Identification and characterization of deoxyguanosine-crotonaldehyde

Toxicol. , 1992, 5 (6), pp 802–808 ... Publication Date: November 1992 ... Journal of the American Chemical Society 2003 125 (19), 5687-5700 .... Fo...
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Chem. Res. Toxicol. 1992,5, 802-808

Identification and Characterization of Deoxyguanosine-Crotonaldehyde Adducts. Formation of 7,8 Cyclic Adducts and 1,N2,7,8 Bis-Cyclic Adducts Erwin Eder* and Christian Hoffman Institute of Toxicology, University of Wiirzburg, Versbacher Strasse 9, 0-8700 Wiirzburg, Germany Received April 27, 1992

Crotonaldehyde, a chemically reactive a,p-unsaturated carbonyl compound, is an important industrial chemical and a ubiquitous environmental pollutant. It has been shown to be carcinogenic and mutagenic. We have studied the reaction of crotonaldehyde with nucleosides and 5’-mononucleotides and found three different types of adducts with deoxyguanosine and 2/-deoxyguanosine 5’-monophosphate. No adducts could be isolated either with nucleosides other than deoxyguanosine or with nucleotides other than 2/-deoxyguanosine5’-monophosphate. With crotonaldehyde, deoxyguanosine produced 1,N2 and 7,8 adducts as well as 1,N2/7,8bisadducts. The 1,N2adducts were mixtures of diastereomers: one pair in which the substituents in the newly formed ring were trans [adduct Ia (6S,8S) and (6R,8R)],about 94%, and another pair Ib in which they were cis. In the case of the 7,8-adducts IIa,b, the ribose was cleaved and a mixture of isomers in which the substituents were cis-IIa and trans-IIb (2:l) in the newly formed tetrahydropyrrole ring was observed. A 3:2 cis-IIIa and trans-IIIb mixture of 1,N2,7,8 bis-adducts was found with the isomerism in the newly formed tetrahydropyrrole ring in analogy to the 7,8 adducts IIa,b. The corresponding bis-adduct with the cis form in the newly formed tetrahydropyrimidine ring was not observed.

Introduction Crotonaldehyde (butenal) is an a,&unsaturated carbonyl compound. This group of chemically reactive compounds includes ubiquitous environmentalpollutants and also substances which are formed endogenously. Crotonaldehyde is an important industrial chemical used as a starting material for the synthesis of several compounds, including plastics and resins, pesticides and dyes, and pharmaceuticals. It is formed during combustion of materials containing carbon and is found in automobile exhausts, tobacco smoke, and flue gases (I). The compound is also formed during humic acid degradation and is a contaminant of surface water and drinking water (2). Like other &substituted acrolein congeners, crotonaldehyde is produced in mammals in some physiological and pathophysiological processes such as lipid peroxidation and arachidonic acid oxidation or in reactions of reactive oxygen species (3,4). The compound has been shown to be carcinogenic (5)and mutagenic (2,6,7).Most probably, these genotoxic activities depend on formation of DNA adducts. Indeed, Chung et al. (8) recently reported the formation of a 1,N2 cyclic deoxyguanosine adduct with crotonaldehyde. For these reasons, crotonaldehyde represents a potential source of DNA damage and is considered to be of great importance in human carcinogenicity (9). However, the severity of this damage and whether and to what extent it is involved in the induction of mutations and tumors have not yet been determined experimentally. Further investigations are necessary for a clear evaluation of the genotoxic and carcinogenic risks associated with this compound; in particular, an exact examination of its interactions with DNA is required. This paper presents a detailed description and characterization of the deoxy* To whom requests for reprints should be addressed.

guanosine and guanine adducts formed by crotonaldehyde with deoxyguanosine and with 5’-deoxyguanosine monophosphate.

Materials and Methods Chemicals. Caution: Crotonaldehyde is very toxic, is a strong irritant to eyes and mucosa, and is mutagenic and carcinogenic. Crotonaldehyde [(E)-2-butenall,purchased from Aldrich (Steinheim, FRG), was distilled under nitrogen, with a “Fischer Spaltrohr” column, immediately before use (104 “C/ 760 Torr). Its identity was checked by lH NMR spectroscopy and its purity by capillary gas chromatography (>99.5%). The nucleosides 2’-deoxyguanosine, 2’-deoxyadenosine,and 2‘-deoxythymidine were bought from Serva Biochemica (Heidelberg, FRG), in the highest purity available, and 2’-deoxycytidine monohydrate was bought from Sigma Chemie GmbH (Deisenhofen, FRG). The nucleotides 2‘-deoxyadenosine5’-monophosphate (free acid), 2’-deoxythymidine 5’-monophosphate (sodium salt), and 2’-deoxyguanosine5’-monophosphate (free acid) were purchased from Sigma Chemie GmbH. Alkaline phosphatase from bovine intestinal mucosa was purchased from Sigma Chemie. All chemicals, solvents, and reagents for the preparation of the buffers, the eluents, etc., were bought in the highest purity available from Merck, Darmstadt; Aldrich, Steinheim; Serva, Heidelberg; or Boehringer, Mannheim GmbH, FRG. Reactions of the Test Substances with Nucleosides a n d Nucleotides. To a solution of 0.412 mmol of nucleoside in 20 mL of 0.1 M sodium phosphate buffer (pH 7) was added 2.0 mmol of crotonaldehyde. The mixture was stirred at 80 “C for 22 h. Crotonaldehyde (2 mmol) was added to a solution of 365 pmol of the respective 5’-mononucleotide in 2.0 mL of 10 mmol of sodium phosphate buffer (pH 7), and the mixture was stirred under nitrogen for 22 h at 90 “C. After cooling to room temperature, the solution was filtered through a 0.45-pm filter and chromatographed on a Sephadex LH-201column as described later. 1 Abbreviations:LH-20,SephadexLH-20gelchromatography; MPLC, medium-pressureliquid chromatography;FT-IR, Fouriertransformation infrared;EI, electron impact; FAB, fast atom bombardment.

