Complete integration of carbene-transfer chemistry into biosynthesis.

Biosynthesis is an environmentally benign and renewable approach that can be used to produce a broad range of natural and, in some cases, new-to-nature products. However, biology lacks many of the reactions that are available to synthetic chemists, resulting in a narrower scope of accessible products when using biosynthesis rather than synthetic chemistry. A prime example of such chemistry is carbene-transfer reactions1. Although it was recently shown that carbene-transfer reactions can be performed in a cell and used for biosynthesis2,3, carbene donors and unnatural cofactors needed to be added exogenously and transported into cells to effect the desired reactions, precluding cost-effective scale-up of the biosynthesis process with these reactions. Here we report the access to a diazo ester carbene precursor by cellular metabolism and a microbial platform for introducing unnatural carbene-transfer reactions into biosynthesis. The α-diazoester azaserine was produced by expressing a biosynthetic gene cluster in Streptomyces albus. The intracellularly produced azaserine was used as a carbene donor to cyclopropanate another intracellularly produced molecule-styrene. The reaction was catalysed by engineered P450 mutants containing a native cofactor with excellent diastereoselectivity and a moderate yield. Our study establishes a scalable, microbial platform for conducting intracellular abiological carbene-transfer reactions to functionalize a range of natural and new-to-nature products and expands the scope of organic products that can be produced by cellular metabolism.

Catalytic asymmetric addition of an amine N-H bond across internal alkenes.

Hydroamination of alkenes, the addition of the N-H bond of an amine across an alkene, is a fundamental, yet challenging, organic transformation that creates an alkylamine from two abundant chemical feedstocks, alkenes and amines, with full atom economy1-3. The reaction is particularly important because amines, especially chiral amines, are prevalent substructures in a wide range of natural products and drugs. Although extensive efforts have been dedicated to developing catalysts for hydroamination, the vast majority of alkenes that undergo intermolecular hydroamination have been limited to conjugated, strained, or terminal alkenes2-4; only a few examples occur by the direct addition of the N-H bond of amines across unactivated internal alkenes5-7, including photocatalytic hydroamination8,9, and no asymmetric intermolecular additions to such alkenes are known. In fact, current examples of direct, enantioselective intermolecular hydroamination of any type of unactivated alkene lacking a directing group occur with only moderate enantioselectivity10-13. Here we report a cationic iridium system that catalyses intermolecular hydroamination of a range of unactivated, internal alkenes, including those in both acyclic and cyclic alkenes, to afford chiral amines with high enantioselectivity. The catalyst contains a phosphine ligand bearing trimethylsilyl-substituted aryl groups and a triflimide counteranion, and the reaction design includes 2-amino-6-methylpyridine as the amine to enhance the rates of multiple steps within the catalytic cycle while serving as an ammonia surrogate. These design principles point the way to the addition of N-H bonds of other reagents, as well as O-H and C-H bonds, across unactivated internal alkenes to streamline the synthesis of functional molecules from basic feedstocks.

Desymmetrization of difluoromethylene groups by C-F bond activation.

Tertiary stereogenic centres containing one fluorine atom are valuable for medicinal chemistry because they mimic common tertiary stereogenic centres containing one hydrogen atom, but they possess distinct charge distribution, lipophilicity, conformation and metabolic stability1-3. Although tertiary stereogenic centres containing one hydrogen atom are often set by enantioselective desymmetrization reactions at one of the two carbon-hydrogen (C-H) bonds of a methylene group, tertiary stereocentres containing fluorine have not yet been constructed by the analogous desymmetrization reaction at one of the two carbon-fluorine (C-F) bonds of a difluoromethylene group3. Fluorine atoms are similar in size to hydrogen atoms but have distinct electronic properties, causing C-F bonds to be exceptionally strong and geminal C-F bonds to strengthen one another4. Thus, exhaustive defluorination typically dominates over the selective replacement of a single C-F bond, hindering the development of the enantioselective substitution of one fluorine atom to form a stereogenic centre5,6. Here we report the catalytic, enantioselective activation of a single C-F bond in an allylic difluoromethylene group to provide a broad range of products containing a monofluorinated tertiary stereogenic centre. By combining a tailored chiral iridium phosphoramidite catalyst, which controls regioselectivity, chemoselectivity and enantioselectivity, with a fluorophilic activator, which assists the oxidative addition of the C-F bond, these reactions occur in high yield and selectivity. The design principles proposed in this work extend to palladium-catalysed benzylic substitution, demonstrating the generality of the approach.

