RESUMEN
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.
Asunto(s)
Biocatálisis , Reacción de Cicloadición , Enzimas , Procesos Fotoquímicos , Biocatálisis/efectos de la radiación , Transferencia de Energía , Estereoisomerismo , Enzimas/genética , Enzimas/metabolismo , Enzimas/efectos de la radiación , Indoles/química , Especificidad por Sustrato , Cristalización , Evolución Molecular Dirigida/métodosRESUMEN
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.
Asunto(s)
Reacción de Cicloadición , Estereoisomerismo , Indoles/química , Indoles/síntesis química , Indoles/metabolismo , Procesos Fotoquímicos , Biocatálisis , Evolución Molecular Dirigida , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/síntesis química , Luz , Escherichia coli/enzimología , Estructura MolecularRESUMEN
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.
Asunto(s)
Naftoles , Proteínas , Ligandos , Proteínas/química , Naftoles/química , FluoresceínaRESUMEN
The market demand for essential oil containing citral is increasing. Our research group identified a rare chemotype of Camphora officinarum whose leaves are high in citral content by examining over 1000 wild trees across the entire native distribution area of C. officinarum in China. Because C. officinarum is suitable for large-scale cultivation, it is therefore seen as a promising source of natural citral. However, the molecular mechanism of citral biosynthesis in C. officinarum is poorly understood. In this study, transcriptomic analyses of C. officinarum with different citral contents revealed a strong positive correlation between the expression of a putative geraniol synthase gene (CoGES) and citral content. The CoGES cDNA was cloned, and the CoGES protein shared high similarity with other monoterpene synthases. Enzymatic assays of CoGES with geranyl diphosphate (GPP) as substrate yielded geraniol as the single product, which is the precursor of citral. Further transient expression of CoGES in Nicotiana benthamiana resulted in a higher relative content of geranial and the appearance of a new substance, neral. These findings indicate that CoGES is a geraniol synthase-encoding gene, and the encoded protein can catalyze the transformation of GPP into geraniol, which is further converted into geranial and neral through an unknown mechanism in vivo. These findings expand our understanding of citral biosynthesis in Lauraceae plants. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-024-01463-4.
RESUMEN
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.
Asunto(s)
Colorantes Fluorescentes , Humanos , Células HEK293 , Supervivencia Celular , Fosfolípido Hidroperóxido Glutatión Peroxidasa/metabolismo , Colorantes Fluorescentes/química , Selenio/metabolismoRESUMEN
DNA nanotechnology has seen large developments over the last 30 years through the combination of solid phase synthesis and the discovery of DNA nanostructures. Solid phase synthesis has facilitated the availability of short DNA sequences and the expansion of the DNA toolbox to increase the chemical functionalities afforded on DNA, which in turn enabled the conception and synthesis of sophisticated and complex 2D and 3D nanostructures. In parallel, polymer science has developed several polymerization approaches to build di- and triblock copolymers bearing hydrophilic, hydrophobic, and amphiphilic properties. By bringing together these two emerging technologies, complementary properties of both materials have been explored; for example, the synthesis of amphiphilic DNA-polymer conjugates has enabled the production of several nanostructures, such as spherical and rod-like micelles. Through both the DNA and polymer parts, stimuli-responsiveness can be instilled. Nanostructures have consequently been developed with responsive structural changes to physical properties, such as pH and temperature, as well as short DNA through competitive complementary binding. These responsive changes have enabled the application of DNA-polymer conjugates in biomedical applications including drug delivery. This review discusses the progress of DNA-polymer conjugates, exploring the synthetic routes and state-of-the-art applications afforded through the combination of nucleic acids and synthetic polymers.
Asunto(s)
Nanoestructuras , Polímeros , ADN/química , Micelas , Nanoestructuras/química , Nanotecnología , Polímeros/químicaRESUMEN
We provide an effective method to create DNA nanostructures below 100 nm with defined charge patterns and explore whether the density and location of charges affect the cellular uptake efficiency of nanoparticles (NPs). To avoid spontaneous charge neutralization, the negatively charged polymer nanopatterns were first created by in situ polymerization using photoresponsive monomers on DNA origami. Subsequent irradiation generated positive charges on the immobilized polymers, achieving precise positively charged patterns on the negatively charged DNA surface. Via this method, we have discovered that the positive charges located on the edges of nanostructures facilitate more efficient cellular uptake in comparison to the central counterparts. In addition, the high-density positive charge decoration could also enhance particle penetration into 3D multicellular spheroids. This strategy paves a new way to construct elaborate charge-separated substructures on NP surfaces and holds great promise for a deeper understanding of the influence between the surface charge distribution and nano-bio interactions.
