Chem. Res. Tonicol. 1989,2, 416-422
416
Structural and Conformational Analyses of 8-Hydroxy-2’-deoxyguanosine Sandra J. Culp,* Bongsup P. Cho, Fred F. Kadlubar, and Frederick E. Evans HFT-100, National Center for Toxicological Research, Jefferson, Arkansas 72079 Received August 25, 1989
Oxidative DNA damage has been shown to involve formation of 8-hydroxy-2’-deoxyguanosine, which may serve as a mispairing lesion during cellular DNA replication. In order to assess the mutagenic potential of this DNA adduct, we examined the possible occurrence of several tautomeric forms and of different base conformations about the deoxyribose. Several spectroscopic and electronic absorption techniques were employed and showed structural changes occurring over a broad p H range. Two pK,’s near pH 8 and 12 were observed by pH-solvent partitioning experiments, ultraviolet absorption spectral analyses, and 13CNMR spectroscopic methods. The presence of two pK,’s suggested the formation of a dianion, with the second being formed in strong alkali. A comparison of ultraviolet absorption band features of 8-hydroxy-2’-deoxyguanosine with that of different CG,C&diketo or enol derivatives supported a C8-keto assignment and also provided evidence that this DNA adduct contains a C6-keto group at physiological pH. I3C NMR data showed marked chemical shifts a t C6 in solutions of p H 7.4-9.3, indicating the location of the first ionization. Increasing basicity produced further shifts at C5 and C8, indicating the C8 position for the second ionization. Multiple infrared bands were observed in the carbonyl region of the neutral compound, but only a single carbonyl band at 1692 cm-l remained a t p H 9.1, implying a keto group at C8. Determination of the chemical shifts and the nuclear Overhauser enhancements of the N1 and N7 resonances in the proton-decoupled 15N NMR spectrum indicated that both nitrogens were indeed protonated a t neutral pH. A study of the N1 and N7 chemical shift patterns of 8-hydroxy-2’-deoxyguanosine, 2’-deoxyguanosine, guanosine, and adenosine provided further support for the pyrrole-like nature of these nitrogens. From the a characteristic downfield shift of lH and 13CNMR spectrum of 8-hydroxy-2’-deoxyguanosine, H2’ and an upfield shift of C2’, as compared to 2‘-deoxyguanosine, indicated a conformational change from anti to syn about the N9-C1’ linkage. Thus, these data establish the structure of 8-hydroxy-2‘-deoxyguanosine as the 6,8-diketo tautomer in the syn conformation and provide a basis for base mispairing and mutations.
Introduction Oxidative DNA damage, induced by ionizing radiation and other oxygen radical generating systems, is known to involve DNA strand scission, base deletion, and formation of modified DNA bases (1-3). One of the major products of oxidative base damage, 8-hydroxy-2’-deoxyguanosine (8-0H-dG),’ has received considerable attention as a consequence of its demonstrated mutagenic potential (4). This hydroxylated product appears to be generated via formation of hydroxyl radicals and has been detected in numerous systems known to produce reactive oxygen species. These systems include oxygen radical initiators such as reducing agents (5),asbestos (6),polyphenols, and metals (7), many of which are also known to be mutagenic or carcinogenic. It has been shown that misreading occurs in DNA templates containing 8-OH-dG, both at the modified base and at adjacent positions ( 4 ) . The basepairing properties of 8-OH-dG during DNA replication may be a direct consequence of its modified structure and thus may determine the accuracy of DNA replication; however, the precise structure of 8-OH-dG has not been established. There are at least four possible tautomeric forms involving the N1, C6, N7, and C8 positions (Figure 1) and two alternative molecular conformations between the base and deoxyribose moieties, anti or syn, none of
* To whom correspondence
should be addressed.
