Repurposing myoglobin into a carbene transferase for a [2,3]-sigmatropic Sommelet-Hauser rearrangement.

New-to-Nature biocatalysis has emerged as a promising tool in organic synthesis thanks to progress in protein engineering. Notably, hemeproteins have been evolved into robust catalysts for carbene and nitrene transfers and related sigmatropic rearrangements. In this work, we report the first example of a [2,3]-sigmatropic Sommelet-Hauser rearrangement initiated by a carbene transfer of the sperm whale myoglobin mutant L29S,H64V,V68F that was previously reported to catalyze the mechanistically similar [2,3]-sigmatropic Doyle-Kirmse rearrangement. This repurposed heme enzyme catalyzes the Sommelet-Hauser rearrangement between ethyl diazoacetate and benzyl thioethers bearing strong electron-withdrawing substituents with good yields and enantiomeric excess. Optimized catalytic conditions in the absence of any reductant led to an increased asymmetric induction with up to 59% enantiomeric excess. This myoglobin mutant is therefore one of the few catalysts for the asymmetric Sommelet-Hauser rearrangement. This work broadens the scope of abiological reactions catalyzed by iron-carbene transferases with a new example of asymmetric sigmatropic rearrangement.

Biocatalytic Strategy for the Highly Stereoselective Synthesis of Fluorinated Cyclopropanes.

Fluorinated cyclopropanes are highly desired pharmacophores in drug discovery owing to the rigid nature of the cyclopropane ring and the beneficial effects of C-F bonds on the pharmacokinetic properties, cell permeability, and metabolic stability of drug molecules. Herein a biocatalytic strategy for the stereoselective synthesis of mono-fluorinated and gem-difluoro cyclopropanes is reported though the use of engineered myoglobin-based catalysts. In particular, this system allows for a broad range of gem-difluoro alkenes to be cyclopropanated in the presence of diazoacetonitrile with excellent diastereo and enantiocontrol (up to 99 : 1 d.r. and 99 % e.e.), thereby enabling a transformation not currently accessible with chemocatalytic methods. The synthetic utility of the present approach is further exemplified through the gram-scale synthesis of a key gem-difluorinated cyclopropane intermediate useful for the preparation of fluorinated bioactive molecules.

Deoxygenation of Oxiranes by λ3 σ3 -Phosphorus Reagents: A Computational, Mechanistic, and Stereochemical Study.

The deoxygenation of parent and substituted oxiranes by λ3 σ3 -phosphorus reagents has been explored in detail, therefore unveiling mechanistic aspects as well as regio- and stereochemical consequences. Attack to a ring C atom is almost always preferred over one-step deoxygenation by direct P-to-O attack. In most cases a carbene transfer occurs as first step, leading to a phosphorane and a carbonyl unit that thereafter react in the usual Wittig fashion via the corresponding λ5 σ5 -1,2-oxaphosphetane intermediate. Betaines rarely constitute true minima after the first C-attack to oxiranes, at least in the gas-phase. Use of the heavier derivatives AsMe3 and SbMe3 as oxirane deoxygenating reagents was also mechanistically studied. The thermodynamic tendency of λ3 σ3 -phosphorus reagents to act as oxygen (O-attack) or carbene acceptors (C-attack) was theoretically studied by means of the thermodynamic oxygen-transfer potential (TOP) and the newly defined thermodynamic carbene-transfer potential (TCP) parameters, that were explored in a wider context together with many other acceptor centres.

Molecular Dynamic Simulations of Aqueous Micellar Organometallic Catalysis: Methane Functionalization as a Case Study.

