Recent advances in repurposing natural enzymes for new-to-nature asymmetric photobiotransformations

Abstract

Photobiocatalysis, which integrates the strengths of visible-light-catalysis and enzymatic catalysis, has established itself as a pivotal tool for asymmetric synthesis. Over the past decade, several naturally occurring enzymes have been repurposed to catalyze diverse unnatural transformations that are notoriously difficult to realize using traditional methods. This emerging review focuses on the advancements in photobiocatalysis published from 2022 to December 2024 and also highlights earlier seminal reports related to the first demonstration. We organize this review by the coupling modes of visible-light and enzymes, including net-reduction photoenzymatic catalysis (typically) through the illumination of enzymatic electron donor–acceptor complexes, redox-neutral photoenzymatic catalysis via direct-visible-light excitation of enzymes, and synergistic dual photo-/enzymatic catalysis. With each section, the discussion is categorized by the type of enzyme, emphasizing the underlying mechanistic aspects, evolutionary trajectories and representative substrate scopes. We anticipate that this review will inspire further developments and application of photobiocatalysis.

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Fulu Liu

Dr Fulu Liu obtained his Ph.D. in Microbiology from the College of Life Sciences, Nankai University in 2021. Then, he joined Huang's group at Nanjing University as a postdoctoral research fellow. Currently, his research interests focus on photoenzymatic catalysis and synthetic biology.

Xichao Peng

Xichao Peng was born in 1996 in Hubei, PR China. He received a BS degree in 2019 at Huanggang Normal University and obtained his MS degree in 2022 under the supervision of Prof. Pengju Feng at Jinan University. Currently he is a PhD student under the supervision of Prof. Xiaoqiang Huang at Nanjing University. His research interest focuses on asymmetric electro- or photobiocatalysis.

Jinhai Yu

Dr Jinhai Yu was born in 1991 in Anhui, PR China. He received a B.S. degree in 2014 and obtained his Ph.D. degree in 2021 under the supervision of Prof. Qincai Jiao at Nanjing University. Then, he started his postdoctoral research at Nanjing University with Prof. Xiaoqiang Huang. His current research focuses on photobiocatalysis and bioorthogonal reactions.

Xiaoqiang Huang

Dr Xiaoqiang Huang was born in 1991 in Fujian, PR China. He received a B.S. degree in 2013 and a M.S. degree in 2015 under the supervision of Prof. Ning Jiao at Peking University. He completed his Ph.D. in 2019 supervised by Prof. Eric Meggers at the University of Marburg. Following postdoctoral research with Prof. Huimin Zhao at the University of Illinois at Urbana–Champaign, he began his independent career as an independent tenure-track PI at Nanjing University in 2021. His research group focuses on integrating chemocatalysis and biocatalysis for asymmetric synthesis.

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1. Introduction

Enzymes have become powerful tools for chemical synthesis due to their attractive features such as exquisite selectivity, evolvability, high efficiency, environmental friendliness, and operation under mild conditions.^1–7 To meet the increasing demands for sustainable asymmetric biomanufacturing, significant efforts have been made to expand the catalytic repertoire of enzymes to perform unnatural reactions of interest to humans.⁸ Elegant strategies, including new enzyme mining,⁹ directed evolution,¹⁰ artificial enzymes,¹¹ chemomimetic,¹² use of synthetic reagents,¹³ and machine learning,¹⁴ have been utilized to achieve new-to-nature biotransformations. In parallel to these, repurposing enzymes with visible-light has emerged as another unique and effective approach, leading to a burgeoning field, named photobiocatalysis.^15–26

On one hand, visible-light catalysis²⁷ is compatible with biocatalysis, as both can be performed under mild conditions, laying the foundation for their integration. More importantly, the diverse mechanistic schemes of visible-light catalysis, such as photoinduced electron transfer,²⁸ energy transfer,²⁹ and atom transfer,³⁰ provide easy access to reactive chemical intermediates like excited states, radicals, ions, and radical ions under biocatalytic systems. These non-natural intermediates^31,32 open up fertile ground for exploring new enzyme reactivity. In turn, the tunability and evolvability of proteins offer unique opportunities to address stereoselectivity and chemoselectivity challenges associated with these reactive intermediates,³³ which are often difficult to manage. Consequently, we have witnessed how photobiocatalysis is pushing the boundary of biocatalysis to asymmetric transformations that are new-to-nature, often difficult to achieve by traditional methods, and hold great potential for applications.

In this Emerging Review, we update recent advances in photobiocatalysis since 2022. While previous examples have been well-documented in excellent review articles,^15–26 seminal reports before 2022 representing milestones will also be highlighted. We will focus on visible-light-reshaped naturally occurring enzymes for unnatural reactivity, as this is among the most fruitful directions. Elegant photobioreactions based on artificial photoenzymes,^34–41 photo/bio cascades utilizing native enzyme reactivity,^42,43 are not included. Based on the synergistic modes of visible-light and enzymes, we organize the review into three sections: net-reduction biocatalysis via repurposing natural enzymes, redox-neutral biocatalysis via visible-light-excitation of proteins,⁴⁴ and synergistic dual photo-/enzyme catalysis. In each part, we will sort the content by the type of enzymes, discuss and emphasize the underlying mechanisms and substrate scopes. This review herein seeks to inspire the exploration of more innovative photoenzymatic asymmetric catalysis.

2. Net-reduction biocatalysis via repurposing natural enzymes

Except for light-driven enzymes involved in photosynthesis, natural enzymes that utilize visible-light to promote chemical reactions are rare. Inspired by the concept of electron donor–acceptor (EDA) complexes (also known as charge transfer (CT) complexes),⁴⁵ researchers established enzymatic EDA schemes to introduce light-driven non-natural reactivity to enzymes. Enzymatic EDA complexes form through the complexation of electron-deficient unnatural radical precursors (electron acceptors) and the reduced enzyme cofactors (electron donors), and are accompanied by the appearance of a new absorption band with longer wavelengths. Upon visible-light irradiation, EDA complexes undergo single electron transfer (SET) events, generating the radical ion pairs capable of initiating further radical reactions. This section mainly discusses how the enzymes with cofactors of naturally occurring oxidoreductases form EDA complexes with unnatural substrates and trigger radical biotransformations with visible-light excitation. Moreover, some net-reduction photoenzymatic reactions that were proposed not to proceed through an EDA pathway are also discussed.

2.1 Nicotinamide-dependent ketoreductases

Nicotinamide-dependent ketoreductases (KREDs) have emerged as indispensable biocatalysts for the stereoselective reduction of ketones to chiral alcohols.⁴⁶ In these enzymes’ native catalysis, the nicotinamide cofactors act as hydride sources. In 2016, Hyster and co-workers utilized the nicotinamide-dependent ketoreductases to achieve an enantioselective radical dehalogenation of lactones, which is the pioneering instance of the photoinduced non-inherent enzymatic activity of enzymes (Scheme 1).⁴⁷ Mechanistic studies revealed the formation of EDA complex between the halolactone and reduced cofactor nicotinamide adenine dinucleotide phosphate (NADPH) in the enzyme active site. Upon irradiation, the SET process followed by the cleavage of the C–Br bond generated NADPH radical cation (NADP˙⁺) and the prochiral radical. Subsequent hydrogen atom transfer (HAT) from NADP˙⁺ to the prochiral radical formed enantioselective products. NADP⁺ was then reduced by cofactor regeneration to complete the catalytic cycle. In this work, an array of different halolactones was amenable to selective dehalogenation activity, providing the (R)-enantiomer when using a short-chain dehydrogenase of the bacterium Lactobacillus kefiri (LKADH) variant, or the (S)-enantiomer when using short-chain dehydrogenases from the bacteria Ralstonia species (RasADH). This study demonstrates a novel strategy for reprogramming existing enzymes’ catalytic machinery through visible-light irradiation and unveiled a fresh pathway to unlock new reactivity in natural enzymes with light.

Scheme 1 Pioneer examples of repurposing ketoreductases by excitation of enzymatic electron donor–acceptor complex for radical dehalogenation. EDA, electron donor–acceptor; GDH, glucose dehydrogenase; Glu, glucose; HAT, hydrogen atom transfer; NADP⁺, nicotinamide adenine dinucleotide phosphate; RasADH, short-chain dehydrogenases from the bacteria Ralstonia species; LKADH, short-chain dehydrogenase of the bacterium Lactobacillus kefiri.