which are easily identified by routine spectroscopic techniques. It is difficult to unambiguously distinguish between the N-H (amide) and 0-H (enolic) bonded protons in lH NMR and between the NH-C=O and N=C-OH carbons in the 13C NMR spectrum. Even infrared (IR) spectroscopy, a normal procedure for determining the presence of the carbonyl functional group, is complicated by NH, deformation bands that appear in the same spectral region. Evidence in support of different tautomers and conformations for &hydroxylated guanine derivatives has been presented previously. Ab initio molecular orbital calculations have suggested that the 6,&diketo and 6-enol-&keto tautomers are the most energetically stable forms of the base derivative 8-hydroxyguanine(8). Another 8-hydroxy analogue, 9-ethyl-&hydroxyguanine,was reported to reside in the 6,8-diketo form by X-ray crystallographic analysis (9). Previous NMR studies have tentatively assigned the structure of the ribose derivative, 8-hydroxyguanosine,as the 6,gdiketo form on the basis of chemical shift arguments (IO,1 1 ) . Kuchino et al. ( 4 ) have also mentioned unpublished studies which imply that 8-OH-dG is prone to adopt the syn conformation, as opposed to the normal anti conformation of unmodified DNA bases. The syn conformation is significant in view of its ability to distort Abbreviations: 8-OH-dG,8-hydroxy-2’-deoxyguanosine;NOE,nuclear Overhauser enhancement.
0 1989 American Chemical Society
Structural and Conformational Analyses of 8-OH-dG
8
Y
dR
6,E-diketo
8
dR
6-enol,&keto QH
dR
dR
6-keto,&enol 6,E-dienol Figure 1. Tautomeric forms of 8-OH-dG. the DNA molecule into a position that is unsuitable for normal base pairing. The growing interest in the biological implications of &OH-dG in cellular DNA and the absence of unequivocal structural information led us to undertake a rigorous characterization of this DNA adduct. By use of indirect methods and model compounds, and conducting experiments with 8-OH-dG in solution at various pH's, the necessary structural information was obtained. The data described herein provide assignment of a 6,B-diketo tautomeric structure for 8-OH-dG at physiological pH and provide spectroscopic evidence for the syn conformation.
Materials and Methods Materials. 2'-Deoxyguanosine,guanosine,and adenosine were purchased from Sigma Chemical Co. (St. Louis, MO). 8-OH-dG was synthesized from 2'-deoxyguanosine according to literature procedures (5,12). The product was characterizedby HPLC using ultraviolet and electrochemical detection (13) and by electronimpact mass spectral analysis: M+ at m/z 283; M+ [pentakis(trimethylsilyl)]at m/z 643. Acid-Base Titration and Solvent Partitioning. For pHsolvent partitioning experiments (14) and UV spectrophotometric studies (I5,16), buffer solutions of pH 2.0-9.0 were prepared by mixing 0.05 M solutions of citric acid, KzHP04/KHzP04,and NaHC03. Buffers outside this range were obtained by adding 1N HCl or 1N NaOH. Stock solutions of 5.6 mM SOH-dG were prepared, and aliquots (12 pL) were added to l-mL buffer solutions to provide a final concentrationof 67 rM. The solutions were partitioned with two separate l-mL volumes of water-saturated 1-butanol. The organic extracts were removed and evaporated to dryness under a stream of argon; and the residues were reconstituted in 1mL of 10mM potassium phosphate buffer, pH 7. The original solutions, the reconstituted fraction, and the remaining aqueous layer were each analyzed by the HPLC/ electrochemical detection system and by ultraviolet spectrophotometry to determine the amount of 80HdG partitioned from each buffer. All experiments were performed in duplicate or triplicate. Instrumentation. The HPLC system consisted of a Waters instrument equipped with a Beckman Analytical 5-pm C18 U1trasphere ODs column (4.6 X 250 mm) with a mobile phase of 12.5 mM sodium citrate-25 mM sodium acetate buffer (pH 5.2)/methanol (8515) and a flow rate of 1 mL/min. Electrochemicaldetedion was accomplished with a Bioanalytical Systems dual LC-4B amperometric detedor, using a glassy carbon working electrode and Ag/AgCl reference electrode (working potential of +0.8 V). For preparative HPLC, a Waters pBondapak C18 Semi-prep reversed-phasecolumn (7.8 X 300 mm) was used; the mobile phase was water/methanol(8515); and the flow rate was 3 mL/min. Ultraviolet spectra were recorded on a Beckman DU-65 spectrophotometer. Infrared spectra were recorded with a Bio-Rad FTS-40 Fourier transform infrared spectrometer, and samples were prepared as KBr pellets (1mg of sample/75 mg of KBr). Alkaline samples of &OH-dG and 2'-deoxyguanosinewere prepared by adjusting the pH of aqueous samples with NaOH,
