Braca

Leaving-group–free electron donor–acceptor (EDA) complexes have emerged as a practical platform for photocatalyst-free radical generation under visible light. This review highlights recent advances in coupling and cyclization reactions driven by such EDA complexes, emphasizing their atom-economical and sustainable feature.

ABSTRACT

Electron donor–acceptor complexes provide a useful platform for photocatalyst-free radical generation under visible light irradiation and has emerged as a sustainable strategy for synthetic applications. Previous synthetic strategies based on EDA-complex formations are typically based on engineering leaving groups into the substrates to suppress back electron transfer that impedes forward reaction. However, such pre-functionalization affects efficiency and atom economy. This review highlights recent developments in the use of strategies for EDA-complex formations to induce photochemical coupling- and cyclization reactions that do not rely on preinstalled sacrificial leaving-groups. Most examples rely on alternative strategies to circumvent back electron transfer such as bond cleavage, oxygen-mediated oxidation, or excitation of EDA complex precursors. These studies illustrate the rising promise of EDA complexes without incorporation of sacrificial leaving-groups and highlight their potential as a broadly applicable, atom-economical platform for photocatalyst-free radical transformations in modern organic synthesis.

ABSTRACT

Fungal unspecific peroxygenases (UPOs) have emerged as powerful catalysts for diverse oxidation reactions. While previous studies have predominantly focused on their native mono(per)oxygenase activity, their full catalytic potential remains underexplored. Herein, we demonstrate that the intrinsic peroxidative activity of UPOs can be effectively leveraged for the straightforward synthesis of benzoxazole compounds. Using the unspecific peroxygenase from Agrocybe aegerita, a broad range of ortho-patterned phenolic substrates were efficiently converted into high-value benzoxazoles with conversions of up to 99% under the neat reaction conditions. The enzyme exhibited superior catalytic performance compared to the well-established horseradish peroxidase. Mechanistic studies demonstrated that phenoxyl radicals generated by UPO's intrinsic peroxidase activity are essential for benzoxazole formation. This work not only presents a mild and efficient synthetic route to benzoxazoles but also expands the known reactivity and oxidative chemistry of UPOs.

In its natural reaction, the fatty acid photodecarboxylase from Chlorella variabilis (CvFAP) catalyzes the light-driven, redox-neutral decarboxylation of fatty acids via carbon-centered radical intermediates. Here, we systematically repurpose this radical pathway to unlock new reactivities, establishing CvFAP as a versatile platform for photobiocatalytic radical transformations. We expand its photocatalytic reaction portfolio by four new reactions: (i) the intermolecular Giese-type coupling of fatty acids with cycloalkenones, (ii) the intramolecular decarboxylative radical cyclization via nucleophilic radicals and isolated C=C bonds of the same philicity, (iii) a cysteine-mediated (Z)→(E) photoisomerization of unsaturated fatty acids, and (iv) a radical carbohydroxylation reaction that demonstrates the enzyme’s capability of olefin bis-functionalization. Enzyme Engineering, using computational modelling as basis, enhanced cyclization efficiency, demonstrating the evolvability of CvFAP towards new reactivities. The study expands the enzymatic repertoire of radical chemistry and demonstrates how photobiocatalysis can channel reactive intermediates to selective, new-to-nature bond-forming transformations, and pave the way toward enzyme-controlled radical couplings using abundant carboxylic acid feedstocks.

Science, Volume 391, Issue 6780, Page 84-89, January 2026.

A non-heme copper protein based on a TM1459 cupin fold was engineered to catalyze the stereoselective Michael addition of 2-nitropropane to 2-azachalcone. Metal-center screening combined with secondary coordination-sphere engineering enabled the initial control of enantioselectivity. X-ray crystallographic analysis further identified nonspecific metal-binding sites on the protein surface, and targeted surface-residue mutations effectively suppressed undesired background reactions. This structure-guided approach significantly enhanced catalytic performance, affording up to 98% enantiomeric excess (ee) (S) and 99% ee (R). These results demonstrate that the visualization-driven selection of mutation sites provides a powerful and generalizable strategy for optimizing artificial metalloenzymes.

