One-pot synthesis of NEMP, a VX surrogate, and reactivation of NeMP-inhibited electrophorus eel acetylcholinesterase by current antidotes

Article abstract of DOI:10.21577/0103-5053.20180246

The use of nerve agents, organophosphorus compounds which irreversibly inhibit acetylcholinesterase, in conflicts and terrorism has gained renewed geopolitical status in recent years. As no “universal antidote” has ever been produced, the development of a

Full text of DOI:10.21577/0103-5053.20180246

http://dx.doi.org/10.21577/0103-5053.20180246
J. Braz. Chem. Soc., Vol. 30, No. 5, 1095-1102, 2019
Short Report
Printed in Brazil - ©2019 Sociedade Brasileira de Química
One-Pot Synthesis of NEMP, a VX Surrogate, and Reactivation of NEMP-Inhibited
Electrophorus Eel Acetylcholinesterase by Current Antidotes
,
a,b
a,c
a
Samir F. A. Cavalcante, * Daniel A. S. Kitagawa, Rafael B. Rodrigues,
a
a
a
Taynara C. Silva, Leandro B. Bernardo, Ana Beatriz A. Correa and
,
b
Alessandro B. C. Simas *
a
Instituto de Defesa Química, Biológica, Radiológica e Nuclear (IDQBRN), Exército Brasileiro,
Avenida das Américas 28705, Área 4, 23020-470 Rio de Janeiro-RJ, Brazil
b
Instituto de Pesquisas de Produtos Naturais Walter Mors (IPPN), Bloco H, HSS-27,
Universidade Federal do Rio de Janeiro (UFRJ), 21941-902 Rio de Janeiro-RJ, Brazil
c
Instituto Militar de Engenharia (IME), Praça General Tibúrcio 80, 22290-270 Rio de Janeiro-RJ, Brazil
The use of nerve agents, organophosphorus compounds which irreversibly inhibit
acetylcholinesterase, in conflicts and terrorism has gained renewed geopolitical status in recent
years. As no “universal antidote” has ever been produced, the development of antidotes to
reactivate enzyme is clearly warranted. Given the fact that nerve agents are restricted substances,
the use of surrogates allows non-qualified laboratories to develop research on novel antidotes.
We herein present an alternative and efficient synthesis of NEMP, O-(4-nitrophenyl) O-ethyl
methylphosphonate, a venomous agent X (VX) surrogate, and Ellman’s test procedures for
inhibition and reactivation of Electrophorus eel acetylcholinesterase involving the NEMP-generated
adduct.
Keywords: nerve agents’ surrogates, VX, Ellman’s test, acetylcholinesterase, drug screening
Introduction
temporary incapacitation or permanent harm to humans or
animals. The related restrictions apply to such chemicals,
regardless of their origin or of their method of production,
and regardless of whether they are produced in facilities,
The period between the Great Wars and later on,
during the Cold War, experienced the development of a
series of organophosphorus compounds initially designed
as pesticides. Due to their high toxicity, they soon were
1
,4
in munitions or elsewhere”.
TheAnnex on Chemicals of CWC classifies compounds
that have “been developed, produced, stockpiled or
used as a chemical weapon”, but CWC allows certified
laboratories to produce small, declarable amounts of
weaponizable substances for research purposes. CWC lists
in its Schedule 1 on the Annex on Chemicals compounds
1-3
deemed as weaponizable compounds.
These compounds are under strict control by
international agreements, being the most important the
Chemical Weapons Convention (CWC), which entered
into force in 1997, and deals with controls and obligations
related to the unlawful use of toxic chemicals, amid other
issues. CWC has led to the creation of Organization
for the Prohibition of Chemical Weapons (OPCW), a
multilateral, international watchdog, overseeing CWC
implementation. Among these toxic chemicals are potent
acetylcholinesterase (AChE) inhibitors, classified as
nerve agents. In the Article II of CWC, toxic chemicals
are referred to as “any chemical which through its
chemical action on life processes can cause death,
1
,4
with little or no peaceful uses. Examples of such
compounds are depicted in Figure 1. Sarin (1), soman (2),
cyclosarin (3), tabun (4) and V-agents (venomous agent X,
4
VX, 5; Russian-VX orVR, 6) are representative examples.
Recently, Novichok agents (A-232, 7), which are also
potent AChE inhibitors, have been confirmed. They pose
a high risk, as they have been created to circumvent CWC,
5
and their toxicity is predicted to be much higher than VX.
