Synthesis of 2'-deoxy-7, 8-dihydro-8-oxoguanosine and 2'-deoxy-7, 8

Veeraiah Bodepudi, Shinya Shibutani, and Francis Johnson*. Department of Pharmacological Sciences, School of Medicine, Health Sciences Center,State...
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Chem. Res. Toxicol. 1992,5, 608-617

608

Synthesis of 2’-Deoxy-7,8- dihydro-8 - oxoguanosine and 2’-Deoxy-7,8-dihydro-8-oxoadenosine and Their Incorporation into Oligomeric DNA Veeraiah Bodepudi, Shinya Shibutani, and Francis Johnson* Department of Pharmacological Sciences, School of Medicine, Health Sciences Center, State University of New York at Stony Brook, Stony Brook, New York 11794 Received February 27, 1992

Reliable methods have been developed for the synthesis of the 3’-0-[(diisopropylamino) (2cyanoethoxy)phosphinol-5’-0-(4,4’-dimethoxytrityl) derivatives of 2‘-deoxy-7,8-dihydr0-8-0~oguanosine (8-oxo-dGuo, 1) and 2’-deoxy-7,8-dihydro-8-oxoadenosine (8-oxo-dAdo, 2), and for the efficient incorporation of the latter into oligomeric DNA. Both methods rely on the conversion of the 2’-deoxy-8-bromopurine nucleosides 3 and 10 to their corresponding 2’-deoxy-8-(benzyloxy) nucleosides 4 and 12 followed by catalytic hydrogenation to generate the 8-oxo function a t the C-8 position. The preparation of the phosphoramidites 8 and 19 required for the synthesis of a series of DNA oligomers was carried out under strictly anhydrous conditions. Failure to keep the systems dry resulted in great difficulties during the purification procedures, and erratic results when DNA synthesis was attempted. In the preparation of the DNA itself, it was found to be extremely important during the ammonia deprotection step to add an antioxidant. Otherwise aerial oxidation resulted in almost complete loss of the oligomer. However, when these special conditions were followed, oligomeric DNA containing 8-oxo-dGuo and 8-oxo-dAdo residues could be prepared in excellent yield. Analysis of selected DNA oligomers by enzymatic degradation and mass spectroscopic analysis confirmed the designated sequences and compositions.

Introduction Recently there has been an upsurge of interest in purine deoxynucleosides having an oxo group at the 8position, because of their occurrence as abnormal nucleotide residues in cellular DNA (1). This type of chemical lesion has significant mutagenic potential and may ultimatelyresult in carcinogenesis (2,3).The abnormal deoxynucleoside residues appear to arise in DNA principally from oxidative events (4). A notable source is ionizing radiation (5-12),but endogenous agents also seem to be implicated (13-15) in their generation. Exogenous substrates such as ferric nitrilotriacetate (16,17) (a known and carcinogen), other “peroxisome generators” (18,19), asbestos (20)in the presence of peroxides have been shown to induce this type of lesion in DNA. Current interest in such lesions centers on (a) biophysical studies where their influence on the microstructure (21-27)of DNA is under scrutiny and (b) research on their miscoding properties (21,28-32)and the cellular mechanisms involved in their repair (33,34).These investigations are frequently best conducted on oligomeric DNA containing such modified nucleoside residues, located site-specifically. In support of these studies (21,28,30,33) our research has centered on the synthesis of DNA oligomers containing (8-oxoeither a 2‘-deoxy-7,8-dihydro-8-oxoguanosine dGuo,l 1) residue or the corresponding adenosine derivative 2 (2’-deoxy-7,8-dihydro-8-oxoadenosine, 8-oxo-

* T o whom correspondence should be addressed.

Abbreviations: 8-oxo-dGuo,2’-deoxy-7,8-dihydro-8-oxoguanosine; 8oxo-dAdo, 2’-deoxy-7,8-dihydro-8-oxoadenosine; DMT, dimethoxytrityl; HMDS, hexamethyldisilazane; DMSO,dimethyl sulfoxide; T H F , tetrahydrofuran; FAB/MS, fast atom bombardment mass spectrometry; TLC, thin-layer chromatography; CPG, controlled pore glass; DCC, dicyclohexylcarbodiimide; HPLC, high-performance liquid chromatograDMF, dimethylformamide. phy; 8-Br-dGuo, 8-bromo-2’-deoxyguanosine;

OH

OH

1 -

-2

dAdo). Previously we outlined in a brief communication (35)a modification of the phosphoramidite procedure (36, 37) which allowed incorporation of 8-oxo-dGuo into oligomeric DNA in excellent yield. Prior to the publication of our results Roelen et al. (38)appear to have tried unsuccessfullyto achieve phosphoramidite formation with the W-acetyl derivative of 5’-0-DMT-8-oxo-dGuo using previously established methods. This failure led them to conclude, albeit erroneously, that protection of the 06and 7-positions was necessary before phosphoramidite formation could be achieved in high yield. In addition, their route was plagued by low yields in its initial steps. This in turn necessitated a lengthier route starting with 8-bromoguanosine. In this paper we now report, in greater detail, simpler but highly reproducible methods for the synthesis of multigram quantities of the dimethoxytrityl (DMT) derivatives of both 8-oxo-dGuo and 8-oxo-dAdo (1and 2, respectively),their conversion to the corresponding phosphoramidites, and the subsequent incorporation of the latter into oligomeric DNA.

Materials and Methods 2’-Deoxyguanosine, 2’-deoxyadenosine,2-cyanoethyl NJ-diisopropylphosphoramidochloridite,and standard 2-cyanoethyl

0893-228~/92/2705-0608$03.00JO 0 1992 American Chemical Society

Incorporation of 8-Oxopurine Deoxynucleosides into DNA

Chem. Res. Toxicol., Vol. 5, No. 5, 1992 609

phosphoramidites were obtained from American Bionetics, Inc. (Hayward, CA). Benzoyl chloride, benzyl alcohol, chlorotrimethylsilane, 4,4’-DMT chloride, hexamethyldisilazane (HMDS), isobutyric anhydride, the Pd-on-charcoal catalyst, and phenoxyacetic acid were obtained from Aldrich Chemical Co., Inc. (Milwaukee, WI). Bromine, Celite, hydrochloric acid (HCl), phosphorus pentoxide (P205),dichloromethane (CHZClZ),methanol (MeOH), dimethyl sulfoxide (DMSO), acetic acid, ether, acetone, pyridine, ethyl acetate (EtOAc), n-butyl alcohol, ethanol, hexane, triethylamine, tetrahydrofuran (THF),and toluene were purchased from Fisher Scientific (Fair Lawn, NJ). Benzene was purchased from J. T. Baker (Phillipsburg, NJ), and silica gel 60 (0.040-0.063 mm) was bought from EM Science (Gibbstown, NJ). The DMSO was dried over CaH2, decanted, and distilled prior to use. Pyridine and CH2C12were heated under reflux under CaH2 and then distilled. Triethylamine was heated with Na wire for 6 h, decanted and then distilled over CaH2. Benzene and toluene were boiled over P205 and then distilled. THF was distilled from sodium benzophenone ketyl. Proton (300 MHz) and 13C (75 MHz) NMR spectra were recorded in DMSO-& on a General Electric QE-300spectrometer unless otherwise noted. Chemical shifts are reported relative to an internal standard of tetramethylsilane. 31PNMR spectra were recorded on a NT-300 spectrometer, and chemical shifts are reported relative to an external standard of P(OCH&. Fast atom bombardment mass spectra (FAB/MS) were recorded on a Kratos MS-890 instrument. Melting points are uncorrected. Thinlayer chromatography (TLC) was performed on EM 5539 silica gel 60 plates, and all column chromatography was performed with EM silica gel 60 (0.040-0.063 nm), with elution under pressure. Oligonucleotides were synthesized on a Dupont CODER-300automated DNA synthesizer using a controlled pore glass (CPG) support with an (aminopropy1)succinatelinker.2Oligonucleotideswere purified by reversed-phase high-performance liquid chromatography (HPLC, Waters 990 photodiode array detector) using pBondapak Cls (0.39 X 30 cm, Waters) column. 8-Bromo-2’-deoxyguanosine (3). To a vigorously stirred suspension of 2‘-deoxyguanosine (5.0 g, 19 mmol) in 30 mL of water, cooled in an ice bath, was added dropwise 120 mL of saturated bromine-water (made by shaking 1.4 mL of Br2 with 120 mL of water a t room temperature), the yellow color being allowed to disappear before each new addition. Initially the suspension became translucent, but it became opaque as the product precipitated. At the end of the addition a yellow color persisted, and stirring was then continued for 5 min at the same temperature. The solid was removed by filtration and washed with cold water (30 mL), and then once with cold acetone (30 mL). Recrystallization from MeOH-H20 (1:l v/v) containing sodium bicarbonate (100 mg) gave 3 in 84% (5.45 g) yield: TLC Rf 0.10 (MeOH-CH2C12,1090); mp 213 “C dec [lit. (39,40)210 “C decl; lH NMR 6 10.82 1 H, NH, s), 6.51 (2 H, NH2, s), 6.16 [ l H, H-C(l’),t, J = 7.35 Hz], 5.27 [ l H, HO-C(3’), d, J = 4.20 Hzl, 4.87 [ l H, HO-C(5’),t , J = 5.85 Hzl,4.40 [ l H, H-C(3’),m], 3.81 [ l H, H-C(4’),m], 3.63-3.42 [2 H, H-C(5’),m], 3.17 and 2.11 [2 H, H-C(2’), 2 ml; 13C NMR 6 156.34, 154.22, 152.89, 121.45, 118.39, 88.80, 85.97, 71.94, 62.96, 37.36. 8-(Benzyloxy)-2’-deoxyguanosine(4). DMSO (200mL)was added to a solution of sodium benzoylate [prepared by stirring freshly distilled benzyl alcohol (75 mL or 78.4 g, 720 mmol) with very small pieces of sodium (2.14 g) at 60 “C until the solution was homogeneous1 under nitrogen. To the resulting mixture was added 3 (10.0g, 29 mmol) in DMSO (80 mL),and the mixture was heated a t 65 “C for 24 h and then cooled to room temperature. After neutralization with glacial acetic acid, the bulk (250 mL) of the DMSO was removed by vacuum distillation at 65 “C (bath temperature). The remaining liquid was then poured slowly into ether (500 mL) with stirring. The ether layer was decanted, and the oily residue remaining was added to acetone (100 mL).

