Characterization of the Alkaline Degradation Products of an

Characterization of the Alkaline Degradation Products of an Oligodeoxynucleotide Containing 8-Oxo-7,8-dihydro-2'-deoxyguanosine by Electrospray Ioniza...
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Chem. Res. Toxicol. 1996, 9, 1313-1318

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Characterization of the Alkaline Degradation Products of an Oligodeoxynucleotide Containing 8-Oxo-7,8-dihydro-2′-deoxyguanosine by Electrospray Ionization Mass Spectrometry M. Cecilia Torres, Robert A. Rieger, and Charles R. Iden* Department of Pharmacological Sciences, The State University of New York at Stony Brook, Stony Brook, New York 11794-3400 Received June 27, 1996X

Oligodeoxynucleotides containing 8-oxo-7,8-dihydro-2′-deoxyguanosine exhibit alkaline sensitivity and undergo cleavage of the phosphodiester backbone. Identification of the major degradation products and unstable intermediates formed in concentrated ammonia was accomplished by HPLC isolation and characterization by electrospray ionization mass spectrometry. Unstable intermediates were reduced in situ with NaBH4 prior to isolation and mass analysis. This technique produced accurate mass data for an oligonucleotide intermediate containing an abasic site, a strand cleavage product containing the 3′-terminus, and two products with the 5′-terminus. 8-Oxoguanine was not present in the product HPLC chromatogram, suggesting rearrangement or degradation of this moiety prior to glycosidic bond cleavage. A scheme for the decomposition of 8-oxo-7,8-dihydro-2′-deoxyguanosine-containing oligonucleotides in 28% ammonia solution is presented.

Introduction The occurrence and chemical characteristics of modified nucleobases generated in DNA by a variety of physical and chemical agents have been investigated for their potentially important role in mutagenesis, carcinogenesis, and aging (1, 2). Oxidation is a common form of DNA damage caused by agents such as ionizing radiation, free radicals, endogenous cellular oxygen species, and other environmental oxidants (3, 4). Among the most widely studied adducts formed by DNA oxidation is 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dGuo).1 It may be formed by reactions of oxygen-containing free radicals (5-7) or singlet oxygen (8) with guanine bases in DNA or by the reaction of water with guanine radical cations formed during photoionization of DNA (9, 10). This substance has been used widely as a biomarker of oxidative damage in DNA (11-13). However, 8-oxo-dGuo lesions may be unstable in basic conditions and may be susceptible to oxidation and modification of the DNA structure. Data on the chemical stability of 8-oxo-dGuo has been derived from experiments with synthetic oligodeoxynucleotides. 8-Oxo-7,8-dihydro-2′-deoxyguanosine can be incorporated into specific positions in oligomers (14, 15) to facilitate the investigation of not only the structure (16) and reactivity of these lesions, but also the enzymatic processes involved in DNA polymerization (17, 18) and base excision repair pathways (19, 20). It is recognized that synthetic oligodeoxynucleotides containing 8-oxodGuo are sensitive to aerial oxidation in 1 N NaOH or concentrated NH4OH (14, 21), and they degrade under * Author to whom correspondence should be directed. Tel: 516-6328867; FAX: 516-632-7394; E-mail: [email protected]. X Abstract published in Advance ACS Abstracts, November 1, 1996. 1 Abbreviations: AP, apurinic; CPG, controlled pore glass; DMT, dimethoxytrityl; ESI, electrospray ionization; FAB, fast atom bombardment; MCA, multichannel averaging; PAGE, polyacrylamide gel electrophoresis; 8-oxo-dGuo, 8-oxo-7,8-dihydro-2′-deoxyguanosine; TEAA, triethylammonium acetate.