The historic and recent use of these compounds in
different conflicts and assassination attempts have attracted
attention of governments, multilateral organizations and
*e-mail: samir.cavalcante@eb.mil.br; abcsimas@nppn.ufrj.br

1
096
One-Pot Synthesis of NEMP, a VX Surrogate, and Reactivation of NEMP-Inhibited Electrophorus Eel
J. Braz. Chem. Soc.
Figure 1. Structures of representative nerve agents.
6
-10
the public. Different research groups have set forth
efforts on the development of novel antidotes, as there
is no “universal antidote” to reactivate inhibited AChE
urination, defecation, gastrointestinal disturbs, emesis,
miosis and muscle spasms), which can be fatal. This is
triggered by overstimulation of cholinergic innervations,
owing to acetylcholine accumulation in the synaptic cleft.
The therapeutic approach to reactivating AChE resorts
to the use of compounds which are able to form nucleophilic
oxygen at physiological conditions. In addition, such active
molecules usually bear a cationic nitrogen atom in order to
mimic the choline moiety, which improves reactivation due
to additional interactions with other enzymatic sites besides
1
1-17
(
EC 3.1.1.7),
a serine-estearase enzyme (8), biological
target of nerve agents. This enzyme is responsible for
the breakdown of acetylcholine (9), the neurotransmitter
in the parasympathetic neurotransmission (Scheme 1),
into acetate (10) and choline (11). Scheme 2 depicts the
formation of sarin-AChE adduct (12), responsible for the
loss of enzymatic function. Broad AChE inhibition may
lead to SLUDGEM syndrome (salivation, lacrimation,
18-27
the catalytic one.
Pyridinium oximes are the only motif
Scheme 1. Breakdown of acetylcholine by AChE.
Scheme 2. Inhibition of acetylcholine by sarin.

Vol. 30, No. 5, 2019
Cavalcante et al.
1097
2
8-30
which has matched these factors.
Figure 2 exemplifies
Nonetheless, non-certified research facilities may
face difficulties in developing novel antidotes given the
impossibility of use of authentic nerve agents, as these
do not comply with requirements to work with these
relevant oximes: pralidoxime (2-PAM, 13), obidoxime
OBD, 14), trimedoxime (TMB, 15), K027 (16), asoxime
HI-6, 17) and HLö-7 (18).
(
(
4
Scheme 3 shows how the oximate (19) generated in vivo
chemicals. In accordance to CWC, facilities that produce
from pralidoxime (13) reacts with phosphonylated enzyme
12), removing the phosphorus moiety from the serine
residue. This reaction yields a phosphonyloxime (20), a
Schedule 1 chemicals must declare their activities and
may be subject of inspection twice a year. To overcome
this limitation, surrogates may be valuable, as they are less
toxic and volatile than the actual nerve agents, but yield
(
31-36
detoxification product, and the reactivated enzyme (8).
Figure 2. Some clinically available antidotes (X may be chloride, bromide, iodide or methanesulfonate).
Scheme 3. Reactivation of sarin-inhibited AChE by pralidoxime.

1
098
One-Pot Synthesis of NEMP, a VX Surrogate, and Reactivation of NEMP-Inhibited Electrophorus Eel
J. Braz. Chem. Soc.
the same enzyme adduct and whose use is not prohibited
according to CWC. For VX, O-(4-nitrophenyl) O-ethyl
methylphosphonate (NEMP, 21, Figure 3) is a known
were purchased from Cambridge Isotopes Laboratories
(Tewksbury, Massachusetts, USA). Obidoxime dichloride
(OBD, 14) and trimedoxime dibromide (TMB, 15) were
4
48,49
surrogate, which has been synthesized by a different
synthesized in accordance to literature and their physical
data were compatible. Purified water was obtained from
Millipore Milli-Q system (18.2 MΩ cm at 25 °C, Millipore
Brazil, São Paulo, Brazil). Biotage Initiator 8 (Charlotte,
North Carolina, USA) was used for synthesis of all oximes.
Sealed tubes (Q-Tube) were purchased from Q-Labtech (East
Lyme, Connecticut, USA). TLC (thin layer chromatography)
aluminum plates coated with silica gel F254 were purchased
from Merck Brazil (São Paulo, Brazil). Camag TLC-MS
3
7
4
approach. In accordance to CWC Annex on Chemicals,
it is classified as Schedule 2.B.04 chemical.
