Oligonucleotide N-alkylphosphoramidates: synthesis and binding to polynucleotides

Abstract

A few different methods for the preparation of oligonucleotide N-alkylphosphoramidates were compared directly. One of these, involving the use of protected nucleoside phosphites as building blocks, provided the requisite N-alkylphosphoramidates via oxidation of the intermediate dinucleoside methyl phosphites with iodine in the presence of the appropriate alkylamine. This method was found to have several attractive features, including the use of building blocks identical with those employed for the synthesis of DNA and compatibility with procedures and instruments employed for the stepwise synthesis of oligonucleotides by solution and solid-phase methods. This procedure was used to make several di-, tri-, and tetranucleotide N-alkylphosphoramidates derived from deoxyadenosine and thymidine; alkyl substituents included N,N-dimethyl, N-butyl, N-octyl, N-dodecyl, and N-(5-aminopentyl). The aminoalkyl derivative of d(TpT) (24) was used to demonstrate the feasibility of introducing an intercalative agent to the alkylphosphoramidate moiety of such derivatives. The oligonucleotide N-alkylphosphoramidates were separated into their component diastereomers and characterized structurally by a number of techniques including circular dichroism, high-field 1H NMR spectroscopy, FAB mass spectrometry, and enzymatic digestion to authentic nucleosides and nucleotides. Physicochemical characterization of several di- and trinucleotide alkyl-phosphoramidates revealed that the adenine nucleotide analogues formed stable complexes with poly-(thymidylic acid). The stabilities of these complexes were found to increase with increasing chain length of the N-alkylphosphoramidate substituents. The finding that N-alkylphosphoramidate substituents can enhance the binding of certain oligonucleotides to their complementary polynucleotides suggests the existence of a novel source of polynucleotide affinity.

Short modified antisense oligonucleotides directed against Ha-ras point mutation induce selective cleavage of the mRNA and inhibit T24 cells proliferation.

“…It is noteworthy that the 9-mer linked only to the dodecanol tail at the 3'-end was also more efficient than the unmodified or acridine-substituted oligomers. Letsinger et al (1986) and Jager et al (1988) have shown that lipophylic substituents may stabilize DNA helical structures. In addition more efficient induction of RNase H cleavage by this oligonucleotide may also result from facilitation of RNase H binding to the hybrid duplex.…”

Strand orientation of [α]‐oligodeoxynucleotides in triple helix structures: Dependence on nucleotide sequence

“…Actually, the potential use of oligonucleotides in uiuo is severely hindered by their degradation by nucleases that hydrolyse the phosphodiester backbone. Therefore, modifications of the backbone have been made in order to improve resistance to nucleases, notably, methylphosphonates (Murakami et al, 1985), phosphotriesters (Pless and Ts'o, 1977) phosphorothioates (Stein ef al., 1988) and phosphoramidates (Jager et al, 1988). Another possible modification consists of substituting the natural 1-anomers by [a]-anomers which change the stereochemistry at the C1' position, placing the base at the location normally occupied by the H1' proton.…”

Triple-helix formation by alpha oligodeoxynucleotides and alpha oligodeoxynucleotide-intercalator conjugates.

“…There are several ways in which oligonucleotides can be made resistant to nucleases. The phosphodiester backbone can be modified: methylphosphonates (11), phosphotriesters (12), phosphorothioates (13), and phosphoramidates (14) have been described. Alternatively, the anomeric configuration of the nucleoside units can be changed.…”