Potential Source of Untypical Aging Off-Flavor in Wine
J. Agric. Food Chem., Vol. 50, No. 15, 2002 4305
12. C12H7NO4. M ) 205.2. Bright yellow crystals, mp 200-202
°C. Purity 95%. H NMR: δ ) 8.52 (1H, d, J ) 8.7 Hz, H-8), 8.31
Table 1. Detection Limits (µg/L) of 1−10 Using the Respective HPLC
Conditions Described under HPLC Analysis (1, Kynurenine; 2,
Indole-3-acetic Acid; 3, 2-Aminoacetophenone; 4, Kynurenamine; 5,
Kynurenic Acid, 6, Skatole; 7, 2-Oxoskatole; 8,
2-Formamidoacetophenone; 9, 2-Oxindole-3-acetic Acid; 10,
3-(2-Formylaminophenyl)-3-oxopropionic Acid)
1
(1H, d, J ) 7.1 Hz, H-5), 8.07 (1H, dd, J ) 7.1 and 8.7 Hz, H-7), 7.86
(1H, dd, J ) 7.1 and 7.1 Hz, H-6), 7.53 (1H, s, H-3) ppm. 13C NMR:
δ ) 161.9 (C-9), 159.8 (C-4), 138.0 (C-2), 136.2 (C-8a), 133.5 (C-7),
129.0 (C-6), 123.5 (C-5), 122.8 (C-4a), 118.1 (C-8), 104.2 (C-3) ppm.
MS (EI): m/z ) 206 [M + H]+ (0.1), 161 (57), 160 (26), 146 (11),
145 (100), 144 (9), 117 (60), 116 (48), 104 (28), 103 (10), 90 (36), 89
(44), 77 (33), 76 (36), 75 (10), 64 (16), 63 (28), 59 (12), 51 (16), 50
(26). MS (FAB): m/z ) 206 [M + H]+ (100). IR (KBr): 3105, 1696,
UV
240nm
UV
260nm
1
2
3
4
5
10
73
48
1603, 1532, 1450, 1381, 1301, 1275, 1241, 1202, 1159, 1115 cm-1
.
5
12
8
Elementary analysis: C ) 57.7% (58.5%), H ) 3.6% (3.4%), N )
6.7% (6.8%).
6
7
47
47
Synthesis of 2-Oxoskatole (7). Compound 7 was prepared according
to Savige and Fontana (23) by treating an ice-cooled solution of 5.0 g
of 6 (38.1 mmol) in 25 mL of glacial acetic acid dropwise with a
mixture of 4.5 mL of dimethyl sulfoxide and 12.5 mL of concentrated
hydrochloric acid (37%). After the solution was stirred for 1 h at room
temperature the pH was adjusted to 3 with ammonia (25%) under ice-
cooling. Subsequently, 30 mL of ethyl acetate was added, and the
organic layer was washed to neutral pH. The solvent was evaporated
to dryness and the residue was purified by column chromatography on
silica gel 60 (70-230 mesh). 1.5 g of 7 (10.2 mmol, 27%) was obtained
using ethyl acetate as eluent. C9H9NO. M ) 147.2. Bright yellow
crystals, mp 121 °C. Purity 98%. 1H NMR: δ ) 7.22 (1H, d, J ) 7.6
Hz, H-4), 7.21 (1H, dd, J ) 7.1 and 7.6 Hz, H-6), 7.03 (1H, dd, J )
7.1 and 7.6 Hz, H-5), 6.90 (1H, d, J ) 7.6 Hz, H-7), 3.44 (1H, q, J )
7.6 Hz, H-3), 1.50 (1H, d, J ) 7.6 Hz, H-8R) ppm. 13C NMR: δ )
181.2 (C-2), 141.1 (C-7a), 131.3 (C-3a), 127.9 (C-6), 123.8 (C-4), 122.4
(C-5), 109.7 (C-7), 41.0 (C-3), 15.2 (C-8) ppm. MS (EI): m/z ) 147
[M + H]+ (100), 132 (24), 128 (8), 119 (73), 104 (13), 47 (13). IR
(KBr): 3197, 2972, 1714, 1678, 1621, 1473, 1402, 1378, 1337, 1232
cm-1. Elementary analysis: C ) 73.1% (73.5%), H ) 6.0% (6.2%),
N ) 9.2% (9.5%).
