Chem. Res. Toxicol. 2007, 20, 1685–1692
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Mechanism of 1,N2-Etheno-2′-deoxyguanosine Formation from Epoxyaldehydes† Katya V. Petrova, Ravikumar S. Jalluri, Ivan D. Kozekov, and Carmelo J. Rizzo* Departments of Chemistry and Biochemistry and Center in Molecular Toxicology, Vanderbilt UniVersity, VU Station B 351822, NashVille, Tennessee 37235-1822 ReceiVed May 1, 2007
Background levels of etheno adducts have been attributed to the reaction of DNA with 2,3epoxyaldehydes, a proposed product of lipid peroxidation. We have examined the reaction of (2R,3S)epoxyhexanal with dGuo to give 7-(1S-hydroxybutyl)-1,N2-etheno-dGuo. We observed that the stereochemistry of the side chain scrambled over time. This process provided insight into the mechanism for the formation of 1,N2-etheno-dGuo from 4,5-epoxy-2-decenal [Lee, S. H., et al. (2002) Chem. Res. Toxicol. 15, 300–304]. The mechanistic proposal predicts that 2-octenal is a by-product of the reaction. The reaction of 4,5-epoxy-2-decenal was reinvestigated, and the 2-octenal adduct of dGuo was identified as a product of this reaction in support of the mechanistic proposal. Also observed are products that appear to be derived from 2,3-epoxyoctanal, which can be formed through Schiff base formation of 4,5-epoxy-2-decenal with the dGuo followed by hydration of the double bond and retro-aldol reaction. Introduction Leonard and coworkers initially investigated the reaction of chloroacetaldehyde with dAdo, Cyt, and dGuo to form the corresponding etheno () adducts in which an ethylene unit bridges two nucleophilic sites of the base (1–3; Figure 1) (1, 2). Some of these modified bases have found utility as fluorescent probes to examine the structure, function, and dynamics of DNA. It was subsequently found that chlorooxirane (5), formed by the in vivo epoxidation of vinyl chloride by a cytochrome P450, is also a source of etheno adducts upon reaction with DNA (3, 4). Exposure to vinyl chloride, which is produced in large quantities in the plastics industry, has been correlated to a unique tumor, hepatic angiosarcoma. Etheno adducts are miscoding and have been attributed at least in part to the carcinogenicity of vinyl chloride and related vinyl monomers (3, 5–9). There has been significant interest in the chemistry and biology of etheno adducts, which are prototypes for socalled exocyclic DNA adducts. Of particular interest, background levels of etheno adducts have been found in unexposed populations (10–13). The endogenous C2 donors were hypothesized to be bis-electrophiles such as 2,3-epoxyaldehydes (6), derived from free radical degradation to polyunsaturated fatty acids initated by reactive oxygen species (14–16). The mechanism of formation of 2,3epoxyaldehydes from lipid peroxidation has not been fully elucidated. The epoxidation of the corresponding R,β-unsaturated aldehyde, which is produced in abundance through lipid peroxidation (17, 18), is a possible source, although the cellular oxidant for this reaction has not been identified (19, 20). Chung postulated that lipid hydroperoxides, a key intermediate in lipid peroxidation, could act as the epoxidation agent for the conversion of R,β-unsaturated aldehydes to the corresponding 2,3-epoxyaldehyde (21, 22). Model studies showed that hydro† This manuscript is dedicated to Professor Lawrence J. Marnett in celebration of his 60th birthday. * To whom correspondence should be addressed. Tel: 615-322-6100. Fax: 615-343-1234. E-mail:
[email protected].