Transition-Metal-Catalyzed Silylation and Borylation of C-H Bonds for the Synthesis and Functionalization of Complex Molecules.

The functionalization of C-H bonds in organic molecules containing functional groups has been one of the holy grails of catalysis. One synthetically important approach to the diverse functionalization of C-H bonds is the catalytic silylation or borylation of C-H bonds, which enables a broad array of downstream transformations to afford diverse structures. Advances in both undirected and directed methods for the transition-metal-catalyzed silylation and borylation of C-H bonds have led to their rapid adoption in early-, mid-, and late-stage of the synthesis of complex molecules. In this Review, we review the application of the transition-metal-catalyzed silylation and borylation of C-H bonds to the synthesis of bioactive molecules, organic materials, and ligands. Overall, we aim to provide a picture of the state of art of the silylation and borylation of C-H bonds as applied to the synthesis and modification of diverse architectures that will spur further application and development of these reactions.

Mechanistic Insights into the Origins of Selectivity in a Cu-Catalyzed C-H Amidation Reaction.

The catalytic transformation of C-H to C-N bonds offers rapid access to fine chemicals and high-performance materials, but achieving high selectivity from undirected aminations of unactivated C(sp3)-H bonds remains an outstanding challenge. We report the origins of the reactivity and selectivity of a Cu-catalyzed C-H amidation of simple alkanes. Using a combination of experimental and computational mechanistic studies and energy decomposition techniques, we uncover a switch in mechanism from inner-sphere to outer-sphere coupling between alkyl radicals and the active Cu(II) catalyst with increasing substitution of the alkyl radical. The combination of computational predictions and detailed experimental validation shows that simultaneous minimization of both Cu-C covalency and alkyl radical size increases the rate of reductive elimination and that both strongly electron-donating and electron-withdrawing substituents on the catalyst accelerate the selectivity-determining C-N bond formation process as a result of a change in mechanism. These findings offer design principles for the development of improved catalyst scaffolds for radical C-H functionalization reactions.

Polymers from Plant Oils Linked by Siloxane Bonds for Programmed Depolymerization.

The increased production of plastics is leading to the accumulation of plastic waste and depletion of limited fossil fuel resources. In this context, we report a strategy to create polymers that can undergo controlled depolymerization by linking renewable feedstocks with siloxane bonds. α,ω-Diesters and α,ω-diols containing siloxane bonds were synthesized from an alkenoic ester derived from castor oil and then polymerized with varied monomers, including related biobased monomers. In addition, cyclic monomers derived from this alkenoic ester and hydrosiloxanes were prepared and cyclized to form a 26-membered macrolactone containing a siloxane unit. Sequential ring-opening polymerization of this macrolactone and lactide afforded an ABA triblock copolymer. This set of polymers containing siloxanes underwent programmed depolymerization into monomers in protic solvents or with hexamethyldisiloxane and an acid catalyst. Monomers afforded by the depolymerization of polyesters containing siloxane linkages were repolymerized to demonstrate circularity in select polymers. Evaluation of the environmental stability of these polymers toward enzymatic degradation showed that they undergo enzymatic hydrolysis by a fungal cutinase from Fusarium solani. Evaluation of soil microbial metabolism of monomers selectively labeled with 13C revealed differential metabolism of the main chain and side chain organic groups by soil microbes.

2-Aminophenanthroline Ligands Enable Mild, Undirected, Iridium-Catalyzed Borylation of Alkyl C-H Bonds.

The catalytic, undirected borylation of alkyl C-H bonds typically occurs at high reaction temperatures or with excess substrate, or both, because of the low reactivity of alkyl C-H bonds. Here we report a new iridium system comprising 2-anilino-1,10-phenanthroline as the ligand that catalyzes the borylation of alkyl C-H bonds with little to no induction period and with high reaction rates. This superior activation and reactivity profile of 2-aminophenanthroline-ligated catalysts leads to broader reaction scope, including reactions of sensitive substrates, such as epoxides and glycosidic acetals, enhanced diastereoselectivity, and higher yields of borylated products. These catalysts also enable the borylation of alkanes, amines, and ethers at room temperature for the first time. Mechanistic studies imply that facile N-borylation occurs under the reaction conditions and that iridium complexes containing N-boryl aminophenanthrolines are competent precatalysts for the reaction.

