新型过氧化物酶荧光底物的合成及其性能的研究

合成了辣根过氧化物酶的５种荧光底物，它们分别是：Ｎ，Ｎ′—二氰甲基邻苯二胺（ＤＣＭ－ＯＰＡ），３，４—二氧喹喔啉－２（１Ｈ）－酮（ＤＨＱ），３－甲基－３，４－二氢喹喔啉－２（１Ｈ）－酮（ＭＤＨＱ），３，４－二氢喹喔啉－２（１Ｈ）－酮－６－羧酸（ＤＨＱ－６－Ａ）及３－甲基－３，４－二氢喹喔啉－２（１Ｈ）－酮－６－羧酸（ＭＤＨＱ－６－Ａ）。其中荧光底物ＤＣＭ－ＯＰＡ，ＤＨＱ和ＭＤＨＱ都可以很容易地进行大量纯品的制备。我们用流动注射分析法对合成底物和已有的最佳荧光底物在ＨＲＰ及其模拟酶氯化血红素催化体系中的性能进行了比较研究。结果表明，ＤＣＭ－ＯＰＡ和ＭＤＨＱ是合成的５种底物中最好的两个，而对羟基苯丙酸（ｐ－ＨＰＰＡ）和对羟基苯乙酸（ｐ－ＨＰＡ）优于高香草酸（ＨＶＡ）和酪氨。底物ｐ－ＨＰＰＡ和ｐ－ＨＰＡ与合成底物ＤＣＭ－ＯＰＡ和ＭＤＨＱ体系在对过氧化氢的分析方法性能相当，最低检测限都在１～１０ｎｍｏｌ／Ｌ之间。对酶的检测，ＤＣＭ－ＯＰＡ是所有研究的底物中最灵敏的，上述两种合成底物保存在４℃的冰箱中都可以稳定一个月以上。

Studies on the Synthesis and Properties of New Fluorogenic Substrates for Peroxidase

Four 3,4-dihydroquinoxalin-2(1H)-one derivatives,i.e.,3,4-dihydroquinoxalin-2(1H)-one(DHQ),3-methyl-3,4-dihydroquinoxalin-2(1H)-one(MDHQ),3,4-dihydroquinoxalin-2(1H)-one-6-acid and 3-methyl-3,4-dihydroquinoxalin-2(1H)-one-6-acid, and N,N′-dicyanomethyl-o-phenylenediamine(DCM-OPA) were systhesised as potential substrates for horseradish peroxidase(HPR). Among these compounds DCM-OPA, DHQ and MDHQ can be prepared by very simple methods in a pure form in large quantities. Their properties for use as fluorogenic substrates for HRP and its mimetic enzyme hemin were comparatively studied with commercially available substrates,i.e., p-hydroxyphenylacetic acid(p-HPPA),p-hydroxypropionic acid(p-HPPA),homovanilic acid(HVA) and tyramine, by a flow injection method. The results showed that DCM-OPA and MDHQ are the best among the five synthesised substrates, and p-HPPA and p-HPA are better than HVA and tyramine. Substrates p-HPPA, p-HPA,DCM-OPA and MDHQ showed comparable ability for H 2O 2 detection in HRP and hemin catalysed reaction systems with the lowest detection limits in the range of 1～10 nmol/L region. For the detection of enzyme DCM-OPA is the most sensitive one of all the substrates studied. The stability of DCM-OPA is better than MDHQ, and both of them are stable at least for a month in a refrigerator.