Deoxyinosine-ethyl-N3-thymidine Interstrand Cross-Link

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Chem. Res. Toxicol. 2008, 21, 686–695

Synthesis and Characterization of DNA Containing an N -2′-Deoxyinosine-ethyl-N3-thymidine Interstrand Cross-Link: A Structural Mimic of the Cross-Link Formed by 1,3-Bis-(2-chloroethyl)-1-nitrosourea 1

Christopher J. Wilds,* Fei Xu, and Anne M. Noronha Department of Chemistry and Biochemistry, Concordia UniVersity, Montreal, Quebec, Canada H4B 1R6 ReceiVed NoVember 30, 2007

Interstrand cross-links, which are generated by chemotherapeutic treatment with bis-alkylating agents, exert their therapeutic effect by connecting the nucleobases of adjacent DNA strands together and represent some of the most threatening forms of damage suffered by genomic DNA. However, one of the reasons for treatment failure using these agents is due to enhanced repair of this DNA damage. The pursuit of understanding the repair of interstrand cross-links by repair systems has necessitated the synthesis of sufficient quantities of such damaged DNA. We report the synthesis of a site-specific interstrand crosslinked duplex containing an ethylene-bridged N1-2′-deoxyinosine-N3-thymidine base pair prepared by solution and solid-phase synthesis as a mimic for the lesion formed by the therapeutic agent 1,3-bis-(2chloroethyl)-1-nitrosourea using both a phosphoramidite and a bis-phosphoramidite approach. UV thermal denaturation experiments revealed that this cross-linked duplex was stabilized by 52 °C relative to the noncross-linked control, and circular dichroism studies indicated little deviation from a B-form structure compared to a duplex that contained a G-C base pair at the same position. Molecular models of the cross-linked duplex that were geometry optimized using the AMBER forcefield also suggest that this lesion induces minimal distortion in B-form DNA. This modified oligonucleotide will be useful for studies related to the investigation of interstrand cross-linked DNA repair. 1. Introduction Bifunctional alkylating agents (BFA) are a class of cancer chemotherapeutic agents that exert their therapeutic effect due to the intrinsic ability of these compounds to form interstrand cross-links (ICLs) within DNA (1). For example, the compound 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) has been shown to covalently link together the N3 atom of 2′-deoxycytidine with the N1 atom of 2′-deoxyguanosine via an ethyl linkage (2). Although they are some of the most pernicious threats to DNA, these very lesions are believed to be mandatory for the efficacy of BFA drug action because they affect both DNA strands and as a consequence interfere with the biological processes of DNA replication and transcription (1, 3). The exact mechanism by which interstrand cross-linked DNA is repaired in cancer cells, which can ultimately lead to treatment failure, continues to be elusive in mammalian systems. While nucleotide excision repair (NER) and recombination have been proposed for ICL repair in E. coli, repair in mammalian cells appears to be more complex with implication of both NER-dependent and independent releasing pathways and even the existence of direct competition among several mechanisms (4–6). In order to increase the efficacy of this category of cancer drugs through an understanding of their repair mechanisms, access to oligonucleotide substrates that contain a stable ICL at specific, unique sites in DNA is needed. In an effort to investigate how repair proteins distinguish interstrand crosslink lesions, the synthesis of clinically relevant interstrand crosslinked duplexes remains important in the armamentarium of * To whom correspondence should be addressed. Tel: 514-848-2424.ext.: 5798. E-mail: [email protected].

DNA repair. Two general approaches have emerged to produce these DNA lesions including the de noVo synthesis of unique site-specific ICLs (7–10) and the production of hybridization triggered ICLs whereby two complementary oligonucleotides are annealed, one of which contains a reactive cross-linked precursor that covalently links the two strands at a unique site after annealing (11, 12). Toward this end, the synthesis of an interstrand cross-link that bridges the N1 atom of 2′-deoxyinosine with the N3 atom of thymidine by an ethyl linkage has been developed (Figure 1). This particular cross-link serves as a structural mimic for the clinically relevant lesion that forms between a G-C base pair by BCNU. The replacement of 2′-deoxyguanosine by 2′deoxyinosine simplifies both solution and solid-phase synthesis procedures as 2′-deoxyinosine lacks an exocyclic amino group, eliminating the necessity of introducing a protecting group at this position. Inosine has been referred to as a universal pairing base because of its ability to base pair with all four nucleobases, with the pairing energies shown to be less dependent than other modified nucleosides (13). The replacement of 2′-deoxycytidine by thymidine allows for efficient alkylation of the N3 atom of the latter under basic conditions. N3-alkylcytosine may undergo a rearrangement to render the N4-substituted derivative (14). Additionally, it has been shown that 2′-deoxycytidine can react at the N3 atom with electrophilic agents such as (1-chloroethenyl)oxirane and undergo a deamination reaction to give the corresponding N3-2′-deoxyuridine adduct (15). Two approaches using solid-phase synthesis were explored to introduce the N12′-deoxyinosine-ethyl-N3-thymidine (N1I-ethyl-N3T) interstrand cross-link into DNA by the use of a phosphoramidite and bisphosphoramidite approach. Bis-phosphoramidites have been

10.1021/tx700422h CCC: $40.75  2008 American Chemical Society Published on Web 02/08/2008

N1-2′-Deoxyinosine-ethyl-N3-thymidine

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Figure 1. Structures of the (a) N1-2′-deoxyguanosine-ethyl-N3-2′-deoxycytidine cross-link induced by BCNU, (b) the N1-2′-deoxyinosine-ethylN3-thymidine cross-link, and oligomers (c) XL 1–1a and (d) XL 1–1b.

employed to produce nucleic acid molecules containing elaborate architectures including dendrimers and branched structures to probe RNA splicing (16–19). Sufficient amounts of interstrand cross-linked DNA material can be produced for biophysical characterization of this DNA lesion, which will ultimately allow for a correlation between structural features present in this molecule with recognition by various DNA repair systems to establish their respective roles in ICL repair.

2. Experimental Procedures 2.1. General Procedures. 5′-O-Dimethoxytrityl-2′-deoxyinosine and 5′-O-dimethoxytritylthymidine were purchased from ChemGenes Inc. (Wilmington, MA). All other chemicals and solvents were purchased from Aldrich Chemical Co. (Milwaukee, WI) and EMD Chemicals (San Diego, CA). Flash column chromatography was performed using silica gel 60 (230–400 mesh) as the adsorbent obtained from Silicycle (Quebec City, QC). Thin layer chromatography (TLC) was performed using precoated TLC plates (Merck, Kieselgel 60 F254, 0.25 mm) purchased from EM Science (Gibbstown, NJ). 5′-O-Dimethoxytrityl-3′-O-phenoxyacetyl-thymidine was synthesized according to published protocols (20). NMR spectra were recorded on a Varian INOVA 300 MHz NMR spectrometer at room temperature. 1H NMR spectra were recorded at a frequency of 300.0 MHz, and chemical shifts were reported in parts per million downfield from tetramethylsilane. 31P NMR spectra (1H decoupled) were recorded at a frequency of 121.5 MHz with H3PO4 used as an external standard. ESI mass spectra for oligonucleotides were obtained at the Concordia University Centre for Biological Applications of Mass Spectrometry (CBAMS) using a Micromass Qtof2 mass spectrometer (Waters) equipped with a nanospray ion source. The mass spectrometer was operated in full scan, negative ion detection mode. ESI mass spectra for small molecules were recorded at the McGill University Department of Chemistry with a Finnigan LCQ DUO mass spectrometer in methanol or acetone. 2.2. Oligonucleotide Preparation and Purification. 5′-ODimethoxytrityldeoxyribonucleoside-3′-O-(β-cyanoethyl-N,N′diisopropyl) phosphoramidites and protected deoxyribonucleoside controlled pore glass (CPG) supports (500 Å) were purchased from ChemGenes Inc. (Wilmington, MA). Protected deoxyribonucleoside polystyrene supports were purchased from Applied Biosystems (CA). The cross-linked duplexes, whose sequences are shown in Figure 1, were assembled on an Applied Biosystems Model 3400 synthesizer on a 1 µmol scale using standard β-cyanoethylphosphoramidite chemistry supplied by the manufacturer with slight modifications to coupling times.

