Solution Stability and Degradation Pathway of Deoxyribonucleoside Phosphoramidites in Acetonitrile

Abstract

The impuritiy profiles of acetonitrile solutions of the four standard O-cyanoethyl-N,N-diisopropyl-phosphoramidites of 5'-O-dimethoxytrityl (DMT) protected deoxyribonucleosides (dG(ib), dA(bz), dC(bz), T) were analyzed by HPLC-MS. The solution stability of the phosphoramidites decreases in the order T, dC>dA>dG. After five weeks storage under inert gas atmosphere the amidite purity was reduced by 2% (T, dC), 6% (dA), and 39% (dG), respectively. The main degradation pathways involve hydrolysis, elimination of acrylonitrile and autocatalytic acrylonitrile-induced formation of cyanoethyl phosphonoamidates. Consequently, the rate of degradation is reduced by reducing the water concentration in solution with molecular sieves and by lowering the amidite concentration. Acid-catalyzed hydrolysis could also be reduced by addition of small amounts of base.

DNA Modification under Mild Conditions by Suzuki–Miyaura Cross‐Coupling for the Generation of Functional Probes

“… 9 This necessitates the synthesis of appropriate novel phosphoramidite building blocks, which are typically oxidation prone. 10 Stability of the desired modification during the synthesis is a prerequisite. Even when these requirements are met, the incorporation of bulky building blocks can decrease the coupling efficiency.…”

High-Density DNA and RNA microarrays - Photolithographic Synthesis, Hybridization and Preparation of Large Nucleic Acid Libraries

“…Stepwise coupling efficiencies above 99% have been calculated for all photosensitive DNA and RNA phosphoramidites, even for very short coupling times (15 s) 24 but lower coupling yields can occasionally occur, particularly in the case of dG amidites. The stability of the solubilized phosphoramidites at room temperature has been investigated before and was shown to depend on the nature of the nucleobase, with guanosine phosphoramidites prone to extensive degradation in only a matter of days 29,30 . But when stored at -25 °C, dG phosphoramidites dissolved in ACN as 30 mM solution were found to be stable for several weeks.…”

Solid-phase synthesis and structural characterisation of phosphoroselenolate-modified DNA: a backbone analogue which does not impose conformational bias and facilitates SAD X-ray crystallography

“…2). Low levels (<4%) of putative H-phosphonate derivatives were also apparent and as this can be associated with autocatalytic degradation, 68,69 the stability of a dimer phosphoramidite in solution was examined by 31 55 Excess reagents were removed following washing with acetonitrile, the CPG dried and suspended in 1 : 70 although somewhat weaker than that found in the corresponding triester.…”