O-Selectivity and Utility of Phosphorylation Mediated by Phosphite Triester Intermediates in the N-Unprotected Phosphoramidite Method

Abstract

Previously, O-selective phosphorylation on polymer supports in the N-unprotected phosphoramidite method could not be carried out because the amino groups of dA and dC have high reactivity toward tervalent phosphorus(III)-type phosphitylating reagents. In this paper, we developed a new coupling strategy named the "activated phosphite method" in which the phosphitylation is mediated by phosphite triester intermediates 1. Application of 1-hydroxybenzotriazole as the promoter to the solid-phase synthesis resulted in excellent O-selectivity of more than 99.7%. This O-selectivity was explained by the frontier molecular orbital interactions between the reactive intermediates and the nucleophiles such as the amino or hydroxyl groups of nucleosides. Furthermore, longer oligonucleotides were synthesized not only by a manual operation but also by a DNA synthesizer. The utility of our new method was demonstrated by the successful synthesis of a base-labile modified oligodeoxyribonucleotide having 4-N-acetyldeoxycytidine residues. Finally, DNA 20-mers containing dA or dC could be synthesized in good yields by use of a combined reagent of 6-trifluoromethyl-1-hydroxybenzotriazole and benzimidazolium triflate.

Formation of N-Branched Oligonucleotides as By-products in Solid-Phase Oligonucleotide Synthesis

“…Whatever is the origin of incomplete N-protection of solid-supported deoxynucleosides, should branched oligonucleotide formation occur during the synthesis of oligonucleotides, a postsynthetic treatment with neat triethylamine trihydrofluoride would selectively and efficiently cleave any phosphoramidate linkage and convert any N-branched oligonucleotide back to the desired compound. This treatment would also improve the recovery of oligonucleotides obtained from the N-unprotected phosphoramidite method (Fourrey and Varenne, 1985;Gryaznov and Letsinger, 1992;Hayakawa and Kataoka, 1998;Ohkubo et al, 2004) that despite considerable progress in the discovery of O-selective activators, still produces detectable amounts of N-branched oligonucleotides.…”

Solid-phase synthesis of base-sensitive oligonucleotides

“…Compatibility with the nucleobase protection chemistry is omitted, but even in that case the CE groups (in the standard phosphoramidite method) have to be removed, for which non-nucleophilic treatments with DBU (e.g. 10% solution in acetonitrile for 1 min) 52 or triethylamine (40% solution in pyridine for 3h) 53 are used. Stability of the linker upon these treatments is desirable, since otherwise the product is liberated into a strong basic environment, in which the resulted acrylonitrile may react with nucleobases.…”

“…Demethylation of the phosphotriesters is accomplished with a treatment of disodium-2-carbamoyl-2-cyanoethylene-1,1,-dithiolate trihydrate (dccdt) (57), the cyanoethyl carbamates are removed by a mixture of triethylamine in DMF (1:1, v/v, 16 h at 55°C) (58) and then the fully deprotected oligonucleotide is released by photolysis. It may be notable that the unsubstituted cyanoethyloxycarbonyl group may be used for the guanine base (54), but the more base labile α,α-dimethylated derivative is required for adenine (53) and cytosine (52). …”

Synthesis and Properties of Oligonucleotides Incorporating Modified Nucleobases Capable of Watson–Crick‐Type Base‐Pair Formation

“…Oligodeoxynucleotides incorporating ac 6 ox 8 A were synthesized using the new strategy [84,85] for DNA synthesis, without base protection in the phosphoramidite approach (Figure 7.11).…”