Studies on the synthesis of a G-rich octaoligoisonucleotide (isoT)₂(isoG)₄(isoT)₂ by the phosphotriester approach and its formation of G-quartet structure

Abstract

The octaoligoisonucleotide (isoT)₂(isoG)₄(isoT)₂ (I), consisting of isonucleoside units 6′-O-allyl-4′-deoxy-4′-(nucleobase)-2′,5′-anhydro-l-mannitol, was synthesized by the phosphotriester approach in solution phase. Based on CD spectra and capillary electrophoresis, it was confirmed that iso-oligomer I could form a parallel intermolecular G-quadruplex structure. K⁺, Na⁺ and Li⁺ can prompt the formation of G-quartet structures and stabilize them. The effective order of these cations is K⁺ > Na⁺ > Li⁺.

INTRODUCTION

Isonucleosides represent a novel class of nucleoside analogs in which the nucleobase is linked to various positions of ribose other than C-l′. Based on the antisense strategy the hybridization properties and enzymatic stability of oligonucleotides bearing some types of such isonucleosides have been studied in our laboratory (1–3). The oligomers are all stable towards snake venom phosphodiesterase and some iso-oligomers exhibit acceptable hybridization properties with complementary sequences. Due to the special structural features and the stability of these iso-oligomers, it might be interesting to study the interaction of such iso-oligomers with RNA and specific proteins.

In this paper, the G-rich sequence (isoT)₂(isoG)₄(isoT)₂ (I) containing isonucleoside units 4 and 12 (see Materials and Methods) was synthesized by a phosphotriester approach in solution phase (Fig. 1). Using CD spectra and capillary electrophoresis, it was confirmed that oligomer I might form a parallel intermolecular G-quadruplex structure.

Figure 1.

Structures of oligomers I and II.

It is well known that at physiological concentrations of monovalent ions G-rich oligonucleotides can form four-stranded structures called G-quadruplexes. G-quadruplex structures comprise stacked tetrads in which four guanines are arranged in a square-planar array and each guanine serves as both hydrogen bond acceptor and donor in a reverse Hoogsteen base pair. Monovalent cations such as K⁺ and Na⁺ have been shown to stabilize G-quadruplex structures, presumably by coordinating with eight carbonyl oxygen atoms present between stacked tetrads (4). Several types of G-quadruplex structures can be classified based on their strand orientation, strand stoichiometry and glycosidic conformation. In the intramolecular chair or edge type structure the dG residues of each quartet alternate syn-anti-syn-anti (5). In the crossover or basket type structure the dG residues alternate syn-syn-anti-anti within each quartet and both intra- and intermolecular basket type structures have been observed (6–8). Intermolecular quadruplex structures formed by four parallel strands have all residues in the anti conformation (9,10).

Several biologically important genomic regions such as telomeres (11), the immunoglobulin switch regions (12), the promoter regions of genes (13) and recombination sites (14,15) were found to have the propensity to form G-quadruplex structures. Several compounds that stabilize G-quadruplex DNA and inhibit telomerase have been reported, making G-quadruplex DNA a promising drug target for cancer therapy and aging research (16–18).

The potential of G-quadruplex DNA as a therapeutic agent is also documented. Wang et al. reported that a 15mer sequence 5′-GGTTGGTGTGGTTGG-3′ has an intramolecular chair type quadruplex struture and can bind to thrombin to inhibit thrombin-catalyzed fibrin clot formation in vitro (19). Jing and Hogan reported that a 17mer sequence 5′-g*tggtgggtgggtggg*t (T30177) and a 16mer sequence 5′-g*ggtgggtgggtggg*t (T30695) were most potent inhibitors of HIV-1 integrase activity (20). Shimada and co-workers showed that 5′-end 3,4-dibenzyloxybenzyl (3,4-DBB) substituted 6mers, 5′-TGGGAG-3′, could form parallel intermolecular G-quadruplex structures and bind to the V3 loop domain of HIV-1 gp120, resulting in anti-HIV-1 activity (21,22). Wyatt et al. reported that a phosphorothioate-type octadeoxyribonucleotide having a 5′-TTGGGGTT-3′ sequence (ISIS5320) formed a parallel-stranded tetrameric G-quartet structure. The tetramer bound to the cationic V3 loop domain of HIV-1 gp120 and inhibited both cell-to-cell and virus-to-cell infection. The G-quartet structure and the phosphorothioate backbone were essential for antiviral activity (23). Since G-quadruplexes have proved useful inhibitors of some proteins and in our laboratory we have found that spin-labeled thrombin can partially bind with (isoT)₂(isoG)₄(isoT)₂ (unpublished data), therefore, this strongly nuclease-resistant analog of guanosine tetrads of (isoT)₂(isoG)₄(isoT)₂ may have enhanced efficacy in vivo.

MATERIALS AND METHODS

General

All solvents were dried and distilled prior to use. Chemical reagents were purchased from Acros and Sigma Co. Thin layer chromatography was performed on silica gel GF-254 (Qin-Dao Chemical Co., China) plates with detection by UV or by heating. Silica gel (200–300 mesh; Qin-Dao Chemical Co.) was used for short column chromatography. NMR spectra were recorded on a Varian VXR-300, Varian Inova-500 or Inova-600 instrument with TMS as internal standard. 2,5:3,4-Dianhydro-l-talofuranose dimethylacetal (1) was synthesized according to Yang et al. (24).

6-O-Allyl-2,5:3,4-dianhydro-l-talofuranose dimethylacetal (2)

A solution of 2,5:3,4-dianhydro-l-talofuranose dimethylacetal (1) (9.65 g, 50.8 mmol) (24) in dry DMF (90 ml) was cooled (ice bath) and sodium hydride added (5.17 g, 210.5 mmol). Allylbromide (14 ml, 157 mmol) was added drop-wise with vigorous stirring. The reaction mixture was kept at ambient temperature for 4 h, then ethanol was added drop-wise at 0°C to hydrolyze NaH. After removal of solvents, the residue was dissolved in dichloromethane. After filtration and evaporation, the residue was subjected to silica gel chromatography (ethyl acetate/petroleum ether) to afford 2 (10.9 g, 93.4% yield).

