Biochem. J. (2013) 450, 231–242 (Printed in Great Britain)
231
doi:10.1042/BJ20121612
Catalytic detoxification of nerve agent and pesticide organophosphates
by butyrylcholinesterase assisted with non-pyridinium oximes
Zoran RADIĆ*1 , Trevor DALE†, Zrinka KOVARIK‡, Suzana BEREND‡, Edzna GARCIA*, Limin ZHANG*, Gabriel AMITAI§,
Carol GREEN, Božica RADIƇ, 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 of
Chemistry, The Scripps Research Institute, La Jolla, CA 92037, U.S.A., ‡Institute for Medical Research and Occupational Health, HR-10001 Zagreb, Croatia, §Department of
Pharmacology, 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, ex
vivo and in vivo study on hydrolytic detoxification of nerve
agent and pesticide OPs (organophosphates) catalysed by purified
hBChE (human butyrylcholinesterase) in combination with novel
non-pyridinium oxime reactivators. We identified TAB2OH (2trimethylammonio-6-hydroxybenzaldehyde oxime) as an efficient
reactivator of OP–hBChE conjugates formed by the nerve
agents VX and cyclosarin, and the pesticide paraoxon. It was
also functional in reactivation of sarin- and tabun-inhibited
hBChE. A 3–5-fold enhancement of in vitro reactivation of
VX-, cyclosarin- and paraoxon-inhibited hBChE was observed
when compared with the commonly used N-methylpyridinium
aldoxime reactivator, 2PAM (2-pyridinealdoxime methiodide).
Kinetic analysis showed that the enhancement resulted from
improved molecular recognition of corresponding OP–hBChE
conjugates by TAB2OH. The unique features of TAB2OH stem
from an exocyclic quaternary nitrogen and a hydroxy group, both
ortho to an oxime group on a benzene ring. pH-dependences
reveal participation of the hydroxy group (pK a = 7.6) forming an
additional ionizing nucleophile to potentiate the oxime (pK a = 10)
at physiological pH. The TAB2OH protective indices in therapy
of sarin- and paraoxon-exposed mice were enhanced by 30–60 %
when they were treated with a combination of TAB2OH and substoichiometric hBChE. The results of the present study establish
that oxime-assisted catalysis is feasible for OP bioscavenging.
INTRODUCTION
bioscavengers, molecules that capture and degrade OPs in blood,
has been introduced [5].
Oxime-assisted recovery of catalytic activity has been studied in
far greater detail for AChE, owing to its physiological importance
in neurotransmission than that of the closely related cholinesterase
found in liver and plasma, BChE (butyrylcholinesterase).
Consequently, most oxime reactivators developed to recover
activity of OP–AChE conjugates are found to be less efficient
in recovering OP–BChE activity [6,7].
Interest in designing potent BChE reactivators has been
stimulated with successful implementation of the ‘stoichiometric
OP bioscavenger’ approach based on administering purified
human BChE to OP-exposed animals [8–10].
Efficient protection of guinea pigs from multiple LD50 doses
of nerve agents is found upon pretreatment with 20–30 mg of
purified human BChE/kg. Similarly, antidotal administration of
BChE to animals following OP exposure was demonstrated to
provide effective protection [10]. The effectiveness in protection
is somewhat overshadowed by a common caveat of stoichiometric
bioscavengers. Multi-milligram quantities of large scavenger
proteins are needed to remove stoichiometric equivalents of
OP molecules of low molecular mass. The associated per
dose cost and administration constraints for such stoichiometric
bioscavengers remain large. By enabling the BChE molecule to
Human exposure to OPs (organophosphates) is extensive and
prevails as a global problem. The World Health Organization
estimates that between 30 000 and 200 000 people die annually
from acute OP poisoning [1], whereas more than 95 % of the
US population was found to be exposed to sublethal levels of
OP-based pesticides [2]. The exposure occurs largely through
occupational exposure to OP-based pesticides, in attempted
suicide with pesticides, by consuming improperly washed fruit
or vegetables, but also from terrorist attacks with nerve gases,
the most recent being one of 1995 in the Tokyo subway. More
recently, several incidents of airplane passengers and crew being
exposed 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 symptomatic
treatment with atropine and reactivation of OP-inhibited
AChE (acetylcholinesterase) by nucleophilic oxime antidotes.
Frequently this therapy is limited by reinhibition of reactivated
AChE by unreacted OPs that remain circulating in the blood
following the exposure. To circumvent this problem the concept of
Key words: butyrylcholinesterase reactivation, catalytic organophosphate bioscavenger, edrophonium analogue, nonpyridinium
oxime reactivator, organophosphate intoxication, oxime reactivation.
Abbreviations used: AChE, acetylcholinesterase; ATCh, acetylthiocholine; BChE, butyrylcholinesterase; ChE, cholinesterase; DTNB, 5,5 -dithiobis-(2nitrobenzoic 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-hydroxybenzaldehyde oxime.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2013 Biochemical Society
232
Z. Radić and others
turnover multiple molecules of the OP, quantities of BChE needed
for the effective protection could be significantly reduced and
catalytic bioscavenger coverage could be provided for a larger
population of OP-exposed individuals. We achieve this objective
in the present study by simultaneous application of an efficient
BChE reactivator, where the reactivating oxime assists BChE
catalytic hydrolysis and turnover of the free OP.
In the present paper we describe development of a prototypic
catalytic OP bioscavenger on the basis of combining purified
human BChE with an efficient oxime reactivator with a distinctive
structural scaffold. Starting with a small directed library of
nonpyridinium, cationic, substituted benzenes, we identified
an efficient reactivator of sarin-, cyclosarin-, VX-, tabunand paraoxon-derived OP–BChE conjugates, and characterized
kinetically the interaction with OP–BChE conjugates in detail.
MOM group by heating in the presence of HCl in methanol.
Finally, the 4-dimethylamino-2-hydroxybenzaldehyde oxime
was quaternarized using an excess of iodomethane in hot
THF (tetrahydrofuran) to yield the desired trimethylammonio
derivative. The 2-dimethylamino-6-hydroxybenzaldehyde oxime
was unreactive in the presence of iodomethane and required
the more reactive electrophilic methyl source methyl triflate to
quaternarize the aniline nitrogen. The triflate salt was anionexchanged for the chloride by exchange in an acetonitrile solution
of 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
for
4PyOHMOH which was synthesized as described previously
[12].
EXPERIMENTAL
Enzyme
Highly purified recombinant monomeric hAChE (human AChE)
was prepared as described previously [7]. Purified human BChE
isolated from human plasma was a gift from Dr David Lenz
and Dr Douglas Cerasoli [USAMRICD (US Army Medical
Research Institute of Chemical Defense), Aberdeen Proving
Ground, MD, U.S.A.]. All enzyme concentrations given refer
to the concentration of catalytic sites, i.e. monomers.
OPs
Low toxicity non-volatile Flu-MPs (fluorescent methylphosphonates) [11] were used in in vitro experiments as analogues of the
nerve agents sarin, cyclosarin and VX. The Flu-MPs differ from
actual nerve agent OPs only by the structure of their respective
leaving groups. Inhibition of hAChE by Flu-MPs results in
OP–hAChE covalent conjugates identical with the ones formed
upon inhibition with nerve agents. Paraoxon was purchased from
Sigma–Aldrich. Nerve agent OPs tabun, VX, sarin and soman
used in in vivo experiments were purchased from NC Laboratory
Spiez.
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-trimethylammonio-6-hydroxybenzaldehyde oxime) and TAB4OH (4-trimethylammonio-2-hydroxy-benzaldehyde oxime) were synthesized
from commercially available 3-dimethylaminophenol (Supplementary Scheme S1 at http://www.biochemj.org/bj/450/
bj4500231add.htm). Protection of the phenol as the MOM
(methoxymethyl) ether was completed in dichloromethane
with chloromethyl methyl ether and DIEA (N,N-di-isopropylethylamine). Product MOM ether was ortho-lithiated using
n-butyllithium in ether at − 40 ◦ C and quenched with DMF
(dimethylformamide) to afford a mixture of 4-dimethylamino-2methoxymethoxybenzaldehyde and 2-dimethylamino-6-methoxymethoxybenzaldehyde in an approximate 1:1.1 ratio. The
regioisomers were separable by column chromatography over
silica gel. Each isomer was separately converted into the oxime
using hydroxylamine hydrochloride followed by removal of the
c The Authors Journal compilation c 2013 Biochemical Society
In vitro oxime reactivation assays
hAChE and hBChE (human BChE) activities were measured
using a spectrophotometric assay [13] at 25 ◦ C in 0.1 M sodium
phosphate buffer (pH 7.4), containing 0.01 % BSA and 1.0 mM
substrate ATCh (acetylthiocholine). OP–hBChE and OP–hAChE
conjugates were prepared, and initial screening and detailed
oxime reactivation experiments were performed [at 37 ◦ C in
0.1 M sodium phosphate buffer (pH 7.4), containing 0.01 % BSA]
as described previously [7,14]. The first-order reactivation rate
constant (kobs ) for each oxime + OP conjugate combination was
calculated by non-linear regression [15]. The dependence of
reactivation rates on oxime concentrations and determination
of maximal reactivation rate constant k2 , Michaelis–Menten
type constant K ox and the overall second-order reactivation rate
constant kr were conducted as described previously [15].
pK a determinations
Protonation of ionizable groups in the compounds was monitored
by using UV–Vis spectrophotometry, by NMR spectrometry or
by monitoring nucleophilic reactivity of the oxime group. A
series of 20 mM phosphate-pyrophosphate buffers at pH values
of 5.0, 6.0, 7.0, 8.0, 8.5, 9.0, 9.5, 10, 10.5 and 11 (containing
0.1 M NaCl) were prepared in either H2 O or 2 H2 O. For 2 H2 O
buffers, p2 H values were determined by correcting the pH reading
by + 0.4 pH units [16]. UV spectra of compounds between 220
and 320 nm wavelengths were recorded on a Cary 1E (Varian)
UV–Vis spectrophotometer at the above pH values.
