Catalytic detoxification of nerve agent and pesticide organophosphates by butyrylcholinesterase assisted with non-pyridinium oximes

Biochem. J. (2013) 450, 231–242 (Printed in Great Britain) doi:10.1042/BJ20121612 231

Catalytic detoxification of nerve agent and pesticide organophosphatesby butyrylcholinesterase assisted with non-pyridinium oximesZoran RADIC*1, Trevor DALE†, Zrinka KOVARIK‡, Suzana BEREND‡, Edzna GARCIA*, Limin ZHANG*, Gabriel AMITAI§,Carol GREEN‖, Bozica RADIC‡, Brendan M. DUGGAN*, Dariush AJAMI†, Julius REBEK Jr† and Palmer TAYLOR**Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, La Jolla, CA 92093, U.S.A., †Skaggs Institute for Chemical Biology and Department ofChemistry, The Scripps Research Institute, La Jolla, CA 92037, U.S.A., ‡Institute for Medical Research and Occupational Health, HR-10001 Zagreb, Croatia, §Department ofPharmacology, Israel Institute for Biological Research, Ness Ziona 74100, Israel, and ‖SRI International, Menlo Park, CA 94025-3493, U.S.A.

In the present paper we show a comprehensive in vitro, exvivo and in vivo study on hydrolytic detoxification of nerveagent and pesticide OPs (organophosphates) catalysed by purifiedhBChE (human butyrylcholinesterase) in combination with novelnon-pyridinium oxime reactivators. We identified TAB2OH (2-trimethylammonio-6-hydroxybenzaldehyde oxime) as an efficientreactivator of OP–hBChE conjugates formed by the nerveagents VX and cyclosarin, and the pesticide paraoxon. It wasalso functional in reactivation of sarin- and tabun-inhibitedhBChE. A 3–5-fold enhancement of in vitro reactivation ofVX-, cyclosarin- and paraoxon-inhibited hBChE was observedwhen compared with the commonly used N-methylpyridiniumaldoxime reactivator, 2PAM (2-pyridinealdoxime methiodide).Kinetic analysis showed that the enhancement resulted fromimproved molecular recognition of corresponding OP–hBChEconjugates by TAB2OH. The unique features of TAB2OH stem

from an exocyclic quaternary nitrogen and a hydroxy group, bothortho to an oxime group on a benzene ring. pH-dependencesreveal participation of the hydroxy group (pKa = 7.6) forming anadditional ionizing nucleophile to potentiate the oxime (pKa = 10)at physiological pH. The TAB2OH protective indices in therapyof sarin- and paraoxon-exposed mice were enhanced by 30–60%when they were treated with a combination of TAB2OH and sub-stoichiometric hBChE. The results of the present study establishthat oxime-assisted catalysis is feasible for OP bioscavenging.

Key words: butyrylcholinesterase reactivation, catalytic organo-phosphate bioscavenger, edrophonium analogue, nonpyridiniumoxime reactivator, organophosphate intoxication, oxime reactiva-tion.

INTRODUCTION

Human exposure to OPs (organophosphates) is extensive andprevails as a global problem. The World Health Organizationestimates that between 30000 and 200000 people die annuallyfrom acute OP poisoning [1], whereas more than 95% of theUS population was found to be exposed to sublethal levels ofOP-based pesticides [2]. The exposure occurs largely throughoccupational exposure to OP-based pesticides, in attemptedsuicide with pesticides, by consuming improperly washed fruitor vegetables, but also from terrorist attacks with nerve gases,the most recent being one of 1995 in the Tokyo subway. Morerecently, several incidents of airplane passengers and crew beingexposed to toxic jet-engine lubricant tri-o-cresylphosphate in‘fume events’ on board commercial aircraft have been reported[3].

Exposure to OPs leads to covalent inhibition of ChEs(cholinesterases) in the peripheral and central nervous systems[4]. Common therapy of OP poisoning consists of symptomatictreatment with atropine and reactivation of OP-inhibitedAChE (acetylcholinesterase) by nucleophilic oxime antidotes.Frequently this therapy is limited by reinhibition of reactivatedAChE by unreacted OPs that remain circulating in the bloodfollowing the exposure. To circumvent this problem the concept of

bioscavengers, molecules that capture and degrade OPs in blood,has been introduced [5].

Oxime-assisted recovery of catalytic activity has been studied infar greater detail for AChE, owing to its physiological importancein neurotransmission than that of the closely related cholinesterasefound in liver and plasma, BChE (butyrylcholinesterase).Consequently, most oxime reactivators developed to recoveractivity of OP–AChE conjugates are found to be less efficientin recovering OP–BChE activity [6,7].

Interest in designing potent BChE reactivators has beenstimulated with successful implementation of the ‘stoichiometricOP bioscavenger’ approach based on administering purifiedhuman BChE to OP-exposed animals [8–10].

Efficient protection of guinea pigs from multiple LD50 dosesof nerve agents is found upon pretreatment with 20–30 mg ofpurified human BChE/kg. Similarly, antidotal administration ofBChE to animals following OP exposure was demonstrated toprovide effective protection [10]. The effectiveness in protectionis somewhat overshadowed by a common caveat of stoichiometricbioscavengers. Multi-milligram quantities of large scavengerproteins are needed to remove stoichiometric equivalents ofOP molecules of low molecular mass. The associated perdose cost and administration constraints for such stoichiometricbioscavengers remain large. By enabling the BChE molecule to

Abbreviations used: AChE, acetylcholinesterase; ATCh, acetylthiocholine; BChE, butyrylcholinesterase; ChE, cholinesterase; DTNB, 5,5′-dithiobis-(2-nitrobenzoic acid); Flu-MP, fluorescent methylphosphonate; hAChE, human AChE; hBChE, human BChE; i.m., intramuscular(ly); i.v., intravenous(ly);LC, liquid chromatography; MDP, maximal dose of poison; MOM, methoxymethyl; OP, organophosphate; 2PAM, 2-pyridinealdoxime methiodide;2PAMOH, 3-hydroxy-2-pyridinealdoxime methiodide; PI, Protective Index; s.c., subcutaneous(ly); TAB2, 2-trimethylammonio benzaldehydeoxime;TAB2OH, 2-trimethylammonio-6-hydroxybenzaldehyde oxime; TAB4, 4-trimethylammonio benzaldehydeoxime; TAB4OH, 4-trimethylammonio-2-hydroxy-benzaldehyde oxime.

1 To whom correspondence should be addressed (email [email protected]).

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232 Z. Radic and others

turnover multiple molecules of the OP, quantities of BChE neededfor the effective protection could be significantly reduced andcatalytic bioscavenger coverage could be provided for a largerpopulation of OP-exposed individuals. We achieve this objectivein the present study by simultaneous application of an efficientBChE reactivator, where the reactivating oxime assists BChEcatalytic hydrolysis and turnover of the free OP.

In the present paper we describe development of a prototypiccatalytic OP bioscavenger on the basis of combining purifiedhuman BChE with an efficient oxime reactivator with a distinctivestructural scaffold. Starting with a small directed library ofnonpyridinium, cationic, substituted benzenes, we identifiedan efficient reactivator of sarin-, cyclosarin-, VX-, tabun-and paraoxon-derived OP–BChE conjugates, and characterizedkinetically the interaction with OP–BChE conjugates in detail.

EXPERIMENTAL

Enzyme

Highly purified recombinant monomeric hAChE (human AChE)was prepared as described previously [7]. Purified human BChEisolated from human plasma was a gift from Dr David Lenzand Dr Douglas Cerasoli [USAMRICD (US Army MedicalResearch Institute of Chemical Defense), Aberdeen ProvingGround, MD, U.S.A.]. All enzyme concentrations given referto the concentration of catalytic sites, i.e. monomers.

OPs

Low toxicity non-volatile Flu-MPs (fluorescent methylphosphon-ates) [11] were used in in vitro experiments as analogues of thenerve agents sarin, cyclosarin and VX. The Flu-MPs differ fromactual nerve agent OPs only by the structure of their respectiveleaving groups. Inhibition of hAChE by Flu-MPs results inOP–hAChE covalent conjugates identical with the ones formedupon inhibition with nerve agents. Paraoxon was purchased fromSigma–Aldrich. Nerve agent OPs tabun, VX, sarin and somanused in in vivo experiments were purchased from NC LaboratorySpiez.

Oximes

2PAM (2-pyridinealdoxime methiodide) and HI-6 (dichloride)were purchased from Sigma–Aldrich and US Biological.

Preparation of novel oximes

The edrophonium-based antidotes TAB2OH (2-trimethylammo-nio-6-hydroxybenzaldehyde oxime) and TAB4OH (4-trimethy-lammonio-2-hydroxy-benzaldehyde oxime) were synthesizedfrom commercially available 3-dimethylaminophenol (Supple-mentary Scheme S1 at http://www.biochemj.org/bj/450/bj4500231add.htm). Protection of the phenol as the MOM(methoxymethyl) ether was completed in dichloromethanewith chloromethyl methyl ether and DIEA (N,N-di-isopro-pylethylamine). Product MOM ether was ortho-lithiated usingn-butyllithium in ether at − 40 ◦C and quenched with DMF(dimethylformamide) to afford a mixture of 4-dimethylamino-2-methoxymethoxybenzaldehyde and 2-dimethylamino-6-metho-xymethoxybenzaldehyde in an approximate 1:1.1 ratio. Theregioisomers were separable by column chromatography oversilica gel. Each isomer was separately converted into the oximeusing hydroxylamine hydrochloride followed by removal of the

MOM group by heating in the presence of HCl in methanol.Finally, the 4-dimethylamino-2-hydroxybenzaldehyde oximewas quaternarized using an excess of iodomethane in hotTHF (tetrahydrofuran) to yield the desired trimethylammonioderivative. The 2-dimethylamino-6-hydroxybenzaldehyde oximewas unreactive in the presence of iodomethane and requiredthe more reactive electrophilic methyl source methyl triflate toquaternarize the aniline nitrogen. The triflate salt was anion-exchanged for the chloride by exchange in an acetonitrile solutionof tetrahexylammonium chloride which caused the product,chloride, to precipitate as a pure white solid.

Synthesis of the remaining seven novel oximes (Figure 1)is described in the Supplementary Online Data (at http://www.biochemj.org/bj/450/bj4500231add.htm), except for4PyOHMOH which was synthesized as described previously[12].

In vitro oxime reactivation assays

hAChE and hBChE (human BChE) activities were measuredusing a spectrophotometric assay [13] at 25 ◦C in 0.1 M sodiumphosphate buffer (pH 7.4), containing 0.01 % BSA and 1.0 mMsubstrate ATCh (acetylthiocholine). OP–hBChE and OP–hAChEconjugates were prepared, and initial screening and detailedoxime reactivation experiments were performed [at 37 ◦C in0.1 M sodium phosphate buffer (pH 7.4), containing 0.01% BSA]as described previously [7,14]. The first-order reactivation rateconstant (kobs) for each oxime + OP conjugate combination wascalculated by non-linear regression [15]. The dependence ofreactivation rates on oxime concentrations and determinationof maximal reactivation rate constant k2, Michaelis–Mententype constant Kox and the overall second-order reactivation rateconstant kr were conducted as described previously [15].

pK a determinations

Protonation of ionizable groups in the compounds was monitoredby using UV–Vis spectrophotometry, by NMR spectrometry orby monitoring nucleophilic reactivity of the oxime group. Aseries of 20 mM phosphate-pyrophosphate buffers at pH valuesof 5.0, 6.0, 7.0, 8.0, 8.5, 9.0, 9.5, 10, 10.5 and 11 (containing0.1 M NaCl) were prepared in either H2O or 2H2O. For 2H2Obuffers, p2H values were determined by correcting the pH readingby + 0.4 pH units [16]. UV spectra of compounds between 220and 320 nm wavelengths were recorded on a Cary 1E (Varian)UV–Vis spectrophotometer at the above pH values.

1H-NMR spectra recorded on a Bruker Avance III 600spectrometer in 20 mM phosphate-pyrophosphate 2H2O buffersat pH values 5.0, 6.0, 7.0, 8.0, 8.5, 9.0, 9.5 and 10 (containing0.1 M NaCl) were overlaid and aligned using benzene as anexternal standard placed in a separate capillary tube within theNMR sample probe. Two-dimensional 1H, 1H-COSY spectra wererecorded on a Bruker Avance III 600 spectrometer.

Nucleophilic reactivity of oximes was determined in the above-described buffers by measuring oxime-induced rates of ATChhydrolysis as detected by the thiol reagent DTNB [5,5′-dithiobis-(2-nitrobenzoic acid)], by following a time course of absorbanceincrease at 412 nm at room temperature (22 ◦C).

UV absorbance at discrete wavelengths, chemical shifts ofdiscrete 1H-NMR peaks or rates of nucleophilic reactions wereplotted as a function of pH yielding pKa values by non-linearregression of eqn (1):

A = Amax/(1 + [H+]/K a) (1)


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Organophosphate detoxification by butyrylcholinesterase and oximes 233

Figure 1 Structures of novel oximes used in the present study compared with structures of the reference oxime 2PAM and the reversible inhibitor edrophonium

Anions for all oximes and edrophonium were chloride, except for 2PAM where the anion was methanesulfonate.

or when the dependence on pH involved two ionization equilibriausing eqn (2):

A = Amax1 /(1 + [H+]/K a1) + Amax

2 /(1 + [H+]/K a2) (2)

Acute oxime toxicity and oxime treatment of OP-exposed mice

Male CD-1 mice of 25–30 g of body mass (purchased from theRudjer Boskovic Institute, Zagreb, Croatia) fed on a standarddiet, had free access to water and were kept in Macrolone cagesat 21 ◦C, exchanging light and dark cycles every 12 h. Mice wererandomly distributed into groups of four for each dose.

Acute i.m. [intramuscular(ly)] toxicity (LD50) of TAB2OHwas based upon 24 h mortality rates upon administration of fourdifferent doses of TAB2OH, one per group of four mice, andcalculated according to Thompson [17] and Weil [18].

The therapeutic efficacy of TAB2OH against OP poisoningwas tested by administering mice (i.m.) with TAB2OH (10 or25 mg/kg) together with atropine sulfate (10 mg/kg), 1 min afters.c. [subcutaneous(ly)] OP exposure [19,20]. Nerve agent stocksolutions were prepared in isopropyl alcohol or in propyleneglycol. Immediately before use, further dilutions were made inphysiological saline.

Alternatively, a combination of pretreatment and therapywas performed by i.v. [intravenous(ly)] application of hBChE(0.5 or 1.0 mg/kg) or a combination of hBChE and TAB2OH(25 mg/kg) 15 or 30 min before s.c. OP exposure and then by i.m.administration of TAB2OH and atropine. The design of individualexperiments is detailed in Table 4.

Antidotal efficacy of the oximes was expressed as a PI(Protective Index) with 95% confidence limits and maximal doseof OP affording protection [MDP (maximal dose of poison)].The PI was the ratio of LD50 exerted by OP with antidote andOP given alone. The MDP was the highest multiple of the OPLD50, which was fully counteracted by the antidotal treatment

applied. The mice were treated in accordance with the approvalof the Ethical Committee of the Institute for Medical Researchand Occupational Health in Zagreb, Croatia.

Oxime pharmacokinetics in mice

Female CD-1 mice 4–8 weeks old (19–27 g of body mass) werepurchased from Charles River Laboratories. Mice were fed PurinaCertified Rodent Chow #5002. Food and purified water wasprovided ad libitum. Mice were kept in hanging polycarbonatecages at 21–23 ◦C, exchanging light and dark cycles every12 h. General procedures for animal care and housing were inaccordance with the NRC (National Research Council) Guide forthe Care and Use of Laboratory Animals (1996) and the AnimalWelfare Standards incorporated in 9 CFR Part 3, 1991.

In the experiments the mice were divided into groups of three.For pharmacokinetic studies, 30 mg of TAB2OH oxime/kg wasadministered i.m. using a 30 mg/ml stock solution in a singledose in the absence of OP. Three animals were injected for everytime point analysed. Brain and plasma were collected at each timepoint. Blood (∼300 μl) was collected from the retro-orbital sinusof mice under isoflurane anaesthesia into tubes containing EDTA,processed to plasma within 30 min of collection, and then storedfrozen at �− 80 ◦C (+−10 ◦C).

Brains were collected from each mouse at each time point(without perfusion of residual brain blood with saline). Brainmass was documented for each animal before storage on dry ice.Brains were stored at �− 80 ◦C (+−10 ◦C) until analysis.

The concentration of the oxime in body compartments wasdetermined by LC (liquid chromatography)-MS using MRM(multiple reaction monitoring) ESI (electrospray ionization)detection in positive-ion mode. The peak transition 194.9→107.8(m/z) at 19 eV collision energy and ∼3.0 min retention time wasmonitored on the Micromass Quatro LC instrument.



