Nucleic Acids Research

General procedures

¹H NMR spectra were measured at 360 MHz with a Bruker AM 360 spectrometer; tetramethylsilane was used as an internal standard. ³¹P NMR spectra were measured at 145.8 MHz with the same spectrometer; 85% orthophosphoric acid was used as an external standard. Merck silica gel 60 F₂₅₄ pre-coated plates (Art 5715 and 5642), which were developed in solvent system A (CH₂Cl₂/MeOH 9:1 v/v), were used for TLC. High performance liquid chromatography (HPLC) was carried out on a 250 × 4.6 mm Hypersil ODS 5µ column, which was eluted with 0.1 mol/dm³ triethylammonium acetate buffer (pH 7.0)/acetonitrile mixtures [programme A: linear gradient of buffer/acetonitrile (95:5-40:60 v/v) over 15 min and then isocratic; programme B: linear gradient of buffer/acetonitrile (97:3-85:15 v/v)]. Peaks were monitored and integrated at 270 nm. Merck Kieselgel H (Art 7729 and 9385) was used for short column chromatography. The general procedure for the chromatography of fully or partially protected oligoribonucleotide phosphorothioates (below under the preparation of DMTr-A[prime]p(s[prime])C[prime]p(s[prime])G[prime]p(s[prime])U[prime]-Lev 23 and the preparation of the fully protected cyclic tetraribonucleoside tetraphosphorothioate 25) was as follows. A suspension of silica gel (~10 g/g of crude product) in dichloromethane (~50 ml) was poured into a column and the silica gel was packed. The diameter of the column was such that the height of the silica gel was ~1-2 cm. A solution of the crude product in dichloromethane (~10 ml) was applied to the column, which was eluted first with ethyl acetate/petroleum ether (b.p. 40-60°C) (50:50-100:0 v/v) in order to remove non-polar impurities. The desired product was eluted with ethyl acetate/acetone (100:0-50:50 v/v). A higher proportion of acetone was required to elute more polar products. Ion exchange chromatography was carried out on a column of DEAE-Sephadex A25, which was eluted with triethylammonium bicarbonate buffer (pH 7.5, linear gradient from 0.01 to 1.00 mol/dm³ over 1000 ml). Triethylamine, pyridine, 1-methylpyrrolidine and 4-methylmorpholine were dried by heating with calcium hydride, under reflux, and were then distilled; TMG was dried by distillation over calcium hydride under reduced pressure; dichloromethane was dried over phosphorus pentaoxide and was then distilled; diethyl ether was dried over sodium wire; toluene was dried by distillation at atmospheric pressure and then discarding the first 20% of distillate. All solvents were stored over 4 Å molecular sieves in sealed containers. Levulinic anhydride was prepared by a previously reported procedure (18). Protected {i.e. N-acyl-5[prime]-O-(4,4[prime]-dimethoxytrityl)-2[prime]-O-[1-(2-fluorophenyl)-4-methoxypiperidin-4-yl]- and N-acyl-3[prime],5[prime]-O-(1,1,3,3-tetraisopropyldi-siloxan-1,3-diyl)-} ribonucleoside derivatives were supplied byCruachem Ltd (Glasgow). Phosphorolytic enzymes and diribo-nucleoside phosphates [r(ApC) and r(GpU)] were purchased from Sigma-Aldrich Co. Ltd. Stock solutions of enzymes were prepared as previously reported (8).

2[prime]-O-[1-(2-fluorophenyl)-4-methoxypiperidin-4-yl]-3[prime]-O-levulinoyluridine (HO-U[prime]-Lev) 20

