D. Beckert

Oxidative damage of human skin lipids: Dependence of lipid peroxidation on sterol concentration

Photoprotection against sunburn and associated irradiation-induced damages of the human skin is mainly attributed to the darkening of the biochrome melanin by its oxidation. Human skin lipids were examined for an additional protection by sterols. Lipid vesicles prepared from extracted human skin lipids as well as from mixtures of typical lipids of the stratum corneum were irradiated by UV light in the presence and absence of oxygen. The oxidative degradation of various lipids was measured by quantitative HPTLC, by the dichlorofluorescein fluorescent assay, by the thiobarbituric acid assay and a novel luminol-based chemiluminescence technique. Electron spin resonance was used to look for certain radical intermediates. The results indicate, that sterols, mainly free cholesterol, with their high concentration in the lipid barrier of the stratum corneum (up to 50 mol%) effectively compete with the peroxidation of other human skin lipids (ceramides and free fatty acids).

Study of radical pairs generated by photoreduction of anthraquinone-2,6-disulfonic acid with thymine by Fourier transform electron paramagnetic resonance

Abstract Using laser photolysis at 308 nm and FT–EPR, the triplet sensitized electron transfer from thymine to 9,10-anthraquinone-2,6-disulfonate in aqueous solution was studied. The anthraquinone radical anion and the deprotonated thymine-1-yl radical are spin-polarized by the CIDEP triplet mechanism and radical pair mechanism. The structure of the anthraquinone radical anion is strongly influenced by the pH of the solution. In weak acidic solution the radical anion dominates whereas the pK of the radical protonation was determined to be 3.2. The deprotonated thymine-1-yl radical shows two different radical pair polarization patterns which are distinguished by the phase of the polarization. This unusual behavior can be attributed to two different states in the primary radical pair.

Fourier transform EPR study of N-centered pyrimidine radicals in the nanosecond time-scale

Abstract Using nanosecond laser photolysis with 308 nm and Fourier transform EPR spectroscopy, the pyrimidine radicals (thymine, uracil and 6-methyluracil) generated by electron transfer from the pyrimidine ground state to spin-polarized anthraquinone-2,6-disulfonate (2,6-AQDS) triplets were studied. The pyrimidine radical cations decay by deprotonation at the N(1) position to the neutral pyrimidine-1-yl radical. No other reaction channel could be found. The rate constant of the electron transfer could be estimated from the triplet spin-polarization (CIDEP) of the 2,6-AQDS radical anions to: k et (thymine) = 4.1 × 10 9 M −1 s −1 , k et (uracil) = 2.4 × 10 9 M −1 s −1 and k et (6-methyluracil) = 4.5 × 10 9 M −1 s −1 .

Radicals formed by electron transfer from cytosine and 1-methylcytosine to the triplet state of anthraquinone-2,6-disulfonic acid. A Fourier-transform EPR study

Radicals generated by electron transfer from cytosine and 1-methylcytosine to the laser-induced triplet state of anthraquinone-2,6-disulfonic acid were studied by time-resolved Fourier-transform (FT) EPR in H2O and D2O at 10°C. The main products observed on the nanosecond timescale were successors of the base radical cations. Their hyperfine couplings were determined by computer simulation of the experimental spectra. Assignment of the radical structures was supported by density functional theory (DFT) quantum mechanical calculations. The experiments with cytosine deprotonation at N1 resulted in the cytosin-1-yl radical 1 with high spin density at N1 and C5 whereas, for 1-methylcytosine, deprotonation at the exocyclic amino group yielded the aminyl radical 2. For both parent compounds, cytosine and 1-methylcytosine, two additional long-lived radicals (3 and 4) with unknown structure were detected on the nanosecond to microsecond timescale. Their spectral parameters were independent of the N1 substituent.

Calculation of spin densities of radicals of pyrimidine-type bases by density functional theory. Influence of solvent and comparison with EPR results

Spin densities and hyperfine coupling (hfc) constants of thymine, 1-methylthymine, uracil, 1-methyluracil, 6-methyluracil, 1,3-dimethyluracil, cytosine and 1-methylcytosine radicals were calculated through the use of density functional theory (DFT) considering the influence of the surrounding solvent molecules by their dielectric properties by applying the Onsager model. The results are compared with experimental hfc constants obtained with Fourier-transform (FT) EPR experiments. The radicals were generated in aqueous solution using the triplet sensitized electron transfer from the pyrimidine-type bases to photoexcited spin-polarized anthraquinone-2,6-disulfonate. It is found that the geometry and the spin density distribution of the radical cations and their deprotonated successor radicals are influenced by a surrounding dielectric solvent. The hfc constants calculated for an aqueous medium are in good agreement with the experimental FT EPR results.

A fourier transform EPR study of uracil and thymine radical anions in aqueous solution

Hydrated electrons are generated by two photon ionization of anthraquinone-1,5-disulfonate in H2O and D2O solutions. The electron attachment to the pyrimidine-type bases thymine and uracil results in the thymine and uracil radical anions. By simulation of the EPR splitting patterns of the protonated and deuterated forms of the radical anions their hyperfine coupling constants are assigned unambiguously. This new assignment of all hfs coupling constants is supported by quantum chemical calculations. The kinetics of electron attachment to the pyrimidine-type bases was studied by the method of kinetic linebroadening effects of the hydrated electron. The rate constants of the electron attachment were determined as katt(thymine) = katt(uracil) = (2.7 ± 0.1) × 109 M−1 s−1.

