Christopher M. Timperley

Analysis of clothing and urine from moscow theatre siege casualties reveals carfentanil and remifentanil use

On October 26, 2002, Russian Special Forces deployed a chemical aerosol against Chechen terrorists to rescue hostages in the Dubrovka theatre. Its use confirmed Russian military interest in chemicals with effects on personnel and caused 125 deaths through a combination of the aerosol and inadequate medical care. This study provides evidence from liquid chromatography-tandem mass spectrometry analysis of extracts of clothing from two British survivors, and urine from a third survivor, that the aerosol comprised a mixture of two anaesthetics--carfentanil and remifentanil--whose relative proportions this study was unable to identify. Carfentanil and remifentanil were found on a shirt sample and a metabolite called norcarfentanil was found in a urine sample. This metabolite probably originated from carfentanil.

Inhibitory Potency against Human Acetylcholinesterase and Enzymatic Hydrolysis of Fluorogenic Nerve Agent Mimics by Human Paraoxonase 1 and Squid Diisopropyl Fluorophosphatase

A wide range of toxic organophosphorus pesticides and nerve agents is effectively hydrolyzed by the structurally related phosphotriesterase enzymes paraoxonase (PON1) from human plasma and diisopropyl fluorophosphatase (DFPase) from the squid Loligo vulgaris. Both enzymes have potential use as medical countermeasures and decontaminants. Enhanced enzymatic activity, stereochemical preference, and substrate variety are still the focus of ongoing research. Derivatives of pesticides and nerve agents bearing a fluorogenic leaving group were introduced for high-throughput screening of mutant libraries recently. We report the inhibitory potency of fluorogenic organophosphorus compounds with three different leaving groups [3-chloro-7-oxy-4-methylcoumarin, 7-oxy-4-methylcoumarin, 7-oxy-4-(trifluoromethyl)coumarin] toward human acetylcholinesterase (AChE) and report kinetic data for the enzymatic hydrolysis of these compounds by PON1 and DFPase. This is the first report of the hydrolysis of a substrate bearing a P-O bond to the leaving group by DFPase (its activity was believed to be restricted to cleavage of P-F and P-CN bonds). The reactivity of the enzymes toward the substrates is explained on the basis of structural reasoning and computational docking studies. We demonstrate that fluorogenic organophosphorus compounds can serve as valuable models for enzyme screening but also show that differences and limitations exist and have to be taken into account. The importance of using protein from human sources to obtain toxicological data for potential in vivo use is highlighted.

Bis(fluoroalkyl)acrylic and methacrylic phosphate monomers, their polymers and some of their properties

Abstract Ten fluoromonomers of structure (RFO)2P(O)OCH2CH2OC(O)CRCH2 were made in 30–64% yield by treating the chloridates (RFO)2P(O)Cl with HOCH2CH2OC(O)CRCH2 in chloroform in the presence of triethylamine [RF=CF3CH2, C2F5CH2, C3F7CH2, C4F9CH2, C4F9CH2CH2 or C6F13CH2CH2; R  H or Me]. The chloromonomer (CCl3CH2O)2P(O)OCH2CH2OC(O)CHCH2 was obtained analogously in 29% yield. Polymerisation of the acrylate monomers, but not the methacrylate monomers, could be effected using α-azoisobutyronitrile as a radical initiator. Acrylic polymers having CF3CH2O, CCl3CH2O and C6F13CH2CH2O side-chains were obtained as translucent rubbers. Specimens of cotton fabric were treated with solutions of the polymers, and average water and oil repellency ratings measured. Fabric coated with the polymer with the C6F13CH2CH2O side-chain afforded protection from penetration of the test liquids. Treated fabrics were subjected to the limiting oxygen index (LOI) test according to BS EN ISO 4589-2 (1999): this test determines the point at which a material just burns in a volumetric flow of oxygen and nitrogen. The treated fabrics were more fire-resistant (LOI 22–29%) than the untreated fabric (LOI 18%). Fabric coated with the CCl3CH2O-based polymer can be considered fire-retardant (LOI 29%). The fluoromonomers were tested for anti-acetylcholinesterase activity and were found to be poor enzyme inhibitors; they are predicted to possess low acute toxicity.

Production of ES1 plasma carboxylesterase knockout mice for toxicity studies.