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the conditions required for nucleobase deprotection and release from the CPG support after synthesis (28% ammonia at 55 °C, 16 h). Under these conditions, scission of the sugar-phosphate backbone occurs at 8-oxodGuo sites (22); however, protection is afforded by an antioxidant such as β-mercaptoethanol (14, 15). The mechanism for this decomposition has not been specified. Treatment of 8-oxo-dGuo-containing oligomers with 1 M piperidine at 90 °C also causes cleavage at the position of the modified 2′-deoxyribonucleoside (23). A single cleavage product containing the 5′-terminus and another containing the 3′-terminus were found and compared to a sequence ladder on denaturing polyacrylamide gel to identify 8-oxo-dGuo as the cleavage site. However, chemical structures of the products were not determined. Cullis et al. examined the effects of hot piperidine on 8-oxo-dGuo and an oligomer (46-mer) containing 8-oxodGuo (10). 8-Oxoguanine was absent as a product of piperidine treatment of 8-oxo-7,8-dihydro-2′-deoxyguanosine. Based on decreased piperidine-induced cleavage of the 8-oxo-dGuo-containing oligomer in the presence of β-mercaptoethanol, it was suggested that strand breaks are produced by an oxidative mechanism. It has also been reported that permanganate oxidation of 8-oxo-dGuo in DNA oligomers causes damage at the site of the lesion and to the neighboring deoxynucleotide residues as well (24). However, products were characterized by PAGE, and the chemical structures are unknown. Chemical characterization of the products and unstable intermediates of alkaline degradation of an 8-oxo-dGuocontaining oligomer would add significantly to our understanding of the stability of this lesion. Electrospray ionization (ESI) mass spectrometry is a useful technique for the analysis of oligodeoxynucleotides (25, 26), and negative ion ESI has been used to confirm the synthesis of oligonucleotides containing modified bases and to identify the products of unstable oligomer degradation © 1996 American Chemical Society

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occurring during the synthesis procedure (27). It has surpassed the utility of fast atom bombardment (FAB) for analysis of oligomers (28, 29) greater than ten deoxynucleotides in length and is considerably more sensitive than the FAB technique. This low energy ionization process produces multiply charged, gas phase ions directly from an aqueous/organic solution of the analyte making it especially suitable for the analyses of biopolymers, thermally unstable substances, and nonvolatile compounds. It provides accurate mass information from very small quantities of sample (10-100 pmol) with a minimum amount of sample preparation. We have used ESI/MS to identify the dimethoxytritylprotected degradation products obtained from the oligomer 5′-DMT-GTTCAXTTGC-3′, (where X ) 8-oxo-dGuo) when it was digested with 28% ammonia in the absence of β-mercaptoethanol (27). In addition, preliminary studies on the identification of the degradation products obtained when the unprotected oligonucleotide, 5′-GTTCAXTTGC-3′, was digested under the same conditions were described (22). We report here the identification of the alkaline degradation products of an 8-oxo-dGuocontaining oligomer resulting from treatment with concentrated ammonia. Of special interest was the isolation of the abasic form of this oligonucleotide, demonstrating that it is an intermediate in the alkaline degradation scheme.

Experimental Procedures All chemicals were purchased from Fisher Scientific (Pittsburgh, PA) unless otherwise indicated. All solvents were HPLC grade, and triethylamine was freshly distilled before buffer preparation. Sodium borohydride (99%) and methylamine (40% solution in H2O) were purchased from Aldrich Chemical Co. (Milwaukee, WI). Oligodeoxynucleotide, 5′-GTTCAXTTGC-3′ where X is 8-oxo-dGuo, was synthesized on an Applied Biosystems Model 394 DNA/RNA synthesizer using the phosphoramidite chemistry. The 8-oxo-dGuo phosphoramidite was synthesized following the procedure of Bodepudi et al. (14, 15). All other synthesis reagents were obtained from Applied Biosystems (Foster City, CA). The DMT-protected oligonucleotide was purified by HPLC according to the procedure reported elsewhere (27). The HPLC system consisted of a Waters 600-MS multisolvent delivery system, a U6K injector, and a 991 photodiode array detector. Samples were dried under vacuum in a SpeedVac SC100 evaporator (Savant Instruments, Farmingdale, NY). Isolation of the Abasic Oligodeoxynucleotide. About 30 µg of oligodeoxynucleotide were digested with 1 mL of 28% NH4OH in the presence of 4 mg of NaBH4 at 4 °C for 60 h. The reaction mixture was taken to dryness, reconstituted with 400500 µL of water, adjusted to pH 7.5-8.0 with 1 N HCl, and desalted with a Bio-Gel P2 column (Bio-Rad Laboratories, Richmond, CA). The eluent was dried, reconstituted in water, and analyzed by HPLC on a Waters Novapak C18 column (0.39 × 300 mm). Elution was started in the isocratic mode at 5% acetonitrile in 0.1 M triethylammonium acetate (TEAA) buffer (pH 6.8) for 5 min at a flow rate of 1.2 mL/min, followed by a linear gradient from 5% to 15% acetonitrile in 0.1 M TEAA buffer (pH 6.8) in 55 min. Ammonia Treatment and Separation of Degradation Products. Quantities of the oligodeoxynucleotide ranging from 30 to 100 µg were digested in sealed Eppendorf tubes in 1.5 mL of 28% NH4OH in the absence of β-mercaptoethanol at 55 °C for different amounts of time, ranging from 1 to 16 h depending on the products that were being isolated. When required, sodium borohydride (4 mg/mL) was added to the digestion mixture to stabilize a degradation product. In one instance, methylamine (40% solution in water) was used instead of 28% NH4OH. After digestion, the samples were immediately dried under vacuum and reconstituted in buffer for HPLC analysis.

