Decoding the mechanism of P450-catalyzed aromatic hydroxylation: Uncovering the arene oxide pathway and insights into the regioselectivity

doi:10.1016/S1872-2067(24)60234-2

Chinese Journal of Catalysis ›› 2025, Vol. 70: 420-430.DOI: 10.1016/S1872-2067(24)60234-2

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Decoding the mechanism of P450-catalyzed aromatic hydroxylation: Uncovering the arene oxide pathway and insights into the regioselectivity

Qun Huang^a^,^b^,^e^,¹, Xuan Zhang^c^,^f^,¹, Guangwu Sun^d^,¹, Rui-ying Qiu^b^,¹, Lan Luo^e, Cuizhen Wang^b, Longwei Gao^a, Bing Gao^d, Bo Chen^b^,^*(), Binju Wang^c^,^*(), Jian-bo Wang^a^,^b^,^*()

^aKey Laboratory of Multiple Organ Failure (Zhejiang University), Ministry of Education, Department of General Intensive Care Unit of the Second Affiliated Hospital and Institute of Pharmaceutical Biotechnology, Zhejiang University School of Medicine, Hangzhou 310058, Zhejiang, China
^bKey Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education) and Key Laboratory of Phytochemical R&D of Hunan Province, College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, Hunan, China
^cState Key Laboratory of Physical Chemistry of Solid Surfaces and Fujian Provincial Key Laboratory of Theoretical and Computational Chemistry, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen361005, Fujian, China
^dState Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, Institute of Chemical Biology and Nanomedicine, Hunan University, Changsha 410082, Hunan, China
^eSchool of Pharmacy and Bioengineering, Chongqing University of Technology, Chongqing 400054, China
^fInstitute of Drug Discovery Technology and Qian Xuesen Collaborative Research Center of Astrochemistry and Space Life Sciences, Ningbo University, Ningbo 315211, Zhejiang, China

Received:2024-11-12 Accepted:2024-12-21 Online:2025-03-18 Published:2025-03-20
Contact: * E-mail: dr-chenpo@vip.sina.com (B. Chen),wangbinju2018@xmu.edu.cn (B. Wang),jwang2023@zju.edu.cn (J. Wang).
About author:¹Contributed equally to this work.
Supported by:
National Key Research and Development Program of China(2023YFA0914100/2023YFA0914102);National Natural Science Foundation of China(22077029);National Natural Science Foundation of China(22477110);National Natural Science Foundation of China(22034002);National Natural Science Foundation of China(22276049);National Natural Science Foundation of China(22073077);National Natural Science Foundation of China(22001065);Fundamental Research Funds for the Central Universities(226202400061);Fundamental Research Funds for the Central Universities(226202300100);Science Fund for Distinguished Young Scholars of Hunan Province(2021JJ10034);Scientific and Technological Research Program of Chongqing Municipal Education Commission(KJQN202401148);Open Fund of Key Laboratory of Phytochemistry R&D of Hunan Province of Hunan Normal University(23010104)

Abstract

Abstract:

P450 enzymes-catalyzed aromatic hydroxylation plays an important role in detoxification, biosynthesis, and potential carcinogenic effect of aromatic compounds. Though it has been explored for decades, the actual process of aromatic hydroxylation and mechanism of regioselectivity catalyzed by cytochrome P450 monooxygenases remained ambiguous. Here, we have resolved these issues. With a stable chiral organofluorine probe, and especially with X-ray data of two isolated arene oxides derivatives, we demonstrate that an arene oxide pathway is definitely involved in P450-catalyzed aromatic hydroxylation. By the capture, isolation, identification and reactivity exploration of the arene 1,2-oxide and arene 2,3-oxide intermediates, together with advanced QM calculations, the mechanism of how two intermediates go to the same product has been elucidated. In addition to the model substrate, we also confirmed that an arene oxide intermediate is involved in the P450-catalyzed hydroxylation pathway of a natural product derivative methyl cinnamate, which indicates that this intermediate appears to be universal in P450-catalyzed aromatic hydroxylation. Our work not only provides the most direct evidence for the arene oxide pathway and new insights into the regioselectivity involved in P450-catalyzed aromatic hydroxylation, but also supplies a new synthetic approach to achieve the dearomatization of aromatic compounds.

Key words: Aromatic hydroxylation, Cytochrome P450, Arene oxide, Regioselectivity, Dearomatization

Qun Huang, Xuan Zhang, Guangwu Sun, Rui-ying Qiu, Lan Luo, Cuizhen Wang, Longwei Gao, Bing Gao, Bo Chen, Binju Wang, Jian-bo Wang. Decoding the mechanism of P450-catalyzed aromatic hydroxylation: Uncovering the arene oxide pathway and insights into the regioselectivity[J]. Chinese Journal of Catalysis, 2025, 70: 420-430.

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URL: https://www.cjcatal.com/EN/10.1016/S1872-2067(24)60234-2

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Figures/Tables 6

Scheme 1. Aromatic hydroxylation catalyzed by cytochrome P450. (a) Mechanism speculations for aromatic hydroxylation catalyzed by cytochrome P450. (b) Regioselectivity of aromatic hydroxylation catalyzed by P450 enzyme: usually occurs at the ortho- or para-sites, while the meta-site is rare. (c) P450-BM3-catalyzed aromatic hydroxylation leading to remote induced defluorination in our previous work.

Fig. 1. Time-scale monitoring of the conversion of 1. The GC analysis of enzymatic conversion of 1 by whole cells harboring P450-BM3 (a) and purified enzyme (b). Details of the reaction systems have been put in the SI.

Fig. 2. Isolation and identification of arene oxides 3 and 5. The GC analysis of purity of 3 (a) and 5 (b) after silica gel column chromatographic separation. (c) The structures of the derivatives of 3 and 5 as determined by X-ray structure analysis.

Fig. 3. Investigation on the arene oxide pathway in aromatic hydroxylation catalyzed by P450. (a) Proposed arene oxide pathway of aromatic hydroxylation catalyzed by P450. The GC analysis of enzymatic conversion of 3 (b) and 5 (c) by purified NemA. The GC analysis of enzymatic conversion of 3 (d) and 5 (e) by purified NemA on time-scale.

Fig. 4. B3LYP/6-311++G(d,p) relative free energies (in kcal/mol) for the rearrangement processes. (a) 3-1 → 7; (b) 3-2 → 7; (c) 5-1 → 7; (d) 5-2 → 7 in aqueous solution (no enzyme) with explicit water molecules in the HCC calculations. R: -COOCH2CH3.

Fig. 5. Verification of the universality of the arene oxide pathway in P450-catalyzed aromatic hydroxylation by non-fluorinated substrate. (a) GC analysis of enzymatic conversion of 8 by purified P450-BM3 (A330Y/T438L). (b) The GC analysis of enzymatic conversion of 8 by purified P450-BM3 (A330Y/T438L) on time-scale. (c) The GC analysis of spontaneous conversion of 9 → 10 on time-scale. (d) The GC analysis of enzymatic conversion of 9 → 10 with purified P450-BM3 (A330Y/T438L) on time-scale. (e) Proposed arene oxide pathway of aromatic hydroxylation of 8 catalyzed by P450.

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Decoding the mechanism of P450-catalyzed aromatic hydroxylation: Uncovering the arene oxide pathway and insights into the regioselectivity

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