Synthesis and properties of H-ras DNA sequences containing O6

Nov 1, 1988 - Richard Coulter , Maureen Blandino , Jessica M. Tomlinson , Gary T. Pauly , Magdalena Krajewska , Robert C. Moschel , Lisa A. Peterson ...
0 downloads 0 Views 1MB Size
Download PDF
Chem. Res. Toaicol. 1988,1, 391-397

391

Synthesis and Properties of H-ras DNA Sequences Containing 06-Substituted 2’-Deoxyguanosine Residues at the First, Second, or Both Positions of Codon 12 Gary T. Pauly,t Marilyn Powers,t Guo K. Pei,? and Robert C. Moschel*>f Laboratory of Chemical and Physical Carcinogenesis, BRI-Basic Research Program, and Program Resources, Inc., NCI-Frederick Cancer Research Facility, Frederick, Maryland 21 701 Received August 26, 1988

Nine 16-base oligodeoxyribonucleotideshaving the sequence of codons 9 through the first base have been of codon 14 of the rodent H-ras gene, Le., 5’-d(GTGGGCGCTG*G*AGGCG)-3’, synthesized containing either an Os-methyl- (G* = m6G), 06-ethyl- (G* = e6G), or the newly described 06-benzyl-2’-deoxyguanosineresidue (G* = b6G) at position 10 and/or 11 from the 5’-end. T h e conversion of the protected 06-substituted 2’-deoxyguanosine derivatives to the corresponding 3’-[0-(2-cyanoethyl) diisopropylphosphoraidites] and their incorporation into oligodeoxyribonucleotides were conveniently accomplished by using an “in situ” activation approach and automated phosphite triester synthetic methods. These oligomers were characterized by enzymatic digestion to their component nucleosides and were shown to be free of detectable contamination by known nucleoside impurities that can be produced during these syntheses. The melting behavior and circular dichroism spectra are described for duplexes of the nine 06-substituted 2’-deoxyguanosine containing oligomers paired with the complementary strand 5’-d(CGCCTCCAGCGCCCAC)-3’, and these data have been compared with those for the “wild-type” unsubstituted duplex.

Following a single dose to rats of the methylating carcinogen N-nitroso-N-methylurea, H-ras activation in mammary carcinomas results from a G-to-A transition mutation in the second position of the normal gene’s 12th codon (GGA) (1,2). This is presumably a consequence of conversion of the second guanine residue to an Osmethylguanine residue by the carcinogen. Loveless (3) first predicted that such lesions could lead to misincorporation of thymidine residues on complementary strands during DNA replication, and this prediction has been shown to be valid in several in vitro (4-6) and cellular (7-13) replication experiments involving oligonucleotides containing Os-methylguanine residues. With respect to H-ras activation, it is interesting that the first guanine residue in the 12th codon is not replaced by an adenine in the Nnitroso-N-methylurea-inducedtumors although in vitro mutagenesis studies indicate that a G-to-A transition in the first base of codon 1 2 also produces an oncogenic ras (14,15) and the Harvey murine sarcoma virus carries this mutation (16). One can speculate from these observations that, in rat mammary gland exposed to the methylating agent, either the first guanine residue at codon 12 is not modified at the Os-position as frequently as the second or, alternatively, that cellular repair systems repair an Ossubstituted guanine residue at the first position more efficiently than at the second (10). In an effort to test these hypotheses, we are evaluating the mutagenic potency of H-ras DNA sequences containing Os-substituted 2’deoxyguanosine residues at the first, second, or both positions of codon 12. We are also evaluating whether or not the steric bulk of the attached Os substituent has any effect on the structure or function of this DNA by comparing the effects of incorporated @-methyl-(m6G,Figure

* Author to whom correspondence should be addressed. BRI-Basic Research Program. *ProgramResources, Inc. f

l),@-ethyl- (e6G,Figure l),or the previously undescribed

06-benzyl-2’-deoxyguanosine(b6G, Figure 1). For the mutagenicity comparisons, an H-ras-containing plasmid has been constructed that will accept carcinogen-modified 16-base sequences in place of the normal sequence that spans codons 9 through the first base of codon 14 in the “sense” strand of the gene (i,e., oligodeoxyribonucleotide 1, Figure 1). For these constructions we have synthesized 1 as well as nine carcinogen-modified oligodeoxyribonucleotides that contain at position 10 and/or 11from the 5’-end either an @-methyl-2’-deoxyguanosine residue (G* = m6G) [i.e., oligomer l(10-m6G),l(ll-m6G),or 1(10,11di-msG),Figure 11, an @-ethyl-2’-deoxyguanosine residue (G* = e6G) [i.e., 1(10-e6G),l(ll-e6G),or l(l0,ll-di-esG)], or an @-benzyl-2’-deoxyguanosine residue (G* = b6G) [i.e., l(10-b6G), l(l1-b6G),or l(l0,ll-di-bsG),Figure 11. In this report we describe the synthesis and characterization of these oligodeoxyribonucleotides as well as the solution properties of their duplexes when they are paired with strand 2 (Figure 1). From this we can establish whether the type of 06-substituted 2’-deoxyguanosine residue or its position within these sequences leads to any significant differential disruption of duplex stability or conformation that might ultimately contribute to a rationale for the apparent selective mutability of the second guanine residue of H-ras codon 12. For these syntheses (Figure l ) , the 06-substituted 2‘-deoxyguanosine derivatives were converted to the correspondingprotected 3’- [0-(2-cyanoethyl) NJV-diisopropylphosphoramidites]and incorporated into the DNA segments by an “in situ” approach (17-20). The resulting oligodeoxyribonucleotides(Figure 1)are among the most complex 06-substituted 2’-deoxyguanosine containing sequences synthesized so far (11, 12, 21-24). Experimental Procedures Materials. The majority of reagents and solvents were from Aldrich Chemical Co., Inc., Milwaukee, WI, American Burdick

0893-228~/88/2701-0391$01.50/0 0 1988 American Chemical Society

392 Chem. Res. Toxicol., Vol. 1, No. 6, 1988 5‘-d(GTGGGCGCTGGAGGCG)-3‘

1 5’-d(CGCCTCCAGCGCCCAC)-3’

2 5 ’ - d ( G T G G G C G C T t G A G G C G ) -3 ‘

Iclo-e, 5 ’ - d ( C T G G G C G C T G t A G G C G ) -3’

& 5‘-d(GTGGGCGCTttAGGCG) -3’

Hd m 6 G . R=CH> e 6 G ; R=CH;CH3 b6G, R=CHBC6HS

l(lOJI-di8)