Stereochemical control of radical conjugate addition is challenging because of the strong racemic background. In 2022, Zhao, Wang and co-workers accomplished an enantioselective intermolecular radical conjugate addition reaction by visible-light excitation on nicotinamide-dependent ketoreductase-associated EDA complexes (Scheme 2).⁴⁸ In this work, N-(acyloxy)phthalimides (NHPI esters) that were easily synthesized from abundant carboxylic acids were used as radical precursors, and α,α-disubstituted terminal alkenes were chosen as radical acceptors. A nicotinamide-dependent ketoreductase (KRED) was engineered via a semi-rational mutagenesis strategy^49,50 to enhance reaction performance through the employment of a compact yet high quality variant library. After several rounds of engineering, the researchers successfully developed a biocatalyst capable of mediating desired reactions with broad substrate scope (up to 78% yield, 92% ee). Mechanistic studies suggested that KRED-bound NADPH formed an EDA complex with the radical precursor. Upon visible-light excitation, leading to the generation of decarboxylated substrate radical. Subsequent radical conjugate addition to the α,α-disubstituted terminal alkenes, and stereoselective HAT of the resultant prochiral radical formed the final product. This work represents a seminal example of photoenzymatic conjugate addition of electron-rich radicals to electron-deficient alkenes.

Scheme 2 Repurposing ketoreductases for intermolecular radical conjugated addition. EWG, electron withdrawing group; KRED, Nicotinamide-dependent ketoreductases; P2-D12, KRED-F147L-L153Q-Y190P; PhthN, N-substituted phthalimides.

2.2 Flavoproteins

Flavin-dependent ene-reductases (EREDs) are highly promiscuous biocatalysts that have great potential in the production of fine and specialty chemicals and are widely used in organic synthesis.⁵¹ In 2019, EREDs were reshaped to catalyze an intramolecular radical cyclization reaction in combination with visible-light excitation by Hyster and co-workers (Scheme 3A).⁵² In this work, α-chloroamides were chosen as substrates to afford a series of enantioenriched 5-, 6-, 7-, and 8-membered lactams with up to 99% yield and 98% ee. Several mechanistic studies were undertaken, among which UV-visible spectra indicated the formation of the EDA complex between the substrate and the reduced cofactor flavin mononucleotide hydroquinone (FMN_hq) in the active site of the enzyme. Single electron transfer occurred between the EDA complex under light illumination, generating carbon-centred radicals. Then, radical cyclization taken place, with the formation of β- or γ-stereocenters. Finally, HAT from the flavin semiquinone to the second C-centred radical terminated the radical reaction, as supported by the isotopic labeling experiments. This work reports the first example of the excitation of ERED-based EDA complexes for intramolecular radical reactions. In their subsequent work, by altering the radical acceptor to a 1,1,2-trisubstituted alkene, the γ-lactam products bearing α-,β-quaternary stereocenter were acquired successfully.⁵³

Scheme 3 Pioneer examples of repurposing flavoprotein by excitation of enzymatic electron donor–acceptor complex. FMN_ox/hq/sq, oxidation/hydroquinone/semiquinone form of FMN; Me, methyl; OYE1, old yellow enzyme isoform I; XenA, ene-reductase from Pseudomonas putida.

In 2021, Zhao and co-workers expanded the photoenzymatic system to the more challenging intermolecular reactivity and reported terminal olefins hydroalkylation using α-bromo ketones as radical precursors catalyzed by EREDs (Scheme 3B).⁵⁴ A series of ketones, amides, or esters containing γ-chiral stereocenters were constructed efficiently and selectively. UV-vis spectroscopy studies demonstrated the EDA complex formed between the α-halo-carbonyls and FMN_hq, which could be excited by visible-light to trigger the SET process. The α-carbonyl carbon-centred radical was then added to the methyl styrene, resulting in the formation of prochiral benzylic radical intermediate. Subsequently, enantioselective HAT formed products with high levels of enantioselectivity. This reaction highlights the unprecedented functional versatility of EREDs as chiral catalysts: under visible-light irradiation, they facilitate diverse asymmetric transformations with reactivities that are different to achieve by standard chemocatalytic systems.

Recent examples of C–C bond formation. In 2021, using α-chloroamide bearing an O-benzyloxime as a substrate, Hyster and co-workers achieved the asymmetric synthesis of chiral tertiary amines (Scheme 4A).⁵⁵ Mechanistic studies indicated that the reaction underwent a similar enzymatic EDA pathway, but using a tethered oximyl C=N bond as the radical acceptor.

Scheme 4 Repurposing ene-reductases with visible-light for asymmetric C–C bond formations. GluER, ene-reductase from Gluconobacter oxydans; OYE1, old yellow enzyme isoform I; KPi, potassium phosphate buffer; TEOA, trolamine; iPrOH, isopropanol; Tris, tris (hydroxymethyl) aminomethane; Tris-HCl, tris (hydroxymethyl) aminomethane hydrochloride.

Recently, building on the ERED-based EDA strategy, Hyster and co-workers reported another radical termination fashion by using allyl silanes as the substrates (Scheme 4B).⁵⁶ In this work, an engineered ene-reductase from Gluconobacter oxydans (GluER), GluER-T36A-K317M-Y343F (GluER-G6), delivered the model product in 92% yield with 98% ee. Ultrafast transient absorption spectroscopy indicated that radical termination proceeds through β-scission of the silyl group.

Diazo compounds are versatile building blocks in organic synthesis, including serving as a photoredox-induced radical precursor.⁵⁷ In 2022, Xu, Jia and co-workers established a light-driven enzymatic system that enables radical-mediated stereoselective hydroalkylation using diazo compounds (Scheme 4C).⁵⁸ Using old yellow enzyme isoform I (OYE1) as a catalyst, upon visible-light irradiation, benzyl 2-diazoacetate could couple with α-methyl styrene to afford the model products in 85% yield with 96% ee. Mechanistic studies indicated that visible-light-illumination of the enzymatic EDA complex containing a diazoester realized the initial radical formation via sequential SET and dinitrogen release. Complementary to the iron-containing cytochromes P450 variants (P411)-mediated carbene transfer reaction,⁵⁹ this photoenzymatic catalysis provides a different application of diazo compounds in enzymatic systems.

Azaarenes are conventionally represented as functional groups in many chiral bioactive molecules. In 2023, Zhao and co-workers achieved remote stereocontrol with a single azaarene group based on the photoenzymatic EDA mechanism (Scheme 4D).⁶⁰ By utilizing OYE1 as a catalyst, bromomethyl azaarene and alkenes as substrates, they obtained the model product in high yield (80%) with 97% ee. Their mechanism studies suggested that the flavin cofactor in ene-reductases was first reduced by the GDH/NADP⁺/Glu system, and subsequently formed an EDA complex with bromomethyl pyridines. In the same year, Hyster and co-workers reported a similar transformation using oxidoreductase from Gluconobacter morbifer (MonstER).⁶¹ Mechanistically, the authors proposed that the light-driven transformation proceeds via the excitation of the enzyme–substrate complex, although the complexes were difficult to observe via UV-visible spectroscopy due to the poor solubility of substrate.

Benzo-fused chiral oxygen-containing heterocycles serve as key structural motifs in pharmaceutical agents. In 2023, Rao, Zhou and co-workers applied a photoenzymatic EREDs-associated EDA strategy to realize the efficient enantioselective synthesis of benzo-fused chiral oxygen-containing heterocycles (Scheme 4E).⁶² Utilizing an engineered GluER-W100H as the biocatalyst, upon visible-light excitation, they obtained diverse desired chiral products such as chromanone, indanone, and benzoxepinones possessing varied benzo-fused ring structures with up to 99% yield and >99% ee. Their mechanistic investigations and molecular docking proved the EDA complex formation, SET process, and HAT in this photoenzymatic reaction.

Fluorinated compounds play pivotal roles in pharmaceutical, agrochemical, and materials science, yet biosynthetic pathways for introducing fluorinated motifs remain remarkably limited in nature. In 2024, Zhao and co-workers reported a photoenzymatic hydrofluoroalkylation platform that is capable of integrating fluorinated motifs into olefins (Scheme 5).⁶³ By employing structurally diverse iodinated fluoroalkanes as radical precursors coupled with an engineered OYE1 variant, this study established an enzymatic platform enabling stereocontrolled functionalization at remote β-, γ-, or δ-positions relative to fluorinated groups, achieving exceptional efficiency (up to 99% yield) and stereoselectivity (>99% ee). Mechanistically, an EDA complex was formed between fluorine agents and FMN_hq, and an independent gradient model (IGM) indicated the formation of hydrogen bonds by tyrosine residues (Y196 and Y375) with the fluorinated motif. Single electron transfer occurred between the EDA complex under light illumination, generating trifluoroethyl radical. Then, trifluoroethyl radical was added to alkenes with the formation of radical intermediate, and following stereocontrolled HAT from semiquinone state flavin cofactor (FMN_sq) to the radical intermediate led to the final products. This study pioneers innovative enzymatic methodologies for the efficient incorporation of fluorinated feedstocks into synthetic pathways while establishing a versatile platform for stereocontrolled biosynthesis of structurally complex fluorinated molecules.

