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1.
Food Chem ; 339: 127898, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-32871303

RESUMO

In this study, lipid oxidation evaluation methods were compared for a krill-oil-in-water emulsion system. With this aim, thiocyanate and DPPP (diphenyl-1-pyrenylphosphine) fluorescence methods were comparatively examined to determine primary oxidation products. 2-thiobarbituric acid reactive substances (TBARS), hexanal and propanal formation were also monitored as secondary oxidations products. All oxidation experiments were performed via both auto-oxidation at 45 °C and light-riboflavin induced photooxidation at 37 °C. The results have shown that thiocyanate method was not suitable to measure lipid hydroperoxides by the both in auto- and photo-oxidation systems. On the other hand, fluorescence intensity of samples containing the DPPP probe increased during incubation period which indicates the formation of lipid hydroperoxides could be detected via this method. TBARS, hexanal and propanal concentrations also increased during storage period and the formation kinetics of secondary oxidation products was confirmed that the DPPP fluorescence method was accurate and reliable at different environmental conditions.


Assuntos
Emulsões/química , Euphausiacea/química , Óleos/química , Água/química , Animais , Antioxidantes/química , Euphausiacea/metabolismo , Compostos Organofosforados/química , Oxirredução , Tamanho da Partícula , Pirenos/química , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta
2.
Food Chem ; 338: 128074, 2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-32950011

RESUMO

A separation and analysis method of fosthiazate stereoisomers was established utilizing supercritical fluid chromatography-tandem mass spectrometry (SFC-MS/MS) with a CHIRALPAK AD-3 column. The determination of the four fosthiazate stereoisomers could be completed within 6 min. The environmental behaviors of fosthiazate stereoisomers were studied in legume vegetables. After applying fosthiazate granules to soil, the concentrations of fosthiazate stereoisomers in the legume vegetables increased with time, reached maximum values in 7-10 days, and then decreased gradually in all legumes except for in Glycine max. No obvious dissipation behaviors were observed in Glycine max. Interestingly, the stereoselective behaviors were species-specific. A-(-), B-(-) and B-(±)-fosthiazate were preferentially enriched in Phaseolus vulgaris Linn and Vigna unguiculata, while A-(+) and A-(±)-fosthiazate preferentially accumulated in Vicia faba Linn, Pisum sativum Linn and G. max. The opposite stereoselectivity of B-(±)-fosthiazate was observed in different growth stage of G. max. No stereoselective dissipation occurred in soil.


Assuntos
Fabaceae/química , Compostos Organofosforados/química , Espectrometria de Massas em Tandem/métodos , Tiazolidinas/química , Cromatografia com Fluido Supercrítico , Fabaceae/metabolismo , Ervilhas/química , Ervilhas/metabolismo , Phaseolus/química , Phaseolus/metabolismo , Soja/química , Soja/metabolismo , Estereoisomerismo , Vicia faba/química , Vicia faba/metabolismo
3.
Sci Rep ; 10(1): 17872, 2020 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-33087770

RESUMO

The FDA-approved prophylactic antimalarial drug atovaquone (ATO) recently was repurposed as an antitumor drug. Studies show that ATO exerts a profound antiproliferative effect in several cancer cells, including breast, ovarian, and glioma. Analogous to the mechanism of action proposed in parasites, ATO inhibits mitochondrial complex III and cell respiration. To enhance the chemotherapeutic efficacy and oxidative phosphorylation inhibition, we developed a mitochondria-targeted triphenylphosphonium-conjugated ATO with varying alkyl side chains (Mito4-ATO, Mito10-ATO, Mito12-ATO, and Mito16-ATO). Results show, for the first time, that triphenylphosphonium-conjugated ATO potently enhanced the antiproliferative effect of ATO in cancer cells and, depending upon the alkyl chain length, the molecular target of inhibition changes from mitochondrial complex III to complex I. Mito4-ATO and Mito10-ATO inhibit both pyruvate/malate-dependent complex I and duroquinol-dependent complex III-induced oxygen consumption whereas Mito12-ATO and Mito16-ATO inhibit only complex I-induced oxygen consumption. Mitochondrial target shifting may have immunoregulatory implications.


