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Imine reductases (IREDs) provide promising opportunities for the synthesis of various chiral amines. Initially, asymmetric imine reduction was reported, followed by reductive aminations of aldehydes and ketones via imines. Herein we present the reductive amination of structurally diverse carbonyls and dicarbonyls with hydrazines (reductive hydrazination), catalyzed by the IRED from Myxococcus stipitatus. In analogy to IRED-catalyzed reductive aminations, various carbonyls and dicarbonyls could react with simple hydrazines to produce substituted acyclic and cyclic N-alkylhydrazines. By incorporating and scaling up hydrogenase cofactor regeneration system, we demonstrated the scalability and atom-efficiency of an H2-driven double reductive hydrazination, highlightling the potential of IREDs in biocatalysis.
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Plant-insect interactions are a driving force into ecosystem evolution and community dynamics. Many insect herbivores enter diapause, a developmental arrest stage in anticipation of adverse conditions, to survive and thrive through seasonal changes. Herein, we investigated the roles of medium- to non-polar metabolites during larval development and diapause in a specialist insect herbivore, Chlosyne lacinia, reared on Aldama robusta leaves. Varying metabolites were determined using gas chromatography-mass spectrometry (GC-MS)-based metabolomics. Sesquiterpenes and steroids were the main metabolites putatively identified in A. robusta leaves, whereas C. lacinia caterpillars were characterized by triterpenes, steroids, fatty acids, and long-chain alkanes. We found out that C. lacinia caterpillars biosynthesized most of the identified steroids and fatty acids from plant-derived ingested metabolites, as well as all triterpenes and long-chain alkanes. Steroids, fatty acids, and long-chain alkanes were detected across all C. lacinia instars and in diapausing caterpillars. Sesquiterpenes and triterpenes were also detected across larval development, yet they were not detected in diapausing caterpillars, which suggested that these metabolites were converted to other molecules prior to the diapause stage. Our findings shed light on the chemical content variation across C. lacinia development and diapause, providing insights into the roles of metabolites in plant-insect interactions.
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Diapausa , Lepidópteros , Sesquiterpenos , Triterpenos , Animales , Cromatografía de Gases y Espectrometría de Masas , Ecosistema , Metabolómica/métodos , Esteroides/metabolismo , Sesquiterpenos/metabolismo , Ácidos Grasos/metabolismo , Alcanos , Triterpenos/metabolismo , LarvaRESUMEN
Primary sclerosing cholangitis (PSC) is a chronic inflammatory disease of the bile ducts that has been associated with diverse metabolic carboxylic acids. Mass spectrometric techniques are the method of choice for their analysis. However, the broad investigation of this metabolite class remains challenging. Derivatization of carboxylic acids represents a strategy to overcome these limitations but available methods suffer from diverse analytical challenges. Herein, we have designed a novel strategy introducing 4-nitrophenyl-2H-azirine as a new chemoselective moiety for the first time for carboxylic acid metabolites. This moiety was selected as it rapidly forms a stable amide bond and also generates a new ketone, which can be analyzed by our recently developed quant-SCHEMA method specific for carbonyl metabolites. Optimization of this new method revealed a high reproducibility and robustness, which was utilized to validate 102â metabolic carboxylic acids using authentic synthetic standard conjugates in human plasma samples including nine metabolites that were newly detected. Using this sequential analysis of the carbonyl- and carboxylic acid-metabolomes revealed alterations of the ketogenesis pathway, which demonstrates the vast benefit of our unique methodology. We anticipate that the developed azirine moiety with rapid functional group transformation will find broad application in diverse chemical biology research fields.
