Formation of s-triazines during aerial oxidation of 8-oxo-7,8-dihydro-2'-deoxyguanosine in concentrated ammonia

M. Cecilia Torres, Chamakura V. Varaprasad, Francis Johnson and Charles R. Iden1

Department of Pharmacological Sciences, State University of New York at Stony Brook, Health Sciences Center, Stony Brook, NY 11794-8651, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
After automated DNA synthesis, oligodeoxynucleotides containing 8-oxoguanine are sensitive to aerial oxidation when subjected to the basic conditions necessary for deprotection and release of the oligomer from the control pore glass support. The major oxidation products of this heterocyclic moiety have been characterized by permitting 8-oxo-7,8-dihydro-2'-deoxyguanosine to react with oxygen in the presence of 28% aqueous ammonia at room temperature. Products were isolated by reverse phase HPLC and analyzed by electrospray ionization-mass spectrometry and gas chromatography–mass spectrometry of the trimethylsilyl-derivatives. 2-Amino-4-hydroxy-s-triazine-6-carboxylic acid and 2-amino-4-hydroxy-6-carbamyl-s-triazine were identified by these techniques and standards were synthesized. In addition, GC–MS analysis revealed other oxidation products, including urea, guanidine and 2-deoxyribose, which were not observed by HPLC because these compounds are transparent in the UV region of the spectrum. Both s-triazines were also observed when a purified, synthetic oligodeoxynucleotide containing a single 8-oxoguanine moiety was exposed to the same conditions. Oxidation of 8-oxoguanine appears to parallel the uric acid oxidation pathway, and a mechanistic scheme is proposed to account for the products of degradation.

Abbreviations: BSTFA, N,O-bis(trimethylsilyl)trifluoroacetamide; CPG, control pore glass; dGTP, 2'-deoxyguanosine triphosphate; dGuo, 2'-deoxyguanosine; ESI, electrospray ionization; GC, gas chromatography; MCA, multichannel averaging; MS, mass spectrometry; 8-oxo-dGTP, 8-oxo-7,8-dihydro-2'-deoxyguanosine triphosphate; 8-oxo-Gua, 8-oxoguanine; 8-oxo-dGuo, 8-oxo-7,8-dihydro-2'-deoxyguanosine; PFK, perfluorokerosene; RT, retention time; TEA, triethylamine; TMCS, trimethylchlorosilane; TSP, sodium salt of 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In nuclear and mitochondrial DNA, the 2'-deoxyguanosine moiety (dGuo) is easily oxidized by reactive oxygen species such as free radicals (1), singlet oxygen (2), hydrogen peroxide (35) and other oxidizing agents (6,7), and the lesions that are created have important functions in mutagenesis, carcinogenesis and aging (8,9). 8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo) has received considerable attention because it is a mispairing lesion during DNA replication giving rise to G->T transversions (10,11). This particular adduct may also be formed during DNA replication when 8-oxo-7,8-dihydro-2'-deoxyguanosine triphosphate (8-oxo-dGTP) is substituted for 2'-deoxyguanosine triphosphate (dGTP) by a polymerase. However, 8-oxo-dGuo is a substrate for several DNA base excision repair systems which include MutM and MutY proteins from Escherichia coli and hOgg1 from human cell lines (9,12,13). The modified nucleobase and the 2'-deoxynucleoside have been used extensively as biomarkers of oxidative damage in DNA and sensitive assays employing HPLC with electrochemical detection and gas chromatography–mass spectrometry (GC–MS) have been developed (1416).

Although 8-oxo-dGuo is a primary oxidation product in DNA, it itself is sensitive to oxygen under basic conditions. One of the present authors found that oligonucleotides containing this residue were sensitive to alkali (17), undergoing degradation and fragmentation in ammonia at 55°C in a normal atmosphere. Preliminary studies on the 2'-deoxynucleoside (1 N NaOH at 37°C) were also carried out (17), but no conclusion was reached because products were not isolated. These observations were clarified by Torres et al. (18), who found that under the basic aerobic conditions that are normally used for the deprotection and release of oligomers from the control pore glass (CPG) support (28% NH3, 55°C, 16 h), there is first formed an abasic site. Because of the alkali sensitivity of this configuration, further degradation occurs with strand cleavage and the formation of multiple products.

