Reactivity and mechanism of α-nucleophile scaffolds as catalytic organophosphate scavengers

Article abstract of DOI:10.1039/c9ob00503j

Despite their unique benefits imparted by their structure and reactivity, certain α-nucleophile molecules remain underexplored as chemical inactivators for the topical decontamination of reactive organophosphates (OPs). Here, we present a library of thirty α-nucleophile scaffolds, each designed with either a pyridinium aldoxime (PAM) or hydroxamic acid (HA) α-nucleophile core tethered to a polar or charged scaffold for optimized physicochemical properties and reactivity. These library compounds were screened for their abilities to catalyze the hydrolysis of a model OP, paraoxon (POX), in kinetic assays. These screening experiments led to the identification of multiple lead compounds with the ability to inactivate POX two- to four-times more rapidly than Dekon 139 - the active ingredient currently used for skin decontamination of OPs. Our mechanistic studies, performed under variable pH and temperature conditions suggested that the differences in the reactivity and activation energy of these compounds are fundamentally attributable to the core nucleophilicity and pK_a. Following their screening and mechanistic studies, select lead compounds were further evaluated and demonstrated greater efficacy than Dekon 139 in the topical decontamination of POX in an ex vivo porcine skin model. In addition to OP reactivity, several compounds in the PAM class displayed a dual mode of activity, as they retained the ability to reactivate POX-inhibited acetylcholine esterase (AChE). In summary, this report describes a rationale for the hydrophilic scaffold design of α-nucleophiles, and it offers advanced insights into their chemical reactivity, mechanism, and practical utility as OP decontaminants.

Full text of DOI:10.1039/c9ob00503j

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DOI: 10.1039/C9OB00503J
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Reactivity and mechanism of -nucleophile scaffolds as catalytic
organophosphate scavengers
Pamela T. Wong,§a,b Somnath Bhattacharjee,§a,b Jayme Cannon,a,b Shengzhuang Tang,a,b Kelly Yang,b Sierra Bowden,b Victoria
Received 00th January 20xx,
Accepted 00th January 20xx
Varnau,b Jessica J. O′Koneka,b and Seok Ki Choi*a,b
DOI: 10.1039/x0xx00000x
Despite their unique benefits imparted by their structure and reactivity, certain -nucleophile molecules remain
underexplored as chemical inactivators for the topical decontamination of reactive organophosphates (OPs). Here, we
present a library of thirty -nucleophile scaffolds, each designed with either a pyridinium aldoxime (PAM) or hydroxamic
acid (HA) -nucleophile core tethered to a polar or charged scaffold for optimized physicochemical properties and
reactivity. These library compounds were screened for their abilities to catalyze the hydrolysis of a model OP, paraoxon
(POX), in kinetic assays. These screening experiments led to the identification of multiple lead compounds with the ability
to inactivate POX two- to four-times more rapidly than Dekon 139—the active ingredient currently used for skin
decontamination of OPs. Our mechanistic studies, performed under variable pH and temperature conditions suggested
that the differences in the reactivity and activation energy of these compounds are fundamentally attributable to the core
nucleophilicity and pKa. Following their screening and mechanistic studies, select lead compounds were further evaluated
and demonstrated greater efficacy than Dekon 139 in the topical decontamination of POX in an ex vivo porcine skin model.
In addition to OP reactivity, several compounds in the PAM class displayed a dual mode of activity, as they retained the
ability to reactivate POX-inhibited acetylcholine esterase (AChE). In summary, this report describes a rationale for the
hydrophilic scaffold design of -nucleophiles, and it offers advanced insights into their chemical reactivity, mechanism,
and practical utility as OP decontaminants.
www.rsc.org/
central nervous system (CNS) bioavailability,3, 4 enzyme-based
OP bioscavengers for systemic use,5-7 and small molecule-
based OP scavengers for topical decontamination.8-10 Here, we
Introduction
Phosphate-based chemical threat agents, collectively referred
to as reactive organophosphates (OPs), cause serious life-
threatening neurotoxicity upon human exposure.1, 2 These OPs
comprise a broad range of phosphoester compounds ranging
from the agricultural pesticides paraoxon (POX), malaoxon and
omethoate to the nerve agents sarin and VX, exploited in
present a new approach for the design and validation of small
molecule scavengers based on -nucleophiles. While -
nucleophiles have demonstrated their therapeutic values as
OP antidotes over several decades, their potential role as OP
scavengers has remained relatively underexplored until
recently.8-11 This article addresses such a significant gap in our
current knowledge on the -nucleophile scavengers by
establishing their structure-reactivity trends, determining their
molecular and kinetic mechanism of OP inactivation, and
evaluating their efficacy in OP decontamination in a skin
model.
chemical terrorism (Figure 1).1, Despite continuing efforts
2
over the past several decades, no single therapeutic agent or
regimen has been proven to be fully effective in the treatment
of OP intoxication.1 Increasing threats of OP exposure
underscores an urgent need for more effective therapeutic
modalities, in particular, by developing OP antidotes with
The biochemical basis of OP neurotoxicity relates to its
specific reactivity with acetylcholinesterase (AChE). As the
target enzyme, AChE is covalently inhibited by OP through O-
phosphorylation at serine-203, the catalytic residue in the
active site pocket.12 This enzyme inhibition leads to the rapid
accumulation of acetylcholine at the synaptic cleft which
results in the symptomatic overstimulation of cholinergic
receptors.1, 13 Current regimens approved for OP intoxication
rely on pralidoxime chloride (2-PAM), a primary antidote
a. Department of Internal Medicine, University of Michigan Medical School, Ann
Arbor, Michigan 48109, United States of America.
b. Michigan Nanotechnology Institute for Medicine and Biological Sciences,
University of Michigan Medical School, Ann Arbor, Michigan 48109, United States
of America.
§ Equal contributions
* E-mail: skchoi@umich.edu
†Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
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Journal Name
its essential role in the treatment of OP intoxication, 2-PAM is
View Article Online
DOI: 10.1039/C9OB00503J
ineffective for preventing neuronal damage due to its lack of
(A)
NO2
CNS bioavailability,3, 4 and due to its short half-life (≤ 2 h) in
plasma.14
O
O
P
O
P
P
N
F
S
O
O
O
O
O
Paraoxon (POX)
Sarin
VX
In addition to systemic antidote administration, topical
decontamination of OPs is essential to effective treatment, as
the exposed surface serves as a depot for extended OP
absorption, and thus promotes sustained toxicity.15 Various
decontamination kits,16 materials,11, 17-20 and methods21 have
been developed that include the FDA-approved reactive skin
decontamination lotion (RSDL) for skin decontamination.22 The
active ingredient in RSDL is Dekon 139 (formulated at 1.2 M,
pH ≥10.8), a potassium salt of diacetyl monooxime (DAM),
which displays strong catalytic reactivity towards OP, leading
to its inactivation through hydrolysis (Figure 2). However,
despite such beneficial reactivity which is attributable to its -
nucleophilic oxime moiety, Dekon 139 is rapidly absorbed
percutaneously as its neutral form, DAM, and can lead to
adverse systemic effects such as skeletal muscle paralysis.23
Here, we wish to advance our fundamental knowledge in
the design and optimization of chemical scavengers for OP
inactivation.8-10, 24 Specifically, we are interested in advancing
the chemical strategy for scavenger design based on two well-
known -nucleophile cores, with the goal of improving their
reactivity while avoiding the high membrane permeability
associated with lipophilic DAM. It involves designing a library
of hydrophilic scaffolds of -nucleophile molecules in which
each -nucleophile core is conjugated to a polar or charged
scaffold that acts as a physicochemical modulator.9 First, we
O
P
O
P
CO2Et
CO2Et
H
N
O
S
O
S
O
O
O
Malaoxon
Omethoate
(B)
Enz Inactivated
OP
O
P
Ser203-OH
AChE
R'
R
AChE
Ser
O
R, R' = Methyl; Alkoxy
2-PAM
Cl-
N
OH
N
2-PAM
2-PAM
8.7 A
Enz Reactivation
O
Oxime
P
OEt
AChE
Ser
O
OEt
hAChE-POX
Fig. 1 (A) Representative reactive organophosphates (OPs). (B) Reactivation of OP-
inactivated acetylcholine esterase (AChE) by pyridine-2-aldoxime methochloride (2-
PAM). Inset: an X-ray crystal structure of POX-inactivated human acetylcholine esterase
(hAChE) in complex with 2-PAM (protein data bank (PDB) code 5HFA12).