Molecular Dynamics (MD) simulations constitute a powerful tool that provides a 3D perspective of the dynamical behavior of chemical systems. Herein the first MD study of the dynamics of a catalytic organometallic system, in micellar media, is presented. The challenging methane catalytic functionalization into ethyl propionate through a silver-catalyzed process has been targeted as the case study. The intimate nature of the micelles formed with the surfactants sodium dodecylsulfate (SDS) and potassium perfluorooctane sulfonate (PFOS) has been ascertained, as well as the relative distribution of the main actors in this transformation, namely methane, the diazo reagent and the silver catalyst, the latter in two different forms: the initial compound and a silver-carbene intermediate. Catalyst deactivation occurs with halide containing surfactants dodecyltrimethylammonium chloride (DTAC) and Triton X-100. Computed simulations allow explaining the experimental results, indicating that micelles behave differently regarding the degree of accumulation and the local distribution of the reactants and their effect in the molecular collisions leading to net reaction.

Synthesis of Trifluoromethylated Monoterpenes by an Engineered Cytochrome P450.

Protein engineering of cytochrome P450s has enabled these biocatalysts to promote a variety of abiotic reactions beyond nature's repertoire. Integrating such non-natural transformations with microbial biosynthetic pathways could allow sustainable enzymatic production of modified natural product derivatives. In particular, trifluoromethylation is a highly desirable modification in pharmaceutical research due to the positive effects of the trifluoromethyl group on drug potency, bioavailability, and metabolic stability. This study demonstrates the biosynthesis of non-natural trifluoromethyl-substituted cyclopropane derivatives of natural monoterpene scaffolds using an engineered cytochrome P450 variant, P411-PFA. P411-PFA successfully catalyzed the transfer of a trifluoromethyl carbene from 2-diazo-1,1,1-trifluoroethane to the terminal alkenes of several monoterpenes, including L-carveol, carvone, perilla alcohol, and perillartine, to generate the corresponding trifluoromethylated cyclopropane products. Furthermore, integration of this abiotic cyclopropanation reaction with a reconstructed metabolic pathway for L-carveol production in Escherichia coli enabled one-step biosynthesis of a trifluoromethylated L-carveol derivative from limonene precursor. Overall, amalgamating synthetic enzymatic chemistry with established metabolic pathways represents a promising approach to sustainably produce bioactive natural product analogs.

Iron-Catalysed Carbene Transfer to Isocyanides as a Platform for Heterocycle Synthesis.

An iron-catalysed carbene transfer reaction of diazo compounds to isocyanides has been developed. The resulting ketenimines are trapped in situ with various bisnucleophiles to access a range of densely functionalized heterocycles (pyrimidinones, dihydropyrazolones, 1H-tetrazoles) in a one-pot process. The electron-rich Hieber anion ([Fe(CO)3 NO]- ) facilitates efficient catalytic carbene transfer from acceptor-type α-diazo carbonyl compounds to isocyanides, providing a cost-efficient and benign alternative to similar noble metal-catalysed processes. Based on DFT calculations a plausible reaction mechanism for activation of the α-diazo carbonyl carbene precursor and ketenimine formation is provided.

YfeX - A New Platform for Carbene Transferase Development with High Intrinsic Reactivity.

Carbene transfer biocatalysis has evolved from basic science to an area with vast potential for the development of new industrial processes. In this study, we show that YfeX, naturally a peroxidase, has great potential for the development of new carbene transferases, due to its high intrinsic reactivity, especially for the N-H insertion reaction of aromatic and aliphatic primary and secondary amines. YfeX shows high stability against organic solvents (methanol and DMSO), greatly improving turnover of hydrophobic substrates. Interestingly, in styrene cyclopropanation, WT YfeX naturally shows high enantioselectivity, generating the trans product with 87 % selectivity for the (R,R) enantiomer. WT YfeX also catalyzes the Si-H insertion efficiently. Steric effects in the active site were further explored using the R232A variant. Quantum Mechanics/Molecular Mechanics (QM/MM) calculations reveal details on the mechanism of Si-H insertion. YfeX, and potentially other peroxidases, are exciting new targets for the development of improved carbene transferases.

Repurposed and artificial heme enzymes for cyclopropanation reactions.

