Substituted Pyrimidine Adducts Formed in Reactions of 4 - American

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Identification of O2-Substituted Pyrimidine Adducts Formed in Reactions of 4-(Acetoxymethylnitrosamino)1-(3-pyridyl)-1-butanone and 4-(Acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanol with DNA Stephen S. Hecht,* Peter W. Villalta, Shana J. Sturla, Guang Cheng, Nanxiong Yu, Pramod Upadhyaya, and Mingyao Wang University of Minnesota Cancer Center Minneapolis, Minnesota 55455 Received December 17, 2003

Metabolic hydroxylation of the methyl group of the tobacco specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its metabolite 4-(methylnitrosamino)-1-(3pyridyl)-1-butanol (NNAL) results in the formation of intermediates that can alkylate DNA. Similarly, metabolic hydroxylation of the 2′-position of the tobacco specific carcinogen N′-nitrosonornicotine gives DNA alkylating intermediates. The resulting pyridyloxobutyl and pyridylhydroxybutyl adducts with dGuo have been characterized, but there are no reports of pyrimidine adducts. Therefore, in this study, we investigated the reactions of 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKCH2OAc) and 4-(acetoxymethylnitrosamino)1-(3-pyridyl)-1-butanol (NNALCH2OAc) with DNA, dCyd, and dThd. NNKCH2OAc and NNALCH2OAc are stable precursors to the products formed upon metabolic methyl hydroxylation of NNK and NNAL. Analysis by LC-ESI-SIM of enzyme hydrolysates of DNA that had been allowed to react with NNKCH2OAc and NNALCH2OAc demonstrated the presence of major adducts with dCyd and dThd. The dCyd adducts were thermally unstable, releasing 4-HPB (18) or 4-hydroxy-1-(3-pyridyl)-1-butanol (25) upon treatment at 100 °C, pH 7.0. The dThd adducts were stable under these conditions. The dCyd adduct of NNALCH2OAc was characterized by its MS and UV and by conversion upon neutral thermal hydrolysis to the corresponding Cyt adduct, which was identified by MS, UV, and NMR. The dCyd and Cyt adducts of NNKCH2OAc were similarly characterized. The dThd adduct of NNKCH2OAc was identified by MS, UV, and NMR. Treatment of this adduct with NaBH4 gave material, which was identical to that produced upon reaction of NNALCH2OAc with DNA or dThd. These data demonstrate that the major pyrimidine adducts formed in the reactions of NNKCH2OAc with DNA are O2[4-(3-pyridyl)-4-oxobut-1-yl]dCyd (26) and O2[4-(3-pyridyl)-4-oxobut-1-yl]dThd (30) while those produced from NNALCH2OAc are O2[4-(3-pyridyl)-4-hydroxybut-1-yl]dCyd (28) and O2[4-(3-pyridyl)-4-hydroxybut-1-yl]dThd (31). Levels of these pyrimidine adducts of NNKCH2OAc in DNA were substantially greater than those of the dGuo adducts of NNKCH2OAc, based on MS peak area. Furthermore, 26 was identified as a major 4-HPB releasing adduct of NNKCH2OAc. These results suggest that pyrimidine adducts of tobacco specific nitrosamines may be important contributors to their mutagenic and carcinogenic activity.

Introduction Tobacco specific nitrosamines are formed from tobacco alkaloids during the curing and processing of tobacco (1, 2). Among these compounds, NNK (2)1 and NNN (5) are the most carcinogenic (3). NNK is a potent pulmonary carcinogen in rats and also induces tumors of the nasal * To whom correspondence should be addressed. Tel: 612-624-7604. Fax: 612-626-5135. E-mail: [email protected]. 1 Abbreviations: 7(POB-1-yl)dGuo, 7[4-(3-pyridyl)-4-oxobut-1-yl]dGuo; HMBC, 1H-13C heteronuclear multibond correlation; 4-HPB, 4-hydroxy-1-(3-pyridyl)-1-butanone; LC-ESI-MS, liquid chromatography-electrospray ionization-mass spectrometry; MS/MS, tandem mass spectrometry; NNAL, 4-(methylnitrosamino)-1-(3-pyridyl)-1butanol; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; NNN, N′-nitrosonornicotine; O2(PHB-1-yl)dCyd, O2[4-(3-pyridyl)-4-hydroxybut-1-yl]dCyd; O2(PHB-1-yl)Cyt, O2[4-(3-pyridyl)-4-hydroxybut-1-yl]Cyt; O2(PHB-1-yl)dThd, O2[4-(3-pyridyl)-4-hydroxybut-1-yl]dThd; O2(POB-1-yl)dCyd, O2[4-(3-pyridyl)-4-oxobut-1-yl]dCyd; O2(POB-1-yl)Cyt, O2[4-(3-pyridyl)-4-oxobut-1-yl]Cyt; O2(POB-1-yl)dThd, O2[4-(3pyridyl)-4-oxobut-1-yl]dThd; PHB, 4-(3-pyridyl)-4-hydroxybutyl; POB, 4-(3-pyridyl)-4-oxobutyl; SIM, selected ion monitoring.

