Identification of Adducts Formed in Reactions of 2 '-Deoxyadenosine

Comparable DNA and chromosome damage in Chinese hamster ovary cells by chlorohydroxyfuranones. Jorma Mäki-Paakkanen , Mariitta Laaksonen , Tony ...
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Chem. Res. Toxicol. 1999, 12, 1205-1212

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Identification of Adducts Formed in Reactions of 2′-Deoxyadenosine and Calf Thymus DNA with the Bacterial Mutagen 3-Chloro-4-(chloromethyl)-5-hydroxy-2(5H)-furanone Tony Munter, Frank Le Curieux, Rainer Sjo¨holm, and Leif Kronberg* Department of Organic Chemistry, A° bo Akademi University, Biskopsgatan 8, FIN-20500 Turku/A° bo, Finland Received July 6, 1999

3-Chloro-4-(chloromethyl)-5-hydroxy-2(5H)-furanone (CMCF) is a strong direct acting bacterial mutagen found in chlorine-disinfected drinking water. We studied the reaction of CMCF with 2′-deoxyadenosine in buffered aqueous solutions and found that three main adducts were formed. The adducts were isolated and purified by C18 column chromatography and HPLC, and characterized on the basis of their UV absorbance, fluorescence emission, 1H and 13C NMR spectroscopic, and mass spectrometric features. The adducts were identified as 3-(2′-deoxy-βD-ribofuranosyl)-7H-8-formyl[2,1-i]pyrimidopurine (pfA-dR), 3-(2′-deoxy-β-D-ribofuranosyl)-7H8-carboxy[2,1-i]pyrimidopurine (pcA-dR), and 4-(N6-2′-deoxyadenosinyl)-3-formyl-2-hydroxy3-butenoic acid (OH-fbaA-dR). In the reactions performed at pH 7.4 and 37 °C, the yields of pfA-dR, pcA-dR, and OH-fbaA-dR were 1.1, 6.7, and 5.5 mol %, respectively. The adduct pfAdR was detected also in calf thymus DNA reacted with CMCF. The yield was about six adducts per 105 bases. To elucidate the mechanisms of formation of the adducts, 13C-3-labeled CMCF was reacted with 2′-deoxyadenosine. The adducts are structurally related to the adducts previously identified in the reactions of structurally analogous chlorohydroxyfuranones with 2′-deoxyadenosine.

Introduction Chlorohydroxyfuranones (CHFs)1 are formed during chlorine disinfection of drinking water as a result of the reaction of chlorine with naturally occurring humic substances (1-3). One of these compounds, 3-chloro-4(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) (Scheme 1), is a very potent direct acting bacterial mutagen and accounts in most cases for about one-third of the mutagenicity of chlorinated drinking water (4-13). Recently, MX was demonstrated to be a multisite carcinogen in rats (14). In studies by Kronberg and Franze´n (1), and Smeds et al. (11), it was found that besides MX, several other structurally related CHFs are present in chlorinated drinking waters. Two of these compounds are 3-chloro4-(chloromethyl)-5-hydroxy-2(5H)-furanone (CMCF) and 3-chloro-4-methyl-5-hydroxy-2(5H)-furanone (MCF). MX, CMCF, and MCF are structurally closely related; MX has a dichloromethyl group, CMCF a chloromethyl group, and MCF a methyl group at C-4 (Scheme 1). In the Ames Salmonella tester strain TA100, CMCF generates about * To whom correspondence should be addressed. E-mail: [email protected]. Phone: +358-2-2154138. Fax: +358-2-2154866. 1 Abbreviations: MX, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)furanone; CMCF, 3-chloro-4-(chloromethyl)-5-hydroxy-2(5H)-furanone; MCF, 3-chloro-4-methyl-5-hydroxy-2(5H)-furanone; pfA-dR, 3-(2′-deoxyβ-D-ribofuranosyl)-7H-8-formyl[2,1-i]pyrimidopurine; pcA-dR, 3-(2′deoxy-β-D-ribofuranosyl)-7H-8-carboxy[2,1-i]pyrimidopurine; OH-fbaAdR, 4-(N6-2′-deoxyadenosinyl)-3-formyl-2-hydroxy-3-butenoic acid; pfcAdR, 3-(2′-deoxy-β-D-ribofuranosyl)-7H-7-carboxy-8-formyl[2,1-i]pyrimidopurine; PEG, polyethylene glycol; COSY, correlation spectroscopy (H-H); HMBC, heteronuclear multiple-bond correlation.

Scheme 1

6 times and MCF generates about 60 times fewer revertants than MX. CMCF has been found to account for a few percent of the total Ames mutagenicity of chlorinated drinking water (1, 11). Numerous studies have demonstrated that genotoxic compounds bind covalently to the base units of DNA (15). Recently, we have reacted MX with nucleosides and found that in the reaction with guanosine an ethenoformyl adduct is formed, and in the reaction with 2′-deoxyadenosine, an N6-propenal adduct and two fluorescent cyclic 1,N6-propenoformyl adducts are obtained (16-18). In the reaction of MCF with adenosine, two products were identified: one with a formylbutenoic acid chain attached to the exocyclic amino group and one where a methylfuranone ring was attached at N6 (19). In the work presented here, we have studied the reaction of CMCF with 2′-deoxyadenosine. The work resulted in the structural identification of the three main adducts.