(thin layer chromatography-mass spectrometry) interface
Figure 3. O-(4-Nitrophenyl) O-ethyl methylphosphonate (NEMP).
was used to follow up reactions (AuTeC, São Paulo, Brazil).
Nuclear magnetic resonance (NMR) spectra was obtained
from Varian Unity 400 MHz (Palo Alto, California, USA)
and Bruker Avance 400 MHz (Billerica, Massachusetts,
Recently, we reported the synthesis of NEMP starting
3
8
from triethyl phosphite, in a two-step procedure. We
herein present a one-pot synthesis of this VX surrogate
from O,O-diethyl methylphosphonate, which does not
require purification steps as it yields NEMP in the required
purity for toxicological and biochemical assessments. We
have also undertaken Ellman’s tests for characterization
of inhibition of commercially available Electrophorus eel
AChE (EeAChE) by herein synthesized NEMP and for
1
13
USA) and referred to tetramethylsilane for H and C NMR
31
spectra and to phosphoric acid for P NMR. GC-MS (gas
chromatography-mass spectrometry) data were obtained
from Agilent 6890 GC system (Santa Clara, California,
USA) equipped with 5975C mass spectrometer detector.
LC-MS (liquid chromatography-mass spectrometry) data
were obtained from Agilent 1210 LC system (Santa Clara,
California, USA) equipped with 6410B triple quadrupole
mass spectrometer detector. Kasvi 96-wells microplates
were purchased from Kasvi Brasil (São José dos Pinhais,
PR, Brazil); Gilson single channel pipettes, from Gilson
Inc. (Middleton, Wisconsin, USA) and Eppendorf 8-channel
pipettes, from Eppendorf Brasil (São Paulo, Brazil). Ellman’s
tests were performed in triplicate, in three different assays,
by at least three different operators, measured at 24 ± 2 °C.
All disposable materials and glassware in contact with
organophosphorus compounds were decontaminated with
aqueous solution containing 10% m/v NaOH and 10% m/v
NaClO for 48 h at room temperature before correct
39,40
enzyme reactivation by known antidotes. Ellman’s test
is a spectrophotometry-based assay to measure thiol groups
in different matrices, useful for evaluation of molecules that
41-47
can interact with AChE.
Experimental
Materials and reagents
Oxalyl chloride was purchased from TCI America
Brazil (São Paulo, Brazil). Acetylthiocholine iodide,
5
,5’-dithiobis-(2-nitrobenzoic) acid (DTNB), lyophilized
acetylcholinesterase from Electrophorus eel (EeAChE,
000 U per mg protein, type V-S, C2888), 2-PAM,
destination and cleaning. Estimations of pK and logP for
a
1
clinical antidotes were obtained from ChemAxon Online
Suite. Solubility and partition coefficient KOW (octanol/
water) estimations were obtained from USEPA EPI Suite 4.1.
Agilent Chemstation E.02.02.1431 was used for area
integration of GC data for purity calculations.
N,N-dimethylformamide (DMF, dry, oxygen-free sealed
bottle), dimethyl sulfoxide (DMSO, biological grade, dry,
oxygen-free sealed bottle), triethylamine (dry, oxygen-free
sealed bottle), diethyl methylphosphonate, dichloromethane
(
dried over 4 Å molecular sieves before use), toluene (dried
over 4 Å molecular sieves before use), sodium hydroxide
pellets), anhydrous sodium sulfate, sodium phosphate
50
Preparation of stock and test solutions
(
-
1
monobasic hydrate and sodium phosphate dibasic dihydrate
were purchased from Sigma-Aldrich (São Paulo, Brazil).
Absolute ethanol was purchased from Tedia (Rio de Janeiro,
Brazil). Hydrochloric acid was purchased from Vetec
Phosphate buffer solution (PBS) at 0.12 mol L ,
pH 7.60 ± 0.10, was prepared with 16.46 g of Na HPO .2H O
2
4
2
and 3.71 g of NaH PO .H O in 1 L of purified water.
pH adjustment was done with aqueous HCl or NaOH at
1 mol L . DTNB was diluted at 0.4 mg mL in PBS and
acetylthiocholineiodide, at1mmolL inPBS. Stocksolutions
2
4
2
-1
-1
(
Rio de Janeiro, Brazil). Deuterated solvents (CDCl and
3
-1
DMSO-d ) containing tetramethylsilane as internal standard
6

Vol. 30, No. 5, 2019
Cavalcante et al.
