Study of Inhibition,Reactivation and Aging Processes of Pesticides Using Graphene Nanosheets/Gold Nanoparticles‐Based Acetylcholinesterase Biosensor

Organophosphate (OP) and carbamate pesticides exert their toxicity via attacking the hydroxyl moiety of serine in the ‘active site’ of acetylcholinesterase (AChE). In this paper we developed a stable AChE biosensor based on self‐assembling AChE to graphene nanosheet (GN)‐gold nanoparticles (AuNPs) nanocomposite electrode for investigation of inhibition, reactivation and aging processes of different pesticides. It is confirmed that pesticides can inhibit AChE in a short time. OPs poisoning is treatable with oximes while carbarmates exposure is insensitive to oximes. The proposed electrochemical approach thus provides a new simple tool for comparison of pesticide sensitivity and guide of therapeutic intervention.     

QSAR for the Organophosphate-Induced Inhibition and ‚Aging‚ of the Enzyme Neuropathy Target Esterase (NTE)

Abstract

QSAR was devised for the neuropathy potency of various organophosphate (OP) compounds. The neuropathy-target-esterase (NTE) inhibition data were either obtained from the literature for a number of OP compounds or were determined experimentally for methamidophos, acephate, coumaphos and EPN. Aging Index that determined whether or not an OP would age NTE, correlated with molecular depth (MD) and the index density dipole-moment (density? Ω) (Eq. (1)). The t_1/2 values that represented the time (min) during which 50% of the OP-inhibited brain NTE undergoes ?aging‘, correlated with the topological indices Dif₃ and 1/Dif₄ (Eq. (2)). Log₁₀I₅₀ for AChE that determined the OP concentration causing 50% inhibition in AChE activity, correlated with E_bond and Charge^?1 (Eq. 3)). Log₁₀I₅₀ for NTE correlated with 1 HS² and H-Bonding (Eq. (4)). The (Log₁₀I₅₀NTE)/(Log₁₀I₅₀AChE) ratio that determined an OPs neuropathy potential relative to its cholinergic toxicity potential, correlated with log P and Log₁₀Polarity (Eq. (6)). Equation (3) accurately predicted AChE inhibition by methamidophos, coumaphos and EPN, but not by acephate. Equations (1), (2), (4)–(6), accurately predicted their respective biological indices. Therefore, it is proposed that the QSAR models developed in this study may accurately predict the neuropathy potential of OP compounds. The only exception is Eq. (3) that did not accurately predict the acephate-induced inhibition of AChE, possibly because acephate and other OPs inhibit the enzyme by distinct mechanisms.     

Molecular Recognition of Nerve Agents and Their Organophosphorus Surrogates: Toward Supramolecular Scavengers and Catalysts

Nerve agents are tetrahedral organophosphorus compounds (OPs) that were developed in the last century to irreversibly inhibit acetylcholinesterase (AChE) and therefore impede neurological signaling in living organisms. Exposure to OPs leads to a rapid development of symptoms from excessive salivation, nasal congestion and chest pain to convulsion and asphyxiation which if left untreated may lead to death. These potent toxins are prepared on a large scale from inexpensive staring materials, making it feasible for terrorist groups or states to use them against military and civilians. The existing antidotes provide limited protection and are difficult to apply to a large number of affected individuals. While new prophylactics are currently being developed, there is still need for therapeutics capable of both preventing and reversing the effects of OP poisoning. In this review, we describe how the science of molecular recognition can expand the pallet of tools for rapid and safe sequestration of nerve agents.     

2-methylindole analogs as cholinesterases and glutathione S-transferase inhibitors: Synthesis,biological evaluation,molecular docking,and pharmacokinetic studies

