A comparison of flexible and constrained haptens in eliciting antibody catalysts for paraoxon hydrolysis

Article abstract of DOI:10.1016/S0968-0896(99)00026-7

A new amine-oxide hapten was employed as an antigen, producing seven monoclonal antibodies (mAbs) from a panel of 20 that catalyzed paraoxon hydrolysis. The current hapten design differs from that previously described in that the molecule is inherently mo

Full text of DOI:10.1016/S0968-0896(99)00026-7

Bioorganic & Medicinal Chemistry 7 (1999) 1145±1150
A Comparison of Flexible and Constrained Haptens in Eliciting
Antibody Catalysts for Paraoxon Hydrolysis
David A. Spivak, Timothy Z. Ho€man, Alisa H. Moore,
Matthew J. Taylor and Kim D. Janda*
The Scripps Research Institute, Department of Chemistry and The Skaggs Institute for Chemical Biology,
10550 North Torrey Pines Road, La Jolla, CA, 92037, USA
Received 11 November 1998; accepted 16 December 1998
AbstractÐA new amine-oxide hapten was employed as an antigen, producing seven monoclonal antibodies (mAbs) from a panel of
20 that catalyzed paraoxon hydrolysis. The current hapten design di€ers from that previously described in that the molecule is
inherently more ¯exible than its constrained predecessor. One of the seven antibody catalysts, mAb 1H9, showed the highest
activity and was selected for detailed study. At pH=8.77, the catalytic hydrolysis of paraoxon by mAb 1H9 followed Michaelis±
1
Menten kinetics a€ording a kcat=3.73Â10 4 min and a Km=1.12 mM with a rate acceleration kcat/kuncat=56. The hapten was
found to be a competitive inhibitor of antibody-catalyzed paraoxon hydrolysis with a Ki=0.54 mM. A comparison of both the
number and pro®ciency of antibody catalysts obtained when utilizing a ¯exible versus constrained hapten indicates that, for para-
oxon hydrolysis, constrained haptens elicit superior catalysts, suggesting that further development should begin with the use of
constrained haptens in producing more pro®cient antibody catalysts for paraoxon hydrolysis. # 1999 Elsevier Science Ltd. All
rights reserved.
Introduction
not been identi®ed.9 In addition to natural phospho-
triesterase, mutated buCHE (butyryl cholinesterase)
and DFPase are being developed for hydrolysis of
phosphotriesters and their derivatives.2
Phosphotriester containing compounds such as para-
oxon, parathion, and diazinon have been widely used as
commercial insecticides (Fig. 1). These compounds act
by inhibiting acetylcholinesterase, the enzyme respon-
sible for regulating the concentration of the neuro-
transmitter acetylcholine.1 In addition to insect control,
phosphotriesters can also poison mammalian systems as
well.1 As a result, structural variants of phosphotriesters
have been developed as potent biological warfare
agents. These compounds, such as sarin and soman, are
referred to as G-series nerve agents or their surrogates
such as DFP (Fig. 2). Methods for the selective neu-
tralization of these phosphotriesters would be of great
value as both protective measures2±5 and therapeutic
treatments.1
In addition to enzyme strategies, antibody catalysts could
in theory be employed for decontamination of toxic
phosphotriesters and their structural variants. Unlike
enzymes that provide for evolutionary determined sub-
strates, antibodies can be tailored for speci®c binding and
catalytic properties. Antibodies can also be utilized for
in vivo as well as in vitro applications. For example,
antibodies have been used therapeutically to treat
exposure to snake venoms10 and cocaine11 by binding
the toxic substances and isolating them from susceptible
tissues. An alternative approach to immunotherapy can
be envisioned by the utilization of antibody catalysts
that decompose the toxin of interest rather than simply
sequestering it. As part of a program directed toward
antibody mediated phosphotriester hydrolysis, we have
focused on developing catalytic antibodies for the
hydrolysis of paraoxon.12±14
Background and Signi®cance
There is considerable interest in developing biocatalysts
for the degradation of phosphotriester derived insecti-
cides and warfare agents.2±6 At present, there is one
known type of phosphotriesterase which utilizes two
zinc ions to e€ect catalysis.7,8 Although phospho-
triesterases exist in nature, their natural substrates have
Antibodies that hydrolyze carboxylate esters have been
elicited against compounds that mimic the tetrahedral
transition state of the respective reactions.15±18 In con-
trast, the analogous phosphotriester hydrolysis occurs
through a trigonal bipyramidal pentacoordinate transi-
tion state intermediate. The pentacoordinate transition
*Corresponding author. Tel.: 619-784-2516; fax: 619-784-2595; e-mail:
kdjanda@scripps.edu
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00026-7

1146
D. A. Spivak et al. / Bioorg. Med. Chem. 7 (1999) 1145±1150
Figure 1. Representative phosphotriester insecticides.
