Structural Influence of C8-Phenoxy-Guanine in the NarI Recognition

Article pubs.acs.org/crt

Structural Influence of C8-Phenoxy-Guanine in the NarI Recognition DNA Sequence Michael S. Kuska,† Aaron A. Witham,† Michael Sproviero,† Richard A. Manderville,*,† Mohadeseh Majdi Yazdi,‡ Purshotam Sharma,‡ and Stacey D. Wetmore*,‡ †

Departments of Chemistry and Toxicology, University of Guelph, Guelph, Ontario, Canada N1G 2W1 Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Alberta, Canada T1K 3M4

‡

S Supporting Information *

ABSTRACT: Phenoxyl radicals can covalently attach to the C8 site of 2′deoxyguanosine (dG) to generate oxygen-linked biaryl ether C8-dG adducts. To assess the structural impact of an O-linked C8-dG adduct in duplex DNA, C8phenoxy-G (PhOG) and C8-4-fluorophenoxy-G (4FPhOG) were incorporated into the G3 position of the 12-mer NarI recognition sequence (5′-CTCGGCXCCATC, where X = G, PhOG, or 4FPhOG) using solid-phase DNA synthesis with O-linked C8-dG phosphoramidites. The modified strands were hybridized to six different complementary strands that include regular base pairing to C [NarI′(C)], mismatches with G, A, T [NarI′(N)], and an abasic site [NarI′(THF)], and a 10-mer sequence to model a −2 deletion duplex [NarI′(−2)]. All duplex structures were characterized using UV−vis thermal melting temperature analysis, and in each instance, the O-linked C8-phenoxy-G adducts were found to destabilize the duplex relative to the unmodified controls. The most stable duplex structures match the O-linked C8-dG adduct against C and a G mismatch, which are comparable in terms of stability. These duplexes were further characterized using circular dichroism, dynamic 19F nuclear magnetic resonance experiments, and molecular dynamics simulations. On the basis of these findings, PhOdG adopts the B conformation opposite C, with the phenoxy moiety residing in the solvent-exposed major groove. However, opposite the G mismatch, PhOdG adopts a “W-type” wedge conformation with the phenoxy group residing in the minor groove. These studies predict that the O-linked C8-dG lesion PhOG will have a weak mutagenic effect, as determined for the corresponding single-ringed nitrogen-linked C8-dG adduct derived from aniline.

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INTRODUCTION Covalent DNA adducts mediate the toxic effects of many carcinogens by generating mutagenesis when the modified DNA is replicated by a DNA polymerase.1−3 Covalent adducts are primarily produced when DNA-reactive species, which include radicals, oxidants, and electrophiles, target nucleobase atoms in DNA. The purine 2′-deoxyguanosine (dG) is particularly susceptible to covalent modifications because it possesses the lowest oxidation potential of the four DNA nucleobases.4 In double-stranded DNA, the non-Watson−Crick edges of the bases are solvent-exposed, making these positions more likely targets for covalent modification.1 Because the major groove is wider and more accessible than the minor groove, O6, C8, and especially N7 in dG are prominent reaction sites for DNA-reactive species.1,3 The C8 position of dG is a preferred target site for electrophilic radicals5,6 and nitrenium ions produced from the metabolism of arylamine carcinogens.7−9 Attachment of a bulky C8 substituent (R in Figure 1) can shift the conformational equilibrium from the preferred anti conformer for dG to the syn conformer for the C8-dG adduct (Figure 1b). The syn conformation reduces the level of steric interaction between the bulky R group and the deoxyribose sugar moiety.10,11 For nitrogen-linked C8-dG adducts produced by arylamine carcinogens, the syn−anti equilibrium of the adduct in duplex © 2013 American Chemical Society

DNA results in three major conformations (depicted in Figure 1a).12−19 The least perturbing conformation is the “B type” (B) in which the C8-dG residue retains an anti conformation and Watson−Crick H-bonding with the opposing C, while the C8 substituent is projected into the major groove and exposed to the solvent. In the base-displaced “stacked” (S) conformer, the C8-dG lesion is present in the syn conformation, the Watson− Crick H-bonding interaction with C is ruptured, and the C8 substituent π-stacks with neighboring base pairs in the helix. An additional trademark of the S conformer is that the opposing C is flipped out of its natural intrahelical position into a solventexposed extrahelical environment. The third so-called “wedge” (W) conformation typically arises when the C8-dG lesion is mispaired opposite a purine base and positions the C8 substituent in the minor groove.20−22 Studies of a variety of N-linked C8-dG lesions have shown that factors affecting conformational preference include the stability of the Watson− Crick H-bonding interaction versus possible Hoogsteen Hbonding and the π-stacking ability of the adducted moiety, which is a factor of its size and planarity.18−22 Because the three DNA conformations vary in helix twisting and π-stacking, the conformations are repaired differently23,24 and have differing Received: July 8, 2013 Published: August 28, 2013 1397

dx.doi.org/10.1021/tx400252g | Chem. Res. Toxicol. 2013, 26, 1397−1408

Chemical Research in Toxicology

Article

Figure 1. (a) Depictions of the three major conformations formed by C8-dG adducts in duplex DNA. (b) anti−syn equilibrium for C8-dG adducts PhO dG (R = phenoxy) and 4FPhOdG (R = 4-fluorophenoxy). Dihedral angle χ [∠(O4′−C1′−N9−C4)] defines the glycosidic bond orientation to be anti (χ = 180 ± 90°) or syn (χ = 0 ± 90°), and θ defines the degree of twist between the nucleobase and the C8 substituent R. (c) DNA sequences used in this study.

Scheme 1. Phosphoramidite Synthesis

features in duplex DNA that are similar to those established for the corresponding N-linked C8-dG adducts. Our recent efforts have focused on the synthesis of oligonucleotides containing C-linked C8-dG adducts,36−39 which are highly emissive and may serve as fluorescent probes for distinguishing syn from anti structures within duplex DNA.38,39 In this paper, we present the structural impact of the O-linked C8-phenoxy-dG (PhOdG) adduct within the 12mer NarI recognition sequence [5′-CTCGGCXCCATC, where X = PhOG or 4FPhOG (see Figure 1)]. Unlike the corresponding biaryl C-linked C8-dG adducts,36−39 O-linked adducts lack fluorescence, and hence, we have incorporated the 4fluorophenoxy-dG (4FPhOdG) analogue into NarI to employ dynamic 19F NMR22,23,40 and thereby determine the adduct conformation in the duplex environment. The biological

propensities to lead to mutagenesis when the adducted DNA is replicated.12−24 Our interest in C8-dG adducts stems from research on DNA adduction by phenolic toxins.25,26 The toxicity of phenols is thought to arise from oxidative metabolism, which can produce phenoxyl radical intermediates in peroxidase-rich tissues.27,28 Phenoxyl radicals are potentially ambident (C vs O) electrophiles,29 and both carbon- and oxygen-linked C8-dG adducts have been observed following the bioactivation of phenolic substrates.30−35 For the O-linked variety, an increased level of chlorination of the phenol ring favors O-linked adduct formation by increasing the electrophilicity of the phenolic radical and decreasing the rate of bimolecular phenolic radical coupling.33 Both C- and O-linked adducts are expected to play a role in phenol-mediated toxicity and may exhibit structural 1398



