R.B. Leveson-Gower

Nature Synthesis, Published online: 26 April 2024; doi:10.1038/s44160-024-00536-2

Development of fluorine rebound processes at an enzymatic Fe(III) centre are a challenge. Now, a plant-derived non-haem iron enzyme, 1-aminocyclopropane-1-carboxylic acid oxidase, is repurposed and evolved to catalyse chemo- and enantioselective C(sp3)–H fluorination, forming a range of enantioenriched organofluorine products.

The assembly of artificial metalloenzymes provides a second coordination sphere around a metal catalyst. Such a well-defined microenvironment can lead to enhancing the activities and selectivity of the catalyst. Herein, we present the development of artificial hydroxylase (ArHase) by embedding a Fe-TAML (TAML = Tetra Amide Macrocyclic Ligand) catalyst into a human carbonic anhydrase II (hCAII). Incorporation of the Fe-TAML catalyst ([BS-Fe-bTAML]–) within hCAII enhanced the Total TurnOver Number (TTON) for the hydroxylation of benzylic C–H bonds. After engineering a thermostable variant of hCAII (hCAIITS), the resulting ArHase, [BS-Fe-bTAML]– · hCAIITS, was subjected to directed evolution using cell lysates in a 384-well format. After three rounds of laboratory evolution, the best-performing variants exhibited 36-fold enhancement in the initial rate (124.4 min-1) and 2.8-fold enhancement in the TTON (2629 TTON) for the hydroxylation of benzylic C–H bonds compared to the free cofactor. We surmise that an arginine residue introduced in the course of directed evolution engages in hydrogen bonding with [BS-Fe-bTAML]–. This study highlights the potential of relying on a thermostable host protein to improve the catalytic performance of the hCAII-based ArMs.

Vanillyl alcohol oxidases (VAOs) are catalytically promiscuous oxidases acting on para-substituted phenols. Engineering of a VAO-type biocatalyst permits a new chemoenzymatic reaction - release of and nucleophilic addition to a reactive electrophilic intermediate, yielding α-aminated and -thiolated para-alkylphenols.

Abstract

Biocatalytic preparation of chiral amines is a large and burgeoning field in organic chemistry. Many enzymes and routes have been published, including transaminases, imine reductases, reductive aminases, amine dehydrogenases and others. However, all these routes rely on some sacrificial substrate, in the form of either amine donor or cofactor regeneration substrate. Herein, we report the direct oxidative amination of p-substituted phenols catalyzed by an evolved flavoprotein oxidase, with the consumption of only substrate and O₂, and release of H₂O₂. The substrate scope of the reaction is studied, and is tolerant of a diverse panel including ammonia, primary and secondary amines, and amino acids. The reaction is later employed at preparative scale to generate aminated products in 50–80 % yield. This report establishes flavoprotein oxidase as a new and economical member of the chemist's toolkit for biocatalytic generation of chiral amines, acting as oxidative aminase.

An artificial metalloenzyme (ArM) based on biotin-streptavidin technology was repurposed for enantioselective nonannulative carboamination of alkenes. The combination of design of experiment (DoE) and genetic optimization led to a >630 % improvement in turnover number (TON).

Abstract

Relying on ubiquitous alkenes, carboamination reactions enable the difunctionalization of the double bond by the concurrent formation of a C−N and a C−C single bond. In the past years, several groups have reported on elegant strategies for the carboamination of alkenes relying on homogeneous catalysts or enzymes. Herein, we report on an artificial metalloenzyme for the enantioselective carboamination of dihydrofuran. Genetic optimization, combined with a Bayesian optimization of catalytic performance, afforded the disubstituted tetrahydrofuran product in up to 22 TON and 85 % ee. X-ray analysis of the evolved artificial carboaminase shed light on critical amino acid residues that affect catalytic performance.

Nature Chemistry, Published online: 17 April 2024; doi:10.1038/s41557-024-01494-0

Despite their intriguing photochemical activities, natural photoenzymes have not yet been repurposed for new-to-nature activities. Now, by leveraging the strongly oxidizing excited-state flavoquinone cofactor, fatty acid photodecarboxylases were engineered to catalyse unnatural decarboxylative radical cyclization with excellent chemo-, enantio- and diastereoselectivities.