Figure 2. Representative G-series nerve agents and their surrogates.
state arises from attack of hydroxide and in-line dis-
placement of the leaving group in apical positions.19±21
Although the tetrahedral intermediate of carboxylate
ester hydrolysis is accurately mimicked by many transi-
tion state analogues,22,23 functionality representing
pentavalent centers24,25 are less common due to
instability or toxicity. The electrostatic features of the
phosphotriester hydrolysis transition state anticipated
along the reaction coordinate, however, are more easily
mimicked. Through a strategy known as `bait and
switch' catalysis, it has been previously shown that
charged haptens can induce complementary antibodies
that can catalyze hydrolysis by stabilization of a polar
transition state, or by eliciting a side chain group cap-
able of acid±base interactions.26±29
In an attempt to obtain antibody catalysts with
improved rates of catalysis, hapten 2 was designed as a
more faithful representation of the paraoxon structure
(Fig. 3). The linker itself incorporates a steric mimic of
one ethyl ester of paraoxon and the propyl group
mimics the other. An important feature was the absence
of the six-membered ring, which may increase the ¯ex-
ibility of the entire structure, allowing a variety of
binding and recognition modes. It was unknown, how-
ever, what a€ect this change would have on the elicita-
tion of antibody catalysts. It was hoped that a more
¯exible hapten would induce more pro®cient catalysts,
though in any event a comparison with our previous
studies may provide for future directions in this area.
Using the principle of hapten-charge complementarity,
it was found that either amine-oxide or quaternary
amine based haptens elicit antibodies with phospho-
triesterase activity.12±14 Initially, the best antibody cata-
lysts were induced by haptens incorporating an amine-
oxide center versus the quaternary amine function-
ality.12 This was presumably due to the ability of the N-
oxide to induce antibodies that not only complemented
the partial positive character of the phosphorous center,
but stabilized the developing negative charge at the
leaving-group oxygen as well. In a subsequent report,
paraoxon was found to be hydrolyzed by an antibody
elicited to compound 1 (Fig. 3), which contained the 4-
nitrophenyl group and the alkyl groups constrained in a
six-membered ring.13,14 The results gave a rate accelera-
tion (kcat/kuncat) of approximately 500, providing a
foundation for the development of more pro®cient
antibody catalysts for this hydrolysis reaction.
Results and Discussion
Hapten 2 was synthesized in four steps as described in
Scheme 1. Reductive amination of 6-amino caproic acid
n-hexyl ester with propionaldehyde gave the secondary
amine 3. Saponi®cation followed by alkylation with 4-
nitrobenzyl bromide gave the amino acid 5. Lastly, oxi-
dation of the amine with mCPBA gave the hapten 2,
which was conjugated to keyhole limpet hemocyanin
(KLH) for immunization.30
A total of 20 monoclonal antibodies were obtained that
bound speci®cally to hapten 2 as determined by enzyme-
linked immunosorbent assay (ELISA). Seven antibodies
were found to accelerate the hydrolysis of paraoxon
with varying degrees of activity. One of these, mAb
1H9, showed the highest activity and was selected for
detailed study. The catalytic hydrolysis of paraoxon by
mAb 1H9 followed Michaelis±Menten kinetics and dis-
played turnover, a€ording kcat values ranging from
1
1
1.21Â10 4 min to 1.25Â10 3 min in the pH range
8.0±10.0. Importantly, at a pH of 9.0, hapten analogue 7
(Scheme 2) was a competitive inhibitor of antibody 1H9
with a Ki of 0.54 mM. Hapten analogue 7 was synthe-
sized by alkylation of dipropylamine with 4-nitrobenzyl
bromide to give intermediate 6, followed by oxidation
with mCPBA (Scheme 2).
