Evidence for direct attack by hydroxide in phosphodiester hydrolysis
Evidence for direct attack by hydroxide in phosphodiester hydrolysis
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DOI：
10.1021/ja020823j
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发表时间：
2002-09-18
影响因子：
15
通讯作者：
Harris, ME
中科院分区：
文献类型：
--
作者：
Cassano, AG;Anderson, VE;Harris, ME
磷酸二酯水解由于其在生物学中的重要性，一直是研究的热点。尽管对磷酸二酯裂解机理有许多重要的分析，但对这些反应中的亲核试剂的了解相对较少。为了确定氢氧化物在胸苷- 5′-对硝基苯基磷酸水解过程中是作为亲核试剂还是一般碱，我们测定了溶剂亲核试剂（18knuc）的溶剂氘同位素效应（D2Ok）、离子强度效应和18o同位素效应。氢氧化物催化的磷酸二酯水解的d20k值是略相反的（0.9±0.1），表明在过渡态中没有发生质子转移。在过氧化氢中观察到明显的α效应，表明氧阴离子在反应中可以作为亲核试剂。此外，氢氧化物和过氧化氢催化的离子强度依赖关系难以区分，表明它们通过相同的机制起作用。最后，氢氧化物催化反应的18指关节为1.068±0.007，远远超过了水和氢氧化物之间的平衡18o同位素效应（1.040±0.003）。总之，这些数据与氢氧根直接亲核攻击最为一致。根据观测到的18节和已知的平衡分量，计算出同位素效应的动力学分量为1.027±0.010。这种大的动力学成分表明，在过渡态中，与亲核试剂的键序很少。
Phosphodiester hydrolysis has been the subject of intense study due to its importance in biology. Despite the numerous significant analyses of phosphodiester cleavage mechansim, comparatively little is known about the nucleophiles in these reactions. To determine whether hydroxide acts as a nucleophile or a general base in the hydrolysis of thymidine-5‘-p-nitrophenyl phosphate,we determined solvent deuterium isotope effects (D2Ok), ionic strength effects, and18O isotope effects on the solvent nucleophile (18knuc). TheD2Okfor hydroxide-catalyzed phosphodiester hydrolysis is slightly inverse (0.9 ± 0.1), suggesting that a proton transfer does not occur in the transition state. A significant α effect is observed with hydroperoxide, demonstrating that oxyanions can act as nucleophiles in the reaction. Additionally, the ionic strength dependencies of hydroxide and hydroperoxide catalysis are indistinguishable, suggesting that they act by the same mechanism. Finally, the18knucfor the hydroxide-catalyzed reaction is 1.068 ± 0.007, well in excess of the equilibrium18O isotope effect between water and hydroxide (1.040 ± 0.003). Together, the data are most consistent with direct nucleophilic attack by hydroxide. From the observed18knucand the known equilibrium component, the kinetic component of the isotope effect was calculated to be 1.027 ± 0.010. This large kinetic component suggests that little bond order to the nucleophile occurs in the transition state.