Characterization of Amino Acid and Glutathione Adducts of cis-2

Metabolic activation of the hepatocarcinogen furan yields metabolites that react covalently with proteins. cis-2-Butene-1,4-dial is a microsomal metab...
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Chem. Res. Toxicol. 1997, 10, 866-874

Characterization of Amino Acid and Glutathione Adducts of cis-2-Butene-1,4-dial, a Reactive Metabolite of Furan Ling-Jen Chen,† Stephen S. Hecht,‡ and Lisa A. Peterson* Division of Chemical Carcinogenesis, American Health Foundation, Valhalla, New York 10595 Received February 5, 1997X

Metabolic activation of the hepatocarcinogen furan yields metabolites that react covalently with proteins. cis-2-Butene-1,4-dial is a microsomal metabolite of furan. This reactive aldehyde is thought to be the toxic metabolite that is responsible for the carcinogenic activity of furan. In order to characterize the chemistry by which this unsaturated dialdehyde could alkylate proteins, the products formed upon reaction of cis-2-butene-1,4-dial with model nucleophiles in pH 7.4 buffer were investigated. NR-Acetyl-L-lysine (AcLys) reacts with cis-2-butene-1,4dial to form N-substituted pyrrolin-2-one adducts. N-Acetyl-L-cysteine (AcCys) reacts rapidly with cis-2-butene-1,4-dial to form multiple uncharacterized products. The inclusion of AcLys in this reaction mixture yielded an N-substituted 3-(S-acetylcysteinyl)pyrrole adduct which links the two amino acid residues. Related compounds were isolated when cis-2-butene-1,4dial and glutathione (GSH) were combined. In this case, cis-2-butene-1,4-dial cross-linked two molecules of GSH resulting in either cyclic or acyclic adducts depending on the relative GSH concentration. Incubation of furan with rat liver microsomes in the presence of [glycine2-3H]GSH led to the formation of radioactive peaks that coeluted with synthetic standards for the bisgluthathione conjugates. These studies demonstrate that the reactive cis-2-butene1,4-dial formed during the microsomal oxidation of furan reacts rapidly and completely with amino acid residues to form pyrrole and pyrrolin-2-one derivatives. Therefore, this metabolite is a likely candidate for the activated furan derivative that binds to proteins. The ease with which cis-2-butene-1,4-dial cross-links amino acids suggests that pyrrole-thiol cross-links may be involved in the toxicity observed following furan exposure.

Introduction Furan (1) is an industrial chemical. It is also present in many foods and beverages (1). Furan is both hepatotoxic and hepatocarcinogenic in rats and mice (2, 3). The carcinogenicity of furan does not appear to arise from direct genotoxicity since it apparently does not react with DNA (4). Furthermore, furan is inactive in Ames tests (5) and does not elicit DNA repair responses in livers of rats and mice (2). However, furan induces liver cell proliferation in experimental animals (2). These data are indicative of a nongenotoxic mechanism for furan carcinogenicity. Currently, the postulated mechanism of furan carcinogenesis involves activation to a chemically reactive metabolite. This reactive intermediate alkylates proteins to elicit a toxic response. This toxicity then stimulates extensive liver cell proliferation which could lead to liver tumor formation (2). Consistently, furan is activated to a protein-binding metabolite both in vivo and in vitro (4, 6). Inhibition and induction studies demonstrated that furan metabolism to a protein-binding intermediate is catalyzed by cytochrome P450 (7, 8). Glutathione (GSH) and, to a lesser extent, semicarbazide block the binding of [14C]furan to microsomal protein (6). In contrast, * To whom requests for reprints should be sent at: Division of Carcinogenesis and Molecular Epidemiology, American Health Foundation, 1 Dana Rd., Valhalla, NY 10595. † Current address: National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709. ‡ Current address: University of Minnesota Cancer Center, Box 806UMHC, 420 Delaware St. SE, Minneapolis, MN 55455. X Abstract published in Advance ACS Abstracts, July 15, 1997.

S0893-228x(97)00017-9 CCC: $14.00

Scheme 1. Proposed Metabolic Activation Pathway of Furan

N-acetyl-L-cysteine (AcCys)1 slightly enhances protein binding (6). Previously, we demonstrated that furan is metabolized by rat liver microsomes to cis-2-butene-1,4dial (2), which could be trapped by semicarbazide (Scheme 1) (9). Therefore, cis-2-butene-1,4-dial is a candidate for the ultimate toxin in furan-treated animals. R,β-Unsaturated dicarbonyl metabolites have been implicated in the metabolic activation of other furancontaining compounds. 4-Oxo-2-pentenal (acetylacrolein) and 2-methylbutene-1,4-dial are the major microsomal metabolites of 2-methylfuran and 3-methylfuran, respectively (10). Reagents that trap these reactive metabolites block the metabolism-dependent protein binding of these two chemicals (10, 11). Likewise, GSH and methoxylamine block the protein binding of the 5-lipoxygenase inhibitor (1S,5R)-3-cyano-1-(3-furyl)-6-{[6-[3-(3R-hydroxy6,8-dioxabicyclo[3.2.1]octanyl)]pyridin-2-yl]methoxyl}naphthalene (L-739,010) (12). Activation of the furan moiety in L-739,010 to 2-butene-1,4-dial is believed to be important for the protein-binding characteristics of this drug. In order to gain insight into the reactivity of cis-2butene-1,4-dial with amino acid residues, we studied the 1 Abbreviations: AcCys, N-acetyl-L-cysteine; AcLys, NR-acetyl-Llysine; 3-NBA, 3-nitrobenzyl alcohol; TFA, trifluoroacetic acid.

© 1997 American Chemical Society

Amino Acid and GSH Adducts of cis-2-Butene-1,4-dial

reactions between cis-2-butene-1,4-dial and several model nucleophiles: AcCys, NR-acetyl-L-lysine (AcLys), and GSH. The results demonstrate that cis-2-butene-1,4-dial rapidly reacts with both sulfhydryl and amino groups and can form cross-links between thiol- and amine-containing compounds.