A solution of 5[prime]-O-(4,4[prime]-dimethoxytrityl)-2[prime]-O-[1-(2-fluoro-phenyl)-4-methoxypiperidin-4-yl]uridine 9; B = 15 (3.77 g, 5.0 mmol), triethylamine (2.78 ml, 20 mmol) and levulinic anhydride (2.14 g, ~10 mmol) was stirred at room temperature. After 1 h, the products were poured into saturated aqueous sodium hydrogen carbonate (50 ml) and the resulting mixture was extracted with dichloromethane (50 ml). The organic extract was dried (MgSO₄) and evaporated under reduced pressure. Redistilled pyrrole (3.47 ml, 50 mmol) and trifluoroacetic acid/dichloromethane (2:98 v/v, 100 ml) were added, with stirring, to the residue at room temperature. After 1 min, 4-methylmorpholine (3.30 ml, 30 mmol) was added with continued stirring. The resulting solution was washed with saturated aqueous sodium hydrogen carbonate (2 × 50 ml), dried (MgSO₄) and evaporated under reduced pressure. The residue was fractionated by short column chromatography on silica gel: the appropriate fractions, which were eluted with CH₂Cl₂/MeOH (99:1-98:2 v/v), were combined and evaporated under reduced pressure to give the title compound 20 as a colourless solid (2.25 g, 82%); R_f 0.60 (system A); [delta]_H [(CD₃)₂SO] 1.63 (1 H, m), 1.85 (3 H, m), 2.12 (3 H, s) 2.59 (2 H, m), 2.75 (3 H, m), 2.88 (1 H, m), 2.96 (3 H, s), 3.00(2 H, m), 3.63 (2 H, m), 4.05 (1 H, s), 4.59 (1 H, dd, J 5.2 and 8.2), 5.21 (1 H, d, J 5.0), 5.50 (1 H, t, J 4.9), 5.83 (1 H, d, J 8.1), 6.07 (1 H, d, J 8.3), 6.96 (2 H, m), 7.11 (2 H, m), 7.97 (1 H, d, J 8.2) and 11.50 (1 H, br.s).

6-O-(2,5-dichlorophenyl)-5[prime]-O-(4,4[prime]-dimethoxytrityl)-2[prime]-O-[1-(2-fluorophenyl)-4-methoxypiperidin-4-yl]-2-N-phenyl-acetylguanosine 9; B = 14

2-N-phenylacetyl-3[prime],5[prime]-O-(1,1,3,3-tetraisopropyldisiloxan-1,3-diyl)guanosine 17 (8) (3.22 g, 5.0 mmol) and 1-(2-fluoro-phenyl)-4-methoxy-1,2,5,6-tetrahydropyridine 16 (19) (2.07 g, 10 mmol) were evaporated together with dry toluene (25 ml) under reduced pressure and the residue was redissolved in dichloromethane (25 ml). Trifluoroacetic acid (1.16 ml, 15 mmol) was added and the solution was stirred at room temperature for 5 h. 4-Methylmorpholine (2.20 ml, 20 mmol) was then added and the products were poured into saturated aqueous sodium hydrogen carbonate (50 ml). The layers were separated and the aqueous layer was extracted with dichloromethane (50 ml). The combined organic layers were dried (MgSO₄) and evaporated under reduced pressure. After it had been co-evaporated with pyridine (25 ml) under reduced pressure, the residue was redissolved in dry pyridine (25 ml). 1-Methylpyrrolidine (7) (2.60 ml, 25 mmol) and mesitylene-2-sulphonyl chloride (2.19 g, 10 mmol) were then added to the cooled (ice-water bath), stirred solution. After 10 min, 2,5-dichlorophenol (2.45 g, 15 mmol) was added and the cooled reactants were stirred for a further period of 3 h. The products were then evaporated under reduced pressure and the residue was partitioned between dichloromethane (50 ml) and saturated aqueous sodium hydrogen carbonate (50 ml). The layers were separated and the aqueous layer was back-extracted with dichloromethane (2 × 25 ml). The combined organic layers were dried (MgSO₄) and then evaporated. The residue was redissolved in a 1.0 mol/dm³ solution of tetraethylammonium fluoride in acetonitrile (35 ml). After 45 min, the products were evaporated under reduced pressure and the residue was partitioned between dichloromethane (50 ml) and saturated aqueous sodium hydrogen carbonate (50 ml). The layers were separated and the aqueous layer was back-extracted with dichloromethane (2 × 25 ml). The residue was fractionated by short column chromatography on silica gel: the appropriate fractions, which were eluted with dichloromethane/methanol (99:1-98:2 v/v), were combined and evaporated to give a colourless solid (3.05 g); R_f (system A) 0.63. A solution of this material (3.02 g) in pyridine (10 ml) was evaporated under reduced pressure and the residue was redissolved in dry pyridine (20 ml) at room temperature. 4,4[prime]-Dimethoxytrityl chloride (1.63 g, 4.8 mmol) was added to the stirred solution. After 5 h, the products were poured into saturated aqueous sodium hydrogen carbonate (50 ml) and the resulting mixture was extracted with dichloromethane (2 × 50 ml). The combined organic layers were dried (MgSO₄) and evaporated under reduced pressure. The residue was fractionated by short column chromatography on silica gel: the appropriate fractions, which were eluted with petroleum ether (b.p. 40-60°C)/ethyl acetate (4:1-1:1 v/v), were combined and evaporated under reduced pressure to give the title compound 9; B = 14 as a colourless solid (4.02 g, 76% overall yield); R_f 0.75 (system A); [delta]_H [(CD₃)₂SO] 1.76 (2 H, m), 1.92 (2 H, m), 2.64 (1 H, m), 2.78 (3 H, s), 2.83(2 H, m), 3.07 (1 H, m), 3.17 (1 H, dd, J 3.0 and 10.3), 3.52(1 H, dd, J 6.6 and 10.2), 3.65 (2 H, d, J 7.5), 3.73 (6 H, s), 4.11 (1 H, m), 4.24 (1 H, m), 5.15 (1 H, m), 5.32 (1 H, d, J 5.3), 6.15 (1 H, d, J 6.6), 6.81 (4 H, dd, J 9.0 and 10.0), 6.93 (2 H, m), 7.0-7.3 (14 H, m), 7.37 (2 H, m), 7.43 (1 H, dd, J 2.4 and 8.7), 7.67 (1 H, d, J 8.7), 7.77 (1 H, d, J 2.5), 8.56 (1 H, s) and 10.58 ( 1 H, br. s).

