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A Mn-based nanozyme, Mn-uNF/Si, with excellent alkali phosphatase-like activity was designed by in-situ growth of ultrathin Mn-MOF on the surface of silicon spheres, and implemented as an effective solid Lewis-Brønsted acid catalyst for broad-spectrum dephosphorylation. H218O-mediated GC-MS studies confirmed the cleavage sites and the involvement of H2O in the new bonds. DRIFT NH3-IR and in-situ ATR-FTIR confirmed the coexistence of Lewis-Brønsted acid sites and the adjustment of adsorption configurations at the interfacial sites. In addition, a green transformation route of "turning waste into treasure" was proposed for the first time ("OPsâPO43-âP food additive") using edible C. reinhardtii as a transfer station. By alkali etching of Mn-uNF/Si, a nanozyme Mn-uNF with laccase-like activity was obtained. Intriguingly, glyphosate exhibits a laccase-like fingerprint-like response (+,-) of Mn-uNF, and a non-enzyme amplified sensor was thus designed, which shows a good linear relationship with Glyp in a wide range of 0.49-750 µM, with a low LOD of 0.61 µM, as well as high selectivity and anti-interference ability under the co-application of phosphate fertilizers and multiple pesticides. This work provides a controllable methodology for the design of bifunctional nanozymes, which sheds light on the highly efficient green transformation of OPs, and paves the way for the selective recognition and quantification of glyphosate. Mechanistically, we also provided deeper insights into the structure-activity relationship at the atomic scale.
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Técnicas Biosensibles , Glicina , Glifosato , Manganeso , Glicina/análogos & derivados , Glicina/química , Manganeso/química , Técnicas Biosensibles/métodos , Compuestos Organofosforados/química , Compuestos Organofosforados/análisis , Estructuras Metalorgánicas/química , Herbicidas/química , Herbicidas/análisis , Nanoestructuras/química , Tecnología Química Verde/métodos , Silicio/química , CatálisisRESUMEN
Brevitaxin was prepared in nine steps from commercially available carnosic acid. The construction of the 1,4-benzodioxin moiety involved an unique stepwise ortho-quinone-engaged [4+2] cycloaddition. Two strategic stages were employed to prepare the highly unsaturated cycloaddition precursor 3: (1) synthesizing the diene moiety (C1-C2 and C10-C20 double bonds) by regioselective ortho-quinone tautomerization, and (2) installing four sp2-hybridized carbon atoms (C3, C5, C6 and C7) in one step using a SeO2-promoted chemo- and regioselective oxidation reaction.
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A series of amides, including α-bromo hydroxamates, N-alkoxyamides, and N-aryloxyamides, were subjected to phosphine-catalyzed ring-opening O-selective addition with cyclopropenones, producing various special α,ß-unsaturated esters containing oxime ether motif, in moderate to excellent yields, with high regioselectivity, and exclusive O-selectivity. The methodology is highly atom-economical, with simple operation procedures, and compatible with a wide substrate scope (more than 44 examples).
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An efficient phosphine-catalyzed dearomative [3+2] annulation of 4-nitroisoxazoles with allenoates or Morita-Baylis-Hillman carbonates has been established for the convenient synthesis of bicyclic isoxazoline derivatives. This reaction approach showed a broad substrate scope, high functional group compatibility, and excellent regioselectivity and diastereoselectivity. Furthermore, the success at the gram-scale and synthetic applications of the obtained compound 3a demonstrate the great potential of this methodology for practical applications in organic synthesis.
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Globins, such as myoglobin (Mb) and neuroglobin (Ngb), are ideal protein scaffolds for the design of functional metalloenzymes. To date, numerous approaches have been developed for enzyme design. This review presents a summary of the progress made in the design of functional metalloenzymes based on Mb and Ngb, with a focus on the exploitation of covalent interactions, including coordination bonds and covalent modifications. These include the construction of a metal-binding site, the incorporation of a non-native metal cofactor, the formation of Cys/Tyr-heme covalent links, and the design of disulfide bonds, as well as other Cys-covalent modifications. As exemplified by recent studies from our group and others, the designed metalloenzymes have potential applications in biocatalysis and bioconversions. Furthermore, we discuss the current trends in the design of functional metalloenzymes and highlight the importance of covalent interactions in the design of functional metalloenzymes.
