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amine in rat striatum after the dopamine releasing drugs dexamphetamine, methylamphetamine, and MPTP. Eur. J. Pharmacol. 132, 65-69. (8) Johnson, M., Elayan, I. M., Hanson, G. R., Foltz, R. L., Gibb, J. W., and Lim, H. K. (1991) Effects of 2,4,5-trihydroxymethamphetamine on the central serotonergic and dopaminergic systems. SOC.Neurosci., Abstract (in press). (9) Elayan, I. M., Johnson, M., Hanson, G. R., Foltz, R. L., Gibb, J. W. and Lim, H. K. (1991) Effect of 2,4,5-trihydroxyamphetamine on monoaminergic systems in the rat brain. SOC.Neurosci., Abstract (in press). (10) Bu'Lock, J. D., and Harley-Mason, J. (1951) Melanin and its precursors. Part 111. New synthesis of 5,6-dihydroxyindole and its derivatives. J. Chem. Soc., 2248-2252. (11) Hoffmann, K. J., and Baillie, T. A. (1988) The use of alkoxycarbonyl derivatives for the mass spectral analysis of drug-thioether metabolites. Studies with the cysteine, mercapturic acid and glutathione conjugates of acetaminophen. Biomed. Enuiron. Mass Spectrom. 15, 637-647. (12) Battaglia, G., Yeh, S. Y., and De Souza, E. B. (1988) Degeneration and recovery of brain serotonin neurons. Pharmacol. Biochem. Behau. 29, 269-274. (13) Powell, W. S., and Heacock, R. A. (1973) The oxidation of 6-hydroxydopamine, J. Pharm. Pharmacol. 25, 193-200. (14) Strife, R. J., Simms, J. R., and Lacey, M. P. (1990) Combined capillary gas chromatography/ion trap mass spectrometry quantitative methods using labeled or unlabeled internal standards. J. Am. SOC.Mass Spectrom. 1, 265-271. (15) De Jong, A. P. J. M., and Cramers, C. A. (1983) Derivatization of catecholamines in aqueous solution for quantitative analysis in biological fluids. J. Chromatogr. 276, 267-278.
(16) Mattock, G . L. (1967) Reactions of adrenochrome with some thiols. Arch. Biochem. Biophys. 120, 170-174. (17) Boobis, A. R., Sesardic, D., Murray, B. P., Edwards, R. J., Singleton, A. M., Rich, K. J., Murray, S., De La Torre, R., Segura, J., Pelkonen, O., Pasanen, M., Kobayashi, S., Zhi-Guang, T., and Davies, D. S. (1990) Species variation in the response of the cytochrome P-450-dependent monooxygenase system to inducers and inhibitors. Xenobiotica 20, 1139-1161. (18) Kobayashi, S., Murray, S., Watson, D., Sesardic, D., Davies, D. S., and Boobis, A. R. (1989) The specificity of inhibition of debrisoquine 4-hydroxylase activity by quinidine and quinine in the rat is the inverse of that in man. Biochem. Pharmacol. 38, 2795-2799. (19) Fonne-Pfister, R.,Bargetzi, M. J., and Meyer, U. A. (1987) MPTP, the neurotoxin inducing Parkinson's disease, is a potent competitive inhibitor of human and rat cytochrome P450 isozymes (P450bufI, P450dbl) catalyzing debrisoquine 4-hydroxylation. Biochem. Biophys. Res. Commun. 148, 1144-1150. (20) Gonzalez, F. J., Skoda, R. C., Kimura, S., Umeno, M., Zanger, U. M., Nebert, D. M., Gelboin, H. M., Hardwick, J. P., and Meyer, U. A. (1988) Characterization of the common genetic defect in humans deficient in debrisoquine metabolism. Nature (London) 331,442-446. (21) Lim, H. K., Su, Zeng, Sakashita, C. O., Chei, D. M., and Foltz, R. L. (1991) Comparison of metabolism of 3,4-(methy1enedioxy)methamphetamine (MDMA) in rats and mice. Toxicologist 11, 50, Abstract 104. (22) Zhao, Z., Ricaurte, G., and Castagnoli, N., Jr. (1990) Evaluation of the neurotoxic potential of 2-hydroxy-4,5-methylenedioxymethamphetamine (BOHMDMA), a reported metabolite of MDMA. SOC.Neurosci., 1031, Abstract 426.2.