Figure 1. Etheno adducts of DNA bases and two-carbon biselectrophiles.
gen peroxide, t-butylhydroperoxide, or lipid hydroperoxides react with trans-4-hydroxynonenal (HNE)1 at neutral pH to give 2,3-epoxy-4-hydroxynonanal (EHN) in modest yield (16, 23, 24). Blair and co-workers demonstrated that 4,5-epoxy-2-decenal is a product of linoleic acid peroxidation, suggesting that epoxyaldehydes may be direct products of lipid peroxidation rather than secondary products (25). The reaction of EHN with dGuo results in a variety of products as shown in Figure 2 (16). These include etheno adducts with a C7-hydroxyalkyl side chain (7) and the parent 1,N2-etheno-2′-deoxyguanosine (1,N2--dGuo; 1). Products from the reaction of dGuo with glycidaldehyde (26, 27), 2,3epoxybutanal (14), 4,5-epoxydec-2-enal (28), and 2,3:4,5diepoxydecanal (29) have also been characterized (21). The mechanism of formation of etheno adducts from 2,3-epoxyaldehydes is shown in Scheme 1. Although an alternative mechanism has been proposed (10, 15), we favor that outlined 1 Abbreviations: 1,N2--dGuo, 3-(2-deoxy-β-D-erythro-pentofuranosyl)3,4-dihydro-9H-imidazo[1,2-a]purin-9-one or 1,N2-etheno-2′-deoxyguanosine; -dAdo, 3-(2-deoxy-β-D-erythro-pentofuranosyl)-3H-imidazo[2,1i]purine or 1,N6-etheno-2′-deoxyadenosine; -dCyd, 6-(2-deoxy-β-D-erythropentofuranosyl)imidazo[1,2-c]pyrimidin-5(6H)one or 3,N4-etheno-2′deoxycytidine; HNE, 4-hydroxy-2-nonenal; EHN, 2,3-epoxy-4hydroxynonanal; ONE, 4-oxo-2-nonenal; EDE, 4,5-epoxy-2-decenal; HPNE, 4-hydroperoxy-2-nonenal; DET, diethyl tartrate; TEMPO, 2,2,4,4-tetramethylpiperidine-N-oxide; UPLC, ultraperformance liquid chromatography; SRM, selected reaction monitoring.
10.1021/tx7001433 CCC: $37.00 2007 American Chemical Society Published on Web 10/02/2007
1686 Chem. Res. Toxicol., Vol. 20, No. 11, 2007
PetroVa et al.
reaction provides insight in the mechanism of 1,N2--dGuo formation from the lipid peroxidation product 4,5-epoxy-2decenal (EDE) (28).
Experimental Procedures
Figure 2. Products from the reaction of 2,3-epoxy-4-hydroxynonanal (EHN) with dGuo.
Scheme 1
by Golding, who studied the reaction of dGuo with glycidaldehyde (26, 27). Condensation of the exocyclic amino group of dGuo with the aldehyde initially gives carbinol amine 9. Epoxide ring opening by N1 gives the cyclized intermediate 10, which can undergo reversible dehydration to imine 11. Tautomerization of the imine provides the C7-(1-hydroxyalkyl)-1,N2--dGuo (12). Alternatively, imine 11 can lose the C7 side chain as the corresponding aldehyde via a retro-aldol reaction, resulting in the formation of the unsubstituted parent 1,N2--dGuo (1). Exposure of the C7-hydroxyalkyl-substituted etheno adduct (12) to alkaline conditions results in quantitative loss of the C7 side chain, presumably through the imine intermediate 11 (16). The bicyclic adduct 8, which is uniquely derived from the reaction of dGuo with EHN, arises by trapping of the cyclic imine (11) by the side chain hydroxyl group. A similar mechanism