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C NMR (100 MHz, DMSO-d6) δ 158.5, 157.3, 157.2, 153.8, 150.4, 148.9, 130.3, 125.8, 120.5, 114.5, 87.9, 82.4, 71.3, 62.4, 41.2, 37.5, 35.1; HRMS (ESI) calcd for C19H23N6O5 415.1730, found 415.1728 (MH+). N2-(Dimethylformamidyl)-C8-(4-fluorophenoxy)-2′-deoxyguanosine (3). C8-(4-Fluorophenoxy)-dG (0.60 g, 1.6 mmol) was placed in 10 mL of dry DMF under argon. N,N-(Dimethylformamidyl) diethyl acetal (1.1 mL, 6.4 mmol) was added, and the mixture was allowed to stir for 5 h. The reaction mixture was then evaporated to dryness and washed with MeOH to give 0.64 g (100%) of 3 as a brown solid: 1H NMR (300 MHz, DMSO-d6) δ 11.34 (s, 1H), 8.53 (s, 1H), 7.40 (m, 2H), 7.29 (m, 2H), 6.30 (t, J = 7.3 Hz, 1H), 5.29 (d, J = 4.2 Hz, 1H), 4.81 (t, J = 5.9 Hz, 1H), 4.38 (m, 1H), 3.78 (m, 1H), 3.51 (m, 2H), 3.15 (s, 3H), 3.02 (s, 3H), 2.20 (m, 1H); 13C NMR (151 MHz, DMSO-d6) δ 157.9, 156.8, 156.7, 153.4, 149.8, 148.4, 129.7, 125.3, 119.9, 114.1, 87.4, 81.9, 70.8, 62.0, 40.7, 37.0, 34.6; HRMS (ESI) calcd for C19H22FN6O5 433.1636, found 433.1628. 5′-O-(4,4′-Dimethoxytrityl)-N2-(dimethylformamidyl)-C8-phenoxy-2′-deoxyguanosine (4). 4,4′-Dimethoxytrityl chloride (DMTCl, 1.2 g, 3.54 mmol) was dissolved in 10 mL of dry pyridine. N2(Dimethylformamidyl)-C8-phenoxy-2′-deoxyguanosine 2 (1 g, 2.41 mmol) was dissolved in 10 mL of dry DMF is a separate flask. The DMT-Cl/pyridine solution was added to the 2/DMF solution dropwise over 45 min under argon at 0 °C. The reaction mixture was allowed to stir at room temperature for 4 h under argon. Upon completion, the reaction mixture was diluted with ethyl acetate (50 mL) and washed with water (2 × 50 mL). The ethyl acetate layer was separated, dried over MgSO4, and evaporated to dryness. The residue was dissolved in CH2Cl2 (2 mL); hexanes (10 mL) were added, and the mixture was stirred overnight. The resulting suspension was filtered, purified on silica gel, and eluted with a MeOH/CHCl3/TEA mixture (5:90:5) to afford a beige solid (1.3 g, 76%): 1H NMR (400 MHz, CD2Cl2) δ 9.09 (s, 1H), 8.51 (s, 1H), 7.37−7.10 (m, 14H), 6.73 (m, 4H), 6.49 (m, 1H), 4.67 (m, 1H), 4.06 (m, 1H), 3.76 (s, 6H), 3.41 (m, 1H), 3.25 (m, 1H), 3.10 (s, 3H), 3.03 (s, 3H), 2.99 (m, 1H), 2.37 (m, 1H); 13C NMR (151 MHz, acetone-d6) δ 159.6, 158.8, 157.7, 157.4, 155.0, 151.1, 149.7, 146.3, 137.0, 136.9, 131.0, 130.9, 130.4, 130.1, 129.0, 128.8, 128.5, 128.4, 127.5, 125.8, 120.7, 115.8, 113.8, 113.7, 87.1, 86.7, 83.1, 72.9, 65.7, 55.5, 41.3, 38.5, 35.1; HRMS (ESI) calcd for C40H41N6O7 717.3027, found 717.3033 (MH+). 5′-O-(4,4′-Dimethoxytrityl)-N2-(dimethylformamidyl)-C8-(4-fluorophenoxy)-2′-deoxyguanosine (5). 4,4′-Dimethoxytrityl chloride (DMT-Cl, 0.60 g, 1.8 mmol) was dissolved in 3 mL of dry pyridine. N2-(Dimethylformamidyl)-C8-(4-fluorophenoxy)-2′-deoxyguanosine (3, 585 mg, 1.4 mmol) was coevaporated from dry pyridine (3 × 5 mL) in a separate flask and reverse filled with argon. To this flask was added 5 mL of dry pyridine, followed by dropwise addition of 3 mL of a DMT-Cl/pyridine solution. The reaction mixture was allowed to stir at room temperature for 3 h under argon and monitored by TLC. Upon completion, the reaction mixture was diluted with ethyl acetate (10 mL) and washed with water (2 × 10 mL). The mixture was then evaporated to dryness, with the resulting solid dissolved in CH2Cl2 (3 mL). Hexanes (10 mL) were added, and the reaction mixture was stirred overnight. The resulting suspension was filtered, purified on silica gel, and eluted with a MeOH/CH2Cl2/TEA mixture (5:90:5) to afford 5 as a white solid (0.76 g, 77%): 1H NMR (300 MHz, CDCl3) δ 8.73 (bs, 1H), 8.50 (s, 1H), 7.33 (d, J = 8.3 Hz, 2H), 7.22−7.16 (m, 7H), 7.03, (m, 2H), 6.93 (t, J = 8.1 Hz, 2H), 6.70 (d, J = 8.3 Hz, 4H), 6.44 (t, J = 8.1 Hz, 1H), 4.63 (m, 1H), 4.01 (m, 1H), 3.73 (s, 6H), 3.39 (m, 1H), 3.20 (t, J = 7.2 Hz, 1H), 3.07 (s, 3H), 3.03 (s, 3H), 2.98 (m, 1H); 13C NMR (151 MHz, CDCl3) δ 160.6, 159.0, 158.5, 157.8, 156.9, 156.0, 150.8, 149.1, 148.7, 144.5, 135.6, 135.5, 129.9, 129.1, 128.0, 127.8, 126.9, 121.7, 116.2, 116.0, 114.5, 113.1, 86.4, 85.0, 81.5, 73.3, 64.3, 41.5, 37.3, 35.1; HRMS (ESI) calcd for C40H40FN6O7 735.2943, found 735.2955 (MH+). 3′-O-[(2-Cyanoethoxy)(diisopropylamino)phosphino]-5′-O-(4,4′dimethoxytrityl)-N2-(dimethylformamidyl)-C8-phenoxy-2′-deoxyguanosine (6). 5′-DMT-N2-(dimethylformamidyl)-C8-phenoxy-2′deoxyguanosine (4, 0.50 g, 0.698 mmol) was coevaporated from dry toluene (3 × 6 mL) followed by dry THF (3 × 6 mL). Dry CH2Cl2 13

implications of the PhOdG lesion are compared to the corresponding N-linked C8-dG adducts that can stabilize a two-base bulge and promote frameshift mutations within the X site of the NarI sequence.17,41

