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1.
Food Chem ; 398: 133869, 2023 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-35964565

RESUMO

This study aims to evaluate the heterocyclic aromatic amine (HAA) formation inhibited by cyanidin and rutin in chemical modeling systems and smoked chicken drumsticks. In the PhIP and ß-carboline chemical modeling systems, cyanidin inhibited the PhIP, Harman, and Norharman formation while rutin inhibited the PhIP formation but promoted the Harman and Norharman formation compared with control sample (P < 0.05). A mechanistic investigation confirmed that inhibiting the PhIP formation by cyanidin was mainly through trapping phenylalanine, creatine, creatinine, glucose, phenylacetaldehyde, and an aldol condensation product. In the smoked chicken drumsticks, cyanidin inhibited the PhIP, Harman, and Norharman formation dose-dependently compared to the control sample (P < 0.05), with 2.0 % (w/v) cyanidin having the highest inhibitory effect. Moreover, cyanidin inhibited the formation of PhIP precursors and intermediates in the smoked chicken drumsticks. These results provide evidence for using phenolic compounds to reduce HAA formation in smoked meat products.


Assuntos
Galinhas , Compostos Heterocíclicos , Aminas/química , Animais , Antocianinas , Culinária/métodos , Compostos Heterocíclicos/química , Temperatura Alta , Carne/análise , Rutina , Fumaça
2.
Food Chem ; 400: 134037, 2023 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-36055146

RESUMO

2-tert-butyl-1,4-benzoquinone (TBBQ) is the major oxidative product of tert-butylhydroquinone which is a widely used antioxidant in edible oils. The biotoxicity of TBBQ is a risk to human health, that the rapid and accurate monitoring of TBBQ is needed. Herein, a specific chromogenic reaction between TBBQ and polyethyleneimine (PEI) could generate adducts with maximum absorption at 478 nm. Amine groups of PEI are prone to link with TBBQ through Michael addition to form colored adducts. A colorimetric method for detecting TBBQ in edible oils was developed based on the aforesaid chromogenic reaction. The linear range for TBBQ was from 3.0 to 100.0 µg g-1, having a limit of detection of 1.8 µg g-1. Recoveries results ranged from 88.4 % to 93.1 %, which had a good agreement with that of high-performance liquid chromatography. Our study provides a rapid and simple strategy for the sensitive detection of TBBQ using commercial chemicals.


Assuntos
Antioxidantes , Colorimetria , Aminas , Antioxidantes/análise , Benzoquinonas , Cromatografia Líquida de Alta Pressão , Humanos , Óleos Vegetais/química , Polietilenoimina
3.
J Enzyme Inhib Med Chem ; 37(1): 2605-2620, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36131624

RESUMO

Twenty-four novel compounds bearing tetrahydroacridine and N-propargyl moieties have been designed, synthesised, and evaluated in vitro for their anti-cholinesterase and anti-monoamine oxidase activities. Propargyltacrine 23 (IC50 = 21 nM) was the most potent acetylcholinesterase (AChE) inhibitor, compound 20 (IC50 = 78 nM) showed the best inhibitory human butyrylcholinesterase (hBChE) profile, and ligand 21 afforded equipotent and significant values on both ChEs (human AChE [hAChE]: IC50 = 0.095 ± 0.001 µM; hBChE: IC50 = 0.093 ± 0.003 µM). Regarding MAO inhibition, compounds 7, 15, and 25 demonstrated the highest inhibitory potential towards hMAO-B (IC50 = 163, 40, and 170 nM, respectively). In all, compounds 7, 15, 20, 21, 23, and 25 exhibiting the most balanced pharmacological profile, were submitted to permeability and cell viability tests. As a result, 7-phenoxy-N-(prop-2-yn-1-yl)-1,2,3,4-tetrahydroacridin-9-amine hydrochloride (15) has been identified as a permeable agent that shows a balanced pharmacological profile [IC50 (hAChE) = 1.472 ± 0.024 µM; IC50 (hBChE) = 0.659 ± 0.077 µM; IC50 (hMAO-B) = 40.39 ± 5.98 nM], and consequently, as a new hit-ligand that deserves further investigation, in particular in vivo analyses, as the preliminary cell viability test results reported here suggest that this is a relatively safe therapeutic agent.


