Diethyl 4-nitrophenyl Phosphate Research Articles

Micellar Effects of Functional Detergents – 1-Cetyl-3-(2-hydroxyiminopropyl)imidazolium Halides – in Reactions with 4-Nitrophenyl Toluenesulfonate and 4-Nitrophenyl Diethyl Phosphate

An anomalously large increase was found in the rate of cleavage of 4-nitrophenyl 4-toluenesulfonate (∼13000 times) and diethylphosphate (∼550 times) by the zwitterionic micelles of 1-cetyl-3-(2-hydroxyiminopropyl)imidazolium chloride and bromide. Detailed kinetic and thermodynamic analysis of these processes made it possible to establish that the main factor responsible for the high rates of reaction in the micellar phase is the concentration of the reagents.

Nucleophilic Substitution at a Four-Coordinate Sulfur Atom: VI. Reactivity of Oximate Ions

The kinetics of the reaction of oximate ions with 2,4-dinitrophenyl p-toluenesulfonate, 4-nitrophenyl p-toluenesulfonate, diethyl 4-nitrophenyl phosphate, and ethyl 4-nitrophenyl ethylphosphonate in H2O-DMSO mixtures (0 to 95 vol % of DMSO) cannot be described in terms of a single Bro/nsted equation. Regardless of the nature of the reaction center and leaving group, both in water and in 80% DMSO fast leveling of the reactivity of oximate ions is observed, the α-effect decreases at pKa ≥ 9.0 and disappears at pKa ≥ 12.0 owing to difference in the solvation of weakly (pKa ≤ 9.0) and strongly basic (pKa ≥ 9.0) oximate ions rather than to change of the transition state. Just unfavorable solvation effects are responsible for the fact that the limiting nucleophilic reactivity of oximate ions (as typical α-nucleophiles) is not higher than the reactivity of strongly basic alkoxide ions.

NMR Evidence for a Short, Strong Hydrogen Bond at the Active Site of a Cholinesterase

Cholinesterases (ChE), use a Glu-His-Ser catalytic triad to enhance the nucleophilicity of the catalytic serine. It has been shown that serine proteases, which employ an Asp-His-Ser catalytic triad for optimal catalytic efficiency, decrease the hydrogen bonding distance between the Asp-His pair to form a short, strong hydrogen bond (SSHB) upon binding mechanism-based inhibitors, which form tetrahedral Ser-adducts, analogous to the tetrahedral intermediates in catalysis, or at low pH when the histidine is protonated [Cassidy, C. S., Lin, J., Frey, P. A. (1997) Biochemistry 36, 4576-4584]. Two types of mechanism-based inhibitors were bound to pure equine butyrylcholinesterase (BChE), a 364 kDa homotetramer, and the complexes were studied by (1)H NMR at 600 MHz and 25-37 degrees C. The downfield region of the (1)H NMR spectrum of free BChE at pH 7.5 showed a broad, weak, deshielded resonance with a chemical shift, delta = 16.1 ppm, ascribed to a small amount of the histidine-protonated form. Upon addition of a 3-fold excess of diethyl 4-nitrophenyl phosphate (paraoxon) and subsequent dealkylation, the broad 16.1 ppm resonance increased in intensity 4.7-fold, and yielded a D/H fractionation factor phi = 0.72+/-0.10 consistent with a SSHB between Glu and His of the catalytic triad. From an empirical correlation of delta with hydrogen-bond length in small crystalline compounds, the length of this SSBH is 2.64+/-0.04 A, in agreement with the length of 2.62+/-0.02 A independently obtained from phi. The addition of a 3-fold excess of m-(N,N, N-trimethylammonio)trifluoroacetophenone to BChE yielded no signal at 16.1 ppm, and a 640 Hz broad, highly deshielded proton resonance with a chemical shift delta = 18.1 ppm and a D/H fractionation factor phi = 0.63+/-0.10, also consistent with a SSHB. The length of this SSHB is calculated to be 2.62+/-0.04 A from delta and 2.59+/-0.03 A from phi. These NMR-derived distances agree with those found in the X-ray structures of the homologous acetylcholinesterase complexed with the same mechanism-based inhibitors, 2.60+/-0.22 and 2.66+/-0.28 A. However, the order of magnitude greater precision of the NMR-derived distances establish the presence of SSHBs. We suggest that ChEs achieve their remarkable catalytic power in ester hydrolysis, in part, due to the formation of a SSHB between Glu and His of the catalytic triad.

