Braca

Nature Catalysis, Published online: 24 September 2025; doi:10.1038/s41929-025-01415-5

The 2025 RepArtZymes conference featured the latest developments in the design and development of artificial and repurposed enzymes for synthetic and biotechnological applications. These contributions illustrate the impact of this rapidly expanding research area towards addressing key challenges in organic synthesis, medicinal chemistry, polymer chemistry, energy conversion, and environmental remediation.

We present a synergistic strategy combining photocatalytic direct C─F borylation of polyfluoroarenes with Suzuki–Miyaura cross-coupling. The high nucleophilicity of amine-ligated boryl radicals enables efficient homolytic aromatic substitution of polyfluoroarenes, forming stable amine–borane adducts that resist protodeboronation and can be directly used in cross-coupling to access functionalized polyfluoroarenes.

Abstract

Polyfluoroarenes are privileged scaffolds in pharmaceutical and materials science, yet their synthesis remains challenging. Aromatic borylation offers a modular entry point for derivatization via Suzuki–Miyaura cross-coupling, but progress is hindered by two persistent issues: the difficulty of direct borylation on electron-deficient polyfluoroarenes, and the pronounced susceptibility of the resulting boron species to rapid protodeboronation under standard cross-coupling conditions. Here, we present an orthogonal strategy that addresses both limitations. Amine-ligated boryl radicals enable direct radical C─F borylation of polyfluoroarenes under visible-light photoredox catalysis. The resulting amine–borane adducts are crystalline, bench-stable, and resistant to protodeboronation, allowing their direct use in Pd-catalyzed Suzuki–Miyaura cross-couplings. This platform provides scalable and broadly applicable access to functionalized polyfluoroarenes and overcomes some of the synthetic constraints associated with these valuable motifs.

Nature Chemistry, Published online: 06 August 2025; doi:10.1038/s41557-025-01886-w

Subcellular lipid transport between organelles and turnover remain poorly explored due to the technical challenges associated with selective lipid labelling. Now a subcellular photocatalytic labelling strategy has been developed that allows organelle-specific lipid analysis and quantitative profiling of lipid transport.

Phosphorus-radical chemistry faces a major challenge in achieving stereochemical control over radical intermediates, a key requirement for accessing diverse P-chirogenic phosphorus compounds that are widely applied in pharmaceuticals, agrochemicals, and materials1–4. Enzymes capable of catalyzing chiral carbon-phosphorus (C-P) bond formation remain scarce5, limiting the biosynthetic development of chiral organophosphorus compounds. Recent advances in non-natural photoenzymatic catalysis expanded the radical reactivity repertoire of biocatalysis, yet P-centered radical transformations remain unexplored6,7. Consequently, the asymmetric synthesis of diverse P- chirogenic or P-/C-bichirogenic phosphorus compounds through highly stereocontrolled phosphorus-radical transformations remains an unmet challenge in both chemical and enzymatic catalysis. In this study, a nicotinamide-dependent imine reductase was repurposed into an asymmetric hydrophosphinylation enzyme (AHP), enabling single-electron oxidation of secondary phosphine oxides to generate P-centered radicals and facilitate subsequent C-P coupling in a redox-neutral, stereoselective fashion. Several series of tertiary phosphine oxide products bearing C-, P-, or P-/C-bistereocenters were prepared using stereodivergent mutants obtained through a focused site-directed evolution strategy. Mechanistic studies reveal a previously unobserved pathway involving single-electron oxidation of the substrate, initiated by photoexcited NADP+. This study advanced enzymatic strategies for chiral P-C coupling and addressed longstanding challenges in achieving stereocontrol over P- and P-/C-bistereocentered radical intermediates in radical chemistry.

Nature Synthesis, Published online: 10 September 2025; doi:10.1038/s44160-025-00882-9

Molecular-dynamics-simulation-guided evolution of flavoenzymes produces efficient catalysts for non-C2-symmetric biaryl synthesis with excellent atroposelectivity, offering promise for natural product synthesis and pharmaceutical applications.

A metallaphotoredox platform enables asymmetric incorporation of amine fragments onto quaternary carbons via photocatalytic generation of α-amino alkyl radicals and nickel-catalyzed coupling with alkene-tethered aryl bromides. The method accesses challenging stereocenters with high enantioselectivity and functional group tolerance, expanding the toolbox for constructing diverse amine-containing quaternary carbons in drug discovery.

