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Chem. Res. Toxicol. 1999, 12, 906-916
Formation and Reactions of N7-Aminoguanosine and Derivatives† F. Peter Guengerich,*,‡ Ralf G. Mundkowski,‡,§ Markus Voehler,| and Fred F. Kadlubar⊥ Departments of Biochemistry and Chemistry and Center in Molecular Toxicology, Vanderbilt University, Nashville, Tennessee 37232, and National Center for Toxicological Research (HFT-100), Jefferson, Arkansas 72079 Received June 1, 1999
Arylamines are mutagens and carcinogens and are thought to initiate tumors by forming adducts with DNA. The major adducts are C8-guanyl, and we have previously suggested a role for guanyl-N7 intermediates in the formation process. N7-Aminoguanosine (Guo) was synthesized and characterized, with the position of the NH2 at N7 established by twodimensional rotating frame Overhauser enhancement NMR spectroscopy. In DMF, N7-NH2Guo formed C8-NH2Guo and the cyclic product C8:5′-O-cycloGuo. In aqueous media, these products were formed along with 8-oxo-7,8-dihydroGuo, N7-NH2guanine, and a product characterized as a purine 8,9-ring-opened derivative (N-aminoformamidopyrimidine). The rate of aqueous decomposition of N7-NH2Guo increased with pH, with a t1/2 of 10 h at pH 7 and a t1/2 of 2 h at pH 9. The rate of migration of NH2 from N7 to C8 is fast enough to explain the formation of C8-NH2Guo from the reaction of 2,4-dinitrophenoxyamine with Guo but not the formation of C8-(arylamino)Guo in the reaction of Guo with aryl hydroxylamine esters; however, the fluorenyl moiety may facilitate the proposed rearrangement by stabilizing an incipient negative charge in the transfer. In the reaction of Guo with N-hydroxy-2-aminofluorene and acetylsalicylic acid, a peak with the mass spectrum expected for N7-(2-aminofluorenyl)Guo was detected early in the reaction and was distinguished from C8-(2-aminofluorenyl)Guo. NMR experiments with [8-13C]Guo also provided some additional support for transient formation of N7-(2-aminofluorenyl)Guo. We conclude that a guanyl-N7 intermediate is reasonable in the reaction of activated arylamines with nucleic acids, although an exact rate of transfer of an N7-arylamine group to the C8 position has not yet been quantified. The results provide an explanation for the numerous products associated with modification of DNA by activated arylamines. However, the contribution of “direct” reaction at the guanine C8 atom cannot be excluded.
Introduction Aryl and heterocyclic amines have long been of interest because of their demonstrated carcinogenicity in experimental animals and humans and their occurrence in industry, pyrolyzed food, and environmental sources such as tobacco smoke (1-3). A large body of evidence has been developed which indicates that these compounds initiate cancer by binding to DNA and producing genotoxic effects such as mutations (1). The amines do not react directly with DNA but require enzymatic hydroxylation and then attachment of a good leaving group (e.g., acetyl or sulfoxy) to the oxygen of the hydroxylamine. Loss of the leaving group (including oxygen) can leave a relatively long-lived nitrenium ion that reacts with a nucleophile; however, † This work was supported in part by U.S. Public Health Service Grants R35 CA44353 and P30 ES00267. * To whom correspondence should be addressed: Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, TN 37232-0146. Telephone: (615) 322-2261. Fax: (615) 322-3141. E-mail: guengerich@ toxicology.mc.vanderbilt.edu. ‡ Department of Biochemistry, Vanderbilt University. § Current address: Abteilung Klinische Pharmakologie, Institut fu ¨r Experimentelle und Klinische Pharmakologie und Toxikologie, Universita¨t Rostock, D-18055 Rostock, Germany. | Department of Chemistry, Vanderbilt University. ⊥ National Center for Toxicological Research.
