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The Large Magellanic Cloud (LMC) and the Small Magellanic Cloud (SMC) are the closest massive satellite galaxies of the Milky Way. They are probably on their first passage on an infalling orbit towards our Galaxy1 and trace the continuing dynamics of the Local Group2. Recent measurements of a high mass for the LMC (Mhalo ≈ 1011.1-11.4 Mâ)3-6 imply that the LMC should host a Magellanic Corona: a collisionally ionized, warm-hot gaseous halo at the virial temperature (105.3-5.5 K) initially extending out to the virial radius (100-130 kiloparsecs (kpc)). Such a corona would have shaped the formation of the Magellanic Stream7, a tidal gas structure extending over 200° across the sky2,8,9 that is bringing in metal-poor gas to the Milky Way10. Here we show evidence for this Magellanic Corona with a potential direct detection in highly ionized oxygen (O+5) and indirectly by means of triply ionized carbon and silicon, seen in ultraviolet (UV) absorption towards background quasars. We find that the Magellanic Corona is part of a pervasive multiphase Magellanic circumgalactic medium (CGM) seen in many ionization states with a declining projected radial profile out to at least 35 kpc from the LMC and a total ionized CGM mass of log10(MH II,CGM/Mâ) ≈ 9.1 ± 0.2. The evidence for the Magellanic Corona is a crucial step forward in characterizing the Magellanic group and its nested evolution with the Local Group.
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Protein engineering often targets binding pockets or active sites which are enriched in epistasis-nonadditive interactions between amino acid substitutions-and where the combined effects of multiple single substitutions are difficult to predict. Few existing sequence-fitness datasets capture epistasis at large scale, especially for enzyme catalysis, limiting the development and assessment of model-guided enzyme engineering approaches. We present here a combinatorially complete, 160,000-variant fitness landscape across four residues in the active site of an enzyme. Assaying the native reaction of a thermostable ß-subunit of tryptophan synthase (TrpB) in a nonnative environment yielded a landscape characterized by significant epistasis and many local optima. These effects prevent simulated directed evolution approaches from efficiently reaching the global optimum. There is nonetheless wide variability in the effectiveness of different directed evolution approaches, which together provide experimental benchmarks for computational and machine learning workflows. The most-fit TrpB variants contain a substitution that is nearly absent in natural TrpB sequences-a result that conservation-based predictions would not capture. Thus, although fitness prediction using evolutionary data can enrich in more-active variants, these approaches struggle to identify and differentiate among the most-active variants, even for this near-native function. Overall, this work presents a large-scale testing ground for model-guided enzyme engineering and suggests that efficient navigation of epistatic fitness landscapes can be improved by advances in both machine learning and physical modeling.
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Dominio Catalítico , Epistasis Genética , Triptófano Sintasa , Dominio Catalítico/genética , Triptófano Sintasa/genética , Triptófano Sintasa/metabolismo , Triptófano Sintasa/química , Ingeniería de Proteínas/métodos , Sustitución de Aminoácidos , Modelos MolecularesRESUMEN
Aromatic amino acids and their derivatives are diverse primary and secondary metabolites with critical roles in protein synthesis, cell structure and integrity, defense and signaling. All de novo aromatic amino acid production relies on a set of ancient and highly conserved chemistries. Here we introduce a new enzymatic transformation for L-tyrosine synthesis by demonstrating that the ß-subunit of tryptophan synthase-which natively couples indole and L-serine to form L-tryptophan-can act as a latent 'tyrosine synthase'. A single substitution of a near-universally conserved catalytic residue unlocks activity toward simple phenol analogs and yields exclusive para carbon-carbon bond formation to furnish L-tyrosines. Structural and mechanistic studies show how a new active-site water molecule orients phenols for a nonnative mechanism of alkylation, with additional directed evolution resulting in a net >30,000-fold rate enhancement. This new biocatalyst can be used to efficiently prepare valuable L-tyrosine analogs at gram scales and provides the missing chemistry for a conceptually different pathway to L-tyrosine.