In this study, we aimed to (i) synthesize new 2-methylindole analogs containing various amino structures, pyrrolidine, piperidine, morpholine, and substituted phenyl groups through structural and molecular modifications, (ii) evaluate the pharmaceutical potential of 2-methylindole analogs via assessing enzyme inhibitory activity against glutathione S-transferase (GST), acetylcholinesterase (AChE), and butyrylcholinesterase (BChE), (iii) predict ADMET and pharmacokinetic properties of the synthesized 2-methylindole analogs, (iv) reveal the possible interactions between the synthesized 2-methylindole analogs with GST, AChE, and BChE enzymes using several molecular docking software. In vitro enzyme inhibition assays showed that the synthesized indole analogs exhibited moderate to good inhibitory activities against GST, AChE, and BChE enzymes. Briefly, the inhibitory activities of the analogs 4b and 4i against AChE, 4a and 4b against BChE, and analogs 1 and 4i against GST were detected to be higher or close to the standard inhibitor compounds. The analog 4b was detected to have the best inhibitory activity against both AChE and BChE enzymes with the lowest IC₅₀ values as 0.648 µM for AChE and 0.745 µM for BChE. The analyses of enzyme inhibition relationship with the synthesized analogs could help to design new analogs for the inhibitors of cholinergic and glutathione pathways based on the indole derivatives.     

膦酰化肟对乙酰胆碱酯酶的抑制作用及中毒酶重活化特性研究——基于效应标志物质谱定量分析技术

将对氧磷(Paraoxon)与肟类重活化剂双复磷(Obidoxime)反应制得膦酰化肟(DEP-obidoxime);利用基于效应标志物的质谱定量技术研究了膦酰化肟对乙酰胆碱酯酶(AChE)的抑制作用及中毒酶的重活化特性.结果表明,膦酰化肟具有极强的AChE抑制毒性,但因膦酰化肟中毒酶与原有机磷毒物中毒酶结构相同,故其膦酰化AChE仍可被经典重活化剂[如氯解磷定(Pralidoxime)、双复磷(Obidoxime)及酰胺磷定(HI-6)]重活化,根据EC_(50)表征结果,这3种重活化剂的重活化效果强弱顺序依次为氯解磷定HI-6双复磷.     

Rapid Detection of Irreversible Acetylcholineasterase Inhibitor by Mass Spectrometry Assay

Here we developed a rapid method to detect acetylcholinesterase (AChE) activity by matrix‐assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI‐FTMS) for screening irreversible AChE inhibitors. Due to its good salt‐tolerance and low sample consumption, MALDI‐FTMS could facilitate rapid detection, especially detection in real application. AChE activity was determined through calculating abundance of substrate and product in mass spectrometry. By this approach, we investigated the relation of organophosphorous (OP) concentrations and AChE inhibition. Shown in different inhibition curves from different OP pesticides, enzyme inhibitions still kept good correlation with concentration of OPs. Finally, this AChE‐inhibited method was applied to screen whole bloods of four decedents and discuss their death reason. In contrast to healthy persons, three of decedents showed low AChE activity, and probably died for irreversible AChE inhibitors. Through the following detecting in GC‐MS/MS, the possible death reason of these three decedents was confirmed, and another decedent actually died for sumicidin, a non‐AChE inhibitor. It demonstrated that screening irreversible AChE inhibitors by detecting enzyme activity in MALDI‐FTMS provided fast and accurate analysis results and excluded another toxicants not functioning on AChE. This method offered alternative choices for indicating the existence of enzyme inhibitors.     

Reversal of Tabun Toxicity Enabled by a Triazole-Annulated Oxime Library—Reactivators of Acetylcholinesterase

Acetylcholinesterase (AChE), an enzyme that degrades the neurotransmitter acetylcholine, when covalently inhibited by organophosphorus compounds (OPs), such as nerve agents and pesticides, can be reactivated by oximes. However, tabun remains among the most dangerous nerve agents due to the low reactivation efficacy of standard pyridinium aldoxime antidotes. Therefore, finding an optimal reactivator for prophylaxis against tabun toxicity and for post-exposure treatment is a continued challenge. In this study, we analyzed the reactivation potency of 111 novel nucleophilic oximes mostly synthesized using the CuAAC triazole ligation between alkyne and azide building blocks. We identified several oximes with significantly improved in vitro reactivating potential for tabun-inhibited human AChE, and in vivo antidotal efficacies in tabun-exposed mice. Our findings offer a significantly improved platform for further development of antidotes and scavengers directed against tabun and related phosphoramidate exposures, such as the Novichok compounds.     

