Formation of Conjugate Adducts in the Reactions of Malonaldehyde

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Chem. Res. Toxicol. 2005, 18, 300-307

Formation of Conjugate Adducts in the Reactions of Malonaldehyde-Acetaldehyde and Malonaldehyde-Formaldehyde with Guanosine Donata Pluskota-Karwatka,† Frank Le Curieux,‡,§ Tony Munter,⊥ Rainer Sjo¨holm,⊥ and Leif Kronberg*,⊥ Department of Organic Chemistry, Åbo Akademi University, Biskopsgatan 8, FIN-20500 Turku/Åbo, Finland, Faculty of Chemistry, Adam Mickiewicz University, Grunwaldzka 6, 60-780 Poznan´ , Poland, De´ partement Toxicologie-Sante´ Publique-Environnement, Faculte´ des Sciences Pharmaceutiques et Biologiques, B.P. 83, 59006 Lille Cedex, France, and Laboratoire de Toxicologie, Institut Pasteur de Lille, 1 rue du Professeur Calmette BP 245, 59019 Lille Cedex, France Received June 14, 2004

The reactions of guanosine with malonaldehyde in buffered aqueous solutions in the presence of acetaldehyde or formaldehyde were studied. The reaction mixtures were analyzed by RPHPLC. Two adducts were formed in the reaction of malonaldehyde and acetaldehyde and one in the reaction of malonaldehyde and formaldehyde. The products were isolated and purified by preparative C-18 chromatography and structurally characterized by UV absorbance, 1H NMR, and 13C NMR spectroscopy and mass spectrometry. The adducts formed in the reaction of malonaldehyde and acetaldehyde were identified as 7-(2,2-diformyl-1-methylethyl)-3-(β-Dribofuranosyl)pyrimido[1,2-a]-purin-10(3H)-one (M2AA-Guo I) and 2-(3,5-diformyl-4-methyl1,4-dihydro-1-pyridyl)-9-(β-D-ribofuranosyl)-purin-6(9H)-one (M2AA-Guo II). In the reaction of malonaldehyde and formaldehyde, the major product was identified as 7-formyl-3-(β-Dribofuranosyl)pyrimido[1,2-a]purin-10(8H)-one (M1FA-Guo). The highest yields of M2AA-Guo I and M2AA-Guo II, 7 and 2 mol %, respectively, were obtained in the reaction performed at pH 7.4 and 37 °C for 6 days, while M1FA-Guo was produced at a yield of 0.3 mol % after 3 days of reaction at pH 7.4 and 37 °C. The products are formed by reactions of malonaldehydeacetaldehyde and malonaldehyde-formaldehyde condensation products with guanosine and are analogous to the previously identified condensation products formed with adenosine, cytidine, and proteins.

Introduction Peroxidation of polyunsaturated fatty acids generates a range of electrophiles, some of which react with proteins and DNA. The most studied compound in this respect is malonaldehyde, an aldehyde known to be mutagenic and carcinogenic (1-4). Malonaldehyde induces cross-links between DNA and proteins (5, 6), and forms stable adducts with nucleic acid bases. The aldehyde reacts most readily with the guanine base to form a 1:1 pyrimido purinone adduct called M1G (7-9). M1G is the major malonaldehyde adduct in DNA and the adduct has been detected in human tissues and is considered to be highly mutagenic (10). Malonaldehyde undergoes self-condensation to afford oligomers that may react with the DNA bases forming adducts (11-13). The formation of mono- and oligomeric malonaldehyde adducts is a consequence of the aldol condensation reaction that malonaldehyde easily undergoes. Acetaldehyde is another very reactive electrophile present in human tissues (14). The compound reacts with * To whom correspondence should be addressed. E-mail: [email protected]. Phone: +358-2-21 54 138. Fax: +358-2-21 54 866. † Adam Mickiewicz University. ‡ Faculte ´ des Sciences Pharmaceutiques et Biologiques. § Institut Pasteur de Lille. ⊥ Åbo Akademi University.

the exocyclic amino group of deoxyribonucleosides and DNA to form unstable Schiff bases (15, 16). The aldehyde also forms stable 1,N2-propano and N2-dimethyldioxane adducts resulting from reactions of more than one molecule of acetaldehyde with deoxyguanosine (17, 18). Acetaldehyde is known to produce interstrand cross-links in DNA (19), as well as DNA-protein cross-links (20). The presence of acetaldehyde in the reaction medium significantly influences the reactivity of malonaldehyde toward nucleosides (21). Moreover, malonaldehyde and acetaldehyde undergo condensation reactions yielding 1:1 or 2:1 malonaldehyde-acetaldehyde conjugates and these conjugates have been shown to bind covalently to proteins (12, 22-26). Previous studies performed in our laboratory have shown that the malonaldehyde-acetaldehyde conjugates react with deoxyadenosine yielding a dihydropyridine and an exocyclic propenoformyl adduct (27). Later, we observed that the analogous adducts are formed when acetaldehyde is replaced with formaldehyde (28). We have reported that the dihydropyridine and the exocyclic propeno adducts are also obtained in the reaction of malonaldehyde with cytidine in the presence of acetaldehyde (29). Further, we showed that replacing acetaldehyde with formaldehyde yields the corresponding propenoformyl cytidine adduct (29).