Photo‐ and Radiation‐Chemical Formation and Electrophilic and Electron Transfer Reactivities of Enolether Radical Cations in Aqueous Solution

In aqueous solution, enolether radical cations (EE.+) were generated by photoionization (lambda < or = 222 nm) or by electron transfer to radiation-chemically produced oxidizing radicals. Like other radical cations, the EE.+ exhibit electrophilic reactivity with respect to nucleophiles such as water or phosphate as well as electron transfer reactivity, for example, towards one-electron reductants such as phenols, amines, vitamins C and E, and guanine nucleosides. The reactivity of these electron donors with the radical cation of cis-1,2-dimethoxyethene.+ (DME.+) can be described by the Marcus equation with the reorganization energy lambda = 16.5 kcalmol(-1). By equilibrating DME.+ with the redox standard 1,2,4-trimethoxybenzene, the reduction potential of DME.+ is determined to be 1.08 +/- 0.02 V/NHE. The oxidizing power of the radical cation of 2,3-dihydrofuran, which can be considered a model for the enolether formed on strand breakage of DNA, is estimated to be in the range 1.27-1.44 V/NHE.

Investigation of the molecular structure of the radical anions of some pyrimidine -type bases in aqueous solution by comparison of calculated hyperfine coupling constants with EPR results

New results of DFT B3LYP calculations in aqueous solution are presented for the radical anions of uracil, thymine, 1-methylthymine, 1-methyluracil and 1,3-dimethyluracil. The most relevant molecular structure of the radical anions in water optimised with extended basis sets using either the Onsager or the CPCM self-consistent reaction field model is the boat conformation. The structure shows pyramidality at the radical centre C6 connected with a deviation of the C6–H atom from the molecular plane up to around 12°. Gas-phase structures, even optimised with extended basis sets, are not able to reproduce the large values of the hyperfine coupling (hfc) constant of the C6–H atom known from the experiments (about 35 MHz). Reliable values for this coupling require optimisations involving the solvent. The CPCM model appears to be superior to the Onsager model. Optimisations with inclusion of up to 10 water molecules, thus modelling hydrogen bonding with the solvent, confirm the results obtained with the continuum models.

Interaction of UV Light‐Induced α–Tocopherol Radicals with Lipids Detected by an Electron Spin Resonance Prooxidation Effect

The reaction rate constants of the interaction between light‐induced α–tocopherol radicals with unsaturated lipids in a heterogeneous system compared to a homogeneous system are of the same order of magnitude. The decay rates of compartmentalized ‐α‐tocopherol radicals were significantly reduced by using negatively charged sodium dodecyl sulfate (SDS) micelles. A partially resolved electron spin resonance (ESR) hyperfine structure was observed under the conditions of both high lipid concentrations in comparison to the α‐tocopherol concentration and of a regular distribution of α‐tocopherol molecules inside the heterogeneous lipid structures. Alphα‐to‐copherol radicals have a considerable prooxidation potential at higher concentrations. Ascorbic acid dissolved in the aqueous medium provokes very fast ‐α‐tocopherol radical recycling through the boundary layer between the aqueous medium and micelles. By contrast, very slow reactions such as those of α‐tocopherol radicals with glutathione through this boundary layer are measurable. Despite using the heterogeneous SDS micellar system, the decay kinetics of the α‐tocopherol radical ESR signal is simply compounded. In addition to the known stabilization effect of cholesterol in membrane systems, cholesterol itself acts as a target molecule attacked by free radicals, e.g. ‐α‐tocopherol radicals. Using stratum corneum extracts that contain unsaturated lipids and cholesterol the ‐α‐tocopherol radical can prooxidatively react with these compounds. Using focused UV light generates a high radical yield in a relatively short time compared to the lifetime of the ‐α‐tocopherol radicals. The decay processes after radical induction can be characterized as consecutive reactions. The compartmentalization of radicals induced in SDS micelles and the close proximity of target molecules are essential if very slow one‐electron reductions are to be measured.

A comparative time-resolved CW EPR and FT EPR investigation of the addition of 2-hydroxy-2-propyl radicals to acrylate and methacrylate monomers

The addition of 2-hydroxy-2-propyl radicals to n-butyl acrylate and n-butyl methacrylate has been investigated by time-resolved continuous wave electron paramagnetic resonance (TR CW EPR) and time-resolved Fourier transform electron paramagnetic resonance (TR FT EPR). The 2-hydroxy-2-propyl radicals were generated by the photolysis of acetone in propan-2-ol (through hydrogen abstraction) and by the photolysis of a ketone 5 (through α-cleavage). The TR CW and TR FT spectra were experimentally equivalent for the addition of 2-hydroxy-2-propyl radicals to n-butyl acrylate to produce 3a. However, there are distinct differences between the TR CW and TR FT spectra for the addition of 2-hydroxy-2-propyl radicals to n-butyl methacrylate which produces the radical adduct 4a. In particular, a number of hyperfine lines clearly present in the TR CW spectra are much weaker in, or are absent from, the TR FT spectra. The differences in the TR CW and TR FT spectra are attributed to hindered rotation, which is important in the spectrum of the adduct of 2-hydroxy-2-propyl radicals to n-butyl methacrylate (4a), but not in the spectrum of the adduct of 2-hydroxy-2-propyl radicals to n-butyl acrylate (3a). The hindered rotation is shown to selectively shorten the spin–spin relaxation time, T2, for certain hyperfine lines in the spectrum of 4a, resulting in broadening or disappearance of these lines and explaining the differences between the TR CW and TR FT spectra.