Abstract

The enantioselective construction of quaternary carbon stereocenters bearing amine functionalities represents a significant challenge in organic synthesis despite their prevalence in pharmaceutically active compounds. Herein, we report a versatile metallaphotoredox platform for the asymmetric incorporation of amine fragments onto quaternary carbons via coupling of alkene-tethered aryl bromides with readily available α-silylamines. This transformation proceeds under mild conditions without requiring organometallic reagents or stoichiometric reductants. Mechanistically, photocatalytic generation of α-amino alkyl radicals enables their enantioselective coupling with chiral quaternary carbon-containing alkyl nickel species. The method delivers exceptional enantioselectivity and exhibits broad functional group tolerance, providing access to a diverse array of complex drug-like molecules bearing amine-functionalized quaternary stereocenters. Mechanistic investigations revealed the intermediacy of cage-escaped, stereodefined quaternary carbon-containing radicals, which guided the development of complementary asymmetric hydrocyclization and difluoroalkenylation protocols. Our unified platform expands the chemical space of three-dimensional quaternary carbon scaffolds, demonstrating the potential of metallaphotoredox catalysis in addressing longstanding synthetic challenges.

Nature Synthesis, Published online: 04 September 2025; doi:10.1038/s44160-025-00860-1

Three-component, iron-based catalytic transformations offer a promising and sustainable approach to building complex molecules in a single step. This Review highlights advances and ongoing challenges in the development of iron-catalysed difunctionalization of alkenes. Mechanistic insights that enhance our understanding and guide the development of new transformations are discussed.

A synergistic photoredox biocatalysis approach was developed to realize new catalytic mechanism of enamine-dependent class I pyruvate aldolase. Both enantiomeric products were obtained in a stereoconvergent fashion through radical alkylation by wild-type and engineered aldolases.

Abstract

Class I aldolases, a unique link among biochemistry, organic chemistry, and computational chemistry are powerful C─C bond-forming enzymes in synthetic chemistry and industry because of their unparalleled selectivity, extensive substrate scope and scalability. However, the types of reactions catalyzed by class I aldolases are restricted and radical reactions have yet to be accomplished. Here, we demonstrate a proof-of-concept study in which a synergistic photoredox biocatalysis strategy can be applied to realize new catalytic functions of enamine-dependent aldolases. This new reactivity enables asymmetric alkylation of a prochiral radical under exclusive stereocontrol, a challenging task for amine catalysts. Both enantiomeric products were obtained in a stereoconvergent fashion from wild-type and engineered aldolases. This synergistic photoredox biocatalysis strategy has resulted in a new-to-nature enzymatic reaction and led to an asymmetric transformation that is not feasible for organocatalysis. We envision that this discovery will motivate the development of enzymatic enamine and iminium catalysis for valuable asymmetric radical transformations, complementing the prevailing organocatalysts.

Iron-based photocatalysis has emerged as a sustainable and versatile platform for facilitating a wide range of chemical transformations, offering an appealing alternative to precious metal photocatalysts. Among the various activation modes, ligand-to-metal charge transfer (LMCT)-driven homolysis of Fe(III)–L(ligand) bonds has garnered considerable attention due to its ability to generate reactive radical species under mild conditions, without requiring the matching of substrates’ redox potentials. In this review, we present a comprehensive overview of recent developments in LMCT-driven iron photocatalysis, with a particular focus on both mechanistic insights and synthetic applications published in the last five years. We classify Fe(III)–L homolysis into four major categories based on the nature of the coordinated ligand: halides, carboxylates, alkoxides, and azide. For a few cases, mechanistic understanding derived from spectroscopic studies, computational modeling, and kinetic investigations is discussed in more detail. We further highlight the expanding repertoire of synthetic transformations enabled by LMCT-driven iron photocatalysis, including C–H functionalization, alkene functionalization, cross-coupling, oxidation, and radical-mediated bond formation. Finally, we provide future perspectives on the continued development of LMCT-based iron photocatalysis as a broadly applicable platform for sustainable organic synthesis. This review aims to serve as a valuable resource for researchers interested in leveraging the full potential of LMCT-mediated iron photocatalysis in modern organic chemistry.

Nature Chemical Biology, Published online: 22 August 2025; doi:10.1038/s41589-025-02004-0

Engineering non-natural functions into enzymes has opened unexpected avenues for chemical synthesis. Whereas past efforts in repurposing natural enzymes have predominantly focused on heme- and flavin-dependent enzymes, latest work further highlights the advantages and potential of non-heme iron enzymes for organic synthesis.

A novel catalytic mode of aldoxime dehydratases for the abiotic radical ring-opening reaction of cyclic ketoximes is reported. Aldoxime dehydratase from Nocardioides simplex was found to efficiently generate iminyl radicals from challenging “non-activated” cycloketone oximes and to promote radical ring-opening reactions to produce γ- and ε-sulfinylated nitriles under mild conditions.