Copper-Mediated Cyanodifluoromethylation of (Hetero)aryl Iodides and Activated (Hetero)aryl Bromides with TMSCF2CN.

Molecules bearing fluorine are increasingly prevalent in pharmaceuticals, agrochemicals, and functional materials. The cyanodifluoromethyl group is unique because its size is closer than that of any other substituted difluoromethyl group to the size of the trifluoromethyl group, but its electronic properties are distinct from those of the trifluoromethyl group. In addition, the presence of the cyano group provides synthetic entry to a wide range of substituted difluoromethyl groups. However, the synthesis of cyanodifluoromethyl compounds requires multiple steps, highly reactive reagents (such as DAST, NSFI, or IF5), or specialized starting materials (such as α,α-dichloroacetonitriles or α-mercaptoacetonitriles). Herein, we report a copper-mediated cyanodifluoromethylation of aryl and heteroaryl iodides and activated aryl and heteroaryl bromides with TMSCF2CN. This cyanodifluoromethylation tolerates an array of functional groups, is applicable to late-stage functionalization of complex molecules, yields analogues of FDA-approved pharmaceuticals and fine chemicals, and enables the synthesis of a range of complex molecules bearing a difluoromethylene unit by transformations of the electron-poor CN unit. Calculations of selected steps of the reaction mechanism by Density Functional Theory indicate that the barriers for both the oxidative addition of iodobenzene to [(DMF)CuCF2CN] and the reductive elimination of the fluoroalkyl product from the fluoroalkyl copper intermediate lie in between those of [(DMF)CuCF3] and [(DMF)CuCF2C(O)NMe2].

Palladium-Catalyzed Amination of Aryl Halides with Aqueous Ammonia and Hydroxide Base Enabled by Ligand Development.

The conversion of aryl halides to primary arylamines with a convenient and inexpensive source of ammonia has been a long-standing synthetic challenge. Aqueous ammonia would be the most convenient and least expensive form of ammonia, but such a palladium-catalyzed amination reaction with a high concentration of water faces challenges concerning catalyst stability and competing hydroxylation, and palladium-catalyzed reactions with this practical reagent are rare. Further, most reactions with ammonia to form primary amines are conducted with tert-butoxide base, but reactions with ammonium hydroxide would contain hydroxide as base. Thus, ammonia surrogates, ammonia in organic solvents, and ammonium salts have been used under anhydrous conditions instead with varying levels of selectivity for the primary amine. We report the palladium-catalyzed amination of aryl and heteroaryl chlorides and bromides with aqueous ammonia and a hydroxide base to form the primary arylamine with high selectivity. The palladium catalyst containing a new dialkyl biheteroaryl phosphine ligand (KPhos) suppresses both the formation of aryl alcohol and diarylamine side products. Mechanistic studies with a soluble hydroxide base revealed turnover-limiting reductive elimination of the arylamine and an equilibrium between arylpalladium amido and hydroxo complexes prior to the turnover-limiting step.

Enantio- and Diastereodivergent Cyclopropanation of Allenes by Directed Evolution of an Iridium-Containing Cytochrome.

Alkylidene cyclopropanes (ACPs) are valuable synthetic intermediates because of their constrained structure and opportunities for further diversification. Although routes to ACPs are known, preparations of ACPs with control of both the configuration of the cyclopropyl (R vs S) group and the geometry of the alkene (E vs Z) are unknown. We describe enzymatic cyclopropanation of allenes with ethyl diazoacetate (EDA) catalyzed by an iridium-containing cytochrome (Ir(Me)-CYP119) that controls both stereochemical elements. Two mutants of Ir(Me)-CYP119 identified by 6-codon (6c, VILAFG) saturation mutagenesis catalyze the formation of (E)-ACPs with -93% to >99% ee and >99:1 E/Z ratio with just three rounds of 96 mutants. By four additional rounds of mutagenesis, an enzyme variant was identified that forms (Z)-ACPs with up to 94% ee and a 28:72 E/Z ratio. Computational studies show that the orientation of the carbene unit dictated by the mutated positions accounts for the stereoselectivity.

Progression of Hydroamination Catalyzed by Late Transition-Metal Complexes from Activated to Unactivated Alkenes.