Structure of Formamidopyrimidine Adducts As Determined by NMR Using Specifically 15N-LabeledGuanosine W. Griffith Humphreys and F. Peter Guengerich* Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146 Received August 9, 1991 Chemical carcinogens are generally electrophilic species that interact with nucleophilic sites on cellular DNA (I). The specific site of interaction is governed by several factors, two of the most important being carcinogen reactivity and the reactivity of the site in question. Of the many nucleophilic sites on DNA the one that is most nucleophilic and thus generally most reactive is the N7position of guanine. Although in many cases the N7-guanyl adducts have been shown to be very weakly mutagenic, there are several cases where these adducts are thought to be important, e.g., the adducts formed by aflatoxins (2) and the ethylene dibromide derived GSH adducts ( 3 , 4 ) . Two of the most common fates that N7-deoxyguanosyl adducts typically undergo are depurination and imidazole ring opening. Acid-catalyzed depurination reactions yield N-guanyl adducts and apurinic sites, and base-catalyzed imidazole ring opening reactions yield FAPY' adducts. Under physiological conditions the relative rates with which specific N-guanyl adducts follow each pathway are quite dependent on the chemical nature of the substituent
Scheme I. FAPY Adduct Structure and Possible Explanations for the Interconversion Observed upon Adduct Isolationa
HO OH
HO
OH
HO OH
'
a Note the change from the purine to the pyrimidine numbering Abbreviations: FAPY, formamidopyrimidine; GSH, reduced glutascheme upon FAPY adduct formation. thione; GSH-FAPY-baae, 5'-[2-[N-formyl-N-(2,6-diamin0-4-0~0-3,4-dihydropyrimidin-5-yl)amino]ethyl]glutathione;GSH-FAPY-glycoside, 5'-[2-[N-formyl-N-[2-amino-6-(N-glycosylamino)-4-oxo-3,4-dihydro- at the N7-position (5). Thus for aflatoxin adducts a major pyrimidin-5-yl]amino]ethyl]glutathione;Me-FAPY-base, 2,d-diaminospecies thought to be present is the FAPY adduct ( 2 , 6 ) 5-(N-formyl-N-methylamino)-4-0~0-3,4-dihydropyrimidine; Me-FAPYglycoside,.2-amino-~~N-fo~yl-N-methylamino)-6-(N-glycosylamino)-4-while for the ethylene dibromide-GSH adduct, S - [2-
oxo-3,4-dihydropyrimidine.
(N-deoxyguanosyl)ethyl]GSH, this does not appear to be
0 1991 American Chemical Society
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important (7) and the predominant pathway is depurination. The two pathways will lead to different end points, either the FAPY adduct or an apurinic site, and these must be considered in the overall mutagenic profiles of these adducts. FAPY adducts are not seen as single species when purified by HPLC but instead are interconverting mixtures of several species (8-11). When observed on the NMR time scale, the individual species are resolved and seen as distinct compounds (8-11). Such mixtures of compounds have been assigned either as a mixture of regioisomers with the formyl group exchanging between the N5- and N6positions (Scheme I, pathway A) or alternatively as a mixture of rotamers (Scheme I, pathways B and C, and other possible rotameric structures). NMR studies of the FAPY adduct of N7-methylguanosine are consistent with the view that the isomerism is rotameric in nature (8-ll), but there has been and still remains considerable confusion in the literature as to the exact nature of the interconverting species for N7-methyl-FAPY and the FAPY adducts derived from other N7-guanyl adducts (5-7,12-14). In this study the nature of the interconversion is examined by use of specifically N7-15N-labeledguanosines. This approach allows the N5- and N6-positionsto be unambiguously distinguished and the exact nature of the interconverting species to be determined. Although the method is applied to only cases in which the N7-position is substituted with a methyl or S-ethyl-GSH substituent, it should be applicable to experiments with all types of FAPY adducts.
