Nuclear Magnetic Resonance Studies on Covalent Modification of

1106

Chem. Res. Toxicol. 2009, 22, 1106–1115

Nuclear Magnetic Resonance Studies on Covalent Modification of Amino Acids Thiol and Amino Residues by Monofunctional Aryl 13 C-Isocyanates, Models of Skin and Respiratory Sensitizers: Transformation of Thiocarbamates into Urea Adducts Olivier Fleischel, Elena Gimeńez-Arnau, and Jean-Pierre Lepoittevin* Laboratoire de Dermatochimie, Institut de Chimie de Strasbourg, ILB, 4 rue Blaise Pascal, 67070 Strasbourg, France ReceiVed February 9, 2009

Exposure to aryl isocyanates, intermediates in the manufacture of polyurethanes, provokes lung sensitization and asthma but also occupational allergic contact dermatitis, sensitization occurring from a single accidental exposure. The initial step in the sensitization process is believed to be the covalent binding of the -NdCdO group with nucleophilic residues on proteins. While a wide knowledge exists on the reactivity of skin sensitizers toward amino acids, little is known about respiratory sensitizers such as aryl isocyanates. 13C-Labeled monofunctional aryl isocyanates were synthesized, and their reactivities toward nucleophilic amino acids, GSH, and a model peptide were studied by 13C and [1H-13C] NMR spectroscopy. An acetonitrile/buffer solution was used as a solvent to avoid the hampering of the follow up of the reactivity by the isocyanate hydrolysis competing reaction. The compounds reacted with thiol groups, through the formation of thiocarbamate bonds and with amino groups to form urea derivatives. The reactivity was confirmed with GSH, containing both free amino and thiol groups, and with a model peptide, particularly in the case of the reaction with lysine. The use of 13C NMR to follow the aryl isocyanates reversible conjugation with thiol groups is also reported. Particularly, it is shown that thiocarbamate adducts can be converted into adducts of the urea kind by reaction with amino groups. These results confirmed the hypothesis by which thiol-containing peptides/proteins may act as carriers of isocyanates for possible reaction at a later time and/or place with other nucleophiles and confirmed the role of lysine as a good competing nucleophilic amino acid. The reactivity of aryl isocyanates with thiol and amino groups needs thus to be considered in their assigned sensitization processes. Introduction Respiratory allergy, typically characterized by rhinitis and asthma, is an important health problem of high incidence and prevalence (1). Occupational asthma is a common workplace lung disease in industrialized countries, and occupational rhinitis is up to three times more frequent, these two conditions occurring together frequently (2, 3). Among the chemicals known to cause sensitization of the respiratory tract, isocyanates are considered one of the most important causes of occupation-related asthma (4, 5). Millions of tons of isocyanates are produced and consumed annually all over the world in a wide variety of work environments. Their largest industrial use is in the manufacture of polyurethanes, which are employed in the production of products such as paints, glues, plastics, surface coatings, adhesives, flexible and rigid foams, and synthetic rubbers, among many others (6). The most commonly used isocyanates for the production of polyurethanes are toluene diisocyanate (TDI)1 and 4,4′-diphenylmethane diisocyanate (MDI) (Chart 1). During the 1970s, TDI was predominantly used. TDI is one of the most volatile diisocyanates. Therefore, there was a shift post-1970s to the use of MDI and prepolymers of TDI. Their lower volatility resulted in much lower air * To whom correspondence should be addressed. Tel: +33 390 24 15 01. Fax: +33 390 24 15 27. E-mail: [email protected]. 1 Abbreviations: MDI, 4,4′-diphenylmethane diisocyanate; GSH, glutathione; HMBC, heteronuclear multiple-bond correlation; HSQC, heteronuclear single-quantum correlation; TDI, toluene diisocyanate.

Chart 1. Chemical Structures of MDI, TDI, Phenyl-[13C]-isocyanate (1) and p-Tolyl-[13C]-isocyanate (2)

exposure in the workplace but, surprisingly, produced little of the expected reduction in occupational asthma (7, 8). Although concerns about the health risks associated with isocyanates have largely focused on respiratory problems, it has been reported that exposure to MDI and TDI can also cause occupational allergic contact dermatitis, skin sensitization occurring from a single accidental exposure (9, 10). While clinical cases were only sporadic in the 1980s, there was a clear increase in the second half of the 1990s as the use of polyurethanes became more widespread. Both the respiratory tract and the skin are potential routes of isocyanate exposure, as this one can occur in the form

10.1021/tx9000539 CCC: $40.75  2009 American Chemical Society Published on Web 04/30/2009

ReactiVity of Aryl Isocyanates

of a liquid, vapor, or aerosol, depending on the isocyanate species and the industrial process employed. Chemicals inducing skin and/or respiratory allergy are low molecular weight compounds that are typically electrophiles or pro-electrophiles capable of reacting with nucleophile functionalities on proteins (11). The conjugated protein constitutes an antigenic structure that will start the stimulation of a specific immune response. Basically, the protein complex is internalized and further processed into peptide fragments by immunocompetent antigen-presenting cells at the site of exposure. The antigen-carrying cells migrate from peripheral tissues to regional lymph nodes for presentation of the altered peptides to naı¨ve T-lymphocytes. This results in the selection and activation of responsive T-lymphocyte subpopulations with T-cell receptors specific for the protein chemical modification. Sensitization is acquired by the clonal expansion of allergen-specific Tlymphocytes that will be activated and trigger allergic hypersensitivity reactions following a second exposure to the allergen (12). The feature that in most instances distinguishes chemical respiratory and skin allergens is that respiratory allergens seem to stimulate Th2 type T lymphocytes, and in contrast, skin allergens seem to induce preferential Th1 type immune responses (13). However, why different chemical allergens are associated with different forms of sensitization and occupational illness continues to be an intriguing issue. The differential distribution of protein modification is thought, at least in part, to determine the ultimate nature of the immunological response. Accordingly, it remains a possibility that different classes of chemical allergens exhibit qualitative differences with respect to association with proteins at the amino acid level. Whereas a quite extensive knowledge exists concerning the reactivity of skin sensitizers toward amino acids (11), little is known for respiratory sensitizers. Allergenic isocyanates are characterized by the very reactive -NdCdO group. The initial step in the sensitization process is thus believed to be the covalent binding of the -NdCdO group with nucleophilic residues on proteins. For some years, we have investigated the reactivity pattern of haptens toward nucleophilic residues on proteins in the case of skin sensitizers. We decided recently to study the reactivity of aromatic compounds bearing a -NdCdO group, these being representative of respiratory allergens for the most part. Experiments were started with model compounds containing a single -NdCdO group. Phenyl isocyanate and p-tolyl isocyanate, trace contaminants of MDI products, have not been associated with clinical cases of sensitization probably because of a low exposure. However, they are known strong respiratory and dermal chemical allergens in animal models, being potent inducers of both cellular and immune responses (14–17). In this paper, we report the synthesis of 13C-labeled phenyl isocyanate 1 and p-tolyl isocyanate 2 (Chart 1) and the study of their reactivity toward nucleophilic amino acids, glutathione (GSH), and a model peptide using 13C and [1H-13C] NMR spectroscopy. Results of the experiments are used to discuss if differences in mechanisms and/or amino acid modifications could exist between skin and respiratory allergens.

Experimental Procedures Caution: Skin and respiratory contact with isocyanates must be aVoided since these are powerful sensitizing substances. As such, they must be handled with care. Chemistry. [13C]-Urea and deuterated solvents were purchased from Euriso-Top (Saint Aubin, France). Ammonium tetrafluoroborate was obtained from Acros Organics (Illkirch, France). All other