10.1021/tx0498455 CCC: $30.25 © 2005 American Chemical Society Published on Web 01/28/2005

Modification of Guanosine with Aldehyde Conjugates

In the work presented here, we have studied the reaction of guanosine with malonaldehyde and acetaldehyde, and with malonaldehyde and formaldehyde. The work resulted in the structural identification of three novel conjugate adducts. The same base modification should also take place in reactions of the aldehyde conjugates with 2′-deoxyguanosine.

Materials and Methods Chemicals. Malonaldehyde was prepared by acid hydrolysis of 1,1,3,3-tetraethoxypropane, as described by Stone et al. (30). Guanosine, 2′-deoxyguanosine and 1,1,3,3-tetraethoxypropane (97% purity) were purchased from Sigma Chemical Co. (St. Louis, MO). Acetaldehyde (purity >99%) and formaldehyde (37% solution stabilized with 10-15% methanol) were obtained from J. T. Baker B. V. (Deventer, Holland). The water used was Aqua Sterilisata from B. Braun Medical Oy (Espoo/Esbo, Finland). Acetonitrile was super purity solvent from Romil Chemicals (England). 1,1,1-Trichloroethane was obtained from Fluka Chemie AG (Buchs, Switzerland). 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- × 125-mm reversed-phase C18 analytical column (Hypersil BDS-C18, Hewlett-Packard, Espoo/ Esbo, Finland). The column was eluted isocratically for 5 min with 0.01 M phosphate buffer, pH 7.1, and then with a gradient from 0% to 30% acetonitrile in 25 min at a flow rate of 1 mL/ min. Preparative isolation of the products was performed by column chromatography on a 4-cm × 4-cm column of preparative C18 bonded silica grade (40 µm, Bondesil, Analytichem International, Harbor City, CA) at a flow rate of 4 mL/min. The products were further purified on a semipreparative 8-µm, 10mm × 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 variable wavelength Shimadzu SPD-6A UV spectrophotometric detector (Shimadzu Europe, Germany). Spectroscopic and Spectrometric Methods. The 1H NMR and 13C NMR spectra were recorded at 30 °C on a JEOL JNMA500 Fourier transform NMR spectrometer at 500 and 125 MHz, respectively (JEOL, 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 determination of the shifts and the coupling constants of the multiplets of the proton signals in the ribose unit were based on a first-order approach and are given with an accuracy of ( 0.3 Hz. The assignment of carbon signals was based on chemical shifts, C-H correlations, and carbon-proton couplings. All chemicals shifts are reported in ppm. The electrospray ionization mass spectra were recorded on a Fisons ZabSpec-oa TOF instrument (Manchester, U.K.). Ionization was carried out using nitrogen as both 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. The isolated compound was introduced by loop injection at a flow rate of 20 µL/min (H2O/CH3CN/acetic acid: 80/20/1). PEG 200 was used as a standard for the exact mass determination. The mass spectrometer was working at a resolution of 7000. The UV spectra of the isolated compounds were recorded with the diode-array detector as the peaks eluted from the HPLC column. A Shimadzu UV-160 spectrophotometer (Shimadzu Europe, Germany) was used to determine the molar extinction coefficients (). Preparation of 7-(2,2-Diformyl-1-methylethyl)-3-(β-Dribofuranosyl)pyrimido[1,2-a]-purin-10(3H)-one (M2AA-