can be envisioned for the formation of the 3-(2-deoxy-β-D-erythropentofuranosyl)-3H-imidazo[2,1-i]purine (-dAdo) adducts from the reaction with 2,3-epoxyaldehydes, but to our knowledge, bicyclic dAdo adducts analogous to 8 have not been observed. EHN was shown to be more tumorgenic than HNE itself (30). The 1,N6--dAdo and 1,N2--dGuo adducts with intact C7 side chains have been observed from calf thymus DNA and the DNA isolated from intact cells that were treated with EHN (24, 31). Etheno adducts with intact side chains are likely to be present in cellular DNA and may have differential biology than parent etheno adducts. In addition, the side chain stereochemistry of these adducts may play a significant role in the structure and mutagenicity of the modified nucleobase. We report here the diastereospecific synthesis of 7-(1S-hydroxybutyl)-1,N2--dGuo from the reaction of dGuo with (2R,3S)-epoxyhexanal and examination of its side chain reactivity. We find that the side chain stereochemistry scrambles over time. The scrambling
All solvents were distilled before use according to standard procedures. Moisture and air sensitive reactions were conducted under a nitrogen atmosphere in oven-dried glassware. Thin-layer chromatography was performed on silica gel glass plates (Merck, Silica Gel 60 F254; layer thickness, 250 µm) and visualized under UV light or by staining with anisaldehyde followed by charring. 1 H NMR spectra were recorded at 600, 400, or 300 MHz, and 13C NMR spectra were recorded at 150.9 MHz in CDCl3 or in DMSOd6. (2S,3S)-Epoxy-1-hexanol (14). In a flame-dried flask under an argon atmosphere were added activated, 4 Å molecular sieves (0.65 g) and anhydrous dichloromethane (75 mL). In a separate flask, (+)-diethyl tartrate [(+)-DET, 1.23 g, 6 mmol] was stirred in anhydrous dichloromethane over activated 4 Å molecular sieves (∼0.2 g) for 15 min and then transferred to the reaction flask via syringe. In a separate flask, Ti(OiPr)4 (1.42 g, 5 mmol) and cumene hydroperoxide (9.55 g, 62.5 mmol) were stirred over activated 4 Å molecular sieves (∼0.2 g) in dichloromethane (10 mL) for 15 min and then transferred to the reaction flask via syringe. The reaction mixture was stirred for 40 min at -40 °C. A solution of trans-2-hexen-1-ol (2.50 g, 25 mmol) in dichloromethane (10 mL) was stirred over activated 4 Å molecular sieves (∼0.2 g) for 15 min and then added to the reaction flask dropwise via syringe. The reaction mixture was stirred at -25 °C for 4 h and then quenched by the addition of ferrous sulfate (15.0 g) in 10% aqueous tartaric acid (50 mL) to the cooled reaction mixture. The mixture was stirred for 1 h after which time the organic phase was separated, washed with water (2 × 100 mL), dried over MgSO4, filtered, and concentrated. The residue was diluted with ether (200 mL) and stirred with 30% NaOH in saturated brine (25 mL) at 0 °C for 30 min. The organic layer was separated, washed with brine, dried over MgSO4, filtered, and evaporated. Purification by flash chromatography on silica, eluting with 20% ethyl acetate in hexanes, afforded 14 (1.66 g, 57%). [R]D24.7 ) -43.7 (c 1.0, CHCl3), [lit. [R]D ) -46.6° (c 1.0, CHCl3), 94% ee] (32). 