Polystyrene solid support was used for the synthesis of XL 1–1a and CPG for XL 1–1b. The nucleoside phosphoramidites containing standard protecting groups were prepared in anhydrous acetonitrile at a concentration of 0.1 M for the 3′-O-deoxyphosphoramidites, 0.05 M for the bis-cross-linked 3′-O-deoxyphosphoramidite, 0.15 M for the cross-linked 3′-O-deoxyphosphoramidite, and 0.2 M for the 5′-O-deoxyphosphoramidites. Assembly of sequences was carried out as follows: (a) detritylation, 3% trichloroacetic acid in dichloromethane; (b) nucleoside phosphoramidite coupling time of 2 min for commercial 3′-O-deoxyphosphoramidites, 3 min for 5′-O-deoxyphosphoramidites, 10 min for the cross-linked phosphoramidite 7 and 30 min for the cross-linked bis-phosphoramidite 8; (c) capping, acetic anhydride/pyridine/ tetrahydrofuran 1:1:8 (v/v/v; solution A) and 1-methyl-imidazole/ tetrahydrofuran 16:84 (w/v; solution B); (d) oxidation, 0.02 M iodine in tetrahydrofuran/water/pyridine 2.5:2:1. For the synthesis of XL 1–1a, the cyanoethyl groups were removed from the polystyrene-linked oligomers by treating the support with 1 mL of anhydrous triethylamine (TEA) for at least 12 h. The support was washed with 30 mL of anhydrous acetonitrile followed by anhydrous tetrahydrofuran. The tert-butyldimethylsilyl group was removed from the partial duplex by treating the support with 2 × 1 mL triethylamine trihydrofluoride for a total of 1 h. The support was then washed with 30 mL of anhydrous tetrahydrofuran and 30 mL of acetonitrile and then dried via high vacuum (20 min). The last segment of the duplex was then synthesized using 5′-Odeoxyphosphoramidites with a total detritylation exposure of 130 s. The 3′-terminal trityl group was removed by the synthesizer. The oligomer-derivatized supports were transferred from the reaction column to screw cap microfuge tubes fitted with Teflon lined caps, and the protecting groups were removed by treatment with a mixture of concentrated ammonium hydroxide/ethanol (0.3 mL: 0.1 mL) for 4 h at 55 °C. The cross-linked duplexes were purified by SAX HPLC using a Dionex DNAPAC PA100 column (0.4 cm × 25 cm) purchased from Dionex Corp, Sunnyvale, CA using a linear gradient of 0–50% buffer B over 30 min (buffer A: 0.1 M Tris HCl, pH 7.5, 10% acetonitrile and buffer B: 0.1 M Tris HCl, pH 7.5, 1 M NaCl, 10% acetonitrile) at 50 °C. The columns were monitored at 260 nm for analytical runs or 290 nm for preparative runs. The purified oligomers were desalted using C-18 SEP PAK cartridges (Waters Inc.) as previously described (21). The amounts of purified oligomers obtained are shown in Table 1. The cross-linked oligomers (0.1 A260 units) were characterized by digestion with a combination of snake venom phosphodiesterase (0.28 units) and calf intestinal phosphatase (5 units) in a buffer containing 10 mM Tris (pH 8.1) and 2 mM magnesium

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Table 1. Amounts, retention times, nucleoside ratios and mass spectral data for cross-linked duplexes nucleoside ratios a

b

mass

cross-linked duplex

A260 units

retention time (minutes)

nucleoside composition

expected

observed

expected

observed

XL 1–1a

116 (46.8)

26.0

24.5

1.00 1.01 1.19 1.22 0.27 1.00 0.99 1.36 1.42 0.27

6080.2

75 (19)

1.00 1.00 1.25 1.25 0.25 1.00 1.00 1.50 1.50 0.25

6080.0

XL 1–1b

dC dG dT dA dI-dT dC dG dT dA dI-dT

6697.0

6697.4

a Amount of crude cross-linked duplex purified by SAX HPLC. The numbers in parentheses indicate the amount of pure duplex obtained. b Retention times (minutes) of cross-linked duplexes on SAX HPLC using a 0.0–0.5 M linear gradient of sodium chloride.

chloride for 16 h at 37 °C as previously described (21). The resulting mixture of nucleosides was analyzed by reversed phase HPLC carried out using a Symmetry C-18 5 µm column (0.46 × 15 cm) purchased from Waters Inc., Milford, MA. The C-18 column was eluted with a linear gradient of 0–30% buffer B over 30 min (buffer A, 50 mM sodium phosphate, pH 5.8 and buffer B, 50 mM sodium phosphate, pH 5.8, 50% acetonitrile). The resulting peaks were identified by coinjection with the corresponding standards and eluted at the following times: dC (4.3 min), dG (7.4 min), dT (8.0 min), dA (9.1 min), and crosslink dimer (12.4 min), and the ratio of nucleosides was determined. The results are given in Table 1. The molecular weights of the cross-linked oligomers were determined by ESI MS, and these were in agreement with the calculated values. Denaturing polyacrylamide gel electrophoresis (PAGE) was carried out on 14 cm × 16 cm × 0.75 cm gels containing 20% acrylamide and 7 M urea in TBE, which contained 89 mM Tris, 89 mM boric acid, and 0.2 mM ethylenediaminetetraacetate. The running buffer was TBE. 2.3. UV Thermal Denaturation Studies. Molar extinction coefficients for the oligonucleotides and cross-linked duplex were calculated from those of the mononucleotides and dinucleotides according to nearest-neighbor approximations (M-1 cm-1) (22). Noncross-linked duplexes were prepared by mixing equimolar amounts of the interacting strands and lyophilizing the resulting mixture to dryness. The pellet was then redissolved in 90 mM sodium chloride, 10 mM sodium phosphate, and 1 mM EDTA buffer (pH 7.0) to give a final concentration of 4.5 µM duplex. The cross-linked duplexes were dissolved in the same buffer to give a final concentration of 2.3 µM. The solutions were then heated to 85 °C for 15 min, cooled slowly to room temperature, and stored at 4 °C overnight before measurements. Prior to the thermal run, samples were degassed by placing them in a speed-vac concentrator for 2 min. Denaturation curves were acquired at 260 nm at a rate of heating of 0.5 °C/min, using a Varian CARY Model 3E spectrophotometer fitted with a 6-sample thermostatted cell block and a temperature controller. The data were analyzed in accordance with the convention of Puglisi and Tinoco (22) and transferred to Microsoft Excel. 2.4. Circular Dichroism (CD) Spectroscopy. Circular dichroism spectra were obtained on a Jasco J-815 spectropolarimeter equipped with a Julaba F25 circulating bath. Samples were allowed to equilibrate for 5–10 min at 10 °C in 90 mM sodium chloride, 10 mM sodium phosphate, 1 mM EDTA (pH 7.0), at a final concentration of 2.3 µM for the cross-linked duplexes and ca. 4.5 µM for control duplexes. Each spectrum was an average of 5 scans. Spectra were collected at a rate of 100 nm/ minute with a bandwidth of 1 nm and sampling wavelength of 0.2 nm using fused quartz cells (Starna 29-Q-10). The CD