0893-228x/92/2705-0802$03.00/0 0 1992 American Chemical Society

Deoxyguanosine-Crotonaldehyde Adducts

Chem. Res. Toxicol., Vol. 5, No. 6,1992 803

Acid hydrolysis of the modified nucleotides was carried out as described previously (IO).

E

Chromatographic Methods. Sephadex LH-20 chromatography (LH-20) was carried out in a 100- X 1.6-cm glass column, wet-filled with Sephadex LH-20, 20-100 pm from Pharmacia (Freiburg, FRG), using double-distilled water as eluent. The flow rates indicated below were regulated with an Ismatec MVCA-4 pump (Wertheim, FRG). The chromatogram was recorded at 254 nm with an LKB 2138 Uvicord UV detector and a Metrawatt Servogar 5b recorder. Thirty-minute fractions were collected with an LKB 2111 Multi Rac fraction collector. MPLC was performed on a Labochrom “FPCG-prepacked” column, 18.5 mm X 28 cm, HD-Sil-l8-30-60,35-70 pm, reversedphase silica gel C8, and a Labomatic MD 80-100 pump fitted with a Labomatic PC-100 pressure control unit at 254 nm with an Isco detector, Model 248, using a stepwisegradient as indicated below. For HPLC, the Waters system consistedof a U6K injection system or a Rheodyne injection valve 1265, two Model 6000 A pumps, a Model 660 gradient solvent programmer, and a Model 450 variable-wavelengthdetector connected to a Hewlett/Packard 3385 A integrator. A reversed-phase C18 column, 5 pm, 0.46 X 25 cm, from Knauer (Homburg, FRG) was used for the analysis and a Bondapak reversed-phase, lO-pm, 0.78- X 30-cm column was used for semipreparative purposes. The solvents, gradients, etc., are described below. Chromatographic Isolation of the Adducts. The chromatographic purification and isolation of the adducts were complex. The reaction mixture obtained with deoxyguanosine was filtered through a 0.45-pm filter and then (in portions) subjected to LH-20. With a flow rate of 30 mL/h, fractions 1618contained adducts I. Several fractions 16-18 were combined, the volume was reduced to 20 mL, and the mixture was subjected to HPLC (Bondapak 10 pm; methanol/H*O; 11%methanol, 33 min; 40% methanol, 10 min; 100% methanol) with a flow rate of 8 mL/min. Under these conditions, adducts I had a retention time of 22 min. After lyophilization, 47 % of the pure mixture of the adducts Ia and Ib were obtained as colorless crystals with a melting point of 144-146 “C. Adducts IIa,b were isolated by using twice LH-20 with a flow rate of 18mL/h in fractions 28-31, with a yield of 1.5%,as colorlesscrystalline powder, decomposing at >250 “C. Fractions 2 6 2 7 under the same conditions contained a mixture of adducts I and 111. They were separated by an additional LH-20 run (20 mL/h). Fractions 22-23 were lyophilized and gave 4.1 % of adducts I11as colorless crystalline powder with a melting point of 187-189 OC. (The yields above are given in percent of the amounts of deoxyguanosineused in the reaction mixture). The 5’-deoxyguanosine monophosphate adducts were also isolated via LH-20 with a flow rate of 14 mL/h. The unreacted nucleotide elutes first, followed by the 1,N2cyclic adducts and later on, the polymers, oligomers, and the unreacted crotonaldehyde. The product-containing fractions were lyophilized and enzymatically hydrolyzed. Also traces (about 0.5 mg) of 7,8 adducts were isolated in a late eluting fraction. After enzymatic hydrolysis, adducts IIa,b were obtained. The reaction with 5’deoxyguanosineyielded (after hydrolysis) 8.3 % and 9.9 % of the Ia diastereomers, 0.4% and 0.9% of the Ib diasteromers, and about 0.3% of adducts IIa,b and traces of adducts 111. Analysis and Characterization. The substances were analyzed and characterized with the following instruments: UV spectroscopy: Kontron Uvikon Model 860 and Model 800 plotter, at pH 1(0.1 N HCl); pH 7 (water);pH 13 (0.1 N NaOH). FT-IR spectroscopy: Nicolet 5 DXC. lH NMR spectroscopy: Bruker AC 250 (250.1 MHz) and a Bruker WM 400 (400.1 MHz); tetramethylsilane (6 = 39.7ppm). l3C NMRspectroscopy Bruker AC 250 (62.9 MHz) and a Bruker WM 400 (100.6 MHz); tetramethylsilane (6 = 39.7 ppm). Mass spectroscopy: (a) Electron impact mass spectrometry (EI): Varian MAT CH7 (70 eV) fitted with a data system 200. (b) Fast atom bombardment (FAB) mass spectra: 8200 Finnigan VAT C 70-eV spectrometer. Xenon and argon were used as ionization gases and glycerol and triethylene or tetraethylene glycol as FAB matrix. ( c ) Ther-

(trans)

IA

c 0

m a 2.

