A New Method for the Postsynthetic Generation of Abasic Sites in

Mihoyo Fujitake, Shinya Harusawa. Mass Spectrometric Determination of Polyfunctionalized Nucleoside Phosphoramidites Using LSI/FAB. YAKUGAKU ZASSHI ...
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Chem. Res. Toxicol. 2000, 13, 907-912

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A New Method for the Postsynthetic Generation of Abasic Sites in Oligomeric DNA Irina G. Shishkina and Francis Johnson* Department of Pharmacological Sciences, The State University of New York at Stony Brook, Stony Brook, New York 11794-3400 Received May 15, 2000

The abasic site is the most common lesion in DNA and is thought to play a critical role in mutagenesis. However, a general chemical method for the site-specific generation of true abasic sites in oligodeoxynucleotides has been lacking. We now describe a procedure which permits the postsynthetic generation of abasic sites in single- or double-stranded DNA oligomers without regard to their base composition. An appropriately protected 3-deoxyhexitol was synthesized and used as the monomer that was incorporated into DNA oligomers using the standard phosphoramidite method for automated DNA synthesis. The resulting stable diol-containing oligonucleotides were purified by HPLC and converted quantitatively into the corresponding abasic DNA sequences by mild periodate oxidation. The abasic site in DNA was found to be relatively stable at room temperature, but was completely cleaved when treated with putrescine at 95 °C. Identification of the major degradation products was accomplished by gel electrophoresis, HPLC isolation, and characterization by electrospray ionization mass spectrometry. The thermal stabilities of duplex oligonucleotides containing a natural abasic site were studied, and the results were compared with those from oligomers containing T/dA, F/dA, or the precursor diol opposite dA at the same site.

Introduction The loss of a heterocyclic base from DNA is a common occurrence. This frequently takes place by a hydrolytic mechanism that is slow (104 depurinations per mammalian cell per day) (1) at 37 °C and neutral pH, but may occur much more rapidly (1, 2) at pH 7 (10-13). Because of this instability, biological studies directly involving the abasic site 1 have been limited and much information has been derived from investigations that used the chemically stable anhydroribitol 2 (6, 14, 15) as a model for 1. The anhydroribitol, commonly termed the “tetrahydrofuran” lesion, is easily incorporated into DNA using standard protocols and automated methods. Despite the fact that it has not proven to be possible to introduce the true abasic site 1 presynthetically into DNA, enzymatic removal of uracil using N-uracil glycosylase (16) and two indirect chemical methods have proven to be successful. The first of the chemical methods involves the carefully-controlled acid hydrolysis of a deoxyadenosine residue that removes only an adenine base followed by stabilization of the resulting abasic site as the methoxyamine derivative (7, 10, 11). After purification, the abasic site can be generated by an exchange * To whom correspondence should be addressed. Telephone: (631) 632-8867. Fax: (631) 632-7394. E-mail: [email protected].

Chart 1

process using acetaldehyde. This method is limited to DNA oligomers having no dG residues and only a single dA group. There is also the risk of forming acetaldehyde adducts with amino groups in other DNA bases. The second chemical method developed by Rayner and his associates (17) uses photocleavage of a 1′-(2-nitrobenzyl) ether 3 (R ) H). Although the required 5′-O-DMT-3′-Ophosphoramidite of 3 (R ) H) can be prepared by an eight-step process from 2-D-deoxyribose, the synthetic route produces a mixture of R- and β-isomers at the 1-position, which complicates purification and manipulation. In addition, the photochemical mode of unmasking the abasic site carries the risk of photodimer formation between adjacent thymine bases in other areas of the molecule. The use of the enzymatic method may be compromised by certain elements of DNA structure such as the proximity of the strand termini, noncanonical structures (hairpins, Holliday junctions, etc.) (18), and the presence of noncanonical moieties among bases, sugar or phosphate groups, or proteins bound to DNA, whereas the peroxidate oxidation is insensitive to such structural elements. This factor may also assume importance in mechanistic studies of the enzymes that correct DNA damage (19). In addition, if inactivation of this glycosylase is necessary, the reaction mixture must be heated for an extended period of time which may lead to abasic site degradation and the generation of artifacts (20). Also the large quantities of DNA required for X-ray structural

10.1021/tx000108s CCC: $19.00 © 2000 American Chemical Society Published on Web 08/25/2000

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studies of abasic site-containing DNA-enzyme complexes would be difficult to prepare using uracil DNA glycosylase. Again, enzymatic reactions in practical terms often do not run to completion, stopping at 80-90% substrate conversion. The method described in this paper is novel and avoids all of the problems described above.