Torres et al. A Waters Novapak C18 column (0.39 × 300 mm) was employed with a linear gradient of 5-15% acetonitrile in 0.1 M TEAA buffer (pH 6.8) in 40 min at a flow rate of 1.0 mL/min. A Polyhydroxyethyl A column (200 × 4.6 mm, Poly LC, Columbia, MD) was used to separate degradation products that were not resolved with the C18 column. Two buffers were used for elution: buffer A was 40 mM TEAA (pH 7.5) in 80% acetonitrile/ water, and buffer B was 400 mM TEAA (pH 7.5) in 5% acetonitrile/water. Elution was started isocratically at 100% buffer A for 5 min followed by a linear gradient of 0-100% buffer B in 40 min at a flow rate of 1.0 mL/min. Fractions were collected, dried under vacuum, and analyzed by ESI/MS as described below. Time Course of Ammonia Degradation of Oligodeoxynucleotide. The oligodeoxynucleotide (3 µg) was incubated at 55 °C in 300 µL of 28% ammonia for ten different time periods (1, 2, 3, 4, 6, 8, 12, 16, 24, and 48 h). Samples were immediately taken to dryness and reconstituted in buffer prior to C18 reverse phase HPLC analysis. HPLC conditions were the same as those described above. ESI Mass Spectrometry. Mass analysis was performed on a TRIO-2000 quadrupole mass spectrometer system (Micromass, Beverly, MA). Samples were diluted to 200 µL/OD with 60% acetonitrile/water and spiked to approximately 1% triethylamine just prior to injection into the electrospray source. The source was supplied with a flow of 60% acetonitrile/water at a rate of 12 µL/min from a Harvard syringe pump. Nitrogen was used as a drying gas (250 L/h) and also as the nebulization gas (15 L/h). The instrument was operated in the negative ion spray mode with the high voltage set to -3.3 kV and the source temperature at 70 °C. The sampling cone was fixed at -54 V, and lenses were adjusted to maximize ion transmission. The data system acquired signal over a m/z range of 200-1500 at 8 s/scan with a 0.06 step. Approximately 8 scans were collected in the multichannel averaging (MCA) mode, baseline adjusted, and digitally filtered to produce a spectrum.

Results Characterization of the Apurinic Oligodeoxynucleotide. Treatment of oligodeoxynucleotide 5′-GTTCAXTTGC-3′, where X is 8-oxo-7,8-dihydro-2′-deoxyguanosine, with 28% NH4OH for 60 h at 4 °C in the presence of NaBH4 produced one major product and several minor products which were separated by C18 reverse phase HPLC (Figure 1a). The major product (A) and the original oligomer (B) could be separated with careful attention to HPLC conditions. The UV spectrum of the product, when compared to that of the original oligomer, showed the loss of the distinctive shoulder at 300-320 nm which is indicative of the 8-oxoguanine moiety in the original oligomer (14, 18). No peak in the HPLC chromatogram was found for 8-oxoguanine, although a pure standard was available for comparison. A pure fraction of the product was collected and analyzed by electrospray ionization mass spectrometry. The mass spectrum (Figure 1b) contains five multiply charged ions with charge states ranging from -3 to -7. The data yielded a molecular mass of 2902.6 Da for the product, corresponding to that expected for an apurinic oligomer formed by the loss of 8-oxoguanine, followed by deoxyribose ring opening and reduction of the aldehyde moiety at C1′ to an alcohol (calculated mass 2902.9 Da). The apurinic oligodeoxynucleotide was also formed and characterized in the same manner when the experiment was repeated with 0.2 N NaOH under the same conditions (data not shown). Products of Basic Degradation. The same oligomer was subjected to treatment with 28% NH4OH at 55 °C for 16 h, standard deprotection conditions after oligode-

