Catalytic antibody mediated hydrolysis of paraoxon

Full text of DOI:10.1021/jo960629d

J . Org. Chem. 1996, 61, 7633-7636
7633
Ca ta lytic An tibod y Med ia ted Hyd r olysis of
P a r a oxon
Brian J . Lavey and Kim D. J anda*
Departments of Chemistry and Molecular Biology,
The Scripps Research Institute and the Skaggs Institute for
Chemical Biology, 10550 North Torrey Pines Road,
La J olla, California 92037
F igu r e 1. Some representative phosphate insecticides.
applying antibody catalysis to phosphate hydrolysis,12 we
chose to study the antibody mediated hydrolysis of
paraoxon.
Received April 4, 1996
Phosphate ester insecticides have become the dominant
form of insect control on the world market. First
synthesized in the 1930s and later developed in the 1940s
and 1950s, tetrasubstituted phosphorus(V) compounds
such as paraoxon, parathion, and diazinon (Figure 1)
became the commercial insecticides of choice after the
problems associated with the persistence in the environ-
ment of aromatic halocarbons such as DDT became
understood. As materials which will decompose slowly
in water,1,2 phosphotriesters such as paraoxon are be-
lieved to be less environmentally hazardous than other
categories of insecticides. Unfortunately, phosphate
insecticides are also most often responsible for the
poisoning of agricultural workers.3,4
The harmful effects of phosphotriester insecticides are
related to their inhibition of mammalian acetylcholinest-
erase, the enzyme responsible for regulating the in vivo
concentration of the neurotransmitter acetylcholine.
Early reports suggested that parathion was unreactive
toward mammalian acetylcholinesterase in vitro and was
presumably less toxic than paraoxon.5 It was rapidly
discovered, however, that parathion is converted into
paraoxon in vivo,6,7 by microsomal P450 enzymes via
oxidative desulfuration of phosphorus.8-10 Given the
broad substrate specificity of P450 enzymes, it is likely
that desulfuration occurs with other phosphorothioate
insecticides as well.
Antibodies have a long history of being used therapeu-
tically to treat poisoning by snake venoms and other
natural toxins by binding the foreign substances and
isolating them from susceptible tissues.11 We believe that
antibodies also have potential as a means of alleviating
the effects of overexposure to phosphate insecticides and
other phosphonate poisons, including nerve agents such
as sarin and tabun. This potential may be clinically
exploited further than has been done previously in
affinity based antibody therapies by using an antibody
to catalytically decompose the toxin of interest rather
than simply immobilize it. As part of a program of
In designing haptens that will induce antibody cataly-
sis, two strategies may be followed. In the first, a
compound which mimics the geometry and charge dis-
tribution of the transition state as closely as possible is
used as the hapten. In the second strategy, which is
referred to as “bait and switch”, a compound which is not
necessarily a transition-state analog is synthesized with
the intent of inducing specific complementary amino acid
residues capable of mechanistic interaction with the
substrate.13-17 Previous demonstrations of this principle
have focused on the placement of a general base in the
active site of an antibody binding pocket. Each strategy
has advantages and disadvantages with regard to a
particular reaction and its mechanism. Unfortunately,
in the case of phosphotriesters, enzymatic systems do not
provide a useful guide for hapten design. There is one
known type of phosphotriesterase18,19 which is a highly
efficient catalyst but requires two zinc ions which are
held in place by four active site histidines for catalytic
activity. Previous work with antibodies has successfully
made use of metal cofactors to bring about catalysis.20-23
However, designing a hapten that will induce an antibody
which is capable of binding two metal cations in a precise
alignment with a substrate remains problematic at this
time, so other strategies were pursued.
The hydrolysis of phosphotriesters is first order in
hydroxide ion and is believed to proceed via an in-line
displacement mechanism through a trigonal bipyramidal
transition state with the nucleophile and leaving group
in the apical positions.24-27 This implies that the hy-
(12) Rosenblum, J . S.; Lo, L.-C.; Li, T. ; J anda, K. D.; Lerner, R. A.
