Synthesis and Reactivity Toward Nucleophilic Amino Acids of 2, 5

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Chem. Res. Toxicol. 2006, 19, 1248-1256

Synthesis and Reactivity Toward Nucleophilic Amino Acids of 2,5-[13C]-Dimethyl-p-benzoquinonediimine Joan Eilstein,† Elena Gimeńez-Arnau,† Daniel Duche´,‡ Franç oise Rousset,‡ and Jean-Pierre Lepoittevin*,† Laboratoire de Dermatochimie, UniVersite´ Louis Pasteur (CNRS-UMR 7177), Clinique Dermatologique CHU, 1 Place de l’Hoˆ pital, 67091 Strasbourg, France, and L’Ore´ al AdVanced Research, 1 AVenue Euge` ne Schueller, 93600 Aulnay sous Bois, France ReceiVed June 23, 2006

2,5-[13C]-Dimethyl-p-benzoquinonediimine was synthesized, and its reactivity toward several nucleophilic amino acids was studied by associated 13C and 1H{13C} NMR spectroscopies, combined with HPLC in tandem with mass spectrometry. A classical electrophile-nucleophile mechanism was observed for the reaction with N-acetyl-Cys. Adducts resulted from the reaction of the amino acid thiol group with the benzoquinonediimine electrophilic positions 3 and 6 as well as with the nitrogen atom of the imino group. However, N-acetyl-Trp and N-acetyl-Lys were chemically modified in the presence of 2,5-[13C]dimethyl-p-benzoquinonediimine through the involvement of oxido-reduction processes. Heteronuclear 1 H{13C} NMR experiments allowed the identification of known oxidation intermediates derived from N-acetyl-Trp, indicating the oxidative strength of the reaction media. An adduct resulted from the reaction between the reduced form of the benzoquinonediimine and N-acetyl-formylkynurenine, which is the most known oxidation derivative of N-acetyl-Trp. In the case of N-acetyl-Lys, 4-amino-2,5-dimethyl-[13C]formanilide and its derivative with N-acetyl-Lys at position 4 were obtained. A reaction mechanism was suggested in which the -NH2 of the amino acid reacted on the electrophilic diimine to form an enamine adduct, which could then induce an oxidative deamination of N-acetyl-Lys. Further oxido-reduction mechanisms on the N-acetyl-R-aminoadipate-δ-semialdehyde formed might afford N,N-acetyl-formyl glutamic semialdehyde, which was considered as the powerful reactive species toward the reduced form of 2,5-[13C]-dimethyl-p-benzoquinonediimine. In the presence of N-acetyl-Tyr or N-acetyl-Met, the hydrolysis of the diimine parent compound was preferred, followed by a reduction to the hydroquinone form. In this study, we have thus shown that p-benzoquinonediimines, the first oxidation derivatives of allergenic p-amino aromatic compounds, can react with nucleophilic residues on amino acids through a set of complex mechanisms and must be seriously considered as potential candidates for the formation of antigenic structures responsible for allergic contact dermatitis. Introduction Aromatic amines are a broad group of chemicals used in a variety of applications such as hair dyes, ink for printers, photographic products, paper and textile industries, among others. Because of their large spectrum of application, skin exposure of the general population to these compounds is high. Safety aspects and toxicity studies have shown that p-amino aromatic compounds and their derivatives are strong skin sensitizers, generally related to dyeing products. One of the most cited examples is p-phenylenediamine (PPD1). During the past few years, reports of severe allergic contact dermatitis (ACD) reactions following the application of temporary black henna tattoos with added PPD to darken the mixture have seriously increased (1, 2). Moreover, PPD is one of the most common primary intermediates of oxidative hair dyes and is usually reported as the main sensitizer in hair dye dermatitis (3, 4). PPD is, therefore, included in the European Standard Series for * Corresponding author. Tel: +33 388 350 664. Fax: +33 388 140 447. E-mail: [email protected]. † Universite ´ Louis Pasteur. ‡ L’Ore ´ al Advanced Research. 1 Abbreviations: ACD, allergic contact dermatitis; p-BQDI, p-benzoquinonediimine; ESI-MS/MS, electrospray ionization mass spectrometry; HMBC, heteronuclear multiple-bond correlation; HSQC, heteronuclear single-quantum correlation; PPD, p-phenylenediamine.

diagnostic patch testing of eczema patients and is generally regarded as the screening agent for contact allergy to p-amino aromatic compounds and azo aromatic compounds used in textile dyes (5-7). ACD is a common disease induced by the modification of skin proteins by haptens (8). Successive processing of such modified proteins by skin dendritic cells such as the antigenpresenting Langerhans cells leads to the selective activation of T-lymphocytes with T-cell receptors specific for the chemical modification (9). Haptens are usually low molecular weight molecules, sufficiently lipophilic to be absorbed by the stratum corneum and penetrate into the epidermis, with a potent chemical reactivity allowing covalent binding to the amino acids side chains of proteins (10). The classic mechanism for this is the reaction of an electrophilic function of the hapten with nucleophilic chemical groups present on the proteins (11). The sensitizing potential of a molecule is, therefore, related to its chemical reactivity toward a few specific amino acids relevant to the sensitization process. Previous studies have shown for instance that nucleophilic amino acids, such as lysine and histidine, play a key role in the induction mechanism of ACD to several chemicals (12, 13). Mayer emphasized, in the 1950s, the importance of oxidized derivatives in connection with sensitization to aromatic amines substituted in the p-position and their relationship to ACD