The white precipitate that formed was removed by filtration and then stirred with water (80 mL) for 0.5 h. The slurry was filtered, and the collected solid was recrystallized from MeOH to give 4 in 70% (7.55 g) yield: TLC Rf 0.20 (MeOH-CHzClZ, 10:90); mp 201 “C dec [lit. (39) >187 “C decl; lH NMR 6 10.69 (1H, NH, br s), 7.39 (5 H, aromatic H, m), 6.38 (2 H, NH2, s), 6.09 [ l H, H-C(l’), t, J = 7.05 Hz], 5.41 (2 H, CHZ-Ph, s), 5.20 [l H, HOC(3’), d, J = 4.10 Hzl, 4.83 [ l H, HO-C(5’), ml, 4.24 [ l H, HC(3’), m], 3.72 [ l H, H-C(4’), ml, 3.41-3.32 [2 H, H-C(5’), ml, 2.86 and 2.02 [2 H, H-C(2’), 2 m]; 13C NMR 6 157.06, 154.21,

* CPG was purchased from CPG Inc. This CPG is covalentlyconnected to 5’-O-DMT-protectednucleosides through an (aminopropy1)succinate linkage.

151.72,150.99,139.00,129.40,129.19,129.09,128.98,111.62,88.32,

82.56, 71.94, 71.55. 8-(Benzyloxy)-N2-isobutyryl-2’-deoxyguanosine (5). Isobutyrylation of 4 was carried out according to the procedure of Ti et al. (41). To a suspension of 4 (2.20 g, 5.89 mmol) in dry pyridine (40 mL) was added trimethylchlorosilane (3.80 mL or 3.25 g, 29.91 mmol). After the solution had been stirred for 15 min, isobutyric anhydride (4.90 mL or 4.67 g, 29.52 mmol) was added and stirring was continued at room temperature for 4 h. The mixture was then cooled in an ice bath, and water (10 mL) was added, followed 5 min later by aqueous ammonia (5 mL, 27 % ) at -0 “C (bath temperature). After stirring for 15 min, the solution was then evaporated to near dryness on a rotary evaporator and the residue was filtered, washed once with a mixture of EtOAc-ether (l:l, 30 mL) and twice with H20 (25 mL), and then recrystallized from MeOH-H20: yield 2.11 g (81%); TLCRf0.45 (MeOH-CH2C12,1090);mp 220 “C; ‘H NMR 6 12.03 (1H, NH, s), 11.52 (1H, NH, s), 7.52-7.31 (5 H, aromatic H, m), 6.21 [ l H, H-C(l’), t, J = 7.05 Hz], 5.48 (2 H, CH2-Ph, s), 5.20 [ l H, HO-C(3’), d, J = 3.90 Hz], 4.70 [ l H, HO-C(5’), t, J = 5.55 Hz], 4.26 [ l H, H-C(3’), m], 3.72 [ l H, H-C(4’), m], 3.443.38 [2 H, H-C(5’),m], 2.90 [lH, H-C(2’),ml, 2.76 [lH, CH(MeI2, m], 2.06 [ l H, H-C(2’),ml, 1.12 (6 H, C-Me2, d, J = 6.90 Hz); 13C NMR 6 180.67, 154.56, 153.29, 148.43, 147.84, 136.35, 129.31, 129.22, 129.07, 115.79, 88.20, 82.57, 71.98, 71.58, 62.78, 36.88, 35.53, 19.69; FAB/MS (+ve ion, thioglycerol matrix), mlz 444 [(M H)+, 71, 328 [(heterocyclic base + 2H)+, 91. 7,8-Dihydro-N2-isobutyryl-8-oxo-2’-deoxyguanosine (6). A solution of 5 (1.60 g, 3.61 mmol) in a mixture of n-butyl alcoholHzO (14:1,60 mL) maintained at 55 “C was hydrogenated over a 10% Pd-on-charcoal catalyst (0.7 g) at 60 psi hydrogen for 12 h. The catalyst was removed by filtration, and the solvent was evaporated to dryness in vacuo to yield pure 6, which was recrystallizedfromethanol: yield 1.2g (95%);TLCRf0.13 (MeOHCH2C12,10:90); mp 238-246 “C dec; lH NMR 6 12.14 (1H, NH, s), 11.59 (1 H, NH, s), 11.28 (1H, NH, s), 6.09 [ l H, H-C(l’), t, J = 6.60 Hz], 5.17 [ l H, HO-C(3’),d, J = 2.40 Hz], 4.73 [l H, HO-C(5’), ml, 4.37 [ l H, H-C(3’), ml, 3.75 [ l H, H-C(4’), ml, 3.59-3.44 [2 H, H-C(5’), ml, 3.07 [ l H, H-C(2’), ml, 2.74 [ l H, CH(Me)2,m], 1.98 [ I H, H-C(2’), m], 1.11 [6 H, C-Me2, d, J = 8.10 Hz]; FAB/MS (+ve ion, thioglycerol matrix), mlz 354 [(M + H)+, 81, 238 [(heterocyclic base + 2H)+, 1001. 7,8-Dihydro-5’0-( 4,4’-dimethoxytrityl)-~-isobutyryl-8oxo-2‘-deoxyguanosine(7). To asolution of 6 (1.2 g, 3.40mmol) in dry pyridine (15 mL) cooled in ice-water was added 4,4’-DMT chloride (1.27g, 3.74 mmol). The cooling bath was then removed, and stirring was continued a t room temperature for 15 min. The reaction mixture was cooled in ice water and quenched with water (50 mL). The solution was extracted with CH2C12 (5 X 20 mL), and the combined organic layers were washed with H20 (2 x 20 mL) and then dried over MgS04. The solvent was evaporated under reduced pressure, and the crude residue was purified by chromatography over silica gel using EtOAc-hexane containing 2% of triethylamine (to inhibit detritylation) as the eluting solvent. The yield of pure 7 was 1.68 g (76%): TLC R, 0.48 (MeOH-CH2C12,10:90); mp 165-170 “C; 1H NMR 6 12.13 (1 H, NH, s), 11.43 (1 H, NH, s), 11.24 (1H, NH, s), 7.34-6.76 (13 H, aromatic-H, m), 6.14 [l H, H-C(l’), t, J = 6.05 Hzl, 5.16 [l H, HO-C(3’), d, J = 4.50 Hz], 4.44 [l H, H-C(3’), ml, 3.91 11 H, H-C(4’), m], 3.72 and 3.71 (6 H, OCH3, 2 s), 3.32 and 3.06 [2 H, H-C(5’), 2 m], 2.98 [l H, H-C(2’), m], 2.74 [l H, CH(Me)Z, m], 2.12 [1H, H-C(2’), ml, 1.12 (6 H, C-Me2, d, J = 6.60 Hz); FAB/

+

Bodepudi et al.

