Chemical Structure and Properties of Interstrand Cross-Links Formed

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Chemical structure and properties of the interstrand cross-link formed by the reaction of guanine residues with abasic sites in duplex DNA Michael J. Catalano, Shuo Liu, Nisana Andersen, Zhiyu Yang, Kevin M. Johnson, Nathan E. Price, Yinsheng Wang, and Kent S. Gates J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.5b00669 • Publication Date (Web): 24 Feb 2015 Downloaded from http://pubs.acs.org on March 5, 2015

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Journal of the American Chemical Society

Chemical structure and properties of the interstrand crosslink formed by the reaction of guanine residues with abasic sites in duplex DNA Michael J. Catalano‡, Shuo Liu†, Nisana Andersen†, Zhiyu Yang‡, Kevin M. Johnson‡, Nathan E. Price‡, Yinsheng Wang† and Kent S. Gates‡,§,* University of Missouri ‡

Department of Chemistry and §

Department of Biochemistry 125 Chemistry Building Columbia, MO 65211

†

University of California-Riverside

Environmental Toxicology Graduate Program and Department of Chemistry Riverside, CA 92521-0403

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ABSTRACT: A new type of interstrand cross-link resulting from the reaction of a DNA abasic site with a guanine residue on the opposing strand of the double helix was recently identified, but the chemical connectivity of the cross-link was not rigorously established. The work described here was designed to characterize the chemical structure and properties of dG-Ap cross-links generated duplex DNA.

The approach involved

characterization of the nucleoside cross-link “remnant” released by enzymatic digestion of DNA duplexes containing the dG-Ap cross-link. We first carried out a chemical synthesis and complete spectroscopic structure determination of the putative cross-link remnant 9b composed of a 2-deoxyribose adduct attached to the exocyclic N2-amino group of dG. A reduced analog of the cross-link remnant was also prepared (11b). LCMS/MS analysis revealed that the retention times and mass spectral properties of the synthetic standards 9b and 11b matched those of the authentic cross-link remnants released by enzymatic digestion of duplexes containing the native and reduced dG-Ap cross-link, respectively. These results establish the chemical connectivity of the dG-Ap cross-link released from duplex DNA and provide a foundation for detection of this lesion in biological samples. The dG-Ap cross-link in duplex DNA was remarkably stable, decomposing with a half-life of 22 d at pH 7 and 23 ˚C. The intrinsic chemical stability of the dG-Ap cross-link suggests that this lesion in duplex DNA may have the power to block DNA-processing enzymes involved in transcription and replication.

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INTRODUCTION Many molecules that contain an aldehyde functional group display mutagenic or cytotoxic properties that stem from their ability to covalently modify DNA.1-17 Reversible attack of DNA nucleophiles on the electrophilic aldehyde carbon typically yields an equilibrating mixture of the hemiaminal and the imine adducts (1 and 2, Scheme 1).1,4-14 In some cases, aldehyde-DNA adducts have been stabilized for analysis via irreversible reduction of the iminium ion 3 to the amine 4 using reagents such as NaCNBH3 under mildly acidic conditions (Scheme 1).4,18-20 We recently identified a special class of aldehyde-DNA adducts derived from the reaction of DNA abasic (Ap) sites with nucleobases on the opposing strand of the double helix.21-23 Ap sites are prevalent lesions in genomic DNA that are generated by a wide variety of endogenous cellular processes,24-28 drugs,25 bioactive natural products,29-34 and environmental carcinogens.25,26 Ap sites exist as an equilibrium mixture of the ringclosed hemiacetal 5 and the ring-opened aldehyde 6 (Scheme 2).35 We showed that the Ap-aldehyde can react reversibly with guanine or adenine residues on the opposing strand of the double helix to generate DNA-DNA interstrand cross-links (Scheme 3).21-23 In the case of the dG-Ap cross-link, treatment with NaCNBH3 generated a reduced, chemically stable analog of the cross-link.21

Interstrand cross-links are extremely deleterious

because they block replication and transcription and present serious challenges to cellular DNA repair systems.36-39

