Chem. Res. Toxicol. 1993,6, 635-648
635
Formation of 1,@- and @,3-Ethenoguanine from 2-Halooxiranes: Isotopic Labeling Studies and Isolation of a Hemiaminal Derivative of @-(2-Oxoethyl)guanine F. Peter Guengerich,’ Magnus Persmark, and W. Griffith Humphreyst Department of Biochemistry and Center in Molecular Toxicology,Vanderbilt Uniuersity School of Medicine, Nashville, Tennessee 37232-0146 Receiued March 5,1993
Vinyl halides are oxidized to 2-halooxiranes, which rapidly rearrange to 2-haloacetaldehydes. Both of these species can react with DNA to generate avariety of adducts, including the potentially mutagenic etheno (e) products. Evidence was provided through kinetic studies that the e-Gua adducts are formed primarily from 2-haloxiranes; consistent with this view, epoxide hydrolase inhibited the formation of N2,3-e-Gua from vinyl chloride but alcohol dehydrogenase did not. Assignments of the NMR shifts of the etheno protons of 1Pand W,3-e-Gua were made with the use of 15N labeling and nuclear Overhauser effects, in revision of the literature. The H-5 proton of N2,3-cGua showed facile exchange in acid or base; the H-7 proton of 1 P - e - G u a was exchanged a t neutral or basic p H but not in acid. Reaction of BrzCHCHzOH (labeled at C1 with 2Hor 13C)with Guo yielded 1J’P-e-Gua andN2,3-e-Gua,presumably through the intermediacy of 2-bromooxirane. ‘H NMR analysis indicated that the labeled carbon was attached to the original Guo N2 atom in both cases. When N2-(2-oxoethyl)Gua was generated from a diethyl acetal or from a glycol, the major product was the cyclic derivative 5,6,7,9-tetrahydro-7-hydroxy9-oxoimidazo[ 1,2-a]purine. This compound was also formed in considerable yield from the reaction of 2-chlorooxirane with Guo, dGuo 5’-phosphate, or DNA and is relatively stable in the presence of acid or mild base. It does not appear to be readily dehydrated to yield the etheno adducts but may be of significance as a DNA adduct in its own right.
Introduction The so-called etheno (t)l nucleic acid adducts contain additional 5-memberedrings and are formed from avariety of chemical carcinogens, at least in in vitro settings. The list includes vinyl and some alkylhalides (1-41,acrylonitrile (5), urethanehinyl carbamate (6,7), nitrosamines (4,8), Michael reagents such as 4-hydroxy-2-nonenal (9), and the hydroxyfuranone mucochloric acid (10). Modified e derivatives also occur as the “Y-bases”in tRNAs (11).Some of these have considerable fluorescence and have found utility in the labeling of nucleoside derivatives such as the pyridine nucleotides and nucleoside triphosphates (12). Their biological significance has been the subject of considerable interest in regard to their ability to induce tumors (13). Although many initial efforts were limited by analytical sensitivity, these adducts have been found in DNA in many exposure conditions (2,14,15).Indeed, the observation has been made that significant levels of these are found even in the absence of exposure to known carcinogens (15).W,3-e-Gua (16-18)and 3JV4-e-Cyt(19) have been reported to be miscoding in site-specific mutagenesis experiments. In rats some of the e adducts (at least W,3-e-Gua) have been reported to be highly persistent and refractory to repair (14).Repair of such *Address correspondence to this author at the Department of Biochemistryand Center in Molecular Toxicology,VanderbiltUniversity School of Medicine, Nashville, TN 37232-0146. f Present address: Bristol-Myers Squibb Pharmaceutical Research Institute, P.O. Box 4000, Princeton, NJ 08543. * Abstract published in Aduance ACS Abstracts, August 15, 1993. Abbreviations: c, etheno; FAB, fast atom bombardmenk NOE, nuclear Overhauser effect. For a list of standard abbreviations of nucleosides and their bases now used in this journal, see: Chem. Res. T O X ~ C6,O5A-9A ~. (1993).
0893-228~/93/2706-0635$04.~0/0
DNA lesions has been reported in bacteria (20)and human cells (21);the apparent discrepancy between rats and humans has yet to be resolved. The chemical and physical properties of the t adducts have been reviewed (12,22). Mechanisms have been presented for the formation of these compounds from haloacetaldehydes (12). However, the more relevant chemicals involved in the formation of t DNA adducts from vinyl monomers are probably the epoxides. 2-Halooxiranes (23,24),2-cyanooxirane ( 5 ) , 2-(N-nitrosomethy1amino)oxirane (4),and 2-carbamoyloxirane (25) have all been shown to react with nucleic acid bases and DNA to generate e adducts. Evidence has been presented that the 2-halooxiranes are considerably more effective than their rearrangement products, the 2-haloacetaldehydes, in forming DNA adducts (26)and specifically 1,P€-Ado (27). We addressed the chemical mechanism of reaction of 2-halooxiraneswith Ado and Cyd to form 1,P€-Adoand 3,N4-e-Cytand concluded that in both cases the mechanism involved attack of the basic endocyclicnitrogen (N1 of Ado or N3 of Cyd) on the unsubstituted methylene carbon of the 2-halooxirane,with subsequent ring closure and dehydration to give the e adduct (28). However, our initial efforts to extend this work to the two known eGua adducts-1 JP-t-Gua and W,3-t-Gua-were not successful, due to the low yields of these products and the propensity for the epoxides to react at the Guo N7 atom. In this study we present a series of experiments on the reaction of Gua derivativeswith 2-halooxiranes. The work was complicated by the low levels of formation of the t compounds of interest and by the unanticipated solvent exchange of the t protons. Isotopic labeling studies were done with 1JP-and W,3-t-Gua. A new, major product of 1993 American Chemical Society
636 Chem. Res. Toxicol., Vol. 6, No. 5, 1993 0
0
0
N *,3-~(d)Guo
the reaction of 2-chlorooxiranewas identified as a cyclized form of N2-(2-oxoethyl)Gua: 5,6,7,9-tetrahydro-7-hydroxy-9-oxoimidazo[1,2-alpurine(I), which has some HO
:
I
properties similar to a characterized one-carbon homolog, a cyclic acrolein adduct (29). This product may be considered a hydrated form of 1,W-c-Gua; although evidence for its role as an intermediate in 1,IV-c-Gua formation is lacking, the compound appears to be a major product and may be of biological interest. Experimental Procedures Chemicals. ClCHzCHO (50% aqueous solution, w/v) and NmethylGua were purchased from Aldrich Chemical Co. (Milwaukee, WI). N-Ethylmorpholine was distilled under vacuum and stored under Ar. 1,iPt-dGuo was a gift of Prof. L. J. Marnett of this department. W-MethylGuo was purchased from Sigma Chemical Co. (St. Louis, MO). Spectroscopy. UV spectra were recorded on a Cary 14/0LIS instrument (On-Line Instrument Systems, Bogart, GA), and the reported A-values were determined using either the peak finder program or second derivative analysis. 'H and 13CNMR spectra were obtained with Bruker AC-300 and AM-400 instruments (Bruker, Billerica, MA). All nuclear Overhauser effect (NOE) work was done in the difference mode. 4,4-Dimethyl-4-silapentane-1-sulfonic acid and (CH&Si were used as standards with aqueous and nonaqueous samples, respectively. MS was done either in the thermospray+ mode with a Nermag R10-1OC instrument (from NH&H~COZsolutions) or in the fast atom bombardment (FAB)+ mode [glycerol, 3-nitrobenzyl alcohol, (CHs)zSO, and/or CF3COzH used as matrices] using either a VG 70-250 instrument (VG, Manchester, U.K.) or a Kratos Concept IIHH instrument (Kratos, Manchester, U.K.). HPLC. All HPLC work involved the use of a Spectra Physics 8700 pumping system (Spectra-Physics, Piscataway, NJ) and a Beckman 5-pm Ultrasphere octadecylsilane column (10 x 250 mm, Beckman, San Ramon, CA) unless noted otherwise. The desired eluents were prepared by mixing 50 mM NH4HCOzbuffer (pH 5.5) with a 9:l (v/v) mixture of CH30H and HzO (30). The effluent was passed through an LDC Spectromonitor 3200 UV detector (Milton Roy, Rivera Beach, FL) and/or a fluorescence detector (Kratos GM 970, Applied Biosystems, Foster City, CA, or McPherson FL-750 HPLC-Plus, McPherson Instruments, Acton, MA). Syntheses. (A)W,3-c-Gua. N2,3-e-Gua was prepared by treatment of 06-benzylGua (31)with CICHzCHOand subsequent acid hydrolysis of the OB-benzyl-W,3+Gua (11)as done previ276 nm; ously in this laboratory (27): UV (H20, pH 7) A, thermospray+ MS (re1abundance in parentheses) mlz 176 (100, [M + HI+); 'H NMR [(C2H3)2SOl6 7.11 (d, lH, H-61, 7.61 (d, 1H,H-5),8.16 (s,lH, H-2) (videinfra for assignment oft protons).