Asunto(s)
Nanopartículas , Nanoestructuras , ADN , Nanopartículas/química , Polimerizacion , Polímeros/química , Propiedades de SuperficieRESUMEN
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-7â 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.
Asunto(s)
Cobre , Oxidorreductasas , Cobre/metabolismo , Estereoisomerismo , Simulación del Acoplamiento Molecular , Oxidorreductasas/metabolismo , Ceruloplasmina/metabolismo , IndolesRESUMEN
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.
Asunto(s)
Catálisis , TemperaturaRESUMEN
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.
Asunto(s)
Productos Biológicos , Flavina-Adenina Dinucleótido , Sistema Enzimático del Citocromo P-450 , Dinitrocresoles , Flavina-Adenina Dinucleótido/química , Flavinas/química , Oxigenasas/químicaRESUMEN
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.
Asunto(s)
Aminas , Indoles , Aminación , CatálisisRESUMEN
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.
Asunto(s)
Ingeniería Genética , Hidrocarburos Halogenados/metabolismo , Hidrolasas/metabolismo , Fármacos Fotosensibilizantes/metabolismo , Biocatálisis , Hidrocarburos Halogenados/química , Hidrolasas/química , Modelos Moleculares , Estructura Molecular , Fármacos Fotosensibilizantes/químicaRESUMEN
Due to its unique physical and chemical characteristics, DNA, which is known only as genetic information, has been identified and utilized as a new material at an astonishing rate. The role of DNA has increased dramatically with the advent of various DNA derivatives such as DNA-RNA, DNA-metal hybrids, and PNA, which can be organized into 2D or 3D structures by exploiting their complementary recognition. Due to its intrinsic biocompatibility, self-assembly, tunable immunogenicity, structural programmability, long stability, and electron-rich nature, DNA has generated major interest in electronic and catalytic applications. Based on its advantages, DNA and its derivatives are utilized in several fields where the traditional methodologies are ineffective. Here, the present challenges and opportunities of DNA transformations are demonstrated, especially in biomedical applications that include diagnosis and therapy. Natural DNAs previously utilized and transformed into patterns are not found in nature due to lack of multiplexing, resulting in low sensitivity and high error frequency in multi-targeted therapeutics. More recently, new platforms have advanced the diagnostic ability and therapeutic efficacy of DNA in biomedicine. There is confidence that DNA will play a strong role in next-generation clinical technology and can be used in multifaceted applications.
RESUMEN
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.
RESUMEN
Using DNA nanostructures as templates to synthesize shape-controlled polydopamine (PDA) is a promising strategy to realize the fabrication of exquisite PDA nanomaterials. However, previous studies using small DNA tiles as templates could only afford very simple structures such as lines and crosses due to the limited space on the template and the relatively low resolution of the PDA nanopatterns. Therefore, the best resolution of the PDA nanostructures that can be achieved by this technique is carefully investigated. And by connecting several DNA tiles together, larger DNA templates are built up and achieve the synthesis of complicated digital nanopatterned PDA structures.
Asunto(s)
Nanoestructuras , Polímeros , ADN , IndolesRESUMEN
PURPOSE: To assess the accuracy of biometric parameters measured by anterior segment optical coherence tomography (AS-OCT) and partial coherence interferometry (PCI) in prediction of effective lens position (ELP) compared with previous formulas in PACG patients. METHODS: 121 PACG eyes were randomly divided into training set (85 eyes) and validation set (36 eyes) with same procedure including AS-OCT, PCI, phacoemulsification and IOL implantation surgery. Preoperative anterior chamber depth (pre-ACD), scleral spur depth (SSD), scleral spur width (SSW), lens vault (LV) and cornea thickness (CT) were measured from AS-OCT image. Axial length (AL) and corneal power (K) were measured by PCI. All the 7 parameters were analyzed by multiple linear regression in training set and a statistic regression formula was developed. In validation set, one-way ANOVA was applied to compare the new regression formula with Sanders-Retzlaff-Kraff theoretic (SRK/T), Holladay 1, Haigis, and a regression formula developed in previous study. RESULTS: The coefficient of determination (R2) of different parameter combinations are 0.19 (pre-ACD, AL), 0.25 (AL, K) and 0.49 (SSD, AL, SSW) in training set. In validation set, the correlation between predicted and measured ELP are: new formula (R2 = 0.50, P = 0.9947) Holladay 1 (R2 = 0.12, P < 0.0001), SRK/T (R2 = 0.11, P < 0.0001) and Haigis (R2 = 0.06, P < 0.0001). CONCLUSION: Among 7 tested parameters, pre-ACD contribute little in ELP prediction. Formula consist of SSD, AL and SSW showed better accuracy than other formulas tested.
