Quantitative electron paramagnetic resonance and spectrophotometric determination of the free radical 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy

The first quantitative estimation of the stable free radical 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy (TEMPOL) simultaneously performed by electron paramagnetic resonance (EPR) and visible spectrophotometric titration is reported. The obtained molar absorptivity of TEMPOL 13.2 +/- 0.1 l(-1) m(-1) cm(-1) at 22,900 cm(-1) may be used as the future criterium about the purity of the EPR active substance. The studied compound may be considered as the second (after 1,1-diphenyl-2-pikrilhydrazil) material calibrated as the primary standard for quantitative EPR spectrometry.     

Effect of substituents,solute–solute–non-polar solvent interactions and temperature on the spectral properties of bis(dithiophosphato)copper(II) complexes

Extended studies by e.p.r. and electronic spectroscopy on the effect of different non-polar solvents, temperature and disulfide concentration on the spectral properties of bis(dithiophosphato)copper(II), Cu[(RO)₂PS₂]₂, complexes [R = Me, Et and i-Pr] are reported. The molar absorptivity and e.p.r. intensity are very sensitive to the shape and size of the remote ligand substituents and increase in the order: Me < Et < i-Pr. The nature of the solvent and time after dissolution are also important regarding the magnitude of the e.p.r. intensity and molar absorptivity which, 1 h after dissolution, do not follow Beer's law. The molar absorptivities obtained at a given Cu^II(R₂–dtp)₂ concentration increase in the solvent order: n-hexane < n-heptane < CCl₄ < PhMe < PhH < CHCl₃. Twenty-four hours after dissolution the same samples exhibit: (i) linearity between absorbance and concentration of Cu^II(R₂–dtp)₂; (ii) a significant increase in molar absorptivity which is not equal for all the complexes studied and follows the same substituent and solvent orders. Beer's law is satisfied above 5 × 10^–4 M for [(RO)₂PS₂]₂Cu (R = Et, i-Pr) and above 3 × 10^–3 M for [(MeO)₂PS₂]Cu. A significant additional increase, ca. 30–40%, of molar absorptivity, is obtained by increasing the solution temperature from 20 to 50 °C. The molar absorbtivity remains unchanged during 2–3 days after reducing the temperature. Further increase of molecular absorbtivity appears after addition of the corresponding disulfide of dithiophosphate [(RO₂)PS₂–S₂P(RO)₂] to Cu^II[(RO)₂PS₂]₂. The molar absorptivity of Cu[(i-PrO)₂PS₂]₂ increases from 4.8 × 10³ cm^–1 M^–1, 1 h after dissolving the complex up to 2 × 10⁴ cm^–1 M^–1 after addition of the corresponding disulfide. The observed effects are explained on the basis of a self-redox reaction taking place in this type of sulfur containing copper(II) complexes.     

Effect of solute-co-ordinating solvent interactions and temperature on the EPR and electronic spectra of bis(dithiophosphato)copper(II)

The self-redox reaction proceeding between two molecules of the complex bis(disubstituted-dithiophosphato)copper(II), CuII(R2-dtp)2, is studied by EPR and UV-VIS spectroscopy in DMFA, DMSO and pyridine. The effect of temperature and disulphide concentration in the solutions is also evaluated. The EPR spectra show that the g-values of CuII(R2-dtp)2 increase when it is dissolved in co-ordinating solvents, whereas the copper hyperfine splitting decreases compared to the corresponding values in non-co-ordinating solvents. Under the same conditions, a hypsochromic shift is observed in the maximal absorption at 420 nm of the electronic spectra which corresponds to the ligand-to-metal charge-transfer (LMCT) transition of the complex. The results are explained with the formation of axial or equatorial adducts between CuII(R2-dtp)2 and the co-ordinating solvents used. On the other hand, the molar absorptivity of the LMCT band and the intensity of the EPR spectrum increase strongly with the nature of the used co-ordinating solvent, the time after dissolution and the quantity of added disulphide. Both also depend on the size and shape of remote ligand substituents and they increase in the order Me < Et < i-Pr. Beer's law is not obeyed immediately after dissolution of copper bis-dithiophosphate complexes. However, after standing for 24 h in the dark, DMFA solutions exhibit linear absorption/concentration dependence with approximately 70% higher molar absorptivity. An additional increase of the LMCT band and EPR intensity is found after heating the solution up to 50 degrees C for a short time, as well as after addition of the corresponding disulphide of dithiophosphate [(RO)2P(S)S-S(S)P(RO)2] to the CuII(R2-dtp)2 solution. As a result, the molar absorptivity value at the maximum of the LMCT band of Cu[(i-PrO)2-dtp]2 increases from 7.9 x 10(3) m(-1) dm3 cm(-1) immediately after dissolution to 2.9 x 10(4) m(-1) dm3 cm(-1). In DMSO and pyridine, the intensity of both the EPR signal and LMCT band of CuII(R2-dtp)2 continuously decrease after the preparation of the solutions. A small increase is only observed immediately after the addition of the corresponding disulphide of dithiophosphate. While DMFA forms stable adducts with Cu[(i-PrO)2-dtp]2, adduct formation with DMSO and pyridine destroys the initial complex.     

The fidelity of spin trapping with DMPO in biological systems

Unlike direct ESR, spin trap methodology depends on the absolute fidelity of the spin trap reaction. Two alternative reactions of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) leading to radical adduct artifacts have been discovered and investigated: inverted spin trapping and the Forrester-Hepburn nucleophilic mechanism. These two alternate pathways to radical adducts are a combination of one-electron oxidation and nucleophilic addition, in either order. In biological systems, serious artifacts have been reported due to the Forrester-Hepburn mechanism, which is initiated by the addition of a nucleophile to DMPO. It has recently been demonstrated that (bi)sulfite (hydrated sulfur dioxide) can react with DMPO via a nonradical, nucleophilic reaction, and it has been further proposed that DMPO/(?)SO(3)(-) formation in biological systems is an artifact and not the result of spin trapping of sulfur trioxide anion radical ((?)SO(3)(-)). The one-electron oxidation of (bi)sulfite catalyzed by horseradish peroxidase (HRP)/hydrogen peroxide (H(2)O(2)) has been reinvestigated by ESR spin trapping with DMPO and oxygen uptake studies to obtain further evidence for the radical reaction mechanism. In the absence of DMPO, the initial rate of (bi)sulfite-dependent oxygen and H(2)O(2) consumption was determined to be half of the initial rate of DMPO/(?)SO(3)(-) radical adduct formation as determined by ESR, demonstrating that, under our experimental conditions, DMPO exclusively forms the radical adduct by trapping the (?)SO(3)(-).