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Azirinas , Hepatopatías , Nitrofenoles , Humanos , Indicadores y Reactivos , Reproducibilidad de los Resultados , Metaboloma , Ácidos Carboxílicos/química , Metabolómica/métodosRESUMEN
Despite being a traditional coursework for pre-medical and medical students around the globe, biochemistry education suffers from a lack of positive appreciation due to the nature of the subject combined with deficiency of teaching modalities. A first semester biochemistry course was designed to include four different teaching modalities: lectures, recitations, case studies, and student presentations. A multi-item, anonymous, and voluntary questionnaire was distributed to students who had just completed the course and to those who had taken it the previous year. The questionnaire asked students to evaluate the course and how the different modalities affected their learning. These questionnaires took place in a two-year period between 2020 and 2021. Eighty-six (46%) of 186 total students responded. The vast majority of respondents agreed with the use of multimodal teaching techniques with respect to its impact on overall preparedness for future coursework, understanding, and enjoyability. Lectures and recitations were found to be the most useful in information retention and learning, although the same were found to be less enjoyable than other modalities. Although case studies and presentations were found to be enjoyable, most students ranked them low in terms of information retention and were the most voted to be removed from the course. There was general agreement between premedical and medical students' perception on the usefulness of the multimodal teaching techniques with respect to medical biochemistry modules and standardized exams. The agreement between cohorts suggests the premedical students accurately evaluated the usefulness of the course for the following year and validates the usefulness of the premedical student surveys. Use of multiple modalities in biochemistry education can be of substantial benefit in engaging and preparing students for further education.
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Educación de Pregrado en Medicina , Estudiantes de Medicina , Humanos , Aprendizaje , Bioquímica/educación , Curriculum , Evaluación Educacional , Educación de Pregrado en Medicina/métodos , Enseñanza , Encuestas y CuestionariosRESUMEN
Hen egg white lysozyme (HEWL) was exploited for the synthesis of ß-amino carbonyl compounds through a direct and three-component Mannich reaction in aqueous, confirming high chemoselectivity toward imine. In order to further extend the applications of the enzyme, HEWL was encapsulated using a metal-organic framework (MOF). The reactivity, stereoselectivity, and reusability of the encapsulated enzyme were investigated. The reaction was significantly enhanced as compared to the non-encapsulated enzyme. A mutated version of the enzyme, containing Asp52Ala (D52A), lacking important catalytical residue, has lost the bacterial site activity against Micrococcus luteus (M. luteus) while the D52A variant displayed an increased rate of the Mannich reaction, indicating a different catalytical residue involved in the promiscuous reaction. Based on site-directed mutagenesis, molecular docking, and molecular dynamic studies, it was proposed that π-stacking, H-bond interactions, and the presence of water in the active site may play crucial roles in the mechanism of the reaction.
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We describe the synthesis and application of a selection of trifunctional reagents for the dual-modality modification of native, solvent accessible disulfide bonds in trastuzumab. The reagents were developed from the dibromomaleimide (DBM) platform with two orthogonal clickable functional groups built around a lysine core. We also describe the development of an aryl diselenide additive which enables antibody disulfide reduction in 4 minutes and a rapid overall reduction-bridging-double click sequence.
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Inmunoconjugados , Lisina , Indicadores y Reactivos , Anticuerpos/química , Inmunoconjugados/química , Disulfuros/químicaRESUMEN
Polyketide natural products have significant promise as pharmaceutical targets for human health and as molecular tools to probe disease and complex biological systems. While the biosynthetic logic of polyketide synthases (PKS) is well-understood, biosynthesis of designer polyketides remains challenging due to several bottlenecks, including substrate specificity constraints, disrupted protein-protein interactions, and protein solubility and folding issues. Focusing on substrate specificity, PKSs are typically interrogated using synthetic thioesters. PKS assembly lines and their products offer a wealth of information when studied in a chemoenzymatic fashion. This review provides an overview of the past two decades of polyketide chemoenzymatic synthesis and their contributions to the field of chemical biology. These synthetic strategies have successfully yielded natural product derivatives while providing critical insights into enzymatic promiscuity and mechanistic activity.
