STEREOCHEMICAL STUDIES OF ISOPRENOID BIOSYNTHESIS

类异戊二烯生物合成的立体化学研究

• 批准号: 3277944
• 负责人: DAVID E CANE
• 金额: $ 22.71万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-01-01 至 1993-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3277944
• 关键词: catalyst; chemical models; cyclization; enzyme mechanism; isomerase; isoprenoid; monoterpenes; pyrophosphates; saturated /unsaturated bonds; sesquiterpenes; stereochemistry; steroid biosynthesis; terpenes

Ongoing studies of cell-free terpenoid cyclizations will be continued and extended. A general stereochemical model of monoterpene and sesquiterpene biosynthesis will be tested by examining the action of a group of sesquiterpene synthetases isolated from a variety of microbial and plant sources. It is expected that the information gained from such studies will provide fundamental understanding of the catalysis of biological carbon- carbon bond formation and the nature of enzymatic catalysis and control in general. 1) We have demonstrated the intermediacy of nerolidyl pyrophosphate (1) in the enzymatic cyclization of farnesyl pyrophosphate (2) to trichodiene (3) and established the detailed stereochemical course of this cyclization. In order to further dissect the complex isomerization - cyclization sequence mediated by trichodiene synthetase, a series of strategically chosen substrate analogs will be incubated with homogeneous trichodiene synthetase, in collaboration with Dr. Thomas Hohn of the USDA. The goal in these experiments will be to disrupt normal bond-making processes, thereby leading to premature release of abortive cyclization products thus revealing the otherwise cryptic bond-forming and rearrangement events which take place during normal cyclization. In parallel with these studies we will also carry out incubations of many of the same substrate analogs with farnesyl pyrophosphate - nerolidyl pyrophosphate isomerase, an enzyme isolated from Gibberella fujikuroi which catalyzes a simple allylic rearrangement corresponding to the initial isomerization step of many terpenoid cyclizations. 2) Two recently isolated new microbial sesquiterpene cyclases, bergamotene (4) synthetase from Pseudeurotium ovalis and aristolochene (5) synthetase from Aspergillus terreus will be studied. The latter enzyme has also been isolated by Dr. Hohn from Penicillium roquefortii and purified to homogeneity. Proposed stereochemical models for each cyclization will be tested using specifically labelled samples of farnesyl and nerolidyl pyrophosphate as well as selected substrate analogs. The study of bergamotene synthetase provides an opportunity to examine cyclizations involving intermediates antipodal to those involved in trichodiene biosynthesis and closely related mechanistically to the formation of the monoterpene beta-pinene, while the aristolochene synthetase reaction provides a platform for the study of the conversion of farnesyl pyrophosphate to all-trans 10-membered ring intermediates. 3) Professor Rodney Croteau of Washington State University has recently succeeded in purifying humulene (6) synthetase isolated from sage (Salvia officinalis). Collaborative studies of the stereochemistry of the formation of this 11-membered ring sesquiterpene, using specifically labelled samples of farnesyl pyrophosphate will be continued. Finally, (- )-gamma-cadinene synthetase will be isolated from A. terreus and the mechanism of cyclization of farnesyl and nerolidyl pyrophosphate to a cis, trans-germacradienyl intermediate will be examined.

正在进行的无细胞萜类环化反应的研究将继续进行， 延期了。单萜和倍半萜的一般立体化学模型 生物合成将通过检查一组 从多种微生物和植物中分离的倍半萜合成酶 消息来源。预计从这类研究中获得的信息将 提供对生物碳的催化作用的基本理解- 碳键的形成和酶催化的性质及其控制 将军。 1)我们证明了橙花内酯焦磷酸(1)的中间体 法尼基焦磷酸(2)的酶促环化反应 并建立了该环化反应的详细立体化学过程。在……里面 为了进一步剖析复杂的异构化-环化序列 在三环二烯合成酶的介导下，一系列策略性选择 底物类似物将与均相的三环二烯孵化 合成酶，与美国农业部的托马斯·霍恩博士合作。中国的目标 这些实验将扰乱正常的债券形成过程，从而 从而导致流产的环化产物过早释放 揭示了原本神秘的成键和重排事件，这些事件 发生在正常的环化过程中。在进行这些研究的同时，我们 还将对许多相同的底物类似物进行孵化 金合欢基焦磷酸酯-橙花酯焦磷酸异构酶 从催化简单烯丙基的藤库里赤霉菌中分离出 与许多异构化起始步骤相对应的重排 萜类环化反应。 2)最近分离到的两个新的微生物倍半萜环化酶，佛手萝卜素 (4)卵形拟青霉和马兜铃烯合成酶(5)合成酶 将对土曲霉进行研究。后一种酶也被 霍恩博士从罗氏青霉中分离并纯化到 同质性。为每个环化反应提出的立体化学模型将是 使用特定标记的法尼基和橙花油基样品进行测试 焦磷酸盐以及选定的底物类似物。的研究。 佛手萝卜素合成酶提供了研究环化反应的机会 涉及与三环二烯相反的中间体 生物合成，并在力学上与 单萜烯-β-蒎烯，而马兜铃烯合成酶反应 为研究法尼基的转化提供了平台 焦磷酸盐到全反式10元环中间体。 3)华盛顿州立大学的罗德尼·克罗托教授最近 鼠尾草草烯(6)合成酶的纯化 药用植物)。人的立体化学的合作研究 该11元环倍半萜的形成，具体使用 标有法尼基焦磷酸盐的样品将继续。最后，(- )-伽马-卡迪烯合成酶将从黄曲霉中分离出来。 法尼基和橙花油基焦磷酸盐环化成顺式化合物的机理， 反式-杰马射线烯基中间体将被检测。