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.

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.

Functional DNA-Polymer Conjugates.

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.

Defined positive charge patterns created on DNA nanostructures determine cellular uptake efficiency.

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.

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.

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.

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.

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.

DNA Transformations for Diagnosis and Therapy.

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.

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.

Digital Numbers Constructed by Fine Patterned Polydopamine on DNA Templates.

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.

Prediction of effective Lens position using anterior segment optical coherence tomography in Chinese subjects with angle closure.

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.

Comparison of Selenium Nanoparticles and Sodium Selenite on the Alleviation of Early Atherosclerosis by Inhibiting Endothelial Dysfunction and Inflammation in Apolipoprotein E-Deficient Mice.

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.

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.

Amphiphilic Polyphenylene Dendron Conjugates for Surface Remodeling of Adenovirus†5.

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 adenovirus 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.

Unraveling In Vivo Brain Transport of Protein-Coated Fluorescent Nanodiamonds.

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.

Hemin-catalyzed biomimetic oxidative phenol-indole [3 + 2] reactions in aqueous media.

A hemin/H2O2 catalytic system for oxidative phenol-indole [3 + 2] coupling in aqueous solution has been developed, enabling benign synthesis of valuable benzofuroindolines under sustainable conditions. Mechanistic studies revealed the dual role of iron porphyrin responsible for both phenol oxidation and Lewis acid activation, which differs from the well-explored chemistry of hemin in carbene and nitrene insertion reactions. A preliminary experiment with cytochrome c showed that the turnover of iron porphyrin was amenable for a macromolecular setting with remarkable efficiency (ca. 13 300 TON).

Fabrication of Defined Polydopamine Nanostructures by DNA Origami-Templated Polymerization.

A versatile, bottom-up approach allows the controlled fabrication of polydopamine (PD) nanostructures on DNA origami. PD is a biosynthetic polymer that has been investigated as an adhesive and promising surface coating material. However, the control of dopamine polymerization is challenged by the multistage-mediated reaction mechanism and diverse chemical structures in PD. DNA origami decorated with multiple horseradish peroxidase-mimicking DNAzyme motifs was used to control the shape and size of PD formation with nanometer resolution. These fabricated PD nanostructures can serve as "supramolecular glue" for controlling DNA origami conformations. Facile liberation of the PD nanostructures from the DNA origami templates has been achieved in acidic medium. This presented DNA origami-controlled polymerization of a highly crosslinked polymer provides a unique access towards anisotropic PD architectures with distinct shapes that were retained even in the absence of the DNA origami template.