1278 J. Agric. Food Chem., Vol. 47, No. 3, 1999
Mercader and Montoya
of 5 mmol of 6-mercaptohexanoic acid (740 mg) and 7.5 mmol
of potassium hydroxide (420 mg) in 20 mL of absolute ethanol,
previously heated until complete dissolution. Fifteen minutes
later, the solvent was removed under reduced pressure and
the residue was dissolved in 5% NaHCO3. After acidification
with 1 N HCl, the precipitate was extracted with ethyl acetate
and the organic phase dried over Na2SO4. Only one spot could
be seen on TLC (Rf 0.48, hexane/ethyl acetate 1:1 with 1%
acetic acid). The product was crystallized twice with hexane/
ethyl acetate (2:1), allowing 216 mg of pure product (28%
yield): 1H NMR (acetone-d6) δ 8.32-7.95 (m, 4H, ArH4), 5.53
(s, 2H, NCH2S), 2.81 (t, 2H, SCH2C), 2.28 (t, 2H, CH2CO2),
1.71-1.40 (m, 6H, 3CH2); 13C NMR (acetone-d6) δ 174.49,
155.00, 145.03, 136.11, 133.62, 129.19, 125.57, 121.00, 51.24,
33.96, 32.41, 30.56, 28.83, 25.13; FAB-MS, m/z 308 (M + H+,
100), 160 (76), 132 (42).
2-(4-Oxo-1,2,3-benzotriazin-3-yl)acetic Acid (BA, Figure 1).
The synthesis of this hapten was carried out in two steps. First,
the benzyl ester of BA was obtained as follows. Ten millimoles
of benzyl bromoacetate (1.6 mL) was added to a solution of
6.8 mmol of 1,2,3-benzotriazin-4(3H)-one (1 g, benzazimide,
CAS 90-16-4) in 30 mL of tetrahydrofuran containing 10 mmol
of triethylamine (1.4 mL). The mixture was refluxed overnight
and then filtered and concentrated. The solution was then
diluted with dichloromethane and washed twice with 1 N HCl.
The product in the organic layer was dried over Na2SO4 and
chromatographed on silica gel column using dichloromethane
as the mobile phase, the benzyl ester of BA being the main
product observed by TLC (Rf 0.4, same solvent, 61.7% yield).
The pure solid product was analyzed by GC/EI-MS: m/z 296
(M + 1, 5), 160 (4), 132 (83), 133 (14), 117 (6), 107 (7), 106
(10), 105 (100), 104 (95), 103 (5), 91 (39), 77 (29), 76 (11), 65
(15); 1H NMR (CDCl3) δ 8.37-7.83 (m, 4H, ArH4), 7.36 (s, 5H,
ArH5), 5.25 (s, 4H, NCH2CO+OCH2Ar); 13C NMR (CDCl3) δ
167.00, 156.00, 144.00, 135.13, 132.65, 128.56, 125.16, 120.00,
67.68, 50.78. Next, the cleavage of the benzyl ester of BA was
performed to obtain BA as follows. 1.36 mmol of the ester (400
mg) was dissolved in 1 mL of dichloromethane, and 2 mL of
hydrogen bromide was added. Reaction was kept at room
temperature for 3 h and the excess of acid was displaced with
a flow of nitrogen. The residue was then dissolved in dichloro-
methane and extracted with 5% NaHCO3. Thereafter, the
aqueous solution was acidified, forming a precipitate, and
extracted with ethyl acetate. Finally, the organic phase was
dried over Na2SO4. BA was purified using a silica column in
hexane/ethyl acetate 1:1 with 1% acetic acid. The pure product
appeared as a single spot on TLC (Rf 0.6, same solvent, 24%
yield): 1H NMR (acetone-d6) δ 8.32-7.99 (m, 4H, ArH4), 5.18
(s, 2H, NCH2CO); 13C NMR (acetone-d6) δ 168.99, 155.90,
145.14, 136.15, 133.62, 129.20, 125.56, 120.54, 51.24; FAB-
MS, m/z 206 (M + H+, 100), 149 (36), 132 (24).
Then, the product was extracted with 5% NaHCO3, the
solution was acidified, forming a precipitate that was dissolved
in ethyl acetate, the organic layer was dried over Na2SO4, and
the solvent was evaporated, leaving a solid residue (130 mg,
92% yield). Only one spot could be seen on TLC (Rf 0.1, hexane/
ethyl acetate 1:1 with 1% acetic acid): 1H NMR (acetone-d6) δ
8.35-7.98 (m, 4H, ArH4), 5.05 (s, 2H, OCH2CO); 13C NMR
(acetone-d6) δ 168.01, 136.23, 133.51, 129.48, 125.92, 123.25,
74.07; FAB-MS, m/z 222 (M + H+, 100).
Type II Haptens. This class of haptens can also be divided
into two subgroups depending on whether they contained the
CH2S moiety or not. MFX-type haptens (MFA, MFP, and
MFH) were prepared from N-(bromomethyl)phthalimide (CAS
5332-26-3) and contained the CH2S group of organodithio-
phosphorus pesticides. FX-type haptens (FA, FP, FB, and FH)
did not contain any heteroatom between the rings and the
spacer arm and were prepared from the corresponding n-amino
acid.
2-(N-Phthalimidoylmethylthio)acetic Acid (MFA, Figure 1).
