Inhibition Of Electric Eel Acetylcholinesterase Research Articles

Acetylcholinesterase Inhibitory Activities of Essential Oils from Vietnamese Traditional Medicinal Plants.

Essential oils are promising as environmentally friendly and safe sources of pesticides for human use. Furthermore, they are also of interest as aromatherapeutic agents in the treatment of Alzheimer’s disease, and inhibition of the enzyme acetylcholinesterase (AChE) has been evaluated as an important mechanism. The essential oils of some species in the genera Callicarpa, Premna, Vitex and Karomia of the family Lamiaceae were evaluated for inhibition of electric eel AChE using the Ellman method. The essential oils of Callicarpa candicans showed promising activity, with IC50 values between 45.67 and 58.38 μg/mL. The essential oils of Callicarpa sinuata, Callicarpa petelotii, Callicarpa nudiflora, Callicarpa erioclona and Vitex ajugifolia showed good activity with IC50 values between 28.71 and 54.69 μg/mL. The essential oils Vitex trifolia subsp. trifolia and Callicarpa rubella showed modest activity, with IC50 values of 81.34 and 89.38, respectively. trans-Carveol showed an IC50 value of 102.88 µg/mL. Molecular docking and molecular dynamics simulation were performed on the major components of the studied essential oils to investigate the possible mechanisms of action of potential inhibitors. The results obtained suggest that these essential oils may be used to control mosquito vectors that transmit pathogenic viruses or to support the treatment of Alzheimer’s disease.

Synthesis and Anticholinesterase Evaluation of Cassine, Spectaline and Analogues

In this work, twelve analogues of piperidine alkaloids (-)-cassine and (-)-spectaline were synthesized, as well as the racemic forms of these natural products. The compounds were evaluated for their inhibition of electric eel acetylcholinesterase (AChEee) and human butyrylcholinesterase (BChEhu) by on-flow mass-spectrometry-based dual-enzyme assay, and the inhibition mechanisms for the most potent analogues were also determined. Our results showed a preference for BChEhu inhibition with compounds 10c (Ki = 5.24 μM), 12b (Ki = 17.4 μM), 13a (Ki = 13.2 μM) and 3 (Ki = 11.3 μM) displaying the best inhibitory activities.

Kinetics of acetylcholinesterase inhibition by hemp seed protein-derived peptides.

The aim of this work was to enhance the acetylcholinesterase (AChE)-inhibitory activity of a pepsin-produced hemp seed protein hydrolysates (HPH) through reverse-phase HPLC separation followed by identification of peptide sequences present in the most active fraction. The HPH was separated into eight fractions (F1-F8) with F7 exhibiting significantly (p<0.05) the strongest (97.5%) in vitro inhibition of electric eel AChE (eeAChE) activity in comparison to 53.8% for HPH. The HPH consisted mostly of low molecular weight peptides of<11 amino acid residues and most contained at least one hydrophobic amino acid. Kinetics of enzyme inhibition revealed a mixed-type inhibition of eeAChE activity by HPH whereas F7 acted through an uncompetitive mode; in contrast inhibition of human AChE by HPH and F7 was uncompetitive. The stronger inhibitory potency of the F7 peptides fraction against both enzymes was confirmed through reduced maximal velocity, catalytic efficiency, and inhibition constant values when compared to the HPH. PRACTICAL APPLICATIONS: The use of natural products for the prevention or treatment of human diseases continues to be an area of intense research activities. Food protein-derived peptides obtained through enzymatic hydrolysis of hemp seed proteins were shown in vitro to be strong inhibitors of activities of both the eel and human forms of acetylcholinesterase (AChE). AChE is an important therapeutic target because excessive activity of this enzyme is a causative factor of neurodegenerative diseases such as dementia and Alzheimer's. This work showed that peptides in the most active fraction are small in sizes, which may favor their absorption into blood circulation and possible permeation of the blood-brain barrier. Therefore, the hemp seed peptides are potential agents that can be used to formulate functional foods and nutraceuticals against neurodegenerative diseases.

Multitarget Therapeutic Leads for Alzheimer's Disease: Quinolizidinyl Derivatives of Bi- and Tricyclic Systems as Dual Inhibitors of Cholinesterases and β-Amyloid (Aβ) Aggregation.