Triethylammonium salt of 5[prime]-O-(4,4[prime]-dimethoxytrityl)-2[prime]-O-[1-(2-fluorophenyl)-4-methoxypiperidin-4-yl]-6-N-pivaloyl-adenosine-3[prime]-H-phosphonate [DMTr-A[prime]p(H)] 10; B = 12

A solution of ammonium p-tolyl H-phosphonate 11a (5) (2.84 g, 15 mmol) and triethylamine (4.18 ml, 30 mmol) in methanol (15 ml) was evaporated under reduced pressure. 5[prime]-O-(4,4[prime]-di-methoxytrityl)-2[prime]-O-[1-(2-fluorophenyl)-4-methoxypiperidin-4-yl]-6-N-pivaloyladenosine 9; B = 12 (4.30 g, 5.0 mmol) was added and the mixture was dissolved in dry pyridine (20 ml). The solution was evaporated under reduced pressure and the residue was redissolved in dry pyridine (30 ml). Pivaloyl chloride (2.46 ml, 20 mmol) was added dropwise over 1 min to the cooled [industrial methylated spirits (IMS)-dry ice bath, -30°C], stirred solution. After 30 min, water (5 ml) was added and the stirred mixture was allowed to warm up to room temperature. After a further period of 1 h, the products were partitioned between dichloromethane (100 ml) and water (100 ml). The organic layer was separated, washed with 0.5 mol/dm³ triethylammonium phosphate buffer (pH 7.0, 3 × 25 ml) and then applied to a short column of silica gel (40 g). The column was eluted with dichloromethane/methanol (95:5-90:10 v/v): the appropriate fractions were combined and evaporated under reduced pressure to give the title compound 10; B = 12 as a colourless solid (4.95 g, 96%); [delta]_H [(CD₃)₂SO] 1.13 (9 H, t, J 7.2), 1.29 (9 H, s), 1.55(1 H, m), 1.74 (1 H, m), 1.99 (1 H, m), 2.57 (4 H, m), 2.73-2.92 (8 H, m), 3.07 (1 H, m), 3.31 (1 H, dd, J 3.8 and 10.0), 3.44(1 H,dd, J 5.6 and 10.1), 3.74 (6 H, s), 4.43 (1 H, m), 4.74 (1 H, dd, J 4.7 and 9.2), 5.34 (1 H, dd, J 4.8 and 7.7), 6.01 (0.5 H, s), 6.23 (1 H, d, J 7.9), 6.83-6.95 (6 H, m), 7.05 (2 H, m), 7.19-7.31 (7 H, m), 7.43 (2 H, d, J 7.3), 7.67 (0.5 H, s), 8.55 (1 H, s), 8.62 (1 H, s) and 10.25 (1 H, br s); [delta]_P [(CD₃)₂SO] 1.75 (dd, J 9.4 and 595.6).

