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.

Efficient Transferase Engineering for SAM Analog Synthesis from Iodoalkanes.

S-Adenosyl-l-methionine (SAM) is an important cosubstrate in various biochemical processes, including selective methyl transfer reactions. Simple methods for the (re)generation of SAM analogs could expand the chemistry accessible with SAM-dependent transferases and go beyond methylation reactions. Here we present an efficient enzyme engineering strategy to synthesize different SAM analogs from "off-the-shelf" iodoalkanes through enzymatic alkylation of S-adenosyl-l-homocysteine (SAH). This was achieved by mutating multiple hydrophobic and structurally dynamic amino acids simultaneously. Combinatorial mutagenesis was guided by the natural amino acid diversity and generated a highly functional mutant library. This approach increased the speed as well as the scale of enzyme engineering by providing a panel of optimized enzymes with orders of magnitude higher activities for multiple substrates in just one round of enzyme engineering. The optimized enzymes exhibit catalytic efficiencies up to 31 M-1 s-1, convert various iodoalkanes, including substrates bearing cyclopropyl or aromatic moieties, and catalyze S-alkylation of SAH with very high stereoselectivities (>99 % de). We further report a high throughput chromatographic screening system for reliable and rapid SAM analog analysis. We believe that the methods and enzymes described herein will further advance the field of selective biocatalytic alkylation chemistry by enabling SAM analog regeneration with "off-the-shelf" reagents.

Controlling Monoterpene Isomerization by Guiding Challenging Carbocation Rearrangement Reactions in Engineered Squalene-Hopene Cyclases.

The interconversion of monoterpenes is facilitated by a complex network of carbocation rearrangement pathways. Controlling these isomerization pathways is challenging when using common Brønsted and Lewis acid catalysts, which often produce product mixtures that are difficult to separate. In contrast, natural monoterpene cyclases exhibit high control over the carbocation rearrangement reactions but are reliant on phosphorylated substrates. In this study, we present engineered squalene-hopene cyclases from Alicyclobacillus acidocaldarius (AacSHC) that catalyze the challenging isomerization of monoterpenes with unprecedented precision. Starting from a promiscuous isomerization of (+)-ß-pinene, we first demonstrate noticeable shifts in the product distribution solely by introducing single point mutations. Furthermore, we showcase the tuneable cation steering by enhancing (+)-borneol selectivity from 1 % to >90 % (>99 % de) aided by iterative saturation mutagenesis. Our combined experimental and computational data suggest that the reorganization of key aromatic residues leads to the restructuring of the water network that facilitates the selective termination of the secondary isobornyl cation. This work expands our mechanistic understanding of carbocation rearrangements and sets the stage for target-oriented skeletal reorganization of broadly abundant terpenes.

Engineering cytochrome P450s for selective alkene to carbonyl oxidation.

The Wacker-Tsuji oxidation is an important aerobic oxidation process to synthesize ethanal from ethene and methyl ketones from 1-alkenes. Current challenges in aerobic alkene oxidation include selective carbonyl product formation beyond methyl ketones. This includes the regioselective oxidation of the terminal carbon atom of 1-alkenes, the regioselective ketone formation with internal alkenes as well as the enantioselective alkene to carbonyl oxidation. Recently, the potential of high-valent metal-oxo species for direct alkene to carbonyl oxidation was explored as carbonyl product formation is frequently reported as a side reaction of alkene epoxidation by cytochrome P450s. It was shown that such promiscuous P450s can be engineered via directed evolution to perform alkene to carbonyl oxidation reactions with high activity and selectivity. Here, we report a protocol to convert promiscuous P450s into efficient and selective enzymes for Wacker-type alkene oxidation. One round of directed evolution is described in detail, which includes the generation and handling of site-saturation libraries, recombinant protein expression, library screening in a 96-well plate format and the rescreening of variants with beneficial mutations. These protocols might be useful to engineer various P450s for selective alkene to carbonyl oxidation, and to engineer enzymes in general.

Comparative S-adenosyl-L-methionine analogue generation for selective biocatalytic Friedel-Crafts alkylation.

Methyltransferases provide excellent specificity in late-stage alkylation of biomolecules. Their dependence on S-adenosyl-L-methionine (SAM) mandates efficient access to SAM analogues for biocatalytic applications. We directly compared halide methyltransferase (HMT) and methionine adenosyltransferase (MAT) to access SAM analogues and explored their utility in cascade reactions with NovO for regioselective, late-stage Friedel-Crafts alkylation of a coumarin. The HMT cascade efficiently provided SAM for methylation, while the MAT cascade also supplied high levels of SAM analogues for alkylation reactions.