1H NMR (CDCl3): δ 3.88–3.92 (m, 1H, C1-H′), 3.62–3.64 (m, 1H, C1-H″), 2.90–2.96 (m, 2H, C2-H, C3-H), 2.13 (br s, 1H, –OH), 1.44–1.56 (m, 4H, 2 CH2), 0.96 (t, J ) 7.2 Hz, 3H, CH3). (2R,3S)-Epoxyhexanal (15). To a solution of 14 (0.58 g, 5 mmol) in dichloromethane (5 mL) was added 2,2,6,6-tetramethylpiperidine-N-oxide (TEMPO, 0.078 g, 0.5 mmol) followed by bisacetoxyiodobenzene (1.77 g, 5.5 mmol). The reaction mixture was stirred at room temperature for 3 h and then diluted with dichloromethane (25 mL). The mixture was washed with a saturated aqueous solution of Na2S2O3 (15 mL), and the aqueous layer was extracted with dichloromethane (3 × 25 mL). The phases were separated, and the aqueous layer was extracted with dichloromethane (3 × 5 mL). The combined organic phases were successively washed with a saturated NaHCO3 solution (25 mL) and saturated brine, dried over MgSO4, filtered, and concentrated under reduced pressure. Purification by flash column chromatography using 10% ethyl acetate in pentane as the eluant afforded 15 (0.346 g, 60%). [R]D24.8 +99.42° (c 0.35, CHCl3). 1H NMR (CDCl3): δ 9.02 (d, J ) 6.3 Hz, 1H, C1-H′), 3.16 (m, 1H, C3-H), 3.06 (dd, J1 ) 6.3 Hz, J2 ) 1.8 Hz, 1H, C2-H), 1.56 (m, 4H, 2 × C4-H, 2 × C5-H), 0.91 (t, J ) 7.2 Hz, 3H, 3 × C6-H). 3-(2-Deoxy-β- D -erythro-pentofuranosyl)-7-(1S-hydroxybutyl)-3,4-dihydro-9H-imidazo[1,2-a]purin-9-one (16). To a suspension of dGuo·H2O (50 mg, 0.18 mmol) in DMF (1 mL) was added K2CO3 (57 mg, 0.41 mmol), and the mixture was stirred for 15 min. A solution of (2R,3S)-epoxyhexanal (61 mg, 0.54 mmol) in DMF (1 mL) was added to the suspension, and the reaction was stirred for 12 h. The reaction was monitored by HPLC (gradient I). The reaction mixture was neutralized to pH 7 with 5% acetic acid and purified by HPLC (gradient II). The fractions collected
Etheno–dGuo Adducts from Lipid Peroxidation Products from HPLC were immediately cooled to -78 °C and lyophilized to afford (S)-16 (36 mg, 55%). Scrambling of the side chain stereochemistry was observed if the HPLC fractions were left for longer periods at room temperature. 1H NMR (DMSO-d6): δ 8.13 (s, 1H, H-2), 7.21 (s, 1H, H-6), 6.22 (dd, J1 ) 7.68, J2 ) 6.28 Hz, 1H, H-1′), 5.28 (d, J ) 4.12 Hz, 1H, 3′-OH), 5.21 (d, J ) 6.04 Hz, 1H, C9-OH), 5.18 (m, 1H, C10-H), 4.94 (t, J ) 5.6 Hz, 1H, 5′OH), 4.36 (m, 1H, H-3′), 3.83 (m, 1H, H-4′), 3.55 (m, 2H, H-5′, H-5″), 2.57 (m, 1H, H-2″), 2.24 (m, 1H, H-2′), 1.8 (m, 1H, C11H′), 1.65 (m, 1H, C11-H″), 1.5–1.33 (m, 2H, C12-H′, H″), 0.89 (t, J ) 8 Hz, 3H, C13-H′, H″,H″′). 13C NMR (DMSO-d6): δ 153.8, 149.5, 146.9, 137.8, 128.3, 116.4, 113.3, 87.7, 83.1, 70.8, 64.6, 61.8, 39.4, 38.5, 18.7, 13.7. LC-ESI-MS m/z calcd for C16H21N5O5 [M + H], 364.15; found, 364.10. 