6

= m 6 G , e 6 G . b6G

Figure 1. Synthetic DNA sequences and structures of the incorporated 06-substituted 2’-deoxyguanosine residues. and Jackson, Muskegon, MI, or Applied Biosystems, Inc., Foster City, CA. Solvents were dried, if necessary, by distillation from CaHz and storage over 3-i% molecular sieves (Alfa Products, Morton Thiokol, Inc., Danvers, MA). Unmodified 2‘-deoxyribonudeosides were from Pharmacia LKB Biotechnology, Inc., Piscataway, NJ. Standard nucleoside phosphoramidites were from Applied Biosystems,Inc. Snake venom phosphodiesterase (type VI1 from Crotalus atrox venom) and bacterial alkaline phosphatase (type111 from Escherichia coli) were from Sigma Chemical Co., St. Louis, MO. Methods. NMR spectra were determined on a Varian XL-200 instrument equipped with an Advance Data System. Negative ion (-ve) fast atom bombardment (FAB)mass spectra (MS) were obtained with a reversed-geometry VG Micromass ZAB-2F spektrometer interfaced to a VG 2035 Data System. A mixture of glycerol and Nfl-dimethylformamide (1:l v/v) was used as the FAB matrix. UV absorption spectra and temperature-dependent absorption changes for DNA solutions were measured with a Gilford Model 250 spectrophotometer equipped with a Model 2527 Theymo-Programmerand an electrically heated cell compartment. The heating rate for the thermal studies was 0.5 “C/min. Circular dichroism spectra were recorded on a Jasco 5-500 A spectropolarimeter equipped with an IF-500 computer interface. Oligodeoxyribonucleotides were purified by highpressure liquid chromatography (HPLC) on a semipreparative 10 mm X 25 cm Beckman Ultrasphere ODS column (5-pm particle size) using two Waters 6000A pumps, a Model 660 solvent programmer, a Model 450 variable-wavelength UV detector, and a Model U6K sample injector. Analytical samples were chromatographed on a 4.6 mm X 25 cm Beckman Ultrasphere ODS column (5-pm particle size) as were enzymatic digests of the oligodeoxyribonucleotides. All HPLC was carried out at room temperature. Synthesis and Purification of 06-Substituted N2-Isobutyryl-2’-deoxyguanosines.These materials were prepared on a 1-3-”01 scale as described by Gaffney and Jones (25). They were purified as described below. When reactions were complete (25),the volatile reagenb and solvents were removed under reduced pressure. Removal of the 3’- and 5‘-O-isobutyryl protecting groups was accomplished by dissolving the resulting residue in 40 mL of methanol at 0 “C, followed by the addition of 40 mL of 2 N NaOH with stirring for 20 min a t 0 “C. The pH of the resulting solution was then adjusted to 7 with concentrated HCl. For the M-isobutyryl derivatives of Oe-methyl- and 06-ethyl2’-deoxyguanosine, the methanol was removed under reduced pressure and the aqueous samples were loaded on a 2.8 X 71 cm Sephadex LH-20 column eluted with HzO a t a flow rate of 1 mL/min. UV absorption was continuouslymonitored at 280 nm, and fractions (10 mL) were collected. P-Isobutyryl-O6methyL2‘-deoxyguanosine eluted in fractions 41-55 following sodium 2,4,6-triisopropylbenzenesulfonate(a byproduct of these syntheses) (25) which eluted in fractions 30-40. Later fractions (e.g., 50-60) contained, in addition to the desired product, a small l-j3-~-2-deoxyriboamount of M-isobutyryl-IP,1VB-dimethyl-9-( furanosyl)-2,6-diaminopurine (a product of a side reaction during these syntheses) (24, 26). Fractions 70-90 contained small amouhb of completely deprotected @-methyl-2’-deoxyguanosine.

Pauly et al. Rechromatography of fractions rich in N?-isobutyryl-06methyl-2’-deoxyguanosine(Le., fractions 41-55) afforded the pure nucleoside. Under identical chromatograhic conditions N2-isobutyryl-06-ethyl-2’-deoxyguanosineeluted in fractions 41-70 followed by the totally deprotected @-ethyl-2’-deoxyguanosine in fractions 80-110. The N2-isobutyryl-I@JP-dimethyl-9-(1-& ~-2-deoxyribofuranosyl)-2,6-diaminopurine produced in these syntheses could not be resolved from the desired N2-isobutyryl-@-ethyl-2’-deoxygosine under these chromatogaphic conditions. They were separable as the corresponding 5‘-0(4,4’-dimethoxytrityl) (4,4’-DMT) derivatives (see below). For N2-isobutyryl-06-benyl-2’-deo~g~e preparations, the crude product obtained in the neutralized MeOH/HzO (1:l)solution (see above) was chromatographed on the 2.8 x 71 cm Sephadex LH-20 column using MeOH/HzO (1:l)as solvent at a flow rate of 1mL/min. NL-Isobutyryl-@-benyl-2’-deoxyguanosine elutes along with benzyl alcohol in fractions (10 mL) 46-60. The benzyl alcohol was removed by repeated evaporation with HzO to afford N 2 - i s o b u t y r y l - @ - b e ~ l - 2 ‘ - d e o ~ ~ o sasi naehard foam in 67% 268; after 5 h at 25 “C in 1 N NaOH, UV yield. UV (HzO) A, A, 247,282; ‘H NMR (MezSO-d6/TMS)6 1.10 [d, 6, CH(CH,),], 2.27 (m, 1, H-2’4, 2.69 (m, 1,H-2’81, 2.88 [m, 1, CH(CH3)z],3.55 (m, 2, H-59, 3.83 (m, 1,H-49, 4.40 (m, 1,H-3’),4.91 (t, 1,OH-5’, exchanges with DzO),5.32 (d, 1,OH-3’,exchanges with DzO),5.62 (5, 2, C&CHz), 6.33 (t, 1,H-l’), 7.30-7.60 (m, 5, C&sCHz), 8.44 (s, 1,H-8), 10.43 (s, 1,NzH, exchanges with DzO);(-ve) FAB MS m / z 426 ([M - HI-), 336 ([M - C6H5CHz]-),310 ([BI-). Fully deprotected @-benzyl-2’-deoxyguanosine eluted from the LH-20 247, 282; ‘H NMR column in fractions 80-110. UV (HzO) ,A, (MezSO-d6/TMS)6 2.20 (m, 1, H-2’4, 2.57 (m, 1,H-2’/3), 3.53 (m, 2, H-5’), 3.82 (m, 1,H-49, 4.35 (m, 1, H-3’),4.98 (t, 1, OH-5’, exchanges with DzO), 5.26 (d, 1,OH-3’,exchanges with DzO),5.50 (s, 2, C&&Hz), 6.21 (t, 1, I-I-l’),6.48 (s, 2, “%Iz,exchange with DzO),7.38 (m, 5, C$Z5CH2),8.09 (s, 1, H-8); (-ve) FAB MS m/z 356 ([M - HI-), 266 ([M - CBH5CHz]-),240 ([BI-). 06-Benzylguanosine (the rihoside) was prepared previously (27). Preparation and Purification of 5’-0-(4,4’-Dimethoxytrityl) Derivatives of OB-SubstitutedN2-Isobutyryl-2’deoxyguanosines. Samples of the 06-substituted N?-isobutyryl-2‘-deoxyguanosineswere converted to the respective 5’-0-(4,4’-dimethoxytrityl)derivative in pyridine following standard procedures (26,223). When reactions were complete, the products were purified by silica gel column chromatography (Kieselgel60, Fluka Chemie) using CHC13/E5N (91) as solvent and were obtained in 70-90% yield. Tritylation of the mixture of nucleosides obtained in the N2-isobutyry1-O6-ethyl-2’-deoxyguanosine preparations leads to formation of the 5’-0-(4,4’-dimethoxytrityl) derivative of both N?-isobutyryl-OB-ethyl-2’deoxyguanosine and N?-isobutyryl-p,p-dimethyl-9-( 1-p-D-2deoxyribofuranosyl)-2,6-diaminopurine. These were not separated under the silica gel chromatographic conditions. Therefore, portions (50-60 mg) of the mixture of these 5’-0-(4,4’-dimethoxytrityl) derivatives were dissolved in 2 mL of CH3CN/H20 (1:l) and loaded on the 10 mm X 25 cm Beckman Ultrasphere ODS column eluted isocratically with CH3CN/Hz0 (57:43) at a flow derivative of rate of 3 mL/min. The 5’-0-(4,4’-dimethoxytrityl) ~-isobutyryl-06-ethyl-2’-deoxyguanosine elutes between 23 and 25 min while the analogous 4,4’-DMT derivative of @-isobutyryl-A@,PP-dimethyl-9-(1-~-~-2-deoxyribofuranosyl)-2,6-diaminopurine elutes between 26 and 28 min. Fractions for the former product were pooled, were made alkaline by the addition of triethylamine,and were evaporated to dryness. Our NMR data for the 5’-0-(4,4’-dimethoxytrityl) derivative of P-isobutyrylOB-methyl-and N2-isobutyryl-@-ethyl-2‘-deoxyguanosineare in agreement with those reported by others (22,24). Data for 5’O-(4,4’-dimethoxytrityl)-N?-isobutyryl-OB-benzyl-2’-deoxyguanosine follow: ‘H NMR (MezSO-dB/TMS)6 1.08 [dd, 6, CH(CH&], 2.35 (m, 1, H-2’4, 2.86 [m, 2, H-2’8 + CH(CH3)z], 3.18 (m, 2, H-5’), 3.69, 3.70 (2 s, 6, OCH,), 3.96 (m, 1,H-49, 4.51 (m, 1, H-39, 5.31 (d, 1, OH-3’, exchanges with DzO), 5.62 (s, 2, C&,CHz),6.37 (t,1, H-l’), 6.69-7.60 (m, 18, DMT-AI + Cd-I,CHz), 8.32 (s, 1,H-8), 10.37 (s, 1, N2H,exchanges with DzO);(-ve) FAB MS m / z 728 ([M - HI-), 638 ([M - CBH~CHZI-), 336 ([M - C6H5CHp - DMTJ-1, 310 ([BI-). Preparation of Protected OB-Substituted 2’-Deoxyguanosine 3’-[ 0 -(2-Cyanoethyl) N , N - d i i s o p r o p y l -