1
H-NMR spectra recorded on a Bruker Avance III 600
spectrometer in 20 mM phosphate-pyrophosphate 2 H2 O buffers
at pH values 5.0, 6.0, 7.0, 8.0, 8.5, 9.0, 9.5 and 10 (containing
0.1 M NaCl) were overlaid and aligned using benzene as an
external standard placed in a separate capillary tube within the
NMR sample probe. Two-dimensional 1 H, 1 H-COSY spectra were
recorded on a Bruker Avance III 600 spectrometer.
Nucleophilic reactivity of oximes was determined in the abovedescribed buffers by measuring oxime-induced rates of ATCh
hydrolysis as detected by the thiol reagent DTNB [5,5 -dithiobis(2-nitrobenzoic acid)], by following a time course of absorbance
increase at 412 nm at room temperature (22 ◦ C).
UV absorbance at discrete wavelengths, chemical shifts of
discrete 1 H-NMR peaks or rates of nucleophilic reactions were
plotted as a function of pH yielding pK a values by non-linear
regression of eqn (1):
A = Amax /(1 + [H+ ]/K a )
(1)
Organophosphate detoxification by butyrylcholinesterase and oximes
Figure 1
233
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 equilibria
using eqn (2):
+
max
+
A = Amax
1 /(1 + [H ]/K a1 ) + A 2 /(1 + [H ]/K a2 )
applied. The mice were treated in accordance with the approval
of the Ethical Committee of the Institute for Medical Research
and Occupational Health in Zagreb, Croatia.
(2)
Acute oxime toxicity and oxime treatment of OP-exposed mice
Oxime pharmacokinetics in mice
Male CD-1 mice of 25–30 g of body mass (purchased from the
Rudjer Bošković Institute, Zagreb, Croatia) fed on a standard
diet, had free access to water and were kept in Macrolone cages
at 21 ◦ C, exchanging light and dark cycles every 12 h. Mice were
randomly distributed into groups of four for each dose.
Acute i.m. [intramuscular(ly)] toxicity (LD50 ) of TAB2OH
was based upon 24 h mortality rates upon administration of four
different doses of TAB2OH, one per group of four mice, and
calculated according to Thompson [17] and Weil [18].
The therapeutic efficacy of TAB2OH against OP poisoning
was tested by administering mice (i.m.) with TAB2OH (10 or
25 mg/kg) together with atropine sulfate (10 mg/kg), 1 min after
s.c. [subcutaneous(ly)] OP exposure [19,20]. Nerve agent stock
solutions were prepared in isopropyl alcohol or in propylene
glycol. Immediately before use, further dilutions were made in
physiological saline.
Alternatively, a combination of pretreatment and therapy
was 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 individual
experiments is detailed in Table 4.
Antidotal efficacy of the oximes was expressed as a PI
(Protective Index) with 95 % confidence limits and maximal dose
of OP affording protection [MDP (maximal dose of poison)].
The PI was the ratio of LD50 exerted by OP with antidote and
OP given alone. The MDP was the highest multiple of the OP
LD50 , which was fully counteracted by the antidotal treatment
Female CD-1 mice 4–8 weeks old (19–27 g of body mass) were
purchased from Charles River Laboratories. Mice were fed Purina
Certified Rodent Chow #5002. Food and purified water was
provided ad libitum. Mice were kept in hanging polycarbonate
cages at 21–23 ◦ C, exchanging light and dark cycles every
12 h. General procedures for animal care and housing were in
accordance with the NRC (National Research Council) Guide for
the Care and Use of Laboratory Animals (1996) and the Animal
Welfare 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 was
administered i.m. using a 30 mg/ml stock solution in a single
dose in the absence of OP. Three animals were injected for every
time point analysed. Brain and plasma were collected at each time
point. Blood (∼300 μl) was collected from the retro-orbital sinus
of mice under isoflurane anaesthesia into tubes containing EDTA,
processed to plasma within 30 min of collection, and then stored
◦
frozen at − 80 ◦ C (+
−10 C).
Brains were collected from each mouse at each time point
(without perfusion of residual brain blood with saline). Brain
mass 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 was
determined 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 was
monitored on the Micromass Quatro LC instrument.
c The Authors Journal compilation c 2013 Biochemical Society
234
Z. Radić and others
Figure 2 Reactivation of paraoxon-, cyclosarin-, VX- and sarin-inhibited hAChE (A) and hBChE (B) by a directed library of ten novel oxime reactivators shown
in 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 reactivation
efficiency. 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 Experimental
section). 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 other
oximes.
RESULTS
Structural design of the oxime library
A small directed library of cationic oxime reactivators was
designed largely on the basis of the base structure of the
established reversible ChE inhibitor edrophonium (Figure 1).
Three structural elements were systematically varied in the eight
compounds of the library, including position of the aldoxime
group ortho or para to the quaternary substitution on the benzene
ring, the presence or absence of an hydroxy group vicinal to the
aldoxime, and the size of the alkyl substitution on the exocyclic
quaternary nitrogen (trimethyl, diethyl methyl or dimethyl ethyl).
Of the two remaining oximes one was based on the structure
of the known reactivator 2PAM where an hydroxy group was
introduced into the meta position of the pyridinium ring vicinal to
the aldoxime group [2PAMOH (3-hydroxy-2-pyridinealdoxime
methiodide); Figure 1]. In the other, two hydroxy groups
were introduced in meta positions, the oxime group in para
and a methyl group in the ortho position (4PyOHMOH;
Figure 1).
Comparative analysis of reactivation for OP–hBChE conjugates
of paraoxon, cyclosarin, VX or sarin, revealed efficacious
reactivation by the oxime TAB2OH, faster than reactivation by
either of the two reference oximes 2PAM or HI-6, for all OPs
except for sarin (Figure 2B). Thus trimethylammonio benzene
substituted with an aldoxime in the ortho position and an hydroxy
group at an adjacent position provided a suitable structural
template for efficient interaction with OP–hBChE conjugates.
The omission of the adjacent hydroxy group [TAB2 (2trimethylammonio benzaldehydeoxime)], the shift of the oxime
to the para position [TAB4OH and TAB4 (4-trimethylammonio
benzaldehydeoxime)] or shift of the exocyclic quaternary nitrogen
into the ring to convert benzene into pyridinium (2PAMOH) all
resulted in the loss of favourable reactivation potency observed
for paraoxon, VX and cyclosarin hBChE conjugates (Figures 1
and 2B). Notably, the favourable influence of the shift of
a delocalized positive charge in the pyridinium ring to an
exocyclic spherically diffused charge distribution suggests that
oxime/phenolate stabilization in OP–hBChE conjugates prefers a
cation– π interaction with aromatic side chains, rather than π–π
stacking of the two delocalized ring systems.
Reactivation screen of OP–hAChE and OP–hBChE conjugates
by oximes of the directed library in vitro
Reactivation kinetics of TAB2OH oxime in vitro
Most oximes of the directed library were less effective than
reference oximes 2PAM and HI-6 in reactivation of OP–hAChE
conjugates resulting from paraoxon, cyclosarin, VX or sarin
inhibition (Figure 2A) as judged by screening procedures [14].
Only TAB4OHmee oxime (Figure 1) was significantly better
than 2PAM in reactivating an OP–hAChE conjugate. An order
of magnitude enhancement in reactivation of cyclosarin-inhibited
hAChE by TAB4OHmee pointed to the importance of the unique
combination of structural elements absent in the other evaluated
oximes (Figure 1) including: bulky symmetrical quaternary
nitrogen substitution, para-substituted oxime group and presence
of a vicinal hydroxy group. Nevertheless reactivation rates
by TAB4OHmee were still less than HI-6, the best hAChE
reactivator in this group of oximes, by an order of magnitude
(Figure 2A).
Comparative analysis of reactivation by a wide range of TAB2OH
concentrations for OP–hBChE conjugates (0.1–10 mM; Figure 3)
and OP–hAChE conjugates (5–50 mM; Supplementary Figure
S1 at http://www.biochemj.org/bj/450/bj4500231add.htm) confirmed the preferences observed in initial screening. Evaluation
of individual reactivation constants k2 and K ox (Table 1) revealed
superior reactivation of OP–hBChE conjugates compared with
2PAM, and faster reactivation of OP–hBChE compared with
OP–hAChE conjugates; both largely reflect an improved binding
interaction of TAB2OH to OP–hBChE conjugates (smaller K ox ).