the reaction can have SN1 or SN2 characteristics depending upon the conditions (4-8). The major DNA adducts formed from these amines are most often formed at the Guo or dGuo N2 and C8 atoms (1, 9). The minor N2-guanyl adducts can be explained by the reaction of the somewhat nucleophilic N2 atom with a delocalized nitrenium ion or its carbenium ion equivalent. The C8-guanyl adducts are predominant, however, and more complex to explain (Scheme 1). The C8 atom of guanine is also a common site for radical reactions (Scheme 1d) (8, 10), but the available evidence does not support a mechanism involving arylamine radicals (4). A caged electron transfer reaction (Scheme 1c) can also be considered but currently has no experimental support. One proposal is that of a guanyl-N7 intermediate (Schemes 1b and 2), which was raised at least as early as 1972 (11, 12). Both this laboratory (13) and subsequently Novak’s group (6) have isolated stable N7arylamine derivatives of C8,N9-Me2Gua, in which migration to the C8 position is blocked (Scheme 3). Kohda’s group has also reported an N7-(4-nitroquinoline N-oxide)Guo adduct formed from Guo, on the basis of 1H NMR (14). The existence of a guanyl-N7 adduct can explain several other phenomena associated with modification of DNA by arylamines. Both depurination (12, 15) and the
10.1021/tx990094u CCC: $18.00 © 1999 American Chemical Society Published on Web 08/20/1999
N7-Aminoguanosine Scheme 1. Some Mechanistic Possibilities for C8-Guanyl Arylamine Adduct Formation
Scheme 2. A Stepwise Mechanism for C8-Guanyl Arylamine Adduct Formation via an N7-Guanyl Intermediate (13)
Scheme 3. Formation of a C8-Me Guanyl-N7-Arylamine Adduct (13)
formation of 8-oxodGuo (16) have been postulated to be the result of N7-guanyl intermediates. Further, Sodum and Fiala isolated N2-NH2Guo, 8-oxoGuo, and C8NH2Guo from RNA treated with NH2OSO3 or from rats treated with 2-nitropropane (as well as the deoxyribose analogues from DNA) and explained these products with a guanyl-N7 intermediate (17). The suggestion has also been made that activated arylamines react with DNA to generate guanine imidazole ring-opened products (1822) [although the products reported by Kriek and Westra (21) may be explained by oxidative degradation (23)]. Although our own work with C8-MeGua derivatives had demonstrated that a guanyl-N7 adduct could be formed at a rate fast enough to support a role as an intermediate in overall C8-arylamination (13), the migration of an amino or arylamino moiety from the N7 to the C8 atom had not been clearly established. We considered the general problem of the existence of guanyl-N7 intermediates using two approaches. First, N7-NH2Guo was generated from the reaction of Guo with 2,4-dinitrophenoxyamine [a model developed by Kohda (24)], and the reactions of N7-NH2Guo were characterized. Second, arylhydroxylamine compounds were reacted with Guo, and efforts were made to detect guanyl-N7-substituted intermediates.
Experimental Procedures Reagents. Guo, dGuo, N7-MeGuo, 8-NH2Guo, C8-oxodGuo, C8-BrGuo, 7-MeGuo, and ribose 1-phosphate were purchased from Sigma Chemical Co. (St. Louis, MO). Ethyl N-hydroxyacetamidate and 1-chloro-2,4-dinitrobenzene were obtained from