Nonheme Fe enzyme isopenicillin N synthase was reprogrammed and evolved as an efficient nitrogen migratase IPNS_Nim, converting diverse azanyl esters to valuable l-arylglycines with up to 16 000 TTN and 97:3 e.r. IPNS_Nim and ACCO_Nim allowed enantiodivergent preparation of both l- and d-arylglycines. Mechanistic studies revealed a change in the rate-determining step and H atom transfer enantioselectivity for these nonheme enzymes.

Abstract

We describe the reprogramming and directed evolution of nonheme Fe enzyme isopenicillin N synthase (IPNS) as an efficient biocatalyst for 1,3-nitrogen migration reactions via an unnatural mechanism. Directed evolution of isopenicillin N synthase from Emericella nidulans furnished a quadruple mutant (EniIPNS V185L I187V S102I R279H, IPNS_Nim), enabling the conversion of a range of azanyl esters into N-protected l-arylglycines. IPNS_Nim achieved a TTN of 16 000 and a TOF of 1200 min⁻¹. This TTN surpassed state-of-the-art small-molecule Fe catalysts by 330-fold and represented the highest TTN value reported for a nonheme Fe enzyme in a new-to-nature reaction. IPNS_Nim and our previously evolved ACCO_Nim (ACCO: 1-aminocyclopropane-1-carboxylic acid oxidase) exhibited complementary enantiopreference, allowing enantioselective synthesis of either l- or d-arylglycines—essential building blocks in clinically important peptide therapeutics. Mechanistic studies revealed a biocatalyst-controlled switch in the rate-determining step (RDS): While the hydrogen atom transfer (HAT) step is the RDS for ACCO_Nim-catalyzed nitrogen migration, it is likely not with IPNS_Nim. Moreover, while ACCO_Nim exhibits almost no enantioselectivity in this HAT step, IPNS_Nim confers excellent enantiocontrol over HAT. Computational studies using density functional theory calculations and molecular dynamics simulations suggested that IPNS and ACCO adopt two different substrate binding modes. Classical MD simulations shed light on important interactions between the substrate and active-site residues that control the substrate binding mode and enantioselectivity.

Organofunctional silanes are valuable in materials science as crosslinkers and adhesion promoters, but their synthesis typically requires costly transition-metal catalysts. We developed a sustainable biocatalytic platform for enantioselective synthesis of ester- and cyano-functionalized silanes. Directed evolution of Thermus amyloliquefaciens protoglobin (TamPgb) yielded enzymes that cyclopropanate vinyl silanes/siloxanes with diazo compounds or N-tosylhydrazones as carbene precursors (up to 2500 TTN, >99% dr/ee). This safer, practical method avoids precious metals and simplifies purification.

Abstract

Organofunctional silanes are highly useful reagents in material science, functioning as effective crosslinking agents and adhesion promoters. Traditionally, their synthesis relies on precious transition-metal catalysts, which require downstream purification and increase overall costs. Herein, we present a green and sustainable biocatalytic platform for the enantioselective synthesis of diverse organofunctional silanes bearing ester and cyano groups. Directed evolution of Thermus amyloliquefaciens protoglobin (TamPgb) produced efficient enzymes for cyclopropanation of vinyl silanes and siloxanes using diazo compounds and N-tosylhydrazones as carbene precursors with excellent diastereo- and enantiocontrol (up to 2500 TTN, >99% dr, >99% ee). Notably, we demonstrate for the first time that N-tosylhydrazones can serve as carbene precursors in enzymatic reactions, providing a safer and more practical alternative for industrial applications.

The development of artificial enzymes through incorporation of new-to-nature catalytic functionality into protein scaffolds has emerged as a powerful approach to expand the biocatalytic repertoire. Inspired by the success of LmrR, a transcriptional regulator protein which unique scaffold has been used for the design of a range of artificial enzymes, we performed a bioinformatics study in an effort to expand the scope of protein scaffolds for artificial enzyme design with other LmrR-like proteins. LmrR belongs to PadR subfamily 2 (PadR-s2) and exhibits an unusual open pore with promiscuous binding capabilities. Using genome mining and homology modeling, we identified six previously uncharacterized PadR s2 proteins and experimentally evaluated them as protein scaffolds for the design of artificial Friedel-Crafts (FC) alkylases. Two of the candidates, Lactococcus fujiensis (LCf)PadR and Brachyspira hampsonii (Bh)PadR, could be applied in the iminium-promoted FC-alkylation using genetically incorporated noncanonical amino acids p aminophenylalanine or 3 aminotyrosine as catalytic residues. Interestingly, contrary to homology models, AlphaFold predictions of the PadR-s2 candidates and X ray crystallography of BhPadR and a variant incorporating 3-aminotyrosine, revealed closed-pore structures. Our findings thus demonstrate that an open-pore structure like LmrR is not a prerequisite for designing artificial FC alkylases, and introduce two new PadR-s2 scaffolds for future application.

