Amide bond formation is widely used in pharmaceutical synthesis, typically involving stoichiometric coupling reagents to activate carboxylic acid substrates for a condensation reaction. As an alternative approach, we repurposed aldehyde dehydrogenases into oxidative amidases by creating a more hydrophobic and spacious catalytic pocket for amines to capture the thioester intermediate. This biocatalyst efficiently facilitates the formation of amide bonds between diverse aldehydes and amines. We also developed a two-step enzymatic cascade to synthesize amides from broadly available aliphatic alcohols. This biocatalytic strategy enabled the redesign of synthetic routes for five drug molecules. Our findings highlight the potential of oxidative amidases in advancing the synthesis of structurally diverse drug molecules through efficient amide bond formation.

Due to the invaluable properties of organofluorine compounds, incorporating a fluorinated unit has become necessary in pharmaceuticals, agrochemicals, and materials. However, achieving asymmetric fluorination such as trifluoromethylation through chemo- or biocatalysis has been a synthetic challenge. Here, we introduce a unique cooperative photoenzymatic catalysis for the enantioselective fluoroalkylation/cyclization cascade. This method, utilizing the engineered flavin-dependent "ene"-reductases (EREDs) and an exogenous photocatalyst (PC), produces a variety of fluorinated cyclic ketones with high yield and enantioselectivity. The discovery of stereocomplementary enzymes that provide access to both enantiomers of the cyclized products further enhances the synthetic applications of our method. The radical-trapping, spectroscopic, and kinetic studies have substantiated the interaction mode between the PC and the enzyme and demonstrated a cascade reaction mechanism involving a unique intermolecular addition of fluorinated radicals and a stereocontrolled intramolecular cyclization. Isotopic labeling experiments support flavin as the source of the hydrogen atom. Molecular dynamics simulations reveal that the binding interaction of the enzyme and the intermediate triggers the photoinduced enantioselective cyclization. This work underscores the potential of enzymes for the asymmetric synthesis of fluorinated compounds.

Aggregation pheromone, 4-vinylanisole (4VA), is specifically released by gregarious migratory locusts, and is crucial in forming locust swarms that cause destructive plagues1. Control of locust plagues relies heavily on the extensive application of chemical pesticides, which has led to severe environmental and health issues2. As pheromones are primary mediators of insect communication and behaviour3, exploring their biosynthesis can provide important cues to develop innovative behavioural regulators, potentially reducing the reliance on chemical pesticides. Here we resolve the biosynthesis of 4VA and behavioural responses of locusts when enzymes in the 4VA biosynthetic pathway are manipulated. The process initiates with phenylalanine derived from food plants and proceeds through three precursors: cinnamic acid, p-hydroxycinnamic acid and 4-vinylphenol (4VP). Notably, the conversion from 4VP to 4VA through methylation is unique to gregarious locusts. This step is catalysed by two crucial methyltransferases, 4VPMT1 and 4VPMT2. Guided by the X-ray co-crystal structure of 4VPMT2 bound with 4VP and S-adenosyl-L-methionine, we developed 4-nitrophenol as a substrate surrogate. We identified several chemicals that can block 4VA production by inhibiting the enzymatic activities of 4VPMT proteins, thereby suppressing locust aggregative behaviour. The findings uncover the chemical logic behind 4VA biosynthesis and pinpoint two crucial enzymes as novel targets for locust swarm management.

Trichothecenes, particularly T-2 toxin (T-2), pose significant threats to food safety as well as to both animal and human health. Although Fhb7 and its variants have been utilized for deoxynivalenol degradation, no enzyme with efficient T-2 degradation activity has been reported. Herein, we generated five enzymes derived from Fhb7 that are capable of T-2 degradation via ancestral sequence reconstruction. Among these, N1, N2, and N4 exhibited superior catalytic activity toward T-2 compared to the parent enzyme Fhb7 and its variants. Structural analyses revealed that residue F27 provides a hydrophobic environment for accommodation of the unique 3-methylbutyryl group of T-2. In addition, the long insertion loop of N2 plays a key role in its improved substrate preference. Furthermore, all the ancestors displayed remarkable thermostability, with N2 and N4 displaying superior thermal tolerance (T m values are 54 and 59 degrees C, respectively, and their half-life times are longer than 90 h), positioning them as a promising candidate for industrial applications. This work introduces a promising enzymatic approach for T-2 degradation and lays a foundation for developing robust biocatalysts for the environmental and industrial bioremediation of mycotoxins.