10.1021/tx0601408 CCC: $33.50 © 2006 American Chemical Society Published on Web 08/18/2006

ReactiVity of 2,5-Dimethyl-p-benzoquinonediimine Scheme 1. Oxidation and Hydrolysis of p-Amino Derivatives

reactions (14). On complete oxidation, p-amino aromatic compounds, which are nonelectrophilic, are indeed converted into p-benzoquinonediimine (p-BQDI) derivatives and, after hydrolysis, into p-benzoquinones, which have been considered for many years as electrophilic sensitizers (Scheme 1). However, it has also been suggested that p-benzoquinones are neither the only nor the major reactive intermediates and that in fact a spectrum of antigenic structures may result from a mixture of oxidation products (15-17). Nucleophilic addition and substitution reactions can occur, for instance, at several electrophilic sites on p-benzoquinonediimines before they become hydrolyzed (18). It is, therefore, of interest to investigate the mechanisms by which p-benzoquinonediimines can react with nucleophilic residues on amino acids to see if this can add some value to the understanding of skin sensitization to p-amino aromatic derivatives. In this article, we report the synthesis of 2,5-[13C]dimethyl-p-benzoquinonediimine (1) and its reactivity toward several nucleophilic amino acids. The studies have been carried out using associated 13C and 1H{13C} NMR spectroscopies, combined with HPLC in tandem with mass spectrometry. The results of these experiments give new insight into the mechanisms of hapten-protein interactions that could account for the sensitizing potential of p-amino aromatic compounds.

Experimental Procedures Caution: Skin contact with p-amino aromatic compounds and oxidation deriVatiVes must be aVoided. Because these are sensitizing substances, they must be handled carefully. Chemistry. PPD was kindly supplied by L’Oreál Recherche (Aulnay sous Bois, France). [13C]-Methyl iodide and deuterated solvents were purchased from Euriso-Top (Saint-Aubin, France). All other chemicals were obtained from Sigma-Aldrich (Saint Quentin Fallavier, France) and used without further purification. Tetrahydrofuran and diethyl ether were dried over sodium and benzophenone. Dichloromethane was dried over P2O5. Dried solvents were freshly distilled before use. All other solvents were used as delivered. All air or moisture-sensitive reactions were conducted in flame-dried glassware under an atmosphere of dry argon. To follow the reactions, TLC analyses were performed on 0.25 mm silica gel plates (60F254; Merck, Darmstadt, Germany). After migration, the TLC plates were inspected under ultraviolet light (254 nm) or sprayed with a solution of phosphomolybdic acid in ethanol (5% w/v), followed by heating. Column chromatography purifications were performed on silica gel (Merck 60, 230-400 mesh). 1H NMR spectra were recorded at 300 or 400 MHz on Bruker Avance 300 and Avance 400 spectrometers. The chemical shifts (δ) are reported in parts per million (ppm) and are indirectly referenced to TMS via the solvent signal (CDCl3, 7.26 ppm; DMSOd6, 2.50 ppm). Multiplicities are denoted as s (singlet), d (doublet), and br (broad). 13C NMR spectra were recorded at 75 or 100 MHz on Bruker Avance 300 and Avance 400 spectrometers. 13C chemical shifts (δ) are reported in ppm and are indirectly referenced to TMS via the 13C isotope signal of the solvent used as the internal reference (CDCl3, 77.03 ppm; DMSO-d6, 39.43 ppm). The different types of carbon in the structures have been identified by the DEPT-135 technique. Melting points were determined on a Bu¨chi Tottoli 510 apparatus and are uncorrected. Positive ion electrospray mass spectra were performed on a LCQ Deca XP (Thermo Finnigan, San Jose´, California) mass spectrometer. N,N′-Bis(tert-butoxycarbonyl)-1,4-diamino-benzene (2). To a suspension of sodium hydride (4.20 g, 175 mmol) in anhydrous