spectra were recorded from 350 to 200 nm at 10 °C. The molar ellipticity was calculated from the equation [φ] ) ε/Cl, where ε is the relative ellipticity (mdeg), C is the molar concentration of oligonucleotides (mol/L), and l is the path length of the cell (cm). The data were processed on a PC using Windows based software supplied by the manufacturer (JASCO, Inc.) and transferred into Microsoft Excel for presentation. 2.5. Molecular Modeling. DNA duplexes ((5′-dCGAAACTTTCG)/(5′-CGAAAGTTTCG) and (5′-dCGAAATTTTCG)/ (5′-CGAAAITTTCG)) and cross-linked duplex XL 1–1b were built using HyperChem molecular modeling software. All duplexes were geometry optimized using the AMBER forcefield. 2.6. Synthesis of Modified Nucleosides. 2.6.1. 5′-O-Dimethoxytrityl-3′-O-tert-butyldimethylsilyl-2′-deoxyinosine (1). To a solution of 5′-O-dimethoxytrityl-2′-deoxyinosine (2.00 g, 3.66 mmol) in anhydrous DMF (38 mL) was added imidazole (1.08 g, 15.9 mmol) followed by tert-butyldimethylsilyl chloride (1.14 g, 7.56 mmol), and the mixture was allowed to stir at room temperature for 12 h. The solvent was removed in vacuo and the crude material taken up in ethyl acetate (100 mL) and washed twice with 5% sodium bicarbonate (100 mL). The organic layer was then dried over Na2SO4 and evaporated. The crude material was purified by silica gel column chromatography using ethyl acetate/methanol (99:1) as eluent to yield the product, which was a colorless foam (2.48 g, 94%). Rf (SiO2): 0.49 in dichloromethane/methanol (4:1). 1H NMR (300 MHz, d6DMSO, ppm): 8.21 (s, 1 H, H2), 7.96 (s, 1 H, H8), 7.29–6.75 (m, 13 H, DMT), 6.30–6.26 (dd, 1 H, J ) 6.3, 6.3 Hz, H1′), 4.64–4.58 (m, 1 H, H3′), 3.86–3.81 (m, 1 H, H4′), 3.68 (s, 6 H, OCH3), 3.22–3.00 (m, 2 H, H5′ and H5′′), 2.82–2.76 (m, 1H, H2′′), 2.33–2.26 (m, 1 H, H2′), 0.77 (s, 9 H, Si-C(CH3)3), 0.00 (s, 3H, Si-CH3), -0.05 (s, 3 H, Si-CH3). ESI-MS: (calcd for C37H44N4O6SiNa): 691.3; found (691.2 M + Na+). 2.6.2. N1-(2-Chloroethyl)-5′-O-dimethoxytrityl-3′-O-(tertbutyldimethylsilyl)-2′-deoxyinosine (2). To a solution of 1 (1.40 g, 2.09 mmol) in DMF (12 mL), was added Cs2CO3 (2.04 g, 6.28 mmol). The mixture was stirred at room temperature for 30 min, and then 1-bromo-2-chloroethane (0.90 g, 6.28 mmol) was added according to a procedure published by Schärer et al. (23). The reaction mixture was stirred at room temperature for 4 h and was then evaporated. The crude material was dissolved in ethyl acetate (100 mL), washed with a saturated solution of NH4Cl (100 mL), water (100 mL), and brine (100 mL), and then dried over Na2SO4. Purification by silica gel column chromatography using hexanes/ethyl acetate (3:1) as eluent produced compound 2 (1.25 g, 82%) as a colorless foam. Rf (SiO2): 0.36 in hexanes/ethyl acetate (2:8). 1H NMR (300 MHz, d6-DMSO, ppm): 8.37 (s, 1 H, H2), 8.31 (s, 1 H, H8), 7.35–6.81 (m, 13 H, DMT), 6.35–6.31 (dd, 1 H, J ) 6.3, 6.3 Hz, H1′), 4.66–4.60 (m, 1 H, H3′), 4.39–4.35 (t, 2 H, J ) 5.7


Hz, -CH2-N), 3.97–3.87 (m, 3 H, -CH2-Cl and H4′), 3.73 (s, 6 H, OCH3), 3.28–3.10 (m, 2 H, H5′ and H5′′), 2.88–2.78 (m, 1H, H2′), 2.39–2.30 (m, 1 H, H2′′), 0.825 (s, 9 H, Si-C(CH3)3), 0.04 (s, 3H, Si-CH3), 0.00 (s, 3 H, Si-CH3). ESI-MS: (calcd for C39H47N4O6SiClNa): 753.3; found (753.2 M + Na+). 2.6.3. N1-(2-Iodoethyl)-5′-O-dimethoxytrityl-3′-O-(tertbutyldimethylsilyl)-2′-deoxyinosine (3). To a stirred solution of 2 (1.22 g, 1.66 mmol) in acetonitrile (17 mL) was added NaI (1.00 g, 6.6 mmol) at room temperature. The reaction mixture was stirred at 70 °C for 18 h, cooled to room temperature, and the solvent removed in vacuo. The crude material was taken up in CH2Cl2 (100 mL), washed with sodium thiosulfate (10%, 3 × 50 mL), dried over Na2SO4, and evaporated to dryness. Purification by silica gel column chromatography using hexanes/ethyl acetate (3:1) as eluent produced 3 (0.775 g, 57%) as an off white foam. Rf (SiO2): 0.69 in hexanes/ethyl acetate (1:9) 1H NMR (300 MHz, d6-DMSO, ppm): 8.41 (s, 1 H, H2), 8.33 (s, 1 H, H8), 7.36–6.84 (m, 13 H, DMT), 6.38–6.34 (dd, 1 H, J ) 5.7, 5.7 Hz, H1′), 4.69–4.63 (m, 1 H, H3′), 4.40–4.35 (t, 2 H, J ) 6.8 Hz, -CH2-N), 3.94–3.91 (m, 1 H, H4′), 3.76 (s, 6 H, OCH3), 3.57–3.52 (t, 2H, J ) 6.8 Hz, -CH2-I), 3.30–3.13 (m, 2 H, H5′ and H5′′), 2.89–2.81 (m, 1H, H2′), 2.42–2.33 (m, 1 H, H2′′), 0.85 (s, 9 H, Si-C(CH3)3), 0.08 (s, 3 H, Si-CH3), 0.03 (s, 3 H, Si-CH3). ESI-MS: (calcd for C39H47N4O6SiINa): 845.2; found (845.2 M + Na+). 2.6.4. 1-{N1-[3′-O-tert-Butyldimethylsilyl-5′-O-dimethoxytrityl-2′-deoxyinosinyl]}-2-{N 3 -[5′-O-dimethoxytritylthymidyl]}-3′-O-phenoxyacetyl]}-ethane (4). To a solution containing compound 3 (0.870 g, 1.06 mmol), and 5′-Odimethoxytrityl-3′-O-phenoxyacetyl-thymidine (0.750 g, 0.885 mmol) in anhydrous acetonitrile (8 mL) was added DBU (0.189 g, 1.24 mmol), and the reaction was allowed to stir at room temperature for 12 h. The solvent was then removed in vacuo, and the crude material taken up in CH2Cl2 (50 mL) and washed with sodium bicarbonate (5%, 50 mL). The organic layer was then dried over Na2SO4 and evaporated. The material was purified by silica gel column chromatography using hexanes/ ethyl acetate (35:65) as eluent to yield the product (0.595 g, 49%) as a colorless foam. Rf (SiO2): 0.58 in hexanes/ethyl acetate (1:9). 1H NMR (300 MHz, d6-DMSO, ppm): 8.26 (s, 1 H, H2), 8.23 (s, 1 H, H8), 7.57 (s, 1H, H6), 7.41–6.82 (m, 31 H, DMT, Pac), 6.34–6.29 (dd, 1 H, J ) 6.3, 6.3 Hz, H1’a), 6.20–6.14 (dd, 1H, J ) 6.0, 7.2 Hz, H1’b), 5.41–5.37 (m, 1H, H3′b), 4.81 (s, 2H, -CH2OAr), 4.61–4.56 (m, 1H, H3′a), 4.21–4.14 (m, 5 H, -N-CH2-CH2-N, H4’b), 3.92–3.88 (m, 1 H, H4’a), 3.75 (s, 12 H, OCH3), 3.28–3.12 (m, 4 H, H5′a, H5′′a, H5′b, H5′′b), 2.79–2.72 (m, 1H, H2’a), 2.49–2.30 (m, 3H, H2′′a, H2’b and H2′′b), 1.41 (s, 3H, C5-CH3), 0.83 (s, 9 H, SiC(CH3)3), 0.04 (s, 3 H, Si-CH3), 0.00 (s, 3 H, Si-CH3). ESIMS: (calcd for C78H84N6O15SiNa): 1395.6; found (1395.5 M + Na+). 2.6.5. 1-{N1-[3′-O-tert-Butyldimethylsilyl-5′-O-dimethoxytrityl-2′-deoxyinosinyl]}-2-{N 3 -[5′-O-dimethoxytritylthymidyl]}-ethane (5). Compound 4 (0.593 g, 0.432 mmol) was dissolved in dichloromethane (50 mL) and excess propylamine (7.20 g, 122.0 mmol) was added. After 12 h, the solvents were removed in vacuo, the crude product was taken up in dichloromethane (100 mL), and the solution was washed with three portions of sodium bicarbonate (3%, 100 mL). The organic layer was dried over Na2SO4 and evaporated to afford the crude product, which was purified via column chromatography using hexanes/ethyl acetate (2:3) as eluent to afford compound 5 (0.471 g, 88%) as a colorless foam. Rf (SiO2): 0.25 in hexanes/ ethyl acetate (2:8). 1H NMR (300 MHz, d6-DMSO, ppm): 8.26


