Mechanistic Proposal for the Formation of Specific Immunogenic

Sep 2, 2009 - Department of Chemistry, Dermatochemistry and Skin Allergy, University of Gothenburg, Gothenburg, Sweden, and Department of Analytical ...
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Mechanistic Proposal for the Formation of Specific Immunogenic Complexes via a Radical Pathway: A Key Step in Allergic Contact Dermatitis to Olefinic Hydroperoxides Staffan Johansson,† Theres Redeby,†,‡ Timothy M. Altamore,† Ulrika Nilsson,‡ and Anna Bo¨rje*,† Department of Chemistry, Dermatochemistry and Skin Allergy, UniVersity of Gothenburg, Gothenburg, Sweden, and Department of Analytical Chemistry, Stockholm UniVersity, Stockholm, Sweden ReceiVed April 16, 2009

The widespread use of scented products causes an increase of allergic contact dermatitis to fragrance compounds in Western countries today. Many fragrance compounds are prone to autoxidation, forming hydroperoxides as their primary oxidation products. Hydroperoxides are known to be strong allergens and to form specific immunogenic complexes. However, the mechanisms for the formation of the immunogenic complexes are largely unknown. We have investigated this mechanism for (5R)-5isopropenyl-2-methyl-2-cyclohexene-1-hydroperoxide (Lim-2-OOH) by studying the formation of adducts in the reaction between this hydroperoxide and 5,10,15,20-tetraphenyl-21H,23H-porphine iron(III) chloride (Fe(III)TPPCl) in the presence of protected cysteine (NAc-Cys-OMe) or glutathione (GSH). Isolated adducts originate from the addition of the thiol group of NAc-Cys-OMe over the carbon-carbon double bonds of carvone. Furthermore, adducts between NAc-Cys-OMe and carveol as well as between GSH and carvone have been identified. The formation of these adducts most likely proceeds via the radical thiol-ene mechanism. The addition of a terpene moiety to cysteine offers an explanation of the specificity of the immune response to structurally different hydroperoxides. These results also explain the lack of cross-reactivity between carvone and Lim-2-OOH. In conclusion, we propose that immunogenic complexes of olefinic hydroperoxides can be formed via the radical thiol-ene mechanism. These complexes will be specific for the individual olefinic hydroperoxides due to the inclusion of a terpene moiety derived from the hydroperoxide. Introducution 1

Allergic contact dermatitis (ACD ) is the clinical manifestation of contact allergy, which is caused by repeated skin exposure to reactive chemicals in the environment that are able to penetrate into the epidermis. These chemicals (haptens) are low molecular weight compounds that can modify macromolecules in the skin, thus forming immunogenic hapten-protein complexes. The immunogenic complexes will be processed to antigens by Langerhan’s cells. At repeated exposure, antigenspecific memory lymphocytes will be activated, causing an inflammation in the skin at the contact site. The classical model for the formation of the immunogenic complex involves hapten-protein interactions where nucleophilic groups of the protein react with the electrophilic hapten (1). The reaction is a nucleophilic substitution or a nucleophilic addition, whereby a covalent bond between the hapten and the protein is created. Both electrophilic and nonelectrophilic haptens are formed when unsaturated hydrocarbons and alcohol ethoxylates are exposed to air (2-6). Among the products formed, the hydro* To whom correspondence should be addressed. Dermatochemistry and Skin Allergy, Department of Chemistry, University of Gothenburg, SE412 96 Gothenburg, Sweden. Phone: +46 31 772 4725. Fax: +46 31 772 3840. E-mail: [email protected]. † University of Gothenburg. ‡ Stockholm University. 1 Abbrevations: ACD, allergic contact dermatitis; CE, collision energy; Fe(III)TPPCl3, 5,10,15,20-tetraphenyl-21H,23H-porphine iron(III) chloride; Lim-2-OOH, (5R)-5-isopropenyl-2-methyl-2-cyclohexene-1-hydroperoxide (limonene-2-hydroperoxide); NAc-Cys-OMe, 2R-acetamido-3-mercaptopropanoic acid methyl ester; TIC, total ion current.

peroxides have been identified as strong allergens (3, 7-9). In recent years, hydroperoxides have also been demonstrated as important allergens among dermatitis patients (10, 11). It has been proposed that hydroperoxides represent an alternative mode of reaction involving radical coupling between the hapten and the protein. Alternatively, intramolecular radical rearrangements may form compounds which act as electrophilic haptens in a classical nucleophilic-electrophilic mechanism (12, 13). A different mode of action could be that the hydroperoxides form nonspecific immunogens, such as oxidized proteins. Since hydroperoxides are oxidizing agents, they may oxidize functional groups in the proteins, e.g., the phenolic groups in tyrosine or the sulfur moieties of cysteine and methionine. In theory, structurally different hydroperoxides could form the same oxidized and immunogenic proteins. Hydroperoxides reacting in this manner would be nonspecific agents, and cross-reactions between hydroperoxides with significantly different structures would be observed in animal experiments or at patch testing. However, in a recent study including human and animal data, we found no support for this theory. Specific allergenic activity caused by specific antigens, where the hapten binds to a host protein, was observed and supported by theoretical chemical calculations (14). R-Limonene is a terpene commonly used as a fragrance compound and flavor additive (Figure 1). Autoxidation of R-limonene forms an allergenic oxidation mixture (4, 15) which causes contact allergy in about 3-4% of consecutive dermatitis patients in European dermatology clinics (10). About half of these patients also react to the pure hydroperoxide fraction of

10.1021/tx9001435 CCC: $40.75  2009 American Chemical Society Published on Web 09/02/2009

Radical Formation of Immunogenic Complexes in ACD

Figure 1. Structures of compounds referred to in this article.

this mixture. Model systems using chemical radical trappers to investigate the mechanisms of radical coupling of hydroperoxides are presented in the literature (13, 14, 16). These studies show that both alkoxy radicals and carbon centered radicals are formed from the investigated hydroperoxides. This suggests that these radicals play an integral part in the formation of immunogenic hapten-protein complexes, the first step for the sensitization of skin to these compounds. The aim of the present study was to investigate the mechanism for the formation of specific immunogenic hapten-protein complexes of olefinic hydroperoxides. This was done by studying the formation of terpene-amino acid adducts formed in the reaction of Lim-2-OOH with Fe(III)TPPCl in the presence of NAc-Cys-OMe or GSH. The structures of isolated adducts were determined, and a radical mechanism for their formation is proposed.

