Generation of 2′-Deoxyadenosine N 6-Aminyl Radicals from the

Dec 10, 2009 - Generation of 2′-Deoxyadenosine N6-Aminyl Radicals from the ... Nitrogen-centered radicals are major species generated by the additio...
3 downloads 0 Views 628KB Size
Download PDF
48

Chem. Res. Toxicol. 2010, 23, 48–54

Generation of 2′-Deoxyadenosine N6-Aminyl Radicals from the Photolysis of Phenylhydrazone Derivatives Vandana Kuttappan-Nair, Francois Samson-Thibault, and J. Richard Wagner* De´partement de Me´decine Nucle´aire et Radiobiologie, Faculte´ de Me´decine, 3001 12e AVenue Nord, UniVersite´ de Sherbrooke, Sherbrooke, Que´bec, Canada J1H 5N4 ReceiVed August 3, 2009

Nitrogen-centered radicals are major species generated by the addition of hydroxyl radicals and the one-electron oxidation of adenine derivatives. Aminyl radicals are also generated in the decomposition of adenine chloramines upon reaction of hypochlorite. Here, we report the photochemistry of modified 2′-deoxyadenosine (dAdo) containing photoactive hydrazone substituents as a model to investigate the chemistry of dAdo N6-aminyl radicals. Derivatives of dAdo containing a phenylhydrazone moiety at N6 displayed UV absorption between 300 and 400 nm. Upon UV photolysis in the presence of a H-donor, that is, glutathione, two major products were formed, dAdo and benzaldehyde, indicating efficient homolytic cleavage to dAdo N6-aminyl radicals and benzylidene iminyl radicals. dAdo N6-phenylhydrazone was photolyzed in the presence of a molar excess of nonmodified dAdo to mimic the reactions taking place in DNA, and the major photoproducts were identified by high-performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance. The formation of 2-(benzylideneamino)-2′-deoxyadenosine as well as a more extensive oxidation product may be explained by the recombination of initial dAdo N6-aminyl and benzylidene iminyl radicals. The formation of 2′-deoxyinosine may be explained by hydrolytic deamination of dAdo N6-aminyl radicals. Interestingly, a dimeric product containing two dAdo moieties was identified in the photolysis mixture. The present studies demonstrate the ability of dAdo N6-aminyl radicals to undergo H-abstraction to give dAdo, deamination to give 2′-deoxyinosine, and addition to the adenine moiety to give dimers. Introduction The oxidation of adenine by ionizing radiation involving either hydroxyl radicals or base radical cations remains poorly understood (1-3). The initial reaction of hydroxyl radicals with adenine derivatives was investigated by pulse radiolysis and product analysis (4, 5). The main reaction pathway involves addition to C4 of the adenine moiety followed by dehydration to oxidizing adenine N6-aminyl radicals. Other reactions include addition to C8, addition to C5, and H-atom abstraction from the 2-deoxyribose moiety. Adenine N6-aminyl radicals are also the main species arising from one-electron oxidation and deprotonation of adenine radical cations. Thus, the reaction of hydroxyl radicals with adenine as well as the one-electron oxidation of adenine generate adenine N6-aminyl radicals as the predominant radical species. Interestingly, the yield of damage arising from the oxidation of adenine derivatives either in solution or in DNA exposed to radiation is much lower than damage to other nucleobases (4, 6, 7). The reason for such a resistance of adenine toward oxidative damage is not clear. It has been suggested that adenine N6-aminyl radicals efficiently undergo chemical repair by reaction with reducing radicals (4); however, this reaction is unlikely in DNA in which radicals become immobilized within the polymer. Alternatively, it is reasonable to propose that adenine N6-aminyl radicals react with other DNA base moieties, leading to intra- and interstrand crosslinks. The photosensitized oxidation of polyA by 2-methyl-1,4naphthoquinone induced an increase of Rayleigh scattering, pointing to the formation of cross-links in polyA (8). The * To whom correspondence should be addressed. Tel: 1-819-820-6868 ext. 12717. E-mail: [email protected].

chemistry of adenine N6-aminyl radicals is very similar to that of cytosine N4-aminyl radicals, which undergo addition to the 5,6-double bond of nonmodified cytosine to give dimers (9, 10). Recent studies suggest that adenine may be a key player in the formation of interstrand cross-links by initial 5-methyl-(uracilyl) radicals (11, 12). Hypochlorite (HOCl) is a strong oxidant, which is generated during inflammation by myeloperoxidase enzyme in the presence of H2O2 and chloride ions. The reaction of adenosine monophosphate (AMP) with HOCl was shown to generate several nitrogen-centered radicals at N1, N3, and N6 of adenine through the decomposition of intermediate chloramines (13). The propensity of radical formation from the chloramines of nucleosides and polynucleotide followed C > A > G > T, as inferred by electron paramagnetic resonance spin trapping (14, 15). The major radical adducts from a mixture of nucleosides appeared to be nitrogen-centered radicals at the exocyclic amino positions of C and A, together with carbon-centered radicals, likely arising from the addition of aminyl radicals to nonmodified nucleosides. HOCl has also been shown to induce single and double strand breaks in plasmid DNA (16). Several studies have demonstrated the ability of free alkyl and aryl aminyl radicals to induce oxidative DNA damage. The formation of 8-oxo-7,8-dihydroguanine and piperidine-labile breaks in double-stranded DNA induced by Cu2+/H2O2 was greatly enhanced in the presence of acrylonitrile, which undergoes oxidation to iminyl radicals in the reaction mixture. Interestingly, the profile of damage shifted from a random distribution without acrylonitrile to one localized on GG and GGG residues in the presence of acrylonitrile (17). Alternatively, the oxidation of formamide to dimethylaminyl radicals by Cu2+/