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Globinas , Mioglobina , Proteínas del Tejido Nervioso , Neuroglobina , Neuroglobina/metabolismo , Neuroglobina/química , Mioglobina/química , Mioglobina/metabolismo , Globinas/química , Globinas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/química , Humanos , Animales , Hemo/química , Hemo/metabolismo , Sitios de Unión , Metaloproteínas/química , Metaloproteínas/metabolismo , Ingeniería de Proteínas/métodosRESUMEN
The development of highly selective Janus Kinase 1 (JAK1) inhibitors is crucial for improving efficacy and minimizing adverse effects in the clinical treatment of autoimmune diseases. In a prior study, we designed a series of C-5 4-pyrazol substituted pyrrolopyridine derivatives that demonstrated significant potency against JAK1, with a 10 â¼ 20-fold selectivity over Janus Kinase 2 (JAK2). Building on this foundation, we adopted orthogonal strategy by modifying the C-5 position with 3-pyrazol/4-pyrazol/3-pyrrol groups and tail with substituted benzyl groups on the pyrrolopyridine head to enhance both potency and selectivity. In this endeavor, we have identified several compounds that exhibit excellent potency and selectivity for JAK1. Notably, compounds 12b and 12e, which combined 4-pyrazol group at C-5 site and meta-substituted benzyl tails, displayed IC50 value with 2.4/2.2 nM and high 352-/253-fold selectivity for JAK1 over JAK2 in enzyme assays. Additionally, both compounds showed good JAK1-selective in Ba/F3-TEL-JAK1/2 cell-based assays. These findings mark a substantial improvement, as these compounds are 10-fold more potent and over 10-fold more selective than the best compound identified in our previous study. The noteworthy potency and selectivity properties of compounds 12b and 12e suggest their potential utility in furthering the development of drugs for autoimmune diseases.
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Enfermedades Autoinmunes , Compuestos Heterocíclicos , Humanos , Relación Estructura-Actividad , Janus Quinasa 1/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Janus Quinasa 2/metabolismoRESUMEN
Using myoglobin (Mb) as a model protein, we herein developed a facial approach to modifying the heme active site. A cavity was first generated in the heme distal site by F46â C mutation, and the thiol group of Cys46 was then used for covalently linked to exogenous ligands, 1H-1,2,4-triazole-3-thiol and 1-(4-hydroxyphenyl)-1H-pyrrole-2,5-dione. The engineered proteins, termed F46C-triazole Mb and F46C-phenol Mb, respectively, were characterized by X-ray crystallography, spectroscopic and stopped-flow kinetic studies. The results showed that both the heme coordination state and the protein function such as H2 O2 activation and peroxidase activity could be efficiently regulated, which suggests that this approach might be generally applied to the design of functional heme proteins.
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Hemo , Mioglobina , Mioglobina/química , Mioglobina/genética , Mioglobina/metabolismo , Dominio Catalítico , Hemo/química , Cinética , Conformación Proteica , Compuestos de SulfhidriloRESUMEN
A Cu-Fe bimetallic hydrogel (2-QF-CuFe-G) was constructed through a simple method. The 2-QF-CuFe-G metallohydrogel possesses excellent peroxidase-like activity to catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H2O2. The catalytic mechanism was confirmed by the addition of â¢OH radical scavenger isopropyl alcohol (IPA), tert-butyl alcohol (TBA) and ËOH trapping agent terephthalic acid (TA). Remarkably, the resultant blue ox-TMB system can be used to selectively and sensitively detect ascorbic acid (AA) with an LOD of 0.93 µM in the range of 4-36 µM through the colorimetric method. Moreover, the assay based on the 2-QF-CuFe-G metallohydrogel can be successfully applied to detect AA in fresh fruits.