610 Chem. Res. Toxicol., Vol. 5, No. 5, 1992

Scheme I 0

(Me,CHCO),O Pyridine, 25 ‘C

OH

OH

OH

NaOCH,Ph DMSO

3

4-

R’

=

Br

R’ = OCH,Ph

0 0

Pyridine, 25 ‘C

DMTOvoJ

w OH 7 -

-8

“ “ Y O 4

r-/ OH

-6

R ; COCHMe2

MS (-ve ion, thioglycerol matrix), m/z 654 [(M - HI-, 1001,584 (28), 352 (12), 236 [(heterocyclic base - 2H)-, 301. 3’-O[(Diisopropylamino)(2-cyanoethoxy)phosphino]-7,8dihydro-5’-0-(4,4’-dimethoxyt rityl)-W-isobutyryl-8-oxo-2’deoxyguanosine (8). To a mixture of 7 (0.58 g, 0.88 mmol; dried over PpO5 in a vacuum desiccator for 24-48 h and then coevaporated with a mixture of dry CHpClp and benzene prior to reaction) and dry Et3N (0.22 g, 2.20 mmol) in dry CH2C12 ( 5 mL) under NOwas added 2-cyanoethyl N,N-diisopropylphosphoramidochloridite (0.25 g, 1.05 mmol). The progress of the reaction was monitored by TLC analysis. The starting material disappeared completely in 20 min, and a new, less polar spot, appeared. The CHzClzthen was evaporated under vacuum, and a mixture of dry THF-benzene (1:4; 25 mL) was introduced. The solution was stirred for 5-10 min and filtered under Nz to remove the Et3NeHCl. The process was then repeated twice using dry benzene as the solvent. The resultant viscous foamy material, when dried over PpO5 in a vacuum desiccator at room temperature overnight, led to a quantitative yield of 8: TLC Rf 0.46 (MeOH-CH2C12-Et3N,4:95:1); 1H NMR 6 11.75-11.13 (3 H, 3 NH, br s), 7.34-6.73 (13 H, aromatic-H, m), 6.12 [ l H, H-C(l’), dd, J = 4.90,5.10 Hz], 4.61 [l H, H-C(3’), ml, 4.03 [3 H, H-C(4’) andOCH2,m],3.71(6H,0CH3,s),3.45(2 H , 2 CHN,m),3.923.34 [2 H, H-C(5’), m], 2.76 (2 H, CHzCN, t, J = 6.00 Hz), 2.63 [ l H, CH(Me)p, ml, 2.31 [2 H, H-C(2’), ml, 1.26 112 H, C(Pri)2, dd], 1.15 (6 H, C-Me2, dd); 31PNMR (121 MHz, CDC13) 6 146.63 and 146.50 (diastereomeric pair); FABIMS (-ve ion, thioglycerol matrix), m / z 854 [(M - H)-, 121, 654 [M - loss of phosphoramidite)-, 141, 236 [(heterocyclic base - 2H)-, 71. 8-(Benzyloxy)-2’-deoxyadenosine(12). A solution of sodium benzoylate was prepared from benzyl alcohol (78.37 g or 75 mL, 0.72 mmol) and sodium (2.0 g). To this solution was added, at room temperature under Nz, solid 8-bromo-2’-deoxyadenosine (10)(42-44) (5g, 15.14mmol)inone portionwithvigorousstirring. The progress of the reaction was monitored by TLC analysis to completion (18 h). The reaction mixture was then cooled to 0 “C (bath temperature) and carefully neutralized with concentrated HCI. EtOAc (100 mL) was added, and the organic layer was separated, washed with saturated NaHC03 solution (2 X 10 mL) and then once with brine (20 mL), and then dried (MgSO,). After filtration the volatile solvents were evaporated and the benzyl alcohol was removed by distillation at 65-70 “C (bath

temperature)/0.2 mm. The residual viscous liquid crystallized from a mixture of MeOH-EtOAc-hexane to give 12 (4.4 g, 81% yield): TLC Rf0.40 (MeOH-CH2C12, 11:89);mp 173-174 “C; 1H NMR 6 8.04 [l H, H-C(2), SI, 7.56-7.39 ( 5 H, aromatic-H, m), 7.00 (2 H, NHp, s), 6.21 [ l H, H-C(I’), t, J = 7.20 Hz], 5.55 (2 H, CH2-Ph, d, J = 3.0 Hz), 5.28 I2 H, HO-C(3‘) and HO-C(5’), m], 4.37 [ l H, H-C(3’), ml, 3.81 [ l H, H-C(4’), ml, 3.56 and 3.42 [2 H, H-C(5’), 2 m], 3.03 and 2.11 [2 H, H-C(2’), 2 ml; I3C NMR 6 154.82, 154.23, 151.38, 149.49, 136.19, 129.32, 129.19, 115.67, 88.69,83.35, 72.16, 72.00, 63.08, 37.74; FABIMS (+ve ion, thioglycerol matrix), miz 358 [(M + H)+,271,242 [(heterocyclic base + 2H)+, 1001. ~-Benzoyld-(benzyloxy)-2’-deoxyadenosine (16). HMDS (8.07g or 10.54 mL, 50 mmol) and 12 (2.0 g, 5.60 mmol) in suspension were heated together at 150 “C under Npfor 12 h (the solution became clear after 9 h). The excess HMDS was then removed under vacuum, and the residue was dissolved in dry pyridine (10 mL). Benzoyl chloride (1.30 mL or 1.57 g, 11.19 mmol) was added dropwise with cooling over 5 min with stirring, and the reaction mixture was allowed to cool to room temperature. After 35 min MeOH-water (20 mL, 3:l) was added, the mixture was stirred for 5 min, and then concentrated ammonia (22 mL) was added. After stirring for 30 min at room temperature, the solvents were removed on a rotary evaporator a t 35 OC and the residual solid was triturated with EtOAc-ether (25 mL, 1:1) for 10 min. The resulting suspension was filtered, and the pale yellow brown solid was crystallized from MeOH: yield 2.3 g (89%);TLC R, 0.51 (MeOH-CHzCl2, 11:89);mp 232-234 “C; ‘H NMR 6 11.06 (1H, NH, s), 8.64 [l H, H-C(2), SI, 8.08-7.41 (10 H, aromatic-H, m), 6.32 [1 H, H-C(l’), t, J = 7-05], 5.56 (2 H, CHp-Ph, d, J = 2.7 Hz), 5.33 [ l H, HO-C(3’), d, J = 4.2 Hz], 4.91 [lH, HO-C(5’),t, J = 5.85 Hz], 4.42 [l H, H-C(3’), m], 3.81 [I H, H-C(4’), ml, 3.55 and 3.41 [2 H, H-C(5’), 2 ml, 3.14 and 2.20 [2 H, H-C(2’), 2 ml; 13C 6 165.86, 156.59, 153.01, 150.00, 149.93, 147.33, 135.52, 134.41, 132.44, 129.00, 128.91, 128.83, 128.77, 124.11, 88.32, 83.01, 72.63, 71.53,62.67, 37.30; FABIMS (+ve ion, thioglycerol matrix), m/z 462 [(M + HI+, 341, 346 [(heterocyclic base + 2H)+, 761. iV-Benzoyl-7,8-dihydro-8-oxo-2’-deoxyadenosine (17). A solution of 16 (0.78 g, 1.69 mmol) in a mixture of MeOH-EtOAc (40 mL, 1.5:l) was hydrogenated over a 10% Pd/C catalyst (0.2 g) at 55-60 psi hydrogen at -50 “C for 1 h. The catalyst was

Incorporation of 8-Oxopurine Deoxynucleosides into DNA removed by filtration through Celite and was washed with hot MeOH (2 x 30 mL). Evaporation of the solvents under reduced pressure left a white solid which was crystallized from MeOH to give an 82% yield of 17 (0.52 g): TLC Rf 0.48 (MeOH-CHzClZ, 11939); mp 227-229 “C; lH NMR 6 11.17 (1 H, NH, br s), 10.80 (1H, NH, br s), 8.49 [ l H, H-C(2), SI, 8.04 (2 H, aromatic-H, d, J = 7.35 Hz), 7.67-7.52 (3 H, aromatic-H, m), 6.25 [ l H, H-C(l’), t, J = 7.2 Hz], 5.27 [ l H, HO-C(3’), d, J = 4.2 Hz], 4.87 11 H, HO-C(5’), t, J = 5.85 Hz], 4.44 [ l H, H-C(3’), m], 3.81 [ l H, H-C(4’), m], 3.63 and 3.47 [2 H, H-C(5’), 2 ml, 3.09 and 2.09 [2 H,H-C(2’),2m];13CNMR6 165.43,151.28,150.20,149.37,138.32, 132.87, 132.28, 128.37, 128.26, 112.60, 87.47, 81.26, 71.11,62.20, 35.63; FAB/MS (+ve ion, thioglycerol matrix), m/z 372 [(M H)+, 261, 256 [(heterocyclic base + 2H)+, 1003. Ng-Benzoyl-5‘-O(4,4‘-dimethoxytrity1)-7,8-dihydro-8-oxo2‘-deoxyadenosine(18). To a cooled solution of 17 (0.2 g, 0.54 mmol) in dry pyridine (3 mL) was added under Nz 4,4’-DMT chloride (0.25 g, 0.74 mmol) in one portion. Stirring was continued for 1.5 h at 25 “C, and the reaction mixture was quenched by the dropwise addition of water (4 mL). After extraction with CH2Clz (4 X 10 mL) the combined organic layers were washed once with water and once with brine and then dried over MgS04. After filtration, the solution was evaporated and the residual material was purified by silica gel column chromatography (MeOH-CHZCl2 containing 2% Et3N, as eluent). The purified material was dissolved in dry THF and then diluted with dry toluene. The solvents were then evaporated. This process was repeated twice to give dry 18 (0.39 g, 91 ’% yield): TLC R, 0.75 (MeOH-CHzC12, 11:89);mp 136-139 “C dec; 1H NMR 6 8.31 [ l H, H-C(2), s], 8.09 (2 H, aromatic-H, d, J = 7.50 Hz), 7.67-6.81 (16 H, aromatic-H, m), 6.29 [ l H, H-C(l’), t, J = 6.6 Hzl, 5.35 [l H, HO-C(3’), d, J = 4.0Hz],4.57 [ l H, H-C(3’), m],4.0 [ l H, H-C(4’), ml, 3.74 and 3.73 (6 H, OCH3,2 s),3.40 and 3.10 [2 H, H-C(5’), 2 ml, 2.54 and 2.18 [2H,H-C(2’),2 m]; l3C NMR6 166.37,158.81,152.37,151.06, 150.01, 145.85, 139.30, 136.70, 136.54, 133.96, 133.04, 130.48,