Laccase is an oxidase of great industrial interest due to its ability to catalyse oxidation processes of phenols and persistent organic pollutants. However, it is susceptible to denaturation at high temperatures, sensitive to pH and in the presence of high concentrations of solvents, which is a problem for industrial use. To solve this problem, this work develops the synthesis in aqueous medium of a new Mn metalloenzyme with laccase oxidase mimetic catalytic activity. To do this, Geobacillus thermocatenulatus lipase (GTL) is used as a "scaffold" enzyme, which is mixed with a manganese salt at 50ºC in an aqueous medium. This produces in situ formation of manganese (IV) oxide nanowires that interact with the enzyme, obtaining the GTL-Mn bionanohybrid. On the other hand, its oxidative activity was evaluated using the ABTS assay, obtaining a catalytic efficiency 300 times greater than the laccase from Trametes versicolor. These new Mn-metalloenzyme turned out to be 2 times more stable at 40 ºC, 3 times more stable in the presence of 10% acetonitrile and 10 times more stable at 20% acetonitrile than laccase Novozym 51003®. Furthermore, the site-selective immobilized GTL-Mn showed much higher stability then the soluble form. Oxidase-like activity of these Mn-metalloenzyme was successfully performed against other substrates such as L-DOPA or phloridzin in oligomerization reactions.

Despite substantial progress made toward elucidating the elegant natural radical enzymology with thiamine pyrophosphate (TPP)-dependent pyruvate:ferredoxin oxidoreductases (PFORs) and pyruvate oxidases (POXs), repurposing naturally occurring two electron TPP-dependent enzymes to catalyze single-electron transformations with significant synthetic value remains a daunting task. Enabled by the synergistic use of visible-light photocatalyst fluorescein and a set of engineered TPP-dependent enzymes derived from benzoylformate decarboxylase (BFD) and benzaldehyde lyase (BAL), we developed an asymmetric photobiocatalytic decarboxylative alkylation of benzaldehydes and a-keto acids to produce highly enantioenriched a-branched ketones. Mechanistically, this dual catalytic radical alkylation involves single-electron oxidation of the enzyme-bound Breslow intermediate and subsequent interception of the photoredox-generated transient alkyl radical. In conjunction with visible light photoredox catalysis, thiamine radical biocatalysis represents a new platform to discover and optimize new asymmetric radical transformations which are unknown to biological systems and not amenable to small-molecule catalysis.

Electrophilic halogenases in nature are typically not efficient. Guided by flavin-dependent halogenase mechanisms and taking advantage of the versatile reactivity of a flavin hydroperoxide adduct and in situ generation of H₂O₂ by flavin-dependent enzymes, it was possible to promote the formation of a hypohalous acid—which is key for electrophilic halogenation—in various non-native halogenases by rerouting the flavin-generated peroxide.

Abstract

Enzymatic electrophilic halogenation is a mild tool for functionalization of diverse organic compounds. Only a few groups of native halogenases are capable of catalyzing such a reaction. In this study, we used a mechanism-guided strategy to discover the electrophilic halogenation activity catalyzed by non-native halogenases. As the ability to form a hypohalous acid (HOX) is key for halogenation, flavin-dependent monooxygenases/oxidases capable of forming C4a-hydroperoxyflavin (Fl_C4a-OOH), such as dehalogenase, hydroxylases, luciferase and pyranose-2-oxidase (P2O), and flavin reductase capable of forming H₂O₂ were explored for their abilities to generate HOX in situ. Transient kinetic analyses using stopped-flow spectrophotometry/fluorometry and product analysis indicate that Fl_C4a-OOH in dehalogenases, selected hydroxylases and luciferases, but not in P2O can form HOX; however, the HOX generated from Fl_C4a-OOH cannot halogenate their substrates. Remarkably, in situ H₂O₂ generated by P2O can form HOI and also iodinate various compounds. Because not all enzymes capable of forming Fl_C4a-OOH can react with halides to form HOX, QM/MM calculations, site-directed mutagenesis and structural analysis were carried out to elucidate the mechanism underlying HOX formation and characterize the active site environment. Our findings shed light on identifying new halogenase scaffolds besides the currently known enzymes and have invoked a new mode of chemoenzymatic halogenation.

Nature, Published online: 10 April 2024; doi:10.1038/s41586-024-07287-2

Citrate synthase from the cyanobacterium Synechococcus elongatus is shown to self-assemble into Sierpiński triangles, a finding that opens up the possibility that other naturally occurring molecular-scale fractals exist.

Science, Volume 384, Issue 6692, April 2024.

Science, Volume 384, Issue 6692, April 2024.

Generation of an artificial enzyme that features a secondary amine residue by genetic code expansion is described. The designer enzyme was evolved to catalyze the asymmetric nitrocyclopropanation of cinnamaldehydes at high conversions with excellent diastereo- and enantioselectivity.

Abstract

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,β-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.

A photocatalytic hydroarylation of dehydroalanine (Dha) and Dha-containing peptides with versatile arylthianthrenium salts was developed in batch and in flow, enabling expedient scale-up. The mild nature of the photocatalytic approach allowed the diversification of peptides featuring various sensitive functional groups and the effective stitching of Dha-containing peptides with a myriad of arenes and drug scaffolds.