Triethylammonium salt of 5[prime]-O-(4,4[prime]-dimethoxytrityl)-2[prime]-O-[1-(2-fluorophenyl)-4-methoxypiperidin-4-yl]-4-N-benzoyl-cytidine-3[prime]-H-phosphonate [DMTr-C[prime]p(H)] 10; B = 13

5[prime]-O-(4,4[prime]-dimethoxytrityl)-2[prime]-O-[1-(2-fluorophenyl)-4-methoxy-piperidin-4-yl]-4-N-benzoylcytidine 9; B = 13 (4.29 g, 5.0 mmol) was converted into the title compound 10; B = 13 (5.00 g, 97%) by the procedure described above for the preparation of DMTr-A[prime]p(H) 10; B = 12; [delta]_H [(CD₃)₂SO] 1.11 (9 H, t, J 7.2), 1.73 (1 H, m), 1.92 (2 H, m), 2.04 (1 H, m), 2.81 (8 H, m), 3.04 (4 H, m), 3.15 (1 H, m), 3.27 (1 H, m), 3.45 (1 H, m), 3.76 (6 H, s), 4.33(1 H, m), 4.70 (2 H, m), 5.98 (0.5 H, s), 6.24 (1 H, d, J 6.3), 6.92 (4 H, m), 6.99-7.17 (4 H, m), 7.23-7.37 (7 H, m), 7.42 (2 H, m), 7.51 (2 H, m), 7.62 (1.5 H, m), 8.01 (2 H, m) and 8.22 (1 H, d, J 7.6); [delta]_P [(CD₃)₂SO] 1.64 (dd, J 8.6 and 595.9).

Triethylammonium salt of 6-O-(2,5-dichlorophenyl)-5[prime]-O-(4,4[prime]-dimethoxytrityl)-2[prime]-O-[1-(2-fluorophenyl)-4-methoxy-piperidin-4-yl]-2-N-phenylacetylguanosine-3[prime]-H-phos-phonate [DMTr-G[prime]p(H)] 10; B = 14

6-O-(2,5-dichlorophenyl)-5[prime]-O-(4,4[prime]-dimethoxytrityl)-2[prime]-O-[1-(2-fluorophenyl)-4-methoxypiperidin-4-yl]-2-N-phenylacetyl-guanosine 9; B = 14 (3.17g, 3.0 mmol) was converted into the title compound 10; B = 14 (3.60 g, 98%) by the procedure described above for the preparation of DMTr-A[prime]p(H) 10; B = 12; [delta]_H [CDCl₃] 1.28 (9 H, t, J 7.3), 1.63 (1 H, m), 1.81 (1 H, m), 2.05(1 H, m), 2.14 (1 H, m), 2.73 (1 H, m), 2.91 (4 H, m), 2.99 (7 H, m), 3.21 (1 H, m), 3.42 (1 H, dd, J 2.4 and 10.5), 3.56 (1 H, dd, J 3.4 and 10.5), 3.77 (6 H, s), 3.81 (2 H, s), 4.56 (1 H, m), 5.07 (1 H, m), 5.41 (1 H, dd, J 4.5 and 7.6), 6.23 (0.5 H, s), 6.30 (1 H, d, J 7.7), 6.78-7.04 (8 H, m), 7.12 (3 H, m), 7.19-7.35 (12 H, m), 7.44 (2 H, m), 7.89 (1 H, s), 7.96 (0.5 H, s), 8.13 (1 H, s) and 12.60 (1 H, br); [delta]_P [CDCl₃] 1.33 (dd, J 9.4 and 625.5).