(A)
-OH
O
O
verify the mechanistic aspects of these compounds for their
Chemical Inactivation
26
reactivity as OP inactivators.25,
Second, we present their
O
P
O
P
O
P
N
N
L
R'
R
O-
R
R
OH
R'
O
chemical reactivity towards the hydrolysis of POX employed as
a model OP measured under pH and temperature-controlled
conditions in a high-throughput (HT) colorimetric screening
assay, and identify multiple lead compounds based on their
rates of POX inactivation. Finally, we evaluate the therapeutic
potential of lead compounds for POX decontamination on
porcine skin in vitro. In summary, we present a rational design
strategy that resulted in multiple -nucleophile scaffolds that
display potent reactivity and practical utility as small molecule
OP scavengers.
R'
L (leaving group)
K+
Oxime
(catalytic)
harmless
Dekon 139
POX Inactivated
OP
-Nucleophile

(B)
O
O
pKa 9.3
N
N
OH
O-

Dekon 139
(Oximate)
DAM (oxime)
pKa 7.8

N
N
O-
N
pH 10.5
pH 7.4
N
OH
Results and discussion
Oximate
O
2-PAM (oxime)
pKa 9.3
O
Design concept of -nucleophile scaffolds

O-
OH
N
N
H
H
pKa >11
We designed thirty -nucleophile compounds of two classes,
each containing two functionally orthogonal elements in their
structure (Table 1 and 2): (i) a reactive -nucleophile core and
(ii) a covalently linked scaffold (R). The core is the reactive
element that engages in the nucleophilic displacement
Hydroxamic acid
(HA)
Hydroxamate
Fig. 2 -Nucleophilic scavengers of organophosphate (OP). (A) Chemical inactivation of
OP by Dekon 139 through a nucleophilic catalytic mechanism. (B) Structures and
protonation states of the three -nucleophiles investigated in this study.
reaction with the phosphoesters for hydrolysis. Of various
27
types of nucleophiles,26,
we selected two -nucleophiles,
known for its ability to reactivate OP-inhibited AChE (Figure 1),
in combination with one or two complementary drugs,1,
which include atropine (a cholinergic receptor antagonist) and
an anticonvulsant for epileptic seizure relief. However, despite
pyridinium aldoxime (PAM)8, 25, 27 and hydroxamic acid (HA),26,
2
28, 29
as the reactive cores of our compounds because of their
relatively strong reactivity due to the -heteroatom adjacent
to the nucleophilic atom (Figure 2).26 This current library
2 | J. Name., 2018, 00, 1-12
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ARTICLE
includes a few compounds, which were previously reported as types, the structure and functionality of the scaVffieowldArtdicloemOnalinine
30, 31
enzyme reactivators,8,
however, their potential as OP had more variations as shown in Tables 1 and 2 including in
DOI: 10.1039/C9OB00503J
inactivators remain unknown.
the spacer length (one to three atoms) and composition
Our design efforts in the PAM core were made with a focus (carbon, nitrogen and/or oxygen). They were then additionally
on three subcores with differences in their orientation such substituted with a polar group for increased hydrophilicity,
that their oxime acidity varies due to electronic effects (pKa: 2- such as with a hydroxyl group for the HA class compounds.
PAM < 4-PAM < 3-PAM). We also included a set of halogen Spacer groups that were too lengthy, bulky or heavily
substituted 4-PAM compounds 8–12, each with an substituted were not considered because of their unfavourable
electronegative Cl or F at the C-3 position. We hypothesized steric congestion to the reaction center.
that its high electronegativity in combination with its proximity
Tables 1 and 2 provide summaries of the calculated
to the oxime moiety would be able to reduce the pKa of the physicochemical properties of the core-scaffold conjugates.
oxime. Thus, the pKa values calculated for these compounds First, each library molecule in the PAM class is larger than DAM
are lowered (7.9–8.0) by approximately 0.3–0.4 units (Mr = 101 g mol−1) in size as suggested by their molecular mass
compared to that of the parent 4-PAM (pKa = 8.3) compound.
(Mr) and calculated molar refractivity (CMR). In addition, each
compound has a negative value of clogP (the partition
coefficient between water and 1-octanol34) and has more than
Rationale for hydrophilic scaffolds
Absorption of molecules across the skin layer is known to five H-bond donors/acceptors, supportive of increased
occur primarily through passive diffusion across hydrophobic hydrophilicity compared to DAM (clogP = 0.332; four H-bond
cellular membranes.32 As an approach to prevent potential donors/acceptors).32, 33 Most compounds in the HA class also
percutaneous absorption associated with small sized or show such improved properties that are considered to help
lipophilic -nucleophile molecules,33 we conjugated each - reduce the permeability of a molecule across the skin layer.33
nucleophile core to a scaffold residue (R) consisting of a spacer Second, the core acidity for both classes as predicted by the
terminated with a polar or charged handle such as a carboxylic
acid, alcohol or primary amine. As compared to the two core
Table 1 Library of pyridinium aldoxime (PAM) scaffold compounds
3-PAM
Non-pyridinium
(neutral)
2-PAM
4-PAM
N
2
OH
3
pKa 10.8
OH
4
OH
N
OH
N
N
N
HO
OH
N
pKa 7.75
pKa 8.3
pKa 9.2
N
N
13
14
Oxime Core
OH
Scaffold (R)
HO2C
HO
OH
CO2H
OH
N
Orientation:
2-, 3-, and 4-PAM
N
Handle Spacer
X
X = Cl, F
Structure
a (oxime)a,b
clogPa
CMRa
pK
Compound Number
Oxime Core (X)
Scaffold (R)
Dekon 139
2-PAM
DAM
2-PAM
2-PAM
3-PAM
3-PAM
3-PAM
4-PAM
4-PAM
4-PAM
-
9.32
7.75
8.0
9.2
9.2
9.2
8.3
8.3
8.3
7.9
7.9
7.9
8.0
8.0
10.8
10.6
0.332 (−0.114)b
−3.666
−6.783
−6.783
−6.287
−3.509
−6.783
−6.287
−3.509
−6.721
−6.226
−3.447
−7.041
−6.546
1.830
2.717
4.142
4.795
4.795
5.259
4.759
4.795
5.259
4.759
5.286
5.75
5.251
4.810
5.274
4.107
4.607
H3C-
1
2
3
4
5
6
7
8
9
10
11
12
13
14
HO2CCH2-
HO2CCH2-
HO2C(CH2)2-
HO(CH2)2-
HO2CCH2-
HO2C(CH2)2-
HO(CH2)2-
HO2CCH2-
HO2C(CH2)2-
HO(CH2)2-
HO2CCH2-
HO2C(CH2)2-
HO-
4-PAM (3-Cl)
4-PAM (3-Cl)
4-PAM (3-Cl)
4-PAM (3-F)
4-PAM (3-F)
Non-pyridinium
Non-pyridinium
HO2C-
−3.159
Abbreviations: pKa = −log(Ka). P = partition coefficient between water and 1-octanol; calculated clogP = log([oxime]1-octanol/[oxime]water). CMR = calculated molar
refractivity (m3 mol−1). Physicochemical parameters calculated by ChemDraw Software (Professional 16.0)a or Advanced Chemistry Development (ACD/Labs) Software
V11.02 (©1994-2017).b
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Table 2 Library of hydroxamic acid (HA) scaffold compounds Library of pyridinium aldoxime (PAM) scaffold compounds
View Article Online
DOI: 10.1039/C9OB00503J
O
OH
NH
Hydroxamic acid (HA)
Core
Scaffold (R)
O
N
Handle Spacer
pKa = 9.3
NH2
CO2H
OH
OH
Handle Spacer
N
NH
OH
H
O
Monomeric HA
Dimeric HA
Structure
Compound
Number
a (HA)a,b
clogPa
CMRa
pK
Scaffold (R)
15
16
17
HO2CCH2-
HO2C(CH2)2-
HO2C(CH2)3-
8.4
9.1
9.3
−1.524 (−0.811)b
−1.562 (−0.711)b
−1.245
2.315
2.779
3.243
(−0.436)a
−3.871
18
19
20
21
22
23
24
25
26
27
28
29
30
HO2CCH2NHCH2-
HO2CCH2N(CH3)CH2-
HO2CCH2OCH2-
HO2CCH2C(CH3)2CH2-
HOCH2-
H2N(CH2)2-
HN(CH3)CH2-
HO(CH2)3-
H2NCH(CH2OH)-
H2NCH(CH2CO2H)-
6.8
6.8
8.9
9.3
9.5
8.6
10.1
9.3
9.0
9.0
8.8
8.8
8.8
3.148
3.611
2.932
4.170
1.816
2.495
2.495
2.743
2.648
3.516
5.560
5.525
6.024
−3.115
−1.562
−0.447
−1.855
−1.909
−1.857
−1.701
−2.928
−3.542
−3.580
−2.12
−4.305
Dimer
Dimer
Dimer
HO2CCH2-
HO(CH2)2-
HO2C(CH2)2-
For abbreviations, see Table 1. Physicochemical parameters calculated by ChemDraw Software (Professional 16.0)a or Advanced Chemistry Development (ACD/Labs)
Software V11.02 (©1994-2017)b
pKa value shows variations depending on the core type,
The synthesis of scaffold compounds 15–30 in the HA class
substitution group and scaffold moiety. In general, the PAM was performed conveniently in a one-step process through the
core family of compounds have a lower range of pKa’s (7.9– hydroxyamidation reaction of an anhydride or ester molecule
9.2) than the HA core family compounds (8.4–9.3). However, with hydroxylamine in methanol (details in the SI). Their
most compounds in the library showed lower pKa values than reactants were selected from cyclic anhydrides (e.g., glutaric,
DAM (9.32) with the exception of only a few compounds in the succinic), /-amino acid esters (N-methyl glycine, -alanine,
HA class.
