Catalytic Detoxification of Organophosphorus Nerve Agents by Butyrylcholinesterase-Polymer-Oxime Bioscavengers

Article abstract of DOI:10.1021/acs.biomac.0c00959

Organophosphorus nerve agents (OPNAs), used in chemical warfare, irreversibly inhibit essential cholinesterases (ChEs) in the cholinergic neurotransmission system. Several potent nucleophilic oximes have been approved for the treatment of acute poisoning by OPNAs, but they are rapidly cleared from blood circulation. Butyrylcholinesterase (BChE) stoichiometrically binds nerve agents, but because the molecular weight of a nerve agent is about 500-fold less than the enzyme, the bioscavenger has had limited utility. We synthesized BChE-polymer-oxime conjugates using atom transfer radical polymerization (ATRP) and azide-alkyne "click"chemistry. The activity of the BChE-polymer-oxime conjugates was dependent on the degree of oxime loading within the copolymer side chains. The covalent modification of oxime-containing copolymers prolonged the activity of BChE in the presence of the VX-and cyclosarin-fluorogenic analogues EMP-MeCyC and CMP-MeCyC, respectively. After complete inactivation by VX and cyclosarin fluorogenic analogues, the conjugates demonstrated efficient self-reactivation of up to 80% within 3-6 h. Repeated inhibition and high-level self-reactivation assays revealed that the BChE-polymer-oxime conjugates were excellent reactivators of OPNA-inhibited BChE. Recurring self-reactivation of BChE-polymer-oxime conjugates following repeated BChE inhibition by fluorogenic OPNAs (Flu-OPNAs) opens the door to developing the next generation of nerve agent "catalytic"bioscavengers.

Full text of DOI:10.1021/acs.biomac.0c00959

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Article
Catalytic Detoxification of Organophosphorus Nerve Agents
by Butyrylcholinesterase-Polymer-Oxime Bioscavengers
Libin Zhang, Hironobu Murata, Gabriel Amitai, Paige N Smith, Krzysztof Matyjaszewski, and Alan J Russell
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.0c00959 • Publication Date (Web): 31 Jul 2020
Downloaded from pubs.acs.org on August 4, 2020
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Catalytic Detoxification of Organophosphorus Nerve
Agents by Butyrylcholinesterase-Polymer-Oxime
Bioscavengers
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Libin Zhang , Hironobu Murata , Gabriel Amitai , Paige N. Smith , Krzysztof
Matyjaszewski1,4,5, and Alan J. Russell1,4,5,
*
1Center for Polymer-Based Protein Engineering,Carnegie Mellon University, 5000 Forbes
Avenue, Pittsburgh, Pennsylvania 15213, United States.
2Wohl Drug Discovery Institute, Nancy and Stephen Grand Israel National Center for
Personalized Medicine (G-INCPM), Weizmann Institute of Science, Rehovot 760001,
Israel
3Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Ave, Pittsburgh,
PA 15213, United States
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4Department of Chemical Engineering, Carnegie Mellon University, 5000 Forbes Avenue,
Pittsburgh, Pennsylvania 15213, United States
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5Department of Chemistry, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh,
Pennsylvania 15213, United States
KEYWORDS: Fluorogenic Organophosphorus nerve agents (Flu-OPNA) analogues,
Butyrylcholinesterase (BChE), Atom Transfer Radical Polymerization (ATRP), Oxime,
Protein-Polymer Conjugate
ABSTRACT
Organophosphorus nerve agents (OPNA), used in chemical warfare, irreversibly inhibit
essential cholinesterases (ChEs) in the cholinergic neurotransmission system. Several
potent nucleophilic oximes have been approved for the treatment of acute poisoning by
OPNAs, but they are rapidly cleared from blood circulation. Butyrylcholinesterase (BChE)
stoichiometrically binds nerve agents, but since the molecular weight of a nerve agent is
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about 500-fold less than the enzyme, the bioscavenger has had limited utility. We have
synthesized BChE-polymer-oxime conjugates using atom transfer radical polymerization
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(ATRP) and azide-alkyne “click” chemistry. The activity of the BChE-polymer-oxime
conjugates was dependent on the degree of oxime loading within the copolymer side
chains. The covalent modification of oxime-containing copolymers prolonged the activity
of BChE in the presence of the VX- and cyclosarin-fluorogenic analogues EMP-MeCyC
and CMP-MeCyC, respectively. After complete inactivation by VX and cyclosarin
fluorogenic analogues, the conjugates demonstrated efficient self-reactivation of up to 80%
within 3-6 hours. Repeated inhibition and high-level self-reactivation assays revealed that
the BChE-polymer-oxime conjugates were excellent reactivators of OPNA-inhibited
BChE. Recurring self-reactivation of BChE-polymer-oxime conjugates following repeated
BChE inhibition by Flu-OPs opens the door to developing the next generation of nerve
agent “catalytic” bioscavengers.
