Chemogenetic Evolution of Diversified Photoenzymes for Enantioselective [2 + 2] Cycloadditions in Whole Cells.

Artificial photoenzymes with novel catalytic modes not found in nature are in high demand; yet, they also present significant challenges in the field of biocatalysis. In this study, a chemogenetic modification strategy is developed to facilitate the rapid diversification of photoenzymes. This strategy integrates site-specific chemical conjugation of various artificial photosensitizers into natural protein cavities and the iterative mutagenesis in cell lysates. Through rounds of directed evolution, prominent visible-light-activatable photoenzyme variants were developed, featuring a thioxanthone chromophore. They successfully enabled the enantioselective [2 + 2] photocycloaddition of 2-carboxamide indoles, a class of UV-sensitive substrates that are traditionally challenging for known photoenzymes. Furthermore, the versatility of this photoenzyme is demonstrated in enantioselective whole-cell photobiocatalysis, enabling the efficient synthesis of enantioenriched cyclobutane-fused indoline tetracycles. These findings significantly expand the photophysical properties of artificial photoenzymes, a critical factor in enhancing their potential for harnessing excited-state reactivity in stereoselective transformations.

Accessing ladder-shape azetidine-fused indoline pentacycles through intermolecular regiodivergent aza-Paternò-Büchi reactions.

Small molecules with conformationally rigid, three-dimensional geometry are highly desirable in drug development, toward which a direct, simple-to-complexity synthetic logic is still of considerable challenges. Here, we report intermolecular aza-[2 + 2] photocycloaddition (the aza-Paternò-Büchi reaction) of indole that facilely assembles planar building blocks into ladder-shape azetidine-fused indoline pentacycles with contiguous quaternary carbons, divergent head-to-head/head-to-tail regioselectivity, and absolute exo stereoselectivity. These products exhibit marked three-dimensionality, many of which possess 3D score values distributed in the highest 0.5% region with reference to structures from DrugBank database. Mechanistic studies elucidated the origin of the observed regio- and stereoselectivities, which arise from distortion-controlled C-N coupling scenarios. This study expands the synthetic repertoire of energy transfer catalysis for accessing structurally intriguing architectures with high molecular complexity and underexplored topological chemical space.

Spatiotemporally Controlled Photolabeling of Genetically Unmodified Proteins in Live Cells.

Selective labeling of the protein of interest (POI) in genetically unmodified live cells is crucial for understanding protein functions and kinetics in their natural habitat. In particular, spatiotemporally controlled installation of the labels on a POI under light control without affecting their original activity is in high demand but is a tremendous challenge. Here, we describe a novel ligand-directed photoclick strategy for spatiotemporally controlled labeling of endogenous proteins in live cells. It was realized with a designer labeling reagent skillfully integrating the photochemistries of 2-nitrophenylpropyloxycarbonyl and 3-hydroxymethyl-2-naphthol with an affinity ligand. Highly electrophilic ortho-naphthoquinone methide was photochemically released and underwent a proximity coupling reaction with nucleophilic amino acid residues on the POI in live cells. With fluorescein as a marker, this photoclick strategy enables time-resolved labeling of carbonic anhydrase subtypes localized either on the cell membrane or in the cytoplasm and a discriminable visualization of their metabolic kinetics. Given the versatility underlined by facilely tethering other functional entities (e.g., biotin, a peptide short chain) via acylation or (in cell) Huisgen cycloaddition, this affinity-driven photoclick chemistry opens up enormous opportunities for discovering dynamic functions and mechanistic interrogation of endogenous proteins in live cells.

Fluorescent Turn-On Probes for Visualizing GPx4 Levels in Live Cells and Predicting Drug Sensitivity.

Glutathione peroxidase 4 (GPx4) is the membrane peroxidase in mammals that is essential for protecting cells against oxidative damage and critical for ferroptosis. However, no live cell probe is currently available to specifically label GPx4. Herein, we report both inhibitory and noninhibitory fluorescent turn-on probes for specific labeling of GPx4 in live cells. With these probes, the GPx4 expression levels and degradation kinetics in live cells could be visualized, and their real-time responses to the cellular selenium availability were revealed. These probes could also potentially serve as staining reagents to predict the sensitivity of GPx4-related ferroptosis drugs. In view of these features, these GPx4-selective probes will offer opportunities for a deeper understanding of GPx4 function in natural habitats and hold great promise for biomedical applications.

