Identification of carbon-centred radicals derived from linalyl hydroperoxide, a strong skin sensitizer: a possible route for protein modifications

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Abstract

Few studies are reported on the formation of reactive carbon-centred radical species from toxic xenobiotics. In this paper the formation of carbon radicals derived from the skin sensitizer linalyl hydroperoxide is described using radical trapping and EPR studies. Radical trapping used TMIO as scavenger agent and light, heat or TPP–Fe3+ as radical inducers. EPR spin trapping was based on the use of the parent alcohol, generating the same allyloxyl radical than the hydroperoxide by photolysis of the corresponding nitrite formed with t-BuONO, also playing the role of the spin trap. It is suggested that the generation of these carbon radical species could play an important role for the binding of the hydroperoxide with skin proteins to form antigenic structures, the first step of the skin sensitization mechanism.

Graphical abstract

The formation of carbon-centred radicals derived from linalyl hydroperoxide is described using radical trapping and EPR studies. These radicals are suspected to play an important role for the binding of the hydroperoxide with skin proteins to form antigenic structures, the first step of the skin sensitization mechanism.

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Introduction

Allergic contact dermatitis (ACD) is a very common disease resulting of epidermal proteins being chemically modified by haptens.1 The processing of such modified proteins by Langerhans cells, the main antigen-presenting cells in the epidermis, generates altered peptides that are subsequently presented, in association with major histocompatibility complex molecules, to naive T-lymphocytes in the lymph nodes. The whole process results in the selection and activation of T-lymphocyte subpopulations with T-cell receptors specific of the chemical modification.1 Haptens are usually a low molecular weight molecules, lipophilic enough to penetrate the epidermis through the Stratum corneum, and with a potent chemical reactivity allowing the formation of a covalent link with protein amino acid side chains. The classical mechanism of antigen formation involved in the ACD pathology is the reaction of an electrophilic function, present on the allergen or hapten, with nucleophilic residues on proteins to form covalent bonds.2, 3 However, over the past few years, the radical mechanism, although it has never been firmly demonstrated, has gained increased interest in the discussion of the mechanism of hapten–protein binding for some specific haptens.4

The high sensitizing potential of allylic hydroperoxides derived from the autoxidation of terpenes is well known. It was already demonstrated, 50 years ago that several hydroperoxides derived from the autoxidation of Δ3-carene were responsible for the allergenic potential of turpentine.5 More recently, Karlberg and co-workers have extensively reported on the allergenic potential of allylic hydroperoxides derived from the autoxidation of d-limonene, used as a fragrance material,6 or from the autoxidation of abietic acid, the main resin acid of colophony.7, 8

Most of the studies reported in the literature on the decomposition of allylic hydroperoxides concern those derived from the oxidation of polyunsaturated fatty acids. By studying the mechanisms of lipid oxidation it has been shown that allylic hydroperoxides, when treated with a variety of radical inducers such as metal complexes, form allyloxyl radicals by easy cleavage of the peroxy bond. These unstable alkoxyl radicals readily lead to the formation of carbon-centred radicals through fragmentation, hydrogen abstraction or intramolecular cyclization that may react with molecular oxygen and then decay to hydroxy derivatives.9, 10 Fragmentation of intermediate dioxetanes, presumably derived from peroxyl radicals formed after cleavage of the peroxy-hydrogen bond, has also been described.11, 12 In the course of our studies on allergenic allylic hydroperoxides we have shown that such kind of carbon-centred radical reactions could be important for the binding of those haptens containing hydroperoxide groups with skin proteins.13, 14 In a previous study, carried out on model molecules potentially derived from the decomposition of linalyl hydroperoxide, we have shown that while hydroperoxide 1 was a very strong sensitizer, potential rearrangement products such as epoxynerol 2, epoxygeraniol 3, furan and pyran derivatives 4 and 5, respectively, were not sensitizing (Fig. 1).15 These results ruled out a possible nucleophilic–electrophilic mechanism involving epoxides as the actual sensitizers. However, the rearrangement of the allylic hydroperoxide is highly probable and, since the rearranged derivatives are not active, it may be supposed that a radical intermediate able to react with skin proteins is formed during the process.

During the last decades, much attention has been denoted to the endogenous or exogenous production of radical reactive oxygen species because of their evident link to the initiation and/or progression of many degenerative and chronic diseases. Research has been focussed on the understanding of their mechanism of action and on the development of compounds with powerful antioxidant activity. However, few studies are reported in the literature on the formation of reactive carbon-centred radical species from toxic xenobiotics that could be responsible for the chemical modification of bioorganic molecules. In this paper we report a complete identification of the carbon-centred radicals derived from the allergen linalyl hydroperoxide 1 using a radical trapping technique with the stable nitroxide 1,1,3,3-tetramethylisoindolin-2-yloxyl (TMIO) and electron paramagnetic resonance (EPR) studies. We suggest that the generation of these radical species could play an important role for the binding of the hydroperoxide with skin proteins to form antigenic structures, the first step of the ACD mechanism.

Section snippets

Radical trapping experiments

The radical trapping technique that we have used relies on the almost diffusion-controlled trapping of carbon-centred radicals by stable nitroxides such as TMIO to form alkoxyamine products (Scheme 1).16 The isoindoline-based aminoxyl TMIO is known to have a variety of advantages over commercially available aminoxyls, including the commonly used pyrrolidine and piperidine species.17 The fused aromatic moiety provides resistance to the ring-opening reactions that are known decomposition pathways

Discussion

The results presented in this paper, coming from a combination of radical trapping and EPR studies, confirmed the formation of reactive carbon-centred radicals from linalyl hydroperoxide 1. The easy cleavage of the hydroperoxide chemical function afforded unstable oxygenated radicals that readily rearranged to form stable carbon radicals. An intramolecular 5-exo cyclization of the alkoxyl radical 10 with the isoprenyl double bond led to the formation of the furan derivative 11 (35–48% yield).

Conclusion

Few studies are reported in the literature on the formation of reactive carbon-centred radical species from toxic xenobiotics. We have demonstrated the formation of carbon radicals from the skin sensitizer linalyl hydroperoxide by using radical trapping and EPR studies. The generation of these carbon radical species could play an important role for the binding of the hydroperoxide with skin proteins to form antigenic structures, the first step of the skin sensitization mechanism. Moreover,

Experimental

Caution: Skin contact with hydroperoxides must be avoided. Since these compounds are skin sensitizing substances, they must be handled with care.

Elemental microanalyses were performed by the microanalytical laboratory of the CRM (Centre de Recherche sur les Macromolécules), Strasbourg, France. IR spectra were recorded on a Perkin–Elmer 1600 Series Fourier Transform spectrophotometer (cm−1 scale) and refer to thin films of liquids (neat). 1H NMR spectra were recorded at 200 MHz on a Bruker AC-200

Acknowledgements

Financial support from the Ministere de l’Education Nationale (France) is gratefully acknowledged for M.B.

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