Ma Chère Elena

Dr Bertrand Vileno

Loris Pr

Pr Brunhilde Grossi

Blömeke

Que Le Dr

Alexandre Specht

Maurice Cope

Bruno Vincent

Marie Betou

Marion Greco

Hassan Srour

Fatma Salhi

François-Marie Moussallieh

Eric Moss

Amélie Godard

Elodie Beroux

etc. Maxime Schlienger

Hervé Dekkiche

Sebastien Jenni

Régis Boehringer

Ryan Djemili

J'aimerais plus particulièrement remercier le jury qui a jugé ce travail de manière objective

Keywords: allergic contact dermatitis, allergen, hapten-protein interaction, radical intermediates, reconstructed human epidermis, electronic paramagnetic resonance, spin trapping « Vous n'y pouvez rien, messieurs, la Science

est et demeure internationale. » Albert Einstein Comment je vois le monde A mes parents, mon frère, mon neveu, mes grands-parents disparues, ma famille, mes amis et à ma femme.

Ce travail de thèse a été réalisé au sein du laboratoire de Dermatochimie de l'université de Strasbourg sous la direction du Dr. Elena Gimenez-Arnau

List of Abbreviations

4.2

Reconstructed human epidermis studies...

Implication des mécanismes de type radicalaire dans les processus de sensibilisation cutanée : compréhension en allant de la molécule au tissu

La peau étant en contact permanent avec l'environnement, elle constitue la première interface de l'organisme vis-à-vis du monde extérieur. C'est par son intermédiaire que se font des échanges avec des xénobiotiques, dont certains sont capables de franchir la couche cornée se retrouvant ainsi dans l'épiderme. L'allergie de contact (ADC) est une réaction d'immunotoxicité cutanée induite par le contact répété de l'individu avec certains de ces xénobiotiques.

Une fois entrés dans l'épiderme et trop petits pour activer le système immunitaire (poids moléculaire inférieur à 1000 Da), ces xénobiotiques allergisants, ou haptènes, doivent réagir avec les protéines de la peau, formant ainsi une entité antigénique qui déclenchera tout le processus immunologique caractérisant l'ADC. Le mécanisme le plus souvent rencontré pour l'interaction haptène-protéine est celui de type nucléophile-électrophile (mécanisme à 2 électrons), mais des mécanismes de type radicalaire (mécanisme à 1 électron) sont également suspectés.

Le sujet de ce travail de thèse a été d'étudier la contribution des réactions radicalaires dans la sensibilisation cutanée aux hydroperoxydes allyliques dérivés de l'autoxydation de terpènes d'origine naturelle, largement utilisés dans la composition de parfums, produits cosmétiques ainsi que dans des produits d'entretien ménagers.

Le travail a été organisé autour de trois axes, comprenant des études de la molécule au tissu :

• in chemico, en étudiant le profil de réactivité chimique d'un hydroperoxyde allylique sensibilisant

• in situ, en étudiant par résonance paramagnétique électronique (RPE) le comportement des espèces radicalaires formées à partir de ces hydroperoxydes sur un modèle 3D d'épiderme humain reconstitué

• in cellulo, en mesurant le stress oxydant induit par les hydroperoxydes allergènes sur l'activation des cellules dendritiques.

Profil de réactivité in chemico au travers de mécanismes radicalaires

Le (5E)-7-hydroperoxy-3,7-diméthylocta-1,5-dièn-3-ol 2, l'un des principaux produits issus de l'autoxydation du linalol 1, est un sensibilisant fort. Il est admis que cet hydroperoxyde réagit avec les protéines cutanées par des mécanismes de type radicalaire.

Une technique utilisée au laboratoire permettant le suivi de la formation d'adduits haptèneprotéine est basée sur l'utilisation de l'haptène contenant un carbone 13 ( 13 C) dans les positions réactives afin de suivre la réactivité vis-à-vis des acides aminés par RMN du 13 C. Plusieurs positions radicalaires potentiellement réactives peuvent se former à partir de 2 (Figure 1). La Les synthèses de l'hydroperoxyde allylique isotopiquement substitué au 13 C dans les positions 4 et 5 ont été réalisées (exemple en Figure 2). Cependant, la synthèse est bloquée en position C, puis réalisées avec l'introduction de 13 C dans les meilleures conditions expérimentales.

Figure 2 : Synthèse de l'hydroperoxyde allylique substitué au 13 C en position 4

Une deuxième partie de ce volet de recherche a consisté en l'étude de la réactivité vis-à-vis des acides aminés par RMN du 13 C.

Les composés synthétisés 4-( 13 C)-LinaOOHs et 5-( 13 C)-LinaOOHs (Figure 3) ont été mis en présence de différents acides aminés et la réactivité suivie par RMN du 13 C 1D , puis on a utilisé le RMN 2D pour essayer de résoudre la structure de l'adduit formé. Les spectres de RMN du 13 C ont illustré la réactivité entre 5-( 13 C)-LinaOOHs ainsi que 4-( 13 C)-LinaOOHs et uniquement avec l'acide aminé ester méthylique de la N-acétyle-L-cystéine avec ou sans initiateur fer (II). Nous avons observé que les pics correspondant au composé substitué au 13 C diminuent avec le temps (sur sept jours) ainsi que la formation de plusieurs nouveaux pics correspondant à des modifications de la cystéine par les hydroperoxydes. Ces modifications correspondent entre autres, à la formation d'adduits dont la structure moléculaire a été estimé grâce aux simulations par ordinateur des déplacements chimiques (Figure 4). Pour conclure, il a été possible de réaliser deux synthèses sur trois de manière reproductible. Il a été aussi observée une réactivité uniquement en présence de l'acide aminé cystéine. Pour finir concernant les études de réactivité, il a été à chaque fois observé que l'isomère de position 6 disparait plus rapidement que le 7, donc est-il plus réactif ou tout simplement moins stable ?

La partie moléculaire in chemico de cette étude étant fini nous allons maintenant voir la partie in situ concernant l'étude dans les épidermes humains reconstitués. L'objectif était d'évaluer le comportement de ces espèces radicalaires dans un modèle plus proche de ce qui peut arriver in vivo.

Investigation in situ par RPE sur un épiderme humain reconstitué

La méthodologie a été développée à l'aide de l'hydroperoxyde de cumène commercial (Cum-OOH) comme composé modèle, référence connue utilisée souvent dans des études de RPE, et des piégeurs de spin tels que le 5-diméthyl-1-pyrroline N-oxyde (DMPO) et le 5diéthoxyphosphoryl-5-méthyl-1-pyrroline N-oxyde (DEPMPO) afin d'avoir une vue large de tous les radicaux générés, oxygénés et aussi carbonés (Figure 6). Une fois la méthode mise en place, les hydroperoxydes dérivés de l'autoxydation du linalol et du limonène ont également été testés. Selon l'environnement, nous avons montré qu'ils génèrent également des radicaux oxygénés et carbonés, cependant il a été montré qu'il faut travailler avec une quantité dix fois supérieure à celle de l'hydroperoxyde de cumène afin d'obtenir un signal. Compte tenu de ce fait et du fait que dans les études en solution aucune différence de réactivité n'avait été observée, on peut émettre l'hypothèse que les hydroperoxydes dérivés du linalol et limonène pénètrent plus en profondeur dans le RHE que le Cum-OOH.

La partie in situ de cette étude étant finie nous allons maintenant voir la partie in cellulo concernant l'étude du stress oxydant.

Mesure du stress oxydant in cellulo

Conclusion générale

Lors de ce travail de thèse, nous avons tout d'abord mis au point la synthèse du 4-( Enfin dans la dernière partie, le potentiel oxydant de ces molécules a été confirmé par l'étude de l'activation de la voie Keap1-Nrf2 à l'aide de cellules de la lignée THP-1.

Pour le moment et en conclusion, l'ensemble de nos études montre que les hydroperoxydes dérivés de l'autoxydation du linalol sont plus réactifs, du point de vue de réactivité chimique et du point de vue d'activation biologique, que ceux dérivés du limonène. Un lien pourrait être donc fait avec la plus grande capacité de ce composé à former plusieurs espèces radicalaires carbonées.

Ces travaux ont ouvert une nouvelle perspective de travail au laboratoire ayant comme objectif principal de faire un lien entre les données de réactivité in chemico et les études de formation de radicaux in situ avec une réponse biologique in cellulo associée à la sensibilisation cutanée, afin de pouvoir mieux évaluer le potentiel sensibilisant de composés réagissant par voie radicalaire.

Allergic contact dermatitis (ACD) is today a common occupational and environmental issue.

Prevalence is rising worldwide, being more important in developed countries. In Europe, 15 to 20 % of the general population is suffering from ACD to at least one chemical allergen. 1 More than one hundred natural or synthetic compounds are today recognized playing a role in ACD as confirmed skin-sensitizers. [2][3][4][5][6] These compounds are found almost everywhere in everyday life, cosmetics, perfumes, textiles, householding products, plants and also in many industrial products, among others. 7,8 Up to now, medical treatments have not been able to heal skin allergy or ACD. Once the person is sensitized, it is for life. Only symptomatic treatments are available to relieve patient's pain. The best way to treat ACD is thus prevention and the eviction of sensitizing compounds.

As an example for prevention, the 7 th Amendment to the European Cosmetics Directive obliges the cosmetics industry to label 26 compounds present in fragrances that are important skin sensitizers. 9 Gell and Coombs classified ACD as a delayed-type hypersensitivity reaction or type IV hypersensitivity. 10 It is a cell-mediated immune reaction that is dependent on the presence of antigen-specific T-cells, where chemistry plays a key role.

ALLERGIC CONTACT DERMATITIS

ACD is the clinical manifestation of an immunotoxicity reaction resulting from skin sensitization to a chemical. It usually manifests with symptoms such as eczema, edema (swelling), erythema (redness), or pruritus (itching). It is induced by skin contact of the individual with a sensitizing substance. After penetration into the epidermis and too small to activate the immune system (molecular weight below 1000 Da), these allergenic substances, also called haptens, must react with skin proteins to form the antigenic immunogenic entity. 11

The skin

The skin is in constant contact with the environment and is the first interface of the human body with the outside. It covers the entire human body and is, by its surface (circa 2 m 2 ), one of the largest organs. It protects us from ultraviolet radiations and shocks while keeping constant the body temperature by retention or elimination of water. It's permeability, constant renewal and elasticity make of it a great natural physical barrier between the organism and external aggressions and xenobiotics. However, the skin is not hermetic and some compounds are able to penetrate into the epidermis and be at the origin of skin sensitization and ACD.

Mammalian skin is composed of three primary layers: the epidermis, the dermis and the hypodermis.

Figure I.1: Anatomy of the skin 12

The epidermis is composing the external layer of the skin. It is a circa 200 micrometers thick nonvascularized tissue. 13 The epidermis is a multistratified keratinized epithelium subdivided into five successively superimposed layers from the surface towards the depth: the stratum corneum, the stratum lucidum, the stratum granulosum, the stratum spinosum and the stratum basal.

Major cells in the epidermis are keratinocytes (approximately 90 % of all cells). Keratinocytes are produced in the stratum basal, then they differentiate through migration from the stratum basal to the stratum corneum.

The stratum corneum, the most superficial layer, consists of a stack of keratinized dead cells, which are eliminated by desquamation, the skin thus renewing itself in a perpetual manner. Other cells composing the epidermis are melanocytes (8 % in the deepest layers), Langerhans cells (3 to 5 % in the deepest layers) and Merkel cells (also in the deepest layers), whose main functions are skin pigmentation, immunity and perception, respectively. [14][15][16] The epidermis and the dermis are separated by the basal lamina, controlling the traffic of cells and molecules between both. It is responsible for the nourishment and waste removal from the dermis and the epidermis. The dermis plays a key role in the thermoregulation of the body and nutrition of the epidermis through the blood vessels. It contains blood and lymph vessels, fibroblasts and an extracellular matrix composed of collagen and elastin fibers that give the skin its strength and elasticity. 13 It is 10 to 40 times thicker than the epidermis.

The hypodermis is the deeper localized part of the skin and also the thicker. Collagen fibers are connecting the hypodermis to the dermis. It is composed of adipocytes, fibroblasts, blood vessels and nerve fibers in a conjunctive tissue. The role of the hypodermis is to stock energy and keep constant the temperature of the body. It stock and release energy trough degradation of fatty acids (lipids), allows cells to obtain energy through b-oxidation of lipids in mitochondria.

ACD mechanism: sensitization and elicitation phases

The sensitization phase

The sensitization phase is the result of a first contact between the allergenic compound and the immune system. At this stage, the immune system is alerted but there are no clinical symptoms.

After penetration of the allergen or hapten into the epidermis, an antigenic complex is formed by its reaction with skin proteins through the formation of stable covalent bonds.

Once the antigenic hapten-protein complex is formed, the immune surveillance system present in the epidermis is able to recognize it as foreign to the organism. At this moment, the antigenic complex is captured by dendritic cells (DCs) playing the role of antigen-presenting cells. They are organized in a cellular network, in a way that any substance penetrating into the epidermis is detected. DCs are the only cells in the epidermis presenting on their surface the major histocompatibility complex of class II (MHC II). The DCs will then indicate the presence of the antigenic complex to the immune system and this way induce proliferation of antigen-specific T lymphocytes. During this phase, there will be production of memory T lymphocytes that will recognize the allergen in question during subsequent exposures.

Two cases are possible for the hapten-protein interaction process. Haptens can either react with extracellular proteins and then DCs take up the antigenic complex by endocytosis, either react with DCs intracellular proteins.

The antigenic complex will then undergo an enzymatic degradation in the cytoplasm. During this process, DCs will maturate and migrate to the local lymph nodes. In the lymph nodes, the peptides containing the chemical modification induced by the hapten will be presented to T cells receptors at the surface of DCs via the MHC II.

Maturation of DCs leads to the production of co-stimulative molecules on their surface such as CD54, CD86 and CD40, playing a key role in the activation of naïve T lymphocytes. 19 As said, once the DCs are in the local lymph nodes they will present to naïve T lymphocytes the haptenized-peptide or antigenic peptide through the MHC II on their surface. Naïve T lymphocytes will then be differentiated in Th1 lymphocytes in the presence of cytokines such as interleukins IL12 and IL18 and also INF-. 20 A clonal expansion of the Th1 lymphocytes occurs, and the newly differentiated cells migrate to the dermis. At this moment, the organism is sensitized to the allergen but is clinically asymptomatic.

The sensitization phase takes 10 to 15 days in humans, leading to the activation and proliferation of antigenic-specific T lymphocytes.

The elicitation phase

The elicitation phase occurs 24 to 48 hours after a second exposure to the same allergen. Initial steps are the same as those happening in the sensitization phase but here Th1 lymphocytes directly recognize the haptenized peptide. Allergen-specific Th1 cells release then cytokines and chemokines as a response to the antigenic complex. This causes an inflammatory reaction and further eczema. It also results in a massive influx of other non-specific T lymphocytes and other inflammatory cells amplifying the reaction even more. The eczematous reaction develops at the site of contact with the allergen.

The allergen

Every day we are exposed to a multitude of chemicals, synthetic or natural. Many of these compounds are able to react with human cells and induce mutagen, carcinogen, toxic and immune system reactions. 21 Allergens such as metallic ions (nickel Ni 2+ , cobalt Co 2+ ) or fragrances are responsible for circa 20 % of ACD developed by the general European population. 1 European regulations are thus controlling exposure to skin allergens for consumers prevention. For example, a European directive from 1994 regulates nickel concentration in jewelry and as a result a decrease of nickel sensitization has been observed in Europe. 22 The risk to develop ACD is also depending on the exposition to skin sensitizers and the way of living. The most concerned occupational sectors are health-care workers, dental workers, hairdressers, construction workers, employees in the food industry, cleaners and painters. 23,24

Chemicals directly reactive with skin proteins: haptens

Haptens are small molecules (molecular weight < 1000 Da) unable to activate themselves the immune system. They need to react with a carrier protein to form an antigenic hapten-protein complex that will then be recognized by the immune system. Landsteiner and Jacobs were the first authors to introduce the hapten concept. 25 Metals react with proteins via the formation of coordinate bonds (Figure I.4).

Figure I.4: Coordinate bond examples for nickel, cobalt and chromium

However, all other haptens react with skin proteins through the formation of stable covalent bonds.

The majority of sensitizing molecules contain an electrophilic chemical function in their molecular structure that is able to react with nucleophilic groups in proteins via two electrons covalent bond formation. 26 In the literature, several electrophilic chemical groups reacting with nucleophilic functions of proteins are described (Table I.1). Amino acids cysteine and lysine are the most described in the literature but also other amino acids having electron-rich heteroatoms, such as histidine, can form covalent bonds with electrophilic haptens. 27

Chemicals needing an activation to become reactive: pre-and pro-haptens

The terms pre-and pro-hapten (Figure I.5) describe a category of molecules initially non-reactive, that become sensitizing after a chemical transformation.

Hapten

Hapten conversion of products generated by phase I enzymes in order to be eliminated (sulfotransferases, acetyltransferases, glutathione-S-transferases, etc.). 28 During these detoxication processes, reactive species can be formed and induce skin sensitization (Figure I.7).

Figure I.7: Eugenol biotic activation

This Ph.D. thesis will focus on pre-haptens and more specifically in terpenes that easily autoxidize to form sensitizing allylic hydroperoxides. Terpenes are natural products used by plants as odorants to communicate with their environment but also as growth regulators. Terpenes are widespread in plants and largely used in perfumery because of their great smell.

The general chemical formula of terpenes is (C 5 H 8 )n as they derive from units of isoprene.

Dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP) are the components in the biosynthesis pathway of terpenes. When n (number of linked isoprene units) is equal to 2 the molecule is called monoterpene (i.e. geraniol, limonene, linalool), when n = 3 sesquiterpene (i.e. farnesol), when n = 4 diterpene (i.e. cafestol), etc.

PREDICTION OF THE SENSITIZING POTENTIAL

Historically, the sensitizing and allergenic potential of chemicals were evaluated by testing on animals. In 1959 Russell and Burch enunciated the deontological concept of the 3 R (replace, reduce, refine). 31 The idea was to "replace" animal testing by in vitro assays, maximally "reduce" the number of animals used and "refine" the techniques used in order to reduce animal suffering.

However, the social pressure and several European directives have ended these practices especially in the cosmetics and fragrances context.

European directives and legislation are today asking the industry to do risk assessment concerning chemicals in order to protect human health and the environment. The REACH legislation (Registration, Evaluation, Authorization, and Restriction of Chemical Substances) of the European Parliament and of the council from the 1 st of June 2007 should allow collecting information on more than 30 000 compounds over eleven years. Moreover, REACH encourages the industry to use animal alternative assays to do toxicity assessment. 32 In 2013, animal testing for the evaluation of the toxicity of cosmetic ingredients was forbidden, as indicated in the 7 th amendment to the European Directive on cosmetic products. 9 Today and as consequence, several animal testing alternative methods have been developed for the evaluation of the sensitizing potential of chemicals and have been approved by the European Center for Validation of Alternative Methods (ECVAM), together with the establishment of OECD guidelines. These methods are based on different physiological steps leading to skin sensitization.

In vivo methods

First tests on allergens were done on human volunteers in 1936 by Landsteiner and Jacobs, they were followed by animal tests such as the guinea pig maximization test (GPMT), the mouse ear swelling test (MEST) and the mouse local lymph node assay (LLNA) developed by Kimber and

Basketter in the 1990's. [33][34][35] The LLNA, based on the sensitization phase of ACD, is today forbidden in the field of cosmetics even if it uses a decreased number of animals. As specified in the OECD guideline, this assay measures the lymphocytes proliferation after exposition to the chemical. 36 The chemical to test is dispensed on to the dorsum of the mouse ear once a day for five days. On the fifth-day, tritiated thymidine is injected into the animal. The animal is then sacrificed, and proximal lymph nodes of the mouse ear are excised. The proliferation is based on the measure of the radioactivity level. If the rate is three times higher compared to that of a not exposed control mouse, the molecule is considered as being a sensitizer. The concentration used to have a threefold factor proliferation of lymphocytes (EC 3 ) allows quantifying the sensitizing potential of the chemical tested and classify it as low, moderate or strong sensitizer. 37 That two softwares continue to be updated by new data.

In silico methods

One of the oldest QSARs is the RAI assay (Relative Alkylation Index), developed in the 1990's.

The RAI is essentially based on electrophilic chemicals, their hydrophobic properties and the concentrations of the molecules tested. 41 As a QSAR method, it is based on chemical structures being linked to a biological response, in this case skin sensitization. It is assumed that the chemical structure of a molecule (chemical functions, geometric and electronic properties, etc.) is responsible for its chemical, physical and biological properties. QSARs give information on the toxicity and also on the intensity of the biological response and are based on a mathematical model activity being function of physicochemical properties. Most of them are recommended by the OECD and even a toolbox has been developed as software providing an approach to predict skin sensitization. All these softwares, based on the reaction mechanisms for the hapten-protein interaction, are very valuable but may be limited by contradictory results coming from in vivo methods such as the LLNA. 42

In chemico methods

As described previously, the first key step in the sensitization process to a chemical is its ability to covalently bind to skin proteins in order to form the antigenic complex. Based on this, it might be supposed that more a molecule is able to react with amino acids in proteins the more this molecule has a sensitizing potential. This was the basis for Gerberick et al. to develop the Direct Peptide Reactivity Assay (DPRA) in 2004, an hapten-peptide based reactivity assay. 43 The test compound is incubated for 24 hours with a peptide, then the depletion of the peptide is measured using highperformance liquid chromatography (HPLC) coupled to a UV detection. At the beginning and to develop the assay, glutathione and three synthetic peptides were used. The main sequence is N-Ac-Arg-Phe-Ala-Ala-X-Ala-Ala, X being cysteine, histidine or lysine. The final assay was developed with cysteine and lysine peptides as they gave the more accurate and reproducible results . A decisional tree was then built allowing the classification of allergens according to their reactivity The study of 82 molecules led to the identification of 89 % of them as allergens with a good correlation between their sensitizing potential and their reactivity against peptides Cys-Pep and Lys-Pep. The DPRA was validated by ECVAM in 2012 and has an OECD guideline. 45 A major drawback of DPRA was that it only could be applied to directly reactive chemicals or haptens, not considering the case of pre-and pro-haptens. To solve this issue, Gerberick et al. developed a second-generation test, the Peroxidase Peptide Reactivity Assay (PPRA) in which an oxidation step was added to the system by using the tandem horseradish peroxidase/hydrogen peroxide. 46 Other in chemico methods exist based on mass spectroscopy or the use of glutathione, but DPRA is today the only validated method representing the first key step of the Adverse Outcome Pathway (AOP) defined for skin sensitization, to say the hapten covalent modification of epidermal proteins. 47

In vitro methods

In vitro validated methods are based on the key steps in the AOP described for skin sensitization considering activation of epidermal keratinocytes and DCs.

The KeratinoSens ® assay is based on genetically modified human cells. 48 Keratinocytes are able to synthesize the enzyme luciferase in presence of sensitizers, as described in its OECD guideline. 49 This enzyme is fluorescent when the substrate luciferin is present. It is based on the human keratinocytes cell line HaCaT, transfected in a stable manner by a gene coding for luciferase. After 48 hours of incubation with the test compound, luciferase substrate is added and luminescence is measured after 20 minutes. Luciferase induction is then correlated to the concentration needed to induce 1.5 induction. An induction rate (EC 1.5) higher to the threshold of 1.5 of luciferase activity (corresponding to a 50 % increase of the gene activity) is considered as predicting a significant positive answer.

The MUSST (Myeloid U937 Skin Sensitization Test) and the h-CLAT (human Cell Line Activation Test) are two methods based on here activation of DCs. 50 In these assays, the cell surface protein expression is measured when exposed to a sensitizer. Indeed, the cell surface proteins CD54 and CD86 are overexpressed when cells are exposed to an allergen. In both tests, after 24 hours in h-CLAT and 48 hours in MUSST, of exposition of cells to the allergen, CD54 and CD86 are overexpressed and measured by flow cytometry. The difference for both tests is the cell type used.

h-CLAT uses the THP-1 cell line, a human monocytic cell-line, and MUSST uses myeloid cells type 937. 51,52 The results show a correlation between the sensitization potential and the overexpression of cell surface proteins.

PRESENTATION OF THE SUBJECT

Hapten-protein interaction: radical mechanisms

As already discussed above, ACD is an adverse reaction of the immune system towards lowmolecular-weight chemicals also called haptens. It is a delayed type hypersensitivity reaction dealing with T-cells. 17 Chemical allergen molecules, too small to be recognized by the immune system, first react with skin proteins through the formation of stable covalent bonds. The so formed antigenic complex will then be recognized by the immune system which will lead to the delayed type hypersensitivity process. Most of the time the hapten-protein interaction occurs via the reaction of an electrophilic chemical function present in the allergen and nucleophilic chemical functions present in the lateral chain of some amino acids via a two-electrons mechanism (Figure I.9). However, some allergenic substances do not fit in this reaction pattern. For these, one electron radical mechanisms are suspected. Terpenes such as limonene and linalool form, after air exposure, limonene hydroperoxides and linalool hydroperoxides as primary oxidation products (Figure I.10). 53,54 These autoxidation products have been described as strong sensitizing compounds. 55 For risk assessment commitments, studies helping to understand all mechanisms leading to allergic reactions by chemical sensitizers are essential in order to cover all categories of molecules.

This Ph.D. work focuses on the identification of free radicals derived from skin sensitizing substances and in the study of their involvement in ACD, from the molecule to the tissue.

Target compounds: allylic hydroperoxides derived from autoxidation of limonene and linalool

Many natural terpenes are used in fine perfumes, cosmetics, household and industrial products due to their odorant properties. [START_REF] Andersch Björkman | Air-Oxidized Linalool Elicits Eczema in Allergic Patients -a Repeated Open Application Test Study: Roat of Oxydized Linalool[END_REF][START_REF] Bråred Christensson | Air-Oxidized Linalool-a Frequent Cause of Fragrance Contact Allergy[END_REF][START_REF] Bråred Christensson | An International Multicentre Study on the Allergenic Activity of Air-Oxidized R -Limonene[END_REF][START_REF] Bråred Christensson | Positive Patch Test Reactions to Oxidized Limonene: Exposure and Relevance: Exposure and allergy to oxidized limonene[END_REF] Target compounds for this study were Limo and Lina hydroperoxides (

Project structure

Radical mechanisms have been poorly considered in the skin allergy field, and therefore little studied to be firmly demonstrated. This project aimed at understanding alternative haptenation processes in skin sensitization involving radical mechanisms. In parallel, skin sensitizers derived from oxidation processes, enzymatic or not, are often potent oxidants able to interfere with the cellular redox balance. There is a real belief that oxidative stress induced by these compounds on epithelial cells may produce a new danger signal for the immune system, synergistic with reactivity, and thus would greatly help to explain the strong allergenic activity. This PhD work was thus based on studies helping to understand radical mechanisms leading to epidermal protein modifications by allylic hydroperoxides and cellular redox homeostasis perturbation because of the inherent oxidative potential related to the chemical structure.

The work has been built on the basis of three complementary approaches: in chemico by conducting reactivity studies, in situ by studying the behavior in reconstructed human epidermis tissue model, and in cellulo by evaluating the response from DCs to the danger signals produced.

Objectives and deliverables of the project were:

1) to understand, from a chemical reactivity profile level, the contribution of radical mechanisms on the formation of antigenic complexes;

2) to examine the in situ production of these danger signals (radical modifications of amino acids, stress induction) on a reconstructed human epidermis (RHE) model;

3) to investigate in cellulo the influence of oxidative stress on the allergenic potential of the chemicals.

For fulfilling the objectives, the work has been built around three major chapters.

Chemical reactivity profile through radical mechanisms

For many years it has been demonstrated in the laboratory the formation of oxygen alkoxyl RO • / peroxyl ROO • and carbon-centered R • free radicals derived from several allylic hydroperoxides, by using chemical trapping and electron paramagnetic resonance spectroscopy (EPR) associated to spin-trapping techniques. [START_REF] Kao | Synthesis of Allylic Hydroperoxides and EPR Spin-Trapping Studies on the Formation of Radicals in Iron Systems as Potential Initiators of the Sensitizing Pathway[END_REF][START_REF] Giménez-Arnau | Identification of Radical Species Derived from Allergenic 15-Hydroperoxyabietic Acid and Insights into the Behaviour of Cyclic Tertiary Allylic Hydroperoxides in Fe(II)/Fe(III) Systems[END_REF][66][67] The generation of these radicals could be thus possible in vivo, in the epidermis, once molecules have penetrated into the skin. This is substantial information indicating that if a certain number of reactive radical intermediates can issue from these compounds, different potentially immunogenic chemical modifications are possible on proteins leading to sensitization. Preliminary studies carried out on their reactivity toward amino acids, followed by NMR and LC-MS, corroborated the formation of adducts originating from radical reactions, together with redox processes producing chemical modifications of the amino acids. 68 However, elucidation of the exact chemical structure of reaction products was difficult, isolation complex, NMR identification not sensitive enough and MS did not discriminate adducts with the same m/z value formed at different radical positions. The use in the laboratory of NMR techniques with 13 C-substituted molecules has been shown to be a very effective tool for the investigation of hapten-protein interaction mechanisms. 69 In here, it was decided to use 13 C-substituted compounds to elucidate the exact molecular structure of hapten-amino acid adducts involving radical mechanisms. Lina-7-OOH, one of the culprits for the allergenic potential of autoxidized linalool was chosen as target compound. Indeed Kao et al. showed previously that Lina-7-OOH decomposes in the presence of ferrous salts affording carbon R • free radicals at different positions (Figure I.16). [START_REF] Kao | Synthesis of Allylic Hydroperoxides and EPR Spin-Trapping Studies on the Formation of Radicals in Iron Systems as Potential Initiators of the Sensitizing Pathway[END_REF] It was thus decided to synthesize Lina-7-OOH 13 C-substituted at carbon positions that are precursors of these potentially reactive radicals.

In situ investigation of danger signals production in reconstructed human epidermis (RHE)

In collaboration with Dr. The major aim was the detection and characterization of free radical spin adducts testimony of radicals derived from R-OOHs in a human skin model, and to probe their behavior and decay.

