Higuchi H, Endo T, Kaji A
Research Institute for Molecular Genetics, Tsumura & Company, Ibaraki, Japan.
Biochemistry. 1990 Sep 18;29(37):8747-53. doi: 10.1021/bi00489a035.
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.
胸苷5'-O-(焦磷酸磷酰甲基膦酸酯)(dTTPαCH3)已通过胸苷5'-O-(甲基膦酸酯)与焦磷酸缩合化学合成。这种新型核苷酸含有一个作为甲基膦酸酯的α-磷原子，在寡核苷酸(5'-GCTGTATCGTCAAGGCACTC-3')作为引发剂存在的情况下，用作末端脱氧核苷酸转移酶(TDTase)的底物。反应产物通过反相高效液相色谱(RP-HPLC)分离为两个组分。这些产物经纯化后，用核酸酶P1和碱性磷酸酶消化，然后通过RP-HPLC分离消化产物。结果分别显示存在2'-脱氧胞苷-3'-甲基膦酰基-5'-胸苷(dCpCH3T)和2'-脱氧胞苷-3'-甲基膦酰基-5'-胸苷酰-3'-甲基膦酰基-5'-胸苷(dCpCH3TpCH3T)的一种异构体。这些产物的快原子轰击质谱进一步支持了二核苷酸和三核苷酸的鉴定。这些结果表明dTTPαCH3用作TDTase的底物，产生甲基膦酸酯键。产生的寡聚物对蛇毒磷酸二酯酶I的水解具有抗性。