Inhibiting the Activity of Calf Pregastric Lipase: Effect of Bile Salts, Lecithin, Liposomes, Phenyl Boronic Acid and Diethyl 4-Nitrophenyl Phosphate

A commercial extract from oropharyngeal tissue of calves has been used as the source of partially purified pregastric lipase. Activity of the enzyme against 4-nitrophenyldecanoate was inhibited by the conjugated bile salts taurocholate, taurodeoxycholate, and tauro- and glyco-chenodeoxycholate in their monomeric form. Although solutions of L-α-lecithin (0-0.75 mg mL -1 ) enhanced the activity of the lipase at all concentrations studied, with maximum rate enhancement (∼190%) occurring within the range (0.11-0.34) mg mL -1 , even this concentration of L-α-lecithin could not remove the inhibitory effect of the bile salts. The Michaelis-Menten parameters, V and K M , were determined for the activity of the enzyme against 4-nitrophenylacetate in the absence and presence of egg phosphatidylcholine (egg-PC) and egg-PC:cholesterol (10:1 mol/mol) liposomes. While values of V decreased slightly (and to the same extent) in the liposomal suspensions, the value of K M was decreased by 50% in the normal liposome but increased by 30% in the cholesterol impregnated liposome. The phenyl boronic acid / lipase dissociation constant was evaluated as 1.9 mM and pronounced inhibition was obtained in the presence of diethyl 4-nitrophenyl phosphate (E600). These inhibition results confirmed the presence of serine in the active site of calf pregastric lipase.

Compatibility of phosphotriesterase from Flavobacterium sp. with detergents

Phosphotriesterase (PTE) activity from Flavobacterium sp. was investigated in the presence of either two types of nonionic, 14 types of anionic, one type of cationic, or teo types of amphoteric detergents. The highest PTE activity toward diethyl 4-nitrophenyl phosphate (Paraoxon) was observed in the presence of 0.01% sodium carboxyl poly-oxyethylene tridecylether , which was 1.7 fold higher than that without detergent. Lyophilized PTE prepared in the presence of 60 mM trehalose retained about 75% of initial preparation after 25 days of storage at 25°C , which was 3 fold higher residual activity of the sample prepared in the absence of trehalose.

Hydrolysis of phosphotriesters promoted by a zinc(II) complex bearing an alcohol pendant

A polyamine–zinc(II) complex with an alcohol pendant promotes the hydrolysis of diethyl(4-nitrophenyl)phosphate via transfer of the diethylphosphate group to its alkoxy pendant.

Nonserine esterases from rat liver cytosol

An effort to identify the major general esterases of rat liver cytosol that are insensitive to the serine esterase inhibitor paraoxon (diethyl 4-nitrophenyl phosphate) has led to the isolation of a dozen enzymes. Four of these are electrophoretically homogeneous. Although purified on the basis of their hydrolytic activity toward 4-nitrophenyl acetate, each of the enzymes has a very broad and overlapping substrate specificity for aromatic esters. Thiol esters serve as substrates but, within the limits of the methods used, amides are not hydrolyzed.

Mechanism and stereochemical course at phosphorus of the reaction catalyzed by a bacterial phosphotriesterase.