Acetylcholinesterase-Inhibiting Activity of Salicylanilide <em>N</em>-Alkylcarbamates and Their Molecular Docking

A series of twenty-five novel salicylanilide N-alkylcarbamates were investigated as potential acetylcholinesterase inhibitors. The compounds were tested for their ability to inhibit acetylcholinesterase (AChE) from electric eel (Electrophorus electricus L.). Experimental lipophilicity was determined, and the structure-activity relationships are discussed. The mode of binding in the active site of AChE was investigated by molecular docking. All the discussed compounds expressed significantly higher AChE inhibitory activity than rivastigmine and slightly lower than galanthamine. Disubstitution by chlorine in C'_(3,4) of the aniline ring and the optimal length of hexyl-undecyl alkyl chains in the carbamate moiety provided the most active AChE inhibitors. Monochlorination in C'₍₄₎ exhibited slightly more effective AChE inhibitors than in C'₍₃₎. Generally it can be stated that compounds with higher lipophilicity showed higher inhibition, and the activity of the compounds is strongly dependent on the length of the N-alkyl chain.     

Adsorption: an easy and efficient immobilisation of acetylcholinesterase on screen-printed electrodes

An amperometric biosensor based on acetylcholinesterase was constructed by simple adsorption of the enzyme on screen-printed electrodes (SPEs). This sensor was used to detect the inhibitory effects of organophosphorus and carbamate insecticides on acetylcholinesterase, and more particularly of chlorpyrifos ethyl oxon (CP-o). We demonstrate that enzyme adsorption on SPEs allows to obtain stable sensors that present good characteristics and are as efficient as other screen-printed biosensors based on covalent binding or entrapment of acetylcholinesterase (AChE).     

Assisted inhibition effect of acetylcholinesterase with n-octylphosphonic acid and application in high sensitive detection of organophosphorous pesticides by matrix-assisted laser desorption/ionization Fourier transform mass spectrometry

A simple and practical approach to improve the sensitivity of acetylcholinesterase (AChE)-inhibited method has been developed for monitoring organophosphorous (OP) pesticide residues. In this work, matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) was used to detect AChE activity. Due to its good salt-tolerance and low sample consumption, MALDI-FTMS facilitates rapid and high-throughput screening of OP pesticides. Here we describe a new method to obtain low detection limits via employing external reagents. Among candidate compounds, n-octylphosphonic acid (n-Octyl-PA) displays assistant effect to enhance AChE inhibition by OP pesticides. In presence of n-Octyl-PA, the percentages of AChE inhibition still kept correlation with OP pesticide concentrations. The detection limits were improved significantly even by 10²–10³ folds in comparison with conventional enzyme-inhibited methods. Different detection limits of OP pesticides with different toxicities were as low as 0.005 μg L⁻¹ for high toxic pesticides and 0.05 μg L⁻¹ for low toxic pesticides. Besides, the reliability of results from this method to analyze cowpea samples had been demonstrated by liquid-chromatography tandem mass spectrometry (LC–MS/MS). The application of this commercial available assistant agent shows great promise to detect OP compounds in complicated biological matrix and broadens the mind for high sensitivity detection of OP pesticide residues in agricultural products.     

O-[7-(1-Methylquinolinium)]-1,3,2-Dioxaphosphorinane 2-Oxide Iodide: A Reversible Anti-acetylcholinesterase and Irreversible Anti-butyrylcholinesterase Organophosphorus Ester

Abstract

Although cyclic organophosphates(OP) esters (I) and their open-chain analogs (II) demonstrate similar reactivity of a P atom towards nucleophilic displacement in aqueous solutions, the open-chain analogs (II) are thousand times more active as inhibitors of acetylcholinesterase(AChE). In order to explain the poor anti-ChE activity of I, a covalent molecular combination,IV, of the cyclic phosphate and an extremely effective leaving group (III) was prepared and evaluated. The new compound, IV, was found to inhibit progressively horse-serum butyrylcholinesterase(BuChE)at t^1/2=15 min.(2.9μM, pH 7.0, 25°) whereas no progressive inhibition could be demonstrated for eel AChE incubated for several hrs under the same experimental conditions.Eel AChE was inhibited reversibly by IV with affinity constant,KI=1.3×10^?6 M. These findings may suggest the following: a. In order to compensate for overcrowding of the AChE active-site by large substituents it is essential to maintain flexibility of the four ligands attached to the P atom. It is further infered that cyclization does not permit such flexibility. b. BuChE differ considerably from AChE in the ability of the enzyme to provide simultaneous four-site interaction with tetrahedral OP inhibitors.     

