Molecular Basis for Chemoselectivity Control in Oxidations of Internal Aryl-Alkenes Catalyzed by Laboratory Evolved P450s.

P450 enzymes naturally perform selective hydroxylations and epoxidations of unfunctionalized hydrocarbon substrates, among other reactions. The adaptation of P450 enzymes to a particular oxidative reaction involving alkenes is of great interest for the design of new synthetically useful biocatalysts. However, the mechanism that these enzymes utilize to precisely modulate the chemoselectivity and distinguishing between competing alkene double bond epoxidations and allylic C-H hydroxylations is sometimes not clear, which hampers the rational design of specific biocatalysts. In a previous work, a P450 from Labrenzia aggregata (P450LA1) was engineered in the laboratory using directed evolution to catalyze the direct oxidation of trans-ß-methylstyrene to phenylacetone. The final variant, KS, was able to overcome the intrinsic preference for alkene epoxidation to directly generate a ketone product via the formation of a highly reactive carbocation intermediate. Here, additional library screening along this evolutionary lineage permitted to serendipitously detect a mutation that overcomes epoxidation and carbonyl formation by exhibiting a large selectivity of 94 % towards allylic C-H hydroxylation. A multiscalar computational methodology was applied to reveal the molecular basis towards this hydroxylation preference. Enzyme modelling suggests that introduction of a bulky substitution dramatically changes the accessible conformations of the substrate in the active site, thus modifying the enzymatic selectivity towards terminal hydroxylation and avoiding the competing epoxidation pathway, which is sterically hindered.

Methylation of Unactivated Alkenes with Engineered Methyltransferases To Generate Non-natural Terpenoids.

Terpenoids are built from isoprene building blocks and have numerous biological functions. Selective late-stage modification of their carbon scaffold has the potential to optimize or transform their biological activities. However, the synthesis of terpenoids with a non-natural carbon scaffold is often a challenging endeavor because of the complexity of these molecules. Herein we report the identification and engineering of (S)-adenosyl-l-methionine-dependent sterol methyltransferases for selective C-methylation of linear terpenoids. The engineered enzyme catalyzes selective methylation of unactivated alkenes in mono-, sesqui- and diterpenoids to produce C11 , C16 and C21 derivatives. Preparative conversion and product isolation reveals that this biocatalyst performs C-C bond formation with high chemo- and regioselectivity. The alkene methylation most likely proceeds via a carbocation intermediate and regioselective deprotonation. This method opens new avenues for modifying the carbon scaffold of alkenes in general and terpenoids in particular.

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

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 effects 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 1,2-hydride 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.

Substrate Profiling of Anion Methyltransferases for Promiscuous Synthesis of S-Adenosylmethionine Analogs from Haloalkanes.

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.

Asymmetric Enzymatic Hydration of Unactivated, Aliphatic Alkenes.

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.