cavity, liver, and pancreas (3). NNN causes tumors of the esophagus and nasal cavity in rats (3). NNK and NNN are also carcinogenic in mice, hamsters, and mink (3). A major metabolite of NNK, NNAL (3), has similar carcinogenic properties to those of NNK (3). Substantial amounts of NNK and NNN are present in both unburned tobacco and its smoke (1, 2, 4-6). Consequently, NNK and NNN are believed to play a significant role as causes of tobaccoinduced cancer in humans (3). NNK, NNAL, and NNN require metabolic activation to form DNA adducts, which are critical in mutagenesis and carcinogenesis by these compounds (3). The metabolic activation of NNK and NNAL is summarized in Scheme 1. Hydroxylation of the carbons R to the N-nitroso group (R-hydroxylation), catalyzed by P450s, is the key reaction leading to DNA adduct formation. R-Hydroxylation of the methylene groups of NNK and NNAL gives intermediates 7 and 8, which spontaneously yield meth-

10.1021/tx034263t CCC: $27.50 © 2004 American Chemical Society Published on Web 04/06/2004

Pyrimidine Adducts of NNKCH2OAc and NNALCH2OAc

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Scheme 1. DNA Adduct Formation from NNK and NNAL

anediazohydroxide (12), a methylating agent that reacts with DNA producing adducts 19-21. R-Hydroxylation of the methyl group of NNK gives compound 6, which spontaneously loses formaldehyde yielding intermediate 11. This intermediate, or electrophiles derived from it, reacts with DNA to give adducts 14-17, in which the POB group is bound at the O6-, 7-, or N2-positions of dGuo. Similarly, R-hydroxylation of the methyl group of NNAL yields intermediates 9 and 13 and ultimately DNA adducts 22-24, in which the PHB group is bound to dGuo. R-Hydroxylation of NNN adjacent to the pyridine ring gives intermediate 10, which undergoes spontaneous ring opening to 11, the same diazohydroxide formed by methyl hydroxylation of NNK. Pyridyloxobutylated DNA has been detected in animals treated with NNK and NNAL, in rat esophagus cultured with NNN, and in lung tissue from smokers (3, 7, 8). Quantitatively major adducts in this DNA release 4-HPB (18) upon neutral thermal hydrolysis, but these had eluded characterization for many years (3, 9). Recently, we characterized 7(POB-1-yl)dGuo (15) as one of the 4-HPB releasing adducts (10). Several other POB-dGuo and PHB-dGuo adducts have also been identified (911) (Scheme 1). Diazohydroxides and their derived electrophiles are known to alkylate pyrimidines as well as purines, and alkylated pyrimidines may have miscoding properties (12). Therefore, in this study, we investigated pyridyloxobutylation and pyridylhydroxybutylation of dCyd and dThd. We used NNKCH2OAc as a stable precursor to intermediates 6 and 11, and NNALCH2OAc as a precursor to 9 and 13.

