2410 J. Agric. Food Chem., Vol. 46, No. 6, 1998
Sanborn et al.
hydrochloric acid, and the product, IV, was collected as a pale
yellow solid (0.016 g 27%): mp 267-269 °C dec; solid probe
MS calcd for C15H6Cl4O4 389.90; m/z (intensity) M+ 390 (34),
391 (9), 392 (42), 393 (8), 394 (20), 355 (100), 356 (23), 357
(92), 358 (13), 292 (45).
4.19 (OCH2, q, J ) 7, 2 H), 5.95 (CHdCHCO2, d, J ) 15, 1 H),
6.77 (CHdCHCO2, dd, J ) 17, 1 H), 6.86 (Ar, s, 1 H), 6.93 (Ar,
s, 2 H), 7.08 (Ar, s, 1 H), 7.10 (ArCHdCH, d, J ) 15, 1 H),
7.36 (ArCHdCH, dd, J ) 17, 1 H); GC-MS (tR ) 22.27 min)
calcd for C19H13Cl3O4 409.99; m/z (intensity) M+ 410 (30), 411
(4), 412 (6), 413 (26), 414 (4), 415 (10), 338 (36), 336 (34), 337
(11), 302 (100), 303 (28), 304 (70).
5-(3,7,8-Tr ich lor od ib en zo-p -d ioxin -2-yl)-tr a n s,tr a n s-
p en ta -2,4-d ien oic Acid (X). To 0.6 mL of methoxyethanol
were added 0.1 mL of water, 0.02 g (0.0003 mol) of potassium
hydroxide, and 0.065 g (0.00016 mol) of IX. The mixture was
refluxed for 1 h and then cooled. After addition of aqueous
hydrochloric acid, a solid, X, was collected (0.045 g, 74%): mp
266-267 °C; FAB-MS (negative mode) (in pyridine/glycerol)
calcd for C17H9Cl3O4 380.95; m/z (intensity) monoisotopic [M
- H]- 381 (83), 37Cl[M - H]- 383 (100), 37Cl2[M - H]- 385
(44).
Eth yl 6-(3,7,8-Tr ich lor od iben zo-p-d ioxin -2-yloxy)h ex-
a n oa te (XIV). 2,3,7-Trichloro-8-hydroxydibenzo-p-dioxin (3.1
mg, 10 µmol) (Mason and Safe, 1986; Singh and Kumar, 1993)
was alkylated with ethyl 6-bromohexanoate (99%, 3.4 mg,15
µmol), with finely grounded potassium carbonate (0.02 g, 0.15
mmol) as base and Aliquat 336 (0.2 mg, 0.5 µmol) as a phase
transfer catalyst (Dehmlow, 1983), in 1 mL of dry DMF. The
mixture was vigorously stirred under nitrogen at 90-100 °C
for a day. The reaction mixture was evaporated in vacuo and
the residue was taken up in acetone. The mixture was filtered,
and the filtrate was concentrated in vacuo. Purification by
preparative TLC (hexane/ethyl acetate/triethylamine, 90:10:
0.5) resulted in 3.8 mg (84%) of ester XIV: mp 92-94 °C; MS
calcd for C20H19Cl3O5 444.03; m/z (intensity) M+ 444 (11), 446
(11), 448 (5), 302 (40), 304 (38), 306 (12), 143 (100).
Eth yl tr a n s-3-(4-Ch lor o-3-n itr op h en yl)-2-m eth ylp r o-
p en oa te (V). To 10 mL of dry THF were added 2.5 g (0.011
mol) of triethyl 2-phosphonopropionate and 0.6 g (0.015 mol)
of 60% sodium hydride in oil. This mixture was cooled with
an ice bath and stirred for 1 h. To this reaction was added
1.83 g (0.01 mol) of 4-chloro-3-nitrobenzaldehyde. The reaction
was stirred for 1 h and then poured into ice water. A yellow
solid, V, was collected (2.4 g, 89%): mp 60-61.5 °C; 1H NMR
(CDCl3) δ 1.36 (CH3, t, J ) 7, 3 H), 2.10 (CH3, d, J ) 3, 3 H),
4.29 (OCH2, q, J ) 7, 2 H), 7.51 (Ar, dd, J ) 2, 1 H), 7.57 (Ar,
s, 1 H), 7.59 (Ar, s, 1 H), 7.88 (ArCH, d, J ) 1.5, 1 H); GC-
MS (tR ) 7.40 min) calcd for C12H12ClNO4 269.05; m/z
(intensity) M+ 269 (32), 270 (5), 271 (11), 224 (62), 225 (12),
226 (19) 149 (42), 150 (19), 151 (16), 115 (100).
