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3
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0023665475
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The final deprotection step in oligonucleotide synthesis is reduced to a mild and rapid ammonia treatment by using labile base-protecting groups
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(1987)
Nucleic Acids Research
, vol.15
, pp. 397
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Schulhof1
Molko2
Téoule3
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4
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84918612731
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D. Molko, J.-C. Schulhof & R. Téoule: Patent requests no EN 86 04 990 for France; 031781 for the USA; 87 400739 6 for Europe; 085 615 for Japon
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7
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84918599185
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The nucleoside (10 mmol) was transiently protected by trimethylchlorosilane in pyridine (50 ml) and acylated by a mixture of phenoxyacetyl chloride and 1-hydroxybenzotriazole (15 mmol each) in acetonitrile (For cytidine acetyl chloride was used alone). After 17 hours at room temperature, the excess acylating agent is hydrolysed, the precipited salts are filtered and the solvent is evaporated to dryness. The residue is taken up in 300 ml water, washed by chloroform (2 × 150 ml) and the aqueous phase is evaporated. Pure base protected nucleosides are obtained by cristallisation in absolute ethanol.
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9
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84918603841
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The protected nucleoside (5 mmol) is reacted with 4,4′-dimethoxytrityl chloride in pyridine (50 ml) overnight at 5° C. The excess tritylating agent is hydrolysed by methanol and the reaction mixture is evaporated to dryness. The gum is dissolved in chloroform (150 ml), washed with saturated sodium hydrogenocarbonate and water. The organic phase is evaporated to dryness and fractionated by preparative HPLC on silica gel. The desired compound is obtained in 50–70 % yield.
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11
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84918607827
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The 5′ and base protected nucleoside (2.5 mmol) is dissolved in 40 ml pyridine. Imidazole (3 eq) and t-butyldimethylchlorosilane (1.5 eq) are added. After 1–3 days at room temperature the excess TBDMS chloride is hydrolysed by water (1 ml) and the solvents are evaporated. The residue is taken up in chloroform (100 ml) and washed with saturated aqueous sodium bicarbonate and water. Pure 2′ isomers are obtained in 30 – 50% yield by preparative HPLC on silica gel columns.
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13
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84918638403
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1H NMR. This showed that pure 3′ phosphoramidite with the exclusion of 2′ isomers were obtained.
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14
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84918602081
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Detritylation (Trichloracetic acid 3% in dichloromethane - 2 min.) - Coupling (Mononucleotide 0.1 M in anhydrous acetonitrile, tetrazole 0.5 M in dry acetonitrile - 20 min.) - Capping (Acetic anhydride or phenoxyacetic anhydride) 0.1 M in lutidine / THF 1/8 + Diméthylaminopyridine 6.5 % wt/vol in THF - 0.5 min., - Oxidation (Iodine 0.1 M in lutidine / THF / water 10 / 40 / 1 - 0.5 min.).
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15
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84918632217
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H. Bazin : Unpublished results
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