gradient from 35 to 50% CH3OH in 10 min. The flow rate was 0.5 mL/min, and detection was by UV (254 nm). System 2 was a 3.9 mm × 300 mm, 10 µm C18 Bondclone (Phenomenex, Torrance, CA) column eluted with convex gradient 5 from 0 to 35% CH3OH in 40 mM ammonium acetate buffer (pH 6.6) at 0.5 mL/min over the course of 60 min, then from 35 to 50% CH3OH in 10 min, and held for 15 min, with detection by UV (254 nm). LC-ESI-MS was carried out with a Thermo Finnigan LCQ Deca instrument (Thermo Finnigan LC/MS Division, San Jose, CA) interfaced with a Waters Alliance 2690 HPLC multisolvent delivery system and equipped with an SPD-10 A UV detector (Shimadzu Scientific Instruments, Columbia, MD). The HPLC columns and elution conditions were the same as above, except that the flow rate was 0.3 mL/min and the final gradient in system 1 was for 20 min. The ESI source was set as follows: voltage, 2.0 kV; current, 10 µA; capillary temperature, 250275 °C. MS/MS data were acquired with the following parameters: isolation width, 1.5 amu; activation amplitude, 30%; activation Q, 0.25; activation time, 30 ms. High-resolution MS were run on a Finnigan MAT 90/95 instrument (Thermo Finnigan MAT GmbH, Bremen, Germany). NMR spectra were recorded in DMSO-d6 using a Varian Unity (Varian, Inc., Palo Alto, CA) spectrometer operating at 800 MHz at 25 °C. A triple resonance 3 mm HCN probe was used. Resonance assignments were made based on 1H-1H COSY and 1H-13C HMBC experiments. The HMBC was acquired using sweep widths of 9000-10 000 and 32 500-37 000 Hz in the t1 and t2 dimensions, respectively. The first delay was set to match a 140 Hz coupling constant, and the second delay was set to match a long-range coupling constant of 4 Hz.

Experimental Procedures

Chemicals and Enzymes. NNKCH2OAc (1) (13), NNALCH2OAc (4) (13), 3-HPB (32) (14), 4-HPB (18) (15), O2ethylCyt (16), and 3-ethylCyt (16) were synthesized. Calf thymus DNA, dThd, dCyd, Cyt, and all enzymes were obtained from Sigma Aldrich Chemical Co. (Milwaukee, WI).

Apparatus and Assay Conditions. HPLC was carried out with Waters Associates (Milford, MA) systems equipped with a model 996 photodiode array detector or a model 440 UV-vis detector set to 254 nm. Solvent elution systems were as follows: System 1 was a 4.6 mm × 25 cm Supelcosil LC-18-DB column (Supelco, Bellefonte, PA) eluted isocratically with 5% CH3OH in 40 mM ammonium acetate buffer, pH 6.6, for 10 min, then a gradient from 5 to 35% CH3OH in 60 min, and finally a

Reactions. 1. NNKCH2OAc (1) and DNA. NNKCH2OAc (132 mg, 0.5 mmol) was allowed to react with calf thymus DNA (20 mg) in 10 mL of 0.1 M phosphate buffer, pH 7.0, in the presence of porcine liver esterase (500 units) at 37 °C for 1.5 h. The mixture was diluted with 10 mL of H2O and extracted twice with 20 mL of CHCl3/isoamyl alcohol (24:1) and 20 mL of ethyl acetate. The DNA was precipitated by addition of ethanol and then washed with 70% aqueous ethanol and ethanol sequentially. The modified DNA was hydrolyzed enzymatically as

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Figure 1. Chromatograms obtained upon LC-ESI-MS analysis (system 1) of an enzyme hydrolysate of DNA that had been allowed to react with NNKCH2OAc. The hydrolysate was analyzed (A) directly or (B) after neutral thermal hydrolysis (100 °C, pH 7.0, 1 h). SIM was carried out at the m/z values indicated in each panel. Identities of the peaks, as determined in the text, and their retention times are indicated by the labels. described (10). For neutral thermal hydrolysis, a portion of the enzyme hydrolysate in 10 mM Tris-HCl/5 mM MgCl2 buffer, pH 7.0, was heated at 100 °C for 1 h. 2. NNKCH2OAc (1) and dCyd or dThd. A mixture of NNKCH2OAc (30 mg, 0.1 mmol), porcine liver esterase (140 units), and dCyd or dThd (0.02 mmol) was allowed to react in 2 mL of 0.1 M phosphate buffer, pH 7, at 37 °C for 1 h. The reaction mixture was washed three times with 2 mL of CHCl3, and the aqueous phase was analyzed by HPLC (system 1) and LC-ESI-MS. Neutral thermal hydrolysis of the dCyd adduct was carried out as described above. For NMR studies, the reaction was carried out with NNKCH2OAc (3.0 mmol) and dThd (0.6 mmol). 3. NNALCH2OAc (4) and DNA. NNALCH2OAc (134 mg, 0.5 mmol) was allowed to react with calf thymus DNA (20 mg) in 10 mL of 0.1 M phosphate buffer, pH 7.0, in the presence of porcine liver esterase (500 units) at 37 °C for 1.5 h. The reaction mixture was extracted with an equal volume of CHCl3. The modified DNA was precipitated by addition of ethanol and then washed with 70% ethanol and ethanol. DNA was hydrolyzed enzymatically or by heating as described (11). 4. NNALCH2OAc (4) and dCyd, Cyt, or dThd. NNALCH2OAc (67 mg, 0.25 mmol) was allowed to react with dCyd, Cyt, or dThd (0.05 mmol) in 5 mL of 0.1 M phosphate buffer, pH 7.0, in the presence of porcine liver esterase (90 units) at 37 °C

