Chem. Res. Toxicol. 1990, 3, 445-452
445
15N Nuclear Magnetic Resonance Studies on the Tautomerism of 8-Hydroxy-2‘-deoxyguanosine, 8-Hydroxyguanosine, and Other C8-Substituted Guanine Nucleosides‘ Bongsup
P. Cho,* Fred F. Kadlubar, S a n d r a J. Culp, and Frederick E. E v a n s
Division of Biochemical Toxicology, National Center for Toxicological Research, Food and Drug Administration, Jefferson, Arkansas 72079 Received June 14, 1990 The favored tautomeric and ionic structures were examined for the oxidative DNA damage adduct 8-hydroxy-2’-deoxyguanosineand its RNA analogue 8-hydroxyguanosine by I5N NMR spectroscopy. In addition, 15N chemical shifts and coupling constants from 13 different guanine nucleosides, including a wide variety of C8 substitutions (OH, SH, Br, OCH2C6H5,O C H , S C H , and S02CH3),have been analyzed with respect to their tautomeric structures. A -98.5-Hz proton-nitrogen coupling constant observed for the N7 resonance of 8-hydroxyguanosine in dimethyl sulfoxide was evidence for 8-keto substitution, which is contrary to the structure implied by the generally used nomenclature. The pH dependence of 15N NMR spectra of 8-hydroxyguanosine in aqueous solution showed downfield shifts of the N1 and N7 resonances that were greater than 50 ppm, which indicated the conversion from a neutral 6,&diketo to a 6-enolate8-keto (pKJ = 8.6) and finally to a 6,8-dienolate structure (pK,2 = 11.7). There was no evidence of an &enol substituent in the absence of ionization. It is proposed that the syn conformation of these oxidized bases in duplex DNA and RNA can be further stabilized by abnormal hydrogen bonding or mispairing that involves N7-H. The combined data show that 15N NMR is a sensitive probe to examine tautomerism of the guanine ring system. The analysis indicates that the change from a single to a double bond for the C8 substituent, and the accompanying removal of the normal double bond between N7 and C8 on the imidazole ring system, has no detectable effect on the tautomerism a t the N1-06 site of the pyrimidine ring system for both the 8-keto and 8-thio substitutions. In addition, large differences in electronegativity of the C8 substituents do not alter the N1-06 tautomerism.
Introduction DNA damage induced by oxygen radicals creates a number of abnormal lesions and appears to play an important role in mutagenesis, carcinogenesis, and aging (1-6). In particular, 8-hydroxy-2’-deoxyguanosine(8-OHdG)213has received considerable attention in recent years (7) as a consequence of its demonstrated mutagenic potential (Chart I) (8). Furthermore, its ribosyl counterpart, 8-hydroxyguanosinetriphosphate, has been studied and exhibits weak substrate properties of uridine triphosphate in the reaction with polynucleotide synthesis (9). Recently, Shigenaga et al. have developed a sensitive, noninvasive in vivo assay for detecting oxidative DNA damage utilizing the high degree of electrochemical sensitivity of 8-OH-dG (10). Detailed characterization of these C8-hydroxylated lesions is thus required for an understanding of their biological consequences, including enzyme repairability. There are at least four possible tautomeric forms (Ia, Ib, IC,and Id) of 8-OH-dG involving the N1, C6, N7, and C8 positions, all of which have implications to base pairing properties (Chart 11). Theoretical calculations on the derivative 8-hydroxyguanine have suggested that the most energetically stable form is the 6,&diketo tautomer (Ia), followed by the 6-enol-8-keto (IC)tautomer ( 1 1 ) . Another was reC8-hydroxy analogue, 9-ethyl-8-hydroxyguanine, ported to reside in the 6,gdiketo form in crystalline form (12). We have shown previously that the Z’-deoxyribose derivative 8-OH-dG exists preferentially in the 6,g-diketo tautomeric form by comparative analyses of UV and IR and initial NMR data (13). In addition, there are two
* To whom correspondence should be addressed. 0893-228x/90/2703-0445$02.50/0
Chart I
1
HO-5
OH R 2
E1 OH OH SH Br SCHi S02CH3 OCH, OCH2C,H, Br OCH~C~HS
8 2 H OH OH OH OH OH OH OH H H
8-OH-dG 8-OH-G 8-SH-G 8-Br-G 8-SMe-G 8-SQMe-G 8-OMe-G 8-OBZ-G 8-Br-dG 8-OBZ-dG
possible molecular conformations about the glycosyl bond, anti and syn, which also could affect base pairing of DNA. Presented in part at the Eightieth Annual Meeting of the American Association for Cancer Research (42). Abbreviations: 8-OH-dG, 8-hydroxy-2’-deoxyguanosine;8-OH-G, 8-hydroxyguanosine;NOE,nuclear Overhauser enhancement. The official IUPAC name of 8-OH-dG is 2-amino-9-(2’-deoxy-&~erythro-pentofuranosyl)purine-6,8(1H,9H)-dione,and it is also the CA name for the 1967-1971 index period. The current CA index name is 2’-deoxy-7,8-dihydro-&oxoguanosine. Both the IUPAC and the current CA name for the enol form are 2’-deoxy-8-hydroxyguanmine,but both tautomers are indexed in CA under the name of the keto form. We thank Dr. K. L. Loening of Chemical Abstracts Service for this information.
0 1990 American Chemical Society
446
Chem. Res. Toxicol., Vol. 3, No. 5, 1990 Chart I1
dR
dR
Ib
la
OH
I
dR
dR
Id
IC
This class of compounds is reported to exist in the syn conformation about the glycosyl bond due to the bulky C8 substituent (14). Herein, we report on a detailed, natural abundance 15N NMR study of the oxidative DNA damage adduct, 8hydroxy-2'-deoxyguanosine (&OH-dG), as well as its RNA analogue, 8-hydroxyguanosine (8-OH-G), and many other C8-substituted guanine nucleosides (Chart I). This is the first 15NNMR analysis on Cgmodified purine nucleosides. The present study explores whether a large range of modifications a t C8 in the imidazole ring alters the tautomerism in the pyrimidine portion of the ring system. This study also considers the possibility of various minor tautomeric forms of 8-OH-dG and 8-OH-G in DMSO and in aqueous solution, since this would have implications for biological functions. On the basis of the results of the tautomeric preference of these adducts, their proposed base pairing properties are examined and compared to those of exocyclic adducts and their biological implications are further discussed.
Experimental Procedures Materials. Nucleosides were purchased from Sigma and were used without further purification. 1,3-Dichloro-l,1,3,3-tetraisopropyldisiloxane was obtained from Aldrich. All other C8-substituted guanine nuclecaides were prepared as previously published (15) or with a slight modification as described below. In addition, the synthesis of a 3',5'-0-disilyl derivative (&OH-TPDS-G, 1) of 8-OH-G is described.