Figure 3. The constrained hapten 1, previously employed to obtain
antibody catalysts for the hydrolysis of paraoxon, and the more ¯ex-
ible hapten 2 used in the current study.

D. A. Spivak et al. / Bioorg. Med. Chem. 7 (1999) 1145±1150
1147
Scheme 1. Synthesis of N-oxide hapten 2.
Scheme 2. Synthesis of hapten analogue 7 used to determine Ki with mAb 1H9.
versus pH plot, and may suggest that antibody catalysis
was operating through stabilization of the transition
state. Transition state stabilization could occur through
complementarity of the antibody combining site to the
developing positive charge on the phosphorous center.
Electrostatic stabilization of the positive phosphorous
center could be achieved with an ionized Glu or Asp
amino acid side chain carboxylate (pKa less than 5.0) in
the combining site. In addition, amino acids with posi-
tively charged side chains (e.g. Arg or Lys) may also
stabilize either the developing negative charge on the
leaving group oxygen atom or the partial negative
charge on the phosphoryl oxygen atom in the transition
state for paraoxon hydrolysis. These interactions would
be expected to lower the energy of the charged transi-
tion state for phosphotriester hydrolysis in the antibody
combining site and may have led to the observed rate
The pH pro®le of catalytic hydrolysis of paraoxon by
mAb 1H9 showed a linear dependence of log kcat on the
concentration of hydroxide ion from pH 8±10 (Fig. 4).
This pH dependence was similar to that seen for anti-
body 3H5.14 Additionally, the uncatalyzed rate of
paraoxon hydrolysis was also found to be linearly
dependent on hydroxide concentration. These observa-
tions argued against general acid±base catalysis, because
no titratable pKa (apparent) was observed in the log kcat
acceleration. A comparison of Km/Ki (2.1) with kcat
/
kuncat (56), however, indicated that other e€ects besides
transition-state stabilization were present. The value
Km/Ki is typically taken to approximate the rate accele-
ration attainable by transition-state stabilization
alone,31 but the observed rate acceleration clearly
exceeds this value. Perhaps general-base catalysis by an
amino acid side chain possessing a pKa outside the pH
range shown in Figure 4 is utilized by mAb 1H9.
Substrate speci®city was assessed utilizing two com-
pounds that are structurally similar to paraoxon and the
hapten (Fig. 5). Compound 8 was synthesized by step-
wise addition of 4-nitrophenol, then 1-butanol to ethyl
dichlorophosphate. Compound 9 was obtained by
Figure 4. Dependence of log kcat antibody 1H9 catalyzed paraoxon
hydrolysis on pH.

1148
D. A. Spivak et al. / Bioorg. Med. Chem. 7 (1999) 1145±1150
found to destabilize the ground state energy of the cyclic
phosphodiester.32 Though it is unknown at this time
whether this is a general phenomenon, it is clear in this
case that the constrained hapten generated fewer, but
more pro®cient antibody catalysts than its ¯exible
counterpart. This will therefore represent a starting
point for improvements in the antibody-catalyzed
hydrolysis of paraoxon. Both examples, however, sup-
port the theory that amine-oxides provide crucial hap-
ten-charge complementarity for lowering the energy of
the developing charges in the transition state for phos-
photriester hydrolysis reactions.
Figure 5. Compounds employed to determine speci®city of hydrolytic
activity by mAb 1H9.
bis-condensation of 4-nitrophenyl phosphorodi-
chloridate. Both compounds 8 and 9 are homologous to
paraoxon and, as a result, mAb 1H9 was found to cat-
alyze the hydrolysis of 8 and 9. A quantitative measure
of substrate speci®city is kcat/Km (Table 1).
4
For paraoxon, kcat/Km=4Â10
at pH 9.0. This is
Experimental
General
approximately ®ve fold lower than for compound 8,
showing little di€erence in substrate speci®city due to a
change in one alkyloxy substituents. However, for
compound 9 a 100-fold decrease in kcat/Km was
observed relative to that for compound 8, suggesting
higher speci®city for the ethyl/butyl versus butyl/butyl
substituted phosphotriester. As in the case of antibody
3H5, the aromatic nitro group is most likely the primary
recognition element for the antibody,14 though it is clear
that the aliphatic substituents are also important deter-
minants for antibody-substrate binding and catalysis.