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.

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

Enantioselective synthesis of chiral alcohols through asymmetric addition of water across an unactivated alkene is a highly sought-after transformation and a big challenge in catalysis. Herein we report the identification and directed evolution of a fatty acid hydratase from Marinitoga hydrogenitolerans for the highly enantioselective hydration of styrenes to yield chiral 1-arylethanols. While directed evolution for styrene hydration was performed in the presence of heptanoic acid to mimic fatty acid binding, the engineered enzyme displayed remarkable asymmetric styrene hydration activity in the absence of the small molecule activator. The evolved styrene hydratase provided access to chiral alcohols from simple alkenes and water with high enantioselectivity (>99 : 1 e.r.) and could be applied on a preparative scale.

Selective Biocatalytic N-Methylation of Unsaturated Heterocycles.

Methods for regioselective N-methylation and -alkylation of unsaturated heterocycles with "off the shelf" reagents are highly sought-after. This reaction could drastically simplify synthesis of privileged bioactive molecules. Here we report engineered and natural methyltransferases for challenging N-(m)ethylation of heterocycles, including benzimidazoles, benzotriazoles, imidazoles and indazoles. The reactions are performed through a cyclic enzyme cascade that consists of two methyltransferases using only iodoalkanes or methyl tosylate as simple reagents. This method enables the selective synthesis of important molecules that are otherwise difficult to access, proceeds with high regioselectivity (r.r. up to >99 %), yield (up to 99 %), on a preparative scale, and with nearly equimolar concentrations of simple starting materials.

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.

Biocatalytic Alkylation Chemistry: Building Molecular Complexity with High Selectivity.

Biocatalysis has traditionally been viewed as a field that primarily enables access to chiral centers. This includes the synthesis of chiral alcohols, amines and carbonyl compounds, often through functional group interconversion via hydrolytic or oxidation-reduction reactions. This limitation is partly being overcome by the design and evolution of new enzymes. Here, we provide an overview of a recently thriving research field that we summarize as biocatalytic alkylation chemistry. In the past 3-4 years, numerous new enzymes have been developed that catalyze sp3 C-C/N/O/S bond formations. These enzymes utilize different mechanisms to generate molecular complexity by coupling simple fragments with high activity and selectivity. In many cases, the engineered enzymes perform reactions that are difficult or impossible to achieve with current small-molecule catalysts such as organocatalysts and transition-metal complexes. This review further highlights that the design of new enzyme function is particularly successful when off-the-shelf synthetic reagents are utilized to access non-natural reactive intermediates. This underscores how biocatalysis is gradually moving to a field that build molecules through selective bond forming reactions.

Fast and easy synthesis of the non-commercially available standard isobutyl monophosphate (ammonium salt).

In an attempt to establish a biosynthetic route towards isobutene, we faced the problem that the first intermediate isobutyl-monophosphate was not commercially available. In order to overcome this limitation we searched in the literature for protocols reporting the synthesis of phosphate monoesters from alcohols. Based on the suitability of the preceding developments for our purposes, we established a customized method for the fast, easy and affordable generation of the pursued molecule. Herein, a prompt and straightforward method for isobutyl-monophosphate (ammonium salt) is provided.•This is a customized method for the production of isobutyl-monophosphate (ammonium salt), using isobutanol as starting compound•Synthesis takes place in a one-pot fashion, under mild reaction conditions, in 2 h•The established sequential strategy requires 8 h at the most, including synthesis and purification steps to obtain the isolated product.

Engineered Enzymes Enable Selective N-Alkylation of Pyrazoles With Simple Haloalkanes.

Selective alkylation of pyrazoles could solve a challenge in chemistry and streamline synthesis of important molecules. Here we report catalyst-controlled pyrazole alkylation by a cyclic two-enzyme cascade. In this enzymatic system, a promiscuous enzyme uses haloalkanes as precursors to generate non-natural analogs of the common cosubstrate S-adenosyl-l-methionine. A second engineered enzyme transfers the alkyl group in highly selective C-N bond formations to the pyrazole substrate. The cosubstrate is recycled and only used in catalytic amounts. Key is a computational enzyme-library design tool that converted a promiscuous methyltransferase into a small enzyme family of pyrazole-alkylating enzymes in one round of mutagenesis and screening. With this enzymatic system, pyrazole alkylation (methylation, ethylation, propylation) was achieved with unprecedented regioselectivity (>99 %), regiodivergence, and in a first example on preparative scale.