Physovenines: Efficient Synthesis of (−)- and (+)-Physovenine and Synthesis of Carbarnate Analogues of (−)-Physovenine. Anticholinesterase Activity and Analgesic Properties of Optically Active Physovenines

Column chromatography of easy available (±)-physovenine ( 2 ) on cellulose triacetate afforded (?)- and (+)-physovenine ( 2a and 2b , resp.). Alkaloids 2a , b required for pharmacological testing were prepared from eserolincs ( 3a , b ) by an improved procedure. Natural (?)-physovenine ( 2a ) was equally potent in inhibiting AChE and BChE in vitro as natural physostigmine (1a), and twice as potent as the unnatural antipode 2b against AChE and 14 times as potent against BChE. Several carbamate analogs of 2a were at least as potent as the former compound in these assays. None of the compounds tested did bind to different opiate receptor or serotonine receptor preparations. Most of the compounds tested had considerable analgesic activity in the Writhing test.     

Myeloperoxidase-mediated oxidation of organophosphorus pesticides as a pre-step in their determination by AChE based bioanalytical methods

In order to improve the sensitivity of assays for inhibitors of the enzyme acetylcholine esterase (AChE), an effective method was developed for the conversion of the organophosphate pesticides (OPs) diazinon, malathion, chlorpyrifos, azinphos-methyl and phorate into more toxic inhibitors. This was accomplished by converting them from the thio form into their oxo form using the enzyme myeloperoxidase. The oxo forms, which are the only products of conversion, were determined by AChE bioassays, using either the free enzyme, or a flow injection analysis manifold with immobilized AChE and spectrophotometric detection. All modified OPs exhibited inhibitory power at ppb levels and within 10 min. The method is considered to represent an excellent means for improving the sensitivity of assays for determination of OPs.     

Design and synthesis of carboxylate inhibitors for matrix metalloproteinases

A series of carboxylate compounds were prepared from N(alpha)-substituted 2,3-diaminopropionic acid and were tested for efficacy as matrix metalloproteinase (MMP) inhibitors. During modeling of the initial compound 10a, we utilized three-dimensional structure modeling software (InsightII/Discover Ver. 2.98). Some of the prepared carboxylate derivatives, such as carbamate compounds (12c,d, 22) and sulfonamide compounds (14b,c), proved to be effective MMP-1 inhibitors (with IC50 values of a 10(-6) M order), depending on the substituent at the N(alpha)-position of 2,3-diaminopropionic acid. Some of them were also evaluated for inhibition of stromelysin-1 (MMP-3), and the sulfonamide compound 14c exceeded the lead compound 5b in its MMP-3 inhibitory potency. For the carbamate compounds, we investigated the minimum molecular size at which the MMP-1 inhibitory potency was maintained, and found that this was P3-P1' compound 10b.     

A mediator-free screen-printed amperometric biosensor for screening of organophosphorus pesticides with flow-injection analysis (FIA) system

A mediator-free amperometric biosensor for screening organophosphorus pesticides (OPs) in flow-injection analysis (FIA) system based on anticholinesterase activity of OPs to immobilized acetylcholinesterase enzyme (AChE) has been developed. The enzyme biosensor is prepared by entrapping AChE in Al₂O₃ sol-gel matrix screen-printed on an integrated 3-electrode plastic chip. This strategy is found not only increase the stability of the embedded AChE, but also effectively catalyze the oxidative reaction of thiocholine, making the Al₂O₃-AChE biosensor detects the substrate at 0.25 V (versus Ag/AgCl), hundreds mini-volt lower than other reported mediator-free ones. The Al₂O₃-AChE biosensor is thus coupled to FIA system to build up a simple and low-cost FIA-EC system for screening OPs in real samples. A wide linear inhibition response for dichlorvos, typical OP, is observed in the range of 0.1-80 μM, corresponding to 7.91-84.94% inhibition for AChE. The detection limit for dichlorvos is achieved at 10 nM in the simulated seawater for 15 min inhibiting time, which allows the biosensor quantitatively detects the ecotoxicological effect of the real samples from the seaports in eastern China, where the OPs pollution is confirmed by GC-MS.     

