Characterization of the Adducts Formed in the Reactions of

Glycidamide (GA) is a mutagenic epoxide metabolite of acrylamide (AM), a high production chemical with many industrial uses. Moreover, recent findings...
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1652

Chem. Res. Toxicol. 2004, 17, 1652-1658

Characterization of the Adducts Formed in the Reactions of Glycidamide with Thymidine and Cytidine Josefin Backman, Rainer Sjo¨holm, and Leif Kronberg* Department of Organic Chemistry, A° bo Akademi University, Biskopsgatan 8, FIN-20500 Turku/A° bo, Finland Received July 1, 2004

Glycidamide (GA) is a mutagenic epoxide metabolite of acrylamide (AM), a high production chemical with many industrial uses. Moreover, recent findings have shown that AM is formed in starchy foods cooked at high temperatures. This has refocused the attention on this chemical and its metabolite and on their possible mutagenicity and carcinogenicity. In this study, we have reacted GA with cytidine and thymidine in aqueous-buffered solutions. The adducts from the nucleosides have been isolated by reversed phase HPLC and characterized by their UV absorbance and 1H and 13C NMR spectroscopic and mass spectrometric features. The reaction with thymidine yielded one adduct, N3-(2-carbamoyl-2-hydroxyethyl)thymidine (N3-GA-dThd), while the reaction with cytidine yielded three adducts. Two adducts were identified as a diastereomeric pair of N3-(2-carboxy-2-hydroxyethyl)cytidine (N3-GA-Cyd-1 and N3-GA-Cyd2). The third adduct from the cytidine reaction was identified as N3-(2-carboxy-2-hydroxyethyl)uridine (N3-GA-Urd).

Introduction

Scheme 1. Structures of AM and GA

(AM;1

Acrylamide Scheme 1) is a vinylic compound used in the synthesis of polyacrylamides, which have a wide variety of industrial applications (1). Recently, AM has emerged as a factor that could be associated with a considerable cancer risk. To¨rnqvist et al. (2) have found that AM is formed during heating of different starchy foodstuffs and showed this to be the major source of AM for humans. The levels of AM could be as high as 1504000 µg/kg foodstuff in carbohydrate-rich foods. AM is formed as a result of the Maillard reaction between amino acids and reducing sugars (3). AM is neurotoxic, clastogenic, and carcinogenic in animal experiments, and it is classified as a probable human carcinogen by the International Agency for Research on Cancer (1). There is, however, no direct evidence for genotoxicity mediated by AM in vivo. Part of the body load of AM is metabolized to a reactive epoxide, glycidamide (GA; Scheme 1) (4, 5). According to studies on hemoglobin adducts in rats, the conversion of AM to GA in vivo is inversely related to the dose (4, 6). About 20-25% of a single injection of AM is converted to GA (7). This epoxide metabolite has been shown to be genotoxic and to exhibit mutagenicity in the Ames test (8). It is therefore assumed that AM is responsible for the neurotoxic effects and GA for the mutagenic and cancer-initiating effects of AM exposure. Previously, it has been shown that AM reacts with the bases in DNA in vitro, but these reactions proceed extremely slowly (9). On the other hand, GA reacts more * To whom correspondence should be addressed. Tel: +358-2-2154138. Fax: +358-2-215-4866. E-mail: [email protected]. 1 Abbreviations: AM, acrylamide; HETCOR, heteronuclear correlation NMR spectroscopy (C-H one-bond correlation NMR spectroscopy); COSY, H-H correlation NMR spectroscopy; GA, glycidamide; HMQC, heteronuclear multiple quantum coherence NMR spectroscopy (C-H one-bond correlation NMR spectroscopy); HMBC, heteronuclear multiple bond connectivity NMR spectroscopy (long-range 1H-13C COSY); LC-DAD liquid chromatography coupled with diode array detection; LC-ESI-MS/MS, liquid chromatography/electrospray ionization tandem mass spectrometry.

readily with the nucleosides. In 1995, Segerba¨ck et al. (10) reported on a major GA-guanine adduct (N7-GAGua) formed in DNA of the mouse and the rat, and recently, Gamboa da Costa et al. (11) reported on the formation of the N7-GA-Gua adduct together with corresponding N3-GA-Ade in DNA of adult and neonatal mice treated with AM and GA. In addition, N1- and N6GA-Ade adducts, along with a cyclic unidentified adduct, were formed in the reactions with 2′-deoxyadenosine and GA. Because other alkyl epoxides, e.g., butadiene monoxide and chloroethylene oxide, are known to form adducts with each of the DNA bases (12-14), we were interested in finding out whether GA forms adducts with bases not previously studied, i.e., thymidine and cytidine. Obviously, further studies on DNA modifications require structural identification of all possible adducts and the availability of reference compounds. Here, we report the isolation and structural determination of novel adducts formed in the reaction of GA with thymidine and cytidine in neutral aqueous solutions at 37 °C.