Cho et al. resuspension of the crude powder in H 2 0 (8 mL) was added saturated bromine-water, which was made by adding 10 mL of H 2 0 to the residual bromine in the same test tube. The colorless slurry was quickly filtered and washed successively with 10 mL of ice-cold H,O and 20 mL of acetone. 'H NMR analysis of the dried powder indicated the reaction was 90-95% complete: 1.67 g (47%); mp >205 "C dec [lit. (17) 210 "C dec]; HPLC retention time 14.7 min (3 mL/min, 1-h, linear gradient, 5-100% aqueous methanol);EIMS m / e (re1intensity) 347 (M', 6%), 345 (M', 6%), 229 (M+ - sugar, loo%), 231 (M' - Sugar, 100%). 8-Hydroxy-2'-deoxyguanosine(8-OH-dG). This compound was prepared by a catalytic hydrogenation of 8-(benzyloxy)-2'deoxyguanosine (8-OBz-dG) as previously described (15). The product was purified either by recrystallization from water or by semipreparative HPLC (1-h linear gradient, 0-100% aqueous methanol, 2 mL/min, HPLC retention time 24.0 min) to give 70 mg of 8-OH-dG as a white solid (49%): mp 219-221 "C [lit. (15) 217-220 "C]; EIMS [pentakis(trimethylsilyl) derivative]n / e 643. 8-(Methylsulfonyl)guanosine(8-S02Me-G). To a suspension of 8-(methy1thio)guanosine(8-SMe-G,0.49 g, 1.5 mmol) in 30 mL of ethanol was added m-chloroperbenzoicacid (0.83 g, 4.8 mmol), and the mixture was stirred for 24 h at room temperature under an argon atmosphere. The solvent was evaporated and the residue was triturated with ether. The product was recrystallized from aqueous ethanol (0.48 g, 89%): mp >200 OC dec [lit. (15) >206 "C); EIMS m / e (re1 intensity) 229 (base H+, 100%).
+
3',5'- 0 -(Tetraisopropyldisiloxane-1,3-diyl)-S-hydroxyguanosine (8-OH-TPDS-G, 1). To 2.77 g (8 mmol) of dried 8-methoxyguanosine (8-OMe-G) (15)suspended in 60 mL of anhydrous dimethylformamide were added 4 mL of dry pyridine and 2.6 mL (8.2 mmol) of 1,3-dichloro-1,1,3,3,-tetraisopropyldisiloxane (18). The mixture was stirred for 18 h under an argon atmosphere at room temperature and then added slowly to 1L of vigorously stirred ice water. The resulting precipitate was collected by filtration and washed with water. This was chromatographed on silica and eluted with chloroform/methanol(91). Evaporation of appropriate fractions and recrystallization of the foamy residue from 95% ethanol gave 1.83 g of product as a white powder (42%). The cleavage of C8-methyl ether during the selective silylation of 8-OMe-G is fortuitous, since it eliminates the necessity for its removal in a subsequent step. The structure of this 3',5'-0-disilyl derivative was confirmed by 'H and 15N NMR, as described in the text, as well as by mass spectral analyxs: EIMS m / e (re1 intensity) 541 (M', 3%), 498 (M+ - iPr, 15%), 167 (B + H', 100%);mp 217-220 "C. Instrumentation. Melting points were determined on a Thomas-Hoover capillary melting point apparatus and were uncorrected. Mass spectra were obtained with a Finnigan 4023 mass spectrometer by electron impact a t 70 eV. The HPLC system consisted of two Beckman/Altex Model llOB pumps controlled by a Beckman System Gold Analog Interface Module 406. Supelco 5-pm ODS (250 X 4.6 mm i.d.) and Beckman 5-pm ODS Ultrasphere columns (250 X 10 mm i.d.) were used for analytical and semipreparative separation, respectively. All NMR spectra were recorded on a Bruker AM 500 NMR spectrometer. The 500-MHz lH NMR spectra were measured at 301 K in deuterated dimethyl sulfoxide (DMSO-$) solutions, unless otherwise indicated. The 'H NMR chemical shifts were determined with the DMSO-d, signal as the reference at 2.50 ppm. Typical acquisitionconditions were as follows: flip angle, 70-80'; data size, 32K; sweep width, 7 kHz; recycle delay, 1.2 s. The chemical shifts of the signals in the 126-MHz 13CNMR spectra were obtained with the DMSO-d, signal assigned as 39.5 ppm. A capillary containing dioxane in DzO was used as a deuterium lock and as an external reference a t 66.58 ppm downfield from TMS. For one-bond and long-range 13C-lH shift-correlated 2D NMR experiments,standard Bruker software (XHCORR and COLOC, respectively) were used. Typical conditions for 13Cspectra were as follows: flip angle, 60-80"; data size, 32K sweep width, 22 kHz; recycle delay, 2 s. The 15NNMR spectra were obtained at 50.7 MHz. For both DMSO-$ and HzO samples, a coaxial capillary containing 0.2 M 15N enriched NaNO, in D20was used as an external reference. Chemical shifts are reported in ppm downfield from anhydrous ammonia by assigning the external reference to 376.53 ppm. For aqueous samples,the D20 in a capillary was also used as a deuterium lock. Aqueous samples for '% and 15NNMR
% -(,:dN 0
+ :o
N
A
N
ysi O'si-0
OH
Y> 1
8-Bromo-2'-deoxyguanosine (8-Br-dG). The following modification was made due to instability of the product under brominating conditions (15). Saturated bromine-water was prepared by mixing bromine (0.5 mL) in 30 mL of H20. The solution is best prepared in a test tube using a Micro-Stirr. Aliquots were then added dropwise to a slurry of 2'-deoxyguanosine (dG, 2.67 g, 0.01 mol) in 8 mL of H,O in a 100-mL beaker, with vigorous stirring. Due to the high viscosity of the solution, a glass rod was used instead of a magnetic stirring bar. The thick slurry was stirred 3 min after the addition and was then quickly filtered and washed with 5 mL of ice-cold H20. Prolonged stirring results in decomposition of the product (16). The reaction is 70-75% complete as determined by 'H NMR analysis. To a
15NNMR of C8-Substituted Guanine Nucleosides Table I. 'H NMR Chemical Shifts (ppm) of compound H1' H2' H2" H3' 8-OH-TPDS-G (1) 5.48 4.60 4.80 8-OH-G 5.56 4.83 4.07 8-SH-G 6.25 4.95 4.22 1.91 4.32 8-OH-dG 6.02 2.97
Chem. Res. Toxicol., Vol. 3, No. 5, 1990 447 8-Keto and 8-Thio Guanine Nucleosides' H4' H5'b H5"b NH2 NH1 3.75 3.88 3.88 6.47 10.78 6.44 10.77 3.77 3.43 3.57 3.79 3.50 3.66 6.52 11.00 3.74 3.43 3.55 6.43 10.74
NH7 10.65 10.75 12.89 10.74