Organic nitriles are significant in pharmaceuticals, agrochemicals, cosmetics, and materials. Although numerous cyanidation methods have been developed, more eco-friendly and green protocols for manufacturing alkyl nitriles are in high demand. Here, we report a photoenzymatic enantioselective intermolecular hydrocyanoalkylation of alkenes catalyzed by flavin-dependent "ene"-reductases. The discovery of stereocomplementary enzymes that provide access to both enantiomers of the high-value nitriles further showcases the synthetic applications of this method. Radical trapping, isotopic labeling, and spectroscopic experiments have elucidated the formation of a charge transfer complex at the protein active site. The single-electron reduction of the cyanoalkyl radical precursor by flavin hydroquinone yields a cyanoalkyl radical, which then undergoes intermolecular radical addition. This active site can stereoselectively control the radical-terminating hydrogen atom transfer, enabling the synthesis of enantioenriched γ-stereogenic nitriles. This work further expands the reactivity repertoire of biocatalytic transformations via non-natural radical mechanisms.

The bisbenzylisoquinoline alkaloids (bisBIAs) have attracted tremendous attention from the synthetic community due to their diverse and intriguing biological activities. Herein, we report the convergent and modular chemoenzymatic syntheses of eight bisBIAs bearing various substitutes and linkages in 5-7 steps. The gram-scale synthesis of various well-designed enantiopure benzylisoquinoline monomers was accomplished through an enzymatic stereoselective Pictet-Spengler reaction, followed by regioselective enzymatic methylation or chemical functionalization in a sequential one-pot process. A modified intermolecular copper-mediated Ullmann coupling enabled the concise and efficient total synthesis of five different linear bisBIAs with either head-to-tail or tail-to-tail linkage. A biomimetic oxidative phenol dimerization selectively formed the sterically hindered, electron-rich diaryl ether bond, and subsequent intramolecular Suzuki-Miyaura domino reaction or Ullmann coupling facilitated the first enantioselective total synthesis of three macrocyclic bisBIAs, including ent-isogranjine, tetrandrine and O-methylrepandine. This study highlights the great potential of chemoenzymatic strategies in the total synthesis of diverse bisBIAs and paves the way to further explore the biological functions of these natural products.

Chronic itch is a debilitating symptom profoundly impacting the quality of life in patients with liver diseases like cholestasis. Activation of the human G-protein coupled receptor, MRGPRX4 (hX4), by bile acids (BAs) is implicated in promoting cholestasis itch. However, the detailed underlying mechanisms remain elusive. Here, we identified 3-sulfated BAs that are elevated in cholestatic patients with itch symptoms. We solved the cryo-EM structure of hX4-Gq in a complex with 3-phosphated deoxycholic acid (DCA-3P), a mimic of the endogenous 3-sulfated deoxycholic acid (DCA-3S). This structure revealed an unprecedented ligand-binding pocket in MRGPR family proteins, highlighting the crucial role of the 3-hydroxyl (3-OH) group on BAs in activating hX4. Guided by this structural information, we designed and developed compound 7 (C7), a BA derivative lacking the 3-OH. Notably, C7 effectively alleviates hepatic injury and fibrosis in liver disease models while significantly mitigating the itch side effects.

In the era of global climate change, the increasingly severe Fusarium head blight (FHB) and deoxynivalenol (DON) contamination have caused economic losses and brought food and feed safety concerns. Recently, an FHB resistance gene Fhb7 coding a glutathione-S transferase (GST) to degrade DON by opening the critical toxic epoxide moiety was identified and opened a new window for wheat breeding and DON detoxification. However, the poor stability of Fhb7 and the elusiveness of the catalytic mechanism hinder its practical application. Herein, we report the first structure of Fhb7 at 2.41 Å and reveal a unique catalytic mechanism of epoxide opening transformation in GST family proteins. Furthermore, variants V29P and M10 showed that 5.5-fold and 266.7-fold longer half-life time than wild-type, respectively, were identified. These variants offer broad substrate scope, and the engineered biosafe Bacillus subtilis overexpressing the variants shows excellent DON degradation performance, exhibiting potential at bacterium engineering to achieve DON detoxification in the feed and biomedicine industry. This work provides a profound mechanistic insight into the enzymatic activities of Fhb7 and paves the way for further utilizing Fhb7-related enzymes in crop breeding and DON detoxification by synthetic biology.

Nitrogen heterocycles are commonly found in bioactive natural products and drugs. However, the biocatalytic tools for nitrogen heterocycle synthesis are limited. Herein, we report the discovery of vanillyl alcohol oxidases (VAOs) as efficient biocatalysts for the one-pot synthesis of 2-aryl thiazolines from various 4-hydroxybenzaldehydes and aminothiols. The wild-type biocatalyst features a broad scope of 4-hydroxybenzaldehydes. Though the scope of aminothiols is limited, it could be improved via semi-rational protein engineering, generating a variant to produce previously inaccessible cysteine-derived bioactive 2-aryl thiazolines using the wild-type VAO. Benefiting from the derivatizable functional groups in the enzymatic products, we further chemically modified these products to expand the chemical space, offering a new chemoenzymatic strategy for the green and efficient synthesis of structurally diverse 2-aryl-thiazoline derivatives to prompt their use in drug discovery and catalysis.

