Computational design of myoglobin-based carbene transferases for monoterpene derivatization.

Carbene transfer reactions have emerged as pivotal methodologies for the synthesis of complex molecular architectures. Heme protein-catalyzed carbene transfer reactions have shown promising results on model compounds. However, their limited substrate scope has hindered their application in natural product functionalization. Building upon the foundation of previously published work on a carbene transferase-myoglobin variant, this study employs computer-aided protein engineering to design myoglobin variants, using either docking or the deep learning-based LigandMPNN method. These variants were utilized as catalysts in carbene transfer reactions with a selection of monoterpene substrates featuring C-C double bonds, leading to seven target products. This cost-effective methodology broadens the substrate scope for heme protein-catalyzed reactions, thereby opening novel pathways for research in heme protein functionalities and offering fresh perspectives in the synthesis of bioactive molecules.

Intramolecular Cyclopropanation of Active Methylene Derivatives Based on FeCl2 or FeCl3-Promoted Radical-Polar Crossover Reactions.

Radical-polar crossover reactions were studied for the intramolecular cyclopropanation of active methylene derivatives. In the presence of FeCl3 as a stoichiometric oxidant and K2HPO4 as a base, the dehydrogenative cyclopropanation of active methylenes proceeded through the FeCl3-promoted oxidative radical cyclization followed by the ionic cyclization to give the bicyclic cyclopropanes. The use of α-chloro-active methylenes leads the subcatalytic cyclopropanation involving two redox pathways. In the presence of K2HPO4, the redox cyclopropanation proceeded by using FeCl2 (20 mol%) in combination with ligand (20 mol%).

Chiral 4-MeO-Pyridine (MOPY) Catalyst for Enantioselective Cyclopropanation: Attenuation of Lewis Basicity Leads to Improved Catalytic Efficiency.

The design of pyridine-derived organocatalysts aims at the increase of their Lewis basicity, however such an approach is not always efficient. For example, strongly Lewis basic DMAP is completely inefficient as catalyst in the cyclopropanation reaction. Herein we disclose an alternative approach that relies on attenuation of DMAP Lewis basicity. Specifically, the replacement of 4-dimethylamino substituent in DMAP for 4-MeO group delivered a highly efficient catalyst for cyclopropanation of electron-deficient olefins with α-bromoketones. Kinetic studies provide compelling evidence that the superior catalytic efficiency of 4-MeO pyridine (MOPY) is to be attributed to the favorable balance between Lewis basicity and leaving group ability. The use of chiral, enantiomerically pure MOPY catalyst has helped to achieve high enantioselectivities (up to 91 : 9 er) in the previously unreported pyridine-catalyzed cyclopropanation 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.

Intramolecular Oxyalkylation of Unactivated Alkenes.

The synthesis of cyclopropanes by the cyclization of allylic diazoesters is well-known. In prior studies toward the sesquiterpenoid euonyminol, we attempted to carry out an intramolecular cyclopropanation of an allylic diazoester containing an electronically-unbiased alkene embedded in a 6-oxa-bicyclo[3.2.1]-oct-3-ene skeleton. We obtained exclusively a product arising from 1,2-addition of oxygen and carbon (oxyalkylation) to the alkene. While oxyalkylation products have been reported when electron-rich alkenes (e.g. enol ethers) are employed, examples derived from electronically-unbiased alkenes are rare. Here, we establish that the oxyalkylation is general for a range of 6-oxa-bicyclo[3.2.1]-oct-3-ene substrates and show that these products form competitively in the cyclization of simpler α-diazo ß-ketoesters. Our data suggest increasing charge separation in the transition state for the addition promotes the oxyalkylation pathway.

Unusual Enzymatic C-C Bond Formation and Cleavage Reactions during Natural Product Biosynthesis.

Natural products from plants and microorganisms provide a valuable reservoir of pharmaceutical compounds. C-C bond formation and cleavage are crucial events during natural product biosynthesis, playing pivotal roles in generating diverse and intricate chemical structures that are essential for biological functions. This review summarizes our recent findings regarding biosynthetic enzymes that catalyze unconventional C-C bond formation and cleavage reactions during natural product biosynthesis.