Assuntos
Atovaquona/farmacologia , Proliferação de Células/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Animais , Antimaláricos/farmacologia , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Arsenicais/farmacologia , Atovaquona/química , Atovaquona/metabolismo , Linhagem Celular Tumoral , Respiração Celular/efeitos dos fármacos , Complexo I de Transporte de Elétrons/efeitos dos fármacos , Complexo III da Cadeia de Transporte de Elétrons/efeitos dos fármacos , Humanos , Camundongos , Mitocôndrias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Compostos Organofosforados/química , Fosforilação Oxidativa , Óxidos/farmacologia , Consumo de Oxigênio/efeitos dos fármacos
4.
J Chromatogr A ; 1632: 461603, 2020 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-33099238

RESUMO

The herbicide glyphosate and its main metabolite aminomethylphosphonic acid (AMPA) are generally studied in environmental samples in the investigation of contamination of soil, plants, water and food. Many analytical methods are based on liquid chromatography or high-performance liquid chromatography, with pre-column or post-column derivatization; in addition, the chromatograph can be coupled to mass spectrometers for detection and quantification. Gas chromatography and spectroscopic and electrochemical methods have also been used. In this work, a simple low-cost method is presented for the analysis of water intended for human consumption with the quantification not only of glyphosate and AMPA, but also of other ions of interest to public health (fluoride, chlorite, bromate, chloride, nitrite, nitrate, sulfate and phosphate). Based on ion chromatography with conductivity detection (chemical suppression of eluent conductivity), the key point in this method is the use of gradient elution with two eluents of different pH and ionic strength, not requiring derivatization. There is no interference from the other ions at higher concentrations. The detection limits obtained for glyphosate and AMPA were 15 µg L-1 and 80 µg L-1, respectively. As the method allows the analysis of a large number of samples, it has been successfully applied to monitoring the quality of tap water in 89 municipalities in the northeast region of the State of São Paulo, Brazil.


Assuntos
Cromatografia por Troca Iônica/métodos , Água Potável/química , Monitoramento Ambiental/métodos , Glicina/análogos & derivados , Saúde Pública , Poluentes Químicos da Água/análise , Glicina/análise , Herbicidas/análise , Humanos , Limite de Detecção , Compostos Organofosforados/química , Padrões de Referência , Soluções
5.
J Med Chem ; 63(20): 11691-11706, 2020 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-32960056

RESUMO

Self-adjuvanting vaccines, wherein an antigenic peptide is covalently bound to an immunostimulating agent, have been shown to be promising tools for immunotherapy. Synthetic Toll-like receptor (TLR) ligands are ideal adjuvants for covalent linking to peptides or proteins. We here introduce a conjugation-ready TLR4 ligand, CRX-527, a potent powerful lipid A analogue, in the generation of novel conjugate-vaccine modalities. Effective chemistry has been developed for the synthesis of the conjugation-ready ligand as well as the connection of it to the peptide antigen. Different linker systems and connection modes to a model peptide were explored, and in vitro evaluation of the conjugates showed them to be powerful immune-activating agents, significantly more effective than the separate components. Mounting the CRX-527 ligand at the N-terminus of the model peptide antigen delivered a vaccine modality that proved to be potent in activation of dendritic cells, in facilitating antigen presentation, and in initiating specific CD8+ T-cell-mediated killing of antigen-loaded target cells in vivo. Synthetic TLR4 ligands thus show great promise in potentiating the conjugate vaccine platform for application in cancer vaccination.


Assuntos
Vacinas Anticâncer/síntese química , Glucosamina/análogos & derivados , Lipídeo A/análogos & derivados , Compostos Organofosforados/química , Ovalbumina/química , Receptor 4 Toll-Like/imunologia , Adjuvantes Imunológicos , Animais , Vacinas Anticâncer/química , Vacinas Anticâncer/imunologia , Citocinas/imunologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Glucosamina/química , Glucosamina/imunologia , Imunoglobulina G/sangue , Ligantes , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Compostos Organofosforados/imunologia , Ovalbumina/imunologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Vacinas Conjugadas
6.
J Med Chem ; 63(19): 11258-11270, 2020 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-32930595

