Self-Complementary Oligodeoxyribonucleotides Incorporating l

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Bioconjugate Chem. 1998, 9, 683−690

683

Self-Complementary Oligodeoxyribonucleotides Incorporating L-Related Isodeoxynucleosides: Synthesis, Physical Characterization, Enzymology, and CD Studies Thomas Wenzel and Vasu Nair* Department of Chemistry, The University of Iowa, Iowa City, Iowa 52242. Received January 15, 1998; Revised Manuscript Received May 22, 1998

The synthesis of self-complementary oligodeoxyribonucleotides (14-16, 18) incorporating the L-related isodeoxynucleotides, isodA (1) and/or isodT (2), is described. For this purpose, the fully protected phosphoramidite building blocks 10 and 12 were synthesized. The conceptually new oligomers exhibit high resistance towards exonucleases. Temperature- and concentration-dependent CD spectral data and accompanying thermodynamic parameters of the oligomers show that isodA-isodT or mixed dAisodT/isodA-dT base pairs participate in duplex stabilization.

INTRODUCTION

Naturally occurring oligonucleotides are readily degraded by extra- and intracellular nucleases that limit their usefulness in in vivo applications. A number of modifications are known that increase the enzymatic stability of oligonucleotides (1-9), and most of them are at the phosphate backbone or at the sugar moiety. Unfortunately, in many cases, the increase in stability toward nucleases is accompanied by a decrease in the binding affinity to the target. Hitherto, unknown are oligodeoxynucleotides which incorporate nucleoside components in which the nucleobase at the natural C-1′ position is transposed to the isomeric 2′-position (also equivalent to transposition of oxygen), while maintaining the cis relationship of the base with the hydroxymethyl group (Scheme 1). Recently, isomeric deoxy- and dideoxynucleosides including (S,S)-isodideoxyadenosine [(S,S)]-isoddA] were synthesized in our laboratory (10-13). [S,S]-IsoddA exhibited very high resistance toward chemical and enzymatic degradation and was also endowed with significant antiviral activity against HIV-1, HIV-2, and HIV clinical isolates (14). Furthermore, (S,S)-isoddA triphosphate was a potent inhibitor of HIV reverse transcriptase with Ki values in the nanomolar range (14). In order to gain a better understanding of the special characteristics that would be acquired by oligodeoxynucleotides incorporating isomeric nucleosides, including those isonucleosides that exhibit antiviral activity through viral DNA chain termination, we have investigated the synthesis, enzymology, and properties of such conceptually new, self-complementary oligodeoxynucleotides. EXPERIMENTAL PROCEDURES

Materials. Melting points are uncorrected and were determined on a Thomas Hoover apparatus fitted with a microscope. 1H- and 13C-NMR spectra were recorded on a Bruker WM 360 in CDCl3 or Me2SO-d6. Chemical shift values are reported in δ, parts per million, relative to the internal standard (TMS). UV spectra were recorded on a Varian Cary 3 spectrophotometer (Melbourne, Australia). CD spectra were measured on an AVIV Circular Dichroism Spectrometer, model 62DS (AVIV, Lakewood, NJ). Elemental analysis was per-

formed at NuMega Resonance Labs, Inc. (San Diego, CA). Thin-layer chromatography (TLC) was carried out on Polygram Sil G/UV254 (0.25 mm, Machery-Nagel, Du¨ren, Germany). TLC plates were visualized by ultraviolet absorbance or charring for several minutes after exposure to a 10% sulfuric acid/ethanol solution. Flash chromatography (FC) was carried out using glass columns packed with 230-400 mesh silica gel. Reversed-phase HPLC separations (4.6 × 250 mm, RP-18, 10 µm, LiChromosorb column, Merck, Darmstadt, Germany) were achieved using a Waters automated 600E system (Milford, MA) interfaced with a photodiode array detector (Waters 990) and a Satellite WISP (Waters 700) for automated sampling. The solid-phase synthesis of oligonucleotides was performed on an automated DNAsynthesizer (Applied Biosystems, Foster City, CA, model AB 391, PCR-MATE for phosphoramidite chemistry). The β-cyanoethyl phosphoramidites of regular 2′-deoxynucleosides and the Fractosil-linked 2′-deoxynucleosides were also products of Applied Biosystems. Snake-venom phosphodiesterase (EC 3.1.4.1., Crotallus durissus), calfspleen phosphodiesterase (EC 3.1.16.1.), alkaline phosphatase (EC 3.1.3.1., Escherichia coli), and Eco R1 (E. coli BS 5) were purchased from Boehringer Mannheim (Mannheim, Germany). Synthesis (Schemes 2 and 3). 4(R)-(6-Amino-9Hpurin-9-yl)-2(R)-(hydroxymethyl)tetrahydrofuran3(R)-ol (1). To an ice-cold solution of 3 (4.00 g, 29.8 mmol) in anhydrous pyridine (80 mL) was added, under N2, tert-butyldiphenylsilyl chloride (7.8 mL, 29.8 mmol). The mixture was stirred for 3 h at 0 °C, quenched with H2O (10 mL), evaporated, and the oily residue applied on the top of a silica gel column (column, 25 × 5 cm; solvent, CHCl3/MeOH 95:5). Evaporation of the separated product yielded 4 (9.50 g, 86%) as a colorless solid. mp 75 °C. TLC (CHCl3/MeOH): Rf 0.3. 1H-NMR (CDCl3): δ 1.09 [s, 9H, (CH3)3C], 3.20 [s (br), 1H, OH-3], 3.39 [s (br), 1H, OH-4], 3.75-3.83 (m, 3H, Hβ-5/CH2), 3.86 (m, 1H, H-2), 4.15 (dd, 1H, J ) 4.7, 9.1 Hz, HR-5), 4.204.26 (m, 2H, H-3/H-4), 7.34-7.42 (m, 6H, arom. H), 7.657.70 (m, 4H, arom. H). 13C-NMR (CDCl3): δ 19.4 [(CH3)3C], 27.0 [(CH3)3C], 64.5 (CH2), 71.7 (C-3), 73.2 (C4, C-5), 83.1 (C-2). A solution of 4 (5.00 g, 13.4 mmol) in dry CH2Cl2 (125 mL) was cooled down to 0 °C. At this

10.1021/bc980009e CCC: $15.00 © 1998 American Chemical Society Published on Web 10/02/1998

684 Bioconjugate Chem., Vol. 9, No. 6, 1998

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Scheme 1

Scheme 2a

a (i) TBDPSCl, pyridine, 0 °C, 3 h. (ii) MsCl, Et N, CH Cl , 0 °C, 3 h. (iii) TBDPSCl, imidazole, DMF, rt, 24 h. (iv) Adenine, K CO , 3 2 2 2 3 18-crown-6, DMF, 120 °C, 60 h. (v) 0.5 M NH4F/MeOH, reflux 8 h.