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Triptófano Sintasa , Tirosina , Triptófano Sintasa/metabolismo , Triptófano Sintasa/química , Tirosina/química , Tirosina/metabolismo , Dominio Catalítico , Modelos Moleculares , Tirosina Fenol-Liasa/metabolismo , Tirosina Fenol-Liasa/química , Tirosina Fenol-Liasa/genética , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Biocatálisis , Triptófano/química , Triptófano/metabolismoRESUMEN
Although abundant in organic molecules, carbon-hydrogen (C-H) bonds are typically considered unreactive and unavailable for chemical manipulation. Recent advances in C-H functionalization technology have begun to transform this logic, while emphasizing the importance of and challenges associated with selective alkylation at a sp3 carbon1,2. Here we describe iron-based catalysts for the enantio-, regio- and chemoselective intermolecular alkylation of sp3 C-H bonds through carbene C-H insertion. The catalysts, derived from a cytochrome P450 enzyme in which the native cysteine axial ligand has been substituted for serine (cytochrome P411), are fully genetically encoded and produced in bacteria, where they can be tuned by directed evolution for activity and selectivity. That these proteins activate iron, the most abundant transition metal, to perform this chemistry provides a desirable alternative to noble-metal catalysts, which have dominated the field of C-H functionalization1,2. The laboratory-evolved enzymes functionalize diverse substrates containing benzylic, allylic or α-amino C-H bonds with high turnover and excellent selectivity. Furthermore, they have enabled the development of concise routes to several natural products. The use of the native iron-haem cofactor of these enzymes to mediate sp3 C-H alkylation suggests that diverse haem proteins could serve as potential catalysts for this abiological transformation, and will facilitate the development of new enzymatic C-H functionalization reactions for applications in chemistry and synthetic biology.
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Biocatálisis , Carbono/química , Carbono/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Hidrógeno/química , Hierro/química , Alquilación , Animales , Coenzimas/química , Coenzimas/metabolismo , Cisteína/metabolismo , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/genética , Evolución Molecular Dirigida , Hemo/química , Hemo/metabolismo , Hidrógeno/metabolismo , Hierro/metabolismo , Masculino , Metano/análogos & derivados , Metano/química , Serina/metabolismo , Especificidad por Sustrato , Vitamina B 12/química , Vitamina B 12/metabolismoRESUMEN
Coronavirus disease 2019 (COVID-19) vaccines reduce severe disease and mortality and may lessen transmission, measured by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral load (VL). Evaluating vaccine associations in VL at COVID-19 diagnosis in 4 phase 3 randomized, placebo-controlled vaccine trials, July 2020 to July 2021, VL reductions were 2.78 log10 copies/mL (95% confidence interval [CI], 1.38-4.18; n = 60 placebo, 11 vaccine) and 2.12 log10 copies/mL (95% CI, 1.44-2.80; n = 594 placebo, 36 vaccine) for NVX-CoV2373 and mRNA-1273, respectively. Associations were not significant for AZD1222 (0.59 log10 copies/mL; 95% CI, -.19 to 1.36; n = 90 placebo, 78 vaccine) or Ad26.COV2.S (0.23 log10 copies/mL; 95% CI, -.01 to .47; n = 916 placebo, 424 vaccine). Thus, vaccines potentially decreased transmission when ancestral SARS-CoV-2 predominated. Clinical Trials Registration. NCT04470427, NCT04505722, NCT04516746, NCT04611802.
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The mechanism of cyclopropanations with diazirines as air-stable and user-friendly alternatives to commonly employed diazo compounds within iron heme enzyme-catalyzed carbene transfer reactions has been studied by means of density functional theory (DFT) calculations of model systems, quantum mechanics/molecular mechanics (QM/MM) calculations, and molecular dynamics (MD) simulations of the iron carbene and the cyclopropanation transition state in the enzyme active site. The reaction is initiated by a direct diazirine-diazo isomerization occurring in the active site of the enzyme. In contrast, an isomerization mechanism proceeding via the formation of a free carbene intermediate in lieu of a direct, one-step isomerization process was observed for model systems. Subsequent reaction with benzyl acrylate takes place through stepwise C-C bond formation via a diradical intermediate, delivering the cyclopropane product. The origin of the observed diastereo- and enantioselectivity in the enzyme was investigated through MD simulations, which indicate a preferred formation of the cis-cyclopropane by steric control.