Experimental Procedures Materials. Male F344 rats (200-400 g) were purchased from Charles River Laboratories (Kingston, NY). Glucose 6-phosphate, glucose 6-phosphate dehydrogenase, and NADP+ were obtained from Sigma Chemical Co. (St. Louis, MO). Furan, AcLys, AcCys, NR-acetyllysine methyl ester HCl, GSH (reduced form), KD2PO4, and K2DPO4 were purchased from Aldrich Chemical Co. (Milwaukee, WI). Furan was freshly distilled before use. Instrumental Analyses. 1H NMR spectra were acquired with a Bruker Model AM360 WB spectrometer and are reported in ppm relative to an external standard. Pyrrole and pyrrolin2-one proton coupling constants were determined in decoupling experiments. HPLC analyses were carried out with a Waters 510 system (Millipore, Waters Division, Milford, MA) connected to a Waters Model 990 photodiode array detector or β-Ram radioflow detector (IN/US Systems, Inc., Tampa, FL). Chemical ionization MS analyses were performed on a Hewlett Packard Model 5988A instrument. Electrospray MS analyses were obtained on a Finnigan Model TSQ 700 tandem quadrapole mass spectrometer with a LC interface. FAB MS were performed by the Washington University Resource for Biomedical and Bio-organic Mass Spectrometry, St. Louis, MO. HPLC Methods. Several different chromatographic systems were employed in these studies. In all cases, the reaction mixtures were eluted from a C18 column (Bondaclone 10, 300 × 3.9 mm; Phenomenex, Torrence, CA) at a flow rate of 1 mL/ min. System 1 utilized solvent A (20 mM NH4OAc, pH 6.8) and solvent B (95% MeOH, 5% H2O). The reaction mixtures were separated on the column using a gradient from 100% solvent A to 50% solvent A over 30 min. In this system, the retention times (min) of the compounds were as follows: 3 (14.5), 5 (16.0), 4 (16.5), and 6 (19.3). System 2 used the same gradient as above substituting 50 mM NH4OAc for solvent A. The retention times (min) of the compounds in this system were as follows: 3 (15.3), 4 (16.8), and 6 (19.3). System 3 used solvent C (50 mM NH4OAc, pH 5.5) and solvent D (CH3CN-H2O, 1:1). The reaction products were eluted with a linear gradient from 100% solvent C to 70% solvent C, 30% solvent D over 30 min and held isocratic for 5 min. The retention times (min) of the compounds in this system were as follows: 8 (12.8), 9 (12.8), 3 (14.5), 17a (15.3), 8 oxidized (16.2), 4 (16.5), 9 oxidized (16.2), 10 (17.6), 10 oxidized (20.8), and 6 (21.5). System 4 employed solvent E (50 mM NH4H2PO4, pH 2.5) and solvent D. A linear gradient from 100% solvent E to 60% solvent E, 40% solvent D over 40 min was used to elute the column. The retention times for the GSH adducts 8 and 9 were 29.2 and 28.3 min, respectively. In system 5, the reaction mixtures were separated using a linear gradient from 100% solvent E to 80% solvent E, 20% solvent D over 80 min. The retention times of 8 and 9 in this system were 62 and 58 min, respectively. General Procedure for Reactions of cis-2-Butene-1,4dial with Model Nucleophiles. cis-2-Butene-1,4-dial was prepared as previously described (9). Except where noted, this acetone solution (0.05 M, 1.6 mL) was immediately added to 1 mL of 0.1-0.4 M potassium phosphate buffer (pH 7.4), and the acetone was removed under reduced pressure prior to the addition of the model nucleophiles. (A) cis-2-Butene-1,4-dial with AcLys. AcLys (15.4 mg, 81.8 µmol) and cis-2-butene-1,4-dial (80 µmol) were incubated in 0.1 M potassium phosphate buffer (pH 7.4) at 37 °C for 1 h (total volume: 1 mL). Analysis using HPLC system 1 indicated the presence of two major UV-absorbing products with retention

Chem. Res. Toxicol., Vol. 10, No. 8, 1997 867 times of 14.5 and 16.5 min, respectively. These compounds were collected. The MeOH was removed under reduced pressure, and H2O and NH4OAc were lyophilized. The compound eluting at 14.5 min was identified as L-2-(acetylamino)-6-(2,5-dihydro-2oxo-1H-pyrrol-1-yl)-1-hexanoic acid (3): 1H NMR (DMSO-d6) δ 7.48 (d, J ) 7.67 Hz, 1H, Lys CONH), 7.25 (dt, J4,3 ) 5.94 Hz, J4,5 ) 1.54 Hz, 1H, C4-H), 6.06 (dt, J3,4 ) 5.94 Hz, J3,5 ) 1.81 Hz, 1H, C3-H), 4.01 (dd, J ) 1.81, 1.54 Hz, 2H, C5-H2), 3.91 (q, J ) 6.5 Hz, 1H, Lys R-CH), 3.27 (t, J ) 7.1 Hz, 2H, Lys -CH2), 1.80 (s, 3H, Lys COCH3), 1.75-1.65 (m, 1H, Lys β-CHa′), 1.651.5 (m, 1H, Lys β-CHb′), 1.5-1.4 (m, 2H, Lys δ-CH2), 1.25-1.1 (m, 2H, Lys γ-CH2); UV λmax 213.5, 236 nm. The compound eluting at 16.5 min was identified as L-2(acetylamino)-6-(2,3-dihydro-2-oxo-1H-pyrrol-1-yl)-1-hexanoic acid (4): 1H NMR (DMSO-d6) δ 6.68 (dt, J5,4 ) 4.87 Hz, J5,3 ) 2.21 Hz, 1H, C5-H), 5.25 (dt, J4,5 ) 4.85 Hz, J4,3 ) 2.29 Hz, 1H, C4H), 2.97 (t, J3,4 and J3,5 ) 2.24 Hz, 2H, C3-H); UV λmax 206, 253 nm. MS analysis of the 10:1 mixture of 3:4 positive ESI-MS/MS (rel intensity): m/z 255 (M + H, 40), 237 (M - OH, 35), 209 (M - COOH, 100), 167 (M - COOH, COCH3, 50). High-resolution positive FAB MS was performed using the methyl ester: calcd for C13H20N2O4, 269.1501; found, 269.1502. The incubation was repeated in 0.1 M potassium phosphate in D2O (pD 7.4), and the 1H NMR spectrum was observed following 1 h at 37 °C. 1H NMR analysis of the reaction mixture indicated that 4 was the initial product. The pyrrolin-2-one protons had undergone deuterium exchange with the solvent. There were only trace levels of cis-2-butene-1,4-dial hydrate and AcLys: 1H NMR (D2O) δ 6.5 (d, 0.3H, J ) 4.8 Hz, C5-H), 5.45 (d, J ) 4.8 Hz, 0.25H, C4-H), 4.1 (dd, J ) 8.9, 4.5 Hz, 1H, Lys R-CH), 3.4 (t, J ) 6.8 Hz, 1.2H, Lys -CH2), 1.9 (s, 3H, Lys COCH3), 1.8-1.6 (m, 1H, Lys β-CHa), 1.6-1.3 (m, 3H, Lys β-CHb, δ-CH2), 1.25-1.1 (m, 2H, Lys γ-CH2). (B) cis-2-Butene-1,4-dial with AcCys. (i) AcCys (13.1 mg, 80 µmol) was reacted with cis-2-butene-1,4-dial (80 µmol) in acetone (total volume: 1.6 mL) at room temperature for 1.5 h. Acetone was removed under reduced pressure, and the residue was extracted with CDCl3 for 1H NMR analysis. The spectrum showed the formation of a mixture of trans-2-butene-1,4-dial (9) and N-acetyl-S-2-furanyl-L-cysteine (5). This experiment was repeated using cis-2-butene-1,4-dial generated from dimethyldioxirane-d6 in acetone-d6. After 22 h at room temperature, the 1H NMR spectrum showed a mixture of trans-2-butene-1,4-dial and 5 in a 5:1 ratio. Trace amounts of unidentified reaction products were also observed. Compound 5 was purified using HPLC system 1: 1H NMR (CDCl3) δ 7.48 (dd, J5,4 ) 1.96 Hz, J5,3 ) 0.92 Hz, 1H, C5-H), 6.53 (dd, J3,4 ) 3.28 Hz, J3,5 ) 0.92 Hz, 1H, C3-H), 6.37 (dd, J4,3 ) 3.27 Hz, J4,5 ) 1.98 Hz, 1H, C4H), 4.79 (dt, J ) 7.5, 4.9 Hz, 1H, Cys R-CH), 3.28 (dd, J ) 14.3, 5.3 Hz, 1H, Cys β-CHa), 3.22 (dd, J ) 14.3, 4.3 Hz, 1H, Cys β-CHb), 1.99 (s, 3H, Cys COCH3); UV λmax 246 nm; positive ESIMS/MS (rel intensity) m/z 230 (M + H, 64), 215 (M - CH3, 49), 199 (M - CH3 - OH, 100), 188 (M - COCH3, 23), 162 (M furan, 28), 130 (M - thiofuran, 60). (ii) AcCys (80 mM) and cis-2-butene-1,4-dial (80 µmol) were incubated in 0.1 M potassium phosphate (pH 7.4) at 37 °C for 1 h (total volume: 1 mL). No new UV-absorbing products were observed using HPLC system 1 with photodiode-array detection. In some cases, semicarbazide HCl (180 µmol) was added. C18 HPLC analysis of the reaction mixtures was performed 20 h later using isocratic 5.5% CH3CN in H2O (9). Controls were performed in the absence of AcCys. The cis-2-butene-1,4-dial-AcCys reaction was repeated in 0.1 M deuterated potassium phosphate (pD 7.4) and analyzed by 1H NMR. The spectrum lacked the signals corresponding to starting materials and indicated the formation of several new products. However, the new signals were not identified. Trace amounts of 5 were detected (see above). (C) cis-2-Butene-1,4-dial with N-Acetyl-L-cysteine Methyl Ester. (i) N-Acetyl-L-cysteine methyl ester was prepared by dissolving AcCys (3.1 g, 0.019 mol) and p-toluenesulfonic acid monohydrate (193.4 mg, 1.02 mmol) in MeOH (30 mL). The