ConspectusCatalytic intermolecular hydroamination of alkenes is an atom- and step-economical method for the synthesis of amines, which have important applications as pharmaceuticals, agrochemicals, catalysts, and materials. However, hydroaminations of alkenes in high yield with high selectivity are challenging to achieve because these reactions often lack a thermodynamic driving force and often are accompanied by side reactions, such as alkene isomerization, telomerization, and oxidative amination. Consequently, early examples of hydroamination were generally limited to the additions of N-H bonds to conjugated alkenes or strained alkenes, and the catalytic hydroamination of unactivated alkenes with late transition metals has only been disclosed recently. Many classes of catalysts, including early transition metals, late transition metals, rare-earth metals, acids, and photocatalysts, have been reported for catalytic hydroamination. Among them, late transition-metal complexes possess several advantages, including their relative ease of handling and their high compatibility of substrates containing polar or sensitive functional groups.This Account describes the progression in our laboratory of hydroaminations catalyzed by late transition-metal complexes from the initial additions of N-H bonds to activated alkenes to the more recent additions to unactivated alkenes. Our developments include the Markovnikov and anti-Markovnikov hydroamination of vinylarenes with palladium, rhodium, and ruthenium, the hydroamination of dienes and trienes with nickel and palladium, the hydroanimation of bicyclic strained alkenes with neutral iridium, and the hydroamination of unactivated terminal and internal alkenes with cationic iridium and ruthenium. Enantioselective hydroaminations of these classes of alkenes to form enantioenriched, chiral amines also have been developed.Mechanistic studies have elucidated the elementary steps and the turnover-limiting steps of these catalytic reactions. The hydroamination of conjugated alkenes catalyzed by palladium, rhodium, nickel, and ruthenium occurs by turnover-limiting nucleophilic attack of the amine on a coordinated benzyl, allyl, alkene, or arene ligand. On the other hand, the hydroamination of unconjugated alkenes catalyzed by ruthenium and iridium occurs by turnover-limiting migratory insertion of the alkene into a metal-nitrogen bond. In addition, pathways for the formation of side products, including isomeric alkenes and enamines, have been identified during our studies. During studies on enantioselective hydroamination, the reversibility of the hydroamination has been shown to erode the enantiopurity of the products. Based on our mechanistic understandings, new generations of catalysts that promote catalytic hydroaminations with higher rates, chemoselectivity, and enantioselectivity have been developed. We hope that our discoveries and mechanistic insights will facilitate the further development of catalysts that promote selective, practical, and efficient hydroamination of alkenes.

Deconvolution and Analysis of the 1H NMR Spectra of Crude Reaction Mixtures.

Nuclear magnetic resonance (NMR) spectroscopy is an important analytical technique in synthetic organic chemistry, but its integration into high-throughput experimentation workflows has been limited by the necessity of manually analyzing the NMR spectra of new chemical entities. Current efforts to automate the analysis of NMR spectra rely on comparisons to databases of reported spectra for known compounds and, therefore, are incompatible with the exploration of new chemical space. By reframing the NMR spectrum of a reaction mixture as a joint probability distribution, we have used Hamiltonian Monte Carlo Markov Chain and density functional theory to fit the predicted NMR spectra to those of crude reaction mixtures. This approach enables the deconvolution and analysis of the spectra of mixtures of compounds without relying on reported spectra. The utility of our approach to analyze crude reaction mixtures is demonstrated with the experimental spectra of reactions that generate a mixture of isomers, such as Wittig olefination and C-H functionalization reactions. The correct identification of compounds in a reaction mixture and their relative concentrations is achieved with a mean absolute error as low as 1%.

Cooperative asymmetric reactions combining photocatalysis and enzymatic catalysis.

