ABSTRACT
A wide variety of modified oligonucleotides have been tested as antisense agents. Each chemical modification produces a distinct profile of potency, toxicity, and specificity. Novel cationic phosphoramidate-modified antisense oligonucleotides have been developed recently that have unique and interesting properties. We compared the relative potency and specificity of a variety of established antisense oligonucleotides, including phosphorothioates (PS), 2'-O-methyl (2'OMe) RNAs, locked nucleic acids (LNAs), and neutral methoxyethyl (MEA) phosphoramidates with new cationic N,N-dimethylethylenediamine (DMED) phosphoramidate-modified antisense oligonucleotides. A series of oligonucleotides was synthesized that targeted two sites in the Xenopus laevis survivin gene and were introduced into Xenopus embryos by microinjection. Effects on survivin gene expression were examined using quantitative real-time PCR. Of the various modified oligonucleotide designs tested, LNA/PS chimeras (which showed the highest melting temperature) and DMED/phosphodiester chimeras (which showed protection of neighboring phosphate bonds) were potent in reducing gene expression. At 40 nM, overall specificity was superior for the LNA/PS-modified compounds compared with the DMED-modified oligonucleotides. However, at 400 nM, both of these compounds led to significant degradation of survivin mRNA, even when up to three mismatches were present in the heteroduplex.
Subject(s)
Ethylenediamines/chemistry , Oligonucleotides, Antisense/pharmacology , RNA, Messenger/antagonists & inhibitors , Xenopus Proteins/antagonists & inhibitors , Amides/chemistry , Animals , Base Pair Mismatch , Base Sequence , Deoxyribonucleases/chemistry , Embryo, Nonmammalian/drug effects , Gene Expression/drug effects , Oligonucleotides, Antisense/chemistry , Oligonucleotides, Antisense/genetics , Phosphoric Acids/chemistry , Survivin , Temperature , Xenopus Proteins/genetics , Xenopus laevisABSTRACT
The 4-pentenoyl group and a number of derivatives have been studied as protecting groups for N(alpha) of the aminoacyl moiety in misacylated tRNAs. The unsubstituted 4-pentenoyl group itself was found to function as efficiently as any of the derivatives studied. Four different N-(4-pentenoyl)aminoacyl-tRNA(CUA)s were prepared and shown to undergo deprotection readily upon admixture of aqueous iodine; the derived misacylated tRNAs all functioned well as suppressors of a nonsense codon in an in vitro protein biosynthesizing system. Also prepared were four N(alpha)-(4-pentenoyl)aspartyl-tRNA(CUA)s that were protected on the side chain carboxylate as the nitroveratryl ester. Following treatment with aqueous iodine, the misacylated suppressor tRNAs incorporated the aspartate derivatives into position 27 of dihydrofolate reductase by suppression of a UAG codon in the mRNA. The suppression yields were significantly better than those obtained when side chain protection was absent. The resulting "caged proteins" were inactive, but full catalytic potential was restored by irradiation under conditions sufficient to effect deprotection of the side chain carboxylate moiety.
ABSTRACT
Regioselective Michael addition of nitro and heterocyclic compounds to levoglucosenone, 1, is effectively catalyzed by amines and also by cathodic electrolysis. In comparison to the base-catalyzed reaction, it was found that under electrochemical conditions the reaction proceeds under milder conditions and with higher yields. Cathodically-initiated Michael addition of thiols to levoglucosenone using small currents produces the previously unknown threo addition product in several instances. The normal erythro isomer, identified as the kinetic product, tends to be formed when large currents are used. In contrast, slow, low current electrolyses promote equilibration of the two forms so that erythro can be converted to threo by the retro reaction and readdition. Addition of 2-naphthalenethiol to (R)-(+)-apoverbenone is also reported.