Di-2-chlorophenyl phosphorochloridate 5

2-Chlorophenol (207.2 ml, 2.0 mol), phosphorus oxychloride (93.2 ml, 1.0 mol) and 1-methylimidazole (1.0 ml) were heated together at 180°C for 15 h. Distillation of the products under reduced pressure (oil pump) gave the title compound 5(265 g, 78%) (found, M⁺, 335.9277; calculated for ¹²C₁₂¹H₈³⁵Cl₃¹⁶O₃³¹P, M⁺, 335.9276) as a colourless liquid, b.p. 172-175°C/0.1 mm Hg; [delta]_C [CDCl₃] 121.9, 125.9, 127.4, 128.1, 131.0 and 145.8; [delta]_P [CDCl₃] -5.0.

2-(4-Methylphenyl)sulphanyl-1H-isoindole-1,3(2H)-dione 6b

p-Thiocresol (26.08 g, 0.21 mol) and phthalimide (29.43 g, 0.20 mol) were dissolved in hot pyridine (80 ml) and acetonitrile (100 ml). The resulting solution was cooled to room temperature and stirred. A solution of bromine (35.2 g, 0.22 mol) in acetonitrile (100 ml) was then added dropwise over a period of 1 h. After a further period of 2 h, methanol (200 ml) was added. The products were cooled (ice-water bath) for 30 min and then filtered to give the title compound 6b as pale yellow crystals (50.1 g, 93%) [found, in material recrystallized from ethyl acetate/petroleum ether (b.p. 40-60°C), C, 67.06; H, 3.96; N, 5.08; calculated for C₁₅H₁₁NO₂S, C, 66.90; H, 4.12; N, 5.20%] m.p. 198-200°C [lit. (9) m.p. 199-200°C]; [delta]_H [(CD₃)₂SO] 2.27 (3 H, s), 7.19 (2 H, d, J 8.1), 7.33 (2 H, d, J 8.2), 7.92 (2 H, m) and 7.97 (2 H, m); [delta]_C [(CD₃)₂SO] 21.0, 124.2, 128.6, 130.4, 131.9, 132.7, 135.5, 138.6 and 167.8.

Preparation of DMTr-A[prime]p(s[prime])C[prime]p(s[prime])G[prime]p(s[prime])U[prime]-Lev 23

(a) The triethylammonium salt of DMTr-G[prime]p(H) 10; B = 14 (1.467 g, 1.20 mmol) and HO-U[prime]-Lev 20 (0.551 g, 1.00 mmol) were co-evaporated with dry pyridine (2 × 10 ml) and the residue was then redissolved in dry pyridine (9 ml). The stirred solution was cooled to -40°C (IMS-dry ice bath) and a solution of di-2-chlorophenyl phosphorochloridate 5 (1.01 g, 3.0 mmol) in dichloromethane (1 ml) was added dropwise over 5 min. After a further period of 5 min, 2-(4-methylphenyl)sulphanyl-1H-iso-indole-1,3(2H)-dione 6b (0.539 g, 2.0 mmol) was added and the reactants were stirred at -40°C for 15 min. Then pyridine/water (1:1 v/v, 1 ml) was added with continuing stirring. After 5 min, the products were evaporated under reduced pressure. The residue was dissolved in dichloromethane (50 ml) and the solution was washed with saturated aqueous sodium hydrogen carbonate (3 × 25 ml). The organic layer was dried (MgSO₄) and evaporated under reduced pressure. After it had been co-evaporated with dry toluene (2 × 20 ml) under reduced pressure, the residue was dissolved in dichloromethane (40 ml) at room temperature and trifluoroacetic acid (0.77 ml, 10 mmol) was added. After 1 min, 4-methylmorpholine (1.65 ml, 15 mmol) was added and the reaction solution was poured into saturated aqueous sodium hydrogen carbonate (50 ml). The layers were separated and the aqueous layer was extracted with dichloromethane (2 × 50 ml). The combined organic layers were dried (MgSO₄) and evaporated under reduced pressure. The residue was purified by short column chromatography on silica gel (General procedures, above): the appropriate fractions, which were eluted with ethyl acetate, were combined and evaporated under reduced pressure to give HO-G[prime]p(s[prime])U[prime]-Lev 21; B = 14 as a colourless solid (1.445 g, 98%); R_f 0.68 (system A); [delta]_P [CDCl₃] 24.1, 25.6.