868 Chem. Res. Toxicol., Vol. 10, No. 8, 1997 MeOH solution was refluxed under air for 2.5 h. Saturated NaHCO3 aqueous solution was added, and the reaction mixture was extracted with ether (40 mL). The ether layer was dried over MgSO4 and concentrated to give a colorless liquid (92.7 mg, 2.8%): 1H NMR (CDCl3) δ 6.36 (br s, 1H, Cys CONH), 4.90 (dt, J ) 7.32, 4.07 Hz, 1H, Cys R-CH), 3.80 (s, 3H, Cys COOCH3), 3.02 (ddd, J ) 9.0, 3.87, 2.0 Hz, 2H, Cys β-CH2), 2.07 (s, 3H, Cys COCH3), 1.33 (t, J ) 9.0 Hz, 1H, Cys SH); positive ESI-MS (rel intensity) m/z 200 (M + Na+). (ii) N-Acetyl-L-cysteine methyl ester (13.5 mg, 76 µmol) was reacted with cis-2-butene-1,4-dial (80 µmol) in acetone (total volume: 1.6 mL) at room temperature overnight. The acetone was removed under reduced pressure, and the residue was extracted with CDCl3. 1H NMR analysis showed the formation of a mixture of trans-2-butene-1,4-dial and N-acetyl-S-2-furanylN-acetylcysteine methyl ester in a 2:1 ratio. N-Acetyl-S-2furanyl-L-cysteine methyl ester was purified by preparative TLC (1 mm silica gel plate, mobile phase ) 10% MeOH in CH2Cl2, Rf ) 0.55): 1H NMR (CDCl3) δ 7.50 (dd, J5,4 ) 1.96 Hz, J5,3 ) 0.93 Hz, 1H, C5-H), 6.52 (dd, J3,4 ) 3.28 Hz, J3,5 ) 0.92 Hz, 1H, C3-H), 6.38 (dd, J4,3 ) 3.13 Hz, J4,5 ) 1.98 Hz, 1H, C4-H), 4.87 (dt, J ) 7.71, 4.44 Hz, 1H, Cys R-CH), 3.67 (s, 3H, Cys COOCH3), 3.25 (d, J ) 4.28 Hz, 2H, Cys β-CH2), 1.99 (s, 3H, Cys COCH3); positive ESI-MS (rel intensity) m/z 266 (M + Na+, 100), 244 (M + H+, 40), 202 (M - COCH3, 45), 176 (M - furan, 15), 144 (M - thiofuran, 60). (D) cis-2-Butene-1,4-dial with AcLys and AcCys. AcLys (29.7 mg, 158 µmol) and AcCys (25.6 mg, 157 µmol) were added to cis-2-butene-1,4-dial (160 µmol) in 0.1 M potassium phosphate (pH 7.4, 2 mL). The reaction mixture was incubated at 37 °C for 1 h. Analysis using HPLC system 1 indicated the presence of one major UV-absorbing reaction product (retention time ) 19.3 min), which was collected. The MeOH was removed under reduced pressure, and H2O and NH4OAc were lyophilized. It was identified as N-acetyl-S-[1-[5-(acetylamino)-5-carboxypentyl]-1H-pyrrol-3-yl]-L-cysteine (6): 1H NMR (DMSO-d6) δ 7.83 (d, J ) 8.08 Hz, 1H, Cys CONH), 7.68 (d, J ) 7.63 Hz, 1H, Lys CONH), 6.82 (dd, J2,5 ) 2.10 Hz, J2,4 ) 1.41 Hz, 1H, C2-H), 6.67 (dd, J5,4 ) 2.57 Hz, J5,2 ) 2.10 Hz, 1H, C5-H), 6.00 (dd, J4,5 ) 2.57 Hz, J4,2 ) 1.41 Hz, 1H, C4-H), 4.07 (td, J ) 8.28, 4.49 Hz, 1H, Cys R-CH), 3.97 (q, J ) 6.31 Hz, 1H, Lys R-CH), 3.79 (t, J ) 6.54 Hz, 1H, Lys -CH2), 2.99 (dd, J ) 13.11, 4.34 Hz, 1H, Cys β-CHa), 2.67 (dd, J ) 13.00, 8.41 Hz, 1H, Cys β-CHb), 1.82 (s, 3H, COCH3), 1.81 (s, 3H, COCH3), 1.63-1.52 (m, 4H, Lys βand δ-CH2), 1.17-1.11(m, 2H, Lys γ-CH2); 1H NMR (D2O) after 20 min δ 6.83 (t, J ) 1.94 Hz, 0.2H, C2-H), 6.74 (d, J ) 2.7 Hz, 1H, C5-H), 6.15 (d, J ) 2.73 Hz, 0.8H, C4-H), 4.28 (dd, J ) 9.12, 4.09 Hz, 1H, Cys R-CH), 4.19 (dd, J ) 8.83, 4.94 Hz, 1H, Lys R-CH), 3.83 (td, J ) 6.61, 2.67 Hz, 2H, Lys -CH2), 3.11 (dd, J ) 14.00, 4.14 Hz, 1H, Cys β-CHa), 2.84 (dd, J ) 14.00, 9.17 Hz, 1H, Cys β-CHb), 1.95 (s, 3H, COCH3), 1.94 (s, 3H, COCH3), 1.781.60 (m, 4H, Lys β- and δ-CH2), 1.26-1.19 (m, 2H, Lys γ-CH2); positive DCI-MS (rel intensity) m/z 400 (M + H+, 50), 382 (M - OH, 10), 271 [M - CH2CH(NHCOCH3)COOH, 100]; highresolution positive FAB MS calcd for C17H26N3O6S 400.1542, found 400.1545. The effect of addition order on product distribution was investigated. In one experiment, AcLys (7.5 mg, 40 µmol) was incubated with cis-2-butene-1,4-dial (40 µmol) in 0.1 M potassium phosphate buffer (pH 7.4) at 37 °C. After 10 min, an aliquot (100 µL) was removed for HPLC analysis using HPLC system 3. AcCys (5.1 mg, 32 µmol) was added to the remaining solution and incubated at 37 °C for an additional 10 min. This solution was stored at 4 °C prior to HPLC analysis. The HPLC chromatogram of the sample removed prior to the addition of AcCys displayed the formation of 4 and 3 in approximately a 11:1 ratio (based on relative peak heights at 254 nm). This ratio changed upon the addition of AcCys, but there was no appearance of any new UV-absorbing compounds, such as 6. In the other experiment, AcCys (6.7 mg, 41 µmol) was incubated with cis-2-butene-1,4-dial (40 µmol) in 0.1 M potassium phosphate buffer (pH 7.4) at 37 °C. After 10 min, an aliquot (100 µL) was removed for HPLC analysis (system 3).