1099
of standard antidotes 2-PAM (13), OBD (14) and TMB (15)
of empty microplate determined. In a clean, 10 mL glass
vial, EeAChE (120 µL, 125 U mL ) was dissolved in
-1
-1
were prepared in DMSO at 10 mmol L and diluted in PBS
-1
-1
to achieve 900 µmol L prior to use (100 µmol L in wells
after all dilutions) to avoid precipitation and vortexed before
use. NEMP test solution was freshly prepared in absolute
6880 µL PBS (enough for 96 wells). Then, the enzyme
-
1
-1
solution (70 µL, 2.14 U mL ), DTNB (80 µL, 0.4 mg mL ),
-1
NEMP solution (1 µmol L , 10 µL, for positive control,
-
1
-1
ethanol at 20 µmol L (1 µmol L in wells after all dilutions).
L (absorbance with inhibitor)) or solvent (10 µL,
i
-1
EeAChE stock solution was prepared at 125 U mL in PBS
0.75 U mL in each well after all dilutions) and stabilized
employed in the inhibitor solution, for negative control,
-1
(
L (absorbance without inhibitor)) and PBS (20 µL) were
0
with 1% bovine serum albumin (BSA). All solutions were
stored at 0 °C prior to use, except for the enzyme solution,
which was stored at –20 °C. This stabilized stored solution
can be used, at least, for six months.
pipetted to each well. After 10 min of incubation, the
acetylthiocholine iodide solution (20 µL) was added and
the absorbance at 412 nm was read at 10, 20 and 30 min
to calculate enzyme inhibition.
Data analysis
Ellman’s test procedure for reactivation of EeAChE inhibited
50
by NEMP
Data are expressed as mean ± standard deviation (SD)
from three different measurements obtained from three
different assays. Microsoft Excel 2007 was used for all
Under the same experimental conditions, to each
-1
well, the enzyme solution (70 µL, 2.14 U mL ), DTNB
®
-1
(80 µL, 0.4 mg mL ), NEMP solution (10 µL, for positive
calculations.
control, absorbance L ) or inhibitor solvent (10 µL, for
i
negative control, absorbance L ) were pipetted, followed
0
One-pot synthesis of NEMP
by incubation for 10 min. Then, the antidote standard
solution (2-PAM (9) or OBD (10) or TMB (11), 20 µL)
was added, followed by incubation for 10 min. At last, the
acetylthiocholine iodide solution (20 µL) was added and the
absorbance at 412 nm was read after 10 min to determine
the enzyme reactivation.
In a round-bottomed reaction flask (dried in oven
at 120 °C for 20 min and cooled down under N flow)
2
equipped with N flow (balloon), 1.0 equiv. of diethyl
2
methylphosphonate (100 mg) and five drops of DMF were
added. This mixture was cooled at 10 °C and oxalyl chloride
(84.6 µL, 1.5 equiv.) was added slowly, with the temperature
Results and Discussion
kept under 40 °C. Then, the solventless reaction mixture
was heated at 50 °C for 2.5 h and, then, was cooled down to
room temperature. The reaction was followed up by GC-MS
One-pot synthesis of NEMP
31
(artifacts as pyrophosphates may be seen) or P NMR (best
The synthetic procedure for NEMP is depicted in
3
7
option). After consumption of the phosphonate, excess of
oxalyl chloride was carefully evaporated and a solution of
dry triethylamine (183 µL, 2.0 equiv.) in toluene (2 mL)
and a solution of 4-nitrophenol (183 mg, 2.0 equiv.) in
toluene (5 mL) were sequentially added. This mixture was
stirred at room temperature for 2 h and, then, diluted with
Scheme 4. In contrast to the literature procedure, we
employed O,O-diethyl methylphosphonate (22) as a starting
material to yield O-ethylmethylphosphonochloridate (23,
Scheme 4), which is less reactive than methylphosphonyl
dichloride, the precursor previously used in the literature.As
we also employed oxalyl chloride, a mild chlorinating agent,
a cleaner reaction and a product of higher purity resulted.
Besides, no solvent was used in the chlorination step. This
methodology, starting from O,O-dialkyl alkylphosphonates,
may also be applicable to the synthesis of other surrogates.