for 1 h. The reaction mixture was extracted with an equal volume of CHCl3. The aqueous phase was injected on HPLC system 2 and analyzed by LC-ESI-MS. For NMR studies, the reaction was carried out with 7.5 mmol of 4 and 1.5 mmol of dCyd. Treatment of Adducts with NaBH4. POB-dCyd or POBdThd adducts were dissolved in 0.5 mL of 40 mM ammonium phosphate buffer, pH 6.6, and NaBH4 (2 mg) was added. The mixture was allowed to stand for 30 min at room temperature. The pH was adjusted to 7 with 1 N HCl prior to analysis by HPLC or LC-ESI-MS.

Results NNKCH2OAc (1) was allowed to react with calf thymus DNA, and then, the mixture was subjected to enzyme hydrolysis. Analysis by LC-ESI-MS (system 1) with SIM at m/z 415, corresponding to POB-dGuo, m/z 390 (POBdThd), and m/z 375 (POB-dCyd), gave the chromatograms illustrated in Figure 1A. The [M + 1]+ ion of 4-HPB (18) (m/z 166) was also monitored in this analysis. POB-dAdo adducts, which may be unstable, were not investigated in this study. The indicated peaks in the dGuo channel (m/z 415) represent O6(POB-1-yl)dGuo (14,



Scheme 2. Summary of Identification of dCyd and Cyt Adducts (EH, Enzyme Hydrolysis)

Table 1. MS Data for dCyd, Cyt, and dThd Adducts 26-31a,b m/z (relative intensity) adduct

HPLC system

M+ or (M + H)+

[(M - deoxyribose) + H]+

other

O2(POB-1-yl)dCyd (26) O2(POB-1-yl)Cyt (27) O2(PHB-1-yl)dCyd (28) O2(PHB-1-yl)Cyt (29) O2(POB-1-yl)dThd (30) O2(PHB-1-yl)dThd (31)

1 1 2 2 1 1

375 (100) 259 (100) 377 (100) 261 (100) 390 (100) 392 (100)

259 (22)

148 [POB]+ (7), 112 [Cyt + H]+ (2) 148 [POB]+ (10), 112 (24) 168 (10) 150 [PHB]+ (24) 148 [POB]+ (14) 150 [PHB]+ (8)

261 (16) 274 (10) 276 (10)

a Obtained by LC-ESI-MS. b High-resolution MS data were obtained for adducts 29 (calcd, 261.1346; found, 261.1361) and 30 (calcd, 412.1479 [M + Na]+; found, 412.1473).

Scheme 1) and 7(POB-1-yl)dGuo (15, Scheme 1), which have been characterized previously (11). The major peaks in the dThd and dCyd (m/z 390 and m/z 375) channels, eluting at 74.0 and 62.7 min, respectively, were investigated here. There were no peaks in these channels in an enzyme hydrolysate of untreated calf thymus DNA. The areas of the POB-dThd (74.0 min) and POB-dCyd (62.7 min) peaks were 18 and six times greater than that of the peak corresponding to 7(POB-1-yl)dGuo (15) (53.1 min), respectively. Neutral thermal hydrolysis of the enzyme hydrolysate, followed by LC-ESI-MS analysis, gave the chromatograms illustrated in Figure 1B. Analysis at m/z 415 demonstrated that 7-(POB-1-yl)dGuo (15) was unstable under these conditions while O6(POB-1-yl)dGuo (14) was stable, consistent with previous results (11). Analysis at m/z 390 and m/z 375 showed that the POB-dThd adduct was stable while the POB-dCyd adduct was not. Analysis at m/z 166 demonstrated an increase of 4-HPB (18) in tandem with the disappearance of 7(POB-1-yl)dGuo (15) and the POB-dCyd adduct. The 4-HPB peak increased 13-fold upon neutral thermal hydrolysis, based on MS peak area. Our approach to the characterization of the dCyd and Cyt adducts is outlined in Scheme 2. This scheme shows that O2(POB-1-yl)dCyd (26) (retention time 62.7 min, Figure 1A), produced in the reaction of NNKCH2OAc with DNA followed by enzyme hydrolysis, was identical to a product formed in the reaction of NNKCH2OAc with dCyd. Neutral thermal hydrolysis of 26 gave O2(POB-1-