Heme enzymes are some of the most versatile catalysts in nature. In recent years it has been found that they can also catalyze reactions for which there are no equivalents in nature. This development has been driven by the abiological catalytic reactivity reported for bio-inspired and biomimetic iron porphyrin complexes. This review focuss es on heme enzymes for catalysis of cyclopropanation reactions. The two most important approaches used to create enzymes for cyclopropanation are repurposing of heme enzymes and the various strategies used to improve these enzymes such as mutagenesis and heme replacement, and artificial heme enzymes. These strategies are introduced and compared. Moreover, lessons learned with regard to mechanism and design principles are discussed.

Noncanonical Heme Ligands Steer Carbene Transfer Reactivity in an Artificial Metalloenzyme*.

Changing the primary metal coordination sphere is a powerful strategy for tuning metalloprotein properties. Here we used amber stop codon suppression with engineered pyrrolysyl-tRNA synthetases, including two newly evolved enzymes, to replace the proximal histidine in myoglobin with Nδ -methylhistidine, 5-thiazoylalanine, 4-thiazoylalanine and 3-(3-thienyl)alanine. In addition to tuning the heme redox potential over a >200 mV range, these noncanonical ligands modulate the protein's carbene transfer activity with ethyl diazoacetate. Variants with increased reduction potential proved superior for cyclopropanation and N-H insertion, whereas variants with reduced Eo values gave higher S-H insertion activity. Given the functional importance of histidine in many enzymes, these genetically encoded analogues could be valuable tools for probing mechanism and enabling new chemistries.

Biocatalytic Strategy for the Highly Stereoselective Synthesis of CHF2 -Containing Trisubstituted Cyclopropanes.

The difluoromethyl (CHF2 ) group has attracted significant attention in drug discovery and development efforts, owing to its ability to serve as fluorinated bioisostere of methyl, hydroxyl, and thiol groups. Herein, we report an efficient biocatalytic method for the highly diastereo- and enantioselective synthesis of CHF2 -containing trisubstituted cyclopropanes. Using engineered myoglobin catalysts, a broad range of α-difluoromethyl alkenes are cyclopropanated in the presence of ethyl diazoacetate to give CHF2 -containing cyclopropanes in high yield (up to >99 %, up to 3000 TON) and with excellent stereoselectivity (up to >99 % de and ee). Enantiodivergent selectivity and extension of the method to the stereoselective cyclopropanation of mono- and trifluoromethylated olefins was also achieved. This methodology represents a powerful strategy for the stereoselective synthesis of high-value fluorinated building blocks for medicinal chemistry, as exemplified by the formal total synthesis of a CHF2 isostere of a TRPV1 inhibitor.

Diversity-Oriented Enzymatic Synthesis of Cyclopropane Building Blocks.

While biocatalysis is increasingly incorporated into drug development pipelines, it is less commonly used in the early stages of drug discovery. By engineering a protein to produce a chiral motif with a derivatizable functional handle, biocatalysts can be used to help generate diverse building blocks for drug discovery. Here we show the engineering of two variants of Rhodothermus marinus nitric oxide dioxygenase (RmaNOD) to catalyze the formation of cis- and tran- diastereomers of a pinacolboronate-substituted cyclopropane which can be readily derivatized to generate diverse stereopure cyclopropane building blocks.

Strategies for the expression and characterization of artificial myoglobin-based carbene transferases.

Myoglobin has recently emerged as a versatile metalloprotein scaffold for the design of efficient and selective biocatalysts for abiological carbene transfer reactions, including asymmetric cyclopropanation reactions. Over the past few years, our group has explored several strategies to modulate the carbene transfer reactivity of myoglobin-based catalysts, including the substitution of the native heme cofactor and conserved histidine axial ligand with non-native porphynoid ligands and alternative natural and unnatural amino acids as the metal-coordinating ligands, respectively. Herein, we report protocols for the generation and reconstitution in vitro and in vivo of myoglobin-based artificial carbene transferases incorporating non-native iron-porphynoid cofactors, also in combination with unnatural amino acids as the proximal ligand. These strategies are effective for imparting these myoglobin-based cyclopropanation biocatalysts with altered and improved function, including tolerance to aerobic conditions and improved reactivity toward electrondeficient olefins.