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Significant efforts have been made in the design of artificial metalloenzymes. Myoglobin (Mb), an O2 carrier, has been engineered to exhibit different functions. Herein, we applied a series of engineered Mb mutants with peroxidase activity for biosynthesis of clofazimine (CFZ), a potential drug with a broad-spectrum antiviral activity, by integration with chemical synthesis. Two of those mutants, F43Y Mb and F43Y/T67R Mb, have been shown to efficiently catalyze the oxidative coupling of 2-N-(4-chlorophenyl) benzene-1,2-diamine (N-4-CPBDA) in the presence of H2O2, with 97% yields. The overall catalytic efficiency (kcat/Km) is 46-fold and 82-fold higher than that of WT Mb, respectively. By further combination of this reaction with chemical synthesis, the production of CFZ was accomplished with an isolated yield of 72%. These results showed that engineered Mbs containing the Tyr-heme cross-link (F43Y Mb and F43Y/T67R Mb) exhibit enhanced activity in the oxidative coupling reaction. This study also indicates that the combination of biocatalysis and chemical synthesis avoids the need for the separation of intermediate products, which offers a convenient approach for the total synthesis of the biological compound CFZ.
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Clofazimina , Mioglobina , Mioglobina/genética , Mioglobina/química , Peróxido de Hidrógeno/química , Modelos Moleculares , Hemo/químicaRESUMEN
We report herein a general and effective system achieving cyclization of ß-trifluoromethyl enones with amidines in the presence of 1,3-diiodo-5,5-dimethylhydantoin (DIH), which affords a range of trifluoromethylated 2-imidazolines in synthetically useful yields with good diastereoselectivities (up to 95% yield, up to 98:2 dr) and good functional group tolerance. Furthermore, the one-pot synthesis of trifluoromethylated imidazoles via sequential cyclization and oxidation is demonstrated. More significantly, the reaction mechanism was verified by ESI-MS studies of possible intermediates, and a reasonable reaction mechanism was proposed.
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Nonenzymatic glycation and the subsequent accumulation of advanced glycation end-products (AGEs) in proteins are factors underlying long-term pathogenesis in diabetes. The study of protein glycation is crucial for elucidating their relationship with diabetes mellitus and related disorders. This study explores the interaction between d-ribose and human myoglobin (HMb), as well as the protective effect of thymoquinone (TQ) on glycation. A time-dependent in-vitro glycation study was performed to investigate the mechanism of d-ribose-induced structural interference of HMb in the absence and presence of TQ. Spectroscopic and proteomic analysis indicated that the presence of TQ significantly reduced the total amount of AGEs while maintaining structural characteristics of HMb. 14 glycated sites on HMb were further identified via liquid chromatography-tandem mass spectrometry (LC-MS/MS) after incubation with d-ribose for 12 h, predominantly interacting with lysine residues. TQ was found to disrupt this interaction, reducing the glycated sites from 14 to 12 sites and the percentage of glycated peptides from 26.50 % to 12.97 %. Additionally, there was a significant decrease in the degree of glycation at the same sites. In summary, our findings suggest that TQ has the potential to act as an anti-glycation agent and provide a comprehensive understanding underlying the inhibition mechanism of glycation.
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Diabetes Mellitus , Reacción de Maillard , Humanos , Productos Finales de Glicación Avanzada/metabolismo , Glicosilación , Ribosa/química , Mioglobina/metabolismo , Cromatografía Liquida , Proteómica , Espectrometría de Masas en TándemRESUMEN
It is desired to design and construct more efficient enzymes with better performance to catalyze carbene N-H insertions for the synthesis of bioactive molecules. To this end, we exploited and designed a series of human neuroglobin (Ngb) mutants. As shown in this study, a double mutant, A15C/H64G Ngb, with an additional disulfide bond and a modified heme active site, exhibited yields up to >99% and total turnover numbers up to 33000 in catalyzing the carbene N-H insertions for aromatic amine derivatives, including those with a large size such as 1-aminopyrene. Moreover, for o-phenylenediamine derivatives, they underwent two cycles of N-H insertions, followed by cyclization to form quinoxalinones, as confirmed by the X-ray crystal structures. This study suggests that Ngb can be designed into a functional carbene transferase for efficiently catalyzing carbene N-H insertion reactions with a range of substrates. It also represents the first example of the formation of quinoxalinones catalyzed by an engineered heme enzyme.