Chen et al. AcLys (6.2 mg, 33 µmol) was added to the remaining solution and incubated at 37 °C for an additional 10 min. This solution was stored at 4 °C prior to HPLC analysis. In this case, the HPLC chromatogram of the sample removed prior to the addition of AcLys contained no significant UV-absorbing peaks. The addition of AcLys led to the formation of a peak that eluted at 21 min, consistent with the formation of 6. (E) cis-2-Butene-1,4-dial with 1 equiv of GSH. GSH (50.4 mg, 164 µmol) and cis-2-butene-1,4-dial (160 µmol) were incubated in 0.1 M potassium phosphate (pH 7.4) at 37 °C for 1 h (total volume: 2 mL). Analysis using HPLC system 3 showed five UV-absorbing products with retention times 15.3, 18.1, 21.4, 24.0, and 26.1 min. These compounds were isolated. The compound eluting at 15.3 min was identified as a cyclic dimer, N-[4-carboxy-4-(3-mercapto-1H-pyrrol-1-yl)-1-oxobutyl]-L-cysteinylglycine cyclic (1f2′),(1′f2)-bis(sulfide) (7; see Chart 2): 1H NMR (D O) δ 6.88 (t, J 2 2,4 ) 1.77 Hz, 1H, C2-H), 6.80 (t, J5,4 ) 2.53 Hz, 1H, C5-H), 6.27 (dd, J4,5 ) 2.64 Hz, J4,2) 1.75 Hz, 1H, C4-H), 4.53 (dd, J ) 11.21, 4.81 Hz, 1H, Glu R-CH), 4.29 (dd, J ) 9.98, 3.93 Hz, 1H, Cys R-CH), 3.64 (s, 2H, Gly R-CH2), 3.14 (dd, J ) 14.24, 4.01 Hz, 1H, Cys β-CHa), 2.79 (dd, J ) 14.24, 10.12 Hz, 1H, Cys β-CHb), 2.43 (m, 1H, Glu β-CHa), 2.24 (m, 1H, Glu γ-CHa), 2.13 (m, 1H, Glu β-CHb), 1.96 (m, 1H, Glu γ-CHb); FAB MS [3-nitrobenzyl alcohol (3-NBA)/glycerol/trifluoroacetic acid (TFA) matrix] m/z 711.4 (M + H+); highresolution FAB MS calcd for C28H35N6O12S2 711.1754, found 711.1771; UV λmax 228.4 nm. The compound eluting at 18.1 min was assigned the structure of the cyclic GSH-cis-2-butene-1,4-dial trimer, N-[4-carboxy4-(3-mercapto-1H-pyrrol-1-yl)-1-oxobutyl]-L-cysteinylglycine cyclic (1f2′),(1′f2′′),(1′′f2)-tris(sulfide): 1H NMR (D2O) δ 6.9 (br s, 1H, C2-H), 6.76 (t, J ) 2.44 Hz, 1H, C5-H), 6.19 (t, J ) 1.90 Hz, 1H, C4-H), 4.48 (dd, J ) 9.91, 4.66 Hz, 1H, Glu R-CH), 4.36 (dd, J ) 9.66, 4.11 Hz, 1H, Cys R-CH), 3.65 (s, 2H, Gly CH2), 3.12 (dd, J ) 14.00, 4.25 Hz, 1H, Cys β-CHa), 2.80 (dd, J ) 13.99, 9.91 Hz, 1H, Cys β-CHb), 2.6-2.3 (m, 1H, Glu β-CHa), 2.3-2.0 (m, 3H, Glu β-CHb and Glu γ-CH2); ESI-MS m/z 532 (M-/2), 354 (M-/3) indicating MW 1066; UV λmax 221 nm. The peak eluting at 21.4 min was identified as the cyclic GSH-cis-2-butene-1,4-dial tetramer, N-[4-carboxy-4-(3-mercapto1H-pyrrol-1-yl)-1-oxobutyl]-L-cysteinylglycine cyclic (1f2′),(1′f2′′),(1′′f2′′′),(1′′′f2)-tetrakis(sulfide): 1H NMR (D2O) δ 6.88 (br s, 1H, C2-H), 6.75 (br s, 1H, C5-H), 6.20 (br s, 1H, C4-H), 4.46 (m, 1H, Glu R-CH2), 4.32 (br dd, J ) 9.25, 3.58 Hz, 1H, Cys R-CH), 3.65 (s, 2H, Gly CH2), 3.10 (br dd, J ) 14.06, 9.95 Hz, 1H, Cys β-CHa), 2.77 (br dd, J ) 13.68, 9.95 Hz, 1H, Cys β-CHb), 2.5-2.3 (m, 1H, Glu β-CH2), 2.3-2.1 (m, 3H, Glu β-CHb and γ-CH2); negative ESI-MS m/z 711.5 (M-/2) indicating MW 1424; UV λmax 218.5 nm. The peak eluting at 24.0 min was identified as the cyclic GSH-cis-2-butene-1,4-dial pentamer, N-[4-carboxy-4-(3-mercyclic capto-1H-pyrrol-1-yl)-1-oxobutyl]-L-cysteinylglycine (1f2′),(1′f2′′),(1′′f2′′′),(1′′′f2′′′′),(1′′′′f2)-pentakis(sulfide): 1H NMR (D2O) δ 6.90 (br s, 1H, C2-H), 6.77 (br s, 1H, C5-H), 6.17 (br s, 1H, C4-H), 4.69 (br s, 1H, Glu R-CH2), 4.32 (br s, 1H, Cys R-CH), 3.65 (br s, 2H, Gly CH2), 3.12 (br s, 1H, Cys β-CHa), 2.76 (m, 1H, Cys β-CHb), 2.5-2.3 (m, 1H, Glu β-CHa), 2.3-2.1 (m, 3H, Glu β-CHb and γ-CH2); negative ESI-MS m/z 591.1 (M-/3), 443.1 (M-/4) indicating MW 1776; UV λmax 218.5 nm. (F) cis-2-Butene-1,4-dial with 10 equiv of GSH. GSH (246.6 mg, 802 µmol) and cis-2-butene-1,4-dial (80 µmol) were incubated in 0.1 M potassium phosphate (pH 7.4) at 37 °C for 1 h (total volume: 10 mL). Analysis using HPLC system 3 indicated the formation of four major products (retention times ) 12.8, 16.2, 17.6, and 20.8 min) which were collected. Reinjection of the peak eluting at 12.8 min using HPLC system 4 revealed that it was a mixture of two compounds with retention times 28.3 and 29.2 min. The compound that eluted at 29.2 min was identified as L-γ-glutamyl-L-cysteinylglycine (2f1′)sulfide with N-[4-carboxy-4-(3-mercapto-1H-pyrrol-1-yl)-1-oxobutyl]-L-cysteinylglycine (8): 1H NMR (D2O) δ 6.91 (dd, J2,5 ) 2.13 Hz, J2,4 ) 1.44 Hz, 1H, C2-H), 6.82 (dd, J5,4 ) 2.67 Hz, J5,2 ) 2.13 Hz, 1H, C5-H), 6.22 (dd, J4,5 ) 2.67 Hz, J4,2 ) 1.44 Hz,