:: ‘ct

Ib (CIS)

t 10 20 30 40 50 min + v)

Figure 1. HPLC of the 1,N25’-deoxyguanosinemonophosphate adducts with crotonaldehyde after hydrolysis with alkaline phosphatase. mospray mass spectrometry (HPLC/MS): Finnigan Mat 450 quadrupolemass spectrometer with Finnigan thermospray LCMS interface connected with a Waters Model 6500 A HPLC pump; water/methanol eluent, flow rate 1.2 mL/min. Ionization was initiated with a 17 mM ammonium acetate solution. The C, H, N elemental analysis was performed at the Institute of Inorganic Chemistry (University of Wiirzburg). Characterizationof the Adducts. The four diastereomers of adducts Ia,b were stable and could be separated by HPLC (Figure 1). The two diastereomers of Ia gave identical lH NMR and 13C NMR spectra (Table I), and Ia and Ib gave identical FT-IR, UV, and mass spectra (Table 11). After acid hydrolysis of la, the respective guanine adducts I’ were obtained in 80% yield as a colorless crystalline powder with a melting point of 191-193 “C. I’eluted in LH-20 (18mL/h) in fractions 22-27 and had a retention time of 18.3 min in HPLC (Knauer column, 5 pm, H2O/methanol 89:11, isocratic, flow rate 1 mL/min). The spectroscopicdata as well as the elemental analysis are presented in Tables I and 11. Adducts IIa,b were also stable and could be separated by HPLC with retention times of 10.6 and 14.8 min (conditions as above). Three diastereomers of adducts IIIa,b could be clearly distinguished by HPLC (Bondapak column, 10 pm, HzO/methanol 9010, isocratic, 2 mL/min) in the ratio 14:2858 with retention times of 6.34,8.18, and 15.0 min. Thus a cis:trans ratio of 58:42 can be calculated. The lH NMR spectroscopic determination using the ratio of the 5”-H resulted in a &:trans ratio of 6040. The spectroscopicdata of IIa,b and IIIa,b are presented in Tables I11 and IV.

Results After the reaction of crotonaldehydewith either deoxyguanosine or ita 5’-monophosphate, we isolated three differenttypes of adducts (see Scheme I). Most probably, the guanine moiety is the most reactive DNA-target for crotonaldehyde since we were not able to isolate any adducts with other nucleosides or nucleotides. UV and lH NMR spectroscopy of the LH-20fractions obtained from the reactionmixturescontainingcrotonaldehyde and other nucleosidesother than deoxyguanosine did not reveal any adducts. A total of 67 % of the unreacted deoxyadenosine was reisolated from the reaction mixture of deoxyadenosine and crotonaldehyde and 97 % deoxycytidine from the reaction mixture of deoxycytidine and crotonaldehyde. The reaction conditions were the same as with deoxyadenosine. The residue was polymers and oligomers.

Eder and Hoffman

804 Chem. Res. Toxicol., Vol. 5, No. 6,1992 Table I. NMR Data of 1,N2Cyclic Deoxyguanosine and Guanine Adducts OH OH 0 OH 0 .

Adduct I’a

Adducts la,b

proton 11-H coupling 7a-H coupling 7b-H 6-H 8-H 8-OH coupling 2-H N(5)-W N(3)-H 2’a-H 2’8-H 5’-H 4’-H 3’-H 5’-OH 3’-OH coupling 1‘-H coupling

‘H NMR (400.1 MHz, [DslDMSO) adducts Ia,b adduct I’a 1.21 d, 3 H 1.21, d, 3 H J11,~ 6.3 HZ 1.42, m, 1H 1.41, td,1H J7&7b = 13.0 HZ J7a,7b = 13.0 HZ J7a,e = 13.0 HZ J7,e 13.0 HZ J7-8 = 2.3 HZ 2.02, br d, 1H 2.13, br d, 1H 3.70, m, 1H 3.70, m, 1H 6.20, m, 1H 6.19, br s (t), 1H 6.58,’ very br 8, 1H 6.65,O d, 1H JOH,8 4.5 HZ 7.91,s, 1H 7.93,” br s, 1H 7.66; s 7.66,bs 12.50,” very br 8 , 1H 2.18, m, 1H 2.51,bm, 1H 3.52, m, 2 H 3.80, m, 1H 4.36, br 8, 1H 4.95,’ br 8 , 1H 5.27,” d, 1H JoH,a = 3.3 HZ 6.10, dd, 1 H J1(,p 7.8 HZ

1

I3C NMR (100.6 MHz, [DalDMSO) C atom c-11 c-7 C-6 C-8 C-lob c-2 C-3b C-4b c-10 c-2‘ c-5’ c-3’

c-1’ c-4’

adducts Ia,b 20.66, g 35.21,’ t 40.30,’ d 69.61, d 115.65,s 135.48, d 150.02, s 150.88, s 153.79, s

adduct I’a 20.77, q 35.45, t 69.61, d 113.80,s 137.20, d 152.80, s 150.54,s 155.42,s

39.60,’ t 61.87, t 70.88, t 82.46, d 87.65, d

a The signal disappears after shaking with DzO. The signals are partly overlapped either by other signals or by the solvent signal. c Values determined by means of a DEPTspectrum (62.9 MHz). The C-3b and C-lob signals are very weak. Only those couplingconstants are presented which could be determined and assigned exactly.

