Solid-Phase Synthesis, Hybridizing Ability, Uptake, and Nuclease Resistant Profiles of Position-Selective Cationic and Hydrophobic Phosphotriester Oligonucleotides

Abstract

Analogues of oligonucleotides and mononucleotides with hydrophobic and/or cationic phophotriester functionalities often generate an improvement in target affinity and cellular uptake. Here we report the synthesis of phosphotriester oligodeoxyribonucleotides (ODNs) that are stable to the conditions used for their preparation. The method has been demonstrated by introducing phosphoramidite synthons where N-benzyloxycarbonyl (Z) protected amino alcohols replace the cyanoethyl group. After synthesis these ODNs were found to be stable to the condition required to remove base labile protecting groups and the ODNs from the solid support. Moreover the use of 1-(4,4-dimethyl-2, 6-dioxocyclohex-1-ylidene) ethyl (Dde) in place of Z protection on the amino alcohol has allowed us to introduce cationic aminoethyl phosphotriester modifications into ODNs. Melting temperatures of duplexes containing cationic or hydrophobic Z modified ODNs indicate that the backbone-phosphotriester modifications minimally affect duplex stability. Nuclease stability assays demonstrate that these phosphotriesters are resistant toward 5'- and 3'-exonucleases. Fluorescently labeled 23-mer ODNs modified with four cationic or hydrophobic Z phosphotriester linkages show efficient cellular uptake during passive transfection in HeLa and Jurkat cells.

Modified internucleoside linkages for nuclease-resistant oligonucleotides

“…In 2015, Caruthers and co-workers developed a general method to obtain oligonucleotides modified with hydrophobic and/or cationic O -alkylated PT internucleoside linkages. 232 The methodology used bis-(diisopropylamino)-3′-phosphorodiamidite derivatives that can be coupled with an alcohol in the presence of 5-ethylthio-1 H -tetrazole, leading to N -protected amino alcohols instead of the classical cyanoethyl protective group. The different O -alkylated phosphoramidites were then incorporated within ODN sequences by solid phase synthesis using 5-ethylthio-1 H -tetrazole as an activator.…”

DNA Analogues Modified at the Nonlinking Positions of Phosphorus

“…These modified siRNAs internalize into cells where thioesterases converted them to natural phosphodiester siRNAs . Based upon these observations, we decided to develop a robust methodology for preparing phosphotriester oligonucleotides having either hydrophobic or cationic internucleotide linkages …”

“…T m values of duplexes containing these oligonucleotides and complementary RNA were reduced 2–6 °C (compared to the unmodified RNA/DNA duplex) even when the oligonucleotide contained only 2 to 6 phosphotriester linkages in a DNA oligomer 22 bases in length. Importantly, these oligonucleotides were found to be resistant to nuclease degradation and were taken up by HeLa and Jurkat cells in the absence of lipid transfection agents …”

“…37 Based upon these observations, we decided to develop a robust methodology for preparing phosphotriester oligonucleotides having either hydrophobic or cationic internucleotide linkages. 38 Phosphotriester linkages are unstable to the ammonia treatment that is required for removing the standard nucleobase protecting groups. Considering this challenge, we developed a phosphoramidite chemistry for synthesizing both hydrophobic and cationic phosphotriester oligonucleotides that is essentially free of this problem.…”

“…Importantly, these oligonucleotides were found to be resistant to nuclease degradation and were taken up by HeLa and Jurkat cells in the absence of lipid transfection agents. 38…”