Measure of oxidative stress in cellulo

In

Chapter 1

Chemical reactivity profile through radical mechanisms:

Synthesis of target compounds and reactivity studies

In this chapter the synthetic pathways developed for the obtention of target compounds 5-( 13 C)-Lina-OOHs, 4-( 13 C)-Lina-OOHs and 2-( 13 C)-Lina-OOHs will firstly be exposed. In the second part of this chapter results on the follow up of their reactivity with different amino acids by 13 C-NMR will be described.

SYNTHESIS OF ALLYLIC HYDROPEROXIDES IN THE LITERATURE

Due to the instability and intrinsic reactivity of the O-O bond hydroperoxides can decompose through highly exothermic processes. 1 Those of low molecular weight can even be explosive.

In fact, the percentage of the total mass formed by the peroxidic oxygen atoms present in the skeleton of hydroperoxides is an indicator of the lability degree of these molecules. Thus, with 33% active oxygen methyl hydroperoxide is known to be a detonating substance, while with 11% active oxygen octyl hydroperoxide is easily purified on a small scale. The high propensity of hydroperoxides to decompose has resulted in the collective opinion that they are far too unstable to be of interest as synthetic intermediates. Hydroperoxides are mostly used as oxidizing reagents. In the case of allylic hydroperoxides, they are used as precursors for the formation of allylic alcohols, which are key intermediates that can be subjected to a wide variety of synthetic transformations.

In the sections below, the processes traditionally used for the synthesis of allylic hydroperoxides will briefly be described. A special focus will be given to the autoxidation of olefins and to their reactions with singlet oxygen.

Basic concepts: autoxidation, triplet and singlet oxygen

Atmospheric oxygen is an oxidizing agent easily reacting with a lot of organic molecules at room temperature. This process is called autoxidation. It has a crucial importance in fatty acid oxidation in the food industry.

The main autoxidation mechanism is a radical chain reaction leading to hydroperoxides as primary oxidation products. It is divided into three different steps: initiation, propagation and termination (Figure 1.1).

The initiation reaction occurs because of heat, light or a catalytic amount of metal, and organic compound radicals R • are generated by abstraction of an atom of hydrogen of the substrate RH.

Then propagation reactions occur, R • can react with atmospheric oxygen to form peroxyl radicals ROO • , that can then abstract a hydrogen atom from RH to form a hydroperoxide ROOH and also a new R • . The termination reaction occurs when two radicals react together to form a non-radical species. The most commonly used method to generate singlet oxygen 1 O 2 is by photosensitization of 3 O 2 in the presence of photosensitizers such as porphyrinic, phthalocyanine pigments, methylene blue or Bengal rose (Figure 1.3), also used in phototherapy in oncology. Initially, the sensitizer absorbs a photon and at the excited state transfers energy to 3 O 2 that becomes 1 O 2 , the sensitizer recovering its initial electronic configuration. 2

Autoxidation on unsaturated molecules

The autoxidation of unsaturated compounds to obtain allylic hydroperoxides is considered of low value as a synthetic tool. It leads very often to the formation of a multitude of oxidized products. In a study on the oxidative degradation of linalool in contact with air for example, in addition to various allylic alcohols, furan and pyran derivatives, several allylic hydroperoxides could be identified. 3

Schenck reaction: ene-reaction with singlet oxygen

In previous studies in the laboratory a way was found to synthesize a proper mixture of hydroperoxides 2 and 3 by employing the so-called Schenck or ene-reaction with singlet oxygen. 4 This reaction, happening between compounds containing hydrogen atoms on allylic positions and singlet oxygen was discovered by Schenck in 1940 and revised by Foot in the 60's. 5,6 Figure bond is a secondary carbon atom. In consequence, the steric hindrance plays a key role. 7 The more the R substituent is a bulky group (Figure 1.8), higher is the percentage of the major product. It is important to stress that the Schenck reaction on linalool 1 produces a different distribution of oxidation products as the one expected. According to the theory above described, the enereaction on linalool 1 using singlet oxygen 1 O 2 should afford Lina-7-OOH 2 as the major product. The steric hindrance of the R substituent at position 4 should promote formation of Lina-7-OOH 2. However, it appears that steric hindrance is minimized in this position due to the free rotation around the C 3 -C 4 bond (Figure 1.10). Thus, the isomer Lina-6-OOH 3 was formed as the major product with a ratio 2:3 for compounds 2 and 3.

On thin layer chromatography these two isomers have the same retention factor, as well as on gel column chromatography on silica or on alumina. Therefore, it is very difficult to isolate 2 and 3 from the obtained Schenck reaction mixture.

Nucleophilic substitution on allylic alcohols

Two different synthetic pathways can in general be used to afford allylic hydroperoxides from primary, secondary or tertiary alcohols.

In the first one, the hydroxyl function is transformed into a good leaving group followed by a nucleophilic substitution with hydrogen peroxide (Figure 1.11). Primary and secondary hydroperoxides are thus mostly prepared via an S N 2 nucleophilic substitution.

In the second one, tertiary hydroperoxides are prepared via a S N 1 nucleophilic substitution using hydrogen peroxide in acidic media, favoring a stable carbocation formation.

SYNTHESIS OF TARGET COMPOUNDS

Introduction of a 13 C-substitution on linalool

The enthalpy of dissociation of the O-O bond of the hydroperoxide chemical function is of a relatively weak order of 175.8 kJ/mol compared to the one of the C-C bond that is around 347-356 kJ/mol. It is therefore generally accepted that the cleavage of O-O bonds is an easy process leading to the formation of alkoxyl radicals. In the case of linalool 1 and due to the presence of double bonds, these radical intermediates are able to further evolve towards the formation of carbon-centered radicals, for example by abstraction of allylic hydrogen atoms, by intramolecular cyclization processes or by β-scission (Figure 1.12). Each of these oxygen-and carbon-centered radical intermediates could be reactive towards the side chains of amino acids having a propensity to participate in radical mechanisms.

Figure 1.12: Lina-7-OOH oxygen and carbon-centered radical intermediates

From these radical rearrangement processes, it can be seen that positions 2, 4 and 5 of Lina-7-OOH are precursors of carbon-centered radicals.

Preliminary reactivity studies in the laboratory between Lina-7-OOH 2 and several amino acids, followed by liquid chromatography combined to mass spectroscopy, corroborated the formation of stable covalent bonds between carbon-centered radicals derived from Lina-7-OOH 2 and the side chain of cysteine, for example. 10 However, it was not possible to exactly elucidate which was the carbon radical position reacting as different hypothetical adducts would have the same observed m/z (Figure 1.13). In this chapter, we will first present the synthetic routes that have been designed to obtain linalool 1 13 C-substituted at position 2, 4 and 5 (Figure 1.14). The Schenck reaction will then be applied to each of them to obtain the mixture of hydroperoxides Lina-7-OOH and Lina-6-OOH ( 13 C)-substituted. In a second part, the reactivity of these compounds towards some tested amino acids will be described. All synthetic pathways were initially developed to set the optimal experimental conditions without 13 C-substitution. Finally, obtained linalool needed to be converted, for all positions, into the hydroperoxides responsible for skin sensitization and at the origin of radical reactions.

Synthesis of 5-( 13 C)-Lina-OOHs

Initially, 13 C-4 was synthesized from 1-( 13 C)-acetic acid, itself used for the introduction of the ). 12 Further reduction of the ester to the allylic alcohol 13 C-7 was carried out by using di-isobutyl aluminum hydride (DIBAL-H) (Figure 1.17).

The product 13 C-7 was used without further purification for the next step, which was a nucleophilic substitution of the alcohol by bromine in the presence of hydrobromic acid leading to 1-( 13 C)-1-bromo-3-methyl but-2-ene 13 C-8, with a yield of 52 %.

Further, deprotonation at the  position of the carbonyl group of ethyl acetoacetate by sodium ethoxide in ethanol and the addition of the anion formed on 13 C-8 afforded ethyl-3-( 13 C)-2acetyl-5-methylhex-4-enoate 13 C-9, with a yield of 67 %. This last reaction needed to be carried out protected from daylight in order to avoid addition of hydrobromic acid on the double bond of 13 C-8.

Figure 1.17: Synthesis of 13 C-9

In order to obtain the ketone intermediate 13 C-10, β-keto ester 13 C-9 was saponified by reaction with sodium hydroxide in order to obtain the corresponding carboxylic acid, that was further decarboxylated in presence of hydrochloric acid. The obtained product was used without further purification for the synthesis of 5-( 13 C)-linalool.

The reaction of non-purified 13 C-10 with vinyl magnesium bromide afforded linalool 13 Csubstituted at position 5 with a yield of 63% (Figure 1.18). Finally, the mixture of isomers 13 C-12 and 13 C-13 was obtained with a yield of 99 % when applying the Schenck ene-reaction to 5-( 13 C)-linalool in which singlet oxygen 1 O 2 was chemically produced by disproportionation of hydrogen peroxide, catalyzed by a sodium molybdate system in a microemulsion.

In summary the final synthetic pathway developed to obtain 5-( 13 C)-Lina-OOHs 13 C-12 and 13 C-13, is a nine-steps synthesis with a global yield of 3.8 %.

Synthesis of 4-( 13 C)-Lina-OOHs

The first step was a classical esterification of 2-( 13 C)-acetic acid in acidic medium to obtain 2- Initially, the synthesis of the next intermediate was tried using MacPhee and Dubois experimental conditions using isopropyl magnesium chloride without success. 13 We then used the experimental conditions described by Debeuckelaere et al. 14 For the synthesis of 2-( 13 C)-ethyl-5-methyl-4-hexenoate 13 C-15, the lithium enolate of 13 C-14 was initially prepared in the presence of diisopropylamine and n-butyllithium and then reacted on 1-bromo-3-methyl-but-2-ene (Figure 1. 19). The non-nucleophilic lithium diisopropylamide formed behaves in solution as a strong base (pKa = 36) and was ideal to deprotonate on the carbon atom of the carbonyl chemical function. The role of hexamethylphosphoramide was to render the lithium enolate more nucleophilic by complexing the lithium ions. This step was carried out with a yield of 52 %.

To continue the synthesis, it was necessary to obtain the ketone 3-( 13 C)-6-methyl-5-hepten-2one from 13 C-15. For this reason, it was decided to revert the electrophilicity of the carbonyl chemical function of 13 C-15 by converting it into a Weinreb amide (Figure 1.19). The method described by Nahm and Weinreb using N-methoxy-N-methyl amide as an acylating agent was here used. 15 This method is described as being very useful for the formation of ketones or aldehydes and increasingly used in total synthesis processes on an industrial scale. We first used the methodology described by Liu et al. using isopropyl magnesium chloride as an activating agent before adding ester 13 C-15, but yields were very low. 16 In a second time, we used conditions described by Guanti and Riva using trimethylaluminum as a coupling agent. 17 Also, the conditions using dimethyl aluminum chloride reported by Shimizu et al. were tested (Table 1.1). 18 Using isopropyl magnesium chloride as activating agent yields were never higher than 40 %, even when varying the temperature and reaction times. Using dimethyl aluminum chloride a better yield of 70 % was obtained by varying the time of the reaction from 30 minutes to three hours. The best yield of the Weinreb amide 13 C-16 83% was however obtained when using trimethylaluminum as activating agent for 5 hours at 0 °C.

The following addition of methyllithium to this particular amide made possible to synthesize the desired ketone In order to obtain 4-( 13 C)-Lina-OOHs 13 C-19 and 13 C-20 from linalool 13 C-18, a Schenck enereaction was used with the same experimental conditions as previously described for the synthesis of 5-( 13 C)-Lina-OOHs.

Synthesis of 2-( 13 C)-Lina-OOHs

This synthesis was initially based on the use of potassium The silane bound to the iodide will be more positively polarized becoming more electrophilic and the nucleophilic cyanide ions reaction will, therefore, be favored. In addition, PEG400

increases the solubility of salts such as potassium cyanide in a consistent manner promoting the reaction. Cao's work suggested to carry out the reaction in acetonitrile to improve its kinetics, in comparison with the reaction rates when carried out in dichloromethane. Based on these studies we have tested the reaction in both solvents, at room temperature for 3 hours, and using either potassium cyanide or sodium cyanide. Unfortunately, we have not been able to reproduce this reaction to obtain trimethylsilyl cyanide. We thought to compare the influence of the purity of the two different potassium cyanides used (97% and 98%) in the experiments on the one hand and to play on the kinetics and reaction temperature on the other hand. Only dichloromethane was used as solvent. The use of acetonitrile was indeed compromised as during the distillation procedure to isolate trimethylsilyl cyanide there was a high risk of formation of an azeotrope between acetonitrile (bp = 82 ° C) and the desired trimethylsilyl cyanide (bp = 112 ° C), thereby compromising purification. But again, assays were still unsuccessful.

Another attempt was then carried out by using trimethylsilyl chloride, potassium cyanide, potassium iodide and N-methyl pyrrolidone, upon the methodology reported in the literature by

Reetz and Chatzhosifidis and Rasmussen and Heilmann. [21][22] Potassium iodide acts now as the catalyst, the iodine ions being good nucleophiles and also good leaving groups. Due to the high boiling point of N-methyl pyrrolidone, it was possible this time to easily isolate the desired trimethylsilyl cyanide by simple distillation at atmospheric pressure.

Initially, potassium cyanide known as "European Pharmacopoeia" (Ph. Eur.) 97% purity was used, without success (Table 1.2). In a second time, with potassium cyanide labeled "BioUltra" 98% purity we were able to obtain the expected product with a yield of 38 %. It turned out that comparing both potassium cyanides, it was noted that traces of metals (Fe, Na, Pb) and other elements (Cl, SO 4 , S) present in potassium cyanide 97% were 100-fold higher than those found in potassium cyanide 98%. These could be the cause of side reactions and therefore disrupt the effectiveness of the reaction. Indeed, carrying out other assays in such a way to compare the reactivity of the two reagents, it was concluded that pure potassium cyanide 98% was most effective. Table 1.2 resumes the different conditions tested. The best experimental conditions were found to be 24 hours at room temperature, with a yield of obtained trimethylsilyl cyanide of 82 %. We have also tested an alternative approach replacing potassium cyanide and potassium iodide by sodium cyanide and sodium iodide. However, very low yields of trimethylsilyl cyanide were obtained. It was observed that the solution passed from light yellow to brown-black. It could be assumed that iodine ions had oxidized to iodide instead of catalyzing the reaction since only 23 % of product was obtained after 24 hours reflux. However, at room temperature after 3 days of reaction, a better yield of trimethylsilyl cyanide of 64% was obtained.

In conclusion, the reaction at room temperature for 24 hours of potassium cyanide (98 %) on trimethylsilyl chloride in N-methyl pyrrolidone and in the presence of potassium iodide were the best conditions for the synthesis of trimethylsilyl cyanide with a yield of 82%. With the use of the 13 C-substituted reagent trimethylsilyl ( 13 C)-cyanide 13 C-21 was also obtained with a yield of 82 %. On the other hand, Evans et al. reported the same type of reaction by refraining from using solvent. 24 The presence of simple iodine allowed the cyano group of trimethylsilyl cyanide to be detached from the silane and to attack the carbonyl chemical function of the ketone. The oxygen anion formed could then capture the trimethylsilyl and thus became protected in the form of a trimethyl silane ether. We also found ourselves that the use of a solvent was not in favor of cyanosilylation, perhaps because of too much dilution of the reagents, even if the yields with solvent remain acceptable. In table 1.3 all experimental conditions tested are resumed. Finally, by following the reaction by thin layer chromatography, the reaction was observed to be very rapid and practically quantitative after only 30 minutes and without solvent.

The nitrile chemical function of 13 C-22 was then reduced to afford aldehyde 13 C-23 by using di-isobutyl aluminum hydride (Figure 1.24). of the order of 90%. [25][26][27] We have therefore tried this reduction step in the two solvents, following the described experimental conditions described in Table 1.4. It appeared that the reaction was as difficult in tetrahydrofuran as in diethyl ether. Indeed, after 3 hours at 0°C and regardless of the solvent used the yields were very low. The reactions were nevertheless stopped in order to attempt a purification of the product on silica gel column chromatography, but the difficulties encountered to find a suitable eluent to separate 13 C-23 from the crude made the purification much more complicated than expected. We next increased the amount of DIBAL-H to 3 equivalents. After 3 hours of reaction, the analysis revealed the absence of 13 C-22 in the reaction medium. Unfortunately, after purification on silica gel column chromatography, very low yields were obtained of the aldehyde. Another solution could be to increase the reaction time ensuring complete conversion of the nitrile to the aldehyde. But even after 24 hours with 2.5 equivalents of DIBAL-H, did not totally reduce 13 C-22. The synthetic pathway containing the 13 C-substitution for the synthesis of 2-( 13 C)-Lina-OOHs seemed to reach in here an impasse. Next steps were therefore only conducted without 13 C-substitution.

Binns et al. reported Wittig reaction conditions using methylentriphenylphosphorane to convert an aldehyde chemical function into an alkene. 28 These experimental conditions were initially employed to prepare linalool 24 from non 13 C-substituted aldehyde 23. was obtained with a yield of only 42%. The presence of a by-product was observed by thin layer chromatography, but it could not be identified.

Thus, for the envisaged synthesis for the obtention of linalool 13 C-substituted in position 2, the three first steps have been successfully realized with a 13 C-substitution, whereas the last two steps are still under optimization.

Conclusion and perspectives

The synthesis of 5-( 13 C)-Lina-OOHs, 13 C-12 and 13 C-13, was carried out with an overall reproducible yield of 3.8 %. This was a relatively long synthetic pathway consisting of nine steps. Moreover, the synthon used for the introduction of the 13 C isotopic substitution was used from the first step which is a drawback. However, the obtained amount of the mixture 13 C-12 -13 C-13 was enough in order to study the reactivity with amino acids by 13 C-NMR.

The synthesis of 4-( 13 C)-Lina-OOHs, 13 C-19 and 13 C-20, was carried out with an overall reproducible yield of 9.5 %. This was an advantageous synthetic route as it was shorter with six steps even if the 13 C isotopic substitution was also introduced in the first step.

The synthesis of 2-( 13 C)-Lina-OOHs could not be accomplished. The problem of the reduction of nitrile 13 C-22 to aldehyde 13 C-23 could not be resolved.

Further reactivity studies were thus only conducted with 5-( 13 C)-Lina-OOHs and 4-( 13 C)-Lina-OOHs.

REACTIVITY STUDIES TOWARDS AMINO ACIDS

In order to study radical-type processes involved in the haptenization of cutaneous proteins by allergenic allylic hydroperoxides, we were interested in the reactivity of 13 C-substituted Lina-OOHs towards various amino acids. Reactions were followed by one dimensional 13 C-NMR.

The analysis of chemical structures of products resulting from the reactivity studies was carried out by two-dimensional 1 H- 13 C heteronuclear NMR experiments HSQC and HMBC.

Several amino acids susceptible to conjugate with radical intermediates were selected. Amino acids having a sulfur atom have been demonstrated to be particularly labile and to be involved in radical reactions. Thus, cysteine was used as a potential spin trap for radicals derived from Also, aromatic amino acids are involved in mono electron reaction mechanisms as shown by

Grossi using peroxynitrite ONOO -on tyrosine in a DEPMPO spin trap environment (Figure 1.27). 30 We also used tryptophan and lysine as they contain lateral chains prompt to radical reactions. 31 methyl-1-pyrroline-N-oxide (DEPMPO) 30

Experimental conditions

All experiments were performed using N-acetylated and esterified amino acids to mimic their chemical structure in a peptide sequence and to let free the potentially reactive side chain. In addition, the protection of the amino group represents a way of preventing the formation of Schiff bases with the carbonyl group of any allylic hydroperoxide degradation product.

Results and discussion

Reactivity profile using N-acetyl-L-cysteine methyl ester

The reaction of 5-( 13 C)-Lina-OOHs (mixture 13 C-12/ 13 C-13, 2/3), (1 eq., 5.37 µM) in CD 3 CN/H 2 O 1/1 with N-acetyl-L-cysteine methyl ester (2 and 10 eq., 10.74 and 53.70 µM) was followed by one dimensional 13 C-NMR at room temperature during one week. The catalytic amount of iron (II) sulfate heptahydrate (0.1 eq., 0. On an other hand, carbon at 31.04 ppm () was coupled to protons at 2.06 ppm characteristic of a mehylene CH 2 carbon atom for compound 28. Also, carbon  saw by HMBC protons at 1.62 ppm () and 6.07 ppm (). The  shift was probably hidden by the pressaturation of the water sift, the band from 4.15 to 4.30 ppm. As seen in the part using 5-( 13 C)-Lina-OOH two compounds can correspond, here by using 4-( 13 C)-Lina-OOH it wasn't possible to gave more precision in. The difficulty here is that the 4-( 13 C)-Lina-7-OOH didn't completely react and also the new compound appearing with pracitacly the same shift as the starting material (Figure 1.31). Unfortunately, this second step using 4-( 13 C)-Lina-6-OOH doesn't entirely confirm the results obtained using 5-( 13 C)-Lina-6-OOH.

Reactivity profile using N-acetyl-L-tyrosine methyl ester

The reaction of 5-( 13 C)-Lina-OOHs (mixture 13 C-12/ 13 C-13, 2/3), (1 eq., 5.37 µM) in CD 3 CN/H 2 O 1/1 with N-acetyl-L-tyrosine methyl ester (2 eq., 10.74 µM) was followed by one dimensional 13 C-NMR at room temperature during one week. The catalytic amount of iron (II) sulfate heptahydrate (0.1 eq., 0.5 µM) was sufficient to induce radical initiation (Figure 1.33).

Control experiments were realized without the presence of iron (II) as initiator. The reaction of 4-( 13 C)-Lina-OOHs (mixture 13 C-12/ 13 C-13, 2/3), (1 eq., 5.37 µM) in CD 3 CN/H 2 O 1/1 with N-acetyl-L-tyrosine methyl ester (2 eq., 10.74 µM) was followed by one dimensional 13 C-NMR at room temperature during a week. The catalytic amount of iron (II) sulfate heptahydrate (0.1 eq., 0.5 µM) was sufficient to induce radical initiation (Figure 1.34).

Control experiments were realized without the presence of iron (II) as initiator.

Reactivity profile using N-acetyl-L-tryptophan methyl ester

The reaction of 5-13 C-Lina-OOHs (mixture 13 C-12/ 13 C-13, 2/3), (1 eq., 5.37 µM) in CD 3 CN/H 2 O 1/1 with N-acetyl-L-tryptophan methyl ester (2 eq., 10.74 µM) was followed by one dimensional 13 C-NMR at room temperature during a week. The catalytic amount of iron (II) sulfate heptahydrate (0.1 eq., 0.5 µM) was sufficient to induce radical initiation (Figure 1.35). Control experiments were realized without the presence of iron (II) as initiator. The reaction of 4-( 13 C)-Lina-OOHs (mixture 13 C-12/ 13 C-13, 2/3), (1 eq., 5.37 µM) in CD 3 CN/H 2 O 1/1 with N-acetyl-L-tryptophan methyl ester (2 eq., 10.74 µM) was followed by one dimensional 13 C-NMR at room temperature during a week. The catalytic amount of iron (II) sulfate heptahydrate (0.1 eq., 0.5 µM) was sufficient to induce radical initiation (Figure 1.36). Control experiments were realized without the presence of iron (II) as initiator.

Reactivity profile using N-acetyl-L-lysine methyl ester

The reaction of 5-13 C-Lina-OOHs (mixture 13 C-12/ 13 C-13, 2/3), (1 eq., 5.37 µM) in CD 3 CN/H 2 O 1/1 with N-acetyl-L-lysine methyl ester (2 eq., 10.74 µM) was followed by one dimensional 13 C-NMR at room temperature during a week. The catalytic amount of iron (II) sulfate heptahydrate (0.1 eq., 0.5 µM) was sufficient to induce radical initiation (Figure 1.37).

Control experiments were realized without the presence of iron (II) as initiator. The reaction of 4-( 13 C)-Lina-OOHs (mixture 13 C-12/ 13 C-13, 2/3), (1 eq., 5.37 µM) in CD 3 CN/H 2 O 1/1 with N-acetyl-L-lysine methyl ester (2 eq., 10.74 µM) was followed by one dimensional 13 C-NMR at room temperature during a week. The catalytic amount of iron (II) sulfate heptahydrate (0.1 eq. 0.5 µM) was sufficient to induce radical initiation (Figure 1.38).

Control experiments were realized without the presence of iron (II) as initiator.

Conclusion:

The In this study only some hypothetical new compounds formed could be proposed.

MATERIALS AND METHODS

Chemicals and reagents

Anhydrous solvents were obtained by distillation under an inert atmosphere of argon and kept on molecular sieves (4 Å):

• Dimethylformamide was dried on phosphorous pentoxide then distilled under reduced pressure

• Ethanol and diisopropylamine were distilled on sodium hydroxide

• Triethylamine was distilled with potassium hydroxide

• Tetrahydrofuran, diethyl ether, toluene, methanol, dichloromethane and acetonitrile were dried through activated alumina columns

All reactions needing anhydrous conditions were carried out under argon atmosphere in flame-dried glassware.

Limonene, linalool, and (-)-carveol were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France) as a mixture of isomers and were used without further purification.

1-( 13 C)-Acetic acid, 2-( 13 C)-acetic acid and potassium-1-( 13 C)-cyanide were purchased from Euriso-top (Saint-Aubin, France).

All other reagents and solvents were purchased from Sigma-Aldrich or Fisher Scientific (Illkirch-Graffenstaden, France) and were used as received.

n-Butyllithium was purchased as a 1.6 M solution in hexane and the solution was titrated using the method described by Suffert 32

Follow-up of the reactions

Reactions were followed by thin layer chromatography (TLC) using silica gel plates 60 F 254 (Merck 60 F 254 ; thickness 0.25 mm). After elution, the plates were revealed under short-wave UV radiation (254 nm), then immersed in phosphomolybdic acid, anisaldehyde or potassium permanganate solutions followed by heating.

Phosphomolybdic acid solution (APM/Ce):

• phosphomolybdic acid (5 g) and cerium sulfate tetrahydrate (2 g) in a mixture of sulfuric acid (12 mL) and distilled water (188 mL)

Anisaldehyde solution (Anis.):

• para-anisaldehyde (0.5 mL) and ortho-anisaldehyde (0.5 mL) in a mixture of sulfuric acid (5 mL), acetic acid (100 mL) and ethanol (8 mL)

Potassium permanganate solution:

• Dissolve 1.5 g KMnO4, 1.25 mL NaOH 10% and 10 g K2CO3 in 200 mL water

Chemical purifications

Column chromatography purifications were performed using silica 60 (Merck; Geduran, 40-63 µm) or previously neutralized silica gel.

Neutralized silica was prepared by adding to a homogeneous water solution of silica 60 a saturated solution of sodium hydrogen carbonate up to pH of about 8. After decantation, the silica precipitate was washed with water to reach a pH around 7, filtered and dried in an oven at least for 24 hours.

NMR characterization

1 H and 13 C-NMR spectra were recorded on a Bruker Avance 300 (300 MHz for 1 H and 75 MHz In a flame-dried double-necked flask under argon, to a solution of 1-( 13 C)-acetic acid 13 C-1 (2.0 g; 33.3 mmol; 1 eq.) and phosphorus tribromide (1.60 mL; 16.66 mmol; 0.5 eq.) was slowly added bromine (6.0 mL; 116.56 mmol; 3.5 eq.). The mixture was stirred during 30 minutes at room temperature, then heated at 60 °C for 6 hours. After that time the mixture was cooled to room temperature before anhydrous ethanol (3.6 mL; 63.28 mmol; 1.9 eq.) was added. After one night at room temperature, the reaction mixture was hydrolyzed with ethanol (10 mL) and distilled water (30 mL). The mixture was extracted with diethyl ether (3 × 30 mL). Collected organic phases were neutralized using a saturated solution of sodium bicarbonate (2 × 30 mL)

and washed with a saturated solution of sodium thiosulfate (30 mL then 20 mL). Organic phases were dried over magnesium sulfate, filtered and concentrated under reduced pressure.

1-( 13 C)-1-Bromo-3-methyl but-2-ene ( 13 C-8)

To a solution of 1-( 13 C)-3-methylbut-2-en-1-ol ( 13 C-7) (1.65 g; 19.17 mmol; 1 eq.) in dichloromethane (7 mL) cooled at 0°C was added hydrobromic acid (48%; 24.3 mL; 214.2 mmol; 11.2 eq.). The mixture, protected from daylight, was vigorously stirred for 2 hours.

Magnesium sulfate (6.69 g; 55.72 mmol; 2.9 eq.) and dichloromethane (30 mL) were then added and the solution was allowed to warm to room temperature. The organic phase was separated, and the aqueous phase was extracted using dichloromethane (2 × 20 mL). The combined organics layers were assembled and dried over magnesium sulfate. The solvent was evaporated under reduced pressure. 1-( 13 C)-1-Bromo-3-methyl but-2-ene ( 13 C-8) (1.44 g) was obtained as a brown oil, used without further purification. In a flame-dried round bottom flask, sodium ethoxide (4.34 mL; 11.62 mmol; 1.2 eq.) was added to a solution of ethyl acetoacetate (2.47 mL; 19.37 mmol; 2 eq.) in anhydrous ethanol (20 mL). The mixture was cooled to 0°C and a solution of 1-( 13 C)-1-bromo-3-methylbut-2-ene ( 13 C-8) (1.44 g; 9.6 mmol; 1 eq.) in ethanol (10 mL) was added to the mixture. After stirring 1 hour at 0 °C, the solution was refluxed for 4 hours. The solvent was evaporated under reduced pressure, then water (5 mL) was added and the aqueous phase was extracted with ethyl acetate (3 × 15 mL). The combined organic phases were dried over magnesium sulfate and evaporated under reduced pressure. The crude product was purified using silica gel chromatography (pentane/Et 2 O 9/1) and ethyl-3-(

4-( 13 C)-6-Methylhept-5-en-2-one ( 13 C-10)

An aqueous solution of sodium hydroxide (10%, 18 mL) was added to a solution of ethyl-3-( 13 C)-2-acetyl-5-methylhex-4-enoate ( 13 C-9) (1.29 g; 6.50 mmol; 1 eq.) in ethanol (20 mL) at 0°C. The mixture was refluxed during 5 hours before allowing to warm to room temperature.