yl)Cyt (27) and 4-HPB (18). Reduction of 27 with NaBH4 produced O2(PHB-1-yl)Cyt (29), which was shown to be identical to products of NNALCH2OAc reactions as summarized in Scheme 2. UV and MS data for adducts 26-29 are summarized in Figure 2 and Table 1. However, the crucial data for identification of O2(PHB-1-yl)Cyt (29), and therefore 26-28 as well, were the 1H and 13 C NMR data summarized in Table 2. The chemical shifts of the carbon at position 1 of the butyl group, 69.5 ppm, and of the protons on this carbon, 4.62 ppm, are consistent with substitution at oxygen, not nitrogen. For example, the chemical shifts of the methylene carbon and protons in 3-ethylCyt (with the ethyl group attached to the ring nitrogen) are 40.2 and 3.86 ppm, respectively. Confirming this assignment, HMBC correlations were observed between the protons on carbon 1 of the butyl group and carbon 2 but not carbon 4 of the pyrimidine ring. These data established the structures of adducts 26-29 (Scheme 2) as O2-substituted dCyd or Cyt derivatives. Hydrolysis experiments were completely consistent with these structural assignments and were informative with respect to the stability of the adducts. Neutral thermal hydrolysis of isolated O2(POB-1-yl)dCyd (26) gave O2(POB-1-yl)Cyt (27) and 4-HPB (18) as illustrated in the LC-ESI-MS chromatograms shown in Figure 3A,B. Figure 3A shows results before neutral thermal hydrolysis while Figure 3B illustrates data obtained after neutral thermal hydrolysis. Figure 3A (top panel) shows SIM for

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Figure 2. UV spectra, obtained in CH3OH/pH 6.6 buffer, of adducts discussed in the text.

m/z 375, the molecular ion of O2(POB-1-yl)dCyd (26). Figure 3A (middle panel) illustrates SIM for the fragment ion with m/z 259, corresponding to [(M-deoxyribose) + H]+ of O2(POB-1-yl)dCyd (26). Figure 3A (bottom panel) monitors m/z 166, which is [M + H]+ of 4-HPB (18). Figure 3B demonstrates that O2(POB-1-yl)dCyd (26) disappears upon neutral thermal hydrolysis with formation of a new peak eluting at 86.4 min (middle panel), corresponding to O2(POB-1-yl)Cyt (27). Figure 3B (bottom panel) shows the appearance of 4-HPB (18) upon neutral thermal hydrolysis of O2(POB-1-yl)dCyd (26). LC-ESIMS experiments established that this material was 4-HPB (18) and not 3-HPB (32).

NNALCH2OAc was allowed to react with DNA, and the mixture was subjected to enzymatic hydrolysis and

analyzed by LC-ESI-MS (system 2, Figure 4A). A portion of this hydrolysate was subjected to neutral thermal hydrolysis and analyzed by LC-ESI-MS (Figure 4B). Analysis by SIM at m/z 377, corresponding to O2(PHB1-yl)dCyd (28), gave the chromatogram illustrated in the upper panel of Figure 4A. Analysis by SIM at m/z 261 (Figure 4A, middle panel) showed a peak at 56 min, corresponding to O2(PHB-1-yl)Cyt (29), partially formed during enzymatic hydrolysis. Analysis by SIM at m/z 168 gave a peak at 28 min, identified as diol 25 (Figure 4A, bottom panel). Figure 4B shows that O2(PHB-1-yl)dCyd (28) disappears upon neutral thermal hydrolysis (upper panel) while O2(PHB-1-yl)Cyt (29) and diol 25 increase (middle and bottom panels). O2(PHB-1-yl)dCyd (28) also slowly depyrimidated (in approximately 40 min) at 37 °C to O2(PHB-1-yl)Cyt (29). Identification of the dThd adducts is summarized in Scheme 3. This scheme shows that reaction of NNKCH2OAc with DNA followed by enzyme hydrolysis, or with dThd, gave a product (retention time 74.0 min, Figure 1A), identified as O2(POB-1-yl)dThd (30). Treat-