INTRODUCTION
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Initially developed in the 1930s, OPNAs have been used as pesticides, tested on criminal
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subjects, used in terrorist acts and employed as chemical warfare agents. Tabun and
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sarin, for example, were used in the Iran-Iraq war in the1980s, sarin was used against
civilians in Syria and by a terrorist sect in the Tokyo subway, and VX was used to
assassinate the North Korean, Kim Jong-nam, in Malaysia (2017).2-5 Accidental and self-
poisoning by organophosphorus pesticides results in around 100,000 deaths and affects
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more than 1 million people each year worldwide. OPNAs inhibit cholinesterases (ChEs)
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in the cholinergic neurotransmission system by phosphorylation of a catalytic serine
residue in the active site. OPNA toxicity results from the accumulation of
neurotransmitters in cholinergic synapses leading to paralysis, hypotension, breathing
failure and death.8-11 Hydrophobic OPNAs cross the blood-brain barrier (BBB) and
inactivate acetylcholinesterase (AChE) in the central nervous system (CNS).
Accumulated OPNAs, retained in the CNS, can also partition back into blood causing
"cholinergic crisis" and irreversible brain damage.12, 13
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Currently approved treatment of acute OPNA poisoning includes administration of a
mixture of a pyridinium aldoxime, an anticonvulsant (benzodiazepine) and a muscarinic
acetylcholine receptor antagonist (atropine).14, 15 Since the seminal work of Wilson and
colleagues in the mid-1950s, thousands of aldoximes have been synthesized and
screened.16, 17 Few molecules, for example pralidoxime (2-PAM), asoxime (HI-6), and
obidoxime (toxogonin), have been developed as OPNA poisoning therapeutics.14, 15
Aldoximes, in the charged oximate nucleophilic state, attack the phosphoryl bond of
covalently-inhibited ChEs at the active site serine, releasing the OP moiety from the active
site and restoring ChE activity.18-20 Over several decades, numerous imidazolium oximes
have been developed as OP-ChE reactivators.12, 21-24 Some of these derivatives were
shown to be efficient reactivators in specific in vitro and in vivo reactivation assays using
VX- and tabun-inhibited ChEs.23-27 Antidotal therapy by oximes, unfortunately, requires
repeated administration because low molecular weight oximes have very short half-lives
in circulation.28, 29 This short half-life also limits the use of oximes as effective long-term
prophylactic drugs.
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A new concept of acute OPNA poisoning treatment, by the administration of
bioscavengers, emerged at the end of the 1980s.30-35 Bioscavengers are proteins that
react with and neutralize OPNAs. Human butyrylcholinesterase (BChE) is effective as a
bioscavenger at both pre- and post-OPNA exposure.36-40 The enzyme is stable in human
blood circulation, with a half-life of 12 days, in concentration ranges between 3.5 and 9.3
mg/L.41-45 BChE is found in plasma, organs, and tissues including liver, skin, striated
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muscle, smooth muscle, lung, and brain. Administration of BChE has proven to be safe
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with no toxic, immunogenic or behavioral effects. As a stoichiometric bioscavenger, with
a molecular weight of about 85 kDa, 500-600 times higher than that of OPNAs, a relatively
high dose of BChE (100-500 mg) is required to effectively protect a 70 kg patient.48
Unfortunately, this dose costs more than $2,000, and therefore BChE therapy has not
been deployed as a prophylactic drug.30
Grafting poly(ethylene glycol) (PEG)49-52 and poly(sialic acid) to the surface of BChE has
been used to increase the half-life of the enzyme in the blood.53-56 Nevertheless, even
extended lifetime BChE does not overcome the inherent limitations of a stoichiometric
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bioscavenger. We have become interested in whether polymers could serve to increase
BChE protein lifetime while also enabling reactivation and protection of the enzyme,
thereby defeating the stoichiometric limitations that drive up the necessary dose. Such a
self-reactivating BChE protein-polymer conjugate could also be used as a critical
component in decontamination of broad areas.
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A “grafting from” high yield approach to protein-polymer conjugate synthesis, involving
the in situ growth of polymers from biomacromolecular initiators, emerged in the early
2000s.57-59 Developed in the early 1990s, atom transfer radical polymerization (ATRP)
has proved to be a controlled and powerful technique to prepare well-defined polymers
and protein-polymer conjugates.51, 60-65 With a long term interest in polymer-based protein
engineering, we have generated a large library of protein-polymer conjugates.66-69
In the past decade, first generation “pseudo-catalytic" bioscavenger systems, composed
of BChE delivered with an efficient oxime reactivator, were developed to reduce the
amount of BChE needed for efficient protection.23, 24, 36, 70 Unfortunately, the fast
elimination of oxime reactivators from blood circulation, requiring continuous infusion of
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the reactivator to sustain reactivation kinetics, limited the use of pseudo-catalytic
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bioscavengers in vivo. Most recently, we have used ATRP to synthesize BChE-polymer
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conjugates with multiple pendant azido groups. Azide-alkyne “click” chemistry was then
used to covalently couple oximes to the surface of the enzyme.71 The BChE-polymer-
oximes exhibited a slowed rate of inactivation by organophosphates and some evidence
of inter- and intramolecular reactivation. Herein, fluorogenic analogues of VX and
cyclosarin (EMP-MeCyC and CMP-MeCyC, respectively)72 were used to study the
kinetics of inactivation and reactivation of next-generation BChE-polymer-oxime
conjugates. The BChE-polymer-oxime conjugate recovered about 85% activity after
complete inactivation induced by fluorogenic analogues of VX in three successive
inactivation-reactivation “catalytic” cycles. The conjugates should also be useful in
surface decontamination of OPNAs.