Then it was acidified using a solution of hydrochloric acid 10% down to pH 4-5. The aqueous phase was extracted with diethyl ether (3 × 50 mL). The combined organic phases were dried over magnesium sulfate and evaporated under reduced pressure. 4-( In a flame-dried round bottom flask, vinyl magnesium bromide (4.12 mL; 4.12 mmol; 2 eq.)

was added to a solution of 4-( 13 C)-6-methylhept-5-en-2-one ( 13 C-10) (0.26 g; 2.06 mmol; 1 eq.) in anhydrous tetrahydrofuran (10 mL) previously cooled to 0 °C. After stirring 1 hour at 0 °C, the reaction was stopped using a saturated solution of ammonium chloride (10 mL). The aqueous phase was extracted with diethyl ether (3 × 30 mL). The combined organic phases were dried over magnesium sulfate and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (pentane/Et 2 O 8/2) and 5-(

Synthesis of 4-( 13 C)-Lina-OOHs

2-( 13 C)-Ethyl acetate ( 13 C-14)

In a round bottom flask combined to a distillation column, to a mixture of 2-(

2-( 13 C)-Ethyl 5-methyl-4-hexenoate ( 13 C-15)

To a solution of diisopropylamine (4.0 mL; 28.60 mmol; 1.5 eq.) in dry tetrahydrofuran (20 mL) at 0 °C, a solution of n-butyllithium (1.6 M in pentane; 17.9 mL; 28.60 mmol; 1.5 eq.) was added very slowly. After 10 minutes stirring at 0 °C 2-( 13 C)-ethyl acetate ( 13 C-14) (1.68 g; 19.07 mmol; 1 eq.) in tetrahydrofuran (20 mL) was very slowly added and the reaction mixture was stirred for 30 minutes at 0 °C. After this time, the reaction mixture was cooled to -78°C and a solution of 3,3-dimethylallyl bromide (3.30 mL; 28.60 mmol; 1.5 eq.) in tetrahydrofuran (17.5 mL) containing hexamethylphosphoramide (5.0 mL; 28.60 mmol; 1.5 eq.) was added to the reaction mixture. After 4 hours at -78 °C, the reaction mixture was allowed to warm to 0 °C. A solution of hydrochloric acid 10% (60 mL) was added and the mixture extracted with diethyl ether (3 × 30 mL). The combined organic phases were dried over magnesium sulfate and evaporated under reduced pressure. The crude product was purified using silica gel column chromatography (petroleum ether/AcOEt 95/5) to give 2-(

2-( 13 C)-N-Methoxy-N,5-dimethylhex-4-enamide ( 13 C-16)

To a solution of N,O-dimethyl hydroxylamine hydrochloride (3.346 g; 34.35 mmol; 3 eq.) in dry tetrahydrofuran (15 mL) at 0 °C, was added a solution of trimethyl aluminum (17.8 mL; 34.35 mmol; 3 eq.) in tetrahydrofuran (10 mL) over 5 minutes. The mixture was allowed to warm up to room temperature and became pink. The solution was then cooled again to 0 °C and 2-( 13 C)-ethyl 5-methyl-4-hexenoate ( 13 C-15) in dichloromethane (5 mL) was very slowly added. The mixture was allowed to warm up to room temperature and was stirred for 5 hours.

After this time a hydrochloric acid solution 10 % was carefully added. The aqueous phase was extracted with dichloromethane (4 × 50 mL), washed with sodium bicarbonate (150 mL) then water (150 mL). The combined organic phases were dried over magnesium sulfate and evaporated under reduced pressure. The crude product was obtained as a yellow oil (1.645 g).

Formula: C 8 13

3-( 13 C)-6-Methyl-5-heptene-2-one ( 13 C-17)

In a flame-dried flask, to a solution of 2-( 13 C)-N-methoxy-N,5-dimethylhex-4-enamide ( 13 C-16) (1.645 g; 9.55 mmol; 1 eq.) in tetrahydrofuran (25 mL) previously cooled to -78 °C was added very slowly a solution of methyllithium (1.6 M in diethyl ether; 7.8 mL; 12.49 mmol;

1.3 eq.). The mixture was stirred for 1 hour 30 minutes at -78 °C and 24 hours at room temperature. The reaction was quenched using a solution of hydrochloric acid 10 % (20 mL).

Diethyl ether (40 mL) and water (40 mL) were then added and the organic phases were recovered. The aqueous phase was extracted with diethyl ether (4 × 80 mL). The combined organic phases were dried over magnesium sulfate and evaporated under reduced pressure. The crude product was purified by silica gel chromatography (petroleum ether/diethyl ether 9/1), to afford 3-( In a flame-dried flask, to a solution of 3-( 13 C)-6-methyl-5-heptene-2-one ( 13 C-17) (0.733 g; 5.81 mmol; 1 eq.) in tetrahydrofuran (20 mL) previously cooled to -78 °C was added a solution of vinyl magnesium bromide (1.6 M in THF; 7.26 mL; 11.62 mmol; 2 eq.) in tetrahydrofuran.

After 2 hours stirring at -20 °C, the reaction was quenched using a saturated ammonium chloride solution (20 mL). The aqueous phase was extracted with diethyl ether (3 × 60 mL), the combined organic phases were dried over magnesium sulfate and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether/diethyl ether 8/2) to give 4-( 13 C)-3,7-dimethylocta-1,6-dien-3-ol ( The semi-solid white product obtained was suspended in dichloromethane (233 mL) and vigorously stirred for 48 hours. The suspension was filtered to recover sodium molybdate and sodium dodecyl sulfate. The filtrate, partially evaporated under reduced pressure, was washed using distilled water (3 × 100 mL) and the aqueous phase was extracted with dichloromethane (100 mL). Combined organic phases were dried over magnesium sulfate, filtered and concentrated under reduced pressure to give a crude yellow oil. A mixture of hydroperoxides hydride (1 M in THF; 5.6 mL; 5.6 mmol; 2.5 eq.) was added very slowly. The mixture was stirred 1 hour at 0 °C than at room temperature for 5 hours. Di-isobutyl aluminum hydride excess was neutralized by adding ethyl acetate (10 mL). The reaction mixture was diluted with diethyl ether (13 mL), then silica (10 g) was added. The slurry was stirred at -78 °C overnight.

Silica was removed by filtration and the solvent of the filtrate was evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (pentane/diethyl ether 95/5).

Linalool (24)

In a flame-dried round bottom flask under argon methyltriphenylphosphonium iodide (0.18 g; 0.44 mmol; 2.0 eq.) previously dried for 24 hours was dissolved in anhydrous tetrahydrofuran

(

Chapter 2 Electron paramagnetic resonance (cw-EPR) investigation of free radicals induced by skin sensitizers: from pure in vitro studies to the use of a reconstructed human epidermis model

As discussed in the introduction of the manuscript, this first step in the development of skin sensitization to a chemical and further contact dermatitis is the binding of the allergen to skin proteins after penetration into the epidermis. The antigenic adduct so formed is then recognized by the immune system as foreign to the body. Allergenic hydroperoxides derived from autoxidation of natural terpenes are believed to form antigens through radical-mediated mechanisms. So far, in vitro investigations on reactive radical intermediates derived from these compounds have been conducted in solution, yet with experimental conditions being far away from real life sensitization. [1][2][3] In the present chapter we propose to probe the radical intermediates issued from ROOHs derived from linalool and R-(+)-limonene, considered as skin sensitizers, and cumene hydroperoxide, using continuous wave Electron Paramagnetic Resonance (cw-EPR) spectroscopy combined with the spin-trapping technique. The objective was to transfer this technology to the investigation of these radicals generation in situ in a reconstructed human epidermis model. After a brief description of the technique and its principal physical principles, a two steps experimental approach is discussed in the following sections: (1) in vitro generation of free radicals and (2) in situ generation using a 3D reconstructed human epidermis (later referred as to RHE) as model system.

ELECTRON PARAMAGNETIC RESONANCE

Introduction

EPR spectroscopy can be divided into two different categories: continuous wave (cw-EPR) and pulsed spectroscopy (p-EPR). 4 Only the first cited one will be discussed here and will later be referred as to EPR. In comparison with nuclear magnetic resonance (NMR) which focus on the behavior of the proton, EPR relies on an unpaired electron within an external magnetic field and a subsequent electromagnetic absorption. EPR has found applications from solid state physics to biomedical use. 5,6 In particular it has been successfully applied to the understanding of many physiological processes ranging from in vivo detection-characterization of reactive nitrogen (RNS) and oxygen species (ROS) and tumors oximetry, to the assessment of redox state in biological tissues.

In 1945 E. Zavoïsky was the first to observe an EPR signal. 7 Free electrons have an intrinsic angular momentum called spin that is present without external forces. Therefore, every free electron induces a magnetic field due to the moving charge and it acts as a small bar magnet / magnetic dipole having a magnetic moment (Figure 2.1).

In contrast to diamagnetic materials where all electrons are paired and share their orbitals with another electron (i.e. net spin = 0), a paramagnetic material possess an unpaired electron and thus exhibits an intrinsic magnetic moment. EPR spectroscopy is adapted for the investigation of materials with unpaired electrons i.e. with magnetic moments that can align either parallel or antiparallel to an external magnetic field (Figure 2.1). In EPR studies, paramagnetic systems with an unpaired electron are seen such as: transition metals, radicals and salts.

Zeeman effect

EPR consists in inducing transitions between two electronic spin states whose degeneracy is lifted when a magnetic field is applied. This is the so-called Zeeman effect, named after the Dutch physicist Pieter Zeeman, who was awarded by the Nobel Prize in Physics in 1902 for this pioneering discovery. 9 If we consider an isotropic system of electronic spin S=½, the resulting spin magnetic moment µ will be given by

𝜇 ⃗ = 𝑔𝛽 𝑒 𝑆 ⃗ (I)
where:

• 𝛽 𝑒 = 𝑒ℏ 2𝑚 𝑒
being the Bohr's magneton (β e = 9.27401.10 -24 A.m 2 ) with m e the electron mass, e its charge and ℏ the reduced Planck constant.

• g is a dimensionless factor also called Landé's factor or "g factor" where g = g e = 2.0023193043617 for a free electron. This value has been predicted by quantum electrodynamics and later verified experimentally. 10 In the isotropic case, g can be assumed as a scalar.

• 𝑆 ⃗ is the electronic spin operator.

In the absence of a magnetic field, the electronic levels are degenerated, but when placed into an external magnetic field 𝐵 0 ⃗⃗⃗⃗⃗ oriented against the z axis, the energy's electron is defined following the equation:

𝐸 = -𝜇 ⃗. 𝐵 0 ⃗⃗⃗⃗⃗ (II)
and the degeneracy is lifted as described by the electronic Zeeman Hamiltonian 𝐻 𝐸𝑍 ⃗⃗⃗⃗⃗⃗⃗⃗ :

𝐻 𝐸𝑍 ⃗⃗⃗⃗⃗⃗⃗⃗ = -𝜇 ⃗. 𝐵 0 ⃗⃗⃗⃗⃗ = 𝛽 𝑒 𝐵 0 𝑆 𝑧 (III)
where dimensionless S z is the projection of the electronic spin operator on the z axis. Such

Hamiltonian acts on the electron spin functions |m> and consequently the two degenerated levels of the spin states split with 𝑚 𝑆 = + (spin anti parallel to the magnetic field), as described in Figure 2.2. The resulting eigen energy of the electronic level becomes: ). Based on these elements, EPR experiment consists in inducing electronic transitions, allowed by the selection rule Δ𝑚 𝑆 = ±1 between the two energy levels, by applying an electromagnetic radiation 𝐵 1 ⃗⃗⃗⃗⃗ oscillating at the frequency , and meeting the following conditions:

𝐸 ± = 𝑔𝜇𝐵 0 𝑚 𝑆 = ± 1 2 𝑔𝜇𝐵 0 (IV)
• 𝐵 1 ⃗⃗⃗⃗⃗ ⊥ 𝐵 0 ⃗⃗⃗⃗⃗
• The resonance condition must be fulfilled, i.e.:

∆𝐸 = ℎ𝜈 = 𝐸 + -𝐸 -= 𝑔𝛽 𝑒 𝐵 0 𝑟𝑒𝑠 (V)
From the practical point of view, EPR is achieved while varying the static magnetic field 𝐵 0 ⃗⃗⃗⃗⃗ until reaching the resonance condition (Figure 2.2), where 𝐵 1 ⃗⃗⃗⃗⃗ is absorbed. Therefore, EPR is an absorption spectroscopy and in the present work, the frequency  of 𝐵 1 ⃗⃗⃗⃗⃗ is of ca. 9.5 GHz which belongs to the microwave range and corresponds to so-called X-band. Also, to enhance the sensitivity of the detection in cw-EPR, 𝐵 0 ⃗⃗⃗⃗⃗ is modulated in amplitude (lock-in detection system, at usually 100 kHz). 11 Hence, the observed EPR signal corresponds to the first derivative of the absorption signal.

Hyperfine interaction

In the case of paramagnetic compounds, if the unpaired electron is in the vicinity of nuclei with spin I with non-zero values, the resulting interaction is called hyperfine interaction between the magnetic moments of the electronic (S) and nuclear spins (I). Such interaction is described by the Hamiltonian spin 𝐻 𝐻𝐹 ⃗⃗⃗⃗⃗⃗⃗⃗ also called Fermi contact:

𝐻 𝐻𝐹 ⃗⃗⃗⃗⃗⃗⃗⃗ = 𝑆 ⃗ 𝐴 𝑖𝑠𝑜 𝐼 ⃗ (VI)
𝐼 ⃗ is the nuclear spin operator and A iso is the isotropic hyperfine coupling constant (hfcc) that represent the energy of interaction between the electronic and the nuclear spin. Such interaction is very much dependent of both the spin density on the nucleus and the gyromagnetic of the nucleus. 12 Stronger are these two parameters stronger is the hyperfine coupling constant. The natural abundance, nuclear spin and gyromagnetic ratio of common nucleus are given in Table 2.1. The Hamiltonian 𝐻 𝑆𝐼 ⃗⃗⃗⃗⃗⃗⃗ describing the electronic (S) and nuclear (I) spin system is the following:

𝐻 𝑆𝐼 ⃗⃗⃗⃗⃗⃗⃗ = 𝐻 𝐸𝑍 ⃗⃗⃗⃗⃗⃗⃗⃗ + 𝐻 𝐻𝐹 ⃗⃗⃗⃗⃗⃗⃗⃗ = 𝑔𝛽 𝑒 𝐵 ⃗⃗ . 𝑆 ⃗ + 𝑆 ⃗ 𝐴 𝑖𝑠𝑜 𝐼 ⃗ (VII)
In fine, such interaction leads to the splitting of each electronic spin level into 2I+1 sub-levels corresponding to each of the m I values whose the electronic energy is:

𝐸(𝑚 𝑆 , 𝑚 𝐼 ) = 𝑔𝛽 𝑒 𝐵 0 𝑚 𝑆 + 𝑚 𝐼 𝐴 𝑖𝑠𝑜 𝑚 𝑆 (VIII)

A simple example is when an electronic spin system (m S = ±½) is in interaction with a neighboring nitrogen nucleus 14 N (m I = 0, ±1). The diagram of the magnetic energy levels is described in Figure 2.3. Consequently, 2I+1=3 transitions can be there observed when applying the selection rules: EPR spectra can be easily complexified whether the paramagnetic probe "is seeing" a multiple set of nuclei with non-zero nuclear spins.

• mS=±1 • mI=0,
Considering a system with a X set of equivalent nuclei interacting with the unpaired electron, as all the involved hyperfine couplings are identical, the EPR spectrum will be composed by 2nI+1 peaks, equally spaced. The relative intensities of the absorption peaks are given by the binomial expansion (1+a) X and the corresponding coefficients by the Pascal's triangle (see (IX)

Spin-trapping approach: from the reactive species to the EPR fingerprint

EPR is a dedicated spectroscopy for the study of free radicals reactive species. However, in our experimental conditions (aqueous solution at room temperature) most of the reactive species are not detectable per se. This is either due to their spin configuration (e.g. 1  g ) and/or mostly to their short lifetime (nano-to milliseconds) which go hand in hand with their great reactivity.

In these cases, the so-called spin-trapping approach is employed. 13 A schematic description of the technique is given in Figure 2.5. The resulting spin-adduct gives valuable information concerning the probed reactive species, as its EPR fingerprint is function of the trapped reactive species. Also, the intensity of the observed signal is directly related to the amount of trapped species. Nevertheless, it should be kept in mind that the spin trapping approach is an indirect method: we are observing spinadducts and not the reactive species itself. Therefore, quantitative analysis is tempered by the spin-adduct lifetime and affinity constant.

A comprehensive analysis of the EPR fingerprint through the characterization of both the g factor(s) and hyperfine coupling(s) constant(s) allows to identify the trapped reactive species(s), by matching the obtained values with existing literature and databases. 17,18 If we take DMPO spin-adduct as an example (Figure 2.7) in aqueous media at room temperature, different scenarios can be envisaged as DMPO is able to trap oxygen-and carboncentered radicals. If a methyl radical (  CH 3 ) is trapped, the resulting unpaired electron of DMPO-CH 3 spin-adduct interacts with neighboring 14 N (I N = 1) and 1 H (I H = ½) in the  position (Figure 2.7A). Such system is assumed to give (2I N +1)(2I H +1) = 6 peaks. Hyperfine coupling constant are here known to be of ca. 16 G and 23 G for the a N and a H respectively. 17 If a hydroxyl (  OH) radical is trapped, the DMPO-OH spin-adduct has another characteristic EPR fingerprint. In this case, a N = a H and is of ca. 15 G. As a direct consequence, only four lines are visible with a typical 1:2:2:1 intensity ratio (Figure 2.7B). 19 If an superoxide radical is trapped (O 2 -), the resulting spin-adduct DMPO-OOH exhibits twelve peaks (see Figure 2.7C).

Such fingerprint was first wrongly attributed to the superposition of the two conformers of the corresponding spin-adduct, but Clement et al. identified it as a contribution of a third nucleus H (located on the pyrroline group in the equatorial position) leading to a relatively smaller a H of ca. 1 G. 20 Unfortunately, the half-life of this spin-adduct is rather short (< 1 min) and it will rapidly convert into DMPO-OH spin-adduct. Consequently, distinguishing from superoxide anion to hydroxyl radical contribution is hardly reachable when using DMPO. Yet in real life scenarios, very often a mixture of different spin-adducts is observed (Figure 2.7D) and the resulting EPR spectrum needs to be deconvoluted to decipher the relative amount of the involved reactive species, this amount will be given in percentage. 21

Limits of the technique and usual artefacts.

The first limit is the quantification of the radicals trapped, also working on the detection limits of the spectrometer due to the low concentrations used. The spectrum interpretation is more difficult in the case of anisotropy and the coupling between electron and orbital moment.

Herein, quantitative assumptions are made here more difficult. It is important to underline the fact that other reaction can form nitroxides such as ene-reaction, Forrester-Hepburn reaction and Inverted Spin Trapping (IST). The ene-reaction occurs when a nitroso function reacts with an olefin (Figure 8.A). A pericyclic reaction produces a hydroxylamine then an oxidation transforms it to a nitroxide function, similar to the spin-adduct of a carbon centered radical. 22 Forrester-Hepburn reaction produces a hydroxylamine by addition of a nucleophile to the spintrap then an oxidation occurs using oxygen for example to synthesize nitroxide (Figure 8.B), similar to the spin-adduct of a hydroxyl radical is observed. 23 IST an oxidation of the spin-trap producing a cation radical using a strong oxidant such as H 2 O 2 , similar to the spin-adduct of a hydroxyl radical is observed. 24 Finally, the purity of the spin trap can also lead to parasitic signals and further purification such as distillation, recrystallization or activated charcoal filtration might be necessary. 25 For all of the above, performing all the necessary control experiment appears crucial.

Data analysisfrom ruler and pencil to simulation and fitting

In front of a spin-adduct EPR spectrum, the first step is to determine roughly the hfccs (a P , a N and a H ) values as well as the g factor by hand with "a ruler and pencil". An example is given in Figure 2.9, for the case of a methyl radical trapped by the DEPMPO spin-trap. (including the microwave source, the attenuator, the reference arm, the circulator and the detector) and a resonant-cavity between two electro-magnets. 28 During EPR measurements, the electro-magnets generating a magnetic field (in Gauss) is applied to the cavity and thus the sample, leading to the Zeeman effect. An EPR scan is carried out by varying the 𝐵 0 ⃗⃗⃗⃗⃗ field at a constant microwave frequency  of ca. 9.5 GHz (i.e. X-band), until reaching the resonance conditions. The coupling between the microwaves arising from the waveguide and the cavity is achieved through an iris whose aperture is adjusted to tune the microwaves entering and reflected from the cavity.

In resonant cavity, a standing electromagnetic wave is created where the electric and magnetic field components are out of phase. By placing the sample in the maximum magnetic field and therefore in the lowest electric field, an optimal signal can be measured. This is especially critical when dealing with system with high dielectric constant (such as aqueous solution at room temperature), where the major part of electrical component is absorbed by the system (as for a regular microwave oven) and significantly deteriorate the sensitivity of the experiment (Figure 2.12). Consequently, specific geometry of the investigated samples is required: (i) for in vitro studies submillimeter quartz or glass capillaries vertically positioned into the cavity are mandatory; (ii) for biological tissue studies a flat cell will be preferred. heating of the sample is not here questioned.

IDENTIFICATION OF RADICALS DERIVED FROM ROOHS: STUDIES IN

SOLUTION

Studies in solution were firstly carried out to set up the optimal experimental conditions providing a good S/N while keeping low ROOHs and spin-trap concentrations, thus suitable for physiological conditions. Radical initiation was triggered by Fe II -induced Fenton-like reaction.

The final aim was to transfer these experimental conditions to a reconstructed human epidermis (RHE) model.

Key players

1. 1

Target compounds

ROOHs that have been chosen for these studies are: cumene hydroperoxide (Cum-OOH), limonene hydroperoxide (Limo-OOH) and a 2:3 mixture of linalool-7-hydroperoxide (Lina-7-OOH) and linalool-6-hydroperoxide (Lina-6-OOH) (Figure 2.13).

Radical initiation: Fe(II) sulfate

It is known that Fe(II) initiates the decomposition of hydroperoxides into alkoxyl radicals and hydroxyl ions, by a mechanism similar to that of Fenton reaction. [29][30] Following such radical initiation, unstable alkoxyl radicals can evolve through different pathways. For example, the abstraction of a hydrogen from the hydroperoxide ROO-H function is possible, leading to an alcohol and peroxyl radicals. These can interact together leading to tetroxide (ROOOOR) compounds able to regenerate alkoxyl radicals together with the release of molecular oxygen.

Peroxyl radicals can also abstract a hydrogen to produce carbon-centered radicals (Figure 2.14).

Figure 2.14: Fenton-like reaction and subsequent radical generation in the case of ROOHs

The spin-traps Spin-traps used were: PBN (N-tert-butyl-α-phenylnitrone), DMPO (5,5-dimethyl-1-pyrroline-

N-oxide, 2,2-dimethyl-3,4-dihydro-2H-pyrrole 1-oxide) and DEPMPO (5- (diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide).
PBN spin-adducts hfccs arise from the interaction of the unpaired electron with the 14 N (I N =1) and the surrounding 1 H (I H = ½) (Figure 2.6). In our experimental conditions (i.e. aqueous solution, neutral pH and room temperature), the g factor does not appear as a discriminatory parameter and will be always of ca. 2.006. This is indeed the case for all spin-traps discussed here. The reactivity of PBN is relatively high and allows the detection of low quantities of radicals. Yet, the relatively low specificity of the hyperfine couplings towards the nature of the trapped species, together with low steric hindrance of the radical within the spin-adduct leading to bio-reduction limit its use in in vivo experiments.

DMPO spin-adducts hfccs arise from the interaction of the unpaired electron with the 14 N (I N =1)

and the 1 H (I H =½) in position  (see Figure 2.6). For this spin-trap, the hyperfine couplings between the electron and the nitrogen and hydrogen nuclei are greatly dependent of the nature of trapped radical. As for PBN, DMPO spin-trap is commonly used in biological media but the low resistance to bio-reductions of its spin-adducts limits the potential use in in vivo applications. 31 DEPMPO spin-adducts hfccs arise from the interaction of the unpaired electron with the 14 N (I N =1), 1 H (I H = ½) in position  and 31 P(I P =½) in position  (see Figure 2.6) and produces spinadducts with longer lifetime, particularly for the superoxide and alkylperoxyl radicals adducts. 32 Thus it presents a pertinent alternative for the experiments within 3D reconstructed human epidermis.

The spin-adduct lifetime depends on the spin-trap as well as its environment (e.g. presence of reductant compounds) and goes from minutes to hours. 33

Solvent and concentration of reagents

Spin-traps, in H 2 O/CH 3 CN (9:1), 10 mM phosphate buffer (PB) or HEPES buffer, were employed in an excess concentration (25 mM) with respect to ROOH (1-10 mM). The spin-trap solution was then mixed with aqueous ferrous sulfate (0-1.7 mM) and the ROOH was lastly added to the mixture which was inserted in a 25 µL glass capillary (Hirschman ringcaps), and investigated by EPR after different reaction times (1 minute to 2 hours).

High concentrations of both the spin-trap (500 mM) and the hydroperoxide (10 mM) were initially used and thereafter gradually decreased (up to 25 and 0.5 mM respectively). We started the studies with Limo-OOH in pure water solution by decreasing the spin-traps and the Limo-OOH concentrations while keeping a decent S/N with a minimum amount of spin-trap and Limo-OOH. The second step was to transpose these conditions into a medium more suitable for biological assays, by using Cum-OOH and phosphate buffer (PB). It was shown that in PB, radical generation was not detected with a catalytic amount of Fe(II) (0.1 mM). Therefore, we decided then to use HEPES buffer in which radical generation occurred with a catalytic amount of Fe(II). Finally, HEPES buffer was chosen to characterize radical generation from Lina -OOHs. Here HEPES buffer was also combined to the cell culture medium (CM): the nutrients medium of the RHE (potential source of antioxidant), with a ratio 4:1 then 1:1 v/v to probe the influence of CM on radical generation. The main idea was to use as much as possible physiological compatible conditions to investigate radicals in the RHE without starving and/or stressing the cells by the sole experimental conditions.

To be clear and concise, we will present the use of DEPMPO allowing the identification of different oxygen and carbon-centered radicals derived from ROOHs. PBN and DMPO complementary studies, respective EPR spectra, hfccs and g values are presented in annexes to this chapter. DEPMPO has been successfully used for the in vivo detection of oxygen-centered radicals due to the high persistency of its superoxide and alkylperoxyl radicals adducts. 34 DEPMPO is also able to distinguish between oxygen and carbon-centered radicals. 16

System Limo-OOH, spin-trap, H 2 O and Fe(II)

Here we will use the example of Limo-OOH in water medium and in the presence of DEPMPO as spin-trap to present the methodology used to identify the formed spin-adducts. Firstly, the "ruler and pencil" approach (see section 1.6) was used to identify the probable spin-adducts together with their approximative hfccs and g values. Then, specific simulation and fitting routines (using Easyspin/Matlab toolbox) were applied to the experimental spectrum (Figure 2.15) in order to refine the hfccs and g for all present spin-adducts together with their relative concentration (when applicable). Worthy of note, relative quantifications of generated radicals remain possible if the spin-adducts lifetimes and efficiency are assumed comparable. For the treatment and characterization of all spectra obtained during this work the identical procedure was applied. In the example presented here, i.e. for Limo-OOH/DEPMPO/H 2 O system, two spin-adducts were identified whose the principal characteristics are summarized in Table 2.2.

According to existing literature , those two spin-adducts were attributed to hydroxyl (DEPMPO-OH) and carbon-centered (DEPMPO-carbon R) radicals. 34 Both the nature and the relative concentration of spin-adducts remain unchanged when decreasing the concentration of Limo-OOH from 10 to 1 mM (Annex 1., Figure A1.1).

Moreover, the hydroxy spin-adduct 1 was formed with ca. 4-fold concentration when compared to the carbon spin-adduct 2 at 1mM Limo-OOH. By decreasing the Limo-OOH concentration (10 mM to 1 mM), both the hydroxyl spin-adduct 1 and carbon spin-adduct 2 were observed. But the relative concentrations remained stable. The decrease of the DEPMPO (from 500 to 25 mM) concentration does not influence the formed radicals (data not shown). By increasing Fe(II) concentrations (0.1 mM to 1.7 mM) in the mixture of Limo-OOH (1 mM) and DEPMPO (25 mM), the carbon spin-adduct ratio increased from ca. 20 to 50 % (Annex 2, Figure A2.1).

In the studies using PBN (50 mM) in a mixture of Limo-OOH (1 mM) and Fe(II) (0.1 mM) in H 2 O, a spin-adduct (3) attributed to a carbon-centered radical was observed (Annex 4, Figure A4.1). The same mixture using acetonitrile as solvent produced a carbon spin-adduct 4 probably due to the decomposition of the acetonitrile in the experimental conditions. 35 summarizes the mechanistical hypotheses for the observed spin-adducts.

In parallel investigations with DMPO, only hydroxyl radical spin-adduct (5) was observed.

Moreover, the relatively short half-life of the DMPO spin-adduct (ca. minutes) leads to its rapid disappearance.