Chem. Res. Toxicol., Vol. 22, No. 6, 2009 1107 chemicals were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). Triethylamine was distilled over potassium hydroxide before use. All other chemicals and solvents were used as delivered. Air or moisture-sensitive reactions were conducted in flame-dried glassware under an atmosphere of dry argon. The reactions were followed by TLC, performed on 0.25 mm silica gel plates (60F254; Merck, Darmstadt, Germany). After migration, the TLC plates were inspected under UV light (254 nm) or sprayed with a solution containing phosphomolybdic acid (5 g), cerium(IV) sulfate (2 g), and sulfuric acid (12 mL) in water (188 mL), followed by heating. Column chromatography purifications were performed on silica gel 60 (Merck, Geduran, 40-63 µm). 1H and 13C NMR spectra were recorded on a Bruker Avance 300 spectrometer at 300 and 75 MHz, respectively. The chemical shifts (δ) are reported in ppm and are indirectly referenced to TMS via the solvent signal (acetone-d6: δ 1H ) 2.05, δ 13C ) 29.84, 206.26; acetonitrile-d3 (CD3CN): δ 1H ) 1.94, δ 13C ) 1.32, 118.26; CDCl3: δ 1H ) 7.26, δ 13C ) 77.16). In the 1H NMR spectra multiplicities are denoted as s (singlet), d (doublet), t (triplet), q (quadruplet), and br (broad). The different types of carbon in the structures were identified by the DEPT-135 technique. Melting points were determined on a Bu¨chi Tottoli 510 apparatus and were uncorrected. Electrospray time-of-flight mass spectrometry (ESI-TOF-MS) was recorded in a micro-TOF LC Bruker Daltonics spectrometer. N-Phenyl-[13C]-urea (5). To a solution of [13C]-urea (1 g, 16.6 mmol) in a mixture of hydrochloric acid (3 N, 5.5 mL) and toluene (80 mL) was added, dropwise and for a period of 1 h, a solution of aniline 3 (1.5 g, 16.6 mmol) in toluene (40 mL). The mixture was refluxed for 24 h and then gradually cooled down to room temperature. The solvent was removed under reduced pressure. The white solid product obtained was purified by column chromatography on silica gel (petroleum ether/diethyl ether 8/2) to give 5 as a white solid (930 mg, 6.78 mmol, 40% yield); mp 145 °C. 1H NMR (300 MHz, acetone-d6): δ 8.50 (s, br, 1H, NH), 7.40 (d, 2H, H-2 and H-6, J ) 8.7 Hz), 7.20 (dd, 2H, H-3 and H-5, J ) 8.4, 7.4 Hz), 6.91 (t, 1H, H-4, J ) 7.4 Hz), 5.82 (s, br, 2H, NH2). 13C NMR (75 MHz, acetone-d6): δ 156.7 (13CdO), 141.5 (C-1), 129.3 (C-3 and C-5), 122.3 (C-4), 119.0 (C-2 and C-6). ESI-TOF-MS: m/z 144.08 [M + Li], m/z 160.05 [M + Na]. N-(4-Methyl-phenyl)-[13C]-urea (6). To a solution of [13C]-urea (500 mg, 8.3 mmol) in a mixture of hydrochloric acid (3 N, 2.8 mL) and toluene (40 mL) was added, dropwise and for a period of 1 h, a solution of p-toluidine 4 (892 mg, 8.3 mmol) in toluene (20 mL). The mixture was refluxed for 24 h and then gradually cooled down to room temperature. The solvent was removed under reduced pressure. The white solid product obtained was purified by column chromatography on silica gel (petroleum ether/diethyl ether 8/2) to give 6 as a white solid (490 mg, 3.24 mmol, 39% yield); mp 180 °C. 1H NMR (300 MHz, acetone-d6): δ 7.95 (s, br, 1H, NH), 7.35 (d, 2H, H-2 and H-6, J ) 8.2 Hz), 7.02 (d, 2H, H-3 and H-5, J ) 8.2 Hz), 5. 42 (s, br, 2H, NH2), 2.23 (s, 3H, CH3). 13C NMR (75 MHz, acetone-d6): δ 156.8 (13CdO), 139.0 (C-1), 131.4 (C-4), 129.8 (C-3 and C-5), 119.2 (C-2 and C-6), 20.7 (CH3). ESI-TOFMS: m/z 152.09 [M + H], m/z 158.09 [M + Li], m/z 174.07 [M + Na]. Ethyl-N-phenyl-[13C]-carbamate (7). To a solution of N-phenyl-[13C]-urea 5 (600 mg, 5.1 mmol) in ethanol (15.5 mL, 25.5 mmol) was added ammonium tetrafluoroborate (5.39 g, 51 mmol) in small fractions. The mixture was stirred vigorously at reflux for 1 week. Then, the temperature was reduced to room temperature, and the solvent was removed under reduced pressure. The crude product was dissolved in water (60 mL) and extracted with diethyl ether (6 × 10 mL). The combined organic layers were washed with brine (80 mL), dried with MgSO4, filtered, and concentrated under reduced pressure. The crude extract obtained was purified by column chromatography on silica gel (petroleum ether/diethyl ether 8/2) to afford compound 7 as a yellowish solid (375 mg, 2.25 mmol, 45% yield); mp 49 °C. 1H NMR (300 MHz, CDCl3): δ 7.38 (d, 2H, H-2 and H-6, J ) 8.3 Hz), 7.29 (dd, 2H, H-3 and H-5, J ) 8.4, 7.1 Hz), 7.05 (t, 1H, H-4, J ) 7.1 Hz), 6.64 (s, br, 1H, NH), 4.22 (qd, 2H, CH2-CH3, J ) 7.1 Hz, 3JHC ) 3.2 Hz), 1.31 (t, 3H,

1108

Chem. Res. Toxicol., Vol. 22, No. 6, 2009

CH2-CH3, J ) 7.1 Hz). 13C NMR (75 MHz, CDCl3): δ 153.7 (13CdO), 138.1 (C-1), 129.2 (C-3 and C-5), 123.5 (C-4), 118.8 (C-2 and C-6), 61.4 (CH2), 14.7 (CH3). ESI-TOF-MS: m/z 173.09 [M + Li], m/z 191.10 [M + Na]. Ethyl N-(4-Methyl-phenyl)-[13C]-carbamate (8). To a solution of N-(4-methyl-phenyl)-[13C]-urea 6 (400 mg, 2.6 mmol) in ethanol (8 mL, 13.2 mmol) was added ammonium tetrafluoroborate (2.8 g, 26.6 mmol) in small fractions. The mixture was stirred vigorously at reflux for 1 week. After this time, the temperature was reduced to room temperature, and the solvent was removed under reduced pressure. The solid crude product obtained was dissolved in water (60 mL) and extracted with diethyl ether (6 × 10 mL). The combined organic layers were washed with brine (80 mL), dried with MgSO4, filtered, and concentrated under reduced pressure. The crude extract obtained was purified by column chromatography on silica gel (petroleum ether/diethyl ether 8/2) to afford compound 8 as a white solid (210 mg, 1.16 mmol, 45% yield); mp 51 °C. 1H NMR (300 MHz, CDCl3): δ 7.26 (d, 2H, H-2 and H-6, J ) 8.3 Hz), 7.12 (d, 2H, H-3 and H-5, J ) 8.3 Hz), 6.53 (s, br, 1H, NH), 4.23 (qd, 2H, CH2-CH3, J ) 7.1 Hz, 3JHC ) 3.2 Hz), 2.30 (s, 3H, Ar-CH3), 1.31 (t, 3H, CH2-CH3, J ) 7.1 Hz). 13C NMR (75 MHz, CDCl3): δ 153.9 (13CdO), 135.5 (C-1), 133.1 (C-4), 129.6 (C-3 and C-5), 118.9 (C-2 and C-6), 61.3 (CH2), 20.9 (Ar-CH3), 14.7 (CH3). ESI-TOF-MS: m/z 187.11 [M + Li], m/z 203.08 [M + Na]. Phenyl-[13C]-isocyanate (1). Ethyl-N-phenyl-[13C]-carbamate 7 (100 mg, 0.6 mmol) was dissolved in pentane (1.5 mL), and triethylamine (0.18 mL, 1.33 mmol) was added. The mixture was stirred at reflux for 30 min. After this time, chlorocatecholborane (211 mg, 1.33 mmol) was added, and the reaction mixture was stirred at reflux for another 30 min. After the addition of chlorocatecholborane, an instantaneous reaction was observed, and a white cloudy solid material appeared in the reaction flask due to the formation of Et3NH+Cl-. The temperature was gradually lowered to room temperature, and pentane was removed under reduced pressure (100 mbar, 5 °C). The remaining material was distilled (2 mbar, room temperature), and the distillation fraction containing 1 was condensed by placing the recuperation roundbottom flask in a dry ice-acetone bath at -78 °C. Compound 1 was obtained as a colorless oil (55 mg, 0.45 mmol, 75% yield). 1H NMR (300 MHz, CD3CN): δ 7.37 (t, 2H, H-3 and H-5, J ) 7.4 Hz), 7.25 (tt, 1H, H-4, J ) 7.4, 1.1 Hz), 7.16 (m, 2H, H-2 and H-6). 13C NMR (75 MHz, CD3CN): δ 134.1 (C-1), 129.6 (C-2 and C-6), 125.8 (C-4), 124.8 (C-3 and C-5), 124.6 (N-13CdO). ESITOF-MS: m/z 121.97 [M + H], m/z 142.09 [M + Na]. p-Tolyl-[13C]-isocyanate (2). Ethyl N-(4-methyl-phenyl)-[13C]carbamate 8 (100 mg, 0.55 mmol) was dissolved in pentane (1.5 mL), and triethylamine (0.17 mL, 1.22 mmol) was added. The mixture was stirred at reflux for 30 min. After this time, boron trichloride (0.21 mL, 0.20 mmol, 1 M solution in heptane) was added, and the reaction mixture was stirred at reflux for another 30 min. After the addition of boron trichloride, an instantaneous reaction was observed, and a white solid material appeared in the reaction flask due to the formation of Et3NH+Cl-. The temperature was lowered to room temperature, and pentane was removed under reduced pressure (100 mbar, 5 °C). The remaining material was distilled (2 mbar, room temperature), and the distillation fraction containing 2 was condensed by placing the recuperation roundbottom flask in a dry ice-acetone bath at -78 °C. Compound 2 was obtained as a colorless oil (60 mg, 0.5 mmol, 90% yield). 1H NMR (300 MHz, CD3CN): δ 7.16 (d, 2H, H-3 and H-5, J ) 8.4 Hz), 7.05 (d, 2H, H-2 and H-6, J ) 8.4 Hz), 2.31 (s, 3H, CH3). 13C NMR (75 MHz, CD3CN): δ 136.8 (C-1), 131.6 (C-4), 131.0 (C-3 and C-5), 125.5 (C-2 and C-6), 125.4 (N-13CdO), 20.9 (CH3). Reaction of Phenyl-[13C]-isocyanate (1) and p-Tolyl-[13C]isocyanate (2) with Nucleophilic Amino Acids. As a general procedure, 1 (1.64 µL, 15 µmol) or 2 (1.90 µL, 15 µmol), dissolved in CD3CN (300 µL), was added to a phosphate buffer solution (300 µL, 0.1 M at pH 7.4) containing the amino acid in its N-acetylated form (10 equiv). The final solution (600 µL) was filtered into a NMR tube. The reaction was followed by 13C NMR. Amino acids tested were N-Ac-Lys, N-Ac-His, N-Ac-Arg, N-Ac-Trp, N-Ac-Ser,