E t h yl
tr a n s-3-(7,8-Dich lor od ib en zo-p -d ioxin -2-yl)-
m eth ylp r op en oa te (VI). To 2 mL of dry DMF under
nitrogen were added 0.56 g (0.002 mol) of V and 0.40 g (0.0022
mol) of 4,5-dichlorocatechol. After the mixture was cooled in
an ice bath, 4.0 mL (0.004 mol) of 1 M potassium tert-butoxide
in tert-butyl alcohol was added. The reaction was heated
(145-150 °C) until the tert-butyl alcohol ceased to distill. After
the reaction cooled, it was poured into water and a solid was
collected. Column chromatography of the solid on silica gel
with hexane/ethyl acetate (9:1) yielded the product, VI (0.165
g, 23%): mp 121-123 °C; 1H NMR (CDCl3) δ 1.35 (CH3, t, J )
7, 3 H), 2.11 (CH3, d, s, 3 H), 4.26 (OCH2, q, J ) 7, 2 H), 6.85-
7.0 (Ar, m, 5H), 7.53 (ArCH, d, J ) 2, 1 H); GC-MS (tR
)
29.44 min) calcd for C18H14Cl2O4 365.21; m/z (intensity) M+
365 (37), 366 (8), 367 (5), 290 (40), 291 (25), 292 (31), 115 (100).
tr a n s-3-(7,8-Dich lor od iben zo-p-d ioxin -2-yl)-2-m eth yl-
p r op en oic Acid (VII). To 1 mL of 2-methoxyethanol were
added 0.037 g (0.0001 mol) of VI and 0.016 g (0.000 26 mol) of
potassium hydroxide dissolved in 0.5 mL of water. The
reaction mixture was refluxed for 1 h. After cooling, aqueous
hydrochloric acid was added. A pale white solid, VII, was
collected by filtration (0.03 g, 89%); mp >280 °C; solid probe
MS calcd for C16H10Cl2O4 336.00; m/z (intensity) M+ 336 (44),
337 (10), 338 (24), 290 (12), 291 (7), 292 (6), 163 (13), 115 (55),
45 (100).
6-((3,7,8-T r ic h lo r o d ib e n zo -p -d io x in -2-y l)o x y )h e x -
a n oic Acid (XV). To 3.3 mg (7.4 µmol) of ester XIV dissolved
in 1 mL of THF was added 0.1 mL of 1 M NaOH. The mixture
was stirred at reflux overnight. The reaction mixture was
concentrated in vacuo, diluted with water, and washed with
dichloromethane (2 × 5 mL). The alkaline aqueous solution
was saturated with sodium chloride, cooled in ice, acidified
by 6 M HCl, and extracted with dichloromethane/ether, 3:1,
mixture (5 × 4 mL). The combined extracts were dried
(Na2SO4) and then evaporated in vacuo to yield a semisolid
acid, XV (2.5 mg, 81%): MS calcd for C18H15Cl3O5 416.00; EI-
MS m/z (relative intensity) M+ 416 (12), 418 (12), 420 (4), 302
(100), 304 (99), 306 (35). This product was conjugated to
proteins without further purification.