Figure 2.16: Hypothetical spin-adducts obtained using Limo-OOH

The next step was to transpose these experimental conditions into a medium more suitable for biological assays and by using Cum-OOH as a reference compound. [36][37][38]

System Cum-OOH, spin-trap, PB or HEPES and Fe(II)

According to Pazos et al., the concentration of Fe(II) is a limiting factor in the generation of radical species in PB, when they used 0.1 mM of Fe(II) and 0.54 mM of Cum-OOH it was sufficient to observe oxidation processes. 30 Yet, in our experiments 0.1 mM of Fe(II) was not enough to start the reaction in 10 mM PB whatever was the spin-trap (at 25 mM). At higher concentrations of Fe(II) (0.5 mM and above), the radical generation rate significantly increased and spin-adducts were already detected after only 1 min of reaction time (Annexes 6-8, Figures A6.1, A7.1, A8.1). The inhibition of Fe(II)-catalyzed CumOOH oxidation at low concentrations is ascribed to the ability of phosphate to catalyze oxidation of Fe(II), forming Fe(III) that is rather insoluble and hence decreasing Fe(II) content in the reaction mixture. 39 PB was thus replaced by 10 mM HEPES buffer, known for its stability at physiological pH. 40 In these conditions, a catalytic amount of Fe(II) (0.1 mM) was enough to start the reaction. Moreover, keeping all other parameters identical the signal intensity observed in HEPES investigations were of an order of magnitude higher than the one observed with PB.

Experiments were then performed in 10 mM HEPES within 0.1-1.7 mM range of Fe(II) together with 25 mM of DEPMPO, DMPO or PBN (Annexes 6-8, Figures A6.2, A7.2, A8.2). Figure 2.17 shows the results obtained for 0.5 mM of Fe(II) in the presence of 0.54 mM of Cum-OOH and 25 mM of DEPMPO. Three distinct spin-adducts were observed in the experimental spectrum. Once deconvoluted the following spin-adducts were identified: DEPMPO-OH (ca. The DEPMPO-OH 1 (Figure 2.17C) originates from the homolytic scission of Cum-OOH (one electron mechanism). 34 The DEPMPO-carbon R 6 (Figure 2.17D) most probably arises from the • CH 3 radical derived from the -scission, and confirmed using both DMPO (Annex 7, Figure A7.2) and PBN (Annex 8, Figure A8.2). 34 The spin-adduct 7 (Figure 2.17E) is ascribed to either • OCH 3 or • OOCum radical following the mechanisms schematized in Figure 2.19. 34 On the one hand, following the -scission the methyl radical can react with molecular oxygen forming peroxymethyl radicals, that can combine into tetraoxodimethyl, and leading to methoxy radical by releasing molecular oxygen. Noteworthy, this methoxy radical issued from the from scission was only reported in the presence of either amino acids or ethanol in the reaction mixtures which was not the case in the present work. 30,41,42 On the other hand, hydrogen abstraction within Cum-OOH forming • OOCum radical can react directly with the spin-trap leading to 7.

EPR spectra shown in Figure 2.17 and Annex 6 (Figures A6.1 and A6.2) highlighted a mixture of spin-adducts formed by DEPMPO trapping of hydroxyl • OH (1), carbon-centered (6), CH 3 O • and/or possible peroxyl CumOO • radicals (7). 34 It was confirmed the direct formation of these radicals at catalytic Fe(II) concentration (0.1 mM) in HEPES buffer (Annex A6, Figure A6.2).

Concerning PBN investigations in HEPES (Annex 8, Figure A8.2), the only spin-adduct observed at low Fe(II) concentration (< 0.5 mM) is testimony of the -scission. Above 1 mM of Fe(II) the hfccs a N of the spin-adduct significantly increased from ca. 15 to 16 G. An explanation could be the trapping of oxaspiro radicals in equilibrium with CumO • , which are suspected intermediates in known O-neophyl rearrangement processes (Figure 2.18). 43 Such rearrangement would be favorized as the electron reservoir is higher (i.e. higher concentration of Fe(II)).

Figure 2.18:

O-Neophyl rearrangement processes described in the literature. 43 As regards the DMPO-OCH 3 spin-adduct 8 was yet detected at catalytic Fe(II) levels (0.1 mM), and CH 3 • radicals also formed and trapped (9) at higher Fe(II) concentrations (Annex A7, Regarding the following section dealing with Lina-OOHs, the aim was to get closer to what could happen in the RHE, using DEPMPO, HEPES buffer later combined with cell culture medium (CM).

System Lina-OOHs, spin-trap, HEPES and Fe(II)

In the case of Lina-7-OOH, one of the isomers deriving from autoxidation of linalool, the The third detected spin-adduct (14, Figure 2.21E) is thought to arise either from the  OCH 3 radical or  OOLina following the same approach described previously in the Cum-OOH section. Some experiments have been also carried out using as solvent the cell culture medium (CM) dedicated for the RHE Episkin TM (HEPES/CM 4:1 then 1:1 v/v). Worthy of note, CM was provided by Episkin to maintain the RHE, lacking any of other information regarding the component of the medium. First, there is a significant difference in the EPR fingerprints intensities when comparing HEPES/CM investigations to pure HEPES ones. Clearly, the presence of CM (and its antioxidant compounds) competes with the spin-trap action and/or with the lifetime of the spin-adduct itself (by reducing it). Nevertheless, it appears that hydroxyl radicals and carbon radicals were equally trapped when using 1 mM of Fe(II). Increasing Fe(II) concentration to 1.7 mM relative carbon radical formation 13 increased while hydroxyl radical formation seemed to decrease. Importantly, the spin-adduct 14 was not observed when solvent HEPES/CM was used whatever was the HEPES/CM ratio (Annexes 10-11 Figures A10.1, A10.2, A11.1 and A11.2). This points to the antioxidative potential of the CM.

Conclusion

The spin-trapping technique using PBN, DMPO, and DEPMPO, combined with EPR spectroscopy, allowed us to characterize radical intermediates issued from Cum-OOH, Limo-OOH, and Lina-OOHs in solution. Using Fe(II) as radical initiator, the formation of alkoxyl, The rational process followed to develop this methodology is described in the following sections.

Cum-OOH as proof of concept

Cum-OOH was initially used as proof of concept. It is often used in EPR studies as a reference compound. [36][37][38] Moreover, CumOOH has been reported as a significant sensitizer in guinea-pigs, and positive specific responses have been reported in allergy cross-reactivity studies with allergenic cyclohexene hydroperoxides. 47

Incubation in the assay medium -application from the bottom

Preliminary attempts were conducted by incubating Cum-OOH (0.54 mM) and DEPMPO (25 mM) in a mixture HEPES/assay medium (AM) (4:1 v/v). AM was provided by Episkin TM , dedicated as its name suggested it for the experiment with the RHE. As for CM, no information regarding the component of the medium was given, except that AM is significantly depleted than compared to CM. Evidently, no Fe(II) was used for in situ approaches with RHE. The aim was to avoid the epidermis barrier function and check at a first sight using absorption from the bottom of RHE if Cum-OOH-mediated radicals could be formed within the epidermis. Even though Episkin TM RHE barrier function has been described to be less developed than that of normal skin (different lipids composition and organization), it may not be neglected. 48 For control purposes, a sample from assay medium was regularly taken (20 L) during incubation (37°C, 5% CO 2 , 1 min to 2 h), inserted into a capillary, sealed and measured by EPR at room temperature. No EPR signal was observed at any time of incubation. After 2 h incubation, the RHE was placed in a EPR tissue cell (Wilmad) equipped with a silica window and EPR spectra were recorded. Although being noisy, a signal was then observed increasing with time (Figure After penetrating the RHE, CumOOH radical initiation was certainly induced by RHE itself as no Fe(II) was included this time. This certainly better mimics real-life skin allergy situation.

One can hypothesize that reaction of organic peroxides in the skin in the presence of oneelectron donor agents (i.e. amino and thiol groups containing amino acids, metals, enzymes) may start electron transfer processes and thus skin proteins haptenation. 49,50 Control experiments without CumOOH were performed (Figure 2.25 A and B), i.e. after only DEPMPO incubation in the RHE and showed no EPR signal, corroborating that most probably this spin-adduct arose from trapping a carbon-centred radical issued from the hydroperoxide.

In the recent OECD guideline on in vitro skin irritation assessment by using this RHE system it is specified that test chemicals must be applied topically to the RHE model. 51 In consequence, with the acquired knowledge that CumOOH radicals can be formed in RHE as described above, and in order to get closer to real life situation, further experiments were based on a topical application procedure.

Incubation by topical application to the RHE.

At the beginning, RHE were treated with Cum-OOH in acetone (1 mM, 20 L) topically and incubated for different time periods (37°C, 5% CO 2 , 1 minute to 24 hours). RHE negative controls were treated only with acetone (20 L). After the incubation time, the RHE were placed in the EPR tissue cell, DEPMPO (50 mM in 10 mM HEPES, 20 L) was applied to the epidermis taking care to ensure that the spin-trap was only applied to the epidermis top. EPR spectra were recorded immediately afterwards. After 5 min of incubation, the fingerprint of a carbon-centered radical DEPMPO adduct was detected as previously observed during incubation protocol (spin-adduct 6), with hfccs a N =15.4 G, a H =22.7 G, a P =48.1 G, and yet with a better signal-to-noise ratio (Figure 2. 26 B; Table 2.4). 2.4, it appeared clearly that a carbon centered radical was obtained in majority in both approaches and most probably a methyl radical. However, despite these very encouraging results, all the numerous attempts using the above described approach with Limo-OOH and Lina-OOHs were vain. Therefore, we decided to invert the order of application of the spin-trap and hydroperoxyde and increase their respective concentrations. Spin trap was topically applied and pre-incubated for a period of time then hydroperoxide was topically added.

LinaOOHs experiments

CM of the RHE was replaced by 10 mM HEPES. The RHE were firstly treated with 20µL of 250 mM DEPMPO in 10 mM HEPES, applied topically and incubated for different time periods (37°C, 5% CO 2 , during 7.5, 15 and 30 minutes). 53 After incubation, RHE were placed in the EPR tissue cell, Lina-OOHs in acetone (10 mM, 20 L) was then topically applied ensuring that the solution was only applied to the epidermis top. EPR spectra were then recorded. If a noisy but detectable spectrum was observed after 7.5 minutes, the best S/N was achieved after 15 minutes of DEPMPO incubation. At 30 minutes a significative decay of the spin-adduct was observed. The observed EPR spectrum correspond to the spin-adduct ascribed to a carboncentered radical (spin-adduct 14) and hydroxyl radical (spin-adduct 1), with the very similar hfccs than in solution investigations (Figure 2.27). G, a H = 22.9 G and a P = 47.5 G. Characterized spin-adducts are resumed in Table 2.5. It appeared that a carbon centered radical was predominantly obtained.

Table 2.5. Spin-adducts formed from Lina-OOHs/DEPMPO system in RHE. 26,34,52 Spin-adducts topical application assignment, g, hfccs (Gauss) DEPMPO-OH 1 g=2.0052 aN=13.9 aH=14.0 aP=47.5

DEPMPO-carbon R 14

g=2.0050 aN=14.9 aH=21.5 aP=48.2

Limo-OOH investigations

An identical protocol as the one used for Lina-OOHs was applied to Limo-OOH. The best results were obtained after 30 minutes of DEPMPO incubation (Figure 2.28). However, the EPR spectrum corresponding to a carbon-centered spin-adduct 2 was observed, with hfccs of a N = 14.6, a H = 22.2 G and a P = 47.2 G. i.e. very much comparable with solution studies.

Conclusion

DEPMPO-carbon R 14

In all cases, oxygen-centered spin-adducts were detected and attributed to hydroxyl radical generation. Even if it could be hypothesized, due to the control experiments, that radicals were directly issued from the ROOHs, it is necessary still to prove if the ROOHs themselves could indirectly generate radical oxygen species (ROS) in the cellular matrices that could eventually also be trapped.

in Matlab software (version 2015b) (http://www.easyspin.org). Hyperfine coupling constants were in agreement with those reported in the literature.

General procedure in solution

Stock solutions were prepared for CumOOH (5.4 In HEPES only a low generation of hydroxy radical was seen. In H 2 O radical hydroxyl was generated but also carbon radicals. By increasing the iron concentration in the mixture, the carbon radical generated ratio increased. Here we can see using literature and databases that it deals with a carbon radical adduct. In those conditions in HEPES or PB, we don't observe any spin-adduct using PBN as a spintrap (data not shown). Some radical identifications were pretty difficult because they had similar hyperfine coupling constants. We were not able to differentiate for example DEPMPO-OCH 3 and DEPMPO-OOCum.

In Figure A6 In Annex 9, by increase iron concentration two radicals were trapped. There are two times more hydroxy radical then carbon radical at 0.1 mM Iron II. After 1 mM of iron II there is two times more carbon radical then hydroxy radical. The same facts are observed at t 30 min. The idea is to see if radical nature changes when in contact with residual CM contained in RHE.

The experiment showed that CM influenced the ratio oxygen/carbon radical generation and but not their nature. The radical generation started at weak iron concentration compared to Cum-OOH in HEPES. Also, the opposite transition 15 to 16 Gauss was seen at high Iron II concentration. In here it becomes clear at 1mM of Iron concentration at t = 30 min conversion is complete. Sensitizers derived from oxidation processes are often potent oxidants that could be able to interfere with the cell redox balance. There is a belief that oxidative stress induced by these compounds on epithelial cells may produce a new danger signal, synergistic with reactivity and thus would help to explain the strong allergenic activity.

The aim here was to evaluate how the inherent oxidative properties of allylic hydroperoxides, together with their reactivity, behave as danger signal and translate into a biological response using the activation of the Keap1-Nrf2 cellular sensor pathway in dendritic cells, involved in detoxification and antioxidant effects.

These studies have been conducted in collaboration with Pr Saadia Kerdine-Römer, Allergy, Immunotoxicology and Immunopathology Unit, Université Paris Sud, INSERM UMR 996.

Previous studies conducted with this team showed that allergens, depending on their chemical structure, may activate the Nrf2 pathway in dendritic cells. 1 Compounds used for the studies were linalool (Lina), limonene (Limo), cumene (Cum), 1-tertbutyl-1-cyclohexene (Mod) and their respective allylic hydroperoxides, the cyclic model hydroperoxide (Mod-OOH) that has been used as model for 15-hydroperoxy abietic acid studies, cumene hydroperoxide (Cum-OOH), limonene hydroperoxide (Limo-OOH) and linalool hydroperoxides (Lina-OOHs) (Figure 3.1). 2 Shortly, without oxidative stress, Nrf2 (Nuclear Factor Erythroid 2-Related Factor 2) is linked to Keap1 (Kelch-like ECH-associated protein 1) by thiol groups from amino acids present in Keap1. 3 These thiol groups are very sensitive to oxidative stress and can recognize it leading to the release of Nrf2. The free Nrf2 is then able to migrate to the nucleus, bind to a Maf (musculoaponeurotic fibrosarcoma) protein and then the heterodimer access the antioxidant response element (ARE). 4 The THP-1 cell line, used as surrogate of dendritic cells, also uses the Keap1-Nrf2 system in order to keep cell homeostasis. Keap1 is the cytosolic repressor of Nrf2. In unstressed conditions Nrf2 is sequestered by the Kelch-like EC-associated protein 1 (Keap1) homodimer in the cytoplasm. Its expression is maintained to be low and ubiquitinated proteasomal degradation occurs. Under oxidative or electrophilic stress, cysteine residues of Keap1 are oxidized changing the homodimer 3D-configuration. Nrf2 degradation is then interrupted and a rapid cytoplasmic accumulation occurs. 5 Nrf2 translocates into the nucleus and combines with small Maf proteins. The new heterodimer activates the antioxidant response element (ARE)dependent gene expression, leading to the transcription of genes involved in detoxification, such as heme oxygenase-1 (ho-1) or NADPH-quinone oxidoreductase 1 (nqo1) and proinflammatory cytokines (il-8). 6 It has been shown that Nrf2 can be activated by different haptens. 1,7 We used the THP-1 cell line as a model of dendritic cells in order to study the activation of the Keap1-Nrf2 pathway by our target compounds. The idea was to follow the genotype and phenotype of these cells exposed to the hydroperoxides and to evaluate the oxidative stress produced by allergenic allylic hydroperoxides on the THP-1 cell line. In order to isolate these two position isomers, a method was developed in the laboratory by Kao et al. 9 It consisted on a selective chemical derivatization of the allylic hydroperoxide Lina-7-OOH by meaning of introducing a protective group. The chemical derivatization was carried out with tert-butyl(chloro)diphenylsilane and only Lina-7-OOH was protected.

Deprotection of the peroxy silane Lina-7-OOTBDPS was carried out in the presence of a methanolic solution of potassium hydroxide making it possible to obtain Lina-7-OOH in a yield of 92% over the two last steps. The methodology was reproducible. However, even if the global yield of the desired hydroperoxide Lina-7-OOH was good enough, a drawback was that it was not possible to obtain Lina-6-OOH inexplicably lost in the reaction.

In order to obtain enough amounts of both hydroperoxides for the in cellulo assays, we The four peaks were collected and stored at -80 ° C (2 times separately). They were extracted with petroleum ether (3 × 20 mL), the organic phase was dried over anhydrous Na 2 SO 4 for 20 minutes, then filtered and evaporated under reduced pressure. The product obtained was submitted to NMR analysis. Fractions HL5-0, HL5-1 and HL5-3 were taken up in deuterated chloroform, the HL5-2 fraction in deuterated methanol and analyzed by NMR. We never observed the spectra of Lina-6-OOH or Lina-7-OOH. It looked like the compounds were degraded. Thus, it was finally decided to use the mixture of Lina-7-OOH and Lina-6-OOH for the biological studies. In the next sections results on the activation of the Keap1-Nrf2 pathway are described.

Nrf2 cytoplasmic accumulation studies: Four molecules and their respective hydroperoxides

The ARE is the promotor region of antioxidant genes. Nrf2 is a transcription factor that will bind to this ARE promoter sequence and induce cytoplasm protective genes expression such as (ho-1) and phase II detoxification genes (nqo-1) as well as pro-inflammatory cytokines (il-8).

After exposure of the THP-1 cell line to the mixture Lina-6-OOH and Lina-7-OOH (1 mM in acetonitrile/water 25/75), an increase of Nrf2 was observed during the first 6 hours, then a decrease at 8 hours (data not shown). In contrast to the parent compound linalool, the hydroperoxides mixture seemed to induce the accumulation of Nrf2 in THP-1 cells (Figure 3.10). A strong protein accumulation of Nrf2 was observed after 6 hours exposure with Lina-6-OOH and Lina-7-OOH (1 mM mixture in acetonitrile/water, ratio 2:3) while we didn't observe this accumulation with Lina in comparison with the vehicle assay (acetonitrile/water).

Anti-oxidant gene expression modulation

Induction of the transcription of Nrf2 target genes ho-1, nqo1 and il-8 by ROOHs was studied.

As described in the section above, the ROOHs led to the cytoplasm accumulation of Nrf2. Nrf2 plays the role of the transcription factor for over-expression of genes such as ho-1, nqo1 and il-8. Lina-6-OOH and Lina-7-OOH (1 mM mixture in acetonitrile/water, ratio 2:3) significantly induced the expression of ho-1 and nqo-1 whereas this induction was not observed for Lina (1 mM). Il-8 was not induced by any molecule in all assays.

Limo-OOH (1.5 mM) didn't induce the expression of ho-1, nqo1 and il-8 genes compared to Limo. Furthermore, the same basal gene expression was observed as in the example on Lina exposure. DNCB and tBHP (tert-butylhydroperoxide) as positive controls underlined the genes overexpression in the case of Lina-OOHs. Cum-OOH (1 mM) significantly induced the expression of ho-1, nqo-1 and il-8 while this induction was not observed for Cum in comparison with vehicle (acetonitrile/water).

Finally, Mod-OOH (1 mM) and Mod (1 mM) significantly induced the expression of ho-1, nqo-1 and il-8. Surprisingly, Mod was reactive even if no hydroperoxide function is present in its that was also activating the Nrf2 pathway surprisingly. According to the results, Lina-OOHs (1 mM), Cum-OOH (1 mM) and Mod-OOH (1 mM) behaved as stronger activators of THP-1 cells than Limo-OOH (1.5 mM). No significant differences between Mod (1 mM) and Mod-OOH (1 mM) were measured. A hypothesis could be that more radical rearrangements could lead to a stronger activation of THP-1 cells and could explain for example why cells are more activated after treatment with Lina-OOHs (1 mM).

MATERIALS AND METHODS:

Different molecules were tested: linalool (Lina), limonene (Limo), cumene (Cum), 1-tert-butyl-1-cyclohexene (Mod) and their respective allylic hydroperoxides, the cyclic model hydroperoxide (Mod-OOH), cumene hydroperoxide (Cum-OOH), limonene hydroperoxide (Limo-OOH) and linalool hydroperoxides (Lina-OOHs)

Chemicals

Tert-butyl hydroperoxide (tBHP), dithiothreitol (DTT), acetonitrile (CH 3 CN), (R)-(+)limonene (LIMO) and linalool (LINA) were all purchased from Sigma-Aldrich (St Quentin-Fallavier, France).

Synthesis of Mod-OH

To 1-acetyl-1-cyclohexene (15.5 g, 125 mmol) in tetrahydrofuran (150 mL) was added, at -78 °C, methyllithium (87.5 mL, 140 mmol, 1.6 M in Et 2 O). After 2 h stirring at -78 °C, the reaction mixture was allowed to warm up to room temperature. Aqueous NH 4 Cl (50 mL) was carefully added and the mixture extracted with ether (3 × 50 mL). Then combined organic layers were dried over magnesium sulfate, filtered, and concentrated under reduced pressure to lead to the crude alcohol, which was purified by column chromatography on silica (petroleum ether/ hexane 9/1) to yield 16.68 g (94 % yield) of pure alcohol (Mod-OH) as a colorless oil. UV for 1 min. Proteins were transferred to PVDF membrane (Bio-Rad Laboratories, Hercules, CA) using the Bio-Rad Trans-Blot Turbo Transfer System for 7 min. Total proteins on membranes were detected using the Stain-free method. 12 For the Nrf2/Keap1 analysis pathway, membranes were incubated with antibodies raised against Nrf2 (H-300; Santa Cruz Biotechnology, Heidelberg, Germany). Immunoreactive bands were detected by chemiluminescence (ECL solution, Amersham Biosciences).

Formula

Total RNA isolation

THP-1 cells (10 6 cells/ml, 2 ml/well) were exposed to test compounds at a subtoxic concentration for 6 hours. This time point was determined in preliminary experiments and was found to be optimal to study the expression of Nrf2 target genes (data not shown). For the RNA extraction, the Macherey Nagel kit was used according to the manufacturer protocol. Treated cells were washed twice with cold PBS. 350 μL of Lysis Buffer was added to the cell pellet and the tubes were vortexed vigorously. The viscosity was reduced and the lysate was cleared by filtration through a filter, the mixture was added to the filter and the tubes were centrifuged for 1 min at 11000 × g. The filter was discarded and 350 μL of ethanol (70%) was added and mixed by pipetting up and down in order to adjust the RNA binding conditions. The lysate was loaded onto a new column which binds to the RNA and the tubes were centrifuged 30 seconds at 11000 × g. The column was placed in a new tube and 350 μL of Membrane Desalting Buffer was added. The tubes were centrifuged 1 minutes at 11000 × g to dry the membrane. Then the silica membrane was washed three times with different buffers and the RNA eluted in 40 μL of RNAse-free water. Total RNA yield was quantified by spectrophotometry using two dilutions (total RNA diluted at 1:100 and 1:200, two independent measurements).

Reverse transcription

RNA concentrations were adjusted (80 ng/μl for THP-1 cells in 11 μl final volume) and combined with 2 μl of deoxynucleotide triphosphate mixture and 2 μl of 50 μM oligo(dT) primers. Mixes were denatured at 70°C for 10 min and then quickly cooled at 4°C in a Biometra thermocycler (Goettingen, Germany). Reverse transcription was carried out in AMV Reverse

Transcriptase Reaction Buffer (Promega, Charbonnières-les-Bains, France) with final concentrations of 2 U/μl of RNase inhibitor (RNasine, Promega) and 0.2 U/μl of AMV Reverse Transcriptase (Promega), and RNase-free water to make up a final volume of 10 μl. The reaction was then performed for 1 h 30 min at 42°C in the thermocycler. A control without reverse transcriptase was performed to confirm that no DNA contamination had occurred. The final product was stored at -20°C and used in the PCR step.

Analysis of gene expression using real-time PCR

Real-time PCR analysis was performed using the SYBR Green technology on a Biorad CFX96 Manager software, and data were analyzed using the delta-delta Ct method (ΔΔCt). The ratio was calculated as the geometrical mean of (1 + E) -ΔΔCt values, where E is the efficiency and ΔΔCt is the target gene expression of treated cells compared with normal levels in untreated cells and corrected using the expression of the reference genes gapdh and β-actin. Results were expressed as fold induction (i.e., ratio of (1 + E) -ΔΔCt of treated cells/(1 + E) -ΔΔCt of vehicletreated cells).

Measurement of Nrf2 DNA binding

DNA binding was analyzed using specific ELISA-based TransAM kits from Active Motif as described by 13 . After being treated THP-1 cells were lysed in a hypertonic buffer constituted of Non-diet P40 (NP40), 20% glycerol, 20 mM HEPES-KOH pH 7.9, 420 mM NaCl, 1 mM DTT, 1mM sodium orthovanadate, 1 mM pyrophosphate sodium, 125 mM okadaic acid, 62.5 mM EDTA, 40 mM EGTA, 0.5 mM PMSF, 1 g/ml aprotinin, 1 g/ml pepstatin and 1 g/ml leupeptin. After being left in ice for 20 min, THP-1 cells were centrifuged 20 min at 4°C at 17600 × g. Total protein concentration was evaluated using bicinchoninic acid assay and 20 μg of protein were used for the assay. Total protein cell extracts (20 mg) were incubated for 1 hour in a 96-well plate to which oligonucleotides, containing consensus binding sites for Nrf2, had been immobilized. After washing, the plate was incubated for 1 hour with the appropriate primary Ab, which specifically detected an epitope accessible only when Nrf2 is activated and bound to its cognate oligonucleotide. The plate was then washed and incubated with horseradish peroxidase-conjugated secondary Ab (1:1000) for 1 hour at room temperature. Colorimetric readout was quantified by spectrophotometry at 450 nm. Independent experiments were repeated at least three times.

  dimethyl-1-pyrroline-N-oxide, 2,2-dimethyl-

  4 MATERIALS AND METHODS ........................................................................................................................ 4.1 Studies in solution ............................................................................................................................... 4.1.1 Chemicals and reagents ......................................................................................... 4.1.2 Buffer Solutions ...................................................................................................... 4.1.3 EPR equipment ....................................................................................................... 4.1.4 General procedure in solution ...............................................................................

4. 3 . 2

 32 Synthesis of Limonene-2-hydroperoxide Limo-OOH .............................................

4. 3 . 3

 33 Synthesis of (5E)-7-Hydroperoxy-3,7-dimethylocta-1,5-dien-3-ol and 5-(13 C)-6hydroperoxy-3,7-dimethylocta-1,7-dien-3-ol (Lina-OOHs)............................................. Annexes:……………………………………………………………………………………………………………………………..156 Literature:....................................................................................................................... CHAPTER 3 ............................................................................................................................... IN-CELLULO STUDIES WITH DENDRITIC CELLS THE KEAP1-NRF2 ACTIVATION PATHWAY ................................................................... 1 THE KEAP1-NRF2 PATHWAY ..................................................................................................................... 2 TARGET COMPOUNDS SYNTHESES ............................................................................................................ 3 RESULTS AND DISCUSSION.......................................................................................................................... 3.1 Nrf2 cytoplasmic accumulation studies: Four molecules and their respective hydroperoxides ................................................................................................................................................... 3.2 Anti-oxidant gene expression modulation .................................................................................. 4 CONCLUSION ................................................................................................................................................... Experimental Chapter 3 .................................................................................................

  liaison O-O de la fonction hydroperoxyde a une faible énergie de dissociation (175 kJ.mol -1 ), elle peut être facilement clivée par un mécanisme de transition électronique et conduire à la formation de radicaux oxygénés tels le radical allyloxyle. En raison de la présence de doubles liaisons allyliques, des réarrangements intramoléculaires ou de l'abstraction d'hydrogène allylique peuvent avoir lieux et ainsi former des radicaux carbonés dans les positions 2, 4 et 5, par exemple.

Figure 1 :

 1 Figure 1 : Structure du linalol 1 et du (5E)-7-hydroperoxy-3,7-diméthylocta-1,5-dièn-3-ol 2 et réarrangements radicalaires L'objectif était de synthétiser l'hydroperoxyde allylique 2 isotopiquement substitué au 13 C dans les positions 2, 4 ou 5, où un radical carboné peut se former par décomposition de l'hydroperoxyde, et d'étudier la réactivité de chacun de ces composés vis-à-vis des acides aminés protéiques. L'objectif principal était d'étudier, de cette façon, s'il existe une spécificité de réaction d'un certain radical carboné vis-à-vis d'un acide aminé précis.

Figure 3 :

 3 Figure 3 : 5-13 C-Lina-OOHs (1 éq., 5.37 µM), N-Acétyle-L-cystéine méthylester (2éq., 10.74 µM) et une quantité catalytique de fer (II) (0.1 éq.) dans un milieu CD 3 CN/H 2 O 1/1 durant une semaine de réactivité

Figure 4 :

 4 Figure 4 : Structures hypothétiques des composes 25, 26 et 27 basées sur l'interprétation du spectre HMBC

Figure 5 :

 5 Figure 5 : Structures hypothétiques des composés 25, 26 et 28 basées sur l'interprétation du spectre HMBC

  En collaboration avec le laboratoire POMAM, Propriétés Optiques et Magnétiques des Architectures Moléculaires, Université de Strasbourg, CNRS-UMR 7177 Nous avons développé dans le deuxième chapitre de ce manuscrit, l'utilisation de la RPE combinée aux techniques de piégeage de spin afin d'étudier la formation et le comportement des radicaux provenant des hydroperoxydes allyliques sensibilisants (Lina-OOHs et Limo-OOH) dans un modèle 3D d'épiderme humain reconstitué (RHE). Nous avons choisi de travailler avec le modèle Episkin TM (Lyon, France). Il se compose d'une culture de kératinocytes humains à couches multiples normales et est similaire histologiquement à l'épiderme humain.