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Diazometano , Hemo , Metano/análogos & derivados , Hemo/química , Modelos Moleculares , Hierro , Ciclopropanos/química , CatálisisRESUMEN
Engineered hemoproteins can selectively incorporate nitrogen from nitrene precursors like hydroxylamine, O-substituted hydroxylamines, and organic azides into organic molecules. Although iron-nitrenoids are often invoked as the reactive intermediates in these reactions, their innate reactivity and transient nature have made their characterization challenging. Here we characterize an iron-nitrosyl intermediate generated from NH2OH within a protoglobin active site that can undergo nitrogen-group transfer catalysis, using UV-vis, electron paramagnetic resonance (EPR) spectroscopy, and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) techniques. The mechanistic insights gained led to the discovery of aminating reagentsânitrite (NO2-), nitric oxide (NO), and nitroxyl (HNO)âthat are new to both nature and synthetic chemistry. Based on the findings, we propose a catalytic cycle for C-H amination inspired by the nitrite reductase pathway. This study highlights the potential of engineered hemoproteins to access natural nitrogen sources for sustainable chemical synthesis and offers a new perspective on the use of biological nitrogen cycle intermediates in biocatalysis.
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Hemoproteínas , Aminación , Hemoproteínas/química , Espectroscopía de Resonancia por Spin del Electrón , Óxido Nítrico/química , Espectrometría de Masa por Ionización de Electrospray , BiocatálisisRESUMEN
Lactones are cyclic esters with extensive applications in materials science, medicinal chemistry, and the food and perfume industries. Nature's strategy for the synthesis of many lactones found in natural products always relies on a single type of retrosynthetic strategy, a C-O bond disconnection. Here, we describe a set of laboratory-engineered enzymes that use a new-to-nature C-C bond-forming strategy to assemble diverse lactone structures. These engineered "carbene transferases" catalyze intramolecular carbene insertions into benzylic or allylic C-H bonds, which allow for the synthesis of lactones with different ring sizes and ring scaffolds from simple starting materials. Starting from a serine-ligated cytochrome P450 variant previously engineered for other carbene-transfer activities, directed evolution generated a variant P411-LAS-5247, which exhibits a high activity for constructing a five-membered ε-lactone, lactam, and cyclic ketone products (up to 5600 total turnovers (TTN) and >99% enantiomeric excess (ee)). Further engineering led to variants P411-LAS-5249 and P411-LAS-5264, which deliver six-membered δ-lactones and seven-membered ε-lactones, respectively, overcoming the thermodynamically unfavorable ring strain associated with these products compared to the γ-lactones. This new carbene-transfer activity was further extended to the synthesis of complex lactone scaffolds based on fused, bridged, and spiro rings. The enzymatic platform developed here complements natural biosynthetic strategies for lactone assembly and expands the structural diversity of lactones accessible through C-H functionalization.
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Sistema Enzimático del Citocromo P-450 , Lactonas , Lactonas/química , Catálisis , Sistema Enzimático del Citocromo P-450/química , MetanoRESUMEN
Boronic acids and esters are highly regarded for their safety, unique reactivity, and versatility in synthesizing a wide range of small molecules, bioconjugates, and materials. They are not exploited in biocatalytic synthesis, however, because enzymes that can make, break, or modify carbon-boron bonds are rare. We wish to combine the advantages of boronic acids and esters for molecular assembly with biocatalysis, which offers the potential for unsurpassed selectivity and efficiency. Here, we introduce an engineered protoglobin nitrene transferase that catalyzes the new-to-nature amination of boronic acids using hydroxylamine. Initially targeting aryl boronic acids, we show that the engineered enzyme can produce a wide array of anilines with high yields and total turnover numbers (up to 99% yield and >4000 TTN), with water and boric acid as the only byproducts. We also demonstrate that the enzyme is effective with bench-stable boronic esters, which hydrolyze in situ to their corresponding boronic acids. Exploring the enzyme's capacity for enantioselective catalysis, we found that a racemic alkyl boronic ester affords an enantioenriched alkyl amine, a transformation not achieved with chemocatalysts. The formation of an exclusively unrearranged product during the amination of a boronic ester radical clock and the reaction's stereospecificity support a two-electron process akin to a 1,2-metallate shift mechanism. The developed transformation enables new biocatalytic routes for synthesizing chiral amines.