Amino Acid and GSH Adducts of cis-2-Butene-1,4-dial 1H, C4-H), 4.52-4.47 (m, 2H, Glu′ R-CH and Cys′ R-CH), 4.35 (dd, J ) 10.04, 4.19 Hz, 1H, Cys R-H), 3.77-3.73 (m, 5H, Gly′ R-CH2, Gly R-CH2, and Glu R-CH), 3.18 (dd, J ) 14.12, 4.24 Hz, 1H, Cys β-CHa), 2.92-2.81 (m, 3H, Cys β-CHb and Cys′ β-CH2), 2.57-2.43 (m, 2H, Glu γ-CH2), 2.43-2.37 (m, 1H, Glu′ γ-CHa), 2.26-2.21 (m, 3H Glu′ γ-CHb and Glu′ β-CH2), 2.13 (q, J ) 7.23 Hz, 2H, Glu β-CH2); UV λmax 223.4 nm in HPLC system 3 and λmax 213.5 nm in HPLC system 4. The compound that eluted at 28.3 min was identified as L-γglutamyl-L-cysteinylglycine (2f1′)-sulfide with N-[4-carboxy-4(2-mercapto-1H-pyrrol-1-yl)-1-oxobutyl]-L-cysteinylglycine (9): 1H NMR (D O) δ 7.04 (dd, J 2 5,4 ) 2.85 Hz, J5,3 ) 1.78 Hz, 1H, C5-H), 6.42 (dd, J3,4 ) 3.55 Hz, J3,5 ) 1.64 Hz, 1H, C3-H), 6.25 (dd, J4,3 ) 2.85 Hz, J4,5 ) 2.85 Hz, 1H, C4-H), 5.02 (dd, J ) 10.94 Hz, 1H, Glu′ R-H), 4.46 (t, J ) 6.00 Hz, 1H, Cys′ R-H), 4.39 (dd, J ) 9.35, 3.40 Hz, 1H, Cys R-H), 3.71-3.67 (m, 5H, Gly′ R-CH2, Gly R-CH2, and Glu R-CH), 2.95 (dd, J ) 14.07, 3.60 Hz, 1H, Cys β-CHa), 2.86-2.80 (m, 3H, Cys β-CHb and Cys′ β-CH2), 2.55-2.47 (m, 1H, Glu′ γ-CHa), 2.41-2.63 (m, 2H, Glu γ-CH2), 2.32-2.16 (m, 3H, Glu′ γ-CHb and Glu′ β-CH2), 2.102.02 (m, 2H, Glu β-CH2); UV λmax 218.5, 248.3 nm in HPLC system 3 and λmax 201.2, 248.3 nm in HPLC system 4; FAB MS of the mixture of 8 and 9 (matrix, 3-NBA/glycerol/TFA) m/z 663.2 (M + H+); high-resolution FAB MS calcd for C24H35N6O12S2 663.1754, found 663.1762; negative ESI-MS/MS (rel intensity) m/z 661 (M - 1, 100), 387 (M - C12H16N3O6, 53). The peak at 17.6 min was assigned the structure L-γglutamyl-L-cysteinylglycine (2f1′),(2f1′′)-bis(sulfide) with N-[4carboxy-4-(3-mercapto-1H-pyrrol-1-yl)-1-oxobutyl]-L-cysteinylglycine (10; see Chart 2): 1H NMR (D2O) δ 6.94 (t, J ) 1.86 Hz, C2-H), 6.91 (t, J ) 1.86 Hz, C2-H), 6.85 (t, J ) 2.55 Hz, C5-H), 6.80 (t, J ) 2.50 Hz, C5-H), 6.23 (dd, J ) 2.59, 1.75 Hz, C4-H), 6.21 (dd, J ) 2.75, 1.60 Hz, C4-H), 4.52-4.47 (m, 3H, Cys′′ R-CH, Glu′ R-CH, and Glu′′ R-CH), 4.40-4.35 (m, 2H, Cys′ R-CH and Cys R-CH), 3.76-3.67 (m, 7H, Gly R-CH2, Gly′ R-CH2, Gly′′ R-CH2, and Glu R-CH), 3.22-3.13 (m, 2H, Cys′ β-CHa and Cys β-CHa), 2.92-2.80 (m, 4H, Cys′′ β-CH2, Cys′ β-CHb, and Cys β-CHb), 2.54-2.37 (m, 4H, Glu γ-CH2, Glu′′ γ-CHa, and Glu′ γ-CHa), 2.26-2.09 (m, 8H, Glu′′ γ-CHb, Glu′ γ-CHb, Glu β-CH2, Glu′′ β-CH2, and Glu′ β-CH2); negative ESI-MS m/z 1016 (M 1). The relative formation of 8 and 9 was determined in reaction mixtures containing cis-2-butene-1,4-dial (2 mM) and [glycine2-3H]GSH (0.7 µCi, 4, 20, or 200 mM) in 0.1 M potassium phosphate buffer (pH 7.4, 1 mL). After 1 h at 37 °C, the reaction mixtures were analyzed by HPLC system 5 with a radioflow detector. Incubation of Furan with Rat Liver Microsomes in the Presence of [glycine-2-3H]GSH. F-344 rat liver microsomal fractions were prepared by the method of Guengerich et al. (13). Duplicate incubations of furan (2 mM) with rat liver microsomes (1 mg/mL) were conducted in 1 mL of 100 mM potassium phosphate buffer (pH 7.4) in the presence of 25 mM glucose 6-phosphate, glucose-6-phosphate dehydrogenase (2 units/mL), 4 mM NADP+, 3 mM MgCl2, 1 mM EDTA, and 4 mM [glycine2-3H]GSH (specific activity ) 0.166 µCi/µmol). Furan was added as an ethanol solution (10 µL). Controls were performed in the absence of NADP+, microsomes, or furan. After 30 min incubation at 37 °C in capped vials, reactions were terminated by addition of 0.3 N Ba(OH)2 and 0.3 N ZnSO4 (0.1 mL each). Following centrifugation, the supernatant was filtered through an Acrodisc (Gelman, Ann Arbor, MI; 0.45 µm, 3 mm) and analyzed directly by HPLC system 5 with UV (225 nm) and radioflow detection. A larger scale incubation (20 mL) of furan (20 mM) in the presence of 4 mM GSH and the required cofactors was conducted to characterize the furan-GSH conjugates. They were purified by HPLC using two different HPLC gradients. Initial isolation of 8 and 9 was acheived by using 0.06% TFA with a linear gradient over 40 min to 12.5% CH3CN. Under these conditions, 8 eluted at 28.6 min and 9 eluted at 27.6 min. These compounds were collected as one fraction, concentrated by lyophilization and repurified using HPLC system 3. Spectral properties: 1H

Chem. Res. Toxicol., Vol. 10, No. 8, 1997 869

Figure 1. HPLC traces (system 1) of the cis-2-butene-1,4-dial (80 mM)-AcLys (82 mM) reaction mixture in 0.1 M potassium phosphate buffer (pH 7.4) after (A) 1 h, (B) 2 h, and (C) 3 h at 37 °C and (D) overnight at room temperature. NMR (D2O) δ 6.9 (t, J ) 1.90 Hz, 1H, C2-H), 6.8 (t, J ) 2.83 Hz, 1H, C5-H), 6.2 (t, J ) 2.24 Hz, 1H, C4-H), 4.5-4.4 (m, 2H, Glu′ R-CH and Cys′ R-CH), 4.35 (dd, J ) 10.36, 4.14 Hz, 1H, Cys R-H), 3.7-3.6 (m, 5H, Gly′ R-CH2, Gly R-CH2, and Glu R-CH), 3.17 (dd, J ) 14.05, 4.01 Hz, 1H, Cys β-CHa), 2.91-2.82 (m, 3H, Cys β-CHb and Cys′ β-CH2), 2.55-2.42 (m, 2H, Glu γ-CH2), 2.42-2.35 (m, 1H, Glu′ γ-CHa), 2.30-2.15 (m, 3H, Glu′ β-CH2 and Glu′ γ-CHb), 2.15-2.05 (q, J ) 7.24 Hz, 2H, Glu β-CH2); negative ESI-MS m/z 661 (M - 1).