Alkaline Degradation of DNA Containing 8-Oxo-dGuo

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Figure 1. (a) HPLC chromatogram of the products of sodium borohydride reduction of the oligodeoxynucleotide 5′-GTTCAXTTGC3′, where X is 8-oxo-dGuo, digest in concentrated ammonia at 4 °C. UV spectra are shown in the inset for the reduced apurinic oligomer (A) and the original oligomer (B). (b) Electrospray mass spectrum of a pure fraction of product (A).

Figure 2. HPLC chromatogram of the products of a 6 h reaction of oligodeoxynucleotide 5′-GTTCAXTTGC-3′, where X is 8-oxo-dGuo, with 28% ammonium hydroxide at 55 °C. Three products (A, B, and C) were well resolved from the original oligomer (D).

oxynucleotide synthesis, without the protective action of β-mercaptoethanol. Three major products of the reaction were separated by reverse phase HPLC (Figure 2), and additional minor products were also present. The first product (A) was collected as a pure fraction and analyzed by electrospray ionization mass spectrometry (Figure 3).

Figure 3. Electrospray mass spectrum of a fraction of product A from the chromatogram in Figure 2 identifying a portion of the original oligomer containing the 3′-terminus.

The molecular weight of the product determined from the spectrum, 1244.7 Da, is consistent with the molecule p-TTGC, the 3′-end of the original oligomer with a 5′terminal phosphate group (calculated mass 1244.8 Da). Products B and C were separated inadequately by the initial HPLC conditions, collected as a single fraction, and rechromatographed on a Polyhydroxyethyl A column. Products B and C were well resolved by this column,

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Figure 4. Electrospray mass spectra of fractions containing (a) product B and (b) product C from the chromatogram in Figure 2 showing two 5′-terminal cleavage products of the oligomer.

collected as pure fractions, and analyzed by ESI/MS (Figures 4a and 4b, respectively). Peak B is a single pure material (1557.5 Da) which corresponds to 5′-GTTCA-p, the 5′-end of the molecule with a 3′-terminal phosphate (calculated mass 1558.0 Da). Two substances appear in the ESI mass spectrum of peak C, indicating further degradation after HPLC purification. One product is the same compound found in peak B, and the second product (molecular mass 1673.5 Da) is a similar product formed after cleavage of the 8-oxoguanine base and β-elimination of the 3′-end of the oligomer (calculated mass 1673.2 Da). It contains an amino sugar moiety from the 8-oxo-dGuo position and continues to degrade after HPLC isolation and/or under the conditions for generating the ESI mass spectrum. Two additional experiments were run to confirm the nature of this unstable intermediate formed after the strand cleavage. First, the mass of the unstable substance in peak C was confirmed by repeating the ammonia digestion at 55 °C in the presence of sodium borohydride to reduce the ring-opened, aldehydic amino sugar to the more stable alcohol. This substance was isolated by HPLC from a number of products; the ESI mass spectrum (Figure 5) of the HPLC fraction containing the reduced product gave the expected molecular mass for this oligomer fragment (measured mass 1674.8 Da; calculated mass 1675.2 Da). In addition, the presence of the amino group in the structure was verified by repeating the reaction with methylamine in place of NH4OH. In this case, ESI/MS confirmed the addition of a methylamino moiety to the product after elimination of the 3′-terminus of the oligomer. A time course for the ammonia degradation of the 8-oxo-dGuo-containing oligomer is shown in Figure 6. Under the conditions of the reaction, the half-life of the oligomer is 2.9 h. Relative concentrations of the major products are also shown. For the construction of this diagram the integrated areas of products B and C were combined and plotted as a single data point for each time period.

Figure 5. ESI mass spectrum of the reduced form of the substance in peak C, Figure 2. Alkaline hydrolysis was done in 28% ammonium hydroxide in the presence of sodium borohydride at 55 °C.