All spectra were obtained in DMSO-d,. See Table I1 for abbreviations. Assignments may be reversed. Table 11. "N NMR Chemical Shifts (ppm) and Coupling Constants (Hz)of Various Guanine Nucleosides' coupling constants, Hzb chemical shifts, ppm concn, compounds M N1 N3 N7 N9 NH, 'JN~.H~ 'JN7.H7 'JNI.HI 3JN3.NH2 G 0.7 148.2 166.7 248.1 170.9 74.2 -89.3 -88.4 2.5 144.9 74.3 -89.7 8-OH-TPDS-G(1) 0.4 147.6 168.0 109.1 -98.2 (73)' 3.2 147.2 109.7 142.3 74.5 -89.7 -98.5 (34)' 3.0 8-OH-G 0.3 168.4 150.1 147.9 167.5 76.2 -90.7 -99.6 -88.0 2.7 169.8 8-SH-G 1.0 253.6 149.4 167.3 167.7 75.9 -90.2 8-Br-G 0.5 -88.0 3.2 242.3 148.5 168.1 165.2 74.7 -89.8 (9)' 3.3 8-SMe-G 0.9 -89.4 263.3 8-S02Me-G 0.3 149.1 167.0 165.0 78.8 -90.5 2.7 145.1 74.0 -89.5 8-OMe-G 0.1 194.0 168.4 150.8 (12)C NDf 194.8 145.7 73.7 -89.6 8-OBZ-G 0.5 148.5 168.6 (40)' 2.7 174.2 160.6 163.2 7-Me-Gd (+) (3) 0.5 149.4 81.2 158.1 167.2 7-Me-Gd (4) 0.3 206.9 169.0 78.9 239.2 137.1 222.2 2e 0.6 214.1 169.6 247.3 148.2 174.6 74.3 -89.6 dG 0.4 166.9 144.2 109.0 150.8 8-OH-dG 0.1 168.5 75.6 -89.8
'All spectra were obtained in DMSO-d,. b A digital resolution of 0.9 Hz. 'Not detected due to broadening, and approximate line width in Hz is in parentheses. dTaken from refs 20 and 21. eTaken from ref 23. 'Not detected due to low signal to noise ratio or line broadening. #Abbreviations: G, guanosine; 8-OH-TPDS-G (11, 3',5'-0-(tetraisopropyldisiloxane-1,3-diyl)-8-hydroxyguanosine; 8-OH-G, 8-hydroxyguanosine; 8-SH-G, 8-mercaptoguanosine; 8-Br-G, 8-bromoguanosine;8-SMe-G, 8-(methylthio)guanosine;8-S02Me-G,8-(methylsulfonyl)guanosine; 8-OMe-G,8-methoxyguanosine;8-OBz-G,8-(benzyloxy)guanosine;7-Me-G (+) (3), 7-methylguanosine(protonated form); 7-Me-G (4), 7-methylguanosine (neutral form); 2, 2-N-(4-tert-butylbenzamido)-6-0[(4-nitrophenyl)ethyl]guanosine;dG, 2'-deoxyguanosine; 8-OHdG, 8-hydroxy-2'-deoxyguanosine. spectra with 10-mm tubes were recorded in 2 mL of deionized H20, and the appropriate pH values were adjusted with dilute NaOH and HCl. Most lSN spectra were obtained at 301 K with no proton decoupling. This is based on the observation that proton decoupling does not usually enhance the signal intensity of pyridine-like nitrogens of nucleosides (19). Proton-decoupled spectra of aqueous samples were obtained at 305 K with either gated decoupling or continuous decoupling using the Waltz-16 sequence. The lSN NMR sample tubes had outside diameters of 10 mm. Typical acquisition conditions were as follows: flip angle, 40'; data size, 64K; sweep width, 30 kHz; recycle delay, 2 s. For coupling constant measurements, the free induction decays were zero-filled to 128K to produce a digital resolution of 0.9 Hz/point. Line width estimates were carried out with Bruker software by fitting the data to the best Lorentzian line shape. Exponential filtering using line broadening of 1-2 Hz wm used. Additional information on 16Ndata acquisition conditions is given in the figure legends.
Results and Discussion (A) NMR Resonance Assignments. The 'H resonances of 8-OH-dG and 8-OH-G were assigned by decoupling experiments and confirmed by 13C-1H heteronuclear shift-correlated 2D NMR experiments. The two exchangeable protons (Nl-H and N7-H) were assigned by 13C-*Hheteronuclear shift-correlated experiments and/or by selective decoupling I5N NMR experiments (vide infra). The 'H NMR results are summarized in Table I. T h e assignments of the I5N NMR resonances are made mainly on the basis of detection of one-bond a n d threebond nitrogen-hydrogen coupling constants. Initially, we chose the silylated derivative 8-OH-TPDS-G (1) as a model compound because it gave the higher quality spectra due to the least amount of broadening from proton exchange. The 'H NMR spectrum of 1 exhibits two sharp proton signals (imino or enolic) in the downfield region (10.78 and 10.65 ppm), whereas 8-OH-dG gives a single broad signal at 10.74 ppm for the two protons (Table I). Figure 1shows
the natural abundance 15NNMR spectrum of 1 in DMSO compared to that of guanosine (G). Both spectra were obtained with no proton decoupling (no NOE). Expansions of those resonances which exhibit fine structure due to long-range coupling are also included in the figure. The I5N spectrum of 1 exhibits five clearly defined resonances: two are doublets, two are triplets, and one resonance is broadened by exchange. The prominent triplet at 74.3 p p m ('&.HZ = -89.7 Hz) was readily assigned to NH2, while the other triplet at 168.0 p p m was assigned to N3 on the basis of a small three-bond N-H coupling with the exocyclic NH2 protons (3JN3.NH2 = 3.2 Hz). The doublet a t 144.9 p p m is assigned to N9 on the basis of a small three-bond coupling to N7-H (3JNsNH7 = 1.8 Hz). The N7 resonance at 109.1 p p m was assigned from its large coupling constant ('JN7.H7 = -98.2 Hz) and its large upfield shift compared to G. The remaining resonance at 147.6 p p m must be N1. This was confirmed by selective decoupling of the most downfield exchangeable proton (10.78 ppm), which changed the broad N1 resonance (147.6 ppm) into a s h a r p singlet. Likewise, the N7 doublet collapsed into a singlet by irradiation of the proton signal at 10.65 ppm. Irradiation of the exocyclic NH, proton resonance at ti.47 p p m sharpened the nitrogen triplets at 74.3 and 167.9 ppm, respectively, thus confirming the ISN assignments of NH2 and N3. All assignments are in accord with chemical shift arguments (vide infra). Similar assignment procedures as well as comparisons of chemical shifts were used to assign resonances for each C8-substituted nucleoside. T h e I5N NMR spectral results of various guanine nucleosides are tabulated in Table 11. The results for G and dG are in agreement with previously reported assignments (29, 22). In general, t h e chemical shifts of N7 a n d N9 are quite sensitive t o t h e nature of C8 substituents. Thus, the conjugative electron donating effects from OMe or OBz groups significantly shield the N7 resonance by the usual
Cho et al.
