Selective aliphatic carbon-hydrogen bond activation of protected alcohol substrates by cytochrome P450 enzymes
Selective aliphatic carbon-hydrogen bond activation of protected alcohol substrates by cytochrome P450 enzymes
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DOI：
10.1039/c3ob42417k
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发表时间：
2014-01-01
影响因子：
3.2
通讯作者：
Wong, Luet-Lok
中科院分区：
文献类型：
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作者：
Bell, Stephen G.;Spence, Justin T. J.;Wong, Luet-Lok
保护环己醇和环己烯醇底物，含有苯醚和苯甲酸酯部分，被设计成适合于细胞色素P450cam Tyr96Ala突变体的活性位点。受保护的环己醇底物被突变酶在环己基环上C-4的反式C-H键上有效和选择性地羟基化。苯甲酸酯保护环己醇的氧化选择性可以通过在P450cam活性位点进行替代氨基酸取代来改变。苯甲酸酯保护环己烯醇的环己基环上添加双键对具有该底物的Tyr96Ala突变体的活性有削弱作用。然而，与Tyr96Ala突变体相比，Phe87Ala/Tyr96Phe双突变体在活性位点的不同位置引入空间，能够有效地在烯丙基C-4位置羟基化1-环己基-2-烯基苯甲酸酯的C-H键。与P450cam的Tyr96突变体相比，Phe87突变体提高了1-苯基- 1环己基乙烯氧化为反式-4-苯基-乙烯基环己醇的选择性（92%）。
Protected cyclohexanol and cyclohex-2-enol substrates, containing benzyl ether and benzoate ester moieties, were designed to fit into the active site of the Tyr96Ala mutant of cytochrome P450cam. The protected cyclohexanol substrates were efficiently and selectively hydroxylated by the mutant enzyme at the trans C-H bond of C-4 on the cyclohexyl ring. The selectivity of oxidation of the benzoate ester protected cyclohexanol could be altered by making alternative amino acid substitutions in the P450cam active site. The addition of the double bond in the cyclohexyl ring of the benzoate ester protected cyclohex-2-enol has a debilitative effect on the activity of the Tyr96Ala mutant with this substrate. However, the Phe87Ala/Tyr96Phe double mutant, which introduces space at a different location in the active site than the Tyr96Ala mutant, was able to efficiently hydroxylate the C-H bonds of 1-cyclohex-2-enyl benzoate at the allylic C-4 position. Mutations at Phe87 improved the selectivity of the oxidation of 1-phenyl1-cyclohexylethylene to trans-4-phenyl-ethenylcyclohexanol (92%) when compared to single mutants at Tyr96 of P450cam.