10.1021/tx990116c CCC: $18.00 © 1999 American Chemical Society Published on Web 11/19/1999

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Further, one of the adducts was observed in calf thymus DNA incubated with CMCF.

Materials and Methods Caution: CMCF has been found to be a direct acting mutagen in the Ames mutagenicity assay with Salmonella typhimurium (TA100). Therefore, caution should be exercised in the handling and the disposal of the compound. Chemicals. 2′-Deoxyadenosine, 1,N6-ethenoadenosine, calf thymus DNA, Dnase from bovine pancreas, nuclease P1 from Penicillium citrinum, alkaline phosphatase from bovine intestinal mucosa, and acid phosphatase from white potato were obtained from Sigma Chemical Co. (St. Louis, MO). 3-Chloro4-(chloromethyl)-5-hydroxy-2(5H)-furanone (CMCF) and 13C-3labeled CMCF were synthesized and purified according to the method of Franze´n and Kronberg (20). The purity of CMCF was at least 98% as estimated by 1H NMR and GC. Chromatographic Methods. HPLC analyses were performed on a Kontron Instruments liquid chromatographic system consisting of a model 322 pump, a 440 diode-array detector (UV), a Jasco FP-920 fluorescence detector, and a KromaSystem 2000 data handling program (Kontron Instruments S. P. A., Milan, Italy). The reaction mixtures were chromatographed on a 5 µm, 4 mm × 125 mm reversed phase C18 analytical column (Spherisorb ODS2, Hewlett-Packard, Espoo/Esbo, Finland). In the spiking experiments, two additional columns were used: a C8 column (5 µm, 4 mm × 125 mm, Lichrospher 100, RP-8; Hewlett-Packard) and a 250 mm long C18 column (5 µm, 4 mm × 250 mm, Spherisorb ODS2). The columns were eluted isocratically for 5 min with 0.01 M phosphate buffer (pH 7.1) and then with a gradient from 0 to 30% acetonitrile over the course of 25 min at a flow rate of 1 mL/min. Preparative isolation of the products from the reaction mixtures was performed by column chromatography on a 2.5 cm × 10 cm column of preparative C18 bonded silica grade Bondesil (40 µm, Analytichem International, Harbor City, CA). The products were further purified by HPLC on a semipreparative 8 µm, 10 mm × 250 mm (Hyperprep ODS, Hypersil, Krotek, Tampere/Tammerfors, Finland) reversed phase C18 column. The column was coupled to a Shimadzu HPLC system, which consisted of two Shimadzu LC-9A pumps and a variablewavelength Shimadzu SPD-6A UV spectrophotometric detector (Shimadzu Europe). Spectroscopic and Spectrometric Methods. The 1H and NMR spectra were recorded at 30 °C on a JEOL JNM-A500 Fourier transform NMR spectrometer at 500 and 125 MHz, respectively (JEOL, Tokyo, Japan). The samples were dissolved in Me2SO-d6, and TMS was used as an internal standard. The 1H NMR signal assignments were based on chemical shifts and H-H and C-H correlation data. The determinations of the shifts and the coupling constants of the multiplets of the proton signals in the deoxyribose unit were based on a first-order approach and are given with an accuracy of (0.3 Hz. Assignment of carbon signals was based on chemical shifts, C-H correlations, and carbon-proton couplings. 13C

The electrospray ionization mass spectra and the LC/MS analyses (electrospray) were performed on a Fisons ZabSpecoaTOF instrument (Manchester, U.K.). In the case of isolated compounds, samples were introduced by loop injection at a flow rate of 20 µL/min (80/20/1 H2O/CH3CN/acetic acid). The LC/MS samples were chromatographed on the analytical column. The column was eluted isocratically for 5 min with 0.05% formic acid and then with a gradient from 0 to 30% acetonitrile over the course of 25 min at a flow rate of 1 mL/min. About 1/25 of the eluate was allowed to enter the ion source. Ionization was carried out using nitrogen as both the nebulizing and bath gas. A potential of 8.0 kV was applied to the ESI needle. The temperature of the pepperpot counter electrode was 90 °C. PEG 200 was used as standard for exact mass determinations. The mass spectrometer was operated at a resolution of 7000.