(s, 1 H, H2), 8.21 (s, 1 H, H8), 7.55 (s, 1H, H6), 7.42–6.81 (m, 26 H, DMT), 6.33–6.29 (dd, 1 H, J ) 6.0, 6.0 Hz, H1’a), 6.14–6.09 (dd, 1H, J ) 6.6, 6.6 Hz, H1’b), 5.35–5.33 (d, 1H, J ) 4.8 Hz, OHb), 4.61–4.57 (m, 1H, H3′b), 4.32–4.20 (m, 5 H, -N-CH2-CH2-N, H3′b), 3.91–3.89 (m, 2 H, H4’a, H4’b), 3.73 (s, 12 H, OCH3), 3.28–3.12 (m, 4 H, H5′a, H5′′a, H5′b, H5′′b), 2.80–2.72 (m, 1H, H2’a), 2.38–2.10 (m, 3H, H2′′a, H2’b and H2′′b), 1.40 (s, 3H, C5-CH3), 0.83 (s, 9 H, Si-C(CH3)3), 0.05 (s, 3 H, Si-CH3), 0.00 (s, 3 H, Si-CH3). ESI-MS: (calcd for C70H78N6O13SiNa): 1261.5; found (1261.4 M + Na+). 2.6.6. 1-{N1-[5′-O-Dimethoxytrityl-2′-deoxyinosinyl]}-2-{N3[5′-O-dimethoxytrityl-thymidyl]}-ethane (6). Compound 5 (0.640 g, 0.517 mmol) was dissolved in THF (5.5 mL), and TBAF (1 M in THF, 0.620 mL, 0.620 mmol) was added. After 30 min, the solvent was removed in vacuo, the crude material was taken up in dichloromethane (50 mL), and the solution was washed with three portions of sodium bicarbonate (3%, 50 mL). The organic layer was dried over Na2SO4 and evaporated to afford the crude product, which was purified via column chromatography using a gradient of ethyl acetate to ethyl acetate/ethanol (19:1) to afford compound 6 (0.553 g, 95%) as a colorless foam. Rf (SiO2): 0.35 in ethyl acetate/ethanol (19:1). 1H NMR (300 MHz, d6-DMSO, ppm): 8.24 (s, 1 H, H2), 8.22 (s, 1 H, H8), 7.58 (s, 1H, H6), 7.45–6.83 (m, 26 H, DMT), 6.38–6.33 (dd, 1 H, J ) 6.3, 6.3 Hz, H1′a), 6.17–6.13 (dd, 1H, J ) 6.3, 6.3 Hz, H1′b), 5.44–5.42 (d, 1H, J ) 4.5 Hz, C3′a-OH), 5.38–5.36 (d, 1H, J ) 4.8 Hz, C3′b-OH), 4.44–4.22 (m, 6 H, -N-CH2-CH2N, H3′a, H3′b), 4.04–4.01 (m, 1 H, H4’a), 3.94–3.91 (m, 1 H, H4′b), 3.76 (s, 12 H, OCH3), 3.25–3.15 (m, 4 H, H5′a, H5′′a, H5′b, H5′′b), 2.78–2.69 (m, 1H, H2′a), 2.41–2.08 (m, 3H, H2′′a, H2′b and H2′′b), 1.42 (s, 3H, C5-CH3). ESI-MS: (calcd for C64H64N6O13Na): 1147.4; found (1147.4 M + Na+). 2.6.7. 1-{N1-[3′-O-tert-Butyldimethylsilyl-5′-O-dimethoxytrityl-2′-deoxyinosinyl]}-2-{N 3 -[5′-O-dimethoxytritylthymidyl]}-3′-O-(β-cyanoethyl-N,N′-diisopropyl)phosphoramidite]}-ethane (7). Compound 5 (0.300 g, 0.242 mmol) was dissolved in tetrahydrofuran (1 mL), and diisopropylethylamine (0.053 g, 0.411 mmol) was added followed by N,N-diisopropylamino cyanoethyl phosphonamidic chloride (0.086 g, 0.363 mmol). The reaction was allowed to stir at room temperature for 2 h. Upon completion, the reaction was quenched by the addition of ethyl acetate (25 mL), and the solution was washed with sodium bicarbonate (3%, 25 mL). The organic layer was dried over sodium sulfate, filtered, and evaporated. The product, a colorless powder, was precipitated from hexanes (0.256 g, 73%). Rf (SiO2): 0.50, 0.61 in hexanes/ethyl acetate (2:8). 31P NMR (161.8 MHz, d6-acetone, ppm): 149.33, 149.29. ESI-MS: (calcd for C79H95N8O14SiPNa): 1461.6; found (1461.6 M + Na+). 2.6.8. 1-{N1-[5′-O-Dimethoxytrityl-2′-deoxyinosinyl]}-3′-O(β-cyanoethyl-N,N′-diisopropyl) phosphoramidite]}-2-{N3[5′-O-dimethoxytrityl-thymidyl]}-3′-O-(β-cyanoethyl-N,N′diisopropyl)phosphoramidite]}-ethane (8). Compound 6 (0.132 g, 0.117 mmol) was dissolved in tetrahydrofuran (0.5 mL), and diisopropylethylamine (0.053 g, 0.411 mmol) was added followed by N,N-diisopropylamino cyanoethyl phosphonamidic chloride (0.083 g, 0.352 mmol). The reaction was allowed to stir at room temperature for 30 min. Upon completion, the reaction was quenched by the addition of ethyl acetate (25 mL), and the solution was washed with sodium bicarbonate (3%, 25 mL). The organic layer was dried over sodium sulfate, filtered, and evaporated. The product, a colorless powder, was precipitated from hexanes (0.140 g, 78%). Rf (SiO2): 0.52, 0.60, 0.66 in hexanes/ethyl acetate/ethanol (3:6.5:0.5). 31P NMR (161.8

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Scheme 1. Synthesis of N1-2′-Deoxyinosine-ethyl-N3-thymidine phosphoramidite (7) and Bis-phosphoramidite (8) a

a Conditions and reagents: (i) tert-Butyldimethylsilyl chloride, imidazole, DMF, 12 h, 94%; (ii) Cs2CO3, 1-bromo-2-chloro-ethane, DMF, 4 h, 82%; (iii) NaI, acetonitrile, 18 h, 70°C, 57%; (iV) 5′-O-dimethoxytrityl-3′-O-phenoxyacetyl-thymidine, DBU, acetonitrile, 12 h, 49%; (V) propylamine, CH2Cl2, 12 h, 88%; (Vi) N,N-diisopropylamino cyanoethyl phosphonamidic chloride, DIPEA, THF, 2 h, 73%; (Vii) Tetrabutylammonium fluoride (1 M in THF), 30 minutes, 95%; (Viii) N,N-diisopropylamino cyanoethyl phosphonamidic chloride, DIPEA, THF, 30 minutes, 78%.

MHz, d6-acetone, ppm): 149.35, 149.28. ESI-MS: (calcd for C82H98N10O15P2Na): 1547.6; found (1547.6 M + Na+).