Guengerich et al. (B) 1,W-t-Guo and 1,N-t-Gua. 1P-e-Guo was prepared by the reaction of Guo with ClCHzCHO (11): UV (HzO) A, (pH 7) 227,285 nm, (pH 13) 231,308 nm; lH NMR [(C2H3)2S016 3.84 (m, 2H, H-5'),4.37 (m, lH, H-4'),4.94 (m, lH, H-3'),5.29 (m, lH, H-2'),6.25 (m, lH, H-l'), 7.42 (d, lH, H-6),7.60 (d, lH, H-7), 8.12 (s, lH, H-2). 1P-e-Gua was prepared by hydrolysis of 1,W-tGuo or 1,W-e-dGuo (0.1-0.5 N HCl, 90-100 "C, 60 minh2 UV (HzO) A, (pH 7) 225,290 nm, (pH 1)A, 226,285 nm, (pH 13) 236, 318 nm; thermospray+ MS mlz 176 (100, [M + HI+); 1H NMR [(C2H3)zSOl6 7.59 (d, lH, H-6, J = 2.4 Hz), 7.75 (d, lH, H-7), 9.11 (d, lH, H-2) (vide infra for t shift assignments). (C) @-Ethyl-W,3-t-Guo. 06-EthylGua (8.1mg, 45pmol) (32) was converted to 06-ethylGuo by enzymatic ribosylation with Escherichia coli purine nucleoside phosphorylase (Sigma) and ribose 1-phosphate (18mg,49pmol) in 15mLof 20 mMpotassium phosphate buffer (pH 7.4) (33-36). The enzyme was removed by the addition of ZnSO4 to 30 mM and centrifugation [(3 X W)g, 10 min]; the resulting supernatant was stirred with 1.0 mL of a 50% (w/v) aqueous ClCHzCHO solution, 3.0 mL of C~HSOH, and 2.0 mL of 1 M sodium acetate buffer (pH 4.5) for 40 h at 37 "C (27,37). HPLC analysis of the reaction product indicated the presence of a large new peak accounting for -80% of Am and which was very fluorescent ( F z l e l ~ ) .This was purified with the same system, and the product @-ethyl-W,3-e-Guowas recovered by repeated lyophilization to remove the NHIHCOZ: UV (CH3OH) A, 222, 270 nm; FAB+ MS [3-nitrobenzyl alcohol/ (CH3),SO] m/z 336 (40, [M + HI+),358 (100, M + Na), 204 (100, M - 132), 176 (96, M - 132-28); 'H NMR (2Hz0) 6 1.43 (t, 3H, CH3), 3.61 (m, lH, H-5"),3.70 (m, lH, H-5'), 4.07 (m, lH, H-4'), 4.15 (m, lH, 29, 4.44 (m, lH, 39, 4.56 (q, ZH, OCHZCH~), 6.22 (d, lH, l', J = 4.4 Hz), 7.48 (d, lH, H-6, J = 1.6 Hz), 7.95 (d, lH, H-5, J = 1.7 Hz), 8.40 (8, lH, H-2) (vide infra for basis of shift assignments). (D) [5-16N]-I,N-t-dGuo. [2-l5N1dGuowas prepared by Dr. B. DeCorte (of this Center) by treatment of 2-fluoro-P-[2(trimethylsilyl)ethyl]deoxyinosinewith lSNH4OH;15 mg of this material(52pmol) wasmixedwith 12.5pLofa50% (w/v)aqueous solution of ClCHzCHO (85pmol) and 10.5 mg of anhydrous KzC03 [in 1.OmLof (CH&NCHO,distilledfromBaO]; after 3 hanother 7 pL of the ClCHzCHO solution (48 pmol) and 7 mg of KzCO3 (50 pmol) were added and the reaction proceeded overnight (all at 23 "C) (38). The product was separated by HPLC (octadecylsilane, 4.5-45% gradient of CHsOH in 50 mM NHdHC02, pH 5.5, over 30 min), and the product was concentrated by repeated lyophilization to give a total of -3 mg of [5-15Nl-1~-e-dGuo: UV (HzO)A,, 227,284 nm (pH 5.5), 279,309 nm (pH 13);FAB+ MS [glycerol/(CH3)~SOlm/z 293 (14, [M + HI+), 315 (90, M + Na), 337 (75, M + 2Na), 177 (100, lJP-eGua), 199 (100, 1.Pe-Gua + Na); see text for 'H NMR (vide infra). (E) Br2CHCH20H. BrzCHCHzOH was prepared by LiAlH, reduction of BrzCHCOzH(28): 'H NMR (CZHC13) d 4.04 (d, 2H, CHzO),5.66 (t, lH, BrzCH). The procedure was repeated with LiAl2b to yieldBrzCHC2Hz0Hin 54 % yield: lH NMR (C2HC13) 6 5.65 (8, BrzCH). BrzCHWH20H was prepared in the same way as previouslydescribed (37% overallyield for the bromination and reduction steps) and showed the same NMR spectra as previous preparations (28). (F)2-Chlorooxirane. 2-Chlorooxirane was synthesized by photochemical chlorination of ethylene oxide in the presence of tert-butyl hypochlorite (39);the oxide (-1 M solution) was quantified with the use of 4-(4'-nitrophenyl)pyidinereagent (23, 39).