We previously introduced a genetically encoded, metal-responsive system for reversible control of protein function based on metal chelation by bipyridylalanine (BpyAla) residues. The efficacy of this linking group approach was demonstrated in two structurally and functionally distinct enzymes, Pyrococcus furiosus prolyl oligopeptidase (Pfu POP) and Photinus pyralis luciferase (Pluc). Here, we investigate the mechanistic basis of this switching in Pfu POP. Fluorescence- based metal competition assays and molecular dynamics (MD) simulations were conducted to quantify Ni(II) binding affinity and evaluate the structural response to Bpy2Ni(II)complex formation. 19F NMR spectroscopy and MD simulations further indicate that linking group- controlled conformational changes near the catalytic triad, particularly within the loop containing H592, drive the observed activity modulation upon metal binding. These findings establish that genetically encoded metal-binding motifs can regulate enzyme function through subtle, localized conformational changes, providing a versatile platform for engineering responsive protein systems in synthetic biology, biosensing, and programmable catalysis.

Vitamin B12 is a structurally unique tetrapyrrole cofactor that catalyzes a wide range of radical and polar reactions. Its unique structure, diverse axial ligands, and ability to access distinct redox states underpin the reactivity of three major classes of cobalamin-dependent enzymes: adenosylcobalamin-dependent isomerases, methylcobalamin-dependent methyltransferases, and reductive dehalogenases. These enzymes leverage precise scaffold-controlled interactions to direct radical rearrangements, methyl group transfers, and reductive bond cleavages with remarkable selectivity and efficiency. Despite these capabilities, the native reactivity of B12 enzymes remain relatively underutilized for biocatalysis, and expansion of their activity toward non-native reactions has only recently emerged as a promising frontier. Mechanistic studies and advances in protein engineering and synthetic biology are beginning to establish B12 enzymes as versatile platforms for selective C-C bond formation, C-H functionalization, alkylation, and other transformations. This perspective summarizes key structural and mechanistic foundations of B12 enzyme catalysis, highlights progress in native biocatalysis, and discusses emerging strategies to exploit B12-dependent enzymes as a platform for non-native biocatalysis.

Despite the remarkable versatility of enzymes for catalyzing complex organic transformations under mild aqueous conditions, no biocatalytic system has been reported for intermolecular C–H fluorination. A radical photoenzymatic approach is introduced that leverages a designed de novo protein scaffold with an unnatural amino acid to achieve precise benzylic monofluorination by a hydrogen atom transfer mechanism.

Abstract

Organofluorine compounds are vital in pharmaceuticals, and enzymes, nature's most efficient catalysts, offer tremendous potential for precise fluorination. However, no enzymatic strategies for intermolecular C–H fluorination have been realized—until now. We present the first radical photoenzymatic system enabling intermolecular C–H fluorination using an unnatural amino acid within a robust de novo protein scaffold. This system achieves chemoselective benzylic monofluorination in aqueous solutions with Selectfluor, driven by hydrogen atom transfer from the photoexcited amino acid. It successfully fluorinated various aromatic compounds and enabled biosynthesis of fluorinated polyketides and chiral fluorinated alcohols. These results establish radical photoenzymatic systems as a powerful new approach for efficient, selective biocatalytic fluorination, with direct relevance to pharmaceuticals.

Developing sustainable methods for C(sp2)–C(sp2) bond formation that avoid transition-metals and prefunctionalized substrates remains a central goal in synthetic chemistry. Phenols and N-heteroarenes (azines) are abundant feedstocks, yet their cross-coupling is hindered by mismatched redox properties and competing pathways. Herein, we report a photochemical strategy that couples phenols with heteroaryl halides under redox-neutral conditions using an organic dye photocatalyst and base. Concurrent oxidation of the phenol component and reduction of the azine component generates complementary radicals that cross-couple efficiently, delivering moderate to high yields (up to 91%) with high functional group tolerance. Mechanistic experiments and density functional theory (DFT) studies elucidate the radical reaction pathways, while substrate clustering, high-throughput experimentation (HTE), and machine learning (ML) enable prediction of C–C versus SNAr reactivity across broad chemical space.