Multitarget therapeutic leads for Alzheimer's disease were designed on the models of compounds capable of maintaining or restoring cell protein homeostasis and of inhibiting β-amyloid (Aβ) oligomerization. Thirty-seven thioxanthen-9-one, xanthen-9-one, naphto- and anthraquinone derivatives were tested for the direct inhibition of Aβ(1-40) aggregation and for the inhibition of electric eel acetylcholinesterase (eeAChE) and horse serum butyrylcholinesterase (hsBChE). These compounds are characterized by basic side chains, mainly quinolizidinylalkyl moieties, linked to various bi- and tri-cyclic (hetero)aromatic systems. With very few exceptions, these compounds displayed inhibitory activity on both AChE and BChE and on the spontaneous aggregation of β-amyloid. In most cases, IC50 values were in the low micromolar and sub-micromolar range, but some compounds even reached nanomolar potency. The time course of amyloid aggregation in the presence of the most active derivative (IC50 =0.84 μM) revealed that these compounds might act as destabilizers of mature fibrils rather than mere inhibitors of fibrillization. Many compounds inhibited one or both cholinesterases and Aβ aggregation with similar potency, a fundamental requisite for the possible development of therapeutics exhibiting a multitarget mechanism of action. The described compounds thus represent interesting leads for the development of multitarget AD therapeutics.

Single and simultaneous exposure of acetylcholinesterase to diazinon, chlorpyrifos and their photodegradation products

In vitro inhibition of electric eel acetylcholinesterase (AChE) by single and simultaneous exposure to organophosphorus insecticides diazinon and chlorpyrifos, and their transformation products, formed due to photoinduced degradation, was investigated. Increasing concentrations of diazinon, chlorpyrifos and their oxidation products, diazoxon and chlorpyrifos-oxon, inhibited AChE in a concentration-dependent manner. IC 50 (20 min) values, obtained from the inhibition curves, were (in mol/l): (5.1 ± 0.3) × 10 −8, (4.3 ± 0.2) × 10 −6 and (3.0 ± 0.1) × 10 −8 for diazoxon, chlorpyrifos and chlorpyrifos-oxon, respectively, while maximal diazinon concentration was lower than its IC 50 (20 min). Calculated K I values, in mol/l, of 7.9 × 10 −7, 9.6 × 10 −6 and 4.3 × 10 −7 were obtained for diazoxon, chlorpyrifos and chlorpyrifos-oxon, respectively. However, 2-isopropyl-4-methyl-6-pyrimidinol (IMP) and 3,5,6-trichloro-2-pyridinol, diazinon and chlorpyrifos hydrolysis products, did not noticeably affect the enzyme activity at all investigated concentrations. Additive inhibition effect was achieved for lower concentrations of the inhibitors (diazinon/diazoxon ⩽1 × 10 −4/1 × 10 −8 mol/l i.e., chlorpyrifos/chlorpyrifos-oxon ⩽2 × 10 −6/3 × 10 −8 mol/l), while an antagonistic effect was obtained for all higher concentrations of the organophosphates. Inhibitory power of 1 × 10 −4 mol/l diazinon irradiated samples can be attributed mostly to the formation of diazoxon, while the presence of non-inhibiting photodegradation product IMP did not affect diazinon and diazoxon inhibitory efficiencies.

Inhibition of electric eel acetylcholinesterase by triarylmethane dyes

The effects of three cationic triarylmethane dyes – pararosaniline (PR), malachite green (MG), methyl green (MetG) – on electric eel AChE (eAChE) activity were tested at 25 °C, in 100 mM MOPS buffer (pH 8) containing 0.125 mM 5-5-dithio-bis(2-nitrobenzoic acid), 20–120 μM acetylthiocholine and 0–20 μM dye. All three dyes caused reversible, linear- or hyperbolic-mixed inhibition of esteratic activity. The respective inhibitory parameters for PR, MG and MetG were K i = 8.4 ± 0.67, 1.9 ± 0.51 and 0.27 ± 0.017 μM; α (competitive coefficient) = 5.8 ± 2.0, 4.8 ± 1.8 and 2.7 ± 0.32; β (noncompetitive coefficient) = 0, 0 and 0.20 ± 0.011. The data were consistent with ligand binding at the peripheral site and a remote effect on substrate binding and turnover.