The LD(50) for soman is 10-20-fold higher for a mouse than a human. The difference in susceptibility is attributed to the presence of carboxylesterase in mouse but not in human plasma. Our goal was to make a mouse lacking plasma carboxylesterase. We used homologous recombination to inactivate the carboxylesterase ES1 gene on mouse chromosome 8 by deleting exon 5 and by introducing a frame shift for amino acids translated from exons 6 to 13. ES1-/- mice have no detectable carboxylesterase activity in plasma but have normal carboxylesterase activity in tissues. Homozygous ES1-/- mice and wild-type littermates were tested for response to a nerve agent model compound (soman coumarin) at 3 mg/kg sc. This dose intoxicated both genotypes but was lethal only to ES1-/- mice. This demonstrated that plasma carboxylesterase protects against a relatively high toxicity organophosphorus compound. The ES1-/- mouse should be an appropriate model for testing highly toxic nerve agents and for evaluating protection strategies against the toxicity of nerve agents.

The steric and electronic effects of aliphatic fluoroalkyl groups

Abstract Ab initio calculations were performed on 18 fluorinated and unfluorinated alcohols at the B3LYP and HF levels with the 6-311G ∗∗ basis set. Molar volumes of the alcohols were computed at each level and averaged to produce a scale of relative size. From this, various isosteric replacements of potential use in drug design were suggested: ethyl by FCH 2 CH 2 or HCF 2 CH 2 , propyl by CF 3 CH 2 , isopropyl by CF 3 (CH 3 )CH or (FCH 2 ) 2 CH, isobutyl or t -butyl by (CF 3 ) 2 CH, and 3-methyl-2-butyl by CF 3 (CH 3 ) 2 C. Calculation of the charge on oxygen and the Wiberg index of the CO bond allowed an electronegativity scale to be constructed for the fluoroalkyl groups. Electronegativity decreased in the order: (CF 3 ) 3 C>(CF 3 ) 2 CH>C 2 F 5 CH 2 >CF 3 CH 2 >CH 3 (CF 3 ) 2 C>HCF 2 CH 2 >CF 3 (CH 3 )CH>(FCH 2 ) 2 CH>FCH 2 CH 2 >CF 3 (CH 3 ) 2 C. This ranking agreed with literature acid dissociation data for the alcohols studied.

Detection of the organophosphorus nerve agent VX and its hydrolysis products in white mustard plants grown in contaminated soil

The Chemical Weapons Convention (CWC) aims to prohibit the development, production, acquisition, stockpiling, retention, transfer or use of chemical weapons by States Parties. Verification of compliance or investigations into allegations of use requires accurate detection of chemical warfare agents (CWAs) and their breakdown products. Detection of CWAs such as organophosphorus nerve agents in the environment relies mainly upon the analysis of soil. Here we present a novel method for the detection of the nerve agent VX and its hydrolysis products through analysis using a combination of gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) of ethanol extracts of contaminated vegetation (white mustard, Sinapis alba), which localised the compounds of interest, and in this study retained them in an extractable form longer than the soil.

Fluorinated phosphorus compounds: Part 6. The synthesis of bis(fluoroalkyl) phosphites and bis(fluoroalkyl) phosphorohalidates

Abstract Reaction of phosphorus trichloride with tert -butanol and fluoroalcohols gave bis(fluoroalkyl) phosphites (R F O) 2 P(O)H in 42–89% yield, where R F =HCF 2 CH 2 , H(CF 2 ) 2 CH 2 , H(CF 2 ) 4 CH 2 , CF 3 CH 2 , C 2 F 5 CH 2 , C 3 F 7 CH 2 , (CF 3 ) 2 CH, (FCH 2 ) 2 CH, CF 3 (CH 3 ) 2 C, (CF 3 ) 2 CH 3 C, CF 3 CH 2 CH 2 , C 4 F 9 CH 2 CH 2 and C 6 F 13 CH 2 CH 2 . Treatment of these with chlorine in dichloromethane gave the bis(fluoroalkyl) phosphorochloridates (R F O) 2 P(O)Cl in 49–96% yield. The chloridate (CF 3 CH 2 O) 2 P(O)Cl was isolated in much lower yield from the interaction of thionyl chloride with bis(trifluoroethyl) phosphite. Heating the latter in dichloromethane with potassium fluoride and a catalytic amount of trifluoroacetic acid gave the corresponding fluoridate (CF 3 CH 2 O) 2 P(O)F in 84% yield. Treatment of bis(trifluoroethyl) phosphite with bromine or iodine gave the bromidate (CF 3 CH 2 O) 2 P(O)Br and iodidate (CF 3 CH 2 O) 2 P(O)I in 51 and 46% yield, respectively. The iodidate is the first dialkyl phosphoroiodidate to have been isolated and characterised properly—its discovery lags behind the first isolation of a dialkyl phosphorochloridate by over 130 years. The fluoroalkyl phosphoryl compounds are generally more stable than known unfluorinated counterparts.