Scheme 5 Photoenzymatic incorporation of fluorinated motifs via asymmetric hydrofluoroalkylation.

Very recently, Hyster and co-workers reported initially photoinduced prochiral C-radical rather than the commonly tamed benzylic radicals that could be well controlled via engineered ERED (Scheme 6).⁶⁴ In this work, through the evolution of GluER, they obtained a mutant that catalyzed the coupling of α,α-dichloro amides with olefins to afford α-chiral α-chloroamides with excellent chemo- and stereoselectivity. The enantioenriched products can serve as linchpins in the synthesis of pharmaceutically valuable motifs. UV-vis spectroscopic studies indicated a ternary EDA complex formed between FMN_hq, amide, and styrene substrates, which was responsible for light absorption and subsequent radical initiation. This work demonstrates that not only the HAT step, but also the intermolecular C–C bond formation step can be rendered asymmetric with engineered enzymes, expanding the synthetic utility of photobiocatalysis.

Scheme 6 Photoenzymatic asymmetric synthesis of α-chloroamides catalyzed by ene-reductases. GluER, ene-reductase from Gluconobacter oxydans; GluER-M6, GluER-T36A-K317M-Y343F; PPh₃, triphenylphosphine; Boc, t-butyloxy carbonyl.

Stereoselective C(sp³)–C(sp³) cross-electrophile couplings are difficult to achieve. In 2022, Hyster and co-workers developed an asymmetric photoenzymatic C(sp³)–C(sp³) formation reaction, using α-chloroamides and nitronates as coupling electrophiles (Scheme 7A).⁶⁵ Upon cyan light irradiation, the ERED from Caulobacter segnis (CsER) or GluER variant (GluER-T36A) catalyzed the desired transformation with up to 98% yield and >99 : 1 er. Mechanistic studies indicated the formation of EDA complex between FMN_hq, the α-chloroamide, and the nitrone. Photoexcitation of the EDA complex enabled the chemoselective reduction of alkyl halide to form an alkyl radical, which was then added to the in situ-generated nitronate, generating a nitro radical anion. Finally, enzyme-mediated homolytic cleavage of the C–N bond generated nitrite and an alkyl radical that can be terminated through HAT to afford the cross-coupled product. This reactivity is uncharacterized in small-molecule catalysis and demonstrates the capability of photoenzymatic catalysis in addressing long-standing synthetic challenges. In their subsequent work, a switched reactivity toward the redox-neutral tertiary nitroalkanes synthesis was discovered when an engineered old yellow enzyme from Geobacillus kaustophilus (GkOYE), GkOYE_D73C-A104H-Y264W (GkOYE-G7) was used as the catalyst (Scheme 7B).⁶⁶ Mechanistically, the corresponding nitro radical anion was proposed to be oxidized by FMN_sq, instead of undergoing homolytic C–N cleavage.

Scheme 7 Photoenzymatic asymmetric C(sp³)–C(sp³) cross-electrophile coupling. DMSO, dimethyl sulfoxide; GkOYE, old yellow enzyme from Geobacillus kaustophilus; GkOYE-G7, GkOYE_D73C-A104H-Y264W; CsER, ene-reductase from Caulobacter segnis.

Recent examples of C–N bond formation. Catalytic enantioselective intermolecular radical hydroamination was always difficult to achieve because of the presence of side reactions and the transient nature of nitrogen-centred radicals. In 2023, Zhao, Wang, Zhong and co-workers reported a photoenzymatic system for the synthesis and control of nitrogen-centred radicals (Scheme 8).⁶⁷ Using a repurposed ERED from Pseudomonas putida (XenB), they accomplished the challenging enantioselective intermolecular radical hydroamination reaction through the combination of biocatalysis and photocatalysis. Then they probed that the reactivity of protected primary amines exhibited a negative correlation with aliphatic chain length, while olefin substrates bearing pare-electron-donating groups promoted the yield (>80% yield). Time-dependent density-functional theory calculations (TDDFT) revealed the formation of FMNH_hq-substrate complex, then photoinduced electron transfer from the excited-state FMNH_hq to the nitrogen-containing precursor generating a nitrogen-centred radical. Followed by the intermolecular radical addition of the nitrogen-centred radical to the pre-anchored olefin substrate, and the enantioselective HAT from FMNH_sq for the production of the chiral amine with up to 96% yield and 97% ee. This work leverages the unique properties of enzyme pockets to effectively inhibit the hydrogen-atom-abstraction process with nitrogen-centred free radicals, and the synergistic activation of enzymatic and photocatalytic processes, thus achieving a challenging enantioselective intermolecular radical hydroamination reaction.

Scheme 8 Photoenzymatic asymmetric C–N bond formation. XenB, one of the Xenobiotic reductase from Pseudomonas putida.

In the subsequent work, Zhao and co-workers extended this strategy to harness the aminium radical cation for asymmetric hydroamination.⁶⁸ Specifically, a mutant of old yellow enzyme from S. cerevisiae (OYE3) not only photocatalytically generated but also precisely controlled the selectivity of aminium radical cations derived from hydroxylamine. Furthermore, it effectively catalyzed the subsequent intermolecular hydroamination, leading to the formation of enantioenriched tertiary amines. The enantioselectivity of this process was achieved through an enzyme-mediated hydrogen atom transfer mechanism.

Recent examples of C–S bond formation. Chiral sulfones are ubiquitously present in medicinal agents and biologically active compounds, and serving as critical structural motifs for enantioselective drug design. In 2023, Xu and co-workers reported a photocatalytic enzymatic system enables stereoselective hydrosulfonylation via light-driven radical initiation (Scheme 9).⁶⁹ Using OYE1 as the catalyst, a collection of β-chiral sulfonyl compounds with up to 92% yield and 98% ee were achieved. Mechanistic studies showed the formation of the EDA complex in the enzyme's binding pocket between the reduced cofactor and benzenesulfonyl chloride. Sulfonyl radicals were then generated upon visible-light irradiation and were captured by alkenes to give prochiral benzylic radicals. Following radical termination through HAT, proposed to mediate by a tyrosine residual, led to the enantioenriched sulfone products. This work provides an alternative approach for the synthesis of chiral sulfonyl motifs prevalent in pharmaceuticals.

Scheme 9 Photoenzymatic asymmetric C–S bond formation.

Recent examples of dehalogenation. In addition to the KRED-based photoenzymatic hydrodehalogenation reactions, Wu, Xu and co-workers reported that flavin-dependent monooxygenase could also have similar activity (Scheme 10).⁷⁰ Upon visible-light excitation, using an engineered cyclohexanone monooxygenase (CHMO), the author achieved an asymmetric reductive dehalogenation reaction with up to 99% yield with 99 : 1 er. Tolerated with GDH/NADP⁺/Glu for flavin adenine dinucleotide (FAD) reduction, mechanistic studies and molecular simulations demonstrated the CHMO-associated EDA complex was formed between the substrate and the enzyme-bound FADH⁻. Isotope incorporation experiments suggest that the radical quenched via electron transfer/proton transfer (ET/PT) process guaranteed the enantioselectivity of products. In 2024, Wu and co-workers extended this biocatalytic system to enable enantioselective reductive dehalogenation, facilitating the asymmetric synthesis of α-deuterated carbonyl compounds with up to 92% yield, 98% ee, and 96% D-incorporation.⁷¹

Scheme 10 Photoenzymatic asymmetric dehalogenation. CHMO, cyclohexanone monooxygenase; PBS buffer, phosphate buffered saline.

Very recently, Wu, Wang, Xu and co-workers achieved non-natural reductive dehalogenation (deacetoxylation) reactivity by reshaping and engineering FAD-dependent fatty acid photodecarboxylase from Chlorella variabilis (CvFAP) (Scheme 11).⁷² Utilizing trolamine (TEOA) as the terminal reductant, a series of chiral α-substituted tetralones were obtained in up to 99% yield and 98% ee. Mechanistic studies demonstrated that enzyme-bound oxidation form of FAD (FAD_ox) was photo-reduced into anionic semiquinone in the presence of the sacrificial electron donor TEOA, which then reduced the substrate to generate an α-carbonyl radical, the prochiral radical was subsequently quenched via ET/PT pathway to furnish the product with the water functioning as the proton source.