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EXPERIMENTAL SECTION

Materials and Methods. Anhydrous 1,4-dioxane was purchased from Sigma-Aldrich (Oakville, ON) and distilled over Na. Xylenes, pyridine, and methylene chloride were purchased from Fisher Scientific (Ottawa, ON), distilled over CaH2, and stored under nitrogen. Dimethylformamide (DMF) and tetrahydrofuran (THF) were obtained from an LC Technology SP-105 solvent purification system at the University of Guelph. Other commercial compounds were used as received. All unmodified oligonucleotides were purchased from Sigma Genosys (Oakville, ON) and were prepared on a 1 μmol scale. Standard phosphoramidites, including the abasic phosphoramidite, were purchased from ChemGenes (Wilmington, MA). Purified water used for buffers and spectroscopic solutions was obtained from a Milli-Q filtration system (18.2 MΩ). The synthesis of 8-bromo-2′-deoxyguanosine (8-Br-dG) was performed, as outlined previously,10,11 by treating dG with N-bromosuccinimide in a water/ acetonitrile mixture. 8-Bromo-3′,5′-O-bis(tert-butyldimethylsilyl)-2′deoxyguanosine was prepared according to literature procedures by treating 8-Br-dG with excess tert-butyl(chloro)dimethylsilane and imidazole in DMF.42 8-Bromo-3′,5′-O-bis(tert-butyldimethylsilyl)-O6(trimethylsilylethyl)-2′-deoxyguanosine (1) (Scheme 1) was prepared by treating 8-bromo-3′,5′-O-bis(tert-butyldimethylsilyl)-2′-deoxyguanosine with triphenylphosphine, 2-(trimethylsilyl)ethanol, and diisopropyl azodicarboxylate (DIAD) in anhydrous dioxane, as outlined previously.43 C8-Phenoxy-2′-deoxyguanosine (PhOdG) was prepared using our general synthetic method for O-linked C8-dG adducts43 by treating 1 with excess phenol and finely ground K3PO4 in xylenes followed by desilylation using tetrabutylammonium fluoride (TBAF) in THF. Chromatography was performed on virgin silica (230−400 mesh) obtained from Silicycle (Québec City, QC). Analytical thin layer chromatography was performed on glass-backed layer plates (60, F254 indicator, Silicycle). Compounds were visualized by UV light (λ = 254 nm). NMR spectra of synthetic samples were recorded at either 300, 400, or 600 MHz on Bruker (Billerica, MA) spectrometers in acetone-d6, DMSO-d6, CD2Cl2, or CDCl3. 19F NMR spectra of oligonucleotide samples were recorded in D2O on a Bruker Avance600 DPX spectrometer (19F, 564 MHz) equipped with a Bruker 5 mm TXO solution probe. Peaks were reported on the δ (parts per million) scale. A Sartorius (Goettingen, Germany) pH meter (UB-10) was used for buffer calibration. High-resolution mass spectrometry was conducted at the McMaster Regional Centre for Mass Spectrometry. C8-(4-Fluorophenoxy)-2′-deoxyguanosine (4FPhOdG). C8-(4-Fluorophenoxy)-2′-deoxyguanosine (4FPhOdG) was synthesized from 1 (1 g, 1.48 mmol), 4-fluorophenol (0.66 g, 5.92 mmol), K3PO4 (0.63 g, 2.96 mmol), and TBAF·3H2O (1.4 g, 4.44 mmol) to yield 0.45 g (81% over two steps): 1H NMR (400 MHz, DMSO-d6) δ 10.42 (bs, 1H), 7.37 (m, 2H), 7.28 (m, 2H), 6.41 (bs, 2H), 6.19 (t, J = 7.3 Hz, 1H), 5.24 (d, J = 4.2 Hz, 1H), 4.81 (t, J = 5.9 Hz, 1H), 4.32 (m, 1H), 3.75 (m, 1H), 3.48 (m, 1H), 3.43 (m, 1H), 2.90 (m, 1H), 2.14 (m, 1H); 13 C NMR (151 MHz, DMSO-d6) δ 155.7, 153.5, 149.9, 149.4, 149.3, 122.0, 121.9, 116.3, 116.2, 110.58, 87.4, 81.9, 70.8, 62.0, 36.6; HRMS (ESI) calcd for C16H17FN5O5 378.1214, found 378.1213 (MH+). N2-(Dimethylformamidyl)-C8-phenoxy-2′-deoxyguanosine (2). C8-Phenoxy-dG (0.4 g, 1.1 mmol) was placed in 25 mL of dry DMF under argon. N,N-(Dimethylformamidyl) diethyl acetal (0.75 mL, 4.4 mmol) was then added, and the mixture was allowed to stir for 12 h. The reaction mixture was evaporated to dryness, washed with MeOH, and purified by silica gel column chromatography eluting with a MeOH/CH2Cl2 mixture (10:90) to afford 2 as a light brown solid (0.324 g, 71%): 1H NMR (300 MHz, DMSO-d6) δ 11.37 (s, 1H), 8.54 (s, 1H), 7.44 (m, 2H), 7.30 (m, 3H), 6.30 (t, J = 9.2 Hz, 1H), 5.29 (d, J = 5.6 Hz, 1H), 4.80 (t, J = 8 Hz, 1H), 4.38 (m, 1H), 3.78 (m, 1H), 3.50 (m, 2H), 3.15 (s, 3H), 3.02 (s, 3H), 2.95 (m, 1H), 2.18 (m, 1H); 1399