Chem. Res. Toxicol., Vol. 12, No. 10, 1999 907 Aldrich Chemical Co. (Milwaukee, WI). [8-13C]Gua (>98 at. %) was purchased from Cambridge Isotopes (Cambridge, MA). 8-OxoGuo was a gift from D. H. Swenson (Saginaw Valley State University, Midland, MI). C8-NH2Gua was prepared by acid hydrolysis of C8-NH2Guo (1 N HCl at 70 °C for 1 h) (25). Recombinant purine nucleoside phosphorylase was a gift from L. J. Marnett (Department of Biochemistry, Vanderbilt University). 2-Fluoro-O6-(trimethylsilylethyl)deoxyinosine was a gift from C. M. Harris (Department of Chemistry, Vanderbilt University). Chromatography and Spectrocopy. (1) HPLC and UV. UV spectra were recorded (in HPLC buffer at pH 5.0) either with a Cary 14/OLIS instrument (On-Line Instrument Systems, Bogart, GA) or on-line with a HPLC system using a ThermoSeparation Products UV3000HR rapid scanning spectrophotometer (Thermo-Separation Products, Piscataway, NJ). HPLC was carried out using a Spectra-Physics 8700 pumping system (Thermo-Separation Products) and a 10 mm × 250 mm Beckman Altex (10 µm) octadecylsilane column (Beckman, Santa Clara, CA), with gradients generated from mixtures of (i) 10 mM NH4CH3CO2 (pH 5.0) and (ii) CH3OH/H2O (9:1, v:v) in the case of analytical and preparative work monitored by UV spectrometry. (2) NMR. NMR spectra were obtained with either a Bruker AM-400 (400.13 MHz for 1H), AC-300 (300.10 MHz), or DRX500 (500.13 MHz) instrument (Bruker, Billerica, MA) in the Vanderbilt facility. Chemical shifts are reported in parts per million for 1H relative to the internal standard (CH3)4Si. The two-dimensional ROESY1 experiment was performed at 500.13 MHz, using a 250 ms pulsed spin lock for the mixing time (26, 27), at 295 K, with 2048 data points in the acquisition domain and 225 increments with 16 scans each. The spectral width was optimized to 5681 Hz in both dimensions, and a relaxation time of 1.5 s was utilized. The data were processed using XWINNMR software, a 1K × 1K matrix, linear prediction in the second dimension, and a squared sine-bell apodization function. 13C spectra were recorded at 125.77 MHz and 298 K with waltz proton decoupling, 16K data points, a 30° carbon pulse, a 1.5 s relaxation delay, and 768 scans. The data were processed with an exponential apodization function of 1 Hz. (3) Mass Spectrometry. In the case of electrospray ionization (ESI) mass spectrometry, HPLC was carried out at ambient temperature using a HP1090 II module (Hewlett-Packard, Palo Alto, CA) coupled to the mass spectrometer, a guarded Partisil C18 column (3.2 mm × 250 mm, 5 µm, Phenomenex, Torrence, CA), and a binary solvent system comprised of 10 mM NH4CH3CO2 (pH 4.5) and CH3OH. Elution was carried out at a flow rate of 200 µL/min by combining isocratic stages and a linear gradient starting at 2% CH3OH (v/v), holding for 3 min, increasing to 95% by 33 min, and holding until 48 min. Mass analysis was performed using a Finnigan TSQ7000 triple-stage instrument (Finnigan, Sunnyvale, CA) interfaced with the HPLC system by an electrospray ion source. N2 was used as the sheath gas (386 kPa/56 psi); no auxiliary stream was applied. Ion source and tune parameters were adapted to the HPLC flow rate and analyte properties by infusing dGuo (0.1 mM) as a standard. The ESI needle was set to a potential of 3.6 kV. The heated capillary was maintained at 195 °C. Collision-induced dissociation (CID) was carried out using Ar at a pressure of 200 mPa (1.5 mTorr) at a collision offset of -12.0 V. In parallel with the mass data, the UV absorbance (A256) was recorded (delay time of m/z registration of 8 s) using an Applied Biosystems 785A variable-wavelength detector (Applied Biosystems/Perkin-Elmer, Foster City, CA) and the signal acquired with the software of the spectrometer. 1 Abbreviations: MS, mass spectrometry; CID, collision-induced dissociation; ESI, electrospray ionization; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; FDNB, 1-fluoro-2,4-dinitrobenzene; 8-oxoGuo, 7,8-dihydro-8-oxoGuo; N-NH2 FAPY glycoside, 2-amino-5-(N-amino-N-formylamino)-6-(N-glycosylamino)-4-oxo-3,4-dihydropyrimidine; ROESY, rotating frame Overhauser enhancement spectroscopy.
