METHYL & METHYLENE ELIMINAT STEREOCHEMISTRY IN EPI ARISTOLOCHENE BIOSYNTHESIS

甲基

• 批准号: 6220455
• 负责人: HIROMI MORIMOTO
• 金额: $ 0.61万
• 依托单位: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-01 至 2000-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6220455
• 关键词: biological products; biomedical resource

The stereochemistry of the proton transfers that terminate the enzyme-catalyzed cyclizations producing isoprenoid natural products reveals a key interaction between the intermediate carbocation and enzyme active-site. Elucidation of the facial specificity of eliminations occurring at methyl groups can be accomplished by use of doubly-labelled substrates bearing 1H, 2H, and 3H at the methyl group in R or S configuration. The experimental approach involves generation of chiral methyl-substituted compounds by stereospecific SN2 displacements with lithium triethylborotritide at high specific activity and 3H NMR analysis of the [2H,3H]terpene product. We propose to elucidate the stereochemistry of the methyl/methylene eliminations associated with enzymatic cyclizations producing germacrene A, a key intermediate in sesquiterpene biosynthesis. Recombinant forms of tobacco epi-aristolochene synthase (TEAS) and Hyoscyamus vetispiradiene synthase (HVS) expressed in the laboratories of Dr. J. Chappell at the University of Kentucky catalyze the conversion of farnesyl diphosphate to their respective sesquiterpene hydrocarbon products, by way of germacrene A as an enzyme-bound intermediate. The X-ray crystal structure of TEAS and TEAS complexes have been solved in the laboratory of Dr. J. Noel at the Salk Institute in California and predictions have been made concerning the stereochemistry of the methyl/methylene elimination in epi-aristolochene (EA) biosynthesis. We have shown that the vinyl protons on the isopropenyl group of EA are resolved in 1H NMR spectra, and we have assigned the resonances by NOE experiments, thus validating the 3 H NMR analysis method for EA. Epi-aristolochene and vetispiradiene are key intermediates in biosynthesis of the sesquiterpene phytoalexins, capsidiol, solavetivone, and rishitin. Initial synthetic steps have been taken on this project, involving the supertritide reduction of a dideuterio-precursor. The tritiated material will be used to study the subsequent chemistry steps, and for preliminary enzyme conversions. Once the chemistry and enzyme procedures are completed with this test material, further tritiations and chemistry will be conducted on the stereospecifically labelled precursors. The eventual aim is to generate both of the chiral methyl stereoisomers of farnesyl diphosphate. With those materials in hand, the enzymatic digestion, isolation of products and NMR assignment of peaks will be straightforward. This project is set to move forward rapidly over the next twelve months.

终止质子转移的立体化学 生产类异戊二烯天然产物的酶催化环化反应 揭示了中间碳阳离子和 酶活性位点 面部特异性的阐明 在甲基上发生的消除可以通过使用 在甲基上带有1H、2 H和3 H的双标记底物 R或S构型。 实验方法包括 通过立体定向制备手性甲基取代的化合物 在高比表面积下用三乙基硼氚化锂置换SN 2 [2 H，3 H]萜烯产物的活性和3 H NMR分析。 我们 建议阐明甲基/亚甲基的立体化学 与酶促环化相关的消除， 大根香叶烯A是倍半萜生物合成的关键中间体。 重组形式的烟草表马兜铃烯合酶（TEAS）和 天仙子草螺烯合酶（HVS）的实验室表达 肯塔基州大学的J. Chappell博士 法呢基二磷酸转化为它们各自的倍半萜烯 烃类产品，通过大根香叶烯A作为酶结合 中间体 TEAS及其配合物的X射线晶体结构 已经在索尔克的J.诺埃尔博士的实验室里解决了 研究所在加州和预测已经作出了关于 甲基/亚甲基消除的立体化学 表马兜铃烯（EA）生物合成。 我们已经证明了乙烯基 EA的异丙烯基上的质子在1H NMR谱中分辨， 我们已经通过NOE实验指定了共振，因此 验证了EA的3 H NMR分析方法。 表马兜铃烯和 香根草螺烯是生物合成的关键中间体， 倍半萜植物抗毒素、辣椒二醇、茄酮和Rishitin。 该项目已采取初步综合步骤， 二氘前体的超氚化物还原。 氚化 材料将用于研究随后的化学步骤， 初步的酶转化。 一旦化学物质和酶 使用该试验材料完成程序后， 化学反应将在立体特异性标记的 前体 最终的目标是生成两种手性甲基 法呢基二磷酸酯的立体异构体。 有了这些材料， 酶解、产物分离和NMR归属 峰值将是直接的。 这个项目将向前推进 在接下来的12个月里，