Chem. Res. Toxicol., Vol. 2, No. 6, 1989 417 followed by lyophilization and mixing with KBr under an argon atmosphere. Deuteration was achieved by dissolving and stirriig in DzO prior to lyophilization. NMR spectra were recorded in the 13C,15N,and 'H configurations on a Bruker AM 500 spectrometer utilizing lo-, lo-, and 5mm probes, respectively. For aqueous samples (HzO),a capillary containing dioxane in DzOwas used as a deuterium lock and an external reference, and the N M R chemical shifts are reported in ppm downfield from tetramethylsilane by assigning the dioxane peak to 66.58 ppm. For 19C NMR measurements obtained in dimethyl sulfoxide, the highest intensitypeak (at 39.5 ppm) was used as an internal reference. Natural abundance 15N NMR measurements were carried out by utilizing a coaxial capillary containing 0.2 M I5N-enrichedNaN03 in D20 as an external reference, with chemical shifts reported in ppm downfield from anhydrous liquid ammonia by assigning the external reference to 376.53 ppm (17). Aqueous samples for 13C and 15NNMR spectra were prepared in HzOand the pH values adjusted with NaOH or HC1. Typical NMR data acquisition conditions for 125.8-MHz 13Cspectra were as follows: data size, 32K; sweep width, 22K; recycle time, 2.0 s. For 50.7-MHz proton-coupled 15N spectra, conditions were as follows: data size, 64K; sweep width, 30K; flip angle, 40"; recycle time, 2.0 s. Proton-coupled and -decoupled NMR spectra were obtained by gated and power-gated decoupling. Proton-coupled 15NNMR spectra were obtained with no decoupling. Proton-decoupled spectra were taken with continuous decoupling (Waltz 16). Spectra were recorded at 28-32 "C.
Results and Discussion Determination of pK,'s. The four tautomeric forms of 8-OH-dG shown in Figure 1would be expected to show marked differences in their respective pKa values. For comparison, 2'-deoxyguanosine has an acidic pKa resulting from protonation of N7, with ionization to the cation in the range of pH 2-3.5, which is typical of guanine derivatives with a pyridine-like nitrogen in the imidazole ring (18, 19). 2'-Deoxyguanosine derivatives containing an N1-H bond also exhibit a basic pKa due to N1 deprotonation and formation of an enolate at C6. Guanine derivatives that contain protons on both N1 and an imidazole nitrogen can undergo dissociation to a dianion. The second pKa is at extremely alkaline pH (>12.0), due to the negative charge of the first ionization suppressing the second one (18, 19). These results allow predictions to be made concerning the ionization of 8-OH-dG. If the imidazole ring of 8OH-dG is in the C8-enol form, then N7 would undergo protonation in acid and an acidic pKa would result. Conversely, if the C8-keto form is already protonated a t N7, no acidic pKa would be observed. For the pyrimidine portion of 8-OH-dG, a C6-keto group, as occurs in 2'deoxyguanosine, would allow deprotonation of N1, resulting in a basic pKa. Likewise, a basic pKa would be observed for dissociation of N7-H as in a C&keto structure or of C6-OH as in a C6-enol structure. To investigate these properties and relate them to the tautomeric forms of 8-OH-dG, the pKa of this compound was determined. One method that has been used to determine pK,'s involves monitoring the change in the partition coefficient as a function of pH (14). As shown in Figure 2, the partition titration of 8-OH-dG at pH 1-12.7 indicated that an ionization occurred between pH 7 and 8, denoting a structural change in this pH range. Attempts to collect evidence for additional pKis occurring above pH 8 were complicated by slow degradation under highly alkaline conditions (>pH 12.0). However, evidence for the existence of a second ionization was obtained under conditions where the amount of 8-OH-dG recovered in the aqueous layer at pH 12.7 after butanol extraction was >go%. The same solvent partitioning experiment was
418 Chem. Res. Toxicol., Vol. 2, No. 6, 1989
Culp et al.