Accordingly, Ap-derived cross-links have the potential to

contribute to aging, sporadic cancers, and the biological effects of the various xenobiotics that induce Ap sites in cellular DNA.36-40

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In earlier work, we proposed that dG-Ap cross-linking involves attachment of the exocyclic N2-amino group of dG to the anomeric carbon of the Ap residue (Scheme 3), but the chemical connectivity of the cross-link was not rigorously determined.21,22 For example, we recognized that the biochemical and mass spectrometric data could not rule out cross-link structures involving attachment of the Ap-aldehyde at an endocyclic nitrogen of dG or conversion of an initial imine adduct to the enamine 10a. Complete understanding of the molecular events underlying the biological consequences of any given DNA lesion requires precise knowledge of chemical structure. Chemical structure determination of a DNA lesion, in turn, requires full spectroscopic characterizations of the lesion.41 The results described here were designed to shed light on the chemical structure and properties of dG-Ap cross-links generated in duplex DNA. Toward this end, we characterized the nucleoside cross-link “remnant” released by enzymatic digestion of a DNA duplex containing the dG-Ap cross-link.21 We first carried out a chemical synthesis and complete spectroscopic structure determination of the putative nucleoside cross-link remnant 9b.

LC-MS/MS analysis was then used to

demonstrate that the properties of the authentic cross-link remnant released by enzymatic digestion of a DNA duplex containing the dG-Ap cross-link matched those of the synthetic material 9b. The reduced cross-link 11 (Scheme 3) was similarly characterized. The results establish the chemical connectivity of the dG-Ap cross-link released from duplex DNA and provide a foundation for detection of this lesion in biological samples. The cross-link remnant 9b was quite stable, decomposing to release dG with a half-life of approximately 17 d at pH 7 and 23 ˚C. NMR spectroscopic data suggests that the stability of this material is due to the fact that the 2-deoxyribose adduct connected at the

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N2-position of dG exists as a mixture of the ring-closed α-pyranose, β-pyranose, αfuranose, and β-furanose isomers, with no detectable amounts of the hydrolytically labile imine present (9b, Scheme 5).

The intrinsic stability of the cross-link attachment

observed in the nucleoside remnant was mirrored in the stability of the actual cross-link in duplex DNA, which dissociated with a half-life of 22 d at pH 7 and 23 ˚C. This suggests that the dG-Ap cross-link may have the power to block critical DNA-processing enzymes.

EXPERIMENTAL PROCEDURES Materials and Methods. Oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA). Uracil-DNA glycosylase (UDG, 5000 U/mL) and UDG buffer were purchased from New England Biolabs (Ipswich, MA). 2′-Deoxyguanosine (dG) monohydrate was purchased from Tokyo Chemical Industry Co., Ltd (Tokyo, Japan). The compounds 2-deoxy-D-ribose (99%), N,N-diisopropylethylamine (redistilled, 99.5%), and tetrabutylammonium fluoride (1 M in THF) were purchased from Sigma-Aldrich (St. Louis, MO). Iodotrimethylsilane (97%) was purchased from Acros Organics (Thermo Fisher) in a 5 g vial and stored at –15 °C in a desiccator; a small amount of metallic copper was added to the vial as a stabilizer. All other reagents used were purchased from Acros Organics in reagent grade and used without further purification unless otherwise noted. Bulk solvents (hexanes, ethyl acetate, and CH2Cl2) were obtained from Sigma-Aldrich. Methanol, THF, and H2O were purchased from Fisher in HPLC grade. When used as reaction solvents, CH2Cl2 and methanol were dried over 4 Å and 3 Å molecular sieves, respectively.

THF was distilled prior to use.

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Anhydrous DMF and dioxane were



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purchased in AcroSeal® bottles from Acros Organics.