6-Amino-n-caproic acid n-hexyl ester p-toluenesulfonate
was purchased from TCI Chemicals. Paraoxon (90%)
was obtained from Aldrich Chemicals and then puri®ed
by ¯ash chromatography using EtOAc:hex (50:50) as
eluant. Flash chromatography was carried out using
Mallinckrodt silica gel 60 (230±400 mesh). Preparative
TLC was performed with Merck glass plates coated
with 1.0 mM silica. HPLC separations were carried out
on a Rainin Preparative HPLC using a Vydac reverse
phase C18 90A pharmaceutical column. 1H and 13C
NMR spectra were obtained on Bruker 300 mHz or
500 mHz instruments. Chromatographic and reagent
solvents were reagent grade and used as received. All
reagents were obtained from commercial sources and
were used without further puri®cation.
The design of ecient biocatalytic hydrolysis of phos-
photriesters and their derivatives is currently an impor-
tant goal for both in vitro and in vivo applications. In
this report, hapten 2 provided seven antibody catalysts
for the hydrolysis of paraoxon. The best of these anti-
bodies, 1H9, provided a rate acceleration of 56, 10-fold
lower than the previous report.14 The di€erences in
performance between antibody 3H5 and 1H9 may be
related to di€erences in hapten design. The design of
hapten 2 incorporated a more faithful representation of
the ground state paraoxon structure and provided
structural ¯exibility for a variety of binding and recog-
nition modes. This structural ¯exibility may be respon-
sible for the high number of antibody catalysts (seven
out of 20) elicited to hapten 2 for paraoxon hydrolysis
as compared to hapten 1 (one out of 25). Alternatively,
the more conformationally restricted alkyl groups of
hapten 1 may have provided a better steric mimic of the
trigonal bipyramidal transition state of paraoxon
hydrolysis, a€ording an antibody catalyst with a higher
rate acceleration (500 versus 56). Perhaps most impor-
tantly, the geometric constraints in hapten 1 may also
provide ground state strain for paraoxon hydrolysis.
With regard to this issue, a relevant comparison can be
found in the small di€erence in ÁÁH (1.2 kcal/mol) of
hydrolysis for the cyclic trimethylene phosphate versus
the acyclic dimethyl phosphate, where ring strain was
Antibody preparation
Hapten 2 was coupled to keyhole limpet hemocyanin
(KLH) and bovine serum albumin using 1-[3-(dimethyl-
amino)propyl]-3-ethylcarbodiimide hydrochloride (EDC)
as previously reported.33 Mice were immunized with the
KLH conjugate of 2, and monoclonal antibodies were
obtained using standard procedures.30 Antibody 1H9
was puri®ed via DEAE anion exchange chromato-
graphy, Protein G anity chromatography, and Mono
Q anion exchange chromatography and was demon-
strated to be >98% homogeneous by SDS acrylamide
gel electrophoresis.
Assays
All paraoxon hydrolysis reactions, as well as for com-
pound 8, were performed in 96-well microtiter plates
containing 235 mL of CHES bu€er with 20 mM antibody
and 2.5% DMSO. For these hydrolysis reactions,
Table 1. Comparison of kinetic parameters for antibody 1H9 catalyzed hydrolysis of compounds 8, 9, and paraoxon at various pH values
1
1
Substrate
pH
kuncat (min
)
kcat (min
)
Km (mM)
kcat/Km
6
5
5
5
6
4
4
3
3
5
4
4
3
3
5
Paraoxon
Paraoxon
8
8
9
8.77
9.25
9.00
9.25
9.00
6.62Â10
1.20Â10
4.32Â10
9.00Â10
1.48Â10
3.73Â10
5.36Â10
1.09Â10
1.25Â10
3.84Â10
1.12
1.06
0.44
0.28
1.03
3.33Â10
5.06Â10
2.48Â10
4.46Â10
3.73Â10

D. A. Spivak et al. / Bioorg. Med. Chem. 7 (1999) 1145±1150
1149
1
kinetic assays for the release of p-nitrophenol were per-
formed at 25 ꢀC on a Molecular Devices Thermomax
ELISA plate reader measured at l=405 nm for detec-
tion of 4-nitrophenolate. For each pH, kinetic para-
meters were obtained from the average of two runs
using substrate concentrations between 500 mM and
5.0 mM of paraoxon. Calibration curves for the absor-
bance of 4-nitrophenol were obtained for each pH.