3. Results 3.1. Syntheses and Characterization of Cross-Linked Duplexes. The structures of the N1I-ethyl-N3T cross-link dimer and the cross-linked duplexes XL 1–1a and XL 1–1b are shown in Figure 1. The synthesis of the cross-linked phosphoramidite 7 and bis-phosphoramidite 8 are shown in Scheme 1. Starting from commercially available 5′-O-dimethoxytrityl-2′-deoxyinosine, the free 3′-alcohol was protected as a tert-butyldimethylsilyl ether to produce 1 in 94% yield. Then, regioselective alkylation at the N1 position was accomplished with 1-bromo2-chloroethane in the presence of Cs2CO3 to produce the chlorinated adduct 2 (24, 25). Replacement of the chloride with

an iodide atom was performed with the use of excess NaI at elevated temperatures (70 °C) to produce iodinated adduct 3. The formation of dimer 4 was achieved by alkylating a slight excess of 5′-O-dimethoxytrityl-3′-O-phenoxyacetyl-thymidine at the N3 atom with iodinated adduct 3 in the presence of DBU overnight to afford the desired dimer in 49% yield. Dimer 4 can then be used to produce either phosphoramidite 7 or bisphosphoramidite 8. Removal of the 3′-O-phenoxyacetyl group from the thymidine nucleoside of dimer 4 with excess propylamine gave dimer 5 in 88% yield. This dimer was converted to phosphoramidite 7 using 1.5 equivalents of N,N-diisopropylamino cyanoethyl phosphonamidic chloride, which was then isolated following hexane precipitation. The isolated phosphoramidite, analyzed by mass spectrometry, gave the expected molecular weight, and



Figure 2. Methodology to construct the cross-linked duplexes XL 1–1a and XL 1–1b via solid phase synthesis using the mono and bis-phosphoramidite approaches. (a) Monophosphoramidite approach with (i) coupling of amidite 7; (ii) extension with 3′-O-deoxyphosphoramidites followed by capping with acetic anhydride; (iii) removal of the 3′-O-tert-butyldimethylsilyl group with TEA·3HF; and (iV) extension with 5′-O-deoxyphosphoramidites and cleavage from the solid support. (b) Bis-phosphoramidite approach with (i) coupling of bis-phosphoramidite 8; (ii) extension with 3′-Odeoxyphosphoramidites; and (iii) cleavage from the solid support. 31 P NMR studies revealed the presence of two signals at 149.33 and 149.29 ppm, in the region diagnostic for a phosphoramidite. The removal of the 3′-O-tert-butyldimethylsilyl group of the 2′-deoxyinosine moiety of dimer 5 using TBAF (1 M) afforded dimer 6 in 95% yield. Dimer 6 was then phosphitylated with excess N,N-diisopropylamino cyanoethyl phosphonamidic chloride (3.0 equivalents) to give bis-phosphoramidite 8 in 78% yield after hexane precipitation. Two different solid phase synthetic strategies were used to prepare the cross-linked duplexes. In both cases, the syntheses were carried out on a 1 µmol scale. The strategy to produce the cross-linked duplex XL 1–1a using phosphoramidite 7 is shown in Figure 2a. The first arm of the duplex was synthesized on a polystyrene support using commercially available 3′-O-deoxyphosphoramidites. The N3T-ethyl-N1I cross-link phosphoramidite 7 was used to introduce the cross-link at the 5′-end of the oligomer to give XL-a. The dimethoxytrityl groups were removed by a brief treatment with 3% trichloroacetic acid in methylene chloride. The second and third arms of the duplex were then synthesized simultaneously by repetitive coupling with protected 3′-O-deoxyphosphoramidites to give, after detritylation and acetylation, the branched XL-b. The 3′-O-tert-butyldimethylsilyl (TBDMS) group was then removed from XL-b by treating the support with anhydrous triethylamine trihydrofluoride in tetrahydrofuran (26). The full length cross-linked duplex XL 1–1a was prepared by repetitive coupling of the 3′-end of intermediate XL-c with 5′-O-deoxyphosphoramidites at a concentration of 0.2 M and coupling time of 3 min. Another approach for the synthesis of the N1I-ethyl-N3T crosslink is illustrated in the synthesis of XL 1–1b, which involves the use of bis-phosphoramidite 8 via the strategy shown in Figure 2b. In this approach, two linear oligonucleotide chains on CPG support were coupled simultaneously at the 5′-end of the two oligomer chains. The dimethoxytrityl groups were removed by a brief treatment with 3% trichloroacetic acid in methylene chloride. Chain extension from both free hydroxyls

was continued simultaneously by repetitive coupling with 3′O-deoxyphosphoramidites to give, after detritylation, the desired XL 1–1b. The cross-linked oligomers were deprotected by treating both supports with a mixture of concentrated ammonium hydroxide/ ethanol (3:1) at 55 °C for 4 h. The duplexes were purified by strong anion exchange (SAX) HPLC using a sodium chloride gradient in buffer that contained 100 mM Tris-HCl and 10% acetonitrile (see Supporting Information). Using the monophosphoramidite strategy, the major product is the fully cross-linked oligonucleotide XL 1–1a. In the case of the bis-phosphoramidite approach, the major side product from the synthesis was the deprotected form of Y, which resulted from incomplete coupling of both phosphoramidite moieties to the linear chains and is itself a mixture of two species (see Supporting Information). This Y-like molecule has been confirmed to be a result of incomplete coupling of both phosphoramidite groups to the linear oligomers on the CPG and not that of incomplete extension after introduction of the cross-link by both mass spectrometry analysis and enzymatic digestion. The PAGE analysis (see Supporting Information) highlights a few important observations for the synthesis of this crosslink via the bis-phosphoramidite approach. Lanes 1, 5, and 6 are standard single strand DNA markers, which contain 20, 11, and 17 nucleotides, respectively. The crude deprotection products are shown in lane 2 with two obvious major products. Despite the fact that Y (lower band) may be partially doublestranded, its mobility is similar to that of the 17-nucleotide marker in lane 6, which is consistent with single-stranded oligomer control containing the same number of bases. The duplexes, whose retention times are shown in Table 1, were purified by SAX HPLC. Analysis of the purified oligomers by SAX HPLC revealed that the oligomers were single peaks and were obtained in approximately 40 and 25% overall yields for the phosphoramidite and bis-phosphoramidite approaches,

692


Figure 3. Absorbance (A260) versus temperature profiles of cross-linked duplex XL 1–1b (s), 5′-dCGAAAGTTTCG-3′/5′-dCGAAACTTTCG3′ containing a central G-C base pair (---), and 5′-dCGAAAITTTCG3′/5′-dCGAAATTTTCG-3′ containing a central I-T mismatch (···). Solutions containing a total strand concentration of 2.3 µM for the crosslinked duplex XL 1–1b and 4.5 µM of the noncross-linked control duplexes in 90 mM sodium chloride, 10 mM sodium phosphate, and 1 mM ethylenediaminetetraacetate buffer at pH 7.0 were heated at 0.5 °C/minute.

respectively. Analysis of these HPLC purified cross-linked duplexes via denaturing PAGE serve to confirm the purity of the isolated cross-linked duplexes. In order to characterize these duplexes, digestion to the constituent nucleosides with a combination of snake venom phosphodiesterase and calf intestinal phosphatase was performed and analyzed by C-18 reversed phase HPLC (see Supporting Information). As shown in Table 1, the ratio of the component deoxynucleosides and the N1I-ethyl-N3T cross-linked nucleoside was consistent with the theoretical composition of the particular duplex. However, compared to linear, unmodified oligonucleotides, the cross-linked oligomers required additional time for digestion (16 h as opposed to 30 min). In addition to this characterization, the duplexes were analyzed by ESI mass spectrometry with the resulting masses consistent with the theoretical values for the cross-linked duplexes. 3.2. UV Thermal Denaturation Studies. Ultraviolet thermal denaturation experiments were carried out to assess the effect of the ethyl linker on duplex stability. The thermal denaturation (Tm) curves for cross-linked duplex XL 1–1b, along with various noncross-linked control duplexes, are shown in Figure 3. All of the duplexes, including cross-linked XL 1–1b, exhibited a sigmoidal denaturation profile, suggesting that these duplexes denature in a highly cooperative manner. The overall hyperchromicities of the transition curves of the cross-linked duplex and the noncross-linked controls are approximately the same. From the denaturation curve of XL1–1b, a Tm value of 78 °C was observed, which is higher than the control duplexes containing an I-T mismatch and a G-C base pair by approximately 52 and 35 °C, respectively. 3.3. Circular Dichroism Spectroscopy and Molecular Modeling Studies. The circular dichroism (CD) spectra of crosslinked duplex XL 1–1b and the noncross-linked controls containing a central G-C base pair and a central I-T mismatch were recorded at 10 °C (Figure 4). Under these conditions, both the cross-linked and noncross-linked oligomers should be in their base-paired, duplex form as shown by the thermal denaturation profiles (Figure 3). In all cases, the CD spectra of the duplexes exhibited signatures characteristic of B-form DNA with a positive maximum peak centered around 280 nm, a negative peak at approximately 245 nm, and a positive peak in the region

Wilds et al.