Experimental Procedures All reagents and solvents, unless otherwise specified, were the best grade commercially available (Aldrich and Fluka) and were used without further purification. NMR (1H, 13C, and 31P) spectra were recorded on a Bruker AC-250 spectrometer. Chemical shifts are reported in parts per million relative to tetramethylsilane in the proton spectra and to the deuterated solvent in the carbon spectra. Mass spectra were recorded on a Micromass TRIO-2000 instrument in fast atom bombardment (FAB)1 mode and on a Micromass Quattro spectrometer in electrospray ionization (ESI) mode. Elemental analyses were performed by Schwarzkopf Microanalytical Laboratory (Woodside, NY). TLC was performed on silica gel sheets (RiendeldeHae¨n, Sleeze, Germany) containing a fluorescent indicator. Flash column chromatographic separation was carried out on 60 Å (230-400 mesh) silica gel (TSI Chemical Co., Cambridge, MA). All experiments dealing with moisture-sensitive compounds were conducted under dry nitrogen. 1,3-(R)-O-Dibenzyl-2-(S)-hydroxy-5-hexene (5). Butyllithium (52 mL of a 2 M solution in pentane) was added to a stirred suspension of methyl triphenylphosphonium bromide (36.6 g, 102.5 mmol) in 400 mL of dry toluene under a nitrogen atmosphere, and the reaction mixture was further stirred at room temperature for 2 h. A solution of compound 4 (13.0 g, 41 mmol) prepared according to the method of Vandenriessche et al. (21) in 50 mL of dry toluene was added slowly to this mixture using a dropping funnel, and after the addition was complete, the reaction mixture was heated at 50 °C for 1 h. The reaction mixture was cooled to room temperature; 100 mL of water was added, and the reaction mixture was extracted with ethyl acetate (2 × 300 mL). The combined organic layers were successively washed with a saturated ammonium chloride solution, and then with water, and finally concentrated in vacuo. The crude reaction mixture was purified by silica gel column chromatography (20:80 ethyl acetate/hexane) to give 7 g of 5 (54%) as an oil. 1H NMR (CDCl3): δ 7.38-7.29 (m, 10H, ArH), 6.00-5.83 (m, 1H, H-5), 5.2-5.08 (m, 2H, H-6), 4.62-4.50 (m, 4H, CH2Ar), 3.85 (m, 1H, H-2), 3.66-3.6 (m, 2H, H-1), 3.6-3.5 (m, 1H, H-3), 2.47-2.45 (m, 3H, H-4, H′-4, OH). 13C NMR (CDCl3): δ 138.2, 137.8, 134.5, 128.3, 127.7, 127.6, 127.5, 117.2, 79.0, 73.4, 72.2, 71.4, 71.0, 34.7. FAB-MS: m/z 313.16 (M + H)+. 4,6-O-Dibenzyl-3-deoxyhexitol (6). To solution of compound 5 (6.4 g, 21.8 mmol) in 14 mL of 6.3% hydrogen peroxide (25 mmol) in tert-butyl alcohol was added 0.3 mL of a 2.5% solution of osmium tetroxide (0.03 mmol) in tert-butyl alcohol. The reaction mixture was stirred for 1 h at room temperature (by which time the initial orange color had changed to yellowgreen) and then concentrated in vacuo. The resulting residue was dissolved in ethyl acetate (100 mL) and washed with aqueous solutions of Na2SO3 (2 × 100 mL), NaHCO3 (2 × 70 mL), and brine (2 × 70 mL). The organic layer was dried (Na2SO4) and concentrated in vacuo. The crude reaction mixture was purified by silica gel column chromatography (10:90 methanol/ dichloromethane) to give 5.2 g of 6 (74%) as a white amorphous solid. TLC: Rf 0.29 (10:90 methanol/dichloromethane), 0.35 (ethyl acetate). 1H NMR (CDCl3): δ 7.33-7.29 (m, 10H, ArH), 4.53 (s, 4H, CH2Ar), 4.00-3.74 (m, 2H, H-1), 3.74-3.65 (m, 1H, H-5), 3.65-3.54 (m, 3H, H-2, H-6, H′-6), 3.45-3.39 (q, 1H, H-4), 3.19 (s, 3H, OH), 1.76-1.70 (t, 2H, H-3). 13C NMR (CDCl3): δ 1 Abbreviations: DMT, 4,4′-dimethoxytrityl; dR, deoxyribose; ESI, electrospray ionization; FAB, fast atom bombardment; F, 1,4-anhydro2′-ribitol; TEAAc, triethylammonium acetate; TBS, tert-butyldimethylsilyl.

