Antisera for the Detection of Organophosphate Pesticides
J. Agric. Food Chem., Vol. 46, No. 8, 1998 3117
MATERIALS AND METHODS
phy (Still et al., 1978) on silica gel (40 mm column, hexane/
ethyl acetate 4:1; Rf ) 0.42) to yield 1.52 g (5.16 mmol, 72%)
of a colorless oil: 1H NMR (330 MHz, CDCl3) δ 7.03 (d, J ) 9
Hz, 2H), 6.75 (d, J ) 8 Hz, 2H), 4.99 (s, br, 1H), 2.57 (t, J ) 8
Hz, 2H), 2.33 (t, J ) 8 Hz, 2H), 1.88 (t, J ) 8.2 Hz, 2H), 0.93
(s, 9H), 0.27 (s, 6H); HRMS (resolution 22 000) theoretical
294.1651 Da, found 294.1648 Da.
Ch em ica ls. Neat pesticide standards were obtained from
Polysciences (Niles, IL). Analytical stock solutions were
prepared in UV-vis grade dimethyl sulfoxide (Fisher Scien-
tific, Fair Lawn, NJ ) and stored frozen at 5 °C. Caprolactone,
4-(4-methoxyphenyl)butyric acid, diethylthiophosphoryl chlo-
ride, tert-butyldimethylsilyl chloride, and N-hydroxysuccin-
imide were obtained from Aldrich Chemical Co. (Milwaukee,
WI). Diethyl ether, hexanes, and ethyl acetate, all of ACS
reagent grade, were obtained from Fisher Scientific. Goat
anti-rabbit/alkaline phosphatase conjugate, p-nitrophenyl phos-
phate substrate tablets, and 1-ethyl-3-[3-(dimethylamino)pro-
pyl]carbodiimide hydrochloride were obtained from Sigma
Chemical Co. (St. Louis, MO).
In str u m en ta tion a n d Equ ip m en t. Proton nuclear mag-
netic resonance (NMR) spectra were obtained with either a
model VXR 300 (300 MHz) or a model EM-360 (60 MHz) NMR
spectrometer (Varian, Sunnyvale, CA). High-resolution mass
spectrometry (HRMS) was carried out on a VG 70-250SE high-
resolution mass spectrometer equipped with a VG 11-250 data
system (Micromass, Ltd., Manchester, U.K.). Melting points
were determined with a Hoover melting point apparatus
(Arthur H. Thomas Co., Philadelphia, PA). Maxisorb II
polystyrene microplates were used for all ELISA procedures
(Nunc, Roskilde, Denmark). ELISA absorbances were read
in the end point mode (450-650 nm) with a Vmax microplate
reader (Molecular Devices, Menlo Park, CA). Microplates were
washed with a SkanWasher300 (Skatron, Lier, Norway).
Microplates were shaken on a Mini-orbital Shaker (Bellco, Inc.,
Vineland, NJ ). Dilutions were performed with a Biomek 1000
robotic workstation (Beckman Instruments, Inc., Palo Alto,
CA). Thin-layer chromatography (TLC) was performed on 250
µm silica gel plates with fluorescent indicator (J . T. Baker,
Irvine, CA).
Syn th esis of Ha p ten s. Compounds 1-5 were synthesized
for use in the preparation of immunogens and coating antigens.
4-(4-Hydroxyphenyl)butyric Acid (1). 4-(4-Methoxyphenyl)-
butyric acid (5.0 g, 25.7 mmol) was added to a 100-mL round-
bottom flask fitted with a condensor, gas inlet, and magnetic
stir bar. To this flask was added 50 mL of 48% HBr, and the
flask was purged with and maintained under dry argon. The
mixture was heated to reflux, at which point the solid
completely dissolved. After 4 h, TLC (hexanes/ethyl acetate,
3:1; Rf ) 0.07 for product) showed complete consumption of
starting material. The reaction mixture was cooled, trans-
ferred to a separatory funnel with 100 mL of water, and
extracted with diethyl ether (3 × 100 mL). The combined ether
extracts were washed with water (3 × 100 mL) and saturated
brine (1 × 100 mL), dried over anhydrous MgSO4, filtered, and
evaporated to yield a yellow oil, which solidified on standing.
The solid was recrystallized from cyclohexane/ethyl acetate to
yield tan crystals (2.84 g, 15.76 mmol, 61%), mp 109-111 °C:
1H NMR (60 MHz, acetone-d6) δ 8.60 (s, br, 2H), 7.11 (d, J )
9.6 Hz, 2H), 6.80 (d, J ) 10 Hz, 2H), 2.6 (t, J ) 7 Hz, 2H),
2.02 (m, 4H).
4-(4-Hydroxyphenyl)butyric Acid tert-Butyldimethylsilyl Es-
ter (2). Compound 2 was prepared according to the method of
Perich and J ohns (1989). To a 100-mL round-bottom flask
equipped with a gas inlet and stir bar was added 1.30 g (7.21
mmol) 4-(4-hydroxyphenyl)butyric acid under an atmosphere
of dry argon. The acid was dissolved with 25 mL of dried
tetrahydrofuran (THF). To this was added 795 µL (731 mg,
7.23 mmol) of dry 4-methylmorpholine, and a solution of tert-
butyldimethylsilyl chloride (1.088 g, 7.22 mmol, in 5.45 mL of
dry THF) was added dropwise. After 30 min, TLC (hexane/
ethyl acetate, 3:1; Rf ) 0.5 for product) showed consumption
of starting acid. The reaction mixture was transferred to a
separatory funnel with 75 mL of ether and 50 mL of water.