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EXPERIMENTAL SECTION
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Materials
Common reagents and solvents were purchased from Fisher Scientific (Pittsburgh, PA)
and used as received unless otherwise specified. Dr. Oksana Lockridge (Eppley Institute,
University of Nebraska Medical Center) kindly provided the human BChE. EMP-MeCyC
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and CMP-MeCyC were prepared we have described previously. MA-PEG -N monomer
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and NHS-Br (ATRP initiator) were prepared in the same way as our previous reports.71,
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Copper(II) bromide (CuBr ), copper(II) sulfate (CuSO ), hydroxylamine hydrochloride,
2
4
triethylamine, sodium ascorbate (NaAsc), 2-imidazolecarboxaldehyde,
S-
butyrylthiocholine iodide (BTC), 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB), N,N-
dimethylacrylamide (DMAA), 1,1,4,7,10,10-hexamethyltriethylenetetramine (HMTETA),
tris[2-(dimethylamino)ethyl] amine (Me TREN), isonicotinaldehyde, 1,4-diiodobutane, 6-
6
iodohex-1-yne, and 4-chlorobenzyl bromide were purchased from Sigma-Aldrich. DMAA,
Me TREN and HMTETA were purified with a basic alumina column before using. 2-(4-
6
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(
(bis((1-(tert-butyl)-1H-1,2,3-triazol-4-yl)methyl)amino)methyl)-1H-1,2,3-triazol-1-yl)
acetic acid (BTTAA) and Azide-PEG-Amine were purchased from Click Chemistry Tools
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LLC. N,N-dimethylprop-2-yn-1-amine was purchased from Fisher Scientific.
Characterization
Nuclear magnetic resonance (NMR) analysis
A 400 MHZ spectrometer (Bruker Avance) in the Center for Molecular Analysis, Carnegie
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Mellon University, Pittsburgh, PA was used to collect H NMR spectra.
Ultraviolet-visible (UV-VIS) and fluorescence spectrophotometry
UV−VIS spectra were analyzed by a UV−VIS spectrophotometer (Lambda 45,
PerkinElmer). Fluorescence spectra were analyzed by a plate reader (Bio-Tek Synergy
H1).
GPC analysis
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The molecular weight (M ) and the polydispersity (Ð) of azide containing copolymers were
n
determined by GPC with 3-columns on a Waters 2695 Series system (Waters
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Ultrahydrogel Linear, 500 and 250) as our previous report. The BChE, BChE-Br, BChE-
PDMAA-N and BChE-PDMAA-OX conjugates were analyzed by the same GPC system
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with 4-columns.
Protein analysis by the Bicinchoninic acid (BCA) assay
The BChE-PDMAA-OX conjugate (1-2 mg/mL, protein, 20 or10 μL) in BP buffer (50 mM,
pH 7.4) was mixed with 200 µL BCA working reagent solution (Reagent A/Reagent B =
50/1). The mixture was incubated at 60°C for 15 min. 562 nm absorbance of the samples
was recorded by a plate reader (Bio-Tek Synergy H1). Standard curves of PEG-IO, PEG-
PO and native BChE were used to determine the protein concentration of the conjugates
(Figure S1).
SDS-PAGE analysis
BChE, BChE-Br, or BChE-polymer-oxime conjugate solution (1.0 mg/mL protein, 10 μL)
was mixed with SDS-PAGE loading buffer (2X, 10 μL) and heated at 95 °C for 10 min. 10
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4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
μL of the sample was loaded onto 4-15% precast gel and run at 200 V for 30 min. The
gel was washed with distilled water three times and stained with 50 mL PageBlue staining
solution. 1 hour later, the gel was de-stained overnight by distilled water.
0
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Methods
Synthesis of alkyne-imidazolium-oxime
Alkyne-imidazolium-oxime was synthesized as described previously.71 Briefly, 2-
imidazolecarboxaldehyde (12 mmol) and K CO (24 mmol) were mixed in 30 mL DMF. 4-
2
3
Chlorobenzyl bromide (24 mmol) was added and stirred at room temperature overnight.
The reaction mixture was filtered, and 250 mL distilled water was added to the filtrate.
The resulting solution was extracted with ethyl acetate (200 mL × 3), then the organic
phase was dried with MgSO . After removing the solvent by rotary evaporator, the
4
alkylimidazole-2-carbaldehyde yield was 74%.