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(10 mL) was then added to a reaction flask, and the mixture was backfilled with argon. 2-Cyanoethyl-N,N-diisopropyl-chloro-phosphoramidite (0.23 mL, 1.1 mmol) was added to the flask along with 0.3 mL of dry TEA. The reaction was allowed to proceed to completion as monitored by TLC (90:5:5 CH2Cl2/MeOH/TEA mixture). To the reaction mixture was added 25 mL of dry CH2Cl2 followed by a wash with 2 × 50 mL of a 5% bicarbonate solution. The organic layer was then dried over Na2SO4 and concentrated in vacuo. The crude product was purified by silica gel chromatography eluting with a MeOH/ CH2Cl2/TEA mixture (5:90:5) to yield phosphoramidite 6 as a white foamy solid (0.38 g, 60%): 1H NMR (600 MHz, acetone-d6) δ 10.12 (s, 1H), 8.57 (s, 1H), 7.42−7.35 (m, 6H), 7.23−7.15 (m, 12H), 6.76 (m, 5H), 6.50 (m, 1H), 4.92 (m, 1H), 4.19 (m, 2H), 4.12 (m, 1H), 3.84 (m, 1H), 3.74 (d, 7H), 3.63 (m, 3H), 3.52 (m, 2H), 3.33 (m, 1H), 3.17 (s, 3H), 3.07 (s, 3H), 2.72 (t, J = 6 Hz, 1H), 2.59 (t, J = 6 Hz, 1H), 1.27−1.19 (m, 10H), 1.15 (m, 9H), 1.06−1.0 (m, 8H); 13C NMR (151 MHz, acetone-d6) δ 159.6, 158.7, 157.7, 157.5, 154.9, 151.0, 149.6, 146.2, 136.8, 131.0, 130.8, 130.4, 129.0, 128.6, 127.5, 125.9, 120.8, 120.7, 119.1, 118.9, 118.6, 115.9, 113.8, 86.9, 85.8, 83.0, 82.9, 75.3, 75.2, 74.7, 74.5, 65.2, 64.8, 59.8, 59.7, 59.6, 59.4, 59.1, 59.0, 55.4, 45.8, 44.1, 44.0, 41.4, 35.1, 24.9, 23.2, 23.1, 20.8, 20.4, 20.3; 31P NMR (121.4 MHz, CDCl3) δ 148.74; HRMS (ESI) calcd for C49H58N8O8P 917.4115, found 917.4123. 3′-O-[(2-Cyanoethyl)(diisopropylamino)phosphino]-5′-O-(4,4′-dimethoxytrityl)-N2-(dimethylformamidyl)-C8-(4-fluorophenoxy)-2′deoxyguanosine (7). 5′-DMT-N2-(dimethylformamidyl)-C8-(4-fluorophenoxy)-2′-deoxyguanosine (5, 0.20 g, 0.27 mmol) was coevaporated from dry toluene (3 × 6 mL) followed by dry THF (3 × 6 mL). Dry CH2Cl2 (10 mL) was then added to a reaction flask and the mixture backfilled with argon. 2-Cyanoethyl-N,N-diisopropyl-chlorophosphoramidite (0.12 mL, 0.54 mmol) was added to the flask along with 0.3 mL of dry TEA. The reaction was allowed to proceed to completion as monitored by TLC (90:5:5 CH2Cl2/MeOH/TEA mixture). To the reaction mixture was added 25 mL of dry CH2Cl2 followed by a wash with 5% aqueous NaHCO3 (2 × 50 mL). The organic was then dried over Na2SO4, and the solvent was removed in vacuo. The resulting solid was then recrystallized from hexanes at −78 °C. The precipitate was filtered yielding 7 as a white solid (0.16 g, 63%): 1H NMR (300 MHz, CDCl3) δ 8.52 (bs, 1H), 8.46 (s, 1H), 7.29 (m, 2H), 7.18 - 7.02 (m, 9H), 6.67−6.61 (m, 4H), 6.40 (m, 1H), 4.78−4.62 (m, 1H), 4.16−4.12 (m, 2H), 3.69 (s, 6H), 3.58−3.40 (m, 4H), 3.25−3.22 (m, 2H), 3.02 (s, 3H), 3.00 (s, 3H), 2.68 (t, J = 7.3 Hz, 1H), 2.52 (t, J = 6.2 Hz, 1H), 1.27−1.19 (m, 10H), 1.10 (q, J = 7.3 Hz, 9H), 0.99 (d, J = 6.8 Hz, 4H), 0.83−0.76 (m, 4H); 13C NMR (151 MHz, CDCl3) δ 159.0, 158.98, 158.4, 158.37, 157.8, 157.77, 156.9, 156.0, 150.84, 150.82, 149.1, 149.0, 148.8, 144.6, 135.73, 135.72, 129.1, 126.7, 117.7, 117.5, 114.73, 114.71, 113.1, 86.2, 86.1, 85.0, 84.99, 84.94, 84.90, 74.3, 74.2, 73.5, 73.4, 64.0, 63.6, 58.6, 58.5, 58.4, 58.2, 58.1, 46.6, 36.9, 36.8, 31.6, 24.6, 22.6, 20.4, 20.2, 20.17, 14.1; 31P NMR (121.4 MHz, CDCl3) δ 148.90, 148.56; HRMS (ESI) calcd for C49H57FN8O8P 935.4021, found 935.4003 (MH+). Oligonucleotide Synthesis. Oligonucleotide synthesis for the C8-G-modified NarI oligonucleotide (5′-CTCGGCXCCATC, where X = PhOG or 4FPhOG) was conducted on a 1 μmol scale on a BioAutomation Corp. MerMade 12 automatic synthesizer using standard β-cyanoethylphosphoramidite chemistry, as outlined previously.39 Following synthesis, oligonucleotides were cleaved from the solid support, deprotected using 2 mL of a 30% ammonium hydroxide solution at 55 °C for 12 h, and purified by HPLC. Purification of Oligonucleotides. Aqueous solutions containing impure oligonucleotides were initially filtered using Mandel syringe filters (0.20 μm PVDF). Oligonucleotide purification was conducted on an Agilent 1200 series HPLC instrument equipped with an autosampler, a diode array detector (monitored at λabs = 258 nm), and an autocollector. Separation was conducted at 50 °C using a Phenomenex clarity 5 μm oligo-RP semipreparative column (100 mm × 10 mm) at a flow rate of 3.5 mL/min using a gradient running from 95% 50 mM aqueous triethylamine acetate (TEAA, pH 7.2) with 5% acetonitrile to 30% 50 mM TEAA with 70% acetonitrile over 30

min. The modified oligonucleotide sample was lyophilized to dryness and obtained as a white foam. DNA Quantification. Stock solutions of DNA (0.50 or 1.00 mL) were prepared in purified water. Consecutive additions of 5 μL of a DNA stock to 1985 μL of purified water were followed by an absorption scan at 260 nm on a Cary 300-Bio UV−visible spectrophotometer equipped with a Peltier block-heating unit and automated temperature controller using standard 10 mm light path quartz glass cells from Hellma GmbH & Co. (Concord, ON). Scans were performed three times to determine the concentration of the stock solution. Molar absorptivities (ε) of the unmodified NarI were used for the modified NarI strand and were calculated online using Integrated DNA Technologies (IDT) OligoAnalyzer version 3.1. Mass Spectrometry. MS experiments for DNA identification were conducted on a Bruker AmaZon quadrupole ion trap SL spectrometer. Masses were acquired in the negative ionization mode with an electrospray ionization source (Bruker Daltronics, Milton, ON). The DNA samples were dissolved in a 90% Milli-Q filtered water/10% MeOH mixture with 0.1% ammonium acetate and injected directly into the electrospray source. Ionization analysis was conducted using the following settings on the ESI: nebulizer gas flow, 40 psi; dry gas, 10 L/min; dry temperature, 200 °C; spray voltage, 4000 V. The scan range was m/z 70−2000, and the scan resolution was 8100 m/z per second. The injection time was 40 μL/s. UV Melting. UV melting experiments were conducted using a Cary 300-Bio UV−vis spectrophotometer equipped with a 6 × 6 multicell Peltier block-heating unit using Hellma 114-QS 10 mm light path cells. Oligonucleotide duplex samples were prepared in 50 mM Na2HPO4 buffer and 100 mM NaCl (pH 7) to a concentration of 6 μM using 1 equiv of complementary strand. The UV absorption at 260 nm was monitored as a function of temperature and consisted of forward− reverse scans from 10 to 90 °C at a rate of 1 °C/min, which was repeated five times. Thermal melting temperatures (Tm) were determined using hyperchromicity calculations provided in the Varian Thermal software. Circular Dichroism (CD). Spectra were recorded on a Jasco J-815 CD spectropolarimeter equipped with a 1 × 6 Multicell Block Peltier, a thermal controller, and a Julabo AWC 100 water circulator unit. CD spectra were recorded using 110-QS cells with a light path of 1 mm. Samples of duplex DNA were prepared in 50 mM Na2HPO4 buffer and 100 mM NaCl (pH 7) to a concentration of 6 μM. Spectra were recorded at 10 °C between 200 and 320 nm, with a bandwidth of 1 nm at a rate of 50 nm/min. The spectra were the averages of four accumulations that were smoothed using the Jasco software. 19 F NMR Studies. Samples for 19F NMR analysis were prepared to a concentration of 0.5 mM with 1 equiv of each strand in 100% D2O containing 50 mM Na2HPO4 and 100 mM NaCl (pH 7) prepared in a total volume of 200 μL in a Shigemi NMR tube. 19F NMR spectra were referenced externally to CFCl3 in CDCl3. Spectra were obtained by collecting 1000 scans with a recycle delay of 1.5 s between acquisitions and processed with a line broadening of 10 Hz. Molecular Modeling. Molecular dynamics (MD) simulations were conducted with the PhOdG adduct placed in the syn and anti conformations at the X position in the NarI sequence (Figure 1c) and paired against either the complementary C or a G mismatch. Two possible orientations of the phenoxyl moiety with respect to the nucleobase, which roughly correspond to θ values of ∼0° and ∼180° (see Figure 1b for a definition of θ), were considered for the syn and anti conformations of the adduct (Table S2 and Figure S20 of the Supporting Information). Initial structures were prepared using the NAB44 module of the AMBER software, and GaussView45 was used to modify the PhOdG residue. Each system was neutralized with 22 sodium ions and solvated with an octahedral (TIP3P)46 water box. Antechamber version 1.447 was used to assign atom types, while RED version III.448 was used to calculate the partial charges of the PhOdG residue. The parmbsc049 modification to the parm9950 force field was used to simulate the natural nucleosides, and the generalized AMBER force field (GAFF)51 was used for the adducted nucleoside. MD simulations were conducted for 40 ns using the PMEMD module of AMBER version 11.52 The total free energy (Gtot) was estimated for 1400