Assuntos
Doença de Alzheimer , Butirilcolinesterase , Acetilcolinesterase/metabolismo , Doença de Alzheimer/tratamento farmacológico , Aminas , Butirilcolinesterase/metabolismo , Inibidores da Colinesterase/farmacologia , Inibidores da Colinesterase/uso terapêutico , Desenho de Fármacos , Humanos , Ligantes , Monoaminoxidase , Inibidores da Monoaminoxidase/farmacologia , Oxirredutases , Relação Estrutura-Atividade , Tacrina/uso terapêutico
4.
Med Sci Monit ; 28: e937784, 2022 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-36068726

RESUMO

BACKGROUND Acute kidney injury (AKI) is one of the most common organ failures. An early diagnosis of AKI using specific biomarkers is essential for effective treatment. This study determined the serum concentrations of selected amino acids and amines using targeted liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) in patients with AKI during sepsis and septic shock treated in the Intensive Care Unit (ICU). MATERIAL AND METHODS A sample of 41 patients was divided into 2 groups: (1) patients with sepsis and septic shock along required continuous renal replacement therapy (CRRT) due to AKI (n=13), and (2) patients with sepsis and septic shock but without AKI (n=28). LC-MS/MS was used to measure a serum concentration of 6 amino acids and amines: arginine, ornithine, asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), dimethylamine (DMA), and citrulline. RESULTS There was a statistically significantly higher median DMA level in AKI patients compared to those without AKI (8.1 vs 5.2 umol/L; P=0.022). The results for the remaining molecules showed no significant differences (P>0.05). Patients with DMA ≥14.95 umol/L (n=5; 100%) and treated with CRRT presented DMA level below the cut-off point (n=7; 20%). Subjects with creatinine levels ≥1.19 mg/dL (n=11; 28%) and treated with CRRT presented creatinine levels below the cut-off point (n=1; 3%). CONCLUSIONS In patients with sepsis, increased serum levels of DMA were significantly associated with AKI requiring CRRT. It remains unclear whether increased DMA concentrations are secondary to sepsis-induced AKI or are a cause.


Assuntos
Injúria Renal Aguda , Terapia de Substituição Renal Contínua , Sepse , Choque Séptico , Injúria Renal Aguda/diagnóstico , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/terapia , Aminas , Aminoácidos , Cromatografia Líquida , Creatinina , Dimetilaminas , Humanos , Unidades de Terapia Intensiva , Terapia de Substituição Renal , Estudos Retrospectivos , Sepse/complicações , Sepse/terapia , Choque Séptico/complicações , Espectrometria de Massas em Tandem
5.
Methods Mol Biol ; 2546: 335-350, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36127602

RESUMO

A patient suspected of an inborn error of metabolism will commonly have urine organic acid analysis performed as part of their workup. The traditional urine organic acid method involves extraction of the acidic fraction from urine samples using an organic solvent, derivatization of extracted compounds, and identification using gas chromatography-mass spectrometry (GC/MS). Unfortunately, the extraction step results in the loss of many neutral and positively charged compounds which may be of interest to metabolic physicians and biochemical geneticists. By replacing the traditional extraction step with an enzymatic treatment of the sample with urease, an abundance of organic molecules is available for separation and quantification by GC/MS. The urease method is a useful adjunct to newborn screening follow-up, and it has the additional benefit of being able to identify many classes of biochemical compounds, such as amino acids, acylglycines, neurotransmitters, and carbohydrates. This method describes the urease treatment, derivatization, and the organic acids and other biochemical metabolites that can be identified.


Assuntos
Erros Inatos do Metabolismo , Urease , Ácidos , Aminas , Aminoácidos , Carboidratos , Cromatografia Gasosa-Espectrometria de Massas/métodos , Humanos , Recém-Nascido , Erros Inatos do Metabolismo/metabolismo , Solventes
6.
Sci Rep ; 12(1): 15691, 2022 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-36127488