448 Chem. Res. Tonicol., Vol. 3, No. 5, 1990 10
Hz
n
N3
NH2 N7 I
I
N9 r 3
I1
N1
N7
N3
N3
1\17
b
1
-
.
...--/I
i
L
r3L
180
>hO
540
>EO
100
EO
PPm
Figure 1. Natural abundance 50.7-MHz 15N NMR spectra in DMSO-dBat 301 K are shown for (a) 0.6 M G and (b)0.4 M 8-OH-TPDS-G(1) with no ‘H noise decoupling.
“b effect”, while the electron withdrawing groups, such as SOzMeand Br, produce moderate deshielding effects. The pyrrole-like sp3 nitrogen, N9, is shielded regardless of substituents, but the shielding is much greater for electron releasing substituents. The C8-enol analogues, 8-OMe-G and 8-OBz-G, exhibit similar effects on the N9 resonance as 8-OH-dG, 8-OH-G, and 8-SH-G but differ at the N7 resonance, reflecting a difference in the tautomeric form at the C8 position (vide infra). The 0 deshielding effect is also seen when the C8 oxygen (8-OH-G, 8-OMe-G) is replaced with sulfur (8-SH-G, 8-SMe-G), which is due to contributions from lower excited states in sulfur compounds (23,24). In addition, small but consistent deshieldings are observed for the exocyclic amines (NH,) of 8-S02Me-G,8-SH-G, and the protonated and neutral forms of 7-Me-G. This may be due to the long-range effect of the electron withdrawing groups attached to the imidazole moiety. The N9 resonances of C8-hydroxy derivatives are influenced less (+1.9 ppm) by the C2’-hydroxyl group effect compared to that (+3.5 ppm) observed for G (19). (B) Tautomerism in Nonaqueous Solution. Analysis of nucleosides and mononucleotides has shown that 15N NMR is a powerful method for establishing the tautomeric equilibrium (24-28). This is due to large chemical shift differences between tautomers and, in selected cases, to the possibility of analyzing nitrogen-hydrogen coupling constants. The I5N chemical shifts should also serve as a tool to investigate the lactam-lactim tautomerism at N7 and O8of C8-substituted guanine nucleosides because N7 participates directly in tautomeric changes. The method offers the possibility of characterizing the effect of C8 substitution on tautomerism of the entire purine ring system. The possible lactam-lactim equilibrium at N7 and Os of 8-hydroxyguanine nucleosides was investigated by 15N NMR spectroscopy. DMSO was initially used as a solvent so that exchange of the directly attached protons could be
minimized. For 8-OH-G and 8-OH-TPDS-G (1)in DMSO (Figure l), the N7 resonance exhibited a well-resolved = -98.2 Hz for 8-OHone-bond N-H coupling (1JN7.H7 TPDS-G), which is characteristic of protons bound to a trigonally hybridized nitrogen atom (29, 30) (Table 11). The line width (1.3 Hz) was virtually the same as that of the signal obtained with proton decoupling. This establishes that a proton is attached to N7 and that exchange is minimal. The N7 chemical shift lies in the range of urea-type nitrogens and exhibits a large upfield shift due to the 8-keto group (+138 ppm) compared to the parent compound, G, indicating a conversion from a pyridine-type nitrogen to a pyrrole-type nitrogen (Figure 1) (31). The N9 resonance exhibits a moderate upfield shift (+26 ppm) compared to G, due to its conversion to an amide type nitrogen. In contrast, the nitrogens of the pyrimidine ring system show little change. Similar results were obtained for 8-OH-dG (Table 11). The possible occurrence of a minor enol tautomer at the O6position of 8-hydroxyguanine nucleosides in DMSO was also investigated by 15N NMR. The situation was complicated because of the broadening of N1. It is significant that the N1 chemical shifts of 8-OH-dG and 8-OH-G are essentially the same as for dG and G, both of which are reported to exist in the 6-keto form (30). This is substantiated by the observation of a sharp doublet for the N1 resonance of G at 148.2 ppm (Figure l),and by previous studies (19,22, 31). Furthermore, the shift of the N1 resonance of l-methylguanosine is reported to be 143 ppm, which is in excellent agreement with that (148 ppm) of dG or G, once the estimated inductive effect (+3.3 ppm) from the methyl group is considered (23). It should be noted that the methyl group of l-methylguanosine prevents C6 enolization. On the other hand, the OWkylated guanosine derivative 2 can be a model compound for the
n
OHOH
2
6-enol tautomer. The pyridine-type N1 of 2 has been reported to resonate at 214.1 ppm (23). The actual shift is estimated as 208 ppm if the substituent effect (p-tertbutylbenzoyl) on the exocyclic NH, (-6 ppm) is taken into consideration. Thus, based on the above chemical shift argument, the percentage of 6-enol isomer (IC)must be very small, if present at all, in DMSO solution. The quantification of this minor tautomer has not been attempted, because of small uncertainties in the chemical shift of the N1 resonance due to its sensitivity toward impurities, concentration, and temperature. These results, together with those on other C8-substituted guanine nucleosides (Table 111, indicate that the modification of the base has little effect on the chemical shift of N1 (less than 3 ppm) as long as the sp3 hybridization at N1 is not disturbed. The constancy of the N1 chemical shift among C8-substituted guanine nucleosides enables several additional conclusions to be drawn. Two of the compounds, 8-
Chem. Res. Toricol., Vol. 3, No. 5, 1990 449
15N NMR of C8-Substituted Guanine Nucleosides