Scheme 11 Repurposing CvFAP to catalyze non-natural reductive dehalogenation (deacetoxylation). CvFAP, FAD-dependent fatty acid photodecarboxylase from Chlorella variabilis.

Recent examples of asymmetric hydrogenation. In 2023, Huang, Wang and co-workers expanded the photoenzymatic strategy for asymmetric reduction of enamides, enabling efficient synthesis of chiral amines (Scheme 12).⁷³ Using OYE1 and mutants, upon visible-light irradiation, they obtained the (R)-products with up to 98% ee. Mechanistically, the proposed direct purple-light-excitation of FMNH⁻ triggers SET to deliver enamide-derived radical anions and FMNH˙. Rapid protonation followed by an enantioselective HAT process finally led to the formation of chiral amine products with regeneration of flavin mononucleotide (FMN). This work expands the reactivity of EREDs to the single-electron reduction of enamides, which is complemented to traditional transition-metal-based approaches.

Scheme 12 Photoenzymatic asymmetric hydrogenation of enamides. FMN, flavin mononucleotide.

2.3 Imine-reductases

As multifunctional biocatalysts, nicotinamide-dependent imine reductases (IREDs) boast a broad substrate scope and applicability for industrial production.⁷⁴ In 2024, Huang, Wang and co-workers repurposed IREDs into a new class of visible-light-responsive enzymes by developing an enantioselective radical hydroalkylation of enamides accessing enantioenriched amines (Scheme 13).⁷⁵ Readily available C(sp³)-hybridized coupling partners, such as amines-derived benzylic amines and cyclohexylamine, carboxylic acids-derived benzylic acids and aliphatic acids, and alkyl halides were utilized to fulfill the radical C–C formation achieving the biocatalytic modular amines’ construction. Mechanistic studies indicated an EDA complex was formed between the radical precursor and NADPH. By irradiation with light, the alkyl radical was generated and captured by IRED-activated enamide to generate the prochiral α-amido radical. Finally, the key stereocontrolled HAT from NADPH˙⁺ to the prochiral radical forms product. The newly developed photo-IRED system holds great potential for expanding the synthetic scope and introducing mechanistic diversity for biosynthesis.

Scheme 13 Repurposing imine-reductases by excitation of enzymatic electron donor–acceptor complex. FG, functional group; PG, protecting group.

3. Redox-neutral biocatalysis via visible-light-excitation

Benefiting from the conjugated structure of quinone-state flavin cofactors, flavoproteins have excellent photophysical properties, enabling them to readily reach excited states with enhanced redox properties upon absorbing visible-light. In 2017, Beisson and co-workers identified the natural flavin-dependent photoenzyme CvFAP that catalyzes the decarboxylation of medium and long-chain fatty acids.⁷⁶ Subsequent mechanistic studies⁷⁷ demonstrated that visible-light-excitation of the FAD cofactor afforded the excited state FAD*, initiating the single electron oxidation of fatty acids anion with the formation of FAD˙⁻ and alkyl radicals. Then a back electron transfer (BET) process from FAD˙⁻ to alkyl radical occurred with the protonation by arginine to form the desired hydrocarbon products.

3.1 CvFAP

To overcome the narrow substrate scope of CvFAP, researchers have made great efforts through protein engineering and tuning substrate specificity and function. Hollmann and co-workers expanded its substrate scope beyond long-chain fatty acid through further engineering.^78–80 Scrutton and co-workers achieved significant progress in biofuel production by overexpressing engineered CvFAP in both Escherichia coli and Halomonas expression systems.⁸¹ This innovative approach enabled light-driven biosynthesis of biopropane and biobutane directly from renewable biomass and industrial waste substrates. Wu, Xu and co-workers reported a photoinduced method for the decarboxylative deuteration⁸⁰ and chemoselective photodecarboxylation of trans fatty acids.^82,83 Gu, Zhou, Gao and co-workers demonstrated applicability of engineered CvFAP by enabling the decarboxylation of bulky secondary and tertiary carboxylic acids.⁸⁴

In 2019, Wu and co-workers reported the first asymmetric biocatalysis with an engineered CvFAP by identifying an efficient variant CvFAP-G462Y that enabled the kinetic resolution of α-hydroxy acids and α-amino acids through a structure-guided protein engineering strategy with up to 99% ee (Scheme 14A).⁸⁵ MD simulations suggested the excited FAD in the catalytic pocket facilitates SET with the carboxylate of the (S) isomer after irradiation by light, while the (R) isomer demonstrates negligible reactivity. This work further demonstrates the power of protein engineering in photobiocatalysis, expanding the reactivity to the asymmetric synthesis of chiral α-functionalized carboxylic acids.

Scheme 14 Direct excitation of CvFAP for non-natural transformations. CvFAP, FAD-dependent fatty acid photodecarboxylase from Chlorella variabilis; FAD, flavin adenine dinucleotide; SET, single electron transfer; FAD, flavin adenine dinucleotide; FAD*, excited state FAD; Rac, racemic mixture.

In their subsequent work, Wu, Wang, Xu and co-workers applied this light-driven kinetic resolution strategy for preparing chiral sec-alcohols and amines in up to 99% ee (Scheme 14B).⁸⁶ Later in 2020, Xue and co-workers expanded the system to prepare enantioenriched phosphinothricin (Scheme 14C).⁸⁷ Through a strategically designed semirational engineering campaign, the catalytic activity and enantioselectivity of CvFAP were significantly enhanced, resulting in CvFAP-G462F-T430R-S573G (CvFAP-M6) with 50% yield and 96% ee. This showcases potential applications of photoenzymatic catalysis in asymmetric synthesis of herbicides, particularly since the D-enantiomer in the commercialized racemic phosphinothricin lacks herbicidal activity and poses environmental hazards.

In 2024, Yang, Liu and co-workers repurposed CvFAP as unnatural radical photocyclases (RAPs) to accomplish a photoenzyme-catalyzed decarboxylative radical cyclization reaction with excellent chemo-, enantio- and diastereoselectivities (Scheme 15).⁸⁸ A set of diastereo- and enantiodivergent RAP variants was engineered through directed evolution, enabling the generation of all four possible stereoisomers of a stereochemical dyad. Mechanistically, the alkyl radical was formed through decarboxylation in the CvFAP active site that was triggered by the SET process. Intramolecular radical conjugate addition then occurred and the resultant α-carbonyl carbon-centred radical underwent proton-coupled electron transfer (PCET) electron-transfer/proton-transfer (ET/PT) to furnish the product in a stereocontrolled manner, with the regeneration the FAD cofactor. This work underscores that integrating protein engineering for unnatural substrates with rational mechanistic designs, capitalizing on the unique redox properties of excited-state flavoquinones is an effective strategy for expanding enzyme reactivity.

Scheme 15 Repurposing natural FAP to catalyze new-to-nature stereoselective radical cyclization.

3.2 Ene-reductases

In 2023, Huang, Wang, Tian and co-workers expanded the reactivity of ene-reductases by introducing a single-electron-oxidation-initiated radical mechanism (Scheme 16).⁸⁹ In this work, the author accomplished stereocontrolled intermolecular radical hydroarylation of alkenes with electron-rich arenes and achieved a series of chiral hydroarylated products in up to 99% yield and 98% ee. Mechanistic studies demonstrated that upon visible-light excitation, the cofactor FMN was excited to FMN_ox*, which then underwent the SET process with the formation of aryl radical cation. Subsequent radical C–C bond formation delivered a prochiral carbon-centred radical intermediate. Finally, electron and proton (or hydrogen atom) transfer furnished the product in a stereocontrolled manner and regenerated the ground-state FMN_ox. This work provides a single-electron-oxidation based catalytic mode for achieving unnatural biotransformations by leveraging the high oxidizing properties of the excited flavin cofactor of ene-reductases.

Scheme 16 Direct excitation of ene-reductase for radical hydroarylation. FMN, flavin mononucleotide; FMN_ox/sq, oxidation/semiquinone form of FMN.

Later, Huang, Zhang, Wang and co-workers extended this strategy to enable radical hydrolactonizations (one C–O bond), lactonization-alkylations, and lactonization-oxygenations (while two C–O bonds) with an additional organic dye Rhodamine 6G (Rh6G) (Scheme 17).⁹⁰ By synergizing photoinduced single-electron oxidation with directed evolution, repurposed ene-reductases catalyzed the synthesis of a diverse array of enantioenhanced 5- and 6-membered lactones featuring vicinal stereocenters was achieved with yields of up to 95%, enantiomeric excess (ee) of 99%, and diastereomeric ratios (dr) of 12.9 : 1. Mechanistic investigations revealed that the highly oxidizing excited state FMN_ox* oxidized the substrates, generating alkene radical cations. This is followed by an intramolecular radical C–O bond formation, which produces a prochiral benzylic radical intermediate. Finally, HAT yielded the target chiral product and regenerated the ground state FMN_ox.