Shishkina and Johnson 137.7, 137.6, 128.4, 128.3, 127.9, 127.8, 77.9, 77.2, 73.4, 72.5, 71.5, 71.8, 71.0, 70.9, 69.2, 69.1, 66.9, 66.8, 33.7, 32.8. FABMS: m/z 347.12 (M + H)+. Anal. Calcd for C20H26O5: C, 69.34; H, 7.56. Found: C, 68.69; H, 7.56. 4,6-O-Dibenzyl-1,2,5-O-tris(tert-butyldimethylsilyl)-3deoxyhexitol (7). The dried product 6 (3.2 g, 9 mmol), tertbutyldimethylsilyl chloride (TBSCl) (5.3 g, 35 mmol), and imidazole (4.8 g, 70 mmol) were dissolved in anhydrous dimethylformamide (14 mL). The reaction mixture was stirred under nitrogen for 16 h at 23 °C. Water was then added to the vigorously stirred reaction mixture, but no precipitate appeared. The reaction mixture was extracted with ethyl acetate (2 × 200 mL), and the organic layers were combined, then washed with water, and concentrated in vacuo. The crude reaction product was purified by silica gel column chromatography (40:60 dichloromethane/hexane) to give 5.6 g of 7 (91%) as an oil. TLC: Rf 0.64 (40:60 dichloromethane/hexane), 0.4 (20:80 chloroform/ hexane), 0.87 (10:90 methanol/dichloromethane). 1H NMR (CDCl3): δ 7.30 (m, 10H, ArH), 4.52 (d, J ) 2.5 Hz, 4H, CH2Ar), 4.02-3.70 (m, 3H, H-1, H-5, H′-1), 3.59-3.44 (m, 4H, H-6, H-2, H′-6, H-4), 1.75 (m, 2H, H-3), 0.90 (s, 27H, C-CH3), 0.04 (s, 18H, Si-CH3). 13C NMR (CDCl3): δ 137.1, 128.3, 127.5, 78.4, 78.0, 74.3, 73.5, 72.4, 71.2, 71.1, 67.9, 67.8, 36.8, 36.9. Anal. Calcd for C38H68O5Si3: C, 66.22; H, 9.94. Found: C, 66.45; H, 10.01. 1,2,5-O-Tris(tert-butyldimethylsilyl)-3-deoxyhexitol (8). A reaction mixture containing 7 (4.5 g, 6.5 mmol) and 10 g of palladium on activated carbon (10% Pd) in 70 mL of methanol was hydrogenated under pressure (50 psi) for 20 h and then filtered through Celite. The solvent was removed in vacuo, and the resulting crude product was purified by silica gel column chromatography (2:98 methanol/dichloromethane) to give pure 8 (1.9 g, 60%), as a white amorphous solid. TLC: Rf 0.42 (2:98 MeOH/dichloromethane), 0.18 (chloroform). 1H NMR (CDCl3): δ 3.95-3.78 (m, 2H, H-1), 3.75-3.42 (m, 5H, H-6, H-5, H′-6, H-2, H-4), 3.16 (bs, 2H, OH), 2.09-1.40 (m, 2H, H-3), 0.92 (s, 27H, C-CH3), 0.09 (s, 12H, Si-CH3), 0.06 (s, 6H, Si-CH3). 13C NMR (CDCl3): δ 75.1, 73.0, 71.2, 66.5, 64.8, 37.4, 36.8, 25.8, 18.0, -4.5. FAB-MS: m/z 509.3 (M + H)+. Anal. Calcd for C24H56O5Si3: C, 56.64; H, 11.09. Found: C, 57.11; H, 11.02. 6-O-(4,4′-Dimethoxytrityl)-1,2,5-O-tris(tert-butyldimethylsilyl)-3-deoxyhexitol (9). Compound 8 (500 mg, 0.98 mmol) was treated with 4,4′-dimethoxytrityl chloride (DMTCl) (406 mg, 1.2 mmol) in 4 mL of pyridine at room temperature over the course of 30 min. The pyridine was removed in vacuo, and the residue was dissolved in dichloromethane (40 mL). This solution was washed with saturated NaHCO3 (2 × 20 mL) and water. The organic layer was dried (Na2SO4) and evaporated in a rotary evaporator. The residue was then purified by silica gel column chromatography (dichloromethane, containing 0.5% triethylamine) to give pure 9 (796 mg, 98%). TLC: Rf 0.49 (dichloromethane containing 0.5% triethylamine). 1H NMR (CDCl3): δ 7.47-7.16 (m, 9H, ArH), 6.85-6.78 (m, 4H, ArH), 4.01-3.87 (m, 2H, H-1), 3.78 (s, 6H, O-CH3), 3.63-3.40 (m, 2H, H-5, H-2), 3.23-2.75 (m, 3H, H-6, H-4, H′-6), 1.75-1.49 (m, 2H, H-3), 0.88 (s, 27H, C-CH3), 0.11-0.01 (m, 18H, Si-CH3). 13C NMR (CDCl3): δ 158.4, 130.1, 128.3, 127.7, 126.7, 113.0, 86.3, 75.8, 71.2, 70.9, 69.3, 67.2, 65.1, 55.2, 36.1, 35.8, 25.9, 18.1, -4.3. ESIMS: m/z 809.2 (M - H)-. 6-O-(4,4′-Dimethoxytrityl)-4-O-[(N,N-diisopropylamino)(2-cyanoethoxy)phosphinyl]-1,2,5-O-tris(tert-butyldimethylsilyl)-3-deoxyhexitol (10). Compound 9 (150 mg, 0.18 mmol) was coevaporated with dry toluene (3 × 5 mL), dissolved in dichloromethane (1.5 mL), and treated with triethylamine (350 µL, 2.5 mmol) and (N,N-diisopropylamino)(2-cyanoethoxy)chlorophosphine (300 µL, 1.26 mmol). The reaction mixture was stirred at 23 °C for 7 h and then diluted with dichloromethane (15 mL). The resulting solution was washed with saturated NaHCO3 (2 × 10 mL), dried (Na2SO4), and then concentrated in vacuo. The crude product was purified by silica gel column chromatography (35:65 hexane/dichloromethane containing 0.5% triethylamine) to obtain the desired product 10 (149 mg, 79%)