(G) N-Methyl-N-(2-hydroxyethyl)Guo.2-Fluorodeoxyinosine (5 mg, gift of Dr. C. M. Harris of this Center) was mixed with a 10-fold molar excess of N-methylethanolamine in 0.5 mL of distilled (CH3)zNCHO at 50 "C overnight. HPLC analysis ( A m ) indicated that the product N2-methyl- N2-(2-hydroxyethy1)dGuo was formed in -80% yield. The material was isolated by preparative HPLC [octadecylsilane, 4-40 % (v/v) linear Extensive decompositionresulted when the hydrolysiswaa done with
2 N HC1 according t o a literature procedure (11).
Ethenoguanine Formation
Chem. Res. Toxicol., Vol. 6, No. 5, 1993 637
the proton at 6 5.59 (H-6) collapsed both the protons at 4.28 (H-7b) and 4.05 (H-7a) (Chart I). Irradiation at 6 4.05 (H-7a) collapsed the peaks at d 4.28 (H-7b) and 5.59 (H-6). Further evidence for the assigned structure IIa (5,6,8,9-tetrahydro-N6methyl-6-hydroxy-9-oxo-3-~-D-ribofuranosylimidazo[ 1,2-a]purine) was obtained with NOE experiments and provided evidence against the alternative structure 111. Irradiation of the proton at 6 3.10 yielded a weak NOE with the proton at 6 5.59 (H-6) and a very weak NOE with the proton at 6 5.98 (H-1') (all NOESseen in this study were positive). Irradiation of the proton at 6 4.05 (H-7a) produced a moderate NOE at 6 4.28 (H-7b). Irradiation at 6 4.28 (H-7b) produced moderate NOES with the protons at 6 4.05 (H-7a)and 5.59 (H-6). Irradiation of the proton H 6 OH at 6 5.59 yielded a moderate NOE at 6 3.10 (NCH3). Irradiation I1 a,b I11 of the protons at 6 3.10 (NCH3) or 6 4.45 did not produce an NOE. With compound IIb, weak NOESat 6 5.97 (H-l'),5.58 (H-6),4.27 (H-7b), and 4.03 (H-7a) were detected when the 6 3.09 proton gradient of CHaOH in 50 mM NH4HCO2 over 30 min, pH 5.5, (NCH3) was irradiated. Irradiation at 6 5.58 (H-6) yielded weak flow rate 4.0 mL min-11 and the solvent and salts were removed NOES with 6 4.27 (H-7b) and 4.03 (H-7a) and a stronger NOE by repeated lyophilization to yield W-methyl-W-(2-hydroxywith 6 3.09 (NCH,), but no NOE was observed with 6 5.97 (H-1'). ethy1)dGuo: UV (HzO) A, (pH 7) 261,291 nm, (pH 11) 261, When the 6 5.97 proton was irradiated, only a weak NOE with 287 nm; FAEP MS mlz 326 (16, [M + HI+), 185 (27, M - 115). 6 3.10 was observed, but other protons were not affected. These The material was converted from the deoxyribonucleosideto the studies support the basic structure IIa(b) as opposed to 111, in ribonucleoside using mild acid hydrolysis and a general enzymatic which a substantial NOE between the methylene protons of the procedure (33-35). The compound was dissolved in 1.0 mL of ethano ring with the ribose H-1' would have been expected (see 0.10 N HC1 and heated at 90 "C for 60 min; HPLC analysis (vide NOE studies with @-ethyl-W,3-~-Guo,vide infra). supra) indicated that hydrolysis was complete: lH NMR (2H20) Further evidence for IIa(b) as opposed to I11 was provided 6 3.19 (8, 3H, CHs), 3.74 (t, 2H, CH20H), 3.87 (t, 2H, NCH2by UV titration studies. Titration of W-methylGuo in the pH CH20H). The solution was added to a 2-mL solution of 20 mM range 1-12 showed a clear pK, near 9, with a decrease in Am potassium phosphate buffer, and the pH of the mixture was (27%)and an increase in A275 (9%). This transition is indicative adjusted to 7.0. Ribose 1-phosphate (2.5 mg) and E. coli purine of the keto-enol transition involving the N1, C6, and 0 6 atoms nucleoside phosphorylase (9 units) were added, and the reaction of Gua and derivatives in which these atoms are unsubstituted. was allowed to proceed overnight at 37 "C under Ar. HPLC When compounds IIa and IIb were examined in this pH range, analysis (vide supra) indicated that coupling was -25% comthe UV spectra were nearly invariant, arguingfor structure IIa(b) plete. The solution was applied to the preparative HPLC column over 111. (vide supra), and the peak fractions were collected and subjected t o repeated lyophilization t o give t h e product (I) NZ-(2,3-Dihydroxypropyl)dGuo.2-Fluorodeoxyinosine 261 W-methyl-W-(2-hydroxyethyl)Guo: UV (H20, pH 7) A, (3 mg, 11 pmol) was stirred with (f)-3-amino-1,2-propanediol nm; FAB+ MS m/z 342 (12, [M + HI+). (36 mg, 0.40 mmol) in 1.0 mL of dry (CH&NC(O)CHs at 50 "C (H) 5,6,7,9-Tetrahydro-lmethyl-6hydroxy-9-oxo-3-8-~- for 14 h (under Ar). HPLC analysis indicated that the reaction had proceeded to -65 % completion as judged by the appearance ribofuranosylimidazo[1,2-a]purine. The carbinolamine prodof two later migrating peaks (octadecylsilane,flow 4.0 mL min-1, ucta of N2-methylguanosine (IIa,b; Chart I) were prepared by 9% CH30H in 50 mM NH4HC02, pH 5.5, t~ 16.5 and 17.8 min). incubation of a mixture of 15 mg of W-methylGuo (50 pmol) These new compounds were separated by preparative HPLC with 0.96 mmol of ClCHzCHO in 15 mL of H2O (pH adjusted to using the same system and are considered to be diastereomers 6.4) for 7 days at 37 "C (under Ar). HPLC analysis using an of (*)-W-(2,3-dihydroxypropyl)dGuobecause of their identical increasing gradient of CHsOH applied to the octadecylsilane spectral properties: UV (H20, pH 7) A, 225 nm; FAB+MS mlz column (10 X 250 mm, vide supra) indicated the presence of 342 (17, [M + HI+),364 (32, M + Na+),386 (10, M + 2Na), 226 three major peaks (A250,plus residual W-methylGuo. Using an (60, M - deoxyribose), 248 (21, M -deoxyribose + Na), 208 (26, eluting solvent of 9% CHaOH (v/v) in 50 mM NH4HC02 (pH 5.5) M - deoxyribose, H2O); lH NMR (2H20)6 2.50 (m, lH, H-29, and flow rate of 4 mL min-1, the major peaks (termed I-IV) were 2.91 (m, lH, H-2'9, 3.46 (dd, lH, side chain CHzNH), 3.62 (m, eluted at t~ 13.9, 18.2, 21.6, and 24.9 min, respectively. The 3H, side-chain CH2NH and CHzOH), 3.79 (m, 2H, H-5', H-5'9, latter two peaks (termed IIa and IIb, eluting after the W 3.97 (m, lH, side-chain CHOH), 4.10 (m, lH, H-4'),4.64 (m, lH, methylGuo peak) were the major ones formed and were isolated H-39, 6.38 (t, lH, H-l'), 7.98 ( 8 , lH, H8). and treated by repeated lyophilization to remove salts. FAB+ MS indicated an (M -k H)+ion at mlz 340 for each (plus mlz 362 (J)N2-(2-Oxoethyl)dGuoDiethyl Acetal. 