Acetic Anhydride → HCID 86588167, compound

Mass modification of nucleotide triphosphates at the 2′ and 3′ amino function: Starting material was 2′-azido-2′-deoxyuridine prepared according to literature (Verheyden et al., J. Org. Chem. 36:250, 1971), which was 4,4-dimethoxytritylated at 5′-OH with 4,4-dimethoxytrityl chloride in pyridine and acetylated at 3′-OH with acetic anhydride in a one-pot reaction using standard reaction conditions. With 191 mg (0.71 mmol) 2′-azido-2′-deoxyuridine as starting material, 396 mg (0.65 mmol; 90.8%) 5′-O-(4,4-dimethoxytrityl)-3′-O-acetyl-2′-azido-2′-deoxyuridine was obtained after purification via silica gel chromatography. Reduction of the azido group was performed (Barta et al., Tetrahedron 46:587-94, 1990). Yield of 5′-O-(4,4-dimethoxytrityl)-3′-O-acetyl-2′-amino-2′-deoxyuridine after silica gel chromatography was 288 mg (0.49 mmol; 76%). This protected 2′-amino-2′-deoxyuridine derivative (588 mg, 1.0 mmol) was reacted with 2 equivalents (927 mg; 2.0 mmol) N-Fmoc-glycine pentafluorophenyl ester in 10 ml dry DMF overnight at room temperature in the presence of 1.0 mmol (174 μl) N,N-diisopropylethylamine. Solvents were removed by evaporation in vacuo and the residue purified by silica gel chromatography. Yield was 711 mg (0.71 mmol; 82%). Detritylation was achieved by a one hour treatment with 80% aqueous acetic acid at room temperature. The residue was evaporated to dryness, co-evaporated twice with toluene, suspended in 1 ml dry acetonitrile and 5′-phosphorylated with POCl3 and directly transformed in a one-pot reaction to the 5′-triphosphate using 3 ml of a 0.5 M solution (1.5 mmol) tetra (tri-n-butylammonium) pyrophosphate in DMF according to literature. The Fmoc and the 3′-O-acetyl groups were removed by a one-hour treatment with concentrated aqueous ammonia at room temperature and the reaction mixture evaporated and lyophilized. Purification also followed standard procedures by using anion-exchange chromatography on DEAE Sephadex with a linear gradient of triethylammonium bicarbonate (0.1 M-1.0 M). Triphosphate containing fractions, checked by thin layer chromatography on polyethyleneimine cellulose plates, were collected, evaporated and lyophilized. Yield by UV-absorbance of the uracil moiety was 68% or 0.48 mmol.

1. customprepared
2. customreaction conditions

ORD record

ord-56df72aa63674d8c9916dbdba75fffeb

Source dataset

uspto-grants-2002_08

DOI

10.6084/m9.figshare.5104873.v1

Patent number

US06436635B1