Materials and Methods Caution: GA has been found to be mutagenic in Salmonella typhimurium. Caution should therefore be exercised in the handling of the compound. Chemicals. GA was purchased from Toronto Research Chemicals (North York, ON, Canada). Thymidine and cytidine were obtained from Sigma Chemical Co. (St. Louis, MO). Acetonitrile (p.a.) was from Labscan Ltd. (Dublin, Ireland). The

10.1021/tx049823i CCC: $27.50 © 2004 American Chemical Society Published on Web 11/24/2004

Thymidine and Cytidine Adducts of Glycidamide water used was distilled water purified with a Millipore system (Simplicity 185, Billerica, MA). Chromatographic Methods. Liquid chromatography/diode array detector (LC-DAD) analyses were performed on an Agilent 1100 Series liquid chromatographic system (Agilent Technologies, Espoo/Esbo, Finland) consisting of a quaternary pump, a vacuum degasser, an autosampler, a thermostated column compartment, and a diode array detector (UV). The reaction mixtures were chromatographed on a 5 µm, 4 mm × 125 mm reversed phase C18 analytical column (Hypersil BDS-C18, Agilent Technologies). For the thymidine reaction, the column was eluted isocratically for 3 min with 1% acetonitrile in ammonium acetate buffer (0.01 M, pH 7) and then with a gradient from 1 to 30% acetonitrile over the course of 12 min at a flow rate of 1 mL/min. For the cytidine reaction, the same elution conditions were used but at a flow rate of 0.5 mL/min. The products were purified on a semipreparative 5 µm, 10 mm × 250 mm reversed phase C18 column (BDS Hypersil C18, Thermo Hypersil-Keystone, Krotek Oy, Tampere/Tammerfors, Finland). The column was coupled to a Varian 5000 Liquid Chromatograph (Varian Aerograph, United States) and a variable wavelength Shimadzu SPD-6A UV spectrophotometric detector (Shimadzu Europe, Germany). Spectroscopic and Spectrometric Methods. The 1H and 13C NMR spectra of the thymidine adduct were recorded at 30 °C on a JEOL JNM-A500 FT NMR spectrometer at 500 and 125 MHz, respectively (JEOL, Japan). The 1H and 13C NMR spectra of the cytidine adducts were recorded on a Bruker Avance 600 NMR spectrometer at 600 and 150 MHz, respectively (Bruker). The samples were dissolved in Me2SO-d6. The 1H NMR signal assignments were based on chemical shifts and H-H and C-H correlation data. The assignment of carbon signals was based on chemical shifts and C-H correlations. The liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) analyses were performed on an Agilent 1100 Series LC/MSD SL Trap instrument (Agilent Technologies) consisting of a quaternary pump, a vacuum degasser, an autosampler, a thermostated column compartment, and a diode array detector (UV). The instrument was equipped with a electrospray source and operated in the positive ion mode. Operation parameters of the ESI ion source were as follows: drying gas temperature, 350 °C; drying gas flow, 12 L/min; nebulizer gas pressure, 40 psi; end plate voltage, -3500 V; end plate offset, -500 V; capillary exit, 115 V; and skim 1 was set at 40 V. Ion trap parameters were as follows: accumulation time, 20 ms; averages, 5; rolling averaging on and ion charge control on. Nitrogen gas was used as the drying and nebulizing gas. Collision-induced dissociation experiments coupled with multiple tandem mass spectrometry (MSn) employed helium as the collision gas. The fragmentation amplitude was varied between 0.7 and 1.0 V. The compounds were introduced through the LC system using the same chromatographic conditions as in the LC-DAD analyses, with the exception of the flow rate, which was adjusted to 0.5 mL/min. The pure compounds were also analyzed by direct inlet infusion to the source by a syringe pump at a rate of 5 µL/min and at a concentration of about 1 µg/mL. The compounds were dissolved in a 1:1 v/v mixture of 0.01 M aqueous ammonium acetate/acetonitrile. The operation parameters were as follows: drying gas temperature, 325 °C; drying gas flow, 5 L/min; and nebulizer gas pressure, 15 psi. The UV spectra of the isolated compounds were recorded with the diode array detector as the peaks eluted from the HPLC column. Preparative-Scale Reaction of GA with Thymidine at pH 9. Preparation of N3-(2-Carbamoyl-2-hydroxyethyl)thymidine (N3-GA-dThd). GA (360 mg, 4.13 mmol) was reacted with thymidine (100 mg, 0.41 mmol) in 30 mL of 0.5 M phosphate buffer solution (pH 9.0). The reaction was performed at 37 °C. The progress of the reaction was followed by LC-DAD and LC-ESI-MS/MS analyses on the C18 analytical column. The reaction was stopped at optimum yield (3 days).The reaction mixture was concentrated by rotary evaporation to about 5 mL,