Munter et al. The UV spectra of the isolated compounds were recorded by the diode-array detector as the peaks eluted from the HPLC column. A Shimadzu UV-160 spectrophotometer was used for the determination of the molar extinction coefficients, . The fluorescence spectra were recorded with a Hitachi F-2000 fluorescence spectrophotometer (Hitachi Ltd.). Large-Scale Reaction of CMCF with 2′-Deoxyadenosine. CMCF (500 mg, 2.7 mmol) was reacted with 2′-deoxyadenosine (345 mg, 1.4 mmol) in 300 mL of a 0.5 M phosphate buffer solution (pH 7.4). The reaction was followed by HPLC analyses on the analytical column. After 4 days, the reaction mixture was filtered and passed through the preparative C18 column. The column was first eluted with 150 mL of water and then with 100 mL of 2, 5, 10, and 20% acetonitrile solutions in water. Fractions (30 mL) were collected. The compounds OHfbaA-dR and pcA-dR eluted from the column with 2 and 5% acetonitrile washes, and pfA-dR eluted with the 10% wash. The fractions containing the compounds were combined and concentrated by rotary evaporation to about 15 mL. The compounds OH-fbaA-dR and pcA-dR were separated and purified by HPLC using the semipreparative column. The same column was also used for the final purification of pfA-dR. The column was eluted isocratically with 6% acetonitrile in a 0.01 M phosphate buffer solution (pH 7.1) for 2 min and then with a gradient from 6 to 30% acetonitrile over the course of 28 min at a flow rate of 4 mL/min. The collected fractions were then desalted by using the preparative C18 column. The desalted solutions were rotary evaporated to dryness, and the residues were subjected to spectroscopic and spectrometric studies. The isolated compounds had the following spectral characteristics. UV spectrum for pcA-dR: UVmax 232, 268 ( ) 7100 M-1 cm-1), 280 nm ( ) 6600 M-1 cm-1); UVmin 251 nm (the HPLC eluent being 7% acetonitrile in 0.01 M phosphate buffer at pH 7.1). UV spectrum for pfA-dR: UVmax 408 ( ) 23 300 M-1 cm-1), 212, 240 nm; UVmin 284 nm (the HPLC eluent being 14% acetonitrile in 0.01 M phosphate buffer at pH 7.1). UV spectrum for OH-fbaA-dR: UVmax 326 ( ) 49 200 M-1 cm-1), 224, 240 nm; UVmin 276 nm (the HPLC eluent being 9% acetonitrile in 0.01 M phosphate buffer at pH 7.1). Fluorescence spectrum (H2O) for pcA-dR: λex 234 nm, λem,max 425 nm. Fluorescence spectrum (H2O) for pfA-dR: λex 379 nm, λem,max 462 nm. OH-fbaA-dR was not fluorescent. In the positive ion electrospray mass spectra, the following ions were observed (m/z, relative abundance, formation): pcAdR, 334 (71, MH+), 218 (100, MH+ - deoxyribosyl + H), with high-resolution mass spectrometry giving a protonated molecular formula of C14H16N5O5 (MH+ 334.1164, calcd 334.1151); pfA-dR 318 (60, MH+), 202 (100, MH+ - deoxyribosyl + H), with high-resolution mass spectrometry giving a protonated molecular formula of C14H16N5O4 (MH+ 318.1195, calcd 318.1202); OH-fbaA-dR, 380 (100, MH+), 264 (65, MH+ - deoxyribosyl + H), with high-resolution mass spectrometry giving a protonated molecular formula of C15H18N5O7 (MH+ 380.1201, calcd 380.1206). The 1H NMR spectroscopic data of pfA-dR were as follows: δ 9.28 (t, 1 H, CHO, J ) 0.6 Hz), 8.49 (m, 1 H, H-2), 8.31 (m, 1 H, H-5), 7.50 (t, 1 H, H-9, J ) 0.8 Hz), 5.00, 5.01 (m, 2 H, H-7a, H-7b, J ) 14.4 Hz), 6.32 (dd, 1 H, H-1′, J ) 7.0, 6.3 Hz), 4.41 (ddd, 1 H, H-3′, J ) 5.9, 3.7, 3.1 Hz), 3.88 (dt, 1 H, H-4′, J ) 4.6, 3.1 Hz), 3.60 (dd, 1 H, H-5′, J ) 11.8, 4.6 Hz), 3.52 (dd, 1 H, H-5′′, J ) 11.8, 4.6 Hz), 2.64 (ddd, 1 H, H-2′, J ) 13.2, 7.0, 5.9 Hz), 2.33 (ddd, 1 H, H-2′′, J ) 13.2, 6.3, 3.7 Hz). The 1H and 13C NMR spectroscopic data of pcA-dR and OHfbaA-dR are presented in Tables 1 and 2, respectively. Small-Scale Reactions of CMCF with 2′-Deoxyadenosine. CMCF (3 mg, 0.016 mmol) was reacted with 2′-deoxyadenosine (2.1 mg, 0.008 mmol) in 2 mL of 0.5 M phosphate buffer solutions at pH 7.4, 6.0, and 4.6. The reactions were performed at 37 °C. The reaction mixtures were analyzed by HPLC using the C18 analytical column. Reaction of 13C-3-Labeled CMCF with 2′-Deoxyadenosine. CMCF (256 mg, 1.4 mmol) mixed with 13C-3-labeled CMCF (64 mg, 0.35 mmol) was reacted with 2′-deoxyadenosine (220