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Table 1. Thermal Parameters of C8-Phenoxy-G-Modified NarI Oligonucleotides duplex

Tm (°C)a

ΔTmb

duplex

Tm (°C)a

ΔTmb

NarI(G):NarI′(C) NarI(PhOG):NarI′(C) NarI(4FPhOG):NarI′(C) NarI(G):NarI′(G) NarI(PhOG):NarI′(G) NarI(4FPhOG):NarI′(G) NarI(G):NarI′(A) NarI(PhOG):NarI′(A) NarI(4FPhOG):NarI′(A)

63.6 52.8 53.8 54.0 52.1 52.7 51.4 44.4 45.8

− −10.8 −9.8 − −1.9 −1.2 − −7.0 −5.6

NarI(G):NarI′(T) NarI(PhOG):NarI′(T) NarI(4FPhOG):NarI′(T) NarI(G):NarI′(THF) NarI(PhOG):NarI′(THF) NarI(4FPhOG):NarI′(THF) NarI(G):NarI′(−2) NarI(PhOG):NarI′(−2) NarI(4FPhOG):NarI′(−2)

53.4 47.1 47.0 45.7 42.3 44.9 39.4 35.0 38.5

− −6.3 −6.4 − −3.4 −0.8 − −4.4 −0.9

Tm values of duplexes (6.0 μM) measured in 50 mM sodium phosphate buffer (pH 7) with 0.1 M NaCl at a heating rate of 1 °C/min. Errors are ±1 °C. bΔTm = Tm(modified duplex) − Tm(control duplex). a

each system using the MM-PBSA method.53 Additional details of the MD simulation protocol and free energy analysis are provided in the Supporting Information. A representative structure from each simulations was chosen for analysis by clustering the entire simulation based on the location of the atoms composing the θ and χ dihedral angles in PhOdG (Figure 1). For comparison, simulations were also conducted on the natural (unmodified) NarI helix with either anti-dG at the X position paired against complementary C or syn-dG at the X position paired against a G mismatch. A representative structure for simulations of the native strands was chosen for analysis by clustering the entire simulation based on the location of the atoms comprising the χ dihedral angle in dG at the X position. DFT calculations were conducted to estimate the H-bonding [B3LPY/6-311+G(2df,p)] and stacking [M06-2X/6-31+G(d,p)] interactions between the nucleoside at the X position and the neighboring bases in the representative structures using Gaussian 09 (revision C.01).54

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natural base C, mismatches with G, A, and T, and opposite an abasic site (THF). NarI′(−2) refers to the 10-mer oligonucleotide 5′-GATGGCCGAG (Figure 1c). When NarI′(−2) is paired with NarI(G), the duplex contains a two-base bulge consisting of 5′-CG3(X). Certain N-linked C8-dG adducts are able to stabilize this two-base bulge when placed in the G3(X) position of the NarI oligonucleotide,17,41 and this observation correlates with the ability of the N-linked lesion to induce −2 frameshift mutations in Escherichia coli.41,56,57 Melting temperatures (Tm) of the modified duplexes were determined using UV−vis spectroscopy by monitoring the absorbance at 260 nm versus temperature (Table 1). In each instance, when the modified C8-phenoxy-G NarI oligonucleotide was matched with the complementary strands, the resulting duplexes were destabilized compared to the unmodified duplex controls, providing negative ΔTm values (Table 1). The largest destabilization occurred when the C8phenoxyl-G adducts were matched with their normal pyrimidine partner C (ΔTm ∼ −10 °C). The adducts were also strongly destabilizing opposite A and T (ΔTm ∼ −6 °C), while the lesions were only slightly destabilizing opposite G (ΔTm ∼ −1.5 °C) relative to the unmodified G:G mismatch. In fact, the modified G:G mismatched duplexes were comparable in stability to the modified NarI oligonucleotides hybridized to NarI′(C). Overall, when hybridized to NarI′(N) (N = C, G, A, and T), NarI(PhOG) and NarI(4FPhOG) provided Tm values that are within experimental error of each other, indicating that the 4-F substituent does not alter the thermal parameters of these duplexes. However, this was not the case when the modified NarI strands were hybridized to NarI′(THF) or NarI′(−2). In these duplexes, PhOG was fairly destabilizing (ΔTm ∼ −4 °C), while 4FPhOG exhibited Tm values within experimental error of those of the unmodified duplex controls. CD Measurements. Figure 2 shows CD spectral overlays of modified NarI(X) [X = PhOG (dotted red lines) and 4FPhOG (dashed green lines)] relative to the unmodified control NarI(G) (solid black lines) hybridized to NarI′(C) (Figure 2a) and NarI′(G) (Figure 2b). All duplexes show characteristics of B form DNA with positive (275 nm) and negative (244 nm) sigmoidal CD curves with a crossover at ∼260 nm.58 When hybridized to NarI′(C), the modified duplexes exhibit considerable hypochromicity compared to that of the unmodified NarI(G):NarI′(C) duplex (Figure 2a). The hypochromicity of a CD band is indicative of partial unwinding of the helix with a loss of stacking interactions.41,59 This observation is consistent with the UV melting data (Table 1), which suggests inclusion of the O-linked C8-phenoxy-dG adduct causes considerable duplex destabilization. Conversely,

RESULTS

Synthesis of Modified NarI. Solid-phase DNA synthesis was employed to incorporate the O-linked C8-phenoxy-dG adducts (PhOdG and 4FPhOdG) into the G3 (X) position of the 12-mer NarI sequence 5′-CTCGGCXCCATC. The synthetic strategy used to prepare the modified phosphoramidites is outlined in Scheme 1. A general method for the synthesis of Olinked C8-phenoxy-dG adducts has been developed by Dahlmann and Sturla55 and involves treating a suitably protected 8-Br-dG analogue with excess phenolate in xylenes at high temperatures (∼130 °C). Employing this strategy, we synthesized PhOdG43 and 4FPhOdG in high yields by treating 1 with the requisite phenolate in xylenes and then employing TBAF for removal of the silyl protecting groups in a single step (Scheme 1). Standard procedures were then employed to convert the nucleoside adducts into the phosphoramidites (see the Supporting Information for NMR spectra of synthetic samples). Negative electrospray ionization mass spectrometry (ESI-MS) analysis confirmed the identity of the C8-phenoxydG-modified NarI oligonucleotides. ESI-MS spectra for the modified NarI oligonucleotides are available in the Supporting Information, and the results of the ESI-MS analysis are summarized in Table S1 of the Supporting Information. UV Thermal Melting Studies. The C8-phenoxy-dGmodified oligonucleotides NarI (X = PhOG or 4FPhOG) were hybridized to six different complementary strands: NarI′(N) (N = C, G, A, T, or THF) and NarI′(−2) (Figure 1c). NarI′(N) refers to the full-length complementary strand in which the four nucleobases (C, G, A, and T) along with an abasic site (THF) have been inserted opposite the C8-phenoxyG lesion upon duplex formation. The stability of these duplexes permitted assessment of the impact of the adduct opposite the 1401