+

129.24,129.08,128.54,128.40,127.30,113.81,86.18,86.12,81.72,

71.87,65.08,55.78,36.51;FAB/MS (+ve ion, thioglycerol matrix), m/z 674 [(M H)+, 0.53, 303 (DMT cation), 1001, 256 [(heterocyclic base + 2H)+, 201.

+

NB-Benzoyl-3’-0-[ (diisopropylamino)(2-cyanoethoxy)phosphino]-5’-0-(4,4’-dimethoxytrity1)-7,8-dihydro8-oxo-2‘-deoxyadenosine (19). To a solution of 18 (0.20 g, 0.30 mmol; dried as described in the previous experiment) and Et3N (0.063 g, 0.62 mmol) in THF (1.5 mL) under NZwas added 2cyanoethyl N,N-diisopropylphosphoramidochloridite(0.074 g, 0.31 mmol). The reaction was complete within 45 min, and the product, when worked up by the procedure used for the isolation of compound 8,gave a quantitative yield of 19 (0.23 g): TLC Rf 0.68 (MeOH-CHZC12,8:92); lH NMR 6 8.29 [lH, H-C(2),SI, 8.11 (2 H, aromatic-H, d, J = 7.53 Hz), 7.61-6.83 (16 H, aromatic-H, m), 6.24 [ l H, H-C(l’), t, J = 6.4 Hzl, 4.55 [ l H, H-C(3’), ml, 4.01 [3 H, H-C(4’) and OCH2, m], 3.72 and 3.70 (6 H, OCH3,2 s), 3.46 (2 H, 2 CHN, m), 3.40 and 3.10 [2 H, H-C(5’), 2 m], 2.73 (2 H, CHzCN, t, J = 5.95 Hz), 2.54 and 2.18 [2 H, H-C(2’), 2 ml, 1.24 [12 H, C(Pri)z, dd]; 31P NMR (121 MHz, CDC13) 6 147.59 and 147.40 (diastereomeric pair). 2’-Deoxy-7,8-dihydro-8-oxoadenosine (2). A solution of 12 (0.50 g, 1.4 mmol) dissolved in ethanol (6 mL) was hydrogenated over a 10% Pd/C catalyst at 60 psi at room temperature for 3.5 h. The reaction mixture was diluted to 20 mL with ethanol, and the catalyst was removed by filtration through Celite. After washing the catalyst once with hot ethanol,the combined organic solutions were evaporated under reduced pressure to give a fluffy white solid which crystallized from EtOAc-MeOH to give 2: yield 0.32 g (87%);TLC Rf 0.17 (MeOH-CHzC12,14:86); mp 192-193 “C; lH NMR 6 10.46 (1H, NH, br s), 8.02 [ l H, H-C(2), 91, 6.58 (2 H, NHz, s), 6.15 [l H, H-C(l’), t, J = 7.35 Hz], 5.19 [2 H, HO-C(3’) and HO-C(5’), m], 4.40 [l H, H-C(3’), m], 3.81 [l H, H-C(4’), ml, 3.61 and 3.44 [2 H, H-C(5’), 2 ml, 2.96 and 2.02 [2 H, H-C(2’), 2 ml; FAB/MS (+ve ion, thioglycerol matrix), mlz 268 [(M + HI+, 251, 152 [(heterocyclic base + 2H)+, 321.

Chem. Res. Toxicol., Vol. 5, No. 5, 1992 611

8-(Benzyloxy)-3’-0,5‘-O-bis(phenoxyacetyl)-2’-deoxyadenosine (13). To a solution of 12 (0.20 g, 0.56 mmol) in dry pyridine (4 mL) was added freshly prepared phenoxyacetic anhydride3(0.96 g, 3.36 mmol) under Nz. The resulting mixture was stirred at 25 “C for 2.5 h and then quenched with water. After extraction with CHzC12 (5 X 10 mL), the combined organic layers were washed once with water, twice withsaturated NaHC03 solution, and finally with brine and then dried over MgS04. The solution was evaporated under reduced pressure, and the residue was purified by column chromatography (silica gel, EtOAc-hexane as eluent) to give 13 in 91% (0.32 g) yield: ‘H NMR 6 8.08 [ I H, H-C(2), SI, 7.58-6.84 (17 H, aromatic-H and NHz, m), 6.25 [ I H, H-C(l’), t, J = 7.2 Hz], 5.58 (2 H, OCHZPh, d, J = 1.8 Hz), 4.87 (2 H, PhOCHzCO, s), 4.70 (2 H, PhOCHZCO, s), 4.62-4.21 [4 H, H-C(3’), H-C(4’), H-C(5’), ml, 3.45 and 2.44 [2 H, H-C(2’), 2 m]; FAB/MS (+ve ion, thioglycerol matrix), mlz 626 [(M + H)+, 111. 8-(Benzyloxy)-3’-0,5’-O-bis( phenoxyacety1)-Ng,Ng-dibenzoyl-2’-deoxyadenosine(14). To a solution of 13 (0.10 g, 0.16 mmol) in dry pyridine (2 mL) in an ice bath was added benzoyl chloride (0.067 g, 0.48 mmol) dropwise. The reaction was quenched with water (2 mL) after 10 min, and the mixture was stirred a t room temperature for 5 min. After extraction with CHZCl2(4 x 10 mL) the combined organic layers were washed once with NaHC03 and once with brine and dried over MgS04. The resulting solution was evaporated to dryness, and the residual material was purified by silica gel column chromatography (EtOAc-hexane) to give 14 in 83% (0.11 g) yield: mp 140-143 “C; lH NMR (CDCl3) 6 8.50 [l H, H-C(2), SI, 7.83-6.85 (25 H, aromatic-H),6.29 [lH, H-C(l’), t, J = 6.9 Hzl, 5.48 (2 H, OCHzPh, s), 4.66 (2 H, PhOCHzCO, s), 4.58 (2 H, PhOCH2C0, s), 4.494.22 [4 H, H-C(3’), H-C(4’), H-C(5‘), m], 3.45 and 2.32 [2 H, H-C(2’), 2 m]; FAB/MS (+ve ion, thioglycerol matrix), m/z 834 [(M + HI+, 51. 8 4 Benzyloxy)-N6,N6-dibenzoyl-2’-deoxyadenosine ( 15). The procedure of Guy et al. (45) was followed. A solution of 14 (0.20 g, 0.24 mmol) in 6 mL of EtsN-pyridine-Ha0 (1:1:3) was stirred for 1.5 h. The solvent was evaporated, water was added, and the mixture was extracted with CHzC12 (5 X 15 mL). The combined extracts were dried over MgS04, filtered, and evaporated under reduced pressure, and the residue was purified by column chromatography (silica gel, MeOH-CHzC12 as eluent) to give pure 15: yield 0.11 g (78%);mp 156-169 OC dec; lH NMR 17 8.45 [ l H, H-C(2), 91, 7.86-7.31 (10 H, aromatic-H, m), 6.36 [ l H, H-C(l’), t, J = 7.2 Hz], 5.47 (2 H, OCHZPh, s), 5.38 [ l H, HO-C(3’), d, J = 4.1],4.72 [l H, HO-C(5’), t, J = 5.761,4.14 [l H, H-C(3’), ml, 3.88 [ I H, H-C(4’), ml, 3.82-3.69 [2 H, H-C(5’), m], 2.89 and 2.25 [2 H, H-C(2’), 2 ml.

