Koerts J, Boeren S, Vervoort J, Weiss R, Veeger C, Rietjens I M
Department of Biochemistry, Agricultural University, Wageningen, The Netherlands.
Chem Biol Interact. 1996 Jan 5;99(1-3):129-46. doi: 10.1016/0009-2797(95)03664-4.
In the present study the in vitro and in vivo aromatic ring hydroxylation of a series of amino and/or methyl containing fluorobenzenes, i.e. 3-fluoro(methyl)anilines, was investigated and compared to the calculated density distribution of the reactive frontier pi-electrons of the aromatic substrate. This was done (1) to study to what extent the regioselectivity of the aromatic ring hydroxylation of the 3-fluoro(methyl)anilines could be predicted on the basis of the calculated chemical reactivity, as was previously observed for a series of fluorinated benzenes and monofluoroanilines, and (2) to investigate which factors contribute to possible deviations from the predictions on the basis of the calculated chemical reactivity. Results obtained show that the in vitro and in vivo aromatic ring hydroxylation of the series of 3-fluoro(methyl)anilines correlates qualitatively with the calculated frontier orbital density distribution for electrophilic attack by the cytochrome P450(FeO)3+ species. These results indicate that the HOMO/HOMO-1 frontier orbital densities, i.e. the chemical reactivity of the carbon centres for an electrophilic attack, predict the preferential as well as the non-reactive sites for cytochrome P450 catalysed aromatic ring hydroxylation of the tested model compounds. The absolute values, however, deviated in a systematic way; C4 para hydroxylation being observed to a higher extent than expected on the basis of chemical reactivity and C2/C6 ortho hydroxylation being observed to a lower extent than expected. Additional experiments were performed using different microsomal preparations and microperoxidase-8. The latter is a mini-heme protein of eight amino acids without a substrate binding site. In incubations of the model compounds with different types of microsomal preparations, as well as with MP-8 and purified reconstructed cytochrome P4502B1, similar systematic deviations between the predicted and observed regioselectivity of aromatic hydroxylation were observed. These results show that the regioselectivity of aromatic ring hydroxylation of the 3-fluoro(methyl)anilines cannot be predominantly ascribed to an interaction between the substrate and the substrate binding site of the cytochromes P450 dictating a specific stereoselective positioning of the substrate in the active site. More likely, the systematic deviations between the observed and predicted regioselectivity of hydroxylation of the tested model substrates should be ascribed to an (orienting) interaction between the substrate and the activated cytochrome P450(FeO)3+ cofactor.
在本研究中，对一系列含氨基和/或甲基的氟苯，即3-氟（甲基）苯胺的体外和体内芳环羟基化反应进行了研究，并与芳族底物反应性前沿π电子的计算密度分布进行了比较。这样做的目的是：（1）研究基于计算出的化学反应性，能在多大程度上预测3-氟（甲基）苯胺芳环羟基化反应的区域选择性，正如之前在一系列氟化苯和一氟苯胺中所观察到的那样；（2）研究哪些因素导致了与基于计算出的化学反应性所做预测的可能偏差。所得结果表明，3-氟（甲基）苯胺系列的体外和体内芳环羟基化反应与细胞色素P450(FeO)3+物种亲电攻击的计算前沿轨道密度分布在定性上相关。这些结果表明，HOMO/HOMO-1前沿轨道密度，即碳中心对亲电攻击的化学反应性，预测了受试模型化合物细胞色素P450催化芳环羟基化反应的优先位点和非反应位点。然而，绝对值存在系统性偏差；观察到C4位的对位羟基化程度高于基于化学反应性预期的程度，而C2/C6位的邻位羟基化程度低于预期。使用不同的微粒体制剂和微过氧化物酶-8进行了额外的实验。微过氧化物酶-8是一种由八个氨基酸组成的微型血红素蛋白，没有底物结合位点。在模型化合物与不同类型的微粒体制剂以及与MP-8和纯化的重组细胞色素P4502B1的孵育实验中，观察到芳环羟基化反应预测的和观察到的区域选择性之间存在类似的系统性偏差。这些结果表明，3-氟（甲基）苯胺芳环羟基化反应的区域选择性不能主要归因于底物与细胞色素P450的底物结合位点之间的相互作用，这种相互作用决定了底物在活性位点的特定立体选择性定位。更有可能的是，受试模型底物羟基化反应观察到的和预测的区域选择性之间的系统性偏差应归因于底物与活化的细胞色素P450(FeO)3+辅因子之间的（定向）相互作用。