RESUMO

Vγ9/Vδ2 T-cells are activated by pyrophosphate-containing small molecules known as phosphoantigens (PAgs). The presence of the pyrophosphate group in these PAgs has limited their drug-like properties because of its instability and polar nature. In this work, we report a novel and short Grubbs olefin metathesis-mediated synthesis of methylene and difluoromethylene monophosphonate derivatives of the PAg (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBP) as well as their aryloxy diester phosphonamidate prodrugs, termed ProPAgens. These prodrugs showed excellent stability in human serum (t1/2 > 12 h) and potent activation of Vγ9/Vδ2 T-cells (EC50 ranging from 5 fM to 73 nM), which translated into sub-nanomolar γδ T-cell-mediated eradication of bladder cancer cells in vitro. Additionally, a combination of in silico and in vitro enzymatic assays demonstrated the metabolism of these phosphonamidates to release the unmasked PAg monophosphonate species. Collectively, this work establishes HMBP monophosphonate ProPAgens as ideal candidates for further investigation as novel cancer immunotherapeutic agents.


Assuntos
Antígenos/imunologia , Imunidade Celular , Compostos Organofosforados/química , Pró-Fármacos/farmacologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Linfócitos T/efeitos dos fármacos , Antígenos/química , Humanos , Pró-Fármacos/química , Linfócitos T/imunologia
7.
Arch Biochem Biophys ; 692: 108516, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32745463

RESUMO

Ketol-acid reductoisomerase (KARI), the second enzyme in the branched-chain amino acid (BCAA) biosynthesis pathway, is an emerging target for the discovery of biocides. Here, we demonstrate that cyclopropane-1,1-dicarboxylate (CPD) inhibits KARIs from the pathogens Mycobacterium tuberculosis (Mt) and Campylobacter jejuni (Cj) reversibly with Ki values of 3.03 µM and 0.59 µM, respectively. Another reversible inhibitor of both KARIs, Hoe 704, is more potent than CPD with Ki values of 300 nM and 110 nM for MtKARI and CjKARI, respectively. The most potent inhibitor tested here is N-hydroxy-N-isopropyloxamate (IpOHA). It has a Ki of ~26 nM for MtKARI, but binds rather slowly (kon ~900 M-1s-1). In contrast, IpOHA binds more rapidly (kon ~7000 M-1s-1) to CjKARI and irreversibly.


Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Campylobacter jejuni/enzimologia , Inibidores Enzimáticos/química , Cetol-Ácido Redutoisomerase/antagonistas & inibidores , Mycobacterium tuberculosis/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Campylobacter jejuni/química , Ciclopropanos/química , Ácidos Dicarboxílicos/química , Ácidos Hidroxâmicos/química , Cetol-Ácido Redutoisomerase/química , Cetol-Ácido Redutoisomerase/metabolismo , Mycobacterium tuberculosis/química , Compostos Organofosforados/química
8.
Ann N Y Acad Sci ; 1480(1): 54-72, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32814367

RESUMO

Organophosphorus compounds (OPs), developed as pesticides and chemical warfare agents, are extremely toxic chemicals that pose a public health risk. Of the different detoxification strategies, organophosphate-hydrolyzing enzymes have attracted much attention, providing a potential route for detoxifying those exposed to OPs. Phosphotriesterase (PTE), also known as organophosphate hydrolase (OPH), is one such enzyme that has been extensively studied as a catalytic bioscavenger. In this review, we will discuss the protein engineering of PTE aimed toward improving the activity and stability of the enzyme. In order to make enzyme utilization in OP detoxification more favorable, enzyme immobilization provides an effective means to increase enzyme activity and stability. Here, we present several such strategies that enhance the storage and operational stability of PTE/OPH.


Assuntos
Substâncias para a Guerra Química/química , Enzimas Imobilizadas , Compostos Organofosforados/química , Praguicidas/química , Monoéster Fosfórico Hidrolases , Engenharia de Proteínas , Catálise , Enzimas Imobilizadas/química , Enzimas Imobilizadas/genética , Humanos , Hidrólise , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/genética
9.
Chemosphere ; 261: 127743, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32721694