Vitamin B12 -Photocatalyzed Cyclopropanation of Electron-Deficient Alkenes Using Dichloromethane as the Methylene Source.

The cyclopropyl group is of great importance in medicinal chemistry, as it can be leveraged to influence a range of pharmaceutical properties in drug molecules. This report describes a Vitamin B12 -photocatalyzed approach for the cyclopropanation of electron-deficient alkenes using dichloromethane (CH2 Cl2 ) as the methylene source. The reaction proceeds in good to excellent yields under mild conditions, has excellent functional group compatibility, and is highly chemoselective. The scope could also be extended to the preparation of D2 -cyclopropyl and methyl-substituted cyclopropyl adducts starting from CD2 Cl2 and 1,1-dichloroethane, respectively.

Aromative Dephosphinidenation of a Bisphosphirane-Fused Anthracene toward E-H (E = H, Si, N and P) Bond Activation.

A bisphosphirane-fused anthracene (5) was prepared by treatment of a sterically encumbered amino phosphorus dichloride (3) with MgA•3THF (A = anthracene). X-ray diffraction analysis revealed a pentacyclic framework consisting of 5 with two phosphirane rings fused to the anthracene in a trans-fashion. Compound 5 has been shown to be an efficient phosphinidene synthon, readily liberating two transient phosphinidene units for subsequent downstream bond activation via the reductive elimination of anthracene under mild conditions. The formal oxidative addition of H2 and E-H (E = Si, N, P) bonds by the liberated phosphinidene provided diphosphine and substituted phosphines. Furthermore, phosphinidene transfer to alkenes and alkynes smoothly yielded the corresponding phosphiranes and phosphirenes. The mechanism of the H2 activation by 5 was investigated by density functional theory (DFT) calculations.

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.

Dichloromethane as C1 Synthon for the Photoredox Catalytic Cyclopropanation of Aromatic Olefins.

Dichloromethane, as a readily available and inexpensive C1 synthon is proposed as a powerful building block for cyclopropanation of alkenes under mild conditions. Herein, we report a highly efficient and versatile dual photoredox system, involving a nickel aminopyridine coordination complex and a photocatalyst, for the cyclopropanation of aromatic olefins using dichloromethane, under visible-light irradiation. The cyclopropanation protocol has been successfully applied at gram scale. Mechanistic studies suggest a Ni(II) pyridyl radical complex as the key intermediate for the homolytic cleavage of the Csp3-Cl bond, generating a chloromethyl radical that is captured by the olefin coupling partner. Our findings also highlight the versatility of this methodology. By directing the radical/polar crossover process, we were able to selectively drive the reaction towards either the formation of cyclopropyl derivatives or the corresponding non-cyclic alkyl chloride products. The methodology also successfully apply to geminal dichloroalkanes, including the formation of spiro[2,2] compounds. Moreover, our methodology extends to the synthesis of deuterium-labelled cyclopropanes, demonstrating its utility in isotopic labelling and broadening its applicability in chemical synthesis and drug development.

Redox Engineering of Myoglobin by Cofactor Substitution to Enhance Cyclopropanation Reactivity.

Design of metal cofactor ligands is essential for controlling the reactivity of metalloenzymes. We investigated a carbene transfer reaction catalyzed by myoglobins containing iron porphyrin cofactors with one and two trifluoromethyl groups at peripheral sites (FePorCF3 and FePor(CF3)2, respectively), native heme and iron porphycene (FePc). These four myoglobins show a wide range of Fe(II)/Fe(III) redox potentials in the protein of +147 mV, +87 mV, +42 mV and -198 mV vs. NHE, respectively. Myoglobin reconstituted with FePor(CF3)2 has a more positive potential, which enhances the reactivity of a carbene intermediate with alkenes, and demonstrates superior cyclopropanation of inert alkenes, such as aliphatic and internal alkenes. In contrast, engineered myoglobin reconstituted with FePc has a more negative redox potential, which accelerates the formation of the intermediate, but has low reactivity for inert alkenes. Mechanistic studies indicate that myoglobin with FePor(CF3)2 generates an undetectable active intermediate with a radical character. In contrast, this reaction catalyzed by myoglobin with FePc includes a detectable iron-carbene species with electrophilic character. This finding highlights the importance of redox-focused design of the iron porphyrinoid cofactor in hemoproteins to tune the reactivity of the carbene transfer reaction.

