RESUMEN
Recombinant human interleukin-2 (rhIL-2) represents one of the most difficult-to-produce cytokines in E. coli due to its extreme hydrophobicity and high tendency to formation of inclusion bodies. Refolding of rhIL-2 inclusion bodies always represents cumbersome downstream processes and low production efficiency. Herein, we disclosed a fusion strategy for efficiently soluble expression and facile production of rhIL-2 in E. coli Origami B (DE3) host. A two-tandem SUMO fusion partner (His-2SUMO) with a unique SUMO protease cleavage site at C-terminus was devised to fuse with the N-terminus of rhIL-2 and the fusion protein (His-2SUMO-rhIL-2) was almost completely expressed in a soluble from. The fusion partner could be efficiently removed by Ulp1 cleavage and the rhIL-2 was simply produced by a two-step Ni-NTA affinity chromatography with a considerable purity and whole recovery. The eventually obtained rhIL-2 was well-characterized and the results showed that the purified rhIL-2 exhibits a compact and ordered structure. Although the finally obtained rhIL-2 exists in a soluble aggregates form and the aggregation probably has been occurred during expression stage, the soluble rhIL-2 aggregates remain exhibit comparable bioactivity with the commercially available rhIL-2 drug formulation.
Asunto(s)
Escherichia coli , Interleucina-2 , Proteínas Recombinantes de Fusión , Solubilidad , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Interleucina-2/genética , Interleucina-2/biosíntesis , Interleucina-2/química , Interleucina-2/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Expresión Génica , Cromatografía de Afinidad , Clonación Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Cuerpos de Inclusión/química , Cuerpos de Inclusión/genética , Cuerpos de Inclusión/metabolismoRESUMEN
Developing fluorogenic probes for simultaneous live cell labeling of multiple targets is crucial for understanding complex cellular events. The emerging [4+1] cycloaddition between tetrazine and isonitriles holds promise as a bioorthogonal tool, yet existing tetrazine probes lack reactivity and fluorogenicity. Here, we present the development of a series of tetrazine-functionalized bioorthogonal probes. By incorporating pyrazole adducts into the fluorophore scaffolds, the post-reacted probes displayed remarkable fluorescence turn-on ratios, up to 3184-fold. Moreover, these modifications are generalizable to various fluorophores, enabling a broad emission range from 473 to 659â nm. Quantum chemical calculations further elucidate the turn-on mechanisms. These probes enable the simultaneous labeling of multiple targets in live cells, without the need for a washing step. Consequently, our findings pave the way for advanced multiplex imaging and detection techniques for cellular studies.
Asunto(s)
Colorantes Fluorescentes , Imagen Óptica , Línea Celular Tumoral , Reacción de Cicloadición , Imagen Óptica/métodosRESUMEN
Over the past decade, bicyclo[1.1.1]pentane (BCP) motifs have come to the fore as valuable pharmaceutical bioisosteres of para-disubstituted benzenes. However, the limited approaches and requisite multistep syntheses of useful BCP building blocks are hampering early discovery research in medicinal chemistry. Herein we report the development of a modular strategy for the divergent preparation of functionalized BCP alkylamines. In this process, a general method to introduce fluoroalkyl groups to BCP scaffolds using readily available and easy-to-handle fluoroalkyl sulfinate salts was also developed. Moreover, this strategy can also be extended to S-centered radicals for incorporation of sulfones and thioethers into the BCP core. Overall, this multicomponent strategy enables rapid construction of BCP-type bioisosteres for applications in drug discovery.
RESUMEN
The development of synthetic strategies for the preparation of bioisosteric compounds is a demanding undertaking in medicinal chemistry. Numerous strategies have been developed for the synthesis of bicyclo[1.1.1]pentanes (BCPs), bridge-substituted BCPs, and bicyclo[2.1.1]hexanes. However, progress on the synthesis of bicyclo[3.1.1]heptanes, which serve as meta-substituted arene bioisosteres, has not been previously explored. Herein, we disclose the first photoinduced [3σ + 2σ] cycloaddition for the synthesis of trisubstituted bicyclo[3.1.1]heptanes using bicyclo[1.1.0]butanes and cyclopropylamines. This transformation not only uses mild and operationally simple conditions but also provides unique meta-substituted arene bioisosteres. The applicability of this method is showcased by simple derivatization reactions.