Fleischel et al. N-Ac-Tyr, N-Ac-Thr, N-Ac-Cys, and N-Ac-Met. In the competition experiment using N-Ac-Cys and N-Ac-Lys, the procedure was the same using 5 equiv of each amino acid. Reaction of Phenyl-[13C]-isocyanate (1) and p-Tolyl-[13C]isocyanate (2) with GSH. Isocyanate 1 (1.64 µL, 15 µmol) or 2 (1.90 µL, 15 µmol), dissolved in CD3CN (300 µL), was added to a phosphate buffer solution (300 µL, 0.1 M at pH 7.4) containing GSH (10 equiv). The final solution (600 µL) was filtered into a NMR tube, and the reaction was followed by 13C NMR. Reversible Conjugation of p-Tolyl-[13C]-isocyanate (2) with Thiol Groups in the Presence of N-Ac-Lys and N-Ac-Tyr. An NMR sample (300 µL) containing the thiocarbamate adduct 12 or 17, formed between 2 and N-Ac-Cys or GSH, respectively, was added to a solution of CD3CN (150 µL) and phosphate buffer (150 µL, 0.1 M at pH 7.4) containing N-Ac-Lys or N-Ac-Tyr (75 µmol). The final solution (600 µL) was filtered into a new NMR tube, and the reaction was followed by 13C NMR. In the case of the experiments with N-Ac-Lys, after 30 days of reaction, the pH was increased by adding a sodium hydroxide solution (50 µL, 1 M), and the reaction was followed by 13C NMR for 30 extra days. Reaction of Phenyl-[13C]-isocyanate (1) and p-Tolyl-[13C]isocyanate (2) with a Model Peptide. Isocyanate 1 (1.64 µL, 15 µmol) or 2 (1.90 µL, 15 µmol), dissolved in CD3CN (300 µL), was added to a phosphate buffer solution (300 µL, 0.1 M at pH 7.4) containing the peptide of sequence H2N-VLSPADKTNWGHEYRMFQIG-CO2H (3.5 mg, 1.5 µmol). The final solution (600 µL) was filtered into a NMR tube, and the reaction was followed by 13 C NMR. The peptide was synthesized manually in our laboratory by using solid-phase synthesis on a polystyrene support and the F-moc strategy. Peptide HPLC purification was performed using a Beckman 128P pump with a Perkin-Elmer 200 UV detector and a semipreparative reverse-phase Nucleodur 100-16 C18 column. The purity of the peptide (>99%) was assessed by mass spectrometry. Structural Characterization of the Products. The reactivity of 1 and 2 with nucleophilic amino acids, GSH, and the model peptide was followed by one-dimensional 13C NMR carried out on a Bruker Avance 300 spectrometer at 75 MHz. The structure of the products formed in the reactions was elucidated by twodimensional heteronuclear [1H-13C] NMR. 1H and 13C NMR data were obtained by heteronuclear single-quantum correlation (HSQC) and heteronuclear multiple-bond correlation (HMBC) experiments recorded on Bruker Avance 400 (1H, 400 MHz; 13C, 100 MHz) and Bruker Avance 500 (1H, 500 MHz; 13C, 125 MHz) spectrometers. Chemical shifts (δ) are reported in ppm with respect to TMS, CD3CN being the internal standard (δ 1H ) 1.94, δ 13C ) 1.32). Structures of the different products were assigned using a combination of HSQC and HMBC data and by comparing the measured chemical shifts with those calculated by ACD/CNMR and ACD/ HNMR Predictor software (version 5.12).

Results Synthesis. NMR techniques in association with 13C-labeled molecules have been shown to be an efficient tool for the investigation of hapten-protein interactions (18–20). Compounds 1 and 2 were synthesized, 13C-labeled at the electrophilic -NdCdO group, and assumed to be reactive sites toward proteins. In general, isocyanates are produced by phosgenation of primary amines. We therefore considered, at the beginning, the use of 13C-phosgene to introduce the isotope label. However, phosgene is highly toxic, and the process coproduces halogen compounds as byproduct. The search of environmentally friendly nonphosgene routes to obtain isocyanates has been considered in the past years (21, 22). Our interest focused on the reported production of alkyl and aryl isocyanates from carbamate intermediates, themselves obtained via the reaction of monosubstituted urea derivatives with aliphatic primary alcohols (23). The synthesis of the substituted urea derivative should allow the introduction of the 13C label by reacting [13C]-urea with a


Chem. Res. Toxicol., Vol. 22, No. 6, 2009 1109

Scheme 1. Synthetic Route for the Preparation of Phenyl-[13C]-isocyanate (1) and p-Tolyl-[13C]-isocyanate (2)

Scheme 2. Reaction of 1 and 2 with N-Ac-Cys and N-Ac-Lys and Characteristic NMR Data

primary amine. Following this strategy, compounds 1 and 2 were obtained in a three-step synthetic approach starting from aniline 3 and p-toluidine 4, respectively (Scheme 1). Treatment of 3 and 4 with [13C]-urea in a refluxed hydrochloric acid and toluene solution gave the corresponding urea derivatives 5 and 6 (24). The synthesis of carbamate derivatives from a monosubstituted urea and an aliphatic alcohol under the deaminating influence of a boron fluoride complex was described in the literature (25). Following this methodology, compounds 5 and 6 were converted into ethyl carbamates 7 and 8 by treatment with ammonium tetrafluoroborate in refluxing ethanol. The use of a boron fluoride complex differed markedly from the use of other boron halides since the latter, under similar conditions, are dehalogenated by the urea derivative with the formation of alkyl borates. Finally, the synthesis of isocyanates 1 and 2 was approached by dealcoholeysis of carbamates 7 and 8. At this point, we found that isocyanate isolation was limited due to its tendency to recombine easily with the alcohol formed. Valli and Alper reported the use of chlorocatecholborane, in the presence of triethylamine, as a reagent to trap the alcohol released by the carbamate (26). On the basis of their studies, refluxing 7 with chlorocatecholborane and triethylamine in pentane afforded successfully phenyl-[13C]-isocyanate 1 with a good yield in a one-pot reaction. As described in the literature, triethylamine was necessary to perform dehydrochlorination from the carbamate and chlorocatecholborane. Compound 1 was then isolated by distillation under reduced pressure (bp 50 °C at 10 mmHg) followed by the side product ethyl catecholborate (bp 86 °C at 10 mmHg). The use of the same reagents for the synthesis of p-tolyl-[13C]-isocyanate 2 from carbamate 8 was unsuccessful due to distillation problems, as the boiling point of 2 (72 °C at 10 mmHg) was very close to that of the expected side product ethyl catecholborate. Interestingly, the synthesis of isocyanates from carbamate esters, using boron trichloride, was reported further on by the same authors (27). They based their studies on the fact that boron trihalides are known for their strong Lewis acid character and their ability to cleave a wide variety of ethers, acetals, and esters under rather mild conditions