Table 11. FT-IR,UV Spectroscopic Data, Mass Spectra, and Elemental Analysis of the 1,N*Cyclic Deoxyguanosine and Guanine Adducts

uv A, (l-lm)(log c) MS ( % I adduct FT-IR (KBr) Y (cm-1) (0.1 N HCl; 0.1 N NaOH) elemental analysis Ia,b 3377 (OH), 2929 (CH), 1691 HzO 259 (4.17), 277;’ Ami, C ~ ~ H ~ Q N(337.3).2.5HzO: JOJ thermospray m / z ( % ) 338.0 (43, MH+), calcd, C 43.89, H 6.33, N 292 (lo), 244 (lo), 222 (100, MH+ (C=O), 1572,1537 (C=N, 227 (3.61) 18.32; found, C 43.86, H - dR), 166 (8), 152 (38, Gua H+); C=C), 1332, 1226,1117, HCI: 202 (4.25), 261 (4.13), 783,642,474 28%’ A- 235 (3.57) 5.79, N 18.33 FAB-MS (70 eV, glycerol) m/z (% ) 338.0 (29, MH+),236.0 (4), 222.0 NaOH: 216 (4.13),260 (70, MH+ - W), 204.0 (9, MH+ (4.12), 277;’ Ami, 243 (370) dRHzO), 178.0 (l8), 152.0 (6, Gua H+),141 (13) I’a 3390 (NH), 3228 (OH), 2975 H20: 252 (4.09), 283 (3.85); C Q H ~ ~ N(221.2)-1.25H~O: JO~ thermospray m/z (%) 222 (100, MH+), 190 (1); ELMS (70 eV) m/z (%) calcd, C 44.35, H 55.58, N Amin 230 (3.74), 272 (3.00) (CH), 1685 (C=O), 1572 28.73; found, C 44.17, H 222.2 (9, M+ + l),221.3 (41, M+), (C=N, C=C), 1331,786, HCI: 202 (4.26), 256 (4.10); Ami, 230 (3.65) 5.59, N 28.74 193.1 (16), 178.1 (70), 151.1 (100, 775,640 Gua+),136.1 (ll),135.2 (38), 134.1 NaOH 222 (4.39), 380 (3.98); (121,110.2 (30), 109.1 (26),70.2 Ami, 248 (3.67) (35), 69.2 (29), 54.3 (20), 53.2 (22), 44.3 (27),43.2 (43),41.1 (49), 39.1 (34) Shoulder.

The reaction conditions were optimized by HPLC analysis of aliquota taken at intervals from reaction mixtures at different temperatures and with different concentrations and amounts of reactanta. The reaction conditionsdescribed in Materials and Methods were found to provide the highest yields as shown in Scheme I. With deoxyguanosine, we isolated a pair of trans diastereomers Ia (51% and 42.5%) and a pair of diaster-

eomers Ib (4.5% and 2-15?,), most probably cis isomers: (6S,8S)-and (6R,8R)-3-(2-deoxy-8-~-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8-hydroxy-6-methylpyimido[1,2-aIpurin-l0(3H)-one. These four diastereomers are stable and do not epimerize at room temperature. Figure 1 shows the HPLC of the enzymatically hydrolyzed nucleotide fractions. Furthermore, we isolated the cis and trans isomers of the 7,8adducts IIa and IIb (2:1),

Chem.Res. Toxicol., Vol. 5, No.6, 1992 805

Deoxyguanosine-Crotonaldehyde Adducts

Table 111. FT-IR and UV Spectroscopic and Mass Spectrometric Data of 7,8 Cyclic Adducts and 1,Na,7,8Bis-Cyclic Adducts adduct IIa,b

FT-IR (KBr) Y (cm-1) 3325,3172 (OH, NH), 2885,2746 (CH), 1683 (C-0), 1540,1466,1377,1111, 777,576

IIIa,b

3367,3296 (OH), 2976,2934 (CH), 1699 (C=O),1582,1553 (C=N, C=C) 1380,1352,1327,1310,1192,1118, 1085,779,558

uv A,,

216, 250, 284

Amin 236, 265 ,A, 223,256,290

ELMS (70 eV) m/z(%) 222.3 (13, M++ l),221.3 (100,M+), 206.1 (10, M+H20 l),204.1 (9), 180.1 (261,179.1(37), 163.1 (lo), 151.1 (18, guanine+),138.1 (151,110.1 (6), 81.1 (6), 69.1 (7), 53.0 913), 42.9 (38) thermosprayMS m/z (%) 222 (100, MH+) 292.1 (30, M+ + l),291.2 (100, M+), 262.1 (16), 249.1 (13), 248.1 (78), 221.1 (84, M+- C A O ) , 206.0 (231,163.0(8). 151.1 (5, guanine+),138.1 (4), 121.0 (7), 83.1 (6), 69.1 (ll),57.1 (M), 43.9 (53)

+

Ami, 241,274

Table IV. 1H NMR Data of the 7,8 Cyclic Guanine Adducts and 1,N2,7,8Bis-Cyclic Adducts (250.1 MHz, [DeIDMSO)