Visible-Light-Induced Radical gem-Iodoallylation of 2,2,2-Trifluorodiazoethane.

A visible-light-induced radical gem-iodoallylation of CF3CHN2 was developed under mild conditions, delivering a variety of α-CF3-substituted homoallylic iodide compounds in moderate to excellent yields. The transformation features broad substrate scope, good functional group compatibility, and operational simplicity. The described protocol provides a convenient and attractive tool to apply CF3CHN2 as CF3-introduction reagent in radical synthetic chemistry.

Expanding the Promiscuity of a Copper-Dependent Oxidase for Enantioselective Cross-Coupling of Indoles.

Herein, we disclose the highly enantioselective oxidative cross-coupling of 3-hydroxyindole esters with various nucleophilic partners as catalyzed by copper efflux oxidase. The biocatalytic transformation delivers functionalized 2,2-disubstituted indolin-3-ones with excellent optical purity (90-99 % ee), which exhibited anticancer activity against MCF-7 cell lines, as shown by preliminary biological evaluation. Mechanistic studies and molecular docking results suggest the formation of a phenoxyl radical and enantiocontrol facilitated by a suited enzyme chiral pocket. This study is significant with regard to expanding the catalytic repertoire of natural multicopper oxidases as well as enlarging the synthetic toolbox for sustainable asymmetric oxidative coupling.

Iodine-Mediated Coupling of 2,2,2-Trifluorodiazoethane and Alkynes To Access Bistrifluoromethylated 1,3,5-Trienes.

Multi-component reaction of higher degree ultilization of diazo molecules for polyene formation is highly intriuging but still underexplored. We present herein an unprecedented coupling of 2,2,2-trifluorodiazoethane and aryl alkynes mediated by iodine under visible light. The multi-component reaction involving two diazo units and two alkyne units provides a straightforward and step-economic access to bistrifluoromethylated 1,3,5-trienes in high stereoselectivity by creation of three C═C bonds in a single step under mild conditions.

Enantioselective [2+2]-cycloadditions with triplet photoenzymes.

Naturally evolved enzymes, despite their astonishingly large variety and functional diversity, operate predominantly through thermochemical activation. Integrating prominent photocatalysis modes into proteins, such as triplet energy transfer, could create artificial photoenzymes that expand the scope of natural biocatalysis1-3. Here, we exploit genetically reprogrammed, chemically evolved photoenzymes embedded with a synthetic triplet photosensitizer that are capable of excited-state enantio-induction4-6. Structural optimization through four rounds of directed evolution afforded proficient variants for the enantioselective intramolecular [2+2]-photocycloaddition of indole derivatives with good substrate generality and excellent enantioselectivities (up to 99% enantiomeric excess). A crystal structure of the photoenzyme-substrate complex elucidated the non-covalent interactions that mediate the reaction stereochemistry. This study expands the energy transfer reactivity7-10 of artificial triplet photoenzymes in a supramolecular protein cavity and unlocks an integrated approach to valuable enantioselective photochemical synthesis that is not accessible with either the synthetic or the biological world alone.

Light-Controlled Traceless Protein Labeling via Decaging Thio-o-naphthoquinone Methide Chemistry.

We report the molecular design of a novel multifunctional reagent and its application for light-controlled selective protein labeling. This molecule integrates functions of protein-ligand recognition, bioconjugation, ligand cleavage, and photoactivation by merging the photochemistries of 2-nitrophenylpropyloxycarbonyl and 3-hydroxymethyl-2-naphthol with an affinity ligand and fluorescein. Highly electrophilic o-naphthoquinone methide was photochemically released and underwent proximity-driven selective labeling with the protein of interest (e.g., carbonic anhydrases), which retains its native function after labeling.

Catalytic Atroposelective Electrophilic Amination of Indoles.

Reported here is the first catalytic atroposelective electrophilic amination of indoles, which delivers functionalized atropochiral N-sulfonyl-3-arylaminoindoles with excellent optical purity. This reaction was furnished by 1,6-nucleophilic addition to p-quinone diimines. Control experiments suggest an ionic mechanism that differs from the radical addition pathway commonly proposed for 1,6-addition to quinones. The origin of 1,6-addition selectivity was investigated through computational studies. Preliminary studies show that the obtained 3-aminoindoles atropisomers exhibit anticancer activities. This method is valuable with respect to enlarging the toolbox for atropochiral amine derivatives.