Chem. Res. Toxicol., Vol. 18, No. 2, 2005 301 Guo I)1 and 2-(3,5-Diformyl-4-methyl-1,4-dihydro-1-pyridyl)9-(β-D-ribofuranosyl)-purin-6(9H)-one (M2AA-Guo II). Guanosine (0.5 g, 1.76 mmol) was dissolved in about 260 mL of a 0.5 M phosphate buffer solution at pH 7.4. For the purpose of increasing the solubility of guanosine, 60 mL of water was added to the phosphate buffer solution. Malonaldehyde was prepared by the hydrolysis of 1,1,3,3-tetraethoxypropane (1.8 g, 8 mmol) with 20 mL of 0.1 M HCl. Acetaldehyde (0.3 g, 8 mmol) was added to the 1,1,3,3-tetraethoxypropane hydrolysate, and this mixture was then added to the solution of guanosine. Following this addition, the pH of the reaction mixture was checked and adjusted to 7.4 with 1 M NaOH. The reaction was performed at 37 °C and the progress of the reaction was followed by HPLC analyses on the C18 analytical column. After 6 days, the reaction mixture was concentrated by rotary evaporation to about 150 mL and filtered, and the adducts were subsequently isolated by chromatography on the preparative C18 column. The column was first eluted with 100 mL of a 0.01 M phosphate buffer at pH 7.1 and then with 100 mL of 5%, 10%, 15% and 20% acetonitrile solutions in 0.01 M phosphate buffer at pH 7.1. Fractions of 30 mL were collected. The compound M2AA-Guo I eluted from the column with 5 and 10% acetonitrile eluent, and the compound M2AA-Guo II eluted with 10 and 15%. The fractions containing each product were combined and concentrated by rotary evaporation to about 20 mL. Both compounds were further purified by using the semipreparative HPLC column. For purification of M2AA-Guo I the column was eluted isocratically with 5% acetonitrile in 0.01 M phosphate buffer solution (pH 7.1) for 2 min and then with a gradient from 5% to 30% acetonitrile over the course of 30 min. For the purification of M2AA-Guo II, the column was eluted isocratically with 7% acetonitrile in 0.01 M phosphate buffer solution (pH 7.1) for 2 min and then with a gradient from 7% to 25% acetonitrile over the course of 30 min. The fractions containing the pure compounds were combined, concentrated to about 30 mL, and then desalted by using the preparative C18 column. The desalted solutions were evaporated to dryness and the residues were subjected to spectroscopic and spectrometric studies. The isolated amounts of compounds were 37 mg (7 mol % yield) for M2AA-Guo I and 11 mg (2 mol % yield) for M2AA-Guo II. The isolated products had the following spectral characteristics: UV spectra, M2AA-Guo I UVmax 220, 260 nm ( 48 400 M-1 cm-1), 312, 352 nm, UVmin 236, 330 nm (HPLC eluent, 11% acetonitrile in 0.01 M phosphate buffer, pH 7.1), M2AA-Guo II UVmax 232, 296 nm ( 58 100 M-1 cm-1), 376 nm, UVmin, 216, 261 and 329 nm, shoulder between 248 and 268 nm (HPLC eluent, 15% acetonitrile in 0.01 M phosphate buffer, pH 7.1). In the positive ion electrospray mass spectra the following ions were observed (m/z, relative abundance, formation): M2AA-Guo I, 418 (42, MH+), 286 (100, MH+ - ribosyl + H); M2AA-Guo II, 418 (61, MH+), 286 (100, MH+ - ribosyl + H). The 1H and 13C NMR spectroscopic data of M2AA-Guo I and M2AA-Guo II are presented in Tables 1 and 2, respectively. Reactions were also performed by using the sodium salt of malonaldehyde instead of the malonaldehyde hydrolysate. The sodium salt of malonaldehyde was prepared according to the procedure described by Go´mez-Sa´nchez et al. (12). Preparation of 7-Formyl-3-(β-D-ribofuranosyl)pyrimido[1,2-a]purin-10(8H)-one (M1FA-Guo). Malonaldehyde was prepared by the hydrolysis of 1,1,3,3-tetraethoxypropane (18 mmol) with 20 mL 0.1 M HCl. One gram of guanosine (3.5 mmol) was dissolved in about 500 mL of a 0.5 M phosphate buffer solution (pH 7.4) and 200 mL of water, and 1,1,3,3-tetraethoxypropane hydrolysate was added to this solution. Formaldehyde (2 g, 66 mmol) was then added to the mixture of malonaldehyde and guanosine. The reaction was performed at 37 °C 1

Abbreviations: M2AA-Guo I, 7-(2,2-diformyl-1-methylethyl)-3-(β-

D-ribofuranosyl)pyrimido[1,2-a]-purin-10(3H)-one; M2AA-Guo II, 2-(3,5diformyl-4-methyl-1,4-dihydro-1-pyridyl)-9-(β-D-ribofuranosyl)-purin6(9H)-one; M1FA-Guo, 7-formyl-3-(β-D-ribofuranosyl)pyrimido[1,2a]purin-10(8H)-one;M1dG,3-(2′-deoxy-β-D-erythro-pentofuranosyl)pyrimido-

[1,2-a]purin-10(3H)-one; PEG, poly(ethylene glycol).

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Table 1. 1H and

13C

protonb

Pluskota-Karwatka et al.