Hydroxylamine hydrochloride (13.5 mmol) was dissolved in 30 mL water, and added to
Na CO (13.5 mmol) and alkylimidazole-2-carbaldehyde (9 mmol). The reaction mixture
2
3
was stirred for 3.5 h at room temperature, and the precipitate was filtered and washed by
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distilled water three times and Et O four times. The product was dried over a vacuum,
2
generating an 87% yield.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
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2
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6
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8
9
0
6-Iodohex-1-yne (1.5 mmol) in 10 mL ACN and 1 mL DMSO were added to the
alkylimidazole-2-carbaldehyde oxime (0.4 mmol). The reaction mixture was stirred at 67
°C for 5 days. Removed the solvent by rotary evaporator. The crude product was purified
by silica gel column (from hexane/acetone = 1/1 to acetone/MeOH = 3/1). White powder,
1
4
8% yield. H NMR (400 MHz, MeOD-d4) δ 8.52 (s, 1H, CH), 7.82 (d, J = 3, 1H, Ar H),
7.74 (d, J = 3, 1H, Ar H), 7.44 (d, J = 3, 2H, Ar H), 7.35 (d, J = 3, 2H, Ar H), 5.63 (s, 2H;
CH ), 4.43 (t, J = 3, 2H; CH ), 3.36 (s, 1H; CH), 2.27 (d, J = 3, 2H, CH ), 2.02-2.00 (m,
2
2
2
2H; CH ), 1.62-1.59 (m, 2H; CH ) (Figure S2).
2
2
Synthesis of PEG-IO
PEG-IO was synthesized as described previously.71 Briefly, BTTAA (2.4 mg in 120 µL
H O) and sodium ascorbate (NaAsc, 2 mg in 100 µL H O) was added to a CuSO solution
2
2
4
(
100 mM, 60 µL). Alkyne-imidazolium-oxime (22 mg) and NH -PEG -N (20 mg in 300 µL
2
6
3
DMF) were added to the solution. The reaction mixture was incubated at room
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2
2
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temperature for 1 hour. The product was purified by precipitation in ether/acetone = 10/1
1
four times and dried by a vacuum. Dark oil, 75% yield. H NMR (400 MHz, MeOD-d4) δ
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4
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8.60 (s, 1H; CH), 7.94 (s, 1H, Ar H), 7.92 (s, 1H, Ar H), 7.81 (d, J = 3, 1H, Ar H), 7.49 (d,
J = 3, 2H, Ar H), 7.33 (d, J = 6, 2H, Ar H), 5.59 (s, 2H; CH ), 4.45 (t, J = 3, 2H; CH ), 4.35
2
2
(
t, J = 3, 2H; CH ), 3.78 (s, 2H; CH ), 3.50-3.44 (m, 24H; 12CH ), 2.62 (d, J = 3, 2H, CH ),
2
2
2
2
1
.81-1.79 (m, 2H; CH ), 1.59 (d, J = 3, 2H, CH ) (Figure S2). PEG-IO was used as a
2
2
standard for the calculation of oxime to BChE ratios of BChE-polymer-oxime conjugates.
Synthesis of alkyne-pyridinium-oxime
Isonicotinaldehyde (50 mmol), hydroxylamine hydrochloride (55 mmol) and K CO (7.6 g,
2
3
55 mmol) were mixed in 250 mL MeOH at room temperature overnight. After removing
the solvent by rotary evaporator, the residue was diluted with water (250 mL) and
extracted with ethyl acetate (5 × 150 mL). The combined extract was washed with brine
(
2 × 150 mL) and dried over MySO . Remove the solvent by rotary evaporator and got
4
the product isonicotinaldehyde oxime, 84% yield.
Isonicotinaldehyde oxime (10 mmol) and 1,4-diiodobutane (30 mmol) were dissolved in
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0 mL ACN and stirred at room temperature for 5 days. The resulting precipitate was
collected by filtration, then concentrated and added to 50 mL ethyl acetate. We next
collected and combined the precipitate, then purified by silica gel (EA/MeOH/HCOOH =
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
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0
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8
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0
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2
3
4
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4
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0
4/1/0.25) to generate the product 4-((hydroxyimino)methyl)-1-(4-iodobutyl)pyridin-1-ium
iodide in 77% yield.
4-((hydroxyimino)methyl)-1-(4-iodobutyl)pyridin-1-ium iodide (0.5 mmol) was dissolved in
5
mL ACN and 400 µL DMSO. N,N-dimethylprop-2-yn-1-amine (1 mmol) was added to
the ACN/DMSO solution and stirred at room temperature for 2 days. The solvent was
removed by rotary evaporation. The product alkyne-pyridinium-oxime was purified by
precipitation in ethyl acetate/acetone (2/1) three time and dried by vacuum. Yellow
1
powder, 65% yield. H NMR (400 MHz, MeOD-d4) δ 8.82 (d, J = 6, 2H, Ar H), 8.35 (s,
1H, Ar H), 8.19 (d, J = 6, 2H, Ar H), 4.68-4.63 (m, 2H; CH ), 4.23 (d, J = 3, 2H, CH ), 3.49-
2
2
3.46 (m, 2H; CH ), 3.23 (t, J = 3, 1H; CH), 3.15 (s, 6H; 2CH ), 2.18-2.07 (m, 2H; CH ),
2
3
2
1.91-1.87 (m, 2H; CH ) (Figure S3).