Chem. Res. Toxicol., Vol. 17, No. 12, 2004 1653 Table 1. 1H and 13C Chemical Shifts (δ) and Spin-Spin Coupling Constants, JH,H (Hz), of Protons in the Thymidine Adduct (N3-GA-dThd) proton

δ (ppm)

multiplicity

JH,H

carbon

H-6a (1 H) H-8a (1 H) H-7aa (1 H) H-7ba (1 H) NHa a(1 H) NHba(1 H) CH3a (3 H) OH-8 (1 H)

7.73, 7.74 4.15, 4.16 3.91, 3.93 4.02, 4.04 7.25, 7.26 7.16, 7.16 1.80, 1.80 5.63

q dd dd dd s s d br d

1.2 9.3, 4.6 12.9, 5.0 12.9, 9.3

C-6 C-8a C-7

134.7 67.4, 67.5 43.9

1.2

CH3

13.0

C-2 C-4a C-5 C-9a C-1′a C-2′b

150.6 162.8, 162.9 108.4 174.39, 174.43 84.70, 84.72

C-3′ C-4′ C-5′

70.2 87.3 61.2

H-1′a (1 H) H-2′ and H-2′′ (2 H) H-2′ and H-2′′ (2 H) H-3′a (1 H) H-4′a (1 H) H-5′a (1 H) H-5′′a (1 H) OH-5 (1 H) OH-3 (1 H)

6.17, 6.17 2.08

t dd

6.9 7.0, 2.8

2.09

dd

6.9, 3.1

4.22, 4.22 3.77, 3.77 3.59, 3.60 3.54, 3.55 5.07 5.27

dt q dd dd br s br s

3.8, 3.0 3.8 11.9, 3.7 11.9, 3.8

δ (ppm)

a Separate shifts due to a mixture of diastereomers. overlapped by solvent.

b

Signal

and the precipitated phosphate salts were removed by filtration. The compound was purified and desalted by using the semipreparative HPLC column. The column was eluted isocratically with 1% acetonitrile in water for 3 min and then with a gradient from 1 to 10% acetonitrile over the course of 7 min, and finally isocratically with 10% acetonitrile for 10 min. The flow rate was 3 mL/min. The solutions containing the pure compound were then combined and rotary evaporated to dryness and dried under vacuum. The residue was subjected to spectroscopic and spectrometric studies. The isolated amount of the compound was 16.7 mg. The yield was 12.3 mol %. The isolated compound had the following spectral characteristics: UV spectrum for N3-GA-dThd, UVmax 270, 210 nm, UVmin 238 nm (HPLC eluent: approximately 23% ACN in ammonium acetate buffer; pH 7). In the positive ion electrospray mass spectrum, the following ions were observed (m/z, relative abundance, formation): MS, 330 (68, MH+), 352 (100, MNa+), 368 (75, MK+), 214 (71, MH+ - deoxyribosyl + H), MS2 of 330, 214 (100, MH+ - deoxyribosyl + H), MS3 of 330f214, 169 (29, MH+ - deoxyribosyl - CONH3 + H), 197 (100, MH+ deoxyribosyl - OH + H), 127 (10, MH+ - deoxyribosyl - C3H6NO2 + H). The 1H and 13C NMR spectroscopic data of the compound are presented in Table 1. Preparative-Scale Reaction of GA with Cytidine. Preparation of the Diastereomers of N3-(2-Carboxy-2-hydroxyethyl)cytidine (N3-GA-Cyd-1 and N3-GA-Cyd-2) and of Adduct N3-(2-Carboxy-2-hydroxyethyl)uridine (N3-GAUrd). GA (500 mg, 5.8 mmol) was reacted with cytidine (140 mg, 0.58 mmol) in 30 mL of 0.5 M phosphate buffer solution (pH 4.6). The reaction was performed at 37 °C. The progress of the reaction was followed by LC-DAD and LC-ESI-MS/MS analyses on the C18 analytical column. The reaction was stopped after 6 days. The reaction mixture was concentrated to about 5 mL by rotary evaporation, and the precipitated phosphate salts were removed by filtration. The adducts were isolated from the mixture by chromatography on the semipreparative column. The column was eluted isocratically with 1% acetonitrile in ammonium acetate buffer (0.01 M, pH 7) at a flow rate of 3 mL/min. The fractions containing the adducts were collected, concentrated by rotary evaporation, and desalted by using the semipreparative column, which was eluted isocratically with 1% acetonitrile in water. The solutions containing the pure adducts were concentrated and dried under vacuum.. The residues were subjected to spectroscopic and spectrometric studies. The isolated amounts of the compounds were as