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Aminas , Biocatálisis , Ácidos Borónicos , Ácidos Borónicos/química , Ácidos Borónicos/metabolismo , Aminas/química , Aminas/metabolismo , Estereoisomerismo , Aminación , Estructura MolecularRESUMEN
Relational savoring (RS) is a brief, strengths-based approach to heightening attentional focus to moments of positive connectedness within relationships. RS can be administered preventatively or within an intervention context when a therapist aspires to foster more optimal relational functioning. Typically administered within a one-on-one therapy setting, RS has demonstrated efficacy in enhancing intra- and interpersonal outcomes. To increase access to mental health services, the developers of RS are committed to engaging in an iterative approach of enhancing the cultural congruence and accessibility of this intervention within various cultural contexts, beginning with Latine groups in Southern California. In this article, we describe relational savoring and its theoretical and empirical support, including the process of culturally adapting the intervention within the context of three major studies, each with a distinct focus on Latine groups, a community that is underserved in mental health care settings. We then provide a vision for future research to improve upon the intervention's compatibility for Latine families and other populations.
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Hispánicos o Latinos , Humanos , Hispánicos o Latinos/psicología , Femenino , Masculino , Relaciones Interpersonales , Adulto , Asistencia Sanitaria Culturalmente Competente , California , Terapia Familiar/métodosRESUMEN
Microcrystal electron diffraction (MicroED) is an emerging technique that has shown great potential for describing new chemical and biological molecular structures. Several important structures of small molecules, natural products, and peptides have been determined using ab initio methods. However, only a couple of novel protein structures have thus far been derived by MicroED. Taking advantage of recent technological advances, including higher acceleration voltage and using a low-noise detector in counting mode, we have determined the first structure of an Aeropyrum pernix protoglobin (ApePgb) variant by MicroED using an AlphaFold2 model for phasing. The structure revealed that mutations introduced during directed evolution enhance carbene transfer activity by reorienting an α helix of ApePgb into a dynamic loop, making the catalytic active site more readily accessible. After exposing the tiny crystals to the substrate, we also trapped the reactive iron-carbenoid intermediate involved in this engineered ApePgb's new-to-nature activity, a challenging carbene transfer from a diazirine via a putative metallo-carbene. The bound structure discloses how an enlarged active site pocket stabilizes the carbene bound to the heme iron and, presumably, the transition state for the formation of this key intermediate. This work demonstrates that improved MicroED technology and the advancement in protein structure prediction now enable investigation of structures that was previously beyond reach.
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Electrones , Proteínas , Proteínas/química , Péptidos , MetanoRESUMEN
Hydroxylamine-derived reagents have enabled versatile nitrene transfer reactions for introducing nitrogen-containing functionalities in small-molecule catalysis, as well as biocatalysis. These reagents, however, result in a poor atom economy and stoichiometric organic waste. Activating hydroxylamine (NH2OH) for nitrene transfer offers a low-cost and sustainable route to amine synthesis, since water is the sole byproduct. Despite its presence in nature, hydroxylamine is not known to be used for enzymatic nitrogen incorporation in biosynthesis. Here, we report an engineered heme enzyme that can utilize hydroxylammonium chloride, an inexpensive commodity chemical, for nitrene transfer. Directed evolution of Pyrobaculum arsenaticum protoglobin generated efficient enzymes for benzylic C-H primary amination and styrene aminohydroxylation. Mechanistic studies supported a stepwise radical pathway involving rate-limiting hydrogen atom transfer. This unprecedented activity is a useful addition to the "nitrene transferase" repertoire and hints at possible future discovery of natural enzymes that use hydroxylamine for amination chemistry.
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Hidroxilaminas , Nitrógeno , Hidroxilamina , AminaciónRESUMEN
In nature and synthetic chemistry, stereoselective [2 + 1] cyclopropanation is the most prevalent strategy for the synthesis of chiral cyclopropanes, a class of key pharmacophores in pharmaceuticals and bioactive natural products. One of the most extensively studied reactions in the organic chemist's arsenal, stereoselective [2 + 1] cyclopropanation, largely relies on the use of stereodefined olefins, which can require elaborate laboratory synthesis or tedious separation to ensure high stereoselectivity. Here, we report engineered hemoproteins derived from a bacterial cytochrome P450 that catalyze the synthesis of chiral 1,2,3-polysubstituted cyclopropanes, regardless of the stereopurity of the olefin substrates used. Cytochrome P450BM3 variant P411-INC-5185 exclusively converts (Z)-enol acetates to enantio- and diastereoenriched cyclopropanes and in the model reaction delivers a leftover (E)-enol acetate with 98% stereopurity, using whole Escherichia coli cells. P411-INC-5185 was further engineered with a single mutation to enable the biotransformation of (E)-enol acetates to α-branched ketones with high levels of enantioselectivity while simultaneously catalyzing the cyclopropanation of (Z)-enol acetates with excellent activities and selectivities. We conducted docking studies and molecular dynamics simulations to understand how active-site residues distinguish between the substrate isomers and enable the enzyme to perform these distinct transformations with such high selectivities. Computational studies suggest the observed enantio- and diastereoselectivities are achieved through a stepwise pathway. These biotransformations streamline the synthesis of chiral 1,2,3-polysubstituted cyclopropanes from readily available mixtures of (Z/E)-olefins, adding a new dimension to classical cyclopropanation methods.