Generation of Abasic Sites in DNA

Figure 1. Time course for the degradation of oligodeoxynucleotide TTdRTT in 0.2 M TEAAc buffer (pH 6). as a pair of isomers. TLC for anomer 1: Rf 0.64 (dichloromethane containing 0.5% triethylamine). 31P NMR for anomer 1 (CDCl3): δ 148.8. 1H NMR for anomer 1 (CDCl3): δ 7.427.18 (m, 9H, ArH), 6.84-6.76 (m, 4H, ArH), 4.21-3.82 (m, 4H, H-1, OCH2CH2CN), 3.78 (s, 6H, O-CH3), 3.75-3.30 (m, 5H, H-5, H-2, H-6, H′-6, H-4), 3.03 [m, 2H, NCH(CH3)2], 2.58 (m, 2H, OCH2CH2CN), 1.86-1.51 (m, 2H, H-3), 1.19-1.03 [m, 12H, NCH(CH3)2], 0.95-0.81 (m, 27H, C-CH3), 0.10-0 (m, 18H, SiCH3). TLC for anomer 2: Rf 0.58 (dichloromethane containing 0.5% triethylamine). 31P NMR for anomer 2 (CDCl3): δ 152.4. 1H NMR for anomer 2 (CDCl ): δ 7.42-7.18 (m, 9H, ArH), 6.853 6.78 (m, 4H, ArH), 4.28-3.91 (m, 4H, H-1, OCH2CH2CN), 3.78 (s, 6H, O-CH3), 3.77-3.22 (m, 5H, H-5, H-2, H-6, H′-6, H-4), 3.01 [m, 2H, NCH(CH3)2], 2.36 (t, J ) 6.9 Hz, 2H, OCH2CH2CN), 1.86-1.47 (m, 2H, H-3), 1.26-1.08 [m, 12H, NCH(CH3)2], 0.96-0.80 (m, 27H, C-CH3), 0.12-0.02 (m, 18H, Si-CH3). FABMS for anomer 2: m/z 1011.4 (M + H)+. Oligonucleotide Synthesis and Purification. The oligodeoxynucleotides TTDTT and CTCCTCDATACCT (where D is the 3-deoxyhexitol residue 13) were synthesized using standard phosphoramidite chemistry on a Perkin-Elmer/ABI 394 DNA/RNA synthesizer (Foster City, CA) at the 1 µmol scale. The coupling yields for DTBS were 66 and 83%, respectively. After synthesis, the oligonucleotides were incubated in NH4OH for 8 h at 36 °C, then evaporated to dryness in a vacuum centrifuge (Savant Instrument, Farmingdale, NY), resuspended in water, and purified by HPLC (Waters model 991, Milford, MA) on a Luna phenyl-hexyl column (Phenomenex, Torrance, CA) using a gradient of acetonitrile (5 to 50% in 50 min) in triethylammonium acetate (TEAAc) buffer (0.1 M, pH 7 for the DMT products, pH 6 for all others). The DMT and TBS protecting groups were removed by treatment with 80% acetic acid for 30 min and then 40% acetic acid for 4 h, respectively. The oligomers were then rechromatographed. Aliquots were taken at each stage of purification and dried for mass analysis. Generation of the Abasic Site in Oligonucleotides and Studies of Its Thermal Stability. The pentamer TTDTT (120 nmol, 0.05-1 A260 unit) containing the 3-deoxyhexitol residue D (13) was treated with 10-100 µL of 5 mM NaIO4 in 0.1 M sodium acetate buffer (pH 6.0) for 5 min, and the product was purified by HPLC. The fractions containing the oligonucleotide TTdRTT having an abasic site were combined and evaporated to half of its volume. Portions of this solution were incubated individually at 22, 37, and 55 °C. Aliquots were taken from each run at appropriate intervals and analyzed by HPLC. A time course for the degradation of the TTdRTT is shown in Figure 1. For the construction of this diagram, the integrated area of TTdRTT was calculated for each time period. At 55 °C, the halflife of the oligomer is 7 h (data not shown). The oligodeoxynucleotide CTCCTCDATTACCT (9 nmol, 0.93 A260 unit) was treated with 60 µL of 5 mM NaIO4 in 0.1 M sodium acetate buffer (pH 6.0) for 5 min and desalted on a SepPak C18 cartridge (Waters). A 1 mL solution of abasic oligo-