Assays for the hydrolysis of compound 9 were carried
out on a Hitachi HPLC using a VYDAC 201TP504
reverse phase column. The mobile phase used was
CH3CN:H2O (34:66) (0.1% TFA) at a ¯ow rate of
1.0 mL/min with UV detection at l=320 nm. Dixon
plot experiments showed the paraoxon analogue 7 to be
a competitive inhibitor of paraoxon hydrolysis by anti-
body 1H9.
0.03 g (22%) of 5 as a yellow oil. H NMR (300 MHz,
CD3OD): d 8.14 (d, J=9 Hz, 2H), 7.47 (d, J=9 Hz, 2
H), 3.59 (s, 2H), 2.31±2.40 (m, 4H), 2.26 (t, J=8 Hz,
2H), 1.52±1.62 (m, 2H), 1.39±1.49 (m, 4H), 1.23±1.32
(m, 4 H), 0.83 (t, J=8 Hz, 3H), 13C NMR (75 mHz,
CD3OD): d 174.2, 148.8, 133.9, 129.1, 123.4, 58.3, 56.1,
53.9, 34.0, 29.7, 26.9, 24.8, 20.3, 11.8; HRMS (FAB+):
calcd 308.2006, found 308.1942.
6-[N-(4-Nitrobenzyl)-N-propyl-N-oxido]-amino caproic acid
(2). Amino acid 5 (0.02 g, 0.07 mmol) was suspended in
1.0 mL CH2Cl2. After mCPBA (60±70%, 0.022 g,
0.07 mmol) was added, the mixture was shaken on a
vortex machine for 30 min. The CH2Cl2 was evaporated
under reduced pressure, and the residue triturated with
Et2O to leave 12 mg of a white solid. The solid was
puri®ed by reversed phase HPLC (¯ow rate=1.0 mL/
min, l=254) using a 35:65 ratio of MeCN:H2O as
eluant. The product eluted at 7.5 min, and lyophiliza-
tion gave 7.5 mg (36%) of pure 2 as a clear oil. 1H
NMR (300 MHz, CD3OD): d 8.25 (d, J=9 Hz, 2H),
7.72 (d, J=9 Hz, 2H), 4.80 (s, 2H), 3.35±3.42 (m, 4H),
2.10 (t, J=7 Hz, 2H), 1.77±1.86 (m, 4H), 1.57 (tt,
J=8 Hz, 7 Hz, 2H), 1.27±1.34 (m, 2H), 0.90 (t,
J=7 Hz, 3H); 13C NMR (75 mHz, CD3OD): d 150.7,
135.4, 132.2, 124.9, 68.1, 66.8, 65.2, 34.3, 26.9, 26.5,
25.5, 23.4, 17.3, 11.2, 10.6; HRMS (FAB+): calcd
324.1685, found 324.1677.
6-N-Propylamino caproic acid n-hexyl ester (3). 6-
Amino-n-caproic acid n-hexyl ester (2.08 g, 9.7 mmol),
propionaldehyde (0.78 g, 13.5 mmol), and acetic acid
(1.18 g, 19.3 mmol) were dissolved in 30.0 mL of MeOH,
followed by addition of 0.05 g 3 angstrom molecular
sieves. Sodium cyanoborohydride was added in one
portion and the reaction mixture was stirred at rt for
48 h. The reaction mixture was brought to pH=2 slowly
using concentrated HCl with concomitant evolution of
gas. After gas evolution stopped, the solution was
brought to pH=13 using solid sodium hydroxide.
Methanol was removed by rotary evaporation, and the
remaining aqueous layer extracted with Et2O
(3Â50 mL). The organic layers were combined and
washed with saturated aqueous sodium chloride
(1Â50 mL), dried over Na2SO4, and evaporated under
reduced pressure. Puri®cation by ¯ash chromatography
using MeOH as eluant gave 0.41 g (16%) of 3 as a clear
residue. 1H NMR (300 mHz, CD3OD): d 4.04 (t,
J=6 Hz, 2H), 3.65 (s, 1H), 2.57 (m, 4H), 2.31 (t,
J=9 Hz, 2H), 1.63 (m, 4H), 1.49 (m, 4H), 1.34 (m, 8H),
0.89 (m, 6H); 13C NMR (75 MHz, CD3OD): d 173.7,
64.3, 51.8, 49.6, 34.1, 31.3, 29.7, 28.4, 26.8, 25.4, 24.7,
23.0, 22.4, 13.8, 11.6; HRMS (FAB+): calcd 257.2355,
found 257.2350.
