title: 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-phosphorus atom as methylphosphonate, was used as a substrate of terminal deoxynucleotidyltransferase (TDTase) in the presence of oligonucleotide (5'-GCTGTATCGTCAAGGCACTC-3') as an initiator. The reaction products were separated into two components by reverse-phase high-performance liquid chromatography (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'-methyl phosphonyl-5'-thymidin e (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.
authors.affiliation: Research Institute for Molecular Genetics, Tsumura & Company, Ibaraki, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Higuchi
authors.forename: T
authors.initials: T
authors.lastname: Endo
authors.forename: A
authors.initials: A
authors.lastname: Kaji
mesh_terms: D001483:Base Sequence; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis*; D009943:Organophosphorus Compounds; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D013237:Stereoisomerism
chemical_list: D009838:Oligodeoxyribonucleotides; D009943:Organophosphorus Compounds; C032627:methylphosphonic acid; D010727:Phosphoric Diester Hydrolases; D043264:Phosphodiesterase I; C470790:phosphodiesterase I, snake venom