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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. Enzymatic Coupling. The purine of interest (0.022 mmol) and thymidine (0.066 mmol) were dissolved in 20 mL of 20 mM potassium phosphate buffer, and the pH 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. Apparatus. NMR spectra were recorded on a Bruker AC 300-MHz instrument. Fast atom bombardment (glycerol matrix),
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Table I. Enzymatic Synthesis of Deoxynucleosides coupling coupling base yield,” % base yield? % 100 7-Me-M1G 61* e-ethylguanine 92 8-methylguanine 75 OS-benzylguanine 0 87 7-methylguanine P-methyladenine S-[2-(A”-guanyl)ethyl]0 78 glutathione IP-(p-hydroxy(guanyl-8-y1amino)0 ethy1)adenine 79 biphenyl 6-chloropurine 0 64b guanyl-8-ylaniline MlG a Yields are reported as isolated. Total coupling yield; N9 and N7 coupling products were obtained in relative yields of 90 and 1070,respectively. positive chemical ionization (direct probe), thermospray, and electrospray mass spectra were recorded on a VG 70-250HF, a Nermag R10-lOC, or a Nemag R30-10 mass spectrometer. HPLC was carried out with either a Varian Vista 5500 or a Varian 9010 pump equipped with a Varian 2050 UV detector or a Hewlett Packard 1040A diode array detector. Radioactive peaks eluting from HPLC columns were detected with a Radiomatic Flo-one Beta detector.
Results and Discussion Purines substituted in the 6-position with small alkyl groups (e.g., 06-ethylguanine, NG-methyladenine, and NG-(6-hydroxyethy1)adeninewere excellent substrates for PNPase (Table I). Likewise, 06-benzylguanine and 6chloropurine were converted to deoxyribonucleosides in high yield. In all cases, NMR spectroscopy revealed that the isolated product was a single diastereomer formed by @-couplingat N-9 of the purine. The yields of the 6-substituted purine deoxyribosides compare quite favorably with syntheses reported in the literature (10-12). In fact, to our knowledge N6-(6-hydroxyethy1)deoxyadenosinehas not been previously prepared. A number of difunctional carbonyl compounds react with deoxyguanosine residues to form 1,N2-cyclicadducts (13). Our laboratory has been particularly interested in a pyrimidopurinone adduct (MIG)formed by reaction with malondialdehyde (9). Previous attempts in our and other laboratories to produce MIG-deoxyriboseby condensation of malondialdehyde with deoxyguanosine led to isolated yields on the order of 2% (9,14). Reaction of MIG with thymidine in the presence of TPaseIPNPase generated the deoxynucleoside in 64% yield (eq 2) (Table I). Similar yields were obtained starting with 7-Me-MIG. In both cases, NMR analysis revealed that the products were a 9O:lO mixture of N-9 and N-7 diastereomers. The two isomers were separated chromatographically, which led to the isolation of the desired N-9 product in 58% yield. This is 30 times higher than previous syntheses.
R=H.MjG:CH~.?*M@
Ho’
(2)
Two different 7-alkylpurines were evaluated as substrates for PNPase. 7-Me-guanine exhibited no reaction even after long incubations (>48 h). This molecule is poorly soluble so coupling was attempted with 5’-[2(N-guanyl)ethyl]glutathione,a molecule that is freely soluble in phosphate buffer. Again, no coupling was observed even after prolonged incubation. Holy reported the coupling of 8-aminoadenine to deoxyribose 1-phosphate in 21% yield using Escherichia coli cells encapsulated in alginate beads as catalyst (6). In the
present study, g-methylguanine was converted to 8methyldeoxyguanosine in 75 3‘ % isolated yield. Thus, PNPase can accommodate small functional groups at the position immediately adjacent to the coupling site. However, when the 8-position was substituted with the larger anilino and aminobiphenylyl groups, no coupling was observed. The latter two reactions were performed in the presence of 5% DMSO to increase solubility. A control experiment with P-(P-hydroxyethyl)adenineindicated that this concentration of DMSO did not inhibit TPase or PNPase. PNPase-catalyzed coupling of substituted purines to deoxyribose 1-phosphate appears to be a viable strategy for synthesis of carcinogen-adducted deoxyribonucleosides. Reaction takes place under mild conditions, and no protecting groups are necessary. The stereochemistry of deoxyriboside coupling is exclusively 0,and the regiochemistry of attack on the purine is predominantly, but not exclusively, N-9. PNPase appears to accept a range of substituted purines, particularly at the 1-, 2-, and 6-positions. The limitations on coupling appear related to poor solubility and steric hindrance, although electrostatics may contribute to the lack of reaction at N-7. For carcinogens in which purine adducts can be prepared in high yield, enzymatic coupling to deoxyribose 1-phosphate may constitute a viable route for synthesis of carcinogen-deoxyribose adducts. Acknowledgment. This work was supported from research grants from the National Institutes of Health (CA-47479 and ES-00267). We are grateful to Thomas Harris for helpful discussions and to Frederick Kadlubar, David Swenson, and W. Griffith Humphreys for samples of purine adducts. Supplementary Material Available: Chromatographic conditions and retention times of all compounds described in this communication along with the NMR and mass spectral properties of the deoxynucleoside products (4 pages). Ordering information is given on any current masthead page.