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(4) Kinetic Analysis. For the formation of amination products of Guo, Guo or dGuo (2.5 mg) was mixed with 9 mg of 2,4-dinitrophenoxyamine in 1 mL of a DMF/H2O mixture (3:1, v:v) in an amber glass vial under Ar at 45 °C. At different times, 25 µL aliquots were analyzed by HPLC, using a Beckman 10 mm × 250 mm octadecylsilane column and increasing the concentration of CH3OH in 10 mM NH4CH3CO2 buffer (pH 5.0): 1% during the first 12 min, increasing from 1 to 4.5% from 12 to 15 min, and increasing from 4.5 to 22.5% from 15 to 30 min (all % v:v). The eluate was monitored by collecting data from 220 to 350 nm with a rapid scanning spectrophotometer. The A254 traces were used in the kinetic analysis, and spectra were used for peak verification. In the analysis of N7-NH2Guo decomposition kinetics, N7NH2Guo was repurified by preparative HPLC, using a slight modification of the system described above for analysis, and diluted directly into buffers at different pHs. The sample was held at room temperature under Ar in amber glass, and at various times, aliquots were analyzed by HPLC using the system described above. Synthesis. (1) 2,4-Dinitrophenoxyamine. 1-Chloro-2,4dinitrobenzene was reacted with ethyl N-hydroxyacetamidate in the presence of 1 equiv of KOH to yield ethyl O-(2,4dinitrophenyl)hydroxamate in 53% yield (28): mp 110-111.5 °C (uncorrected) (lit. 110-112 °C); UV (C2H5OH) 308 ) 10 300 M-1 cm-1; 1H NMR (C2HCl3) δ 1.40 (t, 3H, CH2CH3), 2.25 (s, 3H, NdCCH3), 4.22 (q, 2H, CH2CH3), 7.89 (d, 1H, J ) 9.4 Hz, aryl H-3), 8.41 (dd, 1H, J ) 2.7, 9.3 Hz, aryl H-5), 8.90 (d, 1H, J ) 2.7 Hz, aryl H-6). The product (2.17 g) was treated with 70% HClO4 in 1,4-dioxane on ice to yield 2,4-dinitrophenoxyamine, with the reaction monitored by TLC (silica gel G, 3:1 v:v CH2Cl2/cyclohexane), in 62-84% yield for several preparations (29). The phenoxyamine was carefully crystallized from hot C2H5OH: mp 102-105 °C (lit. 112-113 °C); UV (C2H5OH) 298 ) 10 500 M-1 cm-1; 1H NMR (C2HCl3) δ 6.39 (s, 2H, NH2), 8.06 (d, 1H, J ) 9.4 Hz, aryl H-3), 8.45 (dd, 1H, J ) 2.7, 9.4 Hz, H-5), 8.82 (d, 1H, J ) 2.6 Hz, H-6). The phenoxyamine was mixed with acetophenone to yield the hydroxamate derivative: mp 169-172 °C (uncorrected) [lit. 175 °C (30)]. (2) [8-13C]Guo. [8-13C]Gua (120 mg in 80 mL of 1 N HCl, 10 mM) was added to 400 mL of 0.20 M Tris base and the pH adjusted to 7.0, followed by the addition of 300 mg of ribose 1-phosphate and 9 mg of purine nucleoside phosphorylase (31). HPLC analysis (25) indicated that the reaction was >90% complete in 1 h (23 °C). The reaction mixture was applied to a 4.5 cm × 50 cm Sephadex G-10 column (Pharmacia, Piscataway, NJ), previously equilibrated with 25 mM CH3CO2H (pH 3.5), and eluted with the same solvent (room temperature). Fractions containing Guo, as judged by A254 measurements, were combined and concentrated by lyophilization. [8-13C]Guo was purified by preparative HPLC using a 10 mm × 250 mm Beckman octadecylsilane column (10 µm) using 4% CH3OH (v/v) in 10 mM NH4CH3CO2 (pH 5.0), and the material containing [8-13C]Guo was combined and concentrated by lyophilization. (3) N2-NH2dGuo (2-Hydrazinodeoxyinosine) (32). 