L-serine methyl ester) and linear monoesters terminated with
a carboxylic acid or hydroxyl group at the other end. Dimeric
scaffold compounds 28–30 were prepared by applying the
Synthesis of -nucleophile scaffolds
As methods for PAM compound synthesis, we used the same method from the corresponding dimeric esters.
quaternary N-alkylation of pyridine aldoxime, the approach
Each compound in both classes was fully characterized for
its identity and homogeneity (≥95%) using standard analytical
30, 31
well established for preparing enzyme reactivators.8,
Compounds 1–7 in the PAM series were prepared by the N- methods that included 1H and 13C NMR spectroscopy, mass
alkylation of pyridine-n-aldoxime (n = 2, 3, or 4) with a spectrometry, and ultra-performance liquid chromatography
requisite halogen (Br, I)-terminated alkane scaffold such as (UPLC) as detailed in the SI. Compound stability was tested for
iodoacetic acid, 2-bromoethanol and 3-bromopropionic acid as selected compounds in phosphate buffered saline (PBS) at pH
detailed in the Supporting Information (SI). PAM compounds 10.5, the condition that is used in the formulation of Dekon
8–12, each with a chlorine or fluorine substitution at the C-3 139 in RSDL (Figure S2). UPLC analysis indicated no evidence of
position, were prepared similarly except for the use of 3-chloro compound degradation at pH 10.5 after incubation for at least
or 3-fluoropyridine aldoxime. Two non-pyridinium oxime three days at ambient temperature.
compounds 13, 14, which show lower oxime acidity than the
PAM compounds, were prepared as controls from their
Nucleophilic catalysis of POX hydrolysis
corresponding aldehydes by treatment with hydroxylamine.
Figure 3 shows proposed mechanisms of OP inactivation
applied for POX with Dekon 139.9, 25, 26, 35 The OP inactivation
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O
O
View Article Online
O
O
P
OEt
OEt
DOI: 10.1039/C9OB0O0E5t03J
PAM/HA (8.0 mM)
P
+
O
O
O
OH
OEt
S
Oximate (more
reactive)
Dekon
O
PBS, pD 10.5
O
HS
Rate Determining Step
O
23 o
C
N
O- K+
O
Malaoxon inactivated
[Malaoxon] = 2.5 mM
O
139
NO2
O
P
O
P
HOD
k
1 (slower)
N
POX
O
OEt
EtO
@ 2 h
O
OEt
EtO
4-Nitrophenol
(abs @ 400 nm)
Oxime-OP Adduct
(POX inactivated)
Malaoxon in D2O (ref)
DSS
(pKa 9.3)
N OH
DAM
Oxime
(less reactive)
O
PBS alone, pD 10.5
O
45%
Hydrolysis and Degradation of Oxime-OP Adduct
Dekon 139
N
, k '
(k1 << k2
)
2
OH
O
P
-OH
k2
(faster)
EtO
O-
+
*
*
DAM
(catalytic)
a
*
*
25%
*
*
*
HO
N
*
-OH
PAM 7
N
O
*
*
O
EtO
OH
*
Diethyl phosphate
a
O
P
62%
N
* *
*
O
b
O
OEt
OH
N
17
HA
HO
OEt
H
*
*
-O
O-Ac DAM
O-
N
O
O
k2'
(faster)
b
O
P
O
O
Fig. 4 1H NMR spectral traces of the hydrolysis of malaoxon (2.5 mM) in a deuterated
phosphate buffered saline (dPBS, pD 10.5) alone, or catalysed by Dekon 139 (8.0 mM),
7 (8.0 mM), and 17 (8.0 mM), each in pD 10.5 at 23 °C after 2 h. The fraction (%) of
malaoxon hydrolysed was estimated by the integration of its signals for OMe groups.
N
N
OAc
N
MeCN
Diethyl phosphate
O
OEt
OEt
Fig. 3 Kinetics and mechanism of paraoxon (POX) inactivation by Dekon 139. The rate is
determined by the nucleophilic attack at the first step (rate constant k1) which occurs
much more slowly than the subsequent hydrolysis of an oxime-POX adduct (k2)9 and/or
the potential degradation via Beckmann fragmentation (k2′).35
compounds 7 and 17 in POX hydrolysis (path a) rather than
stoichiometric consumption (path b).
In addition to POX, we performed similar 1H NMR
experiments using two other OP surrogates, malaoxon and
omethoate, each having a P-S bond like VX. As shown in the
overlaid spectra (Figure 4, Figure S3E), compounds 7 and 17
showed evidence for their catalytic reactivity. For example,
malaoxon remained intact in PBS (pD 10.5) for 2 h (up to 24 h;
not shown). However, it was inactivated rapidly by 7 or 17,
each displaying t1/2 (calculated from decay curves) of 2.7 h and
0.6 h, respectively, which is comparable to or better than
Dekon 139 (2.9 h).
occurs in two steps: (i) a slower nucleophilic attack that results
in an OP-oxime (DAM) adduct; (ii) the faster degradation of the
adduct. Thus, the overall rate of OP inactivation shows
dependency on rate constant (k1) in the first step. Compared
to the first step that involves a single mechanism, the second
step could involve two mechanisms as shown in Figure 3
(lower). These include catalytic hydrolysis9 or stoichiometric
35
degradation via Beckmann fragmentation,24,
each
contributing to a variable extent under certain reaction
conditions.
1
In summary, H NMR experiments performed with three
To verify the applicability of such mechanisms to the
current library compounds, we performed 1H NMR
experiments for two representative compounds, 7 and 17, by
reacting POX (2.5 mM) with either compound added at 8.0
mM in deuterated phosphate buffered saline (dPBS, pD 10.5).
Overlaid 1H NMR spectra showed the time-dependent
decrease of POX signals as reflected by the appearance and
increase of new peaks corresponding to 4-nitrophenol (4-NP)
and diethyl phosphate (Figure S3). No POX was detectable
after 24 h of inactivation by compounds 7 and 17, indicating
complete POX inactivation with half-life (t1/2) than Dekon 139,
which still had ~50% intact POX remaining after the same
period. As a control, POX alone (pH 10.5) remained fully stable
over 72 h.
The NMR spectral traces of these compounds show
evidence for catalytic POX hydrolysis because signals
corresponding to 7 or 17 remained largely unchanged in each
spectral set. Certain degradation products such as MeCN or O-
Ac DAM were not detectable that might result from a second-
order Beckmann fragmentation as suggested earlier for Dekon
13924, 35 under non-aqueous conditions (path b; Figure 3). This
lack of spectral changes is supportive of a catalytic function for
OP surrogates (POX, malaoxon, and omethoate) provide new
evidence for the mechanism of OP inactivation by 7 and 17 as
nucleophilic catalysts. This result strongly supports their broad
reactivity for catalytic cleavage of both P-O and P-S bonds.