*I
loot
F
P
16.3
i l5
65 164-
: y.
p i 0
160.
0
. . .
8 a
m 0
6
156-
ln e
-
JU
152148-
._C
I :
E
"
' 4 1406 0
75
90
105
120
135
PH 0
2
6
4
8
10
12
pn
Figure 2. Partitioning titration of gOH-dG using 1-butanol(cf. Materials and Methods).
Figure 4. 13C NMR chemical shifts, in ppm downfield from tetramethylsilane, of the base carbons of 8-OH-dG at pH 6-13 (cf. Materials and Methods). Table I. UV Spectral Parameters of 8-OHIG and Its Derivatives compound deoxyguanosine
8-OH-dG
PH 7.0
12.6
nm"
~IMS?
mM-' 255 (270) 12.9 (8.9) 254-264 9.8 246,293 12.6, 9.7 241, 280 10.2, 8.0 258 (280) 11.6 (10.7) 250,298 13.1, 10.1 250,280 12.2, 10.5 249, 294 11.9, 10.3 A,,,.=,
7.0 8.8 12.9 7-methyl-8-oxoguanosineb 1.0, 7.0 11.0 1-methyl-8-oxoguanosineb 1.0, 7.0 11.0 261,300 1.0, 7.0, 11.0 252, 296 1,7-dimethyl-8-0~0guanosineb
11.9, 9.4
13.7, 10.1
"The parentheses indicate a shoulder or point of inflection. Taken from ref 24.
I
I
200
250
300
Wavelength, nm
Figure 3. UV spectra of 8-OH-dGat different pH's. The pH values are indicated for each spectrum (cf. Materials and Methods). conducted for 2'-deoxyguanosine, and the predicted pKis around pH 2-3 and pH 10 were observed. Ultraviolet spectra of SOH-dG were also recorded (vide infra) from pH 1 to 13. Spectra between pH 1 and 7 showed no significant changes. The spectra recorded for alkaline solutions are shown in Figure 3. Several isosbestic points were obtained, indicating the presence of multiple ionic species; and the spectral changes are again consistent with two pK,'s near 8 and 12. The pK, derived from the spectral method may only be approximate due to the instability of %OH-dG at highly alkaline pH. This difficulty was circumvented by the use of 13C NMR. A series of spectra were recorded in the range of pH 6.0-13.0, in an effort to obtain accurate pK, data at alkaline pH. Figure 4 shows a plot of 13C NMR chemical shifts of the base carbons at various pH's. The pK, values obtained are at pH 8.6 and 11.7, confirming the existence of two basic pK,'s. Of particular significance was the lack of an acidic pK,. Both the 6,Sdienol and the &keto-&enol tautomers would have an acidic pK, resulting from protonation of N7. The N3 of the purine ring in 2'-deoxyguanosine does not readily undergo protonation in acid in comparison to the pyridine-like N7 (20); however, protonation of N1 in the 6-
enol-8-keto tautomer is plausible since the basicity of N7 would be greatly decreased. Only for the 6,8-diketo form would no acidic pK, be expected. Two basic pK,'s would be anticipated from both the 6-enol-8-keto and 6,8-diketo forms, resulting from deprotonation of O6 and N7 and of N1 and N7, respectively. The pK,'s for 8-OH-dG at pH 8.6 and 11.7 observed in our experiment are consistent with data obtained for guanine derivatives that undergo dissociation to a dianion from deprotonation of N1 and an imidazole nitrogen. The second pK, occurs at highly alkaline pH due to the negative charge of the first ionization suppressingthe second one (18,19). It is important to note that the observed pK,, (8.6) is about 1log unit lower than that of 2'-deoxyguanosine (19), pK, = 9.4, reflecting the electron-withdrawing effect of the 8-keto group. For comparison, 7-substitution of guanosine or 2'-deoxyguanosine, which decreases the electron density at N7, results in pK, values that are lowered by 2-3 units (21-23). Thus, the pK, data for &OH-dG most closely follow those expected for the 6,&diketo tautomeric structure. Ultraviolet Absorption Spectra. The ultraviolet spectral bands of 8-OH-dG underwent noticeable alterations as the pH of the solution was increased over the range of pH 1-13. Thus,a comparison of ultraviolet absorption at different pHs, which showed multiple isosbestic points (Figure 3))was consistent with the occurrence of two acidic dissociations at N1-H and N7-H. Specifically, three spectral characteristics were noted: those below pKal, between pK,, and pK,, and above pK, (Table I). Spectra recorded between pH 1and 7 produced no significant band shifts. Ultraviolet spectra (Table I) taken of 2'-deoxyguanosine at pH 7.0 and 12.6 showed distinct band changes; but the features were dissimilar to those of 8-