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Metal complexes, particularly copper(II) complexes, are often used as anticancer drugs due to their ability to generate reactive oxygen species (ROS) in cells. Four copper(II) complexes have been designed based on ligands for triplet pyridine derivatives (complexes 1-4), and their structures have been determined using X-ray single crystal analysis. The interactions of these complexes with calf thymus DNA (CT-DNA) have been investigated using various techniques, including UV-vis absorption, viscosity measurements, and circular dichroism spectroscopy. The results indicate that complexes 1-4 strongly interact with DNA through partial intercalations. Further investigation using agarose gel electrophoresis shows that all four complexes can cleave pBR322 DNA in the presence of ascorbic acid as a reducing agent, and the DNA cleavage mechanism is through the generation of singlet oxygen (1O2). In vitro anticancer activities of these complexes have been evaluated using A549, MDA-MB-231, HeLa, and HepG2 cells. The calculated IC50 values indicate significant efficacy against cancer cells. Additionally, AO/EB staining assays reveal that these complexes induce cell apoptosis in HeLa cell line.
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Antineoplásicos , Complejos de Coordinación , Humanos , Células HeLa , Cobre/química , Ligandos , Complejos de Coordinación/farmacología , Complejos de Coordinación/química , Antineoplásicos/farmacología , Antineoplásicos/química , ADN/química , División del ADN , Cristalografía por Rayos XRESUMEN
The construction of smart materials, especially white light emitting (WLE) hydrogels with multi-stimuli responsive properties, has received widespread attention from researchers. In this study, a WLE hydrogel was obtained by the in situ doping of Eu3+ and Tb3+ into a blue emission low molecular weight gelator (MPF). Remarkably, the prepared WLE hydrogel possessed excellent stimuli responsiveness to pH, temperature and chemicals, and could be used as a soft thermometer and a selective sensor for Cu2+. The correlated color temperature of the WLE hydrogel was calculated to be 5063 K, suggesting a potential application in cool white light. Moreover, a series of metallohydrogels with different colors were obtained by modulating the ratio of MPF, Eu3+ and Tb3+ or changing the excitation wavelength, which was an excellent candidate to construct soft materials of a full-color system. Additionally, the WLE hydrogel could be used for constructing anti-counterfeiting materials. Therefore, this study provides a new approach for preparing smart WLE hydrogels with multiple functions.
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The sequence and structure of human cytochrome c (hCyt c) exhibit evolutionary conservations, with only a limited number of naturally occurring mutations in humans. Herein, we investigated the effects of the naturally occurring S47F/A mutations on the structure and function of hCyt c in the oxidized form. Although the naturally occurring S47F/A mutations did not largely alter the protein structure, the S47F and S47A variants exhibited a small fraction of high-spin species. Kinetic studies showed that the peroxidase activity of the variants was enhanced by â¼2.5-fold under neutral pH conditions, as well as for the rate in reaction with H2O2, when compared to those of wild-type hCyt c. In addition, we evaluated the interaction between hCyt c and human neuroglobin (hNgb) by isothermal titration calorimetry (ITC) studies, which revealed that the binding constant was reduced by â¼8-fold as result of the mutation of the hydrophilic Ser to the hydrophobic Phe/Ala. These findings provide valuable insights into the role of Ser47 in Ω-loop C in sustaining the structure and function of hCyt c.
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Citocromos c , Peróxido de Hidrógeno , Humanos , Citocromos c/química , Cinética , MutaciónRESUMEN
Myoglobin (Mb) was found to undergo self-oxidation when a cysteine residue was engineered at position 67 in the heme distal site. Both the X-ray crystal structure and mass spectrum confirmed the formation of a sulfinic acid (Cys-SO2H). Moreover, the self-oxidation could be controlled during protein purification to yield the unmodified form (T67C Mb). Importantly, both T67C Mb and T67C Mb (Cys-SO2H) were able to be labeled by chemicals, which provided useful platforms to generate artificial proteins.
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Developing selective inhibitors for Janus kinase 1 (JAK1) is a significant focus for improving the efficacy and alleviating the adverse effects in treating immune-inflammatory diseases. Herein, we report the discovery of a series of C-5 pyrazole-modified pyrrolopyrimidine derivatives as JAK1-selective inhibitors. The potential hydrogen bond between the pyrazole group and E966 in JAK1 is the key point that enhances JAK1 selectivity. These compounds exhibit 10- to 20-fold JAK1 selectivity over JAK2 in enzyme assays. Compound 12b also exhibits excellent JAK1 selectivity in Ba/F3-TEL-JAK cellular assays. Metabolism studies and the results of the hair growth model in mice indicate that compound 12b may be a viable lead compound for the development of highly JAK1-selective inhibitors for immune and inflammatory diseases.
