新型吖啶酯类化学发光试剂的合成及其在肿瘤相关酶活性测定的研究

项目名称： 新型吖啶酯类化学发光试剂的合成及其在肿瘤相关酶活性测定的研究

项目编号： No.20805060

项目类型： 青年科学基金项目

立项/批准年度： 2009

项目学科： 化学工业

项目作者： 穆小静

作者单位： 重庆大学

项目金额： 20万元

中文摘要： 本项目合成了两种与癌症的发生相关的酶的化学发光底物。合成了用于对氧磷酶（PON）活性测定的吖啶酯化学发光底物8个。分别以重组人PON和血清催化这些底物的性质研究证明：其中的9-（4-氯代苯基）氧羰基-10甲基吖啶酯三氟甲基磺酸盐、9-（4-叔丁基苯基）氧羰基、9-（4-甲基苯基）氧羰基类似物可用于评价血清PON活性，灵敏度高，特异性好，节约底物用量，毒副作用小等优点；而9-甲氧羰基、而9-乙氧羰基基取代的类似物用于PON活性测定特异性较差；而9-（4-硝基苯基）氧羰基取代、和9-（2，6-二甲基苯基）氧羰基取代的类似物不能用于活性测定，分别因为自水解反应过快和催化水解反应过慢而不适合测定。以上述氯代吖啶酯底物为模型化合物，建立了测定血清PON活性的化学发光方法，考察了最适条件及干扰。方法用于临床病例与健康人的PON活性测定进一步表明，病人体内血清PON活性下降，这与以其它底物如乙酰苯酚、对氧磷为底物进行测定所得结论一致。第二部分设计合成了用于β65293;葡萄糖醛酸酶活性测定的底物β65293;葡萄糖醛酸与吖啶酯形成的糖苷，但初步的筛选研究表明设计的化合物对酶的选择性太小而不适于β65293;葡萄糖醛酸酶活性测定。

中文关键词： 吖啶酯；化学发光；底物；对氧磷酶；β65293;葡萄糖醛酸酶

英文摘要： The two categories of chemiluminescent substrates of enzymes related to cancer have been synthesized and evaluated.Eight acridinium esters as PON substrates were prepared and screened. It showed that 9-(4-chlorophenyloxycarbonyl)-10-methylacridinium triflate, 9-(4-tert-butylphenyloxycarbonyl)-10-methylacridinium trifluoromethanesulfonate, 9-(4-methylphenyloxycarbonyl)-10-methylacridinium trifluoromethanesulfonate,and 9-(3-methylphenyloxycarbonyl)-10-methylacridinium triflate have very good properties for serum PON activity assay. The method for PON activity assay based on these substrates have advantages of high sensitivity, high specificity, small amount of substrate and low toxocity. However, the 9-(methoxycarbonyl) and the 9-(ethoxylcarbonyl) acridinium analogues showed low specificity toward serum PON, while the 9-(4-nitrophenyloxycarbonyl) and the 9-(2,6-dimethylphenyloxycarbonyl) analogues were not eligible for PON assay,due to too quick self-hydrolysis and too slow hydrolysis under catalysis, respectively. Using the Cl-substituted acridinium ester as a model compound, a chemiluminescent method for serum PON activity assay was established, and the interferences were investigated and the conditions were optimised. The method was further used in clinical assay for evaluation PON activity levels in patients, and the results based on the acridinium was consistent with those based on phenyl acetate and paraoxon. Additionally, a glycoside was prepared by linking βlucuronic acid with a hydroxy acridine via a glycoside bond. Unexpectedly, the glucuronidase was not able to hydrolyze the glycoside bond of the compound, and therefore the intended acridinium glycoside cound not be used for the glucuronidase activity assay.

英文关键词： acrdinium esters; chemiluminescence; substrate; paraoxonase; βlucuronidase