Asunto(s)
Compuestos Bicíclicos con Puentes , Heptanos , Compuestos Bicíclicos con Puentes/química , Heptanos/química , Reacción de Cicloadición , Hexanos/química , ButanosRESUMEN
A copper-mediated coupling reaction between ynamides and diazo-compounds to produce N-allenamides is reported for the first time. This method enables facile and rapid access to terminal N-allenamides by using commercially available TMS-diazomethane with wide functional group compatibility on the nitrogen. Furthermore, the ubiquity of molecules containing a fluorine moiety in medicine, in agricultural, and material science requires the continuous search of new building blocks, including this unique surrogate. The CuI/diazo protocol was successfully applied to the synthesis of fluorine-substituted N-allenamides. DFT calculations provided insights in the mechanism involved.
Asunto(s)
Cobre , Flúor , Compuestos Azo , CatálisisRESUMEN
Human interleukin-11 (IL-11) is considered as a difficult-to-express protein in prokaryotic expression systems because of its low expression level and high tendency to form inclusion bodies. The current source of recombinant human IL-11 (rhIL-11) for therapeutic use is mainly obtained from a fusion protein synthesized by Escherichia coli, which requires an additional operation to cleave the fusion tag. Herein, we reported a strategy for the direct expression of tag-free rhIL-11 in E. coli. To explore the soluble expression of rhIL-11 without fusion tags in E. coli, we inserted the rhIL-11 gene into a pBV220 plasmid which is characterized by employing a temperature-sensitive pR/pL promoter to manipulate the transcription and translation of the gene of interest. As a result, the tag free rhIL-11 was efficiently expressed in the soluble form in E. coli. A two-step chromatography method, Capto Butyl-S combined with Capto Q, was developed to efficiently purify the tag-free rhIL-11 from the supernatant of the cell lysate. The resultant rhIL-11 showed a compact and highly ordered structure, as validated by circular dichroism and intrinsic fluorescence emission spectra. Additionally, the biological activity of the purified rhIL-11 was evaluated by TF-1 cell proliferation experiments and the results demonstrated that the E. coli expressed rhIL-11 is biologically active.
Asunto(s)
Escherichia coli , Interleucina-11 , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Humanos , Cuerpos de Inclusión/metabolismo , Interleucina-11/genética , Interleucina-11/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
N-Allenamides, substituted by an ester at the γ-position, were obtained through addition of terminal ynamides with ethyl diazoacetate under copper catalysis for the first time. Regio- and stereoselective hydroamination of those activated N-allenamides provided exclusively E-configured captodative enamimes through a one-pot anti-Michael addition. Numerous ynamides as well as various secondary amines were adapted in this process.
Asunto(s)
Aminas , Cobre , Catálisis , EstereoisomerismoRESUMEN
Increased metabolism is one of the main causes for evolution of herbicide resistance in weeds, a major challenge for sustainable food production. The molecular drivers of this evolution are poorly understood. We tested here the hypothesis that a suitable context for the emergence of herbicide resistance could be provided by plant enzymes with high innate promiscuity with regard to their natural substrates. A selection of yeast-expressed plant cytochrome P450 enzymes with well documented narrow to broad promiscuity when metabolizing natural substrates was tested for herbicide metabolism competence. The positive candidate was assayed for capacity to confer herbicide tolerance in Arabidopsis thaliana. Our data demonstrate that Arabidopsis thaliana CYP706A3, with the most promiscuous activity on monoterpenes and sesquiterpenes for flower defence, can also oxidize plant microtubule assembly inhibitors, dinitroanilines. Ectopic overexpression of CYP706A3 confers dinitroaniline resistance. We show, in addition, that the capacity to metabolize dinitroanilines is shared by other members of the CYP706 family from plants as diverse as eucalyptus and cedar. Supported by three-dimensional (3D) modelling of CYP706A3, the properties of enzyme active site and substrate access channel are discussed together with the shared physicochemical properties of the natural and exogenous substrates to explain herbicide metabolism.
Asunto(s)
Arabidopsis , Herbicidas , Arabidopsis/genética , Sistema Enzimático del Citocromo P-450/genética , Resistencia a los Herbicidas/genética , Herbicidas/farmacología , Malezas/genéticaRESUMEN
In research on hybrid quantum networks, visible or near-infrared frequency conversion has been realized. However, technical limitations mean that there have been few studies involving the ultraviolet band, and unfortunately the wavelengths of the rare-earth or alkaline-earth metal atoms or ions that are used widely in research on quantum information are often in the UV band. Therefore, frequency conversion of the ultraviolet band is very important. In this paper, we demonstrate a quantum frequency conversion between ultraviolet and visible wavelengths by fabricating waveguides in a period-poled MgO:LiTaO3 crystal with a laser writing system, which will be used to connect the wavelength of the dipole transition of 171Yb+ at 369.5 nm and the absorption wavelength of Eu3+ at 580 nm in a solid-state quantum memory system. An external conversion efficiency of 0.85% and a signal-to-noise ratio of greater than 500 are realized with a pumping power of 3.28 W at 1018 nm. Furthermore, we complete frequency conversion of the classical polarization state by means of a symmetric optical setup based on the fabricated waveguide, and the process fidelity of the conversion is (96.13 ± 0.021)%. This converter paves the way for constructing a hybrid quantum network and realizing a quantum router in the ultraviolet band in the future.