Experimental Procedures Caution: Skin contact with hydroperoxides must be aVoided. As these compounds are skin-sensitizing substances, they must be handled with care. Chemicals. 5,10,15,20-Tetraphenyl-21H,23H-porphine iron(III) chloride (Fe(III)TPPCl), (5R)-5-isopropenyl-2-methyl-2-cyclohexenone (carvone, 98%), (5R)-5-isopropenyl-2-methyl-2-cyclohexen1-ol (R-carveol, mixture of isomers, 97%), hydrogen peroxide-urea adduct (30% available H2O2), hydrogen peroxide (35% in water), methanesulfonyl chloride (99.5%), thionyl chloride (99%), triethylamine (99%), and ammonium acetate were purchased from SigmaAldrich (Stockholm, Sweden). All N-acetyl amino acids and GSH were purchased from Bachem (Weil am Rhein, Germany). DMF

Chem. Res. Toxicol., Vol. 22, No. 11, 2009 1775 and acetonitrile were dried over 4 Å molecular sieves. MeOH was distilled from NaOH and stored over 3 Å molecular sieves before use. For the reactions with carvone and NAc-Cys-OMe, (-)-carvone (99%) and N-acetyl-L-cysteine-methyl ester were purchased from Fluka (Sigma-Aldrich, Stockholm, Sweden). Instrumentation and Mode of Analysis. Preparative high performance liquid chromatography (HPLC) was performed using two Gilson pumps model 305, a BAS UV/vis detector model UV116 (Bioanalytical Systems, Inc., W. Lafayette, IN, USA), a CMA 200 Microsampler (CMA Microdialys AB, Stockholm, Sweden) and a Zorbax SB-C18 Semi-Preparative column (250 × 9.4 mm i.d., particle size 5 µm, Agilent). The flow rate was 4 mL/min, each injection was 100 µL, and the compounds were monitored at 202 nm. The mobile phase consisted of 0.4% formic acid and 5% acetonitrile in water (solvent A) and 0.4% formic acid and 5% water in acetonitrile (solvent B). A linear gradient 0-40% of solvent B in A for 3 min was followed by a linear gradient 40-100% of solvent B in A for 8 min, and 1 min of isocratic elution with 100% solvent B. A linear gradient 100-0% of solvent B in A for 2 min was followed by 3 min of isocratic elution with 100% solvent A. Further purification was done employing an isocratic system consisting of 72% A and 28% B, using the same HPLC and column as those for the gradient runs. Liquid chromatography/mass spectrometry (LC/MS) analyses were performed on an Acquity Ultra Performance LC (UPLC) from Waters Corp. (Milford, MA, USA) coupled to a Q-ToF Premier (Micromass, Waters Corp.) used in V-mode. The UPLC column was an Acquity BEH C18 (50 × 2.1 mm i.d., particle size 1.7 µm, Waters Corp.). The mobile phase consisted of solvent A, 10 mM acetic acid and 5% acetonitrile in water, and solvent B, 10 mM acetic acid and 5% water in acetonitrile. When analyzing the crude mixtures and the fractions from the analytical fractionation, a linear gradient of 0-50% B in A for 5 min was followed by a linear gradient of 50-95% B in A for 1.5 min, and 1 min of isocratic elution with 95% B. This was followed by a linear gradient 95-100% of B in A for 0.25 min and a linear gradient 100-0% of B in A for 3.25 min. The flow rate was 0.25 mL/min, and the column temperature was set to 20 °C. The MS was equipped with an electrospray ionization (ESI) interface used in positive mode with the following settings: cone gas flow, 48 L/h; desolvation gas flow, 702 L/h; capillary voltage, 2.8 kV; cone voltage, 35 V; extraction cone, 4 V; ion guide, 2 V; source temperature, 100 °C; and desolvation temperature, 300 °C. Scanning was performed at two different collision energies (CE), 2 and 20 eV for NAc-CysOMe adducts and 3 and 15 eV for GSH adducts, and the scan cycle time was 0.21 s at each CE. Monitoring was performed between 100 and 1000 m/z. Nuclear magnetic resonance (NMR) spectroscopy was performed on a JEOL Eclipse+ 400 instrument at 400 MHz using deuterated chloroform (CDCl3) or Me2SO-d6 as solvent. For CDCl3, chemical shifts (δ) are reported in ppm relative to CHCl3 at 7.26 for 1H, and at 77.0 for 13C. For Me2SO-d6 chemical shifts (δ) are reported in ppm relative to Me2SO at 2.50 for 1H and at 39.4 for 13C. 1H- and 13 C NMR spectra were assigned using 13C distortionless enhancement by polarization transfer (DEPT), 1H-1H correlation spectroscopy (COSY), 1H-13C heteronuclear multiple-quantum coherence (HMQC), 1H-13C heteronuclear single-quantum coherence (HSQC), and 1H-13C heteronuclear multiple-bond correlation (HMBC). Coupling constants are reported in Hz. Column chromatography was performed using Merck silica gel 60 (230-400 mesh ASTM), and TLC was performed using silica plated aluminum sheets (Merck, 60 F254 silica gel) that were developed with anisaldehyde dip (2.1 mL of acetic acid, 5.1 mL of anisaldehyde, and 7 mL of H2SO4 in 186 mL of ethanol) followed by heating. Synthesis: (5R)-5-Isopropenyl-2-methyl-2-cyclohexene-1-hydroperoxide (Lim-2-OOH). The synthesis was performed as reported in the literature (16). General Procedure for Methyl Ester Protection of Selected Amino Acids. Thionyl chloride (1.1 equiv) was added dropwise to a solution of the N-acetyl amino acid (1.0 equiv) in freshly distilled,