Results Reaction of cis-2-Butene-1,4-dial with AcLys. Incubation of cis-2-butene-1,4-dial and AcLys in equimolar amounts at pH 7.4 led to the formation of two UV absorbing compounds (Figure 1). HPLC analysis of this reaction mixture at various times indicated that the relative ratio of these compounds changed with incubation time. The compound eluting at 16.2 min was initially predominant, whereas the compound eluting at 14.5 min was the major product at equilibrium. Collection and reinjection of the peak eluting at 14.5 min indicated that the two compounds were interconvertible. CIMS analysis of the material that eluted at 14.5 min yielded a molecular ion at m/z 255, which corresponds to the formation of a 1:1 adduct between cis-2-butene1,4-dial and AcLys minus H2O. 1H NMR analysis of this fraction demonstrated the presence of a 10:1 equilibrium ratio of two compounds with ring proton signals at 7.25, 6.06, and 4.01 ppm for the major isomer and 6.68, 5.25, and 2.97 ppm for the minor isomer. The chemical shifts of the major isomer (Table 1) were similar to those reported for N-methyl-3-pyrrolin-2-one, while those of the minor isomer were similar to those for N-methyl-4pyrrolin-2-one (14). The spectrum also contained signals attributable to AcLys, but there were no distinguishable

870 Chem. Res. Toxicol., Vol. 10, No. 8, 1997 Table 1. 1H NMR of N-Alkylpyrrolin-2-ones

a

In DMSO-d6. b In CDCl3 (14).

Chart 1. Adducts Formed from Reaction of AcLys and/or AcCys with cis-2-Butene-1,4-dial

differences in chemical shifts of these protons for the two isomers. Consistent with the involvement of the Namino group in the pyrrolin-2-one ring was the downfield shift of the methylene protons attached to the -carbon (3.27 ppm) relative to unadducted AcLys. On the basis of these data, the material that eluted at 14.5 min was identified as L-2-(acetylamino)-6-(2,5-dihydro-2-oxo-1Hpyrrol-1-yl)-1-hexanoic acid (3) and the compound that eluted at 16.2 min was identified as L-2-(acetylamino)6-(2,3-dihydro-2-oxo-1H-pyrrol-1-yl)-1-hexanoic acid (4) (Chart 1). These results were confirmed when the reaction was monitored directly in deuterated phosphate buffer (pD 7.4) with 1H NMR analysis. After 1 h at 37 °C, the reaction mixture contained primarily 4. Relative amounts of isomers 3 and 4 were difficult to determine by 1H NMR analysis since a substantial amount of exchange had occurred in the pyrrolidone ring. However, a comparison of the relative intensity of the signals assigned to the C4 proton of isomer 3 vs 4 indicated that the rate of isomerization of the double bond was not significantly

Chen et al.

affected by the deuterated buffer. Only trace amounts of unreacted cis-2-butene-1,4-dial hydrate and AcLys were detected in the 1H NMR spectrum. Reaction of cis-2-Butene-1,4-dial with AcCys. When AcCys was reacted with cis-2-butene-1,4-dial in acetone-d6, the 1H NMR spectrum indicated the formation of trans-2-butene-1,4-dial (9) and a new compound in a 5:1 ratio; only trace amounts of cis-2-butene-1,4-dial were observed. The unknown compound was purified using HPLC system 1. The 1H NMR spectrum contained three signals in the aromatic region (7.48, 6.53, and 6.37 ppm). The chemical shifts and coupling constants, as shown in Table 2, were similar to those previously reported for 2-substituted furans (15). The appearance of only one furanyl signal downfield (7.48 ppm) from the other two furanyl protons indicated that the substitution was in the 2-position. These signals integrated 1:1 with the signal assigned to R-methine of AcCys (4.79 ppm). The signals assigned to the β-methylene group of AcCys (3.28 and 3.22 ppm) were shifted downfield relative to unadducted AcCys (2.8-2.9 ppm). Therefore, the new compound was identified as N-acetyl-S-2-furanyl-L-cysteine (5; Chart 1). When N-acetylcysteine methyl ester was used in these reactions, a 2:1 mixture of trans-2butene-1,4-dial and N-acetyl-S-2-furanyl-L-cysteine methyl ester was observed. When cis-2-butene-1,4-dial was allowed to react with AcCys in deuterated buffer and the reaction monitored by 1H NMR, the disappearance of starting materials was observed. The presence of new signals indicated that multiple products were formed. However, no trans-2butene-1,4-dial and very little 5 were formed. The HPLC trace of a similar reaction mixture lacked any significant new UV-absorbing peaks, indicating that the reaction products were not stable to HPLC purification. AcCys did react with the aldehyde groups of cis-2-butene-1,4dial since its presence blocked semicarbazone formation (data not shown). Reaction of cis-2-Butene-1,4-dial with AcCys and AcLys. o-Phthalaldehyde (OPA) reacts with thiols and amines to generate 1,2-substituted isoindole products (16). We explored the possibility that the structurally related cis-2-butene-1,4-dial would yield similar products when reacted with primary amines and thiols. An equimolar mixture of AcCys and AcLys was added to a solution of cis-2-butene-1,4-dial in phosphate buffer (pH 7.4) solution. After 1 h at 37 °C, a major product was observed by HPLC analysis with photodiode-array detection. Trace amounts of 3 and 4 were also formed. The new compound was identified as R-(acetylamino)-[3-(Nacetyl-L-cystein-S-yl)-1H-pyrrolyl]-1-hexanoic acid (6; Chart 1) based on the following spectral data. Positive DCIMS analysis of the purified compound yielded a molecular ion at m/z 400. This is consistent with the formation of an adduct containing one molecule each of cis-2-butene1,4-dial, AcLys, and AcCys with the loss of two H2O. The 1 H NMR spectrum displayed three peaks in the aromatic region (6.82, 6.67, and 6.00 ppm), consistent with a substituted pyrrole (17). The chemical shifts of the aromatic protons were consistent with those expected for a 3-substituted pyrrole (Table 2). The signal assigned to the -Lys methylene protons appeared at 3.78 ppm, as expected for pyrrole-N-CH2R (17). The aromatic signals integrated 1:1 with the two amide protons (7.83 and 7.68 ppm) and the R-methine protons of AcCys (4.07 ppm) and AcLys (3.97 ppm). The diastereotopic β-meth-

Amino Acid and GSH Adducts of cis-2-Butene-1,4-dial

Chem. Res. Toxicol., Vol. 10, No. 8, 1997 871

Table 2. 1H NMR Chemical Shifts and Coupling Constants for Ring Protons of 2- and 3-Substituted Pyrroles and Furans

a

CDCl3. b In acetone-d6. c In DMSO-d6.

d

In D2O. e See Chart 1 for full structure. f See Chart 2 for full structure.

ylene protons of cysteine were distinct signals (2.99 and 2.67 ppm), consistent with the adduction at the thiol group. The order of addition of the two nucleophiles in this reaction mixture determined the reaction products. When AcLys was added first, the 3- and 4-pyrrolin-2-one isomers 3 and 4 were formed. The presence of AcCys increased the rate of isomerization of 4 to 3 and also appeared to reduce the product yield, as indicated by HPLC analysis of the reaction mixture (data not shown). Pyrrole 6 was not detected. When AcCys was added prior to AcLys, 6 was the major product observed by HPLC analysis. Only trace amounts of the 3- and 4-pyrrolin2-one derivatives were detected. Reaction of cis-2-Butene-1,4-dial with GSH. Incubation of equimolar amounts of cis-2-butene-1,4-dial

and GSH (80 mM) in phosphate buffer (pH 7.4) at 37 °C for 1 h resulted in the formation of several products. The HPLC chromatogram (system 3) with detection at 225 nm indicated the formation of five new compounds (Figure 2A). The 1H NMR spectra of the purified compounds, which eluted at 15.3, 18.1, 21.4, 23.9, and 26.0 min, were almost identical, except with increasing line broadening. These adducts contained pyrrole rings and were composed of equal amounts of cis-2-butene-1,4dial and GSH. The 1H NMR spectral data supported the conclusion that the R-amino group of GSH reacted with cis-2-butene-1,4-dial to form a pyrrole with a GSH sulfhydryl group in the 3-position (Table 2). MS analysis indicated that these compounds were macrocyclic adducts with increasing ring size, with the smallest compound being a dimer (7; Chart 2).