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.

Anti-Markovnikov alkene oxidation by metal-oxo-mediated enzyme catalysis.

Catalytic anti-Markovnikov oxidation of alkene feedstocks could simplify synthetic routes to many important molecules and solve a long-standing challenge in chemistry. Here we report the engineering of a cytochrome P450 enzyme by directed evolution to catalyze metal-oxo-mediated anti-Markovnikov oxidation of styrenes with high efficiency. The enzyme uses dioxygen as the terminal oxidant and achieves selectivity for anti-Markovnikov oxidation over the kinetically favored alkene epoxidation by trapping high-energy intermediates and catalyzing an oxo transfer, including an enantioselective 1,2-hydride migration. The anti-Markovnikov oxygenase can be combined with other catalysts in synthetic metabolic pathways to access a variety of challenging anti-Markovnikov functionalization reactions.

Squalene hopene cyclases are protonases for stereoselective Brønsted acid catalysis.

For many important reactions catalyzed in chemical laboratories, the corresponding enzymes are missing, representing a restriction in biocatalysis. Although nature provides highly developed machineries appropriate to catalyze such reactions, their potential is often ignored. This also applies to Brønsted acid catalysis, a powerful method to promote a myriad of chemical transformations. Here, we report on the unique protonation machinery of a squalene hopene cyclase (SHC). Active site engineering of this highly evolvable enzyme yielded a platform for enzymatic Brønsted acid catalysis in water. This is illustrated by activation of different functional groups (alkenes, epoxides and carbonyls), enabling the highly stereoselective syntheses of various cyclohexanoids while uncoupling SHC from polycyclization chemistry. This work highlights the potential of systematic investigation on nature's catalytic machineries to generate unique catalysts.

Entropy is key to the formation of pentacyclic terpenoids by enzyme-catalyzed polycyclization.

Polycyclizations constitute a cornerstone of chemistry and biology. Multicyclic scaffolds are generated by terpene cyclase enzymes in nature through a carbocationic polycyclization cascade of a prefolded polyisoprene backbone, for which electrostatic stabilization of transient carbocationic species is believed to drive catalysis. Computational studies and site-directed mutagenesis were used to assess the contribution of entropy to the polycyclization cascade catalyzed by the triterpene cyclase from A. acidocaldarius. Our results show that entropy contributes significantly to the rate enhancement through the release of water molecules through specific channels. A single rational point mutation that results in the disruption of one of these water channels decreased the entropic contribution to catalysis by 60 kcal mol(-1) . This work demonstrates that entropy is the key to enzyme-catalyzed polycyclizations, which are highly relevant in biology since 90 % of all natural products contain a cyclic subunit.

New generation of biocatalysts for organic synthesis.

The use of enzymes as catalysts for the preparation of novel compounds has received steadily increasing attention over the past few years. High demands are placed on the identification of new biocatalysts for organic synthesis. The catalysis of more ambitious reactions reflects the high expectations of this field of research. Enzymes play an increasingly important role as biocatalysts in the synthesis of key intermediates for the pharmaceutical and chemical industry, and new enzymatic technologies and processes have been established. Enzymes are an important part of the spectrum of catalysts available for synthetic chemistry. The advantages and applications of the most recent and attractive biocatalysts--reductases, transaminases, ammonia lyases, epoxide hydrolases, and dehalogenases--will be discussed herein and exemplified by the syntheses of interesting compounds.

Squalene hopene cyclases: highly promiscuous and evolvable catalysts for stereoselective CC and CX bond formation.

We review here how the inherent promiscuous nature, as well as the evolvability of terpene cyclase enzymes enables new applications in chemistry. We mainly focus on squalene hopene cyclases, class II triterpene synthases that use a proton-initiated cationic polycyclization cascade to form carbopolycyclic products. We highlight recent findings to demonstrate that these enzymes are capable of activating different functionalities other than the traditional terminal isoprene C=C-group as well as being compatible with a wide range of nucleophiles beyond the 'ene-functionality'. Thus, squalene hopene cyclases demonstrate a great potential to be used as a toolbox for general Brønsted acid catalysis.