N-(4-Nitrobenzyl)-dipropyl amine (6). Dipropylamine
(3.39 mL, 24.7 mmol) and 4-nitrobenzyl bromide (8.21 g,
38 mmol) were dissolved in 27 mL of DMF. Cesium
carbonate (12.38 g, 38 mmol) was added and the mixture
stirred 1 h. at 0 ꢀC, then 3 h at room temperature. The
reaction mixture was diluted with 5 equiv of H2O, then
extracted with EtOAc (3Â100 mL). The organic layers
were combined, washed with aqueous saturated NaCl
(2Â150 mL), dried over Na2SO4, and evaporated in
vacuo to leave a yellow oil. The oil was redissolved in
100 mL H2O, and acidi®ed to pH=1, and the aqueous
solution washed with EtOAc (2Â100 mL). The aqueous
phase was basi®ed to pH=12 and the product extracted
into EtOAc (3Â100 mL). The organic layers were
combined, washed with aqueous saturated NaCl
(2Â150 mL), dried over Na2SO4, and evaporated in
vacuo to leave 2.0 g of crude product. The crude mate-
rial was puri®ed by ¯ash chromatography using three
eluants in the following order: Hexane:EtOAc (80:20),
EtOAc (100%), MeOH (100%), to give 1.8 g of 6 as a
yellow oil. 1H NMR (500 MHz, CD3OD): d 8.17 (d,
J=10 Hz, 2H), 7.53 (d, J=10 Hz, 2H), 3.63 (s, 2H), 2.38
(t, J=15 Hz, 4H), 1.5±1.4 (m, 4H), 0.86 (t, J=15 Hz, 6
H); 13C NMR (125 MHz, CD3OD): d 150.2, 148.9,
131.2, 124.8, 59.7, 57.8, 21.7, 12.7; HRMS (EI+): calcd
237.1606, found 237.1603.
6-N-Propylamino caproic acid (4). Amine 3 (0.17 g,
0.66 mmol) was dissolved in 1.5 mL MeOH, and an
equal volume of 1M NaOH was added. After stirring
for 1 h, the reaction mixture was puri®ed directly by
¯ash chromatography using MeOH as the eluant to give
1
0.13 g (94%) of 4 as a white solid. H NMR (300 mHz,
CD3OD): d 3.11 (s, 1H), 2.67±2.75 (m, 4H), 2.23 (t,
J=7 Hz, 2H), 1.34±1.54 (m, 6H), 0.78 (t, J=7 Hz, 3 H);
13C NMR (75 MHz, CD3OD): d 165.7, 42.1, 40.5, 25.2,
24.4, 17.0, 15.5, 10.7; HRMS (FAB+): calcd 173.1416,
found 173.1418.
6-[N-(4-Nitrobenzyl)-N-propyl]-amino caproic acid (5).
Amino acid 4 (0.07 g, 0.4 mmol) was dissolved in
3.0 mL MeOH. Triethylamine (0.14 mL, 1.0 mmol) and
4-nitrobenzyl bromide (0.11, 0.5 mmol) were added and
the reaction stirred for 48 h. The reaction was evapo-
rated under pressure, and the product puri®ed by pre-
parative TLC using two eluants: ®rst with a EtOAc:hex
(50:50) mixture, then with pure EtOAc. This a€orded
N-(4-Nitrobenzyl)-dipropyl-N-oxide (7). N-(4-Nitroben-
zyl)-dipropylamine 6 (0.20 g, 0.85 mmol) was dissolved
in 20 mL of CH2Cl2 and the solution brought to 0 ꢀC.
After addition of mCPBA (50±60%, 0.16 g, 0.93 mmol)
the solution was stirred for 1 h at 0 ꢀC, then 4 h at room
temperature. Evaporation of the CH2Cl2 left the crude
product which was redissolved in 50 mL H2O and

1150
D. A. Spivak et al. / Bioorg. Med. Chem. 7 (1999) 1145±1150
brought to pH=12 using saturated sodium carbonate.
