Biocatalytic alkylation reactions can be performed with high chemo‐, regio‐ and stereoselectivity using S‐adenosyl‐l‐methionine (SAM)‐dependent methyltransferases (MTs) and SAM analogs. Currently, however, this methodology is limited in application due to the rather laborious protocols to access SAM analogs. It has recently been shown that halide methyltransferases (HMTs) enable synthesis and recycling of SAM analogs with readily available haloalkanes as starting material. Here we expand this work by using substrate profiling of the anion MT enzyme family to explore promiscuous SAM analog synthesis. Our study shows that anion MTs are in general very promiscuous with respect to the alkyl chain as well as the halide leaving group. Substrate profiling further suggests that promiscuous anion MTs cluster in sequence space. Next to iodoalkanes, cheaper, less toxic, and more available bromoalkanes have been converted and several haloalkanes bearing short alkyl groups, alkyl rings, and functional groups such as alkene, alkyne and aromatic moieties are accepted as substrates. Further, we applied the SAM analogs as electrophiles in enzyme‐catalyzed regioselective pyrazole allylation with 3‐bromopropene as starting material.

The direct enantioselective addition of water to unactivated alkenes could simplify the synthesis of chiral alcohols and solve a long‐standing challenge in catalysis. Here we report that an engineered fatty acid hydratase can catalyze the asymmetric hydration of various terminal and internal alkenes. In the presence of a carboxylic acid decoy molecule for activation of the oleate hydratase from E. meningoseptica , asymmetric hydration of unactivated alkenes was achieved with up to 93 % conversion, excellent selectivity (>99 % ee , >95 % regioselectivity), and on a preparative scale.

Given the widespread importance of piperazines as building blocks for the production of pharmaceuticals, an efficient and selective synthesis is highly desirable. Here we show the direct synthesis of piperazines from 1,2-dicarbonyl and 1,2-diamine substrates using the R-selective imine reductase from Myxococcus stipitatus as biocatalyst.Various N-and C-substituted piperazines with high activity and excellent enantioselectivity were obtained under mild reaction conditions reaching up to 8.1 g per liter.

The aerobic oxidation of alkenes to carbonyls is an important and challenging transformation in synthesis. Recently, a new P450-based enzyme (aMOx) has been evolved in the laboratory to directly oxidize styrenes to their corresponding aldehydes with high activity and selectivity. The enzyme utilizes a heme-based, high-valent iron-oxo species as a catalytic oxidant that normally epoxidizes alkenes, similar to other catalysts. How the evolved aMOx enzyme suppresses the commonly preferred epoxidation and catalyzes direct carbonyl formation is currently not well understood. Here, we combine computational modelling together with mechanistic experiments to study the reaction mechanism and unravel the molecular basis behind the selectivity achieved by aMOx. Our results describe that although both pathways are energetically accessible diverging from a common covalent radical intermediate, intrinsic dynamic ef fects determine the strong preference for epoxidation. We discovered that aMOx overrides these intrinsic preferences by controlling the accessible conformations of the covalent radical intermediate. This disfavors epoxidation and facilitates the formation of a carbocation intermediate that generates the aldehyde product through a fast 1,2hydride migration. Electrostatic preorganization of the enzyme active site also contributes to the stabilization of the carbocation intermediate. Computations predicted that the hydride migration is stereoselective due to the enzymatic conformational control over the intermediate species. These predictions were corroborated by experiments using deuterated styrene substrates, which proved that the hydride migration is cis-and enantioselective. Our results demonstrate that directed evolution tailored a highly specific active site that imposes strong steric control over key fleeting biocatalytic intermediates, which is essential for accessing the carbonyl forming pathway and preventing competing epoxidation.

Ketones are crucial intermediates in synthesis and frequent moieties in many products. The direct regioselective synthesis of ketones from internal alkenes could simplify synthetic routes and solve a long-standing challenge in catalysis. Here we report the laboratory evolution of a cytochrome P450 enzyme for the direct oxidation of internal arylalkenes to ketones with several thousand turnovers. This evolved ketone synthase benefits from 15 crucial mutations, most of them distal to the active site. Computational analysis revealed that all these mutations collaborate to generate and tame a highly reactive carbocation intermediate. This is achieved through a confined, rigid, and geometrically and electrostatically preorganized active site. The engineered enzyme exploits a metal–oxo species for ketone synthesis and enables various challenging alkene functionalization reactions. This includes the catalytic, enantioselective oxidation of internal alkenes to ketones and formal asymmetric hydrofunctionalizations of internal alkenes in combination with other biocatalysts.