Guanine O⁶ and thymine O⁴ phosphitylation in oligonucleotide synthesis

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 reaction cycles. In addition, after a sufficient number of 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.