Abstract

Unnatural amino acids, and their synthesis by the late-stage functionalization (LSF) of peptides, play a crucial role in areas such as drug design and discovery. Historically, the LSF of biomolecules has predominantly utilized traditional synthetic methodologies that exploit nucleophilic residues, such as cysteine, lysine or tyrosine. Herein, we present a photocatalytic hydroarylation process targeting the electrophilic residue dehydroalanine (Dha). This residue possesses an α,β-unsaturated moiety and can be combined with various arylthianthrenium salts, both in batch and flow reactors. Notably, the flow setup proved instrumental for efficient scale-up, paving the way for the synthesis of unnatural amino acids and peptides in substantial quantities. Our photocatalytic approach, being inherently mild, permits the diversification of peptides even when they contain sensitive functional groups. The readily available arylthianthrenium salts facilitate the seamless integration of Dha-containing peptides with a wide range of arenes, drug blueprints, and natural products, culminating in the creation of unconventional phenylalanine derivatives. The synergistic effect of the high functional group tolerance and the modular characteristic of the aryl electrophile enables efficient peptide conjugation and ligation in both batch and flow conditions.

Copper-containing proteins play crucial roles in biological systems. Azurin is a copper-containing protein which has a Type 1 copper site that facilitates electron transfer in the cytochrome chain. Previous research has highlighted the significant impact of mutations in the axial Met121 of the copper site on the reduction potential. However, the mechanism of this regulation has not been fully established. In this study, we employed theoretical modeling to investigate the reduction of the Type 1 copper site, focusing on how unnatural amino acid substitutions at Met121 influence its behavior. Our findings demonstrated a strong linear correlation between electrostatic interactions and the reduction potential of the copper site, which indicates that the perturbation of the reduction potential is primarily influenced by electrostatic interactions between the metal ion and the ligating atom. Furthermore, we found that CF/π and CF…H interactions could induce subtle changes in geometry and hence impact the electronic properties of the systems under study. In addition, our calculations suggest the coordination mode and ion-ligand distance could significantly impact the reduction potential of the copper site. Overall, this study offers valuable insights into the structural and electronic properties of the Type 1 copper site, which could potentially guide the design of future artificial catalysts.

Computational modelling involving density functional theory (DFT) calculations, molecular dynamics (MD) simulations, and hybrid quantum mechanics / molecular mechanics (QM/MM) calculations, are used to investigate and decipher the mechanism for chemoselectivity control achieved by a set of laboratory evolved P450s for selective allylic C−H hydroxylation vs. epoxidation and carbonyl formation of internal aryl-alkenes.

Abstract

P450 enzymes naturally perform selective hydroxylations and epoxidations of unfunctionalized hydrocarbon substrates, among other reactions. The adaptation of P450 enzymes to a particular oxidative reaction involving alkenes is of great interest for the design of new synthetically useful biocatalysts. However, the mechanism that these enzymes utilize to precisely modulate the chemoselectivity and distinguishing between competing alkene double bond epoxidations and allylic C−H hydroxylations is sometimes not clear, which hampers the rational design of specific biocatalysts. In a previous work, a P450 from Labrenzia aggregata (P450_LA1) was engineered in the laboratory using directed evolution to catalyze the direct oxidation of trans-β-methylstyrene to phenylacetone. The final variant, KS, was able to overcome the intrinsic preference for alkene epoxidation to directly generate a ketone product via the formation of a highly reactive carbocation intermediate. Here, additional library screening along this evolutionary lineage permitted to serendipitously detect a mutation that overcomes epoxidation and carbonyl formation by exhibiting a large selectivity of 94 % towards allylic C−H hydroxylation. A multiscalar computational methodology was applied to reveal the molecular basis towards this hydroxylation preference. Enzyme modelling suggests that introduction of a bulky substitution dramatically changes the accessible conformations of the substrate in the active site, thus modifying the enzymatic selectivity towards terminal hydroxylation and avoiding the competing epoxidation pathway, which is sterically hindered.

Science, Volume 384, Issue 6691, Page 106-112, April 2024.

Radical-type carbene transfer catalysis is an efficient method for the direct functionalization of C–H and C=C bonds. However, carbene radical complexes are currently formed via high-energy carbene precursors, such as diazo compounds or iodonium ylides. Many of these carbene precursors require additional synthetic steps, have an explosive nature or generate halogenated waste. Con-sequently, the utilization of carbene radical catalysis is limited by specific carbene precursors to access the carbene radical inter-mediate. In this study, we generate a cobalt(III) carbene radical complex from dimethyl malonate, which is commercially available and bench-stable. EPR and NMR spectroscopy were used to identify the intermediates and showed that the cobalt(III) carbene radical complex is formed upon light irradiation. In presence of styrene, carbene transfer occurred, forming cyclopropane as the product. With this photochemical method, we demonstrate that dimethyl malonate can be used as an alternative carbene precursor in the formation of a cobalt(III) carbene radical complex.