Synthesis of Oligodeoxynucleotides on a Solid Support. The syntheses of the oligodeoxynucleotides containing 1 and 2, site-specifically located, were performed on a CPG support using a fully automated DNA synthesizer and the commerciallyavailable, conventionally-protected deoxynucleoside 2-cyanoethyl phosphoramidites. Syntheses of all these oligonucleotides were carried out at 5-pmol scale. The DNA synthetic cycle consisted essentially of ditritylation, condensation, capping, and oxidation stages. Coupling efficiency of the modified DMT phosphoramidite in the syntheses of oligodeoxynucleotides was found in all cases to be greater than 9876, and the overall yields of the oligomers obtained from the syntheses were about 90%. Upon completion of the synthesis, all base-labile protecting groups on the oligodeoxynucleotide were removed by treatment with concentrated ammonia, in presence of mercaptoethanol (0.25 M), at 55 “C over 15 h. This led to the 5’-O-DMT oligodeoxynucleotide which then was purified by reverse-phase HPLC. Afterward the 5’-DMT group was removed using80% acetic acid. Phenoxyacetic anhydride was prepared fresh from phenoxyacetic acid and dicyclohexylcarbodiimide (DCC) in dry THF at room temperature under NP. The dicyclohexylurea that separated was removed by filtration, and the filtrate was evaporated to dryness. The resulting white solid was dried in vacuo and used as such.

612

Bodepudi et al.

Chem. Res. Toxicol., Vol. 5, No. 5, 1992

Table I. Nucleoside Content of Oligomer 5‘-

.OO

d(CCTTCG(oxo)CTACTTTCTCT)-3’ Containing

dC

.06

nucleoside dC dT dAdo 8-0~0-dGuo

-

-E

-w .w-

ratioc 7.95 (8.0) 7.78 (8.0) 1.00 (1.0)

0.91 (1.0)

3 wg of 8-oxo-dGuoldmer was used for the analysis of nucleoside content. * Nucleoside content was calculated by dividing the integrated HPLC peak for each nucleoside under 260 nm by comparing with authentic standards. Theoretically expected contentsare shown in parentheses. Nucleoside content ratio (relative to dAdo) and theoretical ratio in parentheses.

dT

0

8-0~0-dGu0’ nucleoside contentb(nmol) 4.61 (4.08) 4.51 (4.08) 0.58 (0.51) 0.53 (0.51)

w

u a

z m a

min, evaporated in a vacuum desiccator, then extracted with MeOH (2 X 350 pL), and dried. For analysis, this solution was then injected directly into the HPLC column (pBondapak Cis, 0.39 X 30 cm, Waters).

Results and Discussion

7,8-Dihydro-8-oxo-2’-deoxyguanosine (8-Oxo-dGuo, 20

0

i

40 TIME (min)

Figure 1. HPLC profile of 2’-deoxynucleosides obtained as a result of enzymatic digestion of the oligonucleotide 5’-d(CCTTCXCTACTTTCCTCT)-3’ (9i: X = 8-oxo-dGuo). HPLC conditions: elution with water (pH 5.85) for the first 20 min and then 0-10% acetonitrile (linear gradient) in water over 30 min a t a flow rate 1.0 mL/min. I

1.2,

I 220

240

260

280

300

320

340

360

380

400

WAVELENGTH (nm)

Figure 2. UV spectra of oligomers 5’-d(CCTTCXCTACTTTCCTCT)-3’: (1)9i (X = 8-oxo-dGuo) and (2) 9i (X = dGuo). In the inset diagram are shown the HPLC elution times of the modified and unmodified oligomers. Elution conditions were 10-1576 acetonitrile (linear gradient) in 0.05 M triethylammonium acetate buffer (pH 7.0) over 60 min a t a flow rate of 1.0 mL/min. A second HPLC purification then gave the homogeneous oligomer. Base Composition Analysis. The base composition of the oligodeoxynucleotides with respect to their individualnucleosides was determined by enzymatic hydrolysis using the following procedure (46). T o the oligodeoxynucleotide (3.0 pg) in 100 pl of sodium acetate buffer (0.03 M, pH 5.3) containing 2-mercaptoethanol (10 mM) were added 5 pL of zinc sulfste solution (20 mM) and 2 units of nuclease P1 (Boehringer, W. Germany). The mixture was incubated for 2 h a t 37 “C, and thereafter the pH was adjusted to 8.5 by the addition of 20 p L of Tris-HCl(O.5 M). The sample then was reincubated at 37 “C with 3 units ofbacterial alkaline phosphatase (Sigma, St. Louis, MO) for an additional 2 h. Samples thus obtained were heated in boiling water for 3

1). The overall route to the desired phosphoramidite 8 in the case of 8-oxo-dGuo follows the basic procedure of Lin et al. (39) (Scheme I) which itself was based on the first synthesis of the corresponding ribonucleoside (8-oxo-Guo) by Long and co-workers (40). However, we were able to improve markedly the overall yields of intermediate 6 by modifying the reaction conditions for the synthesis of 3 and 4. In the case of 3 we found that the addition of small amountsof sodium bicarbonate during the recrystallization procedure completely inhibited depurination and improved the yield of pure product from 57 (39)to 84%.A significant yield improvement (from 28 to 6 5 % ) in the preparation of 4 also was realized by first removing almost all of the reaction solvent (DMSO) by high vacuum distillation at 55-60 “C. This then permitted a workup procedure using manageable amounts of ether and acetone, the prescribed isolation solvents (39). Isobutyrylation of the 2-amino group of 4 to give 5 was then accomplished in 7 6 % yield by means of the method of “transient protection” ( 4 1 ) . Finally hydrogenation of 5 over a palladium-on-charcoalcatalyst led to 6 in 95 7% yield. This represents a marked improvement in yield over the reduction of the free 2-amino compound which was realized previously (39) in only 59% yield. The conversion of 6 to its 5’-0-DMT derivative 7, in 76% yield, was carried out according to established literature methods (47,481. The subsequent preparation of pure phosphoramidite 8 from 7 at first gave some difficulty. Initially we attempted to use the established method (36,37)for this conversion, namely, treatment of 7 with 2-cyanoethyl NJV-diisopropylchlorophosphoramid ite, in the presence of N,N-diisopropylethylaminein methylene chloride solution, followed by quenching the reaction mixture in aqueous bicarbonate solution and then chromatographic separation of the product. However, during this purification, extensive decomposition of the desired material occurred. The alternative method used to purify such crude phosphoroamidites, namely, precipitation in hexane at -78 “C from EtOAc solution (49,50), in our hands gave a material that coupled poorly (60% ) during automated oligomer synthesis. After much experimentation it was found that a good quality product, giving high coupling efficiency, could be obtained only when water was rigorously excluded in the latter stages of the synthesis. Tenacious retention of water by the DMT

Chem. Res. Toxicol., Vol. 5, No. 5, 1992 613

Incorporation of 8-Oxopurine Deoxynucleosides into DNA

derivative 7 partially contributed to the problem, but the normal aqueous workup used in phosphoramidite formation was found to constitute the more significant problem. For these reasons, prior to use, 7 was dried over P205 in vacuo for 2 days followed by azeotropic removal of residual moisture using a mixture of CHpCl2-benzene. Once dry, 7 was converted to the desired phosphoramidite under anhydrous conditions. This was accomplished by carrying out the phosphitylation reaction in dry CH2C12 in the presence of dry triethylamine followed by removal of Et3N.HC1 by filtration under nitrogen using first dry THF and then benzene. This procedure gave essentially pure 8,as a mixture of diastereomers in a 1:l ratio. When this material was used in the automated synthesis of a series4 of DNA oligomers (Sa-k;X = 8-oxo-dGuo), in all cases a 5 '-d(GTCACXACTC)-3 '

9a

5 'd(CACTAXTCAC)-3'

9b

5 '-d(CGCXATACGCG)-3'

5 '-d(GTCACTTXACCACrC)-3'

9c 9d 9e 9f

5 '-d(GTCACTCXTACACTC)-3'

b

5'-d(CACTAXTCACTITCCTCT-3' 5 '-d(CClTCXCTACrrrCCrCr)-3 '

9h 9i

5 '-d(CTGGTACCrXAGGATCCAmGAC)-3'

9.i

5 '-d(CTCTCCCITCXCTCCrrrCCTW-3'

9k

5 '-d(CCACTAXTCACC)-3' 5 '-d(CGCATGXGTACGC)-3'

coupling efficiency of >98% was observed at the point of introduction of the 8-oxo-dGuo residue, and excellent yields of the desired products were obtained. 8-Oxo-dGuo derivatives (and oligomers containing this residue), in keeping with 8-(arylamino)-2'-deoxyguanosine (51) and uric acid (52,53), are sensitive to aerial oxidation. Thus if 8-mercaptoethanol(O.25M) is not added to the hot (55 "C) ammonia solution used in the deprotection step, there is a very substantial loss of product. In a quantitative test we found for example that a sample of the decamer Sb was 60% degraded even at 37 "C after 16 h. One of the initial oligomers synthesized was the pentamer 5'-ACG(oxo)AT-3', and its structure was confirmed by negative ion FAB/MS. In the mass spectrum a molecular ion (M - H)- was found at 1501 (calcd MW = 1502) daltons. The sequential fragmentation patterns beginning at the 5'- (1268,979,634, and 321) and 3'- (1277, 964,619, and 330) ends of the oligomer,aresult of cleavage of the carbon-oxygen bonds in the sugar-phosphate backbone, also corroborated the structure (54). One of these oligomers,namely, Si (X = 8-oxo-dGuo),was selected for base composition analysis (46). When it was subjected to sequential enzymatic digestions with nuclease P1 and then bacterial alkaline phosphatase, the individual deoxynucleosides were found to be present in the expected molecular ratios (Figure 1and Table I). The UV spectra of Si (X = 8-oxo-dGuo) and Si (X= dGuo) are shown in Figure 2 together with the HPLC data (inset). As can be seen, the UV spectrum of the oligomer containing the More than 25 oligomers containing8-oxo-dGuo(1) have been prepared without the appearance of complicating side products. 4

#

228 -

WAVELENGTH (220 - 400 nm)

Figure 3. UV spectra of 2'-deoxyguanosine (1) and 2'-deoxy7,8-dihydro-8-oxoguanosine (2).