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Ciclopropanos , Sistema Enzimático del Citocromo P-450 , Ciclopropanos/química , Estereoisomerismo , Sistema Enzimático del Citocromo P-450/metabolismo , Alcoholes , Acetatos , Alquenos/químicaRESUMEN
Recent advances in enzyme engineering and design have expanded nature's catalytic repertoire to functions that are new to biology. However, only a subset of these engineered enzymes can function in living systems. Finding enzymatic pathways that form chemical bonds that are not found in biology is particularly difficult in the cellular environment, as this depends on the discovery not only of new enzyme activities, but also of reagents that are both sufficiently reactive for the desired transformation and stable in vivo. Here we report the discovery, evolution and generalization of a fully genetically encoded platform for producing chiral organoboranes in bacteria. Escherichia coli cells harbouring wild-type cytochrome c from Rhodothermus marinus (Rma cyt c) were found to form carbon-boron bonds in the presence of borane-Lewis base complexes, through carbene insertion into boron-hydrogen bonds. Directed evolution of Rma cyt c in the bacterial catalyst provided access to 16 novel chiral organoboranes. The catalyst is suitable for gram-scale biosynthesis, providing up to 15,300 turnovers, a turnover frequency of 6,100 h-1, a 99:1 enantiomeric ratio and 100% chemoselectivity. The enantiopreference of the biocatalyst could also be tuned to provide either enantiomer of the organoborane products. Evolved in the context of whole-cell catalysts, the proteins were more active in the whole-cell system than in purified forms. This study establishes a DNA-encoded and readily engineered bacterial platform for borylation; engineering can be accomplished at a pace that rivals the development of chemical synthetic methods, with the ability to achieve turnovers that are two orders of magnitude (over 400-fold) greater than those of known chiral catalysts for the same class of transformation. This tunable method for manipulating boron in cells could expand the scope of boron chemistry in living systems.
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Boro/química , Citocromos c/genética , Citocromos c/metabolismo , Evolución Molecular Dirigida , Escherichia coli/metabolismo , Hidrógeno/química , Ingeniería Metabólica , Rhodothermus/enzimología , Biocatálisis , Boro/metabolismo , Escherichia coli/genética , Hidrógeno/metabolismo , Enlace de Hidrógeno , Redes y Vías Metabólicas/genética , Estructura Molecular , Rhodothermus/genética , EstereoisomerismoRESUMEN
We report a computationally driven approach to access enantiodivergent enzymatic carbene N-H insertions catalyzed by P411 enzymes. Computational modeling was employed to rationally guide engineering efforts to control the accessible conformations of a key lactone-carbene (LAC) intermediate in the enzyme active site by installing a new H-bond anchoring point. This H-bonding interaction controls the relative orientation of the reactive carbene intermediate, orienting it for an enantioselective N-nucleophilic attack by the amine substrate. By combining MD simulations and site-saturation mutagenesis and screening targeted to only two key residues, we were able to reverse the stereoselectivity of previously engineered S-selective P411 enzymes. The resulting variant, L5_FL-B3, accepts a broad scope of amine substrates for N-H insertion with excellent yields (up to >99 %), high efficiency (up to 12 300 TTN), and good enantiocontrol (up to 7 : 93 er).
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Metano , Ingeniería de Proteínas , Metano/química , Dominio Catalítico , AminasRESUMEN
Trifluoromethyl-substituted cyclopropanes (CF3 -CPAs) constitute an important class of compounds for drug discovery. While several methods have been developed for synthesis of trans-CF3 -CPAs, stereoselective production of corresponding cis-diastereomers remains a formidable challenge. We report a biocatalyst for diastereo- and enantio-selective synthesis of cis-CF3 -CPAs with activity on a variety of alkenes. We found that an engineered protoglobin from Aeropyrnum pernix (ApePgb) can catalyze this unusual reaction at preparative scale with low-to-excellent yield (6-55 %) and enantioselectivity (17-99 % ee), depending on the substrate. Computational studies revealed that the steric environment in the active site of the protoglobin forced iron-carbenoid and substrates to adopt a pro-cis near-attack conformation. This work demonstrates the capability of enzyme catalysts to tackle challenging chemistry problems and provides a powerful means to expand the structural diversity of CF3 -CPAs for drug discovery.