Scheme 17 Direct excitation of ene-reductase for diastereo and enantioselective lactones synthesis. Rh6G, rhodamine 6G.

In 2024, Ye, Ding, Dai and co-workers accomplished redox-neutral radical C–S bond formations through oxidation-initiated photoenzymatic protocol (Scheme 18A).⁹¹ Utilizing sodium sulfonates as the sulfonyl radical precursors, in combination with a GluER variant as the enzymatic catalyst, the authors achieved the hydrosulfonylation of olefins with high enantioselectivity and broad substrate scope. Concurrently, using sulfonates and hydrazines as substrates, and OYE1 as a catalyst, Xu, Ge, Wang and co-workers developed a similar strategy that affords (R)-configured sulfone products with up to 96% yield and 98% ee (Scheme 18B).⁹² Similar mechanistic schemes were proposed in these two works. Upon visible-light-irradiation, sulfuryl radical was formed through single-electron oxidation by the highly oxidizing excited state FMN_ox*, which was then captured by an olefin to form a prochiral benzylic radical intermediate. The prochiral radical was subsequently quenched by FMN_sq through HAT in the active site of the enzyme. Their oxidation-initiated photoenzymatic platform overcomes the limitations associated with electronsacrificial reagents, which would enable more useful and challenging transformations.

Scheme 18 Direct excitation of ene-reductase for radical hydrosulfonylation.

3.3 Baeyer–Villiger monooxygenases

In 2024, Hyster and co-workers reported an elegant photoenzymatic intermolecular hydroamination, synthesizing enantioenriched 2,2-disubstituted pyrrolidines by repurposing Baeyer–Villiger monooxygenase (Scheme 19).⁹³ They proved the broad substrate scope and prepared α-methylated analog of three aryl pyrrolidine containing active pharmaceutical ingredients (APIs) using this strategy. Interestingly, among the tested different classes of flavoenzymes, including monoamine oxidase (MAO-N-D11C), styrene monooxygenase (StyA1), D-amino acid oxidase (AcDAAO), choline oxidase (AcCO6), cholesterol oxidase (ShCO), and cyclohexanone monooxygenase from Acinetobacter calcoaceticus (AcCHMO), only AcCHMO showed unique catalytic versatility toward the hydroamination reactions. To enhance catalytic efficiency, they isolated a more efficient enzyme from a homologous CHMO library and performed directed evolution through multiple rounds, ultimately engineering the highly optimized A. calcoaceticus hydroaminase (AcHYAM). The authors proposed that the enzyme was photoreduced by buffer with the formation of hydroquinone form of FAD (FAD_hq) and subsequently excited by visible-light. The FAD_hq* then single electron reduced alkene resulting in alkene radical anion formation in the electropositive active site. Finally, the C–N bond was proposed to form by exploiting a through-space interaction of a reductively generated benzylic radical and the nitrogen lone pair. Generally, C–N bond forming especially involving radical intermediates, anti-Markovnikov products were the main products in chemical synthesis. This study showcases the potential of engineered photoenzymes in providing novel mechanistic approaches to long-standing issues in chemical synthesis.

Scheme 19 Direct excitation of ene-reductase for radical hydroaminations. AcHYAM, Acinetobacter calcoaceticus HYdro AMinase; FAD_hq/sq, hydroquinone/semiquinone form of FAD.

3.4 Type I aldolase

In 2024, Melchiorre and co-workers developed a novel photoenzymatic strategy based on the excitation of enzyme-bound catalytic intermediates, rather than the previous reliance on cofactor excitation (Scheme 20).⁹⁴ Specifically, engineered class I aldolase was repurposed using purple light to catalyze unnatural stereospecific cross-couplings of carboxylic acids and enals. When enantiopure carboxylic acids were employed as substrates, the diastereoselectivity of intermolecular radical processes can be effectively controlled through this strategy, leading to the successful synthesis of the desired products. Mechanistic studies indicated that enzyme-bound iminium ions, transiently generated through the condensation of enals with catalytic lysine residual in enzymes, were excited by photo, thereby triggering the SET oxidation of carboxylic acids. Following decarboxylation, the resultant chiral radical undergoes coupling with the β-enaminyl radical, yielding the final products and the configuration of the products consistent with the configuration of carboxylic acid. By exploiting the unique active site microenvironment, this system achieves highly organized transition states stabilized by multiple weak interactions, enabling precise control over both relative diastereoselectivity and absolute enantioselectivity. This work highlights an innovative mechanism by which the enzyme's active site preserves chiral information during radical transformation. Very recently, during the submission of this review, another demonstration on light-repurposing type I aldolases for decarboxylative radical conjugate addition was reported by Saravanan and co-workers.⁹⁵

Scheme 20 Direct excitation of class I aldolase for stereospecific cross-coupling. DERA-MA_S18A, an engineered variant of the natural 2-deoxy-d-ribose-5-phosphate class I aldolase.

4. Synergistic dual photo-/enzyme catalysis

Synergistic dual photo-/enzyme catalysis describes enzymatic transformations with additional photocatalysts to play synergistic roles. The two catalytic cycles twist together in the system, in which the enzyme active site offers a superlative chiral environment and the additional photocatalyst typically fulfills photoinduced electron transfer or energy transfer with substrate or cofactor to drive the enzyme-catalyzed cycle. Based on the type of enzymes, recent synergistic dual photo-/enzyme catalytic modes are discussed as follows.

4.1 Nicotinamide-dependent oxidoreductases

In 2018, Hyster and co-workers developed a synergistic dual catalytic system that enabled enantioselective radical deacetoxylation of α-acetoxyketones through the combination of photocatalyst Rose Bengal (RB) and nicotinamide-dependent double-bond reductase (NtBDR) (Scheme 21).⁹⁶ Mechanism studies demonstrated that upon visible-light irradiation, RB was excited to access an excited state (RB*) capable of oxidizing free NADPH to afford RB˙⁻. RB˙⁻ species then SET reduced the enzyme-bound substrate selectively. The resultant enzyme-bound substrate radical anion underwent fragmentation to deliver prochiral α-carbonyl C-radical. Subsequently, enantioselective HAT from enzyme-bound NADPH to the prochiral radical furnished the enantioenriched product and NADP˙. Oxidation of NADP˙ by an equivalent of RB afforded NADP⁺, which can exchange for an equivalent of NADPH and substrate to terinate the catalytic cycle. The current study elucidates a highly efficient approach leveraging external photoredox mediators to directly activate enzymes for radical transformations, utilizing a user-friendly catalytic module that eliminates complex biohybrid construct preparation.

Scheme 21 Enantioselective deacetoxylation enabled by dual photocatalyst and double-bond reductase. NtBDR, nicotinamide-dependent double-bond reductase; RB, Rose Bengal.

In 2020, Hyster, MacMillan and co-workers described a remarkable work that accomplished a dynamic kinetic resolution (DKR) of β-substituted ketones through the combination of iridium-complex-based photoredox catalysis, enamine catalysis, and nicotinamide-dependent Lactobacillus kefir alcohol dehydrogenase (LK-ADH).⁹⁷

In 2023, Zhu, Wu, Feng and co-workers developed a redox-neutral photo-/enzymatic dual catalytic system combining carbonyl reductase (RasADH) and organic photocatalyst eosin Y (Scheme 22).⁹⁸ Utilizing a set of aldehydes and amino acids as substrates, they accomplished the asymmetric synthesis of chiral 1,2-amino alcohols in 72 to 92% yield, with up to >99% ee. Mechanistic studies suggested that excited-state eosin Y was reduced by N-phenylglycine via a SET process, resulting in an α-amino radical and an eosin radical anion (eosin Y˙⁻). The α-amino radical was then added to the aldehyde with the formation of an oxygen-centred radical. Subsequently, the (R)-enantiomer selectively underwent oxidation to form an amino ketone in the presence of NADP⁺ and the enzyme RasADH-F205N. Concurrently, excited eosin Y was reduced by NADPH, generating eosin Y˙⁻. This was followed by a SET process from eosin Y˙⁻ to the enzyme-bound amino ketone, leading to the formation of a ketyl radical. Finally, a HAT step produced the racemic amino alcohol. This continuous dynamic kinetic resolution process enabled the efficient and highly selective synthesis of 1,2-amino alcohol compounds in the (S)-configuration. This work provided a promising strategy for the effective synthesis of chiral amino alcohols with green chemistry principles.