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Chem. Res. Toxicol., Vol. 17, No. 12, 2004 Table 2. 1H and

13C

Backman et al.

Chemical Shifts (δ) and Spin-Spin Coupling Constants, JH,H (Hz), of Protons in the Two Diastereomers Obtained in the Cytidine Reaction (N3-GA-Cyd)a

δ (ppm)

multiplicity

JH,H

δ (ppm)

proton

I

II

I

II

I

H-6 (1 H) H-5 (1 H) H-7a (1 H) H-7b (1 H) H-8 (1 H)

7.98 6.05 3.98 4.30 3.88

7.94 6.03 3.94 4.31 3.87

d d dd dd m

d d m dd m

7.9 7.9 14.5, 3.5 14.5, 4.5

H-1′ (1 H) H-2′ (1 H) H-3′ (1 H) H-4′ (1 H) H-5′ (1 H) H-5′′ (1 H)

5.75 4.02 3.94 3.88 3.57 3.68

5.74 4.04 3.94 3.87 3.57 3.69

d t t m dd dd

d t m m dd dd

3.86 4.3 5.1

3.11 3.0

12.2, 2.8 12.2, 2.8

12.2, 2.2 12.2, 2.2

a

II 7.9 7.9

carbon

I

C-6 C-5 C-7

138.5 96.5 49.2

H-5, H-1′

HMBC

C-8 C-9 C-4 C-2 C-1′ C-2′ C-3′ C-4′ C-5′

69.6 174.8 158.9 148.6 89.9 73.8 69.0 84.6 60.1

H-7 H-8 H-5, H-6, H-7 H-6, H-7 H-2′

H-8

14.2, 3.9

H-4′, H-5′

I, N3-GA-Cyd-1; II, N3-GA-Cyd-2.

Table 3. 1H and 13C Chemical Shifts (δ) and Spin-Spin Coupling Constants, JH,H (Hz), of Protons in the Uridine Adduct (N3-GA-Urd) proton

δ (ppm)

multiplicity

H-6 (1 H) H-5 (1 H) H-7a (1 H) H-7b (1 H) H-8 (1 H)

7.87 5.69 3.87 3.98 3.89

d d ma mb ma

H-1′ (1 H) H-2′ (1 H) H-3′ (1 H) H-4′ (1 H) H-5′ (1 H) H-5′′ (1 H)

5.81 4.04 3.98 3.86 3.57 3.66

d t t dt dd dd

JH,H 8.0 8.0

4.8 5.0 5.1 5.0, 3.4 12.0, 3.3 12.0, 3.3

carbon

δ (ppm)

C-6 C-5 C-7

138.7 100.9 44.5

C-8 C-2 C-4 C-9 C-1′ C-2′ C-3′ C-4′ C-5′

67.1 150.9c 162.4c 172.1c 89.0 73.4 69.5 84.5 60.6

a Signal overlapped by H-4′. b Signal overlapped by H-3′. c Because of the very small amount of the compound, these carbon signals were assigned from the C-H long-range correlation spectrum.