Table 2. NMR Data for O2(PHB-1-yl)Cyt (29) and O2(POB-1-yl)dThd (30) and for Comparison, Diol 25 and HPB (18) (in DMSO-d6)

adduct O2(PHB-1-yl)Cyt

Pyridyl 3

2 (29)

1H

8.51 (s)

13C

147.4 9.13 (s)

O2(POB-1-yl)dThda (30)

1H

diol 25b

1H

4-HPB (18)b

1H

140.9

8.50 (d), J ) 2.1 9.06 (d), J ) 2.1

4

5

6

7.71 (d), Jc ) 7.1 133.2 8.29 (d), J ) 8.3 7.70 (dt), J ) 7.8, 1.8 8.23 (dt), J ) 8.1, 1.8

7.28 (dd), J ) 7.1, 3.8 123.0 7.56 (d), J ) 8.3 7.33 (dd), J ) 7.8, 4.8 7.51 (dd), J ) 8.1, 5.1

8.43 (d), J ) 3.8 147.9 8.78 (s) 8.42 (dd), J ) 4.8, 1.5 8.73 (dd), J ) 4.8, 1.5

Butyl adduct O2(PHB-1-yl)Cyt (29)

1 1H 13C

O2(POB-1-yl)dThd (30)

1H 13C

diol 25

1H

4-HPB (18)

1H

2

3a

3b

4

4.62 (br) 69.5 4.35 (t), J ) 8.3 67.1 3.35 (m)

1.69 (m) 35.0 2.08 (m)

1.69 (m)

1.60 (m)

4.14 (m) 65.3

3.40 (dt), J ) 5.4, 6.3

1.72 (m)

adduct O2(PHB-1-yl)Cyt (29)

2

adduct O2(POB-1-yl)dThd (30)

Pyrimidyl 4

5

6 7.81 (s) 155.9 7.81 (s) 133.5

164.7

165.1

6.04 (s) 99.1

154.1

169.8

115.5

1H 13C

1′

a 4.39 (t), J ) 5.1

5.41 (d), J ) 4.5 4.55 (dd), J ) 6.3, 11.1 4.48 (t), J ) 5.4

3.04 (t), J ) 7.2

1H 13C

O2(POB-1-yl)dThd (30)

22.0 1.61 (m)

24.8 3.23 (t), J ) 8.3 34.4 1.46 (m) 1.36 (m)

OH 5.38 (br)

2′-Deoxyribosyl 2′

CH3

NH2 6.76 (br)

1.77 (s) 13.3

3′

4′

5a′

5b′

1H

6.16 (m)

2.16 (br)

4.23 (br)

3.77 (br)

3.61 (d), J ) 12.5

3.55 (d), J ) 12.5

13C

a

40.1

a

87.3

60.7

a 13C

resonance values were not obtained for the pyridyl, butyl C4, and 2′-deoxyribosyl 1′ and 3′ carbons of O2(POB-1-yl)dThd. b 1H NMR of 4-HPB (18) and diol 25 were obtained at 300 MHz in DMSO-d6. For ease of comparison, the C-numbering system used for 4-HPB (18) and diol 25 in this table is the same as that used for the adducts but the opposite of standard convention. c Coupling constants (J) are reported in Hz.

ment of 30 with NaBH4 produced O2(PHB-1-yl)dThd (31) as a mixture of diastereomers (Figure 5). These were identical to adducts produced by the reaction of NNALCH2OAc with DNA or dThd (Figure 6). UV and MS data for dThd adducts 30 and 31 are summarized in Figure 2 and Table 1.

attachment at oxygen rather than nitrogen of dThd as in Cyt adduct 29 discussed above. HMBC correlations were observed between the protons on carbon 1 of the butyl group (4.35 ppm) and carbon 2 but not carbon 4 of the pyrimidine moiety.