8
26
9
10
103
104
Elementary analysis: C ) 62.2% (62.8%), H ) 4.8% (4.7%), N )
7.2% (7.3%).
Synthesis of 3-(2-Formylaminophenyl)-3-Oxopropionic Acid (10). 10
was prepared according to Scho¨pf et al. (26) by suspending 5.2 g of 2
(29.7 mmol) in 100 mL of tetrahydrofuran and 3 mL of methanol. The
solution was aerated with oxygen (containing 2 vol-% ozone) for 3 h
at -78 °C. After warming up to 0 °C, a palladium-katalysator (500
mg, 10% on carbon) was added. The solution was stirred for 2 h under
hydrogen, filtered, and the solvent was removed. The residue was
washed with ethyl acetate, and the precipitate was isolated and
recrystallized twice from methanol yielding 2.0 g of 10 (9.7 mmol,
33%). C10H9NO4. M ) 207.2. Colorless crystals, mp 237 °C. Purity
98%. MS (FAB): m/z ) 208 [M + H]+ (15), 154 (100), 136 (73). A
characterization by 1H- and 13C-NMR failed because of the instability
of 10. IR (KBr): 3340, 1734, 1673, 1584, 1519, 1453, 1407, 1326,
1302, 1205, 1151 cm-1. Elementary analysis: C ) 58.5% (58.0%), H
) 4.3% (4.4%), N ) 6.8% (6.8%).
Model Fermentation Medium. A must-like model solution consist-
ing of glucose (95 g/L), fructose (95 g/L), ethanol (5 g/L), malic acid
(3.5 g/L), tartaric acid (3 g/L), MgSO4‚H2O (1.65 g/L), K2HPO4 (1.5
g/L), CaCl2‚H2O (0.5 g/L), and diammoniumphosphate (1 g/L), adjusted
to pH 3.5 with 1 M sodium hydroxide, was spiked with 100 mg/L 1 or
2, respectively. The fermentations were carried out in 1-L flasks, and
incubated on a shaker (100 U min-1) at room temperature after
inoculation with 20 g/hL S. cereVisiae. Samples were taken at different
intervals during fermentation over a period of four weeks. For HPLC
analysis samples were filtered through a 0.2 µm microfilter (Schleicher
& Schuell, Dassel, Germany).
Sulfuration of Model Wines. A wine-like model solution consisting
of ethanol (10 vol%), tartaric acid (5 g/L), and malic acid (5 g/L),
adjusted to pH 3.3 with 1 M sodium hydroxide, was spiked with 200
mg/L of 1, 2, or other potential precursors and sulfurized with 100
mg/L potassium bisulfite. The solutions were stored at room temper-
ature, or at 45 °C to simulate storage at room temperature for several
months. Samples were taken in different intervals over a period of three
weeks.
HPLC Analysis. Determination of 1 and its ReleVant Degradation
Products 3-5 (HPLC-UV240nm). The HPLC-UV240nm system consisted
of a Merck 655A-12 liquid chromatograph, a Merck L-5000 LC
controller, a Merck T-6300 column thermostat, and a LDC/Milton Roy
SpectroMonitor D variable wavelength detector (Milton Roy, Hassel-
roth, Germany), set at 240 nm. Signals were acquired and calculated
using ChromStar software (SCPA, Stuhr, Germany). Chromatographic
separations were carried out on a Nucleosil 120-3 C18 column (250
mm × 4 mm) (CS, Langerwehe, Germany) equipped with a precolumn
(20 mm × 4 mm) using a binary gradient (solvent A, 0.1% trifluoro-
acetic acid in bidistilled water; solvent B, methanol). The following
gradient was used: 0 min, 5% B; 25 min, 40% B. The column was
washed for 2 min with 40% B after each run and equilibrated for 8
min at the starting conditions. The flow rate was set to 0.8 mL/min,
the injection volume was 20 µL, and the temperature of the column
thermostat was 35 °C. Detection limits are given in Table 1.