Biosynthetic enzymes evolutionarily gain novel functions, thereby expanding the structural diversity of natural products to the benefit of host organisms. Diels-Alderases (DAs), functionally unique enzymes catalysing [4 + 2] cycloaddition reactions, have received considerable research interest. However, their evolutionary mechanisms remain obscure. Here, we investigate the evolutionary origins of the intermolecular DAs in the biosynthesis of Moraceae plant-derived Diels-Alder-type secondary metabolites. Our findings suggest that these DAs have evolved from an ancestor functioning as a flavin adenine dinucleotide (FAD)-dependent oxidocyclase (OC), which catalyses the oxidative cyclisation reactions of isoprenoid-substituted phenolic compounds. Through crystal structure determination, computational calculations, and site-directed mutagenesis experiments, we identified several critical substitutions, including S348L, A357L, D389E and H418R that alter the substrate-binding mode and enable the OCs to gain intermolecular DA activity during evolution. This work provides mechanistic insights into the evolutionary rationale of DAs and paves the way for mining and engineering new DAs from other protein families.

Substituted cyclohexanes are common scaffolds found in both natural products and drug molecules. Diels-Alderases that can efficiently catalyze intermolecular Diels-Alder reactions to generate cyclohexene ring systems have received considerable interest. However, the synthetic power of Diels-Alderases is incomparable with chemo-catalysts due to their limited substrate scopes. Here, we report a new chemo-enzymatic strategy for the diversity-oriented syntheses of functionalized cyclohexenes. We first applied focused rational iterative site-specific mutagenesis to generate a natural Diels-Alderase variant M3, which shows a 34-fold increase in catalytic efficiency, broad substrate scope, and good to perfect stereoselectivity. Then, we used diverse transition-metal-catalyzed decarboxylative coupling reactions to functionalize the enzymatic Diels-Alder products. This work offers an efficient synthetic route to structurally diverse cyclohexenes that are not accessible by solely using biocatalysis or chemo-catalysis and illustrates how chemo-catalysis can cooperate with biocatalysis to expand the synthetic application of biocatalysts.

Currently, many countries in the world are entering an aging society. The health of the elderly is the most prominent problem in an aging society. Stem cell exhaustion and aging are some of the most important factors leading to body aging. Therefore, reversing or alleviating stem cell aging is an urgent scientific problem to be solved. Researchers have been looking into biological antiaging molecules, and green and healthy bioactive molecules have great application value and potential. Lutein is a carotene that has a wide range of natural sources. Lutein has been found to have many important biological activities. However, to date, the potential antiaging effect of lutein has not been revealed in stem cell models. In the current study, we used mesenchymal stem cells (MSCs) as a model (in vitro) to evaluate the antiaging potential of lutein. A series of experiments (such as Western blotting and indirect immunofluorescence) were performed to evaluate the antiaging effects of lutein in vivo and in vitro. The results showed that lutein has a good antiaging effect by evaluating Sa-beta-gal and agingrelated markers (such as p16 and p21). Additionally, we further carried out relevant experiments in vivo to evaluate the antiaging effect of lutein and found that lutein could also exhibit an antiaging effect in many tissues and organs in vivo. In conclusion, this work found that lutein could relieve aging in vivo and in vitro, suggesting that lutein is a potential antiaging functional food or drug.

The Diels-Alder (D-A) reaction is one of the most critical chemical transformations to construct C–C bonds with predictable regio- and stereo-selectivities. It has been widely used as a retrosynthetic disconnection in synthetic chemistry. Although significant advances have been made, synthetic challenges remain in how to precisely control the stereochemistry of intermolecular Diels-Alder reaction. Enzymes are well known for their remarkable catalytic efficiency and selectivity compared with chemo-catalysts. Therefore, identifying and designing intermolecular Diels-Alderases that can be used in organic synthesis have received considerable attention from the synthetic community. In this review, we review all the enzymes capable of catalyzing formal intermolecular Diels-Alder reactions in natural product biosynthetic pathways, discuss their catalytic mechanisms in detail, and highlight their synthetic potential in the precise and efficient synthesis of enantiopure D-A products. We also discuss the different strategies that can be used to create new artificial Diels-Alderases, especially RNA-based Diels-Alderases.