(28). Encouraged by their methodology, we successfully synthesized compound 2 by treatment of carbamate 8 with boron trichloride and triethylamine in pentane at reflux. In this case, the byproduct triethylborate (bp 10 °C at 10 mmHg) was easily removed at first when evaporating the solvent under reduced pressure, and 2 was obtained without difficulty by further distillation. Following these results, we did try the use of boron trichloride for the synthesis of 1 from 7. However, we were not able to totally isolate 1 from triethylborate by distillation as mixtures of both compounds were always obtained. Reaction of Phenyl-[13C]-isocyanate (1) and p-Tolyl-[13C]isocyanate (2) with Nucleophilic Amino Acids. The easy hydrolysis reaction of isocyanates, resulting in the formation of carbon dioxide and the corresponding amine, is very wellknown. Therefore, it was important for our studies to consider a solvent in which the hydrolysis reaction was slowed down and thus minimized to favor the direct reactivity of the isocyanates with the amino acids. A good compromise was found with the use of a 1/1 mixture of CD3CN and a phosphate buffer solution (0.1 M; pH 7.4). It is important to note, however, that the hydrolysis reaction was not completely avoided and that site-specific reactions that were observed when reacting 1 and 2 with the amino acids occurred in successful competition with the hydrolysis reaction. The reactivity was followed by 13 C NMR, and the products formed were characterized by 13C and 1H NMR data obtained by heteronuclear [1H-13C] NMR HSQC and HMBC experiments. Aryl monoisocyanates 1 and 2 reacted only with N-Ac-Cys and N-Ac-Lys. Adducts formed are shown in Scheme 2. 1. Reaction with N-Ac-Cys. Compounds 1 and 2 were found to react fast with N-Ac-Cys, as in a few hours the characteristic -Nd13CdO signals of 1 and 2, at 124.6 and 125.4 ppm, respectively, disappeared, and two new signals appeared at 166.2 and 154.1 ppm in the case of 1 and at 166.7 and 155.2 ppm in the case of 2. No evolution of the spectra was observed over time, and peaks at 166.2 and 166.7 were those of higher intensity. The δ of 166.2 and 166.7 ppm corresponded well to that of a carbon atom belonging to a thiocarbamate chemical

1110


Fleischel et al.

Figure 1. Reaction of 1 with N-Ac-Lys. (A) Evolution with time followed by one-dimensional 13C NMR. The spectrum of unreacted N-Ac-Lys is shown at the bottom, and it is followed (from bottom to top) by the 13C NMR spectra obtained after the addition of the isocyanate. Peaks corresponding to the products formed are indicated at day 10. (B) Competition between the isocyanate hydrolysis reaction and the formation of adduct 11. A hydrolysis reaction gave N,N′-diphenyl-urea 10 also evidenced by the observed peak of precursor phenyl-carbamic acid 15. HMBC data (arrows) allowing the structural elucidation of 11 are shown.

function (-NH-CO-S-). HMBC experiments showed that the 13 C signal at 166.2 ppm was correlated via long-range coupling with protons at 3.29 ppm, themselves correlated via 1J(C, H) coupling (HSQC experiments) to a carbon atom at 30.7 ppm, characteristic of the β-methylene group of N-Ac-Cys. Similarly, the 13C signal at 166.7 ppm was correlated via long-range coupling with protons at 3.27 ppm, themselves correlated via 1 J(C, H) coupling to a carbon atom at 31.5 ppm, also characteristic of the β-methylene group of N-Ac-Cys. Also, the carbon atoms at 166.2 and 166.7 ppm were correlated with neighboring NH protons at 9.51 and 9.45 ppm, respectively. These observations were in accordance with adducts 9 and 12 (Scheme 2) formed after attack of the thiol group of N-Ac-Cys on the electrophilic carbon atom of the isocyanate. The chemical structure of 9 was also supported by the existence of long-range couplings between the 13C atom at 166.2 ppm and H-2/H-6 (7.28 ppm) and H-3/H-5 (7.38 ppm), confirming the presence of the aromatic moiety. The same was observed for adduct 12, being the 13C atom at 166.7 ppm correlated via long-range coupling with H-2/H-6 (7.28 ppm) and H-3/H-5 (7.10 ppm). The δ of the new signal at 154.1 ppm in the case of 1, and at 155.2 ppm in the case of 2, corresponded to a carbon atom belonging to a urea chemical function (-NH-CO-NH-). N,N′-Diphenyl-urea 10 was characterized as a side product in the reaction of 1 with N-Ac-Cys. The 13C signal at 154.1 ppm was symmetrically longrange correlated with the adjacent NH protons at 7.88 ppm, H-2/ H-6 and H-2′/H-6′ (7.38 ppm), themselves correlated via 1J(C, H) coupling to 119.7 ppm, and with H-3/H-5 and H-3′/H-5′ (7.27 ppm), correlated via 1J(C, H) coupling to the carbon atom at 129.1 ppm. In the same way, N,N′-di-p-tolyl-urea 13 was characterized as a side product in the reaction of 2 with N-AcCys. From the mechanistic point of view, the competing hydrolysis of 1 afforded aniline, able to react with remaining isocyanate 1 and able to form the urea derivative 10. Similarly, hydrolysis of 2 afforded p-toluidine that also reacted with the remaining isocyanate 2 to form the urea derivative 13. 2. Reaction with N-Ac-Lys. From the first day of reaction, new 13C signals appeared, at 157.2 ppm in the case of 1, at 158.3 ppm in the case of 2, and a common signal at 160.0 ppm for both compounds. These signals remained, and after 10 days of reaction, another signal appeared at 154.1 ppm for 1 and at

155.2 ppm for 2. Peaks at 157.2 and 158.3 ppm increased considerably with time. Figure 1A shows, as an example, the reactivity observed for 1. HSQC and HMBC data indicated the formation of adducts 11 and 14 after nucleophilic attack of the ε-NH2 group of N-Ac-Lys on the electrophilic carbon atom of the isocyanates (Scheme 2). Carbons at 157.2 and 158.3 ppm corresponded well with the typical δ of urea chemical functions (-NH-CO-NH-). For adduct 11, the 13C at 157.2 ppm showed long-range correlations with the NH protons at 8.01 (linked to the aromatic moiety) and 6.01 ppm (linked to a methylene group). However, more conclusive was its correlation with protons at 3.05 ppm, themselves correlated via 1J(C, H) coupling to the carbon atom at 39.1 ppm, characteristic of the ε-methylene group of the lateral chain of N-Ac-Lys. The same for adduct 14, in which the 13C at 158.3 ppm had long-range correlations with NH protons at 7.90 and at 5.98 ppm, and also with the ε-methylene protons of N-Ac-Lys at 3.04 ppm, correlated via 1J(C, H) coupling to the carbon atom at 40.0 ppm. As for the reaction with N-Ac-Cys, the isocyanate hydrolysis competing reaction was confirmed by the formation of urea derivatives 10 (154.1 ppm) and 13 (155.2 ppm). Interestingly, the intermediate phenyl-carbamic acid was visualized this time as evidenced by the quickly appearing signal at 160.0 ppm. For example, Figure 1 shows the precursor 15 in the case of the formation of N,N′-diphenyl-urea 10. 3. Competition Reaction between N-Ac-Cys and N-Ac-Lys. As N-Ac-Cys and N-Ac-Lys were the reactive amino acids, we looked into the reaction of 1 and 2 in the presence of both amino acids together (5 equiv each) in the CD3CN/PBS solution. These experiments confirmed the reactivity of both amino acids in competition and showed a higher and preferential reactivity of N-Ac-Cys as compared to N-Ac-Lys (Figure 2). After 15 days of reaction, the formation of the thiocarbamate adduct 9 (166.2 ppm) was clearly observed, together with, in a minor extent, the formation of the urea-like adduct 11 (157.2 ppm). 4. Reaction with Other Amino Acids. No adducts were observed with the other amino acids tested even after 40 days of reaction but only products such as N,N′-diphenyl-urea 10 and N,N′-di-p-tolyl-urea 13, resulting from the isocyanates hydrolysis.



Figure 2. 13C NMR spectrum of the reaction of 1 with N-Ac-Cys and N-Ac-Lys in competition (5 equiv each) at day 15.