Metalloradical Catalysis: General Approach for Controlling Reactivity and Selectivity of Homolytic Radical Reactions.

Since Friedrich Wöhler's groundbreaking synthesis of urea in 1828, organic synthesis over the past two centuries has predominantly relied on the exploration and utilization of chemical reactions rooted in two-electron heterolytic ionic chemistry. While one-electron homolytic radical chemistry is both rich in fundamental reactivities and attractive with practical advantages, the synthetic application of radical reactions has been long hampered by the formidable challenges associated with the control over reactivity and selectivity of high-energy radical intermediates. To fully harness the untapped potential of radical chemistry for organic synthesis, there is a pressing need to formulate radically different concepts and broadly applicable strategies to address these outstanding issues. In pursuit of this objective, researchers have been actively developing metalloradical catalysis (MRC) as a comprehensive framework to guide the design of general approaches for controlling over reactivity and stereoselectivity of homolytic radical reactions. Essentially, MRC exploits the metal-centered radicals present in open-shell metal complexes as one-electron catalysts for homolytic activation of substrates to generate metal-entangled organic radicals as the key intermediates to govern the reaction pathway and stereochemical course of subsequent catalytic radical processes. Different from the conventional two-electron catalysis by transition metal complexes, MRC operates through one-electron chemistry utilizing stepwise radical mechanisms.

An Artificial Enzyme for Asymmetric Nitrocyclopropanation of α,ß-Unsaturated Aldehydes-Design and Evolution.

The introduction of an abiological catalytic group into the binding pocket of a protein host allows for the expansion of enzyme chemistries. Here, we report the generation of an artificial enzyme by genetic encoding of a non-canonical amino acid that contains a secondary amine side chain. The non-canonical amino acid and the binding pocket function synergistically to catalyze the asymmetric nitrocyclopropanation of α,ß-unsaturated aldehydes by the iminium activation mechanism. The designer enzyme was evolved to an optimal variant that catalyzes the reaction at high conversions with high diastereo- and enantioselectivity. This work demonstrates the application of genetic code expansion in enzyme design and expands the scope of enzyme-catalyzed abiological reactions.

The Rapid Synthesis of Colibactin Warhead Model Compounds Using New Metal-Free Photocatalytic Cyclopropanation Reactions Facilitates the Investigation of Biological Mechanisms.

Herein, we report the synthesis of a series of colibactin warhead model compounds using two newly developed metal-free photocatalytic cyclopropanation reactions. These mild cyclopropanations expand the known applications of eosin within synthesis. A halogen atom transfer reaction mode has been harnessed so that dihalides can be used as the cyclopropanating agents. The colibactin warhead models were then used to provide new insight into two key mechanisms in colibactin chemistry. An explanation is provided for why the colibactin warhead sometimes undergoes a ring expansion-addition reaction to give fused cyclobutyl products while at other times nucleophiles add directly to the cyclopropyl unit (as when DNA adds to colibactin). Finally, we provide some evidence that Cu(II) chelated to colibactin may catalyze an important oxidation of the colibactin-DNA adduct. The Cu(I) generated as a result could then also play a role in inducing double strand breaks in DNA.

Simmons-Smith Cyclopropanation: A Multifaceted Synthetic Protocol toward the Synthesis of Natural Products and Drugs: A Review.

Simmons-Smith cyclopropanation is a widely used reaction in organic synthesis for stereospecific conversion of alkenes into cyclopropane. The utility of this reaction can be realized by the fact that the cyclopropane motif is a privileged synthetic intermediate and a core structural unit of many biologically active natural compounds such as terpenoids, alkaloids, nucleosides, amino acids, fatty acids, polyketides and drugs. The modified form of Simmons-Smith cyclopropanation involves the employment of Et2Zn and CH2I2 (Furukawa reagent) toward the total synthesis of a variety of structurally complex natural products that possess broad range of biological activities including anticancer, antimicrobial and antiviral activities. This review aims to provide an intriguing glimpse of the Furukawa-modified Simmons-Smith cyclopropanation, within the year range of 2005 to 2022.

Diastereoselective Synthesis of Cyclopropanes from Carbon Pronucleophiles and Alkenes.