Identification and protein engineering on an imine reductase (IRED) for both (R)- and (S)-selective synthesis of nonbiaryl amine atropisomers via dynamic kinetic resolution (DKR) have been achieved. Enantiocomplementary ADHs were also identified to catalyze the reductive DKR processes. The axially chiral styrenes were produced in high atroposelectivity and yields with broad substrate scope.

Abstract

Enzymatic synthesis of atropisomers has recently attracted considerable research attention, with most studies focusing on axially chiral biaryls. We report a less explored atroposelective dynamic kinetic resolution (DKR) of nonbiaryl styrenes catalyzed by imine reductases (IREDs) and alcohol dehydrogenases (ADHs). The IR189 wild type enzyme was identified to be highly active and selective; furthermore, the inversion of atroposelectivity was achieved with protein engineering. Additionally, two ADHs with enantio-complementary selectivity for the reductive DKR were identified and applied in the synthesis of axially chiral styrenes. Both IREDs and ADHs exhibited broad substrate scope, affording up to 99:1 e.r. and 99% yields for up to 29 examples. Scaled-up reactions and derivatization of optically pure products demonstrated the synthetic utility of these axially chiral styrenes. Molecular recognition mechanisms were elucidated by molecular dynamics (MD) simulations. The current strategy expands the scope of enzymatic DKR of atropisomeric compounds and significantly advances the field of biocatalytic synthesis of axially chiral compounds.

The conversion of CO₂ into value-added chemicals using sunlight is a major goal in sustainable chemistry. Here, we report the design and chemogenetic optimization of a Re(I)-based artificial metalloenzyme (ArM) for the photocatalytic reduction of CO₂ to CO in aqueous solution. By incorporating a biotinylated Re(I)-phenanthroline catalyst into a tetrameric streptavidin scaffold, we achieved a significant enhancement in catalytic turnover compared to free catalyst. Mutational screening at viable positions Ser112 and Lys121 revealed a range of catalytic activities, highlighting the tunability of the system. Through a combination of molecular dynamics simulations and experimental characterization, we demonstrate that the catalytic turnover number correlates strongly with the accessibility of CO₂ to the catalyst's active site, a factor not considered in free solution catalyst design, and further computation reveals the variable activation of a bound CO2 among mutants. This work establishes a clear design principle for enhancing Re(I)-based photocatalysis by leveraging the second coordination sphere of a protein scaffold to confer aqueous stability and control substrate access.

Nature, Published online: 03 December 2025; doi:10.1038/s41586-025-09746-w

A generative artificial intelligence-powered method enables de novo design of highly active enzymes based on information about the geometry of residues in the active site, without requiring protein backbone or sequence information.

Nature, Published online: 03 December 2025; doi:10.1038/s41586-025-09747-9

A hybrid machine learning and atomistic modelling strategy enables one-shot design of efficient enzymes to catalyse diverse biological and non-biological chemical transformations.

Nature, Published online: 09 December 2025; doi:10.1038/d41586-025-03998-2

Don’t overlook the value of co-supervision in PhD training

The present work showcases a photoenzymatic platform using bromoacetonitrile as a practical cyanide source for asymmetric γ-hydrocyanation of alkenes. The system enables direct activation of inert aliphatic C─Br bonds via engineered ene-reductases (PETNR H182M) under visible light. Remarkably, the enzyme achieves excellent enantiocontrol through stereoselective hydrogen atom transfer within its active site

Abstract

Enzymatic strategies for direct cyano group incorporation remain underdeveloped, creating synthetic bottlenecks for accessing chiral nitrile derivatives in pharmaceuticals and functional materials. Herein, we have developed a photoenzymatic γ-hydrocyanation strategy that addresses this gap. The system demonstrates remarkable efficiency in activating the challenging aliphatic C─Br bond of bromoacetonitrile through engineered ene-reductase catalysis. Upon visible light excitation, the reduced flavin cofactor generates cyanomethyl radicals that are spatially confined and precisely oriented within the enzyme's active site for efficient coupling with α-methylstyrenes. This synergistic photoenzymatic system also demonstrates distinct stereochemical control, with the engineered active site simultaneously governing radical generation and subsequent enantioselective hydrogen atom transfer to afford a large range of γ-stereogenic nitriles in up to 93% yield, 94% ee – a transformation that poses considerable challenges for traditional transition metal catalysis. Beyond providing a robust platform for asymmetric cyanoalkylation, this work significantly advances photoenzymatic catalysis by establishing unactivated alkyl bromides as viable radical precursors for selective bond formations.