Scheme 3. Products Obtained from the Reaction of 1 and 2 with GSHa Figure 3. Monitoring by one-dimensional 13C NMR of the conversion of N-arylcarbamate thioester 12 into adduct 14 after reaction with N-AcLys in an alkaline medium.

a

The most important δ (ppm) are indicated.

Reaction of Phenyl-[13C]-isocyanate (1) and p-Tolyl-[13C]isocyanate (2) with GSH. Compounds 1 and 2 were reacted with GSH in the semiorganic CD3CN/PBS (0.1 M; pH 7.4) medium, using a 10 M excess of GSH to mimic a detoxification situation. The reaction was found to be very fast to form mainly adducts 16-19 with δ characteristic of thiocarbamate and of urea chemical functions (Scheme 3). New 13C peaks at 167.3 and 157.4 ppm appeared in the reaction with 1 and at 166.7 and 156.9 ppm in the reaction with 2. The nucleophilic attack of the GSH thiol group on the -Nd13CdO group afforded adducts 16 and 17. [1H-13C] NMR HSQC and HMBC experiments gave access to the δ of the -CH2S methylene group of GSH, long-range correlated with the 13C atom of 16 and 17 at 167.3 and 166.7 ppm, respectively. On another hand, the electrophilic -Nd13CdO group was able to react with the nucleophilic amino group of GSH, and adducts of the urea kind such as 18 and 19 were observed. The [1H-13C] NMR data correlated, via long-range coupling, the carbon atom of 18 at 157.4 ppm with the NH protons at 8.20 (linked to the aromatic moiety) and 6.30 ppm (linked to GSH), and similarly, the carbon atom of 19 at 156.9 ppm with the NH protons at 8.03 and 6.16 ppm. Signals in the NMR spectra did not allow us to find out if the attack of the amino group of GSH was of the intermolecular kind or if adducts 16 and 17 afforded 18 and 19 by intramolecular attack of the amino group and cyclization followed by opening of the cycle. This kind of intramolecular cyclization has already been observed, for example, in the

reaction of 2-methylisothiazol-3-one with GSH (29). However, in the case of the isocyanates, the signals corresponding to thiocarbamate adducts 16 and 17 at 167.3 and 166.7 ppm did not disappear to the profit of signals attributed to urea derivatives 18 and 19 at 157.4 and 156.9 ppm. Finally, also, the isocyanate hydrolysis reaction was observed by the formation of urea derivatives 10 and 13 (154.1 and 155.2 ppm). Reversible Conjugation of p-Tolyl-[13C]-isocyanate (2) with Thiol Groups in the Presence of N-Ac-Lys and N-Ac-Tyr. When N-arylcarbamate thioesters 12 and 17, resulting from the reaction of 2 with the thiol groups of N-Ac-Cys and GSH, respectively, were in the presence of N-Ac-Lys in an alkaline medium (pH 12.0), the formation of a new adduct resulting from the reaction with the ε-NH2 group of the competing amino acid was observed. Indeed, following the reaction at pH 7.4 during 30 days, no evolution was noticed. However, after increasing the pH of the reaction mixture by adding a sodium hydroxide solution, the characteristic 13C NMR peak of 12 at 166.7 ppm gradually disappeared in 1 week at a profit of a new peak at 157.4 ppm in the range of the δ of carbon atoms belonging to -NH-CO-NH- chemical functions (Figure 3). The chemical structure of 14 in these experiments was corroborated by HSQC and HMBC data. In the same way, the 13C NMR peak of 17 at 166.7 ppm gradually disappeared at a profit of a new peak at 157.6 ppm. It is important to note that in any of the experiments, released isocyanate 2 (125.4 ppm) was detected in the spectra. In the case of the reaction with GSH, the initial NMR sample contained 17 at 166.7 ppm but also 19 at 156.9 ppm. In the presence of N-Ac-Lys at pH 12.0, the signal at 166.7 ppm progressively vanished, a peak at 157.6 ppm appeared, and a peak at 156.9 ppm remained unchanged. These observations would suggest that the new urea derivative formed resulted from the reaction of the amino group of N-Ac-Lys on the thiocarbamate and that the GSH free amino group was not involved in the competing reaction. In the presence of N-Ac-Tyr, the results were not so conclusive even if the reaction evolved at physiological pH. Only the existence in the HMBC spectra of a new single [1H-13C] correlation between a 13C at 154.6 ppm and a NH proton at 8.15 ppm linked to the aromatic moiety suggested the formation of an adduct by reaction of the tyrosyl-hydroxyl group, as the predicted δ for the carbon atom of the theoretically resulting carbamate chemical bond (-NH-CO-O-) was of 152 ppm. However, no other correlation signals neither better resolution of the 13C NMR spectra could help to confirm this hypothesis.

1112


Fleischel et al.

Figure 4. [1H-13C] HMBC for the peptide modified by 2 in a CD3CN/PBS (pH 7.4) medium. The chemical structure of the suggested adducts is shown together with the most important δ (ppm) and the HMBC correlations (arrows) allowing their identification.

Reaction of Phenyl-[13C]-isocyanate (1) and p-Tolyl-[13C]isocyanate (2) with a Model Peptide. The reactivity of 1 and 2 was also studied toward the synthetic peptide of sequence H2N-VLSPADKTNWGHEYRMFQIG-CO2H. This peptide has been used in several reported hapten-protein binding studies for skin sensitizers because it includes all naturally occurring amino acids except cysteine, to prevent the formation of a cystine dimer (29, 30). An additional reported reason to remove cysteine was to facilitate the coupling reaction of the allergen with other potentially reactive amino acids, as it is known that most skin allergenic compounds, weak or strong, are reactive toward thiol chemical functions (31). The peptide was reacted with 1 and 2 in the same semiorganic CD3CN/PBS (0.1 M; pH 7.4) medium. An excess of isocyanate was used, based on previous studies that yielded optimum peptide reactivity conditions for other allergenic compounds (30, 31). After following the reactions by one-dimensional 13C NMR, two-dimensional [1H-13C] NMR experiments were carried out to identify new peaks corresponding to potential adducts. After reaction with 2, for example, three new 13C signals were detected. A first signal at 157.9 ppm that could correspond to an adduct on lysine, a signal at 157.3 ppm also in the characteristic area of urea -NH-CO-NH groups, and the many times mentioned 155.2 ppm signal were always observed and corresponded to N,N′di-p-tolyl-urea 13. For each new 13C signal, 1H long-range correlations were recorded and gave information, in some cases, on the peptide side chain. This was the case for the signal at 157.9 ppm, assigned to a lysine adduct 20 (Figure 4), that was correlated to two NH protons, one at 7.95 ppm linked to the aromatic moiety and another one at 5.80 ppm characteristic of an adduct of the amide type obtained via reaction of the isocyanate with primary amino groups like the ε-NH2 group of lysine. Also, 157.9 ppm was correlated to protons of the ε-methylene group of the lateral chain of lysine (3.04/41.2 ppm), confirming the formation of the adduct. The 13C NMR signal at 157.3 ppm corresponded as well to a carbonyl chemical function of a urea derivative. This one could only be formed by reaction with the other free NH2 group of the peptide, which means the one of valine to form adduct 21 (Figure 4). The signal at 157.3 ppm was long-range correlated with NH protons at 8.03 and 6.12 ppm. However, in this case, no other correlations on the peptide were seen. Similar spectra and correlations were registered in the reaction with 1. The lysine adduct was recognized thanks to the 13C NMR signal at 157.9 ppm, the NH proton at 8.06 ppm (Ar-NH-), the NH proton at 5.88 ppm

(-NH-CH2-), and the ε-methylene group of the lateral chain of lysine at 3.05/43.4 ppm. The valine adduct was recognized thanks to the 13C NMR signal at 157.0 ppm, the NH proton at 8.03 ppm (Ar-NH-), and the NH proton at 6.18 ppm (-NH-valine-). Of course, the possibility to have both adducts formed at the same time within the peptide cannot be excluded. Unfortunately, the NMR experiments presented here did not allow the distinction between the formation of two modified peptides or only one peptide containing the two chemical modifications together.

