ISOPRENOID COMPOUNDS FOR BIOLOGICAL STUDIES

异戊二烯类化合物是在所有生物体中合成的，它们在新陈代谢、细胞结构和细胞内和细胞间的信号传递中发挥着重要作用。在酶分析、药物筛选方案、新陈代谢研究和细胞生物学实验中，需要未标记、稳定同位素标记和放射性标记形式的基本构建反应所需的分子。这些化合物中只有有限数量的化合物可以在商业上获得。生物学家和生物化学家需要除了最常见的衍生品或特殊的标记模式之外的所有东西，他们必须求助于昂贵的“定制”合成。除了少数几个学术实验室外，并不是所有的实验室都有带有荧光或光亲和标记的分子。在第一阶段，(A)我们优化了简单的线性二磷酸C5、C10、C15和C20的“大规模”合成，(B)开发了异戊烯基二磷酸异构酶和法尼基二磷酸合成酶的纯化和储存程序，以及(C)合成了由光敏二苯甲酮基团连接到类异戊二烯二磷酸盐上的光亲和探针。在第二阶段，我们将扩大我们的产品线，以包括更广泛的天然存在的、商业上无法获得的异戊二烯类分子，增加光亲和标签，并增加一系列用于分析、结合测量和预烯基化蛋白质的标签的荧光化合物。将单独提供一套异戊二烯基二磷酸异构酶；一套工程改造的异戊烯基转移酶，用于从简单的C5构建块合成香叶基(C10)、法尼基(C15)、香叶基(C20)和法尼基香叶基(C25)二磷酸；以及蛋白质法尼基转移酶，用于合成标记分子。建议的商业应用：异戊二烯类化合物越来越多地用于动脉粥样硬化和癌症领域的药物发现分析，以研究细胞信号机制，以及研究细胞新陈代谢。ERL的类异戊二烯产品系列将为研究人员提供化学物质和酶，以促进这些研究。

Isoprenoid compounds are synthesized in all living organisms where they play essential roles in metabolism, cellular architecture, and signalling both within and between cells. The molecules required for the fundamental building reactions in the pathway are needed in unlabeled, stable isotope labeled, and radiolabeled form for enzyme assays, drug screening protocols, metabolism studies, and cell biology experiments. Only a limited number of these compounds are available commercially. Biologists and biochemists who require all but the most common derivatives or special labeling patterns must resort to costly "custom" syntheses. Molecules with fluorescent or photoaffinity tags are not available in all but a few academic laboratories. In phase I, (a) we optimized the "large scale" synthesis of simple linear C5, C10, C15, and C20 isoprenoid diphosphates, (b)developed procedures for purification and storage of isopentenyl diphosphate isomerase and farnesyl diphosphate synthase, and (c) synthesized photoaffinity probes consisting of photolabile benzophenone moieties appended to isoprenoid diphosphates. In phase II we will expand our product line to include a wider range of naturally occurring isoprenoid molecules not commercially available, add photoaffinity labels, and add a family of fluorescent compounds for assays, binding measurements, and tags for prenylated proteins. A set of isoprenoid enzymes, including isopentenyl diphosphate, isomerase; an engineered set of prenyltransferases selective for synthesis of geranyl (C10), farnesyl (C15), geranylgeranyl (C20), and farnesylgeranyl (C25) diphosphates from simple C5 building blocks; and protein farnesyltransferase, will be provided individually or as kits for synthesis of labeled molecules. PROPOSED COMMERCIAL APPLICATIONS: Isoprenoid compounds are increasingly used in drug discovery assays, in the areas of atherosclerosis and cancer, to study cell signalling mechanisms, and studies of cellular metabolism. ERL's line of isoprenoid products will provide researchers with chemicals and enzymes to facilitate these studies.