Improved process for the preparation of nucleosidic phosphoramidites usinga safer and cheaper activator

A new, simplified commercial process for the preparation of nucleosidic pho sphoramidites, key raw materials for the automated solid-supported synthesi s of oligonucleotide-based drugs, was developed. Phosphitylation of a varie ty of protected nucleosidic derivatives (1-4) with a small excess of 2-cyan o-ethyl-N,N,N',N'-tetraisopropyl phosphoramidite (5, bis-reagent) and pyrid inium trifluoroacetate (Py . TFA) as the activator in an appropriate solven t at room temperature formed 75-96% of desired nucleosidic phosphoramidite products in less than 2 h, An efficient nonaqueous work-up has been develop ed to further streamline the isolation of moisture-sensitive P(III) nucleos idic compounds, The key finding is the use of Py . TFA, which is effective, inexpensive, stable, less acidic (pK(a) 5.2) than 1H-tetrazole, nontoxic, safe, and highly soluble in organic solvents. The reaction mechanism for ph osphitylation with Py . TFA as an activator has also been studied. An impro ved, robust, and versatile process for the preparation of nucleotide phosph oramidites under very concentrated reaction conditions was developed to sup port commercial manufacture of oligonucleotide-based drugs.