Chemical Modification for the "Off-/On" Regulation of Enzyme Activity.

Enzymes with excellent catalytic performance play important roles in living organisms. Advances in strategies for enzyme chemical modification have enabled powerful strategies for exploring and manipulating enzyme functions and activities. Based on the development of chemical enzyme modifications, incorporating external stimuli-responsive features-for example, responsivity to light, voltage, magnetic force, pH, temperature, redox activity, and small molecules-into a target enzyme to turn "on" and "off" its activity has attracted much attention. The ability to precisely control enzyme activity using different approaches will greatly expand the chemical biology toolbox for clarification and detection of signal transduction and in vivo enzyme function and significantly promote enzyme-based disease therapy. This review summarizes the methods available for chemical enzyme modification mainly for the off-/on control of enzyme activity and particularly highlights the recent progress regarding the applications of this strategy.

Properties and Mechanisms of Flavin-Dependent Monooxygenases and Their Applications in Natural Product Synthesis.

Natural products are usually highly complicated organic molecules with special scaffolds, and they are an important resource in medicine. Natural products with complicated structures are produced by enzymes, and this is still a challenging research field, its mechanisms requiring detailed methods for elucidation. Flavin adenine dinucleotide (FAD)-dependent monooxygenases (FMOs) catalyze many oxidation reactions with chemo-, regio-, and stereo-selectivity, and they are involved in the synthesis of many natural products. In this review, we introduce the mechanisms for different FMOs, with the classical FAD (C4a)-hydroperoxide as the major oxidant. We also summarize the difference between FMOs and cytochrome P450 (CYP450) monooxygenases emphasizing the advantages of FMOs and their specificity for substrates. Finally, we present examples of FMO-catalyzed synthesis of natural products. Based on these explanations, this review will expand our knowledge of FMOs as powerful enzymes, as well as implementation of the FMOs as effective tools for biosynthesis.

Cinchona Alkaloid Derived Iodide Catalyzed Enantioselective Oxidative α-Amination of Carbonyl Compounds toward the Construction of Spiroindolyloxindole.

Novel cinchona alkaloid derived iodide catalysts were developed for the enantioselective oxidative α-amination of 2-oxindoles, providing various functionalized spiropyrrolidine oxindoles in high yields and with good enantioselectivities. This iodide/ROOH catalytic system features a one-step synthesis of a catalyst with multiple functionalities, ease of operation, and good scalability, thereby enriching the repertoire of iodide catalysis for enantioselective oxidative coupling reactions.

Macrocyclic Aromatic Amide Foldamer: Synthesis, Twisted-to-Boxlike Conformational Switching, and Molecular Recognition.

In this study, a twisted macrocycle was synthesized via ring closure of a double-helical aromatic oligoamide foldamer with two disulfide bridges. Single-crystal X-ray structure and NMR spectroscopy demonstrate the twisted conformation of macrocycle both in the solid state and in solution. As a result of the rearrangement of hydrogen bonding preference, the twisted conformation could be transformed to boxlike through protonation of the pyridine segments of macrocycle. In addition, the NMR titration experiments revealed that flat aromatic guests (e. g., coronene and perylene) could bind to the boxlike macrocycle with a 1 : 1 binding stoichiometry. The addition of base to the host-guest complexes resulted in conformational reversal of the macrocycle from boxlike to twisted as well as the release of guest.

Regioselectivity and stereoselectivity of intramolecular [2 + 2] photocycloaddition catalyzed by chiral thioxanthone: a quantum chemical study.

Chiral photosensitizer-catalyzed stereoselective olefin cyclization has shown its significance in organic synthesis. In this work, we investigated the reaction mechanism, regioselectivity and stereoselectivity of photochemical intramolecular [2 + 2] cycloaddition reaction catalyzed by a chiral thioxanthone molecule using quantum chemical calculations. The reaction proceeded via an energy transfer from the triplet thioxanthone to the substrate, involving stepwise and sequential C-C bond formation. The first C-C bond formation was calculated to be the rate-limiting and selectivity-controlling step. The origin of stereoselectivity was found to be interaction-controlled by distortion/interaction analysis. In addition, the catalyst substituent effects (O vs. S vs. Se) on the stereoselectivity of the photocycloadditions were explored, which provides helpful mechanistic information for the design of related photoinduced reactions.