10.1021/tx900268r  2010 American Chemical Society Published on Web 12/10/2009

Generation and Fate of dAdo N6-Aminyl Radicals

Chem. Res. Toxicol., Vol. 23, No. 1, 2010 49

Scheme 1. Synthesis of dAdo N6-Phenylhydrazones (3)

H2O2 induced DNA damage favorably at cytosine and thymine residues as revealed by piperidine-labile breaks in doublestranded DNA (18). The photolysis of p-cyanobenzoyl anthraquinone oxime ester was reported to induce single and double strand breaks in supercoiled plasmid DNA under anaerobic conditions, presumably in part by way of the generation of iminyl radicals of anthraquinone (19). Alkylaminyl radicals also appear to contribute to the formation of breaks in plasmid DNA by near-UV-induced photoionization of naphazoline (20). Thus, freely diffusing aminyl and iminyl radicals are able to oxidize DNA bases to give oxidation products, such as 8-oxo-7,8-dihydroguanine, and to induce strand breaks probably by H-atom abstraction from the sugar moiety. In the present work, we have prepared three novel modifications of 2′-deoxyadenosine (dAdo) containing photoactive hydrazone substituents at N6 as a means to specifically generate N6-aminyl (and tautomeric N1-iminyl) radicals of dAdo. Thereby, we report the chemistry of dAdo N6-aminyl radicals in the presence of a good H-donor, that is, glutathione (GSH), and a high concentration of nucleoside, dAdo, to mimic the possible reactions under biological conditions.

Experimental Procedures All chemicals were purchased from Sigma-Aldrich and were of reagent grade unless stated otherwise. 6-Chloro-2′-deoxypurine (1) was purchased from Berry & Associates, Inc. (Dexter, MI). Highperformance liquid chromatography with ultraviolet detection (HPLC-UV) was carried out using Alliance systems (Waters 2795 or 2690) connected to dual wavelength UV detectors (Waters 2487) and a Millenium workstation (Waters version 4). The standard conditions for the analysis of hydrazones by HPLC-UV with a reversed phase column (5 µm ODS A 250 mm × 6 mm; YMC) included a gradient starting at 100% of mobile phase A [triethylammonium acetate (25 mM) at pH 7 containing 2.5% acetonitrile] and going to 60% A and 40% mobile phase B (95% acetonitrile) in 30 min at a flow rate of 1 mL/min. 1H NMR measurements were obtained using Bruker 300 MHz and Varian 600 MHz spectrometers. Electrospray ionization-tandem mass spectrometry (ESI-MS/ MS) was carried out using a Q-tof-2 instrument, and matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) was carried out on a Tof Spec 2E instrument, both from Micromass. For ESI-MS/MS analysis, samples were dissolved in either 50% CH3CN/H2O or 50% acetonitrile/D2O solutions containing 1 mM NaCl. Continuous injection was done with an automated syringe at a flow of 0.6 µL /min. ESI-MS and ESI-MS/MS analyses of the crude reaction mixture and purified compounds were carried out using a Q-Tof-2 instrument (Waters). The samples were diluted in H2O/25% CH3CN/0.1% HCOOH. Continuous injections were

performed with an automated syringe at a flow of 0.45 µL/min and 2.2 kV electrospray voltage. The cone voltage was set to 45 V, and the instrument was calibrated by infusing a standard NaI solution (0.2 mg/mL) in 50% aqueous isopropanol. All of the experiments were conducted in the positive ionization mode. N6-[Benzaldehyde hydrazone]-2′-deoxyadenosine (3a) [Scheme 1, Method 1 (21)]. Compound 1 (30 mg, 0.11 mmol) was dissolved in dimethyl sulfoxide (DMSO; 0.32 mL), and DIPEA (0.5 mL) was added slowly, followed by benzaldehyde hydrazone (2a, 40 mg, 0.33 mmol). The reaction mixture was stirred at 60 °C for 46 h and the progress of the reaction was monitored by HPLCUV. The final yield of 3a was 39% after purification by HPLC using standard conditions (note: other unidentified products formed in the reaction medium). 1H NMR (300 MHz, acetone-d6) δ 10.55 (s, 1H, NH), 8.41 (s, 1H, NdC2H), 8.33-8.35 (m, 2H, N)CH + NdC8H), 7.84-7.87 (m, 2H, ArH), 7.40-7.48 (m, 3H, ArH), 6.48 (m, 1H, H1′), 5.34 (m, 1H, OH3′), 4.64 (m, 1H, OH5′), 4.44 (m, 1H, H3′), 4.08 (m, 1H, H4′), 3.70-3.83 (m, 2H, H5′ and H5′′), 2.87-2.93 (m, 1H, H2”), 2.33-2.38 (m, 1H, H2′). 13C NMR (100 MHz, DMSOd6) δ 152.2 (C2), 151.7 (NdC(hydrazone)), 150.3 (C4), δ 144.3 (C6), 140.8 (C8), δ 134.9 (C1-benzylidene), 129.3 (C4-benzylidene), 128.7 (C2,C6-benzylidene), 126.7 (C3,C5-benzylidene), 118.9 (C5), 88.0 (C4′), 83.8 (C1′), 70.8 (C3′), 61.8 (C5′), 39.5 (C2′). ESI-MS/MS (50% acetonitrile, 1 mM NaCl, positive mode): m/z 377.0 (M + Na+), m/z 355.0 (M+ H+), m/z 239.0 (M-deoxyribose + Na+). N6-[4-Methoxybenzaldehyde hydrazone]-2′-deoxyadenosine (3b) [Scheme 1, Method 1 (21)]. Compound 1 (18 mg, 0.065 mmol) was dissolved in DMSO (0.32 mL) and DIPEA (0.33 mL) was added slowly followed by 4-methoxybenzaldehyde hydrazone (2b, 32 mg, 0.2 mmol). The reaction mixture was stirred at 60 °C for 46 h and the progress of the reaction was monitored by HPLCUV. The final yield of 3b was 18% after purification by HPLC using standard conditions (Note that several other unidentified products were formed in the reaction medium). 1H NMR (300 MHz, acetone-d6) δ 10.5 (s, 1H, NH), 8.32- 8.35 (m, 3H, N)CH + NdC2H + NdC8H), 7.7- 7.8 (m, 2H, ArH), 7.0- 7.02 (m, 2H, ArH), 6.48 (m, 1H, H1′), δ 5.4 (br, 1H, OH3′), 4.64 (m, 1H, H3′), 4.4 (br, 1H, OH5′), 4.08 (m, 1H, H4′), 3.83- 3.86 (m, 5H, H5′+H5′′ + OCH3, 2.90 (m, 1H, H2”), 2.37 (m,1H, H2′). 13C NMR (100 MHz, DMSOd6) δ 160.4 (C4-benzylidene), 152.2 (C2), 151.7 (C6), 150.2 (C4), 144.4 (NdC(hydrazone)), 140.6 (C8), 128.3 (C2,C6-benzylidene), 127.5 (C1-benzylidene), 118.8 (C5), 114.3 (C3,C5-benzylidene), 88.0 (C4′), δ 83.8 (C1′), 70.9 (C3′), 61.8 (C5′), 55.3 (OCH3benzylidene), 39.5 (C2′). ESI- MS/ MS (50% acetonitrile, 1 mM NaCl, positive mode): m/z 407 (M+ Na+), m/z 291 (M-deoxyribose + Na+), m/z 157 (M-adenine+Na+). N6-[4-Nitrobenzaldehyde Hydrazone]-2′-deoxyadenosine (3c) [Scheme 1, Method 1 (21)]. Compound 1 (30 mg, 0.11 mmol) was dissolved in DMSO (0.32 mL) and DIPEA (0.5 mL) was added slowly followed by 4-nitrobenzaldehyde hydrazone (2c, 182 mg, 1.1 mmol). The reaction mixture was stirred at 60 °C for 4 days.

