore, antioxidant defenses are defective in TP53INP1-deficient mice in correlation with ROS dysregulation. Finally, we show that autophagy is reduced in TP53INP1-deficient cells both at the basal level and upon stress. Altogether, these data show that impaired ROS regulation in TP53INP1-deficient cells is responsible for their sensitivity to induced apoptosis. In addition, they suggest that this sensitivity could rely on a defect of autophagy. Therefore, these data emphasize the role of TP53INP1 in protection against cell injury.

Improving the stability and antioxidant properties of sesame oil: water-soluble spray-dried emulsions from new transesterified phenolic derivatives.

Hydrosoluble sesame oil fatty acid transesters having enhanced antioxidant activities were synthesized in a two-step process. The key step involved the biocatalyzed (lipase from Candida antarctica) acylation of sesame oil methanolic ester with either vanillyl (VNA) or piperonyl benzylic alcohols, or 5-hydroxymethyl resorcinol (5-HMR). These substrates were selected to introduce phenolic or sesamol structurally related frameworks. The VNA and 5-HMR-derived transesters were obtained with 20-40% yields and retained the starting proportions of sesame oil linoleic, oleic, and saturated acids, these fatty acids also being the only constituents of the nonesterified fraction. The VNA-derived transester showed the best antioxidant capacity in standard assays and was processed as the unique lipid phase of spray-dried emulsions containing a high level of linoleic acid phenolic ester. These emulsions provided a high degree of protection to UV-irradiated fibroblasts, through the potential synergy between VNA antioxidant action and replenishment of damaged membranes by unsaturated fatty acids.

Investigation of subcellular acidic compartments using alpha-aminophosphonate 31P nuclear magnetic resonance probes.

The 31P nuclear magnetic resonance (NMR) characteristics, toxicity, and cellular penetration of five linear or cyclic alpha-aminophosphonate highly sensitive pH probes were investigated in Dictyostelium discoideum cells and isolated rat hearts and were compared with three phosphonic acid derivatives. The line width broadening at pH approximately pK(a), which was satisfactorily modelized for all compounds, was significantly limited in biological milieu for the new markers, affording a four- to sixfold better accuracy in pH determination. Cellular uptake or washout of nontoxic concentrations (< 15 mM) of alpha-aminophosphonates occurred by rapid passive permeation, whereas standard probes required a much slower fluid-phase pinocytosis and transport processes that could ultimately lead to trapping. Using mild concentrations (< 4 mM) three alpha-aminophosphonates having 6 < pK(a) < 7 allowed an easy and simultaneous 31P NMR determination of cytosolic, acidic, and extracellular compartments in anoxic-reoxygenated or starving D. discoideum.