Squaric monoamide monoester as a new class of reactive immunization hapten for catalytic antibodies

Article abstract of DOI:10.1016/j.bmcl.2005.06.052

A squaric monoester monoamide motif was employed as an effective reactive immunogen for the discovery of monoclonal antibodies with reactive residue(s) in their combining sites. Two antibodies, 2D4 and 3C8, were uncovered that enhance paraoxon hydrolysis

Full text of DOI:10.1016/j.bmcl.2005.06.052

Bioorganic & Medicinal Chemistry Letters 15 (2005) 4304–4307
Squaric monoamide monoester as a new class of
reactive immunization hapten for catalytic antibodies
Yang Xu, Noboru Yamamoto, Diana I. Ruiz, Diane S. Kubitz and Kim D. Janda*
The Skaggs Institute for Chemical Biology, Departments of Chemistry and Immunology, The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, CA 92037, USA
Received 27 May 2005; revised 15 June 2005; accepted 16 June 2005
Available online 19 July 2005
Abstract—A squaric monoester monoamide motif was employed as an effective reactive immunogen for the discovery of monoclo-
nal antibodies with reactive residue(s) in their combining sites. Two antibodies, 2D4 and 3C8, were uncovered that enhance parao-
xon hydrolysis over background. Kinetic analysis of these antibodies was performed and interestingly both undergo a single
turnover event due to covalent modification within the antibody combining site. Because antibodies 2D4 and 3C8 result in covalent
attachment and thus inactivation of paraoxon, they could be useful probes for investigating paraoxon intoxication.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Reactive immunization, the most recently developed
hapten manifold, has led to the production of new anti-
body catalysts.1–3 In contrast to traditional hapten strat-
egies, where Ônon-reactiveÕ transition state analogs are
employed, reactive immunization relies on an antigen
so highly reactive that a chemical reaction occurs in
the antibody combining site during immunization. The
importance of this approach is that it allows for the iso-
lation of antibodies that have highly reactive residues in
their binding cleft, which, if engaged with an appropri-
ate substrate homologue, can provide proficient turn-
over.3–5 Reactive immunization has been viewed as a
pivotal tool for the recruitment of more sophisticated
catalytic antibodies. However, despite its undeniable
merits, exploration and success of new reactive immuni-
zation haptens have been limited to only two major
manifolds, phosphonate diesters3 and b-diketones.6–8
Clearly, new reactive immunization haptens could pro-
vide additional opportunities for harnessing promising
antibody catalysts.
The toxic effect of this class of insecticide is known to
be associated with their inhibition of mammalian acetyl-
cholinesterase.11–13 Antibodies have a long history of
being used as therapeutic tools for the treatment of poi-
soning via neutralization and elimination of toxins from
susceptible tissues.14–16 Catalytic antibodies would be
especially advantageous for this purpose, as a catalytic
antibody would not only sequester the foreign antigen,
but also decompose and subsequently release innocuous
products, thus regenerating the antibody for further
substrate degradation.
We have mapped out the design and preparation of a
new class of reactive immunization hapten based on
the well-developed chemistry of squaric acid and its
derivatives. The synthesis of squaric acid was first
achieved in 1959 and this initial report spurred the syn-
theses of a vast array of squaric acid derivatives.17–19
Exploration of this class of molecule has been constant
due to its unique chemical properties, wherein the gener-
al reactive motif of squaric acid is due to its two acidic
hydroxyl groups (pKa1 = 0.54, pKa2 = 3.48).20,21 These
functionalities act not only as proton acceptor sites,
but also as nucleophilic binding loci for divalent
metal ions.20,22–25 The reactivity of squaric acid is
exemplified further by its diester derivatives that can
undergo stepwise nucleophilic substitution when treated
with nucleophiles, i.e., amines, giving rise to the
corresponding squaramides.26 This unique reactivity
enables the employment of squarate molecules in multi-
ple research areas such as enzyme inhibition and the
Paraoxon is a representative phosphate insecticide that
is used as the dominant form of insect control world-
wide.9 Unfortunately, these insecticides are often found
responsible for the poisoning of agricultural workers.10
Keywords: Reactive immunization; Squaric monoester monoamide;
Catalytic antibodies.
*
Corresponding author. Tel.: +1 858 784 2516; fax: +1 858 784
2515; e-mail: kdjanda@scripps.edu
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.06.052