Ionic liquid-functionalized graphene for fabricating an amperometric acetylcholinesterase biosensor

This work reports a sensitive amperometric biosensor for organophosphate pesticides (OPs) fabricated by modifying a glassy carbon electrode with acetylcholinesterase (AChE) immobilized on ionic liquid-functionalized graphene (IL-G). The functionalized graphene sheets had good dispersibility and long-term stability in various solvents. The as-prepared biosensor showed high affinity to acetylthiocholine (ATCl) with a Michaelis-Menten constant (K(m)) value of 0.77 mM. Furthermore, based on the inhibition by OPs of the enzymatic activity of the immobilized AChE, and using carbaryl as a model compound, the inhibition of carbaryl was proportional to its concentration ranging from 0.0025 to 0.48 and 0.48 to 1.42 μg mL(-1) with a detection limit of 0.8 ng mL(-1) (S/N = 3). The developed biosensor exhibited a good performance for OPs detection, including good reproducibility and acceptable stability, which provided a new and promising tool for the analysis of enzyme inhibitors.     

Determination of Toxic Organophosphorus Compounds by Specific and Nonspecific Detectors

Abstract

Sample preparation procedures and gas chromatography methodology are presented for the determination of tabun, sarin, soman, and VX in aqueous solutions. Extraction recoveries from chloroform were quantitative. Peak area ratios of organophosphorus compounds (OPs) to internal standard versus concentrations of OP were linear over the range of 10-1000 (μg/ml when determined by the flame ionization detector and 10-800 μg/ml when determined by the flame photometric detector. Imprecision occurring at low ng concentrations of VX was caused by its adsorption on the analytical column. Acceptable precision was regained by the addition of a weak base, such as atropine, to the sample extract prior to its injection onto the gas chromatograph (GC).     

纳米阻燃高分子材料:现状、问题及展望

纳米阻燃体系是一种新型的聚合物阻燃体系,被誉为阻燃技术的革命.极少量(≤5wt%)纳米阻燃剂的加入即能显著降低高分子材料燃烧时的热释放速率(HRR)和烟密度(SEA),延缓其燃烧过程,还能不同程度地提高材料的力学性能.本文总结了近年来国内外纳米阻燃领域的进展,介绍了本课题组在纳米阻燃方面所做的工作,探讨了纳米阻燃研究中存在的问题,并对其未来的发展进行了展望.     

1,3,5-Triazine Nitrogen Mustards with Different Peptide Group as Innovative Candidates for AChE and BACE1 Inhibitors

A series of new analogs of nitrogen mustards (4a–4h) containing the 1,3,5-triazine ring substituted with dipeptide residue were synthesized and evaluated for the inhibition of both acetylcholinesterase (AChE) and β-secretase (BACE1) enzymes. The AChE inhibitory activity studies were carried out using Ellman’s colorimetric method, and the BACE1 inhibitory activity studies were carried out using fluorescence resonance energy transfer (FRET). All compounds displayed considerable AChE and BACE1 inhibition. The most active against both AChE and BACE1 enzymes were compounds A and 4a, with an inhibitory concentration of AChE IC₅₀ = 0.051 µM; 0.055 µM and BACE1 IC₅₀ = 9.00 µM; 11.09 µM, respectively.     

聚乳酸阻燃性能的研究进展

聚乳酸作为一种资源与环境友好材料已得到了广泛深入的研究。如果能够提高聚乳酸的阻燃性能,则能进一步扩大其应用范围。目前对聚乳酸的阻燃改性主要采用添加阻燃剂的方法,并以磷系、氮系、硅系、金属化合物阻燃剂以及多种阻燃成分的复配为主,而聚乳酸的反应型阻燃也在不断研究发展中。本文在介绍阻燃作用机理的基础上,综述了聚乳酸阻燃研究发展现状,并对聚乳酸的阻燃提出展望。