Unexpected rapid aerobic transformation of 2,2,6,6-tetraethyl-4-oxo(piperidin-1-yloxyl) radical by cytochrome P450 in the presence of NADPH: Evidence against a simple reduction of the nitroxide moiety to the hydroxylamine

Abstract

Aminoxyl radicals (nitroxides) are a class of compounds with important biomedical applications, serving as antioxidants, spin labels for proteins, spin probes of oximetry, pH, or redox status in electron paramagnetic resonance (EPR), or as contrast agents in magnetic resonance imaging (MRI). However, the fast reduction of the radical moiety in common tetramethyl-substituted cyclic nitroxides within cells, yielding diamagnetic hydroxylamines, limits their use in spectroscopic and imaging studies. In vivo half-lives of commonly used tetramethyl-substituted nitroxides span no more than a few minutes. Therefore, synthetic efforts have focused on enhancing the nitroxide stability towards reduction by varying the electronic and steric environment of the radical. Tetraethyl-substitution at alpha position to the aminoxyl function proved efficient in vitro against reduction by ascorbate or cytosolic extracts. Moreover, 2,2,6,6-tetraethyl-4-oxo(piperidin-1-yloxyl) radical (TEEPONE) was used successfully for tridimensional EPR and MRI in vivo imaging of mouse head, with a reported half-life of over 80 min. We decided to investigate the stability of tetraethyl-substituted piperidine nitroxides in the presence of hepatic microsomal fractions, since no detailed study of their "metabolic stability" at the molecular level had been reported despite examples of the use of these nitroxides in vivo. In this context, the rapid aerobic transformation of TEEPONE observed in the presence of rat liver microsomal fractions and NADPH was unexpected. Combining EPR, HPLC-HRMS, and DFT studies on a series of piperidine nitroxides -TEEPONE, 4-oxo-2,2,6,6-tetramethyl(piperidin-1-yloxyl) (TEMPONE), and 2,2,6,6tetraethyl-4-hydroxy(piperidin-1-yloxyl) (TEEPOL), we propose that the rapid loss in paramagnetic character of TEEPONE is not due to reduction to hydroxylamine but is a consequence of carbon backbone modification initiated by hydrogen radical abstraction in alpha position to the carbonyl by the P450-Fe (V) =O species. Besides, hydrogen radical abstraction by P450 on ethyl substituents, leading to dehydrogenation or hydroxylation products, leaves the aminoxyl function intact but could alter the linewidth of the EPR signal and thus interfere with methods relying on measurement of this parameter (EPR oximetry).

A Simple Method of Synthesis of 3-Carboxy-2,2,5,5-Tetraethylpyrrolidine-1-oxyl and Preparation of Reduction-Resistant Spin Labels and Probes of Pyrrolidine Series

“…This method was used to prepare a set of new spin labels and probes, which showed higher resistance to reduction with ascorbate than 1. Fast decay of the new spin probes in homogenates of liver and kidney and slow decay in homogenate of heart implies possible participation of cytochrome P450 in nitroxide decay, in analogy to the mechanism suggested by Babić et al [51].…”

“…Taking into account the ascorbate content in tissues [ 46 ] and reduction rate constant shown in the Table 1 , nearly 50% decay of 22 in half an hour cannot be explained by non-enzymatic nitroxide reduction. The recent study by Babić et al showed that NADPH-dependent destruction by cytochrome P450 may be a major pathway of sterically shielded piperidine nitroxides decay in the presence of hepatic microsomal fraction [ 51 ]. The behavior observed here for 22 and 28 is in agreement with the above-mentioned finding.…”

The Cellular and Organismal Effects of Nitroxides and Nitroxide-Containing Nanoparticles

“…Different substituents at the C2 and C4 positions affect the susceptibility of piperidine nitroxides toward ascorbate reduction [50]. In contrast to methyl-substituted nitroxides, tetraethyl-substituted piperidine nitroxides bearing an exocyclic double bond like TEEPONE (Figure 1, #9) are also substrates to fast P450-induced hydrogen atom abstraction in α-position to the sp 2 carbon of the ring, followed by the destruction in vivo [51].…”

Hydrophilic Reduction-Resistant Spin Labels of Pyrrolidine and Pyrroline Series from 3,4-Bis-hydroxymethyl-2,2,5,5-tetraethylpyrrolidine-1-oxyl

“…Stock solutions were usually prepared in DMSO or in water-DMSO mixtures, see for example [20,23]. Recent studies revealed that an alternative metabolic pathway implying cytochrome P450 activity may significantly contribute to the decay of sterically shielded nitroxides in subcellular systems [24,25]. These data coincide with our observations that decay of 2,2,5,5-tetraethylpyrrolidine nitroxides in homogenates of liver and kidney, which are known to be rich of cytochrome P450 enzymes, proceeds much faster than in homogenates of brain, heart or in blood [22].…”