Synthesis of 2-Formamidoacetophenone (8). According to Fu¨rstner
and Jumbam (24), a mixture of 1.0 g of 3 (7.4 mmol), 1.4 mL of acetic
anhydride, and 2.2 mL of formic acid was stirred for 30 min under
ice-cooling. After 2 h, 30 mL of diethyl ether and 15 mL of saturated
sodium hydrogen carbonate solution were added. The aqueous layer
was extracted twice with diethyl ether, and the organic phase was dried
over sodium sulfate. After evaporation, the residue was recrystallized
from ethanol yielding 0.95 g of 8 (5.8 mmol, 78%). C9H9NO2. M )
1
163.2. Colorless crystals, mp 78-79 °C. Purity 99%. H NMR: δ )
11.59 (1H, bs, -NH), 8.71 (1H, d, J ) 7.6 Hz, H-3), 8.43 (1H, s,
-CHO), 7.90 (1H, d, J ) 7.9 Hz, H-6), 7.51 (1H, t, J ) 7.5 and 7.9
Hz, H-5), 7.17 (1H, t, J ) 7.5 and 7.6 Hz, H-4), 2.60 (3H, s, -CH3).
MS (EI): m/z ) 163 [M + H]+ (34), 148 (14), 135 (65), 120 (100), 92
(41), 65 (34). IR (KBr): 3253, 1685, 1647, 1604, 1579, 1516, 1454,
1406, 1390, 1361, 1309, 1254, 1211 cm-1. Elementary analysis: C )
66.2% (66.3%), H ) 5.6% (5.6%), N ) 8.6% (8.6%).
Synthesis of 2-Oxindole-3-acetic acid (9). Compound 9 was syn-
thesized according to Lawson and Witkop (25) by treating 5.0 g of 2
(28.5 mmol) in 250 mL of acetic acid (50%) with 9.2 g of N-
bromsuccinimide (57 mmol) in 25 mL of glacial acetic acid. The
mixture was stirred for 1 h at room temperature. A palladium-katalysator
(500 mg, 10% on carbon) was added, and the solution was stirred for
further 17 h under hydrogen, filtered, and the solvent was removed.
The residue was poured into water, extracted with ethyl acetate, and
dried with sodium sulfate. The solvent was removed, and the crystalline
deposit was recrystallized from a mixture of toluol/diethyl ether (50:
50, v:v) yielding 2.9 g of 9 (15.2 mmol, 52%). C10H9NO3. M ) 191.2.
1
Bright yellow crystals, mp 142 °C. Purity 98%. H NMR: δ ) 7.21
(1H, d, J ) 7.6 Hz, H-4), 7.15 (1H, dd, J ) 7.6 and 7.6 Hz, H-6), 6.90
(1H, dd, J ) 7.6 and 7.6 Hz, H-5), 6.80 (1H, d, J ) 7.6 Hz, H-7), 3.62
(1H, dd, H-3), 2.89 (1H, dd, H-8â), 2.69 (1H, dd, J ) 17.3 Hz, H-8R)
ppm. 13C NMR: δ ) 184.3 (C-9), 178.3 (C-2), 133.1 (C-6), 129.3
(C-4), 127.0 (C-5), 114.9 (C-7), 47.6 (C-3), 40.1 (C-8) ppm. MS (EI):
m/z ) 191 [M + H]+ (20), 145 (100), 117 (64), 99 (19), 90 (38), 78
(51), 69 (9), 63 (14), 56 (23), 51 (21). IR (KBr): 3250, 1735, 1684,
1621, 1487, 1472, 1408, 1341, 1266, 1214, 1155, 1105, 1020 cm-1
.