RESUMO

Organophosphorus insecticides are known to be partly transformed to their respective oxons during the chlorination step of drinking water treatment. For most organophosphorus insecticides, the toxicological endpoint for determining acceptable daily intake levels is inhibition of acetylcholinesterase (AChE). Like the parent insecticides, oxons also inhibit AChE, so the presence of oxons in drinking water is also evaluated. However, no attention is paid to the possible presence of transformation products (TPs) other than oxons. In the present study, we determined whether the anti-AChE activity observed for chlorinated solutions of the organophosphorus insecticides malathion and methidathion could be solely attributed to the parent compounds and their oxons. Upon chlorination, both malathion and methidathion were immediately transformed to their oxons; the maximum transformation ratios were 60% and 30%, respectively, indicating that at least 40% and 70% of these compounds were transformed into other TPs. Before chlorination, malathion- and methidathion-containing solutions exhibited little to no anti-AChE activity, but the solutions showed strong activity after chlorination. The contributions of the parent insecticides and their oxons to the activities of the chlorinated samples were calculated from the concentrations of the compounds in the samples and dose-response curves for chemical standards of the compounds. For both the malathion-containing solution and the methidathion-containing solution, the calculated anti-AChE activities were almost the same as the observed activities at every chlorination time. This suggests that the observed activities could be attributed solely to the parent insecticides and their oxons, indicating that other TPs need not be considered.


Assuntos
Halogenação , Inseticidas/química , Compostos Organofosforados/química , Acetilcolinesterase , Animais , Inibidores da Colinesterase/química , Inseticidas/farmacologia , Malation/química , Compostos Organotiofosforados/química , Purificação da Água/métodos
10.
Chemosphere ; 260: 127461, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32673865

RESUMO

Tris (1-chloro-2-propyl) phosphate (TCPP) is a chlorinated organic phosphate used in various applications as a flame retardant and plasticizer. TCPP is a known suspected carcinogen and is not effectively removed by traditional water treatments such as biological, chlorination, and UV irradiation. In this study, the UV/H2O2 reaction was employed to degrade TCPP in water. TCPP was effectively degraded in the UV/H2O2 reaction by pseudofirst-order kinetics. The second-order rate constant of the reaction between the TCPP and OH radical was determined to be 4.35 (±0.13) × 108 M-1 s-1 using the competition kinetics with nitrobenzene as a reference compound. The degradation of TCPP was affected by the amount of H2O2, pH, and coexisting water components such as HCO3-, NO3-, and humic acid. Approximately 64.2% of total organic carbon (TOC) in TCPP was mineralized in 12 h during the UV/H2O2 reaction, whereas chloride (Cl-) and phosphate (PO43-) ions were identified as ionic byproducts with the recoveries of 96% chlorine (Cl) and 50% phosphorus (P). Five organic transformation products (TPs) of TCPP were also identified using LC-qTOF/MS. Considering the identified TPs, the main degradation pathway of TCPP during the UV/H2O2 reaction was found to be OH-radical-induced hydroxylation. Finally, a 70% decrease in bioluminescence inhibition in Vibrio fischeri was observed during the UV/H2O2 reaction, and the time-toxicity profile was similar to the time-peak area profile of TPs. The result of this study implies that TCPP can be efficiently removed with significant mineralization and toxicity reduction by the UV/H2O2 process.


Assuntos
Compostos Organofosforados/química , Cloretos , Cloro , Retardadores de Chama/análise , Substâncias Húmicas , Peróxido de Hidrogênio/química , Cinética , Organofosfatos/química , Oxirredução , Fosfatos/química , Plastificantes/química , Raios Ultravioleta , Água , Poluentes Químicos da Água/análise , Purificação da Água
11.
Food Chem ; 331: 127352, 2020 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-32652343

RESUMO

The influence of some additives, including metal ions, antioxidants, enzyme inhibitors and organic solvents, on the storage stability of four organophosphorus pesticides in cucumber samples were investigated. It was found that metal ions, including Al3+, Fe3+, and Co2+, increased the stability of dichlorvos, malathion, and chlorpyrifos. Conversely, Al3+, Fe3+, Fe2+, and Co2+ caused catalytic degradation of diazinon. With the addition of organic solvents (CH2Cl2, CHCl3, CCl4, CH3OH and CH3COCH3), remaining of diazinon residues was higher (16-54%) after storage for seven days. CCl4 was associated with the highest retention of malathion, diazinon, and chlorpyrifos (33%, 48% and 44%, respectively) in samples. SDS also stabilized the pesticides since residues were, again, higher (13-38%) after seven days storage. Furthermore, addition of Al3+ and Fe3+ decreased peroxidase (POD) activity and inhibited degradation of dichlorvos and malathion. After 14 days, lyophilization increased the pesticide residues remaining by 36%, 29%, and 58% for diazinon, malathion and chlorpyrifos, respectively. Overall, the stability of these pesticides during storage is impacted by water content and addition of exogenous substances. This could ensure higher quality of pesticide residue data in samples.