50

Chem. Res. Toxicol., Vol. 23, No. 1, 2010

The progress of the reaction and subsequent purification of product was carried out by HPLC using standard conditions. The final yield of 3c after purification was 38%. 1H NMR (300 MHz, acetone-d6) δ 10.9 (s, 1H, NH), 8.1- 8.6 (m, 7H, N)CH + NdC (2) H + ArH), 6.51 (m, 1H, H1′), 5.19 (br, 1H, OH3′), 4.66 (m, 1H, H3′), 4.45 (m, 1H, H3′), 4.1(m, 1H, H4′), 3.71- 3.77 (m, 2H, H5′ + H5′′), 2.94 (m, 1H, H2”), 2.42 (m, 1H, H2′). 13C NMR (100 MHz, DMSOd6) δ 152.1 (C2), 151.5 (C6), 150.7 (C4), 147.3 (C4-benzylidene), 141.6 (CdN(hydrazone)), 141.4 (C1-benzylidene), 141.4 (C8), 127.4 (C2,C6-benzylidene), 124.1 (C3,C5- benzylidene), 119.2 (C5), 88.0 (C4′), 83.7 (C1′), 70.8 (C3′), 61.7 (C5′), 39.5 (C2′). ESI- MS/ MS (50% acetonitrile, 1 mM NaCl, positive mode): m/z 422 (M+ Na+), m/z 306 (M-deoxyribose + Na+). Aldehyde Hydrazones 2a-c (22). Benzaldehyde hydrazone (2a). Two hundred milligrams (1 equiv) of benzaldehdye in DMSO (1.9 mL) was added dropwise to 94 mg (1.88 mmol) of hydrazine monohydrate, and the solution was stirred for 20 min at room temperature. 4-Methoxybenzaldehyde hydrazone (2b). Two hundred milligrams (1 equiv) of benzaldehdye in DMSO (1.9 mL) was added dropwise to 73.1 mg (1.46 mmol; 1 equiv) of hydrazine monohydrate, and the reaction was stirred for 5 h at room temperature. 4-Nitrobenzaldehyde hydrazone (2c). The synthesis of 2c was the same as for 2a except that it required 24 h to complete the reaction. The formation of aldehyde hydrazones was monitored by HPLC using standard conditions. N6-Hydrazino-2′-deoxyadenosine (4) [Scheme 1, Method 2 (23)]. Compound 1 (200 mg, 0.74 mmol) was dissolved in methanol (4 mL), and hydrazine hydrate (1 mL) was added dropwise at room temperature. After it was stirred for 5 min, the reaction mixture was concentrated in vacuo to provide a crude residue, which was suspended in methanol and stirred at room temperature for 30 min. The solid product from the suspension was filtered, washed with methanol, and dried in vacuo to provide 4 (187 mg, 95% yield). This product was used in the subsequent step without further purification. For MS and NMR analyses, it was recrystallized from methanol. N6-[Benzaldehyde hydrazone]-2′-deoxyadenosine (3a) [Scheme 1, Method 2 (23)]. A suspension of 4 (18 mg, 0.07 mmol) and benzaldehyde (0.012 mL, 0.102 mmol) in 2 mL of methanol was stirred at room temperature for 20 min, and the reaction mixture was concentrated in vacuo to provide a crude residue. The mixture was purified by reversed phase HPLC (column: 5 µm ODS-AQ 250 mm × 10 mm; YMC) using a gradient starting at 100% A and going to 70% A and 30% B in 40 min, and then 5% A and 95% B over the next 40 min, at a flow rate of 2.5 mL/min, where solvent A is composed of triethylammonium acetate, pH 7, and solvent B is composed of 95% acetonitrile and 5% water. 2-(Benzylideneamino)-2′-deoxyadenosine (9). Compound 3a (0.10 mM) and dAdo (30 mM) were irradiated for 24 h with BlakRay B-100 lamps until the reduction of 3a to approximately 80%. The photolysis mixture was concentrated and subjected to repeated purification by HPLC-UV (yield ) 10 mg). 1H NMR, 600 MHz (D2O): δ 8.18 (s, 1H, C8H), 7.41-7.74 (m, 5H, ArH), 6.34 (m, 1H, H1′), 4.46 (m, 1H, H3′), 3.99 (m, 1H, H4′), 3.63- 3.65 (m, 2H, H5′ and H5′′), 2.68-2.70 (m, 1H, H2′′), 2.32-2.40 (m, 1H, H2′). The ESI-MS/MS spectrum of product 1 has two major peaks at m/z 393 and 277. 2-Benzamido-2′-deoxyadenosine (10). This product was prepared as described for 9. 1H NMR, 600 MHz (D2O): δ 8.96 (s, 1H, NdCH), 8.35 (s, 1H, C8), 7.66-7.45 (m, 5H, ArH), 6.38 (m, 1H, H1′), 4.60 (s, 2H, D2O), 4.50 (m, 1H, H3′), 3.98 (m, 1H, H4′), 3.63-3.65 (m, 2H, H5′ and H5′′), 2.72-2.75 (m, 1H, H2′′), 2.44-2.47 (m, 1H, H2′). Near-UV Photolysis and Quantum Yield Calculations. Photolysis was carried out using a 1000 W Hg-Xe arc lamp (spectra Physics, Oriel Instruments, Stratford, CT) fitted with an infrared filter and diffracting monochromator (spectral energy Corp., Chester, NY) set at 315 ( 2 nm. The beam of light was focused on a quartz cuvette (1 cm path length). The samples were bubbled with either oxygen or nitrogen for 10 min before photolysis followed by