Adduct IIa

proton

cis-IIa

Adduct

trans-IIb

10-H coupling

&,7

1.50, d, 3 H = 6.3 Hz

J10,7=

1.57, d, 3 H 6.3 HZ

6b-H 6a-H coupling coupling coupling

2.39-2.53,b m, 1H 3.02, d, 1H J&,& = 13.5 HZ Jsa,7= 7.0 HZ Jea,5 7.0 HZ

2.39-2.53,b m, 1H 1.89, dt, 1H J&,& = 13.5 HZ Jh,, = 4.3 HZ J h , 5 = 4.3 HZ

7-H coupling 5-H coupling 5-OH coupling

4.57, ps sext, 1H J = 6.5 Hz 4.97-5.06 m, 1H J5,6b = 4.3 HZ 5-77,”d, 1H J O H , 5 = 5.5 HZ

4.30-4.40, m, 1H 4.90-4.97, m, 1H = 8.01 HZ 5.85,” d, 1H JoH,~ = 5.5 HZ J5,6b

proton 4‘-CH3 coupling 5‘a-H 5”-CH3 coupling 5‘-b-H coupling 4“b-H 4”a-H coupling coupling coupling 4’-H 5”-H coupling 3”-H coupling 3”-OH 6‘-H 6-OH NC3’H

ma

cis-IIIa 1.20, d, 3 H JCHs,4’= 6.3 HZ 1.42, tm, 1H 1.51, d, 3 H JCHs,5” = 6.3 Hz 2.01, br d, 1H J5%,51a = 13 HZ 2.38-2.50: m, 1H 3.03, dt, 1H J4tta,4q, = 13.5 HZ J4”%5” = 7.0 HZ J4ra,3” 7.0 HZ 3.70, m, 1H 4.57, ps sext, 1H J = 6.5 Hz 5.01, m: 1H J3>!,4q, 4.3 HZ 5.75-5.89,” m, 1H 6.17, br s, 1H 6.62,O very br s, 1H 7.65,” S, 1H

trans-IIIb 1.20, d, 3 H Jcb,,r( 6.3 HZ 1.42, tm,1H 1.58, d, 3 H JcH~,~,, 6.3 Hz 2.01, br d, 1H J5$,6ta = 13 HZ 2.38-2.60: m, 1H 1.90, dt, 1H J4,ja,4q = 13.5 HZ J4tpb311 = 4.3 HZ J4(t45” 4.3 HZ 3.70, m, 1H 4.35, m, 1H

4.94, m: 1H J3”,4q,,= 7.3 HZ 5.75-5.89,’ m, 1H 6.17, br s, 1H 6.62,” very br 8, 1H 7.65,’ 8, 1H

6.18,’ s, 2 H N2-Hz 6.18,” s, 2 H 10.83,“very br s, 1H N(l)-H 10.83*, very br s, 1H 4 The signal disappears after shaking with D20.b The signal is overlapped by the solvent signal. J is determined after addition of D20.

2-amino-5,6-dihydro-5-hydroxy-7-methyl-lH-pyrrolo[l,2dlpurin-9(3H)-one,and the 1,N2,7,8bis-adductsIIIa,b (cis/ trans 3:2), (f)-4’-methyl-6‘-hydroxy-3’,4‘,5‘,6‘-tetrahydro3”-hydroxy-5“-methyl-2”,3”,4”,5”-tetrahydro-lH,7N-6oxopyrimido[1,2-a]pyrrolo[ 1,a-Dpurine. According to HPLC analysis, there are at least three stable diastereomers of IIIa,b which do not epimerize (see Characterization of the Adducts). In principle, two different regioisomers of 1,N2adducts are possible, one in which the OH group is vicinal to the N-1 atom of the guanine moiety and the other in which the OH group is vicinal to the N2 atom. In earlier investigations,we found that acrolein forms both types of isomers (1)whereas methyl vinyl ketone and ethyl vinyl ketone forms only the second type (IO). With crotonaldehyde, we found exclusively the first type. As discussed recently in more detail (IO),the two possible regioisomers can be distinguished by lH NMR spectroscopy. In the case of crotonaldehyde, the lH NMR spectra gave clear evidence for the first type of regioisomer: the OH protons of Ia,b and I’ appear at 6 = 6.65 and 6.68 ppm, the 8-H protons at 6 = 6.20 and 6 = 6.19 ppm, and the N-5 protons at 6 = 7.93 and 7.66 ppm. In the case of the other regioisomers, the OH protons are expected to appear at a higher field of 6 = 5.6-6.0 ppm, the 8-H proton between

6 = 4.2 and 4.5 ppm, and the amine proton at 6 = 8.0-8.5 ppm (10). The position of the OH group was also confirmed by Chung et al. (8) by a different technique. These authors reduced the crotonaldehyde-guanine adduct with NaBH4 in 0.5 M NaOH. This reaction leads to ring opening at the N-1 atom unless the OHgroup is vicinal to the N2 atom where no such ring opening is observed. In their publication, Chung et al., reported the separation of 65% of adduct I from the reaction mixture via simple silica gel column chromatography. We could, not however, reproduce this result. The UVand lH NMR data reported by Chung et al., are identical with our data. The three different types of crotonaldehyde adducta 1-111 exhibit some characteristic spectroscopic properties and can be easily distinguished by their chromatographic properties (see above) and their spectra (see Tables I-IV). Thus, structures can be readily assigned to the adducts, for example, by HPLC analysiswith diode array detection. FT-IR techniques have also been used recently to determine the substitution patterns of guanine in binding studies (11,12). WithadductsI1,veryintensiveabsorption bands of the carbonyl vibration at 1683 cm-l and three further characteristic bands at 1540,1465, and 1378 cm-1 are observed. Figure 2 demonstrates that adducts 1’, 11, and I11can be clearly distinguished by their characteristic