Chem. Res. Toxicol., Vol. 2, No. 6, 1989 419
Structural and Conformational Analyses of 8-OH-dG OH-dG, as would be expected from the electronic effects of the C8 carbonyl moiety. Comparison of the ultraviolet absorption spectra of 8-OH-dG with 2'-deoxyguanosine and its derivatives at various p H s supported a number of structural assignments (Table I), including the position ionized at each of the two pK,'s of 8-OH-dG. The ultraviolet spectra of 8-OH-dG at pH 1-7 corresponded favorably with spectra recorded for 7-methyl-8-oxoguanosine, l-methyl-8-oxoguanosine, and 1,7-dimethyl-8-oxoguanosineat pH 7, all of which exist in a GS-diketo form (24). At pH 11,the absorption spectrum for 7-methyl-8-oxoguanosine also showed similar wavelength maxima to that of 8-OH-dG at pH 8.8, which suggested that both can undergo ionization in base at the Nl-C6 position. Furthermore, both of these spectra were comparable to that observed for 06-methyl-2'-deoxyguanosine (maxima at 248 and 277 nm at pH 71, which contains a methoxy instead of a keto group at C6 (25). Thus, the ultraviolet spectrum of 8-OH-dG above pK,, is consistent with deprotonation of N1 and conversion of the 6,8-diketo derivative to a C6-enolate. Since the first ionization occurs at N 1 4 6 , it then follows that the second pK, at pH 11.7 should be at C8. Accordingly, the ultraviolet spectrum of 8-OH-dG at pH 12.9 showed a 11-nm bathochromic shift (Table I), which is consistent with complete aromatization of the purine ring as would be expected for conversion to a 6,gdienolate. 13CNMR Spectroscopy. In order to relate further the ultraviolet spectral and pK, determinations to specific changes in the structure of &OH-dG, the positions affected at each pK, were determined by recording 13C NMR spectra at various pH's (Figure 4). The largest effects are due to Nl-C6 and N7-C8 ionizations, in accordance with the tautomeric changes that accompany these modifications (26, 27). Thus, the C6 resonance exhibited a significant downfield chemical shift of 6.28 ppm from pH 7.4 to 9.3 consistent with the pK,, (8.6), which confirms the locale of the first ionization. A downfield shift of 4.21 ppm of C2 was observed over the same conditions and is consistent with ionization taking place in the form of a keto-enolate conversion at C6-N1. A less likely possibility is abstraction of a proton from a C6-hydroxyl group originating from a C6-enol tautomer. The shift in C2 is better explained by deprotonation and subsequent changes in bond order/excitation energy at the neighboring Nl-C6 which is three bonds away. The reas opposed to 06, maining base carbons exhibit minimal effects in this pH range, with the exception of the C5 resonance, which is shifted 1.55 ppm upfield due to its ortho position to the enolization site. These observations are consistent with chemical shift data for 06-methyl-2'-deoxyguanosine(26), whose aromaticity in the pyrimidine portion of the purine ring is comparable to that of the 6-enolate-8-keto form. Increasing the basicity above pH 10.0 increases deshielding at C8 and is consistent with the second ionization occurring in the imidazole portion of the purine ring. A downfield shift of 5.68 ppm occurs at C8 from pH 10.3 to 13.0, similar to that reported for C6 near pK,,. Likewise, the C8-keto would be favored over the C8-enol structure as the latter would be more readily deprotonated and expected to ionize before C6. In contrast, if C8 was attached to a hydroxyl group, then N7 would be a pyridine-like nitrogen with two readily available electrons for sharing. One would expect N7 to be protonated with relative ease, and as a consequence an acidic pK, would have been observed. Instead, N7 appears to undergo conversion from a pyrrole-like to a pyridine-like nitrogen as basicity is increased. The chemical shift of C5 (112.00 ppm, 10.61 ppm downfield
1800
1400
1800
1400
1800
1400
1800
1400
Wavenumbers (c")
Figure 5. Solid-state IR spectra of the carbonyl region of 8-
OH-dG (cf. Materials and Methods). (A)pH 7.0. (B)pH 9.1. (C) pH 9.1 after DzO. (D)pH 12.7.