Table I. Rates of Imidazole Ring Opening for NT-Guanosyl and N7-Deoxyguanosyl Adducts h i 2 for imidazole ring oDenina, hn 1.2 3.0 2.4 8.7
compound A"-methylguanosine W-methyldeoxyguanosine S-[2-(W-guanosyl)ethyl]GSH S-[2-(W-deoxyguanosyl)ethyl]GSH
Measured by monitoring the decrease in absorbance at 290 nm as described previously (18). Experiments were carried out in 0.1 M 3-(cyclohexylamino)-l-propanesulfonic acid (CAPS) buffer at pH 10.0 and 37 OC. (I
Experimental Procedures 2,4-Diamino-6-hydroxypyrimidineand 1-bromo-2-chloroethane were purchased from the Aldrich Chemical Co. (Milwaukee, WI). Na15N02 was purchased from Isotec, Inc. (Miamisburg, OH). Ribose 1-phosphate, guanosine, and deoxyguanosine were purchased from Sigma Chemical Co. (St. Louis, MO). NMR spectra were recorded on IBM NR/300 and NR/200 NMR spectrometers. Chemical shifts are reported in ppm; 'H NMR spectra were referenced using 2,2-dimethyl-2-silapntane-5sulfonate to measure the position of the water resonance a t 22 "C and then using the residual water resonance as an internal standard. 15N NMR spectra were recorded at 20.287 MHz using a 1 0 " tunable probe and referenced to an external standard of saturated 16NH415N03 in 2H20. Chemical shifts are reported in ppm downfield from anhydrous NH3 by assigning the nitrate signal of the external reference to 376.25 ppm (15). S42-Chloroethyl)GSHwas synthesized as described elsewhere (3). [N7-15N]Guan~sine was synthesized using literature procedures (16)for the synthesis of labeled deoxyguanosine except that ribose 1-phosphate was used to yield the ribose instead of deoxyribose compound. N-Methylguanosine and "-methyldeoxyguanosine were synthesized by standard procedures (17). S-[2-(N7-Guanosyl)ethyl]GSHand S-[2-(N7-deoxyguanosyl)ethyl]GSH were synthesized by mixing 5 mg of guanosine or deoxyguanosine with 50 mg of S-(2-chloroethyl)GSH in 1 mL of a 1:l mixture of (CH3)2SO/H20(v/v) and incubating the mixture at 37 "C for 2 h. The compounds were purified by reverse-phase HPLC (Ultremex, wtadecasilyl, 5 pm, 10 X 250 mm, Phenomenex, Torrance, CA). The column was eluted with 5% CH30H (v/v) in 10 mM NH4CH3C02 (pH 4.5) for 5 min followed by a linear gradient increasing to 40% C H 3 0 H (v/v) over 35 min at a flow rate of 3.0 mL min-I: M-methyldeoxyguanosine, tR 21 min; S-[2-(N'-guanosyl)ethyl]GSH, t~ 19 min; S-[2-("-deoxyguanosyl)ethyl]GSH, t R 19 min. The N-alkylguanosine and -deoxyguanosine derivatives were converted to their FAPY derivatives by incubation in 0.1 N NaOH a t room temperature for 1 h. The deribosylated compound was obtained by heating the compound in 0.1 N HCl a t 100 "C for 30 min. After each step the compounds were purified by reverse-phase HPLC as described above: Me-FAPY-glycoside, tR 5-10 min; Me-FAPY-base, tR 7-10 min; GSH-FAPY-glycoside,
8:4
8:2
8:O 8, PPm
7:8
7:6
Figure 1. A portion of the lH NMR spectrum showing the formyl protons of [N6-15N]Me-FAPY-glycoside derived from [PPJ6N]guanosine (A) and the same compound with substitution of 14N a t the N5-position (B). T h e spectra were recorded in 2Hz0.
t~ 5-10 min; GSH-FAPY-base, t~ 5-6.5 min. All of the adducts, including FAPY derivatives, were characterized by their and 'H NMR and mass spectra, which were identical with those spectra reported previously (7-9).