Cyclopropanes are desirable structural motifs with valuable applications in drug discovery and beyond. Established alkene cyclopropanation methods give rise to cyclopropanes with a limited array of substituents, are difficult to scale, or both. Herein, we disclose a new cyclopropane synthesis through the formal coupling of abundant carbon pronucleophiles and unactivated alkenes. This strategy exploits dicationic adducts derived from electrolysis of thianthrene in the presence of alkene substrates. We find that these dielectrophiles undergo cyclopropanation with methylene pronucleophiles via alkenyl thianthrenium intermediates. This protocol is scalable, proceeds with high diastereoselectivity, and tolerates diverse functional groups on both the alkene and pronucleophile coupling partners. To validate the utility of this new procedure, we prepared an array of substituted analogs of an established cyclopropane that is en route to multiple pharmaceuticals.

Divergent Total Syntheses of (+)-Vulgarisins†A-E.

The asymmetric total syntheses of (+)-vulgarisins A-E, which share a rare and highly oxygenated [5-6-4-5] tetracyclic core structure that were isolated from P. vulgaris Linn., have been described for the first time in a divergent manner. Key transformations include: 1) a catalytic asymmetric intramolecular cyclopropanation to forge the A ring bearing desired stereochemistry at C14; 2) a one-pot borylation/conjugate addition process for creation of the C1-C11 bond; 3) a Wolff ring contraction to assemble the bicyclo[3.2.0]heptane subunit (CD rings); and 4) a stereocontrolled pinacol cyclization for construction of the central B ring of the natural products.

Taming of Furfurylidenes by Chiral Bismuth-Rhodium Paddlewheel Catalysts. Preparation and Functionalization of Optically Active 1,1-Disubstituted (Trifluoromethyl)cyclopropanes.

Although 2-furyl-carbenes (furfurylidenes) are prone to instantaneous electrocyclic ring opening, chiral [BiRh]-paddlewheel complexes empowered by London dispersion allow (trifluoromethyl)furfurylidene metal complexes to be generated from a bench-stable triftosylhydrazone precursor. These reactive intermediates engage in asymmetric [2+1] cycloadditions and hence open entry into valuable trifluoromethylated cyclopropane or -cyclopropene derivatives in optically active form, which are important building blocks for medicinal chemistry but difficult to make otherwise.

Chitin- and Streptavidin-Mediated Affinity Purification Systems: A Screening Platform for Enzyme Discovery.

Affinity purification of recombinant proteins is an essential technique in biotechnology. However, current affinity purification methods are very cost-intensive, and this imposes limits on versatile use of affinity purification for obtaining purified proteins for a variety of applications. To overcome this problem, we developed a new affinity purification system which we call CSAP (chitin- and streptavidin-mediated affinity purification) for low-cost purification of Strep-tag II fusion proteins. The CSAP system is designed to utilize commercially available chitin powder as a chromatography matrix, thereby significantly improving the cost-efficiency of protein affinity purification. We investigated the CSAP system for protein screening in 96-well format as a demonstration. Through the screening of 96 types of purified hemoproteins, several proteins capable of the catalytic diastereodivergent synthesis of cyclopropanes were identified as candidates for an abiotic carbene transfer reaction.

Low Coordination State RhI -Complex as High Performance Catalyst for Asymmetric Intramolecular Cyclopropanation: Construction of penta-Substituted Cyclopropanes.

A simple, broad-scope rhodium(I)/chiral diene catalytic system for challenging asymmetric intramolecular cyclopropanation of various tri-substituted allylic diazoacetates was successfully developed. The low coordination state RhI -complex exhibits an extraordinarily high degree of tolerance to the variation in the extent of substitution of the allyl double bond, thus allowing the efficient construction of a wide range of penta-substituted, fused-ring cyclopropanes bearing three contiguous stereogenic centers, including two quaternary carbon stereocenters, in a highly enantioselective manner with ease at catalyst loading as low as 0.1 mol %. The stereoinduction mode of this RhI -carbene-directed asymmetric intramolecular cyclopropanation was investigated by DFT calculations, indicating that π-π stacking interactions between the aromatic rings of chiral diene ligand and diazo substrate play a key role in the control of the reaction enantioselectivity.