follows: N3-GA-Cyd-1, 2.94 mg; N3-GA-Cyd-2, 1.76 mg; and N3GA-Urd, 0.80 mg. The yields were 1.53, 0.92, and 0.43 mol %. The two diastereomers of N3-GA-Cyd had the following spectral characteristics: UV spectrum, UVmax 282, 210 nm, UVmin 245 nm (HPLC eluent: approximately 2% ACN in ammonium acetate buffer; pH 7). In the positive ion electrospray mass spectrum, the MS2, MS3, and MS4 spectra, the following ions were observed (m/z, relative abundance, formation): MS, 332 (100, MH+), 370 (11, MK+), MS2 of 332, 200 (100, MH+ ribosyl + H), MS3 of 332f 200, 154 (7, MH+ - ribosyl - CH3O2 + H), 182 (1, MH+ - ribosyl - H2O + H), MS4 of 332f200f182, 154 (4, MH+ - ribosyl - CH3O2 + H). The 1H and 13C NMR spectroscopic data of the compound are presented in Table 2. N3-GA-Urd had the following spectral characteristics: UV spectrum, UVmax 265, 206 nm, UVmin 235 nm (HPLC eluent: approximately 2% ACN in ammonium acetate buffer; pH 7). In the positive ion electrospray mass spectrum, the following ions were observed (m/z, relative abundance, formation): MS, 333 (100, MH+), MS2 of 333, 201 (100, MH+ - ribosyl + H), MS3 of 333f201, 155 (100, MH+ - ribosyl - CH3O2 + H), 183 (51, MH+ - ribosyl - H2O + H). The 1H and 13C NMR spectroscopic data of the compound are presented in Table 3. Small-Scale Reactions of GA with Thymidine and Cytidine. Small-scale reactions were performed to find the reaction conditions giving the highest possible yield of the adducts. GA (71 mg, 0.82 mmol) was reacted with thymidine (10 mg, 0.041 mmol) in 3 mL of 0.5 M phosphate buffer solutions at pH 7.0 and 9.0. The reactions were performed at 37 °C. The progress of the reactions was followed daily by LC-DAD and LC-ESI-

MS/MS analyses of aliquots of the reaction mixtures using the C18 analytical column. GA (71 mg, 0.82 mmol) was reacted with cytidine (10 mg, 0.041 mmol) in 3 mL of 0.5 M phosphate buffer solution at pH 4.6 and 7.0. The reaction was performed at 37 °C. The progress of the reactions was followed daily by LC-DAD and LC-ESIMS/MS analyses of aliquots of the reaction mixtures using the C18 analytical column. Small-Scale Reaction of GA with 2′-Deoxycytidine. GA (77 mg, 0.88 mmol) was reacted with 2′-deoxycytidine (10 mg, 0.044 mmol) in 3 mL of 0.5 M phosphate buffer solution at pH 4.6. The reaction was performed at 37 °C. The progress of the reaction was followed daily by LC-DAD and LC-ESI-MS/MS analyses of aliquots of the reaction mixture using the C18 analytical column. Reaction of N3-GA-Cyd-1 with Methyl Iodide. Approximately 200 µg of N3-GA-Cyd-1 was dissolved in 2 mL of methanol, and a few drops of methyl iodide were added to the solution. The solution was stirred for 5 days at room temperature, and the reaction was analyzed with LC-ESI-MS/MS. Aliquots of 100 µL were taken, which were evaporated to dryness in a stream of nitrogen gas. The residue was dissolved in 1:1 acetonitrile/ammonium acetate buffer solution and analyzed on the C18 analytical column. The column was eluted in the same way as described for the cytidine and GA reaction.

Results and Discussion Reaction of GA with Thymidine. A small-scale reaction was performed at various pH conditions to find out the conditions giving the optimal yield of an adduct. LC-DAD and LC-ESI-MS/MS analyses of the small-scale reactions of GA with thymidine showed the formation of one major product peak with a longer retention time than thymidine. The compound marked N3-GA-dThd eluted at 12.5 min from the analytical reversed phase C18 column (Figure 1). The highest yield of the compound was obtained in the reaction carried out for 3 days at pH 9.0

Figure 1. C18 analytical column HPLC chromatogram of the reaction mixture of GA and thymidine held at 37 °C and pH 9.0 for 3 days. The chromatogram was recorded at 254 nm by the UV diode array detector.

Thymidine and Cytidine Adducts of Glycidamide

Figure 2. UV absorbance spectrum of N3-GA-dThd. The UV spectrum was recorded with the diode array detector as the compound eluted from the column (approximately 23% ACN in ammonium acetate buffer solution).

Scheme 2. Structures of the GA-Derived Nucleoside Adducts Identified in This Study

and 37 °C. When the reaction was performed at pH 7, the adduct was formed in significantly lower amounts. For the purpose of determining the structure of the compound, a large-scale reaction was performed at pH 9.0. After 3 days of reaction, the compound was isolated from the reaction mixture by semipreparative C18 column chromatography. The isolated yield was 12.3 mol %. On the basis of data from NMR and UV spectroscopy and mass spectrometry, the structure of the adduct was assigned as N3-GA-dThd (Scheme 2). The UV spectrum of N3-GA-dThd exhibited absorption maxima at 270 and 210 nm and an absorption minimum at 238 nm (Figure 2). These values are close to the absorption maxima and minima of thymidine (UVmax 270, 206 nm, UVmin 233). In the positive ion electrospray mass spectrum of N3GA-dThd, the protonated molecular ion peak was observed at m/z ) 330 and was the most abundant ion. The fragment recorded at m/z ) 214 corresponds to the cleavage of the deoxyribosyl moiety from the protonated molecular ion (followed by the attachment of a proton to N-1). The fragment recorded at m/z ) 127 corresponds to protonated thymine (Scheme 3).