Kuttappan-Nair et al.

Figure 1. UV spectra of phenylhydrazone derivatives of dAdo (0.066 mM) in 10 mM phosphate buffer (pH 7.0) at room temperature. (a) Benzaldehydephenylhydrazone (3a), (b) p-methoxy-benzaldehyde hydrazone (3b), and (c) nitrobenzaldehydehydrazone (3c).

continuous bubbling during photolysis. The three substituted dAdo hydrazones displayed strong UV absorption between 300 and 400 nm in contrast to dAdo, whose absorption cuts off at below 280 nm. Quantum yields were estimated from the loss of parent hydrazone and/or the formation of dAdo as measured by quantitative HPLC divided by the number of incident photons on the solution as determined by a photometer (Industrial Fiber Optics, Inc., United States). The average number of incident photons was 0.85 × 10 16 photons s-1. The initial concentration of hydrazone was adjusted to correspond to an optical density of 1.0 at the wavelength of photolysis (313 nm), for example, the concentration of 3a was 0.05 mM.

Results and Discussion Two methods were employed to synthesize dAdo N6phenylhydrazones (3a-c). Method 1 involved the reaction of 1 with the hydrazone (2) resulting from the addition of hydrazine and the benzaldehyde derivative, whereas method 2 involved the initial conjugation of hydrazine with 1 followed by subsequent addition to the aldehyde (Scheme 1). The presence of phenylhydrazone moieties at N6 of dAdo shifted the absorption band into the near-UV (325-370 nm; Figure 1). In comparison to nonsubstituted 3a (λmax ) 325 nm), the absorption shifted to higher wavelengths for the 4-methyoxy-substituted derivative (3b; λmax ) 330 nm), and this shift was more dramatic for the 4-nitro-substituted derivative (3c; λmax ) 370 nm). Similar absorption properties have been reported for other aryl and alkyl hydrazones (19). The photochemistry of dAdo N6phenylhydrazones (3a-c) was studied by steady state photolysis and product analysis (Table 1). The quantum yield of photodecomposition was in the range from 1.2 to 2.0 × 10-4 for 3a,b but was 100-fold lower for 3c. The relatively low quantum yields suggest that the excited state of 3a-c largely deactivates by nonproductive radiative and/or nonradiative pathways. The main pathway of photodecomposition of 3a-c is believed to involve homolytic cleavage of the N-N bond leading to dAdo N6aminyl radicals (5) and benzylidene iminyl radicals (6) as reported for various phenylhydrazones (Scheme 2) (19, 24). The photolysis of 3a (0.05 mM) in N2-bubbled aqueous solution and neutral pH led to its photodecomposition together with the formation of dAdo and benzaldehyde (8; Table 1). The yield and profile of photoproducts did not significantly change in O2-bubbled solutions. The formation of dAdo likely involves H-atom abstraction by dAdo N6-aminyl radicals, whereas the formation of benzaldehyde involves H-atom abstraction by

Generation and Fate of dAdo N6-Aminyl Radicals

Chem. Res. Toxicol., Vol. 23, No. 1, 2010 51

Table 1. Yields of Products from the Photolysis of dAdo N6-Phenylhydrazones (3) under Various Conditions pmol/minb hydrazone (0.05 mM)

O2a

GSH (50 mM)

-hydrazone

+dAdo

+aldehyde (8)

3a 3a 3a 3b 3c

+ -

+ + +

-4.09 -4.02 -2.58 -3.93 -0.01

0.45 0.15 1.88 2.23 0.01

0.30 0.15 1.27 2.71 0.01

a Solutions were buffered with phosphate buffer at pH 7 and bubbled 10 min before and continuously during photolysis with either O2 (+) or N2 (-). b The quantum yield for the decomposition of 3a in the absence of oxygen and GSH is approximately 2 × 10-4.

