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thyluridine (4): 2,20-Anhydrouridine (452 mg, 2 mmol) was
coevaporated three times each with pyridine, toluene and
dissolved in DMA (4 ml) under argon atmosphere. To the
solution were added 2-cyanoethyl trimethylsilyl ether
(1432 mg, 10 mmol) and BF3ÆOEt (628 ll, 5 mmol). The
mixture was stirred at 120 °C for 15 h, and then MeOH
was added. The solution was mixed with NH-silica gel,
and the resulting mixture was evaporated in vacuo. The
powder was subjected on a silica gel column. Elution was
performed with CHCl3–MeOH (95:5, v/v) to give 20-
O-cyanoethyluridine (4) (383 mg, 65%) as a white solid.
Synthesis of cytidine monomer building block 11. 20-O-(2-
Cyanoethyl)-50-O-(4,40-dimethoxytrityl)uridine 30-[2-cyano-
ethyl (N,N-diisopropyl)phosphoramidite] (8) (250 mg,
0.313 mmol) was coevaporated three times each with
pyridine, toluene and dissolved in dry CH3CN (10 ml)
under argon atmosphere. To the solution were added
triethylamine (1 ml, 7.2 mmol), 1,2,4-1H-triazole (486 mg,
7 mmol) and POCl3 (57 ll, 0.625 mmol). The mixture was
stirred at room temperature for 12 h and then diluted with
ethyl acetate. The solution was extracted twice with
aqueous 5% Na2CO3 solution. The organic layer was
dried over Na2SO4 and filtered. The solution was evapo-
rated under reduced pressure. The residue was dissolved
with pyridine–concd NH3 (4:1, v/v, 15 ml). After being
stirred at room temperature for 2 h, the mixture was
evaporated under reduced pressure. The residue was
chromatographed on an NH-silica gel column with
CHCl3–MeOH (99:1, v/v) to give 20-O-(2-cyanoethyl)-50-
O-(4,40-dimethoxytrityl)cytidine 30-[2-cyanoethyl (N,N-
diisopropyl)phosphoramidite] (10) (200 mg, 80%) as a
white foam. Compound 10 (180 mg, 0.225 mmol) was
coevaporated five times each with pyridine, toluene and
dissolved in dry THF (5 ml) under argon atmosphere. To
the solution were added ethyldiisopropylamine (77 ll,
0.45 mmol) and acetyl chloride (20 ll, 0.27 mmol). After
being stirred at room temperature for 2 h, the mixture was
diluted with CHCl3. The solution was washed with brine
and aqueous NaHCO3. The organic layer was dried over
Na2SO4 and filtered. The solution was evaporated under
reduced pressure. The residue was chromatographed on a
column of silica gel with CHCl3–MeOH (98:2, v/v)
containing 0.5% triethylamine to give 4-N-acetyl-20-O-(2-
cyanoethyl)-50-O-(4,40-dimethoxytrityl)cytidine 30-(N,N-
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22. The original approach required the N3-imido protection
using N3-benzoyl-30,50-(1,1,3,3-tetraisopropylsiloxane-1,3-
diyl)uridine.13 In the 0.5 mmol-scale synthesis of this
compound, this compound could be obtained in more
than 95% yields.25 On a large scale (10–20 mmol), how-
ever, the yield dropped to 70–80% since this reaction was
carried out by a two-phase system where vigorous stirring
was essential. On a laboratory scale, it was difficult to stir
the two-phase mixture efficiently. Therefore, in the large
scale synthesis, the total yield of 20-O-cyanoethyluridine
was ca. 45–50%.
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diisopropyl)phosphoramidite (11) as
a white foam
(160 mg, 85%).