RESUMO

Cooking oil fumes (COFs) represent a major indoor environmental pollutant and exhibit potent mutagenic or carcinogenic health effects caused by containing various heterocyclic aromatic amines (HAAs) and long-chain aldehydes. Despite some evaluation of the cumulative exposure of COFs to cancer cells under high concentration were evaluated, their biological adverse effects with low-dose exposure to healthy cells had been inadequately investigated. Herein, we firstly scrutinized the three selected typically toxic compounds of heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 3,8-dimethylammidazo[4,5-f]quinoxalin-2-amine (MeIQx) and trans, trans-2,4-decadienal (TDA)) emitted from COFs. In vitro studies revealed that the PhIP, MeIQx and TDA aerosol particles were negligible toxicity to cancer cells (A549 and HepG-2) but strong cytotoxicity to normal healthy cells (HelF and L02) under 0.5-4 µg/mL low dose exposure based on the reactive oxygen species (ROS) mechanism. In vivo studies demonstrated that PhIP caused significant lung and liver damage after exposure to PhIP for 30 days with mice. These results indicated the direct proof of healthy cell damage even at low-dose exposure to HAAs and aldehydes.


Assuntos
Culinária , Poluentes Ambientais , Aerossóis , Aldeídos/toxicidade , Aminas , Animais , Gases , Camundongos , Piridinas , Espécies Reativas de Oxigênio
7.
Carbohydr Polym ; 296: 119892, 2022 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-36087967

RESUMO

Inspired by the phenol-amine chemistry and biomineralization of insect cuticles, we developed a green and facile strategy for preparing a bio-adhesive with excellent adhesion properties, mildew resistance, and antibacterial activity. This biomimetic strategy incorporates functional catechol-modified ε-polylysine and vanillin via grafting and Schiff base reactions. The biomineralized cellulose nanocrystals were prepared using a cellulose nanocrystal bio-template by regulating the in-situ biomineralization of inorganic nanoparticles, thereby building an optimized organic-inorganic mineralization framework in the polymer. The bonding strength of composite adhesive was significantly improved by multiple cross-linking networks through sacrificial hydrogen bonds, electrostatic interactions, and dynamic covalent bonds. The adhesion strength of the composite adhesive reached 1.13 MPa, which was 151% higher than the pristine adhesive. As a result of the synergistic effect of the catechol component, cationic ε-polylysine, and aldehyde group, the bio-adhesive also exhibited favorable anti-mildew and anti-bacterial properties.


Assuntos
Celulose , Nanopartículas , Adesivos/química , Aminas , Catecóis/química , Celulose/química , Nanopartículas/química , Polilisina
8.
Methods Mol Biol ; 2541: 13-16, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36083537

RESUMO

Completing a validation process to assess the reactivity of potential building blocks that can be included in a DNA-encoded library is important for ensuring the quality of a library. Here we describe the validation method used to assess the reactivity of potential building blocks to be included in a DNA-encoded library, with HATU acylation of carboxylic acid building blocks with an on-DNA amine substrate as an example.


Assuntos
Ácidos Carboxílicos , DNA , Acilação , Aminas , Biblioteca Gênica
9.
Methods Mol Biol ; 2541: 33-37, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36083540

RESUMO

On-DNA reductive amination (on-DNA aldehyde with amine building blocks) and alkylation (on-DNA amine with aldehyde building blocks) are robust ways to form C-N bond. The large sets of commercially available aldehyde and amine building blocks make reductive amination and alkylation widely used in DEL synthesis.


Assuntos
Aldeídos , Aminas , Aldeídos/química , Alquilação , Aminação , Aminas/química , DNA/química
10.
Methods Mol Biol ; 2541: 49-54, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36083543

RESUMO

DNA-encoded library (DEL) recently has witnessed the swift development which has led to the new chemical entities identified for a wealthy biological interesting target; several small molecular inhibitors have moved into the late-stage clinical development. The success of DEL is heavily dependent on the chemical space which was assembled by the on-DNA chemical reactions. On-DNA C-N cross-coupling has great value for expanding the chemical space of DEL as amines are most abundant organic reagents available. Here we described the first on-DNA C-N cross-coupling reactions promoted by both Pd and Cu.


Assuntos
Aminas , DNA , Aminas/química , Catálise , DNA/química
11.
Methods Mol Biol ; 2541: 67-73, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36083546

RESUMO

A key factor for productive DNA-encoded libraries is the chemical diversity of the small molecule moiety attached to an encoding DNA oligomer. The library structure diversity is often limited to DNA-compatible chemical reactions in aqueous media. Herein, we describe a facile process for reducing aryl nitro groups to aryl amines by using sodium dithionite (Na2S2O4). The new protocol offers simple operation and circumvents the pyrophoric potential of the conventional method (Raney nickel). The utility of this method is demonstrated by the versatile synthesis of benzimidazoles on DNA.