Science, Volume 390, Issue 6777, Page 1050-1056, December 2025.

The past decade has witnessed groundbreaking developments in metalloenzyme-catalyzed free radical transformations which were previously unknown or uncommon in native metalloenzymology. Guided by mechanistic understandings from organic, organometallic and biochemistry, an array of radical reactions has been developed using various metalloprotein catalysts based on first-row transition metal cofactors including Fe, Co and Cu. The structural and functional diversity and the readily tunable active site environment of metalloproteins offer an excellent opportunity to solve the challenging chemo-, regio- and stereoselectivity problems in radical-mediated transformations facing synthetic chemists. In this Review, we summarize metalloprotein-catalyzed radical reactions based on the reactive intermediates involved, including carbon-centered radicals, nitrogen-centered radicals, oxygen-centered radicals, and metal carbenoids and nitrenoids with radical character. We further survey the reaction mechanism, enzyme engineering strategies, and substrate scope of these metalloprotein-catalyzed radical transformations, providing an overview of the current status of metalloenzymology unknown or uncommon in native biochemistry.

Nature, Published online: 03 December 2025; doi:10.1038/s41586-025-09746-w

A generative artificial intelligence-powered method enables de novo design of highly active enzymes based on information about the geometry of residues in the active site, without requiring protein backbone or sequence information.

Nature, Published online: 03 December 2025; doi:10.1038/s41586-025-09747-9

A hybrid machine learning and atomistic modelling strategy enables one-shot design of efficient enzymes to catalyse diverse biological and non-biological chemical transformations.

Herein, we introduce an operationally simple approach for the direct covalent modification of metalloenzymes via Au(III)-mediated S-arylation chemistry. Using a model class of rubredoxin metalloenzymes containing Fe or Ni in the tetrathiolate active site, we show that these biomolecules can be site-selectively tagged with a variety of abiotic functional groups by leveraging a cysteine residue positioned distal to the active site. The bioconjugation proceeds under mild aqueous conditions, rapidly, and in quantitative yields, and most importantly without perturbing the structural or functional integrity of the enzyme, which normally can occur through adventitious demetallation of the active site. Detailed structural studies, including the first reported X-ray crystal structure of a cysteine-arylated metalloprotein, illuminate a clear preference for regioselective arylation at solvent-exposed cysteine residues, offering a blueprint for predictable and programmable bioconjugation in complex biomolecules. This work demonstrates a unique power of “backgroundless” Au(III) arylation chemistry, rendering site-selective bioconjugation of some of the most delicate biomolecules.

The design of artificial photoenzymes by incorporating synthetic chromophores into proteins represents a promising strategy to achieve new-to-nature biocatalytic transformations with high levels of stereocontrol. Selecting an appropriate protein scaffold is a crucial step in this approach, which so far has been limited to naturally occurring proteins. Here, we tested the suitability of computationally designed scaffolds for this purpose. We chose a de novo helical bundle protein that has a central cavity for small molecule binding but no inherent catalytic activity. To generate a starting point for photoenzyme engineering, we installed a thioxanthone-based triplet sensitizer via cysteine bioconjugation. Guided by computational modeling and molecular dynamics (MD) simulations, three rounds of directed evolution toward the [2+2] photocycloaddition of a 3-alkenyloxy-substituted quinolone resulted in highly efficient enzyme variants with opposite enantioselectivity. Upon visible-light irradiation, both product enantiomers were accessible with excellent yield and >90:10 enantiomeric ratio. Furthermore, we obtained high-resolution crystal structures of the evolved designer enzymes. When exposing crystals of substrate-bound protein to blue light, we observed product formation in crystallo and could rationalize the enantioselectivity. Our work highlights the potential of de novo designed protein scaffolds to efficiently generate and evolve stereoselective artificial photoenzymes.