Enzymic synthesis of oligonucleotides containing methylphosphonate internucleotide linkages

Abstract

Thymidine 5''-O-(pyrophosphoryl methylphosphonate) (dTTP.alpha.CH3) has been chemically synthesized by condensation of thymidine 5''-O-(methylphosphonate) with pyrophosphate. This novel nucleotide, which contained an .alpha.-phorphorus atom as methylphosphonate, was used as a substrate of terminal deoxynucleotidyl transferase (TDTase) in the presence of oligonucleotide (5''-GCTGTATCGTCA-AGGCACTC-3'') as an initiator. The reaction products were separated into two components by reverse-phase high-performance liquid chromotography (RP-HPLC). These products were, after purification, digested with nuclease P1 and alkaline phosphatase followed by separation of digested products by RP-HPLC. The result showed the presence of one of the isomers of 2''-deoxycytidyl-3''-methylphosphonyl-5''-thymidine (dCpCH3T) and 2''-deoxycytidyl-3''-methylphosphonyl-5''-thymidyl-3''-methylphosphonyl-5''-thymidine (dCpCH3TpCH3T), respectively. Fast atom bombardment mass spectrometry of these products further supported identification of the dinucleotide and the trinucleotide. These results indicated that dTTP.alpha.CH3 was used as a substrate of TDTase, resulting in methylphosphonate linkages. Produced oligomers were resistant to hydrolysis by snake venom phosphodiesterase I.