After partitioning, the aqueous layer was further extracted
with ether (2 × 50 mL). The combined ether extracts were
quickly washed with water (2 × 50 mL) and saturated brine
(1 × 50 mL), dried over anhydrous Na2SO4, and filtered. The
ether was evaporated to yield 2.15 g (101%) of a clear, faintly
green mobile oil. This oil was purified by flash chromatogra-
O,O-Diethyl, O-[p-(4-Carboxybutyl)phenyl] Phosphorothion-
ate (3). Compound 3 was prepared according to the method
of Valerio et al. (1984). Into a dried 100-mL flask equipped
with a septum and gas inlet were added 1.502 g (5.10 mmol)
of 4-(4-hydroxyphenyl)butyric acid, tert-butyldimethylsilyl es-
ter, and 20 mL of dried p-dioxane. This solution was cooled
to 10-15 °C, and 124 mg (5.16 mmol) of NaH was added in
one portion, followed by stirring for 30 min. Diethylthiophos-
phoryl chloride (1.6 mL, 1.92 g, 10.2 mmol) was added
dropwise over 15 min. The mixture was removed from the
cooling bath and stirred for 3 h at room temperature. TLC
(hexanes/ethyl acetate 3:1; Rf ) 0.40 for starting material)
showed no remaining starting material. To this mixture was
added 10 mL of 3 N HCl, and the resulting solution was stirred
at room temperature for 1 h. The dioxane was evaporated,
and the remaining solution was transferred to a separatory
funnel with 30 mL of water and 25 mL of ether. After
partitioning, the aqueous phase was further extracted with
ether (2 × 25 mL). The combined ether extracts were washed
with water (2 × 25 mL) and saturated brine (1 × 25 mL) and
dried over anhydrous MgSO4. The solution was filtered and
evaporated to yield 2.59 g of an impure oil. This oil was flash
chromatographed on silica gel (40-mm-diameter column, hex-
ane/ethyl acetate/acetic acid, 5:1:1; Rf ) 0.41) to yield 701 mg
of a slightly impure oil, which still contained diethylthiophos-
phoryl chloride. This mixture was rechromatographed on a
20-mm-diameter column using the same conditions described
above. The fractions containing the desired product were
combined in a separatory funnel, diluted with hexane, and
washed with water (3 × 50 mL) and saturated brine (1 × 50
mL). The solution was dried over MgSO4, filtered, and
evaporated to yield 335 mg (1.01 mmol, 20% overall) of the
desired product: 1H NMR (330 MHz, CDCl3) δ 9.97 (s, br, 1H),
7.15 (d, J ) 9.6 Hz, 2H), 7.09 (d, J ) 9.6 Hz, 2H), 4.27 (m,
4H), 2.64 (t, J ) 8.3 Hz, 2H), 2.37 (t, J ) 8.3 Hz, 2H), 1.94 (t,
J ) 7.9 Hz, 2H), 1.36 (t, J ) 7.8 Hz, 6H); HRMS (22 000
resolution) theoretical 332.0847 Da, found 332.0855 Da.
Sodium 6-Hydroxyhexanoate (4). Caprolactone (2.0 mL,
2.06 g, 18.05 mmol) was placed in a 50-mL round-bottom flask
along with 20 mL of water, and to this mixture was added
NaOH (805 mg, 20.13 mmol). The mixture was heated to
reflux for 3 h. The mixture was cooled, and the water was
removed under vacuum. The resulting solid was dissolved in
hot ethanol and precipitated with diethyl ether to yield 2.08 g
(75%) of a white fluffy solid.
O,O-Diethyl O-(6-Carboxyhexyl) Phosphorothionate (5). Com-
pound 5 was prepared following the method of Schrader and
Mu¨hlmann (1956). Into a 50-mL conical flask fitted with a
condenser and gas inlet were placed 411 mg (2.66 mmol) of
sodium 6-hydroxyhexanoate and 5 mL of dry pyridine. The
mixture was stirred vigorously to disperse the salt, and
diethylthiophosphoryl chloride (450 µL, 540 mg, 2.86 mmol)
was added dropwise over 5 min; the resulting mixture was
stirred at 40 °C for 3 h. The mixture was cooled, acidified to
pH 3, and transferred to a separatory funnel with 40 mL of
water and 50 mL of ether. After partitioning, the aqueous
layer was further extracted with ether (2 × 50 mL). The
combined ether extracts were washed with water (3 × 50 mL)
and saturated brine (1 × 50 mL). The ether solution was dried
over anhydrous MgSO4. After filtration, the solution was
evaporated to yield 489 mg of crude product. This product
was purified by flash chromatography on silica gel (40-mm-
diameter column, hexane/ethyl acetate/acetic acid, 5:1:1; Rf )
0.42) to yield 174 mg (0.61 mmol, 23%) of a clear, slightly
yellow liquid: 1H NMR (330 MHz, CDCl3) δ 10.2 (s, br, 1H),
4.16 (m, 6H), 2.4 (t, J ) 8.2 Hz, 2H), 1.68 (m, 4H), 1.32 (m,
8H).