SOLID-PHASE ELECTROPHILE TRAPPING AGENTS

这项提议的目的是为研究 致癌物的代谢激活。该工具将由一个 固相固载的亲核硫醇，例如聚苯乙烯， 聚丙烯酰胺，或受控孔玻璃。亲核者将会是 通过可被切割的连杆固定在固相上 一种特定的试剂，如联氨，以提供可被 经高效液相色谱或气相色谱分析。负载型亲核试剂将与 致癌物和待研究的细胞或亚细胞组分。后 适当的时间段，将固相除去，洗涤干净 所有的粘连材料，亲核剂从其载体上剥离，以及 通过适当的色谱技术对产品进行分析。 这种方法与更常见的 添加可溶的亲核剂，如3，4-二氯苯硫酚。首先， 含有捕获的亲电性的组分可以从 复杂的孵化基质通过简单的过滤步骤，去除各种 可能会对后续产品造成干扰的产品 层析分析。第二，亲核试剂的结构可以是 易于定制，为被捕者提供最佳的层析性能 产品。例如，可以加入卤素来促进GC 用灵敏的EC检测器或荧光发色团进行分析 包括允许荧光检测器与高效液相色谱仪一起使用。第三，在 与整个细胞孵育，所支持的亲核试剂将无法 进入细胞，因此只会检测到足够稳定的亲电体 到达细胞外的培养液。这在以下方面尤其有用： 对被认为需要代谢活化的致癌物的研究 一个器官，通常是肝脏，产生一种针对某些 其他器官。 新试剂将应用于药物代谢的研究。 二甲基亚硝胺，因为有相互矛盾的报道 通常的硫醇亲核剂，2，4-二氯苯硫酚，以捕获 甲基化亚硝胺代谢过程中产生的甲基化物种这个 新的试剂也将用于研究物种差异。 一系列β-氧化亚硝胺的代谢。

The goal of this proposal is to prepare a new tool for the study of metabolic activation of carcinogens. This tool will consist of a nucleophilic thiol supported on a solid phase, such a polystyrene, polyacrylamide, or controlled pore glass. The nucleophile will be anchored to the solid phase by means of a linkage which can be cleaved by a specific agent, such as hydrazine, to give products which can be analyzed by HPLC or GC. The supported nucleophile will be incubated with the carcinogen and cell or subcellular fraction to be studied. After the appropriate time period, the solid phase will be removed, washed free of all adhering material, the nucleophile cleaved from its support, and the products analyzed by the appropriate chromatographic technique. There are several advantages of this approach over the more usual one of adding a soluble nucleophile such as 3,4-dichlorobenzenethiol. First, the fraction containing the trapped electrophiles may be isolated from the complex incubation matrix by a simple filtration step, removing a variety of products which could potentially interfere with subsequent chromatographic analysis. Second, the structure of the nucleophile can be readily tailored to give optimal chromatographic properties to the trapped products. For example, halogen can be incorporated to facilitate GC analysis with a sensitive EC detector, or a fluorescent chromophore can be included to allow a fluorescence detector to be used with HPLC. Third, in incubations with whole cells, the supported nucleophiles would be unable to enter cells, and thus would detect only electrophiles stable enough to reach the extracellular medium. This would be especially useful in the study of carcinogens which are thought to require metabolic activation by one organ, usually liver, to produce an electrophile which targets some other organ. The new agents will be applied to study of metabolism of dimethylnitrosamine, since there are conflicting reports of the ability of the usual thiol nucleophile, 2,4-dichlorobenzenethiol, to trap a methylating species produced during metabolism of this nitrosamine. The new agents will also be used for study of species differences in metabolism of a series of beta-oxidized nitrosamines.