A chemical 5'-phosphorylation of oligodeoxyribonucleotides

To circumvent the use of T4 polynucleotide kinase and ATP for the 5'-phosphorylation of synthetic oligodeoxyribonucleotides after deprotection and purification, we have developed a new chemical phosphorylation reagent that can be used on automated DNA synthesis instruments. The phosphite-derived compound, bis(beta-cyanoethoxy)-N,N-diisopropylaminophosphine, is coupled to a solid-supported fragment and oxidized under the same conditions as nucleoside phosphoramidites. The resultant 5'-phosphate oligomer is then fully deprotected in standard ammonium hydroxide solution at elevated temperature. Reverse-phase, high-pressure liquid chromatography, silica thin-layer chromatography, and 31P-nuclear magnetic resonance spectra of chemically phosphorylated thymidine were compared with a variety of phosphorus derivatives of thymidine. All evidence suggested that the nucleoside was converted to the 5'-phosphate form quantitatively with the reagent. Moreover, an excellent enzymatic ligation reaction was obtained with a chemically phosphorylated oligonucleotide.