8-oxo-dGuo residue is slightly different from that of the normal oligomer, in the 300-nm region. This is in accord with the displacement observed between the spectra of the corresponding deoxynucleosides (Figure 3). 7,8-Dihydro-8-oxo-2'-deoxyadenosine (8-Oxo-dAdo, 2). The synthesis of an 8-oxo-dAdo derivative and its incorporation into DNA oligomers have been reported by Guy and his associates (45). More recently, a second synthesis of 2 has been published (55) but without experimental detail. Initially we followed the procedures of Guy et al. (45), based on an earlier paper by Ikehara (42-44), which starts from the easily available 8-bromo-2'-deoxyadenosine(10). However, in our hands treatment of 10 with the recommended mixture of sodium acetate and phenoxyacetic anhydride gave capricious results. At best we were able to isolate only a 15% yield of the triacylated product 11 after repeated chromatographic purification, rather than the 72% yield reported (45). Part of the variability may be due to the scrambling of acetyl with phenoxyacetyl during the reaction, which could be expected to produce mixtures of acylated products. However, considerable charring also was noted under these reaction conditions. Also, in the 2'-deoxyguanosine series it is interesting to note that Roelen et al. (38)experienced much depurination with this type of reaction, and again yields of the desired 8-oxo-dGuo derivative were only 12%, results that our own experiments confirmed. Attempts also to introduce the 8-oxo group by the reaction of 8-Br-dGuowith sodium trimethylsilanolate (NaOSiMes) in DMF a t 25 "C gave a complex mixture after workup, and even though the presence of 8-oxo-dAdo was evident from the TLC, its low yield did not justify pursuit of this approach. We decided therefore to follow a route analogous to that used in the 8-oxo-dGuo series. Treatment of 10 (Scheme 11) with sodium benzylate in benzyl alcohol led to 12 in 82 % yield: and although we were able to smoothly remove the benzyl group by hydrogenolysis to obtain 2, attempted N6-benzoylationof the latter using the transient protection method gave only a complex mixture. In an alternative approach when 12 was treated with phenoxyacetic anhydride3under much milder conditions than those 5This reaction also proceeds smoothly in DMSO solvent at room temperature in 1.5 h to give 12 in good yield. However, at 65 O C it led to a complex mixture.

614 Chem. Res. Toxicol., Vol. 5, No. 5, 1992

Bodepudi et al.

Scheme I1 NHR

I

OR

OH

11 -

10 -

OH

OR

OH

12 -

2 -

I

OH

13 PhCOCl

OR

14 -

15 R = COCH20Ph R’ = R2 = COPh

used with 10 (see above discussion), there was obtained a 91% yield of the 3’-O15’-0-bis-ester13. Benzoylation of 13 surprisingly gave the WJVG-bis-benzoyl derivative 14 in 8376 yield. Although 14 could be selectivelydeprotected to 15 by an aqueousmixture of pyridine and triethylamine at 25 OC in 1.5h, further work on this route was abandoned because a shorter route emerged. This is based on the benzoylation of 12 (Scheme 111) which was carried out using the transient protection method and led to 16 in 89% yield. Hydrogenation of 16 over a palladium-on-carbon catalyst proved interesting. In aqueous solution at 65-70 OC debenzylation was complete only after 12h and was accompanied by complete debenzoylation to give 2. However, in a mixture of methanol-EtOAc the desired W-benzoylderivative 17 was obtained in good yield within 1h. The remainder of the synthesis proved uneventful. The conversion of 17 to its 4,4’-DMT derivative 18 was accomplished in 91 76 yield under standard conditions. Phosphitylation of 18 using the anhydrous procedure described above in the %oxodGuo series gave the phosphoramidite 19 directly as a diastereomeric pair, in quantitative yield. When the latter material was used directly in an automated synthesis of oligomeric DNA, the coupling efficiencydid not differ from that of the normal commercially-available phosphora-

midites. A selection of some of the oligomers 20a-g (X = 8-oxo-dAdo) synthesized is noted. 5’-d(CCACrAXTCACC)-3’ 5’-d(CCATACXTACTTC)-3’ 5’-d(GlTGXGTACrrrCCrC)-3’

5’-d(CClTCXCTACIl’TCCrCT)-3’ 5’-d(GG’lTGXGTACrrrCCrCr)-3’ 5’-d(CTCTCCCITCXCrCCrrrC(JTCT)-3’

m m 2oc 20d 2oe 20f

5’-d(CrGGTACCrXAGGATCCACTGAC)-3’

Optimal yields (90-95%) of these oligomers were obtained only when concentrations of P-mercaptoethanol were at least 0.25 M during the ammonia deprotection step. The initial oligomer synthesized was again a pentamer, 5’-TGA(oxo)CA-3’. Similarly in this case, negative ion FABIMS established the oligomer composition and the position of the modified base. In this spectrum a parent ion (M - H)- is seen at 1501 (calcd MW = 1502), and confirmatory evidence comes from the fragmentation patterns resulting from the initiation of degradation at the 5’- (1277, 948, 619, and 330) and 3‘-

Chem. Res. Toxicol., Vol. 5, No. 5, 1992 615

Incorporation of 8-Oxopurine Deoxynucleosides into DNA

...,,.........

D

0

3

300

250

2011

B

350

+GO

WAVELENGTH (190 - 400 nm)

dAdo

Figure 6. UV spectra of 2'-deoxyadenosine (1)and 2'-deoxy7,8-dihydro-8-oxoadenosine(2).

Scheme I11 NHR

NHR

I

20

40

TIME (min)

Figure 4. HPLC profile of the 2'-deoxynucleosides obtained as a result of enzymatic digestion of the oligonucleotide 5'd(CCTTCXCTACTTTCCTCT)-3'(20d: X = 8-oxo-dAdo). HPLC conditions: elution with water (pH 5.85) for the first 20 min and then &lo% acetonitrile (linear gradient) in water over 30 min at a flow rate 1.0 mL/min.

HMDS

12

iEe-

16 -

17 -

2 NHCOPh I

NCCH,CH,OP(CI)N(iPr),

NC

-0-P-N

? A Y 19

\ I

220

I

I

240

260

280

300

I

TIME ( m i d

I

I

320

340

I

360

380

I

400

W A V E LENGTH (nm)

F i g u r e 5. UV spectra of oligomers 5'-d(CCTTCXCTACTTTCCTCT)-3': (1) 20d (X = 8-oxo-dAdo) and (2) 20d (X = dAdo). In the inset HPLC diagram are shown the elution times of the modified and unmodified oligomers. Elution conditions were 10-15% acetonitrile (linear gradient) in 0.05 M triethylammonium acetate buffer (pH 7.0) over 60 min at a flow rate of 1.0 mL/min. (1268,979,650,321)ends. One of these oligomers, 20d (X = &oxo-dAdo), was selected for base composition analysis, using t h e same conditions as were described for 9i (X = 8-oxo-dGuo), a n d t h e individual nucleosides were found t o be present in t h e expected molecular ratios (Figure 4 and Table 11). T h e UV spectra of 20d (X = 8-oxo-dAdo) and 20d (X = dAdo) are shown in Figure 5 together with t h e HPLC data (inset). As can be seen, t h e UV spectrum of the oligomer containing t h e 8-oxo-dAdo residue is again slightly different from that of the normal oligomer. T h i s is in accord with t h e displacement observed between t h e spectra of t h e corresponding deoxynucleosides (Figure 6).

OH

10 R

i

COPh

Table 11. Nucleoside Content of Oligomer 5'-

d(CCTTCA(oxo)CTACTTTCCTCT)-3'Containing nu c1eoside dC dT dAdo 8-oxo-dAdo

8-Oxo-dAdo' nucleoside contentb (nmol) 2.85 (2.72) 3.05 (2.72) 0.37 (0.34) 0.36 (0.34)

~~

~

ratioC 7.70 (8.0) 8.24 (8.0) 1.00 (1.0) 0.97 (1.0)

2 pg of 8-oxo-dAdo lamer was used for the analysis of nucleoside content. Nucleoside content was calculated by dividing the integrated HPLC peak for each nucleoside under 260 nm by comparing with authentic standards. Theoretically expected contentsare shown in parentheses. Nucleoside content ratio (relative to dAdo) and theoretical ratio in parentheses.