Y. Xu et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4304–4307
4305
conjugation of oligosaccharides to proteins and polyaza-
macrocycles.27–29 Herein, we report the design, prepa-
agricultural workers. We chose this substrate based on
the following criteria: (A) phosphotriesters are more sus-
ceptible to nucleophilic hydrolysis relative to phospho-
diester and monoester; therefore, paraoxon as
substrate might provide sufficient reactivity toward the
antibody catalyst during initial screening; (B) hydrolysis
of paraoxon leads to the release of 4-nitrophenol, a
compound that can be easily detected quantitatively by
well-established UV assay methods32; (C) antibodies
that catalyze paraoxon hydrolysis could have potential
therapeutic value in the treatment of paraoxon
intoxication.
ration, and immunization results of
a
reactive
immunization hapten based on a squaric monoester
monoamide motif; two of the monoclonal antibodies
(mAbs) elicited during immunization showed the ability
to recognize and cleave paraoxon, a common phosphotri-
ester pesticide.9
Hapten 1 was designed and synthesized as described in
Scheme 1. Ethyl squarate 1 was programmed to trap a
nucelophilic residue at the antibody combining site via
a nucleophilic substitution reaction (Scheme 2). Hapten
1 was coupled to the carrier proteins, bovine serum albu-
min (BSA) and keyhole limpet hemocyanin (KLH), by
prior activation with sulfo-N-hydroxysuccinimide and
1-[3-(dimethylamino)propyl]-3-ethylcarbidiimide hydro-
chloride (EDCI) while the KLH-1 conjugate was utilized
to inoculate Balb-c mice. Following antibody production
by standard hybridoma technology,30,31 17 (mAbs) were
generated that bound to the BSA-1 conjugate. All
(mAbs) were found to belong to the IgG class and were
purified as described previously.2
The reactions were carried out in 50 mM bicine buffer
(pH 8.0) and monitored at 405 nm on a Cary50 UV
spectrometer to detect the release of 4-nitrophenol. In
each reaction, 20 lM antibody was reacted with 1 mM
substrate. From the 17 mAbs that were elicited against
hapten 1, two mAbs, 2D4 and 3C8, were found to accel-
erate the hydrolysis of paraoxon with moderate rate
enhancement over the background reaction. However,
only single turnover was observed for both antibodies.
The pre-steady-state kinetic data are summarized in
Table 1.
The 17 mAbs were screened for phosphotriesterase
activity against the substrate paraoxon (Scheme 3), a
commonly used pesticide often found poisonous among
The observation of a single turnover led to the specula-
tion that the antibodies might be susceptible to product
Scheme 1. Synthesis of the squaric monoester monoamide hapten. Reagents: (a) glutaric anhydride, CHCl3, 84%; (b) Pd/C, H2, MeOH, 84%;
(c) 3,4-diethoxycyclobut-3-ene-1,2-dione, DIPEA, EtOH, 83%; (d) EDCI, sulfo-NHS, BSA/KLH.
Scheme 2. Hypothetical reactive immunization mechanism using hapten 1 to trap a nucleophilic residue within antibody combining site.
Scheme 3. Hydrolysis of paraoxon.

