Aristolochene synthase-catalyzed Cyclization of 2-Fluorofarnesyl-Diphosphate to 2-Fluorogermacrene A
Aristolochene synthase-catalyzed Cyclization of 2-Fluorofarnesyl-Diphosphate to 2-Fluorogermacrene A
复制标题
DOI：
10.1002/cbic.200700219
复制
发表时间：
2007-10-15
期刊：
影响因子：
3.2
通讯作者：
Allemann, Rudolf K.
中科院分区：
文献类型：
--
作者：
Miller, David J.;Yu, Fanglei;Allemann, Rudolf K.
已提出由来自青霉的马兜铃烯合酶催化 (E,E)-法呢基二磷酸 (FPP, 1a) 转化为马兜铃烯 (6) 的机制是通过中性中间体吉马烯 A (4a) 进行的。然而，许多实验证据也与一种机制相一致，其中吉甲烯 A 不是导致马兜铃烯形成的主要机制中的中间体，而是在副反应中形成的旁路产物。因此，为了阐明 FPP 环化的机制，合成了底物类似物 2-氟 FPP (1 b)，并根据 GC-MS 分析，与马兜铃烯合酶一起孵育后转化为单一戊烷可提取产物。根据NMR分析，该产物被鉴定为2-氟香叶烯A (4b)。变温H-1 NMR光谱表明4b存在两种构象异构体，它们在-60℃下缓慢交换，而在90℃下两种异构体产生平均NMR信号。在主要异构体（类似于 75%）中，C3 和 C7 上的甲基最有可能处于从下到下的方向，正如在其他 (E,E)-ger-macranes 中观察到的那样。这项工作表明，在 FPP 与germacryl 阳离子的初始协同环化之后，去质子化导致了germacrene A 的形成，并提供了令人信服的证据，证明germacrene A 确实是马兜铃烯合成糖催化的途径产物。
The mechanism of the conversion of (E,E)-farnesyl diphosphate (FPP, 1a) to aristolochene (6) catalyzed by aristolochene synthase from Penicillium roqueforti has been proposed to proceed through the neutral intermediate germacrene A (4a). However, much of the experimental evidence is also in agreement with a mechanism in which germacrene A is not an intermediate in the predominant mechanism that leads to the formation of aristolochene, but rather an off-pathway product that is formed in a side reaction. Hence, to elucidate the mechanism of FPP cyclisation the substrate analogue 2-fluoroFPP (1 b) was synthesized, and upon incubation with aristolochene synthase was converted to a single pentane extractable product according to GC-MS analysis. On the basis of NMR analyses this product was identified as 2-fluorogermacrene A (4 b). Variable temperature H-1 NMR spectroscopy indicated the existence of two conformers of 4b that were in slow exchange at -60 degrees C, while at 90 degrees C the two isomers gave rise to averaged NMR signals. In the major isomer (similar to 75 %) the methyl groups on C3 and C7 were most likely in the down-down orientation as had been observed for other (E,E)-ger-macranes. This work suggests that after an initial concerted cyclisation of FPP to germacryl cation deprotonation leads to the formation of germacrene A, and provides compelling evidence that germacrene A is indeed an on-pathway product of catalysis by aristolochene synthose.