Reaction kinetics by colorimetric assay
Performing a colorimetric assay enables to measure the
kinetics of POX inactivation due to the release of 4-NP
(absorbance at 400 nm) upon its hydrolysis, as illustrated in
the UV–Vis spectral traces (Figure S4). It also has amenability
to a microplate reader, which offers high-throughput (HT)
capabilities for compound screening.8, 9
Following this HT method, we monitored POX hydrolysis at
37 °C by adding a fixed concentration of POX (30 M) to a test
compound formulated at four different concentrations (0.5–
8.0 mM). The large molar excess of the test compound allows
the determination of the rate constant k1 under pseudo first-
order conditions. Compound screening was performed at pH
7.4 and 10.5 in order to compare the reactivity of each test
compound in the neutral oxime or hydroxamic acid form
(≥95%, pH 7.4) or in the negatively charged oximate or
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(B)
(C)
(A)
16
POX + Dekon
] =
POX +
5
POX +
(A)
View Article Online
DOI: 10.1039/C9OB00503J
pH 10.5
[
[5] =
[16] =
Dekon
k1 = slope (M-1 min-1
)
(E)
(D)
k1 = 0.45
(±0.02)
Compound Number
k1 = 0.44
(±0.11)
(B)
k1 = 0.22 (±0.08)
HA (dimer)
HA (monomer)
4-PAM
3-PAM
2-PAM
Fig. 5 (A–C) Kinetic traces of paraoxon (POX, 30 M) hydrolysis catalysed by Dekon 139,
5 and 16 in PBS, pH 10.5 at 37 °C. (D, E) kobsd and POX half-life t1/2 (= log(2)/kobsd) plotted
as a function of compound concentration. k1 refers to a bimolecular rate constant
obtained from linear regression analysis. Data points are mean ± standard deviation
(SD) (n ≥ 3).
* * *
Compound Number
hydroxamate form (≥95%, pH 10.5) as predicted by the
Henderson-Hasselbalch equation.36
Fig. 6 Rate constants for POX hydrolysis catalysed by library compounds 1–30. (A) kobsd
(min−1) measured for 30 M POX with various concentrations of test compounds (0.5–
4.0 mM, pH 10.5) at 37 (±1) °C. kobsd values at 4.0 mM of the test compound are
indicated in purple. (B) Plots of k1 (M−1 min−1) at pH 10.5 and 7.4. * Not determined due
to compound instability or lack of activity.
Figure
5 shows reaction kinetics of POX hydrolysis
performed at pH 10.5 as illustrated with Dekon 139 and two
representative compounds, 5 and 16. Growth curves in Figure
5A–5C suggest 4-NP production as a function of time and
compound concentrations. Regression analysis of each curve
provided an observed rate constant kobsd (min−1) as the slope of
absorbance (A) growth (A/t) at each concentration, and
calculated the POX half-life (t1/2 = log(2)/kobsd). The plots of
kobsd and t1/2 values presented in Figures 5D and 5E show that
Dekon 139 is less effective for POX inactivation than 5 and 16
given its lower kobsd values and accordingly, longer half-life
values. As defined in Figure 3, a bimolecular rate constant k1
pH conditions. This primary screening led to the identification
of 14 lead compounds in both classes which include 2–8
(PAM), and 16, 17, 21, 23, 25, 29, 30 (HA). Each of these shows
reactivity (k1) ~2 to 4 times greater than Dekon 139 at pH 10.5.
Use of Cl or F-substituted 4-PAM resulted in reactivity not
as effective as would have been anticipated based upon their
lowered pKa. Except 8, these compounds 10–12 displayed
undesirable reaction properties such as non-catalytic
consumption and/or partial degradation even at pH 7.4 as
evident by 1H NMR and mass spectrometry analyses (not
shown). As controls, two non-pyridinium compounds, 13 and
14, showed either a non-detectable or a very low level of
reactivity, emphasizing the essential role played by the core.
The results also show pH-dependent variations in
reactivity.8, 9 At pH 10.5, POX hydrolysis was faster by up to an
order of magnitude as compared to at pH 7.4. This result
confirms our general postulation that the greater reactivity
relates to the oximate or hydroxamate anions, which are the
predominant species (>95%) at pH 10.5 (Figure 2), compared
to the neutral species which predominates at pH 7.4.
was obtained from kobsd-concentration plots under pseudo-
25
first order conditions.8,
Comparison of individual k1 (M−1
min−1) values indicates approximately 2-fold lower reactivity of
Dekon 139 (0.22) compared to 5 (0.45) and 16 (0.44) which
display almost equal reactivity.
In summary, the colorimetric assay illustrated with these
representative library compounds shows that the rate of POX
inactivation is dependent on both compound reactivity and
the test concentration of the compound, which is inversely
correlated with POX half-life. Each linear regression plot for a
test compound (Figure 5E) serves as an important tool for us
to predict an optimal scavenger concentration for faster POX
inactivation, such as 0.3 M to reach a target half-life of ≤10
min, as applied in the topical decontamination of POX as
described later.
Structure-reactivity correlation
We observed certain structure-reactivity trends in the core
and scaffold domain. First, core type appears to account for
generic differences in reactivity observed between the two -
nucleophile classes. At pH 7.4, rate constants are broadly
higher by PAM compounds (Figure 6B), which display lower
pKa values (pKa = 7.75–9.2; Table 1) and thus higher fractions
(Fr) of the oximate anion. Such core-dependent differences
Screening of library compounds
Under the same microplate condition, we performed screening
of all compounds 1–30 for their reactivity of POX hydrolysis.
The rate constants (kobsd, k1) summarized in Figure 6 show
significant variations dependent on -nucleophile types and
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(A)
(B)
disappear at pH 10.5 in which each oxime or HA core is
deprotonatable predominantly to its anionic species
([Oximate/Hydroxamate]/[Oxime/HA] = e[pH – pKa]).36
View Article Online
DOI: 10.1039/C9OB00503J
r = -0.41 (PAM)
r = -0.37 (HA)
r = -0.38 (PAM)
r = -0.34 (HA)
In the PAM class, the core orientation plays a fundamental
role in determining reactivity. Greater reactivity is with the
compounds 6–8 in the 4-PAM subclass than in the 3-PAM or 2-
PAM subclass. Our result is in line with an earlier report which
predicts higher Brønsted-type nucleophilicity for 4-PAM
relative to 2-PAM.27 In addition to this core nucleophilicity and
its associated acidity (pKa), we attribute such greater reactivity
to the lower steric congestion of the 4-PAM core in its
nucleophilic attack on the phosphoester center.
pKa (PAM/HA acidity)
pKa (PAM/HA acidity)
(D)
(C)
Lastly, the scaffold structure most frequently found in lead
compounds from the PAM class is composed of a two-carbon
spacer terminated with a hydroxyl or carboxylic acid group.
However, it appears to have less of an impact on reactivity
than the orientation of the PAM core. Similarly, good scaffolds
that were found in the HA class are composed of a two or
three-carbon spacer. In contrast, scaffolds which are based on
a single carbon spacer were less reactive than those with
longer carbon spacers such as 15 (1 CH2) compared to 16 (2
CH2) and 17 (3 CH2). It is also notable that scaffolds with a
single carbon spacer terminated with an amine or hydroxyl
group such as 18, 19, 22, and 27 were much less reactive than
those with longer carbon spacers, pointing to unfavourable
electronic and steric effects.
CO2H
O
OH
N
N
N
OH
pH
pH
(F)
(E)
CO2
H
N
O
N
HO2
C
NHOH
OH
pH
pH
Fig. 7 Effect of pKa and pH on reactivity. (A, B) Plots of reactivity versus core acidity
(pKa) at pH 10.5 and pH 7.4. (C–F) Plots of k1 (37 °C) and the fraction (Fr) of
deprotonation36 as a function of medium pH for representative compounds. Relative
Role of physicochemical parameters
In our library design, we varied three physicochemical
properties for two independent purposes: i) core acidity (pKa)
as a primary determinant for enhancing OP reactivity; ii)
scaffold hydrophilicity and size, each as a physicochemical
modulator for addressing percutaneous penetration.33 We
determined the extent of the correlation of these properties
with reactivity as presented in Figure 7A, 7B (core acidity) and
Figure S5 (hydrophilicity, size).