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Productos Biológicos , Policétidos , Humanos , Policétidos/química , Productos Biológicos/metabolismo , Sintasas Poliquetidas/metabolismo , Metabolismo Secundario , Especificidad por SustratoRESUMEN
A novel series of 5-sulfinyl(sulfonyl)-4-arylsulfonyl-substituted 1,3-oxazoles has been synthesized, characterized and subjected to NCI inâ vitro assessment. Cancer cell lines of all subpanels were most sensitive to 2-{[4-[(4-fluorophenyl)sulfonyl]-2-(2-furyl)-1,3-oxazol-5-yl]sulfinyl}acetamide (3 l). Its antiproliferative and cytotoxic activity averaged over each subpanel was manifested in a very narrow range of concentrations (GI50 : 1.64-1.86â µM, TGI: 3.16-3.81â µM and LC50 : 5.53-7.27â µM), i. e. practically did not depend on the origin of the cancer cell line. The COMPARE matrix using TGI vector showed a high positive correlation of 3 l (r=0.88) with the intercalating agent aclarubicin, which inhibits topoisomerases. The absence in the database of standard agents that have a high correlation with the cytotoxicity of this compound suggests that it may have a unique mechanism of action. According to the results of the docking analysis, the most promising anticancer target for compound 3 l is DNA topoisomerase IIß. The obtained results indicate the anticancer activity of 5-sulfinyl(sulfonyl)-4-arylsulfonyl-substituted 1,3-oxazoles, which may be useful for the development of new anticancer drugs. 2-{[4-[(4-Fluorophenyl)sulfonyl]-2-(2-furyl)-1,3-oxazol-5-yl]sulfinyl}acetamide (3 l), as the most active, can be recommended for further in-depth studies.
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Antineoplásicos , Oxazoles , Línea Celular Tumoral , Oxazoles/farmacología , Antineoplásicos/farmacología , Relación Estructura-Actividad , Ensayos de Selección de Medicamentos Antitumorales , Proliferación CelularRESUMEN
The anaerobic ammonium oxidation (anammox) process exerts a very vital role in the global nitrogen cycle (estimated to contribute 30%-50% N2 production in the oceans) and presents superiority in water/wastewater nitrogen removal performance. Until now, anammox bacteria can convert ammonium (NH4+) to dinitrogen gas (N2) with nitrite (NO2-), nitric oxide (NO), and even electrode (anode) as electron acceptors. However, it is still unclear whether anammox bacteria could utilize photoexcited holes as electron acceptors to directly oxide NH4+ to N2. Here, we constructed an anammox-cadmium sulfide nanoparticles (CdS NPs) biohybrid system. The photoinduced holes from the CdS NPs could be utilized by anammox bacteria to oxidize NH4+ to N2. 15N-isotope labeling experiments demonstrated that NH2OH instead of NO was the real intermediate. Metatranscriptomics data further proved a similar pathway for NH4+ conversion with anodes as electron acceptors. This study provides a promising and energy-efficient alternative for nitrogen removal from water/wastewater.
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Different from traditional procedures of using solid stabilizers like polymers and surfactants, here we demonstrate that water, as a very "soft" matter, could function as a "spacer" to prevent the aggregation of metal-organic frameworks (MOFs) in aqueous dispersions. Our theoretical calculations reveal in case of an excess of positively charged metal nodes of MOFs, where water molecules are ligated to metal nodes that greatly enhance MOFs' solution dispersibility through electrostatic stabilization. This discovery has motivated us to develop a facile experimental approach for producing a category of "clean" MOF dispersions without foreign additives. Potential application has been demonstrated for the size fractionation of MOFs, which results in small-size MOFs (50-80 nm) characteristic of superior electrocatalytic oxygen evolution activities (256 mV at 10 mA cm-2, Tafel slope of 49 mV dec-1 and durability >30 h). This work would provide new clues for aqueous processing of MOFs for many emerging applications.