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dry MeOH (20 mL/5.0 mmol N-acetyl amino acid) at 0 °C under an atmosphere of nitrogen. The resulting solution was left to stir for 5 h while slowly warming to room temperature. Thereafter, the reaction mixture was concentrated in Vacuo to give sufficiently pure N-acetyl O-methyl protected amino acids. 2S-Acetamido-3-(4-hydroxyphenyl)propanoic Acid Methyl Ester (NAc-Tyr-OMe). Yield 92%, white solid; mp 117-118 °C (lit mp 120-121 °C (17)); 1H NMR (Me2SO-d6) δ 6.92 (d, J ) 8.4 Hz, 2H, H6), 6.72 (d, J ) 8.4 Hz, 2H, H5), 6.10 (br d, J ) 13.4 Hz, 1H, NHAc), 4.86 (dd, J ) 1.8, 5.8 Hz, 1H, H2), 3.73 (s, 3H, H10), 3.07 (dd, J ) 5.5, 13.9 Hz, 1H, H3a), 2.96 (dd, J ) 5.5, 13.9 Hz, 1H, H3b), 1.97 (s, 3H, H9); 13C NMR (Me2SO-d6) δ 172.4 (C1), 170.4 (C8), 155.6 (C7), 130.3 (C5), 127.0 (C4), 115.6 (C6), 53.4 (C2), 52.5 (C10), 37.2 (C3), 23.1 (C9). 2S-Acetamido-3-(1H-indol-3-yl)propanoic Acid Methyl Ester (NAc-Trp-OMe). Yield 93%, white solid; mp 148-149 °C (lit mp 154-155 °C (18)); 1H NMR (Me2SO-d6) δ 10.87 (br. s, 1H, NHAr), 8.32 (d, J ) 7.3 Hz, 1H, ArH), 7.49 (d, J ) 7.7 Hz, 1H, ArH), 7.34 (d, J ) 8.0 Hz, 1H, ArH), 7.15 (br. d, J ) 2.2 Hz, 1H, NHAc), 7.02 (t, J ) 7.4 Hz, 1H, ArH), 6.99 (t, J ) 7.7 Hz, 1H, ArH), 4.49 (dd, J ) 8.0, 13.9 Hz, 1H, H2), 3.58 (s, 3H, H14), 3.14 (dd, J ) 8.5, 14.3 Hz, 1H, H3a), 3.02 (dd, J ) 8.8, 14.6 Hz, 1H, H3b), 2.97 (s, 3H, H13); 13C NMR (Me2SO-d6) δ 173.1 (C1), 169.9 (C12), 136.7 (C6), 127.6 (C11), 124.2 (C5), 121.5 (C9), 119.0, 119.5 (C8, C10), 112.0 (C7), 110.1 (C7), 53.7 (C2), 52.3 (C14), 27.7 (C3), 22.9 (C13). 2S-Acetamido-6-aminohexanoic Acid Methyl Ester (NAc-LysOMe). Yield 94%, white solid; mp 106-108 °C (lit mp 108-114 °C (19)); 1H NMR (Me2SO-d6) δ 8.40 (br d, J ) 7.3 Hz, 1H, NHAc), 5.27 (br s, 2H, NH2), 4.15 (dd, J ) 7.7, 13.9 Hz, 1H, H2), 3.60 (s, 3H, H9), 2.27 (m, 2H, H6), 1.84 (m, 3H, H8), 1.59 (m, 4H, H5, H3), 1.35 (m, 2H, H4); 13C NMR (Me2SO-d6) δ 173.3 (C1), 170.2 (C7), 52.5, 52.4 (C2, C9), 38.8 (C6), 30.7 (C5), 26.9 (C2), 22.9, 22.8 (C8, C3). 2R-Acetamido-3-mercaptopropanoic Acid Methyl Ester (NAcCys-OMe). Yield 68%, white solid; mp 79-80 °C (lit mp 79-81 °C (20)); 1H NMR (Me2SO-d6) δ 6.37 (br s, 1H, NHAc), 4.90 (ddd, J ) 4.3, 4.3, 8.4 Hz, 1H, H2), 3.80 (s, 3H, H6), 3.02 (m, 2H, H3), 2.07 (s, 3H, H5), 1.33 (t, J ) 9.0 Hz, 1H, SH); 13C NMR (Me2SOd6) δ 170.5 (C1), 169.8 (C4), 53.7 (C2), 52.9 (C6), 27.0 (C3), 23.2 (C5). 2S-Acetamido-4-methylpentanoic Acid Methyl Ester (NAc-LeuOMe). Yield 99%, clear oil; 1H NMR (Me2SO-d6) δ 8.25 (br d, J ) 7.3 Hz, 1H, NHAc), 4.24 (m, 1H, H2), 3.61 (s, 3H, H8), 1.84 (s, 3H, H7), 1.52 (m, 3H, H3, H4), 0.88 (d, J ) 6.6 Hz, 3H, H5), 0.83 (d, J ) 6.7 Hz, 3H, H5). 2S-Acetamidopropanoic Acid Methyl Ester (NAc-Ala-OMe). Yield 95%, clear oil; 1H NMR (CDCl3 + 1 drop Me2SO-d6) δ 4.39 (q, 1H, J ) 7.4 Hz, H1), 3.60 (s, 3H, H6), 2.10 (s, 3H, H4), 1.33 (d, 3H, J ) 7.3 Hz, H2); 13C NMR (CDCl3 + 1 drop Me2SO-d6) δ 172.8 (C5), 172.3 (C3), 52.6 (C6), 49.0 (C1), 21.6 (C4), 17.3 (C2). 3-[(2′R)-Methyl-2′-acetamido-3′-thio-propanoate]-5-isopropenyl2-methyl-cyclohexanone (1). (2R)-Methyl-2-acetamido-3-mercaptopropanoate (226 mg, 1.28 mmol) and triethylamine (71 µL, 0.51 mmol) were added to an ovendried flask containing carvone (100 µL, 0.64 mmol). The contents were stirred at room temperature until TLC showed no starting material. The reaction mixture was diluted with dichloromethane/THF and washed three times with 2% HCl (aq) and once with water. The organic phase was dried over MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by flash chromatography to give a white crystalline solid (285 mg, 45%): 1H NMR (CDCl3) δ 1.12 (d, 3H, J ) 6.6 Hz, H7), 1.74 (s, 3H, H9), 1.94-2.04 (m, 1H, H4A), 2.04 (s, 3H, H6′), 2.09-2.17 (m, 1H, H4B), 2.17-2.26 (m, 1H, H2A), 2.43-2.50 (m, 1H, H2B), 2.74-2.82 (m, 1H, H6), 2.78-2.90 (m, 1H, H3), 2.85-3.08 (m, 2H, H1′), 3.43 (m, 1H, H5), 3.75 (s, 3H, H4′), 4.75 (s, 1H, H10A), 4.77-4.83 (m, 1H, H2′), 4.80 (s, 1H, H10B), 6.24 (d, 1H, J ) 7.3 Hz, NH); 13C NMR (CDCl3) δ 12.6 (C7), 20.8 (C9), 23.2 (C6′), 34.1 (C1′), 35.7 (C4), 40.8 (C3), 46.1 (C2), 48.7 (C6), 50.7 (C5), 51.9 (C2′), 52.9 (C4′), 110.5 (C10),