The reaction mechanism for the phosphotriesterase from Pseudomonas diminuta has been examined. When paraoxon (diethyl 4-nitrophenyl phosphate) is hydrolyzed by this enzyme in oxygen-18-labeled water, the oxygen-18 label is found exclusively in the diethyl phosphate product. The absolute configurations for the (+) and (-) enantiomers of O-ethyl phenylphosphonothioic acid have been determined by X-ray diffraction structural determination of the individual crystalline 1-phenylethylamine salts. The (+) enantiomer of the free acid corresponds to the RP configuration. The RP enantiomer of O-ethyl phenylphosphonothioic acid has been converted to the SP enantiomer of EPN [O-ethyl O-(4-nitrophenyl) phenylphosphonothioate]. (SP)-EPN is hydrolyzed by the phosphotriesterase to the SP enantiomer of O-ethyl phenylphosphonothioic acid. The enzymatic reaction therefore proceeds with inversion of configuration. These results have been interpreted as an indication of a single in-line displacement by an activated water molecule directly at the phosphorus center of the phosphotriester substrate. (RP)-EPN is not hydrolyzed by the enzyme at an appreciable rate.

Lymphocyte and brain neurotoxic esterase: Dose and time dependence of inhibition in the hen examined with three organophosphorus esters

Certain organic phosphorus esters produce sensorimotor axonopathy in man and other species. There is an excellent correlation between the capacity of an organophosphorus compound to produce axonopathy and its ability to inhibit brain neurotoxic esterase (NTE) in hens. Because NTE is present in peripheral lymphocytes of both hen and man, it has been suggested that the lymphocyte enzyme might be useful both in experimental and clinical situations as an indicator of exposure to organophosphorus compounds producing axonopathy. Diethyl 4-nitrophenyl phosphate (paraoxon), tri-2-cresyl phosphate (TOCP), methyl 2,5-dichloro-4-bromophenyl phenylphosphonothionate (leptophos), and di- n-butyl-2,2-dichlorovinyl phosphate (di- n-butyl dichlorvos, DBDCV) were used to examine the relationship between lymphocyte and brain NTE inhibition in hens. As expected, paraoxon (0.75 mg/kg) did not inhibit NTE in brain or lymphocytes. TOCP (10 to 100 mg/kg), leptophos (25 to 150 mg/kg), and DBDCV (1.0 to 4.0 mg/kg) inhibited both brain and lymphocyte NTE activity in a doserelated manner with good correlation of inhibition between tissues taken 24 hr after exposure ( r 2 = 0.53 to 0.67; p < 0.020 to 0.001). However, correlation of inhibition between tissues taken from animals killed 48 hr after exposures was poor ( r 2 = 0.15 to 0.30; p < 0.10 to 0.05), with consistently less inhibition of lymphocyte NTE relative to brain NTE. This study indicates that assay of lymphocyte NTE can provide a good monitor of exposure to axonotoxic organophosphorus compounds within 24 hr between exposure and measurement.

The delayed neurotoxic effect of some organophosphorus compounds. Identification of the phosphorylation site as an esterase.

1. Organophosphorus compounds that produce a delayed neurotoxic effect in hens phosphorylate a specific site in the brain soon after administration. 2. Phosphorylation of the specific site by di-isopropyl [(32)P]phosphorofluoridate in vitro is blocked by the prior addition of phenyl phenylacetate. 3. A small proportion of the total activity of hen brain hydrolysing phenyl phenylacetate in vitro was shown to be due to an enzyme different from two others previously described. 4. This enzyme is only slightly inhibited in vitro by concentrations of tetraethyl pyrophosphate and paraoxon (diethyl 4-nitrophenyl phosphate) up to 64mum and is completely inhibited by 6mum-di-isopropyl phosphorofluoridate and 128mum-mipafox. 5. It is also inhibited in vivo by effective doses of neurotoxic organophosphorus compounds but not by high doses of non-neurotoxic analogues. 6. It is deduced that the active site of this enzyme is the phosphorylation site associated with the genesis of delayed neurotoxicity.

Studies of the enzymic mechanism of the metabolism of diethyl 4-nitrophenyl phosphorothionate (parathion) by rat liver microsomes.