H-ras DNA Containing 06-Substituted Guanines Scheme I

Chem. Res. Toxicol., Vol. 1, No. 6,1988 393

samples not containing modified guanine residues. Dried samples were then redissolved in 3 mL of 0.1 M triethylammonium acetate (TEAA). The DNA was then purified by loading three 1-mL portions of the solutions on the semipreparative 10 mm X 25 cm Beckman Ultrasphere ODS column. The eluting solvents were 0.1 M TEAA, pH 7 (A), and acetonitrile (B). The gradient was 10-40% B over 60 min at 3 mL/min. Under these conditions the retention times for the 5’-0-(4,4’-DMT) derivative of all the oligomers except l(l0,ll-di-b6G)fall within the range 26-33 min. Oligomer l(10,11-di-b6G)elutes between 34 and 37 min. The recovered oligodeoxyribonucleotides were dried by lyophilization. These were then detritylated by treatment with acetic acid/H20 (82) for 15 min and redried by coevaporation with EtOH. The recovered samples were redissolved in 3 mL of 0.1 M TEAA, pH D M T - o y + o 7, and this was chromatographed on the semipreparative column 0 using a gradient of 5 4 0 % B over 60 min at 3 mL/min. All the H2N(~-Pr)2 nontritylated oligodeoxyribonucletides except the 06-benzyl-2’NC P deoxyguanosine-containingsamples eluted between 14.5 and 16.5 wO’ ‘N(i-Pr)2 6 min under these chromatographic conditions. Elution times for 5 the ~-benzyl-2’-deoxyguanosine-containing samples 1(10-bBG), l(ll-b6G), and l(l0,ll-di-b6G) were 19.2, 18.9, and 26.5 min, phosphoramidites]. To a dry 0.1 M solution of lH-tetrazole respectively. If necessary, portions of these nontritylated oligomers in acetonitrile (4 mL) was added 150 pL of freshly distilled 2(2C-30 ODzwunits) were further purified by preparative poly(4.7 X cyanoethyl NJVJV’,N’-tetraisopropylphosphorodiamidite acrylamide gel electrophoresis (PAGE) using gels that were 20% lo4 mol) (4, Scheme I) under argon. The appropriate 06-substituted 5’-0-(4,4’-dimethoxytrityl)-~-isobutyryl-2’-deoxy- acrylamide/NJV’-methylenebis(acry1amide) (191), 7 M urea, and 0.1 M Tris-borate, pH 8. guanosine (4 x lo4 mol) (generalized structure 3, Scheme I) was Enzymatic Digestion of Oligodeoxyribonucleotidesand then added, and the resulting solutions were allowed to stand at Nucleoside Composition Analysis. Oligodeoxyribonucleotides room temperature under argon. Thin-layer chromatography (0.2-0.3 ODzsounits) were treated with 0.1 unit of snake venom (TLC) (Kodak Chromagram Sheets, Eastman Kodak Co., phosphodiesterase and 2.5 units of bacterial alkaline phosphatase Rochester, NY) of reaction solutions at various times [TLC in 1 mL of 0.04 M Tris-HC1 and 0.001 M MgC12, pH 7.8, for 3 solvent, dichloromethane/ethyl acetate/triethylamine (45:4510)J h at 37 “C. The digests were then dried by lyophilization, and showed the disappearance of starting nucleoside [Rfvalues for the residue was redissolved in 200 pL of H20. Prior to chrothe 5’-0-(4,4’-DMT)-M-isobutyryl derivative of OB-methyl-, matographic analysis, 20 NL of a 0.50 mM solution of 2‘-deoxy@-ethyl-, and @-benzyl-2’-deoxygunosinewere 0.50,0.56, and uridine was added to the digest as a chromatographic internal 0.58, respectively] and the appearance of the corresponding faster standard. The sample was then loaded on a 4.6 mm X 25 cm migrating 3’- [0-(2-cyanoethyl)NJV-diisopropylphosphoramidite] Beckman Ultrasphere ODS column, eluted with a gradient of [Rfvalues: 0.92,0.96, and 0.96 for the 5’-0-(4,4’-DMT)-M-iso5 4 0 % solvent B over 90 min at a flow rate of 1 mL/min. UV butyryl 3’- [0-(2-cyanoethyl)N,N-diisopropylphosphoramidite] absorbance was continuously monitored at 260 nm, and these data derivative of @-methyl-, @-ethyl-, and Os-benzyl-2’-deoxywere supplied to a Hewlett-Packard 3350 Laboratory Automation guanosine (generalized structure 5, Scheme I), respectively]. System ( U S )for electronic integration of peak areas. Peak areas Reactions were generally complete in 2 h at room temperature. were converted to nanomoles of nucleoside by comparison with If, however, a significant amount of nonphosphitylatednucleoside calibration curves generated with authentic standards. remained after this time, then small volumes of additional Annealing of Oligodeoxyribonucleotide Duplexes. For phosphitylating agent were added to bring about complete conmost experiments 2.5 OD, units each of oligodeoxyribonucleotide version to the 3’-[0-(2-cyanoethyl) NJV-diisopropylphosphor1 and complement 2 (Figure 1)were mixed together in H20 to amidite] after an additional 1-2-h incubation. Proton-decoupled a total volume of 500 pL. This produced a solution in which 2 31Pchemical shifts in CH3CN (relative to 85% phosphoric acid was in slight excess as a consequenceof its lower molar absorptivity as external standard) for the diastereomeric phosphorus in the (see below). The solution was made 0.1 M in NaC1, was heated three OB-substitutedP’-deoxyguanosine phosphoramidite derivto 55-60 “C, and was allowed to cool slowly in a water bath to atives 5 (Scheme I) were 149.4 and 149.6 ppm in each case. When room temperature. The solution was then loaded on the semithe conversion to derivatives 5 was judged complete by TLC, preparative HPLC column eluted with a gradient of 5-40% B solutions were filtered to remove the precipitated diisopropylover 60 min at a flow rate of 3 mL/min. Oligodeoxyribonucleotide ammonium tetrazolide (6) (Scheme I), and the filtrate was used 2 eluted earlier than all the double-stranded duplexes. For studies immediately for DNA synthesis. involving duplex [ l(10,11-di-b6G) + 21, the component single DNA Synthesis, Deprotection, and Purification. Oligostrands were mixed in equimolar quantities and were annealed deoxyribonucleotideswere synthesized on a l0-mol scale by using but were not chromatographed. The higher acetonitrile conan Applied Biosystems, Inc., Model 380B DNA synthesizer. The centration required to elute l(l0,ll-di-b6G)denatured the [ 14-mL solution of 0.1 M modified nucleoside phosphoramidite (10,11-di-b6G)+ 21 duplex and caused the component single prepared as above was sufficient for four couplings on this synstrands to elute separately. AU the annealed samples were dried thesis scale. The standard Applied Biosystems, Inc., 10-pmol-scale by lyophilization and reconstituted in solutions that were either synthesis cycle was used throughout except that the @-substituted 0.01 or 1.0 M in sodium chloride, 0.01 M in sodium phosphate, 2’-deoxyguanosine phosphoramidites were allowed to couple for and 0.001 M in EDTA, pH 7.0. 5 rather than 3 min. At the end of synthesis, the 4,4’-DMT group was not removed from the last nucleoside in the sequence. The oligomers were cleaved from the column solid support by the Results and Discussion standard concentrated NH40H treatment. These were further deprotected by treatment with concentrated NH40H at 55 OC Synthesis and Characterization of Carcinogenfor 18 h. Samples were then evaporated to dryness. The OBModified Oligodeoxyribonucleotides. T h e critical substituted 2’-deoxyguanosine containing oligomers were redisstarting material for t h e synthesis of t h e 06-substituted solved in 20 mL of methanol/l,8-diazabicyclo[5.4.0]undec-7-ene 2’-deoxyguanosine containing oligodeoxyribonucleotides (DBU) (91) and allowed to stand at room temperature for 6 days. d e s c i.bed here is t h e protected OB-substituted2‘-deoxyAt the end of this period the methanol was removed under reduced guai.osine (Figure 1). These materials are now fairly pressure and the DNA was precipitated by the addition of 20 mL readily available through the synthetic sequence described of ether with vigorous mixing. The ether was decanted, and the by Gaffney and Jones (25). This sequence was first used DNA was washed with an additional 20 mL of ether and then air-dried. The methanol/DBU treatment was omitted for DNA t o prepare W-isobutyryl derivatives of 06-methyl-, 06-

394 Chem. Res. Toricol., Vol. 1, No. 6, 1988

Pauly et al.