hydroxy- and 8-mercaptoguanosine, exist in the keto and thio forms, respectively. These structures necessitate a removal of the double bond that normally is positioned between N7 and C8 of the imidazole ring, which might be expected to perturb the tautomerism of the pyrimidine ring system. The chemical shift data indicate that no alteration in tautomerism results from this major structural change. Likewise, it can be concluded that large changes in the electronegativity of the C8 substituent, including both strong electron donating and electron withdrawing groups, do not alter the tautomerism of this class of modified nucleosides. We also note from published chemical shift data (19, 22, 31) that N1 is insensitive to changes in the sugar portion (C2'-hydroxyl or -deoxy and C3'- or C5'-phosphates) of G or dG, as is expected from our analysis. The broadening that is observed for N1 of the 8hydroxyguanine nucleosides, occurring only in the proton-coupled spectra, may be attributed to exchange with water. Repeated purifications failed to further sharpen this resonance. We have experienced a similar problem with most of the other synthetic compounds (Table 11). Contamination with acidic or paramagnetic impurities may play an important role, although the broadening may be due to an intrinsic property. The electron withdrawing effect of the 8-keto group could enhance the exchange rate of the amide proton and thus increase acidity of N1-H (22). This is in fact observed from the pH dependence of 13C NMR chemical shift titration studies that show a decrease in pK, of 0.8 pH unit for 8-OH-dG compared to dG (13). Similar broadening was reported for the N3 resonance of thymidine and uridine and was attributed to the presence of two adjacent flanking carbonyl groups that cause the N3 proton to be more acidic than in a normal amide group (22, 23). An attempt was made to directly detect possible minor tautomeric forms by lowering the temperature (33). 13C NMR measurements were carried out on 8-OH-TPDS-G (1) in methanol due to its higher solubility. Neither subspectra nor unusual broadening was observed in the temperature range of -60 to 25 "C. Likewise, 500-MHz 'H NMR measurements on 8-OH-dG in methanol did not show any evidence of exchange. This may be explained by the predominance of a single tautomeric form, in agreement with the chemical shift arguments. However, the results do not rule out the possibility of small amounts of alternate tautomers since the tautomeric equilibrium may still be too rapid on the NMR time scale. (C) Tautomerism and Ionization in Aqueous Solution. 15NNMR is a direct method for examining ionization and the site of protonation (27). Thus, for example, the acidic N1-H of guanosine 3'-monophosphate is known to deprotonate and tautomerize in basic solution, resulting in a downfield shift of more than 60 ppm, whereas the reverse is observed (50-100 ppm upfield shift) for the protonation of basic N7 under acidic conditions (31, 34, 35). Similarly, the N9 resonance of adenine undergoes a dramatic downfield shift of 56 ppm on formation of the corresponding conjugate anion (36). Therefore, it was expected that 15N NMR would be useful for the investigating ionization of C8-substituted guanine nucleosides. If minor tautomeric forms of the 8-hydroxyguanine nucleosides are present in aqueous solution, they would be expected to be interconverting rapidly. The situation is further complicated since it is possible to have additional pH-dependent ionic species, which in turn may alter tautomerism. We have previously reported that 8-OH-dG has two ionizations values (pK,1 = 8.6 and pK,2 = 11.7)
a
N7
N3
I
NI
, .-,, , -
1
N9
.. .-.,,. .- . .
,,..
.,,.
b
.
,I
..
I .
I
I
200
'
I
180
'
I
160
'
I
140
'
120
I
I
I
'
100
I
~
I
BO
Figure 2. Natural abundance 50.7-MHz lSNNMR spectra of 0.1 M 8-OH-dGare shown with (a) no 'H noise decoupling at pH 9.5 and 301 K, after 76800 transients; (b) 'H noise decoupling at pH 9.5 and 305 K after 25 400 transients; (e) no 'H noise decoupling at pH 7.3 and 301 K after 50440 transients; and (d) 'H noise decoupling at pH 7.3 and 305 K after 50000 transients. The signals of 'H noise-decoupled 16NNMR spectra b and d are phased upright for comparison. Scheme I
dU
la
dR
le
dR
If
(Scheme I) (13). Analogous pK, values (pK,l = 8.5 and pK,2 = 11.2) for 8-OH-G were also obtained by pH dependence of 13C chemical shift measurements (data not shown). Here, the effect of these ionizations on the 15N spectral parameters is investigated and discussed in terms of changes in tautomerism. The 15NNMR spectra of 8-OH-dG in aqueous solution were measured at two different pH values in order to examine the effect of ionization at N1. The spectrum at pH 7.3 recorded with proton decoupling exhibits three singlets (Figure 2d), while the undecoupled spectrum accounts for the remaining two resonances (Figure 2c). Thus, the protonated nitrogens were readily identified as N1, NH2, and N7, from the large negative NOE in the proton-decoupled spectrum. The nonprotonated pyridine-like nitrogens have an unfavorable NOE on the order of -1 or -2, which dramatically lowers the intensity of the signal (19). Since the N3 and N9 atoms do not possess directly bonded hydrogens, their signals are not apparent in the proton-decoupled spectrum. The proton-decoupled spectrum at pH 9.5 exhibits only two nitrogen signals at 113.1 and 71.2 ppm (Figure 2b), corresponding to N7 and NHz, respectively. The N1 resonance at pH 9.5 is seen only in the undecoupled spectrum and is shifted downfield by a 48.5 ppm (Figure 2a). This clearly indicates that the first ionization results in the formation of a 6-enolate-8-keto monoanion (Ie, Scheme I). In other words, at pH 9.5, the protonated N1 was converted to a partially deprotonated nitrogen, which is not detected in proton-decoupled spectrum, due to the unfavorable NOE effect. The observed downfield shift for this ionization is only 48.5 ppm because the N1-H has not been deprotonated completely at the observed pH of 9.5. If the extent of ionization is
450 Chem. Res. Toxicol., Vol. 3, No. 5, 1990 a
Cho et al.
N3
N1
N9
I Ribose
Ribose
3
Figure 3. Natural abundance 50.7-MHz 16NNMR spectra of 0.3 M 8-OH-Gat 301 K, with no 'H noise decoupling, is shown (a) at pH 12.4 in H20 after 12000 transients; (b) at pH 10.5 in H 2 0 after 21 200 transients; and (c) in DMSO-d, after 12000 transients.
Table 111. pH Dependence of 15N NMR Chemical Shifts (Dum) of 8-OH-dG and 8-OH-G" compound solvent (pH) 8-OH-G DMSO-& 8-OH-G HzO (7.3f 8-OH-G Hz0 (10.5) 8-OH-G HzO (12.4) 8-OH-dG DMSO-d6 8-OH-dG HZO (7.3) 8-OH-dG Hz0 (9.5)
N1 147.5 149.8 202.8 206.8 151.0 148.2 196.7
N3 168.4 169.3 173.6 175.6 168.5 169.2 173.2
N7 109.7 110.6 114.9 160.7 109.0 110.5 113.1
N9 142.3 142.0 138.9 142.4 144.2 145.6 142.5
NH2 74.5 74.3 71.1 68.7 75.6 72.5 71.2
I, Concentrations are 0.1 M for 8-OH-dG and 0.3 M for 8-OH-G, both in DMSO-d6 and in H20 as indicated.