First, higher core acidity led to greater reactivity for the
majority of compounds (upper left quadrant). The correlation
analysis of these plots suggests a positive (inverse) correlation
between core acidity (pKa) and reactivity despite relatively low
values for the coefficient (r). Thus, these analyses suggest the
significant role of core acidity in dictating reactivity. Second,
plotting of reactivity versus physicochemical modulators,
cLogP (a predictor for hydrophilicity) and CMR (size), showed a
weak but positive correlation (Figure S5). It suggests that the
optimization of physicochemical properties does not make a
negative impact on the reactivity of the library compounds.
reactivity refers to the ratio of k1(test compound)/k1(Dekon 139, pH 10.5).
r =
correlation coefficient (Pearson)
Figure S6. Each plot shows the rate of POX hydrolysis (k1) as a
function of medium pH (7.4–10.5) along with the predicted
fraction of oximate or hydroxamate species present
(deprotonation fraction = e[pH – pKa]36). Each plots also shows a
fitting curve (blue line) generated to model the reactivity-pH
relationship based on a linear combination of two parallel
catalytic pathways for POX hydrolysis—one through a neutral
species, and the other through an anionic species (Figure 3).
Each compound shows maximal reactivity at pH 10.5 as
anticipated by the greater fraction of oximate or hydroxamate
species (fraction ≥ 0.95; red dotted line). The reactivity
decreases as the fraction of anionic species decreases at the
lower pH. These pH models also show that core pKa does not
solely account for the pH-dependent variation in reactivity
among lead compounds. This is evident by the two orientation
isomers 1 (Figure 7D) and 5 (Figure 7E) in the PAM class.
Despite having identical scaffolds, compound 1 (pKa = 8.0)
shows lower reactivity than 5 (pKa = 8.3) over the entire range
of pH conditions. Thus, these pH models provide evidence that
corroborates the effect of the oxime orientation or perhaps
steric congestion as discussed above. In summary, we built pH
models of POX hydrolysis for the lead compounds that
correlate compound reactivity with the fraction of core
deprotonation.
pH Models of POX hydrolysis
As briefly discussed in the library screening above, the pH of
the reaction media plays a critical role in the rate of POX
hydrolysis because the fraction of reactive oximate or
hydroxamate species varies with pH. To quantitatively
formulate the pH dependence of the rate, we performed
reaction kinetics at variable pH conditions for Dekon 139 and
selected lead compounds in the PAM (1, 3–5, 7) and HA (15–
17, 23, 24) class, as presented in the plots of Figures 7C–7F and
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(B)
(A)
Temperature effect
View Article Online
DOI: 10.1039/C9OB00503J
In addition to compound screening at 37 °C, we evaluated
reaction kinetics at two lower temperatures, 2 °C and 17 °C, to
gain insights into the thermodynamics of POX hydrolysis.
Figure 8 summarizes the results acquired with the selected
compounds, showing that k1 is temperature dependent at
both pH conditions. At pH 10.5, Dekon 139 showed an increase
in k1 by a factor of 2.9 or 2.0 as the temperature was raised
from 2 °C to 17 °C or 17 °C to 37 °C, respectively. This
temperature effect occurred similarly across all of the tested
compounds.
*
*
*
*
PBS
pH 10.5 139
Dekon
17
7
23 30
Time (min)
(D)
(C)
Activation energy (Ea) associated with POX hydrolysis was
calculated according to the Arrhenius equation, ln(k1) = ln(A) −
(Ea/R)(1/T) where A, R and T refers to a pre-exponential factor,
gas constant and absolute temperature (K), respectively. As
summarized in Figure 8C, Ea values are positive, indicating
endothermic process of POX hydrolysis by PAM and HA
compounds. Their values are generally lower at pH 10.5 than
at pH 7.4. These differences are attributable to the higher
reactivity of the deprotonated core species.
*
*
*
*
PBS Dekon
pH 10.5 139
Dekon
PBS
7
17 23 30
7
17 23 30
139
pH 10.5
Fig. 9 POX decontamination on porcine skin ex vivo by treatment with selected lead
compounds in a Franz cell. Fraction of intact POX remaining (% area under curve) (A)
and POX half-life in the donor compartment (B). Amount (nM) of POX detected in the
skin extract and receptor compartment (C), and the rate of skin penetration of POX
after treatment (D). Each value represents a mean ± SD (n = 2–4). *p < 0.05, one-way
ANOVA for each compound compared to Dekon 139.
(A)
pH 7.4
Skin decontamination of POX ex vivo
For validating the practical use of the -nucleophile library
compounds, we selected four lead compounds, 7, 17, 23, and
30, based on their core class, scaffold diversity and reactivity
(Figure 6). We investigated their effectiveness in POX
decontamination in a skin model in a Franz cell (Figure 9). Use
of POX as an OP surrogate for skin penetration offers benefits
because of its similar lipophilicity (clogP = 1.98) close to
prominent OP molecules such as VX (clogP = 2.28). As
described in our previous report, a patch of porcine skin
(dehaired, thickness = 1.5 mm) was clamped between the
donor and receptor chambers (Figure S7).9 The skin surface
was pre-exposed to POX (50 M), and immediately after 2 min,
was treated for 2 h with Dekon 139 or each compound at 0.3
M, or with a vehicle control (PBS, pH 10.5).
Compound Number
(B)
pH 10.5
Compound Number
The kinetics of POX hydrolysis in the donor chamber was
determined by UPLC analysis of the remaining POX in the
donor chamber over the treatment time (Figure 9A, 9B). As
anticipated, treatment with Dekon 139 and each test
compound showed a rapid decay curve for POX as compared
to the PBS treatment (pH 10.5) in which POX remained largely
stable. The effectiveness of the test compounds was evident
with POX half-life (t1/2 = 12.7 min) values calculated from
individual decay curves: 7 (8.2 min); 17 (3.6 min); 23 (4.1 min);
30 (5.1 min). All of the compounds were more effective than
Dekon 139 (t1/2 = 12.7 min) and the PBS vehicle control (720 ±
311 min) (p < 0.05).
(C)
Compound Number
Fig. 8 Effect of temperature on POX hydrolysis kinetics. (A, B) k1 plotted as a function of
temperature at pH 7.4 and pH 10.5. (C) Activation energy (Ea). Each k1 value represents
a mean ± SD (n ≥ 3) at a given temperature (±1 °C).
We quantified the fraction of POX that undergoes skin
penetration, and subsequently flows through to the receptor
chamber by LC MS/MS spectrometry (limit of quantitation or
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(A)
LOQ ≈ 1 nM). Figure 9C shows that the skin treated with the
PBS vehicle contained the highest amount of POX (304 ± 7 nM)
as compared to the skin treated with Dekon 139 (28.1 ± 11.4
nM), 7 (≤1.0 ± 0.3 nM), 17 (≤1.0 ± 0.3 nM), 23 (7.7 ± 0.8 nM) or
30 (≤1.0 ± 0.3 nM). The concentration of POX (nM) in the skin
extract was converted to the rate of skin penetration (= POX
(ng)/[skin weight (g) × h]) as a normalized unit indicative of
decontamination efficacy. As shown in Figure 9D, each lead
compound showed a decrease in the penetration rate larger
than Dekon 139 (4.9 ± 1.6) or the PBS control (58.4 ± 11.1; p
value < 0.05).
View Article Online
DOI: 10.1039/C9OB00503J
AChE alone
+ POX (63 nM)
+ POX (125 nM)
+ POX (250 nM)
+ POX (500 nM)
Time (min)
(B)
(C)
In the receptor chamber, the amount of POX detected was
much lower than that in the skin for each treatment. Such
preferred distribution in the skin could be attributable to the
high lipophilicity of POX.34 In summary, this result
demonstrates the improved efficacy of the four lead
compounds as compared to Dekon 139 in skin
decontamination of POX ex vivo.