Johansson et al. 146.8 (C8), 169.9 (C5′), 171.1 (C3′), 209.5 (C1). MS (API-ES, CE 2 eV) m/z (%): 350 [M + Na] (45), 328 [M + H] (25), 178 [Cys + H] (100), 176 [Cys-H] (62), 151 (44), 136 (45). General Procedure for the Reaction of Lim-2-OOH with Fe(III)TPPCl in the Presence of Protected Amino Acids or Unprotected GSH. Fe(III)TPPCl (0.05 equiv) dissolved in acetone and amino acid or GSH (5 equiv) dissolved in 0.1 M aq. NH4CO2CH3 buffer was added to a solution of Lim-2-OOH (1 equiv) in acetone. The reaction mixture was incubated at 37 °C for 3 h. After the reaction, the acetone was removed by a gentle stream of nitrogen resulting in a dark brown-red clear solution above a black precipitate. In the screening experiments, the reactions were run with 8 mg of Lim-2-OOH in 2 mL of 1:1 acetone/buffer and the dark brown-red solution was directly injected on LC/MS. In the scaled up experiments, the reactions were run with 0.1 g of Lim-2-OOH in 12 mL of 1:1 acetone/buffer and fractionated as described below. Fractionation of Scaled-Up Reactions of Lim-2-OOH with NAc-Cys-OMe. The brown-red solution (8.3 mL) was filtered through a syringe-filter (0.1 µm, Whatman Anotop 25) to remove any remaining Fe(III)TPPCl. The filter was wetted before filtration (1 mL) and rinsed after filtration with MQ-H2O (3.5 mL) that was added to the filtered solution. This 12.8 mL solution was diluted to a total volume of 22.4 mL with HPLC mobile phase A and submitted for preparative HPLC fractionation. The black precipitate was dissolved in acetonitrile (2.0 mL) and filtered through a syringefilter (0.2 µm, Pall Acrodisc Nylon Membrane). The filter was rinsed with acetonitrile (2.0 mL) that was added to the filtrate to give a second dark brown-red clear solution that was fractionated by preparative HPLC. The resulting fractions were analyzed by LC/ MS. Fractions containing compounds with masses corresponding to plausible adducts were further purified by preparative HPLC. Two adducts were isolated and characterized. Characterization data for the first adduct (2.6 mg, 1.1%) agreed with the synthesized reference (1). Characterization data for the second adduct (2, 2.6 mg, 1.1%, mix of diastereomers): 1H NMR (CDCl3) δ 0.97 and 0.99 (s, 3H, H9), 1.62-1.72 (m, 1H, H8), 1.74-1.78 (m, 3H, H7), 2.045 and 2.052 (s, 3H, H6′), 2.09-2.15 (m, 2H, H3 and H4A), 2.14-2.24 (m, 1H, H2A), 2.24-2.33 (m, 1H, H4B), 2.37-2.45 (m, 1H, H10A), 2.40-2.49 (m, 1H, H2B), 2.56-2.64 (m, 1H, H10B), 2.88-3.03 (m, 2H, H1′), 3.767 and 3.771 (s, 3H, H4′), 4.77-4.85 (m, 1H, H2′), 6.19-6.29 (m, 1H, NH), 6.70-6.75 (m, 1H, H5); 13 C NMR δ 15.75 (C7 or C9), 15.95 (C7 or C9), 23.27 (C6′), 28.39 (C4), 35.00 and 35.07 (C1′), 37.13 (C8), 37.64 (C10), 39.05 and 39.09 (C3), 42.50 and 42.53 (C2), 51.89 and 51.95 (C2′), 52.80 and 52.84 (C4′), 135.63 (C6), 144.75 and 144.78 (C5), 169.85 and 169.89 (C5′), 171.41 (C3′), 199.89 and 199.92 (C1). MS (API-ES, CE 2 eV) m/z (%): 350 [M + Na] (40), 328 [M + H] (60), 286 [M + H - CH2CO] (31), 268 [M + H - CH3OH - CO] (35), 176 [Cys-H] (32), 144 [Cys + H-SH2] (100), 131 (13). Reactions of Carvone with NAc-Cys-OMe in the Presence of Fe(III)TPPCl. Fe(III)TPPCl (0.20 mg, 0.28 µmol) dissolved in acetone (0.20 mL) and Ac-Cys-OMe (5 mg, 28 µmol) dissolved in 0.1 M aq. NH4CO2CH3 buffer (1.25 mL) were added to a test tube containing carvone (0.85 mg, 5.6 µmol) dissolved in acetone (0.42 mL). The mixture was incubated at 37 °C for 3 h whereafter the acetone was removed by a nitrogen flow. The reaction mixture was analyzed by LC/MS. Reactions of Carvone with NAc-Cys-OMe in the Absence of Fe(III)TPPCl. Ac-Cys-OMe (5 mg, 28 µmol), dissolved in 0.1 M aq. NH4CO2CH3 buffer (1.25 mL), was added to a test tube containing carvone (0.85 mg, 5.6 µmol) dissolved in acetone (0.42 mL). The mixture was incubated at 37 °C for 3 h in the dark whereafter the acetone was removed by a nitrogen flow. The reaction mixture was analyzed by LC/MS.

