Abstract
In this study, we successfully synthesized boranophosphate (PB), phosphorothioate (PS), and phosphate (PO) chimeric oligonucleotides (ODNs) as a candidate for the antisense oligonucleotides (ASOs). The PB/PS/PO-ODNs were synthesized utilizing H-boranophosphonate, H-phosphonothioate, and H-phosphonate monomers. Each monomer was condensed with a hydroxy group to create H-boranophosphonate, H-phosphonothioate, and H-phosphonate diester linkages, which were oxidized into PB, PS, and PO linkages in the final stage of the synthesis, respectively. As for condensation of an H-phosphonothioate monomer, regulating chemoselectivity was necessary since the monomer has two nucleophilic centers: S and O atoms. To deal with this problem, we used phosphonium-type condensing reagents, which could control the chemoselectivity. In this strategy, we could synthesize PB/PS/PO oligomers, including a 2′-OMe gapmer-type dodecamer. The physiological and biological properties of the synthesized chimeric ODNs were also evaluated. Insights from the evaluation of physiological and biological properties suggested that the introduction of suitable P-modification and sugar modification at proper sites of ODNs would control the duplex stability, nuclease resistance, RNase H-inducing ability, and one base mismatch discrimination ability, which are critical properties as potent ASOs.
Highlights
Many efforts have been devoted to the development of antisense oligonucleotides (ASOs) since it is demonstrated that an oligonucleotide that is complementary to a target mRNA could control the translation of mRNA into a protein
Deoxyribonucleoside 3′-Hboranophosphonate monomers 3a, 3c, 3g, and 3t were synthesized by following our previous report from the nucleosides 1a, 1c, 1g, and 1t
The 3′-H-phosphonate monomers 4a, 4c, 4g, and 4t were synthesized according to a procedure in our preceding publication from the nucleosides 1a, 1c, 1g, and 1t
Summary
Many efforts have been devoted to the development of antisense oligonucleotides (ASOs) since it is demonstrated that an oligonucleotide that is complementary to a target mRNA could control the translation of mRNA into a protein. There are two kinds of ASOs according to a mechanism of translation regulation, namely, a steric blocking type and an RNase H-dependent type. A full PB modification reduces the duplex stability of ASOs with target mRNAs and RNase Hinducing activities.19,22−24 To overcome these problems, Caruthers et al.− and our group introduced both PB and PO linkages in ODNs (PB/ PO chimeric ODNs). The use of condensing reagents such as 1,3-dimethyl-2-(3-nitro1,2,4-triazol-1-yl)-2-pyrrolidin-1-yl-1,3,2-diazaphospholidinium hexafluorophosphate (MNTP) (entry 2) and 3-nitro1,2,4triazol-1-yl-tris(pyrrolidin-1-yl) phosphonium hexafluorophosphate (PyNTP) (entry 3) which have 3-nitro 1,2,4-triazole (NT) as a leaving group afforded PS diester with over 90% HPLC yields These results suggested that the presence of NT was critical for the condensation of the H-phosphonothioate monomer and the 5′-hydroxy group. The RP-HPLC analysis of crude mixture indicated that the desired oligonucleotide was formed as the main product and 25 was isolated in 13% yield (Table 4, entry 7) This result indicated that some 2′-O-modified gapmers would be synthesized by this synthetic strategy. Ratio of Digested Fragmentsa pubs.acs.org/joc entry aFragment A: r(pAUGC); fragment B: r(pAAUGC). bDetermined by the area ratio of A and B in RP-HPLC profile
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