Oxidation and breakdown of the oligonucleotide backbone can be prevented if an antioxidant such as ß-mercaptoethanol is added to the ammonia solution during deprotection and release of the oligomer from the CPG support (19,20). This confirms the oxidative nature of the degradation process that occurs when oligonucleotides containing an 8-oxo-dGuo lesion are exposed to air in an alkaline medium. The use of antioxidants permits the preparation of intact oligonucleotides which are suitable for mutagenesis and enzymatic studies. Aerial oxidation of these oligonucleotides at physiological pH has not been reported unless they are exposed to ionizing radiation or other oxidation agents such as peroxynitrite (21). However, care must be exercised when enzymatic systems are used if the optimum pH for the enzyme is somewhat alkaline.

The fate of the heterocyclic moiety of the 8-oxo-dGuo residue has never been determined. Nevertheless, there are reports on products obtained from the photo-oxidation of 8-oxo-dGuo (2224) and on the aerial oxidation of the structurally related 8-(arylamino)-2'-deoxyguanosine derivatives even under mild alkaline conditions (20,25,26). The oxidation products found in the latter studies parallel to some degree those obtained from the oxidative degradation of uric acid in alkali (2731). Chemically, uric acid is very similar to 8-oxoguanine (8-oxo-Gua), so similar patterns of degradation might be expected. In the present study we have attempted, therefore, to characterize the major oxidative degradation products of 8-oxo-dGuo in concentrated ammonia and to clarify the mechanistic aspects of the degradation pathway.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
HPLC grade acetonitrile, ammonium hydroxide and triethylamine (TEA) were purchased from Fisher Scientific (Pittsburgh, PA). TEA was freshly distilled before each use. 8-Oxo-dGuo was prepared by catalytic hydrogenation of 8-benzyloxy-2'-deoxyguanosine, and a portion was converted to the 5'-dimethoxytrityl-protected phosphoramidite for DNA synthesis (32). An oligodeoxynucleotide, 5'-GTTCAXTTGC-3', where X is 8-oxo-dGuo, was synthesized on a Perkin Elmer/Applied Biosystems (Foster City, CA) Model 394 DNA/RNA synthesizer using conventional phosphoramidite chemistry, and all synthesis reagents were obtained from Applied Biosystems. The oligomer was purified by HPLC (33). N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), 1% trimethylchlorosilane (TMCS), silylation grade acetonitrile and pyridine were purchased from Pierce (Rockville, IL). All chemicals used in the synthesis of standards were purchased from Aldrich (Milwaukee, WI) unless otherwise indicated.

Ammonia treatment and product isolation
8-Oxo-dGuo (100–300 µg) was incubated with 1 ml 28% ammonium hydroxide at room temperature for 16 h, and the reaction mixture was divided into two fractions and dried under vacuum in a Speed-Vac SC100 evaporator (Savant Instruments, Farmingdale, NY). One fraction was reconstituted in water and analyzed on a Waters HPLC system (Milford, MA) consisting of a 600-MS multisolvent delivery system, a U6K injector and a 996 photodiode array detector. A Waters Novapak C18 column (7.8x300 mm) was eluted initially with 100% water for 5 min at a flow rate of 2.8 ml/min. Thereafter, a linear gradient of 1%/min acetonitrile was programmed for the next 20 min. Fractions were collected and dried under vacuum for further investigation. The remainder of the dried reaction mixture was derivatized for GC–MS analysis using a 1:1 mixture of acetonitrile, 2% pyridine:BSTFA, 1% TMCS. The reagents were purged with argon for a few seconds, capped tightly and heated at 100°C for 30 min.