Figure 2 Reactivation of paraoxon-, cyclosarin-, VX- and sarin-inhibited hAChE (A) and hBChE (B) by a directed library of ten novel oxime reactivators shownin Figure 1

Reactivation efficiencies of 0.67 mM oximes (measured in duplicate at 37◦C in 0.10 M phosphate buffer, pH 7.4) were determined using either ‘take 5’ (paraoxon-, cyclosarin- and VX-inhibited hAChE,and VX-inhibited hBChE) or ‘quick-screen’ (paraoxon-, cyclosarin- and sarin-inhibited hBChE, and sarin-inhibited hAChE) assay procedures and are expressed relative to the 2PAM reactivationefficiency. Cyclosarin-, VX- and sarin-inhibited enzymes were formed by reaction with Flu-MPs yielding a conjugate identical with nerve-agent-inhibited enzymes (compare with the Experimentalsection). The bar representing reactivation of cyclosarin-inhibited hAChE conjugate by HI-6 (A) was truncated (the full height was 170) to allow for visualization of less-efficient reactivation by otheroximes.

RESULTS

Structural design of the oxime library

A small directed library of cationic oxime reactivators wasdesigned largely on the basis of the base structure of theestablished reversible ChE inhibitor edrophonium (Figure 1).Three structural elements were systematically varied in the eightcompounds of the library, including position of the aldoximegroup ortho or para to the quaternary substitution on the benzenering, the presence or absence of an hydroxy group vicinal to thealdoxime, and the size of the alkyl substitution on the exocyclicquaternary nitrogen (trimethyl, diethyl methyl or dimethyl ethyl).Of the two remaining oximes one was based on the structureof the known reactivator 2PAM where an hydroxy group wasintroduced into the meta position of the pyridinium ring vicinal tothe aldoxime group [2PAMOH (3-hydroxy-2-pyridinealdoximemethiodide); Figure 1]. In the other, two hydroxy groupswere introduced in meta positions, the oxime group in paraand a methyl group in the ortho position (4PyOHMOH;Figure 1).

Reactivation screen of OP–hAChE and OP–hBChE conjugatesby oximes of the directed library in vitro

Most oximes of the directed library were less effective thanreference oximes 2PAM and HI-6 in reactivation of OP–hAChEconjugates resulting from paraoxon, cyclosarin, VX or sarininhibition (Figure 2A) as judged by screening procedures [14].Only TAB4OHmee oxime (Figure 1) was significantly betterthan 2PAM in reactivating an OP–hAChE conjugate. An orderof magnitude enhancement in reactivation of cyclosarin-inhibitedhAChE by TAB4OHmee pointed to the importance of the uniquecombination of structural elements absent in the other evaluatedoximes (Figure 1) including: bulky symmetrical quaternarynitrogen substitution, para-substituted oxime group and presenceof a vicinal hydroxy group. Nevertheless reactivation ratesby TAB4OHmee were still less than HI-6, the best hAChEreactivator in this group of oximes, by an order of magnitude(Figure 2A).

Comparative analysis of reactivation for OP–hBChE conjugatesof paraoxon, cyclosarin, VX or sarin, revealed efficaciousreactivation by the oxime TAB2OH, faster than reactivation byeither of the two reference oximes 2PAM or HI-6, for all OPsexcept for sarin (Figure 2B). Thus trimethylammonio benzenesubstituted with an aldoxime in the ortho position and an hydroxygroup at an adjacent position provided a suitable structuraltemplate for efficient interaction with OP–hBChE conjugates.The omission of the adjacent hydroxy group [TAB2 (2-trimethylammonio benzaldehydeoxime)], the shift of the oximeto the para position [TAB4OH and TAB4 (4-trimethylammoniobenzaldehydeoxime)] or shift of the exocyclic quaternary nitrogeninto the ring to convert benzene into pyridinium (2PAMOH) allresulted in the loss of favourable reactivation potency observedfor paraoxon, VX and cyclosarin hBChE conjugates (Figures 1and 2B). Notably, the favourable influence of the shift ofa delocalized positive charge in the pyridinium ring to anexocyclic spherically diffused charge distribution suggests thatoxime/phenolate stabilization in OP–hBChE conjugates prefers acation– π interaction with aromatic side chains, rather than π–πstacking of the two delocalized ring systems.

Reactivation kinetics of TAB2OH oxime in vitro

Comparative analysis of reactivation by a wide range of TAB2OHconcentrations for OP–hBChE conjugates (0.1–10 mM; Figure 3)and OP–hAChE conjugates (5–50 mM; Supplementary FigureS1 at http://www.biochemj.org/bj/450/bj4500231add.htm) con-firmed the preferences observed in initial screening. Evaluationof individual reactivation constants k2 and Kox (Table 1) revealedsuperior reactivation of OP–hBChE conjugates compared with2PAM, and faster reactivation of OP–hBChE compared withOP–hAChE conjugates; both largely reflect an improved bindinginteraction of TAB2OH to OP–hBChE conjugates (smaller Kox).Both 2PAM and TAB2OH bind equally well to native hAChE andnative hBChE as reflected in a K i value of 0.2–0.3 mM (Table 1).Covalent OP conjugation differentially affects affinity for thetwo enzyme conjugates, lowering the affinity of TAB2OH forOP–hBChE only several-fold, but by two orders of magnitude




Figure 3 Concentration-dependence of oxime reactivation of sarin (A)-, cyclosarin (B)-, VX (C)- and paraoxon (D)-inhibited (conjugated) hBChE

Dependence for the lead oxime TAB2OH compared with the reference cationic oxime 2PAM (measured in triplicate experiments at 37◦C in 0.10 M phosphate buffer, pH 7.4). Reactivation constantsderived for each concentration dependence are given in Table 1. OP conjugates of sarin, cyclosarin and VX were formed by reaction with Flu-MPs and were identical with nerve-agent-inhibitedenzymes (compare with the Experimental section).

for OP–hAChE. Association of 2PAM is approximately anorder of magnitude weaker in either OP–hAChE or OP–hBChEconjugates. The overall difference in maximal reactivation rateconstants for the two enzyme conjugates was smaller. Except forreactivation of hBChE conjugates of sarin and tabun, maximalreactivation rates for TAB2OH were similar or larger than thosedetermined for 2PAM.

In this analysis we assume that Kox primarily describes initialreversible interaction of oxime reactivator with an OP–hAChEconjugate, whereas k2 refers to the interaction of the reactivatingoxime with the methyl phosphonate or diethyl phosphorateof the OP-conjugated enzymes in forming the transition state ofreactivation.

pK a determinations

Structures of the lead hBChE reactivator TAB2OH and severalstructurally related oximes (Figure 1) include two ionizablegroups in the physiologically relevant pH range with the nucleo-

philic potential to affect the reactivation efficacy, the oximegroup and the phenolic hydroxy group, which form the respectiveoximate and phenolate species. We determined pKa values ofionizable groups for TAB2OH, structurally related oximes,TAB2, TAB4OH and TAB4, the structurally related reversibleinhibitor edrophonium, and a reference oxime 2PAM.

The bathochromic shift in the UV–Vis spectra, resulting fromdeprotonation of oxime and hydroxy groups or from their pro-tonation, enabled pKa value determinations from an incrementalincrease in absorbance at discrete wavelengths (SupplementaryFigure S2 at http://www.biochemj.org/bj/450/bj4500231add.htmand Table 2). For all compounds with single ionizable groups(TAB2, TAB4, 2PAM and edrophonium) the pH-dependence ofthe absorbance change was consistent with a single ionizationequilibrium at all peak wavelengths. For the ring substitutionscontaining the phenolic and aldoxime hydrogens, the pH-dependence of the spectral change showed evidence for twoionization states, and allowed a simultaneous evaluation of pKa

values for both oxime and hydroxy hydrogens (SupplementaryFigure S2 and Table 2).




Table 1 Analysis of kinetic parameters

Kinetic constants for reactivation of paraoxon–, sarin–, cyclosarin–, VX– and tabun–hBChE and hAChE conjugates by the non-pyridinium oxime TAB2OH and the reference oxime 2PAM. Inhibitionconstants [K i and αK i (mM)] of native AChE and BChE (without OP) by two oximes. The maximal reactivation rate constant (k 2, min − 1), apparent dissociation constant of the oxime–OP-hAChEconjugate reversible complex [K ox (mM)] and overall second-order reactivation rate constant [k r (M− 1 · min − 1)] were determined from reactivation curves as presented in Figure 3 and SupplementaryFigure S1 (at http://www.biochemj.org/bj/450/bj4500231add.htm). All constants were determined from triplicate experiments in 0.1 M phosphate buffer (pH 7.4) at 37◦C. Standard errors of determinedkinetic constants were typically less than 30 % of the mean.

Paraoxon Sarin Cyclosarin VX Tabun Without OP

Oxime Enzyme k 2 K ox k r k 2 K ox k r k 2 K ox k r k 2 K ox k r k 2 K ox k r K i αK i

TAB2OH BChE 0.34 0.71 480 0.19 0.79 250 3.9 1.0 3700 2.0 1.5 1300 0.00027 1.3 0.21 0.32 0.742PAM BChE 0.23 2.4 96 2.4 1.8 1300 2.6 5.4 480 1.2 2.5 480 0.0011 1.2 0.91 0.23 18TAB2OH AChE 0.47 12 41 0.92 27 34 0.44 36 12 2.4 23 100 0.0032 8.2 0.39 0.20 4.02PAM AChE 0.27 1.8 150 1.1 0.34 3300 0.73 6.6 110 0.74 0.32 2300 0.0058 1.6 3.8 0.34 1.9

Table 2 Summary of pK a values for ionizable groups in structures of several oximes determined from NMR, spectroscopy and kinetic parameters

pK a values determined in 2H2O (by NMR) are typically higher than those determined in H2O by ∼0.5 (compare with [16]) and were accordingly corrected. All pK a values were determined at roomtemperature. d, doublet; n.d., not determined; s, singlet; t, triplet.

pK a1 pK a2

Oxime Method Hydroxy Oximate

TAB2OH UV 240 nm 7.44 +− 0.05 10.6 +− 0.1UV 275 nm 7.58 +− 0.07 -UV 310 nm 7.52 +− 0.05 10.1 +− 0.3NMR (s) 7.69 +− 0.08 -NMR (t) 7.63 +− 0.09 -NMR (d1) 7.74 +− 0.08 -NMR (d2) 7.70 +− 0.08 -NMR (s2) 7.70 +− 0.08Oximolysis* 7.21 +− 0.14 (k max = 6 +− 1) �9.3 (k max�21)

TAB2 UV 285 nm - 9.47 +− 0.05Oximolysis* - �10.2 (k max�250)

TAB4OH UV 275 nm 7.47 +− 0.12 11.03 +− 0.04UV 340 nm 7.45 +− 0.03 10.93 +− 0.09Oximolysis* 7.54 +− 0.01 (k max=15 +− 1) �10.7 (k max�65)

TAB4 UV 245 nm - 10.13 +− 0.01UV 285 nm - 10.09 +− 0.06Oximolysis* - �10.7 (k max�440)

Edrophonium UV 240 nm 8.22 +− 0.03 -UV 270 nm 8.20 +− 0.06 -UV 295 nm 8.20 +− 0.03 -Oximolysis n.d. (k max∼0) -

2PAM NMR - 8.11 +− 0.03OP oximol - 8.1 +− 0.2Oximolysis - 8.0 +− 0.1 (k max=60)

*pK a2 values determined for oximolysis (using non-linear regression of eqns 1 and 2) and associated k max values were presented as ‘larger or equal’ for pK a2 values higher than 9 since rates ofoximolysis were measured up to pH 10 only.

1H-NMR spectroscopy in 2H2O was also used to monitorpH-dependent chemical shifts in reactivator resonance spectra,and confirmed the pKa values of ionizable groups of thelead hBChE reactivator TAB2OH and the reference oxime2PAM (Supplementary Figure S3 at http://www.biochemj.org/bj/450/bj4500231add.htm and Table 2). The NMR p2H rangeused (5.5–10.5) allowed determination of only one pKa value percompound. The chemical shift and appearance of five peaks inthe TAB2OH spectrum were analysed (Supplementary FigureS4 at http://www.biochemj.org/bj/450/bj4500231add.htm), twosinglets (amido proton and nine equivalent trimethyl quaternary

N protons), two doublets (aromatic benzene protons at ∼7.2p.p.m. and ∼7.42 p.p.m.; at pH 5.0) and a triplet (aromaticbenzene proton at ∼7.52 p.p.m. at pH 5.0). All five peaksshifted to lower p.p.m. values with increasing pH and yieldedpKa values in the range 7.63–7.74 (Supplementary Figures S3and S5 at http://www.biochemj.org/bj/450/bj4500231add.htm, and Table 2), thus reflecting deprotonation of the samegroup, the benzene hydroxy group. In the 1H-NMR spectrum of2PAM in 2H2O, the aldoxime CH and all aromatic proton signalsshifted simultaneously, yielding a pKa value of 8.11 [16]. TheN-methyl peak at 2.8 p.p.m. did not shift (Supplementary









Figure 4 pH-dependence of rates of ATCh hydrolysis by oximes (oximolysis)

Rates of 1 mM ATCh hydrolysis by (A) 100 μM of the oxime TAB2OH, yielding biphasic pH-dependence, and the reference oxime 2PAM, yielding a monophasic dependence, and by(B) 100 μM of oximes TAB4OH and TAB4, yielding respective biphasic and monophasic pH-dependences. Kinetics were measured spectrophotometrically in duplicate or triplicate at 22◦Cin 20 mM phosphate-pyrophosphate buffers (containing 100 mM NaCl) using Ellman’s reagent DTNB. The error of determination was smaller than the size of the symbol, except where indicated byerror bars. Resulting pK a values calculated by non-linear fit [16] are given in the Figure and recorded in Table 2, together with associated errors. pK a2 values higher than 9 were presented as ‘largeror equal’ since rates of oximolysis were measured up to pH 10 or 10.5 only.

Figure S6 at http://www.biochemj.org/bj/450/bj4500231add.htm). Accordingly, the ionization state of the oxime group affectsdelocalization of the aromatic ring system of 2PAM, but notelectron distribution around its N-methyl protons. The ionizationstate of TAB2OH phenolic hydroxy influences the distributionof electrons and charge of the entire molecule, including theoxime group. The π-orbital interactions between delocalizedring electrons of reactivators and aromatic residues in the AChEgorge, and consequently binding orientations of these twostructurally similar reactivators, endo- and exo-cyclic quaternaryamines, thus appear to be different.

pH-dependence of kinetics of catalysis

Nucleophilic reactivity and pKa values for these compoundswere additionally monitored by determining the rates offree oxime-induced catalysis (oximolysis) of ATCh, measuredspectrophotometrically by Ellman’s reagent DTNB (Figure 4and Table 2). The resulting profiles of the pH-dependence foroximolysis exhibited two ionization equilibria for both hydroxyoximes (TAB2OH and TAB4OH), but only a single one forTAB2, TAB4 and 2PAM. No nucleophilic activity was detectablefor edrophonium as judged by the absence of its influence onATCh hydrolysis. The biphasic nucleophilic pH relationships forthe two hydroxy benzadehyde oximes (with pKa values consistentwith those determined for the oxime and hydroxy groups usingdifferent approaches) indicate formation of a nucleophile at a pHvalue close to the physiological range, in addition to the aldoximegroup, and with its intrinsic reactivity significantly higher than thatobserved for the hydroxy substitution alone on the benzene ring(Table 2). As a consequence, the nucleophilic strength of bothhydroxy benzene aldoximes at physiological pH was enhancedin comparison with corresponding non-hydroxylated benzenealdoximes.

pKa values determined for the same compounds using differentmethods were in good agreement. Moreover, pKa values ofthe oxime group for all four benzene aldoximes were notsignificantly different, irrespective of hydroxy substitutions.

Lastly, the hydroxy group pKa values were identical for bothphenolic oximes, in spite of differential substitution in the benzenering. The hydroxy group pKa, on the other hand, appearedat least 0.6 pH units higher in the absence of the aldoximegroup substitution, as evidenced by the edrophonium comparison.Finally, the pyridinium-based 2PAM oximate appeared to be astronger nucleophile at physiological pH than any of the benzene-based oximates (Table 2), due to its lower (by 2 pH units) pKa forthe oxime to oximate dissociation.

