Synthesis and Biophysical Properties of Triazole-Incorporated PMOs (TzPMOs): A Convergent, Click Ligation Approach

Abstract

The synthesis of phosphorodiamidate morpholino oligonucleotides (PMOs) incorporating single or double triazole rings in the backbone has been achieved via Cu(I)-catalyzed azidealkyne cycloaddition (CuAAC). The synthetic approach implemented is fundamentally convergent, involving the ligation of a 5′azide PMO fragment to a 3′-alkyne fragment both in solution and on solid support. To access the 3′-alkyne PMO fragment, we synthesized 3′-N-propargyl chlorophosphoramidate morpholino monomers for all four nucleobases. The resulting triazoleincorporated PMOs (TzPMOs) have exhibited comparable or improved binding affinity toward complementary deoxyribonucleic acid (DNA)/ribonucleic acid (RNA) strands compared to its regular analogues. Finally, a full-length TzPMO was designed to target the Nanog gene, demonstrating almost identical hybridization properties when compared to its regular version. Circular dichroism studies revealed a B-type helical conformation for the duplexes formed by TzPMOs.

Evaluation of Transiently O-6-Protected Guanosine Morpholino Nucleosides in Phosphorodiamidate Morpholino Oligonucleotide Synthesis

“…We have synthesized 4-mer PMO in solution without O-6 protected G, 15a however, we observed that in the present case, it is easy to handle upto 6-mer PMO with O-6 protected guanosine due to its nonpolar character. Based on our experience, [12][13][14][15][16][17] this methodology can improve the yields of longer PMOs having Grich segments; the G-rich blocks can be synthesized in solution phase which can be coupled in the oligomer upon further activation by H-phosphonate 14 or chlorophosphoramidate 15 or click chemistry 19 using convergent approach like regular oligonucleotides. 20 Further evaluation of this approach for the synthesis of longer PMOs, such as the 30-mer Eteplirsen in large scale with improved yield, will be reported in due course.…”