Biochemistry 1987, 26, 2465-247 1 Janzen, E. G. (1971) Acc. Chem. Res. 4, 31-40. Janzen, E. G., Wang, Y. Y., & Shetty, R. V. (1978) J. Am. Chem. SOC.100, 2923-2925. Joshi, A., Rustgi, S., & Riesz, P. (1976) Znt. J . Radiat. Biol. Relat. Stud. Phys., Chem. Med. 30, 151-170. Kay, E. R. M., Simmons, N. S . , & Dounce, A. L. (1952) J. A m . Chem. SOC.74, 1724-1726. Kominami, S . , Rokushika, S., & Hatano, H . (1976) Int. J. Radiat. Biol. Relat. Stud. Phys., Chem. Med. 30, 525-534. Kominami, S., Rokushika, S., & Hatano, H. (1977) Radiat. Res. 72, 89-99. Kuehl, F. A., Jr., & Egan, R. W. (1 980) Science (Washington, D.C.) 210, 978-984. Kuwabara, M., Lion, Y., & Riesz, P. (1981a) Int. J . Radiat. Biol. Relat. Stud. Phys., Chem. Med. 39, 465-490. Kuwabara, M., Lion, Y., & Riesz, P. (198 1 b) Int. J. Radiat. Biol. Relat. Stud. Phys., Chem. Med. 39, 491-514. Kuwabara, M., Zhang, Z.-Y., & Yoshii, G. (1982) Int. J. Radiat. Biol. Relat. Stud. Phys., Chem. Med. 41, 241-259. Kuwabara, M., Yoshii, G., & Itoh, T. (1983) Int. J . Radiat. Biol. Relat. Stud. Phys., Chem. Med. 44, 219-224. Kuwabara, M., Inanami, O., & Sato, F. (1986) Int. J . Radiat. Biol. Relat. Stud. Phys., Chem. Med. 49, 829-844. Lagercranz, C. J. (1971) J . Phys. Chem. 75, 3466-3475. Lagercranz, C. J., & Setaka, M. (1974) Acta Chem. Scand., Ser. B B28, 619-624. Leadon, S . T., & Hanawalt, P. C. (1983) Mutat. Res. 112, 191-200. Lindahl, T. (1982) Ann. Rev. Biochem. 51, 66-87. Morgan, D. D., Mendenhall, C. L., Bobst, A. T., & Rouster, S. D. (1985) Photochem. Photobiol. 42, 93-94. Murota, S . (1984) in Gendai Kagaku, Special Issue 1, pp 3-37, Tokyo Kagaku Dojin, Tokyo.
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Riesz, P., & Rustgi, S. (1979) Radiat. Phys. Chem. 13,21-40. Rustgi, S., & Riesz, P. (1978) Znt. J . Radiat. Biol. Relat. Stud. Phys., Chem. Med. 33, 21-39. Scholes, G., Ward, J. F., & Weiss, J. (1960) J. Mol. Biol. 2, 379-391. Schulte-Frohlinde, D., & Bothe, E. (1984) 2. Naturforsch., C Biosci. 39C, 315-319. Spalletta, R. A., & Bernhard, W. A. (1982) Radiat. Res. 89, 11-24. Teebor, G . W., Frenkel, K., & Goldstein, M. S. (1982) Adv. Enzyme Regul. 20, 39-54. Teebor, G. W., Frenkel, K., & Goldstein, M. S. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 318-321. Ttoule, R., & Cadet, J. (1975) Int. J . Radiat. Biol. Relat. Stud. Phys., Chem. Med. 27, 211-222. Ttoule, R., & Cadet, J. (1978) in Effects of Ionizing Radiation on DNA (Huttermann, J., Kohnlein, W., Ttoule, R., & Bertinchamps, A. J., Eds.) pp 153-170, Springer-Verlag, New York, Heidelberg, and Berlin. von Sonntag, C. (1980) Adv. Carbohydr. Chem. Biochem. 37, 7-77. von Sonntag, C., & Schulte-Frohlinde, D. (1978) in Effects of Ionizing Radiation on DNA (Huttermann, J., Kohnlein, W., Ttoule, R., & Bertinchamps, A. J., Eds.) pp 204-235, Springer-Verlag, New York, Heidelberg, and Berlin. von Sonntag, C., Hagen, U., Schon-Bopp, A., & SchulteFrohlinde, D. (1981) Adv. Radiat. Biol. 9 , 109-142. Ward, J. F., & Kuo, I. (1973) Znt. J . Radiat. Biol. Relat. Stud. Phys., Chem. Med. 23, 543-557. Zhang, Z.-Y., Kuwabara, M., & Yoshii, G. (1983) Radiat. Res. 93, 213-231.