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The general lack of permeability of small molecules observed for Mycobacterium tuberculosis (Mtb) is most ascribed to its unique cell envelope. More specifically, the outer mycomembrane is hypothesized to be the principal determinant for access of antibiotics to their molecular targets. We describe a novel assay that combines metabolic tagging of the peptidoglycan, which sits directly beneath the mycomembrane, click chemistry of test molecules, and a fluorescent labeling chase step, to measure the permeation of small molecules. We showed that the assay workflow was robust and compatible with high-throughput analysis in mycobacteria by testing a small panel of azide-tagged molecules. The general trend is similar across the two types of mycobacteria with some notable exceptions. We anticipate that this assay platform will lay the foundation for medicinal chemistry efforts to understand and improve uptake of both existing drugs and newly-discovered compounds into mycobacteria.
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Mycobacterium tuberculosis , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/metabolismo , Pared Celular/química , Pared Celular/metabolismo , Transporte Biológico , Antibacterianos/química , Antibacterianos/metabolismoRESUMEN
In this study, starting from 2-amino-1,3,4-thiadiazole derivatives (3-5), a new series of 2,6-disubstituted (compounds 7-15) and 2,5,6-trisubstituted (compounds 16-33) imidazo[2,1-b][1,3,4]-thiadiazole derivatives were synthesized using cyclization and Mannich reaction mechanisms, respectively. All synthesized compounds were characterized by 1 H-NMR, 13 C-NMR, FT-IR, elemental analysis, and mass spectroscopy techniques. Also, X-ray diffraction analysis were used for compounds 4, 7, 11, 17, and 19. The cytotoxic effects of the new compounds on the viability of colon cancer cells (DLD-1), lung cancer cells (A549), and liver cancer cells (HepG2) were investigated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method inâ vitro. Compound 15 was found to be the most potent anticancer drug candidate in this series with an IC50 value of 3.63â µM against HepG2 for 48â h. Moreover, the absorption, distribution, metabolism, and excretion (ADME) parameters of the synthesized compounds were calculated and thus, their potential to be safe drugs was evaluated. Finally, to support the biological activity experiments, molecular docking studies of these compounds were carried out on three different target cancer protein structures (PDB IDs: 5ETY, 1M17, and 3GCW), and the amino acids that play key roles in the binding of the compounds to these proteins were determined.
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Antineoplásicos , Sulfuros , Simulación del Acoplamiento Molecular , Relación Estructura-Actividad , Espectroscopía Infrarroja por Transformada de Fourier , Antineoplásicos/químicaRESUMEN
Thiazole has been a key scaffold in antidiabetic drugs. In quest of new and more effective drugs a simple, efficient, high yielding (67-79 %) and convenient synthesis of arylidenehydrazinyl-4-methoxyphenyl)thiazoles is accomplished over two steps. The synthesis involved the condensation of aryl substituted thiosemicarbazones and 2-bromo-4-methoxyacetophenone in absolute ethanol. The structures of the resulting thiazoles are in accord with their UV/VIS, FT-IR, 1 H-, 13 C-NMR and HRMS data. All compounds were evaluated for alpha(α)-amylase inhibition potential, antiglycation, antioxidant abilities and biocompatibility. The compounds library identified 2-(2-(3,4-dichlorobenzylidene)hydrazinyl)-4-(4-methoxyphenyl)thiazole as a lead molecule against α-amylase inhibition with an IC50 of 5.75±0.02â µM. α-Amylase inhibition is also supported by molecular docking studies against α-amylase. All the obtained thiazoles also showed promising antiglycation activity with 4-(4-methoxyphenyl)-2-{2-[2-(trifluoromethyl)benzylidene]hydrazinyl}thiazole exhibiting the best inhibition (IC50 = 0.383±0.001â mg/mL) compared to control. The tested compounds are also biocompatible at the concentration used i. e., 10â µM.