2-FluoroO6-(trimethylethysilyl)deoxyinosine (18 mg, 49 µmol) was reacted with NH2NH2‚H2O (1.0 µmol) in 1.0 mL of DMF at 50 °C for 24 h under Ar. Glacial CH3CO2H (0.25 mL) was added, and the mixture was heated at 50 °C for 2 h. The product was separated by preparative HPLC (5 to 45% CH3OH gradient over the course of 25 min, v:v, in 10 mM NH4CH3CO2, pH 5.0, flow rate of 4 mL/min) and concentrated by lyophilization to yield ∼3 mg of recovered product: UV (HPLC solvent) λmax ) 254, 265-290 nm (shoulder); MS m/z (relative intensity) 283 (MH+, 23), 167 (MH+ - deoxyribose, 100); 1H NMR (Me2SO-d6) δ 2.19 (m, 1H, H-2′), 2.50 (m, 1H, H-2′′), 3.48 (m, 2H, H-5, H-5′′), 3.78 (dd, 1H, H-4′), 4.33 (s, 1H, OH), 4.93 (dd, 1H, H-3′), 5.27 (s, 1H, OH), 6.12 (t, 1H, H-1′), 7.92 (s, 1H, H-8), 8.30 (s, broad, 1H, NH). In typical HPLC systems, the compound was eluted immediately prior to dGuo. (4) 2-NHOH-Fluorene, 2-[15N]NHOH-Fluorene, and 4NHOH-Biphenyl. The N-hydroxyamino derivatives of fluorene
Guengerich et al. and biphenyl were prepared by ammonium bisulfite reduction of their corresponding nitroarenes as described previously (33, 34). 2-[15N]Nitrofluorene was synthesized by nitration of fluorene with Na15NO3. Briefly, 100 mg of fluorene was dissolved in 5 mL of glacial CH3CO2H and added to a solution of 54 mg of Na15NO3 in 3 mL of dry CF3CO2H. After the solution had been mixed, the reaction was allowed to proceed for 24 h at room temperature. The crude product was precipitated by addition of 20 mL of H2O, collected by centrifugation, washed with H2O, and then dried in vacuo. The residue was dissolved in benzene and further purified by column chromatography on neutral alumina using the same solvent as the eluant. The yellow band was collected; the product was crystallized by addition of hexane and dried in vacuo. The yield of 2-[15N]nitrofluorene was 51% and was judged to be >98% pure by HPLC: UV (HPLC solvent) λmax ) 229 nm; MS m/z (relative intensity) 212 (M+, 100), 195 (M+ - OH, 34), 165 (M - 15NO2, 93). (5) C8,N9-Me2-N7-(2-[15N]Aminofluorenyl)Gua. This was prepared from the reaction of 8,9-Me2Gua with 2-[15N]NHOHfluorene and acetylsalicylic acid and isolated by HPLC as described previously (13). (6) C8-(2-[15N]Aminofluorenyl)Guo. This compound was prepared by the reaction of Guo with 2-[15N]NHOH-fluorene and acetylsalicylic acid and isolated by HPLC (13). (7) N7-NH2Guo. The material was prepared by reaction of Guo with excess 2,4-dinitrophenoxyamine in 3:1 v:v DMF/H2O at 45 °C under Ar. For the material used to analyze the kinetics of degradation, 5.8 mg of Guo was incubated with 23 mg of 2,4dinitrophenoxyamine. The sample used to obtain the rotational rotating frame Overhauser enhancement spectroscopy (ROESY) NMR spectrum was prepared from a mixture of 25 mg of Guo and 105 mg of 2,4-dinitrophenoxyamine. The reactions were monitored by analytical HPLC, and after ∼2 days, the solvent was removed by lyophilization following the addition of H2O. The residue was dissolved in HPLC buffer using sonication and purified by preparative HPLC using the Beckman 10 × 250 mm octodecylsilane column and slight modification of the program used for analysis. The peak was collected, concentrated by lyophilization, repurified using the same HPLC system, concentrated by lyophilization, and stored in a desiccator at -20 °C. Spectral Characterization of Reaction Products (2,4Dinitrophenoxyamine and Guo Reaction or Decompostion of N7-NH2Guo). (1) N7-NH2Guo (24): UV λmax 259, 278 nm (shoulder); MS m/z (relative intensity) 299 (M+, 