Results and Discussion Rates of Imidazole Ring Opening. The rates of imidazole ring opening for M-methylguanosine, S-[2-(Nguanosyl)ethyl]GSH,and their deoxyribose analogues were measured using the decrease in Am as described previously (18). The half-lives for ring opening at pH 10.0 and 37 "C are listed in Table I. These results are consonant with published studies showing that ethyl substituents without strong electron-withdrawing groups are less prone to ring opening than are methyl derivatives (5). The results are also in agreement with our own data showing that the FAPY adduct derived from S-[2-(N'-deoxyguanosyl)ethyl]GSH is not formed in DNA either in vivo or in vitro a t neutral pH (7). 'H NMR Studies. The 'H NMR spectra of the MeFAPY-glycoside and the GSH-FAPY-glycoside showed that the samples were mixtures of at least eight compounds (Figures 1 and 2). This multiplicity is due to the facile interconversion of the sugar ring along with the formyl interconversion, giving rise to a mixture of the a- and @-linkedribofuranose forms along with a ribopyranose form (11). The GSH-FAPY-glycoside compound seemed to show preference for only four conformations while MeFAPY-glycoside existed as a mixture of about eight. In both cases the substitution of 15N a t the N7-position of
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634 Chem. Res. Toxicol., Vol. 4, No. 6, 1991
A
0:4
8:s
8:2
6:O
7:O
6:O
8:O
7:O
6:O
5:O
4:O
3:O
2:O
5:0
4:O
3:O
2:O
7:0
0:O
63 PPm
Figure 2. A portion of the 'H NMR spectrum showing the formyl protons of [NS-15N]GSH-FAPY-glycoside derived from [ N 15N)guanosine(A) and the same compound with substitution of 14Nat the N5-position(B). The peak at 6 8.47 is due to a small amount of guanine. The splitting due to the 15N is shown for the four major formyl peaks. The spectra were recorded in *H20.
A
1 LL
6:O
7:O
6:O
7:O
6.0
5:o
5:o
4:O
.
.
4.0
'
3.0 '
6,PPm
Figure 3. 'H NMR spectrum of [MJ5N]Me-FAF'Y-base(A) and the same compound with substitution of "N at the N5-position (B). The resonances at 6 3.0 and 3.1 are due to the methyl protons. The spectra were recorded in 2H20.
'
'
6,PPm
Figure 4. 'H NMR spectrum of [LV'-'~N]GSH-FAPY-~~S~ (A) and the same compound with substitutionof I4Nat the N5-pceition (B).The resonance assignments are as previously described (7). The spectra were recorded in 2H20. Table 11. Coupling Constants for Me-FAPY-base coupling constant conformer 1" conformer 2 2J formyl-I5N methyl-15N 4J formyl-methyl
14.1 1.1 0.4
15.1 1.2 0.7
Conformer 1 is arbitrarily assigned as the conformation whose methyl and formyl resonances are further downfield. The spectra were run in a 5050 mixture of (2H,C)2S0and 2H20.
guanosine, which produces a FAPY adduct with 15N substitution at the N5-position, resulted in the splitting of each formyl signal into a doublet (Figures 1A and 2A), as would be expected for protons geminal to an 15N. If the formyl group were located on the N6-position, it would be five bonds away and thus would not be split by the P J 5 N atom. The coupling constant observed for each of the signals varied between 13.7 and 16.2 Hz (depending on solvent conditions), in good agreement with literature values for formyl proton-15N 2J coupling constants (19). The samples were hydrolyzed with acid to yield MeFAPY-base or the GSH-FAPY-base. In the 'H NMR spectra of these it is even more clear that each of the formyl signals is being split into a doublet by substitution of 15Nat the N5-position (Figures 3 and 4). In the spectrum of Me-FAPY-base the methyl protons appear to not be split. These protons are also two atoms removed from the 15Nand although connected through a saturated system might still be expected to show some splitting. Upon close examination of the peaks a coupling constant of approximately 1 Hz was seen (Figure 5, Table 11). The methyl-15N splitting is complicated by the 4J splitting caused by the formyl proton and can be observed clearly after irradiation of the formyl protons (Figure 5). The value of 1Hz is typical of what is found in the literature
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Chem. Res. Toxicol., Vol. 4, No. 6,1991 635
A
B
A
II
3:2
310
2.9
2%
zoo
150
im
250
C
2m im 6, w m
too
zm
zoo
1%
tm
15N NMR of [N7-15N]guanosine (A) and [N6-lSN]GSH-FAPY-base (B and C). The spectra were recorded in (2H3C)2S0a t 298 K (A and B) or a 50:50 mixture of (2H3C)2S0 and 2H20at 290 K (C) using a flip angle of 45O, a pulse delay of 25 s, and reverse gated decoupling.