Scheme 3a

a (i) Me SiCl, pyridine, rt 0.5 h. (ii) BzCl, 0 °C, 2 h. (iii) NH /H O. (iv) (MeO) TrCl, Et(i-prop) N, pyridine, rt, 2 h. (v) Chloro-(23 3 2 2 2 cyanoethoxy)(diisopropylamine)phosphine, Et(i-prop)2N, rt 1.5 h.

temperature, Et3N (3.9 mL, 26.7 mmol) and methanesulfonyl chloride (1.0 mL, 13.4 mmol) were added and the mixture was stirred for 3 h at 0 °C under N2. Water (10 mL) was added, the solvent was evaporated, and the oily residue was purified by FC (column, 30 × 3 cm; solvent, CHCl3) yielding compound 5 (3.2 g, 53%) as a colorless oil. TLC (CHCl3/MeOH 95:5): Rf 0.6. 1H-NMR (CDCl3): δ 1.09 [s, 9H, (CH3)3C], 2.71 (d, 1H, J ) 3.6 Hz, OH-3), 3.13 (s, 3H, CH3), 3.82 (dd, 1H, J ) 3.3, 11.2 Hz, CH2), 3.89 (m, 1H, CH2), 3.94 (m, 1H, H-2), 4.08 (dd, 1H, J ) 3.6, 10.5 Hz, Hβ-5), 4.24 (dd, 1H, J ) 4.9, 10.5 Hz, HR-5), 4.47 (m, 1H, H-3), 5.22 (m, 1H, H-4), 7.39-7.46 (m, 6H, arom. H), 7.71 (m, 4H, arom. H). 13C-NMR (CDCl3): δ 19.4 [(CH3)3C], 27.0 [(CH3)3C], 38.7 (SO2CH3), 63.9 (CH2), 70.5 (C-5), 72.0 (C-3), 80.4 (C-4), 82.8 (C-2). To a solution of 5 (2.1 g, 4.7 mmol) in dry DMF (35 mL) were added imidazole (930 mg, 14.0 mmol) and tertbutyldiphenylsilyl chloride (1.23 mL, 4.7 mmol) and the

reaction mixture was stirred for 18 h at rt under N2. Then, tert-butyldiphenylsilyl chloride (1.23 mL, 4.7 mmol) was added again and the stirring maintained for another 6 h at rt. The solvent was removed under high-vacuum and the crude product was purified by FC (column, 20 × 3 cm; solvent, (CHCl3). Collection and evaporation of the main zone afforded 6 (2.5 g, 78%) as a viscous, colorless oil. TLC (CHCl3): Rf 0.3. 1H-NMR (CDCl3): δ 0.99 [s, 9H, (CH3)C], 1.12 [s, 9H, (CH3)3C], 2.84 (s, 3H, CH3), 3.33 (dd, 1H, J ) 3.2, 11.4 Hz, CH2), 3.64 (dd, 1H, J ) 2.6, 11.4 Hz, CH2), 4.02 (m, 1H, H-2), 4.15 (m, 2H, H-5), 4.55 (t, 1H, J ) 4.6 Hz, H-3), 4.88 (dd, 1H, J ) 4.9, 9.5 Hz, H-4), 7.31-7.46 (m, 12H, arom. H), 7.54-7.66 (m, 6H, arom. H), 7.74 (m, 4H, arom. H). 13C-NMR (CDCl3): δ 19.3, 19.5 [(CH3)3C], 26.9, 27.0 [(CH3)3C], 38.3 (SO2CH3), 63.8 (CH2), 69.8 (C-5), 72.9 (C-3), 78.3 (C-4), 84.6 (C-2). A mixture of 6 (2.50 g, 3.6 mmol), K2CO3 (990 mg, 7.2 mmol), 18-crown-6 (960 mg, 3.6 mmol), and adenine (970