The aqueous phase was extracted with CH2Cl2 (3Â50 mL)
and the organic layers combined, washed with aqueous
saturated NaCl (2Â150 mL), dried over Na2SO4, and
evaporated in vacuo to leave 60.0 mg of crude solid. Pur-
i®cation of the crude material by ¯ash chromatography
was accomplished using four mobile phases in the follow-
ing order: EtOAc:hex (50:50), EtOAc (100%), MeOH
(100%), MeOH:CHCl3 (90:10). The product 7 (70.0 mg,
References and Notes
1. Ecobichon, D. J. In Casarett and Doull's Toxicology: The
Basic Science of Poisons, 4th ed.; Amdur, M. O.; Doull, J.;
Klaasen, C. D.; Eds.; McGraw-Hill: New York, 1991; p 565.
2. Ember, L. Chem. & Eng. News 1997, 75, 26.
3. LeJeune, K. E.; Wild, J. R.; Russel, A. J. Nature 1998, 395, 27.
4. Cheng, T. C.; Calomiris, J. J. Enzyme Microb. Technol.
1996, 18, 597.
5. LeJeune, K. E.; Russel, A. J. Biotechnol. Bioeng. 1996, 51, 450.
6. Alternative Technologies for the Destruction of Chemical
Agents and Munitions, National Research Council on Engi-
neering and Technical Systems, Washington D.C., 1993.
7. Omburo, G. A.; Kuo, J. M.; Mullins, L. S.; Raushel, F. M.
J. Biol. Chem. 1992, 267, 13278.
1
33%) was obtained as a white solid. H NMR (500 MHz,
CD3OD): d 8.26 (d, J=12 Hz, 2H), 7.85 (d, J=12 Hz, 2
H), 4.47 (s, 2H), 3.0±3.15 (m, 4H), 1.9±1.7 (m, 4 H), 0.95
(t, J=15 Hz, 6H); 13C NMR (125 MHz, CD3OD): d 150.6,
138.9, 135.5, 124.7, 70.1, 68.8, 18.1, 11.6; HRMS (EI+):
calcd 253.1546, found 253.1552.
8. Lewis, V. E.; Nonarski, W. J.; Wilk, J. R.; Raushel, F. M.
Biochemistry 1988, 27, 1591.
9. Caldwell, S. R.; Newcomb, J. R.; Schlect, K. A.; Raushel,
F. M. Biochemistry 1991, 30, 7438.
Butyl-ethyl-(4-nitrophenyl) phosphate (8). Ethyl dichloro-
phosphate (1.0 g, 6.14 mmol) was dissolved in 10.0 mL
CH2Cl2 and Et3N (2.56 mL, 18.42 mmol) was added.
para-Nitrophenol (0.854 g, 6.14 mmol) was dissolved in
another 10.0 mL CH2Cl2 and added dropwise to the
vigorously stirring reaction mixture over 10 min and
allowed to stir at rt for 20 h. Afterward, 1-butanol
(0.454, 6.14 mmol) was added in one portion and stir-
ring continued for another 15 h. The reaction mixture
was evaporated under reduced pressure then puri®ed by
¯ash chromatography and preparative TLC using
EtOAc:hex (50:50) as eluant to give 0.38 g (20%) of 8 as
10. Butler, V. P., Jr. Pharmacol. Rev. 1982, 34, 109.
11. Carrera, M. R.; Ashley, J. A.; Parsons, L. H.; Wirsching,
P.; Koob, G. F.; Janda, K. D. Nature 1995, 378, 727.
12. Rosenblum, J. S.; Lo, L.-C.; Li, T.; Janda, K. D.; Lerner,
R. A. Angew. Chem. 1995, 107, 2448; Angew. Chem., Int. Ed.
Engl. 1995, 34, 2275.
13. Lavey, B. J.; Janda, K. D. Bioorg. Med. Chem. Lett. 1996,
6, 1523.
14. Lavey, B. J.; Janda, K. D. J. Org. Chem. 1996, 61, 7633.
15. Tramantano, A.; Janda, K. D.; Lerner, R. A. Science
1986, 234, 1566.
16. Tanaka, F.; Linoshita, K.; Tanimura, R.; Fujii, I. J. Am.
Chem. Soc. 1996, 118, 2332.
17. Li, T.; Hilton, S.; Janda, K. D. J. Am. Chem. Soc. 1995,
117, 2123.