Chem. Res. Toxicol., Vol. 13, No. 9, 2000 909 nucleotide CTCCTCdRATACCT in acetonitrile and water (1:1) was obtained and evaporated to 100 µL. Preparation of the Double-Stranded Oligonucleotide Substrate. Oligonucleotide CTCCTCDATACCT was 5′-endlabeled by treatment with T4 polynucleotide kinase and [γ-32P]ATP. The mixture of an equimolar amount of this labeled oligonucleotide and the complementary strand in 100 µL of buffer [10 mM Tris-HCl (pH 7.5) and 10 mM MgCl2] was heated at 95 °C for 1 min, and the two DNA strands were annealed, first at 37 °C for 5 min and then at 4 °C for 1 h. Generation of an Abasic Site in Duplex DNA. Reactions to show the generation of abasic sites were performed with 2 µL of the oligonucleotide duplex in 10 µL of solvents, containing a 10 mM solution of NaIO4 (in the case where the diol in duplex was oxidized to abasic site) and/or a 10% solution of putrescine (to cleave the oligonucleotide at the abasic site). The samples with putrescine were incubated at 95 °C for either 15 or 30 min. Then 1 µL of glycerol and 5 µL of xylene cyanol and bromophenol blue were added to all samples, and 4 µL aliquots were analyzed by denaturing gel electrophoresis in 20% polyacrylamide and 8 M urea followed by visualization on a Molecular Dynamics Phosphor Imager. Thermal Stability. The thermal stabilities of oligonucleotide duplexes were determined using a Perkin-Elmer Lambda 2 spectrophotometer with a six-cell changer/six-cell holder based on the Peltier effect and a programmable Peltier block PTF-6. Quartz cuvettes with a path length of 1 cm were used to maintain absorbance values near 0.3 at 270 nm. The computer recorded the absorbance and temperature every 30 s over a period in which the heating rate was 0.4 °C/min. This resulted in more than 370 points for an annealing curve ranging from 10 to 85 °C. The optical and calorimetric melting profiles were analyzed by the nonlinear least-squares method to extract the melting temperatures (Tm).