Natural Diels–Alderases that selectively form endo or exo products are increasingly well known but generally form one stereoisomer with limited substrate scope. Here we report the discovery of two homologous groups of flavin-adenine-dinucleotide-dependent enzymes that catalyse intermolecular Diels–Alder reactions on the same substrates with opposite endo / exo selectivity and high enantioselectivity. We show that these enzymes are effective biocatalysts with a wide range of diene and dienophile substrates. The crystal structure of an exo -selective Diels–Alderase was determined at 2.94 Å resolution. Based on the structure and computational investigation of the catalytic mechanism, we designed and prepared mutant enzymes that reverse the stereoselectivity from exo to endo . A combination of structure-based comparison, computational and mutational studies have revealed two different catalytic mechanisms that control the endo / exo selectivity in these enzymatic Diels–Alder reactions.

beta-Lactam antibiotic resistance has become a critical global health threat. One of the major reasons for drug resistance is the expression of beta-lactamases especially metallo-beta-lactamases such as New Delhi metallo-beta-lactamase (NDM-1) by Gram-negative bacteria. The fungal natural product aspergillomarasmine A (AMA) was found to be a promising inhibitor of NDM-1 to potentiate currently used beta-lactam antibiotics to overcome drug resistance both in vitro and in vivo. Although several chemical synthesis and chemoenzymatic synthesis approaches to access AMA have been reported, the biosynthesis of AMA was still elusive. Herein, we identified the key enzyme responsible for the biosynthesis of AMA in Aspergillus oryzae. AMA synthase is a PLP-dependent cysteine synthase homologous protein which utilizes O-acetyl-L-serine/Ophospho-L-serine and L-aspartic acid as its substrates. Remarkably, this enzyme catalyzes two consecutive C-N bond formations to produce AMA efficiently which may be attributed to the spacious substrate-binding pocket. PLP is covalently bound to Lys61 by an internal aldimine from the PLP re face, and the si face of PLP pyridine ring is accessible to the substrates to promote the nucleophilic addition of amino acids to the double bond of the external adiminine and ultimately to generate chiral C-alpha with S configuration. The catalytic mechanism was proposed based on molecular docking and biochemical experiments. In addition, we have further investigated the substrate scope of AMA synthase and identified a variant enzyme which shows promising potential in producing structurally diverse molecules containing the C-N bond.

Diels-Alder reaction is one of the most important transformations used in organic synthesis, with the ability to construct two new CC bonds and up to four chiral centers simultaneously. However, the biggest synthetic challenge in Diels-Alder reaction lies in controlling its regio-, diastereo-, and enantioselectivity. Using Stille cross-coupling and enzymatic Diels-Alder reaction as the key steps, the first chemoenzymatic total synthesis of artonin I is achieved in 30% overall yield over only seven steps. This enzymatic Diels-Alder reaction catalyzed by MaDA is featured with excellent endo- and enantioselectivity and high catalytic efficiency (kcat /KM = 362 ± 54 mm-1  s-1 ). These successful chemoenzymatic total syntheses of artonin I and dideoxyartonin I demonstrated the remarkable potential of the intermolecular Diels-Alderase MaDA in biocatalysis.

A series of benzenesulfonamide derivatives were synthesized and evaluated for their anti-proliferative activity and interaction with tubulin. These new derivatives showed significant activities against cellular proliferative and tubulin polymerization. Compound BA-3b proved to be the most potent compound with IC50 value ranging from 0.007 to 0.036 μM against seven cancer cell lines, and three drug-resistant cancer cell lines, which indicated a promising anti-cancer agent. The target tubulin was also verified by dynamic tubulin polymerization assay and tubulin intensity assay.

Reported here is a formal synthesis of gracilamine using Rh(I)-catalyzed [3 + 2 + 1] reaction of yne-VCP (±)-4 and CO. The key reaction gave the cycloadduct (±)-trans-3 with the A-B-C core structure of gracilamine. This advanced intermediate was further transformed to Gao's intermediate (±)-2 via regular transformations to realize the formal synthesis of gracilamine. The present strategy was used to accomplish the asymmetric formal synthesis of gracilamine using chiral substrate (+)-4.

A series of 1-sulfonyl indolines was synthesized and evaluated for antiproliferative activity. The most potent compounds 9a and 9e showed significant cytotoxicity (IC50 in the range of 0.055–0.105 and 0.039–0.112μM, respectively) against four human cancer cell lines HCT116, PC3, HepG2 and SK-OV-3. The structure–activity relationship of this series of sulfonamides, including the influence of azaheterocycle rings, substituent at the different positions of indoline, and the cyclopropane moiety, was described.