2-PAM
Obidoxime
5
6
7
Reactivation of POX-inhibited AChE
[Oxime], M
[Oxime], M
In the experimental designs above, we focused on screening
and identifying lead -nucleophile scaffolds as potent OP
scavengers. These results provide clear evidence supportive of
their potent utility for topical OP decontamination. In addition
to this main objective, we were also interested in certain -
nucleophiles for their inherent ability that pertains to the
reactivation of OP-inactivated AChE.
Of the two nucleophilic cores employed here, the PAM
core has the potential ability to reactivate OP-inhibited AChE
as well established with 2-PAM and obidoxime.37-39 Here, we
investigated whether hydrophilic PAM compounds retain such
activity using AChE (Electrophorus electricus; 0.1 U/mL) in PBS,
pH 8.0 at 23 °C according to Ellman’s method.40, 41 As shown in
Figure 10A, POX showed dose-dependent inhibition of AChE
activity with IC50 of 50 nM (calculated) as reported in the
literature.42 2-PAM and obidoxime were then tested for their
ability to reactivate AChE pre-inhibited by treatment with POX
(500 nM) for 60 min. As anticipated, they showed activity for
AChE reactivation in a dose-dependent manner to a maximal
level of 67% by 2-PAM (0.1 mM) and 70% by obidoxime (0.01
mM) (Figure 10B).
A dozen of library compounds were similarly tested in this
enzyme assay, and only three compounds 5–7, all in the 4-
PAM subclass, were found to show AChE reactivation activity
to a various degree (Figure 10B). Observation of their activity is
notable, as other PAM compounds including 3 in the 3-PAM
subclass and 29 and 30 in the HA class failed to show any
activity at the same dose range (not shown). Among the three
compounds, 7 was most active, and though less active than 2-
PAM or obidoxime, it continued to increase its reactivation
activity up to 37% at 1.0 mM. Like 2-PAM and obidoxime, 7
showed enzyme inhibition in a dose-dependent manner
(Figure 10C), which could be attributable to their intrinsic
Fig. 10 Reactivation of POX-inhibited AChE (Electrophorus electricus) by oximes 5–7. (A)
Kinetic traces of enzyme activity showing dose-dependent inhibition by POX. (B)
Compound efficiency in the reactivation of AChE pre-inhibited by POX (500 nM). (C)
Effects of oximes alone on AChE activity.
neutral hydroxyl scaffold (7) is more effective for reactivation
activity than a negatively charged scaffold (6). In summary,
compounds in the 4-PAM subclass retain the ability to
reactivate AChE inhibited by POX. This activity may serve as an
additional benefit in their use for skin decontamination.
Having such activity is not necessary for OP decontamination,
but its determination would be valuable for expanding our
knowledge base in the design of enzyme reactivators, and
potentially as an early lead in the design of multifunctional
antidotes for treating OP exposure.45
Conclusion
This study offers significant advances in the design of -
nucleophile compounds as the chemical scavengers of OPs.
Despite its essential role in skin decontamination, Dekon 139
suffers from adverse systemic effects arising from the rapid
percutaneous absorption of its neutral form, DAM.23 In this
work, we investigated a core-scaffold binary approach in the
design of potent small molecule scavengers of OPs in which
the scaffold involves to improve a physicochemical property
while each linked -nucleophile core directly engages in the
catalytic reaction. We prepared and tested 30 library
compounds, which show improvements in their molecular size,
hydrophilicity, and/or core acidity than DAM. We presented
NMR evidence supportive of their catalytic reactivity against
three OP surrogates (POX, malaoxon, omethoate), each having
a P-O or P-S bond. We identified 14 reactive compounds highly
effective for catalysing POX hydrolysis using a colorimetric
assay under variable pH and temperature conditions. Some of
ability to inhibit the enzyme via competitive occupation in the
44
catalytic pocket.43,
Differences in the reactivation activity
among the three 4-PAM compounds are attributable to the
structural difference in their scaffold. Conjugation with a
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these further displayed greater efficacy for POX As a primary method for compound screening of POX
View Article Online
DOI: 10.1039/C9OB00503J
hydrolysis, a colorimetry assay was performed in a plate
decontamination in a skin model as compared to Dekon 139.
Oure results offer significant insights into the practice of reader (SynergyTM, BioTek). In a representative experiment,
OP decontamination. Our pH models established with multiple Dekon 139 or a test compound formulated in PBS at either pH
lead compounds suggest that compound formulation should 10.5 or 7.4 was loaded in a 96-well microplate (180 L; n = 3)
be performed at or above the pH that leads to the significant and serially diluted 2-fold using the same buffer to a range of
fraction of core deprotonation (≥95%) for high reactivity. test concentrations (0.5–8.0 mM). A POX solution (20 L, 0.30
Variable temperature models, which verified the endothermic mM) freshly prepared in the test PBS condition was then
pathway associated with POX hydrolysis catalysed by - added to each test well. Immediately after mixing, the
nucleophile compounds, provide solid evidence for applying microplate was read in an absorbance mode (400 nm). After
decontaminant solutions at temperatures as high as permitted this first read (At = 1), the plate was incubated in a temperature-
by the skin.
controlled environment such as at 37 (± 1) °C, and it was taken
Finally, the chemical handle terminating each scaffold out shortly for reading at variable time points (At = 2 to At = 8) as
allows further modifications of the lead compound into a specified in Figure 5.
much larger construct through its covalent attachment to a
Rate constants of POX hydrolysis
polymer molecule or nanoscale carrier. As reported in the
46-48
design of delivery systems for antidote drugs8,
and Data analysis for determining rate constants was performed as
bioscavengers,17 such oxime/HA-polymer conjugates can be previously reported.8 First, absorbance at a specific time point
highly advantageous in topical applications as they provide an (At =n) was calculated by subtraction of the first read (At = n
−
excellent route to further reduce percutaneous permeation At = 1). Second, standard calibration curves of 4-NP absorbance
and facilitate extended duration of action via polymer vs. concentration were acquired in the range of 0.1–100 M in
pharmacokinetics.49 This aspect of nanoscale scavenger design PBS at two different pH conditions: A (400 nm) = slope × [4-
will constitute the subject of future studies.
NP]; slope = 1.01 (±0.02) × 10−4 at pH 10.5, and 1.46 (±0.03) ×
Overall, we identified multiple lead compounds from the 10−4 at pH 7.4. Each curve was used to calculate the
current library of -nucleophile scaffolds that offer substantial concentration of 4-NP produced ([4-NP]t n) and thus POX
=
benefits as OP scavengers based on their structure-reactivity remaining ([POX]t = n = 3.0 × 10-5 M − [4-NP]t = n). Rate constants
trends, their catalytic mechanism of OP inactivation, and their for POX hydrolysis were extracted according to a second-order
potent efficacy demonstrated for POX decontamination in the rate law under pseudo-first order conditions ([PAM/HA] >>
skin model. These results strongly support their development [POX]; Eq 1–Eq 4). The bimolecular rate constant k1 was
potential as the topical decontaminant of OPs.
determined as the slope of the linear plot of kobsd versus
PAM/HA concentration (Eq 2). The values of the kinetic
parameters including kobsd and POX half-life (t1/2) are reported
Experimental section
as
a mean (±SD) value obtained from at least three
independent measurements (n ≥ 3).
Synthesis of -nucleophile scaffolds
Full details for compound synthesis and characterization (1–
30) are provided in SI with references cited therein. Copies of
spectral data are also provided for the selected compounds
including 1H NMR spectra, mass spectra and UPLC traces.
Rate = −d[POX]/dt ≈ k1[POX][PAM/HA] where k1 << k2
Rate = kobsd[POX] where k1[PAM/HA] ≈ kobsd
Eq 1
Eq 2
Eq 3
Eq 4
ln[POX]t = ln[POX]t = 0 − kobsd
t
1H NMR spectroscopy
The NMR experiments for POX hydrolysis were performed at
t1/2 = ln(2)/kobsd
o
room temperature (23 ± 1 C).8, 9, 48 In a typical experiment,
pH model of POX hydrolysis
Dekon 139 or a test compound, each prepared at 8.0 mM in a
deuterated PBS buffer (pD 10.5), was mixed with POX at 2.5
mM. The kinetics was monitored under a standard acquisition
condition46, 47 by collecting spectra at variable time points as
specified in Fig. S3.