Column chromatography was

performed on silica gel 60 (Sigma-Aldrich) under positive pressure. Glass-backed TLC plates with a 254 nm fluorescent indicator were purchased from Sigma-Aldrich and stored in a dessicator. Compounds on developed TLC plates were visualized using a 254 nm UV lamp or by dipping in a solution of 10% phosphomolybdic acid hydrate (PMA) in ethanol solution, followed by charring with a heat gun. Unless otherwise specified, reactions were conducted under an atmosphere of dry N2 gas. 1H and 13C NMR spectra were recorded on a Bruker DRX500 at 298 K in CDCl3, DMSO-d6 or D2O (Cambridge Isotope Laboratories, Inc.).

Synthesis of O6-(trimethylsilylethyl)-3′,5′-bis-O-(tert-butyldimethylsilyl)-2′deoxyguanosine (12). To a solution containing triphenylphosphine (1.91 g, 7.28 mmol) and 2-(trimethylsilyl)ethanol (1.04 ml, 7.28 mmol) in dry dioxane (12 mL) was added 3′,5′-bis-O-(tert-butyldimethylsilyl)-2′-deoxyguanosine (1.44 g, 2.91 mmol), which was prepared as described previously.42 Diisopropylazodicarboxylate (1.43 mL, 7.28 mmol) was added slowly by syringe, causing the white slurry to turn translucent yellow upon complete addition. After stirring for 2 h at 23 ˚C, the solvent was evaporated and the crude oil redissolved in diethyl ether (12 mL).

Triphenylphosphine oxide was

crystallized from the mixture by submersion of the flask in liquid N2 and then removed by vacuum filtration.

The filtrate was concentrated to a yellow oil and column

chromatography on silica gel eluted with 5:1 hexane/ethyl acetate gave 12 (1.18 g, 68%, Rf = 0.21 5:1 hexane/ethyl acetate) as a sticky yellow solid: 1H NMR (500 MHz, CDCl3) δ 7.86 (1H, s, H8), 6.30 (1H, t, J = 6.5, H1′), 4.92 (2H, br s, NH2), 4.59-4.56 (1H, m,

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H3′), 4.56-4.52 (2H, m, ROCH2CH2TMS), 3.96 (1H, q, J = 3.5, H4′), 3.79 (1H, dd, J = 4.5, 11, H5a′), 3.73 (1H, dd, J = 3.3, 11.3, H5b′), 2.55 (1H, dt, J = 6.5, 13.3, H2a′), 2.33 (1H, ddd, J = 3.5, 6, 13, H2b′), 1.24-1.19 (2H, m, ROCH2CH2TMS), 1.00-0.77 (18H, m, SiC(CH3)3), 0.08 (6H, s, Si(CH3)2), 0.06 (6H, s, ROEtSi(CH3)3), 0.06 (3H, s, ROEtSi(CH3)3), 0.05 (6H, s, Si(CH3)2); 13C NMR (126 MHz, CDCl3) δ 161.3 (C6), 159.2 (C2), 153.3 (C4), 137.3 (C8), 115.9 (C5), 87.6 (C4′), 83.5 (C1′), 71.9 (C3′), 64.8 (ROCH2CH2TMS), 62.8 (C5′), 40.8 (C2′), 25.9, 25.7 (SiC(CH3)3), 18.4, 18.0 (SiC(CH3)3), 17.5 (ROCH2CH2TMS), -1.5 (ROEtSi(CH3)3), -4.7, -4.8, -5.4, -5.6 (Si(CH3)2).