Nature, Published online: 27 March 2024; doi:10.1038/d41586-024-00958-0

Posting about a paper on X seems to boost engagement but not citations. Plus, researchers pinpoint humans’ first home outside Africa and what the science says about the Baltimore bridge collapse.

Nature, Published online: 28 March 2024; doi:10.1038/s41586-024-07341-z

Copper-catalyzed dehydrogenation or lactonization of C(sp³)−H bonds

Due to the scarcity of C–F bond forming enzymatic activities in nature and the contrasting ubiquity of organofluorine moieties in bioactive compounds, developing new biocatalytic fluorination reactions represents a preeminent challenge in enzymology, biocatalysis, and synthetic biology. Additionally, catalytic asymmetric C(sp3)–H fluorination remains a challenging problem facing synthetic chemists. Although many nonheme Fe halogenases have been discovered to promote C(sp3)–H halogenation reactions, to date, efforts to convert these Fe halogenases to fluorinases have remained unsuccessful. We repurposed a plant-derived natural nonheme enzyme 1-aminocyclopropane-1-carboxylic acid oxidase (ACCO) to catalyze unnatural enantioselective C(sp3)–H fluorination via a radical rebound mechanism. Directed evolution afforded C–H fluorinating enzyme ACCOCHF displaying 200-fold higher activity, substantially improved chemoselectivity and excellent enantioselectivity, converting a range of substrates into enantioenriched organofluorine products. Notably, almost all the beneficial mutations were found to be distal to the Fe centre, underscoring the importance of substrate tunnel engineering in nonheme Fe biocatalysis. Computational studies revealed that the radical rebound step with the Fe(III)–F intermediate has an exceedingly low activation barrier of 3.4 kcal/mol, highlighting a new avenue to expand the catalytic repertoire of enzymes to encompass asymmetric C–F bond formation.

The importance of protein templates in artificial enzyme design is illustrated through genetic code expansion. Incorporation of a secondary amine into the nucleotide-binding DHFR and multidrug-binding LmrR resulted in catalytic entities, with the former favoring the use of NADPH as the hydride source for reactions, whereas the latter required biomimetic 1-benzyl-1,4-dihydronicotinamide (BNAH).

Abstract

Secondary amines, due to their reactivity, can transform protein templates into catalytically active entities, accelerating the development of artificial enzymes. However, existing methods, predominantly reliant on modified ligands or N-terminal prolines, impose significant limitations on template selection. In this study, genetic code expansion was used to break this boundary, enabling secondary amines to be incorporated into alternative proteins and positions of choice. Pyrrolysine analogues carrying different secondary amines could be incorporated into superfolder green fluorescent protein (sfGFP), multidrug-binding LmrR and nucleotide-binding dihydrofolate reductase (DHFR). Notably, the analogue containing a D-proline moiety demonstrated both proteolytic stability and catalytic activity, conferring LmrR and DHFR with the desired transfer hydrogenation activity. While the LmrR variants were confined to the biomimetic 1-benzyl-1,4-dihydronicotinamide (BNAH) as the hydride source, the optimal DHFR variant favorably used the pro-R hydride from NADPH for stereoselective reactions (e.r. up to 92 : 8), highlighting that a switch of protein template could broaden the nucleophile option for catalysis. Owing to the cofactor compatibility, the DHFR-based secondary amine catalysis could be integrated into an enzymatic recycling scheme. This established method shows substantial potential in enzyme design, applicable from studies on enzyme evolution to the development of new biocatalysts.

Nature Synthesis, Published online: 28 March 2024; doi:10.1038/s44160-024-00514-8

Non-canonical amino acids are important building blocks in the synthesis of natural products, peptides and drugs. Now, a one-pot chemoenzymatic approach to synthesize branched azacyclic non-canonical amino acids is reported. This method combines enzymatic transamination of 2,n-diketoacids and stereocontrolled chemical reduction to provide the desired products with high stereoselectivity.

Nature Synthesis, Published online: 28 March 2024; doi:10.1038/s44160-024-00507-7

Methods for enzymatic C–F bond formation are rare. Now an enzymatic method for enantioselective C(sp3)–F bond formation is reported, through reprogramming non-haem iron enzyme (S)-2-hydroxypropylphosphonate epoxidase. Mechanistic studies reveal that the process proceeds through an iron-mediated radical fluorine transfer process.