Oxime-assisted OP hydrolysis by hBChE in vitro and ex vivo

Hydrolysis of 5 μM solutions of racemic fluorescent nerveagent OP analogues (Flu-MPs) by combinations of 500 nMhBChE + 100 μM oxime), as monitored by release of fluorescentOP leaving groups, revealed substantial breakdown of all fournerve agent analogues tested (cyclosarin, VX, sarin and soman)within the first few minutes of the reaction (Figure 5). Theinitial fast phases of hydrolysis by cyclosarin and VX probablycorrespond to complete degradation of more toxic fast-inhibitingand fast-reactivating OP enantiomers with PS configuration[21,22], whereas the slow phase represents the sum of non-enzymatic OP oximolysis and slow hBChE inhibition/reactivationby less reactive OP enantiomers with PR configuration. For thesoman analogue, the short initial fast phase corresponds to OPdegradation before completion of aging of the soman–hBChEconjugate. OP hydrolysis assisted by TAB2OH was substantiallyfaster than hydrolysis assisted by 2PAM, for cyclosarin and VX.The hydrolysis of the sarin analogue was slowest (except forthe soman analogue). It was slightly better assisted by 2PAM,consistent with reactivation experiments (Figure 3), and largelymonophasic. Non-enzymatic OP oximolysis, largely equivalentto slow reaction phases (Figure 5), was approximately 2-foldfaster for 2PAM compared with TAB2OH (results not shown),consistent with its stronger nucleophilic reactivity observedagainst ATCh at pH 7.4 (Figure 4 and Table 2).

Thus the combination of 500 nM hBChE and 100 μMTAB2OH was capable of catalysing the complete degradation





Figure 5 Time course of nerve agent OP analogue (5 μM) catalytic hydrolysis by 0.1 mM, TAB2OH + 500 nM hBChE (A) and 0.1 mM 2PAM + 500 nMhBChE (B)

Continuous measurements of the increase in fluorescence intensity of fluorescent leaving groups at 37◦C in 0.1 M phosphate buffer, pH 7.4. The fluorescent OP leaving group for cyclosarin, sarinand soman analogues was 3-cyano-4-methyl-7-hydroxy coumarin, whereas for the VX analogue it was 7-hydroxy-9H-1,3-dichloro-9,9-dimethylacridine-2-one. Hydrolysis in the absence of oximeor in the absence of hBChE was significantly slower and equivalent to or slower than the slow phase of soman or VX analogue hydrolysis.

of 2.5 μM VX and cyclosarin analogues (half of 5 μM racemicOP) within 20 min, under physiological conditions (Figure 5and Supplementary Table S1 at http://www.biochemj.org/bj/450/bj4500231add.htm). Hydrolysis assisted by a combination of500 nM hBChE and 100 μM 2PAM was approximately 4–5-foldslower (Supplementary Table S1).

OP hydrolysis ex vivo monitored in human blood showssignificant OP degradation and consequential recovery of totalChE activity within 10 min following the addition of 100 μMTAB2OH. The blood was initially supplemented with 300 or60 nM purified hBChE and subsequently exposed to 0.5 μMnerve agent Flu-MP OP analogue or paraoxon (Figure 6). Theoxime, hBChE and OP concentrations in this experiment wereselected to mimic respective concentrations likely to be found inthe blood of an OP-exposed person. Here 0.5 μM VX representsan equivalent of 935 μg of VX in a 7.0 litre blood volume fora ∼70 kg adult, or an equivalent of a 13 μg/kg i.v. dose whichis 1.7-fold greater than the extrapolated LD50 value for humans[23] (assuming that all injected OP is transiently retained inblood or vascular space). For hBChE, the range 60–300 nMis an equivalent of an actual 0.5–2.5 mg/kg i.v. dose, and this isapproximately 10- to 50-fold lower than the dose recommendedfor administration of hBChE as a stoichiometric OP bioscavenger[24] in the treatment of an OP exposure. Within the initial 10 minof TAB2OH-assisted catalysis, the VX analogue concentrationdeclined most rapidly (50–70 % recovered activity), followed bythe cyclosarin analogue and paraoxon (∼30% recovered activity),and the sarin analogue (∼15% recovered activity).

Acute TAB2OH toxicity and treatment of OP-exposed mice

We determined the acute i.m. toxicity for mice of the leadhBChE reactivator TAB2OH to be 100 mg/kg, comparable withthat of 2PAM (LD50 = 106 mg/kg; [16]), but more toxic thanthe common oxime reactivator HI-6 (LD50 = 450 mg/kg) or our

lead centrally active N-substituted hydroxyimino acetamido alkylamine, RS194B (LD50 = 500 mg/kg) [16].

OP-exposed mice were treated i.m. with TAB2OH (a doseequal to 25% of its LD50 where no signs of intoxicationwere observed) in conjunction with atropine 1 min after s.c.OP exposure. The mice were 5.0-fold less sensitive to thedose of VX, 10-fold less sensitive to paraoxon, 2.9-foldless sensitive to sarin and 1.6-fold less sensitive to tabun(Table 3 and Supplementary Table S3 at http://www.biochemj.org/bj/450/bj4500231add.htm). The corresponding indices for2PAM were slightly better, with respective PIs for VX, paraoxon,sarin and tabun of 9.3, 47, 6.7 and 1.3 [16]. This therapeuticefficacy appears surprisingly good and better by (on average)one order of magnitude than would be expected solely fromcomparison of in vitro OP–hAChE reactivation kinetics for 2-PAM and TAB2OH (Supplementary Figure S1 and Table 1). Therespective second-order reactivation rate constants of 2PAM were23-fold, 3.7-fold, 97-fold and 9.7-fold larger than the TAB2OHreactivation rate constants for the respective OP–AChE conjug-ates, VX, paraoxon, sarin and tabun (Table 1), whereas ratios ofrespective PIs between 2PAM and TAB2OH therapies were 1.9,4.7, 2.3 and 0.81 (Table 3). Thus a question could be raised onthe vital importance of reactivation of OP–hBChE conjugates inperipheral tissues accessible to the charged reactivators TAB2OHand 2PAM, for survival in OP intoxication. Pretreatment of micewith a combination of 25 mg of TAB2OH/kg and 1 mg of purifiedhBChE/kg 15 min before OP exposure followed by therapeuticadministration of another 25 mg of TAB2OH/kg in conjunctionwith atropine (10 mg/kg i.m.) 1 min post-OP did not providenotable protection in VX- and tabun-exposed mice. However,significantly (judged by 95% confidence limits; Tables 3 and4) higher PIs were observed for paraoxon (59% increase) andsarin (38% increase) when compared with the oxime therapyalone (in conjunction with atropine). The in-vitro-determined Kox

values for TAB2OH and OP–hAChE conjugates are in the 8–36 mM range, approximately 1000-fold higher than the measured10 μM TAB2OH concentrations in plasma. Accordingly, the







Figure 6 Recovery of total ChE activity in whole human blood supplemented with 300 or 60 nM hBChE and then exposed to nerve agent OP analogues(0.5 μM), upon the addition of 100 μM TAB2OH measured at 37◦C in duplicate

Grey symbols and lines refer to 60 nM hBChE, and black symbols and lines refer to 300 nM hBChE experiments. VX analogue exposure (diamonds), cyclosarin analogue exposure (circles), sarinanalogue exposure (squares) and paraoxon exposure (triangles) is shown.

observed PI increase cannot be ascribed to the protective effectof the native hAChE from OP inhibition by TAB2OH. Rather,we ascribe the increase to catalytic degradation of OP in plasmamediated by the combination of sub-stoichiometric amounts ofhBChE and TAB2OH. To verify this conclusion we varied thetiming and administration regime of hBChE and TAB2OH tomice during and after OP exposure (Table 4). As expected,pretreatment of mice by sub-stoichiometric doses of hBChEalone did not influence PIs, except when it was administeredtogether with atropine. On the other hand, whenever sub-stoichiometric hBChE was administered together with TAB2OH(and with atropine), either as a pretreatment before paraoxonor as a therapy post-paraoxon exposure, the resulting PIs werehigher (significantly considering 95 % confidence limits) by 30–60% than in the treatment without hBChE (Table 4) but withatropine.

TAB2OH pharmacokinetics in mice

The highest concentration of TAB2OH determined in plasmaupon i.m. administration of a single dose of 30 mg/kg was2.2 μg/ml (or ∼10 μM), observed at the initial 15 min collectionpoint (Supplementary Figure S7 and Supplementary TableS2 at http://www.biochemj.org/bj/450/bj4500231add.htm). Thedecline from the plasma is rapid and appears multiphasic,precluding an initial estimate of the volume of distribution afteri.m. dosing. As expected for a quaternary cation, BBB (blood–brain barrier) penetration appeared minimal. The maximal brainconcentration determined at the 15 min collection point was0.13 μg/ml (equivalent to ∼0.56 μM) or approximately 18-foldlower than in plasma. Retention of the low concentrations ofTAB2OH that appear to have penetrated into the brain, however,was prolonged. Although kinetics of elimination by mice istypically more rapid than in humans, the high plasma clearancerate of TAB2OH may necessitate either multiple dosing or a single

dose of a pharmaceutical formulation of the oxime, affordingsustained release from the administration site.

DISCUSSION

The results of the present study describe the initial design,synthesis and detailed in vitro and in vivo characterization ofa novel effective reactivator of phosphorylated hBChE: cationicquaternary oxime TAB2OH. Identified from a directed library ofcationic oxime reactivators based on the ChE reversible inhibitoredrophonium, it proved 4–5-fold more effective than the referenceoxime 2PAM for in vitro reactivation of OP–hBChE conjugatesof cyclosarin, VX and paraoxon. Dissection of reactivation rateconstants revealed improved molecular recognition (reduced Kox)to contribute most to the superior reactivation when comparedeither with 2PAM for reactivation of OP–hBChE conjugates orwith TAB2OH reactivation of OP–hAChE conjugates.

Determinations of oxime group pKa values for the tworeactivators, however, reveal a much less dissociated oximenucleophile of the TAB2OH reactivator (with an averagepKa = 10) compared with 2PAM (with average pKa = 8.1).Measurements of oxime nucleophilic reactivities by hydrolysisof both ATCh and Flu-MPs (Figure 4 and Table 2, andSupplementary Table S1) in the absence of enzyme at pH 7.4show only a 2–3-fold difference between the two oximes dueto biphasic dependence on pH for TAB2OH reactivation. Thelower pKa of 7.21 evaluated from this biphasic dependencecoincides with the lower of the two pKa values observed frompH-dependence of UV spectra of TAB2OH and its homologoushydroxy oxime TAB4OH. This was not observed in monophasicpH-dependences of their non-hydroxy analogues TAB2 andTAB4 (Table 2 and Figure 4) that reveal only a single pKa value of∼10 corresponding to the oxime group. The observed enhancednucleophilic reactivity of TAB2OH (and TAB4OH) at pH 7.4




Table 3 Treatment of OP-exposed mice with the oxime TAB2OH

The oxime was administered either alone (Therapy: i.m., 25 mg/kg with atropine 1 min after an OP) or in combination with hBChE [Pretreatment + Therapy: i.v., 1 mg of hBChE/kg + 25 mg ofTAB2OH/kg 15 min before OP followed by TAB2OH (25 mg/kg with atropine) administered i.m. 1 min after an OP]. PI is the ratio of LD50 for OP-exposed animals with pretreatment/therapy andanimals exposed to OP only. Shown in parentheses are the 95 % confidence limits of PI and MDP (the highest multiple of the OP LD50, which was fully counteracted by the antidotal treatment).

VX* Paraoxon† Sarin‡ Tabun§

Treatment PI (95 % confidence limit) MDP PI (95 % confidence limit) MDP PI (95 % confidence limit) MDP PI (95 % confidence limit) MDP

Therapy 5.0 (2.2–11.7) 3.2 10 (7.5–13.3) 6.3 2.9 (2.3–3.7) 2.0 1.6 (1.2–2.1) 1.3Pretreatment + Therapy 5.0 (3.3–7.7) 3.2 16 (13.5–18.7) 13 4.0 (2.5–6.4) 2.0 1.3 (1.0–1.6) 1.3

*s.c. LD50 of VX was 28 μg/kg.†s.c. LD50 of paraoxon was 740 μg/kg.‡s.c. LD50 of sarin was 240 μg/kg.§s.c. LD50 of tabun was 570 μg/kg.

Table 4 Antidotal efficacy of the oxime TAB2OH and human BChE in OP-exposed mice

PI is the ratio of LD50 for OP-exposed animals with pretreatment/therapy and animals exposed to OP only. In parentheses are the 95 % confidence limits of PI and MDP (the highest multiple of theOP LD50, which was fully counteracted by the antidotal treatment). n.d., not detected.

Pretreatment (i.v.) Therapy (i.m.) VX* Paraoxon†

15 min 30 min 1 min after OP PI (95 % confidence limits) MDP PI (95 % confidence limits) MDP

25 mg of TAB2OH/kg 1.3 (0.7–2.1) 1.310 mg of TAB2OH/kg with

atropine2.7 (2.5–3.0) 2.5

25 mg of TAB2OH/kg withatropine

5.0 (2.2–11.7) 3.2 10.0 (7.5–13.3) 6.3

0.5 mg of hBChE/kg 1.0 (0.9–1.1) 0.791 mg of hBChE/kg 1.1 (0.8–1.4) n.d. <1.0 (n.d.) n.d.0.5 mg of hBChE/kg 10 mg of TAB2OH/kg with

atropine3.4 (2.7–4.4) 3.4

1 mg of hBChE/kg in atropine 2.5 (1.3–4.8) 1.31 mg of hBChE/kg 25 mg of TAB2OH/kg with

atropine5.0 (3.3–7.7) 3.2 14.1 (12.0–16.7) 10

1 mg of hBChE/kg with 25 mgof TAB2OH/kg

<1 (n.d.) n.d. <1 (n.d.) n.d.

1 mg of hBChE/kg with25 mg of TAB2OH/kg

<1 (n.d.) n.d.

1 mg of hBChE/kg with 25 mgof TAB2OH/kg

25 mg of TAB2OH/kg withatropine

5.0 (3.3–7.7) 3.2 16 (13.5–18.7) 13

1 mg of hBChE/kg + 25 mg ofTAB2OH/kg with atropine

13 (8.2–19.1) 7.9

*s.c. LD50 of VX was 28 μg/kg.†s.c. LD50 of paraoxon was 740 μg/kg.

thus appears as a consequence of deprotonation of the ortho-substituted phenolic hydroxy. Direct participation of nucleophilicreactivity of this hydroxy itself in those reactions is, however,unlikely since the hydroxy group of the structurally relatededrophonium remains completely unreactive, even at pH 10 in itsnearly completely deprotonated form. Thus a new non-aldoximenucleophilic species or an activated aldoxime moiety due toan intramolecular interaction with the adjacent hydroxy moiety,formed upon deprotonation of the hydroxy ortho to the aldoxime,contributes to the unusual reactivity of TAB2OH and TAB4OHat physiological pH values.

One possible mechanism that could bridge between these twodifferent pKa values and nucleophilicity of phenolate comparedwith oxime group could be the formation of an intermediate toa stabilized bicyclic ring comprising two adjacent carbons of thebenzene ring, the phenolate O− and two atoms of the aldoximeC = N-O-H. Such a transition state or meta-stable intermediate

ring cyclization to form an isoxazole could form at pH 7.4.Alternatively the phenolate O− in the active site might react witha terminal hydrogen from the CH = NOH group, leading to partialdeprotonation of CH = NO-H and converting it into an oximateanion CH = NO− at physiological pH, the active nucleophilein reactivation of OP–ChE conjugates by aldoximes. Additionalstudies will be needed to identify the precise structural speciesand mechanistic nature of this functionally beneficial nucleophilicreaction. Enhanced nucleophilicity of ortho hydroxy-substitutedbenzene aldoximes, but with monophasic pH-dependence andonly limited in vitro reactivation potency has been observedelsewhere [25]. Additionally, hydroxy groups positioned orthoto oxime reactivators enhance decomposition of phosphonyl-oximes, probably through intramolecular formation of anisoxazole ring [26,27], and minimize eventual reinhibitionof enzymes observed for some phosphonyl-oxime adducts[28].



A reasonable in vitro potency of TAB2OH for reactivation ofOP–hBChE conjugates raises new considerations for establishinga catalytic OP bioscavenger on the basis of TAB2OH-assistedOP hydrolysis by hBChE. Our initial experiments demonstratethat both in vitro in physiologically conditioned buffers andex vivo in the whole human blood, low micromolar and sub-micromolar OP concentrations (that could be expected toaccumulate in the blood of OP-exposed individuals) are degradedsignificantly within 5–20 min of administration of 100 μMTAB2OH and sub-stoichiometric (60–300 nM) hBChE. Theseresults and moderate acute toxicity of TAB2OH (LD50 of100 mg/kg i.m.) in mice allowed us to test this catalytic OPbioscavenger in vivo in nerve-agent- and OP-pesticide-exposedmice. Under various administration regimens of TAB2OH andhBChE to the OP-exposed mice, we observed 30–60 % enhancedoxime PIs whenever the BChE + TAB2OH combination wasadministered to paraoxon-exposed mice, either before paraoxonexposure (pretreatment) or after paraoxon exposure (therapy).No protection was observed for mice administered with sub-stoichiometric amounts of hBChE alone (without atropine), eitherin pretreatment or when administered therapeutically post-OP. Weassume that enhanced PIs reflect catalytic turnover of paraoxonby the hBChE + TAB2OH combination acting as a catalyticbioscavenger.