ethyl-, and 0B-n-butyl-2’-deoxyguanosine (25),and more recently, Borowy-Borowski and Chambers (24) employed essentially the same sequence to prepare several additional 06-substituted 2’-deoxyguanosines bearing branched-chain alkyl groups at the OB-position. We also used the method I I of Gaffney and Jones (25) to prepare W-isobutyryl deE 051 . rivatives of OB-methyl-,@-ethyl-, and the previously undescribed 06-benzyl-2’-deoxyguanosine.The latter was isolated in 67% yield by Sephadex LH-20 column chromatography of synthesis reaction mixtures. Spectroscopic data for this material are presented under Experimental Procedures. Similar Sephadex LH-20 column chromatographic procedures were used to isolate the W-isobutyryl derivative of 06-methyl-and @-ethyl-2’-deoxyguanosine. This chromatographicstep simplified the separation of the desired W-isobutyryl derivative from the respective fully 0 10 5 deprotected 06-substituted 2‘-deoxyguanosine that is in10 15 20 25 30’ 55 60 65 evitably produced during alkaline hydrolysis of the 3‘- and Mlnutes 5’-O-isobutyryl protecting groups (25). In addition, for the Figure 2. HPLC separation of 2’-deoxyribonucleosides: (A) chromatography of 2’-deoxyribonucleoside standards; (B) nupreparations of the W-isobutyryl derivatives of @-methylcleoside mixture resulting from enzymatic digestion of oligoand 06-benzyl-2‘-deoxyguanosine, Sephadex LH-20 chrodeoxyribonucleotide l(10,11-di-m6G);(C) digest of oligodeoxymatography also resolved the desired product from a side ribonucleotide l(10,11-di-e6G);(D) digest of oligodeoxyriboproduct of these reactions, namely, W-isobutyrylnucleotide l(10,11-di-b6G).A known amount of 2‘-deoxyuridine NG,NG-dimethyl-9-(1-~-~-2-deoxyribofuranosy1)-2,6-di-is included in these digestions as an internal standard. Chroaminopurine (24, 26). For W-isobutyryl-OB-ethyl-2’matography was carried out on a 4.6 mm X 25 cm Beckman Ultrasphere ODS column (5-pm particle size) eluted with a linear deoxyguanosine syntheses, it was not possible to resolve gradient of 5 4 0 % acetonitrile in 0.1 M triethylammoniumacetate the desired 2‘-deoxyguanosine derivative from the side (pH 7) over 90 min at a flow rate of 1 mL/min. product by using LH-20 chromatography. However, following conversion of the mixture of these two nucleosides storage of these air- and moisture-sensitive materials can to their respective 5’-0-(4,4’-dimethoxytrityl) derivative, be avoided, this “in situ” activation approach can be highly the two products were readily separated by HPLC (see conserving of precious starting materials, and it seems the Experimental Procedures). In our hands, the extent of formation of W-isobutyryl-NG,NG-dimethyl-9-(1-(3-~-2- approach of choice for converting small quantities of difficultly accessible modified nucleosides to reactive dedeoxyribofuranosyl)-2,6-diaminopurinenever exceeded 5 % rivatives suitable for phosphite triester DNA synthesis. of that for the W-isobutyryl derivative of 06-methyl-, DNA samples prepared in this way were cleaved from 06-ethyl-, or 06-benzyl-2‘-deoxyguanosine, but since a the polymer support by a standard automated concenphosphoramidite of this side product can compete with trated NH40H treatment while the majority of the base06-substituted 2’-deoxyguanosine derivatives for incorpoprotecting groups were subsequently removed by treating ration in synthetic DNA, it is important that even trace the samples with concentrated NH40H at 55 OC for 18 h. amounts of the NG,NG-dimethyl-2,6-diaminopurine nuTo remove the W-isobutyryl protecting groups from the cleoside be removed from protected 06-substituted 2‘06-substituted 2’-deoxyguanosine residues in the carcinodeoxyguanosine preparations prior to their conversion to gen-modified oligodeoxyribonucleotides(Figure l),samples phosphoramidites. were also treated with solutions of MeOH/DBU ( 9 1 v/v) Following conversion of the W-isobutyryl derivatives of for 6 days at room temperature. This treatment was all the 06-substituted 2’-deoxyguanosines to their respecchosen since an alternative deprotection protocol involving tive 5’-0-(4,4’-DMT) derivative (generalized structure 3, treatment with concentrated NHIOH at high temperature Scheme I) (26,28),we used an “in situ” method (Scheme for extended times has been shown to convert OB-substiI) to generate the respective 3’- [ 0-(2-cyanoethyl) diisotuted guanine residues to 2,6-diaminopurine residues (13, propylphosphoramiditee](generalized structure 5, Scheme 22, 24). I) for DNA synthesis. Others have shown (17-20) that All the DNA segments (Figure 1) were purified by similar approaches can be used to successfully convert HPLC to afford between 200 and 300 ODzeounits of normal protected 2’-deoxyribonueleosidesto reactive 3’chromatographically homogeneous oligodeoxyribo0-phosphoramidites for automated DNA synthesis. In our nucleotide from a single 10-pmol-scalesynthesis. If necadaptation of these methods (Scheme I), samples of 3 were essary, portions of these were further purified by PAGE. treated with a 10-20% molar excess of 2-cyanoethyl N,These oligomers were characterized by enzymatic digestion N,N’,N’-tetraisopropylphosphorodiamidite(4) in the to their component 2’-deoxyribonucleosideswith snake presence of 1 equiv of 1H-tetrazole in dry acetonitrile venom phosphodiesterase and bacterial alkaline phossolution under argon. When conversion to product 5 was phatase followed by chromatographic analyses of their complete, the precipitated diisopropylammonium tetranucleoside composition (Figure 2). The elution profile zolide (6) was filtered and the filtrate was used immedipresented in Figure 2A shows the chromatographic reately for automated DNA synthesis. Coupling efficiencies tention time of the four common 2’-deoxyribonucleosides for the 06-substituted2’-deoxyguanosine phosphoramidites together with that for 2’-deoxyuridine (used as an internal prepared in this manner were in the range of 92-97% in performance calibration standard) (see Experimental all cases, and these were very similar to those for the Procedures) and the OB-methyl-, 06-ethyl-, and 06normal nucleoside phosphoramidites. Since only a minibenzyl-2’-deoxyguanosines.The small peak immediately mum amount of 5‘-0-(4,4‘-DMT)-protected nucleoside following that for @-ethyl-2’-deoxyguanosine in Figure 2A need be converted to a 3‘- [ 0-(2-cyanoethyl)diisopropylis due to a small amount of NG,P-dimethyl-9-(1-p-D-2phosphoramidite] by this method, and since isolation and deoxyribofuranosyl)-2,6-diaminopurine which was included

b

1 4

H-ras D N A Containing @-Substituted Guanines

Chem. Res. Toxicol., Vol. 1, No. 6, 1988 395

Table I. Nucleoside ComDosition of Synthetic Oliaodeoxyribonucleotides empirical nucleoside composition found" (empirical nucleoside composition expected) oligodeoxyribodThd dAdo 08-MedGuo 08-EtdGuo 08-BzlGuo em--- (X1O-b)b nucleotide dCvd dGuo 1.44 1 1.95 (2.00) 0.98 (1.00) nPC 3.02 (3.00) 10.07 (10.00) nP nP 1.13 1.05 (1.00) 1.97 (2.00) nP 2 9.91 (10.00) 3.08 (3.00) nP nP 1.37 1.02 (1.00) 0.99 (1.00) nP 1(10-msG) 2.03 (2.00) 3.00 (3.00) 8.99 (9.00) nP 1.97 (2.00) 1.36 1.05 (1.00) 0.97 (1.00) 1(11-msG) 8.90 (9.00) 3.08 (3.00) nP nP 1.94 (2.00) 1.27 1.07 (1.00) 1.91 (2.00) l(10,11-di-m6G) 3.11 (3.00) 7.96 (8.00) nP nP 1.97 (2.00) 0.98 (1.00) nP 0.93 (1.00) nP 1.37 3.05 (3.00) 9.09 (9.00) 1(10-e8G) 1.02 (1.00) nP 0.98 (1.00) 1.34 2.01 (2.00) 3.11 (3.00) 8.90 (9.00) l(ll-eeG) nP 1.27 1.92 (2.00) 0.97 (1.00) nP 1.92 (2.00) nP 3.00 (3.00) 8.19 (8.00) l(lO,ll-di-esG) 1.06 (1.00) 1.34 8.92 (9.00) 2.06 (2.00) 0.98 (1.00) nP 2.97 (3.00) 1(10-bsG) nP 1.03 (1.00) 1.95 (2.00) 0.98 (1.00) nP 1.34 9.03 (9.00) 3.01 (3.00) 1(1l-b'G). nP 1.29 0.95 (1.00) nP 2.18 (2.00) 2.12 (2.00) 7.83 (8.00) 2.92 (3.00) 1(10,ll-dl-b'G) nP 'Results are the average of three determinations. bCalculated from the total number of nanomoles of nucleoside liberated in a 1-mL digest of a known ODpmvalue of oligodeoxyribonucleotide(see Experimental Procedures). The number of nanomoles of oligodeoxyribonucleotide digested per milliliter was then calculated. B was obtained by multiplying ODm.mL/nmol by lo6. Data were determined in 0.05 M Tris-HC1, pH 8, and they are the average of three determinations. np = not present. ~~