Chem. Res. Toxicol., Vol. 19, No. 9, 2006 1249 tetrahydrofuran (250 mL) was added p-phenylenediamine (5.17 g, 47.8 mmol). The mixture was refluxed for 1 h and then gradually cooled down to room temperature. Di-tert-butyl dicarbonate (25 g, 115 mmol) was then added. The reaction mixture was stirred for 30 min. After that time, extra sodium hydride was added (4.20 g, 175 mmol), and the solution was heated at reflux overnight. The reaction was hydrolyzed with water (150 mL) and extracted with ethyl acetate (12 × 200 mL). The combined organic layers were dried over MgSO4, filtered, and concentrated under reduced pressure. Compound 2 was obtained as a white crystalline solid (12.77 g, 41.4 mmol, 87% yield) from the crude product by recristallization with acetone; mp 230-234 °C. 1H NMR (300 MHz, DMSO-d6): δ 9.14 (s, 2H, NH), 7.30 (s, 4H, ArH), 1.45 (s, 18H, 2 × C(CH3)3). 13C NMR (75 MHz, DMSO-d6): δ 152.8 (2 × COOC(CH3)3), 134.0 (2 × NH-CAr), 118.5 (4 × CHAr), 78.7 (2 × C(CH3)3), 28.2 (2 × (CH3)3). ESI-MS: m/z 331.16 [M + Na]+. SCI Finder registry number [121680-23-7]. N,N′-Bis(tert-butoxycarbonyl)-1,4-diamino-2,5-bis(trimethylsilyl)-benzene (3). To a solution of compound 2 (2 g, 6.5 mmol) in anhydrous diethyl ether (9 mL) and tetrahydrofuran (9 mL), cooled down to -45 °C, was added dropwise tert-butyllithium (19 mL, 32.3 mmol, 1.7 M solution in pentane). The reaction mixture was stirred at -45 °C for 3 h. After that time, trimethylsilyl chloride (2 mL, 15.9 mmol) was added dropwise at -55 °C. The mixture was stirred overnight at room temperature. The reaction was then treated with hydrochloric acid 10% (10 mL), and the resulting aqueous solution was extracted with ethyl acetate (3 × 50 mL). The combined organic layers were dried over MgSO4, filtered, and concentrated under reduced pressure. The yellow-orange crude extract obtained was purified by column chromatography on silica gel (Hex/AcOEt 95/5) to give 3 as a white solid (1.71 g, 3.9 mmol, 61% yield); mp 170-174 °C. 1H NMR (300 MHz, DMSO-d6): δ 8.36 (s, 2H, NH), 7.10 (s, 2H, ArH), 1.42 (s, 18H, 2 × C(CH3)3), 0.23 (s, 18H, 2 × Si(CH3)3). 13C NMR (75 MHz, DMSO-d6): δ 154.6 (2 × COOC(CH3)3), 139.9 (2 × NH-CAr), 139.2 (2 × (CH3)3Si-CAr), 134.1 (2 × CHAr), 78.3 (2 × C(CH3)3), 28.2 (2 × (CH3)3), -0.5 (2 × Si(CH3)3). ESI-MS: m/z 475.24 [M + Na]+. SCI Finder registry number [159624-14-3]. N,N′-Bis(tert-butoxycarbonyl)-1,4-diamino-2,5-dibromo-benzene (4). To a solution of compound 3 (1.71 g, 3.9 mmol) in dichloromethane (15 mL) was added N-bromosuccinimide (1.53 g, 8.4 mmol). The mixture was stirred at room temperature for 3 h. Water (5 mL) was then added, the aqueous layer was extracted with dichloromethane (3 × 20 mL), and the combined organic layers were dried over MgSO4, filtered, and concentrated under reduced pressure. Compound 4 was obtained as a white crystalline solid (1.58 g, 3.4 mmol, 87% yield) from the crude extract by recristallization with ethyl acetate; mp 196-198 °C (lit. 199-201 °C). 1H NMR (300 MHz, CDCl3): δ 8.31 (s, 2H, NH), 6.81 (s, 2H, ArH), 1.46 (s, 18H, 2 × C(CH3)3). 13C NMR (75 MHz, CDCl3): δ 152.2 (2 × COOC(CH3)3), 132.0 (2 × NH-CAr), 122.8 (2 × CHAr), 111.6 (2 × Br-CAr), 81.4 (2 × C(CH3)3), 28.3 (2 × (CH3)3). SCI Finder registry number [159624-12-1]. N,N′-Bis(tert-butoxycarbonyl)-1,4-diamino-2,5-[13C]-dimethylbenzene (5). To a solution of compound 4 (908 mg, 2 mmol) in anhydrous diethyl ether (9.7 mL) was added dropwise methyllithium (2.5 mL, 4 mmol, 1.6 M solution in diethyl ether). The solution was stirred at room temperature for 10 min and afterward cooled down to -78 °C. At this temperature, tert-butyllithium was added dropwise (6.9 mL, 11.7 mmol, 1.7 M solution in pentane). The reaction mixture was then warmed up to 0 °C and stirred for 3 h. After that time, 13C-methyl iodide (1.1 mL, 17.6 mmol) was added dropwise. The solution was warmed up to room temperature, stirred overnight, and treated with water (10 mL). The aqueous layer was extracted with dichloromethane (3 × 50 mL), and the combined organic layers were dried over MgSO4, filtered, and concentrated under reduced pressure. Compound 5 was obtained as a white crystalline solid (480 mg, 1.4 mmol, 73% yield) from the crude extract by recristallization with ethyl acetate; mp 226-228 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.37 (s, 2H, NH), 7.06 (d, 2H, ArH, 3JHC ) 4.9 Hz), 2.10 (d, 6H, 2 × 13CH3, 1JHC ) 126.6 Hz);

1250 Chem. Res. Toxicol., Vol. 19, No. 9, 2006

Eilstein et al.