Scheme 22 Redox-neutral decarboxylative asymmetric C–C coupling. Eosin Y, organic photocatalyst.

In 2024, Zhu, Wu and co-workers reported a scalable asymmetric synthesis platform for the asymmetric carbohydroxylation of alkenes through the combination of photocatalyst Ir(ppy)₃ and RasADH (Scheme 23A).⁹⁹ In this work, a diverse array of bulky chiral alcohols were synthesized by aryl alkenes and N-hydroxyphthalimide esters, yielding products with up to 75% isolated yield and 99% ee. Recently, Borowiecki and co-workers reported a “one-pot sequential two-step” concurrent oxidation–reduction photobiocatalytic strategy via the combination of photocatalyst 9-fluorenone and alcohol dehydrogenases (Scheme 23B).¹⁰⁰ This work integrates enantioselective photocatalytic oxidation of racemic alcohols with sequential one-pot biocatalytic reduction using stereocomplementary alcohol dehydrogenases (ADHs), they obtained a series of optically pure alcohols with up to 99% conversion and >99% ee.

Scheme 23 Repurposing nicotinamide-dependent oxidoreductases by synergistic photoenzymatic catalysis. ADH, alcohol dehydrogenases.

4.2 Flavin-dependent oxidoreductases

In 2018, Hartwig, Zhao and co-workers developed a cooperative photo-/enzymatic catalysis consisting of [Ir(dmppy)₂(dtbbpy)]PF₆ or FMN as the photocatalyst and ene-reductases with a nicotinamide cofactor regeneration system (Scheme 24).¹⁰¹ This system targeted asymmetric reduction of E/Z mixtures of olefins with the formation of various enantioenriched precursors to biologically active compounds, in which the excited photocatalysts converted Z alkenes into E alkenes via energy transfer, meanwhile ene-reductases selectively recognized E alkenes and catalyzed the reduction of asymmetric C=C bond. Mechanistically, the excited-state photocatalyst facilitated the E/Z isomerization of alkenes through energy transfer pathway, followed by enantioselective reduction of the E-isomer catalyzed by an ene-reductase in a GDH-facilitated cofactor regeneration system. Synergistically, stereoconvergent reduction of E/Z mixtures was achieved. This work demonstrates the compatibility of photocatalysts with nonimmobilized enzymes, which would offer opportunities for the development of new cooperative chemoenzymatic transformations. In Zhao and co-workers’ subsequent work, an updated cooperative chemoenzymatic transformation was designed by utilizing a photoactivated FMN reduction strategy without adding extra expensive nicotinamide cofactors.¹⁰² Very recently, using dicyanopyrazine (DPZ)-type photosensitizer for biocompatible olefin E/Z isomerization, Jiang, Guo and co-workers developed a convenient ene-reductase-based photobiocatalytic platforms for stereoconvergent synthesis of β-fluoromethylated chiral ketones.¹⁰³

Scheme 24 Stereoconvergent reduction of alkenes by combining photocatalysis and ene-reductase. [Ir(dmppy)₂(dtbbpy)]PF₆; dmppy, 4-methyl-2-(4-methylphenyl)pyridine; dtbbpy, 4,4′-di-tert-butyl2,2′-bipyridine.

In 2023, Hyster and co-workers developed a dual photo-/enzymatic platform capable of harnessing nitrogen-centred radicals (NCRs) for (5-exo, 6-endo, 7-endo, and 8-endo) hydroamination reactions (Scheme 25).¹⁰⁴ Using two engineered ene-reductases from Bacillus subtilis (YqjM), YqjM-Y28A-I69N-Y169H-A252G-R336 W (YqjM-S) and YqjM-M25L-C26S-I69T (YqjM-R), they accessed the inversion of the enantioselectivity of the enzyme, and when Ru(bpy)₃Cl₂ was absent, wild type YqjM could afford the desired hydroaminated product with only <5% yield, that was attributed to nitrogen-centred radicals was reductively quenched by FMN_sq. Mechanistically, the authors proposed that the excited-state photocatalyst (*Ru^II) was first reduced to form Ru^I species, which then transferred an electron to enzyme-bound hydroxamic ester with the generation of NCRs and Ru^II. The NCRs then underwent radical cyclization to form a benzylic radical and subsequent HAT process from FMN_hq to the radical intermediate leading to the final products with high enantioselectivity. The resulting FMN_sq underwent SET with the excited-state *Ru^II photocatalyst to form FMN_ox. Finally, FMN_ox was reduced to FMN_hq by the cofactor regeneration system. The research demonstrates a novel strategy leveraging enzyme-directed NCR manipulation, and establishes a dual-catalytic framework that unlocks latent enzymatic activities via multifaceted catalytic synergy, overcoming inherent functional limitations.

Scheme 25 Asymmetric radical C–N bond formation enabled by photocatalysis and ene-reductase. bpy, 2,2′-bipyridine; ET, electron-transfer; FMN_hq, hydroquinone form of FMN; GDH-105, glucose dehydrogenase; OBz, benzoyloxy group; YqjM, ene-reductases from Bacillus subtilis.

In their subsequent work, a method for the enantiodivergent decarboxylative alkylation of amino acids using α-heterocyclic olefins was developed by synergistically combining EREDs with photoredox catalysis (Scheme 26).¹⁰⁵ Mechanistic studies indicated that excited-state photocatalyst (*Ru^II). Which was proposed to bind nearby enzyme's active site, oxidized the carboxylate substrate to afford an α-amino radical and Ru^I. Following conjugate addition of α-amino radical to vinylpyridine afforded a prochiral benzylic radical. Then, this radical underwent enzyme-mediated enantioselective HAT to generate the final products. Based on this new biotransformation, they synthesized an anti-human cytomegalovirus (HCMV) compound starting from 8-aminoquinoline in three steps in 41% yield with 82% ee. Overall, this work provides an intriguing mechanism for radical generation and again demonstrates that enzymatic HAT can address the difficulty in asymmetric radical conjugate additions.

Scheme 26 Enantiodivergent decarboxylative alkylation of amino acids enabled by photocatalysis and ene-reductase. HCMV, an anti-human cytomegalovirus compound; OYE3, old yellow enzyme from S. cerevisiae.

In 2022, Poelarends, Saravanan and co-workers unveiled a dual-catalytic system comprising BaNTR1 (a flavin-dependent nitroreductase) and Ru(bpy)₃Cl₂ photocatalyst, where the enzyme's promiscuous reactivity is harnessed for asymmetric reduction of diverse ketone substrates (Scheme 27A).¹⁰⁶ Desired alcohol products were achieved with high conversion (up to >99%) and outstanding enantiopurity (up to >99 : 1 er). In 2023, Poelarends and co-workers further extended the synergistic system to the integration of nitroreductase BaNTR1 and chlorophyll (Scheme 27B).¹⁰⁷ In this work, a series of aromatic amino, azoxy, and azo products were obtained with excellent yields through the selective conversions of electronically-diverse nitroarenes. These studies demonstrated the utility of flavin-dependent nitroreductases as catalyst in photo-/biocatalysis systems, while highlighting chlorophyll's exceptional role as a non-toxic photosensitizer for sustainable asymmetric synthesis of high-value chemicals, enabling sunlight-driven unnatural transformations with minimal environmental impact.

Scheme 27 Repurposing flavoprotein by synergistic photoenzymatic catalysis. BaNTR1, flavin-dependent nitroreductase; MOPS, S3-(N-morpholino)propanesulfonic acid.

In 2024, Huang and co-workers developed a dual photo-/biocatalysis system through the combination of organic dye rhodamine B (RhB) and ene-reductase (Scheme 28).¹⁰⁸ Diverse enantioenriched vicinal diamines were accessed through NCRs in addition to enamides with up to 82% yield and 99% ee. Mechanistically, upon green light irradiation, RhB was excited to access an excited state (RhB*), which was proposed to transfer energy or induce the SET process to the EDA complex. Then, following fragmentation triggered the formation of NCRs, together with semiquinone state flavin cofactor FMN_sq. NCRs were subsequently captured by enzyme-activated enamides. Later, the resulting prochiral α-amido radicals were terminated through HAT to afford the enantioenriched product.

Scheme 28 Enantioselective biosynthesis of vicinal diamines enabled by photocatalysis and ene-reductase. RhB, rhodamine B.