Monochlorination of O,O-diethyl methylphosphonate
(22) with oxalyl chloride and DMF (nucleophilic catalyst),
in a solventless medium, under mild conditions, afforded
O-ethyl methylphosphonochloridate (23, not isolated).
After removal of excess of the chlorinating agent by careful
evaporation, this intermediate reacted with 4-nitrophenol
and triethylamine in toluene, a less toxic solvent, instead
2
0 mL of ethyl acetate and carefully washed with 20 mL
of 2% aqueous sodium bicarbonate. The organic layer
was separated, dried over anhydrous sodium sulfate and
filtered under mild vacuum (washing with 20 mL of ethyl
acetate). Finally, careful evaporation yields crude NEMP as
a yellow oil. As it is a potential AChE inhibitor, it must be
manipulated with precaution. Therefore, it was not purified
37
to avoid increasing toxicity risks.
50
Ellman’s test procedures
37
Ellman’s test procedure for inhibition of EeAChE by NEMP
of benzene, employed in previous reports. The produced
crude NEMP displayed a higher purity compared to the
The instrument temperature was set and the absorbance

1
100
One-Pot Synthesis of NEMP, a VX Surrogate, and Reactivation of NEMP-Inhibited Electrophorus Eel
J. Braz. Chem. Soc.
Scheme 4. One-pot synthesis of NEMP via methylphosphonochloridate (23): (i) oxalyl chloride (1.5 equiv.), DMF (catalytic), 50 °C, 2.5 h; (ii) 4-nitrophenol
2 equiv.), triethylamine (2 equiv.), toluene, 2 h, rt.
(
previously described procedure, being the major impurity
measuring the enzyme activity using Ellman’s test.AsAChE
is conserved across species, we decided to use commercially
available EeAChE. After a 10 min-incubation of EeAChE
with 1 µmol L of NEMP, up to 95% of enzyme inhibition
was attained, confirming the synthesized NEMP as a potent
inhibitor. The inhibition level (%I) was calculated by
1
4
-nitrophenol (by GC-MS and H NMR analyses), similarly
37
to the literature method. Nevertheless, we reckon that the
use of a less reactive starting material, mild chlorinating
agent and less toxic solvent makes this procedure more
appealing. Recently, we had achieved the synthesis of
NEMP from diethyl methylphosphonate obtained via
Michaelis-Arbuzov reaction with triethyl phosphite in a
-1
equation 1, wherein L stands for the absorbance without
0
inhibitor and L , for the absorbance with inhibitor. Results for
i
38
pressure tube (vide supra), which we have explored for
EeAChE inhibition in different times are shown in Table 1.
2
8,29,31,32
syntheses of other surrogates.
Concentration was chosen in accordance to literature,
-5
-9
-1
NEMP was obtained as a yellow oil in higher yield. In its
which ranges from 10 to 10 mol L . It is noteworthy that
the enzyme is not to be fully inhibited, so as to prevent
misinterpretation of reactivation data.
1
H NMR spectrum a doublet can be seen at 1.74 ppm (CH P,
3
2
JHP 17.74 Hz).At 4.23 ppm, a complex pattern related to the
methylene moiety from O-ethyl group is observed. This is
due to the diastereotopic nature of methylene protons at the
ethyl group, as the phosphorus atom is chiral.Additionally,
(
1)
3
two aromatic doublets at 7.38 ppm (2H, J 9.14 Hz) and
HH
3
8
.25 ppm (2H, J 9.15 Hz), related to the 4-nitrophenyl
group, are present. P NMR of this compound shows an
intense signal at 30.35 ppm. Finally, the mass spectrum
HH
3
1
a
Table 1. Enzyme inhibition results (mean ± standard deviation, SD)
Reading time / min
NEMP
displayed the m/z 245, molecular ion peak at t (retention
time) 17.526 min. The major impurity was identified as
R
10
20
30
4
-nitrophenol, by both GC-MS (t = 14.993 min, m/z 139)
Enzyme inhibition
(%I ± SD) / %
R
9
5 ± 2
95 ± 2
94 ± 2
1
and H NMR (aromatic protons at 6.90 and 8.10 ppm).
Purity was accessed either by GC or H NMR. By Agilent
Chemstation GC MS algorithm, we achieved 84% of purity,
while H NMR analysis retrieved 80%.Again, comparison
with the purity achieved by the literature methods (69%)
clearly shows improvement.