The moderate extent of PI enhancement for paraoxon, and theabsence of the enhancement in the treatment of VX-exposed miceprobably reflect low tissue accumulation and rapid clearanceof TAB2OH from plasma. A maximal determined plasmaconcentration of 10 μM TAB2OH 15 min following a single30 mg/kg i.m. administration (Supplementary Figure S5) is likelyto be insufficient to provide optimal assistance to hBChE in theOP turnover. To enhance efficacy in catalytic OP bioscavenging,a compound with lower toxicity and lower clearance than theTAB2OH lead would be beneficial. Nevertheless, the results ofthe present study with this lead show the ‘proof of principle’for effective catalytic OP bioscavenging based on combinedadministration of sub-stoichiometric amounts of purified hBChEand an oxime reactivator. Such an approach would allow forsignificant cost reduction and more practical implementation ofantidotal administration in treatment of OP exposure in high-risksettings.

AUTHOR CONTRIBUTION

Zoran Radic, Julius Rebek Jr, Dariush Ajami, Trevor Dale and Palmer Taylor conceptuallydeveloped the project. Zoran Radic, Palmer Taylor, Julius Rebek Jr, Zrinka Kovarik, GabrielAmitai, Suzana Berend, Dariush Ajami, Carol Green and Brendan Duggan contributedto writing the paper. Julius Rebek Jr, Trevor Dale, Dariush Ajami and Gabriel Amitaidesigned and synthesized the oximes and fluorescent non-volatile OPs. Zoran Radic,Palmer Taylor, Julius Rebek Jr, Zrinka Kovarik, Bozica Radic and Carol Green contributedto the experimental design of the study. Zoran Radic, Edzna Garcia, Limin Zhang, SuzanaBerend, Zrinka Kovarik and Brendan Duggan performed experiments and analysed thedata.

ACKNOWLEDGEMENTS

We thank Mr Vedran Micek and Ms Jasna Milekovic for assistance with in vivo experiments,and Ms Valeria Gerardi for recording the TAB2OH UV–Vis spectra.

FUNDING

This work was supported by the CounterACT Program, the National Institutes of HealthOffice of the Director, and the National Institute of Neurological Disorders and Stroke[grant numbers U01 NS058046 and R21NS072086].

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Received 22 October 2012/4 December 2012; accepted 6 December 2012Published as BJ Immediate Publication 6 December 2012, doi:10.1042/BJ20121612


Biochem. J. (2013) 450, 231–242 (Printed in Great Britain) doi:10.1042/BJ20121612

SUPPLEMENTARY ONLINE DATACatalytic detoxification of nerve agent and pesticide organophosphatesby butyrylcholinesterase assisted with non-pyridinium oximesZoran RADIC*1, Trevor DALE†, Zrinka KOVARIK‡, Suzana BEREND‡, Edzna GARCIA*, Limin ZHANG*, Gabriel AMITAI§,Carol GREEN‖, Bozica RADIC‡, Brendan M. DUGGAN*, Dariush AJAMI†, Julius REBEK Jr† and Palmer TAYLOR**Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, La Jolla, CA 92093, U.S.A., †Skaggs Institute for Chemical Biology and Department ofChemistry, The Scripps Research Institute, La Jolla, CA 92037, U.S.A., ‡Institute for Medical Research and Occupational Health, HR-10001 Zagreb, Croatia, §Department ofPharmacology, Israel Institute for Biological Research, Ness Ziona 74100, Israel, and ‖SRI International, Menlo Park, CA 94025-3493, U.S.A.

1H-NMR and 13C-NMR spectra were recorded at 600 and150.9 MHz respectively, using a Bruker DRX-600 spectrometerequipped with a 5 mm QNP probe. Chemical shifts of 1H NMRand 13C NMR are given in p.p.m. by using deuterated solvents asreferences. Standard abbreviations indicating multiplicity wereused as follows: s (singlet), b (broad), d (doublet), t (triplet), q(quartet) and m (multiplet). MALDI–TOF (matrix-assisted laser-desorption ionization–time-of-flight) spectra and HRMS (high-resolution mass spectra) were recorded on an Applied BiosystemsVoyager STR (2) apparatus and an Agilent ESI-TOF massspectrometer respectively. Anhydrous dichloromethane, N,N-diethylethanamine and diethyl ether were taken from a solventdrying system (SG Water USA).

SYNTHETIC PROCEDURES

(3-Methoxymethoxy-phenyl)-dimethylamine (4.15)

To a stirring solution of 3-dimethylaminophenol (1.2 g, 8.8 mmol)in dichloromethane (12 ml) at 0 ◦C was added DIEA (3.0 ml,17.2 mmol) and chloromethyl methyl ether (1.2 ml, 15.8 mmol)and the solution was stirred at room temperature for 3 h. Thereaction was quenched with a saturated solution of sodiumbicarbonate and stirred for 30 min. The layers were separatedand the aquous layer was extracted twice with dichloromethane,the fractions combined, dried with magnesium sulfate, filteredand the solvent was removed under reduced pressure. The crudematerial was purified twice by flash chromatography eluting oncewith dichloromethane and the second time with 9:1 hexane/ethylacetate to afford 660 mg of pure product as a colourless oil(42%). 1H-NMR (600 MHz, [2H]chloroform) δ 7.13–7.16 (m, 1H), 6.40–6.44 (m, 3 H), 5.17 (s, 2 H), 3.49 (s, 3 H), 2.94 (s, 6 H).13C-NMR (150.9 MHz, [2H]chloroform) δ 158.4, 152.0, 129.7,106.7, 103.9, 101.1, 94.5, 55.9, 40.5. HRMS (MH+ ) expected,182.1175; found, 182.1179.

2-Dimethylamino-6-methoxymethoxy-benzaldehyde (4.16) and4-dimethylamino-2-methoxymethoxy-benzaldehyde (4.17)

To a stirring solution of 4.15 (600 mg, 3.3 mmol) andfreshly distilled N,N,N ′,N ′-tetramethylethylenediamine (0.5 ml,3.3 mmol) in dry ethyl ether (10 ml) cooled to − 40 ◦C underargon was added n-butyllithium (2.0 ml of 1.8 M in hexanesolution, 3.6 mmol) and the reaction was allowed to warm toroom temperature and stirred for 1 h. The reaction was re-cooled to − 40 ◦C and dry DMF (0.5 ml, 6.5 mmol) was addedand the reaction was allowed to warm to room temperaturewith stirring over 1 h before being quenched with a diluteammonium chloride aqueous solution. The mixture was extractedthree times with ethyl acetate, the organic fractions combined,dried over magnesium sulfate, filtered and then the volatileswere removed under reduced pressure. The crude material waspurified by flash chromatography over silica gel eluting with4:1 hexane/ethyl acetate to separate the isomers and then eachone was individually purified by flash chromatography oversilica gel eluting with dichloromethane to afford each pureisomer.

4-Dimethylamino-2-methoxymethoxy-benzaldehyde (4.16)

281 mg, pale yellow solid, 41%. 1H-NMR (600 MHz,[2H]chloroform) δ 10.19 (s, 1 H), 7.73 (d, J 8.9 Hz, 1 H), 6.36(dd, J 8.9, 2.0 Hz, 1 H), 6.33 (d, J 2.0 Hz, 1 H), 5.28 (s, 2 H), 3.52(s, 3 H), 3.06 (s, 6 H). 13C-NMR (150.9 MHz, [2H]chloroform) δ187.4, 161.8, 155.8, 130.1, 115.2, 105.7, 96.3, 94.7, 56.4, 40.1.HRMS (MH+ ) expected, 210.1125; found, 210.1125.


317 mg, yellow oil, 46%. 1H-NMR (600 MHz, [2H]chloroform)δ 10.37 (s, 1 H), 7.32 (t, J 8.3 Hz, 1 H), 6.63 (d, J 8.3 Hz,2 H), 5.25 (s, 2 H), 3.51 (s, 3 H), 2.89 (s, 6 H). 13C-NMR (150.9MHz, [2H]chloroform) δ 188.6, 161.1, 155.2, 134.9, 115.8, 110.4,105.1, 95.0, 56.5, 44.7. HRMS (MH+ ) expected, 210.1125; found,210.1123.

4-Dimethylamino-2-methoxymethoxy-benzaldehyde oxime

To a stirring solution of 4.16 (130 mg, 0.62 mmol) inethanol (10 ml) was added hydroxylamine hydrochloride (77 mg,



Z. Radic and others

1.1 mmol) and the reaction was stirred at room temperature for30 min. A saturated solution of sodium bicarbonate and ethylacetate were added and the layers were separated. The aqueouslayer was extracted twice with ethyl acetate, the organic fractionscombined, dried over magnesium sulfate, filtered and then thevolatiles were removed under reduced pressure. The solventwas removed under reduced pressure and the crude material waspurified by flash chromatography over silica gel eluting with1:3:1 hexane/dichloromethane/ethyl acetate to afford 89 mg ofpure product as a white solid (64 %). In addition 21 mg of asecond product was isolated that appears to be the oxime isomer(15%). 1H-NMR (600 MHz, [2H]chloroform) δ 8.41 (s, 1 H),7.78 (bs, 1 H), 7.57 (d, J 8.8 Hz, 1 H), 6.43 (d, J 2.4 Hz,1 H), 6.33 (dd, J 8.8, 2.4 Hz, 1 H), 5.22 (s, 2 H), 3.50 (s,3 H), 2.99 (s, 6 H). 13C-NMR (150.9 MHz, [2H]chloroform)δ 156.9, 152.8, 146.9, 127.5, 109.4, 106.3, 98.1, 94.8, 56.2,40.3. HRMS (MH+ ) expected, 225.1234; found, 225.1233.

4-Dimethylamino-2-hydroxy-benzaldehyde oxime (4.18)

To a stirring solution of 4-dimethylamino-2-methoxymethoxybenzaldehyde oxime (80 mg, 0.36 mmol) in methanol (2 ml) atroom temperature was added a 4 M solution of hydrogen chloridein dioxane (0.3 ml, 1.2 mmol). The reaction was heated to 70 ◦Cfor 1 h and then cooled to room temperature and the solvent wasblown off with nitrogen. The crude material was purified by flashchromatography over silica gel eluting with dichloromethane toafford 50 mg of pure product as a white solid (82%). 1H-NMR(600 MHz, [2H]chloroform) δ 9.83 (s, 1 H), 8.11 (s, 1 H), 7.00 (d,J 8.2 Hz, 1 H), 6.93 (s, 1 H), 6.27 (m, 2 H), 2.99 (s, 6 H). 13C-NMR(150.9 MHz, [2H]chloroform) δ 158.7, 153.1, 152.7, 131.7, 105.5,104.2, 99.0, 40.1. HRMS (MH+ ) expected, 181.0971; found,181.0969.

4-Trimethylammonio-2-hydroxy-benzaldehyde oxime iodide(TAB4OH)

To a stirring solution of 4.18 (32 mg, 0.18 mmol) indichloromethane (0.7 ml) at room temperature was addediodomethane (0.1 ml, 1.6 mmol). The reaction was stirred atroom temperature for 40 h as a precipitate formed. The productwas collected by filtration washing with excess dichloromethaneyielding 21 mg of pure product as a white solid (37%). 1H-NMR(600 MHz, [U-2H]methanol) δ 8.36 (s, 1 H), 7.61 (d, J 8.7 Hz, 1H), 7.47 (d, J 2.7 Hz, 1 H), 7.44 (dd, J 8.7, 2.7 Hz, 1 H), 3.69(s, 9 H). 13C-NMR (150.9 MHz, [U-2H]methanol) δ 159.5, 150.2,149.5, 133.6, 121.0, 111.8, 109.6, 57.7. HRMS (M + ) expected,195.1133; found, 195.1133.


To a stirring solution of 4.17 (163 mg, 0.78 mmol) inethanol (10 ml) was added hydroxylamine hydrochloride (85 mg,1.2 mmol) and the reaction was stirred at room temperature for30 min. A saturated solution of sodium bicarbonate and ethylacetate were added and the layers were separated. The aqueouslayer was extracted twice with ethyl acetate, the organic fractionscombined, dried over magnesium sulfate, filtered and then thevolatiles were removed under reduced pressure. The solventwas removed under reduced pressure and the crude material waspurified by flash chromatography over silica gel eluting with1:3:1 hexane/dichloromethane/ethyl acetate to afford 160 mg ofpure product as a white solid (91 %). 1H-NMR (600 MHz,[2H]chloroform) δ 9.93 (s, 1 H), 8.46 (s, 1 H), 7.24 (t, J 8.2Hz, 1 H), 6.88 (d, J 8.2 Hz, 1 H), 6.76 (d, J 8.2 Hz, 1 H),5.25 (s, 2 H), 3.50 (s, 3 H), 2.75 (s, 6 H). 13C-NMR (150.9MHz, [2H]chloroform) δ 156.4, 154.8, 146.0, 130.4, 114.5, 112.1,108.9, 94.7, 56.3, 44.9. HRMS (MH+ ) expected, 225.1234; found,225.1234.


To a stirring solution of 2-dimethylamino-6-methoxymethoxy-benzaldehyde oxime (14 mg, 0.06 mmol) in methanol (0.2 ml) atroom temperature was added a 4 M solution of hydrogen chloridein dioxane (0.2 ml, 0.8 mmol). The reaction was heated to 70 ◦Cfor 1 h and then cooled to room temperature and the solvent wasblown off with nitrogen. The crude material was purified by flashchromatography over silica gel eluting with dichloromethane toafford 10 mg of pure product as a white solid (91%). 1H-NMR(600 MHz, [2H]chloroform) δ 10.06 (s, 1 H), 8.65 (s, 1 H), 7.21 (t,J 8.1 Hz, 1 H), 7.11 (s, 1 H), 6.66 (d, J 8.2 Hz, 1 H), 6.60 (d, J 8.0Hz, 1 H), 2.73 (s, 6 H). 13C-NMR (150.9 MHz, [2H]chloroform)δ 158.5, 154.8, 151.4, 131.6, 111.3, 110.2, 109.8, 45.6. HRMS(MH+ ) expected, 181.0971; found, 181.0983.

2-Trimethylammonio-6-hydroxy-benzaldehyde oxime chloride(TAB2OH)

To a stirring solution of 4.19 (90 mg, 0.50 mmol) in dichlorometh-ane (0.2 ml) at 0 ◦C was added methyl trifluoromethansulfonate(62 μl, 0.55 mmol). The reaction was allowed to warm to roomtemperature and stirred for 40 h. The precipitate formed wascollected by filtration washing with excess dichloromethaneto afford 101 mg of the pure triflate salt as a white solid(59%). The salt was dissolved in acetonitrile and a solutionof tetrahexylammonium chloride in acetonitrile was added toprecipitate the pure chloride salt as a white solid. 1H-NMR (600MHz, d6-DMSO) δ 11.74 (s, 1 H), 10.95 (s, 1 H), 8.34 (s, 1H), 7.45 (t, J 8.4 Hz, 1 H), 7.39 (d, J 8.4 Hz, 1 H), 7.26 (d, J8.2 Hz, 1 H), 3.68 (s, 9 H). 13C-NMR (150.9 MHz, d6-DMSO)


Organophosphate detoxification by butyrylcholinesterase and oximes

δ 158.9, 145.6, 145.5, 130.6, 117.5, 113.5, 111.3, 57.2. HRMS(M + ) expected, 195.1133; found, 195.1130.

4-Dimethylethylammonio-2-hydroxy-benzaldehyde oxime chloride(TAB4OHmme)

To a stirring solution of 4.18 (53 mg, 0.29 mmol) in dichlorometh-ane (1.5 ml) at 0 ◦C was added ethyl trifluoromethansulfonate(42 μl, 0.32 mmol). The reaction was allowed to warm to roomtemperature and stirred for 24 h. The mixture was tritratedwith dichloromethane and then the triflate salt was dissolved inacetonitrile and a solution of tetrahexylammonium chloridein acetonitrile was added to precipitate 46 mg of the pure chloridesalt as a white solid (64%). 1H-NMR (600 MHz, d6-DMSO) δ11.72 (s, 1 H), 10.94 (s, 1 H), 8.37 (s, 1 H), 7.71 (d, J 8.8 Hz, 1H), 7.44 (m, 1 H), 7.39 (dd, J 8.8, 2.6 Hz, 1 H), 3.90 (q, J 7.2 Hz,2 H), 3.53 (s, 6 H), 1.00 (t, J 7.2 Hz, 3 H). 13C-NMR (150.9MHz, d6-DMSO) δ 156.5, 145.7, 145.0, 128.8, 119.9, 112.0,109.4, 63.8, 53.0, 8.4. HRMS (M + ) expected, 209.1284; found,209.1294.