in the mixture of standard nucleosides. This material is 1 the fully deprotected analogue of the side product produced in the OB-substituted2'-deoxyguanosine syntheses 0.8 (see above) (24,26). Figure 2B-D shows representative chromatograms of digests of portions of the disubstituted + 0.6 oligodeoxyribonucleotides 1(10,ll-di-m6G),1(10,11-di-e6G), and l(10,11-di-b6G), respectively (Figure 1). These chro0.4 matograms indicate that these DNA samples are free of a detectable contamination by the P,P-dimethyl-2,6-di0.2 aminopurine nucleoside. This is also true for digests of all six of the other monosubstituted oligodeoxyribo0- nucleotides (Figure 1). In addition, these digests and those 20 40 60 80 100 illustrated in Figure 2B-D are also free of detectable Temperature ("C) contamination by 9-(1-~-~-2-deoxyribofuranosyl)-2,6-diaminopurine,the product of ammonolysis of 06-substituted Figure 3. Normalized absorbance versus temperature profiles 2'-deoxyguanosine derivatives (13,22,24). To establish for synthetic DNA duplexes: (curve a) duplex [ l(l0,ll-di-esG) + 21; (curve b) duplex [l(10,11-di-b6G) + 21; (curve c) duplex this, we prepared a sample of this material by heating [l(10-e6G)+ 21; (curved) duplex [l(ll-b%) + 21; (curve e) duplex 06-methyl-2'-deoxyguanosine(2.5 mg) in 2 mL of con[ 1 + 21. Melting profiles were determined at duplex concentrations centrated NH40H to 55 "C for 11days. The ammonolysis of 10 pM in 1.0 M NaCl, 0.01 M NaH2P04,and 0.001 M EDTA, product was purified by HPLC, and samples were chrovalue pH 7.0. Absorbance was monitored at 280 nm. The T,,, matographed under conditions used for the DNA digests is the temperature at which (A, - A,)/AA = 0.5. to determine its retention time in relation to those for the standard 2'-deoxyribonucleosides. Under these chromaTable 11. T, Values for Oligodeoxyribonucleotide tographic conditions 9-(1-~-~-2-deoxyribofuranosy1)-2,6Du~lexes" diaminopurine elutes at 13 min between dThd and dAdo. oligodeoxyribooligodeoxyriboNo detectable peak for this material appears in the digest nucleotide dudex T, ("C) nucleotide dudex 7'- ("(2) chromatograms illustrated in Figure 2B-D or in those of [I + 21 79.8 [l(ll-eeG) + 21 71.4 the other synthetic oligodeoxyribonucleotides. This in[l(lO-msG)+ 21 70.4 [l(lO,ll-di-esG)+ 21 64.5 dicates that the 2,6-diaminopurine nucleoside is not a [l(ll-meG) + 21 71.3 [l(lO-beG)+ 21 72.8 [l(lO,ll-di-meG)+ 21 63.7 [l(ll-b6G) + 21 74.2 significant contaminant of these oligomers when depro[1(10-e6G)+ 21 71.3 [l(lO,ll-di-bsG)+ 21 68.3 tected and purified by our regimen (see Experimental Procedures). Chromatograms of digest such as those in aDetermined in 1.0 M NaCl, 0.01 M NaH2P04, and 0.001 M Figure 2B-D also indicate that the empirical nucleoside EDTA, pH 7.0, at a duplex concentration of 10 pM. composition determined for these oligomers (Figure 1)is in excellent agreement with expected values. These data ical studies, each of the carcinogen-modified olgomers was are presented in Table I. Also included in Table I are annealed with either a slight excess or a precisely detervalues for the molar absorptivities of these oligomers at mined equimolar amount of oligomer 2 (Figure 1). The 260 nm in aqueous solution. These data were calculated excess of 2 was easily separated from the majority of dufrom the known ODzso value of the oligodeoxyriboplexes by HPLC employing the chromatographyconditions nucleotide digested and the number of nanomoles of nuused to purify non-dimethoxytrityl-containingoligocleoside produced by its digestion. Consistent with the deoxyribonucleotides (see Experimental Procedures). This lower absorptivity at 260 nm of 06-substituted relative to ensured that the component strands of all duplexes were unsubstituted 2'-deoxyguanosine residues, the oligopresent in a 1:l molar ratio. The melting behavior of deoxyribonucleotides containing one and two 06-substiseveral of these duplexes in 1.0 M NaC1,O.Ol M NaH2P04, tuted 2'-deoxyguanosine residues exhibit molar absorpand 0.001 M EDTA, pH 7.0, is shown in Figure 3, and a tivities approximately 7600 and 16000 units less, respecsummary of T, values is presented in Table 11. The tively, than that for the unsubstituted oligomer 1 (Table melting curve for the "wild-type" duplex [I + 21 (T, = 79.8 "C, Table 11) is shown by curve e, Figure 3. Curve d shows I). the melting profile for the duplex of an 06-benzyl-2'Solution Properties of Carcinogen-Modified Oligodeoxyribonucleotide Duplexes. For physical-chemdeoxyguanosine-containing oligomer paired with 2 (i.e.,