2-Fluoro-P-[2and 378 for the Na+ and K+ adducts and mlz at 268 for M - 132, (trimethylsilyl)ethy1]-deoxyinosine(15 mg, 40 pmol, gift of Dr. B. DeCorte of this Center) was mixed with 68 mg (74 pL, 500 base peak) consistent with the presence of stable carbinolamine pmol) of 2-aminoacetaldehyde diethyl acetal (Aldrich, distilled derivatives. The first peak was a mixture as determined by under aspirator vacuum) in 1.0 mL of (CH&NHCHO (distilled subsequent NMR and MS and was not characterized further. Peaks IIa and IIb showed identical UV spectra (A, 252 nm over BaO) at 50 "C overnight. HPLC analysis (vide infra) with a 286-nm shoulder at pH 5.5). lH NMR analysis of indicated that the reaction was essentially complete. Glacial CHSCO~H(0.25 mL) was added and the mixture was heated at compounds IIa and IIb indicated that they are probably both diastereomers of the structure 5,6,7,9-tetrahydro-P-methyl-6- 50 "C for 2 h to remove the trimethylsilylethyl protecting group. hydroxy-9-oxo-3-~-~-ribofuranosylimidazo[ 1,2-a]purine, as they The product was purified by preparative HPLC [octadecylsilane, have nearly superimposable spectra (in 2H20, 400 MHz). IIa: 4.5-45% (v/v) linear gradient of CH30H in 50 mM NHrHC02, pH 5.5, over 30 min, with the product eluting just at the end of 6 3.10 (8, 3H, NCHs), 3.85 (dd, lH, H-57, 3.91 (dd, lH, H-59, the gradient]. The product accounted for -50% of the Am; 4.05 (dd, lH, H-7a, J = 2.1 Hz, 12.8 Hz), 4.23 (m, lH, H-3'),4.28 (m, lH, H-7b), 4.45 (m, lH, H-2'), 5.59 (dd, lH, H-6, J = 2.1 Hz, residual product still containingthe trimethylsilylethylprotecting 7.1 Hz), 5.98 (d, lH, H-l'), 8.00 ( 8 , lH, H-2); IIb: 6 3.09 (5,3H, group was eluted by increasingthe CH30H concentration to 90%, v/v, as judged by MS. The product W-(2-oxoethyl)dGuo diethyl NCHs), 3.88 (m, 2H, H-5 and H-5'), 4.03 (dd, lH, H-7a, J = 2.1, acetal was recovered after preparative HPLC and lyophilization: 12.8 Hz), 4.23 (m, lH, H-39, 4.27 (m, lH, H-7b), 4.44 (m, lH, H-2'), 5.58 (dd, lH, H-6, J 2.1,7.1 Hz), 5.97 (d, lH, H-1'), 8.00 UV (HzO-CH~OH, pH 5.5) A, 254 nm; FAB+ MS mlz 389 (12, (s, lH, H-2). In decoupling experiments with IIa, irradiation of [M + HI+),406 (66, M + Na+),422 (100, M + K+), 444 (10, M
Chart I. Postulated Structure of Diastereomeric Carbinolamines Derived from Reaction of N1-MethylGuo with ClCH2CHO (IIa,b; NOES Shown) and an Alternate Structure (111)
Guengerich et al.
638 Chem. Res. Toxicol., Vol. 6, No. 5, 1993
Scheme I. Enzymatic Oxidation of Vinyl Chloride to 2-Chlorooxirane and Rearrangement to ClCH&HO and Design of Experiments for in Situ Destruction of Reactive Intermediates
m
a
0
N2,3-e-Guanine
0
E
0
50
1
1
n
alcohol dehydrogenase
epoxide hydrolase
100
150
200
pmol 2-Chlorooxirane or ClCHzCHO
Figure 1. Comparison of formation of 1,W-e-Guo and N2,3-rGuo from 2-chlorooxiraneand ClCHZCHO. Incubations (2.0 mL) contained 200 mM potassium phosphate buffer (pH 7.4),30 mg of herring sperm DNA mL-l, and either 105mM 2-chlorooxirane (0,A)or ClCHzCHO (0, A)and were analyzed as described under Experimental Procedures: 1JP-c-Guo, circles; p,3-e-Guo, triangles.
HO-o
a-OH
Results and Discussion Comparison of Rates of Formation of t-Guo Prod-
ucts from Guo with 2-Chlorooxirane and ClCH2CHO. We previously reported that the t1/2 of 2-chlorooxirane was -20minat5°Cin0.10Mpotassiumphosphate buffer + K + Na), 268 (36, M - deoxyribose), 222 (54, M - deoxyribose (pH 7.4) (28). An aqueous solution of DNA was reacted - CHs), 176 (61, M - deoxyribose - 2 X [OCzH&; lH NMR with 2-chlorooxirane and ClCHzCHO preparations under [(C2Hs)zSO)]6 1.20 (t, 3H, CHzCHs), 1.22 (t,3H, CHZCH~), 2.49 these conditions (1 t1/2), and the rates of formation of t (m,lH,H-2’), 2.92 (m, lH, H-2’9, 3.56 (8, 2H,NHCH2), 3.69 (m, derivatives were compared, after the t-Guo products were 2H,H-5’,H-5”),3.82(m,4H,CH(OCH~CH3)2),4.09(m,lH,H-4’), hydrolyzed to yield the e-Gua derivatives for analysis. As 4.63 (m, lH, H-39, 4.87 (t, lH, CH(OCzH5)z). reported previously (27), 2-chlorooxirane forms W,3+ Incubation Conditions. (A) Reaction of 2-Chlorooxirane and ClCH&HO with DNA.Reactions containing the indicated Guo more effectively than does ClCHzCHO (Figure 1). components proceeded for 20 min at 5 “C (28) and were Even under the conditions of these chemical experiments, terminated by the addition of 5 volumes of cold CzH50H to the analysis of 1,W-t-Gua required special considerations precipitate the DNA. DNA was recovered by centrifugation [(3 because of the insensitivity of detection. The HPLC X lO3)g, 15 min] and hydrolyzed in 1.0 mL of 0.10 N HC1 at 70 effluent in the region of 1JV-e-Gua was collected, and the OC for 60 min to release Gua, l,N2-t-Gua, and W,3-e-Gua. Each material was concentrated by lyophilization. The material sample was treated with 5 volumes of CzH&H, and apurinic acid was dissolved and injected onto an analytical HPLC was precipitated by centrifugation as before. The supernatants column operating at pH 10.5, with the effluent passing were concentrated to near dryness under an Nz stream and through a fluorescence monitor. This approach takes dissolved in 50 mM NH4HC02 buffer (pH 5.5). Aliquots were advantage of the weak characteristic fluorescence of lflinjected onto a 10 X 250 mm Beckman Ultrasphere 5-pm HPLC column, which was eluted with a linear gradient of 4.5-45% e-Gua at alkaline pH (381, even though the separation on CHsOH (v/v) in 50 mM NHrHCOz (pH 5.5) at a flow rate of 4.0 HPLC at pH 10.5is not optimal. In contrast to the report mL min-1. N1,3-e-Gua and 1JP-cGua eluted at 11.7 and 12.2 of Kusmierek and Singer (38), we found that the fluomin, respectively, as monitored by FZlS/mand Am measurements. rescence of 1,W-eGua was considerably less than that of The Fz16,m measurements were used to calculate W,3-eGua W,3-t-Gua. formation, using the fluorescence of external standards. The Effects of Epoxide Hydrolase and Alcohol Dehyfractions containing the 1,W-e-Guapeaks were impure and were drogenase on Formation of N1,3-t-Gua. We have collected, reduced to dryness by lyophilization, and dissolved previously used the approach of adding purified epoxide immediately in 2.0 mL of 50 mM (NH4)2HPO4 buffer that had hydrolase or alcohol dehydrogenase/NADH to destroy been adjusted to pH 10.5 with (C&&N. Aliquots (1.0 mL) were 2-chlorooxirane or ClCH2CH0, respectively, generated applied to a 4.6 X 150 mm Zorbax