Scheme 2. Synthetic Route for the Preparation of 2,5-[13C]-Dimethyl-p-benzoquinonediimine (1)

1.44 (s, 18H, 2 × C(CH3)3). 13C NMR (100 MHz, DMSO-d6): δ 153.6 (2 × COOC(CH3)3), 133.1 (d, 2 × NH-CAr, 2JCC ) 4.0 Hz), 129.2 (d, 2 × 13CH3-CAr, 1JCC ) 43.3 Hz), 126.5 (2 × CHAr), 78.4 (2 ×C(CH3)3), 28.1 (2 ×(CH3)3), 17.3 (2 × 13CH3-CAr). ESIMS: m/z 361.20 [M + Na]+. 2,5-[13C]-Dimethyl-p-phenylenediamine (6). To a solution of compound 5 (480 mg, 1.4 mmol) in anhydrous dichloromethane (10 mL) was added trifluoroacetic acid (15 mL). The reaction was stirred for 4 h at room temperature and, after that time, treated with a saturated solution of sodium hydrogencarbonate (2 mL) previously deaerated. The aqueous layer was extracted with deaerated dichloromethane (3 × 20 mL), and the combined organic layers were dried over MgSO4, filtered, and concentrated under reduced pressure. The crude product obtained was purified by column chromatography on silica gel (Hex/AcOEt 10/90) to give 6 as a yellow solid (136.2 mg, 1 mmol, 71% yield); mp 149-150 °C. 1H NMR (300 MHz, CDCl3): δ 6.45 (d, 2H, ArH, 3JHC ) 4.2 Hz), 3.06 (s, br, 4H, 2 × NH2), 2.10 (d, 6H, 2 × 13CH3, 1JHC ) 126.0 Hz). 13C NMR (75 MHz, CDCl3): δ 136.6 (d, 2 × NH2CAr, 2JCC ) 3.7 Hz), 121.5 (d, 2 × 13CH3-CAr, 1JCC ) 44.4 Hz), 117.9 (2 × CHAr), 17.0 (2 × 13CH3-CAr). ESI-MS: m/z 139.11 [M + H]+. 2,5-[13C]-Dimethyl-p-benzoquinonediimine (1). To a solution of compound 6 (10 mg, 73.5 µmol) in anhydrous diethyl ether (50 mL) were added anhydrous sodium sulfate (1 g, 7 mmol) and silver (I) oxide (1 g, 4.3 mmol). The mixture was placed in an ultrasonic bath at 0 °C for 4 h, and afterward filtered on Celite545. It was then concentrated under reduced pressure to obtain 1 as beige crystals (9.9 mg, 73.5 µmol, 100% yield); mp 101-102 °C. 1H NMR (400 MHz, CDCl3): δ 6.54 (d, 2H, 13CH3-CdCH, 3JHC ) 5.6 Hz), 2.12 (dd, 6H, 2 × 13CH3, 1JHC ) 128.2 Hz, 4JHH ) 1.4 Hz). 13C NMR (100 MHz, CDCl3): δ 167.5 (2 × NHdC), 139.8 (d, 2 ×13CH3-C, 1JCC ) 45.1 Hz), 132.3 (2 × 13CH3-CdCH), 16.7 (2 × 13CH3-C). ESI-MS: m/z 137.10 [M + H]+. Reaction of 2,5-[13C]-Dimethyl-p-benzoquinonediimine (1) with Nucleophilic Amino Acids. As a general procedure, 2,5-[13C]dimethyl-p-benzoquinonediimine (1) (1 mg) was added to a phosphate buffer solution (600 µL; mixture of 450 µL of H2O and 150 µL of D2O; 0.1 M at pH 7.4) containing the amino acid in its N-acetylated form (10 equiv). The solution was filtered into an NMR tube, a trace of acetonitrile was added as an internal reference, and the reaction was followed by 13C NMR at 75 MHz on a Bruker AC 300 spectrometer. Structure Assignement. The different products formed were characterized by a combination of NMR and MS data. 13C and 1H NMR data were obtained by {1H}-decoupled 13C NMR, 1H{13C} heteronuclear single-quantum correlation (HSQC) and heteronuclear multiple-bond correlation (HMBC) experiments carried out on Bruker Avance 400 and Avance 500 spectrometers. Chemical shifts (δ) are reported in ppm with respect to TMS, using acetonitrile as the internal standard (1H, δ ) 2.06; 13C, δ ) 119.65) and are indicated in Schemes 3, 5, and Chart 2. The measured chemical