Results and Discussion Synthesis. To be used as the reference, we synthesized compound 1 (Figure 1) from carvone and NAc-Cys-OMe via a Michael addition reaction utilizing triethylamine as a base. The

Radical Formation of Immunogenic Complexes in ACD

Figure 2. LC/MS chromatogram of the reaction mixture from the reaction of Lim-2-OOH with Fe(III)TPPCl in the presence of NAcCys-OMe. Structures of compounds 1-3 are found in Figure 3, compounds 4-5 are unidentified, structures of compounds 6-9 are found in Figure 4, and compound 10 is the NAc-Cys-OMe dimer.

amino acids included in the screening study were purchased as the N-acetyl derivatives and protected as the corresponding methyl esters. Difficulties were encountered in the synthesis of N-acetyl histidine methyl ester; therefore, the similar analogue N-benzoyl histidine methyl ester was used as purchased. We did not believe that the bulkier protecting group would adversely affect the proposed experiments. Screening Experiments with Amino Acids and GSH. Screening experiments were performed in order to detect possible adduct formation between the amino acids/GSH and Lim-2OOH, carvone, or carveol. Amino acids were selected to comprise nucleophilic and non-nucleophilic amino acids as well as amino acids expected to participate in radical reactions. The reaction mixtures were analyzed by LC/MS. No adducts were identified when using protected alanine, leucine, lysine, tryptophan, or tyrosine. Tyrosine formed relatively large amounts of a tyrosine-tyrosine dimer, while histidine formed small amounts of likely adducts. Several terpene adducts were detected by LC/MS in the experiments using protected cysteine (NAcCys-OMe) or GSH; therefore, further investigations were focused on these reactions. Reactions with Lim-2-OOH and NAc-Cys-OMe. As a model for the formation of immunogenic hapten-protein complexes of olefinic hydroperoxides, the possible formation of adducts between Lim-2-OOH and NAc-Cys-OMe was studied. Lim-2OOH was incubated with Fe(III)TPPCl and NAc-Cys-OMe in a mixture of acetone and buffer at 37 °C for three hours. Preliminary tests indicated that all hydroperoxide was consumed after 2 h. The small scale screening reaction was scaled-up in order to isolate sufficient material for NMR analysis. LC/MSanalysis of the unfractionated reaction mixtures revealed a complex mixture of compounds as shown by the TIC-chromatogram in Figure 2. In our previous investigation of radical formation from Lim-2-OOH, using Fe(III)TPPCl as radical initiator, the main product was carvone together with small amounts of carveol (16). In a control experiment using only Lim-2-OOH and Fe(III)TPPCl (results not shown), the two main products were carvone and carveol. Thus, the current investigation focused on the identification of adducts between NAc-CysOMe and Lim-2-OOH, carvone, or carveol. Peaks with masses corresponding to the addition of carvone (m/z 328, 1-6) or carveol (m/z 330, 7) to NAc-Cys-OMe were present. In addition, peaks corresponding to the addition of carvone with one or two additional oxygens, m/z 344 (8 and 9) and 360, respectively, were present. Because of the complexity of the mixture, further analysis was focused on the m/z 328 peaks, compounds 1-6 in the extracted ion chromatogram (Figure 2, m/z 328). Five of those (2-6) were formed only in the hydroperoxide reaction.

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Figure 3. Structures of identified m/z 328 adducts isolated from the reaction mixture of Lim-2-OOH with Fe(III)TPPCl in the presence of NAc-Cys-OMe.