Figure 6.
after adduction, serve as an extremely useful reporter of
I
3.2
3:l
FAPY formation and structure within the oligonucleotide. Acknowledgment. This research was supported by USPHS Grants CA 44353 and ES 00267. W.G.H. is the recipient of USPHS Fellowship ES 05473. We thank Prof. T. M. Harris for help in obtaining some of the NMR
3.0
1(
C
spectra.
References (1) Lawley, P. D. (1984) In Chemical Carcinogens (Searle, C. E.,
$2
3.1
3.0
219
6 PPm Figure 5. A portion of the 'H NMR spectrum showing the methyl proton region of [N6-1SN]Me-FAPY-base. The spectra were recorded either without irradiation (A) or with irradiation at 6 8.18 (B)or 6 7.92 (C).
for substituted formamides; i.e., for N-methylformamide the 2J for the methyl-15N splitting is 1.0 Hz while the 2J is 15 Hz for the formyl-15N coupling constant (19). The ratio of the two formyl protons for both sets of compounds in 2H20is almost 1:l as seen in Figures 3 and 4. This is very dependent on solvent, and when the spectra are recorded in (2H3C)2S0,an 8:l ratio is seen, more typical of the reported literature (10, 11). 15N NMR Studies. The 15N NMR spectra of MeFAPY-base was recorded, and a single 15N resonance was found a t 6 109.7, as opposed to 6 249.1 seen for [W-15N]guanosine when the spectrum was recorded in (2H3C)2S0 (Figure 6). The spectrum was also recorded in a 50:50 mixture of (2H3C)2S0and 2H20,conditions under which there is an equal population of two species, and two signals at b 110.6 and 111.7 were seen (Figure 6C).
Conclusions This study provides an unambiguous assignment of the structure of the interconverting species of two different FAPY adducts derived from W-guanyl adducts. The structures must be those corresponding to rotameric isomerization shown in pathway B or C of Scheme I. Although this was the conclusion inferred from some previous studies, there was never any unequivocable proof that the isomerism observed was of a rotameric nature and not regioisomerism. The method described here should be applicable to other "-adducts where it is important to determine the nature of the FAPY structure. Although the work would be somewhat laborious, [N7-15N]deoxyguanosine could be built into oligonucleotides and could,
Ed.) 2nd ed., pp 325-484, American Chemical Society, Washington, DC. (2) Groopman, J. D., Croy, R. G., and Wogan, G. N. (1981) In vitro reactions of aflatoxin Bl-adducted DNA. Roc. Natl. A d . Sci. U.S.A. 78,5445-5449. (3) Humphreys, W. G., Kim, D.-H., Cmarik, J. L., Shimada, T., and Guengerich, F. P. (1990) Comparison of the DNA alkylating properties and mutagenic responses caused by a series of S42haloethyl)-substituted cysteine and glutathione derivatives. Biochemistry 29, 10342-10350. (4) Cmarik, J. L., Lloyd, R. S., Bruner, K. L., Tibbetts, C., and Guengerich, F. P. (1991) Mutational spectrum resulting from DNA adducts of 1,2-dibromoethane. h o c . Am. Assoc. Cancer Res. 32, 102. (5) Muller, N., and Eisenbrand, G. (1985) The influence of "-substituents on the stability of "-alkylated guanosines. Chem.-Biol. Interact. 53, 173-181. (6) Baertachi, S. W., Raney, K. D., Shimada, T., Harris, T. M., and Guengerich, F. P. (1989) Comparison of rates of enzymatic oxidation of aflatoxin Bl, aflatoxin GI, and sterigmatocystin and activities of the epoxides in forming guanyl-N7 adducts and inducing different genetic responses. Chem. Res. Toxicol. 2, 114-122. (7) Kim, D.-H., Humphreys, W. G., and Guengerich, F. P. (1990) Characterization of S-[2-(Wadenyl)ethyl]glutathione formed in DNA and RNA from 1,2-dibromoethane. Chem. Res. Toxicol. 3, 587-594. (8) Beranek, D. T., Weis, C. C., Evans, F. E., Chetsanga, C. J., and Kadlubar, F. F. (1983) Identification of M-methyl-M-formyl2,5,6-triamino-4-hydroxypyrimidine as a major adduct in rat liver DNA after treatment with the carcinogens,NJV-dimethylnitrosamine or 1,2-dimethylhydrazine. Biochem. Biophys. Res. Commun. 