shifts were compared with those calculated by ACD/CNMR and ACD/HNMR Predictor (version 5.12). When necessary, further HPLC and electrospray ionization mass spectrometry (ESI-MS/MS) analyses were performed in order to confirm the chemical structure of the obtained adducts. The HPLC analyses were performed on a Surveyor HPLC system (Thermo Finnigan, San Jose´, California) equipped with a quaternary pump, an automatic sample injector, and a diode array absorbance detector scanning from 200 to 700 nm. A C18 reverse phase column (4.6 × 150 mm, i.d. 5 µm (Waters, XTerra)) protected by two serial 10 µm Kromasil C18 cartridges as guard columns was used. The elution solvent was a gradient of methanol (B) in ammonium acetate (20 mM) (A) at a flow rate of 0.8 mL/min. In the experiments with N-acetyl-Cys, the gradient system consisted of 2% to 10% B for the first 10 min, to 17% B for 25 min, ending with 40% B in 5 min. In the experiments with N-acetyl-Lys and N-acetyl-Trp, the gradient system consisted of 10% B to 45% B over 30 min. ESI-MS/MS data were acquired in a LCQ Deca XP mass spectrometer (Thermo Finnigan, San Jose´, California) fitted with an electrospray interface. The analysis employed the positive ion mode (m/z [M + H]+). Preliminary analyses were carried out using full scan, datadependent MS/MS scanning from m/z 50-600. The capillary temperature was 300 °C, sheath gas and auxiliary gas were 70 and 20 units/min, respectively, and source voltage was 4 kV. MS/MS fragmentation was carried out with 25 to 35% of collision energy. The Xcalibur software (version 1.4, Thermo Electron Corporation) was used for instrument control and data acquisition.

Results and Discussion Synthesis. The use of 13C-labeled molecules in association with NMR techniques is a powerful tool for the investigation of hapten-protein interactions. Best results are obtained using direct labeling at the reactive sites of the molecule. In the case of p-BQDI, such labeling at the C-1, C-2, and C-3 ring positions is complex. Previous studies have shown that the introduction of a 13C methyl group on the aromatic ring of alkylcatechol derivatives helped the study of their reactivity toward proteins by using specific NMR techniques (19). It was indeed possible to obtain the signal of the 13C methyl group directly without having to subtract other residual signals. Moreover, the large range of chemical shifts of methyl groups in both 13C and 1H was favorable for a precise identification of substitutions. We, therefore, decided to synthesize the p-BQDI derivative 1 containing two 13C-labeled methyl groups at positions C-2 and C-5. The labeled positions were close to all of the potential reactive sites, and at the same time, many reactive positions on p-BQDI were reduced, facilitating the follow-up of the reactivity (Chart 1).

ReactiVity of 2,5-Dimethyl-p-benzoquinonediimine

Chem. Res. Toxicol., Vol. 19, No. 9, 2006 1251

Scheme 3. Reaction of 1 with Nucleophilic Amino Acids: Combination of NMR and MS Data

Scheme 4. Formation of Adduct 7

Chart 1. Sites of Nucleophilic Attack on p-BQDI and 1

Chart 2. HSQC (δ) and HMBC (Arrows) Data for Compound 24

Scheme 5. Oxidative Degradation of N-Acetyl-Trp

Compound 1 was obtained in a six steps (Scheme 2). Initially, both PPD amino substituents were protected as tert-butoxy carbamate groups by reaction with sodium hydride and di-tertbutyl dicarbonate in anhydrous tetrahydrofuran in a yield of 87%. The tert-butoxy carbamate group is a very well-known

ortho-metalation director of aromatic compounds with wide applications in the field of organic synthesis (20). For instance, ortho-lithiation of N-(tert-butoxycarbonyl)-aniline and subsequent reaction with various electrophiles simply yield 2-substituted products (21). Thus, we attempted a bis-ortho-lithiation directly on compound 2, and subsequently, the reaction of the intermediate formed with 13C-methyl iodide. However, the reactions were repeatedly low-yielding. Therefore, we turned our attention to halogen-lithium exchange reactions. Direct halogenation of PPD and 2 was not possible because the nature of the substituents in both compounds allowed the aromatic electrophilic substitution to occur predominantly at C-2 and C-6. Another appropriate method to prepare halogen functionalized arenes is based on the cleavage of aryl silicon bonds by halogen electrophiles (22). We successfully managed this strategy. Compound 2 was first converted into the bis-trimethylsilyl derivative 3. Treatment with tert-butyllithium in anhydrous diethyl ether and tetrahydrofuran gave the C-2 and C-5 ortho-


Eilstein et al. Scheme 6. ESI-MS/MS Analysis Allowing the Elucidation of 9

Figure 1. Reaction of 1 with N-acetyl-Cys.