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The NMR spectra showed that two diastereomers of the compound were present in the sample. Separate shifts of the two diastereomers were observed for all protons and for some of the carbons (Table 1). The 1H NMR spectrum of N3-GA-dThd displayed a oneproton quartet at δ ) 7.73 ppm and a one-proton doublet at δ ) 1.80 ppm. An H-H long-range correlation was observed between these signals, and they were assigned to H-6 and to the methyl group of the thymidine unit, respectively. In the 13C spectrum, the resonance signal of the methyl group was found at δ ) 13.01 ppm and that of C-6 at δ ) 134.7 ppm. The 1H NMR spectrum showed no signal for the proton at the endocyclic thymidine nitrogen. The 13C NMR spectrum displayed two signals at δ ) 150.6 and 162.8 ppm, which according to their low field chemical shifts, were assigned to the carbonyl carbons C-2 and C-4, respectively (15). The presence of signals due to carbonyl groups and the lack of the N3 proton signal show that the side chain is attached to N3. The C-5 signal was found at δ ) 108.4 ppm. Additional signals in the 1H NMR spectrum were oneproton doublets of doublets at δ ) 4.15, 4.02, and 3.91 ppm, a broad one-proton doublet at δ ) 5.63 ppm, and one-proton signals at δ ) 7.16 and 7.25 ppm. The signals at δ ) 3.91 and 4.02 ppm were assigned to the protons H-7a and H-7b, respectively. A correlation between these signals and a carbon signal at approximately δ ) 43.9 ppm was observed in the one-bond C-H correlation spectrum (HETCOR), and accordingly, the carbon signal was assigned to C-7. In the H-H correlation NMR spectroscopy (COSY) spectrum, a very strong correlation was observed between the H-7 proton signal and the signal at δ ) 4.15 ppm. This one-proton signal was assigned to H-8, and the corresponding carbon signal was found at δ ) 67.4 ppm. The signals at δ ) 5.63, 7.16, and 7.25 ppm lacked one-bond C-H correlations. The resonance signal at δ ) 5.63 ppm was assigned to OH-8 and showed correlations to H-8. The broad singlets at δ ) 7.16 ppm and at δ ) 7.25 ppm were assigned to the chemically inequivalent amide protons. The carbon of the amide function (C-9) was found at δ ) 174.4 ppm. Besides the above-discussed signals, the spectra displayed signals from the sugar unit. Reaction of GA with Cytidine. LC-DAD and LCESI-MS/MS analyses of the small-scale reactions of GA with cytidine showed three product peaks (N3-GA-Cyd1, N3-GA-Cyd-2, and N3-GA-Urd; Figure 3) with shorter retention times than cytidine. The three compounds eluted at 3.2, 3.4, and 3.6 min, respectively. The highest yield of these compounds was obtained in the reaction

Scheme 3. Ions Observed in the Positive Electrospray Mass Spectrum and Proposed Fragment Structures

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Figure 3. C18 analytical column HPLC chromatogram of the reaction mixture of GA and cytidine held at 37 °C and pH 4.6 for 6 days. The chromatogram was recorded at 254 nm by the UV diode array detector. Figure 5. UV absorbance spectrum of N3-GA-Urd. The UV spectrum was recorded with the diode array detector as the compound eluted from the column (approximately 2% ACN in ammonium acetate buffer).

Figure 4. UV absorbance spectrum of N3-GA-Cyd-1 and N3GA-Cyd-2. The UV spectrum was recorded with the diode array detector as the compound eluted from the column (approximately 2% ACN in ammonium acetate buffer).