A Study of Side Reactions Occurring during Synthesis of Oligodeoxynucleotides Containing @-Alkyldeoxyguanosine Residues at Preselected Sitest Henryk Borowy-Borowski and Robert W. Chambers* Department of Biochemistry, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada Received May 23, 1986; Revised Manuscript Received December 12, 1986
As part of our studies on the molecular mechanisms of mutation by carcinogens we have synthesized 1.2 oligonucleotides (1 5-mers) containing an 06-alkylguanine residue at a preselected position for use as primers in the enzymatic synthesis of biologically active DNA. Ten of these oligonucleotides are derived from a minus strand sequence carrying the modified nucleotide in the third codon of gene G of bacteriophage GX174 DNA. Two others are derived from plus strand sequences carrying the modification in the 12th codon of the human Ha-ras protooncogene. During this work several potentially serious side reactions, which could complicate interpretation of mutagenesis data, were observed. This paper describes a detailed study of these reactions. Since we were unable to avoid undesirable side products, we developed simple chromatographic methods for detecting and removing them. ABSTRACT:
06-Alkylguanine moieties in DNA have received considerable attention ever since Loveless suggested that they were important premutagenic and precarcinogenic lesions (Loveless, 1969). As part of our studies on the molecular mechanisms f
This work was supported by a Terry Fox Special Initiatives Award
from the National Cancer Institute of Canada * Correspondence should be addressed tothis author
0006-2960/87/0426-2465$01.SO10
of mutation by carcinogens we have synthesized 12 oligonucleotides (15-mers) containing an @-alkyl group at a preselected position for use as primers in the enzymatic synthesis of biologically active DNA. Ten of these oligonucleotidesare derived from a minus strand sequence carrying the modified in the third condon Of gene Of bacteriophage cPX174 DNA. Two others are derived from plus strand sequences carrying the modification in the 12th condon of the 0 1987 American Chemical Society
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BI O CH EM ISTRY
human Ha-ras protooncogene. Side reactions encountered in these syntheses are potentially serious. A mutation arises from a single change in a single DNA molecule in a single cell, so the mutants one finds are biologically amplified. Since most of our experiments involve biological selection (Bhanot et al., 1979; Chambers et al., 1985), the mutants scored could, in principle, arise from an impurity rather than the desired adduct. This problem is most serious when the mutation frequency is low. In this paper we will describe our studies of the side reactions that occurred during the synthesis of 12 oligonucleotides containing an 06-alkylguanine moiety at a preselected position. We will show that in most cases side products cannot be avoided with current methodology. We will describe how we detected and removed these side products before using the modified oligonucleotide for enzymatic synthesis of DNA.
EXPERIMENTAL PROCEDURES Materials Unless otherwise noted, all reagents and solvents were from commercial sources and were used without further purification. Methods Preparative thin-layer Chromatography was carried out on 0.5 mm thick layers of Si1 G-25 UV254 on glass plates (Brinkman Instruments) with the following solvent systems: (A) n-propyl alcohol/NH40H/H20, 55/10/35 v/v/v: (B) CH,OH/ethyl acetate, 10/90 v/v; (C) CH,OH/CHCI,, 10/90 v/v. Analytical thin-layer chromatography was carried out on silica gel 60 F254backed with plastic (Merck). Column chromatography on silica gel was carried out with the following solvent systems: (D) CH30H/ethyl acetate, 1/99 v/v: (E) ethyl acetate/benzene, 15/85 v/v. Analytical HPLCI and purification of oligonucleotides were performed by reversephase chromatography using a 4.6 mm X 75 mm column (Beckman RPSC, 5-pm average particle size) monitored with an Altex Model 322 absorption meter. Vapor-phase chromatography was carried out on a 6 ft X in. Poropak Q column at 25 mL/min N2.2 The UV absorption spectra were measured with a Cary 14 spectrophotometer. The N M R spectra were measured on a Nicolet 360-MHz spectrometer at the Atlantic Regional NMR Facility, Department of Chemistry, Dalhousie University. Nucleoside analysis was carried out on a 0.5-nmol scale by digestion of the oligonucleotide with snake venom phosphodiesterase and Escherichia coli alkaline phosphatase as reported previously (KuEan et al., 1971). The resulting nucleosides were fractionated by reverse-phase chromatography on a 4.5 cm X 25 cm column (Lichrosorb RP-18, 10-pm particle size) with either isocratic elution or a linear gradient of CH,CN in 0.1 M triethylammonium acetate, pH 7.0, as indicated in the appropriate figure legends. Calibration curves
‘
Abbreviations: TLC, thin-layer chromatography; HPLC, highperformance liquid chromatography; Tris, tris(hydroxymethy1)aminomethane; DMT, dimethoxytrityl; iBu, isobutyryl; MsSC1, mesitylenesulfonyl chloride; 2-NH2-dAdo, 2-amino-2’-deoxyadenosine or 2’deoxyribosyl-2,6-diaminopurine; DBU, 1,8-diazabicyclo[5.4.O]undec-7ene. +X mutants are described by designating the gene, the nature of the mutation (ms = missense, am = amber, etc.), the codon number, the DNA sequence for the codon, the amino acid coded for by the codon, and the phenotype (wt = wild type; ts = temperature sensitive; le = lethal), ~) in that order. For example, + X G ~ S ~ ( T C G ) ~ ”is‘ a( ~temperaturesensitive missense mutant carrying the serine codon, TCG, as the third codon of gene G. * We are indebted to Dr. Carl Breckenridge of this department for the analyses.