taken into account, the shift is comparable to the 60 ppm shift that is expected for conversion of a pyrrole- to a pyridine-like structure (31). Small changes in the chemical shift of the N3 (-4.0 ppm) and the NH2 (+1.3 ppm) resonances are observed in the 15N spectra, suggesting that the ionization at N1-H increases aromaticity of the pyrimidine moiety. These results are further evidence for the formation of a 6-enolate. The spectrum of 8-OH-dG at a pH higher than 1 2 was not obtained because of its basic instability in a long-term acquisition. The ribose derivative, however, was sufficiently stable in strong base to enable natural abundance 15N measurements to be made over a wider pH range, and thus to examine all three ionization states. Figure 3 shows the 15N NMR spectrum of 8-OH-G at pH 12.4 (Figure 3a) compared to that at pH 10.5 (Figure 3b) and to the neutral form in DMSO (Figure 312). The spectrum obtained in DMSO is used here for the sake of visual comparison, because of a poor signal to noise ratio encountered at pH 7.3. The 15N NMR chemical shifts are summarized in Table 111. The first and second ionizations at N1 and N7 each induce about 57 and 50 ppm downfield shifts at N1 and N7, respectively, indicating the formation of a 6,8dienolate at high pH (Scheme I). The formation of the 6-enolate-8-keto form (Ie) from the 6,8-diketo form (Ia) is also illustrated by comparison with the conversion of protonated 7-Me-G (3) to the neutral form (4) (20). It is noted that their pyrimidine moieties are isoelectronic. Thus, the N1 chemical shift (149.4 ppm) of 3 is strikingly similar to that (149.8 ppm) of 8-OH-G at neutral pH. The N1 resonance (206.9 ppm) of the neutral form of 4 matches
4
that (202.8 ppm) of 8-OH-G at pH 10.5. This is comparable to the 60 ppm downfield shift observed for the full deprotonation of guanosine 3'-monophosphate at N7 (31). It should be mentioned that the I5N NMR experiments described here by way of pH dependence are a direct observation of monoenolate and dienolate formation. It is interesting to note that while the N9 resonance is shielded (+3.1 ppm) in the range of pH 7.3-10.5, the direction of the shift is reversed (-3.5 ppm) during the course of the second ionization at higher pH. However, the pHdependent chemical shift pattern is different for the N3 and NH2 resonances, which are shifted downfield by 6.3 ppm and upfield by 5.6 ppm, respectively, in the range of pH 7.3-12.4. It may be that the N9 behaves as an exocyclic nitrogen in the first ionization, resulting in upfield shifts of both the NH2 and N9. The N3 resonance is expected to be deshielded under these conditions, because of the increase in aromaticity of the pyrimidine ring (Ie, Scheme I). During the second ionization, however, both the N9 and the N3 nitrogens are deshielded because they are part of the purine ring system (If), whereas the NH2 remains as an exocyclic nitrogen, being shielded for both ionizations. During the second ionization, the N1 resonance is shifted further downfield by 4 ppm. This is probably explained by the fact that the ionization at the N7-H delocalizes electrons into the pyrimidine moiety. The N7 and N9 resonances of 8-OH-G at pH 12.4 are also quite comparable with those found in 8-enol or 8-enolate model compounds (8-OMe-G and 8-OBz-G), if the substituent effects are taken into the consideration. The usual p effects are clearly observed when C8 oxygen is substituted by sulfur (23). Thus, the N7 resonances of both 8-SH-G and 8-SMe-G are deshielded by 40.4 and 48.3 ppm, respectively, from those of the corresponding C8 oxygen counterpart (8-OH-G and 8-OMe-G). The coupling constants of the N7 and N1 resonances of = -88.0 Hz and lJN,.H, 8-SH-G observed in DMSO (lJNlSH1 = -99.6 Hz) undoubtedly establish that the base exists in the thio rather than the mercapto tautomeric form, as has been shown for 8-OH-G, and that O6is in the keto form as well. This compound gives additional evidence that removal of the normal double bond between N7 and C8 does not alter the tautomerism in the pyrimidine part of the guanine ring system. The 15N NMR spectral patterns of 8-OH-dG and 8OH-G in both aqueous solution and DMSO are virtually identical, with corresponding nitrogen signals being within a few ppm (Table 111). The insignificant solvent effect on the chemical shift of N1 strongly suggests that the neutral form of 8-hydroxyguanine nucleosides in aqueous solution does not contain pyridine-like nitrogens. In contrast, pyridine-like nitrogens can exert a substantial upfield shift in a strongly hydrogen-bonded solvent such as H20. For example, the N7 resonance of guanosine 3'-monophosphate in H 2 0 is 12.6 ppm upfield of that observed for G in DMSO (19). The stepwise ionization of 8-OH-dG and 8-OH-G in aqueous solution is also evident from their solubility behavior. The water solubility dramatically increases as the pH goes up, while the compound is precipitated by the reverse titration. Although we experienced some difficulty
15N NMR of CB-Substituted Guanine Nucleosides
in handling 8-OH-dG in base, 8-OH-G exhibited much better stability and solubility. Due to the relatively high solubility of these compounds in aqueous solution, it was possible to obtain natural abundance I5N NMR spectra. In the past, such natural abundance I5N studies have been recorded on labeled nucleosides or nucleotides due to their greater solubility in aqueous solution (19, 31, 36). I t has been shown that a number of carcinogens covalently bind to the N7 position of the guanine ring, resulting in a lowering of the pK, at N1 (37). This indicates that the pK, value for the guanine N1-H ionization is dictated by electronic effects from imidazole substitution. Similarly, the 8-keto group increases the exchange rate of the N1 proton, thereby making it more acidic than that of the parent compound. The effect is quite clear in 'H NMR spectra of the isoelectronic 8-SH-G, insofar as its N1-H and N7-H resonances are deshielded noticeably from that of other C8-substituted guanine nucleosides (Table I). The results on the neutral forms of 8-OH-dG and 8OH-G and the parent compounds show that C8 substituents have no detectable direct effect on the tautomeric structure at N1. However, the C8-keto substituent lowers the pK, from 9.4 to 8.6 for the deoxyribose and from 9.2 to 8.5 for the ribose compound. It may be significant, in terms of possible biological relevance of tautomers, that a t physiological pH the C6-enolate is present at 7%. It is concluded that introduction of a C8-keto group on to the guanine ring does not alter the tautomerism at N1, except for the change that accompanies partial ionization in aqueous solution. (D) Biological Implications. Mispairing of purine and pyrimidine bases has been invoked for a variety of DNA adducts as a mechanism for mutation (38). A bulky substituent at the purine C8 position of a nucleoside generally induces a change in the glycosyl linkage, leading to the syn conformation in order to relieve the steric crowding between the sugar moiety and the C8 substituent (14,39). In the case of the &oxo substituent, additional base pairing possibilities involving the proton at N7 and possibly the oxygen at C8 are present. The availability of these positions for base pairing with the complementary strand would depend on the glycosyl torsion angle of an 8hydroxyguanosine. The conformational preference for 8-OH-G was determined to be preferentially syn both in DMSO and in aqueous solution, on the basis of 'H and I3C NMR chemical shift analyses, as has been shown previously for 8-OH-dG (13). There are several possibilities for abnormal mispairing dependent on the orientation about the glycosyl bond (13). Thus, for example, 8-OH-dG and 8-OH-G in the syn conformation would be capable of base pairing with adenine by formation of two hydrogen bonds, one linking the N7-H of guanine with N1 of adenine (5). This is strikingly n
n
5
6
similar to that which has been observed for the protonated form of a 1,W-propanodeoxyguanosineexocyclic adduct opposite adenosine (6)in a duplex and characterized by NMR spectroscopy (40). This example differs from that of 8-OH-dG since mild acid was needed to protonate N7 of the exocyclic adduct and the bulky exocyclic moiety may more strongly destabilize the anti conformation in a A or B form helix. One would expect, from the studies pres-
Chem. Res. Toxicol., Vol. 3, No. 5, 1990 451
ented in the previous section, that the N7 of 8-OH-dG in DNA would already have a hydrogen attached a t physiological pH. Thus, the conformation and base pairing of &OH-dG in DNA may have a different dependence on pH and sequence from that of the exocyclic adducts. There is also precedence for another type of purine-purine mismatch, with a syn adenine and an anti guanine (41).