Living organisms rely on simultaneous reactions catalysed by mutually compatible and selective enzymes to synthesize complex natural products and other metabolites. To combine the advantages of these biological systems with the reactivity of artificial chemical catalysts, chemists have devised sequential, concurrent, and cooperative chemoenzymatic reactions that combine enzymatic and artificial catalysts1-9. Cooperative chemoenzymatic reactions consist of interconnected processes that generate products in yields and selectivities that cannot be obtained when the two reactions are carried out sequentially with their respective substrates2,7. However, such reactions are difficult to develop because chemical and enzymatic catalysts generally operate in different media at different temperatures and can deactivate each other1-9. Owing to these constraints, the vast majority of cooperative chemoenzymatic processes that have been reported over the past 30 years can be divided into just two categories: chemoenzymatic dynamic kinetic resolutions of racemic alcohols and amines, and enzymatic reactions requiring the simultaneous regeneration of a cofactor2,4,5. New approaches to the development of chemoenzymatic reactions are needed to enable valuable chemical transformations beyond this scope. Here we report a class of cooperative chemoenzymatic reaction that combines photocatalysts that isomerize alkenes with ene-reductases that reduce carbon-carbon double bonds to generate valuable enantioenriched products. This method enables the stereoconvergent reduction of E/Z mixtures of alkenes or reduction of the unreactive stereoisomers of alkenes in yields and enantiomeric excesses that match those obtained from the reduction of the pure, more reactive isomers. The system affords a range of enantioenriched precursors to biologically active compounds. More generally, these results show that the compatibility between photocatalysts and enzymes enables chemoenzymatic processes beyond cofactor regeneration and provides a general strategy for converting stereoselective enzymatic reactions into stereoconvergent ones.

Carbonic Anhydrase Variants Catalyze the Reduction of Dialkyl Ketones with High Enantioselectivity.

Human carbonic anhydrase II (hCAII) naturally catalyzes the reaction between two achiral molecules - water and carbon dioxide - to yield the achiral product carbonic acid through a zinc hydroxide intermediate. We have previously shown that a zinc hydride, instead of a hydroxide, can be generated in this enzyme to create a catalyst for the reduction of aryl ketones. Dialkyl ketones are more challenging to reduce, and the enantioselective reduction of dialkyl ketones with two alkyl groups that are similar in size and electronic properties, is a particularly challenging transformation to achieve with high activity and selectivity. Here, we show that hCAII, as well as a double variant of it, catalyzes the enantioselective reduction of dialkyl ketones with high yields and enantioselectivities, even when the two alkyl groups are similar in size. We also show that variants of hCAII catalyze the site-selective reduction of one ketone over the other in an unsymmetrical aliphatic diketone. Computational docking of a dialkyl ketone to the double variant containing the zinc hydride provides insights into the origins of the reactivity of various substrates and the high enantioselectivity of the transformations and show how a confined environment can control the enantioselectivity of an abiological intermediate.

Progress, Challenges, and Opportunities with Artificial Metalloenzymes in Biosynthesis.

In this Perspective, we present progress, outstanding challenges, and opportunities for the incorporation of artificial metalloenzymes (ArMs) into biosynthetic pathways. We first explain discoveries within the field of ArMs that led to the potential inclusion of these enzymes in biosynthesis. We then describe the specific barriers that our laboratory, in collaboration with the laboratories of Keasling and Mukhopadhyay, addressed to establish a biosynthetic pathway containing an ArM. This biosynthesis produced an unnatural cyclopropyl terpenoid by combining heterologous production of the terpene with modification of its terminal alkene by an ArM built from a cytochrome P450. Finally, we describe the remaining challenges and opportunities related to the application of ArMs in synthetic biology.

Remote Hydroamination of Disubstituted Alkenes by a Combination of Isomerization and Regioselective N-H Addition.

Remote hydrofunctionalizations of alkenes incorporate functional groups distal to existing carbon-carbon double bonds. While remote carbonylations are well-known, remote hydrofunctionalizations are most common for addition of relatively nonpolar B-H, Si-H, and C-H bonds with alkenes. We report a system for the remote hydroamination of disubstituted alkenes to functionalize an alkyl chain selectively at the subterminal, unactivated, methylene position. Critical to the high regioselectivity and reaction rates are the electronic properties of the substituent on the amine and the development of the ligand DIP-Ad-SEGPHOS by evaluating the steric and electronic effects of ligand modules on reactivity and selectivity. The remote hydroamination is compatible with a broad scope of alkenes and aminopyridines and enables the regioconvergent synthesis of amines from an isomeric mixture of alkenes. The products can be derivatized by nucleophilic aromatic substitution on the amino substituent with a variety of nucleophiles.

Iridium-Catalyzed, Site-Selective Silylation of Secondary C(sp3)-H Bonds in Secondary Alcohols and Ketones.

