376 J. Agric. Food Chem., Vol. 57, No. 2, 2009
Ramo´n-Azco´n et al.
protocol as described above for hapten 2 preparation, to obtain hapten
4 (50 mg, 90% yield).
3-((Isopropoxycarbonyl)bis(4-bromophenyl)methoxy)propyl 3-Io-
dopropionate (Hapten 6). Purified BP (1 g, 2.3 mmol, 1 equiv) was
placed in a round-bottom flask provided with a magnetic stirring bar
and a condenser. SOCl2 (700 µL, 9.3 mmol, 4 equiv) was added
dropwise under Ar atmosphere and the mixture left to react for 30 min
until the disappearance of the starting material by TLC. The crude
product was diluted with hexane, washed with 1 M NaOH, and extracted
with AcOEt. The organic layer was then dried with anhydrous MgSO4,
filtered, and evaporated under reduced pressure to obtain the ester
isopropyl 2,2-bis(4-bromophenyl)-2-chloroacetate (0.93 g, 90% yield).
Next, a solution of this ester (900 mg, 2 mmol, 1 equiv) in
1,3-propanediol (2 mL) was gently heated under Ar atmosphere. Then,
anhydrous Cs2CO3 (167 mg, 0.51 mmol, 0.2 equiv) was added to the
solution, and the mixture was left to react for 4 h at 120 °C. The crude
product was diluted in AcOEt, washed with a saturated solution of
NaCl, dried with MgSO4, filtered, and evaporated under reduced
pressure to obtain the ester isopropyl 2-(3-hydroxypropoxy)-2,2-bis(4-
bromophenyl)acetate (5) (506 mg, 41% yield). Subsequently, a solution
of 5 in anhydrous CH2Cl2 (2 mL) was added under Ar atmosphere to
iodoacetic acid (338 mg, 1.8 mmol, 3 equiv) The mixture was left to
react for 24 h at 50 °C. Then, the crude product was diluted in AcOEt,
washed with a saturated solution of NaCl, dried with MgSO4, filtered,
and evaporated. The crude was purified silica gel flash chromatography
using hexane/AcOEt (4:1) to obtain hapten 6 (204 mg, 62% yield).
Isopropyl 2-(2-Formylethoxy)-2,2-bis(4-bromophenyl)acetate (Hap-
ten 7). A solution of pyridinium chlorochromate (PCC) (414 mg, 1.9
mmol, 2 equiv) in anhydrous CH2Cl2 (4 mL) was added to 5 (467 mg,
0.96 mmol, 1 equiv) under Ar atmosphere. The mixture was left for
2 h at room temperature, then diluted in AcOEt, filtered with zeolite,
and concentrated to dryness. The crude was purified by silica gel flash
chromatography using hexane/AcOEt (4:1) as mobile phase to obtain
hapten 7 (278 mg, 60% yield).
Methyl 2-(2-Formylethoxy)-2,2-bis(4-bromophenyl)acetate (Hapten
9). A solution of isopropyl 2-(3-hydroxypropoxy)-2,2-bis(4-bromophe-
nyl)acetate (5) (500 mg, 1.03 mmol, 1 equiv) in MeOH (2 mL) was
hydrolyzed with 1 M NaOH (2 mL) as described above to obtain 2-(3-
hydroxypropoxy)-2,2-bis(4-bromophenyl)acetic acid (324 mg, 71%
yield). To a solution of this acid (100 mg, 0.96 mmol) in anhydrous
MeOH (2 mL) was added dropwise SOCl2 (200 µL, 2.6 mmol, 4 equiv)
under Ar atmosphere. The mixture was heated for 30 min, then diluted
with hexane, washed with 1 M NaOH, and extracted with AcOEt. The
organic layer was dried with MgSO4, filtered, and evaporated. The crude
product was purified by silica gel flash chromatography using hexane/
AcOEt (4:1) as mobile phase to obtain methyl 2-(3-hydroxypropoxy)-
2,2-bis(4-bromophenyl)acetate (8) (105 mg, 32% yield). Following the
same protocol as described above for hapten 7 preparation, hapten 9
(96 mg, >95% yield) was obtained from PCC (97 mg, 0.45 mmol, 2
equiv) from product 8 (105 mg, 0.22 mmol, 1 equiv).
Immunochemistry. Instrumentation. The matrix-assisted laser
desorption ionization time-of-flight mass spectrometer (MALDI-TOF-
MS) used for analyzing the protein conjugates was a Perspective
BioSpectrometry Workstation provided with the software Voyager-DE-
RP (version 4.03) developed by Perspective Biosystems Inc. (Framing-
ham, MA) and Grams/386 (for Microsoft Windows, version 3.04, level
III) developed by Galactic Industries Corp. (Salem, NH). The pH and
conductivity of all buffers and solutions were measured with a pH-
meter (pH 540 GLP) and a conductimeter (LF 340), respectively (both
from WTW, Weilheim, Germany). Polystyrene microtiter plates were
purchased from Nunc (Maxisorp, Roskilde, Denmark). The vacutainer
blood collection set was acquired from Becton Dickinson (Meylon
Ce´dex, France). Washing steps were performed on an SLY96 PW
microplate washer (SLT Labinstruments GmbH, Salzburg, Austria).