1. The metabolism of parathion by rat liver microsomes is affected by various enzyme inhibitors in a manner quite typical of the ;mixed-function oxidase' enzyme systems. 2. With many of these inhibitors (p-chloromercuribenzoate, Cu(2+), 8-hydroxyquinoline) the conversion of parathion into diethyl hydrogen phosphorothionate is less inhibited than conversion into diethyl 4-nitrophenyl phosphate (paraoxon). 3. Compounds containing reduced sulphur stimulate the overall metabolism of parathion. However, the conversion of parathion into diethyl hydrogen phosphorothionate is stimulated more than its conversion into paraoxon. 4. The metabolism of parathion to diethyl hydrogen phosphorothionate is also stimulated by EDTA, Ca(2+) and Ba(2+), but these stimulatory effects are not additive. 5. The electron acceptors FAD, riboflavine, menadione and methylene blue exhibit a concentration-dependent differential inhibition of the metabolism of parathion to diethyl hydrogen phosphorothionate and to paraoxon. 6. The concentration of parathion required for the half-maximal rate of production of diethyl hydrogen phosphorothionate is significantly different from the concentration required for half-maximal rate of production of paraoxon. 7. The results are discussed in terms of either two separate enzyme systems metabolizing parathion to diethyl hydrogen phosphorothionate and to paraoxon or two different binding sites for parathion, which share a common electron-transport pathway.

Studies on the metabolism of diethyl 4-nitrophenyl phosphorothionate (parathion) in vitro.

1. The metabolism of the phosphorothionate parathion in vitro was examined by using [(32)P]parathion and microsomes isolated from the livers of various animal species. 2. The major metabolic products of parathion in this system in vitro were identified as diethyl 4-nitrophenyl phosphate (paraoxon), diethyl hydrogen phosphate, diethyl hydrogen phosphorothionate and p-nitrophenol. 3. The reaction leading to the formation of diethyl hydrogen phosphorothionate and p-nitrophenol requires the same cofactors (NADPH and oxygen) required for metabolism of parathion to its active anti-acetylcholinesterase paraoxon. 4. The enzyme activity towards parathion per unit weight of liver is increased some 65-130% by pretreatment of male rats with phenobarbital and 3,4-benzopyrene. 5. The metabolism of parathion is inhibited by incubation in a nitrogen atmosphere and in an atmosphere containing carbon monoxide. Pure oxygen is also inhibitory. These results are discussed in terms of a deficiency of oxygen for maximal activity as well as the lability of some component of the system to oxidation.

The reactivation by pyridinium aldoximes of phosphorylated acetylcholinesterase in the central nervous system

The in vivo reactivation of phosphorylated acetylcholinesterase in the brain was studied in rats injected with a sub-lethal dose of Paraoxon (diethyl 4-nitrophenyl phosphate) and 30 min later with 0.1 m-mole/kg of a pyridinium aldoxime, e.g. N,N'-trimethylenebis (pyridinium-4-aldoxime) (TMB-4) dibromide or dichloride, or N-methyl pyridinium-2-aldoxime iodide (P-2-AM). Calculations based on acetylcholinesterase (AChE) activities of homogenates (to which both functional and non-functional AChE contribute) showed that reactivation took place but was not uniform in all parts of the brain. On the other hand, calculations based on the hydrolysis of acetyl-β-methyl-choline by larger pieces of intact tissue (to which only functional AChE contributes) gave degrees of reactivation which were generally higher than those determined by the previous method, and which were similar throughout the brain. TMB-4 and P-2-AM reactivated approx. one third and one fifth, respectively, of phosphorylated functional acetylcholinesterase. The results indicate that in the brain pyridinium aldoximes can reactivate phosphorylated functional AChE, but have little effect on phosphorylated non-functional AChE. This difference is not seen with the lipoid soluble oxime monoisonitrosoacetone (MINA). The results also indicate that homogenates of the cerebellum are more useful than other homogenates for screening the in vivo reactivation of phosphorylated functional AChE in the brain. The relationship between the antidotal action of oximes and their effect on phosphorylated AChE is discussed, and it is concluded that the interpretation still holds that the antidotal action of pyridinium aldoximes in mice and rats depends at least largely on the reactivation of phosphorylated AChE at peripheral sites.