43), 167 (M+ - ribose, 100); 1H NMR (Me2SO-d6) δ 3.57 (m, 1H, H-3′), 3.68 (m, 1H, H-5′′), 3.95 (m, 1H, H-4′), 4.11 (m, 1H, H-3′), 4.41 (dd, 1H, H-2′), 5.73 (d, 1H, H-1′), 6.33 (s, broad, 2H, 2-NH2), 6.94 (s, broad, 2H, N7-NH2), 9.08 (s, 1H, H-8) (see Figure 3 for NMR). (2) C8:5′-O-CycloGuo (24): UV λmax 252, 280 nm; MS m/z (relative intensity) 282 (MH+, 57), 304 (M + Na+, 100), 321 (M + K+, 75); CID [m/z 282 (relative intensity)] daughter ion at m/z 168 (MH+ - ribose, 30); 1H NMR (Me2SO-d6) δ 3.85 (m, 1H, H-5′), 3.92 (m, 1H, H-5′′), 4.13 (m, 1H, H-4′), 4.40 (m, 1H, H-3′), 4.50 (m, 1H, H-2′), 5.75 (d, 1H, H-1′). (3) 8-OxoGuo (35, 36): UV λmax 247, 294 nm; MS m/z (relative intensity) 300 (MH+, 22), 168 (MH+ - ribose, 100); 1H NMR (Me2SO-d6) δ 3.55 (m, 2H, H-5′, H-5′′), 3.78 (m, 1H, H-4′), 4.09 (m, 1H, H-3′), 4.82 (t, 1H, H-2′), 5.58 (d, 1H, H-1′), 6.48 (s, broad, NH), 6.58 (s, 1H, NH). (4) C8-NH2Guo: UV λmax 256, 291 nm; MS m/z (relative intensity) 299 (MH+, 91), 321 (M + Na+, 95), 337 (M + K+, 45), 167 (M - ribose, 100); 1H NMR (Me2SO-d6) δ 3.60 (m, 2H, H-5′, H-5′′), 3.86 (m, 1H, H-4′), 4.07 (m, 1H, H-3′), 4.53 (m, 1H, H-2′), 5.02 (d, 1H, 5′-OH), 5.26 (d, 1H, 3′-OH), 5.50 (m, 1H, 2′-OH), 5.73 (d, 1H, H-1′), 5.96 (s, 2- or 8-NH2), 6.14 (s, 2- or 8-NH2), 6.58 (s, 1H, N1H). (5) N7-NH2Gua: UV λmax 244, 286 nm; MS m/z (relative intensity) 167 (MH+, 100), 149 (MH+ - H2O, 35); CID [m/z 167 (relative intensity)] m/z 150 (MH+ - NH3). (6) 2-Amino-5-(N-amino-N-formylamino)-6-(N-glycosylamino)-4-oxo-3,4-dihydropyrimidine (N-NH2 FAPY glyco-
N7-Aminoguanosine
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Figure 1. HPLC separation of products of the reaction of 2,4dinitrophenoxyamine with Guo. The reaction time was 154 h (3:1 v:v DMF/H2O at 45 °C).
Figure 2. UV spectra of products of the reaction of 2,4dinitrophenoxyamine with Guo. side): UV λmax 272 nm; MS m/z (relative intensity) 317 (MH+, 64), 316 (M+, 100), 299 (M+ - H2O, 35); CID [m/z 317 (relative intensity)] m/z 300 (M+ - NH2, 30), 168 (M+ - ribose, M+ H2O, 100).
Results Reaction of Guo with 2,4-Dinitrophenoxyamine. Preliminary experiments indicated that 2,4-dinitrophenoxyamine was a more effective reagent for forming derivatives than NH2OSO3. The separation of some products is shown in Figure 1. When dGuo was used instead of Guo, the profiles were dominated by products with shorter retention times, indicative of depurination products (vide infra). Therefore, further work was focused on Guo. The major products were characterized by their UV (Figure 2), mass, and NMR spectra (Scheme 4). Standard 8-oxoGuo and C8-NH2Guo were available for direct comparisons, and their UV spectra are also relatively characteristic. As reported by Sodum and Fiala (32), both N2-NH2Guo and N2-NH2dGuo elute immediately prior to Guo and dGuo, respectively, in similar HPLC systems. N2-NH2dGuo was synthesized and used in comparisons. The spectrum of N2-NH2(d)Guo is similar to that of Guo but did not match the major product eluted immediately prior to Guo (Figures 1 and 2). The compound was identified as C8:5′-O-cycloGuo by its spectral properties (24). We presume that some N2-NH2Guo is also formed (32) but is obscured by the C8:5′-O-cycloGuo peak. The structure of the polar compound is assigned as N-NH2 FAPY glycoside on the basis of UV and mass spectra; the 1H NMR spctra of such compunds are very complex (37).