¹H NMR (300 MHz, CDCl₃): δ 3.38 (s, 3H, 1-OCH₃), 3.40 (s, 3H, 1-OCH₃), 3.50 (d, J = 6.0 Hz, 2H, H-6), 3.72 (d, J = 3.3 Hz, 1H, H-3), 3.75 (d, J = 3.3 Hz, 1H, H-4), 3.96 (m, 2H, -CH₂-O), 4.04 (d, J = 4.2 Hz, 1H, H-2), 4.12 (t, J = 6.0 Hz, 1H, H-5), 4.24 (d, J = 3.9 Hz, 1H, H-1), 5.10 (m, 2H, CH₂=), 5.70 (m, 1H, =CH-). ¹³C NMR (300 MHz, CDCl₃): δ 55.4 (C-4), 56.5 (C-3), 56.7 (1-OCH₃), 56.9 (1-OCH₃), 68.6 (C-6), 72.4 (-CH₂-O), 76.6 (C-5), 78.2 (C-2), 105.0 (C-1), 117.1 (CH₂=), 134.4 (=CH-). FAB-HRMS m/z: 231.1232 [M + H]⁺. Calculated for C₁₁H₁₉O₅: 231.1232.

6′-O-Allyl-4′-deoxy-4′-(thymin-1-yl)-2′,5′-anhydro-l-mannofuranose dimethyl acetal (3)

A mixture of thymine (4.26 g, 33.8 mmol), DBU (12 ml, 80 mmol) and 2 (5.98 g, 26 mmol) was dissolved in anhydrous DMF (80 ml). The clear solution was stirred at 95°C for 77 h. After removal of DMF in vacuo, the resulting brown residue was diluted with dichloromethane and applied to silica gel chromatography eluting with EtOAc/petroleum ether to recover unreacted 2, then with CH₂Cl₂/CH₃OH to afford 3 (2.37 g, 45.1%).

¹H NMR (500 MHz, DMSO-d₆): δ 1.79 (s, 3H, 5-CH₃), 3.35 (s, 6H, 1′-OCH₃), 3.43 (t, 2H, H-6′), 3.75 (t, J = 6.0 Hz, 1H, H-2′), 3.93 (m, 2H, -CH₂-O), 4.09 (p, J = 5.0 Hz, 1H, H-5′), 4.35 (q, J = 6.0 Hz, 5.5 Hz, 1H, H-3″), 4.47 (d, J = 5.5 Hz, 1H, H-1′), 4.59 (q, J = 7.5 Hz, 1H, H-4′), 5.10 (m, 2H, CH₂=), 5.52 (d, J = 6.5 Hz, 1H, 3′-OH), 5.80 (m, 1H, =CH-), 7.55 (s, 1H, H-6), 11.3 (s, 1H, 3-NH). FAB-HRMS m/z: 357.1584 [M + H]⁺. Calculated for C₁₆H₂₅N₂O₇: 357.1662.

6′-O-Allyl-4′-deoxy-4′-(thymin-1-yl)-2′,5′-anhydro-l-mannitol (4)

Compound 3 (2.37 g, 6.66 mmol) was dissolved in 1% hydrochloric acid in water (55 ml) and refluxed for 3 h. After cooling to 0°C and neutralization with 2 M NaOH, sodium borohydride (610 mg, 4.49 mmol) was added. The solution was stirred at room temperature for 5 h then neutralized with 2 M HCl at 0°C. The water was removed under reduced pressure and the residue was dissolved in C₂H₅OH. After filtration, the mixture was purified by silica gel chromatography eluting with CH₂Cl₂/CH₃OH to obtain 4 (1.39 g, white solid, 67%).

¹H NMR (300 MHz, DMSO-d₆): δ 1.77 (s, 3H, 5-CH₃), 3.42 (m, 2H, H-6′), 3.47 (q, 1H, 1′-CH₂), 3.57 (d, 1H, 1′-CH₂), 3.69 (m, 1H, H-2′), 3.93 (d, J = 5.4 Hz, 2H, -CH₂-O), 4.04 (p, J = 4.8 Hz, 1H, H-5′), 4.16 (q, J = 7.5 Hz, 5.7 Hz, 1H, H-3′), 4.67 (t, J = 8.4 Hz, 1H, H-4′), 4.77 (t, J = 5.1 Hz, 1′-OH), 5.08 (m, 2H, CH₂=), 5.50 (d, J = 5.7 Hz, 1H, 3′-OH), 5.77 (m, 1H, =CH-), 7.57 (d, J = 2.0 Hz, 1H, H-6), 11.3 (s, 1H, 3-NH). FAB-HRMS m/z: 313.1399 [M + H]⁺.

6′-O-Allyl-1′-O-dimethoxytrityl-4′-deoxy-4′-(thymin-1-yl)-2′,5′-anhydro-l-mannitol (5)

Compound 4 (817 mg, 2.63 mmol) was dissolved in dry pyridine (15 ml) and dimethoxytrityl (DMT) chloride (1080 mg, 3.19 mmol) was added. The solution was stirred at room temperature for 24 h. After evaporation, the mixture was purified by silica gel chromatography, eluting with petroleum ether/acetone (3:2) (1% Et₃N added), to afford compound 5 (867 mg) as a white foam in 53.8% yield.

¹H NMR (300 MHz, DMSO-d₆): δ 1.71 (s, 3H, 5-CH₃), 3.10 (m, 2H, 1′-CH₂), 3.49 (d, J = 4.5 Hz, 2H, H-6′), 3.73 (s, 6H, 1′-OCH₃), 3.92 (m, 1H, H-2′), 3.99 (m, 2H, -CH₂-O), 4.14 (m, 2H, H-5′, H-3′), 4.66 (t, 1H, H-4′), 5.12 (m, 2H, CH₂=), 5.53 (d, J = 6.0 Hz, 1H, 3′-OH), 5.82 (m, 1H, =CH-), 6.86 (d, J = 8.7 Hz, 4H, Ph), 7.21 (m, 7H, Ph), 7.41 (d, J = 8.4Hz, 2H, Ph), 7.57 (s, 1H, H-6), 11.3 (s, 1H, 3-NH).

Triethylammonium salt of 6′-O-allyl-1′-O-DMT-4′-deoxy-4′-(thymin-1-yl)-2′,5′-anhydro-l-mannitol-3′-(2-chlorophenyl) phosphate (6)

2-Chlorophenyl phosphorodichloridate (0.65 ml, 4 mmol), 1,2,4-triazole (600 mg, 8.8 mmol), dry triethylamine (1.2 ml, 8 mmol) and anhydrous THF (20 ml) were stirred together at room temperature. After 30 min, a solution of compound 5 (794 mg, 1.3 mmol) and 1-methylimidazole (1 ml, 13 mmol) in THF (10 ml) was added and the resulting mixture was stirred for 2 h. Et₃N (1 ml) and enough water were then added to give a clear solution. The resulting solution was stirred for 30 min and then concentrated under reduced pressure. The residue was dissolved in CH₂Cl₂ and the solution was extracted with 5% aqueous sodium hydrogen carbonate. The organic layer was dried with Na₂SO₄ and concentrated under reduced pressure. A solution of the residue in CH₂Cl₂ (5 ml) was added drop-wise to stirred light petroleum ether (bp 30–60°C, 250 ml) to give compound 6 as a solid (1.04 g, 88.9%).