872 Chem. Res. Toxicol., Vol. 10, No. 8, 1997

A

Chen et al. Chart 2. Adducts Isolated from Reactions of GSH and cis-2-Butene-1,4-dial

B

Figure 2. Representative HPLC chromatograms (UV detection at 225 nm, system 3) obtained for reaction mixtures of (A) 1:1 GSH (82 mM):cis-2-butene-1,4-dial (80 mM) and (B) 10:1 GSH (80 mM):cis-2-butene-1,4-dial (8 mM) in 0.1 M potassium phosphate buffer (pH 7.4) following incubation for 1 h at 37 °C.

These reactions were repeated with a 10-fold excess of GSH, since GSH should be in large excess in the biological situation. The macrocyclic GSH adducts were not observed under these conditions. An HPLC chromatogram of the reaction mixture (detection at 225 nm using system 3) contained peaks with retention times 6, 12.8, 16.2, and 17.6 min (Figure 2B). The peak eluting at 12.8 min was identified as a mixture of 8 and 9 (Chart 2), based on the following data. The 1H NMR indicated a pyrrole-containing adduct composed of cis-2-butene-1,4dial and GSH in a 1:2 ratio. FAB MS showed a molecular ion at m/z 663. These data are consistent with an adduct formed by reaction of one molecule of cis-2-butene-1,4dial and two molecules of GSH followed by the loss of two H2O. The 1H NMR spectrum displayed three signals at 6.91, 6.82, and 6.22 ppm, indicating the presence of a 3-substituted pyrrole moiety (Table 2). One of the cysteine R-methine protons (4.35 ppm) was coupled to a β-methylene group that resonated as two distinct signals, consistent with alkylation of the thiol group (18). The other cysteine signals had chemical shifts similar to unadducted GSH. The R-methine of one glutamate appeared at 4.5 ppm, indicating the involvement of the GSH amino group in a pyrrole ring. The 1H NMR spectrum of the 12.8 min peak also showed a minor product that had signals at 7.04, 6.42, and 6.25 ppm. These chemical shifts and the corresponding coupling constants were similar to a 2-substituted pyrrole (Table 2). These two positional isomers could be separated from one another using HPLC system

4. These compounds were purified, and structural assignments were confirmed by 1H NMR analysis. Reanalysis of the isolated bis-GSH conjugates by HPLC system 3 indicated that they could be converted into the compound that eluted at 16.2 min. NMR analysis of this peak demonstrated that it contained the oxidized forms of the two bis conjugates as indicated by the alteration of the coupling pattern and chemical shifts of the β-methylene of the unalkylated Cys. Similar changes were observed upon the oxidation of GSH to GSSG (19). 1H NMR analysis of the compound that eluted at 17.6 min indicated the presence of two molecules of pyrrole and three molecules of GSH. This compound was assigned structure 10 (Chart 2). Microsomal Metabolism of Furan in the Presence of [glycine-2-3H]GSH. In order to see which GSH conjugate would be formed, furan (2 mM) was incubated with microsomes in the presence of cofactors and 4 mM [glycine-2-3H]GSH. These conditions were comparable to those reported by Parmar and Burka (6). HPLC analysis indicated the formation of radioactive metabolites that coeluted with standards for GSH adducts 8 and 9. The formation of the GSH adducts required the presence of NADPH, GSH, and microsomes. The structural assignments were confirmed by preparative isolation of these metabolites. Their NMR properties were identical to the standards. The yield of the bis-GSH conjugates 8 and 9 was 15 ( 1 nmol/mL. This compares

Amino Acid and GSH Adducts of cis-2-Butene-1,4-dial

Figure 3. Representative HPLC radiogram (system 5) obtained upon incubation of 2 mM furan with rat liver microsomes (1 mg/mL) in the presence of 4 mM [glycine-2-3H]GSH and required P450 cofactors for 30 min at 37 °C.

Scheme 2. Proposed Mechanisms of Reaction between cis-2-Butene-1,4-dial and Thiols and/or Amines

well to the amount of cis-2-butene-1,4-dial bis-semicarbazone detected in microsomal incubations of 2 mM furan when semicarbazide was the trapping agent (16 ( 2 nmol/mL) (9). The ratio of adduct 8:9 was 4:1 (Figure 3). A similar ratio (5:1) was observed when cis-2-butene1,4-dial (2 mM) was reacted with [glycine-2-3H]GSH (4 or 20 mM) at pH 7.4. A 10:1 ratio of 8:9 was observed when the GSH concentration was 100 times that of cis2-butene-1,4-dial. No evidence for production of cyclic adducts such as 7 was obtained.

Discussion The results demonstrate that cis-2-butene-1,4-dial rapidly reacted with amino acids to form pyrrole-related compounds. The major products formed by reaction with AcLys were 3 and 4 (Chart 1). The initial product was 4 which rearranged to 3. This type of rearrangement is similar to what has been reported for other ∆3- and ∆4N-alkylpyrrolin-2-ones (14, 20). The reactions with AcCys did not yield isolable products when performed in H2O. However, the reaction in acetone yielded trans-2-butene-1,4-dial and N-acetyl-S2-furanyl-L-cysteine. These results indicate that the thiol adds reversibly to cis-2-butene-1,4-dial via either 1,2addition to the aldehyde group, leading to N-acetyl-S-2furanyl-L-cysteine, or 1,4-addition to the double bond (Scheme 2). 1,4-Addition caused isomerization to trans2-butane-1,4-dial. This isomerization was not observed

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in aqueous solutions. In H2O, cis-2-butene-1,4-dial exists as a cyclic hydrate (9) which probably stabilizes the cisorientation. Thiol adducts could be trapped as the corresponding pyrroles when an amine was present (Scheme 2). Incubation of cis-2-butene-1,4-dial with either GSH or a mixture of AcCys and AcLys led to the formation of predominately 3-(thioalkyl)pyrroles, such as 6-10, where the pyrrole nitrogen came from either GSH or AcLys. In the case of the GSH reactions, the 2-substituted pyrrole was also observed. The mechanism for formation of these products appears to involve initial 1,4- or 1,2-addition of the thiol to cis-2-butene-1,4-dial. This is followed by reaction of the amino group with the free aldehyde and subsequent rearrangement and dehydration to form the pyrrole adducts. The initial adduction of the thiol group to the unsaturated system is comparable to the mechanism of OPA-mediated cross-linking of amine and thiol groups to yield 1,2-substituted isoindole adducts (16, 21). The proposal that rapid addition of the thiol to cis-2butene-1,4-dial occurs prior to the reaction of the amine is supported by the inability of N-alkylpyrrolin-2-one derivatives 3 and 4 to form the 3-substituted pyrrole derivatives upon addition of thiol. However, the N-alkylpyrrolin-2-one compounds are still potentially reactive. Michael addition of a nucleophile would produce 4-substituted 1-alkylpyrrolidin-2-one derivatives (22). Further studies are planned to investigate this possibility. The formation of the GSH conjugates in GSH-supplemented microsomal incubations of furan provides further evidence for the intermediacy of cis-2-butene-1,4-dial in the activation of furan (9). It is likely that these conjugates are formed in vivo. Disposition studies using [14C]furan indicated that 25% of the dose was excreted in the urine and 20% in the feces. HPLC analysis of the urine demonstrated the presence of several polar metabolites which could be GSH adducts or mercapturic acid derivatives (4). Furan depletes GSH in hepatocytes (7). cis-2-Butene-1,4-dial could be involved in this toxic response since one molecule of cis-2-butene-1,4-dial reacts with two molecules of GSH. Further studies in our laboratory will explore whether this conjugative pathway occurs in vivo. The chemical studies described in this report also help explain why GSH, but not AcCys, effectively blocks protein binding of furan (6). In fact, AcCys modestly enhanced protein binding. We found that in the absence of a free amino group, thiols can react with cis-2-butene1,4-dial to form unstable adducts that still retain reactivity toward nucleophiles. The addition of an amine group then allows formation of stable pyrrole adducts. Since GSH contains both amine and sulfhydryl moieties, it is an effective trap for cis-2-butene-1,4-dial. On the other hand, AcCys increases the protein binding of the furan metabolite by activating the metabolite for reaction with protein amine residues to form relatively stable pyrrole adducts. It is likely that the adducts formed upon furan activation are thiol-amine pyrrole cross-links. Crosslinked pyrrole adducts have been implicated in the neurotoxicity of hexane (18, 23, 24). Future studies will investigate the chemical nature of the furan protein adducts in order to determine the involvement of cis-2butene-1,4-dial in the activation of this carcinogen in vivo. Note: The nomenclature used for the GSH compounds was chosen based on discussions with Dr. Warren Powell at Chemical Abstracts Service (Columbus, OH). We used