The pH-rate (k1) curves were simulated under an assumption
that POX hydrolysis occurs via two parallel catalytic
pathways—one through a neutral species, and the other
through an anionic species (Figure 3). We applied the weighted
linear combination of the two pathways, each dependent on
the fraction (Fr) of either a neutral species (oxime/HA) or an
anionic species (oximate/hydroxamate) (Eq 5–Eq 7). The
fraction of core deprotonation for a test compound was
UV–Vis spectrometry
The kinetics of POX hydrolysis by UV–Vis spectrometry was
performed as reported earlier.8, 9 POX (30 M) was added to a
test solution prepared at 4.0 mM in PBS, pH 10.5 at 37 °C. The
fraction of POX hydrolysed was determined by measuring an
increase in 4-NP absorbance at 400 nm (Figure S4).
calculated according to its core pKa-pH approximation.36
A
curve fitting of k1 versus pH was made through a linear
combination of two fractional rate constants using k1, oxime (≈
k1, pH 7.4) and k1, oximate (≈ k1, pH 10.5) (Eq 7).
Colorimetric screening of compound reactivity
10 | J. Name., 2018, 00, 1-12
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Organic & Biomolecular Chemistry
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Rate = rate (Oximate) + rate (Oxime)
Eq 5
Eq 6
Eq 7
phase A = 0.1% formic acid in 98:2 water:methVaienwoAl;rticMleoObnliinlee
phase B = 0.1% formic acid in methanol. DOI: 10.1039/C9OB00503J
POX was detected using a method of multiple reaction
monitoring (MRM) parameters (positive ionization mode;
Rate = −d[POX]/dt ≈ k1,oximate[Oximate][POX] +
k1,oxime[Oxime][POX]
o
source temperature = 150 C; desolvation temperature = 400
Rate ≈ (k1,oximate•Froximate + k1,oxime•Froxime)[Oxime +
Oximate]total[POX] where (Froximate + Froxime) = 1
oC; cone voltage = 27 V; collision energy = 20 eV). POX was
identified as the species at tr = 6.0 min in the LC trace which
produced a parent mass (m/z) of 276.036. A calibration curve
for POX was generated in the range of 1.0 nM to 1000 nM:
Skin decontamination ex vivo
Porcine skin was used as the test membrane for POX AUC = slope × [POX] (slope = 1640.6, R2 = 0.996).
decontamination in a Franz cell device with a water jacket
controlled at 37 ºC. The dehaired skin (1.5 mm thick; Stellen
Acetylcholine esterase (AChE) assay
Medical, LLC) was clamped between the donor and receptor The enzymatic assay used AChE from Electrophorous electricus
chambers. A solution of POX (0.4 mL, 50 M) in PBS pH 10.5 (Sigma-Aldrich) and thioacetylcholine as the substrate in PBS,
50, 51
containing 0.5% DMSO was added to the donor chamber. After pH 8.0 at 23 °C.40,
Reaction kinetics were measured
2 min exposure, a solution (0.4 mL) of Dekon 139 or a test spectrophotometrically at 412 nm using a 96-well microplate
compound (7, 17, 23, 30), each formulated at 0.3 M in PBS pH in a plate reader. For enzyme reactivation, AChE (0.1 U/mL)
10.5, was added to the POX solution in the donor side. The PBS was completely inhibited to ≥95% by pre-incubation with POX
pH 10.5 vehicle alone was also tested as a control. After the (500 nM) for 60 min prior to treatment with 2-PAM or a test
decontaminant addition, analyte samples were taken from the compound over a range of test concentrations (10−7–10−3 M).
solution in the donor side at specific time points (t = 0, 6, 15,
The efficiency of enzyme reactivation is reported as %
30, 60, and 120 min) and from the receptor side at t = 120 min. activity as previously established:52 AChE activity (%) = 100 ×
After 2 h of treatment, each skin sample was recovered, [(koxime – kPOX-inactive)/(kactive – kPOX-inactive)] where each of the
thoroughly rinsed with PBS, and weighed. Skin was cut into terms refers to a first-order rate constant k (dA/dt; min−1)
small pieces, and POX was extracted in 100% ethanol (1.0 mL) determined from a fully active enzyme (kactive), the POX-
on a shaker at room temperature overnight. Samples were inactivated enzyme (kPOX-inactive), or the POX-inactivated
centrifuged at 2,000 rpm, and the supernatant was filtered enzyme after an oxime treatment (koxime). The activity of
through a nylon cell strainer to obtain clarified extract.
enzyme inhibition by an oxime compound alone is reported as
Sample solutions from the donor side were analysed by % inhibition which was calculated as: Enzyme Inhibition (%) =
UPLC as detailed in the SI. The concentration of intact POX in 100 × (koxime/kactive).
each sample was determined from the area under curve (AUC)
Hazardous Materials
analysis of its peak relative to its calibration curve prepared
from a series of standards with known POX concentrations (50 Caution: The following chemicals are hazardous and should be
M–0.1 M). The POX half-life was calculated from the handled carefully: paraoxon-ethyl (POX), malaoxon, and
exponential decay of the AUC values over time.
omethoate.1
Samples of skin extracts and the flow-through in the
receptor chamber were analysed by LCMS mass spectrometry
Statistical analysis
using an injection volume of 10 µL as described below. The Analysis of statistical significance was performed by one-way
concentration of POX was determined from the AUC value of ANOVA with GraphPad Prism 7 software. Differences with p
the POX peak compared to its standard calibration curve. The values of <0.05 are reported as statistically significant.
total amount of POX (ng) was calculated, and used to calculate
the rate of skin penetration (unit = POX ng/(skin tissue g × h))
measured in a skin sample (g) after 2 h exposure.
Supporting information
Details of materials and analytical methods not detailed above,
synthetic methods, copies of NMR and UV–Vis spectra,
supplementary figures and tables.
POX analysis by mass spectrometry
LCMS spectrometry spectrometry (Waters Acquity UPLC
system equipped with
a
Waters TQ detector mass
spectrometer) was employed for quantitative analysis of POX
from samples collected in the skin decontamination
experiment. The chromatographic system had an ODS column
(Acquity UPLC BEH C18 1.7 m; 2.1 × 100 mm, with an XBridge
C18 2.5 m guard column, Waters) at a column temperature of
40 °C with a flow rate of 0.45 mL/min. Column elution was
performed using a gradient mode with two mobile phases:
95% A (0–0.25 min), 95–0% A (0.25–7.75 min), 0% A (7.75–8.5
min), 0–95% A (8.5–8.51 min) over a 10 min cycle. Mobile
Conflicts of interest
The authors declare no competing financial interest.
Acknowledgments
This work was supported by the US Defense Threats Reduction
Agency–DOD (HDTRA1-17-C-00001).
This journal is © The Royal Society of Chemistry 20xx
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29 R. Swidler and G. M. Steinberg, J. Am. Chem. Soc., 1956, 78,
Notes and references
View Article Online
3594–3598.
30 S. B. Bharate, L. Guo, T. E. Reeves, D.DMOI.: 1C0e.1r0a3s9o/lCi 9aOnBd0C05.0M3J.
Thompson, Bioorg. Med. Chem. Lett., 2009, 19, 5101–5104.
31 E. J. Poziomek, B. E. Hackley and G. M. Steinberg, J. Org.
Chem., 1958, 23, 714–717.
32 J. Hadgraft and W. John Pugh, J. Invest. Dermatol. Symp.
Proc., 1998, 3, 131–135.
1
J. Bajgar, in Advances in Clinical Chemistry, ed. S. M. Gregory,
Elsevier, 2004, vol. Volume 38, pp. 151–216.
2
3
J. A. Vale, Toxicology, 2007, 240, 141–142.
J. C. DeMar, E. D. Clarkson, R. H. Ratcliffe, A. J. Campbell, S.
G. Thangavelu, C. A. Herdman, H. Leader, S. M. Schulz, E.
Marek, M. A. Medynets, T. C. Ku, S. A. Evans, F. A. Khan, R. R.
Owens, M. P. Nambiar and R. K. Gordon, Chem.-Biol.
Interact., 2010, 187, 191–198.
R. K. Sit, Z. Radić, V. Gerardi, L. Zhang, E. Garcia, M. Katalinić,
G. Amitai, Z. Kovarik, V. V. Fokin, K. B. Sharpless and P.
Taylor, J. Biol. Chem., 2011, 286, 19422–19430.