carried out for 6 days at pH 4.6 and 37 °C. The smallscale reaction that was performed at pH 7 showed that adducts were formed also at neutral conditions but in slightly smaller amounts than at pH 4.6. A small-scale reaction with 2′-deoxycytidine and GA at pH 4.6 was also performed. On the basis of the UV data and on the MS fragmentation pattern recorded on the product peaks in the LC chromatogram obtained from the analyses, it was concluded that the corresponding dCyd adducts including the uridine adduct (see the following text) were formed. For the purpose of determining the structure of the compounds, a large-scale reaction was performed at pH 4.6. After 6 days of reaction, the compounds were isolated from the reaction mixture by semipreparative C18 column chromatography. The isolated yields of the three compounds were 1.53, 0.92, and 0.43 mol %. Data from NMR and UV spectroscopy and mass spectrometry showed that the two compounds eluting first from the column were diastereomers, and their structure was assigned as N3-GA-Cyd-1 and N3-GA-Cyd-2 (Scheme 2). The structure of the third compound was assigned as N3GA-Urd (Scheme 2). The UV spectra of the two diastereomers of N3-GACyd were identical and exhibited absorption maxima at 282 and 210 nm and an absorption minimum at 245 nm (Figure 4). The UV spectra of the diastereomers of N3GA-Cyd are consistent with the spectral shape and maxima of other N3-substituted cytidine adducts (16, 17). The UV spectrum of N3-GA-Urd exhibited absorption maxima at 265 and 206 nm and an absorption minimum at 235 nm (Figure 5). The UV spectrum of N3-GA-Urd is consistent with the spectral shape and maxima of other N3-substituted uridine adducts (16, 17). In the positive ion electrospray mass spectrum of the diastereomers of N3-GA-Cyd, the protonated molecular ion peak was observed at m/z ) 332 (Scheme 3). This was also the most abundant ion. Also observed was an ion at m/z ) 200, which corresponded to the loss of the

ribosyl moiety from MH+. Furthermore, an ion at m/z ) 182 was observed, and this corresponds to the loss of water from [MH+ - ribosyl]. A small ion at m/z ) 154 corresponds to the loss of CH3O2 and the ribosyl unit from MH+. The fragment recorded at m/z ) 112 corresponds to protonated cytosine. In the positive ion electrospray mass spectrum of N3GA-Urd, the protonated molecular ion was observed at m/z ) 333 (Scheme 3). The fragment recorded at m/z ) 201 corresponds to the cleavage of the ribosyl moiety from the protonated molecular ion (followed by the attachment of a proton to N-1). The fragment recorded at m/z ) 155 corresponds to the cleavage of both the ribosyl moiety and the part of the GA tail from the protonated molecular ion. Also observed was an ion at m/z ) 113 which corresponds to protonated uracil. The 1H NMR spectrum of N3-GA-Cyd-1 displayed, besides the signals from the protons of the ribose moiety, two one-proton doublets at δ ) 7.98 and 6.05 ppm (Table 2). Moreover, the spectrum showed one-proton doublets of doublets at δ ) 4.30 and 3.98 ppm and a one-proton multiplet at δ ) 3.88 ppm. The signals at δ ) 7.98 and 6.05 ppm were assigned to the H-6 and H-5 of the cytosine unit, respectively. An H-H short-range correlation was observed between these signals. The one-bond C-H correlation spectrum (heteronuclear multiple quantum coherence, HMQC) showed the carbon signals at δ ) 138.5 ppm and at δ ) 96.5 ppm to be due to C-6 and C-5, respectively. The signals at δ ) 4.30 and 3.98 ppm were assigned to the protons H-7a and H-7b, respectively. A strong correlation between these signals was observed in the COSY spectrum. These signals correlated also to the signal at δ ) 3.88 ppm in the COSY spectrum and consequently, the signal was assigned to H-8. On the basis of C-H correlation, the carbon signals at δ ) 49.2 ppm and at δ ) 69.6 ppm were assigned to C-7 and C-8, respectively. In the long-range C-H correlation spectrum (HMBC), correlations were observed between the H-7 protons and the C-8 carbon and between the H-8 proton and the C-7 carbon. This further confirmed the right assignment of H-7 and H-8. Furthermore, a correlation between the H-8 proton and a carbon signal at δ ) 174.8 ppm was observed. On the basis of this low field chemical shift, which can be expected for a carboxylic acid group, and on the observed correlation, this carbon signal was assigned to C-9. The 13C NMR spectrum of N3-GA-Cyd-1 showed additional signals at δ ) 148.6 and 158.9 ppm. These were assigned to C-2 and C-4, respectively. These chemical shifts are in accordance with previously reported chemi-