The UV spectrum of the product identified as N7NH2Guo is displaced from that of Guo by 7 nm (Figure 2) and resembles that of authentic N7-methylGuo (not shown, obtained under the same conditions). The mass spectrum, which only exhibited peaks at m/z 299 (relative abundance M+ or MH+) and 167 (base peak, - ribose), clearly established the addition of only NH2 or O (16 amu), and several other possibilities could be excluded (e.g., C8-NH2 and 2-NH2). The 1H NMR spectrum (Figure 3) indicated that the H-8 proton was still present when the material was dissolved in Me2SO-d6 but readily exchanged in 2H2O, as expected for an N7-substituted Guo derivative. An extra set of NH2 protons was present (δ 6.95). Efforts to elucidate the linkage of these with oneand two-dimensional nuclear Overhauser effect experiments were unsuccessful, but the ROESY NMR clearly showed that these protons interacted with the H-8 proton at δ 9.1, which in turn interacted with the δ 5.8 proton, which can only be assigned as H-1′ (Figure 3B). Kinetics of Formation of Products from Guo and 2,4-Dinitrophenoxyamine. The kinetics of the reaction were monitored over the course of 250 h (45 °C). The 254 values of the compounds are rather similar (Figure 2), and a plot of the A254 peak integral units versus time is presented as a reasonable approximation of the rates of formation (Figure 4). The peak identified as N7-NH2Guo appears earlier, and the other products are subsequently formed. The level of 8-oxoGuo increases only after the level of N7-NH2Guo reaches a steady state, and the level of C8-NH2Guo increases after the level of N7-NH2Guo declines. The pattern is consistent with the role of N7NH2Guo as a precursor of the other products, including C8:5′-O-cycloGuo. Decomposition of N7-NH2Guo. N7-NH2Guo was stable enough to be isolated, concentrated, and used in spectroscopy experiments (e.g., Figure 3). The t1/2 of N7NH2Guo in DMF was 190 h at room temperature, as judged by HPLC measurements. When N7-NH2Guo was isolated by HPLC (NH4CH3CO2/ CH3OH), concentrated by lyophilization, and rechromatographed (HPLC), the major products were C8-NH2Guo, 8-oxoGuo, and C8:5′-O-cycloGuo, the latter previously reported by Kohda (24) (Figure 5). C8-NH2Guo may be derived in part from the NH4+ in the buffer, because in DMF the major products formed were C8:5′-O-cycloGuo and C8-NH2Guo, with the amount of the former being ∼2 times greater than the amount of the latter. The transformation of N7-NH2Guo to C8-NH2Guo was more rapid when N,N-diisopropyl-N-ethylamine was added to the DMF solution. The stability of N7-NH2Guo was examined in aqueous media (room temperature) as a function of pH (Figure 6). The compound was considerably more stable at acid pH. At pH 7, the t1/2 was 10 h, and at pH 9, the t1/2 was 2 h. The product distribution varied with pH. HPLC of products and the time courses for the pH 4.3 and 7.2 reactions are shown in Figure 7. Yields are relative, based upon A254 measurements in the absence of accurate standards for some of the products. However, the sum of the A254 areas of the products was roughly equal to that of the N7-NH2Guo that decomposed. At pH 4.3 (Figure 7), N7-NH2Gua predominated, followed by C8:5′-O-cycloGuo. At pH 7.2 (Figure 7B), the N-NH2 FAPY glycoside product predominated (also at pH 9.3). In the region of pH 5.0-6.1, C8:5′-O-cycloGuo was the predominant prod-
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Scheme 4. Products of the Reaction of Guo with 2,4-Dinitrophenoxyamine, with N7-NH2Guo as a Postulated Intermediate
Figure 3. 1H NMR spectrum of N7-NH2Guo. The ROESY spectrum is shown in the inset.