Figure 6 :

 6 Figure 6 : Formation de spin adduit du système Cum-OOH/spin trap/fer (II)

Figure 7 :

 7 Figure 7 : Formation de spin adduit du système Limo-OOH/spin trap/fer (II)

Figure 8 :

 8 Figure 8 : Spectre RPE obtenu dans les RHE par application topic : expérience utilisant du Cum-OOH (1 mM dans l'acétone), DEPMPO (50mM dans l'HEPES): (A) expérience control avec uniquement du DEPMPO et le véhicule; (B) Spectre RHE avec application du CumOOH et incubation (5 min, 37 °C, 5% CO 2 ) suivie par l' addition du DEPMPO; (C) simulation des spectres par ordinateur (B); (D-E) déconvolution de DEPMPO-carbone R et DEPMPO-OH.

  En collaboration avec le laboratoire de Signalisation en Immunotoxicologie et en Immunopharmacologie, Université de Paris Sud, INSERM-UMR 996 Le but ici a été d'évaluer le stress oxydant produit par des hydroperoxydes allyliques allergisants comparés à leur molécule d'origine non sensibilisante sur la lignée cellulaire THP-1, modèle reconnu des cellules dendritiques présentatrices d'antigène jouant un rôle clé dans l'ADC. La voie de signalisation Keap1-Nrf2, impliquée dans la réponse cellulaire antioxydante, a été utilisée afin de détecter le stress oxydant induit par ces composés. Quatre hydroperoxydes allyliques de structure chimique différente ont été testés : un modèle cyclique, le Cum-OOH, et les hydroperoxydes de linalol et de limonène (Figure 9).

Figure 9 :

 9 Figure 9 : Hydroperoxydes allyliques pour l'étude d'activation de la voie Keap1-Nrf2

Figure 10 :

 10 Figure 10 : Accumulation cytoplasmique de Nrf2 suivant l'exposition aux Lina, Limo, Lina -OOHs et Limo-OOH en comparaison avec le véhicule (acétonitrile/eau) comme contrôle négatif et aussi au « loading » control

Figure 11 :

 11 Figure 11 : Surexpression des gènes ho-1, nqo-1 et il-8 après exposition aux LinaOOHs et Limo-OOH comparé à leurs molécules parentes et aux véhicules

Figure I. 2

 2 Figure I.2 resumes the mechanism inducing ACD to a chemical after skin penetration. It is based on two distinct phases: sensitization and elicitation.

Figure I. 2 :

 2 Figure I.2: ACD mechanism based on the sensitization and elicitation phases

Figure I. 3 :

 3 Figure I.3: Photography of an ACD patient (courtesy Pr Ann Goossens)

Figure I. 5 :

 5 Figure I.5: Activation of different types of haptens 28

Figure I. 6 :

 6 Figure I.6: Example of geraniol autoxidation: generation of an allylic skin sensitizing hydroperoxide among other sensitizers

(

  Figure I.8).44

Figure I. 8 :

 8 Figure I.8: Decisional tree from DPRA classifying sensitizers according to their reactivity toward Cys-Pep and Lys-Pep

Figure I. 9 :

 9 Figure I.9: Hapten-protein reaction mechanisms via two-electrons and radical pathways

Figure I. 10 :

 10 Figure I.10: Limonene and linalool autoxidation primary products

Figure I. 11 :

 11 Figure I.11: Radical intermediates derived from linalool hydroperoxide 2

  Figure I.14). Commercial cumene hydroperoxide (Cum-OOH) and a synthetic monocycle hydroperoxide (Mod-OOH) have been used as reference compounds for some of the studies developed here. Cum-OOH is a reference molecule widely used in Electron Paramagnetic Resonance studies and Mod-OOH is a model molecule for the study of allergenic 15-hydroperoxiabietic acid.[START_REF] Mutterer | Synthesis and Allergenic Potential of a 15-Hydroperoxyabietic Acid-like Model: Trapping of Radical Intermediates[END_REF][START_REF] Pazos | Amino Acid and Protein Scavenging of Radicals Generated by Iron/Hydroperoxide System: An Electron Spin Resonance Spin Trapping Study[END_REF]

Figure I. 14 :

 14 Figure I.14: Target compounds: limonene hydroperoxide, linalool hydroperoxides, cumene hydroperoxide and monocycle hydroperoxide

Figure I. 15 :

 15 Figure I.15: Different types of radical intermediate issued from Lina-7-OOH based on Kao et al.[START_REF] Kao | Synthesis of Allylic Hydroperoxides and EPR Spin-Trapping Studies on the Formation of Radicals in Iron Systems as Potential Initiators of the Sensitizing Pathway[END_REF]

Figure I. 16 :

 16 Figure I.16: 13 C-target compounds: 5-( 13 C)-Lina-OOH, 4-( 13 C)-Lina-OOH and 2-( 13 C)-Lina-OOH

Figure 1 . 1 :

 11 Figure 1.1: General mechanism for autoxidation processes based on three steps: initiation, propagation and termination

Figure 1 . 2 :

 12 Figure 1.2: Molecular orbitals diagram of the triplet oxygen state 3 O 2, the two-red spins are in two orbitals  px * and  py * and in the singlet oxygen state 1 O 2 , the two-red spins are in the same orbital  px *

Figure 1 . 3 : 2 Figure 1 . 4 :

 13214 Figure 1.3: Examples of photosensitizers used to produce 1 O 2 from 3 O 2

Figure 1 . 5 : 4 ;

 154 Figure 1.5: Autoxidation of linalool: 45 weeks of oxygen exposure gave the best yield 15 % of compounds 2 and 3; after 50 weeks of oxygen exposure was obtained the best yield 20% of compound 4; after 79 weeks of oxygen exposure was obtained the best yield 4 % of compound 5; 6 and 7 compounds are secondary oxidation products

1 . 6 : 7 Figure 1 . 7 :

 16717 Figure 1.6: Schenck reaction: general representation

Figure 1 . 8 :

 18 Figure 1.8: Steric hindrance influence on the transition states in the Schenck ene-reaction with 1 O 2

Figure 1 . 9 :

 19 Figure 1.9: Hydrogen peroxide disproportionation in the presence of sodium molybdate

Figure 1 . 10 :

 110 Figure 1.10: Schenck reaction on linalool 1 and obtention of Lina-7-OOH 2 / Lina-6-OOH 3

Figure 1 . 11 :

 111 Figure 1.11: Synthesis of hydroperoxides from tertiary, secondary and primary alcohols (R = allylic chain; R' = H, alkyl; R'' = alkyl; Lg = leaving group)

Figure 1 . 13 :

 113 Figure 1.13: Example of adducts with same m/z value

Figure 1 . 14 :

 114 Figure 1.14: Chemical structures of ( 13 C)-substituted linalool target compounds

Figure 1 . 15 :

 115 Figure 1.15: Proposed retrosynthesis for the obtention of linalool ( 13 C)-substituted in positions 5, 4 and 2

( 13 C 11 Figure 1 . 16 : 6 A

 13111166 Figure 1.16: Synthesis of 13 C-6

Figure 1 . 18 :

 118 Figure 1.18: Synthesis of 13 C-12 and 13 C-13

( 13 C

 13 )-ethyl acetate with a yield of 58 % (Figure1.19).

Figure 1 . 19 :

 119 Figure 1.19: Synthesis of ( 13 C-16)

  13 C-17 with a yield of 70 % (Figure1.20). The conditions described by Reetz et al. using organometallic reagents to realize this step were employed.19

Figure 1 . 20 : 20 Finally, 4 -

 120204 Figure 1.20: Synthesis of 13 C-19 and 13 C-20

  ( 13 C)-cyanide as reagent introducing the13 C-substitution. The key step consisted in the synthesis of trimethylsilyl ( 13 C)-cyanide 13 C-21 starting from potassium-( 13 C)-cyanide 13 C-3. In order to carry out the synthesis of trimethylsilyl ( 13 C)-cyanide 13 C-21, better experimental conditions were set up without13 Csubstituted reagents.A method based on Cao et al. work suggested the use of potassium cyanide or sodium cyanide, with zinc iodide and polyethylene glycol 400 (PEG 400), either in acetonitrile or in dichloromethane (Figure1.21).20 Iodide ions have an excellent catalytic effect for S N 2 reactions, zinc iodide being a Lewis acid which facilitates the cleavage of the Si-Cl bond.

Figure 1 . 21 :

 121 Figure 1.21: Me 3 SiCN synthesis using zinc iodide and PEG400 and the experimental conditions described by Cao et al.20

Figure 1 . 22 : 23 Figure 1 . 23 :

 12223123 Figure 1.22: Trimethylsilyl cyanide synthesis containing a 13 CN group

Figure 1 . 24 :

 124 Figure 1.24: Reduction of 13 C-22 nitrile to aldehyde 13 C-23

Figure 1 . 25 :

 125 Figure 1.25: Synthesis of linalool 24 from aldehyde 23 using methylentriphenylphosphorane, parent member of phosphorous ylides

  linalool hydroperoxides. Gardner et al. reported in the literature the formation of an adduct between N-acetyl-cysteine and 13-hydroperoxylinoleic acid (Figure1.26).29

Figure 1 . 26 :

 126 Figure 1.26: Adduct formation between 13-hydroperoxylinoleic acid and N-acetyl-cysteine in presence of a catalytic amount of iron chloride (III)29

Figure 1 . 27 :

 127 Figure 1.27: Spin trapping of a tyrosine carbon-centered radical by 5-diethoxyphosphoryl-5-

13 C-

 13 Substituted Lina-OOHs (1 mg, 5.37 µM) in the presence of an excess of a methylic ester of the N-acetyl amino acids cysteine, tryptophan, tyrosine or lysine (2 to 10 eq. for each one), in a degassed mixture of deuterated acetonitrile and water (1/1, v/v) were treated with a catalytic amount of iron (II) sulfate at room temperature. The progress of the reaction was monitored by 13 C-NMR (on a Brucker Avance 500 MHz spectrometer) until complete disappearance of Lina-OOHs or no more evolution of the reaction.

  5 µM) was sufficient to induce radical initiation (Figure 1.28). Control experiments were realized without the presence of iron (II) as initiator.

Figure 1 .

 1 Figure 1.28: 5-13 C-Lina-OOHs (1 eq., 5.37 µM), N-Acetyl-L-cysteine methyl ester (2eq., 10.74 µM) and a catalytic amount of iron (II) (0.1 eq.) in CD 3 CN/H 2 O 1/1 medium during one week reaction time

Figure 1 . 29 :

 129 Figure 1.29: Hypothetical chemical structures of compounds 25, 26 and 27 based on HMBC interpretation

Figure 1 .

 1 Figure 1.30: 5-13 C-Lina-OOHs (1 eq., 5.37 µM), N-Acetyl-L-cysteine methyl ester (2eq., 10.74 µM) without iron (II) in CD 3 CN/H 2 O 1/1 medium during one week reaction time

Figure 1 .

 1 Figure 1.31: 4-13 C-Lina-OOHs (1 eq., 5.37 µM), N-Acetyl-L-cysteine methyl ester (2eq., 10.74 µM) and a catalytic amount of iron (II) (0.1 eq.) in CD 3 CN/H 2 O 1/1 medium during one week reaction time

Figure 1 . 32 :

 132 Figure 1.32: Hypothetical chemical structures of compounds 25, 26 and 28 based on HMBC/HSQC interpretation

Figure 1 .

 1 Figure 1.33: 5-13 C-Lina-OOHs (1 eq., 5.37 µM), N-acetyl-L-tyrosine methyl ester (2eq., 10.74 µM) and a catalytic amount of iron (II) (0.1 eq.) in CD 3 CN/H 2 O 1/1 medium during one week reaction time

Figure 1 .

 1 Figure 1.34: 4-13 C-Lina-OOHs (1 eq., 5.37 µM), N-acetyl-L-tyrosine methyl ester (2eq., 10.74 µM) and a catalytic amount of iron (II) (0.1 eq.) in CD 3 CN/H 2 O 1/1 medium during one week reaction time

Figure 1 .

 1 Figure 1.35: 5-13 C-Lina-OOHs (1 eq., 5.37 µM), N-Acetyl-L-tryptophan methyl ester (2eq., 10.74 µM) and a catalytic amount of iron (II) (0.1 eq.) in CD 3 CN/H 2 O 1/1 medium during one week reaction time

Figure 1 .

 1 Figure 1.36: 4-13 C-Lina-OOHs (1 eq., 5.37 µM), N-Acetyl-L-tyrosine methyl ester (2eq., 10.74 µM) and a catalytic amount of iron (II) (0.1 eq.) in CD 3 CN/H 2 O 1/1 medium during one week reaction time

Figure 1 .

 1 Figure 1.37: 5-( 13 C)-Lina-OOHs (1 eq., 5.37 µM), N-acetyl-L-lysine methyl ester (2eq., 10.74 µM) and a catalytic amount of iron (II) (0.1 eq.) in CD 3 CN/H 2 O 1/1 medium during one week reaction time

Figure 1 .

 1 Figure 1.38: 4-13 C-Lina-OOHs (1 eq., 5.37 µM), N-acetyl-L-tyrosine methyl ester (2eq., 10.74 µM) and a catalytic amount of iron (II) (0.1 eq.) in CD 3 CN/H 2 O 1/1 medium during one week reaction time

19 and 13 C- 20 , 6 -

 1913206 reactivity of 5-( 13 C)-Lina-OOHs, 13 C-12 and 13 C-13, and of 4-( 13 C)-Lina-OOHs, 13 Cwas carried out with four different protected amino acids. This study showed transformation of 5-( 13 C)-Lina-OOHs ( 13 C-12 and 13 C-13) and 4-( 13 C)-Lina-OOHs ( 13 C-19 and 13 C-20) only in presence of N-acetyl-L-cysteine methyl ester. The 5-( 13 C)-Lina-OOHs ( 13 C-12 and 13 C-13) 13 C peaks disappeared completely in a week reaction time whereas 4-( 13 C)-Lina-OOHs ( 13 C-19 and 13 C-20) didn't completely disappear after a week reaction time. Furthermore, 5-( 13 C)-Lina-6-OOHs 13 C-13 disappeared faster than 13 C-12, also 4-( 13 C)-Lina-OOHs 13 C-20 disappeared faster than 4-( 13 C)-Lina-7-OOHs 13 C-19, so is Lina-6-OOH hypothetically more reactive then Lina-7-OOH.

for 13 C

 13 ) and a Bruker Avance 500 (500 MHz for 1 H and 125 MHz for 13 C) spectrometers. The chemical shifts (δ) are reported in ppm and coupling constants (J) are reported in Hz. The chemical shifts (δ) are indirectly referenced to TMS via the solvent signal CHCl 3 (CDCl 3  1 H = 7.26 ppm;  13 C = 77.2 ppm). Multiplicity was denoted as s (singlet), sl (singlet large), d (doublet), dd (doublet of doublets), t (triplet) and m (multiplet).

4. 2

 2 Synthesis of 5-( 13 C)Lina-OOHs 2-Bromo-1-( 13 C)-ethyl acetate ( 13 C-4)

NMR 13 C 5 (Ethyl- 1 -( 13 C)- 3 -methyl but- 2 -enoate ( 13 C- 6 ) 1 CAS (non 13 C 8 TLC: 4 NMR 1 H 2 ( 1 CAS (non 13 C 1 NMR 1 H 3 J 1 J

 1351133213611384121131131 (CDCl 3 , δ, ppm) : 14.1 (OCH 2 CH 3 ),16.4 (d, 2 × OCH 2 CH 3 , 3 J CP = 6.5 Hz), 34.dd, CH 2 , 1 J CC = 59.0 Hz,1 J CP = 134.0 Hz), 61.6 (OCH 2 CH 3 ), 62.7 (d, 2 × OCH 2 CH 3 , 2 J CP = 6.5 Hz), 166.0 (d,13 C=O,2 J CP = 6.1 Hz).Sodium hydride (60 % in mineral oil, 0.87 g; 22.10 mmol; 1 eq.) was introduced in a flamed dried flask under argon. 2-(Diethoxyphosphoryl)-1-( 13 C)-ethyl acetate ( 13 C-5) (4.95 g; 22.10 mmol; 1eq.) dissolved in anhydrous tetrahydrofuran (50 mL) was added at 0 °C. The mixture was stirred for 15 minutes. Anhydrous acetone (1.57 mL; 20.1 mmol; 0.95 eq.) was added to the reaction and the mixture was stirred during 30 minutes at 0°C then allowed to warm to room temperature overnight. The reaction was quenched using a saturated solution of ammonium chloride (25 mL). The aqueous phase was extracted with diethyl ether (3 × 80mL), organic fractions collected, dried over magnesium sulfate , filtered then concentrated under reduced pressure. The product was purified by silica gel column chromatography (pentane/diethyl ether 97/3), to obtain 13 C-6 as a yellow oil (1.46 g; 51 %). Formula: C 6 13 CH 12 O 2 Molar mass: 129.16 g.mol --substituted): 638-10-Rf = 0.3 (pentane/diethylether 97/3): KMnO (CDCl 3 , δ, ppm): 1.25 (t, 3H, OCH 2 CH 3 , 3 J HH = 7.1 Hz), 1.88 (s, 3H, CH 3 cis), 2.14 (s, 3H, CH 3 trans), 4.15 (dd, 2H, CH 2 , 3 J HH = 7.1 Hz, 3 J HC = 3.1 Hz), 5.67 (m, 1H, =CH). NMR 13 C (CDCl 3 , δ, ppm): 13.9 (OCH 2 CH 3 ), 19.9 (CH 3 cis), 27.7 (CH 3 trans), 59.OCH 2 CH 3 ), 116.0 (d, =CH, 1 J CC = 75.4 Hz), 156.5 ((CH 3 ) 2 C=), 166.7 ( 13 C=O). 1-( 13 C)-3-Methylbut-2-en-1-ol ( 13 C-7) A diisobutylaluminum hydride solution in dichloromethane (27.50 mL; 27.50 mmol; 2.4 eq.)was introduced into a flame-dried round bottom flask equipped with an addition funnel and cooled at -78°C. 13 C-6 (1.46 g; 11.43 mmol; 1 eq.) dissolved in anhydrous dichloromethane (20 mL) was slowly added. The reaction mixture was allowed to warm to room temperature overnight. The reaction was stopped by adding ethyl acetate (10 mL) and water (10 mL). The solution was filtered on celite and the organic phase was separated. The aqueous phase was extracted with diethyl ether (2 × 10 mL). The combined organic phases were dried over magnesium sulfate, filtered and concentrated under reduced pressure . 1-( 13 C)-3-Methyl but-2en-1-ol ( 13 C-7) (1.65 g) was obtained as a transparent oil and used without further purification. Formula: C 4 13 CH 10 O Molar mass: 87.13 g.mol --substituted): 556-82-(CDCl 3 , δ, ppm): 1.69 (s, 3H, CH 3 cis), 1.75 (s, 3H, CH 3 trans), 4.10 (dd, 2H, 13 CH 2 , HH = 7.1 Hz, 1 J HC = 142.1 Hz), 5.41 (m, 1H, =CH).NMR 13 C (CDCl 3 , δ, ppm): 17.8 (CH 3 cis), 25.7 (CH 3 trans), 59.3 (HO 13 CH 2 ), 123.6 (d, =CH, CC = 47.0 Hz), 136.3 ((CH 3 ) 2 C=).

Formula: C 4 13 CH 9 Br 3 NMR 1 H 3 J 1 J

 93131 Molar mass: 150.02 g.mol -1 CAS (non 13 C-substituted): 870-63-(CDCl 3 , δ, ppm): 1.73 (s, 3H, CH 3 cis), 1.78 (s, 3H, CH 3 trans), 4.15 (dd, 2H, CH 2, HH = 8.5 Hz, 1 J HC = 153.0 Hz), 5.48-5.56 (m, 1H, =CH).NMR 13 C (CDCl 3 , δ, ppm): 17.5 (CH 3 cis), 25.8 (CH 3 trans), 29.7 ( 13 CH 2 ), 121.6 (d, =CH, CC = 47.0 Hz), 137.9 ((CH 3 ) 2

ALinalool ( 13 C- 11 ) 3 1 CAS (non 13 C 4 TLC 13 CH 2 (

 131131134132 microemulsion was prepared by slowly adding a sodium molybdate solution (0.239 g in 1.27 mL distilled water) to a suspension of sodium dodecyl sulfate (1.975 g) in butanol (2.41 mL) and dichloromethane(11.76 mL). At the end of the addition, the microemulsion became clear after 5 minutes stirring.5-( 13 C)-Linalool 13 C-11 (0.198 g; 1.26 mmol; 1 eq.) was introduced in the microemulsion at room temperature. Hydrogen peroxide (aqueous solution 35%; 71 µL one fraction) was then added. The solution was instantly red colored. After 20 minutes stirring the solution became yellow. Fourteen other fractions of hydrogen peroxide (aqueous solution 35%; 71 µL EACH) were added successively by an interval of 10 minutes each. The resulting yellow solution was stirred overnight at room temperature. When the reaction was thoroughly translucent, 5-( 13 C)was completely oxidized. The solvent was removed under reduced pressure at 40 °C. The semi-solid white product obtained was suspended in dichloromethane(65 mL) and vigorously stirred for 48 hours. The suspension was filtered to recover sodium molybdate and sodium dodecyl sulfate. The filtrate, partially evaporated under reduced pressure, was washed using distilled water (3 × 50 mL) and the aqueous phase was extracted with dichloromethane (50 mL). Combined organic phases were dried over magnesium sulfate, filtered and concentrated under reduced pressure to give a crude yellow oil. A mixture of hydroperoxides ( 13 C-12) and ( 13 C-13) (ratio 2/3) was obtained using silica gel column chromatography (petroleum ether/AcOEt 8/2, then 6/4) as a yellow oil (0.238 g; 1.25 mmol; 99%). Formula: C 9 13 CH 18 O Molar mass: 187.24 g.mol --substituted): Isomer ( 13 C-12): 51276-32-5 -Isomer ( 13 C-13): 51276-31-= 0,24 (petroleum ether/AcOEt 7/3): APM/Ce NMR 1 H (CDCl 3 , δ, ppm) Isomer ( 13 C-12) (mixture of two enantiomers): 1.27 (s, 3H, CH 3 C(OH)), 1.31 (s, 6H, (CH 3 ) 2 C(OOH)), 2.28 (m, 2H, CH 2 C(OH)), 5.05 (dd, 1H, CH 2 (CH)C(OH), 3 J HH = 1.1 Hz, 2 J HH = 10.8 Hz), 5.21 (dd, 1H, CH 2 (CH)C(OH), 3 J HH = 1.1 Hz, 2 J HH = 17.4 Hz), 5.50-5.64 (m, 2H, 13 CHCH), 5.92 (dd, 1H, CHC(OH), 3 J HH = 10.7 Hz, 2 J HH = 17.3 Hz). Isomer ( 13 C-13) (mixture of four diastereoisomers) : 1.29 (s, 6H, 2 × CH 3 C(OH)), 1.51-1.66 (m, 4H, 2 × 13 CH 2 CH 2 ), 1.73 (s, 6H, 2 × CH 3 (C)CH(OOH)), 4.29 (m, 2H, 2 × CH(OOH)), 5.00 (m, 4H, 2 × CH 2 (C)CH(OOH)), 5.07 (dd, 2H, 2 × CH 2 (CH)C(OH), 3 J HH = 1.1 Hz, 2 J HH = 10.8 Hz), 5.17 (dd, 1H, CH 2 (CH)C(OH), 3 J HH = 1.1 Hz, 2 J HH = 17.4 Hz), 5.20 (dd, 1H, CH 2 (CH)C(OH), 3 J HH =1.6 Hz, 3 J HH = 17.4 Hz), 5.86 (dd, 1H, CHC(OH), 3 J HH = 10.8 Hz, 2 J HH = 17.4 Hz), 5.88 (dd, 1H, CHC(OH), 3 J HH =10.8 Hz, 2 J HH = 17.4 Hz).NMR 13 C (CDCl 3 , δ, ppm): Isomer 13 C-12 (mixture of two enantiomers): 24.4 (2 × (CH 3 ) 2 C(OOH)), 27.8 (CH 3 C(OH)), 45.1 (d, CH 2 C(OH), 1 J CC = 43.5 Hz), 72.8 (C(OH)), 82.1 (C(OOH)), 112.3 (CH 2 (CH)C(OH)), 126.7 (CH 2 ( 13 CH)CH), 138.0 (d, CHC(OOH), 1 J CC = 70.9 Hz), 144.8 (CHC(OH)) Isomer 13 C-13 (mixture of four diastereoisomers) : 17.1 (2 × CH 3 (C)CH(OOH)), 25.1 (d, 2 × CH)OOH, 1 J CC = 3.5 Hz), 28.2 and 28.4 (2 × CH 3 C(OH)), 37.7 and 37.8 (d, 2 × CH 2 C(OH), 1 J CC = 23.8 Hz), 73.1 (2 × C(OH)), 89.2 and 89.7 (d, 2 × CH(OOH), 1 J CC = 20.5 Hz), 112.3 (2 × CH 2 (CH)C(OH)), 114.2 and 114.3 (2 × CH 2 (C)CH(OOH)), 143.6 (2 × C(CH)OOH), 144.7 and 144.8 (2 × CHC(OH)).

NMR 13 C 13 CH 3 -

 13133 (CDCl 3 , δ, ppm): 14.1 (CH 3 ), 20.7 ( 13 CH 3 -C=O), 60.5 (CH 3 -CH 2 -O), 170.2 (d, C=O, 1 J CC = 60 Hz).

4-( 13 C)- 3 , 7 -

 1337 Dimethylocta-1,6-dien-3-ol or 4-( 13 C)-Linalool ( 13 C-18)

  prepared by slowly adding a sodium molybdate solution (0.876 g in 5 mL distilled water) to a suspension of sodium dodecyl sulfate (7.47g) in butanol (8.8 mL) and dichloromethane(45 mL). At the end of the addition, the microemulsion became clear after 5 minutes stirring.4-( 13 C)-Linalool 13 C-18 (0.712 g; 4.62 mmol; 1 eq) was introduced in the microemulsion at room temperature. Hydrogen peroxide (aqueous solution 35%; 260 µL one fraction) was then added. The solution was instantly red colored. After 20 minutes stirring, it became yellow.Fourteen other fractions of hydrogen peroxide (aqueous solution 35%; 260 µL each) were added successively by an interval of 10 minutes each. The yellow solution was stirred overnight at room temperature. When the reaction was thoroughly translucent, it meant that 4-( 13 C)-linalool 13 C-18 was completely oxidized. The solvent was removed under reduced pressure at 40 °C.

Formula: C 11 13 CH 23 NOSi 23 )

 2323 Molar mass: 226.40 g.mol -1 CAS (non 13 C-substituted): 58422-84-7 CCM : Rf = 0.90 (pentane/AcOEt 9/1) : APM/Ce NMR 1 H (CDCl 3 , δ, ppm): 0.21 (s, 9H, -Si(CH 3 ) 3 ), 1.55 (s, 3H, -C(CN)(OTMS)-CH 3 ), 1.61 (d, 3H, -CH=C-CH 3 cis, 4 J HH = 1.1 Hz,), 1.67 (d, 3H, -CH=C-CH 3 trans, 4 J HH = 1.3 Hz), 1.70 (m, 2H, -CH 2 -CH 2 -CH=C(CH 3 ) 2 ), 2.15 (m, 2H, -CH 2 -CH=C(CH 3 ) 2 ), 5.08 (m, 1H, -CH=C(CH 3 ) 2 ) NMR 13 C (CDCl 3 , δ, ppm): 0.1 (-Si(CH 3 ) 3 ), 16.4 (-CH=C-CH 3 cis), 21.8 (-CH=C-CH 3 trans), 24.4 (-CH 2 -CH=C(CH 3 ) 2 ), 27.7 (-C( 13 CN)(OTMS)-CH 3 ), 42.0 (-CH 2 -CH 2 -CH=C(CH 3 ) 2 ), 68.1 (d, -C( 13 CN)(OTMS)-CH 3 , 1 J CC = 57.5 Hz), 120.8 (-13 C≡N), 121.2 (-CH=C(CH 3 ) 2 ), 131.5 (-CH=C(CH 3 ) 2 ) In a flame-dried round bottom flask under argon 1-( 13 C)-2,6-dimethyl-2-((trimethylsilyl)oxy)hept-5-enenitrile 13 C-22 (0.57 mL; 2.2 mmol; 1.0 eq.) was dissolved in anhydrous diethyl ether (13 mL), cooled to -78 °C and a solution of diisobutylaluminium

Formula: C 11 1 CAS (non 13 C 6 CCM:

 111136 13 CH 24 O 2 Si Molar mass: 228.41 g.mol --substituted): 78-70-Rf = 0.80 (pentane/diethyl ether 95/5): APM/Ce NMR 1 H (CDCl 3 , δ, ppm): 0.14 (s, 9H, -Si(CH 3 ) 3 ), 1.26 (s, 3H, -C(CO)(OTMS)-CH 3 ), 1.56 (s, 3H, -CH=C-CH 3 cis), 1.64 (s, 3H, -CH=C-CH 3 trans), 1.51-1.68 (m, 2H, -CH 2 -CH 2 -CH=C(CH 3 ) 2 ), 1.97 (m, 2H, -CH 2 -CH=C(CH 3 ) 2 ), 5,01 (m, 1H, -CH=C(CH 3 ) 2 ), 9.51 (s, 1H, -CH=O).

NMR 13 C

 13 (CDCl 3 , δ, ppm): 0.1 (-Si(CH 3 ) 3 ), 15.4 (-CH=C-CH 3 cis), 19.8 (-CH 2 -CH=C(CH 3 ) 2 ), 20.8 (-CH=C-CH 3 trans), 23.4 (-C(CO)(OTMS)-CH 3 ), 37.0 (-CH 2 -CH 2 -CH=C(CH 3 ) 2 ), 78.5 (-C(CO)(OTMS)-CH 3 ), 121.6 (-CH=C(CH 3 ) 2 ), 130.0 (-CH=C(CH 3 ) 2 ), 202.9 (-CH=O).