Angew. Chem. 1995, 107, 2448; Angew. Chem., Int. Ed. Engl. 1995,
34, 2275. For other work on antibody-mediated hydrolysis of phos-
phates and phosphonates, see: Scanlan, T. S.; Prudent, J . R.; Schultz,
P. G. J . Am. Chem. Soc. 1991, 113, 9397. Brimfield, A. A.; Lenz, D. E;
Maxwell, D. M.; Broomfield, C. A. Chem. Biol. Interact. 1993, 87, 95.
For recent surveys of the catalytic antibody literature, see: Lavey, B.
J .; J anda, K. D. In Antibody Expression and Engineering; Wang, H.
Y., Imanaka, T., Eds; ACS Symposium Series 604; American Chemical
Society: Washington, DC, 1995; Chapter 10. Schultz, P. G.; Lerner,
R. A. Science 1995, 269, 1835.
* Author to whom correspondence should be addressed. Phone:
International code + (619)-554-4318. Fax: International code + (619)-
554-6068. E-mail: kdjanda@scripps.edu.
(13) J anda, K. D. Abstracts of Papers, 198th National Meeting of
the American Chemical Society; American Chemical Society: Wash-
ington, DC, 1987; ORGN 196.
(14) J anda, K. D.; Weinhouse, M. I.; Schloeder, D. M.; Lerner, R.
A.; Benkovic, S. J . J . Am. Chem. Soc. 1990, 112, 1274.
(15) Shokat, K. M.; Leumann, C. J .; Sugasawara, R.; Schultz, P. G.
Nature 1989, 338, 269.
(1) Coates, H. Ann. Appl. Biol. 1949, 36, 156.
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(3) 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; pp 565.
(16) J anda, K. D. Biotechnol. Prog. 1990, 6, 178.
(4) Murphy, S. D. In Casarett and Doull’s Toxicology: The Basic
Science of Poisons, 3rd ed.; Amdur, M. O., Doull, J ., Klaasen, C. D.,
Eds.; McGraw-Hill: New York, 1986; pp 519.
(5) Aldridge, W. N.; Davison, A. N. Biochem. J . 1952, 52, 663.
(6) Dubois, K. P. ; Doull, J .; Coon, J . M. Fed. Proc. 1948, 7, 216.
(7) Diggle, W. M.; Gage, J . C. Nature 1951, 168, 998.
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25.
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1974, 23, 1733.
(17) J anda, K. D.; Weinhouse, M. I.; Dannon, T.; Pacelli, K. A.;
Schloeder, D. M. J . Am. Chem. Soc. 1991, 113, 5427.
(18) Omburo, G. A.; Kuo, J . M.; Mullins, L. S.; Raushel, F. M. J .
Biol. Chem. 1992, 267, 13278.
(19) Lewis, V. E.; Donarski, W. J .; Wilk, J . R.; Raushel, F. M.
Biochemistry 1988, 27, 1591.
(20) Iverson, B. L.; Lerner, R. A. Science 1989, 243, 1184.
(21) Wade, W. S.; Ashley, J . A.; J ahangiri, G. K.; McElhaney, G.;
J anda, K. D.; Lerner, R. A. J . Am. Chem. Soc. 1993, 115, 4906.
(22) Cochran, A. G.; Schultz, P. G. Science 1990, 249, 781.
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Chem. Soc. 1995, 117, 5627.
(11) Butler, V. P., J r. Pharmacol. Rev. 1982, 34, 109.
S0022-3263(96)00629-9 CCC: $12.00 © 1996 American Chemical Society

7634 J . Org. Chem., Vol. 61, No. 21, 1996
Sch em e 1. Syn th esis of N-Oxid e Ha p ten 1
Notes
F igu r e 2. Change in kcat with pH. The slope of 1.1 suggests
that the rate of hydrolysis is proportional to hydroxide ion
concentration.
drolysis of paraoxon should be susceptible to specific base
catalysis. Paraoxon hydrolysis also might be accelerated
by either the protonation of the leaving-group alkoxide
or the stabilization of developing negative charge on the
PdO bond.