Chemical Shifts (δ)a and Spin-Spin Coupling Constants (JH,H and JC,H (Hz)) of Protons and Carbons in M2AA-Guo I δ

multiplicity

JH, H

H-6

(1H)

9.00

d (br)

2.7

H-8 H-a CH3 H-b CHO

(1H) (1H) (3H)

dm qt d

2.7 7.2; 1.5 7.2

(2H)

9.05 4.14 1.52 c 8.51

H-2 H-1′ H-2′ H-3′ H-4′ H-5′ H-5′′ OH-2′ OH-3′ OH-5′

(1H) (1H) (1H) (1H) (1H) (1H) (1H) (1H) (1H) (1H)

8.50 6.01 4.59 4.20 3.99 3.71 3.59 5.6 5.3 5.2

s d t dd q dd dm br br br

m

5.6 5.3 5.0; 3.7 3.7 11.9; 3.4 11.9

carbon

δ

multiplicity

1J C,H

C-6 C-7 C-8 C-a CH3 C-b CHO C-4a C-3a C-10a C-10 C-2 C-1′ C-2′ C-3′ C-4′ C-5′

163.6 130.0 130.9 28.3 16.9 117.7 185.3 148.0 149.5 117.6 152.1 140.7 87.3 73.8 70.3 85.6 61.3

dddd dd dd d dq tm ddd dd dd d dd ddd d d d d t

183.6 188.5 126.7 127.0 154.6

215.2 166.5 147.9 149.2 148.2 139.9

>1J C,H

7.7; 4.4; 0.8 7.2; 4.4; 2.3 5.6; 4.4; 2.3 5.4 18.7 4.9; 3.9 15.5; 4.4 4.9; 2.9 11.6 3.1; 1.0 4.3; 1.7

a Relative to TMS. b H-1′-H-5′ and OH are the protons in the ribosyl unit. The determination of the shifts and the coupling constants in the ribosyl unit was based on a first-order approach. c Not detected. Due to the acidity of the proton, the signal undergoes exchange with the residual water present in the sample.

Table 2. 1H and

13C

protonb

Chemical Shifts (δ)a and Spin-Spin Coupling Constants (JH,H and JC,H (Hz)) of Protons and Carbons in M2AA-Guo II δ

multiplicity

CHO CHO

(1H) (1H)

9.47 9.46

s s

H-2/H-6 H-4 CH3

(2H) (1H) (3H)

8.50 3.68 1.01

s q d

H-8p H-1′ H-2′ H-3′ H-4′ H-5′ H-5′′ OH-2 OH-3′ OH-5′

(1H) (1H) (1H) (1H) (1H) (1H) (1H) (1H) (1H) (1H)

7.86 5.79 4.59 4.17 3.89 3.65 3.55 5.38 5.16 4.95

s d dd dd dd m m d d t

JH,H

6.6 6.6

5.3 4.9; 9.9 3.9; 7.3 4.2; 8.0

carbon

δ

multiplicity

1J C,H

>1J C,H

CHO CHO C-3/C-5 C-2/C-6 C-4 CH3 C-2p C-4p C-5p C-6p C-8p C-1′ C-2′ C-3′ C-4′ C-5′

190.5 190.4 123.4 142.4 22.8 21.5 151.2 149.7 122.8 166.1 135.7 87.2 73.3 70.5 84.9 61.6

ddd ddd ddd ddd dm qm t dd d s dd d d d d t

172.8 172.8

5.8; 2.5 5.8; 2.0 23.8; 5.9; 4.1 9.6; 4.6

180.0 132.9 127.4

2.7 4.5; 3.4 10.5 210 161.7 147.1 148.5 146.4 140.4

4.1

5.5 3.5 5.6

a Relative to TMS. b H-1′-H-5′ and OH are the protons in the ribosyl unit. The determination of the shifts and the coupling constants in the ribosyl unit was based on a first-order approach.

and the progress of the reaction was followed by HPLC analyses on the C18 analytical column. After 3 days, the reaction mixture was concentrated and filtered, and the adduct was subsequently isolated by chromatography on the preparative C18 column. The column was eluted with 200 mL of 0.01 M phosphate buffer at pH 7.1 and then with 100 mL of 5%, 10%, and 15% acetonitrile solutions in 0.01 M phosphate buffer (pH 7.1). Fractions of 30 mL were collected. The compound M1FA-Guo eluted from the column with the 10% acetonitrile eluent. The fractions containing the product were combined and concentrated by rotary evaporation to about 20 mL. The product was further purified by use of the semipreparative HPLC column. The column was eluted isocratically with 5% acetonitrile in water for 2 min, and then with a gradient from 5% to 25% over the course of 30 min at a flow rate of 4 mL/min. The collected fractions were combined and evaporated to dryness, and the obtained residue was subjected to spectroscopic and spectrometric studies. The isolated amount of the compound was 4 mg (0.3 mol % yield). M1FA-Guo had the following spectral characteristic: UV spectrum (HPLC eluent, 14% acetonitrile in 0.01 M phosphate

