Directed Evolution of a Ketone Synthase for Efficient and Highly Selective Functionalization of Internal Alkenes by Accessing Reactive Carbocation Intermediates

Abstract

The direct regioselective oxidation of internal alkenes to ketones could simplify synthetic routes and solve a longstanding challenge in synthesis. This reaction is of particular importance because ketones are predominant moieties in valuable products as well as crucial intermediates in synthesis. Here we report the directed evolution of a ketone synthase that oxidizes internal alkenes directly to ketones with several thousand turnovers. The evolved ketone synthase benefits from more than a dozen crucial mutations, most of them distal to the active site. Computational analysis reveals that all these mutations collaborate to facilitate the formation of a highly reactive carbocation intermediate by generating a confined, rigid and preorganized active site through an enhanced dynamical network. The evolved ketone synthase fully exploits a catalytic cycle that has largely eluded small molecule catalysis and consequently enables various challenging functionalization reactions of internal alkenes. This includes the first catalytic, enantioselective oxidation of internal alkenes to ketones, as well as the formal asymmetric hydration and hydroamination of unactivated internal alkenes in combination with other biocatalysts.

Enzymatic Control over Reactive Intermediates Enables Direct Oxidation of Alkenes to Carbonyls by a P450 Iron-Oxo Species

“…The dynamic behavior of such intermediates which indeed can be (pro-)chiral 52 has a large impact on the chemoselectivity of the reaction. Mechanistic insights obtained in this work provide useful guidance that may help to further expand this new biocatalytic direct oxidation of alkenes to carbonyl compounds toward more challenging substrates, including internal alkenes 66 or unactivated, aliphatic alkenes. We envision that other biological or abiological reactions might use similar mechanisms to outcompete intrinsic dynamic effects of reactive intermediates to access alternative catalytic cycles that are challenging to achieve otherwise.…”

Enlightening the Path to Protein Engineering: Chemoselective Turn-On Probes for High-Throughput Screening of Enzymatic Activity

“…The group of Hammer recently optimized a ketone synthase for the regioselective anti -Markovnikov oxidation of aromatic alkenes. Starting from a P450 wild-type, 12 rounds of evolution identified 18 mutations that were beneficial for the stereoselective oxidation of styrene derivatives 145 (Scheme a) …”

Chiral Alcohols from Alkenes and Water: Directed Evolution of a Styrene Hydratase

“…This enzymatic reaction involves a p ‐quinone methide intermediate and the process is thus a conjugate addition to an activated alkene (unsaturated carbonyl compound), a feature that explains the limitation of this reaction to 4‐hydroxystyrenes as substrates (Figure S2). In addition, anti‐Markovnikov hydration of styrenes to produce 2‐arylethanols has recently been reported with a promiscuous monooxygenase [12] and through redox hydration using engineered P450s and ADHs in a two‐enzyme cascade [13, 14] …”

“…In addition, anti-Markovnikov hydration of styrenes to produce 2-arylethanols has recently been reported with a promiscuous monooxygenase [12] and through redox hydration using engineered P450s and ADHs in a two-enzyme cascade. [13,14] Several classes of natural enzymes (termed hydratases) are known that add water to unactivated alkenes with outstanding regio-and enantioselectivity. [15][16][17][18][19][20][21][22][23] Alkene hydration by hydratases is proposed to be achieved through precise positioning of the alkene and water substrates in combination with cooperative Brønsted acid-base catalysis, which simultaneously activates the alkene electrophile and the water nucleophile (Figure 1b).…”