¹H NMR (300 MHz, DMSO-d₆): δ 1.10 (t, J = 7.5 Hz, 9H, 3CH₃), 1.53 (s, 3H, 5-CH₃), 2.97 (q, J = 7.5 Hz, 6H, 3CH₂), 3.49 (m, 2H, 1′-CH₂), 3.69 (s, 2H, H-6′), 3.71 (d, 6H, 1′-OCH₃), 3.98 (d, 2H, -CH₂-O), 4.16 (m, 2H, H-5′, H-2′), 4.48 (t, 1H, H-4′), 4.80 (q, J = 6.6 Hz, 1H, H-3′), 5.10 (m, 2H, CH₂=), 5.86 (m, 1H, =CH-), 6.81–7.39 (18H, H-6 and Ph), 7.96 (s, 1H, NH), 11.07 (s, 1H, 3-NH). ³¹P NMR (200 MHz, DMSO-d6): δ –5.92. FAB-MS m/z: 803.1 [M – ⁺NHEt₃]^–.

6′-O-Allyl-4′-deoxy-4′-(guanin-9-yl)-2′,5′-anhydro-l-mannofuranose dimethyl acetal (7) and 6′-O-allyl-4′-deoxy-4′-(guanin-7-yl)-2′,5′-anhydro-l-mannofuranose dimethyl acetal (8)

A mixture of guanine (4.76 g, 31.5 mmol), dry potassium carbonate (5.54 g, 40.1 mmol), 18-crown-6 (3.2 g, 12.1 mmol) and 2 (5.56 g, 24.2 mmol) were dissolved in anhydrous DMSO (120 ml). The mixture was heated at 100°C for 100 h. After filtration and evaporation, the residue was applied to a silica gel column eluting with EtOAc/petroleum ether to recover unreacted 7, then with CH₂Cl₂/CH₃OH to afford an unseparable mixture of 7 and 8 (4.77 g, 51.8%).

¹H NMR (300 MHz, DMSO-d₆): δ 3.33–3.37 (q, 6H, 1′-OCH₃), 3.39 (t, J = 4.2 Hz, 2H, H-6′), 3.79 (m, 1H, H-2′), 3.88 (m, 2H, -CH₂-O), 4.37–4.50 (m, 1H, H-5′), 4.53–4.66 (m, 3H, H-1′, H-3′, H-4′), 5.07–5.19 (m, 2H, CH₂=), 5.57–5.63 (d, J = 5.7 Hz, 1H, 3′-OH), 5.76 (m, 1H, =CH-), 6.22 (s, 0.82H, 2-NH₂), 6.44 (s, 1H, 2-NH₂), 7.78 (s, 0.56H, H-8), 8.03 (s, 0.44H, H-8), 10.64 (s, 0.53H, 1-NH), 10.89 (s, 0.39H, 1-NH). FAB-MS m/z: 382.2 [M + H]⁺. Calculated for C₁₆H₂₃N₅O₆: C, 50.39; H, 6.08; N, 18.36. Found: C, 50.63; H, 6.31; N, 18.66.

6′-O-Allyl-1′,3′-di-O-propionyl-4′-deoxy-4′-(N²-propionyl-guanin-9-yl)-2′,5′-anhydro-l-mannitol (9) and 6′-O-allyl-1′,3′-di-O-propionyl-4′-deoxy-4′-(N²-propionyl-guanin-7-yl)-2′,5′-anhydro-l-mannitol (10)

A mixture of 7 and 8 (4.77 g, 12.5 mmol) was dissolved in 1% hydrochloric acid in water (120 ml) and refluxed for 4 h. After cooling to 0°C and neutralization with 2 M NaOH, NaBH₄ (1100 mg, 29 mmol) was added. The solution was stirred at room temperature overnight and then neutralized with 2 M HCl at 0°C. After removal of the water, the mixture was dried completely by oil pump.

The mixture was suspended in dry pyridine (80 ml) and propionic anhydride (16 ml, 125 mmol) and DMAP (330 mg, 27 mmol) were added. The mixture was heated with stirring at 70°C for 3 h. Then Na₂CO₃ solution (1 M, 120 ml) was added drop-wise with external cooling until the evolution of CO₂ ceased. The aqueous solution was extracted with CH₂Cl₂. The organic extracts were combined, washed with 5% NaHCO₃, dried over MgSO₄, filtered and evaporated under reduced pressure. The residue was co-evaporated several times with toluene to remove traces of pyridine and applied to a silica gel column eluting with petroleum ether/acetone (3:1–3:2) to give 9 (316 mg), 10 (785 mg) and a mixture of 9 and 10 (2.96 g). The total yield of two steps was 90%.

Compound 9 ¹H NMR (300 MHz, DMSO-d₆): δ 0.88 (t, J = 7.5 Hz, 3H, N²-CH₃), 0.99 (p, 6H, 1′-CH₃, 3′-CH₃), 2.26 (m, 4H, 1′-CH₂, 3′-CH₂), 2.47 (q, J = 7.5 Hz, 2H, N²-CH₂), 3.50 (m, 2H, H-6′), 3.91 (d, J = 3.9 Hz, 2H, -CH₂-O), 4.24 (q, J = 6.0 Hz, 5.4 Hz, 1H, H-2′), 4.34 (d, J = 5.1 Hz, 2H, 1′-CH₂), 4.63 (p, J = 4.8 Hz, 4.2 Hz, 3.6 Hz, 1H, H-5′), 4.96 (t, J = 8.4 Hz, 7.8 Hz, 1H, H-4′), 5.06 (m, 2H, CH₂=), 5.51 (t, J =7.2 Hz, 6.6 Hz, 1H, H-3′), 5.75 (m, 1H, =CH-), 8.19 (s, 1H, H-8), 11.54 (s, 1H, 1-NH), 12.06 (s, 1H, 2-NH). ¹³C NMR (300 MHz, DMSO-d₆): δ 8.54 (CH₃), 8.73 (CH₃), 26.3 (CH₂), 26.6 (CH₂), 29.2 (N²-CH₂), 59.9 (C-1′), 63.6 (C-6′), 68.9 (C-4′), 71.3 (-CH₂-O), 76.8 (C-3′), 77.9 (C-5′), 78.5 (C-2′), 116.4 (CH₂=), 120.2 (C-5), 134.6 (=CH-), 138.3 (C-8), 147.6 (C-4), 148.7 (C-2), 154.7 (C-6), 173.0 (C=O), 173.3 (C=O), 176.8 (N²-C=O). FAB-HRMS m/z: 506.2251 [M + H]⁺. Calculated for C₂₃H₃₂N₅O₈: 506.2251.

