Generation of guanine-amino acid cross-links by a free radical combination mechanism.
Generation of guanine-amino acid cross-links by a free radical combination mechanism.
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通过自由基结合机制产生鸟嘌呤-氨基酸交联。
DOI：
10.1039/c4cp00675e
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发表时间：
2014
期刊：
影响因子：
--
通讯作者：
Shafirovich,Vladimir
中科院分区：
文献类型：
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作者：
Uvaydov,Yuriy;Geacintov,NicholasE;Shafirovich,Vladimir
一种直接的方法已被开发用于体外合成稳定的DNA-蛋白质交联（DPC）之间的鸟嘌呤和氨基酸（赖氨酸和精氨酸）。该方法采用鸟嘌呤中性自由基、G（-H）stec和侧链C-中心氨基酸自由基的组合。后者是在用纳秒级308 nm强激光脉冲选择性电离5′-d（CC[2AP]TCGCTACC）中的2-氨基嘌呤（2AP）后间接产生的。2AP自由基阳离子快速去质子化以形成2AP（-H）stec中性自由基，2AP（-H）stec中性自由基继而氧化附近的鸟嘌呤以形成中性鸟嘌呤G（-H）stec自由基，如先前所述（Shafirovich等人，J. Phys. Chem. B，2001，105，8431）。同时，由2AP的光电离产生的水合电子被一氧化二氮清除以产生羟基自由基。在存在大量过量氨基酸的情况下，羟基自由基氧化后者以产生C-中心氨基酸自由基，该C-中心氨基酸自由基与G（-H）stecradicals联合收割机结合以形成鸟嘌呤-氨基酸交联寡核苷酸产物。用266 nm Nd：YAG激光脉冲辐照游离核苷，产生类似产物2′，3 ′，5 ′-三-O-乙酰基鸟苷（tri-O-Ac-Guo）。通过HPLC方法分离由此产生的位点特异性定位在寡核苷酸中或游离核苷三-O-Ac-Guo中的鸟嘌呤-氨基酸交联，并通过高分辨率LC-TOF/MS和LC-MS/MS方法鉴定。类似的鸟嘌呤-氨基酸交联的产品可以在体内形成的可能性进行了讨论，在氧化应激过程中使用单击自由基生成机制。
A direct method has been developed for the in vitro synthesis of stable DNA–protein cross-links (DPC's) between guanine and amino acids (lysine and arginine). This method employs the combination of guanine neutral radicals, G(–H)˙, and side-chain C-centered amino acid radicals. The latter were generated indirectly after first causing the selective photoionization of 2-aminopurine (2AP) embedded in the oligonucleotide, 5′-d(CC[2AP]TCGCTACC), by intense nanosecond 308 nm excimer laser pulses. The 2AP radical cation deprotonates rapidly to form the 2AP(–H)˙ neutral radical which, in turn, oxidizes the nearby guanine to form the neutral guanine G(–H)˙ radical, as described previously (Shafirovich et al., J. Phys. Chem. B, 2001, 105, 8431). In parallel, the hydrated electrons, generated by the photoionization of 2AP, are scavenged by nitrous oxide to generate hydroxyl radicals. In the presence of a large excess of the amino acids, the hydroxyl radicals oxidize the latter to produce C-centered amino acid radicals that combine with the G(–H)˙ radicals to form the guanine–amino acid cross-linked oligonucleotide product. Analogous products were generated by photoionizing the free nucleoside, 2′,3′,5′-tri-O-acetylguanosine, (tri-O-Ac-Guo), using intense nanosecond 266 nm Nd:YAG laser pulse irradiation. The guanine–amino acid cross-links thus produced site-specifically positioned either in oligonucleotides, or in the free nucleoside tri-O-Ac-Guo were isolated by HPLC methods and identified by high resolution LC-TOF/MS and LC-MS/MS methods. The possibility that analogous guanine–amino acid cross-linked products could be formed in vivo using single hit radical generation mechanisms during oxidative stress is discussed.