Both 2PAM and TAB2OH bind equally well to native hAChE and
native hBChE as reflected in a K i value of 0.2–0.3 mM (Table 1).
Covalent OP conjugation differentially affects affinity for the
two enzyme conjugates, lowering the affinity of TAB2OH for
OP–hBChE only several-fold, but by two orders of magnitude
c The Authors Journal compilation c 2013 Biochemical Society
Organophosphate detoxification by butyrylcholinesterase and oximes
Figure 3
235
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 constants
derived 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-inhibited
enzymes (compare with the Experimental section).
for OP–hAChE. Association of 2PAM is approximately an
order of magnitude weaker in either OP–hAChE or OP–hBChE
conjugates. The overall difference in maximal reactivation rate
constants for the two enzyme conjugates was smaller. Except for
reactivation of hBChE conjugates of sarin and tabun, maximal
reactivation rates for TAB2OH were similar or larger than those
determined for 2PAM.
In this analysis we assume that K ox primarily describes initial
reversible interaction of oxime reactivator with an OP–hAChE
conjugate, whereas k2 refers to the interaction of the reactivating
oxime with the methyl phosphonate or diethyl phosphorate
of the OP-conjugated enzymes in forming the transition state of
reactivation.
pK a determinations
Structures of the lead hBChE reactivator TAB2OH and several
structurally related oximes (Figure 1) include two ionizable
groups in the physiologically relevant pH range with the nucleo-
philic potential to affect the reactivation efficacy, the oxime
group and the phenolic hydroxy group, which form the respective
oximate and phenolate species. We determined pK a values of
ionizable groups for TAB2OH, structurally related oximes,
TAB2, TAB4OH and TAB4, the structurally related reversible
inhibitor edrophonium, and a reference oxime 2PAM.
The bathochromic shift in the UV–Vis spectra, resulting from
deprotonation of oxime and hydroxy groups or from their protonation, enabled pK a value determinations from an incremental
increase in absorbance at discrete wavelengths (Supplementary
Figure S2 at http://www.biochemj.org/bj/450/bj4500231add.htm
and Table 2). For all compounds with single ionizable groups
(TAB2, TAB4, 2PAM and edrophonium) the pH-dependence of
the absorbance change was consistent with a single ionization
equilibrium at all peak wavelengths. For the ring substitutions
containing the phenolic and aldoxime hydrogens, the pHdependence of the spectral change showed evidence for two
ionization states, and allowed a simultaneous evaluation of pK a
values for both oxime and hydroxy hydrogens (Supplementary
Figure S2 and Table 2).
c The Authors Journal compilation c 2013 Biochemical Society
236
Table 1
Z. Radić and others
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. Inhibition
constants [K i and α K 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-hAChE
conjugate 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 Supplementary
Figure 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 determined
kinetic constants were typically less than 30 % of the mean.
Paraoxon
Sarin
Cyclosarin
VX
Tabun
Without OP
Oxime
Enzyme
k2
K ox
kr
k2
K ox
kr
k2
K ox
kr
k2
K ox
kr
k2
K ox
kr
Ki
αK i
TAB2OH
2PAM
TAB2OH
2PAM
BChE
BChE
AChE
AChE
0.34
0.23
0.47
0.27
0.71
2.4
12
1.8
480
96
41
150
0.19
2.4
0.92
1.1
0.79
1.8
27
0.34
250
1300
34
3300
3.9
2.6
0.44
0.73
1.0
5.4
36
6.6
3700
480
12
110
2.0
1.2
2.4
0.74
1.5
2.5
23
0.32
1300
480
100
2300
0.00027
0.0011
0.0032
0.0058
1.3
1.2
8.2
1.6
0.21
0.91
0.39
3.8
0.32
0.23
0.20
0.34
0.74
18
4.0
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 2 H2 O (by NMR) are typically higher than those determined in H2 O by ∼0.5 (compare with [16]) and were accordingly corrected. All pK a values were determined at room
temperature. d, doublet; n.d., not determined; s, singlet; t, triplet.
Oxime
Method
pK a1
Hydroxy
pK a2
Oximate
TAB2OH
UV 240 nm
UV 275 nm
UV 310 nm
NMR (s)
NMR (t)
NMR (d1)
NMR (d2)
NMR (s2)
Oximolysis*
UV 285 nm
Oximolysis*
7.44 +
− 0.05
7.58 +
− 0.07
7.52 +
− 0.05
7.69 +
− 0.08
7.63 +
− 0.09
7.74 +
− 0.08
7.70 +
− 0.08
7.70 +
− 0.08
+
7.21 +
− 0.14 (k max = 6 − 1)
-
10.6 +
− 0.1
10.1 +
− 0.3
9.3 (k max 21)
9.47 +
− 0.05
10.2 (k max 250)
TAB4OH
UV 275 nm
UV 340 nm
Oximolysis*
7.47 +
− 0.12
7.45 +
− 0.03
+
7.54 +
− 0.01 (k max =15 − 1)
11.03 +
− 0.04
10.93 +
− 0.09
10.7 (k max 65)
TAB4
UV 245 nm
UV 285 nm
Oximolysis*
UV 240 nm
UV 270 nm
UV 295 nm
Oximolysis
8.22 +
− 0.03
8.20 +
− 0.06
8.20 +
− 0.03
n.d. (k max ∼0)
10.13 +
− 0.01
10.09 +
− 0.06
10.7 (k max 440)
-
NMR
OP oximol
Oximolysis
-
8.11 +
− 0.03
8.1 +
− 0.2
8.0 +
− 0.1 (k max =60)
TAB2
Edrophonium
2PAM
*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 of
oximolysis were measured up to pH 10 only.
1
H-NMR spectroscopy in 2 H2 O was also used to monitor
pH-dependent chemical shifts in reactivator resonance spectra,
and confirmed the pK a values of ionizable groups of the
lead hBChE reactivator TAB2OH and the reference oxime
2PAM (Supplementary Figure S3 at http://www.biochemj.
org/bj/450/bj4500231add.htm and Table 2). The NMR p2 H range
used (5.5–10.5) allowed determination of only one pK a value per
compound. The chemical shift and appearance of five peaks in
the TAB2OH spectrum were analysed (Supplementary Figure
S4 at http://www.biochemj.org/bj/450/bj4500231add.htm), two
singlets (amido proton and nine equivalent trimethyl quaternary
c The Authors Journal compilation c 2013 Biochemical Society
N protons), two doublets (aromatic benzene protons at ∼ 7.2
p.p.m. and ∼7.42 p.p.m.; at pH 5.0) and a triplet (aromatic
benzene proton at ∼ 7.52 p.p.m. at pH 5.0). All five peaks
shifted to lower p.p.m. values with increasing pH and yielded
pK a values in the range 7.63–7.74 (Supplementary Figures S3
and S5 at http://www.biochemj.org/bj/450/bj4500231add.
htm, and Table 2), thus reflecting deprotonation of the same
group, the benzene hydroxy group. In the 1 H-NMR spectrum of
2PAM in 2 H2 O, the aldoxime CH and all aromatic proton signals
shifted simultaneously, yielding a pK a value of 8.11 [16]. The
N-methyl peak at 2.8 p.p.m. did not shift (Supplementary
Organophosphate detoxification by butyrylcholinesterase and oximes
Figure 4
237
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 ◦ C
in 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 by
error 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 ‘larger
or 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 affects
delocalization of the aromatic ring system of 2PAM, but not
electron distribution around its N-methyl protons. The ionization
state of TAB2OH phenolic hydroxy influences the distribution
of electrons and charge of the entire molecule, including the
oxime group. The π-orbital interactions between delocalized
ring electrons of reactivators and aromatic residues in the AChE
gorge, and consequently binding orientations of these two
structurally similar reactivators, endo- and exo-cyclic quaternary
amines, thus appear to be different.
Lastly, the hydroxy group pK a values were identical for both
phenolic oximes, in spite of differential substitution in the benzene
ring. The hydroxy group pK a , on the other hand, appeared
at least 0.6 pH units higher in the absence of the aldoxime
group substitution, as evidenced by the edrophonium comparison.
Finally, the pyridinium-based 2PAM oximate appeared to be a
stronger nucleophile at physiological pH than any of the benzenebased oximates (Table 2), due to its lower (by 2 pH units) pK a for
the oxime to oximate dissociation.
Oxime-assisted OP hydrolysis by hBChE in vitro and ex vivo
pH-dependence of kinetics of catalysis
Nucleophilic reactivity and pK a values for these compounds
were additionally monitored by determining the rates of
free oxime-induced catalysis (oximolysis) of ATCh, measured
spectrophotometrically by Ellman’s reagent DTNB (Figure 4
and Table 2). The resulting profiles of the pH-dependence for
oximolysis exhibited two ionization equilibria for both hydroxy
oximes (TAB2OH and TAB4OH), but only a single one for
TAB2, TAB4 and 2PAM. No nucleophilic activity was detectable
for edrophonium as judged by the absence of its influence on
ATCh hydrolysis. The biphasic nucleophilic pH relationships for
the two hydroxy benzadehyde oximes (with pK a values consistent
with those determined for the oxime and hydroxy groups using
different approaches) indicate formation of a nucleophile at a pH
value close to the physiological range, in addition to the aldoxime
group, and with its intrinsic reactivity significantly higher than that
observed for the hydroxy substitution alone on the benzene ring
(Table 2). As a consequence, the nucleophilic strength of both
hydroxy benzene aldoximes at physiological pH was enhanced
in comparison with corresponding non-hydroxylated benzene
aldoximes.
pK a values determined for the same compounds using different
methods were in good agreement. Moreover, pK a values of
the oxime group for all four benzene aldoximes were not
significantly different, irrespective of hydroxy substitutions.