Compound 10 ¹H NMR (300 MHz, DMSO-d₆): δ 0.90 (t, J = 7.5 Hz, 3H, N²-CH₃), 0.93 (p, 6H, 1′-CH₃, 3′-CH₃), 2.26 (m, 4H, 1′-CH₂, 3′-CH₂), 2.44 (q, J = 7.5 Hz, 2H, N²-CH₂), 3.47 (t, 2H, H-6′), 3.89 (p, J = 3.0 Hz, 2H, -CH₂-O), 4.22 (m, 1H, H-2′), 4.28–4.45 (m, 2H, 1′-CH₂), 4.55 (p, J = 4.5 Hz, 4.2 Hz, 1H, H-5′), 5.06 (m, 2H, CH₂=), 5.16 (t, J = 8.1 Hz, 8.7 Hz, 1H, H-4′), 5.66 (t, J = 6.9 Hz, 6.6 Hz, 1H, H-3′), 5.75 (m, 1H, =CH-), 8.34 (s, 1H, H-8), 11.59 (s, 1H, 1-NH), 12.21 (s, 1H, 2-NH). ¹³C NMR (300 MHz, DMSO-d₆): δ 8.57 (CH₃), 8.64 (CH₃), 8.76 (N²-CH₃), 26.4 (CH₂), 26.6 (CH₂), 29.2 (N²-CH₂), 62.8 (C-1′), 63.5 (C-6′), 69.0 (C-4′), 71.3 (-CH₂-O), 77.4 (C-3′), 78.8 (C-2′, C-5′), 111.0 (C-5), 116.4 (CH₂=), 134.7 (=CH-), 144.6 (C-8), 147.1 (C-4), 152.5 (C-2), 158.2 (C-6), 173.1 (C=O), 173.4 (C=O), 176.8 (N²-C=O). FAB-MS m/z: 506.1 [M + H]⁺. Calculated for C₂₃H₃₁N₅O₈: C, 54.65; H, 6.18; N, 13.85. Found: C, 54.71; H, 6.22; N, 13.60.

6′-O-Allyl-1′,3′-di-O-propionyl-4′-deoxy-4′-(N²-propionyl-O⁶-N,N′-diphenylcarbamoyl-guanin-9-yl)-2′,5′-anhydro-l-mannitol (11)

The above mixture of 9 and 10 (2.43 g, 4.82 mmol) was dissolved in dry pyridine (23 ml), and diphenylcarbamoyl chloride (2.21 g, 9.55 mmol) and diisopropylethylamine (0.84 ml, 4.77 mmol) were added. The mixture was stirred at room temperature for 3 h and then diluted with EtOH at 0°C. After 10 min, the solvent was removed under reduced pressure and the residue co-evaporated several times with toluene. The residue was submitted to silica gel chromatography, eluting with petroleum ether/EtOAc (1:1) to afford 11 (2.11 g) and then eluted with petroleum ether/acetone (1:1) to recover unreacted 10 (0.866 g).

¹H NMR (500 MHz, DMSO-d₆): δ 0.90 (t, J = 7.5 Hz, 3H, N²-CH₃), 1.01 (t, J = 7.5 Hz, 3H, 1′-CH₃), 1.05 (t, J = 7.5 Hz, 3H, 3′-CH₃), 2.27 (q, J = 7.5Hz, 2H, 1′-CH₂), 2.33 (q, J = 7.5 Hz, 2H, 3′-CH₂), 2.50 (m, 2H, N²-CH₂), 3.53 (t, J = 5.0 Hz, 3.5 Hz, 2H, H-6′), 3.91 (d, J = 5.5 Hz, 2H, -CH₂-O), 4.30 (m, 1H, H-2′), 4.39–4.49 (m, 2H, 1′-CH₂), 4.77 (p, J = 4.0 Hz, 4.5 Hz, 1H, H-5′), 5.04–5.14 (m, 3H, H-4′, CH₂=), 5.69 (t, J = 6.0 Hz, 6.5 Hz, 1H, H-3′), 5.75 (m, 1H, =CH-), 7.31 (t, J = 6.5 Hz, 7.5 Hz, 2H, p-Ph), 7.44–7.48 (m, 8H, Ph), 8.56 (s, 1H, H-8), 10.68 (s, 1H, 2-NH). FAB-MS m/z: 701.2 [M + H]⁺. Calculated for C₃₆H₄₀N₆O₉: C, 61.70; H, 5.75; N, 11.99. Found: C, 62.10; H, 6.15; N, 11.69.

6′-O-Allyl-4′-deoxy-4′-(N²-propionyl-O⁶-N,N′-diphenylcarbamoyl-guanin-9-yl)-2′,5′-anhydro-l-mannitol (12)

To a solution of compound 11 (1.75 g, 2.49 mmol) in EtOH (25 ml) 2 M NaOH (10 ml) was added with cooling. After 10 min, acetic acid was added to neutralize the solution. After removal of solvent, the residue was submitted to silica gel chromatography eluting with CH₂Cl₂/CH₃OH to obtain 12 (1.37 g, 93.2%).

¹H NMR (300 MHz, DMSO-d₆): δ 1.03 (t, J = 7.5 Hz, 3H, N²-CH₃), 2.50 (q, J = 7.5 Hz, 2H, N²-CH₂), 3.48 (d, J = 4.5 Hz, 2H, H-6′), 3.57–3.63 (m, 2H, 1′-CH₂), 3.82 (m, 1H, H-2′), 3.89 (m, 2H, -CH₂-O), 4.51 (p, J = 4.5 Hz, 1H, H-5′), 4.61 (q, J = 5.1 Hz, 1H, H-3′), 4.75 (t, J = 8.4 Hz, 1H, H-4′), 4.82 (t, J = 5.4 Hz, 6.0 Hz, 1H, 1′-OH), 5.01 (m, 2H, CH₂=), 5.73 (m, 1H, =CH-), 5.74 (d, J = 5.4 Hz, 1H, 3′-OH), 7.29 (t, 2H, p-Ph), 7.41 (m, 8H, Ph), 8.56 (s, 1H, H-8), 10.67 (s, 1H, 2-NH). FAB-HRMS m/z: 589.2411 [M + H]⁺. Calculated for C₃₀H₃₃N₆O₇: 589.2411.

6′-O-Allyl-1′-O-DMT-4′-deoxy-4′-(N²-propionyl-O⁶-N,N′-diphenylcarbamoyl-guanin-9-yl)-2′,5′-anhydro-l-mannitol (13)

The title compound 13 (451 mg, 76.8%) was prepared from compound 12 (461 mg, 0.785 mmol) in the same way that compound 5 was obtained from compound 4.