Assuntos
Cucumis sativus/química , Contaminação de Alimentos/análise , Armazenamento de Alimentos , Inseticidas/química , Compostos Organofosforados/química , Alumínio/química , Clorpirifos/análise , Clorpirifos/química , Diazinon/análise , Diazinon/química , Diclorvós/análise , Diclorvós/química , Aditivos Alimentares/química , Liofilização , Inseticidas/análise , Malation/análise , Malation/química , Compostos Organofosforados/análise , Oxirredutases/química , Peroxidases/química , Peroxidases/metabolismo , Resíduos de Praguicidas/análise , Resíduos de Praguicidas/química , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Dodecilsulfato de Sódio/química , Água/química
12.
AAPS PharmSciTech ; 21(5): 160, 2020 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-32476084

RESUMO

The aim of this present study was to investigate the ability of different dissolution methods to predict the in vivo performance of efonidipine hydrochloride (EFH). The solid dispersions of EFH were prepared by solvent evaporation method with HPMC-AS as matrix and urea as a pH adjusting agent. The paddle method, the open-loop, and the closed-loop flow-through cell methods were studied. In the study, Weibull's model was the best fit to explain release profiles. The pharmacokinetics behaviors of two kinds of solid dispersions with different release rate were investigated in comparison to the EFH after oral administration in rats. In vivo absorption was calculated by a numerical deconvolution method. In the study, the level A in vivo and in vitro correlation (IVIVC) was utilized. The correlation coefficient was calculated and interpreted by means of linear regression analysis (Origin.Pro.8.5 software). As a result, excellent IVIVC for solid dispersions and crude drug (r2 = 0.9352-0.9916) was obtained for the dissolution rate determined with flow-through cell open-loop system in phosphate buffer solution with 0.1% (w/v) polysorbate 80 at pH 6.5, the flow-rate of 4 mL/min. In addition, the self-assembled flow cell system had good repeatability and accuracy. The dissolution rate of the solid dispersion could be slowed down by the flow-through method, and the difference caused by preparation was significantly distinguished. The study demonstrated that flow-through cell method of the open-loop, compared with paddle method, was suitable for predicting in vivo performance of EFH solid dispersions.


Assuntos
Bloqueadores dos Canais de Cálcio/química , Di-Hidropiridinas/química , Nitrofenóis/química , Animais , Bloqueadores dos Canais de Cálcio/farmacocinética , Di-Hidropiridinas/farmacocinética , Técnicas In Vitro , Masculino , Nitrofenóis/farmacocinética , Compostos Organofosforados/química , Compostos Organofosforados/farmacocinética , Preparações Farmacêuticas , Ratos , Ratos Sprague-Dawley , Solubilidade , Solventes , Água
13.
Nature ; 583(7817): 548-553, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32480398

RESUMO

Tertiary stereogenic centres containing one fluorine atom are valuable for medicinal chemistry because they mimic common tertiary stereogenic centres containing one hydrogen atom, but they possess distinct charge distribution, lipophilicity, conformation and metabolic stability1-3. Although tertiary stereogenic centres containing one hydrogen atom are often set by enantioselective desymmetrization reactions at one of the two carbon-hydrogen (C-H) bonds of a methylene group, tertiary stereocentres containing fluorine have not yet been constructed by the analogous desymmetrization reaction at one of the two carbon-fluorine (C-F) bonds of a difluoromethylene group3. Fluorine atoms are similar in size to hydrogen atoms but have distinct electronic properties, causing C-F bonds to be exceptionally strong and geminal C-F bonds to strengthen one another4. Thus, exhaustive defluorination typically dominates over the selective replacement of a single C-F bond, hindering the development of the enantioselective substitution of one fluorine atom to form a stereogenic centre5,6. Here we report the catalytic, enantioselective activation of a single C-F bond in an allylic difluoromethylene group to provide a broad range of products containing a monofluorinated tertiary stereogenic centre. By combining a tailored chiral iridium phosphoramidite catalyst, which controls regioselectivity, chemoselectivity and enantioselectivity, with a fluorophilic activator, which assists the oxidative addition of the C-F bond, these reactions occur in high yield and selectivity. The design principles proposed in this work extend to palladium-catalysed benzylic substitution, demonstrating the generality of the approach.