Conclusions 15N NMR has been shown to be a powerful method for analysis of tautomerism of C8-substituted guanine nucleosides. The use of pH dependence of I5N NMFt spectra has provided insights into the tautomerism and ionization of the C8-hydroxylated guanine nucleosides in solution. Two basic ionizations have been observed and characterized for 8-OH-dG and 8-OH-G. The C8-keto substituent does not alter the tautomerism at the C6 position, except by partial ionization, which causes partial conversion from a 6-keto to a 6-enolate structure at physiological pH. High alkaline pH results in a 6,Sdienolate structure. In addition to predominance of the 6,&diketo tautomer (Ia), these adducts are preferentially in the syn conformation about the glycosyl bond at physiological pH. As such, the modified guanine is expected to be able to form stable base pair structures in DNA by mispairing that involves hydrogen bonding with the N7-H. A keto or thio group at C8 does not alter the tautomerism of the pyrimidine ring, in spite of the removal of the double bond between N7 and C8. Likewise, large differences in electronegativity of the C8 substituent do not alter tautomerism. Finally, we propose to use the term *~-oxo-"instead of "&hydroxy-" and "&thio-" instead of "8-mercapto-" to describe these compounds as the latter tend to be misleading regarding the actual structure and its biological implications. Acknowledgment. We thank Drs. James F. Freeman and Matthew S. Bryant for obtaining mass spectra. Technical assistance provided by Mr. Robert A. Levine is greatly appreciated. B.P.C. was supported in part by an appointment to the ORAU Postgraduate Research Program at the National Center for Toxicological Research, which is administered by the Oak Ridge Associated Universities through an interagency agreement between the U.S. Department of Energy and the US. Food and Drug Administration. Registry No. 1, 128600-03-3;8-OH-dG, 88847-89-6;8-OH-G, 88847-89-6; 8-SH-G, 3868-31-3; 8-Br-G, 26001-38-7; 8-SMe-G, 4016-63-1;8-OMe-G, 2104-66-7; 8-OBz-G, 7057-53-6;8-OBz-dG, 3868-36-8; 8-S02Me-G, 96964-90-8; 8-Br-dG, 7057-50-3; dG, 13389-03-2;1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane, 961-07-9, 69304-37-6.
References (1) Ward, J. F. (1988)D N A damage produced by ionizing radiation
in mammalian cells: identities, mechanisms of formation, and repairability. Prog. Nucleic Acids Res. 35, 95-125. (2) O'Brien, P. J. (1988) Radical Formation during the peroxidase catalyzed metabolism of carcinogens and xenobiotics: the r e activity of these radicals with GSH, D N A , and unsaturated lipid. Free Radical Biol.Med. 4, 169-183. (3) Cross, C. E., Halliwell, B., Borish, E. T., Pryor, W. A., Ames, B. N., Saul, R. L., McCord, J. M., and Harman, D. (1987) Oxygen radicals and human disease. Ann. Int. Med. 107, 526-545. (4) Chuaqui, C. A., and Pekau, A. (1987) Chemical reactivity and biological effects of superoxide radicals. Radiat. Phys. Chem. 30, 365-373. (5) Sies, H. (1986)Biochemistry of oxidative stress. Angew. Chem., Int. Ed. Engl. 25, 1058-1071. (6) Imlay, J. A., and Linn, S. (1988) D N A damage and oxygen radical toxicity. Science 240, 1302-1309. (7) Floyd, R. A., West, M. S., Eneff, K. L., and Schneider, J. E. (1989) Methylene blue plus light mediates 8-hydroxyguanine
452 Chem. Res. Toxicol., Vol. 3, No.5, 1990 formation in DNA. Arch. Biochem. Biophys. 273, 106-111 and references cited therein. (8) Kuchino, Y., Mori, F., Kasai, H., Inoue, H., Iwai, S., Miura, K., Ohtsuka, E., and Nishimura, S. (1987) Misreading of DNA templates containing 8-hydroxydeoxyguanosine at the modified base and at adjacent residues. Nature 327, 77-79. (9) Bruskov, V. I., and Kuklina, 0. V. (1988) Manifestation by 8-hydroxy-GTP of the substrate properties of UTP in the reaction of polynucleotide synthesis by escherichia coli RNA polymerase on a poly[d(AT).d(AT)] template. Mol. Biol. 22, 580-584. (10) Shigenaga, M. K., Gimeno, C. J., and Ames, B. N. (1989) Urinary 8-hydroxy-2‘-deoxyguanosineas a biological marker of in vivo oxidative DNA damage. Proc. Natl. Acad. Sci. U.S.A. 86, 9697-9701. (11) Aida, M., and Nishimura, S. (1987) An ab initio molecular orbital study on the characteristics of 8-hydroxyguanine. Mutat. Res. 192, 83-89. (12) Kasai, H., Nishimura, S., Toriumi, Y., Itai, A., and Iitaka, Y. (1987) The crystal structure of 9-ethyl-8-hydroxyguanine.Bull. Chem. SOC.Jpn. 60, 3799-3800. (13) Culp, S. J., Cho, B. P., Kadlubar, F. F., and Evans, F. E. (1989) Structural and conformational analyses of 8-hydroxy-2’-deoxyguanosine. Chem. Res. Toxicol. 2, 416-422. (14) Davies, D. B. (1978) Conformations of nucleosides and nucleotides. hog. Nucl. Magn. Reson. Spectrosc. 12, 135-225. (15) Lin, T.-S., Cheng, J.-C., Ishiguro, K., and Sartorelli, A. C. (1985) 8-Substituted guanosine and 2’-deoxyguanosine derivatives as potential inducers of the differentiation of Friend erythroleukemia cells. J. Med. Chem. 28, 1194-1198. (16) Shapiro, R., and Agarwal, S. (1966) Oxidation of guanine and guanosine by bromine. Biochem. Biophys. Res. Commun. 