Pregnane-Type Steroidal Alkaloids ofSarcococca saligna: a New Class of Cholinesterase Inhibitors

Phytochemical investigation of Sarcococca saligna by extensive bioassay-guided fractionation resulted in the isolation of the pregnane-type steroidal alkaloids 1–15, i.e. of the five new compounds 1–5 and the ten known alkaloids 6–15. The structures of the new alkaloids salignenamide C (1), salignenamide D (2), 2β-hydroxyepipachysamine D (3), salignenamide E (4), and salignenamide F (5) were elucidated with the help of modern spectroscopic techniques, while the known alkaloids axillarine C (6), axillarine F (7), sarcorine (8), N3-demethylsaracodine (9), saligcinnamide (10), salignenamide A (11), vaganine A (12), axillaridine A (13), sarsalignone (14), and sarsalignenone (15) were identified by comparing their spectral data with those reported earlier. Inhibition of electric-eel acetylcholinesterase (EC 3.1.1.7) and horse-serum butyrylcholinesterase (EC 3.1.1.8) by alkaloids 1–15 were investigated. These new cholinesterase inhibitors may act as potential leads in the discovery of clinically useful inhibitors for nervous-system disorders, particularly by reducing memory deficiency in Alzheimer's disease patients by potentiating and effecting the cholinergic transmission process. These compounds were found to inhibit both enzymes in a concentration-dependent fashion with the IC50 values ranging from 5.21–227.92 μM against acetylcholinesterase and 2.18–38.36 μM against butyrylcholinesterase.

New cholinesterase inhibiting steroidal alkaloids from the leaves of Sarcococca coriacea of Nepalese origin.

From the leaves of Sarcococca coriacea two new steroidal alkaloids, epoxynepapakistamine-A [(20S)-20-(N-methylamino)-3beta-(tigloylamino)-5alpha-pregna-16alpha,17alpha-epoxy-2beta,4beta-di-O-acetate] (1), and epoxysarcovagenine-D [(20S)-20-(N-methylamino)-3beta-(tigloylamino)-5alpha-pregna-2-en-16alpha,17alpha-epoxy-4-one] (2), and two known compounds funtumafrine C [(20S)-20-(N,N-dimethylamino)-5alpha-pregna-3-one] (3) and N-methylfuntumine (4) were isolated. Their structures were elucidated on the basis of their spectral properties. The compounds 1, 3 and 4 were found to have cholinesterase inhibitory activity when tested for the inhibition of electric eel acetylcholinesterase and horse serum butyrylcholinesterase. They inhibited both enzymes in a concentration dependent fashion.

Inhibition of acetylcholinesterase by (1S,3S)-isomalathion proceeds with loss of thiomethyl: kinetic and mass spectral evidence for an unexpected primary leaving group.

Previous work demonstrated kinetically that inhibition of mammalian acetylcholinesterase (AChE) by (1S)-isomalathions may proceed by loss of thiomethyl instead of the expected diethyl thiosuccinate as the primary leaving group followed by one of four possible modes of rapid aging. This study sought to identify the adduct that renders AChE refractory toward reactivation after inhibition with the (1S, 3S)-stereoisomer. Electric eel acetylcholinesterase (EEAChE) was inhibited with the four stereoisomers of isomalathion, and rate constants for spontaneous and oxime-mediated reactivation (k(3)) were measured. Oxime-mediated k(3) values were >25-fold higher for enzyme inhibited by (1R)- versus (1S)-stereoisomers with the greatest contrast between the (1R,3R)- and (1S,3S)-enantiomers. EEAChE inactivated by (1R,3R)-isomalathion reactivated spontaneously and in the presence of pyridine-2-aldoxime methiodide (2-PAM) with k(3) values of 1.88 x 10(5) and 4.18 x 10(5) min(-)(1), respectively. In contrast, enzyme treated with the (1S,3S)-enantiomer had spontaneous and 2-PAM-mediated k(3) values of 0 and 6.05 x 10(3) min(-)(1), respectively. The kinetic data that were measured were consistent with those obtained for mammalian AChE used in previous studies. Identification of the adduct that renders EEAChE stable toward reactivation after inhibition with (1S,3S)-isomalathion was accomplished using a peptide mass mapping approach with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). A peak with a mass corresponding to the active site peptide containing the catalytic Ser with a covalently bound O-methyl phosphate adduct was found in the mass spectra of (1S, 3S)-treated EEAChE but not control samples. Identities of the modified active site peptide and adduct were confirmed by fragmentation in MALDI-TOF-MS post-source decay (PSD) analysis, and peaks corresponding to the loss of an adduct as phosphorous/phosphoric acid methyl ester were observed. The results demonstrate that inhibition of EEAChE by (1S,3S)-isomalathion proceeds with loss of thiomethyl as the primary leaving group followed by rapid expulsion of diethyl thiosuccinate as the secondary leaving group to yield an aged enzyme.