We believed that both of these catalytic processes
might be accessible using a bait and switch approach with
N-oxide 1 as the hapten. The partial positive charge on
the tetrasubstituted nitrogen could induce a general base
that would assist in the formation of hydroxide in the
appropriate location to attack the phosphorus atom. The
partial negative charge on the N-oxide oxygen can induce
either a residue capable of protonating the leaving group
according to a bait and switch strategy or a residue that
would stabilize the developing negative charge on the
phosphoryl oxygen as the transition state is approached.
We hoped to define whether antibodies raised against 1
would act to hydrolyze paraoxon primarily by stabilizing
transient charges in the transition state or by general
acid and general base mechanisms. Hapten 1 was
synthesized by treating piperidone with nitrobenzyl
bromide and then forming amine 3 via reductive amina-
tion (Scheme 1). Attachment of the linker by treatment
with glutaric anhydride followed by oxidation with
mCPBA gave 1.
Hapten 1 retains the nitrophenyl group and general
shape of paraoxon. Previous work has shown that
antibodies raised against the p-amino analog of paraoxon
would bind to the insecticide.28,29 However, the use of a
phosphate triester as a hapten would make the incorpo-
ration of useful charged functionality difficult. The
retention of a nitroaromatic moiety in the hapten was
also believed to be desirable for induction of a strong
immune response. As a result, the linker was attached
on the cyclohexyl side of the molecule rather than the
aromatic portion.
F igu r e 3. Compounds examined for hydrolytic activity by
antibody 3H5.
Ta ble 1. Kin etic P a r a m eter s for th e Hyd r olysis of
P a r a oxon betw een p H 7.8 a n d 9.1
pH
kuncat (min-1
)
kcat (min-1
)
KM (mM)
7.80
8.25
8.51
8.75
9.15
7.97 × 10-7
1.80 × 10-6
2.23 × 10-6
2.95 × 10-6
5.64 × 10-6
3.99 × 10-4
7.00 × 10-4
1.15 × 10-3
1.30 × 10-3
1.95 × 10-3
1.60
1.92
3.73
4.40
5.05
hydroxide ion concentration in the pH range 7.8-9.1
(Figure 2, Table 1). The linear dependence of the rate of
catalysis on hydroxide ion concentration implies that
antibody 3H5 mediates the reaction primarily through
transition-state stabilization rather than general acid or
base catalysis. It is unclear whether this effect is due to
the inability of an N-oxide to induce a general base in
the antibody combining site, or whether it is simply that
any side chains capable of acting as a general base are
incorrectly positioned for the necessary in-line displace-
ment by hydroxide. Unfortunately, kinetic measure-
ments alone will not distinguish between these two
posssibilities.
In order to determine the substrate specificity of
antibody 3H5, four additional compounds were prepared
(Figure 3). Compound 5 was prepared by treating diethyl
phosphorochloridate with p-methoxyphenol in the pres-
ence of base. Compound 6 was synthesized from paraox-
on by hydrogenolysis over platinum on carbon followed
immediately by acetylation with acetic anhydride and a
catalytic amount of DMAP. Compounds 7 and 8 were
prepared by reacting p-nitrophenyl phosphorodichlori-
date with 1,3-propanediol and N-acetylserinol, respec-
tively. Antibody 3H5 did not appreciably accelerate the
rate of hydrolysis of any of these compounds. This
suggests that the aromatic nitro group is one of the
primary recognition sites for the antibody. The fact that
Of 25 monoclonal antibodies raised against 1, one
catalyzed the hydrolysis of paraoxon. Antibody 3H5
exhibited Michaelis-Menten kinetics and displayed turn-
over. At a pH of 8.25, hapten 1 is a competitive, tight-
binding inhibitor of antibody 3H5 with a Ki of 0.98 µM.
The kcat values of antibody 3H5 ranged from 3.8 × 10-4
min-1 to 1.9 × 10-3 min-1 and increased linearly with
(24) Knowles, J . R. Annu. Rev. Biochem. 1980, 49, 877.