Biocatalytic Cross-Coupling of Aryl Halides with a Genetically Engineered Photosensitizer Artificial Dehalogenase.

Devising artificial photoenzymes for abiological bond-forming reactions is of high synthetic value but also a tremendous challenge. Disclosed herein is the first photobiocatalytic cross-coupling of aryl halides enabled by a designer artificial dehalogenase, which features a genetically encoded benzophenone chromophore and site-specifically modified synthetic NiII(bpy) cofactor with tunable proximity to streamline the dual catalysis. Transient absorption studies suggest the likelihood of energy transfer activation in the elementary organometallic event. This design strategy is viable to significantly expand the catalytic repertoire of artificial photoenzymes for useful organic transformations.

Hemin-Catalyzed Oxidative Phenol-Hydrazone [3+3] Cycloaddition Enables Rapid Construction of 1,3,4-Oxadiazines.

Herein, we present a hemin-catalyzed oxidative phenol-hydrazone [3+3] cycloaddition that accommodates a broad spectrum of N-arylhydrazones, a class of less exploited 1,3-dipoles due to their significant Lewis basicity and weak tendency to undergo 1,2-prototropy to form azomethine imines. It renders expedient assembly of diversely functionalized 1,3,4-oxadiazines with excellent atom and step economy. Preliminary mechanistic studies point to the involvement of a one-electron oxidation pathway, which likely differs from the base-promoted aerobic oxidative scenario.

Harnessing structurally unbiased ortho-benzoquinone monoimine for biomimetic oxidative [4+2] cycloaddition with enamines.

Reported herein is the first catalytic oxidative [4+2] cycloaddition of 2-aminophenols with cyclic enamines. This biomimetic catalytic oxidative strategy expediently accommodates the very labile structurally unbiased ortho-quinone monoimine intermediate for cycloaddition by controlling its formation rate, thus refraining from otherwise prerequisite steric or electronic stabilization and allowing efficient assembly of various tricyclic 1,4-benzoxazines in a step and atom economic fashion.

Intermolecular Vicinal Diaminative Assembly of Tetrahydroquinoxalines via Metal-free Oxidative [4 + 2] Cycloaddition Strategy.

Reported herein is the first metal-free oxidative [4 + 2] coupling of o-phenylenediamines with various alkenes. Differing from the known strategy that hinged on reactive π-allyl Pd intermediates from restrained allylic alcohol/acetate and diene substrates, this metal-free method features easy accessibility of starting materials, step economy, benign reaction conditions, and more importantly broad C-C double bonds (styrenes, vinyl (thio)ethers, benzofurans, indoles) with diastereospecificities. Mechanistic studies suggest the intermediacy of the benzoquinone diimides, a class of useful but yet underexploited synthons. Of note, they efficiently furnished functionalized tetrahydroquinoxalines and complement the well-studied alkene vicinal diamination typically toward acyclic diamine derivatives.

Manganese(II) Oxidizing Bacteria as Whole-Cell Catalyst for ß-Keto Ester Oxidation.

Manganese oxidizing bacteria can produce biogenic manganese oxides (BMO) on their cell surface and have been applied in the fields of agriculture, bioremediation, and drinking water treatment to remove toxic contaminants based on their remarkable chemical reactivity. Herein, we report for the first time the synthetic application of the manganese oxidizing bacteria, Pseudomonas putida MnB1 as a whole-cell biocatalyst for the effective oxidation of ß-keto ester with excellent yield. Differing from known chemical protocols toward this transformation that generally necessitate the use of organic solvents, stoichiometric oxygenating agents and complex chemical catalysts, our strategy can accomplish it simply under aqueous and mild conditions with higher efficiency than that provided by chemical manganese oxides. Moreover, the live MnB1 bacteria are capable of continuous catalysis for this C-O bond forming reaction for several cycles and remain proliferating, highlighting the favorable merits of this novel protocol for sustainable chemistry and green synthesis.