benzylidene iminyl radicals followed by hydrolysis of the resulting imine in aqueous solution (Scheme 2). Thus, the low yields of dAdo (11% of total) and benzaldehyde (7% of total), in the absence of additives, suggest that dAdo N6-aminyl radicals and benzylidene iminyl radicals, generated by initial photocleavage, readily react with 3a leading to its decomposition. The nature of decomposition products was not pursued because of their number and relatively low yield. Previously, the photolysis of benzaldehyde phenylhydrazone was shown to induce homolytic cleavage followed by H-atom transfer to give benzonitrile and aniline (24). However, we can rule out H-atom transfer in the photochemistry of dAdo N6-phenylhydrazines in view of the low yield of dAdo in the absence of additives and lack of formation of benzonitrile. Photolysis in the Presence of GSH. The initial yields of dAdo N6-aminyl radicals and benylidene iminyl radicals were estimated by the photolysis of 3a in the presence of a good H-donor, that is, GSH. The loss of 3a and formation of dAdo were monitored as a function of time of photolysis (Figure 2 and Table 1). The reactions of dAdo N6-aminyl radicals and benzylidene iminyl radicals in the presence of GSH are shown in Scheme 2. The photodecomposition of 3a decreases in the presence of GSH because it scavenges dAdo N6-aminyl radicals (5) and benzylidene iminyl radicals (6), which react with 3a in the absence of other substrates. In contrast, the formation of both dAdo and benzaldehyde increases because GSH readily donates H-atoms to precursor radicals of these products. The yield of dAdo reaches 73% of the total decomposition of 3a at a concentration of GSH of 50 mM (1.88/2.58; Table 1). Thus, the generation of dAdo N6-aminyl radicals and benzylidene iminyl radicals is the major pathway of UVA-induced decomposition of dAdo N6-phenylhydrazones. Effect of Substitutents on Phenylhydrazone Photochemistry. The photochemistry of three derivatives of dAdo N6phenylhydrazones (3a-c) was examined (Table 1). In comparison to nonsubstituted phenylhydrazone (3a), the

Figure 2. Decomposition of 3a and formation of dAdo as a function of photolysis time. Decomposition of 3a (solid squares). Formation of dAdo (solid circles). Compound 3a (0.05 mM, 1 OD) was irradiated in the presence of GSH (50 mM) at pH 7 with N2 bubbling. Aliquots of 50 µL were injected for analyses by HPLC-UV.

photodecomposition of the methoxy-substituted phenylhydrazone (3b) was 2-fold more efficient, whereas the nitrophenylhydrazone derivative (3c) did not significantly decompose under identical conditions. Similarly, the most efficient arylhydrazone derivatives that induce strand breaks in DNA were previously shown to be those with a methoxy group in the para-position, whereas those with a nitro group at the same position were much less efficient (19). The methoxy group probably enhances homolytic cleavage of the hydrazone because it pushes electrons into the phenyl ring, thereby stabilizing the formation of electron-deficient iminyl radicals. Photolysis in the Presence of an Excess of dAdo. The photolysis of 3a was examined in the presence of an excess of dAdo to mimic the reactions of nitrogen-centered radicals in DNA. The mixture of photoproducts was separated by HPLC, and the major products were identified by chemical analysis (Figure 3). On the basis of these analyses, we propose four pathways of decomposition of dAdo N6-aminyl radicals (5): recombination with benzylidene iminyl radicals (6) to give 9 and 10, H-atom abstraction to dAdo, deamination to 2′deoxyinosine (11), and addition with dAdo to dAdo-dAdo dimers (Scheme 3). The contribution of each reaction was estimated from the formation of products divided by the decomposition of 3a as a function of irradiation time as determined by HPLC-UV. The formation of the above products is blocked in the presence of 50 mM GSH, indicating that they arise from dAdo N6-aminyl radicals. Because 10 and dAdo-dAdo elute together under standard HPLC-UV conditions, the indi-

Scheme 2. Photolysis of dAdo N6-Phenylhydrazones (3) in the Presence of GSH

52

Chem. Res. Toxicol., Vol. 23, No. 1, 2010

Figure 3. HPLC-UV analysis of the mixture from the photolysis of 3a (0.05 mM) with dAdo (30 mM) in phosphate buffer (10 mM, pH 7). Photolysis time: 0 (black line), 30 (blue line), and 60 min (red line).

Scheme 3. Reactions of dAdo N6-Aminyl Radicals in the Presence of dAdo

vidual yield of these products was estimated from the corresponding MS ion signals of the purified HPLC peak. The ion abundance of dAdo-dAdo dimer is 5-fold less than that of 10, suggesting that it is less than 1% of the total decomposition of 3a. The rate of formation of dAdo was determined in parallel experiments using an excess of the ribonucleoside (adenosine) in place of dAdo. Together, products 9-11, dAdo, and dAdo-dAdo account for about 90% of the total decomposition of 3a. Reactions of Benzylidene Iminyl Radicals. Two products were identified by MS and NMR analyses, and they have a similar structure: 2-(benzylideneamino)-2′-deoxyadenosine 9 and 2-benzamido-2′-deoxyadenosine (10; Scheme 4). The ESI-MS spectrum of 9 displayed the molecular ion and corresponding Na+ adduct at m/z 355 and m/z 377, respectively. Fragmentation of m/z 377 gave m/z 261 (loss of 2-deoxyribose), and subsequent fragmentation of m/z 239 gave m/z 136 (loss of adenine). Indeed, the MS features of 9 were nearly identical to those of 3a. Analysis of 9 equilibrated in D2O, however, revealed that this compound contained four exchangeable protons, while 3a showed only three exchangeable protons. The position of attachment between dAdo and benzylidene amine was confirmed by NMR analyses. The 1H NMR spectrum depicted only one

Kuttappan-Nair et al.