Discussion It is well-known that isocyanates are susceptible to nucleophilic attack at the carbon atom by nucleophiles present on biological macromolecules. For instance, the diverse reactivity of isocyanates toward nucleophiles made them attractive, many years ago, as potential molecular “rulers” to measure structural features of proteins (32, 33). Although virtually every nucleophilic group can give a defined product with an isocyanate under some defined set of conditions, those studies were mostly centered on products expected to be formed at physiological pH. This narrowed the reactive amino acid studied to those having a hydroxyl or a thiol group. Reactions with amino acids containing an amino group in the lateral chain, even if possible, were not considered at that time, as under physiological conditions, they are less probable, the amine function being mainly protonated and nonreactive. On another hand, it is also known that the reaction of isocyanates with these functional groups competes with the hydrolysis to the corresponding amine and the release of carbon dioxide. It has been reported that the relative hydrolysis rates of aryl monoisocyanates are 5-1400fold faster over alkyl monoisocyanates, depending also on the solvent chosen for the study (34). We now report our results on the reactivity, followed by 13C NMR, of 13C-labeled phenyl isocyanate 1 and p-tolyl isocyanate 2 toward nucleophilic amino acids in a semiorganic CD3CN/PBS (0.1 M; pH 7.4) medium. In this solvent, the hydrolysis reaction did not hinder the monitoring of the formation of adducts with the amino acids. On the contrary, tests using dimethylsulfoxide instead of acetonitrile showed that the hydrolysis reaction was much more favored and hampered the follow-up of the reactivity (data not shown). Moreover, the chemical shifts of the 13C atoms of N,N′diphenyl-urea 10 and N,N′-di-p-tolyl-urea 13, side products derived from the hydrolysis of 1 and 2, did not overlay with

ReactiVity of Aryl Isocyanates 13

C atoms signing the formation of amino acid adducts and thus confirmed the use of 13C NMR as a helpful tool for the investigation of isocyanate-protein interactions. Aryl monoisocyanates 1 and 2 reacted with N-Ac-Cys and N-Ac-Lys (Scheme 2). N-Ac-Cys was linked to the isocyanate group through a thiocarbamate bond, forming adducts 9 and 12. In the case of N-Ac-Lys, the ε-NH2 group was linked to the isocyanate through the formation of urea derivatives 11 and 14, confirming that in the CD3CN/PBS (0.1 M; pH 7.4) medium N-Ac-Lys was reactive. Adducts of 2 with N-Ac-Cys and N-AcLys had already been synthesized in organic medium and fully characterized (35), confirming the assigned structures. In both cases, side products 10 and 13 derived from the hydrolysis competing reaction were observed. However, only when using N-Ac-Lys the transition through a phenyl-carbamic acid was visualized (Figure 1). It has been reported in the literature that the kinetics of N-arylcarbamates decarboxylation is a function of pH and buffer concentration (36). The decarboxylation of aryl compounds is generally catalyzed in acidic media, whereas it is inhibited in strong alkaline media. Arguments reported in the literature suggest that the rate-controlling step for the decarboxylation process in the presence of amines with high pKa values is nitrogen protonation (36, 37). One could thus assume that, in the presence of an excess of an amine compound such as N-Ac-Lys [10 equiv; pKa (ε-NH2) ) 10.5], the pH of the solution becoming more alkaline, the kinetics of the decarboxylation of the carbamic acid deriving from hydrolysis of the isocyanate is slowed down. This way, it was possible to observe the 13C NMR signal corresponding to the aryl carbamic acid before it releases carbon dioxide to give the corresponding amine (i.e., signal at 160.0 ppm for compound 15, Figure 1). At neutral pH, the decarboxylation is much faster in a way that the 13C NMR detection of the carbamic acid intermediate was not possible; the amine formed after release of carbon dioxide, also reacting quickly with the remaining isocyanate to give ureas 10 and 13. Looking quickly into the behavior of 1 and 2 in the presence of N-Ac-Cys and N-Ac-Lys together (5 equiv each) in the CD3CN/PBS solution, the reactivity of both amino acids was confirmed, and a preferential reactivity of N-Ac-Cys as compared to N-Ac-Lys was observed. When performing the experiments with GSH, which contains both the free amino and the thiol groups, it was also shown that 1 and 2 were able to react with the two nucleophilic chemical functions to form adducts of the thiocarbamate ester and urea kind. Therefore, all of the results concluded that in the experimental conditions that we tested 1 and 2 were highly reactive toward thiol but also amino chemical functions. The majority of bonds formed between electrophilic haptens and nucleophilic functions present in proteins are considered irreversible under physiological conditions. However, an exception is the reaction of isocyanates with thiol groups, whose products are stable under acidic conditions but fully reversible under slightly alkaline pH conditions. It was shown in the 1960s that thiol groups reacted reversibly with cyanate at pH 7 (38). The resulting carbamate thioester adducts decomposed rapidly in bicarbonate solution but were stable at pH values of 5 or less. Further studies on the chemistry of N-arylcarbamate thioesters provided mechanistic information relevant to the reversible conjugation with thiol groups. Kinetic evidence on the hydrolysis of N-arylcarbamate thioesters through an E1cB mechanism (unimolecular elimination via the conjugate base) and elimination of an isocyanate was reported (39). The hydrolysis rate constant was found to be much higher than that


of the corresponding carbamate ester, and the difference was attributed to the higher acidity of the N-H proton in the thioester and to the role of sulfur in stabilizing the negative charge in the transition state, leading to the reaction intermediate. On this basis, we expected reactions of 1 and 2 with GSH, yielding carbamate thioesters, to be facile and reversible. The in vivo formation of thiocarbamates from administered isocyanates was demonstrated in several cases by finding GSH and cysteine adducts in bile and urine of rats (40). Also, GSH has been hypothesized as being capable of capturing isocyanates through the formation of a thioester on the cysteine side chain and of transporting them in an inert form to a distal location where it can reverse releasing the isocyanate functional group, which can further react with another nucleophile (41, 42). This hypothesis has never been in fact definitely proved in the sense that, even if thought that the isocyanate is newly released, the reaction with a competing nucleophile, other than water, most probably occurs through a direct addition-elimination process on the carbonyl chemical function of the thioester adduct (Bac2 mechanism) without real release of the isocyanate (E1cB mechanism). However, because allergenic isocyanates are able to cause covalent modifications at sites in the body that have not been directly exposed, there is no doubt suggesting that a sort of “transport mechanism” for the isocyanate chemical function certainly exists. Nowadays, adducts of aliphatic monoisocyanates and aromatic diisocyanates with GSH have been synthesized, and their stabilities in aqueous solution have been studied, indicating a reversible base-catalyzed degradation through an E1cB mechanism (42, 43). Adding to these studies, we now report the use of 13C NMR to follow the evolution of aryl monoisocyanate adducts with thiol groups in the presence of other nucleophiles apart from water. p-Tolyl-[13C]-isocyanate 2 was used, and its thiocarbamate adduct formed with either N-Ac-Cys or GSH was put into competition with other nucleophilic amino acids such as N-Ac-Lys and N-Ac-Tyr. The evolution of the thiocarbamate adduct was followed by 13C NMR, together with the formation of new adducts with the competing amino acid. In the case of N-Ac-Lys, the reaction showed clear pH dependence. It was observed that the N-AcCys or GSH thiocarbamate adducts were converted under alkaline pH into irreversible N-Ac-Lys urea adducts. The nucleophilic character of the ε-NH2 group of lysine was increased as it was deprotonated at high pH, and thus, its reactivity was favored. A 13C signal corresponding to released isocyanate was never detected in the spectra during the time that the reactions evolved. Therefore, the formation of urea adducts from thiocarbamates and lysine happened most probably by direct attack of the amino group of lysine on the thiocarbamates through a Bac2 mechanism and not by previous reverse reaction to an isocyanate as in a E1cB mechanism. Indeed, in the addition-elimination Bac2 mechanism, nucleophilicity is dominating and attack on the electrophilic carbonyl is the first step, followed by elimination of the best leaving group. The E1cB mechanism would first involve a base-catalyzed deprotonation of the nitrogen, followed by elimination of a nucleophile to release the isocyanate, which seemed not to be likely in our experiments. In the case of N-Ac-Tyr, results were not conclusive. Even if, as expected, the reaction evolved at physiological pH, in which the amino acid exists in its neutral form, the elucidation of the most probable tyrosyl-hydroxyl adduct could not be completely certain due to NMR resolution problems. These studies confirmed the role of lysine as a good competing nucleophilic amino acid during the conjugation of isocyanates with thiol-containing peptides. Clearly, the use of