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Inhibidores de las Cinasas Janus , Pirazoles , Ratones , Animales , Relación Estructura-Actividad , Janus Quinasa 1 , Pirazoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de las Cinasas Janus/farmacología , Janus Quinasa 2 , Janus Quinasa 3RESUMEN
Natural and artificial nucleases have extensive applications in biotechnology and biomedicine. The exploration of protein with potential DNA cleavage activity also inspires the design of artificial nuclease and helps to understand the physiological process of DNA damage. In this study, we engineered four human cytochrome c (Cyt c) mutants (N52S, N52A, I81N, and I81D Cyt c), which showed enhanced DNA cleavage activity and degradation in comparison with WT Cyt c, especially under acidic conditions. The mechanism assays revealed that the superoxide (O2â¢-) plays an important role in the nuclease reaction. The kinetic assays showed that the peroxidase activity of the I81D Cyt c mutant enhanced up to 9-fold at pH 5. This study suggests that the mutations of Ile81 and Asn52 in Ω-loop C/D are critical for the nuclease activity of Cyt c, which may have physiological significance in DNA damage and potential applications in biomedicine.
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Citocromos c , Superóxidos , Humanos , Citocromos c/genética , Citocromos c/metabolismo , Oxidación-Reducción , Mutación , Estrés OxidativoRESUMEN
A facile and dual fluorescent chemosensor (named 7-IDF) based on a phenylalanine derivative with an indole group was designed and synthesized. 7-IDF can selectively and sensitively detect Zn2+ via obvious fluorescence enhancement in an aqueous solution. Remarkably, the 7-IDF-Zn complex with blue luminescence has higher selectivity toward cysteine (Cys) and histidine (His) than for other amino acids. Intriguingly, 7-IDF can also be used as an excellent probe to detect Zn2+ in real water samples. Moreover, 7-IDF and 7-IDF-Zn possess excellent biocompatibility and cell permeability, and 7-IDF can consecutively detect Zn2+ and Cys/His in Hela cells through fluorescence imaging experiments. This study suggests that the phenylalanine-based chemosensor possesses great potential applications for the sequential detection of Zn2+ and Cys/His in biosystems.
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Cisteína , Colorantes Fluorescentes , Humanos , Colorantes Fluorescentes/química , Cisteína/química , Células HeLa , Histidina , Fenilalanina , Espectrometría de Fluorescencia , ZincRESUMEN
Enzyme-based biocatalytic treatment has been known as an effective measure to biologically degrade organic pollutants. Advantageously, enzymes could be immobilized on solid supports, and such fact enables reuse/prolong the enzymatic capability. It could be of great importance to functionalize a support material for enhancing the immobilization efficiency/stability of enzymes. As such, this study laid great emphasis on covalent bonding to immobilize horseradish peroxidase (HRP) on a functionalized rice straw biochar with glutaraldehyde (GA) as a crosslinker. Biochar was pretreated by the electrochemical method and the acid treatment respectively to enrich the oxygen-containing functional groups. These led to the enhanced immobilizing ability of biochar. The HRP immobilized on the electrochemically-functionalized biochar (HRP-EBC) showed three times as much enzyme activity as the HRP directly adsorbed onto biochar. The HRP immobilized on the acid-functionalized biochar (HRP-ABC) showed activity similar to that of HRP-EBC. It was concluded that both the (acid/electrochemical) pretreatments are effective to enhance enzyme immobilization. Nevertheless, the electrochemical functionalized method of biochar is chemical oxidant-free, and one important lesson from a series of tests was that the pretreatment of biochar through the electrochemical method could be more environmentally benign. Moreover, employing HRP-EBC could be beneficial from a perspective of a real environmental practice considering its higher pH, thermal stability, and good reusability. 80% of phenol was degraded in 1 h in the presence of HRP-EBC when pH was 7.0 and a ratio of H2O2 to phenol was 1:1.5.