Absorbances were read using a SpectramaxPlus microplate reader
(Molecular Devices, Sunnyvale, CA) at a single wavelength of 450
nm. The competitive curves were analyzed with a four-parameter
logistic equation using the software SoftmaxPro v2.6 (Molecular
Devices) and GraphPad Prism (GraphPad Sofware Inc., San Diego,
CA). Unless otherwise indicated, the data presented correspond to the
average of at least two well replicates.
Figure 1. Hapten design.
BP. These antibodies have been used to develop an immunoas-
say to determine BP.
MATERIALS AND METHODS
Chemistry. General Methods and Instruments. Thin-layer chro-
matography (TLC) was performed on 0.25 mm, precoated silica gel
60 F254 aluminum sheets (Merck, Darmstadt, Germany). Unless
otherwise indicated, purification of the reaction mixtures was ac-
complished by “flash” chromatography using silica gel as the stationary
1
phase. H and 13C NMR spectra were obtained with a Varian Unity-
1
300 (Varian Inc., Palo Alto, CA) spectrometer (300 MHz for H and
75 MHz for 13C) or with a Varian Inova-500 spectrometer (500 MHz
1H and 125 MHz for 13C). Chemical reagents were purchased from
Aldrich Chemical Co. (Milwaukee, WI). Formulated BP was obtained
from a commercial supplier (Sygenta). Log Pow was calculated using
ACD/LogP DB version 3.00.
Synthesis of the Haptens. Spectroscopic and spectrometric data are
given as Supporting Information (see Figures 1-3).
2,2-Bis(4-bromophenyl)-N-2-hydroxyacetamide-butanoic acid (Hap-
ten 2). A 1 M solution of NaOH (5 mL) was added dropwise to a
solution of purified BP (1 g, 2 mmol, 1 equiv) in MeOH (5 mL). The
mixture was vigorously stirred for 1 h at room temperature until the
disappearance of the starting material by TLC. The crude product was
washed with hexane, acidified with HCl, and extracted with AcOEt.
The organic layer was then dried with MgSO4, filtered, and evaporated
under reduced pressure to obtain 2,2-bis(4-bromophenyl)-2-hydroxy-
acetic acid (930 mg, 92% yield). Subsequently, the acid (660 mg, 1.72
mmol, 1 equiv) and 1,1′-carbonyldiimidazole (CDI) (337 mg, 2.07
mmol, 1.2 equiv) were placed in a two-neck round-bottom flask
provided with a magnetic stirring bar and diluted in anhydrous CH2Cl2
(10 mL). Then, a solution of methyl 3-aminoproanoate chlorhydrate
(265 mg, 1.72 mmol, 1 equiv) in anhydrous CH2Cl2 (2 mL) was slowly
added under Ar atmosphere to the reaction mixture. The reaction was
left for 24 h at room temperature. The CH2Cl2 was evaporated, and
the remaining crude was dissolved in EtO2 and washed with saturated
NaHCO3. The organic layer was dried with MgSO4, filtered, and
evaporated. Finally, the crude product was purified by silica gel flash
chromatography using hexane/EtOAc (1:1) as mobile phase to obtain
the ester methyl 2,2-bis(4-bromophenyl)-N-2-hydroxyacetamide-bu-
tanoate (1) (380 mg, 42% yield). Subsequently, ester 1 (140 mg, 0.41
mmol, 1 equiv) was hydrolyzed with a 1 M aqueous solution of NaOH
(5 mL) in MeOH (5 mL) at room temperature until the complete
disappearance of the ester by TLC. The crude product was washed
with hexane, acidified with HCl, and extracted with AcOEt. The organic
layer was dried with MgSO4, filtered, and evaporated under reduced
pressure to obtain hapten 2 (126 mg, 95% yield).
4-(2,2-Bis(4-bromophenyl)-2-chloroacetamido)butanoic Acid (Hap-
ten 4). Ester 1 (112 mg, 0.23 mmol, 1 equiv) was placed in a round-
bottom flask provided with a magnetic stirring bar and Dimroth
refrigerant under Ar atmosphere. SOCl2 (165 µL, 2.2 mmol, 10 equiv)
was added dropwise, and the mixture was heated until reflux for 1 h.
The crude product was diluted with hexane, washed with 1 M NaOH,
and extracted with AcOEt. The organic layer was dried with MgSO4,
filtered, and evaporated under reduced pressure to obtain the ester
methyl 4-(2,2-bis(4-bromophenyl)-2-chloroacetamido)butanoate (3) (59
mg, 50% yield). Next, ester 3 (59 mg, 0.11 mmol, 1 equiv) was
hydrolyzed with a 1 M aqueous solution of NaOH, following the same