Eth yl 5-(2,4-Dich lor o-5-n itr op h en yl)-tr a n s,tr a n s-2,4-
p en ta d ien oa te (VIII). To 10 mL of dry THF were added 0.2
g (0.005 mol) of 60% sodium hydride in oil and 1.25 g (0.005
mol) of triethyl 3-phosphonocrotonate. The solution was cooled
to 10-15 °C, and 0.96 g (0.0044 mol) of 2,4-dichloro-5-
nitrobenzaldehyde (Aldous et al., 1974) was added in 5 mL of
THF. The reaction mixture was allowed to warm to room
temperature and then heated to reflux. After cooling, the THF
was removed and the solid was dissolved in ethyl acetate. This
solution was first washed with water, then dried over sodium
sulfate, and filtered. The reaction mixture was adsorbed on
silica gel. Following column chromatography with ethyl
acetate/hexane (1:10), a solid, VIII, was obtained (0.40 g,
16%): mp 95-101 °C; 1H NMR (CDCl3) δ 1.25 (CH3, t, J ) 7,
3 H), 4.25 (OCH2, q, J ) 7, 2 H), 6.13 (CHdCHCO2, d, J ) 15,
1 H), 6.94 (CH)CHCO2, dd, J ) 12, 1 H), 7.23 (ArCHdCH, d,
J ) 15, 1 H), 7.43 (ArCHdCH, dd, J ) 12, 1 H), 7.62 (Ar, s, 1
2,3,7-Tr ich lor o-8-m eth yld iben zo-p-d ioxin (TMDD) was
synthesized according to the methods of Romkes et al. (Romkes
et al., 1987a,b) and Denomme et al. (Denomme et al., 1985).
The product had NMR and GC-MS data that were consistent
with the assigned structure.
P r ep a r a tion of Con ju ga tes. Haptens containing car-
boxylic acids were activated by the mixed anhydride method.
Haptens IV, VII, X, XI, XII, and XIII (0.03 mmol) were
dissolved in dry p-dioxane. Isobutyl chloroformate and tri-n-
butylamine were added in slight molar excess. The solution
was stirred at room temperature for 30 min. Fifty milligrams
of each protein (BSA or LPH) was dissolved in 30 mL of 0.2 M
borate buffer, pH 8. The protein solution was ice-cooled. To
improve the solubility of the activated hapten in the aqueous
protein solution, 2 mL of p-dioxane was added to the protein
solution. The addition of p-dioxane to the protein solution
caused a slight cloudiness. The activated hapten solution was
then added to the protein solution dropwise with stirring.
Stirring was continued on ice for 30 min to an hour. To remove
unreacted small molecules, the protein conjugates were pre-
cipitated with ice cold 100% ethanol. The precipitated protein
was pelleted by centrifugation at 4 °C for 10 min, 4500g. The
supernatant containing unreacted small molecules was de-
canted. The pellet was resuspended with cold ethanol three
times, centrifuging between resuspensions. The supernatants
were discarded, and the pellet was resuspended in distilled
water to a concentration of approximately 5 mg of protein/
mL. Conjugates of hapten XV were prepared similarly, except
the hapten was dissolved in dry DMF and the reaction carried
H), 8.17 (Ar, s, 1 H); GC-MS (tR ) 15.76 min) calcd for C13H11
-
Cl2NO4 315.00; m/z (intensity) M+ 315 (80), 316 (24), 317 (54),
318 (17), 270 (76), 271 (18), 272 (48), 161 (50), 126 (100).
E t h yl 5-(3,7,8-Tr ich lor od ib en zo-p -d ioxin -2-yl)-tr a n s,
tr a n s-p en ta -2,4-d ien oa te (IX). To 2 mL of dry DMF under
nitrogen were added 0.20 g (0.00063 mol) of VIII and 0.19 g
(0.0011 mol) of 4,5-dichlorocatechol. The mixture was cooled
with an ice bath, and 2.0 mL (0.002 mol) of 1 M potassium
tert-butoxide in tert-butyl alcohol was added. The mixture was
heated (120-140 °C) until the tert-butyl alcohol ceased to
distill. After cooling, water was added and a tan solid was
collected. Column chromatography over silica gel with ethyl
acetate/hexane (1:10) yielded a solid, IX (0.075 g, 27%): mp
136-141 °C; 1H NMR (CD2Cl2) δ 1.30 (CH3, t, J ) 7, 3 H),