3928 J. Agric. Food Chem., Vol. 47, No. 9, 1999
Tessier and Clark
phenol, collected as the remaining fraction after removal of
phenol and related byproducts, was recrystallized from petro-
leum ether to yield 3.8 g of white, needlelike crystals with a
distinct phenolic odor: mp 77 °C; purity, 99.9% (GC/MS); IR
(KBr, cm-1) 3320-3180 (s, b, OH), 1600, 1450 (aryl ring); GC/
MS tr ) 10.27 min, m/z 150 (M+), 135 (M - CH3), 121 (M -
C2H5).
3,5-Diethyl-4-nitrosophenol (10). 3,5-Diethylphenol (9; 3.6
g, 24 mmol) was dissolved in ethanol, and an equal volume of
concentrated HCl was added with stirring. The reaction
mixture was cooled to 0 °C, and NaNO3 (2.5 g, 30 mmol),
dissolved in dH2O, was added dropwise. The mixture was
stirred for 2 h at 10 °C and then poured onto ice-water and
filtered to yield 3.4 g of rust-colored crystals: mp 126 °C (dec);
IR (KBr, cm-1) 1500 (s, NdO), 1140 (s, COH), 1000 (CN).
4-Amino-3,5-diethylphenol (11). 3,5-Diethyl-4-nitrosophenol
(10; 3.4 g in THF) was hydrogenated at atmospheric pressure
in the presence of palladium catalyst (380 mg). [CAUTION!
The palladium catalyst can ignite solvent vapors. The hydro-
genation apparatus must be flushed with inert gas prior to
addition of catalyst.] The hydrogenation reaction continued for
3 h, after which time, the reaction solution was filtered through
Celite 545. Removal of the solvent yielded 3 g of orange
crystals: mp 118 °C; purity, 97.6% (GC/MS); GC/MS tr ) 8.4
min, m/z 165 (M+), 150 (M - CH3), 135. Elemental analysis
(theoretical values in parentheses): C, 72.38% (72.73); H, 9.3%
(9.1); N, 8.36% (8.5); O, 9.96% (9.7).
Ethyl 5-(4-Amino-3,5-diethyl)phenoxypentanoate (12). 4-
Amino-3,5-diethylphenol (11; 1 g, 6 mmol) was dissolved in
dimethyl sulfoxide. The stirred solution was cooled to 15 °C,
and KOH (0.5 g, 9 mmol) added. The solution was returned to
ambient temperature, and ethyl 5-bromopentanoate (1.9 g, 9
mmol) was slowly added over the course of 70 min, after which
time, the reaction was stopped by pouring the solution into 4
M HCl at 0 °C. The acidic solution was extracted with diethyl
ether and then basified with 2 M NaOH. The basic solution
was extracted again with diethyl ether, and the combined
ethereal extracts were washed with dH2O and brine and dried
over Na2SO4. The solvent was removed to yield 0.3 g of a brown
oil: purity, 99.7% (GC/MS); IR (neat, cm-1) 1725 (CdO, ester),
1590 (-NH); GC/MS tr ) 14.14 min, m/z 293 (M+), 248, 165/
164, 129, 101.
F igu r e 5. Direct coupling of CDA to protein carrier molecules
via the coupling reagent AHT. The carrier protein was either
BSA (immunogen) or OVA (plate-coating antigen).
to the filtrate, which was washed with dH2O and brine and
dried (Na2SO4). Removal of solvent yielded an amber, oily
crystalline residue. The esters were purified by thin-layer
chromatography (20 × 20 cm, 2000 µm silica, 25% ethyl
acetate/hexane). C2-CDA: IR (neat, cm-1) 3320 (w, NH), 1830,
1795, 1750, 1690 (s, CdO), 1460-1370 (m, CN), 1240-1200
(s, -CO‚OR); 1H NMR (CDCl3) δ 1.2 (t, 6H, -CH3), 2.6 (q, 4H,
alkyl -CH2), 2.7 (s, 4H, imide -CH2), 3.7 (s, 2H, -NCH2-
COOR), 4.5 (s, 2H, ClCH2CO‚R), 7.2 (m, 3H, aryl CH); MS
(direct probe) m/z 380 (M+), 266, 238, 216, 188, 162. Elemental
analysis (theoretical values in parentheses): C, 57.13% (56.77%);
H, 5.85% (5.56%); N, 7.0% (7.36%); O, 21.08% (21.00%); Cl,
8.93% (9.30%). C4-CDA: IR (neat, cm-1) 3400-3300 (w, NH),
1795, 1760, 1740, 1650 (s, CdO), 1460-1320 (m, CN), 1200
(s, -CO‚OR). Elemental analysis (theoretical values in paren-
theses): C, 59.58% (59.04%); H, 6.43% (5.70%); N, 6.69%
(6.89%); O, 18.85% (19.66%); Cl, 8.45% (8.71%). Phe-CDA IR
(neat, cm-1) 3290 (s, NH), 1795, 1760, 1670, 1630 (CdO), 1210
(s, CO‚OR).