(25) Khan, S. A.; Kirby, A. J . J . Chem. Soc. B 1970, 1172.
(26) Cox, J . R.; Ramsay, O. B. Chem. Rev. 1964, 64, 317.
(27) Caldwell, S. R.; Raushel, F. M.; Weiss, P. M.; Cleland W. W.
Biochemisty 1991, 30, 7444.
(28) Sternberger, L. A.; et al. US Army Sci. Conf. Proc. 1972, 3, 429.
(29) Hunter, K. W., J r.; Lenz, D. E. Life Sci. 1982, 30, 355.

Notes
J . Org. Chem., Vol. 61, No. 21, 1996 7635
123.6, 61.1, 52.9, 41.1. HRMS (ESI+): calcd 235.1083, found
235.1076. Anal. Calcd for C12H14N2O3: C, 61.52; H, 6.02; N,
11.96. Found: C, 61.51; H, 6.13; N, 11.85.
the hydrolysis of compounds 7 and 8 was not accelerated
suggests that the cyclohexyl and N-acyl portions of
hapten 1, which are closest to the KLH protein, are not
recognized by the antibody.
4-P ip er id in a m in e, 1-[(4-Nitr op h en yl) m eth yl]- (3). (Ni-
trobenzyl)piperidone 2 (0.852 g, 3.64 mmol) was dissolved in 20
mL of MeOH, and 3 Å molecular sieves (1.5 g), NH4OAc (2.845
g, 37.0 mmol), and NaBH3CN (0.127 g, 2.02 mmol) were added.
The reaction mixture was stirred at rt for 16 h and then
concentrated. H2O (10 mL) was added, followed by 1 M HCl to
pH 2. After gas evolution had ceased, the flask was placed in
an ice bath and KOH (s) was added until the pH was 7. The
solvent was evaporated to give 6.36 g of solid, which was
subjected to sinter funnel chromagraphy using MeOH/NH4OH/
CH2Cl2 as the mobile phase to give 711 mg (83%) of yellow oil
as the product. HPLC (λ ) 254 nm) indicated that this material
was 96% pure. It was used in the next step without further
purification. 1H NMR (300 MHz, CD3OD): δ 8.14 (d, J ) 8 Hz,
2 H), 7.53 (d, J ) 8 Hz, 2 H), 3.57 (s, 2 H), 2.82 (d, J ) 12 Hz,
2 H), 2.70-2.65 (m, 1 H), 2.55-2.35 (m, 1 H), 2.13 (t, J ) 12
Hz, 2 H), 1.82 (d, J ) 12 Hz, 2 H), 1.43-1.33 (m, 2 H). 13C
NMR: δ 146.9, 146.7, 129.3, 123.3, 61.9, 52.4, 48.4, 35.6. HRMS
(ESI+): calcd 236.1399, found 236.1392.
P en ta n oic Acid , 5-[[1-[(4-Nitr op h en yl)m eth yl]-4-p ip er -
id in yl]a m in o]-5-oxo- (4). Amine 3 (521 mg, 2.21 mmol) and
glutaric anhydride (0.386 g, 3.38 mmol) were dissolved in 4 mL
of CH2Cl2 and then stirred at rt for 6.5 h. Evaporation of the
solvent followed by flash chromatography (NH4OH/MeOH/CH2-
Cl2) afforded 633 mg (82% yield) of yellow solid. 1H NMR (300
MHz, CD3OD): δ 8.14 (d, J ) 9 Hz, 2 H), 7.53 (d, J ) 9 Hz, 2
H), 4.86 (s, 1 H), 3.85 (s, 1 H), 3.85-3.70 (m, 1 H), 3.0 (d, J ) 12
Hz, 2 H), 2.05 (t, J ) 12 Hz, 2 H), 1.89 (d, J ) 12 Hz, 2 H),
1.64-1.39 (m, 2 H). 13C NMR: δ 178.4, 175.4, 175.0, 148.8,
145.7, 131.6, 124.5, 62.5, 53.3, 52.0, 47.4, 36.4, 35.4, 34.0, 32.0,
22.9, 21.8. HRMS (FAB): calcd 350.1716, found 350.1719.