RESUMEN
We demonstrate a laser frequency stabilization method with large tuning range to stabilize a UV laser by installing piezoelectric ceramic actuators into a Fabry-Pérot cavity with an ultra-low expansion spacer. To suppress piezoelectric drift, a two-layer symmetrical structure is adopted for the piezoelectric actuator, and a 14.7 GHz tuning range is achieved. The short-term drift of the piezoelectric ceramics caused by temperature and creep is eliminated, and the long-term drift is 0.268 MHz/h when the Fabry-Pérot cavity is sealed in a chamber without a vacuum environment. The long-term frequency drift is mainly caused by stress release and is eliminated by compensating the cavity voltage with an open loop. Without the need for an external reference or a vacuum environment, the laser frequency stabilization system is greatly simplified, and it can be extended to wavelengths ranging from ultraviolet to infrared. Owing to its simplicity, stability, and large tuning range, it is applicable in cold atom and trapped ion experiments.
RESUMEN
In cold atomic systems, fast and high-resolution microscopy of individual atoms is crucial, since it can provide direct information on the dynamics and correlations of the system. Here, we demonstrate nanosecond-scale two-dimensional stroboscopic pictures of a single trapped ion beyond the optical diffraction limit, by combining the main idea of ground-state depletion microscopy with quantum-state transition control in cold atoms. We achieve a spatial resolution up to 175 nm using a NA=0.1 objective in the experiment, which represents a more than tenfold improvement compared with direct fluorescence imaging. To show the potential of this method, we apply it to observe the secular motion of the trapped ion; we demonstrate a temporal resolution up to 50 ns with a displacement detection sensitivity of 10 nm. Our method provides a powerful tool for probing particle positions, momenta, and correlations, as well as their dynamics in cold atomic systems.
RESUMEN
Breast cancer is the second leading cause of cancer-related deaths in women. Ligand-modified liposomes are used for breast tumor-specific drug delivery to improve the efficacy and reduce the side effects of chemotherapy; however, only a few liposomes with high targeting efficiency have been developed because the mono-targeting, ligand-modified liposomes are generally unable to deliver an adequate therapeutic dose. In this study, we designed biotin-glucose branched ligand-modified, dual-targeting liposomes (Bio-Glu-Lip) and evaluated their potential as a targeted chemotherapy delivery system in vitro and in vivo. When compared with the non-targeting liposome (Lip), Bio-Lip, and Glu-Lip, Bio-Glu-Lip had the highest cell uptake in 4T1 cells (3.00-fold, 1.60-fold, and 1.95-fold higher, respectively) and in MCF-7 cells (2.63-fold, 1.63-fold, and 1.85-fold higher, respectively). The subsequent cytotoxicity and in vivo assays further supported the dual-targeting liposome is a promising drug delivery carrier for the treatment of breast cancer.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Biotina/química , Neoplasias de la Mama/tratamiento farmacológico , Sistemas de Liberación de Medicamentos , Glucosa/química , Paclitaxel/farmacología , Animales , Antineoplásicos Fitogénicos/química , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Portadores de Fármacos/síntesis química , Portadores de Fármacos/química , Femenino , Humanos , Ligandos , Liposomas/química , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Ratones , Estructura Molecular , Paclitaxel/química , Relación Estructura-ActividadRESUMEN
The success of a structure-based drug is highly dependent on a known binding pose of the protein-ligand system. However, this is not always available. In this study, we set out to explore the applicability of the popular and easy-to-use MD-based MM/GBSA method to determine the binding poses of known FGFR inhibitors. It was found that MM/GBSA combined with 100 ns of MD simulation significantly improved the success rate of docking methods from 30-40% to 70%. This work demonstrates a way that the MM/GBSA method can be more accurate than it is in ligand ranking, filling a gap in structure-based drug discovery when the binding pose is unknown.
Asunto(s)
Descubrimiento de Drogas/métodos , Simulación de Dinámica Molecular , Receptores de Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Simulación del Acoplamiento Molecular , Unión Proteica , Receptores de Factores de Crecimiento de Fibroblastos/metabolismoRESUMEN
A concise, efficient one-pot synthesis of functionalized chromeno[4,3-b]pyridine derivatives via a three-component reaction of 4-oxo-4H-chromene-3-carbaldehydes, malononitrile or cyanoacetates, and aromatic amines under catalyst-free conditions in an environmentally friendly medium (ethanol-water, 3:1 v/v) is described. This synthesis involves a group-assisted purification process, which avoids traditional recrystallization and chromatographic purification methods.
