Synthesis of oligonucleotides with 3'-terminal 5-(3-acylamidopropargyl)-3'-amino-2',3'-dideoxyuridine residues and their reactivity in single-nucleotide steps of chemical replication.

Oligonucleotides with a 3'-terminal 5-alkynyl-3'-amino-2',3'-dideoxyuridine residue were prepared, starting from 2'-deoxyuridine. The optimized route employs a 2',3'-dideoxy-3'-trifluoroacetamido-5-iodouridine 5'-phosphoramidite as building block for DNA synthesis and involves on-support Sonogashira coupling with N-tritylpropargylamine to generate oligonucleotides. The amino group of the propargylamine side chain was acylated to accelerate primer extension reactions involving the 3'-amino group. Three acyl groups were identified that decrease the half-life for DNA-templated extension steps with 7-azabenzotriazole esters of 2'-deoxynucleotides. The residue of 4-pyrenylbutyric acid was found to accelerate primer extension reactions and to render them more selective than those of the control primer. With this substituent, primer extension is also faster than previously measured for three-strand systems involving template, aminoprimer, and a downstream-binding helper oligonucleotide. Fast-reacting primers might become useful for genotyping single nucleotides.