Abstract

The iminyl radical is a distinctive N-centered radical which serves as a versatile synthon in preparation of nitrogen-containing compounds. In principle, iminyl radicals can be directly generated by single electron reduction of oximes through elimination of OH group. However, due to the low reactivity of the oxime N─OH bonds, direct conversion of the oximes does not proceed efficiently, thereby enforcing chemical activation of the oxime OH group which results in the formation of stoichiometric by-products. To overcome this problem, we are developing a new biocatalytic system using aldoxime dehydratases. Through a series of enzyme screenings, we identified an aldoxime dehydratase from N. simplex (NsOxd) which is capable of catalyzing iminyl radical-mediated ring-opening reactions. Notably, NsOxd efficiently converts the “non-activated” 2-phenylcyclobutanone oxime within 10 min under ambient conditions and quantitatively produces the corresponding γ-sulfinylated nitrile in >95% yield. This enzyme activity is even faster than that of previously-reported chemo-catalysts. Furthermore, evaluation of the scope of potential substrates indicates that NsOxd has a versatile N─O bond cleaving activity which efficiently generates iminyl radicals from various “non-activated” oximes. These findings highlight the utility of aldoxime dehydratases for managing the reactivity of “non-activated” oximes and for achieving challenging iminyl radical-mediated catalytic reactions.

Thioxanthone is an efficient visible-light photosensitizer and facilitates reactions that are not known from natural enzymes. It is now possible to incorporate thioxanthone as a noncanonical amino acid (thioX) into proteins by an engineered amino acyl tRNA synthetase. With this approach we created a photoenzyme that catalyzes the photo-E/Z isomerization of a hydroxycinnamate ester.

Abstract

Photocatalysis in biocatalytic systems provides a promising approach for achieving selective and efficient chemical transformations under mild conditions. Naturally occurring photoactive cofactors are rare. To overcome this limitation, genetic code engineering can be applied to equip proteins with additional functionalities beyond those known in the 20 canonical amino acids. Here, we report the engineering of an aminoacyl-tRNA synthetase (thioXRS) that allows the incorporation of a thioxanthone-bearing noncanonical amino acid (thioX). As proof-of-concept, we utilized the versatile biocatalyst LmrR as a protein scaffold. We identified an active variant able to catalyze the E/Z-photoisomerization of a cinnamate ester derivative into coumarin. The reaction design allows direct monitoring through fluorescence measurements, as the fluorescent substrate is converted into a non-fluorescent product. This work demonstrates that thioXRS is a versatile tool for the future development of new-to-nature photoenzymes, expanding the synthetic capabilities of biocatalysis towards light-driven transformations.

Nature Synthesis, Published online: 19 August 2025; doi:10.1038/s44160-025-00866-9

Combining hydrogen-atom transfer for prochiral radical formation, organic-dye-modulated single-electron transfer and an engineered thiamine-dependent enzyme, a photobiocatalytic platform is developed for assembling C(sp2)–C(sp3) bonds via benzylic C(sp3)–H and aldehyde C(sp2)–H oxidative cross-coupling under mild conditions.

Integrating new metal-catalysed transformations into enzymes is a key objective in biocatalysis. This study introduces photoinduced ligand-to-metal charge transfer (LMCT) as a new strategy for enabling abiotic cross-coupling reactions in metalloenzymes. By tailoring the primary coordination sphere to establish a 2-histidine metal binding site and replacing the iron center with nickel, the ethylene-forming enzyme from Pseudomonas savastanoi (PsEFE) was activated for nickel-catalysed C(sp2)‒S cross-coupling between aryl bromides and thiols. Directed evolution of PsEFE produced highly active variants capable of generating over 50 thioether products in up to 98% yield and 530 total turnover numbers. Mechanistic investigations suggest that this photoenzymatic reaction involves a Ni(II)/Ni(I)/Ni(III) catalytic cycle with generation of a reactive Ni(I) species and thiyl radical via photoinduced LMCT. We anticipate that these findings will inspire further exploration of integrating abiotic cross-coupling transformations into enzymatic catalysis.

Mechanism of benzyl alcohol to benzaldehyde peroxidation by de novo-designed Artificial Cu Proteins (ArCuPs) is investigated. A PCET step involving H⁺ transfer from benzylic C─H bond to Cu^II─OH and ET from O-centered substrate radical to Cu^II is found to be the RDS. Outer-sphere steric modifications alter reactivity. The most active variant produces the least amount of detrimental OH and is the most thermostable.