Results and Discussion Synthesis. As mentioned earlier, we have now developed a risk-free procedure that generates the natural abasic site cleanly and without complication. This uses the same type of periodate diol cleavage protocol that we used previously to generate an acrolein adduct of dG, in oligomeric DNA (22). In the current case, the method utilizes an appropriately protected 3-deoxyhexitol (11) whose 1,2-glycol group is cleaved postsynthetically to produce the desired C-1′ aldehyde of a D-ribose residue. Although formaldehyde is cogenerated in the process, in our experience, this does not interfere with the DNA, possibly because of its very low concentration. The DMT-phosphoramidite (10) required for incorporation into oligomeric DNA was synthesized according to Scheme 1. 3,5-O-Dibenzyl-2-deoxy-D-ribose (4), synthesized from 2-deoxy-D-ribose by the procedure of Vandendriessche et al. (21), was used as the starting material. Treatment of this compound with methyl triphenylphosphonium bromide in toluene led to the olefin 5 in 54% yield (23). When the latter was oxidized with hydrogen peroxide in tert-butyl alcohol in the presence of a catalytic quantity of OsO4, the required triol 6 was obtained after chromatography in 74% yield as an approximately 50: 50 mixture of isomers differing in orientation at the newly introduced secondary hydroxyl group (24). This mixture presented no difficulty in manageability or in further synthetic work. Treatment of triol 6 with tertbutyldimethylsilyl chloride (TBSCl) in DMF containing imidazole gave 7 in 91% yield. Debenzylation of 7 to give 8 was accomplished in 60% yield by catalytic hydrogenation over a 20% Pd-on-carbon catalyst. The synthesis of 10 from 8 was then carried out by the standard two-step protocol of dimethoxytritylation to give 9 (98% yield)

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Shishkina and Johnson Scheme 1

Chart 2

followed by treatment of the latter with the (diisopropylamino)(2-cyanoethoxy)chlorophosphine. The final step proceeded in 79% yield, and product 10 was shown by 31 P NMR spectroscopy to be a mixture of separable diastereomers related to the new asymmetric center at the phosphorus atom. This mixture was then used in standard automated DNA syntheses to generate oligomers containing the abasic residue 13, derived from the 3-deoxyhexitol (11). All oligomers were characterized by both HPLC analysis and electrospray mass spectroscopy. Conditions for the conversion of 12 to 1 (R ) DNA) were developed using the pentamer TTDTBSTT. It was found that the latter could be converted smoothly to TTDTT containing the desired abasic site 13, by modifying the aqueous acetic acid conditions (25) that were used to remove the terminal DMT group. Oxidation of TTDTT by sodium periodate immediately gave the pentamer TTdRTT (in which dR represents the natural abasic site 1) in essentially quantitative yield. Studies on the Abasic Site. The abasic site proved to be stable almost indefinitely at 5 °C if kept in 0.2 M triethylamine acetate (TEAAc) buffer (pH 6). However, at room temperature (22 °C), it has a half-life of ∼30 days (Figure 1), whereas at 55 °C, the half-life decreases to 7 h. Evaporation of the solution to dryness caused complete degradation. A larger oligodeoxynucleotide, namely, the 13-mer CTCCTCDATACCT, was also synthesized by exactly the same procedure as was used for TTDTT. The oxidative

Figure 2. Creation of a true abasic sites in single- and doublestranded DNA fragments and their cleavage by putrescine. Analysis of oxidation and cleavage products by 8 M urea-20% polyacrylamide gel electrophoresis. The oligonucleotide containing an abasic diol residue (D) was 5′-32P-labeled.

cleavage of the 32P-end-labeled 13-mer to CTCCTCdRATACCT and the putrescine-induced degradation of the latter in both single- and double-stranded forms were then examined by gel electrophoresis. The results are summarized in Figure 2. From these data, it is evident that the initial diol (3-deoxyhexitol-derived) abasic site (13) is stable (lanes 1 and 4) and that putrescine at 95 °C has no effect on it (lanes 5 and 6). However, treatment of the DNA with NaIO4 followed by putrescine at 95 °C (lanes 3, 8, and 9) leads essentially to complete chain cleavage. Three degradation products were separated by HPLC and identified by electrospray mass spectrometry. Their structures are shown in Scheme 2 and are completely in accord with previous studies (12, 13, 26) on the patterns of fragmentation, and the products that are

Generation of Abasic Sites in DNA

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Scheme 2. Decomposition of an Oligodeoxynucleotide with an Abasic Site in a Putrescine Solution in 15 min at 95 °C

Table 1. Comparison of Melting Temperatures Obtained with Oligonucleotide Duplexes duplex (X‚Y)

Tm (°C)

duplex (X‚Y)

