(DIFLUOROMETHYLENE)PHOSPHATES OF GUANINE NUCLEOSIDES AS PROBES OF DNA-POLYMERASES AND G-PROTEINS
(DIFLUOROMETHYLENE)PHOSPHATES OF GUANINE NUCLEOSIDES AS PROBES OF DNA-POLYMERASES AND G-PROTEINS
复制标题
DOI：
10.1021/bi00481a010
复制
发表时间：
1990-07-24
期刊：
影响因子：
2.9
通讯作者：
WRIGHT, GE
中科院分区：
文献类型：
--
作者：
ARABSHAHI, L;KHAN, NN;WRIGHT, GE
合成了N_2-（对正丁基苯基）-2 ″-脱氧鸟苷和N_2-脱氧鸟苷的5 ″-多磷酸酯，它们含有一个二氟亚甲基取代磷酸酐氧。5“-[β，.伽马（二氟亚甲基）三磷酸盐]，包括2“-脱氧鸟苷的三磷酸盐，通过用1，1”-羰基二咪唑活化的相应5“-磷酸盐与二氟甲烷二膦酸盐反应来制备。N2-（对正丁基苯基）鸟苷的5“-[（二氟亚甲基）二磷酸]通过用二氟甲烷二膦酸酯处理保护的5”-甲苯磺酰基核苷，然后脱保护来制备。用1，1“-羰基二咪唑活化的该核苷酸与正磷酸盐缩合，得到N2-（对正丁基苯基）鸟苷5”-[（α，.贝塔二氟亚甲基）三磷酸盐]。产物通过31 P和19 F NMR光谱进行表征。测试膦酸酯从GTP结合蛋白细胞（EC）和致癌（Leu-61）Ha-ras p21置换[3 H]GDP的能力，以及它们抑制DNA聚合酶α的能力。中国仓鼠卵巢细胞。当CF 2基团在β，.γ的位置，但不是当它在α，。β的位置，并且它们不与相应的（二氟亚甲基）二磷酸结合。相反，CF 2组对DNA聚合酶α的抑制没有影响。通过N2-（对正丁基苯基）-2“-脱氧鸟苷5”-[（β，.伽马二氟亚甲基）三磷酸盐]。2“-脱氧鸟苷5”-[（β，.伽马二氟亚甲基）三磷酸]被发现是几种DNA聚合酶的真正底物，并且与枯草芽孢杆菌DNA聚合酶III相比具有比dGTP更低的表观Km。
5''-Polyphosphates of N2-(p-n-butylphenyl)-2''-deoxyguanosine and -guanosine which contain a difluoromethylene group in place of a phosphoanhydride oxygen have been synthesized. 5''-[.beta.,.gamma.-(Difluoromethylene)triphosphates], including that of 2''-deoxyguanosine, were prepared by reaction of the corresponding 5''-phosphates, activated by 1,1''-carbonyldiimidazole, with difluoromethanediphosphonate. The 5''-[(difluoromethylene)diphosphate] of N2-(p-n-butylphenyl)guanosine was prepared by treatment of a protected 5''-tosyl nucleoside with difluoromethanediphosphonate, followed by deprotection. Condensation of this nucleotide, activated with 1,1''-carbonyldiimidazole, with orthophosphate gave N2-(p-n-butylphenyl)guanosine 5''-[(.alpha.,.beta.-difluoromethylene)triphosphate]. Products were characterized by 31P and 19F NMR spectroscopy. The phosphonates were tested for their ability to displace [3H]GDP from the GTP binding proteins cellular (EC) and oncogenic (Leu-61) Ha-ras p21, and for their ability to inhibit DNA polymerase .alpha. from Chinese hamster ovary cells. The p21s bound weakly to a triphosphonate when the CF2 group was in the .beta.,.gamma. position, but not when it was in the .alpha.,.beta. position, and they did not bind to the corresponding (difluoromethylene)diphosphate. In contrast, the CF2 group had no effect on inhibition of DNA polymerase .alpha. by N2-(p-n-butylphenyl)-2''-deoxyguanosine 5''-[(.beta.,.gamma.-difluoromethylene)triphosphate]. 2''-Deoxyguanosine 5''-[(.beta.,.gamma.-difluoromethylene)triphosphate] was found to be a bona fide substrate for several DNA polymerases and had a lower apparent Km than dGTP with Bacillus subtilis DNA polymerase III.