¹H NMR (500 MHz, DMSO-d₆): δ 0.96 (t, J = 7.5 Hz, 3H, N²-CH₃), 2.50 (q, J = 7.5 Hz, 2H, N²-CH₂), 3.18–3.24 (m, 2H, 1′-CH₂), 3.57 (d, J = 3.2 Hz, 2H, H-6′), 3.73 (s, 6H, 1′-OCH₃), 3.97 (d, J = 4.5 Hz, 2H, -CH₂-O), 4.06 (t, 1H, H-2′), 4.56 (m, 2H, H-5′, H-3′), 4.79 (t, J = 8.5 Hz, 1H, H-4′), 5.06 (m, 2H, CH₂=), 5.77 (d, J = 5.2 Hz, 1H, 3′-OH), 5.79 (m, 1H, =CH-), 6.88 (d, 4H, ph), 7.22 (t, 1H, ph), 7.29 (m, 8H, ph), 7.43 (m, 9H, ph), 8.58 (s, 1H, H-8), 10.6 (s, 1H, 2-NH). FAB-MS m/z: 891 [M + H]⁺. Calculated for C₅₁H₅₁N₆O₉: 891.37.

Triethylammonium salt of 6′-O-allyl-1′-O-DMT-4′-deoxy-4′-(N²-propionyl-O⁶-N,N′-diphenylcarbamoyl-guanin-9-yl)-2′,5′-anhydro-l-mannitol-3′-(2-chlorophenyl) phosphate (14)

Compound 14 was obtained from 13 as a colorless solid and in 98% yield by using the methodology for the preparation of 6 from 5.

¹H NMR (300 MHz, DMSO-d₆): δ 0.93 (t, J = 7.5 Hz, 3H, N²-CH₃), 1.01 (t, J = 7.2 Hz, 9H, 3CH₃), 2.44 (q, J = 7.5 Hz, 2H, N²-CH₂), 2.86 (q, J = 7.5 Hz, 6H, 3CH₂), 3.43–3.54 (m, 2H, 1′-CH₂), 3.69 (s, 6H, 1′-OCH₃), 3.71 (d, J = 3.0 Hz, 2H, H-6′), 3.93 (q, 2H, -CH₂-O), 4.25 (q, 1H, H-2′), 4.48 (p, 1H, H-5′), 4.94 (t, J = 7.5 Hz, 1H, H-4′), 5.02 (m, 3H, CH₂=, H-3′), 5.75 (m, 1H, =CH-), 6.65–7.48 (m, 27H, Ph), 8.01 (s, 1H, NH), 8.44 (s, 1H, H-8), 10.46 (s, 1H, 2-NH). ³¹P NMR (200 MHz, DMSO-d₆): δ –6.35. FAB-MS m/z: 1080.2 [M – Et₃N⁺H + H]. Calculated for C₅₇H₅₃ClN₆O₁₂P: 1079.31.

Synthesis of 1′-DMT-(isoT)₂-OH-3′ (15)

A solution of compound 6 (605 mg, 0.669 mmol), compound 4 (197 mg, 0.63 mmol) and 1-methylimidazole (0.51 ml, 6.33 mmol) in dry pyridine (5 ml) was concentrated to small volume under reduced pressure (bath temperature <25°C). This process was repeated and the residue was dissolved in dry pyridine (8 ml). 2,4,6-Triisopropylbenzenesulfonyl chloride (608 mg, 2.0 mmol) was added. The reaction mixture was stirred at room temperature for 12 h, then quenched with saturated aqueous NaHCO₃. After removal of solvent, the residue was co-evaporated with toluene and dissolved in CH₂Cl₂, filtered and the resulting solution extracted with 5% aqueous NaHCO₃. The organic layer was dried with NaSO₄. After filtration and evaporation, the residue was submitted to silica gel chromatography eluting with CH₂Cl₂/CH₃OH (70:1) (1% Et₃N added), to give the fully protected dinucleotide 15 (606 mg, 75%).

¹H NMR (300 MHz, DMSO-d₆): δ 1.59 (d, 3H, 5-CH₃), 1.75 (s, 3H, 5-CH₃), 4.68 (t, 1H, H-4′), 4.83 (t, 1H, H-4′), 5.06–5.34 (m, 6H, 2CH₂=, H-3′), 5.71 (d, 1H, 3′-OH), 5.76 (m, 2H, =CH-), 6.81–7.54 (19H, H-6 and Ph), 11.31 (s, 2H, 3-NH). ³¹P NMR (200 MHz, DMSO-d₆): δ 10.28.

Synthesis of 1′-DMT-(isoG)₂-OH-3′ (16)

The title compound 16 (254 mg, 47%) was prepared by the method described above from the coupling of compound 14 (360 mg, 0.305 mmol) with compound 12 (180 mg, 0.305 mmol) except that NMI was replaced by 1H-tetrazole (70 mg, 1 mmol).

¹H NMR (300 MHz, DMSO-d₆): δ 0.91 (m, 6H, N²-CH₃), 2.39 (m,4H, N²-CH₂), 3.43–3.54 (m, 2H, 1′-CH₂), 3.66 (d, 6H, 1′-OCH₃), 3.83 (m, 4H, -CH₂-O), 4.16 (m, 1H, H-2′), 4.97 (m, 4H, CH₂=), 5.68 (m, 2H, =CH-), 5.93 (d, 1H, 3′-OH), 6.77–7.55 (m, 27H, Ph), 8.49 (s,2H, H-8), 10.53 (q, 2H, 2-NH). ³¹P NMR (200 MHz, DMSO-d₆): δ 9.89.

General procedure for removal of the DMT group from fully protected isonucleotides

A fully protected oligoisonucleotide was dissolved in 3% TCA solution in CH₂Cl₂. After 10 min, saturated aqueous NaHCO₃ was added to neutralize the solution. The resulting solution was extracted with saturated aqueous NaHCO₃. The organic layer was dried over Na₂SO₄, filtered and concentrated. A solution of the residue in CH₂Cl₂ (5 ml) was added drop-wise to stirred light petroleum ether (bp 30–60°C, 250 ml) to give the corresponding hydroxyl component.

General procedure for preparation of triethylammonium salts of isonucleotide phosphate

The general procedure for phosphorylation of di-isonucleotides and tetra-isonucleotides was the same as the process for preparation of isonucleoside 3′-phosphates except that the phosphorylating agent 2-chlorophenyl phosphorodichloridate was used in greater excess.

Synthesis of [1′-DMT-(isoT)₂(isoG)₄(isoT)₂-OH-3′] (26)

The fully protected octaoligoisonucleotide 26 (49 mg, crude yield 53%) was prepared using standard conditions to condense compound 24 (54 mg, 0.019 mmol) with compound 25 (38 mg, 0.016 mmol).