Hydrolysis of 5 μM solutions of racemic fluorescent nerve
agent OP analogues (Flu-MPs) by combinations of 500 nM
hBChE + 100 μM oxime), as monitored by release of fluorescent
OP leaving groups, revealed substantial breakdown of all four
nerve agent analogues tested (cyclosarin, VX, sarin and soman)
within the first few minutes of the reaction (Figure 5). The
initial fast phases of hydrolysis by cyclosarin and VX probably
correspond to complete degradation of more toxic fast-inhibiting
and fast-reactivating OP enantiomers with PS configuration
[21,22], whereas the slow phase represents the sum of nonenzymatic OP oximolysis and slow hBChE inhibition/reactivation
by less reactive OP enantiomers with PR configuration. For the
soman analogue, the short initial fast phase corresponds to OP
degradation before completion of aging of the soman–hBChE
conjugate. OP hydrolysis assisted by TAB2OH was substantially
faster than hydrolysis assisted by 2PAM, for cyclosarin and VX.
The hydrolysis of the sarin analogue was slowest (except for
the soman analogue). It was slightly better assisted by 2PAM,
consistent with reactivation experiments (Figure 3), and largely
monophasic. Non-enzymatic OP oximolysis, largely equivalent
to slow reaction phases (Figure 5), was approximately 2-fold
faster for 2PAM compared with TAB2OH (results not shown),
consistent with its stronger nucleophilic reactivity observed
against ATCh at pH 7.4 (Figure 4 and Table 2).
Thus the combination of 500 nM hBChE and 100 μM
TAB2OH was capable of catalysing the complete degradation
c The Authors Journal compilation c 2013 Biochemical Society
238
Z. Radić and others
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 nM
hBChE (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, sarin
and 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 oxime
or 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 racemic
OP) within 20 min, under physiological conditions (Figure 5
and Supplementary Table S1 at http://www.biochemj.org/bj/
450/bj4500231add.htm). Hydrolysis assisted by a combination of
500 nM hBChE and 100 μM 2PAM was approximately 4–5-fold
slower (Supplementary Table S1).
OP hydrolysis ex vivo monitored in human blood shows
significant OP degradation and consequential recovery of total
ChE activity within 10 min following the addition of 100 μM
TAB2OH. The blood was initially supplemented with 300 or
60 nM purified hBChE and subsequently exposed to 0.5 μM
nerve agent Flu-MP OP analogue or paraoxon (Figure 6). The
oxime, hBChE and OP concentrations in this experiment were
selected to mimic respective concentrations likely to be found in
the blood of an OP-exposed person. Here 0.5 μM VX represents
an equivalent of 935 μg of VX in a 7.0 litre blood volume for
a ∼ 70 kg adult, or an equivalent of a 13 μg/kg i.v. dose which
is 1.7-fold greater than the extrapolated LD50 value for humans
[23] (assuming that all injected OP is transiently retained in
blood or vascular space). For hBChE, the range 60–300 nM
is an equivalent of an actual 0.5–2.5 mg/kg i.v. dose, and this is
approximately 10- to 50-fold lower than the dose recommended
for administration of hBChE as a stoichiometric OP bioscavenger
[24] in the treatment of an OP exposure. Within the initial 10 min
of TAB2OH-assisted catalysis, the VX analogue concentration
declined most rapidly (50–70 % recovered activity), followed by
the 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 lead
hBChE reactivator TAB2OH to be 100 mg/kg, comparable with
that of 2PAM (LD50 = 106 mg/kg; [16]), but more toxic than
the common oxime reactivator HI-6 (LD50 = 450 mg/kg) or our
c The Authors Journal compilation c 2013 Biochemical Society
lead centrally active N-substituted hydroxyimino acetamido alkyl
amine, RS194B (LD50 = 500 mg/kg) [16].
OP-exposed mice were treated i.m. with TAB2OH (a dose
equal to 25 % of its LD50 where no signs of intoxication
were observed) in conjunction with atropine 1 min after s.c.
OP exposure. The mice were 5.0-fold less sensitive to the
dose of VX, 10-fold less sensitive to paraoxon, 2.9-fold
less 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 for
2PAM were slightly better, with respective PIs for VX, paraoxon,
sarin and tabun of 9.3, 47, 6.7 and 1.3 [16]. This therapeutic
efficacy appears surprisingly good and better by (on average)
one order of magnitude than would be expected solely from
comparison of in vitro OP–hAChE reactivation kinetics for 2PAM and TAB2OH (Supplementary Figure S1 and Table 1). The
respective second-order reactivation rate constants of 2PAM were
23-fold, 3.7-fold, 97-fold and 9.7-fold larger than the TAB2OH
reactivation rate constants for the respective OP–AChE conjugates, VX, paraoxon, sarin and tabun (Table 1), whereas ratios of
respective PIs between 2PAM and TAB2OH therapies were 1.9,
4.7, 2.3 and 0.81 (Table 3). Thus a question could be raised on
the vital importance of reactivation of OP–hBChE conjugates in
peripheral tissues accessible to the charged reactivators TAB2OH
and 2PAM, for survival in OP intoxication. Pretreatment of mice
with a combination of 25 mg of TAB2OH/kg and 1 mg of purified
hBChE/kg 15 min before OP exposure followed by therapeutic
administration of another 25 mg of TAB2OH/kg in conjunction
with atropine (10 mg/kg i.m.) 1 min post-OP did not provide
notable protection in VX- and tabun-exposed mice. However,
significantly (judged by 95 % confidence limits; Tables 3 and
4) higher PIs were observed for paraoxon (59 % increase) and
sarin (38 % increase) when compared with the oxime therapy
alone (in conjunction with atropine). The in-vitro-determined K ox
values for TAB2OH and OP–hAChE conjugates are in the 8–
36 mM range, approximately 1000-fold higher than the measured
10 μM TAB2OH concentrations in plasma. Accordingly, the
Organophosphate detoxification by butyrylcholinesterase and oximes
239
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), sarin
analogue exposure (squares) and paraoxon exposure (triangles) is shown.
observed PI increase cannot be ascribed to the protective effect
of the native hAChE from OP inhibition by TAB2OH. Rather,
we ascribe the increase to catalytic degradation of OP in plasma
mediated by the combination of sub-stoichiometric amounts of
hBChE and TAB2OH. To verify this conclusion we varied the
timing and administration regime of hBChE and TAB2OH to
mice during and after OP exposure (Table 4). As expected,
pretreatment of mice by sub-stoichiometric doses of hBChE
alone did not influence PIs, except when it was administered
together with atropine. On the other hand, whenever substoichiometric hBChE was administered together with TAB2OH
(and with atropine), either as a pretreatment before paraoxon
or as a therapy post-paraoxon exposure, the resulting PIs were
higher (significantly considering 95 % confidence limits) by 30–
60 % than in the treatment without hBChE (Table 4) but with
atropine.
TAB2OH pharmacokinetics in mice
The highest concentration of TAB2OH determined in plasma
upon i.m. administration of a single dose of 30 mg/kg was
2.2 μg/ml (or ∼10 μM), observed at the initial 15 min collection
point (Supplementary Figure S7 and Supplementary Table
S2 at http://www.biochemj.org/bj/450/bj4500231add.htm). The
decline from the plasma is rapid and appears multiphasic,
precluding an initial estimate of the volume of distribution after
i.m. dosing. As expected for a quaternary cation, BBB (blood–
brain barrier) penetration appeared minimal. The maximal brain
concentration determined at the 15 min collection point was
0.13 μg/ml (equivalent to ∼0.56 μM) or approximately 18-fold
lower than in plasma. Retention of the low concentrations of
TAB2OH that appear to have penetrated into the brain, however,
was prolonged. Although kinetics of elimination by mice is
typically more rapid than in humans, the high plasma clearance
rate of TAB2OH may necessitate either multiple dosing or a single
dose of a pharmaceutical formulation of the oxime, affording
sustained 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 of
a novel effective reactivator of phosphorylated hBChE: cationic
quaternary oxime TAB2OH. Identified from a directed library of
cationic oxime reactivators based on the ChE reversible inhibitor
edrophonium, it proved 4–5-fold more effective than the reference
oxime 2PAM for in vitro reactivation of OP–hBChE conjugates
of cyclosarin, VX and paraoxon. Dissection of reactivation rate
constants revealed improved molecular recognition (reduced K ox )
to contribute most to the superior reactivation when compared
either with 2PAM for reactivation of OP–hBChE conjugates or
with TAB2OH reactivation of OP–hAChE conjugates.