Abstract

De novo-designed artificial Cu protein (ArCuP), 3SCC, featuring a trigonal Cu(His)₃ binding environment, activates H₂O₂, O₂, and benzylic C─H bonds of abiotic substrates. We outline the mechanism of ArCuP-catalyzed C─H peroxidation of one such abiotic substrate, the peroxidation of benzyl alcohol (BA) to benzaldehyde. The Cu^I(H₂O₂) complex undergoes homolytic cleavage, producing Cu^II-OH and ^•OH, akin to the lytic polysaccharide monooxygenases (LPMOs). The rate-limiting step is found in a PCET process, where the Cu^II-OH species accepts the benzylic C─H proton, accompanied by electron transfer from the O-centered substrate radical, to produce benzaldehyde. The C─H peroxidation is modulated by outer-sphere modifications, with the I12A-3SCC variant having the highest catalytic proficiency. Combined ^•OH monitoring and proteomics analysis reveal that the I12A variant produces the least amount of ^•OH, indicating no oxidation of the active site His residues, unlike other ArCuPs. The chemical and thermal stability of the I12A variant contributes to its superior reactivity. The presence of substrate significantly lowers protein-level oxidation, similar to LPMOs and other Cu and heme-based enzymes.

Science, Volume 389, Issue 6761, Page 741-746, August 2025.

Nature Catalysis, Published online: 12 August 2025; doi:10.1038/s41929-025-01390-x

Expanding the methods for constructing artificial enzymes is of high interest. Now a photoactive cofactor is designed that mimics NAD+, allowing its insertion into a range of NAD+-binding protein scaffolds to catalyse inter- and intramolecular [2 + 2] cycloaddition reactions.

Nonheme iron enzyme, 4-hydroxymandelate synthase from Amycolatopsis orientalis (AoHMS), was engineered to catalyze enantioselective amino azidation via a stepwise nitrene addition and radical azide transfer.

Abstract

Alkene difunctionalization represents an important category of reactions in organic synthesis, with a diverse array of transformations developed over the past decades for various synthetic applications. Nevertheless, the scope and diversity of biocatalytic alkene difunctionalization have been limited, constraining its synthetic utility. In this study, we repurposed nonheme iron enzymes to generate iron nitrene intermediates for alkene difunctionalization. 4-hydroxymandelate synthase from Amycolatopsis orientalis (AoHMS) was successfully engineered for direct alkene aminoazidation to produce chiral 2-azidoamines. Directed evolution was performed on AoHMS to provide evolved variants that could utilize O-pivaloylhydroxylamine triflic acid as the nitrene precursor and produced various primary aminoazidation products with up to 44% yield, 44 total turnover number (TTN), and 98.5:1.5 enantiomeric ratio (e.r.). Mechanistic studies indicated that this new biocatalytic transformation proceeds through a stepwise radical addition and azide recombination pathway. This work expands the catalytic toolbox of metalloenzymes and opens up new opportunities for biosynthesis by introducing nonnatural olefin difunctionalization reactions into biocatalysis.

Functional group migration (FGM) reactions represent a fundamental class of transformations in organic chemistry, enabling the repositioning of functional moieties in non-obvious ways. However, catalytic asymmetric radical-mediated FGMs re-main rare, due to the inherent challenges of achieving catalyst-controlled enantioselectivity over free radical intermediates. Herein, we repurpose imine reductases (IREDs), a class of biotechnologically important enzymes known for their substrate promiscuity, to enable the first examples of catalytic asymmetric cyano group migration via a radical mechanism. An orthog-onal set of radical enzymes, including PbaIREDCym and SmiIREDCym, were engineered, allowing both 1,4- and 1,5-cyano group migrations reactions to occur in an enantiodivergent fashion. The use of nonionic surfactant TPGS-1000 was found to improve both the yield and enantioselectivity of these cyano migration reactions. This biocatalytic process exhibited a broad substrate scope and is readily scalable, affording a rare example of chiral non-amine product assembly with imine reductases. More broadly, stereoselective radical biocatalysis with engineered IREDs and other versatile enzymes provide a potentially general solution to challenging asymmetric FGM reactions.

Alkene hydrogenation is a cornerstone of chemical synthesis, yet enzymatic strategies remain limited to electron deficient substrates via hydride transfer. With heme enzymes, we unlock an unprecedented hydrogenation pathway – termed biocatalytic cooperative metal hydrogen atom transfer – for the asymmetric reduction of unactivated olefins. A silane promoted, heme-cysteine redox cycle in the active site catalyzes sequential hydrogen atom transfer to challenging scaffolds including 1,1-disubstituted as well as tri- and tetrasubstituted alkenes. The evolved enzymes are promiscuous, oxygen-tolerant, utilize earth-abundant iron, and can operate on gram scale under ambient conditions. Orthogonal hydrogen atom sources enable site-divergent asymmetric isotope labeling. Mechanistic and computational studies support a stepwise radical process, highlighting the potential for independent stereocontrol during the delivery of each hydrogen atom. Our work introduces a fundamentally new biochemical logic for stereoselective olefin reduction and provides a platform for next-generation biocatalytic hydrogenation.