shift) in strong alkali is close to the value of 8-methoxyguanosine (110.82 ppm; Cho et al., unpublished data), which can be attributed to the gain of aromaticity of the imidazole ring system by ionization at C8. Solid-state Infrared Spectral Analyses. Solid-state infrared spectra also provided important information in assigning the structure of 8-OH-dG. The infrared spectrum of a neutral 8-OH-dG pellet showed a complex region between 1600 and 1800 cm-' that can be attributed to one or more carbonyl bands, and to NH2 deformation, NH bending, or C=N stretching vibrations, with the carbonyl signals presumed to be the most intense (Figure 5A). Based on infrared structural investigations of 2'-deoxyguanosine, guanine, and their derivatives (28) and on known spectral frequencies of the various functional groups (29), the following assignments were made. The C6-carbonyl peak of the neutral parent compound, 2'-deoxyguanosine, is revealed in the spectrum at 1690 cm-', followed by a less intense band at 1594 cm-l attributed to NH2 deformation (28). When 2'-deoxyguanosine is made basic at pH 12.7, there is loss of the 1690-cm-' band (data not shown). This loss in intensity upon increasing the pH corresponds to conversion of the C6-keto group to the enolate via deprotonation of N1. The infrared spectrum of 8-OH-dG in base would also be expected to lead to the loss of the C6- or C8-keto groups. Accordingly, the spectrum of 8-OH-dG recorded at pH 9.1 (Figure 5B) showed only two major peaks remaining in the carbonyl region. The peak at 1692 cm-' corresponded to the most intense peak of the neutral spectrum and is consistent with the C8-keto group. The second peak at 1617 cm-' may be assigned to NH2 deformation, although it is not clear why there was an increase in the intensity of the band in alkali. Upon deuteration, the 1692-cm-' peak was essentially unaffected; however, the 1617-cm-' band exhibited a loss of intensity and frequency change (1598 cm-') that was indicative of hydrogen-deuterium exchange at NH2 (Figure 5C). The spectrum of 8-OH-dG at pH 12.7 (>pKa2)revealed a complete loss of band features in the carbonyl region, which corresponds to the expected loss of keto groups at C6 and C8 due to the formation of a dienolate (Figure 5D). These data provide further support for the C6,C8-diketo structure of 8-OH-dG at physiological pH, and they specifically exclude the possibility of 8-enol tautomers. 15NNMR Spectroscopy. The direct involvement of N1 and N7 in the ionization of 8-OH-dG made 15NNMR analyses an important approach to complete the characterization of this structure as the 6,8-diketo tautomer. A
420 Chem. Res. Toxicol., Vol. 2, No. 6, 1989
Gulp et al. H
Table 11. *‘NNMR Chemical Shifts of 8-OH-dG and Its Derivativesa compound solvent (M) N1 N3 N7 N9 NHz HzOb (0.10) 148.2 169.2 110.5 145.6 72.5 8-OH-dG 8-OH-dG DMSO (0.10) 150.8 168.5 109.0 144.2 75.6 deoxyguanosine DMSO (0.37) 148.2 166.9 247.3 174.6 74.3 guanosine DMSO (0.64) 148.2 166.7 248.1 170.9 74.2 adenosine DMSO (1.0) 236.1 223.2 240.9 170.3 82.7 “Expressed in ppm downfield from NHB (cf. Materials and Methods). bpH 7.3.
