Insertion of Chemical Handles into the Backbone of DNA during Solid‐Phase Synthesis by Oxidative Coupling of Amines to Phosphites

Abstract

Conjugation of molecules or proteins to oligonucleotides can improve their functional and therapeutic capacity. However, such modifications are often limited to the 5' and 3' end of oligonucleotides. Herein, we report the development of an inexpensive and simple method that allows for the insertion of chemical handles into the backbone of oligonucleotides. This method is compatible with standardized automated solid-phase oligonucleotide synthesis, and relies on formation of phosphoramidates. A unique phosphoramidite is incorporated into a growing oligonucleotide, and oxidized to the desired phosphoramidate using iodine and an amine of choice. Azides, alkynes, amines, and alkanes have been linked to oligonucleotides via internally positioned phosphoramidates with oxidative coupling yields above 80 %. We show the design of phosphoramidates from secondary amines that specifically hydrolyze to the phosphate only at decreased pH. Finally, we show the synthesis of an antibody-DNA conjugate, where the oligonucleotide can be selectively released in a pH 5.5 buffer.

Influence of Combinations of Lipophilic and Phosphate Backbone Modifications on Cellular Uptake of Modified Oligonucleotides

“…Considering the chemical approach of phosphorothioate modification synthesis, there is a crucial drawback since only one representative of this class of altered phosphate groups could be obtained using the developed sulfurizing reagents [ 23 ], which are not intended to vary functional groups in their structure. Over the past decade, several chemical approaches utilizing alternative oxidation steps to obtain novel structures of modified phosphate groups, which allow one not only to change the nature of the phosphate backbone but also to apply the chemistry of its introduction to varying desirable functional moieties, have been proposed [ 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 ]. In particular, we have adopted the method of using electron-deficient azides in the Staudinger reaction during the oxidation step to efficiently obtain various phosphate derivatives [ 34 , 35 , 36 , 37 , 38 , 39 ].…”

Influence of Combinations of Lipophilic and Phosphate Backbone Modifications on Cellular Uptake of Modified Oligonucleotides

“…Considering the chemical approach of phosphorothioate modification synthesis, there is a crucial drawback since only one representative of this class of altered phosphate group could be obtained using the developed sulfurizing reagents [23] which doesn't mean to vary functional groups in its structure. Over the past decade, several chemical approaches utilizing alternative oxidation step to obtain novel structures of modified phosphate group which allow one not only to change the nature of phosphate backbone, but also to apply chemistry of its introduction for varying desirable functional moieties, have been proposed [24][25][26][27][28][29][30][31][32][33]. Particularly, we have adopted the method of use of electron-deficient azides in the Staudinger reaction during oxidation step for the efficient obtaining various phosphate derivatives [34][35][36][37][38][39].…”