An Enzymatic Platform for the Highly Enantioselective and Stereodivergent Construction of Cyclopropyl-δ-lactones.

Abiological enzymes offers new opportunities for sustainable chemistry. Herein, we report the development of biological catalysts derived from sperm whale myoglobin that exploit a carbene transfer mechanism for the asymmetric synthesis of cyclopropane-fused-δ-lactones, which are key structural motifs found in many biologically active natural products. While hemin, wild-type myoglobin, and other hemoproteins are unable to catalyze this reaction, the myoglobin scaffold could be remodeled by protein engineering to permit the intramolecular cyclopropanation of a broad spectrum of homoallylic diazoacetate substrates in high yields and with up to 99 % enantiomeric excess. Via an alternate evolutionary trajectory, a stereodivergent biocatalyst was also obtained for affording mirror-image forms of the desired bicyclic products. In combination with whole-cell transformations, the myoglobin-based biocatalyst was used for the asymmetric construction of a cyclopropyl-δ-lactone scaffold at a gram scale, which could be further elaborated to furnish a variety of enantiopure trisubstituted cyclopropanes.

Organic solvent stability and long-term storage of myoglobin-based carbene transfer biocatalysts.

Recent years have witnessed a rapid increase in the application of enzymes for chemical synthesis and manufacturing, including the industrial-scale synthesis of pharmaceuticals using multienzyme processes. From an operational standpoint, these bioprocesses often require robust biocatalysts capable of tolerating high concentrations of organic solvents and possessing long shelflife stability. In this work, we investigated the activity and stability of myoglobin (Mb)-based carbene transfer biocatalysts in the presence of organic solvents and after lyophilization. Our studies demonstrate that Mb-based cyclopropanases possess remarkable organic solvent stability, maintaining high levels of activity and stereoselectivity in the presence of up to 30%-50% (v/v) concentrations of various organic solvents, including ethanol, methanol, N,N-dimethylformamide, acetonitrile, and dimethyl sulfoxide. Furthermore, they tolerate long-term storage in lyophilized form, both as purified protein and as whole cells, without significant loss in activity and stereoselectivity. These stability properties are shared by Mb-based carbene transferases optimized for other type of asymmetric carbene transfer reactions. Finally, we report on simple protocols for catalyst recycling as whole-cell system and for obviating the need for strictly anaerobic conditions to perform these transformations. These findings demonstrate the robustness of Mb-based carbene transferases under operationally relevant conditions and should help guide the application of these biocatalysts for synthetic applications.

Highly Stereoselective Synthesis of Fused Cyclopropane-γ-Lactams via Biocatalytic Iron-Catalyzed Intramolecular Cyclopropanation.

We report the development of an iron-based biocatalytic strategy for the asymmetric synthesis of fused cyclopropane-γ-lactams, which are key structural motifs found in synthetic drugs and bioactive natural products. Using a combination of mutational landscape and iterative site-saturation mutagenesis, sperm whale myoglobin was evolved into a biocatalyst capable of promoting the cyclization of a diverse range of allyl diazoacetamide substrates into the corresponding bicyclic lactams in high yields and with high enantioselectivity (up to 99% ee). These biocatalytic transformations can be performed in whole cells and could be leveraged to enable the efficient (chemo)enzymatic construction of chiral cyclopropane-γ-lactams as well as ß-cyclopropyl amines and cyclopropane-fused pyrrolidines, as valuable building blocks and synthons for medicinal chemistry and natural product synthesis.

A de novo peroxidase is also a promiscuous yet stereoselective carbene transferase.