Figure 2 . 1

 21 Figure 2.1 Schematic view of (a) an unpaired electron with a spin angular momentum (curved arrow) and magnetic moment (tilted arrow); (b) paramagnetic system in the presence of an external magnetic Field: the classical (up) and quantum approach (bottom) 8

1 2(

 1 spin parallel to the magnetic field) and 𝑚 𝑆 = -1 2

Figure 2 . 2 2 )

 222 Figure 2.2 Schematic description of the Zeeman effect for an isotropic electronic spin system S=½ placed into an external magnetic field B 0 and the resulting microwave absorption and EPR signal when the resonance condition (∆𝐸 = ℎ𝜈 = 𝐸 + -𝐸 -= 𝑔𝛽 𝑒 𝐵 0 𝑟𝑒𝑠 ) is verified.

Figure 2 . 3

 23 Figure 2.3 Schematic description of the magnetic energy level diagram and the allowed transitions for an unpaired electron in interaction with a nitrogen nucleus ( 14 N, I=1), leading to the classical 3 lines EPR spectrum e.g. obtained for a nitroxide derivative compound.

Figure 2 . 4 :

 24 Figure 2.4: Two examples of electron delocalized on 2 equivalents (left side two protons) and 2 nonequivalent (right side a proton and a nitrogen) nucleus, nuclear spin I and n number of nucleus (red numbers indicates the relative intensity of the signal)

Figure 2 . 5 :

 25 Figure 2.5: Schematic description of the spin-trapping approach: free radicals lifetimes are usually in the micro-to nanosecond time scale. Therefore, direct EPR characterization is not often possible. By reacting with a diamagnetic substrate (the spin-trap), the targeted reactive specie produces a paramagnetic spin-adduct with much longer lifetime (minutes to hours), whose the EPR signature is related to the nature of the trapped specie.

Figure 2 . 6 :

 26 Figure 2.6: Different spin-trapping with their respective spin-adducts representation: ,  and  indicate the positions of the nuclear spin (I ≠ 0) interacting with the electron spin.

Figure 2 . 7 :

 27 Figure 2.7: Example of DMPO spin-adducts EPR fingerprints: EPR signature (DMPO-X mix) with deconvoluted simulation of two oxygen radical and a carbon radical trapped. (A). methyl radical trapped, (B). hydroxyl radical trapped and (C). peroxyl radical trapped EPR fingerprints.

Figure 2 . 8 :

 28 Figure 2.8: Nitroxide formation from nitrone or nitroso compounds without radical trapping. a) ene-reaction: MNP reacting with an olefin; b) Forrester-Hepburn reaction: addition of a nucleophile and oxidation; c) IST oxidation of the nitrone forming a radical cation followed by a nucleophile addition 4

Figure 2 . 9 :

 29 Figure 2.9: Schematic interpretation of a simple EPR spectrum: DEPMPO-CH 3 is the spinadduct represented here. It deals with a carbon radical trapped characterized by three hfccs a N , a H , a P and g-factor in the middle of the spectra

Figure 2 . 11 :

 211 Figure 2.11: Schematic description of an EPR spectrometer composed of a microwave bridge

Figure 2 . 12 :

 212 Figure 2.12: Schematic illustration of the Magnetic (left) and electric field (right) in a standard EPR cavity containing a flat cell.

Figure 2 . 13 :

 213 Figure 2.13: Chemical structures of the target hydroperoxides used in the studies in solution.

Figure 2 . 15 :

 215 Figure 2.15: (A) EPR spectrum of DEPMPO (25 mM) spin-adducts formed from Limo-OOH (1 mM)/Fe(II) (0.1 mM) in H 2 O; (B) computer simulation and (C and D) deconvolution of DEPMPO-OH (ca. 80 %) 1 and DEPMPO-R (ca. 20 %) 2 (more available data in Annex 1, Figure A1.1), (* cavity contamination).

Figure 2 . 16

 216

50 %) 1 ,

 1 DEPMPO-carbon R (ca. 20 %) 6 and DEPMPO-OCH3/OOCum 7 (ca. 30 %).

Figure 2 . 17 :

 217 Figure 2.17: (A) EPR spectrum of DEPMPO (25 mM) spin-adducts formed from Cum-OOH (0.54 mM) / Fe(II) (0.5 mM) in HEPES, (B) computer simulation and (C, D and E) deconvolution of DEPMPO-OH (ca. 50 %) 1, DEPMPO-carbon R (ca. 20 %) 6 and DEPMPO-OCH 3 /OOCum 7 (ca. 30 %) (more data available in Annex 6, Figure A6.2).

Figures A7. 1

 1 Figures A7.1 and A7.2).44

Figure 2 . 19 :

 219 Figure 2.19: Formation of CumOOH/Fe(II) spin-adducts and related mechanisms.

  literature shows that oxygen-centered radicals can produce carbon-centered radicals by different rearrangement processes. Allyloxyl radicals formed from cleavage of the O-OH bond can follow β-scission and 3-exo-trig cyclization for example that can further evolve to form several other oxygen-and carbon-centered radicals (Figure2.20).45

Figure 2 . 20 :

 220 Figure 2.20: Radicals generated from Lina-7-OOH and different involved mechanisms. 45

Figure 2 .

 2 Figure 2.21 shows the experimental data and subsequent simulations for the DEPMPO investigations when using Lina-OOHs mixture (Lina-7-OOH and Lina-6-OOH). Three spinadducts were clearly distinguished. The first one corresponds to DEPMPO-OH 1 (Figure 2.21C), ascribed to hydroxyl radical as previously discussed in the Cum-OOH section. The second one is attributed to DEPMPO-carbon R 13 (Figure 2.21D). It was indeed confirmed by the PBN and DMPO investigations (Annexes 12, 13 and Figures A12.1, A13.1&2 respectively).

Figure 2 . 21 :

 221 Figure 2.21: (A) EPR spectrum of DEPMPO (25 mM) spin-adducts formed from Lina-OOHs (1 mM) / Fe(II) (0.1 mM) in HEPES, (B) computer simulation and (C, D and E) deconvolution of DEPMPO-OH (ca. 70 %) 1, DEPMPO-carbon R (ca. 15 %) 13 and DEPMPO-OCH 3 /OOLina 14 (ca. 15 %) (more data are available in the Annex 9, Figure A9).

Figure 2 . 22 :

 222 Figure 2.22: Schematic description of the potential mechanisms leading to the formation of the spin-adducts arising from Lina-OOHs/Fe(II).

Figure 2 . 23 :

 223 Figure 2.23: Episkin TM model RHE

Figure 2 . 24 :

 224 Figure 2.24: Experimental methodology by topical application developed for RHE EPR studies

2. 25 C

 25 -E). Major peaks assigned to a carbon-centered radical DEPMPO adduct (spin-adduct 6) based on the set of hfccs a N =15.4 G, a H =22.5 G, a P =48.3 G (Figure2.25 F), the minor peaks assigned to oxygen centered radicals whose the hfccs were hardly definable in a precise manner (Figure2.25 F) and comparable with our data in solution (Annex A6).

Figure 2 . 25 :

 225 Figure 2.25: Preliminary trials incubating CumOOH (0.54 mM) and DEPMPO (25mM in HEPES) in the assay medium: (A) sample from the assay medium after 2 h incubation with DEPMPO and CumOOH; (B) spectrum of RHE when the assay medium is incubated 2 h with

Figure 2 . 26 :deconvolution of 6 and 1 .Figure 2 . 26 B

 2261226 Figure 2.26: EPR spectra obtained in RHE topical application experiments by using Cum-OOH (1 mM in acetone) and DEPMPO (50mM in HEPES): (A) RHE control experiment with single DEPMPO and vehicle; (B) RHE spectra after CumOOH application and incubation (5 min, 37 °C, 5% CO2) followed by DEPMPO addition; (C) computer simulation of spectrum (B); (D-E) deconvolution of 6 and 1.

Figure 2 . 27 :

 227 Figure 2.27: EPR spectra obtained in RHE topical application experiments by using Lina-OOH (10 mM in acetone) and DEPMPO (250mM in HEPES): (A) EPR spectrum spin-adducts formed, (B) computer simulation and (C and D) deconvolution of DEPMPO-OH 1 and DEPMPO-carbon R 14

Figure 2 . 28 : 2

 2282 Figure 2.28: EPR spectra obtained in RHE topical application experiments by using Limo -OOH (10 mM in acetone) and DEPMPO (250mM in HEPES): (A) EPR spectrum spin-adducts

  EPR spin-trapping allowed us for the first time to study the formation of carbon radicals issued from organic hydroperoxides in a 3D epidermis model. This will open perspectives and new insights for the molecular understanding of skin sensitization processes to autoxidized natural fragrance terpenes.Cum-OOH produced a better signal to noise ratio when compared to Limo-OOH and Lina-OOHs, having they probably different reactivities in the RHE environment. It was necessary to increase the concentration of those hydroperoxides and of the spin-trap to see a well resolved signal to noise ratio.Concerning the initiation of the radical generation the question of who gave the first electron is still open. It could be residual iron of the RHE or amino acids themselves. For this purpose, a system Lina-OOHs/N-acetyl-L-cysteine methyl ester/DEPMPO, was tested in solution. This system produced spin-adducts as defined in Figure2.29. Therefore, it seems that this amino acid can induced radical generation.

Figure 2 . 29 :

 229 Figure 2.29: EPR spectra obtained in solution experiments: (A) EPR spectrum of DEPMPO (50 mM) spin-adducts formed from Lina-OOHs (1 mM)/N-acetyl-L-cysteine methyl ester (10 mM) in HEPES, (B) computer simulation and (C and D) deconvolution of DEPMPO-OH 1,

Figure A1. 1 :

 1 Figure A1.1: Spin-adducts 1 and 2 formed in H 2 O with different Limo-OOH concentrations

Figure A2. 1 :Figure A2. 2 :Annex 3 .

 123 Figure A2.1: Spin-adducts in HEPES, Limo-OOH (1 mM) / DEPMPO (25 mM), Fe II concentration varying

Figure A3. 1 :

 1 Figure A3.1: Spin-adducts in HEPES / Limo-OOH (1 mM) / DMPO (25 mM), Fe(II) concentration varying

Figure A4. 1 :

 1 Figure A4.1: Spin-adducts in H 2 O spectra Limo-OOH (1 mM) / PBN (50 mM) with Fe(II) varying concentrations

Figure A7. 1 :Figure A7. 2 :Figure A8. 1 :

 121 Figure A7.1: Spin-adducts in PB, Cum-OOH (0.54 mM) / DMPO (25 mM), Fe(II) varying concentrations

Figure A8. 2 :

 2 Figure A8.2: Cum-OOH (0.54 mM) / PBN (25 mM) system in PB and HEPES with Fe(II) varying concentrations spectra at t = 0 minutes (above) and 30 minutes (bellow) respectively

At t = 24 Figure A9. 1 :Figure A9. 1 :

 2411 Figure A9.1: Spin-adducts formed in HEPES / Lina-OOHs (1 mM) / DEPMPO (25 mM) system, Fe(II) varying concentrations at 0 min

Figure A11. 1 :Figure A11. 2 :

 12 Figure A11.1: Spin-adducts HEPES-CM (1-1/v-v), Lina-OOHs (1 mM) /DEPMPO (25 mM), Fe(II) varying concentrations at 0 min

Figure A13. 1 :Figure A13. 2 :

 12 Figure A13.1: Spin-adducts in HEPES spectra at t = 0 min

Figure 3 . 1 : 1 1Figure 3 .

 3113 Figure 3.1: Allylic hydroperoxides used for Keap1-Nrf2 cellular sensor pathway activation studies

Figure 3 . 2 :

 32 Figure 3.2: Keap1-Nrf2 signaling pathway under unstressed and oxidative stress conditions 1

2 2 .

 2 TARGET COMPOUNDS SYNTHESESTarget compounds are shown in Figure3.1. Their oxygenated and carbon radical intermediates could trigger the Keap1-Nrf2 antioxidant response in THP-1 cells.Cum, Cum-OOH, Limo, Lina and Mod were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). Mod-OOH was synthesized as described by Lepoittevin and Karlberg.8 The synthesis started from 1-acetyl-1-cyclohexene which was transformed to the tertiary alcohol Mod-OH by addition of methyllithium. Then, a solution of H 2 O 2 (35 % in water) in acidic conditions gave the hydroperoxide Mod-OOH (Figure3.3).

Figure 3 . 3 :

 33 Figure 3.3: Mod-OOH synthesis as described by Lepoittevin and Karlberg 8

Figure 3 . 4 :

 34 Figure 3.4: Synthesis of Limo-OOH derived from autoxidation of R-(+)-limonene (Limo)

Figure 3 . 5 :

 35 Figure 3.5: Chemical derivatization allowing to obtain single isomer Lina-7-OOH 9

  established a collaboration with Dr. Aurélie Urbain form the Laboratoire d'Innovation Thérapeutique (Faculté de Pharmacie, Université de Strasbourg CNRS UMR 7200) to try to isolate the compounds from the mixture by high performance liquid chromatography (HPLC). An HPLC analytical method published by Calandra et al. was applied to the mixture of linalool hydroperoxides and detected Lina-7-OOH and Lina-6-OOH. An HPLC chemiluminescence detection method was employed with a C 18 Luna column and a gradient combining methanol and acetonitrile (Figure 3.6).

Figure 3 . 6 :Figure 3 . 7 :Figure 3 . 8 :Figure 3 . 9 :

 36373839 Figure 3.6: Calandra et al. HPLC analytical methodology gradient 10

Table 3 . 1 :

 31 The accumulation of protein Nrf2 and the expression of antioxidant genes (ho-1, il8, and nqo1) were measured by Western Blot and polymerase chain reaction (PCR) respectively. During the two and a half weeks I spent in the laboratory Allergy, Immunotoxicology and Immunopathology (INSERM, UMR 996) in Paris, we worked on the development of the experimental conditions of the model and the cytotoxicity of the hydroperoxides (Limo-OOH and Lina-OOHs) on the THP-1 cell-line. Cytotoxicity for the terpene chemicals based on trypan blue assays

Figure 3 . 10 :

 310 Figure 3.10: Nrf2 cytoplasmic accumulation following exposure of Lina, Limo, Lina-OOHs and Limo-OOH in comparison to vehicle (acetonitrile/water) as negative control and the loading control

Figure 3 . 11 :

 311 Figure 3.11: Nrf2 cytoplasmic accumulation following exposure of Limo and Limo-OOH, in comparison to Bach-1 (Nrf2 repressor), 2,4-dinitrochlorobenzene (DNCB) as positive control, vehicle (acetonitrile/water) as negative control and the loading control p38 tot

Figure 3 . 12 :

 312 Figure 3.12: Nrf2 cytoplasmic accumulation following exposure of Cum and Mod and their respective hydroperoxides Cum-OOH and Mod-OOH, in comparison cinnamaldehyde (CinA) as positive control in DMSO (vehicle), vehicle (acetonitrile/water) as negative control and the loading control

Figure 3 . 13 :

 313 Figure 3.13: Nrf2 protein binding ARE, in comparison with cinnamaldehyde (CinA) as positive control, vehicle (acetonitrile) as negative control and the loading control DMSO

Figure 3 . 14 :

 314 Figure 3.14: Lina, Limo and their respective hydroperoxides Lina-OOHs and Limo-OOH inducing ho-1, nqo-1 and il-8 genes overexpression

  system. Each reaction consisted of cDNA (1:50 diluted for THP-1 cells in 4 μl final volume of nuclease-free water), 0.5 μM each of the forward and reverse primers (ho-1 [5′-GGCCTGGCCTTCTTCACCTT-3′ and 5′-GAGGGGCTCTGGTCCTTGGT-3′], nqo1 [5′-GGGCAAGTCCATCCCAACTG-3′ and 5′-GCAAGTCAGGGAAGCCTGGA-3′], il-8 [5′-TCTCTTGGCAGCCTTCCTGA-3′ and 5′-TGGGGTGGAAAGGTTTGG AG-3′], gapdh [5′-GGGCAAGTCCATCCCAACTG-3′ and 5′-GCAAGTCAG-GGAAGCCTG GA-3′], or β-actin [5′-GGCATCCTCACCCTGAAGTA-3′ and 5′-GCACACGCAGCTCATTGTAG′]), and Sso Fast EvaGreen Supermix (Bio-rad, Marnes la Coquette, France) in a total reaction volume of 10 μl. After 30 seconds at 95°C for Sso7d-fusion polymerase activation, amplification was allowed to proceed up to 44 cycles, each consisting of denaturation at 95°C for 5 seconds and annealing/extension at 62°C for 5 seconds. Six-fold serial dilutions of mixed cDNA (from different samples) were analyzed for each target gene and allowed us to construct linear standard curves, from which the efficiency (E) of each PCR reaction was evaluated and taken into account. The SYBR green fluorescence was detected at the end of each elongation cycle, after which a melting curve was assessed to confirm the specificity of the PCR products. The quantification was performed with the Biorad CFX

  Incubation by topical application to the RHE. ....................................................... LinaOOHs experiments ..................................................................................................................... 3.3 Limo-OOH investigations ................................................................................................................ 3.4 Conclusion .............................................................................................................................................

	DMSO DNCB J KCN MUSST mW THF TLC 3.1.2 3.2	dimethyl sulfoxide 2,4-dinitrochlorobenzene NMR coupling constant potassium cyanide Myeloid U937 Skin Sensitization Test milliwatt tetrahydrofuran thin layer chromatography
	DPRA Keap1 NMR TOPKAT	direct peptide reactivity assay Kelch-like ECH-associated protein 1 nuclear magnetic resonance toxicity Prediction by Computer Assisted Technology
	DTT KJ nqo1 TPP	dithiothreitol kilojoule NADPH quinone oxidoreductase 1 5,10,15,20-tetraphenyl-21H,23H-porphyrine
	ECVAM KMnO 4 Nrf2	European center for validation of alternative methods potassium permanganate Nuclear Factor Erythroid 2-Related Factor 2
	eq. Limo Nu	equivalent limonene nucleophilic function
	Et Limo-OOH limonene hydroperoxyde ethyl PB phosphate buffer
	et al. Lina PBN	et alii linalool N-tert-butyl-α-phenylnitrone
	Fe (II) Lina-6-OOH 6-hydroperoxy-3,7-dimethyl octa-1,7-dien-3-ol iron (II) p-EPR pulsed Electron Paramagnetic Resonance
	G Lina-7-OOH (5E) -7-hydroperoxy-3,7-dimethyl octa-1,5-dien-3-ol gauss Ph phenyl
	g Lina-OOHs linalool hydroperoxyde gram ppm parts per million
	g factor LLNA PPRA	Landé's factor mouse local lymph node assay peroxidase peptide reactivity assay
	GHz M QSARs	gigahertz molar quantitative structure-activity relationships
	GPMT m RAI	guinea pig maximization test multiplet relative alkylation index
	h-CLAT m/z REACH	human cell line activation test ratio mass on charge registration, evaluation, authorization, and restriction of chemical substances
	hfccs Maf RHE	hyperfine coupling constants musculoaponeurotic fibrosarcoma reconstructed human epidermis
	HMBC MC RNS	heteronuclear multiple bond correlation cell culture media reactive nitrogen
	ho-1 Me 2 AlCl ROAT	heme oxygenase-1 dimethyl aluminum chloride repeated open application test study
	HOMO Me 3 Al R-OOHs	highest occupied molecular orbital trimethylaluminum hydroperoxides
	HPLC MEST ROS	high-performance liquid chromatography mouse ear swelling test reactive oxygen species
	HSQC MHC II rt	heteronuclear single quantum correlation major histocompatibility complex of class II room temperature
	Hz min s	hertz minute singlet
	IL12 mL S/N	interleukine 12 milliliter signal to noise ratio
	IL18 mM sl	interleukine 18 millimolar singlet large
	il-8 mM S N 1	pro-inflammatory cytokines millimole nucleophilic substitution
	INF-  Mod-OOH S N 2	interferon  monocycle hydroperoxide nucleophilic substitution
	IPP mol t	isopentenyl pyrophosphate mole triplet
	iPrMgCl ms tBHP	isopropyl magnesium chloride millisecond tert-butyl hydroperoxide	3,4-dihydro-2H-pyrrole 1-oxide

  ................................................................................. 4.2.1 Chemicals and reagents ......................................................................................... 4.2.2 EPR equipment ....................................................................................................... 4.2.3 General procedure in RHE.....................................................................................

4.3 Synthesis.................................................................................................................................................. 4.3.1 Synthesis DEPMPO................................................................................................

  13

  11

Table I . 1

 I1 Classification of the main families of allergenic compounds referred as to the electrophilic chemical function encountered in the molecular structure and the mechanism by

	which they bind covalently to nucleophilic sites (Nu) of amino acids in proteins (	= electrophilic
	chemical function, Nu = nucleophile).

  Different chemical and physical properties of compounds linked to a specific biological answer are the basis of computer models. In silico methods in skin allergy are essentially databases, expert

	softwares and studies on quantitative structure-activity relationships (QSARs). Databases compile
	information on observations of allergy cases in different hospitals, but a low number of cases are
	reported. Benezra et al. developed a complete general observation issued in The Journal of
	Investigative Dermatology from 1975 to 1982. 38 The resulting program was called PROPHET and
	allowed to search compounds by chemical structures. However, PROPHET collects just atypical
	ACD cases and it does not reflect the most relevant allergens in the general population.
	Expert softwares search is based on molecular structures supposed to be protein reactive. 39,40
	Among them, DEREK (Deduction and Estimation of Risk from Existing Knowledge) and
	TOPKAT (Toxicity Prediction by Computer Assisted Technology). DEREK was developed on the
	basis of 294 molecules assigned as allergenic by the GPMT. Heterocycle or aromatic cycles,
	ionizable functions and reactive chemical functions are considered. TOPKAT is also based on
	GPMT studies. It is able to predict if a poly-aromatic molecule is a skin sensitizer and for aliphatic
	structures it can distinguish the degree of the allergen potency from low to moderate and strong.

Figure I.12: Autoxidation general mechanism

	Among them, limonene (Limo) and linalool (Lina) are probably the
	most used. Both belong to the pre-haptens category of skin sensitizers as they need to autoxidize
	to form reactive and sensitizing derived products. The general mechanism for autoxidation
	processes is described in Figure I.12. The first step or initiation, probably induced by daylight, heat
	or a catalytic amount of metal, generates radicals R • from organic compounds. Figure I.13: Limonene and linalool secondary autoxidation products
	Limo and Lina allylic hydroperoxides have been classified as moderate to strong sensitizers in the
	mouse local lymph node assay (LLNA), with LLNA EC 3 values between 0.3 and 1.6 % (Table I.2),
	whereas Limo and Lina are classified as non-sensitizers. 28,57,58
	Then, in a second step called propagation, radicals R • can react with triplet state oxygen 3 O 2 to form
	peroxyl radicals ROO • , which are able to abstract a hydrogen from an organic compound to form
	a hydroperoxide and a new radical R • . Hydrogen abstraction is a slower step favorized by the
	presence of labile hydrogens such as allylic hydrogens in terpenes. The final step or termination
	concerns two radical interactions such as R • and ROO • forming a non-radical substance or
	diamagnetic products.
	As it was shown in Figure I.10 primary autoxidation derivatives of Limo and Lina, are allylic
	hydroperoxides. Secondary autoxidation products are epoxides, ketones and diols (Figure I.13).

Table I .2: LLNA

 I EC 3 values for Limo, Lina and derived autoxidation allylic hydroperoxide a) The EC 3 value is the estimated concentration of chemical necessary to cause a 3-fold increase in its proliferating activity. Skin sensitization classification up to EC 3 values: < 0.1 % extreme, 0.1-< 1 % strong, 1-< 10 % moderate and 10-< 100 % non-sensitizer.Clinical data have also shown frequent allergic reactions to Lina and Limo hydroperoxides. In an

	European multicenter study, patch tests on 2900 consecutive patients resulted in 5.2 % positive
	reactions to oxidized limonene 3 % in petrolatum (Limo-OOHs 0.33 %) and 6.9 % positive
	reactions to oxidized linalool 6 % in petrolatum (Lina-OOHs 1 %). Also, a repeated open
	application test study (ROAT) showed that repeated exposure to low concentrations of oxidized
	linalool elicited ACD in sensitized patients.

  Identification of Alkyl Radicals Derived from an Allergenic Cyclic Tertiary Allylic Hydroperoxide by Combined Use of Radical Trapping and ESR Studies. Tetrahedron 2002, 58 (27), 5535-5545.

	collaboration with Pr. Saadia Kerdine-Römer, Allergy, Immunotoxicology and
	Immunopharmacology, University Paris Sud-INSERM UMR 996
	The Keap1-Nrf2 cellular sensor pathway, implicated in the antioxidant response of cells, can detect
	chemical and oxidative stresses. Under normal conditions, the Kelch-like ECH-associated protein
	1 (Keap1) sequesters the transcriptional regulator nuclear factor-erythroid 2-related factor (Nrf2)
	in the cytoplasm provoking its proteasomal degradation. In the presence of chemicals or oxidants,
	cysteine residues of Keap1 are chemically modified leading to its dissociation from Nrf2. Nrf2
	translocate then to the nucleus and forms heterodimers with MAF proteins inducing the
	transcription of antioxidant genes such as heme oxygenase-1 (ho-1) and NADPH quinone
	oxidoreductase 1 (nqo1). Keap1-Nrf2 is thus one of the cellular sensor mechanisms that recognize
	intrinsic chemical reactivity of molecules. A collaboration conducted with the Allergy
	Immunotoxicology and Immunopharmacology Unit at INSERM UMR 996 shows that allergens,
	depending on their chemical structure, may also activate the Nrf2 pathway in DCs. 73 The aim herein
	was to evaluate how the inherent oxidative properties of allylic hydroperoxides, together with their
	reactivity through radical mechanisms, behave as danger signals and translate into a biological
	response using the activation of the Nrf2 pathway, assessed by the induction of Nrf2-target genes

(ho-1, nqo1 and il8) involved in detoxification and anti-oxidant effects. Four allylic hydroperoxides of different chemical structures were used the monocyclic model (Mod-OOH), cumene hydroperoxide (Cum-OOH) and the allergenic hydroperoxides derived from autoxidation of linalool (Lina) and limonene (Limo)) and tested in a model of human dendritic cells, measuring target antioxidant genes production by real-time PCR (Figure I.18). The DCs phenotype status and Nrf2 cytoplasm accumulation were studied by Western Blot.

Figure I.18: Allylic hydroperoxide models for Keap1-Nrf2 activation study (66) Giménez Arnau, E.; Haberkorn, L.; Grossi, L.; Lepoittevin, J.-P. (67) Johansson, S.; Giménez-Arnau, E.; Grøtli, M.; Karlberg, A.-T.; Börje, A. Carbon-and Oxygen-Centered Radicals Are Equally Important Haptens of Allylic Hydroperoxides in Allergic Contact Dermatitis. Chem. Res. Toxicol. 2008, 21 (8), 1536-1547. (68) Kao, D.; Chaintreau, A.; Lepoittevin, J.-P.; Giménez-Arnau, E. Mechanistic Studies on the Reactivity of Sensitizing Allylic Hydroperoxides: Investigation of the Covalent Modification of Amino Acids by Carbon-Radical Intermediates. Toxicol. Res. 2014, 3 (4), 278-289. (69) Eilstein, J.; Giménez-Arnau, E.; Duché, D.; Rousset, F.; Lepoittevin, J.-P. Synthesis and Reactivity Toward Nucleophilic Amino Acids of 2,5-[13 C]-Dimethyl-p -Benzoquinonediimine. Chem. Res. Toxicol. 2006, 19 (9), 1248-1256. (70) Biomedical EPR. Eaton, S. S., Eaton, G. R., Berliner, L. J., Eds.; Biological magnetic resonance; Kluwer Academic/Plenum Publishers: New York, 2004. (71) Jurkiewicz, B. A.; Buettnerf, G. R. EPR Detection of Free Radicals in UV-Irradiated Skin: Mouse Versus Human. Photochem. Photobiol. 1996, 64 (6), 918-922. (72) Netzlaff, F.; Lehr, C.-M.; Wertz, P. W.; Schaefer, U. F. The Human Epidermis Models EpiSkin®, SkinEthic® and EpiDerm®: An Evaluation of Morphology and Their Suitability for Testing Phototoxicity, Irritancy, Corrosivity, and Substance Transport. Eur. J. Pharm. Biopharm. 2005, 60 (2), 167-178. (73) Migdal, C.; Botton, J.; El Ali, Z.; Azoury, M.-E.; Guldemann, J.; Giménez-Arnau, E.; Lepoittevin, J.-P.; Kerdine-Römer, S.; Pallardy, M. Reactivity of Chemical Sensitizers Toward Amino Acids In Cellulo Plays a Role in the Activation of the Nrf2-ARE Pathway in Human Monocyte Dendritic Cells and the THP-1 Cell Line. Toxicol. Sci. 2013, 133 (2), 259-274.