In the control reactions between carvone and NAc-Cys-OMe (with and without Fe(III)TPPCl), compound 1 was the major product; only minute amounts of compounds 2 and 3 were formed. Compounds 1-3 and 6 were isolated and analyzed by NMR, thereby identifying four different adducts between NAcCys-OMe and carvone. The amounts of compounds 4 and 5 were insufficient for NMR analysis. In the reactions of both Lim-2-OOH and carvone with Fe(III)TPPCl in the presence of NAc-Cys-OMe, large amounts of the NAc-Cys-OMe dimer were also identified (10, Figure 2). This is consistent with the finding that thiols rapidly form disulfides in the presence of a Fe(III)TPPCl and a Lewis base, e.g., acetone (21). Structural Analysis of NAc-Cys-OMe Adducts. The structures of the NAc-Cys-OMe adducts were determined using a combination of LC/MS fragmentation, one-dimensional (1D) and two-dimensional (2D) NMR experiments (1H, 13C, DEPT, COSY, HMQC, HSQC, and HMBC). In all adducts, the integrity of the NAc-Cys-OMe residues were supported by the following correlations in the NMR analysis (Figure 3): The 4′ hydrogens displayed a 3J(C, H) coupling to the 3′ carbon. The 3′ carbon correlated to the 2′ and 1′ hydrogens via 2J(C, H) and 3J(C, H) couplings. The 2′ hydrogens displayed a 3J(H, H) coupling to the amide proton. The 1′ and 2′ hydrogens and carbons displayed full connectivity. The 5′ carbon displayed a 2J(C, H) coupling to the 6′ hydrogens. Compound 1 in the LC/MS chromatogram (Figure 2, m/z 328) was identified by NMR as NAc-Cys-OMe added to the endocyclic double bond of carvone (1, Figure 3). This compound was also formed as the major compound in the control reactions with carvone and is therefore not unique for the hydroperoxideNAc-Cys-OMe reaction. All NMR-shifts correlated with the synthesized reference compound. The protonated molecule showed an intense fragmentation to 178 (base peak), which corresponds to protonated NAc-Cys-OMe. The carvone residue was represented by terpene fragments, such as m/z 109, 123, and 151. The intense peak at m/z 178, formed by the loss of 150 Da, is probably due to the fact that this specific fragmentation regenerates the stable conjugated carbonyl system in carvone. Compound 2 in the LC/MS chromatogram (Figure 2, m/z 328) was identified by NMR as NAc-Cys-OMe added to the double bond of the isopropenyl group in carvone (2, Figure 3). The attachment of the NAc-Cys-OMe residue to the terpene skeleton was confirmed by the 3J(C, H) correlation between the 1′ hydrogens and the carbon in position 10. The hydrogens in position 10 displayed a 3J(H, H) coupling to the hydrogens in position 8 as well as a 3J(C, H) correlation with the carbon

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in position 9. The attachment of the isopropenyl residue to the terpene ring was supported by the 3J(H, H) coupling between hydrogens in positions 3 and 8 as well as a 2J(C, H) correlation between the hydrogens in position 8 and the carbon in position 3 of the ring. Hydrogen and carbon atoms in the ring together with the methyl group in position 7 displayed full connectivity. The preservation of the R,β-unsaturated carbonyl system was manifested by the 3J(C, H) couplings from the hydrogen in position 5 to the carbons in position 1 and 7, the 3J(C, H) and 2 J(C, H) correlations between the hydrogens in position 7 to the carbons in positions 1, 5, and 6, and the 4J(H, H) coupling from the vinylic hydrogen in position 5 to the methyl hydrogens in position 7. LC/MS fragmentation showed a pattern similar to that for compound 1, except that m/z 176 (NAc-Cys-OMe H) was a major fragment, while m/z 178 could not be observed. This difference is probably due to the fact that no conjugated double bond formation is involved in the fragmentation of 2. In the fragmentation of 1, the regeneration of the R,β-unsaturated carbonyl system of carvone gives the intense m/z 178 peak. In the fragmentation of 2, the double bond of the isopropenyl group is not regenerated, which leads to the relatively intense m/z 176 peak. Compound 3 in the LC/MS chromatogram displayed LC/ MS and NMR characteristics almost identical to 2. Most likely, compounds 2 and 3 are diastereomers (Figure 3). The fraction corresponding to compound 6 in the LC/MS chromatogram (Figure 2) was subjected to the full set of NMRexperiments. This revealed not only a strong similarity to compounds 1 and 2 but also the absence of any signal corresponding to olefinic atoms. The LC/MS analysis showed a spectrum similar to that for the other m/z 328 peaks, except that in this case m/z 328 was a fragment from the protonated molecule m/z 505. This indicates the formation of an adduct where two NAc-Cys-OMe have added to the carbon-carbon double bonds of carvone (6, Figure 4). The LC/MS chromatogram displayed peaks with m/z 330 (7, Figure 2), which corresponds to the addition of NAc-Cys-OMe to carveol (7, Figure 4). The fragmentation pattern of compound 7 strongly resembles that of compound 1, with m/z 178 as the base peak. This indicates that NAc-Cys-OMe is added to the endocyclic bond of carveol. LC/MS fragmentation of the adducts with one extra oxygen (m/z 344, corresponding to NAc-Cys-OMe + carvone + O, 8 and 9, Figure 2) or two extra oxygens (m/z 360, corresponding to NAc-Cys-OMe + carvone + 2O), yielded ions at m/z 167 and m/z 186, respectively. Adducts with m/z 344 could correspond to the addition of NAc-Cys-OMe to unreacted Lim-2OOH. However, the reaction time was set to 3 h to ensure that no unreacted hydroperoxide functional groups would remain; therefore, adducts of this kind are unlikely. Instead, the formation of these adducts may indicate oxidation in the carvone moiety (8 and 9, Figure 4). No fragments could be detected corresponding to sulfur oxidation in NAc-Cys-OMe. Oxidation of the carvone moiety may have occurred in the reaction with Fe(III)TPPCl after adduct formation (22). Reactions with Lim-2-OOH and GSH. To investigate if similar adducts as with NAc-Cys-OMe were formed in the reaction of Lim-2-OOH with Fe(III)TPPCl in the presence of unprotected GSH, a crude reaction mixture was analyzed by LC/MS. Further purification and NMR analysis was not performed. Ions with m/z 458 (corresponding to GSH + carvone + H) and m/z 474 (corresponding to GSH + carvone + O + H) were monitored. No other ions were studied. In contrast to NAc-Cys-OMe, GSH formed only two terpene adducts corresponding to the addition of carvone (m/z 458). The peaks were