Value of ED50 testing in assessing hazards of acute poisoning by carbamates and organophosphates.

It is shown from the kinetics of inhibition of cholinesterase by <i>N</i>-methylcarbamates and organophosphates that the LD₅₀ dose is likely to be a much greater multiple of the dose causing signs of poisoning in 50% of the animals (the ED₅₀) for the carbamates than for the organophosphates. The expected difference was demonstrated by a comparison of the LD₅₀s and ED₅₀s, intravenous and intramuscular, of five carbamates (2-<i>iso</i>propoxyphenyl <i>N</i>-methylcarbamate, 3-<i>iso</i>propylphenyl <i>N</i>-methylcarbamate, 6-chloro-3,4-xylyl <i>N</i>-methylcarbamate, 3,4,5-trimethylphenyl <i>N</i>-methylcarbamate, and 3-methyl-5-<i>iso</i>propylphenyl <i>N</i>-methylcarbamate) and two organophosphorus compounds (diethyl 4-nitrophenyl phosphate and dimethyl 4-nitrophenyl phosphate). The slightest evoked tremor was chosen as the most reliable sign of poisoning from which to estimate the ED₅₀ values. Carbamates gave much greater LD₅₀/ED₅₀ ratios than organophosphorus compounds. It is likely that occupational exposure to carbamates will produce incapacitating symptoms at doses well below lethal levels.

Studies on the Percutaneous Absorption of Parathion and Paraoxon: Vi.In Vivo Decomposition of Paraoxon During the Epidermal Passage

In five earlier papers of this series various aspects of the percutaneous absorption of the well-known insecticide parathion (E 605, or diethyl 4-nitrophenyl thionophosphate) and its more toxic oxygen analogue paraoxon (E 600, or diethyl 4-nitrophenyl phosphate) were discussed. These studies covered such topics as the in vitro metabolism and enzymic degradation within the skin (8) the distribution of 32P-labelled parathion within the skin (2); the rate of absorption of parathion as investigated with the aid of labelled material and disappearance technic (3); decontamination of human skin from parathion (4); and also the rate of absorption of paraoxon (5). The method used in the last mentioned case was a dose-response technic, i.e. the rate of inhibition of plasma cholinesterase activity was determined following percutaneous absorption and continuous intravenous infusion in two separate groups of cats. The intravenous infusion was adjusted so that the rate of enzyme inhibition in plasma was approximately the same as following topical application. Since the rate of infusion and the area of absorption were known the rate of percutaneous absorption could easily be calculated.

Effect of paraoxon (diethyl 4-nitrophenyl phosphate) on axone reflex sweating produced by alpha-lobeline.

Paraoxon beeinflusst die Wirkung vonα-Lobelin uber den Axonreflex, der Schwitzen menschlicher Haut auslost. Geringe Konzentrationen vonα-Lobelin werden durch Paraoxon verstarkt, wahrend hohere gehemmt werden. Daraus wird gefolgert, dass endogenes Acetylcholin an der Auslosung des Axonreflexes beteiligt sein kann.

Action of paraoxon (diethyl 4-nitrophenyl phosphate) on human sweat glands and the sympathetic axone reflex.

Action of paraoxon (diethyl 4-nitrophenyl phosphate) on human sweat glands and the sympathetic axone reflex.

Pentamethonium as an adjuvant to atropine in the therapy of paraoxon poisoning.

Paton and Perry1 demonstrated a competitive antagonism between acetyl oholine and penta-methyline bis-trimethyl ammonium dibromide (pentamethonium), at ganglia, and this suggested that pentamethonium might be of value in treating poisoning with antioholinesterases such as diethyl 4-nitrophenyl phosphate (paraoxon). Douglas and Matthews2 used pentamethonium in cats poisoned with tetraethyl pyrophosphate (TEPP), and showed that it improved the contractile power of the diaphragm. We now have evidence that pentamethonium bromide significantly increases survival of fully atropinized animals—mice, rabbits and cats— poisoned with paraoxon.