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Ciclopropanos , Metano , Ciclopropanos/química , Estereoisomerismo , Metano/química , CatálisisRESUMEN
We report enantioselective one-carbon ring expansion of aziridines to make azetidines as a new-to-nature activity of engineered "carbene transferase" enzymes. A laboratory-evolved variant of cytochrome P450BM3, P411-AzetS, not only exerts unparalleled stereocontrol (99:1 er) over a [1,2]-Stevens rearrangement but also overrides the inherent reactivity of aziridinium ylides, cheletropic extrusion of olefins, to perform a [1,2]-Stevens rearrangement. By controlling the fate of the highly reactive aziridinium ylide intermediates, these evolvable biocatalysts promote a transformation which cannot currently be performed using other catalyst classes.
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Azetidinas , Aziridinas , Carbono , Catálisis , EstereoisomerismoRESUMEN
Biocatalytic carbene transfer from diazo compounds is a versatile strategy in asymmetric synthesis. However, the limited pool of stable diazo compounds constrains the variety of accessible products. To overcome this restriction, we have engineered variants of Aeropyrum pernix protoglobin (ApePgb) that use diazirines as carbene precursors. While the enhanced stability of diazirines relative to their diazo isomers enables access to a diverse array of carbenes, they have previously resisted catalytic activation. Our engineered ApePgb variants represent the first example of catalysts for selective carbene transfer from these species at room temperature. The structure of an ApePgb variant, determined by microcrystal electron diffraction (MicroED), reveals that evolution has enhanced access to the heme active site to facilitate this new-to-nature catalysis. Using readily prepared aryl diazirines as model substrates, we demonstrate the application of these highly stable carbene precursors in biocatalytic cyclopropanation, N-H insertion, and Si-H insertion reactions.
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Diazometano , Metano , Compuestos Azo , Biocatálisis , Catálisis , Metano/análogos & derivados , Metano/químicaRESUMEN
Propargyl amines are versatile synthetic intermediates with numerous applications in the pharmaceutical industry. An attractive strategy for efficient preparation of these compounds is nitrene propargylic C(sp3)-H insertion. However, achieving this reaction with good chemo-, regio-, and enantioselective control has proven to be challenging. Here, we report an enzymatic platform for the enantioselective propargylic amination of alkynes using a hydroxylamine derivative as the nitrene precursor. Cytochrome P450 variant PA-G8 catalyzing this transformation was identified after eight rounds of directed evolution. A variety of 1-aryl-2-alkyl alkynes are accepted by PA-G8, including those bearing heteroaromatic rings. This biocatalytic process is efficient and selective (up to 2610 total turnover number (TTN) and 96% ee) and can be performed on preparative scale.
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AlquinosRESUMEN
Selective functionalization of aliphatic C-H bonds, ubiquitous in molecular structures, could allow ready access to diverse chemical products. While enzymatic oxygenation of C-H bonds is well established, the analogous enzymatic nitrogen functionalization is still unknown; nature is reliant on preoxidized compounds for nitrogen incorporation. Likewise, synthetic methods for selective nitrogen derivatization of unbiased C-H bonds remain elusive. In this work, new-to-nature heme-containing nitrene transferases were used as starting points for the directed evolution of enzymes to selectively aminate and amidate unactivated C(sp3)-H sites. The desymmetrization of methyl- and ethylcyclohexane with divergent site selectivity is offered as demonstration. The evolved enzymes in these lineages are highly promiscuous and show activity toward a wide array of substrates, providing a foundation for further evolution of nitrene transferase function. Computational studies and kinetic isotope effects (KIEs) are consistent with a stepwise radical pathway involving an irreversible, enantiodetermining hydrogen atom transfer (HAT), followed by a lower-barrier diastereoselectivity-determining radical rebound step. In-enzyme molecular dynamics (MD) simulations reveal a predominantly hydrophobic pocket with favorable dispersion interactions with the substrate. By offering a direct path from saturated precursors, these enzymes present a new biochemical logic for accessing nitrogen-containing compounds.