Determinations of oxime group pK a values for the two
reactivators, however, reveal a much less dissociated oxime
nucleophile of the TAB2OH reactivator (with an average
pK a = 10) compared with 2PAM (with average pK a = 8.1).
Measurements of oxime nucleophilic reactivities by hydrolysis
of both ATCh and Flu-MPs (Figure 4 and Table 2, and
Supplementary Table S1) in the absence of enzyme at pH 7.4
show only a 2–3-fold difference between the two oximes due
to biphasic dependence on pH for TAB2OH reactivation. The
lower pK a of 7.21 evaluated from this biphasic dependence
coincides with the lower of the two pK a values observed from
pH-dependence of UV spectra of TAB2OH and its homologous
hydroxy oxime TAB4OH. This was not observed in monophasic
pH-dependences of their non-hydroxy analogues TAB2 and
TAB4 (Table 2 and Figure 4) that reveal only a single pK a value of
∼10 corresponding to the oxime group. The observed enhanced
nucleophilic reactivity of TAB2OH (and TAB4OH) at pH 7.4
c The Authors Journal compilation c 2013 Biochemical Society
240
Table 3
Z. Radić and others
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 of
TAB2OH/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 and
animals 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
Pretreatment + Therapy
5.0 (2.2–11.7)
5.0 (3.3–7.7)
3.2
3.2
10 (7.5–13.3)
16 (13.5–18.7)
6.3
13
2.9 (2.3–3.7)
4.0 (2.5–6.4)
2.0
2.0
1.6 (1.2–2.1)
1.3 (1.0–1.6)
1.3
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 the
OP LD50 , which was fully counteracted by the antidotal treatment). n.d., not detected.
Pretreatment (i.v.)
15 min
30 min
0.5 mg of hBChE/kg
1 mg of hBChE/kg
0.5 mg of hBChE/kg
1 mg of hBChE/kg in atropine
1 mg of hBChE/kg
Therapy (i.m.)
VX*
1 min after OP
PI (95 % confidence limits)
MDP
25 mg of TAB2OH/kg
10 mg of TAB2OH/kg with
atropine
25 mg of TAB2OH/kg with
atropine
1.3 (0.7–2.1)
2.7 (2.5–3.0)
1.3
2.5
5.0 (2.2–11.7)
10 mg of TAB2OH/kg with
atropine
25 mg of TAB2OH/kg with
atropine
1 mg of hBChE/kg with 25 mg
of TAB2OH/kg
Paraoxon†
PI (95 % confidence limits)
MDP
3.2
10.0 (7.5–13.3)
6.3
1.0 (0.9–1.1)
1.1 (0.8–1.4)
3.4 (2.7–4.4)
0.79
n.d.
3.4
<1.0 (n.d.)
n.d.
2.5 (1.3–4.8)
5.0 (3.3–7.7)
1.3
3.2
14.1 (12.0–16.7)
10
<1 (n.d.)
n.d.
<1 (n.d.)
n.d.
<1 (n.d.)
n.d.
16 (13.5–18.7)
13
13 (8.2–19.1)
7.9
1 mg of hBChE/kg with
25 mg of TAB2OH/kg
1 mg of hBChE/kg with 25 mg
of TAB2OH/kg
25 mg of TAB2OH/kg with
atropine
1 mg of hBChE/kg + 25 mg of
TAB2OH/kg with atropine
5.0 (3.3–7.7)
3.2
*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 orthosubstituted phenolic hydroxy. Direct participation of nucleophilic
reactivity of this hydroxy itself in those reactions is, however,
unlikely since the hydroxy group of the structurally related
edrophonium remains completely unreactive, even at pH 10 in its
nearly completely deprotonated form. Thus a new non-aldoxime
nucleophilic species or an activated aldoxime moiety due to
an 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 TAB4OH
at physiological pH values.
One possible mechanism that could bridge between these two
different pKa values and nucleophilicity of phenolate compared
with oxime group could be the formation of an intermediate to
a stabilized bicyclic ring comprising two adjacent carbons of the
benzene ring, the phenolate O − and two atoms of the aldoxime
C = N-O-H. Such a transition state or meta-stable intermediate
c The Authors Journal compilation c 2013 Biochemical Society
ring cyclization to form an isoxazole could form at pH 7.4.
Alternatively the phenolate O − in the active site might react with
a terminal hydrogen from the CH = NOH group, leading to partial
deprotonation of CH = NO-H and converting it into an oximate
anion CH = NO − at physiological pH, the active nucleophile
in reactivation of OP–ChE conjugates by aldoximes. Additional
studies will be needed to identify the precise structural species
and mechanistic nature of this functionally beneficial nucleophilic
reaction. Enhanced nucleophilicity of ortho hydroxy-substituted
benzene aldoximes, but with monophasic pH-dependence and
only limited in vitro reactivation potency has been observed
elsewhere [25]. Additionally, hydroxy groups positioned ortho
to oxime reactivators enhance decomposition of phosphonyloximes, probably through intramolecular formation of an
isoxazole ring [26,27], and minimize eventual reinhibition
of enzymes observed for some phosphonyl-oxime adducts
[28].
Organophosphate detoxification by butyrylcholinesterase and oximes
A reasonable in vitro potency of TAB2OH for reactivation of
OP–hBChE conjugates raises new considerations for establishing
a catalytic OP bioscavenger on the basis of TAB2OH-assisted
OP hydrolysis by hBChE. Our initial experiments demonstrate
that both in vitro in physiologically conditioned buffers and
ex vivo in the whole human blood, low micromolar and submicromolar OP concentrations (that could be expected to
accumulate in the blood of OP-exposed individuals) are degraded
significantly within 5–20 min of administration of 100 μM
TAB2OH and sub-stoichiometric (60–300 nM) hBChE. These
results and moderate acute toxicity of TAB2OH (LD50 of
100 mg/kg i.m.) in mice allowed us to test this catalytic OP
bioscavenger in vivo in nerve-agent- and OP-pesticide-exposed
mice. Under various administration regimens of TAB2OH and
hBChE to the OP-exposed mice, we observed 30–60 % enhanced
oxime PIs whenever the BChE + TAB2OH combination was
administered to paraoxon-exposed mice, either before paraoxon
exposure (pretreatment) or after paraoxon exposure (therapy).
No protection was observed for mice administered with substoichiometric amounts of hBChE alone (without atropine), either
in pretreatment or when administered therapeutically post-OP. We
assume that enhanced PIs reflect catalytic turnover of paraoxon
by the hBChE + TAB2OH combination acting as a catalytic
bioscavenger.
The moderate extent of PI enhancement for paraoxon, and the
absence of the enhancement in the treatment of VX-exposed mice
probably reflect low tissue accumulation and rapid clearance
of TAB2OH from plasma. A maximal determined plasma
concentration of 10 μM TAB2OH 15 min following a single
30 mg/kg i.m. administration (Supplementary Figure S5) is likely
to be insufficient to provide optimal assistance to hBChE in the
OP turnover. To enhance efficacy in catalytic OP bioscavenging,
a compound with lower toxicity and lower clearance than the
TAB2OH lead would be beneficial. Nevertheless, the results of
the present study with this lead show the ‘proof of principle’
for effective catalytic OP bioscavenging based on combined
administration of sub-stoichiometric amounts of purified hBChE
and an oxime reactivator. Such an approach would allow for
significant cost reduction and more practical implementation of
antidotal administration in treatment of OP exposure in high-risk
settings.
AUTHOR CONTRIBUTION
Zoran Radić, Julius Rebek Jr, Dariush Ajami, Trevor Dale and Palmer Taylor conceptually
developed the project. Zoran Radić, Palmer Taylor, Julius Rebek Jr, Zrinka Kovarik, Gabriel
Amitai, Suzana Berend, Dariush Ajami, Carol Green and Brendan Duggan contributed
to writing the paper. Julius Rebek Jr, Trevor Dale, Dariush Ajami and Gabriel Amitai
designed and synthesized the oximes and fluorescent non-volatile OPs. Zoran Radić,
Palmer Taylor, Julius Rebek Jr, Zrinka Kovarik, Božica Radić and Carol Green contributed
to the experimental design of the study. Zoran Radić, Edzna Garcia, Limin Zhang, Suzana
Berend, Zrinka Kovarik and Brendan Duggan performed experiments and analysed the
data.
ACKNOWLEDGEMENTS
We thank Mr Vedran Micek and Ms Jasna Mileković 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 Health
Office of the Director, and the National Institute of Neurological Disorders and Stroke
[grant numbers U01 NS058046 and R21NS072086].
241
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Received 22 October 2012/4 December 2012; accepted 6 December 2012
Published as BJ Immediate Publication 6 December 2012, doi:10.1042/BJ20121612
c The Authors Journal compilation c 2013 Biochemical Society
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Biochem. J. (2013) 450, 231–242 (Printed in Great Britain)
doi:10.1042/BJ20121612
SUPPLEMENTARY ONLINE DATA
Catalytic detoxification of nerve agent and pesticide organophosphates
by butyrylcholinesterase assisted with non-pyridinium oximes
Zoran RADIĆ*1 , Trevor DALE†, Zrinka KOVARIK‡, Suzana BEREND‡, Edzna GARCIA*, Limin ZHANG*, Gabriel AMITAI§,
Carol GREEN, Božica RADIƇ, 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 of
Chemistry, The Scripps Research Institute, La Jolla, CA 92037, U.S.A., ‡Institute for Medical Research and Occupational Health, HR-10001 Zagreb, Croatia, §Department of
Pharmacology, Israel Institute for Biological Research, Ness Ziona 74100, Israel, and SRI International, Menlo Park, CA 94025-3493, U.S.A.