lithiate intermediate, which was quenched with trimethylsilyl chloride in a 61% yield. Further bromodesilylation of compound 3 was achieved by using N-bromosuccinimide in dichloromethane, the ipso substitution affording dibromo derivative 4 with a yield of 87%. This compound was subjected to a bishalogen-lithium exchange reaction by treatment with an excess of tert-butyllithium in anhydrous diethyl ether. To avoid the deprotonation of the tert-butoxy carbamate protected amino groups by the resulting ortho-lithiate intermediate, compound 4 was previously treated with 2 equiv of methyllithium (23). The the bis-ortho-lithiate intermediate was obtained quickly, probably favored by the reaction of the excess tert-butyllithium with the tert-butylbromide byproduct, forming inert lithium bromide and shifting, in this manner, the equilibrium to the product side (24). The bis-ortho-lithiate intermediate was in the end quenched by 13C-methyl iodide to give 5 with a yield of 73%. After this, the tert-butoxy carbamate groups were readily cleaved by use of trifluoroacetic acid in dichloromethane to afford 2,5-[13C]-dimethyl-p-phenylenediamine (6) in a 71% yield. In order to prevent its rapid degradation, compound 6 was stored under its hydrochlorate form, obtained by bubbling hydrochloric acid in a diethyl ether solution. To recover 6 each time before utilization, the hydrochlorate derivative was neutralized with a sodium hydrogenocarbonate solution, and 6 was extracted with deaerated diethyl ether. Finally, the desired 2,5[13C]-dimethyl-p-benzoquinonediimine (1) was prepared in a 100% yield by simple oxidation of 6 with silver (I) oxide in a diethyl ether solution containing sodium sulfate (25). Because of its high instability, compound 1 needed to be used immediately after it was obtained. Reaction of 2,5-[13C]-Dimethyl-p-benzoquinonediimine (1) with Nucleophilic Amino Acids. The chemical reactivity of 1 toward nucleophilic amino acids was followed by 13C NMR. The products formed (Scheme 3) were generally characterized by 13C and 1H NMR data obtained by heteronuclear 1H{13C} HSQC and HMBC experiments. When necessary, further HPLC combined with ESI-MS/MS analyses were performed in order to completely elucidate their chemical structure. 1. Reaction with N-acetyl-Cys. N-Acetyl-Cys was found to react very quickly with 1 in the experimental conditions used. From the first day of the reaction, four new peaks appeared in the methyl region at 15.7, 16.4, 17.0, and 17.6 ppm (Figure 1). No parent compound 1 remained, and no evolution of spectra was observed over time. Heteronuclear 1H{13C} NMR experiments showed that the new signals corresponded to several products (Scheme 3). HSQC experiments allowed, via 1J(C, H), the correlation of new methyl carbons to protons at 2.46 (J ) 130.4 Hz), 2.23 (J ) 130.8 Hz), 2.10 (J ) 126.0 Hz), and 2.19 ppm (J ) 130.4), respectively. HMBC experiments showed that the methyl protons at 2.46 ppm were correlated via long-range coupling with quaternary carbons at 119.0, 121.9, and 134.0

ppm, suggesting for one of the adducts the loss of H-3 and H-6 of 1 as well as aromatization. The double addition of the thiol group of N-acetyl-Cys at positions 3 and 6, affording symmetrical adduct 7, was then corroborated by the characteristic chemical shift of the carbons bearing the sulfur atom, in addition to long-range couplings observed between 119.0 ppm and the β methylene group of N-acetyl-Cys (35.7/3.02-3.23 ppm). Mechanistically, a first addition of N-acetyl-Cys formed intermediate 14, which was subsequently oxidized by residual 1 to form 15, able to follow a second addition of N-acetyl-Cys (Scheme 4). The identification of 6 in the NMR spectra, with the methyl groups at 17.0/2.10 ppm, also supports this mechanism. Methyl groups at 16.4/2.23 and 17.6/2.19 ppm corresponded to another adduct in which H-3 and H-6 were conserved. Indeed, long-range couplings existed between protons at 2.23 ppm and an aromatic C-H at 123.3/7.03 ppm, and between protons at 2.19 ppm and another aromatic C-H at 125.6/ 7.08 ppm. Two quaternary carbons at 146.7 and 128.3 ppm were also long-correlated with this system. However, the NMR data were not conclusive, and the structure of adduct 8 was finally elucidated by ESI-MS/MS. The HPLC profile of the reaction showed three peaks, whose m/z values corresponded to 6 (m/z 139), 7 (m/z 461), and 8 (m/z 300). ESI-MS/MS analysis of m/z 461 confirmed the structure of 7 by obtaining [14-H]+ with m/z 299. ESI-MS/MS analysis of m/z 300 confirmed the structure of 8 by obtaining m/z 136 corresponding to the N-SCys fragmentation. Adduct 8 was formed after an attack of the thiol group of N-acetyl-Cys on the nitrogen atom of the imino group, in favor of the following aromatization of the system (18). 2. Reaction with N-Acetyl-Trp. From the first day of the reaction, a new set of 13C signals appeared around 16.5 ppm. This area of the spectrum was better resolved over time, but no adduct with N-acetyl-Trp could be identified by NMR. The HSQC and HMBC data only informed about the presence in a new compound of the aromatic system with the two labeled methyl groups at 16.5/2.13 and 16.9/2.06 ppm, correlated with H-3 and H-6 at 6.82 and 6.30 ppm, respectively. However, the heteronuclear 1H{13C} NMR experiments allowed the identification of 16, 17, and N-acetyl-formylkynurenine (18), resulting



Figure 2. ESI-MS/MS fragmentation of m/z 167.