The Bacterial Mutagen CMCF Forms Adducts with dAdo Table 1. 1H and proton

13C

Chemical Shifts (δ)a and Spin-Spin Coupling Constants JH,H and JC,H of Protons and Carbons in pcA-dR

δ (ppm)

H-2 (1 H) H-5 (1 H)

multiplicity

8.47 9.12

carbon

δ (ppm)

multiplicity

139.2 136.5 137.3 139.9 122.9 129.6 123.1 34.1 170.9 83.9 39.7

dd d ddd ddd d dt dt t m d dd

213.6 214.7

0.8

C-2 C-5 C-3a C-10a C-10b C-9 C-8 C-7 COOH C-1′ C-2′

d d td

148.5 147.4 136.6

s s

7.20

t

H-7a, H-7b (2 H) COOH (1 H) H-1′ (1 H) H-2′ (1 H) H-2′′ (1 H) H-3′ (1 H) H-4′ (1 H) H-5′ (1 H) H-5′′ (1 H) OH (1 H) OH (1 H)

3.63; 3.64 nob 6.47 2.73 2.36 4.45 3.90 3.63 3.54 5.50 5.09

AB

16.8

dd ddd ddd dt dt dd dd br br

7.3; 6.3 13.2; 7.3; 5.9 13.3; 6.3; 3.6 5.6; 3.5 4.6; 3.2 11.9; 4.9 11.8; 4.6

C-3′ C-4′ C-5′

70.5 87.8 61.5

C,H

(Hz)

>1J

C,H

(Hz)

4.1 12.9; 5.4; 2.6 11.4; 4.7; 1.0 11.4 4.1 15.0; 5.7

187.2 127.9 166.6 134.5; 132.9

3.6

Relative to TMS. b no, not observed.

Table 2. 1H and proton H-8 (1 H) H-2 (1 H) N-H (1 H)

13C

Chemical Shifts (δ)a and Spin-Spin Coupling Constants JH,H and JC,H of Protons and Carbons in OH-fbaA-dR

δ (ppm) 8.66 8.52 nob

multiplicity

JH,H (Hz)

s s

Ha

8.39

br

Hc (1 H) OHc (1 H) COOH (1 H) CHO (1 H) H-1′ (1 H) H-2′ (1 H) H-2′′ (1 H) H-3′ (1 H) H-4′ (1 H) H-5′ (1 H) H-5′′ (1 H) OH (1 H) OH (1 H)

4.82 nob nob 9.29 6.43 2.73 2.34 4.44 3.90 3.62 3.53 5.4 5.1

dd

a

1J

JH,H (Hz)

H-9 (1 H)

a

Chem. Res. Toxicol., Vol. 12, No. 12, 1999 1207

d td ddd ddd dt dt dd ddd br br

0.7; 0.3 0.3 6.7; 2.0 13.4; 7.3; 6.1 13.3; 6.2; 3.4 6.0; 3.2 4.4; 3.1 11.8; 4.3 11.8; 4.2; 1.8

carbon

1J

δ (ppm)

multiplicity

C-8 C-2

142.6 152.0

dt dd

C-4 C-5 C-6

150.8 120.7 148.9

dm d dm

Ca Cb Cc

144.5 123.3 65.9

d ddd ddd

148.5

COOH CHO C-1′ C-2′

174.7 190.2 83.9 39.5

d ddd d t

170.7 166.0 133.6

d d td

149.5 147.9 140.7

C-3′ C-4′ C-5′

70.7 88.0 61.6

C,H

(Hz)

214.7 204.3

>1J C,H

(Hz)

3.9 1.0 12.9 11.4 10.9

174.3 23.0; 5.2; 1.8 6.0; 3.1 4.7 7.2; 3.1

3.4

Relative to TMS. b no, not observed.

mg, 0.9 mmol) in 200 mL of a 0.5 M phosphate buffer solution at pH 7.4. The reaction was allowed to proceed at 37 °C for 4 days. The reaction products pfA-dR, pcA-dR, and OH-fbaA-dR were isolated from the reaction mixture and purified in the same way as described for the compounds in the large-scale reaction. Determination of Product Yields. Quantitative 1H NMR analysis, using 1,1,1-trichloroethane as an internal standard, was performed on aliquots of the adducts. Standard solutions were made for HPLC analyses by taking an exact volume of the NMR sample and diluting it with an appropriate volume of water. The quantitative determination of the adducts in the reaction mixtures was made by comparing the peak area of the adducts in the standard solutions with the area of the adduct peaks in the reaction mixtures. The adducts were quantified using UV detection at 230, 325, and 400 nm. The molar yields were calculated from the original amount of 2′-deoxyadenosine in the reaction mixture. Reaction of CMCF with Calf Thymus DNA. CMCF (50 mg) was reacted with double-stranded calf thymus DNA (10 mg) in 10 mL of 0.5 M phosphate buffer at pH 7.4 and 5.5. The mixtures were stirred and incubated at 37 °C for 4 days. During the first 12 h of reaction and then twice a day, the pHs of the incubation mixtures were monitored and readjusted when necessary.