BOROWY-BOROWSKI AND CHAMBERS
relating peak height to nanomoles were prepared from authentic standards. The column calibration was checked before each series of analyses with a standard mixture of nucleosides. When analyses are performed in duplicate, the deviation from the average is approximately 5%. Alkylation of 5‘-O-(Dimethoxytrityl)-N%obutyryl-2‘deoxyguanosine. An ethereal solution of the appropriate diazoalkane (generated from the appropriate N-alkyl-N’nitro-N-nitrosoguanidinewith KOH) was added in two or three portions to a previously cooled (0 “C) solution of the blocked nucleoside 3 (1 mmol) in 50 mL of methanol. After each addition, the reaction mixture was examined by TLC; the main product (depending upon the alkyl group) in solvent B had Rf = 0.45-0.55 and in solvent C had Rr 0.55-0.75. After 30-60 min, when only a small amount of unreacted material remained, the reaction was stopped to avoid prolonged exposure to diazoalkane. The mixture was fractionated by column chromatography on silica gel (2 cm X 30 cm) using solvent system D. The products were precipitated with cold hexane; yields were 45-65%. For characterization small samples were deprotected first with 80% acetic acid (20 min at 25 “C) to remove the dimethoxytrityl group and then with concentrated ammonia (50 OC, 48 h) to remove the isobutyryl group. After purification by TLC in solvent B, each sample was checked by HPLC and characterized by measuring the ultraviolet spectrum in methanol. The wavelengths corresponding to the maxima and minima for the 06-alkyl-2’-deoxyguanosinesare as follows (in nm): methyl, A, 247 and 282, A,, 225 and 262; ethyl, A, 247 and 282, A,, 227 and 262; n-propyl, A,, 248 and 282, A, 228 and 262; n-butyl,,,A 248 and 282: A,, 228 and 262. The values for @-methyl agree well with those reported in the literature (Friedman, 1963; Farmer et al., 1973). The IH N M R spectra of each 5’-0-(dimethoxytrity1)-Nisobutyryl-06-alkyl-2’-deoxyguanosinederivative was measured in Me2SO-d6. Our peak assignments are based on (1) comparison with authentic compounds of increasing complexity (2’-deoxyguanosine, 5’-O-(dimethoxytrityl)-2’-deoxyguanosine, fl-isobutyryl-2’-deoxyguanosine,5’-0-(dimethoxytrity1)fl-isobutyryl-2’-deoxyguanosine),(2) values reported for 5’-O(dimethoxytrityl)-~-isobutyryl-06-methyl-2f-deoxyguanosine in CDCl, (Kuzmich et al., 1983), and (3) spectral changes produced by spin decoupling. 5 ’-( Dimethoxytrityl)-h%sobutyryl-06-methyl-2 ’-deoxyguanosine: 6 1.06, 1.08 [2 d, 6, J = 6.4 Hz, (CH,),C], 2.38 (m, 1, H 2 9 , 2.84-2.90 (m, 2, H2’, -CHMe,), 3.08-3.29 (m, 2, H5’5”), 3.70, 3.71 (2 s, 6, Ar-OCH,), 3.96 (m, 1, H4’), 4.08 (s, 3, -OCH3), 4.53 (m, 1, H3’), 5.31 (d, 1, J = 4.3 Hz, 3’OH), 6.37 (t, 1, J = 6.3 Hz, Hl’), 6.69-7.30 (m, 13, Ar), 8.32 (s, 1, H8), 10.30 (s, 1, N2-H). 