806 Chem. Res. Toxicol., Vol. 5, No.6,1992

Eder and Hoffman

Scheme I. Reaction of Crotonaldehyde with Deoxyguanosine OH

c H3 L

OH 0

o

phosphate buffer 80°C/22h

1.5% * II A,B cisltrans 2:l

4.1%

111 A,B cidtrans 3:2

H

H

,

,

,

~

~

,

~

l

1 3200 2400 1900 1500 1100 850

l

l

650

l

I l

l

~

l

400

Wavelength ( c m -’)

Figure 2. FT-IR spectra (KBr) of the crotonaldehyde adducts 1’, IIab, and IIIa,b.

FT-IR spectra. It must, however, be stated that the IR spectra are not applicable to the discrimination of the different regioisomers (see above). The EI-MS of adducts

IIa,b gives the same molecule peak as 1’; however, the mass fragments are clearly different. This is indicative of two different structures for the 1:l crotonaldehyde guanine adducts I’ and 11. The structure of the 7,&adducts IIa,b can be derived from the lH NMR spectrum. The free amine protons of N(l)-Hand N2-Happear at 6 = 10.83 and 6.18 ppm. This is proof that adducts IIa,b can be neither N-1 nor N2 adducts (and of course also not N-1,N2cyclic adducts). The absence of a signal from the 8-H proton of the guanine moiety is evidence for a C-C bond at this position (C-4b and C-5 of the new nomenclature, see formulas, Table IV, adduct IIa). The deoxyribose muat be cleaved since no deoxyriboseprotons are observed. Evidently the OH group is located on the C-5 atom. Both the cis and the trans signals of the 5-H and 7-H protons are observed in the range 4.57-5.06 ppm. The difference in the chemical shift is only about 0.5 ppm. This difference would expected to be higher if the OH protons were located on the N-7of the guanine moiety (N-8 in the new nomenclature) since in this case the neighboring heteroatoms would produce a strong low-field shift. The proposed structure is in accordance with the following reaction mechanism: the first step must be a Michael addition of the guanine N-7 atom to the double bond of crotonaldehyde (see Discussion). The conformation of the adducts is dealt with under Discussion. The structure of the adducts IIIa,b was also ascertained by spectroscopic methods. The EI-MS gave the molecular ion (291.2) as the base peak, indicating the presence of a 2:l adduct formed from two molecules of crotonaldehyde and one molecule of deoxyguanosine without deoxyribose. The mass fragments (Table 111) are also consistent with the proposed structure. Although the lH NMR spectrum is complex, the adducts could be clearly identified by comparison with the spectra of I’ and IIa,b. The bisadducts IIIa,b bear the structural elements

Deoxyguanosine-Crotonaldehyde Adducts

Chem. Res. Toxicol., VoE. 5, No.6, 1992 807 1.42

of the 1,N2 cyclic adduct I’ and those of the 7,8 cyclic adducts IIa,b. The cis form of the tetrahydropyrimidine ring was not observed. However, a 3:2 cis:trans mixture IIIa,b with the isomerism in the tetrahydropyrrole ring was isolated (see Characterization of the Adducts).

Discussion Crotonaldehyde is a carcinogenic and mutagenic compound of great importance in industry and as an environmental pollutant (see the Introduction). Most probably its carcinogenic and genotoxic effects depend on interactions with DNA components which have not been previously explored in detail. The structures of the putative adducts of this compound must be established before highly sensitive detection methods for the determination of such adducts in humans can be developed. This is a prerequisite for a clear risk evaluation for crotonaldehyde. It is not yet clear to what extent the various adducts 1-111 described here are responsible for mutations and contribute to the initiation of cancer. The adducts I are the main adducts, and they represent a significant DNA lesion because the N-1 and N2atoms which are of essential significance for base pairing are blocked. In the case of adducts 11,there is no such blocking; however, the adduct formation leads to cleavage of the deoxyribose which can result in depurination and strand breaks. It is possible that adducts I11 represent the most severe DNA lesion since they combine both genotoxic effects. Adduct I has been described by Chung et al. (81, who published the UV and ‘H NMR spectra of 1’. We can confirm the results of Chung et al. and have provided additional data which could be important for the understanding of the genotoxic mechanism as well as for risk assessment. Furthermore, we additionally found the respective cis adduct in which both the OH group and the CH3 group are in the axial position (see Scheme I). To our knowledge, the isolation of this cis adduct has not yet been described. Chung et al. (13) reported recently the detection of 7,8 adducts in hepatic DNA of rats treated with N-nitrosopyrrolidine and in calf thymus DNA reacted with a-acetoxy-N-nitrosopyrrolidine. They also provided spectroscopic data which demonstrate that these 7,8 adducts are identical with the 7,8 adducts which they isolated in a control experiment with crotonaldehyde and deoxyguanosine. The structures of adducts 2 and 3 described by Chung et al. are identical with those of our adduct IIa,b isomers isolated after reaction of crotonaldehyde with deoxyguanosine, and the spectroscopic data presented by these authors are consistent with ours. Thus the finding of Wang et al. (14) that crotonaldehyde is a metabolite of N-nitrosopyrrolidine and Chung’shypothesis that crotonaldehyde plays a role in the carcinogenesis of N-nitrosopyrrolidine is supported by our results. Adducts IIIa,b also had not yet been described for crotonaldehyde; however, Shapiro et al. (15)recently presented a similar bis-adduct for acrolein obtained in the reaction of acrolein and guanosine a t pH 4. However, these authors described another regioisomer, namely, one in which the OH group is vicinal to the N2 atom. Their result is consistent with our finding that acrolein forms both regioisomers (see above). For steric reasons, crotonaldehyde forms only the regioisomer in which the OH group is vicinal to the N-1 atom. Consequently, in the