2
Synthesis of PEG-PO
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1
PEG-PO was synthesized as described previously. Briefly, BTTAA (13 mg in 300 µL
H O) and sodium ascorbate (NaAsc, 20 mg in 200 µL H O) were added to a CuSO
2
2
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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1
2
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4
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9
0
solution (100 mM, 300 µL). Alkyne-pyridinium-oxime (52 mg) and NH -PEG -N (55 mg
2
3
3
in 600 µL DMF) were then added to the solution and the reaction mixture was incubated
at room temperature overnight. The product was purified by precipitation in ether/acetone
1
=
10/1 four times and dried by a vacuum. Dark oil, 53% yield. H NMR (400 MHz, MeOD-
d4) δ 8.82 (s, 1H; Ar H), 8.80 (s, 1H; Ar H), 8.34 (d, J = 3, 2H, Ar H), 8.17 (d, J = 6, 2H,
Ar H), 4.69-4.62 (m, 6H; 3CH ), 3.95 (t, J = 3, 2H, CH ), 3.70 (t, J = 3, 2H, CH ), 3.65-
2
2
2
3
.59 (m, 8H; 4CH ), 3.35-3.32 (m, 2H; CH ), 3.16 (t, J = 3, 2H, CH ), 3.04 (s, 6H; 2CH ),
2
2
2
3
2.08-2.03 (m, 2H; CH ), 2.01-1.95 (m, 2H; CH ) (Figure S3). PEG-PO was used as a
2
2
standard for the calculation of oxime to BChE ratios of BChE-polymer-oxime conjugates.
Synthesis of BChE-polymer-oxime conjugates
BChE-PDMAA-N3 conjugate was synthesized as described previously.71 Alkyne-
imidazolium-oxime or alkyne-pyridinium-oxime was attached to the side chains of BChE-
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PDMAA-N conjugate by “click” chemistry. NaAsc (10 mM × 35 µL), CuSO (10 mM ×
3
4
1
7.6 µL), and BTTAA (10 mM × 35 µL) in distilled water were added into a BChE-PDMAA-
1
1
1
1
1
1
1
1
1
1
2
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0
N (27 µM BChE, 650 µL) solution. Alkyne-imidazolium-oxime or alkyne-pyridinium-oxime
3
(
50 mM) in distilled water was added in different oxime to BChE ratios (40-, 80- or 160-
fold). The reaction mixture was incubated at room temperature for 3 h. The BChE-
PDMAA-OX conjugates were purified by ultrafiltration (50 kDa cut-off membrane) four
times. The number of oximes per BChE monomer of the conjugates was determined by
BCA assay and UV absorbance at 280 nm (Figure S1). BChE-PDMAA-OX conjugates
were characterized by GPC and SDS-PAGE analysis.
Synthesis of PDMAA-IO and PDMAA-PO
To synthesize the azide containing copolymer, PDMAA-N , DMAA (1.20 mmol), MA-
3
PEG -N (60 µmol) and ATRP initiator (1 µmol) were mixed in 2 mL of PB buffer (50 mM,
6
3
pH 7.4), sealed, and bubbled with argon for 30 min at room temperature. Deoxygenated
Me TREN (8 µmol), CuBr (3 µmol) and NaAsc (4 µmol) in distilled water (1 mL) were
6
2
added to the initiator-monomer solution, sealed, and stirred for 2 h at room temperature.
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The copolymer PDMAA-N was purified by dialysis (8 kDa cut-off membrane) in distilled
3
water and lyophilization. The molecular weight (M ) and dispersity (Ð) of PDMAA-N were
n
3
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1
2
3
4
5
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1
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0
64.4 kDa and 1.87, respectively (Figure S4 and S5). GPC calibration was based on
poly(ethylene glycol) standards.
Alkyne-imidazolium-oxime or alkyne-pyridinium-oxime was attached to the side chains of
74
PDMAA-N conjugate by “click” chemistry to prepare PDMAA-IO or PDMAA-PO. NaAsc
3
(
10 mM × 80 µL), CuSO (10 mM × 40 µL), and BTTAA (10 mM × 80 µL) in distilled water
4
were added into a PDMAA-N (14 mg in 650 µL H O) solution. Alkyne-imidazolium-oxime
3
2
or alkyne-pyridinium-oxime (2 mg in 80 µL DMSO) was added and incubated at room
temperature overnight. PDMAA-IO or PDMAA-PO was purified by ultrafiltration (30 kDa
cut-off membrane) four times.