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Y. Xu et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4304–4307
Table 1. Kinetic data for mAb, 2D4, and 3C8 that accelerate the
paraoxon hydrolysisa
ered the catalytic activities of both antibodies. Again,
this evidence strongly suggests covalent modification at
a reactive residue in the combining site of these
antibodies.
mAb kcat (minÀ1
)
Km (mM) kcat/kuncat kcat/Km (MÀ1minÀ1
)
2D4 2.28 · 10À3 1.18
2.31 · 103 1.93
3C8 2.03 · 10À3 1.29
2.06 · 103 1.57
Putting this current research effort in perspective, a cat-
alytic antibody was previously disclosed by our labora-
tory to decompose paraoxon.32 Here, the N-oxide 3
was utilized as the hapten, following a Ôbait-and-switchÕ
strategy33,34 (Fig. 1). The hapten retains the nitrophenyl
group and general geometry of paraoxon. The partial
positive charge on the N-oxide nitrogen was placed to
induce a general base in the antibody combining site
to assist in the formation of hydroxide in proximity to
the phosphorus atom, while the partial negative charge
on the N-oxide oxygen atom could induce a general acid
capable of either protonating the leaving oxide group or
stabilizing the developing negative charge on the P@O
bond during the transition state. Out of 25 mAbs, anti-
body 3H5 was uncovered as a catalyst against paraoxon
hydrolysis. However, a linear dependence of the rate of
catalysis upon hydroxide ion concentration implied that
antibody 3H5 catalyzed the reaction primarily through
transition-state stabilization rather than a Ôbait-and-
switchÕ general acid/base catalysis mechanism.
a Determined in 50 mM bicine with 50 mM NaCl, pH 8.0, 5% DMSO
cosolvent at 23 ꢁC. The kuncat (paraoxon) = 9.86 · 10À7 minÀ1
.
inhibition. Thus, the possibility of active site product
inhibition was further investigated as follows. Both anti-
bodies 2D4 and 3C8 were incubated with a stoichiome-
tric amount of 4-nitrophenol, one of the products of
paraoxon hydrolysis. Neither antibody was affected by
4-nitrophenol, as both maintained hydrolytic activity
against paraoxon, suggesting the other product of
hydrolysis might be an inhibitor. Accordingly, both
antibodies were incubated with readily available diethyl
phosphorochloridate, which immediately hydrolyzes to
the diethyl hydrogen phosphate under the assay condi-
tions; again, neither antibodyÕs hydrolytic activity was
impaired.
The observation of a single turnover and a lack of prod-
uct inhibition strongly suggested that paraoxon could
participate in a covalent modification of the antibody,
inactivating it toward further hydrolysis. To investigate
this hypothesis, both antibodies were incubated with
paraoxon in large excess at 37 ꢁC for 60 h. After incuba-
tion, both antibodies were subjected to exhaustive dial-
ysis against bicine and assayed for hydrolytic activity.
Neither antibody was capable of further hydrolysis of
paraoxon. The inactivated antibodies were subsequently
treated with hydroxylamine in 50-fold excess at 37 ꢁC
for 4 h. After dialysis, about 40% of the hydrolytic activ-
ity was recovered for both antibodies. This evidence
strongly suggested that paraoxon was a covalent active
site inhibitor of the antibodies 2D4 and 3C8.
In our current study, we rely on a reactive immunogen to
elicit antibody catalysts that could decompose paraoxon
via covalent catalysis. In our approach, we took advan-
tage of the susceptibility of the squaric acid monoester
monoamide motif toward nucleophilic substitution to
trap a nucleophile residue in the antibody combining site.
A total of 17 mAbs were raised against hapten 1 and two
mAbs, 2D4 and 3C8, accelerated the hydrolysis of parao-
xon. Kinetic studies demonstrated that this event is med-
iated by a reactive residue in the antibody cleft. However,
it is likely based on our kinetic investigation that both
antibodies are subjected to irreversible inhibition form-
ing an unreactive phosphoryl-antibody intermediate.
This phosphoryl-antibody intermediate is stable in
buffer; however, in the presence of hydroxylamine the
To probe further possible Ôreactive immunizationÕ and
its mechanism, we investigated hapten analog 2 (Scheme
4) as an inhibitor. A stoichiometric amount of this
molecule was incubated with both antibodies, and to
the mixture was added 1 mM paraoxon. After 2 h incu-
bation, the reactivity of both 2D4 and 3C8 was fully
quenched. This same observation was made for incuba-
tion times as short as 10 min, indicating that the binding
of 2 is much more favored over that of paraoxon. For
this reason, a time-dependent paraoxon hydrolysis pro-
file for this inhibitor could not be obtained. Exhaustive
dialysis of analog 2-inhibited antibody did not regener-
ate antibody hydrolytic reactivity. However, treatment
of the inactivated antibody with 50-fold excess hydrox-
ylamine at 37 ꢁC for 4 h followed by dialysis fully recov-
Figure 1. N-Oxide hapten designed and synthesized by Lavey and
Janda.
Scheme 4. Synthesis of analog inhibitor 2: (a) 3,4-diethoxycyclobut-3-ene-1,2-dione, DIPEA, EtOH, 93%.

Y. Xu et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4304–4307
4307
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We have shown that the squaric monoester monoamide
motif is an effective reactive immunogen for the recovery
of antibodies with reactive residue(s) in their binding
pockets. It is important to note that the initial substrate
we used in our study to assay antibody activity was in
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Acknowledgments
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