4.3 PLP-dependent oxidoreductases

Pyridoxal 5′-phosphate (PLP)-dependent enzymes operate through a unique carbonyl-based catalytic mechanism to facilitate C–C and C-heteroatom bond-forming reactions at the α-, β-, and γ-positions of amino acids. In 2023, Yang, Liu and co-workers reprogrammed the natural PLP-dependent Pyrococcus furiosus tryptophan synthase β subunit (PfPLP^β) by the synergistic merger with photocatalyst rhodamine B (RhB) (Scheme 29).¹⁰⁹ This photo-/biocatalytic system realized the asymmetric radical C–C coupling reaction of benzyltrifluoroborate salts with β-hydroxy-α-amino acids and accessed a broad spectrum of noncanonical α-amino acids (ncAAs) with diastereo- and enantiocontrol. Mechanistically, the excited-state photocatalyst promoted the SET oxidation of benzyltrifluoroborate salts to alkyl radical intermediates. Concurrent with this photoredox catalytic cycle, an engineered enzyme converted serine and other β-hydroxy-α-amino acids into an electrophilic aminoacrylate via PLP catalytic pathway. The alkyl radical then entered the active site and added to the aminoacrylate to form an azaallyl radical intermediate. Subsequent electron transfer/proton transfer (ET/PT) or protoncoupled electron transfer (PCET) furnished an external aldimine, which upon hydrolysis released target products. This work introduces PLP-dependent enzymes for photobiocatalysis and provides an effective biosynthetic strategy for the asymmetric construction of a series of unprotected ncAAs.

Scheme 29 NcAAs preparation by combining photocatalysis and PLP-dependent enzyme. PLP, pyridoxal 5′-phosphate; ET/PT, electron transfer/proton transfer; PCET, proton-coupled electron transfer; PfPLP^β, Pyrococcus furiosus tryptophan synthase β subunit; ncAAs, noncanonical α-amino acids.

In 2024, Yang, Liu and co-workers reported another elegant dual-catalytic system through the synergistic merger of photoredox catalysis and PLP-dependent threonine aldolases (Scheme 30).¹¹⁰ In this work, they accomplished asymmetric C(sp³)–C(sp³) oxidative cross-coupling between organoboron reagents and glycine or α-branched amino acid substrates. Through strategic enzyme engineering, primary and secondary radical precursors including benzyl, allyl, and alkylboron reagents, were adaptable to give enantioenriched α-tri- and tetrasubstituted ncAAs in an enantio- and diastereocontrolled fashion. Mechanistically, stoichiometric oxidant [Co(NH₃)₆]³⁺ oxidized the excited-state photocatalyst (fac)-Ir(ppy)₃* to provide (fac)-Ir(ppy)₃⁺ as a potent oxidant to single-electron oxidized organoboron substrate, affording a transient carbon-centred radical. Concurrent with this photoredox catalytic cycle, threonine aldolases converted the selected amino acid substrate into quinonoid intermediate. The carbon-centred radical was proposed to travel into the enzyme's active site, where it undergoes enantioselective addition to the α-carbon of the quinonoid, subsequently generating a nitrogen-centred radical. Subsequent single-electron oxidation generated an external aldimine, which released the C(sp³)–C(sp³) coupled ncAAs products and regenerated the PLP biocatalyst. This work provides a new platform for photobiocatalytic C(sp³)–C(sp³) oxidative coupling.

Scheme 30 Asymmetric C(sp³)–C(sp³) oxidative cross-coupling for the synthesis of ncAAs. ET/PT, electron transfer/proton transfer; PCET, proton-coupled electron transfer; TmPLP^α, PLP α-radical enzyme.

At the same time, Hyster and co-workers also developed a dual-catalytic system by combining the PLP-dependent threonine aldolases and photocatalyst RB (Scheme 31).¹¹¹ In this work, redox-neutral α-alkylation of unprotected alanine and glycine was achieved using pyridinium salts, giving various α-tertiary amino acids with high enantioselectivity. Mechanistically, the authors proposed a unique ternary interaction between the RB, pyridinium salt, and the enzyme that helped localize radical formation to the protein active site. RB's triplet excited state was responsible for converting the pyridinium salt into alkyl radical, which was then added to the enzymatic quinonoid that formed through condensation of PLP and glycine or alanine. The resultant radical quinonoid intermediate was then single-electron oxidized by the RB radical cation to furnish the product-associated external aldimine and ground-state RB. Subsequently, the external aldimine underwent Schiff base exchange with the lysine residual of enzyme active site to release the enantioenriched product and reformed the enzymatic internal aldimine.

Scheme 31 Synthesis of α-tertiary amino acids by combining photocatalysis and PLP-dependent enzyme. ET, electron transfer; LTAs, L-threonine aldolases; TmLTA, thermophilic LTA from Thermotoga maritima; TeLTA, enzyme from Aeromonas tecta.

Very recently, Yang, Zhu and co-workers reported a related enzyme-catalyzed radical reaction, which emplyed PLP-dependent threonine aldolase to mediate α-C–H alkylation of abundant amino acids, utilizing Katritzky pyridinium salts as alkylating agents.¹¹² In these studies, different types of PLP enzymes have been redesigned to enable diverse unnatural reactions by integrating them with externally added photoredox catalysts. Given the vast array of naturally occurring PLP enzymes available, there exists immense potential for further exploration.

4.4 Thiamine-dependent oxidoreductases

Thiamine diphosphate (ThDP)-dependent enzymes are widely distributed in all organisms, and are naturally catalyzing a variety of C–C bond-forming (or bond-breaking) reactions via enzymatic Breslow intermediates. In 2023, Huang, Liang, Tian and co-workers developed a dual photo-/enzymatic system consisting of an organic photocatalyst eosin Y and an engineered ThDP-dependent enzyme (Scheme 32).¹¹³ In this fashion, a ThDP-dependent benzaldehyde lyase was repurposed into a radical acyltransferase (RAT) to enable an unnatural and highly enantioselective radical acylation of prochiral radicals. Diverse chiral ketones were prepared from aldehydes and redox-active esters (35 examples, up to 97% ee). Mechanistic studies demonstrated that excited state eosin Y* triggered the single-electron oxidation of enzyme-bound Breslow intermediate, leading to the generation of ThDP-derived ketyl radical within the enzyme's active site and eosin Y radical anion. Meanwhile eosin Y˙⁻ reduced the radical precursor to generate the prochiral benzylic radical (initially outside the active site). Finally, radical–radical cross-coupling of the prochiral benzylic radical and ketyl radical in the active site of the enzyme, formed the C(sp²)–C(sp³) bond enantioselectively and released the final products upon fragmentation of catalytic active ThDP-carbene cofactor. This work proposed an enzymatic strategy for controlling free prochiral radicals with exceptional enantioselectivity, thereby expanding the methodological landscape of bio-inspired radical chemistry to address limitations in current synthetic protocols. In 2024, Yang and co-workers also reported a similar transformation with the synergistic use of photocatalyst fluorescein and ThDP-dependent enzymes, which accomplished asymmetric alkylation of benzaldehydes and α-keto acids.¹¹⁴

Scheme 32 Repurposing natural benzaldehyde lyase with light for enantioselective radical acylation. RAT, radical acyltransferases.

More recently, Huang, Wang and co-workers further expanded this dual photoredox/thiamine-dependent enzymatic catalysis to an enantioselective three-component radical cross-coupling (Scheme 33).¹¹⁵ Utilizing three variable and easily accessible starting materials, aldehydes as acyl precursors, α-acyl bromides as electrophilic radical precursors, and alkenes as radical acceptors, an engineered thiamine-dependent 3-component radical enzyme (3CRE) gave access to diverse enantioenriched ketones. Mechanistically, the reaction of 3CRE with aldehyde proceeded via formation of the Breslow intermediate, which underwent single-electron oxidation by the photoexcited [Ru]* complex to generate the enzymatic ketyl radical. Concurrently, the reduced [Ru]˙⁻ species initiated the single-electron reduction of α-acyl bromide, generating an electrophilic radical, which subsenquently underwent philicity-matched radical addition to alkene substrate, yielding a prochiral nucleophilic radical. Ultimately, the enzyme's active site directed chemo- and enantioselective radical–radical cross-coupling, furnished enantioenhanced ketones. This synergistic system further pushed the boundary of biocatalysis to 3-component transformation, and also provides a unique strategy for sorting multiple radicals complementing existing chemical tools.

Scheme 33 Enantioselective triple radical sorting enabled by photocatalysis and benzaldehyde lyase. 3CRE, three-component radical enzymes; PfBAL, benzaldehyde lyase from Pseudomonas fluorescens.