Tm (°C)

T‚dA D‚dA F‚dA dR‚dA

46 31.2 30.7 31.3

dR‚dG dR‚T dR‚dC

30.5 28.2 24.5

observed when an abasic site undergoes base-catalyzed degradation by a primary amine. The availability of an oligomer containing a natural abasic site prompted us to examine the thermal stability of the double-stranded forms in which the dR residue was paired individually with the four normal deoxynucleoside residues. These Tm values are shown in Table 1 and are compared with those for the normal base pair T‚dA, the diol D (13) opposite dA, and the model abasic site F (2) opposite dA. It is interesting and significant that there is little difference between the Tm for dR‚dA and that for F‚dA, especially considering the extensive use of F as a model abasic site. In conclusion, we have developed a relatively simple method for the generation, postsynthetically, of the natural 2′-deoxyribose abasic site in oligomeric DNA having any base composition. The method involves the very mild periodate oxidation of the homologous 3-deoxyhexitol residue in a reaction that is quantitative and without side products. The 3-deoxyhexitol is completely resistant to the chemistry of DNA synthesis, and its 6-ODMT-4-O-phosphoramidite 10 is easily incorporated into DNA. This new approach to the production of an abasic site embedded in DNA has distinct advantages when compared with other chemical methods that have been used and is superior, we belive, to the traditional use of uracil DNA glycosylase to produce such sites. By contrast, periodate oxidation is nearly 100% efficient and may be quenched simply by adding an external diol, and complete buffer exchange is easily achieved by gel filtration. DNA containing an abasic site generated by this diol cleavage method was found to be relatively stable at room

temperature, but underwent rapid degradation when it was heated, when it was treated with a nitrogen base at elevated temperature, or when an attempt was made to isolate the oligomer from the solution containing it, by evaporation to dryness.

Acknowledgment. We thank Mr. Robert A. Rieger for obtaining the mass spectral data, Ms. M. C. Torres for synthesizing the oligodeoxynucleotides, Dr. S. Lokhov for the determination of the thermal stabilities of the oligonucleotide duplexes, and Dr. D. Zharkov and Dr. N. Bulychev for their very helpful discussions and advice. This research was supported by National Institutes of Health Grant CA47995 awarded by the National Cancer Institute.

References (1) Loeb, L. A., and Preston, B. D. (1986) Mutagenesis by apurinic/ apyrimidinic sites. Annu. Rev. Genet. 20, 201-230. (2) Lindahl, T., and Nyberg, B. (1972) Rate of depurination of native deoxyribonucleic acid. Biochemistry 11, 3610-3617. (3) Lindahl, T. (1982) DNA repair enzymes. Annu. Rev. Biochem. 51, 61-87. (4) Friedberg, E. C. (1985) DNA Repair, Freeman and Co., New York. (5) Demple, B., and Harrison, L. (1994) Repair of oxidative damage to DNA: Enzymology and biology. Annu. Rev. Biochem. 63, 915948. (6) Takeshita, M., Chang, C. N., Johnson, F., Will, S., and Grollman, A. P. (1987) Oligodeoxynucleotides containing synthetic abasic sites. Model substrates for DNA polymerases and apurinic/ apyrimidinic endonucleases. J. Biol. Chem. 262, 10171-10179. (7) Vasseur, J. J., Rayner, B., and Imbach, J.-L. (1986) Preparation of a short synthetic apurinic oligonucleotide. Biochem. Biophys. Res. Commun. 134, 1204-1208. (8) Wilde, J. A., Bolton, P. H., Monoharan, M., Ransom, S. C., and Gerlt, J. A. (1989) Characterization of the equilibrating forms of the aldehydic abasic sites in duplex DNA by 17O NMR. J. Am. Chem. Soc. 111, 1894-1895. (9) Wang, K. Y., Parker, Sh. A., Goljer, I., and Bolton, P. H. (1997) Solution structure of a duplex DNA with an abasic site in a dA tract. Biochemistry 36, 11629-11639. (10) Bertrand, J.-R., Vasseur, J.-J., Rayner, B., Imbach, J.-L., Paoletti, J., Paoletti, C., and Malvy, C. (1989) Synthesis, thermal stability and reactivity towards 9-aminoellipticine of double-stranded

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(11) (12) (13)

(14) (15) (16)

(17) (18) (19)

