Phosphoramidite Oligonucleotide Synthesis
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The cyanomethyl esters of the succinic, glutaric, adipic acids were introduced into
the 2’-position of 2’-amino-2’-deoxyuridine, and the cyanomethyl ester of the glutaric
acid into the 2’-position of 9-(2-amino-2-deoxy-b-D-arabinofuranosyl)-N6-benzoyl
adenine[6] (Fig. 1A). The 2’-amino group in compound 1 reacted with bis-cyanomethyl
esters of varies dicarbonic acids prepared in situ in the presence of diisopropylehyl-
amine (DIPEA) at room temperature. Bis-cyanomethyl esters of succinic, glutaric,
adipic acids were prepared as follow (Fig. 1B): potassium carbonate and bromoace-
tonitrile were added to a solution of dicarbonic acid in anhydrous CH3CN. The reaction
mixture was stirred at room temperature for 12 h, the precipitate was removed and then
washed with CH3CN. Filtrates were used for reaction with 2’-amino-2’-deoxyuridine
without additional purification. The reaction with 9-(2-amino-2-deoxy-b-D-arabinofur-
anosyl)-N6-benzoyl adenine 2 was analogous. Nucleosides 3–7 were obtained in yields
of 40, 30, 46, 47, 30% respectively. In this series we also synthesized the derivative of
2’-amino-2’-deoxyuridine bearing the monomethyl ester of malonic acid. Its reactivity is
lower than the cyanomethyl esters, though it is reactive enough for successful
functionalisation by primary amines.
GENERAL METHOD OF PREPARATION OF NUCLEOSIDES
BEARING REACTIVE LINKER GROUPS
The nucleoside bearing an amino group at the 2’-position (1 mmol) was added to
the bis-cyanomethyl ester of the corresponding dicarbonic acid prepared in situ (1.2 eq,
0.5 M CH3CN solution) in the presence of DIPEA (2.4 eq) at room temperature. After
68 h, the reaction was complete (as monitored by TLC). The reaction mixture was
evaporated to dryness in vacuo. The crude product was purified by silica-gel column
chromatography using CH2Cl2 as eluent. The corresponding phosphoramidites were
prepared using a standard procedure (Table 1).
5’-O-DMT-2’-deoxy-2’-(O-cyanomethyl)succinamidouridine 3: Rf 0.6 [CH2Cl2–
CH3OH (9:1)]; mp 98–102°C; UV (EtOH), lmax/nm: 202 (73022), 234 (19584), 266
(8919). The yield of phosphoramidite 8 is 35%; Rf 0.7 [CH2Cl2–CH3OH–Et3N
(9.4:0.5:0.1)]; 31P NMR [(CD3)2CO]: d 151.4, 152.4.
5’-O-DMT-2’-deoxy-2’-(O-cyanomethyl)glutaramidouridine 4: Rf 0.56 [CH2Cl2–
CH3OH (9:1)]; mp 90–95°C; UV (EtOH), lmax/nm: 203 (76009), 234 (22519), 265
(10925). The yield of phosphoramidite 9 is 88%, Rf 0.63 [CH2Cl2CH3OH–Et3N
(9.4:0.5:0.1)]; 31P NMR [(CD3)2CO]: d 151.2, 152.3.
5’-O-DMT-2’-deoxy-2’-(O-cyanomethyl)adipinamidouridine 5: Rf 0.7 [CH2Cl2–
CH3OH (9:1)]; mp 95–97°C; UV (EtOH), lmax/nm: 201 (94372), 235 (24376), 266
(12450). The yield of phosphoramidite 10 is 47%, Rf 0.92 [CH2Cl2–CH3OH–Et3N
(9.4:0.5:0.1)]; 31P NMR [(CD3)2CO]: d 151.3, 152.4.
9-[5’-O-DMT-2’-deoxy-2’-(O-cyanomethyl)glutaramido-b-D-arabinofuranosyl]-N6-
benzoyladenine 6: Rf = 0.43 [CH2Cl2–CH3OH–Et3N (9.4:0.5:0.1)]; UV (EtOH),
lmax/nm: 206 (60080), 237 (27091), 277 (13571). The yield of phosphoramidite 11 is