Synthesis

of

1,3,5-tris-O-(tert-butyldimethylsilyl)-2-deoxy-D-ribofuranose

(13). The compound 2-deoxy-D-ribose (1.50 g, 11.18 mmol), tert-butyldimethylsilyl chloride (5.60 g, 37.16 mmol), and imidazole (3.00 g, 44.07 mmol) were dissolved in anhydrous DMF (15 mL) and stirred at 23 ˚C. After 19 h, the reaction mixture was diluted with hexane (100 mL), washed with water (3 x 50 mL) and brine (1 x 50 mL), dried over Na2SO4, and the solvent evaporated under reduced pressure to give a light yellow oil. Column chromatography on silica gel eluted with 3% ethyl acetate in hexane gave 13 (3.83 g, 72%) as a colorless gel (Rf = 0.37 in 3% ethyl acetate-hexane): 1H NMR (500 MHz, CDCl3, α and β = anomeric isomers, p and f = pyranose and furanose isomers) δ 5.60 (0.40H, t, J = 4.3, H1, β-f), 5.46 (0.37H, dd, J = 2.5, 5, H1, α-f), 5.20 (0.21H, dd, J = 2.3, 5, H1, β-p), 4.75 (0.02H, dd, J = 2.5, 7.5, H1, α-p), 4.36 (0.40H, dt, J = 3.2, 5.1, H3, β-f), 4.18 (0.37H, dt, J = 4.8, 7.8, H3, α-f), 4.14-4.10 (0.02H, m, H3, α-p), 4.09-4.05 (0.21H, m, H3, β-p), 3.97 (0.37H, q, J = 4.3, H4, α-f), 3.89-3.86 (0.02H, m,

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H4, α-p), 3.84 (0.40H, ddd, J = 3, 5, 7.5, H4, β-f), 3.75-3.69 (0.42H, m, H4, H5a, β-p), 3.67-3.59 (1.37H, m, H5a, β-f; H5, α-f; H5b, β-p; H5a, α-p), 3.57 (0.40H, dd, J = 7.5, 10.5, H5b, β-f), 3.31 (0.02H, dd, J = 1.5, 12, H5b, α-p), 2.22 (0.37H, ddd, J = 5.4, 7.6, 13.1, H2a, α-f), 2.05-1.97 (1H, m, H2, β-f; H2a, β-p), 1.97-1.92 (0.02H, m, H2a, α-p), 1.82 (0.37H, ddd, J = 2.4, 4.4, 13.1, H2b, α-f), 1.72-1.68 (0.02H, m, H2b, α-p), 1.58 (0.21H, ddd, 3.5, 5, 12.5, H2b, β-p), 0.93-0.85 (27H, m, SiC(CH3)3), 0.11-0.03 (18H, m, Si(CH3)2); 13C NMR (126 MHz, CDCl3) δ 99.1 (C1, β-f), 98.5 (C1, α-f), 94.7 (C1, α-p), 93.0 (C1, β-p), 87.0 (C4, β-f), 85.4 (C4, α-f), 73.3 (C3, β-f), 71.9 (C3, α-f), 70.0 (C4, βp), 69.9 (C4, α-p), 69.4 (C3, α-p), 68.1 (C3, β-p), 65.8 (C5, α-p), 64.8 (C5, β-f), 64.7 (C5, α-f), 63.0 (C5, β-p), 44.6 (C2, β-f), 44.4 (C2, α-f), 38.7 (C2, β-p), 38.5 (C2, α-p), 26.0, 25.9, 25.8, 25.8, 25.7, 25.7 (SiC(CH3)3), 18.4, 18.3, 18.1, 18.0, 18.0, 18.0, 17.9, 17.9 (SiC(CH3)3), -4.1, -4.2, -4.2, -4.4, -4.5, -4.6, -4.7, -4.7, -4.7, -4.8, -4.8 (Si(CH3)2).

Synthesis of N2-[3,5-bis-O-(tert-butyldimethylsilyl)-2-deoxy-D-ribofuranos-1yl]-O6-benzyl-3′,5′-bis-O-(tert-butyldimethylsilyl)-2′-deoxyguanosine (14). Compound 13 (248 mg, 0.520 mmol) was dissolved in dry CH2Cl2 (2 mL) in an ovendried round-bottom flask. The flask was purged with dry N2 and cooled in a –78 °C dry ice-acetone bath. Iodotrimethylsilane (71 µL, 0.499 mmol) was added via syringe to generate the glycosyl iodide, and the resulting light yellow liquid stirred for 10 min. A solution of 12 (50 mg, 0.084 mmol) and diisopropylethylamine (DIPEA, 120 µL, 0.689 mmol) in dry CH2Cl2 (250 µL) was added by syringe and the mixture stirred for 15 min at –78 °C, then for an additional 15 min while warming to room temperature. Unreacted glycosyl iodide was quenched by the addition dry MeOH (4 mL). The mixture was

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evaporated to dryness under reduced pressure, redissolved in 7:1 hexane-ethyl acetate and washed with H2O to remove the diisopropylethylammonium hydroiodide salt.