By constructing an in vivo-assembled, catalytically proficient peroxidase, C45, we have recently demonstrated the catalytic potential of simple, de novo-designed heme proteins. Here, we show that C45's enzymatic activity extends to the efficient and stereoselective intermolecular transfer of carbenes to olefins, heterocycles, aldehydes, and amines. Not only is this a report of carbene transferase activity in a completely de novo protein, but also of enzyme-catalyzed ring expansion of aromatic heterocycles via carbene transfer by any enzyme.

Enantioselective Synthesis of Chiral Amines via Biocatalytic Carbene N-H Insertion.

Optically active amines represent highly valuable building blocks for the synthesis of advanced pharmaceutical intermediates, drug molecules, and biologically active natural products. Hemoproteins have recently emerged as promising biocatalysts for the formation of C-N bonds via carbene transfer, but asymmetric N-H carbene insertion reactions using these or other enzymes have so far been elusive. Here, we report the successful development of a biocatalytic strategy for the asymmetric N-H carbene insertion of aromatic amines with 2-diazopropanoate esters using engineered variants of myoglobin. High activity and stereoinduction in this reaction could be achieved by tuning the chiral environment around the heme cofactor in the metalloprotein in combination with catalyst-matching and tailoring of the diazo reagent. Using this approach, an efficient biocatalytic protocol for the synthesis of a broad range of substituted aryl amines with up to 82% ee was obtained. In addition, a stereocomplementary catalyst useful for accessing the mirror-image form of the N-H insertion products was identified. This work paves the way to asymmetric amine synthesis via biocatalytic carbene transfer, and the present strategy based on the synergistic combination of protein and diazo reagent engineering is expected to prove useful in the context of these as well as other challenging asymmetric carbene transfer reactions.

Aziridinium Ylides: Underutilized Intermediates for Complex Amine Synthesis.

Harnessing the chemistry of onium ylide intermediates generated from transition metal catalysis is a powerful strategy to convert simple precursors into complex scaffolds. While the chemistry of onium ylides has been studied for over three decades, transformations of aziridinium ylides have just recently emerged as a versatile way to exploit the strain of these reactive intermediates to furnish densely functionalized N-heterocycles in a highly stereocontrolled manner. Herein, we provide a short overview of the key concepts and recent developments in this area, with a focus on how mechanistic studies to delineate the factors controlling the reactivity of aziridinium ylides can stimulate fruitful future investigations.

Synthesis of gem-Difluoro Olefins through C-H Functionalization and ß-fluoride Elimination Reactions.

A palladium catalyzed C-H functionalization and consecutive ß-fluoride elimination reaction between indole heterocycles and fluorinated diazoalkanes is reported. This approach provides for the first time a facile method for the rapid synthesis of gem-difluoro olefins using fluorinated diazoalkanes under mild reaction conditions. Cyclopropanation products were obtained when N-arylated rather than N-alkylated indoles were applied in this reaction. Mechanistic studies reveal the importance of the ß-fluoride elimination step in this transformation. This method presents a new concept for the simple and direct transfer of a 1-aryl-(2,2-difluorovinyl) group to access gem-difluoro olefins.

Biocatalytic Strategy for Highly Diastereo- and Enantioselective Synthesis of 2,3-Dihydrobenzofuran-Based Tricyclic Scaffolds.

2,3-Dihydrobenzofurans are key pharmacophores in many natural and synthetic bioactive molecules. A biocatalytic strategy is reported here for the highly diastereo- and enantioselective construction of stereochemically rich 2,3-dihydrobenzofurans in high enantiopurity (>99.9% de and ee), high yields, and on a preparative scale via benzofuran cyclopropanation with engineered myoglobins. Computational and structure-reactivity studies provide insights into the mechanism of this reaction, enabling the elaboration of a stereochemical model that can rationalize the high stereoselectivity of the biocatalyst. This information was leveraged to implement a highly stereoselective route to a drug molecule and a tricyclic scaffold featuring five stereogenic centers via a single-enzyme transformation. This work expands the biocatalytic toolbox for asymmetric C-C bond transformations and should prove useful for further development of metalloprotein catalysts for abiotic carbene transfer reactions.