buffer pH 7.1) UVmax 204, 274 and 344 nm ( 20 000 M-1 cm-1), UVmin 240 and 296 nm. In the positive ion electrospray mass spectrum the following ions were observed (m/z, relative abundance, formation): 350 (85, MH+), 218 (100, MH+ - ribosyl + H). The 1H NMR spectroscopic data of M1FA-Guo were as follows: 9.28 (d, 1H, CHO, J ) 1.6 Hz), 8.15 (d, 1H, H-2, J ) 1.1 Hz), 7.50 (d, 1H, H-6, J ) 0.6 Hz), 4.57, 4.56 (dd, 2H, H-8a, H-8b, J ) 21.5; 16.0 Hz), 5.73 (d, 1H, H-1′, J ) 6.5 Hz), 4.44 (m, 1H, H-2′), 4.11 (dd, 1H, H-3′, J ) 4.1; 8.4 Hz), 3.89 (dd, 1H, H-4′, J ) 3.9; 7.9 Hz), 3.62 (dd, 1H, H-5′, J ) 5.0; 12.4 Hz), 3.54 (dd, 1H, H-5′′, J ) 5.0; 12.4 Hz). The 13C NMR chemical shifts were as follows: 187.0 (CHO), 137.7 (C-2), 143.3 (C-6), 119.4 (C-7), 146.5 (C-4a), 147.9 (C-3a), 156.0 (C-10), 110.8 (C-10a), 86.5 (C-1′), 73.8 (C-2′), 70.4 (C-3′), 85.4 (C-4′), 61.3 (C-5′). C-8 shift could not be observed due to overlapping with the solvent signal (DMSO). Small-Scale Reactions. Guanosine (11.3 mg, 0.04 mmol) and malonaldehyde hydrolyzed from 1,1,3,3-tetraethoxypropane (40 mg, 0.18 mmol) were mixed with acetaldehyde (8 mg, 0.18


Chem. Res. Toxicol., Vol. 18, No. 2, 2005 303

mmol) or with formaldehyde (20 mg, 0.67 mmol) in 3 mL of 0.5 M phosphate buffer at pH 7.4. The appropriate aldehyde (acetaldehyde or formaldehyde) was added to malonaldehyde, and this mixture was then added to the solution of guanosine. The reactions were performed at 37 °C, and the progress of the reactions were followed daily by HPLC analyses of aliquots of the reaction mixtures. Transformation of M2AA-Guo I into M2AA-Guo II. M2AA-Guo I (5 mg, 0.02 mmol) was dissolved in 0.05 M NaOH and the solution was incubated at 45 °C. The transformation of M2AA-Guo I to M2AA-Guo II was determined by HPLC analysis of aliquots of the solution. Determination of Product Yields. Quantitative 1H NMR analysis, using 1,1,1-trichloroethane as an internal standard, was performed on aliquots of the pure adducts. By comparing the intensity of the proton signal of the methyl group in the standard and the proton signals of the adduct in question, it was possible to calculate the exact molar amount of the adduct present in the NMR sample. HPLC standard solutions were prepared by taking an exact volume of the NMR sample (i.e. an exact amount of the adduct) and diluting it with an appropriate volume of water. In this way, the peak areas found in the HPLC chromatogram of the standard solutions could be directly related to the amounts of the compound injected. The quantitative determination of adducts in the reaction mixtures was made by comparing the peak area of adducts in the HPLC standard solutions with the peak area of adducts in the reaction mixtures. The adduct M2AA-Guo I was quantified using UV detection at 254 nm. For M2AA-Guo II and M1FA-Guo, UV detection at 325 and 350 nm, respectively, was used. The molar yields were calculated from the original amount of guanosine in the reaction mixtures.