t

6 60

3 70

Figure 3. Chair conformation of the newly formed tetrahydropyrimidine ring of the trans adduct Ia. Geminal coupling is only found between the 7-H protons and axial, axial vicinal coupling only between 6-H and 7a-H.

1,N2,7,8 bis-adduct of crotonaldehyde the OH group is also adjacent to the N-1 atom. Another interesting feature is the conformation of the adducts. In the chair configuration of the tetrahydropyrimidine ring (see Figure 3) of the trans adduct Ia, the alkyl group is in the energetically favored equatorial position, whereas the OH group is in the axial position. In general, the equatorial position is favored; however, polar groups in the a-position to a hetero ring atom frequently take an axial position (anomeric effect). In the cis isomers Ib both the OH group and the CH3 group presumablytake the axial position. For energetic reasons, only very low amounts of the cis isomers Ib are formed. Unfortunately, the amounts of the cis products Ib isolated were insufficient for us to record lH NMR spectra. The FT-IR, UV, and mass spectra of these isomers are, however, identical with those of the main products Ia. In an earlier paper (IO), we reported that the conformation of the tetrahydropyrimidine ring can be deduced by consideration of the coupling constants. The geminal coupling constants are about 13 Hz. Three different vicinal coupling constants are observed in the chair form of the tetrahydropyrimidine ring: the axial, axial coupling constant (3Ja,J,which was in the range of 12-14 Hz throughout our experiments, and the axial, equatorial (Va,& and the equatorial, equatorial coupling constants ( 3 J e , e ) , which were about 2.3 Hz. The only geminal coupling (13 Hz) observed is between the two 7-H protons. An axial coupling of 14 Hz is observed between the 6-H proton and the 7a-H proton of the trans adduct. In no case is axial coupling observed between the 8-H and the 7-H protons. For this reason the 8-OH group must be located in the axial position (anomeric effect) and the alkyl group of the trans adduct in the equatorial position (see Figure 3). The conformation of the 7,8-tetrahydropyrrole ring in adducts I1 and I11 is possibly an envelope type in which the atom C-6 is not in the plane of the ring. In the cis configuration of IIa and IIIa, both substituents are in the energetically favored (pseudo-) equatorial position. In the case of the trans adducts IIb and IIIb the OH group presumably takes the equatorial position; however, the pseudo-axialposition (anomeric effect) cannot be excluded. We did not find any indications that regioisomers other than those described in the Results were formed. The position of the OH substituents of adducts I was ascertained by our group and by Chung et al. (8). A formation of a 4,N2 cyclic adduct can be excluded since the N( 1)-H proton was not found. This proton appears at 10.83 in the 7,8 cyclic adducts IIa,b but not in the adducts I and 111. Furthermore, we found a characteristic strong low-field shift for the equatorial (8-H) proton (6 = 6.20 ppm) due to a combination of the influence of the OH group and that of the carbonyl function (on C-6 of the guanine moiety). Carbonyl functions, generally, have a strong

808 Chem. Res. Toxicol., Vol. 5, No. 6,1992

Scheme I1

descreening effect on vicinal protons which are situated in the nodal plane of the n-bonds (see also Figure 3). In contrast to the low-field shift of the C-8H proton, a much higher field is observed for the C-6H protons of the regioisomers with the OH group vicinal to the N2atom of acrolein (1) or of methyl (ethyl) vinyl ketone (10) which appear in the range 6 = 4.2-4.5 ppm. The presence of the characteristic C-8H proton is proof against the formation of a4,N2adduct. Furthermore, in the case of a 4,N2adduct an additional proton, namely, the C-3H proton (of the guanine moiety), would be observed. It may be that, for steric reasons, the deoxyribose moiety hinders the formation of such adducts. The positions of the OH group of the 7,8 adducts IIa,b were also ascertained by lH NMR spectroscopy (see Results). The formation of these regioisomers is in accordancewith the reaction mechanism shown in Scheme 11. a,@-Unsaturatedcarbonyl compounds are, in general, good Michael systems. The first step in the reaction must be a Michael addition in which the N-7 atom attacks the C-3 atom of crotonaldehyde. A Michael addition to the C-8 atom is unlikely under these reaction conditions. The second step is an attack on C-8 atom by the carbonyl atom of crotonaldehyde, leading to cyclization. As a consequence of this reaction, the deoxyribose is cleaved. We did not isolate any precursors of the adducts I1 such as an N-7(9)+/C-8-zwitterion and did not examine whether the loss of the deoxyribose occurs before or after cyclization at the C-8 atom. For the genotoxic consequences, it is important that the deoxyriboseis cleaved, and the question as to when thiscleavage occurs is only of secondaryinterest. A first attack at the carbonyl function of crotonaldehyde is very improbable since the activation of the double bond as described above (Scheme 11)would be abolished in this case and thus asubsequent Michael addition would become impossible. We have described the formation of the deoxyguanosine adducts and presented their detailed structures and properties. These new data provide for a better understanding of the genotoxic and carcinogenic activities of crotonaldehydeand form a basis for better risk assessment. However, further investigations, in particular, the development of sensitive detection methods for these adducts, are required before the role of this industrially important and environmentally ubiquitous carcinogen and mutagen can be evaluated.