(3-Methoxymethoxyphenyl)-diethylamine (4.21)

To a stirring solution of 3-diethylaminophenol (1.29 g, 7.8 mmol)in dichloromethane (12 ml) at 0 ◦C was added DIEA (2.0 ml,11.5 mmol) and chloromethyl methyl ether (0.75 ml, 9.9 mmol)and the solution was stirred at room temperature for 15 h.The reaction was quenched with a 10% solution of sodiumhydroxide and stirred for 30 min. The layers were separatedand the aquous layer extracted twice with dichloromethane, thefractions combined, dried with magnesium sulfate, filtered andthe solvent removed under reduced pressure. The crudematerial was purified by flash chromatography eluting with 9:1hexane/ethyl acetate to afford 900 mg of pure product as acolourless oil (55%). 1H-NMR (600 MHz, [2H]chloroform) δ7.09–7.12 (m, 1 H), 6.35–6.36 (m, 3 H), 5.16 (s, 2 H), 3.49 (s, 3H), 3.34 (q, J 7.1 Hz, 4 H) 1.16 (t, J 7.1 Hz, 6 H). 13C-NMR (150.9MHz, [2H]chloroform) δ 158.6, 149.2, 129.9, 106.0, 102.6, 100.2,94.5, 55.9, 44.4, 12.6. HRMS (MH+ ) expected, 210.1488; found,210.1492.

4-Diethylamino-2-methoxymethoxy-benzaldehyde (4.22)

To a stirring solution of 4-diethylaminosalicylaldehyde (436 mg,2.3 mmol) in DMF (6 ml) at 0 ◦C was added DIEA (0.6 ml,3.5 mmol) and chloromethyl methyl ether (0.2 ml, 2.6 mmol)

and the solution was stirred at room temperature for 3 h.The reaction was quenched with a 5% sodium hydroxidesolution and stirred for 30 min. Ethyl acetate was added andthe layers were separated and the aqueous layer was extractedtwice with ethyl acetate, the fractions combined, dried withmagnesium sulfate, filtered and the solvent removed underreduced pressure. The crude material was purified twice byflash chromatography eluting once with dichloromethane andthe second time with 9:1 dichloromethane/ethyl acetate toafford 250 mg of pure product (47%). 1H-NMR (600 MHz,[2H]chloroform) δ 10.16 (s, 1 H), 7.71 (d, J 9.0 Hz, 1 H),6.33–6.35 (m, 2 H), 5.26 (s, 2 H), 3.52 (s, 3 H), 3.41 (q, J7.1 Hz, 4 H), 1.21 (t, J 7.1 Hz, 6 H). 13C-NMR (150.9 MHz,[2H]chloroform) δ 187.1, 162.1, 153.7, 130.3, 114.7, 105.4, 95.9,94.7, 56.3, 44.8, 12.5. HRMS (MH+ ) expected, 238.1438; found,238.1438.

4-Diethylamino-2-methoxymethoxy-benzaldehyde (4.22) and2-diethylamino-6-methoxymethoxy-benzaldehyde (4.23)

To a stirring solution of 4.21 (840 mg, 4.0 mmol) andfreshly distilled N,N,N ′,N ′-tetramethylethylenediamine (0.61 ml,4.1 mmol) in dry ethyl ether (15 ml) cooled to − 40 ◦C underargon was added n-butyllithium (2.5 ml of 1.8 M in hexanesolution, 4.5 mmol) and the reaction was allowed to warm toroom temperature and stirred for 1 h. The reaction was re-cooledto − 40 ◦C and dry DMF (0.6 ml, 7.7 mmol) was added andthe reaction was allowed to warm to room temperature withstirring over 1 h before being quenched with a dilute ammoniumchloride aqueous solution. The mixture was extracted three timeswith ethyl acetate, the organic fractions combined, dried overmagnesium sulfate, filtered and then the volatiles were removedunder reduced pressure. The crude material was purified by flashchromatography over silica gel eluting with dichloromethane. Theisomers were separated by eluting with 7:3 hexane/ethyl acetateto afford each pure isomer.


386 mg, pale yellow oil, 41 %. 1H-NMR (600 MHz,[2H]chloroform) δ 10.16 (s, 1 H). 7.72 (d, J 8.8 Hz, 1 H), 6.33–6.35 (m, 2 H), 5.26 (s, 2 H), 3.52 (s, 3 H), 3.41 (q, J 7.1 Hz, 4 H),1.21 (t, J 7.1 Hz, 6 H). 13C-NMR (150.9 MHz, [2H]chloroform)δ 187.1, 162.1, 153.7, 130.3, 114.7, 105.4, 95.9, 94.7, 56.3, 44.8,12.6. HRMS (MH+ ) expected, 238.1438; found, 238.1438.


239 mg, yellow oil, 25%. 1H-NMR (600 MHz, [2H]chloroform)δ 10.25 (s, 1 H). 7.35 (t, J 8.3 Hz, 1 H), 6.77 (dd, J 8.3, 2.3Hz, 2 H), 5.25 (s, 2 H), 3.51 (s, 3 H), 3.19 (q, J 7.1 Hz, 4 H),1.07 (t, J 7.1 Hz, 6 H). 13C-NMR (150.9 MHz, [2H]chloroform) δ190.1, 159.5, 155.0, 134.1, 120.3, 114.5, 108.1, 95.1, 56.5, 48.2,12.3. HRMS (MH+ ) expected, 238.1438; found, 238.1440.


Z. Radic and others

4-Diethylamino-2-hydroxy-benzaldehyde oxime (4.25)

To a stirring solution of 4-diethylamino-salicylaldehyde (494 mg,2.6 mmol) in ethanol (10 ml) was added hydroxylaminehydrochloride (220 mg, 3.2 mmol) and the reaction was stirredat room temperature for 15 h. A saturated solution of sodiumbicarbonate and ethyl acetate were added and the layers wereseparated. The aqueous layer was extracted twice with ethylacetate, the organic fractions combined, dried over magnesiumsulfate, filtered and then the volatiles were removed under reducedpressure. The solvent was removed under reduced pressure andthe crude material was purified by flash chromatography oversilica gel eluting with dichloromethane to afford 432 mg ofpure product as an off-white solid (81 %). 1H-NMR (600 MHz,[2H]chloroform) δ 9.79 (s, 1 H), 8.09 (s, 1 H), 6.96 (m, 1 H), 6.83(bs, 1 H), 6.22–6.24 (m, 2 H), 3.36 (q, J 7.1 Hz, 4 H), 1.18 (t,J 7.1 Hz, 6 H). 13C-NMR (150.9 MHz, [2H]chloroform) δ 159.0,153.1, 150.3, 131.9, 104.7, 103.7, 98.2, 44.4, 12.6. HRMS (MH+ )expected, 209.1284; found, 209.1285.

4-Diethylmethylammonio-2-hydroxy-benzaldehyde oxime chloride(TAB4OHmee)

To a stirring solution of 4.25 (98 mg, 0.47 mmol) indichloromethane (0.7 ml) at 0 ◦C was added MeOTf (methyltrifluoromethansulfonate; 62 μl, 0.55 mmol). The reaction wasallowed to warm to room temperature and stirred for 23 h. Thevolatiles were then blown off with nitrogen and the residuepurified twice by flash chromatography over silica gel by elutingwith a gradient of 19:1 to 17:1 dichloromethane/methanol toafford the pure tosylate salt. The salt was dissoved in methanoland eluted through DOWEX 1-2×200 resin to exchange for thechloride anion and the material was recrystallized from ethanolto afford 38 mg of the pure chloride salt as a white solid (31 %).1H-NMR (600 MHz, [U-2H]methanol) δ 8.37 (s, 1 H), 7.63 (d, J8.7 Hz, 1 H), 7.33 (d, J 2.7 Hz, 1 H), 7.29 (dd, J 8.7, 2.7 Hz, 1H), 4.06 (m, 2 H), 3.86 (m, 2 H), 3.52 (s, 3 H), 1.19 (t, J 7.1 Hz,6 H). 13C-NMR (150.9 MHz, [U-2H]methanol) δ 159.7, 150.2,143.4, 132.7, 121.0, 113.7, 111.4, 65.6, 46.7, 8.8. HRMS (M + )expected, 223.1446; found, 223.1446.

4-Dimethylamino-benzaldehyde oxime (4.28)

To a stirring solution of 4-dimethylamino-benzaldehyde (457 mg,3.1 mmol) in ethanol (17 ml) was added hydroxylaminehydrochloride (290 mg, 4.2 mmol) and the reaction was stirred

at room temperature for 15 h as a yellow colour developed. Asaturated solution of sodium bicarbonate and ethyl acetate wereadded and the layers were separated. The aqueous layer wasextracted twice with ethyl acetate, the organic fractions combined,dried over magnesium sulfate, filtered and then the volatiles wereremoved under reduced pressure. The solvent was removed underreduced pressure and the crude material was purified by flashchromatography over silica gel eluting with a gradient of 1:0 to 4:1dichloromethane/ethyl acetate to afford 470 mg of pure productas a white solid (93%). 1H-NMR (600 MHz, [2H]chloroform) δ8.05 (s, 1 H), 7.45 (d, J 8.9 Hz, 2 H), 6.69 (d, J 8.9, 2 H), 3.00(s, 9 H). 13C-NMR (150.9 MHz, [2H]chloroform) δ 151.5, 150.6,128.3, 119.7, 111.9, 40.2. HRMS (MH+ ) expected, 165.1022;found, 165.1028.

4-Trimethylammonio-benzaldehyde oxime chloride of TAB4

To a stirring solution of 4.28 (75 mg, 0.46 mmol) in dichlorometh-ane (3 ml) at 0 ◦C was added methyl trifluoromethansulfonate(57 μl, 0.50 mmol). The reaction was allowed to warm to roomtemperature and stirred for 19 h. The formed precipitate wascollected by filtration washing with excess dichloromethaneto afford 128 mg of the pure triflate salt as a white solid(85%). The salt was dissolved in acetonitrile and a solutionof tetrahexylammonium chloride in acetonitrile was added toprecipitate the pure chloride salt as a white solid (58 %). 1H-NMR (600 MHz, d6-DMSO) δ 11.64 (s, 1 H), 8.24 (s, 1 H),8.03 (d, J 9.1 Hz, 2 H), 7.81 (d, J 9.1 Hz, 2 H), 3.64 (s, 9H). 13C-NMR (150.9 MHz, d6-DMSO) δ 147.3, 146.4, 134.6,127.4, 121.0, 56.2. HRMS (M + ) expected, 179.1179; found,179.1180.


To a stirring solution of 2-dimethylamino-benzaldehyde (266 mg,1.8 mmol) in ethanol (5 ml) was added hydroxylaminehydrochloride (137 mg, 2.0 mmol) and the reaction was stirredat room temperature for 30 min as the yellow colour faded tocolourless. A saturated solution of sodium bicarbonate and ethylacetate were added and the layers were separated. The aqueouslayer was extracted twice with ethyl acetate, the organic fractionscombined, dried over magnesium sulfate, filtered and then thevolatiles were removed under reduced pressure. The solventwas removed under reduced pressure and the crude material waspurified by flash chromatography over silica gel eluting with2:2:1 hexane/dichloromethane/ethyl acetate to afford 270 mg ofpure product as a white solid (92 %). 1H-NMR (600 MHz,[2H]chloroform) δ 8.61 (bs, 1 H), 8.47 (s, 1 H), 7.68 (d, J 7.7 Hz,1 H), 7.34 (t, J 8.5 Hz, 1 H), 7.07 (d, J 8.1 Hz, 1 H), 7.03 (t, J 7.5Hz, 1 H), 2.75 (s, 6 H). 13C-NMR (150.9 MHz, [2H]chloroform)δ 153.1, 149.0, 130.5, 127.5, 125.4, 122.5, 118.5, 45.2. HRMS(MH+ ) expected, 165.1022; found, 165.1025.




To a stirring solution of 4.30 (69 mg, 0.42 mmol) in dichlorometh-ane (2 ml) at 0 ◦C was added methyl trifluoromethansulfonate(50 μl, 0.44 mmol). The reaction was allowed to warm to roomtemperature and stirred for 36 h. After the solvent was blownoff under nitrogen, the salt was dissoved in methanol and elutedthrough DOWEX 1-2×200 resin to exchange for the chlorideanion. After drying under reduced pressure, the crude materialwas dissolved in a mixture of ethyl acetate and water and the layerswere separated. The organic layer was extracted once with waterand then the aqueous solution was freeze-dried. The materialwas then tritrated with dichloromethane and acetonitrile to afford16 mg of a white solid that was 85% pure, a mixture of thechloride salt with an unknown impurity (18 %). 1H-NMR (600MHz, [U-2H]methanol) δ 8.75 (s, 1 H), 7.99 (d, J 7.7 Hz, 1 H), 7.77(t, J 8.5 Hz, 1 H), 7.68 (m, 2 H), 3.80 (s, 9 H). 13C-NMR (150.9MHz, [U-2H]methanol) δ 148.4, 146.5, 135.1, 132.3, 132.0, 128.2,122.3, 58.5. HRMS (M + ) expected, 179.1179; found, 179.1182.

4-Dimethylethylammonio-benzaldehyde oxime chloride(TAB4mme)

To a stirring solution of 4.28 (66 mg, 0.40 mmol) in dichloro-methane (2 ml) at 0 ◦C was added ethyl trifluoromethansulfonate(57 μl, 0.44 mmol). The reaction was allowed to warm to roomtemperature and stirred for 24 h. The formed precipitate wascollected by filtration washing with excess dichloromethane toafford 90 mg of the pure triflate salt (65%). The salt was dissolvedin acetonitrile and a solution of tetrahexylammonium chloride inacetonitrile was added to precipitate 42 mg of the pure chloridesalt as a white solid (46%). 1H-NMR (600 MHz, d6-DMSO) δ11.63 (s, 1 H), 8.24 (s, 1 H), 7.94 (d, J 9.0 Hz, 2 H), 7.82 (d, J 9.0Hz, 2 H), 3.96 (q, J 7.2 Hz, 2 H), 3.59 (s, 6 H), 0.99 (t, J 7.2 Hz,3 H). 13C-NMR (150.9 MHz, d6-DMSO) δ 146.4, 144.2, 134.6,127.5, 121.8, 63.9, 53.1, 8.5. HRMS (M + ) expected, 193.1335;found, 193.1335.

4-Diethylamino benzaldehyde oxime (4.32)

To a stirring solution of 4-diethylamino-benzaldehyde (417 mg,2.4 mmol) in ethanol (5 ml) was added hydroxylaminehydrochloride (171 mg, 2.5 mmol) and the reaction was stirredat room temperature for 1 h. A saturated solution of sodiumbicarbonate and ethyl acetate were added and the layers wereseparated. The aqueous layer was extracted twice with ethylacetate, the organic fractions combined, dried over magnesiumsulfate, filtered and then the volatiles were removed under reducedpressure. The solvent was removed under reduced pressure and

the crude material was purified by flash chromatography oversilica gel eluting with a gradient of 3:1 to 2:1 hexane/ethyl acetateto afford 250 mg of pure product as an off-white solid (55%).1H-NMR (600 MHz, [2H]chloroform) δ 8.04 (s, 1 H), 7.79 (bs,1 H), 7.42 (d, J 8.7 Hz, 1 H), 6.65 (bd, J 7.3 Hz, 1 H), 3.38 (q,J 7.1 Hz, 4 H), 1.18 (t, J 7.1 Hz, 6 H). 13C-NMR (150.9 MHz,[2H]chloroform) δ 150.5, 149.0, 128.5, 118.6, 111.3, 44.4, 12.5.HRMS (MH+ ) expected, 193.1341; found, 193.1334.

4-Diethylmethylammonio-benzaldehyde oxime chloride (TAB4mee)

To a stirring solution of 4.32 (71 mg, 0.37 mmol) in dichlorometh-ane (1 ml) at 0 ◦C was added methyl trifluoromethansulfonate(50 μl, 0.44 mmol). The reaction was allowed to warm to roomtemperature and stirred for 24 h. After the solvent was blownoff under nitrogen, the salt was dissoved in methanol and elutedthrough DOWEX 1-2×200 resin to exchange for the chlorideanion and the material was tritrated with dichloromethane toafford 15 mg of the pure chloride salt as a white solid (17%).1H-NMR (600 MHz, [U-2H]methanol) δ 8.17 (s, 1 H), 7.88 (d, J9.1 Hz, 1 H), 7.78 (d, J 9.1 Hz, 1 H), 4.09 (m, 2 H), 3.87 (m, 2H), 3.54 (s, 3 H), 1.15 (t, J 7.2 Hz, 6 H). 13C-NMR (150.9 MHz,[U-2H]methanol) δ 147.7, 142.6, 137.1, 129.6, 123.5, 65.6, 46.8,8.9. HRMS (M + ) expected, 207.1492; found, 207.1492.

3-Hydroxypyridine-2-carboxaldehyde (4.37)

A suspension of 2-(hydroxylmethyl)-3-hydroxy-pyridine (1.0 g,8.0 mmol) in chloroform (20 ml) was added to a stirringsuspension of manganese dioxide (4.0 g) in chloroform (20 ml)at 50 ◦C. The reaction was heated to 60 ◦C for 3 h and thenthe suspension was filtered through celite washing with excesschloroform. The solvent was removed under reduced pressure toafford 430 mg of pure product (43%). 1H-NMR (600 MHz, d6-DMSO) δ 10.77 (s, 1 H), 10.10 (s, 1 H), 8.30 (d, J 4.3 Hz, 1 H),7.55 (dd, J 8.5, 4.2 Hz, 1 H), 7.47 (d, J 8.5 Hz, 1 H). 13C-NMR(150.9 MHz, d6-DMSO) δ 193.9, 156.8, 141.6, 137.9, 129.7,125.8. HRMS (MH+ ) expected, 124.0393; found, 124.0395.