The purified oligodeoxynucleotide containing a single 8-oxo-dGuo (OD260 = 15) was incubated with 28% ammonium hydroxide for 16 h at room temperature. The resulting mixture was dried under vacuum and reconstituted in water for HPLC analysis. Products were collected as a single fraction from 1 to 8 min of analysis, dried and derivatized for GC–MS as described above. In a separate control experiment, the reaction with 8-oxo-dGuo was repeated, and the products of interest were collected as a single fraction from 1 to 8 min during the HPLC analysis. Unreacted 8-oxo-dGuo was excluded. After drying under vacuum, the mixture was derivatized and analyzed by GC–MS.

Spectroscopic measurements
Electrospray ionization (ESI)-mass spectrometry analysis was performed on each of the collected fractions using a TRIO-2000 mass spectrometer (Micromass, Beverly, MA). Dried samples collected from the HPLC were dissolved in a 1:1 mixture of acetonitrile:2% formic acid and infused into the source with a syringe pump (Harvard Apparatus, Holliston, MA) at 10 µl/min. Nitrogen was used as the drying gas at 300 l/h and also employed for nebulization at 5 l/h. The samples were analyzed in the positive ion mode with the probe voltage set at 3.48 kV. Data were acquired in the multichannel averaging (MCA) mode over an m/z range of 100–500.

GC–MS analysis was performed on a Hewlett-Packard 5970B mass selective detector (EI mode, 70 eV, ion source temperature, 200°C) connected to a Hewlett-Packard 5890A gas chromatograph equipped with a DB-5ms capillary column (25 mx0.2 mm i.d., 0.33 µm film thickness; J&W Scientific, Folsom, CA). The injector port was maintained at 240°C, and the mass spectrometer interface was set at 280°C. The column temperature, initially at 60°C for 2 min, was increased to 140°C at a rate of 30°C/min and further raised to 280°C at a rate of 20°C/min. Helium was used as the carrier gas (0.75 ml/min), and mass spectra were obtained in the full scan mode using 1 µl splitless injections.

Low resolution EI mass analysis (70 eV) was carried out on a HP5980A mass spectrometer using a solid probe for sample introduction. Exact mass measurements (EI mode, 70 eV) were performed on a Kratos MS-890/DS-90 mass spectrometer at a resolution of 10 000. The ion source temperature was 180°C and perfluorokerosene (PFK) was used as a reference.

13C NMR spectroscopy was performed on a Varian Unity 500 NMR spectrometer. Spectra were recorded in D2O using 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid, sodium salt (TSP) as the internal reference. UV absorption spectra were obtained with a Hewlett-Packard 8452A diode array spectrophotometer and FTIR spectra were recorded on a Mattson Galaxy Series 3000 spectrometer.

Preparation of 2-amino-4-hydroxy-s-triazine-6-carboxylic acid
An authentic standard was prepared by oxidation of guanine with H2O2 in alkaline medium using the procedure of Moschel and Behrman (34). UV/vis {lambda}max (H2O) 200.6 nm ({varepsilon} = 27961), 252.2 nm ({varepsilon} = 4236). IR {upsilon}max (KBr), cm–1: 3600–2200 (broad), 1752, 1677, 1602, 1525, 1459, 1377, 1348, 925, 780, 770. 13C NMR [sodium salt, (500 MHz, {delta}, D2O)], p.p.m.: 175.3, 175.2, 174.9, 172.1. EI-MS (70 eV) m/z (relative intensity) 112 [(M-CO2)+, 100], 85 (8), 69 (35), 53 (7). ESI-MS (M+H)+: calculated 157.1 Da; found 157.2 Da. HRMS [trimethylsilyl derivative, (M+ 3TMS)+]: calculated for C13H28O3N4Si3 372.1469, found 372.1454.