Hydrolysis and Oxidation Products of the Chemical Warfare Agents 1,2-Bis[(2-chloroethyl)thio]ethane Q and 2,2′-Bis(2-chloroethylthio)diethyl Ether T

Syntheses of diols of structure [HOCH2CH2S]2(CH2)n in 86–95% yield from the sodium salt of 2-mercaptoethanol and Br(CH2)nBr (n = 1 to 5) or in 60–90% yield from 2-chloroethanol and NaS(CH2)nSNa (n = 2 to 5) are described. The diol [HOCH2CH2SCH2CH2]2O was prepared in 82% yield from the sodium salt of 2-mercaptoethanol and [ClCH2CH2]2O, and in 88% yield from 2-chloroethanol and [HSCH2CH2]2O. Mono- and bis-sulfoxides and bis-sulfones of these species were prepared in generally high yield by treatment with an equivalent of KIO4 in aqueous methanol, two equivalents of NaIO4 in aqueous methanol, or four equivalents of H2O2 in trifluoroacetic acid respectively. The compounds are important analytical standards for investigating the fate of the chemical warfare agents sesquimustard Q and oxygen mustard T in environmental samples.

Toxicity and medical countermeasure studies on the organophosphorus nerve agents VM and VX

To support the effort to eliminate the Syrian Arab Republic chemical weapons stockpile safely, there was a requirement to provide scientific advice based on experimentally derived information on both toxicity and medical countermeasures (MedCM) in the event of exposure to VM, VX or VM–VX mixtures. Complementary in vitro and in vivo studies were undertaken to inform that advice. The penetration rate of neat VM was not significantly different from that of neat VX, through either guinea pig or pig skin in vitro. The presence of VX did not affect the penetration rate of VM in mixtures of various proportions. A lethal dose of VM was approximately twice that of VX in guinea pigs poisoned via the percutaneous route. There was no interaction in mixed agent solutions which altered the in vivo toxicity of the agents. Percutaneous poisoning by VM responded to treatment with standard MedCM, although complete protection was not achieved.

Phosphotriesterase variants with high methylphosphonatase activity and strong negative trade-off against phosphotriesters

The most lethal organophosphorus nerve agents (NA), like sarin, soman, agent-VX and Russian-VX, share a methylphosphonate moiety. Pseudomonas diminuta phosphotriesterase (PTE) catalyses the hydrolysis of methylphosphonate NA analogues with a catalytic efficiency orders of magnitude lower than that towards the pesticide paraoxon. With a view to obtaining PTE variants that more readily accept methylphosphonate NA, ~75,000 PTE variants of the substrate-binding residues Gly-60, Ile-106, Leu-303 and Ser-308 were screened with fluorogenic analogues of the NA Russian-VX and cyclosarin. Seven new PTE variants were isolated, purified and their k(cat)/K(M) determined against five phosphotriesters and five methylphosphonate analogues of sarin, cyclosarin, soman, agent-VX and Russian-VX. The novel PTE variants exhibited as much as a 10-fold increase in activity towards the methylphosphonate compounds--many reaching a k(cat)/K(M) of 10⁶ M⁻¹ s⁻¹--and as much as a 29,000-fold decrease in their phosphotriesterase activity. The mutations found in two of the variants, SS0.5 (G60V/I106L/S308G) and SS4.5 (G60V/I106A/S308G), were modelled into a high-resolution structure of PTE-wild type and docked with analogues of cyclosarin and Russian-VX using Autodock 4.2. The kinetic data and docking simulations suggest that the increase in activity towards the methylphosphonates and the loss of function against the phosphotriesters were due to an alteration of the shape and hydrophobicity of the binding pocket that hinders the productive binding of non-chiral racemic phosphotriesters, yet allows the binding of the highly asymmetric methylphosphonates.