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Complicaciones de la Diabetes , Diabetes Mellitus Tipo 2 , Humanos , Simulación del Acoplamiento Molecular , Relación Estructura-Actividad , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Espectroscopía Infrarroja por Transformada de Fourier , alfa-Amilasas , Tiazoles/química , Estructura MolecularRESUMEN
Methods for intracellular protein photoactivation have been studied to elucidate the spatial and temporal roles of proteins of interest. In this study, an intracellular protein photoactivation method was developed using sterically bulky caging. The protein of interest was modified with biotin via a photocleavable linker, and then conjugated with streptavidin to sterically block the protein surface for inactivation. The caged protein was transduced into cells and reactivated by light-induced degradation of the conjugates. A cytotoxic protein, saporin, was caged and photoactivated both inâ vitro and in living cells with this method. This method achieved control of the cytotoxic activity in an off-on manner, introducing cell death selectively at the designed location using light. This simple and versatile photoactivation method is a promising tool for studying spatio-temporal cellular events that are related to intracellular proteins of interest.
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Biotina , Proteínas , Proteínas/metabolismo , EstreptavidinaRESUMEN
A novel series of 1,3-oxazol-4-yltriphenylphosphonium salts has been synthesized and functionalized. Oxazole derivatives were subjected to NCI inâ vitro assessment. Seven most active derivatives have been selected for five-dose assay. Among them, compounds 9 ([2-(4-methylphenyl)-5-[(4-methylphenyl)sulfanyl]-1,3-oxazol-4-yl]triphenylphosphonium perchlorate), 1 ([5-(4-methylphenyl)amino]-2-phenyl-1,3-oxazol-4-yl]triphenylphosphonium perchlorate) and 4 ([5-phenyl-2-[(4-methylphenyl)amino]-1,3-oxazol-4-yl]triphenylphosphonium perchlorate) were the most active against all tested cancer subpanels. Statistical analysis of the total panel data showed average values of parameters of anticancer activity in the range of 0.3-1.1â µM (GI50 ), 1.2-2.5â µM (TGI) and 5-6â µM (LC50 ). It was found that the presence of phenyl or 4-methylphenyl groups at C(2) and C(5) in the oxazole ring is of critical importance for the manifestation of the anticancer activity. Matrix COMPARE analysis using LC50 vector showed a high positive correlation of compound 9 with standard anticancer agents that can directly disrupt mitochondrial function, causing programmed death of cancer cells. The obtained results indicate the anticancer activity of 1,3-oxazol-4-ylphosphonium salts, which could be useful for developing new anticancer drugs. The most active of them can be recommended for further in-depth studies and synthesis of new derivatives with antitumor activity on their basis.
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Antineoplásicos , Sales (Química) , Sales (Química)/farmacología , Percloratos , Relación Estructura-Actividad , Antineoplásicos/farmacología , Oxazoles/farmacología , Línea Celular Tumoral , Ensayos de Selección de Medicamentos AntitumoralesRESUMEN
Alzheimer's disease (AD) is a chronic and progressive neurodegenerative disease, characterized by irreversible cognitive impairment, memory loss, and behavioral disturbances, ultimately resulting in death. The critical roles of glycogen synthase kinase-3ß (GSK-3ß) in tau pathology have also received considerable attention. Based on molecular docking studies, a series of novel α-carboline derivatives were designed, synthesized, and evaluated as GSK-3ß inhibitors for their various biological activities. Among them, compound ZCH-9 showed the most potent inhibitory activity against GSK-3ß, with an IC50 value of 1.71 ± 0.09 µM. The cytotoxicity assay showed that ZCH-9 had low cytotoxicity toward the cell lines SH-SY5Y, HepG2, and HL-7702. Moreover, Western blot analysis indicated that ZCH-9 effectively inhibited hyperphosphorylation of the tau protein in okadaic acid-treated SH-SY5Y cells. The binding mode between ZCH-9 and GSK-3ß was analyzed and further clarified throughout the molecular dynamics simulations. In general, these results suggested that the α-carboline-based small-molecule compounds could serve as potential candidates targeting GSK-3ß for the treatment of AD.