Preparation of 2-amino-4-hydroxy-6-carbamyl-s-triazine
bis-Trichloromethyl-1,3,5-triazine-carboxylic acid ethyl ester was prepared in good yield according to the procedure of Grundmann et al. (35), followed by substitution of the trichloromethyl groups and formation of the amide using the method of Kreutzberger (36) with some modifications. After incubation of the ethyl monoester with concentrated ammonia (5–10 mg in 20–40 µl 28% ammonium hydroxide) at 100°C for 4 h, the yield of the desired product was very low. However, using milder conditions (concentrated ammonia at 55°C for 1 h), the yield increased, and the desired product, 4-amino-2-hydroxy-6-carbamyl-s-triazine, was isolated by HPLC [retention time (RT) 5 min using the conditions described above]. UV/vis {lambda}max (H2O) 205.2 nm, 271.1 nm. IR {upsilon}max (KBr), cm–1: 3600–2200 (broad), 1701, 1686, 1655, 1577, 1499, 1437, 1322, 1085, 1000, 930, 800, 698. EI-MS (70 eV) m/z (relative intensity) 155 (M+, 25), 111 [(M+-CONH2), 95], 69 (100), 55 (60). ESI-MS (M+H)+: calculated 156.1 Da; found 156.1 Da. HRMS [trimethylsilyl derivative, (M+ 3TMS)+]: calculated for C13H29O2N5Si3 371.1629, found 371.1632.


    Results
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 Abstract
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 Materials and methods
 Results
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 References
 
Reverse phase HPLC analysis of the products resulting from treatment of 8-oxo-dGuo with concentrated ammonia at room temperature (Figure 1Go) disclosed two major products (A and B) and several minor components. These very polar products were eluted from a Novapak C18 column with 100% water within the first 8 min of the analysis. The only significant peak observed thereafter was unreacted 8-oxo-dGuo, which was eluted at ~15 min. Each of the major products was collected as a pure fraction, rechromatographed and dried. Products in fractions A and B were stable in water at room temperature. ESI-MS was used to determine the molecular masses for the products in these fractions, which were 156.2 and 155.1 Da, respectively.



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Fig. 1. HPLC chromatogram of the products obtained from digestion of 8-oxo-dGuo in 28% NH4OH at room temperature for 16 h. The eluant for the Novapak C18 column was 100% H2O for the first 8 min and a linear gradient of 1%/min CH3CN thereafter.

 
When the reaction mixture was dried, trimethylsilylated and analyzed by GC–MS, the total ion chromatogram contained many products (Figure 2Go). These data were compared with that from the trimethylsilylation of the individual fractions, A and B. Peaks with retention times (RT) of 10.97 and 11.82 min corresponded to the compounds found in fractions A and B from the HPLC analysis. These derivatives had molecular ions at m/z 372 and 371, respectively, indicating the addition of three trimethylsilyl groups to each product. Other products in the reaction mixture were discerned directly from their mass spectra using the library search facility. Nevertheless, the identity of these substances was confirmed by the preparation of trimethylsilylated standards followed by GC–MS analysis. Substances identified in this manner were urea (RT 6.78 min), diastereomers of 2-deoxyribose (RT 8.51 and 8.63 min) and guanidine (RT 8.31 min), none of which absorb in the UV region of the spectrum and which were not detected during the HPLC analysis.



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Fig. 2. Total ion chromatogram from the GC–MS analysis of the trimethylsilylated reaction mixture obtained after incubation of 8-oxo-dGuo in 28% NH4OH at room temperature for 16 h. Derivatization conditions are described in Materials and methods. Column: DB-5ms, 25 m, EI mode, 70 eV, ion source temperature 200°C.

 
Potential structures for substances A and B were developed upon consideration of the oxidation mechanism of uric acid, a chemical analog of 8-oxo-Gua, and two compounds were synthesized by unambiguous routes. 2-Amino-4-hydroxy-s-triazine-6- carboxylic acid was made by the method of Moschel and Behrman (34) and data which support proof of structure for this compound are provided in Materials and methods. The HPLC retention time, UV spectrum and ESI mass spectrum were precisely the same as those for the substance in peak A. Furthermore, the GC retention time and mass spectrum of the trimethylsilyl derivative were also identical. The second compound, 2-amino-4-hydroxy-6-carbamyl-s-triazine, was prepared with minor modifications to a literature preparation (35,36) and the chromatographic and spectroscopic data obtained for this compound and the trimethylsilyl derivative were precisely the same as those generated by the substance in peak B.