Results and Discussion Following a reaction time of 1 day, HPLC analysis of the small-scale reactions of malonaldehyde hydrolysate with guanosine in the presence of acetaldehyde (pH 7.4 and 37 °C) showed the formation of the adduct M2AAGuo I. After a reaction time of 2 days, the adduct M2AAGuo II was also detected. In the chromatogram, the two peaks representing M2AA-Guo I and M2AA-Guo II were found at longer retention times than guanosine (Figure 1 A). The highest yields, 7 and 2 mol % for M2AA-Guo I and M2AA-Guo II, respectively, were obtained after 6 days of reaction (Figure 2A). Prolonged reaction resulted in a drop in the yields. The same products were formed in reactions where the malonaldehyde hydrolysate was replaced by the sodium salt of malonaldehyde and in later experiments it was found that the yields were higher in the reaction using the malonaldehyde sodium salt (data not shown). For the purpose of isolating sufficient amounts of the products for structural determination by spectrometric and spectroscopic methods, a large-scale reaction was performed. The compounds were isolated and purified by preparative C18 column chromatography and semipreparative HPLC. On the basis of data collected from UV, NMR spectroscopy and electrospray mass spectrometry the compounds were identified as 7-(2,2-diformyl1-methylethyl)-3-(β-D-ribofuranosyl)pyrimido[1,2-a]purin10(3H)-one (M2AA-Guo I) and 2-(3,5-diformyl-4-methyl1,4-dihydro-1-pyridyl)-9-(β-D-ribofuranosyl)-purin-6(9H)one (M2AA-Guo II), (Scheme 1). The UV spectrum of M2AA-Guo I exhibited absorption maxima at 220, 260, 312 and 352 nm, and absorption minima at 236 and 330 nm (Figure 3A). This spectrum resembled the UV spectrum of M1G (8) but displayed a bathochromic shift as compared to M1G. The UV spec-

Figure 1. C18 analytical column HPLC chromatogram of the reaction mixture of (A) malonaldehyde and acetaldehyde with guanosine held at 37 °C and pH 7.4 for 6 days and (B) malonaldehyde and formaldehyde with guanosine held at 37 °C and pH 7.4 for 3 days. For analysis conditions, see Materials and Methods.

trum of M2AA-Guo II exhibited absorption maxima at 232, 296 and 376 nm, absorption minima at 216, 261 and 329 nm, and a weak shoulder between 248 and 268 nm (Figure 3A). In the positive ion electrospray mass spectrum, both compounds gave rise to a protonated molecular ion peak at m/z 418 and an ion peak at m/z 286 that corresponded to the loss of the ribosyl unit from MH+. The 1H NMR spectrum of M2AA-Guo I displayed, besides the signals from the protons of the ribose moiety, a two-proton multiplet at δ ) 8.51 ppm, a one-proton singlet at 8.50 ppm, a one-proton doublet of multiplets at δ ) 9.05, and a one-proton doublet at 9.00 ppm (Table 1). Moreover, the spectrum exhibited a three-proton doublet at δ ) 1.52 ppm and a one-proton quartet at δ ) 4.14 ppm. The two-proton signal at δ ) 8.51 ppm was assigned to the formyl protons on the basis of the downfield chemical shift and the one bond C-H correlation with the carbon signal at δ ) 185.3 ppm. The singlet at δ ) 8.50 ppm was assigned to the proton in the purine ring (H-2) and this signal showed an H-H long-range correlation with the H-1′ signal in the ribose unit at δ ) 6.01 ppm. The formyl protons displayed H-H correlations with the three-proton doublet at δ ) 1.52 ppm and with the one-proton quartet at δ ) 4.14 ppm. On the basis of the chemical shift and the coupling constants (J ) 7.2

304


Figure 2. Formation of (A) M2AA-Guo I and M2AA-Guo II at 37 °C and pH 7.4 in the reaction of guanosine with malonaldehyde and acetaldehyde and of (B) M1FA-Guo at 37 °C and pH 7.4 in the reaction of guanosine with malonaldehyde and formaldehyde.

Scheme 1. Structures of Guanosine Adducts Obtained in the Reaction of the Mixture of Malonaldehyde and Formaldehyde (M1FA-Guo) and Malonaldehyde and Acetaldehyde (M2AA-Guo I and M2AA-Guo II)

Hz), the signals at δ ) 1.52 and 4.14 ppm were assigned to the methyl group and to the H-a protons, respectively. The olefinic proton, H-6, appeared at δ ) 9.00 ppm and it showed coupling (J ) 2.7 Hz) to the signal at δ ) 9.05 ppm which in turn was assigned to H-8. Seto et al. (31) reported chemical shifts at δ ) 9.08 and 9.38 ppm and a coupling constant of J ) 2.0 Hz for these protons (H-6 and H-8, respectively) in the corresponding unsubstituted pyrimidopurine. The 13C NMR spectrum of M2AA-Guo I showed seven carbon signals, in addition to the 10 signals arising from


Figure 3. UV absorbance spectra of (A) M2AA-Guo I and M2AA-Guo II and (B) M1FA-Guo. The spectra were recorded with the diode-array detector as the compounds eluted from the HPLC column.