References (1) Singer, B., and Grunberger, D. (1983)Molecular biology of mu-
tagens and carcinogens, Plenum, New York. (2) Basu, A. K., and Essigmann, J. M. (1988) Site-specifically modified oligonucleotides as probes for the structural and biological effects of DNA-damaging agents. Chem. Res. Toricol. 1, 1-18. (3) Srivastava, P. C., Robins, R. K., and Meyer, R. B. (1988) Synthesis and properties of purine nucleosides and nucleotides. In Chemistry of nucleosides and nucleotides (Townsend, L. B., Ed.) pp 113-281, Plenum Press, New York. (4) Krenitsky, T. A,, Koszalka, G. W., and Tuttle, J. V. (1981) Purine nucleoside synthesis, an efficient method employing nucleoside phosphorylases. Biochemistry 20, 3615-3621. (5) Krenitsky, T. A., Rideout, J. L., Chao, E. Y., Koszalka, G . W., Gurney, F., Crouch, R. C., Cohn, N. K., Wolberg, G., and Vinegar, R. (1986) Imidazo[4,5-c]pyridines (3-deazapurines) and their nucleosides as immunosuppressive and antiinflammatory agents. J. Med. Chem. 29, 138-143. (6) Holy, A., and Votruba, I. (1987)Facile preparation of purine and by biotransformation on pyrimidine 2-deoxy-~-D-ribonucleosides encapsulated cells. Nucleic Acids Symp. Ser. 18, 69-72. (7) Massefski, W., Jr., and Redfield, A. (1990) [7-15N]Guanosinelabeled oligonucleotides as nuclear magnetic resonance probes for J. Am. motein-nucleic acid interaction in the major - eroove. Chem. S O ~112, . 5350-5351. (8) . . Gaffnev. B. L.. Kune. P.-P.. and Jones. R. A. (1990) Nitroeen15-labeled deoxynucleosides. 2. Synthesis of [7:*6N]-labeleideoxyadenosine, deoxyguanosine,and related deoxynucleosides. J. Am. Chem. SOC.112, 6748-6749. (9) Basu, A. K., O’Hara, S. M., Valladier, P., Stone, K., Mols, O., and Marnett, L. J. (1988) Identification of adducts formed by reaction of guanine nucleosides with malondialdehyde and structurally related aldehydes. Chem. Res. Toricol. 1, 53-59. ’
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(10) Farmer, P. B., Foster, A. B., Jarman, M., and Tisdale, M. J. (1973) The alkylation of 2'-deoxyguanosineand of thymidine with diazoalkanes. Biochem. J. 135, 203-213. (11) Jones, J. W., and Robins, R. K. (1963) Purine nucleosides. 111. Methylation studies of certain naturally occurring purine nucleosides. J . Am. Chem. SOC.85, 193. (12) Robins, M. J., and Basom, G. L. (1973) Nucleic acid related compounds. 8. Direct conversion of 2'-deoxyinosine to 6-chloropurine 2'-deoxyriboside and selected 6-substituted deoxy-
nucleosides and their evaluation as substrates of adenosine deaminase. Can. J . Chem. 51, 3161. (13) Singer, B., and Bartach, H. (1986) The role of cyclic nucleic acid adducts in carcinogenesis and mutagenesis, IARC, Lyon. (14) Seto, H., Seto, T., Takesue, T., and Ikemura, T. (1986) Reaction of malonaldehyde with nucleic acid. 111. Studies of the fluorescent substances released by enzymatic digestion of nucleic acids modified with malonaldehyde. Chem. Pharm. Bull. 34, 5079-5085.