Synthesis of [1′-DMT-(isoT)₈-OH-3′] (29)

The fully protected octaisothymine 29 (96 mg, crude yield 68%) was obtained using standard conditions to couple compound 28 (81 mg, 0.034 mmol) with compound 27 (60 mg, 0.034 mmol).

Deprotection of the fully protected octamer 26 and purification of oligomer I

Crude compound 26 (49 mg) was dissolved in a 0.3 M solution of N¹,N¹,N³,N³-tetramethylguanidium salt of 2-pyridine-syn-carboxaldoximate in dioxane/water (7:1 v/v), and the solution was kept at room temperature for 24 h. After removal of solvent, the residue was dissolved in aqueous ammonia (29%) and the reaction mixture allowed to stand at 60°C for 24 h. After cooling, the aqueous solution was evaporated. The residue was treated with 2 M HCl for 15 min to remove the 1′-DMT group. After neutralization with 2 M NaOH and removal of the solvent, the residue was dissolved in water and extracted successively with CH₂Cl₂ and ether. The resulting aqueous solution was evaporated under reduced pressure.

The final crude product was chromatographed on DEAE–Sephadex A-25 with a linear gradient of 0.001–1.0 M triethylammonium bicarbonate buffer (pH 7.0). The appropriate fractions were mixed and concentrated. The residue was co-evaporated several times with water to remove buffer. It was then dissolved in the desired chromatographic buffer and purified by reverse phase HPLC. Buffer A, 0.1 M triethylammonium acetate (TEAA), pH 7.0; buffer B, 20% 0.1 M TEAA and 80% acetonitrile; gradient 0–50% B over 15 min, flow rate 3 ml/min. The appropriate peaks were mixed and desalted by HPLC with water and CH₃CN to give pure product (20 OD A₂₆₀ units). The pure oligomer I was lyophilized and stored at –20°C. Allyl groups were still retained in oligomer I.

¹H NMR (600 MHz, D₂O): δ 7.45, 7.47, 7.56 [3s, 4T (H-6)], 7.83, 7.87, 7.94, 7.97 [4s, 4G (H-8)]. ³¹P NMR (600 MHz, D₂O): δ 0.386, –0.477, –0.677, –1.170. ESI-TOF MS m/z: 2911.48 (M – 3 × allyl, observed), 2910.68 (M – 3 × allyl, calculated).

Deprotection of the fully protected octamer 29 and purification of oligomer II

Oligomer II was obtained after deprotection and purification of 29 following a similar deblocking process except that aqueous NH₃ was not used.

¹H NMR (500 MHz, D₂O): δ 7.43 (s, 1H, H-6). ³¹P NMR (600 MHz, D₂O): δ –0.453. MALDI-TOF MS m/z: 3621.167 (observed), 3638.591 (calculated).

Deprotection of compound 15 and purification of compound 30

Compound 15 (50 mg) was dissolved in methanol and palladium (II) chloride was added. The reaction mixture was stirred at 60°C for 3 h, filtered and evaporated. The resulting residue was then subjected to the general conditions for removal of 2-chlorophenyl groups to give compound 30.

¹H NMR (600 MHz, D₂O): δ 1.25 (t, 9H, CH₃), 1.88 (d, 6H, 5-CH₃), 3.16 (q, 6H, CH₂), 3.60 (m, 2H, H-6′), 3.70 (m, 2H, H-6′), 3.82 (m, 2H, H-2′), 3.89 (m, 3H, 1′-CH₂, H-5′), 4.15 (m, 1H, 1′-CH₂), 4.28 (p, 1H, 1′-CH₂), 4.49 (t, 1H, H-4′), 4.89 (m, 2H, H-3′, H-4′), 4.99 (q, 1H, H-3′), 7.55 (d, 2H, H-6). ³¹P NMR (600 MHz, D₂O): δ 0.507, –0.490. FAB-HRMS m/z: 607.1626 [M + H]⁺. Calculated for C₂₂H₃₂N₄O₁₄P: 607.1653.

CD spectra

CD spectra were measured on a Jasco J-715 spectropolarimeter in a 0.2 cm path length cell. The samples were dissolved in a buffer solution containing 0.1 M KCl, pH 7.0. The solution containing each sample was heated at 80°C for 5 min, then cooled gradually to 4°C and used for the CD experiment. Spectra were recorded at a strand concentration of 20 µM at 25°C. All CD data represent an average of four scans, baseline corrected for signals due to the buffer and cell.

Capillary electrophoresis

Capillary electrophoresis experiments were carried out at 20°C on a Beckman P/ACE MDQ capillary electrophoresis system operated in the reverse polarity mode, with detection by UV absorbance at 254 nm. A fused silica capillary 37 cm long with a 100 µm inner diameter and a surface coating 0.1 µm thick (DB-17; J&W Scientific, Rancho Cordova, CA) was used. Before each experiment the capillary was rinsed with several column volumes of H₂O. Electrophoresis was performed in the presence of a neutral polymer sieving agent, methylcellulose (MC) (4000 cP), dissolved in the electrophoresis buffer (1%). All electrophoresis experiments were performed in buffer containing 25 mM Tris–borate (pH 8.0), 1% MC and the appropriate concentration of KCl, NaCl and LiCl; sample concentrations were 20 µM in strand. The oligodeoxynucleotide dT₈ was used in all experiments as an internal marker. Buffer containing polymer MC was injected under high pressure (85.0 p.s.i.) for 20 min followed by a low pressure (2.0 p.s.i.) injection of the sample solution for 60 s. Electrophoresis was then carried out at constant voltage (7 kV). All experiments were repeated twice.

RESULTS AND DISCUSSION

Synthesis of isonucleoside units 4 and 12

Isonucleosides were obtained by the reaction of epoxide 2 with nucleobases. Epoxide 1 was synthesized from d-glucose via six steps as described (24). In our previous work, the 6′-OH on the sugar moiety of oligomer I was protected by a benzoyl group after the isonucleoside was constructed. But for the synthesis of an oligomer containing this isonucleoside on an automated DNA synthesizer, it was found that steric hindrance caused by the benzoyl group resulted in lower coupling yields. We therefore changed the synthetic strategy using a smaller allyl group to protect the 6′-OH of 1. Because of its ready availability and stability under reasonably strong acidic and basic conditions, the allyl group has been widely used for protection of hydroxyl groups in carbohydrate chemistry (25) and for protection of the phosphate ester bond, nucleobase carbonyl group or hydroxyl group (26,27) in the synthesis of oligonucleotides. In our laboratory, we found that allyl groups can be used to protect the hydroxyl group of the sugar moiety in a nucleoside and can be removed with PdCl₂ in methanol in high yield (28). These properties make the allyl group a promising group to block the 6′-OH of 1.