GC–MS analysis of the reaction mixture resulting from incubation of 5'-GTTCAXTTGC-3', where X is 8-oxo-dGuo, in 28% ammonium hydroxide at room temperature revealed, among other products, the same two substituted symmetrical triazines reported above. The total ion chromatograms of the trimethylsilylated reaction products from the modified oligodeoxynucleotide (Figure 3aGo) and from the monomer, 8-oxo-dGuo (Figure 3bGo), contained identical peaks corresponding to the two s-triazines described above. In each case, the reaction mixture was subjected to HPLC prior to derivatization and GC–MS analysis. Peaks with RT ~9.44 and 10.25 min are the trimethylsilylated s-triazines, 2-amino-4-hydroxy-s-triazine-6-carboxylic acid and 2-amino-4-hydroxy-6-carbamyl-s-triazine, respectively. Their structure and corresponding mass spectra are shown in Figure 4Go.



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Fig. 3. Peaks containing the tris–trimethylsilyl-s-triazines appeared at ~9.44 and 10.25 min in (a) the total ion chromatogram from the GC–MS analysis of the trimethylsilyl products obtained after incubation of 5'-GTTCAXTTGC-3', where X is 8-oxo-dGuo, in 28% NH4OH at room temperature for 16 h (the reaction mixture was partially purified by HPLC before derivatization and products eluting from 0–8 min were collected and dried as a single fraction to yield the analytical sample), and (b) the total ion chromatogram of the trimethylsilylated reaction mixture obtained after incubation of 8-oxo-dGuo in 28% NH4OH at room temperature for 16 h and subsequent partial purification by HPLC. Products eluting from 0–8 min (Figure 1Go) were collected and dried as a single fraction before derivatization. GC column: DB-5ms, 20 m, EI mode, 70 eV, ion source temperature 200°C.

 


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Fig. 4. Mass spectra of trimethylsilylated HPLC products A and B (Figure 1Go). (a) Mass spectrum of isolated and derivatized product A. (b) Mass spectrum of isolated and derivatized product B. Fragmentation patterns of these two products match exactly the mass spectra of derivatized standards 2-amino-4-hydroxy-s-triazine-6- carboxylic acid and 2-amino-4-hydroxy-6-carbamyl-s-triazine, respectively.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
8-Oxo-dGuo, located site-specifically in synthetic oligodeoxynucleotides, has shown sensitivity to aerial oxidation under the strongly basic conditions necessary for nucleobase deprotection and release of the oligomer from the CPG support (17). Although formation of an abasic site at the location of the 8-oxo-Gua has been demonstrated (18), the disposition of the 8-oxo-Gua moiety has never been determined. We have identified two substituted s-triazines, urea, guanidine and 2-deoxyribose as products of aerial oxidation of 8-oxo-dGuo. One of these compounds, 2-amino-4-hydroxy-s-triazine-6-carboxylic acid, was reported in the oxidation of 8-oxo-dGuo by 1 N KOH at 40°C using much stronger oxidizing conditions, i.e. peroxodisulfate, potassium permanganate or hydrogen peroxide (34). Urea and guanidine were also found as products under these conditions. Identification of the s-triazine carboxylic acid was based solely on comparison of its infrared spectrum with that of an authentic sample (34). We have unequivocally characterized 2-amino-4-hydroxy-s-triazine-6-carboxylic acid as one of the products resulting from the aerial oxidation of 8-oxo-dGuo. The second product, 2-amino-4-hydroxy-6-carbamyl-s-triazine, has not been described previously as an oxidation product of 8-oxo-dGuo.