7,7′-Butylidene-bis[3-(2-deoxy-β-D-erythro-pentofuranosyl)3,4-dihydro-9H-imidazo[1,2-a]purin-9-one (18a). (S)-16 (3.6 mg, 0.01 mmol) was stirred in pH 9 buffer (boric acid–KCl–NaOH, 0.1 M, 1 mL) for 72 h. The reaction mixture was neutralized to pH 7 with 5% acetic acid and purified by HPLC (gradient II) to afford 18a (1.9 mg, 59 %). 1H NMR (DMSO-d6): δ 8.02 (d, 2H, 2 × H-2), 6.89 (d, 2H, 2 × H-6), 6.28 (t, 1H, C10-H, J ) 6.8 Hz), 6.19 (t, 2H, 2 × H-1′, J ) 6.8 Hz), 5.27 (d, 2H, 2 × 3′-OH, J ) 4.0 Hz), 4.97 (d, 1H, 2 × 5′-OH, J ) 5.6 Hz), 4.35 (m, 2H, 2 × H-3′), 3.82 (m, 2H, 2 × H-4′), 3.55 (m, 4H, 2 × H-5′, 2 × H-5″), 2.57 (m, 2H, 2 × H-2″), 2.22 (m, 2H, 2 × H-2′), 1.98 (q, 2H, C11-H′, H″, J ) 7.2 Hz), 1.5 (m, 2H, C12-H′, H′ ), 0.93 (t, 3H, C13-H′, H″,H″′, J ) 7.2 Hz). LC-ESI-MS m/z calcd for C28H32N10O8 [M + H], 637.25; found, 637.21. General Procedure for Epimerization. (S)-16 (1 mg, 0.0028 mM) was stirred in pH 5.5, 7.4, and 9.0 buffer (potassium biphthalate–NaOH, 0.05 M, 1 mL) for up to 7 days at room temperature. The scrambling reaction was monitored by HPLC. In addition to the epimerization of (S)-16, etheno adduct 1 and dimer 18a were observed. EDE. EDE was prepared in two steps starting from 2-octenal as previously described (28, 33). The final product and intermediate were purified by flash chromatography. The EDE was analyzed by GC-MS and judged free of any starting 2-octenal.2 3-(2-Deoxy-β-D-erythro-pentofuranosyl)-4,6,7,8-tetrahydro8-hydroxy-6-pentyl-pyrimido[1,2-a]purin-10(3H)-one (26). A solution of trans-2-octenal (2.52 mg, 20 µmol) in 100 µL of degassed acetonitrile was added to a suspension of dGuo·H2O (2.85 mg, 10 µmol) and L-arginine (3.48 mg, 20 µmol) in bicine buffer (100 mM, pH 8.0, 450 µL). The reaction mixture was heated at 60 °C for 60 h. Additional trans-2-octenal (1.89 mg, 15 µmol) in acetonitrile (100 µL) and L-arginine (2.61 mg, 15 µmol) in bicine buffer (100 µL) was added to the reaction mixture every 15 h (three additions). The total amount of 2-octenal and L-arginine added was 65 µmol. The reaction was monitored by HPLC gradient II. The reaction mixture was extracted with hexanes and acidified to pH 5.0 with 1 N HCl, and the resulting solution was purified by HPLC using gradient system I (flow rate, 5.0 mL/min) to give 26 (1.85 mg, 47% yield) as a white solid. The purity of the product was judged to be >99% by HPLC (gradient II). 1H NMR (600 MHz, DMSO-d6): δ 7.88 (s, 1H, H2), 7.49 (s, 1H, N2-H), 6.58 (br s, 1H, C8-OH), 6.20 (s, 1H, H8), 6.1 (t, J ) 6.6 Hz,1H, H-1′), 5.23 (br s, 1H, C3′-OH), 4.89 (br s, 1H, C5′-OH), 4.32 (s, 1H, H3′), 3.78 (s, 1H, H4′), 3.57 (m, 1H, H6), 3.52 (m, 2H, H5′, H5″), 2.5 (m, 1H, H2′), 2.16 (m, 1H, H2″), 2.05 (m, 1H, H7), 1.36 (m, 1H, H7), 1.31 (m, 8H, 4CH2), 0.88 (t, J ) 6.6 Hz, 3H, CH3). 13C NMR (DMSOd6): δ 156.0, 151.3, 150.3, 135.7, 115.9, 87.9, 82.6, 71.1, 69.7, 62.1, 44.8, 34.5, 32.9, 31.7, 24.4, 22.5, 14.3. UV λmax () 260 nm (14020). Positive ESI-MS m/z 394 [M + H]+. MS/MS of m/z 394 2 The purity of EDE was examined by GC-SIM-MS. 2-Octenal could not be detected in the sample (