The

organic layer was dried over Na2SO4 and evaporated under reduced pressure to yield a yellow oil. Column chromatography on silica gel eluted with 7:1 hexane-ethyl acetate (v/v) gave 14 (43 mg, 54%, Rf = 0.25) as a white foam: TOF-MS/ES+ 940.5687 M+; 1H NMR (500 MHz, CDCl3) δ 7.86 (0.2H, s, H8, β-f), 7.84 (0.8H, s, H8, α-f), 6.49 (0.8H, d, J = 10.5, NH, α-f), 6.37 (0.2H, m, H1′), 6.31 (0.8H, t, J = 6.5, H1′), 6.29-6.26 (0.2H, m, H1′′, β-f), 6.21 (0.8H, dd, J = 6.5, 10.5, H1′′, α-f), 5.39 (0.2H, d, J = 10, NH, β-f), 4.604.55 (1H, m, H3′), 4.55-4.47 (2H, m, OCH2CH2TMS), 4.45 (0.8H, d, J = 4.5, H3′′, α-f), 4.44-4.43 (0.2H, m, H3′′, β-f), 4.11 (0.8H, dd, J = 3.8, 7.3, H4′′, α-f), 3.98-3.91 (1H, m, H4′), 3.89 (0.2H, ddd, J = 2.4, 2.4, 4.6, H4′′, β-f), 3.79 (1H, dd, J = 5, 11, H5a′), 3.75 (1H, dd, J = 3.8, 11, H5b′), 3.72-3.67 (0.2H, m, H5a′′, β-f), 3.67 (0.8H, dd, J = 3.8, 10.8, H5a′′, α-f), 3.58 (0.2H, dd, J = 5, 10.5, 5b′′, β-f), 3.36 (0.8H, dd, J = 7.5, 10.5, H5b′′, α-f), 2.652.49 (1H, m, H2a′), 2.39-2.35 (0.2H, m, H2b′, β-f), 2.33 (0.8H, ddd, J = 3.9, 6.1, 13.1, H2b′, α-f), 2.27-2.20 (0.8H, m, H2a′′, α-f), 2.20-2.16 (0.2H, m, H2a′′, β-f), 1.99-1.95 (0.2H, m, H2b′′, β-f), 1.93 (0.8H, d, J = 13, H2b′′, α-f), 1.28-1.18 (2H, m, OCH2CH2TMS), 0.98-0.88 (36H, m, SiC(CH3)3), 0.15-0.04 (33H, m, Si(CH3)2; OEtSi(CH3)3);

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C NMR (126 MHz, CDCl3) δ 161.1 (C6), 157.9, 157.6 (C2), 153.3,

153.0 (C4), 137.5, 137.3 (C8), 116.3, 116.2 (C5), 87.7, 87.5 (C4′), 87.0 (C4′′α-f), 86.7 (C4′′, β-f), 84.1 (C1′), 83.6 (C1′′), 83.4 (C1′), 74.5 (C3′′, α-f), 73.1 (C3′′, β-f), 72.1 (C3′), 64.4 (OCH2CH2TMS), 63.9 (C5′′, β-f), 63.7 (C5′′, α-f), 63.0 (C5′), 26.0, 26.0, 25.9, 25.8, 25.8 (SiC(CH3)3), 18.4, 18.4, 18.3, 18.2, 18.0, 18.0, 18.0, 17.8, 17.5 (SiC(CH3)3), 17.5 (OCH2CH2TMS), -1.4 (OEtSi(CH3)3), -4.7, -4.7, -4.7, -4.8, -4.9, -5.3, -5.4, -5.5, -5.5, -5.5

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(Si(CH3)2).

NMR analysis indicated the presence of a small amount (

Chemical Structure and Properties of Interstrand Cross-Links Formed

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