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Figure 4. Possible structures of m/z 330, 344, 505, and 458 adducts identified in the reactions of Lim-2-OOH with Fe(III)TPPCl in the presence of NAc-Cys-OMe or GSH (m/z 458).

of significant intensities (similar heights at 2.37 and 2.41 min), and their MS spectra were very similar. They revealed stable protonated molecules and almost no fragmentation when applying a CE of 3 V. When increasing CE to 15 V, both adducts fragmented by a loss of GSH to m/z 151 corresponding to [M + H]+ of carvone. Another typical terpene fragment observed was m/z 107. Neutral losses were 129 (Glu) and 75 (Gly), confirming the site of the terpene bond being the cysteine. The absence of a neutral loss corresponding to carvone as well as the rest of the fragmentation pattern reveals several similarities to that observed for adduct 2. This indicates that the two peaks may be diastereomers of an adduct where GSH has added over the double bond of the isopropenyl group (11, m/z 458, Figure 4). One peak was observed for m/z 474, eluting slightly earlier than the m/z 458 peaks. The m/z 474 ion corresponds to a terpene adduct including two oxygens. This was confirmed by observing the ion m/z 167 ([M + H]+ corresponding to carvone + O + H) when applying a CE of 15 V. The fragmentation pattern was similar to that for the m/z 458 adducts, i.e., the loss of Gly and Glu. Formation of Specific Immunogenic Complexes of Olefinic Hydroperoxides. In order for the immune system to respond, a hapten has to react with macromolecules, e.g., proteins, thereby forming specific immunogenic hapten-protein complexes that

Radical Formation of Immunogenic Complexes in ACD Scheme 1. Mechanism That Accounts for the Formation of the Identified m/z 328 Adducts Isolated from the Reaction Mixture of Lim-2-OOH with Fe(III)TPPCl in the Presence of NAc-Cys-OMea

a Cys represents protected cysteine (NAc-Cys-OMe), and R• represents any of the radicals formed in the reaction of Lim-2-OOH with Fe(III)TPPCl (16).

will be processed to antigens. If two haptens generate the same or very similar antigens, which do not allow the memory T-cells to distinguish one from the other, the two haptens are said to cross-react. Clinically, this is observed when an individual sensitized to one hapten will react to the other. For the majority of haptens, formation of the immunogenic complex follows an electrophilic-nucleophilic mechanism, whereas, e.g., hydroperoxides are believed to react via a radical mechanism. The reaction of Lim-2-OOH with Fe(III)TPPCl has been investigated (16). In this reaction, the oxygen-oxygen bond is cleaved homolytically, resulting in a cascade of radicals and the formation of both carvone and carveol. In the present work, the reaction is performed in the presence of NAc-Cys-OMe or GSH. If any of the formed radicals abstracts a hydrogen atom from the thiol group of NAc-Cys-OMe or GSH, a thiyl radical is formed (Scheme 1). Thiyl radicals are known to add to olefinic double bonds in the thiol-ene reaction (23). The first step of the addition generates a carbon radical and occurs so as to form