Table 1 . 1 :

 11 Experimental conditions tested for the conversion of 13 C-15 into the Weinreb amide 13 C-16 using N,O-dimethylhydroxylamine hydrochloride

	Experimental conditions: time, temperature, agent	Yield (%)
	1 hour, -20 °C, iPrMgCl	10
	1 hour, -78 °C, iPrMgCl	38 %
	30 minutes, -20 °C, iPrMgCl	40 %
	30 minutes, 0 °C then room temperature, Me 2 AlCl	70 %
	3 hours, 0 °C then room temperature, Me 2 AlCl	63 %
	5 hours, 0 °C, Me 3 Al	83 %

Table 1 . 2 :

 12 Experimental conditions tested for the synthesis of Me 3 SiCN from different KCN

	Experimental conditions: time, temperature and KCN	Yield in % (± 5)
		82 %

Reagents: TMSCl (1eq), KCN (1eq), KI (0.1 eq) and NMP

Table 1 .3: Synthesis of 13 C-22 using 13 C-21 as a synthon Experimental conditions: temperature, time, solvent Yield % (± 5)

 1

	-78 °C then r.t., 30 min, CH 2 Cl 2

Table 1 . 4 :

 14 Experimental conditions tested for the reduction of 13 C-22 to obtain aldehyde 13 C-

	23
	Experimental conditions: DIBAL-H, temperature, time, solvent	Yield % (± 5)
	1.2 eq. DIBAL-H, -78 °C then 0 °C, 3 h, diethyl ether	20 %
	1.2 eq. DIBAL-H, -78 °C then 0 °C, 15 h, tetrahydrofuran	39 %
	2.5 eq. DIBAL-H, -78 °C then 0 °C, 24 h, diethyl ether	31 %
	3 eq. DIBAL-H, -78 °C then 0 °C, 3 h, tetrahydrofuran	-
	5.0 eq. DIBAL-H, -78 °C then 0 °C, 3 h, tetrahydrofuran	20 %
	5.0 eq. DIBAL-H, -78 °C then 0 °C, 48 h, diethyl ether	35 %

(CDCl 3 , δ, ppm): 1

  .28 (t, 3H, OCH 2 CH 3 , 3 J HH = 7.5 Hz), 3.81 (d, 2H, CH 2 , 2 J HC = 5.0 Hz), 4.22 (qd, 2H, OCH 2 CH 3, 3 J HH = 7.0 Hz, 3 J HC = 3.0 Hz). : 1.26 (t, 3H, OCH 2 CH 3 , 3 J HH = 7.0 Hz), 1.32 (t, 6H, 2 × OCH 2 CH 3 , HH = 7.5 Hz), 2.94 (dd, 2H, CH 2 , 2 J HP = 22.0 Hz, 2 J HC = 7.5 Hz), 4.06-4.20 (m, 6H, 3 × OCH 2 CH 3 ).

	2-Bromo-
	1-( 13 C)-ethyl acetate 13 C-4 (3.67 g) was obtained as an orange oil and was used without further
	purification.
	Formula: C 3 13 C H 7 BrO 2
	Molar mass: 167.99 g.mol -1
	CAS (non 13 C-substituted): 105-36-2
	NMR 1 H Formula: C 7 13 CH 17 O 5 P
	Molar mass: 225.19 g.mol -1
	CAS (non 13 C-substituted): 864-13-0

NMR 13 C (CDCl 3 , δ, ppm): 14.1 (CH 3 ), 26.3 (d, CH 2 Br, 1 J CC = 64.6 Hz), 62.4 (OCH 2 CH 3 ), 167.4 ( 13 C=O). 2-(Diethoxyphosphoryl)-1-( 13 C)-ethyl acetate ( 13 C-5) In a flame-dried round bottom flask under argon triethyl phosphite (4.7 mL; 26.21 mmol; 1.2 eq.) was added to 2-bromo-1-( 13 C)-ethyl acetate ( 13 C-4) (3.67 g; 21.84 mmol; 1eq.). The mixture was heated at 140°C for 3 hours and then cooled to room temperature. The product was purified by silica gel chromatography (AcOEt/pentane 8/2). 2-(Diethoxyphosphoryl)-1-( 13 C)ethyl acetate ( 13 C-5) was obtained as a yellow oil (4.95 g; 90 %). TLC: Rf = 0.24 (AcOEt/pentane 8/2): APM/Ce NMR 1 H (CDCl 3 , δ, ppm) 3 J

(CDCl 3 , δ, ppm

  ): 1.25 (t, 3H, OCH 2 CH 3 , 3 J HH = 7.1 Hz), 1.63 (s, 3H, CH 3 cis), 1.67 (s, 3H, CH 3 trans), 2.22 (s, 3H, COCH 3 ), 2.53 (m, 2H, 13 CH 2 ), 3.40 (m, 1H, CH), 4.19 (q, 2H, OCH 2 CH 3 , 3 J HH = 7.1 Hz), 4.98-5.06 (m, 1H, =CH). CH 3 ), 17.6 (CH 3 cis), 25.8 (CH 3 trans), 27.1 ( 13 CH 2 ), 59.8 (d, CH, 1 J CC = 34.2 Hz), 60.4 (COCH 3 ), 61.4 (OCH 2 CH 3 ), 120.1 (d, =CH, 1 J CC = 44.1 Hz), 134.9 ((CH 3 )

	Formula: C 10 13 CH 18 O 3
	Molar mass: 199.25 g.mol -1
	CAS (non 13 C-substituted): 1845-52-9
	TLC : Rf = 0.33 (Pentane/Et 2 O 9/1) : APM/Ce
	NMR 1 H

13

C)-2-acetyl-5-methylhex-4-enoate ( 13 C-9) was obtained as a colorless oil (1.289 g; 67%). NMR 13 C (CDCl 3 , δ, ppm): 14.0 (OCH 2 2 C=), 169.6 ((O)C=O), 203.0 (C=O).

(CDCl 3 , δ, ppm): 17

  HH = 1.1 Hz, 2 J HH = 10.8 Hz), 5.11 (m, 1H, =CH), 5.20 (dd, 1H, CH=CH 2 cis, 3 J HH = 1.1 Hz, 2 J HH = 17.3 Hz), 5,90 (dd, 1H, CH 13 CH 2 , 3 J HH = 10.8 Hz, 2 J HC = 17.3 Hz). .7 (CH 3 cis), 22.8 ( 13 CH 2 CH), 25.7 (CH 3 trans), 27.9 (CH 3 ), 42.0 (d, CH 2 COH, 1 J CC = 34.4 Hz), 73.5 (COH), 111.7 (=CH 2 ), 124.3 (d, =CH, 1 J CC = 43.6 Hz)

	Formula: C 9 13 CH 18 O Molar mass: 155.25 g.mol -1 CAS (non 13 C-substituted): 78-70-6 TLC: Rf = 0.30 (pentane/Et 2 O 8/2) : APM/Ce NMR NMR 13 C 131.9 ((CH 3

13 C)-linalool ( 13 C-11) was obtained as a yellow oil (0.198 g; 63 %). 1 H (CDCl 3 , δ, ppm): 1.27 (s, 3H, CH 3 ), 1.52-1.57 (m, 2H, =CHCH 2 CH 2 ), 1.60 (s, 3H, CH 3 cis), 1.67 (s, 3H, CH 3 trans), 1.92-2.09 (m, 2H, =CH 13 CH 2 CH 2 ), 5.05 (dd, 1H, CH=CH 2 trans, 3 J

  CH 17NO 2

	Molar mass: 172.23 g.mol -1
	CAS (non 13 C-substituted): 1110631-07-6
	TLC: Rf = 0.24 (pethroleum ether/AcOEt 95/5): APM/Ce
	NMR

1 H (CDCl 3 , δ, ppm): 1.70 (s, 3H, CH 3 cis), 1.82 (s, 3H, CH 3 trans), 2.26 (m, 2H, CH 2 ), 2.28 (m, 2H, 13 CH 2 ), 3.16 (s, 3H, CH 3 ), 3.66 (s, 3H, OCH 3 ), 5.11 (m, 1H, =CH). NMR 13 C (CDCl 3 , δ, ppm): 17.9 (CH 3 trans), 23.3 (CH 3 cis), 26.9 (d, CH 2 , 1 J CC = 11.4 Hz), 32.1 ( 13 CH 2 ), 34.6 (NCH 3 ), 61.2 (OCH 3 ), 123.1 (=CH), 132.7 ((CH 3 ) 2 C=), 171.2 ((O)C-N).

  : 1.26 (d, 3H CH 3 , 3 J HC = 4.0 Hz), 1.50-1.55 (m, 2H, =CHCH 2 13 CH 2 ), 1.58 (s, 3H, CH 3 cis), 1.66 (s, 3H, CH 3 trans), 1.92-2.09 (m, 2H, =CHCH 2 13 CH 2 ), 5.05 (d, 1H, CH=CH 2 trans, 3 J HH = 10.8 Hz), 5.11 (m, 1H, =CH), 5.20 (d, 1H, CH=CH 2 cis, 3 J HH = 17.4 Hz), 5.90 (dd, 1H, CH=CH 2 cis, 3 J HH = 10.8 Hz, 3 J HH = 17.3 Hz).

	NMR 13 C (CDCl 3 , δ, ppm): 17.5 (CH 3 cis), 22.8 (d, CH 2 CH, 1 J CC = 4.1 Hz), 25.7 (CH 3 trans),
	27.9 (CH 3 ), 41.7 ( 13 CH 2 COH), 73.5 (d, COH, 1 J CC = 38.1 Hz), 111.7 (=CH 2 ), 124.3 (=CH),
	131.9 ((CH 3 ) 2 C=), 145.0 (CH 2 =CH).
	13 C-18) as a yellow oil
	(0.712 g; 85 %).
	Formula: C 9 13 CH 18 O
	Molar mass: 155.25 g.mol -1
	CAS (non 13 C-substituted): 78-70-6

TLC: Rf = 0.9 (pethroleum ether/Et 2 O 8/2): APM/Ce NMR 1 H (CDCl 3 , δ, ppm)

13 C)-2,6-Dimethyl-2-((trimethylsilyl)oxy)hept-5-enenitrile ( 13 C-22)

	4-( 13 C)-(5E)-7-Hydroperoxy-3,7-dimethylocta-1,5-dien-3-ol	( 13 C-19)	and	4-( 13 C)-6-
	hydroperoxy-3,7-dimethylocta-1,7-dien-3-ol ( 13 C-20) (ratio 2/3) was purified using silica gel
	column chromatography (petroleum ether/ethyl acetate 4/1, then 3/2) as a yellow oil (0.588 g;
	3.14 mmol; 69%).
	Formula: C 9 13 CH 18 O 3
	Molar mass: 187.24 g.mol -1
	CAS (non 13 C-substituted): Isomer ( 13 C-19): 51276-32-5 -Isomer ( 13 C-20): 51276-31-4
	TLC = 0.24 (petroleum ether/ethyl acetate 7/3): APM/Ce
	In a flame-dried round bottom flask under argon 6-methyl-5-hepten-2-one (3.62 mL; 24.25
	mmol; 1 eq) and anhydrous iodine (0.492 g; 1.94 mmol; 0.08 eq) were introduced in
	NMR NMR 13 C (CDCl 3 , δ, ppm):
	Isomer ( 13 C-19) (mixture of two enantiomers): 24.4 (2 × (CH 3 ) 2 C(OOH)), 27.8 (CH 3 C(OH)),
	45.4 (s, 13 CH 2 C(OH)), 72.7 (C(OH)), 82.2 (C(OOH)), 112.3 (CH 2 (CH)C(OH)), 126.8 (d,
	CH 2 (CH)CH, 1 J CC = 43.4 Hz), 138.0 (s, CHC(OOH)), 144.8 (CHC(OH))
	Isomer ( 13 C-20) (mixture of four diastereoisomers) : 17.6 (2 × CH 3 (C)CH(OOH)), 25.1 (s, 2 ×
	CH 2 (CH)OOH), 28.2 and 28.3 (2 × CH 3 C(OH)), 37.7 (d, 2 × 13 CH 2 C(OH), 1 J CC = 23.8 Hz),

1 H (CDCl 3 , δ, ppm) Isomer ( 13 C-19) (mixture of two enantiomers): 1.28 (s, 3H, CH 3 C(OH)), 1.32 (s, 6H, (CH 3 ) 2 C(OOH)), 2.29 (m, 2H, 13 CH 2 C(OH)), 5.07 (dd, 1H, CH 2 (CH)C(OH), 3 J HH = 1.1 Hz, 2 J HH = 10.8 Hz), 5.21 (dd, 1H, CH 2 (CH)C(OH), 3 J HH = 1.1 Hz, 2 J HH = 17.4 Hz,), 5.50-5.64 (m, 2H, CHCH), 5.91 (dd, 1H, CHC(OH), 3 J HH 10.7 Hz, 2 J HH = 17.3 Hz). Isomer ( 13 C-20) (mixture of four diastereoisomers) : 1.29 (s, 6H, 2 × CH 3 C(OH)), 1.51-1.66 (m, 4H, 2 × CH 2 13 CH 2 ), 1.73 (s, 6H, 2 × CH 3 (C)CH(OOH)), 4.29 (m, 2H, 2 × CH(OOH)), 5.00 (m, 4H, 2 × CH 2 (C)CH(OOH)), 5.07 (dd, 2H, 2 × CH 2 (CH)C(OH), 3 J HH = 1.1 Hz, 2 J HH = 10.8 Hz), 5.20 (dd, 1H, CH 2 (CH)C(OH), 3 J HH = 1.1 Hz, 2 J HH = 17.4 Hz), 5.21 (dd, 1H, CH 2 (CH)C(OH), 3 J HH =1.6 Hz, 2 J HH = 17.4 Hz), 5.87 (dd, 1H, CHC(OH), 3 J HH = 10.8 Hz, 2 J HH = 17.4 Hz), 5.88 (dd, 1H, CHC(OH), 3 J HH =10.8 Hz, 2 J HH = 17.4 Hz,). 73.1 (2 × C(OH)), 89.7 (d, 2 × CH(OOH), 1 J CC = 20.5 Hz), 112.3 (2 × CH 2 (CH)C(OH)), 114.2 and 114.3 (2 × CH 2 (C)CH(OOH)), 143.7 (2 × C(CH)OOH), 144.7 et 144.8 (2 × CHC(OH)) 1-( dichloromethane (10 mL) and cooled to -78 °C. Trimethylsilyl ( 13 C)-cyanide ( 13 C-21) (2.4 g; 24.

25 mmol; 1 eq) was then added very slowly. The mixture was stirred for 30 minutes at room temperature. Water (100 mL) was added and the solution color changed from brown to red. The aqueous phase was extracted with dichloromethane (2 × 100 mL). The combined organic phases were washed with a saturated solution of sodium thiosulfate (100 mL), dried over magnesium sulfate, filtered, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (pentane/AcOEt 9/1) to give 1-( 13 C)-2,6-dimethyl-2-((trimethylsilyl)oxy)hept-5-enenitrile 13 C-22 as a light-yellow oil (4.378 g; 80 %).

Table 2 .

 2

1: Natural abundance, nuclear spin (I) and gyromagnetic ratio of common nucleus

Nucleus Natural abundance (%) Nuclear spin (I) Gyromagnetic ratio (rad.s -1 .T -1 )

	1 H	99.987	1/2	267.513
	13 C	1.07	1/2	67.262
	14 N	99.634	1	19.338
	17 O	0.038	5/2	-36.279
	19 F	100	1/2	25.181
	31 P	100	1/2	108.291

Table 2 .

 2 1 and Figure 2.4).

Table 2 . 2 :

 22 Hfccs, g and relative quantification for spin-adducts identification in system Limo-OOH/DEPMPO/H 2 O.

Table 2 . 4

 24 Spin-adducts formed from CumOOH/DEPMPO system in RHE.26,34,52

	Incubation in assay medium	Topical application
	assignment, g, hfccs (Gauss)	assignment, g, hfccs (Gauss)
	DEPMPO-OH	DEPMPO-carbon R	DEPMPO-OH	DEPMPO-carbon R
	1	6	1	6
	g=2.0055	g=2.0050	g=2.0050	g=2.0052
	aN=14.6	aN=15.4	aN=15.0	aN=15.4
	aH=13.2	aH=22.6	aH=13.4	aH=22.7
	aP=46.2	aP=48.1	aP=44.8	aP=48.0

Table 2

 2 .6.

Table 2 . 6 .

 26 Spin-adducts formed from Limo-OOH/DEPMPO system in RHE.26,34,52

	Spin-adducts topical application
	assignment, g, hfccs (Gauss)
	DEPMPO-OH	DEPMPO-carbon R
	1	2
	g=2.0055	g=2.0053
	aN=13.8	aN=14.7
	aH=14.4	aH=22.1
	aP=44.6	aP=47.2

Diethyl-(2-methyl-2-pyrrolidinyl)phosphonate synthesis

  mM, PBS or HEPES/CH 3 CN 9/1), the spintraps (100 mM in PBS or HEPES) and FeSO 4 .7H 2 O (10 mM in deionized water). 12.5 L of spin-trap solution were mixed with FeSO 4 .7H 2 O (i.e. 0.5 L for a final concentration in the reaction mixture of 0.1 mM), 5 L CumOOH solution added and final volume completed to 50 L. This way, final concentrations in the reaction mixture were 25 mM spin-trap and 0.54 mM CumOOH. The reaction mixture was subjected to stirring, further introduced into the EPR quartz capillary tube, sealed on both ends, and EPR spectra registered. CumOOH (200 L of 5.4 mM HEPES/CH 3 CN 9/1 stock solution) and DEPMPO (100 L of 500 mM stock solution in HEPES) were introduced in a mixture of 300 L assay medium and 1400 L of HEPES buffer. The RHE model was then placed in a well containing this mixture. After 2 hours incubation, RHE was placed in the EPR tissue cell and EPR spectra recorded.

	1°K) as soon as possible after incubating the reagents. Treated RHE were placed in an EPR was then added. The solution was washed with dichloromethane (3 × 80 mL). The aqueous
	tissue cell equipped with a silica window (Bruker Biospin GmbH #ER162TC-Q) and EPR phase was basified with a saturated solution of sodium carbonate up to pH 9. Then the product
	spectra recorded. EPR spectra were treated and hyperfine splitting assignments obtained by was extracted with chloroform (3 × 100 mL). Organic phases were dried over magnesium
	means of computer simulation using Easyspin launched in Matlab software (version 2015b) sulfate, filtered and concentrated under reduced pressure to obtain diethyl-(2-methyl-2-
	(http://www.easyspin.org). pyrrolidinyl)phosphonate as a brown oil (30,2 g ; 136 mmoles ; 67 %).
	4.2.3 General procedure in RHE Formula: C 9 H 20 NO 3 P
	Incubation in the assay medium Molar mass: 221.23 g.mol -1
	CAS: 157230-68-7
	CCM: Rf = 0.60 (dichloromethane/ethanol 9/1): PMA
	4.2 Reconstructed human epidermis studies NMR 1 H (CDCl 3 , δ, ppm): 1,10 (td, 6H, 2 × OCH 2 CH 3, 3 J HH = 7.1 Hz, 3 J PH = 2.4 Hz); 1,29 (d,
	4.2.1 Chemicals and reagents Incubation by topical application to the RHE 3H, CCH 3, 3 J PH = 15.2 Hz); 1,34-1,76 (m, 4H, CCH 2 CH 2 ); 2.21-2.39 (m, 1H, NH); 2,71-2,79
	Episkin TM (Lyon, France) is an in vitro reconstructed human epidermis (RHE) from normal RHE were topically treated with DEPMPO in HEPES (250 mM, 20 L) and post-incubated (m, 1H, NHCH 2 ); 2,86-2,95 (m, 1H, NHCH 2 ); 3,98-4,16 (m, 4H, 2 × OCH 2 CH 3 )
	human keratinocytes cultured for 13 days on a collagen matrix at the air-liquid interface. It is a (37 °C, 5% CO 2 ) different time periods (7 to 30 minutes). After the incubation time, the RHE
	3D epidermis model histologically similar to human epidermis. The small 0.38 cm 2 format was were placed in the EPR tissue cell and ROOH (1 to 10 mM in HEPES buffer, 20 L) was
	chosen for the studies. Immediately after arrival in the laboratory, the 3D reconstructed applied to the epidermis taking care to ensure that the solution was only applied to it. EPR NMR 13 C (CDCl 3 , δ, ppm):16,9 (d, 2 × OCH 2 CH 3 , 3 J PC = 5.6 Hz); 24,4 (d, CCH 3 , 2 J PC = 6.2
	epidermis was removed from the agarose-nutrient solution in the shipping multiwell plate under spectra were then recorded. Hz); 26,3 (d, CCH 2 CH 2 , 3 J PC = 4,3 Hz); 35,3 (d, CCH 2 CH 2 , 2 J PC = 2.5 Hz); 47,5 (d, NHCH 2 ,
	a sterile airflow. Then, they were immediately placed on a plate in which each well was 3 J PC = 7.4 Hz); 59,9 (d, CCH 3 , 1 J PC = 166.5 Hz); 62,0 (d, OCH 2 CH 3 , 2 J PC = 4.4 Hz); 62,3 (d,
	previously filled with 2 mL Episkin TM maintenance or growth medium at room temperature. It 4.3 Synthesis OCH 2 CH 3 , 2 J PC = 4,3 Hz)
	was necessary to act quickly as the tissue cultures dry out rapidly when not in contact with the medium. Samples were placed in the incubator at 37 °C, 5% CO 2 and saturated humidity at 4.3.1 Synthesis DEPMPO 52 5
	least 24 hours before incubation. Episkin TM (Lyon, France) furnished the assay medium used
	for the incubations.
	4.2.2 EPR equipment
	EPR spectra were recorded on an EPR X-band spectrometer (ESP300E, Bruker Biospin GmbH,
	Germany), equipped with a high sensitivity resonator (8950opt300, Bruker Biospin GmbH, In a round bottom flask under argon, diethyl phosphite (98%; 32.4 mL; 246 mmol; 1.2 equiv.)
	Germany). The g calibration standard was a strong pitch of known g factor 2.0028. The was slowly added to a solution of 2-methylpyrroline (95%; 20.0 mL; 201 mmol) in ethanol (60
	spectrometer was operated at 9.8 GHz and spectrometer settings were 100 kHz modulation mL). The mixture was stirred for 7 days at room temperature. Then the solvent was evaporated
	frequency, microwave power 5.1 mW, sweep width 160 G, sweep time 327.7 s, modulation under reduced pressure to obtain a brown oil. A solution of hydrochloric acid (10 %; 200 mL)
	amplitude 1 G and time constant 163.8 s. Spectra were recorded at room temperature (295°K ±

-Diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide DEPMPO

	stirred for 15 minutes, then a clear green color appeared. Organic phases were washed with a using neutralized silica gel chromatography (pentane/diethyl ether 8/2) as a clear oil (2.7 g; 16.0 ANNEXES
	sodium chloride saturated solution (150 mL), dried over magnesium sulfate, filtered and the mmol; 42 %).
	solvent removed under reduced pressure. The crude product was purified by silica gel Formula: C 10 H 16 O 2
	chromatography (dichloromethane/ethanol 9/1) and DEPMPO was obtained as a yellow oil (4.8 Annexes 1-12 resume all the experimental data in PB and in HEPES for all spin-traps and all
	g; 20.4 moles; 29 %). Molar mass: 168.23 g.mol -1 spin-adducts, including g (g = +/-0.0005) values, hfccs and ratios of spin-adducts formed in the
	Formula: C 9 H 18 NO 4 P CAS: 1045725-54-9 mixtures.
	Molar mass: 235.22 g.mol -1 CCM: Rf = 0.21 (petroleum ether/ethyl acetate 95:5): Anisaldehyde. Annex 1. Fe(II) (0.1 mM) / DEPMPO (25 mM) system in H 2 O with Limo-OOH varying
	CAS: 157230-67-6 NMR 1 H (500 MHz; CDCl 3 ): concentrations
	Limo-OOH Spin-adducts in H2O assignment, g,
	CCM: Rf = 0.30 (dichloromethane/ethanol 9/1) : PMA Compound (mixture of two diastereoisomers) : 1,42-1,51 (m, 2 × 1H, 2 × CH 2 (CH)OOH); 1,75 hfccs (Gauss)
	and 1,76 (s, 2 × 3H, 2 × CH 3 C(CH 2 )); 1,76 and 1,81 (s, 2 × 3H, 2 × CH 3 (C)CH(OOH)); 2,14-1 mM 1 (80%) 2 (20%)
	2,40 (m, 2 × 4H, 2 × (CH 2 (CH)C(CH 2 ), CHC(CH 2 ) and CH 2 (CH)OOH)); 4.37 (s, 1H, DEPMPO-OH DEPMPO-R
	NMR 1 H (CDCl 3 , δ, ppm): 1,02 (t, 3H, OCH 2 CH 3 , 3 J HH = 7.0 Hz); 1,14 (t, 3H, OCH 2 CH 3 , 3 g=2.0055 g=2.0056 CH(OOH)); 4,55 (m, 1H, CH(OOH)); 4,76 (s, 2 × 2H, 2 × CH 2 C(CH 3 )); 5,76 (d, 1H, aN=14.0 aN=14.1 J HH = 7.0 Hz); 1,33-1,52 (m, 1H, CCH 2 CH 2 ); 1,59 (d, 3H, CCH 3 , 3 J PH = 14.6 Hz); 1,59-1,68 × OOH) (m, 1H, CCH 2 CH 2 ); 2,21-2,37 (m, 1H, CCH 2 CH 2 ); 2,49-2,63 (m, 1H, CCH 2 CH 2 ); 3,83-4,02 aH=13.6 aH=22.3 CH(C)CH(OOH), 3 J HH = 5.0 Hz); 5,65 (m, 1H, CH(C)CH(OOH)); 7,99 and 7,65 (s, 2 × 1H, 2 aP=47.7 aP=47.1
	2 mM (m, 2H, OCH 2 CH 3 ); 4,24-4,38 (m, 1H, OCH 2 CH 3 ); 4,41-4,55 (m, 1H, OCH 2 CH 3 ); 6,34 (q, 1H, 1 (80%) 2 (20%) DEPMPO-OH DEPMPO-R NMR 13 C (75 MHz; CDCl 3 ): NOCH, 3 J HH = 2.9 Hz, 4 J PH = 2.9 Hz) g=2.0055 g=2.0057 Compound (mixture of two diastereoisomers) : 19,4 and 21,5 (2 × CH 3 (C)CH(OOH)); 20,6 aN=14.0 aN=14.7
	aH=13.6 and 21,1 (2 × CH 3 C(CH 2 )); 31,1 and 31,4 (2 × CH 2 (CH)C(CH 2 ) or 2 × CH 2 (CH)OOH); 32,8 aH=22.2 aP=47.6 aP=46.9
	NMR 13 C (CDCl 3 , δ, ppm): 16,3 (d, OCH 2 CH 3 , 3 J PC = 6.2 Hz); 16,4 (d, OCH 2 CH 3 , 3 J PC = 6.2 (2 × CH 2 (CH)C(CH 2 ) or 2 × CH 2 (CH)OOH); 35,4 and 40,8 (2 × CHC(CH 2 )); 82,8 and 84,5 (2 5 mM 1 (80%) 2 (20%)
	Hz); 21,1 (s, CCH 3 ); 25,4 (s, CCH 2 CH 2 ); 31,1 (s, CCH 2 CH 2 ); 61,7 (d, OCH 2 CH 3 , 2 J PC = 7.5 × CH(OOH)); 109,3 and 109,4 (2 × CH 2 C(CH 3 )); 127,3 and 129,9 (2 x CH(C)CH(OOH); 129,6 DEPMPO-OH DEPMPO-R
	and 132,8 (2 × C(CH)OOH); 148,8 and 149,3 (2 × C(CH 2 )) g=2.0055 g=2.0058 Hz); 64,2 (d, OCH 2 CH 3 , 2 J PC = 5.6 Hz); 75,2 (d, CCH 3 , 1 J PC = 154.1 Hz); 132,1 (s, NOCH) aN=14.0 aN=14.6
	4.3.2 Synthesis of Limonene-2-hydroperoxide Limo-OOH aH=13.6 aH=24.6 4.3.3 Synthesis of (5E)-7-Hydroperoxy-3,7-dimethylocta-1,5-dien-3-ol and 5-( 13 C)-aP=47.6 aP=46.8
	6-hydroperoxy-3,7-dimethylocta-1,7-dien-3-ol (Lina-OOHs) 10 mM 1 (80%) 2 (20%)
	DEPMPO-OH	DEPMPO-R
	g=2.0055	g=2.0058
	aN=14.2	aN=15.1
	aH=13.3	aH=24.4
	In a round bottom flask under argon, diethyl-(2-methyl-2-pyrrolidinyl)phosphonate (15.0 g; aP=47.8 aP=45.7
	To an aqueous solution of hydrogen peroxide (50%; 85 mL) were added at 0°C some drops of 67.8 moles) was dissolved in chloroform (150 mL). The temperature of the solution was sulfuric acid and (-)-carveol (97%; 6.0 g; 38.2 mmol). The mixture at 0°C was stirred for 2 days A microemulsion was prepared by slowly adding a sodium molybdate solution (3.38 g in 18 maintained under -5°C. A suspension of 3-chloroperbenzoic acid (77%; 31.0 g; 135 moles; 2 then extracted with pentane (5 × 200 mL). Organic phases assembled were dried over mL distilled water) to a suspension of sodium dodecyl sulfate (27.9 g) in butanol (34 mL) and equiv.), in chloroform (250 mL) was slowly added. The color passed from green to white. A magnesium sulfate, filtered, and evaporated under reduced pressure. Limo-OOH was obtained dichloromethane (166 mL). At the end of the addition, the microemulsion became clear after 5 saturated solution of sodium carbonate (130 mL) was added and the solution was vigorously minutes stirring.

Annex 2 .

 2 Limo-OOH (1 mM) / DEPMPO (25 mM) system in HEPES and H 2 O with Fe(II) varying concentrations

	Fe(II)	Spin-adducts in HEPES	Spin-adducts in H2O
		assignment, g, hfccs (Gauss)	assignment, g, hfccs (Gauss)
	0.1 mM	1	1 (80%)	2 (20%)
		DEPMPO-OH	DEPMPO-OH	DEPMPO-R
		g=2.0057	g=2.0055	g=2.0054
		aN=14.0	aN=14.1	aN=14.7
		aH=14.0	aH=13.6	aH=21.7
		aP=47.5	aP=47.6	aP=46.8
	0.5 mM	1	1 (70%)	2 (30%)
		DEPMPO-OH	DEPMPO-OH	DEPMPO-R
		g=2.0057	g=2.0056	g=2.0056
		aN=14.2	aN=14.1	aN=14.6
		aH=13.5	aH=13.5	aH=21.7
		aP=47.8	aP=47.7	aP=46.9
	1.0 mM	1	1 (60%)	2 (40%)
		DEPMPO-OH	DEPMPO-OH	DEPMPO-R
		g=2.0057	g=2.0056	g=2.0056
		aN=14.2	aN=14.1	aN=14.6
		aH=13.5	aH=13.6	aH=21.8
		aP=47.7	aP=47.7	aP=46.9
	1.7 mM	-	1 (50%)	2 (50%)
			DEPMPO-OH	DEPMPO-R
			g=2.0056	g=2.0057
			aN=14.1	aN=14.5
			aH=13.6	aH=21.6
			aP=47.6	aP=46.7

Annex 5 .

