Guanine O6 and Thymine O4 Phosphitylation in Oligonucleotide Synthesis

Abstract

The guanine O6 phosphitylation side reaction, which can lead to the formation of the intermediates – abasic sites and O6 phosphate derivatives – responsible for internucleotide bond cleavage, and G-to-2,6-diaminopurine (D), or G-to-A after PCR amplification, substitution error, respectively, represents a significant barrier to the synthesis of long oligonucleotides (ONs) and limits the use of phosphoramidite capping in the synthesis of sensitive ONs. To confirm this side reaction and determine the degree to which it occurs, we synthesized the sequence 5′-T6GT18-3′, and subjected its 5′-capped version to repeated coupling and oxidation reaction cycles. Subsequent deprotection and cleavage with neat nBuNH2 and LCMS analysis of the resulting products confirmed that both intermediates can form simultaneously, with quantities increasing with the increase of the number of the reaction cycles. In addition, after a sufficient number of the cycles, a substantial amount of thymine O4 phosphate derivative, which can cause T-to-C substitution errors and has not been reported in the literature, was also observed.