Synthesis, Hybridization, and Nuclease Resistance Properties of 2'-O-AMINOOXYETHYL Modified Oligonucleotides (original) (raw)

1999, Nucleos Nucleot Nucleic Acids

Abstract

The novel RNA mimic 2'-O-AOE has been incorporated into antisense oligonucleotides. This 2'-O-modification significantly enhances hybridization against target RNA, and furthermore, exhibits specificity for RNA over DNA. The nuclease resistance (SVPD) of 2'-O-AOE modified phosphodiester oligonucleotides is significantly higher than the unmodified DNA and comparable to the 2'-O-MOE oligonucleotides.

Figures (3)

by standard chemistries gave phosphoramidite 4 in modest yield. The synthesis of 2’-O-AOE-adenosine entailed a direct alkylation of adenosine with ethyl 2-bromoacetate to give the 2’-O-alkylated nucleoside (Scheme 2) along with the putative 3’-O-isomer. The product mixture was not purified at this stage but was protected as the disiloxanylidene!! derivative to provide the 2’-O-ethylester 5 in 22% over two steps. Subsequent chemistries were analogous to that of the 2’-O-AOE-5-methyluridine synthesis (Scheme 1) to afford moderate yields of the phosphoramidite 7. Finally the modified give the alcohol which on subsequent Mitsunobu’¥ reaction with N-hydroxyphthalimide provided 3 in good

by standard chemistries gave phosphoramidite 4 in modest yield. The synthesis of 2’-O-AOE-adenosine entailed a direct alkylation of adenosine with ethyl 2-bromoacetate to give the 2’-O-alkylated nucleoside (Scheme 2) along with the putative 3’-O-isomer. The product mixture was not purified at this stage but was protected as the disiloxanylidene!! derivative to provide the 2’-O-ethylester 5 in 22% over two steps. Subsequent chemistries were analogous to that of the 2’-O-AOE-5-methyluridine synthesis (Scheme 1) to afford moderate yields of the phosphoramidite 7. Finally the modified give the alcohol which on subsequent Mitsunobu’¥ reaction with N-hydroxyphthalimide provided 3 in good

ARES RUE ieee NR Me Oe ee AST EEC NS wee ORAM SENOS SERIE Se UEDA V ene NWS ite UST SAR ccnwen SReer RESON Se Hybridization. 12 The 2’-O-AOE modified oligonucleotides showed impressive enhancement of T,, against RNA of 1.0 and 1.2° C/substitution (relative to DNA standard) when four or ten consecutive substitutions, respectively, were incorporated (Table I). In comparison to the 2’-O-MOE modification, the stabilizing effects of ten 2’-O-AOE substitutions were essentially equivalent. This translates to nearly +2.0 °C increase/modification when compared to the first generation phosphorothioate (P=S) drugs. Hybridization of the oligonucleotide with 10 substitutions against DNA resulted in a destabilized duplex (relative to DNA standard) as evidenced by the decrease in T, of -0.94° C/substitution indicating the 2’-O-AOE modified oligonucleotides display a high specificity toward RNA vs. DNA.

ARES RUE ieee NR Me Oe ee AST EEC NS wee ORAM SENOS SERIE Se UEDA V ene NWS ite UST SAR ccnwen SReer RESON Se Hybridization. 12 The 2’-O-AOE modified oligonucleotides showed impressive enhancement of T,, against RNA of 1.0 and 1.2° C/substitution (relative to DNA standard) when four or ten consecutive substitutions, respectively, were incorporated (Table I). In comparison to the 2’-O-MOE modification, the stabilizing effects of ten 2’-O-AOE substitutions were essentially equivalent. This translates to nearly +2.0 °C increase/modification when compared to the first generation phosphorothioate (P=S) drugs. Hybridization of the oligonucleotide with 10 substitutions against DNA resulted in a destabilized duplex (relative to DNA standard) as evidenced by the decrease in T, of -0.94° C/substitution indicating the 2’-O-AOE modified oligonucleotides display a high specificity toward RNA vs. DNA.

Conclusions. In summary we have synthesized the novel 2’-O-AOE- 5-methyluridine and —adenosine nucleosides and successfully incorporated them into oligonucleotides. The novel 2’-O-modifications significantly enhance hybridization against RNA, and furthermore, exhibit specificity for RNA over DNA. Although the nuclease resistance (SVPD) of 2’-O-AOE modified oligonucleotides is comparable to that of 2’- O-MOE, the presently well-accepted antisense RNA mimetic, we desire further improvements. In this regard the primary aminooxy group of 2’-O-AOE nucleosides is currently being elaborated into other derivatives to optimize the properties of significantly enhance hybridization against RNA, and furthermore, exhibit specificity for RNA over DNA. nucleosides and successfully incorporated them into oligonucleotides. The novel 2’-O-modifications

Conclusions. In summary we have synthesized the novel 2’-O-AOE- 5-methyluridine and —adenosine nucleosides and successfully incorporated them into oligonucleotides. The novel 2’-O-modifications significantly enhance hybridization against RNA, and furthermore, exhibit specificity for RNA over DNA. Although the nuclease resistance (SVPD) of 2’-O-AOE modified oligonucleotides is comparable to that of 2’- O-MOE, the presently well-accepted antisense RNA mimetic, we desire further improvements. In this regard the primary aminooxy group of 2’-O-AOE nucleosides is currently being elaborated into other derivatives to optimize the properties of significantly enhance hybridization against RNA, and furthermore, exhibit specificity for RNA over DNA. nucleosides and successfully incorporated them into oligonucleotides. The novel 2’-O-modifications

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  12. Each sample contained 100 mM Na', 10 mM phosphate (pH 7), 0.1 mM EDTA, 4 ~tM modified oligonucleotide and 4 IIM complementary, length matched RNA.