110,625-631. (9) Kadlubar, F. F., Beranek, D. T., Weis, C. C., Evans, F. E., Cox, R., and Irving, C. C. (1984) Characterization of the purine ring opened 7-methylguanine and its persistence in rat bladder epithelial DNA after treatment with the carcinogen N-methylnitrosourea. Carcinogenesis 5, 587-592. (10) Boiteux, S., Belleney, J., Roques, B. P., and Laval, J. (1984) Two rotameric forms of open ring 7-methylguanineare present in alkylated polynucleotides. Nucleic Acids Res. 12, 5429-5439. (11) Tomasz, M., Lipman, R., Lee, M. S., Verdine, G. L., and Nakanishi, K. (1987) Reaction of acid-activated mitomycin C with calf thymus DNA and model guanines: elucidation of the basecatalyzed degradation of A"-alkylguaninenucleosides. Biochemistry 26, 2010-2027. (12) Chetsanga, C. J., Bearie, B., and Makaroff, C. (1982) Alkaline opening of imidazole ring of 7-methylguanosine. 1. Analysis of the resulting pyrimidine derivatives. Chem.-Biol. Interact. 41, 217-233.
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(13) Hertzog, P. J., Smith, J. R. L., and Garner, R. C. (1982) Characterization of the imidazole ring-opened forms of trans-8,9dihydro-8-(7-guanyl)9-hydroxy aflatoxin B,. Carcinogenesis 3, 723-725. (14) Wood, M. L., Lindsay Smith, J. R., and Garner, R. C. (1988) Structural characterization of the major adducts obtained after reaction of an ultimate carcinogen aflatoxin B,-dichloride with calf thymus DNA in vitro. Cancer Res. 48, 5391-5396. (15) Levy, G. C. and Lichter, R. L. (1979) Nitrogen-I5 Nuclear Magnetic Resonance Spectroscopy, Wiley, New York.
(16) Massefski, W., Jr., Redfield, A., Sarma, U. D., Bannerji, A., and Roy, S. (1990) [ 7-15N]Guanosine-labeledoligonucleotides as nuclear magnetic resonance probes for protein-nucleic acid interaction in the major groove. J. Am. Chem. SOC.112, 5351-5353. (17) Jones, J. W., and Robbins, R. K. (1963) Synthesis of W-methyl guanosine. J. Am. Chem. SOC.85, 193-200. (18) Singer, B. (1972) Reaction of guanosine with ethylating agents. Biochemistry 11, 3939-3947. (19) Martin, G. R., Martin, M. L., and Gouesnard, J.-P. (1981) I5N NMR Spectroscopy, Springer-Verlag, New York.
Enzymatic Synthesis of Purine Deoxynucleoside Adducts Marie-Christine Chapeau and Lawrence J. Marnett" A. B. Hancock, Jr., Memorial Laboratory for Cancer Research, Departments of Biochemistry and Chemistry, Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 Received August 9, 1991
Introduction Adducts between electrophiles and nucleic acid bases are believed to play a key role in chemically induced mutation and cancer (1). Chemical synthesis of deoxynucleoside adducts provides authentic standards for comparison to biologically derived material and reagents for the preparation of adducted oligonucleotides (2). The preparation of certain classes of deoxynucleoside adducts is problematic because of the instability of intermediates to the conditions of synthetic transformations (e.g., the acid lability of purine deoxyribosides). An approach to the synthesis of sensitive deoxynucleosides is coupling of adducted bases to activated deoxyribose derivatives (3). This approach has found limited application because of problems of yield and stereochemistry and the need for multiple protecting groups. Enzymatic coupling of purine analogues to deoxyribose has been employed for the synthesis of isotopically substituted deoxynucleosides, antitumor agents, and biologically active molecules (4-8). Purine nucleoside phosphorylase (PNPase)' catalyzes displacement of phosphate from deoxyribose 1-phosphate by purines and purine analogues (eq 1). The stereochemistry of purine attachment produces the naturally occurring p-isomers. Although deoxyribose l-phosphate is commercially available, it is conveniently generated in situ by phosphorolysis of thymidine [catalyzed by thymidine phosphorylase (TPase)]. TPase and PNPase are commercially available, which provides an opportunity for a convenient synthesis of deoxynucleoside derivatives from thymidine (eq 1). In the present communication, we describe the utility of this method for the preparation of a variety of carcinogendeoxynucleoside adducts.