NMR data (Table 2) were critical for the structural assignment of O2(POB-1-yl)dThd (30) and therefore 31. Carbon resonances of the dThd moiety were consistent with literature data (17). HMBC correlations observed between the methyl protons and the carbons 4, 5, and 6 of dThd confirmed these assignments. The spectral characteristics of the oxobutyl moiety demonstrated that it was a but-1-yl, not a but-2-yl substituted dThd, as the latter would have had a methyl doublet. Furthermore, the NMR data excluded the possibility of a cyclized tetrahydrofuranyl structure. The chemical shift of carbon 1 of the butyl moiety, 67.1 ppm, was consistent with

Discussion Our data clearly demonstrate that the O2-positions of dCyd and dThd are major sites of pyridyloxobutylation and pyridylhydroxybutylation of pyrimidines in DNA reacted with NNKCH2OAc and NNALCH2OAc, respectively. A consistent set of structural data support this conclusion. The dCyd adducts were thermally unstable, which suggested that substitution had occurred at either the O2- or the N-3-positions (12, 18). In both cases, the molecule is susceptible to depyrimidation and loss of 4-HPB as observed here (Figures 1 and 3). The UV

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Figure 3. Chromatograms obtained upon LC-ESI-MS analysis (system 1) of O2(POB-1-yl)dCyd (26) collected by HPLC (system 1) from the reaction of NNKCH2OAc with dCyd. The analyses were carried out (A) directly or (B) after neutral thermal hydrolysis (100 °C, pH 7.0, 1 h). SIM was carried out at the m/z values indicated in each panel. Identities of the peaks, as determined in the text, and their retention times are indicated by the labels.

Figure 4. Chromatograms obtained upon LC-ESI-MS analysis (system 2) of an enzyme hydrolysate of DNA that had been allowed to react with NNALCH2OAc. The hydrolysate was analyzed (A) directly or (B) after neutral thermal hydrolysis (100 °C, pH 7.0, 1 h). SIM was carried out at the m/z values indicated in each panel. Identities of the peaks, as determined in the text, and their retention times are indicated by the labels.



Scheme 3. Summary of Dientification of dThd Adducts (EH, Enzyme Hydrolysis)

Figure 5. Chromatogram obtained upon HPLC analysis (system 1) of the reaction mixture of NaBH4 and O2(POB-1-yl)dThd (30), which was collected by HPLC (system 1) from the reaction of NNKCH2OAc with dThd. The indicated peaks are diastereomers of O2(PHB-1-yl)dThd (31).

spectrum of O2(PHB-1-yl)Cyt (29) (Figure 2D) resembled that of O2-ethylCyt (Figure 2E) more closely than that of 3-ethylCyt (λmax 279) (12). Substitution at the O2position of dCyd was confirmed by NMR data. The chemical shifts of carbon-1 of the butyl group and the corresponding methylene protons were compatible only with substitution at oxygen, and this was supported by HMBC data. Unlike the dCyd adducts, the major dThd adducts were thermally stable, consistent with the lack of a positive charge on the pyrimidine ring. The UV spectra of O2(POB-1-yl)dThd (30) and O2(PHB-1-yl)dThd (31) (Figure 2F-H) were quite similar to those of O2(POB-1-yl)dCyd (26) and O2(PHB-1-yl)dCyd (28) (Figure 2A,C), respectively. The UV spectrum of O2(PHB-1yl)dThd (31) (Figure 2G,H) had λmax 258.1 nm, similar to that of O2-ethyldThd (256 nm) but different from those of 3-ethyldThd (267 nm) and O4-ethyldThd (280 nm) (19). NMR data were completely consistent with substitution at O2 of dThd. Finally, acid hydrolysis (0.1 N HCl, 85 °C) of O2(PHB-1-yl)dThd (31) resulted in depyrimidation,

Figure 6. Chromatograms obtained upon (A) LC-ESI-MS analysis (system 2) of an enzyme hydrolysate of DNA that had been allowed to react with NNALCH2OAc. SIM was carried out at m/z 392 [M + H]+ of O2(PHB-1-yl)dThd (31). In panel B, selected reaction monitoring of m/z 392f276 [(M-deoxyribose) + H]+ is shown. Diastereomers of 31 were not separated under these conditions.

