Multi Labeling of Oligonucleotides
It has been shown that the 5-position of pyrimidine and the 7-position of 7-deazapurine nucleosides are the ideal positions to introduce functionalities, as these sites lie in the major groove of the DNA providing steric freedom. In order to enable efficient click-chemistry labeling of alkyne modified oligonucleotides, our nucleosides provide a 5-(octa-1,7-diynyl) sidechain. Phosphoramidites of nucleosides 1-4 were shown to be incorporated into DNA oligomers by solid-phase synthesis with excellent coupling efficiency (e.g. 1: > 99 %). Another feature of the octadiynyl-sidechain is their stabilizing effect on DNA duplexes (e.g. 1: Tm increase of 1-2 °C).
The Cu(I)-catalyzed Huisgen reaction enables the multiple postsynthetic labeling of alkyne modified DNA as well. Complete high-density functionalization of several alkyne moieties can be achieved without the formation of by-products.
22mer, five internal alkynes reacted with 5 equivalents PEG-Azide, 4 h at 37 °C. Ethanol precipitation with 86% recovery of the labelled oligo. MALDI-mass analysis of the crude product -> 100% oligo-dye conjugate.
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