Preparation of fully protected cyclic tetraribonucleoside tetraphosphorothioate 25

Trifluoroacetic acid (0.39 ml, 5 mmol) was added to a stirred solution of DMTr-A[prime]p(s[prime])C[prime]p(s[prime])G[prime]p(s[prime])U[prime]-Lev 23 (1.613 g, 0.50 mmol) at room temperature. After 1 min, 4-methylmorpholine (0.83 ml, 7.5 mmol) was added and the products were poured into saturated aqueous sodium hydrogen carbonate (50 ml). The layers were separated and the aqueous layer was extracted with dichloromethane (2 × 50 ml). The combined organic layers were dried (MgSO₄) and evaporated under reduced pressure. The residue was fractionated by short column chromatography on silica gel: the appropriate fractions, which were eluted with dichloromethane/methanol (98:2-95:5 v/v) were combined and evaporated under reduced pressure to give HO-A[prime]p(s[prime])-C[prime]p(s[prime])G[prime]p(s[prime])U[prime]-Lev as a colourless solid (1.435 g, 98%); R_f 0.45 (system A); [delta]_P [CDCl₃] 24.3-26.3 (12 signals).

A solution of ammonium p-tolyl H-phosphonate 11a (0.604 g, 3.2 mmol) and triethylamine (0.90 ml, 6.5 mmol) in methanol (10 ml) was concentrated under reduced pressure and then co-evaporated with pyridine (10 ml). HO-A[prime]p(s[prime])C[prime]p(s[prime])-G[prime]p(s[prime])U[prime]-Lev (1.17 g, 0.40 mmol) was added and the mixture was dissolved in dry pyridine (10 ml). The solution was evaporated under reduced pressure and the residue was redissolved in dry pyridine (10 ml). Pivaloyl chloride (0.50 ml, 4 mmol) was added dropwise over 1 min to the cooled (IMS-dry ice bath, -30°C), stirred solution. After 30 min, water (1 ml) was added and the stirred mixture was allowed to warm up to room temperature. After a further period of 1 h, the products were partitioned between dichloromethane (50 ml) and water (50 ml). The organic layer was separated, washed with 0.5 mol/dm³ triethylammonium phosphate buffer (pH 7.0, 3 × 25 ml) and then applied to a short column of silica gel. The column was eluted with dichloromethane/methanol (95:5-90:10 v/v): the appropriate fractions were combined and evaporated under reduced pressure to give the triethylammonium salt of H(p)A[prime]p(s[prime])C[prime]p(s[prime])G[prime]p(s[prime])U[prime]-Lev as a colourless solid (1.19 g, 96%); [delta]_P [CDCl₃] 4.6 (d, J_P,H 623.4), 4.9 (d, J_P,H 621.0), 24.6-26.4 (12 signals). Hydrazine monohydrate (0.5 ml, 10 mmol) was added to a stirred solution of the latter material (0.618 g, 0.2 mmol) in pyridine (16 ml) and glacial acetic acid (4 ml) at room temperature. After 10 min, the products were partitioned between water (50 ml) and dichloromethane (50 ml). The organic layer was separated and washed first with water(2 × 25 ml) and then with 0.5 mol/dm³ triethylammonium phosphate buffer (pH 7.0, 25 ml); it was then dried (MgSO₄) and applied to a short column of silica gel. The appropriate fractions, which were eluted with dichloromethane/methanol (95:5-90:10 v/v) were combined and evaporated under reduced pressure to give the triethylammonium salt of H(p)A[prime]p(s[prime])C[prime]p(s[prime])G[prime]p(s[prime])U[prime]-OH 24 as a colourless solid (0.54 g, 90%); [delta]_P [CDCl₃] 4.3 (d, J_P,H 623.1), 4.6 (d, J_P,H 624.1), 5.0 (d, J_P,H 624.6), 24.7-26.0 (8 signals).