Self-Complementary Oligodeoxyribonucleotides

mg, 7.2 mmol) in dry DMF (120 mL) was heated for 60 h at 120 °C. The solvent was evaporated under reduced pressure. The residue was dissolved in H2O (400 mL) and extracted with CHCl3 (3 × 200 mL). Then, the combined organic layers were extracted with brine (2 × 200 mL), dried (Na2SO4), evaporated and purified by FC (column, 20 × 3 cm, solvent, CHCl3/MeOH 95:5). The faster migrating zone furnished 7a (580 mg, 22%) as a light yellow oil. TLC (CHCl3/MeOH 95:5): Rf 0.5. 1HNMR (CDCl3): δ 0.98 [s, 9H, (CH3)3C], 1.01 [s, 9H, (CH3)3C], 3.63 (dd, 1H, J ) 4.4, 11.6 Hz, CH2), 3.75 (dd, 1H, J ) 2.4, 11.6 Hz, CH2), 4.08 (m, 1H, H-2′), 4.13 (dd, 1H, J ) 4.5, 9.7 Hz, Hβ-5′), 4.29 (dd, 1H, J ) 6.5, 9.7 Hz, HR-5′), 4.78 (m, 1H, H-3′), 5.13 (m, 1H, H-4′), 6.13 [s (br), 2H, NH2-6], 7.07-7.47 (m, 16H, arom. H), 7.54-7.62 (m, 4H, arom. H), 7.74 (m, 4H, arom. H), 8.01 (s, 1H, H-2), 8.22 (s, 1H, H-8). 13C-NMR (CDCl3): δ 19.1, 19.3 [(CH3)3C], 26.9, 27.0 [(CH3)3C], 63.0 (C-3′), 63.2 (CH2), 71.2 (C-5′), 78.0 (C-4′), 86.9 (C-2′), 119.4 (C-5), 138.8 (C8), 149.8 (C-4), 152.7 (C-2), 155.6 (C-6). The slower migrating zone afforded 7b (470 mg, 27%) as light yellow foam. TLC (CHCl3/MeOH 95:5): Rf 0.4. 1H-NMR (CDCl3): δ 1.02 [s, 9H, (CH3)3C], 3.88 (dd, 1H, J ) 4.1, 11.3 Hz, CH2), 3.93 (dd, 1H, J ) 3.5, 11.4 Hz, CH2), 4.06 (m, 1H, H-2′), 4.33 (dd, 1H, J ) 6.4, 9.4 Hz, Hβ-5′), 4.49 (dd, 1H, J ) 6.6, 9.4 Hz, HR-5′), 4.65 (m, 1H, H-3′), 4.88 (m, 1H, H-4′), 5.75 [s (br), 1H, OH-3′], 5.98 [s (br), 2H, NH2-6], 7.32-7.43 (m, 6H, arom. H), 7.65 (m, 4H, arom. H), 7.75 (s, 1H, H-2), 8.28 (s, 1H, H-8). 13C-NMR (CDCl3): δ 19.4 [(CH3)3C], 27.0 [(CH3)3C], 63.3 (CH2), 64.0 (C-3′), 69.7 (C-5′), 77.0 (C-4′), 85.1 (C-2′), 119.7 (C-5), 138.6 (C-8), 150.2 (C-4), 152.8 (C-2), 155.9 (C-6). To a solution of 7a (500 mg, 0.68 mmol) in MeOH (10 mL) was added 0.5M NH4F (20 mL). The mixture was refluxed for 18 h and the solvent was evaporated and the residue was purified by FC (column, 10 × 3 cm, solvent: CHCl3/MeOH 8:2) to give 1 (120 mg, 71%). Compound 7b (400 mg, 0.82 mmol) was similarly deprotected to give 1 (160 mg, 78%): mp 227 °C (EtOH). TLC (CHCl3/MeOH 8:2), Rf 0.2. UV (MeOH): λ 260 nm ( 14 800). 1H-NMR (Me2SO-d6): δ 3.57-3.68 (m, 2H, CH2), 3.70 (m, 1H, H-2′), 4.10 (dd, 1H, J ) 4.9, 9.6 Hz, Hβ-5′), 4.49 (dd, 1H, J ) 6.6, 9.6 Hz, HR-5′), 4.40 (m, 1H, H-3′), 4.85-4.91 (m, 2H, H-4′/CH2OH), 5.76 (d, 1H, J ) 3.9 Hz, OH-3′), 7.23 [s (br) 2H, NH2-6], 8.15 (s, 1H, H-2), 8.19 (s, 1H, H-8). 1HNOE-difference NMR (D2O): Irradiation at H-4′: Observed NOEs at H-1′R (2.8%), H-2′ (3.2%), H-2 (0.6%), H-8 (4.3%). 13C-NMR (Me2SO-d6): δ 60.9 (CH2), 61.9 (C3′), 69.5 (C-5′), 75.7 (C-4′), 85.9 (C-2′), 118.7 (C-5), 139.2 (C-8), 149.4 (C-4), 152.4 (C-2), 156.0 (C-6). Anal. Calcd for C10H13N5O3: C, 47.81; H, 5.22; N, 27.87. Found: C, 47.46; H, 5.34; N, 27.76. 4(R)-(6-Benzoylamino-9H-purin-9-yl)-2(R)-(hydroxymethyl)tetrahydrofuran-3(R)-ol (8). To a solution of 1 (200 mg, 0.80 mmol) in dry pyridine (20 mL) was added, under N2, Me3SiCl (510 µL, 3.97 mmol). The mixture was stirred for 30 min at rt. After cooling down to 0 °C, benzoyl chloride (460 µL, 3.97 mmol) was added and the reaction mixture was stirred for 2 h. Excess of benzoyl chloride was destroyed by adding H2O (5 mL). After 10 min, 25% aqueous NH3 (10 mL) was added, and the reaction mixture was stirred for another 30 min. The solution was evaporated and the residue was purified by FC (column, 15 × 3 cm; solvent, CHCl3/MeOH 9:1). Isolation of the main zone yielded 8 (230 mg, 81%): mp 198-199 °C (colorless plates from acetone/MeOH). TLC (CHCl3/MeOH 9:1): Rf 0.4. UV (MeOH): λ 280 nm ( 19 000). 1H-NMR (Me2SO-d6): δ 3.57-3.66 (m, 2H, CH2), 3.73 (m, 1H, H-2′), 4.21 (m, 2H, H-5′), 4.44 (m, 1H, H-3′),