Asunto(s)
Piridinas/química , Piridinas/síntesis química , Técnicas de Química Sintética , Etanol/química , Tecnología Química Verde , Agua/químicaRESUMEN
With the aim of broadening the versatility of lentiviral vectors as a tool in nucleic acid research, we expanded the genetic code in the propagation of lentiviral vectors for site-specific incorporation of chemical moieties with unique properties. Through systematic exploration of the structure-function relationship of lentiviral VSVg envelope by site-specific mutagenesis and incorporation of residues displaying azide- and diazirine-moieties, the modifiable sites on the vector surface were identified, with most at the PH domain that neither affects the expression of envelope protein nor propagation or infectivity of the progeny virus. Furthermore, via the incorporation of such chemical moieties, a variety of fluorescence probes, ligands, PEG and other functional molecules are conjugated, orthogonally and stoichiometrically, to the lentiviral vector. Using this methodology, a facile platform is established that is useful for tracking virus movement, targeting gene delivery and detecting virus-host interactions. This study may provide a new direction for rational design of lentiviral vectors, with significant impact on both basic research and therapeutic applications.
Asunto(s)
Código Genético , Vectores Genéticos , Lentivirus/genética , Aminoácidos/química , Azidas/química , Línea Celular , Diazometano/química , Marcación de Gen , Vectores Genéticos/química , Humanos , Glicoproteínas de Membrana/química , Mutagénesis Sitio-Dirigida , Polietilenglicoles/química , Transfección , Proteínas del Envoltorio Viral/químicaRESUMEN
Pd-catalyzed regioselective coupling of γ-C(sp(3))-H bonds of oxalyl amide-protected amino acids with heteroaryl and aryl iodides is reported. A wide variety of iodides are tolerated, giving the corresponding products in moderate to good yields. Various oxalyl amide-protected amino acids were compatible in this C-H transformation, thus representing a practical method for constructing non-natural amino acid derivatives.
RESUMEN
A method for palladium-catalyzed oxalyl amide-directed arylation of α-unsubstituted aliphatic amines with aryl iodides has been developed. A wide variety of aryl iodides are tolerated in this transformation, affording various γ-arylpropylamine derivatives. Heterocyclic iodides can also be competent reagents in this γ-C(sp(3))-H bonds transformation.
RESUMEN
Strategies to regulate gene function frequently use small interfering RNAs (siRNAs) that can be made from their shRNA precursors via Dicer. However, when the duplex components of these siRNA effectors are expressed from their respective coding genes, the RNA interference (RNAi) activity is much reduced. Here, we explored the mechanisms of action of shRNA and siRNA and found the expressed siRNA, in contrast to short hairpin RNA (shRNA), exhibits strong strand antagonism, with the sense RNA negatively and unexpectedly regulating RNAi. Therefore, we altered the relative levels of strands of siRNA duplexes during their expression, increasing the level of the antisense component, reducing the level of the sense component, or both and, in this way we were able to enhance the potency of the siRNA. Such vector-delivered siRNA attacked its target effectively. These findings provide new insight into RNAi and, in particular, they demonstrate that strand antagonism is responsible for making siRNA far less potent than shRNA.
Asunto(s)
Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Transporte Activo de Núcleo Celular , Animales , Núcleo Celular/metabolismo , Células Cultivadas , Técnicas de Silenciamiento del Gen , Precursores del ARN/metabolismo , Transducción GenéticaRESUMEN
Low permeability is a key geological factor constraining the development of shale gas, and reservoir modification to improve its permeability is a prerequisite. Controlled shock wave fracturing can induce the formation of complex fractures in reservoirs and is expected to become an important means of reservoir modification. However, the mechanism of controlled shock wave fracturing in shale and the geological engineering control factors are unclear. Therefore, this article reveals the mechanism and effect of shock wave modification through small-scale experiments and large-scale numerical simulations. Results show that as the impact number increases, a significant increase in large fractures and fracture connectivity within the shale samples is observed, while the correlation between the geometric parameters of the fractures and the number of impacts is weak. High-energy input in the model will cause a larger range of damage to the rock, accompanied by a smaller attenuation index, indicating that the speed of energy attenuation plays a decisive role in rock damage. The influence of crustal stress is greater than the speed of energy attenuation, and higher crustal stress will inhibit the formation of fractures. A moderate increase in the number of controllable shock waves is beneficial for the fracturing effect; however, further increasing the loading number of controllable shock waves will weaken the strengthening effect of the fracturing effect.