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.

Anchoring a Structurally Editable Proximal Cofactor‐like Module to Construct an Artificial Dual‐center Peroxygenase

“…The peroxygenase activity of P450BM3 was evaluated in the presence of Im-C6-Phe and Im-DFSM-dipeps with styrene epoxidation, ethylbenzene hydroxylation, and thioanisole sulfoxidation as model reactions under standard conditions, as previously reported (Figure S43). [33,39] Two beneficial mutants, namely, F87A for styrene and thioanisole and F87A/T268 V for ethylbenzene, were selected as previously reported. [27,33] The catalytic activity of the P450BM3 F87A mutant was enhanced with increased Im-DFSM-dipeps concentration, consistent with Im-C6-Phe observed previously.…”

“…[33,39] Two beneficial mutants, namely, F87A for styrene and thioanisole and F87A/T268 V for ethylbenzene, were selected as previously reported. [27,33] The catalytic activity of the P450BM3 F87A mutant was enhanced with increased Im-DFSM-dipeps concentration, consistent with Im-C6-Phe observed previously. Compared with Im-C6-Phe, Im-DFSM-dipeps generally tends to achieve its highest catalytic TON at lower concentration, which was demonstrated using concentration gradient experiments (Figure 4B).…”

Post‐Nickelation of a Crystalline Trinuclear Copper Organic Framework for Synergistic Photocatalytic Carbon Dioxide Conversion

“…[7] In comparison with the metal-free COFs, metal-containing COFs prepared by either direct integration of metal coordination modules or postsynthetic modification (PSM) with metal ions have been demonstrated to show excellent catalytic properties due to the combination of active metal sites with highly porous structures and unique photonic/electronic characteristics. [8] In this direction, the chemistry of COFs and metal-organic framework (MOFs) has been demonstrated to generate interesting frameworks materials with both coordination linkages and covalent linkages. [9] However, investigations over fabrication of heterometallic COFs and mixed linkage frameworks still remain extremely rare, limited to PAE-NiCuPcF8, NiPc-NH-CoPcF 8 , [3h] CuPcF 8 -CoNPc, CdS@COF, and MCOF-Ti 6 Cu 3 [9e] afforded through the pre-design synthesis by directly integrating two coordination modules possessing different metal ions together, [10] NiÀ Ir@Tp-Bpy via PSM, [3k] and ReCoCOF by combination of pre-design synthesis with PSM, [11] to the best of our knowledge.…”

A Simple Access to γ‐ and ε‐Keto Arenes via Enzymatic Divergent C─H Bond Oxyfunctionalization

“…For example, evolved P450 enzymes have been applied to provide direct access to the oxidation of less reachable C─H bonds in artemisinin, [ 11 ] sclareol, [ 12 ] terpenes [ 13 ] or steroids. [ 14 ] The recent synergistic use of protein engineering and exogenous decoy molecules enabled P450 peroxygenases to perform divergent hydroxylation of alkylarenes at multiple sp 3 C─H bonds [ 15 ] (Scheme 1b). Also, both the wild‐type and engineered fatty acid hydroxylases P450 BM3 [ 16 ] and CYP152 [ 17 ] have demonstrated to hydroxylate the fatty acids at divergent sites from the carboxylate group for valorizing the renewable fatty acids.…”