The modified DNA was recovered by precipitation with cold ethanol. To 1 volume of the incubation mixture were added 0.1 volume of 5 M NaCl and 2 volumes of cold 96% ethanol. This mixture was centrifuged (10 min at 3000 rpm), and the supernatant was collected. The supernatant was concentrated about 40-fold and analyzed by HPLC to search for modified purines and pyrimidines. The precipitated DNA was washed with 1 volume of 70% ethanol and then redissolved in 1 volume of water. This precipitation/washing procedure was performed (at least twice) until there was no more unreacted CMCF left in the supernatant (controlled by HPLC analyses). The enzymatic hydrolysis of the DNA was carried out following the procedure described by Martin et al. (21). Briefly, the modified DNA was dissolved in 2.5 mL of 0.1 M phosphate buffer (pH 7.4) containing 5 mM MgCl2. DNase I (dissolved at a concentration of 10 mg of Dnase/mL in 0.9% NaCl) was added to obtain a concentration of 0.1 mg of DNase/mL. The mixture was incubated and stirred for 3 h at 37 °C. Nuclease P1 (dissolved at a concentration of 0.5 mg of nuclease P1/mL in 1 mM ZnCl2) was added to obtain 20 µg of nuclease/mL as the final concentration. Finally, alkaline phosphatase (87 units/mL in water) and acid phosphatase (20 units/mL in water) were added to give final concentrations of 0.5 and 0.3 unit/mL, respectively. The mixture was then incubated and stirred at 37 °C for 18 h. The

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Figure 1. C18 analytical column HPLC chromatogram of the reaction mixture of CMCF and 2′-deoxyadenosine held at 37 °C and pH 7.4 for 6 days. For analysis conditions, see Materials and Methods. mixture of the hydrolyzed DNA was rotary evaporated to near dryness. The residue was extracted three times with 3 mL of 1/1 ethanol/methanol. The washes were combined, and insoluble particles were removed by centrifugation (20 min at 3000 rpm). Finally, the solution was evaporated to near dryness, and an appropriate amount of water (250 µL for 10 mg of DNA) was added. Fifty microliters of the solution was injected on the HPLC columns. Blanks and HPLC Checks. A blank sample was prepared by allowing calf thymus DNA to stand for 2 days at 37 °C. The precipitation was performed following the procedure described above. Enzymatic hydrolysis and HPLC sample preparation were then carried out exactly as described above. HPLC analysis of the blank sample showed that no peaks were observed that could represent the adducts formed by CMCF. Following reaction of CMCF with DNA, enzymatic hydrolysis of the DNA, and evaporation of the hydrolysates, the adducts in the residues were extracted with ethanol/methanol. No adducts were observed upon HPLC analyses of the redissolved residues. The extraction was thus considered to be quantitative.

Munter et al.

Figure 2. Formation of the adducts at 37 °C and various pH conditions in the reaction of CMCF with 2′-deoxyadenosine: ([) pH 4.6, (9) 6.0, and (O) 7.4.

Figure 3. UV absorbance spectra of the adducts. The UV spectra were recorded with the diode-array detector as the compounds eluted from the HPLC column.

Chart 1

Results and Discussion Reaction of CMCF with 2′-deoxyadenosine at pH 7.4 resulted in the formation of three major product peaks as detected by analytical HPLC (Figure 1). The compounds represented by the peaks marked pcA-dR and OH-fbaA-dR were formed at higher yields under neutral conditions (6.7 and 5.5%, respectively) than under acid conditions. In contrast, the compound marked pfA-dR was obtained in the highest yield (6%) in the reaction carried out at pH 4.6 (Figure 2). For the purpose of determining the structures of the compounds, a large-scale reaction was performed at pH 7.4. Following reaction for 4 days, the compounds were isolated from the reaction mixture by preparative C18 column chromatography and semipreparative HPLC. On the basis of data collected from UV, fluorescence and NMR spectroscopy, and electrospray mass spectrometry, the compounds were identified as 3-(2′-deoxy-β-D-ribofuranosyl)-7H-8-formyl[2,1-i]pyrimidopurine (pfA-dR), 3-(2′deoxy-β-D-ribofuranosyl)-7H-8-carboxy[2,1-i]pyrimidopu-

rine (pcA-dR), and 4-(N6-2′-deoxyadenosinyl)-3-formyl-2hydroxy-3-butenoic acid (OH-fbaA-dR) (Chart 1). The three compounds had very different UV absorption spectra (Figure 3). The UV spectrum of pcA-dR exhibited absorption maxima at 232, 268, and 280 nm. This spectrum is very similar to the spectrum of 1,N6ethenoadenosine (22, 23). The compound OH-fbaA-dR had an intense absorption maximum at 326 nm. It has been previously reported in the literature that compounds with an R,β-unsaturated aldehyde moiety at-