3-Hydroxypyridine-2-carboxaldehyde oxime (4.38)

To a stirring solution of 4.37 (225 mg, 1.8 mmol) in ethanol(10 ml) was added hydroxylamine hydrochloride (137 mg,2.0 mmol) and the reaction was stirred at room temperature for2 h. The solvent was removed under reduced pressure and thecrude material was purified by flash chromatography over silicagel eluting with 40:60:0.5 hexane/ethyl acetate/triethanolamine toafford 105 mg of product as a white solid (41%). An analyticallypure sample was repurified by flash chromatography oversilica gel eluting with 19:1 dichloromethane/methanol. 1H-NMR(600 MHz, d6-DMSO) δ 11.87 (s, 1 H), 10.29 (s, 1 H), 8.31 (s, 1


Z. Radic and others

H), 8.15 (dd, J 4.3, 1.2 Hz, 1 H), 7.34 (dd, J 8.3, 1.1 Hz, 1 H),7.28 (dd, J 8.3, 4.4 Hz, 1 H). 13C-NMR (150.9 MHz, d6-DMSO) δ153.1, 150.6, 140.9, 136.6, 124.7, 123.4. HRMS (MH+ ) expected,139.0502; found, 139.0503.

3-Hydroxy-2-pyridinealdoxime methiodide (2PAMOH)

To a stirring solution of 4.38 in tetrahydrofolate (2 ml) was addediodomethane (0.1 ml, 1.6 mmol) and the reaction was heated to70 ◦C in a sealed tube for 60 h. After cooling, the precipitate wascollected by filtration to yield 29 mg of pure product as a paleyellow solid (60%). 1H-NMR (600 MHz, d6-DMSO) δ 8.77 (s,1 H), 8.42 (d, J 5.5 Hz, 1 H), 7.99 (d, J 8.6 Hz, 1 H), 7.81 (m, 1 H).13C-NMR (150.9 MHz, d6-DMSO) δ 159.3, 143.4, 139.7, 134.8,133.4, 128.4. HRMS (M + ) expected, 153.0659; found, 53.0664.

Figure S1 Concentration-dependence of oxime reactivation of (A) sarin, (B) cyclosarin, (C) VX and (D) paraoxon-inhibited (conjugated) hAChE

Dependence for the lead oxime TAB2OH compared with the reference cationic oxime 2PAM (measured at 37◦C in 0.1 M phosphate buffer, pH 7.4). Data from two to four experiments are shown withassociated S.E.M. of determination. For TAB2OH errors were smaller than the symbol size.



Figure S2 pH-dependence of (A) UV spectra of 50 μM TAB2OH and pH-dependences of (B) A 240nm of 50 μM TAB2OH, (C) A 275nm of 50 μM TAB2OH and (D)A 310nm of 50 μM TAB2OH, along with corresponding pK a values calculated by non-linear regression of eqn 2 (black curves in B and D) or eqn 1 (grey curves inB and D) of the main text [1]


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Figure S3 pH-dependence of 1H-NMR spectra of 10 mM TAB2OH in 2H2O buffers (A, B and C) along with corresponding pK a values calculated from theobserved pH-induced difference in chemical shifts (D, E and F) by non-linear regression [1]

Spectra from the single experiment were aligned using the benzene external standard singlet at 4.55 p.p.m. Resonating protons are highlighted in the TAB2OH structure for each of the peaks.

Figure S4 Peak assignment in the 1H-NMR spectrum of 10 mM TAB2OH in 20 mM phosphate-pyrophosphate ( + 0.1 M NaCl) 2H2O buffer, pH 5



Figure S5 pH-dependence of 1H-NMR spectra of 10 mM TAB2OH in 2H2O buffers, pH 5–10

(A) Expanded view of the spectrum in the chemical-shift region 6.9–7.3. along with the pH-dependent change in chemical shifts for the 7.2 p.p.m. doublet and (B) expanded view of the spectrum inthe chemical shift region 7.0–7.5. along with the pH-dependent change in chemical shifts for the 7.42 p.p.m. doublet. The corresponding pK a values were calculated from the observed pH-induceddifference in chemical shifts (C and D) by non-linear regression [1]. Spectra from the single experiment were aligned using a benzene external standard singlet at 4.55 p.p.m.


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Figure S6 pH-dependence of 1H-NMR spectra of 2.0 mM 2PAM in 2H2Obuffers, pH 5–10

(A) Expanded view of the spectrum in the chemical-shift region 2.80–2.86. (B) pH-dependentchange in chemical shifts for the 2.83 p.p.m. singlet. Spectra from the single experiment werealigned using a benzene external standard singlet at 7.16 p.p.m.

Figure S7 Pharmacokinetics of TAB2OH in mice

Brain (grey symbols and lines) and plasma (white symbols and black lines) compound concentrations were determined at discrete time points upon single 30 mg/kg doses administered to mice i.m.Values are means +− S.D. for three mice.



Scheme S1 Synthesis of TAB2OH and TAB4OH

Scheme S2 Synthesis of TAB4OHmme

Scheme S3 Synthesis of TAB4OHmee


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Scheme S4 Synthesis of TAB2 and TAB4

Scheme S5 Synthesis of TAB4mme

Scheme S6 Synthesis of TAB4mee

Scheme S7 Synthesis of 2PAMOH



Table S1 The first-order rate constants k (10− 5 s− 1) for hydrolysis of 5.0 μM nerve agent OP analogues (Flu-MPs) by oximes alone or by combination of500 nM hBChE and an oxime

Constants refer to the fast phase of hydrolysis shown in Figure 5 of the main text, representing degradation of a fraction of total racemic OP. Constants calculated in representative experiments aregiven. The experiments were replicated at least once.

Oxime (1.0 mM) + OP [BuChE + oxime (1.0 mM)] + OP Oxime (0.1 mM) + OP [BuChE + oxime (0.1 mM)] + OP

Nerve agent OP analogue TAB2OH 2PAM TAB2OH 2PAM TAB2OH 2PAM TAB2OH 2PAM

Sarin 5.5 11 15 27 4.1 4.6 8.5 12Cyclosarin 6.4 11 530 (30 % total OP) 37 3.4 3.7 180 37VX 15 28 430 (50 % total OP) 64 11 13 150 40

Table S2 Maximal oxime concentrations in brains and plasma of micedetermined upon i.m. administration of a single oxime dose (compare withFigure S5)

Distribution coefficients (logD) were calculated from oxime structures using the ChemAxonsoftware package (http://www.chemaxon.com).

[Oxime]max

μg/ml μM

Oxime i.m. dose (mg/kg) Brain Plasma Brain Plasma Brain/plasma logD

TAB2OH 30 0.13 2.2 0.56 10 0.056 − 2.0


http://www.chemaxon.com

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Table S3 Antidotal efficacy of oxime TAB2OH and human BChE in VX-, paraoxon- or sarin-exposed mice

BChE (1 mg/kg) and oxime (25 mg/kg) were administered i.v. 15 min before OP (s.c.). Oxime (25 mg/kg) in therapy was administered i.m. together with atropine (10 mg/kg) 1 min after OP exposure.

Pretreatment 15 min before OP and therapy 1 min after OP exposure

VX (s.c. LD50 = 28.3 μg/kg) Paraoxon (s.c. LD50 = 740.8 μg/kg) Sarin (s.c. LD50 = 238.3 μg/kg)

n×LD50 Survived/treated Symptoms Survived/treated Symptoms Survived/treated Symptoms

1.0 4/4 No visible symptoms.1.26 4/4 One mouse had light tremor during the

first 10 min on application.1.59 4/4 Light tremor 3–4 min on application.2.0 4/4 Tremor and light salivation 20 min on

application.4/4 Tremor 20 min on application.

Surviving animals were weak after24 h.

2.52 4/4 Strong tremor, light salivation andrespiratory disturbance. All symptomsdisappeared after 2 h.

3/4 Light tremor 3–4 min uponapplication. Surviving animalswere weak after 24 h.


2/4 Strong tremor immediately onapplication. Surviving animalswere weak and apathic after 24 h.

4.0 3/4 Strong tremor and salivation 3 min onapplication. Mice were weak withrespiratory disturbance. Survivinganimals were weak after 24 h.


5.0 2/4 Strong tremor and respiratorydisturbance immediately onapplication. Surviving animals wereweak after 24 h.

2/4 Cramps and strong tremorimmediately on application.Surviving animals were very weakafter 24 h.

6.3 1/4 Strong tremor, salivation and respiratorydisturbance immediately onapplication. Surviving mouse wasweak after 24 h.

0/4 Cramps and strong tremorimmediately on application.Animals died between 5 and60 min after application.

7.9 2/4 Strong tremor, paralysis. Survivinganimals were weak after 24 h.

12.6 4/4 Lacrimation, dyspnoea, tremor, oedemaof the eyelids.

15.9 1/4 Strong tremor, lacrimation, dyspnoea.Surviving mouse was weak after 24 h.

20.0 1/4 Three mice died 3–4 min on application.The surviving mouse was very weak,had respiratory disturbance andclosed eyes after 24 h.

25.2 0/4 Strong tremor immediately onapplication. Animals died after2–3 min of application

REFERENCE

1 Radic, Z., Sit, R. K., Kovarik, Z., Berend, S., Garcia, E., Zhang, L., Amitai, G., Green, C.,Radic, B., Fokin, V. V. et al. (2012) Refinement of structural leads for centrally acting oximereactivators of phosphylated cholinesterases. J. Biol. Chem. 287, 11798–11809



Biochem. J. (2013) 450, 231–242 (Printed in Great Britain) doi:10.1042/BJ20121612

SUPPLEMENTARY ONLINE DATACatalytic detoxification of nerve agent and pesticide organophosphatesby butyrylcholinesterase assisted with non-pyridinium oximesZoran RADIC*1, Trevor DALE†, Zrinka KOVARIK‡, Suzana BEREND‡, Edzna GARCIA*, Limin ZHANG*, Gabriel AMITAI§,Carol GREEN‖, Bozica RADIC‡, Brendan M. DUGGAN*, Dariush AJAMI†, Julius REBEK Jr† and Palmer TAYLOR**Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, La Jolla, CA 92093, U.S.A., †Skaggs Institute for Chemical Biology and Department ofChemistry, The Scripps Research Institute, La Jolla, CA 92037, U.S.A., ‡Institute for Medical Research and Occupational Health, HR-10001 Zagreb, Croatia, §Department ofPharmacology, Israel Institute for Biological Research, Ness Ziona 74100, Israel, and ‖SRI International, Menlo Park, CA 94025-3493, U.S.A.

1H-NMR and 13C-NMR spectra were recorded at 600 and150.9 MHz respectively, using a Bruker DRX-600 spectrometerequipped with a 5 mm QNP probe. Chemical shifts of 1H NMRand 13C NMR are given in p.p.m. by using deuterated solvents asreferences. Standard abbreviations indicating multiplicity wereused as follows: s (singlet), b (broad), d (doublet), t (triplet), q(quartet) and m (multiplet). MALDI–TOF (matrix-assisted laser-desorption ionization–time-of-flight) spectra and HRMS (high-resolution mass spectra) were recorded on an Applied BiosystemsVoyager STR (2) apparatus and an Agilent ESI-TOF massspectrometer respectively. Anhydrous dichloromethane, N,N-diethylethanamine and diethyl ether were taken from a solventdrying system (SG Water USA).

SYNTHETIC PROCEDURES

(3-Methoxymethoxy-phenyl)-dimethylamine (4.15)

To a stirring solution of 3-dimethylaminophenol (1.2 g, 8.8 mmol)in dichloromethane (12 ml) at 0 ◦C was added DIEA (3.0 ml,17.2 mmol) and chloromethyl methyl ether (1.2 ml, 15.8 mmol)and the solution was stirred at room temperature for 3 h. Thereaction was quenched with a saturated solution of sodiumbicarbonate and stirred for 30 min. The layers were separatedand the aquous layer was extracted twice with dichloromethane,the fractions combined, dried with magnesium sulfate, filteredand the solvent was removed under reduced pressure. The crudematerial was purified twice by flash chromatography eluting oncewith dichloromethane and the second time with 9:1 hexane/ethylacetate to afford 660 mg of pure product as a colourless oil(42%). 1H-NMR (600 MHz, [2H]chloroform) δ 7.13–7.16 (m, 1H), 6.40–6.44 (m, 3 H), 5.17 (s, 2 H), 3.49 (s, 3 H), 2.94 (s, 6 H).13C-NMR (150.9 MHz, [2H]chloroform) δ 158.4, 152.0, 129.7,106.7, 103.9, 101.1, 94.5, 55.9, 40.5. HRMS (MH+ ) expected,182.1175; found, 182.1179.

2-Dimethylamino-6-methoxymethoxy-benzaldehyde (4.16) and4-dimethylamino-2-methoxymethoxy-benzaldehyde (4.17)

To a stirring solution of 4.15 (600 mg, 3.3 mmol) andfreshly distilled N,N,N ′,N ′-tetramethylethylenediamine (0.5 ml,3.3 mmol) in dry ethyl ether (10 ml) cooled to − 40 ◦C underargon was added n-butyllithium (2.0 ml of 1.8 M in hexanesolution, 3.6 mmol) and the reaction was allowed to warm toroom temperature and stirred for 1 h. The reaction was re-cooled to − 40 ◦C and dry DMF (0.5 ml, 6.5 mmol) was addedand the reaction was allowed to warm to room temperaturewith stirring over 1 h before being quenched with a diluteammonium chloride aqueous solution. The mixture was extractedthree times with ethyl acetate, the organic fractions combined,dried over magnesium sulfate, filtered and then the volatileswere removed under reduced pressure. The crude material waspurified by flash chromatography over silica gel eluting with4:1 hexane/ethyl acetate to separate the isomers and then eachone was individually purified by flash chromatography oversilica gel eluting with dichloromethane to afford each pureisomer.


281 mg, pale yellow solid, 41%. 1H-NMR (600 MHz,[2H]chloroform) δ 10.19 (s, 1 H), 7.73 (d, J 8.9 Hz, 1 H), 6.36(dd, J 8.9, 2.0 Hz, 1 H), 6.33 (d, J 2.0 Hz, 1 H), 5.28 (s, 2 H), 3.52(s, 3 H), 3.06 (s, 6 H). 13C-NMR (150.9 MHz, [2H]chloroform) δ187.4, 161.8, 155.8, 130.1, 115.2, 105.7, 96.3, 94.7, 56.4, 40.1.HRMS (MH+ ) expected, 210.1125; found, 210.1125.


317 mg, yellow oil, 46%. 1H-NMR (600 MHz, [2H]chloroform)δ 10.37 (s, 1 H), 7.32 (t, J 8.3 Hz, 1 H), 6.63 (d, J 8.3 Hz,2 H), 5.25 (s, 2 H), 3.51 (s, 3 H), 2.89 (s, 6 H). 13C-NMR (150.9MHz, [2H]chloroform) δ 188.6, 161.1, 155.2, 134.9, 115.8, 110.4,105.1, 95.0, 56.5, 44.7. HRMS (MH+ ) expected, 210.1125; found,210.1123.


To a stirring solution of 4.16 (130 mg, 0.62 mmol) inethanol (10 ml) was added hydroxylamine hydrochloride (77 mg,



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1.1 mmol) and the reaction was stirred at room temperature for30 min. A saturated solution of sodium bicarbonate and ethylacetate were added and the layers were separated. The aqueouslayer was extracted twice with ethyl acetate, the organic fractionscombined, dried over magnesium sulfate, filtered and then thevolatiles were removed under reduced pressure. The solventwas removed under reduced pressure and the crude material waspurified by flash chromatography over silica gel eluting with1:3:1 hexane/dichloromethane/ethyl acetate to afford 89 mg ofpure product as a white solid (64 %). In addition 21 mg of asecond product was isolated that appears to be the oxime isomer(15%). 1H-NMR (600 MHz, [2H]chloroform) δ 8.41 (s, 1 H),7.78 (bs, 1 H), 7.57 (d, J 8.8 Hz, 1 H), 6.43 (d, J 2.4 Hz,1 H), 6.33 (dd, J 8.8, 2.4 Hz, 1 H), 5.22 (s, 2 H), 3.50 (s,3 H), 2.99 (s, 6 H). 13C-NMR (150.9 MHz, [2H]chloroform)δ 156.9, 152.8, 146.9, 127.5, 109.4, 106.3, 98.1, 94.8, 56.2,40.3. HRMS (MH+ ) expected, 225.1234; found, 225.1233.