Asunto(s)
Lentes Intraoculares , Tomografía de Coherencia Óptica , Biometría , China , Córnea , Humanos , Óptica y FotónicaRESUMEN
Atherosclerosis and related cardiovascular diseases represent the greatest threats to human health, worldwide. Previous animal studies showed that selenium nanoparticles (SeNPs) and Na2SeO3 might have anti-atherosclerotic activity, but the underlying mechanisms are poorly elucidated. This study compared the anti-atherosclerotic activity of SeNPs stabilized with chitosan (CS-SeNPs) and Na2SeO3 and the related mechanism in a high-fat-diet-fed apolipoprotein E-deficient mouse model of atherosclerosis. The results showed that oral administration of both CS-SeNPs and Na2SeO3 (40 µg Se/kg/day) for 10 weeks significantly reduced atherosclerotic lesions in mouse aortae. Mechanistically, CS-SeNPs and Na2SeO3 not only alleviated vascular endothelial dysfunction, as evidenced by the increase of serum nitric oxide level and the decrease of aortic adhesion molecule expression, but also vascular inflammation, as evidenced by the decrease of macrophage recruitment as well as the expression of proinflammatory molecules. Importantly, these results were replicated within in-vivo experiments on the cultured human endothelial cell line EA.hy926. Overall, CS-SeNPs had a comparable effect with Na2SeO3 but might have more potential in atherosclerosis prevention due to its lower toxicity. Together, these results provide more insights into the mechanisms of selenium against atherosclerosis and further highlight the potential of selenium supplementation as a therapeutic strategy for atherosclerosis.
Asunto(s)
Apolipoproteínas E/metabolismo , Aterosclerosis/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Nanopartículas/administración & dosificación , Selenio/farmacología , Selenito de Sodio/farmacología , Animales , Antioxidantes/metabolismo , Aterosclerosis/metabolismo , Línea Celular , Quitosano/química , Glutatión Peroxidasa/metabolismo , Humanos , Inflamación/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico/metabolismo , Estrés Oxidativo/efectos de los fármacosRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Biocatálisis , Ésteres/metabolismo , Compuestos de Manganeso/metabolismo , Óxidos/metabolismo , Oxidorreductasas/metabolismo , Pseudomonas putida/enzimología , Microbiología Industrial/métodosRESUMEN
Amphiphilic surface groups play an important role in many biological processes. The synthesis of amphiphilic polyphenylene dendrimer branches (dendrons), providing alternating hydrophilic and lipophilic surface groups and one reactive ethynyl group at the core is reported. The amphiphilic surface groups serve as biorecognition units that bind to the surface of adenovirusâ 5 (Ad5), which is a common vector in gene therapy. The Ad5/dendron complexes showed high gene transduction efficiencies in coxsackie-adenovirus receptor (CAR)-negative cells. Moreover, the dendrons offer incorporation of new functions at the dendron core by in situ post-modifications, even when bound to the Ad5 surface. Surfaces coated with these dendrons were analyzed for their blood-protein binding capacity, which is essential to predict their performance in the blood stream. A new platform for introducing bioactive groups to the Ad5 surface without chemically modifying the virus particles is provided.
Asunto(s)
Adenoviridae/química , Dendrímeros/química , Polímeros/química , Adenoviridae/fisiología , Animales , Proteínas Sanguíneas/química , Proteínas Sanguíneas/metabolismo , Células CHO , Supervivencia Celular/efectos de los fármacos , Cricetinae , Cricetulus , Reacción de Cicloadición , Dendrímeros/síntesis química , Dendrímeros/farmacología , Interacciones Hidrofóbicas e Hidrofílicas , Liposomas/química , Unión Proteica , Propiedades de SuperficieRESUMEN
Nanotheranostics, combining diagnostics and therapy, has the potential to revolutionize treatment of neurological disorders. But one of the major obstacles for treating central nervous system diseases is the blood-brain barrier (BBB) preventing systemic delivery of drugs and optical probes into the brain. To overcome these limitations, nanodiamonds (NDs) are investigated in this study as they are a powerful sensing and imaging platform for various biological applications and possess outstanding stable far-red fluorescence, do not photobleach, and are highly biocompatible. Herein, fluorescent NDs encapsulated by a customized human serum albumin-based biopolymer (polyethylene glycol) coating (dcHSA-PEG) are taken up by target brain cells. In vitro BBB models reveal transcytosis and an additional direct cell-cell transport via tunneling nanotubes. Systemic application of dcHSA-NDs confirms their ability to cross the BBB in a mouse model. Tracking of dcHSA-NDs is possible at the single cell level and reveals their uptake into neurons and astrocytes in vivo. This study shows for the first time systemic NDs brain delivery and suggests transport mechanisms across the BBB and direct cell-cell transport. Fluorescent NDs are envisioned as traceable transporters for in vivo brain imaging, sensing, and drug delivery.