The Bacterial Mutagen CMCF Forms Adducts with dAdo

Figure 4. Fluorescence emission spectrum of pcA-dR.

tached to N6 of adenine nucleosides have characteristic UV spectra with an absorption maximum at 320-330 nm (19, 24-26). The UV spectrum of pfA-dR exhibited a longwavelength maximum at 408 nm, and both the UV and fluorescence spectra were in all features identical with the spectra of the compound pfA-dR that we have previously reported to be formed in the reaction of the chlorohydroxyfuranone MX with 2′-deoxyadenosine (18). The fluorescence spectrum of pcA-dR exhibited an emission maximum at 425 nm when excited at 234 nm (Figure 4). These fluorescence characteristics are like those reported for 1,N6-ethenoadenosine (23). When the fluorescence detector signal intensities for equal molar amounts of 1,N6-ethenoadenosine, pcA-dR, and pfA-dR were compared, it was found that 1,N6-ethenoadenosine gave a slightly stronger signal (1.6 times stronger) than pcA-dR, while the signal intensity of pfA-dR was about 5% of that of pcA-dR. The compound OH-fbaA-dR was not fluorescent. In the positive ion electrospray mass spectrum of pcAdR, the protonated molecular ion was observed at m/z 334. The most abundant ion was recorded at m/z 218, and it corresponded to the loss of the deoxyribosyl unit from MH+. The mass spectrum of OH-fbaA-dR exhibited a protonated molecular ion peak at m/z 380, and the fragment peak at m/z 264 resulted from the loss of the sugar moiety from MH+. The protonated molecular ion peak in the mass spectrum of pfA-dR was observed at m/z 318, and the loss of the sugar unit resulted in a peak at m/z 202. The 1H NMR spectrum of pcA-dR exhibited, besides the signals from the protons of the deoxyribose moiety, one-proton signals at δ ) 9.12, 8.47, and 7.20 ppm (Table 1). Moreover, signals exhibiting an AB pattern were centered at δ ) 3.635 ppm. The signal at δ ) 8.47 ppm was assigned to H-2 on the basis of the observed H-H correlation (COSY) with the H-1′ signal in the deoxyribose moiety at δ ) 6.47 ppm. The signals of the AB protons (δ ∼ 3.64 ppm) both exhibited a one-bond shift correlation with the carbon signal at δ ) 34.1 ppm and were assigned to the two geminal protons of the methylene group (H-7a and H-7b). The triplet at δ ) 7.20 ppm was assigned to H-9, and it exhibited a long-range H-H correlation with the two methylene protons at δ ) 3.64 ppm. The proton at position 5 in the purine ring appeared at δ ) 9.12 ppm, and it exhibited long-range H-H correlations with the H-2 proton and the H-7 protons. Protons H-2′-H-5′ of the deoxyribose unit were assigned using H-H correlation data. The 13C NMR spectrum of pcA-dR exhibited, besides the signals from the purine and deoxyribose moieties, four carbon signals at δ ) 170.9, 129.6, 123.1, and 34.1 ppm (Table 1). The signal at δ ) 129.6 ppm was assigned

