The properties of phosphodiester oligodeoxynucleotides (ODNs) containing 4′R-C-aminomethyl, -ethyl, -propyl, and -N-(2-aminoethyl)carbamoylthymidines (1, 2, 4, and 5) as potential antisense molecules are investigated in detail. We developed new radical chemistry with a vinylsilyl or an allylsilyl group as a temporary radical acceptor tether to synthesize the required 4′R-branched thymidines. Thus, an intramolecular radical cyclization of 4′-phenylseleno nucleosides 7a and 7b, which have a dimethylvinylsilyl and a dimethylallylsilyl group at the 3′-hydroxyl, respectively, with Bu 3 SnH/AIBN and subsequent Tamao oxidation (8b). Compounds 8a and 8b were then converted into 4′R-C-(2-trifluoroacetamidoethyl)thymidine 12a and 4′R-C-(3-trifluoroacetamidopropyl)thymidine 12b, which were phosphitylated to give the phosphoramidite units 14a and 14b. The phosphoramidite units of 1 and 5 were prepared by previous methods. The nucleosides 1, 2, 4, and 5 were incorporated into the 18-mer, 5′-d[MTMTMTMTMTMTMT-MTMT]-3′, where M is 5-methyl-2′-deoxycytidine, instead of T at various positions. We also prepared a 21-mer ODN 29 with a mixed sequence containing five residues of 2. The ODNs containing the modified nucleosides formed more stable duplexes with complementary DNA than the corresponding unmodified ODN. These ODNs also formed stable duplexes with the complimentary RNA. The ODNs containing the modified nucleosides were significantly resistant to nucleolytic hydrolysis by both snake venom phosphodiesterase (a 3′-exonuclease) and DNase I (an endonuclease) and were also very stable in PBS containing 50% human serum. It is worthwhile to note that these ODNs contain natural phosphodiester linkages. Furthermore, the duplexes formed by the ODNs containing the modified nucleosides and their complementary RNAs were good substrates for Escherichia coli RNase H and HeLa cell nuclear extracts as a source of human RNase H. Thus, these ODNs were identified as candidates for antisense molecules.
The synthesis and properties of oligodeoxynucleotides (ODNs) containing 4′-C- [2-[[N-(2-aminoethyl)carbamoyl]oxy]ethyl]thymidine (3) are described. 4′R-(2-Hydroxyethyl)thymidine (4), which is a precursor for phosphoramidite 5, was synthesized using a newly developed intramolecular radical cyclization reaction at the 4′-position of thymidine derivative 7. The radical reaction of 4′ -(phenylseleno)-3′-O-(dimethylvinylsilyl)thymidine derivative 7, which was prepared from thymidine in several steps, with Bu 3 SnH and AIBN, followed by Tamao oxidation, gave either 4′R-(2-hydroxyethyl) derivative 6 or 4′R-(1-hydroxyethyl) derivative 13, respectively. With a low Bu 3 -SnH concentration, the reaction gave 6, via 6-endo-radical-cyclized product 11, as a sole product in 87% yield. The reaction of 7 in the presence of excess Bu 3 SnH gave 13 in 75% yield, via 5-exocyclized product 12, as a diastereomeric mixture. The 4′R-(2-hydroxyethyl) derivative 6 was then converted into a 4′-C-[2-[[N-(2-aminoethyl)carbamoyl]oxy]ethyl]thymidine derivative 14, which was phosphitylated to give phosphoramidite 5 in 72% yield. In this study, 3 was incorporated into a nonadecamer, d[CTGGCTCAGCTCGTCTCAT]-3′, and a heptadecamer, d[CTCGTACCATTCCGCTC]-3′, instead of T at various positions. ODNs containing 3 were more resistant to nucleolytic hydrolysis by both snake venom phosphodiesterase (a 3′-exonuclease) and DNase I (an endonuclease) than unmodified parent ODNs, although ODNs containing 3 only slightly destabilized duplex formation with both complementary DNA and RNA strands. Furthermore, the duplex formed by an ODN containing 3 and its complementary RNA was a good substrate for Escherichia coli RNase H.
An efficient method for the synthesis of 4'alpha-branched 2'-deoxyadenosines starting from 2'-deoxyadenosine has been developed utilizing a novel radical cyclization reaction with a silicon tether. The radical reaction of 4'beta-(phenylseleno)-3'-O-diphenylvinylsilyl adeninenucleoside derivative 17 with Bu(3)SnH and AIBN, followed by Tamao oxidation, gave selectively either the 4'alpha-(2-hydroxyethyl) derivative 21 or 4'alpha-(1-hydroxyethyl) derivative 19, depending on the reaction conditions. With a lower Bu(3)SnH concentration, the reaction gave the 4'alpha-(2-hydroxyethyl) derivative 21, via a 6-endo-radical cyclized product 20, as the sole product in 72% yield. The reaction of 17 in the presence of excess Bu(3)SnH gave 19 quantitatively, via a 5-exo-cyclized product 18, as a diastereomeric mixture. The reaction mechanism was examined using Bu(3)SnD. The results demonstrated that the 5-exo cyclized (3-oxa-2-silacyclopentyl)methyl radical (C) was formed initially which was trapped when the concentration of Bu(3)SnH(D) was high enough. With lower concentrations of Bu(3)SnH(D), radical C rearranged into the ring-enlarged 4-oxa-3-silacyclohexyl radical (D) which was then trapped with Bu(3)SnH(D) to give endo-cyclized product F.
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