Assuntos
Carbono/química , Flúor/química , Alcenos/química , Catálise , Cátions , Halogenação , Hidrogênio/química , Irídio/química , Compostos Organofosforados/química , Oxirredução , Paládio/química
14.
Chem Biol Interact ; 326: 109111, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32413347

RESUMO

Human scalp hair is a biological matrix that can trap chemical vapours from explosives (TNT), drugs (THC) and chemical weapons (yperite). The external contamination of human's hair following exposure to organophosphorus (OP) nerve agent was simulated by model compounds: triethyl phosphate (TEP) and diisopropyl fluorophosphate (DFP). In this work were exposed strands of hair to vapours of TEP and DFP (3 and 7 ppmv) to model sorption kinetics. Sorption isotherms were also investigated at several contamination levels (80-3000 mg min.m-3). OP nerve agent simulants were extracted from hair by soaking in DCM. Raw extracts were analysed in GC-MS/MS to quantify each simulant content in hair. Results were fitted by applying isotherm or kinetic equations. The best model was found to be bimodal first-order, suggesting the co-existence of two different mechanisms of sorption. The best equation to describe OP vapours incorporation on hair was Freundlich model. Thus hair can be used as a passive sensor able to trap chemical G-agents and can also offer valuable information regarding both individual contamination and proof of exposure to chemical weapons.


Assuntos
Substâncias para a Guerra Química/química , Cabelo/química , Couro Cabeludo/química , Humanos , Isoflurofato/química , Gás de Mostarda/química , Agentes Neurotóxicos/química , Organofosfatos/química , Compostos Organofosforados/química
15.
Chemosphere ; 253: 126698, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32302901

RESUMO

Organophosphates insecticides (OPs) are one of the major environmental pollutants and their interaction with human serum albumin (HSA) has been shown to have significant effects on their bioavailability which is related to toxicokinetics and toxicodynamics in human body. In this research, solid-phase microextraction methods were developed to analyse the free concentrations of three OPs (chlorpyrifos, parathion-methyl and malathion) in buffered HSA solution and that provide a useful method for the determination of binding affinity constants (Ka), binding forces and binding location. Polydimethylsiloxane fibers were selected for analysing the free concentrations of OPs, with an external calibration approach. Good linearities conducted in PBS solution were observed in the range of 0.0025-1.7 µmol L-1 (R2 = 0.9975) for chlorpyrifos, 1.0-27 µmol L-1 (R2 = 0.9974) for parathion-methyl, and 0.5-70 µmol L-1 (R2 = 0.9973)for malathion, respectively. The LODs for instrument response were 1 ng, 5 ng and 10 ng for chlorpyrifos, parathion-methyl and malathion, respectively. The Ka values for chlorpyrifos, parathion-methyl and malathion showed that they were positively correlated with hydrophobicity and negatively correlated with temperature. The OP binding sites on HSA were confirmed by site marker competition test and further proven by computational approaches. The recognition region of parathion-methyl was situated within residues 199-292 in subdomain IIA. Malathion bonded to residues 404-558 in subdomain IIIA. The mode of action between HSA-parathion-methyl and HSA-malathion is found to involve mainly by H-bonds, π-π stacking and hydrophobic effects. These results clearly demonstrate the noncovalent binding of OPs with HSA and provide new insight into solid-phase microextraction, thermodynamics and computational approaches.


Assuntos
Inseticidas/toxicidade , Compostos Organofosforados/toxicidade , Clorpirifos , Dimetilpolisiloxanos , Humanos , Inseticidas/química , Inseticidas/metabolismo , Malation/análise , Metil Paration , Compostos Organofosforados/química , Compostos Organofosforados/metabolismo , Albumina Sérica Humana/química , Albumina Sérica Humana/metabolismo , Microextração em Fase Sólida , Temperatura , Termodinâmica
16.
J Chromatogr A ; 1622: 461127, 2020 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-32331778

RESUMO

Gas chromatographic columns based on ionic liquids (ILs) are very promising since the selectivity of these columns can be tuned by both the cation and the anion chemical nature. In this paper, efficiencies of capillary columns based on four phosphonium ionic liquids were studied. The performance of seven columns containing the cation trihexyl(tetradecyl)phosphonium and the anions bromide, chloride, and bis(trifluoromethylsulfonyl)imide was evaluated by measuring the solute band broadening as a function of gas velocities at three temperatures. Hence, classical height equivalent to a theoretical plate (H) against gas velocity (u) plots corresponding to those columns were generated and the data were fitted to the Golay-Guiochon equation with the aim of seeking the optimum conditions to be operated each of them. Band broadening at practical gas velocities is mainly due to poor mass transfer properties of solutes in the (viscous) liquid phases, which limits the achieved efficiencies. These H/u plots proved to be necessary to characterize the column quality at a given temperature, to interpret the band broadening phenomena and thus, to establish the lower temperature limits and the expected plate counts at that temperature.