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to C-9, and it exhibited a strong one-bond C-H coupling (1J ) 187.2 Hz) and was further split into a triplet due to coupling to the two methylene protons (3J ) 4.1 Hz). The signal for C-8 appeared at δ ) 123.1 ppm and was split into a doublet of triplets due to two bond couplings to H-9 (2J ) 15 Hz) and the methylene protons (2J ) 5.7 Hz). Further, the corresponding C-H correlations could be observed (HMBC). The signal of the methylene carbon, C-7, appeared at δ ) 34.1 ppm as a triplet (1J ) 127.9 Hz). The presence of a carboxyl group was indicated by a signal at δ ) 170.9 ppm. The resonance signals at δ ) 139.2 ppm and δ ) 136.5 ppm were assigned to C-2 and C-5, respectively, on the basis of their strong one-bond couplings to the purine protons. The C-3a resonance signal appeared at δ ) 137.3 ppm, and it was split into three doublets due to couplings to H-2, H-1′, and H-5. The signal at δ ) 122.9 ppm was assigned to C-10b on the basis of the chemical shift and the three-bond coupling (3J ) 11.4 Hz) to H-2. C-10a gave a resonance signal at δ ) 139.9 ppm, and it was split into three doublets due to coupling to H-9, H-5, and H-2. The 1H NMR spectrum of OH-fbaA-dR exhibited five one-proton signals at δ ) 9.29, 8.66, 8.52, 8.39, and 4.82 ppm, in addition to the signals from the protons of the deoxyribose moiety (Table 2). The signal at δ ) 9.29 ppm was assigned to the formyl proton on the basis of the downfield chemical shift and the one-bond shift correlation with the carbon signal at δ ) 190.2 ppm. The olefinic proton, Ha, appeared at δ ) 8.39 as a broad singlet. The signal at δ ) 4.82 ppm was assigned to Hc, and a longrange H-H correlation with the formyl proton was observed. The signal at δ ) 8.66 ppm was assigned to H-8 on the basis of the long-range H-H correlation with the signal of H-1′ in the deoxyribose moiety. The H-2 proton appeared as a singlet at δ ) 8.52 ppm. The carboxyl, OHc, and amino protons were not observed, because of exchange with residual water in the sample. In the 13C NMR spectrum of OH-fbaA-dR, five carbon signals at δ ) 190.2, 174.7, 144.5, 123.3, and 65.9 ppm were found in addition to the ten carbon signals from the purine and deoxyribose moieties (Table 2). The signal observed at δ ) 190.2 ppm was assigned to the formyl carbon. It exhibited a strong one-bond C-H coupling (1J ) 170.7 Hz), and C-H long-range correlations with Ha and Hc (HMBC). The magnitude of the coupling to Ha (3J ) 7.2 Hz) suggests a cis relation of the formyl carbon and Ha (27). Ca was found at δ ) 144.5 ppm, and it exhibited a one-bond C-H coupling 1J of 174.3 Hz and long-range C-H correlations with the formyl and Hc protons. The signal at δ ) 123.3 ppm exhibited longrange C-H correlations with the formyl proton and Hc. The strong coupling (2J ) 23.0 Hz) indicated that the carbon was located at the R-position to the formyl group (28). The signal of the methine carbon, Cc, was found at δ ) 65.9 ppm. It exhibited a one-bond C-H coupling 1J of 148.5 Hz and a long-range C-H correlation with the formyl proton. The carboxyl carbon was found as a doublet at δ ) 174.7 ppm, and a long-range correlation was observed between the carbon signal and Hc. The chemical shifts for the purine carbons were very similar to those reported previously for N6-propenal or N6butenal adenosine adducts (19, 25). The UV, fluorescence, 1H NMR, and mass spectra of the third compound formed in the reaction were identical with those of the compound pfA-dR we previously have reported to be formed in the reaction of MX with

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Scheme 2

Figure 5. C8 analytical column (125 mm) HPLC separation of calf thymus DNA incubated with CMCF and enzymatically hydrolyzed. The chromatogram of the spiked hydrolysate is superimposed on the original chromatogram.

2′-deoxyadenosine (18). Further, a spiking experiment showed the compounds to coelute in the HPLC chromatogram. Thus, we conclude that pfA-dR is formed in the reaction of MX and of CMCF with 2′-deoxyadenosine. To gain knowledge about the mechanisms for the formation of the adducts, we reacted 13C-3-labeled CMCF with 2′-deoxyadenosine and analyzed the isolated adducts by 13C NMR spectroscopy. In the 13C NMR spectra of pcAdR and pfA-dR, we found that the signal for the methylene carbon was markedly more intense than the signals for the other carbons. The 13C spectrum of OH-fbaA-dR showed that the signal of the methine carbon, Cc, was the most intensive signal. On the basis of these findings, we propose that the mechanisms for the formation of the adducts are as depicted in Scheme 2. Initially, a proton transfer takes place from the chloromethyl carbon in the open form of CMCF to the labeled carbon atom. In the next step, the exocyclic amino group of 2′-deoxyadenosine attacks the double-bonded carbon atom at the β-position to the aldehyde group and an enol is obtained. Upon formation of the keto form, the chlorine bound to the carbon located next to the exocyclic nitrogen will be lost. The keto form is the key intermediate giving rise to the adducts. The propeno bridge is formed through displacement of the remaining chlorine by a conjugate attack via the endocyclic nitrogen (N-1) of adenine. Decarboxylation of the intermediate pfcA-dR will yield pfA-dR. The formation of pcA-dR involves an oxidation of the formyl group to a carboxyl group. However, experimentally it was found that the compound pfA-dR was not oxidized under the prevailing reaction conditions, and thus, we propose it is the keto intermediate that is oxidized. The compound OH-fbaA-dR is formed by replacement of the chlorine at the labeled carbon with a hydroxyl group. The presence of the intermediate pfcA-dR could be observed in the reaction mixtures. The compound was represented in the HPLC chromatograms as a fluorescent peak (retention time of 13.5 min) with UV characteristics (UVmax ) 400 nm) similar to those of pfA-dR. In the mass spectrum of the peak, obtained by LC/MS analysis of the mixture, two abundant ion peaks were observed. The peak at m/z 362 corresponded to the protonated molecular