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4.92 (t, 1H, J ) 5.5 Hz, CH2OH), 5.03 (m, 1H, H-4′), 5.81 (d, 1H, J ) 5.4 Hz, OH-3′), 7.52-7.65 (m, 3H, arom. H), 8.04 (d, 2H, J ) 7.1 Hz, arom. H), 8.53 (s, 1H, H-2), 8.74 (s, 1H, H-8), 11.14 [s (br), 1H, NH-6]. 13C-NMR (Me2SO-d6): δ 60.7 (CH2), 62.2 (C-3′), 69.4 (C-5′), 75.7 (C-4′), 86.0 (C-2′), 125.4 (C-5), 143.0 (C-8), 150.2 (C-4), 151.3 (C6), 152.3 (C-2). Anal. Calcd for C17H17N5O4: C, 57.46; H, 4.82; N, 19.71. Found: C, 57.19; H, 4.97; N, 19.61. 4(R)-(6-Benzoylamino-9H-purin-9-yl)-2-[O-(4,4′-dimethoxytriphenylmethyl)-2(R)-(hydroxymethyl)tetrahydrofuran-3(R)-ol (9). To a solution of 8 (200 mg, 0.56 mmol) in anhydrous pyridine (5 mL) were added, under N2, ethyldiisopropylamine (370 µL, 2.28 mmol) and 4,4′-dimethoxytrityl chloride (290 mg, 0.86 mmol). The mixture was stirred for 2 h at rt. The excess of acid chloride was destroyed with 5% aqueous NaHCO3 solution (5 mL). The aqueous layer was extracted with CH2Cl2 (5 × 5 mL), the solvent was evaporated, and the oily residue was applied on the top of a silica gel column (column, 20 × 3 cm; solvent, CHCl3/MeOH 95:5). Evaporation of the main zone furnished 9, which precipitated from light petroleum ether as a colorless solid (230 mg, 62%): mp 119-122 °C. TLC (CHCl3/MeOH 95:5): Rf 0.4. UV (MeOH): λ 270 nm ( 26 000). 1H-NMR (Me2SO-d6): δ 3.19 (m, 2H, CH2), 3.73 (s, 3H, OCH3), 3.74 (s, 3H, OCH3), 3.94 (m, 1H, H-2′), 4.30-4.37 (m, 2H, H-5′), 4.56 (m, 1H, H-3′), 5.05 (m, 1H, H-4′), 5.81 (d, 1H, J ) 5.4 Hz, OH-3′), 6.88 (d, 4H, J ) 8.9 Hz, arom. H), 7.20-7.40 (m, 7H, arom. H), 7.42 (m, 2H, arom. H), 7.55 (t, 2H, J ) 7.2 Hz, arom. H), 7.63 (t, 1H, J ) 7.3 Hz, arom. H), 8.05 (d, 2H, J ) 7.3 Hz, arom. H), 8.50 (s, 1H, H-2), 8.74 (s, 1H, H-8), 11.16 [s (br), 1H, NH-6]. 13C-NMR (Me2SOd6): δ 54.9, 55.0 (OCH3), 62.1 (C-3′), 63.5 (CH2), 68.5 (C5′), 75.0 (C-4′), 83.3 (C-2′), 125.7 (C-5), 143.2 (C-8), 150.3 (C-4), 151.3 (C-6), 152.4 (C-2). Anal. Calcd for C38H35N5O6: C, 69.39; H, 5.36; N, 10.65. Found: C, 68.28; H, 5.31; N, 10.79. 4(R)-(6-Benzoylamino-9H-purin-9-yl)-2-[O-(4,4′-dimethoxytriphenylmethyl)]-2(R)-(hydroxymethyl)tetrahydrofuran-3(R)-yl-(2-cyanoethyl-N,N-diisopropylphosphoramidite (10). A solution of 9 (200 mg, 0.30 mmol) in dry CH2Cl2 (7 mL) was kept under N2. Then, chloro(2-cyanoethoxy) (diisopropylamino)phosphine (200 µL, 0.91 mmol) together with ethyldiisopropylamine (160 µL, 0.93 mmol) was added under N2. After stirring for 90 min at rt, 5% aqueous NaHCO3 solution (7 mL) was added and the mixture was extracted with CH2Cl2 (5 × 5 mL). The extract was dried (Na2SO4), evaporated, and purified by FC (column, 10 × 3 cm; solvent, CH2Cl2/ EtOAc/Et3N 49:49:2). Isolation of the overlapping main zones yielded the diastereoisomeric mixture 10 (220 mg, 84%) as an oil. TLC (CH2Cl2/EtOAc/Et3N 49:49:2): Rf 0.3, 0.4. 1H-NMR (CDCl3): δ 0.96-1.12 [m, 14H, (CH3)2CH)], 2.28 (m, 2H, CH2CH2CN), 2.53 (m, 2H, CH2CH2CN), 3.44-3.57 (m, 2H, CH2), 3.76 (s, 3H, OCH3), 3.77 (s, 3H, OCH3), 4.10-4.18 (m, 1H, H-2′), 4.38 (m, 2H, H-5′), 4.80 (m, 1H, H-3′), 5.24 (m, 1H, H-4′), 6.77-6.83 (m, 4H, arom. H), 7.18-7.32 (m, 7H, arom. H), 7.387.44 (m, 2H, arom. H), 7.53 (t, 2H, J ) 7.7 Hz, arom. H), 7.60 (t, 1H, J ) 7.3 Hz, arom. H), 8.03 (d, 2H, J ) 7.1 Hz, arom. H), 8.23 (s, 1H, H-2), 8.80 (s, 1H, H-8), 9.02 [s (br), 1H, NH-6]. 31P-NMR (CDCl3): δ 151.2, 151.4. Anal. Calcd for C47H52N7O7P: C, 65.80; H, 6.11; N, 11.43. Found: C, 64.74; H, 6.30; N, 11.32. 4(R)-[3,4-Dihydro-2,4-dioxo-5-methyl-1(2H)-pyrimidinyl]-2-[O-(4,4′-dimethoxytriphenylmethyl)]-2(R)(hydroxymethyl)-tetrahydrofuran-3(R)-ol (11). Compound 2 (200 mg, 0.83 mmol) was dissolved in dry pyridine (10 mL) and stirred with 4,4′-dimethoxytri-

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Figure 1. (a) Temperature-dependent CD-spectra of d(G-T-A-G-iA-iA-iT-iT-C-T-A-C) (15) in 60 mM sodium-cacodylate (pH 7.0, 100 mM MgCl2, 1 M NaCl); oligomer concentration, 5.0 µM; (0) 10 °C; (4) 40 °C; (O) 70 °C. (b) CD spectra of d(A-T)6 (17, 0) and d(A-iT)6-T (18, 4) at 10 °C; conditions, see panel a; oligomer concentration, 5.6 µM.