Scheme 7. Mechanism Proposed for the Formation of 10

from the known oxidation of N-acetyl-Trp (Scheme 5) (26). The presence of these derivatives in the reaction media was confirmed by HPLC and ESI-MS/MS. Values of molecular ions at m/z 263 and m/z 279 were found for the HPLC peaks corresponding to the ambiguously NMR attributed 16 and 17. A value of m/z 279 was found for the HPLC peak related to 18. In addition, HPLC combined to ESI-MS permitted the identification of adduct 9 (Scheme 3). The peak of highest intensity in the HPLC chromatogram contained the molecular ion of 9 with m/z 399. MS/MS analysis of m/z 399 gave one fragment at m/z 381, indicating a loss of water, and another at m/z 261, resulting from the loss of compound 6’s distinctive 138 mass unit (Scheme 6). These fragmentations were confirmed when the reaction with N-acetyl-Trp was carried out using unlabeled 2,5-dimethyl-p-benzoquinonediimine. In this case, the highest HPLC peak had a molecular ion of m/z 397, which was split by MS/MS into m/z 379 (loss of water) and the previous ion at m/z 261, proving the loss of 2,5-dimethyl-p-phenylenediamine. These results confirmed the structure of adduct 9, which can result from the reaction between the reduced form of 1 and N-acetyl-formylkynurenine 18. 3. Reaction with N-Acetyl-Lys. As before, from the first day of the reaction, a new set of 13C signals appeared around

16.5 ppm. Once more, even if this area of the spectrum was better resolved over time, the HSQC and HMBC data basically informed us about the presence in a new compound of the aromatic system with the two labeled methyl groups at 17.0/ 2.10 ppm and 16.4/2.10, correlated with H-3 and H-6 at 6.73 and 7.03 ppm, respectively. Signals characteristic of an aldehydic chemical function (164.0 and 8.23 ppm) and sodium formiate (171.5 and 8.44 ppm) were also present. Although no complete elucidation of an adduct with N-acetyl-Lys could be identified by 13C NMR, additional HPLC and MS analysis of the reaction mixture showed the presence of two major peaks with associated m/z 167 and m/z 338 values. ESI-MS/MS analysis of m/z 167 (Figure 2) allowed the identification of 4-amino-2,5-[13C]-dimethylformanilide (10) (Scheme 3), in which the calculated 1H and 13C NMR chemical shifts corresponded exactly with the experimental 1H{13C} NMR data indicated above. A reaction mechanism can be suggested for obtaining 10 on the basis of an oxidative deamination of N-acetyl-Lys (Scheme 7). It is known in the literature that NRprotected Lys can be oxidatively deaminated to yield a N-protected-R-aminoadipate-δ-semialdehyde, which is spontaneously converted by intramolecular cyclization into a N-protected-1-piperideine-6-carboxylic acid (27). Interestingly, it has


Eilstein et al.

Figure 3. ESI-MS/MS fragmentation of m/z 338.

been reported that this oxidative deamination of Lys can be induced by quinone-imine derivatives (28). Also, it is known that N-protected-1-piperideine-6-carboxylic acids can be converted into glutamic acid derivatives through oxidation processes similar to those shown for N-acetyl-Trp in Scheme 5 (29). Initially, adduct 19 can be formed by the reaction between the -NH2 group of N-acetyl-Lys and diimine 1 (Scheme 7). A nucleophilic attack of the -NH2 on one of the imine carbon atoms followed by the release of a NH3 molecule gives a first intermediate, which can then eliminate a hydrogen of the R-carbon atom and form, after aromatization of the cycle, the Schiff base type intermediate 19. This adduct can progressively release N-acetyl-R-aminoadipate-δ-semialdehyde (20) through hydrolysis, thereby yielding the reduced form of 1, the 2,5[13C]-dimethyl-p-phenylenediamine (6) (28). Further oxidation of N-acetyl-1-piperideine-6-carboxylic acid (21), itself obtained by easy intramolecular cyclization of 20, can afford derivative 22, which is the precursor of N,N-acetyl-formyl glutamic semialdehyde (23). Finally, a nucleophilic attack of released 6 on intermediate 23 can explain how adduct 10 was obtained. In order to verify this hypothetical mechanism in which C-6 of N-acetyl-Lys becomes the formyl carbon of 10, unlabeled 2,5dimethyl-p-benzoquinonediimine was reacted with Lys 13Clabeled at C-6. The formation of 4-amino-2,5-dimethyl-[13C]formanilide (24), characterized by heteronuclear 1H{13C} NMR experiments (Chart 2) as well as the identification of 13C-labeled sodium formiate (171.5 and 8.44 ppm), came to support the suggested mechanism. Still, additional reactivity and HPLC/ MS studies are currently in progress and should provide us with extra information (results unavailable). ESI-MS/MS analysis of m/z 338 (Figure 3) allowed the identification of adduct 11 (Scheme 3). The presence of the C-4-attached amino acid was, moreover, proved by further MS of m/z 278, which afforded the characteristic m/z 84 immonium ion resulting from the known fragmentation of N-acetyl-Lys

Figure 4. Reaction of 1 with N-acetyl-Tyr. HSQC (δ) and HMBC (arrows) data for 12 and 13.