The oxime content of PDMAA-IO and PDMAA-PO, determined from the UV-VIS spectra,
were 12.5 wt% and 4.5 wt%, respectively (Figure S1 and S4).
Activity assay of BChE and BChE-PDMAA-OX conjugates
BTC was used as a substrate to determine the activity of BChE and BChE-PDMAA-OX
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conjugates at room temperature. 1 mL of BTC (1 mM) and DTNB (Ellman assay reagent,
0.1 mM) in PB buffer (50 mM, pH 7.4) was added to a 1.5 mL cuvette. Native BChE or
1
1
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1
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1
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2
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1
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9
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1
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9
0
BChE-PDMAA-OX conjugates (20 nM protein, 100 μL) was then added to the cuvette.75
The activity of BChE or BChE-PDMAA-OX conjugates was evaluated by monitoring BTC
hydrolysis, resulting in an increase in absorbance at 412 nm from TNB (extinction
-1
-1
coefficient of 14,000 M cm ), using a Lambda 2 PerkinElmer UV-VIS spectrophotometer.
Inhibition assay of BChE and BChE-polymer-oxime conjugates
BChE or BChE-PDMAA-OX conjugate (20 or 10 nM) was incubated with a 5-fold
stoichiometric excess of EMP-MeCyC or CMP-MeCyC at room temperature. An aliquot
was diluted 10 or 20 times in PB buffer (50 mM, pH 7.4) at specified time intervals. After
addition of BTC (1 mM) and DTNB (0.1 mM), we measured the enzymatic activity of each
group. The uninhibited BChE or BChE-PDMAA-OX conjugate was treated as 100%
activity at the time zero. Activity evaluation was performed in PB buffer (50 mM, pH 7.4)
by the Ellman assay using a Lambda 2 PerkinElmer UV-VIS spectrophotometer.75
Flu-OPNAs degradation assay
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6
A mixture of BChE, BChE-PDMAA-IO172 conjugate (or BChE-PDMAA-PO216 conjugate)
and PDMAA-IO (or PDMAA-PO) at various ratios (protein 50 nM; Oxime/BChE = 43 or
0
1
2
3
4
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9
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0
54) were incubated with a 5-fold excess of EMP-MeCyC (or CMP-MeCyC) in PB buffer
at pH 7.4. The total volume of the mixture was 100 µL. The fluorescence intensity (ex 400
nm; em 446 nm) was measured using a Bio-Tek Synergy H1 plate reader at specified
time intervals.
Reactivation assay of BChE and BChE-PDMAA-OX conjugates
A reactivation assay was carried out with BChE or BChE-PDMAA-OX conjugates (1 µM)
in 1 mg/mL bovine serum albumin (BSA) solution in PB buffer (50 mM, pH 6.0) that we
mixed with a 10-fold molar excess of EMP-MeCyC or CMP-MeCyC in PB buffer (50 mM,
pH 6.0) for 10 min. The completely inactive BChE or conjugates were transferred into an
ultrafiltration tube (50 kDa cut-off membrane) to remove the excess of EMP-MeCyC or
CMP-MeCyC (10,000 × g for 5 min, three times), then diluted 50-fold in PB buffer (50 mM,
pH 8.0) at room temperature. At specified time intervals, the activities of BChE or
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conjugates were measured by Ellman assay using BTC (1 mM) and DTNB (0. 1 mM).
The activity of uninhibited BChE or conjugates were used to determine the 100% activity.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
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3
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3
4
4
4
4
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4
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4
4
4
5
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5
5
5
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5
5
5
6
0
1
2
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6
7
8
9
0
1
2
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8
9
0
1
2
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4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Inactivation and reactivation recycle of BChE-PDMAA-OX conjugates
BChE-PDMAA-OX conjugates 1, 2 or 3 (1 µM) in 1 mg/mL bovine serum albumin (BSA)
in PB buffer (50 mM, pH 6.0) solution was mixed with a 10-fold molar excess of EMP-
MeCyC in PB buffer (50 mM, pH 6.0) for 10 min. The completely inactive conjugates were
purified by ultrafiltration tube (50 kDa cut-off membrane, 10,000 × g for 5 min, three times),
then diluted 50-fold in PB buffer (50 mM, pH 8.0) and incubated at room temperature for
200 min. The conjugates were buffer-exchanged with PB buffer (50 mM, pH 6.0) and
concentrated to 1 µM, then mixed with a 10-fold molar excess of EMP-MeCyC in PB buffer
(50 mM, pH 6.0) and incubated at room temperature for 10 min. The completely inactive
conjugates were purified by ultrafiltration tube (50 kDa cut-off membrane, 10,000 × g for
5
min, three times), then diluted 50-fold in PB buffer (50 mM, pH 8.0) and incubated at
room temperature for 200 min. The inactivation and reactivation cycle were repeated
three times. At specified time intervals, the conjugate activity was measured by the Ellman
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assay using BTC (1 mM) and DTNB (0.1 mM) as substrates. The activity of uninhibited,
or activity recovered conjugates at pH 8.0 after 200 min, were used to determine the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
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9
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1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
100% activity value.
