Regiodivergent and Enantioselective Hydroxylation of C-H bonds by Synergistic Use of Protein Engineering and Exogenous Dual-Functional Small Molecules

Jie Chen; Sheng Dong; Wenhan Fang; Yiping Jiang; Zhifeng Chen; Xiangquan Qin; Cong Wang; Haifeng Zhou; Longyi Jin; Yingang Feng; Binju Wang; Zhiqi Cong

doi:10.1002/anie.202215088

Regiodivergent and Enantioselective Hydroxylation of C-H bonds by Synergistic Use of Protein Engineering and Exogenous Dual-Functional Small Molecules

Angew Chem Int Ed Engl. 2023 Jan 23;62(4):e202215088. doi: 10.1002/anie.202215088. Epub 2022 Dec 14.

Authors

Jie Chen^{1

2

3}, Sheng Dong^{1

2

3}, Wenhan Fang⁴, Yiping Jiang^{1

3}, Zhifeng Chen^{1

5}, Xiangquan Qin^{1

6}, Cong Wang¹, Haifeng Zhou⁵, Longyi Jin⁶, Yingang Feng^{1

2

3}, Binju Wang⁴, Zhiqi Cong^{1

2

3}

Affiliations

¹ CAS Key Laboratory of Biofuels and Shandong Provincial Key Laboratory of Synthetic Biology, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, 266101, Qingdao, China.
² University of Chinese Academy of Sciences, 100049, Beijing, China.
³ Shandong Energy Institute, 266101, Qingdao, China.
⁴ State 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, 361005, Xiamen, China.
⁵ Hubei Key Laboratory of Natural Products Research and Development, Key Laboratory of Functional Yeast, China National Light Industry, College of Biological and Pharmaceutical Sciences, China Three Gorges University, 443002, Yichang, China.
⁶ Department of Chemistry, Yanbian University, 133002, Yanji, China.

PMID: 36417593
DOI: 10.1002/anie.202215088

Abstract

It is a great challenge to optionally access diverse hydroxylation products from a given substrate bearing multiple reaction sites of sp³ and sp² C-H bonds. Herein, we report the highly selective divergent hydroxylation of alkylbenzenes by an engineered P450 peroxygenase driven by a dual-functional small molecule (DFSM). Using combinations of various P450BM3 variants with DFSMs enabled access to more than half of all possible hydroxylated products from each substrate with excellent regioselectivity (up to >99 %), enantioselectivity (up to >99 % ee), and high total turnover numbers (up to 80963). Crystal structure analysis, molecular dynamic simulations, and theoretical calculations revealed that synergistic effects between exogenous DFSMs and the protein environment controlled regio- and enantioselectivity. This work has implications for exogenous-molecule-modulated enzymatic regiodivergent and enantioselective hydroxylation with potential applications in synthetic chemistry.

Keywords: Cytochrome P450 Enzymes; Divergent Hydroxylation; Enantioselectivity; Enzyme Catalysis; Regioselectivity.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Catalytic Domain
Hydroxylation
Molecular Dynamics Simulation*
Protein Engineering*
Stereoisomerism
Substrate Specificity