(30). From a mechanistic point of view and on the basis of the above-described Scheme 7, it can be assumed that adduct 11 is also formed via an oxidative deamination of Lys that follows, this time, an initial double attack of the -NH2 group of N-acetylLys in positions 1 and 4 of diimine 1. 4. Reaction with N-Acetyl-Tyr, N-Acetyl-Met, or N-AcetylHis. When using N-acetyl-Tyr or N-acetyl-Met, a new 13C peak at 15.2 ppm was immediately observed. After 3 days, another 13C peak appeared at 15.4 ppm, and its intensity increased overtime to the detriment of the first signal obtained. Heteronuclear 1H{13C} NMR data allowed the characterization, in the beginning, of 2,5-[13C]-dimethyl-p-benzoquinone (12) and, with


time, 2,5-[13C]-dimethyl-p-hydroquinone (13) (Figure 4). Simply, 1 was hydrolyzed instantly to get 12, which was then reduced to hydroquinone 13. Thus, compared to the other nucleophilic amino acids tested, no adduct was observed between 1 and N-acetyl-Tyr or N-acetyl-Met. The hydrolysis of 1 was faster than any other reaction, and both amino acids played an essential role in reducing the quinone intermediate. Actually, in the case of the reaction with N-acetyl-Met, the participation of oxido-reduction processes was confirmed by the presence of N-acetyl-Met-sulfoxide in the 2D NMR spectra. Finally, unexplained fast polymerization of 1 was observed when the reaction was performed in the presence of N-acetyl-His.

Conclusion 2,5-[13C]-Dimethyl-p-benzoquinonediimine (1) was found to have very different behaviors toward the nucleophilic amino acids tested. It reacted easily and quickly with all of them, but several reaction mechanisms were implicated. The reactivity observed matched the one that we expected only in the case of N-acetyl-Cys. The nucleophilic thiol group reacted in a classical way with the electrophilic positions 3 and 6 of 1 to form 7 and also with the nitrogen of the imino group to form 8. Interestingly, in the case of N-acetyl-Trp and N-acetyl-Lys, major modifications were induced on the amino acid themselves, always with the involvement of oxido-reduction processes. The presence in the reaction media of known oxidation derivatives of N-acetylTrp, including N-acetyl-formylkynurenine 18, was an explicit indication of the oxidative strength of the aqueous medium containing the p-benzoquinonediimine. Looking at the chemical structure of adduct 9, it could be assumed that diimine 1 was involved in an oxido-reduction cycle furnishing the real reactive intermediate 6 toward the modified amino acid. This was confirmed by the observed oxidative deamination of N-acetylLys and obtaining adduct 11. The oxidative stress of proteins induced by 1 is, therefore, an additional reactivity element to consider. To this initially unexpected chemistry was added the fact that in the presence of other amino acids such as N-acetylTyr or N-acetyl-Met the hydrolysis of 1 was preferred, followed by a reduction to the hydroquinone form. At this point, it is interesting to note that compound 1 did not follow the same behavior when the aqueous solution did not contain the amino acid. Instead of hydrolysis to form a p-quinone and reduction to the hydroquinone, fast polymerization was observed. In conclusion, using the model of 2,5-dimethyl-p-benzoquinonediimine, we have been able to show that p-benzoquinonediimines, which are the first oxidation derivatives of p-amino aromatic compounds, can react with nucleophilic residues on amino acids and this through a set of complex and complementary mechanisms. To our knowledge, p-benzoquinonediimine has not been tested on patients who are allergic to p-phenylenediamine. This is most likely due to its high instability as well as to its presumably low ablitity to penetrate the skin. Most probably, p-benzoquinonediimine is formed by oxidation in the skin, enzymatic or not, of the more lipophilic parent compound p-phenylenediamine. Even if the results obtained are, thus, just qualitative, this study, however, confirms that p-benzoquinonediimines must be seriously considered as potential candidates for the formation of antigenic structures responsible for ACD to p-amino aromatic compounds. Acknowledgment. We thank L’Oreál for their financial support to J.E.. We also gratefully acknowledge the excellent technical assistance of Pierre-Alain Meunier during the MS studies.


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Synthesis and Reactivity Toward Nucleophilic Amino Acids of 2, 5

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