phenylmethyl chloride (420 mg, 1.26 mmol) in the presence of ethyldiisopropylamine (540 µL, 3.32 mmol) for 3 h at rt under N2. The solution was quenched with 5% aqueous NaHCO3 (10 mL), extracted with CH2Cl2 (5 × 10 mL), and the combined organic layers were dried (Na2SO4), and the solvent was evaporated. The residue was purified by FC (column, 15 × 3 cm; CHCl3/MeOH 95:5). Evaporation of the main zone afforded 11 (270 mg, 60%) as a colorless foam. TLC (CHCl3/MeOH 95:5): Rf 0.4. UV (MeOH): λ 232, 273 nm, ( 24 400, 11 900). 1H-NMR (Me2SO-d6): δ 1.60 (s, 3H, CH3), 3.12 (dd,1H, J ) 5.8, 10.4 Hz, CH2OH), 3.18 (dd, 1H, J ) 2.5, 10.4 Hz, CH2OH), 3.71 (s, 3H, OCH3), 3.72 (s, 3H, OCH3), 3.75 (m, 1H, H-2′), 3.94 (dd, 1H, J ) 5.0, 10.1 Hz, Hβ-5′), 4.04 (dd, 1H, J ) 7.3, 10.1 Hz, HR-5′), 4.13 (m, 1H, H-3′), 4.77 (m, 1H, H-4′), 5.61 (d, 1H, J ) 5.8 Hz, OH-3′), 6.87 (d, 4H, J ) 7.8 Hz, arom. H), 7.19-7.31 (m, 7H, arom. H), 7.38 (d, 2H, J ) 7.1 Hz, arom. H), 7.44 (s, 1H, H-4), 11.29 (s, 1H, NH-1). 13C-NMR (Me2SO-d6): δ 55.0, 55.1 (OCH3), 62.5 (C-3′), 63.2 (CH2), 68.5 (C-5′), 75.3 (C-4′), 83.5 (C2′), 109.3 (C-5), 137.6 (C-6), 151.2 (C-2), 163.7 (C-4). Anal. Calcd for C31H32N2O7: C, 68.37; H, 5.92; N, 5.14. Found: C, 67.97; H, 5.64; N, 4.99. 4(R)-[3,4-Dihydro-2,4-dioxo-5-methyl-1(2H)-pyrimidinyl]-2-[O-(4,4′-dimethoxytriphenylmethyl)]-2(R)(hydroxymethyl)-tetrahydrofuran-3(R)-yl-(2-cyanoethyl-N,N-diisopropylphosphoramidite) (12). A solution of 11 (200 mg, 0.37 mmol) and ethyldiisopropylamine (190 µL, 1.08 mmol) in dry CH2Cl2 (10 mL) was treated with chloro(2-cyanoethoxy) (diisopropylamino)phosphine (230 µL, 1.02 mmol) at rt under N2. After stirring for 1 h, the reaction was quenched by adding 5% aqueous NaHCO3 solution (10 mL). The mixture was extracted with CH2Cl2 (5 × 10 mL), the organic layer was dried (Na2SO4) and evaporated and the residue was purified by FC (column, 10 × 3 cm; solvent, CH2Cl2/ EtOAc/Et3N 49:49:2). Isolation of the overlapping main zones furnished 12 (180 mg, 66%) as a diastereoisomeric mixture. TLC (CH2Cl2/EtOAc/Et3N 49:49:2): Rf 0.6, 0.8. 1 H-NMR (CDCl3): δ 1.04-1.25 [m, 14H (CH3)2CH], 1.54 (s, 3H, CH3), 2.20 (m, 2H, CH2CH2CN), 2.56 (m, 2H, CH2CH2CN), 3.41-3.51 (m, 2H, CH2OH), 3.71 (s, 3H, OCH3), 3.72 (s, 3H, OCH3), 3.82-3.88 (m, 1H, H-2′), 3.98-4.15, (m, 2H, H-5′), 4.52 (m, 1H, H-3′), 5.09 (m, 1H, H-4′), 6.75 (m, 4H, arom. H), 7.14-7.26 (m, 7H, arom. H), 7.34 (m, 3H, H-4/arom. H), 8.30 (s, 1H, NH-1). 31PNMR (CDCl3): 151.6, 151.9. Anal. Calcd for C40H49N4O8P: C, 64.50; H, 6.63; N, 7.52. Found: C, 63.63; H, 6.46; N, 7.45. Solid-Phase Synthesis of the Oligonucleotides 13-18. Synthesis of the oligomers 13-18 was carried

out on a 1 µmol scale using the 3′-cyanoethyl phosphoramidites of the following protected deoxynucleosides: [(MeO)2Tr]dT, [(MeO)2Tr]Bz4dC, [(MeO)2Tr]Bz6dA, [(MeO)2Tr]ib2dG, and compounds 1 and 2. Deprotection of the oligonucleotides was performed in 25% aqueous NH3 at 60 °C for 24 h. The 5′-[(MeO)2Tr]-oligomers were purified, detritylated and desalted using OPC (ABI, Masterpiece). The oligonucleotides were lyophilized, dissolved in H2O (200 µL), and stored frozen at -20 °C. Enzymatic Hydrolysis of the Oligomers. The selfcomplementary oligonucleotides 13-17 (0.2 A260 units) were dissolved in 0.1 M Tris-HCl buffer (pH 8.3; 200 µL) and treated with snake-venom phosphodiesterase (6 µg) for 24 h at 37 °C followed by alkaline phosphatase (2 µg) for 30 min at 37 °C. The mixture was analyzed using RP-18 HPLC (gradient II, followed by gradient I, see below). The quantification of the oligomers was made on the basis of the peak areas which were divided by the extinction coefficients of the nucleoside constituents. Enzymatic Hypochromicity of the Oligonucleotides 13-17. The enzymatic hypochromicities were determined by enzymatic degradation of 0.2 A260 units of the corresponding oligomer as described above. Time Course of Phosphodiester Hydrolysis of the Oligomers 13-18. For these experiments, the corresponding oligonucleotide (2.25 A260 units) was dissolved in 0.1 M Tris-HCl buffer (900 µL, pH 8.3), and alkaline phosphatase (22.5 µg) was added; two stock solutions were prepared. The reaction was started by addition of either snake-venom phosphodiesterase (67.5 µg) or calfspleen phosphodiesterase (135 µg). From the stock solutions, samples (100 µL) were taken after a certain interval of time. An aliquot (80 µL) was examined by HPLC (gradient II, followed by I, see below). The UV absorbance was measured at 260 nm and quantification was made on the basis of peak areas and extinction coefficients. HPLC Separation. The following solvent systems consisting of 0.1 M (Et3NH)OAc (pH 7)/MeCN 95:5 (A) and MeCN (B) were used: gradient I, 20 min 0-30% B in A, 5 min 30% B in A, flow rate, 1 mL/min; gradient II, 30 min 100% A, flow rate, 0.6 mL/min. CD Spectral Data. Temperature-dependent CD spectra were obtained under the conditions described in Figures 1 and 2. RESULTS AND DISCUSSION