To a stirring solution of 4-dimethylamino-2-methoxymethoxybenzaldehyde oxime (80 mg, 0.36 mmol) in methanol (2 ml) atroom temperature was added a 4 M solution of hydrogen chloridein dioxane (0.3 ml, 1.2 mmol). The reaction was heated to 70 ◦Cfor 1 h and then cooled to room temperature and the solvent wasblown off with nitrogen. The crude material was purified by flashchromatography over silica gel eluting with dichloromethane toafford 50 mg of pure product as a white solid (82%). 1H-NMR(600 MHz, [2H]chloroform) δ 9.83 (s, 1 H), 8.11 (s, 1 H), 7.00 (d,J 8.2 Hz, 1 H), 6.93 (s, 1 H), 6.27 (m, 2 H), 2.99 (s, 6 H). 13C-NMR(150.9 MHz, [2H]chloroform) δ 158.7, 153.1, 152.7, 131.7, 105.5,104.2, 99.0, 40.1. HRMS (MH+ ) expected, 181.0971; found,181.0969.

4-Trimethylammonio-2-hydroxy-benzaldehyde oxime iodide(TAB4OH)

To a stirring solution of 4.18 (32 mg, 0.18 mmol) indichloromethane (0.7 ml) at room temperature was addediodomethane (0.1 ml, 1.6 mmol). The reaction was stirred atroom temperature for 40 h as a precipitate formed. The productwas collected by filtration washing with excess dichloromethaneyielding 21 mg of pure product as a white solid (37%). 1H-NMR(600 MHz, [U-2H]methanol) δ 8.36 (s, 1 H), 7.61 (d, J 8.7 Hz, 1H), 7.47 (d, J 2.7 Hz, 1 H), 7.44 (dd, J 8.7, 2.7 Hz, 1 H), 3.69(s, 9 H). 13C-NMR (150.9 MHz, [U-2H]methanol) δ 159.5, 150.2,149.5, 133.6, 121.0, 111.8, 109.6, 57.7. HRMS (M + ) expected,195.1133; found, 195.1133.


To a stirring solution of 4.17 (163 mg, 0.78 mmol) inethanol (10 ml) was added hydroxylamine hydrochloride (85 mg,1.2 mmol) and the reaction was stirred at room temperature for30 min. A saturated solution of sodium bicarbonate and ethylacetate were added and the layers were separated. The aqueouslayer was extracted twice with ethyl acetate, the organic fractionscombined, dried over magnesium sulfate, filtered and then thevolatiles were removed under reduced pressure. The solventwas removed under reduced pressure and the crude material waspurified by flash chromatography over silica gel eluting with1:3:1 hexane/dichloromethane/ethyl acetate to afford 160 mg ofpure product as a white solid (91 %). 1H-NMR (600 MHz,[2H]chloroform) δ 9.93 (s, 1 H), 8.46 (s, 1 H), 7.24 (t, J 8.2Hz, 1 H), 6.88 (d, J 8.2 Hz, 1 H), 6.76 (d, J 8.2 Hz, 1 H),5.25 (s, 2 H), 3.50 (s, 3 H), 2.75 (s, 6 H). 13C-NMR (150.9MHz, [2H]chloroform) δ 156.4, 154.8, 146.0, 130.4, 114.5, 112.1,108.9, 94.7, 56.3, 44.9. HRMS (MH+ ) expected, 225.1234; found,225.1234.


To a stirring solution of 2-dimethylamino-6-methoxymethoxy-benzaldehyde oxime (14 mg, 0.06 mmol) in methanol (0.2 ml) atroom temperature was added a 4 M solution of hydrogen chloridein dioxane (0.2 ml, 0.8 mmol). The reaction was heated to 70 ◦Cfor 1 h and then cooled to room temperature and the solvent wasblown off with nitrogen. The crude material was purified by flashchromatography over silica gel eluting with dichloromethane toafford 10 mg of pure product as a white solid (91%). 1H-NMR(600 MHz, [2H]chloroform) δ 10.06 (s, 1 H), 8.65 (s, 1 H), 7.21 (t,J 8.1 Hz, 1 H), 7.11 (s, 1 H), 6.66 (d, J 8.2 Hz, 1 H), 6.60 (d, J 8.0Hz, 1 H), 2.73 (s, 6 H). 13C-NMR (150.9 MHz, [2H]chloroform)δ 158.5, 154.8, 151.4, 131.6, 111.3, 110.2, 109.8, 45.6. HRMS(MH+ ) expected, 181.0971; found, 181.0983.

2-Trimethylammonio-6-hydroxy-benzaldehyde oxime chloride(TAB2OH)

To a stirring solution of 4.19 (90 mg, 0.50 mmol) in dichlorometh-ane (0.2 ml) at 0 ◦C was added methyl trifluoromethansulfonate(62 μl, 0.55 mmol). The reaction was allowed to warm to roomtemperature and stirred for 40 h. The precipitate formed wascollected by filtration washing with excess dichloromethaneto afford 101 mg of the pure triflate salt as a white solid(59%). The salt was dissolved in acetonitrile and a solutionof tetrahexylammonium chloride in acetonitrile was added toprecipitate the pure chloride salt as a white solid. 1H-NMR (600MHz, d6-DMSO) δ 11.74 (s, 1 H), 10.95 (s, 1 H), 8.34 (s, 1H), 7.45 (t, J 8.4 Hz, 1 H), 7.39 (d, J 8.4 Hz, 1 H), 7.26 (d, J8.2 Hz, 1 H), 3.68 (s, 9 H). 13C-NMR (150.9 MHz, d6-DMSO)



δ 158.9, 145.6, 145.5, 130.6, 117.5, 113.5, 111.3, 57.2. HRMS(M + ) expected, 195.1133; found, 195.1130.

4-Dimethylethylammonio-2-hydroxy-benzaldehyde oxime chloride(TAB4OHmme)

To a stirring solution of 4.18 (53 mg, 0.29 mmol) in dichlorometh-ane (1.5 ml) at 0 ◦C was added ethyl trifluoromethansulfonate(42 μl, 0.32 mmol). The reaction was allowed to warm to roomtemperature and stirred for 24 h. The mixture was tritratedwith dichloromethane and then the triflate salt was dissolved inacetonitrile and a solution of tetrahexylammonium chloridein acetonitrile was added to precipitate 46 mg of the pure chloridesalt as a white solid (64%). 1H-NMR (600 MHz, d6-DMSO) δ11.72 (s, 1 H), 10.94 (s, 1 H), 8.37 (s, 1 H), 7.71 (d, J 8.8 Hz, 1H), 7.44 (m, 1 H), 7.39 (dd, J 8.8, 2.6 Hz, 1 H), 3.90 (q, J 7.2 Hz,2 H), 3.53 (s, 6 H), 1.00 (t, J 7.2 Hz, 3 H). 13C-NMR (150.9MHz, d6-DMSO) δ 156.5, 145.7, 145.0, 128.8, 119.9, 112.0,109.4, 63.8, 53.0, 8.4. HRMS (M + ) expected, 209.1284; found,209.1294.

(3-Methoxymethoxyphenyl)-diethylamine (4.21)

To a stirring solution of 3-diethylaminophenol (1.29 g, 7.8 mmol)in dichloromethane (12 ml) at 0 ◦C was added DIEA (2.0 ml,11.5 mmol) and chloromethyl methyl ether (0.75 ml, 9.9 mmol)and the solution was stirred at room temperature for 15 h.The reaction was quenched with a 10% solution of sodiumhydroxide and stirred for 30 min. The layers were separatedand the aquous layer extracted twice with dichloromethane, thefractions combined, dried with magnesium sulfate, filtered andthe solvent removed under reduced pressure. The crudematerial was purified by flash chromatography eluting with 9:1hexane/ethyl acetate to afford 900 mg of pure product as acolourless oil (55%). 1H-NMR (600 MHz, [2H]chloroform) δ7.09–7.12 (m, 1 H), 6.35–6.36 (m, 3 H), 5.16 (s, 2 H), 3.49 (s, 3H), 3.34 (q, J 7.1 Hz, 4 H) 1.16 (t, J 7.1 Hz, 6 H). 13C-NMR (150.9MHz, [2H]chloroform) δ 158.6, 149.2, 129.9, 106.0, 102.6, 100.2,94.5, 55.9, 44.4, 12.6. HRMS (MH+ ) expected, 210.1488; found,210.1492.


To a stirring solution of 4-diethylaminosalicylaldehyde (436 mg,2.3 mmol) in DMF (6 ml) at 0 ◦C was added DIEA (0.6 ml,3.5 mmol) and chloromethyl methyl ether (0.2 ml, 2.6 mmol)

and the solution was stirred at room temperature for 3 h.The reaction was quenched with a 5% sodium hydroxidesolution and stirred for 30 min. Ethyl acetate was added andthe layers were separated and the aqueous layer was extractedtwice with ethyl acetate, the fractions combined, dried withmagnesium sulfate, filtered and the solvent removed underreduced pressure. The crude material was purified twice byflash chromatography eluting once with dichloromethane andthe second time with 9:1 dichloromethane/ethyl acetate toafford 250 mg of pure product (47%). 1H-NMR (600 MHz,[2H]chloroform) δ 10.16 (s, 1 H), 7.71 (d, J 9.0 Hz, 1 H),6.33–6.35 (m, 2 H), 5.26 (s, 2 H), 3.52 (s, 3 H), 3.41 (q, J7.1 Hz, 4 H), 1.21 (t, J 7.1 Hz, 6 H). 13C-NMR (150.9 MHz,[2H]chloroform) δ 187.1, 162.1, 153.7, 130.3, 114.7, 105.4, 95.9,94.7, 56.3, 44.8, 12.5. HRMS (MH+ ) expected, 238.1438; found,238.1438.

4-Diethylamino-2-methoxymethoxy-benzaldehyde (4.22) and2-diethylamino-6-methoxymethoxy-benzaldehyde (4.23)

To a stirring solution of 4.21 (840 mg, 4.0 mmol) andfreshly distilled N,N,N ′,N ′-tetramethylethylenediamine (0.61 ml,4.1 mmol) in dry ethyl ether (15 ml) cooled to − 40 ◦C underargon was added n-butyllithium (2.5 ml of 1.8 M in hexanesolution, 4.5 mmol) and the reaction was allowed to warm toroom temperature and stirred for 1 h. The reaction was re-cooledto − 40 ◦C and dry DMF (0.6 ml, 7.7 mmol) was added andthe reaction was allowed to warm to room temperature withstirring over 1 h before being quenched with a dilute ammoniumchloride aqueous solution. The mixture was extracted three timeswith ethyl acetate, the organic fractions combined, dried overmagnesium sulfate, filtered and then the volatiles were removedunder reduced pressure. The crude material was purified by flashchromatography over silica gel eluting with dichloromethane. Theisomers were separated by eluting with 7:3 hexane/ethyl acetateto afford each pure isomer.


386 mg, pale yellow oil, 41 %. 1H-NMR (600 MHz,[2H]chloroform) δ 10.16 (s, 1 H). 7.72 (d, J 8.8 Hz, 1 H), 6.33–6.35 (m, 2 H), 5.26 (s, 2 H), 3.52 (s, 3 H), 3.41 (q, J 7.1 Hz, 4 H),1.21 (t, J 7.1 Hz, 6 H). 13C-NMR (150.9 MHz, [2H]chloroform)δ 187.1, 162.1, 153.7, 130.3, 114.7, 105.4, 95.9, 94.7, 56.3, 44.8,12.6. HRMS (MH+ ) expected, 238.1438; found, 238.1438.


239 mg, yellow oil, 25%. 1H-NMR (600 MHz, [2H]chloroform)δ 10.25 (s, 1 H). 7.35 (t, J 8.3 Hz, 1 H), 6.77 (dd, J 8.3, 2.3Hz, 2 H), 5.25 (s, 2 H), 3.51 (s, 3 H), 3.19 (q, J 7.1 Hz, 4 H),1.07 (t, J 7.1 Hz, 6 H). 13C-NMR (150.9 MHz, [2H]chloroform) δ190.1, 159.5, 155.0, 134.1, 120.3, 114.5, 108.1, 95.1, 56.5, 48.2,12.3. HRMS (MH+ ) expected, 238.1438; found, 238.1440.


Z. Radic and others

4-Diethylamino-2-hydroxy-benzaldehyde oxime (4.25)

To a stirring solution of 4-diethylamino-salicylaldehyde (494 mg,2.6 mmol) in ethanol (10 ml) was added hydroxylaminehydrochloride (220 mg, 3.2 mmol) and the reaction was stirredat room temperature for 15 h. A saturated solution of sodiumbicarbonate and ethyl acetate were added and the layers wereseparated. The aqueous layer was extracted twice with ethylacetate, the organic fractions combined, dried over magnesiumsulfate, filtered and then the volatiles were removed under reducedpressure. The solvent was removed under reduced pressure andthe crude material was purified by flash chromatography oversilica gel eluting with dichloromethane to afford 432 mg ofpure product as an off-white solid (81 %). 1H-NMR (600 MHz,[2H]chloroform) δ 9.79 (s, 1 H), 8.09 (s, 1 H), 6.96 (m, 1 H), 6.83(bs, 1 H), 6.22–6.24 (m, 2 H), 3.36 (q, J 7.1 Hz, 4 H), 1.18 (t,J 7.1 Hz, 6 H). 13C-NMR (150.9 MHz, [2H]chloroform) δ 159.0,153.1, 150.3, 131.9, 104.7, 103.7, 98.2, 44.4, 12.6. HRMS (MH+ )expected, 209.1284; found, 209.1285.

4-Diethylmethylammonio-2-hydroxy-benzaldehyde oxime chloride(TAB4OHmee)

To a stirring solution of 4.25 (98 mg, 0.47 mmol) indichloromethane (0.7 ml) at 0 ◦C was added MeOTf (methyltrifluoromethansulfonate; 62 μl, 0.55 mmol). The reaction wasallowed to warm to room temperature and stirred for 23 h. Thevolatiles were then blown off with nitrogen and the residuepurified twice by flash chromatography over silica gel by elutingwith a gradient of 19:1 to 17:1 dichloromethane/methanol toafford the pure tosylate salt. The salt was dissoved in methanoland eluted through DOWEX 1-2×200 resin to exchange for thechloride anion and the material was recrystallized from ethanolto afford 38 mg of the pure chloride salt as a white solid (31 %).1H-NMR (600 MHz, [U-2H]methanol) δ 8.37 (s, 1 H), 7.63 (d, J8.7 Hz, 1 H), 7.33 (d, J 2.7 Hz, 1 H), 7.29 (dd, J 8.7, 2.7 Hz, 1H), 4.06 (m, 2 H), 3.86 (m, 2 H), 3.52 (s, 3 H), 1.19 (t, J 7.1 Hz,6 H). 13C-NMR (150.9 MHz, [U-2H]methanol) δ 159.7, 150.2,143.4, 132.7, 121.0, 113.7, 111.4, 65.6, 46.7, 8.8. HRMS (M + )expected, 223.1446; found, 223.1446.


To a stirring solution of 4-dimethylamino-benzaldehyde (457 mg,3.1 mmol) in ethanol (17 ml) was added hydroxylaminehydrochloride (290 mg, 4.2 mmol) and the reaction was stirred

at room temperature for 15 h as a yellow colour developed. Asaturated solution of sodium bicarbonate and ethyl acetate wereadded and the layers were separated. The aqueous layer wasextracted twice with ethyl acetate, the organic fractions combined,dried over magnesium sulfate, filtered and then the volatiles wereremoved under reduced pressure. The solvent was removed underreduced pressure and the crude material was purified by flashchromatography over silica gel eluting with a gradient of 1:0 to 4:1dichloromethane/ethyl acetate to afford 470 mg of pure productas a white solid (93%). 1H-NMR (600 MHz, [2H]chloroform) δ8.05 (s, 1 H), 7.45 (d, J 8.9 Hz, 2 H), 6.69 (d, J 8.9, 2 H), 3.00(s, 9 H). 13C-NMR (150.9 MHz, [2H]chloroform) δ 151.5, 150.6,128.3, 119.7, 111.9, 40.2. HRMS (MH+ ) expected, 165.1022;found, 165.1028.


To a stirring solution of 4.28 (75 mg, 0.46 mmol) in dichlorometh-ane (3 ml) at 0 ◦C was added methyl trifluoromethansulfonate(57 μl, 0.50 mmol). The reaction was allowed to warm to roomtemperature and stirred for 19 h. The formed precipitate wascollected by filtration washing with excess dichloromethaneto afford 128 mg of the pure triflate salt as a white solid(85%). The salt was dissolved in acetonitrile and a solutionof tetrahexylammonium chloride in acetonitrile was added toprecipitate the pure chloride salt as a white solid (58 %). 1H-NMR (600 MHz, d6-DMSO) δ 11.64 (s, 1 H), 8.24 (s, 1 H),8.03 (d, J 9.1 Hz, 2 H), 7.81 (d, J 9.1 Hz, 2 H), 3.64 (s, 9H). 13C-NMR (150.9 MHz, d6-DMSO) δ 147.3, 146.4, 134.6,127.4, 121.0, 56.2. HRMS (M + ) expected, 179.1179; found,179.1180.