We report the iridium-catalyzed, stereoselective conversion of secondary alcohols or ketones to anti-1,3-diols by the silylation of secondary C-H bonds γ to oxygen and oxidation of the resulting oxasilolane. The silylation of secondary C-H bonds in secondary silyl ethers derived from alcohols or ketones is enabled by a catalyst formed from a simple bisamidine ligand. The silylation occurs with high selectivity at a secondary C-H bond γ to oxygen over distal primary or proximal secondary C-H bonds. Initial mechanistic investigations suggest that the source of the newly achieved reactivity is a long catalyst lifetime resulting from the high binding constant of the strongly electron-donating bisamidine ligand.

Hybrid Machine Learning Approach to Predict the Site Selectivity of Iridium-Catalyzed Arene Borylation.

The borylation of aryl and heteroaryl C-H bonds is valuable for the site-selective functionalization of C-H bonds in complex molecules. Iridium catalysts ligated by bipyridine ligands catalyze the borylation of the C-H bond that is most acidic and least sterically hindered in an arene, but predicting the site of borylation in molecules containing multiple arenes is difficult. To address this challenge, we report a hybrid computational model that predicts the Site of Borylation (SoBo) in complex molecules. The SoBo model combines density functional theory, semiempirical quantum mechanics, cheminformatics, linear regression, and machine learning to predict site selectivity and to extrapolate these predictions to new chemical space. Experimental validation of SoBo showed that the model predicts the major site of borylation of pharmaceutical intermediates with higher accuracy than prior machine-learning models or human experts, demonstrating that SoBo will be useful to guide experiments for the borylation of specific C(sp2)-H bonds during pharmaceutical development.

Chemical Modification of Oxidized Polyethylene Enables Access to Functional Polyethylenes with Greater Reuse.

Polyethylene is a commodity material that is widely used because of its low cost and valuable properties. However, the lack of functional groups in polyethylene limits its use in applications that include adhesives, gas barriers, and plastic blends. The inertness of polyethylene makes it difficult to install groups that would enhance its properties and enable programmed chemical decomposition. To overcome these deficiencies, the installation of pendent functional groups that imbue polyethylene with enhanced properties is an attractive strategy to overcome its inherent limitations. Here, we describe strategies to derivatize oxidized polyethylene that contains both ketones and alcohols to monofunctional variants with bulk properties superior to those of unmodified polyethylene. Iridium-catalyzed transfer dehydrogenation with acetone furnished polyethylenes with only ketones, and ruthenium-catalyzed hydrogenation with hydrogen furnished polyethylenes with only alcohols. We demonstrate that the ratio of these functional groups can be controlled by reduction with stoichiometric hydride-containing reagents. The ketones and alcohols serve as sites to introduce esters and oximes onto the polymer, thereby improving surface and bulk properties over those of polyethylene. These esters and oximes were removed by hydrolysis to regenerate the original oxygenated polyethylenes, showing how functionalization can lead to materials with circularity. Waste polyethylenes were equally amenable to oxidative functionalization and derivatization of the oxidized material, showing that this low- or negative-value feedstock can be used to prepare materials of higher value. Finally, the derivatized polymers with distinct solubilities were separated from mechanically mixed plastic blends by selective dissolution, demonstrating that functionalization can lead to novel approaches for distinguishing and separating polymers from a mixture.

Discovery of the Azaserine Biosynthetic Pathway Uncovers a Biological Route for α-Diazoester Production.

Azaserine is a bacterial metabolite containing a biologically unusual and synthetically enabling α-diazoester functional group. Herein, we report the discovery of the azaserine (aza) biosynthetic gene cluster from Glycomyces harbinensis. Discovery of related gene clusters reveals previously unappreciated azaserine producers, and heterologous expression of the aza gene cluster confirms its role in azaserine assembly. Notably, this gene cluster encodes homologues of hydrazonoacetic acid (HYAA)-producing enzymes, implicating HYAA in α-diazoester biosynthesis. Isotope feeding and biochemical experiments support this hypothesis. These discoveries indicate that a 2-electron oxidation of a hydrazonoacetyl intermediate is required for α-diazoester formation, constituting a distinct logic for diazo biosynthesis. Uncovering this biological route for α-diazoester synthesis now enables the production of a highly versatile carbene precursor in cells, facilitating approaches for engineering complete carbene-mediated biosynthetic transformations in vivo.