C. A. Broomfield, O. Lockridge and C. B. Millard, Chem.-Biol.
Interact., 1999, 119–120, 413–418.
O. Cohen, C. Kronman, L. Raveh, O. Mazor, A. Ordentlich and
A. Shafferman, Mol. Pharmacol., 2006, 70, 1121–1131.
D. Jun, L. Musilová, M. Link, M. Loiodice, F. Nachon, D.
Rochu, F. Renault and P. Masson, Chem.-Biol. Interact., 2010,
187, 380–383.
33 J. D. Bos and M. M. H. M. Meinardi, Exp. Dermatol., 2000, 9,
165–169.
4
34 J.
Sangster,
Octanol-Water
Partition
Coefficients:
Fundamentals and Physical Chemistry., John Wiley & Sons
Ltd, Chichester, 1997.
5
6
7
35 C. A. Boulet and A. S. Hansen, Phosphorus, Sulfur, and Silicon
and the Related Elements, 1991, 57, 147–161.
36 H. N. Po and N. M. Senozan, J. Chem. Educ., 2001, 78, 1499.
37 F. Worek, G. Reiter, P. Eyer and L. Szinicz, Arch. Toxicol.,
2002, 76, 523–529.
38 F. Worek, H. Thiermann, L. Szinicz and P. Eyer, Biochem.
Pharmacol. (Amsterdam, Neth.), 2004, 68, 2237–2248.
39 J. A. Berberich, T. R. Stouch, S. Manepalli, E. X. Esposito and
J. D. Madura, Chem. Res. Toxicol., 2016, 29, 1534–1540.
40 G. L. Ellman, K. D. Courtney, V. Andres jr and R. M.
Featherstone, Biochem. Pharmacol. (Amsterdam, Neth.),
1961, 7, 88–95.
41 P. Demerseman, D. Kiffer, L. Debussche, C. Lion, R. Royer and
H. Sentenac-Roumanou, Eur. J. Med. Chem., 1988, 23, 63–68.
42 K. B. Wallace and U. Herzberg, Toxicol. Appl. Pharmacol.,
1988, 92, 307–314.
43 D. Jun, L. Musilova, K. Musilek and K. Kuca, Int. J. Mol. Sci.,
2011, 12, 2077–2087.
44 G. A. Petroianu, K. Arafat, S. M. Nurulain, K. Kuča and J.
Kassa, J. Appl. Toxicol., 2007, 27, 168–175.
45 T. Zorbaz, A. Braïki, N. Maraković, J. Renou, E. deꢀlaꢀMora, N.
MačekꢀHrvat, M. Katalinić, I. Silman, J. L. Sussman, G.
Mercey, C. Gomez, R. Mougeot, B. Pérez, R. Baati, F. Nachon,
M. Weik, L. Jean, Z. Kovarik and P.-Y. Renard, Chem. - Eur. J.,
2018, 24, 9675–9691.
46 S. K. Choi, P. Leroueil, M.-H. Li, A. Desai, H. Zong, A. F. L. Van
Der Spek and J. R. Baker Jr, Macromolecules, 2011, 44, 4026–
4029.
47 S. K. Choi, T. P. Thomas, P. R. Leroueil, A. Kotlyar, A. F. L. Van
Der Spek and J. R. Baker, J. Phys. Chem. B, 2012, 116, 10387–
10397.
8
9
S. Bharathi, P. T. Wong, A. Desai, O. Lykhytska, V. Choe, H.
Kim, T. P. Thomas, J. R. Baker and S. K. Choi, J. Mater. Chem.
B, 2014, 2, 1068–1078.
S. Tang, P. T. Wong, J. Cannon, K. Yang, S. Bowden, S.
Bhattacharjee, J. J. O'Konek and S. K. Choi, Chem.-Biol.
Interact., 2019, 297, 67–79.
10 M. Fentabil, M. Gebremedhin, J. G. Purdon, L. Cochrane and
V. S. Goldman, Toxicol. Lett., 2018, 293, 241–248.
11 N. Singh, Y. Karpichev, R. Sharma, B. Gupta, A. K. Sahu, M. L.
Satnami and K. K. Ghosh, Org. Biomol. Chem., 2015, 13,
2827–2848.
12 M. C. Franklin, M. J. Rudolph, C. Ginter, M. S. Cassidy and J.
Cheung, Proteins: Struct., Funct., Bioinf., 2016, 84, 1246–
1256.
13 B. t. Sanson, F. Nachon, J.-P. Colletier, M.-T. r. s. Froment, L.
Toker, H. M. Greenblatt, J. L. Sussman, Y. Ashani, P. Masson,
I. Silman and M. Weik, J. Med. Chem., 2009, 52, 7593–7603.
14 D. Jovanović, Arch. Toxicol., 1989, 63, 416–418.
15 J. Misik, R. Pavlikova, D. Josse, J. Cabal and K. Kuca, Toxicol.
Mech. Methods, 2012, 22, 520–525.
16 S. Bjarnason, J. Mikler, I. Hill, C. Tenn, M. Garrett, N. Caddy
and T. Sawyer, Hum. Exp. Toxicol., 2008, 27, 253–261.
17 P. Li, S.-Y. Moon, M. A. Guelta, L. Lin, D. A. Gómez-Gualdrón,
R. Q. Snurr, S. P. Harvey, J. T. Hupp and O. K. Farha, ACS
Nano, 2016, 10, 9174–9182.
18 G. Amitai, H. Murata, J. D. Andersen, R. R. Koepsel and A. J.
Russell, Biomaterials, 2010, 31, 4417–4425.
19 J. I. Morales, R. Figueroa, M. Rojas, D. Millán, R. A. Tapia and
P. Pavez, Org. Biomol. Chem., 2018, 16, 7446–7453.
20 J. S. W. Tsang, A. A. Neverov and R. S. Brown, Org. Biomol.
Chem., 2004, 2, 3457–3463.
21 P. Jacquet, D. Daudé, J. Bzdrenga, P. Masson, M. Elias and E.
Chabrière, Environ. Sci. Pollut. Res., 2016, 23, 8200–8218.
22 E. H. Braue, K. H. Smith, B. F. Doxzon, H. L. Lumpkin and E. D.
Clarkson, Cutaneous Ocul. Toxicol., 2011, 30, 15–28.
23 M. W. Fryer, P. W. Gage, I. R. Neering, A. F. Dulhunty and G.
D. Lamh, Pfluegers Arch., 1988, 411, 76–79.
48 J. Mukherjee, P. T. Wong, S. Tang, K. Gam, A. Coulter, J. R.
Baker and S. K. Choi, Mol. Pharmaceutics, 2015, 12, 4498–
4508.
49 M. J. Vicent, H. Ringsdorf and R. Duncan, Adv. Drug Delivery
Rev., 2009, 61, 1117–1120.
50 R. Cochran, J. Kalisiak, T. Küçükkılınç, Z. Radić, E. Garcia, L.
Zhang, K.-Y. Ho, G. Amitai, Z. Kovarik, V. V. Fokin, K. B.
Sharpless and P. Taylor, J. Biol. Chem., 2011, 286, 29718–
29724.
51 M. C. de Koning, G. Horn, F. Worek and M. van Grol, Eur. J.
Med. Chem., 2018, 157, 151–160.
52 G. E. Garcia, A. J. Campbell, J. Olson, D. Moorad-Doctor and
V. I. Morthole, Chem.-Biol. Interact., 2010, 187, 199–206.
24 G. Becker, A. Kawan and L. Szinicz, Arch. Toxicol., 1997, 71,
714–718.
25 J. C. Lamb, G. M. Steinberg, S. Solomon and B. E. Hackley,
Biochemistry, 1965, 4, 2475–2484.
26 E. J. Behrman, M. J. Biallas, H. J. Brass, J. O. Edwards and M.
Isaks, J. Org. Chem., 1970, 35, 3069–3075.
27 F. Terrier, P. Rodriguez-Dafonte, E. Le Guevel and G.
Moutiers, Org. Biomol. Chem., 2006, 4, 4352–4363.
28 B. E. Hackley, R. Plapinger, M. Stolberg and T. Wagner-
Jauregg, J. Am. Chem. Soc., 1955, 77, 3651–3653.
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