The work of CNR group within the SULFRAD network has dealt with two aspects: (i) thiyl radical damage caused to unsaturated lipids, focusing on the conversion of cis geometry of methylene-interrupted double bonds to corresponding trans isomers (cis/trans isomerization), and (ii) thiols as hydrogen donors to carbon-centered radicals on the sugar or base moieties of nucleosides.
Thiyl radical induced isomerization of fatty acids in membranes
The research, directed in interdisciplinary areas related to chemistry and biology since the network beginning, has been implemented and updated as follows:
a) Synthetic aspects of the lipid isomerization. A photolytical method for selective generation of thiyl radical has been developed. Based on this methodology, a general approach towards the TRANS LIPID LIBRARY has been offered using economical starting materials such as natural oils and with enormous potential for application to lipidomic researches. Gathering analytical data on trans lipids is important for the characterization of all lipid components in living organisms, which is the lipidomics target, and it has been demonstrated that nuclear magnetic resonance (NMR) is meaningful for the differences between cis and trans isomers. The analytical part has been also coupled with new aspects of gas chromatography (GC) related also to the examination of biological samples. Another aspect has dealt with the all-trans PUFA strategy, which can be useful for pharmacological applications due to the relevance of trans fatty acids as enzyme inhibitors. All-trans fatty acids isomers are readily accessible by our methodology. Indeed, the synthesis of all-trans arachidonic acid has been achieved in high yield and this new compound was found to be active as inhibitor of platelet aggregation, at the level of aggregation given by PAF but not given by thrombin.
b) Biomimetic model of liposomes. The occurrence of a regioselective cis/trans isomerization has been demonstrated in model membranes with particular emphasis to the arachidonic acid isomers. It is now possible to distinguish between the dietary contribution of trans fatty acids (deriving from foods and partially hydrogenated oils) and the in vivo transformation, which can occur in cell under radical stress conditions. A model for protein/lipid clustered radical damage has also been described. The model is based on g-irradiation of cis lipid vesicles containing RNase A, and shows that protein degradation is accompanied by formation of trans lipids. Thiyl radicals are the isomerizing agent deriving exclusively or nearly so from the degradation of the methionine residues, diffusing through the lipid bilayer and converting the cis double bond to its geometrical trans isomer. This model also envisages the role of trans lipids as markers of protein damage and inactivation. Several applications on this area are in progress in the group.
c) Endogenous formation of trans lipids. The objective is to obtain evidence that in the normal cell metabolism of human cells the formation of thiyl radicals occurs and it can induce the isomerization of unsaturated lipids. This goal is of great relevance in biology, since up to now it is known that trans fatty acids cannot be formed in human cells, but only in bacteria. In collaboration with Leipzig and Poznan partners, cell membrane phospholipids of haematopoietic human cells (types U937 – HeLa60 – THP-1) have been monitored by growing the cells under different conditions and in the presence of different thiol compounds, then extracting and analysing the hydrophobic fatty acid components of cell membranes. In a parallel investigation, trans lipids have been found also in tissues isolated from mice that were fed a trans-free diet for three months, and in some case a N-acetylcysteine diet. All results are in favour of a trans fatty acid formation during the cell life cycle by the thiyl radical path. A thorough study on the repair systems that involve the protection of the cis double bond geometry in Nature is currently carried out.
d) Properties of vesicle. Studies for the individuation how the selection for the cis geometry took place in Nature are in progress. The formation and properties of vesicles obtained from phospholipids with different percentages of trans fatty acid residues are investigated by dynamic light scattering (DLS), by UV and by fluorescence detection of specific probes inserted in the vesicles. A study of the isomerization in vesicles enclosing a fluorescent probe and using the stopped-flow technique for spectrofluorimetric measurements is in progress.
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Interaction of thiols with nucleoside-derived radicals
These reactions might be of interest from synthetic a point of view or could be considered as mimic of DNA or RNA repair. Indeed, the research has been focused in both aspects:
a) The reaction of thiols with sugar radicals. Many of the proposed mechanisms for the strand-break of DNA or RNA include the initial formation of sugar radicals through hydrogen atom abstraction from one of the five available positions. Selective generation of 2’-adenosinyl radical allowed to obtain relative rate constants between the hydrogen abstraction from glutathione (GSH) and the unimolecular process that liberates adenine. Selective generation of pseudo-C4’ by thermal decomposition of C5’-carboxythimidine tert-butyl perester was applied in order to study its reactivity in the presence of GSH under aerobic conditions. A detailed mechanistic picture has been obtained. Theoretical investigation on the reactivity of sugar radicals with thiols is in progress.
b) Synthesis of 5’,8-cyclopurine and 5’,8-cyclopyrimidine nucleosides. Cyclopurine and cyclopyrimidine lesions are observed among the decomposition products of DNA, when exposed to ionizing radiations. Their chemical synthesis and incorporation on specific sites of DNA is of considerable importance in order to investigate the biochemical and biophysical features of such lesions. Synthetic methodologies based on C5’ radical attack on the C6 position of the thymine or on the C8 position of the adenine moiety have been developed. In particular, the cyclization of 5’-carboaldehydes using BuSH/(TMS)3SiH as reducing system under standard free radical conditions afforded the desired products with high diastereoselectivity. Kinetic information on the addition of C5’ radical to the base moieties in competition with the hydrogen atom abstraction from n-butanethiol have been obtained in some details. The measurements of rate constants for the hydrogen abstraction from a variety of alkyl- and aryl-substituted thiols by primary alkyl radicals is in progress using the radical clock methodology and RSH/(TMS)3SiH as reducing system.
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