Extension of these methods (phosphoramiditeformation a n d their use in t h e synthesis of oligomeric DNA) t o a series of other substrates, including t h e model abasic site (56-591,the 1,3-propylene a n d 1,2-ethylene glycols (59), lJ\n-(1,3-propano)-dGuo (601,a n d more recently a 54hydroxymethyl)-2'-deoxyuridine derivative: has in general given much superior results t o those obtained by previously published procedures. 6

B. Veeraiah and F. Johnson, unpublished results.

616 Chem. Res. Toxicol., Vol. 5, No. 5, 1992

Acknowledgment. This work was supported by NIH Grants CA17395 and CA47995 from the National Cancer Institute. We are grateful to Dr. C. R. Iden and Robert A. Rieger for the automated synthesis of oligodeoxynucleotides containing site-specificallymodified nucleosides, and for mass spectral data. We also thank Drs. A. P. Grollman and M. Takeshita for their interest in this program. References (1) Kasai, H., Crain, P. F., Kuchino, Y., Nishimura, S., Ootsuyama, A., and Tanooka, H. (1986) Formation of 8-hydroxyguanine moiety in cellular DNA by agents producing oxygen radicals and evidence for ita repair. Carcinogenesis 7, 1849-1851. (2) Breimer, L. H. (1990) Review: Molecular mechanisms of oxygen radical carcinogenesis and mutagensis: The role of DNA base damage. Mol. Carcinog. 3, 188-197. (3) Floyd, R. A. (1990) Commentary on the role of 8-hydroxyguanine in carcinogenesis. Carcinogenesis 11, 1447-1450. (4) Kasai, H., and Nishimura, S. (1991) Formation of b-hydroxydeoxyguanosine in DNA by oxygen radicals and ita biological significance. In Oxidative Stress: Oxidants and Antioxidants (Sies, H., Ed.) pp 99-116, Academic, London. (5) Mariaggi, N., and TBoule, R. (1976) Acides NuclBiques. Caract&isation de la dihydr0-7,8-0~0-8-d6soxy-2’-adBnosine, produite par irradiation gamma de la d6soxy-2’-ad8nosine en solution. Bull SOC. Chim. Fr., 1595-1598. (6) Mariaggi, N., Cadet, J., and TBoule, R. (1976) Cyclisation radicalaire de la desoxy-2’-adenosine en solution aqueuse, sous l’effet du rayonnement gamma. Tetrahedron 32, 2385-2387. (7) Kasai, H., Tanooka, H., and Nishimura, S. (1984) Formation of 8-hydroxyguanineresidues in DNA by X-irradiation. Gann 75,10371039. (8) Cadet, J., and Berger, M. (1985) Radiation-induced decomposition of the purine bases within DNA and related model compounds. Int. J. Radiat. Biol. 47, 127-143. (9) Dizdaroglu, M. (1986) Characterization of free radical-induced damage to DNA by the combined use of enzymatic hydrolysis and gas chromatography-mass spectrometry. J . Chronatogr. 367,357366. (10) Duplaa, A. M., Guy, A., and TBoule, R. (1989) Measurement of the radiosensitivity of a radiation-induced adenine defect in a single stranded DNA chain. Radiat. Enuiron. Biophys. 28, 169-176. (11) Lesiak, K. B., and Wheeler, K. T. (1990) Formation of a-deoxyadenosine in polydeoxynucleotides exposed to ionizing radiation under anoxic conditions. Radiat. Res. 121, 328-337. (12) Gajewski, E., Rao, G., Nackerdien, Z., and Dizdaroglu, M. (1990) Modification of DNA bases in mammalian chromatin by radiationgenerated free radicals. Biochemistry 29, 7876-7882. (13) Fraga, C. G., Shigenaga, M. K., Park, J.-W., Degan, P., and Ames, B. N. (1990) Oxidative damage to DNA during aging: &Hydroxy2’-deoxyguanosine in rat organ DNA and urine. Proc. Natl. Acad. Sci. U.S.A. 87,4533-4537. (14) Shigenaga, M. K., Gimeno, C. J., and Ames, B. N. (1989) Urinary 8-hydroxy-2’-deoxyguanosineas a biological marker of in vivo oxidative DNA damage. Proc. Natl. Acad. Sci. U.S.A. 86, 96979701. (15) Richter, C., Park, J.-W., and Ames, B. N. (1988) Normal oxidative damage to mitochondrial and nuclear DNA is extensive. Proc. Natl. Acad. Sci. U.S.A. 85, 6465-6467. (16) Umemura, T., Sai, K., Takagi, A., Hasegawa, R., and Kurokawa, Y. (1990) Formation of 8-Hydroxydeoxyguanosine (8-OH-dG) in rat kidney DNA after intraperitoneal administration of ferric nitrilotriacetate (Fe-NTA). Carcinogenesis 11, 345-347. (17) Umemura, T., Sai, K., Takagi, A,, Hasegawa, R., and Kurokawa, Y. (1990)Oxidative DNA damage, lipid peroxidation and nephrotoxicity induced in the rat kidney after ferric nitrilotriacetate administration. Cancer Lett. 54, 95-100. (18) Takagi, A., Sai, K., Umemura, T., Hasegawa, R., and Kurokawa, Y. (1990) Relationship between hepatic peroxisome proliferation and 8-hydroxydeoxyguanosine formation in liver DNA of rats following long-term exposure to three peroxisome proliferators; di(2-ethylhexyl) phthalate, aluminum clofibrate and simfibrate. Cancer Lett. 53, 33-38. (19) Takagi, A., Sai, K., Umemura, T., Hasegawa, R., and Kurokawa, Y. (1990)Significant increase of 8-hydroxydeoxyguanosinein liver DNA of rats following short-term exposure to the peroxisome proliferators di(2-ethylhexy1)phthalate and di(2-ethylhexy1)adipate. J p n . J. Cancer Res. 81, 213-215. (20) Kasai, H.,and Nishimura,S. (1984)DNAdamage induced by asbestos in the presence of hydrogen peroxide. Gann 75, 841-844.