Inhibition of electric eel acetylcholinesterase by porphin compounds.

Synthetic porphin compounds have been found to be reversible inhibitors of acetylcholinesterase from electric eel with Ki values of microM range. It seems that the number and position of fluorine on the phenyl ring and metal of an inhibitor play an important role for binding of an inhibitor to the enzyme active site.

Quantitative structure-activity analysis of acetylcholinesterase inhibition by oxono and thiono analogues of organophosphorus compounds.

A comparison of the bimolecular rate constants (ki) for inhibition of electric eel acetylcholinesterase (AChE) by the oxono (i.e., P=O) and thiono (i.e., P=S) analogues of parathion, methylparathion, leptophos, fonofos, sarin, and soman revealed that the oxono/thiono ratios of ki values varied from 14 for soman to 1240 for parathion. Analysis of the relative importance of the dissociation equilibrium constant and the phosphorylation rate constant in producing this variation in ki values indicated that the oxono analogues and the phosphorylation rate constant values that varied in a narrow range from 8- to 14-fold greater than their thiono counterparts, while the oxono/thiono ratios for dissociation constants varied widely from 1 for soman to 82 for fonofos. The lower affinities of thiono analogues for AChE probably resulted from differences in the hydrophobic binding of oxono and thiono analogues to the active site of AChE, inasmuch as the hydrophobicities (i.e., octanol/water partition coefficients) of thiono organophosphorus compounds were much greater than the hydrophobicities of their oxono analogues. Quantitative structure--activity analysis indicated that the hydrophobic effects of oxono and thiono moieties correlated with log ki for AChE inhibition to a greater extent (r2 = 0.79) than their electronic effects (r2 less than or equal to 0.48). These observations suggest that the differences in hydrophobicity of oxono and thiono analogues of organophosphorus compounds may be as important as their electronic differences in determining their effectiveness as AChE inhibitors.

Anatoxin-a(s), an anticholinesterase from the cyanobacterium Anabaena flos-aquae NRC-525-17

N. A. Mahmood and W. W. Carmichael. Anatoxin-a(s), an anticholinesterase from the cyanobacterium Anabaena flos-aquae NRC-525-17. Toxicon 25, 1221 – 1227, 1987. — Anatoxin-a(s) [antx-a(s)] given intraperitoneally to Sprague-Dawley rats at different doses (0.1 – 1.0 mg/kg) caused signs of severe cholinergic overstimulation. Assays of rat blood acetylcholinesterase (AChE) revealed a dose-dependent inhibition. The in vitro inhibition of electric eel acetylcholinesterase (AChE, E.C. 3.1.1.7) and horse serum butyrylcholinesterase (BUChE, E.C. 3.1.1.8) by antx-a(s) was time- and concentration-dependent. The inhibition of electric eel AChE follows first order kinetics, indicative of irreversible inhibition. The irreversibility of electric eel AChE inhibition was confirmed by a plot of V max versus total enzyme concentration [ E T ]. The kinetics of inhibition of cholinesterase by antx-a(s) supports the previous pharmacological findings that antx-a(s) is an anticholinesterase and that signs of intoxication by it are primarily due to cholinesterase inhibition.

The anticholinesterase activity of mefloquine.

The characteristics of the inhibition of electric eel acetylcholinesterase (AChE) and horse serum butyrylcholinesterase (BChE) by the antimalarial agents mefloquine, chloroquine, amodiaquine and amopyroquine were determined. The antimalarials were found to be non-competitive inhibitors of both AChE and BChE. In both enzyme systems, inhibitory potencies were in the order amodiaquine greater than amopyroquine greater than chloroquine greater than mefloquine. The low inhibitory potency of mefloquine may account in part for the appearance of gastrointestinal and central nervous system disturbances only at high doses of the drug.