Discussion Negative ion electrospray ionization mass spectrometry has the unique advantage of accurately determining molecular masses of oligodeoxynucleotides, or portions thereof, from solutions or directly from the column effluent of an HPLC. We have used ESI and HPLC offline to determine accurate molecular masses of intermediates in the degradation of an oligomer containing the modified deoxynucleoside, 8-oxo-7,8-dihydro-2′-deoxyguanosine. The scheme, shown in Figure 7, proceeds through the formation of an abasic site by the elimination of 8-oxoguanine under alkaline conditions. This was suggested initially by the nature of the major degradation

Alkaline Degradation of DNA Containing 8-Oxo-dGuo

Figure 6. Time course for the alkaline degradation of oligodeoxynucleotide 5′-GTTCAXTTGC-3′, where X is 8-oxo-dGuo (peak D, Figure 2). The oligomer decays with a half-life of 2.9 h; relative concentrations of products (A) and (B + C) from Figure 2 are also shown.

products and the UV spectrum of the product of sodium borohydride reduction. While the abasic oligodeoxynucleotide is not found in the HPLC chromatogram of an alkaline oligomer digest at 55 °C, it can be isolated from a reaction at 4 °C by sodium borohydride reduction of the ring-opened, aldehydic form of the abasic site. ESI/ MS confirmed the formation of the reduced form of the abasic oligomer. The residual portion of the oligomer, 8-oxoguanine, is not found in the HPLC chromatogram of a digest at 55 or 4 °C, indicating a possible rearrangement of this moiety prior to glycosidic bond cleavage. With our HPLC conditions, a synthetic standard of 8-oxoguanine elutes within the first 5 min of the chromatogram. Minor peaks in this region of the chromatogram did not show the

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distinctive UV absorption for 8-oxoguanine. Thus, the ultimate disposition of the modified nucleobase has not been established, although an oxidative process is suspected since β-mercaptoethanol effectively retards oligomer decomposition (10, 14, 15). Products analogous to those formed in the degradation of oligomers containing deoxyguanosine with (acetylamino)fluorene substituted at the C8-position (30) may have little or no UV absorption and, if formed, may not be observed in the HPLC chromatogram generated by a UV absorbance detector. Other potential compounds include oxidation products characterized from reactions of singlet oxygen (1O2) with 8-oxo-dGuo (31). Further degradation of the abasic oligomer in the presence of concentrated ammonia followed a mechanism similar to that reported for the decay of oligodeoxynucleotides containing aldehydic abasic sites (32, 33). However, we have characterized two different products containing the 5′-terminus of the oligomer. Under alkaline conditions, β-elimination from the deoxyribose moiety released the 3′-terminus of the oligomer, leaving the 5′fragment of the oligomer with an unsaturated sugar moiety. When digestion is accomplished in concentrated ammonia or methylamine, Michael addition occurs at the double bond of the modified deoxyribose to form an amino sugar or methylamino sugar derivative. These substances, moderately unstable in basic solution, were isolated by reverse phase HPLC, and the molecular masses determined by ESI/MS coincided with that of the expected products. In addition, the reduced form of the amino sugar was also characterized by ESI mass spectrometry, indicating addition of ammonia to the C2′ double bond and not Schiff base formation at the C1′ aldehydic site. Finally, the 5′-terminus of the oligomer with a 3′-terminal phosphate, a stable substance easily characterized by ESI/MS, was released by δ-elimination at the deoxyribose moiety. In summary, our data present compelling evidence for the formation of an abasic site during the decomposition

Figure 7. Scheme for the decomposition of an 8-oxo-dGuo-containing oligomer in concentrated ammonia. Calculated masses are shown for the intermediates. R ) 5′-GTTCA; R′ ) TTGC-3′.

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of an 8-oxo-dGuo-containing oligodeoxynucleotide in alkaline solution despite the fact that 8-oxoguanine is not a product of the reaction. Similar results were found in reactions in concentrated ammonia and 0.2 N sodium hydroxide. In addition, two products containing the 5′terminus of the oligomer were isolated by reverse phase HPLC and characterized by ESI/MS. We have demonstrated that these two analytical techniques are invaluable for examining reaction products at the oligodeoxynucleotide level, providing a high degree of chromatographic resolution, facile isolation of pure components, and accurate mass analysis.

Acknowledgment. The authors would like to acknowledge many helpful discussions with Professor Francis Johnson. This research was supported by the National Institutes of Health Grants CA47995 and ES04068.

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