signal at low field in the region of C2 and C8 protons for an intact adenine ring. The low field signal was assigned to the C8 proton in view of the presence of cross-peaks between C8 and the anomeric proton in the nuclear Overhauser effect spectroscopy (NOESY) spectrum. Thus, the structure of 9 consists of benzylidene amine attached to the C2 position of dAdo. The mechanism of formation of 9 in the presence of an excess of dAdo may involve the recombination of dAdo N6-aminyl and benzylidene iminyl radicals (Scheme 4) and/or the addition of benzylidene radicals to nonmodified dAdo. Two lines of evidence point to the recombination of radicals as the main pathway. First, there was no significant change in the yield of 9 when 3a was irradiated in the presence of either 10 or 30 mM dAdo. Second, the photolysis of 3a in the presence of an excess of the ribonucleoside derivative, adenosine in place of dAdo, led predominantly to the formation of 9 and not to the corresponding ribose derivative. Although the ribose derivative of 9 was identified in the photolysis mixture [retention time ∼25 min, m/z 371 (MH+) and m/z 239 (MH+ minus ribose)], its yield was 15-fold less than the corresponding 2-deoxyribose derivative. These results indicate that the addition of benzylidene iminyl radicals to dAdo is a minor pathway in the formation of 9. As a probable mechanism of formation, we propose that dAdo N6-aminyl and benzylidene iminyl radicals recombine at the C2 position of dAdo followed by rearrangement of the resulting biradical (Scheme 4). The recombination reaction appears to involve diffusible radicals in the bulk solution because the formation of 9 is blocked by the addition of GSH. However, we cannot rule out the possibility that the reaction takes place within a radical cage in which GSH cannot scavenge the initial radicals but interferes with the chemistry of subsequent radicals in the formation of 9, such as 12 and 13. The structure of 10 was deduced by MS and NMR analyses. The mass of 10 [m/z 371 (MH+)] was 16 mass units higher than that of 9 [m/z 355 (MH+)]. The MS spectra of 9 and 10 showed similar features, including the loss of 2-deoxyribose from the molecular ion and the presence of five exchangeable protons. Of the exchangeable protons, three were present on the exocyclic amino group and two on the sugar moiety. The 1 HNMR spectra of 9 and 10 were also very similar except for one major difference: The imine proton of 9 was not present in the spectrum of 10. These results are consistent with a structure in which the imine group of 9 undergoes oxidation to an amide group as in product 10 (Scheme 4). The proposed structure is consistent with other NMR features. The phenyl group displayed five protons, excluding the possibility that the aromatic ring was hydroxylated. Similar to the analysis of 9, the 1H NMR, correlation spectroscopy, and NOESY spectra of 10 indicated the presence of a proton at C8 but not at C2. The formation of 10 may be explained by photooxidation of product 9, which,

Scheme 4. Proposed Mechanism of Formation of 9 and Its Oxidation Product 10

Generation and Fate of dAdo N6-Aminyl Radicals

Chem. Res. Toxicol., Vol. 23, No. 1, 2010 53

Figure 4. ESI-MS/MS spectrum of a dAdo-dAdo dimer.

similar to dAdo hydrazones, absorbs UV light at 313 nm. Alternatively, 9 may undergo autoxidation to product 10 because it was observed as a minor product in the NMR spectrum of purified 9. Reactions of dAdo N6-Aminyl Radicals. The reactivity of dAdo N6-aminyl radicals was examined by the photolysis of 3a (0.05 mM) in the presence of a molar excess of dAdo (30 mM). From the photolysis mixture, two photoproducts were identified, which likely arise from the reaction of dAdo N6aminyl radicals. The peak at 14 min was identified as 11 on the basis of HPLC-UV and MS analyses. The formation of 11 may be explained by the addition of water to dAdo N6-aminyl radicals followed by deamination. Similarly, dAdo N6-aminyl radicals have been implicated in the formation of 11 by the radiolysis of deaerated aqueous solutions of dAdo and the UVA photolysis of dry films of dAdo and 7-methylpyrido[3,4-c]psoralen (25, 26). The formation of 11 is very similar to the formation of 2′-deoxyuridine from the hydrolysis of cytosine N4-aminyl radicals generated in the UVA photosensitized oxidation of 2′deoxycytidine by 2-methyl-1,4-naphthoquinone (27). The conversion of cytosine N4-aminyl radicals to 2′-deoxyuridine was the subject of a theoretical study supporting the addition of H2O to the electrophilic C4 followed by loss of the exocyclic amino group (28). For cytosine and adenine nitrogen-centered radicals, the deamination reaction may take place via the iminyl tautomer, which, similar to ground state imines, should be more susceptible to hydrolysis. A novel product consisting of two dAdo moieties was tentatively identified by MS. This product eluted at 22 min on HPLC-UV with the same retention as 10 (Figure 3). The MS spectrum of the product displayed a molecular ion peak at m/z 523, which corresponded to the molecular ion of two dAdo moieties (m/z 502) plus Na+ (m/z 23) minus 2 mass units. The MS/MS spectrum of m/z 523 showed ion fragments at m/z 407 and 291, indicating the consecutive loss of two 2-deoxyribose moieties (minus m/z 116) (Figure 4). The yield of dAdo-dAdo dimer increased upon an increase in the concentration of nonmodified dAdo in the photolysis solution from 10 to 30 mM. Furthermore, the formation of dimer was not detected when the photolysis was carried out in the presence of an excess of nonmodified adenosine instead of dAdo. In contrast, an analogous product was observed in the photolysis mixture with the mass of adenosine and dAdo and the loss of ribose (m/z minus 132). These results suggest that dAdo-dAdo dimer is formed by the addition of dAdo N6-aminyl radicals to nonmodified