octadecylsilane HPLC column (5pm, MacModd, Chadds Ford, PA) equilibrated with the buffer from the microsomal oxidation of vinyl chloride in order in which the samples had been dissolved. The column was eluted to determine which of the two electrophiles was reacting with the same buffer, and Fw/mwas measured in the effluent; with DNA or protein (Scheme I) (26, 27, 39). This the alkaline pH was required for the fluorescence (38).1 , W - e approach was extended to study the formation of W,3Gua eluted at t~ 2.5 min (-3X void volume); quantitation was e-Gua. It was not technically feasible to use DNA in these based upon external standards. studies because of the low rates of epoxide formation and (B) Microsomal Incubations. Incubations containing the the limited trapping ability of DNA relative to the highly indicated components proceeded for 2 h at 37 OC and were then soluble dGuo 5’-phosphate (and cleaner reaction product); quenched by the addition of 60 pL of 0.6 M ZnSO4. The protein preliminary studies with calf thymus and herring sperm was precipitated by centrifugation [(3 X lO3)g, 10 min] and the DNA, Guo, and dGuo 5’-phosphate indicated that the best supernatant was transferred to a 3.0-mL volume Reacti-vial (Pierce Chemical Co., Rockford, IL). The pH of each sample sensitivity (- 20X background) was achieved with dGuo was lowered to 1.0 by the addition of 60 pL of 6 N HCl, and the 5’-phosphate. Detection of 1fl-e-Gua or a derivative was samples were heated at 70 “C for 60 min. Aliquots of each sample still not feasible under these conditions. (0.80 mL) were injected on to a 10 X 250mmBeckmanUltrasphere The addition of epoxide hydrolase inhibited the foroctadecylsilaneHPLC column ( 5 pm), and W,3-e-Guawas eluted mation of W,3-t-Gua but alcohol dehydrogenase did not with a 4.5-45% (v/v) linear gradient of CH3OH in 50 mM (Figure 2). On the basis of this result and direct comNH4HCOz (pH 5.5) over 30min with the effluent passing through parison of rates of adduct formation (Figure 1)we conclude both a UV ( A ~ w and ) a fluorescence detector (F216,m). W,3-tthat the 2-halooxirane is the vinyl halide product directly Gua eluted at 11.9 min under these conditions and was quantified involved in the formation of W,3-e-Gua, as in the cases of by comparison of the peaks to those generated with external l,iV6-e-Ade (27) and total DNA adducts (26). standards.
Chem. Res. Toxicol., Vol. 6, No. 5, 1993 639
Ethenoguanine Formation
o
f
H
The one-dimensional NMR and mass spectra do not distinguish between structures IIa(b) and 111. The assignment of the structure as IIa(b) is based primarily on (i) the lack of significant NOES between the methylene protons and the ribose H-1' protons and (ii) the lack of change of the UV spectra as a function of pH, suggesting that the Nl-C6-06 atom grouping (nomenclature of Gua) is not ionized. An alternative structure, the W-methyl derivative of I, cannot be unambiguously ruled out but seems less likely because of the lack of resemblance of the UV spectrum to that of I (vide infra). W-MethylGuo (10 mM) was incubated with either 2-chlorooxirane or ClCHzCHO (105 mM) at 5 "C, and several HPLC analyses were done with each incubation up to a period of 60 min (3 tI/z's of 2-chlorooxirane). The dominant product derived from 2-chlorooxirane appears as expected (Scheme to be N7-(2-oxoethyl)-W-methylGuo, 11,path a), since the reaction of Guo derivatives with S N ~ alkylating agents is generally directed to the N7 atom (vide infra). This product eluted before Guo in the HPLC and showed considerable fluorescence, as expected, although the compound was not rigorously characterized. Neither compound IIa nor IIb was detected a t a level of >0.02 % formation from either 2-chlorooxirane or ClCHzCHO within the 60 min time period (Scheme 11, path b). In a separate set of such incubations with 2-chlorooxirane, aliquota were removed at varying periods of time and added to alkaline NaBHr in aqueous CH30H. HPLC analysis of these products revealed the transient formation of W methyl-W-(2-hydroxyethyl)Guoafter 1min, as judged by cochromatographywith authentic material (Figure 3). The level of this product declined after the initial burst, as might be expected if the aldehyde were attacked by one of the ring nitrogens. The maximum level of formation of W-methyl-W-(2-oxoethyl)guanosinewas -0.2 % , detected using this procedure (Figure 3). The compound was not detected in similar incubations containing ClCHzCHO instead of 2-chlorooxirane. It should be
1
601
20
tl 0
-I 0.2
0.4
0.6
0.8
1.0
[~nzyme],mg ml"
Figure 2. Effects of the addition of purified epoxide hydrolase and alcohol dehydrogenase on the formation of N2,3-e-Gua in a system containing rat liver microsomes, vinyl chloride, and dGuo 5'-phosphate. The indicated concentrations of purified rat liver epoxide hydrolase ( 0 )or horse liver alcohol dehydrogenase ( 0 ) (plus 1.0 mM NADH) were added to systems containing 50 mM potassium phosphate buffer (pH 7.4), liver microsomesprepared from isoniazid-treated rata (5.4 mg of protein mL-I) (3), 15 mM dGuo 5'-phosphate, and an NADPH-generating system (26,27) in a total liquid volume of 1.0 mL in Teflon-sealed vials of 4.0mL total volume; the head space (3.0 mL) contained 0.50% vinyl chloride (v/v; i.e., 5000 ppm). See Experimental Procedures for details of analysis.
Products of Reactions of N2-MethylGuo with 2-Chlorooxirane and ClCH2CHO. In our previous studies on the mechanism of formation of 1 P - e A d e we utilized the approach of reacting Ns-methylAdo with 2-chlorooxirane and ClCHzCHO to form stable carbinolamine products, which could not readily dehydrate because of the presence of the tertiary nitrogen (28).The analogous W-methylGua-based carbinolamines had not been reported in the literature. Prolonged reaction (7 days at 37 "C) of W-methylGuo with ClCHzCHO yielded a mixture of products, the major two of which were assigned as the diasteromers of structures IIa and IIb (videsupra).
Scheme 11. Possible Reactions of N2-MethylGuo with 2-Chlorooxirane 0
D' +
0
I7ib N
Rib
CH3
0 HO
I CH3
8
H
Aib
dH3
H6
I 0
II
640 Chem. Res. Toxicol., Vol. 6, No.5, 1993 0
0
Guengerich et al. 2',2"--
0
' " [ ; a , I
CY
I
I
Rib
CY
4'--
I
A
11
~
.
