Park Soobong, Anderson Christopher, Loeber Rachel, Seetharaman Mahadevan, Jones Roger, Tretyakova Natalia
Cancer Center and the Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455, USA.
J Am Chem Soc. 2005 Oct 19;127(41):14355-65. doi: 10.1021/ja051979x.
1,2,3,4-Diepoxybutane (DEB) is a bifunctional electrophile capable of forming DNA-DNA and DNA-protein cross-links. DNA alkylation by DEB produces N7-(2'-hydroxy-3',4'-epoxybut-1'-yl)-guanine monoadducts, which can then form 1,4-bis-(guan-7-yl)-2,3-butanediol (bis-N7G-BD) lesions. All three optical isomers of DEB are produced metabolically from 1,3-butadiene, but S,S-DEB is the most cytotoxic and genotoxic. In the present work, interstrand and intrastrand DNA-DNA cross-linking by individual DEB stereoisomers was investigated by PAGE, mass spectrometry, and stable isotope labeling. S,S-, R,R-, and meso-diepoxides were synthesized from l-dimethyl-2,3-O-isopropylidene-tartrate, d-dimethyl-2,3-O-isopropylidene-tartrate, and meso-erythritol, respectively. Total numbers of bis-N7G-BD lesions (intrastrand and interstrand) in calf thymus DNA treated separately with S,S-, R,R-, or meso-DEB (0.01-0.5 mM) were similar as determined by capillary HPLC-ESI(+)-MS/MS of DNA hydrolysates. However, denaturing PAGE has revealed that S,S-DEB produced the highest number of interchain cross-links in 5'-GGC-3'/3'-CCG-5' sequences. Intrastrand adduct formation by DEB was investigated by a novel methodology based on stable isotope labeling HPLC-ESI(+)-MS/MS. Meso DEB treatment of DNA duplexes containing 5'-[1,7, NH(2)-(15)N(3),2-(13)C-G]GC-3'/3'-CCG-5' and 5'-GGC-3'/3'-CC[(15)N(3),2-(13)C-G]-5' trinucleotides gave rise to comparable numbers of 1,2-intrastrand and 1,3-interstrand bis-N7G-BD cross-links, while S,S DEB produced few intrastrand lesions. R,R-DEB treated DNA contained mostly 1,3-interstrand bis-N7G-BD, along with smaller amounts of 1,2-interstrand and 1,2-intrastrand adducts. The effects of DEB stereochemistry on its ability to form DNA-DNA cross-links may be rationalized by the spatial relationships between the epoxy alcohol side chains in stereoisomeric N7-(2'-hydroxy-3',4'-epoxybut-1'-yl)-guanine adducts and their DNA environment. Different cross-linking specificities of DEB stereoisomers provide a likely structural basis for their distinct biological activities.
1,2,3,4-二环氧丁烷（DEB）是一种双功能亲电试剂，能够形成DNA-DNA和DNA-蛋白质交联。DEB对DNA的烷基化作用会产生N7-（2'-羟基-3'，4'-环氧丁-1'-基）-鸟嘌呤单加合物，该单加合物随后可形成1,4-双-（鸟嘌呤-7-基）-2,3-丁二醇（双-N7G-BD）损伤。DEB的所有三种旋光异构体均由1,3-丁二烯代谢产生，但S,S-DEB具有最强的细胞毒性和遗传毒性。在本研究中，通过聚丙烯酰胺凝胶电泳（PAGE）、质谱分析和稳定同位素标记法，对单个DEB立体异构体的链间和链内DNA-DNA交联进行了研究。S,S-、R,R-和内消旋二环氧物分别由L-二甲基-2,3-O-异亚丙基酒石酸酯、D-二甲基-2,3-O-异亚丙基酒石酸酯和内消旋赤藓糖醇合成。通过对DNA水解产物进行毛细管高效液相色谱-电喷雾电离（+）-串联质谱分析（HPLC-ESI(+)-MS/MS）测定，分别用S,S-、R,R-或内消旋DEB（0.01 - 0.5 mM）处理的小牛胸腺DNA中双-N7G-BD损伤（链内和链间）的总数相似。然而，变性PAGE显示，S,S-DEB在5'-GGC-3'/3'-CCG-5'序列中产生的链间交联数量最多。基于稳定同位素标记的HPLC-ESI(+)-MS/MS新技术，对DEB的链内加合物形成进行了研究。用内消旋DEB处理含有5'-[1,7, NH(2)-(15)N(3),2-(13)C-G]GC-3'/3'-CCG-5'和5'-GGC-3'/3'-CC[(15)N(3),2-(13)C-G]-5'三核苷酸的DNA双链体，产生了数量相当的1,2-链内和1,3-链间双-N7G-BD交联，而S,S-DEB产生的链内损伤较少。R,R-DEB处理的DNA主要含有1,