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peptide names based on current CAS policies. The numbers indicating the sulfide connections are the numbers assigned to the amino acids in peptides. The “2” refers to the second amino acid (cysteine) of the primary chain (GSH) and the “1′“ refers to the systematically named first “amino acid” (for example, N-[4-carboxy4-(3-mercapto-1H-pyrrol-1-yl)-1-oxobutyl]) of the secondary peptide chain. The peptide chain with the sulfide attachment to the pyrrole was chosen as the primary peptide. The peptide containing the group attached to the pyrrole nitrogen is the minor peptide.

Acknowledgment. We would like to thank the AHF Research Animal Facility for isolation of tissues and Mr. Dennis Ng for the preparation of microsomes. Highresolution MS analyses were performed by the Washington University Resource for Biomedical and Bioorganic Mass Spectrometry, St. Louis, MO. This study was supported by Grant CA-44377 from the National Cancer Institute. The Research Animal Facility and the Instrument Facility at AHF are partially supported by National Cancer Institute Cancer Center Support Grant CA-17613.

References (1) Maga, J. (1979) Furans in foods. Crit. Rev. Food Sci. Nutr. 11, 355-366. (2) Wilson, D. M., Goldsworthy, T. L., Popp, J. A., and Butterworth, B. E. (1992) Evaluation of genotoxicity, pathological lesions, and cell proliferation in livers of rats and mice treated with furan. Environ. Mol. Mutagen. 19, 209-222. (3) National Toxicology Program. (1993) Toxicology and carcinogenesis studies of furan in F344/N rats and B6C3F1 mice, NTP Technical Report No. 402, U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC. (4) Burka, L. T., Washburn, K. D., and Irwin, R. D. (1991) Disposition of [14C]furan in the male F344 rat. J. Toxicol. Environ. Health 34, 245-257. (5) Mortelmans, K., Haworth, S., Lawlor, T., Speck, W., Tainer, B., and Zeiger, E. (1986) Salmonella mutagenicity tests. II. Results from the testing of 270 chemicals. Environ. Mutagen. 7 (Suppl.), 1-119. (6) Parmar, D., and Burka, L. T. (1993) Studies on the interaction of furan with hepatic cytochrome P-450. J. Biochem. Toxicol. 8, 1-9. (7) Carfagna, M. A., Held, S. D., and Kedderis, G. L. (1993) Furaninduced cytolethality in isolated rat hepatocytes: Correspondence with in vivo dosimetry. Toxicol. Appl. Pharmacol. 123, 265-273. (8) Kedderis, G. L., Carfagna, M. A., Held, S. D., Batra, R., Murphy, J. E., and Gargas, M. L. (1993) Kinetic analysis of furan biotransformation by F-344 rats in vivo and in vitro. Toxicol. Appl. Pharmacol. 123, 274-282. (9) Chen, L. J., Hecht, S. S., and Peterson, L. A. (1995) Identification of cis-2-butene-1,4-dial as a microsomal metabolite of furan.

Chen et al. Chem. Res. Toxicol. 8, 903-906. (10) Ravindranath, V., Burka, L. T., and Boyd, M. R. (1984) Reactive metabolites from the bioactivation of toxic methylfurans. Science 224, 884-886. (11) Ravindranath, V., and Boyd, M. R. (1985) Metabolic activation of 2-methylfuran by rat microsomal systems. Toxicol. Appl. Pharmacol. 78, 370-376. (12) Zhang, K. E., Naue, J. A., Arison, B., and Vyas, K. P. (1996) Microsomal metabolism of the 5-lipoxygenase inhibitor L-739,010: Evidence for furan bioactivation. Chem. Res. Toxicol. 9, 547-554. (13) Guengerich, F. P. (1982) Microsomal enzymes involved in toxicology-Analysis and separation. In Principles and Methods of Toxicology (Hayes, A. W., Ed.) pp 609-634, Raven Press, New York. (14) Baker, J. T., and Sifniades, S. (1979) Synthesis and properties of pyrrolin-2-ones. J. Org. Chem. 44, 2798-2800. (15) Fringuelli, F., Gronowitz, S., Hornfeldt, A. B., Johnson, I., and Taticchi, A. (1974) Nuclear magnetic resonsance of aromatic heterocyclics. VII. A comparative study of 1H and 13C spectra of some 2-substituted furans, thiophenes, selenophenes and tellurophenes. Acta Chem. Scand. B28, 175-184. (16) Alvarez-Coque, M. C. G., Hernandez, M. J. M., Caman˜as, R. M. V., and Ferna`ndez, C. M. (1989) Formation and instability of o-phthalaldehyde derivatives of amino acids. Anal. Biochem. 178, 1-7. (17) Gronowitz, S., and Kada, R. (1984) Sulfur derivatives of 1-methylpyrrole. J. Heterocycl. Chem. 21, 1041-1043. (18) Zhu, M., Spink, D. C., Yan, B., Bank, S., and DeCaprio, A. P. (1995) Inhibition of 2,5-hexanedione-induced protein cross-linking by biological thiols: chemical mechanisms and toxicological implications. Chem. Res. Toxicol. 8, 764-771. (19) Koga, N., Inskeep, P. B., Harris, T. M., and Guengerich, F. P. (1986) S-[2-(N7-Guanyl)ethyl]glutathione, the major DNA adduct formed from 1,2-dibromoethane. Biochemistry 25, 2192-2198. (20) Ribo´, J. M., and Valle´s, A. (1987) Tautomerism of 2-hydroxypyrrole and some related derivatives studied by MINDO/3 and MNDO methods. J. Chem. Res. (S), 284-285. (21) Simons, S. S., Jr., and Johnson, D. F. (1978) Reaction of ophthalaldehyde and thiols with primary amines: formation of 1-alkyl(and aryl)thio-2-alkylisoindoles. J. Org. Chem. 43, 28862891. (22) Fiorenza, M., Reginato, G., Ricci, A., and Taddei, M. (1984) Regioselective functionalization of heterocyclic rings: synthesis and reactions of 1-methyl-2-(trimethylsiloxy)pyrrole and 2-(trimethylsiloxy)thiophene. J. Org. Chem. 49, 551-553. (23) DeCaprio, A., and O’Neill, E. A. (1985) Alterations in rat axonal cytoskeletal proteins induced by in vitro and in vivo 2,5-hexanedione exposure. Toxicol. Appl. Pharmacol. 78, 235-247. (24) Graham, D. G., Anthony, D. C., Boekelheide, K., Maschmann, N. A., Richards, R. G., Wolfram, J. W., and Shaw, B. R. (1982) Studies of the molecular pathogenesis of hexane neuropathy. II. Evidence that pyrrole derivatization of lysyl residues leads to protein crosslinking. Toxicol. Appl. Pharmacol. 64, 415-422. (25) Fukui, H., Shimokawa, S., Sohma, J., Owadare, T., and Esumi, N. (1971) Analysis of the NMR spectrum of 3-methyl-pyrrole. J. Mol. Spectrom. 39, 521-524.

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