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Enfermedad de Alzheimer , Neuroblastoma , Enfermedades Neurodegenerativas , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Carbolinas , Glucógeno Sintasa Quinasa 3 beta , Humanos , Simulación del Acoplamiento Molecular , Ácido Ocadaico/metabolismo , Fosforilación , Relación Estructura-Actividad , Proteínas tau/metabolismoRESUMEN
Inflammation is the natural defense mechanism against any external stimuli in the human body and it saves us from foreign entities that may alter our bodies' normal functioning. Any anomaly in this natural defense system leads to the development of different pathological conditions associated with chronic inflammation like rheumatoid arthritis, osteoarthritis, atopy, asthma, allergic rhino-conjunctivitis, coronary artery disease, cardiac arrhythmias, obesity, insulin resistance and type-2 diabetes, depression, and aging. These disorders impair the quality of life of affected people in different ways. Among these, rheumatoid arthritis and osteoarthritis are the most common chronic inflammation-associated disorders. Different therapeutic strategies are already available for the treatment of rheumatoid arthritis and osteoarthritis, but all with pros and cons. Here, we discuss the emergence of several antiarthritic analogs developed by different researchers which could provide the basis for the evolution of newer therapeutic strategies with better activity and safety profiles.
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Artritis Reumatoide , Osteoartritis , Humanos , Calidad de Vida , Relación Estructura-Actividad , Artritis Reumatoide/tratamiento farmacológico , Osteoartritis/tratamiento farmacológico , Osteoartritis/complicaciones , InflamaciónRESUMEN
Biogenic amine organocatalysts have transformed the field of synthetic organic chemistry. Yet despite their use in synthesis and to label biomolecules inâ vitro, amine organocatalysis inâ vivo has received comparatively little attention - despite the potential of such reactions to be interfaced with living cells and to modify cellular metabolites. Herein we report that biogenic amines derived from L-tyrosine catalyze the self-aldol condensation of butanal to 2-ethylhexenal - a key intermediate in the production of the bulk chemical 2-ethylhexanol - in the presence of living Escherichia coli and outperform many amine organocatalysts currently used in synthetic organic chemistry. Furthermore, we demonstrate that cell lysate from E. coli and the prolific amine overproducer Corynebacterium glutamicum ATCC 13032 catalyze this reaction inâ vitro, demonstrating the potential for microbial metabolism to be used as a source of organocatalysts for biocompatible reactions in cells.
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Escherichia coli , Tiramina , Aldehídos , Aminas/química , DimerizaciónRESUMEN
To mimic the levels of spatiotemporal control that exist in nature, tools for chemically induced dimerization (CID) are employed to manipulate protein-protein interactions. Although linker composition is known to influence speed and efficiency of heterobifunctional compounds, modeling or inâ vitro experiments are often insufficient to predict optimal linker structure. This can be attributed to the complexity of ternary complex formation and the overlapping factors that impact the effective concentration of probe within the cell, such as efflux and passive permeability. Herein, we synthesize a library of modular chemical tools with varying linker structures and perform quantitative microscopy in live cells to visualize dimerization in real-time. We use our optimized probe to demonstrate our ability to recruit a protein of interest (POI) to the mitochondria, cell membrane, and nucleus. Finally, we induce and monitor local and global phase separation. We highlight the importance of quantitative approaches to linker optimization for dynamic systems and introduce new, synthetically accessible tools for the rapid control of protein localization.
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Transporte de Proteínas , Membrana Celular , DimerizaciónRESUMEN
Stimulation of the body's immune system toward tumor cells is now well recognized as a promising strategy in cancer therapy. Just behind cell therapy and monoclonal antibodies, small molecule-based strategies are receiving growing attention as alternatives to direct immune response against tumor cells. However, the development of small-molecule approaches to modulate the balance between stimulatory immune factors and suppressive factors in a targeted way remains a challenge. Here, we report the cell surface functionalization of LS174T cancer cells with an abiotic hapten to recruit antibodies to the cell surface. Metabolic glycoengineering followed by covalent reaction with the hapten results in antibody recognition of the target cells. Microscopy and flow cytometry studies provide compelling evidence that metabolic glycoengineering and small molecule stimulators can be combined to direct antibody recognition.