Abbreviations: PNPase, purine nucleoside phosphorylase; TPase, thymidine phosphorylase; 7-Me-MIG,7-methylpyrimido[1,2-a]purin-l0(W-one; MIG, pyrimido[1,2-a]purin-l0(3H)-one;HPLC, high-performance liquid chromatography;MPLC,medium-performanceliquid chromatography; dR, deoxyribose.
Materials and Methods Chemicals. Thymidine, p-(@hydroxyethyl)adenine, pmethyladenine, 7-methylguanine, TPase (EC 2.4.2.4),and PNPase (EC 2.4.2.1) were purchased from Sigma Chemical Co. (St.Louis, MO). 6-Chloropurine was obtained from Aldrich Chemical Co. (Milwaukee, WI). The commercial purines were used without further purification. ( [3H]Guanyl-8-ylamino)biphenyland [3H]guanyl-8-ylaniline were kindly provided by F. F. Kadlubar (National Center for Toxicological Research, Jefferson, AR). They were in excess of 95 % radiochemical purity. 06-Benzylguanine was kindly provided by Dr. David Swenson (Louisiana State University). 06-Ethylguanine, S-[2-(N"-guanyl)ethyl]glutathione, and 8-methylguanine were kindly provided by Dr. W. G. Humphreys (Vanderbilt University, Nashville, TN), and their purities were estimated to be greater than 99% by HPLC. 7-Me-MIG and MIG were synthesized by reaction of a-methylmalondialdehyde and malondialdehyde, respectively, with guanine (9). They were in excess of 98% pure by NMR analysis. Buffer salts and solvents were purchased from Fisher Scientific (Atlanta, GA) and were used without purification. E n z y m a t i c Coupling. The purine of interest (0.022 mmol) and thymidine (0.066 mmol) were dissolved in 20 m L of 20 mM potassium phosphate buffer, and the p H was adjusted to 7.3. TPase (2.2 units) and PNPase (3.3 units) were added, and the solution was shaken at 37-39 "C for varying times. The solutions were filtered through 0.45-pm filters, and the products were purified by HPLC (Beckman Ultrasphere C18-5 pm, 10 X 250 mm). The different solvent systems and the retention times of all the compounds are listed in the supplementary material. The general order of elution from reversed-phase columns was thymine, thymidine, starting base, and the adducted deoxynucleoside. All yields are reported as isolated. Syntheses of MIG-dR and 7-Me-M1G-dR were performed on a larger scale (0.3 m o l of purine). Purification was accomplished by open-column silica gel chromatography followed by MPLC. The silica gel column was eluted with a gradient of methanol in dichloromethane (for MIGdR 1:15 to 1.3:15 and for 7-MeMIGdR 1:15 to 1.8:15). The MPLC column (Baker cls-40 pm, 30 X 500 mm) was eluted with 20% methanol in water. Fluorescent fractions were collected and concentrated. The general procedure was slightly modified for ([3H]guanyl-8-y1amino)biphenyl and [3H]guanyl-8-ylaniline. One hundred microliters of a 1mM solution of the base in DMSO was added to 2 mL of 20 mM potassium phosphate buffer (pH 7.3) containing 0.7 pmol of thymidine. TPase (0.7 unit) and PNPase (1.5 units) were added, and the mixture was shaken a t 39 "C. A p p a r a t u s . NMR spectra were recorded on a Bruker AC 300-MHz instrument. Fast atom bombardment (glycerolmatrix),
0893-228~/91/2704-0636$02.50/0 0 1991 American Chemical Society