3

f

396 Chem. Res. Toxicol., Vol. 1, No. 6, 1988

[1(11-b6G)+ 21, T, = 74.2 “C, Table 11). Curve c is for the 06-ethyl-2’-deoxyguanosine-containing duplex [ 1(10e6G) + 21 (T, = 71.3 “C) while curves b and a are for the di-@-benzyl- and di-@-ethyl-2’-deoxyguanosine-containing duplexes [l(10,11-di-b6G)+ 21 (T, = 68.3 “C) and [l(10,ll-di-e6G) 21 (T, = 64.5 “C), respectively. The melting c w e s for the other duplexes, like those illustrated in Figure 3, are monophasic in every case, indicative of a simple two-state helix to single-strand transition where no significant amount of other conformation (e.g., singlestrand hairpin loop) develops with increasing temperature. The somewhat broader melting curves observed for duplexes containing adjacent carcinogen-modified bases (e.g., curves a and b, Figure 3) probably reflect the presence of a region of greater localized instability in these duplexes relative to the monosubstituted analogues (22). Under identical solution conditions, no significant difference in T, was observed for duplexes containing the same 06substituted 2‘-deoxyguanosine residue a t either position 10 or 11of sequence 1 (Table 11). T, values for duplexes containing the same 06-substituted 2’-deoxyguanosine residue at both positions 10 and 11 (i.e., [l(lO,ll-di-m6G) 21, T , = 63.7 OC; [l(lO,ll-di-e6G) 21, T , = 64.5 “C; [l(lO,ll-di-b6G) 21, T, = 68.3 “C) are further reduced but by an amount slightly less than the sum of the reductions resulting from incorporation of the modified base a t either position 10 or 11 (Table 11). While it is not surprising that the duplexes comprised of an 06-substituted 2‘-deoxyguanosine containing strand melt at lower temperatures than that for the “wild-type”duplex [ 1 + 21, it is interesting that the decrease in duplex T, as a function of alkyl group structure follows the sequence “wild-type”> @-benzyl substituted > @-ethyl substituted 06-methylsubstituted. The factors responsible for the slightly greater stability of the 06-benzyl-substituted duplexes relative to the 06-methyl- or ethyl-substituted duplexes are uncertain at present. We observed only small decreases in T, (i-e., 1-3 “C) over a duplex concentration range of 10-2.5 pM (23,29-32). This is due, in part, to the limited concentration range examined and in part to the high GC content of these duplexes. This GC content would be expected to produce a fairly high value for the enthalpy of strand dissociation (AH”) (29). Since l/Tmis proportional to (RIM”) In C (where C = duplex concentration), a large value for AH” would cause plots of l/Tmvs In C to exhibit fairly shallow slopes reflecting the relative insensitivity of T, values to duplex concentration. Circular dichroism (CD) spectra for representative duplexes as a function of NaCl concentration are shown in Figure 4. Data for the other six duplexes are not readily distinguishable from these. Panel A, Figure 4, shows spectra for the “wild-type”duplex [l + 21. Panel B, Figure 4, contains spectra for the single 06-methylguanine-containing duplex [ 1(10-m6G)+ 21. Panels C and D contain data for duplexes [l(ll-e6G)+ 21 and [l(lO,ll-di-b6G)+ 21, respectively. In solutions of low NaCl concentration (i.e., 0.01 M, curves a, Figure 4) the CD spectra indicate that all the duplexes exist in the right-handed helical B-DNA conformation (33-35). Increases in NaCl concentration (i.e., curves b and c, Figure 4) bring about a transition to the C-DNA conformation for the “wild-type” duplex as well as all the 06-substituted 2’-deoxyguanosine containing duplexes as evidenced by the decrease in intensity of the positive band in the 270-290-nm region of each duplex spectrum. This is a common property of double-stranded DNA samples (33). However, the positive band in the 270-290-nm region of the spectrum for the

Pauly et al.

+

+

+

+

I

I I I I I , I I I I I 1 1 1 220 240 260 280 300 320 220 240 260 280 300 320

Wavelength (nm) Figure 4. Circular dichroism spectra of syntheticDNA duplexes as a function of NaCl concentration. Spectra were determined for solutions that were 0.01 M in NaH2P04and 0.001 M in EDTA, pH 7.0. Curves a in each panel are for solutions that were 0.01 M in NaC1, curves b were 1.0 M in NaC1, and curves c were 5.0 M in NaC1. Panel A: Duplex [I + 21. Panel B: Duplex [1(10m6G) + 21. Panel C: Duplex [l(ll-esG)+ 21. Panel D: Duplex

[l(10,11-di-b6G)+ 21.