Thymidine and Cytidine Adducts of Glycidamide

cal shifts for the carbonyl carbons in N-3-substituted cytidine adducts (15). In the long-range C-H correlation spectrum, correlations between C-4 and H-5, H-6 and H-7, and C-2 and H-6 and H-7 were observed. This confirmed the position for the alkylation to be N3. Further proof for the N3 alkylation was the observation of the proton signals due to H-5 and H-6 and the lack of the signal for the N3 proton. The OH signals could not be observed in the spectra, due to a small amount of water in the sample. The NMR spectroscopy data of N3-GA-Cyd-2 are almost identical to the data of N3-GA-Cyd-1 (Table 2). To further prove the presence of a carboxyl function in the N3-GA-Cyd-1 adduct, the compound was transformed to the methyl ester. In the mass spectrum of the ester, the protonated molecular ion peak was observed at m/z ) 346 (Scheme 3). Also observed was an ion peak at m/z ) 214, which corresponds to the loss of the ribosyl moiety from MH+. Further proof that the methylation reaction occurred in the carboxyl tail of the adduct was the loss of 102 mass units, which corresponds to the loss of the methyl ester tail from the pyrimidine base. The 1H NMR spectrum of N3-GA-Urd displayed, besides the signals from the protons of the ribose moiety, two one-proton doublets at δ ) 7.87 and 5.69 ppm (Table 3). These signals correlated strongly in the COSY spectrum, and they were assigned to H-6 and H-5, respectively. Moreover, the spectrum showed one-proton signals at δ ) 3.87, 3.98, and 3.89 ppm. The signals were assigned to H-7a, H-7b, and H-8, respectively. Although the signals were overlapped by the H-4′ and H-3′ signals of the sugar unit, the assignment was possible by means of the C-H one-bond correlation spectrum and by comparison of the chemical shift data recorded for the N3GA-Cyd adducts and the N3-GA-dThd adduct. A C-H correlation was observed between the H-7 protons and a carbon signal at δ ) 44.5 ppm. This high field chemical shift can be expected for a -CH2 group, and similar shifts were observed in the spectra of both N3-GA-dThd and N3-GA-Cyd. Consequently, the carbon signal was assigned to C-7. Furthermore, a correlation was also observed between the H-8 proton and a carbon signal at δ ) 67.1 ppm. This signal was assigned to C-8. The cytidine and the uridine adducts were found to have a carboxyl function instead of the amide function, which was found in the thymidine adduct and which was originally present in GA. The carboxyl function is also present in the N1 and N6 dA adducts but not in the N3 adenine and N7 guanine adducts (10, 11). The conversion of the amide to the carboxyl function has been assumed to be due to the exocyclic amino group of the cytosine and adenine bases. Solomon et al. have proposed a mechanism for the transformation where the proton on the exocyclic amino group catalyzes the hydrolysis of the amide group (18). Alternatively, a transamidation could occur by means of the exocyclic amino group yielding a cyclic intermediate (Scheme 4). The intermediate undergoes hydrolysis, and the carboxyl function is obtained. The formation of the uridine adduct in the GA-cytidine reaction mixture is most likely a consequence of deamination of the corresponding cytidine adducts (N3-GACyd). Studies of epoxide adducts have shown that when alkylation takes place at N-3, the cytidine adduct undergoes deamination. Several mechanisms have been proposed for this reaction (15, 17, 19, 20).

Chem. Res. Toxicol., Vol. 17, No. 12, 2004 1657 Scheme 4. Transamidation Yielding a Ring Intermediate, Followed by Hydrolysis

In our studies, we could not detect any cyclic GAcytidine adducts although cyclic adducts have previously been proposed to form in the GA-cytidine reaction (21). Gamboa da Costa et al. (11) recently reported the detection of a cyclic adduct formed in the reaction of 2′deoxyadenosine with GA. This adduct was, however, found to be rapidly hydrolyzed to the ring-opened 2′deoxyadenosine adduct and could not be isolated for further structural studies.

Conclusions In this study, we characterized the reaction products of GA with cytidine and thymidine. Thymidine was found to produce the N3-GA-dThd adduct in comparatively high yield (12.3 mol %) in the reaction performed at pH 9. The yield dropped significantly when the reaction was carried out at pH 7. The cytidine adducts were produced in approximately 10 times lower yields. In the cytidine adduct, the unit derived from GA contained a carboxyl function instead of the amide function originally present in GA. The hydrolysis of the amide function may occur through a transamidation. The cytidine adducts underwent deamination yielding the uridine adduct. When 2′-deoxycytidine was reacted with GA, the corresponding deoxy adducts, including the deoxyuridine adduct, were formed. The adducts characterized in this work may be used as reference compounds in studies aiming at the identification of adducts in laboratory animals exposed to GA.

Acknowledgment. This work was financially supported by the Foundation of Magnus Ehrnrooth and the Foundation of the Research Institute at Åbo Akademi University. Supporting Information Available: 1H and 13C spectra of all of the adducts. This material is available free of charge via the Internet at http://pubs.acs.org.

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