Assuntos
Cromatografia Gasosa/métodos , Líquidos Iônicos/química , Compostos Organofosforados/química , Ânions , Cátions , Difusão , Dióxido de Silício/química , Temperatura
17.
Biochem Pharmacol ; 177: 113980, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32305437

RESUMO

Human Cathepsin A (CatA) is a lysosomal serine carboxypeptidase of the renin-angiotensin system (RAS) and is structurally similar to acetylcholinesterase (AChE). CatA can remove the C-terminal amino acids of endothelin I, angiotensin I, Substance P, oxytocin, and bradykinin, and can deamidate neurokinin A. Proteomic studies identified CatA and its homologue, SCPEP1, as potential targets of organophosphates (OP). CatA could be stably inhibited by low µM to high nM concentrations of racemic sarin (GB), soman (GD), cyclosarin (GF), VX, and VR within minutes to hours at pH 7. Cyclosarin was the most potent with a kinetically measured dissociation constant (KI) of 2 µM followed by VR (KI = 2.8 µM). Bimolecular rate constants for inhibition by cyclosarin and VR were 1.3 × 103 M-1sec-1 and 1.2 × 103 M-1sec-1, respectively, and were approximately 3-orders of magnitude lower than those of human AChE indicating slower reactivity. Notably, both AChE and CatA bound diisopropylfluorophosphate (DFP) comparably and had KIDFP = 13 µM and 11 µM, respectively. At low pH, greater than 85% of the enzyme spontaneously reactivated after OP inhibition, conditions under which OP-adducts of cholinesterases irreversibly age. At pH 6.5 CatA remained stably inhibited by GB and GF and <10% of the enzyme spontaneously reactivated after 200 h. A crystal structure of DFP-inhibited CatA was determined and contained an aged adduct. Similar to AChE, CatA appears to have a "backdoor" for product release. CatA has not been shown previously to age. These results may have implications for: OP-associated inflammation; cardiovascular effects; and the dysregulation of RAS enzymes by OP.


Assuntos
Catepsina A/antagonistas & inibidores , Compostos Organofosforados/química , Compostos Organotiofosforados/química , Sarina/química , Soman/química , Acetilcolinesterase/química , Acetilcolinesterase/genética , Acetilcolinesterase/metabolismo , Sítios de Ligação , Catepsina A/química , Catepsina A/genética , Catepsina A/metabolismo , Linhagem Celular , Substâncias para a Guerra Química/química , Substâncias para a Guerra Química/toxicidade , Inibidores da Colinesterase/química , Inibidores da Colinesterase/toxicidade , Cristalografia por Raios X , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Expressão Gênica , Células HEK293 , Humanos , Isoflurofato/química , Isoflurofato/farmacologia , Cinética , Modelos Moleculares , Compostos Organofosforados/toxicidade , Compostos Organotiofosforados/toxicidade , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sarina/toxicidade , Soman/toxicidade , Especificidade por Substrato , Fatores de Tempo
18.
Chemistry ; 26(41): 8857-8861, 2020 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-32166818

RESUMO

We demonstrate the first mechanochemical synthesis of DNA fragments by ball milling, enabling the synthesis of oligomers of controllable sequence and length using multi-step, one-pot reactions, without bulk solvent or the need to isolate intermediates. Mechanochemistry allowed for coupling of phosphoramidite monomers to the 5'-hydroxyl group of nucleosides, iodine/water oxidation of the resulting phosphite triester linkage, and removal of the 5'-dimethoxytrityl (DMTr) protecting group in situ in good yields (up to 60 % over three steps) to produce DNA dimers in a one-pot manner. H-Phosphonate chemistry under milling conditions enabled coupling and protection of the H-phosphonate linkage, as well as removal of the 5'-DMTr protecting group in situ, enabling a one-pot process with good yields (up to 65 % over three steps, or ca. 87 % per step). Sulfurization of the internucleotide linkage was possible using elemental sulfur (S8) or sulfur transfer reagents, yielding the target DNA phosphorothioate dimers in good yield (up to 80 % over two steps). This work opens the door to creation of solvent-free synthesis methodologies for DNA and RNA therapeutics.