dAdo. However, we were not able to confirm the structure of the cross-link by NMR analysis due to its relatively low yield. Finally, dAdo is a major product of dAdo N6-aminyl radicals (32%) (Scheme 3). The formation of dAdo likely involves H-atom abstraction of dAdo N6-aminyl radicals from GSH or other possible sources such as dAdo in the absence of GSH and presence of an excess of dAdo. These results agree with the tendency of primary aminyl radicals to undergo H-atom abstraction rather than addition to a double bond when compared to alkylamino or iminyl radicals (29-31). Nevertheless, the situation may be complicated by the contribution of tautomeric dAdo N1-iminyl radicals (4, 32). According to natural population analysis, the distribution of spin densities of neutral dAdo N6-aminyl radical is greatest at the N6 position (-0.59) as compared to other positions: N1 (-0.16), N3 (-0.22), C5 (-0.22), and C8 (-0.14) (33). The photosensitized oxidation of several dinucleoside monophosphates by 2-methyl-1,4naphthoquinone, including d(ApA), d(CpA), and d(ApC), led to the formation of N6-formyladenine and N6-acetyladenine modifications (34, 35). When 2-methyl-1,4-naphthoquinone tethered oligonucleotides were exposed to UVA light, the formation of interstrand cross-links occurred between the adenine and the quinone moieties, which may be explained from the condensation of dAdo N6-aminyl radical and semiquinone radicals (36).

Conclusions 6

dAdo N -aminyl radicals are the main species generated by the reaction of hydroxyl radicals with adenine and the deprotonation of adenine radical cations. Furthermore, the deprotonation of adenine radical cations at N6 is favored in doublestranded DNA due to the lack of H-bonding and contact with H2O in the major groove (33). The present study suggests that dAdo N6-aminyl radicals will undergo three reactions: H-atom abstraction, hydrolytic deamination, and addition to nonmodified adenine. H-atom abstraction in DNA may lead to strand breaks as has been reported for various alkyl and aryl aminyl radicals. Deamination of adenine produces inosine, which should not significantly affect DNA structure or its coding ability. In contrast, the addition of dAdo N6-aminyl radicals to another adenine moiety, or another DNA base, may cause intra- or interstrand cross-links in DNA, which may have severe consequences in cell death and mutagenesis. Future studies are in progress to assess the fate of dAdo N6-aminyl radicals in duplex DNA.

54

Chem. Res. Toxicol., Vol. 23, No. 1, 2010

Acknowledgment. We thank Natural Science and Engineering Research Council of Canada (NSERC) for funding. We also thank Prof. Klaus Klarskov for mass spectrometry analyses and Profs. Pierre Lavinge and Luc Tremblay for NMR analyses. V.K.-N. thanks Mahatma Gandhi University, Kottayam, for sanctioning leave for this project.

References (1) von Sonntag, C. (2006) Free-Radical Induced DNA Damage and Its Repair. A Chemical PerspectiVe, Springer, Heidelberg. (2) Cadet, J., Berger, M., Douki, T., and Ravanat, J. L. (1997) Oxidative damage to DNA: Formation, measurement, and biological significance. ReV. Physiol. Biochem. Pharmacol. 131, 1–87. (3) Cadet, J., Delatour, T., Douki, T., Gasparutto, D., Pouget, J. P., Ravanat, J. L., and Sauvaigo, S. (1999) Hydroxyl radicals and DNA base damage. Mutat. Res., Fundam. Mol. Mech. Mutagen. 424, 9–21. (4) Vieira, A., and Steenken, S. (1990) Pattern of OH radical reaction with adenine and its nucleosides and nucleotidessCharacterization of 2 types of isomeric oh adduct and their unimolecular transformation reactions. J. Am. Chem. Soc. 112, 6986–6994. (5) Cadet, J., and Berger, M. (1985) Radiation-induced decomposition of purine bases within DNA and related model compounds. Int. J. Radiat. Biol. 47, 127–143. (6) Cadet, J., Douki, T., Frelon, S., Sauvaigo, S., Pouget, J. P., and Ravanat, J. L. (2002) Assessment of oxidative base damage to isolated and cellular DNA by HPLC-MS/MS measurement. Free Radical Biol. Med. 33, 441–449. (7) Frelon, S., Douki, T., and Cadet, J. (2002) Radical oxidation of the adenine moiety of nucleoside and DNA: 2-Hydroxy-2 ′-deoxyadenosine is a minor decomposition product. Free Radical Res. 36, 499– 508. (8) Melvin, T., Bothe, E., and Schultefrohlinde, D. (1996) The reaction of triplet 2-methyl-1,4-naphthoquinone (menadione) with DNA and polynucleotides. Photochem. Photobiol. 64, 769–776. (9) Liu, Z., Gao, Y., and Wang, Y. (2003) Identification and characterization of a novel cross-link lesion in d(CpC) upon 365-nm irradiation in the presence of 2-methyl-1,4-naphthoquinone. Nucleic Acids Res. 31, 5413–5424. (10) Liu, Z. J., Gao, Y., Zeng, Y., Fang, F., Chi, D., and Wang, Y. S. (2004) Isolation and characterization of a novel cross-link lesion in d(CpC) induced by one-electron photooxidation. Photochem. Photobiol. 80, 209–215. (11) Ding, H., Majumdar, A., Tolman, J. R., and Greenberg, M. M. (2008) Multinuclear NMR and kinetic analysis of DNA interstrand crosslink formation. J. Am. Chem. Soc. 130, 17981–17987. (12) Hong, I. S., and Greenberg, M. M. (2005) Efficient DNA interstrand cross-link formation from a nucleotide radical. J. Am. Chem. Soc. 127, 3692–3693. (13) Bernofsky, C., Bandara, B. M. R., Hinojosa, O., and Strauss, S. L. (1990) Hypochlorite-modified adenine-nucleotidessStructure, spintrapping, and formation by activated guinea-pig polymorphonuclear leukocytes. Free Radical Res. Commun. 303–315. (14) Hawkins, C. L., and Davies, M. J. (2001) Hypochlorite-induced damage to nucleosides: Formation of chloramines and nitrogen-centered radicals. Chem. Res. Toxicol. 14, 1071–1081. (15) Hawkins, C. L., and Davies, M. J. (2002) Hypochlorite-induced damage to DNA, RNA, and polynucleotides: Formation of chloramines and nitrogen-centered radicals. Chem. Res. Toxicol. 15, 83–92. (16) Hawkins, C. L., Pattison, D. I., and Davies, M. J. (2002) Reaction of protein chloramines with DNA and nucleosides: Evidence for the formation of radicals, protein-DNA cross-links and DNA fragmentation. Biochem. J. 365, 605–615. (17) Murata, M., Ohnishi, S., and Kawanishi, S. (2001) Acrylonitrile enhances H2O2-mediated DNA damage via nitrogen-centered radical formation. Chem. Res. Toxicol. 14, 1421–1427.