24, 401-405. (17) Long, R. A., Robins, R. K., and Townsend, L. B. (1967) Purine nucleosides. XV. The synthesis of 8-amino- and 8-substituted aminopurine nucleosides. J. Org. Chem. 32, 2751-2756. (18) Robins, M. J., Wilson, J. S., and Hansake, F. (1983) Nucleic acid related compounds. 42. A general procedure for the efficient deoxygenation of secondary alcohols. Regiospecific and stereospecific conversion of ribonucleosides to 2’-deoxynucleosides. J. Am. Chem. SOC. 105, 4059-4065. (19) Markowski, V., Sullivan, G. R., and Roberts, J. D. (1977) Nitrogen-15 nuclear magnetic resonance spectroscopy of some nu99, 714-718. cleosides and nucleotides. J. Am. Chem. SOC. (20) Barbarella, G., Bertoluzza, A., and Tugnoli, V. (1987) Nitrogen-15 NMR characterization of 7-methylguanosine and its base-pair with cytidine in dimethyl sulphoxide. Magn. Reson. Chem. 25,864-868. (21) Barbarella, G., Bertoluzza, A., and Tugnoli, V. (1988) Nitrogen-15 NMR characterization of the neutral form of 7-methylguanosine. Nucleic Acids Res. 16, 7202. (22) Hawkes, G. E., Randall, E. W., and Hull, W. E. (1977) Natural abundance nitrogen-15 nuclear magnetic resonance spectroscopy. The pyrimidine and purine nucleosides. J. Chem. SOC., Perkin Trans. 2, 1268-1275. (23) Remaud, G., Zhou, X., Welch, C. J., and Chattopadhyaya, J. (1986) An assessment of the reactivity of guanosine and some of its derivatives to electrophiles by 15N-NMRspectroscopy. Tetrahedron 42,4057-4067. (24) Witanowski, M., Stefaniak, L., and Webb, G. A. (1977) Nitrogen NMR spectroscopy. In Annual Reports in NMR Spectroscopy (Webb, G. A., Ed.) Vol. 7, pp 117-244, Academic Press, London. (25) Levy, G. C., and Lichter, R. L. (1979) Nitrogen-I5 Nuclear Magnetic Resonance Spectroscopy, Wiley, New York.
Cho et al. (26) Witanowski, M., and Webb, G. A. (1973) Nitrogen NMR, Plenum Press, London. (27) Buchanan, G. W. (1989) Applications of 15NNMR spectroscopy to the study of molecular structure, stereochemistry and binding phenomena. Tetrahedron 45, 581-604. (28) Roberts, J. D., and Kanamori, K. (1983) 15NNMR studies of biological systems. Acc. Chem. Res. 16, 35-41. (29) Axenrod, T., Pregosin, P. S., Wieder, M. J., Becker, E. D., Bradley, R. B., and Milne, G. W. A. (1971) Nitrogen-15 nuclear magnetic resonance spectroscopy. Substituent effects on 15N-H coupling constants and nitrogen chemical shifts in aniline derivatives. J. Am. Chem. SOC. 93,6536-6541. (30) Evans, F. E., and Levine, R. A. (1986) Conformation and configuration at the central amine nitrogen of a nucleotide adduct of the carcinogen 2-(acety1amino)fluoreneas studied by 13Cand 15N NMR spectroscopy. J. Biomol. Struct. Dyn. 3, 923-934. (31) Buchner, P., Maurer, W., and Ruterjans, H. (1978) Nitrogen-15 nuclear magnetic resonance spectroscopy of 16N-labelednucleotides. J. Magn. Reson. 29, 45-63. (32) Cantor, C. R. (1984) Principles of Nucleic Acid Structure, pp 105-116, Springer-Verlag, New York. (33) Neidle, S., Kuroda, R., Broyde, S., Hingerty, B. E., Levine, R. A., Miller, D. W., and Evans, F. E. (1984) Studies on the conformation and dynamics of the C8-substituted guanine adduct of the carcinogen acetylaminofluorene; model for a possible 2-DNA modified structure. Nucleic Acids Res. 12, 8219-8233. (34) Schumacher, M., and Gunther, H. (1983) Contributions to 15N NMR spectroscopy. Protonation and tautomerism in purines: purine, 7- and 9-methylpurine. Chem. Ber. 116, 2001-2104. (35) Barbarella, G., Capobianco, M. L., Carcuro, A., Colonna, F. P., and Tugnoli, V. (1988) 15Nand 13CNuclear magnetic resonance of deoxydinucleotide monophosphates. I. Protonation of deoxycytidyl-(3’,5’)-guanosinein dimethylsulfoxide. Can. J. Chem. 66, 2492-2497. (36) Gonnella, N., Nakanishi, H., Holtwick, J. B., Horowitz, D. A,, Kanamori, K., Leonard, N. J., and Roberts, J. D. (1983) Studies of tautomers and protonation of adenine and its derivatives by nitrogen-15 nuclear magnet.ic resonance spectroscopy. J. Am. Chem. Soc. 105, 2050-2055. (37) Vodicka, P., and Hemminki, K. (1988) Depurination and imidazole ring-opening in nucleosides and DNA alkylated by styrene oxide. Chem.-Biol. Interact. 68, 117-126. (38) Loechler, E. L. (1989) Adduct-induced base-shifts: a mechanism by which the adducts of bulky carcinogens might induce mutations. Biopolymers 28, 909-927. (39) Evans, F. E., Miller, D. W., and Beland, F. A. (1980) Sensitivity of the conformation of deoxyguanosine to binding at the C-8 position by N-acetylated and unacetylated 2-aminofluorene. Carcinogenesis 1, 719-725. (40) Kouchakdjian, M., Marinelli, E., Gao, X., Johnson, F., Grollman, A., and Patel, D. (1989) NMR Studies of exocyclic 1,Mpropanodeoxyguanosine adducts (X) opposite purines in DNA duplexes: protonated X(syn).A(anti) pairing (acidic pH) and X(syn).G(syn) pairing (neutral pH) at the lesion site. Biochemistry 28, 5647-5657. (41) Kennard, 0. (1985) Structural studies of DNA fragments: the G:T wobble base pair in A, B and 2 DNA; the G:A base pair in DNA. J. Biomol. Struct. Dyn. 3, 205-226. (42) Cho, B. P., et al. (1989) Carcinogenesis 8, Abstract 797; Eightieth Annual Meeting of the American Association for Cancer Research, Minisymposium on Oxidative Damage and Free Radicals, San Francisco, CA, May 1989.