Assuntos
Benzimidazóis , Bibliotecas de Moléculas Pequenas , Aminas/química , Benzimidazóis/química , DNA/química , DNA/genética , Biblioteca Gênica , Bibliotecas de Moléculas Pequenas/química
12.
Huan Jing Ke Xue ; 43(9): 4674-4683, 2022 Sep 08.
Artigo em Chinês | MEDLINE | ID: mdl-36096608

RESUMO

Atmospheric nitrogen deposition has a crucial impact on the structure and function of soil microorganisms of wetland ecosystems. Therefore, carrying out a study on the effects of soil carbon metabolism capacity has a great significance for the protection and utilization of wetland ecosystems. In this study, the effects of simulated nitrogen deposition on the carbon metabolic capacity of soil microorganisms in Calamagrostis angustifolia wetland for five consecutive years was investigated using Biolog-Eco technology. The results showed:① soil water content (SMC), pH, nitrate nitrogen (NO3-), ammonium nitrogen (NH4+), dissolved organic carbon (DOC), and total nitrogen (TN) contents were significantly different (P<0.05) under different nitrogen deposition conditions. ② The average well color development (AWCD) values of soil microorganisms within different N depositions were in the order of CK (control)>HN (high nitrogen treatment)>LN (low nitrogen treatment). LN significantly reduced the Shannon diversity index of soil microorganisms, and HN significantly reduced the Pielou index of soil microorganisms (P<0.05). ③ LN significantly inhibited the intensity of the utilization of carbohydrates, alcohols, amines, and acids by soil microorganisms (P<0.05); HN significantly promoted the utilization of esters by microorganisms, but HN caused soil microorganisms to inhibit the carbon sources of carbohydrates, amines, and acids (P<0.05). ④ Redundancy analysis showed that NH4+, DOC, and pH were the main environmental factors affecting the functional diversity of soil microbial communities in Calamagrostis angustifolia wetland in the Sanjiang Plain. Long-term nitrogen deposition will lead to the reduction in soil microbial functional diversity; the microbial activity related to the utilization of carbon source substrates is also significantly reduced, and the ability of microorganisms to utilize a single carbon source substrate also changes.


Assuntos
Nitrogênio , Solo , Aminas/metabolismo , Carboidratos , Carbono/química , Ecossistema , Nitrogênio/análise , Poaceae , Solo/química , Microbiologia do Solo , Áreas Alagadas
13.
Sci Rep ; 12(1): 15488, 2022 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-36109536

RESUMO

For the first time, a bio-photo-catalyst is synthesized in a microfluidic platform. The microchannel, which is wall-coated by in situ synthesized bio-photo-catalyst is used as an opto-fluidic reactor for amoxicillin degradation. Analyses including SEM, XRD, FTIR, Raman, UV-Vis spectra, and DLS have been used to characterize samples. The structure and morphology of TiO2 in microfluidic assisted synthesis are studied at 70-120 °C. The results show that both single-crystalline anatase sample and two-phase samples of anatase and rutile can be attained. According to SEM images, the smallest size and the narrowest particle size distribution (0.86 nm [Formula: see text]) is achieved by synthesis at 70 °C. Elemental mapping of Ti shows a uniform coating layer on inner walls. Raman signals besides the primary amines in FTIR results show the biological activity of the cross-linked Glucose oxidase (GOx), which is aimed for situ generation of H2O2. FTIR comparison of bulk and spiral microfluidic synthesized ZnO indicates identical bonds. SEM-coupled with performance experimentation reveal that by regulating the flowrate of spiral micromixer for ZnCl2 at 25 µl/min and NaOH at 50 µl/min, the narrowest size distribution and best the bio-photo-catalytic performance of ZnO nanoparticles is observed.