1114 13


C-labeled isocyanates at the reactive site presented an advantage for future studies, as it should help the monitoring of this reactivity in the presence of biomolecules, in vitro and in vivo, being the 13C NMR enhanced signal of the adducts easy to be distinguished by certain 13C and [1H-13C] NMR spectroscopy techniques. The reactivity with amino acid residues included in a peptide sequence could be different from the one observed with isolated amino acids. Indeed, proteins should be regarded as a mixture of nucleophiles having different electronic characteristics, which are also modified by their environment and accessibility, among others. It was therefore of interest to evaluate if there was any different selectivity in the reactivity of 1 and 2 with nucleophilic amino acids when these are located in a peptide environment. The use of the sequence H2N-VLSPADKTNWGHEYRMFQIGCO2H confirmed again, without ambiguity, the high reactivity of isocyanates 1 and 2 toward amino groups (ε-NH2 of lysine-7 and -NH2 of valine-1) to form adducts of the urea kind in the experimental conditions tested, to say in the semiorganic CD3CN/PBS (0.1 M; pH 7.4) medium. No reactions with other nucleophilic amino acids were detected. Certainly, further studies will be needed to assess this reactivity in the context of larger proteins as the environment and, therefore, the access to the reactive lysine amino acids, will be modified. We have thus shown that 1 and 2 react essentially with amino and thiol groups when they are isolated in lysine or cysteine, respectively, when they are put into competition, for example, in GSH and also when lysine is surrounded by other nucleophilic amino acids in a model peptide that included all naturally occurring amino acids except cysteine. Skin allergens, even if each one has its own chemical reactivity pattern, very often react with the same nucleophilic chemical groups, although not exclusively. Thus, most skin allergens, weak, moderate, or strong, are mainly reactive toward thiol chemical groups (31). In parallel, the ability in some cases to modify lysine residues has been launched as a possible, but not unique, reason to explain differences in the skin-sensitizing potential of a compound when compared to that of other molecules (20, 29, 44). The results of our studies on aryl isocyanates such as 1 and 2 reveal that they are reactive toward thiol groups but also significantly toward free amino groups. We have also shown that intermediate thiocarbamate adducts resulting from reaction with thiol groups can be converted into thermodynamically more stable adducts of the urea type by reaction with amino groups. This isocyanate chemistry has already been described in organic solvents but never in a semiorganic medium (45, 46). In the studies presented here, the isocyanates were reactive toward lysine in the experimental conditions tested and thus can, from now, be considered reactive toward amino groups at physiological pH in a semiorganic medium. In this study, 1 and 2 were chosen as representative models for MDI and TDI. MDI and TDI, known respiratory sensitizers, can also cause skin contact allergy. Until now, the fundamental difference described distinguishing respiratory and skin allergens is that chemical respiratory allergens seem to stimulate Th2 lymphocytes, and skin allergens seem to induce preferential Th1 type immune responses (13). It has been longtime considered that induced skin or respiratory allergy to a compound was exclusively related to the way of exposition, through the skin or the respiratory tract. However, it has been proposed recently that the stimulation of Th-1/Th-2 immune responses depends exclusively on the nature of the hapten and not on the way of exposition (47). Relationships between the nature/reactivity of

Fleischel et al.

haptens toward amino acids and the later immune responses are not yet well understood. Some authors have suggested that haptens inducing type I allergy would selectively react with lysine residues, whereas haptens inducing type IV allergy would react with cysteine residues preferentially (48). Type IV hypersensibility reactions are generally specific to skin allergens and related to Th-1 type lymphocytes, while type I reactions are mainly associated to respiratory allergens and to immunoglobulin E and Th-2 immune responses. Isocyanates such as MDI and TDI have been reported to provoke either Th-1 and Th-2 immune responses (49). We have shown that model isocyanates 1 and 2 react strongly with thiol groups (cysteine, GSH) but also with amino groups (lysine, GSH, model peptide). Maybe this reactivity could be related to the Th-1/Th-2 immune response duality for isocyanates acting as respiratory but also skin sensitizers. Of course, even if this hypothetic explanation still needs to be demonstrated, cysteine and lysine are key amino acids to be considered in the sensitization process to isocyanates, and the aptitude of the isocyanates to form urea derivatives with lysine, if not directly then by further reaction of the initial thiocarbamate adduct, could play a valuable role.

Conclusion The reactivity of aromatic isocyanates 1 and 2, models of respiratory sensitizers, bearing the very reactive -NdCdO chemical group, was studied toward nucleophilic amino acids, GSH, and a model peptide. Reactions were carried out in a semiorganic CD3CN/PBS (0.1 M; pH 7.4) medium in which the known isocyanate competing hydrolysis reaction did not hinder the monitoring of the formation of adducts with the amino acids. Phenyl isocyanate 1 and p-tolyl isocyanate 2 reacted essentially with the nucleophilic thiol and amino groups to form adducts of the thiocarbamate ester and urea kind, respectively. In parallel, the role of lysine as a good competing nucleophilic amino acid during the reversible conjugation of isocyanates with thiol-containing peptides was confirmed. The reactivity of aryl isocyanates with thiol and amino groups needs thus to be considered in their assigned sensitization processes. Finally, the use of 13C-labeled isocyanates at the reactive site is an advantage for future studies, as it should help the monitoring of this reactivity in the presence of biomolecules, in vitro and in vivo. Acknowledgment. This work was supported by a grant from the 6th Framework Programme for Research and Technological Development of the European Commission as part of the integrated project “Novel testing strategies for in vitro assessment of allergens (Sens-It-Iv)” (LSHB-CT-2005-018681). Note Added after ASAP Publication. A citation was inadvertently left out of the second paragraph of the Discussion in the version published ASAP April 30, 2009. The citation and corresponding reference were published in the correct version June 2, 2009.

References (1) Bernstein, I. L., Chang-Yeung, M., Malo, J.-L., and Bernstein, D. I. (2006) Asthma in the Workplace, 3rd ed., p 621, Taylor & Francis, New York. (2) Blanc, P. D., and Toren, K. (1999) How much adult asthma can be attributed to occupational factors? Am. J. Med. 107, 580–587. (3) Siracusa, A., Desrosiers, M., and Marabini, A. (2000) Epidemiology of occupational rhinitis: Prevalence, aetiology and determinants. Clin. Exp. Allergy 30, 1519–1534. (4) Redlich, C. A., and Karol, M. H. (2002) Diisocyanate asthma: clinical aspects and immunopathogenesis. Int. Immunopharmacol. 2, 213–224.