Chromium Bound to DNA Alters Cleavage by Restriction Endonucleases Kim M. Borgesti* and Karen E. Wetterhahn* Department of Biochemistry, Dartmouth Medical School, and Department of Chemistry, Dartmouth College, Hanover, New Hampshire 03755 Received June 10, 1991
Introduction Chromium(V1) compounds are known carcinogens in humans and animals (1) and mutagens in bacterial and eukaryotic systems (2). Chromium(V1)-induced DNA damage has been observed in the form of chromium-DNA complexes, DNA interstrand cross-links, DNA-protein cross-links, and DNA strand breaks (3-9). In vitro, significant interaction between chromium and purified DNA has been shown to occur only in the presence of substances able to reduce chromium(V1) to chromium(II1); e.g., reaction of chromium(V1) with DNA in the presence of microsomal enzymes led to chromium-DNA binding and DNA-protein cross-linking (10). Reaction of chromium(VI) with thiols, i.e., glutathione, cysteine, dithiothreitol, or 0-mercaptoethanol, in the presence of DNA resulted in chromium-DNA binding, as well as formation of glutathione-chromium(II1)-DNA and cysteine2-4-chromium(111)-DNA complexes (11,12). Chromium-DNA adduct formation, which appears to occur preferentially at guanine bases (10,12-14), induced aggregation and other conformational changes in DNA (11). Cross-linking of protein to DNA in CHO cells treated with chromium(V1) was shown to be mediated by chromium(III), and it was suggested that DNA replication, as well as RNA transcription and processing, would be affected by the DNA-protein cross-links (8). There is both in vitro and in vivo evidence for the ability of chromium-DNA complexes to affect normal DNAprotein interactions. Chromium(II1) increased the processivity of the Klenow fragment of DNA polymerase I and thereby enhanced the incorporation of nucleotides into newly synthesized DNA during replication of a singlestranded DNA template in vitro (15). Binding of chromium(II1) to plasmid DNA resulted in the inhibition of cleavage by the restriction enzyme HaeII, and it was suggested that modification of DNA structure by chromium(II1) could influence the interaction of regulatory proteins with specific DNA recognition sequences (16). Treatment of chick embryos in vivo with chromium(V1) resulted in differential effects on the expression of inducible and constitutive genes in liver, and the changes in expression of targeted genes correlated with chromium-
* To whom correspondence should be addressed at the Department of Chemistry, Steele Hall, Dartmouth College, Hanover, NH 03755. Department of Biochemistry, Dartmouth Medical School. *Presentaddress: Department of Cellular and Molecular Biology, University of Auckland, Auckland, New Zealand.
(VI)-inducedDNA damage in the form of chromium-DNA binding and DNA cross-links (3, 17). These studies indicate that chromium-DNA complexes may affect normal DNA-protein and DNA-enzyme interactions and thus potentially could interfere with both replication and transcription processes. Restriction endonucleases provide a well-characterized model system for studying sequence-specific DNA-protein interactions that are important in fundamental cellular processes (18). In order to determine whether glutathione-chromium(II1)-DNA complexes and other chromium-DNA adducts can alter the ability of enzymes to interact with DNA, we studied cleavage of chromiummodified DNA by restriction endonucleases. We report that the presence of chromium-DNA adducts can inhibit as well as enhance cleavage by restriction endonucleases, depending on the endonuclease involved, the DNA substrate, and the extent of chromium-DNA binding.
Experimental Procedures Supercoiled (form I) or PstI-linearized (form III) pBR322 DNA, or EcoRI-linearized SV40 DNA (form 111),was purified of contaminating metal ions by dialysis for 12 h a t 4 "C against 0.05 M Tris-HC1 (pH 7.0) containing 1 mM diethylenetriaminepentaacetic acid (Aldrich Chemical Co., Milwaukee, WI), followed by dialysis against the buffer only for 12 h at 4 "C. DNA (48 pM DNA-P') was incubated with 480 pM chromium(V1) (240 pM potassium dichromate, Fisher Scientific, Medford, MA) and dithiothreitol(O.48 or 2.4 mM, Bethesda Research Laboratories, Gaithersburg, MD), P-mercaptoethanol (2.4 or 4.8 mM, Sigma Chemical Co., St. Louis, MO), or glutathione (9.6 mM, Sigma Chemical Co.) at 37 OC for 30 min in 0.05 M Tris-HC1 (pH 7.0). Reactions containing 1.8 mM chromium(V1) and 18 mM glutathione were also prepared. Chromium(V1) solutions contained 0.12 pCi of [51Cr]sodiumchromate/nmol of chromium (15.631.2 Ci/mmol of Cr, New England Nuclear-Du Pont, Boston, MA). The reactions were stopped by cooling 0 OC, and chromium-DNA complexes were isolated by NENsorb 20 column (New England Nuclear-Du Pont) chromatography (11). DNA was assayed fluorimetrically using a modification of the diaminobenzoic acid technique of Kissane and Robbins (19),and levels of chromium were measured by scintillation counting in Aquasol-2 (New England Nuclear-Du Pont) using a Packard 1900CA liquid scintillation counter. The counting efficiency for [61Cr]chromium was 35% based on a quench curve. The residual chromium levela (typically less than 5%) in control samples lacking thiol, chromium, or DNA were subtracted from the levels in the complete Abbreviations: bp, DNA base pair, BME, 8-mercaptoethanol;DTT, dithiothreitol; DNA-P, DNA nucleotide: EDTA, disodium ethylenediaminetetraacetic acid; GSH, reduced glutathione.
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