-
- 7 - 6
7.0
6.0
5.0
4.0
3.0
2.0
WI , PPM
1
I_h
PPM
. 2
8.0
0 2 4 6 8 0 2 4
80
Rlb
6 8 1 0 1 2 0 2 4 6 8 1 0 1 2 0 2 4 6 8 1012
tR,
min
Figure 3. Evidence for transient formation of N2-methyl-N2(2-oxoethy1)guanosine in the reaction of N2-methylGuo and 2-chlorooxirane. The reaction mixture included 200 mM potassium phosphate buffer (pH 7.4),10 mM NZ-methylGuo, and 105mM 2-chlorooxirane [at 5 OC, where the tllz of 2-chloroxirane is 20 min (27)]. At varying time points, 25-pL aliquots were withdrawn and analyzad for the formation of stable carbinolamine products using HPLC with the standard 10 X 250 mm octadecylsilane column (27% CHaOH, v/v, in 50 mM NKHC02, pH 5.5, flow rate 7.0 mL min-l, Am). Aliquota of 50 pL were also added to 50 p L of a 1M CHsOH solution of NaBH4; another 100 pL of HzO was added, and reduction proceeded for 60 min. At that time 100 pL of 50 mM NKHCOz buffer (pH 5.5) was added to each sample, and 50 pL of the resulting mixture was analyzed by HPLC using the standard column and the elution system of 18% CHIOH, v/v, in 50 mM NH4HCOz (flow rate 4.0 mL min-l, Am).. (A) Standard ~-methyl,W-(2-hydroxyethyl)Guo;(B) reaction product to which NaBH4 was added after 1 min of incubation; (C)same injectionas sample B but with the coinjection (D)reaction of authentic N2-methyl-N2-(2-hydroxyethyl)Guo; product to which NaBHd was added after 8 min of incubation.
emphasized that the yield of this product would be expected to be low because of the dominance of the reaction at the Gua N7 atom. Compounds derived from initial attack at the N3 atom (Scheme 11,path d) were not prepared in this study, and it is therefore not possible to draw many conclusions about the feasibility of this pathway. However, with regard to the other three reactions presented in Scheme I1the order of preference is a > c > b, and the N2 atom of W methylGuoappears to attack the unsubstituted methylene carbon of 2-chlorooxirane. (Attempts to prepare authentic NS-methyl-l,W-e-Guo,a potential product in this work, were not successful.)^ Assignment of e Protons in NMR Spectra. For studies on isotopic labeling, the correct assignment of the chemical shifts of the two e protons of 1,W-e-Gua and W,3-cGua is critical. The assignments made by Sattsangi e t al. (11) are based largely upon labeling studies with CIC2H2CHO and also assume the mechanism. This (11) and other studies (8) also made some comparisons with model compounds; however, the absolute shifts of the two e protons vary considerably with the solvent environment, and the shiftsrelative to each other are of more significance. The assignment of the NMR shifts of the two e protons sone approach involved methylation of lJV1-s-Guowith CHsI in (CH&NCHO. Analpie of the products indicated that the reaction was dominated by methylation of the N1 nitrogen (correspondingto N7 in Guo) and that there waa extensive deribosylation and alkylation at that nitrogen (N3) by the excess CHJ. Another route involved reaction of tram-2-butenal (crotonaldehyde)epoxide with W-methylGuo to form 7-(hydroxymethyl)-lJVa-c-Guo, which was characterized by UV, NMFt, and MS. This was expected to generate the derived product by mild treatment with base (9,40). However, no significant reaction occurred even at 50 O C in 0.10 N NaOH for 14 h. This result suggests reevaluation of the proposed retroaldol cleavage mechanism proposed by others for the nonmethylated compound (9).
3 160 150
140
130
120 110
100
W
Bo
70
Bo
50
PPM
Figure 4. NMR spectra of 1,W-[5-16N]-e-dGuo. Spectra were recorded in (C2H&SO (Bruker AM-400). Individualaseignments are shown (see Table I). (A) Expansion of the individual regions of the 13CNMR spectrum. (B)Correlation spectroscopy (COSY) diagram showing W-lH attachments. (C) Full one-dimensional 19CN M R spectrum.
of 1,W-e-Guais at variance in the literature (contrast refs 11, 9, and 41 with 8 and 10). Therefore, we felt the assignments needed to be examined, particularly since our preliminary experiments revealed selective exchange of hydrogen atoms (uide infra). The shifts of the two t protons of OB-ethyl-W,3-t-Gua were determined using NOE measurements (an OB group was necessary to form the W,3-e-Guo adduct but difficult to remove without losing the ribose, which was necessary for the NOE work). Irradiation of the H-1' sugar proton at 6 6.22 produced an 11%positive NOE for the proton at 6 7.95 but did not affect the proton at 6 7.48. Thus the 6 7.95 proton (downfield) is considered to be H-5, in line with the assignment of Sattsangi et al. (11). In the assignment for 1,W-e-Gua the use of NOE methods is not straightforward. Therefore, [W-1sN]-lJv2e-Gua was prepared as described in the Experimental Procedures, and through-bond coupling was analyzed. [W15Nl-1,W-e-G~ohas previously been reported as an impurity in the labeled 7-(hydroxymethyl) derivative, which was used to obtain NMR spectra (42). However, spectra of [W-lSNl-l,W-e-G~o itself were not reported, and the resonances of the other compund cannot be used to make direct comparisons. The natural abundance NMR spectrumwas recorded in (CH&SO and is shown in Figure 4 (assignments in Table I). In the lH NMR spectrum, both the H-6 and H-7 protons were split with similar J values (3.3,4.7 Hz), in line with other protons a and j3 to IsN in unsaturated ring systems (43). However, the C6 13CNMR signal was split by the geminal 15Natom while the C7 signalwas not (Figure 4B,C), also in line with literature precedents for 1sN-13C splitting in unsaturated ring systems (43). The C7 carbon was shown to be attached to the proton at 7.62 ppm, clearly downfield of H-6 at 6 7.44 (Figure 4B). A further study was done with l,W-e-dGuo, utilizing the exchangeable NH(5) proton in (C2H3)2S0 solution (Figure 5). That proton is far downfield (6 12.5, cf. ref 9). When the upfield (6 7.4) e proton was irradiated, a strong (positive) NOE was seen with the downfield (6 7.6) proton and a weak (positive)NOE was seen with the NH(5) proton (Figure 5B,C) (the weak NOE due in part to exchange with 2H20). However, irradiation of the downfield (6 7.6)
Chem. Res. Toricol., Vol. 6, No. 5, 1993 641
Ethenoguanine Formation
Table I. Chemical Shifts and Coupling Constants for [5-1SN]-1fl-c-Guo coupling constants (J,Hz) chemical shift (6, ppm)" position
'H
9 2 6 7 1' 2', 2" 3' 4' 5', 5'' a
JnH
'3C
146.23 150.38 115.82 151.71 137.38 116.90 107.30 83.35 (0.50) -40 71.12 (0.68) 88.05 (0.60) 62.09 (0.31)
4a 3a 9a
8.142 7.442 7.622 6.266 2.265,2.611 4.382 3.851 3.526,3.596
3 J = 2~.6 ~ 3 J= 2.6 ~ ~ 3 J 6.2,7.6 ~ ~
JNH
JCN
2Jm = 3.3 3Jm 4.7
'JCN = 11.1
(C2H&S0.
A
7
6 ,I
H-2 1
1 7 6
0
1'
7.75
7.65
7.55
7.45
A , . -
7.35
915
910
815
S!O
715
710
615
610
6, PPM I
'
I
~
I
'
I
~
I
'
I
13.0 12.0 11.0 10.0 9.0 8.0
'
I
'
7.0 6.0
I
'
I
'
I I_ '
I
5.0 4.0 3.0 2.0
PPM
!
B
Irradiate at S 7.4
-r., ,
I
.