1
H-NMR and 13 C-NMR spectra were recorded at 600 and
150.9 MHz respectively, using a Bruker DRX-600 spectrometer
equipped with a 5 mm QNP probe. Chemical shifts of 1 H NMR
and 13 C NMR are given in p.p.m. by using deuterated solvents as
references. Standard abbreviations indicating multiplicity were
used as follows: s (singlet), b (broad), d (doublet), t (triplet), q
(quartet) and m (multiplet). MALDI–TOF (matrix-assisted laserdesorption ionization–time-of-flight) spectra and HRMS (highresolution mass spectra) were recorded on an Applied Biosystems
Voyager STR (2) apparatus and an Agilent ESI-TOF mass
spectrometer respectively. Anhydrous dichloromethane, N,Ndiethylethanamine and diethyl ether were taken from a solvent
drying system (SG Water USA).
SYNTHETIC PROCEDURES
(3-Methoxymethoxy-phenyl)-dimethylamine (4.15)
To a stirring solution of 4.15 (600 mg, 3.3 mmol) and
freshly distilled N,N,N ,N -tetramethylethylenediamine (0.5 ml,
3.3 mmol) in dry ethyl ether (10 ml) cooled to − 40 ◦ C under
argon was added n-butyllithium (2.0 ml of 1.8 M in hexane
solution, 3.6 mmol) and the reaction was allowed to warm to
room temperature and stirred for 1 h. The reaction was recooled to − 40 ◦ C and dry DMF (0.5 ml, 6.5 mmol) was added
and the reaction was allowed to warm to room temperature
with stirring over 1 h before being quenched with a dilute
ammonium chloride aqueous solution. The mixture was extracted
three times with ethyl acetate, the organic fractions combined,
dried over magnesium sulfate, filtered and then the volatiles
were removed under reduced pressure. The crude material was
purified by flash chromatography over silica gel eluting with
4:1 hexane/ethyl acetate to separate the isomers and then each
one was individually purified by flash chromatography over
silica gel eluting with dichloromethane to afford each pure
isomer.
4-Dimethylamino-2-methoxymethoxy-benzaldehyde (4.16)
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. The
reaction was quenched with a saturated solution of sodium
bicarbonate and stirred for 30 min. The layers were separated
and the aquous layer was extracted twice with dichloromethane,
the fractions combined, dried with magnesium sulfate, filtered
and the solvent was removed under reduced pressure. The crude
material was purified twice by flash chromatography eluting once
with dichloromethane and the second time with 9:1 hexane/ethyl
acetate to afford 660 mg of pure product as a colourless oil
(42 %). 1 H-NMR (600 MHz, [2 H]chloroform) δ 7.13–7.16 (m, 1
H), 6.40–6.44 (m, 3 H), 5.17 (s, 2 H), 3.49 (s, 3 H), 2.94 (s, 6 H).
13
C-NMR (150.9 MHz, [2 H]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.
281 mg, pale yellow solid, 41 %. 1 H-NMR (600 MHz,
[2 H]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). 13 C-NMR (150.9 MHz, [2 H]chloroform) δ
187.4, 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.
2-Dimethylamino-6-methoxymethoxy-benzaldehyde (4.17)
317 mg, yellow oil, 46 %. 1 H-NMR (600 MHz, [2 H]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). 13 C-NMR (150.9
MHz, [2 H]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
2-Dimethylamino-6-methoxymethoxy-benzaldehyde (4.16) and
4-dimethylamino-2-methoxymethoxy-benzaldehyde (4.17)
To a stirring solution of 4.16 (130 mg, 0.62 mmol) in
ethanol (10 ml) was added hydroxylamine hydrochloride (77 mg,
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2013 Biochemical Society
Z. Radić and others
1.1 mmol) and the reaction was stirred at room temperature for
30 min. A saturated solution of sodium bicarbonate and ethyl
acetate were added and the layers were separated. The aqueous
layer was extracted twice with ethyl acetate, the organic fractions
combined, dried over magnesium sulfate, filtered and then the
volatiles were removed under reduced pressure. The solvent
was removed under reduced pressure and the crude material was
purified by flash chromatography over silica gel eluting with
1:3:1 hexane/dichloromethane/ethyl acetate to afford 89 mg of
pure product as a white solid (64 %). In addition 21 mg of a
second product was isolated that appears to be the oxime isomer
(15 %). 1 H-NMR (600 MHz, [2 H]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). 13 C-NMR (150.9 MHz, [2 H]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)
2-Dimethylamino-6-methoxymethoxy-benzaldehyde oxime
To a stirring solution of 4.17 (163 mg, 0.78 mmol) in
ethanol (10 ml) was added hydroxylamine hydrochloride (85 mg,
1.2 mmol) and the reaction was stirred at room temperature for
30 min. A saturated solution of sodium bicarbonate and ethyl
acetate were added and the layers were separated. The aqueous
layer was extracted twice with ethyl acetate, the organic fractions
combined, dried over magnesium sulfate, filtered and then the
volatiles were removed under reduced pressure. The solvent
was removed under reduced pressure and the crude material was
purified by flash chromatography over silica gel eluting with
1:3:1 hexane/dichloromethane/ethyl acetate to afford 160 mg of
pure product as a white solid (91 %). 1 H-NMR (600 MHz,
[2 H]chloroform) δ 9.93 (s, 1 H), 8.46 (s, 1 H), 7.24 (t, J 8.2
Hz, 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). 13 C-NMR (150.9
MHz, [2 H]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.
2-Dimethylamino-6-hydroxy-benzaldehyde oxime (4.19)
To a stirring solution of 4-dimethylamino-2-methoxymethoxy
benzaldehyde oxime (80 mg, 0.36 mmol) in methanol (2 ml) at
room temperature was added a 4 M solution of hydrogen chloride
in dioxane (0.3 ml, 1.2 mmol). The reaction was heated to 70 ◦ C
for 1 h and then cooled to room temperature and the solvent was
blown off with nitrogen. The crude material was purified by flash
chromatography over silica gel eluting with dichloromethane to
afford 50 mg of pure product as a white solid (82 %). 1 H-NMR
(600 MHz, [2 H]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). 13 C-NMR
(150.9 MHz, [2 H]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) in
dichloromethane (0.7 ml) at room temperature was added
iodomethane (0.1 ml, 1.6 mmol). The reaction was stirred at
room temperature for 40 h as a precipitate formed. The product
was collected by filtration washing with excess dichloromethane
yielding 21 mg of pure product as a white solid (37 %). 1 H-NMR
(600 MHz, [U-2 H]methanol) δ 8.36 (s, 1 H), 7.61 (d, J 8.7 Hz, 1
H), 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). 13 C-NMR (150.9 MHz, [U-2 H]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.
c The Authors Journal compilation c 2013 Biochemical Society
To a stirring solution of 2-dimethylamino-6-methoxymethoxybenzaldehyde oxime (14 mg, 0.06 mmol) in methanol (0.2 ml) at
room temperature was added a 4 M solution of hydrogen chloride
in dioxane (0.2 ml, 0.8 mmol). The reaction was heated to 70 ◦ C
for 1 h and then cooled to room temperature and the solvent was
blown off with nitrogen. The crude material was purified by flash
chromatography over silica gel eluting with dichloromethane to
afford 10 mg of pure product as a white solid (91 %). 1 H-NMR
(600 MHz, [2 H]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 8.0
Hz, 1 H), 2.73 (s, 6 H). 13 C-NMR (150.9 MHz, [2 H]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 dichloromethane (0.2 ml) at 0 ◦ C was added methyl trifluoromethansulfonate
(62 μl, 0.55 mmol). The reaction was allowed to warm to room
temperature and stirred for 40 h. The precipitate formed was
collected by filtration washing with excess dichloromethane
to afford 101 mg of the pure triflate salt as a white solid
(59 %). The salt was dissolved in acetonitrile and a solution
of tetrahexylammonium chloride in acetonitrile was added to
precipitate the pure chloride salt as a white solid. 1 H-NMR (600
MHz, d6 -DMSO) δ 11.74 (s, 1 H), 10.95 (s, 1 H), 8.34 (s, 1
H), 7.45 (t, J 8.4 Hz, 1 H), 7.39 (d, J 8.4 Hz, 1 H), 7.26 (d, J
8.2 Hz, 1 H), 3.68 (s, 9 H). 13 C-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 dichloromethane (1.5 ml) at 0 ◦ C was added ethyl trifluoromethansulfonate
(42 μl, 0.32 mmol). The reaction was allowed to warm to room
temperature and stirred for 24 h. The mixture was tritrated
with dichloromethane and then the triflate salt was dissolved in
acetonitrile and a solution of tetrahexylammonium chloride
in acetonitrile was added to precipitate 46 mg of the pure chloride
salt as a white solid (64 %). 1 H-NMR (600 MHz, d6 -DMSO) δ
11.72 (s, 1 H), 10.94 (s, 1 H), 8.37 (s, 1 H), 7.71 (d, J 8.8 Hz, 1
H), 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). 13 C-NMR (150.9
MHz, 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 sodium
hydroxide and stirred for 30 min. The layers were separated
and the aquous layer extracted twice with dichloromethane, the
fractions combined, dried with magnesium sulfate, filtered and
the solvent removed under reduced pressure. The crude
material was purified by flash chromatography eluting with 9:1
hexane/ethyl acetate to afford 900 mg of pure product as a
colourless oil (55 %). 1 H-NMR (600 MHz, [2 H]chloroform) δ
7.09–7.12 (m, 1 H), 6.35–6.36 (m, 3 H), 5.16 (s, 2 H), 3.49 (s, 3
H), 3.34 (q, J 7.1 Hz, 4 H) 1.16 (t, J 7.1 Hz, 6 H). 13 C-NMR (150.9
MHz, [2 H]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.
and the solution was stirred at room temperature for 3 h.