Using NaH as a base, epoxide 1 reacted with allyl bromide in dry DMF to give 2 in 93.4% yield (Fig. 2). Compound 2 reacted with thymine in dry DMF and a regioselective epoxide opening took place in the presence of DBU to give compound 3. Compound 3 was hydrolyzed by 1% HCl to remove the dimethylacetal, followed by reduction with NaBH₄ to give the desired isonucleoside unit 4 in 67% yield. Then the 1′-OH of 4 was protected with a DMT group to obtain compound 5.

Figure 2.

Synthesis of isonucleoside unit 4 and its phosphodiester 6. Reagents and conditions: (i) allyl bromide, NaH, DMF, room temperature; (ii) thymine, DBU/DMF, 95°C; (iii) 1% HCl, reflux, 2 M NaOH, NaBH₄, room temperature; (iv) DMTrCl, Py, room temperature; (v) 2-ClC₆H₄OPOCl₂, 1H-1,2,4-triazole, NMI, Et₃N, THF.

Compound 2 reacted with guanine in anhydrous DMSO in the presence of K₂CO₃ and 18-crown-6 to produce mixtures of N⁹-isoguanosine 7 and N⁷-isomer 8 in reasonable yield (Fig. 3). ¹H NMR analysis of mixtures indicated that the N⁷:N⁹ isomer ratio was 4:5. It was difficult to separate compounds 7 and 8 because of their similar chromatographic mobilities. We also failed to separate the N⁷/N⁹ isoguanosine mixtures after hydrolytic and reductive reaction of mixtures of compounds 7 and 8 because of their high polarity. Fortunately, when the O⁶-amido and N²-amino groups of guanosine were masked with the diphenylcarbamoyl and propionyl groups, respectively, the N⁷/N⁹ isomeric mixtures can be easily separated. Therefore, after hydrolytic and reductive reactions, the whole mixtures were dried completely and reacted with 15 equivalent of propionic anhydride in anhydrous pyridine to afford N⁷/N⁹ isomers 9 and 10 (29). Then the mixture of 9 and 10 was reacted with 2 equivalent of diphenylcarbamoyl chloride in dry pyridine to protect the O⁶-amido group of the guanine heterocycle. It was found that only compound 9 reacted with this reagent to afford compound 11, whereas compound 10 did not react with it. Therefore, through these two protective steps, the N⁷/N⁹ isomers were isolated completely. In 9 the O⁶-amido group is distant from the sugar, but in 10 the O⁶-amido group is proximate to the sugar. Thus, the large steric hindrance of the O⁶-amido group in the N⁷ isomer resulted in the failure of N⁷ isomer 10 to react with this reagent. The structures of 9 and 10 were identified by ¹H NMR spectra (30). Treatment of compound 11 with 2 M NaOH in ethanol to hydrolyze the two propionyl groups gave compound 12. Protection of 12 with a DMT group afforded compound 13.

Figure 3.

Synthesis of isonucleoside unit 12 and its phosphodiester 14. Reagents and conditions: (i) K₂CO₃/18-Crown-6/DMF, 100°C; (ii) 1% HCl, reflux, 2 M NaOH, NaBH₄, room temperature; (iii) propionic anhydride, Py, DMAP, 70°C; (iv) diphenylcarbamoyl chloride, DIPEA, Py, room temperature; (v) 2 M NaOH, C₂H₅OH, ice bath; (vi) DMTrCl, Py, room temperature; (vii) 2-ClC₆H₄OPOCl₂, 1H-1,2,4-triazole, NMI, Et₃N, THF.

Synthesis of building blocks 6 and 14

Treatment of compounds 5 and 13 with an excess of 2-chlorophenyl dichlorophosphate in the presence of 1,2,4-triazole and NMI gave the required 3′-phosphodiester building blocks 6 and 14 in high yields (31). Their structures were supported by ¹H NMR, ³¹P NMR and FAB-MS.

Solution phase synthesis of oligomers I and II

Condensation reactions between the isonucleoside 3′-phosphodiesters 6 or 14 and free 1′-hydoxyl isonucleosides 4 or 12 were carried out using the solution phase phosphotriester approach to afford fully protected dimers (isoT)₂ and (isoG)₂ (31). The yields were 75 and 47%, respectively. The lower coupling yields were probably due to the steric hindrance of the 3′-phosphate ester group caused by the profound changes in conformation of the sugar in such isonucleosides compared with regular nucleosides.

By fragment condensations, three tetramers (isoT)₂(isoG)₂, (isoG)₂(isoT)₂ and (isoT)₄ were synthesized likewise in yields of 56.7, 48.6 and 45%, respectively (Fig. 4). Then repetition of the same procedures gave the fully protected octamer (isoT)₂(isoG)₄(isoT)₂ (26) and (isoT)₈ (29) in yields of 53 and 68%, respectively. During these condensations, we did not observe by- products resulting from 3′–3′ coupling or the sulfonation of 1′-OH with condensing agents.

Figure 4.

Synthesis of oligomers I and II. Reagents and conditions: (i) TPSCl, 1H-tetrazole, Py, room temperature; (ii) 3% TCA (in CH₂Cl₂); (iii) 2-ClC₆H₄OPOCl₂, 1H-1,2,4-triazole, NMI, Et₃N, THF; (iv) N¹,N¹,N³,N³-tetramethylguanidinium salts of syn-pyridine-2-carboxaldoxime/dioxane-H₂O, concentrated aqueous NH₃, 2 M HCl; (v) PdCl₂/CH₃OH, 60°C.

After the usual deprotecting procedure (32), purification of oligomers I and II with Sephadex A-25 and by RP-HPLC, pure lyophilized products were isolated from two crude samples. The composition of oligomer I was confirmed by 600 MHz ¹H NMR and ³¹P NMR. Four hydrogen peaks in the region from 7.97 to 7.83 p.p.m. were assigned to the signals of four H-8 of the four guanine moieties and four hydrogen peaks from 7.56 to 7.45 p.p.m. were assigned to the signals of four H-6 of the four thymine moieties. Four phosphorus signals in the ³¹P NMR spectra were also consistent with the symmetric base sequence of oligomer I. The composition of oligomers I and II were characterized by mass spectroscopy.