5 ‘-0-(Dimet hoxytrityl)-N%obutyry l- 06-ethyl-2’-deoxyguanosine: 6 1.06, 1.08 [2 d, 6, J = 6.5 Hz, (CH,),C], 1.41 (t, 3, J = 7.09 Hz, -CH3), 2.37 (m, 1, H2”), 2.83-2.89 (m, 2, H2’, -CHMe2), 3.09-3.29 (m, 2, H5’5”), 3.70, 3.71 (2 s, 6, Ar-OCH3), 3.96 (m, 1, H4’), 4.53 (m, 1 , H3’), 4.57 (4, 2, J = 7.03 Hz, -OCH2-), 5.310 (d, 1, J = 4.3 Hz, 3’OH), 6.36 (t, 1, J = 6.3 Hz, Hl’), 6.70-7.30 (m, 13, Ar), 8.31 (s, 1, H8), 10.27 ( s , 1, N2-H). 5 ‘-0-(Dimethoxytrityl)-N2-isobutyryl-O6-n-propy1-2 ’deoxyguanosine: 6 1.00 (t, 3, J = 7.4 Hz, - CH,), 1.06, 1.07 (2 d, 6, J = 6.4 Hz, Me&), 1.82 (m, 2, -CH,-), 2.36 (m, 1, H 2 9 , 2.82-2.87 (m, 2, H2’, -CHMe,), 3.07-3.26 (m,2 , H5’5’’), 3.70, 3.71 (2 s, 6, Ar-OCH3), 3.96 (m,1, H49, 4.48 (t, 2, J = 6.7 Hz, -OCH2-), 4.53 (m, 1, H3’), 5.32 (d, 1, J = 4.5 Hz, 3’OH), 6.37 (t, 1, J = 6.3 Hz, Hl’), 6.71-7.30 (m,
OLIGODEOXYNUCLEOTIDES CONTAINING O~-ALKYLGUANINE
13, Ar), 8.30 (s, 1, H8), 10.26 (s, 1, N2-H).
\
N
2467
Scheme I“
5’-O-(Dimethoxytrityl)-N2-isobutyryl-~-n-butyl-2‘deoxyguanosine: 6 0.95 (t, 3, J = 7.3 hz, -CH3), 1.05, 1.07 (2 d, 6, J =t 6.3 Hz,- Me2C), 1.45 (m, 2, -CH2CH3), 1.78b (m, N Y ) r ‘ 2, -CH,-), 2.35 (m, 1, H2”), 2.83-2.89 (m, 2, H2’, -CHMe2), 3.09-3.29 (m, 2, H5’5’’)) 3.70, 3.71 (2 s, 6, Ar-OCH,), 3.96 (m, 1, H4’), 4.53 (t, 2, J = 6.7 Hz, -OCH2-, m, 1, H3’), 5.31 (d, 1, J = 4.3 Hz, 3’OH), 6.36 (t, 1, J = 6.3 Hz, Hl’), 6.70-7.29 (m, 13, Ar), 8.30 (s, 1, H8), 10.26 (s, 1, N2-H). Preparation of @-Alkyl-2‘-deoxyguanosines via 06-Mesitylenesulfonyl Derivatives. 2’-Deoxyguanosine was converted to ~,3’,5’-triisobutyryl-06-(mesitylenesulfonyl)-2’-deoxyguanosine essentially as described by Gaffney and Jones (1982) except that mesitylenesulfonyl chloride instead of triisopropylbenzenesulfonyl chloride was used. After chromatographic purification using solvent system E, the product was obtained as a white foam by evaporation of the solvent (at 70% yield). This was reacted with the appropriate alcohol (n-propyl alcohol, isopropyl alcohol, n-butyl alcohol, isobutyl alcohol, sec-butyl alcohol, tert-butyl alcohol, or neopentyl alcohol; 2.5 mmol) at 0 OC (Gaffney & Jones, 1982). Before use each alcohol was examined for purity by vapor-phase chromatography. No isomers or other impurities were detected in the n-propyl alcohol or n-butyl alcohol at a level that would have detected 0.5%. Small amounts of impurities (98% normal isomer., Thus, in methanol very little, if Data supporting this statement were submitted for review but are not shown.
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BOROWY-BOROWSKI A N D CHAMBERS
BIOCHEMISTRY
Scheme 11’ 0*05