The reaction was quenched with a 5 % sodium hydroxide
solution and stirred for 30 min. Ethyl acetate was added and
the layers were separated and the aqueous layer was extracted
twice with ethyl acetate, the fractions combined, dried with
magnesium sulfate, filtered and the solvent removed under
reduced pressure. The crude material was purified twice by
flash chromatography eluting once with dichloromethane and
the second time with 9:1 dichloromethane/ethyl acetate to
afford 250 mg of pure product (47 %). 1 H-NMR (600 MHz,
[2 H]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, J
7.1 Hz, 4 H), 1.21 (t, J 7.1 Hz, 6 H). 13 C-NMR (150.9 MHz,
[2 H]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) and
2-diethylamino-6-methoxymethoxy-benzaldehyde (4.23)
To a stirring solution of 4.21 (840 mg, 4.0 mmol) and
freshly distilled N,N,N ,N -tetramethylethylenediamine (0.61 ml,
4.1 mmol) in dry ethyl ether (15 ml) cooled to − 40 ◦ C under
argon was added n-butyllithium (2.5 ml of 1.8 M in hexane
solution, 4.5 mmol) and the reaction was allowed to warm to
room temperature and stirred for 1 h. The reaction was re-cooled
to − 40 ◦ C and dry DMF (0.6 ml, 7.7 mmol) was added and
the reaction was allowed to warm to room temperature with
stirring over 1 h before being quenched with a dilute ammonium
chloride aqueous solution. The mixture was extracted three times
with ethyl acetate, the organic fractions combined, dried over
magnesium sulfate, filtered and then the volatiles were removed
under reduced pressure. The crude material was purified by flash
chromatography over silica gel eluting with dichloromethane. The
isomers were separated by eluting with 7:3 hexane/ethyl acetate
to afford each pure isomer.
4-Diethylamino-2-methoxymethoxy-benzaldehyde (4.22)
386 mg, pale yellow oil, 41 %. 1 H-NMR (600 MHz,
[2 H]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). 13 C-NMR (150.9 MHz, [2 H]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.
4-Diethylamino-2-methoxymethoxy-benzaldehyde (4.22)
2-Diethylamino-6-methoxymethoxy-benzaldehyde (4.23)
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)
239 mg, yellow oil, 25 %. 1 H-NMR (600 MHz, [2 H]chloroform)
δ 10.25 (s, 1 H). 7.35 (t, J 8.3 Hz, 1 H), 6.77 (dd, J 8.3, 2.3
Hz, 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). 13 C-NMR (150.9 MHz, [2 H]chloroform) δ
190.1, 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.
c The Authors Journal compilation c 2013 Biochemical Society
Z. Radić 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 hydroxylamine
hydrochloride (220 mg, 3.2 mmol) and the reaction was stirred
at room temperature for 15 h. A saturated solution of sodium
bicarbonate and ethyl acetate were added and the layers were
separated. The aqueous layer was extracted twice with ethyl
acetate, the organic fractions combined, dried over magnesium
sulfate, filtered and then the volatiles were removed under reduced
pressure. The solvent was removed under reduced pressure and
the crude material was purified by flash chromatography over
silica gel eluting with dichloromethane to afford 432 mg of
pure product as an off-white solid (81 %). 1 H-NMR (600 MHz,
[2 H]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). 13 C-NMR (150.9 MHz, [2 H]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) in
dichloromethane (0.7 ml) at 0 ◦ C was added MeOTf (methyl
trifluoromethansulfonate; 62 μl, 0.55 mmol). The reaction was
allowed to warm to room temperature and stirred for 23 h. The
volatiles were then blown off with nitrogen and the residue
purified twice by flash chromatography over silica gel by eluting
with a gradient of 19:1 to 17:1 dichloromethane/methanol to
afford the pure tosylate salt. The salt was dissoved in methanol
and eluted through DOWEX 1-2×200 resin to exchange for the
chloride anion and the material was recrystallized from ethanol
to afford 38 mg of the pure chloride salt as a white solid (31 %).
1
H-NMR (600 MHz, [U-2 H]methanol) δ 8.37 (s, 1 H), 7.63 (d, J
8.7 Hz, 1 H), 7.33 (d, J 2.7 Hz, 1 H), 7.29 (dd, J 8.7, 2.7 Hz, 1
H), 4.06 (m, 2 H), 3.86 (m, 2 H), 3.52 (s, 3 H), 1.19 (t, J 7.1 Hz,
6 H). 13 C-NMR (150.9 MHz, [U-2 H]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 hydroxylamine
hydrochloride (290 mg, 4.2 mmol) and the reaction was stirred
c The Authors Journal compilation c 2013 Biochemical Society
at room temperature for 15 h as a yellow colour developed. A
saturated solution of sodium bicarbonate and ethyl acetate were
added and the layers were separated. The aqueous layer was
extracted twice with ethyl acetate, the organic fractions combined,
dried over magnesium sulfate, filtered and then the volatiles were
removed under reduced pressure. The solvent was removed under
reduced pressure and the crude material was purified by flash
chromatography over silica gel eluting with a gradient of 1:0 to 4:1
dichloromethane/ethyl acetate to afford 470 mg of pure product
as a white solid (93 %). 1 H-NMR (600 MHz, [2 H]chloroform) δ
8.05 (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). 13 C-NMR (150.9 MHz, [2 H]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 dichloromethane (3 ml) at 0 ◦ C was added methyl trifluoromethansulfonate
(57 μl, 0.50 mmol). The reaction was allowed to warm to room
temperature and stirred for 19 h. The formed precipitate was
collected by filtration washing with excess dichloromethane
to afford 128 mg of the pure triflate salt as a white solid
(85 %). The salt was dissolved in acetonitrile and a solution
of tetrahexylammonium chloride in acetonitrile was added to
precipitate the pure chloride salt as a white solid (58 %). 1 HNMR (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, 9
H). 13 C-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.
2-Dimethylamino-benzaldehyde oxime (4.30)
To a stirring solution of 2-dimethylamino-benzaldehyde (266 mg,
1.8 mmol) in ethanol (5 ml) was added hydroxylamine
hydrochloride (137 mg, 2.0 mmol) and the reaction was stirred
at room temperature for 30 min as the yellow colour faded to
colourless. A saturated solution of sodium bicarbonate and ethyl
acetate were added and the layers were separated. The aqueous
layer was extracted twice with ethyl acetate, the organic fractions
combined, dried over magnesium sulfate, filtered and then the
volatiles were removed under reduced pressure. The solvent
was removed under reduced pressure and the crude material was
purified by flash chromatography over silica gel eluting with
2:2:1 hexane/dichloromethane/ethyl acetate to afford 270 mg of
pure product as a white solid (92 %). 1 H-NMR (600 MHz,
[2 H]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 7.5
Hz, 1 H), 2.75 (s, 6 H). 13 C-NMR (150.9 MHz, [2 H]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.
Organophosphate detoxification by butyrylcholinesterase and oximes
2-Trimethylammonio-benzaldehyde oxime chloride of TAB2
To a stirring solution of 4.30 (69 mg, 0.42 mmol) in dichloromethane (2 ml) at 0 ◦ C was added methyl trifluoromethansulfonate
(50 μl, 0.44 mmol). The reaction was allowed to warm to room
temperature and stirred for 36 h. After the solvent was blown
off under nitrogen, the salt was dissoved in methanol and eluted
through DOWEX 1-2×200 resin to exchange for the chloride
anion. After drying under reduced pressure, the crude material
was dissolved in a mixture of ethyl acetate and water and the layers
were separated. The organic layer was extracted once with water
and then the aqueous solution was freeze-dried. The material
was then tritrated with dichloromethane and acetonitrile to afford
16 mg of a white solid that was 85 % pure, a mixture of the
chloride salt with an unknown impurity (18 %). 1 H-NMR (600
MHz, [U-2 H]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). 13 C-NMR (150.9
MHz, [U-2 H]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)
the crude material was purified by flash chromatography over
silica gel eluting with a gradient of 3:1 to 2:1 hexane/ethyl acetate
to afford 250 mg of pure product as an off-white solid (55 %).