4.5 Non-haem iron enzymes

In 2024, Huang, Garcia-Borràs, Guo and co-workers developed an adaptive biocatalytic platform that combines nonheme iron enzyme and photoredox-driven radical generation for enantioselective decarboxylative azidation/thiocyanation of redox-active NHPI esters (Scheme 34).¹¹⁶ Specifically, the authors engineered 4-hydroxyphenylpyruvate dioxygenase from Strepto myces avermitilis (SavHPPD) and obtained a final quadruple mutant SavHPPD-PC after 4 rounds of sitesaturation mutagenesis (SSM), which enabled the target reactions with up to 77% yield and 94% ee. Mechanistic studies indicated that excited-state photocatalyst (eosin Y*) firstly oxidized the Fe(II) centre with the generation of Fe(III)–N₃/Fe(III)–NCS intermediate. Meanwhile the reduced photocatalyst eosin Y˙⁻ activated NHPI esters to generate the prochiral C-radical, which was then captured by Fe(III)–N₃/Fe(III)–N₃ intermediate via radical rebound mechanism with the formation of C–N₃ and C–SCN bond. While, the authors mentioned another pathway that the excited-state photocatalyst initially reduced NHPI ester to generate the prochiral C-radical, followed by a SET between Fe(II) center and eosin Y radical cation was also possible. This work demonstrates a pioneering approach to repurpose metalloenzymes through visible-light irradiation, enabling enantioselective biocatalysis, which holds promise for broad applications.

Scheme 34 Repurposing non-haem iron enzymes for enantioselective decarboxylative azidation/thiocyanation. NPhth, phthalimide.

4.6 Others

Lipases. Lipases are a class of hydrolases that perform their catalysis via the catalytic triad in their active sites, usually exhibiting diverse substrate scope and superior robustness in organic solvents. In 2018, He, Guan and co-workers reported a concurrent photo-/bio system combining wheat germ lipase (WGL) with ruthenium-based photoredox catalysis for enantioselective synthesis of 2,2-disubstituted indol-3-ones from 2-arylindoles (Scheme 35A).¹¹⁷ Initially, the SET oxidation of substrate 2-phenylindole to excited state Ru(II)* led to the enamine cation radical, which was then proposed to be captured by a superoxide anion. The subsequent dehydration process yielded a ketoimine intermediate, which underwent WGL-catalyzed protonation to form a protonated imine intermediate. Meanwhile, acetone underwent in situ enolization through proton transfer to WGL's active site, forming a stabilized enolate anion intermediate. Finally, the enolate anion attacked the protonated imine intermediate to generate the desired products. In 2023, they demonstrated the extension of this dual-catalytic systems to enable enantioselective α-alkylation of secondary acyclic amines using simple ketones as alkylating agents, obtained various β-amino ketones successfully (Scheme 35B).¹¹⁸

Scheme 35 Repurposing lipases by synergistic photoenzymatic catalysis. DPEPhos, bis[2-(diphenylphosphino)phenyl] ether; WGL, wheat germ lipase; PPL, lipase from Aspergillus melleus; Novozym 435, commercially available immobilized lipase; CalB, Candida antarctica lipase B.

In 2018, Zhou and co-workers reported a photo/biocatalytic DKR of amines via combining photocatalyzed racemization of amines and enzymatic resolution, leading to the formation of the enantioenriched amides with up to 96% yield and 99% ee (Scheme 35C).¹¹⁹ In this work, Candida antarctica lipase B (Novozym 435) was chosen as the biocatalyst, while n-OctSH acted as a proper HAT catalyst, and Ir(ppy)₂(dtb-bpy)PF₆ served as the photocatalyst. A reaction pathway mechanism was proposed, which included an enzymatic DKR driven by fast acylation reaction with methyl β-methoxypropionate as the acyl donor, leading to the rapid accumulation of the enzyme-preferred enantiomer of the amide. For the racemization of amines, the SET process took place between the n-OctSH and excited state Ir(III)* firstly to produce Ir(II) and thiyl radicals. Owing to the polaritymatching effect, HAT and re-HAT between thiyl radical/the electron-rich α-C–H bond of substrate, resulted in the racemization of amines.

Recently, Collins and co-workers extended the substrate of photobiocatalytic DKR to secondary alcohols and amines employing lipase and heteroleptic copper complexes (Scheme 35D).¹²⁰ In this work, copper-based photocatalysis responded to racemization of the substrate through promoting hydrogen atom transfer processes employing thiyl radicals, solid-supported Candida antarctica lipase B (CALB) performed enzymatic DKR of racemic secondary alcohol with isopropenyl acetate as the acyl donor.

Methionine sulfoxide reductases. Methionine sulfoxide reductases (Msrs) are extensively characterized biocatalysts that stereoselectively reduce one enantiomer of racemic sulfoxides through a conserved cysteine in their protein active site. And Msr from Pseudomonas montelii (paMsr) was proven to catalyze the stereoselective reduction of methyl aryl sulfoxides. Protochlorophyllide is a green photo-organocatalyst isolated from Rhodobacter capsulatus, which can catalyze unselective light-driven sulfide oxidation. In 2022, Glueck, Winkler and co-workers described a concurrent reduction-oxidation sequence for deracemization of sulfoxides through the combination of these two steps, providing between 43% and 91% of recovered starting material with >99% ee (Scheme 36).¹²¹

Scheme 36 Synthesis of enantiopure sulfoxides by concurrent biocatalytic reduction and photocatalytic oxidation. paMsr, (S)-selective methionine sulfoxide reductases from Pseudomonas alcaliphila; DTT, dithiothreitol.

5. Summary and outlook

Photobiocatalysis has made remarkable progress in recent years. A series of innovative strategies have been developed to broaden the substrate spectrum and reaction types catalyzed by enzymes, and many enantioselective reactions that can't be achieved by photo- or enzyme catalysts alone have been realized. This paves the way for establishing a green synthetic platform for efficient production of bioactive molecules with exceptional stereocontrol.

As updated in this emerging review, redox-active co-factor-dependent enzymes have been effectively reshaped with visible-light-driven pathways. These include nicotinamide-dependent, flavin-dependent, pyridoxal 5′-phosphate-dependent, thiamine-dependent, and non-haem iron enzymes. More recently, type I aldolases bearing a catalytically active lysine residue have also been repurposed to be unnatural photodecarboxylases. Given the vast number of enzymes on record, mining nature for new photoenzymes is necessary and will allow us to achieve more versatile non-natural photobiocatalysis.

A prime example is ene-reductase: its reduced cofactor FMN_hq forms EDA complexes with substrates, which upon photoexcitation facilitates a net-reduction mechanism. Conversely, light irradiation of the oxidized cofactor FMN_ox allows for the development of a redox-neutral mechanism. The reaction pathway can thus be modulated by adjusting cofactor redox states—for instance, through the presence or absence of a reducing system (e.g., GDH and glucose). This principle is expected to extends to other cofactor-dependent enzymes, such as thiamine diphosphate (ThDP) and PLP-dependent families, where varying cofactor states may unlock unexplored photoenzymatic mechanisms.

To address unmet challenges in photobiocatalysis, rational enzyme selection and cofactor photochemical analysis are essential. Non-photoactive cofactors like PLP and ThDP lack intrinsic light-absorbing capacity; however, unnatural reactive intermediates can be harnessed by integrating exogenous photocatalysts into synergistic systems. In contrast, FMN and FAD exhibit direct light absorption or EDA complex-forming abilities, making them versatile for designing net reduction, redox-neutral, or synergistic catalytic platforms.

Nevertheless, despite these significant advancements, photobiocatalysis remains in its early stages, facing challenges and limitations. For instance, key areas requiring further exploration include novel non-natural transformations, such as inert bond activation (e.g., C(sp³)–H bonds), heteroatom-involved bond formation (e.g., C–P bonds), and the development of more sophisticated multi-radical sorting and synergistic regulation mechanisms. Additionally, current limitations—including constrained (photo)enzyme diversity and reaction scope, poor stability and scalability, and biocompatibility issues—hinder broader applications of photobiocatalysis.

Overcoming these challenges demands interdisciplinary collaboration. Strategies such as the design of artificial enzymes, de novo protein design, and cofactor redesign hold promise for expanding the chemical space and reactivity capabilities of photobiocatalysis. Furthermore, rationally guided directed evolution, high-throughput screening platforms, and process optimization are accelerating the translation of photoenzymatic catalysis into practical applications in green chemistry and biotechnology. Meanwhile, the integration of these approaches is expected to enable the construction of photo-driven microbial cell factories, ultimately facilitating large-scale biomanufacturing of high-value functional molecules.

Data availability

This is a review manuscript that does not generate new data.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

Financial support from the National Key Research and Development Program of China 2022YFA0913000, the National Natural Science Foundation of China (22277053), the Fundamental Research Funds for the Central Universities 0205/14380346 is gratefully acknowledged.

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Footnote

† These authors contributed equally.

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