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

Abstract

It is a great challenge to optionally access diverse hydroxylation products from a given substrate bearing multiple reaction sites of sp 3 and sp 2 CÀ 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.

Artificial Small Molecules as Cofactors and Biomacromolecular Building Blocks in Synthetic Biology: Design, Synthesis, Applications, and Challenges

“…An even more exciting advancement in this design cofactor system is that the DFSM-facilitated system enables access to over half of all possible hydroxylated products from each given alkylbenzenes substrate, with excellent regioselectivity (up to 99%) and enantioselectivity (up to 99% e.e.) and high total TON (up to 80963) [151]. These results indicate that the synergistic use of an exogenous DFSM and protein engineering constitutes an efficient strategy for controlling the regio-and enantioselectivity of P450BM3 for non-native substrates.…”

Unraveling the Catalytic Mechanism of Taxadiene-5α-hydroxylase from Crystallography and Computational Analyses

“…The QM/MM coupling was treated by the electronic embedding scheme, while the QM/MM boundary was treated with hydrogen link atoms with the charge-shift model. UB3LYP functionals were used for the QM region, − which were demonstrated to be reliable for Fe-containing enzymes. ,− The double-ζ basis set def2-SVP­(B1) was used to optimize the geometry, while the larger basis set of def2-TZVP­(B2) was used to correct the energies . Grimme’s D3 method was employed to account for dispersion corrections in all quantum mechanical calculations. − The QM region in the system included the substrate, the propionate-truncated heme, and the side chain of Cys383.…”