Figure 4. Circular dichroism spectra of cross-linked duplex XL 1–1b (s), 5′-dCGAAAGTTTCG-3′/5′-dCGAAACTTTCG-3′ containing a central G-C base pair (---), and 5′-dCGAAAITTTCG-3′/5′-dCGAAATTTTCG-3′ containing a central I-T mismatch (···). Solutions contained a total strand concentration of 2.3 µM for the cross-linked duplex XL 1–1b and 4.5 µM of the noncross-linked control duplexes in 90 mM sodium chloride, 10 mM sodium phosphate, and 1 mM ethylene-diaminetetraacetate buffer at pH 7.0. Spectra are the average of 5 scans and were recorded at 10 °C.

of 220 nm (27). Various 11-bp duplexes containing a central G-C base pair, an I-T mismatch, and duplex XL 1–1b with a central N1-2′-deoxyinosine-ethyl-N3-thymidine cross-link were geometry optimized using the AMBER force field and are shown in Figure 5. The geometry optimized model containing the central I-T mismatch exhibits very minor structural differences from a B-form duplex with hydrogen bonds formed between the N1 atom of 2′-deoxyinosine and O2 atom of thymidine as well as the O6 atom of 2′-deoxyinosine with the N3 atom of thymidine. To enable this hydrogen bond arrangement, the thymine base is displaced toward the major groove. The presence of the ethyl cross-link bridging the N1 atom of 2′deoxyinosine and N3 atom of thymidine with an ethyl group abolishes the formation of any hydrogen bonds between the two bases with some minor structural differences between the I-T wobble pair and the N1I-ethyl-N3T cross-linked duplex. For example, the distance between the N1 and N3 atoms of 2′-deoxyinosine and thymidine is approximately 3.7 Å for the cross-linked duplex, whereas the distance is approximately 4.0 Å for the noncross-linked base pair. Overall, the molecular model cross-link does not appear to induce any gross conformational distortions around the site of this lesion nor from the global structure of a B-form duplex.

4. Discussion The synthesis of chemically stable interstrand cross-linked DNA duplexes by both solution and solid-phase methodologies enables access to substrates for DNA repair studies as these molecules contain well-defined lesions in precise orientations within the duplex. An alternative approach to prepare such interstrand cross-linked substrates involves the treatment of DNA with bifunctional alkylating agents. The antitumor drug 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) is an example of a bifunctional alkylating agent that acts upon DNA to form an ethyl cross-link bridging the N1 atom of guanine with the N3 atom of cytosine. The proposed mechanism for this cross-link formation involves the formation of an O6,N1-ethanoguanine intermediate, which then reacts with the N3 atom of cytidine on the adjacent strand to afford the cross-link. The sequence specificity of this cross-link was investigated by Hopkins on



Figure 5. Geometry optimized models of (a) 5′-dCGAAAGTTTCG-3′/5′-dCGAAACTTTCG-3′, (b) 5′-dCGAAAITTTCG-3′/5′-dCGAAATTTTCG3′ (containing a central I-T mismatch), and (c) cross-linked duplex XL 1–1b. (The central base pairs are colored in green.)

the basis of experiments with chemically synthesized oligonucleotides and excess BCNU (2). Relatively low yields (0.4 – 3.7%) of cross-linked duplex were obtained after isolation by gel electrophoresis. Enzymatic digest of the cross-linked duplex to the nucleosides followed by ESI MS analysis revealed the presence of 1-[N3-2′-deoxycytidyl]-2-[N1-2′-deoxyguanosyl] ethane, helping to establish the identity of this cross-link (2). However, experiments requiring ample amounts of this crosslinked material, such as structural studies by NMR or DNA repair studies, have not been reported. It has been demonstrated that O6-alkylguanine alkyltransferase can remove the chloroethyl group from the O6 atom of guanine, the first step toward the formation of this interstrand cross-link (28). The chemical preparation of 1-[N3-2′-deoxycytidyl]-2-[N12′-deoxyguanosyl]ethane has been reported, in amounts sufficient for identification of this lesion in DNA by various analytical procedures such as mass spectrometry (29). This article describes a chemical synthesis of a 1-[N3-thymidyl]-2[N1-2′-deoxyinosinyl]ethane cross-linked dimer employing mild alkylation reactions that proceed in high yield, enabling the preparation of a phosphoramidite that can be used in solid-phase oligonucleotide synthesis for site-specific incorporation of this lesion in a DNA duplex. From a synthetic perspective, 2′deoxyinosine is a simpler nucleoside than 2′-deoxyguanosine as it lacks an exocyclic amino functionality at the C2 position of the heterocycle, eliminating the need for the introduction of a protecting group employed in solid-phase oligonucleotide synthesis. In addition, it has been shown that 2′-deoxyinosine is capable of forming stable base pairs with all four DNA bases as assessed by UV thermal denaturation experiments with the stability of the base pairs having the following order of I-C > I-A > I-T > I-G (30–32). Reactions that alkylate specifically at the N3 position of thymidine (20) and N1 position of 2′-deoxyinosine (24, 25) have been shown to proceed in high yield. The synthesis of a mono and bis-phosphoramidite of the 1-[N3-thymidyl]-2-[N1-2′-deoxyinosinyl]ethane dimer is shown

in Scheme 1 starting with alkylation of the N1 atom of 5′-Odimethoxytrityl-3′-O-tert-butyldimethylsilyl-2′-deoxyinosine (1) with 1-bromo-2-chloroethane. A second alkylation reaction with either 5′-O-dimethoxytrityl-3′-O-phenoxyacetyl-thymidine or 5′O-dimethoxytrityl-3′-O-tert-butyldimethylsilyl-thymidine produces after removal of the 3′-O protecting groups, either a monoamidite (7) or bis-amidite (8). NMR analyses and MS data were used to confirm the identity of the intermediates and final compounds. Cross-linked duplex XL 1–1a was prepared according to slight modifications of procedures used previously (20). One key modification involves the removal of a 3′-O-tert-butyldimethylsilyl group from the 2′-deoxyinosine at the site of the cross-link in XL-b. Previously, our group and others have employed the use of TBAF for desilylation in the synthesis of cross-linked and branched oligonucleotides; however, lower yields of the desired oligonucleotide products were observed with longer TBAF exposure times, which has been attributed to the cleavage of the oligonucleotide from the solid support (20, 33). The use of CPG as solid-support makes it necessary to use TBAF as the fluoride source rather than triethylamine trihydrofluoride because of the reactivity of the latter with the glass beads. We herein employ a polystyrene-based solid support for the synthesis of the oligomer requiring TBDMS removal, which now permits the use of triethylamine trihydrofluoride as the desilylating agent. Because fluoride-mediated desilylation can result in the cleavage of adjacent β-cyanoethylphosphotriester linkages, XL-b was first treated with anhydrous triethylamine to convert the triester groups to phosphodiester linkages (33). To determine the extent of removal of the silyl group, a small amount (approximately 1 mg) of support was treated with a mixture of concentrated ammonium hydroxide/ethanol (3:1) at 55 °C for 4 h. These conditions are typically used to deprotect RNA without removing the silyl group (34). The hydrolyzate was analyzed by C-18 reversed phase HPLC. The desilyated oligomer XL-c has a reduced retention time on the reversed phase column and was easily distinguished from the slower