1
a
-1
Condition: 10 min-incubation with 1 µmol L of NEMP (O-(4-nitrophenyl)
O-ethyl methylphosphonate).
1
We chose an enzyme source (commercial EeAChE)
different from literature ones, extracted from tissues or
37-39
37
direct analysis in vivo. Therefore, it exhibited different
activity. Nevertheless, based on results described in Table 1,
we can state that the crude NEMP by our route was able to
similarly inhibit the enzyme in high level in comparison
with published data. This proves its usefulness as a tool
for toxicological and biochemical studies.
3
7-39
Similarly to the literature procedure,
we decided
to use crude NEMP, without further purification.
Given its predictable toxic properties, as a pentavalent
organophosphorus compound, we tried to avoid
manipulation risks. Our aim was to obtain a compound
that yields the sameVX-adduct withAChE, but employing
a non-Schedule I CWC chemical, for a more realistic
evaluation of our antidote candidates.
37
Reactivation of NEMP-inhibition EeAChE with standard
antidotes
Inhibition of EeAChE with NEMP
EeAChE reactivation (%R) was calculated by using
the equation 2, wherein L stands for the absorbance after
r
We tested this crude synthetic sample asAChE inhibitor,
addition of reactivator. The results for enzyme reactivation

Vol. 30, No. 5, 2019
Cavalcante et al.
1101
Table 2. Enzyme reactivation (mean ± standard deviation, SD)
a
-1
Antidote
-PAM
rAChE (%R ± SD) / %
pKa
logP
−3.26
−6.93
−7.04
KOW
1.22
2.60
1.95
Water solubility (25 °C) / (mg L )
2
21 ± 1
46 ± 1
7.63
83941
77.105
5031.3
OBD
7.51, 8.11
8.63, 9.24
TMB
39 ± 1
a
-1
All oximes were tested at 100 µmol L . EeAChE was previously inhibited over 10 min by NEMP (O-(4-nitrophenyl) O-ethyl methylphosphonate) at
-
1
1
µmol L . rAChE: AChE reactivation (in percentage); KOW: octanol/water partition coefficient; 2-PAM: pralidoxime iodide; OBD: obidoxime dichloride;
TMB: trimedoxime dibromide.
using standard antidotes 2-PAM (13), OBD (14) and TMB
major impurity detected, 4-nitrophenol) is available free of
charge at http://jbcs.sbq.org.br as PDF file.
-1
(
15) at 100 µmol L , the highest attainable concentration
51,52
-1
in vivo, following inhibition by NEMP (1 µmol L ) over
0 min, are shown in Table 2.
1
Acknowledgments
Authors would like to thank Dr M. Pohanka and Dr K.
Kuča for providing relevant literature and advice; Central
Analítica (IPPN/UFRJ) and Núcleo de Competência para
o Desenvolvimento de Tecnologias de Carbono (NCDTC)
for NMR spectra. S. F. A. C. gratefully acknowledges to
Organisation for the Prohibition of Chemical Weapons
(OPCW) for research grant (L/ICA/ICB/201062/15) and
Brazilian Army for providing infrastructure.
(
2)
The data in Table 2 further confirm the formation of
the VX-adduct via our synthesized NEMP. The observed
reactivation levels, with OBD and TMB surpassing
-PAM, are consistent with the published data. The
lower OBD pK , combined with additional interactions
3
7
2
a
by the second cationic moiety, possibly justifies its higher
activity. The other physicochemical data (logP, KOW and
water solubility) relate to the ease of hematoencephalic
barrier crossing, a relevant issue in the pharmacokinetics
of these drugs.
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Conclusions
We herein report a straightforward, one-pot synthesis
of NEMP, O-(4-nitrophenyl) O-ethyl methylphosphonate,
VX surrogate. The obtained crude material was shown
to successfully inhibit EeAChE. Assays of reactivation
of NEMP-inhibited EeAChE by three known antidotes
were undertaken. For such experiments, we followed a
set of guidelines which we had previously disclosed for
the involved Ellman’s tests, which enables a safer first
approach to the technique. We hope, by presenting these
methodologies, to encourage other groups to perform
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One-Pot Synthesis of NEMP, a VX Surrogate, and Reactivation of NEMP-Inhibited Electrophorus Eel
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