Con ju ga tion of Ha p ten s to Ca r r ier P r otein s. The
activated esters of C2-CDA, C4-CDA, and Phe-CDA, dissolved
in THF, were stirred with carrier protein [bovine serum
albumin (BSA), human serum albumin (HSA), or ovalbumin
(OVA)] in phosphate-buffered saline (PBS) at 0 °C, at a 50:1
molar ratio. The reaction was continued for 48 h at 4 °C. The
conjugate solutions were dialyzed against dH2O (8-10 changes)
at 4 °C and then lyophilized.
CDA was also directly coupled to carrier protein via N-acetyl
homocysteinethiolactone (AHT; Figure 5; Feng et al., 1992).
BSA or OVA (200 mg) was dissolved in dH2O. AHT (18.4 mg,
115 mmol) was added to the protein with stirring and the
solution chilled to 0 °C. CDA dissolved in dioxane was added
dropwise and the reaction mixture adjusted to pH g10 with
carbonate-bicarbonate buffer (1.0 M, pH 11). After stirring
at 0 °C for an additional 15 min, the solution was transferred
to a 50 °C water bath and the reaction continued for 2 h. The
reaction solution was neutralized with phosphate buffer (1.0
M, pH 7.2) and dialyzed against dH2O (eight changes).
The immunogens utilized BSA as the carrier protein,
whereas the plate-coating antigens utilized OVA or HSA as
the carrier protein. Hapten densities were determined by
titrating free amine moieties with trinitrobenzene sulfonic acid
(TNBS; Habeeb, 1965).
Ethyl 5-(4-Chloroacetamido-3,5-diethyl)phenoxypentanoate
(13). Ethyl 5-(4-amino-3,5-diethyl)phenoxypentanoate (12; 0.3
g, 1 mmol) was dissolved in methylene chloride. Chloroacetyl
chloride (170 mg, 1.5 mmol, diluted in methylene chloride) was
added at room temperature to the stirred solution of 12 in 100
µL aliquots at 2 min intervals, and the reaction continued for
30 min after the last chloroacetyl chloride addition. The
reaction mixture was washed with dH2O and brine and dried
(Na2SO4), and the solvent was removed to yield 0.33 g of a
brown solid: purity, 99.9% (GC/MS); IR (Nujol, cm-1) 3240,
2940, 1740 (s, CdO); GC/MS tr ) 15.00 min, m/z 369 (M+),
129, 101 (M - C2H5CO‚C2H5). Elemental analysis (theoretical
values in parentheses): C, 61.55% (61.7); H, 7.57% (7.63); N,
3.68% (3.79); Cl, 9.38% (9.58); O, 17.82 (17.30).
5-(4-Chloroacetamido-3,5-diethyl)phenoxypentanoic Acid (14).
Ethyl 5-(4-chloroacetamido-3,5-diethyl)phenoxypentanoate (13;
163 mg, 0.4 mmol), dissolved in acetone, was stirred in the
presence of 2 M KOH at ambient temperature for 24 h. The
reaction solution was neutralized with 4 M HCl and extracted
with diethyl ether. The combined extracts were washed with
dH2O and brine and dried over Na2SO4. The solvent was
removed to yield 152 mg of a brown semisolid. Crude 14 was
purified by thin-layer chromatography (silica gel, 2000 µ, ethyl
acetate/hexane/acetic acid, 25:70:5) to yield 103 mg of white
crystals: mp 132 °C (dec); purity, 99% (TLC); IR (KBr, cm-1
)
3680-2500 (m, b, OH), 3285 (s, NH), 1720 (s, CdO); GC/MS
An tibod y P r od u ction . Immunizations and blood collec-
tions were performed by Animal Care and Research Services,
Graduate School, University of Massachusetts, Amherst, MA.
Standard immunization protocols were followed (Coligan et
al., 1995). Female white New Zealand rabbits (2-3 kg) were
initially immunized with 250 µg of hapten-protein conjugate
solubilized in 250 µL of PBS (pH 7.2) and emulsified 1:1 with
Freund’s complete adjuvant. Subsequent boosts of 250 µg of
(methyl ester) tr ) 10.9, m/z ) 355 (M+), 192, 115.
P r ep a r a tion of Ha p ten -Activa ted Ester s. C2-CDA, C4-
CDA, or Phe-CDA (0.1-0.6 g) and N-hydroxysuccinimide (0.1-
0.26 g) were dissolved in THF. The solution was cooled to 0
°C, and N,N′-dicyclohexylcarbodiimide (50-500 mg) was added
with stirring. The reaction was continued at -20 °C for 24 h.
The cold solution was filtered and methylene chloride added