真菌漆酶催化Domino反应制备多羟基苯并呋喃类物质机理研究

Natural compounds with a polyhydroxylated benzofuran skeleton are well known for their broad range of biological activities. Due to scarce in natural resources, many of them have become the star molecules of total synthesis. But it was complicated usually involving multisteps and expensive. Using fungal laccase in preparation of polyhydroxylated benzofurans had never been reported until 2007. The mechanism of this Dominio reaction is nothing but proposed. For the first time the intermediate of Domino reaction was isolated by the applicant in initial experiments. And this intermediate was quickly converted to the product without the help of fungal laccase at room temperature. So it seemed that these findings were not in agreement with former proposed mechanisms. In this project mechanism is to be deduced based on EPR technology, selection of specific raw materials and accurate isolation of the intermediate. And this surely will offer a theoretical basis for designing polyhydroxylated benzofurans. At the same time, the intrinsic mechanism will be disclosed when digging into laccase (from culture of Daldinia eschscholzii IFB-TL01 residing in the gut of the mantis species Tenodera aridifolia) structural basis using homology model or crystallization strategy. It is to lay the foundation for future application research on this laccase owning proprietary intellectual property rights.

多羟基苯并呋喃类天然产物因具有广泛的生理活性而备受关注。由于天然来源稀少，其中许多已成为全合成的明星分子，然而全合成制备繁琐，成本高昂。2007年发现可使用绿色环保的漆酶简单高效催化Domino反应制备此类物质，但该酶催化反应的机理研究始终处于假设推断阶段。我们前期实验中首次分离到Domino 反应的中间体，并发现该中间体常温时无需漆酶作用便可迅速转化成目标产物，与此前提出的假设机理不一致。本项目拟通过EPR(电子顺磁共振)技术结合特定原料的选择及中间体的分离和结构鉴定，明确真菌漆酶催化Domino反应的控制和影响，阐明真菌漆酶催化Domino反应的机理，为今后真菌漆酶催化Domino反应制备多羟基苯并呋喃类物质的设计提供理论基础。同时拟通过同源模建、结构解析等方法阐明螳螂肠道内生真菌IFB-TL01来源漆酶与其催化功能相关的结构基础，为这一拥有自主知识产权漆酶的应用研究奠定基础。

多羟基苯并呋喃类天然产物因具有广泛的生理活性而备受关注。由于天然来源稀少，其中许多已成为全合成的明星分子，然而全合成制备繁琐，成本高昂。2007年发现可使用绿色环保的漆酶简单高效催化Domino反应制备此类物质，但该酶催化反应的机理研究始终处于假设推断阶段。我们前期实验中首次分离到Domino 反应的中间体，并发现该中间体常温时无需漆酶作用便可迅速转化成目标产物，与此前提出的假设机理不一致。本项目探讨了不同漆酶的酶学性质，合成了系列漆酶底物，然而由于复杂分子的难溶性，在催化Domino反应时并没有得到期望的中间体及产物。但是我们发现这些化合物在特定的条件下能够对金属离子实现单一选择性识别，因而对其识别条件进行筛选，同时其在优化条件下应用于活体细胞中，发现同样能在活体中识别。化合物RBMAB，可用于金属离子Fe3+的检测。RBMAB对Fe3+具有高灵敏度单一性选择，加入Fe3+离子的配体溶液颜色会在很短的时间内变红，可实现“裸眼”识别，得出了该化合物对金属离子Fe3+的检出限是0.021µM。RBTA通过荧光选择性实验可以看出RBTA对Fe3+和Sn2+有较强的荧光响应对Fe3+的检测极限为0.008 μM；对Sn2+的检测极限为0.003 μM。PYPA，对Zn2+的检测极限为0.030 μM。FLTC，通过荧光选择性实验可以看出FLTC对Hg2+有较强的荧光响应；检测极限为0.21 μM；通过阳离子竞争性实验可以看出在以Hg2+或者Ag+作为背景离子时对应的Ag+或者Hg2+有较强的荧光响应。FLTC对Hg2+的检测有很好的专一性和灵敏性。是一种很好的检测Hg2+的荧光材料。