Given the chemical similarity of 8-oxo-Gua to uric acid, analogous patterns of oxidation might be expected. For example, oxonic acid or potassium oxonate has been reported in the alkaline oxidation of uric acid using either potassium hydroxide, potassium permanganate or hydrogen peroxide (2731). By analogy, the corresponding 2-amino-4-hydroxy-s-triazine-6-carboxylic acid might be expected from the oxidation of 8-oxo-dGuo. However, in the case of 2-amino-4-hydroxy-6-carbamyl-s-triazine, the analogous compound from the oxidation of uric acid, 2,4-dihydroxy-6-carbamyl-s-triazine, has not been reported, even when oxidation was performed in concentrated ammonia with potassium ferricyanide (37).

Based on the structures of the s-triazine compounds and by analogy to uric acid oxidation, a mechanism for the aerial oxidation of 8-oxo-dGuo in 28% ammonium hydroxide at room temperature is proposed in Figure 5Go. 8-Oxo-dGuo is oxidized rapidly to products III and IV by a mechanism similar to that reported for the aerial oxidation of 8-(arylamino)-2'-deoxyguanosine adducts (25,26). Pyrimidine ring-opening and loss of carbon dioxide (28,29,38) would follow to afford allantoin- and 4-iminoallantoin-type compounds, V and VI, respectively. 4-Imino analogs of allantoin have been reported as intermediates in the oxidation of uric acid in the presence of ammonia (37) or primary or secondary amines (39). Similar products were also described in the electrochemical oxidation of uric acid when ammonium acetate was used in the course of the analysis (40). The authors postulated that intermediates of this kind, produced during aerial oxidation of uric acid in basic media, result from cleavage of the pyrimidine but not of the imidazole portion of the purine ring. However, further alkaline hydrolysis and oxidation of V and VI would open the imidazole ring, leading to unstable compounds VII and VIII. Recyclization affords the triazine derivatives IX and X, respectively. s-Triazine formation from ring rearrangement reactions of allantoin-type intermediates formed during alkaline oxidation of uric acid have been suspected and reported (27,28,30,38,41), but the exact mechanism is still unclear. It is also possible that under alkaline conditions, the amide X is hydrolyzed to the acid IX, giving rise to a second route of formation for the latter substance, although this is more likely to occur at higher temperatures.



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Fig. 5. Scheme for the formation of s-triazines from the decomposition of 8-oxo-dGuo in 28% NH4OH at room temperature.

 
In conclusion, two highly substituted s-triazines, 2-amino-4-hydroxy-s-triazine-6-carboxylic acid and 2-amino-4-hydroxy-6-carbamyl-s-triazine, have been unequivocally identified as products of aerial oxidation of 8-oxo-dGuo at room temperature in 28% ammonium hydroxide. Although these are the most stable products, other compounds absorbing in the 230–250 nm region of the spectrum are present in the reaction mixture and can be detected by HPLC and GC–MS. Some are unstable in NH4OH and H2O and difficult to characterize fully. Finally, the finding that compounds IX and X are also found when oligonucleotides containing 8-oxo-dGuo are digested in concentrated ammonia at room temperature is a significant step towards elucidating the previously unknown fate of 8-oxo-Gua when it is oxidized and released from the oligodeoxynucleotide backbone.


    Notes
 
1 To whom correspondence should be addressed Email: charlie{at}pharm.sunysb.edu Back


    Acknowledgments
 
The authors would like to thank Dr Sivaprasad Attaluri for the synthesis of 8-oxo-7,8-dihydro-2'-deoxyguanosine and Mr Leon Kachan and Mr Robert Rieger for mass spectrometer analyses. We also thank Mr Francis Picart for acquiring the NMR spectra and Dr James Marecek for obtaining the infrared spectra. This research was supported by the National Institutes of Health under grants ES04068 and CA47995.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received June 19, 1998; revised September 28, 1998; accepted October 2, 1998.





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