Chem. Res. Toxicol., Vol. 13, No. 9, 2000 oligonucleotides containing a true abasic site. Nucleic Acids Res. 17, 10307-10319. Bailly, V., and Verly, G. (1987) Escherichia coli endonuclease III is not an endonuclease but a β-elimination catalyst. Biochem. J. 242, 565-572. Sugiyama, H., Hosoda, M., and Saito, I. (1990) Covalent alkylation of DNA with duocarmycin A. Identification of abasic site structure. Tetrahedron Lett. 31, 7197-7200. Mazumder, A., Gerlt, J. A., Absalon, M. J., Stubbe, J., Cunningham, R. P., Withka, J., and Bolton, P. H. (1991) Stereochemical studies of the β-elimination reactions at aldehydic abasic sites in DNA: endonuclease III from Escherichia coli, sodium hydroxide, and Lys-Trp-Lys. Biochemistry 30, 1119-1126. Randall, S. K., Eritja, R., Kaplan, B. E., Petruska, J., and Goodman, M. F. (1987) Nucleotide insertion kinetics opposite abasic lesions in DNA. J. Biol. Chem. 262, 6864-6870. Takeuchi, M., Lillis, R., Demple, B., and Takeshita, M. (1994) Interactions of Escherichia coli endonuclease IV and exonuclease III with abasic sites in DNA. J. Biol. Chem. 269, 21907-21914. Manoharan, M., Ransom, S. C., Mazumder, A., Gerlt, J. A., Wilde, J. A., Withka, J. M., and Bolton, P. H. (1988) Enzymatic synthesis of abasic sites in DNA heteroduplexes and their characterization by site specific labeling with 13C. J. Am. Chem. Soc. 110, 16201622. Peoc’h, D., Meyer, A., Imbach, J.-L., and Rayner, B. (1991) Efficient chemical synthesis of oligodeoxynucleotides containing a true abasic site. Tetrahedron Lett. 32, 207-210. Kumar, N. V., and Varshney, U. (1994) Inefficient excision of uracil from loop regions of DNA oligomers by E. coli uracil DNA glycosylase. Nucleic Acids Res. 22, 3737-3741. Piersen, C. E., McCullough, A. K., and Lloyd, R. S. (2000) AP lyases and dRPases: commonality of mechanism. Mutat. Res. 459, 43-53.

Shishkina and Johnson (20) Uracil-DNA glycosylase. Technical bulletin published by New England Biolabs (1999). (21) Vandendriessche, F., Snoeck, R., Janssen, G., Hoogmartens, J., Van Aerschot, A., De Clercq, E., and Herdewijn, P. (1992) Synthesis and antiviral activity of acyclic nucleosides with a 3(S),5-dihydroxypentyl or 4(R)-methoxy-3(S),5-dihydroxypentyl side chain. J. Med. Chem. 35, 1458-1465. (22) Khullar, S., Varaprasad, C. V., and Johnson, F. (1999) Postsynthetic generation of a major acrolein adduct of 2′-deoxyguanosine in oligomeric DNA. J. Med. Chem. 42, 947-950. (23) Hossain, N., Blaton, N., Peeters, O., Rozenski, J., and Herdewijn, P. A. (1996) Synthesis of homo-N-nucleosides, a series of C1′ branched-chain nucleosides. Tetrahedron 52, 5563-5578. (24) Desilylation of compound 8 gave a mixture of the D-arabino- and D-ribo-3-deoxyhexitols as was evident from the optical rotation ([R]20D -18.3° in water, c 0.82). The known isomers have rotations of -27° and -10°, respectively, under essentially the same conditions [Wood, H. B., and Fletcher, H. G. (1961) 2-Deoxy-Dribose. VI. The preparation of derivatives of 3-deoxy-D-ribohexonic acid and 3-deoxy-D-arabino-hexonic acid therefrom. Some observations on the Kiliani Synthesis. J. Org. Chem. 26, 19691973]. The 13C NMR of 11 (not reported) also confirmed this conclusion. (25) Magdalena, J., Fernandez, S., Ferrero, M., and Gotor, V. (1999) Synthesis of novel carbazoyl linked pyrimidine-pyrimidine and pyrimidine-purine dinucleotide analogues. Tetrahedron Lett. 40, 1787-1790. (26) Torres, M. C., Rieger, R. A., and Iden, C. R. (1996) Characterization of the alkaline degradation products of an oligodeoxynucleotide containing 8-oxo-7,8-dihydro-2′-deoxyguanosine by electrospray ionization mass spectrometry. Chem. Res. Toxicol. 9, 1313-1318.

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