A solution of the triethylammonium salt of H(p)A[prime]p(s[prime])-C[prime]p(s[prime])G[prime]p(s[prime])U[prime]-OH 24 (0.229 g, 0.10 mmol) in dichloromethane (4 ml) was added dropwise over a period of 15 min to a stirred solution of di-2-chlorophenyl phosphorochloridate 5 (0.68 g, 2.0 mmol) in dry pyridine (20 ml) at -40°C (IMS-dry ice bath). After a further period of 5 min, 2-(4-methylphenyl)sulphanyl-1H-isoindole-1,3(2H)-dione 6b (0.135 g, 0.5 mmol) was added and the reactants were stirred at -40°C for 15 min. Water/pyridine (1:1 v/v, 1 ml) was then added. The products were stirred at -40°C for an additional 5 min and were then evaporated under reduced pressure. The residue was dissolved in dichloromethane (50 ml) and the solution was washed with saturated aqueous sodium hydrogen carbonate (3 × 25 ml), dried (MgSO₄) and evaporated under reduced pressure. The residue was fractionated by short column chromatography on silica gel: the appropriate fractions, which were eluted with dichloromethane/methanol (98:2-95:5 v/v), were combined and evaporated under reduced pressure to give the fully protected cyclic tetraribonucleoside tetraphosphorothioate 25 (0.275 g, 92%) as a colourless solid;R_f 0.40 (system A); R_t 11.01 min (programme A); [delta]_P [CDCl₃] 23.8-27.7 (numerous signals).

Complete unblocking of fully protected oligonucleotide phosphorothioates

(a) DMTr-A[prime]p(s[prime])C[prime]p(s[prime])G[prime]p(s[prime])U[prime]-Lev 23 (0.025 g, ~7.7 µmol) was added to a stirred solution of E-2-nitrobenzaldoxime 7 (0.083 g, 0.5 mmol) and TMG 8 (0.056 ml, 0.45 mmol) in acetonitrile (0.5 ml) at room temperature. After 12 h, the products were evaporated under reduced pressure and concentrated aqueous ammonia (d 0.88, 5 ml) was added. The resulting mixture was heated in a closed vessel at 50°C for 5 h and the products were then evaporated under reduced pressure. The residue was dissolved in methanol (2 ml) and ethyl acetate (40 ml) was added. The resulting precipitate was collected by centrifugation, washed with ethyl acetate (2 × 20 ml) and dried to give an off-white solid (0.015 g). This material was dissolved in 0.5 mol/dm³ sodium acetate buffer (pH 4.0, 5 ml) and the solution was maintained at 40°C for 5 h. The aqueous products were extracted with dichloromethane (3 × 5 ml), neutralized with 1.0 mol/dm³ triethylammonium bicarbonate buffer (pH 7.5, 2.5 ml) and then applied to a column (20 × 2 cm diameter) of DEAE-Sephadex A 25, which was eluted with triethylammonium bicarbonate buffer (pH 7.5, linear gradient from 0.01 to 1.0 mol/dm³ over 1000 ml). Appropriate fractions (eluted with ~0.6 mol/dm³ buffer) were combined and evaporated under reduced pressure to give the triethylammonium salt of r(ApCpGpU) 18 (210 A₂₆₀ units) as a colourless solid; R_t 13.06 min (programme B); [delta]_H [D₂O] includes the following signals: 5.62 (1 H, d, J 7.3), 5.66 (2 H, m), 5.73(1 H, d, J 3.9), 5.84 (1 H, d, J 4.2), 5.96 (1 H, d, J 4.2), 7.60 (1 H, d, J 7.5 ), 7.76 (1 H, d, J 8.2), 7.81 (1 H, s), 8.06 (1 H, s) and 8.24 (1 H, s); [delta]_P (D₂O) 0.04 (Fig. 1a).

Enzymatic digestion of r(ApCpGpU) 18 and the cyclic tetraribonucleotide 19

(a) Crotalus adamanteus snake venom phosphodiester stock solution (8) (30 µl) was added to a solution of the substrate(~2 A₂₆₀ units) in sterile water (20 µl) and the resulting solution was incubated at 37°C for 20 h. Escherichia coli alkaline phosphatase stock solution (8) (30 µl) was then added and, after 14 h, the products were analysed by HPLC (programme B). Both substrates were fully digested to their constituent nucleosides. The adenosine (R_t 12.3 min):cytidine (R_t 4.4 min):guanosine (R_t 8.7 min):uridine (R_t 5.5 min) ratios were found to be 1.00:1.00:1.08:1.00 and 1.00:1.03:1.04:1.00 for 18 and 19, respectively.