Synthesis of Monomers. While two methods are available for the synthesis of isomeric deoxyadenosine

Self-Complementary Oligodeoxyribonucleotides

Bioconjugate Chem., Vol. 9, No. 6, 1998 687

Figure 2. (a) Normalized melting profiles of d(G-T-A-G-iA-iA-T-T-C-T-A-C) (14, 0) and d(G-T-A-G-iA-iA-iT-iT-C-T-A-C) (15, 4); conditions, see Figure 1a; oligomer concentation 5 µM. (b) -ln c vs 1/Tm plot, 14 (0) and 15 (4). (c) Normalized melting profiles of d(G-iT-iA-G-A-A-T-T-C-iT-iA-C) (16, 0) and d(A-iT)6-T (18, 4); conditions, see Figure 1a. (d) -ln c vs 1/Tm plot, 16 (0) and 18 (4).

(15, 16), these either give low yields of product or have been developed for very small scale synthesis. As the synthesis of oligodeoxyribonucleotides requires the availability of isodeoxyadenosine in gram quantities, we developed a synthesis strategy for isodA (1) starting from 1-deoxyribose (3) (17). This approach was based on previous work from our laboratory (18). Selective protection of the primary hydroxyl group by treatment of 3 with tert-butyldiphenylsilyl chloride (TBDPS-Cl) in anhydrous pyridine at 0 °C afforded 4 in 86% yield. Methanesulfonylation of 4 at 0 °C led to a mixture of mesylates from which 5 was isolated in 53% yield. The 3-mesylate was the minor product (21%). Following protection of the 3-hydroxyl group in 5 (78%), the resulting compound 6 was coupled with adenine in the presence of K2CO3 and 18-crown-6 in dry DMF. However, this alkylation step required drastic conditions (120 °C, 60 h) and compond 7a and its partially deprotected derivative 7b were obtained in only 49% yield. Deprotection of nucleosides 7a and 7b was carried out using 0.5 M methanolic NH4F solution to furnish 1 in high yields. The isomeric thymine analog, isodT, could not be prepared in good yields using the alkylation step described above because of complications associated with O-alkylation. However, this intermediate 2 was prepared using modification of a procedure developed previously in our laboratory recently (18). Nucleosides 1 and 2 were converted into the β-cyanoethyl phosphoramidite building blocks 10 and 12. As N6benzoyldeoxyadenosine has a half-life of 71 min under mild alkaline conditions (25% aqueous NH3 solution (19), we protected the exocyclic amino group of 1 with the benzoyl group. Reaction of 1 with benzoyl chloride, after temporary protection of the sugar hydroxyls with Me3Si

residues (transient protection) (20), afforded compound 8 in 81% yield. Compounds 8 and 2 were converted into the 4,4′-dimethoxytrityl [(MeO)2Tr] derivatives 9 and 11 under standard conditions (60-62%). Subsequent phosphonylation of 9 and 11 with chloro(diisopropylamino)β-cyanoethoxyphosphine furnished the phosphoramidites 10 and 12 as diastereoisomeric mixtures in good yields. These new compounds were characterized by 1H-, 13C-, and 31P-NMR spectral data (see Experimental Procedures). Assignment of the protons was made on the basis of COSY spectra, whereas the unequivocal assignment of the carbons was aided by [1H,13C]-2D-correlation or [1H,13C]-gated decoupled spectra. The anomeric configuration and the alkylation position of compound 1 were assigned by 1H-NOE difference spectroscopy (see Experimental Procedures) (21). Synthesis of Oligonucleotides. The β-cyanoethyl phosphoramidites 10 and 12 were used in automated solid-phase synthesis of oligonucleotides (22). However, the incorporation of the isomeric DNA building blocks 10 and 12 required 10 times longer coupling times than the corresponding 2′-deoxyribophosphoramidites. Furthermore, the coupling yields determined on the liberation of the 4,4′-dimethoxytrityl cation at 498 nm were significantly lower than those of the regular DNAbuilding blocks (10, 70%; 12, 80%). After treatment with concentrated aqueous NH3 solution (24 h at 60 °C), the 5′-(MeO)2Tr-protected oligomers were purified, detritylated, and desalted on oligonucleotide-purification cartridges (Perkin Elmer, Applied Biosystem Division) to yield the oligomers 13-18 (Table 1). The content of the modified oligonucleotides was determined by tandem hydrolysis with snake-venom phosphodiesterase followed by alkaline phosphatase (Table 2).

688 Bioconjugate Chem., Vol. 9, No. 6, 1998

Wenzel and Nair

Table 1. Yields and Retention Times of the Oligomers 13-18

oligomer

yield A260

units (%)

retention timea (min)

d(G-T-A-G-A-A-T-T-C-T-A-C) (13) d(G-T-A-G-iA-iA-T-T-C-T-A-C) (14) d(G-T-A-G-iA-iA-iT-iT-C-T-A-C) (15) d(G-iT-iA-G-A-A-T-T-C-iT-iA-C) (16) d(A-T)6 (17) d(A-iT)6T (18)

34.4 24.0 14.8 10.8 36.8 7.7

36.5 21.9 12.7 9.5 33.3 5.0

18.6 19.5 20.5 21.4 21.6 22.2

a

Gradient I; flow rate, 1.0 mL/min, column: 4.6 × 250 mm.