Eder and Hoffman

Acknowledgment. We are grateful to Dr. G. Lange for recording the mass spectra, Mrs. J. Colberg for performing the thermospray mass spectra, and Mrs. E. Ruckdeschel for recording the NMR spectra. We thank Mrs. C. Grimm,Mrs. D. Deininger, and Mr. E. Weinfurtner for excellent technical assistance. We are indebted to Dr. A. Dunlop for linguistic assistance. References Eder, E., Hoffman, C., Bastian, H. Deininger, C., and Scheckenbach, S. (1990) Molecular mechanisms of DNA damage initiated by a,@unsaturated carbonyl compounds as criteria for genotoxicity and mutagenicity. Environ. Health Perspect. 88, 99-100. Neudecker, T., Lutz, D., Eder, E., and Henschler, D. (1981) Crotonaldehyde is mutagenic in a modified Salmonella typhimurium mutagenicity testing system. Mutant. Res. 91, 27-31. Esterbauer, H., Eckl, P., and Ortner, H. (1990) Possible mutagens derived from lipids and lipid precursors. Mutat. Res. 238,223-233. Comporti, M. (1989) Three models of free radical-inducedcell injury. Chem.-Biol Interact. 72, 1-56. Chung, F. L., Tanaka, T., and Hecht, S. S. (1986) Induction of liver tumorsin F 344rats bycrotonaldehyde. Cancer Res. 46,1285-1289. Eder, E., Henschler, H., and Neudecker, T. (1982) Mutagenic properties of allylic and a,fl-unsaturated carbonyl compounds: consideration of alkylating mechanisms. Xenobiotica 12,831-848. Neudecker, T., Eder, E., Deininger, C., and Henschler, D. (1989) Crotonaldehyde is mutagenic in Salmonella typhimurium TA100. Environ. Mol. Mutagen. 14, 146-148. Chung, F. L., Young, R., and Hecht, S. S. (1984) Formation of cyclic 1,N2-propanodeoxyguanosine adducts in DNA upon reaction with acrolein or crotonaldehyde. Cancer Res. 44,9!30-995. Eder, E., Deininger, C., Neudecker, T., and Deininger, D. (1992) Mutagenicity of @-alkylsubstitutedacrolein congeners in the Salmonella typhimurium strain TAl00 and genotoxicity-testing in the SOS chromotest. Environ. Mol. Mutagen. 19, 338-345. Eder, E., Hoffman, C., and Deininger, C. (1992) Identification and characterizationof deoxyguanosine adducts of methyl vinyl ketone and ethyl vinyl ketone. Genotoxicity of the ketons in the SOS chromotest. Chem. Res. Toxicol. 4, 5C-57. McGuinness, B. F., Nakamishi, K., Lipman, R., and Tomasz, M. (1988) Synthesis of guanine derivatives substitutedinthe 06-position by mitomycin C. Tetrahedron Lett. 29,4673-4676. Tomasz, M., Lipman, R., Mc Guinness, B. F., and Nakanishi, K. (1988) Characterization of a major adduct between mytomycin C and DNA. J. Am. Chem. SOC. 110, 5892-5896. Chung, F. L., Wang, M., and Hecht, S. S. (1989) Detection of exocyclic guanine adducts in hydrolysates of hepatic DNA of rata treated with N-nitrosopyrrolidine and in calf thymus DNA reacted with a-acetoxy-N-nitrosopyrrolidine.Cancer Res. 49, 2034-2071. Wang, M., Chung, F. L., and Hecht, S. S. (1988) Identification of crotonaldehyde as a hepatic microsomal metabolite formed by a-hydroxylation of the carcinogen N-nitroeopyrrolidine. Chem. Res. Toxicol. 1, 28-31. Shapiro, R., Sodum, R. S., Everett, D. W., and Kundu, S. K. (1986) Reactions of nucleosides withglyoxal and acrolein (Singer, B., and Bartach, H., Eds.) IARC Scientific Publications No. 20, pp 165173, IARC, Lyon.

Registry No. (GS,BS)-Adductla, 85405-01-2;(GR,BR)-adduct la, 85352-97-2; (GR,BS-adductlb, 143441-78-5;(GS,BR)-adduct lb, 143441-79-6; cis-IIa, 120917-73-9; trans-IIb, 120917-74-0; adduct I11 isomer 1, 143371-41-9;adduct I11 isomer 2, 14344180-9; crotonaldehyde, 4170-30-3; deoxyguanosine, 961-07-9; 2'deoxyguanosine 5'-monophosphate, 902-04-5.