18. Gibbs, R. A.; Benkovic, P. A.; Janda, K. D.; Lerner, R.
A.; Benkovic, S. J. J. Am. Chem. Soc. 1992, 114, 3528.
19. Caldwell, S. R.; Raushel, F. M.; Weiss, P. M.; Cleland, W.
W. Biochemistry 1991, 30, 7444.
1
a yellow oil. H NMR (300 MHz, CDCl3) d 8.22 (d,
J=9 Hz, 2H), 7.35 (d, J=9 Hz, 2H), 4.14±4.26 (m, 4 H),
1.63±1.69 (m, 2H), 1.33±1.42 (m, 4H), 0.91 (t, J=7 Hz,
3H); 13C NMR (125 MHz, CDCl3): d 155.8, 144.9,
125.9, 120.7, 69.1, 65.4, 32.4, 18.8, 16.3, 13.7; HRMS
(Fab+): calcd 304.0950, found 304.0946.
20. Knowles, J. R. Ann. Rev. Biochem. 1980, 49, 877.
21. Khan, S. A.; Kirby, A. J. J. Chem. Soc. B. 1970, 1172.
22. Lerner, R. A.; Benkovic, S. J.; Schultz, P. G. Science 1991,
252, 659.
23. Janda, K. D.; Chen, Y.-C. J. The Pharmacology of Mono-
clonal Antibodies, Vol. 113, Springer, Berlin, 1994, 209.
24. Chen, Y.-C. J.; Janda, K. D. J. Am. Chem. Soc. 1992, 114,
1488.
25. Moriarty, R. M.; Hiratake, J.; Liu, K. J. Am. Chem. Soc.
1990, 112, 8575.
26. Shokat, K.; Uno, T.; Schultz, P. G. J. Am. Chem. Soc.
1994, 116, 2261.
Dibutyl-(4-nitrophenyl) phosphate (9). 4-Nitrophenyl
phosphorodichloridate (1.0 g, 3.91 mmol) was dissolved
in 20.0 mL CH2Cl2 and Et3N (1.2 mL, 8.6 mmol) was
added. 1-Butanol was added in one portion at room
temperature, and the reaction mixture stirred for 23 h.
The reaction mixture was evaporated under reduced
pressure then puri®ed by ¯ash chromatography using
the mobile phase EtOAc:hex (50:50) to give 0.75 g
(65%) of product 9 as a yellow oil. 1H NMR (300 MHz,
CDCl3) d 8.17 (d, J=9 Hz, 2H), 7.32 (d, J=9 Hz, 2H),
4.12 (dt, J=6 Hz, 6 Hz, 4H), 1.63 (tt, J=6 Hz, 7 Hz,
4H), 1.34 (dq, J=6 Hz, 6 Hz, 4H), 0.87 (t, J=7 Hz, 6H);
13C NMR (125 MHz, CDCl3): d 155.8, 144.7, 125.7,
120.6, 68.9, 32.1, 18.6, 13.6; HRMS (FAB+): calcd.
332.1263, found 332.1258.
27. Khalaf, A. I.; Proctor, G. R.; Suckling, C. J.; Bence, L.
H.; Irvine, J. I.; Stimson, W. H. J. Chem. Soc. Perkin Trans. 1
1992, 1475.
28. Janda, K. D.; Weinhouse, M. I.; Dannon, T.; Pacelli, K.
A.; Schloeder, D. M. J. Am. Chem. Soc. 1991, 113, 5427.
29. Janda, K. D. Biotechnol. Prog. 1990, 6, 178.
30. Kohler, G.; Milstein, C. Nature 1975, 256, 495.
31. Lienhard, G. E. Science 1973, 180, 149±154.
32. Marsh, F. J.; Weiner, P.; Douglas, J. E.; Kollman, P. A.;
Kenyon, G. L.; Gerlt, J. A. J. Am. Chem. Soc. 1980, 102, 1660.
33. Janda, K. D.; Weinhouse, M. I.; Schloeder, D. M.; Ler-
ner, R. A.; Benkovic, S. J. J. Am. Chem. Soc. 1990, 112, 1274.
Acknowledgements
This work was supported in part by the National Insti-
tutes of Health (GM 43858), and an Individual National
Research Service Award to D.A.S. (NIGMS GM17822).