Parallel and competitive pathways for substrate desaturation, hydroxylation, and radical rearrangement by the non-heme diiron hydroxylase AlkB.
Parallel and competitive pathways for substrate desaturation, hydroxylation, and radical rearrangement by the non-heme diiron hydroxylase AlkB.
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DOI：
10.1021/ja3059149
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发表时间：
2012-12-19
影响因子：
15
通讯作者：
Groves JT
中科院分区：
文献类型：
--
作者：
Cooper HL;Mishra G;Huang X;Pender-Cudlip M;Austin RN;Shanklin J;Groves JT
使用诊断探针底物降冰片烷研究了非血红素二铁羟化酶AlkB的纯化和高活性形式。反应得到C2（26%）和C3（43%）羟基化和去饱和产物（31%）。初始C-H裂解在C2导致7%的C2羟基化和19%的3-羟甲基环己烯，一个自由基重排途径的重排产物特征。氘代底物类似物，3，3，4，4-降碳烷-d4，提供了急剧减少的C3醇（8%）和去饱和产物（5%），而自由基重排的醇现在是主要产物（65%）。产物比率的这种变化表明对于C3处的C-H羟基化和去饱和途径两者，约20的大动力学氢同位素效应，其中所有去饱和都源自C3处的氢提取而不是C2处。数据表明AlkB与降冰片烷的反应是通过C2或C3的C-H氢提取进行的，并且C3羟基化、C3去饱和和C2羟基化/自由基重排这三条途径是平行和竞争的。因此，C3处的初始自由基或者与铁-氧代中心反应形成醇，或者通过第二次H-提取沿着去饱和途径进行沿着，得到2-降冰片烯和3-降冰片烯。这些降卡烯的后续反应导致可检测量的羟基化产物和甲苯。相比之下，2-norcaranyl自由基中间体导致C2羟基化和诊断自由基重排，但这种自由基显然不提供去饱和产物。结果表明，C-H羟基化和去饱和遵循类似的分步反应通道，通过碳自由基，在产品形成步骤发散。
A purified and highly active form of the non-heme diiron hydroxylase AlkB was investigated using the diagnostic probe substrate norcarane. The reaction afforded C2 (26%) and C3 (43%) hydroxylation and desaturation products (31%). Initial C-H cleavage at C2 led to 7% C2 hydroxylation and 19% 3-hydroxymethylcyclohexene, a rearrangement product characteristic of a radical rearrangement pathway. A deuterated substrate analog, 3,3,4,4-norcarane-d4, afforded drastically reduced amounts of C3 alcohol (8%) and desaturation products (5%), while the radical rearranged alcohol was now the major product (65%). This change in product ratios indicates a large kinetic hydrogen isotope effect of ~20 for both the C-H hydroxylation at C3 and the desaturation pathway, with all of the desaturation originating via hydrogen abstraction at C3 and not C2. The data indicates that AlkB reacts with norcarane via initial C-H hydrogen abstraction from C2 or C3 and that the three pathways, C3 hydroxylation, C3 desaturation and C2 hydroxylation/radical rearrangement, are parallel and competitive. Thus, the incipient radical at C3 either reacts with the iron-oxo center to form an alcohol or proceeds along the desaturation pathway via a second H-abstraction to afford both 2-norcarene and 3-norcarene. Subsequent reactions of these norcarenes lead to detectable amounts of hydroxylation products and toluene. By contrast, the 2-norcaranyl radical intermediate leads to C2 hydroxylation and the diagnostic radical rearrangement, but this radical apparently does not afford desaturation products. The results indicate that C-H hydroxylation and desaturation follow analogous stepwise reaction channels via carbon radicals that diverge at the product-forming step.
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影响因子：
16.6
作者：
Austin, Rachel N.;Luddy, Kate;Groves, John T.
通讯作者：
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影响因子：
3.9
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作者：
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通讯作者：
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通讯作者：
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作者：
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