Isolation, in vitro activity, and acute toxicity in mice of the four stereoisomers of soman

We report the isolation, on a 0.1- to 2-mg scale, of the four stereoisomers of the nerve agent pinacolyl methylphosphonofluoridate (soman), assigned as C(+)P(+), C(+)P(−), C(−)P(+), and C(−)P(−), with more than 99% optical purity. This result was realized by means of (i) complete optical resolution of pinacolyl alcohol, (ii) synthesis of C(+)- and C(−)-soman from the (+)- and (−)-enantiomers of the alcohol, (iii) optimalization of conditions for stereospecific inhibition of α-chymotrypsin with the P(−)-isomers of C(+)- and C(−)-soman, followed by isolation of the C(+)P(+)- and C(−)P(+)-isomers, (iv) isolation of the C(+)P(−)- and C(−)P(−)-isomers after incubation in rabbit plasma of C(+)- and C(−)-soman, respectively, which hydrolyzes stereospecifically the P(+)-isomers. Solutions (0.2 m m) of the steroisomers in organic solvents are optically stable for at least a month at −20°C. The bimolecular rate constants for inhibition of electric eel acetylcholinesterase (AChE) at pH 7.7, 25°C, are at least 3.6 × 10 4 larger for the P(−)- than for the P(+)-isomers. AChE from electric eel as well as from mouse erythrocytes inhibited with C(+)P(−)-soman is much more effectively reactivated with the oximes HI-6, HGG-42, and obidoxime than these enzymes inhibited with C(−)P(−)-soman. Similarly, the neuromuscular transmission (NMT) in mouse diaphragm strips poisoned with C(+)-soman is more easily restored with HI-6 than NMT of such muscle preparations poisoned with C(−)-soman. The LD50 values (mice, sc) are in accordance with the P(−) P(+) -ratio of inhibition rates of AChE, i.e. 99, 38, >5000, >2000, 214, 133, and 156 μg/kg for C(+)P(−)-, C(−)P(−)-, C(+)P(+)-, C(−)P(+), C(+)-, C(−)-soman, and “soman,” respectively. We suggest that mice challenged with a lethal dose of soman (sc) are killed primarily by the C(−)P(−)-isomer.

Isolation, anticholinesterase properties, and acute toxicity in mice of the four stereoisomers of the nerve agent soman

The four stereoisomers of the nerve agent pinacolyl methylphosphonofluoridate (soman), designated as C(+)P(+), C(+)P(−), C(−)P(+), and C(−)P(−), have different toxicologic properties due to stereospecific interactions in living organisms. We report the isolation of these stereoisomers with more than 99% optical purity. This result was realized by means of (i) complete optical resolution of pinacolyl alcohol, (ii) synthesis of C(+)- and C(−)-soman from the (+)- and (−)-enantiomers of the alcohol, (iii) optimalization of conditions for stereospecific inhibition of α-chymotrypsin with the P(−)-isomers of C(+)- and C(−)-soman, followed by isolation of the C(+)P(+)- and C(−)P(+)-isomers, (iv) isolation of the C(+)P(−)- and C(−)P(−)-isomers after incubation of C(+)- and C(−)-soman, respectively, in rabbit plasma, which hydrolyzes stereospecifically the P(+)-isomers. The bimolecular rate constants for inhibition of electric eel acetylcholinesterase (AChE) at pH 7.7, 25°C, are at least 3.6 × 10 4 larger for the P(−)- than for the P(+)-isomers. The enzyme inhibited with C(+)P(−)-soman is much more effectively reactivated with the oximes HI-6, HGG-42, and obidoxime than AChE inhibited with C(−)P(−)-soman. The LD50 values (sc, mice) are in accordance with the P(−) P(+) ratio of inhibition rates of AChE, i.e. 99, 38, >5000, >2000, 214, 133, and 156 μg/kg for C(+)P(−)-, C(−)P(−)-, C(+)P(+)-, C(−)P(+)-, C(+)-, C(−)-soman, and “soman,” respectively. The relative LD50 values of the C(−)P(−)- and C(+)P(−)-isomers do not correspond with the small differences in their rates of inhibition of AChE, indicating that such small rate ratios may be overruled by other stereospecific effects, e.g., in vivo rates of detoxification.