1
H-NMR (600 MHz, [2 H]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). 13 C-NMR (150.9 MHz,
[2 H]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 dichloromethane (1 ml) at 0 ◦ C was added methyl trifluoromethansulfonate
(50 μl, 0.44 mmol). The reaction was allowed to warm to room
temperature and stirred for 24 h. After the solvent was blown
off under nitrogen, the salt was dissoved in methanol and eluted
through DOWEX 1-2×200 resin to exchange for the chloride
anion and the material was tritrated with dichloromethane to
afford 15 mg of the pure chloride salt as a white solid (17 %).
1
H-NMR (600 MHz, [U-2 H]methanol) δ 8.17 (s, 1 H), 7.88 (d, J
9.1 Hz, 1 H), 7.78 (d, J 9.1 Hz, 1 H), 4.09 (m, 2 H), 3.87 (m, 2
H), 3.54 (s, 3 H), 1.15 (t, J 7.2 Hz, 6 H). 13 C-NMR (150.9 MHz,
[U-2 H]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)
To a stirring solution of 4.28 (66 mg, 0.40 mmol) in dichloromethane (2 ml) at 0 ◦ C was added ethyl trifluoromethansulfonate
(57 μl, 0.44 mmol). The reaction was allowed to warm to room
temperature and stirred for 24 h. The formed precipitate was
collected by filtration washing with excess dichloromethane to
afford 90 mg of the pure triflate salt (65 %). The salt was dissolved
in acetonitrile and a solution of tetrahexylammonium chloride in
acetonitrile was added to precipitate 42 mg of the pure chloride
salt as a white solid (46 %). 1 H-NMR (600 MHz, d6 -DMSO) δ
11.63 (s, 1 H), 8.24 (s, 1 H), 7.94 (d, J 9.0 Hz, 2 H), 7.82 (d, J 9.0
Hz, 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). 13 C-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.
A suspension of 2-(hydroxylmethyl)-3-hydroxy-pyridine (1.0 g,
8.0 mmol) in chloroform (20 ml) was added to a stirring
suspension of manganese dioxide (4.0 g) in chloroform (20 ml)
at 50 ◦ C. The reaction was heated to 60 ◦ C for 3 h and then
the suspension was filtered through celite washing with excess
chloroform. The solvent was removed under reduced pressure to
afford 430 mg of pure product (43 %). 1 H-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). 13 C-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.
4-Diethylamino benzaldehyde oxime (4.32)
3-Hydroxypyridine-2-carboxaldehyde oxime (4.38)
To a stirring solution of 4-diethylamino-benzaldehyde (417 mg,
2.4 mmol) in ethanol (5 ml) was added hydroxylamine
hydrochloride (171 mg, 2.5 mmol) and the reaction was stirred
at room temperature for 1 h. A saturated solution of sodium
bicarbonate and ethyl acetate were added and the layers were
separated. The aqueous layer was extracted twice with ethyl
acetate, the organic fractions combined, dried over magnesium
sulfate, filtered and then the volatiles were removed under reduced
pressure. The solvent was removed under reduced pressure and
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 for
2 h. The solvent was removed under reduced pressure and the
crude material was purified by flash chromatography over silica
gel eluting with 40:60:0.5 hexane/ethyl acetate/triethanolamine to
afford 105 mg of product as a white solid (41 %). An analytically
pure sample was repurified by flash chromatography over
silica gel eluting with 19:1 dichloromethane/methanol. 1 H-NMR
(600 MHz, d6 -DMSO) δ 11.87 (s, 1 H), 10.29 (s, 1 H), 8.31 (s, 1
c The Authors Journal compilation c 2013 Biochemical Society
Z. Radić 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). 13 C-NMR (150.9 MHz, d6 -DMSO) δ
153.1, 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 added
iodomethane (0.1 ml, 1.6 mmol) and the reaction was heated to
70 ◦ C in a sealed tube for 60 h. After cooling, the precipitate was
collected by filtration to yield 29 mg of pure product as a pale
yellow solid (60 %). 1 H-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).
13
C-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 with
associated S.E.M. of determination. For TAB2OH errors were smaller than the symbol size.
c The Authors Journal compilation c 2013 Biochemical Society
Organophosphate detoxification by butyrylcholinesterase and oximes
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 in
B and D) of the main text [1]
c The Authors Journal compilation c 2013 Biochemical Society
Z. Radić and others
Figure S3 pH-dependence of 1 H-NMR spectra of 10 mM TAB2OH in 2 H2 O buffers (A, B and C) along with corresponding pK a values calculated from the
observed 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 1 H-NMR spectrum of 10 mM TAB2OH in 20 mM phosphate-pyrophosphate ( + 0.1 M NaCl) 2 H2 O buffer, pH 5
c The Authors Journal compilation c 2013 Biochemical Society
Organophosphate detoxification by butyrylcholinesterase and oximes
Figure S5
pH-dependence of 1 H-NMR spectra of 10 mM TAB2OH in 2 H2 O 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 in
the 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-induced
difference 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.
c The Authors Journal compilation c 2013 Biochemical Society
Z. Radić and others
Figure S6 pH-dependence of 1 H-NMR spectra of 2.0 mM 2PAM in 2 H2 O
buffers, pH 5–10
(A) Expanded view of the spectrum in the chemical-shift region 2.80–2.86. (B) pH-dependent
change in chemical shifts for the 2.83 p.p.m. singlet. Spectra from the single experiment were
aligned 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.
c The Authors Journal compilation c 2013 Biochemical Society
Organophosphate detoxification by butyrylcholinesterase and oximes
Scheme S1
Synthesis of TAB2OH and TAB4OH
Scheme S2
Synthesis of TAB4OHmme
Scheme S3
Synthesis of TAB4OHmee
c The Authors Journal compilation c 2013 Biochemical Society
Z. Radić and others
Scheme S4
Synthesis of TAB2 and TAB4
Scheme S5
Synthesis of TAB4mme
Scheme S6
Synthesis of TAB4mee
Scheme S7
Synthesis of 2PAMOH
c The Authors Journal compilation c 2013 Biochemical Society
Organophosphate detoxification by butyrylcholinesterase and oximes
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 of
500 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 are
given. 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
Cyclosarin
VX
5.5
6.4
15
11
11
28
15
530 (30 % total OP)
430 (50 % total OP)
27
37
64
4.1
3.4
11
4.6
3.7
13
8.5
180
150
12
37
40
Table S2 Maximal oxime concentrations in brains and plasma of mice
determined upon i.m. administration of a single oxime dose (compare with
Figure S5)
Distribution coefficients (logD) were calculated from oxime structures using the ChemAxon
software 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
c The Authors Journal compilation c 2013 Biochemical Society
Z. Radić and others
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
1.26
4/4
4/4
1.59
2.0
4/4
4/4
2.52
4/4
3.18
4/4
4.0
3/4
5.0
2/4
6.3
1/4
7.9
2/4
No visible symptoms.
One mouse had light tremor during the
first 10 min on application.
Light tremor 3–4 min on application.
Tremor and light salivation 20 min on
application.
4/4
Strong tremor, light salivation and
respiratory disturbance. All symptoms
disappeared after 2 h.
Strong tremor, light salivation and
respiratory disturbance. All symptoms
disappeared after 2 h.
Strong tremor and salivation 3 min on
application. Mice were weak with
respiratory disturbance. Surviving
animals were weak after 24 h.
Strong tremor and respiratory
disturbance immediately on
application. Surviving animals were
weak after 24 h.
Strong tremor, salivation and respiratory
disturbance immediately on
application. Surviving mouse was
weak after 24 h.
Strong tremor, paralysis. Surviving
animals were weak after 24 h.
3/4
2/4
2/4
2/4
0/4
12.6
4/4
15.9
1/4
20.0
1/4
25.2
0/4
Lacrimation, dyspnoea, tremor, oedema
of the eyelids.
Strong tremor, lacrimation, dyspnoea.
Surviving mouse was weak after 24 h.
Three mice died 3–4 min on application.
The surviving mouse was very weak,
had respiratory disturbance and
closed eyes after 24 h.
Strong tremor immediately on
application. Animals died after
2–3 min of application
REFERENCE
1 Radić, Z., Sit, R. K., Kovarik, Z., Berend, S., Garcia, E., Zhang, L., Amitai, G., Green, C.,
Radić, B., Fokin, V. V. et al. (2012) Refinement of structural leads for centrally acting oxime
reactivators of phosphylated cholinesterases. J. Biol. Chem. 287, 11798–11809
Received 22 October 2012/4 December 2012; accepted 6 December 2012
Published as BJ Immediate Publication 6 December 2012, doi:10.1042/BJ20121612
c The Authors Journal compilation c 2013 Biochemical Society
Tremor 20 min on application.
Surviving animals were weak after
24 h.
Light tremor 3–4 min upon
application. Surviving animals
were weak after 24 h.
Strong tremor immediately on
application. Surviving animals
were weak and apathic after 24 h.
Strong tremor immediately on
application. Surviving animals
were weak and apathic after 24 h.
Cramps and strong tremor
immediately on application.
Surviving animals were very weak
after 24 h.
Cramps and strong tremor
immediately on application.
Animals died between 5 and
60 min after application.