d(G-T-A-G-A-A-T-T-C-T-A-C) (13) d(G-T-A-G-iA-iA-T-T-C-T-A-C) (14) d(G-T-A-G-iA-iA-iT-iT-C-T-A-C) (15) d(G-iT-iA-G-A-A-T-T-C-iT-iA-C) (16) d(A-T)6 (17) d(A-iT)6-T (18) Duplex Stability of Oligodeoxyribosides Incorporating L-Related Isodeoxynucleosides. Figure 1a displays the temperature-dependent CD spectra of d(GT-A-G-iA-iA-iT-iT-C-T-A-C) (15) measured in 1 M NaCl. As can be seen, the change from the low- to the hightemperature profile led to significant decrease of the ellipticity of the CD minima and maxima, whereas increasing of the temperature did not alter the wavelength of the Cotton effects. Furthermore, the temperature-dependent CD profiles exhibit well-defined isodichroic points, an experimental finding which is in good agreement with our results on the dinucleotide, 2′deoxyadenylyl-(3′f5′)-isodideoxyadenosine (23). The CD spectra of the self-complementary alternating oligomers 17 and 18 were also measured (Figure 1b). While the unmodified oligomer 17 shows a B1u transition at 246 nm with a negative sign and a B2u transition at 268 nm with positive sign, the corresponding π-π* transitions of oligonucleotide 18 exhibit similar Cotton effects but with a strong bathochromic shift of the B1u and B2u transitions (8 nm). Moreover, the ellipticity value of the B1u transition is markedly reduced, whereas the intensity of the B2u band is almost the same.

The structural stability of the modified oligonucleotides 14-16 and 18 using temperature-dependent CD spectroscopy was also investigated by measuring both π-π* transitions (B1u, B2u) as a function of temperature (10 and 70 °C). In all cases, sigmoidal monophasic melting profiles were obtained (Figure 2, panels a and c). Furthermore, both π-π* transitions exhibited almost the same Tm value. To discriminate between duplex and hairpin formation, the thermal denaturation curves of 14-16 and 18 as a function of oligomer concentration were determined (Figure 2, panels b and d). Reduction of the oligomer concentration from 10 to 2 µmol led to a significant decrease of the Tm values by 4-6 °C. Therefore, in all cases, duplex melting was observed; the formation of hairpin structures could be excluded. Surprisingly, the modified oligonucleotide d(A-iT)6-T (18) showed exactly the same Tm value as determined for the regular counterpart 17. Moreover, the oligomers 14-16 exhibited only a moderate decrease of the Tm values compared to the parent duplex 13. These experimental findings encouraged us to study the thermodynamic parameters for the conceptually novel oligonucleotides. From the ln c vs 1/Tm plot (Figure 2, panels b and d), ∆H and ∆S of the melting process can be determined according to Borer et al. (Table 3) (24). The experimentally determined ∆H values of the duplexes 15 and 16 were slightly reduced compared to the parent duplex 13. Surprisingly, the incorporation of two isodA residues opposite of dT within the oligonucleotide 14 led to an increase of the enthalpy. The measured ∆H values of all self-complementary oligonucleotides (13-16) were in good agreement with calculated values (∆H ) -95 kcal/mol for 13) using published increments (25). These results indicate that incorporation of Lrelated isodeoxynucleosides does not disturb duplex formation to any significant extent; the isodA-dT base pair (oligomer 14) is enthalpically favored but entropically unfavored. The incorporation of 4 isodA-isodT base pairs in the innermost part (15) or at the termini (16) of 13 led to stable duplex structures. However, the contribution of each isodA-isodT base pair to ∆H (-4.0 to -4.5 kcal/mol) is lower than that of the dA-dT base pair (-6 kcal/mol). Evaluations of the thermodynamics of duplex formation of the alternating self-complementary oligomers 17and 18 exhibited a significant difference. While the enthalpy of duplex formation for d(A-iT)6-T (18) is 16% lower than that of the regular counterpart, d(A-T)6

Table 2. Composition of the Oligomers 14-16 and 18 Determined by Tandem-Hydrolysis with Snake-Venom Phosphodiesterase and Alkaline Phosphatase in 0.1 M Tris-HCl Buffer, pH 8.3

a

oligomer

dA/iAd

dC

dG

dT/iTd

d(GTAGiAiATTCTAC) (14) d(GTAGiAiAiTiTCTAC) (15) d(GiTiAGAATTCiTiAC) (16) d(AiT)6Ta (18)

4.3 (4.0) 4.0 (4.0) 3.6 (4.0)

1.8 (2.0) 2.2 (2.0) 1.8 (2.0)

1.9 (2.0) 2.0 (2.0) 2.0 (2.0)

4.0 (4.0) 3.7 (4.0) 3.7 (4.0)

No significant enzymatic hydrolysis.

Table 3. Tm Values and Thermodynamic Data of the Oligomers 13-18a oligomer

henzymeb (%)

Tm (°C)

∆H (kcal/mol)

∆S (cal/K mol)

∆G° (kcal/mol)

d(GTAGAATTCTAC) (13) d(GTAGiAiATTCTAC) (14) d(GTAGiAiAiTiTCTAC) (15) d(GiTiAGAATTCiTiAC) (16) d(AT)6 (17) d(AiT)6T (18)

31 18 12 16 28 c

48 45 39 40 32 32

-90.1 -97.7 -86.2 -88.6 -59.5 -50.0

-280 -307 -276 -283 -195 -164

-6.6 -6.2 -3.9 -4.3 -1.4 -1.1

a Measured in 1 M NaCl containing 100 mM MgCl and 60 mM sodium-cacodylate (pH 7.0); single strand concentration 5 µM. b Enzymatic 2 hypochromicity (snake-venom phosphodiesterase). c No significant hydrolysis of the phosphodiester backbone.

Self-Complementary Oligodeoxyribonucleotides

Bioconjugate Chem., Vol. 9, No. 6, 1998 689

Figure 3. Time course of phosphodiester hydrolysis of the oligonucleotides 13-16 in 0.1 M Tris-HCl buffer (pH 8.3) at 37 °C; (- - -) d(G-T-A-G-A-A-T-T-C-T-A-C) (13), (0) d(G-T-A-G-iA-iA-T-T-C-T-A-C) (14), (O) d(G-T-A-G-iA-iA-iT-iT-C-T-A-C) (15), (4) d(G-iT-iAG-A-A-T-T-C-iT-iA-C) (16); (a) snake-venom phosphodiesterase (3′,5′-exonuclease); (b) calf-spleen phosphodiesterase (5′,3′-exonuclease), both followed by alkaline phosphatase treatment.