the guanine and ribose moieties (Table 1). The signal observed at δ ) 185.3 ppm was due to the two formyl carbons and displayed a strong one-bond C-H coupling (1J ) 154.6 Hz). The signal was further split into two weak doublets and by selective decoupling experiments one of the couplings (J ) 4.9 Hz) was found to be due to coupling to the H-a proton. One-bond correlations were observed between the signal at δ ) 163.6 and the proton at δ ) 9.00 (H-6) and between the signal at δ ) 130.9 and the proton at δ ) 9.05 ppm (H-8). Consequently, these signals were assigned to C-6 and C-8, respectively. The carbon signal at δ ) 130.0 ppm exhibited long-range correlations to the proton H-6 and to the protons of the methyl group. The signal was assigned to C-7. The assignment of the signal at δ ) 117.7 ppm to C-b was confirmed by its strong two-bond coupling (2J ) 18.7 Hz) to the formyl proton, a coupling common for protons located R to a formyl group (32). The signal at δ ) 28.3 ppm was assigned to C-a, and the assignment was confirmed by the one-bond coupling (1J ) 126.7 Hz) to the proton signal at δ ) 4.14 ppm. Further, the signal of C-a showed C-H correlation to the formyl protons. The methyl carbon signal appeared at δ ) 16.9 ppm and the signal was split into a quartet of doublets due to couplings to the methyl protons (1J ) 127.0 Hz), and to H-a (2J ) 5.4 Hz). The carbons originating from the guanine unit gave signals at δ ) 152.1, 149.5, 148.0, 140.7 and 117.6 ppm. The signal at δ ) 152.1 ppm exhibited long-range correlation to H-8 and was assigned to C-10. The carbon signal at δ ) 149.5 ppm was assigned to C-3a and it displayed correlations to H-2 and to the H-1′ ribosyl proton. The signal at δ ) 148.0 ppm was split into two doublets and exhibited couplings to H-6 and H-8, and was thus assigned to C-4a. The carbon signal at δ ) 140.7



Scheme 2. Proposed Mechanism for the Formation of Guanosine Adducts M1FA-Guo, M2AA-Guo I, and M2AA-Guo II

ppm (1J ) 215.2) was assigned to C-2, and the signal exhibited connectivity with the H-1′ proton signal of the ribosyl unit. The purine signal, C-10a, appeared at δ ) 117.6 ppm and displayed a long-range correlation to the guanine H-2 proton. The 1H NMR spectrum of M2AA-Guo II displayed, besides the signals from the protons of the ribose moiety, one-proton singlets at δ ) 9.47, 9.46, and 7.86 ppm, and a two-proton singlet at δ ) 8.50 ppm (Table 2). Moreover, the spectrum displayed a three-proton doublet at δ ) 1.01 ppm and a one-proton quartet at δ ) 3.68 ppm. The 13C NMR spectrum of the compound exhibited besides the signals from the purine and the ribose units, carbons signals at δ ) 190.5, 190.4, 123.4.0, 142.4, 22.8 and 21.5 ppm (Table 2). The 1H and 13C NMR chemical shifts are almost identical to those we have reported for the dihydropyridine units connected to adenosine and cytidine (27, 29), with the exception that we now observe two different signals for the protons and carbons in the formyl groups. The reason for the chemical unequivalency of the formyl groups is probably the hindered rotation of the dihydropyridine unit around the bond between the C-2p and the dihydropyridine nitrogen. In previous work carried out in our laboratory, it has been shown that malonaldehyde generates conjugate adducts with cytidine and 2′-deoxyadenosine not only in the presence of acetaldehyde, but also when acetaldehyde is replaced by formaldehyde (28, 29). Consequently, we studied the reaction between guanosine and a mixture of malonaldehyde and formaldehyde. HPLC analyses of the small-scale reactions revealed the formation of one product peak marked M1FA-Guo (Figure 1B). The adduct M1FA-Guo was formed at the maximum yield of 0.32 mol % in reactions proceeding for 3 days, and prolonged reaction resulted in lower yields (Figure 2B).

The product was isolated from a large-scale reaction by preparative C18 column chromatography and purified by HPLC. In the positive ion electrospray mass spectrum the protonated molecular ion was observed at m/z 350. The UV spectrum of M1FA-Guo, (Figure 3B), showed absorption maxima at 204, 274 and 344 nm, and absorption minima at 240 and 296 nm. These data and the 1H and 13C NMR spectra are fully identical to those recorded for the unsaturated pyrimidopurine derivative found to be formed in the reaction mixture of guanosine and triformylmethane (33). Thus the same derivative is formed in reaction of guanosine with malonaldehyde and formaldehyde. The corresponding derivative, which would result from reaction of malonaldehyde-acetaldehyde 1:1 conjugate (M1AA, Scheme 2) could not be observed. Neither could we observe any derivatives resulting from reaction of the 2:1 malonaldehyde-formaldehyde conjugate. In previous studies, it was reported that the dihydropyridine derivatives of deoxyadenosine and cytidine and of amino groups in proteins are strongly fluorescent. Also, the cyclic propenoformyl derivatives of deoxyadenosine and cytidine have been reported to be strongly fluorescent (25, 28, 29). However, none of the guanosine adducts presented in this paper exhibited marked fluorescent properties. The reason for this is unclear, but obviously the purine moiety affects the electron distribution of the dihydropyridine unit and as a consequence, the adduct does not absorb or emit energy in such a way that it would have fluorescence properties. Tuma et al. (25) suggested that the dihydropyridine adducts found in proteins are formed by dissociation of one malonaldehyde - acetaldehyde conjugate moiety from an amino acid nitrogen and transfer of this moiety to a malonaldehyde adducted amino acid nitrogen (present