 5 Spin-adducts formed from Fe(II) (0.1 mM) / PBN (50 mM) system in H 2 O and CH 3 CN with Limo-OOH varying concentrations Comparing those spectra, a weak ratio signal/noise increasing by hydroperoxide concentration in H 2 O or in CH 3 CN was observed, using PBN spin-trap.

	Annex 6. Spin-adducts formed from Cum-OOH (0.54 mM) /DEPMPO (25 mM) system in PB
	and HEPES with Fe(II) varying concentrations
	Fe(II)	Limo-OOH Spin-adducts in PB assignment, g, hfccs Spin-adducts in H2O	Spin-adducts in CH3CN Spin-adducts in HEPES
				assignment, g, hfccs (Gauss) (Gauss)	assignment, g, hfccs (Gauss) assignment, g, hfccs (Gauss)
	0.1 mM	-	1 mM	-	3 PBN-R g=2.0051 -	1 (60%) DEPMPO-OH PBN-CH2CN 6 (20%) 4 DEPMPO-g=2.0057 carbon R	7 (20%) DEPMPO-OCH3/OOCum
					aN=15.4 aH=3.3	g=2.0059 aN=14.1	aN=14.2 g=2.0058 aH=2.2 aN=15.2	g=2.0059 aN=13.6
			2 mM		3	aH=13.3		4 aH=22.4	aH=9.4
					PBN-R	aP=47.5	PBN-CH2CN aP=48.0	aP=48.2
					g=2.0051			g=2.0057
	0.5 mM	1 (40%) DEPMPO-OH	6 (30%) DEPMPO-carbon R	aN=15.3 aH=3.2 7 (30%) DEPMPO-OCH3/OOCum	1 (60%) DEPMPO-OH aN=14.2 6 (20%) DEPMPO-aH=2.1 carbon R	7 (20%) DEPMPO-OCH3/OOCum
			5 mM		3			4
		g=2.0058	g=2.0056	PBN-R g=2.0058	PBN-CH2CN g=2.0059 g=2.0057	g=2.0059
		aN=14.2	aN=15.3	g=2.0052 aN=13.8	aN=14.1		g=2.0057 aN=15.3	aN=13.5
		aH=13.5	aH=22.6	aN=15.3 aH=8.9	aH=13.3		aN=14.2 aH=22.5	aH=9.3
		aP=47.5	aP=47.8	aH=3.1 aP=48.1	aP=47.5		aH=2.1 aP=47.9	aP=48.2
	1.0 mM	10 mM 1 (40%)	6 (60%)	3	1 (50%)		4 6 (30%)	7 (20%)
		DEPMPO-	DEPMPO-	PBN-R	PBN-CH2CN DEPMPO-OH DEPMPO-	DEPMPO-
		OH	carbon R	g=2.0053			g=2.0057 carbon R	OCH3/OOCum
					aN=15.2			aN=14.1
		g=2.0058	g=2.0055	aH=3.1	g=2.0059	aH=2.1 g=2.0057	g=2.0053
		aN=14.3	aN=15.4		aN=14.2		aN=15.3	aN=13.4
		aH=13.4	aH=22.6		aH=13.5		aH=22.8	aH=9.1
		aP=47.5	aP=47.9		aP=47.4		aP=48.0	aP=48.2
	1.7 mM	1 (20%)	6 (80%)		1 (70%)		6 (30%)
		DEPMPO-	DEPMPO-		DEPMPO-OH	DEPMPO-
		OH	carbon R				carbon R
						g=2.0059
		g=2.0058	g=2.0055		aN=14.2		g=2.0057
		aN=14.2	aN=15.3		aH=13.4		aN=15.3
		aH=13.3	aH=22.7		aP=47.6		aH=22.6
		aP=47.5	aP=48.0				aP=47.9

Figure A5.1: Fe(II) (0.1 mM) / PBN (50 mM) system in H 2 O with Limo-OOH varying concentrations Figure A5.2: Fe(II) (0.1 mM) / PBN (50 mM) system in CH 3 CN with Limo-OOH varying concentrations

  .1, no spin-adducts are observed at 0.1 and 0.5Mm. It is possible that PB chelates Fe II which therefore prevents radical generation at low concentration. Spin-adducts formed from Cum-OOH (0.54 mM) / DMPO (25 mM) system in PB and HEPES with Fe(II) varying concentrations

	Annex 7. Fe(II)	Spin-adducts in PB		Spin-adducts in HEPES
		assignment, g, hfccs (Gauss)	assignment, g, hfccs (Gauss)
	0.1 mM	-	-	-	-	-
	0.5 mM	-	-	-	8 (60%)	9 (20%)	10 (20%)
					DMPO-OCH3	DMPO-	DMPO-OH
					g=2.0055	carbon R	g=2.0057
					aN=14.65	g=2.0053	aN=15.30
					aH=10.77 (1.27)	aN=16.40	aH=15.02
						aH=23.87
	1.0 mM	8 (50%)	9 (40%)	10 (10%)	8 (70%)	9 (15%)	10 (15%)
		DMPO-	DMPO-carbon	DMPO-	DMPO-OCH3	DMPO-	DMPO-OH
		OCH3	R	OH	g=2.0055	carbon R	g=2.0057
		g=2.0056	g=2.0054	g=2.0056	aN=14.61	g=2.0055	aN=15.30
		aN=14.55	aN=16.61	aN=15.13	aH=10.97 (1.44)	aN=16.65	aH=15.02
		aH=10.80	aH=23.70	aH=15.01		aH=24.00
		(1.32)
	1.7 mM	8 (49%)	9 (40%)	10 (11%)	8 (80%)	9 (10%)	10 (10%)
		DMPO-OCH3	DMPO-	DMPO-	DMPO-OCH3	DMPO-	DMPO-OH
		g=2.0056	carbon R	OH	g=2.0055	carbon R	g=2.0057
		aN=14.63	g=2.0054	g=2.0057	aN=14.61	g=2.0053	aN=15.30
		aH=10.81	aN=16.56	aN=15.30	aH=10.97 (1.44)	aN=16.45	aH=15.02
		(1.34)	aH=23.65	aH=15.02		aH=23.64

Annex 9 .

 9 Spin-adducts formed from Lina-OOHs (1 mM) /DEPMPO (25 mM) system in HEPES with Fe(II) varying concentrations

	Fe(II)	Spin-adducts in HEPES	Spin-adducts in HEPES
		assignment, g, hfccs (Gauss) t = 0 min	assignment, g, hfccs (Gauss) t = 30 min
	0.1 mM	1 (70%)	13 (15%)	14 (15%)	1 (70%)	13 (15%)	14 (15%)
		DEPMPO-	DEPMPO-	DEPMPO-	DEPMPO-	DEPMPO-	DEPMPO-
		OH	carbon R	OCH3/OOLina	OH	carbon R	OCH3/OOLina
		g=2.0055	g=2.0054	g=2.0059	g=2.0055	g=2.0054	g=2.0059
		aN=14.0	aN=15.2	aN=13.5	aN=14.1	aN=15.3	aN=13.5
		aH=13.6	aH=22.4	aH=9.6	aH=13.3	aH=22.5	aH=9.6
		aP=47.6	aP=47.7	aP=48.2	aP=47.7	aP=47.7	aP=48.2
	0.5 mM	1 (60%)	13 (20%)	14 (10%)	1 (40%)	13 (50%)	14 (10%)
		DEPMPO-	DEPMPO-	DEPMPO-	DEPMPO-	DEPMPO-	DEPMPO-
		OH	carbon R	OCH3/OOLina	OH	carbon R	OCH3/OOLina
		g=2.0055	g=2.0054	g=2.0059	g=2.0055	g=2.0053	g=2.0059
		aN=14.0	aN=15.1	aN=13.5	aN=14.1	aN=15.1	aN=13.5
		aH=13.6	aH=22.4	aH=9.3	aH=13.3	aH=22.3	aH=9.3
		aP=47.6	aP=47.6	aP=48.2	aP=47.6	aP=47.8	aP=48.2
	1.0 mM	1 (35%)	13 (50%)	14 (15%)	1 (40%)	13 (40%)	14 (20%)
		DEPMPO-	DEPMPO-	DEPMPO-	DEPMPO-	DEPMPO-	DEPMPO-
		OH	carbon R	OCH3/OOLina	OH	carbon R	OCH3/OOLina
		g=2.0055	g=2.0053	g=2.0053	g=2.0053	g=2.0053	g=2.0053
		aN=14.1	aN=15.2	aN=13.6	aN=14.1	aN=15.2	aN=13.6
		aH=13.5	aH=22.4	aH=9.2	aH=13.3	aH=22.8	aH=9.1
		aP=47.7	aP=48.2	aP=48.2	aP=47.6	aP=48.0	aP=48.2
	1.7 mM	1 (30%)	13 (50%)	14 (20%)	1 (35%)	13 (50%)	14 (15%)
		DEPMPO-	DEPMPO-	DEPMPO-	DEPMPO-	DEPMPO-	DEPMPO-
		OH	carbon R	OCH3/OOLina	OH	carbon R	OCH3/OOLina
		g=2.0055	g=2.0053	g=2.0059	g=2.0055	g=2.0053	g=2.0059
		aN=14.1	aN=15.2	aN=13.6	aN=14.1	aN=15.1	aN=13.6
		aH=13.4	aH=22.6	aH=9.4	aH=13.7	aH=22.3	aH=9.4
		aP=47.6	aP=48.0	aP=48.2	aP=47.6	aP=47.6	aP=48.2

  Here, radical hydroxy and carbon radical were trapped. A catalytic amount of Iron II an equal proportion of them is observed but at t = 30 min carbon radical decreased and hydroxy radical increased. By increasing Iron II concentration carbon radical formation was increased and hydroxyl radical formation was decreased up to disappear at 1.7 mM Iron II. At t = 30 min same behavior is observed. At 1.7 mM Iron II and t = 0 min only carbon radical was observed. CM seemed to have a role in to the formation of carbon radical because here the ration oxygen/carbon radical was increased compared to this ratio without using CM.

	Annex 11. Spin-adducts formed from Lina-OOHs (1 mM) /DEPMPO (25 mM) system in
	HEPES-CM (1-1/v-v) with Fe(II) varying concentrations
	Fe(II)	Spin-adducts in HEPES	Spin-adducts in HEPES
		assignment, g, hfccs (Gauss) t = 0 min	assignment, g, hfccs (Gauss) t = 30 min
	0.1 mM	-	-	1 (70%)	13 (30%)
				DEPMPO-OH	DEPMPO-carbon R
		50%) DEPMPO-OH	13 (50%) DEPMPO-carbon R	1 (60%) DEPMPO-OH g=2.0055 aN=14.05	13 (40%) g=2.0054 DEPMPO-carbon R aN=15.14
		g=2.0055 aN=14.1	g=2.0053 aN=15.3	g=2.0055 aN=14.1 aH=13.77 aP=47.65	aH=22.28 g=2.0054 aN=15.3 aP=47.81
	0.5 mM	aH=13.5 aP=47.5 1 (40%) DEPMPO-OH	aH=22.1 aP=47.8 13 (60%) DEPMPO-carbon R	aH=13.5 aP=47.7 1 (50%) DEPMPO-OH	aH=22.3 13 (50%) aP=47.7 DEPMPO-carbon R
	0.5 mM	1 (40%) DEPMPO-OH g=2.0055 aN=14.08	13 (60%) DEPMPO-carbon R g=2.0054 aN=15.07	1 (50%) DEPMPO-OH g=2.0055 aN=14.10	13 (50%) g=2.0054 DEPMPO-carbon R aN=15.01
		g=2.0055 aN=14.1 aH=13.40 aP=47.61	g=2.0054 aN=15.3 aH=21.85 aP=47.74	g=2.0055 aN=14.1 aH=13.54 aP=47.54	aH=22.03 g=2.0054 aN=15.3 aP=47.45
	1.0 mM	aH=13.4 aP=47.5 1 (25%) DEPMPO-OH	aH=22.3 aP=47.6 13 (75%) DEPMPO-carbon R	aH=13.5 aP=47.6 1 (30%) DEPMPO-OH	aH=22.4 13 (70%) aP=47.4 DEPMPO-carbon R
	1.0 mM Fe(II)	1 (25%) DEPMPO-OH g=2.0055 aN=14.12	13 (75%) DEPMPO-carbon R g=2.0054 aN=15.17	1 (25%) DEPMPO-OH g=2.0055 aN=14.07	13 (75%) g=2.0054 DEPMPO-carbon R aN=15.20
		g=2.0055 aN=14.1 aH=13.69 aP=47.31	g=2.0053 aN=15.2 aH=22.14 aP=47.54	g=2.0055 aN=14.1 aH=13.70 aP=47.53	aH=22.16 g=2.0053 aN=15.0 aP=47.67
	1.7 mM	aH=13.7 aP=47.3 DEPMPO-OH 1 (10%)	aH=22.1 aP=47.7 DEPMPO-carbon R 13 (90%)	aH=13.7 aP=47.5 -	aH=22.0 aP=47.4 -
	1.7 mM Fe(II)	-g=2.0055 aN=14.45	13 g=2.0055 DEPMPO-carbon R aN=15.18	-	-
		aH=13.73 aP=46.55	aH=22.26 g=2.0053 aN=15.2 aP=47.62
			aH=22.3
	aP=47.9 Radical generation at low concentration of Iron II was not observed at t = 0 min whereas it was
	at t = 30 min. The same radicals were obtained in HEPES, HEPES-CM 8/2 and HEPES-CM
	5/5 but the carbon radical generated seemed to increase by adding CM. We studied those
	proportion in order to mimic what could happen on RHE.

(CDCl 3 , δ, ppm): 1

  : C 9 H 16 O .28 (s, 3H, CH 3 ), 1.31 (s, 3H, CH 3 ), 1.50-1.67 (m, 5H, CH 2 + OH), 2.02-2.06 (m, 4H, 2 × =CCH 2 ), 5.73 (dd, l H, 3 J HH = 2.0 Hz, 3 J HH = 3.6 Hz, =CH).To a solution of hydrogen peroxide (250 mL, 35% in water) was added, at 0 °C, one drop of sulfuric acid (98 %) and Mod-OH (10 g; 71.4 mmol). The reaction mixture was vigorously stirred for 3 hours at °C then extracted with pentane (3 × 200 mL). Combined organic layers were dried over magnesium sulfate, filtered, and evaporated under reduced pressure to give the crude hydroperoxide, which was purified by column chromatography on silica gel (petroleum ether/ hexane 9/1) to give 4.48 g (42 % yield) of the pure hydroperoxide Mod-OOH as a colorless oil.

	NMR 1 H NMR 13 C (CDCl 3 , δ, ppm):
	22.5 (s, =CHCH2CH2); 23.5 (s, =CCH2CH2); 24.6 (s, 2 × CH3); 25,5 (s, =CHCH2); 29.3 (s, =CCH2);
	78.3 (s, C(CH3)2OH); 119.1 (s, =CH); 144.1 (s, C=CH);
	Synthesis of Mod-OOH
	Formula: C 9 H 16 O 2
	Molar mass: 156.23 g.mol -1
	Cas N°: Not exist
	TLC: Rf = 0.3 (petroleum ether/ethyl acetate 9/1): UV, APM/Ce
	Molar mass: 140.23 g.mol -1 NMR 1 H (CDCl 3 , δ, ppm):
	Cas N°: 25910-97-8 1.31 (s, 6H, 2 × CH3); 1.56-1.68 (m, 4H, =CHCH2CH2 + =CCH2CH2); 2.00-2.09 (m, 4H, =CCH2 +
	TLC: Rf = 0.26 (petroleum ether/ethyl acetate 9/1): UV, APM/Ce =CHCH2); 5.75 (m, lH, =CH); 7.25 (s, lH, OOH)
	NMR 13 C (CDCl 3 , δ, ppm):

22.2 (s, =CHCH2CH2); 23.0 (s, =CCH2CH2); 23.8 (s, 2 × CH3); 24,0 (s, =CHCH2); 25.2 (s, =CCH2); 84.6 (s, =CH); 123.1 (s, C=CH); 139.8 (s, C(CH3)2OOH)

MATERIALS AND METHODS:....................................................................................................................... Literature........................................................................................................................

hours, reflux 110 °C, KCN (97 %) -3 hours, reflux 110 °C, KCN (98 %) 38 % 24 hours, room temperature, KCN (97 %) -24 hours, room temperature, KCN (98 %)

In a flame-dried round bottom flask under argon 1-( 13 C)-potassium cyanide ( 13 C-3) (98 %; 2.0 g; 31 mmol; 1 eq) and potassium iodide (0.55 g; 3.3 mmol; 0.11 eq) were dried at 120 °C under a vacuum pump for 24 hours. Anhydrous N-methyl-2-pyrrolidone (2.0 mL) and anhydrous trimethylsilyl chloride (3.8 mL; 30 mmol; 0.97 eq.) were added very slowly. The solution color changed from white to yellow. The mixture was stirred for 24 hours at room temperature. 1 Trimethylsilyl ( 13 C)-cyanide 13 C-21 was obtained by distillation (bp = 112 °C) as a colorless oil (2.4 g; 82 %).

Formula: C 3 13 CH 9 NSi Molar mass: 100.20 g.mol -1 CAS (non 13 C-substituted): 7677-24-9 NMR 1 H (CDCl 3 , δ, ppm): 0.35 (s, 9H, -Si(CH 3 ) 3 ).

NMR 13 C (CDCl 3 , δ, ppm): 0.1 (Si(CH 3 ) 3 ), 124.9 (-13 C≡N).

Literature

For this reason, labmade routine to simulate EPR experimental data were developed, based on Easyspin, a computational EPR toolbox developed by Dr. Stefan Stoll et al., under Matlab environment. 27 After a comprehensive simulation and fitting to the experimental data, the program can deconvolute the EPR mixed signature and determine the ratio of the different spinadducts composing the reaction mixture. Both the hfccs and g values for each system are thus obtained and can be compared to the existing literature, spin databases, and/or DFT calculations. 17,18 This approach is especially useful when dealing with weak S/N (Figure 2.10). The above described protocol based on "ruler and pencil" data analysis together with computational approaches paved the way of the analysis working method especially developed in the context of this PhD work.

EPR instrumentation

A schematic representation of an EPR spectrometer is shown in Figure 2.11. Major components are: an electro-magnet, a microwave bridge, the resonant cavity, the console and the work station.

Optimized simulation

Experimental spectrum peroxyl and carbon-centered radicals has been observed. When the solvent was exclusively acetonitrile, the proportion of carbon radicals trapped considerably increased, probably due to the decomposition of the solvent. Moreover, it seems that ROOH undergo initially a homolytic scission. Hydroxyl radical spin-adduct were detected using DEPMPO and DMPO. Peroxy cumyl and peroxy linalyl radicals were also identified by using DEPMPO as well as methoxy radicals that were detected by both DEPMPO and DMPO. Finally, carbon radicals were also identified by using PBN, DMPO, and DEPMPO. Interestingly, it appeared that Cum-OOH and Lina-OOHs exhibit a similar behavior rather different from that observed from Limo-OOH as summarized in Table 2.3 Such studies revealed that several reactive radical intermediates issued from the ROOHs are possible depending on their chemical structure, suggesting that specific immunogenic protein chemical modifications could lead to sensitization. However, such studies were carried out in solution (aqueous buffer or semi-organic), far from mimicking what may actually happen in real-life scenario, i.e. in the skin.

IDENTIFICATION OF RADICALS DERIVED FROM ROOHS: IN SITU APPROACH IN RHE

The objective of the present work was to develop an EPR spin-trapping methodology with the potential to investigate the formation and behavior of radicals issued from ROOHs in a reconstructed human epidermis 3D model (RHE). RHE Episkin TM (Lyon, France) is an in vitro reconstructed human epidermis from normal cultured keratinocytes (13-days stratification) on the collagen matrix at the air-liquid interface. 46 Keratinocytes are the major cell type in the epidermis and play a key role in skin inflammatory reactions.

Spin-traps

Limo-OOH Cum-OOH Lina-OOHs DEPMPO

Linalool (2.83 g; 17.8 mmol; 1 eq.) was introduced in the microemulsion at room temperature. Hydrogen peroxide (aqueous solution 35%; 1 mL one fraction) was then added.

The solution was instantly red colored. After 20 minutes stirring the solution became yellow.

Fourteen other fractions of hydrogen peroxide (aqueous solution 35%; 1 mL each) were added successively by an interval of 10 minutes each. The resulting yellow solution was stirred overnight at room temperature. When the reaction was thoroughly translucent, linalool was completely oxidized. The solvent was removed under reduced pressure at 40 °C. The semi-solid white product obtained was suspended in dichloromethane (900 mL) and vigorously stirred for 48 hours. The suspension was filtered to recover sodium molybdate and sodium dodecyl sulfate.

The filtrate, partially evaporated under reduced pressure, was washed using distilled water (3 × 100 mL) and the aqueous phase was extracted with dichloromethane (100 mL). Combined organic phases were dried over magnesium sulfate, filtered and concentrated under reduced pressure to give a crude yellow oil. A mixture of hydroperoxides Lina-OOHs (ratio 2/3) was obtained using silica gel column chromatography (petroleum ether/AcOEt 4/1, then 3/2) as a yellow oil (2.97 g; 15.97 mmol; 90%). In Figure A7.1 and 2 shows that the same kind of radicals were generated. In HEPES buffer radical generation stared at catalytic amount compared to PB, that is the reason why we will use HEPES buffer for the further tests and also in RHE.

Annex 8. Spin-adducts formed from Cum-OOH (0.54 mM) / PBN (25 mM) system in PB and HEPES with Fe(II) varying concentrations 17,30,41,43 Fe(II) Spin-adducts in PB assignment, g, hfccs (Gauss) Spin-adducts in HEPES assignment, g, hfccs (Gauss)

CONCLUSION

This study underlined that the tested allylic hydroperoxides seemed not to be equal for DC activation in regard to Nrf2 cytoplasmic accumulation. Results showed that Lina and Limo need to be oxidized to activate THP-1 cells. Lina-OOHs (1 mM) seemed to be stronger activators of THP-1 cells than Limo-OOH (1.5 mM). Only the hydroperoxides were able to activate the Nrf2 pathway and to induce the transcription of target genes ho-1, nqo1 and il-8, except Mod (1 mM)

Experimental Chapter 3 Synthesis of Limo-OOH

See Chapter 2. Section 4.3.2

Synthesis of LinaOOHs

See Chapter 2. Section 4.3.3

Chemical treatment

THP-1 cells were cultured for at least 12 hours at 8 × 10 5 cells/ml in complete medium without 2-mercaptoethanol. On the next day, the cells were seeded in complete medium at 1 × 10 6 cells/ml and treated with chemicals. CinA was diluted in DMSO and tBHP, terpenes and allylic hydroperoxides in CH 3 CN as they were water-insoluble. Final concentrations of solvent in culture media did not exceed 0.1%.

Measurement of cell viability

THP-1 cells were seeded in 24-well plates (1 × 10 6 cells/ml, 1 ml/well) with various concentrations of each chemical ranging from 0.25 to 1.5 mM for 24 hours (Table 3.1). Cells were washed twice with PBS and harvested cells were incubated with annexin-V-PE and 7aminoactinomycin D (7-AAD) according to the manufacturer's protocol (BD Biosciences). Cell viability was measured using the FL-3 wavelength on a FACScalibur flow cytometer (Becton Dickinson, Le Pont de Claix, France). Data acquisition was performed using the CellQuest software (BD Biosciences). At least four independent experiments were performed for each chemical.

Immunoblotting

Western blot was performed as previously described. and left in ice for 20 min. Then, they were centrifuged at 17600 × g for 20 min at 4°C, before the supernatants were collected. Total protein concentration was evaluated using bicinchoninic acid assay, and equal amounts of denatured proteins (30 μg) were loaded on 12% polyacrylamide gels (TGX Stain-Free FastCast; Bio-Rad Laboratories, Hercules, CA) for approximately 30 min at 300V in running buffer. Stain-free gels were activated by exposure to ACD is a disease widespread in the population, induced by repeated contact with a chemical compound that is able, after penetration into the epidermis, to chemically modify skin proteins. 1 Chemically modified proteins are treated by DCs, playing the role of antigen presenting cells in the epidermis. DCs, expressing the MHC II, will present the modified peptides to T lymphocytes in the local lymph nodes. This will generate selective activation of T lymphocytes having the specific receptor of the presented peptide modification on their surface. After a second contact with the same allergen specific T lymphocytes are reactivated and will generate an immune reaction that will produce clinical symptoms such as edema eczema and erythema in the zone where allergen was in contact with the epidermis. 2 Allergen or haptens are xenobiotics with low molecular weight (less than 1000 Da), capable of penetrating the first layer of the epidermis. In the epidermis these molecules can react with side chains amino acids through the formation of stable covalent bond. The mechanism commonly accepted for this step, forming the hapten-protein complex, is a two electrons mechanism. In general, allergenic compounds contain electrophilic chemical functions that react with nucleophilic side chains of amino acids. However, in the case of allergenic allylic hydroperoxides no electrophilic function is present and one electron radical mechanisms are suspected. 3 This Ph.D.

work focused on the comprehension of skin sensitization mechanisms to allylic hydroperoxides derived from autoxidation of terpenes. More precisely to Lina-OOHs and Limo-OOH identified as potent skin sensitizers derived from autoxidation of linalool and limonene, respectively, widely used for their odorant properties. 4,5 The main goal was to evaluate the influence of radical mechanisms in the interaction between these allylic hydroperoxides and proteins.

The first part of this work was to synthesize 5-( 13 C)-Lina-OOHs and 4-( 13 C)-Lina-OOHs to study their behavior in presence of some amino acids prompt to react by radical mechanisms. The synthetic pathways developed in order to synthesize 5-( 13 C)-Lina-OOHs and 4-( 13 C)-Lina-OOHs are exposed in the first chapter. The mixture of 5-( 13 C)-Lina-OOHs was synthesized in a nine-steps synthesis with a global yield of 3.8 %. The mixture of 4-( 13 C)-Lina-OOHs synthesized in six-steps synthesis with a global yield of 9.5 %. Reactivity studies with several amino acids were then followed up by mono-dimensional 13 C-NMR and heteronuclear bi-dimensional 1 H-13 C-NMR (HSQC and HMBC). Only reaction with the side chain of N-acetyl-L-cysteine methyl ester was observed, and a variety of hypothetical compounds was obtained. Some hypothesis of the exact structures of these compounds have been proposed.

The second part of this work, was focused on the evaluation and characterization of free radicals (oxygen and carbon-centered) generation from target compounds. EPR spectroscopy combined with the spin-trapping technique was used. Spin-traps used were PBN, DMPO and DEPMPO. First trials were done in solution by using Fe (II) as radical initiator as it is suspected to play a role in the in vivo hydroperoxides degradation. 6 In second part of the study, we developed the model to study the in situ generation of these radicals on a 3D-reconstructed human epidermis (RHE) model (Episkin TM ). No Fe (II) radical initiator was used here to be closer to a real-life scenario exposure of the skin. Carbon-centered radicals, peroxyl and allyloxyl radicals, derived from allylic hydroperoxides (Lina-OOHs, Limo-OOH and Cum-OOH) were identified in solution and in the RHE environment. In solution, a more extensive generation of radicals was seen compared to RHE.

However in both systems oxygen-centered, and carbon-centered radicals were observed, proving the crucial role of radical mechanisms. 7 Control experiments in RHE suggest that radicals observed are derived from the ROOHs themselves and not from biomolecules radical degradation in RHE.

However, further investigations are necessary to confirm this. These RHE results could be in agreement with reported animal and clinical experimentations that have ruled out the existence of cross-reactivity between different allergenic hydroperoxides. 8 These studies confirmed that immune responses to the antigenic hapten-protein complex are necessarily specific. Carboncentered, allyloxyl and organic peroxyl radicals observed in the RHE could form specific complexes which is not the case of the hydroxyl radical.

The third part of this work was focused on the activation of the Nrf2-Keap1 cellular pathway in DCs by allylic hydroperoxides. Lina-OOHs, Limo-OOH, Mod-OOH and Cum-OOH seemed to activate this pathway. However, it also appeared that they were not equal for DCs activation regarding Nrf2 cytoplasmic accumulation because Limo-OOH seemed not to be activating it. They also induced the transcription of target genes ho-1, nqo1 and il-8, except Mod that was also activating the Nrf2 pathway. 9 The futures perspectives of the present work are as various as promising to decipher how molecular phenomena occur in ACD caused by allylic hydroperoxides. Firstly, reactivity studies between 5- Secondly, the EPR original methodology that we successfully developed in this work made possible to investigate in situ situations closer to a real-life skin allergy situation. 7 The principal aim of this manuscript was to set up the technology transfer from studies in solutions to the model in situ using RHE. Now the challenge will be to apply the novel approach to important skin allergens reacting through radical mechanisms and check all different secondary reactions that could occur because of the radical initiation, the potential oxidation of biomolecules in RHE skin cells, and/or induced by the buffer. A substantial question that remains is where the radical initiation occurs. Our assumption based on the investigations presented in chapter 2, is that the spin-adducts issued from Cum-OOH would be formed on cell surfaces whereas for Lina-OOH and Limo-OOHs the reaction would occur within the cells. As a matter of fact, the cell cytoplasm is known to constitute an efficient reductive environment, and this could explain both the lower S/N ratio and the weaker lifetime observed for Lina-OOH and Limo-OOH spin-adducts when compared to Cum-OOH experiments. For this purpose, CD-DEPMPO spin-trap described by Abbas et al.

would lead to a better localization of the hydroperoxide-mediated radical generation. 10 Indeed, such spin-trap is assumed not to penetrate within the cells whereas DEPMPO probably does. On the other hand, Hardy et al. described mitochondrial target spin-trap (Mito-DEPMPO) which could be tested to probe potential hydroperoxide radical generation within the cells. 11 Furthermore, the 13 C substitution on e.g. of Cum-OOH's methyl would allow to distinguish whether the methyl radical observed stem for the hydroperoxide or not. Finally, a variety of molecules susceptible to undergo a radical intermediate during the reactivity process can be tested such as antioxidants, colour dye, peptides etc.