Povirk L F, Goldberg I H
Nucleic Acids Res. 1982 Oct 25;10(20):6255-64. doi: 10.1093/nar/10.20.6255.
The nonprotein chromophore of neocarzinostatin forms a variety of adducts with DNA. The predominant adduct recovered from nuclease digests of chromophore-treated poly(dA-dT). poly(dA-dT) is a compound with structure chromophore-d(TpApT). Mild acid hydrolysis of this compound released free adenine, while snake venom exonuclease (pH 6.5) released 5'-dTMP leaving in both cases adducts of slightly altered chromatographic mobility. These results eliminate adenine and 5'-dTMP as possible sites of covalent chromophore attachment. Electrophoresis data suggest that the adduct is not a phosphotriester. At pH 8.6, chromophore-d(TpApT) spontaneously hydrolyzed, releasing chromophore and 3'-dTMP, leaving a modified d(ApT) which contained deoxyadenosine-5'-aldehyde. Deoxyadenosine-5'-aldehyde was released from the modified d(ApT) by snake venom exonuclease, and identified by a series of derivatizations including 1) mild oxidation to deoxyadenosine-5'-carboxylic acid, 2) NaBH4 reduction to deoxyadenosine, and 3) formation of a hydrazone with phenylhydrazine. Since deoxyadenosine-5'-aldehyde cannot exist as such in the chromophore-d(TpApT) adduct, we suggest that the chromophore may be covalently attached to the C-5' of deoxyadenosine as a phosphorylacetal or similar structure. Hydrolysis of the chromophore-acetal bond at pH 8.6 would leave a phosphorylhemiacetal on C-5', which would be expected to spontaneously decompose to yield the observed 3'-phosphate and 5'-aldehyde groups.
新制癌菌素的非蛋白质发色团与DNA形成多种加合物。从经发色团处理的聚（dA-dT）·聚（dA-dT）的核酸酶消化产物中回收的主要加合物是一种具有发色团-d（TpApT）结构的化合物。该化合物经温和酸水解释放出游离腺嘌呤，而蛇毒外切核酸酶（pH 6.5）释放出5'-dTMP，在这两种情况下，加合物的色谱迁移率均略有改变。这些结果排除了腺嘌呤和5'-dTMP作为发色团共价连接的可能位点。电泳数据表明该加合物不是磷酸三酯。在pH 8.6时，发色团-d（TpApT）自发水解，释放出发色团和3'-dTMP，留下一种修饰的d（ApT），其中含有脱氧腺苷-5'-醛。脱氧腺苷-5'-醛通过蛇毒外切核酸酶从修饰的d（ApT）中释放出来，并通过一系列衍生化反应进行鉴定，包括：1）温和氧化为脱氧腺苷-5'-羧酸；2）用NaBH4还原为脱氧腺苷；3）与苯肼形成腙。由于脱氧腺苷-5'-醛在发色团-d（TpApT）加合物中不能以这种形式存在，我们认为发色团可能以磷酰缩醛或类似结构共价连接到脱氧腺苷的C-5'上。在pH 8.6时，发色团-缩醛键的水解会在C-5'上留下磷酰半缩醛，预计会自发分解产生观察到的3'-磷酸基团和5'-醛基团。