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heated and slowly cooled to allow for annealing of the two strands, followed by the acquisition of data. The NMR experiments were conducted at 25, 35, 45, 55, and 60 °C with proton decoupling to characterize the duplex to single strand (SS) transition, as previously implemented by Cho and co-workers for N-linked arylamine-modified duplexes.22,23,40,41 Each modified duplex showed a single 19F resonance at 25 °C, suggesting the uptake of a single conformation of the modified base within the duplex. Interestingly, the 19F resonances for NarI(4FPhOG):NarI′(C) and NarI(4FPhOG):NarI′(G) duplexes were observed at −117.5 and −116.3 ppm, respectively, indicating discrete conformations when C or G is opposite the modification in the complementary strand. This is supported by the temperature-dependent migration of the 19F chemical shift. Upon thermal denaturation of NarI(4FPhOG):NarI′(C) (Figure 3b), the 19F chemical shift migrates downfield from −117.5 to −117.2 ppm (19F resonance of the SS). In contrast, the mismatched NarI(4FPhOG):NarI′(G) duplex showed an upfield migration from −116.3 to −117.2 ppm under analogous conditions (Figure 3a). MD Simulations. MD simulations were conducted with PhO dG placed at the X position in the NarI sequence to provide complementary structural information about the adduct in DNA duplex environments. Specifically, two orientations of both the syn and anti PhOdG conformers with respect to rotation about the C8−O linkage were paired opposite C or G in the complementary strand. These two orientations roughly correspond to θ values of ∼0° and ∼180°, respectively, but with deviations from planarity of 10−37° to eliminate steric clashes with neighboring residues. When PhOdG is paired opposite C, the calculated free energies suggest that the anti conformation of the adduct is more stable than the syn conformation (Table 2 and Table S2

Figure 2. CD spectral overlays of (a) NarI(G or X):NarI′(C) and (b) NarI(G or X):NarI′(G): unmodified control duplexes (black lines), NarI(PhOG) duplexes (dotted red lines), and NarI(4FPhOG) duplexes (dashed green lines).

the CD spectra of the mismatched NarI(X):NarI′(G) duplexes showed little deviation from those of the unmodified NarI(G):NarI(G) mismatched duplex (Figure 2b). Again, this observation is consistent with the UV melting data, which showed the modified duplexes are almost equal in stability to the unmodified control duplex. This suggests retention of the global and local DNA structure at the lesion site between modified and unmodified helices. Dynamic 19F NMR. To gain insight into the conformation of the O-linked C8-phenoxy-G adduct at the G3(X) position of NarI, 19F NMR studies were performed on both NarI(4FPhOG):NarI′(C) and NarI(4FPhOG):NarI′(G) duplexes (Figure 3). For these duplexes, the 4-F substituent does not

Table 2. Free Energy Analysis of the Opposite C and Ga adduct PhO

dG

PhO

dG

PhO

dG Adduct Paired

opposite base

adduct conformation

initial θ (deg)

GTotb (kJ/mol)

Grelc (kJ/mol)

C

anti (θ ∼ 0°) anti (θ ∼ 180°) syn (θ ∼ 0°) syn (θ ∼ 180°) anti (θ ∼ 0°) anti (θ ∼ 180°) syn (θ ∼ 0°) syn (θ ∼ 180°)

∼0 ∼180

−23851.3 −23854.7

3.3 0.0

∼0 ∼180 ∼0 ∼180

−23830.0 −23826.2 −23692.7 −23670.1

24.7 28.5 34.3 56.9

∼0 ∼180

−23724.2 −23727.0

2.9 0.0

G

a

All energies are in kilojoules per mole. bTotal free energy. cRelative free energy with respect to the same adduct conformation paired opposite the same nucleobase.

Figure 3. Dynamic 19F NMR spectra of NarI(4FPhOG) hybridized to (a) NarI′(G) or (b) NarI′(C). SS denotes single strand (spectra recorded at 25 °C).

of the Supporting Information), as known for natural dG. In this preferred structure, the adduct adopts a B conformer with an average χ close to 235° (Table 3), which is comparable to the average χ (∼249°) adopted by dG at the X position in the same sequence. Furthermore, in the representative structure containing PhOdG, the phenoxyl moiety is located in the major groove, does not stack with the neighboring bases, is exposed to solvent, and is slightly nonplanar with respect to the nucleobase [average θ of ∼145° (Table 3)]. Three strong Watson−Crick H-bonds between PhOdG and C remain intact throughout the simulation [∼100% occupancy (Table 3)], which leads to a

significantly impact thermal stability (Table 1) or duplex conformation (Figure 2), suggesting that the 19F NMR data would provide valuable insight into the preferred conformation of the unsubstituted PhOG lesion. Samples for the NMR study were prepared in a 1:1 NarI(4FPhOG):NarI′(C/G) ratio to a concentration of ∼0.5 mM in 50 mM sodium phosphate (pH 7) and 100 mM NaCl in 100% D2O. The solutions were then 1402



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Table 3. Hydrogen Bonding Occupancies for the Hydrogen Bonds between the Adduct and the Opposing Base over the Duration of the MD Simulations conformation

opposing base

χava

θavb

H-bond

%c

dG

anti

C

248.6

−

dG

syn

G

53.4

−

N2-H(dG)···O2(C) N1-H(dG)···N3(C) N4-H(C)···O6(dG) N1-H(G)···O6(dG) N2-H(G)···N7(dG) N2-H(G)···O6(dG) N2-H(PhOdG)···O2(C) N1-H(PhOdG)···N3(C) N4-H(C)···O6(PhOdG) N1-H(G)···O6(PhOdG) N2-H(G)···N7(PhOdG) N2-H(G)···O6(PhOdG)

99.9 100.0 99.1 86.2 90.7 19.8 99.8 100.0 98.6 82.0 73.8 41.9

X

PhO

dG

anti

C

235.5

145.1

PhO

dG

syn

G

55.7

165.8

Average χ dihedral angle (degrees) over the entire 40 ns MD simulation. bAverage θ dihedral angle (degrees) over the entire 40 ns MD simulation. The implemented H-bond distance cutoff was a 3.40 Å heavy atom separation and a 120° X−H−X angle. Only H-bonds with occupancy of >15% are reported. a c