306



Scheme 3. Transformation of M2AA-Guo I to M2AA-Guo II

in the enamine form). A prerequisite for the transfer is that the enamine and the conjugate are positioned on protein nitrogens of close proximity to each other. This mechanism is probably not involved in the formation of adducts generated from single nucleoside bases. Instead, a mechanism where the nucleobases are attacked by malonaldehyde-acetaldehyde conjugates formed in the reaction mixture seems to be more likely (Scheme 2) (2729). Support for this mechanism is provided by the studies that have shown that the conjugation products are formed at the reaction conditions employed in our work and that these conjugates react with amino groups yielding dihydropyridine derivatives (12, 26). An alternative route to the adducts could be that they were derived from M1G, or the ring-opened form of M1G [N2-(3-oxopropenyl)guanosine]. However, this route was ruled out, since we could not detect the adducts in reaction mixtures of M1G and malonaldehyde and acetaldehyde (pH 7.4, and 37 °C). On the other hand, at least in part, M2AA-Guo II could have been obtained from M2AA-Guo I. HPLC analyses of a basic solution (0.05 M NaOH) of 5 mg pure M2AAGuo I held at 45 °C showed that the amount of the compound decreased, while at the same time M2AA-Guo II was formed. After 4 h storage, the transformation had produced 0.4 mg M2AA-Guo II (Figure 4 A and B). Prolonged storage resulted in degradation of the adducts. The transformation took also place at neutral conditions,

but at a slower rate (results not shown). The identification of M2AA-Guo II in these solutions was based on comparison of retention time, UV and mass spectra to that of M2AA-Guo II isolated from the preparative reaction. The transformation goes most likely through a ring-opening of the propeno ring at the endocyclic nitrogen of guanosine followed by an attack of the guanosine amino nitrogen on one of the terminal carbonyl groups and finally the dihydropyridine structure is obtained by dehydration (Scheme 3). Riggins et al. (34, 35) have recently studied in depth the equivalent ring opening in M1dG. In conclusion, the study shows that when guanosine is mixed with malonaldehyde and acetaldehyde, or formaldehyde, adducts are formed which contain units derived from condensation reactions of the aldehydes. The identified adducts are analogous to those formed when deoxyadenosine or cytidine are reacted with mixtures of the aldehydes (27, 28, 29). The adducts were formed at neutral pH conditions and thus their formation in DNA in vitro and in vivo cannot be ruled out. Recently, Wang et al. found stable calf thymus DNA adducts that consist of acetaldehyde self-condensation products (36, 37). As far as we now, there are no reports on the formation of conjugate adducts in DNA in vivo. If such adducts are detected in DNA, they could play a significant role in the carcinogenic and mutagenic effects of malonaldehyde, formaldehyde and acetaldehyde.

Acknowledgment. We are very grateful to Mr. Markku Reunanen for the mass spectra. This work was financially supported by the Foundation of Jenny and Antti Wihurin in Helsinki and by State Committee for Scientific Research (Grant No. 3 T09A 035 26, D. Pluskota-Karwatka), by the European Commission (Contract No. ERBFMBICT961394, F. Le Curieux), and by the Graduate School of Bio-Organic Chemistry at Åbo Akademi University (T. Munter). Supporting Information Available: The 500 MHz 1H NMR spectrum of M1FA-Guo, the 125 MHz 13C NMR spectrum of M1FA-Guo, the 500 MHz 1H NMR spectrum of M2AA-Guo I, the 125 MHz 13C NMR of M2AA-Guo I, 500 MHz 1H NMR spectrum of M2AA-Guo II, and the 125 MHz 13C NMR of M2AA-Guo II. This material is available free of charge via the Internet at http://pubs.acs.org.

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Figure 4. (A) Degradation of M2AA-Guo I in 0.05 M NaOH at 45 °C. (B) Formation of M2AA-Guo II from M2AA-Guo I in 0.05 M NaOH at 45 °C.

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TX0498455

Formation of Conjugate Adducts in the Reactions of Malonaldehyde

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