Investigating the Activation Kinetics of Phosphoramidites for Oligonucleotide Synthesis

Solid-phase synthesis of oligonucleotides relies on a repeating cycle of rapid and well-defined synthetic steps to generate the target sequence. It is recognized that the underlying skeleton of the nucleotide influences the rates of the coupling reactions when the phosphoramidite coupling method is used. Using both in-line mid-IR spectroscopy and rapid NMR spectroscopy, we studied the rates of activation of traditional deoxyribonucleoside phosphoramidites and their chemically modified constrained ethyl (cEt) ribose locked nucleic acid analogues. This article reports our findings, which demonstrate a striking difference in the kinetics of activation for the two nucleotide structures. It was found that the cEt phosphoramidites required a larger excess of activator to achieve complete activation compared with their deoxyribonucleoside analogues. However, even with double the relative activator charge, the observed rate was over 10 times lower.