natural abundance, proton-decoupled 15NNMR, with full nuclear Overhauser enhancement (NOE),l of &OH-dG (pH 7.3) in aqueous solution was obtained and exhibited three singlets at 148.2, 110.5, and 72.5 ppm (Table 11). The spectrum recorded with no decoupling (no NOE) produced two additional resonances at 145.6 and 169.2 ppm. Protonated nitrogens are known to yield the full negative NOE, and signal intensities can be increased as much as four times; while the nonprotonated pyridine-like nitrogen has an unfavorable NOE on the order of -1, which dramatically lowers the intensity of the signal (17). Therefore, the first three resonances were assigned to the exocyclic NH, and the two protonated nitrogens. Since N3 and N9 are not attached to hydrogens, their signals were not apparent in the decoupled spectrum. Thus, assignments for the 148.2, 110.5, and 72.5 ppm resonances were limited to N1, N7, and NH,; while the 145.6 and 169.2 ppm resonances must have originated from N3 and N9. The 15NNMR spectrum of 8-OH-dG in DMSO showed essentially the same chemical shift pattern as that in H,O, suggesting the absence of a solvent effect. This also indicated that N7 was protonated, since a pyridine-like N7 would experience a substantial upfield shift in strongly hydrogen-bonded solvents such as H 2 0 (30). In order to assign further the resonances of 8-OH-dG, the 15N NMR spectrum of the parent compound, 2’deoxyguanosine, was obtained in DMSO, and its chemical shifts were assigned by comparison to the 15N NMR spectra of guanosine and adenosine, which have been previously characterized in DMSO (30). The N1, NH,,and N3 resonances of 2‘-deoxyguanosine in DMSO exhibited chemical shifts that were virtually identical with three of the nitrogen resonances of 8-OH-dG in either solvent (Table 11). Therefore, the resonances at 148.2 ppm (150.8 ppm in DMSO), 72.5 ppm (75.6 ppm in DMSO), and 169.2 ppm (168.5 ppm in DMSO) were assigned to N1, NH2,N3, respectively. Thus,the remaining resonances at 110.5 ppm (109.0 ppm in DMSO) and 145.6 ppm (144.2 ppm in DMSO) were assigned to N7 and N9, respectively. The upfield shifts exhibited by these nitrogens, in comparison to 2’-deoxyguanosine, are consistent with the pyrrole-like N7 of 8-OH-dG and the effect of the neighboring 8-keto group. No one-bond N-H coupling constant at N1 of 8-OH-dG could be observed due to considerable line broadening. Repeated purifications in an effort to remove possible acidic or paramagnetic impurities did not split the resonance, suggesting the broadening may be due to intrinsic properties of the molecule, i.e., the increased acidity of N1-H as compared to that of the parent compounds, 2’deoxyguanosine and guanosine. The electronic effect of the C&keto group may be responsible for the enhancement of the N1-H exchange rate, which is substantiated by the lower pKal (8.6) at N1 of 8-OH-dG. Similar broadening has been observed for the thymidine N3 signal since the presence of two adjacent flanking carbonyl groups make the N3 proton more acidic than in a normal amide function (31). Nonetheless, the N1 chemical shift of the 8-OH-dG
dR‘y-H .....