Table 4. DFT Interaction Energies (kilojoules per mole) between Natural dG or Calculated Using a Representative Structurea X dG dG PhO PhO

dG dG

PhO

dG and Neighboring Nucleobases

conformation

opposite base

ΔEHbondb

ΔEintra5′c

ΔEintra3′d

ΔEinter5′e

ΔEinter3′f

anti syn anti syn

C G C G

−100.6 −38.4 −96.7 −57.5

−31.8 −15.9 −22.1 −20.4

−41.1 −2.2 −44.9 −7.5

−22.1 −2.4 −25.6 −5.0

+10.8 −7.9 +19.4 −10.7

Each representative structure was obtained by clustering the entire MD simulation with respect to the χ (natural dG) or χ and θ (PhOdG) dihedral angles. bΔEHbond is the counterpoise-corrected B3LYP/6-311+G(2df,p) hydrogen bond strength in the dimer consisting of the nucleobase at the X position and the opposing base. cΔEintra5′ is the M06-2X/6-31+G(d,p) stacking interaction energy between the nucleobase at the X position and the intrastrand base at the 5′-side of the adduct. dΔEintra3′ is the M06-2X/6-31+G(d,p) stacking interaction energy between the nucleobase at the X position and the intrastrand base at the 3′-side of the adduct. eΔEinter5′ is the M06-2X/6-31+G(d,p) stacking interaction energy between the nucleobase at the X position and the base in the opposing strand at the 5′-side of the adduct. fΔEinter3′ is the M06-2X/6-31+G(d,p) interaction energy between the nucleobase at the X position and the base on the opposite strand stacked at the 3′-side of the adduct. a

base pairing energy of ∼97 kJ/mol (Table 4). In fact, the Hbonding between PhOdG and C is comparable to the interaction between natural dG and C at the same position in the NarI sequence in terms of occupancy [∼100% (Table 3)] and base pairing energy [∼101 kJ/mol−1 (Table 4)]. Because of the helical twist, intrastrand stacking (Table 4) at the 3′-side of the adduct (−45 kJ/mol) is stronger than at the 5′-side (−22 kJ/ mol). Furthermore, steric interactions between the adduct and the 5′-cytosine decrease the strength of the stacking interaction by ∼10 kJ/mol compared to that of the unmodified duplex. Finally, as observed for natural dG at the X position, significant interstrand stacking is observed at the 5′-side of the adduct (−26 kJ/mol), but the interstrand contact at the 3′-side (inter3′) is more repulsive (by ∼9 kJ/mol) than in the unmodified duplex. When PhOdG is paired against a G mismatch, the calculated free energies suggest that the syn conformation of PhOdG is preferred, while the opposing dG remains in an anti conformation. This observation is consistent with G:G base pairs within duplex DNA, where one dG adopts a syn conformation and the other an anti conformation.60 The average χ value adopted during the simulation is ∼56°, which is very close to the average χ adopted by natural dG at the X position in the NarI sequence (∼54°). In the representative structure of adducted DNA, the phenoxyl moiety is located in the minor groove but does not stack in the helix or displace the opposing base. Thus, the adduct adopts a W-type wedge

conformation. Nevertheless, the orientation of the phenoxyl moiety with respect to the nucleobase is slightly nonplanar [θ ∼ 165° (Table 3)] because of steric interactions with the opposing G. Two strong Hoogsteen H-bonds [N1-H···O6 (82% occupancy) and N2-H···N7 (74% occupancy)] are formed between the adduct and guanine, while a third Hbond (N2-H···O6) is present for a smaller portion of the simulation (42% occupancy). Although the N2-H···O6 H-bond also appears over the course of the simulation of the natural sequence, the occupancy is greater in adducted DNA because steric interactions between the phenoxyl moiety and the opposing G are slightly alleviated when the G shifts toward the major groove and forms the N2-H···O6 H-bond with the adduct. Nevertheless, the strength of Hoogsteen H-bonding is enhanced by ∼19 kJ/mol compared to that of the natural strand, which is likely mainly due to the greater planarity of paired bases in the adducted helix in the representative structure (Figure 4). In addition, the stacking interactions are slightly enhanced (by ∼3−5 kJ/mol) in the adducted helix compared to those in the natural helix because of changes in the relative arrangement of the flanking bases, which help accommodate the additional phenoxyl moiety in the duplex. Overall, the level of steric interactions caused by the phenoxyl group is elevated when the adduct adopts the syn conformation opposite G compared to when the adduct adopts the anti conformation against C. However, despite these steric contacts and slight changes in the Hoogsteen H-bonding 1403



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Figure 4. Portion of the representative structure from MD simulations with (a) anti-G paired against cytosine in the B-DNA conformation, (b) synG Hoogsteen hydrogen bonded with the guanine mismatch, (c) anti-PhOG paired against cytosine showing the B-type conformation, and (d) syn-PhOG paired against guanine showing the W conformation.

regard, initial duplex structures were built to search for the S conformation with PhOG paired against C or G (Figure S20 of the Supporting Information). In these structures, PhOG is present in the syn conformation (θ ∼ 180) with the phenoxyl moiety oriented to permit stacking within the helix. Following energy minimization, the W-type (wedged) structures were obtained, as shown for the PhOdG:G mismatch in Figure 4. Our previous 20 ns MD study of the C8-(2″-benzo[b]thienyl)-dG ( BthdG) adduct showed that a conversion from a W conformation to an S conformation is possible in a 20 ns MD time frame.38 Given that no such structural transition was evident within the 40 ns simulation in our work, we believe that the S conformation for the PhOdG adduct is not stable. For the NarI(4FPhOG):NarI′(C) duplex, 19F NMR evidence suggests one conformation due to a single peak at −117.5 ppm that was 0.3 ppm upfield of the 19F resonance for 4FPhOG in the single strand (Figure 3b). In comparison, B conformer duplexes as characterized by Cho and co-workers for N-linked C8-dG adducts have rendered chemical shifts in the range of −115.5 to −117.5 ppm, which were dependent on adduct structure and flanking sequence.15,22,23,40 Although this is a substantial range, the B conformer chemical shift is consistently slightly upfield compared to that of the single strand.15,22,23,40 Molecular mechanics minimizations on the structurally similar N-linked C8-dG aniline adduct (AN-dG) also favor the exclusive uptake of the B conformer in 9-mer duplexes when the adduct is matched with its normal pyrimidine partner.62 UV−vis thermal melting (Tm) studies of AN-dGmodified duplexes with a C base pairing complement rendered destabilizations of 10%63 and 16%.64 In comparison, PhOdG and 4FPhO dG induced a destabilization of 15% in the NarI:NarI′(C) duplex (Table 1). The reduction in duplex stability in such cases can be rationalized as the solvation energy required for the lipophilic aromatic ring in the aqueous environment of the

relative to that of the corresponding natural sequence, the introduction of the phenoxyl moiety does not significantly disrupt the helical conformation when the adduct adopts the syn conformation.

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DISCUSSION Biaryl ether O-linked adducts have been observed following metabolism of phenols by peroxidase enzymes in vitro.31−34 For the first time, we have incorporated O-linked C8-dG adducts, PhO dG and 4FPhOdG, into an oligonucleotide substrate and determined their structural impact in duplex DNA. The unsubstituted PhOdG lesion represents the simplest member of the O-linked adduct class. While PhOdG itself has not been detected following bioactivation of phenol in the presence of DNA substrates, its structural impact within duplex DNA can serve as a comparison to other O-linked adducts that have been detected in vitro and contain larger aryl rings and chlorine substituents.31−34 Our choice of the 12-mer NarI substrate stems from its extensive use as an oligonucleotide substrate for N-linked C8-dG adducts.17,19,41,61 This utility is derived from the finding that the G3 (X) position of NarI is a hot spot for −2 frameshift mutations mediated by N-linked C8-dG adducts.41,56,57 Thus, it was our desire to make a direct comparison between O-linked and N-linked adducts in terms of their structural impact within duplex DNA, which can be related to probable mutagenic outcome. Opposite the normal pyrimidine partner C, our data suggest that both PhOG and 4FPhOG adopt an anti conformation with the duplex present in a B conformer. This hypothesis is supported by free energy analysis following MD simulations of the NarI(PhOG):NarI′(C) duplex, which indicates that the syn conformation of the adduct is at least 24 kJ/mol less stable than the anti conformation when the adduct is paired opposite C (Table 2 and Table S2 of the Supporting Information). In this 1404