Calcd for C17H23N3O5: C, 58.43; H, 6.63; N, 12.03. Found: C,
58.53; H, 6.86; N, 11.82.
P en ta n oic Acid , 5-[[1-[(4-Nitr op h en yl)m eth yl]-4-p ip er -
id in yl]a m in o]-5-oxo- (1). Amino acid 4 (43 mg, 0.123 mmol)
was dissolved in 5 mL of MeOH, and mCPBA (Aldrich 50-60%,
60 mg) was added. The reaction mixture was stirred at rt for 1
h. Purification by preparative TLC using 2% AcOH/10% MeOH/
88% CH2Cl2 as the mobile phase gave 38 mg (84%) of white solid
as product. HPLC (λ ) 254 nm) indicated that this material
was 99% pure. 1H NMR (300 MHz, CD3OD): δ 8.13 (d, J ) 9
Hz, 2 H), 7.80 (d, J ) 9 Hz, 2 H), 4.55 (s, 2 H), 3.75-3.9 (m, 1
H), 3.65-3.50 (t, J ) 12 Hz, 2 H), 3.47-3.35 (d, J ) 12 Hz, 2
H), 2.26 (t, J ) 7 Hz, 2 H), 2.19 (t, J ) 7 Hz, 2 H), 2.15-2.00 (m,
2 H) 1.95-1.75 (m, 4 H). HRMS (FAB): calcd 366.1665, found
366.1656.
P h osp h or ic Acid , Dieth yl 4-Meth oxyp h en yl Ester - (5).
Hunig’s base (1mL) and p-methoxyphenol (255 mg, 2.06 mmol)
were dissolved in 3 mL of CH3CN, and diethyl phosphorochlo-
ridate was added (290 µL, 2.01 mmol). The reaction mixture
was stirred at rt for 15 h and then concentrated and redissolved
in 50 mL of EtOAc. After washing with citric acid (3 × 5 mL)
and brine (2 × 5 mL), the solution was evaporated and subjected
to flash chromatography using EtOAc/hexanes as the mobile
phase to give 345 mg of the known34 5 as a clear oil (66%). 1H
NMR (500 MHz, CDCl3): δ 7.12 (d, J ) 10 Hz, 2 H), 6.82 (d, J
) 10 Hz, 2 H), 4.22-4.16 (m, 4 H), 3.76 (s, 3 H), 1.33 (t, 7 Hz,
6 H). 13C NMR: δ 156.6, 144.2, 120.8, 114.5, 64.4, 55.5, 16.0.
HRMS (FAB): calcd 261.0892, found 261.0900.
The ability of antibody 3H5 to hydrolyze paraoxon
suggests that catalytic antibodies may be an appropriate
means of decomposing toxins such as insecticides in vivo.
The advantage of using a catalytic antibody rather than
an antibody which only binds to the toxin is that
hydrolysis of the toxin and the subsequent release of
relatively innocuous decomposition products leaves the
antibody free for further substrate binding. To be fully
useful in clinical therapy, higher rates of catalysis and
lower KM values than observed with 3H5 will be needed.
We believe, however, that this work makes a significant
first step toward developing antibodies for use in treat-
ment of organophosphate insecticide poisoning. In the
future, this principle may be applied to other materials.
Exp er im en ta l Section
Gen er a l. 13C NMR spectra were obtained at 125 MHz.
Melting points were determined on open slides and are uncor-
rected. Flash and sinter funnel30 chromatography were carried
out with Mallinckrodt silica gel 60 (230-400 mesh). Analytical
TLC was performed on Merck glass plates coated with 0.25 mm
silica. Preparative TLC was performed with Merck glass plates
coated with 1 mm silica. Chromatographic and reagent solvents
were reagent grade and were used as received. All reagents
were obtained from commercial sources and were used without
further purification. Brine refers to a saturated solution of
sodium chloride. Elemental analyses were performed by Gal-
braith Laboratories Inc.