The whole procedure of deprotection of 15 was simplified as a two-step one-pot reaction: treatment of 15 with palladium (II) chloride in methanol at 60°C for 3 h removed both the O-allyl protecting groups and the DMT group and then cleavage of the 2-chlorophenyl protectors gave compound 30. No cleavage of internucleotide linkages was observed during these operations. These results suggest that the allyl group is an effective protecting group for the sugar moiety in the synthesis of oligonucleotides because it can be introduced in high yield and removed under neutral and mild conditions in good yield. In order to promote the permeability and hybridization in further biological studies, we retained the allyl groups in oligomers I and II.

CD spectra

Figure 5A displays the CD spectra of oligomer II and the normal octamer (dT)₈. The regular oligomer (dT)₈ showed a positive signal at 277 nm and the modified oligomer II exhibited opposite Cotton effects with a negative signal at 280 nm. Moreover, the ellipticity value of oligomer II was markedly increased. This result is in agreement with the conclusion that the configuration of this type of isonucleoside sugar is ‘l-’, in accord with molecular modeling. Furthermore, we supposed that the hydrophobic interaction of allyl groups might increase the base stacking of oligomer II and result in the observed strong Cotton effects.

Figure 5.

(A) CD spectra of d(T)₈ and (isoT)₈ at 25°C (C_o = 20 µM, in 0.14 M NaCl, 0.01 M Na₂HPO₄ and 1.0 mM EDTA, pH 7.2). Red squares, d(T)₈; blue triangles, (isoT)₈. (B) CD spectra of d(T₂G₄T₂) and (isoT)₂(isoG)₄(isoT)₂ tetramers at 25°C (C_o = 20 µM, in 0.1 M KCl, pH 7.0). Red squares, d(T₂G₄T₂) tetramers; blue triangles, (isoT)₂(isoG)₄(isoT)₂ tetramers.

Figure 5B shows the CD spectra of oligomer I and normal octamer T₂G₄T₂. The CD spectrum of the regular octamer displayed a typical spectrum of parallel tetra-stranded G-quartet structures reported by others (33,34), with a strongly positive band at 260 nm and a weakly negative band at 240 nm. The shape of the spectrum of oligomer I was very similar to that of the control octamer. However, the intensity of the positive and negative peaks in the spectrum of oligomer I was much decreased compared with those for the control octamer. These results indicate that oligomer I can form the parallel tetramer G-quartet structure in the presence of K⁺ cation. However, formation of cyclic reverse Hoogsteen hydrogen bonding arising from planar association of four guanines might be weak owing to the torsion of the backbone in such an oligomer, and this leads to reduced association of four oligomer Is.

Capillary electrophoresis

G-quadruplex DNAs migrate faster on non-denaturing polyacrylamide gels than do single and duplex DNAs containing the same number of nucleotides, and gel mobility results have often been used as evidence for the presence of G-quadruplex DNA (23,35). A preliminary capillary electrophoresis experiment on d(G₃T₄G₃) in the presence of KCl revealed that the quadruplex structure migrated as a single species and more rapidly than the unstructured single strand, suggesting a very compact nature of the G-quartet structure (36).

We used capillary electrophoresis to study the electrophoretic mobility of oligomer I in order to gain further evidence for the presence of the G-quartet structure. Figure 6 shows an electrophoretogram for oligomer I in the presence of 100 mM KCl, 100 mM NaCl and 100 mM LiCl or in the absence of monovalent metal cations. Since the migration time for all species changed when the salts were added, marker III, (dT)₈, was used in these experiments. It can be seen from Figure 6A and C that, in the absence of cations, oligomer I migrated faster than (dT)₈ and oligomer II migrated a little slower than (dT)₈. After addition of 100 mM KCl (Fig. 6B and D) the migration time of oligomer I was much greater than that of (dT)₈. Oligomer II still migrated slower than (dT)₈, although the separation time between them became longer, and it appeared a little faster than oligomer I. In the presence of 100 mM NaCl and 100 mM LiCl, the interval time between (dT)₈ and oligomer I became even longer (Fig. 6E and F).

Figure 6.

Capillary electrophoresis traces of oligomer I, internal marker (III) and oligomer II at 20°C. (A) Oligomer I and internal marker (III) in the absence of monovalent metal cations. (B, E and F) Oligomer I and internal marker (III) in the presence of 0.1 M KCl, 0.1 M NaCl and 0.1 M LiCl, respectively. (C) Oligomer I, internal marker (III) and oligomer II in the absence of monovalent metal cations. (D) Oligomer I, internal marker (III) and oligomer II in the presence of 0.1 M KCl.

The electrophoresis was carried out in a capillary coated on the inside to eliminate electro-osmotic flow and filled with a neutral polymer sieving agent. DNA fragments can be separated by using linear polymers, such as methylcellulose, hydroxypropylmethylcellulose or polyethylene glycol, as additives in the HPCE buffer (37,38). The electrophoretograms presented in Figure 6 indicate that the apparent size of oligomer I became bigger in the presence of K⁺, Na⁺ and Li⁺ cations and clearly showed the formation of a new polymeric species. Combined with the results of the CD experiments, it was concluded that oligomer I formed G-quadruplex structures in the presence of KCl, NaCl and LiCl. In normal parallel tetramer G-quartet structures, the cyclic reverse Hoogsteen hydrogen bonding is strong, therefore, G-quadruplex structures being comprised of stacked tetrads are more compact than single strands. However, in our case, in the parallel G-quartet structure formed by four oligomer I monomers, the cyclic reverse Hoogsteen hydrogen bonding might be weak because of the profound changes in conformation of the sugars in such an oligomer, which causes loose G-tetrads and poor association of the four monomer I units. Monovalent cations such as K⁺, Na⁺ and Li⁺ can prompt the formation of G-quadruplex structure and stabilize it. According to the electrophoretograms in Figure 6B, E and F, the K⁺ form structure might be more compact than either the Na⁺ form or the Li⁺ form structures, while the Li⁺ form structure was the weakest of the three. These results suggest that the order of these cations as structure makers is K⁺ > Na⁺ > Li⁺.

CONCLUSION

In summary, two isonucleosides 4 and 12 have been synthesized. Using a combination of propionyl and DPC groups to protect the N²-amino and O⁶-amido groups of guanine moieties, we succeeded in isolating the N⁹ and N⁷ isomers of isoguanosine. Using the phosphotriester approach in solution phase, we synthesized specific G-rich sequence oligomers I and II built from isonucleoside units 4 and 12. The results of CD spectra and capillary electrophoresis experiments suggested that oligomer I can form a parallel intermolecular G-quadruplex structure in the presence of monovalent cations. K⁺, Na⁺ and Li⁺ promote formation of the G-quartet structure. The order of the cation effect is K⁺ > Na⁺ > Li⁺. Since G-quadruplexes have proved useful inhibitors of some proteins, this strongly nuclease-resistant analog of the guanosine tetrad, oligomer I, may have enhanced efficacy in vivo.