unsubstituted duplex [ 1 + 21 decreases to a lower value in response to increasing NaCl concentration than does the same band in the spectra for the carcinogen-modified duplexes (e.g., Figure 4B-D). This suggests that 06-substitution on guanine residues may somewhat inhibit the salt-induced transition from B to C conformational forms. Others have observed an equally subtle inhibition of salt-induced B to Z transitions as a result of 06-methyl2‘-deoxyguanosine substitution in synthetic DNA (22). Our data demonstrate that the incorporation of one or two 06-substituted 2’-deoxyguanosine residues in these duplexes decreases the T , relative to that for the unmodified duplex, as expected. However, the incorporation of the same 06-substituted guanine residue a t either position 10 or 11 in the “sense” strand of these duplexes brings about virtually the same diminution in T,. Similarly, no readily distinguishable conformational changes result from incorporation of any of the 06-substituted guanines at position(s) 10 and/or 11. While it is understood that these observations are made on a very abbreviated model for the H-ras gene, they suggest that no major differential structural perturbation would result from the presence of an 06-substituted 2‘-deoxyguanosine at either the fiist or second position of the gene’s 12th codon. Thus, if repair in rat mammary gland of an Os-methylguanine residue at the first position is more rapid than repair a t the second position, it seems unlikely that this would result from gross structural effects induced by the modified base that cause it not to be recognized at the second position by repair systems. Other more subtle factors related to base sequence may be involved (10, 36). On the other hand, differential repair of this damage may not occur in certain mammalian cells. In this event, the selective Nnitroso-N-methylurea-induced G-to-A transition mutation for the second G of H-ras codon 12 would most likely be a consequence of a DNA sequence effect that directs methylation to the 06-position of the second guanine in preference to the first. DNA sequence can influence the

H-ras DNA Containing @-Substituted Guanines

extents and types of products produced with alkylating agents (37-39).

Acknowledgment. We are indebted to Drs. B. Hilton and G. Chmurney and Mr. John Klose for determining the NMR spectra and Drs. Y. Tondeur and C. Metra1 for determining the FAB mass spectra. This work was supported by the National Cancer Institute, DHHS, under Contract N01-74101 with Bionetics Research, Inc., and Contract N01-74102 with Program Resources, Inc. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US. Government.

References (1) Sukumar, S., Notario, V., Martin-Zanca, D., and Barbacid, M. (1983) Induction of mammary carcinomas in rata by nitrosomethylurea involves malignant activation of H-ras-1 locus by single point mutations. Nature (London) 306, 658-661. (2) Zarbl, H., Sukumar, S., Arthur, A. V., Martin-Zanca, D., and Barbacid, M. (1985) Direct mutagenesis of Ha-ras-1 oncogenes by N-Nitroso-N-methylureaduring initiation of mammary carcinogenesis in rata. Nature (London) 315,382-385. (3) Loveless, A. (1969) Possible relevance of OB-alkylationof deoxyguanosine to the mutagenicity and carcinogenicity of nitrosamines and nitrosamides. Nature (London) 223, 206-207. (4) Gerchman, L. L., and Ludlum, D. B. (1973) The properties of @-methylguanine in templates for RNA polymerase. Biochim. Biophys. Acta 308, 310-316. (5) Mehta, J. R., and Ludlum, D. B. (1978) Synthesis and properties of 08-methyldeoxyguanylic acid and its copolymers with deoxycytidylic acid. Biochim. Biophys. Acta 521, 770-778. (6) Abbott, P. J., and Saffhill, R. (1979) DNA synthesis with methylated poly(dC-dG) templates. Evidence for a competitive nature to miscoding by @-methylguanine. Biochim. Biophys. Acta 562, 51-61. (7) Green, C. L., Loechler, E. L., Fowler, K. W., and Essigmann, J. M. (1984)Construction and characterization of extrachromosomal probes for mutagenesis by carcinogens: Site-specificincorporation of @-methylguanine into viral and plasmid genomes. Proc. Natl. Acad Sci. U.S.A. 81,13-17. (8) Loechler, E. L., Green, C. L., and Essigmann, J. M. (1984) In uiuo mutagenesis by OB-methylguaninebuilt into a unique site in a viral genome. Proc. Natl. Acad. Sci. U.S.A. 81, 6271-6275. (9) Chambers, R. W., Sledziewska-Gojska,E., Hirani-Hojatti, S., and Borowy-Borowski, H. (1985) uurA and recA mutations inhibit a site-specifictransition produced by a single @-methylguanine in gene G of bacteriophage 4x174. Proc. Natl. Acad. Sci. USA. 82, 7173-7177. (10) Topal, M. D., Eadie, J. S., and Conrad, M. (1986) @-Methylguanine mutation and repair is nonuniform. J. Biol. Chem. 261, 9879-9885. (11) Bhanot, 0. S., and Ray, A. (1986) The in uiuo mutagenic frequency and specificity of OB-methylguaninein 4x174 replicative form DNA. Proc. Natl. Acad. Sci. U.S.A. 83, 7348-7352. (12) Hill-Perkins, M., Jones, M. D., and Karran, P. (1986) Sitespecific mutagenesis in vivo by single methylated or deaminated purine bases. Mutat. Res. 162,153-163. (13) Basu, A. K., and Essigmann, J. M. (1988) Site-specifically modified oligodeoxynucleotides as probes for the structdral and biological effects of DNA-damaging agents. Chem. Res. Tozicol. 1, 1-18. (14) Barbacid, M. (1986) Human Oncogenes. In Important Aduances In Oncology (DeVita, V. T., Hillman, s.,and Rosenberg, S. A., Eds.) pp 3-22, J. B. Lippincott, Co., Philadelphia. (15) Barbacid, M. (1987) ras genes. Annu. Reu. Biochem. 56, 779-827. (16) Dhar, R., Ellis, R. W., Shih, T. Y., Oroszlan, S., Shapiro, B., Maizel, J., Lowy, D., and Scolnick, E. (1982) Nucleotide sequence of the p21 transforming protein of Harvey murine sarcoma virus. Science 217,934-937.

