Nature's Ingenuity in Stereoselective (Phenol) Coupling

9631980刘易斯自然界中最重要但知之甚少的生物事件之一是双分子苯氧基自由基偶联。令人惊讶的是，苯氧基自由基偶联反应长期以来一直没有得到完美的表征，并且经常被描述为缺乏基于其体外催化性质的立体选择性。然而，即使对自然界中这种反应的粗略分析也表明，由于产物是立体专一性的，因此一定存在某种赋予立体专一性的机制。为了了解这种立体选择性是如何产生的，研究人员研究了E-松柏醇的两个非手性分子偶联得到(+)-松脂醇，这是自然界中已知的最简单的二聚体木脂素产品之一。他们确定，一个78 kDa的蛋白质是赋予产品区域和立体特异性所必需的。这种立体选择性偶联只有在通过添加氧化酶或自由基引发剂(如过二硫酸铵或FMN)来提供辅助氧化能力时才会发生。这项调查将试图确定这种独特控制模式的生化机制。将研究78 kDa蛋白、底物和辅助氧化酶/氧化剂之间的关系。也就是说，作用机制是否涉及两个E-松柏醇分子与78 kDa蛋白质的初始结合，或者结合的底物实际上是通过单电子氧化获得的自由基物种？第三种可能性是存在某种电荷转移机制，尽管没有任何可检测到的活性部位(假体基团)，这是不太可能的。研究人员将对偶联反应进行详细的动力学分析，并确定结合/偶联的底物特异性和假定的抑制剂的效果。这样，苯氧基自由基偶联的立体选择性机制就被确定了，并将作为一个起点来确定这一机制在自然界中的普遍性。自然界中最重要但也是最不为人所知的生物事件之一是双分子苯氧基自由基偶联，即含有苯酚基团的两个分子的连接。它具有非常重要的意义，因为它最终导致木质和软木组织、昆虫角质层、真菌子实体、蚜虫色素和一系列防御分子的形成，特别是在植物中。总而言之，酚类偶联反应约占所有有机植物材料的35%-40%。两个苯酚基团通常可以以多种方式连接在一起，各个产品是彼此的镜像。在这些研究人员取得进展之前，体外反应只产生各种镜像产品的混合物。然而，在体内的反应通常只产生一种产物，而且是以一种镜像形式出现的。这种特殊性是如何实现的？研究人员已经分离出一种蛋白质，他们称之为“说明符”，它赋予了这些反应如此特殊的特性。本项目的目标是确定这种新蛋白质的生化机制。***

9631980 Lewis One of the most important yet poorly understood biological events in nature is that of bimolecular phenoxy radical coupling. Surprisingly, phenoxy radical coupling reactions have long been imperfectly characterized, and are frequently described as lacking stereoselectivity based on their catalytic properties in vitro. Yet even a cursory analysis of such reactions in nature indicates that some mechanism for conferring stereospecificity must exist, since the products are stereospecific. Seeking to understand how this stereoselectivity is produced, the investigators examined the coupling of two achiral molecules of E-coniferyl alcohol to give (+)-pinoresinol, one of the simplest dimeric lignan products known in nature. They established that a 78kDa protein is necessary to confer both regio- & stereospecificity on the products. This stereoselective coupling only occurs when auxiliary oxidative capacity is provided through the addition of either an oxidase or a free-radical initiator, such as ammonium peroxydisulfate or FMN. This investigation will seek to define the biochemical mechanism of this unique mode of control. The relationships between the 78 kDa protein, the substrate and an auxiliary oxidase/oxidant will be studied. That is, does the operative mechanism involve initial binding of two E-coniferyl alcohol molecules to the 78 kDa protein, or is the substrate for binding actually the free-radical species obtained via one electron oxidation? A third possibility is that some charge-transfer mechanism is in place, although the absence of any detectable active site (prosthetic group) makes this unlikely. The investigators will conduct a detailed kinetic anaylsis of the coupling reaction as well as defining the substrate specificity for binding/coupling and the effect of putative inhibitors. In this way the mechanism for conferring stereoselectivity in phenoxy radical coupling will be determined and will serve as a starting point to determine the generality of this m echanism in nature. %%% One of the most important, and yet most poorly understood, biological events in nature is that of bimolecular phenoxy radical coupling, the joining of two molecules containing phenol moieties. It is highly significant, since it eventually leads to the formation of woody and cork tissues, insect cuticles, fungal fruiting bodies, aphid pigments, and a host of defense molecules, particularly in plants. Taken together, phenolic coupling reactions account for roughly 35-40% of all organic plant material. Two phenol moieties can often be joined in numerous ways, with the individual products being mirror images of each other. Before the advances by these investigators, in vitro reactions only yielded mixtures of various mirror image products. In vivo reactions, however, often yield only one product and in one mirror image form. How is that specificity attained? The investigators have isolated a protein, which they have called a "specifier", which confers such specificity on the reactions. The goal of this project is to define the biochemical mechanism of this novel protein. ***