ion, and the peak at m/z 246 corresponded to the loss of the deoxyribosyl unit from the molecule. On the basis of these observations, we propose that pfcA-dR is formed in the reaction of CMCF with 2′-deoxyadenosine. During isolation of the compound from the reaction mixture, decarboxylation took place and pfA-dR was obtained. Reaction of CMCF with Calf Thymus DNA. HPLC analyses of the hydrolysate of the calf thymus DNA reacted with CMCF showed the presence of a peak at exactly the same retention time as pfA-dR. Spiking experiments of the DNA hydrolysate with pure pfA-dR demonstrated that the compounds coeluted when chromatographed on a short (125 mm) and a longer (250 mm) C18 column, and on a short C8 column (Figure 5). Moreover, the UV spectra and the fluorescence characteristics of the adduct in the hydrolysate and of the reference pfA-dR were in all essential features identical. In addition, the DNA hydrolysate was analyzed using a HPLC system coupled to a mass spectrometry detector, and a product peak was observed with a retention time of about 16 min with an ion peak at m/z 318. We concluded that pfA-dR was formed in the reaction of CMCF with calf thymus DNA at pH 7.4. The yield of formation of pfA-dR in DNA was about six adducts per 105 nucleotides. The compound pfA-dR was not observed in DNA when the reaction was performed at pH 5.5. This finding is rather surprising because in the reaction of CMCF with 2′-deoxyadenosine pfA-dR was formed at higher yield at lower pH. Another unexpected observation was that pcAdR was not detected in the DNA hydrolysate at pH 7.4 or 5.5, although this compound is strongly fluorescent and its yield is 6.7% in the reaction with 2′-deoxyadenosine at pH 7.4. We cannot explain these results at present, and they show that the reactions with DNA do not always reflect exactly the mechanisms observed in the reaction with monomeric nucleosides. The CMCF adduct OHfbaA-dR was not detected in the DNA hydrolysate. The limit of detection of our HPLC analytical method was about four adducts per 1010 bases for pcA-dR, four adducts per 109 bases for pfA-dR, and four adducts per

The Bacterial Mutagen CMCF Forms Adducts with dAdo

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107 bases for OH-fbaA-dR. The yield of formation of pfAdR in DNA at pH 7.4 is 10 times higher in the reaction with CMCF (six pfA-dR adducts per 105 bases) than in the reaction with MX (0.6 pfA-dR adduct per 105 bases) (18). This is not in agreement with the higher genotoxicity of MX.

(5) LaLonde, R. T., Cook, G. P., Pera¨kyla¨, H., and Dence, C. W. (1991) Effect on mutagenicity of the stepwise removal of hydroxyl group and chlorine atoms from 3-chloro-4-(dichloromethyl)-5-hydroxy2(5H)-furanone: 13C NMR chemical shifts as determinants of mutagenicity. Chem. Res. Toxicol. 4, 35-40.

Conclusions Structural characterization of nucleoside adducts of genotoxic chlorohydroxyfuranones is important in determining the nature of their possible chemical interaction with DNA, and in clarifying the underlying mechanisms of the genotoxicity of these compounds. The results of this study demonstrate that CMCF reacts with 2′-deoxyadenosine, giving rise to two stable cyclic propeno adducts (pfA-dR and pcA-dR), and one open-chain hydroxyformylbutenoic acid adduct (OH-fbaA-dR). These adducts are structurally related to the adducts formed in the reactions of MX and MCF with 2′-deoxyadenosine. It has been found that CMCF and MX cause the same type of mutations in the Ames tester strain TA100 (29). In this work, we show that both compounds form the adduct pfA-dR in reactions with 2′-deoxyadenosine and calf thymus DNA. It would be tempting to explain the TA100 genotoxicity of MX by the formation of the pfAdR adduct. However, the yields of the adduct in calf thymus DNA reacted with MX and with CMCF do not correlate with the difference in the mutagenicity potencies of the compounds, and further, it has been reported that the mutational specificity of MX and CMCF in S. typhimurium cannot be explained by the formation of adenosine adducts, but by the formation of guanosine adducts (29). On the other hand, it is possible that the identified adenosine adducts play a role in the genotoxicity of CMCF in other biological systems.

Acknowledgment. This work was supported by the Graduate School of Bio-Organic Chemistry at A˙ bo Akademi University (T.M.) and by the European Commission (contract ERBFMBICT961394, F.L.C.). We are grateful to Mr. Markku Reunanen for the mass spectra. Supporting Information Available: The 500 MHz 1H NMR spectrum of pfA-dR, the 125 MHz 13C NMR and protoncoupled spectrum of pfA-dR, the positive ion electrospray mass spectrum of pfA-dR, the 500 MHz 1H NMR spectrum of pcAdR, the 125 MHz 13C NMR and proton-coupled spectrum of pcAdR, the long-range COSY H-H spectrum of pcA-dR, the longrange HMBC C-H correlation spectrum of pcA-dR, the positive ion electrospray mass spectrum of pcA-dR, the 500 MHz 1H NMR spectrum of OH-fbaA-dR, the 125 MHz 13C NMR and proton-coupled spectrum of OH-fbaA-dR, and the positive ion electrospray mass spectrum of OH-fbaA-dR. This material is available free of charge via the Internet at http://pubs.acs.org.

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