I
,
I
,
,
14131211109
,
it
,ll.
6
I
7
.
I
6
.
I
5
.
I
4
.
I
3
.
I
.
,
2 1
., 0
PPM Figure 5. 1H NMR spectra of 1,W-t-dGuo. Spectra were recorded in (C2Ha)zSO without exchange of protons. (A) Onedimensional spectrum. All assignments are labeled. The inset shows expansion of the e proton region. (B)Difference NOE spectra, with expansion of the NH(5) region.
proton produced a strong NOE with the other e proton but none with the NH(5) proton. Hydrogen Exchange of 1,N-and N2,3-e-Gua. The yields of the c-Gua adducts from 2-halooxiranes are very low (27,401,and initial plans were made to take advantage of MS to study the mechanism of formation. For instance, the facileexchange of the methylene protons of ClCH2CHO (28; t 1 / 2 shown to be -20 min) could, in principle, be used with 2Hz0 experiments to determine the role of this
~
Figure 6. 1H NMR spectrum of W,3-e-Gua after heating in 2Hz0 and 1.0 N HC1 at 100 "C for 60 min. The spectrum was recorded at 300 MHz in (CZH3)zSO. In this spectrum the assignments are 6 7.52 (s, l H , H-61,7.97 (s, lH,H-5), and 8.38 ( 8 , lH,H-2).
compound-as a rearrangement product of 2-chlorooxirane-in the formation of c-guanyl adducts. However, initial studies yielded rather unexpected labeling patterns. These were attributed to rather facile hydrogen exchange reactions that are not seen in the case of lP-e-Ado (28). The first exchange reaction observed was the selective loss of the H-5 proton of iV,3-c-Guaupon heating in mild aqueous HCl (Figure 6). This exchange explained our difficulties in preparing [5-2Hl-iV,3-e-Guafrom ClC2H2CH0 (initial reaction in 2H20),as the last step in the sequence is acid hydrolysis (HZO/HCl) of O6-benzyl-W,3t-Gua (11). Further studies were done at pH 7.7 (typical enzyme incubation pH) and 9.2 (used in the i n situ generation of 2-bromooxirane from BrzCHCH20H) (28). The H-5 proton of iV,3-c-Guo and the H-7 proton of lJV2-e-dGuo were selectively exchanged at either pH upon incubation for 7 days (in both cases considerable exchange at H-2 also occurred under the conditions used here) (Scheme 111). The 'H NMR spectra of 1,W-e-dGuo before and after incubation at pH 7.7 are shown in Figure 7. Much of the hydrogen at H-2 and even more at H-7 are exchanged; the H-6 proton is not exchanged (cf. deoxyribose protons) and now appears as asinglet instead of a doublet. The kinetics of these exchange reactions have not been examined in detail, but in other experiments it was noticed that samples exchanged protons even when mixed with and lyophilized from 2H20. Plausible mechanisms for acid- and base-catalyzed reactions are presented in Scheme 111. The mechanisms are hypothetical; careful kinetic studies at several pH values have not been done. The H-6 proton of 1 P - c dGuo was not exchanged in 0.10 N HCl, even upon heating for 60 min at 95 "C.
642
Chem. Res. Toxicol., Vol. 6, No. 5, 1993
Guengerich et al.
Scheme 111. Proposed Mechanisms of Acid- and Base-Catalyzed Hydrogen Exchange in e-Gua Derivatives
A
OD
/
H
'H
Formation of 1,N-and N,3-e-Guo by Reaction of Guo and Br2CHCH20H. In our previous studies on the mechanism of formation of 1,IP-e-Ado and 3,N4-e-Cyt,we found that reaction with BrzCHCHzOH at pH 9.2 provided a useful means of generating 2-bromooxiranein situ (28). This approach also proved to be applicable to the formation of the e-Gua adducts, although the yields were considerably lower (Figure8). The optimal pH forW,3-eGuoformation was -9; considerably less W,3-t-Guo was formed with C12CHCH20H. No W,3-cGua was formed from BrCH2CH20H (Figure 8);this information is important because traces of BrCH2CH20H contaminate Br2CHCH20H (28) and the e Guo products are recovered in low yield. Initial labeling studies were done with Br2CHC2H20H at pH 9.2. Both 1,W- and W,3-e-Guo products were obtained and analyzed by 'H NMR, since MS had been shown not to be useful because of the exchange problem. In each case, both of the two e protons were missing (e.g., Figure 9). However, the H-7 proton of lJV2-e-Gua and the H-5 proton of W,3-e-Gua had apparently been exchanged during the workup of the NMR samples in 2H20and actual analysis (Scheme 111). Attempts were not made to work in 'H2O as solvent and use suppression techniques because of the limited amounts of the samples available (Figure 9). Incubations were done with Br2CH13CH20H and Guo at pH 9.2, and the c-guanyl products were obtained and analyzed as the free bases, in (C2H3)2S0. The amounts of material recovered were not sufficient for l3C NMR analysis, so the analysis involved the W-lH splitting in the 'H NMR spectra. W,3-e-Gua was obtained in considerably higher yield than 1,ZP-cGua (Figure 10A). The H-6 proton (6 7.08) was split by the geminal 13C (J = 190 Hz) and the H-5 atom was split with a coupling constant typical for vicinal 13C-lH splitting (J = 9.2 Hz). These assignments were verified by decoupling analysis. The yield of 1,ZP-e-Gua in this experiment was considerably less, and the product had to be treated with 2H20 during the course of preparation for 1H NMR analysis (Figure
HF
D
10B). Several impurities are also present. However, the only 1 P - t - G u a protons observed were H-2 (6 9.06) and H-6, a doublet centered a t 6 7.32 and split with J = 140 Hz, indicative of 13C-lH geminal splitting. The H-7 proton is considered to have exchanged with 2H20. Thus, both the 2H and 13C labeling experiments with Br2CHCH20H support the view that the Guo N2 atom reacts with the unsubstituted methylene of 2-bromooxirane, generated in situ from Br2CHCH20H, to yield 1,Wt-Guo or fl,3-cGuo with the indicated labeling patterns (Scheme IV-A,B). The second t proton is readily exchanged during analysis (Schemes I11 and IV-A). These labeling patterns are not consistent with lJV2-t-Guo or W,-3-t-Guo formation proceeding from initial attack of the Guo N1 or N3 atom on the unsubstituted methylene of 2-bromooxirane. The presumed pattern for the preparation of 1JV2-eGua and W,3-e-Gua from ClCHzCHO in the work of Sattsangi et ai. (11) is shown in Scheme IV-D, with the view that the carbonyl would react with the exocyclic (N2) nitrogen (see also Scheme IV-C). If the putative 2-bromooxirane rearranged to BrCHzCHO and then reacted with Guo, then a labeling pattern opposite to that seen here should result for both 1PandN2,3-e-Gua.However, the possibilitiesfor exchange of deuterium before and after ring formation with C1C2H2CH0could preclude definition of that mechanism (involving ClCH2CHO) (Scheme IVD), and further study is needed to reach a conclusion as to its validity Ci.e., Sattsangi et al. (11)reported the loss of the NMR upfield etheno proton of 1,N2-eGua in a labeling study done with ClC2H2CH0, which is unexplained by the new NMR assignment (Table I) or the exchange results (Figure 7B) since the upfield proton is H-6 (Table I, Figures 4 and 5)l. Preparation of N2-(2-Osoethyl)dGuo and Formation of 1,N-and N,3-e-Cfua. The above evidence is consistent with the view that the principal initial reaction involved in formation of 1fl-t-Gua and N2,3-e-Guafrom 2-halooxiranes involves the initial attack of the Gua N2
Chem. Res. Toricol., Vol. 6, No. 5, 1993 643
Ethenoguunine Formation
A H-2
I
H-6
PH
u
Figure 8. Formation of N2,3-t-Guafrom Guo and 2-halo alcohols as a function of pH. Guo (5 mM) waa mixed with 0.20 M N-ethylmorpholineacetate (pH 9.2) and a 0.10 M concentration of either BrCHzCHzOH (A),Cl2CHCHzOH (A),or BraCHCH2OH ( 0 ) (total volume 1.0 mL). The pH of each incubation was adjusted to the value indicated on the axis. Vials were purged with Ar, sealed with Teflon liners, and incubated for 4 days at 37 OC. The buffer was then removed by lyophilization, and samples were dissolved in 1.0 mL of 0.10 N HC1 and heated for 60 min at 95 O C . Aliquota of 25-400 p L were analyzed for W,3t-Gua by HPLC using the standard octadecylsilane column, with an elution buffer consisting of 9% CHsOH (v/v) in 50 mM NHJ-ICOz (pH 5.5, flow rate 4.0 mL min-l, fluorescence detection with excitation at 216 nm and emission at 389 nm). 1JVZ-c-Gua formation was not detectable under these conditions due to the lack of sensitivity.