Kuttappan-Nair et al. (18) Midorikawa, K., Murata, M., Oikawa, S., Tada-Oikawa, S., and Kawanishi, S. (2000) DNA damage by dimethylformamide: Role of hydrogen peroxide generated during degradation. Chem. Res. Toxicol. 13, 309–315. (19) Hwu, J. R., Lin, C. C., Chuang, S. H., King, K. Y., Sue, T. R., and Tsay, S. C. (2004) Aminyl and iminyl radicals from arylhydrazones in the photo-induced DNA cleavage. Bioorg. Med. Chem. 12, 2509– 2515. (20) Sortino, S., Giuffrida, S., and Scaiano, J. C. (1999) Phototoxicity of naphazoline. Evidence that hydrated electrons, nitrogen-centered radicals, and OH radicals trigger DNA damage: A combined photocleavage and laser flash photolysis study. Chem. Res. Toxicol. 12, 971– 978. (21) Wang, H., Marnett, L. J., Harris, T. M., and Rizzo, C. J. (2004) A novel synthesis of malondialdehyde adducts of deoxyguanosine, deoxyadenosine, and deoxycytidine. Chem. Res. Toxicol. 17, 144– 149. (22) Nenajdenko, V. G., Reznichenko, A. L., Lenkova, O. N., Shastin, A. V., and Balenkova, E. S. (2005) A new synthetic approach to alphachlorocinnamaldehydes. Synthesis-Stuttgart 605–609. (23) Prakash, J., Casaba, S., and Andrew, L. S. (2005) International Application Published under the Patent Cooporation Treaty, 117910 A117912. (24) Binkley, R. W. (1970) Photochemistry of unsaturated nitrogencontaining compounds. VII. Photolysis of phenylhydrazones. J. Org. Chem. 35, 2796–2801. (25) Raoul, S., Bardet, M., and Cadet, J. (1995) Gamma-irradiation of 2′deoxyadenosine in oxygen-free aqueous-solutionssIdentification and conformational features of formamidopyrimidine nucleoside derivatives. Chem. Res. Toxicol. 8, 924–933. (26) Voituriez, L., and Cadet, J. (1999) Isolation and characterization of two furan-side photoadducts of 7-methylpyrido[3,4-c] psoralen to the sugar moiety of 2′-deoxyadenosine. Photochem. Photobiol. 70, 152– 158. (27) Wagner, J. R., Decarroz, C., Berger, M., and Cadet, J. (1999) Hydroxyl radical-induced decomposition of 2′-deoxycytidine in aerated aqueous solutions. J. Am. Chem. Soc. 121, 4101–4110. (28) Labet, V., Grant, A., Cadet, J., and Erriksson, L. A. (2008) Deamination of the radical cation of the base moiety of 2′-deoxycytidine: A theoretical study. ChemPhysChem 9, 1195–1203. (29) Lalevee, J., Gigmes, D., Bertin, D., Graff, B., Allonas, X., and Fouassier, J. P. (2007) Comparative reactivity of aminyl and aminoalkyl radicals. Chem. Phys. Lett. 438, 346–350. (30) Le Tadic Biadatti, M.-H., Callier-Dublanchet, A.-C., Horner, J. H., Quiclet-Sire, B., Zard, S. Z., and Newcomb, M. (1997) Absolute rate constants for iminyl radical reactions. J. Org. Chem. 62, 559–563. (31) Zard, S. Z. (2008) Recent progress in the generation and use of nitrogen-centred radicals. Chem. Soc. ReV. 37, 1603–1618. (32) Steenken, S. (1989) Purine-bases, nucleosides, and nucleotidessAqueoussolution redox chemistry and transformation reactions of their radical cations and e- and oh adducts. Chem. ReV. 89, 503–520. (33) Reynisson, J., and Steenken, S. (2002) DFT calculations on the electrophilic reaction with water of the guanine and adenine radical cations. A model for the situation in DNA. Phys. Chem. Chem. Phys. 4, 527–532. (34) Wang, Y. S., and Liu, Z. J. (2002) Mechanisms for the formation of major oxidation products of adenine upon 365-nm irradiation with 2-methyl-1,4-naphthoquinone as a sensitizer. J. Org. Chem. 67, 8507– 8512. (35) Wang, Y. S., Liu, Z. J., and Dixon, C. (2002) Major adenine products from 2-methyl-1,4-naphthoquinone-sensitized photoirradiation at 365 nm. Biochem. Biophys. Res. Commun. 291, 1252–1257. (36) Bergeron, F., Klarskov, K., Hunting, D. J., and Wagner, J. R. (2007) Near-UV photolysis of 2-methyl-1,4-naphthoquinone-DNA duplexes: Characterization of reversible and stable interstrand cross-links between quinone and adenine moieties. Chem. Res. Toxicol. 20, 745–756.

TX900268R