Assuntos
DNA/síntese química , Organofosfonatos/síntese química , Compostos Organofosforados/química , Fosfatos/química , Fosfitos/química , Indicadores e Reagentes , Nucleosídeos , Fosfatos/síntese química , RNA
19.
Talanta ; 212: 120735, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32113527

RESUMO

Changes in expression of Klotho gene are associated with chronic kidney disease and its potential as early biomarker is being studied. We report, for the first time, the detection of Klotho gene by a biosensor platform. Self-assembled mixed monolayers (SAMs) as DNA immobilization method in screen-printed gold electrodes and a sandwich format detection were used in the development of an electrochemical genosensor for the detection of a 100-mer DNA fragment, copy of the partial region of the mRNA Klotho gene. The use of different binary and ternary SAMs based on aliphatic (mercaptohexanol, MCH, and hexanedithiol, HDT) and aromatic (mercaptophenylacetic acid, MPAA) thiol diluents and capture probe (CP) as sensing phases was evaluated by cyclic voltammetry and electrochemical impedance spectroscopy. Multiple configurations were studied, changing the order of component addition and comparing co-immobilization and two-step immobilization processes. The procedure for binary SAM preparation consisting of sequential addition of a thiol diluent followed by CP was found to have the least detrimental impact on electrochemical performance. The signal-to-blank ratios increased considerably in the case of thioaromatic binary DNA monolayers, MPPA/CP, compared to the values obtained for aliphatic SAMs. Ternary monolayers formed by MCH and HDT rendered good fractional coverage levels and generated more reversible redox reactions at the surface, mostly when CP was firstly immobilized, CP/HDT/MCH. A significant reduction of the blank and non-specific (non-complementary sequence) signals was obtained with this ternary SAM, compared to binary SAMs and an increase of 2.42-fold of the S/B ratio (10 nM of target) compared with MPAA/CP SAMs. A linear response in the range of 5·10-10 to 5·10-8 M was obtained with CP/HDT/MCH monolayer, with a detection limit of 0.5 nM and RSD of 8.10%.


Assuntos
Técnicas Biossensoriais/métodos , DNA/análise , Técnicas Eletroquímicas/métodos , Glucuronidase/genética , Compostos de Sulfidrila/química , Fosfatase Alcalina/química , DNA/química , DNA/genética , Sondas de DNA/química , Sondas de DNA/genética , Fluoresceínas/química , Corantes Fluorescentes/química , Humanos , Ácidos Nucleicos Imobilizados/química , Ácidos Nucleicos Imobilizados/genética , Limite de Detecção , Naftalenos/química , Hibridização de Ácido Nucleico , Compostos Organofosforados/química
20.
Sensors (Basel) ; 20(5)2020 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-32131482

RESUMO

Pesticides represent some of the most common man-made chemicals in the world. Despite their unquestionable utility in the agricultural field and in the prevention of pest infestation in public areas of cities, pesticides and their biotransformation products are toxic to the environment and hazardous to human health. Esterase-based biosensors represent a viable alternative to the expensive and time-consuming systems currently used for their detection. In this work, we used the esterase-2 from Alicyclobacillus acidocaldarius as bioreceptor for a biosensing device based on an automated robotic approach. Coupling the robotic system with a fluorescence inhibition assay, in only 30 s of enzymatic assay, we accomplished the detection limit of 10 pmol for 11 chemically oxidized thio-organophosphates in solution. In addition, we observed differences in the shape of the inhibition curves determined measuring the decrease of esterase-2 residual activity over time. These differences could be used for the characterization and identification of thio-organophosphate pesticides, leading to a pseudo fingerprinting for each of these compounds. This research represents a starting point to develop technologies for automated screening of toxic compounds in samples from industrial sectors, such as the food industry, and for environmental monitoring.


Assuntos
Técnicas Biossensoriais/métodos , Organofosfatos/química , Compostos Organofosforados/química , Robótica/métodos , Alicyclobacillus/química , Bioensaio/métodos , Monitoramento Ambiental/métodos , Esterases/química , Fluorescência , Limite de Detecção , Praguicidas/química
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