LL
6, PPM
J-,L B
I LO
I
,I
,
,
LO
83
L
ID
,I
IO
48
40
LO
6, PPM
Figure 7. 1H NMR spectra of 1JVZ-e-dGuobefore and after incubation at pH 9.2 for 7 days at 37 O C . Both were recorded in (C2H3)zS0at 400 MHz. (A) 1JVZ-e-dGuowithout incubation. Assignments: 6 3.54 (m, lH, H-5'),3.59 (m, lH, H-5"),3.86 (m, lH, H-4'), 4.38 (m, lH, H-3'), 4.87 (solvent), 5.22 (d, 2H, H-29, 6.38 (t, lH, H-l'), 7.39 (d, lH, H-6), 7.59 (d, lH, H-7), 8.11 ( 8 , lH, H-2). Note splitting of the H-6 and H-7 protons in the offset expansion of the region between 7.3 and 8.2 ppm. (B) 1JVZ-tdGuo after incubation in 50 mM N-ethylmorpholine acetate buffer (in 2Hz0,pH 9.2) under Ar for 7 days at 37 O C (no further purification except removal of N-ethylmorpholine acetate by lyophilization). Proton assignmenta are indicated on the spectrum. Note loss of H-2 and H-7 but persistence of H-6; also note lack of splitting of H-6 even though the ribose protons maintain their multiplicity.
atom on the unsubstituted methylene of t h e 2-halooxirane. If this hypothesis is correct, then it might be possible to prepare an N2-(2-oxoethyl)Gua derivative and observe its transformation to lJP-e-Gua and N2,3-e-Gua. T h e route depicted in Scheme V was employed: N2-(2-oxoethyl)dGuo diethyl acetal was synthesized and characterized by UV, NMR, and MS. T h e material was treated overnight with 1.0 N HC1 at 23 "C t o cleave the acetal (which also caused deribosylation), and HPLC analysis indicated essentially complete conversion t o a new product which eluted very early from t h e column [octadecylsilane, flow 4.0 m L min-l, 4.5% CH30H (v/v) in 50 m M NHdHC02 (pH 5.51, tR 8 minl. FAB+ MS of this compound indicated the loss of t h e deoxyribose and the acetal: mlz 194 (23, [M + HI+),216 (97, M Na), 238 (95, M 2Na),260 (51, M 3Na), 176 (100, M - HzO H+). T h e pronounced loss of H2O in the mass spectrum is similar to t h e behavior of t h e 1,W-cyclo addition product formed by the addition of crotonaldehyde to dGuo reported by Chung and Hecht (44) and Eder and Hoffman (45). Further, the UV spectra were more complex than those of simple W-substituted Gua derivatives but resembled those reported for the crotonaldehyde addition product 5,6,7,8-tetrahydro-8-hydroxy-6-methylpyrimi-
+
+
+
+
H
dRlb
1'
2
I 910
815
8!0
715
710
6:s
610
515
510
415
410
315
310
PPM Figure 9. 1H NMR spectrum of 1JVZ-e-Gua prepared by incubation of Guo with Br&HC2Hz0H at pH 9.2. The preparation (27) was isolated using HPLC, and the product showed the characteristic t~ and UV spectrum including the red shift in alkalai (11). The sample was prepared for NMR analysis by repeated lyophilization from 2H20 and recorded in 2Hz0 (400 MHz). Proton assignmenta are indicated on the spectrum. Note the appearance of all ribose protons and H-2 (6 8.49) and absence of both e protons.
do[l,2-alpurin-10(3H)-one (44) and also N-methylGua (Figure 11). T h e UV spectrum also resembled t h a t reported for the glyoxal-Gua condensation product, 6,7dihydroxy-1JP-e-Gua (46): A, (pH 5) 246 nm (e 10.6 mM-l cm-l) a n d 279 nm (e 6.3 mM-l cm-l) with minima a t 249 and 263 nm. For comparison, I has A, at 249 and 273 (e ratio to t h e glyoxal product) and minima at 227 and 263 nm (Figure 11). T h e N M R spectrum [(C2H3)2SOl did not show an aldehydic proton; a downfield triplet (6 5.99, 1H) was coupled t o an apparent doublet of doublets at 6 3.72 (1H). T h e other expected methylene proton appeared t o be obscured by t h e large peak centered a t 3.3 ppm. T h e (C2H&SO was removed in uucuo, and t h e sample was dissolved in [2H~lpyridine to move t h e interfering signals. T h e carbinol proton (H-7) now appeared as a doublet of doublets a t 6.71 ppm and was coupled to t h e two individual (H-6) methylene protons, which appeared as apparent doublets of doublets a t 6 3.84 and 3.94 (H-2 was at 6 8.12 in pyridine). T h e 6 6.71 signal was collapsed by irradiation of either of t h e upfield protons and vice versa. From the
-
644 Chem. Res. Toxicol., Vol. 6, No. 5, 1993
Guengerich et al.
I
I
10.0
I
I
B
I
9.5
I
9.0
I
8.5
200
240
280
320
200
240
280
320
Wavelength, nm Figure 11. UV spectra of (A) authentic W-MeGua and (B)the compound assigned the structure I, derived from HCl hydrolysis of N242-oxoethyl diethyl acetal) dGuo. Spectra are shown for pH 1 (“H+”),pH 5.5, and pH 13 (“OH-”) in 50 mM NHlHCOz buffer. I
8.0
I
I
7.5
7.0
I
6.5
I
6.0
along with some other unidentified products (results not presented). No 1,W-t-Gua or N,3-c-Gua was present in the N2-(2oxoethy1)dGuo diethyl acetal preparation after overnight treatment with 1.0 N HC1. An aliquot of the purified product was left in 0.10 MN-ethylmorpholine buffer (pH 9.2) for 5 days at 37 “C, the conditions used in the Br2CHCH20H labeling studies. At this point or after an additional 6 days at 23 “C, there was no evidence of formation of l,W-eGua (