An tibod y P r ep a r a tion . Hapten 1 was coupled to keyhole
limpet hemocyanin (KLH) and bovine serum albumin using 1-[3-
(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC)
as previously reported.14 Mice were immunized with the KLH
conjugate of 1, and monoclonal antibodies were obtained via
standard procedures.31,32 Antibody 3H5 was purified via DEAE
anion exchange chromatography, Protein G affinity chromatog-
raphy, and Mono Q anion exchange chromatography and was
demonstrated to be >98% homogeneous by SDS acrylamide gel
electrophoresis.
Assa ys. Paraoxon (90%) was purchased from Aldrich and
then purified via flash column chromatography using EtOAc/
CH2Cl2/hexanes as the mobile phase. Assays for the hydrolysis
of 6 and 7 were carried out on a Hitachi HPLC using a reverse
phase column and UV detection at λ ) 286 nm. All kinetic
assays for the release of p-nitrophenol were performed at 25 (
0.1 °C on a Molecular Devices Thermomax ELISA plate reader
using the absorbance of light at 405 nm to observe the formation
of p-nitrophenolate. Calibration curves for the absorbance of
p-nitrophenol were obtained for each pH.
All paraoxon hydrolysis reactions were performed in 96-well
microtiter plates containing 235 µL of 50 mM bicine with 20 µM
antibody and 5% DMSO. For each pH, kinetic parameters were
obtained from the average of two runs using substrate concen-
trations between 250 µM and 5 mM of paraoxon. Initial Dixon
plot experiments showed the hapten to be a tight-binding
inhibitor of antibody 3H5. As a result, the Ki was determined
using the Copeland33 variation of the Henderson plot.
P h osp h or ic Acid , 4-(Acetyla m in o)p h en yl Dieth yl Ester -
(6). A Parr hydrogenation bottle was placed under an Ar
blanket, and 5% Pt on C (20 mg) was added, followed by MeOH
(10 mL) and a solution of paraoxon (365 mg, 1.33 mmol) in 5
mL of MeOH. The flask was placed under 25 psi of H2 and
shaken at rt for 4 h. The resulting mixture was filtered, and
the solvent was evaporated to give 320 mg of brown oil. This
material was redissolved in 20 mL of CH2Cl2, and Ac2O (195
µL, 2.07 mmol), Hunig's base (340 µL, 1.95 mmol), and DMAP
(12 mg, 0.098 mmol) were added. The reaction mixture was
stirred at rt for 2 h, then diluted with CH2Cl2, washed with citric
acid, NaHCO3, and brine, then dried with Na2SO4, and evapo-
rated to give 350 mg of a brown oil. Flash chromatography using
4-P ip er id in on e, 1-[(4-Nitr op h en yl)m eth yl]- (2). p-Ni-
trobenzyl bromide (7.35 g, 3.40 mmol) and 4-piperidone (5.00 g,
3.25 mmol) were suspended in 13 mL of DMF and 8 mL of Et3N.
The mixture was stirred at rt for 17 h, then filtered through a
celite pad, concentrated, and chromatographed using an IPA/
CH2Cl2 gradient to give 5.07 g (66%) of 2 as a white solid, mp
116-118. 1H NMR (300 MHz, CDCl3): δ 8.14 (d, J ) 8 Hz, 2
H), 7.53 (d, J ) 8 Hz, 2 H), 3.68 (s, 2 H), 2.72 (t, J ) 6 Hz, 4 H),
2.43 (t, J ) 6 Hz, 4 H). 13C NMR: δ 208.4, 147.2, 146.1, 129.2,
(30) Harwood, L. M. Aldrichimica Acta 1985, 18, 25.
(31) Ko¨hler, G.; Milstein, C. Nature 1975, 256, 495.
(32) Engvall, E. Methods Enzymol. 1980, 70, 419.
(33) Copeland, R. A.; Lombardo, D.; Giannaras, J .; Dediddo, C. P.
Bioorg. Med. Chem. Lett. 1995, 5, 1947.
(34) van Hooidonk, C.; Ginjaar, L. Recl. Trans. Chim. Pays-Bas
1967, 86, 449.