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eluting silyated oligomer XL-b. HPLC analysis revealed almost complete removal of the TBDMS group. The full length crosslinked duplex XL 1–1a was prepared by repetitive coupling of the 3′-end of intermediate XL-c with 5′-O-deoxyphosphoramidites at a concentration of 0.2 M and coupling time of 3 min. One advantage of the bis-phosphoramidite approach used in the synthesis of XL 1–1b is that it is not necessary to have an additional deprotection step of the oligomer attached to the solid support nor does it employ the use of more expensive 5′-Odeoxyphosphoramidites. Additionally, the bis-phosphoramidite approach requires a lower concentration of the cross-linked nucleoside dimer in order to afford optimal coupling of both phosphoramidite moieties to the linear chains to afford the crosslinked duplexes. However, the major synthetic limitation of the dimer used in this methodology results from having dimethoxytrityl groups at both 5′ alcohol groups of thymidine and 2′deoxyinosine. As a result, only symmetrical sequences can be prepared around the cross-link site. Using either approach, with efficient coupling of the phosphoramidites, cross-linked duplexes containing up to 20 base pairs have been successfully synthesized. Purification of these cross-linked oligomers by SAX HPLC afforded final yields of 40 and 25% for the phosphoramidite and bis-phosphoramidite approaches, respectively, on the basis of OD measurements of the amount of isolated and purified oligomer from a known aliquot. It is surmised that longer cross-linked oligonucleotides could be prepared in this fashion with limitations similar to those of linear DNA synthesis. Strategies such as employing CPG support with a larger pore size (1000 Å) may enable the synthesis of longer oligomers (35). Overall, either procedure provides sufficient quantities of the N1I-ethyl-N3T interstrand cross-link containing duplexes to conduct biological and physical studies, including structural studies by high resolution NMR spectroscopy. As was seen previously with other interstrand cross-linkcontaining duplexes, the denaturation temperature of the N1Iethyl-N3T cross-linked duplex was higher than the melting temperature of the noncross-linked control (20). Indeed, an increase in Tm of approximately 52 °C was observed compared to that of an I-T containing mismatch, representing one of the most stabilizing cross-links we have prepared to date (7, 20, 21). This increased thermal stability is most likely a consequence of covalently linking the two strands of the duplex with the denaturation/renaturation of the cross-linked duplex being a unimolecular process, which is independent of the strand concentration, whereas for noncross-linked duplexes these processes are bimolecular. The enhanced thermal stability of the cross-linked duplex revealed by the observed denaturation temperatures is reflected by the electrophoretic mobilities on a denaturing polyacrylamide gel (see Supporting Information). Cross-linked duplex XL 1–1b (lane 3) had a greater electrophoretic mobility compared to that of the 20-mer standard (lane 1), suggesting that the cross-linked duplex was not denatured in the presence of 7 M urea in the polyacrylamide gel. The CD spectra of noncross-linked controls containing a central G-C base pair and I-T mismatch illustrate some differences, particularly a reduction of the signal at 280 nm for the latter, which suggests reduced base stacking (36). Crystallographic studies of an A-form octanucleotide duplex containing an I-T mismatched pair revealed that this mismatch formed a wobble base pair that is linked by two hydrogen bonds between the O6 and N1 atoms of 2′-deoxyinosine with the N3 and O2 atoms of thymidine, respectively (37). Moreover, this hydrogen

Wilds et al.

bonding arrangement is similar to that observed in the crystal structure of a B-form duplex containing a G-T mismatch between the O6 and N1 atoms of 2′-deoxyguanosine with the N3 and O2 atoms of thymidine (38). In both cases, these mismatches were well accommodated into the duplex with little overall adjustment of the sugar–phosphate backbone. The global features of the CD spectrum for the cross-linked duplex suggest that the N1I-ethyl-N3T cross-link did not significantly affect the duplex structure from that of a B-form DNA. Similarly, the molecular model of a duplex containing the N1I-ethyl-N3T interstrand cross-link suggested little effect on the duplex in terms of global structure with minimal distortion of the stacking or hydrogen bonding of the flanking base pairs. Indeed, the CD spectrum more closely resembles that of the noncross-linked control containing a G-C pair rather than the I-T mismatch. Both crystallographic and NMR data of duplexes containing an I-T mismatch have revealed that the 2′-deoxyinosine base is displaced toward the major groove, whereas the thymidine is displaced toward the minor groove (37, 39). In addition, there is some effect upon base stacking above and below the I-T mismatch (37). It is reasonable to assume that the presence of the ethyl linkage between the N1 atom of 2′-deoxyinosine and N3 atom of thymidine will have an effect in the displacement of these bases in the cross-linked duplex relative to the noncross-linked I-T duplex. Investigation of the structure of this cross-linked duplex by high-field NMR experiments, which are currently underway, should reveal more details on the impact that this lesion has on nucleic acid structure, which in turn can be correlated with DNA repair studies. The current study of the N1I-ethyl-N3T cross-link provides a simpler, more synthetically accessible structural mimic of N1Gethyl-N3C, which forms as a result of the treatment of DNA by the chemotherapeutic agent BCNU. Solid-phase synthesis can be used to produce ample quantities of material for structural and repair studies. Ultraviolet thermal denaturation experiments have revealed that the placement of this two-carbon alkyl linker between the N1 atom of 2′-deoxyinosine and the N3 atom of thymidine imparts thermal stability. Preliminary structural studies by CD experiments of sequences containing the N1Iethyl-N3T cross-link illustrate that the global structure of the duplex is similar to that of a noncross-linked duplex containing a C-G pair at the same site, which are correlated by molecular modeling studies. Since this lesion appears to have little impact on structure, it will be interesting to study whether this crosslink will be recognized or repaired by various DNA repair systems. For example, it has been shown that duplexes containing a 2′-deoxyinosine/thymidine mismatch were bound to the E. coli mutS protein involved in mismatch repair, albeit to a lesser extent than a 2′-deoxyguanosine/thymidine mismatch (40). Given the structural similarity suggested by CD experiments between the N1I-ethyl-N3T cross-linked duplex and the noncrosslinked control containing a G-C pair at the same site, it remains to be seen the extent to which this cross-linked duplex is recognized by repair proteins such as mutS. Further characterization such as high resolution NMR analyses, which is currently underway, will provide more details regarding the structure of the duplex and therefore give more insight to aid in our understanding of the repair mechanism of the therapeutically relevant cross-links in biological systems that are believed to play an important role in the resistance to chemotherapeutic agents.


Acknowledgment. We thank Mr. Nadim Saadeh and Dr. Bruce Lennox of McGill University for ESI-MS analysis. This project was supported financially by the Natural Sciences and Engineering Research Council of Canada (NSERC), the Canada Foundation for Innovation (CFI), and the Canada Research Chair (CRC) program. Supporting Information Available: 1H NMR spectra of compounds 1 to 6 and 31P NMR spectra of compounds 7 and 8; SAX HPLC chromatograms, RP HPLC chromatogram of the nuclease digest, PAGE analysis and mass spectra of purified oligonucleotides XL 1–1a and XL 1–1b. This material is available free of charge via the Internet at http://pubs.acs.org.

