Post‐Synthetic Modification of Triplex‐Forming Oligonucleotides Containing 2‐Aminoethyl‐2′‐Deoxynebularine Derivatives

This article describes the detailed synthetic protocol for the preparation of oligonucleotides containing 2-guanidinoethyl-2'-deoxynebularine and 2-ureidoethyl-2'-deoxynebularine nucleoside derivatives. These derivatives are obtained by a post-synthetic modification of triplex-forming oligonucleotides (TFOs) containing 2-aminoethyl-2'-deoxynebularine, which is useful for forming stable triplex DNA with duplex DNA sequences containing 5m CG and CG interrupting sites. The hydroxyl groups of the sugar moiety of commercially available 2'-deoxyguanosine are acetyl-protected, the 6-position is chlorinated and reduced to give a 2-substituted nebularine derivative, and then the sugar moiety is deprotected. The hydroxyl groups of the sugar moiety are silyl-protected and the amino group at the 2-position is iodinated before being coupled with diethyl malonate. The ethyl ester is reduced and the resulting alcohol converted to an amino group for protection. The compound is then converted to a phosphoramidite unit and incorporated into a TFO. Subsequent modification of the aminoethyl group on the TFO completes the synthesis of the oligonucleotides containing 2-guanidinoethyl-2'-deoxynebularine and 2-ureidoethyl-2'-deoxynebularine. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Preparation of the phosphoramidite unit of the 2-aminoethyl-2'-deoxynebularine derivative (14) Basic Protocol 2: Post-synthetic modification of oligonucleotides containing 2-aminoethyl-2'-deoxynebularine derivatives Basic Protocol 3: Determination of the triplex-forming ability of oligonucleotides containing 2-aminoethyl-2'-deoxynebularine derivatives.