Figure 4. Time course of phosphodiester hydrolysis of d(A-iT)6-T (18); conditions, see Figure 3; (a) snake-venom phosphodiesterase; (b) calf-spleen phosphodiesterase, both followed by alkaline phosphatase treatment.

(17), the entropical term is favored by about 18%. In that case, the contribution of each dA-isodT base pair to ∆H is still approximately -4.2 kcal/mol. Finally, these studies show that the novel synthesized oligonucleotides 14-16 and 18 form stable Watson-Crick duplex structures in which formation of the hydrogen bonds between isodA-dT or isodA-isodT or dA-isodT is not disturbed. Enzymology. Nuclease stability of the phosphodiester backbone is an important requirement for the development of therapeutic oligonucleotides. To test this behavior of the modified oligomers, 14-16 and 18, we carried out enzymatic studies using snake-venom phosphodiesterase as well as calf-spleen phosphodiesterase. Figure 3 shows the rates of phosphodiester bond hydrolysis of the oligonucleotides 13-16 in the presence of the exonucleases. As can be seen, incorporation of L-related isodeoxynucleosides within the parent oligomer 13 led to significant resistance of the phosphodiester bonds towards exonucleases. Furthermore, the resistance increased with the number of isomeric nucleosides within the self-complementary oligonucleotides. Exchange of the innermost part of the oligomer by isomeric nucleosides (oligomer 15) exhibited the highest stability toward exonucleases. The enzyme kinetics follows a two-step mechanism in which the unmodified nucleotides are hydrolyzed first with rates almost identical to the parent sequence, 13. In the second step the modified inner part is hydrolyzed at a much lower rate. Figure 4 shows the enzymatic degradation of the phosphodiester backbone of the alternating self-complementary d(A-iT)6-T (18). The kinetic studies show that

oligomer 18 is very resistant toward enzymatic hydrolysis, toward either snake-venom or calf-spleen phosphodiesterase. Even after 100-120 h (Figure 4) only minimal amounts of oligonucleotide 18 had hydrolyzed. Summary and Conclusion. In summary, the Lrelated isodeoxynucleosides 1 and 2 were successfully incorporated within self-complementary oligonucleotides, (14-16, 18). These conceptually novel oligomers, and especially the alternating self-complementary oligonucleotide, d(A-iT)6-T (18), exhibit high resistance toward nucleases. Comprehensive CD spectral studies and the resulting thermodynamic parameters indicate the participation of the isodA-isodT or mixed dA-isodT/isodAdT base pairs in duplex stabilization. ACKNOWLEDGMENT

We thank Dr. G. Pearson for technical assistance with several 2D-NMR spectra and Dr. R. Forsyth of the Protein Structure Facility for help with the operation of the CD spectrometer. This research work was supported by the National Institutes of Health (NIAID). LITERATURE CITED (1) De Mesmaeker, A., Ha¨ner, R., Martin, P., and Moser, H. E. (1995) Antisense oligonucleotides. Acc. Chem. Res. 28, 366374. (2) Van Aerschot, A., Verheggen, I., Hendrix, C., and Herdewijn, P. (1995) 1,5-Anhydrohexitol nucleic acids, a new promising antisense construct. Angew. Chem., Int. Ed. Engl. 34, 13381339. (3) Seela, F., Heckel, M., and Rosemeyer, H. (1996) Xylose-DNA containing the four natural bases. Helv. Chim. Acta 79, 14511461.

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Wenzel and Nair (15) Montgomery, J. A. and Thomas, H. J. (1978) Isonucleosides. 2. Purine and pyrimidine derivatives of 1,4-anhydro-2-deoxyD-arabinitol. J. Org. Chem. 43, 541-544. (16) Kakefuda, A., Shuto, S., Nagahota, T., Seki, J., Sasaki, T., and Matsuda, A. (1994) Nucleosides and nucleotides. 132. Synthesis and biological evaluations of ring-expanded oxetanocin analogues: purine and pyrimidine analogues of 1,4anhydro-2-deoxy-D-arabitol and 1,4-anhydro-2-deoxy-3-hydroxymethyl-D-arabitol. Tetrahedron 34, 10167-10182. (17) Barker, R., and Fletcher, H. G., Jr. (1961) 2,3,5-Tri-Obenzyl-D-ribosyl and -L-arabinsoyl bromides. J. Org. Chem. 26, 4605-4609. (18) Purdy, D. F., Zintek, L. B., and Nair, V. (1994) Synthesis of isonucleosides related to AZT and AZU. Nucleosides Nucleotides 13 (1-3), 109-126. (19) Seela, F., and Wenzel, T. (1992) 1,7-Dideaza-2′-deoxyadenosine: building blocks for solid-phase synthesis and secondary structure of base-modified oligodeoxyribonucleotides. Helv. Chim. Acta 75, 1111-1122. (20) Ti, G. S., Gaffney, B. L., and Jones, R. A. (1982) Transient protection: efficient one-flask syntheses of protected deoxynucleosides. J. Am. Chem. Soc. 104, 1316-1319. (21) Rosemeyer, H., Toth, G., and Seela, F. (1989) Assignment of anomeric configuration of D-ribo-, arabino-, 2′-deoxyribo-, and 2′,3′-dideoxyribonucleosides by NOE difference spectroscopy. Nucleosides Nucleotides 8 (4), 587-597. (22) Beaucage, S. L., and Caruthers, M. H. (1981) Deoxynucleoside phosphoramidites. A new class of key intermediates for deoxypolynucleotide synthesis. Tetrahedron Lett. 22, 18591862. (23) Jahnke, T. S., and Nair, V. (1995) 2′-Deoxyadenylyl-(3′f5′)isodideoxyadenosine, a unique dinucleotide: synthesis, enzymology, and conformational studies. Bioorg. Med. Chem. Lett. 5, 2235-2238. (24) Borer, B. N., Dengler, B., Uhlenbeck, O. C., and Tinoco, I., Jr. (1974) Stability of ribonucleic acid double-stranded helices. J. Mol. Biol. 86, 843-853. (25) Breslauer, K. J., Frank, R., Blo¨cker, H., and Marky, L. A. (1986) Predicting DNA duplex stability from the base sequence. Proc. Natl. Acad. Sci. U.S.A. 83, 3746-3750.

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