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may place the fluorine atom in a different region of the minor groove, and therefore, the atom could possess a different electronic environment. This phenomenon has been observed for minor groove binding antibiotics such as distamycin65 and covalent adducts such as styrene oxide adducts that reside in the minor groove through attachment to the exocyclic NH2 group of dG.66 In these examples, select protons within distamycin and styrene shift upfield, while others shift downfield upon binding, because of their positioning at the edges of the aromatic nucleobases. Likewise, the aromatic DNA protons can shift downfield upon exposure to the edge of the distamycin and styrene aromatic residues.65,66 The representative structure of the NarI(PhOG):NarI′(G) duplex from MD simulations (Figure 4d) provides insight for the observed downfield shift in the 19F resonance. Specifically, the phenoxyl moiety is located in the minor groove and does not stack in the helix, which would typically place the 4-F substituent in a shielding environment. Instead, however, the phenoxyl ring is twisted from planarity and is directed toward the edges of the nucleobases, primarily with G on the complementary strand. Such positioning of the phenoxyl ring in the minor groove could place the 4-F substituent in a deshielding environment and would therefore cause the observed downfield shift of the 19F resonance for the W conformation. The biological relevance of the C8-phenoxy-dG adduct in the context of conformation-induced toxicity renders the unsubstituted O-linked phenolic adduct as a suspected weak mutagen. The lack of stabilization of the two-base bulge resulting in no uptake of the promutagenic S conformation makes the phenolic adduct an unlikely candidate to cause frameshift mutations. The uptake of the W conformation within the G:G mismatched duplex gives some evidence that G → C transversions could occur. However, these mutations would likely occur at a low frequency because the stability of this modified duplex was equal to, rather than greater than, that of the unmodified sequence. These predictions of mutagenicity for the PhOdG lesion are not particularly surprising given that other single-ring major groove adducts, such as the N-linked aniline C8-dG adduct62 and adenine N6 lesions of styrene oxide,67 tend to lack mutagenicity.

major groove,37 as well as steric interactions with the DNA backbone that weaken π-stacking interactions (Figure 2 and Table 4). Certain N-linked C8-dG adducts will exhibit all three conformers (B, S, and W) upon hybridization to the normal pyrimidine partner C.23,41 For example, at G3 of the NarI duplex paired with C, the N-linked fluorinated N-acetylaminofluorene (FAAF) adduct exhibits populations of 13% B, 61% S, and 26% W.23 The 2-acetylaminofluorene (AAF) moiety is a bulky, lipophilic, planar aromatic system, and both the S and W conformers shield the aromatic moiety from the solvent. The preferred S conformer has the added advantage of stacking the aromatic system with bases of the helix.62 Adducts that prefer an S conformation have also been shown to stabilize the twobase bulge produced by hybridization to the complementary NarI′(−2) 10-mer sequence.17,41,61 For the N-linked C8-dG adducts, a two-base bulge stabilization with a ΔTm of 3.3−15 °C has been observed,17,41,61 where the adducts with the greater proportion of the S conformation were found to induce greater stabilization of the bulge.41 For the C8-phenoxy-G-modified duplexes studied here, no stabilization of the bulge was observed [ΔTm = −4.4 and −0.9 °C (Table 1)], which indicates that it is not energetically favorable for the adducted phenolic moiety to flip into a base-displaced S conformation. Further evidence of the argument made above is the lack of stabilization of the duplex containing an abasic site opposite the modification [NarI(X):NarI′(THF)]. These results strongly support a B-type duplex with the C8-phenoxy-G adducts in an anti conformation, as noted for the single-ring N-linked AN-dG adduct.62 The duplexes containing the C8-phenoxy-G lesion basepaired opposite G [NarI(X):NarI′(G)] were comparable in stability to the unmodified G:G mismatch and the modified NarI duplexes base-paired to C (Table 1). MD simulations indicated that the preferred syn structure of the adduct against G (Table 2 and Table S2 of the Supporting Information) involves Hoogsteen H-bonding with the anti-G in the opposite strand, which is similar to the structure adopted by natural G against a G mismatch.60 In other words, the modification does not alter the structure of the duplex. Supporting the lack of structural change are the Tm values, which showed a very slight adduct destabilization (ΔTm = −1.9 and −1.2 °C), and overlays of the CD spectra, which showed very few differences between the unmodified and modified duplexes containing a G:G mismatch (Figure 2b). The retention of this conformation, as characterized by CD, Tm, and MD studies, is strongly supportive of a W-type conformation for the NarI(X):NarI′(G) duplex. Furthermore, the W conformation is known to be favored for N-linked C8-dG adducts mismatched with dA.20−22 The 19 F resonance at −116.3 ppm for the NarI(4FPhOG):NarI′(G) duplex was shifted downfield by 0.9 ppm relative to the 19F resonance for 4FPhOG in the single strand (Figure 3a). This downfield shift is atypical for a W conformation. Work conducted by Cho and co-workers on N-linked C8-dG adducts consistently reported the W conformation as being the most upfield, typically at ∼118 ppm, approximately 1−1.5 ppm upfield from the single-strand 19 F resonance.21,39 Although this seems not to support the W conformation for the C8-phenoxy-G adducts, all fluorinated adducts studied by Cho et al. differ structurally from the adduct studied here by both the linking atom (O-linked vs N-linked) and the size of the aromatic ring (monocyclic vs polycyclic).21,39 Therefore, in this case, uptake of the W conformer

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CONCLUSIONS

The results of our studies suggest that the single-ring oxygenlinked C8-phenoxy-dG adduct ( PhO G) adopts an anti conformation opposite C within the G3 (X) position of the 12-mer NarI recognition sequence (5′-CTCGGCXCCATC). This conformation positions the phenoxy moiety in the major groove of B form DNA, which is also the preferred conformation of the single-ring nitrogen-linked C8-dG adduct derived from aniline. No evidence of a stacked conformation with PhOG in a syn conformation opposite C was obtained. However, within a PhOG:G mismatch, our data suggest that PhO G adopts a syn conformation, which positions the phenoxy moiety in the minor groove of B form DNA. Our results are the first to describe the influence of an oxygen-linked biaryl ether C8-dG adduct within duplex DNA and provide a structural basis for comparison to other O-linked C8-dG adducts derived from phenolic toxins. 1405



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ASSOCIATED CONTENT

S Supporting Information *

NMR spectra of synthetic samples, ESI-MS analysis and spectra of modified oligonucleotides, additional computational details, free energy analysis of PhOdG paired against C and G, and rootmean-square deviations for MD simulations on duplexes considered. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Funding

Support for this research was provided by the Natural Sciences and Engineering Research Council (NSERC) of Canada, the Canada Research Chairs program, the Canada Foundation for Innovation, and the Ontario Innovation Trust Fund. Computer calculations were conducted on the New Upscale Cluster at Lethbridge for Enabling Innovative Chemistry (NUCLEIC) computing cluster and WestGrid, part of the Compute/Calcul Canada high-performance computing platform. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS M.S.K. and R.A.M. thank Dr. Andy Lo (University of Guelph) for assistance with the dynamic 19F NMR experiments. R.A.M. also thanks the reviewers of this manuscript for their helpful suggestions.

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ABBREVIATIONS dG, 2′-deoxyguanosine; PhOdG, C8-(phenoxy)-2′-deoxyguanosine; 4FPhOdG, C8-(4-fluorophenoxy)-2′-deoxyguanosine; THF, tetrahedrofuran; TEA, triethylamine; CD, circular dichroism; MD, molecular dynamics; W, wedge; S, stacked

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REFERENCES

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Structural Influence of C8-Phenoxy-Guanine in the NarI Recognition

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