Isolation, in Vitro Activity, and Acute Toxicity in Mice of the Four Stereoisomers of Soman

Isolation, in Vitro Activity, and Acute Toxicity in Mice of the Four Stereoisomers of Soman. BENSCHOP, H. P., KONINGS, C. A. G., VAN GENDEREN, J., AND DE JONG, L. P. A. (1984). Fundam. Appl. Toxicol. 4, S84–S95. We report the isolation, on a 0.1-to 2-mg scale, of the four stereoisomers of the nerve agent pinacolyl methylphosphonofluoridate (soman), assigned as C(+)P(+), C(+)P(−), C(−)P(+), and C(−)P(−), with more than 99% optical purity. This result was realized by means of (i) complete optical resolution of pinacolyl alcohol, (ii) synthesis of C(+)- and C(−)-soman from the (+)- and (−)-enantiomers of the alcohol, (iii) optimalization of conditions for stereospecific inhibition of α-chymotrypsin with the P(−)-isomers of C(+)- and C(−)-soman, followed by isolation of the C(+)P(+)- and C(−)P(+)-isomers, (iv) isolation of the C(+)P(−)- and C(−)P(−)-isomers after incubation in rabbit plasma of C(+)- and C(−)-soman, respectively, which hydrolyzes stereospecifically the P(+)-isomers. Solutions (0.2 mM) of the steroisomers in organic solvents are optically stable for at least a month at −20°C. The bimolecular rate constants for inhibition of electric eel acetylcholinesterase (AChE) at pH 7.7, 25°C, are at least 3.6 × 104 larger for the P(−)- than for the P(+)-isomers. AChE from electric eel as well as from mouse erythrocytes inhibited with C(+)P(−)-soman is much more effectively reactivated with the oximes HI-6, HGG-42, and obidoxime than these enzymes inhibited with C(−)P(−)-soman. Similarly, the neuro-muscular transmission (NMT) in mouse diaphragm strips poisoned with C(+)-soman is more easily restored with HI-6 than NMT of such muscle preparations poisoned with C(−)-soman. The LD50 values (mice, sc) are in accordance with the P(−)/P(+)-ratio of inhibition rates of AChE, i.e. 99, 38, >5000, >2000, 214, 133, and 156 μg/kg for C(+)P(−)-, C(−)P(−)-, C(+)P(+)-, C(−)P(+), C(+)-, C(−)-soman, and “soman,” respectively. We suggest that mice challenged with a lethal dose of soman (sc) are killed primarily by the C(−)P(−)-isomer.

Isolation, anticholinesterase properties and acute toxicity of the four stereoisomers of the nerve agent soman

Abstract The four stereoisomers of pinacolyl methylphospho-nofluoridate (soman) were isolated with more than 99% optical purity. The bimolecular rate constants for inhibition of electric eel acetylcholinesterase and the LD50-values (sc, mice) of the stereoisomers were determined.

Formation of an unstable covalent intermediate during the inhibition of electric-eel acetylcholinesterase with 1,3,2-dioxaphosphorinane 2-oxides

The kinetics of interaction of eel acetylcholinesterase (EC 3.1.1.7) with 1,3,2-dioxaphosphorinane 2-oxides were investigated. It was demonstrated that the rate of spontaneous re-activation as well as the re-activation profile in the presence of 2-pyridine aldoxime methiodide of the inhibited enzyme are irrespective of the leaving group of three inhibitors and exhibit the same values. The dissociation constant of the corresponding Michaelis complex was evaluated by two independent methods and the results were found to be in close agreement. It was shown that the active site is essential for interaction between the enzyme and the various dioxaphosphorinanes. The mixed anhydride of diethyl phosphoric acid and 2-hydroxy-1,3,2-dioxaphosphorinane 2-oxide behaves exactly as would be predicted from a typical diethyl phosphate inhibitor. Enxyme that was incubated with the cyclic acid or the corresponding methyl ester recovered immediately upon extensive dilution. Inhibition of enzyme in the presence of high concentratasions of the corresponding 2-chloro and 2-fluoro derivatives decreased the regeneration rates as well as the maximal amount of the re-activated enzyme. This observation could not be explained in terms of a classical aging process. On the basis of the kinetics observations it is suggested that an unstable covalent phospho-enzyme intermediate is formed during the reaction between acetylcholinesterase and 1,3,2-dioxaphosphorinane 2-oxides.