ELISA for Sulfonamide Antibiotics in Milk
J. Agric. Food Chem., Vol. 57, No. 2, 2009 387
(125 MHz, CDCl3) δ 16.4 (-CH3), 37.7 (-CHCOO), 39.1 (-CH2),
51.5 (-OCH3), 116.8 (CH2d), 135.4 (dCH-), 176.5 (-COO-).
2.43 (m, 1H, CH), 2.49 (t, J ) 8 Hz, 2H, CH2), 3.64 (s, 3H, COOCH3),
7.52 (s, 1H), 7.57 (d, J ) 9 Hz, 2H), 7.68 (d, J ) 9 Hz, 2H), 8.13 (s,
1H), 8.28 (s, 1H).
Methyl 5-(4-Amino-3-pyridinyl)-2-methyl-4-pentenoate (3). A solu-
tion of tetrabutylammonium chloride (160 mg, 0.57 mmol) and
Pd(AcO)2 (13 mg, 0.06 mmol) in anhydrous dimethylformamide (anh
DMF, 3 mL) was slowly added to a solution of 2-amino-5-iodopyridine
(500 mg, 2.27 mmol) and potassium formate (153 mg, 1.82 mmol) in
the same solvent (3 mL). Subsequently, a solution of the ester 2 (1.3
g, 11.4 mmol) in anh DMF (2 mL) was added to the mixture, and the
reaction was kept at 65 °C under Ar atmosphere for 6 h. Additional
equivalents of potassium formate (50 mg, 0.59 mmol) and the ester 2
(300 mg, 2.63 mmol) were added, and the progress of the reaction
was monitored by TLC using ethyl acetate (AcOEt) as mobile phase.
After the reaction was finished, the crude material was cooled and the
solvent evaporated. The oil obtained was redissolved in MeOH (25
mL) and filtered through Celite to remove all of the palladium formed,
and the solvent was evaporated. The residue obtained was dissolved
again with AcOEt (50 mL) and washed with saturated NaHCO3 (3 ×
25 mL). The organic layer was separated, dried with anhydrous MgSO4,
filtered, and evaporated to dryness under reduced pressure. The red oil
obtained was purified by silica gel column chromatography using a
1:1 to 1:3 polarity gradient of hexane/AcOEt as mobile phase to obtain
a yellow oil (250 mg) of a 4:1 mixture of the methyl 5-(4-amino-3-
pyridinyl)-2-methyl-4-trans-pentenoate 3 (186 mg, 36% yield) and
methyl 4-(4-amino-3-pyridinyl)-2-methyl-4-methylene-butanoate 4 (62
5-[6-(4-Aminobenzenesulfonylamino)pyridin-3-yl]-2-methylpentano-
ic acid (SA1). A solution of the sulfonamide 7 (165 mg, 0.39 mmol)
in 1 N NaOH (5 mL, 5 mmol) was kept under reflux for 4 h under
argon atmosphere. The crude was cooled, acidified with concentrated
HCl to pH 3, and extracted with AcOEt (3 × 5 mL). The organic layer
was dried with anhydrous MgSO4, filtered, and evaporated to dryness
under reduced pressure to obtain the desired hapten SA1 (105 mg, 73%
yield) as a pale yellow oil: 1H NMR (500 MHz, CD3OD) δ 1.13 (d, J
) 7 Hz, 3H, CH3), 1.40 (m, 1H, CH2), 1.58 (m, 2H, CH2), 1.62 (m,
1H, CH2), 2.41 (m, 1H, CH), 2.52 (t, J ) 7.5 Hz, 2H, CH2), 6.61 (d,
J ) 8.5 Hz, 2H), 7.12 (d, J ) 8.5 Hz, 1H), 7.53 (dd, J ) 8.5 Hz, J )
2.5 Hz, 1H), 7.56 (d, J ) 8.5 Hz, 2H), 7.88 (d, J ) 2.5 Hz, 1H); 13C
NMR (125 MHz, CD3OD) δ 17.6 (CH(CH3)), 29.7 (CH2CH2COOH),
32.6 (CH2CH2Pyr), 34.2 (CH2COOH), 40.4 (CH2Pyr), 114.2 (C3), 114.7
(Cmeta SO NH ), 127.9 (Cortho SO NH ), 130.1 (C5), 132.6 (CSO NH ), 140.9
2
2
2
2
2
2
(C4), 145.5 (C6), 152.2 (C2), 154.1(CNH ), 180.5 (COOH). HRMS (+EI)
2
calcd for C17H22N3O4S (M+): 364.1331; found, 364.1319.