Assuntos
Glucose Oxidase , Óxido de Zinco , Aminas , Amoxicilina , Peróxido de Hidrogênio , Microfluídica , Hidróxido de Sódio , Temperatura , Titânio , Óxido de Zinco/química
14.
Sci Rep ; 12(1): 15499, 2022 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-36109538

RESUMO

The jeopardy of the synthetic dyes effluents on human health and the environment has swiftly aggravated, threatening human survival. Hence, sustained studies have figured out the most acceptable way to eliminate this poisonous contaminant. Thereby, our investigation aimed to fabricate montmorillonite/magnetic NiFe2O4@amine-functionalized chitosan (MMT-mAmCs) composite as a promising green adsorbent to remove the cationic methylene blue (MB) and crystal violet (CV) dyes from the wastewater in neutral conditions. Interestingly, MMT-mAmCs composite carries high negative charges at a wide pH range from 4 to 11 as clarified from zeta potential measurements, asserting its suitability to adsorb the cationic contaminants. In addition, the experimental study confirmed that the optimum pH to adsorb both MB and CV was pH 7, inferring the ability of MMT-mAmCs to adsorb both cationic dyes in simple process conditions. Furthermore, the ferromagnetic behavior of the MMT-mAmCs composite is additional merit to our adsorbent that provides facile, fast, and flawless separation. Notably, the as-fabricated composite revealed an auspicious adsorbability towards the adsorptive removal of MB and CV, since the maximum adsorption capacity of MB and CV were 137 and 118 mg/g, respectively. Moreover, the isotherm and kinetic investigatins depicted that the adsorption of both cationic dyes fitted Langmuir and Pseudo 2nd order models, respectively. Besides, the advanced adsorbent preserved satisfactory adsorption characteristics with maximal removal efficacy exceeding 87% after reuse for ten consecutive cycles. More importantly, MMT-mAmCs efficiently adsorbed MB and CV from real agricultural water, Nile river water and wastewater samples at the neutral pH medium, reflecting its potentiality to be a superb reusable candidate for adsorptive removal cationic pollutants from their aquatic media.


Assuntos
Quitosana , Poluentes Ambientais , Purificação da Água , Aminas , Bentonita/química , Cátions , Corantes/química , Violeta Genciana , Humanos , Azul de Metileno/química , Compostos Organometálicos , Águas Residuárias/química , Água
15.
J Org Chem ; 87(18): 12214-12224, 2022 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-36053202

RESUMO

Practical copper-promoted N-alkylation and bromination of arylamines/indazoles with alkyl bromides are described; the N-alkylation-C-4-bromination and N-dialkylation-C-4-bromination of arylamines, and N-alkylation-C-3-bromination of indazoles, with alkyl bromides have been analyzed. The full use of alkyl bromides as alkylating and brominating building blocks without atom wastage, indicating excellent atom and step economy, has been highlighted. Eco-friendly oxygen and water are the reaction oxidant and byproduct, respectively.


Assuntos
Brometos , Cobre , Alquilação , Aminas , Halogenação , Indazóis , Indicadores e Reagentes , Oxidantes , Oxigênio , Água
16.
J Am Chem Soc ; 144(36): 16535-16544, 2022 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-36053726

RESUMO

The development of unconventional strategies for the activation of ammonia (NH3) and water (H2O) is of capital importance for the advancement of sustainable chemical strategies. Herein we provide the synthesis and characterization of a radical equilibrium complex based on bismuth featuring an extremely weak Bi-O bond, which permits the in situ generation of reactive Bi(II) species. The ensuing organobismuth(II) engages with various amines and alcohols and exerts an unprecedented effect onto the X-H bond, leading to low BDFEX-H. As a result, radical activation of various N-H and O-H bonds─including ammonia and water─occurs in seconds at room temperature, delivering well-defined Bi(III)-amido and -alkoxy complexes. Moreover, we demonstrate that the resulting Bi(III)-N complexes engage in a unique reactivity pattern with the triad of H+, H-, and H• sources, thus providing alternative pathways for main group chemistry.