Tertiary structure-based prediction of NHS-ATRP initiator modification
The tertiary structure of BChE was downloaded from Protein Data Bank (PDB code:
6I2T). In order of influence on initiator-amino group specificity, the following properties
for each lysine residue and N-termini were determined: exposed surface area (ESA),
76
steric hindrance, pK , secondary structure, local charge, and H-bonding (Table S1).
a
RESULTS AND DISCUSSION
Synthesis and Characterization of BChE-polymer-oxime conjugates
To synthesize BChE-polymer-oxime conjugates, an N-hydroxysuccinimide (NHS) ester
modified ATRP initiator (NHS-Br) was reacted with accessible amine groups on the
surface of BChE generating the macroinitiator BChE-Br. On average, 6.5 initiators were
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attached to each BChE monomer.71 To pinpoint where these modification sites were
located, we employed a tertiary structure-based predictive model that we had previously
developed to identify highly reactive amine groups on the surface BChE.76 This approach
enabled us to isolate the N-termini and a small group of lysine residues (K9, K60, K105,
K190, and K558) as the likely sites of reaction with the ATRP initiators (Table 1). Azide-
containing copolymers were grown from BChE-Br at fast-modified lysine residues and the
N-termini by in situ ATRP with a neutral monomer, N,N-dimethylacrylamide (DMAA), in
the presence of an azido monomer (MA-PEG -N ), to generate BChE-PDMAA-N
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71
conjugates, as described previously. Copper catalyzed azide-alkyne cycloaddition “click”
chemistry was then used to couple an alkyne-imidazolium-oxime or an alkyne-pyridinium-
oxime to the azido-containing copolymers of BChE-PDMAA-N conjugates (Figure 1).
3
Stoichiometric alkyne-imidazolium-oxime (IO) or alkyne-pyridinium-oxime (PO) excesses
of 40-fold, 80-fold or 160-fold were used to modify BChE-PDMAA-N in the preparation
3
of BChE-PDMAA-OX conjugates (BChE-PDMAA-IO and BChE-PDMAA-PO). The BChE-
PDMAA-OX conjugates were purified by ultrafiltration (100 kDa cut-off membrane) and
the oxime to BChE ratios were determined using the bicinchoninic acid (BCA) assay and
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absorbance at 280 nm (Figure S1). The number of imidazolium-oximes per BChE
tetramer was 28, 84 and 172 for BChE-PDMAA-IO conjugates (we refer to these
conjugates as BChE-PDMAA-IO , BChE-PDMAA-IO , and BChE-PDMAA-IO ,
0
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28
84
172
respectively). The number of pyridinium-oximes per BChE tetramer was 44, 104 and 216
for BChE-PDMAA-PO conjugates BChE-PDMAA-PO , BChE-PDMAA-PO , and
44
104
BChE-PDMAA-PO , respectively. The molecular weights and purities of the BChE-
216
PDMAA-OX conjugates were compared by gel permeation chromatography (GPC) and
sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis (Table
1, Figure 2). It is important to note that there are no GPC standards for these kinds of
protein-polymer conjugates, so we must be cautious when interpreting observed
conjugate molecular weights. That’s said, GPC can be useful when interpreting the
differences in apparent molecular weight between various conjugates.
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NHS-ATRP initiator modification
SI-ATRP of DMAA + MA-PEG -N
6
3
H
H
Br
N
N
O
O
BChE
BChE-Br
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nBr
N
y
nBr
Oximes (IO) Attachment by
O
x
NH
O
N
NH
O
click reaction
O
m
O
N
O
NH
O
N
N3
N
O
N3
N
I
N
N
BChE-PDMAA-N3
HO
BChE-PDMAA-IO
Cl
nBr
N
nBr
Oximes (PO) Attachment by
click reaction
y
O
x
O
N
NH
O
m
O NH
O
N
O
NH
O
N3
N
O
N
N3
N
I
BChE-PDMAA-N3
N
I
BChE-PDMAA-PO
N
N
OH
Oximes:
OH
O
O
H
Br
N
Cl
N
O
HO
N
N
O
N
N
N
N3
N
6
H
N
I
O
O
O
I
O
I
NHS-ATRP Initiator
DMAA
MA-PEG -N
Alkyne-imidazolium-oxime (IO)
Alkyne-pyridinium-oxime (PO)
6
3
Figure 1. Synthesis of butyrylcholinesterase-polymer-oxime conjugates using atom-
transfer radical polymerization (ATRP) and “click” chemistry. Additional acronyms: N-
hydroxysuccinimide
(NHS),
N,N-dimethylacrylamide
(DMAA),
N-(20-azido-
3
,6,9,12,15,18-hexaoxaicosyl)methacrylamide (MA-PEG -N ), butyrylcholinesterase
6
3
(
BChE).