Synthesis of the Competitor Hapten SA2. N-(4-(Aminosulfonyl)phe-
nyl)acetamide (9). A 30% aqueous solution of NH3 (5.6 mL, 4.3 mmol)
was added slowly to a freshly prepared solution of N-acetyl-p-
aminobenzenechlorosulfonic acid 8 (2 g, 8.6 mmol) in anhydrous
acetonitrile (10 mL), and the mixture was kept for 2 h until the complete
disappearance of the starting material by TLC analysis (1:1 AcOEt/
CH2Cl2). The solvent was removed under reduced pressure, and the
residue redissolved in water (20 mL) and extracted with AcOEt (3 ×
10 mL). The organic layer was dried with anhydrous MgSO4, filtered,
and evaporated to dryness to obtain the desired compound 9 (1.5 g,
1
mg, 12% yield). 3: H NMR (500 MHz, CDCl3) δ 1.19 (d, J ) 7 Hz,
3H, CH3), 2.31 (m, 1H, CH2), 2.53 (m, 1H, CH2), 2.58 (m, 1H, CH),
3.67 (s, 3H, COOCH3), 5.95 (dt, J ) 16 Hz, J ) 7 Hz, 1H, dCH-),
6.28 (d, J ) 16 Hz, 1H, -CHd), 6.45 (d, J ) 8.5 Hz, 1H, Armeta),
7.49 (dd, J ) 8.5 Hz, J ) 2.5 Hz, 1H, Arortho), 7.99 (d, J ) 2.5 Hz,
1
1
1H, Arortho). 4: H NMR (500 MHz, CDCl3) δ 1.12 (d, J ) 7 Hz, 3H,
76% yield) as a white solid: H NMR (500 MHz, CDCl3/CD3OD, 14:
CH3), 2.45 (m, 1H, CH2), 2.90 (m, 1H, CH2), 2.56 (m, 1H, CH), 3.63
(s, 3H, COOCH3), 4.99 (d, J ) 1 Hz, 1H, CH2d), 5.20 (d, J ) 1 Hz,
1H, CH2d), 6.50 (d, J ) 8.5 Hz, 1H, Armeta), 7.48 (dd, J ) 8.5 Hz, J
) 2 Hz, 1H, Arortho), 8.12 (d, J ) 2 Hz, 1H, Arortho).
1) δ 2.17 (s, 3H, CH3-), 7.71 (dd, J ) 9.2 Hz, J ) 2.1 Hz, 2H, Armeta),
7.84 (dd, J ) 9.2 Hz, J ) 2.1 Hz, 2H, Arortho); 13C NMR (125 MHz,
CDCl3/CD3OD, 14:1) δ 24.6 (CH3), 119.5 (Cmeta SO Cl), 127.51
2
(Cortho SO Cl), 139.6 (CSO NH ), 143.6 (CNHCOCH ), 171.9 (CO).
2
2
2
3
Methyl 5-(4-Amino-3-pyridinyl)-2-methylpentanoate (6). The mixture
of esters 3 and 4 (250 mg) in MeOH (12 mL) was reduced under H2
at atmospheric pressure in the presence of Pd/C (23 mg, 10% Pd, 0.018
mmol of Pd) for 12 h at room temperature. The reaction was passed
through Celite, and the solvent was evaporated until dryness. The yellow
oil obtained was purified by column chromatography using a 1:1 to
1:3 polarity gradient of hexane/AcOEt as mobile phase to isolate 180
mg of a yellow oil containing a 5:1 mixture of the desired ester 6 and
methyl 4-(4-amino-3-pyridinyl)-2,4-dimethylbutanoate. Further purifica-
tion of the mixture by preparative HPLC using a C18 column (25 × 1
cm, 5 µm, Kromasil 100) as stationary phase and ACN/H2O (Et3N/
CH3COOH, 0.2 M, pH 7.6) 30:70 as mobile phase allowed us to isolate
Methyl 5-(4-(Acetylamino)phenylsulfonamide-5-oxopentatanoate (10).