Assuntos
Amônia , Bismuto , Aminas , Amônia/química , Bismuto/química , Água/química
17.
J Org Chem ; 87(18): 12334-12341, 2022 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-36066498

RESUMO

Ferrocene derivatives have a wide range of applications, including as ligands in asymmetric catalysis, due to their chemical stability, rigid backbone, steric bulk, and ability to encode stereochemical information via planar chirality. Unfortunately, few of the available molecular mechanics force fields incorporate parameters for the accurate study of this important building block. Here, we present a MM3* force field for ferrocenyl ligands, which was generated using the quantum-guided molecular mechanics (Q2MM) method. Detailed validation by comparison to DFT calculations and crystal structures demonstrates the accuracy of the parameters and uncovers the physical origin of deviations through excess energy analysis. Combining the ferrocene force field with a force field for Pd-allyl complexes and comparing the crystal structures shows the compatibility with previously developed MM3* force fields. Finally, the ferrocene force field was combined with a previously published transition-state force field to predict the stereochemical outcomes of the aminations of Pd-allyl complexes with different amines and different chiral ferrocenyl ligands, with an R2 of ∼0.91 over 10 examples.


Assuntos
Aminas , Compostos Ferrosos , Compostos Ferrosos/química , Ligantes , Metalocenos
18.
J Org Chem ; 87(18): 12148-12163, 2022 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-36069394

RESUMO

A copper(II)/HOBt-catalyzed procedure for the synthesis of dipeptides and "general" amides has been developed using microwave irradiation to considerably hasten the reaction. As an alternative to using traditional carboxylic acid activation, the method relies on the use of N-acyl imidazoles as activated amino partners. By doing so, a nonconventional way to reach dipeptides and amides has been proposed through the challenging and less studied N → C direction synthesis. A series of dipeptides and "general" amides have been successfully synthesized, and the applicability of the method has been illustrated in gram-scale syntheses. The mild reaction conditions proposed are completely adequate for couplings in the presence of sensitive amino acids, affording the products without detectable racemization. Furthermore, experimental observations prompted us to propose a plausible reaction pathway for the couplings.


Assuntos
Aminas , Dipeptídeos , Amidas/química , Aminoácidos/química , Ácidos Carboxílicos/química , Cobre/química , Dipeptídeos/química , Imidazóis
19.
Org Lett ; 24(36): 6619-6624, 2022 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-36069423

RESUMO

An electrochemical amidation of benzoyl hydrazine/carbazate and primary/secondary amine as coupling partners via concomitant cleavage and formation of C(sp2)-N bonds has been achieved. This methodology proceeds under metal-free and exogenous oxidant-free conditions producing N2 and H2 as byproducts. Mechanistic studies reveal the in situ generations of both acyl and N-centered radicals from benzoyl hydrazines and amines. The utility of this protocol is demonstrated through a large-scale, and synthesis of bezafibrate, a hyperlipidemic drug.


Assuntos
Aminas , Bezafibrato , Aminas/química , Hidrazinas/química
20.
ACS Chem Biol ; 17(9): 2448-2460, 2022 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-36069699

RESUMO

Pulsed electron-electron double resonance (PELDOR) spectroscopy, X-ray scattering interferometry (XSI), and single-molecule Förster resonance energy transfer (smFRET) are molecular rulers that provide inter- or intramolecular pair-wise distance distributions in the nanometer range, thus being ideally suitable for structural and dynamic studies of biomolecules including RNAs. The prerequisite for such applications requires site-specific labeling of biomolecules with spin labels, gold nanoparticles, and fluorescent tags, respectively. Recently, site-specific labeling of large RNAs has been achieved by a combination of transcription of an expanded genetic alphabet containing A-T/G-C base pairs and NaM-TPT3 unnatural base pair (UBP) with post-transcriptional modifications at UBP bases by click chemistry or amine-NHS ester reactions. However, due to the bulky sizes of functional groups or labeling probes used, such strategies might cause structural perturbation and decrease the accuracy of distance measurements. Here, we synthesize an α-thiophosphorylated variant of rTPT3TP (rTPT3αS), which allows for post-transcriptional site-specific labeling of large RNAs at the internal α-phosphate backbone via maleimide-modified probes. Subsequent PELDOR, XSI, and smFRET measurements result in narrower distance distributions than labeling at the TPT3 base. The presented strategy provides a new route to empower the molecular rulers for structural and dynamic studies of large RNA and its complex.


Assuntos
Ouro , Nanopartículas Metálicas , Aminas , Espectroscopia de Ressonância de Spin Eletrônica , Ésteres , Ouro/química , Maleimidas , Fosfatos , RNA , Marcadores de Spin
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