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the more stable carbon radical, i.e., in an anti-Markovnikov fashion (24). This carbon radical then abstracts a hydrogen atom to form the final product. The formation of adduct 1, in which NAc-Cys-OMe is added over the endocyclic double bond of carvone, can be achieved through both the radical thiol-ene mechanism and an ionic mechanism. The ionic mechanism known as a Michael addition corresponds to the addition of a nucleophile in the electrophilic β-position of an R,β-unsaturated carbonyl, followed by regeneration of the carbonyl group. Thus, the identification of this adduct from the reaction of Lim-2-OOH with NAc-Cys-OMe does not reveal by which mechanism it is formed. The formation of adduct 2, where NAc-Cys-OMe is added over the isopropenyl double bond, cannot be explained by an ionic mechanism. The isopropenyl carbon-carbon double bond does not contain an electrophilic carbon; thus, it is not prone to react with the nucleophilic thiol group in an ionic reaction. However, in the thiol-ene reaction, the electron rich carboncarbon double bond readily reacts with the thiyl radical. The formation of diastereomers 2 and 3 (Figure 3) is consistent with the formation of a carbon-centered radical localized on the quaternary carbon of the isopropenyl group (Figure 3, position 8). This radical and the adjacent carbon atoms define a plane. The subsequent hydrogen abstraction can take place on both sides of the plane, resulting in diastereomers. As the thiol-ene reaction is dependent on the formation of thiyl radicals, adduct 2 is only formed in the experiments with Lim-2-OOH and Fe(III)TPPCl in the presence of NAc-Cys-OMe. In the control experiments with carvone and Fe(III)TPPCl in the presence or absence of NAc-Cys-OMe, no radicals are formed, and the only adduct identified is 1, presumably formed via an ionic reaction mechanism. Thus, the radical thiol-ene mechanism offers an explanation for the formation of adducts where NAc-Cys-OMe or GSH is added over any or both of the carbon-carbon bonds of carvone or carveol (compounds 1, 2, 3, 6, 7, and 11). We propose the formation of protein thiyl radicals and the addition of compounds derived from the hydroperoxide as a possible mechanism for the formation of immunogenic complexes of olefinic hydroperoxides. The TIC-chromatogram of the unfractionated reaction mixture in Figure 2 reveals the complexity of the reaction between Lim2-OOH and Fe(III)TPPCl in the presence of NAc-Cys-OMe. Our previous work on the reaction between Lim-2-OOH and Fe(III)TPPCl in the presence of radical trapper TMIO gave carvone as the major product together with small amounts of carveol (16). This made us focus the present investigation on the formation of adducts between NAc-Cys-OMe and hydroperoxide, carvone, or carveol. The complex reaction mixture indicates the possibility of other, so far unidentified, intermediates or products that could form adducts. It is possible that small amounts of such adducts have been formed but not identified. It should be noted that any adduct formed in the amino acid model system does not necessarily correspond to an immunogenic complex formed in ViVo. However, in order to elicit an immune response, a stable immunogenic complex must be formed in ViVo. According to theory, this is accomplished by the binding of the hapten to a protein (25, 26).Therefore, it is plausible that the hapten would also form a stable adduct. The isolation and identification of adducts 1 and 2 do not prove the formation of an immunogenic complex in ViVo. However, the results indicate the possibility to form an immunogenic complex that corresponds to adduct 2 via the radical thiol-ene reaction. This is in accordance with the hypothesis that immunogenic complex formation of hydroperoxides takes place

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via a radical mechanism (12, 13, 27-29) and that specificity is required in the antigen formation of hydroperoxides (14). It would also explain the lack of cross-reactivity between Lim2-OOH and carvone (see below). The formation of an immunogenic complex corresponding to adduct 2 offers an explanation for the lack of cross-reactivity between carvone and limonene-hydroperoxides (8). In a previous sensitization study in animals, it was shown that carvone analogues with a methyl group in the β-position or without the endocyclic double bond do not cross-react with carvone (30). Thus, it can be assumed that the formation of an immunogenic complex of carvone includes nucleophilic addition in the β-position. Furthermore, dermatitis patients with positive reactions to the hydroperoxide fraction of oxidized limonene do not react to carvone (10). This lack of cross-reactivity indicates that the antigens formed from carvone and Lim-2-OOH must be markedly different, which will be the case with an immunogenic complex of Lim-2-OOH formed by radical addition over the isopropenyl double bond. The lack of cross-reactivity between carvone and limonene-hydroperoxides also implies that immunogenic complexes corresponding to adduct 1 are not formed from Lim-2-OOH in sufficient amounts in ViVo to trigger an immune response. This further indicates the formation of specific immunogenic complexes of olefinic hydroperoxides. It is known that hydroperoxides form specific immunogenic complexes (14), but the exact mechanism of formation is so far unknown. When hydroperoxides react with Fe(III)TPPCl, used as a model for metabolizing P450 enzymes, large amounts of radicals are formed (16). Elevated levels of radicals formed from hydroperoxides in the skin will increase the oxidative stress by consumption of antioxidants (31, 32). This would increase the possibility for radical formation on macromolecules such as proteins and lipids. Reactions between protein radicals and compounds derived from the hydroperoxide (e.g., carvone) would result in a specific immunogenic hapten-protein complex and ACD. However, the reaction of protein and lipid radicals with molecular oxygen results in the formation of reactive oxygen species (ROS) that will deplete antioxidant reserves even further. Thus, the action of hydroperoxides in ACD results in increased oxidative stress due to the formation of high amounts of radicals, which facilitates the formation of specific immunogenic complexes. This form of action could explain why all hydroperoxides are strong sensitizers with small differences in their sensitizing capacities.

Conclusions We propose that the formation of specific immunogenic hapten-protein complexes from olefinic hydroperoxides can proceed via the radical thiol-ene mechanism. In this work, adducts between carvone and NAc-Cys-OMe or GSH have been identified from the reaction of Lim-2-OOH and Fe(III)TPPCl in the presence of NAc-Cys-OMe or GSH. The structure of these adducts indicates that their formation most likely proceeds via the radical thiol-ene mechanism. On the basis of the combined results of our previous investigation of allylic hydroperoxides (16) and this study, we propose the following mechanism for the formation of specific immunogenic complexes of olefinic hydroperoxides: The formation of large amounts of radicals from the hydroperoxide weakens the antioxidant defenses (31, 32). This facilitates the addition of a compound derived from the hydroperoxide to a protein via the radical thiol-ene reaction, resulting in a specific immunogenic complex. Acknowledgment. We thank Professor Ann-Therese Karlberg at Department of Chemistry, Dermatochemistry and Skin

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Allergy, University of Gothenburg, for valuable discussions and comments on the manuscript and Assistant Professor Mate Erdelyi at Department of Chemistry, Organic Chemistry, University of Gothenburg, for valuable advice with NMRexperiments. We also thank Jo¨rgen Magne´r and Tomas Alsberg at Department of Applied Environmetal Science, Stockholm University, for their skilful help with QToF analyses. The work was performed within the Go¨teborg Science Centre for Molecular Skin Research. Supporting Information Available: 1H- and 13C NMR spectra for adducts 1 and 2. This material is available free of charge via the Internet at http://pubs.acs.org.

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