To a stirring solution of 2-dimethylamino-benzaldehyde (266 mg,1.8 mmol) in ethanol (5 ml) was added hydroxylaminehydrochloride (137 mg, 2.0 mmol) and the reaction was stirredat room temperature for 30 min as the yellow colour faded tocolourless. A saturated solution of sodium bicarbonate and ethylacetate were added and the layers were separated. The aqueouslayer was extracted twice with ethyl acetate, the organic fractionscombined, dried over magnesium sulfate, filtered and then thevolatiles were removed under reduced pressure. The solventwas removed under reduced pressure and the crude material waspurified by flash chromatography over silica gel eluting with2:2:1 hexane/dichloromethane/ethyl acetate to afford 270 mg ofpure product as a white solid (92 %). 1H-NMR (600 MHz,[2H]chloroform) δ 8.61 (bs, 1 H), 8.47 (s, 1 H), 7.68 (d, J 7.7 Hz,1 H), 7.34 (t, J 8.5 Hz, 1 H), 7.07 (d, J 8.1 Hz, 1 H), 7.03 (t, J 7.5Hz, 1 H), 2.75 (s, 6 H). 13C-NMR (150.9 MHz, [2H]chloroform)δ 153.1, 149.0, 130.5, 127.5, 125.4, 122.5, 118.5, 45.2. HRMS(MH+ ) expected, 165.1022; found, 165.1025.




To a stirring solution of 4.30 (69 mg, 0.42 mmol) in dichlorometh-ane (2 ml) at 0 ◦C was added methyl trifluoromethansulfonate(50 μl, 0.44 mmol). The reaction was allowed to warm to roomtemperature and stirred for 36 h. After the solvent was blownoff under nitrogen, the salt was dissoved in methanol and elutedthrough DOWEX 1-2×200 resin to exchange for the chlorideanion. After drying under reduced pressure, the crude materialwas dissolved in a mixture of ethyl acetate and water and the layerswere separated. The organic layer was extracted once with waterand then the aqueous solution was freeze-dried. The materialwas then tritrated with dichloromethane and acetonitrile to afford16 mg of a white solid that was 85% pure, a mixture of thechloride salt with an unknown impurity (18 %). 1H-NMR (600MHz, [U-2H]methanol) δ 8.75 (s, 1 H), 7.99 (d, J 7.7 Hz, 1 H), 7.77(t, J 8.5 Hz, 1 H), 7.68 (m, 2 H), 3.80 (s, 9 H). 13C-NMR (150.9MHz, [U-2H]methanol) δ 148.4, 146.5, 135.1, 132.3, 132.0, 128.2,122.3, 58.5. HRMS (M + ) expected, 179.1179; found, 179.1182.

4-Dimethylethylammonio-benzaldehyde oxime chloride(TAB4mme)

To a stirring solution of 4.28 (66 mg, 0.40 mmol) in dichloro-methane (2 ml) at 0 ◦C was added ethyl trifluoromethansulfonate(57 μl, 0.44 mmol). The reaction was allowed to warm to roomtemperature and stirred for 24 h. The formed precipitate wascollected by filtration washing with excess dichloromethane toafford 90 mg of the pure triflate salt (65%). The salt was dissolvedin acetonitrile and a solution of tetrahexylammonium chloride inacetonitrile was added to precipitate 42 mg of the pure chloridesalt as a white solid (46%). 1H-NMR (600 MHz, d6-DMSO) δ11.63 (s, 1 H), 8.24 (s, 1 H), 7.94 (d, J 9.0 Hz, 2 H), 7.82 (d, J 9.0Hz, 2 H), 3.96 (q, J 7.2 Hz, 2 H), 3.59 (s, 6 H), 0.99 (t, J 7.2 Hz,3 H). 13C-NMR (150.9 MHz, d6-DMSO) δ 146.4, 144.2, 134.6,127.5, 121.8, 63.9, 53.1, 8.5. HRMS (M + ) expected, 193.1335;found, 193.1335.

4-Diethylamino benzaldehyde oxime (4.32)

To a stirring solution of 4-diethylamino-benzaldehyde (417 mg,2.4 mmol) in ethanol (5 ml) was added hydroxylaminehydrochloride (171 mg, 2.5 mmol) and the reaction was stirredat room temperature for 1 h. A saturated solution of sodiumbicarbonate and ethyl acetate were added and the layers wereseparated. The aqueous layer was extracted twice with ethylacetate, the organic fractions combined, dried over magnesiumsulfate, filtered and then the volatiles were removed under reducedpressure. The solvent was removed under reduced pressure and

the crude material was purified by flash chromatography oversilica gel eluting with a gradient of 3:1 to 2:1 hexane/ethyl acetateto afford 250 mg of pure product as an off-white solid (55%).1H-NMR (600 MHz, [2H]chloroform) δ 8.04 (s, 1 H), 7.79 (bs,1 H), 7.42 (d, J 8.7 Hz, 1 H), 6.65 (bd, J 7.3 Hz, 1 H), 3.38 (q,J 7.1 Hz, 4 H), 1.18 (t, J 7.1 Hz, 6 H). 13C-NMR (150.9 MHz,[2H]chloroform) δ 150.5, 149.0, 128.5, 118.6, 111.3, 44.4, 12.5.HRMS (MH+ ) expected, 193.1341; found, 193.1334.

4-Diethylmethylammonio-benzaldehyde oxime chloride (TAB4mee)

To a stirring solution of 4.32 (71 mg, 0.37 mmol) in dichlorometh-ane (1 ml) at 0 ◦C was added methyl trifluoromethansulfonate(50 μl, 0.44 mmol). The reaction was allowed to warm to roomtemperature and stirred for 24 h. After the solvent was blownoff under nitrogen, the salt was dissoved in methanol and elutedthrough DOWEX 1-2×200 resin to exchange for the chlorideanion and the material was tritrated with dichloromethane toafford 15 mg of the pure chloride salt as a white solid (17%).1H-NMR (600 MHz, [U-2H]methanol) δ 8.17 (s, 1 H), 7.88 (d, J9.1 Hz, 1 H), 7.78 (d, J 9.1 Hz, 1 H), 4.09 (m, 2 H), 3.87 (m, 2H), 3.54 (s, 3 H), 1.15 (t, J 7.2 Hz, 6 H). 13C-NMR (150.9 MHz,[U-2H]methanol) δ 147.7, 142.6, 137.1, 129.6, 123.5, 65.6, 46.8,8.9. HRMS (M + ) expected, 207.1492; found, 207.1492.

3-Hydroxypyridine-2-carboxaldehyde (4.37)

A suspension of 2-(hydroxylmethyl)-3-hydroxy-pyridine (1.0 g,8.0 mmol) in chloroform (20 ml) was added to a stirringsuspension of manganese dioxide (4.0 g) in chloroform (20 ml)at 50 ◦C. The reaction was heated to 60 ◦C for 3 h and thenthe suspension was filtered through celite washing with excesschloroform. The solvent was removed under reduced pressure toafford 430 mg of pure product (43%). 1H-NMR (600 MHz, d6-DMSO) δ 10.77 (s, 1 H), 10.10 (s, 1 H), 8.30 (d, J 4.3 Hz, 1 H),7.55 (dd, J 8.5, 4.2 Hz, 1 H), 7.47 (d, J 8.5 Hz, 1 H). 13C-NMR(150.9 MHz, d6-DMSO) δ 193.9, 156.8, 141.6, 137.9, 129.7,125.8. HRMS (MH+ ) expected, 124.0393; found, 124.0395.

3-Hydroxypyridine-2-carboxaldehyde oxime (4.38)

To a stirring solution of 4.37 (225 mg, 1.8 mmol) in ethanol(10 ml) was added hydroxylamine hydrochloride (137 mg,2.0 mmol) and the reaction was stirred at room temperature for2 h. The solvent was removed under reduced pressure and thecrude material was purified by flash chromatography over silicagel eluting with 40:60:0.5 hexane/ethyl acetate/triethanolamine toafford 105 mg of product as a white solid (41%). An analyticallypure sample was repurified by flash chromatography oversilica gel eluting with 19:1 dichloromethane/methanol. 1H-NMR(600 MHz, d6-DMSO) δ 11.87 (s, 1 H), 10.29 (s, 1 H), 8.31 (s, 1


Z. Radic and others

H), 8.15 (dd, J 4.3, 1.2 Hz, 1 H), 7.34 (dd, J 8.3, 1.1 Hz, 1 H),7.28 (dd, J 8.3, 4.4 Hz, 1 H). 13C-NMR (150.9 MHz, d6-DMSO) δ153.1, 150.6, 140.9, 136.6, 124.7, 123.4. HRMS (MH+ ) expected,139.0502; found, 139.0503.

3-Hydroxy-2-pyridinealdoxime methiodide (2PAMOH)

To a stirring solution of 4.38 in tetrahydrofolate (2 ml) was addediodomethane (0.1 ml, 1.6 mmol) and the reaction was heated to70 ◦C in a sealed tube for 60 h. After cooling, the precipitate wascollected by filtration to yield 29 mg of pure product as a paleyellow solid (60%). 1H-NMR (600 MHz, d6-DMSO) δ 8.77 (s,1 H), 8.42 (d, J 5.5 Hz, 1 H), 7.99 (d, J 8.6 Hz, 1 H), 7.81 (m, 1 H).13C-NMR (150.9 MHz, d6-DMSO) δ 159.3, 143.4, 139.7, 134.8,133.4, 128.4. HRMS (M + ) expected, 153.0659; found, 53.0664.

Figure S1 Concentration-dependence of oxime reactivation of (A) sarin, (B) cyclosarin, (C) VX and (D) paraoxon-inhibited (conjugated) hAChE

Dependence for the lead oxime TAB2OH compared with the reference cationic oxime 2PAM (measured at 37◦C in 0.1 M phosphate buffer, pH 7.4). Data from two to four experiments are shown withassociated S.E.M. of determination. For TAB2OH errors were smaller than the symbol size.



Figure S2 pH-dependence of (A) UV spectra of 50 μM TAB2OH and pH-dependences of (B) A 240nm of 50 μM TAB2OH, (C) A 275nm of 50 μM TAB2OH and (D)A 310nm of 50 μM TAB2OH, along with corresponding pK a values calculated by non-linear regression of eqn 2 (black curves in B and D) or eqn 1 (grey curves inB and D) of the main text [1]


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Figure S3 pH-dependence of 1H-NMR spectra of 10 mM TAB2OH in 2H2O buffers (A, B and C) along with corresponding pK a values calculated from theobserved pH-induced difference in chemical shifts (D, E and F) by non-linear regression [1]

Spectra from the single experiment were aligned using the benzene external standard singlet at 4.55 p.p.m. Resonating protons are highlighted in the TAB2OH structure for each of the peaks.

Figure S4 Peak assignment in the 1H-NMR spectrum of 10 mM TAB2OH in 20 mM phosphate-pyrophosphate ( + 0.1 M NaCl) 2H2O buffer, pH 5



Figure S5 pH-dependence of 1H-NMR spectra of 10 mM TAB2OH in 2H2O buffers, pH 5–10

(A) Expanded view of the spectrum in the chemical-shift region 6.9–7.3. along with the pH-dependent change in chemical shifts for the 7.2 p.p.m. doublet and (B) expanded view of the spectrum inthe chemical shift region 7.0–7.5. along with the pH-dependent change in chemical shifts for the 7.42 p.p.m. doublet. The corresponding pK a values were calculated from the observed pH-induceddifference in chemical shifts (C and D) by non-linear regression [1]. Spectra from the single experiment were aligned using a benzene external standard singlet at 4.55 p.p.m.


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Figure S6 pH-dependence of 1H-NMR spectra of 2.0 mM 2PAM in 2H2Obuffers, pH 5–10

(A) Expanded view of the spectrum in the chemical-shift region 2.80–2.86. (B) pH-dependentchange in chemical shifts for the 2.83 p.p.m. singlet. Spectra from the single experiment werealigned using a benzene external standard singlet at 7.16 p.p.m.

Figure S7 Pharmacokinetics of TAB2OH in mice

Brain (grey symbols and lines) and plasma (white symbols and black lines) compound concentrations were determined at discrete time points upon single 30 mg/kg doses administered to mice i.m.Values are means +− S.D. for three mice.



Scheme S1 Synthesis of TAB2OH and TAB4OH

Scheme S2 Synthesis of TAB4OHmme

Scheme S3 Synthesis of TAB4OHmee


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Scheme S4 Synthesis of TAB2 and TAB4

Scheme S5 Synthesis of TAB4mme

Scheme S6 Synthesis of TAB4mee

Scheme S7 Synthesis of 2PAMOH



Table S1 The first-order rate constants k (10− 5 s− 1) for hydrolysis of 5.0 μM nerve agent OP analogues (Flu-MPs) by oximes alone or by combination of500 nM hBChE and an oxime

Constants refer to the fast phase of hydrolysis shown in Figure 5 of the main text, representing degradation of a fraction of total racemic OP. Constants calculated in representative experiments aregiven. The experiments were replicated at least once.

Oxime (1.0 mM) + OP [BuChE + oxime (1.0 mM)] + OP Oxime (0.1 mM) + OP [BuChE + oxime (0.1 mM)] + OP

Nerve agent OP analogue TAB2OH 2PAM TAB2OH 2PAM TAB2OH 2PAM TAB2OH 2PAM

Sarin 5.5 11 15 27 4.1 4.6 8.5 12Cyclosarin 6.4 11 530 (30 % total OP) 37 3.4 3.7 180 37VX 15 28 430 (50 % total OP) 64 11 13 150 40

Table S2 Maximal oxime concentrations in brains and plasma of micedetermined upon i.m. administration of a single oxime dose (compare withFigure S5)

Distribution coefficients (logD) were calculated from oxime structures using the ChemAxonsoftware package (http://www.chemaxon.com).

[Oxime]max

μg/ml μM

Oxime i.m. dose (mg/kg) Brain Plasma Brain Plasma Brain/plasma logD

TAB2OH 30 0.13 2.2 0.56 10 0.056 − 2.0


http://www.chemaxon.com

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Table S3 Antidotal efficacy of oxime TAB2OH and human BChE in VX-, paraoxon- or sarin-exposed mice

BChE (1 mg/kg) and oxime (25 mg/kg) were administered i.v. 15 min before OP (s.c.). Oxime (25 mg/kg) in therapy was administered i.m. together with atropine (10 mg/kg) 1 min after OP exposure.

Pretreatment 15 min before OP and therapy 1 min after OP exposure

VX (s.c. LD50 = 28.3 μg/kg) Paraoxon (s.c. LD50 = 740.8 μg/kg) Sarin (s.c. LD50 = 238.3 μg/kg)

n×LD50 Survived/treated Symptoms Survived/treated Symptoms Survived/treated Symptoms

1.0 4/4 No visible symptoms.1.26 4/4 One mouse had light tremor during the

first 10 min on application.1.59 4/4 Light tremor 3–4 min on application.2.0 4/4 Tremor and light salivation 20 min on

application.4/4 Tremor 20 min on application.

Surviving animals were weak after24 h.


3/4 Light tremor 3–4 min uponapplication. Surviving animalswere weak after 24 h.



4.0 3/4 Strong tremor and salivation 3 min onapplication. Mice were weak withrespiratory disturbance. Survivinganimals were weak after 24 h.


5.0 2/4 Strong tremor and respiratorydisturbance immediately onapplication. Surviving animals wereweak after 24 h.

2/4 Cramps and strong tremorimmediately on application.Surviving animals were very weakafter 24 h.

6.3 1/4 Strong tremor, salivation and respiratorydisturbance immediately onapplication. Surviving mouse wasweak after 24 h.

0/4 Cramps and strong tremorimmediately on application.Animals died between 5 and60 min after application.

7.9 2/4 Strong tremor, paralysis. Survivinganimals were weak after 24 h.

12.6 4/4 Lacrimation, dyspnoea, tremor, oedemaof the eyelids.

15.9 1/4 Strong tremor, lacrimation, dyspnoea.Surviving mouse was weak after 24 h.

20.0 1/4 Three mice died 3–4 min on application.The surviving mouse was very weak,had respiratory disturbance andclosed eyes after 24 h.

25.2 0/4 Strong tremor immediately onapplication. Animals died after2–3 min of application

REFERENCE

1 Radic, Z., Sit, R. K., Kovarik, Z., Berend, S., Garcia, E., Zhang, L., Amitai, G., Green, C.,Radic, B., Fokin, V. V. et al. (2012) Refinement of structural leads for centrally acting oximereactivators of phosphylated cholinesterases. J. Biol. Chem. 287, 11798–11809



Catalytic detoxification of nerve agent and pesticide organophosphates by butyrylcholinesterase assisted with non-pyridinium oximes

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