Enzymatic Oxidation Of Drugs To Toxic And Carcinogenic Metabolites

我们的实验室一直专注于合成方法的发展，使可能的PAH-加合核苷，以及位点特异性加合寡核苷酸在任何所需的序列背景下，在一个多毫克的规模，适合于NMR和X-射线晶体学研究以及生物化学调查相对容易的合成。 所采取的方法是制备适当保护形式的适当加合核苷，并将其转化为亚磷酰胺用于固相寡核苷酸合成。 今年，我们报道了高立体选择性的方法来合成脱氧腺苷（dAdo）和脱氧鸟苷（dGuo）加合物和它们的亚磷酰胺衍生的原型致癌物PAH，苯并（a）芘（BaP）。 一种这样的方法涉及使用氟化醇作为溶剂，用于通过受保护的嘌呤核苷的环外氨基使BaP二醇环氧化物开环（1）。 通过改变三氟乙醇（TFE）、全氟-叔丁醇或六氟-2-丙醇（HFP）与二醇环氧化物的摩尔比，可以实现在BaP的苄基C10位上保护的dGuo的环外2-氨基的顺式与反式加成的立体选择性的大的变化。通过使用这种方法与新开发的高效HPLC分离方法相结合，BaP N2-dGuo加合物的四种可能的亚磷酰胺（顺式/反式，R/S）可以在高达1克的规模上制备用于寡核苷酸合成。 我们还采用了一种新的，高度区域选择性的取代反应（2）的C10乙酰氧基的四醇四乙酸酯（乙酰化水解产物的BaP二醇环氧化物）在TFE或HFP的保护dGuo得到C10加合物。 该反应以高产率进行，为制备PAH加合物寡核苷酸结构单元提供了一种新的策略。 氟化醇方法还用于从相应的3，4-二醇二乙酸酯制备环戊并（cd）芘3，4-氧化物的C3处的dGuo加合物，产率为75-85%，并且具有优异的区域选择性（3）。 利用我们的合成方法，在DNA中的主要BaP二醇环氧化物加合物，BaP-N2-脱氧鸟苷，被放置在合成的寡核苷酸中的模板-引物连接处。 这种与Y家族DNA聚合酶Dpo 4复合的修饰DNA的晶体结构（4）揭示了大体积PAH加合物与聚合酶相互作用的三种可能后果：复制阻断、延伸越过错配病变和-1移码突变。在生产性结构中，庞大的加合物从DNA螺旋中翻转/环出，进入酶的小指和核心结构域之间的结构间隙。这些观察结果，结合复制和诱变数据，提出了一个模型，其中该缺口提供了一个结合位点，稳定的extrahelical核苷酸，并允许病变旁路产生的碱基取代和-1移码突变。

Our laboratory has focused on the development of synthetic methods which make possible the relatively facile synthesis of PAH-adducted nucleosides, as well as site-specifically adducted oligonucleotides in any desired sequence context on a multi-milligram scale, suitable for NMR and X-ray crystallographic studies as well as for biochemical investigations. The approach taken is to prepare the appropriate adducted nucleosides in suitably protected form and to convert them to phosphoramidites for use in solid phase oligonucleotide synthesis. This year we have reported highly stereoselective methods for the synthesis of deoxyadenosine (dAdo) and deoxyguanosine (dGuo) adducts and their phosphoramidites derived from the prototypical carcinogenic PAH, benzo(a)pyrene (BaP). One such method involves the use of fluorinated alcohols as solvents for the ring opening of BaP diol epoxides by the exocyclic amino groups of protected purine nucleosides (1). By varying the molar ratios of trifluoroethanol (TFE), perfluoro-tert-butanol or hexafluoro-2-propanol (HFP) to diol epoxide, large changes in the stereoselectivity for cis vs. trans addition of the exocyclic 2-amino group of protected dGuo at the benzylic C10 position of BaP could be achieved. By use of this approach in combination with a newly developed and highly efficient HPLC separation method, the four possible phosphoramidites (cis/trans, R/S) of the BaP N2-dGuo adducts could be prepared on scales up to a gram for use in oligonucleotide synthesis. We have also employed a novel, highly regioselective substitution reaction (2) of the C10 acetoxy group of tetraol tetraacetates (acetylated hydrolysis products of the BaP diol epoxides) in TFE or HFP by protected dGuo to give the C10 adducts. This reaction, which proceeds in high yield, provides a new strategy for the preparation of PAH-adducted oligonucleotide building blocks. The fluorinated alcohol approach was also used to prepare dGuo adducts at C3 of cyclopenta(cd)pyrene 3,4-oxide from the corresponding 3,4-diol diacetates in 75-85% yield and with excellent regioselectivity (3). Utilizing our synthetic methodology, the major BaP diol epoxide adduct in DNA, BaP-N2-deoxyguanosine, was placed at a template-primer junction in a synthetic oligonucleotide. Crystal structures (4) of this modified DNA in complex with the Y-family DNA polymerase Dpo4 revealed three possible consequences of interaction of the bulky PAH adduct with a polymerase enzyme: replication blockage, extension past a mismatched lesion, and a -1 frameshift mutation. In the productive structures, the bulky adduct was flipped/looped out of the DNA helix into a structural gap between the little finger and core domains of the enzyme. These observations, in combination with replication and mutagenesis data, suggest a model in which this gap provides a binding site that stabilizes the extrahelical nucleotide and permits lesion bypass by generation of base substitutions and -1 frameshift mutations.