0‘
iR
-.
syn6,8.dikelo: dC pairing
synB,B.dikelo: dT mispairing
n
H
syn6.8-dikelo: dA mispairing
ant! B-enolate.8.kelo: dG mispairing
Figure 6. Base pairing and mispairing resulting from the syn 6,8-diketoand anti 6-enolate-&keto forms of 8-OH-dG.
adduct is essentially the same as N1 of guanosine and 2’-deoxyguanosine, both of which are known to exist in the 6-keto form (18). In addition, the same chemical shift argument favoring a pyrrole-like N7 can be applied to N1. If N1 of the adduct were in a pyridine-like form, (Le., 6-enol form), then a chemical shift similar to the N1 of adenosine would be expected (Table 11). Therefore, on the basis of chemical shifts and NOE results, the 15N NMR spectral data provide conclusive evidence for the 68-diketo tautomeric form at neutral pH. NMR Conformational Analyses. The anti conformation is typical of most normal nucleosides. However, modification at C8 of purine nucleosides by bulky carcinogens has been shown to induce a change in the glycosidic linkage leading to the syn conformation (32, 33). This conformational change is particularly prone to occur during replication when the DNA helix is unwound. As a result, the O6and N7 atoms of the modified base are placed in position to mispair with N1 and N2 of a guanine or with N6 or N1 of an adenine in the complementaryDNA strand (34). A number of characteristic ‘9 and ‘H NMR patterns have been described relating the effect of C8-substitution on purine base conformation (11,35,36). In comparison to the parent nucleoside, C8-substitution resulted in a significant upfield shift of the C2’ signal and a small downfield shift of C5’ (11,37). We found that the 13C NMR spectrum of 8-OH-dG is in agreement with these observations. The 13C NMR shifts for C2’ and C5’ of 2’-deoxyguanosine in DMSO are 39.89 and 62.08 ppm, respectively. The corresponding chemical shifts for 8OH-dG are 35.36 and 62.24 ppm. Thus, the C2’ of 8-OHdG shifted upfield 4.53 ppm, while C5’ exhibited a 0.16 ppm downfield shift. Similar results were obtained in aqueous solution (below pK,). The ‘H NMR spectrum of 8-OH-dG also showed a characteristic downfield shift (0.47 ppm) of the H2’ resonance compared to that of 2’deoxyguanosine in DMSO, which is in accordance with theoretical predictions for the syn conformation (38). These results are uniquely consistent with those found in several other C8-substituted guanines and demonstrate that the 8-OH-dG adduct in either the 6,8-diketo or 6enolate-&keto form exists preferentially in the syn conformation. Conclusions. In view of the origin and role of 8-OH-dG in oxidative DNA damage, the structural characterization of this DNA adduct has several important consequences for base mispairing and mutations (39). As the 6,B-diketo tautomer in the syn conformation,8-OH-dGwould still be capable of pairing with cytosine resulting in normal base insertion. However, abnormal base pairing (e.g., Hoogsteen pairs) may also occur with the syn conformation (Figure
Structural and Conformational Analyses of 8-OH-dG 6). Thus, mispairing with adenine or thymine could result in a guanine to thymine transversion or a guanine to adenine transition, respectively (Figure 6). Of further significance is that C8-keto substitution of 2’-deoxyguanosine serves to appreciably lower the pK, of N1-H in comparison to that of the parent compound. Therefore, 8% of the molecules should exist in the 6-enolate-8-keto form at physiological pH (pH 7.4), on the basis of the pH dependence of NMR chemical shifts (Figure 4). Although this form of the molecule is also capable of pairing with cytosine, adenine, or thymine, the possibility of an additional mispairing lesion arises from the equilibrium between the syn and anti conformers. In the anti 6-enolate-8-keto form, the modified base could mispair with guanine, resulting in a guanine to cytosine transversion (Figure 6). Thus, the complementary base mispairing reported by Kuchino et al. ( 4 ) in DNA templates containing 8-OH-dG can be rationalized in terms of the structures described in this study. As a result of these observations, it is evident that the 8-OH-dG adduct has biological implications as both a potential mutagenic lesion and a relevant marker for oxidative DNA damage.
Acknowledgment. We thank Drs. Dwight W. Miller and S. Mark Billedeau for their assistance in obtaining FTIR spectra for 8-OH-dG and 2’-deoxyguanosine. One of us (B.P.C.) was supported in part by an appointment to the ORAU Postgraduate Research Program at the National Center for ToxicologicalResearch, which is administered by the Oak Ridge Associated Universities through an interagency agreement between the US. Department of Energy and the US. Food and Drug Administration.
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