a characteristic of O2- but not O4-substituted dThd (18) (data not shown). Our data are consistent with previous studies demonstrating that alkylnitrosoureas such as ethylnitrosourea readily react with oxygens of pyrimidines (20). Singer showed that O2-ethyldThd was formed to a similar extent as O6-ethyldGuo in reactions with DNA (20). Smaller amounts of O2-ethyldCyd were also observed in those reactions. Ethylation of the O2-position of both dCyd and dThd exceeded reactions at the N-3-positions of these pyrimidines and at O4 of dThd (20). Patterns of DNA alkylation by alkylnitrosoureas and other alkylating agents have been reviewed (12, 21). Alkylnitrosoureas, similar to compounds 1 and 4 used here, generate diazohydroxides and diazonium ions upon solvolysis. However, there may be important differences in reactivity between simple alkanediazohydroxides and those formed here, which have POB and PHB groups. It is possible that noncovalent minor groove binding of these pyridine-containing intermediates occurs. It is known that minor groove ligands prefer AT rich sites, and this could lead to increased covalent binding to T as seen here.

596


Such noncovalent interactions have been observed with 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, which also contains a pyridine ring (22-24). A potentially notable result of this study was the apparently high extent of formation of the pyrimidine adducts as compared to dGuo adducts in the reaction of NNKCH2OAc with DNA. Because we do not yet have quantified standards of the pyrimidine adducts, we cannot be certain of the relative amounts. However, on the basis of MS area alone, the level of O2(POB-1-yl)dThd (30) was 18 times greater and that of O2(POB-1yl)dCyd (26) six times greater than the amount of 7(POB1-yl)dGuo (15). This suggests that pyrimidines in DNA may be much more reactive than dGuo toward pyridyloxobutylating agents, which could be due to the noncovalent interactions referred to above. Furthermore, the stability of O2(POB-1-yl)dThd (30) relative to O2(POB1-yl)dCyd (26) and 7(POB-1-yl)dGuo (15) suggests that it may be biologically more important. Studies with diethylnitrosamine and ethylnitrosourea have shown that dThd adducts can be highly persistent in vivo, thus increasing the probability of miscoding events (25-27). The stability of O2(POB-1-yl)dThd (30) may also be significant with respect to its possible use as a biomarker for DNA damage by tobacco specific nitrosamines. DNA exposed to pyridyloxobutylating agents releases 4-HPB (18) upon neutral thermal hydrolysis (3). This property has been known since 1988, but the adducts that release this 4-HPB had not been identified. We recently reported that 7(POB-1-yl)dGuo (15) is one of the 4-HPB releasing adducts in pyridyloxobutylated DNA, accounting for an estimated 30-35% of these adducts (10). The results of this study demonstrate that O2(POB-1-yl)dCyd (26) is another major 4-HPB releasing adduct in pyridyloxobutylated DNA. The quantitative aspects are unclear, but on the basis of the relative MS peak areas of this adduct and 7(POB-1-yl)dGuo (15), it seems likely that O2(POB-1-yl)dCyd (26) makes a major contribution to 4-HPB released upon neutral thermal hydrolysis. The origin of this phenomenon, resulting in hydrolytic cleavage of the POB group in competition with depurination or depyrimidation, likely relates to anchimeric assistance by the carbonyl group of the POB residue as discussed previously (10). In summary, this study has demonstrated that the O2positions of dCyd and dThd in DNA are major targets of pyridyloxobutylation and pyridylhydroxybutylation by NNKCH2OAc and NNALCH2OAc. The extent of formation of O2(POB-1-yl)dThd (30), a thermally stable adduct, appears to be particularly high as compared to other DNA adducts of NNKCH2OAc. We are currently carrying out studies directed toward quantification of the pyrimidine adducts. Furthermore, this study has shown that O2(POB1-yl)dCyd (26) is a thermally unstable 4-HPB releasing adduct in pyridyloxobutylated DNA.

Acknowledgment. This study was supported by Grant CA-81301 from the National Cancer Institute. S.S.H. is an American Cancer Society Research Professor, supported by ACS Grant RP-00-138. S.J.S. is a fellow of the American Cancer Society supported by the Bowman Family Foundation. We thank Priyanka A. Rao for her assistance in this study and Bob Carlson for editorial assistance.

Hecht et al.

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Substituted Pyrimidine Adducts Formed in Reactions of 4 - American

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