16-aza-ent-beyerane and 16-aza-ent-trachylobane:: Potent mechanism-based inhibitors of recombinant ent-kaurene synthase from Arabidopsis thaliana
16-aza-ent-beyerane and 16-aza-ent-trachylobane:: Potent mechanism-based inhibitors of recombinant ent-kaurene synthase from Arabidopsis thaliana
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DOI：
10.1021/ja072447e
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发表时间：
2007-10-17
影响因子：
15
通讯作者：
Coates, Robert M.
中科院分区：
文献类型：
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作者：
Roy, Arnab;Roberts, Frank G.;Coates, Robert M.
二次碳正离子(7)是使许多四环和五环二萜的环结构合理化的机械方案中的关键分支点中间体，包括叔丁二烯、叔丁烯、叔丁烷和叔丁烯，它们被认为是1000个已知二萜的前体。为了验证这些机理假说，我们通过汞(Il)和铅(IV)诱导的环化反应，在C-8含氨甲酰甲基和氨甲基的三环中间体的A12双键上进行环化反应，合成了杂环类似物16-氮杂-对苯乙烷(12)和16-氮杂-对映异构体(13)。13，16-Seco-16-去甲氨基甲酸酯(20a)经Beckmann裂解、水解、Curtius重排等反应制得。氮杂类似物对来自拟南芥的重组t-Kaurene合成酶(GST-rAtKS)具有抑制作用，其抑制常数(IC50=1×10(-7)和1×10(-6)M)与双环磷脂酰磷酯底物的假结合常数(K-m=3×10-7M)相似。在1 mM焦磷酸盐存在下，结合亲和力显著增强(ick=4×10(-9)和2×10(-8)M)，这与该二萜合成酶催化环化重排反应中紧密结合的对-β-乙基(+)/焦磷酸-离子对相一致。对位-16-异氧基二磷酸(111)的抑制作用较弱(IC50=1×10(-5)M)，且不能发生桥式重排反应生成高岭烯，这似乎排除了共价环磷酸盐作为游离中间体的可能性。16-氮杂苯乙烷被认为是一种有效的碳正离子模拟化合物，具有潜在的应用前景：作为多种二萜环化酶的活性位点探针，以及作为植物赤霉素生物合成的一种新的选择性抑制剂。
The secondary ent-beyeran-16-yl carbocation (7) is a key branch point intermediate in mechanistic schemes to rationalize the cyclic structures of many tetra- and pentacyclic diterpenes, including ent-beyerene, ent-kaurene, ent-trachylobane, and ent-atiserene, presumed precursors to >1000 known diterpenes. To evaluate these mechanistic hypotheses, we synthesized the heterocyclic analogues 16-aza-ent-beyerane (12) and 16-aza-ent-trachylobane (13) by means of Hg(Il)- and Pb(IV)-induced cyclizations onto the A12 double bonds of tricyclic intermediates bearing carbamoylmethyl and aminomethyl groups at C-8. The 13,16-seco-16-norcarbamate (20a) was obtained from ent-beyeran-16-one oxime (17) by Beckmann fragmentation, hydrolysis, and Curtius rearrangement. The aza analogues inhibited recombinant ent-kaurene synthase from Arabidopsis thaliana (GST-rAtKS) with inhibition constants (IC50 = 1 X 10(-7) and 1 X 10(-6) M) similar in magnitude to the pseudo-binding constant of the bicyclic ent-copalyl cliphosphate substrate (K-m = 3 x 10-7 M). Large enhancements of binding affinities (ICK = 4 x 10(-9) and 2 x 10(-8) M) were observed in the presence of 1 mM pyrophosphate, which is consistent with a tightly bound ent-beyeranyl(+)/ pyrophosphate- ion pair intermediate in the cyclization -rearrangement catalyzed by this diterpene synthase. The weak inhibition (IC50 = 1 x 10(-5) M) exhibited by ent-beyeran-16-exo-yl diphosphate (111) and its failure to undergo bridge rearrangement to kaurene appear to rule out the covalent cliphosphate as a free intermediate. 16-Aza-ent-beyerane is proposed as an effective mimic for the ent-beyeran-1 6-yl carbocation with potential applications as an active site probe for the various ent-diterpene cyclases and as a novel, selective inhibitor of gibberellin biosynthesis in plants.