We next investigated whether the BChE-PDMAA-OX conjugates retained bioactivity.
Using S-butyrylthiocholine iodide (BTC) as a substrate (10 µM, pH 8.0), we compared
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conjugate activity to that of native BChE. For BChE-PDMAA-IO , BChE-PDMAA-IO ,
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and BChE-PDMAA-IO , activity was inversely proportional to oxime content (76%, 67%
172
0
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and 54%, respectively). The BChE-PDMAA-PO conjugates also exhibited reduced
activity, but with a less pronounced inverse dependence on oxime content (63%, 48%
and 48% for BChE-PDMAA-PO , BChE-PDMAA-PO , and BChE-PDMAA-PO
44
104
216
respectively) (Figure 3). Previous work lead us to hypothesize that the activity loss of
BChE-PDMAA-OX conjugates resulted from a combination of polymer attachment and
reversible direct oxime inhibition of the enzyme.23, 71 In addition, Cu2+ and Cu-ligand
complexes can inhibit cholinesterase activity.77, 78 The alkyne-imidazolium-oxime had a
BChE IC of approximately 1 µM (Figure S6) and the alkyne-pyridinium-oxime BChE IC
50
50
was > 50 µM (Figure S7). The BChE-PDMAA-OX conjugates reported herein were the
highest activity BChE-polymer-oxime conjugates that we have studied to date. We have
recently determined the Michaelis-Menten kinetics for the BChE, BChE-Br, BChE-
71
PDMAA-N starting materials.
3
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Figure 2. Characterization of BChE, BChE-Br, BChE-PDMAA-N and BChE-PDMAA-OX
3
conjugates. A) GPC analysis of BChE (black curve), BChE-Br (red curve), BChE-PDMAA-
N (green curve), BChE-PDMAA-IO (yellow curve) and BChE-PDMAA-PO104 (blue
3
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curve) conjugates. B) SDS-PAGE analysis of BChE, BChE-Br, BChE-PDMAA-N and
3
BChE-PDMAA-IO conjugates. Lane 1: BChE; Lane 2: BChE-Br; Lane 3: BChE-PDMAA-
N ; Lane 4: BChE-PDMAA-IO ; Lane 5: BChE-PDMAA-IO ; Lane 6: BChE-PDMAA-
3
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IO ; Lane 7: Marker. C) SDS-PAGE analysis of BChE, BChE-Br, BChE-PDMAA-N and
172
3
BChE-PDMAA-PO conjugates. Lane 1: BChE-PDMAA-PO ; Lane 2: BChE-PDMAA-
44
PO ; Lane 3: BChE-PDMAA-PO ; Lane 4: BChE-Br; Lane 5: BChE; Lane 6: Marker;
104
216
Lane 7: BChE-PDMAA-N3.
Since only negatively charged oximates can reactivate BChE active sites, it was important
to determine the pK ’s for the monomeric alkyne-imidazolium-oxime and alkyne-
a
pyridinium-oxime.76 We used a spectroscopic assay and thereby surmised that the
oximes groups on BChE-PDMAA-IO and BChE-PDMAA-PO had pK ’s of 8.35 and 8.38,
a
respectively (Figure S8). Thus, at pH 6.0 the conjugates would be in their fully protonated
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oxime form, and therefore would be unable to undergo either inter- or intramolecular
reactivation. Therefore, in order to minimize any reactivation, we inhibited the BChE-
polymer-oxime conjugates by fluorogenic OPNAs (Flu-OPNA) analogues at pH 6.0 (vide
infra).
0
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Figure 3. Enzymatic activity of BChE, BChE-PDMAA-IO and BChE-PDMAA-PO
conjugates at a single concentration of BTC in PB (50 mM) buffer at pH 8.0. Results are
presented as mean values ± standard deviation (n = 3).
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Oxime containing copolymers protect BChE against OPs
Two racemic Flu-OPNAs analogues, which were designed to be analogues of VX or
cyclosarin after reaction with BChE, were used to inhibit BChE-polymer-oxime conjugates.
The Flu-OPNAs contained either an ethyl (E) or cyclohexyl (C) O-alkyl attached to a
methyl-phosphoryl (MP) moiety. The fluorescent moiety, 3-cyano-4-methyl-7-hydroxy
coumarin (MeCyC-OH), was released upon reaction between the BChE active site serine
and Flu-OPNAs (EMP-MeCyC or CMP-MeCyC) resulting in a significant and measurable
increase in fluorescence intensity.72 BChE-PDMAA-OX conjugates (10 or 20 nM) were
exposed to a 5-fold stoichiometric excess of Flu-OPNA in sodium phosphate buffer (pH
7.4, 50 mM) and the activity loss was measured over time by removing aliquots and
performing an activity assay as described above. Following the addition of Flu-OPNAs,
native BChE lost all activity within 10 min (Figure S9A). After exposure to EMP-MeCyC,
BChE-PDMAA-IO , BChE-PDMAA-IO , and BChE-PDMAA-IO retained 7%, 10% and
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