7636 J . Org. Chem., Vol. 61, No. 21, 1996
Notes
1% AcOH, 5% absolute EtOH, and 94% CH2Cl2 as the mobile
phase afforded 234 mg of 6 (61%) which HPLC (λ ) 283 nm)
indicated was >99% pure. 1H NMR (500 MHz, CDCl3): δ 8.44
(s, 1 H), 7.40 (d, J ) 9 Hz, 2 H), 7.05 (d, J ) 9 Hz, 2 H), 4.20
(pseudoquintet, 4 H), 2.12 (s, 3 H), 1.35 (t, J ) 7 Hz, 6 H). 13C
NMR: δ 168.9, 146.4, 135.4, 121.3, 120.2, 64.7, 24.1, 16.1.
HRMS (FAB): calcd 288.1001, found 288.0995.
1,3,2-Dioxaph osph or in an e, 2-(4-Nitr oph en oxy)-, 2-Oxide-
(7). A 100 mL flask equipped with a stir bar was flame dried,
cooled under N2, and placed in a rt water bath. p-Nitrophenyl
phosphorodichloridate (2.060 g, 8.048 mmol) was added, followed
by 20 mL of dry THF and 2 mL of dry pyridine. A solution of
1,3-propanediol (0.615 g, 8.082 mmol) dissolved in 4 mL of THF
was added, and the reaction mixture was stirred at rt for 14 h.
The resulting mixture was diluted with Et2O and filtered
through a pad of Celite. The resulting solution was concentrated
and subjected to flash chromatography using an EtOH/CH2Cl2
gradient as the mobile phase to give 0.898 mg of white solid as
product (43% yield). HPLC (λ ) 254 nm) indicated that this
material was >99% pure. 1H NMR (300 MHz, CDCl3): δ 8.26
(d, J ) 9 Hz, 2 H), 7.43 (d, J ) 9 Hz, 2 H), 4.65-4.45 (m, 4 H),
2.55-2.35 (m, 1 H), 1.87 (d of q, 14.9 Hz, 4.9 Hz, 2.4 Hz). HRMS
(FAB): calcd 260.0324, found 260.0321.
(1.054 g, 4.12 mmol). The reaction mixture was stirred at rt for
11 h. The pyridine was evaporated, and EtOAc (45 mL) and
citric acid (10 mL) were added. The layers were separated, and
the organic layer was washed with 3 × 6 mL of citric acid and
6 mL of brine. Evaporation of the solvent afforded 352 mg of
white solid. After flash chromatography using a gradient of IPA/
CH2Cl2 as the mobile phase, 95 mg of white solid was obtained
as product (7% yield). 1H NMR (300 MHz, CDCl3): δ 8.28 (d, J
) 9 Hz, 2 H), 7.55 (d, J ) 8 Hz, 1 H), 7.42 (d, J ) 9 Hz, 2 H),
4.71 (d, J ) 12 Hz, 2 H), 4.99 (d of d, J ) 12 Hz, 2 H), 4.35 (d,
J ) 8 Hz, 1 H), 2.09 (s, 3 H). HRMS: calcd M + Cs+ 449.9515,
found 449.9523.
Ack n ow led gm en t. We gratefully acknowledge fi-
nancial support from the Alfred P. Sloan Foundation
and the NIH (GM48351). We thank J ulie Coakley for
technical support in raising antibodies.
Su p p or tin g In for m a tion Ava ila ble: Eadie Hofstee plots
of the paraoxon hydrolysis reactions (1 page). This material
is contained in libraries on microfiche, immediately follows this
article in the microfilm version of the journal, and can be
ordered from the ACS; see any current masthead page for
ordering information.
Acet a m id e, N-[2-(4-Nit r op h en oxy)-2-oxid o-1,3,2-d ioxa -
p h osp h or in a n -5-yl]- (8). A 100 mL flask was prepared as for
8, and N-acetylserinol (0.543 g, 4.078 mmol) was added, followed
by 10 mL of pyridine and p-nitrophenyl phosphorodichloridate
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