Methyl 4-(chloroformyl) butyrate (0.65 mL, 4.69 mmol) was added slowly
to a freshly prepared solution of 9 (1 g, 4.69 mmol) in pyridine (2 mL),
and the mixture was kept under argon atmosphere for 2 h. Then, the
reaction was stopped by adding concentrated HCl in ice (30 mL). The
aqueous layer was extracted with AcOEt (3 × 15 mL), and the organic
layer was dried with anhydrous MgSO4, filtered, and evaporated to dryness
under reduced pressure to obtain a pale yellow solid. Isolation of compound
10 was performed by crystallization with 9:1 2-propanol/water (0.85 g,
1
50% yield): H NMR (500 MHz, CDCl3/CD3OD, 14:1) δ 1.79 (m, J )
7.2 Hz, 2H, -CH2-), 2.17 (s, 3H, -CH3), 2.25 (t, J ) 7.2 Hz, 2H,
CH2CO), 2.27 (t, J ) 7.2 Hz, 2H, -CH2Ph), 3.65 (s, 3H, COOCH3), 7.75
(dd, J ) 9.2 Hz, J )2.1, Hz 2H, 2H, Armeta), 7.95 (dd, J ) 9.2 Hz, J )
2.1 Hz, 2H, 2H, Arortho); 13C NMR (125 MHz, CDCl3/CD3OD, 14:1) δ
19.2 (CH3), 23.6 (CH2), 32.4 (NHCOCH2), 34.5 (CH2COOH), 51.4
(-OCH3), 119.1 (Cmeta SO NH ), 127.1 (Cortho SO NH), 132.8 (CSO NH), 143.4
1
the pure ester 6 (81 mg, 40% yield): H NMR (500 MHz, CDCl3) δ
1.13 (d, J ) 7 Hz, 3H, CH3), 1.42 (m, 1H, CH2), 1.53 (m, 2H, CH2),
1.65 (m, 1H, CH2), 2.44 (m, 1H, CH), 2.45 (t, J ) 8 Hz, 2H, CH2),
3.65 (s, 3H, COOCH3), 6.44 (d, J ) 8.5 Hz, 1H, Armeta), 7.24 (dd, J )
2
2
2
2
8.5 Hz, J ) 2.5 Hz, 1H, Arortho), 7.85 (d, J ) 2.5 Hz, 1H, Arortho); 13
C
(CNHAc), 170.19 (CONH), 171.1 (COOH).
NMR (125 MHz, CDCl3) δ 17.4 (CH3CH), 29.3 (CH2CH2COO), 32.2
(CH2CH2Pyr), 33.9 (CH2Pyr), 39.6 (CHCOO), 51.5 (COOCH3), 108.7
(C3), 127.9 (C5), 138.2 (C4), 147 (C6), 156.9 (C2), 176.9 (COOCH2).
Preparation of 5-[4-(Amino)phenylsulfonamide]-5-oxopentanoic Acid
(SA2). A solution of compound 10 (130 mg, 3.9 mmol) in 1 N NaOH
(8 mL) was heated at 75 °C for 6 h until the total disappearance of the
starting material was observed by TLC (9:1:1 EtAc/CH2Cl2/MeOH;
1% AcOH). The crude mixture was acidified with 1 N HCl to pH 2
and extracted with AcOEt (3 × 10 mL). The organic layer was finally
washed with water, dried with anhydrous MgSO4, filtered, and
evaporated to dryness under reduced pressure. Isolation of SA2 was
performed using a silica gel column and 1:2 AcOEt/CH2Cl2 (1% acetic
Methyl 5-[6-(4-Acetylaminobenzenesulfonylamino)pyridin-3-yl]-2-
methylpentanoate (7). Ester 6 (250 mg, 1.13 mmol) in anhydrous
dioxane (2 mL) was added to a solution of N-acetyl-p-aminoben-
zenechlorosulfonic acid (290 mg, 1.24 mmol) in the same solvent (8
mL). Subsequently, triethylamine (175 µL, 1.24 mmol) was added
slowly, and the mixture was kept under argon atmosphere for 15 h at
room temperature. When the reaction was finished, the solvent was
evaporated, and the oil obtained was redissolved with AcOEt (10 mL)
and washed with saturated NaHCO3 (3 × 5 mL). The organic layer
was dried with anhydrous MgSO4, filtered, and evaporated to dryness
under reduced pressure to obtain yellow oil. Purification of the product
was performed using a silica gel column, with 1:4 hexane/AcOEt as
mobile phase to obtain the desired sulfonamide 7 (239 mg, 35% yield):
1H NMR (500 MHz, CDCl3) δ 1.13 (d, J ) 7 Hz, 3H, CH3), 1.41 (m,
1H, CH2), 1.53 (m, 2H, CH2), 1.65 (m, 1H, CH2), 2.20 (s, 3H, CH3CO),
1
acid) as mobile phase to obtain SA2 (71 mg, 65% yield): H NMR
(500 MHz, CD3OD) δ 1.79 (m, J ) 7.2 Hz, 2H, -CH2-), 2.25 (t, J )
7.2 Hz, 2H, CH2COOH), 2.27 (t, J ) 7.2 Hz, 2H, -CH2Ph), 6.67 (dd,
J ) 7.2, J ) 2.1 Hz, 2H, 2HAr meta), 7.66 (dd, J ) 7.2, J ) 2.1 Hz, 2H,
2H, Arortho); 13C NMR (125 MHz, CDCl3/CD3OD, 9:1) δ 20.74 (CH2),
32.4 (NHCOCH2), 34.5 (CH2COOH), 113.8 (Cmeta SO NH ), 125.8
2
2
(Cortho SO2NH2), 131.6 (CSO2NH2), 154.9 (CNH2), 172.9 (CO).
HRMS (+EI) calcd for C11H15N2O5S (M+): 287.0702; found,
287.0693.