Chem. Res. Toxicol., Vol. 1 , No. 6,1988 397 (17) Beaucage, S. L. (1984) A simple and efficient preparation of deoxynucleoside phosphoramidites in situ. Tetrahedron Lett. 25, 375-378. (18) Barone, A. D., Tang, J.-Y., and Caruthers, Itk H. (1984)In situ activation of bis-dialkylaminophosphines-d new method for synthesizing deoxyoligonucleotideson polymer supports. Nucleic Acids Res. 12, 4051-4061. (19) Moore, M. F., and Beaucage, S. L. (1985) Conceptual basis of the selective activation of bis(dialky1amino)methoxyphosphines by weak acids and ita application toward the prepaiation of deoxynucleoside phosphoramidites in situ. J. Org. Chem. 50, 2019-2025. (20) Nielsen, J., Marugg, J. E., Thgaard, M., van Boom, J. H., and Dhal, 0. (1986) Polymer-supported synthesis of deoxyoligonucleotides using in-situ prepared deoxynucleoside 2-cyanoethyl phosphoramidites. Recl. Trav. Chim. Pays-Bas 105, 33-34. (21) Fowler, K. W., Buchi, G., and Essigmann, J. M. (1982) Synthesis and characterization of an oligonucleotide containing a carcinogen-modified base: @-methylguanine. J. Am. Chem. SOC. 104, 1050-1054. (22) Kuzmich, S., Marky, L. A., and Jones, R. A. (1983) Specifically alkylated DNA fragments. Synthesis and physical characterization of d[CGC(OBMe)GCG]and d[CGT(06Me)GCG]. Nucleic Acids Res. 11, 3393-3404. (23) Gaffney, B. L., Marky, L. A., and Jones, R. A. (1984) Synthesis and characterization of a set of four dodecadeoxyribonucleoside undecaphosphates containing @-methylguanineopposite adenine, cytosine, guanine, and thymine. Biochemistry 23, 5686-5691. (24) Borowy-Borowski,H., and Chambers, R. W. (1987) A study of side reactions occurring during synthesis of oligodeoxynucleotides containing OB-alkyldeoxyguanosineresidues at preselected sites. Biochemistry 26, 2465-2471. (25) Gaffney, B. L., and Jones, R. A. (1982) Synthesis of OB-alkylated deoxyguanosine nucleosides. Tetrahedron Lett. 23,2253-2256. (26) Jones, R. A. (1984) Preparation of protected deoxyribonucleosides. In Oligonucleotide Synthesis: A Practical Approach (Gait, M. J., Ed.) pp 23-24, IRL Press Limited, Oxford, U.K. (27) Gerster, J. F., and Robins, R. K. (1965) Purine nucleosides. X. The synthesis of certain naturally occurring 2-substituted amino-9-@-D-ribofuranosylpurin-6( lw-ones (N2-substituted guanosines). J. Am. Chem. SOC. 87,3752-3759. (28) Narang, S. A,, Brousseau, R., Hsiung, H. M., and Michniewicz, J. J. (1980) Chemical synthesis of deoxyoligonucleotides by the modified triester method. Methods Enzymol. 65, 610-620. (29) Borer, P. N., Dengler, B., Tinoco, I., and Uhlenbeck, 0. C. (1974) Stability of ribonucleic acid double-stranded helices. J. Mol. Bid. 86, 843-853. (30) Albergo, D. D., Marky, L. A., Breslauer, K. J., and Turner, D. H. (1981) Thermodynamics of (dG-dQS double-helix formation in water and deuterium oxide. Biochemistry 20, 1409-1413. (31) Albergo, D. D., and Turner, D. H. (1981) Solvent effects on thermodynamics of double-helix formation in (dG-dQ. Biochemistry 20, 1413-1418. (32) Kierzek, R., Caruthers, M. H., Longfellow, C. E., Swinton, D., Turner, D. H., and Freier, S. M. (1986) Polymer-supported RNA synthesis and its application to test the nearest-neighbor model for duplex stability. Biochemistry 25, 7840-7846. (33) Ivanov, V. I., Minchencova, L. E., Schyolkina, A. K., and Poletayev, A. I. (1973) Different conformations of double-stranded nucleic acid in solution as revealed by circular dichroism. Biopolymers 12, 89-110. (34) Pohl, F. M. (1976) Polymorphism of a synthetic DNA in solution. Nature (London) 260, 365-366. (35) Saenger, W. (1984) Principles of Nucleic Acid Structure, Springer-Verlag, New York. (36) Topal, M. D. (1988) DNA repair, oncogenes and carcinogenesis. Carcinogenesis 9, 691-696. (37) Briscoe, W. T., and Cotter, L.-E. (1985) DNA sequence has an effect on the extent and kinds of alkylation of DNA by a potent carcinogen. Chem.-Biol. Interact. 56, 321-331. (38) Hartley, J. A., Gibson, N. W., Kohn, K. W., and Mattes, W. B. (1986) DNA sequence selectivity of guanine-N7 alkylation by three antitumor chloroethylating agents. Cancer Res. 46, 1943-1947. (39) Mattes, W. B., Hartley, J. A., and Kohn, K. W. (1986) DNA sequence selectivity of guanine-N7 alkylation by nitrogen mustards. Nucleic Acids Res. 14, 2971-2987.