Interception of the enzymatic conversion of farnesyl diphosphate to 5-epi-aristolochene by using a fluoro substrate analogue:: 1-fluorogermacrene a from (2E,6Z)-6-Fluorofarnesyl diphosphate
Interception of the enzymatic conversion of farnesyl diphosphate to 5-epi-aristolochene by using a fluoro substrate analogue:: 1-fluorogermacrene a from (2E,6Z)-6-Fluorofarnesyl diphosphate
复制标题
DOI：
10.1002/cbic.200700398
复制
发表时间：
2007-10-15
期刊：
影响因子：
3.2
通讯作者：
Coates, Robert M.
中科院分区：
文献类型：
--
作者：
Faraldos, Juan A.;Zhao, Yuxin;Coates, Robert M.
烟草5-表马兜铃烯合成酶(TEAS)催化(E，E)-法尼基二磷酸(PP)通过一个紧密结合的(+)-杰马烯A作为去质子化中间体，催化(E，E)-法尼基二磷酸(PP)的环化和重排成双环倍半萜碳氢化合物。对于天然酶，在催化循环中，只有几个百分点的假定的Germacene A中间体从活性部位释放出来。为了阻止5-表马兜铃烯的环化重排反应，设计合成了6-氟甲酰基PP。事实上，将(2e，6z)-6-氟芳基PP与重组茶孵育，可得到(-)-1-氟锗A，产率为58%。法尼基PP和6-氟类似物的稳态动力学实验表明，两种底物的总催化效率(k(Cat)/K-m)基本相当。通过(TLC、GC)、MS、旋光度、UV、IR和H-1核磁共振数据以及热诱导Cope重排生成(+)-1-氟-β-榄香烯等方法对其进行了表征。室温下的H-1核磁共振谱表明，这种(E，E)构型的氟环十碳二烯以UU和UD构象的7：3混合形式存在于溶液中。1-氟基茂铁烯A在三氟乙酸催化下环化得到三个1-α-氟有机硒异构体，其环化速度比(+)-基茂铁烯A与母体硒的环化反应速度慢约1000倍。
Tobacco 5-epi-aristolochene synthase (TEAS) catalyzes the Mg-II-dependent cyclizations and rearrangements of (E,E)-farnesyl diphosphate (PP) to the bicyclic sesquiterpene hydrocarbon via a tightly bound (+)-germacrene A as a deprotonated intermediate. With the native enzyme, only a few percent of the putative germacrene A intermediate is released from the active site during the catalytic cycle. 6-Fluorofamesyl PP was designed and synthesized with the aim of arresting the cyclization-rearrangement mechanism en route to 5-epi-aristolochene. Indeed, incubation of (2E,6Z)-6-fluorofarnesyl PP with recombinant TEAS afforded (-)- 1-fluororogermacrene A as the sole product in 58 % yield. Steady-state kinetic experiments with farnesyl PP and the 6-fluoro analogue showed that the overall catalytic efficiencies (k(cat)/K-m) are essentially the some for both substrates. 1-Fluorogermacrene A was characterized by chromatographic properties (TLC, GC), MS, optical rotation, UV, IR and H-1 NMR data, and by heat-induced Cope rearrangement to (+)-1-fluoro-beta-elemene. H-1 NMR spectra at room temperature revealed that this (E,E)-configured fluorocyclodecadiene exists in solution as a 7:3 mixture of UU and UD conformers. 1-Fluorogermacrene A underwent trifluoroacetic acid-catalyzed cyclization to give three 1 alpha-fluoroselinene isomers at a rate estimated to be about 1000 times slower than that of the similar cyclization of (+)-germacrene A to the parent selinenes.