Design of New Derivatives of Nitrone DEPMPO Functionalized at C-4 for Further Specific Applications in Superoxide Radical Detection

Abstract

A general synthetic route to prepare derivatives of the DEPMPO nitrone (5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide) functionalized at C-4 was established via the synthesis of 4-HMDEPMPO nitrone (5-diethoxyphosphoryl-4-hydroxymethyl-5-methyl-1-pyrroline-N-oxide) that was obtained from reduction of the nitro compound 1. (4R*,5S*)-4-HMDEPMPO was successfully separated from its minor diastereoisomer and could be used to generate various substituted analogues. Among them, 4-NHSDEPMPO, 5-diethoxyphosphoryl-4-succinimidyloxycarbonyloxymethyl-5-methyl-1-pyrroline-N-oxide, constitutes a NH2-reactive precursor for further conjugation to relevant moieties such as targeting groups, labels, or drugs. From 4-NHSDEPMPO, a biotinylated nitrone was synthesized offering new perspectives for targeted delivery applications. A short study of the trapping behaviors of the (4R*,5S*)-isomer of these 4-HMDEPMPO analogues proved that they are as good as DEPMPO for detecting superoxide. For each isomer, only one diastereoisomer adduct was obtained, resulting from the addition of superoxide on the less hindered face of the nitrone, that is, trans to the phosphoryl group and the C-4 substituent. From spectra simulation and experiments in various solvents, we proved that ESR patterns of each adduct corresponded to the superimposed signals of two sets of conformers in a sufficiently slow chemical exchange to induce a widening and a dissymmetry of some of the signal lines. This phenomenon was drastically reduced when compared with that observed for DEPMPO superoxide and attributed to a similar chemical exchange, and it did not hamper spectrum assignment. Determination of the decay rate of the superoxide adduct of (4R*,5S*)-4-HMDEPMPO proved that it has a 25% longer half-life time than the superoxide adduct of DEPMPO.

The Selenium-independent Inherent Pro-oxidant NADPH Oxidase Activity of Mammalian Thioredoxin Reductase and Its Selenium-dependent Direct Peroxidase Activities

“…The KO 2 /18-crown-6 ether system as described by (40,41) to 1.7 mM. After 1 min at room temperature, both catalase and SOD were added to 1000 units/ml each.…”

Synthesis and characterization of 5-hydroxymethyl-5-methyl-pyrroline N-oxide and its derivatives

“…On the other hand, previous studies with hydroxyl-substituted DEPMPO compounds revealed higher superoxide adduct stabilities, [8][9][10] although the reported synthetic procedure is rather complex. In view of the low stabilities of the superoxide adducts we investigated the stabilizing effect of b-cyclodextrin and compared it to the values obtained with DMPO and EMPO.…”

“…Previous studies about DEPMPO and EMPO derivatives [1][2][3][4][5][6][7] were focused on the stability of superoxide adducts as well as investigations of mitochondria targeted spin traps [8][9][10] bearing positively charged phosphonium groups. Based on these studies we were aiming at synthesizing EMPO derivatives with a function to which a mitochondria targeting group could be readily attached in a single step.…”

“…Based on these studies we were aiming at synthesizing EMPO derivatives with a function to which a mitochondria targeting group could be readily attached in a single step. Unfortunately, the synthesis of 5-ethoxycarbonyl-4-hydroxymethyl-5-methyl-pyrroline N-oxide (the analogue of the previously reported 5-diethoxyphosphoryl-4-hydroxymethyl-5-methyl-1-pyrroline N-oxide, 4-HMDEPMPO 8,9 ) is not straightforward, and several attempts to synthesize this compound in pure form have failed in our hands. 11 We therefore decided to first develop strategies involving different protective groups for the hydroxyl groups starting from simple related model compounds.…”

ESR study of the spin adducts of three analogues of DEPMPO substituted at C4or C3

“…5 Earlier studies showed that the pseudorotation occurring within the ring of stable -phosphorylated pyrrolidinyloxyl radicals was slowed down by the ring substitution with a phenyl group. 6 In the DEPMPO series, synthesis of the 3-phenyl 7 and 4-phenyl 8 and 4-hydroxymethyl 9 DEPMPO analogue named 3-PhDEPMPO, 4-PhDEPMPO and 4-HMDEPMPO respectively (Scheme 1), were recently reported. The presence of both kinds of substituents led to an important steric effect on the course of addition of the superoxide radical.…”