ReactiVity of Aryl Isocyanates (5) Bakerly, N. D., Moore, V. C., Vellore, A. D., Jaakkola, M. S., Robertson, A. S., and Burge, P. S. (2008) Fifteen-year trends in occupational asthma: data from the Shield surveillance scheme. Occup. Med. (London) 58, 169–174. (6) Klees, J. E., and Ott, M. G. (1999) Diisocyanates in polyurethane plastics applications. Occup. Med. 14, 759–775. (7) Tanser, A. R., Bourke, M. P., and Blandford, A. G. (1973) Isocyanate asthma: respiratory symptoms caused by diphenyl-methane di-isocyanate. Thorax 28, 596–600. (8) Meredith, S. K., Bugler, J., and Clark, R. L. (2000) Isocyanate exposure and occupational asthma: A case-referent study. Occup. EnViron. Med. 57, 830–836. (9) Estlander, T., Keskinen, H., Jolanki, R., and Kanerva, L. (1992) Occupational dermatitis from exposure to polyurethane chemicals. Contact Dermatitis 27, 161–165. (10) Goossens, A., Detienne, T., and Bruze, M. (2002) Occupational allergic contact dermatitis caused by isocyanates. Contact Dermatitis 47, 304– 308. (11) Lepoittevin, J.-P. (2006) Molecular aspects of allergic contact dermatitis. In Contact Dermatitis (Frosch, P. J., Menne´, T., and Lepoittevin, J.-P., Eds.) pp 45-68, Springer-Verlag, Berlin, Heidelberg, New York. (12) Kimber, I., and Dearman, R. J. (2003) What makes a chemical an allergen? Ann. Allergy Asthma Immunol. 90, 28–31. (13) Kimber, I., and Dearman, R. J. (2005) What makes a chemical a respiratory sensitizer? Curr. Opin. Allergy Clin. Immunol. 5, 119– 124. (14) Karol, M. H., and Kramarik, J. A. (1996) Phenyl isocyanate is a potent chemical sensitizer. Toxicol. Lett. 89, 139–146. (15) Karol, M. H., Dixon, C., Brady, M., and Alarie, Y. (1980) Immunologic sensitization and pulmonary hypersensitivity by repeated inhalation of aromatic isocyanates. Toxicol. Appl. Pharmacol. 53, 260–270. (16) Stevens, M. A. (1967) Use of the albino guinea pig to detect the skinsensitizing ability of chemicals. Br. J. Ind. Med. 24, 189–202. (17) Pauluhn, J., Ru¨ngeler, W., and Mohr, U. (1995) Phenyl isocyanateinduced asthma in rats following a 2-week exposure period. Fundam. Appl. Toxicol. 24, 217–228. (18) Eilstein, J., Gimeńez-Arnau, E., Duche´, D., Rousset, F., and Lepoittevin, J.-P. (2006) Synthesis and reactivity toward nucleophilic amino acids of 2,5-[13C]-dimethyl-para-benzoquinonediimine. Chem. Res. Toxicol. 19, 1248–1256. (19) Alvarez-Sańchez, R., Divkovic, M., Basketter, D., Pease, C., Panico, M., Dell, A., Morris, H., and Lepoittevin, J.-P. (2004) Effect of glutathione on the covalent binding of the 13C-labeled skin sensitizer 5-chloro-2-methylisothiazol-3-one to human serum albumin: identification of adducts by nuclear magnetic resonance, matrix-assisted laser desorption/ionization mass spectrometry, and nanoelectrospray tandem mass spectrometry. Chem. Res. Toxicol. 17, 1280–1288. (20) Meschkat, E., Barrat, M. D., and Lepoittevin, J.-P. (2001) Studies of the chemical selectivity of hapten, reactivity, and skin sensitization potency. 2. NMR studies of the covalent binding of the 13C-labeled skin sensitizers 2-[13C]- and 3-[13C]hex-1-ene- and 3-[13C]hexane-1,3sultones to human serum albumin. Chem. Res. Toxicol. 14, 118–126. (21) Katada, N., Fujinaga, H., Nakamura, Y., Okumura, K., Nishigaki, K., and Niwa, M. (2002) Catalytic activity of mesoporous silica for synthesis of methyl N-phenyl carbamate from dimethyl carbonate and aniline. Catal. Lett. 80, 47–51. (22) Valli, V. L. K., and Alper, H. (1993) Reductive carbonylation of monoand dinitroarenes catalyzed by montmorillonitebipyridinylpalladium (II) acetate and ruthenium carbonyl. J. Am. Chem. Soc. 115, 3778– 3779. (23) Wang, J., Li, Q., Dong, W., Kang, M., Wang, X., and Peng, S. (2004) A new non-phosgene route for synthesis of methyl N-phenyl carbamate from phenyl urea and methanol. Appl. Catal., A 261, 191–197. (24) Davis, T. L., and Blanchard, K. C. (1923) The urea dearrangement. II. J. Am. Chem. Soc. 45, 1816–1820. (25) Sowa, F. J. (1960) Chemical process for the production of esters of carbamic and carbonic acids. GB 840866 Patent. (26) Valli, V. L. K., and Alper, H. (1995) A simple, convenient, and efficient method for the synthesis of isocyanates from urethanes. J. Org. Chem. 60, 257–258. (27) Butler, D. C. D., and Alper, H. (1998) Synthesis of isocyanates from carbamate esters employing boron trichloride. Chem. Commun. 23, 2575–2576. (28) Gerrard, W., and Lappert, M. F. (1958) Reactions of boron trichloride with organic compounds. Chem. ReV. 58, 1081–1111. (29) Alvarez-Sańchez, R., Basketter, D., Pease, C., and Lepoittevin, J.-P. (2004) Covalent binding of the 13C-labeled skin sensitizers 5-chloro-


(30)

(31)

(32) (33) (34)

(35) (36)

(37) (38) (39)

(40)

(41) (42)

(43)

(44)

(45) (46) (47) (48)

(49)

2-methylisothiazol-3-one (MCI) and 2-methylisothiazol-3-one (MI) to a model peptide and glutathione. Bioorg. Med. Chem. Lett. 14, 365– 368. Eilstein, J., Gimeńez-Arnau, E., Duche´, D., Cavusoglu, N., Hussler, G., Rousset, F., and Lepoittevin, J.-P. (2008) Sensitization to p-amino aromatic compounds: study of the covalent binding of 2,5dimethyl-p-benzoquinonediimine to a model peptide by electrospray ionization tandem mass spectrometry. Bioorg. Med. Chem. 16, 5482–5489. Gerberick, F. G., Vassallo, J. D., Bailey, R. E., Chaney, J. G., Morrall, S. W., and Lepoittevin, J.-P. (2004) Development of a peptide reactivity assay for screening contact allergens. Toxicol. Sci. 81, 332– 343. Twu, J.-S., and Wold, F. (1973) Butyl isocyanate, an active site specific reagent for yeast alcohol dehydrogenase. Biochemistry 12, 381–386. Brown, W. E. (1975) Alkyl isocyanates as active site specific reagents for serine proteases. Location of alkyl binding site in chymotrypsin by x-ray diffraction. Biochemistry 14, 5079–5084. Brown, W. E., Green, A. H., Cedel, T. E., and Cairns, J. (1987) Biochemistry of protein-isocyanate interactions: A comparison of the effects of aryl vs. alkyl isocyanates. EnViron. Health Perspect. 72, 5–11. Sabbioni, G., Lamb, J. H., Farmer, P. B., and Sepai, O. (1997) Reactions of 4-methylphenyl isocyanate with amino acids. Biomarkers 2, 223–232. Johnson, S. L., and Morrison, D. L. (1972) Kinetics and mechanism of decarboxylation of N-arylcarbamates. Evidence for kinetically important zwitterionic carbamic acid species of short lifetime. J. Am. Chem. Soc. 94, 1323–1334. Caplow, M. (1968) Kinetics of carbamate formation and breakdown. J. Am. Chem. Soc. 90, 6795–6803. Stark, G. R. (1964) On the reversible reaction of cyanate with sulfhydryl groups and the determination of NH2-terminal cysteine and cystine in proteins. J. Biol. Chem. 239, 1411–1414. Bourne, N., Williams, A., Douglas, K. T., and Penkava, T. R. (1984) The E1cB mechanism for thiocarbamate ester hydrolysis: equilibrium and kinetic studies. J. Chem. Soc., Perkin Trans. 2 11, 1827– 1832. Pearson, P. G., Slatter, J. G., Rashed, M. S., Han, D. H., Grillo, M. P., and Baillie, T. A. (1990) S-(N-Methylcarbamoyl)glutathione: A reactive S-linked metabolite of methyl isocyanate. Biochem. Biophys. Res. Commun. 166, 245–250. Baillie, T. A., and Slatter, J. G. (1991) Glutathione: a vehicle for the transport of chemically reactive metabolites in vivo. Acc. Chem. Res. 24, 264–270. Reisser, M., Schmidt, B. F., and Brown, W. E. (2002) Synthesis, characterization, and solvolysis of mono- and bis-S-(glutathionyl) adducts of methylene-bis-(phenylisocyanate) (MDI). Chem. Res. Toxicol. 15, 1235–1241. Day, B. W., Ruhzi, J., Basalyga, D. M., Kramarik, J. A., and Karol, M. H. (1997) Formation, solvolysis, and transcarbamoylation reactions of bis(S-glutathionyl)adducts of 2 4-and 2,6-diisocyanotoluene. Chem. Res. Toxicol. 10, 424–431. Alvarez-Sańchez, R., Basketter, D., Pease, C., and Lepoittevin, J.-P. (2003) Studies of chemical selectivity of hapten, reactivity, and skin sensitization potency. 3. Synthesis and studies on the reactivity toward model nucleophiles of the 13C-labeled skin sensitizers, 5-chloro-2methylisothiazol-3-one (MCI) and 2-methylisothiazol-3-one (MI). Chem. Res. Toxicol. 16, 627–636. Iwakura, Y., and Okada, H. (1960) The kinetics of the tertiary-aminecatalyzed reaction of organic isocyanates with thiols. Can. J. Chem. 38, 2418–2424. Oh, H. K., Park, J. E., Sung, D. D., and Lee, I. (2004) Nucleophilic substitution reactions of aryl-N-phenyl thiocarbamates with benzylamines in acetonitrile. J. Org. Chem. 69, 3150–3153. Kimber, I., Basketter, D. A., and Roggeband, G. (2001) Chemical respiratory allergy: Classification and labeling. Toxicology 167, 159– 162. Ahlfors, S. R., Kristiansson, M. H., Lindh, C. H., Jo¨nsson, B. A. G., and Hansson, C. (2005) Adducts between nucleophilic amino acids and hexahydrophtalic anhydride, a structure inducing both types I and IV allergy. Biomarkers 10, 321–335. Dearman, R. J., Smith, S., Basketter, D. A., and Kimber, I. (1996) Classification of chemical allergens according to cytokine secretion profiles of murine lymph node cells. J. Appl. Toxicol. 17, 53–62.

TX9000539

Nuclear Magnetic Resonance Studies on Covalent Modification of

Recommend Documents