A Chemical 5′-Phosphorylation of Oligodeoxyribonucleotides

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Abstract
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(β-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.