Figure 5. Decomposition products of N7-NH2Guo. N7-NH2Guo was purified by HPLC, concentrated by lyophilization, and analyzed by HPLC in the same system [10 mM NH4CH3CO2 (pH 5.0)/CH3OH].
Figure 4. Time course of the formation of products from the reaction of Guo with 2,4-dinitrophenoxyamine (3:1 v:v Me2SO/ H2O at 45 °C). R-O-NH2 is 2,4-dinitrophenoxyamine.
Figure 6. Decomposition of N7-NH2Guo as a function of pH in aqueous media.
uct, with 8-oxoGuo also being significant (Figure 7). At all pH values, C8-NH2Guo was a minor product. Source of Oxygen in 8-OxoGuo. The formation of 8-oxo(d)Guo is usually associated with processes involving partially reduced and other reactive oxygen species (38, 39). Most of the work reported here was carried out under Ar, to minimize such processes, as well as potential oxidation reactions occurring with guanyl-C8 amino derivatives (23). To determine the source of the oxygen in 8-oxoGuo, N7-NH2Guo was isolated by HPLC, diluted with an equal volume of H218O (>95 at. %, thus diluted
to 47%), and left standing for 72 h at room temperature. Analysis by MS (HPLC/ESI/full scan and also peak integration from m/z traces) indicated a 39% excess of the m/z 302 ion (cf. m/z 300 ion being MH+), indicating that most of the oxygen (∼90%) had come from H2O. Control experiments with 8-oxoGuo in H218O indicated incorporation of 99.9% of any contaminating Guo and reported that the result could not be attributed to the presence of residual Guo (13), Kennedy et al. (6) correctly pointed out that we had used a 50-fold excess of Guo over 2-NHOH-fluorene and the results could still be explained by contaminating Guo. To address this issue, we purified C8-BrGuo by HPLC and then repurified the material using the same system (98 at. % 15N. A similar set of studies was designed with [8-13C]Guo and 2-NHOHfluorene (Figure 8). Two small peaks with shifts expected for an N7-aminoGuo derivative were observed in repeated NMR experiments. These were seen at ∼15 min and disappeared with time. The roles of these as intermediates cannot be elucidated in these experiments, because the other 13C product peaks were seen earlier and persisted, including the C8-(2-aminofluorenyl)Guo signal (143.3 ppm). However, the exact temporal relationship is unclear because of the low sensitivity of the 141 ppm region signals. An early eluting HPLC peak with the expected m/z value for N7-NH2Guo was observed at early times in the reaction, and this peak disappeared with further reaction time. Conclusions. N7-NH2Guo has been prepared according to a method described by Kohda (24) and characterized, and its reactions exhibit many of the properties characteristic of arylamine reactions with DNA. Transfer of the N7-NH2 group to C8 occurs but is not fast enough for us to conclude that such transfer must be obligatory in the formation of C8-arylamine adducts. However, the presence of an aryl moiety may be expected to facilitate transfer considerably. In the reaction of Guo and (acetylated) 2-NHOH-fluorene, some evidence was obtained for a transient product considered to be a guanyl-N7 adduct, which might rearrange to the C8 adduct. If guanyl-C8 arylamine adducts are formed in this way, then the aryl substituent would probably alter the step that is ratelimiting.
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We have now provided evidence that guanyl-N7 adduct formation is feasible in the case of arylamines (13) and that N7 to C8 transfer is also, at the very least in the case of N7-NH2Guo. This mechanism may be viable for arylamines. Nevertheless, we cannot rule out the possibility that a fraction of a “direct” reaction occurs (Schemes 1a and 8). However, the N7 scheme is certainly preferred in explaining the side products of arylamine reactions, which appear to be considerable on the basis of our work and the literature (Scheme 8).
Acknowledgment. We thank Dr. M. Persmark for his assistance with the 15N NMR spectroscopy.
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