Benzo[<i>a</i>]pyrene Diol Epoxide Forms Covalent Adducts with Deoxycytidylic Acid by Alkylation at Both Exocyclic Amino N⁴ and Ring Imino N-3 Positions

The carcinogen 7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) alkylates DNA at dGuo, dAdo, and dCyd. dCyd adducts, formed in small amounts, elute near the more abundant dGuo adducts. We isolated the dCyd adducts formed with dCMP. Each BPDE enantiomer forms three major adducts with dCMP, two cis and one trans. The trans adduct and one of the cis adducts form by alkylation at exocyclic N4, while the second cis adduct is a dUrd adduct formed by alkylation at ring N-3 followed by deamination. Epoxide ring-opening geometries were assigned on the basis of halide and temperature effects on adduct yield, the sign of the major CD band, and benzo ring proton NMR coupling constants. One of each set of cis adducts is fluorescent (FL), and the other is nonfluorescent (NF). The trans and FL cis adducts have fluorescence quantum yields 40−50% of that of the BPDE hydrolysis product. The long wavelength UV maxima of the FL and NF cis adducts are red-shifted 1 and 3 nm relative to the trans adduct. 1H NMR deuterium exchange experiments indicate that in the trans and FL cis adducts N4−H is coupled to C10−H. Adduct formation experiments with methyl-protected Cyd derivatives show that NF cis adducts result from alkylation at N-3. MS results, pKa measurements, and dUrd alkylation experiments indicate that the N-3 dCyd adducts spontaneously deaminate to dUrd adducts. NMR coupling constants show that in the NF cis adduct the C7 and C8 substituents are quasi equatorial and the C9 substituent is quasi axial, unlike in other cis BPDE adducts. 1H NOESY spectra of the (−)-BPDE NF cis adduct reveal that it exists in two conformers. Molecular modeling shows that the conformers result from two low-energy conformations of very similar energies with the pyrimidine in opposite orientations, separated by significant barriers to rotation of the uracil moiety.致癌物7R,8T-二羟基-9T,10T-环氧-7,8,9,10-四氢苯并[a]芘（anti-BPDE）可在脱氧鸟苷（dGuo）、脱氧腺苷（dAdo）与脱氧胞苷（dCyd）位点与DNA发生烷基化修饰。少量生成的脱氧胞苷加合物，其洗脱峰紧邻丰度更高的脱氧鸟苷加合物。我们分离了与脱氧胞苷一磷酸（dCMP）结合形成的脱氧胞苷加合物。每一种BPDE对映体均可与dCMP形成三种主要加合物，其中两种为顺式加合物，一种为反式加合物。反式加合物与其中一种顺式加合物通过外环N4位的烷基化反应生成，而第二种顺式加合物为脱氧尿苷（dUrd）加合物，其形成过程为先在环N-3位发生烷基化，随后发生脱氨基反应。我们基于卤离子与温度对加合物产率的影响、主要圆二色（CD）谱带的符号以及苯环质子核磁共振（NMR）耦合常数，确定了环氧开环的立体构型。每一组顺式加合物中，一种具有荧光性（FL），另一种则无荧光性（NF）。反式加合物与具有荧光性的顺式加合物的荧光量子产率为BPDE水解产物的40%~50%。相较于反式加合物，具有荧光性与无荧光性的顺式加合物的长波长紫外吸收峰分别红移了1 nm与3 nm。氘交换质子核磁共振（1H NMR）实验结果表明，在反式加合物与具有荧光性的顺式加合物中，N4-H与C10-H存在自旋耦合作用。使用甲基保护的胞苷衍生物开展的加合物形成实验表明，无荧光性的顺式加合物源于N-3位的烷基化反应。质谱（MS）分析结果、酸解离常数（pKa）测定结果以及脱氧尿苷烷基化实验均证实，N-3位的脱氧胞苷加合物会自发发生脱氨基反应，转化为脱氧尿苷加合物。核磁共振耦合常数显示，与其他BPDE顺式加合物不同，无荧光性的顺式加合物中C7与C8取代基为准平伏键，C9取代基为准直立键。(−)-BPDE的无荧光性顺式加合物的1H核Overhauser效应谱（NOESY）结果显示，该加合物以两种构象体形式存在。分子模拟结果表明，这两种构象体源于两种能量极为相近的低能构象：嘧啶环取向相反，且尿嘧啶部分的旋转存在显著能垒，从而将两种构象体分隔开来。