N-(Bromomethyl)phthalimide (2.5 mmol; 600 mg) dissolved in
40 mL of absolute ethanol (heating was needed to completely
dissolve the compound) was slowly added to a solution of 5
mmol of mercaptoacetic acid (348 µL) and 10 mmol of potas-
sium hydroxide (560 mg) in 20 mL of absolute ethanol. After
45 min of stirring, the mixture was filtered and the solvent
removed. The residue was dissolved with 5% NaHCO3, the
solution washed with dichloromethane, the product precipi-
tated with acid and extracted with ethyl acetate, and the
organic layer dried over Na2SO4. The crude product was
chromatographed on silica gel using hexane/ethyl acetate 1:1
with 1% acetic acid. Those fractions showing only one spot on
TLC were collected (Rf 0.4, same solvent) and crystallized with
hexane/ethyl acetate (1:1.5, 16% yield): 1H NMR (acetone-d6)
δ 7.88 (br s, 4H, ArH4), 4.90 (s, 2H, NCH2S), 3.60 (s, 2H, SCH2-
CO); 13C NMR (acetone-d6) δ 171.18, 168.07, 135.21, 133.04,
123.97, 40.07, 34.40; FAB-MS, m/z 252 (M + H+, 91), 160 (100).
3-(N-Phthalimidoylmethylthio)propanoic Acid (MFP, Figure
1). This hapten was obtained in a similar manner as MFA,
but 5 mmol (435 µL) of 3-mercaptopropanoic acid was used
instead of mercaptoacetic acid. The reaction was kept under
stirring for 20 min, and the solvent was then removed under
reduced pressure. After precipitation with acid and extraction
with ethyl acetate, the crude extract was crystallized with
ethyl acetate. Two hundred and seventeen milligrams of pure
product was obtained (14% yield), and only one spot could be
seen on TLC (Rf 0.38, hexane/ethyl acetate 1:1 with 1% acetic
acid): 1H NMR (acetone-d6) δ 7.89 (br s, 4H, ArH4), 4.79 (s,
2H, NCH2S), 2.94 (t, 2H, SCH2C), 2.64 (t, 2H, CCH2CO2); 13
C
NMR (acetone-d6) δ 172.82, 168.04, 135.29, 132.98, 123.98,
39.44, 34.73, 27.75; FAB-MS, m/z 266 (M + H+, 76), 160 (100).
6-(N-Phthalimidoylmethylthio)hexanoic Acid (MFH, Figure
1). The synthesis of MFH was performed according to the
strategy followed for MBH preparation, but N-(bromomethyl)-
phthalimide was used instead of CMB. The reaction was
stirred for 30 min at room temperature and then filtered.
Afterward, the solvent was removed under reduced pressure
and the residue dissolved in 5% NaHCO3. The solution was
then washed with dichloromethane and precipitated with acid.
The pellet formed was dissolved with ethyl acetate and the
organic layer dried over Na2SO4. The concentrated product was
chromatographed on silica gel using hexane/ethyl acetate 1:1
with 1% acetic acid as the mobile phase. Fractions showing
only one spot on TLC (Rf 0.57, same solvent) were collected
and the residue was crystallized twice with a mixture of
hexane/ethyl ether (1:1), rendering 80 mg of pure product (10%
yield): 1H NMR (acetone-d6) δ 7.89 (br s, 4H, ArH4), 4.76 (s,
2H, NCH2S), 2.73 (t, 2H, SCH2C), 2.29 (t, 2H, CH2CO2), 1.69-
1.43 (m, 6H, 3CH2); 13C NMR (acetone-d6) δ 175.04, 168.52,
135.77, 133.46, 124.46, 39.90, 39.57, 34.47, 33.00, 25.67, 25.64;
FAB-MS, m/z 308 (M + H+, 100), 307 (M, 92), 294 (34), 290
(46), 289 (42), 176 (23).
2-[(4-Oxo-1,2,3-benzotriazin-3-yl)oxy]acetic Acid (HBA, Fig-
ure 1). This hapten was synthesized in a similar manner as
BA. First, the benzyl ester of HBA was prepared by overnight
incubation under reflux of 5 mmol of 3-hydroxy-1,2,3-benzo-
triazin-4(3H)-one (814 mg, hydroxybenzazimide, CAS 28230-
32-2) in 30 mL of dichloromethane containing 7.5 mmol of
benzyl bromoacetate (1.2 mL) and 5.5 mmol of triethylamine
(765 µL). Afterward, the solution was diluted with dichloro-
methane, washed once with 5% NaHCO3, twice with 1 N HCl,
and again with 5% NaHCO3. The organic layer was dried over
Na2SO4 and concentrated. The residue was subjected to silica
gel chromatography using dichloromethane with 1% ethyl
acetate as the mobile phase. The combination of those fractions
showing only one spot on TLC (Rf 0.4, same solvent) provided
the benzyl ester of HBA (70% efficiency): 1H NMR (CDCl3) δ
8.37-7.83 (m, 4H, ArH4), 7.35 (s, 5H, ArH5), 5.25 (s, 2H, OCH2-
CO), 5.09 (s, 2H, OCH2Ar); 13C NMR (acetone-d6) δ 167.16,
151.00, 145.03, 136.56, 136.11, 133.41, 129.43, 129.28, 129.14,
129.07, 125.91, 123.42, 73.98, 67.38; FAB-MS, m/z 312 (M +
H+, 100). Next, the acid was deprotected as for BA. The benzyl
ester of HBA (0.64 mmol; 200 mg) in 2 mL of dichloromethane
containing 2 mL of hydrogen bromide was stirred for 1.5 h at
room temperature. Excess of acid was removed under a flow
of nitrogen and the residue was dissolved in dichloromethane.
N-Phthaloylglycine (FA, Figure 1). This hapten is a com-
mercial product and was purchased from Aldrich Qu´ımica
(Madrid, Spain) (CAS 4702-13-0).