Synthesis and characterization of oligonucleotides containing 2′-fluorinated thymidine glycol as inhibitors of the endonuclease III reaction
Synthesis and characterization of oligonucleotides containing 2′-fluorinated thymidine glycol as inhibitors of the endonuclease III reaction
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DOI：
10.1093/nar/gkl061
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发表时间：
2006-01-01
影响因子：
14.9
通讯作者：
Iwai, S
中科院分区：
文献类型：
--
作者：
Doi, Y;Katafuchi, A;Iwai, S
核酸内切酶 III (Endo III) 是一种碱基切除修复酶，可识别包括胸腺嘧啶乙二醇在内的氧化嘧啶碱基。该酶是一种糖基化酶/裂合酶，在去除受损碱基后与底物形成希夫碱型中间体。为了通过X射线晶体学研究其底物识别机制，我们合成了含有2'-氟胸苷二醇的寡核苷酸，期望2'位置的吸电子氟原子能够稳定共价中间体，正如我们在之前的研究中对T4核酸内切酶V（Endo V）所观察到的那样。用 OsO4 氧化 5'-和 3'-保护的 2'-氟胸苷产生胸腺嘧啶二醇的两种异构体。在羟基官能团保护后，通过核磁共振波谱法确定它们的构型。 (5R,6S)和(5S,6R)异构体的比率为3:1，而在未修饰的糖的情况下该比率为6:1。两种胸苷二醇异构体均转化为相应的亚磷酰胺结构单元，并掺入寡核苷酸中。当含有2'-氟化5R-或5S-胸苷二醇的双链体用大肠杆菌endo III处理时，无论C5处的立体化学如何，都没有观察到稳定的共价中间体。发现 5S 异构体形成酶-DNA 复合物，但切口可能受到氟诱导的糖苷键稳定性的抑制。
Endonuclease III (Endo III) is a base excision repair enzyme that recognizes oxidized pyrimidine bases including thymine glycol. This enzyme is a glycosylase/lyase and forms a Schiff base-type intermediate with the substrate after the damaged base is removed. To investigate the mechanism of its substrate recognition by X-ray crystallography, we have synthesized oligonucleotides containing 2'-fluorothymidine glycol, expecting that the electron-withdrawing fluorine atom at the 2' position would stabilize the covalent intermediate, as observed for T4 endonuclease V (Endo V) in our previous study. Oxidation of 5'- and 3'-protected 2'-fluorothymidine with OsO4 produced two isomers of thymine glycol. Their configurations were determined by NMR spectroscopy after protection of the hydroxyl functions. The ratio of (5R,6S) and (5S,6R) isomers was 3:1, whereas this ratio was 6:1 in the case of the unmodified sugar. Both of the thymidine glycol isomers were converted to the corresponding phosphoramidite building blocks and were incorporated into oligonucleotides. When the duplexes containing 2'-fluorinated 5R- or 5S-thymidine glycol were treated with Escherichia coli endo III, no stabilized covalent intermediate was observed regardless of the stereochemistry at C5. The 5S isomer was found to form an enzyme-DNA complex, but the incision was inhibited probably by the fluorine-induced stabilization of the glycosidic bond.