References (1) Dronkert, M. L., and Kanaar, R. (2001) Repair of DNA interstrand cross-links. Mutat. Res. 486, 217–247. (2) Fischhaber, P. L., Gall, A. S., Duncan, J. A., and Hopkins, P. B. (1999) Direct demonstration in synthetic oligonucleotides that N,N′-bis(2chloroethyl)-nitrosourea cross links N1 of deoxyguanosine to N3 of deoxycytidine on opposite strands of duplex DNA. Cancer Res. 59, 4363–4368. (3) de Silva, I. U., McHugh, P. J., Clingen, P. H., and Hartley, J. A. (2000) Defining the roles of nucleotide excision repair and recombination in the repair of DNA interstrand cross-links in mammalian cells. Mol. Cell. Biol. 20, 7980–7990. (4) Kuraoka, I., Kobertz, W. R., Ariza, R. R., Biggerstaff, M., Essigmann, J. M, and Wood, R. D. (2000) Repair of an interstrand DNA crosslink initiated by ERCC1-XPF repair/recombination nuclease. J. Biol. Chem. 275, 26632–26636. (5) Niedernhofer, L. J., Odijk, H., Budzowska, M., van Drunen, E., Maas, A., Theil, A. F., de Wit, J., Jaspers, N. G. J., Beverloo, H. B., Hoeijmakers, J. H. J., and Kanaar, R. (2004) The structure-specific endonuclease ERCC1-XPF is required to resolve DNA interstrand cross-link-induced double-strand breaks. Mol. Cell. Biol. 24, 5776– 5787. (6) Bessho, T. (2003) Induction of DNA replication-mediated double strand breaks by psoralen DNA interstrand cross-links. J. Biol. Chem. 278, 5250–5254. (7) Noronha, A. M., Noll, D. M., Wilds, C. J., and Miller, P. S. (2002) N(4)C-ethyl-N(4)C cross-linked DNA: synthesis and characterization of duplexes with interstrand cross-links of different orientations. Biochemistry 41, 760–771. (8) Bhat, B., Leonard, N. J., Robinson, H., and Wang, A.H.-J. (1996) Deoxyadenosine and thymidine bases held proximal and distal by means of a covalently-linked dimensional analogue of dA·dT: intramolecular vs intermolecular hydrogen bonding. J. Am. Chem. Soc. 118, 10744–10751. (9) Harwood, E. A., Sigurdsson, S. T., Edfeldt, N. B. F., Reid, B. R., and Hopkins, P. B. (1999) Chemical synthesis and preliminary structural characterization of a nitrous acid interstrand cross-linked duplex DNA. J. Am. Chem. Soc. 121, 5081–5082. (10) Li, H. Y., Qiu, Y. L., Moyroud, E., and Kishi, Y. (2001) Synthesis of DNA oligomers possessing a covalently cross-linked Watson-Crick base pair model. Angew. Chem., Int. Ed. 40, 1471–1475. (11) Ferentz, A. E., Keating, T. E., and Verdine, G. L. (1993) Synthesis and characterization of disulfide cross-linked oligonucleotides. J. Am. Chem. Soc. 115, 9006–9014. (12) Dooley, P. A., Tsarouhtsis, D., Korbel, G. A., Nechev, L. V., Shearer, J., Zegar, I. S., Harris, C. M., Stone, M. P., and Harris, T. M. (2001) Structural studies of an oligodeoxynucleotide containing a trimethylene interstrand cross-link in a 5′-(CpG) motif: Model of a malondialdehyde cross-link. J. Am. Chem. Soc. 123, 1730–1739. (13) Watkins, N. E., Jr., and SantaLucia, J., Jr. (2005) Nearest-neighbor thermodynamics of deoxyinosine pairs in DNA duplexes. Nucleic Acids Res. 33, 6258–6267. (14) Ueda, T., and Fox, J. J. (1964) Pyrimidines. IV.1 The interconversion of N4-methylcytosine and 3-methylcytosine. J. Org. Chem. 29, 1770– 1772. (15) Munter, T., Cottrell, L., Hill, S., Kronberg, L., Watson, W. P., and Golding, B. T. (2002) Identification of adducts derived from reactions of (1-chloroethenyl)oxirane with nucleosides and calf thymus DNA. Chem. Res. Toxicol. 15, 1549–1560. (16) Hudson, R. H. E., Robidoux, S., and Damha, M. J. (1998) Divergent solid-phase synthesis of nucleic acid dendrimers. Tetrahedron Lett. 39, 1299–1302.

Chem. Res. Toxicol., Vol. 21, No. 3, 2008 695 (17) Hudson, R. H. E., and Damha, M. J. (1993) Nucleic acid dendrimers: Novel biopolymer structures. J. Am. Chem. Soc. 115, 2119–2124. (18) Damha, M. J., Ganeshan, K., Hudson, R. H. E., and Zabarylo, S. V. (1992) Solid-phase synthesis of branched oligoribonucleotides related to messenger RNA splicing intermediates. Nucleic Acids Res. 20, 6565–6573. (19) Carriero, S., Braich, R. S., Hudson, R. H. E., Anglin, D., Friesen, J. D., and Damha, M. J. (2001) Inhibition of in vitro pre-mRNA splicing in S. cerevisiae by branched oligonucleotides. Nucleosides, Nucleotides, Nucleic Acids 20, 873–877. (20) Wilds, C. J., Noronha, A. M., Robidoux, S., and Miller, P. S. (2004) Mispair-aligned N3T-alkyl-N3T interstrand cross-linked DNA: synthesis and characterization of duplexes with interstrand cross-links of variable lengths. J. Am. Chem. Soc. 126, 9257–9265. (21) Noll, D. M., Noronha, A. M., and Miller, P. S. (2001) Syntheses of DNA duplexes containing a C-C interstrand cross-link. J. Am. Chem. Soc. 123, 3405–3411. (22) Puglisi, J. D., and Tinoco, I. J. (1989) Absorbance melting curves of RNA. Methods Enzymol. 180, 304–325. (23) Alzeer, J., and Schärer, O. D. (2006) A modified thymine for the synthesis of site-specific thymine-guanine DNA interstrand crosslinks. Nucleic Acids Res. 34, 4458–4466. (24) Vincent, S. P., Mioskowski, C., and Lebeau, L. (1999) Regioselective N1-alkylation of guanosine derivatives protected at N2 by an N,Ndialkyl amidine group. Nucleosides, Nucleotides 18, 2127–2139. (25) Huang, Y., and Johnson, F. (2002) Regioselective 1-alkylation of 2′deoxyguanosine. Nucleosides, Nucleotides, Nucleic Acids 21, 435– 447. (26) Miller, P. S. private communication. (27) Johnson, W. C., Jr. (1985) Circular Dichroism and Its Empirical Application to Biopolymers. In Methods of Biochemical Analysis (Glick, D., Ed.) Vol. 31, pp 61–163, Wiley, New York. (28) Pegg, A. E. (1990) Mammalian O6-alkylguanine-DNA alkyltransferase: regulation and importance in response to alkylating carcinogenic and therapeutic agents. Cancer Res. 50, 6119–6129. (29) Bodell, W. J., and Pongracz, K. (1993) Chemical synthesis and detection of the cross-link 1-[N3-(2′-deoxycytidyl)]-2-[N1-(2′-deoxyguanosinyl)]ethane in DNA reacted with 1-(2-chloroethyl)-1-nitrosourea. Chem. Res. Toxicol. 6, 434–438. (30) Martin, F. H., Castro, M. M., and Aboul-ela, F., Jr. (1985) Base pairing involving deoxyinosine: Implications for probe design. Nucleic Acids Res. 13, 8927–8938. (31) Kawase, U., Iwai, S., Inoue, J., Miura, K., and Ohtsuka, E. (1986) Studies on nucleic acid interactions. I. Stabilities of mini-duplexes (dG2A4XA4G2-dC2T4YT4C2) and self-complementary d(GGGAAXYTTCCC) containing deoxyinosine and other mismatched bases. Nucleic Acids Res. 14, 7727–7736. (32) Kawase, Y., Iwai, S., and Ohtsuka, E. (1989) Synthesis and thermal stability of dodecadeoxyribonucleotides containing deoxyinosine pairing with four major bases. Chem. Pharm. Bull. 37, 599–601. (33) Braich, R. S., and Damha, M. J. (1997) Regiospecific solid-phase synthesis of branched oligonucleotides. Effect of vicinal 2′,5′- (or 2′,3′-) and 3′,5′-phosphodiester linkages on the formation of hairpin DNA. Bioconjugate Chem. 8, 370–377. (34) Damha, M. J., and Ogilvie, K. K. (1993) Oligoribonucleotide Synthesis. In Protocols for Oligonucleotides and Analogues: Synthesis and Properties, Methods in Molecular Biology (Agrawal, S., Ed.) pp 84– 114, Humana Press, Totowa, NJ. (35) Pon, R. T., and Yu, S. (2005) Tandem oligonucleotide synthesis using linker phosphoramidites. Nucleic Acids Res. 33, 1940–1948. (36) Johnson, W. C., Jr. (1996) Determination of the Conformation of Nucleic Acids by Electronic CD. In Circular Dichroism and the Conformational Analysis of Biomolecules (Fasman, G. D., Ed.) pp 433–468, Plenum Press, New York. (37) Cruse, W. B. T., Aymani, J., Kennard, O., Brown, T., Jack, A. G. C., and Leonard, G. A. (1989) Refined crystal structure of an octanucleotide duplex with I.T. mismatched base pairs. Nucleic Acids Res. 17, 55–72. (38) Hunter, W. N., Brown, T., Kneale, G., Anand, N. N., Rabinovich, D., and Kennard, O. (1987) The structure of guanosine-thymidine mismatches in B-DNA at 2.5 Å resolution. J. Biol. Chem. 262, 9962– 9970. (39) Carbonnaux, C., Fazakerley, G. V., and Sowers, L. C. (1990) An NMR structural study of deaminated base pairs in DNA. Nucleic Acids Res. 18, 4075–4081. (40) Jiricny, J., Su, S.-S., Wood, S. G., and Modrich, P. (1988) Mismatchcontaining oligonucleotide duplexes bound by the E. coli mutSencoded protein. Nucleic Acids Res. 16, 7843–7853.

TX700422H

Deoxyinosine-ethyl-N3-thymidine Interstrand Cross-Link

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