Bodepudi et al. (21) Grollman, A. P. (1991) Structural basis for the mutagenic specificity of 8-oxoguanine-DNA, In Structure and Function, Volume I: Nucleic Acids, Proceedings of the Seventh Conversation in Biomolecular Stereodynamics, June, 1991, pp 165-170, Academic Press, Schenectady, NY. (22) Kasai, H., Nishimura, S., Toriumi, Y., Itai, A., and Iitaka, Y. (1987) The crystal structure of 9-ethyl-8-hydroxyguanine. Bull. Chem. SOC. J p n . 60, 3799-3800. (23) Aida, M., and Nishimura, S. (1987) An ab initio molecular orbital study on the characteristics of 8-hydroxyguanine, Mutat. Res. 192, 83-89. (24) Culp, S.J., Cho, B. P., Kadlubar, F. F., and Evans, F. E. (1989) Structural and conformational analyses of b-hydroxy-2’-deoxyguanosine. Chem. Res. Toxicol. 2,416-422. (25) Uesugi, S., and Ikehara, M. (1977) Carbon-13 magnetic resonance spectra of 8-substituted purine nucleosides. Characteristic shifts for the syn conformation. J . Am. Chem. SOC. 99, 3250-3253. (26) Cho, B. P., Culp, S. J., Evans, F. E., and Kadlubar, F. F. (1989) Structural characterization of 8-hydroxy-2’-deoxyguanosine.Proc. Am. Assoc. Cancer Res. 30, 201, No. 797. (27) Kouchakdjian, M., Bodepudi, V., Shibutani, S., Eisenberg, M., Johnson, F., Grollman, A. P., and Patel, D. J. (1991) NMR structural studies of the ionizing radiation adduct 8-oxodeoxyguanosine (8oxodG) opposite adenosine in a DNA duplex: b-oxoG(syn) A(anti) alignment at lesion site. Biochemistry 30, 1403-1412. (28) Moriya, M., Ou, C., Bodepudi, V., Johnson, F., Takeshita, M., and Grollman, A. P. (1991) Site-specific mutagenesis using a gapped duplex vector: A study of translesion synthesis past 8-oxodeoxyguanosine in E. coli. Mutat. Res. 254, 281-288. (29) Wood, M. L., Dizdaroglu, M., Gajewski, E., and Essigmann, J. M. (1990) Mechanistic studies of ionizing radiation and oxidative mutagenesis: genetic effects of a single 8-hydroxyguanine (7-hydro8-oxoguanine) residue inserted at a unique site in a viral genome. Biochemistry 29, 7024-7032. (30) Shibutani, S., Takeshita, M., and Grollman, A. P. (1991) Insertion of specific bases during DNA synthesis past the oxidation damaged base 8-oxodG. Nature 349, 431-434. (31) Kuchino, Y., Mori, F., Kasai, H., Inoue, H., Iwai, S., Miura, K., Ohtauka, E., and Nishimura, S.(1987) Misreadingof DNAtemplates containing 8-hydroxydeoxyguanosine a t the modified base and a t adjacent residues. Nature 327,77-79. (32) Jones, D. S., Nemoto, F., Kuchino, Y., Ohtsuka, E., and Nishimura, S. (1990) 8-Hydroxydeoxyguanosine in DNA inhibits restriction endonuclease digestion. Nucleosides Nucleotides 9, 223-233. (33) Tchou, J., Kasai,H., Shibutani,S., Chung, M.-H.,Laval, J.,Grollman, A. P., and Nishimura, S. (1991) 8-Oxoguanine (8-hydroxyguanine) DNA elvcosvlase and its substrate sDecificitv. Proc. Natl. Acad. Sci. U:S.A. 88, 4690-4694. (34) Chune. M.-H.. Kasai, H., Jones, D. S.,Inoue, N., Ishikawa. H., Ohtsika, E., and Nishimura, S. (1991) An activity of Escherichia coli that specifically removes 8-hydroxyguanine residues from DNA. Mutat. Res. 254, 1-12. (35) Bodepudi, V., Iden, C. R., and Johnson, F. (1991) An improved method for the preparation of the phosphoramidites of modified 2’-deoxynucleotides: Incorporation of 8-oxo-2’-deoxy-7H-guanosine into synthetic oligomers. Nucleosides Nucleotides 10, 755-761. (36) Sinha,N. D., Biernat, J., McManus, J., andKoster, H. (1984)Polymer support oligonucleotide synthesis: Use of j3-cyanoethyl-N,N-dialkylamino-/N-morpholino phosphoramidite of deoxynucleosides for the synthesis of DNA fragments simplifying deprotection and isolation of the final product. Nucleic Acids Res. 12,4539-4557. (37) Sinha, N. D., Biernat, J., and Koster, H. (1983) j3-Cyanoethyl N,NdialkylaminoiN-morpholinomonochlorophosphoamiditea, new phosphitylating agent facilitating ease of deprotection and work-up of synthesized oligonucleotides. Tetrahedron Lett. 24, 5843-5846. (38) Roelen, H. C. P. F., Saris, C. P., Brugghe, H. F., Van den Elst, H., Westra, J. G., Van der Marel, G. A., and Van Boom, J H. (1991) Solid-phase synthesis of DNA fragments containing the modified Nucleic Acids Res. 19,4361base 7-hydr0-8-0~0-2’-deoxyguanosine. 4369. (39) Lin, T.-S., Cheng, J.-C., Ishiguro, K., and Sartorelli, A. C. (1985) 8-Substituted guanosine and 2’-deoxyguansine derivatives as potential inducers of the differentiation of friend erythroleukemia cells. J . Med. Chem. 28, 1194-1198. (40) Long, R. A., Robins, R. K., and Townsend, L. B. (1967) Purine nucleosides: The synthesis of 8-amino- and 8-substituted aminopurine nucleosides. J . Org. Chem. 32, 2751-2756. (41) Ti, G. S.,Gaffney,B. L.,and Jones,R. A. (1982)Transientprotection: Efficient one-flask syntheses of protected deoxynucleosides. J. Am. 104, 1316-1319. Chem. SOC. (42) Ikehara, M., Tada, H., and Muneyama, K. (1965) Synthesis of 8-hydroxypurine nucleosides. Chem. Pharm. Bull. 13,1140-1142. (43) Ikehara, M., Tada, H., and Kaneko, M. (1968) Purine cyclonucleosides: Synthesis of purine cyclonucleoside having 8,2’-0-anhydro linkage and its cleavage reactions. Tetrahedron 24, 3489-3498.

Incorporation of 8-Oxopurine Deoxynucleosides into D N A (44)Ikehara, M., and Maruyama, T. (1975)Purine cyclonucleosides: A versatile method for the synthesis of purine 0-cyclo-nucleosides. The first synthesis of 8,2’-anhydro-8-oxy-9-~-~arabinofuranosylguanine. Tetrahedron 31, 1369-1372. (45)Guy, A,, Duplaa, A.-M., Harel, P., and TBoule, R. (1988)Synthesis and characterization of DNA fragmenta bearing an adenine radiation product: 7,8-Dihydroadenine-S-one. Helu. Chim. Acta 71,15661572. (46) Gehrke, C. W., McCune, R. A., Gama-Sosa, M. A., Ehrlich, M., and Kuo, K. C. (1984)Quantitative reversed-phase high-performance liquid chromatography of major and modified nucleosides in DNA. J. Chromatogr. 301, 199-219. (47) Schaller, H., Weimann, G., Lerch, B., and Khorana, H. G. (1963) The step-wise synthesis of specific deoxyribopolynucleotides. Protected derivatives of deoxyribonucleosides and new synthesis of deoxyribonucleoside-3’-phosphates.J . Am. Chem. SOC.85, 38213827. (48) Smith, M., Rammler, D. H., Goldberg, I. H., and Khorana, H. G. (1962)Specific synthesis of the CB’-Cs,inter ribonucleotide linkage. Synthesis of uridylyl-(3’+5’)-uridine and uridylyl-(3’--5’)-adenosine. J. Am. Chem. SOC. 84,430-440. (49) Beaucage, S. L., and Caruthers, M. H. (1981) Deoxynucleoside phosphoamidites-A new class of key intermediates for deoxypolynucleotide synthesis. Tetrahedron Lett. 22, 1859-1862. (50) Barone, A. D., Tang, J.-Y., and Caruthers, M. H. (1984)In situ activation of bis-dialkylaminophosphines-A new method for synthesizing deoxyoligonucleotideson polymer support. Nucleic Acids Res. 12,4051-4061. (51)Shibutani, S.,Gentles, R. G., and Johnson, F. (1990)Aerial oxidation of acetylaminofluorene-derived DNA adducts. In Nitroarenes: Occurrence, Metabolism and Biological Impact (Howard, P. C., Hecht, S.S., and Beland, F. A., Eds.) pp 135-147,Plenum Press, New York.

Chem. Res. Toxicol., Vol. 5, No. 5, 1992 617 (52) Poje, M., and Sokolic-Maravic, L. (1986)The mechanism for the conversion of uric acid into allantoin and dehydro-allantoin. Tetrahedron 42,747-751. (53) Poje, M., and Sokolic-Maravic, L. (1988)The mechanism for the conversion of uric acid into uroxanate and allantoin. Tetrahedron 44,6723-6728. (54) Rieger, R.A., Walther, D. M., Bodepudi, V., and Iden, C. R. (1990) Fast atom bombardment mass spectrometry of oligodeoxynucleotides containing 8-oxo-2’-deoxyguanosine.Proceedings of the 38th ASMS Conference on Mass Spectrometry and Allied Topics, Tucson, AZ, June 3-8, 1990,pp 1417-1418. (55) Berger, M., Anselmino, C., Mouret, J.-F., and Cadet, J. (1990)High performance liquid chromatography-electrochemical assay for monitoring the formation of 8-oxo-7,S-dihydroadenine and ita related 2’-deoxyribonucleoside. J. Liq. Chromatogr. 13,929-940. (56) Millican, T. A., Mock, G. A., Chauncey, M. A,, Patel, T. P., Eaton, M. A. W., Gunning, J., Cutbush, S. D., Neidle, S., and Mann, J. (1984)Synthesis and Biophysical studies of short oligodeoxynucleotides with novel modifications: A possible approach to the problem of mixed base oligodeoxynucleotide synthesis. Nucleic Acids Res. 12,7435-7453. (57) Takeshita, M., Peden, K. W. C., Cheng, C.-N., Will, S. G., Johnson, F., and Grollman, A. P. (1986)J. Enuiron. Mutat. SOC.8, 84. (58) Eritja,R., Walker, P. A.,Randall,S. K.,Goodman, M. F., and Kaplan, B. E. (1987)Synthesis of oligonucleotides containing the abasic site model compound. 1,4-Anhydro-2-deoxy-~-ribitol. Nucleosides Nucleotides 6 , 803-814. (59) Takeshita, M., Chang, C.-N., Johnson, F., Will, S., and Grollman. A. P. (1987)Oligodeo&ucleotides containing synthetic ebasic sites: J.Biol. Chem. 262,10171-10179. (60) Marinelli, E. R.,Johnson, F., Iden, C. R., and Yu, P.-L. (1990) Synthesis of 1,W( 1,3-propano)-2’-deoxyguanosine and incorporation into oligodeoxynucleotides: A model for exocyclic acrolein-DNA adducts. Chem. Res. Toricol3, 49-58.